ANALYSIS OF THE STRUCTURE AND FUNCTION OF A GALANIN RECEPTOR GENE

Arie Steven Jacoby

A thesis submitted in fulfilment of the requirements for the degree of Doctor of Philosophy

Garvan Institute of Medical Research Faculty of Medicine The University of New South Wales

October, 2000 ABSTRACT

The neuropeptide galanin is distributed throughout the nervous system and endocrine tissues of mammalian species. It is involved in numerous functions such as modulation of neurotransmitter and hormone release, pain transmission and appetite. Galanin is secreted by neurons and endocrine cells and exerts its effects by activating receptors of the G protein­ coupled receptor family. Three subtypes of galanin receptor have been characterised by molecular cloning but their specific in viva roles remain undefined. The aim of this project was to investigate the structure and function of the gene encoding the galanin receptor, GALRl, with a focus on the development of a genetically modified mouse model in which the receptor was selectively ablated.

The gene encoding GALRl was isolated from a mouse genomic library and the structure and sequence of this gene were determined. This information was used to design and construct a gene targeting vector and a line of GALRl knockout mice was derived by standard procedures. To provide a framework for analysis of the GALRl knockout mouse, a preliminary study of the expression of the genes encoding the galanin receptors in mouse tissues was undertaken. The mRNAs encoding the three receptors were widely expressed in the adult mouse, as well as in the embryo and placenta. Interestingly, the absence of ligand, examined in tissues of the galanin knockout mouse, had no major, consistent effect on receptor gene expression. The GALRl knockout mice were viable, healthy and able to reproduce normally. Their growth rate was similar to wild-type mice. However, a subset of GALRl knockout mice were susceptible to generalised epileptic seizures induced by minor stressful stimuli. Dark-cycle filming revealed that the mice also suffered spontaneous seizures, usually upon awakening from a period of sleep. These results implicate GALRl as an essential component of an inhibitory pathway controlling neuronal excitability in the mammalian limbic system.

The findings presented increase our knowledge of the function and expression of the galanin receptors. They provide a basis for the possible involvement of galanin in the pathogenesis of epilepsy and suggest that GALRl is a potential target for novel anti-convulsants.

ii ACKNOWLEDGMENTS

For the completion of this saga, I was reliant upon the expertise and willing assistance of numerous people, without whom I would definitely not have reached this point.

First and foremost, I have been privileged to benefit from the supervision of two most dedicated scientists. I am indebted to Dr Tiina Iismaa for her exceptional guidance, her constant support and, above all, her patience. I also thank my co-supervisor, Prof John Shine, not only for his patience, but for his much-appreciated insight and encouragement.

I am extremely grateful to Dr Peter Noakes for taking the time to teach me everything I needed to know about embryonic stem cells and for providing valuable reagents and an infectious enthusiasm for the subject. I would also like to thank Prof Ashley Dunn for his advice in the early stages of the project and Dr Graham Webb for his excellent chromosomal localization data. I am grateful to Dr Chris Ormandy for helpful discussions and to Dr David Wynick for his encouragement and for providing the galanin knockout mice. Several crucial experiments could not have been undertaken without the irradiation services provided by Dr Peter Cross and the staff of the Department of Radiation Oncology, St. Vincent's Hospital, and I acknowledge their assistance. I would also like to thank Sharon Fielder, Irene Michas and Gabrielle McCoy for help with tissue culture and Marjorie Liu for her DNA sequencing expertise.

Sincere thanks to my lab buddy Yvonne Hort for her friendship and conversation, her tolerance of my moods and mumblings and her tireless genotyping efforts. I am also grateful to the other members of the Neurobiology Program for their interest and support, in particular, Drs Linda Adams, Renee Badenhop, Bret Church, Kharen Doyle and Trevor Lewis for their proofreading efforts and Dr Barbara Depczynski for her willingness to answer endless queries on all matters medical and hypophysial.

The encouragement of my family and friends over a long period has always been greatly appreciated. For her inexhaustible support and understanding, my best friend, Simone, deserves special mention, but I know of no words which could express my gratitude. I have simply been very lucky. Finally, I reserve my most heartfelt thanks for my parents, the strongest people I know and a constant inspiration, and my Omama, who is proud of me even though I haven't discovered a cure for a single one of her ailments.

This project was supported by a Dora Lush Biomedical Postgraduate Scholarship from the National Health and Medical Research Council of Australia and a Garvan Institute Postgraduate Scholarship.

iii TABLE OF CONTENTS

ABSTRACT ...... ii ACKNOWLEDGMENTS ...... iii

TABLE OF CONTENTS ...... i V LIST OF FIGURES AND TABLES ...... ix PUBLICATIONS ARISING FROM THIS WORK ...... xi ABBREVIATIONS ...... xii

CHAPTERl GENERAL INTRODUCTION...... 1 1.1 GALANIN ...... 2 (i) Galanin peptide ...... 2 (ii) Galanin gene structure ...... 9 (iii) Distribution of galanin ...... 9 (iv) Regulation of galanin gene expression ...... 12 1.2 BIOLOGICAL EFFECTS OF GALANIN ...... 15 (i) Memory and learning ...... 15 (ii) Seizure modulation ...... 16 (iii) Nociception and analgaesia ...... 17 (iv) Peripheral nerve injury and regeneration ...... 18 (v) Appetite and body weight ...... 19 (vi) Neuroendocrine function ...... 21 (vii) Gastrointestinal motility and hormone release ...... 23 (viii) Other actions ...... 24 1.3 G PROTEIN-COUPLED RECEPTORS ...... 26 (i) Structure and mechanism of G protein-coupled receptors (GPCRs) ...... 26 (ii) Gene structure of GPCRs ...... 29 1.4 GALANIN RECEPTORS ...... 29 1.5 GALRl ...... 30 (i) Molecular cloning of GALRl ...... 30 (ii) Pharmacology of GALRl ...... 32 (iii) Structure-function studies of GALRl...... 33 (iv) Distribution and regulation of GALRl expression...... 35 1.6 OTHER GALANIN RECEPTORS ...... 43 (i) GALR2 ...... 43

iv (ii) GALR3 ...... 47 (iii) Evidence for further galanin receptor subtypes ...... 48 1.7 OVERVIEW OF THESIS AIMS ...... 50

CHAPTER2 GENERAL METHODS ...... 53 2.1 MATERIALS ...... 54 (i) Chemicals and enzymes ...... 54 (ii) Common reagents and media ...... 55 (iii) Kits ...... 55 (iv) Tissue culture reagents ...... 55 2.2 RECOMBINANT LIBRARY SCREENING ...... 56 2.3 PREPARATION OF DNA ...... 56 (i) Preparation of bacteriophage 'A DNA ...... 56 (ii) Small scale preparation of plasmid DNA ...... 57 (iii) Large scale preparation of plasmid DNA ...... 58 (iv) Small scale extraction of genomic DNA from embryonic stem (ES) cells ...... 58 ( v) Large scale extraction of genomic DNA from ES cells ...... 59 (vi) Extraction of DNA from mouse tail biopsies ...... 60 2.4 ELECTROPHORESIS AND TRANSFER OF DNA ...... 60 (i) Agarose gel electrophoresis ...... 60 (ii) Polyacrylamide gel electrophoresis ...... 61 2.5 RADIOACTIVE LABELLING OF DNA ...... 61 (i) Random priming ...... 61 (ii) 5' end labelling ...... 62 (iii) Preparation of spin columns ...... 62 2.6 HYBRIDIZATION OF DNA ...... 62 (i) Hybridization of radioactively labelled DNA fragments ...... 62 (ii) Hybridization of oligonucleotide probes ...... 63 2.7 CLONING OF DNA ...... 63 (i) Restriction digestion ...... 63 (ii) Gel purification of DNA fragments ...... 64 (iii) Ligation of DNA into plasmid vectors ...... 64 (iv) Linker ligation ...... 64 (v) Generation of blunt-ended restriction fragments ...... 65 (vi) Preparation of competent cells ...... 65

V (vii) Bacterial transformation ...... 65 (viii) Transfer and immobilization of plasmid and bacteriophage DNA ...... 66 2.8 DNA SEQUENCING ...... 66 2.9 PREPARATION OF RNA ...... 66 (i) Total RNA preparation ...... 66 (ii) Poly-A+ RNA preparation...... 67 2.10 QUANTITATION OF NUCLEIC ACIDS ...... 67 2.11 POLYMERASE CHAIN REACTION AND VARIATIONS ...... 67 (i) Polymerase chain reaction (PCR) ...... 67 (ii) Reverse transcriptase (RT-) PCR...... 68 (iii) 5'/3'-Rapid Amplification of cDNA Ends (RACE) ...... 68

CHAPTER3 CLONING AND CHARACTERIZATION OF THE MOUSE Galnr1 GALANIN RECEPTOR GENE ...... 70 3.1 INTRODUCTION ...... 71 3.2 METHODS ...... 73 (i) Library screening ...... 73 (ii) Subcloning ...... 73 (iii) Cloning of partial cDNA ...... 73 (iv) Restriction Mapping ...... 74 (v) DNA Sequencing ...... 74 (vi) 5'- and 3'-RACE ...... 75 (vii) PCR amplification of the 3'-UTR ...... 76 (viii) Chromosomal localization of Galnrl ...... 76 3.3 RESULTS ...... 77 (i) Cloning and characterization of the mouse Galnrl gene ...... 77 (ii) Cloning of the 5' end of the GALRl cDNA ...... 86 (iii) Characterization of the 3' end of the GALRl cDNA ...... 88 (iv) Chromosomal localization of the Galnrl gene ...... 88 3.4 DISCUSSION ...... 89

CHAPTER4 DEVELOPMENT OF A GALRl "KNOCKOUT" MOUSE STRAIN...... 95 4.1 INTRODUCTION ...... 96

vi 4.2 METHODS ...... 98 (i) Construction of targeting vector ...... 98 (ii) Primary embryonic fibroblast (PEF) preparation and inactivation ...... 99 (iii) ES cell culture ...... 100 (iv) Electropora tion of ES cells ...... 101 (v) Colony picking and culture ...... 101 (vi) Freezing ES cells ...... 102 (vii) Thawing ES cells ...... 102 (viii) PCR screening of ES cell clones ...... 102 (ix) Southern hybridisation screening of ES cell clones and chimaeras ...... 103 (x) Blastocyst injection ...... 105 (xi) Breeding and screening of chimaeras ...... 105 (xii) PCR genotyping ...... 106 (xiii) Analysis of gene expression by RT-PCR...... 106 (xiv) Other procedures ...... 107 4.3 RESULTS ...... 109 (i) Isolation of a targeted ES cell clone ...... 109 (ii) Breeding of GALRl knockout mice ...... 112 (iii) Genotyping of GALRl knockout mice ...... 115 (iv) Expression of galanin and galanin receptor genes in GALRl knockout mice ...... 115 (v) Growth and development of GALRl knockout mice ...... 121 (vi) Seizure behaviour in GALRl knockout mice ...... 121 4.4 DISCUSSION ...... 123

CHAPTERS EXPRESSION OF GALANIN RECEPTOR-ENCODING GENES IN THE MOUSE ...... 133 5.1 INTRODUCTION ...... 134 5.2 METHODS ...... 136 (i) Mice ...... 136 (ii) RT-PCR for detection of gene expression ...... 136 5.3 RESULTS ...... 137 (i) Expression of galanin receptor-encoding genes in wild-type mouse ...... 137

vii (ii) Differences in expression between wild-type and galanin knockout mice ...... 139 (iii) Expression of galanin and its receptors in wild-type mammary gland and pituitary...... 139 (iv) Developmental expression of galanin and galanin receptor genes ...... 140 (v) Developmental expression in galanin knockout mice ...... 148 5.4 DISCUSSION ...... 148

CHAPTER6 SUMMARY AND CONCLUSIONS ...... 154

REFERENCES ...... 160

SPECIAL ENCLOSURE CD-ROM containing Quicktime video of three episodes of seizure behaviour exhibited by GALRl knockout mice ...... REAR POCKET

APPENDIX Jacoby A.S., Webb G.C., Liu M.L., Kofler B., Hort Y.J., Fathi Z., Bottema C.D.K., Shine J. and Iismaa T.P. (1997) Structural organization of the mouse and human GALRl galanin receptor genes (Galnr and GALNR) and chromosomal localization of the mouse gene. Genomics, 45: 496-508 ...... 195

viii LIST OF FIGURES AND TABLES

FIGURE 1.1 Alignment of the amino acid sequence of mature human galanin with (a) galanin of other species, (b) galanin-like peptide (GALP) and (c) tuna galanin and invertebrate neuropeptides ...... 4 FIGURE 1.2 Alignment of amino acid sequence of preprogalanin in human, rat, mouse, pig, cow and dog ...... 7 FIGURE 1.3 Structural organisation of the human GALN gene ...... 8 FIGURE 1.4 Representation of the three dimensional structure of a G protein-coupled receptor ...... 27 FIGURE 1.5 Comparison of the structural organisation of the genes encoding the human galanin receptors GALNR1, GALNR2 and GALNR3 ...... 31 FIGURE 1.6 Signal transduction pathways of the cloned galanin receptors ...... 34 FIGURE 1.7 Molecular model of the human GALRl receptor-ligand complex as viewed from the plane of the membrane ...... 36 TABLE 1.1 Tissue distribution of galanin receptor subtypes ...... 38 TABLE 3.1 Sequence of DNA sequencing primers ...... 75 FIGURE 3.1 Genomic organisation of GALRl galanin receptor genes in mouse (Galnrl) and human (GALNRl) ...... 78 FIGURE 3.2 Nucleotide and deduced amino acid sequence of the mouse gene encoding GALRl...... 80 FIGURE 3.3 Alignment of mouse, rat and human GALRl amino acid sequences ...... 84 FIGURE 3.4 Alignment of the nucleotide sequences immediately upstream of the translation initiation codon of the Galnrl gene in mouse and rat...... 87 FIGURE 3.5 Silver grains over mouse Chromosome 18 hybridised in situ with tritiated cDNA derived from the Galnrl gene ...... 90 FIGURE 3.6 Alignment of the deduced amino acid sequences of mouse GALRl, GALR2 and GALR3 ...... 93 TABLE 4.1 Electroporation conditions for transformation of ES cells ...... 101 FIGURE 4.1 Approach used to disrupt the Galnrl gene ...... 104 TABLE 4.2 Oligonucleotides used for the amplification and detection of mRNA encoding galanin and its receptors ...... 108 FIGURE 4.2 Screening of ES cell clones by PCR...... 110

ix FIGURE 4.3 Screening of ES cell clones by Southern hybridization ...... 113 FIGURE 4.4 Agarose gel showing PCR products from a genotyping assay ...... 116 FIGURE 4.5 Expression of GALRl mRNA in the Galnrl -I- mouse ...... 118 FIGURE 4.6 Expression of genes encoding components of the galaninergic system in tissues of + / + and Galnrl -/- mice ...... 120 FIGURE 4.7 Mean body weight of GALRl knockout females (a) and males (b) ...... 122 FIGURE 5.1 Expression of genes encoding the galanin receptors in tissues of wild-type and galanin knockout mice ...... 138 FIGURE 5.2 Expression of the genes encoding galanin and the galanin receptors in virgin mouse mammary gland and pituitary gland ...... 140 FIGURE 5.3 Expression of the gene encoding galanin in the (a) wild- type and (b) galanin knockou t mouse embryo ...... 142 FIGURE 5.4 Expression of the genes encoding GALRl, GALR2 and GALR3 in wild-type and galanin knockout mouse embryo ...... 144 FIGURE 5.5 Expression of the genes encoding galanin, GALRl, GALR2 and GALR3 in the wild-type and galanin knockout mouse placenta ...... 146

X PUBLICATIONS ARISING FROM THIS WORK

Jacoby A.S., Webb G.C., Liu M.L., Kofler B., Hort Y.J., Fathi Z., Bottema C.D.K., Shine J. and Iismaa T.P. (1997) Structural organization of the mouse and human GALRl galanin receptor genes (Galnr and GALNR) and chromosomal localization of the mouse gene. Genomics 45: 496-508.

Jacoby A.S., Hort Y.J., Shine J. and Iismaa T.P. (2000) An essential role for galanin receptor subtype GALRl in tonic modulation of neuronal excitability. (In preparation)

xi ABBREVIATIONS

ACh Acetylcholine bp Base pair BSA Bovine serum albumin cAMP Adenosine 3',5'-cyclic monophosphate cDNA Complementary deoxyribonucleic acid CHO Chinese hamster ovary CNS Central nervous system cpm Counts per minute DMEM Dulbecco's modification of Eagle's Medium DMSO Dimethyl sulphoxide DNA Deoxyribonucleic acid DNase Deoxyribonuclease DRG Dorsal root ganglia En Day n of embryonic development EC Extracellular loop EDTA Ethylenediaminetetraacetic acid ES Embryonic stem FCS Foetal calf serum FISH Fluorescence in situ hybridisation G protein GTP-binding protein GABA g-Aminobutyric acid galanin-LI Galanin-like immunoreactivity GALP Galanin-like peptide GALRl Type 1 galanin receptor GALR2 Type 2 galanin receptor GALR3 Type 3 galanin receptor GDP Guanosine 5'-diphosphate GH Growth hormone GHRH Growth hormone-releasing hormone GMAP Galanin message-associated peptide GMAP-LI Galanin message-associated peptide-like immunoreactivity GnRH Gonadotrophin-releasing hormone GPCR G protein-coupled receptor GTP Guanosine 5'-triphosphate HEK Human embryonic kidney HMCB Human Bowes melanoma cell line

xii IC Intracellular loop kb Kilobase pair Kir G protein-coupled, inwardly rectifying K+ channel subunit LB Luria-Bertani medium LH Leuteinizing hormone LIF Leukaemia inhibitory factor MAPK Mitogen-activated protein kinase mRNA messenger ribonucleic acid NGF Nerve growth factor NPY Neuropeptide tyrosine OD Optical density PBS Phosphate-buffered saline PCR Polymerase chain reaction PEF Primary embryonic fibroblasts PEG Polyethylene glycol pfu plaque forming units PGK Phosphoglycerate kinase PNS Peripheral nervous system POMC Pro-opiomelanocortin PRL Prolac tin PTZ Pentylenetetrazole PVN Paraventricular nucleus of the hypothalamus QTL Quantitative trait locus RACE Rapid amplification of cDNA ends RNA Ribonucleic acid RNase Ribonuclease rpm Revolutions per minute RT Room temperature RT-PCR Reverse transcriptase-PCR S.E.M Standard error of the mean SDS Sodium dodecyl sulphate sec Seconds SM Suspension medium (for bacteriophage) SON Supraoptic nucleus of the hypothalamus SSC Saline sodium citrate sst2 Type 2 somatostatin receptor sst3 Type 3 somatostatin receptor TAE Iris-acetate EDTA buffer

xiii TE Tris-EDTA buffer TEMED N,N,N',N'-tetramethylethylenediamine TM Transmembrane domain UTR Untranslated region X-gal 5-bromo-4-chloro-3-indoyl-B-D-galactopyranoside

XIV CHAPTER 1

GENERAL INTRODUCTION Chapter 1

Understanding the functioning of the mammalian nervous system is one of the greatest challenges facing modern science. This most complex of organ systems is responsible for the co-ordination of innumerable neuronal pathways, processing of ceaseless streams of sensory information and providing the capacity for creative accomplishments of higher function and conscious thought. The brain exerts tight control over our physiology by directing the flux of a diverse array of chemical signals between cells. The communication of chemical messages between neurons directs the ion flow which underlies cellular alteration, metabolism and growth. The signalling molecules carrying out these tasks range in structure from the simplest chemicals to membrane-bound glycoproteins. All have specific modulatory roles which are integrated to coordinately regulate the development and function of neurons, neuronal circuits and whole nervous systems.

The release and uptake of chemical messengers, and the sensitivity and strength of response of cells and tissues to these signals, are tightly controlled at a number of different levels, starting with the gene. An imbalance in any component of the system can lead to the serious pathologies characterising mental illness or nervous system disorders. A deep understanding of each signalling pathway, and how the pathway integrates with the whole system, is the key to the elucidation of the basis of nervous system pathology. Neuropeptides comprise a large class of signalling molecule which evokes relatively slow but long-lasting effects in neurons and endocrine cells. Neuropeptides can be as small as three amino acids long or larger than 100 amino acids. In particular, we know of several relatively small peptides which are produced throughout the nervous system and possess a broad range of activities, including regulation of neurotransmission, hormone release, nerve repair and appetite. One of these neuropeptides, galanin, is the focus of this dissertation.

1.1 GALANIN

(i) Galanin peptide

The galanin peptide was initially isolated from porcine small intestine using a procedure for detecting peptides with a C-terminal amide group, a feature of many bioactive peptides (Tatemoto et al., 1983). It was named for its

2 Chapter 1 amino- (N-) terminal glycine and carboxy- (C-) terminal alanine residues. The cDNA encoding rat galanin was cloned, independently of the peptide sequence, as a pituitary tumour transcript strongly induced by chronic estrogen treatment (Vrontakis et al., 1987). In the past 17 years galanin has been shown to be involved in many physiological processes, in both the nervous system and the neuroendocrine system of mammals (Crawley, 1995). It is also found in birds, reptiles, amphibia and fish, while invertebrates such as molluscs, blowfly and sea cucumber display galanin­ like immunoreactivity (galanin-LI) (Iismaa and Shine, 1999).

The amino acid sequence of galanin is known for 16 vertebrate species, ranging from humans to teleost fish (Figure l.l(a)) (Iismaa and Shine, 1999; Wang et al., 1999). Comparison of these sequences reveals a very strong conservation within the N-terminal half of the peptide. Indeed, apart from the alanine in position six of tuna galanin, the first 14 amino acids are identical in all species examined. The C-terminal portion of the peptide is somewhat more divergent, and it has been well demonstrated that, in most instances, this region is not crucial to the binding of galanin to its known receptors. A distinctive difference is found in the human peptide, which is one residue longer than the 29 amino acids found to constitute all other galanin orthologues (Evans and Shine, 1991). The 30 amino acid human galanin is also non-amidated, but this difference appears to have no significant effect on its functional capability (Carey et al., 1993).

The galanin family has recently expanded with the discovery of a novel galanin-like peptide, GALP (Ohtaki et al., 1999). GALP is the only known mammalian peptide sharing amino acid identity with galanin. It is a processed 60 amino acid peptide isolated from porcine hypothalamus, and residues 9-21 of GALP are identical to the N-terminal 13 amino acids of galanin (Figure 1.l(b)). The functional relationship between galanin and GALP is not yet known, although they can interact with a common receptor, as will be discussed (Section 1.6(i)). Interestingly, a family of insect neuropeptides, with similarity to the N-terminal eight residues of galanin, has been discovered (Figure 1.l(c)). These include Lom-MIP and Bom-PTSP, identical peptides isolated from the locust and silkworm, respectively (Hua et al., 1999; Schoofs et al., 1991). Lom-MIP has myoinhibitory properties while Bom-PTSP is involved in the hormonal control of steroidogenesis.

3 FIGURE 1.1 Alignment of the amino acid sequence of mature human galanin with (a) galanin of other species, (b) galanin-like peptide (GALP) and (c) tuna galanin and invertebrate neuropeptides. Dashes represent identity with human galanin; a gap has been introduced into the human and tuna sequences in (c) to optimise the alignment. The sequence of dogfish galanin is known only for the first 20 amino acids. GRB-Ast-B1 was isolated from the cricket, Gryllus bimaculatus; LOM-MIP was isolated from the locust, Locusta migratoria; Bom-PTSP was isolated from the silkworm Bombyx mori; MAS-MIP I and MAS-MIP II were isolated from the tobacco hornworm, Manduca sexta.

4 Chapter 1

(a)

Human GWTLNSAGYLLGPHAVGNHRSFSDKNGLTS Rat, Mouse ------ID------H---(amide) Pig ------ID-----H--Y--A(amide) Cow ------LDS----Q--H--A(amide) Sheep ------ID-----H--H--A(amide) Dog ------ID-----HE-P---(amide) Chicken, Quail ------D-----N--H-F-(amide) Alligator ------ID-----NE-H-IA(amide) Frog ------ID-----N--H--A(amide) Tortoise ------D----LI--H--A(amide) Bowfin ------D----LN--H--A(amide) Dogfish ------D--- Sturgeon ------DG---LS--H--P(amide) Trout ------GIDG--TL---H--A(amide) Tuna -----A------GIDG--TLG--P--A(amide)

(b)

GAL ...... GWTLNSAGVLLGPHAVGNHRSFSDKNGLTS GALP APAHRGRG------VLHLPQMGDQ-GKRE-A LEILDLWKAIDGLPYSHPPQPD

(c)

Human galanin GWT LNSAGYLLGPHAVGNHRSFSDKNGLTS Tuna galanin --- --A------GIDG--TLG--P--A(amide) Grb-AST-B1 --QD--GGW(amide) LOM-MIP A-QD--A-W(amide) Bom-PTSP A-QD----W(amide) Mas-MIP I A-QD----W(amide) Mas-MIP II --QD----W(amide)

5 Chapter 1

Other inhibitory peptides closely related to these two include Mas-MIP I, Mas-MIP II and Grb-AST Bl from the tobacco hornworm and cricket, respectively (Blackburn et al., 1995; Lorenz et al., 1995). The significance of these peptides in relation to vertebrate galanin is unclear, but an evolutionary link, both structurally and functionally, is conceivable.

Galanin is synthesised as a precursor peptide, preprogalanin, which is post­ transla tionall y processed to its active form by proteolytic cleavage. Preprogalanin consists of a 23 amino acid hydrophobic signal peptide, followed by a prepeptide of nine amino acids, the 29-30 amino acid galanin peptide and a C-terminal peptide 59-60 amino acids long known as galanin message-associated peptide (GMAP) (Figures 1.2 and 1.3) (Iismaa and Shine, 1999). Lys-Arg dibasic motifs flank the galanin sequence within the precursor, presenting a substrate for proteolytic cleavage by a trypsin-like endoprotease activity (Rokaeus, 1994). The C-terminal cleavage site is preceded by a glycine residue in all species studied to date except human. This glycine acts as the amide donor in the conversion of galanin to a C­ terminally a-amidated peptide by the enzyme peptidyl-glycine a-amidating monooxygenase (Eipper et al., 1992). The human galanin sequence has a serine in place of the glycine at the C-terminus, resulting from a G to A nucleotide substitution in the first position of the corresponding codon, and thus the mature peptide escapes this processing step to remain a 30 residue non-amidated species (Evans and Shine, 1991).

The GMAP sequence, while less highly conserved than galanin, does retain an overall acidic nature in those sequences characterised. There is also strong homology in the C-terminal 30-35 residues (Figure 1.2). A definitive role for GMAP has not yet been ascertained. Comparative studies of the distribution of galanin- and GMAP-like immunoreactivity have predominantly demonstrated similarities in abundance (Hokfelt et al., 1992). GMAP and its fragments have also been shown to have inhibitory effects in rat spinal cord (Xu et al., 1995a), suggesting that a specific receptor may exist to mediate the actions of the peptide. The identity of this receptor is unknown, but it may couple to inhibition of stimulated adenylyl cyclase activity (Andell-Jonsson and Bartfai, 1998).

6 Chapter 1

1 32 Human MARGSALLLASLLLAAALSASAGLWSPA-KEKR Rat MARGSVILLAWLLLVATISATLGLGMPT-KEKR Mouse MARGSVILLGWLLLVVTLSATLGLGMPT-KEKR Pig MPRGCALLLASLLLASALSATLGLGSPVVKEKR Cow MPRGSVLLLASLLLAAALSATLGLGSPV-KEKR Dog KEKR

MATURE GALANIN 33 62 Human GWTLNSAGYLLGPHAVGNHRSFSDKNGLTS Rat GWTLNSAGYLLGPHAIDNHRSFSDKHGLT­ Mouse GWTLNSAGYLLGPHAIDNHRSFSDKHGLT­ Pig GWTLNSAGYLLGPHAIDNHRSFHDKYGLA­ Cow GWTLNSAGYLLGPHALDSHRSFQDKHGLA­ Dog GWTLNSAGYLLGPHAIDNHRSFHEKPGLT-

GMAP 63 92 Human -KRELRPE-DDMKPGSFDRSI--PENNIMRTIIE Rat GKRELPLEVEEGRLGSVAVPL--PESNIVRTIME Mouse GKRELQLEVEERRPGSVDVPL--PESNIVRTIME Pig GKRELEPE-DEARPGGFDRLQ--SEDKAIRTIME Cow GKRELEPE-DEARPGSFDRPL--AENNVVRTIIE Dog GKRELPPE-DEGRSGGFAGPLSLSENAAVRTIME

93 123 Human FLSFLHLKEAGALDRLLDLPAAASSEDIERS Rat FLSFLHLKEAGALDSLPGIPLATSSEDLEQS Mouse FLSFLHLKGYRALDSLPGIPLATSSEDLEKS Pig FLAFLHLKEAGALGRLPGLPSAASSEDAGQS Cow FLYFLHLKDAGALERLPSLPTAESAEDAERS Dog FL

FIGURE 1.2 Alignment of amino acid sequence of preprogalanin in human, rat, mouse, pig, cow and dog (Iismaa and Shine, 1999). Numbering of residues is given for human preprogalanin. The reported sequence of dog preprogalanin is incomplete. Gaps introduced to optimise the alignment are indicated by dashes.

7 Chapter 1

1 23 33 62 123 AATAAA ATG STOP

FIGURE 1.3 Structural organisation of the human GALN gene. Diagram illustrating the genomic structure of GALN and its relationship to the preprogalanin cDNA. The portion of the peptide encoded by each of the six exons is indicated. Numbering corresponds to the amino acids of human preprogalanin. The positions of the translation initiation codon (ATG), termination codon (STOP) and polyadenylation site (AATAAA) are shown, as are the dibasic cleavage sites (Lys-Arg).

Several structural studies of synthetic galanin have been undertaken. It has been demonstrated that, while principally adopting a random structure in aqueous solution, a-helical regions can be induced in hydrophobic solvents such as trifluoroethanol or negatively charged reversed micelles (Ohman et al., 1998; Rigler et al., 1991). The helical regions are separated by a kink provided by the invariant proline at position 13 of the galanin sequence. Some short-range structural features were identified in rapid equilibrium with random coil conformations in human galanin in aqueous conditions. Most notably, the region between residues 3-11 is capable of forming a nascent helix incorporating a hydrophobic core (Morris et al., 1995). The helix is stabilised in the presence of helix-promoting solvents and may represent a structure recognised by the galanin receptors. This is consistent with findings that the determinants of galanin's activity reside within the N-terminal half of the peptide. Specifically, the major pharmacophores of galanin, as revealed by the activity of alanine-substituted analogues, were identified as Trp2, Asn5 and Tyr9, residues which would reside on the same face of an a-helix (Fathi et al., 1998b). This finding further supports the physiological relevance of an N-terminal helical structure. The peptide exists as a monomer at concentrations up to 1 mM (Morris et al., 1995), suggesting that galanin interacts with its receptors in a monomeric form.

8 Chapter 1

(ii) Galanin gene structure

The human gene encoding preprogalanin, denoted GALN, spans 6.5 kilobase pairs (kb) and is located at chromosome position llql3.3-13.5 (Evans et al., 1993). The mouse gene (Gain) has also been cloned (Kofler et al., 1996) and has been mapped to mouse proximal chromosome 19 (Guida et al., 1998). GALN is located within a genomic region which is known to be amplified in bladder, breast and small cell lung cancers and in squamous cell carcinoma (Ormandy et al., 1998a). The gene consists of six exons (Figure 1.3). The first exon encodes the 5'-untranslated region (UTR) of the preprogalanin transcript. Exon 2 encodes the majority of the signal sequence of the precursor peptide, ending before a Lys-Arg proteolytic cleavage site. Exon 3 starts with this dibasic motif and includes the first 13 amino acids of galanin itself. The fourth and fifth exon encode the remainder of galanin and the majority of GMAP, and exon 6 codes for the C-terminal third of GMAP and the 3'-UTR region. This genomic structure is conserved in mouse, rat and cow (Corness et al., 1997; Kofler et al., 1996; Rokaeus and Waschek, 1994).

It appears that an ancestral exon has been duplicated and inserted into different loci to encode exon 3 of GALN and a portion of GALP. This can be deduced from the DNA sequence of these exons, which are more highly conserved among species variants of GALP than between GALP and galanin (Ohtaki et al., 1999). Further comparison of GALN and the gene encoding GALP awaits a full description of the structure of the latter gene.

(iii) Distribution of galanin

The distribution of galanin mRNA and peptide has been extensively mapped in a range of vertebrate species (Merchenthaler et al., 1993). Of the mammals, most is known about galanin localisation in the rat, although studies have been carried out in man, monkey and mouse. Galanin mRNA has been detected by various methods such as Northern hybridisation analysis and in situ hybridisation. The galanin peptide has been visualised by immunohistochemistry or quantified by radioimmunoassay. Galanin is present in many regions of the mammalian central and peripheral nervous systems and endocrine tissues.

9 Chapter 1

Galanin in the CNS is produced primarily by neurons and secreted at synaptic terminals, although it has also been detected in glial cells in vivo following colchicine treatment (Xu et al., 1992). In the brain, the highest levels of galanin were detected in the hypothalamus, hippocampus, nucleus accumbens, amygdala, locus coeruleus and septum. In the spinal cord of rats, galanin-LI is seen at all levels. Immunoreactive perikarya are present in the superficial layers of the dorsal horn and in lamina X, while immunoreactive fibre networks are also seen in the ventral horn (Merchenthaler et al., 1993). Galanin is synthesised by a small proportion of primary sensory and motor neurons. Some galanin-LI is also detected in scattered fibres throughout the grey matter (Merchenthaler et al., 1993).

Galanin coexists with numerous neurotransmitters and other neuropeptides in neurons in various regions of the CNS. For example, in the basal forebrain and motor neurons of the spinal cord, galanin coexists with acetylcholine (Merchenthaler et al., 1993). In the hypothalamus, galanin may coexist with dopamine, y-aminobutyric acid (GABA), gonadotrophin-releasing hormone (GnRH) and vasopressin, depending on the region. In the brainstem, the majority of norepinephrine neurons contain galanin, while in dorsal root ganglia (DRG), galanin and calcitonin gene-related peptide coexist. Several other instances of coexistence of galanin and neurotransmitters or neuropeptides are known (Merchenthaler et al., 1993). As is the case for the other neuropeptides, the physiological function of galanin in the nervous system is believed to primarily relate to the modulation of the release or action of the coexisting neurotransmitter.

Outside the CNS, sites of expression of galanin include the pituitary gland, cardiovascular system, gastrointestinal and genitourinary tracts, liver, pancreas, thyroid and adrenal glands, eye and respiratory tract (Crawley, 1995; Iismaa and Shine, 1999; Rokaeus, 1994). At these sites, galanin may be found in autonomic ganglia, afferent fibres, intrinsic interneurons or secretory endocrine cells. Galanin is also found at low concentrations in the peripheral circulation, being detected at 700 pg/ml in rat serum. It has been determined that approximately 30% of serum galanin is secreted from the pituitary gland with the rest originating in the gut (Crawley, 1995). Galanin expression has even been detected in immune cells in a rat model of dermal inflammation (Ji et al., 1995)

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The distribution of GMAP has been assessed by a specific antiserum and, for the most part, overlaps with galanin-LI. Some exceptions have been observed in the periphery: in the retinal cones and anterior pituitary, GMAP-like immunoreactivity (GMAP-LI) was detected in some cells not immunoreactive for galanin; in pancreatic beta cells, GMAP-LI was found to be more intense than GAL-LI (Hokfelt et al., 1992). The basis of these discrepancies is not known at present but may possibly involve a selective post-translational processing mechanism.

The ontogeny of galanin synthesis has been observed in the rat by in situ hybridization and immunohistochemistry. Galanin expression was detected in the conceptus by Northern analysis from day 5 of pregnancy (ES), soon after implantation of the embryo takes place (Vrontakis et al., 1992). This expression was shown to be localised to the decidual cells. Levels of galanin mRNA in the conceptus peak at Ell then gradually declined, becoming undetectable by Northern analysis by El8, at which time the decidua has regressed considerably. Consistent with this, serum levels of galanin-LI rise to a peak at El2, a seven-fold increase over levels in non-pregnant females. This suggests that decidual galanin may be secreted into the peripheral circulation and may act on distal tissues. It was speculated that galanin produced by decidual cells could act as an autocrine growth factor or may regulate placental prolactin (PRL) or PRL-like hormone production in a manner similar to pituitary galanin (see Section l.2(vi)) (Vrontakis et al., 1992).

In the rat embryo, galanin-LI has been detected in the brain at the earliest time point sampled, E14 (Xu et al., 1996a). Expression in DRG and trigeminal ganglia was also seen from El4, and by E19 was weaker in DRG. Galanin-LI was seen in neurons in the ventral horn of the embryonic spinal cord. Immunoreactive fibres were seen at E15-E17 in peripheral tissues, extending into the epithelium. Sensory systems displaying galanin expression from around ElS included the inner ear, the eye and the nasal mucosa. Other sites displaying galanin mRNA included intestine from E14 and developing bone at El9. The broad distribution of galanin expression in embryonic rat may indicate multiple developmental roles for the peptide, possibly including trophic actions (Xu et al., 1996a). Postnatally, galanin expression levels in rat brain have been reported to increase in intensity

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from day 1, reaching mature levels by day 28 (Giorgi et al., 1995; Ryan et al., 1997).

Of relevance to the current project, a study specifically addressing galanin gene expression in the mouse found levels of mRNA detectable by Northern hybridisation analysis in the hypothalamus/thalamus region, medulla, pons and spinal cord (Rokaeus and Waschek, 1998). Expression outside the CNS was widespread, with varied levels detected in most tissues examined. A recent reverse transcriptase- polymerase chain reaction (RT­ PCR) study done as part of a contribution to the mouse Gene Expression Database identified widespread expression of the galanin gene (Freeman et al., 1998). Of 45 tissue samples examined, including whole embryo and several brain regions, galanin mRNA was detected in every one. High levels of expression were seen in brain, spinal cord, pituitary, gastrointestinal tract, adrenal gland, pancreas and testis.

(iv) Regulation of galanin gene expression

The galanin cDNA was first isolated as an estrogen-regulated anterior pituitary transcript (Vrontakis et al., 1987) and, since that time, many other factors have been identified as having the capacity to alter galanin gene expression (lismaa and Shine, 1999). In rats, for example, galanin expression is upregulated by nerve growth factor (NGF) in the basal forebrain, by sympathetic activation in the adrenal medulla and by nerve injury or viral infection in the DRG and trigeminal ganglia (Rokaeus et al., 1998). It is inhibited by dopamine in the anterior pituitary gland (Hyde and Keller, 1991). Studies have shown upregulation of galanin mRNA in central neuronal populations following terminal field lesion (Holmes and Crawley, 1996). This induction in neurons following injury is thought to depend on both a stimulatory effect of leukaemia inhibitory factor (LIF) and the removal of a target-derived signal, NGF (Corness et al., 1998; Shadiack et al., 1998). Physiological disturbances shown to alter galanin expression in the hypothalamus include salt loading (Meister et al., 1990), hypophysectomy (Villar et al., 1990) and high fat intake (Mercer et al., 1996).

Much of the research into the regulation of galanin gene expression in vivo has concentrated on the pituitary and hypothalamus. Expression in both of these areas is influenced by circulating gonadal steroids and corticosteroids

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(Selvais et al., 1995). In the rat anterior pituitary, estrogen treatment elevates galanin mRNA levels 4000-fold and galanin-LI 50-fold, primarily affecting lactotrophs (Vrontakis et al., 1989). Galanin expression in hypothalamic GnRH neurons is altered by estrogen status in a sexually dimorphic manner (Finn et al., 1996). The human galanin gene is also estrogen-responsive (Howard et al., 1997b), however, in the human pituitary, galanin is only produced by corticotrophs and is not found in lactotrophs after estrogen stimulation (Hsu et al., 1991; Vrontakis et al., 1990). In the mouse pituitary, upregulation of galanin expression by one week of estradiol treatment has been demonstrated by ribonuclease protection assay (Shen et al., 1999), but a single injection of estradiol had no detectable effect (Lundkvist et al., 1995). The single injection protocol, which was reported to be effective in stimulating galanin expression in rat pituitary, was also ineffective in upregulating hypothalamic galanin expression in the mouse (Lundkvist et al., 1995). Moreover, seven days of dexamethasone treatment also had no significant stimulatory effect on galanin expression in the mouse (Lundkvist et al., 1995), a finding not consistent with the presence of a glucocorticoid response element in the mouse preprogalanin gene promoter (Kofler et al., 1996). Both acute and chronic administration of dexamethasone increased galanin mRNA levels in rat pituitary and uterus, without affecting levels of the peptide (Vrontakis et al., 1996). The reasons behind these inconsistencies are not clear but may indicate that galanin gene expression is regulated differently in rat and mouse. Galanin expression in other regions of the CNS may also be sensitive to estrogen, as locus coeruleus galanin gene expression was increased by estrogen in the rat (Tseng et al., 1997).

Thyroid hormone is another circulating factor that plays an important role in regulating galanin gene expression, in both the anterior pituitary (Hooi et al., 1997) and hypothalamus (Hyde et al., 1996). It is required for the full effect of estrogen on galanin expression as well as for basal levels of expression. Hypothyroid rats display diminished levels of galanin mRNA in anterior pituitary, while treatment of primary cultures of pituitary cells with thyroid hormone results in a dose-dependent increase in galanin rnRNA levels (Hooi et al., 1997).

Experiments in tissue culture systems have addressed galanin promoter function and responsiveness to growth factors and mediators. As predicted

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by in vivo studies, functional estrogen response elements have been found in the promoters of the rat (Corness et al., 1997), bovine (Rokaeus and Waschek, 1994) and human (Howard et al., 1997b) preprogalanin genes. A variety of primary cultures and cell lines have been used to demonstrate upregulation of galanin gene expression by NGF, LIF, phorbol esters and forskolin (Corness et al., 1997; Rokaeus et al., 1998). These last two effects suggest the involvement of protein kinase C (PKC) and protein kinase A signalling pathways. Basal enhancer activity has been found to reside within the first exon of the bovine galanin gene, while the regions of the promoter containing elements responsive to factors such as phorbol esters and NGF have been delineated (Rokaeus et al., 1998). A role for transcription factors of the activator protein-1 and cAMP response element­ binding protein families in the phorbol ester-induced activation of the gene has recently been demonstrated (Jiang et al., 1998).

Five kilobases of the bovine preprogalanin gene promoter was sufficient to achieve high levels of expression of a reporter gene in many regions of the central nervous system (CNS) of transgenic mice. However, it appeared to lack elements necessary for expression in some peripheral tissues where galanin is known to be expressed, such as the pituitary gland and intestine (Rokaeus and Waschek, 1998). A shorter promoter fragment of 131 base pairs (bp) enabled widespread expression of a reporter gene, including tissues expressing little or no endogenous galanin mRNA. Thus, the region of the galanin promoter between 131 bp and 5 kb upstream of the coding sequence contains inhibitory elements restricting expression in a manner approximating, but not reproducing, endogenous galanin gene expression (Rokaeus and Waschek, 1998).

In summary, numerous physiological alterations and biochemical factors are known to alter galanin gene expression in neurons and in the pituitary gland. In particular, tight regulation by changes in estrogen levels suggest that galanin may be important for maintaining neuroendocrine homeostasis in the female reproductive axis. The sensitivity to neuronal injury also indicates that galanin may have regenerative or trophic roles in both the central and peripheral nervous systems, as will be detailed in the following section.

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1.2 BIOLOGICAL EFFECTS OF GALANIN

Many biological actions of galanin have been described in the mammal. These include both responses to exogenously applied peptide and putative roles for endogenous galanin. An overview of some of these actions is presented, with particular reference to the mouse as a model for research in this field.

(i) Memory and learning

Galanin is colocalised with acetylcholine (ACh) in neuronal cell bodies in the basal forebrain of rats and several species of monkey (Crawley, 1996). The cholinergic circuitry originating in this region is intimately involved in memory functions. Research on rats spanning several years has provided strong evidence for an inhibitory role for galanin in these functions. Administration of galanin, into either the cerebral ventricles or the hippocampus, results in deficits in a variety of experimental memory tasks in rats (Malin et al., 1992; Sundstrom et al., 1988). These effects are more pronounced when cholinergic transmission is pharmacologically impaired (Robinson and Crawley, 1993). Lesions of cholinergic fibres projecting to the hippocampus induce deficits in memory acquisition tasks in rats, which can be partially reversed by injection of ACh into the ventral hippocampus. Co­ administration of galanin antagonises this restoration of memory performance (Mastropaolo et al., 1988). At relatively high doses, galanin injected alone can impair the acquisition of spatial learning tasks, implying an inhibitory effect on learning at pharmacological concentrations. Conversely, a galanin functional antagonist improves memory acquisition when administered intracerebroventricularly (Ogren et al., 1992). Therefore, at physiological doses, galanin may impinge on memory pathways by tonic inhibition of ACh release.

In humans, the co-existence of galanin and ACh in basal forebrain is not seen, however, small galanin-immunoreactive interneurons are found in close proximity to cholinergic cell bodies (Crawley, 1996). This may have functional significance in degenerative brain disorders, as evidence is accumulating that galanin may contribute to the cognitive deficits characteristic of Alzheimer's dementia and Parkinson's disease. Post­ mortem brain samples from sufferers of these neurodegenerative diseases

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show hyperinnervation of cholinergic neurons by galaninergic axon terminals, specifically in the frontal cortex (Chan-Palay, 1988). There is also an apparent up-regulation of galanin-LI in these terminals compared to age­ matched controls. Galanin concentration in the nucleus basalis of Meynert is higher in Alzheimer's brains, a region in which galanin binding sites persist despite depletion of cholinergic neurons in Alzheimer's disease (Beal et al., 1990). This increase in galanin synthesis may be a response to the axonal damage which occurs following plaque deposition. It is thought that galanin may contribute to memory deficits by inhibiting tonic release of ACh, which is already affected by degeneration of cholinergic neurons in the basal forebrain (Crawley, 1996). Galanin receptor antagonists may be potential therapeutic agents for alleviating the memory deficits that develop in Alzheimer's disease, possibly in combination with cholinesterase inhibitors or muscarinic receptor agonists.

(ii) Seizure modulation

Evidence is emerging that galanin may have inhibitory activity in hippocampal seizure states. In the rat, fibres displaying galanin-LI are normally present throughout the hippocampus whereas no galanin-LI is seen in neuronal cell bodies (Melander et al., 1986; Xu et al., 1998). The majority of galanin-containing fibres project from the medial septum, locus coeruleus and hypothalamus (Mazarati et al., 1998; Merchenthaler et al., 1993). There is also a high density of galanin binding sites in the hippocampus (Fisone et al., 1989; Skofitsch et al., 1986). The galanin-LI in hippocampal fibres disappears within a few hours of the onset of experimentally induced seizures, reappearing after one week. Galanin-LI appears in neurons as soon as three hours after status epilepticus, decreasing gradually in the following days but still present after one week (Mazarati et al., 1998). These changes may reflect depletion of galanin stores and protective neuronal adaptation, respectively. The changes may be confined to the hippocampal formation, as a study of galanin in the hypothalamus of chronically seizing female rats found no differences in immunoreactivity over control samples (Amado et al., 1993). This would indicate that the peptide is not involved in the endocrine and reproductive alterations seen in temporal lobe epilepsy and related animal models.

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Results of studies using various rat models of convulsive syndromes implicate galanin in the inhibition of seizure activity. The most extensive investigation, to date, into the properties of galanin in seizure modulation revealed that the peptide exerts a significant anti-convulsant effect in the hippocampus (Mazarati et al., 1998). In this study, galanin injected into the hippocampus was able to attenuate seizure induction by electrical stimulation, and this response was blocked by co-administration of galanin functional antagonists. Previously, it was demonstrated that galanin had an inhibitory effect on picrotoxin-induced convulsions when injected into the lateral ventricles, caudate putamen, substantia nigra and nucleus accumbens. Injection of galanin into the ventral tegmental area was not effective (Mazarati et al., 1992). The dose-dependency of the anticonvulsive effect varied between brain regions and may suggest that galanin has region­ specific mechanisms of action. Galanin administered intraperitoneally was also effective in reducing neonatal hyperthermia-induced seizures in rats, a model of childhood febrile convulsions (Chepurnov et al., 1997a). It has recently been reported that mice with a targeted deletion of the galanin gene, i.e. galanin "knockout" mice, have a shorter latency period and more severe responses in PTZ-induced seizures. Conversely, transgenic mice overexpressing galanin in noradrenergic neurons are more resistant to convulsive agents than wild-type mice (Mazarati et al., 1999).

The mechanisms involved in this seizure inhibition have not been elucidated. Galanin has inhibitory properties in the ventral hippocampus, as demonstrated by the presynaptic inhibition of cholinergic neurotransmission in the CAl region (Dutar et al., 1989). Galanin has also been shown to inhibit the release of the excitatory amino acids glutamate and aspartate in hippocampal and striatal slices, possibly via the activation of ATP-sensitive potassium channels (Ellis and Davies, 1994; Zini et al., 1993). This has been proposed as an endogenous neuroprotective mechanism against the excessive release of excitatory amino acids that may result from ischaemic or epileptic trauma (Ben-Ari and Lazdunski, 1989). It could plausibly explain the anti-convulsant action of galanin.

(iii) N ociception and analgaesia

Galanin is present in neurons of the DRG and dorsal horn of the spinal cord. Galanin binding sites are also found in the dorsal horn and deep

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spinal laminae (Kask et al., 1995). A series of functional and electrophysiological studies have implicated galanin in nociception and pain transmission in both the intact and injured state (Bartfai et al., 1993). For example, intrathecally administered galanin inhibits the flexor reflex to painful stimuli in rats and mice (Wiesenfeld-Hallin et al., 1989). The C­ terminal peptide GMAP has also been shown to have moderate effects on facilitation of the flexor reflex (Xu et al., 1995b). The analgaesic properties of morphine are potentiated by galanin, suggesting that morphine may act via a galanin-sensitive pathway (Wiesenfeld-Hallin et al., 1990).

The role of galanin in nociception may be more relevant following nerve injury, a state in which expression of the peptide in DRG is dramatically increased (Hokfelt et al., 1987). Chronic intrathecal infusion of galanin functional antagonists after sciatic nerve transection increases autotomy behaviour, a manifestation of neuropathic pain in rodents (Verge et al., 1993). Furthermore, the application of galanin antisense oligonucleotides to the injured nerve results in an increase in pain sensation and autotomy (Ji et al., 1994). This suggests a role for endogenous galanin in the inhibition of pain perception following nerve injury. However, this is far from a settled issue, as galanin knockout mice are hypoalgaesic following peripheral nerve injury and autotomy does not occur (Holmes et al., 1997; Wynick, 1997). These observations may be attributed to a developmental role for galanin in sensory neurogenesis. This series of findings has led to suggestions that galanin receptor agonists may be useful in the control of chronic pain, and further work is therefore required to reveal the receptor subtypes involved in the antinociceptive actions of galanin.

(iv) Peripheral nerve injury and regeneration

Under normal conditions, galanin is present in only very few neurons of the peripheral ganglia. An increase in the levels of galanin gene expression and peptide has been demonstrated as one of the specific neuronal responses to injury. Galanin expression is induced in sympathetic, sensory and motor neurons under pathological conditions (Crawley, 1995). The response to galanin in DRG neurons is also heightened (Xu et al., 1997). A variety of animal models of axotomy and inflammatory and neuropathic pain have been used to study the role of galanin in these processes. For example, ligation of the sciatic nerve induces a strong upregulation of galanin levels

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in the DRG (Hokfelt et al., 1987). In a partial ligation model, this occurs in both the injured neurons and, to a lesser extent, the uninjured neurons at the site of nerve injury (Ma and Bisby, 1999).

The induction of galanin gene expression is strongly dependent on LIF, as the response is greatly attenuated in LIP-deficient mice (Sun and Zigmond, 1996). Interleukin-6 is another cytokine released in response to injury that may be involved in the process, as it interacts with the same class of receptor as LIF and is able to stimulate galanin expression in intact sensory neurons (Thompson et al., 1998). The withdrawal of the retrogradely-transported factor NGF is also necessary for the axotomy response, as the combination of NGF-neutralising antiserum and LIF is able to partially mimic the effects of axotomy in intact neurons (Shadiack et al., 1998). This cannot be achieved by LIF treatment alone. Other unidentified factors are also involved as this combination did not achieve the same levels of galanin expression seen in axotomised neurons.

The complete role of galanin under conditions of nerve injury has not been elucidated, but it appears to be complex. The general profile of changes that occur in neurons in these circumstances suggests a switch from neurotransmission to survival and regenerative functions. In galanin­ deficient mice, functional recovery of sensory neurons following sciatic nerve crush is impaired (Wynick, 1997). In addition, recent data show that galanin may have trophic activity in cultures of dispersed DRG cells, with positive effects on neurite initiation and elongation (Mahoney et al., 1999). However, the addition of galanin to cultures of rat sympathetic neurons isolated from the superior cervical ganglion had no effect on the survival of these cells, suggesting cell type-specific actions (Klimaschewski et al., 1995). As discussed above, it is also believed that galanin may inhibit the development of neuropathic pain resulting from nerve damage.

(v) Appetite and body weight

Galanin is one of a large repertoire of neuropeptides known to regulate feeding behaviour and appetite via the hypothalamus (Schwartz et al., 2000). In satiated rats, injection of galanin into the paraventricular nucleus (PVN) of the hypothalamus, amygdala or nucleus of the solitary tract can stimulate feeding (Rowland and Kalra, 1997). This is a fast-acting but short-lived

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effect, lasting around 30 mins (Crawley et al., 1990). However, the chronic administration of galanin has no significant effect on body weight and does not alter overall daily food intake (Smith et al., 1994). In certain feeding paradigms, galanin can also affect nutrient choice, inducing preference for fat over high-protein or chow meal formulations (Tempel et al., 1988).

Compared to lean rats, obese rats had higher galanin gene expression in the PVN, while a high fat diet reduced the mRNA levels in these rats (Mercer et al., 1996). Another study showed increased galanin mRNA and peptide secretion in normal rats fed a high-fat diet (Leibowitz et al., 1998). This response has been localised to a group of PVN neurons projecting to the median eminence. Injection of galanin antisense oligonucleotides into the region containing these neurons leads to a decline in fat ingestion and a reduction in body weight (Akabayashi et al., 1994). Following on from these studies, a role for galanin in the development of obesity resulting from a high-fat diet has been postulated (Leibowitz et al., 1998).

Galanin knockout mice display normal feeding behaviour and body weight, indicating that the peptide is not required for normal feeding under conditions of unrestricted food supply. However, these mice are more sensitive to the anorectic effects of exogenous leptin than wild-type controls, suggesting that galanin may be involved in regulating leptin responses (Hohmann et al., 1999). The involvement of galanin in the central leptin pathway is further supported by the findings that leptin reduces hypothalamic galanin gene expression (Sahu, 1998a) and antagonises the hyperphagic response to galanin in the rat (Sahu, 1998b). The presence of leptin receptors in galaninergic neurons has been demonstrated in the PVN (Hakansson et al., 1998). The actions of galanin on feeding behaviour may be exerted through the stimulation of noradrenaline release in the PVN, as suggested by microdialysis experiments (Kyrkouli et al., 1992) and studies using adrenergic receptor antagonists and catecholamine synthesis inhibitors (Kyrkouli et al., 1990).

The contribution of galanin to appetite-related syndromes in humans is unclear. A small number of clinical studies on obese and anorexic patients have been inconclusive (Crawley, 1999b), while genetic linkage studies have reported no association between body fat and polymorphisms in the genes encoding either galanin or one of the galanin receptors (Kofler et al., 1998;

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Lapsys et al., 1999). The regulation of appetite and energy metabolism are finely balanced systems with a high degree of redundancy. Further work on the physiological role of galanin is required for a complete understanding of the role of peptide mediators in these processes. This, in turn, has important consequences for the management of obesity and related diseases.

(vi) Neuroendocrine function

The hypothalamic-pituitary axis exerts primary control over physiology, development and reproduction by the tightly controlled release of an array of hormones. Galanin influences the functioning of this axis at multiple levels by both modulating the release and activity of hypophysiotrophic factors and by acting directly on the anterior pituitary gland. Extensive evidence supports the view that galanin is an important autocrine and trophic factor in the pituitary gland.

Galanin is strongly expressed in the pituitary and can modulate the activity of a number of endocrine cell types. For example, galanin has been shown to be a trophic factor for lactotrophs in cultures of dispersed anterior pituitary cells (Wynick et al., 1993a) and in vivo in mice (Cai et al., 1999). It also stimulates secretion of PRL, with galanin knockout mice displaying defects in lactation and mammary gland development linked to low pituitary PRL levels (Wynick et al., 1998). Conversely, overexpression of galanin in lactotrophs leads to pituitary hyperplasia and enhanced prolactin secretion in female transgenic mice (Cai et al., 1999). Galanin may have an important role in pituitary growth and modulation in high estrogen states as it is strongly up-regulated in lactotrophs under these conditions. Following two weeks of estrogen treatment, one-third of pituitary lactotrophs express galanin mRNA (Cai et al., 1998). Galanin-expressing lactotrophs secrete significantly more PRL than those which do not secrete galanin. Galanin is also required for estrogen-induced pituitary hyperplasia. These data establish rodent galanin as an autocrine and paracrine lactotroph modulator. The relevance of these findings for humans is unclear, as it has been reported that in the normal human pituitary, galanin is found only in corticotrophs (Vrontakis et al., 1990). However, infusion of galanin at high doses does evoke a significant increase in plasma PRL levels in humans (Carey et al., 1993; Todd et al., 2000).

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As previously mentioned, galanin infusion into man results in the secretion of GH from the pituitary (Carey et al., 1993; Todd et al., 2000). This response may involve modulation of both somatostatin (Tanoh et al., 1993) and growth hormone releasing hormone (GHRH) release (Aguila et al., 1992; Murakami et al., 1989), although a direct effect of galanin on the pituitary has been reported (Gabriel et al., 1988). Experiments using transgenic mouse models have also demonstrated an important modulatory role for galanin in pituitary somatotrophs. GHRH-overexpressing mice develop somatotroph adenomas and show augmented production of galanin and growth hormone (GH) in the pituitary (Moore Jr. et al., 1999). Similar to lactotrophs, the somatotrophs expressing galanin in these mice secrete significantly more GH than the non-expressing somatotrophs. Therefore, it is thought that galanin may mediate the hyperplastic and hypersecretory effects of elevated GHRH levels (Moore Jr. et al., 1999).

The reproductive organs are regulated by a hypothalamo-pituitary-gonadal axis originating in hypothalamic GnRH neurons. GnRH stimulates release of luteinizing hormone (LH) from gonadotrophs in the pituitary, a function which can be modulated by galanin at the level of both the pituitary and hypothalamus. In rats, galanin is expressed by a subset of GnRH neurons and and is also located in nerve terminals surrounding GnRH cell bodies (Lopez et al., 1991). Galanin can induce the release of GnRH when applied to isolated rat hypothalamic fragments (Lopez and Negro-Vilar, 1990). It can also potentiate the effects of GnRH on LH secretion and directly stimulate LH release in pituitary cell cultures (Lopez et al., 1991). Galanin expressed in GnRH neurons is upregulated by estrogen and the full magnitude of the preovulatory LH surge is dependent upon endogenous galanin (Cheung et al., 1996). In fact, recent findings suggest an interplay between hypothalamic and pituitary galanin, with their combined actions on LH secretion dependent on the stage of the oestrous cycle (Todd et al., 1998). The involvement of galanin in ovulation appears to be species-specific, as infusion of galanin into humans has no observable effect on basal LH levels (Todd et al., 2000).

The levels of several hypothalamic and pituitary hormones are normal under basal conditions in galanin knockout mice (Wynick et al., 1998). This observation would tend to support the assertion that, in a broad sense, the major role of neuropeptides is in restoring homeostasis in response to

22 Chapter 1 periods of imbalance or disturbance (Hokfelt et al., 2000). Aside from its essential role in lactotroph function, it may only be under these circumstances that the neuroendocrine roles of galanin become apparent.

(vii) Gastrointestinal motility and hormone release

Porcine small intestine was the original source of galanin (Tatemoto et al., 1983) and multiple functions of the peptide in the gastrointestinal tract have since been described. The principal effects of galanin in the enteric nervous system include increasing passage time through the gut, autonomic effects on smooth muscle contraction and neuroendocrine effects on the secretion of hormones and components of the digestive milieu (Rokaeus, 1994). Most of the galanin present in the gastrointestinal tract is found in enteric neurons. In the mammalian species studied, including man, myenteric galanin-immunoreactive neurons send long projections which terminate in the myenteric ganglia or the smooth muscle (Rattan, 1990). Galanin-LI is also found in nerve fibres innervating the submucosal plexus, the muscle layers and the mucosa surrounding blood vessels (Rokaeus, 1994). The majority of galanin-immunoreactive neurons in the gut are intrinsic, but there is some input from the dorsal motor nucleus of the vagal nerve, and galanin is present in vagal afferents (Rokaeus, 1994). This localisation would suggest that galanin may act to transmit signals from the gut through the vagus, as well as being a modulator of enteric neuronal activity.

Galanin is thought to be an important transmitter in the enteric nervous system, with both a direct action on smooth muscle and modulatory activity on neuronal ACh release (Rattan, 1990). In the human gastrointestinal tract, galanin delays gastric emptying and increases the mouth to caecum transit time (Rokaeus, 1994). In mammalian model systems, both in vitro and in vivo, galanin has effects on smooth muscle contraction at many regions from the oesophageal sphincter to the anal sphincter. These effects can differ between regions and species and it is therefore difficult to extrapolate findings. For example, galanin inhibits contraction of cat oesophageal sphincter elicited by a variety of agonists (Lichtenstein et al., 1994) but stimulates contraction and suppresses relaxation of opossum oesophageal sphincter (Rattan and Goyal, 1987). In addition, galanin inhibits nerve­ stimulated contractions of canine pyloric sphincter but induces contraction in jejunal longitudinal muscle of human, rat and dog (Crawley, 1995).

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The effects of galanin on fluid and hormone secretion in the gastrointestinal tract have also been observed in several species. Intravenous galanin administration in man stimulates salivation and increases postprandial secretion of bicarbonate and electrolytes. Plasma levels of glucose, insulin, glucagon, peptide YY, pancreatic polypeptide, neurotensin and somatostatin are all suppressed (Rokaeus, 1994). In the rat, galanin exerts an inhibitory effect on the secretion of gastric acid, gastrin, somatostatin and amylase, while in the dog, bombesin-stimulated gastric acid production is inhibited (Rokaeus, 1994). Galanin has also been shown to diminish ion transport in the porcine jejunal mucosa (Brown et al., 1990). However, it induces Cl­ secretion in human colonic epithelium, suggesting that it may contribute to diarrheal syndromes (Benya et al., 1999). Other potential roles for galanin in gastrointestinal pathogenesis are suggested by evidence such as increased levels of the peptide in the colon of patients with chronic slow transit constipation (Sjolund et al., 1997) and higher levels of expression in enteric neurons of rats with partial obstruction of the ileum (Ekblad et al., 1998).

(viii) Other actions

Galanin has been shown to inhibit glucose-stimulated insulin release both in vitro and in viva. The elicitation of hyperglycaemia in dogs infused with galanin was among the first activities associated with the peptide (Tatemoto et al., 1983), and this effect has also been reported in mice (Lindskog and Ahren, 1987). In vitro, the effect has been demonstrated with isolated rat pancreatic islets (Leonhardt et al., 1989) and in the rat insulinoma cell line, RINmSF (Amiranoff et al., 1988). In viva, inhibition of secretion has been reported in several species (Rokaeus, 1994). This appears to be a species­ specific effect, however, as glucose-stimulated insulin release is not altered by galanin in humans in viva (Carey et al., 1993), but is inhibited in vitro (Ahren et al., 1991). Differing effects have also been observed depending on the particular analog or species of galanin peptide utilised in studies (Rokaeus, 1994). Galanin-LI is present in nerve fibres surrounding pancreatic islets (McDonald et al., 1992), where it may colocalise with noradrenaline (Ahren et al., 1990; Scheurink et al., 1992). Galanin is released by vagal stimulation from these nerves to bind to receptors present on the ~­ cell and activate pertussis-toxin sensitive signalling pathways, leading to a reduction in intracellular Ca2+ concentration (Bartfai et al., 1993). The

24 Chapter 1 peptide also inhibits pancreatic somatostatin secretion in rat and dog (Rokaeus, 1994).

A potential role for galanin in endocrine and certain other cancers has been proposed. Galanin stimulates mitogenesis in the rat prolactinoma cell line 235-1 and in dispersed anterior pituitary cultures (Hammond et al., 1996; Wynick et al., 1993a). Small cell lung cancer cells also show a mitogenic response to exogenously applied galanin (Sethi and Rozengurt, 1991). This response is mediated by the activation of the p42 isoform of mitogen­ activated protein kinase (MAPK) (Seufferlein and Rozengurt, 1996). Depending on the galanin receptor subtype involved, the stimulation of a mitogenic pathway involving MAPK can occur via a PKC-dependent or PKC-independent mechanism (Wang et al., 1998b). Galanin mRNA is expressed by several human breast cancer cell lines and is strongly regulated by estradiol in those lines which also express estrogen receptors (Ormandy et al., 1998b). Galanin expression by other hyperplastic cells has been documented, including neuroblastoma (Tuechler et al., 1998) and phaeochromocytoma (Bauer et al., 1986b), and corticotroph (Vrontakis et al., 1990) and somatotroph (Bennet et al., 1991) adenomas. The GALRl galanin receptor was initially cloned from a melanoma cell line, indicating that melanoma cells are responsive to galanin (Habert-Ortoli et al., 1994). The significance of galanin as an endocrine factor in tumourigenesis has not been clarified and conflicting results have been reported in different cancer models. For example, prolonged administration of galanin inhibits gastric carcinogenesis in rats (lishi et al., 1994).

Numerous other important neural and endocrine effects of galanin have been described. In addition to the pancreas, galanin is present in the sympathetic nerves innervating peripheral organs such as the heart, liver, adrenal glands and genital tract (Crawley, 1995). Modulatory effects of galanin released by stimulation of these nerves have been demonstrated at all of these sites. They include the inhibition of cardiac vagal action in man, where infusion of human galanin caused an increase in heart rate and a decrease in sinus arrhythmia (Carey et al., 1993). Similar responses have been seen in the cat and possum (Potter, 1998). In the lung, galanin inhibits the bronchoconstriction induced by tachykinins (Takahashi et al., 1994), while it is also released from the liver upon sympathetic stimulation, possibly influencing hepatic glucose output (Kowalyk et al., 1992).

25 Chapter 1

Elsewhere, galanin stimulates uterine contraction in the rat (Shew et al., 1992) and inhibits human urinary bladder contraction (Maggi et al., 1987). It may also play a role in initiating copulatory behaviour in rats and in anxiety-related behaviour (Crawley, 1995).

1.3 G PROTEIN-COUPLED RECEPTORS

(i) Structure and mechanism of G protein-coupled receptors (GPCRs)

Most neuropeptides, including galanin, exert their effects by activating receptors which are members of the superfamily of G protein-coupled receptors (GPCRs) (lismaa et al., 1995). A great sequence diversity within this class of proteins underlies a common structure. These receptors consist of a single polypeptide chain with seven a-helical hydrophobic domains which are thought to traverse the plasma membrane in a serpentine fashion (Ji et al., 1998). The receptor is oriented such that the N-terminus domain protrudes from the extracellular surface of the membrane while the C­ terminus resides cytoplasmically. The transmembrane (TM) domains, which form a cluster, are connected by loops which alternately reside intra­ and extracellularly (Figure 1.4). The ligand binding pocket may be formed by residues in the extracellular and TM regions of the peptide.

In addition to neuropeptides, a wide variety of molecules bind and activate GPCRs, including bioamines, glycoprotein hormones, lipids and odorants. It is estimated that the human genome may contain genes encoding several thousand different GPCRs, including the olfactory and pheromone receptors (Iismaa et al., 1995). The GPCR superfamily has been divided into subclasses on the basis of conserved structural and sequence features. In many cases, this classification has grouped receptors also related by type of ligand. For example, the majority of peptide receptors are classed as "Family lb" GPCRs (Bockaert and Pin, 1999). The galanin receptors share similarities with known receptors in this subclass of GPCRs, based not only on type of ligand but also on location of ligand binding site (Bockaert and Pin, 1999).

26 FIGURE 1.4 Representation of the three dimensional structure of a G protein­ coupled receptor. Putative transmembrane domains are represented as cylinders and their relative positions within the membrane are shown. They are numbered 1-7 in an anti­ clockwise direction. N-linked glycosylation of residues, depicted in green, may occur in the extracellular region. Attachment of the C-terminal domain to the cyto­ plasmic face of the plasma membrane by palmitoylation is shown.

27 Chapter 1

Signalling through a GPCR involves a trimeric protein complex known as a GTP-binding protein, or G protein. This complex consists of a, f3 and y subunits and associates with the receptor on the cytoplasmic surface of the plasma membrane (lismaa et al., 1995). The intracellular portions of the receptor, usually the third intracellular loop and C-terminal tail, are involved in binding G proteins. Current models of GPCR behaviour propose that the receptor, in an unoccupied state, fluctuates between inactive and active conformations. Ligand binds to the active conformation of the receptor and stabilises this conformation (Gether and Kobilka, 1998). This activation triggers the exchange of GDP for GTP on the G protein a subunit and the subsequent dissociation of the trimeric G protein complex into Ga and G13y subunits. These can both propagate intracellular signalling cascades initiated by binding of the ligand. The a subunit, which possesses inherent GTPase activity, hydrolyses the bound GTP to revert to its inactive GDP-bound form and terminate the activation of second messenger systems (Iismaa et al., 1995).

There are several different a and f3y subunits coupling to a variety of signal transduction mechanisms. These include the activation or inhibition of enzymes and ion channels and the subsequent generation of second messenger molecules in the cell (lismaa et al., 1995). Enzymes such as adenylyl cyclase, phospholipase C and phosphodiesterase catalyse the production of cAMP, inositol phosphates and arachadonic acid, respectively. Other effectors include ion channels such as the G protein-coupled inwardly rectifying potassium (Kir) channels, which can be activated directly by binding of G13y to initiate a faster response than that associated with enzymatic activity (Karschin, 1999).

As discussed, neuropeptides such as galanin have a multitude of in viva activities. There are various ways in which this diversity of action may be generated. Firstly, a single ligand may activate multiple GPCR subtypes. This is true for neurotransmitters such as ACh and dopamine and is also the case for many neuropeptides, including galanin, neuropeptide Y (NPY) and somatostatin (lismaa et al., 1995). Secondly, a single receptor may associate with more than one class of G protein, each with its own set of downstream events. Thirdly, as mentioned above, both the a and f3y subunits of the G protein can independently activate a signalling cascade. These mechanisms all depend on the cellular context and relative

28 Chapter 1

availability of components of the effector systems in the tissue or cell type in question. In a general sense, this may explain how a peptide such as galanin can display effects ranging from modulation of neurotransmitter and hormone release (both stimulation and inhibition), to mitogenicity. The coupling of multiple GPCR subtypes to multiple signalling events resulting in an array of cellular responses underlies the complexity of neuroendocrine control of physiology and metabolism.

(ii) Gene structure of GPCRs

The structure of many genes encoding GPCRs has been elucidated. A variety of genomic structures has been observed, from intronless to multiple-intron-containing genes (Iismaa et al., 1995). The abundance of GPCR-encoding genes lacking introns suggests a significant role for retrotransposition in the evolution of genes encoding GPCRs (Brosius, 1999). The conservation of specific genomic structures for receptor subtypes or receptors for related ligands also suggests the importance of gene duplication in generating receptor diversity. Many GPCR-encoding genes have been described which possess introns in the upstream region of the gene. Examples include the human NPY Yl receptor gene and the mouse somatostatin receptor sst2 gene, which both possess multiple untranslated 5' exons separated by large introns (Ball et al., 1995; Kraus et al., 1998). Alternative splicing in the untranslated portion of the transcript represents a mechanism by which multiple promoters can control the expression of a gene. Alternative splicing of coding exons has also been observed as a mechanism for the synthesis of multiple receptor isoforms from a single gene (Kilpatrick et al., 1999). Elucidation of the genomic structure of a GPCR can reveal clues regarding regulation of expression, possible alternative splicing and evolutionary relationships with other genes.

1.4 GALANIN RECEPTORS

Prior to the molecular cloning of the galanin receptors, a number of studies had characterised the galanin binding sites in the nervous system and other tissues using radio-labelled galanin (Merchenthaler et al., 1993). These binding sites were found to be very widely distributed and often co-localised with endogenous galanin. Biochemical experiments had identified a protein of 54 kDa in rat brain and insulinoma cells which bound 1251-

29 Chapter 1

labelled galanin (Bartfai et al., 1993). This receptor was shown to be a glycoprotein which occurred in complex with a G protein. Other studies demonstrating an association between galanin receptors and GJ G0 proteins further supported the classification of galanin receptors as members of the GPCR family (Bartfai et al., 1993). Three distinct galanin receptor subtypes have now been characterised by molecular cloning and expression in recombinant systems (Habert-Ortoli et al., 1994; Howard et al., 1997a; Wang et al., 1997b) (Figure 1.5). The overall distribution of expression of the three receptor genes appears to account for most of the observed galanin binding sites in the nervous system (Branchek et al., 2000; lismaa and Shine, 1999). However, some binding studies and pharmacological data point to the existence of other galanin receptor species, as will be discussed.

1.5 GALRl

(i) Molecular cloning of GALRl

The first galanin receptor was identified by expression cloning from the human Bowes melanoma (HMCB) cell line and was designated GALRl (Habert-Ortoli et al., 1994). The cloned cDNA was found to encode a protein of 349 amino acids, possessing the sequence hallmarks of a typical rhodopsin-like GPCR. The peptide sequence displays around 30% identity with the somatostatin and opioid receptors (Habert-Ortoli et al., 1994). GALRl has also been cloned from rat and mouse (Burgevin et al., 1995; Jacoby et al., 1997; Parker et al., 1995; Wang et al., 1997c) and, although the size of the proteins varies slightly (348 amino acids in rat, 346 amino acids in mouse), the sequence identity between the species homologues is around 92%. The genomic structure of GALNR1 has been elucidated and will be described in detail in Chapter 3.

The GALNR1 gene, encoding human GALRl, has been mapped by fluorescence in situ hybridisation (FISH) and radiation hybrid analysis to chromosome 18q23 (Crawford et al., 1999; Nicholl et al., 1995). This localization places GALNR1 within the critical region for a childhood disorder of growth hormone insufficiency associated with a deletion of 18q23 (Cody et al., 1997). The critical region has recently been narrowed to 2 Mb of DNA containing only one other known gene, MBP, encoding myelin basic protein. Patients with this disorder have short stature due to low

30 GALA

GALNR2

GALNR3

FIGURE 1.5 Comparison of the structural organisation of the genes encoding the human galanin receptors GALNRl, GALNR2 and GALNR3. The structure of each gene is shown, along with its relationship to the galanin receptor protein (GALR). The approximate size of each intron is indicated and the predicted transmembrane (TM) domains of the receptor are numbered. Solid boxes represent coding regions of each exon and striped boxes represent non-coding regions. The extent of the non-coding regions of the GALNR2 and GALNR3 genes is not known.

31 Chapter 1

circulating levels of GH. Their clinical characteristics are consistent with a hypothalamic defect as the primary cause. As galanin is known to be a potent stimulant of GH secretion in humans (Carey et al., 1993), GALNRl is currently a strong candidate for the gene underlying this disorder. However, further studies are needed to address the role of galanin signalling in these patients. An animal model of GALRl deficiency would also contribute to investigation of this disorder.

(ii) Pharmacology of GALRl

The pharmacology and signalling mechanisms of recombinant GALRl expressed in cell culture systems have been well studied (Figure 1.6). Initially, human GALRl transfected into African green monkey kidney (COS) cells was shown to bind galanin with an affinity of 0.8 nM. Activation of the receptor was able to inhibit the forskolin-stimulated accumulation of cAMP in a dose-dependent manner, with a half-maximal effective concentration of 0.9 nM. Maximum inhibition reached 50% inhibition of cAMP production (Habert-Ortoli et al., 1994). Subsequently, similar properties have been displayed by rat GALRl cloned from brain (Burgevin et al., 1995; Sullivan et al., 1997) and Rin14B insulinoma cells (Parker et al., 1995), and by mouse GALRl also cloned from brain (Wang et al., 1997c). The rat GALRl expressed in Chinese hamster ovary (CHO.Kl) cells is able to inhibit both basal and forskolin-stimulated cellular cAMP production (Parker et al., 1995). Coupling of GALRl to co-expressed inwardly rectifying K+ channels composed of Kir3.1 and Kir3.4 subunits has been demonstrated in Xenopus oocytes (Smith et al., 1998), resulting in membrane depolarisation in the presence of high extracellular K+ concentrations.

The coupling of GALRl to inhibition of adenylyl cyclase and activation of K+ channels is pertussis toxin-sensitive, which suggests that it is mediated by

Gi/G0 proteins (Parker et al., 1995; Smith et al., 1998). When stably expressed in CHO.Kl cells, GALRl is also known to couple to activation of mitogen­ activated protein kinase (MAPK) (Wang et al., 1998b). This response, which is also pertussis toxin-sensitive, is directed through the G protein J3y subunit, as it was shown to be sensitive to co-expression of the C-terminus of J3r adrenergic receptor kinase. This peptide is known to inhibit Gsy-mediated signalling by binding to the J3y subunit. The activation of MAPK by GALRl is also protein kinase C-independent, further distinguishing it from the

32 Chapter 1

protein kinase C-dependent activation of MAPK through the a subunit of

G0 (Wang et al., 1998b). The coupling of GALRl to multiple effector systems suggests that the receptor may impinge on an array of cellular functions.

GALRl binds a range of galanin analogues and truncated peptides with high affinity but has low affinity for galanin(2-29), which is missing the normal N-terminus, [D-Trp2]galanin(l-29) and galanin(3-29), demonstrating the importance of the N-terminal two residues of galanin as pharmacophores for GALRl. GALRl has no affinity for C-terminal fragments such as galanin(l0-29) (Parker et al., 1995; Wang et al., 1997c). A range of chimaeric peptides has been designed, consisting of galanin(l-13) coupled to fragments of other neuropeptides or random peptide sequence (Bartfai et al., 1992). These peptides antagonise several galanin-elicited functional responses in the brain, spinal cord and cardiovascular system (Alexander and Peters, 2000). The chimaeric peptides behave as full agonists at GALRl expressed in heterologous cell lines (Smith et al., 1997; Smith et al., 1998), which implies that they have a complex mode of action or are acting on a different receptor in vivo. The order of relative potency for galanin and its fragments at GALRl is galanin(l-29) > galanin(l-16) >> galanin(2-29) >>> [D­ Trp2]galanin(l-29) == galanin(3-29).

(iii) Structure-function studies of GALRl

The mode of interaction of galanin with GALRl has been studied by a combination of site-directed mutagenesis and molecular modelling. These approaches have identified a number of residues within human GALRl which contribute to the binding of ligand. The specific residues are His264 and His267 in the sixth transmembrane (TM6) domain, Phe11 5 in TM3 and Glu271 and Phe282 in the third extracellular loop (EC3) (Berthold et al., 1997; Kask et al., 1996). Experiments using a fluorescein-labelled galanin analog demonstrate that the N-terminal region of galanin binds to a hydrophobic pocket in GALRl, in contrast to the hydrophilic interaction of other neuropeptides with their cognate receptors (Wang et al., 1998c). Recent evidence supports a role for Phe186 in EC2 in mediating a hydrophobic interaction with galanin (Jones, 2000).

33 HYPERPOLARISATION

'fCAMP 'f cAMP 'fCAMP

FIGURE 1.6 Signal transduction pathways of the cloned galanin receptors. Included are the G proteins to which each receptor is known to couple. The downstream signalling pathways activated by the receptors are also shown, including inhibition of adenylyl cyclase activity, activation of phospholipase C and mitogen-activated protein kinase, and hyperpolarisation of the cell via inwardly rectifying potassium channels. Abbreviations used: AC, adenylyl cyclase; cAMP, cyclic AMP; DAG, diacylglycerol; IP3, inositol 1,4,5- triphosphate; MAPK, mitogen-activated protein kinase; PIP2, phosphatidyl inositol 4,5-bisphosphate; PKC, protein kinase C; PLC, phospholipase C.

34 Chapter 1

A few specific interactions with the major pharmacophores of galanin account for most of the free energy of binding. In particular, the N-terminal Cly of galanin is proposed to bind in the vicinity of Glu271, while Trp2 interacts with His264 in TM6. In addition, Ala7 or Leull of galaninmay interact with Phe186 in EC2, while Tyr9 of galanin may interact with Phe282 in EC3. Phe115 is proposed to maintain the structure of the ligand-binding pocket by its involvement in an intrahelix interaction (Jones, 2000). Finally, His267 may be important for stabilising the active state of GALRl, as mutation of this residue severely impairs the signalling ability of the bound receptor (Kask et al., 1996). A series of models of GALRl incorporating the experimental evidence have thus evolved (Figure 1.7) (Berthold et al., 1997; Jones, 2000; Kask et al., 1996; Kuiper, 1998). The models predict the main interactions occurring between the N-terminal half of the ligand and a pocket formed by the extracellular loops and extracellular surface of the TM helices of the receptor.

Additional studies have addressed certain aspects of GALRl signalling. The role of the third intracellular loop (IC3) of GALRl in its function was examined using an IC3-mimicking peptide. This peptide was able to inhibit galanin binding and induction of signalling in rat hippocampal membranes, demonstrating that the third intracellular loop is involved in the interaction of GALRl with G protein (Saar et al., 1997). This finding is consistent with studies of several other GPCRs. Using the fluorescein­ labelled galanin analogue mentioned above, it was also found that the binding of galanin to rat GALRl expressed in CHO.Kl cells precedes a rapid internalization event that is energy-dependent and extensive (Wang et al., 1998c). These studies all contribute to our understanding of the functioning of GALRl and to efforts to develop specific ligands for this receptor.

(iv) Distribution and regulation of GALRl expression

The expression of GALRl is widespread in human and rat (Table 1.1). Human GALRl cDNAs have been isolated from a variety of sources: HMCB cells (Habert-Ortoli et al., 1994), foetal brain (Sullivan et al., 1997), hypothalamus (Jacoby et al., 1997) and small intestine (Lorimer and Benya, 1996). Northern hybridisation analyses have detected transcript sizes of 9 kb, 5 kb and 3.2 kb in human, with the smaller mRNA sizes predominant in peripheral tissues and most strongly expressed in heart, prostate and testes

35 FIGURE 1.7 Molecular model of the human GALRl receptor-ligand complex as viewed from the plane of the membrane. Ribbons represent the protein backbone. The GALRl helix backbone is shown in dark green with the final residue in each transmembrane domain shown in light green. The white ribbons represent the loops, with the position of residues partic­ ipating in a putative disulphide-bond shown in yellow. The galanin ligand is rep­ resented by the orange ribbon. The numbering of the helices is depicted in the inset.

36 Chapter 1

as well as small intestine. The 9 kb transcript predominates in brain, where expression has been broadly detected but is strongest in cerebral cortex, amygdala and substantia nigra (Sullivan et al., 1997). In other studies, the human GALRl mRNA has been estimated at 1.4-1.6 kb with a larger transcript of 2.7-3.6 kb (Habert-Ortoli et al., 1994; Lorimer and Benya, 1996). The reasons for variation in these estimates have not been determined, but no alternative splicing of the GALRl message has been reported.

Estimates of the size of the rat GALRl mRNA range from 2 kb to 9 kb, with multiple bands seen in Northern analysis of certain tissues (Parker et al., 1995; Sullivan et al., 1997). The strongest signals are seen in brain, spinal cord, heart, large intestine, testes, kidney, stomach, lung and the rat insulinoma cell lines, Rinm5F and Rin14B (Parker et al., 1995; Sullivan et al., 1997). Analysis of expression in rat by RT-PCR has also detected GALRl mRNA in bladder, pancreas and uterus (Iismaa and Shine, 1999). A quantitative study using ribonuclease protection assays found relatively high levels of GALRl mRNA in the CNS, with the highest levels detected in the hypothalamus (Waters and Krause, 2000). This sensitive assay unexpectedly failed to detect any signal outside the CNS, but all other published studies have demonstrated GALRl expression in peripheral tissues of human, rat and mouse.

In situ hybridization studies of GALRl expression in the rat CNS have demonstrated widespread expression in the brain, with strong signals observed in the hypothalamus, thalamus, hippocampus, amygdala and brainstem (Gustafson et al., 1996; Parker et al., 1995). Expression was also evident in the pons and medulla and in the dorsal horn at all levels of the spinal cord (Gustafson et al., 1996). Two recent studies detailing the fine mapping of hypothalamic GALRl expression in rat described expression in virtually the entire hypothalamus (Landry et al., 1998; Mitchell et al., 1997), with the highest relative levels of expression found in the medial preoptic area (POA) (Mitchell et al., 1997). Expression appeared to be restricted to neurons (Landry et al., 1998). Using double-labelling techniques, GALRl mRNA was found in galanin-expressing neurons in the PVN supraoptic nucleus (SON), the dorsomedial nucleus and medial POA. However, GALRl and galanin were expressed by separate cellular populations in the anteroventral preoptic nucleus. In the PVN and SON, subpopulations of GALRl-expressing neurons also contained vasopressin mRNA (Landry et

37 Chapter 1

hGALRl rGALRl hGALR2 rGALR2 rGALR3 Brain + + + + + Hippocampus + + + + - Cerebellum - - + + + Hypothalamus + + + + Pituitary + - + + + Spinal cord + + - + + Neuroblastoma + Heart + + + + + Kidney + + + + + Liver + + - + + Lung + + + + + Small cell 1ung cancer + + Skeletal muscle + + - + - Stomach + + + + + Small intestine + + + + + Large intestine + + - Colon + + Pancreas + + - + + Spleen + -- + + Thymus + - Leukocytes - - Adrenal - + Prostate + + - + + Testis + + + + + Vas deferens + + - Ovary + + - + + Uterus + + + + - Placenta + - +

TABLE 1.1 Tissue distribution of galanin receptor subtypes. Detection of expression of human (h) or rat (r) GALRl, GALR2 or GALR3 by Northern hybridisation, ribonuclease protection, RT-PCR analysis or in situ

11 11 hybridisation is denoted + ", lack of signal is denoted -".

38 Chapter 1

al., 1998). Further studies found GALRl mRNA expressed by a subset of proopiomelanocortin (POMC) neurons in the arcuate nucleus (Bouret et al., 2000) of male rats and by a subset of GnRH neurons in the rostral preoptic area of female rats (Mitchell et al., 1999). The expression of GALRl in several hypothalamic nuclei is consistent with a direct role in modulating the activity of cells which secrete neuroendocrine peptides including vasopressin, POMC and GnRH.

A limited number of studies have addressed the plasticity of central GALRl expression in viva and its relationship to regulation of galanin synthesis. In the hypothalamus, the expression of GALRl can be altered by physiological and experimental manipulation (Landry et al., 1998). For example, salt loading by adding 2% NaCl to drinking water increases GALRl mRNA levels in magnocellular neurons. This increase peaked at 10 days of salt loading and returned to control levels after two weeks of rehydration. GALRl mRNA levels are also higher in the SON and PVN of the vasopressin-deficient Brattleboro rat, an alternative model of osmotic stimulation (Landry et al., 1999). This parallels the increased expression of galanin in magnocellular vasopressinergic neurons in this model (Rokaeus et al., 1988). Expression of GALRl mRNA is also increased in these regions by pharmacological inhibition of glucose and fatty acid metabolism (Gorbatyuk and Hokfelt, 1998). In contrast, hypophysectomy in male rats causes a strong down-regulation of expression in the magnocellular region, which persists after 14 days. Furthermore, the response of GALRl to lactation matches that of galanin, with a small decrease in expression of the order of 15% by day five of lactation, returning to control levels after two weeks (Landry et al., 1998). These effects on GALRl mRNA expression are consistent with an inhibitory role for this receptor on vasopressin and oxytocin release in the magnocellular region of the hypothalamus.

The expression of GALRl is sensitive to hormonal influences, as circulating gonadal steroids are effective in regulating levels of GALRl mRNA in the hypothalamus. The density of GALRl mRNA-expressing cells is lower in the POA of male rats compared to females. The density and intensity of GALRl mRNA in the POA of female rats also varies during the oestrous cycle, in both GnRH and non-GnRH neurons (Faure-Virelizier et al., 1998; Mitchell et al., 1999). This variation does not correlate with reported changes in galanin mRNA and appears to be influenced more by

39 Chapter 1

progesterone than estradiol. Progesterone plus estradiol treatment is able to decrease the levels of GALRl mRNA in the POA of ovariectomized animals whereas estradiol alone has no significant effect (Faure-Virelizier et al., 1998). Progesterone totally abolishes GALRl mRNA expression in GnRH neurons (Mitchell et al., 1999). In male rats, androgens may contribute to the regulation of hypothalamic GALRl expression. GALRl mRNA levels in the arcuate nucleus are reduced by castration, but this effect is eliminated by testosterone replacement (Bouret et al., 2000). Hence, testosterone stimulates expression of GALRl (and GALR2) mRNA in the arcuate nucleus, including POMC neurons which project to the POA (Bouret et al., 2000). These findings provide a potential mechanism by which galanin contributes to the regulation of the reproductive axis by sex hormones. The regulation of GALRl mRNA levels by gonadal steroids is also consistent with observations of pubertal changes in galanin binding sites in the rat brain (Planas et al., 1995).

Injection of colchicine, an agent which disrupts microtubules, has a variable and complex effect on galanin expression. While colchicine treatment increases galanin expression in the PVN and SON, GALRl expression is strongly down-regulated in these nuclei and in some dorsal mid-thalamic nuclei. However, in the lateroanterior hypothalamus, GALRl mRNA levels are increased and in other nuclei are unaffected (Landry et al., 1998). These studies also suggest plasticity or adaptation of GALRl expression under different physiological environments in which expression of the ligand itself is altered. Certain conditions appear to influence the expression of ligand and receptor in the same manner. Other conditions induce divergent alteration of the complementary components. Alternate galanin receptor subtypes are thus implicated in the response to increased galanin production under those conditions in which GALRl expression is decreased (Landry et al., 1998).

A role for GALRl in the complicated feedback control of GH secretion has been proposed (Chan et al., 1996). Double-fabelling in situ hybridization experiments in the rat show the expression of GALRl mRNA in a subset of. somatostatin neurons in the periventricular nucleus of the hypothalamus. Galanin may act through these receptors to inhibit somatostatin secretion, thereby resulting in enhanced secretion of GH by the action of GHRH in the anterior pituitary. This may explain the well-documented stimulatory

40 Chapter 1

effects on GH secretion resulting from administration of exogenous galanin (Murakami et al., 1989; Ottlecz et al., 1986).

The receptor(s) which mediates galanin's orexigenic effect in the rat has not been directly determined, and all three cloned galanin receptors are expressed in the hypothalamus. One study addressing this question used galanin analogs in an experimental feeding assay (Wang et al., 1998a). Of the candidates, including a putative galanin(3-29) receptor and the three cloned receptors, the results were consistent with GALRl as the subtype responsible. Confirmation of this finding requires GALRl-specific ligands, in vivo antisense nucleic acid studies or experiments using receptor knockout mice.

A double-labelling in situ hybridization technique has been employed to determine the means by which galanin inhibits ACh release in the rat basal forebrain (Miller et al., 1997). While an abundance of GALRl-expressing cells occurs in this region, none of these are cholinergic neurons. This indicates that GALRl may mediate the inhibition of ACh release via an intermediate neurotransmitter. If galanin does act directly on cholinergic neurons within the basal forebrain, then it does so through another galanin receptor subtype. In contrast, the vast majority of extrahypothalamic galanin/vasopressin-expressing neurons are positive for GALRl mRNA. These neurons reside in the bed nucleus of the stria terminalis and the medial amygdala and project to septo-hippocampal regions. This result suggests that GALRl acts as an autoreceptor, or acts post-synaptically in response to galanin inputs from elsewhere, to regulate the secretion pattern of vasopressin and galanin into the septo-hippocampal area (Miller et al., 1997). This could provide a further pathway by which GALRl participates in regulation of cholinergic neurotransmission.

A role for GALRl in nociception is suggested by its expression in the rat DRG (Xu et al., 1996b). The expression in DRG was quantified at more than 20% of all neurons, consisting mainly of small- and medium-sized neurons. However, a second study found DRG expression of GALRl confined mainly to large neurons (O'Donnell et al., 1999). The majority of GALRl-expressing cells also contain mRNA encoding calcitonin gene-related peptide, a gene expressed by most DRG neurons. Almost 30% of GALRl-positive DRG cells also express GALR2 mRNA (Shi et al., 1997). Expression of GALRl in DRG

41 Chapter 1

is transiently diminished by inflammation, with partial recovery after five days. Peripheral axotomy also affects expression, with very few positive cells seen seven days after injury (Xu et al., 1996b). This response is reminiscent of the effects of hypophysectomy on GALRl expression in the hypothalamus (Landry et al., 1998). The down-regulation of GALRl mRNA under conditions which increase expression of galanin, and the neuronal response to galanin (Xu et al., 1997), cast doubt on the importance of this receptor following nerve injury. In a novel study by Pooga et al. (1998), antisense peptide nucleic acid constructs were used to inhibit expression of GALRl in the spinal cord of rats. Intrathecal injection of the constructs results in reduction in galanin binding in.. the dorsal horn of the spinal cord, accompanied by an alteration in the galanin-induced inhibition of sensory excitation. This study demonstrates a role for GALRl in transmission of the flexor reflex at the level of the spinal cord.

The expression of GALRl in the human gastrointestinal tract has been closely examined by semi-quantitative RT-PCR (Lorimer and Benya, 1996). This study found GALRl expression in mucosa! epithelial cells throughout the gut, with highest relative expression in the duodenum. Further evidence for expression of GALRl mRNA in colonic epithelium has come from in situ hybridization experiments on colonic biopsies from children (Engelis et al., 1998). The role of GALRl in the gastric mucosa was addressed in another study, using the T84 colon cancer cell line as a model of human colonic epithelium (Benya et al., 1999). It was found that activation of GALRl by galanin in this cell line causes a Ca2+-dependent increase in c1- secretion. This suggests that galanin released by enteric nerve terminals in vivo may be acting, through GALRl, as a secretagogue in colonic epithelial cells. The possible significance of this process in relation to diarrhoeal infection has also been demonstrated by the up-regulation of GALRl expression following infection by pathogenic bacteria in vitro and in mouse colon (Hecht et al., 1999). Elsewhere in the periphery, the expression of GALRl peptide in ~-cells of the mouse pancreas has been revealed by immunohistochemistry, implicating the receptor in the modulation of insulin release (Hecht et al., 1999).

The embryonic expression of GALRl in the rat has been reported from day 14 (E14) of development (Xu et al., 1996a), when expression is seen in the brain and intestine. In spinal cord, GALRl mRNA is present in the ventral

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horn at E17 and the dorsal horn at El9. The trigeminal ganglia also display expression at E17 while the DRG express GALRl mRNA at E21. Another developmental study on sections of whole E20-21 rat embryos confirms the expression of GALRl in brain, spinal cord, DRG and small intestine (Parker et al., 1995). These results suggest that GALRl has a role in embryogenesis from as early as 14 days of gestation.

The only report, to date, of the distribution of GALRl mRNA in the mouse shows relatively abundant expression of a 9 kb transcript in brain. Intermediate levels of a 7-7.5 kb transcript are present in heart and skeletal muscle. No expression is detected in liver, kidney, testes, lung or spleen (Wang et al., 1997c). In this study, detection of gene expression was performed by Northern analysis, which may not be sensitive enough to detect low levels of mRNA in peripheral tissues.

1.6 OTHER GALANIN RECEPTORS

(i) GALR2

A second subtype of galanin receptor, GALR2, has been cloned from human (Bloomquist et al., 1998; Borowsky et al., 1998; Fathi et al., 1998a), rat (Fathi et al., 1997; Howard et al., 1997a; Smith et al., 1997; Wang et al., 1997a) and mouse (Pang et al., 1998) by expression cloning and homology cloning approaches. Human GALR2 is a 387 residue protein sharing 40% amino acid identity with GALRl. It is encoded by the GALNR2 gene located on chromosome 17q25.3 (Fathi et al., 1998a). In human and rat, this gene consists of two coding exons separated by an intron approximately 1.4 kb in length. Exon 1 encodes the N-terminal portion of the receptor up to the end of the third TM domain. Exon 2 encodes the remainder of the receptor protein (Figure 1.5). The amino acid sequence of the two orthologues is highly conserved, sharing 87% overall sequence identity (Fathi et al., 1998a). The gene structure is conserved in the mouse Galnr2 gene, which has an intron of 1.1 kb and amino acid sequence identity of 94% with rat GALR2 (Pang et al., 1998).

The overall pharmacology of GALR2 is similar to GALRl. The exceptions to this are that GALR2 has a markedly higher affinity for the N-terminally

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truncated ligand, galanin(2-29), as well as for the analog D-[Trp2]galanin (Fathi et al., 1997; Howard et al., 1997a; Smith et al., 1997). The differential affinity for galanin(2-29) has been used to implicate GALR2 in the effect of galanin on contraction of rat jejuna! longitudinal muscle (Wang et al., 1998a). GALR2 does not bind galanin(3-29). The galanin homologue GALP is a novel endogenous GALR2 agonist. Porcine GALP has been shown to possess high affinity for rat GALR2 but not GALRl (Ohtaki et al., 1999). This is interesting in light of the fact that the 13 residues common to both galanin and GALP are important in the binding of galanin to both receptors. It is too early to speculate on the basis of the selective affinity of GALP, or whether GALR2 is the only receptor for this peptide. It is likely, however, that GALR2 is involved in mediating the activity of endogenous GALP.

Rat and human GALR2 have been shown to couple to multiple signalling pathways when expressed in heterologous culture systems (Figure 1.6). In CHO.Kl, COS-7 and human embryonic kidney- (HEK-) 293 cells, activation of rat GALR2 stimulates inositol phosphate production by the activation of phospholipase C, mediated via a Gq111-type G protein (Fathi et al., 1997; Smith et al., 1997; Wang et al., 1998b). This response, which is pertussis toxin-insensitive, is also observed in CHO.Kl and HEK-293 cells expressing human GALR2 (Borowsky et al., 1998; Fathi et al., 1998a; Kolakowski et al., 1998b). Similarly, mouse GALR2 expressed in COS-7 cells was shown to couple to Gq/11 to increase production of inositol triphosphate but not to signal through Gi to inhibit cAMP accumulation triggered by the activation of co-expressed ~-adrenergic receptors (Pang et al., 1998). However, in CHO.Kl and COS-1 cells, rat GALR2 is capable of coupling via Gi to the pertussis toxin-sensitive inhibition of forskolin stimulated cAMP production (Wang et al., 1998b; Wang et al., 1997a). It has also been demonstrated that rat GALR2 expressed in CHO.Kl cells can activate the

MAPK pathway via G0 (Wang et al., 1998b). Thus GALR2 interacts with multiple G proteins in various cell types, a property which is not intrinsic to GALRl or GALR3.

The expression of GALR2 mRNA has been examined by several groups and, similar to GALRl, is widespread in human and rat tissues (Table 1.1). Using RT-PCR, human GALR2 was found to be expressed both centrally and peripherally (Borowsky et al., 1998; Fathi et al., 1998a; Iismaa and Shine, 1999). Strongest peripheral expression is found in the heart, kidney, liver

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and small intestine, with lower levels in the retina and testes. Expression is also seen in two human cell lines, HMCB cells and NCI-H69 small cell lung carcinoma cells (Fathi et al., 1998a). Two studies have reported differing results of RT-PCR experiments (Borowsky et al., 1998; Fathi et al., 1998a). In the brain, expression is strong in the hippocampus, thalamus and amygdala, with some expression also in the cerebellum (Borowsky et al., 1998; Fathi et al., 1998a). Expression of GALR2 in the hypothalamus is seen only in the second of the two studies (Borowsky et al., 1998), while only the first study detects GALR2 in cerebral cortex, pituitary, stomach and lung (Fathi et al., 1998a). The reason for the discrepancies in these results is not known, but may relate to the relative sensitivities of the protocols used and should be resolved by in situ hybridisation experiments in the future. Northern analysis additionally identified a 1.4-2.0 kb human GALR2 transcript in rectum and colon (Bloomquist et al., 1998; Kolakowski et al., 1998b).

A number of studies have used Northern hybridisation methods to determine which tissues express GALR2 in the rat (Howard et al., 1997a; Smith et al., 1997; Wang et al., 1998a; Wang et al., 1997a). A transcript of 1.8- 2.4 kb has been identified in brain, uterus, ovary, prostate, vas deferens, testis, heart, lung, spleen, kidney, liver, large intestine, jejunum and skeletal muscle. Expression is also seen in the insulinoma cell line RINmSF. In addition to some of these tissues, rat GALR2 mRNA is detected in DRG, spinal cord, stomach and pancreas by RT-PCR (Iismaa and Shine, 1999; Waters and Krause, 2000) and in hypothalamus, anterior pituitary (of rat and mouse) and the GH3 pituitary cell line by ribonuclease protection analysis (Fathi et al., 1997; Moore Jr. et al., 1999). In situ hybridisation analysis has additionally been carried out to map regions of GALR2 expression in the rat nervous system. In general, expression of GALR2 mRNA is restricted than expression of GALRl mRNA, but with regions in common. The strongest signal is seen in the DRG, corresponding mostly to small and medium-sized cells (O'Donnell et al., 1999). Levels of expression ranging from strong to weak are detected in numerous areas of the brain and in all laminae of the spinal cord (O'Donnell et al., 1999). Apart from the hypothalamus, regions of the brain displaying GALR2 expression include the hippocampus, brainstem, thalamus and cerebellum (Fathi et al., 1997; Kolakowski et al., 1998b; Xu et al., 1998). Expression has also been readily detected by in situ hybridisation in the anterior pituitary gland (Fathi et al., 1997).

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The identity of cells expressing GALR2 mRNA has been studied using dual­ labelling histochemical techniques. In the rat hypothalamus, co-localisation of GALR2 mRNA with somatostatin immunoreactivity is observed in the arcuate nucleus and median eminence. Co-localisation with tyrosine hydroxylase immunoreactivity, a marker of dopamine biosynthesis, is observed in a proportion of neuronal cell bodies in the zona incerta (Nichol et al., 1999). Therefore, GALR2 mRNA is expressed in subsets of somatostatin and dopaminergic neurons. As mentioned above for GALRl, a subset of POMC neurons in the arcuate nucleus express GALR2 mRNA, suggesting that galanin can directly modulate the activity of these neurons via multiple signalling pathways (Bouret et al., 2000). Expression of GALR2 mRNA in the arcuate nucleus of the hypothalamus is positively regulated by testosterone (Bouret et al., 2000). While the hypothalamic expression of GALR2 mRNA is consistent with a role in food intake, a recent study with the GALR2-selective agonist galanin(2-29) suggests that this subtype does not directly mediate the orexigenic response to galanin (Wang et al., 1998a).

In the rat pituitary gland, GALR2 is expressed widely and strongly in the intermediate lobe, and by a subset of cells in the anterior lobe (Depczynski et al., 1998). No GALR2 expression is seen in the posterior pituitary. In determining the identity of pituitary cells expressing GALR2, it was shown that the majority of intermediate lobe melanotrophs express GALR2. In the anterior pituitary, subsets of somatotrophs, lactotrophs, thyrotrophs and gonadotrophs express GALR2 (Depczynski et al., 1998). Expression of GALR2 mRNA in multiple cell types raises the possibility that this receptor subtype contributes to the regulation of the secretion of most major pituitary hormones.

An investigation into the expression and regulation of GALR2 mRNA in rat spinal cord has implicated this receptor in sensory transmission (Shi et al., 1997). It was found that approximately 25% of DRG neurons contain GALR2 mRNA and that these are mostly small in size. Similarly to GALRl expression, most of the GALR2-positive cells also contain calcitonin gene­ related peptide mRNA. In fact, analysis of adjacent tissue sections showed that 20% of GALR2-expressing cells also express GALRl mRNA. As with GALRl expression, axotomy was found to decrease the intensity of GALR2 expression and also the total number of GALR2-positive cells, a change

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which persisted up to 28 days post-injury. In contrast, peripheral inflammation results in an increase in GALR2 expression up to a week after induction of inflammation, and an increase in the number of expressing cells after three days (Shi et al., 1997). Another recent study documented changes in GALR2 mRNA following facial nerve crush in the rat (Burazin and Gundlach, 1998). Induction of GALR2 mRNA, but not GALRl rnRNA, is detected in motor neurons of the facial nucleus up to seven days post­ injury. This finding demonstrates the differential regulation of galanin receptor subtype gene expression in a specific neuronal population, and suggests a role for GALR2 in the potential regenerative activity of galanin in motor neurons (Burazin and Gundlach, 1998). However, the complex expression pattern and regulation of GALR2 mRNA and their implications for pain transmission under conditions of nerve damage are not well understood.

(ii) GALR3

The most recent galanin receptor subtype to be characterised has been designated GALR3. The gene encoding human GALR3 was initially identified in a large genomic BAC clone as a sequence with homology to other galanin receptors (Kolakowski et al., 1998b; Wang et al., 1997b). Rat GALR3 was independently cloned from hypothalamic cDNA by a combination of expression and homology cloning approaches (Smith et al., 1998). The deduced human GALR3 peptide sequence shares only 36% amino acid identity with GALRl and 58% identity with GALR2. It also shares with GALR2 a two exon genomic structure with an intron of 954 base pairs (bp) located in the same position, after TM3 (Iismaa et al., 1998) (Figure 1.5). The GALNR3 gene encoding this receptor subtype has been localised to 22q12-22ql3.1, in the vicinity of SSTR3, a gene encoding another GPCR, viz. the somatostatin receptor subtype, sst3. A Pstl polymorphism has been identified in the intron of human GALNR3 but its significance, if any, is unknown (Lapsys et al., 1999).

GALR3 expression has been reported in the brain and several other tissues of the rat, including pituitary, DRG, heart, spleen, testes, liver, kidney, stomach, adrenal cortex, lung, small intestine, pancreas, prostate and ovary (Iismaa and Shine, 1999; Kolakowski et al., 1998b; Smith et al., 1998; Wang et al., 1997b; Waters and Krause, 2000) (Table 1.1). In the CNS, highest levels of

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GALR3 transcript are seen in the hypothalamus, with lower levels detected in the spinal cord, olfactory bulb, cerebral cortex, medulla oblongata, caudate putamen and cerebellum. Human GALR3 expression has been examined by Northern analysis, with signals detected in the brain and spinal cord, thyroid, adrenal gland, testis, skeletal muscle, pancreas, small and large intestine, rectum and placenta (Kolakowski et al., 1998b). While expression in human pituitary was not examined, a ribonuclease protection analysis on baboon tissue did detect low levels of GALR3 expression in the pituitary, other neuroendocrine tissues and in several brain regions (Kolakowski et al., 1998b).

When expressed heterologously, GALR3 is capable of binding galanin with a high affinity similar to GALRl and GALR2. The pharmacology of GALR3 with respect to the binding of galanin peptides does exhibit some distinguishing features. Like GALR2, rat GALR3 has a higher affinity for galanin(2-29) than does GALRl (Smith et al., 1998; Wang et al., 1997b). However, its affinity for the fragment galanin(l-16) is lower than that of the other receptors. In addition, rat and human GALR3 bind human galanin with marginally lower affinity than rat or porcine galanin (Smith et al., 1998). This is in contrast to both GALRl and GALR2 and has been suggested by Smith et al. (1998) as evidence for the possible existence of an additional galanin-like peptide in humans. It is not yet known whether GALR3 can bind GALP. It has been demonstrated in Xenopus oocytes that human and rat GALR3 can couple to co-expressed Kir channels. These results strongly suggest that GALR3 activates a pertussis toxin-sensitive Gi/G0 pathway. In another study utilising a Xenopus melanophore pigmentation assay, activation of human GALR3 by galanin resulted in pigment aggregation, providing further evidence for coupling to Gi and signalling through the inhibition of adenylyl cyclase activation (Kolakowski et al., 1998b) (Figure 1.6).

(iii) Evidence for further galanin receptor subtypes

Evidence has emerged, from a variety of approaches, for the existence of further putative galanin receptor subtypes. Firstly, pharmacological experiments using several peptide antagonists in vivo have revealed a number of functional responses to galanin which are antagonised by these agents. As these antagonists all behave as full agonists at the cloned galanin

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receptors, it suggests that they may be acting in viva at undiscovered subtype(s) or with unidentified receptor-modulating proteins. Secondly, a receptor which is activated by a C-terminal galanin fragment (galanin(3-29)) has been described in rat pituitary, hypothalamus and guinea pig gastric smooth muscle cells (Gu et al., 1995; Kinney et al., 1998; Todd et al., 1998; Wynick et al., 1993b). This receptor also remains to be isolated, as the pituitary receptors cloned to date, GALR2 and GALR3, do not bind galanin(3-29) with high affinity. Thirdly, a recent study by Xu et al. (1999) describes the selective induction of a hyperpolarizing current in rat hippocampal neurons by galanin(l-15), another ligand for which the receptor is not known. Autoradiographic binding studies have also identified populations of binding sites for 1251-labelled galanin(l-15) in the dorsal hippocampus, neocortex and neostriatum, regions where binding of full-length galanin is not seen (Hedlund et al., 1992). Finally, a truncated variant of galanin, galanin(S-29), which may be a degradation product, has been isolated from porcine brain (Sillard et al., 1992). This peptide has biological activity in the intestine (Rossowski et al., 1990) and binding affinity in hippocampus (Fisone et al., 1989) yet has not been established as a ligand for the cloned receptors.

Experiments reported by Shi et al. (1997) provide evidence for the expression of multiple galanin receptors in the same cell, raising the spectre of receptor heterodimeriza tion. Although heterodimeriza tion of galanin receptors has not been studied, the formation of distinct receptor species by heterodimerization of subtypes has been demonstrated for members of the same class of GPCRs, such as the opioid, serotonin and somatostatin receptors (Jordan and Devi, 1999; Rocheville et al., 2000; Xie et al., 1999). While speculative at present, the possibility does exist that co-expressed galanin receptor subtypes may interact at the membrane to form a novel subtype. The discovery of the galanin homologue GALP also hints at a greater complexity in the galanin system than previously suspected (Ohtaki et al., 1999). While GALR2 binds GALP with high affinity, it is unknown whether GALR2 is the major GALP receptor, or indeed whether undiscovered GALP receptors may also be activated by galanin. As the amino acid sequence common to both peptides does participate in binding to the galanin receptors, this latter consideration may be likely.

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1.7 OVERVIEW OF THESIS AIMS

A range of studies has implicated a role for galanin in conditions such as obesity, Alzheimer's disease, epilepsy and chronic pain. The galaninergic system may well be a rational therapeutic target for intervention in some of these disorders. Moreover, the relatively low degree of sequence similarity between galanin receptor subtypes increases the likelihood that specific pharmacological agents could be developed that activate or antagonise single subtypes. In order to evaluate the potential utility of such agents in clinical medicine, it is essential to establish the roles played by each subtype in mediating the functions of galanin in viva.

A number of complementary approaches can be used to achieve this. Firstly, pharmacological studies can investigate the consequences of temporarily interrupting the function of specific receptor subtypes in viva. At present, pharmacological approaches to galanin receptor function are limited by a scarcity of subtype-specific ligands. Only one non-peptide galanin receptor ligand has been reported and its action and specificity are unknown (Chu et al., 1997). No antagonists of the cloned galanin receptors have been reported. Secondly, localisation of receptor gene and protein expression is important in establishing a framework for general inferences of receptor function. In this respect, GALRl has been the subject of several studies in the rat and is therefore the best understood of the receptors. However, the lack of specific antibodies has also limited the exploration of receptor peptide distribution. Thirdly, genetic approaches, which have the advantage of high specificity, are becoming increasingly used in the study of gene function. The large number of knockout mouse strains so far developed demonstrates the usefulness of these methodologies in providing whole-animal models of gene function and dysfunction (Brandon et al., 1995).

For many years, the mouse has been used as an important model organism for the study of mammalian physiology (Hogan et al., 1994). However, the recent development of transgenic and embryo manipulation techniques has resulted in unprecedented opportunities to exploit the mouse for the study of gene function and developmental processes. In particular, the advent of knockout approaches now allows the in viva function of any gene to be revealed by targeted disruption or mutation. To date, the knockout

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approach has been used to good effect in the field of neuroendocrinology. This approach has provided novel data on the physiological roles of many neurotransmitters and neuropeptides and their receptors (Rohrer and Kobilka, 1998). In some cases, this approach has complemented pharmacological studies with specific or partially specific antagonists. In other cases, as with the galanin system, specific antagonists have not yet been developed and so knockouts have provided an alternative avenue of investigation. The specificity and conceptual simplicity of the genetic approaches have made the knockout mouse an attractive option for researchers in the field of GPCR biology.

The knockout approach has already been used to generate a galanin­ deficient mouse strain. As mentioned, the phenotypic features reported for this strain include failure of mammary gland development and lactation in females, and impaired functional regeneration of sensory neurons and hypoalgaesia following peripheral nerve injury (Wynick, 1997; Wynick et al., 1998). Other findings include an increased responsiveness to leptin (Hohmann et al., 1999), a heightened sensitivity to convulsants (Mazarati et al., 1999) and a loss of cholinergic neurons from the basal forebrain (Ma et al., 1999). These findings reinforce the importance of galanin in promoting prolactin secretion, in regulating sensory transmission and as a central neuromodulator. Following this line of investigation further, the knockout approach can be used to pinpoint which galanin receptor or receptor combination mediates these essential roles of the peptide. As GALRl is not expressed in rat pituitary, it may not be directly involved in the effects exerted by galanin on the rodent lactotroph. However, GALRl is expressed in brain, spinal cord and DRG, making it a candidate for involvement in galanin's effects in these regions. A mouse strain deficient in GALRl can be used to further investigations into the role of this receptor in vivo. Thus, the aim of this project was to develop a novel tool with which to study the physiological role of GALRl, primarily by a standard gene-targeting approach in the mouse.

The isolation, characterisation and chromosomal localisation of the mouse Galnrl gene is described in Chapter 3. These data provide information regarding the comparative genomic structure of mouse GALRl as well as forming the basis for further studies into the function of this gene. Most significantly, the structure and sequence of Galnrl provide the necessary

51 Chapter 1

data for the design and construction of a gene targeting vector for the development of a Galnrl knockout mouse. The development and preliminary characterisation of this mouse strain is described in Chapter 4.

As discussed, the expression of the genes encoding galanin and its receptors has been explored by various means in human and rat. Although many similarities may exist between these expression patterns and those in mouse, it should not be assumed that they are identical. In order for the mouse to fulfil its potential as a model for the study of galanin function, the expression of the neuropeptide and its receptors must be investigated in this species. The expression of these genes during embryonic development will similarly require elucidation in order to assess the developmental functions of the galanin system. The factors regulating the expression of these genes also remain largely unexplored. To complement the development of a GALRl knockout mouse, these issues are addressed in Chapter 5. The availability of the galanin knockout mouse (Wynick et al., 1998) facilitated the investigation of the role that the ligand plays in expression of the genes encoding the galanin receptors, both during development and in the adult mouse.

In short, the purpose of this study is to use genetic modification in the mouse to define the major in vivo roles of GALRl. Furthermore, investigation of the expression of genes encoding components of the galaninergic system will provide a molecular context within which to extend the phenotypic characterisation of the mouse strain developed.

52 CHAPTER 2

GENERAL METHODS Chapter 2

2.1 MATERIALS

(i) Chemicals and enzymes

The restriction endonucleases and T4 polynucleotide kinase were supplied by both Promega (Madison, WI) and Roche Diagnostics GmbH (Mannheim, Germany). Calf intestinal alkaline phosphatase was manufactured by New

England Biolabs (Beverley, MA). AmpliTaq™ and AmpliTaq Gold TM thermostable DNA polymerase, 10 x PCR Buffer and MgCl2 Solution came from Perkin Elmer (Branchburg, NJ). Promega also supplied T4 DNA ligase, T7 DNA polymerase, deoxynucleotide triphosphates and the pGEM®-T and pGEM®-3z cloning vectors. Gibco BRL-Life Technologies (Gaithersburg, MD) supplied T4 DNA ligase, SuperScript™ II reverse transcriptase and a 1kb DNA ladder. The pBluescript® II SK ( +) phagemid vector was manufactured by Stratagene (La Jolla, CA). Proteinase K, DNase I, RNase A, lysozyme, ampicillin, Expand™ Reverse Transcriptase, RNase inhibitor, dithiothreitol, CsCl and formamide were all supplied by Roche Diagnostics GmbH. Klenow fragment of DNA polymerase I, random hexanucleotides, Sephadex® G-25 and Hybond-N+ nylon membrane were supplied by Amersham Pharmacia Biotech (Buckinghamshire, England).

The EcoRI linkers, DNA Grade Agarose and High Resolution Agarose were made by Progen Industries (Darra, Australia). Salmon sperm DNA, bovine serum albumin (BSA; Fraction V), N,N,N',N'-tetramethylethylenediamine (TEMED) and Tri Reagent™ were supplied by Sigma (St. Louis, MI). Synthetic oligonucleotide primers were synthesised by Beckman Instuments (Sydney, Australia). RNAlater™ was supplied by Ambion (Austin, TX). Radioactively labelled deoxynucleotide triphosphates were made by NEN Research Products-Du Pont (Boston, MA) and Amersham Pharmacia Biotech. NEN-Du Pont also supplied the Colony /Plaque Screen nylon disk membranes. BIOMAX™ and X-OMAT™ Scientific Imaging Film was manufactured by Eastman Kodak Co. (Rochester, NY) and the L4 nuclear emulsion was made by ILFORD Imaging (Paramus, NJ). Phenol came from Amresco (Solon, OH) and acrylamide from National Diagnostics (Atlanta, GA). Other laboratory chemicals were obtained at analytical grade from Sigma, Ajax Chemicals (Auburn, Australia) and BDH (Kilsyth, Australia).

54 Chapter 2

(ii) Common reagents and media

Common reagents were prepared as described (Sambrook et al., 1989) and included the following: 100 x Denhardt's solution (2% (w /v) Ficoll 400, 2% (w /v) polyvinylpyrrolidone, 2% (w /v) BSA); 1 x SM buffer (100 mM NaCl, 50 mM Tris.HCl, 10 mM MgSO4, 0.01 % (w /v) gelatin); 20 x SSC (3 M NaCl, 300 mM trisodium citrate); 20 x SSPE (600 mM NaCl, 400 mM NaH2PO4, 4 mM ethylenediaminetetraacetic acid (EDTA)); 50 x TAE buffer (2 M Tris acetate, 50 mM EDTA pH 8.0); 1 x TE (10 mM Tris.HCl, 1 mM EDTA, pH 8.0); 10 x TBE (450 mM Tris borate, 40 mM EDTA); chloroform for DNA extraction was mixed 24:1 with isoamyl alcohol; phenol for DNA extraction was equilibrated with 100 mM Tris.HCl pH 8.0; Luria-Bertani (LB) liquid medium (1 % (w /v) bactotryptone, 0.5% (w /v) yeast extract, 1 % (w /v) NaCl); LB agar (1.5% (w /v) agar in LB); LB soft-top agarose (0.6% (w /v) agarose in LB); NZYDT medium was supplied as a pre-formulated powder (Difeo Laboratories, Detroit, MI) and was prepared by dissolving in water and autoclaving; SOC medium (2% (w /v) bactotryptone, 0.5% (w /v) yeast extract, 20mM glucose, 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl2, 10 mM MgSO4); Terrific Broth (1.2% (w /v) bactotryptone, 2.4% (w /v) yeast extract, 0.4% (v /v) glycerol, 17 mM KH2PO4, 72 mM K2HPO4).

(iii) Kits

The Wizard® PCR Preps kit, the fmol® DNA Cycle Sequencing System and PolyAT tract® mRNA Isolation System IV were manufactured by Promega. The Megaprime DNA labelling system was from Amersham Pharmacia Biotech. The 5'/3'-RACE kit was supplied by Roche Diagnostics GmbH.

(iv) Tissue culture reagents

Gibco BRL-Life Technologies supplied the Dulbecco's Modification of Eagle's Medium (DMEM), phosphate-buffered saline (PBS), trypsin-EDTA, non­ essential amino acids and G418. The mitomycin C and nucleosides were supplied by Sigma. The ESGRO™ recombinant leukemia inhibitory factor was manufactured by AMRAD Biotech (Boronia, Australia). The foetal calf serum (FCS) was supplied by Commonwealth Serum Laboratories (North Ryde, Australia). Suppliers of other chemicals used are as described in Section 2.l(i) above.

55 Chapter 2

2.2 RECOMBINANT LIBRARY SCREENING

A mouse genomic library in bacteriophage vector A FIXII (Stratagene) was screened by standard methods (Sambrook et al., 1989). The library was titred by plating a serial dilution series and counting the plaques arising to obtain a value for the concentration of plaque forming units (pfu). A culture of Escherichia coli strain LE392 was grown overnight at 37°C in LB broth containing 10 mM MgSO4 and 0.2% (w /v) maltose. The bacterial cells were infected with 6 x 10s pfu (1.8 genome equivalents) of bacteriophage by mixing, followed by incubation at 37°C for 15 min. The infected bacteria were then mixed with molten LB containing 0.6% (w /v) agarose at 50°C and poured over LB agar plates. Plaques formed on the bacterial lawn during overnight incubation of the plates at 37°C. The plates were cooled to 4°C before the bacteriophage DNA was transferred to nylon membranes for hybridisation to a radioactive DNA probe. Duplicate filters were made from each plate in order to verify the position of positively hybridising plaques. Plaques which hybridised to the 32P-labelled DNA probe were detected by autoradiography. Plugs of agar underlying these positive plaques were removed into 1 ml SM containing 5 µl chloroform. The isolates were titered by plating of serial dilution series of bacteriophage suspensions. The bacteriophage were purified by a further two rounds of dilution and screening, resulting in the isolation of several individual hybridising bacteriophage clones. High titre lysate stocks of recombinant clones were prepared essentially as described (Sambrook et al., 1989).

2.3 PREPARATION OF DNA

(i) Preparation of bacteriophage A DNA

Large scale preparations of bacteriophage A DNA were purified by extraction of bacteriophage stocks collected from plate lysates. For each of five large plates, 1-2 µl of a high titre lysate were used as inoculum to produce plate lysates as described above. Total lysis of the bacterial lawn was evident after overnight incubation at 37°C. SM (10 ml) was added to each plate and the plates were gently agitated for 3-5 hat room temperature (RT). The SM was collected and pooled and 2 ml of chloroform was added. This lysate was shaken at 37°C for 30 min to ensure complete lysis of bacterial cells. Debris

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were then pelleted by centrifugation at 8000 xg for 10 min. The supernatant solution was collected and incubated with RNase A and DNasel for 1 h at 37°C to digest bacterial nucleic acids. Bacteriophage particles were precipitated by the addition of NaCl to 1 Mand PEG 8000 to a concentration of 10% (w /v), followed by chilling on ice for 1 h. The bacteriophage were collected by centrifugation at 9000 xg for 20 min. The pellet was resuspended in 5 ml SM. SDS and EDTA were added to this suspension to a final concentration of 0.5% (w /v) and 20 mM, respectively. Proteinase K (100 µg/rnl) was added and the suspension was incubated at 65°C for 30 min to degrade viral proteins. The DNA was extracted sequentially with equal volumes of phenol, phenol:chloroform:isoamyl alcohol (25:24:1) and chloroform:isoamyl alcohol (24:1) and then precipitated with 0.5 volumes 7.5 M ammonium acetate and 2.5 volumes absolute ethanol. The precipitate was collected by centrifugation at 10 000 xg for 15 min, washed in 70% (v /v) ethanol and resuspended in 4 ml of 0.4 M NaCl/6.5% (w /v) PEG 8000. After incubation on ice for 60 min, the suspension was spun as above and the pellet was again washed with 70% (v /v) ethanol. This pellet was vacuum­ dried and resuspended in 100 µl TE.

(ii) Small scale preparation of plasmid DNA

Small scale preparations of plasmid DNA were purified using a variation of the alkaline lysis method (Birnboim and Doly, 1979). By this method, 1.5 ml of an overnight bacterial culture grown in LB or Terrific Broth with 50 µg/ml ampicillin was transferred to an eppendorf tube. Tubes were centrifuged at 16 000 xg in a microfuge for 2 min to pellet the cells. The supernatant solution was removed and the cell pellet was resuspended in 200 µl of Solution I (50 mM glucose, 25 mM Tris.HCl, pH 8.0, 10 mM EDTA). Cells were lysed by the addition of 400 µl of Solution II (0.2 M NaOH, 0.1 % (w /v) SDS). Precipitation of cellular debris and chromosomal DNA was achieved by the addition of 300 µl of ice-cold Solution III (3M potassium acetate, 2 M acetic acid) and spinning at 16 000 xg in a rnicrofuge for 5 min. The supernatant solution was collected and 0.6 volume of isopropanol was added to precipitate plasmid DNA. The DNA was pelleted by centrifugation for 2 min, washed with 70% (v /v) ethanol, vacuum-dried and resuspended in 50 µl TE buffer or H20. If digestion of contaminating RNA was required, 1 µl of a 10 mg/ml solution of RNase A was added and the preparation was incubated at 37°C for 15 min.

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(iii) Large scale preparation of plasmid DNA

Large amounts of plasmid DNA were prepared by large scale plasmid purification (Sambrook et al., 1989). A starter culture was grown by inoculating 10 ml of LB containing 100 µg/ml ampicillin with 200 µl of a saturated culture of E. coli containing the plasmid of interest. This culture was incubated for several hours and then the entire 10 ml was used to inoculate 500 ml of LB containing 100 µg/ml ampicillin and 10 mM MgSO4 for overnight incubation. The bacteria were pelleted by centrifugation at 6370 xg for 10 min. Cells were then resuspended in 20 ml of Solution I (see above) containing 10 mg/ml lysozyme and allowed to stand for 10 min at RT. Solution II (40 ml) was added, the bacterial suspension was mixed and left at RT for 5 min. Ice-cold Solution III (40 ml) was then added and the mixture was chilled on ice for 15 min to precipitate bacterial cell debris. Water (5 ml) was added to the mixture and the precipitated material was collected by centrifugation at 11300 xg for 10 min. The supernatant solution was transferred to a 200 ml centrifuge bottle and 45 ml of isopropanol was added to precipitate the DNA. The bottle was centrifuged at 9820 xg for 12 min to pellet the DNA. This pellet was vacuum-dried and resuspended in 6.5 ml of TE. Then 0.5 M EDTA (150 µl) was added to the DNA, followed by CsCl to 1 mg/ml. Finally, 350 µl of ethidium bromide (10 mg/ml) was added to the suspension, which was then loaded into OptiSeal™ centrifuge tubes (Beckman Instruments, Palo Alto, CA). A CsCl gradient was established by ultracentrifugation at 342 000 xg for at least 16 h. The plasmid band was collected and extracted four times with an equal volume of isopropanol saturated with SM NaCl in TE. Two volumes of TE were added to the preparation, followed by 0.6 volumes of isopropanol in order to precipitate the plasmid DNA. The DNA was pelleted by centrifugation at 10 000 rpm 11 000 xg for 20 min, washed in 70% (v /v) ethanol, vacuum-dried and resuspended in 1000 µl TE.

(iv) Small scale extraction of genomic DNA from embryonic stem (ES) cells

Genomic DNA for PCR screening was purified from ES cells by the method of Hogan et al. (Hogan et al., 1994). Briefly, cells were allowed to overgrow in 96-well plates, then rinsed twice with PBS. Lysis buffer (10 mM Tris.HCl pH 7.7, 10 mM EDTA, 10 mM NaCl, 0.5% (w /v) Sarkosyl, 1 mg/ml

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proteinase K; 50 µl) was added to each well and the plates were incubated in a humid atmosphere at 60°C overnight. The following day, an NaCl/ ethanol mixture was prepared by adding 150 µl of 5 M NaCl per 10 ml of ice-cold absolute ethanol. One hundred µl of this mixture was added to each well and left at RT for 30 min to allow the DNA to precipitate. The precipitated DNA was washed with 70% (v /v) ethanol, the ethanol was removed and the DNA allowed to dry by evaporation. The DNA was dissolved in 100 µl H2O and stored at -20°C.

(v) Large scale extraction of genomic DNA from ES cells

One confluent 10 cm2 dish of ES cells was used for each preparation. Cells were lifted from the dish by overlaying with 2 ml trypsin solution (0.05% (w /v) containing 0.53 mM EDTA) and incubating for 5-10 min at 37°C. Once the cells had begun lifting from the culture dish, the trypsin was inactivated by the addition of medium and the cells were broken up into small clumps by vigorous pipetting. The cells were washed once in PBS, pelleted by centrifugation and then suspended in 1 ml TE followed by a further 1 ml TE containing 1% (v /v) Nonidet P-40. Cells were lysed by vortexing and the lysates were centrifuged at 2700 rpm for 5 min to pellet the nuclei. Nuclear pellets were carefully suspended in 1 ml of Buffer 1 (100 mM NaCl, 50 mM Tris.HCl pH 7.5, 1 mM EDTA) and then 1 ml of Buffer 2 (Buffer 1 plus 1% (w /v) SDS) was added slowly. Finally, proteinase K was added to a concentration of 125 µg/ml and the mixture was incubated for several hours at 37°C with mixing every 30 min. Proteins were extracted by adding 1 ml of phenol, followed by 1 ml of chloroform:isoamyl alcohol (24:1), with mixing in between. The extraction proceeded overnight with shaking at RT. The following day, the mixture was centrifuged at 3500 rpm for 10 min and the aqueous layer was transferred to a fresh tube. An equal volume of chloroform:isoamyl alcohol (24:1) was added to this aqueous layer and the tubes were shaken for four hours at RT. The tubes were centrifuged as previously and the chloroform layer was discarded. Genomic DNA was precipitated by the addition of sodium acetate to 0.2 M, followed by 2 volumes of absolute ethanol. The precipitate was transferred using a sealed yellow tip to 500 µl of TE and allowed to resuspend. An additional organic extraction was carried out to remove residual protein, using Phase Lock Gel tubes (5Prime-3Prime, Boulder, CO) according to the manufacturer's instructions.

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(vi) Extraction of DNA from mouse tail biopsies

Genomic DNA was extracted from tail tips for genotyping knockout mouse lines (Shehee et al., 1997). The volumes given are for 2 mm lengths of tail. For 5 mm lengths all volumes were doubled except the final volume for resuspension of DNA. Tail tips were digested overnight at 42°C in 200 µl of salt lysis buffer (100 mM NaCl, 100 mM EDTA, 50 mM Tris.HCl pH 8, 1% (w /v) SDS) with 10 µl of 10 mg/ml proteinase K added just prior to incubation. Following digestion, 100 µl of saturated NaCl solution was added and the tubes were mixed vigorously and spun at 16 000 xg in a microfuge for 20 min. The supernatant solution was transferred to a fresh tube and the DNA was precipitated by the addition of 0.6 volumes of absolute ethanol. The precipitate was either picked out with a sealed yellow tip or pelleted by centrifugation and then washed with 70% (v /v) ethanol. The DNA was resuspended in 100 µl H2O. In general, 1 µl of tail DNA was used for PCR analysis and 15-20 µl for restriction digestion.

2.4 ELECTROPHORESIS AND TRANSFER OF DNA

(i) Agarose gel electrophoresis

Agarose was dissolved by heating in 1 x TAE buffer at concentrations of 0.5%-2.5% (w /v) depending on the size of DNA fragments to be resolved. For preparative gels, from which DNA fragments were to be excised, agarose with a low melting temperature was used. Ethidium bromide was often added to the molten agarose before pouring, at a final concentration of 100 µg/ml. In other cases, gels were stained after electrophoresis in 1 x TAE buffer containing ethidium bromide (100-200 µg/ml). Prior to loading onto the gel, samples were mixed with loading buffer (6x buffer: 30% (v /v) glycerol, 0.25% (w /v) bromophenol blue, 0.25% (w /v) xylene cyanol). A ladder of standard DNA fragments was run alongside the samples to determine the size of the DNA in the samples. Gels were submerged in 1 x TAE buffer in a horizontal apparatus and electrophoresis was carried out at 7 V / cm for analytical gels or 5-5.4 V / cm for low melting temperature gels. DNA was visualised on a UV transilluminator (UVP Inc., San Gabriel, CA) and gel images were captured with a gel documentation apparatus (Bio-Rad

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Gel Doc 1000, Bio-Rad, Hercules, CA) using the Molecular Analyst software package.

DNA separated by electrophoresis in agarose gels was immobilised on charged nylon membranes by means of alkali blotting (Sambrook et al., 1989). For transfer of large (>3 kb) DNA fragments, gels were soaked in 0.25 M HCl for 15 min, followed by rinsing in H2O. Transfer of DNA onto the membranes was carried out using 0.4 N NaOH overnight at RT. Membranes were rinsed in 2 x SSC before pre-hybridization.

(ii) Polyacrylamide gel electrophoresis

Denaturing polyacrylamide gels were used for electrophoresis of DNA cycle sequencing products. The gel was prepared with 6% (v /v) bis-acrylamide and 5.4 M urea in 1 x TBE buffer. The acrylamide was polymerised by addition of ammonium persulphate and TEMED to a final concentration of 0.08% (w /v) and 0.06% (v /v), respectively. The gels were prepared at a thickness of 0.5 mm by pouring between two glass plates. Cycle sequencing products were denatured by heating to 95°C for 2 min and then loaded into wells at the top of the gel. Electrophoresis was carried out in a Base Runner vertical apparatus (International Biotechnologies Inc., New Haven, CT) in 1 x TBE buffer at a setting of 45 W. Following electrophoresis, gels were transferred to paper for vacuum-drying. The dried gel was apposed to X-ray film for autoradiography.

2.5 RADIOACTIVE LABELLING OF DNA

(i) Random priming

Radioactively labelled probes were synthesised from DNA fragments such as PCR products and restriction fragments by extension of random hexamers annealed to the DNA template. The Megaprime DNA labelling system was used, with 50 µCi of [a-32P]-dCTP and 25-50 ng of DNA, according to manufacturer's instructions. After incubating the labelling reaction for 10 min at 37°C, the labelled product was separated from unincorporated nucleotides by centrifugation through a 1 ml Sephadex® G-25 spin column.

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Prior to adding the labelled DNA probe to the hybridization buffer it was denatured by heating to 95°C for 2-3 min.

(ii) 5' end labelling

Oligonucleotides were radioactively labelled by the transfer of a 32P-labelled phosphate group to the 5' terminus of the oligonucleotide. Twenty pmol of oligonucleotide was labelled in a reaction containing 10 U of T4 polynucleotide kinase, reaction buffer provided with the enzyme and 50 µCi of [y-32P]-ATP. After incubation at 37°C for 30 min, the labelled product was purified by centrifugation through a 1 ml Sephadex® G-25 spin column.

(iii) Preparation of spin columns

Sephadex® G-25 spin columns were used to purify radioactively labelled probes. The columns were prepared by first plugging a 1 ml syringe barrel with siliconised glass wool, then packing the syringe with a slurry of Sephadex® G-25 in TE. The slurry was compacted by centrifugation at 160 xg for 5 min after placing the syringe in a 15 ml centrifuge tube. The column was washed with 50 µl TE and re-centrifuged before the sample was added. Purified probe was collected into a 1.5 ml microfuge tube by centrifugation as before.

2.6 HYBRIDIZATION OF DNA

(i) Hybridization of radioactively labelled DNA fragments

DNA probes radioactively labelled by random priming were hybridized to target DNA immobilized on nylon filters. The filters were pre-hybridized in buffer containing 5 x SSPE, 5 x Denhardt's reagent, 0.5% (w /v) SDS and 100 µg/ml sheared, denatured salmon sperm DNA, for a minimum of two hours at 65°C for homologous probes or 50°C for cross-species hybridizations (genomic library screening). The probe was added to the buffer and hybridization proceeded for at least 16 h with shaking at the same temperature. Filters were then washed to remove non-specific probe binding. Washes were initially carried out in 2 x SSC at RT with two changes of buffer. The buffer was then changed to 1 x SSC containing 0.05%

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(w /v) SDS for genomic library screening or 0.1 x SSC containing 0.1 % (w /v) SDS for other filters, and the wash carried out at the hybridization temperature. A single wash at elevated temperature was generally sufficient for genomic Southern blots, while other filters required an additional two changes of buffer. Finally, the filters were air-dried and exposed to X-ray film or phosphor screens. Phosphor images were developed and analysed on a Phosphorlmager™ 445SI with ImageQuant software (Molecular Dynamics, Sunnyvale, CA).

(ii) Hybridization of oligonucleotide probes

Southern hybridizations with radioactively labelled oligonucleotides were carried out to detect membrane-bound restriction digest or PCR products containing sequences complementary to the oligonucleotide. DNA fragments immobilised on nylon membranes were first prehybridized in 20- 50 ml of hybridization buffer for a minimum of two hours at 42°C. Hybridization buffer was 20% (v /v) formamide containing 5 x SSC, 5 x Denhardt's reagent, 1% (w /v) SDS and 50 µg/ml sheared, denatured salmon sperm DNA. Following the pre-incubation, 32P-labelled oligonucleotide was added to the buffer and hybridization proceeded for at least 16 h, with shaking at 42°C. Non-specifically bound oligonucleotide was then removed by washing membranes in 6 x SSC containing 0.1 % (w /v) SDS at 42°C. Filters were then partially air-dried and exposed to X-ray film or phosphor screens.

2.7 CLONING OF DNA

(i) Restriction digestion

DNA was digested with restriction endonucleases, singly or in combination, according to manufacturers' recommendations. The DNA was incubated in the appropriate pre-formulated buffer with approximately 4 U of enzyme per µg of DNA. Incubation periods varied from 1-2 h for plasmid or A DNA to overnight for genomic DNA. Restriction digestion was generally carried out at 37°C.

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(ii) Gel purification of DNA fragments

DNA fragments to be cloned or radioactively labelled were purified by electrophoresis through low melting-point agarose and elution with the Wizard® PCR Preps kit. Briefly, following electrophoresis, the DNA was visualised on a UV transilluminator and a wedge of agarose containing the band of interest was removed with a scalpel blade. The agarose was melted by heating at 65°C and then mixed with a DNA binding matrix. This mixture was passed through a column in which the DNA was retained. The column was washed with 80% (v /v) isopropanol and the DNA then eluted by the addition of 30-50 µl of TE or H20 and collected by centrifugation.

(iii) Ligation of DNA into plasmid vectors

DNA fragments were sub-cloned by ligation to cloning vectors prepared with compatible termini. For ligation to genomic DNA fragments, vectors were prepared by restriction digestion. For sub-cloning of PCR products, the T-tailed vector pGEM®-T was used with no preparation required. Vector DNA (50-100 ng) was incubated with an approximately two-fold molar excess of insert DNA in a reaction containing 1-3 U of T4 DNA ligase and 1 x ligase buffer (supplied by the manufacturer) in a total volume of 10-20 µl. The reaction was incubated overnight at 37°C or for several hours at 15°C. If the DNA was contained in an agarose gel slice, the gel was melted at 65°C for 10 min before the reaction components were added. In this case, 5 U of enzyme was used. In general, following ligation, reaction products were introduced into competent cells by transformation.

(iv) Linker ligation

In order to create compatible ends for the cloning of restriction fragments EcoRI linkers were ligated to fragments which had previously been blunted by treatment with Klenow polymerase (see Section 2.7(v) below). Water plus a one hundred-fold molar excess of 12-mer linker was added to a dessicated pellet of DNA, to a total volume of 45 µl. The tube was incubated at 65°C for 10 min to resuspend the DNA. DNA ligase buffer and 1 U of T4 DNA ligase were added to a final volume of 50 µl. The ligation reaction was incubated at 16°C for 5 h. The entire reaction was then extracted with phenol:chloroform:isoamyl alcohol (25:24:1) using phase-lock gel tubes. The

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ligation products were precipitated using 3M sodium acetate (0.1 vol) and 100% ethanol (2.5 vol) to remove excess linkers before being digested with restriction endonuclease.

(v) Generation of blunt-ended restriction fragments

The Klenow fragment of DNA polymerase I was used to extend recessed ends of restriction fragments. The reaction consisted of an aqueous mixture of DNA fragment, 1 x Klenow buffer (provided with the enzyme), 12.5 nM of each deoxynucleotide triphosphate and 10 U of Klenow fragment in a total volume of 20 µl. The reaction was incubated for 30 min at 37°C before being terminated by extraction with organic solvents.

(vi) Preparation of competent cells

Competent E. coli cells (strain DH5a) were prepared essentially as described (Sambrook et al., 1989), except for some differences in the buffer components. One ml of a saturated bacterial culture was used to inoculate 100 ml of LB broth. The culture was grown with agitation at 37°C until its absorbance at 550 nm reached 0.45-0.55. The culture was placed on ice for 10 min, then centrifuged at 860 xg for 10 min. The medium was discarded and the bacterial pellet was suspended in 30 ml of cold PB (100 mM KCl, 50 mM CaCh, 10 mM potassium acetate pH 7.5, 10% (v /v) glycerol, pH adjusted to 6.2) and placed on ice for 20 min. The suspension was centrifuged again at 3440 xg for 10 min and the pellet was resuspended in 4 ml of PB. The cells were then aliquoted into cryogenic vials, snap frozen and stored at -80°C for future use.

(vii) Bacterial transformation

Plasmid DNA was introduced into bacteria by a standard transfection protocol (Sambrook et al., 1989). Competent E. coli cells (100 µl) were mixed with a minimal volume (up to 50 µl) of DNA and chilled at 0°C for 30 min. The cells were then heat-shocked at 42°C for 45 sec and chilled for a further 2 min. A volume of 0.9 ml of SOC or LB medium was added to the cells and they were incubated at 37°C for 1 h with shaking. Volumes of cell suspension in the range 10-100 µl were spread onto LB agar plates containing 50 µg/ml ampicillin and X-gal (40 µg/ml) for growth overnight

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at 37°C. Under these conditions, cells containing recombinant plasmid grew as white colonies while other colonies were coloured blue.

(viii) Transfer and immobilization of plasmid and bacteriophage DNA

Identification of recombinant plasmids or bacteriophage containing sequence of interest was done by hybridization to radioactively labelled DNA probes. The target DNA, grown in bacterial colonies or plaques on a bacterial lawn, was first immobilized by transfer of the colony or plaque to nylon filters and fixation of the DNA. For transfer of bacteriophage DNA, agar plates were first cooled to 4°C for one hour. The filters were overlayed on the agar plates and left for 2-3 min. The filters were lifted off and the DNA was denatured by placing on 0.5 N NaOH-soaked filter paper for 10 min. The filters then underwent two washes of 3 min each in neutralising solution comprising 1.5 M NaCl, 0.5 M Tris.HCl pH 7.2 and 1 mM EDTA. The filters were left to air-dry for several hours prior to hybridization.

2.8 DNA SEQUENCING

DNA sequencing was carried out by the Sanger "dideoxy" chain termination method (Sambrook et al., 1989). The fmo[® kit was used for cycle sequencing of double-stranded plasmid DNA with 33P-labelled oligonucleotide primers. The DNA template was prepared by small-scale purification as described (Section 2.3(ii)). Sequencing primer (10 pmol) was labelled with [y-33P]-ATP as recommended. Following a standard cycle sequencing program, the reactions were stopped by addition of STOP solution supplied with the kit. Sequencing reaction products were separated by eletrophoresis through polyacrylamide gels (Section 2.4(ii)).

2.9 PREPARATION OF RNA

(i) Total RNA preparation

Mouse tissues and embryos were either snap frozen in liquid nitrogen or immersed in RNAlater fixative or Tri Reagent (pituitary only). All samples except pituitary tissue were homogenised in 2 ml Tri Reagent and total

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RNA was extracted according to the manufacturers' protocol. Pituitaries collected in 0.5 ml Tri Reagent were dispersed by passing through a 21G needle, followed by a 25G needle. Total RNA was suspended in H 20 and stored at-80°C.

(ii) Poly-A+ RNA preparation

Poly-A+ RNA was purified from total RNA preparations using the Promega PolyATtract® mRNA Isolation System IV kit according to manufacturer's instructions.

2.10 QUANTITATION OF NUCLEIC ACIDS

The concentration of solutions of nucleic acids was determined by absorbance at 260 nm using a Pharmacia GeneQuant spectrophotometer. An absorbance of 1.0 was considered equivalent to a concentration of 50 µg/ml of double-stranded DNA, 40 µg/ml of RNA and 37 µg/ml of oligonucleotide.

2.11 POLYMERASE CHAIN REACTION AND V ARIATI ONS

(i) Polymerase chain reaction (PCR)

PCR was employed for the detection or amplification of DNA in a variety of settings. A standard reaction was prepared with 0.2 µM of each primer, 1.5 mM MgCh, 1 x concentration of PCR buffer, 200 µM of each deoxynucleotide, 2.5 U Taq polymerase and DNA template in a total volume of 50 µl. The amount of template DNA used was approximately 1 µg of genomic DNA or approximately 50 ng of plasmid DNA. The number of cycles used was dependent on the initial amount of target DNA: 30 cycles for plasmid and 35 cycles for genomic DNA. When AmpliTaq Gold™ DNA polymerase was used, an initial 9 min incubation at 94°C was carried out to activate the enzyme. The PCR program was carried out in either a Corbett (Sydney, Australia) or Perkin Elmer Cetus (Norwalk, CT) thermocycler.

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(ii) Reverse transcriptase (RT-) PCR

RT-PCR was used to amplify specific mRNAs in preparations of total RNA or poly-A+ RNA from mouse tissues. The methodology employed a two­ step system: a cDNA synthesis reaction using reverse transcriptase, followed by a conventional PCR to amplify the cDNAs of interest. Reverse transcription was carried out with Expand™ Reverse Transcriptase according to the protocol accompanying the enzyme. One µg of total RNA was used as template material, unless otherwise described. The RNA was mixed with 50 pmol of random hexamer oligonucleotides and heated at 65°C for 10 min to eliminate secondary structure in the template, then placed on ice to enable primer annealing. The final reaction volume of 20 µl also consisted of 10 mM dithiothreitol, 1 mM of each deoxynucleotide, 20 U RNase inhibitor, 50 U Expand™ Reverse Transcriptase and 1 x concentration of reaction buffer (provided with the enzyme). The reaction was incubated at 30°C for 10 min, followed by 45 min at 42°C. Five µl of cDNA was then used as template in a PCR reaction with gene-specific primers as described (Section 2.ll(i)).

(iii) 5'/3'-Rapid Amplification of cDNA Ends (RACE)

5'-RACE was carried out using 2 µg of mouse brain total RNA with a 5' /3'­ RACE kit. For 5'-RACE, a gene-specific primer annealing near the 5' end of the coding sequence and AMV reverse transcriptase were used as recommended to produce first-strand cDNA. Following tailing of the first­ strand cDNA with poly-A using terminal transferase, one-fifth of the poly A-tailed cDNA was incubated with 250 nM oligonucleotide primers, 200 µM dNTPs and 0.5 units AmpliTaq Gold™ DNA polymerase in 10 mM Tris.HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl2 in a total volume of 50 µl. The oligonucleotide primers used were an oligo-dT anchor primer provided with the kit (5'-GACCACGCGTATCGATGTCGACTTTTTTTTTTTTTTTT-3') and a second antisense gene-specific primer. PCR amplification was performed with the following temperature parameters: incubation at 94 °C for 12 min to activate AmpliTaq® Gold DNA polymerase, followed by 10 cycles of 94 °C for 15 sec, 50 °C for 30 sec and 72 °C for 40 sec, then 25 cycles of 94 °C for 15 sec, 50 °C for 30 sec and 72 °C for 40 sec with an additional 20 sec at 72 °C for each successive cycle, then incubation at 72°C for 7 min. Reamplification was done under the same reaction and cycling conditions

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using 1 µl of a 1:20 dilution of the original PCR reaction and a PCR anchor primer (5'-GACCACGCGTATCGATGTCGAC-3') supplied with the kit instead of the oligo-dT anchor primer. 5'-RACE products were analysed by electrophoresis in 1.5% (w /v) agarose gels.

3'-RACE was carried out using the same kit. cDNA synthesis used the oligo dT anchor primer described above and conditions recommended. Amplification of the cDNA by PCR used the PCR anchor primer described and a gene specific primer proximal to the 3' end of the coding region of the message. Cycling conditions were as for 5'-RACE.

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CLONING AND CHARACTERIZATION OF THE MOUSE Galnr1 GALANIN RECEPTOR GENE Chapter 3

3.1 INTRODUCTION

This chapter describes the cloning, genomic structure and sequence of the mouse Galnrl gene encoding the GALRl galanin receptor. These data serve two main purposes. Firstly, they provide the basic information crucial to our understanding of the structure of this gene and future analysis of its function. Secondly, within the context of this project, they enable the design of constructs for targeted mutagenesis of the Galnrl locus in embryonic stem cells.

The human and rat GALRl cDNAs were isolated by expression cloning and were classified as members of the superfamily of GPCRs (Burgevin et al., 1995; Habert-Ortoli et al., 1994; Parker et al., 1995; Sullivan et al., 1997). They possess conserved residues characteristic of the rhodopsin subfamily of GPCRs. The human GALNRl gene encoding GALRl consists of 3 coding exons and extends approximately 15-20 kb on chromosome 18q23 (Jacoby et al., 1997; Nicholl et al., 1995). As well as providing the basis for gene knockout studies, it is of interest to determine whether the orthologous mouse gene shares this genomic structure and relative chromosomal position. Furthermore, if any documented mutant mouse strains have mutations linked to the chromosomal location of Galnrl, the phenotypic effect of the mutation could give us insight into the function of the gene.

At present, very little is known about the regulation of the gene encoding GALRl. The mRNA is widely distributed in the nervous system of man and rat. It is present in the brain and spinal cord, intestine, pancreas, testis, ovary and several other tissues (Burgevin et al., 1995; Lorimer and Benya, 1996; Parker et al., 1995; Sullivan et al., 1997). During embryonic development in the rat, GALRl mRNA is found in the brain and intestine from E14 and sensory ganglia and spinal cord from E17 (Xu et al., 1996a). It has also been demonstrated that GALRl mRNA expression is down­ regulated in response to peripheral nerve injury and inflammation in rat dorsal root ganglia (Xu et al., 1996b) and by hypophysectomy or lactation in rat hypothalamus (Landry et al., 1998). In contrast, salt loading in rats increases GALRl mRNA levels in magnocellular neurons of the hypothalamus (Landry et al., 1998). Some manipulations, such as peripheral axotomy (Xu et al., 1996b), induce opposing changes in galanin and GALRl expression, implying a complex response of the galaninergic system to

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physiological disturbances. A better understanding of the factors and conditions which regulate the expression of the Galnrl gene may give us further clues as to its role in galanin signalling.

The analysis of sequences beyond the boundaries of the coding region of the Galnrl gene may uncover features involved in regulation of expression of the gene. Determining the site or sites of transcription initiation on the genomic DNA will delineate the promoter region, possibly containing sequence motifs which reveal the factors that control expression. Analysis of the 5'- and 3'-UTRs of the GALRl transcript may reveal untranslated exons which have not previously been reported but have been found in other GPCR genes (Ball et al., 1995; Kraus et al., 1998). In some GPCR genes, alternative splicing of upstream exons places the coding region under the control of different promoters which may be activated in specific tissues or physiological states or during different stages of development. We aim to determine whether this may occur in the Galnrl gene.

The development of approaches for the specific manipulation of the mouse genome has opened new avenues for understanding the functional role of individual genes. The use of targeted mutagenesis of the Galnrl gene in order to probe the role of galanin in mammalian physiology becomes a possibility upon physical characterization of the gene in the mouse. The initial steps in targeted mutagenesis experiments involve replacement, by homologous recombination, of an endogenous allele of the gene with a targeting construct in embryonic stem cells. Basic targeting constructs consist of two genomic fragments flanking a selectable marker which is inserted within, or replacing, coding sequence. The relative orientation of the genomic fragments must be homologous with the endogenous allele so as to align with it when the construct is introduced into the cells.

Most commonly, targeting vectors are constructed by subcloning genomic restriction fragments, of a length in the range of one to several kilobases, on both sides of the selectable marker which is already present in the cloning vector. This is carried out in such a way that, upon homologous recombination with the endogenous locus, the marker gene will disrupt or replace an essential coding region of the gene. The physical data necessary to design such a vector include a detailed restriction map of the gene of interest and sequence of the coding exons to be disrupted or deleted. Elucidating a

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complete genomic structure and coding sequence will aid in determining the regions critical for protein function. In principle, the disruption of these critical regions in vivo will result in a null allele. Study of the functional consequences of disruption of the Galnrl gene in the mouse should provide insights into the role of GALRl in galanin signalling in vivo.

3.2 METHODS

(i) Library screening

A custom mouse 129 /SvEv genomic library in A FIXII was screened with the full-length coding sequence of human GALRl. The DNA probe was radioactively labelled by random priming and the hybridization was carried out at 50°C. Aproximately 1.8 genome equivalents (6 x 105 pfu) were screened in duplicate. Non-specific binding of probe was removed by washing in 1 x SSC at 50°C. Following three rounds of screening, seven independent clones were isolated. DNA was purified from these clones and digested with several restriction endonucleases. Agarose gel electrophoresis of these digests demonstrated that all seven isolates were identical.

(ii) Subcloning

Southern analysis using the full-length human GALRl coding region as a probe identified restriction fragments of the genomic clone containing coding sequence. Further Southern hybridization analysis with fragments specific to exons 1 and 3 of the human gene identified a 3 kb BamH I fragment and a 4.5 kb HincII fragment containing exon 1-related and exon 3- related sequences, respectively. Both restriction fragments were subcloned into pBluescript® II for DNA sequencing.

(iii) Cloning of partial cDNA

RT-PCR was carried out to amplify a partial mouse GALRl cDNA containing exon 2-derived sequence. The procedure followed was as described in Chapter 2, with some variations. Reverse transcription was carried out on 4 µg brain total RNA from the FVB /N mouse strain, using 15 pmol of random hexamer primers and 200 U Superscript™ II reverse

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transcriptase. The reaction was incubated for 1 h at 42°C. For amplification of the cDNA, two rounds of PCR were carried out, using primers derived from human GALRl exons 1 (#452; Table 3.1) and 3 (#864; Table 3.1) and a reaction volume of 100 µl. Amplification products in the predicted size range of 440 bp were obtained and subcloned into pGem-T® for sequencing.

Using vector primers, the sequence of the partial cDNA containing exon 2 was determined for FVB /N mouse brain GALRl. Based on this sequence, an oligonucleotide probe specific for exon 2 was designed and used to isolate from the genomic clone, a 194 bp Hindi restriction fragment containing exon 2 of the Galnrl gene. This restriction fragment was subcloned into pBluescript® II and sequenced with vector primers.

(iv) Restriction Mapping

Digests with standard restriction endonucleases, singly or in combination, were carried out on genomic clone DNA. Digestion products were separated by electrophoresis on agarose gels and the DNA was transferred by alkali blot to nylon membranes. The membranes were hybridized with either full­ length or partial human GALRl cDNA, or with oligonucleotides based on human or mouse genomic sequence. A restriction map of the lambda clone was compiled for the enzymes Apal, BamHI, EcoRI, Kpnl, Sacl and Spel. The positions of several restriction sites within or flanking coding exons were confirmed by DNA sequencing.

(v) DNA Sequencing

DNA sequencing was carried out on genomic sub-clones, initially using vector primers and primers derived from the human GALRl sequence (#452, #864; Table 3.1). Sequencing was completed with the use of primers based on additional sequence as it was obtained (#1048, #1061, #1072, #1107, #6862; Table 3.1). The positions of the primers used are displayed in Figure 3.2.

74 Chapter 3

PRIMER SEQUENCE 452 5'-CGCCTTCATCTGCAAGTTCA-3' 864 5'-CAGAACTGTCTGTGCAGTC-3' 1048 5'-AATGGCCACGTAGCGATCCA-3' 1061 5'-TACAGCGCTGTATTCTCCTG-3' 1072 5'-GCTCCCATTCCCTTCACTGA-3' 1107 5'-CTTTTTCTTGGATGCTTCAG-3' 6862 5'-CAACTGCACCCACGTGTGAAGG-3'

TABLE 3.1 Sequence of DNA sequencing primers

(vi) 5'- and 3'-RACE

RACE was used in an attempt to define the extent of cDNA non-coding regions. 5'-RACE uses a series of steps to amplify the 5' end of cDNAs. Firstly, reverse transcriptase is used to extend a primer annealed to a region of mRNA whose sequence is known. This strand of cDNA should terminate at the 5' end of the transcript. A poly-A tail is then added to the cDNA using terminal transferase and dATP. A two-round PCR strategy is used to amplify the tailed cDNA. In the first round, a nested gene-specific primer is used in combination with an oligo dT-anchor primer. A second round of PCR is then carried out with a nested PCR anchor primer in combination with the same gene-specific primer, to produce enough specific product for downstream analysis. 3'-RACE is similar but requires fewer steps as the 3' end of the mRNA naturally possesses a poly-A tail. In this case, reverse transcription from the oligo dT-anchor primer and a single PCR reaction should produce sufficient product for analysis and cloning.

5'-RACE was carried out on mouse brain total RNA using a 5' /3'-RACE kit according to the manufacturer's instructions. The primer used for reverse transcription of the 5' end of the transcript corresponded to human GALRl coding sequence, nucleotides 109-90 (#799; 5'­ GCGTGACGAAGTTCTCCACG-3'). The antisense PCR primer used corresponded to the mouse GALRl coding sequence, nucleotides 42-23 (#1072; 5'-GCTCCCATTCCCTTCACTGA-3'). Products were separated on agarose gels and transferred to nylon membranes. The major product was visible on the gel by ethidium bromide staining and was subcloned for DNA

75 Chapter 3

sequencing. Larger products were detected by hybridization to an oligonucleotide corresponding to mouse genomic sequence, nucleotides -190 to -169 (#5962; 5'-CGCTCTTCCAGGCTTTCTTGCG-3') and were subcloned for DNA sequencing.

3'-RACE was also attempted on mouse brain total RNA using the kit described. The sense primer used to amplify the cDNA in combination with the provided anchor primer, corresponded to mouse GALRl sequence, nucleotides 1029-1050 (#6862; 5'-CAACTGCACCCACGTGTGAAGG-3'). An internal oligonucleotide was used to identify 3'-RACE products by hybridization. This oligonucleotide corresponded to the mouse GALRl cDNA, nucleotides 1070-1089 (#6899; 5'-TCCAGCTCCATGTGTGTTAG-3'). On another occasion this oligonucleotide was used as the sense PCR primer in amplifying the cDNA. Two other oligonucleotides were used to identify specific products, at nucleotides 1456-1475 (#7445; 5'­ GTTTCTCTGTGTAGCCCTGG-3') and 1358-1377 (#7775; 5'­ TTCCAGTCTGCCTCAGTATC-3') relative to the start codon.

(vii) PCR amplification of the 3'-UTR

In order to characterise sequences downstream of the coding region at the Galnrl locus, PCR amplification of cloned DNA and of genomic DNA from mouse strains 129 (three sub-strains) and FVB/N was performed. The forward primer used (#6862) annealed at the 3' end of the GALRl coding region, nucleotides 1029-1050, as shown in Figure 3.2. Oligonucleotides corresponding to either the cloned sequence or a published Galnrl sequence (Wang et al., 1997c) were used as alternate reverse primers. The primer designed to amplify the cloned sequence corresponded to the sequence 546- 525 nucleotides 3' of the STOP codon (#13342; 5' - GCAGTTAAAACAATGAGGCAGG-3'). The primer designed to amplify the reported sequence corresponded to nucleotides 1533-1514 according to the numbering of the published cDNA (#13543; 5'­ GTGCCGGTCCACAAAGGTTC-3').

(viii) Chromosomal localization of Galnr1

The 440 bp partial cDNA described in Section 3.2(ii) above was used as a probe for chromosomal localization of Galnrl. This fragment was too short

76 Chapter 3

to be detected by use of fluorescent in situ hybridization, so it was detected using tritium labelling and autoradiography. The probe was labelled to a specific activity 3.6 x 107 cpm/µg by nick translation with tritiated dATP, dCTP and dTTP (Amersham). Preparation of mitotic chromosomes from splenic lymphocytes of C57BL and BALB/c male mice, in situ hybridization of the probe to the slides, and scoring, were as previously described (Webb et al., 1989). The slides were exposed to L4 nuclear emulsion for 75 days. This work was carried out by Dr. Graham C. Webb (University of Adelaide, Adelaide, Australia).

3.3 RESULTS

(i) Cloning and characterization of the mouse Galnr1 gene

A mouse lambda genomic DNA library was screened using full-length human GALRl coding sequence as a probe. Several clones were isolated and shown by Southern hybridization analysis to be identical. The inserts of these clones were approximately 20 kb. A restriction map was constructed and indicated that the gene encoding GALRl spans approximately 15-20 kb (Figure 3.1).

A 3 kb BamHI restriction fragment and a 4.5 kb Hindi restriction fragment were identified by hybridization as containing coding sequence and were subcloned into pBluescript® II. These sub-clones were partially sequenced, initially using human primers specific for exons 1 and 3 of the human GALNRl gene, then primers based on the additional sequence as it was obtained. The positions of the primers used are displayed in Figure 3.2. A 440 bp partial cDNA isolated from mouse brain by RT-PCR included 66 bp of sequence with strong similarity to the human exon 2 sequence. As this cDNA was cloned from a different mouse strain to the source of the genomic library (strain FVB/N), it was necessary to confirm the exon 2 sequence of the genomic clone, derived from strain 129 /SvEv. A 194 bp Hindi restriction fragment from the genomic clone was identified using an oligonucleotide probe that corresponded to part of the putative exon 2 sequence. This restriction fragment did include 66 bp of sequence identical to exon 2 of the partial cDNA.

77 Mouse Galnr1 ~

N Sc N K E 1 kb

A B

GALR1 (NH2) "M 1 2 3

E E E V ell / E E ~ i ~ ~ ~------~r I I 111I I ~------1tf7£t------~ p p p PP ,c: > Pst I fragments Human GALNR1 (2.4, 1.2, 0.9, 0.55, 0.3, 0.25) kb

FIGURE 3.1 Genomic organisation of GALRl galanin receptor genes in mouse (Galnrl) and human (GALNR1, described in Jacoby et al., 1997). The upper and lower lines depict the restriction endonuclease maps of mouse and human genes, respectively. The cloned mouse Galnrl DNA is delimited by vector-derived Natl restriction endonuclease cleavage sites. Solid boxes represent coding sequence. Lightly hatched boxes represent non-coding exon sequence, with the S'-upstream limit of the shorter mouse GALRl transcript indicated with a filled arrowhead, and the S'-upstream limit of a human hypothalamic cDNA clone indicated with an unfilled arrowhead. The dashed segments of human GALNRl DNA represent sections of the map where the DNA has not been mapped for BamHI or Pstl cleavage sites (upstream of exon 1), EcaRI cleavage sites (downstream of exon 3), or where the relative order of Pstl fragments has not been defined (intron 2). The central part of the figure is a schematic representation of GALRl coding sequence. Putative transmembrane domains are shown as purple boxes and are numbered 1-7, other parts of the coding region are shown as coloured boxes, and segments of the mature GALRl receptor protein encoded by each of the exons are indicated. A, Apa!; B, BamHI; E, EcaRI; K, Kpnl; N, Natl; P, Pstl; S, Spel; Sc, Sac!. Chapter 3

When assembled, the translated exon sequences predicted a peptide of 348 amino acids with a characteristic seven transmembrane domain structure (Figure 3.2). The genomic structure was found to be identical with that of the gene encoding human GALRl, consisting of three coding exons with exact conservation of the position of exon:intron boundaries (Figure 3.1). The exon:intron junctions exhibited good agreement with consensus splice donor and acceptor sites (Mount, 1982). The first exon encodes 221 amino acids, from the N-terminus of the predicted receptor to the end of the fifth transmembrane domain. The second exon encodes the third intracellular loop of 22 amino acids. The third coding exon encodes the sixth and seventh transmembrane domains and the C-terminal tail of the receptor, a total of 105 amino acids. Exons 1 and 2 are separated by approximately 5 kb while exons 2 and 3 are separated by approximately 8 kb (Figure 3.1).

Alignment of the deduced amino acid sequence of mouse GALRl with human and rat GALRl sequences indicated sequence identities of 93% and 96%, respectively (Figure 3.3). The mouse nucleic acid sequence encodes a protein one residue shorter than the human receptor, with Pro170 in the second extracellular loop of the human receptor having no counterpart in the mouse. There are 26 other amino acid differences between the two orthologues, with the transmembrane regions displaying the highest degree of conservation and the C-terminal tail exhibiting the most variation. Of the 26 differences in amino acid sequence, nine are conservative differences.

In comparison, rat GALRl consists of only 346 amino acids with a 96% identity to the mouse sequence. This difference in peptide length arises from an extra residue in both the N-terminal extracellular domain and the C-terminal tail of mouse GALRl, in addition to the absence of a counterpart to Pro170 of the human GALRl sequence. Potential N-linked glycosylation sites at Asn residues 7, 12 and 182 are strictly conserved between receptor sequences of all three species, as is a predicted cAMP / cGMP-dependent phosphorylation site at Seq43 in mouse and human sequences, which corresponds to Seq42 in the rat GALRl sequence.

79 FIGURE 3.2 Nucleotide and deduced amino acid sequence of the mouse gene encoding GALRl. Exon sequence is in upper case, presumed intron sequence is in lower case, and gt/ ag residues flanking splice donor and acceptor sites are in bold lettering. Nucleotides are numbered, with +1 corresponding to the first nucleotide of the methionine translation initiation codon. Untranslated sequence as defined by 5'-RACE analysis of mouse brain RNA is shown in dark blue up to nucleotide -164 and thence in purple up to nucleotide -645 relative to the translation initiation codon, signifying the two identified transcription start sites in the mouse genomic DNA. The deduced amino acid sequence is shown in one-letter code below the nucleotide sequence. Amino acids are numbered from the methionine translation initiation site and putative transmembrane domains are underlined and coloured light blue. Numbered pink arrows represent sequencing primers, as described in the text (Section 3.2(v)). The nucleotide sequence has been deposited in the GenBank/EMBL Data Base under Accession Nos U90655-57.

80 -1040

1

16

121 -940

-840 -

-340 -

-640 46 -

-140 -540

41 -440

240

40 740

ACCCTCTICACCATIGAAGG'IG'IGCATCGCroGGCTCTCGGACGTI'CGGGAAGAAGAGCTCAAAGCAACAGG'IGCAACCTCAAGGCAC'IGAAAGCAAGGG

CG'roAGAGAGGC'lX:GCCCTGCAGAGGACCCGGGACTAAGAGGGAGCCGCAGGCCAGCGCAGCGAQX:AGGGAGG'IGGATC'ITAG'!GmGGAAGCTCAGCG

GCGCGC'lX:GCTC'lX:GCCGCTC'IGTCC'IGGGCCACTCCG'IGATCCTAGGCTACCTCCAGAGCCAG'ITTTCCC'IGGC'IGGCACAACTCTCCAGGGCGCTCCG

GTCCGT'IGCACAGCGCCCCAAGGGGGTATCCCAGI'AAG'IG GACGCAGCTCACAAGGGCCAAGGGATI'GAACCCATAACCGCTCAGAAGATTCTCCGCC'IGCGGAGAGC'IGCGGAGGAGTCCCACCCGI'CCAGCT'IGC'IGA gttacccggtacctacatgcaagagcaggtctttcttttggactgctagaagcagtggctcatagttggtgtggcccccgcctcccttctcagcagagcg

gcctttcaccacagtcaaatgactcttcacaaacacttcactcgtcaaggcttccgtttgtgtcactcagatgagtaatgtctggtggaaatgat

ggtaaagtggctcctgcctcaccattgcgcatccttgcctgcagggctggactgtgcaacccgcactggtgcatctccacagagctttcccaacgagcac

GGC cctgtcctacccttctgtgcacaatctgtttctggccttgtgtctagcagttgtggtcactcaccgctggaggatcgctggcgttttggaaagcgatata

C'IGCGAGCAG'IGAGAGTCGCCTAGACCCGTACCTC'IG'IGTTC'IGGAGCC'IGCCGCCCCCGCACGGGAAAGGC'ITAGCTCGGGACT'IGCAGCACCGCCTCC CCA

TC'ITTAGCCAGGCCAGGCACGAGGATAG'IG'IGATCGGGCACAGCCAGGGTCGCTCTTCCAGGC'ITTCTI'GCGGG

L I L G

E P P S P F I V N E V G I G F L P R S E P A P P E P

GAG

C'IG

*

A'IT

CCG

* * * *

* * * * *

* *

* * * * *

*

*

*

*

CCA

TTC

*

A F

GCC

GCG

*

*

* * * *

* * * *

*

*

*

*

*

A'IG

CCG

M

GAG

GGC

G

*

G'IG

TCC

*

L N L V L S N G L V

*

*

* * * * * * * *

*

*

AGG

C'IG

*

GGC

CCG

*

*

* * * * *

* * *

*

*

*

* * * * *

AAC

CTC

A'IG

E A V L E N S D S G N G E S L N V M A L E M

AGC

TTC

GAA

*

GGC

C'IG

*

*

C'IG *

* *

*

*

ATT G'IG

*

GCT

*

ATC

GGC

A'IG

I

*

*

*

*

*

*

*

ACC

G'IG

GTG

V L V T

GTG

GAG

AAC

*

*

AAC

C'IG

CTC

*

*

*

* *

*

* * *

~

GCG

TTC

*

R K G K G P K S R A

AGT

I L V F V V L T I F

T'IGCGGGAGGTACTAGTIGGAGACGC

A'IT

CGC

GAA

*

*

*

*

*

* *

* *

*

*

ACG

AGC

GGG

AAA

C'IG

1072

AAT

*

GI'A

CCA

GGG

*

*

* *

*

*

* *

*

*

GGC

G'IG

*

AGC

*

GGATC

AAG

TTT

GAC

*

*

*

* *

*

*

*

*

00

~

715 715

216 216 239 239

222 222

196 196

664 664

191 191

166 166

271 271

646 646

141 141

346 346

116 116

496 496 66 66

571 571 421 421

91 91

ATG ATG

GCA GCA

atgtctccgcgacact------intron atgtctccgcgacact------intron

ACC ACC

CGG CGG

aacccgcttcatccggttatgttttgc

CCC CCC

TGC TGC

ctggtgtgtctgaagactgttacagtgaactgcacatataataataagtaaatttttaaaaaagaaagttttttaaataaataaatgctacagaggtgta ctggtgtgtctgaagactgttacagtgaactgcacatataataataagtaaatttttaaaaaagaaagttttttaaataaataaatgctacagaggtgta

TIT TIT

CCG CGC CGC CCG

------intron ------intron

A S K K K K K K S A

M A S P V V P S A M

R R S S R R

C F C Y A K K A Y C F C

T V V T

P N K L H K K A A K K H L K N P

F F

P R S T T N N T T S R P

GTG GTG

AAC AAC

GCC GCC

CGC CGC

'ICC 'ICC

CAG CAG

TTT TTT

O O

* *

* *

* *

* *

* *

AAG AAG

GCC GCC

TCG TCG

TCC TCC

AAG AAG AGC AGC

TCC TCC

TGC TGC

A A

S S

ATG ATG

ACC ACC

ere ere

CCG CCG

'ICC 'ICC

AAA AAG AAG AAA ACC ACC

* *

* *

TAT TAT

* *

M L V S I F T L L T F I S V L M

s s

T V Y A A Y V T

GTG GTG GTG GTG

'ICC 'ICC

ACC ACC

CAC CAC CTG CTG

GCC GCC

S L R V S R N N R S V R L S

* *

* *

* *

* *

AAG AAG AAG AAG

GCC GCC

GTG GTG

CTC CTC

AAC AAC

TAT TAT

gt

AAG AAG

A Y H Q R L F H R D S N N S D R H F L R Q H Y A

aaattcacacacagatgcggttcctgcccattttcgagagcttagttgattgttgttttagattattttcacatccag aaattcacacacagatgcggttcctgcccattttcgagagcttagttgattgttgttttagattattttcacatccag

AGG AGG

AGC AGC

GCA GCA

TAC TAC

CTG CTG

gt

L F I I F L

* *

* *

* *

gagtgaggagcgctggccaggctcctatgcagctcttagtgaccggtgacctgacctggagcttctggagcttc gagtgaggagcgctggccaggctcctatgcagctcttagtgaccggtgacctgacctggagcttctggagcttc

ag ag

GCT GCT

ATC ATC

G'IG G'IG

CAC CAC

C'IG C'IG

TIT TIT

L P P L

GTC GTC

V L N H L H K K L K N M S K K S S K K S M N K L K K H L H N L V

ATC ATC

TAC TAC

CAG CAG CCA CCA

'ICC 'ICC

TTC TTC

* *

* *

* *

Y Y

CTT CTT

GTG GTG

ACC ACC

ACC ACC

CGC CGC

CGT CGT

CTC CTC

V V C C V V

T W V L G A F I C K K C I F A G L V W T

L L

AAT AAT

* * * *

* *

* *

AAC AAC

G'IG G'IG

CTT CTT

AAT AAT

CTG CTG

TGG TGG

N L L N

CAT CAT

GCA GCA

GCC GCC

TGC TGC

GTG GTG

TTC TTC

CTG CTG

A L L G G L L A

A A

* *

* *

* *

CTG CTG

ACT ACT

GCG GCG

CAT CAT

CTG CTG

AGC AGC

CTG CTG

2, 2,

1, 1,

A A

T F F T

s s

CAT CAT

* *

8 8

TTC TTC

ATG ATG

CGG CGG

GGC GGC

CTG CTG

ATC ATC

5 5

* *

* *

* *

M M

I I

kb------

kb------

AAA AAG AAG AAA

GAC GAC

GGC GGC

GTC GTC

GCC GCC

GCA GCA

TCT TCT

V V

A A

S V D D V S

* *

* *

* *

AGC AGC

GTG GTG

TTT TTT GTG GTG

GAC GAC

TTC TTC

V G G V

F G Y Y G F

D L L D

......

CTG CTG

mG mG

AAC AAC

GGC GGC

GAT GAT

ATC ATC

CTG CTG

* *

* *

* *

* *

AAA AAA

GCC GCC

TAC TAC

CAG CAG

TTC TTC

CGC CGC

TGC TGC

Q Q

F I I F

A A

R Y V A I V H S S H V I A V Y R

4 4

AAC AAC

52 52

ACC ACC

ATC ATC

AAG AAG

CTT CTT

TAC TAC

TAC TAC

* *

* *

* *

T F C C F T

L L P L L L L L L P L L

Y Y

J J

A'IG A'IG

048 048

GTG GTG

TTC TTC

TGG TGG

CTG CTG

TIT TIT

CTG CTG

W W

F I H Y F F F F Y H I F

L L F C I I C F L L

* * * *

* *

*

TCA TCA

......

ATA ATA

GCG GCG

CCC CCC GCC GCC

TGC TGC

CTC CTC

A L L A

AAA AAA

ATT ATT

TTA TTA

CAC CAC

TGG TGG

CTG CTG

TTC TTC

W W

* *

* *

* *

AAG AAG

GAG GAG

CTG CTG GTG GTG

TAC TAC

'ICC 'ICC

TGC TGC

E E

S S

TCT TCT

*

ATC ATC ATC ATC

CTC CTC

CAG CAG

TTC TTC TTC TTC

CAC CAC

~ ~

* *

* *

* *

Q W W Q

I A A I

GAA GAA

E E

ATC ATC

GCC GCC

TGG TGG

CCT CCT

TCG TCG

1107 1107

I I

P P

* *

* *

* *

N N 00 00 ('(') 00

1061 ~ * 864 730 ttttacagcgctgtattctcc 't'gatataactggtgtattcctgcagctcctgtcccctctccaaccctcttccattactctctcttcc ag ACT GCA 244 .'.L___A

* * * * * * * * 736 CAG ACC GTC CIG G'IG GTC G'IT GTA GTA TTT GGC ATA TCC '1GG CTG CCC CAT CAT GTC GI'C CAC CTC '1GG GCT GAG 246 0 T V L V V V V V F G I S W L P H H V V H L W A E_

* * * * * * * 812 TTT GGA GCC TTC CCA CTG ACG CCA GCT TCC TTC TTC TTC AGA ATC ACC GCC CAT TGC CIG GCA TAC AGC AAC TCC 271 F G A F P L T P A S F F F R I T A H C L A Y S N S

* * * * * * * * 887 TCA GTG AAC CCC ATC ATA TAT GCC TTT CTC TCA GAA AAC TTC CGG AAG GCG TAC AAG CAA GTG TTC AAG TGT CAT 296 S V N P I I Y A F L S E N F R K A Y K Q V F K C H

* * * * * * * 962 GTT TGC GAT GAA TCT CCA CGC AGT GAA ACT AAG GAA AAC AAG AGC CGG ATG GAC ACC CCG CCA TCC ACC AAC TGC 321 V C D E S P R S E T K E N K S R M D T P P S T N C

* 6862 ,,: * * * * * * * * 1037 ACC CAC GTG TGA AGGITIGCGGGAGCCTCCCGACTTCCAGCTCCCATGTGTGTTAGAGAGAGGAGGGCGGAGCGAATTATCAAGTAACA'IGGCAGC 346 T H V ***

* * * * * * * * * * 1133 TTATTC'It:CACAGCAATTCCTATCGATCCAACTACATTCCACAG'IGGTAAAAGGACGTTGATTGTTCAGGGAACTCGTGGGTCTACTGAAGATCATTTTC

* * * * * * * * * * 1233 CAATTTCATTTTACTCTATAATTGTATATATGAGACAAAAGAAACTTCIGTATAGTTTCTAGCTCTCAAGGAATGAAAGTCCTACAGCACTCTGCAAATG

1333 TTTTGAT FIGURE 3.3 Alignment of mouse (m), rat (r) and human (h) GALRl amino acid sequences. Identical residues are boxed and sites of non-conservative amino acid substitution are indicated with an asterisk. Putative transmembrane domains are shaded and are numbered 1-7. Symbols used: +, potential sites for N-linked glycosylation; .A., potential site for phosphorylation by cAMP / cGMP-dependent protein kinase (protein kinase A).

84 ..----,*. • * **.------, mGALR1 MELAMVNLSEGNGSDPEPPAPESRPLFGIGVEN ~ rGALR1 MELAPVNLSEGNGSDPEPPAQEPRPLFGIGVEN ~ hGALR1 MELAVGNLSEGNAS PEPPAPEPGPLFGIGVEN ~ TM1 * mGALR1 FITLVVFGLIFAMGVLGNSLVITVL ARSKPGKP ~ rGALR1 FITLVVFGLIFAMGVLGNSLVITVL ARSKPGKP ~ hGALR1 FVTLVVFGLIFALGVLGNSLVITVL ARSKPGKP ~ TM2 mGALR1 RSTTN LFILNLSIADLAYLLFCIPFQATVYA LP w rGALR1 RSTTN LFILNLSIADLAYLLFCIPFQATVYA LP oo hGALR1 RSTTN LFILNLSIADLAYLLFCIPFQATVYA LP w TM3 mGALR1 TWVLGAFICK FIHYFFTVSMLVSIFTLAAMSYD 1~ rGALR1 T WV L G A F I C KF I H Y F F T V S M L V S I F TA L AM S V D 131 hGALR1 TWVLGAFICK FIHYFFTVSMLVSIFTLAAMSVD 1~ _.... * TM4 mGALR1 R Y V A I V H S R R S S S L R VRN S 'A L L GVG F I WA L S I A 165 rGALR1 R Y V A I V H S R R S S S L R VRN S 'A L L GVG F I WA L S I A 164 hGALR1 R Y V A I V H S R R S S S L R VRN S 'A L L GVG C I WA L S I A 165

mGALR1 ,-.M;;;;;A;;..;;S~ P-'v;..,.,--,-A....,.Y..,....,H""'.""""'-::Q---:R:--,-L* --=F,...,.H..,, - R *.O S N Q T F C W E Q W p ***N K..... L_H_K~K 197 rGALR1 MA$ p V A v[y]a R L F H- RD $ N Q T F CW E[B}N p N Q L H K K 196 hGALR1 MAS p VAY HQ G L F Hp RAS N QT F CW E aw p D p RH K K 198

mGALR1 230 rGALR1 229 hGALR1 231 TM6 mGALR1 KNMSKKSEASKKK ~ AQTVLVVVVVFGISWLPHH ~ rGALR1 KNMSKKSEASKKK AQTVLVVVVVFGISWLPHH ~ hGALR1 KNMSKKSEASKKK JAQTVLVVVVVFGISWLPHH 264 * TM7 mGALR1 VVH LWAEFGAFPLTP 'ASFFFRITAHCLAYSNSS ~ rGALR1 VIH LWAEFGAFPLTP ASFFFRI T AHCLAYSNSS ~ hGALR1 I IH LWAEFGVFPLTP 'ASFLFRITAHCLAYSNSS ~

mGALR1 rGALR1 hGALR1

.------.. .------, mGALR1 T K E N K S R*M D T P P S T N CT H V 348 rGALR1 A KED K~ R I D Tp p S T N C T H V 346 hGALR1 T K E N K S R I D T P P S T N C T H 349V

85 Chapter 3

Two other mouse GALRl cDNA sequences have been reported (Wang et al., 1997c), unpublished; GenBank Accession No. E12486). One sequence is identical to the compiled exon sequences obtained here. The other sequence has two nucleotide differences: a C in place of G195, predicting a Asn residue where our sequence predicts Lys6s, and a C in place of G19s which does not alter the predicted amino acid sequence. It is not known if these differences are true sequence polymorphisms or sequencing errors in the GenBank direct database submission.

(ii) Cloning of the 5' end of the GALRl cDNA

5'-RACE and DNA sequencing were undertaken to analyse the 5' end of the GALRl cDNA. Using the 3 kb BamHI subclone containing exon 1, approximately 1 kb upstream of the translation start site was sequenced. 5'­ RACE was carried out on mouse brain total RNA to determine the extent of the 5'-untranslated region of the mouse GALRl transcript. PCR amplification of the mouse GALRl first-strand cDNA gave rise to two 5'- RACE products which were cloned and sequenced. Both 5'-RACE products were identical to the genomic sequence upstream of the translation initiation codon. The major product extended 164 bp upstream and the minor product 645 bp upstream of the translation initiation codon (Figs 3.1 and 3.2). No TATA or CAAT boxes were identified in the genomic sequence immediately upstream of either 5'-RACE product. A number of putative transcription factor recognition motifs were found in these regions, including glucocorticoid response elements and Spl binding sites. An AP-1 and an AP-2 site were identified upstream of the shorter 5'-RACE product.

In comparing cDNA sequences, it was found that an 84% identity exists between mouse and rat sequences in the 243 nucleotides immediately upstream of the translation initiation codon (Figure 3.4). This is the full extent of the rat GALRl upstream sequence as contained in the GenBank database. The corresponding region of the human GALRl 5' untranslated sequence is more divergent, sharing only 57% identity with the mouse sequence. No significant regions of strong identity with mouse sequence were apparent in a human cDNA containing 782 nucleotides of 5' untranslated region, with an overall identity of 49% (Jacoby et al., 1997).

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* * * * * MOUSE 1 GCCAGGCCAGGCACGAGGATAGTGTGATCGGGCACAGCCAGGGTCGCTCT 50 11111111111 I 11111111 I 1111 111111111 11111 11 I RAT 1 GCCAGGCCAGGGAAGAGGATAGCGAGATCAGGCACAGCCCGGGTCACTGT 50

* * * * * MOUSE 51 TCCAGGCTTTCTTGCGGGTTGCGGGAGGTACTAGTTGGAGACGCGCGCGC 100 I 111 111111 1111111111111 1111 1111111111 RAT 51 CCTCTGCTGTCTTGCCGGTTGCGGGAGGTTCTAGCTGGAGACGCG ..... 95

* * * * * MOUSE 101 TCGCTCTCGCCGCTCTGTCCTGGGCCACTCCGTGATCCTAGGCTACCTCC 150 1111111 1111 11111 111111 I 1111 11111111111111 RAT 96 .CGCTCTCTCCGCGCTGTCTAGGGCCATCCTGTGACCCTAGGCTACCTCC 144

* * * * * MOUSE 151 AGAGCCAGTTTTCCCTGGCTGGCACAACTCTCCAGGGCGCTCCGGTCCGT 200 11111 I I I I I I I I I I I 111111111111111 11 1111111111 I RAT 145 AGAGCTGGCTTTCCCTGGCTGGCACAACTCTCCAAGGAGCTCCGGTCCAT 194

* * * MOUSE 201 TGCACAGCGCCCCAAGGGGGTATCCCAGTAAGTGATG 237 I I I I I I 111111111111111 11 111111111111 RAT 195 TGCACAGCGCCCCAAGGGGGTGTCTCAGTAAGTGATG 231

FIGURE 3.4 Alignment of the nucleotide sequences immediately upstream of the translation initiation codon of the Galnrl gene in mouse and rat. Gaps in the alignment are indicated by dots. The last three nucleotides represent the translation initiation codon ATG.

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(iii) Characterization of the 3' end of the GALRl cDNA

The 3' end of the GALRl transcript was examined for possible untranslated exons and a polyadenylation signal. 3'-RACE was carried out on mouse brain RNA but no specific products were isolated. Sequencing of 1.1 kb of the 3'-untranslated region of the cloned mouse Galnrl gene revealed a number of features. The first 290 nt of 3'-untranslated region displays an 83% identity with the equivalent region of rat GALRl cDNA. A 100 nt stretch of sequence within this region also shares 73% identity with the 3'­ untranslated region of the human GALRl cDNA, beginning 143 nt downstream of the STOP codon of the GALNR1 gene. Beyond this, a 130 bp B1-like repeat sequence (Krayev et al., 1980) and 12 consecutive elements of the hexamer 5'-GGGAGA-3' were also found.

To confirm the presence of these unexpected sequence elements at the Galnrl locus, PCR was carried out on genomic DNA from the inbred mouse strains 129 and FVB/N to amplify this region of the 3'-UTR. A PCR product was amplified from our genomic clone, as expected, but not from any sample of genomic DNA tested. At this time, an alternate genomic sequence of Galnrl was published (Wang et al., 1997c). This sequence showed divergence from that described here, in the 3' untranslated region 292 bp downstream of the STOP codon. The published sequence included a putative polyadenylation signal 359 bp downstream of the STOP codon and was 82% identical to the rat GALRl cDNA up to this point. This difference may have been sub-strain specific or the result of a rearrangement in our genomic clone. PCR carried out using primers specific to the published sequence yielded a product from the 129 genomic DNA but not from our genomic clone. The absence, from mouse genomic DNA and from another reported clone, of the sequence 292 bp downstream of the coding region in our genomic clone, suggests that this portion of the sequence of our clone was artefactual.

(iv) Chromosomal localization of the Galnrl gene

In situ hybridization on mitotic mouse chromosomes was carried out to determine the chromosomal localization of Galnrl (Figure 3.5(a)). The 440 bp exon 2-containing cDNA was labelled with tritium and used as the probe. A total of 260 silver grains was scored to an idiogram (Nesbitt and Francke,

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1973). Of all mouse chromosomes in approximately 200 cells, 88 of the grains (34%) were scored over Chromosome 18, with a tallest column, of 47 grains, lying over the distal sub-band, 18E4 (data not shown). Small peaks of grains, to a maximum column height of 3 grains, occurred on Chromosomes 1, 16, 19 and the Y, but none of these were regarded as indicative of the occurrence of a second gene or pseudogene.

Having established Chromosome 18 to be the major target for the cDN A probe, the result was confirmed by scoring a total of 108 grains over Chromosome 18 from approximately 90 high quality cells, using a more detailed idiogram of Chromosome 18 (Evans, 1989); 69 (64%) of the grains formed the three tallest columns, which were over, or adjacent to, the sub­ band 18E4 (Figure 3.S(b)). For all scoring, the two mouse strains C57BL and BALB/c showed similar patterns of grain distribution. The results of in situ hybridization showed that Galnrl was almost certainly located at sub-bands 18E3-4 on mouse Chromosome 18, with a strong probability of extremely distal point localization to sub-band 18E4.

3.4 DISCUSSION

The high degree of sequence identity of the coding region of the mouse gene described in this chapter to both the human and rat GALRl sequences confirms the isolated clone as the mouse homologue of the gene encoding the GALRl galanin receptor (GALNR1). The gene is thus designated Galnrl. Characterization of Ga 1n r 1 genomic clones revealed a structural organization conserved between human and mouse, and unique among genes described to date encoding G protein-coupled receptors.

The coding sequences of Galnrl are divided into three exons, with exon 1 encoding the N-terminal end of the receptor and the first five transmembrane domains. Exon 2 encodes the third intracellular loop and exon 3 encodes the remainder of GALRl, from transmembrane domain 6 to the C-terminus of the receptor protein. Coding of the third intracellular loop of GALRl on a separate exon is noteworthy. It has been demonstrated for a large number of G protein-coupled receptors that this domain is involved in the interaction of the receptor with G proteins. There is some

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a b 40

30

~ 20 //'

10

' 5 " 18

' -E3-4

FIGURE 3.5 Silver grains over mouse Chromosome 18 hybridised in situ with tritiated cDNA derived from the Galnrl gene. (a) Six examples of Chromosome 18 showing individual grains (indicated by arrows). The top two chromosomes are from the same cell; the bottom one is from a BALB / c and the rest are from C57BL mice. (b) Plot of grains over approximately 100 Chromosomes 18 showing highly probable localization to sub-bands 18E3-4 (marked with a bar). The arrowhead at the base of the tallest column indicates the probable point localization of Galnrl to sub­ band 18E4.

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evidence that this is also the case for GALRl (Saar et al., 1997). In some G protein-coupled receptor genes, this domain of the receptor is the site of alternative splicing of the transcript to generate receptor subtype diversity, and in the case of dopamine D2 and pituitary adenylyl cyclase activating peptide receptors, splice variants differing in the third intracellular loop exhibit differential efficiency of coupling to intracellular signalling pathways (Hayes et al., 1992; Spengler et al., 1993). Encoding of the third intracellular loop of GALRl on a discrete exon raises the possibility of alternative splicing of exon 2 to generate functionally distinct GALRl transcripts. However, no evidence for this has yet emerged from the isolation of several human and rat GALRl cDNA clones and the partial mouse cDNA described here. In addition, analysis of several mouse tissues was carried out by RT-PCR amplification of cDNAs spanning exon 2 and no evidence of novel coding sequence was obtained (data not shown).

Alignment of the coding sequences reveals a close identity between the deduced amino acid sequences of mouse and human GALRl. However, the 5' and 3' untranslated regions of the cDNA display significant divergence. This sequence divergence may reflect species-specific differences in GALRl gene expression. Although some differences in the GALRl expression pattern between rodent and human have been reported, conflicting data exist and a detailed systematic comparison is yet to be published. Differences in regulation and sites of expression between species may imply functional differences. Species-specific differences in galanin activity have been described and it is also possible that some functional differences exist between human and mouse GALRl. This issue will be addressed by comparative studies when specific GALRl antagonists are developed but it is relevant when considering the implications for man of transgenic studies of GALRl function performed in the mouse.

No consensus TATA or CCAAT boxes were identified closely preceding the 5'-untranslated region of the mouse Galnr1 gene. Absence of these motifs from the promoter region has been reported for many other GPCR-encoding genes, including the genes encoding the rat bradykinin B2, human neuropeptide Y Yl, dopamine D2 and adenosine Al receptor subtypes (Ball et al., 1995; Pesquero et al., 1994; Ren and Stiles, 1994; Valdenaire et al., 1994). Several transcription factor binding sites were identified on the basis of consensus sequence identity. As little is known about the regulation of

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Galnrl expression, promoter studies are required to determine if any of these sites are active in viva. The existence of alternatively spliced exons in the 5'-UTR region has been observed in transcripts encoding a number of G protein-coupled receptors, including the porcine muscarinic acetylcholine, rat endothelin ETA and human neuropeptide Y Yl receptors (Ball et al., 1995; Cheng et al., 1993; Peralta et al., 1987). The existence of alternatively spliced exons in the 5'-untranslated region of the gene encoding GALRl has not been fully investigated to date. However, the 5'-RACE products identified in mouse brain GALRl transcripts were identical, in length and sequence, to the Galnrl genomic sequence up to 646 nucleotides upstream of the translation initiation codon. Hence, no untranslated exons were found in the GALRl transcripts detected.

The two other galanin receptors identified to date, GALR2 and GALR3, are encoded by genes which differ markedly from Galnrl in both sequence and structure. Mouse GALRl shares only 40% amino acid identity with mouse GALR2 (Pang et al., 1998) and only 36% with mouse GALR3 (unpublished; GenBank Accession No. AF042783) (Figure 3.6). In contrast, GALR2 and GALR3 share 55% amino acid identity. The genes encoding GALR2 and GALR3 also share a common genomic structure distinct from Galnrl (see Figure 1.5). These genes each comprise two coding exons. Exon 1 encodes the N-terminal extracellular domain and the first three transmembrane domains, and exon 2 encodes the remainder of the receptor (lismaa et al., 1998; Kolakowski et al., 1998a). This common structure and sequence similarity indicates that GALR2 and GALR3 are more closely related to one another than either is to GALRl. These findings also suggest that GALRl evolved independently of GALR2 and GALR3, and that the latter two may have evolved by duplication of an ancestral gene.

The physical localization of Galnrl to mouse Chromosome 18E4 (Figure 3.5) is similar to that of the gene encoding nuclear factor of activated T-cells, cytoplasmic 1 (Nfatcl), but Nfatcl has not been mapped genetically (Johnson and Davisson, 1996). The nearest gene to Galnrl which has been mapped both genetically and physically is the gene encoding myelin basic protein (Mbp), which gives rise to the shiverer phenotype when mutated. Mbp has been mapped to 55 cM on Chromosome 18 and localized to 18E2-3 (Johnson and Davisson, 1996). By comparison with Mbp, Galnrl should map by linkage

92 GALR1 ME L A M V N L S E G N G S D P E P P A P E S R P L F G I G V E N 33 GALR2 ------MN GSD S QG AEDSSQEGGGGWQPE AV ~ GALR3 ------M ADIQN I S LDSP GSVG AV 18

GALR1 34 FIT LV VFGLIFAMG VLGNSLVITV L ARSKPG-- 64 GALR2 26 L VPL F FALIFL VGAVGN ALVLAVLL RG G ----- 53 GALR3 19 AV P V VF AL I F L LG MVG NG L V L AV L L Q P G PS AWQ 51

GALR1 65 K P R S T T N L F I L N L S I A D L A Y L L F C I P F Q A T V Y A 97 GALR2 54 QAV STTNLF I LNL G VADLCF I LCCVPFQAT I YT 86 GALR3 52 E PGSTT D LF I LNL AVADLCF I LCCVPFQA A I YT 84

GALR1 98 L PTWV LGAF I CK FI HYFFTVS MLV S I FTLAA MS 130 GALR2 87 LD DWVFG SLL CK AVH F LI F LTM HASSFTLAAVS 119 GALR3 85 LD AWL FGAF VCK T VH L LI YLTM YASSFTLAAVS 117

GALR1 131 VDRY VAI VHSR RS SS LR VS RNAL LG VG F IW ALS 163 GALR2 120 LDRYLAIR YP MH SR E LRTPRNALAA I GLIWG LA 152 GALR3 118 VDRYLA VRHP L RSR ALRTPRNA RAAVGL VWL LA 150

GALR1 164 IAMAS P VAYHQRLFHRDS NQT F CWEQWPNKLHK 196 GALR2 153 L LFS G PYLSYYSQSQL - ANLT VC H PAWSAP- RR 183 GALR3 151 ALFS APYLSYY GTVRY - GA L EL C VPAW EDA- RR 181

GALR1 197 K A Y V VC T F V F G Y L L P L L L I C F C Y A K V L N H L H K K 229 GALR2 184 RA MD LCTFVF S YLLPVLV LSL T YARTL HY LW RT 216 GALR3 182 RA LDV ATF AA GYLLPV T VVSL AYG RTL CF LW AA 214

GALR1 230 L K N MS K KS E A S K --- - K K T A Q T V L V V V V V F G I S 258 GALR2 217 V D P V A AG S G S Q R -- A K R K V T ~ M I V I V A V L F C L C 247 GALR3 215 VG P AG AAAAEA RRR ATGRAG AM L AV AL YA LC ~

GALR1 259 W L P H H V V H L WA E F G A F P L T P AS F F F R I T A H C L A 291 GALR2 248 WMP H H A L I L C VW F G R F P L T R A T Y A L R I L S H L VS 200 GALR3 248 WG PHHALILC FWYGRF AFS PATYA C RLA SHCLA ~

GALR1 292 Y S N S S VN P I I Y A F L S E N F R K A Y KQ V F KC - - - - - 319 GALR2 281 YAN SC VN P I VY AL VS K H FR KG F R -- K I C A G L L R 311 GALR3 281 Y AN S C L N P L V Y S L AS RH F R A R F R R L W PC G H R RH 313

GALR1 320 - H VC D E S P R S E T K E N - - - KS R MD T P P S T N C T H V 348 GALR2 312 RAPRRA S G RVCILAPGNH SG GMLE P EST DL TQ V ~ GALR3 314 RH HHHRLH RALRRVQPAS SG PAGY P GDARPRGW~

GALR1 348 348 GALR2 345 S EAAGPLVPAPALPNCT TLS - RTLDPAC 371 GALR3 347 S MEPRGDALRGGETRL - TLS ARGPQ 370

FIGURE 3.6 Alignment of the deduced amino acid sequences of mouse GALRl, GALR2 and GALR3. Identical residues are shaded yellow. Gaps have been introduced to optimise the alignment.

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between 55 cM and the present limit for Chromosome 18, of 60 cM. This localization has recently been confirmed by further genetic mapping, which also places Galnrl at 55 cM, with complete linkage to Mbp (Simoneaux et al., 1997). The shiverer mutant is the only spontaneous mouse mutant mapped to date to this chromosomal location. Galnrl is co-localised with a quantitative trait locus (QTL) for susceptibility to kainic acid-induced seizures, mapped to an interval bordered by microsatellite markers at 52 cM and 57 cM (Ferraro et al., 1997). This is relevant when considering the finding, to be described in Chapter 4, that the targeted disruption of Galnrl leads to spontaneous seizures in mice.

The human GALNRl gene has been physically and genetically localized to a similar position on human chromosome 18, in the vicinity of MBP and the gene encoding peptidase A (PEPA) (Crawford et al., 1999; Nicholl et al., 1995). Peptidase A is called peptidase-1 (Pepl) in the mouse and has also been localized to Chromosome 18 of the mouse genome (Lalley and McKusick, 1985). The chromosomal localization of GALRl is therefore homoeologous in human and mouse.

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DEVELOPMENT OF A GALRl "KNOCKOUT" MOUSE STRAIN Chapter 4

4.1 INTRODUCTION

Galanin elicits a diverse array of actions in the nervous system and many other tissues, such as the pituitary gland, pancreas and gut. A response is initiated upon binding of the peptide to GPCRs in the plasma membrane of target cells. The wide range of galanin's activities may be explained, at least partly, by multiple receptor subtypes, each responsible for a discrete or overlapping subset of galanin's functions. Three galanin receptor subtypes have been identified by molecular cloning (Habert-Ortoli et al., 1994; Howard et al., 1997a; Wang et al., 1997b) and the existence of further subtypes has been postulated on the basis of pharmacological evidence (Iismaa and Shine, 1999). The diversity of responses elicited by galanin may be further accounted for by the involvement of multiple signalling pathways downstream of receptor activation (Kolakowski et al., 1998b; Smith et al., 1998; Wang et al., 1998b).

As the galanin receptors are potential drug targets for the treatment of a range of disorders, it is essential to determine the contribution of each receptor subtype within the spectrum of galanin actions. Traditionally, elucidation of receptor function has relied on the use of specific pharmacological probes. However, in the absence of galanin receptor subtype-specific agonists and antagonists, targeted mutagenesis can provide an alternative approach to resolving this question. Ideally, a complete assessment of the role of a particular subtype should take into account data from pharmacological and transgenic (both overexpression and deletion) experiments. A transgenic deletion approach may initially provide novel insights from which further studies of receptor subtype function can be developed. This was the approach taken with the work presented here.

Transgenic deletion or, more precisely, targeted mutagenesis, is a strategy for the selective inactivation of a single gene. The functional consequences of this inactivation are studied in vivo to help reveal the physiological role of the gene product. This strategy is widely acknowledged as a valuable approach to elucidation of gene function in many areas of mammalian biology (Brandon et al., 1995). Thus far, targeted mutagenesis of the Gain gene in the mouse has revealed the crucial contribution of galanin to several processes, including regulation of PRL secretion and mammary

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gland development (Wynick et al., 1998), neuropathic pain and functional recovery following nerve injury (Wynick, 1997), central cholinergic modulation (Ma et al., 1999) and control of seizure activity (Mazarati et al., 1999). However, while it has been invaluable in revealing galanin's essential functions, genetic deletion of the ligand does not allow us to assign functions to its various receptor subtypes.

An analogous example is neuropeptide Y (NPY), for which deletion of the Yl, Y2 and Y5 receptors has revealed the role these receptors play in vasoconstriction and feeding (Kushi et al., 1998; Marsh et al., 1998; Naveilhan et al., 1999; Pedrazzini et al., 1998), roles not readily apparent from the phenotype of the NPY null mouse strain (Erickson et al., 1996). Other examples are emerging of this approach being applied to individual GPCR subtypes in neuronal and neuroendocrine signalling. These include the dopamine receptors (Accili et al., 1996; Baik et al., 1995; Drago et al., 1994; Kelly et al., 1997; Rubinstein et al., 1997; Xu et al., 1994), the opioid receptors (Matthes et al., 1996; Nishi et al., 1997; Simonin et al., 1998; Sora et al., 1997), the serotonin receptors (Parks et al., 1998; Saudou et al., 1994; Tecott et al., 1995) and the melanocortin receptors (Chen et al., 1997; Huszar et al., 1997). Targeted mutagenesis of single galanin receptor subtypes may likewise help to define the roles played by these proteins in viva. It may be expected that disruption of the GALRl galanin receptor will reproduce some of the phenotypic features of the galanin knockout mouse. This evidence will then link those consequences of galanin deficiency to GALRl.

The development of a strain of targeted mutant mouse involves a number of distinct steps (Galli-Taliadoros et al., 1995). First the gene of interest must be isolated and characterised such that a gene targeting vector may be designed and constructed. A conventional targeting vector consists of two arms of homology with the gene, flanking a selectable marker positioned so as to disrupt or replace a critical coding region. This construct is introduced by electroporation into totipotent mouse embryonic stem (ES) cells. The cells are then subject to selection and only those cells which have integrated the vector will survive this period in culture. In a small fraction of these cells, the construct will have undergone homologous recombination with an endogenous allele of the target gene. These targeted cells are identified by DNA analysis.

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The targeted cells are microinjected into 3.5 day blastocysts isolated from the uterus of female mice, where they aggregate with the inner cell mass. The blastocysts are then implanted into pseudopregnant foster females whereupon they can continue a normal developmental regime. The injected ES cells are totipotent and so are able to colonise all tissues of the developing embryo. Hence, the mice which develop from these chimaeric blastocysts are a mix of two strains, those being the strains from which the ES cells and the blastocysts were derived. In general, the two strains differ in coat colour and therefore the contribution of the ES cells to the chimaeric mice can be determined on this basis. For example, in this project the ES cells were derived from an agouti mouse, while the blastocyst donor was a black mouse. The desired outcome is the contribution of the ES cells to the germline of the chimaera, so that the targeted mutation can be transmitted to subsequent generations. If this has occurred, the chimaeras can be crossed to wild-type mice to produce offspring heterozygous for the mutant allele. Interbreeding these heterozygotes will yield animals homozygous for the targeted mutation. This chapter describes the application of this approach to the development of a strain of mouse lacking functional GALRl.

4.2 METHODS

(i) Construction of targeting vector

The PGK-neor selection cassette, inserted in the general purpose cloning vector pGEM®-7z, was a gift of Prof. Ashley Dunn, Ludwig Institute of Cancer Research, Melbourne, Australia. This cassette consists of the phosphoglycerate kinase promoter and poly-A addition signal flanking a bacterial neomycin phosphotransferase coding sequence. The selection cassette confers resistance to neomycin and analogues such as G418 and it extends 1.8 kb in total. The PGK-neor cassette was excised from the vector by digestion with Sacl. It was ligated to the Sad-digested vector pGEM®-3z to generate pPGK-neor.

A short arm of homology for the targeting vector ptar-1 was isolated from the 3 kb BamHI subclone described in the previous chapter (3.2(ii)) as containing exon 1 of the Galnrl gene. Digestion of this subclone with both Spel and KpnI liberated a 780 bp fragment containing the majority of exon 1

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plus some 5' flanking sequence. This fragment was used as the short arm of the targeting construct. It was incubated with Kienow enzyme to fill in the overhanging ends. It was then ligated to EcoRI linkers and the ligated product was digested with EcoRI. This fragment was then ligated to the pPGK-neor vector which had also been digested with EcoRI. This last step generated pmGalRl-T.sh, containing the short arm of the targeting construct inserted adjacent to the 3' end of the PGK-neor selection cassette.

The long arm of homology was assembled from two contiguous fragments of DNA. A 1.7 kb Kpnl-BamHI fragment, containing the 3' end of exon 1 and the 5' end of intron 1 of the Galnrl gene, was isolated from the 3 kb BamHI subclone containing the first exon of the Galnrl gene. This fragment was directionally cloned into pBluescriptII® to generate pBs-KBl.7. A 2 kb BamHI-Spel fragment, identified by restriction mapping as containing part of intron 1, was isolated from the A genomic clone described previously (see Figure 3.1). The 2 kb fragment was directionally cloned into pBs-KBl.7, which had been digested with BamHI and Spel. This step completed the assembly of the long arm clone, designated pBs-KS3.7. The complete insert of 3.7 kb was excised from the vector by digestion with Kpnl and Spel. The partial construct pmGalRl-T.sh was digested with Kpnl and Xbal to expose compatible termini for the directional cloning of the long arm fragment. The long arm fragment was ligated to the partial construct to complete the construction of the targeting vector ptar-1 (Figure 4.1).

(ii) Primary embryonic fibroblast (PEF) preparation and inactivation

PEFs were isolated from 13-14 day near mouse embryos, after dissecting the uterus and rinsing in PBS to remove blood and mucous. The head and liver of the embryos were discarded and the remaining tissue was minced with scalpels in 2-3 ml PBS. The minced tissue was placed in trypsin (0.05% (w /v) containing 0.53 mM EDTA; 5 ml per embryo) overnight at 4°C to allow the protease to diffuse into the tissue. The following day, most of the trypsin solution was aspirated and the minced tissue was placed at 37°C for 30 min. The medium used for culturing embryonic fibroblasts was DMEM containing 10% (v /v) FCS and 100 µM ~-mercaptoethanol. A 10 ml volume of this medium was added to the embryo suspension which was then triturated through a 10 ml pipette to dissociate remaining fragments of tissue. The suspended cells were transferred to 10 cm dishes and allowed to

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adhere to the plastic before the medium was changed. Fibroblasts were routinely cultured in either 10 cm2 dishes or 75 cm2 flasks, under standard conditions of 37°C and 5% CO2.

The PEFs were mitotically inactivated by y-irradiation or treatment with mitomycin C. Prior to irradiation, confluent flasks of cells were treated with 2-5 ml trypsin (5 min at 37°C) to resuspend the cells which were then collected into culture medium in 50 ml tubes. The tubes were placed upright in a plastic rack and subjected to 3000 Rads of y-irradia tion, delivered by a Siemens Mevatron linear accelerator. Following irradiation, cells were frozen in liquid N2 or plated as needed. Mitomycin C treatment was carried out on confluent monolayers of cells in 75 cm2 culture flasks. Medium was removed and fresh medium containing 10 µg/ml mitomycin C was added to the cells, which were then incubated at 37°C for 3-4 h. Following treatment, the cells were washed several times in PBS, treated with trypsin and frozen in liquid N2, unless required immediately as feeder cells.

Monolayers of mitotically inactivated PEFs on 10 cm2 dishes were used as feeder layers for the culture of ES cells. Dishes were coated with 0.1 % (w /v) gelatin prior to addition of cells.

(iii) ES cell culture

The W9.5 ES cell line and associated methods were provided by Prof. Ashley Dunn. This cell line was derived from a strain of 129/Sv.C3-+c+r mice (Szabo and Mann, 1994). Cells were grown on monolayers of mitotically inactivated PEFs in DMEM containing 15% (v /v) FCS, 100 µM ~­ mercaptoethanol, 1000 U /ml LIF, 100 µM non-essential amino acids and nucleosides (2.0 mg/ml adenosine, 2.1 mg/ml guanosine, 1.8 mg/ml cytidine, 1.8 mg/ml uridine, 0.6 mg/ml thymidine). The ES cells were plated at a density of 1 x 106 cells per 10 cm2 dish and passaged every few days, such that individual colonies were not allowed to grow too large. Cells were maintained under standard conditions of 37°C and 5% CO2. When cells were ready for passaging, the medium was removed and the cells were rinsed with PBS. The PBS was also removed and the cells were overlayed with a small volume of trypsin (0.05% w /v, containing 0.53 mM EDTA} and incubated for 5-10 min at 37°C. Once the cells had begun lifting from the

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culture dish, the trypsin was inactivated by the addition of medium. Complete dissociation of ES cell colonies was achieved by vigorous trituration with a pasteur pipette until single cells or small aggregates predominated. Cells were counted with a haemocytometer and then plated onto inactivated PEFs.

(iv) Electroporation of ES cells

W9.5 ES cells were transfected by electroporation, one day after passaging. A single-cell suspension was prepared as described above. Approximately 70 x 106 cells were divided into four electroporation cuvettes in 0.8 ml PBS/cuvette. Linearised ptar-1 was added to the cuvette and the cells underwent electroporation in a Bio-Rad Gene-Pulser®II (Bio-Rad Laboratories, Hercules, CA). Each cuvette was subject to different electroporation parameters in order to increase the probability of optimising the conditions. The settings used are shown in Table 4.1. Following electroporation, the cells were allowed to recover by incubation on ice for five minutes. Cells from each cuvette were then overlayed onto feeder cells in a 10 cm2 culture dish. The following day, selection was initiated with the addition of 175 µg/ml G418 to the culture medium. Selection was carried out for 10 days, by which time the majority of cells had died and been removed and the resistant cells had formed colonies which were visible by eye.

Cuvette DNA added (µg) Voltage (V) Capacitance (µF) 1 15 270 500 2 60 270 500 3 15 200 800 4 15 400 25

TABLE 4.1 Electroporation conditions for transformation of ES cells

(v) Colony picking and culture

Following selection in G418, surviving ES cell colonies were picked for screening to identify targeted clones. Medium was removed from the cells, which were washed with DMEM, then overlayed with 2 ml fresh DMEM.

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Individual ES cell colonies were sucked into a yellow pipette tip using a Gilson P20 pipettor set at 5 µI. The colonies were ejected into 50 µl of trypsin (0.05% w /v, containing 0.53 mM EDTA) in individual wells of a 96-well plate. After picking 48 colonies, the plate was incubated for 5 min at 37°C. The trypsin was then inactivated by the addition of 100 µl of medium to each well. The colonies were dispersed by up to 20 passages through a yellow pipette tip and the cells were then transferred to 96-well plates containing feeder layers of PEFs. When confluent, these cells were passaged and 50% of the contents of each well was frozen. The remaining 50% was allowed to continue growing and was then used for DNA extraction.

(vi) Freezing ES cells

ES cells were frozen in ES medium containing 10% (v /v) DMSO. Cells were placed in cryogenic vials at a density of 5 x 106 cells/ml. The vials were first frozen overnight at -80°C in styrofoam racks and then transferred to liquid nitrogen. Cells in 96-well plates were overlayed with 100 µl of freezing medium, insulated in styrofoam and frozen and stored at -80°C.

(vii) Thawing ES cells

Cryogenic vials were thawed at 37°C for 5 min and the contents were diluted in warm ES medium and transferred to 6 cm2 or 10 cm2 dishes pre-prepared with PEF feeder layers. The medium was changed after several hours to remove the DMSO. Frozen 96-well plates of ES cells were thawed at 37°C for approximately 10 min. Cells from individual wells were transferred to single wells of 24-well plates pre-prepared with PEFs. The cells were then expanded as required.

(viii) PCR screening of ES cell clones

A PCR amplification protocol was developed to detect the targeted Galnrl allele in ES cells. The forward primer was a 22-mer (5' - CGCTCTTCCAGGCTTTCTTGCG-3'; #5869) designed to anneal within the 5' region of the Galnrl gene, approximately 10-30 bp upstream of the short arm of ptar-1. The reverse primer was also a 22-mer (5' - CCGCTTCCTCGTGCTTTACGGT-3'; #5727) annealing at the 3' end of the PGK-neo selection cassette (Mann et al., 1993). A PCR reaction using these

102 Chapter 4

primers was predicted to amplify a fragment of 1.3 kb from a correctly targeted allele (Figure 4.1).

DNA was isolated from ES cell clones grown in 96-well plates (see Section 2.3(iv)) and used as template for PCR amplification. Initially, the clones were screened by PCR amplification in pools of three. DNA solution (20 µl) from each of three single clones was combined and diluted with water to a total volume of 210 µI. A 20 µl portion of this pooled diluted DNA solution was then used as the template in a 50 µl PCR reaction. PCR amplification was repeated on individual clones from those pools which yielded a PCR product. In this instance, 5 µl of DNA from each clone was used in the PCR reaction. The PCR was prepared as described (Section 2.ll(i)). Following thermal activation of the DNA polymerase, the reaction underwent 40 cycles of the following program: 94°C for 60 sec, 61 °C for 60 sec, 72°C for 150 sec. A final elongation step of 72°C for 10 min completed the PCR. The samples were analysed by electrophoresis of 10 µl of each reaction on a 0.8% (w /v) agarose gel.

(ix) Southern hybridisation screening of ES cell clones and chimaeras

Southern hybridisation was used to confirm the presence of correctly targeted alleles in individual ES cell clones. Clones which were positive by PCR analysis were thawed into individual wells of a 24-well plate, prepared with PEF monolayers. The clones were sequentially expanded for DNA analysis and preparation of frozen cell stocks. ES cells for DNA extraction were harvested by trypsin treatment from one confluent 10 cm dish. The cells were washed once in 10 ml PBS and genomic DNA was purified as described (Section 2.3(v)). DNA concentration was assessed by spectrophotometry and 20-40 µg was used in digests with the restriction enzymes Kpnl and Sacl. The digested DNA was fractionated by agarose gel electrophoresis on 0.5% gels and then transferred to nylon membranes by alkali blotting. DNA from each ES cell clone was hybridized with three different probes: a 5' external probe, a 3' external probe and the neor coding region (Figure 4.1)

103 Sc K 1 kb 8 Targeting vector -----+• PGK-neol I 1------rl s B \ ! s Sc K K Sc Galnr1 allele I -t-\\ II ,. I I I B S B s 5' PROBE- 3' PROBE- 5869 5727 > Sc K sc • K Sc n Targeted allele PGK-neo -t-\\ II 1.- j I I I I I B S B s neoPROBE-

1.5 kb I 5' PROBE wild-type allele I Kpnl digest 3.3 kb targeted allele I I

- 20 kb 3' PROBE wild-type allele t-\\ 11 kb Sacl digest targeted allele

FIGURE 4.1 Approach used to disrupt the Galnrl gene. The design of the targeting vector ptar-1 is shown, above the structure of the wild-type and predicted targeted Galnrl allele. Dashed lines represent plasmid DNA. Coloured boxes symbolise exons of the Galnrl gene, as per Figure 3.1. The positions of restriction endonuclease recognition sites are represented by vertical bars (B, BamHI; K, Kpnl; S, Spel; Sc, Sac!). The positions of PCR primers used to screen ES cell clones are illustrated by light blue arrowheads. The light blue bars indicate the location of DNA probes used in Southern blot screening of clones. The predicted sizes of restriction fragments hybridizing to these probes are illustrated below the allele structures. Chapter 4

The 5' probe used was a 362 bp PCR-generated fragment of the Galnrl upstream region, 5' of the short arm of ptar-1. The forward primer used to amplify this fragment corresponded to nucleotides -526 to -508 relative to the translation start site (5'-TGAAGGTGTGCATCGCTGG-3'). The reverse primer corresponded to nucleotides -165 to -183 relative to the translation start site (5' -AACCCGCAAGAAAGCCTGG-3'). The 3' probe used was the 194 bp Hincll subclone containing exon 2 of the Galnrl gene (see Section 3.3(i)). The neor probe used was the 0.8 kb coding region of the neomycin phosphotransferase gene isolated by Pstl digestion of the pPGK-neo plasmid.

(x) Blastocyst injection

Blastocyst injection of ES cells and subsequent breeding and screening of chimaeras were carried out in the Genetically Modified Mouse Laboratory at the Walter and Eliza Hall Institute, Melbourne, Victoria.

ES cells were injected over four sessions into blastocysts from C57BL/6 females at 3.5 days postcoitum. The injected blastocysts were transferred to the uteri of pseudopregnant C57BL/ 6 recipients and carried to term. Pups were assessed for chimaerism on the basis of coat colour.

(xi) Breeding and screening of chimaeras

The agouti progeny of matings of coat-colour chimaeras to C57BL/6 females were screened for the targeted allele by Southern hybridisation analysis. DNA was isolated from tail biopsies collected at weaning. The DNA was digested with BamHI and analysed by Southern hybridisation using the 5' probe described above. Mice that were shown to possess a targeted Galnrl allele were used as founders for the GALRl knockout mouse colony. The genetic background of these mice was a combination of C57BL/6 and 129/Sv.

Chimaeric males that had been shown to transmit the targeted allele through their germline were also bred with females of the 129T2/SvEmsJ strain, obtained from the Animal Resources Centre (Perth, Australia). This was done to initiate the transfer of the targeted allele onto an inbred genetic background. All progeny of these matings were genotyped as above and

105 Chapter 4

those offspring inheriting the targeted allele were used to start the 129 GALRl knockout mouse colony.

(xii) PCR genotyping

The genotype of the offspring of GALRl + / - x + /- crosses was determined by PCR amplification of genomic DNA isolated from tail biopsies. Three PCR primers were used in combination to distinguish wild-type and targeted alleles, a single forward primer and two reverse primers. The forward primer was a 20-mer corresponding to nucleotides 248-267 of Galnrl exon 1 (5'-CCTACCTGCTCTTCTGCATC-3'; #4464). This primer annealed upstream of the site of incorporation of the neo' cassette. The reverse primer, predicted to generate a PCR product from a wild-type allele, was designed to anneal just downstream of the 3' end of Galnrl exon 1, at position 704-685 (5'-CTAAGAGCTGCATAGGAGCC-3'; #20971). The alternate reverse primer, used to identify a targeted allele, was the primer #5727, also used in screening ES cell transfectants (see Section 4.2(viii)).

Of the 100 µl tail DNA preparation, 1 µl was used as template in a 50 µl PCR reaction. The PCR conditions used were an initial DNA denaturation/polymerase activation at 94°C for 9 min, followed by 35 cycles of 94°C for 50 sec, 63°C for 50 sec and 72°C for 1 min 30 sec, then a final extension of 72°C for 10 min. The sizes of the predicted PCR products were 457 bp for a wild-type allele and approximately 1 kb for a targeted allele. Under the conditions described, no product would be generated from a targeted allele with primers #4464 and #20971 as such a product would exceed 2 kb in length. PCR products were separated by electrophoresis on 1.5% agarose gels.

(xiii) Analysis of gene expression by RT-PCR

The expression of genes encoding components of the galaninergic system in the GALRl knockout mouse was examined, and compared to wild-type mouse. This was carried out by RT-PCR on total RNA or poly-A+ RNA isolated from mouse tissues. The genes examined were those coding for galanin, GALRl, GALR2 and GALR3. The detection of 13-actin mRNA was used as a control for efficiency of cDNA synthesis. The tissues analysed were brain, large intestine, spinal cord and uterus. Total RNA was prepared

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from each tissue and poly-A+ RNA was purified from large intestine. cDNA was synthesised as described (Section 2.ll(ii)) using 1 µg of total RNA or approximately 500 ng of poly-A+ RNA as template. Two negative controls were used to detect contamination during RNA extraction or cDNA synthesis. The first control was the RNA extraction procedure without starting material. The second control was a cDNA synthesis reaction without template RNA.

The details of oligonucleotides used for the detection of gene expression are shown in Table 4.2. For each gene, the PCR amplification primers are listed first as forward (-f) and reverse (-r) primers, followed by an internal (-i) oligonucleotide if the PCR product was detected by Southern hybridization. Also included in Table 4.2 is the region of DNA sequence corresponding to each oligonucleotide, and the size of the PCR products generated by each primer set. Multiple primer pairs were used to amplify GALRl and ~-actin mRNA in a single PCR reaction, and GALR2 and GALR3 mRNA in a single reaction. PCR reactions were carried out in a volume of 50 µl, and the following cycling conditions were used: DNA denaturation/polymerase activation step of 94°C for 9 min, followed by 35 cycles of 94°C for 45 sec, 55°C for 60 sec, 72°C for 60 sec; a final 10 min elongation step at 72°C completed the PCR. A third negative control was employed at the amplification step, viz. a PCR reaction without cDNA template.

The PCR products were separated by electrophoresis on 1.5% or 2.5% agarose gels, depending on size. For the detection of GALRl, GALR2 and GALR3 mRNAs, the PCR products were transferred to nylon membranes by alkali blotting (Section 2.4(i)) and then hybridized with a 32P-labelled internal oligonucleotide. The other PCR products, including the GALRl 5' end amplified with primers 4464 and 4463, were visible after staining the gel with ethidium bromide.

(xiv) Other procedures

GALRl knockout mice with a 129 strain background were housed under standard conditions and provided with laboratory chow and water ad libitum. The mice were weighed fortnightly from four weeks of age. Mean body weights ± standard error of the mean (S.E.M) were plotted with n=6 for each data point.

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Dark cycle filming of mice under infrared light was carried out using a Sony video camera, model CCD-TRV87E, connected to a Sony SL V-EZ44 video cassette recorder. The camera was set up at the level of the mouse cage to film through the perspex side of the cage.

All procedures involving mice were conducted in accordance with the National Health and Medical Research Council's Code of Practice for the Care and Use of Animals for Scientific Purposes, and with the approval of the Animal Experimentation Ethics Committee of the Garvan Institute of Medical Research.

OLIGO. NAME SEQUENCE (5'-+3') REGION SIZE

341 mGAL-f TGC AGT AAG CGA CCA TCC AG 5'-utr, -108 to -88 512bp 320 mGAL-r AGC ACA GGA CAC ACG TGC AC 3'-utr, 404-385

452 mGALRl-f CGC CTT CAT CTG CAA GTT TA exon 1, 312-331 440bp 864 mGALRl-r CAG GAC GGT CTG TGC AGT exon 3, 747-731 1048 mGALRl-i AAT GGC CAC GTA GCG ATC CA exon 1, 408-389

4464 GALR1-f2 CCT ACC TGC TCT TCT GCA TC exon 1, 248-262 200bp 4463 GALR1-r2 GGA CAC CCT GAG GGA GGA GGA GC exon 1, 447-425

15338 mGALR2-f TGC CTT TCC AGG CCA CCA TC exon 1, 233-252 580bp 15337 mGALR2-r GCG T AA GTG GCA CGC GTG AG exon 2, 812-793 5666 rGALR2-i GTA GCT GCA GGC TCA GGT TCC exon 2, 658-678

13190 rGALR3-f CCT GGC TCT TTG GGG CTT TCG TG exon 1, 260-282 478bp 8437 rGALR3-r AGC GCG TAG AGC GCG GCC ACT G exon 2, 737-718 8436 rGALR3-i GTG GCC GTG GTG AGC CTG GCC T exon 2, 589-610

19030 ml3-Actin-f GTG GGC CGC TCT AGG CAC CAA cDNA 25-45 540bp 19031 ml3-Actin-r CTCTTTGATGTCACGCACGATTTC cDNA 565-542

TABLE 4.2 Oligonucleotides used for the amplification and detection of mRNA encoding galanin and its receptors.

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4.3 RESULTS

(i) Isolation of a targeted ES cell clone

Gene targeting constructs were designed with the aim of replacing an endogenous Galnrl allele by homologous recombination in ES cells. The construct ptar-1 was constructed by subcloning genomic fragments into the pPGK-near vector. The construct contained two Galnrl genomic fragments: a 0.8 kb BamHI-Kpnl fragment 5' of the PGK-nea' selection cassette and a 3.7 kb Kpnl-Spel fragment 3' of the cassette (Figure 4.1). Relative to the genomic fragments, the selection cassette was in a reverse transcriptional orientation in order to minimise the possibility of "read-through" from the near transcript. The junction between the 5' arm of homology and the near cassette in ptar-1 was sequenced to locate in-frame stop codons in the construct. The most proximal in-frame stop codon was found 88 bp downstream of the junction.

W9.5 ES cells were cultured and electroporated with ptar-1 as described. Following electroporation and selection in G418, a total of 288 clonal ES cell colonies were picked and seeded into separate wells of three 96-well culture dishes. Colonies were picked from the four electroporation plates. Although the total number of surviving colonies was not determined, it was estimated that treatments 3 and 4 in Table 4.1 resulted in higher transfection efficiencies than treatments 1 and 2. After regrowth, the clones were screened for the presence of a targeted allele, initially by PCR amplification in pools of three clones. Two positive pools were identified, Pll/42 and Pll/70 (Figure 4.2(a)). A second PCR was then carried out on the individual clones from the two pools. This identified two clones, 11 / 42 and 11 /262, as putative targeted clones (Figure 4.2(b )).

In order to confirm the presence of a correctly targeted Galnrl allele, Southern hybridisation analysis of genomic DNA from the PCR-positive clones was carried out. DNA was purified from the clones 11 / 42 and 11 /262, as well as the wild-type clone 11/138 as a control. DNA (40 µg) from each of the three clones was digested with Kpnl and Southern blots were made to probe the 5' end of the Galnrl gene and the integrated PGK-nea' cassette. A 20 µg amount of DNA from each clone was also digested with Sacl in order to probe the gene 3' of the construct integration site.

109 FIGURE 4.2 Screening of ES cell clones by PCR. (a) Clones were initially screened in pools of three. Shown are the two agarose gels on which PCR reaction products were analysed. Apart from the outermost lanes, into which DNA markers were loaded, each lane represents three ES cell clones. The PCR product is approximately 1.3 kb in size. The positive pools were identified as Pll/42 and Pll/70, and the PCR products are boxed and labelled. (b) PCR reactions were repeated on the individual clones which made up the positive pools. The products were analysed by electrophoresis, alongside their "parent" pools. Explanation of labels: M, DNA size marker; 1, Positive plasmid control; 2, negative (no template) control; 3, Pool Pll/42; 4, Clone 11/42; 5, Clone 11/138; 6, Clone 11/234; 7, Pool Pll/70; 8, Clone 11/70; 9, Clone 11/166; 10, Clone 11/262.

110 a

P11/42

P11 /70

b M 1 2 3 4 5 6 7 8 9 10

111 Chapter 4

The results of the Southern hybridisation screening are shown in Figure 4.3. The 5' probe hybridized to the 1.5 kb wild-type Kpnl band in all three samples, but also to a 3.3 kb band in 11/262 DNA, indicative of the presence of the PGK-neor selection cassette at one Galnrl allele in this clone. A correctly targeted allele in clone 11 /262 was confirmed with the 3' probe, which hybridized to an 11 kb band as well as the normal 20 kb Sacl band that was detected in all samples. The smaller band resulted from the introduction into the allele of a Sacl site in the PGK-neor cassette in ptar-1. Hybridization with the near probe resulted in detection of a single band, providing evidence that no extra copies of the targeting construct had been integrated into the 11 /262 genome. Clone 11 / 42 displayed only the wild­ type band with both external probes. The PCR amplification result for clone 11/42 was therefore a false positive, probably resulting from contaminating DNA in the PCR reaction. A second DNA preparation from 11 / 42 cells yielded no product when screened by PCR amplification (not shown).

A second targeting vector was constructed, in which the majority of exon 1 of Galnrl was deleted and replaced with the PGK-neor cassette. Multiple transfections were carried out with this vector and a total of 248 ES cell transfectants were screened. However, no targeted clones were identified and it was decided to proceed with blastocyst injections using clone 11/262.

(ii) Breeding of GALRl knockout mice

Cells of the targeted clone 11/262 were injected into C57BL/6 blastocysts which were then implanted into pseudopregnant females, also of the C57BL/6 strain. A total of 40 chimaeric mice were born. Several of these mice had a high proportion of agouti pigmentation in the coat, indicative of a strong contribution of the introduced cells to the tissues of these mice.

Male chimaeras with an approximately 90% agouti coat were crossed with C57BL/6 females to initiate transmission of the targeted allele. The first F1 litters were entirely of an agouti coat colour, representing transmission of the 11/262 genome. DNA from tail biopsies of these mice was digested wi thBamHI and hybridised with the 5' external probe. In addition to the wild-type 3 kb band, a 4.5 kb band hybridised with the probe in 13 of 28 DNA samples, demonstrating the presence of the targeted Galnrl allele (not

112 FIGURE 4.3 Screening of ES cell clones by Southern hybridization. ES cell clones were screened using three different probes. (a) The structure of the wild type and predicted targeted alleles are shown with the positions of the probes used and predicted fragment sizes in matching colours. (b) Phosphor images of Southern hybridisation experiments on the putative targeted clones using the three probes described. The two clones flank a non-targeted wild-type control ES cell clone (w.t.). Only clone 11/262 displays hybridizing restriction fragments indicative of a targeted allele. Clone 11 / 42 is identical to the wild-type control.

113

b b

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Wild-type Wild-type

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11/42 11/42

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11/262 11/262

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shown). Some of these mice, which were heterozygous for the targeted allele, were inter-bred to generate F2 homozygotes for analysis.

Heterozygotes have now been back-crossed to C57Bl/ 6 mice over several generations, with the aim of producing a congenic GALRl knockout mouse strain in this background. In addition, the mutant allele has been bred into a 129 strain background, by mating male germline chimaeras with 129T2/SvEmsJ females. Due to the genetic variation between the ES cell donor strain and the sub-strain into which the mutation has been bred (Simpson et al., 1997), back-crossing to the 129T2/SvEmsJ sub-strain has also been initiated. The aim of this strategy is to maximise the uniformity of genetic background of the 129 GALRl knockout line.

(iii) Genotyping of GALRl knockout mice

The genotype of litters is routinely determined by PCR of DNA isolated from tail biopsies. A three primer reaction is used, in which a product of 0.45 kb is generated from a wild-type allele and a 1 kb product is generated from a mutant allele (Figure 4.4). Of 131 offspring of GALRl heterozygous breeding pairs, 70 (53.4%) were heterozygous, 28 (21.4%) were wild-type and 33 (25.2%) were homozygous for the disrupted allele. The production of offspring in approximate Mendelian ratios indicates that absence of a normal Galnrl allele does not affect survival of the embryo. A total of 171 homozygous mice have been born to date, of which 82 (48%) are male and 89 (52%) are female. No gender bias has therefore been observed among mice lacking a functional Galnrl gene.

(iv) Expression of galanin and galanin receptor genes in GALRl knockout mice

The specificity of the gene disruption with respect to the expression of genes involved in the galaninergic system was investigated. Primer sets specific to each gene were used to amplify cDNA from individual tissues and PCR products were detected on agarose gels or by Southern transfer and hybridization to oligonucleotides complementary to sequence within the PCR products. RT-PCR experiments were used initially to confirm the absence of normal GALRl mRNA in homozygous knockout mice. As seen in Figure 4.5(a), Southern transfer and hybridization of RT-PCR products

115 M123 4 5 6 78 9

1.0 kb T

0.4 kb w

FIGURE 4.4 Agarose gel showing PCR products from a genotyping assay. Amplification of normal and targeted alleles generates a 0.4 kb and a 1 kb product, respectively. Among this litter of nine pups, from a + / - x + /­ breeding pair, three are+/-, two are+/+ and four are-/-. Abbreviations used: M, 1 kb DNA ladder; T, targeted allele; W, wild-type allele.

116 Chapter 4

was able to detect GALRl mRNA in + / + brain, spinal cord and uterus. However, no normal GALRl mRNA was present in those tissues taken from GALRl -/- mice. The detection of (3-actin mRNA was used as a control for the fidelity of cDNA synthesis and equal loading of PCR products onto the agarose gel (Figure 4.S(a)).

In addition, the 5' end of the GALRl mRNA was amplified from poly-A+ RNA extracted from+/+ and-/- large intestine (Figure 4.S(b)). This was carried out to determine whether the expression of the gene encoding GALRl had been up-regulated consequent to the absence of normal gene product. Relative to levels of (3-actin mRNA, there appeared to be approximately equal levels of normal and mutant transcript produced in large intestine, a tissue in which GALRl mRNA is readily detected.

The disruption of the gene encoding GALRl could conceivably have an effect on the regulation of other components of the galanin signalling system. By RT-PCR, the expression of the genes encoding galanin and the receptors GALR2 and GALR3 was therefore compared between tissues of +I+ and -I- mice. Galanin mRN A was clearly detected in brain, large intestine and spinal cord but no amplification product from uterus was visible (Figure 4.6(a)). Levels of galanin mRNA appeared to be identical in +I+ and -/- mice. GALR2 and GALR3 mRNA was detected in all four tissues by Southern hybridization of PCR products (Figure 4.6(b )). Expression of GALR2 mRNA varied greatly between tissues, from very weak expression in whole brain to strong expression in intestine, but no major differences were evident between -/- and + / + samples. In contrast, expression of GALR3 mRNA was uniformly weak. In comparison to the +I+ samples, it appears there may be some loss of expression of GALR3 in brain and uterus of the GALRl -/- mouse, and possibly some up-regulation in spinal cord. However, this result will need to be confirmed by other approaches, as phosphorescent image detection of very weak signals can be unreliable. No signal was detected from any of the negative control reactions.

117 FIGURE 4.5 Expression of GALRl mRNA in the Galnrl -I- mouse. (a) Expression of normal GALRl and J3-actin mRNA in mouse tissues. Lanes 1, 3, 5: tissue from + / + mouse; lanes 2, 4, 6: tissue from -/- mouse. 1,2: brain; 3,4: spinal cord; 5,6: uterus; 7: "no tissue" control; 8: "no RNA" control; 9: PCR negative control. (b) RT-PCR amplification of a region of exon 1 from large intestine, 5' of the gene disruption. Amplification of ~­ actin mRNA is used as a control. Symbols used: +, sample from a wild-type mouse;-, sample from a knockout mouse.

118 a 1 2 3 4 5 6 7 8 9

GALR1

______, ~-Actin

b + GALR1 m ~-Actin •

119 a brain large spinal uterus intestine cord + + + +

GAL

- . ·--:~1~~~:t~·~w'~j~,;,.,,~,' .... '... , B-Actin

..,;=#"~ ;.2,%,.. -

b brain 1 large I spinal 1 uterus I : intestine : cord I I I I + • I + • I+ • I+

GALR2 : :- GALR3 : :

FIGURE 4.6 Expression of genes encoding components of the galaninergic system in tissues of +/+ (+) and Galnrl -I- (-) mice. (a) Expression of galanin and ~-actin mRNA in brain, large intestine, spinal cord and uterus. (b) Expression of GALR2 and GALR3 mRNA in these same tissues. Refer to (a) for ~-actin control bands for these samples.

120 Chapter 4

(v) Growth and development of GALRl knockout mice

Mice of all genotypes were weighed fortnightly from four weeks of age. The growth rate of GALRl -/- mice was compared to that of+/+ and +/- mice (Figure 4.7). Although the initial weight was slightly higher in the -/- mice (15.7±0.3 g vs. 13.7±0.2 g for females; 17.9±0.5 g vs. 15.5±0.5 g for males; n=6), no major difference between the genotypes was observed in the growth rate of males or females. The -/- mice were outwardly indistinguishable from +I+ or + /- littermates, indicating that functional GALRl is not essential for growth or development under normal laboratory housing conditions.

It has previously been reported that female galanin knockout mice are unable to lactate (Wynick et al., 1998). The female GALRl knockout mice showed no evidence of such a deficiency. They were observed to nurse their litters normally. The pups generally appeared healthy, showing no signs of dehydration, and consistently survived to weaning.

(vi) Seizure behaviour in GALRl knockout mice

A partially penetrant phenotype displaying seizure activity was initially observed in homozygous knockout mice of mixed genetic background. The behaviour has been observed in 30 of 117 -/- mice of mixed background; an incidence of 26%. Of these 30 mice, 22 are male and eight are female. The seizures have also been seen in six + / - mice out of 104. Of more than 30 +I+ mice of this line, none have displayed this behaviour. The seizures most often occur from approximately 11 weeks of age, although in some mice the age of onset was as young as three weeks. They are generally triggered merely by removing the lid of the cage under strong overhead lighting or by gentle handling. The seizures vary in severity, ranging from mild forelimb clonus with rearing and tail rigidity to complete loss of normal motor control accompanied by violent running and jumping, salivation, urination and vocalisation. A brief period of apparent respiratory distress usually follows the more severe seizures. The typical seizure lasts approximately one minute and is characterised by rearing and falling, forelimb clonus and tail rigidity. These episodes are followed by a quiescent phase and then a period of grooming. The mice appear to recover completely within a few minutes, with recovery time being proportional to seizure severity. Please refer to enclosed CD-ROM, Segments 1-2.

121 a Female 30

25

...... __..0) ~ +/+ ..c -0) 20 -<>-- +/- ·a; ~ --0- -/-

15

10 0 5 10 15 20

Age (weeks)

b Male 30

25

...... __..0) --tr- +I+ ..c -0) 20 ·a; -<>-- +/- ~ --0- -/-

15

10 0 5 10 15 20

Age (weeks)

FIGURE 4.7 Mean body weight of GALRl knockout females (a) and males (b). Mice were weighed up to four months of age. Data for each genotype are plotted in a different colour, as indicated in the legend. n=6 for each point; values plotted ± S.E.M.

122 Chapter 4

A higher rate of mortality is seen among the affected knockout mice. The rate of premature death among affected mice is 33%, compared to 12% for all homozygote null mice of this lineage. The cause of premature death has not been definitively determined, although fatal seizure episodes were observed on two occasions. The mice involved were knockout females, one close to term and the other nursing a litter. In both instances the seizure proceeded to a terminal stage, with rigid extension of fore- and hindlimbs, within 30 sec. Video recording of mice at night under infra-red illumination revealed spontaneous seizures during the normal sleep-wake cycle. The seizures have the same characteristics as those observed when the cage lid is removed. They most often occur as the mouse is waking and have not been observed during nocturnal periods of locomotor activity (CD, Segment 3). The seizure phenotype appears to be dependent on genetic background. It is seen on the mixed background and after two generations of back-crossing to the C57BL/6 strain, but not on the 129 strain background.

4.4 DISCUSSION

The development of the strain of GALRl knockout mice described in this chapter provides us with a novel tool for the study of GALRl function in vzvo. Initial observations of the phenotype of mice lacking functional GALRl has shown that, for the most part, the mice are viable and healthy with normal reproductive capabilities. However, a subset of these mice exhibit spontaneous limbic seizures and are prone to premature death. Although further investigations are required, this observation implies a critical role for GALRl in the modulation of neuronal excitability. A range of studies are planned to probe for deficits present in these mice that may be attributed to the absence of GALRl. Initially, these may be based on experiments that have revealed defects in galanin knockout mice (Hohmann et al., 1999; Ma et al., 1999; Mazarati et al., 1999; Wynick, 1997; Wynick et al., 1998). As GALRl expression has been reported in regions of the nervous system and peripheral tissues which mediate numerous functions, the investigations may ultimately branch out into studies designed to detect neural, neuroendocrine and behavioural deficits.

A number of factors are likely to impact on the outcome of a targeting experiment. As discussed below, these include targeting construct design,

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compensatory mechanisms and genetic background factors. In most cases, the aim of a gene targeting project is to create a null allele and this should be considered when designing the targeting construct. To ensure a null allele is produced, researchers often delete portions of the coding region of the gene of interest, replacing them with a selectable marker gene. In other cases, the selectable marker gene is inserted into a coding exon, thereby producing a mutant allele encoding a truncated non-functional gene product. Depending on the gene being studied, deleting coding sequence is often preferable as it is more likely to result in a null, rather than a hypomorphic, allele. During the course of this project, both strategies were attempted. The two types of targeting vector were introduced into ES cells and a targeted clone was successfully obtained with ptar-1, the "insertion" vector. Due to time constraints, further experiments with the second vector were not pursued.

Based on an extensive literature of GPCR structure and activity, the successful construct was predicted to give rise to a null allele upon homologous recombination at the Galnrl locus. If a peptide was expressed from this mutant allele, it would possess only four transmembrane domains and would terminate in several residues encoded by the antisense strand at the 3' end of the PGK-neor cassette. It has been well documented that GPCRs require all seven transmembrane domains and an intact third intracellular loop in order to be functional (Heymann and Subramaniam, 1997; Ji et al., 1998). It has also been demonstrated that the residues His264 and His267 in the sixth transmembrane domain and Phe282 in the seventh transmembrane domain of GALRl are essential for ligand binding (Kask et al., 1996). Any putative mutant receptor protein would thus be bereft of galanin binding activity. Nevertheless, when a reliable GALRl antibody becomes available for histochemical applications, it will be important to establish whether immunoreactive mutant peptide is detectable in tissues of the knockout mice. The accumulation of mutant peptide may be detrimental to the tissue and could contribute to any observed phenotype.

The targeted deletion of a gene could conceivably result in compensatory alterations in the expression of genes with related functions. In the case of

GPCR subtypes, this has been seen in mice lacking the B2 bradykinin receptor. In the superior cervical ganglia of these mice, mRNA encoding the B1 receptor subtype appears more abundant than in the wild-type mouse

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(Seabrook et al., 1997). However, this is not necessarily a common mechanism as it is not seen in mice lacking single NPY receptor subtypes. Deletion of the NPY YS receptor had no effect on expression of NPY or NPY Yl receptor mRNAs (Marsh et al., 1998). Similarly, deletion of the NPY Yl receptor did not result in noticeable changes in expression of the genes encoding the YS or Y2 receptors (Pedrazzini et al., 1998). Finally, mice lacking NPY Y2 receptors display normal expression levels of NPY, Yl and YS mRNAs (Naveilhan et al., 1999).

The expression of the genes encoding galanin and the other known receptors GALR2 and GALR3 was found to not be significantly up-regulated in GALRl knockout mice. As expected, normal GALRl mRNA, which was clearly present in wild-type mice, was not detected in the homozygotes. By detection of a region of GALRl transcript upstream of the disruption, it was also shown that the expression of Galnrl itself in homozygotes is not altered by absence of normal gene product. These RT-PCR experiments were a relatively crude means of approaching this question and may need to be followed up by an in situ hybridization approach, focusing on those tissues in which an aberrant phenotype is observed. The experiments carried out here do provide some evidence that the phenotype results from the absence of GALRl rather than the mis-regulation of the expression of related genes.

The issue of genetic background has been the subject of much recent discussion in the field of mouse mutagenesis (Banbury Conference, 1997; Gerlai, 1996a). In knockout studies, the ES cell lines used have predominantly been of 129 strain origin while the host blastocysts have generally been isolated from C57BL/6 mice. Analysis of the phenotype of homozygous mutants is often performed on mice of mixed 129:C57BL/6 genetic background (Gerlai, 1996b). However, some studies have demonstrated that the genetic background can have a major impact on the expression of a mutant phenotype. The presence of modifier genes in a particular background can exacerbate or diminish the effect of an introduced mutation. Also, in these situations, litter-mates used as controls may have a very dissimilar combination of background genes.

We have sought to avoid the complications of a hybrid background by breeding germline-transmitting chimaeras to 129 /SvEmsJ female mice, thereby transferring the targeted allele directly onto a 129 genetic

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background. While the ES cell line used was from a different 129 sub-strain, a few generations of back-crossing to the 129 /SvEmsJ sub-strain will integrate the mutant allele into a background considered to be inbred (Simpson et al., 1997). This strategy also has the advantage of rendering our results comparable to the analysis of galanin knockout mice, which was carried out with mice of 129 background (Wynick et al., 1998). Concurrently, heterozygous GALRl mice are being back-crossed to C57Bl/ 6 mice to establish a congenic mutant line. The C57Bl/6 strain is considered more suitable for behavioural studies that are planned (Crawley et al., 1997).

Galanin fulfils an essential role in regulating lactotroph function, as its absence in mice results in lactational defects associated with depleted pituitary PRL levels (Wynick et al., 1998). This activity may not be directly mediated by GALRl as the homozygous knockout females are competent in nursing their litters and show no overt evidence of insufficient milk production. In addition, in vitro evidence suggests that the receptor responsible for transducing galanin's signal to the lactotroph is responsive to the N-terminally truncated peptide, galanin(3-29) (Wynick et al., 1993b). This analogue is ineffective at activating GALRl in vitro. However, until PRL levels and mammary gland development are assessed in the GALRl knockout mice, a role for GALRl in the regulation of PRL secretion cannot be discounted. There are a number of reasons for this. Firstly, it should be noted that lactational failure in the galanin knockout mice is largely substrain-dependent (D. Wynick, personal communication). Absence of galanin in mice with a 129 /OlaHsd strain background results in lactational insufficiency and delayed mammary gland maturation. When a hybrid 129 /OlaHsd-129 /Sv background is present, lactation develops normally even though PRL secretion and mammary gland defects remain. Absence of functional GALRl may also influence lactational capacity in a substrain­ dependent manner. Secondly, as will be shown in Chapter 5, GALRl mRNA appears to be abundant in mouse pituitary, although it may only be present in cell types other than the lactotroph. Thirdly, GALRl expressed in the hypothalamus may conceivably play an indirect role in lactotroph function, by modulating release of hypothalamic releasing factors or by being involved in feedback control of galanin release into the hypophysial portal circulation.

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The administration of galanin elicits a robust GH secretary response in humans and rats (Bauer et al., 1986a; Carey et al., 1993; Ottlecz et al., 1986), and it is therefore considered a modulator of the GH axis. However, its role in normal growth is unknown, and galanin knockout mice reportedly have a normal growth rate (Wynick et al., 1998). The GALNRl gene has been proposed as a candidate for the GH insufficiency syndrome associated with a deletion of chromosomal region 18q23 (Cody et al., 1997). A possible anatomical basis for this proposal lies in the expression of GALRl mRNA in a subset of hypothalamic somatostatin neurons (Chan et al., 1996), as discussed in Chapter 1. The GALRl knockout mouse is an ideal in viva model to assess the role of GALRl in regulating GH secretion and growth rate.

Basal growth rate did not appear to be affected by absence of functional GALRl. The weight at four weeks of age appeared to be somewhat higher in both homozygote females and males in comparison to wild-type and heterozygous mice. This result would need to be confirmed with larger numbers of animals before it could be deemed significant. The weight of mice at this immature stage could depend on a number of variables, including position in the womb, size of the litter and age at weaning. These are factors which are difficult to control and therefore could contribute to the observed differences. It may be worthwhile to monitor weights at earlier stages, including at birth, to determine whether the differences at four weeks can be extrapolated. However, separating the litter from the mother prior to weaning, even for short periods, would present technical difficulties as it may result in rejection of the pups. The next stage in this line of investigation may be to quantify serum GH levels in the mice, initially to determine basal levels, but also to measure the response to exogenous galanin. If GH secretion following galanin administration is diminished in GALRl knockout mice, it would provide strong evidence for the involvement of this receptor in galanin's GH secretagogue activity.

The generalised motor seizures exhibited by a subset of GALRl knockout mice is an exciting and unprecedented finding. Compared to wild-type mice, galanin knockout mice have a shorter latency to seizure and increased severity in response to the convulsant pentylenetetrazole (PTZ) (Mazarati et al., 1999), but have not been reported to experience uninduced seizures. Galanin has also been shown to have anti-convulsive properties in a variety

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of rodent seizure models when administered exogenously (Chepurnov et al., 1997b; Mazarati et al., 1992; Mazarati et al., 1998). It is known to be an inhibitor of evoked glutamate and aspartate release in the hippocampus, thereby modulating neuronal excitability in this region (Zini et al., 1993). Although GALRl has not been directly implicated in this activity, the expression of GALRl mRNA in ventral hippocampus has been demonstrated in a number of studies (Burgevin et al., 1995; Gustafson et al., 1996; O'Donnell et al., 1999; Parker et al., 1995; Planas et al., 1998). Galanin's inhibitory effect may result from activation of neuronal K+ channels, which is in accord with the ability of GALRl to couple to co-expressed Kir channels in Xenopus oocytes (Smith et al., 1998). It is also worth noting that mice with a genetic deletion of the Kir3.2 channel subunit exhibit sporadic seizures with features common to those exhibited by GALRl knockout mice, such as induction by stressful environmental triggers, motor effects and rapid recovery (Signorini et al., 1997). Taken together, this diverse experimental evidence strongly implicates GALRl in modulation of hippocampal neuronal excitability.

A mechanism integrating our observations with previous data can thus be envisaged. In the mammalian brain, galanin released into the ventral hippocampus may act via GALRl coupling to K+ channels to pre­ synaptically modulate glutamate release. In the absence of GALRl, this inhibitory input may be eliminated, and the resultant postulated elevation of glutamate release may be sufficient to trigger the observed seizures. Initially, histological analysis of the brains of susceptible mice will be necessary to determine whether the seizures are a result of a gross developmental abnormality rather than a specific biochemical dysregulation of hippocampal excitability. Other mechanisms underlying the observed seizures are also possible, such as those involving cholinergic pathways, as galanin is known to inhibit the release of acetylcholine in the ventral hippocampus (Crawley, 1996). Much experimentation remains to be done to elucidate the seizure pathway in these mice. However specific GALRl agonists may eventually prove to be promising candidates for the development of novel anti-convulsant agents.

One study soon to be initiated will be to explore the sensitivity of the GALRl knockout mouse to convulsive agents. Given that a proportion of homozygotes display uninduced seizures, and that galanin knockout mice

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are more susceptible to PTZ, we predict that, compared to wild-type mice, the homozygous mice would be hyperexcitable in response to convulsant. If GALRl is the major site of action of galanin's anti-convulsant activity, then the reported effect of exogenous galanin in ameliorating the actions of convulsants such as PTZ should be nullified in the homozygotes. Various other experimental approaches could shed light on the seizure mechanism in these mice. Brains are being collected from seizure-susceptible mice for histochemical analysis, including mice perfused within 30 min of a seizure episode. Detection of early gene products such as c-fos may be informative in localising brain regions activated by seizure initiation. The recording of electrographic activity associated with the seizures, both in vivo and in hippocampal slice culture, may be useful in relating these seizures to human epilepsy syndromes and other mouse epilepsy models, such as the Kir3.2 knockout mouse (Signorini et al., 1997).

Among the homozygotes susceptible to spontaneous seizures, there appears to be a gender bias, with approximately twice as many males as females exhibiting this phenotype. This implies a sexually dimorphic expression of the trait, and may suggest that female mice have a partially protective innate mechanism. An obvious contributing factor may be the action of female sex hormones in the brain, as estrogen has a well documented stimulatory effect on galanin expression, at least in the hypothalamus and locus ceruleus (Gabriel et al., 1992; Tseng et al., 1997). It will be of interest to determine whether any evidence for a protective mechanism is seen in the relative susceptibility to convulsants in the female homozygotes.

It is of significant interest that a QTL for susceptibility to kainic acid-induced seizures in mice has previously been localised to an interval on Chromosome 18 encompassing Galnrl (Ferraro et al., 1997). The interval is defined by two microsatellite markers which have been assigned positions at 52 and 57 cM (Mouse Genome Database, 2000)). As discussed in Chapter 3, Galnrl is located at 55 cM on Chromosome 18 (Simoneaux et al., 1997). Although a region of 5 cM may be likely to contain well over 100 genes, Galnrl is currently a good candidate for this locus. It could equally be argued that the seizure susceptibility locus represents a neighbouring pro­ convulsant gene, the expression of which is altered by the selection cassette active at the mutant Galnrl allele, as discussed below. A so-called "seizure­ related gene" of indeterminate function, PTZ17, has been mapped to

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Chromosome 18 in the mouse, but a specific location was not provided (Wakana et al., 2000). However, the diagrammatic representation of results in the report implies that this gene is distant from Galnrl. The study by Ferraro et al. (1997) used recombinant inbred strains of mice, with the DBA/2 strain harbouring the susceptibility locus on Chromosome 18. It may therefore be worthwhile to sequence the Galnrl gene from the DBA/2 strain in a search for polymorphisms in the coding or regulatory regions. Any polymorphisms present which negatively affect activity or expression of the GALRl protein would provide strong evidence that the described QTL resides at Galnrl.

Susceptibility to seizures in mice is a trait that is highly strain-dependent (Kosobud and Crabbe, 1990). It is therefore not alarming that seizures have not been observed in GALRl knockout mice with a 129 genetic background. They have been observed on a mixed background (50% 129/Sv:50% C57Bl/6) and after back-crossing to the C57Bl/ 6 strain (<25% 129 /Sv). Although the C57Bl/ 6 strain is considered relatively resistant to convulsive stimuli, it is a more susceptible strain than the 129 strain (Royle et al., 1999). It is possible that increasing the proportion of C57Bl/6 background in the GALRl knockout mice by backcrossing will result in increased penetrance of the seizure trait. At this stage it is too early to predict the outcome of this approach. Another strategy for increasing the penetrance of the trait is to transfer the targeted mutation to a strain considered more susceptible to seizures, such as DBA/2 (Kosobud and Crabbe, 1990). The 20% penetrance of seizures in GALRl knockout mice also indicates the presence of modifier genes. This is perhaps to be expected, as the majority of heritable epilepsy syndromes are polygenic in aetiology (McNamara, 1999). Molecular genetic studies of seizure susceptibility in mice have found several loci to be involved, largely dependent on the seizure-inducing agent used (Ferraro et al., 1997; Ferraro et al., 1999). Placing the targeted mutation into a more favourable genetic milieu may increase the impact of the mutation on the overall incidence of seizures. If complete penetrance of the seizure phenotype was seen on a DBA/2 background, it would suggest that genetic background does exert a strong influence on expression of the null phenotype. For practical purposes, this strategy would also enable us to breed larger numbers of mice suitable for experimentation.

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A complex balance of inhibitory and excitatory components maintains control over neuronal excitability. A recent review of mouse models of epilepsy reported over 30 genes which, when mutated or absent, resulted in a seizure phenotype (Puranam and McNamara, 1999). The majority of these genes encoded components of ion channels or neurotransmitter pathways, or were associated with more generalised brain abnormalities. Two exceptions are the genes encoding NPY and the S-HT2c serotonin receptor (Erickson et al., 1996; Tecott et al., 1995). Like GALRl, the serotonin receptor is a GPCR, while NPY is another neuropeptide with inhibitory functions in the CNS. These examples serve to illustrate that disruption of the normal functioning of neurochemical inputs that are analogous, in some ways, to the galaninergic system, can lead to unbalanced neuronal excitability.

Mechanisms of spontaneous seizure induction not involving galanin, but rather resulting from the knockout process, cannot yet be ruled out. As a consequence of the gene targeting process, the PGK-neo' expression cassette remains active at the mutant Galnrl locus in neurons and, indeed, all cells of the knockout mouse. The expression of the neor gene is under the control of the phosphoglycerate kinase promoter, a very strong promoter which may be able to affect the expression of neighbouring genes. This has previously been reported for other knockout strains in which genes closely linked to the targeted gene have been affected (Olson et al., 1996; Taylor et al., 1998). In this scenario, a gene involved in intensifying neuronal excitability that is tightly linked to Galnrl may be inappropriately up­ regulated by the PGK promoter, resulting in the observed seizures. The availability of DNA sequence from the relevant region of human Chromosome 18 (GenBank Accession No. AP001933) may soon clarify the likelihood of this scenario, by facilitating a search for putative genes physically linked to GALNRl.

Among the range of experiments which could be undertaken investigate the null phenotype of the GALRl knockout mouse are those which have already revealed phenotypic defects in galanin knockout mice (Mazarati et al., 1999; Wynick et al., 1998). Some of these have already been mentioned, but other examples include the study of pain transmission and nerve regeneration following peripheral axotomy. Galanin knockout mice do not develop neuropathic pain and are severely impaired in their peripheral nerve regenerative capability (Wynick, 1997). The possible role of GALRl in

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these defects is unclear, as levels of spinal cord GALRl mRNA are reduced following inflammation or axotomy (Xu et al., 1996b). However, the receptor may have an inhibitory role in the propagation of the flexor reflex, as pharmacological inhibition of GALRl expression in the spinal cord is accompanied by a reduction in the inhibition of the reflex that is seen in response to exogenous galanin (Pooga et al., 1998). The use of experimental designs already applied to the galanin knockout mouse could clarify some of these issues when applied to the GALRl knockout mouse strain.

The widespread expression of GALRl mRNA in the brain is suggestive of a role in functions such as learning, anxiety and appetite control. Behavioural experiments will be used to detect abnormalitites associated with the loss of GALRl in the brain. These will initially consist of testing of general neurological reflexes and motor function. A well-established battery of tests will then be used to assess functions such as cognitive ability, memory acquisition and feeding behaviour (Crawley, 1999a; Crawley and Paylor, 1997).

In summary, this chapter describes the development and initial characterisation of a strain of GALRl knockout mouse. This strain, developed using standard gene targeting procedures, is viable and exhibits normal growth and reproductive capabilities. While we await the development of specific GALRl antagonists, these mice present another avenue to explore and extend our understanding of the physiological role of GALRl with a range of in vivo studies now possible. The phenotype of spontaneous motor seizures exhibited by a proportion of homozygotes indicates that GALRl may have an essential inhibitory role in the modulation of neuronal excitability. This could have important implications for our understanding of the mechanisms underlying epileptic syndromes, and suggests a potential novel target for the development of anti-convulsant drugs.

132 CHAPTER 5

EXPRESSION OF GALANIN RECEPTOR­ ENCODING GENES IN THE MOUSE Chapter 5

5.1 INTRODUCTION

The components of the galaninergic system which have been cloned to date consist of the genes encoding the peptides galanin and GALP and three GPCRs. As a basis for understanding the function and coordination of the system as a whole, it is important to determine the expression throughout the body of each of these components. The distribution of galanin in a range of species has been addressed in some depth, largely through molecular studies (Merchenthaler et al., 1993). The distribution of mRNAs encoding the galanin receptors has also been reported, predominantly in the rat but in some studies, in human (Bloomquist et al., 1998; Borowsky et al., 1998; Fathi et al., 1998a; Kolakowski et al., 1998b; Lorimer and Benya, 1996; Sullivan et al., 1997) and baboon (Kolakowski et al., 1998b).

As a result of the widespread application of whole-animal genetic and transgenic studies, the mouse is becoming the primary model organism for in vivo studies in neuroscience. Consequently, it is important to determine patterns of gene expression in the mouse, not only for comparative purposes, but also to facilitate the interpretation of the outcomes of genetic manipulation experiments. There is some evidence for differences in expression of galanin and galanin receptor genes between mouse and rat. For example, a comparison of Northern blot analyses has indicated that galanin gene expression may be more widely distributed in peripheral tissues of mouse than rat (Rokaeus and Waschek, 1998). Galanin mRNA has been detected by RT-PCR in the mammary glands of mouse (Freeman et al., 1998) but not of rat (Chen et al., 1999). There is also some evidence that the galanin gene may be more sensitive to the stimulatory effects of estrogen in the rat than in the mouse (Lundkvist et al., 1995). In addition, GALRl mRNA is absent from rat cerebellum, but detected at low levels in mouse cerebellum by in situ hybridization (Gundlach et al., 1999).

Recently, an extensive survey of galanin mRNA distribution in the mouse was undertaken (Freeman et al., 1998). This RT-PCR experiment, part of a larger study of 517 mouse genes, detected expression in every one of 46 tissues analysed, including mid-term foetus and placenta. This result supports and extends the findings of an earlier study reporting widespread detection of galanin gene expression by Northern analysis, including in several regions of the CNS (Rokaeus and Waschek, 1998). In contrast, no

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comprehensive data have been published on expression of galanin receptors in the mouse, as the rat has been the model of choice for gene expression studies to date. The work described in this chapter is intended as a first step towards addressing this issue. It aims to provide a starting point for in situ hybridization experiments, and to begin to establish a basis to rationally investigate mouse models of defective galanin signalling such as the GALRl knockout strain developed as part of this project.

Little is currently known about the regulation of the genes encoding the galanin receptors. An in vitro assay of mouse Galnrl promoter activity showed that expression of this gene is up-regulated by increases in cAMP (Zachariou and Picciotto, 1999). As mentioned in Chapter 1, in viva studies in the rat have demonstrated that GALRl expression is plastic, and affected by factors such as sex hormones, inflammation (Hecht et al., 1999) and physiological stress or injury (Bouret et al., 2000; Faure-Virelizier et al., 1998; Gorbatyuk and Hokfelt, 1998; Landry et al., 1999; Landry et al., 1998; Xu et al., 1996b). One factor to be investigated in this chapter is the relationship between availability of a peptide ligand and regulation of expression of the genes encoding its receptors. The galanin knockout mouse provides a useful model for the in viva study of galanin receptor gene regulation under conditions of chronic galanin depletion. For this reason, it was used as a source of tissue for the experiments performed.

The literature related to embryonic expression of the genes involved in galanin signalling is very limited. Yet, such studies are essential to our understanding of the peptide's developmental role. Galanin mRNA is detectable by Northern blot analysis in the rat conceptus between days 5 and 18 of pregnancy, peaking at embryonic day 11 (Ell) (Vrontakis et al., 1992). Galanin expression in the embryo has been shown to be widespread from E14 (Xu et al., 1996a). GALRl mRNA was also detected from E14 in brain and intestine, from E17 in spinal cord and trigeminal ganglia, and in dorsal root ganglia at E21 (Parker et al., 1995; Xu et al., 1996a). In this chapter, analysis of whole embryo extracts will attempt to extend these findings to the mouse, assess expression of GALRl mRNA at an earlier developmental stage and provide some initial data on expression of the genes encoding the other receptor subtypes.

135 Chapter 5

5.2 METHODS

(i) Mice

The galanin knockout mouse line, on a predominantly 129 /Sv background, was obtained from Dr D. Wynick, Department of Medicine, University of Bristol, U.K. Mice were maintained under standard laboratory conditions and all manipulations were approved by the Garvan Institute of Medical Research Animal Experimentation Ethics Committee and conducted in accordance with the National Health and Medical Research Council's Code of Practice for the Care and Use of Animals for Scientific Purposes. Mice were genotyped by PCR, according to protocols provided by Dr Wynick (Wynick et al., 1998). Eight week old, age-matched wild-type and homozygous galanin knockout mice were used in experiments as a source of adult tissue.

(ii) RT-PCR for detection of gene expression

Mice were euthanased by cervical dislocation and tissues or embryos were collected. Tissues analysed, in at least two animals of each genotype, were brain, spinal cord, small intestine, large intestine, heart, skeletal muscle and uterus. For a preliminary experiment, the pituitary gland and inguinal mammary gland from a single virgin female wild-type mouse were collected.

Embryos were collected following timed matings, at three stages of development: El0.5, E14.5 and El9.5. For each developmental stage, embryos were collected from two females of each genotype. At El0.5, each litter was pooled prior to RNA extraction to obtain sufficient amounts of material for analysis. A crude dissection was carried out on embryos collected at E14.5 and E19.5, separating the head from the rest of the embryo, and the specimens were then processed separately. At these two later stages, placentae were also collected.

Total RNA was extracted from tissues as described (Section 2.9). The RT­ PCR and Southern analysis for the detection of mRNA encoding 13-actin, galanin and the galanin receptors was carried out as described in Section 4.2 (xiii). In addition to the oligonucleotides detailed in Table 4.2, an internal

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oligonucleotide was used for the detection of galanin mRNA in mammary and pituitary glands by Southern hybridization. The sequence of this oligonucleotide (#1490) is 5'-ATGGCCAGGGGCAGCGTTAT-3' and corresponds to the gene encoding rat galanin, nucleotides 1-20. This sequence differs from the mouse gene sequence at one nucleotide only.

5.3 RESULTS

(i) Expression of galanin receptor-encoding genes in wild-type mouse

The expression of the genes encoding galanin and the galanin receptors in mouse tissues was determined by RT-PCR analysis, using the identical reagents and conditions as were described in Chapter 4. Expression of the~­ actin gene was again used as a control for the efficiency of cDNA synthesis and for sample loading. The tissues examined in initial experiments were brain, spinal cord, small and large intestine, heart, smooth muscle and uterus (Figure 5.1). Although the RT-PCR assays were not considered quantitative, an equivalent amount of total RNA from each sample was used as starting material. Therefore, a comparison of relative mRNA levels for each gene could be made between different tissues, and expression was categorised as either low, intermediate or high. Each receptor displayed a unique gene expression pattern.

GALRl GALRl mRNA was detected in all tissues examined. Expression levels were low to intermediate, with mRNA most abundant in brain, spinal cord and uterus, and very low in heart.

GALR2 GALR2 mRNA was also detected in all seven tissues. Expression levels were lowest in brain and heart and highest in large intestine, skeletal muscle and uterus.

GALR3 The expression of the gene encoding GALR3 was found to be highly variable. No GALR3 mRNA was detected in heart, and levels in skeletal muscle were only barely within levels of detection. In contrast, expression

137 +-+- +- +-+- +- +-

GALR1

GALR2

GALR3

~-actin

FIGURE 5.1 Expression of the genes encoding the galanin receptors in tissues of wild-type and galanin knockout mice. RT-PCR analysis of gene expression in eight week old wild-type (+) and knockout (-) mice. Panels are labelled according to the tissues for which the results were obtained. Expression of ~-actin mRNA was used as a control for cDNA synthesis and to compare sample amounts loaded on gels.

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was relatively high in uterus and intermediate in brain.

(ii) Differences in expression between wild-type and galanin knockout mice

The expression of the genes encoding the galanin receptors was examined in tissues of the galanin knockout mouse to ascertain whether complete developmental absence of ligand affected receptor gene expression. Some differences were seen in a direct comparison between wild-type and knockout tissues. However, these differences showed no consistent pattern among tissues and also showed considerable variability between duplicate samples. That is, when duplicate samples were assessed, differences in signal intensity were observed that could not be accounted for by differences in amounts of PCR product loaded on the gel (not shown).

Differences in RT-PCR product intensity between wild-type and knockout samples that exceeded differences in J3-actin intensity were seen in small intestine, skeletal muscle and uterus. In small intestine and skeletal muscle, levels of GALRl and GALR2 mRNA appeared higher in knockout tissue than wild-type tissue. In uterus, the opposite appeared the case for both receptors (Figure 5.1). The analysis of skeletal muscle and uterus expression was not performed in duplicate. No noticeable differences were seen in GALR3 mRNA levels, although expression was so minimal in most tissues as to render this comparison unreliable.

(iii) Expression of galanin and its receptors in wild-type mammary gland and pituitary

The lactational deficiency of the galanin knockout mouse implicates the pituitary and, potentially, the mammary gland as sites of action of galanin in the mouse. A preliminary experiment was carried out to detect the expression of galanin and its receptors in these tissues. Large intestine was used as a positive control as it expresses detectable levels of all genes analysed. RT-PCR analysis demonstrated that galanin mRNA was present at relatively high levels in pituitary but was not detected in mammary gland (Fig 5.2). Transcripts of all three galanin receptor subtypes were detected in both tissues (Fig. 5.2). GALRl mRNA was very abundant in pituitary gland,

139 1 2 3 4 5 6

GAL ..

GALR1

GALR2 -

GALR3

.~. - ~-Actin ......

FIGURE 5.2 Expression of the genes encoding galanin and the galanin receptors in virgin mouse mammary gland and pituitary gland. Large intestine was used as a positive control tissue and three negative con­ trol samples were also used, as described in Section 4.2 (xiii). Explanation of labels: 1, large intestine; 2, mammary gland; 3, pituitary gland; 4, "no tissue" control; 5, "no RNA" control; 6, PCR negative control. The lower band seen in Lane 3 of the b-actin panel is the GALRl RT-PCR product amplified in the same reaction.

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while GALR2 and GALR3 mRNAs were also detected at high levels. The galanin receptors were only weakly expressed in mammary gland.

(iv) Developmental expression of galanin and galanin receptor genes

The expression of the genes encoding galanin (Figure 5.3) and the galanin receptors (Figure 5.4) was investigated in wild-type and galanin knockout embryos and placentae. Embryos were collected at El0.5, E14.5 and E19.5 (and placentae at E14.5 and El9.5) and RT-PCR was carried out following RNA extraction. At E14.5 and E19.5 the head and torso of the embryos were dissected and analysed separately.

Galanin The Gain gene was expressed as early as El0.5 in the embryo and expression continued at detectable levels throughout prenatal development (Figure 5.3(a)). Relative to expression of ~-actin mRNA, levels of steady-state galanin mRNA were approximately equal at each time point. Expression of galanin mRNA was also seen in placenta and was very abundant in this tissue at E19.5 (Figure 5.5). As expected, no galanin mRNA was detected in the galanin knockout mouse, as the corresponding gene has been deleted (Figure 5.3(b)).

GALRl GALRl mRNA was detected at El0.5 and thereafter. Levels of expression were highest in the embryonic head at El4.5 and El9.5 (Figure 5.4). Expression of GALRl was barely detectable in placenta (Figure 5.5).

GALR2 GALR2 mRNA was present in all embryonic and placental samples analysed (Figures 5.4 and 5.5). Expression levels in the embryo may have been slightly lower at E14.5 than at the other time points. In contrast to galanin mRNA, placental expression of GALR2 was constant at a relatively high abundance (Figure 5.5).

GALR3 Embryonic expression of GALR3 was very low and therefore its detection was somewhat unreliable. GALR3 mRNA was present at all three stages of development, peaking in the torso of the E19.5 embryo (Figure 5.4). In the

141 FIGURE 5.3 Expression of the gene encoding galanin in the (a) wild-type (GAL+/+) and (b) galanin knockout (GAL-/-) mouse embryo. Duplicate embryo preparations were analysed at three stages of development, El0.5, E14.5 and E19.5. For the latter two stages, embryos were crudely dissected into head (h) and torso (t). Expression of the f3-actin gene was used as a control for efficiency of cDNA synthesis. The galanin RT-PCR product is 520 bp and the f3-actin product is 540 bp. No galanin mRNA was detected in the galanin knockout embryos. The negative control samples for this experiment are shown in Figure 5.5.

142 a

Days E10.51 E14.5 I E19.5 I I Development I I I I I I 1htht1htht

GAL

GAL+/+

~-Actin

b

GAL

GAL-/-

~-Actin

143 FIGURE 5.4 Expression of the genes encoding GALRl, GALR2 and GALR3 in wild-type and galanin knockout mouse embryo. Duplicate embryos were analysed at three stages of development, El0.5, E14.5 and E19.5. For the latter two stages, embryos were crudely dissected into head (h) and torso (t). Expression of the ~-actin gene was used as a control for efficiency of cDNA synthesis. For each gene, expression in wild-type ( + / +) embryos is displayed in the upper panel and expression in galanin knockout (- /-) embryos is displayed in the lower panel. The negative control samples for this experiment are shown in Figure 5.5.

144 I 1 E14.5 E19.5 Days E10.5 I Development I

1 htht 1 htht

... +/+

GALR1 -/-

+/+

GALR2 -/- •

- +/+ GALR3

-/-

+/+

~-Actin -/-

145 FIGURE 5.5 Expression of the genes encoding galanin (GAL), GALRl, GALR2 and GALR3 in the wild-type and galanin knockout mouse placenta. Duplicate placentae were analysed at E14.5 and E19.5. Expression of the~­ actin gene was used as a control for efficiency of cDNA synthesis. For each gene, expression in wild-type ( + / +) embryos is displayed in the upper panel and expression in galanin knockout (-/-) embryos is displayed in the lower panel. Symbols used: Cl, "no RNA control"; C2, PCR negative control.

146 I Days E14.s: E19.5 :c1 C2 Development

+/+ GAL -/-

+/+ GALR1 -/-

+/+ GALR2 ------/-

+/+ GALR3 -- -/-

'~,;?,fa~~ti~r~z-'*;,:'..;._ ~;i «,:,,t"/ - __ +/+ ~-Actin -/-

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placenta, GALR3 was detected weakly at E14.5 but much more strongly at E19.5, resembling the expression of galanin mRNA.

(v) Developmental expression in galanin knockout mice

Embryonic expression of the galanin receptor-encoding genes was not significantly altered by absence of ligand. Any differences in intensity between sample images generally paralleled corresponding changes in the intensity of control J3-actin mRNA bands. This indicates variation in gel loading or cDNA yield. The exceptions to this are the levels of GALRl and GALR2 RT-PCR product in the torso of El9.5 galanin knockout embryos (Figure 5.4). In duplicate samples, the expression of these mRNAs appeared to be substantially lower than in the wild-type embryos. In the placental samples, the only noticeable difference between the genotypes was in the level of GALR3 mRNA at El9.5. The increase in GALR3 expression from El4.5 to El9.5 seen in the wild-type placenta was not apparent in the knockout placenta (Figure 5.5). Unfortunately, the RT-PCR reaction on one of the duplicate samples was unsuccessful, so this result will need verification.

5.4 DISCUSSION

In order to understand the role of galanin and its receptors in mammalian systems, it is important to ascertain the sites of expression of each the genes encoding them. The predominant expression of a particular galanin receptor in a certain tissue is evidence that this subtype may be responsible for transducing galanin's activities at that site. While most of the gene expression studies in this field have focused on rat, it is critical, in light of continuing advances in mouse genetic manipulation, to extend these studies to include the mouse.

Our initial experiments on the CNS and peripheral tissues demonstrated that the genes encoding the three galanin receptor subtypes are widely expressed. Although the technique used was not quantitative, it was apparent that the amount of mRNA detected, per microgram of whole tissue total RNA, varied between tissues. This was true of all the receptors, such that over the seven tissues examined, the subtypes displayed a unique

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and overlapping gene expression pattern. For example, GALR2 mRNA was most abundant in peripheral tissues such as intestine and skeletal muscle but only weakly expressed in whole brain. In contrast, GALR3 mRNA levels were relatively low in all tissues examined with the exception of uterus. The role of galanin in the mammalian uterus is unknown, but may relate to an effect on smooth muscle contraction (Shew et al., 1992). On the evidence presented here, GALR2 and GALR3 may be important in galanin signalling in the uterus of the mouse.

In a general sense, these observations are consistent with the results of studies on subtype gene expression in rat and human. That is, most reports on the three cloned receptors detail widespread gene expression (Branchek et al., 2000; Iismaa and Shine, 1999), although some putative species differences do exist. For some tissues, a lack of consensus among published reports may make a meaningful comparison difficult. The only tissue in which all three receptor mRNAs were not detected was the heart, in which GALR3 mRNA was undetectable. One recent gene expression study in rat also did not detect GALR3 mRNA in rat heart but additionally detected no GALR3 expression in large intestine (Waters and Krause, 2000), a tissue which does display some GALR3 expression in mouse. The results of another analysis confirmed the absence of detectable GALR3 expression in rat heart but also reported no expression in uterus. Moreover, it differed from the first study in failing to detect GALR3 mRNA in DRG (Smith et al., 1998). Complicating the comparison further, a Northern blot analysis has been published, reporting expression of rat GALR3 mRNA in heart but not brain or skeletal muscle (Wang et al., 1997b). Some of these differences are likely to result from the different sensitivities of the methods employed, with RT-PCR studies having to be compared to Northern blot analyses and solution hybridization techniques. The main conclusion to be drawn from these comparisons is that there may be important species differences in the tissue distribution of galanin receptor gene expression. Varying expression levels between tissues in the adult mouse also suggests specific and complex roles for each receptor subtype.

Galanin receptor gene expression was examined in tissues of the galanin knockout mouse to determine the role that the ligand may have in regulating the expression of the cognate receptors. In other GPCR pathways, such as that involving noradrenaline (Bengtsson et al., 2000) or the peptide

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neurotensin (Najimi et al., 1998), both in vitro and in vivo studies have shown that acute ligand stimulation can influence the transcription of the corresponding receptor gene. However, the consequences of chronic ligand manipulation have not been well examined. Duplicate samples were analysed and results compared to ~-actin mRNA levels in an attempt to look for reproducible patterns of expression. Some reproducible differences in mRNA levels were detected between wild-type and knockout samples. In some cases, there were differences between samples of the same genotype, possibly indicative of variation in expression between animals. The most striking difference was the increase in GALR2 mRNA level over wild-type seen in knockout small intestine. This response was highly tissue-specific as no difference in expression was observed in large intestine. A similar difference was seen in GALRl mRNA levels.

This result is difficult to interpret without further experiments being done. A more quantitative assay of gene expression, such as real-time PCR, as well as in situ hybridization analysis, would add meaning to the findings. One possible interpretation is that the activation of galanin receptors in small intestine transfers a signal to the cell nucleus to suppress expression of the gene encoding GALR2 and, possibly, GALRl. In the absence of galanin, this suppression fails to occur and GALR2 expression is increased, as a potential mechanism to enhance sensitivity to low concentrations of ligand. The presence of GALP, a peptide with homology to galanin and activity at galanin receptors, would add a level of complexity to this system (Ohtaki et al., 1999). GALP is presumably still produced in the galanin knockout mouse, however, it is not yet known whether it is present in the intestine. The PCR primers used in these experiments were specific to galanin and would not amplify GALP cDNA. The main conclusion from this section of work is that galanin is not essential for the maintenance of expression of its receptors in adult mouse, as no major universal alteration in receptor gene expression was observed in the galanin knockout mouse.

Galanin mRNA was not detected in the mammary gland under the conditions described. This finding contradicts results published electronically in the mouse Gene Expression Database (Freeman et al., 1998). It also differs from a report of galanin gene expression by human breast cancer cell lines (Ormandy et al., 1998b) but is consistent with a study on neuropeptide gene expression in rat mammary gland (Chen et al., 1999).

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The difference in result between the two mouse RT-PCR experiments cannot be easily explained. Although the PCR primers and mouse strains used were different, the experimental conditions were similar, and detection of PCR products in the published report was by staining of agarose gels rather than by Southern hybridization, as was carried out here. This may indicate that galanin mRNA levels were significantly higher in the samples of Freeman et al. (1998), suggesting possible strain-specific expression. All three receptor genes were expressed in mammary gland, demonstrating that this tissue is responsive to galanin. Thus, although the activity of galanin in the pituitary gland may have a major influence on development of the mammary gland (Wynick et al., 1998), it would appear that it can also act directly on this tissue, possibly by an endocrine mode of action in the absence of local expression of the gene.

In agreement with previous studies (Freeman et al., 1998; Rokaeus and Waschek, 1998), a preliminary experiment showed the gene encoding galanin to be strongly expressed in the pituitary gland. The three genes encoding the galanin receptors were also strongly expressed, with GALRl mRNA detected at the highest levels. In fact, the amount of GALRl mRNA detected in the pituitary far exceeded that of any other tissue analysed. This result is unexpected, as GALRl mRNA has not been found in rat pituitary, and it implies a role for this receptor subtype in neuroendocrine modulation in the mouse. This role may not include direct involvement in lactotroph activation, as mice lacking functional GALRl are proficient with respect to lactation (see Section 4.3(v)). The roles of the receptor subtypes, including the putative galanin(3-29)-selective receptor, in the pituitary are likely to be complex. Double-labelling in situ hybridization and/ or immunohistochemical analysis is required to determine the identity of the pituitary cells expressing each receptor.

The developmental role of galanin and its receptors has not been investigated to any great degree. Previously, the earliest stage at which galanin was known to be produced in mammals was at ES of rat embryonic development, although the whole conceptus, including extraembryonic tissue, was analysed in this study (Vrontakis et al., 1992). The experiments described here show that the gene encoding galanin is expressed at 10.5 days of mouse development and that mRNAs encoding the three receptor subtypes are also detectable at this developmental stage. This raises the

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possibility that any defects observed in the galanin and GALRl knockout mouse strains may originate from developmental deficiencies relatively early in embryogenesis.

The development of the specific tissues which are known to express the genes encoding galanin and GALRl at later stages of rat embryogenesis, such as brain and gut, is already well underway at El0.5 (Hogan et al., 1994). To gain further insight into the role of this system during development, it is important not only to ascertain where the expression occurs, but also at what stage galanin is first produced. It is possible that the major role of galanin in embryogenesis is as an autocrine or paracrine factor. Its mitogenic activity in the rat pituitary gland and certain cancer cell lines may represent a general function of the peptide in a range of cell types. Both GALRl and GALR2 have been shown to activate mitogenic signalling pathways in vitro (Wang et al., 1998b). Although speculative, it is possible that certain cell lineages may be dependent on galanin for normal development. Hence, the relative decrease in GALRl and GALR2 mRNA seen in the torso of the E19.5 galanin knockout embryo, and in GALR3 mRNA in placenta, may actually indicate a loss or absence of galanin­ responsive cells rather than a down-regulation of expression of the receptor­ encoding genes. This possibility could also be addressed by in situ hybridization.

The differential expression of galanin receptor mRNAs in placenta is an interesting finding. To our knowledge, it is the first description of the expression of these genes in this tissue. By E14.5, the placenta has developed into a complicated structure containing both extraembryonic and maternally-derived cells. It produces many peptide hormones which regulate embryo and maternal physiology (Hogan et al., 1994). Galanin may be contributing to this regulation, or it may have a paracrine or autocrine trophic role in the placenta (Vrontakis et al., 1992).

As mentioned above, the interpretation of these experiments becomes more complicated with the recent dicovery of a putative additional endogenous GALR2 ligand, GALP (Ohtaki et al., 1999). The extent of GALP expression in the rodent is not yet known, nor whether its expression is increased in the galanin knockout mouse. Its presence, however, may negate any influence of galanin deficiency on the expression of GALR2. The same may be true of

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GALR3, as the affinity of GALP for this receptor has not yet been reported. The discovery of GALP also raises the spectre of further undiscovered galanin family members, possibly including other endogenous GALRl ligands. This should be recognised when evaluating the role of ligand in the regulation of expression of cognate receptors.

The results detailed in this chapter were intended to provide basic data on gene expression within the galanin signalling system. As whole tissue extracts were used, certain limitations were inherent to this approach. For example, although GALRl expression has been reported to be strong in certain brain regions, such as hypothalamus and amygdala (Burgevin et al., 1995; Parker et al., 1995), only low levels of GALRl mRNA were detected in whole brain. This is likely to be a result of the diluting effect of using extracts from a tissue in which expression may have been localised to small regions rather than distributed throughout the tissue. It may be informative to follow the experiments described with analysis of dissected brain regions, concentrating on those regions where galanin is proposed to act, such as hypothalamus, amygdala and frontal cortex. These results could then be correlated with in situ hybridization studies to provide a more complete picture of the extent and relative levels of expression of the genes of interest in the nervous system.

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SUMMARY AND CONCLUSIONS Chapter 6

Since its discovery in 1983, the neuropeptide galanin has been shown to be involved in a wide range of neuronal and physiological processes. These include inhibition of neurotransmitter release with resultant effects on learning and memory, modulation of pituitary hormone secretion and regulation of appetite, gastrointestinal motility and pain transmission (Crawley, 1995; Iismaa and Shine, 1999). In recent years, the molecular cloning and characterisation of three distinct galanin receptor subtypes has begun to reveal the basis of galanin's pleiotropic modality (Habert-Ortoli et al., 1994; Howard et al., 1997a; Wang et al., 1997b). A number of approaches have been used to gain insight into the specific roles of each receptor subtype in mediating the activities of galanin. Gene expression studies, pharmacological analysis of heterologously expressed receptors, and gene structure and mapping approaches have contributed to our understanding of the galanin receptors, particularly GALRl. However, the lack of subtype­ specific antagonists has limited the study of galanin receptor function in

VlVO.

In the absence of specific antagonists, gene deletion approaches represent a powerful alternative and a valuable complementary tool when receptor antagonists eventually become available. The major aim of this project has been to develop an in vivo model to study the physiological function of the galanin receptor GALRl. This has been achieved with the development of a mouse strain harbouring a disruption in the Galnrl gene. The analysis of the phenotypic characteristics of this mouse will contribute to the elucidation of the essential roles of this receptor subtype in communicating galanin's effects in mammalian systems. In addition, a survey of galanin and galanin receptor gene expression has been carried out in the mouse, both during development and in the mature animal. These data will help to establish a context for the study of the GALRl knockout within the galanin signalling system in this organism. As species specificity has previously been reported in galanin's actions and expression of its receptors, it is important to determine expression patterns in the mouse if the observed phenotype is to be interpreted meaningfully.

The first step towards development of the GALRl knockout mouse was isolation of the corresponding gene. This was accomplished by screening a genomic library with cloned human GALRl coding sequence. Several

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identical clones were isolated and found to contain the Galnrl coding region plus 5' and 3' flanking sequences. The genomic structure of this gene was elucidated and the coding exons were sub-cloned and sequenced. Sizeable upstream and downstream regions were also sequenced, in an attempt to locate non-coding exons or motifs contributing to the control of gene expression.

The structural organisation of Galnrl was found to be identical to the human orthologue, GALNR1 (Jacoby et al., 1997). The genomic structure, comprising three coding exons, is distinct from the structure of the two other galanin receptors characterised and is unique among GPCRs cloned to date. This has implications for the molecular evolution of this family of receptors and suggests that the ancestry of the GALNR1 gene is distinct from that of GALNR2 and GALNR3. While GALNR2 and GALNR3 may have evolved by duplication of a predecessor gene, it is likely that GALNR1 evolved from a different ancestor. Further insights into the genomic organisation of this class of receptors are likely to be forthcoming from the data emanating from the Human Genome Project.

The Galnrl gene was physically mapped by radioactive in situ hybridization to Chromosome 18E3-4, a location consistent with that of a recent genetic mapping study (Simoneaux et al., 1997), and homoeologous with localization of the human gene at Chromosome 18q23 (Crawford et al., 1999; Nicholl et al., 1995). Elucidation of the structure of Galnrl adds to the body of knowledge on the genomics of the G protein-coupled receptor superfamily, while the chromosomal localization extends the region of Chromosome 18 which is known to be conserved between human and mouse.

The characterisation of Galnrl enabled the design and construction of a gene targeting vector to disrupt this gene in ES cells. A selection cassette was inserted into the first exon of the gene to interrupt the coding sequence. Transfection of ES cells by electroporation with the targeting construct led to the isolation of numerous clones which had integrated the introduced DNA. A two stage screening process, involving PCR followed by verification by Southern hybridization analysis, identified a clone with one Galnrl allele correctly targeted. The cells of this targeted clone were injected into blastocysts. These blastocysts were implanted into foster females and

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developed into chimaeric mice. Several of the chimaeras were capable of transmitting the mutant Gal n r 1 allele through their germline, demonstrating that the ES cells had retained pluripotency during the gene targeting experiments. From these chimaeras, lines of GALRl knockout mice were bred.

Experiments on tissues using RT-PCR showed that normal GALRl mRNA is absent in homozygous GALRl deficient mice and that there is no up­ regulation of the expression of the disrupted gene. Additionally, expression of the genes encoding galanin, GALR2 and GALR3 was shown to be largely unaffected. The inheritance of the targeted allele by offspring of heterozygous breeding pairs was in a close approximation of Mendelian ratios. This indicated that the absence of functional GALRl does not affect embryonic development or peri-natal survival. The growth rate as determined by weight gain up to four months of age was identical between wild-type, heterozygous and homozygous null mice. Homozygous mice reproduced normally and females showed no evidence of the lactation defects reported in galanin knockout mice. This would suggest that the effect of galanin on lactotroph function in mice is not mediated by GALRl, a conclusion supported by previous in vitro experiments (Hammond et al., 1996; Wynick et al., 1993b).

The major novel finding of this study is that mice lacking normal GALRl are susceptible to spontaneous generalised motor seizures. The seizures range from relatively minor to quite severe, are seen in approximately 20% of homozygotes of mixed, or predominantly C57Bl/6, genetic background and have a mean age of onset of 8-11 weeks. The seizures also occur during the normal active period, as recorded by infrared video. The high mortality rate among the fitting mice suggests that the seizures may occasionally be so severe as to be fatal. Death resulting directly from a seizure was indeed observed on two occasions.

The study of galanin's role in epileptogenesis is an emerging field in which much basic experimental work still needs to be done. The finding of spontaneous generalised seizures in the GALRl knockout mouse is an exciting addition to this body of research. It raises the possibility that GALRl may be a potential target for novel anti-convulsant drugs. Future experiments will need to address the nature of the mechanism which is

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disrupted in this seizure model. Another challenge arising from this finding is to demonstrate its relevance to human clinical syndromes. In this respect, it is encouraging that the analysis of a cohort of subjects with 18q terminal deletions encompassing GALNRl reported seizures in 31 % of cases (Strathdee et al., 1995).

The final section of experimental work was aimed at generating some basic data on the expression, in the mouse, of genes encoding components of the galanin circuitry. The majority of reports in this field have focused on the rat and, with the emergence of the mouse as a prominent experimental model, comparative studies are warranted. By using the galanin knockout mouse, we were also able to assess the role of ligand in regulation of receptor gene expression. RT-PCR analysis was used to detect gene expression in tissues and embryos of wild-type and galanin knockout mice. The genes encoding the three known galanin receptors were all found to be widely expressed at varying levels in adult tissues. The absence of ligand appeared to have a complex effect on levels of GALRl and GALR2 mRNA in certain tissues. For example, GALRl and GALR2 mRNA levels in galanin knockout tissues were higher in small intestine and skeletal muscle, but lower in uterus, compared to wild-type tissues. However, presence of galanin was certainly not required for the induction or maintenance of receptor gene expression. Surprisingly, GALRl mRNA was detected at high levels in pituitary gland, a result which will need verification as it contradicts findings in the rat. If reproducible, this raises the possibility of species specificity in galanin receptor function.

The expression of the genes encoding galanin and all three receptor subtypes was detected as early as 10.5 days of embryonic development. This novel result suggests that multiple receptors mediate galanin's developmental roles. The same may be true of the placenta, where mRNA encoding GALR2 and GALR3 was present at E14.5 and E19.5. This tissue had not previous! y been analysed for galanin receptor expression and therefore increases the range of tissues known to be a target for the peptide. The limitations of the methodology used in Chapter 5 preclude wide-ranging conclusions being drawn. However, the results do establish a basis upon which more intricate in situ hybridization and immunohistochemical studies can build.

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Galanin has a functional role in numerous neuronal and neuroendocrine processes. The widespread expression of the gene encoding GALRl would suggest that this receptor is involved in many of these. The work detailed in this thesis has culminated in the development of a novel and exciting tool to facilitate research into the physiological importance of GALRl in the mammal. The overall objective of future studies of the GALRl knockout mouse will be to interpret the results with respect to human physiology, to increase our understanding of the contribution of galanin and GALRl to maintaining health or precipitating disease. Our preliminary observations of the seizures exhibited by these mice already suggest that GALRl may be an integral component in the control of neuronal excitability and, in certain circumstances, may contribute to epilepsy. Ultimately, analysis of the GALRl knockout mouse may point to new therapies for human disorders in which GALRl is currently implicated.

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194 APPENDIX

Jacoby A.S., Webb G.C., Liu M.L., Kofler B., Hort Y.J., Fathi Z., Bottema C.D.K., Shine J. and lismaa T.P. (1997) Structural organization of the mouse and human GALR1 galanin receptor genes (Galnr and GALNR) and chromosomal localization of the mouse gene. Genomics 45: 496-508.

195 GENOMJCS 45, 496-508 (1997) ARTICLE NO. GE974960

Structural Organization of the Mouse and Human GALR1 Galanin Receptor Genes (Galnr and GALNR) and Chromosomal Localization of the Mouse Gene

Arie S. Jacoby,* Graham C. Webb, t·t Marjorie L. Liu,* Barbara Kofler,§ Yvonne J. Hort,* Zahra Fathi, ~ Cynthia D. K. Bottema, t John Shine,* and Tiina P. lismaa*· 1

*Neurobiology Program, The Garvan Institute of Medical Research, 384 Victoria Street, Sydney, New South Wales 2010, Australia; tDepartment of Animal Science and t-Department of Obstetrics and Gynaecology (The Queen Elizabeth Hospital), The University of Adelaide, Waite Campus, Glen Osmond, South Australia 5064, Australia; §Children's Hospital, Landeskrankenanstalten, Salzburg A-5020, Austria; and ~Neuroscience Drug Discovery, Bristol-Myers Squibb Pharmaceutical Research Institute, 5 Research Parkway, Wallingford, Connecticut 06492

Received March 3, 1997; accepted August 5, 1997

terminally amidated in all species except human, in The neuropeptide galanin elicits a range of biologi­ which it is 30 amino acids long and nonamidated. The cal effects by interaction with specific G-protein-cou­ primary amino acid sequence of galanin has been pled receptors. Human and rat GALRl galanin recep­ highly conserved throughout evolution, particularly tor cDNA clones have previously been isolated using the N-terminal 15 amino acids, which are invariant expression cloning. We have used the human GALRl in mammals, birds, reptiles, and amphibia (Crawley, cDNA in hybridization screening to isolate the gene 1995; Chartrel et al., 1995). In mammals, galanin is encoding GALRl in both human (GALNR) and mouse widely distributed in the central and peripheral ner­ (Galnr). The gene spans approximately 15-20 kb in vous system, where it elicits numerous biological ef­ both species; its structural organization is conserved fects. These include inhibition of neurotransmitter re­ and is unique among G-protein-coupled receptors. The lease, regulation of pituitary hormone secretion, modu­ coding sequence is contained on three exons, with exon 1 encoding the N-terminal end of the receptor lation of heart rate and blood pressure, and effects on and the first five transmembrane domains. Exon 2 en­ appetite, memory, nociception, and sexual activity codes the third intracellular loop, while exon 3 en­ (Bartfai et al., 1993; Carey et al., 1993; Crawley, 1995). codes the remainder of the receptor, from transmem­ The spectrum of biological activities ascribed to ga­ brane domain 6 to the C-terminus of the receptor pro­ lanin indicates that it plays an important role as a tein. The mouse and human GALRl receptor proteins neurotransmitter and neuromodulator. Aberrant ga­ are 348 and 349 amino acids long, respectively, and lanin activity has also been implicated in a range of display 93% identity at the amino acid level. The mouse disorders, including dementia associated with both Alz­ Galnr gene has been localized to Chromosome 18E4, heimer and Parkinson diseases and proliferation of homoeologous with the previously reported localiza­ small cell lung carcinoma cells (Bartfai et al., 1993; tion of the human GALNR gene to 18q23 in the same Crawley, 1995). Galanin receptors therefore show syntenic group as the genes encoding nuclear factor promise as targets for the development of therapeutic of activated T-cells, cytoplasmic 1, and myelin basic agents. They are members of the superfamily of protein. © 1997 Academic Press G-protein-coupled receptors and have been shown to activate a variety of intracellular second-messenger pathways, including inhibition or stimulation of intra­ INTRODUCTION cellular cAMP accumulation, stimulation of inositol Galanin is a 29-amino-acid neuropeptide found ubiq­ phosphate accumulation, blockage of voltage-depen­ uitously in the animal kingdom. Mature galanin is C- dent Ca2 + channels, and activation of ATP-sensitive and ATP-insensitive K+ channels (Bartfai et al., 1993). Sequence data from this article have been deposited with the Pharmacological data have suggested the existence EMBUGenBank Data Libraries under Accession Nos. U90655- of galanin receptor subtypes, and expression cloning U90657 and U90658-U90660. 1 To whom correspondence should be addressed. Telephone: 61 2 has been used to isolate cDNA clones encoding the hu­ 9295 8293. Fax: 61 2 9295 8281. E-mail: [email protected]. man and rat species homologs of a G-protein-coupled edu.au. galanin receptor that has been designated GALRl (Ha-

496 0888-7543/97 $25.00 Copyright © 1997 by Academic Press All rights of reproduction in any form reserved. GALRl GALANIN RECEPTOR GENE (GALNR) STRUCTURE IN MOUSE AND HUMAN 497 bert-Ortoli et al., 1994; Parker et al., 1995). Hybridiza­ bation at 42°C for 16 h, followed by washing twice at 42°C for 15 min tion screening approaches have subsequently been each time in 6X SSC containing 0.1% (w/v) SDS. Detailed restriction endonuclease mapping was carried out on selected mouse and human used to isolate a second galanin receptor subtype, des­ genomic DNA clones, and hybridizing fragments of a suitable size ignated GALR2 (Howard et al., 1997; Fathi et al., 1997). were subcloned from these isolates into pBluescript II SK ( +) (Stra­ Both GALRl and GALR2 exhibit conservation of amino tagene) or pUC119 (Sambrook et al., 1989) for sequencing. acid residues characteristic of the subfamily of G-pro­ Construction of a human hypothalamic cDNA library. Total RNA tein-coupled receptors exemplified by rhodopsin and was prepared from frozen human hypothalamus tissue (The National the adrenergic receptors (Iismaa et al., 1995). In situ Disease Research Interchange, Philadelphia, PA) using the Tri Re­ agent kit (Molecular Research Center, Cincinnati, OH), and poly(A)+ hybridization studies and Northern analysis have mRNA was isolated by two cycles ofoligo(dT) selection [Poly(A) Quik; shown GALRl to be widely expressed in the brain and Stratagene]. A random-primed cDNA library was constructed as de­ spinal cord, as well as in peripheral sites such as the scribed (Davis et al., 1994) with minor modifications. Briefly, 5 µg small intestine and heart (Parker et al., 1995; Burgevin poly(A)+ mRNA was used to synthesize hexamer-primed first-strand cDNA. After second-strand synthesis and adaptor (EcoRI-Notl, Cat. et al., 1995; Gustafson et al., 1996; Sullivan et al., No. 901111; Stratagene) addition, double-stranded cDNA was size 1997). selected by a combination of gel filtration chromatography (cDNA We report here the structural organization of the Size Fractionation Columns; Life Technologies, Gaithersburg, MD) gene encoding GALRl in mouse (Galnr) and human and sucrose density gradient centrifugation as described (Kieffer, (GALNR) and chromosomal localization of the mouse 1991). Fractions from the sucrose gradient containing cDNA larger than 2 kb were concentrated by precipitation and subsequently used Galnr gene. The structure of the gene encoding GALRl for ligation into ~gtlO (Lambda gtlO/EcoRI/CIAP-Treated Vector kit; has been conserved between the two species. Its geno­ Stratagene). Products of the ligation reaction were then packaged mic organization is different from that of the gene en­ in vitro using Gigapack III Gold Packaging Extract (Stratagene). coding GALR2 and is unique among G-protein-coupled Primary recombinant phage clones were titered on E. coli host strain receptors examined to date. Its chromosomal localiza­ NM514. tion in the mouse genome is homoeologous with the Isolation of a human GALRJ hypothalamic cDNA clone. Primary recombinant plaques (1 X 106) from the ~gtlO human hypothalamus human localization. cDNA library propagated in E. coli host strain NM514 were screened on nitrocellulose filters (Schleicher & Schuell, Keene, NH) with a full­ 32 MATERIALS AND METHODS length human GALRl coding sequence labeled with P (Amersham) according to instructions provided with the Nick-Translation System kit (Life Technologies). Hybridization was in a solution containing Isolation and characterization of mouse and human GALRJ geno­ 4x SSC, lx Denhardt's solution, 20 mM Tris-HCl, pH 7.4, 10% (w/ mic clones. Plaques (6 X 105) of a x.FIXII mouse 129 strain genomic v) dextran sulfate, 40% (v/v) formamide, and 20 µg/ml sheared and library (Stratagene, La Jolla, CA) propagated in Escherichia coli denatured salmon sperm DNA at 37°C overnight, followed by wash­ host strain LE392 and colonies (4 X 105 ) of cosmid pWE15 human ing at RT four times, twice for 5 min and another two times for 25 lymphocyte genomic library (Stratagene) propagated in E. coli host min, each time in 0.lx SSC containing 0.1% (w/v) SDS, then twice strain NM544 were screened on Colony/Plaque Screen nylon filters at 37°C for 30 min each time in 0.lx SSC containing 0.1% (w/v) SDS. (NEN-DuPont, Boston, MA) with full-length human GALRl coding A plaque-purified positive clone that withstood high stringency wash sequence (Habert-Ortoli et al., 1994; Nicholl et al., 1995) labeled with conditions [65°C in 0.lx SSC containing 0.1 % (w/v) SDS] was further 32 32 P ([a- P]dCTP, 3000 Ci/mmol; Amersham, Buckinghamshire, UK) characterized by subcloning its 3 kb insert into pGEM7zf( +) (Pro­ using random nonamer oligonucleotide primers and Klenow frag­ mega) for sequencing. ment of DNA polymerase I (Amersham; Cat. No. RPN 1607). For DNA sequencing. Sequencing was carried out using the Sanger isolation of mouse genomic DNA clones, hybridization was in a solu­ dideoxy chain termination method (Sanger et al., 1977) using T7 tion containing 5x SSPE (20x SSPE: 3M NaCl, 200 mM NaH2PO4 , 33 20 mM EDTA, pH 7.4), 5X Denhardt's solution [lO0x Denhardt's: polymerase (Promega) or cycle sequencing with P-end-labeled oligo­ 2% (w/v) bovine serum albumin, 2% (w/v) Ficoll 400, 2% (w/v) polyvi­ nucleotide primers as recommended by the manufacturer (Promega; nylpyrrolidone], 0.5% (w/v) SDS, and 200 µg/ml sheared and dena­ Cat. No. Q4100). Synthetic oligonucleotide primers (Beckman Instru­ tured salmon sperm DNA at 50°C for 16 h, followed by washing twice ments, Sydney, Australia) based on the human GALRl cDNA se­ at room temperature (RT) for 15 min each time in 2x SSC (20x SSC: quence, and upon additional sequence information as it was obtained, 150 mM NaCl, 15 mM sodium citrate, pH 7.0), then three times at were used to sequence exons and flanking regions. Sequences were 50°C for 15 min each time in lx SSC containing 0.05% (w/v) SDS. analyzed using MacVector (Oxford Molecular Group, Inc., CA) and For isolation of human genomic DNA clones, hybridization was in a the Australian National Genome Information Service. solution containing 5x SSPE, 5x Denhardt's solution, 0.5% (w/v) PCR amplification. For standard PCR amplification, 0.5 ng cos­ SDS, and 200 µg/ml sheared and denatured salmon sperm DNA at mid or plasmid DNA or 100 ng human genomic DNA was incubated 65°C for 16 h, followed by washing twice at RT for 15 min each time with 20 µM oligonucleotide primers, 200 µM dNTPs, and 0.5 unit in 2x SSC, then three times at 65°C for 15 min each time in 0.lx AmpliTaq Gold DNA polymerase (Perkin-Elmer Cetus Corp, Nor­ SSC containing 0.1% (w/v) SDS. walk, CT) in 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgC1 2 DNA isolated from positively hybridizing clones (Sambrook et al., in a total volume of 100 µI. A Perkin-Elmer Cetus DNA thermal 1989) was characterized by restriction endonuclease digestion, with cycler was used with the following temperature parameters: incuba­ electrophoresis in 0. 7% agarose gels and blotting to Hybond N+ mem­ tion at 94°C for 5 min to activate AmpliTaq Gold DNA polymerase branes (Amersham) using 0.4 M NaOH, as recommended by the (Perkin-Elmer Cetus), followed by 30 cycles of 94°C for 1 min, 49- manufacturer, for Southern blot analysis. Hybridizing fragments 540C for 1 min, and 72°C for 1.5-2.5 min. For long-range PCR ampli­ were identified using either 32P-labeled full-length human GALRl fication, 10 ng cosmid DNA was incubated with 200 nM oligonucleo­ coding sequence as above or oligonucleotide probes end-labeled with tide primers, 500 µM dNTPs, and 2.6 units Expand Long Template 32P ((-y-32P]ATP, 3000 Ci/mmol; NEN-DuPont) using T4 polynucleo­ DNA polymerases (Taq and Pwo DNA polymerases; Boehringer tide kinase (Promega Corp, Madison, WI) in a solution containing Mannheim, Mannheim, Germany) in 50 mM Tris-HCl (pH 9.2), 16 20% (v/v) formamide, 5X SSC, 5X Denhardt's solution, 1% (w/v) SDS, mM (NH4) 2SO4 , 2.25 mM MgC1 2 buffer containing 2% (v/v) dimethyl and 50 µg/ml sheared and denatured salmon sperm DNA, with incu- sulfoxide and 0.1 % (v/v) Tween 20 in a total volume of 50 µI. An 498 JACOBY ET AL.

OmniGene thermal cycler (Hybaid, Middlesex, UK) was used with and 20 cycles of94°C for 10 sand 68°C for 3.5 min. This was followed the following temperature parameters: incubation at 94°C for 2 min, by nested PCR amplification, with the following temperature param­ followed by 30 cycles of94°C for 10 s, 50°C for 30 s, and 68°C for 8 min, eters: 94°C for 1 min, followed by 30 cycles of 94°C for 10 s, 53°C for then incubation at 68°C for 8 min. Gene-specific sense oligonucleotide 10 s, and 68°C for 2 min. The 3 '-RACE products were analyzed by primers used in long-range PCR amplification corresponded to hu­ electrophoresis in 1.2% agarose gels and hybridization using a 32p. man GALRl coding sequence nucleotides 312-331 (5'-CGCCTTCAT­ end-labeled oligonucleotide probe corresponding to human GALRl CTGCAAGTTCA-3 '), nucleotides 648-666 (5'-CTGCTTCTGCTA­ coding sequence, nucleotides 1053-1003 (5'-TTATCACACATGAGT­ TGCCAAG-3'), and nucleotides 676-695 (5'-CACTTGCATAAA­ ACAATTGGTTGATGGTGGGGTGTCTATTCGACTTTT-3') as de­ AAGTTGAA-3' ), and antisense oligonucleotide primers scribed above for oligonucleotide hybridization. corresponded to human GALRl coding sequence nucleotides 723- Chromosomal localization of the mouse Galnr gene. A 440-bp 704 (5'-GGATGCTTCAGACTTCTTTG-3') and nucleotides 1053- mouse GALRl partial cDNA was used as a probe for chromosomal 1029 (5'-TTATCACACATGAGTACAATTGG-3 '). localization of Galnr. The cDNA was generated by reverse tran­ Characterization of the 5' and 3' ends ofGALRl mRNA. 5'- and scriptase-PCR

1.5 mM MgCl2 in a total volume of 50 µI. The sense/antisense primer 20 and 35 kb, respectively. Hybridization with primers pairs used for amplification were a gene-specific sense primer corre­ specific to the 5' and 3' ends ofGALRl coding sequence sponding to human GALRl coding sequence, nucleotides 850-876 established that both clones contained the entire (5'-TTCAGAATCACCGCCCACTGCCTGGCG-3'), and the primer APl (5'-CCATCCTAATACGACTCACTATAGGGC-3') supplied by GALRl coding region. Restriction maps of the region the manufacturer. A second nested PCR amplification was conducted encoding GALRl in each clone were constructed using using a sense primer corresponding to human GALRl coding se­ a combination of restriction endonuclease digestion, quence, nucleotides 916-932 (5'-CTCTCTGAAAATTTCAG-3'), and hybridization analysis with both full-length GALRl the primer AP2 (5' -ACTCACTATAGGGCTCGAGCGGC-3 'l supplied coding sequence and specific oligonucleotide probes, by the manufacturer. An OmniGene thermal cycler (Hybaid) was used, with the following temperature parameters for the first round PCR amplification of cloned and genomic DNA, and of amplification: 94°C for 1 min, followed by 5 cycles of 94°C for 10 Southern blot hybridization analysis of genomic DNA sand 72°C for 3.5 min, 5 cycles of94°C for 10 sand 70°C for 3.5 min, (data not shown). The GALRl coding region spanned GALRl GALANIN RECEPTOR GENE (GALNR) STRUCTURE IN MOUSE AND HUMAN 499

Mouse Galnr

N Sc N E

B s A

GALR1

E E E B ~HJ----+-+-++--+--+----+t------J.______.:::i:~;;~;; ------~ p p p p p PP Human GALNR Pst I fragments (2.4, 1.2, 0.9, 0.55, 0.3, 0.25) kb

1 kb

FIG. 1. Genomic organization ofGALRl galanin receptor genes in mouse (Galnr) and human (GALNR). The top and bottom lines depict the restriction endonuclease maps of mouse and human genes, respectively. The cloned mouse Galnr DNA is delimited by vector-derived Natl restriction endonuclease cleavage sites. Heavily hatched boxes represent coding sequence. Lightly hatched boxes represent noncoding exon sequence, with the 5' -upstream limit of the shorter mouse GALRl transcript indicated with a filled arrowhead and the 5' -upstream limit of the human hypothalamic cDNA clone indicated with an unfilled arrowhead. The dashed segments of human GALNR DNA represent sections of the map for which the DNA has not been mapped for BamHI or Pstl cleavage sites (upstream of exon 1) or EcoRI cleavage sites (downstream of exon 3) or for which the relative order of Pstl fragments has not been defined (intron 2). In the center is a schematic representation of GALRl coding sequence. Putative transmembrane domains are shown as black boxes and are numbered 1-7, other parts of the coding region are shown as white boxes, and segments of the mature GALRl receptor protein encoded by each of the exons are indicated. A, Apa!; B, BamHI; E, EcoRI; K, Kpnl; N, Natl; P, Pstl; S, Spel; Sc, Sacl. approximately 15-20 kb in both mouse and human isolated from HMCB cells (Habert-Ortoli et al., 1994) clones. and from human small intestine (Lorimer and Benya, The mouse and human GALRl genes encode proteins 1996) encode a Cys residue at position 15 of the pre­ of 348 and 349 amino acids, respectively, with a pre­ dicted protein sequence, while the sequence of genomic dicted seven-transmembrane-domain structure charac­ DNA predicts a Trp residue in this position, due to the teristic of members of the G-protein-coupled receptor presence of the nucleotide T instead ofG at position 45 superfamily. The coding sequence is contained on three of the coding sequence (Fig. 2b). A cloned human exons (Fig. 1). The first exon encodes the receptor from GALRl coding sequence isolated by RT-PCR amplifi­ the N-terminus to the end of the fifth transmembrane cation of fetal brain RNA (Sullivan et al., 1997) and domain. The second exon encodes the third intracellu­ a cDNA encoding GALRl isolated from human brain lar loop. The third coding exon encodes the sixth and (unpublished; GenBank Accession U23854) also predict seventh transmembrane domains and the C-terminal a Trp at position 15, consistent with both the sequence tail of the receptor. Exons 1 and 2 are separated by of genomic DNA reported here and the sequence of a approximately 5 kb in both mouse and human genes, partial hypothalamic cDNA clone also isolated in this while exons 2 and 3 are separated by approximately 8 work. The brain GALRl cDNA sequence exhibits an and 11 kb in mouse and human genes, respectively. additional difference that does not alter the sequence The genomic organizations of mouse and human of the encoded protein, with the Gly residue at position GALRl genes are identical, with exact conservation of 47 being encoded by the codon GGT instead of the GGC the position of exon:intron boundaries. The exon:intron that is observed in all other human GALRl sequences junctions exhibit good agreement with consensus splice reported to date, including the hypothalamic cDNA donor and acceptor sites (Mount, 1982) (Fig. 2). clone isolated here. These minor differences in se­ The amino acid sequence of human GALRl deduced quence could represent sequencing artifacts or a poly­ from cloned genomic DNA exhibits minor differences morphism in the GALNR gene, as high-stringency from the sequences reported for human GALRl cDNA Southern blot hybridization analysis of human genomic clones isolated to date. Human GALRl cDNA clones DNA (data not shown) was consistent with the map of 500 JACOBY ET AL. a -1040 gtaaagtggctcctgcctcaccattgcgcatccttgcctgcagggctggactgtgcaacccgcactggtgcatctccacagagctttcccaacgagcacc

-940 ctgtcctacccttctgtgcacaatctgtttctggccttgtgtctagcagttgtggtcactcaccgctggaggatcgctggcgttttggaaagcgatatag

-840 ttacccggtacctacatgcaagagcaggtctttcttttggactgctagaagcagtggctcatagttggtgtggcccccgcctcccttctcagcagagcgg T -740 cctttcaccacagtcaaatgactcttcacaaacacttcactcgtcaaggcttccgtttgtgtcactcagatgagtaatgtctggtggaaatgatGGATCC

-640 GTGAGAGAGGCTCGCCCTGCAGAGGACCCGGGACTAAGAGGGAGCCGCAGGCCAGCGCAGCGAGGCAGGGAGGTGGATCTTAGTGCGGGAAGCTCAGCGA

-540 CCCTCTTCACCATTGAAGGTGTGCATCGCTGGGCTCTCGGACGTTCGGGAAGAAGAGGCTCAAAGCAACAGGTGCAACCTCAAGGCACTGAAAGCAAGGG

-440 GACGCAGCTCACAAGGGCCAAGGGATTGAACCCATAACCGCTCAGAAGATTCTCCGCCTGCGGAGAGCTGCGGAGGAGTCCCACCCGTCCAGCTTGCTGA

-340 CTGCGAGCAGTGAGAGTCGCCTAGACCCGTACCTCTGTGTTCTGGAGCCTGCCGCCCCCGCACGGGAAAGGCTTAGCTCGGGACTTGCAGCACCGCCTCC

* T * -240 TCTTTAGCCAGGCCAGGCACGAGGATAGTGTGATCGGGCACAGCCAGGGTCGCTCTTCCAGGCTTTCTTGCGGGTTGCGGGAGGTACTAGTTGGAGACGC

-140 GCGCGCTCGCTCTCGCCGCTCTGTCCTGGGCCACTCCGTGATCCTAGGCTACCTCCAGAGCCAGTTTTCCCTGGCTGGCACAACTCTCCAGGGCGCTCCG

-40 GTCCGTTGCACAGCGCCCCAAGGGGGTATCCCAGTAAGTG ATG GAA CTG GCT ATG GTG AAC CTC AGT GAA GGG AAT GGG AGC GAC 1 M E L A M V N L s E G N G s D

46 CCA GAG CCG CCA GCC CCG GAG TCC AGG CCG CTC TTC GGC ATT GGC GTG GAG AAC TTC ATT ACG CTG GTA GTG TTT 16 p E p p A p E s R p L F G I G V E N F I T L V V F

121 GGC CTG ATT TTC GCG ATG GGC GTG CTG GGC AAC AGC CTG GTG ATC ACC GTG CTG GCG CGC AGC AAA CCA GGC AAG 41 G L I F A M G V L G N s L V I T V L A R s K p G K

196 CCG CGC AGC ACC ACC AAC CTG TTT ATC CTC AAT CTG AGC ATC GCA GAC CTG GCC TAC CTG CTC TTC TGC ATC CCT 66 p R s T T N L F I L N L s I A D L A y L L F C I p

FIG. 2. Nucleotide and deduced amino acid sequences of(a) mouse and (b) human genes encoding GALRl. Exon sequence is in uppercase, presumed intron sequence is in lowercase, and gt/ag residues flanking splice donor and acceptor sites are in bold lettering. Nucleotides are numbered, with + 1 corresponding to the first nucleotide of the methionine translation initiation codon. Transcription start sites for the mouse GALRl transcript defined by 5' -RACE analysis of mouse brain RNA are denoted by filled arrowheads at nucleotides -163 and -646 upstream of the translation initiation codon in the mouse genomic DNA, and the start of the human GALRl hypothalamic cDNA clone isolated in this work is shown by an unfilled arrowhead at nucleotide -772 upstream of the translation initiation codon in the human genomic DNA sequence. A Bl sequence element present in the 3' untranslated region of the mouse Galnr gene (nucleotides 1569-1439) is bordered by hash marks and a region comprising 12 units of a 6-nucleotide repeat sequence (nucleotides 1803-1874) is boxed. The polyadenylation signal in mouse and human genes is underlined. The deduced amino acid sequence is shown in one-letter code below the nucleotide sequence. Amino acids are numbered from the methionine translation initiation site and putative transmembrane domains are indicated by underlining. The nucleotide sequences have been deposited in the GenBank/EMBL Data Libraries under Accession Nos. U90655-57 (mouse) and U90658-60 (human).

GALNR shown in Fig. 1 and did not indicate the exis­ most variation. Of the differences in amino acid se­ tence of more than one gene encoding GALRl. quence, 9 are conservative differences. Alignment of the mouse and human GALRl coding In comparison, rat GALRl consists of only 346 amino sequences reveals a 93% amino acid identity (Fig. 3). acids with a 96% identity to the mouse sequence (Par­ The mouse sequence encodes a peptide one residue ker et al., 1995). This size difference arises from an shorter than the human receptor, with Pro170 in the extra residue in both the N-terminal extracellular do­ second extracellular loop of the human receptor having main and the C-terminal tail of mouse GALRl in addi­ no counterpart in the mouse. There are 26 other amino tion to the absence of a counterpart to Pro170 of the acid differences between the two homologs, with the human GALRl sequence (Fig. 3). Potential N-linked transmembrane regions displaying the highest degree glycosylation sites at Asn residues 7 and 12 are strictly of conservation and the C-terminal tail exhibiting the conserved between receptor sequences of all three spe- GALRl GALANIN RECEPTOR GENE (GALNR) STRUCTURE IN MOUSE AND HUMAN 501

271 TTT CAG GCC ACC GTG TAT GCA CTG CCA ACC TGG GTG CTG GGC GCC TTC ATC TGC AAG TTT ATA CAC TAC TTC TTC 91 F Q A T V Y A L P T W V L G A F I C K F I H Y F F

346 ACC GTG TCC ATG CTG GTG AGC ATC TTC ACC CTG GCC GCG ATG TCT GTG GAT CGC TAC GTG GCC ATT GTG CAC TCG 116 T V S M L V S I F T L A A M S V D R Y V A I V H S

421 CGG CGC TCC TCC TCC CTC AGG GTG TCC CGC AAC GCA CTG CTG GGC GTG GGC TTC ATC TGG GCG CTG TCC ATC GCC 141 R R S S S L R V S R N A L L G V G F I W A L S I A

496 ATG GCC TCG CCG GTG GCC TAC CAC CAG CGT CTT TTC CAT CGG GAC AGC AAC CAG ACC TTC TGC TGG GAG CAG TGG 166 M A S P V A Y H Q R L F H R D S N Q T F C W E Q W

571 CCC AAC AAG CTC CAC AAG AAG GCT TAC GTG GTG TGC ACT TTC GTC TTT GGG TAC CTT CTG CCC TTA CTG CTC ATC 191 P N K L H K K A Y V V C T F V F G Y L L P L L L I

646 TGC TTT TGC TAT GCC AAG gtgagtgaggagcgctggccaggctcctatgcagctcttagtgaccggtgacctgacctggagcttctggagcttc 216 C F C Y A K

atgtctccgcgacact------intron 1, 5 kb------

664 aacccgcttcatccggttatgttttgcag GTC CTT AAT CAT CTG CAT AAA AAG CTG AAA AAC ATG TCA AAA AAG TCT GAA 222 V L N H L H K K L K N M S K K S E

715 GCA TCC AAG AAA AAG gtaaattcacacacagatgcggttcctgcccattttcgagagcttagttgattgttgttttagattattttcacatccag 239 A S K K K

------intron 2, 8 kb------

ctggtgtgtctgaagactgttacagtgaactgcacatataataataagtaaatttttaaaaaagaaagttttttaaataaataaatgctacagaggtgta

730 ttttacagcgctgtattctcctgatataactggtgtattcctgcagctcctgtcccctctccaaccctcttccattactctctcttccag ACT GCA 244 T A

736 CAG ACC GTC CTG GTG GTC GTT GTA GTA TTT GGC ATA TCC TGG CTG CCC CAT CAT GTC GTC CAC CTC TGG GCT GAG 246 Q T V L V V V V V F G I s w L p H H V V H L w A E

811 TTT GGA GCC TTC CCA CTG ACG CCA GCT TCC TTC TTC TTC AGA ATC ACC GCC CAT TGC CTG GCA TAC AGC AAC TCC 271 F G A F p L T p A s F F F R I T A H C L A y s N s

FIG. 2-Continued cies, as is a predicted cAMP/cGMP-dependent phos­ boxes or putative transcription factor recognition sites phorylation site at Ser143 in mouse and human se­ were identified in the genomic DNA sequence up to 1 quences, which corresponds to Ser142 in the rat GALRl kb upstream of the translation initiation codon of the sequence. Galnr gene. Precise mapping of the transcription start site of the Cloning of the 5' End of GALRl cDNA human GALRl transcript in the HMCB cell line was 5'-RACE was carried out on mouse brain total RNA not undertaken. However, a partial cDNA clone iso­ to determine the extent of the 5' untranslated region lated from a human hypothalamic cDNA library, which of the mouse GALRl transcript. PCR amplification of encompassed exons 1 and 2 of GALRl coding sequence, the mouse GALRl first-strand cDNA gave rise to two contained 772 nucleotides of sequence upstream of the 5'-RACE products identical in sequence to the genomic translation initiation codon. This sequence was identi­ sequence upstream of the translation initiation codon cal to human GALRl genomic sequence upstream of (Fig. 2a). The major product extended 163 bp upstream the translation initiation codon, suggesting that the of the translation initiation codon, while the minor start of transcription of the human GALRl transcript product extended 646 bp upstream of the translation in hypothalamic neurons occurs at least 772 nucleo­ initiation codon. No consensus TATA and CCAAT tides upstream of the translation initiation codon. Ge- 502 JACOBY ET AL.

886 TCA GTG AAC CCC ATC ATA TAT GCC TTT CTC TCA GAA AAC TTC CGG AAG GCG TAC AAG CAA GTG TTC AAG TGT CAT 296 s V N p I I y A F L s E N F R K A y K Q V F K C H

* 961 GTT TGC GAT GAA TCT CCA CGC AGT GAA ACT AAG GAA AAC AAG AGC CGG ATG GAC ACC CCG CCA TCC ACC AAC TGC 321 V C D E s p R s E T K E N K s R M D T p p s T N C

* * * * * * 1036 ACC CAC GTG TGA AGGTTTGCGGGAGCCTCCCGACTTCCAGCTCCCATGTGTGTTAGAGAGAGGAGGGCGGAGCGAATTATCAAGTAACATGGCAGC 346 T H V ***

* * * * * 1132 TTATTCTCCACAGCAATTCCTATCGATCCAACTACATTCCACAGTGGTAAAAGGACGTTGATTGTTCAGGGAACTCGTGGGTCTACTGAAGATCATTTTC

1232 CAATTTCATTTTACTCTATAATTGTATATATGAGACAAAAGAAACTTCTGTATAGTTTCTAGCTCTCAAGGAATGAAAGTCCTACAGCACTCTGCAAATG

1332 TTTTGATGCATGCCCCAGCCTTCCCTCCCAGTCTGCCTCAGTATCCTCGGGCTTCCGCCACTCGTGCCTCATTGTTTTTTGTTTGTTTGTTTGTTTTTGG

#* * * * * * * * * 1432 TATTTTGAAATAGGGTTTCTCTGTGTAGCCCTGGCTGTCCTGAACTCACTCTGTAGACCAGGCTGGCCTAGAACTCAGAAATCTGCCTGCCTCTGCCTCC

* * #* * 1532 CAAGTGCTGGGATTAAAGGTGTGCACCACCACTGCCCGGCCCTGCCTCATTGTTTTAACTGCATGTGGGACAGTCTCTAGTGGGCCACATCCTTTTGTGG

1632 GTGGAGTCTCCTGTGAGCTCAGATCTGCTCTGTTCAGAAATACAGAATCAAACAGAAGGGGCTGCTGTAGTTGGCCAACTAATCTCTACTCGAATCACCC

1732 GGCGATGCTGGGGTGTTCCTCACTATTCACCCCAATGTTCCAGGTTCCCGAATGAAATACACACAGACAGf€GGAGAGGGAGAAGGAGAGGGAAAGGGAG

* 1832 AGGGAGAGGGAGAGGGAGAGGGAGAGGGAGAGGGAGAGGGAijJ:;ccATATTTTTTAATGTTTTTTAACAGCTCAACGGCTGGGCTACTTCTTAGCCTCCG

1932 TGCGGCTAACACACTCTTTCAATATTCCTGACTCACCACTTACTAAAATCCTGGGCACCCCAGACCCAGACCTGTAGCCCTTCTGGGCCATGCCCCCGAC

2032 TCTTACACAGTGGTTCTGTGCTCTTCTGTACCTCTCACGTCTGGTTTTTACTACTTTCCTGGCATGGCAGCCCCTCCCCTGTCCCTTGCCCAAGAATCTT

2132 AAAAGTACTGCCTTTGTCTCCGTACCCAGCCATTTGCTGCTGGCAACTTTATTTACCAATAAAAACCAACTTGGGGCAAGGA

FIG. 2-----Continued nomic DNA sequence up to 1.1 kb upstream of the GALNR gene. A 130-bp Bl-like repeat sequence translation initiation codon contained no consensus (Krayev et al., 1980), spanning nucleotides 1569-1439 TATA, CCAAT, or putative transcription factor recog­ on the noncoding strand of the mouse Galnr genomic nition sequence motifs. DNA sequence, was identified (Fig. 2a). A region com­ An 84% identity exists between mouse and rat se­ prising 12 elements of a 6-nucleotide repeat (5' -GGG­ quences in the 243 nucleotides immediately upstream AGA-3 ') was also observed, corresponding to nucleo­ of the translation initiation codon (Parker et al., 1995), tides 1803-1874 in the mouse Galnr genomic DNA se­ while the corresponding region of the human 5' un­ quence (Fig. 2a). This hexamer repeat has previously translated sequence is dramatically different from the been identified in at least two other mouse genes, the rodent sequences (Fig. 2). germ-line IgM gene (Richards et al., 1983) and the do­ pamine transporter gene (unpublished; GenBank Ac­ Cloning of the 3' End of GALRl cDNA cession No. U12313), but its significance is unknown. Sequencing of 1.1 kb of the 3' untranslated region A putative polyadenylation signal (5'-AATAAA-3') of the cloned mouse Galnr gene revealed a number of was identified 1140 nt downstream of the termination features. The first 290 nucleotides of 3' untranslated codon TGA. region displays an 83% identity with the equivalent 3'-RACE carried out on HMCB poly(A)+ mRNA and region ofrat GALRl cDNA (Parker et al., 1995). A 100- initiated using a primer located within the coding re­ nucleotide stretch of sequence within this region also gion of the GALRl transcript resulted in the amplifica­ shares 73% identity with the 3 1 untranslated region of tion of a fragment of 1.4 kb, which comprised 111 nucle­ the human GALRl cDNA, beginning 143 nucleotides otides of GALRl coding sequence and 1234 nucleotides downstream of the translation termination codon of the of 3' untranslated sequence followed by a poly(A) tail GALRl GALANIN RECEPTOR GENE (GALNR) STRUCTURE IN MOUSE AND HUMAN 503

b -1098 gggtgagggtgggattagcggccaattttacaaaactccagggacgtcgccagcctcgatttcctggggttattcctgggagagaggcgttctcacggac

-998 agccaccctggggaggaggaggaggaaaggcactaatggatgaggaggcccgcgcacccctccgcctcccaccccggcgcgccctggaacgccctggagc

-898 gcgcccggcttccctcgcccgcctggcccgcggcatccggcagccccgccttcagcccgccgggcagggccgcactccgcggaggcgagcgcgctccggt "v* -798 tccagccgggaggtgggcggcgacccATCCCGCTAGMTCCGTCCAGTCTCTGCTCGCGCACCGTGACTTCTAAGGGGCGCGGATTTCAGCCGAGCTGTT

-698 TTCGCCTCTCAGTTGCAGCAGAGAAGCCCCTGGCACCCGACTCTATCCACCACCAGGAAGCCTCCCMAAGAGCTCTCGCCCTGTGGACGACTCGGAATC

-598 CCTGGAMAGCCGGGAGGGAGTCGGAGGCGCCAGCCCACTGGGGAGGTGGCGCTGGGCGCGCGGGATGCGCGGGGAGCCTTCTCTGCAGGAGCCGCACAG

-498 TGCACTGCTGCGCGCTGGGCAGTGCGGGGAAGCGCCGCGGGAAGGAGCGGCTCCGAGCMCAGGTGCAGCACGCAGCCGCTCCGGGAGCCAGGGAMACC

-398 GCCGGCGAAGATCTGGAGCGGTMGGCGGAGAGAAGGGTCTTTCCACCTGCGCGGCTGCAGCCGGCGGATCCCTCTTCCCAGGCTCCGTGGTCGCGCAGC

-298 GGGCGGAGGCGCCCGGGCAGGGGACCCCAGTGCTCTCGAGATCACCGTCCCTTCCCGAGAAGGTCCAGCTCCGGGCTCCCGMCCCACCCTCTCTCAGAA

-198 GGTCGCGGCGCAMGACGGTGCCACCAGGCACGGCCACCGGATCCCCGCTCCCGCTGGCTCGCGCCTCGGGGGMGCTCAGACTCCTMACTCGCACTCT

-98 CCGTGCTTTGCGCCGGGACCCCTGGCCACCCCCGGCGCCTGCTATCCCGCCCTCCCTCCCCGCGCGCCCCGCCGCTCGCCGGGACAGCCCCGCGGGCC

* * * 1 ATG GAG CTG GCG GTC GGG MC CTC AGC GAG GGC MC GCG AGC TGG CCG GAG CCC CCC GCC CCG GAG CCC GGG CCG 1 M E L A V G N L s E G N A s w p E p p A p E p G p

76 CTG TTC GGC ATC GGC GTG GAG MC TTC GTC ACG CTG GTG GTG TTC GGC CTG ATC TTC GCG CTG GGC GTG CTG GGC 26 L F G I G V E N F V T L V V F G L I F A L G V L G

* 151 MC AGC CTA GTG ATC ACC GTG CTG GCG CGC AGC MG CCG GGC MG CCG CGG AGC ACC ACC AAC CTG TTC ATC CTC 51 N s L V I T V L A R s K p G K p R s T T N L F I L

* * 22 6 MC CTG AGC ATC GCC GAC CTG GCC TAC CTG CTC TTC TGC ATC CCC TTC CAG GCC ACC GTG TAC GCG CTG CCC ACC 76 N L s I A D L A y L L F C I p F Q A T V y A L p T

301 TGG GTG CTG GGC GCC TTC ATC TGC AAG TTC ATC CAC TAC TTC TTC ACC GTG TCC ATG CTG GTG AGC ATC TTC ACC 101 w V L G A F I C K F I H y F F T V s M L V s I F T

FIG. 2-Continued of 32 residues. The sequence of the 3 '-RACE product lated region of 1.23 kb, the 5' untranslated region of corresponded to genomic DNA sequence up to the site the major GALRl transcript in HMCB cells may be of polyadenylation, consistent with the absence of alter­ estimated to be approximately 0.4-0.9 kb in length. native splicing in the 3' untranslated region of the GALNR gene. Northern blot analysis of poly(A)+ Chromosomal Localization of the Galnr Gene mRNA isolated from HMCB cells allowed the identifi­ A total of260 silver grains were scored to an idiogram cation of two human GALRl transcripts, comprising a (Nesbitt and Francke, 1973) of all mouse chromosomes predominant transcript of 3.4 kb and a minor tran­ in approximately 200 cells. Eighty-eight of the grains script of 1.8 kb (data not shown). This is consistent (34%) were scored over Chromosome 18, with a tallest with previous reports of GALRl transcripts in HMCB column, of 4 7 grains, lying over the distal subband, cells (2. 7 and 1.3 kb; Habert-Ortoli et al., 1994), in hu­ 18E4 (data not illustrated). Small peaks of grains, to man small intestinal tissue (3.6 and 1.4 kb; Lorimer a maximum column height of 3 grains, occurred on and Benya, 1996), and in a range of human peripheral Chromosomes 1, 16, 19, and Y, but none of these were tissues including heart (3.2 kb; Sullivan et al., 1997). regarded as indicative of the occurrence of a second Given a coding sequence of 1.05 kb and a 3' untrans- gene or pseudogene. 504 JACOBY ET AL.

376 CTG GCC GCG ATG TCC GTG GAC CGC TAC GTG GCC ATC GTG CAC TCG CGG CGC TCC TCC TCC CTC AGG GTG TCC CGC 126 L A A M S V D R Y V A I V H S R R S S S L R V S R

451 AAC GCG CTG CTG GGC GTG GGC TGC ATC TGG GCG CTG TCC ATT GCC ATG GCC TCG CCC GTG GCC TAC CAC CAG GGC 151 N A L L G V G C I W A L S I A M A S P V A Y H Q G

526 CTC TTC CAC CCG CGC GCC AGC AAC CAG ACC TTC TGC TGG GAG CAG TGG CCC GAC CCT CGC CAC AAG AAG GCC TAC 176 L F H P R A S N Q T F C W E Q W P D P R H K K A Y

601 GTG GTG TGC ACC TTC GTC TTC GGC TAC CTG CTG CCG CTC CTG CTC ATC TGC TTC TGC TAT GCC AAG gtgcacgccggt 201 V V C T F V F G Y L L P L L L I C F C Y A K

cgcggggccgagacgcgcga------intron 1, 5 kb------

667 tcag GTC CTT AAT CAC TTG CAT AAA AAG TTG AAG AAC ATG TCA AAG AAG TCT GAA GCA TCC AAG AAA AAG gtaatga 223 V L N H L H K K L K N M S K K S E A S K K K

tcacaaatatatatatatatgttacttttca------intron 2, 11 kb------­

ctcatagaagtgactatttggagcaaagtccagcgctcctccgagatgcattgctggtgatacagctgcggtgggtgagatgcttgaacagttttgctag

tttaaaagttacagtctctaactttgtacttttctagttccatgacttgaagcatacttcctataatgcatttcacatatatcacaaaattattactatg

gaaggtaatgacaggtcttggaaaagttatgaacaaaattattatggggagtgaaggttaacacttaaagcagatgtagtatccatttttgatctcatga

aaatatacataaaattagcttaggatgtattttaaaatgctggattccttttgtgttgtaatcaatgaatttccttccttcttcttctcccttaccgcct

733 cctcctcctcctcctcctcctcctcttcttcttttctag ACT GCA CAG ACA GTT CTG GTG GTG GTT GTG GTG TTT GGA ATC TCC 245 T A Q T V L V V V V V F G I S

778 TGG CTG CCG CAC CAC ATC ATC CAT CTC TGG GCT GAG TTT GGA GTT TTC CCG CTG ACG CCG GCT TCC TTC CTC TTC 260 W L P H H I I H L W A E F G V F P L T P A S F L F

853 AGA ATC ACC GCC CAC TGC CTG GCG TAC AGC AAT TCC TCC GTG AAT CCT ATC ATT TAT GCA TTT CTC TCT GAA AAT 285 R I T A H C L A y s N s s V N p I I y A F L s E N

928 TTC AGG AAG GCC TAT AAA CAA GTG TTC AAG TGT CAC ATT CGC AAA GAT TCA CAC CTG AGT GAT ACT AAA GAA AAT 310 F R K A Y K Q V F K C H I R K D S H L S D T K E N

1003 AAA AGT CGA ATA GAC ACC CCA CCA TCA ACC AAT TGT ACT CAT GTG TGA TAAAAGATAGAGTATCCTTATGGTTGAGTTTCCATA 335 K S R I D T P P S T N C T H V

1087 TAAGTGGACCAGACACAGAAACAAACAGAATGAGCTAGTAAGCGATGCTGCAACTTGTTATCTTAACAAGAATTCAAGTCGTTTTAATTAAATCCCACGT

1187 GTGTTAAAAAGTACTTTGATCCATTTAGGAAATTCCTAGGTCTAGTGAGAATTATTTTTCAATTTTATTTTAGTTCTAAATTATGTTTCAGAAACAAAAG

FIG. 2-Continued

Having established Chromosome 18 to be the major on mouse Chromosome 18, with a strong probability of target for the Galnr probe, we confirmed the result by extremely distal point localization to subband 18E4 scoring a total of 108 grains over Chromosome 18 (Fig. (Fig. 4b). 4a) from approximately 90 high-quality cells, using a more detailed idiogram of Chromosome 18 (Evans, DISCUSSION 1989); 69 (64%) of the grains formed the three tallest columns, which were over, or adjacent to, the subband The high degree of sequence homology of the coding 18E4 (Fig. 4b). For all scoring, the two mouse strains region of the cloned mouse Galnr gene to both the hu­ C57BL and BALB/c showed similar patterns of grain man and the rat GALRl sequences confirms the iso­ distribution. lated clone as the mouse homo log of the GALRl galanin The results from in situ hybridization show that receptor gene. Isolation and characterization of geno­ Galnr is almost certainly located at subbands 18E3-4 mic clones spanning mouse and human GALRl reveal GALRl GALANIN RECEPTOR GENE (GALNR) STRUCTURE IN MOUSE AND HUMAN 505

1287 ACAATGCTGTACAGTTTTATTCCTCTTCAGACATGAAAGGGAACATATATATTCCATATATATGTTCAACTCTTCATAGATTGTGAACTGGCCCATCAAT

1387 ATGGTCAGGAATATTTGCAGTCTACATTTTAAAGCCAATTTATTTAGAAAAAAAATTTGAGCTTTAATTCTTTAATTTTAAGAGAAGTAATATTGTGAAC

1487 TATGTATTTTAAAATATGATCATGGACACACAATGATGAATTTTTTGGCCATTTACATAGACATATCTATTAAGTGGAAAGAAGGCTTTCTGAAGTCTGT

1587 TTGCACAGGTGGCATTTGCTTCCAATTGTAGCTAGCGCACAGAGCTTTGGAAGCCTGTCATTATGAGATACAGTCGGTTTACCTCAGGAGTCAATTCAGT

1687 GTTGTACTGGTGACCTGGGATGCAGTAGTAGGCACTGTTGATTCAAATTTATCCTGTGAAACTGGCTTTATAGAGTTAACAAAACAGAGTCAGAGACCAC

1787 TGTCTTAACAGTGGAAGATGCAAATAAGTTTTTGAGAATAAAACTGGATTTTGAAATTTTACATTAGTACTTGACAAAAGTTTTCATTTTGCCTTGAATG

1887 GAACCTACTAAAAAGAGAGATGAAAAAAAATCAGCGAGGTTGATGTAGATAATAATTTCTATGGGACCAAAGACTAGACAGAATTCAGTAAGTCACATGA

1987 AGTAATGGTCATGCCTGTACATAAAGCATATTTCATGTTTGATTTAGATGACATTCAAAAAAAATCATGGGACTGAATATACCTGGGGTATCCTATCTTG

2087 TACAAATGCATGCTTTTTCATTAAATTTGTAATGATGTTTAATGAACATTTCCACCAAACATTATTTCCTCTAAAAATGTTAATTTGGGGTTAAAACCAT

2187 CACCATTTGAATTTCAAATGTAGTTTTCATGACAATTTTATATTGATGTGTGTTTACAATGAGAAAATGGCATGAAAATATTAAATTGTCTTGTATCG

FIG. 2-Continued a structural organization conserved between the two the isolation of several human and rat GALRl cDNA species that is different from the structural organiza­ clones (Habert-Ortoli et al., 1994; Lorimer and Benya, tion of the gene encoding GALR2 in the rat and is 1996; Parker et al., 1995; Burgevin et al., 1995), includ­ unique among genes encoding G-protein-coupled recep­ ing the partial GALRl hypothalamic cDNA clone iso­ tors. lated in this work. The coding sequences of GALRl are divided into Alignment of the coding sequences reveals a close three exons, with exon 1 encoding the N-terminal end identity between the deduced amino acid sequences of of the receptor and the first five transmembrane do­ mouse and human GALRl. However, the 5' and 3' mains. Exon 2 encodes the third intracellular loop, and untranslated regions of the cDNA display significant exon 3 encodes the remainder of GALRl, from trans­ divergence. This sequence divergence may reflect spe­ membrane domain 6 to the C-terminus of the receptor cies-specific differences in GALRl gene expression. protein. This is in contrast to the structural organiza­ While no definitive differences in the expression of tion of the gene encoding GALR2, which comprises two GALRl mRNA between rodents and humans have been exons separated by an intron of 490 bp (Howard et al., reported to date (Sullivan et al., 1997), cell-type-specific 1997). Exon 1 encodes the N-terminal end of GALR2 differences in expression of the peptide galanin are and the first three transmembrane domains, and exon known to occur between species, for example, in cells 2 encodes the remainder of the receptor. Coding of the of the anterior pituitary gland (Vrontakis et al., 1991). third intracellular loop of GALRl on a separate exon With the availability of cloned rodent and human is noteworthy. It has been demonstrated for a large GALRl sequences, studies addressing the anatomical number of G-protein-coupled receptors that this do­ distribution of this receptor subtype in different species main is involved in the interaction of the receptor with and in experimental paradigms known to affect expres­ G proteins. In some G-protein-coupled receptor genes, sion of the peptide galanin can now be undertaken. this domain of the receptor is the site of alternative No consensus TATA or CCAAT boxes were identified splicing of the transcript to generate receptor subtype closely preceding the 5' untranslated region of the diversity, and in the case of dopamine D2 and pituitary mouse Galnr gene. Absence of these motifs from the adenylyl cyclase activating peptide receptors, splice promoter region has been reported for many other G­ variants differing in the third intracellular loop exhibit protein-coupled receptor genes, including the genes en­ differential efficiency of coupling to intracellular sig­ coding the rat bradykinin B2, human neuropeptide Y naling pathways (Hayes et al., 1992; Spengler et al., Yl, dopamine D2, and adenosine Al receptor subtypes 1993). Encoding of the third intracellular loop of (Pesquero et al., 1994; Ball et al., 1995; Valdenaire et GALRl on a discrete exon raises the possibility of alter­ al., 1994; Ren and Stiles, 1994). The existence of alter­ native splicing of exon 2 within the GALRl transcript. natively spliced exons in the 5' untranslated region has However, no evidence for this has yet emerged from also been observed in transcripts encoding a number mGALR1 -M-E_L_A-.MVNLSEGNGSDPEPPAPES=R-P~L-F~G-I_G_V--=E-N~*. • * ** ~ rGALR1 MELAPVNLSEGNGSDPEPPA[JEPRPLFGIGVEN ~ hGALR1 MELAVGNLSEGNAS PEPPAPEPGPLFGIGVEN ~ TM1 * mGALR1 FITLVVFGLIFAMGVLGNSLVITVLARSKPGKP ~ rGALR1 FlTLVVFGLIFAMGVLGNSLVITVLARSKPGKP ~ hGALR1 l!:.fvlT L V VF G L I F A LG V L G N S L V I T V L A R S K P G K P 66 TM2 mGALR1 RS T TN LFI LNLSIADLAYLLFClPFQATVYA L p 99 rGALR1 RS T TN LF I LNLSIADLAYLLFC I PFQATVYA L p 98 hGALR1 RS T TN LFJLNLSI ADLAYLLFC JPFQATVYA L p 99 TM3 mGALR1 T WV L G A F I C K F I HY F ·F TVS M L V S I F T L A A MS VD 132 rGALR1 T WV L G A F I C K F I H Y r FT V S M L V S I F T L A A MS VD 131 hGALR1 TWVLGAFICKFIHYFFTVSMLVSfFTLAAMSVD 1~ ..... * TM4 mGALR1 R Y V A I V H S R R S S S L R V S RN A L L G VG F I WA L S I A 165 rGALR1 R Y V A I V H S R R S S S L R V S RN A L L G VG F I WA L S I A 164 hGALR1 R Y V A I V H S R R S S S L R V S RN A L L G VG C I WA L S I A 165

mGALR1 F.==M=A:;::::;::S:::;;P;=V:::;:.1-A-Y...,..,..,.H--=O,....,R:,,;....,...L--=F,,..,H,..,.,* * - RDS NOT F CW E OW P *N **K~L_H_K_K_, 197 rGALR1 l\.-1A $ PV A Y[y]O R L F H - FfD SNOT F CW E[ffiw P NOL H K K 196 hGALR1 MA 8 PVA Y HOG L F H p RAS NOT F CW E ow PD PR H K K 198 TM5 mGALR1 A Y V V C T F V FG Y L L P L L L IC F C Y A K VLNHLHKKL 230 rGALR1 A YVVCTFVFGYLLPLLL ICFCYAK VLNHLHKKL 229 hGALR1 A YVVCTFVFGYLLPLLL I CFC YAK VLNHLHKKL 231 TM6 mGALR1 KN MS K KS E ASK K KT AQJVL VVVV V FG I SWL PHH 263 rGALR1 K N MS K KS E A S K K K TAOT V L V V VV VF G IS W L P H H 262 hGALR1 K N MS K KS E A S K K KT AO T V L VVV V VF G I SW L P H H 264 * TM7 mGALR1 V V H L W A E F G A F P L T P AS F F F R I T AH C L A Y S N S S 296 rGALR1 V I H L WA E F G A F P L T P AS F FF R I TA H C L A Y S N S S 295 hGALR1 I I H L W A E F G V F P L T P AS F L F R T T A H C L A Y S N S S 297

mGALR1 rGALR1 hGALR1

mGALR1 =r-K--=E-N-,--,K---=s---="R *M~D_T_P_P_S_T_N_C_T_H_V_, 348 rGALR1 AK E[JKIH]R I DTP PST N C TH V 346 hGALR1 T K E N K S R I D T P P S T N C T H V 349

FIG. 3. Alignment of mouse (m}, rat (r), and human (h) GALRl amino acid sequences. Identical residues are boxed and sites ofnonconservative amino acid substitution are indicated with an asterisk. Putative transmembrane domains are boxed and shaded and are numbered 1-7. ( +} Potential sites for N-linked glycosylation, (&) potential site for phosphorylation by cAMP/cGMP-dependent protein kinase (protein kinase A).

506 GALRl GALANIN RECEPTOR GENE (GALNR ) STRUCTURE IN MOUSE AND HUMAN 507

a b with Mbp, Galnr should map by linkage between 55 40 cM and the present limit for Chromosome 18 of 60 cM. The human GALNR gene has been physically localized to a similar position on human chromosome 18, in the

30 vicinity of MBP and the gene encoding peptidase A (PEPA) (Nicholl et al., 1995). Peptidase A is called pep­ tidase-1 (Pep 1 ) in the mouse and has been localized to - 20 the whole of mouse Chromosome 18 (Lalley and McKu­ sick, 1985). The chromosomal localization of GALRl is therefore homoeologous in human and mouse, in the

10 same syntenic group as NFATC and MBP and, per­ haps, PEPA. ' 5 In summary, this work describes the structural char­ I acterization of the mouse and human homologs of the 18 gene encoding GALRl, the first known galanin receptor \ gene. The cloning of these genes should allow elucida­ A tion of the regulatory characteristics of the promoter in the two species. In addition, cloning of the mouse E3-4 - Galnr gene will further facilitate transgenic ap­ FIG. 4. Silver grains over mouse Chromosome 18 probed in situ proaches to analysis of the physiological role of galanin with tritiated cDNA derived from the Ga.lnr gene. (a ) Six examples in mammals. of Chromosome 18 showing grains (indicated by arrows). The top two chromosomes are from the same cell ; the bottom one is from a BALB/c mouse and the rest are from C57BL mice. (b ) Plot of grains ACKNOWLEDGMENTS over approximately 100 Chromosomes 18 showing highly probable localization to subbands 18E3- 4 (marked with a bar). The arrow­ We thank Melanie Drummond and Lawrence G. Iben for technical head at the base of the tallest column indicates the probable point assista nce. This work was supported by the National Health and localization of Ga.lnr to subband 18E4. Medical Research Council (NHMRC) of Australia, the R. T. Hall Trust, the J . S. Davies Bequest (C.D.B. and G.C.W.), and the Chil­ dren's Cancer Foundation Salzburg (Austria; B.K.). A.S.J. is the re­ of G-protein-coupled receptors, including the porcine cipient of a Dora Lush (Biomedical) Postgraduate Scholarship from muscarinic acetylcholine, rat endothelin ETA , and hu­ the NHMRC of Australia and G.C.W. ofa pa rt-time Senior Research man neuropeptide Y Yl receptors (Peralta et al., 1987; Fellowship from The Queen Elizabeth Hospita l. Cheng et al., 1993; Ball et al., 1995). While the exis­ tence of alternatively spliced exons in the 5' untrans­ Note added in proof A recently published sequence for the mouse lated region of the gene encoding GALRl has not been Ga.lnr gene [Wang et a.l. (1997) FEES Lett. 411: 225-230) differs investigated rigorously to date, the identity in sequence from the sequence described above in the 3' untranslated region. As the published sequence retains homology to rat GALRl beyond the of 5'-RACE products of the mouse brain GALRl tran­ point at which the sequence described in this work diverges (position script with Galnr genomic DNA sequence for 646 nucle­ 1440 in Fig. 2a), it is possible that our clone may contain a rearrange­ otides upstream of the translation initiation codon sug­ ment or a substrain-specific difference in this region of Ga.lnr. gests the absence of alternatively spliced exons in this region of the mouse Galnr gene. Similarly, identity of REFERENCES sequence of the 5' untranslated region of the human hypothalamic cDNA clone reported in this work with Ball, H. J ., Shine, J ., and Herzog, H. (1995). Multiple promoters regu­ GALNR genomic DNA sequence for 772 nucleotides up­ late tissue-specific expression of the human NPY-Yl receptor gene. stream of the translation initiation codon is consistent J. Biol. Chem. 270: 27272- 27276. with the absence of alternatively spliced exons within Bartfai, T., Hokfelt, T., a nd Langel, D. (1993). Galanin- A neuroen­ this region of the human GALNR gene. docrine peptide. Crit. Rev. Neurobiol. 7: 229- 274. The physical localization of the Galnr gene to mouse Burgevin, M.-C., Loquet, I., Quarteronet, D. , and Habert-Ortoli, E. (1995). Clonjng, pharmacological characterization, and anatomical Chromosome 18E4 (Fig. 4b) is similar to that of the distribution of a rat cDNA encoding for a galanin receptor. J . Mol. gene encoding nuclear factor of activated T-cells, cyto­ Neurosci. 6: 33- 41. plasmic 1 (Nfatcl ), but Nfatcl has not been mapped Carey, D. G., Iismaa, T. P., Ho, K. Y., Rajkovic, I. A. , Kelly, J., genetically (Johnson and Davisson, 1996); its human Kraegen, E. W., Ferguson, J ., Inglis, A. S., Shine, J ., and Chisholm, homolog, NFATC, maps to 18q23. The gene nearest D. J . (1993). Potent effects of human galanin in man: Growth hor­ to Galnr that has been mapped both genetically and mone secretion and vagal blockade. J. Clin. Endocrinol. Meta.b. 77: 90-93. physically is the gene encoding myelin basic protein Chartrel, N., Wang, Y. , Fournier, A., Vaudry, H., and Conlon, J . M. (Mbp ), which when mutated gives rise to the shiverer (1995). Frog vasoactive intestinal polypeptide and galanin: Pri­ phenotype. Mbp has been mapped to 55 cM on Chromo­ mary structures and effects on pituitary adenylate cyclase. Endo­ some 18 and localized to 18E2- 3 (Johnson and Davis­ crinology 136: 3079-3086. son, 1996) and, in humans, to 18q23. By comparison Cheng, H. F., Su, Y. M., Yeh, J . R., and Chang, K. J . (1993). Alterna- 508 JACOBY ET AL.

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CD-ROM containing video in .mov format of three episodes of seizure behaviour exhibited by GALRl knockout mice. Refer to Section 4.3 (vi) for details.

Requires Quicktime Player or equivalent. Running time 4 min 48 sec. On-screen arrows indicate affected mice.

SEGMENT 1: INDUCED SEIZURE 1:40

SEGMENT 2: SEVERE SEIZURE 1:31

SEGMENT 3: SPONTANEOUS SEIZURE 1:29