CHARACTBRISATION OF THE PYRUVATE t\. &'qfl CARBOXYLASE GENE AND STUDIES ON REGULATION OF ITS EXPRESSION IN RAT by Sarawut Jitrapakdee M.Sc. (Biochemistry) A thesis submitted to the University of Adelaide for the degree of Doctor of Philosophy t*tt t Department of BiochemistrY University of Adelaide Adelaide, South Australia March, 1999 TABLE OF CONTENTS 1 SUMMARY iv STATEMENT ACKNOWLEDGEMENTS vi ABBREVIATIONS púnr,Ic¡,TIoNS ARISING FROM THESIS vii CHAPTER 1 INTRODUCTION AND LITERATURE REVIEW 1.1 GLUCONEOGENESIS AND GLYCOLYSIS t.z cõononrATE REGULATIoN oF GLYCoLYTIC AND GLUCONEOGENIC ENZYME GENES IN LIVER 1 1.3 TRANSCRIPTIONAL REGULATION OF GLUCONEOGENIC ENZYME GENE EXPRESSION 5 1.3.1PEPCK 5 1 1 .3 .2 Fructose- 1,6-bisphosphatase 1 I . 3 . 3 Glucose-6-PhosPhatase t.4 PYUVATE CARBOXYLASE (PC) 8 BIOLOGICAL ROLES OF PC 8 1.5 9 1 .5.1 Gluconeogenesis 9 L 5.2 Lipogenesis 10 1 .5.3 Räte"of PC in insulin signalling in pancreatic islets 11 I .5.4 Role of PC in astrocYtes 12 SYNTHESIS AND DEGRADATION 1.6 t4 1.1 BIOTIN CARBOXYLASE FAMILY 1.8 REACTION MECHANISM 15 T,g STRUCTURE OF PYRUVATE CARBOXYLASE 18 1.9.1 Primary and domain structure 18 t9 I . 9. 2 Three -dimensional structure 1.10 PHYSIOLOGICAL STATES THAT ALTER PC EXPRESSION 2T 21 1.10.1 Nutrition and xenobiotics 22 1.10.2 Diabetes 23 1. I 0. 3 Hormonal alterations 25 I . 1 0.4 P ostnatal gluc oneo genesis 1.10.5 Genetic obesity 25 26 1.1 1 PC DEFICIENCY 28 1.1 2 GENE STRUCTURE OF PC 1.1 3 TRANSCRIPTIONAL REGULATION OF YEAST PCl AND PC2 GENES 29 1.14 ALTERNATIVE SPLICING IN THE CONTROL OF GENE EXPRESSION 30 1.15 PROMOTER SWITCHING IN BIOTIN CARBOXYLASE GENE 31 1.16 PROJECT RATIONALE 32 CHAPTER 2 MATERIALS AND GENERAL MOLECULAR BIOLOGY METHODS 34 2.1 CFIEMICALS 2.2 RADIOCHEMICALS 34 2.3 OLIGONUCLEOTIDES 34 2.4 MOLECULAR BIOLOGY KITS 35 35 2.5 ENZYI\4ES AND PROTEIN MARKERS 36 2.6 BACTERIAL STRAINS 36 2.7 BACTERIAL GROV/TH MEDIA 36 2.8 MAMMALIAN CELL LINES 31 2.9 CELL CULTURE MEDIA 31 GENOMIC LIBRARIES 2.t0 38 2.lr PLASMIDS 2.12 DNA AND RNA TECHNIQUES 38 DNA restriction enzyme 2 . 12. I Small scale preparation of plasmid for digestion, sequencing and in vitro transcription 38 2.12.2 tire, scale preparolion of plasmid DNA by phenol/chloroþrm/PEG 39 p r e c ip r t ran sfe ction itation "o¡fo s gel G ene cle an kit 40 2. 1 2. 3 Þ urffi catio" ovl, iragm zls from agaro e using 2.12.4 Ligation 40 40 2. 12.5 Bacterial transþrmation 2.12.6 Isolation of high molecularweight DNAfromtissues 4l 2.12.7 Labelling of DNAwith"P 41 2.12.7.1 Endfilling of DNA markers 4l 2.12.7.2 Labelling S'-end of oligo by kinasing 42 42 2. I 2.7. 3 Random Priming 42 2. I 2.8 Southern blot hybridisation 2.12.9 DNA sequencing 43 2.12.9.1 .fmol" DNA sequencing 43 2.12.9.2 DYe Primer sequencing 44 2.12.10 Isolation of RNA 44 2.12.10.1 Isoiatíon of RNA using guanidine method 44 2.12.10.2 Isolation of nLl, using-RNAqueous" kit 45 2.12.10.3 Quantitation of DNA and RNA by spectrophotometry 46 CHAPTER 3 ISOLATION OF FULL LENGTH CDNA AND DOMAIN STRUCTURE OF THE RAT PC 3.1 INTRODUCTION 41 3.2 SPECIFIC METHODS 49 3.2.1RACE-PCR 49 3.2. 1. I Reverse transcriPtion 49 3.2.1.2 cDNA purification andAnchor ligation 49 3.2. 1. 3 P CR amplification 50 3.2.2 Sequence analyses by computer 50 3.3 RESULTS 51 51 3 .3. I Isolation of cDNA by RACE-PCR 3.3.2 cDNA sequence and inferued amino acid sequence of rat PC 52 3.4 DISCUSSION 55 3.4.I Domain structure of rat PC 55 3.4.1.1 Biotin carboxylase (BC) domain 55 3.4. 1.2 Transcarboxylation domain 58 3.4. 1.3 Biotinyl domain 60 CHAPTER 4 IDENTIFICATION AND CHARACTERISATION OF MULTIPLE TRANSCRIPTS OF RAT AND HUMAN PC GENES 4.lINTRODUCTION 63 4,2 SPECIFIC METHODS 65 4.2.I Northern blot analysis 65 4. 2 . 2 Multiplex reverse trans criptase p olymeras e chain reaction ( RT- P CR) 65 4.3 RESULTS AND DISCUSSION 61 4.3.1 Multiple rat tissue Northern blot analysis 61 4.3.2ldenffication of multiple PC ruRNA tsoforms in dffirent rat tissues by RACE-PCR 61 4.3 .3 Cloning and sequencing of RACE products 68 4.3.4 Distríbution of rat PC %RNA isoþrms in various tissues 69 4.3.5 Muttiple rat PC nRNA isoþrms are transcribed 11 from a single coPY Sene 4.3.6ldentificatlon of multiple PC %RNA isoþrms in human liver by RACE-PCR 12 CHAPTER 5 STRUCTURAL ORGANISATION' ALTERNATE SPLICING AND CHROMOSOMAL LOCALISATION OF THE RAT PC GENE 5.l INTRODUCTION 14 5.2 SPECIFIC METHODS 71 5.2 .I Screening of lambda genomic libraries 1l 5.2 .2 Smatt scãeþreparation of lambdabacteriophage DNA 7l 5.2 .3 Screening of cosmid genomic library 18 79 5.2 .4 Long distance PCR (LD-PCR) 5.3 RESULTS 80 5.3. I Organisation of coding exons 80 5.3.2 S'-Úntranslated region exons and alternative splicing 83 5.3.3 Chromosomal localisation of the rat PC gene 88 5.4 DISCUSSION 89 CHAPTER 6 PARTIAL CHARACTERISATION OF ALTERNATE PROMOTERS OF THE RAT PC GENE 6.1 INTRODUCTION 94 6.2 SPECIFIC METHODS 94 6.2. I Transient transfection of reporter gene 94 95 6.2.2 Luciftrase and þgalactosidase assays 6.3 RESULTS 91 6.3.1 91 6.3.2 sion Plasmids 97 6.3.3 ene fusion Plasmids 98 6.3.4 the distal Promoter 99 6.3 .5 Construction of -reporter gene fusion plasmids 100 plasmids 100 6.3.6 Expression of ih moter-reporter .gene fusion 6.3 .7 Eipression of th oter and the distal promoter in dffirent cell lines 101 6.3.8Regulation of PC promoters by insulin 102 6.4 DISCUSSION 104 CHAPTER 7 REGULATION OF RAT PC EXPRESSION /N VIVO 7.1 INTRODUCTION 110 7.2 SPECIFIC METHODS 111 7.2.1Animals 111 7.2.2 Cell culture 111 7 . 2. 3 Ribonuclease Protection Assay t12 7.2.4 Reverse transcriptase polymerase chain reaction (RT-PCR) 113 7.2.5 Preparation of tissue homogenate and PC assay 113 7. 2. 6 SDS-PAGE and Western analysis 113 7.2.7 In vitro translation 114 7.2 .8 Isolation of polysome bound RNAs 115 7.3 RESULTS il6 7.3 . I Establishment of linearity of the RNase protection assay and PC mRNAs and PC protein 116 Western analysis for detecting - 7.3.2 Developmental regulation of PC expression in postnatal rat liver 116 7.3.3 Overexpression of PC in genetically obese rats 118 7.3.4 Expresiion pattern of PC transcripts in cultured cells and in pancreatic islets 119 7.3.5 S'-UTils of dffirent PC transcripts modulate their translation 119 7.4 DISCUSSION t2l CHAPTER 8 EXPRESSION AND PURIFICATION OF RECOMBINANT HUMAN PC 8.1 INTRODUCTION 130 8.2 SPECIFIC METHODS 132 t32 8,2.I Transient and stable transþction of 293T cells 132 8.2.2 Preparation of PC from mitochondrial extracts s o g r r33 8 . 2 . 3 M onome ric av idin- S e phar o e chr omat aphy 133 8.2.4 PC assay 13s 8.3 RESULTS 135 8.3.1 Generation of lengthhuman PC clone fuII 136 8. 3.2 Transient exPression human PC from dffirent expression vectors 8.3.2.1 expression vector t36 136 8. 3.2.2 Bicistronic vectors. 293T t36 8. 3 . 3 Expression of hPC from stably transfected .cells r39 S. 3 .4 Pirffication änd châracterisation of recombirønt hPC 141 8.4 DISCUSSION CHAPTER 9 FINAL DISCUSSION, CONCLUSIONS AND FUTURE DIRECTIONS t46 REFERENCES 153 SUMMARY belonging to Pyruvate carboxylase (PC) IEC 6.4.1.1.1 is a multi-functional enzyme a mobile carboxy-group the biotin carboxylase family, enzymes that use the biotin moiety as mitochondrial carrier between the two catalytic domains. Mammalian PC is located in the matrix where it plays a vital role in gluconeogenesis, lipogenesis and other biosynthetic pathways. Although cDNA or genomic sequences encoding PC from a number of the molecular organisms have been reported they have not improved our understanding of molecular basis mechanisms that control the PC expression. This study aimed to unravel the of what controls PC expression at the gene level' Full length rar PC çDNA has been isolated by performing 5' RACE-PCR' Full length cDNA contains an open reading frame of 3531 bp encoding a polypeptide with a calculated Mr of 129,848. The inferred protein sequence shows a high degree of sequence similarity to PC from other species. Three functional domains of rat PC i.e. the biotin carboxylation domain, the transcarboxylation domain and the biotinyl domain have been from determined by a combination of limited proteolysis, sequence comparisons with PC other organisms and with other biotin enzymes. Comparison with the known structure of the biotin carboxylation subunit of Escherichia coli acetyl-CoA carboxylase, suggested the functional importance of l1 highly conserved residues including Cys265, His21l, Lys273, Glu31l,Glu324, Asn326, Ãrg328, Gln330, Val337,Glu332 and Arg377. The rat PC gene spans over 40 kb comprising nineteen coding exons and four 5'-untranslated region exons, Organisation of exons in the gene is consistent with the domain structure of the enzyme, suggesting a close relationship between exons and protein domains.