Genetic^and Physical Mapping Studies on Mouse Chromosome 2 by Stavros Males A thesis submitted for the degree of Doctor of Philosophy at the University of London May 1995 The Galton Laboratory Department of Genetics and Biometry University College London ProQuest Number: 10018553 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. uest. ProQuest 10018553 Published by ProQuest LLC(2016). Copyright of the Dissertation is held by the Author. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code. Microform Edition © ProQuest LLC. ProQuest LLC 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106-1346 Abstract This thesis describes two genetic maps of mouse chromosome 2 (MMU2), genetic maps relative to the wasted {wsf) and Ragged (Ra) mutations on distal MMU2 and a physical map of the region likely to contain the latter two genes. The first two maps include 18 new PCR markers which were isolated from a subgenomic library constructed from a mouse-hamster somatic cell hybrid line whose main mouse-genome component was MMU2. Fourteen of these loci define microsatellite sequences and four represent randomly chosen DNA sequences. The maps were constructed using two interspecific backcrosses established using a laboratory mouse strain and mice from the related species Mus spretus. The inheritance pattern on MMU2 reveals loci that exhibit significant segregation distortion (SD) in males. The genes responsible for the wasted (wsf) and ragged (Ra) phenotypes are very closely linked (0.2 cM) on the extreme end of MMU2. Two interspecific backcrosses segregating wst and Ra were set up in order to define the genetic position of these loci relative to currently available molecular markers. In total, 167 wstA/vst and 329 Ra/+ or +/+ mice were phenotyped for 5 microsatellites and two genes which, on the basis of the consensus map, would be expected to map near wst and Ra. The gene order established is; D2Ucl1, Gnas- 1.8 cM + 1.0, D2Mit200- 1.8 cM + 1.0, D2Mit230 0.6 cM + 0.6, D2Mit74- 0.6 cM + 0.6, Acra4- 0.6 cM + 0.6, D2Mit266, wst D2Ucl1, Gnas- 1.2 cM + 0.6, D2Mit200- 0.6 cM + 0.4, D2Mit230- 0.6 cM + 0.4, D2Mit74, Acra4, D2Mit266, Ra In an attempt to isolate flanking markers for both mutations, four overlapping yeast artificial chromosomes (YACs) were isolated and analysed by way of fluorescence in situ hybridisation (FISH), Southern blot and PCR analysis. These clones span a region of at least 360 kb and encompass the markers D2Mit74, Acra4 and D2Mit266\ the contig extends at least 200 kb proximal and 160 kb distal to Acra4. In the context of this work, a framework physical map extending 200 kb proximal and 310 kb distal to this gene was also established. Two YACs were found to span the T(2;16)28H translocation breakpoint. On the basis of these results, D2Mit74 is tentatively proposed to reside proximal to the breakpoint. All the genes which map to human chromosome 20 also map to distal MMU2 and the order appears to be conserved. The human homologues of Ra and wst would be expected to map on 20q13.1^ qter. The human homologue of the rat gene for vitamin D3 24-hydroxylase (CYP24) maps to 20q13.1^ qter. In this study, the mouse homologue of this gene {Cyp24) was localised to distal MMU2 9 cM proximal to Ra. II “Exri CTDÇuyo pou Kai aiouç yoveiç pou” III Table of contents Title I Abstract II Dedication III Table of contents IV List of figures IX List of tables XI Abbreviations XII Acknowledgments XIV Chapter 1 : Introduction Part 1. Aims of the project Part 2. The mouse in genetic analysis 2.1. Why mice 2.2. The establishment of inbred strains 7 2.2.1. Derivation of inbred strain 8 2.2.2. The development of congenic lines a 2.3. Interspecific backcrosses 10 2.3.1. Uses of interspecific backcrosses 12 2.3.2. Limitations of interspecific backcrosses 13 Part 3. Molecular genetic analysis 14 3.1. The establishment of genetic maps 14 3.2. The discovery of DNA sequence variation 14 3.3. Microsatellites in genetic mapping 14 3.3.1. Structural features of microsatellites 15 3.3.2. Isolation and analysis of microsatellites sequences 16 3.3.3. Distribution of microsatellite sequences 17 3.3.4. Function of microsatellites 19 3.3.5. Uses of microsatellites 19 3.3.5.1. Tackling multifactorial diseases 20 3.4. Pathogenic microsatellites 22 Part 4. The isolation of developmental mutations in the mouse 24 IV 4.1. Positioning a mutant gene on the genetic and physical map 24 4.2. The isolation of molecular markers 26 4.2.1. Somatic cell hybrids 26 4.2.2. Chromosome microdissection 28 4.3. The story so far 29 Part 5. The wasted and ragged mouse mutants 31 5.1. The wasted mouse 31 5.1.1. Immunological abnormalities 31 5.1.2. Neurological abnormalities 34 5.2. The ragged mouse 36 Chapter 2: Materials and Methods Part 1. General Techniques 39 1.1. Bacterial media, strains, antibiotics 39 1.1.1. Liquid and solid bacteriology media 39 1.1.2. Bacterial strains 39 1.1.3. Antibiotic stock solutions and working concentrations 39 1.2. Growth of bacterial cultures, storage media 39 1.2.1. Growth in liquid media 39 1.2.2. Growth on solid media 40 1.2.3. Preparation of competent cells 40 1.3. Propagation of bacteriophage X 40 1.3.1. Preparation of plate lysate stocks 40 1.3.2. Growth of bacteriophage X clones in a microtiter-format arrangement 41 1.3.3. Storage of bacteriophage X lysates 41 1.4. Propagation of yeast cultures 41 1.4.1. Growth medium 41 1.4.2. Expansion of yeast cultures 41 1.4.2.1 Liquid cultures 42 1.4.2.2 Growth on solid media 42 1.4.3. Storage of yeast cultures 42 1.5. DNA extraction protocols 42 1.5.1. Extraction of total mouse/hamster genomic DNA 42 1.5.2. Extraction of total yeast DNA 43 1.5.3. Extraction of plasmid and bacteriophage X DNA 44 1.5.4. Immobilisation of high molecular weight DNA in agarose blocks for PFGE 44 1.5.4.1. Mouse DNA 44 1.5.4 2. Yeast DNA 44 1.5.5. Quantification of DNA 45 1.6. DNA modification reactions 45 1.6.1. Restriction of DNA with bacterial endonucleases type II 45 1.6.1.1. Restriction of low molecular weight DNA 45 1.6.1.2. Restriction of high molecular weight DNA in agarose blocks 46 V 1.6.2. Déphosphorylation of DNA 46 1.6.3. Ligation of DNA 46 1.6.3.1. Subcloning of bacteriophage X DNA 47 1.6.31.1. Non-directional cloning 47 1.6.3.1.2. Directional cloning 47 16.3.1.3. Cloning of PCR products 47 1.7. Transformation of competent E.Coli cells 47 1.8. Immobilisation of colonies/plaques onto nylon membrane 48 1.8.1. Bacterial colonies 48 1.8.2. Bacteriophage X, plaques 48 1.8.3. Processing of membranes 48 1.9. In vlto DNA replication 49 1.9.1. Exponential amplification using the Polymerase Chain Reaction 49 1.9.2. Radioactive labelling of DNA 49 1.9.3. DNA sequencing 50 1.10. Electrophoretic analysis of the DNA 51 1.10.1. Agarose gel electrophoresis of low molecular weight DNA 51 1.10.2. Pulse field gel electrophoresis (PFGE) of high molecular weight DNA 52 1.10.3. Polyacrylamide gel electrophoresis 52 1.10.3.1. Non-denaturing polyacrylamide gels (native gels) 52 1.10.3.2. Denaturing polyacrylamide gels 53 1.11.Southern blotting 53 1.11.1. Salt transfer protocol 54 1.11.2. Alkaline DNA transfer 54 1.12. Hybridization of radlolabelled probes to Immobilised DNA 54 1.12.1. Hybridisation to plaque/colony DNA 55 1.12.2. Hybridisation to low and high molecular weight DNA 56 Part 2. The Isolation of molecular markers from mouse chromosome 2 57 2.1. Construction of a subgenomic library from hybrid line EBS-18/AZ 57 2.1.1. Partial fill-in, ligation and packaging reaction 59 2.2. Screening the mouse DNA-containIng clones for the presence of (AC)n dinucleotlde repeats 60 2.3. Subcloning, cloning, screening and sequencing of the clones with an (AC)n repeat 60 2.4. Genetic mapping of markers Isolated 61 2.4.1. The interspecific backcrosses and mapping of the markers isolated 61 Part 3. Genetic and physical mapping near wst and Ra 62 3.1. Genetic maps 62 3.1.1. The interspecific crosses 62 3.1.2. The construction of the genetic maps 63 VI 3.2. Screening of Yeast Artificial Chromosome (VAC) library 65 3.2.1. Screening of the library stacks 65 3.2.2. Screening individual stacks to determine the coordinates 65 3.2.3. Construction of vectorette libraries 67 3.2.3.1. Isolation of hybridisation probes by vectorette PCR 67 3.3 The mapping of the vitamin D 24-hydroxylase in the mouse 69 genome Chapter 3: Results Part 1 : The isolation of markers from MMU2 71 1.1. The isolation of mouse clones from hybrid line EBS-18/AZ 71 1.2.