Analysis of a Gene Amplification Event in Rat Cells Edith Heard Thesis presented in partial fulfillment of the degree of Doctor of Philosophy to the University of London Imperial Cancer Research Fund Department of Biochemistry Laboratories Imperial College of Science Lincoln's Inn Fields and Technology London, WC2A3PX London, SW7 2AZ September 1990 For Vincent, who shared this experience. ABSTRACT. The 3B transformed rat cell line was isolated as part of an experiment to try and clone mouse enhancer elements by transfecting immortalized rat cells (the Rat-2 cell line) with a ligation mixture of total restricted mouse DNA and a polyoma virus transforming gene. When the polyoma insert was cloned out of the genomic DNA of the 3B transformant, it was found to be in the form of a large inverted duplication, consisting of both the mouse and polyoma sequences as well as rat DNA. The palindromic sequences were also found to be amplified 20-40 fold in 3B genomic DNA. Thus, the amplified DNA in this cell line existed in the form of large inverted repeats. This has subsequently been found to be a common feature of mammalian amplified DNA sequences. Furthermore, the finding that amplification units are organized as inverted rather than tandem repeats has certain implications on the possible mechanisms by which amplification occurs in mammalian cells. This thesis extends the analysis of the amplified DNA in the 3B cell line with the purpose of elucidating the nature of the processes by which it arose. The structure and the stability of the amplification have been studied, at both a molecular and a karyotypic level. Molecular cloning and long range restriction mapping have shown that the amplified units (each unit being one side of the inverted duplication) seem to be homogeneous, and are approximately 450 kb in size. No rearrangements are detected between the amplified rat sequences and the unamplified alleles of the same region in rat cells, and the structure and copy number of the amplification units appear to be completely stable. Furthermore, the units appear to be clustered in an array of 10-11 Mb. Cytogenetic analysis by fluorescence in situ hybridisation of 3B metaphase spreads with a biotinylated probe which detects only the amplified DNA, has shown that all the amplified DNA is stably located at a single chromosomal site. Similar analysis of both Rat-2 and 3B cells, using a rat DNA probe that detects both the amplified and unamplified copies of the region, has revealed the chromosomal origin of the inverted duplication. Karyotypic analysis has shown that the amplified DNA is located on a rearranged chromosome, and that the amplification event probably led to the disruption of the chromosome from which it arose. On the basis of all these observations, the possible mechanism which gave rise to the amplified DNA in 3B cells is considered. ACKNOWLEDGEMENTS Many thanks to members of the TVG / EGOE lab., whose solidarity and humour carried me through my time here. Special thanks to Moira Read for her patience, and her expert help with cultured cells; and to Brendan Davies, for being my constant source of advice and competent cells, and for being a tolerant neighbour. I am also greatly indebted to Sarah Williams for being generous with both her expertise in cytogenetics and her time; and to Zoia Larin and Tony Monaco for their constant encouragement and help with YAC cloning. Finally, my thanks to Mike, for providing a unique experience, for teaching me about amplifiction, and for initiating me in the ways of science. ABBREVIATIONS. ABR abnormal banded region AMPD adenylate deaminase bp base pair CAD carbamyl phosphate synthetase, aspartate transcarbamylase, dihydroorotase. DHFR dihydrofolate reductatase DM double minute DNA deoxyribonucleic acid DTT dithiothreitol ECR extended chromosomal region EDTA diaminoethane tetra-acetic acid (disodium salt) FCS foetal calf serum hrs hours HSR homogeneously staining region kb kilo base LMP low melting point Mb mega base MDR multi-drug resistance mins minutes MTX methotrexate O/N overnight PALA N-(phosphonacetyl)-L-aspartate PCR polymerase chain reaction PFGE pulsed field gel electrophoresis Py polyoma virus rpm revolutions per minute SDS sodium dodecyl sulphate secs seconds Tris Tris [hydroxymethyl] aminomethane YAC yeast artificial chromosome CONTENTS PAGE CHAPTER 1 GENERAL INTRODUCTION 11 1.1 Developmentally Programmed Gene Amplification in Lower 11 Eukaryotes. 1.2 Gene Amplification in Pesticide Resistance. 13 1.3 Gene Amplification in the Germ Line of Mammals. 15 1.4 Gene Amplification in Somatic Cells. 16 1.5 Systems and Strategies Used for the Investigation of 18 Mammalian Gene Amplification. 1.6 Karyotypic Hallmarks of Amplification. 21 1.7 Submicroscopic Circular DNA. 22 1.8 Location of Amplified DN A. 23 1.9 Gene Amplification Associated with Chromosomal 25 Rearrangement. 1.10 Amplification and Chromosomal Deletion. 28 1.11 The Molecular Structure of Amplified DNA. 29 1.12 Novel Joints and the Organisation of Amplified DNA. 31 1.13 Stability and Evolution of Amplified DNA. 34 1.14 Mechanisms of Amplification in Mammalian Cells. 37 1.15 Models 40 1.16 Questions to be answered. 58 1.17 The Amplified Inverted Duplication in the 3B Rat Cell Line. 59 OBJECTIVES 64 CHAPTER 2 67 2.1 INTRODUCTION 67 2.2 Isolation of a 15 kb BamHI Rat DNA Clone from the Inverted 67 Duplication. 2.3 Isolation of Further Overlapping Sau3A Clones from the Inverted 72 Duplication. 2.4 DISCUSSION 76 CHAPTER 3 80 3.1 INTRODUCTION 80 3.2 The Identification of Infrequently Cutting Restriction Enzyme 82 Sites which Lie Within the Inverted Duplication. 3.3 The use of 5-azacytidine to Reveal the Presence of Multiple 87 Methylated Sail and Xhol sites in the Amplified Units. 3.4 The Use of Infrequently cutting Restriction Enzymes which 96 do not Cleave within the Amplified Rat sequences in 3B Cells in Determining the Size of the Amplification Units. 3.5 The Amplified Units may be Clustered in a Single Region of 3B 100 DNA. 3.6 Detection of Sites which could be Useful for Chromosome 108 Jumping Along the Inverted Duplication. 3.7 DISCUSSION 117 CHAPTER 4 123 4.1 INTRODUCTION 123 4.2 Construction of a YAC Library of 3B EcoRI Partial Fragments 129 Without DNA Size Fractionation. 4.3 Stability of a Large Inverted Duplication Present as a YAC in 136 yeast cells. 4.4 Attempted Cloning of the 240 kb Clal Fragment Spanning the 141 Centre of the Inverted Duplication. 4.5 Attempted Cloning of the 180 kb Xhol Fragment Spanning the 155 Centre of the Inverted Duplication. 4.6 Construction of a Size-Selected YAC library with EcoRI 164 Overlapping Fragments of 3B DNA. 4.7 DISCUSSION 177 CHAPTER 5 184 5.1 INTRODUCTION 184 5.2 In Situ Hybridisation of 3B Cell Chromosome Spreads with the 186 Amplification-specific A,3B Probe. 5.3 In Situ Hybridisation of 3B and Rat 2 Chromosome Spreads 192 with the M3 am Rat Probe. 5.4 Characterisation of 3B and Rat 2 Chromosomes by G-banding 198 Followed by In Situ Hybridisation with the XBam probe. 5.5 Karyotypic Analysis of the 3B and Rat 2 Cell Lines. 212 5.6 Investigation of the Translocated Marker Chromosome Ml/t(2;3). 237 5.7 DISCUSSION 244 CHAPTER 6 CONCLUSIONS AND FUTURE PROSPECTS 253 CHAPTER 7 MATERIALS AND METHODS 258 Materials 258 Methods 264 REFERENCES 298 APPENDIX 1 308 FIGURES. PAGE 1.1 Unequal sister chromatid exchange. 41 1.2 The disproportionate replication model. 45 1.3 The extrachromosomal double rolling circle model. 49 1.4 The chromosomal spiral model. 52 1.5 Restriction map of the centre of the inverted duplication and the X.3B clone. 60 2.2.1 Restriction map of the cloned region of the inverted duplication in 3B 68 cells and a summary of the lambda clones isolated. 2.2.2 Southern blotting analysis of 3B and Rat 2 genomic DNA using the 70 3.9 kb RI-Bam rat DNA probe. 2.2.3 Southern blotting analysis of 3B and Rat 2 genomic DNA using a 74 0.8 kb KpnI-Sall rat DNA probe. 3.2.1 Analysis by PFGE of the amplified DNA in 3B cells cut with a range of 83 rare cutter restriction enzymes. 3.2.2 Analysis by PFGE of 3B genomic DNA cut with a range of rare cutter 86 restriction enzymes using the Py and RI-Bam probes. 3.3.1 Analysis by PFGE of Sall-digested genomic DNA from 3B cells grown 88 in the absence or presence of 5-azacytidine. 3.3.2 Analysis by PFGE of Sail and Xhol digested genomic DNA from 3B 92 and Rat 2 cells grown in the presence of different concentrations of 5-azacytidine. 3.3.3 Long range restriction map of the inverted duplication in 3B cells and of 94 region of rat genome from which it has been derived. 3.4.1 Analysis by PFGE of 3B and Rat 2 genomic DNA cut with the enzymes 97 Narl and Eagl. 3.5.1 Analysis of the overall organization of the amplification units in 3B cells 101 by PFGE resolution of DNA up to 3 Mb in length. 3.5.2 Further analysis of the organization of the amplified units in 3B cells 104 through the resolution of DNA up to approximately 10 Mb in length. 3.5.3 The organization of the amplified array in 3B cells and of the native locus 106 in Rat 2 cells. 3.6.1 Rare cutter enzymes located within the insert of the A,3B clone. 110 3.6.2 Ability of rare cutter enzyme sites at the centre of the inverted duplication 112 to be cleaved in 3B cellular DNA.