The Molecular Cloning and Characterisation
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THE MOLECULAR CLONING AND CHARACTERISATION OF NORMAL AND BETA-PLUS THALASSAEMIC HUMAN' GLOBIN GENES ' \ . ' by DAVID ANTHONY WESTAWAY a thesis submitted for the degree of Doctor of Philosophy in the University of London December, 1980 Department of Biochemistry St Mary's Hospital Medical School London W2 IPG VI ABSTRACT ^thalassaemia is a human hereditary diseases characterised by decreased output of p globin chains. Human & ~rp~r andy - globin cDNA plasmids, whose identity was confirmed by sequence analysis, were used to investigate the p- globin locus in j3 -thalassaemia. No gross alteration of the globin locus is associated-' with the disease. In order to identify the mutation conferring the thai phenotype, a chromosomal p globin gene has been cloned in phage lambda. Starting material was DNA from a Turkish Cypriot compound heterozygote, with a P gene .on one chromosome and a 6p fusion gene on the other chromosome. The DNA was digested with the restriction endonuclease Hsu 1 which generates a 7.5kb fragment from the p globin gene, and a 16kb fragment from the fusion gene. The Hsu 1 cleaved DNA was enriched for the 7.5kb size fraction and ligated to the lambda cloning vector ANEM788. 160,000 recombinants were screened and a positive scoring phage isolated. Restriction analysis confirmed that this phage contained a P globin gene. The 7.5kb insert was subcloned into the plasmid pAT153 and digestion with seven restriction endonucleases gave a pattern indistinguishable from that of a normal p globin gene. Thus within the 7.5kb of DNA cloned, which includes the p globin gene plus almost 6kb of flanking DNA, there have been no insertions or deletions greater than about 100 base-pairs. Also no point mutations occur within the hexanucleotide recognition sequences of the restriction endonucleases used here. The complete nucleotide sequence of the coding blocks and introns of the P gene are presented and compared to a normal P globin gene. The gene has an G->A transition in the first intron. This sequence variant+ is also found in the P gene of a Greek Cypriot homozygous p thalassaemic described by Spritz et al (Spritz et al, 1980). To my.Parents/ iii Acknowledgements. To Bob Williamson Peter Little, and Ian Jackson for endless f useful discussions (Figure 19 is the Peter Little Memorial Diagram). To Ned Mantei and Charles Weissmann for help with DNA sequencing. To Binie Klein and Ken Murray for help with in vitro packaging. To Noreen Murray, David Sherratt, and Tom Maniatis for phage and bacterial strains. To Bernadette Modell for pointing out the compound heterozygote patient in our stocks of human DNA. To Julian Crampton for photographic help. To Emma Whitelaw for reading the manuscript, and to my sister for typing it. I was supported by an MRC studentship. ( To all the Biochemistry department at St. Mary's. A special mention to all the hard-core globinologists (real or honorary) Jack, Tim, Adam, Auntie Janet, Sue, Gill, Mike, Raymond, Rob, Charles, Lesley, and Derek. To my family. To my Croydonian friends for their acerbic wit. ABBREVIATIONS bp base-pai r kb 10^1 base-pairs Bis NN -methylenebisacrylamide BSA bovine serum albumin cpm counts per minute DEAE diethylaminoethyl D.melanogaster Drosophila melanogaster (a fruit fly) DNA deoxyribonucleic acid DNase deoxyribonuclease cDNA complementary DNA dpm . - disintegrations per minute E.coli Escherichia coli (a bacterium) EDTA ethylene diamine tetraacetic acid EtOH ethanol g acceleration due to gravity (terran units) Hb haemoglobin ug microgram ng nanogram P9 picogram M mola r moi multiplicity of infection pfu plaque forming units RNA ribonucleic acid hnRNA heterogeneous nuclear RNA mRNA messenger RNA tRNA transfer RNA RNase r ibonuclease rpm revolutions per minute SDS sodium dodecyl sulphate TCA trichloroacetic acid TEMED N'N'N'N' tetramethylethylene diamine Thai thalassaemia Tris 2-amino-2(hydroxymethyl)- propane-1,3-diol UV ultraviolet w/v weight per volume X.laevis Xenopus laevis (a toad) / Experiments that failed' too many times, And transformations that were too hard to find Blue Oyster Cult, 1974 VI Figures Page 1. The Structure of the Human a- and/}- Globin mRNAs• 7 2. The Human a- and /}-like Globin Gene Clusters. 10 3. Structure of the Humana- and /} -Globin Genes. 12 4. Putative Recognition Sequences of the Human/}- 17 Globin Gene. 5. Gene Deletions in /? Thalassaemia 26 6. Human a-,/}-, and y - Globin Plasmids pHa//}/yGl. 54 7. A Sequencing Gel of the a-Globin cDNA Plasmid pHaGl 56 8. Partial Nucleotide Sequences of pHa//}/yGl. 58 + 9. The/} Thalassaemic Patient. 61 10. Southern Transfers of the Patients DNA. 62 11. Protocol for Cloning the Hsu 1 /}Globin Gene Fragment. 65 12. Optimising UV Irradiation of In Vitro Packaging 68 Lysogens. 13. Analysis of Size-Fractionated Human DNA. 71 14. The Hind 111 (Hsu 1) Lambda Vector NEM788. 73 15. Restriction Endonuclease Analysis of Recombinant 79 Phage. 16. "Benton and Davis" screening of Recombinant Phage. 81 17. Plaque-Purification of a Positive-Scoring 82/83 Recombinant Phage, Apaddington 1. 18. Southern Transfers of the Recombinant Phage 85/86 A Paddington 1. 19. Mapping the/}Globin cDNA Hybridising Fragments in 87 A Paddington 1. 20. Restriction Digests of the Pst 1 and Hsu 1 Subclones 92 + 21. Restriction Digests of the 7.5/} Subclone. 94 * (« /II + 22. Restriction Maps of the Plasmids 4.4//and 7.5 /J . 97 + 98/99 23. Parallel Restriction Mapping of the Normal and p Thalassaemic Globin Genes. 103 24. The "Shotgun" Sequencing Protocol. 105 25. Starting Fragments for Shotgun Sequencing of the fj Thalassaemic Human p Globin Gene. 26. Sequencing Gel of an Rsa 1 Fragment spanning the 3' 107 end of the large intron. 27. The Sequencing Protocol. 109 28. Sequencing Gel of a Hinf 1 Fragment spanning the 1L0 Gene's 3' flanking region. 29. Sequencing Gel of a Hinf 1 Fragment spanning the 111 region approximately 80 nucleotides downstream from the Gene's polyadenylation site. 30. Sequencing Gels of the coding and anti-coding 112 strands of an Hph 1 fragment spanning the gene's small intron. Yin Tables Page 1. Packaging Efficiencies of Lambda DNAs. 69 2. Packaging Efficiencies of restricted and 75 unrestricted Vector DNAs. 3. Construction of \ NEM788/Human recombinants In Vitro. 77 4. cDNA Hybridising Restriction Fragments of Lambda 89 Paddington 1. 5. Restriction Fragments generated by double-digests 95 + of the Hsu 1 Subclone (7.50 ). ix CONTENTS PAGE Abstract i Dedication ii Acknowledgements iii Abbreviations iv Quotation v List of Figures vi List of Tables viii 1. Introduction 1.1 Recombinant DNA and Molecular Cloning 1 1.2 The Southern Transfer Technique 3 1.3 The Human Globin Proteins and Messenger RNAs 4 1.4 Globin Gene Linkage 9 1.5 Introns and Precursor RNAs 9 1.6 Globin Pseudogenes and Repeated DNA Sequences 15 1.7 Fine Structure of the Globin Genes 16 1.8 The Proteins of Globin Gene Expression 18 1.9 Inherited Diseases of the Globin Genes 20 1.10 The Molecular Basis of Thalassaemia 22 Gene Deletions in Thalassaemia 23 Non-Deletion Thalassaemias 28 1.11 Summary and Aims 31 2. Materials and Methods 2.1 Materials 34 VI 2.2 Bacterial and Phage Strains 36 2.3 General Methods 2.3.1 Restriction Enzyme Digestion 36 2.3.2 Ethanol Precipitation of Nucleic Acids 37 2.3.3 Agarose Gel Electrophoresis 38 2.3.4 Acrylamide Gel Electrophoresis 38 2.3.5 Visualisation of DNA Restriction Fragments 38 2.3.6 Autoradiography 39 2.3.7 Recovery of Restriction Fragments 39 2.3.8 Gel Filtration 40 2.3.9 Determination of Radioactivity 40 2.4 Specific Methods 2.4.1 Preparation of Plasmid DNA 41 2.4.2 Transformation of HB101 43 2.4.3 Colony Electrophoresis 43 2.4.4 Labelling Plasmid DNA by Nick-Translation 44 2.4.5 Southern Transfers 44 2.4.6 Grow'th of Phage Lambda in liquid culture 46 2.4.7 Growth of Phage Lambda on Agar plates 46 2.4.8 Preparation of Phage DNA 47 2.4.9 Preparation of Phage Vector "arms" 48 2.4.10 In Vitro Packaging of Lambdoid DNA 49 2.4.11 Miniprep Analysis of Lambdoid DNA 49 2.4.12 Screening of Lambda Recombinants 50 2.4.13 Maxam and Gilberting in Zurich 50 xi 2.4.14 Maxam and Gilberting in Paddington 51 3. Results. 3.1 Partial Sequencing of Human a-, andy Globin 53 cDNA Plasmids 3.2 Molecular Diagnosis of p Thalassaemia in 59 association with Hb Lepore 3.3 In Vitro Packaging of Lambdoid DNA - 64 3.4 Making Recombinant Phage 3.4.1 Enrichment for Human Globin Sequences 70 3.4.2 Manipulation of Vector DNA 72 3.5 Screening Recombinant Phage derived from the 80 P /Lepore Human DNA 3.6 Mapping the Recombinant Phage 84 3.7 Subcloning of the cloned J3 Globin Gene into the 90 Plasmid pAT153 3.8 Restriction mapping of the Subcloned Recombinant 93 Recombinant Plasmids + 3.9 The Sequence of the /? Thalassaemic P Globin Gene 3.9.1 Rationale 100 3.9.2 Technical Aspects 101 3.9.3 The Sequence 108 4. Discussion 4.1 The Analysis of Control Sequences in Eukaryotic 114 Genes 4.2 Naturally occuring mutants of the Human Globin 118 XII Genes 4.3 The map position of the/}+Thalassaemia Gene 119 4.4 Molecular Cloning of the. /}+Thalassaemic Globin 122 Gene 4.5 Structural Comparison of Normal and [J Thalassaemic 124 Globin Genes 5. Summary and Perspectives 130 6. References 132 Errata 142 Section 1 Introduction. 1 1 INTRODUCTION 1.1 Recombinant DNA and Molecular Cloning The genome of a higher eukaryote consists of vast amounts of deoxyribonucleic acid (DNA).