Molecular and evolutionary investigation of the phosphoglucomutase gene family. by Janine Tomkins July 1996 A thesis submitted for the degree of Doctor of Philosophy in the University of London MRC Human Biochemical Genetics Unit, Galton Laboratory, University College London. ProQuest Number: 10106760 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 10106760 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 molecular and evolutionary investigations of the phosphoglucomutase (PGM) gene family. The PGM loci {PGM1, PGM2an6 PGM3) widely expressed in man are thought to be the products of a diverged gene family. Following the cloning of PGM1 in 1992, the primary aim of this project was to investigate approaches for cloning the other members of the gene family. The strategies investigated include the use of anti-PGMI antibodies, low stringency PGR, degenerate primer PGR and searching EST databases. A variety of resources were used, including the human cell line K562. This cell line is devoid of PGM1 activity and the deficiency was found to be associated with a marked reduction in PGM1 mRNA, thereby providing a useful resource. Two novel DNA sequences, hyhbfanô human ESTI have been partially characterized. Hyhbf was identified by degenerate primer PGR of human cDNA. Although it is a member of the PGM gene family, no evidence could be obtained to confirm the sequence was human and it is suspected to be of bacterial origin. The human ESTI sequence, however, represents a widely expressed gene, which shows alternative transcripts and a related sequence. Evidence suggests it is a candidate for PGM2. Evolution of the PGM1 gene was investigated in mammals. Nucleotide analysis of the great apes showed the PGM1*1+ is ancestral since the ape homologues have the same characteristic amino acid substitutions as man. Extensive phylogenetic analysis of prokaryotic and eukaryotic sequences identified through conserved functional protein domains was undertaken. Eight distinct evolutionary pathways were identified, two of which, represented by Mycoplasma pirum PMManb Saccharomyces cerevisiae AGM are thought to reflect the divergent evolution of PGM2 and PGM3. ACKNOWLEDGEMENTS I would first like to thank David for all his advice, encouragement and support throughout my PhD, both in his role as a supervisor and a friend. I would also like to thank Hoppy for his ideas and comments, particuarly regarding the production of this thesis. I would like to thank Jenny Farrington and Margaret Fox for carrying out the cytogenetic analysis of K562, and Owen McMillan for his help and advice on the phylogenetic analysis. I thank Jane Sowden for providing the K562 cell line, Ben Carrit for the ROD primers, Kay Taylor for the primate DNA and Michael Miles for the Typanosome cruzi DNA. I am most grateful to Jenny for all the help and support she has provided during the last four years, and also to other members of the Galton, both past and present, who have provided friendship and inspiration, particularly Helen, Alex, Martine, Jane, Alex and Rod. I would also like to thank those in Newcastle who have been supportive over the last few months. I am very grateful. Finally, I would like to thank Kate, Clare, and my family for their patience and understanding throughout it all. This work was funded by an MRC Research Studentship. ABBREVIATIONS A adenine bp base pairs C cytosine cDNA complementary DNA chr chromosome cps counts per second der derivative DNA deoxyribonucleic acid dNTPs 2' deoxyribonucleotide triphosphate EDTA ethylenediaminetetraacetic acid G guanine GDP guanosine diphosphate hnRNP heterogeneous nuclear ribonucleoprotein particles igG immunoglobulin G IVS intervening sequence kb kilobase mRNA messenger RNA mw molecular weight nt nucleotide CD optical density pi isoelectric point RNase ribonuclease RNA ribonucleic acid rRNA ribosomal RNA T thymine Tris tris(hydroxymethyl)aminomethane UDP uridine diphosphate UV ultraviolet CONTENTS Page Number CHAPTER ONE: INTRODUCTION 18-48 1.1 Evolution of proteins 18-21 1.1.1 Gene families 19-20 1.1.2 Gene sharing 20-21 1.1.3 Convergent evolution 21 1.2 Phosphoglucomutase 21-32 1.2.1 Early studies of PGM 21-22 1.2.2 PGM loci in man 22 - 31 1.2.2.1 Polymorphic and variant alleles of PGM 24 - 28 1.2.2.2 Chromosomal localization of the PGM loci 28 1.2.2.3 Properties of the PGM loci 29 - 31 1.2.3 PGM loci in other species 31 - 32 1.3 Molecular analysis of phosphoglucomutase 32-44 1.3.1 PGM1 in man 33-38 1.3.1.1 Cloning of PGM7 33 1.3.1.2 The genomic structure of PGM1 in man 33 1.3.1.3 Molecular basis of the PGM1 protein polymorphism 36 - 37 1.3.1.4 The 3' untranslated region polymorphism 37 1.3.1.5 The Taq\ polymorphism 37 - 38 1.3.2 PGM in other species 38 - 40 1.3.2.1 Isoforms of PGM1 in eukaryotes 38 - 40 1.3.2.2 PGM in bacteria 40 1.3.3 Phosphomannomutase in bacteria 40 - 42 1.3.4 Conservation of protein motifs 42 - 44 1.4 Divergence of function 44 - 46 1.4.1 N-acetylglucosamine-phosphate mutase in S.cerevisiae 44 - 45 1.4.2 Parafusin in Paramecium tetraurelia 45 1.4.3 Aciculin in man 46 1.5 Convergent evolution of phosphomannomutase 46 - 47 1.6 Summary of aims 47-48 CHAPTER TWO: MATERIALS AND METHODS 49-68 2.1 Materials 49-51 2.1.1 General reagents 49 2.1.2 Cell culture 49 2.1.3 Electrophoresis materials 49 2.1.4 Commonly used solutions 49-50 2.1.5 Cell culture media 50 2.1.6 Microbiological media 50 2.1.7 PGM samples 50-51 2.1.8 Bacterial strains 51 2.2 Methods 51-68 2.2.1 Cell Culture 51 2.2.2 Preparation of placental and cell extracts 51 2.2.3 Protein electrophoresis techniques 52 - 53 2.2.3.1 Starch gel electrophoresis of PGM 52 2.2.3.2 Starch gel electrophoresis of PGD 52 2.2.3.3 Isoelectric focusing 52 2.2.3.4 Sodium-dodecyl-sulphate polyacrylamide gel electrophoresis 53 2.2.4 Protein detection methods 53 - 54 2.2.4.1 PGM activity stain 53 2.2.4.2 PGD activity stain 53 - 54 2.2.4.3 Immunoblot detection 54 2.2.4.3.1 Electroblotting of starch and SDS-PAGE gels 54 2.2.4.3.2 Passive blotting of lEF gels 54 2.2.4.3.3 Detection of antigen 54 2.2.5 Preparation of genomic DNA and RNA 56 - 57 2.2.5.1 Preparation of K562 genomic DNA 56 2.2.5.2 Preparation of E co //DNA 56 2.2.5.3 Preparation of total RNA 56 2.2.5.4 Preparation of pA+ RNA 57 2.2.6 Estimation of nucleic acid concentration 57 2.2.6.1 Spectrophotometry 57 2.2.6.2 Molecular weight standards 57 2.2.7 Agarose gel electrophoresis 57 - 58 2.2.7.1 Standard agarose gels 57 2.2.7.2 Nusieve agarose gels 58 2.2.7.3 Hybrid agarose gels 58 2.2.8 Polymerase chain reaction 58 - 62 2.2.8.1 Genomic DNA PCR 58 2.2.8.2 Reverse transcriptase PCR (RT-PGR) of PGM1 58 - 61 2.2.8.3 Low stringency RT-PGR 61 2.2.8.4 Degenerate primer PGR 61 2.2.8.5 Touchdown' PGR 61 - 62 2.2.8.6 Primers 62 2.2.9 Restriction enzyme digests 62 2.2.10 Southern blot analysis 62-64 2.2.10.1 Transfer of DNA to nitrocellulose 62 2.2.10.2 Preparation of hybridization probes 62 - 63 2.2.10.2.1 Electroelution 63 2.2.10.2.2 Spinning through glass wool 63 2.2.10.2.3 Wizard DNA PCR prep purification kit 63 2.2.10.3 Prehybridization of filter 63 2.2.10.4 Labelling of probe 64 2.2.10.5 Hybridization 64 2.2.10.6 Stringency washes 64 2.2.11 Determination of the PGM1 polymorphism 64 - 65 2.2.11.1 SSCP analysis 64-65 2.2.11.2 Restriction endonuclease analysis 65 2.2.12 Cloning of degenerate primer PCR products 65 2.2.13 Preparation of cloned DNA 65 - 66 2.2.13.1 The "Quick mini-preps" 65-66 2.2.13.2 Preparation of DNA for sequencing 66 2.2.14 Sequencing of PCR products 66-67 2.2.14.1 Sequencing of cloned DNA 66 2.2.14.2 Direct sequencing of PCR products 66-67 2.2.14.3 Polyacrylamide gel electrophoresis 67 2.2.15 Computer analysis 67 - 68 2.2.15.1 GCG 67-68 2.2.15.2 Phylogenetic analysis 68 CHAPTER THREE: CHARACTERIZATION OF THE CELL LINE K562 69 - 94 3.1 Characterization of K562 at the protein level 69 - 79 3.1.1 Detection of PGM activity 69 - 70 3.1.1.1 Starch gel electrophoresis and isoelectric focusing 70 3.1.1.2 Estimation of the sensitivity of the PGM activity stain 70 3.1.2 Detection of PGM antigen 70 - 79 3.1.2.1 Immunoblot detection using anti-rabbit PGM antibodies 74 3.1.2.2 Estimation of the sensitivity of the anti-rabbit PGM 74 3.1.2.3 Immunoblot detection using anti-human PGM1 antibodies 74 - 79 3.2 Characterization of the PGM1 gene 79 - 88 3.2.1 Fluorescence/n-s/Yu hybridization 79 3.2.2 Southern blot analysis 83 3.3 Characterization of PGM1 mRNA 83 3.4 Determination of the PGM1 phenotype in K562 89 3.4.1 SSCP analysis 89 3.4.2 Restriction enzyme analysis 89 3.5 Analysis of PGM1 alleles in K562 mRNA 89-92 3.6 Summary 92 3.7 Conclusions 94 CHAPTER FOUR: PCR-BASED SEARCH FOR MEMBERS OF THE PGM GENE FAMILY 95-119 4.1 Low stringency PCR 95 - 99 4.1.1 Optimization and results of low stringency RT-PCR 97 - 99 4.2 Degenerate primer PCR 99 - 115 4.2.1 Degenerate primer PCR strategy 99 - 100 4.2.2 PGM and PMM sequence based degenerate primer PCR 100 - 113 4.2.2.1 The degenerate primers 103 4.2.2.2 Selection of template DNA samples 106 4.2.2.3