The Genetic Significance of Enzymes in Tissues
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THE GENETIC SIGNIFICANCE OF ENZYMES IN TISSUES. by PEGGY CLARK, Master of Science, (Sydney). Thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philos ophy in the University of New South Wales. Submitted. lAtyr. , 1970 KENSINGTON E/a ADDITIONAL WORK UNDERTAKEN IN FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILO SOPHY IN THE SCHOOL OF HUMAN GENETICS, UNIVERS ITY OF NEW SOUTH WALES. "ABO, Rh and MNS Blood Typing Results and other Biochemical Traits in the People of the Yap Islands"., Jane Hainline, Peggy Clark and R.J. Walsh, published in Arch eology and Physical Anthropology in Oceania, 4, 1969, pp. 64 - 71. "A Genetic Study of the Maring People of the Simbai Valley, Bismarck Mountains, New Guinea", in conjunction with Georgeda Bick, Columbia University - in preparation. TABLE OF CONTENTS. A Page No. List of illustrations. i-v. Summary 1,2. Chapter 1, General Introduction. 3-32. 1.1.1. Historical survey of genetics. 3* 2. Beginnings of modern genetics. 3»4. 3. Complementary contributions of breeding experiments and cytology. 5* 4. Contributions of human studies to genetics. 6,7* 5. Structure of DNA and the development of modern genetics. 7*8. 6. Enzymes as.genetic markers and the introduction of the isozyme concept. 8-11. 1.2.1. Suitability of enzymes for the study of the evolution of proteins - primitive nature of enzymes. 11-13* 2. Epigenetic factors in the expression of enzyme phenotypes. 13*14. 3* Relationship between electrophoretic enzyme patterns and the functions of isozymes in the cell. 14-16. 4. Contributions of enzyme investigations in systematics and population studies. 16-19* TABLE OF CONTENTS, (continued.) B Page No 1.3*1. The meaning of the term"isozymen - biochemical and genetic significance of isozymes, 20-23. 2. Effects of epigenetic factors upon the expression of isozyme phenotypes. 23-25. 3. Molecular structure of isozymes of lactic acid , malic acid, isocitric acid dehyd rogenases and amylase. 25-29. Additional references, Chapter 1. 30-32. Chapter 2, Methods and Techniques. 33-58. 2.1.1. Electrophoresis using solid supporting media. 33-3$. 2*. Interpretation of protein electrophoresis. 38,39- 3. Method of electrophoresis. 39-43. 4. Advantages of disc electrophoresis. 44. 5. Disadvantages of disc electrophoresis. 44, 45 2.2.1. Collection of blood samples. 46. 2. Transport and storage of samples. 46,47. 3. Preparation of samples for electrophoresis. 47. 2.3.1. Staining techniques - principles. 48-57. Additional references, Chapter 2. 58. Chapter 3$ Studies with Human Material. 59-109* 3.1.1. Introduction 59 TABLE OF CONTENTS, (continued.) C Page No, 3*2.1. Lactic acid dehydrogenase - collection and storage of blood. 60. 2. LDH in red cells. 60-63* 3* Reproducibility of LDH patterns. 63-69* A. Occurrence of LDH types 1 and 2 in populations. 69-74. 5* LDH patterns in human serum, red cells and white cells. 74-80. 6. Occurrence of malic, isocitric and succinic acid dehydrogenases in human red cells. 80-94. 3*3*1* Glucose 6 phosphate dehydrogenase. 95-106. Additional references, Chapter 3* dehydrogenases, 106. 3*4.1. Serum amylase. 107-109* Note on the use of capital letters for non- zoological names for animals or groups of animals, 110. Chapter 4, Comparative Studies of Red Cell Dehyd- enases in Some Animals and Birds. 111-139* 4.1.1. Introduction. 111. 2. Lactic acid dehydrogenase. 111-127* 3* Malic acid dehydrogenase. 127-133* 4. Isocitric acid dehydrogenase. 133»134. TABLE OF CONTENTS, (continued.) D Page No. 5. General conclusions. 135-138. Additional references, Chapter 4. 139. Chapter 5» Red Cell Dehydrogenases in Marsupials. 140-179* 5.1.1. Introduction. 140 2. Acknowledgements. 140,141 3. Classification of marsupials. 141 5.2.1. Methods. 142 2*. Results. 142-168 3. Discussion. 168-179 Chapter 6 , A Study of Red Cell LDH in Grey Kangaroos. 180-194 6.1.1. Introduction and Acknowledgement. 180-183 2. Methods. 183-185 3. Results and conclusions. 186-194 Chapter 7 • Red Cell Lactic and Malic Acid De- hydrogenases in Hybrids in the Macropodidae. 195-218 7.1.1. Introduction. 195 2. Results. 196-212 3. Discussion. 212-218 Chapter 8 , Summary of Conclusions. 219-226 * — OO • • • Aims and conclusions of the investigation .219-226 TABLE OF CONTENTS, (continued,) E Page No, Acknowledgements, 227-229. Appendix I, Zoological names of mammals mentioned in Chapter 4. 230,231. Appendix II, Map of Australia and nearby islands, showing places mentioned in the text. 232. Glossary, 233-233. Bibliography. 236-270. LIST OF ILLUSTRATIONS. i Page No. Chapter 2. 2-1. Disc electrophoresis - complete gel. 37• 2-2. Disc electrophoresis apparatus - layout. 40. 2-3. Electrophoresis bath - close-up view. 40. 2-4. Hydrogen pathway in cellular respiration. 50* 2-5. Reduction of tetrazolium dyes. 52. 2- 6. Glycolytic and tricarboxylic acid cycles. 54. Chapter 3« 3- 1. LDH patterns in human red cells - disc electrophoresis. 62. 3-2. LDH types in human red cells. 64. 3-3* Abnormal LDH types in two human pop ulations. 71. 3-4. LDH in red and white cells of five blood donors. 76. 3-5• Range of LDH patterns in human serum. 77* 3-6. Most common serum LDH patterns - disc acrylamide electrophoresis. 78. 3-7. Patterns produced using malic acid as substrate. 83. 3-8. White cell MDH pattern in six blood donors.86. 3-9. LDH, MDH and IDH in one individual. 88. LIST OF ILLUSTRATIONS, (continued.) ii Page No, 3-10. Pentose phosphate pathway in red cells. 96. 3-11. Distribution of G6PD values in two human populations. 100. 3-12. Distribution of G6PD values in males and females in two populations. 101. 3-13. G6PD values and age. 102. 3- 14. Amylase patterns in human sera. 107. Chapter 4. 4- 1. Evolution of vertebrate LDH. 112. 4-2. LDH patterns in red cells of certain mammals and birds. 115. 4-3. Red cell LDH and MDH in rabbits. 118. 4-4. LDH and MDH in chicken haemolysates. 121. 4-5* LDH and MDH in red cells of chickens and humans. 122. 4-6. Red cell LDH in domestic cats. 124. 4-7. LDH in stored haemolysates of several mammals. 125. 4-8. MDH patterns in some animals and birds. 129. 4-9. Red cell MDH in several mammals. 130. LIST OF ILLUSTRATIONS, (continued.) iii Page No 4- 10. Isocitrate dehydrogenase in red cells of humans, cats and sheep. 133-134 Chapter 5« 5- 1. Red cell LDH and MDH in marsupials and humans. 143* 5-2. Range of red cell LDH patterns in Macropodidae. 145. 5-3. Red cell LDH types A, B and D. 151• 5-4. Red cell LDH and MDH in Potorous tridactylus. 152. 5-5. LDH and MDH in several Macropodidae. 155» 5-6. LDH type Em in western grey kangaroos. 156. 5-7* LDH and MDH in western grey kangaroos and a wallaroo. 157* 5-8. Red cell dehydrogenases in the potoroo. 158. 5-9. Red cell MDH in Macropodidae. 160. 5-10. LDH in red cells and diaphragm muscle of a euro (M. r. erubescens). 163. 5-11. Red cell LDH in Macropodidae from New Guinea and one mainland genus. 165. 5-12. Variations in LDH types from New Guinean and Australian genera. 166. • LIST OF ILLUSTRATIONS, (continued.) iv Page No, 5-13* LDH in Dasyuridae, Peramelidae and Phalangeridae. 168. 5-14. Red cell LDH in Macropodidae and Peramelidae. 169. 5-15« A dendrogram of the Macropodidae. 172* 5- 16. LDH and MDH in two wallaroos (M. robustus robustus). 178. Chapter 6. 6- 1. Geographical distribution of grey kangaroos. 181. 6-2. Geographical distribution of red cell LDH types in grey kangaroos. 182. 6-3. Red cell LDH in grey kangaroos. 183* 6-4. Red cell LDH in grey kangaroos (photograph). 187. 6-5* Variations in red cell LDH in grey kangaroos. 188. Chapter 7» 7-1. Hybrid LDH patterns in inter-generic and inter-specific crosses. 199- 7-2. Red cell LDH and MDH in red kang aroo/wallaroo hybrid. 200. • 7-3- LDH and MDH in red kangaroo father and red kangaroo/wallaroo hybrid. 201 LIST OF ILLUSTRATIONS, (continued.) v Page No. 7-^. LDH and MDH in red kangaroo/grey kangaroo hybrids, 202. 7-5* Parental and hybrid LDH and MDH in red kangaroo/grey kangaroo crosses. 20k. 7-6. Red cell LDH and MDH patterns in pretty face wallaby/E. grey kangaroo hybrid and parents. 206. 7-7• LDH and MDH in pretty face wallaby/ E. grey kangaroo and parents, showing •'hybrid'* MDH protein. 207. 7-8. Red cell LDH in euro mother and euro/ wallaroo hybrid. 211. 7- 9* LDH patterns produced by XXyy and xxYY genotypes. 218. Chapter 8. 8- 1. Suggested mode of formation of LDH phenotypes 222 SUMMARY: The purposes of the work reported in this thesis are (1) to test the suitability of the disc acrylamide electrophoresis technique for the investig ation of certain markers in population genetics and (2) to use the technique for a study of dehydrogenases in human and other vertebrate populat ions . (1) Isozymes of the lactate dehydrogenase series proved to be the most suitable of the enzymes studied. The presence in red cells of dehydrogenases of the tricarboxylic acid cycle is discussed. The stability of LDH and reproducibility of patterns in humans were also studied and a quantitative variation involving the slowest LDH isozyme in red cells was observed. Variations in G6PD electrophoretic patt erns are known to be rare but a semi-quantitative method has been used to observe the range and pattern of G6PD values in red cells of members of two populations.