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.
(2) Red cell LDH and, to a lesser extent, malate dehydrogenase, proved to be variable in members of the family Macropodidae (Marsupialia). Few sub bands occur in LDH isozymes when disc acrylamide electro 2 phoresis is used and minor differences in electrophoretic mobility are not detected but there are variations in
the amounts of different LDH isozymes in red cells.
These have been discussed in terms of species differences and the determination of phylogenetic relationships.
Hybrids have been used to investigate the
factors affecting the production of LDH and MDH patt erns in Macropodids.
Red cell LDH patterns in some vertebrates indicate that, although minor differences in mobility may be present, LDH isozymes are combinations of A and
B polypeptides. Patterns are determined by epigenetic factors acting upon the genes responsible for the prod uction of A and B polypeptides. These result in patt erns varying in amounts of different isozymes. There is no obvious relationship between red cell LDH patt erns and evolutionary affinities between major mamm alian Orders. 3
Chapter 1. General Introduction.
1.1.1. Historical Survey of the Development of Genetics.
The practical application of v/hat are now known as genetic principles to the improvement of domestic plants and animals is of great antiquity. These principles were certainly practised in China 6,000 years ago and a stone tablet at least 4,000 years old illustrates that the Chaldeans distinguished several inherited variations in the head shapes and mane characteristics of horses (Winchester, 1961). The recessive nature of coat colours in cattle and sheep was known to the writers of Genesis (Langenauer, 1966). The ancient Egyptians understood the necessity for cross pollinating date palms and the possibilities of varying crop quality and yield.
Such improvements in stock lines as occurred were the result of trial and error but a large amount of practical knowledge was accumulated, much of which is incorporated in the present day discipline of agriculture, its allied subjects and animal husbandry.
1.1.2. The Beginnings of Modern Genetics. Ravin, in his book, "Evolution of Genetics", (1965) points out that genetics 4 is one of the few scientific disciplines for which a "birth" date can be assigned. The first work which reported carefully controlled experiments leading to the form ulation of theoretical concepts capable of verification was that of Gregor Mendel, published in 1866. At the same time the conclusions of Charles Darwin were posing questions about the origins of observed variations in organisms. Attempts to answer these questions prepared the way for the development of modern genetics, which therefore dated from 1900 or 1901, when the significance of Mendel's work became apparent.
Several organisms proved useful for the class ical investigations of the early years and included the vinegar fly, Drosophila melanogaster, (T.H. Morgan and his associates, 1910-1920) sweet peas, (Bateson and
Punnett, 1906) and the Ascomycete, Neurospora crassa,
(Beadle and Tatum, 19^5)• These organisms provided evidence for many of the basic principles which were at first applied universally. Later it was appreciated that,although many genetic rules were applicable to organisms at all levels of evolutionary development, there were important exceptions. One of the most fundamental misconceptions was that the effect of the Y chromosome was the same in Drosophila sp. as in Man. 5
1.1«5» The Complementary Contributions of Breeding
Experiments and Cytology.
The work of Mendel and Darwin was completed before the existence of the chromosome was known. Breed ing programmes were aimed at perpetuating favourable characteristics such as increased yield or fertility but there was no comprehension of the factors respons ible for the expression of these characteristics. In choosing certain easily distinguishable alternate char acters, Mendel was able to observe and describe rules which required the existence of particulate entities, later to be called "genes" by Wilhelm Johanssen (Win chester, ibid.)
The discovery of chromosomes led to the theory formulated by Sutton and by Boveri (1902) that they were the physical structures by which genetic characters could be passed from pairs of individuals to their offspring.
It was realised that the linear arrangement of genes on the chromosome accounted for apparent departures from
Mendelian ratios. (Sinnott, Dunn and Dobzhansky, 1958.)
Cytological studies have consistently complemented the investigations of the experimental geneticist. 6
1,1.4. The Contribution of Human Studies to Genetics.
In spite of the classical studies on hereditary
genius by Galton, (18 66) human blood groups by Landsteiner,
(1900) and inborn errors of metabolism by Garrod, (1909)i
humans were considered to be unsuitable for genetic studies.
The reasons most often advanced were (1) the life span of
the investigator and the subject are of the same duration
(2) it is difficult to acquire sufficient family data and impossible to con duct controlled breeding experiments (3) samples of human material available for investigation are limited and often restricted to post mortem material. The difficulties
inherent in (2) and (3) can be avoided and the consequences of (1) minimised by the use of statistical methods whereby conditions in large populations can be inferred from the correct use of sampling techniques. In some cases human material has proved to be more suitable than that from animals. This is apparent in the development of blood group genetics which followed the discovery of the ABO blood groups. A large body of data on blood group genetics has been ably collated by Race and Sanger (1962), Wiener
(1940 et seq.) and Mourant (1954).
In 1955 Smithies developed the technique of starch gel electrophoresis and he and subsequent investigators extended the number of genetic differences which could be 7 detected in human blood. Many of the variations in human characters are the result of single alleles segregating according to Mendelian principles.
1.1,5» The Structure of DNA and the Development of
Molecular Genetics. In 1953 Watson and Crick proposed their model for the structure of DNA. For the first time it was possible to understand how four bases, adenine, guanine, cytosine and thymine within the chromo some could be responsible for the sequence of amino acids assembled in the cytoplasm. This development stimulated research in molecular genetics - a field in which a large amount of information is accumulating. Molecular genetics concerns itself with the ultrastructure of the chromosomes and with inherited changes in cell components resulting in differences in the products of protein synthesis. With some organisms such as Neurospora crassa it has been possible to study transformations within the genes themselves.
(Catcheside, 1960).
A further stimulus to the study of molecular gen etics was the work of Ingram (Review 1963)» Kendrew and
Perutz (1957) and Pauling et al. (19^9)* During the course of these studies the structure of haemoglobin and amino 8 acid sequences of the two types of polypeptide in normal human haemoglobin were determined. The identification of a single substitution in the amino acid sequence of one of the polypeptides of Haemoglobin S (sickle cell haemo globin) illustrated for the first time a link between an observed genetic defect and an "error" in the primary gene product* which in this case is the globin molecule. 1 2 Investigation of the two haptoglobin genes, Hp and Hp 2 1 has indicated that Hp has developed from Hp by gene duplication*. The evidence is based partly upon the fact 2 that the protein coded by the Hp gene is almost twice 1 the molecular weight of the protein coded by the Hp gene and is a valuable contribution to the knowledge of the evolution of proteins (Smithies and Connell, 1959;
Smithies, Connell and Dixon, 1962).
1.1,6. Enzymes as Genetic Markers and the Introduction
* of the Isozyme* Concept. Starch gel electro phoresis had enabled the proteins of tissue fluids to be separated and studied. In 1959» Markert and Hunter successfully adapted certain histological staining techniques for use with several electrophoretic
* Words or phrases which are used in a special sense are marked by an asterisk throughout and are defined in a glossary at the end of the work. * See note overleaf. 9
* The Use of the Word Isozyme*
The word "isozyme" was first used by Markert.
In 196^- the following statement was published in "Nature" by E.C. Webb, Chairman of a committee formed to discuss the nomenclature of multiple forms of enzyme,
" Multiple enzyme forms in a single species should be known as iso-enzymes, although since either form is readily intelligible this recommendation is not to be interpreted as excluding the use of "isozyme" if any individual prefers it".
Markert's terminology has been used in this work and the term "isozyme" has been retained. 10 media. Markert first demonstrated the multiple bands of lactic acid dehydrogenase, LDH (1.1.1.27**). Since that time at least nine other dehydrogenases have been located using minor modifications of existing histological tech niques. Two hydrolases, alkaline phosphatase (3.1.3.1.) and acid phosphatase (3.1.3.2.) and the transferases adenylate kinase (2.7-^.3.) and phosphoglucomutase (2.7.5*1.) have been used for population studies.
In 1952 Nielands, using biochemical extraction methods, had established that LDH activity v/as located in beef heart muscle and Kaplan consequently called this
"H" type LDH. The other was predominant in pigeon breast muscle and was named "M" type LDH (19&1). Agar gel electrophoresis demonstrated that there were five proteins with specificity for the same substrate (Wieme, 1959).
These five forms were isomeric enzymes* and were called
'’isozymes'’ by Markert. A sixth LDH was later found in human sperm (Goldberg, 19&3) anc* human testes at maturity
** The number 1.1.1.27. is that allotted to LDH by the Enzyme
Commission. (Reference, Dixon and Webb, 2nd. edition, 1966.)
Wherever an enzyme is mentioned for the first time the EC number will follow 11
(Blanco and Zinkham, 19&3)• This LDH is believed by
Zinkham to be produced by a separate gene from the two responsible for the production of the other five LDH proteins (1968). There is therefore some doubt whether the sixth LDH enzyme is an isozyme. Some of the genetic
implications of the isozyme concept are discussed in
Chapter 1, section 3«
1.2. The Suitability of Enzymes for the Study of the
Evolution of Proteins.
1.2,1, The Primitive Nature of Enzymes. In 1938 Oparin published "The Origin of Life" in which he propounded the
theory that living material arose in an atmosphere devoid of oxygen by the condensation of carbon and nitrogen.
Support for this theory has recently been obtained by
Matthews and Moser (1968) who have synthesised amino acids in vitro, starting with hydrocyanic acid (HCN) which is believed to have been present in the atmosphere. It had previously been accepted that life could not have arisen in the absence of oxygen.
Zuckerkandl and Pauling (1965) say "it may well be that all polypeptide chains are endowed with weak enzymic act ivities of some kind, especially in the presence of trace metals”. Fox (1965) suggests that the first synthesis of a polypeptide may have coincided with the first synthesis of an enzyme 12
In view of their role in the synthesis of living matter it is logical to conclude that enzymes were among the first biological substances. They must also have been stable molecules if metabolic processes were to be maintained, part icularly in respect of their active sites. Margoliash and
Smith (1965)1 in their discussion on cytochrome c in evol ution, distinguish only "microbial” cytochrome oxidase, isolated from micro-organisms and "mammalian” cytochrome oxidase•
The study of cellular processes is synonymous with the study of enzymes. All proteins, fats, carbohydrates and other components of living material must be synthes ised enzymatically and the nature of the DNA itself must be controlled by enzymes at some stage. Enzyme proteins are likely to be primary gene products and because of this are good indicators of changes in the DNA sequence. Tatum expressed the relationship between DNA and the kinds of proteins which are synthesised as "one gene - one protein”.
(Tatum's views are presented by him in an introductory address to the New York Academy of Sciences, 1965)* The work of Kaplan (1963) and Markert (1959)1 among others, revealed the isomeric form of many enzymes, due to the aggregation of polypeptides to form multimers*. For example, LDH is composed of four polypeptides which are the products of two separate pairs of genes. It was necessary to modify the theory to "one gene - polypeptide”. A 13
further alteration in interpretation was required when it
was realised that the resultant phenotypic expression of
an enzyme is not always due only to the gene or genes
responsible for the synthesis of the enzyme polypeptides
themselves. At present the most correct statement of
the theory is "one cistron* - one polypeptide".
1.2.2. Epigenetic* Factors in the Expression of Enzyme
Phenotypes. The phenotypic expression of multiple molecular forms of enzymes is not the result of the expression of primary* genes alone. There are many reports of enzyme patterns which cannot be interpreted simply as random arrangements of the sub-units determined by one or more genes. The polypeptides which are the products of the genes are assembled in the cytoplasm and the way in which they are finally arranged may be influenced by other genes or even by the whole genome* of the individual. Waddington developed the concept of the control of genetic mechanisms distant in space and time from the immediate gene product in his books (19^0 and
1962) and called the effects "epigenetic".
In 1968 Schlesinger and Anderson presented evidence that in Escherichia coli the final form of the enzyme alkaline phosphatase can be influenced by physiological conditions in the cell after the sub-units have been made. There are several accounts of the changes which occur in enzyme patterns with the growth and development of the individual. These include the work of Goldberg and Cather on the snail Agrobuccinium oregonense, Redfield
(1963) and that of Goldberg and Hawtrey (19&7) anc* Zinkham
(1968) on the changes in testicular LDH patterns with development in mice and in humans. Baglioni and Ingram
(1961) have described an individual with four different haemoglobin types and have suggested that the sub-units aggregate after synthesis according to the configuration of the available polypeptide molecules. This would clearly be an epigenetic effect influencing the phenotype of the individual.
1,2.3« The Relationship between Electrophoretic Enzyme
Patterns and the Functions of Isozymes in the Cell,
Epigenetic expression of enzyme phenotypes must be influenced by the spatial arrangement of enzymes taking part in metabolic reactions. Enzymes may appear to be deficient because they cannot react with their substrates and not because they are incorrectly coded by DNA. In addition, although some enzymes may display several different forms after electrophoresis they may not have such diversity 15
in vivo. The multiple bands after electrophoresis may
be artefacts but they can be artefacts which represent
genetically determined differences. This can be verified
in populations by applying the Hardy Weinberg equation to
the observed number of phenotypes or by examining family data. A similar situation may apply to human haptoglobins
which have been studied more extensively than any other serum protein (see Giblett, 1969). It has not yet been
shown that the three genetically determined types of haptoglobin, 1-1, 2-1 and 2-2, function differently in vivo (Himaguchi and Nakajina, 1968). However,the protein patterns indicate genetic differences which show up in the electrophoretic pattern.
Biochemical extraction of LDH isozymes detected only two proteins but electrophoresis has revealed six in higher animals. There are up to nine bands in the tissues of some fish (Goldberg, 1965 a.) Given that each gene produces a different gene product the multiple bands provide direct evidence of the number of genes involved in the production of the enzyme.
Isozyme patterns vary from tissue to tissue in the one organism (Wachsmuth et al., 1964, Walter and
Selby, 1966 and Hule, 1966). There is some evidence that slower moving LDH bands are associated with nucleated 16 cells (Vesell, 1965 and Vesell and Bearn, 1962). In the case of LDH, Kaplan et al. (1968) have suggested a rel ationship between the distribution of isozymes in tissues and their physiological role in the cells but Vesell,
(1968) expresses some doubt about the relevance of the results because the experiments were conducted under unphysiological conditions.
1,2,4, The Contribution of Enzyme Investigations to the
Understanding of Systematics and Population Studies.
In 1958 Haupt and Giersberg drew attention to the possibility of using differences in isozymes for the study of evolution and systematics. Opinions are divided on the value of inferring phylogeny or inter-relationship of organisms from biochemical differences. Zuckerkandl and Pauling (1965)1 using the differences in the amino acid sequences of mammalian haemoglobins, maintain that
"the evaluation of the amount of differences between two organisms as derived from sequences in structural* genes or in their polypeptide translation is likely to lead to quantities different from those obtained on the basis of observations made at any other higher level of biological integration". They also state that "the largest conc entration of information present in an organism and 17
perhaps also the largest amount of information and the
only organically transmissable information is in its
semantides*". Evidence in support of this view includes
ti the work of Blomback et al. (1962) on the amino acid
sequences of fibrinopeptides and of Fitch and Margoliash
(1967) and Fitch (1968) on amino acid sequences in cyto
chrome c. On the other hand, Simpson (1964) believes
that "characters far removed from the genes are better
than characters of genes themselves or of closely rel
ated polypeptide chains". In this he is supported by the
systematist, Mayr (1964) and by Buettner-Janusch and Hill
(1965). These authors point out some obvious anomalies,
principally in the haemoglobins, where amino acid diff
erences are not invariably directly proportional to their
systematic relationship.
It is clear that more data must be obtained
before it can be decided where biochemical markers can
be used to interpret systematic affinities or evolutionary
developments. Enzymes are suitable for the accumulation
of such information. Dixon and Webb (1966) list 842
enzymes which were described by the Enzyme Commission up to 1962. Over a hundred of these are isozymic in
form (Markert, 1968). Many of these enzymes have been
investigated by electrophoretic or immunological methods. 18
(See Giblett, 1969, for an extensive list of enzymes
detected in human blood. See also Beckman, 1966 and
Latner and Skillen, 1966, also Lush, 1966, for a survey
of genetic markers in non human tissues). Modes of
inheritance vary from single proteins inherited as
Mendelian co-dominants to multimers* of up to six
units determined by one or more pairs of genes and
assembled epigenetically. Enzymes therefore represent
a most versatile tool for testing the validity of
using primary gene products* to solve the problems associated with the evolution of organisms. Chapters 3i
4, 5 and 6 deal with the application of enzyme studies
to the problem of affinities and relationships between animals about which limited systematic evidence has been obtained.
Examinations of enzyme differences in populations have been extended to many enzymes, the most fruitful being glucose 6 phosphate dehydrogenase (EC 1.1.1.49), acid phosphatase (EC 3«1«3»2.) and phosphoglucomutase
(EC Population studies are required to be done urgently because of the accelerated disappearance of isolated groups of people who provide the most useful data for determining genetic drift. To date the lability of many enzymes has inhibited their use as genetic markers 19 but methods of collection and transport are being standardised as more becomes known about the stability of individual enzymes. In spite of the disadvantage of the lability of many enzymes they are so numerous that they represent one of the most promising areas of investigation for the future. 20
Chapter 1.5» The Meaning of the Term, "Isozyme11.
1.3»1» Biochemical and Genetic Significance of Isozymes.
In 1959 Markert and Miller introduced the word
"isozyme" to describe "the multiple molecular forms of
an enzyme found within a single organism or an individual
within a single species". It was, as Markert described
in 1968," designed to have operational utility and not
to define the molecular basis for any particular set of
isozymes".
The new word became necessary because of the
realisation that many enzymes exist in more than one molecular form. The concept of isozymes resulted from
observations on the electrophoretic behaviour of proteins reacting with the same substrate. Methods of identification were similar to those used for many years in histology.
The proteins were made visible after electrophoresis by
forming a coloured complex with a dye as a result of their enzymic activity (Hunter and Markert, 1957; Hunter and
Burstone, 195$)• It soon became apparent that the term could have different meanings depending upon whether one was considering biochemical, functional or genetic homogeneity.
The criteria for biochemical identity are 21 identical substrate specificity and the same susceptibility to inhibitors and activators (Beckman, 1966). It is customary to compare the Michaelis Constants (k^) of the different enzymes using the same substrate and chemical analogues of the substrate. The Michaelis Constant defines the rate of reaction of an enzyme by reference to the concentration (expressed as moles per litre) of the substrate which gives half the numerical maximum velocity at a given temperature and with a given substrate conc entration. ("Principles of Biochemistry", White et al.,
3rd. Ed., 1964). A further test of identity is that of immunological identity which has been used for amylase
(EC 3.2.1.1., oc amylase) Mc.Geachin and Reynolds, 1961,
Mc.Geachin, 1968), lactic acid dehydrogenase, (Mansour et al. , 1954, Nisselbaum and Bodansky,- 1959) and phosphatases
(Schlamowitz, 1954, Schlamowitz and Bodansky, 1959) •
If these criteria are used, it is clear that electrophoretic identity is not necessarily the same as biochemical identity. This is illustrated by the esterases, the "zymogram", which was one of the first examples of multimolecular forms of enzymes to be described (Hunter and Markert, 1957)- The kinetic and inhibitory properties of the set of enzymes made visible by the use of one substrate ( /3 naphthyl acetate, Gomori, 1952) are quite 22 different. (See also Allen, 19^1). Another example is that of lactic acid dehydrogenase where two protein forms were first found. They were called ”H" and "M" forms by
Kaplan (1963)• These two proteins have different kinetic properties and degrees of inhibition by excess pyruvic acid (Kaplan, 1965)* When subjected to electrophoresis, they were found to differ in mobility and were resolved into five macromolecules which proved to be combinations of two types of polypeptides.
In addition to differences in kinetic properties, there are differences in isozymes from different tissues.
LDII enzymes vary in the proportions of each enzyme type or '’species1’* obtained from tissue to tissue. With the exception of testicular LDH, all are combinations of the products of the same two genes, a and b. Testicular LDH is believed to be the product of a separate gene, c. This is an example of a set of enzymes which act upon the same substrate but which cannot be treated as isozymes for the purpose of genetic analysis. The same problem exists in defining amylase isozymes. When dealing with analyses of population data by means of the Hardy Weinberg equation, it is essential that the enzymes being considered should be immunologically indistinguishable, since this is a good indication that they are the products of the same gene or 23
genes.
1.3*2. The Effects of Epigenetic Factors Upon the
Expression of Isozyme Phenotypes.
In genetic analyses, those phenotypes which
are the result of epigenetic effects must be recognised.
Allen (1968) found that epigenetic factors, as yet not
clearly defined, produce variable phenotypes according
to the availability of polypeptide units and probably because of physiological differences within the cytoplasm.
Ogita (1968) has investigated genetic mechanisms res ponsible for isozyme formation and has suggested the
following classification,
(a) Unigenic Isozymes.;- an enzyme set* with several distinguishable molecular species which are under the control of a single gene. A single polypeptide is originally produced but gives rise to other molecular species by polymerisation or change in molecular con
figuration,
(b) Multigenic Isozymes;- an enzyme set encoded by two or more genes,
(i) Allelic Isozymes:- each isozyme is under 24
the control of a co-dominant allelic gene. At least two
enzyme types are found in a heterozygote,
(ii) Non Allelic Isozymes:- two or more isozymes
controlled by two or more genes at different loci. LDH
falls into this category.
In genetic surveys it is important to know which kind of isozyme is being investigated. Serum can contain
isozymes from several tissues and if organs are diseased,
enzymes from another enzyme set* may be present. The
electrophoretic patterns of amylase (Dreiling et al., 1963)* leucine amino peptidase, also known as aryl amidase, EC
3*4.1.1. (Kowlessar et al. , 1961, Beckman et al., 1966) and lactic acid dehydrogenase (Goldman et al., 1964, Stark weather et al., Dioguardi et al., 1966, Cohen, 1967 and
Wieme et al., 1968) in serum are altered in disease states which cause leakages of enzymes into the blood.
For the purpose of the investigations conducted in the following chapters, isozymes are considered to be those acting upon the one substrate, using the same co enzyme if applicable, and obtained from the one tissue by similar extraction techniques. 25
1,5»5« The Molecular Structure of the Isozymes of Lactic
Acid Dehydrogenase, Malic Acid Dehydrogenase,
Isocitric Acid Dehydrogenase and Amylase,
(a.) Lactic Acid Dehydrogenase:- In the cells of tissues there are two genes, a and b, responsible for the production of polypeptide chains A and B (Markert) or
M and H (Kaplan). It is not known if these genes are linked. The two polypeptides differ in their electrophoretic mobility and this is undoubtedly due to differences in amino acid composition (Kaplan and Ciotti, 19&1; Markert, 19&3 and Markert, 1968).
In most organisms the isolated enzymes were found to consist of four polypeptides arranged as tetramers. LDH isolated and crystallised from beef heart moved furthest and consisted of four B sub-units, BBBB (or HHHH). The form crystallised from chicken breast muscle migrated only a short distance and contained four A sub-units, AAAA (or
MMMM)• The random arrangement of these sub-units could result in five polymeric forms with the following comp ositions, AAAA (MMMM), AAAB (MMMH), AABB (MMHH) , ABBB (MHHH) and BBBB (HHHH). Markert (1968) demonstrated that these five forms were produced in vitro when mixtures of purified fast and slow LDH were frozen in a 1.0 Molar NaCl solution 26
in Phosphate buffer, pH 7.0 and thawed. All five bands
have been found in some tissues of animals and plants
although not necessarily all together. Markert named the
five bands LDH 1, 2, 3i ^ and LDH 1 being the fastest
moving LDH. Markert*s terminology has been used in this
thesis.
In 1963 Blanco and Zinkham described an LDH
from post-pubertal testes which migrated between LDH 3
and LDH 4 and they called the enzyme LDH MXf* • By 1968
Zinkham had concluded that production of this LDH was due
to the operation of a gene c, which produced a tetramer,
CCCC. In pigeons Zinkham found an allelic gene c*, which
resulted in a set of five LDH bands, CCCC, CCCC', CCC’C',
CC'C'C* and C’C’C'C*. No evidence was found that LDH C
polypeptides hybridised with LDH A or LDH B polypeptides
in vivo but AC and BC hybrids could be formed in vitro.
More BC hybrids were produced than AC hybrids, suggesting
that B polypeptides resembled C polypeptides more than
A polypeptides did.
In several animals, including Man, more than five
LDH bands have been found with starch gel electrophoresis.
Additional proteins usually take the form of sub-bands associated with one or more of the major bands. Kaplan
(1968) illustrated the mechanism by which these sub-bands
could be produced. In certain mouse tissues there were two 27 allelic forms of the M (A) polypeptide, resulting in fifteen LDH bands with the following constitutions
+
LDH 1 HHHH BBBB
LDH 2 MaHHH AaBBB
MbHIiH AaBBB
LDH 3 MaMaHH AaAaBB
M^HH AaAbBB
MbMbHH AbAbBB
LDH 4 MaMaMaH AaAaAaB
MaMaMbH AaAaAbB
m^Vh AaAbAbB »b. b.b_ MbMbMbH A A A B
A a.a.a.a LDH 5 MaMaMaMa A A A A
AaAaAaAb
MaMaMbMb AaAaAbAb
AaAbAbAb
MbMbMbMb AbAbAbAb 28
The pattern on the previous page would be
obtained if two alleles were operating to produce the
slow polypeptides M& (Aa) and (A^). If they v/ere
producing two fast polypeptides H& (Ba) and (B^)
there would also be fifteen sub-bands but LDH 1 would have five and LDH 5 would have a single band. If both
polypeptides had two alleles, thirty five sub-bands would be produced. LDH isozymes would only show a multiplicity of bands if the altered allelic genes produced polypeptides with altered electrophoretic mobilities•
(b) Malic and Isocitric Dehydrogenases. (EC
1.1.1.37, DPN dependent MDII, 1.1.1.40, TPN dependent MDH;
1.1.1.41, DPN dependent IDH, 1.1.1.42, TPN dependent IDH).
The molecular constitutions of these enzymes are not as well understood as that for LDH. In some organisms both enzymes are found in supernatant cell fractions and in mitochondria. TPN and DPN dependent enzymes are probably produced by different gene systems (Henderson,
1968). Henderson also believes that vertebrate, invert ebrate and plant malic dehydrogenases have evolved as separate systems.
Most investigations have been carried out on TPN 29
dependent MDH and IDH. Kaplan suggests that MDH isozymes
are conformational*, that is, they are polypeptides with
identical amino acid sequences which become folded so that
different charged groups are exposed at the surface of the molecule. In this way the electrophoretic mobility of
isozymes can be altered epigenetically (Epstein and
Schech.ter, 1968). Munkres (1968) reports that in Neurospora sp., mitochondrial MDH is a heteropolymorphic* protein with sub-units. Henderson (1968) is uncertain whether the hybrid molecule produced by crossing two strains of mice has three or five sub-bands and consequently has not decided whether vertebrate TPN dependent MDH is a dimer or a tetramer. Other opinions favour a dimeric structure.
Henderson also states that TPN dependent IDH forms a hybrid enzyme with three bands and is therefore a dimer.
(c) Amylase:- Little has been published about the molecular structure of the amylases. It is not certain whether the systems studied consist of allelic isozymes.
Using immunological methods, Me. Geachin (1968) reports that pig liver and pancreatic amylases are isozymes of salivary amylase. Berk et al. (1965) found three amylase fractions in rabbit serum and Muus and Vnenchak (1964) describe four fractions in crystallised human salivary amylase which leads them to suggest that salivary amylase is a trimer 30 CHAPTER 1, Additional References.
1.1.4. Contribution of human-studies.
Giblett et al. , 1959* Blake et al., 1969* Davidson et al., 1965, Kraus and Neely, 1964, Poulik, 1962, Rob son and Harris, 1965? Spencer et al., 1964, Hopkinson and Harris, 1966, Hopkinson et al., 1964, Robson and
Harris, 1965* Tashian et al., 1963, Lie-injo, 1967*
1.1.3« DNA and molecular genetics.
Fincham, 1939, Huehns and Shooter, 1965* Muirhead and Perutz, 1963? Anfinson, 1960, Yanofsky, 1963* Jacob and Monod, 1961, 19631 Monod et al., 1963
1.1.6. The isozyme concept.
Joshi et al., 1965,Leventhal et al., 1962, Markert and Appella, 1961, Mysels and Scholten, 1962, Wieland and Pfleiderer, 1961 , Wieme, 1962, Wiess and Ephrussi, 1966.
1.2, Enzymes and the evolution of proteins.
Bernal, 1963* Allison and Kaplan, 1964, Rutter,
1964, 1963? Rutter and Groves, 1964, Granick, 1965?
Horowitz, 1963
1.2.2. Epigenetic factors and enzyme phenotypes.
Allen, J., 1961 ,
1.2.4. Systematics and population studies.
(See 1.1.4 and Parr, 1966, Parr and Fitch, 1967, 31
Chapter 1, Additional References (cont'd.)
1.3»3« Subunit structure of dehydrogenases.
LDH. Schwartz, 1960, 1964, Kornberg and Pricer, 1951,
Appella and Markert, 1961, Costello and Kaplan, 1963,
Goldberg, 1965, (a) and (b), Fritz and Jacobson, 1963,
Harris, 1964, Schlesinger and Levinthal, 1963, Shaw,
1965, Shaw and Barto, 1963, Markert, 1962.
MDH, Baker and Mintz, 1969, Kitto et al., 1966,
Shows and Ruddle, 1968.
IDH. Henderson, 1965, 1968.
General.
LDH. (a) Tissue specificity.
Vesell, 1963, Vesell and Bearn, 1958, Wilson et al.,
1963.
(b) Testicular.
Blanco and Zinkham, 1963, Goldberg, 1964, Zinkham et al., 1964.
(c) In development.
Nace et al., 1961, Rosa and Schapira, 1965, Wald,
1961.
(d) In disease.
11 Delbruck et al., 1959, Gerhardt and Clausen, 1963, 32
Chapter 1, Additional References (cont'd.)
Goldman et al., 1964, VeseTl et al. , 1962, Vesell and Brody, 1964, Wieme, 1959? Wrobleski et al., 1960,
Wrobleski and Gregory, 1961.
(e) In pregnancy.
Friedman and Lapan, 1961•
(f) Substrate inhibition.
Kaplan et al., 1968, Meister, 1950, Stambaugh and
Post, 1966 (b), Umbarger, 1961.
(g) Dilution effects.
Vesell, 1962.
(h) Temperature.
Stambaugh and Post, 1966 (a), Vesell and Yielding,
1968, Zondag, 1963*
MDH.
Englard et al., 1966, Harrison, 1963, Henderson,
1966, Thorne et al., 1960, Grimm and Doherty, 1961.
Multiple molecular forms of enzymes.
Fredrick, 1962, Kaplan, 1963* Kaplan et al., i960,
Nisselbaum et al., 1961, Pfleiderer et al., 1968, Popp and Popp, 1962, Wieland et al., 1961. 33
Chapter 2. Methods and Techniques.
2.1.1. Electrophoresis Using Solid Supporting Media.
Electrophoresis is the separation of charged molecules by subjecting them to an electric current while they are suspended in a buffer solution. Proteins can be separated in this way because they have excess positive or negative charges depending upon the amino acids they contain. Proteins or small charged structures such as red blood cells can be separated in solution but for the identification and typing of soluble proteins a solid supporting medium is preferred. Such media include filter paper, cellulose acetate membranes or gels, starch gel, starch paste, agar gel and acrylamide gel.
(a) Filter Paper. The first successful type of electrophoresis used filter or chromatography paper as a supporting medium. Prior to this, proteins had been separated and purified by chemical means which took advantage of their differing solubilities in concentrated salt solutions. These latter methods were time consuming but by filter paper electrophoresis, human serum could be separated into six or seven proteins in approximately sixteen hours. The method was suit- able for investigating particular proteins such as trans ferrins and haptoglobins. It was not sufficiently sens itive to detect genetically determined variants of these two proteins.
(b) Cellulose Acetate. The use of a cellulose acetate membrane enabled smaller quantities of serum to be used. The proteins are not absorbed by the membrane but flow along the surface in the buffer layer. This method is most useful in clinical work as the time for electrophoresis is shortened to less than two hours.
A recent modification uses a cellulose acetate gel, sold as •’Cellogel" (Chemetron, Milano). Enzymes have been successfully separated using cellulose acetate but its use does not increase the number of proteins which can be detected in human serum.
(c) Starch Gel. In 1955 Smithies developed a method by which human serum could be separated into fifteen or sixteen different fractions. Hydrolysed starch in buffer is boiled, then poured into a mould and allowed to cool. Starch gel electrophoresis has been the most widely used method for separating proteins and especially for enzymes. The running time ranges from six to twenty two hours. Samples can be run vert ically or horizontally, the vertical method being the 35 more sensitive. Starch paste electrophoresis does not use soluble starch but a mixture of starch granules in buffer. It is used as a preparative technique rather
than an analytical one. Its main advantage is that large amounts of sample can be run and the separated proteins can be removed in blocks and eluted for invest
igation.
(d) Agar Gel. A thin sheet of agar gel spread on glass plates was used by Wieme (1959). Two advantages are that the running time is short and the background is colourless and transparent. The widest use of agar gel is for immuno-electrophoresis.
(e) Acrylamide Gel. The use of a synthetic gel, acrylamide monomer, has been developed. There are two ways in which acrylamide gels are used.
(i) As a flat gel on which many samples may be run at the one time (Raymond et al., 1962). The acrylamide preparation, "Cyanogum 41" (BDH) is mixed with two polymerising agents, DMAPN (dimethylamino- propionitrile) and ammonium persulphate. It is then poured into a mould as a flat slab.
(ii) As a number of cylinders, each set separately in small tubes. This method is known as
"disc electrophoresis" and was developed by Davis and 36
Ornstein (1964). Figure 2-1 illustrates a complete gel.
The running gels are first allowed to polymerise in
/ glass tubes approximately two inches long and three
sixteenths of an inch in internal diameter (A). This
gel needs ammonium persulphate as a polymerising agent
and does not require ultra-violet light. Acrylamide
monomer is combined with bis-acrylamide in such prop
ortions as to make a gel with a pre-determined pore size.
Pore size may be varied within wide limits but physical
considerations limit the sizes which can be used. Gels
become softer as pore size increases and more brittle as
pore size decreases. The texture of the gel also limits
the pH range in which the gels can be run and successfull;
removed from the tubes afterwards for staining. A small
amount of water is carefully placed on the upper surface
of the running gel so that it will set in a straight
line •
After the running gel has polymerised, the water is poured off and a small amount of '’large pore"
gel is layered on the flat surface of the running gel.
(B) Water is also pipetted onto this gel to provide a
flat interface between it and the next layer. Polymer
isation takes place only in the presence of ultra-violet light. The function of the large pore gel is to arrange 37 the protein molecules in "stacks" prior to the electro phoresis run. Consequently a clear separation can be
/ achieved in a short distance.
The solution to be examined is then placed on top of the "stacking" or "large pore " gel, either in "large pore" gel or some viscous solution such as
*f.O Molar sucrose. This gel is known as the "sample" gel (C). It is not essential that the sample gel be polymerised.
FIGURE 2-1.
Complete Gel for Disc Electrophoresis.
C
B —
A
HOLDER 38
The protein bands do not exhibit any tendency to "tail" as they do with both starch and acrylamide sheet gels. It is necessary to fix the proteins as soon as possible after the run as there is rapid diffusion when the gels are removed from the electric field. A maximum of thirty five proteins have been demonstrated with disc electrophoresis.
2.1.2. Interpretation of Protein Electrophoresis. All electrophoretic methods make use of the amphoteric prop erties of proteins, although movement of proteins through a porous medium in an electric field is not entirely due to their residual surface charges. Molecular size, the accessibility of hydrophobic groups and the buffer used contribute to the protein pattern obtained. The time taken for the electrophoresis run can be important if it causes unfolding or denaturation of the proteins. The nature of the supporting medium has an effect upon the final protein pattern.
All electrophoretic patterns are artefacts and care must be taken in interpreting them. For this reason it is necessary to consider patterns obtained with disc electrophoresis independently from those found with any other medium.
It is obvious that the one type of electroph- 39 oresis will not be suitable for all proteins. The gel slab method of Raymond is better for studies in which the mobility of the proteins is different, not the pattern.
Transferrins are such proteins and disc acrylamide is not a satisfactory method for typing transferrins.
2.1.3. Method of Electrophoresis. The method of choice for the present investigations is that of disc electrophoresis and follows closely the procedure described by Davis (1964). The apparatus used is illustrated in Figures 2-2 and 2-3. The electrophoresis baths are based upon a design used at the Prince of Wales Hospital at Randwick, New South Wales. It is possible to run thirty two samples at one time with the equipment illust rated but there is no reason why more units cannot be connected in parallel up to the current capacity of the power unit.
The electrophoresis run takes from one hour to eighty minutes at room temperature. Brom-phenol Blue is incorporated with the buffer in the cathode bath. Some of the dye attaches to any albumin present but the free dye runs with the buffer front. The dye is allowed to run a distance of one and a half inches. The voltage drop across the terminals of each bath is 240 volts, which is the same as the voltage reading of the power source. The current passing through each bath at the beginning is 3^ milliamps, FIGURE 2-2. 40
Disc Electrophoresis Apparatus.
Lay-out of disc electrophoresis apparatus,
set up to run 32 samples.
FIGURE 2-3.
Electrophoresis Bath.
UPPER BATH LOWER BATH negative positive
Close up of one electrophoresis bath.
Eight samples can be run in each b;ath.
Electrodes are carbon, but platinuim can be used. 41 falling to 19.5 milliamps by the end of the run. Immed iately after the completion of the run, which is carried out at room temperature, the gels are removed from the tubes by running a needle between the gels ar.d the glass while the tubes are submerged in water. The gels slip out of the tubes and are then placed in small test tubes to be stained for the appropriate enzyme.
Composition of Gels and Buffer. Stock sol utions were similar to those recommended by Davis and described in Part II of a "Canalco" Bulletin on Disc
Electrophoresis, "Materials and Methods". The only diff erence is in the ammonium persulphate used for polymer ising the small-pore gel.
(a) Small pore or running gel.
Stock Solution A
1 Normal HC1. 48 ml.
2-amino-2-(hydroxymethyl)-1,3-propanediol,
(Tris), (Sigma) 36.6 g.
N ,N,N',N’-tetramethylethylenediamine, (TEMED),
(Eastman) 0.46 ml.
H20 dist. to 100 ml. (pH 8.9)
Stock Solution C
Acrylamide monomer, (Tokyo Kasei) 30«0 g«
N ,N'-methylenebisacrylamide,(Eastman)
0.8 g 42
(Stock Solution C, continued.)
Potassium ferricyanide, K^Fe(CN)^ 15-0 mg
H2Q dist to 100 ml
Small pore solution No. 1 is made by combining
1 part Solution A
2 parts Solution C
1 part H20
Small pore solution No. 2 is made as follows,
Ammonium persulphate, 0.28 g.
H20 dist. to 100 ml.
Immediately before use, the running gel is made by mixing equal volumes of small pore solution 1 and small pore solution 2. The pH of the running gel is between pH 8.7 and 9.0.
(b) Large pore gel, used as spacer gel and sample
gel.
Stock Solution B
1 Normal HC1 48 ml
Tris, (Sigma) 5.98 g.
H20 dist. to 100 ml. (pH 6.7)
Stock Solution D
Acrylamide monomer (Tokyo Kasei) 10.0 g.
N,N’-methylenebisacrylamide,
(Eastman) 2.5 g
H20 dist to 100 ml 43
Stock Solution E
Riboflavine, (BDH) 4.0 mg
to 100 ml.
Large pore gel is made by combining
1 part Solution B
3 parts Solution D
1 part Solution E
3 parts H^O dist.
It is necessary to expose large pore gel to ultra violet light in order to polymerise the gel. The final pH is between pH 6.5 and 6.8.
(c) Reservoir Buffer
Tris 6.0 g Glycine 28.8 g
H2O dist. to 100 ml diluted 1 in 10 2.1.4 Advantages of Disc Electrophoresis (a) The distinctive properties of the disc electrophoresis tech nique produce a series of compact protein bands in a short time.
(b) A readable pattern can be obtained with small amounts of sample. This is an obvious advantage in survey work.
(c) There is a high degree of reproducibility if care is taken in preparing and running the samples.
There is also an absence of the multiplicity of minor bands which are found even in normal samples when they are run on starch gels.
(d) Most enzyme locating agents contain exp ensive ingredients such as TPN+ and tetrazolium salts.
Using disc electrophoresis , a hundred samples can be tested using the same amount of reagent as would be required for one starch gel.
2,1.3. Disadvantages of Disc Electrophoresis.
(a) Since samples are run on individual gels it is difficult to compare mobilities. If there is a question of differing mobilities it is necessary to make many duplicate runs.
(b) Diffusion rate of proteins is rapid and ^5 gels must be fixed and stained immediately. It is also necessary to keep a photographic record of all gels as they cannot be stored. Diffusion occurs even if the enzymes have been stained, but it can be slowed down by storage in a refrigerator.
(c) It has not been possible to slice disc gels satisfactorily so only one substance can be located with each gel. This disadvantage is lessened by the fact that many gels can be run at one time. 46
2.2.1, Collection of Blood Samples. Most blood samples
were collected in Acid-Citrate-Dextrose solution with
added inosine (ACDI). This anti-coagulant is recommended
by the World Health Organisation for use with blood samples
collected for glucose 6 phosphate dehydrogenase estimations.
(W.H.O., 1967). Heparin was also used, especially when
it was not convenient to carry ACDI which must be stored
at low temperature. EDTA Na^ or EDTA K were not used with
the dehydrogenases because they chelate metal ions and
many dehyrogenases are activated by metals.
Human blood samples were obtained by venepuncture.
Animal blood was obtained in several ways, marsupials
being bled from the tail vein. Blood was obtained from most other animals post-mortem.
2.2.2. Transport and Storage of Samples. Blood samples were put into cold polystyrene containers immediately after collection. They were either transferred to a refrigerator at 4-6°C. at the end of the day or despatched by air in the polystyrene containers packed with fresh ice.
The samples were kept refrigerated at the airport upon arrival. Immediately after they were received at the laboratory, the samples were centrifuged and the plasma removed and stored in a deep freeze at -20°C. The red cells were washed three times with physiological saline
(0.85% w/v. )
Storage. (a) If the red cells were fresh, they were placed in a mixture of glycerol ((OH) v) and phos- 5 Z) 5 phate-citrate buffer and stored at -20°C. The final glycerol concentration was approximately 35%. This method of preserving red cells is described in the third edition of Mollison's book, "Blood Transfusion in Clin ical Medicine”, Blackwell, Oxford, 1962.
(b) Washed red cells were placed directly into the deep freeze at -20°C. and stored as haemolysates if there was excessive haemolysis in the sample, since they could not be stored in glycerol as whole cells.
2.2.3. Preparation of Samples for Electrophoresis.
Ten drops of distilled water were added to two drops of packed cells. After haemolysis had occurred the blood was centrifuged at 2,300 x g. as it was found that the presence of cellular material interfered with the running of the sample in acrylamide disc,gels. Haemolysate was drawn into a small piece of transparent PVC tubing fitted over a sawn-off hypodermic needle attached to an automatic pipette set to take up 0.*f ml. A mark on the tubing ensures that a fixed (but unknown) amount of haemolysate, (approximately 0. 015 ml.) is mixed with 48
0.4 ml. of "large pore" gel.
Sample gels were allowed to polymerise and this took approximately one hour. The presence of red colour in the haemolysate slowed down the rate of poly merisation which took approximately half an hour with serum.
2.1» Staining Techniques.
Principles. General reference, "Histochemistry,
Theoretical and Applied", A.G. Everson Pearse, second edition, J. and A. Churchill Ltd., London, '\96'\.
Dehydrogenases. All dehydrogenases except succ inic dehydrogenase (EC 1.3»99«1) act in the same way.
Hydrogens are removed from the substrate and passed to a co-enzyme. This may be diphosphopyridine nucleotide (DPN+) which was first named "co-enzyme I" (Harden and Young) and is now called nicotinamide adenine diphosphate (NAD+), or triphosphopyridine nucleotide (TPN^), first known as
"co-enzyme II" (Warburg) and now called nicotinamide adenine diphospho phosphate (NADP+)• The terms NAD+ and
NADP+ were proposed in 19&1 by the Commission on Enzymes of the International Union of Biochemistry as conforming more accurately to the conventions of chemical nomenclature.
(Cited in Conn and Stumpf, "Outlines of Biochemistry", 49
John Wiley and Sons Inc., New York and London, 19&5* p. 130) but the symbols DPN and TPN will be retained in this text.
The hydrogens are passed to other substances which may include flavin(e) adenine dinucleotide (FAD) via the "electron chain", the cytochromes and cytochrome oxidase. Dixon and Webb (1966) prefer to consider the movement of hydrogens rather than electrons (pp. 262,
263). During the alternate oxidations and reductions of the cytochromes the energy level falls and the lib erated energy is stored, mainly in the ATP molecule.
The hydrogens are finally passed to cytochrome oxidase, where they combine directly with molecular oxygen to form water.
Figure 2-4 is a diagram showing how hydrogens are passed from the substrate to a suitable dye which will make visible the dehydrogenase activity of the enzyme. Cyanide is specific for inhibiting the activity of cytochrome oxidase in vivo and this prevents the passage of hydrogens to molecular oxygen to form water, thus making them available to reduce some other hydrogen acceptor. In vitro it is necessary to substitute hydrogen acceptors with higher redox potentials than that of the system from which it must accept electrons. Such sub- FIGURE 2-A. 50
HYDROGEN PATHWAY IN CELLULAR RESPIRATION.
(Adapted from Pearse, p. 561).
SUBSTRATE TPNH dehydr Dgenase _— -> MTT DPNH
PRODUCT FAlf (FMN) FORMAZAN cytochrome b (blue)
♦ cytochrome c
cyanide__
2H+ * cytochrome oxidase (a and a^)
* Pathway of hydrogens with succinic dehydrogenase which does not require TPN or DPN. Pathway followed by hydrogens (electrons) in cellular
respiration. Solid lines show the pathway in vivo,
dotted lines show pathway iri vitro when the cytochrome
oxidase is poisoned with cyanide and tetrazolium dyes
are substituted as hydrogen acceptors. 51 stances are the tetrazolium salts, which have the prop erty of forming coloured compounds when reduced to formazans (Pearse). Figure 2-5 illustrates the reaction which takes place when a diformazan is formed from a ditetrazolium salt by reduction. The tetrazolium salt used in the present work is MTT, (5-(^i5-dimethyl- thiazolyl-2)-2,5-diphenyl tetrazolium bromide, Sigma) which has a higher redox potential than that involved in the oxidation of lactic acid to pyruvic acid.
Phenazine Methosulphate (PMS) is a soluble redox dye which has been found to assist in passing hydrogens
(electrons) to the tetrazolium dye although the manner in which it does this is not known (Pearse). One suggestion made by Singer et al. (1957) is that PMS can accept electrons from the Fe++ ions in the iron- flavin(e) molecule, whereas tetrazolium salts can accept electrons only from the flavin(e) part of the molecule.
Cyanide is incorporated in the incubation mixture but its role is obviously not that of inhibiting cytochrome oxidase. It forms an addition compound with
DPN + which is enzymically inactive (Dixon and Webb, p. 570) and it is included to inactivate the DPN+ once the hydrogens have been passed to the tetrazolium salt. FIGURE 2-5. 52
REDUCTION OF DITETRAZOLIUM DYES.
Ditetrazolium salt Diformazan (colourless) (blue)
5 c6h5 C—M-Nh-C6H
In
- OCM
-V- 4-H
c— ■N-NH- \ ^6
Mode of action of reduction of a ditetrazolium salt to form a diformazan. (After Pearse, p. 558). 53
Lactic Acid Dehydrogenase. The reaction carried out by LDH occurs at point "a" in the respiratory cycle, illustrated in Figure 2-6. Aerobically, the overall reaction
lactic acid pyruvic acid
HO 0
"\/C C x ^ | H C OH c—0 + I I CH., CS 5
DPN + DPNH + H is heavily weighted kinetically to proceed from left to right, although anaerobically in muscle the direction is towards the formation of lactic acid and the oxidation of DPNH + H. Iri vitro the conditions are maintained in the direction of pyruvic acid formation and the reduction of DPN + .
Incubation Mixture. (Modified from that of Allen, 1963).
0.05M. Tris-HCl Buffer, pH 7.4 ; ; .. 36 ml.
dl Lactic acid (Sigma, 98-99% w/v)
(Sodium salt);...... 0.1 ml.
DPN+ (Sigma);...... 20.0 mg.
PMS (Phenazine methosulphate,
2 mg./ml.), (Sigma)...... 0.8 ml.
0.06m. KCN in aqueous solution.#..10.0 ml.
MTT (2 mg./ml.), (Sigma)...... 12.0 ml. (for 32 gels.) FIGURE 2-6
GLYCOLYTIC AND TRICARBOXYLIC ACID CYCLES.
Glycolytic cycle Tricarboxylic acid cycle
GLUCOSE
V G 6 P
* cis-ACONITATE PYRUVATE CITRATE
ACETYL CoA iso-CITRATE
OXALOACETATE LACTIC ACID <* KETOGLUTARATE
MALATE SUCCINATE
FUMARATE
Diagram of cellular respiratory cycles, showing the sites of action of the dehydrogenases mentioned in the text (dotted lines). Adapted from Pearse, p. 573- 55
Malic Acid Dehydrogenase. MDH takes part in the tri
carboxylic acid or Krebs’ cycle of oxidative respiration.
There are DPN and TPN dependent forms which are found
in cytoplasm and mitochondria. The DPN dependent enzyme
is known to act at point ”b” in Figure 2-6 and catalyses
the reaction
1-malic acid oxaloacetic acid
COOH COOH
H C H
H C OH C=0 | + 2H
COOH COOH
DPN + DPNH + H
TPN + TPNH + H
The physiological significance of the TPN dependent MDH has not yet been made clear although it presumably catalyses the same reaction as the DPN dependent form.
Incubation Mixture. This is the same as for LDH except that the substrate is 1-malic acid (Sigma) 56
Isocitric Acid Dehydrogenase. IDH occurs in DPN and
TPN dependent forms and the enzyme most often studied uses TPN as co-enzyme. The reaction utilising IDH occurs at point "c" in Figure 2-6. The reaction is
isocitric acid oxalosuccinic acid
H2C—COOH H2C—COOH
H C —COOH H C- COOH
HO C- COOH 0— C— COOH
H
TPN+ TPNH + 2H
DPN+ DPNH + 2H
The dehydrogenase reaction results in the formation of oxalosuccinic acid but in the presence of Mn ions and under physiological conditions, decarboxylation takes place and oc keto-glutaric acid is formed without the intervention of another enzyme.
Incubation. The incubation medium is the same as that for LDH and MDH. DPN is used so that the bands produced when isocitric acid (Sigma) is used as substrate can be compared v/ith those produced with lactic acid and malic acid 57
Amylase. The amylase occurring in animals is o(. amylase
(«<-1 ,4-glucan 4-glucano-hydrolase) . It hydrolyses the
amylose chains of starch in a series of steps which
first produce dextrins and finally maltose molecules.
The cleavage occurs at the 1,4 glycosidic linkage.
Amylase may be made visible after electrophoresis in
three ways.
(a) If starch gels are used, amylase may
be detected by its hydrolytic action on the starch,
the "amyloclastic" method. (Ashton, 1965)*
(b) Amylase converts starch to sugars and
these can be identified on the gels using n-methyl-
p-phenylene diamine.
(c) Starch forms a blue-black compound with
iodine (Van Loon et al.,1952; Mc.Geachin and Lewis,
1959; Mc.Geachin and Potter, 1961; Muus and Vnenchak,
1964; Dreiling et al., 1963 and Berk et al., 1965).
In the work reported in Chapter 3» 5% hydrolysed
starch is incorporated in the acrylamide running gel.
After electrophoresis the gel is left overnight, during which time the amylase digests the starch. The gel is
then incubated in a 2% Kl/l^ solution. The areas of amylase activity show up as colourless lines with a deep blue background. 58
Chapter 2. Additional References.
2.1.1. Electrophoretic techniques.
Acrylamide. Barka, 1961, Clarke, 1964, Ferris et al,
Maizel, 1964, Nakamichi and Raymond, 1962, Raymond and
Nakamichi, 1962, Raymond and Way, 1960, Raymond and Wein- traub, 1959, Reisfeld et al., 1962, Williams and Reis- feld, 1964, Raleigh, 1964, Zingale et al., 1963.
Starch. Poulik and Smithies, 1998, Poulik and Edel- man, 1961, Poulik, 1957i Lawrence et al., 1960.
Paper. Poulik and Smithies, 1958, Sayr and Hill,
1957.
General. Chang and Steward, 1962, Abrahamson et al.,
1942, Allen and Hyncik, 1963* Ressler et al., 1963? Cann and Goad, 1968.
2.3.1. Staining techniques.
Hunter and Burstone, 1958, Hunter and Markert, 19571
Lehrer and Ornstein, 1959. 59
CHAPTER 3« Studies with Human Material,
5.1. Introduction. The aims of the study were
(a) to use disc acrylamide electrophoresis to investigate patterns produced by certain isozymes and
(b) to conduct a quantitative survey of an enzyme which had been shown by others to exhibit few electrophoretic variants.
In both cases it was desired to find suitable means of detecting genetic variations using enzymes which were easily obtained and methods which were economical and practical for field survey work.
The enzymes which were studied were
(a) (i) lactic acid dehydrogenase in red cells, white cells and serum,
(ii) malic acid dehydrogenase in red cells, white cells and serum,
(iii) isocitric acid dehydrogenase in red cells, white cells and serum,
(iv) amylase in serum
(b) (i) glucose 6 phosphate dehydrogenase in red cells 6o
3.2. Lactic Acid Dehydrogenase,
3.2.1. Collection and Storage of Blood. LDH is a water soluble enzyme located in the cytoplasm and it
is desirable to preserve the red cells intact until
just before they are to be tested. Haemolysis should be avoided when collecting blood. If cells are to be stored they should be placed in buffered glycerol solution and kept in the deep freeze at -20°C. (see
Methods and Techniques, Chapter 2). Otherwise the blood should be collected in an anti-coagulant and the red cells kept in contact with their own plasma until haemolysates are to be prepared for electrophoresis.
Anti-coagulants. (i) ACDI, a combination of citric acid, tri-potassium citrate, dextrose and inosine (W.II.O.
Report, 1967)? 0.19 ml. added to each 1 ml. of whole blood. (ii) Heparin, (Dumex), 0.04 ml. to
4 ml. of whole blood.
(iii) Ethylene diamine tetracetic acid, disodium salt, (BDH), approximately 0.005 milli grams to 1 ml. of whole blood.
3»2.2. LDH in Red Cells. The LDH pattern of freshly 61
prepared haemolysates comprised three or four bands. The
three bands which were always present'corresponded with
LDH 1, LDH 2 and LDH 3 according to Markert’s terminology.
For the purpose of the present study, four red
cell LDH types were distinguished. The differences were
not in electrophoretic mobility but were quantitative
differences. The four LDH types are illustrated in
Figure 3-1•
Type 1, LDH 1 is the heaviest band with a regular
decrease in the amounts of LDH 2 and LDH 3i the latter
being the lightest band.
Type 1 mod. LDH 1, LDH 2 and LDH 3 are the same as in
Type 1 but the fourth minor band is present immediately
behind LDH 2.
Type 2. The relationship between the intensities of
LDH 1 and LDH 2 are not markedly different from those
of Type 1 but LDH 3 is much lighter and may be almost
invisible.
Type 2 mod. The same as Type 2 but possessing the
fourth minor band behind LDH 2.
A careful inspection of approximately three hundred samples of fresh blood was made and it was decided that Type 2 was not an artefact caused by a dilution effect (Vesell, 1962).
No differences in electrophoretic mobility were observed. FIGURE 3-1 62
DISC ELECTROPHORESIS LDH PATTERNS
IN HUMAN RED CELLS. 63
LDH types 1 and 2 mod. are shown in Figure 3-2 (a and b).
3.2.3. Reproducibility of LDH Patterns. It has been established that the electrophoretically slow LDH bands
(LDH 4 and LDH 5) are less stable than the fast LDH bands
(Vesell, 1965). It is necessary, therefore, to determine if the differences between LDH Type 1 and LDH Type 2 are artefacts. The following tests were applied.
(a) Effects of Anti-coagulants and Storage on LDH Patterns.
Blood from the one subject was collected in (i) ACDI,
(ii) Heparin, (iii) EDTANa^. One series was washed immed iately with 0.85% NaCl (physiological saline) and the washed cells were haemolysed with ten volumes of distilled water. The haemolysates were then subjected to electro phoresis on disc acrylamide gels. The elapse of time between collection of the blood and the commencement of electrophoresis was two hours. The remainder of the blood sample was stored at -20°C. A second series of blood samples was left at room temperature for 72 hours after collection to simulate field conditions. The cells were then washed, one aliquot was subjected to electrophoresis and the remainder stored at -20°C. Glycerol was not added to either of the stored bloods.
Samples of haemolysates from both treatments FIGURE 3-2. 64
LDH PATTERNS IN HUMAN RED CELLS.
3
2
1
ABC
A. LDH Type 2; B. LDH Type 1 mod.
C. Malic Acid Dehydrogenase. 65 v/ere tested at intervals of one week, one month, three months and four months. The results arfe shown in Table
3-a.
TABLE 5 - a.
Effects of Anti-coagulants and Storage on
Red Cell LDH Patterns.
Anti No one one three four coagulant storage week month months months ACDI 1 mod. 1 1* 1* 1*
Hep. T mod. 1 1* A EDTA 1 mod. 1 1* 1* yj *
ACDI 1 mod. 1 1* 1*
Hep. 1 mod. 1 2 2 B 1* EDTA 1 mod. 1 1* 2 2
* LDH 5 is fainter and it is difficult to determine if
the LDH type is 1 or 2.
A- sample tested immediately, then placed in the deep freeze
B- sample kept at room temperature for 72 hours before being
tested and placed in the deep freeze.
Results:- 1. The anti-coagulants used do not affect the
LDH pattern of fresh blood or of blood kept
at room temperature for 72 hours. 66
2. The pattern is not altered when blood is
left at room temperature•for 72 hours,
3. The fourth band, running behind LDH 2, has
disappeared after one week’s storage in
the deep freeze.
4. When ACDI is used as anti-coagulant, the
LDH is still typed as LDH Type 1 after
four months. When heparin and SDTANa^ are
used, the LDH is typed as LDH Type 2 at
three months in the samples which were
left for 72 hours at room temperature.
3. LDH 1 and LDH 2 remained unchanged in
intensity throughout the period of observ
ation but in those samples marked with an
asterisk, LDH 3 decreased in intensity with
time of storage.
6. Sub-bands other than the fourth band already
mentioned, were not produced by storage
when run on disc electrophoresis.
Conclusions:- When fresh blood samples are used, the
LDH pattern is reproducible and is not altered by the use of ACDI, heparin or EDTANa^ as anti-coagulant.
Changes occur during storage at -20°C. and these result in the loss of the slowest LDH 3* 67
(b) Repeated Samplings from the Same Individual.
Blood from ten volunteers was collected in ACDI at weekly intervals for five weeks. Haemolysates of washed cells were subjected to electrophoresis on disc acrylamide gels on the same day as the blood was collected.
The results are shown in Table 3-b.
TABLE 3 - b,
REPEATED TYPING OF LDH FROM THE SAME SUBJECT.
Subject LDH type
1 2 3 4 3
1 E.M. ? 2 m 2 2 2 2
2 E.N. 3 s.s. cT 1 m* 1 m* 1 m 1 m 1 m 4 P.C. 9 1 m 1 m 1 m 1 m 1 m 5 P.R. ? 1 m 1 m 1 m 1 1 m 6 J.G. ? 1 m* 1 m 1 m 1 m 1 m 7 R.W. Cf 2 m 2 2 m 2 m 2 m 8 L.L. Cf 1 1 m 1 * 1 m 1 m 9 J.M. ? 1 * 1 1 - 1 10 S.St. 5 1 m abn* * 1 -- * Additional minor bands are present. (see * * See results 1 68 Results. 1. Six of the ten bloods showed changes in LDH patterns during the course of the experiment. Only one of these (10 - 2) could not be typed as LDH Type 1 or LDH Type 2. The band at the LDH 2 position was heavier than that at the LDH 1 position, producing the following pattern instead of It is probable that there was a large amount of the minor (mod.) band and that this caused the widening of the LDH 2 band. The sample would therefore be Type 1 mod. 2. Extra minor bands appeared irregularly in 3? 6, 8 and 9. These are marked * in Table 3-b. They did not correspond with any known LDH band, became visible much later and were faint. There is little chance that an experienced observer would confuse them with LDH bands. 3. In samples 2, 3» 6 and 9 the LDH typing was the same in each of the five tests of freshly drawn blood. In the other five, the minor (mod.) band was some- 69 times present and sometimes absent. There was no change in typing from LDH-Type 1 to LDH Type 2 or from LDH Type 2 to LDH Type 1. Conclusions. LDH patterns from fresh red cells of the same individual show minor differences from sampling to sampling. These differences are due to the behaviour of the minor (mod.) band which may not be consistently present or absent in the blood of the one individual. It is suggested that the appearance of the mod. band is due to a conformational* rearrangement of the polypeptides of the molecule ini vitro or when running on disc acrylamide gels. It is similar to the sub-bands which appear in LDH patterns run on starch gels, with the difference that the sub-band only occurs with LDH 2, the ABBB tetramer, in acrylamide gels. 3.2.*f. The Occurrence of LDH Types 1 and 2 in Populations. The results obtained in (a) and (b) of Section 3.2.3. suggest that red cell LDH Types 1 and 2 represent two phenotypes differing in a quantitative character, namely the amount of LDH 3 produced relative to the amounts of LDH 2 and LDH 1. The difference could be (a) in the stability of the respective LDH 3 tetramers or 70 (b) in the ability of the individual to make slow tetramers. LDH 1 is BBBB and LDH 2 is ABBB while LDH 3 is AABB. If few A polypeptides are made they will go preferentially to ABBB tetramers. Thus the amount of LDH 3 produced would be genetically determined by the ability of the individual to make A polypeptides. The results obtained in the storage experiment (Table 3~a) do not support (a) , while the results obtained in this and the repeated sampling experiment (Table 3-b) indicate that one of the two types is found consistently in the one individual. (1) Studies with Fresh Blood Samples. Blood was obtained from 189 blood donors, 107 of whom were born in Great Britain or Australia of parents of British stock (Populat ion A). The remaining 82 were born in Greece, Italy, Malta or Australia and came from entirely Greek or Italian stock (Population B). Blood was tested for LDH within 2k hours of collection. The results are shown in Table 3-c. Results. Three samples in Population A and two in Pop ulation B were abnormal and are illustrated in figure 3-3 TABLE 3 - c 71 LDH TYPES IN TWO DIFFERENT POPULATIONS. Population A Population B (of British stock) (of Greek or Italian stock) LDH type No. % LDH type No. % 1 41 1 30 62 38 5^ 66 1 mod. 21 1 mod. 24 2 31 2 12 42 49 26 31 2 mod. 11 2 mod. 14 X 1 Y 1 3 3 2 3 X mod. 2 Y mod. 1 Totals 107 100 82 100 LDH Types 1 and 1 mod. and 2 and 2 mod. have been combined for the reasons stated in Section 3*2.J>. FIGURE 3-3. ABNORMAL LDH TYPES IN POPULATIONS A AND B. ■? ■ ,'p. I p LDH X LDH X mod LDH Y LDH Y mod 72 2 Using 3x2 contingency tables, = 1.25 for two degrees of freedom. This represents a probability that the results could have been obtained by chance between twenty and thirty times in every hundred. The proportions of LDH Type 1 to LDH Type 2 are not signif icantly different in the two populations. (2) Studies with Stored Blood. The results obtained in the previous section demonstrated that LDH Type 1 and LDH Type 2 occurred in two different populations in similar proportions. It has also been shown (3.2.3.) that the LDH type did not change when haemolysates were stored at -20°C. for three weeks, although after one month’s storage, LDH 3 became fainter. Blood samples which had been stored for three weeks were available from (a) 107 New Guinea indigenes (Bainings) and (b) 39 Australian aborigines from the state of Queensland. The proportions of LDH types in these two groups are shown in Table 3- Results. (1) There are more LDH Type 2 in the Bain ings samples than LDH Type 1. (2) There is no LDH Type 2 in the thirty nine Australian aboriginal bloods. 73 (3) Two abnormal LDH types were found in the Bainings population. One was LDH Type. X and the other LDH Type Y (Figure 3-3). TABLE 3-d. LDH TYPES IN STORED BLOOD. Bainings, N.G. Aust. aborigines LDH type No. % No. % 1 38 23 41 38.5 39 100 1 mod. 3 14 2 64 0 64 59.8 0 0 2 mod. 0 0 Abn. 2 1.9 0 0 Totals 107 100.0 39 100 Using a 3 x 2 contingency table to compare the Bainings population with populations A and B described 2 in Section 3.2,4. (1), = 16.5 for two degrees of freedom. This corresponds with a probability of less than 0.001/6 and represents a highly significant differ ence. Conclusions. LDH Type 1 and LDH Type 2 occur in three of the four populations and 97/6 or more of the samples 74 in these populations are one of these two types. In the fourth population no LDH Type 2 was found in 39 bloods. The increase in LDH Type 2 in the Bainings population could be due to storage effects, although the evidence of the storage experiment and the absence of LDH Type 2 in the Australian aboriginal bloods, both of which have been stored, do not support this view. There are quantitative differences in human red cell LDH which can be detected by disc acrylamide electrophoresis and can be interpreted as being due to an inability to make large numbers of slow (A or M) polypeptide chains. A similar condition has been observed in members of the marsupial Order, Macropodidae. The genetic significance of these quantitative differences is examined in Chapters 3» 6 and 7* An alternative explanation for the differences in two of the four populations is that some non-genetic cause such as disease or malnutrition has altered the phenotypic expression of LDH in the red cells but there is no evidence to support this. 3.2.3. LDH Patterns in Human Serum, Red Cells and White Cells. (a) Red Cells and White Cells from the Same Person. Method;- Red cells and white cells from 75 five blood donors were provided by the N.S.W. Red Cross Blood Transfusion Service.* Disc acrylamide electrophoresis was performed on haemolysates and lysed white cells. The patterns obtained are shown in Figure 3-4. Results. (1) All five red cell patterns are LDH Type 1 mod. (see Fig. 3-1 b). (2) There are two patterns in the aqueous extract from the white cells of the five donors. In bloods from donors 2, 3 and 4 the most prominent band is LDH 3, with LDH 2 and LDH 1 decreasing in intensity. In bloods from donors 1 and 5» LDH 2 is the most prominent band with LDH 3 and LDH 1 appearing as minor bands. (3) White cells do not contain the minor (mod.) band which is present in the red cells. Conclusions. White cells make more slow polypeptides than red cells, resulting in higher densities of LDH 3 (AABB, MMHH) and LDH 2 ( ABBB, MHHH). Red cells have a preferential production of fast LDH polypeptides. These observations support the results reported in "Isoenzymes in Biology and Medicine", Latner and Skillen, 1968,pp. 7 and 8, using starch gel electrophoresis. (b) LDH in Serum. Serum LDH was studied in two groups of people. * Concentrated white cell preparations were provided by Dr. G. T. Archer FIGURE 3-4 76 LDH PATTERNS OF RED CELLS AMD WHITE CELLS FROM FIVE BLOOD DONORS. Donor 1 Donor 2 Donor 3 LDH band 3 mod 2 1 R W R W R W Donor 4 Donor 5 3 mod 2 1 R W R V/ R . Red cells, W White cells 77 (a) Fresh serum from 46 blood donors, (b) Serum from 55 indigenous schoolchildren from Kieta, Bougainville, which had been stored at ~20°C. for three weeks. The range of LDH patterns found and the numbers of each in the two populations (a and b) are shown in Figure 5-5• Figure 5-6 is a photographic reproduction of the three most common LDH types. FIGURE 5-5. RANGE OF LDH PATTERNS IN SERUM. Pop. Serum LDH Pattern 12 5 4 5 6 7 5 4 5 2 r Blood Donors1715 5 5 7 1 2 Kieta child.2252 0 0 1 0 0 FIGURE 3-6 78 MOST COMMON SERUM LDH PATTERNS with DISC ACRYLAMIDE GELS. 1 2 3 4 5 6 7 8 Y 79 Results. (1) Serum LDH patterns show great quantit ative variation, seven being distinguished in the blood of 46 blood donors. (2) There is no predominance of slow or fast LDH bands. For example, in types 1 and 7i (Fig. 3-5) there are more fast moving polypeptides than slow, resulting in heavy production of LDH 1 and LDH 2, while in types 3 and 6, more slow polypeptides are produced, resulting in more LDH 3 and LDH 2 than LDH 1. (3) One of the 46 fresh serum samples has only two bands, LDH 1 and LDH 2, while two have an extra band at the LDH 5 (AAAA,MMMM) position. Conclusions. LDH patterns in human serum are variable and this is what might be expected if LDH from different tissues can leak into the plasma when tissue breakdown occurs. In rats there is an increase of LDH bands after exercise (Garbus et al., 1964). Changes in serum LDH also occur in many diseases and some have been shown to be useful in diagnosis. Some of these conditions are myocardial infarction (Vesell and Bearn, 1957), rheumatoid arthritis (Wieme, 1963) and abdominal em ergencies of several kinds (Vesell and Bearn, 1961)• In viev; of the above alterations in serum LDH 80 and the results recorded in 3*2.5» (b) , it is evident that serum LDH patterns are not suitable for genetic studies, especially if the phenotype is expressed as a quantitatively altered LDH pattern. 3.2,6, (a) The Occurrence of Malic, Isocitric and Succinic Acid Dehydrogenases in Human Red Cells. Introduction. The sites of operation of these three enzymes are marked "b", McM and Mdu respectively in Figure 2-6. Malic and isocitric acid dehydrogenases + -f transfer hydrogens to a co-enzyme, DPN or TPN but succinic acid dehydrogenase passes the hydrogens dir ectly to flavin(e) adenine dinucleotide (FAD+). (See Figure 2-4). All three enzymes can therefore be located by using the same dye-coupling technique. The incub ating media differ only in the substrates which are malic acid, isocitric acid and sodium succinate and in the absence of co-enzyme in the medium for succinic dehydrogenase. In some organisms, MDH and IDH exist in both DPN and TPN dependent forms (Siegel and Englard, 1962; Henderson, 1966, 1968; Villee, 1968; Plaut, 1963 and Kornberg and Pricer, 1951)* They are found in mit ochondrial and supernatant fractions when cells are 81 ruptured and centrifuged at high speed (50,000 x g. ) . The physiological significance of these two forms has not yet been recognised but it is probable that the two forms of MDH and IDII are coded at two different loci and are not isozymes within the definition used in this thesis. All three dehydrogenases act in the tricarb oxylic acid cycle (Figure 2-6). MDH is described as being the DPN dependent enzyme while IDH is the TPN dependent enzyme. These are presumably the enzymes present in mitochondria. Succinic acid dehydrogenase is believed to be physically associated with the cyto chrome system in the mitochondrion to form the "succinic oxidase" or "succinoxidase" system (Keilin and Hartree, 1939; Singer, Kearney and Massey, 1956). Dixon and Webb (1968) do not feel that succinic acid dehydrogenase is indivisibly connected v/ith the cytochrome system and presumably does function apart from the cytochromes. It is of interest that SDH was the first dehydrogenase to be demonstrated histologically using the dye coupling method and this involves the uncoupling of the cytochrome system from the dehydrogenase. Mature human red cells do not have mitochondria and should not contain DPN dependent MDH, TPN dependent IDH or SDH. In addition, red cell enzymes are not syn- 82 thesised once the red cells have matured. It is of interest to determine whether the tetrazolium dye coupling technique indicates that DPN dependent MDH and IDH, or SDH are present in mature red cells and if it does, to state the genetic implications and to see if they are suitable for use as genetic markers. Results. (a) DPN Dependent Malic Acid Dehydrogenase. Red cells from 84 donors were tested for DPN dependent MDH within 48 hours of the collection of the blood samples. (i) 42 samples contained one broad blue staining band which did not coincide in position with any LDH band but ran just ahead of (or anodic to) LDH 3. (See Figure 3-7 * 2). (ii) 41 samples had a minor band running at the same position as LDH 1 (Figure 3-7? 3)• (iii) 1 sample had three bands, including the two mentioned in (ii) above and an additional minor band running at the same position as LDH 3« (Fig. 3-7i 4) Reproducibility of Minor Bands. Blood from nine of the ten subjects who were tested for LDH at five weekly intervals (3«2.3»ib) were examined for the presence of DPN dependent MDH. The results indicated that the occurrence of minor bands varied from week to week in the same individual although the broad band FIGURE 5-7. 83 BANDS PRODUCED WITH MALIC ACID AS SUBSTRATE. LDH MDH (i) (ii) (iii) 3 4 1 2 3 b The substrate used for detecting MDH i, ii and iii is 1-malic acid. The positions of the LDH bands are shov/n in column 1 for comparison. 84 with an electrophoretic mobility between those of LDH 3 and LDH 2 was always present in the same position. (See Table 3-e). TABLE 3 - e. REPRODUCIBILITY OF BANDS WITH MALIC ACID AS SUBSTRATE. Subject Week No. * 1 2 3 4 5 E.M. 1+1 1 + 1 1 1 1 P.R. 1 + 1 1 1 1 1 E.N. 1 + 1 1 1 + 1 1 1 R.W. 1+1 1 1 1 + 1 1+2 P.C. 1 + 1 1 1 1 1 J.M. 1 + 1 1 1 1 1 S.S. 1 + 1 1 1 1 1 J.G. 1+1 1 1 1 1 L.L. 1 + 1 1 1 1 1 1 + 1 - 1 major band + 1 minor band at LDII position 1 1 - 1 major band only 1+2 - 1 major band + 2 minor bands at LDH positions 1 and 3. * For a note on the results in this column, see overleaf 85 Note on the results recorded in Table J>-e, week 1. In the first week, MDH patterns consisted of a major band and a minor band in the same position as LDH 1. In the following weeks the minor band appeared infrequently and there was no consistency in their appearance. It is suggested, from evidence with human and other red cells, that the minor bands are conform ational artefacts. It is therefore concluded that the appearance of the minor band in all samples in week 1 is due to such a change, possibly brought about by some irregularity in the gels. Minor bands make up only a small fraction of the total material. MDH is believed to consist of one band when run on disc acrylamide gels (See the remainder of Section 5*2.6.). 86 White Cells. White cell preparations from six subjects v/ere tested , using 1-malic acid as substrate. These preparations v/ere obtained from the N.S.W.Red Cross Blood Transfusion Service (Section 3-2.5-). No major bands v/ere detected using disc acrylamide gels but minor bands were stained at positions corresponding with LDH 1, LDH 2 and LDII 3- The results are shown in Figure 3-8, each pattern being represented in tv/o samples. FIGURE 3-8. WHITE CELL PATTERNS IN SIX BLOOD DONORS, USING " MALIC ACID AS SUBSTRATE. LDH MDH ABC 3 k 2 ------ 1 ESI ------ No. of 2 2 2 samples 87 DPN Dependent MDH in Serum. Sera from four subjects were tested for MDH activity.and variable results were obtained. The serum from one subject had a faint band corresponding with the MDH band in red cells, two sera had one band only, one corresponding with LDH 2 and the other corresponding with LDH 3i while serum from the fourth subject had two bands corresponding with LDH 1 and LDH 2. For reasons discussed in the conclusion to this section it is probable that the minor bands are artefacts and do not represent MDH activity, so it was decided not to continue testing of serum for this enzyme. (b) Patterns Obtained Using DPN as Co-enzyme and dl-Isocitric Acid as Substrate. Isocitric acid dehydrogenase involved in the tricarboxylic acid cycle is the TPN dependent enzyme, but a DPN dependent form of IDH has also been reported in the tissues of some animals (Plaut, 19&3; Kornberg and Pricer, 1931)- Blood samples from forty nine individuals were examined for IDH activity. 88 38 samples contained two minor bands which corresponded with LDH bands 1 and 2. 11 samples did not show any bands. (c) Succinic Dehydrogenase. Two haemolysates and five sera were tested for SDH. The substrate was sodium succinate (AR) and no co-enzyme was included in the incubation medium. No staining bands were observed when disc acrylamide gels were used. The relationship between LDH, MDH and IDH in one individual is shown in Figure 3-9 and a summary of the results obtained in Section 3*2.6. is shown in Table 3-f* FIGURE 3-9* LDH, MDH AND IDH PATTERNS IN ONE INDIVIDUAL. 5 4 3 2 1 - LDH MDH IDH TABLE 3 - f 89 MDH, IDH AND SDH IN HUMAN BLOOD. Enzyme Red Cells White Cells Serum No. No. No. tested tested tested MDH 84 + 6 ? 4 ? IDH 49 ? n.t. n.t. SDH 2 n.t. 5 - + enzyme detected. - no enzyme detected. ? bands which coincide with LDH bands, n.t. not tested. Conclusions. (1) DPN dependent Isocitric acid dehyd rogenase does not produce consistent patterns when haem olysates are subjected to electrophoresis on disc acryl amide gels. (2) Succinic acid dehydrogenase is not found in the samples tested and this result would be expected if SDH is associated with the mitochondrial membrane 90 (3) Malic acid dehydrogenase is present in red cells and appears on disc acrylamide gels as one band with an electrophoretic mobility between that of LDH 2 and LDH 3. (4) Minor staining bands occur with both malic acid and isocitric acid as substrates. A band with the same electrophoretic mobility as LDH 1 (BBBB,HHHH) occurs most frequently, although bands with the same mobilities as LDH 2 and occasionally LDH 3? ocdur in red cells and white cells with Malate substrate. The presence of the minor bands and the irregular nature of their appearance present diff iculties in interpretation. They could be (a) IDH and MDH proteins-- with the same electrophoretic mobility as LDH proteins, (b) due to conformational changes occurr ing in vitro during electrophoresis or (c) due to a portion of each dehydrogenase molecule having a common reactive site, permitting some activity with all substrates undergoing dehydrogenation and using TPN or DPN as hydrogen acceptors. In. vivo each enzyme would have access to its preferred substrate but under the experimental conditions, only one substrate is available. It is difficult to accept (a) as an 91 explanation of the bands because of the irregularity of their appearance. (b) implies that, during electro phoresis on disc acrylamide gels, some degree of conform ational change occurs, so that the LDH multimers become less specific and act on several suitable substrates. This would account for the irregular appearance of bands because differences in pore size and in the loading of the gels could alter the degree of conformational re arrangement of the molecules. (c) suggests that dehydrogenases have evolved from a ’’primitive” polypeptide . This idea has been developed by Kaplan (’’Evolution of the Dehydrogenases” in ’’Evolving Genes and Proteins, 1969). It is logical to suppose that an amino acid sequence in a polypeptide could provide an active site for the dehydrogenation of certain organic acids. Modifications could occur by mutations and/or polymerisation, resulting in a number of enzymes with. a specificity for a particular substrate. Kaplan bases his argument in part upon the similarity in the proportions of certain amino acids in LDH "M” and LDH "H" (Table 3-g) and the constant ratio between the molecular weights and the number of sub units in DPN dependent dehydrogenases (Table 3-h)• Kaplan says, ’’These data suggest the possibility that all DPN TABLE 3 - C 92 AMINO ACID CONTENT OF "M" AND "H" LDH ISOZYMES. (Reproduced, from ’’Evolution of the Dehydrogenases" Kaplan, 1965)* No. of molecules Amino acid LDH "H” LDH "M" oo Valine V>l 115 Isoleucine 85 91 Leucine 143 156 Cystine 17 26 Histidine * 26 53 * The presence of more of the basic amino acid, histidine, in LDH "M” probably accounts for its slower electrophoretic mobility. TABLE 3 - h 93 SUB-UNIT STRUCTURE ALP MOLECULAR WEIGHTS OF DPN LINKED DEHYDROGENASES. (Reproduced from "Evolution of the Dehydrogenases", Kaplan, 1965)• Enzyme Molecular No. of weight sub-units Glutamic dehydrogenase 1,000,000 circa 30 Lactic dehydrogenase 145,000 4 Triose phosphate dehydrogenase 150,000 4 Yeast alcohol dehydrogenase i4o,ooo 4 Liver alcohol dehydrogenase 68,000 2 Malic dehydrogenase 67,000 2 The molecular weight/sub-unit relationship strongly suggests that there is a basic polypeptide unit with a molecular weight approximately equal to 35*000. It is not known if any dehydrogenase exists with only one polypeptide unit. 94 linked dehydrogenases arose from a common ancestral dehydrogenase" (ibid., 1965, p. 249)., It is of interest that all the enzymes mentioned in Table 3-h have even numbers of sub-units, suggesting that a minimum of two polypeptides is necessary for dehydrogenase activity. The occurrence of minor bands when malic acid and isocitric acid are used as substrates is considered to be due to conformational changes occurring in. vitro. Therefore DPN dependent IDH has not been demonstrated in human red cells. DPN dependent MDH is present in red cells but, contrary to expectation, is not found in white cell preparations. There is no explanation for this observ ation unless the MDH is retained within the mitochondria in white cells but is released into the cytoplasm of the red cells as they mature and lose their mitochondria. It is still unexplained why red cells should retain large amounts of MDH which they apparently cannot use. 95 3»3» Glucose 6 Phosphate Dehydrogenase. 3»3«1. Introduction. GoPD catalyses the oxidation of glucose 6 phosphate to 6 phosphogluconolactone, using TPN as co-enzyme. The reaction can be represented as follows H C OH I H C OH H V I ___ i____ . HO C H HO H CI OH H H cI ----- H I h2copo3h2 h2copo3h2 TPN+ TPNH + H+ It operates in the oxidative "hexose monophosphate shunt” or pentose phosphate pathway which is not located in the mitochondrion. The effect of the ’’shunt" is to release hydrogens to TPN. In red cells the hydrogens are used to reduce any methaemoglobin which may be formed during the metabolism of the cell. The reduction of methaemoglobin involves reactions by two other enzymes, glutathione reductase (EC 1.6.4.2.) and methaemoglobin 96 reductase ( EC number not given by Dixon and Webb, 1962). On rare occasions, apparent G6PD deficiency has proved to be a deficiency of one or other of the two enzymes just mentioned. Figure 3-10 is a diagram of the pentose phosphate pathway, showing the site of action of G6PD. It is a modification of a diagram in "Genetic Markers in Human Blood", 1969? by Giblett (p. kk^). FIGURE 3 ~ 10. PENTOSE PHOSPHATE PATHWAY IN RED CELLS. (Modified from Giblett, 1969)• From anaerobic breakdown of glucose Glucos< 6 Phosphogluconate Lacticv acid 97 Genetics of G6PD. This enzyme has been studied more extensively than any other and much is known about the genetics of G6PD deficiency and the racial distribution of G6PD variants. It has also proved to be of interest because it is sex-linked. The current extent of know ledge has been clearly summarised by Giblett, (ibid., 1969, pp. 44} - 482). Structure of G6PD. Human G6PD is believed to be an isomeric hexamer (Kirkman and Hanna, 1968). The negro variant, A+, has been found to be a molecule of the same size as the normal enzyme, B+ and Yoshida (in a footnote to the article of Kirkman and Hanna, above) suggests that the alteration in the electrophoretic mobility of A+ is due to a single step mutation in DNA. This mut ation results in the replacement of one asparagine res idue by an aspartic acid residue. Purpose of the Investigation. At the time of commence ment of the study (1965), it had been established that electrophoretic variants were rarely found in Caucasians. It was therefore decided that an investigation of quantitative variations in populations should be made. Populations A and B, whose bloods were used for LDH 98 investigations (Section 3-2.4. (1), were used. Population A consists of subjects born in Australia or the British Isles, both of whose parents were of British stock and Population B consists of subjects born in Australia, Greece, Italy or Malta, both of whose parents were of Greek or Italian stock. The aims were to use a method suitable for field work (a) to determine the distribution of G6PD levels in two populations and (b) to compare G6PD values of males and females within the two populations. Method. A convenient method for field use is the "Sigma” Kit No. 400 for G6PD determination and the semi-quantit ative estimation described in Technical Bulletin No. 400 (Sigma) has been used. A colour change occurs when the hydrogens released by the dehydrogenase reduce a blue dye. This method is similar to the screening method of Motulsky and Campbell-Kraut, 19&1, which uses brilliant cresyl blue. 0.05 ml. of whole blood is haemolysed in 2.30 ml. distilled water. 1.00 ml. of the haemolysate 99 is incubated in a water-bath at 37°C. in the dark with 0.5 ml. of buffer (pH = 7.4) in which is dissolved a fixed amount of dye placed in the vial by the manufact urers. Air is excluded by covering the solution with a layer of mineral oil. The time taken for the dye to change from bright blue to plum colour is observed, so that the longer the time taken, the less the G6PD act ivity. Results. The results obtained are shown in Table 3-i and in Figures 3-11, 3-12 and 3-13* TABLE 3 - i. g6pd activity in males and females from POPULATIONS A AND B. Mean time X - X Population No c T) f m (minutes). S.E. of diff.* males 72 40.3 - 10.6 A 3.1 females 35 49.4 - 15.6 B Greek males 21 39.9 i 14.1 C\J 0 1 * * • females 5 58.4 - 19.4 B Italian males 58 41.3 i 12.0 2.4 females 16 49.4 - 11.7 * See appendix No.7» "Use and Abuse of Statistics", FIGURE 3-11 Distribution o f G6PD V alues 100 •sivnaiAiONi jo aaswnM MSl.011-.020 ______• ^^ 1 . . . . 111-.120 041:050 . . 031-.040 021-.0 031-.040 011-020 30 RATE OF REACTION FIGURE 3 -1 2 DISTRIBUTION OF G6PO VALUES-POPULATIONS 101 SlVnaiAIQNI do ON RATE OF REACTION FIGURE 3 -1 3 G6PD VALUES / AGE US < (S«V3A) 102 •f 39V o o c* CNi o o o o o o o to p o o CO o o CD o p o o o cr> o RATE OF REACTION 103 Reichmann, Pelican, 1964. The "t" test for unpaired experiments was also used to test the significances of differences between the following groups, (i) males of Greek stock and males of Italian stock, (both from population B) , t^^ orCO) = 0*39 (ii) males from population A and males from the pop ulation of Greek stock, B, t,„„ \ = 0.12 (91 ore©) (iii) males from population A and males from the pop ulation of Italian stock, B, \ = 0.4-9 (12o or oo) (iv) females from population A and females from the population of Greek stock, B, t,-.0 N = 0.99 (3o oroc) (v) females from Greek stock and females from Italian stock, both population B, t^^) = 0.98 * * A G6PD deficient subject from the male population of Greek stock. The subject was born-in Greece. Summary of Results. G6PD activity is measured as time taken for a colour change to be completed. Therefore higher numbers imply lower enzyme activity (e.g., a sub ject with a value of 46 (min.) has a higher G6PD activity than a subject with a value of 30 (min.) ). In Figures 3-11, 3-12 and 3-13i G6PD values have been represented as rates. (1) There is a significant differ- 104 ence between males and females in all three populations (Table 3-i). (2) There is no significant difference between values for males nor for females in the three populations (see footnotes to Table 3-i)» (3) Among 22 men of Greek stock, there is one G6PD deficient subject. This finding was verified by A. Czuppon, using a quantitative method. (4) In view of the findings in (2), all male values and all female values were combined to obtain Figures 3-111 3-12 and 3-13* Figure 3-11 shows that the distribution of female values does not coincide with male values. Figure 3-12 indicates that both male and female values form broadly based curves of normal distribution, with a wide range. Figure 3-13 is a scatter diagram representing the relationship between G6PD values and age and shows no relationship between them. All three figures confirm that female values are lower than male values. Conclusions (1) A simple, semi-quantitative method 105 for G6PD determination has been used to make a survey of two groups of people. (2) G6PD values fall within a curve of normal distribution and there is a wide range of values among normal persons. (5) The lower mean G6PD values for females is unexpected. It is interesting to compare these re sults with values for normal males and females used as controls by Harris et al. (1963)i using a quantitative method. Activities were expressed as units of enzyme activity/gram haemoglobin, Sex No. G6PD Value Males 14 8.38 - 1.83 Females 2b 8.02 i 1.45 The same trend is indicated in these results but the difference is not statistically significant. The semi-quantitative method relates G6PD activity to whole blood and not to grams of haemoglobin and this may account for the difference in results, since haem- atocrits are lower for females than for males. Results obtained with the semi-quantitative method used here support the generally accepted theory of the random inactivation of one X chromosome in the 106 female (Lyon, 1961, Ohno et al., 1959, Ohno and Makino, 1961, Grumbach et al., 1962, Nitowsky and Childs, 1963, Harris et al., 1965, Ohno, 1963, Beutler, 1966 and Shaw and Koen, 1968). Additional References. 3-2, LHP variants in populations. Blake et al., 1969, Davidson et al., 1965, Kraus and Neely, 1964. 3-3. Glucose 6 phosphate dehydrogenase. Davidson et al., 1963, Kirkman and Hanna, 1968, Parr, 1966, Phosphogluconate dehydrogenase. Parr and Fitch, 1957. 107 3.4. Serum Amylase, 3.4. 1. Introduction. A simple, inexpensive test is available to detect the presence of amylase after electrophoresis. It makes use of the fact that starch stains blue in the presence of iodine, while the break down products of starch (sugars) do not. The method is described in Section 2.3.1. Ninety six serum samples from a Chinese pop ulation from Singapore were selected to test the method. The sera had been frozen for some time at -20°C. The results are shown in Figure 3-14. FIGURE 3 - 14. AMYLASE PATTERNS IN HUMAN SERA. Origin 1 . 2 3 4 5 6 No result Total Eld □ EZ3 E3 ID I No. 42 16 16 1 4 16 1 96 The bands were just ahead of the origin in the region of the haptoglobins. 108 Results. One of the 96 samples did not contain an amylase band* 42 of the other 95 had a single broad band which ran just ahead of the origin. The remainder of the samples had minor white bands in addition to the main band. The minor bands are shown in Figure 3-1^- Conclusions. Serum contains one broad band in the position equivalent to the f globulin bands. The nature of the minor bands is unknown. It is obvious from the work of Berk et al., 1965 and Dreiling et al., 1965 that there is some activity associated with the main serum protein fractions. The following results are reproduced from Berk et al., % globulin, 66.9% of total amylase activity globulin, 7*6% ©^globulin, 8.4% o£( globulin, 6.4% albumin, 10.5%« In the present study no activity was located in assoc iation with albumin. The minor bands occur at positions occupied by the proteins of the haptoglobin series. It is not known whether the apparent amylase activity of these bands is due to an amylase isozyme or an artefact due to adsorption 109 The results indicate that disc acrylamide gel electrophoresis is not the method of choice for genetic investigations of serum amylase. 110 Note on the Use of Capital Letters for Non Zoological Names for Animals or Groups of Animals, In chapters 4, 6 and 7> small letters have been used for (1) common names used descriptively; e.g. "grey kangaroos” or "humans”, (2) the use of the word as an adjective; e.g. "rat" blood or "human red cells". Capital letters have been used if a non- zoological term has been used in place of a zoological family or generic name, even if only one animal of that type has been discussed; e.g. "the Cat family", meaning the family Felidae or "Man", meaning Homo sp. 111 Chapter 4. Comparative Studies of Red Cell Dehydro genases in Some Animals and Birds. 4.1.1. Introduction. In the examination of red cell dehydrogenases in humans (Chapter 3), the possibility that there are genetically determined quantitative diff erences in the isozymes has been introduced. It is therefore of interest to examine red cell dehydrogenase patterns from as many species as possible to determine whether similarity between patterns denotes phylo genetic affinity. Alterations in mobility or changes in the relative amounts of each isozyme type could ind icate points at which mutations occur. 4.1.2. Lactic Acid Dehydrogenase. There have been several comparative studies of LDH, one of the most extensive being that of Wilson et al., (1964), using starch gel electrophoresis. They examined vertebrate animals ranging from the Cyclostomes (Lampreys) to Mammals and including Cartilaginous and Bony Fishes, Amphibians, Lower and Higher Reptiles, Paleognathous and Neognathous Birds and Marsupials. From the data of Wilson et al., Kaplan(1965) has proposed an "evol utionary tree" for vertebrate LDH which is reproduced in Figure 4-1. FIGURE 4 - 1 112 EVOLUTION OF VERTEBRATE LDH. (Reproduced from Kaplan, "Evolution of the De hydrogenases", in "Evolving Genes and Proteins", ed. Bryson and Vogel. Acad. Press, N.Y. and Lond., 1965, p. 271.) NEOGNATHOUS BIRDS (stable, slow) MAMMALS (unstable, very fast) PALEOGNATHOUS BIRDS (stable, fast) HIGHER REPTILES LOWER REPTILES (unstable,t fast) AMPHIBIANS - (unstable, fast) t FISHES (unstable, fast) 113 It can be inferred from Figure 4-1 that LDH in mammals and Neognathous birds has evolved from the lower Reptiles along completely different paths. It is also implicit in the diagram that there are no major struct ural differences in LDH within the mammals which are likely to result in changes in electrophoretic mobil ities. The theory is based upon differences in kinetic properties as well as electrophoretic mobilities but in the same article, Kaplan quotes data indicating that the ratios of certain amino acid residues in birds are different from those of mammals. Data for LDH H^ and LDH M^ are shown in Tables 4-a and 4-b. TABLE 4 - a. AMINO ACID RESIDUES IN H, LDH. (Number per mole.) Animal Arginine Isoleucine Lysine Chicken 55 66 99 Rabbit 55 84 97 Man 50 88 96 Pesce, A., reproduced from Kaplan, ’’Evolution of Dehydrogenases”, from "Evolving Genes and Proteins", p. 247. TABLE 4 - b 114 AMINO ACID RESIDUES IN M, (AAAA) LDH. (Data from Pesce , ibid.) (Number per mole.) Animal Histidine Phenylalanine Chicken 63 27 Duck 57 27 Rabbit 41 26 Aim of the Investigation. Disc acrylamide electroph oresis was used to make a comparative study of red cell LDH patterns in mammals and in two Neognathous birds, namely ducks and chickens. Results. Figure 4-2 indicates the patterns observed in 16 kinds of ammals and two kinds of birds. Samples were tested as they became available from Zoo post mortems or from field workers. Marsupials of the family Macropodidae were also studied and these results are presented in Chapters 5* 6 and 7« In some cases only one animal was available for study and the patterns seen may not be the only ones found in the red cells of these animals. Rabbits, merino sheep, dogs, cats FIGURE 4 - 2 115 LDH PATTERNS JN CERTAIN MAMMALS & BIRDS. A C B M E D 0 K J F P L S Q N H G U T R V w I 0 5 4 B [ 0 1 0 s 0 I 0 I I 0 I 2 0 0 a □ 1 3 1 I I 1 1 1 1 1 i 1 (For DUCK CHICKEN JAGUAR CAT MOUSE LION RAT GUINEA DOG ELEPHANT RABBIT POLAR MERINO BARBARY HYENA MAN WHITE-TAILED. CHIMPANZEE notes BEAR PIG X SHEEP - SHEEP see No, DEER overleaf.) 350 tested 153 20 17 86 66 3 4 1 6 1 1 1 1 1 1 1 1 116 NOTES ON FIGURE 4-2. (a) All LDH patterns are those found in freshly prepared haemolysates• (b) M0” is the origin. Cross-hatched areas behind the origin represent LDH remaining in the Large Pore gel. This is thought to be LDH 5 which is less stable than the other LDH isozymes. (c) The cross-hatched area at LDH 4 position (I), represents a diffuse staining area rather than a discrete band. (d) The dotted lines represent staining bands which are not present in all individuals. (e) The widths of the isozyme bands do not rep resent quantitative differences between species but relative differences in the intensities of the isozymes in each individual pattern have been recorded as accurately as possible. 117 and chickens represent varietally different animals. For example, dogs include dingoes, Papuan native dogs, an Indian jackal, a timber wolf, huskies, foxes and several varieties of domestic dog (Clark and Ryan, unpublished data). (1) All five expected LDH tetramers are found in the series, the patterns ranging from one band in the jaguar and some rabbits to five bands in some chickens and the Rat. When the slowest LDH iso zyme, LDH 5i is present it frequently does not enter the running gel but remains in the Large Pore gel where it can be detected by heavy purple staining. There are two exceptions, that of some chickens (Fig ure 4-2) and the Grey Kangaroo (Chapter 5)* (2) There are no obvious differences in the electrophoretic mobilities of the LDH bands with the possible exceptions of LDH 1 in the Hyena and the Rabbit Figure 4-3 is a photograph of rabbit red cell LDH (1) and MDH (2). The positions of the bands are illustrated below LDH MDH haemoglobin bands. H m (cross-hatched) FIGURE 4-3 118 RED CELL LDH AND MDH IN RABBITS. (1) Rabbit LDH, consisting of one band at LDH 1 position. The diffuse band just behind the LDH band is haemoglobin. (2) The "fast” MDH of rabbits, running just ahead of haemoglobin. The minor MDH band is ahead of the major MDH band and also ahead of LDH 1, The position of this band is indicated by an arrow 119 LDH is represented by one band running just ahead of the diffuse haemoglobin band. MDH has a major band which runs just behind that of LDH 1 and a minor band which runs just ahead of it. In Chapter 3 it was shown that minor MDH bands occupy positions with the same electrophoretic mobilities as LDH bands. By anal ogy with human material, the finding with rabbit blood means (a) that rabbit LDH has a slower electrophoretic mobility than that of other animals or (b) that the minor MDH band in rabbits is a true conformational rearrangement of the polypeptides comp rising the main MDH band and thus has a different electro phoretic mobility from any of the LDH bands. This finding emphasises the difficulties encountered in using disc acrylamide gels to determine differences in electrophoretic mobilities. It is interesting to note that Kaplan reports that rabbit LDH H^ (BBBB or 1) has a slower mobility on starch gels than that of Man, Rat, Mouse and a marsupial, Macropus robustus. The results are shown in Table k-c. TABLE 4 - c 120 ELECTROPHORETIC MOBILITY OF LDH H, . Animal Electrophoretic mobility LDH H^ (in cm.) Man 15 Laboratory Rabbit 12 Laboratory Rat 15 Laboratory Mouse 15 (Reproduced from Kaplan, ibid.) (3) As reported by many workers, duck and chicken erythrocytes contain mainly slow moving enzymes. (See Figures 4-2, A, B and C and Figure 4-4). Kaplan has called the major band isolated from heart muscles of birds LDH H^ which, by analogy with his own and Markert' terminology, makes this LDH HHHH or BBBB. However, in these experiments, bird LDH from red cells has the same electrophoretic mobility as mammalian LDH 4, which suggests that the bird LDH is MMMH or AAAB. In Figure 4-5 chick en LDH and MDH are compared with human LDH and MDH and minor LDH bands can be seen in the chicken haemolysate which correspond with LDH 1, 2 and 3 of the human sample. (4) The mouse red cell LDH pattern using FIGURE 4-4 121 LDH AND MDH IN CHICKEN HAEMOLYSATES. 12 3 4 (1 and J) Chicken red cell LDH patterns. This band is at LDH 4 position (see Fig. 4-5). Both sample; gels are heavily stained with what is believed to be LDH 5 remaining in the gel. (2 and 4) Chicken red cell MDH which runs ahead of chicken red cell LDH FIGURE 4 - 5. 122 LDH AND MDH IN RED CELLS OF CHICKENS AND HUMANS. 1 2 3 4 5 6 (1) and (2), Human LDH Types 2 and 1 mod. (3) Human MDH with a major band running between LDH 2 and LDH 3 and a minor band coinciding with LDH 1. (4) and (3) Chicken red cell LDH. Minor bands can be seen at LDH 1, 2 and 3 but the major LDH band corresponds with LDH 4. The minor bands are only present if the sample gel has been overloaded and are probably due to alterations in conformational arrangement. They are therefore artefacts. (6) Chicken red cell MDH, with a major band running between LDH positions 3 and 4 and a minor band running at LDH 4 position. 123 the disc acrylamide method is different from that of any other animal tested. It is composed mainly of slow moving LDH tetramers which form a broad, diffuse band at the position of LDH 4 (Figure 4-2, I). It is diff icult to interpret this finding but the diffuse nature of the main band suggests the presence of sub-bands. Markert, 1968, reports the formation of sub-bands in mice (see also Section 1.3«3*)« (5) Elephant red cell XDH has a high prop ortion of slow tetramers (Figure 4-2, N and Figure 4-6, 8). All other mammalian species shown are composed mainly of LDH 1, 2 and 3, with LDH 1 (BBBB) and LDH 2 (ABBB) predominating. Where there is more than one patt ern within a species, the difference is that one type has a large amount of LDH 2 and the other a large amount of LDH 1. (See Rabbit, 4-2, L & M and Sheep, 4-2, R & S.) (6) The White-tailed Deer, Merino Sheep, Barbary Sheep, Hyena, Rabbit, Guinea Pig and Dog have one major band which is either LDH 1 or LDH 2 and one or occasionally more minor bands. (7) Three members of the Cat family are represented and the red cell LDH patterns are variable (Figure 4-2, D, E and F). Figure 4-6 is a photograph FIGURE 4 - 6 124 RED CELL LDH PATTERNS IN DOMESTIC CATS. (1) and (2) have similar LDH patterns, although a minor band can be seen running just ahead of LDH 3 in (1) The major band in all three animals is LDH 2. (3) has a band running in the LDH 1 position. FIGURE 4-7 125 LDH PATTERNS IN STORED HAEMOLYSATES OF SEVERAL MAMMALS. 1 2 3* 5 6 7 8 (1) Man, (2) Chimpanzee, (5) Hyena, (4) Barbary Sheep, (5) Jaguar, (6) White-tailed Deer, (7) Guinea Pig, (8) Elephant. As mentioned in the text, these samples have been stored for some months and LDH act ivity is low in (4). The difference between Man and the Chimpanzee can be seen, although the mod. band is not present in stored cells. The prominent LDH 4 (slow) band can be seen in the elephant sample (8). The heavy blue band at the bottom of each tube is the tracking dye and marks the length of the run. 126 showing LDH patterns in three domestic cats. Cat 14, (No. 1 in Figure 4-6) has a minor band running just ahead of LDH 2. The Jaguar has one band at LDH 1, while the Lion has multiple bands. (8) Human and chimpanzee red cell LDH patterns both show a minor band just behind LDH 2. This band has been discussed in connection with the work on human samples (Chapter 3)* It is not found in any of the other animals tested. In Man the heaviest staining band is LDH 1 (BBBB), while the heaviest band in the chimpanzee pattern is LDH 2 (ABBB). LDH 3 (AABB) is light in humans but heavy in the Chimpanzee. Figure 4-7 is a colour photograph of red cell LDH patterns of certain of the animals discussed in the previous paragraphs. Some of the haemolysates had been stored for several months and there is some loss of activity ( e.g. that of the Barbary Sheep, No. 4, which does not show any LDH activity) but the difference bet ween Man and the Chimpanzee (1 and 2) can be seen and the predominantly slow LDH bands of the Elephant (8) are still present. Conclusions. There is no overall relationship between the phylogenetic affinities of the mammals investigated and the LDH patterns of their red cells. Man and the Chimpanzee both have the minor "mod*" band which is 127 absent in all other animals. There is a predominance of slow tetramers in the red cells of birds. The major bands correspond with LDH k of the mammalian isozymes which have the structure MMMH or AAAB. Vesell and Bearn (1962) believe that the production of M or A containing tetramers is associated with the presence of a nucleus. Wilson et al., 1963? have recognised differences in substrate and substrate analogue specificity in breast muscles of birds with different flight habits and suggest that isozymes should be ident ified by kinetic experiments as well as by electrophor esis. The problems raised by the above interpretations emphasise the importance of distinguishing between alt erations in phenotypes due to epigenetic effects and those resulting from gene mutations. 4.1»3« Malic Acid Dehydrogenase. Introduction. MDH does not produce multiple molec ular forms when subjected to electrophoresis and is believed to be a dimer (Kaplan, 1968). Kaplan states that MDH from Bacillus subtilis has a molecular weight of 97,000 compared with 67,000 for MDH from higher org anisms and suggests that the former is a trimer. The position is made more complex because of the presence 128 of soluble (or cytoplasmic) DPN and TPN dependent MDH and insoluble (or mitochondrial) DPN and TPN dependent MDH (Henderson, 1968). The enzyme in red cells is be lieved to be the cytoplasmic DPN dependent form (EC 1.1.1.37-). Results. Figure 4-8 is a diagrammatic representation of MDH bands recorded for 16 different mammalian and bird species. The positions of the main MDH bands have been ascertained by running blood samples from several species at the same time. In order to minimise the errors introduced by the use of individual disc acryl amide gels, mobilities are compared with those of human MDH and with the positions of the five LDH bands. It is also helpful to observe the relationship between the positions of the haemoglobin bands and the MDH bands, Figure 4-9 is a colour photograph of MDH in stored cells from different animals. In all cases there is one major band and the mobilities of these bands can be compared by their relationship to the haemoglobin band. This can be seen in tubes 1,2, 3 and 8, where there is a space between the MDH and the haemoglobin and in tubes 4 and 7» where the MDH and haemoglobin bands almost coincide. (Mobilities cannot 0 129 No. tested oSl 1 23b 5 i i i Positions of LDH i 1 i CO i i bands A < DUCK 3 2 B CHICKEN 17 WHITE-TAILED < C DEER 1 2 B BARBARY SHEEP 1 Ll! E MERINO X 20 oo 2 o F RAT 4 CO • CO G DOG 86 * X 0 CHIMPANZEE 1 o P MAN 84 * Positions occupied by the five LDH bands included to check the mobilities of the MDH FIGURE 4-9. 130 RED CELL MDH PATTERNS IN SEVERAL MAMMALS. (1) Man, (2) Chimpanzee, (3) Hyena, (A) White-tailed Deer, (3) Jaguar, (6) Barbary Sheep, (7) Elephant, (8) Guinea Pig. As described in the text, mobilities can only be determined by repeated running of the samples to obtain runs of equal lengths and by comparing the relative positions of MDH and haemoglobin bands. From the picture the "slow" MDH of (1), (2) and (3) can be detected by the space between the orange Hb. band and the purple MDH band. Similarly, the "fast" MDH can be seen in (4) and (6). The "fast" MDH of the Elephant and the "slow" MDH of the Guinea Pig can be seen in (7) and (8). 131 be determined from this picture because the electroph oretic runs are of different lengths). Conclusions. (1) Most mammalian haemolysates contain one major band when malic acid is used as substrate and DPN + is used as co-enzyme. In all mammals except the Jaguar the band has an electrophoretic mobility which is clearly different from the mobilities of any of the LDH bands. In the mammals from C to I in Figure *f-8 the MDH bands occur between the positions of LDH 1 and LDH 2 and have been called "fast" MDH for convenience here and in later chapters. Mammals represented in Figure *f-8, L to P, have an MDH whose mobility is between those of LDH 2 and LDH 3 and has been called "slow" MDH. The Jaguar and Cat MDH bands lie between the slow and fast MDH bands of other mammals. The Jaguar appears to have an MDH whose electrophoretic mobility almost coincides with LDH 2 but only one animal has been avail able for testing and it is impossible to state whether Jaguar MDH has the same electrophoretic mobility as LDH 2 or is slightly slower. (2) There is a second minor MDH band in red blood cell samples from the Rabbit, Guinea pig, Hyena, Chimpanzee and Man. In all except the Rabbit, 132 which has already been discussed in this chapter, the minor band has the same electrophoretic mobility as LDH 1. In Man and the Rabbit, the only two mammals with two bands which have been tested in large numbers, the minor band does not occur in every individual, (3) Red cell MDH bands from ducks and chickens are slower than those of mammals, having electrophoretic mobilities slightly slower than that of LDH 3. In the Chicken there is a minor MDH band corr esponding with LDH 4 (Figure 4-3) but this minor band is not always present (Figure 4-4). Discussion. Mammalian and bird red cells both contain an enzyme which is characterised as Malic acid dehydrog enase. Birds have nucleated red cells which retain their ability to use MDH by means of their tricarbox ylic acid cycles while mammalian red cells lack nuclei and do not possess the elements of the tricarboxylic acid cycle. The function of MDH in mammalian red cells remains obscure. It is of interest that bird red cells contain slow LDH isozymes and slow MDH, while mammalian red cells contain fast LDH isozymes and fast MDH. The findings suggest a relationship betv/een the polypeptides 133 of LDH and MDH but such a conclusion cannot be sub stantiated without an analysis of amino acid sequences in the two molecules. 4.1.A-. Isocitric Acid Dehydrogenase. Introduction. Since mammalian red cells lack mitochondria they do not have a tricarboxylic acid cycle and there is no obvious function for IDH. In addition, the IDH which operates in the tricarboxylic acid cycle is the TPN + dependent form. However, when dl isocitric acid was used as a substrate and DPN + as co-enzyme, IDH act ivity was observed in some of the human bloods tested (Section 3*2.6.b). The same test was applied to some other mammals. Results. The results obtained with Man, cats and sheep are shown in Figure *t-10, (a), (b) and (c). FIGURE 4-10. (a) Man Number tested = 37* LDH MDH IDH No bands positions 3 >-----> i------1 2 1 No. of individuals 27 2 8 FIGURE 4 - 10. 134 (b) Cat Number tested = 11 LDH MDH IDH No bands positions 3----- 2---- No. of individuals 9 1 1 (c) Sheep Number tested = 10 LDH MDH IDH No bands positions 3 -- 2____ ----- * ____ 1 ______* ' No. of individuals 3 2 5 Conclusions. IDH activity in humans, cats and sheep is variable. When present in blood samples it is assoc iated with bands having the same electrophoretic mobilities as the LDH isozymes or MDH bands. 4.1General Conclusions. (1) Similarity of red cell dehydrogenase patterns does not indicate phylo genetic affinity in mammalian orders but is rather the result of the similar nature of the sub-units making up the enzymes and the ways in which the enzyme pheno types are expressed. (2) The Chimpanzee and human red cell samples are the only ones to contain the minor "mod." LDH band just behind LDH 2, which has been discussed in Chapter (3) In general, mamm alian red cells have predominantly fast moving LDH bands indicating a preferential production of B (or H) poly peptides. The exceptions are the Elephant, which must produce almost equal amounts of A and B (or M and H) polypeptides (Fig. k-2, N) and the Mouse, which prod uces mainly slow moving polypeptides (A or M) in its red cells (Fig. ^-2, I). (4) Chicken and duck red cells contain slow moving LDH bands, confirming the results of many other workers. When disc acrylamide gels are used, the major red cell LDH of birds has the same electrophoretic mobility as LDH 4-, indicating that its structure is AAAB (or MMMH). 136 (5) Red cell MDH does not produce multiple isozyme bands but a variation in the electrophoretic mobility of a single major band. There is no obvious correlation between the phylogen etic relationship of mammals and the mobility of MDH, except in the case of the domestic Cat and the Jaguar, both of which have an MDH with an electrophoretic mob ility intermediate between the ’’fast” and "slow" MDH bands of the other mammals. (6) Bird red cell MDH has a slower electrophoretic mobility than that of any mammalian MDH. In this respect it resembles the sit uation with red cell LDH. (7) The results obtained indicate that there is some doubt about the presence of true Isocitric acid dehydrogenase in mammalian red cells. Apparent IDH activity occurs irregularly and is not found in some samples even when they have been freshly collected. Where bands do appear with isocitrate as substrate, they always have the same electrophoretic mobilities as LDH or MDH bands. The author suggests that IDH activity and the activity associated with the minor MDH bands are the result of non specific dehydrogenase activity. The lack of specificity may be due to conform ational changes in the enzyme molecule resulting from 137 haemolysis of the red cells and/or electrophoresis. Such a theory is feasible if, as Kaplan contends (1965)* the dehydrogenases have evolved from a common ancestral molecule. (8) Variations in LDH phenotypes are not commonly expressed by alterations in electrophoretic mobility but by the relative amounts of slow and fast polypeptides produced and by the ways in which they are assembled. This type of variation is less hazardous than one producing mutations in protein structure which could destroy enzyme activity. The phenotypic expression of LDH isozymes does not appear to be due to a random production of A and B (or M and H) polypeptides but is produced epigenetically by a mechanism controlling the production of these two polypeptides and the frequency with which different tetramers are assembled. This mechanism requires the presence of a regulator system whose nature is not yet known. The hypothesis is ex amined and developed in the next three chapters, using red cell LDH and MDH from members of the marsupial family, Macropodidae 138 Author's Note, Since the work reported in Chapter 4 was completed, the paper by Koen and Goodman (1969) has become available to the author. These investigators, using starch gel electrophoresis, have described qual itative and quantitative differences in the slower moving LDH isozymes in tissues of Primates. They have suggested that these differences are caused by the instability of the M or A polypeptide in evolution compared with that of the H or B polypeptide. A possible criticism is that conclusions are drawn using few samples (2 - 10 animals). However, there is corroborative evidence in the disc acrylamide electrophoresis results (see Figure 4-2, L, Rabbit, P, Hyena and R, Merino X Sheep). In addition, in Figure 5-1 » LDH 1 is level in both wallaby (1) and human (2) red cell patterns but the slower bands in the human pattern become progressively slower than corresp onding bands in the wallaby sample (see also figure below) RED CELL LDH PATTERNS. Wallaby Human 139 Chapter 4, Additional References. LDH. Species differences. Paul and Fottrell, 1961, Pesce et al. , 1964, Gold berg and Wuntch, 1964, Salthe, 1969i Moyer et al., 1968, Kurata, 1963- 1^0 Chapter 3» Red Cell Dehydrogenases in Marsupials. 3«1 • 1» Introduction. In chapters 3 and 4, LDH was shown to exhibit quantitative variation in the amounts of LDH tetramers or isozymes found in red cells of diff erent animal and bird species. There is no evidence of a relationship between red cell LDH patterns and gener ally accepted evolutionary affinities within animal orders but within species one or more patterns constantly occur, suggesting that the production of LDH tetramers is under genetic control. In the next three chapters red cell dehydrogenases within a marsupial family, the Macropod- idae, are examined. 3.1.2. Acknowledgements. The work described in this section would not have been possible without the help of zoologists specialising in the study of marsupials. These workers have been mentioned in the acknowledgents at the end of the thesis but it is appropriate to mention here the assistance given by G.B. Sharman, lately Professor of Zoology at the University of N.S.W. and now at Mac quarie University, Sydney, who has provided the dendrogram shown in Figure 3-15* Messrs. B. Richardson and P. Johnston of the University of N.S.W. have provided blood samples from many of the marsupials tested and have given 141 assistance in helping the author to appreciate the prob lems associated with classification of animals in the family Macropodidae. In the following chapters, genetic aspects of LDH isozyme formation which result in the production of inherited LDH patterns are examined. The findings have been used to determine whether a relationship exists between LDH patterns and phylogeny in the Macropodidae. While acknowledging the assistance of others, the author accepts responsibility for any inaccuracies which may have been introduced because of lack of familiarity with the difficulties encountered in this field of investigation. 5.1.3* Classification of Marsupials. The larger Aust ralian marsupials were described soon after the country was settled and they still bear the descriptive names given to them at that time. For example, the east ern grey kangaroo, Macropus giganteus, was named in 1790 by Shaw and the red kangaroo, Megaleia rufa by Desmarest in 1822. The classification of marsupials has been based upon external characteristics (Iredale and Troughton, 1934, Troughton, 1957 and Marlow, 1962) or skull measurements (Raven and Gregory, 194.6) • The relationships between the many species 142 included in the Hacropodidae are still not established. In 1966 Calaby stated, "the phylogeny of the family is far from clear" and suggested that many members which had been separated in the past should be placed in one large group until further evidence is obtained which will enable a more precise division into genera to be made • In 19^8i Tate published a work on the anatomy and phylogeny of the Macropodidae. Kirsch and Poole (1967) and Kirsch (1968) have described differences in certain Macropodidae which are based upon physiological differences and biochemical traits such as serum trans ferrins . In 1961, Sharman used cytological studies to describe a possible phylogenetic relationship within the Order, Marsupialia. 9.2.1. Methods. Blood collection, preparation of haemolysates and disc acrylamide electrophoresis were carried out as described in the General Methods, (Chapter 2). All LDH patterns were identified using fresh blood. 9.2.2, Results. (1) Lactic Dehydrogenase in Red Cells. All red cell LDH patterns in Marsupials have at least four bands corresponding with LDH 1, 2, 9 and and some have the fifth band, LDH 9« Figure 9-1 is an illustrat- FIGURE 5-1. 143 COMPARISON OF RED CELL LDH AND MDH IN MARSUPIALS AND HUMANS. 12 3 4 (1) Red cell LDH type C from a "pretty face" wallaby (2) Human red cell LDH type 1 mod. ( the minor band is just visible behind LDH 2). (3) Fast MDH in the "pretty face" wallaby. (4) Slow MDH and the fast minor band at the LDH 1 position. 144 ion of LDH and MDH in a member of the Macropodidae comp ared with human LDH and MDH. LDH 4 is absent in the human pattern. Table 5-a is a list of the marsupials tested, to gether with the number of each species. The range of red cell LDH patterns in the Macropodidae is illustrated * in Figure 5-2 . The patterns may be divided into two main groups on the basis of the relative amounts of slow and fast tetramers. GROUP 1. is composed of types A, B and F (Figure 5-2. LDH 1 is faint, indicating that few BBBB tetramers are assembled. GROUP 2. consists of types C, D, E, E^ and G (Fig- * ure 5-2 ). LDH 1 is the heaviest band in all these patterns with the exception of type G, where LDH 1, 2 and 3 are all heavy and have equal intensity. Description of Red Cell LDH Types in Groups 1 and 2. Type A. On development of the gel for one hour in the dark, LDH 2 and 3 are of equal intensity, while LDH 1 and ♦ Since the printing of Figure 5-2, another pattern has been distinguished which has been called type G. This is illustrated as a separate drawing accompanying Fig ure 5-2 FIGURE 3-2 145 RANGE OF RED CELL LDH PATTERNS IN MEMBERS OF THE MACROPODIDAE. ^-0 + A B C F TYPE G Group 1 Types A, B and F. -- 4 Group 2 Types C, D, E, E^ and G. MR 3 ■■n 2 E2283K 1 Dotted line in type A indicates that a faint LDH 5 band has been found in some samples. This may be an overloading effect. Shaded bands denote diffuse bands of LDH 5» ( Photograph by courtesy of the Dept, of Medical Illust ration, University of N.S.W.) TABLE 5 - a 146 MEMBERS OF THE MACROPODIDAE TESTED FOR RED CELL LDH AND MDH. NAME No. LDH MDH 1. Aepyprymnus rufescens (Gray) , 1837 1 F S 2. Bettongia cuniculus (Ogilby), 1838 2 1A, 1B s 3. *Potorous tridactylus (Kerr) 1793 (Potoroo) 100 2A, 88B s 4. Dendrolagus lumholtzi (Collett), 1884, (Tree kangaroo)• 2 B s D. goodfellowi 2 C s Dorcopsulus macleayi 2 1A, 1B s 6. Dorcopsis hageni 1 a s 7. Setonix brachyurus (Quoy & Gaimard), 1830, (Quokka). 10 s 8. Megaleia rufa rufa (Desmarest), 1822. (Red kangaroo). 63 35A, 8b s M. rufa pallidus 2 A s 9. Macropus robustus robustus (Gould), 1841. (Wallaroo) 13 A s 10. M. r. erubescens (Gould), 1841 (Euro). 44 20A, 24B s 11. M. r. reginae (Schwarz), 1910. 1 A F 12. M. r. cervinus (Thomas), 1900. 1 C s 13. M. antilopinus (Gould), 1842 16 C s (Continued overleaf.) TABLE 5 - a, (continued). 147 MEMBERS OF THE MACROPODIDAE TESTED FOR RED CELL LDH AND MDH. NAME No. LDH MDH 14. M. alligatoris++ (Thomas), 1901. 17 15A, 2C S 15. M. rufogriseus (Desmarest), 1817 7 C S 16. M. dorsalis (Gray), 1857 2 C S 17. M. bernardus (Rothschild), 1904. 1 C S 18. M. eugenii (Desmarest), 1817, (Tammar)+++ 18 5A, 15C S 19. M. parma (Waterhouse), 1846 4 C + F 20. M. giganteus (Shaw), 1790, East- ern grey kangaroo 45 50D, 15E F, M. g. tasmaniensis 11 8d, 5E F, 21. M. fuliginosus fuliginosus (Des- marest) , 1817 » Kangaroo Is. western grey kangaroo 26 26e F M. f. melanops (Gould), 1842, 50 11D, 19E S M. f. ocydromus (Gould), 1842 15 1D, 14E S (mainland western greys) 22. M. parryi (Bennett), 1855 ^ (Pretty face wallaby). 5 C s 25. M. irma (Jourdan), 1857 i (Black gloved wallaby). 5 2A, 1B s 24. Wallabia bicolor (Desmarest), 1804 (Swamp wallaby) 7 C F TABLE 5 - ai (continued). 148 MEMBERS OF THE MACROPODIDAE TESTED FOR RED CELL LDH AND MDH. NAME No. LDH MDH 25« W. rufogrisea frutica (Ogilby), 1838 1 C 26. Petrogale inornata (Gould), 1842, (Rock wallaby l‘ C S P. pearsoni 1 B S P. xanthopus (Gray), 1855 1 A F 27«H Thylogale billardierii (Desmarest), 1822, (Tasmanian pademelon). 15 A S T. brunii brownii (N.G. pademelon) 2 A S T. stigmatica wilcoxi (M’Coy), 1 C S 1866 T. s. coxenii (Gray), 1866 1 C S T. thetis (Lesson) , 1827 1 C S (last four are mainland pademelons) * Results included with the permission of P. Johnston. C+ An extreme form of LDH pattern C which is similar to pattern D. ++ Two animals from Arnhem Land (N.T.) were typed C but one of these was an atypical C pattern intermediate between A and C. (See discussion). + + + Type C LDH found in Western Australian tammars, Type A in South Australian tammars TABLE 5 - a, (continued.) 149 e Tasmanian and N.G. pademelons are LDH type A; mainland species are type C. It is unusual for "fast" and "slow'1 MDH to be found in the one species but have been recorded for these two species. The problems associated with this finding are discussed in the text. References: Iredale and Troughton, 1934, Marlow, 1962, Tate, 1948 and Raven and Gregory, 1946. 150 LDII 4 are faint and appear later than the other two bands Type B. There is a difference in intensity between LDH 2 and 3* the former being the heavier. LDH 1 and 4 are also uneven in intensity, LDH 4 being heavier than LDH 1 which is always the lightest band. Care must be taken in distinguishing between types A and B, both of which are found in red kangaroos, pot- oroos, wallaroos and euros. When acrylamide gels are left overnight, the slower isozymes diffuse more widely in the gels than the faster isozymes, so that type A patterns left overnight resemble type B. At this time it is not clear whether types A and B are extremes of expression of the one phenotype or whether they are truly polymorphic forms. Examples of the two types, together with a type found in grey kangaroos and discussed below under type D, are shown in Figure 5-3 • It is of interest to observe (Figure 5-b ) that potoroo types are mostly B, while type A predominates in red kangaroos, euros and wallaroos. Type C. This is one of the red cell LDH patterns belonging to Group 2. The heaviest band is LDH 1, with 2, 3 and 4 gradually decreasing in intensity. Type C is seen in Figure 5-^ and is typical of the wallabies although it occurs in some other genera. These other FIGURE 5-3. 151 RED CELL LDH TYPES A, B AND D. * i ' v So SI* *NN» ^ I. 1 2 3 4 5 6 7 8 Type D. (1) from an eastern grey kangaroo. Type A. (6), (7) and (8) from red kangaroos Type B. (3)» (^) and (5) from red kangaroos FIGURE 5-4. 152. RED CELL LDH AND MDH PATTERNS IN POTOROOS. 1 2 3 4 5 6 7 8 (1) , (3) ■» (5) and (?)• LDH type B patterns from red cells of potoroos. LDH 3 is the heaviest band, LDH 1 the lightest and LDH 2 and 4 are heavy. (2) , (4), (6) and (8). A single "slow” MDH band with a mobility intermediate between those of LDH 2 and LDH 3« Minor dehydrogenase activity is present at LDH 1 position in (2), (4) and (6), 153 genera include parma v/allabies (M. parma) and quokkas (Setonix brachyurus) whose LDH pattern has been shown as C+ in Table 5-a. It is similar to type D but LDH 2 is more intense in the former. Type D, LDH type D represents the most extreme patt ern in Group 2 because the LDH 1 tetramer (BBBB) is most prominent and LDH 2, 3 and 4 are minor bands. LDH 2 is heavier than LDH 3 and 4. This LDH pattern is shown in Figure 5“5 where it is compared with type A from a euro and type E from a grey kangaroo. Type D can also be seen in Figure 5-3 (1)* Types E and E . Both of these LDH patterns belong to Group 2 and the heaviest staining band is that of the LDH 1 tetramer. Like type D they are found in grey kangaroos. Repeated testing with fresh blood samples has confirmed that type D is not an artefact produced by loss of activity of slower LDH tetramers. The most obvious difference between types D and E is the heavy staining associated with LDH 5 in type E. As discussed in Chapter 4, LDH 5 is a diffuse band and much activity remains in the sample gel (see Figure 5-6 (1) ). By increasing the amount of ammonium persulphate in the running gel to 0.28 per cent and increasing the volt age at the beginning of the electrophoretic run to 300 v. it is possible to concentrate LDH 5 into a discrete band. Type E patterns produced by this method are shown in Figure 5-7, together with type C. In addition to the presence of LDH 5» LDH 4 is more intense than LDH 3 or 2 (see figures 5-5 (1), 5-6 and 5-7)• The sub division into types E and E are made on the basis of the relative intensities of LDH 2 and 3» LDH 5 is lighter than LDH 2 in type E and heavier than LDH 2 in type E^. Type F. This is one of the patterns belonging to Group 1 and has been found in Aepyprymnus sp., a rare genus represented in this study by a single specimen. It has two equally intense bands at LDH 4 and 5 posit ions, LDH 2 is lighter and LDH 1 is the lightest band. Type G. An LDH pattern which has been found in a New Guinea genus, Dorcopsis hageni (see Fig. 5-11 » 6). It has been included in Group 2 but is more accurately placed in a position between Groups 1,-and 2. LDH 1, 2 and 3 are equally heavy and LDH 4 is lighter. (2) Malic Acid Dehydrogenase. The Macro- podidae contain examples of "fast” and "slow" MDH bands which have been described in Chapter 4. Minor MDH bands were seen in some genera. Usually the minor bands had the same electrophoretic mobility as LDH 1 but a second minor band was present in one potoroo at LDH position 4 (See Figure 5-8). MDH patt- FIGURE 3-3* 155 LDH AND MDH TYPES IN MEMBERS OF THE MACROPODIDAE. 12 3^56 78 9 10 (1) LDH type E, (2) Slow MDH, Kangaroo Island west ern grey kangaroo, Macropus fuligino>sus fuligin- osus. (5)i (5) and (9) LDH type D, (4), (6) and (10) Slow MDH, eastern grey kangaroo, Macropus giganteus. * (7) LDH type A,* (8) Slow MDH, wallaroo, Macropus robustus robustus. * * Acrylamide gels were left overnight before being photographed. LDH 4 has diffused so that the pattern could be type B. (See discussion in Section 5.2.2.) FIGURE 3-6. 156 LDH TYPE E FROM WESTERN GREY KANGAROOS. ------m------ As in all grey kangaroos, LDH 1 is the heaviest band LDH 4 is heavier than LDH 2 or 3 and LDH 5 is present LDH 2 is lighter than LDH 3i which defines it as LDH type E^ and not E. These patterns were reproduced in subsequent tests of the same samples. FIGURE 5-7 157 LDH AND MDH PATTERNS IN WESTERN GREY KANGAROOS AND A WALLAROO. 1 2 3 4 5 6 (1), (5)i LDH type E, (2), (4) Fast MDH from two western grey kangaroos, Macropus fuliginosus ocydromus. (5)1 Slow MDH, (6) LDH type "C” in wallaroo from the Kimberley Ranges in N.W. Western Australia. These samples contain variations which are discussed in Section 5*2.5*i b FIGURE 3-8 158 RED CELL DEHYDROGENASES IN THE POTOROO. 1 2 3 4 (1) LDH type B. (2) Slow MDH, with a minor band at LDH position 1 and a second minor MDH band at LDH position 4. (3) A faint band at LDH 1 position - substrate Isocitric acid. (4) A faint band at LDH 1 position - substrate Sodium succinate 159 * erns in Macropodidae are illustrated in Figure 5~9» (3) Dehydrogenases in Red Cells of the Potoroo. Figure 5-8 demonstrates patterns from red cells of a potoroo, using the following substrates, (1) Lactic acid, (2) Malic acid, (3) Isocitric acid and (4) Sodium succinate. DPN+ was used as co-enzyme, except for succinic acid dehydrogenase, which does not require a co-enzyme. Results. LDH is type B; MDH has a major band in the "slow" position and two minor bands, one with the same mobility as LDH 1 and the other with the same mob ility as LDH 4. IDH and SDH have one faint band with the same electrophoretic mobility as LDH 1. This result is int eresting because these two activities are not expected in cells v/hich lack nuclei and mitochondria. Succinic acid dehydrogenase activity is believed to depend upon * MDH results are given with the following qualification. Identification is made using electrophoretic mobility and may later be revised for genera from which isolated samples have been obtained and tested at different times. (See "disadvantages of disc electrophoresis", 2.1.4.) — ”1" I 160 in I I • I I I LDH positions I I f Aepyprymnus 0 BettongU Q Potorous 0 Dendrolagus (D Dorcopsulus CD 1 TJ Dorccpsis •q I o a. 1 Setonix CD o 1 Megaleia ID (0 Macropus spp. 1 Euros & Wallaroo spp. 2 M. reginae <=■ LU • —» M. parma M. f. fuliginosus M.f.ocydromus CD I o M. f. melanops *o (D M. giganteus a: M. g. tasmaniensis M. parryi M. irma Waliabia bicolor Petrogale ino rnata 1 P. pearson i P. xanthoput Thylogale spp. 161 maintaining the structure of mitochondrial membranes. The finding of IDH and SDH activities in the red cells of this animal and similar results with human red cells (Chapter 3) indicate that some isozymes can have non specific dehydrogenase activity, associated with LDH 1 (BBBB) tetramers. MDH activity is also found in a band at the LDH k position (AAAB). (4) LDH Patterns in Red Cells and Diaphragm Muscle of the Euro, Macropus robustus erubescens. Red cell LDH patterns in genera within the Macro- podidae are confined to one or two typical patterns, suggesting that they are genetically determined. In other animals, including Man, LDH patterns are consist ent in the same tissues within species but vary from tissue to tissue. Fresh blood and diaphragm muscle tissue were avail able from a euro. Haemolysate was prepared as on pre vious occasions. Diaphragm muscle, kept on ice after removal from the animal, was homogenised in a small volume of physiological saline (0.85% NaCl, w/v.), using a ground glass homogeniser. The homogenate was centrif uged at 5,000 x g. for 25 minutes and the supernatant solution used for electrophoresis. LDH patterns are 162 shown in Figure 5-10 (2) and (3)» together with a type G LDH from red cells of a swamp wallaby, Wallabia bicolor. Euro red cell LDH is represented by type A, in which LDH 2 and LDH 3 are the heaviest bands, while the pattern in diaphragm muscle is the reverse. LDH 2 and LDH 3 are light but LDH 1 and LDH k are heavy. This represents a change in phenotypic expression due to differing re quirements in the cells of blood and diaphragm muscle. (3) Dehydrogenase Patterns in Members of the Macro- podidae from New Guinea, Tasmania and New Zealand. Some members of the Macropodidae have representatives on the nearby islands as well as the mainland of Australia. All agile wallabies are included in one species, Macropus agilis, while pade- melons are divided into at least four species, including Thylogale thetis and T. stigmatica from the mainland, T. brunii brownii from New Guinea and T. billardierii from Tasmania. A list of the animals tested is given in Table 5-b and examples of red cell LDH types are shown in Figures 5-11 and 5-12. LDH type G is shown in Figure 5-11, (6) and, as described in Section 5*2.2., it is inter mediate between Groups 1 and 2. It has heavy LDH 2 and 3 bands like type A but LDH 1 is also heavy as in type C. The animal, Dorcopsis hageni, is a New Guinea wallaby. FIGURE 5-10. 163 LDH PATTERNS IN RED CELLS AND DIAPHRAGM MUSCLE. 1 2 3 (1) Red cell LDH type C from the swamp wallaby, V/allabia bicolor. (2) Red cell LDH type A from the euro, Macropus robustus erubescens. (3) LDH pattern from diaphragm muscle of the euro, M. r. erubescens. TABLE 5 - b 164 RED CELL LDH AND MDH IN MACROPODIDAE FROM NEW GUINEA, TASMANIA AND NEW ZEALAND. * Name Location No. LDH MDH Dorcopsulus macleayi+ N.G. 2 A Slow *4* Dorcopsis hageni N.G. 1 G Slow it Dendrolagus goodfellowi N.G. 2 C Slow JJ Dendrolagus lumholtzi^ N.G. 2 B, A Slow Thylogale brunii brownii N.G. 2 A Slow Thylogale billardierii Tas. 13 A Slow * * Thylogale stigmatica ML 2 C Slow Thylogale thetis ML. 1 C Slow Macropus agilis N.G. 2 C Fast Macropus agilis ML. 3 C Fast Macropus parma N.Z. 2 C Fast Macropus parma ML. 1 C Fast * Blood was collected from most of these animals at Gosford (courtesy Eric Worrell). * Mainland. + N.Guinea wallabies # N.G. Tree kangaroos FIGURE ^ - 11. 165 RED CELL LDH PATTERNS IN NEW GUINEA MACROPODIDAE AND IN ONE MAINLAND GENUS. 1 2 3 4 5 6 7 (1) N.G. wallaby, Dorcopsulus macleayi, typed as A. (2) Northern swamp wallaby (mainland), Wallalbia bicolor, type C. (3) and (7) N.G. pademelons, Thylogale brunjj brownij, type A. LDH 1 is abnormal and the nature of this abnormality is discussed in Section 5*2.3«1 (b). (4) N.G. agile wallaby, Macropus agilis, type C. (5) Cuscus (N.G. possum) Phalanger orientalis, 5 bands. (6) N.G. wallaby, Dorcopsis hageni, type G FIGURE ^ - 12. 166 VARIATIONS IN LDH TYPES OF AUSTRALIAN AND NEW GUINEA GENERA. (1) Agile wallaby (M. agilis) from the mainland and (5) Agile wallaby (M. agilis) from New Guinea. Both are type C and are indistinguishable. (2) N.G. tree kangaroo, Dendrolagus goodfellowi4 type C. D. lumholtzi has type A or B. (This type is illustrated in Section 5-2.3*» b) . (3) Tasmanian pademelon, Thylogale billardierij. This is type A, since LDH 2 & 3 are heavier than LDH 1. (4) Parma wallaby, Macropus parma. This is an extreme form of type C, in which LDH j> and 4 are reduced. The same pattern is found in quokkas (Setonix brachyurus) and is similar to type D. 167 (6) Red Cell Dehydrogenases in Members of the Marsupial Families, Dasyuridae, Peramelidae and Phalangeridae. The animals tested are listed in Table 5-c and LDH patterns are drawn in Figure LDH patterns in all members of the Phal angeridae tested and the one representative genus of the Peramelidae, the long-nosed bandicoot, Perameles nasuta, contain five isozymes (Fig. 3 and Fig. 5-1^, 1). TABLE 3 - c. RED CELL DEHYDROGENASES IN DASYURIDAE, PERAMELIDAE AND PHALANGERIDAE. NAME. No. LDH MDH Dasyuridae, (1) Sminthopsis sp., Marsupial mouse 1 1 band Fast (2) Sarcophilus harrisi (Boitard) 1841 Tasmanian devil 1 4 bands Fast Peramelidae, (1) Perameles nasuta, Geoffroy, 1804 Long-nosed bandicoot 4 3 bands Slow Phalangeridae, (1) Phalanger orientalis (Pallas) 1766 Cuscus (New Guinea) 2 5 bands Slow (2) Trichosurus vulpecula (Kerr) 1792 Brush-tailed possum 3 5 bands Slow (3) Phascolarctos cinereus (Goldfuss) 1817 Koala 1 5 bands Slow FIGURE 3-13 168 LDH PATTERNS IN DASYURIDAE, PERAMELIDAE AND PHALANGERIDAE. mmm 5 MM Mm ^ 4 (1) (2) : (3) (1) Marsupial mouse - 1 specimen, (Dasyuridae). (2) Tasmanian devil - 1 specimen. (Dasyuridae). (3) Long-nosed bandicoot (Peramelidae), Cuscus, Brush-tailed possum, Koala (Phalangeridae). 5.2.3. Discussion. (a) LDH and MDH Typing and Affin ities in the Macropodidae. Biochemical markers are presumed to have adv antages over polygenic traits such as skull measurements or morphological characters (i) because the resultant phenotype is the product of the interaction of a small number of genes. LDH tetramers are produced by two gene pairs and MDH is probably the product of one pair of genes. FIGURE 5-14. 169 RED CELL LDH PATTERNS IN MACROPODIDAE AND PERAMELIDAE. 5 4 3 2 1 (1) LDH with 5 bands. LDH 1 and 2 are reduced and there is an intense LDH 5 band. This sample is from a long-nosed bandicoot (Perameles nasuta) The same LDH pattern is present in the Phalang- eridae. (2) LDH type D from an eastern grey kangaroo. (3), (4) and (5) Type B in three red kangaroos 170 (ii) they are less susceptible to variations due to environment. The expression of LDH genes a and b does vary with cellular environment but, as far as can be determined, conditions in the one tissue are constant at the same period in the life cycle. The following observations can be made from results described in this chapter. 1. LDH of all members of the Order Marsupialia which have been tested contain combinations of A and B polypeptides. 2. A genetic mechanism controls the rate of production of each polypeptide and the ways in which polypeptides are assembled into tetramers. The form ation of tetramers is not random and the phenotypes result from epigenetic factors which are characteristic of the species in which the LDH phenotypes occur. 3. LDH patterns vary from tissue to tissue. In this respect the Macropodidae resemble other animals, including Man. 4. All red cell LDH patterns in the Macropod idae can be assigned to one of two groups, Group 1, which produces slow tetramers rather than fast tetramers and Group 2, which produces more fast tetramers than slow tetramers e.g. 171 Group 1 Group 2 LDH type A LDH type B LDH type C LDH type D AAAB k i . .. >. AABB —* 3 ABBB 2 — BBBB 1 F O EII3 These groups differ in the relative numbers of A and B polypeptides present. 5. "Slow" and "fast" MDH bands are present in the Macropodidae and other marsupial families. The Distribution of LDH and MDH Types and their Relation ship to a Dendrogram of the Macropodidae. A dendrogram of the family Macropodidae, provided by G.B. Sharman and based on results of studies on the chromosomes, reproduct ive systems and reproductive patterns of macropodid mar supials, is presented in Figure The numerals "i", "ii", "iii" and Miv", together with LDH and MDH data have been added by the author. The author^ interpretation of the Dendrogram. Members of the family Macropodidae have arisen from an unknown ancestor. At an early stage, section (i), which includes Aepyprymnus sp. and Potorous sp., split off and has not separated into further genera. Most of the more recently separated genera can be separated into three major sections, two exceptions being Lagostrophus sp. and Setonix sp FIGURE 5-15 172 A DENDROGRAM OF THE MACROPODIDAE LDH MDH F — Acpyprymnus...... F t S — Bettongia...... A,B i S — Hypsipry mnodon...... K0 sample — Potorous...... A f B 1 S — Lagostrophus^.. ho sample -OendrolagUs.;;“w. *CB ’ | — Dorcopsulus...... A.B 1 S — DorCOpsiS...... G inter. S — Setonix...... C+ z S — Megalcia...... A.B \ S Mr erubescrns... A.B 1 S M r robustus...... A 1 S ““Osphranter Melligatons. A C 1.2 S III M. antilopinus-...... C 2 S — Macropus rufoqrieeus...... C l S fci pirmi:v;:.I* l I ""-eugenii...... ac 1.2 s — iiM. __gi ga, *rtteu*. ------,-parryi_------...... D.Ec 22 F.Ss -M. irm'rrr.:...... A.B , S — M. agilis...... G 2 F — Wallabia bicolor...... C 2 F — Onychogalea...... s*Mpi-£ Petrogale spp...... A.B t S.F P. inornata...... C * s Thylogale billardierii...... A 1 S T.brunii brownii...... A a S T. stigmatica...... C 2 S T.thetis...... C 2 S Lagorchestes hirsulus...... N° sample Denotes whether LDH patterns are from groups 1 or 2 173 Section ii, containing the tree kangaroos, Dendrolagus spp., Dorcopsulus sp. and Dorcopsis sp., is the second group to be separated, followed in time by section iii, which is a diverse group containing several genera and many species. It is divided into red kangaroos (Megaleia sp.), euros (Macropus sp.) and wallaroos (Macropus sp.) on the one hand and grey kan garoos (Macropus spp.) and wallabies (Macropus spp.), (Wallabia sp.) on the other hand. The most recently derived genera, comprising rock wallabies (Petrogale spp.) and pademelons (Thylogale spp.) make up the greater part of section iv. Conclusions. (i) Group 1 patterns appear first in the family Macropodidae and it is probable that the red cell LDH type of the unknown ancestor belonged to this group. (ii) Group 2 LDH patterns first occur in section ii (Dendrolagus sp.) and thereafter are rep resented in all sections. They are most abundant in section iii where they range from patterns in which the LDH activity is almost completely confined to one band (type D) to ones in which three of the four LDH bands have equal and intense activity (type G). (iii) The most primitive MDH is the slow form and "fast" MDH first appears in section iii. There is one example of a "fast" MDH in section iv (Petrogale xanthopus) which is represented by one animal. As with LDH, the altered MDH form is most often found among grey kangaroos and wallabies. (iv) Red cell LDH patterns are capable of var iation of expression but all can be interpreted within the accepted theory that LDH isozymes are composed of tetramers containing one or two types of polypeptide, A or B (or M or H). It can be concluded that, in general, changes in LDH and MDH patterns can be correlated with the class ification of the Macropodidae which has been compiled using other criteria. There are some cases in which LDH and MDH patterns suggest divergences within genera and where an investigation of other markers may support or contradict the evidence of these two enzymes in det ermining affinities or phylogenetic succession. Some of these divergences and difficulties of interpretation are described below. (a) Macropus irma. This wallaby differs from its nearest neighbours in Figure 5-^5 in both LDH and MDH types - a difference reflected in other markers- 175 both biochemical and morphological (Sharman, Richard son, Johnston and Clark, unpub.). (b) M. alligatoris and M. antilopinus are two wallaroos which are found in overlapping areas in the Northern Territory and are distinguishable by diff erences in skull measurements and biochemical markers (Richardson - personal communication). All M. antil opinus tested were LDH type C and M. alligatoris were predominantly LDH type A. Two type C LDH individuals were found. One of these was abnormal, having less LDH 1 than a normal type C and being intermediate be- tween A and C, e,g. Normal C Aberrant Normal A JBHM ■■■ praaa In previous work Clark and Richardson (1968) recorded one animal as M. ? antilopinus since it had LDH type A and the morphological features were not clearly those of M. antilopinus. The appearance of these two abberrant animals suggests that limited inter breeding may occur but the persistent separation of the two LDH types and the differences in other genetic markers indicate that M. antilopinus and M. alligatoris 176 are separate species. ( See also Figure 7-1i (4) and (5) ) (c) M. eugenii, (Tammars). LDH patterns from Groups 1 and 2 are reported within a single species. Animals with type C LDH are from Garden Is. and the Abrolhos Is. off the coast of Western Australia and those with type A LDH are from Kangaroo Is. off the coast of South Australia (See map, appendix ii). (d) Dendrolagus spp. and Thylogale spp. These two genera have species with LDH type A and LDH type C. It is not appropriate to comment upon the two animals representing Dendrolagus spp. because nothing is known of their history except that they came from New Guinea. It is known that Thylogale spp. with LDH type C are found in New South Wales and Queensland, while T. brunii brownii, which is type A, comes from New Guinea and T. billardierii, which is also type A is from Tasmania. (e) Both types of MDH have been recorded for members of the species, Macropus giganteus. The difficult ies associated with determining MDH mobilities have been discussed and in this case, the author feels that further work is necessary to confirm the presence of the two MDH types in the one species. 177 (f) MDH in two wallaroo species. Figure 5-16 shows that Macropus robustus robustus has a "slow” MDH and M. r. reginae has a "fast" MDH. (See also Fig. 5-9)• It differs from all other wallaroos in this respect. General Conclusions. Red cell LDH patterns are cap able of wide variation but some genetic factor or factors determine that patterns do not vary randomly over the range of possible patterns. Although closely related species have similar LDH patterns, some genera (and possibly two species) include LDH patterns from Groups 1 and 2, indicating that a mechanism involving other genes is responsible for the preferential pro duction of fast or slow tetramers. LDH and MDH patterns vary ind ependently.. LDH and MDH are useful genetic markers for investigating possible phylogenetic rel ationships in marsupials but it is necessary to dem onstrate consistent differences with several markers. The presence of two LDH types in red cells of grey kangaroos indicates that a poly morphism exists and this in turn suggests that there FIGURE 5-16 178 LDH AND MDH PATTERNS IN TWO WALLAROOS. 12 3 4 (1) Type A LDH in Macropus robustus robustus, the eastern wallaroo. (2) ’’slow" MDH in M. r. robustus. (3) Type A LDH in Macropus r. reginae. (k) "fast" MDH in M. r. reginae. 179 are two different genetic or epigenetic mechanisms res ponsible for LDH production. Kirsch and Poole, (19&1) have presented serological evidence of species differences in grey kangaroos and several species are recognised. These have been divided into "eastern” and "western” grey kangaroos. A number of blood samples from all types was made available by W.E. Poole and several others, and in the next chapter the distribution of the two phenotypes is examined (a) to determine whether a polymorphism exists in red cell LDH from grey kangaroos and (b) to see if any information can be obtained about the origin of different species of Grey Kangaroo. Chapter 6 A Study of Red Cell LDH in Grey Kangaroos. 6.1.1. Introduction and Acknowledgement. Grey kan garoos are distributed throughout the eastern and south ern mainland of Australia and neighbouring islands and have been divided into eastern and western types. Sub divisions in grey kangaroos and their geographic dist ribution are seen in Figures 6-1 and 6-2, which are reproduced with the permission of W.E. Poole of C.S.I.R.O. Division of Wildlife Research, Canberra, Australia, without whose co-operation this work could not have been done • Morphological, physiological and biochemical differences have been observed in the two types (Kirsch and Poole, 1967; Kirsch, 1968). In chapter 9 (Table 5- a and Figure 5-15) two major red cell LDH types were described for grey kangaroos. They are types D and E. A minor modification of E, called E was distinguished but for the purpose of this study both type E patterns are combined. The three patterns are drawn in Figure 6- 9. The use of fresh blood samples and the ability to obtain reproducible patterns indicate that type D is not produced by a loss of activity in the slower LDH tetramers with storage. FIGURE 6-1. 181 SUB-DIVISION OF GREY KANGAROOS AND THEIR GEOGRAPHICAL DISTRIBUTION. Grey Kangaroos eastern western Macropus giganteus Shaw, 1790 M. g. giganteus M. g. major Shaw, 1800 N. E. Aust., S,E. Aust., (Cooktown) (Sydney) M. f, melanops M. f. '.Gould, 18^2 ■f-“ligin°5us s.w. N.S.W., Kangaroo Is S.A., W.A. M. g. tas'maniensis Le Souef, 1923 Tasmania M. f. ocydromus Gould, 1842 W.A. Reproduced by permission of W.E. Poole FIGURE 6-2. 182 GEOGRAPHIC DISTRIBUTION OF GREY KANGAROOS AND OF RED CELL LDH TYPES. (Reproduced from a figure drawn by W.E. Poole and presented with his permission.) f.melanops £E = ♦ fD 0.0667 f. fuliginosus ^ fD = 0.0000 fE = 1.0000 tasmaniensis + Distribution of eastern grey kangaroos. 0 Distribution of western grey kangaroos. FIGURE 6-5. 183 RED CELL LDH PATTERNS IN GREY KANGAROOS. W//, W/A Origin D and E LDH types are found in the same species and have been used to determine if (a) the phenotypes are inherited unchanged or (b) there are differences in the frequency of D and E phenotypes in several species of Grey Kangaroo. 6.1.2. Methods. Blood samples were obtained from 129 grey kangaroos from C.S.I.R.O. herds, zoos, sanct uaries and private sources. Where possible the history of each animal was obtained 184 Blood samples were kept refrigerated and despatched by air to the laboratory where LDH patterns were det ermined within 48 hours of their arrival. Table 6-a shows the kinds and numbers of animals tested. TABLE 6-a. GREY KANGAROOS AVAILABLE FOR LDH TESTING. NAME NUMBER Macropus giganteus,(Shaw) 1790 (eastern grey) 45 Macropus giganteus tasmaniensis (Tasmanian eastern grey) 13 M. fuliginosus fuliginosus, (Desmarest) 1817» (K.I. western grey) 26 M. f. melanops, (Gould) 1842, (mainland western grey) 30 M. f. ocydromus, (Gould) 1842, (mainland western grey) 15 Total 7610901876 * K.I. is Kangaroo Island, off the coast of South Aust ralia. M. giganteus extends from North Queensland down the eastern part of Australia, including most of New South Wales and Vistoria and extending into the northern part of Tasmania. Poole divides the eastern grey into M. giganteus, found around Cooktown in northern Queens land, M. g. major, (Shaw, 1800) from S.E. Australia and including those found around Sydney and M. g. tasman- iensis, (Le Souef, 1923)i the species found in northern Tasmania (Figure 6-1). In this study mainland eastern grey kangaroos have been included as one group. There is a region of overlap between eastern and western grey kangaroo niches in western N.S.W. and Victoria and the mainland western greys extend from this area to Western Australia across southern South Aust ralia. Poole has divided these into M.f. melanops, extending from western N.S.W., through S. Australia into Western Australia and M. f. ocydromus, found in Western Australia. A third group of western grey kangaroos is morphologically distinct and was confined to Kangaroo Island, off the S. Australian coast. It is possible that individuals are now free on the Australian main land. Interbreeding between all these species has been found in captivity but has not been observed in the 186 field. 6.1.3. Results. (1) The Existence of Two Red Cell LDH Types in Grey Kangaroos. Figure 6-4 shows LDH types E, (1), (2) and D (3)i which were ob served in freshly prepared haemolysates. LDH 3 and LDH 4 are present in higher concentration in type E and there is a heavy band in the LDH 5 position. Types D and E were found in all species except M. f. fulig- inosus, where no type D LDH was found in 26 samples. (2) Variation in Expression of the Two Phenotypes. Although it is possible to type LDH patterns as D or E, the types tend to form a graded series rather than two distinct and unchanging phenotypes. Degrees of variation can be seen in Figure 6-5. Samples (1), (3) and (4) are typical LDH E pat terns, sample (6) is typical LDH D but samples (2) and (5) are intermediate. In these two samples LDH 3 and LDH 4 are light, as in type D, but there is a heavy band at the LDH 5 position as in type E. (3) Family Data Illustrating the Genetic Nature of LDH Types in Grey Kan garoos . Thirteen sets of parents and their offspring were tested and the results are shown in Table 6-b. The mothers are on the left. FIGURE 6-4. 187 RED CELL LDH TYPES IN GREY KANGAROOS. (1), (2), LDH type E, illustrating heavy band at LDH 5. (3) LDH type D FIGURE 6-5. 188 VARIATIONS IN RED CELL LDH PATTERNS IN GREY KANGAROOS. 1 2 3 ^ 5 6 (1) , (3) and (A). Typical LDH type E. (6) Typical LDH type D. (2) and (3) LDH patterns with intermediate char acteristics. (See text). TABLE 6 - b. 189 * (1) G 115 (g) X G 51 (g) offspring G 190 2 ' ■ E+ D D it (2) G 120 (g) X G99 (g) G 243 2 D D D it (3) G 129 (g) X G99 (g) G 245 0" E D E 11 (4) G X G 60 (g) 76 (g) G 163 2 E E E ti (3) G 3 (g) X G 60 (g) G 76 2 E E E u (6) G 1 (g) X G 35 (g) G 96 $ E EE it 2 X (7) G (g) G 35 (g) G 128 2 D E E it (8) G 92 (0) X G 90 (0) G 222 cf E D E 11 (9) G 94 (0) X G 90 (0) G 233 cf E D E 11 (10) G (m) X 63 G 13 (m) G 227 2 E E E n (11) G 45 (m) X G 40 (m) G 231 Cf X E E n (12) G 125 (m) X G 15 (m) G 234 cf E E E 11 (13) G 83 (m) X G 40 (m) G 228 2 x E E 190 * (g) = M. giganteus, (m) = M. f. melanops, (0) = M• f. ocydromus. + LDH type. (1) One D x D mating was observed and the result ant offspring was D. (ii) Five E x E matings occurred and all offspring were E. (iii) Five matings were D x E and of these, four produced E offspring and one, D. (iv) LDH types of two mothers were not available. Conclusions. No new LDH patterns resulted from mat ings between DxD, DxEorExE individuals. All were grouped as LDH type D or LDH type E. In all cases but one (1), type E LDH was found in offspring of matings in which at least one of the parents was LDH type E. Type E has more slow tetramers (AAAA and AAAB) than type D which is composed mainly of LDH 1, (BBBB). If the production of LDH patt erns depends in part upon the epigenetic assembly of available polypeptides into tetramers, the presence in 191 one parent of a mechanism for the production of A poly peptides in large amounts favours the expression of type E LDH. In a D x D mating the ability to make a large number of A polypeptides is not present and the LDH type in the offspring should be type D. This is the case in the one D x D mating. If expression of the LDH phenotype is pleiotropic, the effects of other genes could cause large numbers of slow (A) polypeptides to be produced in the offspring although neither parent does so. Inter-specific hybrids draw on different gene pools and could produce LDH patterns which are appar ently incompatible more frequently than intra-specific offspring. (See Chapter 7)• (4) Frequency of Red Cell LDH Types in Grey Kangaroos. There is evidence (Kirsch and Poole, 1968) that eastern and western grey kangaroos maintain their identities when occupying the same region and this is evidence that they are different species. (See Figure 6-2). Western grey kangaroos have been separated into M.f. ocydromus and M.f. mel- anops on the mainland and M.f. fuliginosus, which until recently was confined to Kangaroo Island. Eastern grey kangaroos have also been divided into mainland and ins ular species 192 Table 6-c shows the distribution of LDH types in eastern and western grey kangaroo species and phenotype frequencies• TABLE 6 - c. RED CELL LDH PHENOTYPES IN GREY KANGAROOS. Phenotype freq Species Locality D E Total D E M. giganteus E. Aust. 50 15 45 0.6667 0.5555 M. g. tasman- iensis N. Tas. 10 5 15 0.7692 0.2508 M. f. melanops N.S.W. , Vic., S.A. 11 19 50 O.5667 0.6555 M. f. ocydromus W.A. 1 14 15 O.O667 0.9555 M. f. fuliginosus K.I. 0 26 26 0.0000 1.0000 Total greys 52 77 129 0.4051 0.5969 When contingency tests are applied to this data p (Ractliffe, 1967)^. for ^ degrees of freedom is 47.59* which corresponds with a probability of less than 0.1% that the observed differences arose by chance. This value is statistically significant. It is not possible to determine gene frequencies be cause two structural genes are responsible for the pro- 193 duction of tetramers and because the expression of pheno types is influenced by factors other than structural genes. Phenotype frequencies have been added to the map in Figure 6-2 so that their geographical distribution can be seen. Conclusions, (1) There is evidence that grey kan garoo populations described here comprise different gene pools for LDH production with a high incidence of type E in western grey kangaroos and a high incidence of type D in eastern grey kangaroos. (2) With the exception of M. f. fulig- inosus which is an isolated population, LDH types D and E are represented in each group and this is a reflection of their common origin and relatively recent separation (Figure 3-15)• (3) Eastern grey kangaroos from the mainland and from Tasmania have similar phenotype freq uencies . (4) The highest frequency of LDH D phenotypes in western grey kangaroos occurs in M. f. melanops which share some territory with eastern greys. In the absence of evidence of interbreeding from field observations or other genetic markers it is not possible 19k to infer that the genome favouring D type LDH production has "leaked” from one population to the other. Differ ences in phenotype frequencies may be due to the oper- * ation of the "founder principle". The isolation of populations for geographic, physiological or genetic reasons has perpetuated random gene differences which were present when the populations were first separated. 195 Chapter 7« Red Cell Lactic Acid Dehydrogenase and Malic Acid Dehydrogenase in Hybrids. 7.1.1. Introduction. In the two preceding chapters, LDH patterns in members of the family Macropodidae have been described. Patterns are inherited and have been divided into two categories, Group 1, in which slower LDH bands (5> ^ and 5) predominate and Group 2, in which LDH 1 is the heaviest band. Group 1 patterns are char acteristic of red kangaroos, wallaroos, pademelons and rock wallabies. However, Group 1 and Group 2 LDH patt erns are present in several genera and, in two cases, within species (M. alligatoris and M. eugenii, see Fig. 5-15)* These differences have arisen independently. It has been stated that, in marsupials, LDH phenotypes are the result of epigenetic factors influencing the production of polypeptides and their assembly into tetramers. Constant patterns are produced in animals having similar genomes. In hyb rids two different genomes contribute to the phenotypes and in the remainder of this chapter LDH patterns in offspring of several inter-generic and inter-specific hybrids are discussed 196 7.1.2, Results, All hybrids were produced in cap tivity, although one inter-specific hybrid has been reported in the field (B. Richardson, personal commun ication). Inter-specific hybrids are usually sterile but one female M. f. melanops / M. giganteus hybrid crossed with a M. f. melanops male produced a male (W.E. Poole, personal communication). The available inter-generic and inter-specific hybrids are listed in Table 7-a. A. (1) Red kangaroo - euro. (Megaleia rufa rufa - Macropus robustus erubescens,) Offspring of two matings between red kangaroo fathers and euro mothers produced red cell LDH patterns shown in Figure 7-1, (6) and (7). Blood was not available from parents but both genera have LDH type A or B. Patterns in both offspring are similar to LDH type B but have an increased of LDH k. This pattern is indistinguishable from LDH type F (See Figure 5-2), a pattern seen in Aepyprymnus sp. (2) Red kangaroo - wallaroo. (M. r. rufa - Macropus robustus robustus)• A red kangaroo male mated with a wallaroo female. The parental types were LDH type B, "slow" MDH x LDH type A, "slow" MDH. LDH and MDH in the hybrid is shown in Figure 7-2, (5) and (6). There is an increase in LDH 1 and the pattern does not resemble TABLE 7 - a. LIST (OF HYBRIDS. Parental types No (A) Inter-generic crosses (1) Red kangaroo - euro 2 (2) Red kangaroo - wallaroo 1 (3) Red kangaroo - E. grey 2 w Red kangaroo - W. grey 1 (5) P.F. wallaby - E. grey 1 (B) Inter-specific crosses (6) E. grey - W. grey (M. f. melanops) 4 (7) E. grey - W. grey (M. f. ocydromus) 2 (8) W. grey - W. grey (M. f. (M . f. fuliginosus) inelanops) 1 (9) Euro - wallaroo 1 (10) Thylogale Thylogale stigmatica thetis 1 (C) M. f. melanops/ - M. f. melanops 1 M. giganteus 198 Explanatory Note on Figure 7-1, (overleaf). As described in ’’Disadvantages of Disc Electrophoresis” (Section 2.1.5, (b)), enzyme patterns must be photo graphed immediately and it is not always possible to arrange photographs so that only patterns being discussed are included. This applies to Figure 7-1, where tubes (1), (2) and (3), (left to right) are patterns found in an interspecific cross and (6) and (7) are patterns from two offspring of intergeneric crosses. Tubes (4) and (5) contain gels with red cell LDH patterns from two wallaroos and are not part of the work described in Chapter 7. They are discussed in the conc lusions to Chapter 5, section (b). FIGURE 7 - 1 19>9 HYBRID RED CELL LDH PATTERNS IN INTER-GENERIC AND INTER-SPECIFIC CROSSES. ( See explanatory note, previous page.) 1 2 3 4 5 6 7 (a) Inter-specific Cross. (1) Type D in M. f. ocydromus parent. (2) Type D in M. f. ocydromus / M. giganteus hybrid, (3) Type D in M. giganteus parent. (b) Inter-generic Cross. (6) Modified type B in red kangaroo/euro cross, 1. (7) Modified type B in red kangaroo/euro cross, 2. (c) Non hybrids. (4) is type C LDH from M. antilopinus, (3) is type A from an animal referred to as M. ? antilopinus by Clark and Richardson, 1968. This animal had other aberrant characteristics and its identity has still not been established. FIGURE 7 - 2. 200 RED CELL LDH AND MDH IN RED KANGAR«00/WALLAR00 HYBRID. (1) , (3) Type A LDH found in wallaro (3) Atypical LDH pattern in red kang;aroo/wallaroo hybrid. (7) Type B LDH from red kangaroo fatlher. (2) , (k) MDH patterns in wallaroos (6) MDH in the hybrid. (8) MDH in red kangaroo father. FIGURE 7-3 201 LDH AND MDH IN RED KANGAROO FATHER AND RED KANGAROO/ WALL- AROO HYBRID = ONE HOUR AFTER ELECTROPHORESIS. 12 3 4 (1) LDH type B in red kangaroo father. (2) MDH in red kangaroo father. (3) Atypical LDH pattern in red kangaroo/wallaroo hybrid. (*0 MDH in red kangaroo/wallaroo hybrid. FIGURE 7-4. 202 RED CELL LDH AND MDH IN RED KANGAROO/GREY KANGAROO HYBRIDS. 1 2 3 4 5 6 7 8 (1) LDH type A in red kangaroo parent. (2) Slow MDH in red kangaroo (3») LDH type D in eastern grey kangaroo (4) Fast MDH in eastern grey kangaroo. (5) LDH type B in red/grey kangaroo, hybrid 1 cT (6) Slow MDH in red/grey kangaroo hybrid 1. cf (7) LDH type C in red/grey kangaroo hybrid 2. 9 (8) Intermediate MDH in red/grey kangaroo hybrid 2. ^ 203 any pattern illustrated in Figure 5-2. MDH has the same electrophoretic mobility in all samples (Figure 7-2, (2), (4), (6) and (8). Blood was not available from the wallaroo parent but the generic LDH type is A or B. Figure 7-3 is a photograph of LDH and MDH from the red kangaroo father, (1) and (2) and the red kangaroo-wallaroo hybrid described above and was taken one hour after a second electrophoretic separation on disc acrylamide gels. Haemoglobin has not diffused out of the gel and can be seen as the second, more diffuse band running ahead of the MDH in (2) and (4). (3) Red kangaroo- eastern grey kangaroo. (M. r. rufa - Macropus giganteus). LDH types could not be ascertained in the parents but the red kangaroo type is A or B (Group 1) and the grey kangaroo type would be D or E (Group 2). Figure 7-4 shows type A from a red kangaroo (1) and type D from an eastern kangaroo (3). LDH patterns are different in the two hybrids. A male, (3) is type B, while a female-* (7) is type C, which is a Group 2 pattern. MDH types. Red kangaroo MDH(2) is "slow" and grey kangaroo MDH is "fast", (4). (6) Female hybrid - "slow" MDH. (8) Male hybrid - ’’intermediate” MDH. The results are shown in Figure 7-5)• 204 Since this work was done both "slow" and "fast” MDH bands have been found in eastern grey kangaroos (see Figure 5-9), so the parental types may have been (i) 0 , "slow" MDH x "slow” MDH, (ii) 0 , "slow” MDH x "fast" MDH. FIGURE 7-5. PARENTAL AND HYBRID LDH AND MDH TYPES IN RED KANGAROO- GREY KANGAROO CROSSES. Origin k WHm: Red Red/grey Red/grey Grey kangaroo hybrid hybrid kangaroo d* 9 205 (k) Red kangaroo - western grey kangaroo. (M. r• rufa - M. f. melanops). Blood from both parents was available for testing and the results were as follows; (j) M• r• rufa x O'7* M• f• melanops LDH type B LDH type E (j) hybrid LDH type B (5) Pretty face wallaby - eastern grey kangaroo. (Macropus parryi - M. giganteus). Figures 7-6 and 7-7 show LDH and MDH patterns from a pretty face wallaby, an eastern grey kangaroo and a hybrid between these two. Pretty face wallabies have LDH type C patterns and a "slow” MDH. Eastern grey kangaroos from this colony had type D LDH patterns and "fast" MDH. The hybrid LDH pattern is intermediate between the two parental types and coincides with LDH type C+ ( see Chapter 5)» This pattern has been found in quokkas and parma wallabies. The hybrid MDH has a diffuse band with an intermediate electrophoretic mobility and a narrow band at the "fast" position. This is shown in Figure 7-7 (5)• B.(6) Four inter-specific crosses between eastern grey FIGURE 7-6 206 RED CELL LDH AND MDH PATTERNS IN PRETTY FACE WALLABY/ EASTERN GREY KANGAROO HYBRID AND PARENTS. (1) Type C LDH in pretty face wallaby. (2) Slow MDH in pretty face wallaby. (3) Type C+ LDH in pretty face wallaby/eastern grey kangaroo hybrid. (4) Intermediate MDH pattern in pretty face wallaby/ eastern grey kangaroo hybrid. (5) Type D LDH in eastern grey kangaroo. (6) Fast MDH in eastern grey kangaroo. FIGURE 7-7. 20? RED CELL LDH AND MDH IN PRETTY FACE WALLABY/EASTERN GREY KANGAROO HYBRID AND PARENTS. (1) Type C LDH in pretty face wallaby. (2) Type C+ LDH in pretty face wallaby/eastern grey kangaroo hybrid. (3) Type D LDH in eastern grey kangaroo. W Slow MDH in pretty face wallaby (3) "Hybrid" MDH in pretty face wallaby/eastern grey kangaroo hybrid. There are two bands, one fast MDH band and a wide band with an intermediate electrophoretic mobility. (6) Fast MDH in eastern grey kangaroo 208 kangaroo females and western grey kangaroo males were tested for red cell LDH patterns and the results are shown in Table 7-b. TABLE 7 - b. CROSSES BETWEEN EASTERN AND WESTERN GREY KANGAROOS. M. giganteus x M• f• melanops 1. G 96 X G 40 hybrid G 183 0" E E D h 2. G 36 X G 40 G 160 9 D E E it 3. G 13 X G 40 G 196 9 D E D 4. G 147 X G 13 it G 181 9 ? E E M. giganteus and M. f, melanops are represented by both D and E type LDH and the four hybrid offspring are one or other of these two types. The first three crosses involve the same father, G 40, who is type E, (2) and (3) in Table 7-b are D x E crosses but one hybrid (2) is type E and the other (3) is type D. In (1) the LDH type of the hybrid is different from either parent and in this respect it differs from the other three 209 and from any of the intra-specific matings mentioned in Chapter 6. (7) Macropus giganteus - Macropus f. ocydromus. Parents and offspring from two matings were investigated and the results are shown below. M. giganteus x M. f. ocydromus ? 0* (1) G 1 x G 90 hybrid G 223 2 D D E (2) G 2 x G 90 n G 194 0* D D D In the two crosses, G 90 is the father. Both mothers are LDH type D but one hybrid is type D, which is the expected result and the other is LDH type E. Two of six hybrid offspring from inter-specific crosses between eastern and western grey kangaroos produced "new" phenotypes not present in either parent. These "new” phenotypes do not depart from the types already found in eastern and western grey kangaroos. (8) M. f. fuliginosus - M. f. melanops. This is an inter-specific cross between two western grey kan garoos. The female M. f. fuliginosus is from Kangaroo Island and the male M, f. melanops is from the South 210 Australian mainland (personal communication, W.E. Poole). The results are shown below, M. f. fuliginosus x M. f. melanops G 103 £ x G 15 Cf hybrid G 113 EE E (9) Euro - wallaroo. (Macropus robustus erubescens - M. r. robustus). M. r. erubescens £> x M. r. robustus hybrid robustus d* E 8, Type B x E 5i Type A E 16 cf Type B LDH patterns from the female parent and the hybrid are shown in Figure 7-8. The hybrid LDH pattern (2) has a heavier LDH band than normal type B LDH and resembles type F. (See also section (1) of the results in this chapter). (10) An Inter-specific Cross Between Two N.S.W. Pademelons. (Thylogale stigmatica wilcoxi - Thylogale thetis). The cross was between a female T. thetis and a male T. stigmatica wilcoxi. The LDH pattern of both parents and the hybrid was LDH C. C. ( 11) A Back-cross Between a Female M. giganteus/ M. f. melanops Hybrid and a M. f. melanops Male. The details of this back-cross are shown in the FIGURE 7-8. 211 RED CELL LDH IN EURO MOTHER AND EURO/WALLAROO HYBRID. 1 2 (1) Type B LDH in euro (M. r. erubescens) mother. (2) Type F LDH in euro/ wallaroo (M. r. robustus) hybrid 212 following pedigree, which was provided by W.E. Poole of the C.S.I.R.O. Division of Wildlife Research, Canberra, who also supplied the blood samples. M • gigant eus G 13i type D G 15i-type E hybrid ^ x M. f. melanops O'* G 68 (n.s.) G 40, type E M. g. , _ / M. f. melanops M. f. m. ------£— G 212, type E 7 •1»3« Discussion. (1) Hybrid Types. The hybrids described in this chapter are the products of matings ranging from inter-generic crosses, such as those be tween red and grey kangaroos (3) and a wallaby and a grey kangaroo (5) to inter-specific crosses between two western grey kangaroos (8). They are the expression of interactions between genomes which differ from each other in many respects or which are similar. LDH patterns of some parents of inter-generic crosses belong to different groups (Group 1 or Group 2, see Chapter 5)1 but others have identical or similar LDH patterns. No examples 213 of inter-specific crosses involving LDH types from diff erent groups were found. (2) Effects of Hybridisation on Polypeptide Production. It has been stated in previous chapters that LDH patterns are the result of different rates of production of A and B polypeptides. The grouping of LDH polypeptides into tetramers is an epi-genetic process which is regulated so that animals from the same species produce only one or two LDH patterns. Table 7-c summ arises the results from all crosses. LDH patterns in red kangaroo/grey kangaroo hybrids (3) are intermediate between parental types but one phenotype belongs to Group 1 and the other to Group 2. Both animals have red kangaroo fathers and eastern grey kangaroo mothers but in one hybrid there is a suppression of slow tetramers and an increase of fast tetramers resulting in type C LDH and in the other a suppression of fast tetramers and an increased production of slow tetramers, forming type B LDH. In case No. 5 the hybrid pattern is intermed iate between the two parental patterns which both belong to Group 2. Group 1 and Group 2 are represented in the parents of (4) and the LDH in the hybrid resembles that of 214 o Pi p o p • O bO d t3 *H d P OJ OJ to cr5 Ph v--' '—' P CO /^N /^N /* N /^N CO P > o P + ■H M EH S pq CQ O Q q W pq O w bO « •H O-i W I—I CQ Q p fci t-q H O M • K O OJ V- OJ r~ C“ -rf- OJ r- r- r~ V" S N •> pq s OJ CO >H '--- a • W E to /—\ /^N /—\ /-—V ,—N /—*. /—^ /-- X ,—« o CO 0 « r *— OJ OJ OJ OJ OJ OJ v— OJ OJ p o __' '—/ v-/ v—X v—/ v_/ S-H ■-_h V_X Trf p 1 < Pi o p pq w O o CQ o < W P w P W < o W :0 CO Eh p •* •» cm •* p w Js; O < p II CO p X W C Pi -p PQ « t3 P <<—V O d < < d X X X X X X XX X X X bO o P erf EH Pi erf V_' erf bO W P •rH p *-*» ,—\ /—* ✓—\ /—V ,-- N ^~N /—\ /—tl P ■ r- P bO M Ph V- V r~ r~ OJ OJ OJ OJ r~ OJ OJ P w B >5 N_/ v—' ---- V—^ s-' -_* p. • CQ EH o» o CO S W pq pq w II t p n PH W pq PQ o p w pq o W P>H erf CO I >H Q •* ** »• X I—I o Eh hP < < p P •- p p CQ w E •H cm « E bO o « o • v-' «• II •H d hQ • o f>> >> CO p i—I erf CQ p P P P ✓—* /—\ /-—V P P P erf P P >> CO E o P CO •~ E P p CO o i—l bO bO p CO v_ '—/ P •H o s_/ E • CO P i—l p o bO •P • CO CO O P erf • • bO p >5 P E • r- P t -P P p £ W 3: o p p • • p P Pi P o \\ \ \ • p p o -p tp o O p o o O O W o bO bO \ o • *- erf P • p o o o O o \ •H ^^ P • c the mother. Where LDH types are similar or the same in parents of inter-generic hybrids, the pattern in the offspring is modified and may be different from parent al types. For example, in (1) the hybrid pattern is type F, a modified Group 1 pattern which was seen in Aepyprymnus sp. The LDH of the hybrid in (2) is diff erent from any described in Chapter 5« Hybrid LDH patterns in inter-specific crosses between individuals with similar types resemble one or other of the parental types. New patterns are not seen although there is one case where a D x D cross results in a hybrid with type E LDH and an E x E cross produces a type D LDH in the offspring. There is no evidence of hybridisation of LDH tetramers to form sub-bands with different electrophor etic mobilities from bands already present. (3) Factors Responsible for LDH Phenotypes. (a) Epigenetic, There is evidence that LDH patt erns in marsupials are influenced by components of the genome other than the structural genes a and b. The strongest evidence is that of the two red kangaroo/ grey kangaroo hybrids where two different patterns were 216 produced although the LDH types of the two mothers and of the two fathers were similar. (b) Heterozygosity in Structural Genes. Quant itative changes could occur by different means if the structural genes responsible for LDH polypeptide pro duction exist as "dominant" and "recessive" alleles. It is usual to describe LDH structural genes as a and b and the polypeptide products as A and B. In order to avoid confusion regarding the meaning of A, a, B and b and for the purpose of the following discussion, let X represent a "dominant" gene for making many A polypeptides, x represent a "recessive" gene for making few A polypeptides, Y represent a "dominant" gene for making many B polypeptides and y represent a "recessive" gene for making few B polypeptides. Thus LDH 1 = BBBB,(Markert) = YYYY LDH 2 zz ABBB " = XYYY LDH 3 = AABB " = XXYY LDH k = AAAB " =: XXXY LDH 5 = AAAA " — XXXX If there is heterozygosity in the genes for making 217 A and B polypeptides, an individual could have any of the following genotypes, LDH 1 (5), YYYY, YYYy, YYyy, Yyyy, yyyy. LDII 2 (8), XYYY, XYYy, XYyy, Xyyy, xYYY, xYYy, xYyy, xyyy. LDH 3 (9), XXYY, XXYy, XXyy, XxYY, XxYy, Xxyy, xxYY, xxYy, xxyy. LDH U (8), XXXY, XXxY, XxxY, xxxY, XXXy, XXxy, Xxxy, xxxy. LDH 5 (5)» XXXX, XXXx, XXxx, Xxxx, xxxx. XXXX, XXXY, XXYY, XYYY and YYYY would produce heavy bands corresponding with LDH 5, LDH A, LDH 3* LDH 2 and LDH 1; xxxx, xxxy, xxyy, xyyy and yyyy would produce light bands. If the genotype is XXyy, LDH bands with many A polypeptides will be heavy and those with B polypeptides will be light. If the genotype is xxYY, the bands containing many B polypeptides will be heavy and those with A bands will be light. Patterns produced are shown in Figure 7-9* This mechanism allows for varia bility in patterns having the same structural genes. Conclusions. The results with hybrids suggest that phenotypic expressions of LDH patterns are influenced by epigenetic factors which determine the amounts of FIGURE 7-9. 218 LDH PATTERNS PRODUCED BY XXyy AND xxYY GENOTYPES. XXyy xxYY Origin 5 EH 4 essssisa 3 I'lru'-.-A-.*™ 2 — 1 1 - ..T*] ^ t slow and fast tetramers. Numbers of different patterns are possible but each species expresses a limited num ber. LDH types are increased when different genomes are combined in hybrids and this flexibility can account for the appearance of Group 1 and Group 2 phenotypes in closely related species. It also suggests, but does not prove, that certain LDH patterns may have arisen by random fertile hybridisations in the past and the stability of the resultant genome has perpetuated the pattern for that species. 219 Chapter 8. Summary of Conclusions, 8.1.1. The aims of the investigation were (1) to determine the suitability of disc acrylamide electrophoresis for the detection of genet ically determined enzyme variations and (2) to use disc acrylamide electrophoresis to study the DPN dependent dehydrogenases in red cells of many animals. (1) Disc acrylamide electrophoresis proved to be an economical and reliable method for investigat ion of enzymes in which differences in electrophoretic mobility are not the only means of detecting phenotypic heterogeneity. One disadvantage is that proteins which do not move towards the anode at pH 8.9 cannot be de tected. Disc acrylamide electrophoresis is suitable for the investigation of enzymes (i) because of the small volumes of substrates and co-factors required to make the enzymes visible and (ii) multiple sub-bands are not produced as they are after prolonged electrophoresis in starch gels. The only method used was the standard 7«5 per cent gel with a pH of 8.9 and a tris-glycine buffer in the anodal and cathodal compartments. (Davis, 1964). 220 Under these conditions, (a) amylase was associated with the and ^ globulin region just ahead of the origin. Separation was inferior to that reported by Mc.Geachin (1959)i Mc.Geachin and Potter (19&1) and Mc.Geachin and Reynolds (1961). The problem of the source of amylases in serum has remained unsolved and it is not clear if the enzymes are isozymes (see also Dreiling et al., 1965)* (b) localisation of glucose 6 phosphate de hydrogenase was not satisfactory. Bands were diffuse and differences in electrophoretic mobility were not easily detected. A semi-quantitative method suitable for use in the field was tested on 208 human blood samples and values for males and females conformed to broadly based curves of normal distribution. A difference was observed between G6PD values for males and females and may be due to the use of whole blood instead of constant volumes of packed red cells or constant haemoglobin val ues . (c) DPN dependent dehydrogenases in red cells are the most suitable of the enzymes tested, part icularly lactic acid dehydrogenase, which exists in multi- molecular forms. Differences in malic acid dehydrogenases involve electrophoretic mobility and disc acrylamide elect rophoresis is not as useful for this enzyme as starch gel electrophoresis or acrylamide slab electrophoresis. 221 (2) Human red cell LDH consists of three bands corresponding with LDH 1 (BBBB), LDH 2 (ABBB) and LDH (AABB). Patterns are reproducible and do not form multiple sub-bands such as those found in some normal blood samples when run on starch gels (Blake et al. , 1969)• Two LDH patterns are found, the difference being in the amounts of LDH 3 in fresh samples. In 208 haemolysates there were no LDH bands with altered elec trophoretic mobility. There is a wide range of red cell LDH patterns in the animal kingdom, varying from a single band (usually composed of fast tetramers, BBBB or ABBB, in mammals and slow tetramers, AAAA or AAAB in birds) to five bands in grey kangaroos. LDH in red cells of mem bers of the Macropodidae, from which most marsupial samples were obtained, have quantitative differences and these are genetically determined. LDH patterns, combined with other biochemical markers, morphometric, cytological and physiological characters can be used to study possible affinities of members of the family. Polymorphism is present in some species but it is not possible to apply the Hardy-Weinberg form ula to results nor to determine gene frequencies since an unknown number of factors operate between the form- 222 ation of the primary gene products and the final pheno types* (See Figure 8-1*) FIGURE 8-1. SUGGESTED MODE OF FORMATION OF LDH PHENOTYPES* Group 1 Group 2 a b a b AAAA BBBB AAAA BBBB AAAA BBBB AAAA BBBB AAAA BBBB AAAA BBBB AAAA BBBB AAAA BBBB 5 AAAA (6) (1) (3) (2) k AAAB (6) (3) (1) 3 AABB O) (1) (3) (1) 2 ABBB (1) (2) (3) 1 BBBB (2) (6) (3) (3) V wlf▼ * * i >iIr 5 AAAA E3 — V k AAAB El Bff — 3 AABB — — Bfll — 2 ABBB — EEHB nsa 1 BBBB m BB and n^ represent unknown numbers of * V n21 n3 epigenetic factors which result in the available peptides being arranged in tetramers. Some factors may be common to all four systems. 223 Genes a and b are responsible for the production of polypeptides A and B in the cytoplasm and in this theoretical system polypeptides are made at different rates. Tetraraers are assembled in the cytoplasm and their composition depends upon the kinds of polypeptides available at the sites of tetramer production. The work of Wilson et al. (1963)1 Latner et al. (1966) and Stambaugh and Post (1966, a and b) suggests that the presence of different LDH types is related to the met abolic role of the tissue. Figure 8-1 shows some theoretical patterns which can be made from the polypeptides present (e.g., 24 A polypeptides and 8 B polypeptides in Group 1 on the left and 8 A polypeptides and 24 B polypeptides in Group 2 on the right). It is clear that, although any of the LDH tetramers can be made, those from Group 2 will contain predominantly fast-moving proteins and those from Group 1 will have mainly slow-moving ones. In marsupials AAAA tetramers are rarely observed and it is not clear whether this is due to inhibition mechanisms or to the instability of A containing tet ramers. The first possibility is favoured because grey kangaroo LDH type E contains LDH 5 (AAAA). In some types of animal only one tetramer is produced in red 224 cells although the animal retains the ability to make other tetramers in different tissues. Heterozygosity of genes a and b with respect to the amounts of polypeptide synthesised is another poss ible means of producing different LDH patterns and this has been discussed at the end of Chapter 7. MDH. DPN dependent malic acid dehydrogenase activity is present in red cells of mammals and birds, although there is no obvious role for MDH in anucleate mammalian red cells which do not respire by means of the tricarboxylic acid cycle. Enzymes, including MDH, are synthesised in immature red cells which possess nuclei and cytoplasm and these must last for the life time of the cell. It appears that an excess of MDH is made which persists in the mature red cell. When sep arated by disc acrylamide electrophoresis, MDH behaves as though it is a dimer. At least two proteins with MDH activity are detected on the basis of electrophoret ic mobility which does not correspond with that of any LDH tetramer. Other Dehydrogenases. Minor staining bands occur in some animals when malic acid is used as substrate. These have the same electrophoretic mobility as LDH bands, usually that of LDH 1. Isocitric acid and succ- 225 inic acid dehydrogenase activity are also associated with LDH bands, particularly LDH 1 (BBBB). An explan ation for these findings could be that preparation and/ or electrophoresis cause conformational changes in dehydrogenase proteins so that they assume a '’primitive" non-specific configuration. Kaplan (1965) suggests that all DPN dependent dehydrogenases have arisen from an "ancestral" protein or polypeptide. Since non specific dehydrogenase activity is most often associated with LDH 1 (BBBB or HHHH) it is of interest that, in a report of a discussion following a paper on lactic acid dehydrogenase activity presented by Moyer, Speaker and Wright (1968), Yoshida, from Seattle, pointed out that Bacillus subtilis contains one LDH which resembles heart-type enzyme (H sub-units according to Kaplan's terminology or B sub-units according to Markert). He states, "It seems therefore most likely that the muscle type sub-unit (M or A) differentiated from heart-type sub-unit at a later stage of evolution". LDH Patterns as Indicators of Phylogenetic Relat ionship. LDH patterns in red cells of different animals are variable and represent the epigenetic effects of different genomes upon the production of A and B poly peptides. In the family Macropodidae, similarities of 226 LDH patterns broadly reflect affinities which are inferred from other characters although unlike patterns can arise fortuitously in closely related species. For this reason it is advisable to use several markers. ACKNOWLEDGEMENTS. 227 All biochemical testing, the application of stat istical treatments and photography were carried out by the author unless otherwise stated but the length of the acknowledgements listed below emphasises the impossibility of conducting a study of this kind without the co-oper ation of many others, to whom thanks are due. Professor R.J. Walsh, Head of the School of Human Genetics, Univ. of N.S.W., for encouragement and interest throughout. Professor G.B. Sharman, lately Head of the School of Zoology, Univ. of N.S.W., for supply of data and samples for marsupial studies. (Present address, School of Biology, Macquarie Univ., Sydney). The late Professor Ed. Horn, Duke University, N. Carolina, U.S.A., for supply of tammar samples. Mr. Barry Richardson, post-graduate student in the School of Zoology, Univ. of N.S.W., for macropodid samples. Mr. P. Johnston, post-graduate student in the School of Zoology, Univ. of N.S.W., for permission to include potoroo results. Members of the Zoology School, Univ. of N.S.W. at Kensington and Cowan, for collection of samples. 228 Mr. Eric Worrell, Reptile Park, Gosford, N.S.W., for supply of marsupial samples, including those of New Guinea animals. Mr. W. E. Poole of the C.S.I.R.O. Division of Wild life Research, Canberra and his colleagues for samples of marsupial blood, especially grey kangaroo material. Mr. G. Ryan of the N.S.W. Department of Agri culture, for supply of dog blood and for permission to include results. Dr. L. Lai of the School of Human Genetics, Univ. of N.S.W. , for providing blood samples from Chinese and Australian Aboriginal subjects. Mr. A. E. Stark of the Faculty of Medicine, Univ. of N.S.W. for advice on the application of statistical methods• Mr. R. Straughan, Director of Taronga Park Zoo and Dr. E.M. Nicholls of the School of Human Genetics, Univ. of N.S.W., for blood samples from Zoo animals. Dr. J. Carmody and Mr. Rowe of the School of Phys iology and Dr. R. Cummings from the School of Pathology, for the supply of cat, rabbit and rat blood. Dr. Myers, C.S.I.R.O. Div. of Wildlife Research, Canberra, for permission to use rabbit blood samples sent 229 to Professor Walsh. Members of the Biochemistry School, Univ. of N.S.W., for mouse blood. Mr. A. Czuppon and members of the technical staff of the School of Human Genetics for valuable assistance and for supplying blood samples. Mrs. P. Rosenthal, secretary to the School of Human Genetics, for clerical help in the preparation of the thesis. Special thanks are due to Dr. K. Clemens and the staff of the N.S.W. Blood Transfusion Service, Sydney, for the collection of blood samples from Greek, Italian, British and Australian blood donors and the Director for permission to use blood from blood donors. APPENDIX I. 230 ZOOLOGICAL NAMES OF MAMMALS MENTIONED IN TABLE 4-2. (The numbers in brackets indicate the source or sources of the names and these are listed on the next page.) 1. Rabbit .. 2. Mouse .., 3. Rat .... or Rattus rattus norvegicus (4), var. albino. 4. Guinea Pig...... Cavia porcellus, L. (3)* * 5. Dog .... 6. Cat .... 7. Lion .... leo (4). 8. Jaguar .. onca (4). 9. Hyena ... 10. Polar Bear ...... Thalarctos maritimus (1), (4) or Thalassarctos maritimus (2). 11. Elephant 12. Sheep ... 13. Barbary Sheep ...... Ammotragus lervia (1). 14. White-tailed Deer.... Odocoileus virginianus, Zimmerman, (3) Buddaert, (5)• APPENDIX I, (continued). 231 (1) "Wildlife of the World - Illustrated"., advisory- editor, M. Burton, Odham's Press Ltd., Lond. (2) "Mammals of Eastern Asia"., G.H.H.Tate, Macmillan Co., N.Y., 19^7. (3) "Vertebrates of the United States, 2nd. Edit., Blair, W.F., Blair, A.P., Brodkorb, P., Cagle, F.R. and Moore, G.A., Mc.Graw Hill, N.Y. (4) "The Mammals"., D. Morris, Hodder and Stoughton, Lond., 1965» (5) "The Biochemical Genetics of Vertebrates Except Man"., I.E. Lush, North-Holland Pub. Co., Amster dam, 1966. * The binomial nomenclature appears to be inconsistent in this case and should be Cavia porcella. (P.C.) APPENDIX El 232 MAP OF AUSTRALIA AND NEARBY ISLANDS,SHOWING PLACES MENTIONED IN THE TEXT. 1. Kangaroo Island. 2. Garden Island. 3* Abrolhos Islands. k. Arnhem Land. 5* New Britain (Bainings). 6. Bougainville. 7- Cooktown. GLOSSARY. 233 Specialised terminology has proliferated in most disciplines. Many words which have previously had general meanings have been used in a special sense by certain workers and this has occasionally led to confusion. The glossary lists such words which are used in this thesis and the definitions are ones which the author understands the special phrases to imply. Cistron: The total genetic material responsible for the expression of a trait. It may consist of one or more genes. Conformational: A rearrangement of the folding of polypeptides so that physical and/or electrophor etic properties are altered although amino acid sequences are unchanged. Epigenetic: The appearance of a phenotype which is due to the operation of factors in addition to structural genes. Founder Principle: The principle whereby the freq uencies of genes in populations depends upon genes possessed by two or few originators of the popul ation. Gene Duplication: The acquisition of duplicate portions of gene material by unequal crossing over GLOSSARY, (Continued). or other irregularities in cell divisions. Genetically Stable: Used in this thesis to refer to LDH patterns in Macropodidae which are reproducible and constant for the species being studied. Genome: The haploid gene complement of an organism. Heteropolymorphic: A multimeric protein which may exist in different forms, presumably due to epi genetic factors. Informational Molecules: The meaning of this phrase is open to several interpretations. It refers to DNA sequences which are responsible for coding the molecule but may also refer to messenger RNA, sol uble RNA or ribosomal RNA. Isomeric Enzymes: Enzymes with the same substrate specificity but different physical and/or kinetic properties. Multimers: Molecules consisting of more than one polypeptide chain held together by weak bonds such as Van der Waal forces. Primary Gene Product: The first product, a polypep tide or protein, which is produced in the ribosome in response to a message coded by DNA and relayed to the ribosomes by messenger RNA. 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