Genetics - Theses

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Studies on minor non-metrical skeletal variants in the mouse and man

Kellock, Wendy Lorraine. (University of Melbourne, 1970)

This thesis consists of papers presenting the results of studies on the genetical, developmental and anthropological aspects of minor non-metric al variants in man and the house mouse. The work is mainly on variants of the skeleton, particularly the cranium, but includes a limited discussion of published data on minor non-metrical variants of the muscular and vascular systems. Each study is based on a number of variants, and, where applicable, single measures have been obtained to express the overall difference in skeletal variability between populations or the overall effect on skeletal variability of certain environmental factors. Investigations into the role of genotype and environment in the determination of minor skeletal variants in mice and man indicate that most of them are under some genetic control but that maternal physiology and other non-genetic factors may influence the frequency of individual variants. Data presented here (Publication 1) on 25 minor skeletal variants in inbred strains of mice and their hybrids suggest that genotype is more important than environment in determining skeletal variability. Although the frequency of a few individual variants was found to be significantly affected by certain non-genetic factors, when many variants were considered together the environment had no overall significant effect. In contrast, large differences, due mainly to genetic factors, were observed between inbred strains and hybrids. Further studies on inbred strains of mice and hybrids (Publication 2) indicate that stabilizing mechanisms operate during the formation of the skeleton. For most of the 29 bilateral minor non-metrical variants studied , the frequency of asymmetrical mice (i.e., those with the variant present on only one side) was less than expected on the assumption that the number of mice with the variant present on both, one or neither sides depends solely on the frequency of the variant on each side. This tendency for the development of the skeleton to be canalized against asymmetry has been described as a form of morphogenetic homeostasis. The same phenomenon has been observed for bilateral minor non-metrical variants in man (Publication 3) for the skeletal, muscular and vascular systems (based on data published by Danforth in 1924) and for the skeletal system of Australian Aborigines. Studies on inbred strains of mice (e.g., Publication l) indicate that genotype plays the major role in determining the frequency of minor non-metrical variants. If these findings can be extrapolated to man, minor non-metrical variants may be of use in anthropological work. A general survey of skeletal variation, based on 30 such variants, was carried out on Aboriginal crania from many parts of Australia (Publication 4). Regional differences in the pattern of cranial morphology were observed which appear to culminate in two extreme populations: one in the north and north-west of the continent, the other in south-eastern Australia. These results were considered in relation to some current theories on the origin and ethnic composition of the Australian Aborigines.
Mutagenesis by X-rays and [125I] iododeoxyuridine in mammalian cells

Gibbs, Richard (Richard A.) (University of Melbourne, 1985)

Ionising radiation causes death and gene mutations in mammalian cells by the induction of DNA damage. The range of damage includes DNA double strand breaks, single strand breaks, base modifications and cross-linking to other molecules. Several studies have implicated DNA double strand breaks as the critical lesions leading to cell death, but relatively little is known about the nature of radiation-induced premutagenic lesions. This is primarily because of the difficulty of observing any one class of DNA damage in isolation. One way to expose cells to radiation is to incorporate radionuclides into DNA during S-phase using nucleotide analogues. When the radionuclide is iodine-125, incorporated as the thymidine analogue 5-iodo-2'-deoxyuridine (I-dUrd), there is on average, one DNA double strand break produced for each radioactive decay event. Thus, when compared with external sources of radiation, 125I decay allows the study of a high yield of DNA double strand breaks, relative to other classes of DNA damage. In this study the mutagenic and cytotoxic effects of X-rays and 125I decays have been compared in cultured mammalian cells. Asynchronous, exponentially growing Chinese hamster ovary (CHO) cells were either irradiated with 250 kVp X-rays at 37�C, or labelled for 1.2 cell population doublings with [125I] I-dUrd and stored at 4�C for 48 hours to accumulate 125I decays. X-ray survival was fitted to a linear-quadratic expression: -InS = ?.D + ?.D2, which yielded ? and ? values of 0.107 (� 0.042) Gy-1 and 2.321 (� 0.543) X 10-2 Gy-2, respectively. The l25I survival was of the simple exponential type with a Do = 34.1 � 0.95 decays. Both agents induced 6-thioguanine resistant mutants which can arise either by point mutations or by rearrangements or deletions leading to inactivation of the hypoxanthine phosphoribosyl transferase (HPRT) gene. For X-rays, there were 8.66 (� 0.0154) X 10-6 mutants Gy-1 and for 125I decays 6.22(� 0.89) X 10-7 mutants per decay. When compared on a direct plot of the logarithm of survival against induced mutation frequency, the mutagenic efficiencies of the two agents were indistinguishable. Neither X-rays nor 125I decays induced mutations at the Na+/K+ ATPase locus, suggesting that the radiation treatments did not induce point mutations. To further analyse the molecular nature of the radiation-induced HPRT mutations, ten X-ray-induced, six 125I-induced and four spontaneous independent HPRT mutant clones were isolated and examined at the enzyme, chromosome and DNA levels. None of the mutants exhibited residual HPRT enzyme activity. The arm, of the X chromosome bearing the HPRT did not show consistent chromosomal G-banding changes. Southern blotting analysis using a cloned mouse HPRT cDNA probe showed that 5/10 X-ray and 2/6 125I-induced HPRT mutants had lost all HPRT coding DNA sequences. A further 2/10 X-ray and 4/6 125I-induced mutants had altered Southern blotting patterns, but retained some HPRT coding sequences. Only one of the four spontaneous HPRT mutants had an altered Southern blotting pattern. The identification of mutants with complete loss of HPRT coding sequences indicates that both the X-rays and 125I decays can cause mutational inactivation by gene deletions involving more than 25 kilobases, which is the approximate size of the hamster HPRT gene. The identification of mutants which had altered Southern blotting patterns, but retained some HPRT coding sequences, indicated that the radiation- induced DNA deletions were probably not much larger than the size of the HPRT gene.
Copper dependent enzymes in mottled mouse mutants

Phillips, Alison Marie. (University of Melbourne, 1984)

The activities of two copper dependent enzymes, cytochrome c oxidase and superoxide dismutase, were determined, and related to intracellular copper concentrations, in six tissues of mice carrying the x-linked defects in copper homeostasis, brindled and blotchy, at the mottled locus. The mutants were assayed throughout post natal development in the presence and absence of a sub-cutaneous copper load. Copper replete and copper deficient genetically normal mice were used as experimental controls. Mutant tissues could be divided into enzyme deficient, that is brain, heart and skeletal muscle, and tissues with normal enzyme activity, liver, kidney and lung. Copper concentration was highly correlated with enzyme activity in deficient tissues. In mutant tissues with normal activity intracellular copper could be reduced, (liver) greatly increased (kidney) or close to normal (lung). Comparisons with genetically normal copper deprived mice suggest that copper availability to the cell, rather than within the cell, is responsible for the enzyme deficiency in brain, heart and skeletal muscle and that mutant gene expression and the accompanying copper accumulation phenotype is confined to epithelial rich tissues, at least in the brindled mouse. However, the possibility of intracellular expression of the basic defect in these enzyme deficient tissues has not been excluded. The response of copper dependent enzymes to subcutaneous copper injection (50 ?g copper) differed in skeletal muscle in the two mutants and studies on the distribution of radioactive isotopes of copper showed small but consistent tissue specific differences between the brindled and blotchy hemizygote. These results, together with previously published data on lysyl oxidase activity suggest that different polypeptides are defective in the two mutants. In early post natal development the activity of both copper dependent enzymes studied increased significantly in a number of tissues in copper replete mice. An inability to meet the increased copper requirements that accompany these developmental changes, particularly in the maturing brain, is believed to be responsible for the death of the brindled mouse at the mid-suckling stage. In the more viable blotchy mutant the compensatory effects of stunted growth may decrease the magnitude of the developmentally related enzyme deficiency in some tissues but cannot account for the phenotypic differences between the mutants. A number of hypotheses are proposed to explain the experimental results, and the objectives and directions of future research projects are indicated.
A sex-influenced protein in Chironomus: detection and characterization

Cheong, Mei Foong. (University of Melbourne, 1984)

The synthesis of a protein in Chironomus was indicated to be sex-influenced by its female-specific occurrence detected electrophoretically in a range of geographically distinct species. These species included Ch. tentans derived from Canada, Ch. duplex from Australia and Ch. species a from New Zealand. The sex-influenced protein was found to contain two subunits of similar molecular weight at approximately 17,800 dal tons. By virtue of its positive staining with benzidine, this protein was indicated to be one of the haemochironomins, a class of predominant haemolymph proteins synthesized during the larval stage. The presence of this sex- influenced protein in the female haemolymph was correlated to the stages of development. It was found to be absent in haemolymph of the female fourth instar larvae younger than phase 3 in Ch. tentans and phase 5 in Ch. duplex. It was present throughout the later phases and persisted in the haemolymph of the female pupa. Tissue distribution studies indicated that the sex-influenced protein was found solely in the haemolymph in the female larva. However, pupal ovaries containing yolky oocytes as well as adult eggs were indicated electrophoretically to contain the sex-influenced protein. An additional and reliable method for identifying the sex-influenced protein in female larval haemolymph and in eggs of the various species was based on the criterion of antigenic specificity to an antiserum raised against the protein isolated in the present study. The results suggested that the female-specific synthesis of the sex-influenced protein may be correlated to. female-specific requirement for it in egg formation.
Characterization of the DNA of normal and transformed cells of the black-tailed wallaby, protomnodon bicolor

Newton, Gail. (University of Melbourne, 1977)
Patterns of protein synthesis in larval and imaginal tissues of Calliphora : an analysis of gene activity during differentiation and development in a holometabolous insect

Martin, Marjorie-Dore (University of Melbourne, 1975)

This study attempts to develop a logically consistent approach to the analysis of the genetic basis of complete metamorphosis in holometabolous insects,using the Australian brown blowfly, Calliphora stygia (Diptera), as the experimental organism. The following aspects of the problem have been examined: 1. Definition of the protein and enzyme spectra of selected larval and imaginal tissues during development. 2. Identification of the homologous gene products amongst various tissues with the view to establishing the uniqueness or overlap of gene readout in the series of tissues. 3. Identification of the times of synthesis of gene products as distinct from the duration of their occurrence leading to a definition of overall patterns of gene activities in the series of tissues. 4. Investigation of a specific model system for hormone action in vitro, namely with fat-body tissue and moulting hormone (MH). Current genetic models of insect metamorphosis have been examined in the light of the findings in this study of C. stygia.
Contributions to the development of hormonal insecticides : the biosynthesis and mode of action of the ecdysones

Thomson, J. A. (John Alexander), 1934- (University of Melbourne, 1974)

During the last 10 years, the candidate's interests have centered on problems of the regulation of gene activity during cytodifferentiation in insect tissues. Certain higher Diptera offer especially good opportunities for studies of this kind: developmental pattern of the Holometabola permit the same genome to be studied in larval and imaginal differentiation and morphogenesis, many larval and pupal tissues have well developed polytene chromosomes so that biochemical and cytological correlations are facilitated, the individuals are of reasonable size and the generation time is short. These features led the candidate to select the Australian brown blowfly, Calliphora stygia, as the experimental material for an extensive and broadly based study of gene action during development. While this experimental system was being developed, Dr. D. H. S. Horn of the Division of Applied Chemistry, CSIRO, was expanding his project on the chemistry and biosynthesis of the insect moulting hormones, with the long-term goal of developing insecticides based on moulting hormones, their analogues, inhibitors or competing compounds. Dr. Horn's team required biological support for their chemical studies along three main lines: (i) preparation of accurately aged samples for studies of endogenous hormone levels and the biosynthetic pathway of moulting hormone in a holometabolous insect (ii) a reliable bioassay to permit comparison of the relative activities of various ecdysones and their analogues (iii) a physiologically defined test-preparation in which isotopically labelled compounds could be tested for ability to serve in vivo as precursors for moulting hormone biosynthesis. Each of these requirements was compatible with the aims of our study on Calliphora at the University of Melbourne, so that collaboration with the CSIRO group rapidly developed. In addition, the availability of quantities of carefully purified moulting hormone through Dr. Horn's work provided the basis for our studies on the mode of action of the moulting hormones on individual larval tissues, especially in relation to protein and RNA synthesis. The contributions of the various workers involved in these projects to the publications presented here are summarized below: Section 1 Introduction: Ideas presented here were developed by Thomson in an invited lecture contributed to Symposium 4, 14 International Congress of Entomology, Canberra, 1972. Paper 1 represents the published abstract of this lecture. Section 2. The Bioassay of Insect Moulting Hormones: Papers 2-3 are based on experimental work conceived by Thomson, carried out in collaboration with Imray in Thomson's laboratory and were written up by Thomson. Pure crystalline ecdysones were provided by Horn. Section 3. The Mode of Action of Insect Moulting Hormones: Papers 4-8 represent work planned and executed in Thomson's laboratory using pure hormone samples provided by Horn. All papers were written by Thomson. Experimental work for paper 4 was done by Thomson. For paper 5, much of the laboratory work on the time-course of response of protein synthesizing activity in individual tissues was done by Neufeld in consultation with Thomson; the latter did the subcellular fractionation and collected one set of dose-response data. For paper 6, Thomson carried out the incorporation experiments, made the squash preparations and sections for radioautography, and interpreted the results. Gunson contributed cytological observations on the nuclear membrane, and Rogers did the electron microscopy. Pure radio-labelled ?-ecdysone was prepared by Horn from extracts of animals treated in Thomson's laboratory with �-ecdysone of high specific activity. Paper 7 is based on experimental work planned by Thomson and carried out jointly with Kinnear and Martin who were largely responsible for the electrophoretic part of the study; the results were analyzed by Thomson. Paper 8 represents a synthesis of the studies grouped in this section. Section 4. The Biosynthesis and Catabolism of Insect Moulting Hormones: Papers 9-20 are based on work arising largely at the initiative of Horn and his collaborators, principally Galbraith and Middleton. Experiments were planned in consultation with Thomson, who provided, treated, timed and harvested all experimental animals in his laboratory, but whose role in this work was clearly subsidiary. All purified hormone preparations and analogues were made, and chemical syntheses done, by Horn and colleagues at CSIRO; these workers were also responsible for the major portion of writing of the resulting papers. Thomson performed all bioassays, and contributed those paragraphs to each paper which are primarily concerned with the animals used, the response of individual tissues, and with certain aspects of the biological significance of the results.
Parthenogenesis, chromosomal polymorphism and morphological variation in Chironomids

Porter, David Laurence. (University of Melbourne, 1973)

Parthenogenesis, in the forms of arrhenotoky, deuterotoky, or thelytoky, is a quite common phenomenon in the animal kingdom. Thelytoky, in which females produce exclusively female progeny in the absence of genetic fertilization, is the most widespread and most mechanistically diverse form of parthenogenesis. It does not follow that thelytoky is the most common ; arrhenotoky, which only occurs in one rotifer, one arachnid and four insect orders (Hartl 1971), is probably the most successful form of parthenogenesis in terms of numbers, due mainly to the presence of over 100,000 species of hymenopterans, the vast majority of which are arrhenotokous. Thelytoky itself is present in a wide variety of forms. The mechanism for the maintenance of thelytoky may be automictic, in which at least the first meiotic division is normal, the chromosomes pairing at prophase and forming bivalents. The zygoid phase is restored by the restitution of anaphase I or metaphase II chromosome plates, fusion of second division products or endomitosis in cleavage nuclei. Alternatively the mechanism may be apomictic, in which meiotic features may be partly or wholly absent, the one or two maturation divisions being equational. Thelytoky may be complete, it being the only manner of reproduction; or it may be cyclical, where it alternates either regularly, or under the influence of environmental factors, with amphimixis or arrhenotoky. Thelytoky may also be either facultative or obligatory. Facultative thelytoky is the situation whereby reproduction is normally bisexual, however a percentage of eggs may develop without fertilization. Obligatory thelytokous forms produce all their offspring without genetic fertilization, reproduction can therefore never be bisexual. There are many thelytokous forms in which the eggs require penetration by the sperm of the same or related species before they develop. In this case, gynogenesis, the sperm makes no chromosomal contribution to the embryo, although there is a cytoplasmic contribution which may have some effect. In this chapter there are two main areas to be examined. The first is to investigate the cytology of two members of the chironomid subfamily Chironominae, Lundstroemia parthenogenetica and Lauterbornia sp. , and to compare them to other thelytokous forms with analogous maturation mechanisms, especially the members of the subfamily Orthocladiinae described by Scholl (1956, 1960). The second area is the discussion of the evolution and properties of thelytokous organisms. Most review articles tend to be limited to a broad presentation of the magnitude of the phenomenon (Oliver 1971), but also order the paper from the point of view of mechanism or animal groupings. Here the discussion is ordered from the point of view of the importance of various phenomena in the evolution and maintenance of thelytokous forms, e.g. hybridization, genetic and ecological considerations. This may not be a more systematic mode of presentation, but it seems to me to be more logical.
Determination and differentiation in imaginal discs of holometabolous insects, literature review ; changes in some specific proteins during development of third instar larvae of the blowfly Calliphora stygia, research report

Dugan, P. R. (Patrick R.) (University of Melbourne, 1971)
Chromosome studies on institutionalised persons

Sutherland, Grant R. (University of Melbourne, 1971)

Genetics - Theses

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