"January 2004" / Includes bibliographical references (leaves 178-201) / Accompanying CD-ROM contains raw data for the thesis / Systems requirements for accompanying CD-ROM: IBM PC or compatible; CD-ROM drive; Adobe acrobat reader / xv, 201 leaves : ill. (some col.), plates (col.) ; 30 cm. + 1 CD-ROM (col. ill. ; 4 3/4 in.) / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, Dept. of Obstetrics and Gynaecology, 2004

Thesis (Ph.D.)--University of Adelaide, Dept. of Obstetrics and Gynaecology, 2004. / "January 2004" Includes bibliographical references (leaves 178-201).

Thesis (M.S.)--University of Wisconsin--Madison, 1982. / Typescript. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references (leaves 28-37).

Mosquito borne diseases continue to be a big threat to human health worldwide. Despite using various vector control methods, we lose a great number of lives to this malicious disease in tropical and subtropical countries each year. Not surprisingly, mosquito is considered as the deadliest animal on the earth, because mortality rates from mosquito-vectored infections only lag behind other major diseases such as HIV and tuberculosis. Current approaches of vector control are mostly limited to using insecticidal bed nets, thus novel techniques are required to prevent a staggering loss to human health and quality of life. Advances in the genome sequencing in the past decade have helped to uncover numerous secrets of diverse genomes. The genome of malaria mosquito Anopheles gambiae was first sequenced in 2002 and since then has been updated to include additional scaffolds, their orientations and correction of mis-assemblies. Yet, the greatest challenge remains in assembling the heterochromatin regions, that are repeat rich part and contain relatively low-gene density. Although previously neglected by scientific studies due to its characteristic paucity of genes, heterochromatin is now recognized to be crucial for several processes such as cell viability, chromosome pairing, meiosis, longevity etc. It is therefore not surprising that heterochromatin comprises of a significant portion of the genome in many species. The efforts to analyze the genome of malaria mosquito in order to identify potential new leads for vector control warrant a better understanding of the heterochromatin. Mosquitoes diploid chromosome number equal 6. While autosomes 2 and 3 are submetacentric and present in both sexes, females are homogametic with XX and males are heterogametic with XY sex chromosomes. To achieve a better understanding of the Anopheles heterochromatin, we investigated heterochromatic region of the X chromosome. Despite one arm of the X chromosome being completely heterochromatic, few studies have investigated the molecular content of this region. Protocols were developed for performing fluorescent in situ hybridization (FISH) on mitotic X chromosomes in An. gambiae. Using cytogenetics and molecular biology techniques, we characterized the X chromosome heterochromatin in members of the An. gambiae complex. Specific satellite DNA and 18S ribosomal DNA probes (major components of heterochromatin) were mapped to X chromosomes enabling their differentiation and characterization in the An. gambiae complex. Microarray studies have highlighted the importance of X chromosome during investigation of nascent species An. gambiae and An. coluzzii. Here for the first time qualitative differences in heterochromatin in between nascent species are described. Cytogenetic idiograms are developed as to include the molecular and qualitative differences between the species of the An. gambiae complex. These idiograms are expected to provide a better resolution of the X chromosome heterochromatin for comparison in major malaria vectors, closing some of the gaps present due to poor sequencing of unassembled repeat rich regions in An. gambiae complex. The current understanding of Y chromosome for transgenic manipulation is poor and limited to very few genes. Due to its near total heterochromatic composition, it is the hardest part of the genome to assemble. In collaboration with other researchers, the Y chromosome content was characterized among sibling species of the An. gambiae complex. Our data revealed the swift changes the Y chromosome has undergone in a relatively short evolutionary time period. These include a rapid rate of turnover not only in heterochromatin but also in euchromatin. In addition to previously described repeats, a novel highly repetitive element called Zanzibar was discovered and mapped to the males of various Anopheles sibling species. Our data can form the basis for evolutionary studies in heterochromatin for male mosquitoes within the An. gambiae complex while also help identify novel targets to create successful transgenic male populations. Along with the X chromosome heterochromatin, to our knowledge this is the most extensive contribution to improve the understanding of mitotic chromosome heterochromatin in malaria mosquitoes. This study also investigated if epigenetics play role in mosquito development, fecundity and heterochromatin formation. DNA methylation, histone modifications and small noncoding RNAs are among the epigenetic mechanisms scrutinized in mammals. However, knowledge about epigenetic mechanisms and their effects is sparse in mosquitoes. A protocol for testing the various effects of epigenetics on different stages of malaria mosquito was developed. An epigenetic drug was utilized to probe the effects on immature and adult malaria mosquitoes. Different concentrations of DZNep, a histone methyltransferase inhibitor, were administered to An. coluzzii larvae. Total survivorship and pupation were compared for treated and untreated groups. The drug was also administered to adult blood feeding females to determine any effects on fecundity and egg morphology, revealing a negative association with an increase in drug concentration. A dose dependent decrease in SAH hydrolase concentration in An. coluzzii was also noticed. These results suggest epigenetics plays a critical role in mosquito pupation and ovarian development. Our work lays the groundwork for future investigations into the field of epigenetics in mosquitoes by revealing its effect on several important developmental stages in malaria mosquitoes. Although genomics and next-gen sequencing technology have come a long way in the last decade since the first Anopheles genome was sequenced, considerable gaps still exist in case characterization of heterochromatin function in an organism. Through our work, we have endeavored to elucidate a few of the major roles that heterochromatin may play in organization, evolution and adaptation of the malaria mosquitoes. / Ph. D.

During the screening process 5,20 8 X chromosomes of -Drosophila melanogaster were analyzed for the presence of temperature-sensitive (ts) lethal mutations (i.e. mutants which die at 29°C but are viable at 22°C) in short proximal and distal segments of the chromosome. Seven ts and 16 non-ts lethals were recovered in both regions combined. A new class of mutations (class-3), which failed to survive at 29°C with either proximal or distal duplication and showed ts lethality with one, was found and extensively analyzed. These mutants were initially interpreted to be dominant ts's, although the heterozygotes of each mutant showed this not to be so. It was decided that these might more probably be chromosomes carrying a lethal mutation covered by the duplication, and a ts lethal mapping elsewhere. By masking the non-conditional lethal with a duplication, developmental studies of the ts mutant were made. The temperature-sensitive period (TSP) and lethal phase (LP) were characterized for each. All TSP’s spanned the early pupal interval, though an individual TSP might extend to either side of this interval. The pattern of temperature-sensitivity of C3-3 suggested that once formed at permissive temperature, its product was not affected by 29°C. The experiments suggest that the temperature-sensitive process occurs at transcription or translation. A lethal allele of the dor locus was recovered, and, in analysis of this mutant with other dor alleles and several variegating duplications, dor itself was found to be a ts lethal. "Warped" wing, a new phenotype of the dor locus which occurred only with the variegating duplications, was described. This paper further describes a method for developmental analysis of non-ts lethal mutations, involving the use of variegating rearrangements. / Science, Faculty of / Zoology, Department of / Graduate

The X and Z chromosomes have unique characteristics that lead to unique evolutionary consequences. Lepidopterans have a well-known, disproportionately large-Z effect for behavioral and morphological traits that distinguish closely related species. A potential explanation for the Large-X effect is the faster evolution of the sex chromosome (Faster-X evolution). We use whole genome re-sequencing of Heliconius erato races and of the incipient species H. himera to test for faster-Z evolution between hybridizing populations at different reproductive isolation levels, by calculating divergence and nucleotide diversity. We show evidence for Faster-Z evolution in Heliconius butterflies at the early stages of speciation and along the speciation continuum. Evidence of higher divergence and lower nucleotide diversity suggests not only selection but also nonaptive process, like demographic changes, may be driving faster-Z evolution, especially in the incipient species.

A form of familial sex-linked mental retardation has been associated with the expression of a fragile site near the terminal end of the long arm of the X chromosome. Previous reports on the fragile X chromosome showed expression of the fragile site to be limited to chromosome preparations from peripheral blood lymphocytes of mentally retarded males and their female relatives in families in which the disorder was segregating. Fragile site expression has also been shown to be a function of the medium employed in cell culture. The fragile X chromosome could only be demonstrated in lymphocytes cultured in medium 199 or media deprived of folic acid. This study was undertaken to develop a method for demonstrating the fragile X chromosome in cultured skin fibroblasts. Fibroblast cell lines from five patients (two mentally retarded males, two obligate carrier females, and a potential carrier female) from a family in which familial sex-linked mental retardation was known to be segregating were established and routinely maintained in a complete culture medium. Forty-three hours prior to chromosome harvest, cells from each patient were transferred to media deficient in folic acid. Under conditions of folic acid deprivation, it was possible to elicit expression of the fragile X chromosome in skin fibroblasts from all five patients studied. No fragile X chromosomes were detected in fibroblasts from three normal control subjects. In a preliminary assessment of the reliability of the fibroblast method, three patients (two mentally retarded males and a potential carrier female) from a second unrelated family in which the disorder is known to be segregating were studied with this method. The fragile X chromosome could be demonstrated in fibroblasts from both of the retarded male patients but could not be. demonstrated in fibroblast chromosome preparations from the potential carrier female. Lymphocytes for all patients studied were grown under similar folate deprived conditions for the purpose of comparing the effectiveness of fibroblast culture with lymphocyte culture in demonstrating the expression of the fragile X chromosome. Neither tissue was shown to consistently provide a higher frequency of expression of the fragile X chromosome. In addition to folate deprivation, it was shown that two other features of the fibroblast method influenced the frequency of expression of the fragile X chromosome. The fragile site was expressed at a significantly higher frequency in chromosome preparations in which the chromosomes were not severely contracted. The frequency of expression in fibroblasts was also shown to be significantly higher with a hypotonic treatment at chromosome harvest using 1% NaCitrate rather than 0.075M KC1. Because fragile site expression was shown to be a function of the degree of chromosome condensation, two agents, 5-BrdU and actinomycin-D, were studied to examine their decondensation effects on the frequency of expression. Neither BrdU nor actinomycin D proved effective in accentuating the frequency of expression. Since fibroblasts behave much like amniocytes in terms of cell culture and chromosome harvest, the development of a method for demonstrating the fragile X chromosome in cultured skin fibroblasts is a step toward the prospect of reliable antenatal diagnosis of familial sex-linked mental retardation associated with a fragile X chromosome. / Medicine, Faculty of / Medical Genetics, Department of / Graduate

Thesis--University of Turku, 1984. / At head of title: From the Institute of Dentistry and the Institute of Biomedicine, University of Turku. Extra t.p. with thesis statement inserted. Also published in: Proceedings of the Finnish Dental Society, Vol. 80, 1984, Suppl. V. Includes bibliographical references.

No description available.

Most vertebrates have separate sexes, and sex-specific traits that are regulated by genes with sex-biased expression patterns. In many species with genetic sex determination system, genetic recombination is suppressed in genomic regions linked to the master regulator of sex determination – the gene or set of linked loci that orchestrate sexual differentiation. Natural selection may favour alleles with sex-specific effects - including those with sexually antagonistic (SA) fitness effects (e.g., beneficial to females but harmful to males) – to become fixed in or be translocated to these non-recombining regions of sex chromosomes, because sex-specific or sex-biased modes of inheritance can resolve genomic conflict associated with SA. Sexually antagonism may also be resolve by sex-biased gene expression, and in theory these two mechanism (sex-linkage and sex-biased gene expression) could operate synergistically. However, there are relatively few empirical studies that test whether genes with sex biased expression patterns are indeed more abundant on sex chromosomes – and especially on newly evolved sex chromosomes. We explored this question with an African frog species Xenopus borealis, whose sex chromosome evolved within the last 25 million years (my) and have a large (~50Mbp) region of suppressed recombination, making it a young sex chromosome system compared to many other intensively studied systems, such as the sex chromosomes of mammals. We tested the possibility that a higher proportion of genes with sex-biased expression would be located on the sex-linked region of the sex chromosome of this species. By examining gene expression in adult liver and gonad and also tadpole gonad/mesenephros at two developmental stages, we found that the sex-linked region of these sex chromosome do have a higher proportion of sex biased genes compared to the non-sex-linked region of the same sex chromosomes, compared to (i) a homeologous genomic region in the tetraploid genome of X. borealis, and also (ii) the autosomes of this species. We did not observe the same pattern in a closely related frog species, Xenopus laevis, which has sex chromosome that are not homologous to those of X. borealis and, unlike X. borealis, lacks a large region of suppressed recombination on its sex chromosome. Using Brownian Motion model, we found as well that expression divergence evolution of genes in the sex-linked region of X. borealis is faster compared to its non-sex-linked homeologs (within X. borealis), and also compared to orthologous regions that are also non-sex-linked. One possible explanation for these observations is that natural selection favoured an expansion of recombination suppression (via unknown mechanisms) on chromosome such that polymorphic regulatory regions became linked (or unlinked) to the sex determining locus in such a way to resolve SA. Alternatively, it is possible that these sex-biased expression pattern evolved rapidly after recombination suppression. / Thesis / Master of Science (MSc) / Sexual selection favours the evolution of distinctive traits in each sex in order to optimize the reproductive success of each one. However, because most of the genome is shared between the sexes, sexual selection may result in genomic conflict when mutations are beneficial to one sex but harmful to the other; this conflict is known as sexual antagonism. Genomic conflict associated with alleles with sexually antagonistic (SA) fitness effects can be resolved via the origin of sex-biased expression patterns and this may be catalyzed by genetic linkage to a sex-determining locus on a sex chromosome. Consequently, one might predict there to be an enrichment of genes with sex-biased expression patterns on the sex chromosome as compared to the autosomes. We tested this expectation in an African frog species Xenopus borealis, which has a relatively young sex chromosomes and a large region of recombination suppression on the female-specific W-chromosome. We found enrichment of sex-biased genes on the nonrecombining region of the sex chromosomes of this species in adult liver and gonad tissue and also tadpole mesenephros/gonads at two developmental stages. Additionally, we found that expression divergence of genes in the non-recombining region have a faster rate of evolution as compared to the rate of expression divergence of genes in other genomic regions. One possible explanation for these observations is that natural selection favours an expansion of recombination suppression (via unknown mechanisms) on sex chromosome such that polymorphic regulatory region become linked (or unlinked) to the sex determining locus in such a way as to resolve SA.