The optimisation of a simple, reliable and practical method of interphase FISH which allows prenatal diagnosis of major chromosome aneuploidies using a minimum volume of amniotic fluid sample was the overall objective of this study. When all the probes required were available, the study continued by developing the technique of ratio-mixing FISH for simultaneous detection of the five major chromosome aneuploidies. The technique of five-colour ratio mixing FISH which has been presented here is simple and straightforward, since only two haptenisation and detection systems have been employed to visualise simultaneously five different targets in five distinguishable colours. The steps of denaturation, hybridisation and detection are the same as those used in a uni-colour FISH experiment. The results obtained from hybridisation of an unselected series of 20 uncultured lymphocytes and 27 uncultured amniocytes indicate that the technique is reliable and can be used for simultaneous detection of major chromosome aneuploidies. In order to provide a practical strategy for clinical diagnostic purposes, the use of a three colour ratio-mixing FISH and a dual colour was investigated to visualise the five probe sets on two slides from the same sample. A total unselected series of 45 uncultured lymphocytes and 60 uncultured aminocytes were hybridised with different probe combinations using three colour ratio-mixing FISH. The results indicate that the major chromosome aneuploidies can be simply and reliably identified on two slides from the same sample, using a three colour ratio-mixing FISH to detect the chromosomes X, Y and 21 and a dual colour to detect chromosomes 13 and 18. The failure rate was reduced to 4 per cent using this approach.

DiGeorge syndrome (DGS) is a developmental defect associated with deletions in chromosomal region 22q11.2. Recently, other syndromes (Velo-Cardio-Facial syndrome, Conotruncal Anomaly Face syndrome, isolated conotruncal cardiopathy) with overlapping phenotypes have been found to be associated with deletions of a similar extent in this chromosomal region. All these syndromes have been grouped under the acronym CATCH 22 (Cardiac defect, Abnormal facies, Thymic hypoplasia, Cleft palate, Hypocalcemia, chromosome 22q11.2 deletions). In order to characterize genetically this group of syndromes, we have searched for deletions in the 22q11.2 chromosomal region by fluorescence in situ hybridization (FISH). A set of 6 cosmid probes dispersed within the whole length of the DGS deleted region was used to screen 23 patients. A 22q11.2 deletion was observed in 96% of the patients studied. Furthermore, there does not seem to exist any correlation between the size of the deletion and the phenotype observed, since the majority of patients studied, although widely divergent in their clinical manifestation of DGS, appeared to present the same extent of deletion in this genomic region. / There appears to be a predominance of deletion-bearing mothers in familial CATCH 22 when published pedigrees are examined. Furthermore, our own familial cases and the sporadic cases where the parental origin of the deletion could be deduced using a chromosome 22 short arm heteromorphisms seem to confirm this tendency. Because we had isolated a CA-repeat locus mapping within the DGS deleted region, the parental origin of the deletion in sporadic DGS/VCFS cases was studied by assessing the inheritance pattern of this microsatellite marker. The deleted portion of chromosome 22 was of maternal origin in 16 out of 22 cases (72%). When cases of sporadic, familial and unbalanced translocation inheritance reported in the literature were pooled with these results, there appears to be a net tendency for the deletions to be of maternal origin in CATCH 22 (70 deletions of maternal origin, 21 of paternal origin, X$ sp2$ = 26.4, p $<$ 0.0001). / In order to identify the molecular defect underlying DGS, we embarked on a positional cloning approach. A detailed physical map of the 22q11.2 region was made using one- and two-color FISH on metaphases and G$ sb0$ interphase nuclei, and by hybridization to a chromosome 22 hybrid panel. This permitted delineation of a critical region, within which the breakpoint of a balanced translocation carrier affected with DGS was mapping. This breakpoint was cloned by the construction of cosmid contigs, and a novel gene mapped to this region was isolated. The gene potentially encodes an adhesion receptor, and is not interrupted by the balanced translocation breakpoint. Possible mechanisms through which this gene can be involved in the pathogenesis of DGS are presented. / This research project has contributed toward the understanding of the genetics of DGS and related syndromes. Furthermore, a candidate gene for the CATCH 22 syndromes has been isolated and further work will confirm whether it plays a major role in the pathogenesis of these syndromes.

Prp20, the Saccharomyces cerevisiae homolog to the mammalian regulator of chromosome condensation, RCC1, binds to double-stranded (ds) DNA in vitro through a multi-component complex. Three members of this complex bind GTP in vitro. The Prp20 complex specifically loses its ability to bind dsDNA during DNA replication as determined by an in vitro assay using cell extracts from arrested cdc mutants. This loss of dsDNA-binding activity does not affect the proper organization of the nucleoplasm as was the case for the prp20-1 and prp20-7 mutants, suggesting a segregation exists in the biochemical activities of the Prp20 protein. Detailed analysis of Prp20 demonstrates that specific and highly conserved amino acids coordinate these distinct activities of the Prp20 complex. These essential residues are mainly located in the second and the third repeats of the amino terminus and the last two repeats of the carboxyl terminus of Prp20. Furthermore, mutations in the last two repeats are suppressed by Gsp1, one of the GTP-binding components of the Prp20 complex, but not the mutations in the amino-terminus of Prp20.

Thesis (Ph.D.)--Kent State University, 2007. / Title from PDF t.p. (viewed Aug. 6, 2008). Advisor: Daniel L Ely. Includes bibliographical references (p. 229-251).

Thesis (Ph.D.) -- Australian National University, 2008.

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).

A dissertation submitted to the Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, in fulfilment of the requirements for the degree of Master of Science in Medicine Johannesburg, 2014 / Evolution and historical events over the past 300 000 years have contributed in shaping the gene pool of sub-Saharan African populations. By examining patterns of Y chromosome variation, through the screening of single nucleotide polymorphisms (SNPs) and short tandem repeats (STRs), the present study aimed to characterise the phylogeography of ancient African Y chromosome haplogroups found in populations across sub-Saharan Africa, as well as understand the genetic affinities of these populations. In order to screen the large number of the markers required, seven multiplex single base extension assays were developed. These were used to refine the resolution of Y chromosomes commonly found in Africa, but also included a few markers to delineate the common non-African Y chromosome haplogroups, following a hierarchical screening process. In total, 1667 males were screened, and these data were compiled together with comparative published data. The resultant SNP and STR dataset was used in illustrating, more specifically, the phylogeographies of haplogroups A and B. The wide geographic distribution of haplogroup A, together with its position at the root of the phylogeny and high diversity, support an early diversification of the haplogroup into its subclades, which subsequently spread across Africa. The distribution of major haplogroup B subclades, however, are possibly due to post-glacial migrations in the case of haplogroup B-M112, and recent population expansions, leading to the common presence of haplogroup B-M152 across sub-Saharan Africa. The spread of haplogroup E, however, created the biggest impact on African populations; with its expansion likely resulting in the diminished presence of many of the subclades of haplogroups A and B. The Y chromosome compositions of present sub-Saharan African populations are, thus, the result of several diversification events, followed by migration, and mixing of population groups, over the course of modern human existence.

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