The Isolation and Characterisation of Y Homologous and XY Homologous Human DNA Sequences

Mitchell, Michael John (1989) The Isolation and Characterisation of Y Homologous and XY Homologous Human DNA Sequences. PhD thesis, University of Glasgow.

Abstract

The human Y chromosome has been estimated to be around 60Mb in length of which 50-70% is composed of non-functional heterochromatin. Meiotic pairing is observed between the distal end of Xp and Yp. This region was termed the pairing segment although the exact nature of the synapsis was unknown. Karyotype-phenotype correlations have led to the assignment of seven putative Y chromosome genes which include the primary testis determinants, suppressor(s) of the Turner phenotype, and factor(s) involved in the promotion of gonadoblastoma in streak gonads. The aim of this project was to further characterise the Y chromosome at the molecular level. The basis of the work was the isolation of 47 Y homologous inserts from a flow-sorted Y chromosome specific library. These were identified as Y homologous using the Y-only somatic cell hybrid 3E7. They were further assigned to Yp or to Yq by positive or negative hybridisation, respectively, to DNA from an iso Yp male. Nineteen probes detected Yq specific restriction fragments, eighteen detected Yp specific restriction fragments and nine detected both Yp and Yq restriction fragments. To obtain a finer deletion map of the chromosome these probes were then used in conjunction with a number of other probes to analyse 23 XX males, 8 XY females, 2 XO males and 15 dicentric Yp individuals. These individuals carry varying amounts of the Y chromosome and from this analysis a 30 interval deletion map of the Y euchromatin was created. This map was constructed on the basis that the individuals analysed had terminal deletions of the Y chromosome arising from a single breakpoint. Only five individuals were found not to possess a contiguous piece of Y chromatin based on this consensus ordering of Y sequences. With the aim of detecting testis transcripts directly thirty of the probes were used in Northern analysis of testis and placenta RNA. Only one probe, GMGY28, gave a distinct hybridisation in the testes track. This probe detects five distinct autosomal bands on Southern analysis and it is therefore possible that the Y sequence isolated is part of a pseudogene of a highly expressed autosomal testes transcript. Eight of the probes, GMGXY4-GMGXY7, GMGXY9-GMGXY11 and GMGXY19 detected both X and Y restriction fragments. The X fragments were mapped using somatic cell hybrids, and individuals, carrying deleted X chromosomes or one product of an X:autosome translocation. At least three regions of XY homology were defined between Xp22.3/Yq (GMGXY19), Xq21/Yp (GMGXY4-GMGXY7 and GMGXY9) and Xq27-28/Yp/Yq (GMGXY10). Probes with a similar pattern of hybridisation to the first two homologies have already been described but the GMGXY10 homology has not previously been defined. Five of the eight probes were of the Xq21/Yp class suggesting that such sequences form a large part of Yp. These sequences together with other similarly hybridising probes were mapped to adjacent intervals on Yp. GMGXY19 and GMGXY3 (the latter isolated by L. Florentin) were used to analyse males with steroid sulphatase (STS) deficiency, as a high percentage have deletions of the STS gene which also maps to Xp22.3. It was found that 17/23 of the males analysed were deleted for both these probes and one for only GMGXY19. These 18 deletions have been shown to include the STS Neither probe detected a transcript in placental RNA indicating that they are not part of the major STS transcripts. The XY homologous probes were used to analyse Southern blots of 59 individuals from five different ethnic origins cut with up to 24 restriction enzymes. This allowed an estimate to be made of the degree of conservation in these regions between the two chromosomes through the XY differences observed at the restriction sites. The Xp22.3/Yq and Xq27-28/Yp/Yq homologies were estimated to be less than 94% conserved while the Xq21/Yp homology was estimated to be 98% conserved. Nine different RFLPs were also revealed for these probes. GMGXY4 detected two different X polymorphisms and the only Y polymorphism found in the whole study. GMGXY5 detected three different X polymorphisms that showed complete linkage disequilibrium while GMGXY9 revealed a four allele X polymorphism. GMGXY7 and GMGXY10 revealed infrequent X polymorphisms with a single enzyme. An estimate of the polymorphic frequency of the X and Y chromosomes revealed that the Y chromosome is as much as 10 times less polymorphic than the X chromosome in these regions. (Abstract shortened by ProQuest.).

• Item Type: Thesis (PhD)
• Qualification Level: Doctoral
• Keywords: Genetics
• Date of Award: 1989
• Depositing User: Enlighten Team
• Unique ID: glathesis:1989-77907
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 28 Feb 2020 12:09
• Last Modified: 28 Feb 2020 12:09
• URI: https://theses.gla.ac.uk/id/eprint/77907