In Situ Hybridisation for Human and Comparative Gene Mapping

Kalaitsidaki, Marianna (1989) In Situ Hybridisation for Human and Comparative Gene Mapping. PhD thesis, University of Glasgow.

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The overall aim of this project was to develop the technique of in situ hybridisation (ISH) for the localisation of cloned DNA sequences on the Y and other chromosomes in man and other primates using both 3H- and biotin labelling. Using tritium labelling two problems were encountered and circumvented, high non-specific labelling and loss of chromosome morphology following hybridisation. Following this, seven anonymous DNA sequences cloned in plasmid vectors were localised to specific chromosome sites in the human: GMGY10 (DYS59) and GMGY7 (DYS58) to the short of the Y chromosome (Yp), P2F2 (DY25) to chromosome X band q21 and Yp, pY3.4 (DYZ1) to chromosome Y distal band Yq12, GMGY3 (DYS13) to Yp and chromosome 9 region p23-pter, GMGY4b (DYS51) to the pericentromeric region of chromosomes Y, 15, 21, 22, JG73 to pericentromeric region of chromosomes 19, 21, 22 , Y and (14, 15, and 20) and finally JG51 (D21S89) to the pericentromeric region of chromosomes 13, 14, 15, 20, 21, and 22. Under conditions of high stringency the last two probes were localised to chromosome 21, and chromosomes 13, 14 and 22 respectively, contrary to filter hybridisation data where these two sequences appear to be identical. Conventional in situ hybridisation techniques using radioactive probes suffer the serious disadvantages of prolonged autoradiographic exposure times and limited spatial resolution. To overcome these problems several non-isotopic methods have been introduced using a variety of different immunogenic, fluorescent and enzymatic labels. The possibility of using biotin-labelling for gene mapping was explored during this project using a recently described technique that employs a streptavidin-alkaline phosphatase detection system. The method was shown to be simple, reliable, rapid and sufficiently sensitive to detect single copy DNA sequences, as was indicated by the localisation of a 3.2kb DNA sequence, p72 (D21S92), to chromosome 21 proximal band q21. Results were obtained in 24 hours as compared with 1 week autoradiographic exposure for repetitive probe and 3 weeks for single copy probe. Another probe, GMGXY8 (DXYS34) was mapped to chromosome Yp. All probes mentioned above had been regionally mapped by Southern analysis using somatic cell hybrids. ISH not only confirmed but also extended the previous findings by indicating autosomal homologies for probes GMGY3, GMGY4b and regions of extended homology for JG73 and JG51. The homology between short arm of chromosomes Y and 9 revealed for probe GMGY3 is of special interest in view of the fact that GMGY3 resides in the sex-determining region of the Y chromosome. This finding coupled with observations that 9p monosomy is sometimes associated with anomalous sex differentiation could suggest a functional homology between Yp and distal 9p. ISH was also used to determine the chromosomal location of Y-specific sequences in eleven patients with paradoxical sex chromosome complements previously shown to possess such sequences. Eight XX males were studied with 3H-labelled GMGY10 and/or GMGY7 and in all cases it could be clearly demonstrated that these Y-specific sequences were located on distal short arm of the X chromosome (band Xp22.3) as predicted by the X-Y interchange hypothesis (Ferguson-Smith, 1966). The ninth XX male was investigated with probe P2F2 and although signal was recorded on the short arm of the X chromosome it could not be shown conclusively that this was due to an X-Y. interchange. Using GMGY10 or GMGY7 probes in two apparent XO males with an additional minute chromosomal fragment present in their peripheral blood lymphocytes it was demonstrated that the fragment had originated from a Y chromosome thus explaining the male phenotype of these individuals. Apart from its value in gene mapping studies, ISH has an enormous potential as a tool in clinical diagnosis. This was demonstrated in the present study by the confirmation of a suspected Yp:15q translocation in amniotic fluid cells and in paternal lymphocytes with a chromosome 15 polymorphism by using 3H-labelled probe pY3.4. The last part of the project involved comparative mapping in the gorilla (Gorilla gorilla), chimpanzee (Pan troglodytes) and orangutan (Pongo pygmaeus) of a human transfer RNA gene for glutamic acid (tRNA Glu ) and probes GMGY10 and GMGY7. The tRNA Glu gene was localised to distal long arm of chromosome 1 (band 1p36 according to ISCN nomenclature) in all three species, providing further evidence for homology between distal long arm of the ape chromosome 1 and distal short arm of human chromosome 1 where this gene has been previously localised.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Keywords: Genetics
Date of Award: 1989
Depositing User: Enlighten Team
Unique ID: glathesis:1989-77993
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 30 Jan 2020 15:44
Last Modified: 30 Jan 2020 15:44

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