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Cloning of large regions of mammalian genomes: The molecular genetics of X-linked hypophosphatemic rickets

Francis, Fiona Mary; (1995) Cloning of large regions of mammalian genomes: The molecular genetics of X-linked hypophosphatemic rickets. Doctoral thesis (Ph.D), UCL (University College London). Green open access

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Abstract

This thesis describes two main projects, first the development of an alternative method to construct large insert libraries in a P1 vector, and this project led to the construction and use of P1 libraries made from the genomes of Schizosaccharomyces pombe, mouse and human, and second, the progress in a positional cloning project for the X-linked hypophosphatemic rickets gene. The P1 cloning system allows the propagation of maximally 95kb fragments of genomic DNA and represents an intermediate level of resolution between yeast artificial chromosomes (YACs) and cosmid clones. A protocol for P1 library construction was developed, which involves the use of pulsed field gel electrophoresis for size selection of insert DNA. A P1 library of 16-fold coverage was constructed from the fission yeast S. pombe, which is of interest biologically since it is likely to have many functions that are conserved in higher eukaryotes. A human P1 library representing a 1-fold genome coverage (47000 clones) and a C57BL/6 mouse P1 library representing a 3-fold genome coverage (120000) were also prepared. Each of these libraries has been arrayed in microtitre dishes, gridded on Nylon membranes and screened by hybridization. In parallel to this work, two YAC contigs were constructed in the human Xp22 region, which, in the early stages of this project, was poorly characterized compared to other regions of the X chromosome. Radiation hybrids were used to isolate cosmid and YAC clones giving rise to a 2Mb YAC contig spanning the ZFX-POLA region. Single copy probes were used to isolate YACs from the main area of interest, the X-linked dominant hypophosphatemic rickets region and a 1.5Mb YAC contig was assembled. Further characterization of the latter region by isolation of cosmid, P1, and P1-derived artificial chromosome clones, and the analysis of gene transcripts derived from exon-trapping and cDNA enrichment experiments are described.

Type: Thesis (Doctoral)
Qualification: Ph.D
Title: Cloning of large regions of mammalian genomes: The molecular genetics of X-linked hypophosphatemic rickets
Open access status: An open access version is available from UCL Discovery
Language: English
Additional information: Thesis digitised by ProQuest.
Keywords: Biological sciences; Positional cloning
URI: https://discovery.ucl.ac.uk/id/eprint/10100880
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