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Title: Microcloning and molecular mapping of the mouse X chromosome
Author: Fisher, Elizabeth Mary Claire
Awarding Body: University of London
Current Institution: Imperial College London
Date of Award: 1987
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Microdissection and microcloning have been applied to generate banks of chromosome specific probes for molecular analysis of the mouse X chromosome. For the purposes of microdissection the CD strain of wild mouse (Mus musculus) was used - in which the X chromosome is clearly distinguishable from the rest of the karyotype. X chromosome fragments were microdissected from unstained metaphase spreads and were collected in a nanolitre microdrop. Following collection of chromosome fragments DNA was purified and after restriction enzyme digestion was cloned into a lambda vector, all using special microprocedures. Two microdissections of the whole X chromosome generated banks of 2000 and 1000 microclones. In addition a region specific microdissection was carried out. The mouse X chromosome was divided into four equally sized regions, designated centromeric, proximal, distal and telomeric regions. The ’proximal’ region was chosen for microdissection because it contains a number of interesting genetic loci. The regional microdissection resulted in a bank of 650 microclones. Microclones from a whole X chromosome microdissection and from the regional microdissection have been analysed for size and repeat sequence content. Mean average microclone size from both banks was observed to be smaller than expected - probably due to acid fixation of chromosomes during preparation of metaphase spreads for microdissection. A number of microclones, mainly from the regional microdissection, were further analysed for X chromosome specificity and restriction fragment length variation between Mus musculus DNA and Mus spretus DNA. Suitable microclones have been mapped on the mouse X chromosome by classical genetic analysis, utilising a mouse pedigree derived from a Mus musculus female, heterozygous for two semi-dominant X linked coat mutations, crossed to a wild type Mus spretus male. Resulting female progeny were ’backerossed' to male Mus musculus mice, resulting in over 230 progeny for genetic analysis of microclone positions. X chromosome specific probes were mapped in relation to the segregating coat mutations, Harlequin and Tabby and to the other probes, thus distancing and ordering clones. Six microclone probes displaying suitable restriction fragment length variation have been positioned on the mouse X chromosome and span the expected genetic area. Individual markers display unusual physical characteristics - two probes appear to detect localised repeat sequence islands. The set of X chromosome specific molecular markers provide a basis from which further analysis of the mouse X chromosome may be undertaken.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available