Cloning of a Multi-Tissue Tumour Suppressor/Replcative Senescence Gene on Human Chromosome 7q31

Hurlstone, Adam Felix Lloyd (1998) Cloning of a Multi-Tissue Tumour Suppressor/Replcative Senescence Gene on Human Chromosome 7q31. PhD thesis, University of Glasgow.

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The q31 region of human chromosome 7 is frequently deleted in a broad spectrum of human cancers, and is believed to harbour a multi-tissue tumour suppressor gene. I found loss of heterozygosity at one or more microsatellite markers from 7q31 in 36% of breast carcinomas. The smallest common deleted region was between two CA.GT microsatellite markers, D7S522 and 17TA-5/17B-RE3 in the CFTR locus. Loss of 7q31 markers in two non-tumourigenic, human fibroblast cell lines, SUSM-1 and KMST-6 (both immortality complementation group D), has been associated with the emergence of an immortal phenotype. This phenotype can be suppressed by re-introduction of an intact copy of chromosome 7. We conjectured that the multi-tissue tumour suppressor and immortality complementation group D gene, which we named SEND, are one and the same. A physical and functional cloning strategy was adopted to isolate SEND. Intact copies of a hygromycin-resistance tagged human chromosome 7 were introduced into SUSM-1 cells by microcell-mediated monochromosome transfer. This induced replicative senescence in a significant proportion of the hygromycin-resistant colonies recovered. Occasional immortal segregants also arose, most likely as a result of inactivating SEND on the introduced chromosome. The sites of inactivation were mapped by analysing polymorphic microsatellite markers that differed between donor and recipient chromosomes. This also entailed my generating novel polymorphic markers. Using this strategy, I defined three 'hot spots' of allele loss on chromosome 7 in immortal segregants. One, an approximately 500Kbp interval between 724CA and 786CA, two novel CA.GT dinucleotide repeats, was nested within the smallest common region of allele loss determined for breast cancers. The putative tumour suppressor gene/SEND may reside in this interval. I assembled a YAC, cosmid, and PAC clone contig for the smallest region of allele loss in breast tumours and mapped a number of genes to it by a combination of approaches, including exon-trapping, sequencing, and EST-content mapping. Certain candidate genes were further characterised. This analysis included cloning of full length cDNAs, determining the genomic (exon-intron) structure, determining expression in tumour cell lines by northern and western blot analysis, and looking for mutations by SSCP analysis. Perhaps the most interesting candidate, CAVEOLIN-1, although not mutated in human cancers, was found to be transcriptionally silenced in a number of tumour-derived cell lines by methylation of 5'-sequences.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Additional Information: Adviser: Ken Parkinson
Keywords: Genetics
Date of Award: 1998
Depositing User: Enlighten Team
Unique ID: glathesis:1998-74738
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 27 Sep 2019 16:43
Last Modified: 27 Sep 2019 16:43

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