U096127.pdf (10.78 MB)
Isolation and characterisation of Telomere and Telomere-related sequences in the chicken genome
thesisposted on 2014-12-15, 10:33 authored by Philippa. Thomson
Telomeres are the physical ends of eukaryotic chromosomes, and are essential for chromosome stability and complete replication.;Chicken telomere and telomere-related sequences were characterised using a variety of approaches including: cosmid libraries, anchored PCR strategies, and fluorescence in situ hybridisation (FISH). The results suggested that chicken chromosomes terminate in 6.7-14.0 kb tandem arrays of the vertebrate telomere repeat, (TTAGGG)n. The proximal boundaries of these arrays are degraded, and include many variant repeat types which differ from the telomere repeat by one base change. Southern analysis suggested that the degraded repeats can be digested with restriction enzymes, increasing the probability of locating telomere-related sequences in standard cosmid libraries.;Complex repeats associated with the terminal arrays and the degraded repeats were isolated, and shown to be located proterminally on metaphase chromosomes. Hybridisation of a telomere repeat probe to genomic DNA separated by pulse field gel electrophoresis (PFGE) suggested that complex subtelomeric repeats may form large satellite blocks at the ends of a subset of chromosomes, resulting in terminal MboI restriction fragments of up to 620kb.;Fluorescence in situ hybridisation (FISH) experiments indicated that the distribution of subterminal and terminal sequences within the genome were correlated with distance from the chromosome end. Sequences located towards the ends of chromosomes (i.e. interspersed telomere repeats) were found on more chromosomes than those from more proximal locations (i.e. complex repeats).;Screening of a large insert cosmid library (Stratagene) with a telomere repeat probe resulted in the isolation of 59 clones. Single-locus restriction fragment length polymorphisms (RFLPs) were identified from ten of these clones, and the inserts were localised by FISH on metaphase chromosomes. The cosmid probes showed no bias towards location on macrochromosomes. The results suggested that some of the clones may contain DNA from subtelomeric locations, but that others clearly identify interstitial loci.
Date of award1997-01-01
Awarding institutionUniversity of Leicester