As an EC funded programmewe have established the use of cloned telomeres to produce single chromosome hybrids containing terminal deletions of the long arm of the X chromosome (TACF, Nature Genetics 2 275). These hybrids are stable, easily propagated and can provide a source of additional mapped markers for the chromosome being deleted.
Rapid progress in genome analysis has resulted in almost complete YAC Contig maps of two small chromosomes, a large fraction of the human genome present in unassigned contigs, a rapidly improving genetic map and may thousands oE anonymous cDNA partial sequences. Assembly of this data into an integrated whole is the next stage of the process. For small chromosomes much of this task can be accomplished on YAC contigs of the whole chromosome. In the case of larger chromosomes our technology of chromosome fragmentation, producing a distributable resource of terminally deleted chromosomes, will provide a means of giving a chromosomal order to YAC or cosmid contigs assembled by comparison of their restriction profiles. Southern blots of such hybrid cell panels can also be used to provide map locations for cDNAs without the intricacies of in situ hybridization or the expense of PCR primer synthesis. our approach to the creation of hybrids is particularly necessary in the case of Chromosome 1 because of a dearth of existing hybrid lines. In a rodent/human hybrid line we will target +/- selectable markers to the angiotensinogen locus in 1q42-43 and to the G418 gene present on distal chromosome lP. This will provide counterselectable markers on both arms of the chromosome. Following back selection, broken chromosomes will be rescued and stabilised by the introduction of cloned telomeres. The products of breakage will be analysed by in situ hybridization to check the chromosomal integrity of each cell line.
In cooperation with Genethon we will select YACs using previously genetically mapped CA repeat sequences and use in situ hybridization to map them within the panel of deletion hybrids. These YACs, selected to be un rearranged, will then provide anchor and orientation sites for YAC contigs developed by others. Southern blots for panels of hybrids will be made and used for mapping cDNAs to establish the utility of this approach.
Topic(s)Data not available
Call for proposalData not available
Funding SchemeCSC - Cost-sharing contracts
CB2 3EH Cambridge