PubMed · 9108517
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K K Singh, J A Heinemann. 1997. Yeast plasmids.. https://doi.org/10.1385/0-89603-480-1%3A113
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We have developed an approach to identify and localize cDNAs encoded by YACs. In this scheme, a YAC truncation vector containing a cDNA library is used to interrupt the YAC by homologous recombination in yeast. This approach generates YACs truncated at the site of recombination between the cDNA and the cognate YAC sequence and thus localizes the gene in the YAC. This method results in the production of a large percentage of true recombinants identifying gene encoding regions of the genome. This approach is shown to identify an unique EST sequence from a YAC in Xp22, the recently described transketolase-like gene in a YAC from Xq28 and a putative kinesin-like gene in Xq13. This system should also be useful in the mapping of YACs by targeted integration. We have constructed a new telomere truncation vector, pGR8, which incorporates two selectable markers, HIS5 and LYS2. This vector overcomes problems of previous vectors including: incompatibility with most YAC libraries, vector homology with the YAC arms and high backgrounds resulting from the use of a single selectible marker. A third counterselection with 5-fluoroorotic acid (5FOA) against yeast clones retaining the URA3 gene was also employed to reduce background further. Therefore, this vector and approach should be useful to the transcriptional analysis of YAC maps of any genome.
A recurrent reciprocal chromosomal translocation, t(12;14)(q15;q24) is frequently observed in uterine leiomyoma. Chromosome 12 breakpoints have been shown to occur in a region of approximately 150 kb that contains the gene for a high mobility group protein (HMGI-C). The breakpoint region on chromosome 14 has not been precisely defined. We have generated a contig of overlapping yeast artificial chromosome (YAC) clones approximately 3 Mb in size. Fluorescence in situ hybridization (FISH) analysis showed that this contig spanned the t(12;14) breakpoints in three uterine leiomyomas and that the breakpoints in these tumors occurred within a 1 Mb region. A 30 kb cosmid spanning one of the breakpoints was isolated to set the stage for identifying regions on chromosome 14 that may cause this region to be a preferential site for chromosomal translocation.
A region of chromosome 13q14.3, telomeric to the Retinoblastoma gene RB-1 is frequently deleted in patients with B-cell chronic lymphocytic leukemia (B-CLL). A cosmid and P1-derived artificial chromosome (PAC) contig spanning over 600 kb has been constructed, which encompasses this locus. The contig clones have been used to order a number of markers along the minimally deleted region and to localize a series of CpG islands corresponding to possible candidate genes. A novel polymorphic dinucleotide repeat, 6E3.2, present in one of the ordered cosmid clones has been isolated for use in deletion mapping studies of patient DNA. Leukemic samples from 229 CLL patients have been screened for loss of heterozygosity using microsatellite markers and analyzed for hemizygous and homozygous deletions by Southern blot techniques using genomic probes selected from cosmids across the region. Hemizygous deletions were found in 31% of cases with an additional 10% showing homozygous loss. The use of these probes has defined the commonly deleted area to less than 130 kb, centromeric to the locus D13S272.