The isolation of cDNAs within the Huntington disease region by hybridisation of yeast artificial chromosomes to a cDNA library

Abstract

Flanking recombination events have defined the Huntington Disease gene candidate region to between D4S10 and D4S98, about 2.2Mb. Because of the large size of the candidate region and the likely large number of genes within it we decided to screen cDNA libraries with probes generated from whole Yeast Artificial Chromosomes (YACs) containing parts of this region. We have thus far used 4 YACs ranging in size from 180kb to 600kb covering 880kb and have isolate 13 cDNA clones, 7 of which are unique. Three of the 13 clones contain parts of the 3' untranslated region of the alpha-adducin gene. One YAC of 600kb could not be purified from two yeast chromosomes, therefore this YAC probe had a net complexity of 1.8Mb. Even so this probe identified a cDNA from the HD candidate region indicating that very large YACs may be used as probes.

The Huntington's Disease Gene Discovery

“…1,3 After concluding that the gene mutation would be located on chromosome 4, they studied HD families for further recombinants, to define the candidate region more precisely. Based on previous reports describing linkage disequilibrium with the markers D4S95 and D4S98, [24][25][26][27][28] they extended the study around D4S98 and cloned some of that region, placing the HD gene close to the D4S98 locus. 28 Thereafter, Dr. Marcy MacDonald, along with her team at Harvard Medical School, employed exon amplification to acquire candidate coding sequences for the proximal section of the 500 kb segment.…”

“…Based on previous reports describing linkage disequilibrium with the markers D4S95 and D4S98, [24][25][26][27][28] they extended the study around D4S98 and cloned some of that region, placing the HD gene close to the D4S98 locus. 28 Thereafter, Dr. Marcy MacDonald, along with her team at Harvard Medical School, employed exon amplification to acquire candidate coding sequences for the proximal section of the 500 kb segment. In this process, they identified a sizable gene, IT15, spanning 210 kb, which codes for a previously undescribed protein of 348 kb.…”

Integrated genetic and physical map of the 1q31→q32.1 region, encompassing the RP12 locus, the F13B and HF1 genes, and the EEF1AL11 and RPL30 pseudogenes

“…PAC inserts, to be used as probe, were retrieved in LMT-agarose after NotI digestion and preparative PFGE of the PAC DNA. Probe labeling and preassociation conditions were adapted from Snell (1993) and Elvin (1990). In brief, approximately 100 ng of PAC insert was radioactively labeled with 4.5 MBq [·-32 P]dCTP (Amersham) for 16 h at 37°C in a 200-Ìl volume by multiprime labeling (Amersham).…”

Delimiting the Wolf-Hirschhorn syndrome critical region to 750 kilobase pairs

“…Owing to the abundant cloned resources from this region of the genome, a large number of potential coding sequences were identified [Gilliam et al, 1987;John et al, 1994;Rommens et al, 1993;Snell et al, 1993]. Overall, the region appears to be gene dense.…”

“…Therefore, we conclude that genes that contribute to the WHS phenotype reside in the interval distal to D4S43 and proximal to D4S168. This localization effectively eliminates several genes (MYL5, ZNF141, PDE␤, and IDUA) mapped to 4p16.3 previously suggested as having a role in the etiology of WHS.Several potentially expressed sequences have been found throughout the WHSCR but these have not been studied in detail [Gilliam et al, 1987;John et al, 1994;Rommens et al, 1993;Snell et al, 1993]. The exception is the FGFR3 gene, located near the distal end of the refined WHSCR, FGFR3 has been studied both for its role in a number of growth related disorders [Bellus et al, 1995;Meyers et al, 1995;Shiang et al, 1994;Tavormina et al, 1995].…”