Isolation of SMA candidate genes from a YAC contig by direct selection of cDNA clones from normalized cDNA libraries
- Columbia Univ., New York, NY (United States); and others
A YAC contig has been constructed across the spinal muscular atrophy (SMA) region of chromosome 5 (5q11-13). Further definition by pedigree analysis has yielded a minimal genetic region of 400 kb. For isolation of candidate genes in this region, the following cDNA selection method was hybridized to directionally cloned normalized (Cot 1 DNA-preannealed) cDNA libraries in the form of single-stranded circles. The libraries used were constructed from SMA infant brain and normal fetal liver+spleen. Hybridizing circles were eluted off the filter, partially converted into duplexes and electroporated into bacteria. The selected clones were then sequentially hybridized with a human Cot 1 DNA probe (BRL), and a probe made from the corresponding YAC DNA. Clones that hybridized only to the YAC DNA probe were verified to map to the critical region by genomic Southern analyses. Ten different cDNA clones have been isolated by this procedure so far. Three of them have been definitively mapped back to the region. Four of the ten clones are now completely sequenced. One clone shows sequence homology to a transcriptional initiation factor; another has homology to a prokaryotic attachment site sequence for the lipid moiety of membrane lipoproteins. Two clones show no homology to sequences represented in the public databases. We are continuing the full characterization of the cDNA clones as candidates for the SMA gene.
- OSTI ID:
- 134464
- Report Number(s):
- CONF-941009-; ISSN 0002-9297; TRN: 95:005313-1198
- Journal Information:
- American Journal of Human Genetics, Vol. 55, Issue Suppl.3; Conference: 44. annual meeting of the American Society of Human Genetics, Montreal (Canada), 18-22 Oct 1994; Other Information: PBD: Sep 1994
- Country of Publication:
- United States
- Language:
- English
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