A cross-reference strategy for EST isolation from the human Y chromosome
- Univ. of California, San Francisco, CA (United States)
- Massachusetss General Hospital, Boston, MA (United States)
The Y chromosome constitutes a portion of the human genome in which, apart from sex determination, little information on its functionality is available. Using a combination of exon trapping and DNA-mediated gene transfer techniques, we have initiated a study to isolate transcribed sequences from this chromosome with the aim to eventually construct a human Y transcription map. Two exon libraries were generated from a flow-sorted human Y cosmid library (>4,600 clones). The first one was constructed from 96-well pools using the general exon amplification vector, pSPL3. A total of 2304 clones were arrayed in microtiter plates. The second exon library was constructed from a 4,608-cosmid pool with the 3{prime} exon trapping vector, pTAG4. A total of 576 clones of the 3,000-colony library were arrayed. Hybridization analysis indicated that both TSPY and YRRM exons were trapped. They constituted about 7-12% of the clones in these libraries. Preliminary DNA sequence analysis confirmed the presence of both genes. To generate a source of Y-specific transcripts, L cells were co-transfected with the same cosmid pool and a neomycin selectable marker gene. Northern and RT-PCR analyses indicated that Y-specific genes, such as TSPY, YRRm and MIC2, were expressed in these transfected cells. A subtracted cDNA library was constructed between the L cells transfected with the Y cosmids about those transfected with the selectable marker alone. This and other cDNA libraries (e.g. human testis) were used in hybridization screening of cDNAs corresponding to unique Y-specific trapped exons. Positive hybridization was indeed detected with this approach. The characterization of these cDNA clones are in progress and will be discussed. This cross-reference strategy, therefore, demonstrates the functionality of the trapped exons, isolates the corresponding cDNAs and is an extremely valuable tool for the identification of chromosome specific ESTs and construction of transcription maps.
- OSTI ID:
- 134431
- Report Number(s):
- CONF-941009-; ISSN 0002-9297; TRN: 95:005313-1165
- 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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