Construction and screening of a cosmid library generated from a somatic cell hybrid bearing human chromosome 15.
A cosmid library has been constructed with DNA isolated from a mouse/human hybrid cell line designated A15, which was previously characterized and shown to retain chromosome 15 as the only human material. The library was generated and stored as 34 independent pools of primary colonies at 8-10,000 colonies per pool. Screening colonies representing pools of this library by hybridization with a human-specific repetitive probe has facilitated the identification of random clones bearing human inserts. To data, 43 unique clones have been isolated and the inserts mapped by fluorescence in situ hybridization (FISH) to chromosome 15. An STS was generated for each of these clones by end sequencing of the inserts. Two of these clones, c36 and MR23, were mapped by FISH to 15q26.1, and end-sequence data revealed homology to different regions of the FES protooncogene. Sequence generated from the other end of the c36 insert was found to match the previously identified and unmapped coding sequence EST00075. In addition to the identification of such random clones, establishment of the library as pools was expected to facilitate the PCR-based identification of unique clones representing specific regions of chromosome 15. Screening of the library pools with primers specific to the FES region led to the recovery of five independent clones facilitating the development of a cosmid contig encompassing the FES gene. One of the cosmids isolated by PCR-based analysis was derived from the same pool as M23 and was subsequently shown to be identical to M23. The data offer the first report of a chromosome 15 cosmid library and demonstrate the value of utilizing pools to evaluate libraries generated from complex sources like somatic cell hybrids.
McDaniel
L D
LD
McDermott Center for Human Growth and Development, University of Texas Southwestern Medical Center, Dallas 75235, USA.
Zhang
B
B
Kubiczek
E
E
Ritter
M
M
Huang
J
J
Berard
C
C
Leana-Cox
J
J
Schwartz
S
S
Schultz
R A
RA
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002884:Chromosomes, Human, Pair 15; D003360:Cosmids; D015723:Gene Library; D006801:Humans; D006822:Hybrid Cells; D017404:In Situ Hybridization, Fluorescence; D051379:Mice; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D016133:Polymerase Chain Reaction; D016324:Sequence Tagged Sites