Construction and analysis of an hn-cDNA library derived from the p-arm of pig chromosome 12.
Our aim is to find unidentified genes on specific pig chromosomes or chromosome fragments. Our approach has involved the construction of a heterogeneous nuclear complementary (hn-c) DNA library of the p-arm of pig Chromosome (Chr) 12, the only pig chromosome present in the pig x hamster hybrid cell line 8990. Total RNA was extracted from the cells and first-strand synthesis of hn-cDNA carried out with random and oligo dT primers. Pig hn-cDNA was isolated by amplification of first-strand synthesized hn-cDNA with primers specific for Short Interspersed Repeat Elements (SINEs) via the polymerase chain reaction (PCR). Hn-cDNAs were size selected and cloned in E. coli XL-1 blue cells with PCR-Script as the vector. The library consisted of 6000 clones. Clone inserts were amplified by PCR with vector-specific primers, and randomly picked inserts greater than 600 bp were sequenced. Homology searches were carried out with the FASTA search program on the GenEmbl database. Thirty clones were sequenced, and of these three showed strong homologies to GenEmbl sequences: (1) to sheep, mouse, human, and rat mammary gland factor (MGF); (2) to MLN-50, a gene that is amplified in human familial breast cancer and is present on human Chr 17; the latter is homologous to pig chromosome 12; (3) to a family of unassigned overlapping human ESTs. Of the other sequenced clones, seven were over 80% homologous with pig SINE sequences; three were over 75% homologous to human LINE sequences; six displayed open reading frames over a mean distance equivalent to 50 amino acids, although these showed no significant similarities with sequences in the databases. Using this approach, we have been able to identify several new genes on the p-arm of pig Chr 12. This is the first report of gene isolation from a library derived from a pig chromosome fragment.
Anderson Dear
D V
DV
Babraham Institute, Babraham Hall, Cambridge, UK CB2 4AF.
Miller
J R
JR
D000818:Animals; D002467:Cell Nucleus; D002874:Chromosome Mapping; D006224:Cricetinae; D004247:DNA / Q000235:genetics; D017931:DNA Primers; D018076:DNA, Complementary; D015723:Gene Library; D006801:Humans; D006822:Hybrid Cells; D007256:Information Systems; D051379:Mice; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D051381:Rats; D013552:Swine / Q000235:genetics*
D017931:DNA Primers; D018076:DNA, Complementary; D004247:DNA