Identification of fragments of human transcripts froma defined chromosomal region: representational difference analysis of somatic cell hybrids.
We have tested representational difference analysis of cDNAs from somatic cell hybrids as a means to directly isolate expressed sequences derived from a defined chromosomal region. To this end, the hamster-human somatic cell hybrid Q1Z, which carries Xq28 as the only human chromosomal fragment, was used as Tester and the parental hamster cell line Y21 as Driver. After two rounds of subtraction, two major products of 510 and 307 bp were obtained, derived from the highly expressed human Xq28-derived QM gene and from a hamster repeat sequence strongly up-regulated in Q1Z, respectively. In a second subtraction experiment these fragments were added to the driver, to prevent their reappearance. After three rounds of subtraction a more complex difference product was obtained. Of 26 different fragments analysed, 12 fragments were derived from Xq28-derived genes, 10 of which were from known genes. One fragment was derived from a hamster gene strongly up-regulated in Q1Z. These results demonstrate that cDNA RDA can be used to isolate gene fragments from defined chromosomal regions and that suppression with major products, derived from highly expressed genes, is advantageous to isolate larger number of fragments, presumably derived from rarer transcripts.
Groot
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PC
Department of Clinical Sciences of Companion Animals, Faculty of Veterinary Medicine, University of Utrecht,PO Box 80154, 3508 TD Utrecht, The Netherlands.
van Oost
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BA
D000818:Animals; D002877:Chromosomes, Human / Q000235:genetics*; D006224:Cricetinae; D018076:DNA, Complementary / Q000235:genetics*; D005786:Gene Expression Regulation; D006801:Humans; D006822:Hybrid Cells; D014158:Transcription, Genetic
D018076:DNA, Complementary