Construction and evaluation of a hncDNA library of human 12p transcribed sequences derived from a somatic cell hybrid.
An arrayed library of human heterogeneous nuclear complementary (hnc) DNA was constructed from a somatic cell hybrid (M28) containing an i(12p) marker as the sole human chromosome. Heterogeneous nuclear (hn) RNA of M28 was used to synthesize first-strand hncDNA with a primer (RT) containing a random hexanucleotide at its 3' end. Specific amplification of human sequences from this hncDNA was performed using Alu primers in combination with the RT primer. The products were directionally cloned and an arrayed library was constructed. Experiments indicated that all clones were derived from transcribed sequences. A number of randomly isolated clones were evaluated by Southern and Northern experiments, sequence analysis, and PCR. At least 80% of these clones were of human 12p origin. The number of independent clones in the library was estimated to be approximately 550. Using 60 hncDNA clones as probes, 6 showed positive signals on Northern blots. For 3 of these, the corresponding cDNAs were isolated: clone CD60A1 codes for the cation-dependent mannose 6-phosphate receptor, clone CC6 is a human homologue of the bovine 39-kDa nuclear-encoded NADH:ubiquinone oxidoreductase subunit, and CD18 belongs to the family of tumor necrosis factor receptor proteins. Southern experiments showed the 3 cDNAs to map to human chromosome 12p as expected. Taken together these results show that the generation of a hncDNA library is a useful tool for the isolation of unknown genes located on a human chromosome (fragment) present in a somatic cell hybrid.
Baens
M
M
Center for Human Genetics, University of Leuven, Belgium.
Chaffanet
M
M
Cassiman
J J
JJ
van den Berghe
H
H
Marynen
P
P
D000818:Animals; D001483:Base Sequence; D002874:Chromosome Mapping; D002881:Chromosomes, Human, Pair 12; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics*; D015342:DNA Probes; D015723:Gene Library; D005819:Genetic Markers; D006801:Humans; D006822:Hybrid Cells; D051379:Mice; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000235:genetics; D012091:Repetitive Sequences, Nucleic Acid; D014158:Transcription, Genetic
D015342:DNA Probes; D005819:Genetic Markers; D012333:RNA, Messenger; D004247:DNA