Analysis of DNA sequences present in complexes of v-Myc and cellular DNA.
We have isolated cellular DNA sequences from the myelocytomatosis virus-transformed quail fibroblastic cell-line (MC29-Q8) by indirect immunoprecipitation of v-Myc-DNA complexes and subsequent cloning of the DNA. The v-Myc-DNA complexes were obtained from isolated nuclei pretreated with 150 mM salt and indirect immunoprecipitation of the p110 Gag-Myc protein with the IgG of a Gag-specific monoclonal antibody. A non-specific monoclonal IgG was used as control to account for non-specific interactions. The DNA from the precipitates was isolated, cloned and characterized. 21 positive and 13 control clones with inserts ranging in size from 25 to 330 nucleotides were sequenced and analyzed for sequence homologies to known DNA-motives. Some of the sequences over-represented in the specific DNA fragments corresponded to elements which have been previously described to promote DNA amplification. Six of these DNA fragments were tested for their ability to promote DNA amplification by insertion into a plasmid containing the HSV-1 tk gene with a truncated promoter. These constructs were transfected into mouse Ltk- cells which grow under HAT selection only upon amplification of the tk-carrying plasmids. Two of the six DNA fragments showed the capability to amplify their plasmids in cis and create stable cellular clones. Copy numbers of the amplified plasmids in these Ltk+ clones ranged from 470 to 680 whereby the amplified sequences were integrated as large clusters of head-to-tail tandems. The data presented here suggest that the Myc protein may be involved in DNA amplification and therefore may play a role in DNA replication.
Lorenz
U
U
Max-Planck-Institut für Molekulare Genetik, Abt. Schuster, Berlin, Federal Republic of Germany.
Sock
E
E
Grummt
F
F
Moelling
K
K
D000818:Animals; D001483:Base Sequence; D015139:Blotting, Southern; D003001:Cloning, Molecular; D003198:Computer Simulation; D016259:Genes, myc; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D011233:Precipitin Tests; D011784:Quail / Q000235:genetics; D014162:Transfection; D014170:Transformation, Genetic