title: Molecular cloning of transcripts that accumulate during the late G1 phase in cultured mouse cells.
abstract: To identify previously undetected genes that might be involved in later stages of the transition from a quiescent state (G0) to the DNA synthetic phase (S) of murine cells, we set out to isolate cDNA clones derived from mRNAs that appear late in G1 phase in serum-stimulated cells. A lambda-cDNA library was prepared using poly(A)+ RNA from chemically transformed Balb/c 3T3 cells (BP/A31) that had been brought to quiescence and subsequently stimulated for 12 h with serum. From the first screening of approximately 21,000 recombinant phage plaques, about 100 clones were isolated that hybridized to a single-stranded cDNA pool derived from stimulated-cell RNA but not to DNAs made from resting-cell RNA. Eventually, six different clones were identified. The mRNAs from five of these genes increased gradually during the G0 to S transition, in contrast to the "immediate-early" rise of c-myc mRNA or the later rise of thymidine kinase mRNA. These six clones were sequenced and compared to the GenBank database. Clones LG-80, LG-2, and LG-69 are highly homologous to beta-actin, lactate dehydrogenase, and alpha-tubulin. Clones LG-5, LG-61, and LG-74 had no significant homologies to known sequences. A subtractive cDNA library was used to isolate two additional clones, Sub-S1 and Sub-S2; these have homologies to enolase and ribosomal protein L32. Additional studies that examine the function and regulation of these newly identified "late response" genes in the pre-DNA synthesis pathway are in progress.
authors.affiliation: Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts.
authors.forename: T
authors.initials: T
authors.lastname: Nikaido
authors.forename: D W
authors.initials: DW
authors.lastname: Bradley
authors.forename: A B
authors.initials: AB
authors.lastname: Pardee
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002461:Cell Line, Transformed; D002478:Cells, Cultured; D003001:Cloning, Molecular; D003513:Cycloheximide / Q000494:pharmacology; D016193:G1 Phase / Q000235:genetics*; D015870:Gene Expression; D015723:Gene Library; D007700:Kinetics; D051379:Mice; D008969:Molecular Sequence Data; D012333:RNA, Messenger / Q000235:genetics; D016192:Resting Phase, Cell Cycle / Q000235:genetics; D014158:Transcription, Genetic / Q000187:drug effects / Q000502:physiology*
chemical_list: D012333:RNA, Messenger; D003513:Cycloheximide