Cell cycle phase-specific cDNA libraries reflecting phase-specific gene expression of Ehrlich ascites cells growing in vivo.
Asynchronous populations of Ehrlich ascites tumor cells grown in vivo were separated by centrifugal elutriation into fractions of G1-, S-, and G2/M-phase cells with less than 10% cross-contamination. Cytoplasmic mRNA from phase-synchronous cells was used to prepare cDNA which was ligated with bacteriophage lambda gt10 arms and amplified in Escherichia coli C600 hfl-. EcoRI digests of DNA isolated from the sublibraries (G1, S, G2/M) were submitted to Southern hybridizations with radiolabeled probes either (a) for genes whose phase-specific expression is clearly documented, thymidine kinase, dihydrofolate reductase, and thymidylate synthase, or (b) for genes whose change of expression during the cell cycle is likely, lamin C, beta-actin, alpha- and beta-tubulin, c-myc, c-fos, p53. The cDNA sequences for genes of group (a) were found to be significantly enriched in DNA of the S-phase library indicating that the cell cycle phase-specific patterns of the respective mRNA levels are conserved in the sublibraries. Sequences belonging to group (b) were also found to be enriched in DNA isolated from the sublibraries: c-fos in G1 phase, lamin C, beta-actin, tubulins, c-myc in S phase, and p53 in G1/S phase. The unexpected prevalence of c-myc and alpha-tubulin in the S-phase library is supported by Northern analysis of RNA from phase-synchronous cells. Non-phase-specific, randomly chosen sequences hybridized equally strong with DNA isolated from the different sublibraries. No significant changes of the patterns of hybridization signals were observed with DNA from different amplifications of the sublibraries when analyzed with the same DNA probe indicating that the cDNA complexities are well conserved during amplifications. Consequently, the sublibraries are useful to obtain information about the cell cycle phase-specific expression of mRNAs for other genes of interest. Since the sublibraries reflect mRNA levels of the cells growing in vivo they supply data on the physiological in vivo pattern of gene expression undisturbed by potentially unphysiological in vitro conditions.
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Institute of Cell and Tumor Biology, German Cancer Research Center, Heidelberg.
Kopun
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Werner
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D000818:Animals; D002286:Carcinoma, Ehrlich Tumor / Q000473:pathology*; D002453:Cell Cycle; D003001:Cloning, Molecular; D003598:Cytoskeletal Proteins / Q000235:genetics; D004247:DNA / Q000378:metabolism*; D005796:Genes; D007399:Interphase; D051379:Mice; D008815:Mice, Inbred Strains; D009693:Nucleic Acid Hybridization; D009857:Oncogenes; D012334:RNA, Neoplasm / Q000235:genetics; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics; D014158:Transcription, Genetic
D003598:Cytoskeletal Proteins; D012334:RNA, Neoplasm; D004247:DNA; D013762:Tetrahydrofolate Dehydrogenase