Differential screening of murine ascites cDNA libraries by means of in vitro transcripts of cell-cycle-phase-specific cDNA and digital image processing.
Cell-cycle-phase-specific cDNA libraries were prepared in the lambda gt10 vector and in the in vitro transcription vector, pBluescript. Plaques of the cDNA libraries prepared in the lambda gt10 vector were differentially screened with (a) in vitro transcripts of the cell-cycle-phase-specific cDNAs cloned in the transcription vector and (b) with first-strand cDNA of mRNA from phase-synchronous cells. The results suggest that first-strand cDNA can be replaced, at least in prescreening experiments, by in vitro transcripts of representative cDNA libraries prepared in in vitro transcription vectors. The fractions of differential clones detected with in vitro transcripts (1.2%) and with first-strand cDNA (1%) were in the same order. Individual clones selected by differential hybridization with in vitro transcripts could be verified by differential hybridization with cell-cycle-phase-specific first-strand cDNA. This indicates that the pattern of stage-specific prevalences of cDNA clones is essentially retained during careful amplifications of large cDNA libraries. The application of in vitro transcripts of stage-specific cDNA for differential screening experiments is of interest in cases where the amount of biological material is either limited or difficult to prepare. It also allows standardization of the probes in repeated screening experiments. Three clones reflecting cell-cycle-phase-specific mRNA prevalences were chosen and analyzed on the sequence level. Two sequences with S-phase prevalences were identified. They code for elongation factor EF1 alpha and for glyceraldehyde-3-phosphate dehydrogenase, respectively. The third sequence reflects the first cDNA of a mRNA with significant prevalence in G2-phase cells.(ABSTRACT TRUNCATED AT 250 WORDS)
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Institut für Zell- und Tumorbiologie, Deutsches Krebsforschungszentrum, Heidelberg, F.R.G.
Dengler
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Rothbarth
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Werner
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D000595:Amino Acid Sequence; D000818:Animals; D002286:Carcinoma, Ehrlich Tumor / Q000235:genetics* / Q000473:pathology; D002453:Cell Cycle; D002999:Clone Cells; D004273:DNA, Neoplasm / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics; D005784:Gene Amplification; D015723:Gene Library; D005821:Genetic Techniques; D005822:Genetic Vectors; D008565:Membrane Proteins / Q000235:genetics; D051379:Mice; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D021382:Protein Sorting Signals / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics / Q000302:isolation & purification; D014158:Transcription, Genetic
D004273:DNA, Neoplasm; D008565:Membrane Proteins; D021382:Protein Sorting Signals; D012333:RNA, Messenger