Transcriptional activity of the human thymidine kinase gene determined by a method using the polymerase chain reaction and an intron-specific probe.
We have used the technique of reverse transcription coupled to the polymerase chain reaction to detect mRNA precursors [heterogeneous nuclear RNA (hnRNA)] transcribed from the thymidine kinase (TK) gene of human diploid fibroblasts. With this method, the amplification products of both hnRNA (containing the introns) and mature mRNA can be detected on Southern blots with appropriate hybridization probes. With the experimental conditions used, the sensitivity of the technique is such that TK mRNA can be detected in as few as 20 S-phase cells. TK hnRNA is maximally expressed early in the S phase of the cell cycle after quiescent human fibroblasts are stimulated to proliferate. At this point, the ratio of TK hnRNA to TK mRNA is 1:155. A small amount of TK hnRNA can be detected in populations of cells that appear to be quiescent. However, the presence of the precursor in these populations correlates with the number of cells still cycling. No TK hnRNA can be detected in truly quiescent human diploid fibroblasts, suggesting that in these cells, the TK gene is not transcribed in G0.
Lipson
K E
KE
Department of Pathology, Temple University School of Medicine, Philadelphia, PA 19140.
Baserga
R
R
D001483:Base Sequence; D015139:Blotting, Southern; D002460:Cell Line; D004261:DNA Replication; D004259:DNA-Directed DNA Polymerase; D005796:Genes; D006801:Humans; D007438:Introns; D008969:Molecular Sequence Data; D008979:Moloney murine leukemia virus / Q000201:enzymology; D021141:Nucleic Acid Amplification Techniques; D015345:Oligonucleotide Probes; D016133:Polymerase Chain Reaction / Q000379:methods*; D012322:RNA Precursors / Q000235:genetics; D012332:RNA, Heterogeneous Nuclear / Q000235:genetics; D012194:RNA-Directed DNA Polymerase; D019914:Taq Polymerase; D013937:Thymidine Kinase / Q000235:genetics*; D014158:Transcription, Genetic
D015345:Oligonucleotide Probes; D012322:RNA Precursors; D012332:RNA, Heterogeneous Nuclear; D013937:Thymidine Kinase; D019914:Taq Polymerase; D012194:RNA-Directed DNA Polymerase; D004259:DNA-Directed DNA Polymerase