TY - JOUR
T1 - Growth-regulated antisense transcription of the mouse thymidine kinase gene
AU - Sutterluety, Hedwig
AU - Bartl, Stefan
AU - Doetzlhofer, Angelika
AU - Khier, Harald
AU - Wintersberger, Erhard
AU - Seiser, Christian
N1 - Funding Information:
We thank T.Sauer for performing the FACS analysis and part of the nucleotide sequence analysis and Dr M.Hengstschlaeger for providing the ribonucleotide reductase cDNA. We are grateful to Dr A.Infante, F.M.Yeong and Dr H.Rotheneder for critically reviewing this manuscript. This work was supported in part by the Austrian Science Foundation (grant P11179-GEN to C.S. and grant P10873-GEN to E.W.), the Austrian National Bank, the Austrian Ministry of Science (grant 70.003/2-Pr/4/95) and the Herzfelder Foundation.
PY - 1998/11/1
Y1 - 1998/11/1
N2 - The expression of the salvage pathway enzyme thymidine kinase (TK) is very low in resting mammalian cells, but increases dramatically when growth-stimulated cells enter S phase. The 30-fold rise in TK mRNA levels in response to growth factors is due to a well-characterized transcriptional activation and less defined post-transcriptional mechanisms. A minigene containing the murine TK promoter and the TK cDNA showed a 3-fold increase in TK mRNA levels after growth induction in stably transfected mouse TK-deficient L fibroblasts. Introduction of the first three TK introns resulted in a 10-fold regulation of TK expression which was predominantly due to repressed TK mRNA levels in serum-deprived cells. Removal of intron 3 from this construct or replacement of the TK promoter by a constitutive SV40 promoter led to a reduced, but still significant increase in TK mRNA levels during the onset of proliferation. These results indicate that both the TK promoter and specific TK introns contribute independently to the growth-dependent regulation of TK mRNA expression. To examine the regulatory mechanisms in more detail we analyzed TK transcription rates and steady-state levels of nuclear transcripts from an SV40 promoter-driven minigene that contains introns 2 and 3 of the TK gene. Using a set of single-stranded probes we detected TK-specific antisense transcription that was up-regulated in resting cells. Similarly, antisense transcription of the endogenous TK gene in Swiss 3T3 cells rose during serum deprivation while sense transcription was regulated in the opposite way. Luciferase reporter assays revealed the presence of a putative antisense promoter in intron 3 of the murine TK gene. These results suggest a negative role for intron-dependent antisense transcription in the regulation of TK mRNA expression in mouse fibroblasts.
AB - The expression of the salvage pathway enzyme thymidine kinase (TK) is very low in resting mammalian cells, but increases dramatically when growth-stimulated cells enter S phase. The 30-fold rise in TK mRNA levels in response to growth factors is due to a well-characterized transcriptional activation and less defined post-transcriptional mechanisms. A minigene containing the murine TK promoter and the TK cDNA showed a 3-fold increase in TK mRNA levels after growth induction in stably transfected mouse TK-deficient L fibroblasts. Introduction of the first three TK introns resulted in a 10-fold regulation of TK expression which was predominantly due to repressed TK mRNA levels in serum-deprived cells. Removal of intron 3 from this construct or replacement of the TK promoter by a constitutive SV40 promoter led to a reduced, but still significant increase in TK mRNA levels during the onset of proliferation. These results indicate that both the TK promoter and specific TK introns contribute independently to the growth-dependent regulation of TK mRNA expression. To examine the regulatory mechanisms in more detail we analyzed TK transcription rates and steady-state levels of nuclear transcripts from an SV40 promoter-driven minigene that contains introns 2 and 3 of the TK gene. Using a set of single-stranded probes we detected TK-specific antisense transcription that was up-regulated in resting cells. Similarly, antisense transcription of the endogenous TK gene in Swiss 3T3 cells rose during serum deprivation while sense transcription was regulated in the opposite way. Luciferase reporter assays revealed the presence of a putative antisense promoter in intron 3 of the murine TK gene. These results suggest a negative role for intron-dependent antisense transcription in the regulation of TK mRNA expression in mouse fibroblasts.
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U2 - 10.1093/nar/26.21.4989
DO - 10.1093/nar/26.21.4989
M3 - Article
C2 - 9776764
AN - SCOPUS:0032212212
SN - 0305-1048
VL - 26
SP - 4989
EP - 4995
JO - Nucleic acids research
JF - Nucleic acids research
IS - 21
ER -