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Growth-regulated antisense transcription of the mouse thymidine kinase gene.

ABSTRACT: 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.

SUBMITTER: Sutterluety H

PROVIDER: S-EPMC147947 | biostudies-other | 1998 Nov

REPOSITORIES: biostudies-other

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Growth-regulated antisense transcription of the mouse thymidine kinase gene.

Sutterluety H H Bartl S S Doetzlhofer A A Khier H H Wintersberger E E Seiser C C

Nucleic acids research 19981101 21

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 ...[more]

PMID: 9776764

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Project description:BACKGROUND: The Thymidine kinase (Tk) mutants generated from the widely used L5178Y mouse lymphoma assay fall into two categories, small colony and large colony. Cells from the large colonies grow at a normal rate while cells from the small colonies grow slower than normal. The relative proportion of large and small colonies after mutagen treatment is associated with a mutagen's ability to induce point mutations and/or chromosomal mutations. The molecular distinction between large and small colony mutants, however, is not clear. RESULTS: To gain insights into the underlying mechanisms responsible for the mutant colony phenotype, microarray gene expression analysis was carried out on 4 small and 4 large colony Tk mutant samples. NCTR-fabricated long-oligonucleotide microarrays of 20,000 mouse genes were used in a two-color reference design experiment. The data were analyzed within ArrayTrack software that was developed at the NCTR. Principal component analysis and hierarchical clustering of the gene expression profiles showed that the samples were clearly separated into two groups based on their colony size phenotypes. The Welch T-test was used for determining significant changes in gene expression between the large and small colony groups and 90 genes whose expression was significantly altered were identified (p < 0.01; fold change > 1.5). Using Ingenuity Pathways Analysis (IPA), 50 out of the 90 significant genes were found in the IPA database and mapped to four networks associated with cell growth. Eleven percent of the 90 significant genes were located on chromosome 11 where the Tk gene resides while only 5.6% of the genes on the microarrays mapped to chromosome 11. All of the chromosome 11 significant genes were expressed at a higher level in the small colony mutants compared to the large colony mutants. Also, most of the significant genes located on chromosome 11 were disproportionally concentrated on the distal end of chromosome 11 where the Tk mutations occurred. CONCLUSION: The results indicate that microarray analysis can define cellular phenotypes and identify genes that are related to the colony size phenotypes. The findings suggest that genes in the DNA segment altered by the Tk mutations were significantly up-regulated in the small colony mutants, but not in the large colony mutants, leading to differential expression of a set of growth regulation genes that are related to cell apoptosis and other cellular functions related to the restriction of cell growth.

Dataset Information

Growth-regulated antisense transcription of the mouse thymidine kinase gene.

Publications

Growth-regulated antisense transcription of the mouse thymidine kinase gene.

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