Regulation of dihydrofolate reductase gene transcription in methotrexate‐resistant mouse fibroblasts

Dihydrofolate reductase (DHFR) synthesis is regulated in a growth-dependent fashion. Dividing cells synthesize DHFR at a 10-fold-higher rate than do stationary cells. To study this growth-dependent synthesis, DHFR genes have been constructed from a DHFR cDNA segment, the adenovirus major late promoter, and fragments of simian virus 40 (SV40) which provide signals for polyadenylation. These genes have been introduced into Chinese hamster ovary cells. The DHFR mRNAs produced in different transformants are identical at their 5' ends, but differ in sequences in their 3' ends as different sites are utilized for polyadenylation. Three transformants that utilize either DHFR polyadenylation signals or the SV40 late polyadenylation signal exhibit growth-dependent DHFR synthesis. The level of DHFR mRNA in growing cells is approximately 10 times that in -stationary cells for these transformants. This growth-dependent DHFR mRNA production probably results from posttranscriptional events. In contrast, three transformants that utilize the SV40 early polyadenylation signal and another transformant that utilizes a cellular polyadenylation signal do not exhibit growthdependent DHFR synthesis. In these three cell lines, the fraction of mRNAs polyadenylated at different sites in a tandem array shifts between growing and stationary cells. These results suggest that the metabolic state of the cell is important in determining either the efficiency of polyadenylation at various sites or the stability of mRNA polyadenylated at various sites.

mentioning

confidence: 90%

Growth-dependent expression of dihydrofolate reductase mRNA from modular cDNA genes.

Kaufman¹,

Sharp²

1983

“…The RNA of the proto-oncogenes c-myc and c-fos can also be induced by increases in cAMP (5,19,38,43). Genes coding for key metabolic enzymes, including tyrosine aminotransferase, lactate dehydrogenase, tryosine hydroxylase, phosphoenol pyruvate carboxykinase, and dihydrofolate reductase (21,25,32,41,45,50), are regulated at the level of transcription by cAMP in either a positive or a negative way. In addition, the mRNA levels of tubulin and actin, the main protein constituents of microtubules and microfilaments, are modulated by cAMP (16).…”

mentioning

confidence: 99%

Levels of RNA from a family of putative serine protease genes are reduced in Drosophila melanogaster dunce mutants and are regulated by cyclic AMP.

Yun¹,

Davis²

1989

We have isolated several genes expressed at abnormal levels in the memory mutant, dunce (dnc), of Drosophila melanogaster. These mutants have an elevated cyclic AMP (cAMP) content due to a mutation in the structural gene for cAMP phosphodiesterase, so the isolated genes are potentially ones regulated by cAMP. Here, we describe the characterization of a genomic clone and corresponding cDNA clones which contain sequences that are underexpressed in dnc mutants. Sequence analysis of portions of the genomic clone and representative cDNAs revealed the presence of two uninterrupted and complete open reading frames (SER1 and SER2) and part of a third (SER3). The predicted amino acid sequences of all of these were found to be homologous to the serine protease family of enzymes. The genomic clone was localized to the polytene chromosome region 99C-D, although genome-blotting experiments indicated the existence of several other genes related to the cloned serine protease-like genes. Hybridization experiments with probes representing each of the three sequenced genes showed that only the SERl-related genes were differentially expressed in dnc mutants. The putative serine protease genes were abundantly expressed in the larval gut, suggesting a major function in digestion. Feeding normal flies cAMP, isobutylmethylxanthine, or forskolin resulted in a decreased RNA level of the SERl-related genes. Thus, RNA levels of this serine protease gene family are negatively regulated by cAMP.Cyclic AMP (cAMP) has been shown to mediate many biological processes in diverse organisms. In procaryotes, cAMP and its receptor protein, CRP, modulate the transcriptional activity of a variety of genes (2). In eucaryotes, the molecule serves as an intracellular second messenger of external stimuli, including hormones and neurotransmitters. One important area of cAMP action currently under intensive study is its role in eucaryotic gene expression. cAMP has been shown, for example, to regulate the mRNA levels of the genes encoding the hormones preproenkephalin (10), gonadotropin (23), prolactin (34), somatostatin (36), vasoactive intestinal polypeptide (44), and growth hormone (47). The mRNA levels of these genes are regulated in a positive manner at least partly at the level of transcription. The RNA of the proto-oncogenes c-myc and c-fos can also be induced by increases in cAMP (5,19,38,43). Genes coding for key metabolic enzymes, including tyrosine aminotransferase, lactate dehydrogenase, tryosine hydroxylase, phosphoenol pyruvate carboxykinase, and dihydrofolate reductase (21,25,32,41,45,50), are regulated at the level of transcription by cAMP in either a positive or a negative way. In addition, the mRNA levels of tubulin and actin, the main protein constituents of microtubules and microfilaments, are modulated by cAMP (16). Thus, cAMP modulates the activity of various genes by altering mRNA abundance, either positively or negatively, and in most cases this modulation occurs at least in part at the level of transcription. The * Corresponding auth...

“…In the case of the histone genes, there is good evidence that both transcriptional and post-transcriptional controls occur (1,7,21,22). Post-transcriptional control of DHFR expression is well documented (14,15), but the existence of transcriptional control has been controversial (20,26). A recent report suggests that there is a transient burst of DHFR transcription at the G1-S boundary, but that the increased transcriptional level is not maintained throughout the S phase (2).…”

mentioning

confidence: 99%

Evidence for transcriptional and post-transcriptional control of the cellular thymidine kinase gene.

Stewart¹,

Ito²,

Conrad³

1987

109

We have studied the cell cycle-regulated expression of the thymidine kinase (TK) gene in mammalian tissue culture cells. TK mRNA and enzyme levels are low in resting, GO-phase cells, but increase dramatically (10-to 20-fold) during the S phase in both serum-stimulated and simian virus 40-infected cells. To determine whether an increase in the rate of TK gene transcription is responsible for this induction, nuclear run-on transcription assays were performed at various times after serum stimulation or simian virus 40 infection of growth-arrested simian CV1 cells. When assays were performed at 12-h intervals, a small (two-to threefold) but reproducible increase in TK transcription was detected during the S phase. When time points were chosen to span the G1-S interface a larger (six-to sevenfold) increase in transcriptional activity was observed in serum-stimulated cells but not in simian virus 40-infected cells. The large increase in TK mRNA levels and the relatively small increase in transcription rates in growth-stimulated cells suggest that TK gene expression is controlled at both a transcriptional and post-transcriptional level during the mammalian cell cycle. To identify the DNA sequences required for cell cycle-regulated expression, several TK cDNA clones were transfected into Rat-3 TK-cells, and their expression was examined in resting and serum-stimulated cultures. These experiments indicated that the body of the TK cDNA is sufficient to insure cell cycle-regulated expression regardless of the promoter or polyadenylation signal used.