Size heterogeneity in the 3′ end of dihydrofolate reductase messenger RNAs in mouse cells

Setzer, David R.; McGrogan, Michael; Nunberg, Jack H.; Schimke, Robert T.

doi:10.1016/0092-8674(80)90346-3

Cited by 296 publications

(139 citation statements)

References 38 publications

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“…Thus, S3-500 cells produced the same RNAs as do normal cells. Furthermore, the variation in 3' processing or polyadenylation, or both, observed for this gene in other amplified and nonamplified cell lines (10,29,37) was also observed in the S3-500 line.…”

Section: Resultsmentioning

confidence: 99%

Control of cellular gene expression during adenovirus infection: induction and shut-off of dihydrofolate reductase gene expression by adenovirus type 2.

et al. 1983

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Infection of human cells by adenovirus results in multiple alterations of host gene expression. To examine the effects of viral infection on the expression of a single gene, a line of human cells was developed which is resistant to growth in methotrexate and which contains amplified RNA and protein specific for dihydrofolate reductase (DHFR). Cytogenetic evidence indicated the presence of amplified DNA. Adenovirus infection of these cells caused an induction and subsequent decline in the synthesis of DHFR protein. The maximum DHFR induction occurred 16 to 19 h after infection and reached a level 2.5-fold greater than that observed in uninfected cells. Induction of DHFR protein synthesis was accompanied by concomitant increases in the level of steady-state DHFR-specific cytoplasmic RNA. The relative rate of DHFR mRNA production (i.e., the appearance of DHFR-specific mRNA sequences in the cytoplasm) also increased 2.5-fold during induction. Later in infection, the relative rate of DHFR protein synthesis declined, reaching a level below that observed in uninfected cells. This decline was accompanied by a similar decline in the steady-state levels of DHFR RNA and in the relative rate of synthesis of DHFR mRNA. These data suggest that adenovirus infection controls DHFR gene expression by increasing and subsequently decreasing the relative rate at which DHFR-specific mRNA sequences appear in the cytoplasm and enter the pool of mRNA available for translation.

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Section: Resultsmentioning

confidence: 99%

Control of cellular gene expression during adenovirus infection: induction and shut-off of dihydrofolate reductase gene expression by adenovirus type 2.

et al. 1983

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“…To address these questions, we have chosen to study the transcription unit for the mouse dihydrofolate reductase (dhfr) gene. The mouse dhfr transcription unit is particularly interesting from a structural standpoint in that it uses seven different polyadenylation [poly(A)] sites to produce seven polyadenylated mRNAs that vary by more than 20-fold in relative concentration; the four smallest mRNAs (0.75, 1.0, 1.2, 1.6 kilobases [kb]) are present in very high concentrations relative to the three largest mRNAs (2.0, 4.2, 5.6 kb) (23,24). The seven poly(A) sites used in the formation of these mRNAs are distributed throughout a 5-kb region of genomic DNA located at the 3' end of the structural gene.…”

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confidence: 99%

“…Transcription termination occurred within a discrete region (900 base pairs) located 1 kilobase beyond the last of seven functional polyadenylation sites and near a repetitive DNA sequence element. The results imply that a distinct genetic signal may be associated with the process of transcription termination.The 5' ends of mature mRNAs in higher eucaryotes correspond to sites of transcription initiation (7,20 (23,24). The seven poly(A) sites used in the formation of these mRNAs are distributed throughout a 5-kb region of genomic DNA located at the 3' end of the structural gene.…”

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confidence: 99%

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Transcription of the mouse dihydrofolate reductase gene proceeds unabated through seven polyadenylation sites and terminates near a region of repeated DNA.

Frayne¹,

Leys²,

Crouse³

et al. 1984

Mol. Cell. Biol.

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We observed equimolar transcription throughout the 35-kilobase mouse dihydrofolate reductase structural gene. Transcription termination occurred within a discrete region (900 base pairs) located 1 kilobase beyond the last of seven functional polyadenylation sites and near a repetitive DNA sequence element. The results imply that a distinct genetic signal may be associated with the process of transcription termination.The 5' ends of mature mRNAs in higher eucaryotes correspond to sites of transcription initiation (7,20 (23,24). The seven poly(A) sites used in the formation of these mRNAs are distributed throughout a 5-kb region of genomic DNA located at the 3' end of the structural gene. Therefore, to determine the number, location, and efficiency of transcription termination sites relative to the seven poly(A) sites within the dhfr transcription unit, we have measured the level of transcriptional activity throughout this region. These studies were made possible by the use of dhfr gene amplification mutants which permitted the detection of transcriptional activity from a gene expressed at a level too low to measure accurately in normal cells. In the S180-500R cell line (1, 11) used in the present study, the amplified unit extends over 10 kb 3' of the dhfr gene and is unaltered in this region relative to the S180 parental cell line; therefore, these cells serve as a suitable model for studying the dhfr transcription unit (6,8,22).

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“…200 nucleotides of untranslated region. A few other cases of mRNAs with large untranslated regions have been reported (18,24,29,30), but the functional significance of these tracts is not known. Some studies suggest that noncoding sequences can play a role in regulating mRNA levels (17,38).…”

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Xenopus fibrinogen: characterization of the mRNAs for the three subunits.

Holland¹,

Wangh²

1984

Mol. Cell. Biol.

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We purified and characterized the mRNAs coding for each of the three subunits of Xenopus fibrinogen. Purification was accomplished by electrophoretic separation of liver polyadenylated RNA in a fully denaturing gel, followed by recovery of the RNA from the gel via transfer to an ion-exchange membrane. This procedure yielded fractions which were highly enriched for the mRNAs for each of the fibrinogen chains. The fibrinogen mRNAs were identified by two methods: (i) in vitro translation followed by subunit-specific cleavage with the proteases thrombin and batroxobin; and (ii) cross-hybridization with cDNA clones for individual subunits of rat fibrinogen. The results demonstrate that the Aca and -y chains of frog fibrinogen are each coded by a single mRNA species. The Aca mRNA is ca. 3,100 nucleotides in length, which is nearly twice the minimum size required to code for the Aca precursor polypeptide. The y chain mRNA comprises about 1,600 bases and includes only a small untranslated region. In contrast, the BP subunit is synthesized from two mRNAs, one of which is 2,500 and the other 1,800 nucleotides long. The 2,500-base mRNA includes a large noncoding region, whereas the smaller one is near the minimum required size. The larger BP8 mRNA is ca. fivefold more abundant that the smaller species.Fibrinogen, which is synthesized and secreted by the liver, is found in the plasma of all vertebrates. The intact molecule comprises two sets of three nonidentical subunits designated Aax, BP, and -y. During the process of blood coagulation, the proteolytic enzyme thrombin cleaves the NH2-terminal A and B fibrinopeptides from the Aa and BP chains, thus generating fibrin which forms the structural matrix of a blood clot.We have previously described the polypeptide chain composition of fibrinogen from the frog Xenopus laevis (14,33 inducing the acute-phase response in mammals (16,19), was used in an effort to increase fibrinogen production in frogs. Polyadenylated RNA was purified (13) from the liver offrogs that had received a single injection of pure spirits of turpentine (Parks) into the leg muscle at a dose of 83 ,Il/50 g of body weight 12 to 24 h before sacrifice. This mRNA preparation was slightly enriched for the fibrinogen mRNAs relative to albumin mRNA, based on the in vitro translation assay, and was therefore used for the experiments described here. The sizes of all of the fibrinogen translation products and mRNAs were identical whether the RNA had been isolated from normal or turpentine-treated animals.To isolate the mRNAs coding for the Aa, BP, and -y chains of fibrinogen, liver mRNA was fractionated in a methylmercuric hydroxide agarose gel and recovered from the gel by transfer to and elution from a DEAE membrane (13). The high resolution of the RNA achieved during electrophoresis was preserved by slicing the DEAE membrane into 1-mm sections. The RNA eluted from each membrane slice was translated in vitro, and the products were resolved in a sodium dodecyl sulfate (SDS)-polyacrylamide gel (Fig. 1). All translated...

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Size heterogeneity in the 3′ end of dihydrofolate reductase messenger RNAs in mouse cells

Cited by 296 publications

References 38 publications

Control of cellular gene expression during adenovirus infection: induction and shut-off of dihydrofolate reductase gene expression by adenovirus type 2.

Control of cellular gene expression during adenovirus infection: induction and shut-off of dihydrofolate reductase gene expression by adenovirus type 2.

Transcription of the mouse dihydrofolate reductase gene proceeds unabated through seven polyadenylation sites and terminates near a region of repeated DNA.

Xenopus fibrinogen: characterization of the mRNAs for the three subunits.

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