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N C Jones

Publications and source records attributed to N C Jones.

At least 55 records · Page 3Linked to original sources
PubMed1988

Rapid turnover of adenovirus E1A is determined through a co-translational mechanism that requires an aminoterminal domain.

The product of the adenovirus E1A 13S mRNA can both stimulate and repress the expression of certain viral and cellular genes. As with several other regulatory proteins, E1A has a short half-life, approximately 40 min. Although this short half-life is observed in cells expressing the E1A gene, it is not the case with cells injected with E1A protein, where its half-life is very long, generally greater than 15 h. We have sought to reconcile these apparent differences in E1A stability. Using Xenopus oocytes, we find that E1A exhibits its characteristic short half-life when it is synthesized from injected mRNA while it has a very long half-life when it is injected as a protein synthesized originally in Escherichia coli or reticulocyte lysates. In order to delineate the amino acids responsible for rapid E1A turnover, several deletion mRNAs were constructed, injected into oocytes, and E1A half-life determined. Carboxyl-terminal deletions and an internal deletion of residues 38-86 failed to increase the half-life of E1A. In contrast, amino-terminal deletions of 70 and 14 residues resulted in very stable E1A proteins (t1/2 greater than 20 h). Furthermore, deletion of the second amino acid, an arginine, resulted in a stable E1A protein. The amino-terminal region of E1A was able to induce the rapid turnover of a normally stable protein, beta-globin, in oocytes injected with an E1A-globin chimeric mRNA. This E1A-induced instability of globin was abolished, however, when the protein was first synthesized in reticulocyte lysates and then injected into oocytes. The amino-terminal region of E1A is also important in governing halflife in adenovirus-infected HeLa cells. These results demonstrate that the half-life of E1A is established cotranslationally through a mechanism involving sequences within the amino-terminal 37 residues.

Adenovirus Early Proteins↗
PubMed1988

Heterogeneity of adenovirus type 5 E1A proteins: multiple serine phosphorylations induce slow-migrating electrophoretic variants but do not affect E1A-induced transcriptional activation or transformation.

The 289-amino-acid product encoded by the adenovirus E1A 13S mRNA has several pleiotropic activities, including transcriptional activation, transcriptional repression, and when acting in concert with certain oncogene products, cell transformation. In all cell types in which E1A has been introduced (except bacteria), E1A protein is extensively posttranslationally modified to yield several isoelectric and molecular weight variants. The most striking variant is one that has a retarded mobility, by about Mr = 2,000, in sodium dodecyl sulfate gels. We have investigated the nature of this modification and have assessed its importance for E1A activity. Phosphorylation is responsible for the altered mobility of E1A, since acid phosphatase treatment eliminates the higher apparent molecular weight products. By using several E1A deletion mutants, we show that at least two seryl residues, residing between residues 86 and 120 and 224 and 289, are the sites of phosphorylation and that each phosphorylation can independently induce the mobility shift. However, E1A mutants lacking these seryl residues transcriptionally activate the adenovirus E3 and E2A promoters and transform baby rat kidney cells to near wild-type levels.

Adenovirus Early Proteins↗
PubMed1988

Polyoma middle T abrogates TPA requirement of murine melanocytes and induces malignant melanoma.

We have transfected the polyoma middle T oncogene into an immortal murine melanocyte cell line, Mel-ab. This highly pigmented line is phorbol ester (TPA) dependent for in vitro growth, suggesting activation and/or down regulation of Protein Kinase C (PKC) is essential for mitogenesis. Moreover, cells of this line do not form tumours when injected subcutaneously into immunocompetent or immunoincompetent mice. Here we show that PyMT alone is sufficient to produce TPA-independence and transformation to the tumourigenic state in transfected Mel-ab cells. Western blot analysis shows that middle T overcomes the TPA requirement by a mechanism independent of PKC down regulation though this does appear to occur when Mel-ab cells are grown continuously in TPA. These results suggest that PyMT is not exerting its transforming effect by PKC down regulation, but conceivably at some later stage of second messenger signalling, possibly through PyMT-c-src protein kinase activity.

Animals↗
PubMed1987

Mutational analysis of the adenovirus E1a gene: the role of transcriptional regulation in transformation.

To determine whether the transcription regulatory activities of the adenoviral E1a gene play a role in its ability to transform primary cells we have constructed an extensive series of mutations within the E1a gene. The mutants have been characterized for their ability to transactivate the adenoviral early promoters, repress the transcriptional stimulation of the polyoma virus enhancer, establish primary baby rat kidney cells in culture and cooperate with the activated Ha-ras oncogene in morphologically transforming these cells. The mutant phenotypes reveal that: (i) the two transcription regulatory activities of E1a are separable since essential protein domains map within different regions of the protein; (ii) transactivation is unlikely to contribute significantly to E1a-mediated transformation since several isolated mutants lost the ability to transactivate but were nevertheless efficient at transformation; and (iii) both establishment and oncogene cooperation are linked to enhancer repression suggesting that E1a transforms cells by the repression of a cellular enhancer.

Adenoviridae↗
PubMed1987

A cellular protein, activating transcription factor, activates transcription of multiple E1A-inducible adenovirus early promoters.

We have examined the relationship between sequence-specific DNA-binding proteins that activate transcription of E1A-inducible adenovirus early promoters. Factors previously referred to as E4F1 and E2A-EF bind to the E4 and E2A promoters, respectively. We demonstrate here that E4F1 and E2A-EF have identical DNA-binding specificity. Moreover, E4F1 and E2A-EF both activate transcription of the E4 and E2A promoters in vitro. These findings demonstrate that E4F1 and E2A-EF are the same factor, which we have designated activating transcription factor, or ATF. In addition to the E4 and E2A promoters, ATF binds to an important functional element of the E1A-inducible E3 promoter. Interaction of a common activator protein, ATF, with multiple E1A-inducible early viral promoters, suggests a significant role for ATF in E1A-mediated transcriptional activation.

Adenovirus Early Proteins↗
PubMed1987

Identification of factors that interact with the E1A-inducible adenovirus E3 promoter.

We have investigated the E1A-inducible E3 promoter of adenovirus type 5 with respect to its ability to bind specific nuclear proteins. Four distinct nucleoprotein-binding sites were detected, located between positions-7 to -33, -44 to -68, -81 to -103, and -154 to -183, relative to the E3 cap site. These sites contain sequences previously shown to be functionally important for efficient E3 transcription. No major qualitative or quantitative differences were found in the binding pattern between nucleoprotein extracts prepared from uninfected or adenovirus-infected HeLa cells. Competition experiments suggest that the factors binding to the -154 to -183 and -81 to -103 sites are the previously identified nucleoproteins, NF1 and AP1, respectively. The factor binding to the -44 to -68 site, which we term ATF, also interacts with other E1A-inducible promoters and is very similar and probably identical to the factor that binds to the cAMP-responsive element of somatostatin. We have purified this factor, which is a protein of 43 kD in size.

Adenovirus Early Proteins↗
PubMed1987

Adenovirus E1A requires synthesis of a cellular protein to establish a stable transcription complex in injected Xenopus laevis oocytes.

The Escherichia coli-expressed adenovirus E1A 13S mRNA product injected into Xenopus oocytes was active, as assessed by its ability to stimulate the transcription of an injected gene which is normally responsive to E1A in mammalian cells. In the presence of the protein synthesis inhibitors pactamycin or cycloheximide, E1A was correctly posttranslationally modified (phosphorylated) and transported to the nucleus; but it failed to stimulate the transcription of an injected gene containing the human heat shock protein 70 promoter. The basal (unstimulated) level of transcription of the gene was unaffected by these inhibitors. If oocytes were cultured in the presence of cycloheximide after E1A stimulated transcription, however, the high level of transcription was maintained for several hours without new protein synthesis. Results of competition studies with the same promoter (the heat shock protein 70 promoter) linked to two marked genes demonstrated that once the induction of transcription by E1A took place, the stimulated levels of transcription were maintained, even when they were challenged with excess competitor DNA. Results of these studies suggest that E1A requires the synthesis of a cellular protein to form a stable transcription complex.

Adenovirus Early Proteins↗
PubMed1986

The E1A 13S product of adenovirus 5 activates transcription of the cellular human HSP70 gene.

Expression of the human gene encoding the major heat shock protein, HSP70, was induced during cell growth by serum stimulation and after infection with adenovirus 5. In this study we showed that HSP70 gene expression could be induced by adenovirus 5 infection, even in the absence of exogenous serum factors. Whereas serum stimulation induced the expression of the endogenous HSP70 gene, it had no effect on early adenovirus promoters. However, expression of both the cellular HSP70 gene and the adenovirus E3 promoter were activated during adenovirus infection. By using a collection of reconstructed mutant viruses, we identified the 13S product of the E1A region as the specific transcriptional trans-activator of the HSP70 gene.

Adenoviruses, Human↗
PubMed1986

Use of tracheloscopy and directed biopsy for evaluating cervical involvement in endometrial carcinoma.

Accurate assessment of cervical involvement in endometrial carcinoma (stage II) is often difficult. The value of tracheloscopy as a possible method for improving accuracy was assessed. One hundred one patients with endometrial carcinoma were studied; 28 were suspected of having cervical involvement on endocervical curettage, and 26 were evaluable. Only five patients were documented to have cervical involvement using tracheloscopy. This finding suggests that there is general overtreatment of patients with false-positive endocervical curettage. However, a definitive statement concerning the incidence of stage II disease in the series cannot be made because many patients received traditional preoperative radiation therapy, which may have eradicated intracervical disease missed by tracheloscopy, therefore falsely confirming stage 1 disease on history from subsequent hysterectomy specimens.

Adult↗
PubMed1985

Adenovirus E3-early promoter: sequences required for activation by E1A.

To identify sequences within the adenovirus-5 E3 promoter necessary for E1A trans-activation, a series of promoter deletion mutants were constructed and analysed. A region between positions -82 and -105 was shown to be critical both for E1A induced expression as well as uninduced expression. The importance of this region was confirmed by constructing hybrid promoters consisting of E3 and Herpes simplex virus thymidine kinase sequences. The E1A insensitive tk promoter could be converted to an E1A sensitive promoter by replacing sequences upstream of position -79 with the corresponding region of the E3 promoter. This critical region of the E3 promoter contains a sequence 5' AGATGACTA3' which is also present in important upstream regions of the E2A and E4 promoters.

Adenoviruses, Human↗
PubMed1985

A first exon-encoded domain of E1A sufficient for posttranslational modification, nuclear-localization, and induction of adenovirus E3 promoter expression in Xenopus oocytes.

The purified Escherichia coli-expressed human subgroup C adenovirus E1A 13S mRNA product induces expression from the adenovirus type 5 E3 promoter when injected into Xenopus oocytes. In the present communication, the E. coli-expressed E1A 13S and 12S mRNA products are shown to undergo a posttranslational modification in microinjected Xenopus oocytes, which causes a 2- to 4-kDa increase in apparent molecular size, identical to that occurring in HeLa cells expressing the E1A gene. The E. coli-expressed E1A proteins are similarly modified in vitro in a soluble rabbit reticulocyte lysate. The modified form of the E1A proteins preferentially localizes to the oocyte nucleus following cytoplasmic microinjection. The use of various deleted forms of E1A protein synthesized in E. coli shows that a first exon-encoded domain of E1A, residing between amino acid residues 23 and 120, is sufficient for the posttranslational modification and nuclear localization of E1A and also for the trans-activation of the E3 promoter by E1A in Xenopus oocytes. These results suggest that the posttranslational modification of E1A protein may be important for its function.

Acetyltransferases↗
PubMed1984

Transformation properties of type 5 adenovirus mutants that differentially express the E1A gene products.

Type 5 adenovirus mutants that differentially express E1A 13S, 12S, or 9S mRNAs were constructed to study the role of their gene products in transformation. H5dl520 expresses the 243-amino-acid (AA) protein encoded in the 12S mRNA but not the 13S mRNA-encoded 289-AA protein. This mutant transformed both cloned rat embryo fibroblast (CREF) cells and baby rat kidney (BRK) cells at a frequency 40-100 times greater than did wild-type viruses. In addition, all of the foci produced were fibroblastic and grew very slowly at 32 degrees C. In contrast, H5dl21, which was mutated so that only the 54-AA protein encoded by the 9S mRNA was synthesized, did not transform either cell type. DNA transfection studies with plasmids from which these mutants were constructed demonstrated that the differences in transformation frequencies were not as marked. The plasmid pD1/D2, which directs the synthesis of the 54-AA protein only, was found to transform baby rat kidney cells at low frequency, provided the gene was linked to a fragment from the simian virus 40 genome containing the transcriptional enhancer element.

Adenoviruses, Human↗
PubMed1983

Regulation of adenovirus transcription by an E1a gene in microinjected Xenopus laevis oocytes.

The regulation of adenovirus type 5 gene expression by the E1a gene product was examined in microinjected Xenopus laevis oocytes. Chimeric genes were constructed which included the promoter region of early adenovirus type 5 gene 3 and the structural sequence which codes for the bacterial enzyme chloramphenicol-3-O-acetyltransferase (CAT). A plasmid containing this chimeric gene as well as plasmids containing the E1a gene were coinjected into oocyte nuclei. The presence of the E1a gene was shown to increase CAT activity by up to 8.5-fold over basal levels. Synthesis of the functional product from the E1a gene requires the removal of intron sequences by RNA splicing. The E1a gene and a derivative that precisely lacks the intron were equally effective in increasing CAT activity, suggesting that splicing of the primary E1a transcript is efficiently accomplished in the oocyte nucleus. This was confirmed by directly examining the E1a mRNAs by the S1 mapping procedure. A protein extract from adenovirus type 5-infected HeLa cells enriched for the E1a protein may supplant the E1a plasmid in enhancing CAT activity. Synthesis of the CAT enzyme after gene injection is invariant in oocytes from the same frog, but oocytes from different frogs show a high degree of variability in their ability to synthesize the CAT enzyme. Microinjected X. laevis oocytes appear to be an extremely useful system to study the effects of protein elements on transcription.

Acetyltransferases↗
PubMed1983

E1A control of gene expression is mediated by sequences 5' to the transcriptional starts of the early viral genes.

A product of the adenovirus E1A gene is a positive regulator of early viral gene expression. In this report we show that E1A regulates at the transcriptional level and that sequences located 5' to the early viral regions contain sites which confer regulation by the E1A gene product. We constructed chimeric genes in which the sequences at the 5' end of the E2A, E3, and E4 regions were fused to the structural sequences of either the herpes simplex virus thymidine kinase gene, the bacterial gene encoding the enzyme neomycin phosphotransferase, or the chloramphenicol acetyltransferase gene. In all cases, expression of the chimeric genes was induced by a product of the E1A region. It was also found that the insertion of a fragment from the left-hand end of the adenovirus type 5 genome into a plasmid harboring the thymidine kinase gene resulted in elevated frequencies of transformation of TK- cells to TK+. The elevated transformation frequencies were only detected when the insert and tk gene were covalently joined. This effect occurred even when the insert was several kilobase upstream from, and regardless of its orientation to, the transcriptional initiation site of the tk gene. We propose that this region of the adenovirus type 5 genome harbors a cis-acting enhancer of transcription.

Adenoviridae↗
PubMed1982

Stimulation of Xenopus oocyte protein synthesis by microinjected adenovirus RNA.

The ability of injected heterologous mRNAs to compete with endogenous mRNAs in Xenopus oocytes was assayed. In confirmation of previous reports, globin mRNA translation in oocytes results in a concomitant decrease in endogenous protein synthesis. In contrast, injection of adenovirus 5 mRNA into the oocyte results in a stimulation of endogenous protein synthesis. The stimulation is dose dependent and does not require nuclear transcription in the oocyte. Preliminary mapping data suggest that the stimulatory RNA is a product of one of the viral immediate early genes.

Adenoviruses, Human↗