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H R Pelham

Publications and source records attributed to H R Pelham.

106 records · Page 6Linked to original sources
PubMed1982

A synthetic heat-shock promoter element confers heat-inducibility on the herpes simplex virus thymidine kinase gene.

Previous deletion analysis of the Drosophila hsp70 heat-shock promoter has identified a sequence upstream of the TATA box that is required for heat induction. This region contains homology to other heat-shock promoters, and it was proposed that the common sequence is an important element in the regulation of the heat-shock genes. We have constructed sequences similar to the consensus CT-GAA-TTC-AG from synthetic oligonucleotides and placed them upstream of the TATA box of the herpes virus thymidine kinase gene, in place of the normal upstream promoter element. The resultant genes are heat-inducible both in monkey COS cells and in Xenopus oocytes. We conclude that the transcriptional heat-shock response is mediated by some factor that interacts with this sequence.

Animals↗
PubMed1982

Expression of a Drosophila heat-shock protein in Xenopus oocytes: conserved and divergent regulatory signals.

On injection of cloned Drosophila hsp70 heat-shock genes into Xenopus oocytes, heat-inducible expression is observed: the level of correctly initiated transcripts is increased 20- to 100-fold on heat shock at 34 degrees C. We show that this induction is due to activation of the heat-shock gene promoter, and that the DNA sequences required for induction lie between 10 and 66 bases upstream from the transcription start site. Most heat-induced transcripts have a correct 3' end, and hsp70 mRNA activity is detectable after extraction of the RNA from oocytes and subsequent in vitro translation. Drosophila heat-shock protein (hsp70) is synthesised in the injected oocytes after heat-shock, but only at low temperature. Under heat-shock conditions, Drosophila hsp70 mRNA translation is reduced 10-fold as is translation of the normal (25 degrees C) mRNAs, whereas translation of the endogenous Xenopus hsp70 mRNA is strongly induced. Translation of Drosophila heat-shock mRNAs extracted from flies and injected into the oocytes is also reduced by heat-shock. This suggests that Xenopus oocytes do not recognise the translational regulatory signals of Drosophila heat-shock mRNAs. In contrast, the signals for heat-induced transcription must be strongly conserved between Xenopus and Drosophila.

Animals↗
PubMed1982

A regulatory upstream promoter element in the Drosophila hsp 70 heat-shock gene.

Deletion mutants of the Drosophila hsp 70 heat-shock gene have been assayed in COS cells using a vector that contains an SV40 replication origin. COS cells are SV40-transformed monkey cells that support high-level replication of the vector. S1 mapping of transcripts shows that the hsp 70 gene is heat-inducible in these cells, whereas the herpes virus thymidine kinase gene is not. Residues -10 to -66 of the hsp 70 gene are apparently sufficient for heat-inducible promotion, and residues between -47 and -66 are necessary. This region upstream of the TATA box contains sequence features common to other heat-shock genes. Expression of the hsp 70 gene can be forced at low temperature by SV40 sequences that include the 72 bp repeat, but only if these are present on the 5' side of the gene. It seems that the upstream element of the hsp 70 promoter is analogous to that of other promoters, but is only functional in heat-shocked cells.

Animals↗
PubMed1981

Related 5S RNA transcription factors in Xenopus oocytes and somatic cells.

Xenopus oocytes contain an abundant protein that binds specifically to the center of 5S RNA genes and directs their transcription by RNA polymerase III. This protein also binds to 5S RNA. We show here that transcription of cloned 5S RNA genes in extracts derived from Xenopus tissue culture cells is dependent on the intragenic control region and is inhibited by 5S RNA and by antibodies raised against the previously characterized oocyte transcription factor. Somatic cells contain a protein that is similar to the oocyte factor in charge, affinity for heparin-agarose, and antigenicity but has an apparent molecular mass about 2000 daltons greater than that of the oocyte protein. Our experiments strongly suggest that this larger protein is the transcription factor for 5S RNA genes in somatic cells. The 5S RNA may regulate its own synthesis in somatic cells by binding to this protein, which is present at a low concentration. The presence of two different proteins responsible for 5S RNA synthesis in oocytes and in somatic cells cannot by itself explain the developmental control of oocyte and somatic 5S RNA genes, because somatic cell extracts transcribe both types of gene.

Animals↗
PubMed1980

A specific transcription factor that can bind either the 5S RNA gene or 5S RNA.

5S ribosomal RNA specifically inhibits transcription of cloned repeating units of 5S DNA in a nuclear extract of Xenopus oocytes. The inhibition can be explained by the interaction of 5S RNA with a transcription factor that binds specifically to a control region located within the 5S RNA gene. This transcription factor is identical to an abundant cytoplasmic protein that is known to be complexed with 5S RNA in immature Xenopus oocytes. Thus the presence of large amounts of this protein in these cells can account for both the high rate of synthesis and the subsequent storage of 5S RNA to ribosome synthesis.

Animals↗
PubMed1978

Translation of encephalomyocarditis virus RNA in vitro yields an active proteolytic processing enzyme.

In contrast to other cell-free translation systems, the mRNA-dependent reticulocyte lysate can translate encephalomyocarditis virus RNA efficiently and completely when supplemented with heterologous tRNA. Cleavage of the nascent polypeptide chain occurs, and one of the translation products appears to be a specific proteolytic enzyme which correctly processes the primary products. The identity of the proteins made in vitro was verified by comparison with infected cell proteins on dodecylsulphate/polyacrylamide gels, and by mapping their coding sequences on the viral genome.

Animals↗
PubMed1978

Characteristics of a coupled cell-free transcription and translation system directed by vaccinia cores.

1. A coupled transcription and translation system is described in which protein synthesis is directed by mRNA synthesised in situ by vaccinia virus cores. The cell-free system is based on a micrococcal-nuclease-treated reticulocyte lysate. 2. The polypeptides made in vitro include many authentic early vaccinia proteins, but also other proteins which were not detected in infected cells. 3. Concentrations of cores which inhibit host cell protein synthesis in vivo caused a delayed inhibition of translation in vitro; this was partly, but not entirely, due to dsRNA associated with the cores. 4. The mRNA made was methylated by core enzymes. Inhibition of methylation reduced the rate of translation tenfold; unmethylated RNA bound ribosomes poorly, but was nevertheless translated faithfully.

Animals↗
PubMed1978

Heat inactivation of vaccinia virus particle-associated functions: properties of heated particles in vivo and in vitro.

The heat inactivation characteristics of several vaccinia virus particle-associated functions known to be involved in the transcription of the genome were examined. All functions were more resistant to heat than infectivity. Noninfectious particles were generated which exhibited significant levels of activity of all enzymes examined, and their properties were investigaed both in vitro and in vivo. RNA was synthesized in vitro by such particles, although transport of the RNA into the surrounding medium was defective. This RNA was larger than that made in normal particles but it was polyadenylated and functioned in vitro as a message coding for normal early proteins. The sequences transcribed were similar to those transcribed in normal particles, and we suggest that the production of abnormally large RNA is due to a defect in transcriptional termination. We could not detect any virus-specific protein or RNA synthesis in cells exposed to these inactivated particles and conclude that the loss of infectivity caused by heating is due to a general decline in the activities of a number of particle functions.

Cell Line↗
PubMed1976

An efficient mRNA-dependent translation system from reticulocyte lysates.

A simple method is described for converting a standard rabbit reticulocyte cell-free extract (lysate) into an mRNA-dependent protein synthesis system. The lysate is preincubated with CaCl2 and micrococcal nuclease, and then excess ethyleneglycol-bis(2-aminoethylether)-N,N'-tetraacetic acid is added to chelate the Ca2+ and inactivate the nuclease. Lysates treated in this way have neglibible endogenous amino acid incorporation activity, but 75% of the activity of the original lysate can be recovered by the addition of globin mRNA. The efficiency of utilisation of added mRNA and the sensitivity of the system are both very high. No residual nuclease activity could be detected, and the tRNA is functionally unimpaired. Several different species of mRNA have been shown to be translated efficiently into full-sized products of the expected molecular weight up to about 200000, and there is no detectable accumulation of incomplete protein products. The efficient translation of RNA from two plant viruses (tobacco mosaic virus and cowpea mosaic virus) required heterologous tRNA.

Animals↗
PubMedDate not supplied

Association of an S1 nuclease-sensitive structure with short direct repeats 5' of Drosophila heat shock genes.

The potential for a DNA molecule to assume different tertiary structures has been suggested as a possible determinant of transcriptional regulation in eukaryotes. Such forms include cruciforms transitions from B to Z DNA in the same molecule and short unpaired stretches of DNA in an alternating copolymer. All such structures contain regions which are preferentially sensitive to S1 nuclease. We report here an additional class of S1 nuclease sensitive sites associated with short direct repeats of DNA found in the 5' flanking regions of certain Drosophila melanogaster heat shock genes.

Chromosome Mapping↗
PubMedDate not supplied

Heat shock factor is regulated differently in yeast and HeLa cells.

When cells are exposed to elevated temperatures, transcription of a small set of genes, the heat-shock genes, is activated. This response is mediated by a short DNA sequence, the heat-shock element (HSE), which is thought to be the binding site for a specific transcription factor. Studies with Drosophila show that this protein binds to HSEs only in heat-shocked cells, suggesting that changes in factor binding are responsible for gene activation. We have investigated the properties of HSE-binding proteins from yeast and HeLa cells. In HeLa cells, binding activity is present only after heat shock. In contrast, control and heat-shocked yeast cells yield the same amount of HSE-binding activity; however, the mobility of protein-HSE complexes on polyacrylamide gels is altered following heat shock. This mobility difference can be significantly reduced by treatment of crude extracts with phosphatase. We propose that the yeast heat-shock factor binds constitutively to DNA but only activates transcription after heat-induced phosphorylation.

Gene Expression Regulation↗