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B Wasylyk

Publications and source records attributed to B Wasylyk.

At least 91 records · Page 5Linked to original sources
PubMed1986

The immunoglobulin heavy chain enhancer is stimulated by the adenovirus type 2 E1A products in mouse fibroblasts.

In contrast with our previous results (Hen, R., Borrelli, E. & Chambon, P. (1985) Science 230, 1391-1394), which demonstrated that the mouse immunoglobulin heavy chain transcriptional enhancer is repressed in lymphoid cells by the products of the adenovirus type 2 E1A transcription unit, we show here that these products activate the same enhancer in mouse fibroblast L cell lines that contain stably integrated copies of a recombinant in which the enhancer is inserted upstream from the chicken conalbumin promoter. In addition, competition experiments suggest that the activity of the heavy chain enhancer may be repressed by a trans-acting factor in mouse L cells. We speculate that the E1A products may prevent the action of this cellular repressor in these cells.

Adenovirus Early Proteins↗
PubMed1984

The MLV and SV40 enhancers have a similar pattern of transcriptional activation.

Activation of transcription by the Moloney Murine Leukemia Virus (MLV) and Simian Virus (SV40) enhancers was compared by transfecting recombinants containing these enhancers in either mouse or human cell-lines, and analysing RNA 48 h later by quantitative S1 nuclease mapping. The enhancers share the following properties. They stimulate transcription in an orientation-independent manner from the same startsites on the natural heterologous conalbumin (+62 to -102) or SV40 early promoter elements as well as on substitute promoter elements. The enhancers are most efficient when they are located directly upstream from the conalbumin (+62 to -102) promoter element, but they still stimulate transcription when they are either immediately downstream from the promoter element, or further upstream. Increasing the distance by interposing DNA sequences between the enhancers and the conalbumin promoter fragment results in decreased activation. Both enhancers show some cell-line specificity for activation of transcription. However, in all cell-lines and constructions tested the MLV enhancer was always less efficient than the SV40 enhancer. These results suggest that the MLV and SV40 enhancers stimulate transcription by similar mechanisms.

Animals↗
PubMed1984

Short and long range activation by the SV40 enhancer.

Activation of transcription by the SV40 enhancer decreases in an apparently biphasic manner when DNA sequences are interposed between the SV40 enhancer and either the homologous SV40 early or the heterologous conalbumin promoter elements. With increasing lengths of short DNA fragments (up to about 150 bp) activation of transcription decreases to less than 10% of the maximum. This short range effect is observed for both the SV40 early and conalbumin promoter elements and for either orientation of the SV40 enhancer. With the conalbumin promoter, increasing the length of the interposing DNA to 275 bp decreases activation to approximately 4%. Larger inserts, of 650 or 3737 bp, lead to an activation of 0.5%. However, in these recombinants, transcription is still activated at least 10 fold compared to an enhancerless recombinant. The implication of these results is discussed.

Chromosome Mapping↗
PubMed1984

Stimulation of in vitro transcription from heterologous promoters by the simian virus 40 enhancer.

Insertion of the simian virus 40 enhancer upstream from the +33 to -34 adenovirus major late promoter element or the +62 to -102 conalbumin promoter region causes a 10-fold stimulation of specific transcription using a whole cell extract but not an S100 extract. Many of the in vivo effects of the enhancer were mimicked in vitro. This stimulation occurred only in cis, with either orientation of the 72-base-pair repeat, and was markedly decreased by deletions known to diminish the enhancer activity in vivo. However, in vitro, the 72-base-pair repeat did not stimulate at a distance or in the presence of a strong upstream promoter element.

Adenoviruses, Human↗
PubMed1984

A novel eukaryotic promoter element: the simian virus 40 72 base pair repeat.

Using both clones of mouse LMTK- cells cotransformed with various chimeric conalbumin promoter simian virus 40 (SV40) early gene recombinants and the herpes thymidine kinase gene, and HeLa cells transfected with the same chimeric recombinants, we show that the SV40 72 base pair (bp) repeat sequence is a bidirectional potentiator of initiation of transcription from adjacent T-A-T-A box-dependent and -independent start sites. These results are consistent with our previous model based mainly on the results of T antigen gene expression assays that the 72-bp repeat acts as a bidirectional entry site for RNA polymerase B. We also show that the conalbumin T-A-T-A box is an important element for efficient and accurate in vivo initiation of transcription.

Conalbumin↗
PubMed1983

Transcription from the SV40 early-early and late-early overlapping promoters in the absence of DNA replication.

Transcription for a hybrid SV40 promoter-beta globin coding sequence recombinant initiates from both early-early (EE) and late-early (LE) SV40 start sites (EES and LES) in the absence of DNA replication. The 72-bp repeat is essential to potentiate the elements of the two overlapping EE and LE promoters (EEP and LEP). Two current models, which can account for the EE to LE shift in RNA chain initiation during the SV40 replication cycle, are that LE transcription is linked to replication and occurs on newly replicated DNA molecules or that there are two promoter elements, a stronger EEP and a weaker LEP, T antigen repressing the EEP late in infection. Our results support the second model. A 5'-TATTTAT-3' to 5'-TATCGAT-3' mutation in the putative SV40 TATA box decreases transcription from EES, increases transcription from LES, and inhibits DNA replication. Therefore, this element acts as a classical TATA box for transcription, and yet is also important for DNA replication.

Animals↗
PubMed1983

The SV40 72 bp repeat preferentially potentiates transcription starting from proximal natural or substitute promoter elements.

Activation of gene expression by the SV40 72 bp repeat was studied at the transcriptional level by quantitative S1 nuclease mapping of total RNA isolated from Hela cells transfected with chimeric conalbumin promoter-SV40 early gene recombinants. Our results demonstrate that, irrespective of its orientation, the 72 bp repeat is a potentiator of initiation of transcription from "TATA"-box-dependent and -independent "natural" or "substitute" promoter elements. In addition, we show that potential proximal promoter sequences are activated in preference to more distal ones. These results are consistent with the bidirectional entry site model for transcription activation by the 72 bp repeat.

Antigens, Viral↗
PubMed1981

The SV40 72 base repair repeat has a striking effect on gene expression both in SV40 and other chimeric recombinants.

By introduction of recombinant plasmids into monkey CV1 cells, we have unambiguously demonstrated that sequences entirely within the 72 bp repeat, which is located upstream of the SV40 early region, are crucial for T-antigen expression in vivo. We have also shown that a DNA fragment containing the 72 bp repeat, inserted directly before chicken conalbumin or adenovirus-2 major late promoter sequences in chimeric plasmids where these promoters replace that of the SV40 early genes, caused a dramatic increase in the expression of T-antigen in vivo. This effect was independent of the orientation of the 72 bp repeat, but was sensitive to its location within the plasmid, when the 72 bp repeat was separated from the promoter sequences, T-antigen expression was reduced. Insertion of the 72 bp repeat into equivalent plasmids containing no known eukaryotic promoter sequences (plasmids which were not detectably expressed in vivo) gave rise to a measurable, but smaller level of expression. The stimulation of expression by the 72 bp repeat is cis-acting : it required covalent linkage to the recombinant. We discuss the possibility that the 72 bp repeat region in SV40 may act as a bi-directional entry site for RNA polymerase B such that promoter sequences linked to the repeat are more efficiently utilised.

Animals↗
PubMed1981

A T to A base substitution and small deletions in the conalbumin TATA box drastically decrease specific in vitro transcription.

We have previously shown that a T to G transversion at the second T of the conalbumin "TATA" box drastically decreases specific initiation of transcription by RNA polymerase B (1). We now report that a T to A transcription at the same nucleotide also drastically decreases transcription, suggesting that these mutations influence transcription mainly by altering nucleotide recognition rather than helix stability. Small deletions of 2, 3 and 4 nucleotides in the "TATA" box also drastically decrease specific initiation of transcription.

Animals↗
PubMed1981

The isolation, mapping and transcription in vitro of a beta 0-thalassaemia globin gene.

The red blood cell precursors of a patient with homozygous beta 0-thalassaemia have previously been shown to contain nuclear, but not cytoplasmic, beta-globin-specific transcripts. We describe the isolation of a beta-globin gene from this patient as a recombinant bacteriophage chromosome. Restriction-enzyme cleavage-site mapping experiments demonstrate no detectable deletions, insertions or major rearrangements in this thalassaemia gene. Two different techniques show that the gene isolated is transcribed as efficiently in vitro as the normal beta-globin gene.

Base Sequence↗
PubMed1980

Promoter sequences of eukaryotic protein-coding genes.

In vitro genetic techniques were used to study the sequence requirements for the initiation of specific transcription. Deletion mutants were constructed around the putative promoter of the adenovirus-2 major late and chicken conalbumin genes. Specific transcription in vitro by RNA polymerase B together with a HeLa cell cytoplasmic extract was used as the test for promoter function. With this approach sequences which are essential for the initiation of specific transcription in vitro, were shown to be located between 12 and 32 base pairs upstream from the 5' end of these genes.

Base Sequence↗
PubMed1980

Specific in vitro initiation of transcription on conalbumin and ovalbumin genes and comparison with adenovirus-2 early and late genes.

Specific initiation of transcription of cloned conalbumin and ovalbumin genes has been obtained in a cell-free system in the presence of purified calf thymus RNA polymerase B. Comparison with the adenovirus-2 early (E1A) and major late genes reveals marked variations in transcriptional efficiencies. Specific fragmentation of the conalbumin gene DNA indicates that a short DNA segment 5' to the initiation site is involved in specific transcription.

Adenoviruses, Human↗
PubMed1980

Specific in vitro transcription of conalbumin gene is drastically decreased by single-point mutation in T-A-T-A box homology sequence.

A single-point mutation, consisting of a T-to-G transversion, was made in the third nucleotide of the conalbumin gene T-A-T-A-A-A-A homology sequence (the T-A-T-A or Goldberg-Hogness box). In an in vitro system, specific transcription of the mutant DNA was drastically decreased compared to the normal gene. This down-mutation is consistent with the idea that the T-A-T-A box is an important element for specific initiation of transcription.

Animals↗
PubMed1980

Studies on the mechanism of transcription of nucleosomal complexes.

The mechanism of transcription by Escherichia coli RNA polymerase holoenzyme of chromatin assembled in vitro has been studied by two approaches. Using digestion with endodeoxyribonuclease EcoRI as a probe of mobility, it was found that nucleosome movement is slow compared to the time taken for RNA polymerase to transcribe through regions organised into nucleosomes. However, transcription leads to at least some displacement of nucleosomes relative to their original site on the DNA. In the second approach chromatin was reconstituted from extensively crosslinked histone octamers and simian virus 40 DNA. RNA chain elongation on this template is inhibited relative to non-crosslinked chromatin. This can be related to a decrease in the ability of the cross-linked histone octamers to dissociate from the DNA.

Chromatin↗
PubMed1979

Preferential in vitro assembly of nucleosome cores on some AT-rich regions of SV40 DNA.

We have found that nucleosomes reconstituted from histone octamers and SV40 DNA Form I by progressively decreasing the salt concentration from 2 M NaCl are formed preferentially around 0.27, 0.37, 0.50 and 0.85 on SV40 DNA (relative to the EcoRI site). When SV40 DNA Form III is used, the nucleosomes form mainly at 0.28, 0.38, 0.61 and 0.83. These sites are very close to both the sites of RNA chain initiation by calf thymus RNA polymerase B on SV40 DNA Form I (0.25, 0.35, 0.42 and 0.88) and the regions of the supercoiled DNA which are readily denaturable by T4 gene 32 protein (0.25, 0.47 and 0.88), and correspond to AT-rich regions as deduced from the nucleotide sequence of SV40 DNA. The physiologically important region around 0.67 is an unfavourable site for all three types of proteins, and corresponds to a GC-rich region surrounding a 17 base pair AT cluster.

Base Composition↗