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W S Dynan

Publications and source records attributed to W S Dynan.

At least 55 records · Page 3Linked to original sources
PubMed1990

Interaction of host cell proteins with the human T-cell leukemia virus type I transcriptional control region. II. A comprehensive map of protein-binding sites facilitates construction of a simple chimeric promoter responsive to the viral tax2 gene product.

We present a map describing the binding of cellular proteins to a 300-base pair (bp) region of the human T-cell leukemia virus type I (HTLV-I) long terminal repeat. The map accounts for nearly all of the DNase I protection reported in a previous study using crude nuclear extracts. Notable features include a complex arrangement of overlapping binding sites encompassing the 21-bp repeat elements (see accompanying paper) as well as binding sites for the transcription factors Sp1 and NF-I that significantly deviate from the previously defined consensus recognition sequences. Based on the binding results, we constructed simple chimeric promoters containing 21-bp repeat elements, Sp1-, and nuclear factor I-binding sites upstream of a TATA box. Transient transfection experiments show that these promoters are expressed in T-cells and are regulated by the viral tax2 gene product. Deletion of the Sp1 and nuclear factor I sites abolishes tax-induction, suggesting that one or both of these proteins play a role in mediating the tax-responsiveness conferred by the 21-bp repeat element.

Base Sequence↗
PubMed1990

Interaction of cellular proteins with the human T-cell leukemia virus type I transcriptional control region. Purification of cellular proteins that bind the 21-base pair repeat elements.

Nuclear extracts of cultured human cells contain multiple proteins that recognize specific sequence elements within the transcriptional control region of the human retrovirus, human T-cell leukemia virus type I (HTLV-I). Here we report the purification of host expression factor 1T (HEF-1T), a DNA-binding protein from human T-lymphocytes that binds to each of the three 21-base pair repeat enhancer elements in the proviral long terminal repeats of HTLV-I and the related virus, HTLV-II. HEF-1T is composed of two polypeptides of 41 and 59 kDa. We show that HEF-1T is distinct from a second protein, present in non-lymphoid cells, that binds to overlapping sites in and near the 21-base pair repeats. This second protein is composed of a single 47-kDa polypeptide and appears to be identical to the previously described transcription factor AP-2. A third protein, also distinct from HEF-1T, binds within the first repeat. The present results suggest that there may be multiple modes of HTLV-I promoter recognition involving distinct sets of cellular proteins.

Base Sequence↗
PubMed1990

Promoter evolution in BK virus: functional elements are created at sequence junctions.

The archetypal strain of BK virus directed very little early gene expression compared with rearranged laboratory strains of the virus. One laboratory strain that was analyzed in detail contained newly created AP-1 binding enhancer modules spanning the junction between adjacent sequence repeats. Introduction of these sequences into the archetype activated the previously quiescent early promoter.

BK Virus↗
PubMed1990

A downstream-element-binding factor facilitates assembly of a functional preinitiation complex at the simian virus 40 major late promoter.

Recent work has shown that many promoters recognized by eucaryotic RNA polymerase II contain essential sequences located downstream of the transcriptional initiation site. We show here that the activity of a promoter element centered 28 base pairs downstream of the simian virus 40 major late initiation site appears to be mediated by a DNA-binding protein, which was isolated by affinity chromatography from HeLa cell nuclear extracts. In the absence of the other components of the transcriptional machinery, the protein bound specifically but weakly to its recognition sequence, with a Kd of approximately 10(-8) M. Analysis of kinetic data showed that mutation of the downstream element decreased the number of functional preinitiation complexes assembled at the promoter without significantly altering the time required for half the complexes to assemble. This suggests that in the absence of the downstream activating protein, preinitiation complexes are at least partially assembled but are not transcriptionally competent.

Base Sequence↗
PubMed1989

Measurement of the binding of transcription factor Sp1 to a single GC box recognition sequence.

The equilibrium constant was determined for the binding of the transcription factor Sp1 to a single consensus GC box DNA recognition site, (5'-GGGGCGGGGC-3'). For these experiments, single copies of the recognition site were synthesized and cloned in a standard plasmid background. Binding was measured either by a footprinting assay modified so that the binding reaction was at equilibrium, or by a gel mobility shift assay. The concentration of active Sp1 in the reactions and the dissociation constant were determined by computer-assisted fitting to theoretical curves. Values for the dissociation constant obtained in different experiments ranged from 4.1 X 10(-10) M to 5.3 X 10(-10) M. Several variants of the consensus recognition site were also tested. An A-substituted variant (5'-GGGGAGGGGC-3') and a T-substituted variant (5'-GGGGTGGGGC-3') were bound 3-fold and 6-fold more weakly than the consensus site, respectively. A G-substituted variant (5'-GGGGGGGGGC-3') was bound at least 30-fold more weakly than the consensus site. These findings help distinguish between alternative models for Sp1-DNA recognition. They are consistent with the presence of specific hydrogen-bond contacts between Sp1 and the central C-G base pair, but provide no particular evidence to support a model where local DNA structure is the dominant factor in the interaction.

Base Composition↗
PubMed1989

Promoter-dependent transcription by RNA polymerase II using immobilized enzyme complexes.

DNA fragments containing the adenovirus 2 major late or simian virus 40 early promoters were attached to a solid support via a biotin-streptavidin linkage at one end of the fragment, upstream of the RNA start site. Templates immobilized in this manner were incubated with HeLa cell nuclear extracts to form preinitiation complexes containing RNA polymerase II and accessory proteins required for faithful in vitro transcription. These complexes did not require ATP or dATP for assembly, were sensitive to 0.25% Sarkosyl, and were stable to extensive washing. Their incubation with specific combinations of nucleoside triphosphates resulted in the initiation of RNA chain polymerization in situ, while addition of the remaining nucleoside triphosphates was necessary to produce a full length runoff RNA. Transcriptional activity associated with preinitiation complexes was purified approximately 300-fold, relative to the unfractionated nuclear extract. The use of immobilized template permits considerable flexibility in experimental design, as substrates and inhibitors can be added and washed out of the reaction at each step. We exploited this property of the system to dissect the temporal substrate requirements for initiation of RNA synthesis. It is known from prior work that at least one step in the promoter-dependent RNA synthesis reaction requires an adenosine nucleotide that is hydrolyzable at the beta, gamma-position. This requirement is independent of the initiating nucleotide and can be satisfied by dATP, which is not ordinarily incorporated into the RNA product. We show here that the beta, gamma-hydrolyzable adenosine nucleotide must be present simultaneously with the initiating nucleoside triphosphates. No reaction occurred when complexes were incubated with dATP, washed to remove dATP, and incubated subsequently with the two initiating nucleotides.

Adenine Nucleotides↗
PubMed1989

Characterization of a minimal simian virus 40 late promoter: enhancer elements in the 72-base-pair repeat not required.

A 272-base-pair (bp) portion of the simian virus 40 regulatory region containing the replication origin, Sp1-binding region, and part of the 72-bp direct repeats makes up a minimal late promoter that is able to direct late-direction RNA synthesis in vivo and in vitro. Fourteen linker-scan mutants within this region were characterized. Mutations in the Sp1-binding region decreased late expression both in vivo and in vitro. By contrast, mutations that eliminate genetically defined elements of the early transcriptional enhancer or that prevent binding of the transcription factors AP-1, AP-2, and AP-3 in the 72-bp repeat region had little or no effect on late-direction expression. These results argue that, at least under certain circumstances, the early transcriptional enhancer sequences are not required for simian virus 40 late gene expression.

Biological Evolution↗
PubMed1988

Binding of host-cell factors to DNA sequences in the long terminal repeat of human T-cell leukemia virus type I: implications for viral gene expression.

Efficient expression of human T-cell leukemia virus type I (HTLV-I) genes requires both host and viral proteins and is dependent on DNA sequences in the proviral long terminal repeats (LTRs). We have used DNase I-protection assays (footprinting) to construct a map of protein-DNA interactions over a 250-nucleotide region of the LTR upstream of the start site for viral RNA synthesis. We find that a host factor (host expression factor 1, or HEF-1) binds to the imperfect 21-nucleotide repeats that have previously been implicated in HTLV-I gene expression. HEF-1 binding activity is present in preparations from both lymphoid and nonlymphoid cell lines. However, the boundaries of the protected regions and the presence of a flanking DNase-hypersensitive site vary with cell type. Several regions of binding are detected in addition to the HEF-1 sites, including a complex group of sites 40-90 nucleotides upstream of the RNA start site. A comparison of HTLV-I-transformed T lymphocytes that do and do not express the viral trans-activating protein p40xI shows that none of the observed features of the DNase I footprint pattern correlate directly with the presence of this protein in the extract. These results suggest (i) that the primary recognition of promoter elements in the HTLV-I LTR involves specific interactions with host-cell proteins and (ii) that p40xI influences the activity of one or more of these proteins, rather than interacting directly with the DNA.

Binding Sites↗
PubMed1988

Binding of cellular proteins to the regulatory region of BK virus DNA.

The human papovavirus BK has a noncoding regulatory region located between the divergently transcribed early and late coding regions. Many strains of BK virus (BKV) have direct DNA sequence repeats in the regulatory region, although the number and extent of these repeats varies widely between independent isolates. Until recently, little was known about the individual functional elements within the BKV regulatory region, and the biological significance of the variable repeat structure has been unclear. To characterize the interaction between sequences in the BKV regulatory region and host cell transcription factors, we have carried out DNase I footprinting and competitive binding experiments on three strains of BKV, including one strain that does not contain direct sequence repeats. We have used relatively crude fractions from HeLa cell nuclear extracts, as well as DNA affinity-purified preparations of proteins. Our results demonstrate that BK(Dunlop), BK(WW), and BK(MM) each contain multiple binding sites for a factor, NF-BK, that is a member of the nuclear factor 1 family of transcription factors. We predict the presence of three to eight binding sites for NF-BK in the other strains of BKV for which a DNA sequence is available. This suggests that the binding of this protein is likely to be required for biological activity of the virus. In addition to NF-BK sites, BK(WW) and BK(MM) each contain a single binding site for transcription factor Sp1, and BK(Dunlop) contains two binding sites for transcription factor AP-1. The AP-1 sites in BK(Dunlop) span the junction of adjacent direct repeats, suggesting that repeat formation may be an important mechanism for de novo formation of binding sites not present in a parental strain.

BK Virus↗
PubMed1988

Simian virus 40 major late promoter: a novel tripartite structure that includes intragenic sequences.

Unlike most genes transcribed by RNA polymerase II, the simian virus 40 late transcription unit does not have a TATA box. To determine what sequences are required for initiation at the major late mRNA cap site of simian virus 40, clustered point mutations were constructed and tested for transcriptional activity in vitro and in vivo. Three promoter elements were defined. The first is centered 31 base pairs upstream of the cap site in a position normally reserved for a TATA box. The second is at the cap site. The third occupies a novel position centered 28 base pairs downstream of the cap site within a protein-coding sequence. The ability of RNA polymerase II to recognize this promoter suggests that there is greater variation in promoter architecture than had been believed previously.

Base Sequence↗
PubMed1985

Transcription factor Sp1 recognizes promoter sequences from the monkey genome that are simian virus 40 promoter.

A 440-base-pair fragment of African green monkey genomic DNA shares homology with the transcriptional regulatory region of simian virus 40 (SV40) and has been reported to direct transcription in vivo. We find that two regions within this fragment bind the promoter-specific cellular transcription factor Sp1 and are protected in DNase protection ("footprinting") experiments. As in SV40, binding occurs in regions containing multiple copies of the sequence GGGCGG. These regions, when fused to the proximal, or "TATA box," element of the herpes simplex virus thymidine kinase promoter, are able to direct Sp1-dependent transcription in vitro. The finding that Sp1 is capable of productive interaction with sequences taken from a cellular promoter supports the idea that Sp1 may play a role in modulating transcription of cellular genes.

Animals↗
PubMed1984

Affinity-purified antibody as a probe of RNA polymerase II subunit structure.

Antibodies reactive against mammalian RNA polymerase II were isolated by affinity chromatography. Serum from a goat immunized with Drosophila RNA polymerase II was passed over a column containing covalently coupled calf thymus RNA polymerase II, and reactive antibodies were eluted at low pH. The affinity-purified antibody was reactive against two large subunits (Mr = 140,000-220,000) and four small subunits (Mr = 16,000-35,000) of RNA polymerase II purified from calf, human, chicken, and Drosophila, as well as two large subunits of wheat germ RNA polymerase II. These findings confirm that RNA polymerase II subunits share conserved antigenic determinants over wide evolutionary distances. The affinity-purified antibody was also used to investigate the presence and structure of RNA polymerase II in human cell extracts capable of carrying out promoter-selective transcription in vitro. The subunit structure of RNA polymerase in the extract appears to be the same as that of purified human RNA polymerase II. In both cases, the human enzyme differs from the calf thymus enzyme in that it contains a large (Mr = 220,000) form of the largest subunit.

Animals↗
PubMed1983

Isolation of transcription factors that discriminate between different promoters recognized by RNA polymerase II.

A new fractionation procedure separates a whole-cell HeLa extract into three components required for accurate in vitro transcription. One component (Sp1) is a promoter-specific factor required for transcription of the SV40 early and late promoters, but not for transcription of other promoters we have tested. The second component (Sp2) is a general factor required for transcription of the SV40 promoters and a series of others, including the adeno-virus 2 major late promoter, the human beta-globin promoter and the avian sarcoma virus promoter. The third component is a fraction containing the endogenous RNA polymerase II. When SV40 and adenovirus templates were present simultaneously in an in vitro transcription reaction, addition of the Sp1 factor stimulated SV40 early promoter transcription 40-fold, and inhibited adenovirus major late promoter transcription by 40%. This finding suggests that Sp1 is involved in promoter selection, and is not merely a general transcription stimulatory factor.

DNA-Directed RNA Polymerases↗
PubMed1983

The promoter-specific transcription factor Sp1 binds to upstream sequences in the SV40 early promoter.

Fractionation of HeLa cell extracts reveals the presence of a promoter-specific transcription factor, Sp 1, which activates a class of promoters that includes the SV40 early promoter but not several others that have been tested. We analyzed SV40 early-promoter deletion mutants and determined that transcriptional activation by Sp 1 requires sequences within tandem 21 bp repeats located 70-110 bp upstream of the transcription initiation sites. In a DNAase footprinting assay, Sp 1 protected sequences in this same 21 bp repeat region, thus indicating the presence of a specific site for Sp 1 binding. During purification of Sp 1, there was a correlation between transcription-stimulatory activity and promoter-binding activity. These results suggest that direct binding of Sp 1 to sequences in the upstream promoter element is the mechanism by which this factor activates transcription by RNA polymerase II at the SV40 early promoter.

Binding Sites↗
PubMed1981

Purification and characterization of wheat germ DNA topoisomerase I (nicking-closing enzyme).

Wheat germ contains an enzyme capable of removing supercoils from circular DNA. We have purified this enzyme using Polymin P fractionation, ammonium sulfate precipitation, and chromatography on Bio-Rex 70 and phenyl-Sepharose. Renaturation after electrophoresis on sodium dodecyl sulfate-polyacrylamide gels shows that topoisomerase activity is associated with a polypeptide with a Mr = about 111,000. The enzyme is similar to other eukaryotic type I DNA topoisomerases (nicking-closing enzymes) by the following criteria: it is capable of increasing or decreasing the topological linking number of covalently closed DNA substrate; it is capable of restoring an equilibrium distribution of linking numbers to DNA substrate with a single unique linking number; and it does not require magnesium ion or ATP for activity.

Cations, Divalent↗
PubMed1981

In vitro transcription by wheat germ RNA polymerase II. Initiation of RNA synthesis on relaxed, closed circular template.

We have developed a novel method for studying the ability to wheat germ RNA polymerase II to initiate transcription in vitro on unnicked duplex DNA template. Our template is a plasmid which has been relaxed in vitro with wheat germ DNA topoisomerase I. The plasmid consists of SV40 cloned in pBR322. We transcribe briefly to synthesize highly labeled nascent RNA, then fractionate the resulting ternary complexes on an agarose gel to separate relaxed, closed circular DNA from other DNA species contaminating the template preparation. Under optimal conditions, we recover about half of the total RNA synthesized in complex with relaxed, closed circular DNA. The remaining RNA is in association with other DNA species or is free in solution. The principal conclusions we draw from these studies are: 1) relaxed, closed circular DNA can serve as a template for in vitro transcription, although the relative efficiencies of transcription are such that even a few per cent contamination with nicked or supercoiled template can cause transcription from these forms to dominate the reaction. 2) Initiation occurs on approximately 1 in 25 cloned circular molecules in the course of a 5-min reaction, under optimal conditions. This transcription is not predominantly from SV40 in vivo promoters and is either random or from a sufficient number of sites as to appear random. 3) There is a striking dependence of initiation frequency on purine nucleoside triphosphate concentration, especially with GTP.

DNA Topoisomerases, Type I↗