Biomedical subjects

R Tjian

Publications and source records attributed to R Tjian.

At least 181 records · Page 10Linked to original sources

PubMed1985

Transcription of herpes simplex virus tk sequences under the control of wild-type and mutant human RNA polymerase I promoters.

We studied RNA polymerase I transcription in cells transfected with a plasmid, prHuTK, containing the herpes simplex virus tk gene fused to a human rRNA promoter. Primer extension analysis of tk RNA isolated from COS cells transfected with prHuTK reveals that transcription from the RNA polymerase I promoter is highly efficient and initiates at the same position used for the synthesis of endogenous rRNA in HeLa cells. The RNA products derived from prHuTK are distinguishable from normal RNA polymerase II transcripts of tk in that they are not polyadenylated, are extremely unstable, and are found predominantly in the nucleus. Moreover, the transcription observed is resistant to 300 micrograms of alpha-amanitin per ml. These results strongly suggest that prHuTK transcription is under the control of the human rRNA promoter and RNA polymerase I. To further characterize the activity of the human rDNA promoter in vivo, a series of 5' and 3' deletion mutants was tested in this transfection assay. The deletion analysis indicates that a core region of ca. 40 base pairs overlapping the initiation site is critical for transcription. In addition, a region between nucleotides -234 and -131 upstream from the core sequence serves to modulate the efficiency of transcription. Insertion into prHuTK of additional ribosomal nontranscribed spacer DNA or the simian virus 40 enhancer element has no apparent effect on the promoter activity. Surprisingly, RNA polymerase II transcripts synthesized at low levels from two start sites within the core control element of the wild-type RNA polymerase I promoter are activated upon deletion of upstream RNA polymerase I promoter sequences. However, these RNA polymerase II transcripts are not expressed from the endogenous rRNA promoter.

Amanitins↗

PubMed1985

Purification and characterization of a transcription factor that confers promoter specificity to human RNA polymerase I.

A whole-cell HeLa extract was fractionated into two components required for accurate in vitro transcription of human rRNA. One fraction contained endogenous RNA polymerase I, and the second component contained a factor (SL1) that confers promoter selectivity to RNA polymerase I. Analysis of mutant templates suggests that the core control element of the rRNA promoter is required for activation of transcription by SL1. We purified SL1 approximately 100,000-fold by column chromatography and have shown that the addition of SL1 can reprogram the otherwise nonpermissive mouse transcription system to recognize and initiate accurate RNA synthesis from human rDNA. Antibodies raised against SL1 bind preferentially to a protein localized in the nucleolus of primate cells and specifically inhibit in vitro transcription initiating from the human rRNA promoter. By contrast, anti-SL1 does not react with the nucleolus of rodent cells and has no effect on the in vitro synthesis of mouse rRNA by a transcription system derived from mouse cells. These findings suggest that SL1 is a selectivity factor present in the nucleolus that imparts promoter recognition to RNA polymerase I and that can discriminate between rRNA promoters from different species.

Animals↗

PubMed1984

Mutational analysis of simian virus 40 large T antigen DNA binding sites.

We have tested the effects of various mutations within SV40 T antigen DNA recognition sites I and II on specific T antigen binding using the DNase footprint technique. In addition, the replication of plasmid DNA templates carrying these T antigen binding site mutations was monitored by Southern analysis of transfected DNA in COS cells. Deletion mapping of site I sequences defined a central core of approximately 18 bp that is both necessary and sufficient for T antigen recognition; this region contains the site I contact nucleotides that were previously mapped using methylation-interference and methylation-protection experiments. A similar deletion analysis delineated sequences that impart specificity of binding to site II. We find that T antigen is capable of specific recognition of site II in the absence of site I sequences, indicating that binding to site II in vitro is not dependent on binding of T antigen at site I. Site II binding was not diminished by small deletion or substitution mutations that perturb the 27-bp palindrome central to binding site II, whereas extensive substitution of site II sequences completely eliminated specific site II binding. Analysis of the replication in COS7 cells of plasmids that contain these mutant origins revealed that sequences both at the late side of binding site I and within the site II palindrome are crucial for viral DNA replication, but are not involved in binding T antigen.

Antigens, Viral, Tumor↗

PubMed1984

Interaction of AD2+D2 protein and simian virus 40 large T antigen with the large tumor antigen binding site I.

In a lytic infection of a permissive host by SV40, the large tumor antigen (T antigen), which is a product of early transcription of the SV40 A gene, has been previously shown to autoregulate its own transcription by binding to SV40 DNA. The DNA region to which T antigen binds most tightly was synthesized and subsequently introduced into the bacterial plasmid pUC8. The interaction of SV40 T antigen with the DNA duplexes, derived from both chemical synthesis and the recombinant plasmid, were examined by using nitrocellulose filter binding assays. An SV40-adenovirus hybrid protein, AD2+D2 protein, was also tested. The SV40 T antigen was found to bind more tightly than the hybrid protein. Kinetic assays demonstrated that the association rates for the two proteins with the DNA binding site were equivalent; however, once formed, the T antigen-DNA complex dissociated more slowly than the AD2+D2 protein-DNA complex.

Antigens, Viral, Tumor↗

PubMed1984

Essential contact residues within SV40 large T antigen binding sites I and II identified by alkylation-interference.

Essential nucleotide contacts between the SV40 large T (tumor) antigen and binding sites I and II on the SV40 genome have been inferred from in vitro methylation- and ethylation-interference experiments. Each site contains two clusters of guanine residues that reduce the specific binding of T antigen when modified. Methylation at any one of nine guanines within site I or any one of five guanines within site II severely interferes with the interaction of T antigen with each respective site. Methylation at any one of a second group of five guanines within site II results in an appreciably weaker effect on the binding of T antigen. A similar inhibitory effect on binding is observed upon ethylation of adjacent phosphate residues. Although there are significant differences in the nucleotide sequence of the two binding sites, the pattern of protein contacts is strikingly similar between sites I and II. Three-dimensional projection reveals that the guanine contacts within each binding site are localized so that the specific binding interactions are accessible from only one face of the DNA helix.

Alkylation↗

PubMed1984

Human DNA sequences homologous to a protein coding region conserved between homeotic genes of Drosophila.

Several human DNA sequences were isolated by virtue of homology to a highly conserved region that has been identified in a number of homeotic genes in Drosophila. Structural analysis of the human DNAs indicate that two separate and distinct regions sharing a high degree of homology with the homeo box sequences of Drosophila are separated by only 5 kb in the human genome. Sequence determination of these regions reveals that both human DNA sequences contain a region capable of coding 61 amino acids, which shares greater than 90% homology with the peptide sequences specified by the homeo box domain of Drosophila homeotic genes, Antennapedia, fushi tarazu, and Ultrabithorax. By contrast, the human DNA sequences lying outside of the 190 nucleotide homeo box region share virtually no sequence homology, either with the flanking sequences of the other human clones or with flanking regions of the known Drosophila homeotic genes.

Animals↗

PubMed1984

Multiple control elements involved in the initiation of SV40 late transcription.

Mutants with deletions in the control region of simian virus 40 (SV40) were tested for their ability to direct late transcription in a nuclear extract derived from HeLa cells. Primer extension analysis revealed that late SV40 transcription initiates predominantly at two sites in vitro, one of which corresponds to the major in vivo start site at nucleotide 325, while the other site is located at nucleotide 170. A series of 5',3', and internal deletions of the putative promoter region were used to define two distinct control elements that appear to function independently of each other and that are located upstream from each of the in vitro initiation sites. In addition, transcription from the initiation site at 325 is also influenced by GC-rich sequences (CCGCCC) found within a regulatory region that consists of two 21 bp perfect repeats and a third degenerate repeat located 250 bp upstream from the major late initiation site. These six upstream GC blocks, which lie directly upstream from the initiation site at 170, also affect transcription from this start site. Although these 21 bp repeats are known to be an important part of the SV40 early promoter, our findings suggest that they are also involved in modulating the levels of late transcription in vitro.

Base Sequence↗

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↗

PubMed1983

SV40 T antigen binding site mutations that affect autoregulation.

Deletion and substitution mutations in the control region of simian virus 40 (SV40) were used to study regulation of early transcription by T antigen. A mutant, pIN4, containing substitutions within T antigen binding site II, is transcribed in a cell-free extract at wild-type efficiency but is unable to be repressed in vitro by purified T antigen under conditions that fully repress wild-type transcription. These results suggest a functional role for T antigen binding site II in the repression of early SV40 transcription. To investigate autoregulation in vivo, transcription from the SV40 early promoter was quantitated in COS7 monkey cells transfected with plasmid vectors carrying the mouse dihydrofolate reductase gene (SV-dhfr vectors). Mutant SV-dhfr vectors lacking T antigen site I or site II sequences overproduce dhfr RNA from the SV40 early promoter three to four fold, whereas deletion of both sites I and II or the presence of a temperature-sensitive T antigen (tsA209) results in an eight to ten fold increase in dhfr RNA. Our results indicate that binding of T antigen to both sites I and II plays a role in autoregulation.

Animals↗

PubMed1983

Translational control of SV40 T antigen expressed from the adenovirus late promoter.

We have constructed four novel adenovirus-SV40 hybrid viruses that contain the SV40 A gene at different positions downstream from the adenoviral major late promoter, within the region that encodes the second and third segments of the late tripartite RNA leader. The SV40 insert was precisely positioned in preselected regions of the adenoviral genome by using a combination of in vitro and in vivo recombination. As expected, all four recombinants produce equally high levels of SV40-encoded RNA that initiates at the adenovirus late promoter and contains two or three leader segments at the 5' end. Yet, in spite of this efficient transcription, only one virus, Ad-SVR284, directs the synthesis of high levels of SV40 large T antigen in infected cells; the other recombinants all produce approximately 20-fold less T antigen. This differential expression is, however, not seen in vitro, where equal amounts of hybrid T mRNA direct the synthesis of equal amounts of SV40 T antigen. Thus, some form of translational regulation is present in adenovirus-infected cells that is missing from the in vitro translation reaction.

Adenoviridae↗

PubMed1983

Regulation of human ribosomal RNA transcription.

We have used a cell-free polymerase I transcription system derived from HeLa cells to study the regulation of human rRNA synthesis. Analysis of deletion mutants spanning the start site of transcription at nucleotide +1 indicates that the control region affecting initiation of human rRNA synthesis is contained within sequences from nucleotides -158 to +18. This promoter region can be subdivided into (i) a central segment of approximately 40 base pair that is required for transcription and (ii) flanking sequences that influence the efficiency of transcription in vitro. We have examined the in vitro transcriptional activity of the human extract under various conditions that are thought to modulate rRNA synthesis in vivo. Cell-free extracts prepared from HeLa cells infected with adenovirus 2 synthesize human rRNA at levels greatly decreased relative to uninfected cell extracts. By contrast, in vitro transcription of human rRNA is stimulated 2- to 3-fold by the addition of purified simian virus 40 large tumor antigen to the transcription reaction. Moreover, a mutant tumor antigen known to be defective for rRNA activation in vivo is incapable of stimulating rRNA synthesis in vitro. The ability to detect these different regulatory phenomena in vitro provides us with an experimental basis for investigating the molecular mechanisms that control rRNA synthesis.

Cell Transformation, Neoplastic↗

PubMed1983

Topography of simian virus 40 A protein-DNA complexes: arrangement of pentanucleotide interaction sites at the origin of replication.

Investigation of the DNA binding properties of the simian virus 40 (SV40) A protein (large T antigen) and the hybrid adenovirus-SV40 D2 protein revealed that both viral proteins protect similar regions of SV40 DNA from digestion by DNase I or methylation by dimethyl sulfate. However, the interaction of D2 protein with DNA was more sensitive to increases of NaCl concentration than was the interaction of wild-type SV40 A protein. Dimethylsulfate footprinting identified 13 DNA pentanucleotide contact sites at the viral origin of replication. The sequences of these sites corresponded to the consensus family 5'-(G greater than T) (A greater than G)GGC-3'. The pentanucleotides were distributed in three regions of origin DNA. Region I contained three pentanucleotide contact sites arranged as direct repetitions encompassing a span of 23 base pairs. In region II, four pentanucleotides were oriented as inverted repetitions that also spanned a total of 23 base pairs. Region III had six recognition pentanucleotides arranged as direct repetitions in a space of 59 base pairs. These fundamental variations in DNA arrangement are likely to determine different patterns of protein binding in each region.

Antigens, Viral↗

PubMed1983

Biochemical activities of T-antigen proteins encoded by simian virus 40 A gene deletion mutants.

We have analyzed T antigens produced by a set of simian virus 40 (SV40) A gene deletion mutants for ATPase activity and for binding to the SV40 origin of DNA replication. Virus stocks of nonviable SV40 A gene deletion mutants were established in SV40-transformed monkey COS cells. Mutant T antigens were produced in mutant virus-infected CV1 cells. The structures of the mutant T antigens were characterized by immunoprecipitation with monoclonal antibodies directed against distinct regions of the T-antigen molecule. T antigens in crude extracts prepared from cells infected with 10 different mutants were immobilized on polyacrylamide beads with monoclonal antibodies, quantified by Coomassie blue staining, and then assayed directly for T antigen-specific ATPase activity and for binding to the SV40 origin of DNA replication. Our results indicate that the T antigen coding sequences required for origin binding map between 0.54 and 0.35 map units on the SV40 genome. In contrast, sequences closer to the C terminus of T antigen (between 0.24 and 0.20 map units) are required for ATPase activity. The presence of the ATPase activity correlated closely with the ability of the mutant viruses to replicate and to transform nonpermissive cells. The origin binding activity was retained, however, by three mutants that lacked these two functions, indicating that this activity is not sufficient to support either cellular transformation or viral replication. Neither the ATPase activity nor the origin binding activity correlated with the ability of the mutant DNA to activate silent rRNA genes or host cell DNA synthesis.

Adenosine Triphosphatases↗

PubMed1982

Construction of adenovirus expression vectors by site-directed in vivo recombination.

We developed a method for conveniently positioning foreign DNA at many preselected sites in the adenoviral genome by a combination of in vitro and in vivo recombination. Using this technique, we constructed a set of recombinant viruses that contain the SV40 A gene downstream from the adenovirus tripartite leader. One of these hybrid viruses, Ad-SVR26, contains the A gene close to and downstream from both the major late promoter and the first segment of the tripartite leader. The transcripts encoded by the inserted SV40 DNA are highly overproduced in infected cells; they initiate at the adenoviral late promoter and terminate at the SV40 polyadenylation site. Several novel splice acceptor sites in the SV40 sequences are used in the processing of the primary transcript to produce six different species of spliced RNA. The synthesis of T antigen in Ad-SVR26-infected cells requires the use of novel AUG initiation codons present within the SV40 coding region or adenoviral sequences that normally form part of the intron between the first and second segments of the tripartite leader. The level of T antigen expression is not as high as the level of mRNA production. The usage of these new AUG triplets or the absence of the complete adenovirus tripartite leader sequence may account for the low efficiency of translation.

Adenoviruses, Human↗

PubMed1982

In vitro transcription of human ribosomal RNA genes by RNA polymerase I.

We have studied the initiation of human ribosomal RNA synthesis in vitro using a cell-free polymerase I transcription system derived from HeLa cells and cloned human ribosomal DNA containing the site of initiation for ribosomal RNA synthesis. Mapping of the RNA products by run-off assays and high-resolution S1 nuclease analysis indicated that transcription in vitro initiates at a unique site and that the RNA has the same 5' terminus as in vivo precursor ribosomal RNA isolated from nuclei of HeLa cells. To provide additional evidence for the initiation site of ribosomal RNA transcription, dinucleoside monophosphates complementary to a sequence on the template have been used as primers for ribosomal RNA synthesis. When the concentrations of the four nucleoside triphosphates in the transcription reaction were reduced to 10 microM, [alpha-32 P]GTP was no longer incorporated into the run-off transcript. Under these conditions, we then tested a variety of dinucleotides for their ability to initiate promoter-specific RNA synthesis, and found that GpC exhibited maximum stimulation. We have also determined that the human cell-free system exhibits a significant degree of template specificity and is able to transcribe ribosomal DNA derived from human and rhesus monkey cells but not from mouse cells.

Cell-Free System↗

PubMed1981

Use of monoclonal antibodies as probes of simian virus 40 T antigen ATPase activity.

We have investigated the ATPase activity of simian virus 40 (SV40) large T antigen by using monoclonal antibodies as specific probes of enzymatic activity. Three hybridoma cell lines secreting anti-T antigen antibodies were derived from mice that were immunized with D2 T antigen, an SV40 T antigen-related protein. Monoclonal antibodies secreted by these hybridomas bind to three distinct T-antigen determinants. In order to bind to T antigen, the three antibodies required amino acid residues coded by the region of the A gene between 0.37 and 0.29 map unit. Two of these antibodies (DL3C3 and DL3C4) strongly inhibited T antigen ATPase activity. The third antibody (DL3C5) only weakly inhibited the ATPase activity possibly by decreasing the affinity of T antigen for ATP. These results demonstrate that the ATPase activity is intrinsic to T antigen and suggest that the ATPase function of T antigen maps on the SV40 A gene between 0.37 and 0.29 map unit. A T antigen-specific ATPase assay capable of detecting low levels of T antigen in crude extracts of SV40 infected cells was developed by using 3C5 to immobilize an active form of the enzyme. These results indicate that monoclonal antibodies can be used as probes of enzyme structure and function.

Adenosine Triphosphatases↗