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R Tjian

Publications and source records attributed to R Tjian.

At least 163 records · Page 9Linked to original sources
PubMed1986

Purification and biochemical characterization of the promoter-specific transcription factor, Sp1.

The biochemical analysis of cellular trans-activators involved in promoter recognition provides an important step toward understanding the mechanisms of gene expression in animal cells. The promoter selective transcription factor, Sp1, has been purified from human cells to more than 95 percent homogeneity by sequence-specific DNA affinity chromatography. Isolation and renaturation of proteins purified from sodium dodecyl sulfate polyacrylamide gels allowed the identification of two polypeptides (105 and 95 kilodaltons) as those responsible for recognizing and interacting specifically with the GC-box promoter elements characteristic of Sp1 binding sites.

Animals↗
PubMed1986

Binding of the Sp1 transcription factor by the human Harvey ras1 proto-oncogene promoter.

Members of the ras gene family encode proteins that when overproduced or mutated can transform immortalized mammalian cells. It is therefore important to understand the mechanisms by which the ras genes are regulated. The promoter region of the human Harvey ras proto-oncogene c-Ha-ras1 initiates RNA transcription at multiple sites and contains repeated copies of the hexanucleotide GGGCGG and its inverted complement CCGCCC, referred to as GC boxes. These GC boxes consist of sequences identical to those found in the SV40 early promoter, where the human cellular transcriptional factor Sp1 binds. Footprinting analysis with deoxyribonuclease I was used to show that Sp1 binds to six GC box sequences within the c-Ha-ras1 promoter. An in vivo transfection assay showed competition between the 21-base pair repeats of the SV40 promoter and the c-Ha-ras1 promoter for common regulatory factors. In this system the presence of Sp1 is apparently required for c-Ha-ras1 transcription. Analysis of deletions of the c-Ha-ras1 promoter region by means of a transient expression assay revealed that the three Sp1 binding sites closest to the RNA start sites were sufficient for full transcriptional activity.

Base Sequence↗
PubMed1986

Activation of the AIDS retrovirus promoter by the cellular transcription factor, Sp1.

The nature and position of transcriptional control elements responsible for the expression of genes encoded by the retrovirus associated with acquired immune deficiency syndrome (AIDS) have not been precisely defined. In this study it is shown that the mammalian Sp1 transcription factor binds to promoter sequences within the AIDS retrovirus long terminal repeat (LTR) and activates RNA synthesis five- to eightfold in reconstituted reactions in vitro. Experiments in which regions of DNA were protected from added reagents by specifically bound proteins (footprinting) indicated that the upstream promoter region of the AIDS virus LTR lies between -45 and -77 (relative to the RNA start site, +1) and contains three tandem, closely spaced SP1 binding sites of variable affinity. Base-substitution mutations targeted to one or all three Sp1 binding sites were found both to eliminate the binding of Sp1 and to cause up to a tenfold reduction in transcriptional efficiency in vitro. These findings suggest that one important component of the AIDS virus transcriptional control region interacts with a cellular transcription factor, Sp1, and that this factor must function in conjunction with transcriptional elements located downstream of the RNA cap site to mediate the response of the LTR to viral trans-activation.

Acquired Immunodeficiency Syndrome↗
PubMed1986

Affinity purification of sequence-specific DNA binding proteins.

We describe a method for affinity purification of sequence-specific DNA binding proteins that is fast and effective. Complementary chemically synthesized oligodeoxynucleotides that contain a recognition site for a sequence-specific DNA binding protein are annealed and ligated to give oligomers. This DNA is then covalently coupled to Sepharose CL-2B with cyanogen bromide to yield the affinity resin. A partially purified protein fraction is combined with competitor DNA and subsequently passed through the DNA-Sepharose resin. The desired sequence-specific DNA binding protein is purified because it preferentially binds to the recognition sites in the affinity resin rather than to the nonspecific competitor DNA in solution. For example, a protein fraction that is enriched for transcription factor Sp1 can be further purified 500- to 1000-fold by two sequential affinity chromatography steps to give Sp1 of an estimated 90% homogeneity with 30% yield. In addition, the use of tandem affinity columns containing different protein binding sites allows the simultaneous purification of multiple DNA binding proteins from the same extract. This method provides a means for the purification of rare sequence-specific DNA binding proteins, such as Sp1 and CAAT-binding transcription factor.

Base Sequence↗
PubMed1986

Properties of the simian virus 40 (SV40) large T antigens encoded by SV40 mutants with deletions in gene A.

The biochemical properties of the large T antigens encoded by simian virus 40 (SV40) mutants with deletions at DdeI sites in the SV40 A gene were determined. Mutant large T antigens containing only the first 138 to 140 amino acids were unable to bind to the SV40 origin of DNA replication as were large T antigens containing at their COOH termini 96 or 97 amino acids encoded by the long open reading frame located between 0.22 and 0.165 map units (m.u.). All other mutant large T antigens were able to bind to the SV40 origin of replication. Mutants with in-phase deletions at 0.288 and 0.243 m.u. lacked ATPase activity, but ATPase activity was normal in mutants lacking origin-binding activity. The 627-amino acid large T antigen encoded by dlA2465, with a deletion at 0.219 m.u., was the smallest large T antigen displaying ATPase activity. Mutant large T antigens with the alternate 96- or 97-amino acid COOH terminus also lacked ATPase activity. All mutant large T antigens were found in the nuclei of infected cells; a small amount of large T with the alternate COOH terminus was also located in the cytoplasm. Mutant dlA2465 belonged to the same class of mutants as dlA2459. It was unable to form plaques on CV-1p cells at 37 or 32 degrees C but could form plaques on BSC-1 monolayers at 37 degrees C but not at 32 degrees C. It was positive for viral DNA replication and showed intracistronic complementation with any group A mutant whose large T antigen contained a normal carboxyl terminus. These findings and those of others suggest that both DNA binding and ATPase activity are required for the viral DNA replication function of large T antigen, that these two activities must be located on the same T antigen monomer, and that these two activities are performed by distinct domains of the polypeptide. These domains are distinct and separable from the domain affected by the mutation of dlA2465 and indicate that SV40 large T antigen is made up of at least three separate functional domains.

Adenosine Triphosphatases↗
PubMed1986

Two distinct promoter elements in the human rRNA gene identified by linker scanning mutagenesis.

A cell-free RNA polymerase I transcription system was used to evaluate the transcription efficiency of 21 linker scanning mutations that span the human rRNA gene promoter. Our analysis revealed the presence of two major control elements, designated the core and upstream elements, that affect the level of transcription initiation. The core element extends from -45 to +18 relative to the RNA start site, and transcription is severely affected (up to 100-fold) by linker scanning mutations in this region. Linker scanning and deletion mutations in the upstream element, located between nucleotides -156 and -107, cause a three- to fivefold reduction in transcription. Under certain reaction conditions, such as the presence of a high ratio of protein to template or supplementation of the reaction with partially purified protein fractions, sequences upstream of the core element can have an even greater effect (20- to 50-fold) on RNA polymerase I transcription. Primer extension analysis showed that RNA synthesized from all of these mutant templates is initiated at the correct in vivo start site. To examine the functional relationship between the core and the upstream region, mutant promoters were constructed that alter the orientation, distance, or multiplicity of these control elements relative to each other. The upstream control element appears to function in only one orientation, and its position relative to the core is constrained within a fairly narrow region. Moreover, multiple core elements in close proximity to each other have an inhibitory effect on transcription.

Base Sequence↗
PubMed1986

T-antigen-DNA polymerase alpha complex implicated in simian virus 40 DNA replication.

We have combined in vitro DNA replication reactions and immunological techniques to analyze biochemical interactions between simian virus (SV40) large T antigen and components of the cellular replication apparatus. First, in vitro SV40 DNA replication was characterized with specific origin mutants. Next, monoclonal antibodies were used to demonstrate that a specific domain of T antigen formed a complex with cellular DNA polymerase alpha. Several antibodies were identified that coprecipitated T antigen and DNA polymerase alpha, while others were found to selectively prevent this interaction and concomitantly inhibit DNA replication. DNA polymerase alpha also bound efficiently to a T-antigen affinity column, confirming the immunoprecipitation results and providing a useful method for purification of the complete protein complex. Taken together, these results suggest that the T-antigen-polymerase association may be a key step in the initiation of SV40 DNA replication.

Antibodies, Monoclonal↗
PubMed1986

Downstream sequences affect transcription initiation from the adenovirus major late promoter.

We analyzed a set of adenovirus-simian virus 40 (SV40) hybrids in which the SV40 T antigen coding sequences are inserted downstream from the adenovirus major late promoter within the first, second, and third segments of the tripartite leader. In infected cells, these viruses give rise to a matched set of hybrid SV40 mRNAs that differ only in the number of tripartite leader segments attached to the complete SV40 T antigen coding region. We found that the number of tripartite leader segments present at the 5' end of the hybrid SV40 mRNAs had little effect on the efficiency of T antigen translation. Surprisingly, insertion of SV40 sequences within the first leader segment, at +33 relative to the start of transcription, significantly reduced the frequency of transcription initiation from the major late promoter. The 3' boundary of this downstream transcriptional control element was mapped between +33 and +190 by showing that insertion of SV40 sequences within the intron after the first leader segment at +190 had very little effect on transcription initiation from the late promoter. A transient expression assay was used to show that the effect of downstream sequences on transcription initiation from the major late promoter is dependent on a trans-acting factor encoded or induced by adenovirus.

Adenoviridae↗
PubMed1985

Bidirectional SV40 transcription mediated by tandem Sp1 binding interactions.

The 21-base pair repeat elements of the SV40 promoter contain six tandem copies of the GGGCGG hexanucleotide (GC-box), each of which can bind, with varying affinity, to the cellular transcription factor, Sp1. In vitro SV40 early RNA synthesis is mediated by interaction of Sp1 with GC-boxes I, II, and III, whereas transcription in the late direction is mediated by binding to GC-boxes III, V, and VI.

Autoradiography↗
PubMed1985

A novel strategy for constructing clustered point mutations.

We have modified the synthetic linker mutagenesis procedure (1,2) in order to facilitate both the construction and the analysis of deletions, insertions and clustered point mutations generated in DNA in vitro. The protocol as originally described by McKnight and Kingsbury (1) involved attaching a synthetic linker sequence to each 5' or 3' deletion endpoint in DNA. We have designed specific plasmid vectors that can be used to generate nested sets of deletion mutations in the DNA being analyzed. The utility of these vectors is that a linker sequence of choice can be inserted at the endpoint of a deletion in a single intramolecular ligation without the use of synthetic linker DNA. In a second modification of the original procedure, we have adopted a rapid method for sequencing supercoiled plasmid DNAs from 10 ml cultures by primer extension. The site-directed mutagenesis strategy outlined here is suited for studying regulatory regions of DNA, such as origins of DNA replication, transcriptional promoters, enhancer elements, and activator binding sites. We have used this rapid and efficient strategy to generate deletions, insertions, and clustered point mutations in the transcriptional control region of a gene encoding the major human ribosomal RNAs.

Base Sequence↗
PubMed1985

A mammalian host-vector system that regulates expression and amplification of transfected genes by temperature induction.

SV40-transformed simian cells that permit temperature-dependent regulation of vector DNA replication were isolated and characterized. These cell lines (ts COS cells) produce high levels of thermolabile large T antigen under the transcriptional control of the Rous sarcoma virus long terminal repeat. The ts COS cell lines can complement SV40 A gene mutants and support replication of SV40-origin containing vectors at 33 degrees C but not at 40 degrees C. It should now be possible to regulate the copy number of transfected plasmid DNA's and also maintain selectable vector sequences either as integrated DNA or as autonomously replicating episomes by modulating T antigen activity in ts COS cells.

Animals↗
PubMed1985

Expression of homologous homeo-box-containing genes in differentiated human teratocarcinoma cells and mouse embryos.

Human chromosome 17 contains a cluster of at least three distinct homeo box regions, Hu1, Hu2, and Hu5, within a 20 kb stretch of DNA. A mouse homeo box region, Mu1, which maps to chromosome 11, was isolated and found to contain extensive nucleotide homology with a 4 kb region encompassing the Hu1 homeo box. The conservation of these chromosomal regions between man and mouse was confirmed by nucleotide sequence analysis: approximately 380 bp of DNA are more than 90% homologous and the 61 amino acids of the homeo box domain are perfectly conserved. We found that a human teratocarcinoma cell line expresses high levels of Hu1 homeo-box-containing mRNA only after differentiation of the cells following treatment with retinoic acid. In contrast, mouse teratocarcinoma cells did not express Mu1 homeo-box-containing mRNA at any stage of differentiation, whereas the expression of such transcripts was detected in mouse embryos from 10 to 17 days of gestation.

Amino Acid Sequence↗
PubMed1985

Two distinct transcription factors bind to the HSV thymidine kinase promoter in vitro.

We have characterized an in vitro transcription system derived from uninfected HeLa cells that accurately initiates RNA synthesis at the herpes virus thymidine kinase (TK) promoter. Analysis of linker-scanning, single-site, and promoter-inversion mutants reveals that the TK upstream elements previously mapped in vivo are accurately recognized in vitro. A protein fraction required for TK transcription in reconstitution experiments was found to contain multiple protein species that bind specifically to the TK promoter. DNAase I footprint experiments with wild-type and mutant promoters reveal that the TK upstream elements contain three distinctive protein binding sites, two of which appear to be recognized by the Sp1 transcription factor and one which interacts with a cellular protein that binds to "CCAAT" sequences. Optimal expression of the thymidine kinase gene appears to require the coordinate interaction of these two types of transcription factors with the three upstream elements of the promoter.

Base Sequence↗
PubMed1985

Characterization of Drosophila transcription factors that activate the tandem promoters of the alcohol dehydrogenase gene.

Fractionation of a nuclear extract derived from Drosophila tissue culture cells reveals the presence of multiple components involved in accurate transcription of both distal and proximal promoters of the alcohol dehydrogenase (Adh) gene. Transcription of deletion mutants indicates that a region between -24 and -85 upstream of the distal start site contains sequences required for RNA synthesis in vitro. Moreover, sequences that overlap this same upstream control region are specifically bound and protected from DNAase digestion by a promoter-specific transcription factor, Adf-1. Analysis of proximal promoter mutants identified multiple upstream elements that influence transcription, and DNAase footprint analysis detected three specific binding regions. Adf-1 binds at least one of these proximal promoter regions but interaction at this site is not specifically required for transcription. Our results suggest that multiple sequence-specific DNA binding proteins interact differentially with the proximal and distal promoters of Adh to activate transcription.

Alcohol Dehydrogenase↗
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↗
PubMed1985

An adenovirus vector system used to express polyoma virus tumor antigens.

We have used a generalized adenovirus vector system to express the three polyoma tumor (T) antigen proteins under the control of the adenovirus major late promoter. One hybrid virus, Ad-PySVR498, expresses high levels of polyoma middle and small T antigens. A second hybrid virus, Ad-LTSVR545, which contains a cDNA copy of the polyoma A gene, overproduces large T antigen. The T antigens produced are indistinguishable from their authentic polyoma counterparts as determined by immunoprecipitation and partial cleavage by V8 protease. Analysis of polyoma mRNAs encoded by the recombinant viruses showed that they initiate from the adenovirus major late promoter and contain the tripartite leader at their 5' ends. Large T antigen isolated from Ad-LTSVR545-infected cells by immunoaffinity was shown to bind selectively to polyoma DNA sequences that contain the origin of viral DNA replication as well as the sites for transcription initiation.

Adenoviridae↗