Showing papers by "Phillip A. Sharp published in 1989"

Cell lines established by a temperature-sensitive simian virus 40 large-T-antigen gene are growth restricted at the nonpermissive temperature.

Abstract: The thermolabile large T antigen, encoded by the simian virus 40 early-region mutant tsA58, was used to establish clonal cell lines derived from rat embryo fibroblasts. These cell lines grew continuously at the permissive temperature but upon shift-up to the nonpermissive temperature showed rapidly arrested growth. The growth arrest occurred in either the G1 or G2 phase of the cell cycle. After growth arrest, the cells remained metabolically active as assayed by general protein synthesis and the ability to exclude trypan blue. The inability of these cell lines to divide at the nonpermissive temperature was not readily complemented by the exogenous introduction of other nuclear oncogenes. This finding suggests that either these genes establish cells via different pathways or that immortalization by one oncogene results in a finely balanced cellular state which cannot be adequately complemented by another establishment gene.

Identification and purification of a 62,000-dalton protein that binds specifically to the polypyrimidine tract of introns.

Abstract: A protein of molecular size 62,000 daltons (p62) was detected in HeLa cell nuclear extracts by UV cross-linking to mRNA precursors. p62 binds specifically to the polypyrimidine tract of the 3' splice site region of introns. p62 purified to homogeneity binds the polypyrimidine tract of pre-mRNAs. This binding does not require the AG dinucleotide at the 3' splice site. Alterations in the polypyrimidine tract that reduce the binding of p62 yield a corresponding reduction in the efficiency of formation of a U2 snRNP/pre-mRNA complex and splicing. The p62 protein is retained in the spliceosome, where it remains bound to the pre-mRNA. This polypyrimidine tract binding protein (pPTB) is proposed to be a critical component in recognition of the 3' splice site during splicing.

Yeast TATA-binding protein TFIID binds to TATA elements with both consensus and nonconsensus DNA sequences

Abstract: The DNA binding properties of the yeast TATA element-binding protein TFIID were investigated. The affinity (apparent equilibrium dissociation constant) of TFIID for the adenovirus major late promoter consensus TATA element is 2 x 10(-9) M, a value similar to the affinity of gene-specific regulatory proteins for their binding sites. TFIID binding is highly specific and recognizes nonspecific sites with approximately 10(5)-fold lower affinity. Despite this specificity, TFIID also binds with high affinity to several TATA elements that do not match the consensus TATA sequences (TATAAA and TATATA): the yeast LEU2 TATA (TATTATTTA), the simian virus 40 TATA (CTTATTTAT), and the yeast CYC1 -10 TATA (TTATACATT) all bound TFIID. Furthermore, TFIID was active in promoting transcription in vitro from the nonconsensus TATA elements. Thus, contrary to previous suggestions, the existence of nonconsensus TATA elements does not itself indicate the existence of multiple TATA-binding factors.

The octamer-binding proteins form multi-protein--DNA complexes with the HSV alpha TIF regulatory protein.

Abstract: The herpes simplex virus transactivator, alpha TIF, stimulates transcription of the alpha/immediate early genes via a cis-acting site containing an octamer element and a conserved flanking sequence. The alpha TIF protein, produced in a baculovirus expression system, nucleates the formation of at least two DNA--protein complexes on this regulatory element. Both of these complexes contain the ubiquitous Oct-1 protein, whose POU domain alone is sufficient to allow assembly of the alpha TIF-dependent complexes. A second member of the POU domain family, the lymphoid specific Oct-2 protein, can also be assembled into similar complexes at high concentrations of alpha TIF protein. These complexes contain at least two cellular proteins in addition to Oct-1. One of these proteins is present in both insect and HeLa cells and probably recognizes sequences in the cis element. The second cellular protein, only present in HeLa cells, probably binds by protein-protein interactions.

Positive genetic selection for gene disruption in mammalian cells by homologous recombination

Abstract: Efficient modification of genes in mammalian cells by homologous recombination has not been possible because of the high frequency of nonhomologous recombination. An efficient method for targeted gene disruption has been developed. Cells with substitution of exogenous sequences into a chromosomal locus were enriched, by a factor of 100, using a positive genetic selection that specifically selects for homologous recombination at the targeted site. The selection is based on the conditional expression of a dominant selectable marker by virtue of in-frame gene fusion with the target gene. The dominant selectable marker was derived by modification of the Escherichia coli neo gene so that it retains significant activity in mammalian cells after in-frame fusion with heterologous coding sequences. In the example presented here, homologous recombinants were efficiently recovered from a pool in which the targeted gene was disrupted in 1 per 10,000 cells incorporating exogenous DNA.

The Oct-2 protein binds cooperatively to adjacent octamer sites.

Abstract: Recombinant proteins derived from the cloned human oct-2 gene were used to investigate cooperative binding by Oct-2 to adjacent DNA-binding sites. Oct-2, a B-cell-specific transcription factor, binds tightly to the octamer sequence in immunoglobulin promoters. A second apparently unrelated consensus sequence in heavy chain promoters, the heptamer site, also is recognized by the Oct-2 protein but with 1000-fold lower affinity. Simultaneous occupancy of both the octamer and heptamer sites is favored by cooperative interactions. The heptamer site is probably recognized by the same binding surface in the Oct-2 protein as the octamer site and thus is conserved as a lower-affinity binding site. This permits the immunoglobulin promoter to respond to a much broader range of levels of Oct-2 protein. Substitution of prototype octamer sequences for heptamer sequences yields a probe with two octamer sites spaced by 2 nucleotides, which also binds Oct-2 protein cooperatively. Only the POU domain in the Oct-2 protein is required for this cooperative interaction. Similar protein-protein interactions between bound Oct-2 proteins may promote promoter-enhancer synergism in the heavy chain gene.

Identification of a yeast protein homologous in function to the mammalian general transcription factor, TFIIA.

Abstract: The yeast homolog of the mammalian RNA polymerase II general transcription factor TFIIA has been identified by complementation of a mammalian in vitro transcription system depleted for TFIIA. Like the mammalian factor, the yeast protein does not bind DNA, alters the size of the TFIID DNase I footprint at the adenovirus major late promoter, and forms specific TFIIA-TFIID-DNA complexes which are stable during electrophoresis in native acrylamide gels. The partially purified yeast factor was used to investigate its effect on the binding of TFIID to the major late promoter. Contrary to earlier models, we find that TFIIA does not significantly change the affinity or kinetics of TFIID binding, suggesting that it acts by altering the conformation of TFIID and/or by serving as a bridge between TFIID and the other general transcription factors.

Nuclear factors that bind to the enhancer region of nondefective Friend murine leukemia virus.

Abstract: Nondefective Friend murine leukemia virus (MuLV) causes erythroleukemia when injected into newborn NFS mice, while Moloney MuLV causes T-cell lymphoma. Exchange of the Friend virus enhancer region, a sequence of about 180 nucleotides including the direct repeat and a short 3'-adjacent segment, for the corresponding region in Moloney MuLV confers the ability to cause erythroid disease on Moloney MuLV. We have used the electrophoretic mobility shift assay and methylation interference analysis to identify cellular factors which bind to the Friend virus enhancer region and compared these with factors, previously identified, that bind to the Moloney virus direct repeat (N. A. Speck and D. Baltimore, Mol. Cell. Biol. 7:1101-1110, 1987). We identified five binding sites for sequence-specific DNA-binding proteins in the Friend virus enhancer region. While some binding sites are present in both the Moloney and Friend virus enhancers, both viruses contain unique sites not present in the other. Although none of the factors identified in this report which bind to these unique sites are present exclusively in T cells or erythroid cells, they bind to three regions of the enhancer shown by genetic analysis to encode disease specificity and thus are candidates to mediate the tissue-specific expression and distinct disease specificities encoded by these virus enhancer elements.

Construction, stable transformation, and function of an amber suppressor tRNA gene in Drosophila melanogaster

Abstract: Drosophila melanogaster strains with a stably incorporated amber suppressor tRNA gene have been generated. A tRNATyr gene was site specifically mutated to produce an anticodon sequence that recognizes the amber codon and then introduced into Drosophila by using P-element-mediated transformation. Transformants from four integration events were recovered. Two integrations resulted in both male and female sterility, whereas the other two resulted in male sterility but female fertility. Strains derived from the two female-fertile integration events were shown to have a low level of amber-suppressing activity by their ability to suppress an amber mutation in a chloramphenicol acetyltransferase gene.

A yeast protein possesses the DNA-binding properties of the adenovirus major late transcription factor.

Abstract: The adenovirus major late transcription factor (MLTF), or upstream stimulatory factor, is a human promoter-specific transcription factor which recognizes the near-palindromic sequence GGCCACGTGACC (R. W. Carthew, L. A. Chodosh, and P. A. Sharp, Cell 43:439-448, 1985; L. A. Chodosh, R. W. Carthew, and P. A. Sharp, Mol. Cell. Biol. 6:4723-4733, 1986; M. Sawadogo and R. G. Roeder, Cell 43:165-175, 1985). We describe here a protein found in the yeast Saccharomyces cerevisiae which possesses DNA-binding properties that are virtually identical to those of human MLTF. These two proteins recognize the same DNA-binding site, make the same purine nucleotide contacts, and are affected in the same manner by mutations in the MLTF-binding site.

Gene Activation by Induced DNA Rearrangements

Abstract: A murine cell line (EN/NIH) containing the retroviral vector ZIPNeoSV(x)1 that was modified by deletion of the enhancer elements in the viral long terminal repeats has been used as an assay system to detect induced DNA rearrangements that result in activation of a transcriptionally silent reporter gene (neomycin phosphotransferase, neo ) encoded by the viral genome. The spontaneous frequency of G418 resistance is less than 10 -7 , whereas exposure to the tumor promoter 12- O -tetradecanoylphorbol-13-acetate (TPA) or the combination of UV irradiation plus TPA resulted in the emergence of drug resistant cell lines at a frequency of 5 per 10 6 and 67 per 10 6 cells, respectively. In several of the cell lines that were analyzed a low level of amplification of one of the two parental retroviral integrants was observed, whereas in others no alteration in the region of the viral genome was detected. To determine the effect of the SV40 large T antigen on induced DNA rearrangements, EN/NIH cells were transfected with a temperature sensitive ( ts ) mutant of SV40 T. Transfectants were maintained at the permissive temperature (33°C) for varying periods of time (1–5 days) in order to vary SV40 T antigen exposure, after which they were shifted to 39.5°C for selection in G418. The frequency of emergence of drug resistant cell clones increased with duration of exposure to large T antigen (9–52 per 10 6 cells over 1–5 days, respectively), and all cell lines analyzed demonstrated DNA rearrangements in the region of the neo gene. A novel 18-kilobase pair Xba I fragment was cloned from one cell line which revealed the presence of a 2.0-kilobase pair Eco RI segment containing an inverted duplication which hybridized to neo sequences. It is likely that the observed rearrangement was initiated by the specific binding of large T antigen to the SV40 origin of replication encoded within the viral genome. The investigations with phorbol esters, UV light, and the SV40 large T antigen demonstrate the utility of the EN/NIH cell lines for the study of induced DNA rearrangements and support the future use of this system to investigate the mechanism by which varied stimuli or specific gene functions promote DNA rearrangements.

36 – In Vitro Transcription: Whole-Cell Extract

Abstract: Publisher Summary This chapter focuses on three classes of nuclear DNA-dependent RNA polymerases that have been identified in eukaryotic cells. Synthesis of mature RNA molecules requires additional enzymes and factors other than those needed to bring about accurate transcriptional initiation. For analysis by hybridization and SI nuclease digestion, it is important first to remove the template DNA. Titrations both of DNA and of extract yield nonlinear responses. At a constant extract concentration, measuring runoff transcription as a function of DNA concentration yields a threshold DNA concentration below which no transcription occurs and an inhibitory effect of high DNA concentration. For a given promoter, short runoff transcripts have a higher optimum DNA concentration than longer runoff transcripts. A number of inhibitory activities can be removed by fractionation on phosphocellulose, yielding a more efficient transcription extract.