Effect of insulin on the cyclic AMP response of cultured glomerular mesangial cells.
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1-Methyl-3-isobutylxanthine↗
Biomedical subjects
P A Sharp
Publications and source records attributed to P A Sharp.
Positive genetic selection for gene disruption in mammalian cells by homologous recombination.
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.
Animals↗
Yeast TATA-binding protein TFIID binds to TATA elements with both consensus and nonconsensus DNA sequences.
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.
Base Sequence↗
Construction, stable transformation, and function of an amber suppressor tRNA gene in Drosophila melanogaster.
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.
Animals↗
The Oct-2 protein binds cooperatively to adjacent octamer sites.
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.
Base Sequence↗
Identification and purification of a 62,000-dalton protein that binds specifically to the polypyrimidine tract of introns.
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.
Base Sequence↗
The DNA-binding homeo domain of the Oct-2 protein.
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Amino Acid Sequence↗
Nuclear factors that bind to the enhancer region of nondefective Friend murine leukemia virus.
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.
Animals↗
A yeast protein possesses the DNA-binding properties of the adenovirus major late transcription factor.
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.
Adenoviridae↗
Cell lines established by a temperature-sensitive simian virus 40 large-T-antigen gene are growth restricted at the nonpermissive temperature.
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.
Animals↗
RNA splicing and genes.
The splicing of long transcripts of RNA (copied from DNA in the cell nucleus) into smaller, specific mRNA (ready for export to the protein-producing machinery in the cytoplasm) is an important event in the regulation of gene expression in eukaryotic cells. The splicing reaction occurs as a late step in the nuclear pathway for synthesis of mRNAs. This pathway commences with initiation of transcription by RNA polymerase II and probably involves an integrated series of steps each dependent on previous events. Splicing of precursors to mRNAs involves the formation of a spliceosome complex containing the 5' and 3' splice sites. This complex contains the evolutionarily highly conserved small nuclear RNAs (snRNAs) U2, U4, U5, and U6. The most abundant snRNA, U1, is required to form the spliceosome and may be a part of the spliceosome. Analogues of these snRNAs have been identified in yeast. Assembly of the spliceosome probably involves the binding of a multi-snRNA complex containing U4, U5, and U6 snRNAs. Several observations suggest that the association of snRNAs in such complexes is quite dynamic. It is argued that the snRNAs in the spliceosome form a catalytic RNA structure that is responsible for the cleavage and ligation steps during splicing.
Animals↗
Formation of transcription preinitiation complexes with an amanitin-resistant RNA polymerase II.
Accurate transcription by RNA polymerase II has been shown to require multiple factors which participate in a number of intermediate steps prior to initiation. The last detectable step before initiation is the formation of an activated or rapid start complex which is template-associated. In this study we used two phenotypically distinguishable forms of mammalian RNA polymerase II to examine the requirement for specific factors in the formation of the activated complex. RNA polymerase II was purified from a mutant cell line which was resistant to levels of alpha-amanitin that are toxic for normal cells. When added to a polymerase-dependent transcription system consisting of HeLa factors and adenovirus DNA, the mutant polymerase accurately transcribed the template in an amanitin-resistant fashion. This amanitin-resistant transcription was competitively inhibited when wild-type polymerase was also added to the system. Preincubation of amanitin-resistant polymerase with DNA and factors produced a polymerase-associated complex, defined by its resistance to exchange for the amanitin-sensitive polymerase. Complex formation required the presence of polymerase, DNA, and the HeLa factors during the preincubation but did not require the presence of nucleotides. Complexes were template-associated, as shown by their inability to exchange onto a second template. Thus, prior to initiation, RNA polymerase II forms a stable association with the DNA template in an activated complex.
Amanitins↗
Cloning of a lymphoid-specific cDNA encoding a protein binding the regulatory octamer DNA motif.
An octamer DNA sequence plays a critical role in directing transcription of immunoglobulin genes in B lymphocytes. A new technique of direct binding of radioactive DNA was used to screen a complementary DNA expression library from the BJAB cell line in lambda gt11 phage to derive molecular cDNA clones representing a putative B lymphocyte-specific octamer binding protein. The plaques were screened with DNA containing four copies of the octamer sequence and positive phage recombinants were identified. The fusion protein produced on inducing a lysogen of one phage bound to a monomeric octamer probe. The cDNA insert from this phage hybridized to messenger RNA found in B lymphocytes, but not in most other cells. Thus, this cDNA derives from a gene (oct-2) that specifies an octamer binding protein expressed preferentially in B lymphocytes, proving that, for at least one gene, a cell-specific transcription factor exists and its amount is controlled through messenger RNA availability.
Cloning, Molecular↗
Function of a yeast TATA element-binding protein in a mammalian transcription system.
Saccharomyces cerevisiae contains a protein which is functionally similar to the mammalian TATA element-binding transcription factor, TFIID. The yeast factor substitutes for TFIID in a mammalian RNA polymerase II in vitro transcription system, forms a stable preinitiation complex on the Adenovirus-2 major late promoter, and binds specifically to the TATA boxes of the viral promoter and the yeast CYC1 promoter. Interestingly, the yeast factor promotes initiation at a distance from the TATA element typical of a mammalian system.
DNA-Binding Proteins↗
Human CCAAT-binding proteins have heterologous subunits.
We have characterized three distinct proteins present in HeLa cell extracts that specifically recognize different subsets of transcriptional elements containing the pentanucleotide sequence CCAAT. One of these CCAAT-binding proteins, CP1, binds with high affinity to CCAAT elements present in the human alpha-globin promoter and the adenovirus major late promoter (MLP). A second protein, CP2, binds with high affinity to a CCAAT element present in the rat gamma-fibrinogen promoter. Finally, the third CCAAT-binding protein is nuclear factor I (NF-I), a cellular DNA-binding protein that binds to the adenovirus origin of replication and is required for the initiation of adenoviral replication. CP1, CP2, and NF-I are distinct activities in that each binds to its own recognition site with an affinity that is at least three orders of magnitude higher than that with which it binds to the recognition sites of the other two proteins. Surprisingly, CP1, CP2, and NF-I each appear to recognize their binding site with highest affinity as a multisubunit complex composed of heterologous subunits. In the case of CP1, two different types of subunits form a stable complex in the absence of a DNA-binding site. Moreover, both subunits are present in the CP1-DNA complex. We thus propose the existence of a family of related multisubunit CCAAT-binding proteins that are composed of heterologous subunits.
Animals↗
A yeast and a human CCAAT-binding protein have heterologous subunits that are functionally interchangeable.
The S. cerevisiae HAP2 and HAP3 gene products have been shown to recognize CCAAT-containing transcription elements as a HAP2/HAP3 multisubunit heterologous complex, or heteromer. Recently, we have demonstrated that the human sequence-specific DNA-binding protein, CP1, also recognizes CCAAT-containing transcription elements as a heteromer. Mammalian cells contain at least three distinct CCAAT-binding proteins, all of which appear to be multisubunit complexes composed of heterologous subunits. One of these proteins, CP1, has DNA-binding properties that are virtually identical to the yeast HAP2/HAP3 complex. These two proteins bind to the same target sequences, make the same DNA contacts, and are affected in a similar manner by mutations in the CCAAT element. Most surprisingly, the subunits of CP1 and HAP2/HAP3 are functionally interchangeable. That is, the yeast/human hybrid complexes that are formed retain the ability to specifically recognize CCAAT elements.
Biological Evolution↗
Molecular cloning of an enhancer binding protein: isolation by screening of an expression library with a recognition site DNA.
A novel strategy has been used to isolate a cDNA clone that encodes a DNA binding domain whose recognition properties overlap those of the mammalian transcription factors H2TF1 and NF-kappa B. These two factors are distinguished by their cell type distributions and their relative affinities for related sequence elements in the enhancers of the major histocompatibility complex (MHC) class I and immunoglobulin kappa chain genes. The human cDNA clone was detected by screening a lambda phage expression library with a binding site probe derived from the MHC enhancer. The phage encoded fusion protein binds specifically to both the MHC and kappa gene enhancers. The cDNA hybridizes to a single copy gene that is expressed as a 10 kb mRNA in both B and non-B cells. The strategy used in this study may prove generally useful in the cloning and analysis of sequence-specific DNA binding proteins.
Animals↗
Processing at immunoglobulin polyadenylation sites in lymphoid cell extracts.
We have developed an in vitro system for polyadenylation of RNA substrates in cell-free nuclear extracts prepared from murine cells of lymphoid origin. RNA substrates containing the adenovirus L3, murine immunoglobulin (IgM) secreted and membrane polyadenylation sites were accurately polyadenylated in these extracts. Kinetic analysis showed that the rate of polyadenylation in vitro responds proportionally to the substrate concentration. Quantitation of the initial rate of polyadenylation at the three sites permitted comparison of the activities of extracts prepared from HeLa cells, B cells (Wehi 231) and plasmacytoma cells (P9.37.11). From this analysis, we concluded that in all three extracts the polyadenylation activity at the L3 site was higher than that of either of the IgM sites. In contrast to the preferential utilization of the secreted site in vivo in plasmacytomas, this site was not selectively processed in plasmacytoma as compared to B cell extracts. The efficiency of polyadenylation at both IgM sites in the plasmacytoma extract was significantly lower than that in the B cell extract. The common low activity at the IgM sites in the plasmacytoma cell extract suggests that the rate-limiting step for polyadenylation at these two sites differs from that at the L3 site.
Animals↗