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Showing papers by "Phillip A. Sharp published in 1988"


Journal Article•DOI•
TL;DR: The POU domain is a novel bipartite DNA-binding structure in which the POU homoeo and POU-specific regions form two subdomains that are both required for DNA binding but are held together by a flexible linker.
Abstract: The POU domain1 (pronounced 'pow') is a highly charged 155–162-amino-acid (aa) region of sequence similarity contained within three mammalian transcription factors. Pit-1 (ref. 2), Oct-1 (ref. 3) and Oct-2 (ref. 4), and the product of the nematode gene unc-86 (ref. 5) which is involved in determining neural cell lineage. This domain consists of two subdomains, a C-terminal homoeo domain and an N-terminal POL -specific region separated by a short nonconserved linker; the sequence relationship shows that the POU homoeo domains form a distinct POU-related family. In the ubiquitous and lymphoid-specific octamer-motif binding proteins Oct-1 and Oct-2, the POU domain is sufficient for sequence-specific DNA binding3,4. Homoeobox domains contain a helix-turn-helix DNA-binding motif6,7, first identified in bacterial repressers8. The helix-turn-helix region of the POU domain is important for DNA binding3,9 and, in other classes of homoeo-containing proteins, the entire homoeo domain is sufficient for DNA binding10–12; thus the new POU-specific region could be involved in other functions such as protein–protein interactions. Nevertheless, we show here that in fact the POU domain is a novel bipartite DNA-binding structure in which the POU homoeo and POU-specific regions form two subdomains that are both required for DNA binding but are held together by a flexible linker.

689 citations


Journal Article•DOI•
12 Feb 1988-Cell
TL;DR: 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.

603 citations


Journal Article•DOI•
08 Apr 1988-Cell
TL;DR: A family of related multisubunit CCAAT-binding proteins that are composed of heterologous subunits are proposed that are related to each other and to the adenovirus origin of replication and is required for the initiation ofadenoviral replication.

596 citations


Journal Article•DOI•
01 Jan 1988
TL;DR: A He La cell nuclear transcription extract that is approximately 20 times more efficient than standard HeLa cell transcription extracts was developed, and exhibits an increase of approximately twofold on transcription of a closed circular, as opposed to a linear, major late promoter template.
Abstract: A HeLa cell nuclear transcription extract that is approximately 20 times more efficient than standard HeLa cell transcription extracts was developed. Transcription of the strong adenovirus II major late promoter by this extract results in the synthesis of 1.5-4 molecules of product RNA per molecule of template, indicating that the extract is capable of multiple rounds of initiation. Standard HeLa cell nuclear extracts transcribe closed circular and linear adenovirus major late promoter templates with equal efficiency. In contrast, the new extract exhibits an increase of approximately twofold on transcription of a closed circular, as opposed to a linear, major late promoter template.

540 citations


Journal Article•DOI•
TL;DR: The sequence of a cDNA encoding a B-cell-specific protein, Oct-2, has been determined and it is proposed that the oct-2 gene is expressed as multiple mRNAs that vary in splicing patterns.
Abstract: Transcription of promoters of immunoglobulin genes is controlled by an octanucleotide sequence element. The sequence of a cDNA encoding a B-cell-specific protein, Oct-2, has been determined. This protein specifically recognizes the octanucleotide element and is part of the previously identified NF-A2 family of proteins. The DNA-binding domain of Oct-2 is structurally related to the homeo box consensus and thus contains a potential helix-turn-helix sequence. Oct-2 also possesses a potential 'leucine zipper' domain, where four leucines are each separated by exactly seven residues. Comparisons of Oct-2 with protein Oct-1, which also recognizes the octanucleotide element but is constitutively expressed in all cell types, show high sequence conservation through the 60-residue DNA-binding domain, as well as an adjacent tract of 75 residues. The latter conserved region is also found in regulatory genes expressed in pituitary cells and nematodes and has been termed a POU box. Because two different cDNAs were isolated, it is proposed that the oct-2 gene is expressed as multiple mRNAs that vary in splicing patterns. Most interestingly, the oct-2 cDNA contains a second overlapping open reading frame, 278 residues in length, which might also specify a protein important for B-cell development.

366 citations


Journal Article•DOI•
07 Jul 1988-Nature
TL;DR: Saccharomyces cerevisiae contains a protein which is functionally similar to the mammalian TATA element-binding transcription factor, TFIID, which promotes initiation at a distance from the TATA elements typical of a mammalian system.
Abstract: 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 CYCl promoter. Interestingly, the yeast factor promotes initiation at a distance from the TATA element typical of a mammalian system.

305 citations


Journal Article•DOI•
29 Jul 1988-Science
TL;DR: 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.
Abstract: 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.

255 citations


Journal Article•DOI•
TL;DR: It is shown here that NF-kappa B, an inducible B-cell-specific factor that binds the kappa immunoglobulin light chain gene enhancer, also binds the H2TF1 regulatory sequence.
Abstract: A sequence centered 166 nucleotides upstream of the mouse H-2Kb class I major histocompatibility gene binds a nuclear factor, H2TF1, found in many cell types. Previous studies have shown that binding of H2TF1 to this sequence stimulates class I gene expression. Furthermore, this factor binds a similar sequence in the 72-base-pair repeat enhancer element of simian virus 40. We show here that NF-kappa B, an inducible B-cell-specific factor that binds the kappa immunoglobulin light chain gene enhancer, also binds the H2TF1 regulatory sequence. Methylation-interference experiments demonstrate that NF-kappa B closely interacts with six of the eight symmetrically positioned guanines that contact H2TF1. These experiments suggest that NF-kappa B may play a role in class I major histocompatibility gene expression and that H2TF1 and NF-kappa B may be related DNA-binding proteins.

254 citations


Journal Article•DOI•
TL;DR: A pathway of spliceosome assembly was deduced that has at least three stages and U2 small nuclear ribonucleoprotein alone binds to sequences of mRNA upstream of the 3' splice site.
Abstract: Splicing complexes that form a rabbit beta-globin precursor mRNA (pre-mRNA) have been analyzed for their small nuclear RNA (snRNA) content by both affinity chromatography and specific probe hybridization of replicas of native electrophoretic gels. A pathway of spliceosome assembly was deduced that has at least three stages. (i) U2 small nuclear ribonucleoprotein (snRNP) alone binds to sequences of mRNA upstream of the 3' splice site. (ii) U4, U5, and U6 snRNPs bind, apparently simultaneously. (iii) U4 snRNP is released to generate a spliceosome that contains U2, U5, and U6 snRNPs together with the RNA intermediates in splicing. U1 snRNP was not detected in association with any of these complexes. A parallel analysis of the spliceosome found with an adenovirus precursor mRNA substrate yielded an identical snRNP composition with one additional, unidentified RNA species, called X. This latter RNA species was not detected in the spliceosome bound to the beta-globin substrate.

141 citations


Journal Article•DOI•
TL;DR: It is suggested that the constitutive OCTA-binding factor NF-A1 can activate transcription of the Ig promoter and that B-cell-specific transcription of this promoter, at least in vitro, is partially due to a quantitative difference in the amount of OCTa-binding protein.
Abstract: The B-cell-type specificity of the immunoglobulin (Ig) heavy-chain and light-chain promoters is mediated by an octanucleotide (OCTA) element, ATGCAAAT, that is also a functional component of other RNA polymerase II promoters, such as snRNA and histone H2B promoters. Two nuclear proteins that bind specifically and with high affinity to the OCTA element have been identified. NF-A1 is present in a variety of cell types, whereas the presence of NF-A2 is essentially confined to B cells, leading to the hypothesis that NF-A2 activates cell-type-specific transcription of the Ig promoter and NF-A1 mediates the other responses of the OCTA element. Extracts of the B-cell line, BJA-B, contain high levels of NF-A2 and specifically transcribe Ig promoters. In contrast, extracts from HeLa cells transcribed the Ig promoter poorly. Surprisingly, addition of either affinity-enriched NF-A2 or NF-A1 to either a HeLa extract or a partially purified reaction system specifically stimulates the Ig promoter. This suggests that the constitutive OCTA-binding factor NF-A1 can activate transcription of the Ig promoter and that B-cell-specific transcription of this promoter, at least in vitro, is partially due to a quantitative difference in the amount of OCTA-binding protein. Because NF-A1 can stimulate Ig transcription, the inability of this factor to activate in vivo the Ig promoter to the same degree as the snRNA promoters probably reflects a difference in the context of the OCTA element in these two types of promoters.

104 citations


Journal Article•DOI•
TL;DR: Formation of this U2-U4-U5-U6 (U2/4/5/6) complex, the pseudospliceosome, suggests that the basic structure of the splicingosome is specified by snRNP-snRNP interactions.
Abstract: Small nuclear ribonucleoprotein particles (snRNPs) associate to form multi-snRNP complexes during splicing of mRNA precursors. A vast majority of the three snRNPs U4, U5, and U6 are present in a nuclear extract in a single complex, while U1 and U2 snRNPs exist as separate particles. Under conditions optimal for splicing in vitro the U4-U5-U6 (U4/5/6) complex dissociates to release free snRNPs, suggesting that the interactions between its components are dynamic. Several forms of splicing complexes assemble on precursor RNA during splicing in vitro. One of these forms, spliceosome B, contains U2, U4, U5, and U6 snRNPs bound to the precursor RNA. This same set of snRNPs associates efficiently in the absence of precursor RNA during incubation of the extract at high salt concentration. Formation of this U2-U4-U5-U6 (U2/4/5/6) complex, the pseudospliceosome, suggests that the basic structure of the spliceosome is specified by snRNP-snRNP interactions.

Journal Article•DOI•
TL;DR: In this paper, the presence of a dominant repressor element within the mu enhancer was found to activate transcription in fibroblasts but not in myeloma cells, and the possible biological roles of this phenomenon were discussed.
Abstract: The immunoglobulin heavy-chain enhancer is a cis-acting element which activates transcription of nearby genes only in cells of the lymphoid lineage. To identify the minimal sequences necessary to impart cell type transcriptional specificity, we tested the activity of several deletions and internal mutations in the mu enhancer. Experiments involving measurement of both chloramphenicol acetyltransferase activity and RNA levels indicated the presence of a dominant repressor element within the mu enhancer. This repressive activity was detected in fibroblasts but not in myeloma cells. Removal or disruption of this repressor element revealed the presence of elements within the mu enhancer that activate transcription in fibroblasts. Thus, enhancer tissue specificity is in part due to the composite of both constitutive activation and cell-type-specific repressive activity. The possible biological roles of this phenomenon are discussed.

Journal Article•DOI•
TL;DR: Kinetic studies suggest that the 50S complex is an intermediate in the polyadenylation reaction and forms at an early stage in the reaction and at later times contains both poly(A)+ RNA as well as unreacted precursor.
Abstract: Precursor RNA containing the adenovirus L3 polyadenylation site is assembled into a 50S complex upon incubation with HeLa nuclear extract at 30 degrees C. The cofactor and sequence requirements for 50S complex formation are similar to those of the in vitro polyadenylation reaction. Assembly of this complex requires ATP but is not dependent upon synthesis of a poly(A) tract. In addition, a 50S complex does not form on substrate RNA in which the AAUAAA hexanucleotide upstream of the poly(A) site has been mutated to AAGAAA or on RNA in which sequences between +5 and +48 nucleotides downstream of the site have been removed. These mutations also prevent in vitro processing of substrate RNA. Kinetic studies suggest that the 50S complex is an intermediate in the polyadenylation reaction. It forms at an early stage in the reaction and at later times contains both poly(A)+ RNA as well as unreacted precursor. U-type small nuclear ribonucleoprotein particles are components of the 50S complex, as shown by immunoprecipitation with antiserum specific to the trimethyl cap of these small nuclear RNAs.

Journal Article•DOI•
25 Nov 1988-JAMA
TL;DR: 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.
Abstract: 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. ( JAMA 1988;260:3035-3041)

Journal Article•DOI•
TL;DR: Two phenotypically distinguishable forms of mammalian RNA polymerase II were used to examine the requirement for specific factors in the formation of the activated complex of an activated or rapid start complex.

Journal Article•DOI•
TL;DR: In all three extracts the polyadenylation activity at the L3 site was higher than that of either of the IgM sites, and this site was not selectively processed in plasmacytoma as compared to B cell extracts.
Abstract: 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 (P93711) 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

Journal Article•DOI•
TL;DR: The results of the studies of expression of a Saccharomyces cerevisiae amber suppressor tRNA(Leu) gene (SUP53) in mammalian cells in vivo and in cell extracts in vitro suggest that processing of pre-tRNAs in CV1 cells is blocked at the level of 5'- and 3'-end maturation.
Abstract: We describe the results of our studies of expression of a Saccharomyces cerevisiae amber suppressor tRNA(Leu) gene (SUP53) in mammalian cells in vivo and in cell extracts in vitro. Parallel studies were carried out with the wild-type (Su-) tRNA(Leu) gene. Extracts from HeLa or CV1 cells transcribed both tRNA(Leu) genes. The transcripts were processed correctly at the 5' and 3' ends and accurately spliced to produce mature tRNA(Leu). Surprisingly, when the same tRNA(Leu) genes were introduced into CV1 cells, only pre-tRNAs(Leu) were produced. The pre-tRNAs(Leu) made in vivo were of the same size and contained the 5'-leader and 3'-trailer sequences as did pre-tRNAs(Leu) made in vitro. Furthermore, the pre-tRNAs(Leu) made in vivo were processed to mature tRNA(Leu) when incubated with HeLa cell extracts. A tRNA(Leu) gene from which the intervening sequence had been removed yielded RNAs that also were not processed at either their 5' or 3' termini. Thus, processing of pre-tRNA(Leu) in CV1 cells is blocked at the level of 5'- and 3'-end maturation. One possible explanation of the discrepancy in the results obtained in vivo and in vitro is that tRNA biosynthesis in mammalian cells involves transport of pre-tRNA from the site of its synthesis to a site or sites where processing takes place, and perhaps the yeast pre-tRNAs(Leu) synthesized in CV1 cells are not transported to the appropriate site.