Biomedical subjects

P A Sharp

Publications and source records attributed to P A Sharp.

At least 55 records · Page 3Linked to original sources

PubMed1996

Sequence-specific DNA binding of the B-cell-specific coactivator OCA-B.

B-cell-specific transcription of immunoglobulin genes is mediated by the interaction of a POU domain containing transcription factor Oct-1 or Oct-2, with the B-cell-specific coactivator OCA-B (Bob-1, OBF-1) and a prototype octamer element. We find that OCA-B binds DNA directly in the major groove between the two subdomains of the POU domain, requiring both an A at the fifth position of the octamer element and contact with the POU domain. An amino-terminal fragment of OCA-B binds the octamer site in the absence of a POU domain with the same sequence specificity. Coactivator OCA-B may undergo a POU-dependent conformational change that exposes its amino terminus, allowing it to recognize specific DNA sequences in the major groove within the binding site for Oct-1 or Oct-2. The recognition of both the POU domain and the octamer sequence by OCA-B provides a mechanism for differential regulation of octamer sites containing genes by the ubiquitous factor Oct-1.

B-Lymphocytes↗

PubMed1996

A hyperphosphorylated form of the large subunit of RNA polymerase II is associated with splicing complexes and the nuclear matrix.

A hyperphosphorylated form of the largest subunit of RNA polymerase II (pol IIo) is associated with the pre-mRNA splicing process. Pol IIo was detected in association with a subset of small nuclear ribonucleoprotein particle and Ser-Arg protein splicing factors and also with pre-mRNA splicing complexes assembled in vitro. A subpopulation of pol IIo was localized to nuclear "speckle" domains enriched in splicing factors, indicating that it may also be associated with RNA processing in vivo. Moreover, pol IIo was retained in a similar pattern following in situ extraction of cells and was quantitatively recovered in the nuclear matrix fraction. The results implicate nuclear matrix-associated hyperphosphorylated pol IIo as a possible link in the coordination of transcription and splicing processes.

Animals↗

PubMed1996

Sin3 corepressor function in Myc-induced transcription and transformation.

Many basic-helix-loop-helix-leucine zipper (b-HLH-LZ) proteins, including the Myc family and non-Myc family, bind a common DNA sequence CACGTG, yet have quite different biological actions. Myc binds this sequence as a heterodimer with Max in the activation of both transcription and transformation. The Myc family members Mad and Mxi1 are known to suppress Myc-induced transcription and transformation and to dimerize with Max to form ternary complexes with the mammalian Sin3 transcriptional corepressor (mSin3). The b-HLH-LZ domain of TFEB, which cannot heterodimerize within the Myc family, does not suppress Myc-induced transcription or transformation. However, transfer of a 25- to 36-aa region from Mad or Mxi1, which interacts with mSin3, to the b-HLH-LZ of TFEB, mediated profound suppression of Myc-induced transcription and transformation. These results suggest that the DNA binding specificities of the Myc family and non-Myc family b-HLH-LZ proteins, in the context of the cellular genes involved in Myc-induced transformation, are shared. The results also demonstrate that targeting mSin3 to CACGTG sites via a non-Myc family DNA binding domain is sufficient to oppose Myc activity in growth regulation.

Amino Acid Sequence↗

PubMed1996

A negative cofactor containing Dr1/p19 modulates transcription with TFIIA in a promoter-specific fashion.

An activity that modulated the relative levels of transcription from the adenovirus major late promoter (MLP), and the immunoglobulin heavy chain mu promoter (mu) was purified as a 90-kDa factor. This factor is suggested to be a heterotetramer of two subunits: a 20-kDa polypeptide identical to the previously described Dr1/p19 and a novel 30-kDa polypeptide. The Dr1/p19 protein has been characterized as a repressor of transcription, and the 30-kDa protein is related to a recently identified yeast gene proposed to encode a repressor of transcription. The 90-kDa factor forms a complex with TATA-binding protein on DNA and at high concentrations of both factors protects over a 150-base pair region around the promoter from DNase I cleavage. The conformation of this complex as assayed by footprinting analysis is altered by the transcription factor TFIIA on the MLP but not on the mu promoter. Similarly, TFIIA reverses the repression of transcription by the 90-kDa factor on the MLP but not on the mu promoter. Thus, the interactions of TATA-binding protein, TFIIA, and the 90-kDa factor are promoter-specific.

Amino Acid Sequence↗

PubMed1996

Three recognition events at the branch-site adenine.

An adenosine at the branch site, the nucleophile for the first transesterification step of splicing, is nearly invariant in mammalian pre-mRNA introns. The chemical groups on the adenine base were varied systematically and assayed for formation of early spliceosome complexes and execution of the first and second steps of splicing. Recognition of constituents of the adenine is critical in formation of a U2 snRNP-containing complex on a minimal branch-site oligonucleotide. Furthermore, the efficiencies of the first and second chemical steps have different dependencies on the functional groups of the adenine. In total, the chemical groups on the adenine base at the branch site are differentially recognized during at least three different processes in the splicing of pre-mRNA. Moreover, a protein, p14, interacts with the adenine in a base-specific fashion and may mediate early recognition of this base.

Adenine↗

PubMed1996

Rapid enhancement of brush border glucose uptake after exposure of rat jejunal mucosa to glucose.

BACKGROUND: Increased jejunal glucose transport after ingestion of carbohydrate rich diets may reflect higher concentrations of lumenal glucose. Normal processing of carbohydrate causes wide fluctuations in glucose concentration in the jejunal lumen and this raises the question of whether the high lumenal concentrations seen at peak digestion affect glucose uptake. AIMS: To study the effects of 30 minute exposure of rat jejunal mucosa to glucose on sodium-glucose transporter (SGLT1) mediated glucose transport across the brush border membrane. METHODS: Jejunal mucosa was exposed in vitro or in vivo to 25 mM glucose or 25 mM mannitol for 30 minutes. In addition, isolated villus enterocytes were incubated with mannitol or glucose for the same time. Brush border membrane vesicles were isolated from these preparations and phlorizin sensitive 3H-D-glucose accumulation was measured. RESULTS: Lumenal glucose in vivo significantly enhanced SGLT1 mediated glucose uptake by 49.2-57.2%. For jejunal loops in vitro, the increase was 32.0-85.2%. Kinetic analysis disclosed a 50% greater Vmax for glucose uptake in each preparation. The facilitated and passive components of uptake were, however, unaffected by prior exposure to glucose. Incubation of villus enterocytes with 25 mM glucose did not influence glucose uptake by brush border membranes. Finally, exposure of intact mucosa to 20 mM galactose, a nonmetabolised sugar also transported by SGLT1, did not alter glucose transport. CONCLUSIONS: Lumenal glucose promotes glucose transport by brush border membrane within 30 minutes. An intact mucosa is necessary for upregulation and evidence suggests that the response is mediated by locally acting mechanisms.

Animals↗

PubMed1996

Cis-acting elements distinct from the 5' splice site promote U1-independent pre-mRNA splicing.

We have identified a class of pre-mRNAs that are spliced in HeLa extracts depleted for U1 snRNP (delta U1 extracts). Previously, we described pre-mRNAs that can be spliced in delta U1 extracts only when high concentrations of SR splicing factors are added. In contrast, the substrates characterized here are efficiently processed in delta U1 extracts without the addition of excess SR proteins. The members of this class comprise both a naturally occurring pre-mRNA, from the Drosophila fushi tarazu gene, and a chimera containing sequences from two different pre-mRNAs that individually are dependent upon U1 snRNP or excess SR proteins. Several sequence elements account for the variations in dependence on U1 snRNP and SR proteins for splicing. In one pre-mRNA, a single element was identified adjacent to the branch site. In the other, two elements flanking the 5' splice site were found to be critical. This U1-independent splicing reaction may provide a mechanism for cells to control the extent of processing of different classes of pre-mRNAs in response to altered activities of SR proteins, and furthermore suggests that U1 snRNP-independent splicing may not be uncommon.

Base Sequence↗

PubMed1995

Analysis of homeodomain function by structure-based design of a transcription factor.

The homeodomain is a 60-amino acid module which mediates critical protein-DNA and protein-protein interactions for a large family of regulatory proteins. We have used structure-based design to analyze the ability of the Oct-1 homeodomain to nucleate an enhancer complex. The Oct-1 protein regulates herpes simplex virus (HSV) gene expression by participating in the formation of a multiprotein complex (C1 complex) which regulates alpha (immediate early) genes. We recently described the design of ZFHD1, a chimeric transcription factor containing zinc fingers 1 and 2 of Zif268, a four-residue linker, and the Oct-1 homeodomain. In the presence of alpha-transinduction factor and C1 factor, ZFHD1 efficiently nucleates formation of the C1 complex in vitro and specifically activates gene expression in vivo. The sequence specificity of ZFHD1 recruits C1 complex formation to an enhancer element which is not efficiently recognized by Oct-1. ZFHD1 function depends on the recognition of the Oct-1 homeodomain surface. These results prove that the Oct-1 homeodomain mediates all the protein-protein interactions that are required to efficiently recruit alpha-transinduction factor and C1 factor into a C1 complex. The structure-based design of transcription factors should provide valuable tools for dissecting the interactions of DNA-bound domains in other regulatory circuits.

Amino Acid Sequence↗

PubMed1995

A U6 snRNA:pre-mRNA interaction can be rate-limiting for U1-independent splicing.

The full set of consensus sequences at the 5' splice site is recognized during splicing of pre-mRNA in extracts depleted of U1 snRNP. High concentrations of HeLa SR proteins or purified SC35 alone promote the splicing of specific RNA substrates, bypassing the requirement for U1 snRNP in formation of the U2 snRNP-pre-mRNA complex. Under these conditions, mutations in the substrate that increase the sequence complementarity between U6 snRNA and the 5' splice site region can facilitate splicing. This provides additional strong evidence that U1 snRNP is not essential for splicing. Thus, the consensus sequence at the 5' splice site is probably recognized twice during splicing of most introns; however, some pre-mRNAs could potentially be processed in the absence of interactions with U1 snRNP in regions of the nucleus containing high concentrations of SR protein.

Base Sequence↗

PubMed1995

Activation of the TFIID-TFIIA complex with HMG-2.

The nonhistone chromosomal protein HMG-2 was identified as a factor necessary for activation in a defined transcription reaction in vitro containing RNA polymerase II and purified factors. Activation occurred on all promoters assayed except that of the immunoglobulin IgH gene. TFIIA was required for stimulated levels of transcription. The activation process depended on the presence of TAFs in the TFIID complex and generated a preinitiation complex from which TFIIB dissociated more slowly. However, titration of TFIIB over three orders of magnitude did not obviate the requirement of activator and HMG-2 to achieve stimulated levels of transcription. Analysis of the activated reaction identified the TFIID-TFIIA complex as the first stage of modification during activation. These results suggest that activation can occur solely in the presence of the basal factors, activator protein, and an "architectural" HMG factor, which probably stabilizes an activated conformation of the TFIID-TFIIA-promoter complex.

Base Sequence↗

PubMed1995

A kinase-deficient transcription factor TFIIH is functional in basal and activated transcription.

Phosphorylation of the carboxyl-terminal domain (CTD) of the large subunit of RNA polymerase II has been suggested to be critical for transcription initiation, activation, or elongation. A kinase activity specific for CTD is a component of the general transcription factor TFIIH. Recently, a cyclin-dependent kinase-activator kinase (MO15 and cyclin H) was found to be associated with TFIIH preparations and was suggested to be the CTD kinase. TFIIH preparations containing mutant, kinase-deficient MO15 lack CTD kinase activity, indicating that MO15 is critical for polymerase phosphorylation. Nonetheless, these mutant TFIIH preparations were fully functional (in vitro) in both basal and activated transcription. These results indicate that CTD phosphorylation is not required for transcription with a highly purified system.

Bone Neoplasms↗

PubMed1995

The cellular C1 factor of the herpes simplex virus enhancer complex is a family of polypeptides.

The alpha/immediate early genes of herpes simplex virus are regulated by the specific assembly of a multiprotein enhancer complex containing the Oct-1 POU domain protein, the viral alpha-transinduction factor alpha TIF, (VP16, ICP25), and the C1 cellular factor. The C1 factor from mammalian cells is a heterogeneous but related set of polypeptides that interact directly with the alpha-transinduction factor to form a heteromeric protein complex. The isolation of cDNAs encoding the polypeptides of the C1 factor suggests that these proteins are proteolytic products of a novel precursor. The sequence of the amino termini of these polypeptide products indicate that the proteins are generated by site-specific cleavages within a reiterated 20-amino acid sequence. Although the C1 factor appears to be ubiquitously expressed, it is localized to subnuclear structures in specific cell types.

Amino Acid Sequence↗

PubMed1995

Pre-bending of a promoter sequence enhances affinity for the TATA-binding factor.

TATA-binding protein (TBP) binds the minor groove of the TATA element with the DNA bent 80 degrees towards the major groove. A constrained minicircle strategy has been used to test the effect of DNA topology on the affinity of TBP for the TATA element. We report here that TBP bound to DNA which was slightly pre-bent towards the major groove with 100-fold higher affinity than unbent (linear) DNA of identical sequence and 300-fold higher affinity than DNA pre-bent towards the minor groove. Similar discrimination was observed with the holo-TFIID transcription complex. DNA topology, particularly bending, is determined by many factors including chromatin in cells and may, through changes in the affinity of the TATA factor, be important in the control of transcription.

DNA↗

PubMed1995

Novel mechanism and factor for regulation by HIV-1 Tat.

Tat regulation of human immunodeficiency virus (HIV) transcription is unique because of its specificity for an RNA target, TAR, and its ability to increase the efficiency of elongation by polymerase. A reconstituted reaction that is Tat-specific and TAR-dependent for activation of HIV transcription has been used to identify and partially purify a cellular activity that is required for trans-activation by Tat, but not by other activators. In the reaction, Tat stimulates the efficiency of elongation by polymerase, whereas Sp1 and other DNA sequence-specific transcription factors activate the rate of initiation. Furthermore, while TATA binding protein (TBP)-associated factors (TAFs) in the TFIID complex are required for activation by transcription factors, they are dispensable for Tat function. Thus, Tat acts through a novel mechanism, which is mediated by a specific host cellular factor, to stimulate HIV-1 gene expression.

Fungal Proteins↗

PubMed1995

Structure-based design of transcription factors.

Computer modeling suggested that transcription factors with novel sequence specificities could be designed by combining known DNA binding domains. This structure-based strategy was tested by construction of a fusion protein, ZFHD1, that contained zinc fingers 1 and 2 from Zif268, a short polypeptide linker, and the homeodomain from Oct-1. The fusion protein bound optimally to a sequence containing adjacent homeodomain (TAATTA) and zinc finger (NGGGNG) subsites. When fused to an activation domain, ZFHD1 regulated promoter activity in vivo in a sequence-specific manner. Analysis of known protein-DNA complexes suggests that many other DNA binding proteins could be designed in a similar fashion.

Amino Acid Sequence↗

PubMed1995

The effects of streptozotocin diabetes on sodium-glucose transporter (SGLT1) expression and function in rat jejunal and ileal villus-attached enterocytes.

Rats treated with streptozotocin for 17 days were used to determine the cellular origin of enhanced brush border glucose transport in the diabetic small intestine. In the jejunum of both normal and diabetic rats, phlorizin-sensitive (SGLT1-mediated) glucose transport was shown, by section autoradiography, to take place in upper villus enterocytes. The distribution of brush border SGLT1 transporters along villi, determined using immunogold cytochemistry, was similar to that found for glucose uptake. Longer villi, supporting a larger number of absorbing enterocytes in the diabetic jejunum, appeared to be responsible for increased glucose uptake in this condition. SGLT1 protein and SGLT1-mediated glucose transport were undetectable in normal distal ileal villi. However, following treatment with streptozotocin, both SGLT1 protein and SGLT1-mediated glucose transport were found to be present in basal ileal villus enterocytes. SGLT1 protein and SGLT1-mediated glucose transport both increased during enterocyte migration to the villus tip. Cellular induction of the SGLT1 transporter, as well as longer villi contribute to enhanced glucose transport in diabetic rat distal ileum. Close correlation between the positional expression of SGLT1 protein and absorptive function suggests that transporter density is an important determinant for up-regulation of sodium-dependent glucose transport in diabetes.

Animals↗

PubMed1995

New proteins related to the Ser-Arg family of splicing factors.

A family of six highly conserved proteins that contain domains rich in alternating serine/arginine residues (SR proteins) function in the regulation of splice site selection and are required for splicing. Using a selective precipitation method, more than 35 proteins were detected in nuclear extracts of HeLa cells that co-fractionate with the defined SR family. Many of these proteins were recognized by three monoclonal antibodies that bind to distinct phosphoepitopes on SR proteins. Two of these SR-related proteins were identified as the nuclear matrix antigens B1C8 and B4A11, which previously have been implicated in splicing. A subset of SR proteins, in their phosphorylated state, are associated with spliceosome complexes through both steps of the splicing reaction, remaining preferentially bound to complexes containing the exon-product. In contrast, other SR-related proteins appear to remain specifically associated with the intron-Iariat complex. The results indicate the existence of a potentially large group of SR-related proteins, and also suggest possible additional functions of SR proteins at a post-splicing level.

Antibodies, Monoclonal↗

PubMed1995

The branch site adenosine is recognized differently for the two steps of pre-mRNA splicing.

The functional groups responsible for branch site identity in the two steps of pre-mRNA splicing as well as for spliceosome assembly were tested by incorporation of site-specific modifications at the branch site of a pre-mRNA. These results show that recognition of the adenosine occurs early in complex formation and that the branch site adenosine is recognized differently before the first step and for the second step. Further, direct UV cross-linking with these modified RNAs was used to identify a factor whose interaction was dependent upon the identity of the branch site nucleotide.

Adenosine↗