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Biomedical subjects

P A Sharp

Publications and source records attributed to P A Sharp.

At least 91 records · Page 5Linked to original sources
PubMed1993

The basic domain of Rev from human immunodeficiency virus type 1 specifically blocks the entry of U4/U6.U5 small nuclear ribonucleoprotein in spliceosome assembly.

Human immunodeficiency virus type 1 (HIV-1) encodes a regulatory protein, Rev, which is required for cytoplasmic expression of incompletely spliced viral mRNA. Rev binds to a cis-acting Rev-responsive element (RRE) located within the env region of HIV-1. It has previously been shown that a 17-amino-acid peptide, corresponding to the basic domain of Rev, specifically inhibited in vitro the splicing of mRNAs containing the RRE. In this reaction, the peptide acts after an ATP-dependent step in the spliceosome assembly resulting in an accumulation of a 45-50S splicing-deficient complex. Characterization of this complex revealed that the basic domain of Rev does not interfere with U1 small nuclear ribonucleoprotein binding but blocks the entry of U4, U5, and U6 small nuclear RNAs into the spliceosome. Binding of U2 small nuclear ribonucleoprotein was partially inhibited. The critical nature of the oligomeric structure of RRE has been investigated both in vitro and in vivo. Reporter genes that contained one, three, or six repeated-monomer high-affinity Rev binding sites (IIB) within an intron yielded a correlation among the oligomeric state of bound Rev; inhibition of splicing; ability to block the assembly of U4, U5, and U6 small nuclear RNAs in the spliceosome in vitro; and level of Rev response in vivo.

Base Sequence↗
PubMed1993

TATA-binding protein and associated factors in polymerase II and polymerase III transcription.

Transcription by RNA polymerase I (pol I), pol II, and pol III requires the TATA-binding protein (TBP). This protein functions in association with distinct TBP-associated factors (TAFs) which may specify the nature of the polymerase selected for initiation at a promoter site. In the pol III transcription system, the TBP-TAF complex is a component of the TFIIIB factor. This factor has been resolved into a TBP-TAF complex and another component, both of which are required for reconstitution of transcription by pol III. Neither the TBP-TAF complexes B-TFIID and D-TFIID, which were previously characterized as active for pol II transcription, nor TBP alone can complement pol III transcription reactions that are dependent upon the TBP-TAF subcomponent of TFIIIB. Surprisingly, the TBP-TAF subcomponent of TFIIIB is active in reconstitution of pol II transcription.

DNA-Binding Proteins↗
PubMed1992

Myc/Max and other helix-loop-helix/leucine zipper proteins bend DNA toward the minor groove.

A distinct family of DNA-binding proteins is characterized by the presence of adjacent "basic," helix-loop-helix, and leucine zipper domains. Members of this family include the Myc oncoproteins, their binding partner Max, and the mammalian transcription factors USF, TFE3, and TFEB. Consistent with their homologous domains, these proteins bind to DNA containing the same core hexanucleotide sequence CACGTG. Analysis of the conformation of DNA in protein-DNA complexes has been undertaken with a circular permutation assay. Large mobility anomalies were detected for all basic/helix-loop-helix/leucine zipper proteins tested, suggesting that each protein induced a similar degree of bending. Phasing analysis revealed that basic/helix-loop-helix/leucine zipper proteins orient the DNA bend toward the minor groove. The presence of in-phase spacing between adjacent binding sites for this family of proteins in the immunoglobulin heavy-chain enhancer suggests the possible formation of an unusual triple-bended structure and may have implications for the activities of Myc.

Base Sequence↗
PubMed1992

Composition of transcription factor B-TFIID.

Initiation of transcription by RNA polymerase II requires a TFIID factor, which can recognize the TATA element common to many promoters. Two distinct multisubunit TFIID factors can be resolved from extracts of mammalian cells, and both of them contain the well-characterized TATA-binding protein (TBP) and are capable of supporting RNA polymerase II transcription in an in vitro reaction system. The smaller complex, B-TFIID, was purified and its subunit composition was determined. B-TFIID consists of two subunits: the TBP and a TBP-associated factor (TAF) of 170 kDa. This TAF is specific for B-TFIID and appears not to be present in the D-TFIID complex. Furthermore, it was found that the highly purified B-TFIID fractions have (d)ATPase activity.

Adenosine Triphosphatases↗
PubMed1992

The p50 subunit of NF-kappa B associates with the NF-IL6 transcription factor.

The NF-kappa B-p50 polypeptide, a member of the Rel family of transcription factors, was produced as a fusion protein containing amino-terminal peptide additions that facilitate purification and detection with a monoclonal antibody and specific radiolabeling by phosphorylation in vitro. The 32P-labeled NK-kappa B-p50 fusion polypeptide was used as the probe in Western blotting experiments and in screenings of a bacteriophage expression library to isolate cDNAs encoding interacting protein domains. As expected, cDNAs encoding proteins of the Rel family were identified. Surprisingly, the 32P-labeled NF-kappa B protein also specifically bound to proteins encoded by cDNAs for the human NF-IL6 transcription factor. The NF-kappa B-p50 and NF-IL6 proteins directly interact, and the Rel homology domain and leucine-zipper motif, respectively, are important for this interaction. Since induction of the NF-kappa B and NF-IL6 factors are important events in immune and acute-phase responses, this interaction could permit coregulation of genes.

Amino Acid Sequence↗
PubMed1992

Maternal inheritance of atopic IgE responsiveness on chromosome 11q.

Atopy is a common familial state underlying allergic asthma and rhinitis. Lately, we have assigned a gene for atopy to chromosome 11q by linkage to the marker D11S97. Since previous studies have suggested that the risk of atopy is higher for children of atopic mothers than for those of atopic fathers, we sought differences between maternal and paternal patterns of transmission at the 11q13 locus among pairs of siblings in families affected by atopy. When we defined atopy as the presence of a positive skinprick test (greater than or equal to 2 mm) to any of a panel of common allergens, a higher than normal concentration of total serum IgE, or a positive radioallergosorbent test for a specific IgE, we found that 125 (62%) of the sibling-pairs affected by atopy shared the maternal 11q13 allele and 78 (38%) did not. This distribution differs significantly from the expected 50/50 distribution (p = 0.001). Of paternally derived alleles, 83 (46%) were shared and 96 (54%) were not (not significantly different from 50/50). The result was similar whatever definition of atopy was used and with other genetic markers on 11q. These findings show that transmission of atopy at the chromosome 11q locus is detectable only through the maternal line. The pattern of inheritance is consistent either with paternal genomic imprinting or with maternal modification of developing immune responses.

Alleles↗
PubMed1992

A dominant mutation in the Wilms tumor gene WT1 cooperates with the viral oncogene E1A in transformation of primary kidney cells.

Wilms tumor is a pediatric kidney cancer that has been linked to the inactivation of a tumor-suppressor gene at chromosome locus 11p13. The WT1 gene, mapping to this locus, is developmentally regulated in the kidney and encodes a putative transcription factor that has been shown to be mutated in Wilms tumor specimens. We have suggested that one such altered product of the WT1 gene may be capable of trans-dominant suppression, since the mutated allele was found to be coexpressed with the wild-type allele in a sporadic Wilms tumor. We therefore tested the ability of this mutant WT1 allele, containing an in-frame deletion within the DNA-binding domain, to transform primary baby rat kidney cells. The mutant WT1 gene was found to cooperate with the adenoviral E1A gene in transforming baby rat kidney cells, as demonstrated by growth in soft agar and tumorigenicity in nude mice. The wild-type WT1 gene in all of its alternatively spliced forms neither suppressed E1A-induced focus formation nor cooperated with E1A. Our results indicate that impairment of DNA binding of the WT1 tumor-suppressor gene product can result in a dominant negative mutation.

Adenovirus Early Proteins↗
PubMed1992

Site-specific modification of pre-mRNA: the 2'-hydroxyl groups at the splice sites.

A simple and efficient method for synthesizing long, site-specifically modified RNA molecules was developed whereby segments of RNA were joined with the use of bacteriophage T4 DNA ligase. A single hydrogen or O-methyl group was substituted for the 2'-hydroxyl group at either splice site of a nuclear pre-messenger RNA substrate. Splicing of the modified pre-messenger RNA's in vitro revealed that, although a 2'-hydroxyl is not absolutely required at either splice site, the 2'-hydroxyl at the 3' splice site is important for the second step of splicing. These results are compared to previous studies of analogous 2'-hydroxyl groups in the self-splicing Tetrahymena group I intron.

Animals↗
PubMed1992

Promoter specificity of basal transcription factors.

Regulation of expression of protein-encoding genes in eukaryotes is frequently mediated by sequence-specific transcription factors that control the activities of the basal factors and RNA polymerase II. Basal factors have been considered to be essential for all polymerase II promoters. Studies of the basal factor requirements for transcription from the immunoglobulin heavy chain gene (IgH) core promoter and the adenovirus major late gene core promoter (MLP) suggest that this paradigm is too simple. Basal transcription from the IgH promoter was reconstituted by TFIID, TFIIB, TFIIF, and polymerase, whereas basal transcription from the MLP is highly dependent upon TFIIE in addition to the above factors. Two novel protein activities, referred to as 700 kd and 90 kd, further stimulated the basal reaction from the MLP. Thus, these data indicate that not all basal factors are in fact general.

Base Sequence↗
PubMed1992

Specific binding of a basic peptide from HIV-1 Rev.

Human immunodeficiency virus type I (HIV-1) encodes a regulatory protein, Rev, which is required for cytoplasmic expression of incompletely spliced viral mRNA. Rev activity is mediated through specific binding to a cis-acting Rev responsive element (RRE) located within the env region of HIV-1. A monomer Rev binding site corresponding to 37 nucleotides of the RRE (IIB RNA) was studied by RNA footprinting, modification interference experiments and mutational analysis. Surprisingly, a 17 amino acid peptide, corresponding to the basic domain of Rev, binds specifically to this site at essentially identical nucleotides and probably induces additional base pairing. The Rev protein and related peptide interact primarily with two sets of nucleotides located at the junction of single and double stranded regions, and at an additional site located within a helix. This suggests that the domains of proteins responsible for specific RNA binding can be remarkably small and that the interaction between RNA and protein can probably induce structure in both constituents.

Amino Acid Sequence↗
PubMed1992

Recognition of the surface of a homeo domain protein.

Homeo domain proteins exhibit distinct biological functions with specificities that cannot be predicted by their sequence specificities for binding DNA. Recognition of the surface of the Oct-1 POU homeo domain provides a general model for the contribution of selective protein-protein interactions to the functional specificity of the homeo domain family of factors. The assembly of Oct-1 into a multiprotein complex on the herpes simplex virus alpha/IE enhancer is specified by the interactions of its homeo domain with ancillary factors. This complex (C1 complex) is composed of the viral alpha TIF protein (VP16), Oct-1, and one additional cellular component, the C1 factor. Variants of the Oct-1 POU homeo domain were generated by site-directed mutagenesis, which altered the residues predicted to form the exposed surface of the domain-DNA complex. Proteins with single amino acid substitutions on the surface of either helix 1 or 2 of the Oct-1 POU homeo domain had decreased abilities to form the C1 complex. The behavior of these mutants in a cooperative DNA-binding assay with alpha TIF suggested that the Oct-1 POU homeo domain is principally recognized by alpha TIF in the C1 complex. The preferential recognition of Oct-1 over the closely related Oct-2 protein is critically influenced by a single residue on the surface of helix 1 because the introduction of this residue into the Oct-2 POU homeo domain significantly enhanced its ability to form a C1 complex.

Amino Acid Sequence↗
PubMed1992

Factors confounding genetic linkage between atopy and chromosome 11q.

The results of testing for linkage between atopy and the chromosome 11 marker D11S97 is shown for all the 723 subjects genotyped by us up to January 1992. Lod score estimations were confounded by the high population prevalence of atopy, maternal inheritance of atopy at the 11q locus, genetic heterogeneity, and excess of atopy in families not ascertained through a single proband. Affected sib-pair analysis shows evidence for linkage which is not dependent on the definition of atopy or model specification. We suggest that presentation of sib-pair data will be suitable for meta-analysis of the different studies of genetic linkage and atopy.

Alleles↗
PubMed1992

Rapid stimulatory effect of bradykinin on glucose transport across the brush-border and basolateral membranes of rat jejunal enterocytes.

Bradykinin has been shown to stimulate sugar uptake by intact intestine, but the cellular locus of this action is unknown. In this study on rat we show that bradykinin (10(-7) M) stimulates both sodium-dependent glucose uptake at the brush-border membrane and carrier-mediated movement across the basolateral membrane of upper villus enterocytes. These results suggest a role for bradykinin in the local control of glucose transport though the mechanisms underlying these effects have still to be elucidated.

Animals↗
PubMed1992

Confirmation of genetic linkage between atopic IgE responses and chromosome 11q13.

Genetic linkage between atopic IgE responses and chromosome 11q13 (D11S97) has been previously reported in a limited number of extended families. Difficulties of phenotyping in the older family members, poor family structure in some families, and genetic heterogeneity were proposed as possible explanations for the variability in lod scores. To test this finding a second linkage study of 64 young nuclear families was undertaken and gave a two point lod score of 3.8 at theta = 0.07 (assuming theta m = theta f). A test of genetic heterogeneity in the nuclear families shows that atopic IgE responses are linked to this locus in 60 to 100% of families (approximate 95% confidence limits).

Adolescent↗
PubMed1991

Identification of novel factors which bind specifically to the core promoter of the immunoglobulin heavy chain gene.

Nuclear extracts from HeLa cells and the B-cell line, BJA-B, generated two protein-DNA complexes which bound specifically to sequences in the TATA box of the immunoglobulin heavy chain gene (IgH) promoter. Complex A also bound the core promoter of a retroviral long terminal repeat but did not bind to five other promoters including the adenovirus major late promoter. Of these seven promoters, complex B bound only to the IgH promoter. Footprinting analysis revealed that both complexes A and B bound sequences which include the TATA element, and complex A additionally contacted sequences downstream to +28. Mutation of the IgH TATA element from ATTAATATA to GCTA-TAAAA, the optimal TATA sequence as found in the major late promoter, resulted in a 10-fold decrease in binding to complex A and a 25-fold decrease in binding complex B. Surprisingly, both transfection experiments in HeLa cells and in vitro transcription experiments with whole nuclear extract demonstrated that mutation of the TATA box in the core IgH promoter to this consensus sequence resulted in a 2-fold decrease in the level of transcription. These data suggest that the specific sequence of the TATA region is important, and factors which recognize these sequences, such as complex A and B, may modulate the level of transcription from the IgH promoter.

Base Sequence↗