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Biomedical subjects

P A Sharp

Publications and source records attributed to P A Sharp.

At least 109 records · Page 6Linked to original sources
PubMed1991

Specific regulation of mRNA splicing in vitro by a peptide from HIV-1 Rev.

The Rev protein of HIV-1 regulates the synthesis of partially spliced forms of cytoplasmic viral mRNA by binding to a cis-acting RNA sequence, the Rev response element (RRE). We have investigated the regulation of splicing in vitro and have shown that Rev specifically inhibits splicing of pre-mRNAs containing an RRE by 3- to 4-fold. A synthetic peptide of 17 amino acids containing the RNA-binding domain of Rev is highly functional and specifically inhibits splicing by up to 30-fold. Other peptides that bind to the RRE with high affinity, but with low specificity, do not specifically inhibit splicing. Six repeated monomeric binding sites for the peptide can substitute for the RRE, indicating that regulation by Rev requires interactions with multiple sites. The peptide acts at a step in the assembly of splicing complexes, suggesting that one of the functions of the basic region of Rev is to prevent formation of a functional spliceosome.

Amino Acid Sequence↗
PubMed1991

RNA polymerase II-associated proteins are required for a DNA conformation change in the transcription initiation complex.

Proteins purified on the basis of their affinity for RNA polymerase II effectively substitute for previously defined transcription initiation factors. In two assays, formation of initiation complexes and transcription in vitro, the RNA polymerase II-associated proteins behaved identically to a fraction containing transcription factors IIE and IIF. Both fractions greatly stabilized the association of polymerase with the promoter and were required for the formation of complete initiation complexes. By using the DNA-cleaving reagent phenanthroline.copper in footprinting reactions, the RNA polymerase II-associated proteins were shown to be required for a DNA conformation change near the initiation site of the promoter. Based on similarity to the prokaryotic transcription complex, this conformation change is likely to represent a transition from a closed to an open complex.

Cellulose↗
PubMed1991

Exon amplification: a strategy to isolate mammalian genes based on RNA splicing.

We have developed a method, exon amplification, for fast and efficient isolation of coding sequences from complex mammalian genomic DNA. This method is based on the selection of RNA sequences, exons, which are flanked by functional 5' and 3' splice sites. Fragments of cloned genomic DNA are inserted into an intron, which is flanked by 5' and 3' splice sites of the human immunodeficiency virus 1 tat gene contained within the plasmid pSPL1. COS-7 cells are transfected with these constructs, and the resulting RNA transcripts are processed in vivo. Splice sites of exons contained within the inserted genomic fragment are paired with splice sites of the flanking tat intron. The resulting mature RNA contains the previously unidentified exons, which can then be amplified via RNA-based PCR and cloned. Using this method, we have isolated exon sequences from cloned genomic fragments of the murine Na,K-ATPase alpha 1-subunit gene. We have also screened randomly selected genomic clones known to be derived from a segment of human chromosome 19 and have isolated exon sequences of the DNA repair gene ERCC1. The sensitivity and ease of the exon amplification method permit screening of 20-40 kilobase pairs of genomic DNA in a single transfection. This approach will be extremely useful for rapid identification of mammalian exons and the genes from which they are derived as well as for the generation of chromosomal transcription maps.

Animals↗
PubMed1991

Negative regulation of the major histocompatibility complex class I promoter in embryonal carcinoma cells.

Transcription of major histocompatibility complex (MHC) class I genes is negatively regulated in undifferentiated F9 mouse embryonal carcinoma cells via the conserved upstream regulatory region. This region contains constitutive enhancers and an inducible enhancer, the interferon consensus sequence (ICS), that is responsible for interferon-induced transcription. A series of mutations in the ICS, but not in the enhancer elements, resulted in an increase in expression of the MHC class I promoter in F9 cells. However, these ICS mutants did not increase promoter activity in F9 cells differentiated after retinoic acid treatment. Results of mobility-shift DNA-binding assays and methylation interference experiments showed that undifferentiated F9 cells contained a factor(s) that bound to a sequence within the 5' and central part of the ICS. This binding site, termed the MHC negative regulatory element (NRE), coincided with the site of mutations that increased promoter activity in F9 cells and was distinct from the element to which interferon-response factors bind. The factor(s) that binds to the MHC NRE was not detected in differentiated F9 cells treated with retinoic acid or in other cells expressing MHC class I genes. Finally, introduction of concatenated, double-stranded NRE oligomers, but not oligomers of unrelated sequences, into F9 cells abolished negative regulation of the MHC class I promoter activity, providing evidence that the NRE binding factor is responsible for repression of the MHC class I genes in F9 cells.

Animals↗
PubMed1991

Structural analysis of the interaction between the human immunodeficiency virus Rev protein and the Rev response element.

The specific interaction between a defined structural element of the human immunodeficiency virus mRNA (RRE, the Rev response element) and the virus-encoded protein Rev has been implicated in the regulation of the export of unspliced or singly spliced mRNA from the nucleus to the cytoplasm. Rev protein was expressed and purified from insect cells using the baculovirus expression system. Chemical and RNase probes were used to analyze the structure of the RRE and the regions involved in Rev binding. Increased reactivity to single-strand-specific probes of nucleotides in two helical domains indicates that Rev binding induces conformational changes in the RRE. Binding of Rev to the RRE primarily protects helical segments and adjacent nucleotides in domain II. A Rev unit binding site is proposed that consists of a six-base-pair helical segment and three adjacent nucleotides. The data also suggest that multiple Rev proteins bind to repeated structural elements of the RRE.

Animals↗
PubMed1991

HIV-1 Tat protein promotes formation of more-processive elongation complexes.

The Tat protein of HIV-1 trans-activates transcription in vitro in a cell-free extract of HeLa nuclei. Quantitative analysis of the efficiency of elongation revealed that a majority of the elongation complexes generated by the HIV-1 promoter were not highly processive and terminated within the first 500 nucleotides. Tat trans-activation of transcription from the HIV-1 promoter resulted from an increase in processive character of the elongation complexes. More specifically, the analysis suggests that there exist two classes of elongation complexes initiating from the HIV promoter: a less-processive form and a more-processive form. Addition of purified Tat protein was found to increase the abundance of the more-processive class of elongation complex. The purine nucleoside analog, 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB) inhibits transcription in this reaction by decreasing the efficiency of elongation. Surprisingly, stimulation of transcription elongation by Tat was preferentially inhibited by the addition of DRB.

Dichlororibofuranosylbenzimidazole↗
PubMed1991

The mammalian TFIID protein is present in two functionally distinct complexes.

The TFIID activity recognizes a TATA-box element and supports formation of an initiation complex containing RNA polymerase II. Antisera specific for the 38-kD human TFIID protein were used to determine whether this protein cofractionated with the TFIID activity. Surprisingly, the TFIID activity in HeLa whole-cell extracts was resolved into two different size complexes, one of 300 kD and one of greater than 700 kD. Cofractionation studies suggest that both complexes contain the 38-kD protein; thus, this component of the large complexes is probably responsible for recognition of the TATA sequence and interaction with the other general transcription factors in formation of the initiation complex. Interestingly, in contrast to the TFIID activity characterized previously, the 300-kD form of TFIID activity, B-TFIID, does not support stimulation of transcription by factors containing acidic or glutamine-rich activating motifs. We propose that the functional and physical differences between these two forms of TFIID activity are caused by differences in the protein composition of the TFIID complexes of which the 38-kD hTFIID protein is an integral part.

Enzyme-Linked Immunosorbent Assay↗
PubMed1991

TFEB has DNA-binding and oligomerization properties of a unique helix-loop-helix/leucine-zipper family.

The DNA-binding factor TFEB contains adjacent helix-loop-helix (HLH) and leucine zipper (LZ) domains flanked by an upstream basic region. This arrangement of interactive motifs has recently been observed in several other transcription factors and in the Myc family of oncogenes. TFEB was isolated by virtue of its binding to the major late promoter of adenovirus. DNA binding by a soluble protein was achieved by deleting a hydrophobic amino-terminal domain and permitted the structural analysis of the oligomerization and DNA-binding properties of TFEB. TFEB specifically bound DNA as both a homodimer and a heterodimer with another b-HLH-LZ protein TFE3. The LZ domain was essential for homo- or hetero-oligomerization and high-affinity DNA binding. In the absence of DNA a tetramer-sized form of TFEB was observed that dissociates to bind added DNA as a dimer. Binding by TFEB and TFE3 to related, but different, naturally occurring DNA target sequences was observed with distinct binding preferences. Analysis of basic domain residues in this family of proteins revealed a pattern of sequence conservation predictive of an interacting alpha-helical face. Common oligomerization and DNA-binding features suggest the b-HLH-LZ domain structure to define a distinct family of DNA-binding factors.

Adenoviridae↗
PubMed1991

Characterization of cDNAs encoding the polypyrimidine tract-binding protein.

The polypyrimidine tract of mammalian introns is recognized by a 62-kD protein (pPTB). Mutations in the polypyrimidine tract that reduce the binding of pPTB also reduce the efficiency of formation of the pre-spliceosome complex containing U2 snRNP. The PTB protein was purified to homogeneity by affinity chromatography on a matrix containing poly(U), and peptide sequence was used to isolate several cDNAs. Because a variety of cell types express mRNA complementary to these cDNAs, PTB may be a ubiquitous splicing factor. Three classes of cDNAs were identified, on the basis of the presence of additional sequences at an internal position. This variation in sequence probably reflects alternative splicing of the PTB pre-mRNA and produces mRNAs encoding the prototype PTB protein, a form of PTB protein containing 19 additional residues, and a truncated form of PTB protein with a novel carboxyl terminus. A murine homolog of pPTB has been characterized previously as a DNA-binding protein. Sequence comparisons indicate that pPTB is distantly related to the hnRNP L protein and that these two proteins should be considered as members of a novel family of RNA-binding proteins.

Amino Acid Sequence↗
PubMed1990

HIV-1 Tat protein trans-activates transcription in vitro.

Tat protein of human immunodeficiency virus 1 is a potent trans-activator of viral gene expression. We show that purified Tat protein stimulates transcription from viral promoters greater than 10-fold in vitro. A Tat protein mutant that does not trans-activate in vivo did not stimulate transcription in vitro. Tat trans-activation required a functional TAR RNA sequence; trans-activation was competed by the addition of in vitro synthesized wild-type TAR RNA but not by mutant TAR RNAs. That Tat protein directly interacts with the TAR RNA during trans-activation in vitro was suggested by competition with Tat peptides. Preliminary evidence suggests the involvement of a cellular factor in recognition of TAR RNA during Tat trans-activation. Analysis of Tat trans-activation in vitro will provide new mechanistic insights into this process and allow a more detailed study of the relationship between Tat protein structure and function.

Amino Acid Sequence↗
PubMed1990

Structure of RNAs replicated by the DNA-dependent T7 RNA polymerase.

The DNA-dependent RNA polymerase of bacteriophage T7 efficiently and specifically replicates two structurally related RNAs, termed X and Y RNAs. Replication of both RNAs involves synthesis of complementary strands initiated with pppC and pppG. RNAs transcribed from DNA template containing the established sequences of X and Y RNAs were efficiently replicated by T7 RNA polymerase. Both RNAs possess palindromic sequences with a dual axis of symmetry, permitting formation of hairpin-, dumbbell-, or cloverleaf-type structures. The template must consist of RNA and not DNA sequence, and the terminal unpaired dinucleotides of the RNA are necessary for replication. Nucleotidyl transferase activity of E. coli adenylates the unpaired CCOH dinucleotide at the 3' end of a C strand of X RNA. This feature, as well as the length (64 nucleotides) and compact structure of X and Y RNAs, suggests that they may resemble tRNA molecules and tRNA-like structures at the 3' termini of many plant viral RNA genomes.

Base Sequence↗
PubMed1990

Human estrogen receptor forms multiple protein-DNA complexes.

A baculovirus expression system was used to overproduce the human estrogen receptor in insect cells. The estrogen receptor made in this system is full-length, binds estrogen specifically, and is recognized by a monoclonal antibody to the human estrogen receptor. The recombinant estrogen receptor binds the estrogen response element (ERE) in both the absence and presence of estrogen if the binding is carried out in the absence of Mg2+. In the presence of Mg2+, the estrogen receptor binds the ERE in a hormone-dependent fashion. This effect is more pronounced at higher temperatures. Tamoxifen, a nonsteroidal anti-estrogen, is able to stimulate ERE binding to the same extent and under the same conditions as estradiol. Estradiol stimulates formation of an estrogen receptor-ERE complex with an increased mobility in native gels as compared with the complex formed without hormone or with tamoxifen. These results demonstrate that specific DNA binding of the estrogen receptor is not absolutely dependent on the presence of hormone and that estradiol but not tamoxifen is able to induce a change in the estrogen receptor. This differential effect of estradiol and tamoxifen may be important in understanding the role of the receptor to activate target genes differentially.

Animals↗
PubMed1990

Comparison of immune-mediated models of acute and chronic otitis media.

The distribution of immunoglobulin-bearing cells and the pattern of histopathological changes in the middle ear (ME) mucosa, round window membrane (RWM), and inner ear were compared during acute and chronic immune-mediated otitis media with effusion (OME) in the guinea pig as an animal model. In both acute and chronic immune responses (IRs), mucosal hyperplasia, edema, neovascularization, and cellular infiltration were observed. IgG+ cells were predominant in both the acute and chronic IRs. The number of IgA+ cells, however, increased in the mucosa and RWM during chronic IRs. Only the chronic IR resulted in gland formation within the ME and inflammation within the cochlea. These results indicate that the chronic IR was more similar to reports of clinical OME than the acute IR. The cochlear inflammation associated with chronic OME can lead to sensorineural hearing loss, as reported in clinical studies.

Acute Disease↗
PubMed1990

A mammalian protein of 220 kDa binds pre-mRNAs in the spliceosome: a potential homologue of the yeast PRP8 protein.

A mammalian protein of approximately 220 kDa (p220) was UV-crosslinked to precursor mRNAs (pre-mRNAs) under splicing conditions. The kinetics and biochemical requirements of the UV-crosslinking of p220 corresponded to the kinetics and biochemical requirements of spliceosome formation. On Western blots, antibodies against the yeast splicing factor PRP8 recognized a doublet of proteins, the faster migrating of which comigrated with p220. Furthermore, UV-crosslinked p220 was immunoprecipitated with anti-PRP8 antisera. These results suggest structural conservation of the splicing factor PRP8 from yeast to mammals and show that this protein is in close proximity to the pre-mRNA in the spliceosome.

Blotting, Western↗