Showing papers by "Phillip A. Sharp published in 1991"

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

Abstract: 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.

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

Abstract: 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.

Five easy pieces

Abstract: Why should wait for some days to get or receive the five easy pieces book that you order? Why should you take it if you can get the faster one? You can find the same book that you order right here. This is it the book that you can receive directly after purchasing. This five easy pieces is well known book in the world, of course many people will try to own it. Why don't you become the first? Still confused with the way?

Characterization of cDNAs encoding the polypyrimidine tract-binding protein.

Abstract: 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.

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

Abstract: 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.

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

Abstract: 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.

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

Abstract: 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.

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

Abstract: 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 phenanthrolinecopper 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

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

Abstract: 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.