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Biomedical subjects

D C Rio

Publications and source records attributed to D C Rio.

At least 19 recordsLinked to original sources
PubMed1992

P element transposition in vitro proceeds by a cut-and-paste mechanism and uses GTP as a cofactor.

We have developed an in vitro reaction system for Drosophila P element transposition. Transposition products were recovered by selection in E. coli, and contained simple P element insertions flanked by 8 bp target site duplications as observed in vivo. Transposition required Mg+2 and partially purified P element transposase. Unlike other DNA rearrangement reactions, P element transposition in vitro used GTP as a cofactor; deoxyGTP, dideoxyGTP, or the nonhydrolyzable GTP analogs GMP-PNP or GMP-PCP were also used. Transposon DNA molecules cleaved at the P element termini were able to transpose, but those lacking 3'-hydroxyl groups were inactive. These biochemical data are consistent with genetic data suggesting that P element transposition occurs via a "cut-and-paste" mechanism.

Animals↗
PubMed1992

RNA processing.

Significant progress has been made over the last year in our understanding of the roles that RNA-binding proteins play in pre-mRNA splicing, the components of the spliceosome and how these components relate to the mechanism of splicing. Of particular importance has been the sequence analysis of the first mammalian splicing factors and structural determination of an RNA-binding domain.

Animals↗
PubMed1992

The mechanism of somatic inhibition of Drosophila P-element pre-mRNA splicing: multiprotein complexes at an exon pseudo-5' splice site control U1 snRNP binding.

Somatic inhibition restricts splicing of the Drosophila P-element third intron (IVS3) to the germ line. We have exploited this simple system to provide a model for a mechanism of alternative pre-mRNA splicing. Biochemical complementation experiments revealed that Drosophila somatic extracts inhibited U1 snRNP binding to the 5' splice site. Using sensitive RNase protection and modification-interference assays, we found that U1 snRNP bound to a pseudo-5' splice site in the 5' exon and that multiprotein complexes bound to an adjacent site. Binding of these factors appeared to mediate the inhibitory effect, because mutations in the pseudo-5' splice sites blocked binding and activated splicing in vitro. Likewise, wild-type, but not mutant, 5' exon RNA titrated inhibitory factors away from the pre-mRNA and activated splicing. Thus, we have defined the pseudo-5' splice sites as crucial components of the regulatory element, correlated the inhibitory activity with specific RNA binding factors from Drosophila somatic cells, and provided a mechanistic description of somatic inhibition. Because the inhibitory activity involves general splicing functions such as protein recognition of 5' splice site sequences and changes in the distribution of bound U1 snRNP, our data may also provide insights into how splice sites are selected.

Animals↗
PubMed1991

Drosophila P-element transposase is a transcriptional repressor in vitro.

Mobility of P transposable elements in Drosophila melanogaster depends on the 87-kDa transposase protein encoded by the P element. Transposase recognizes a 10-base-pair DNA sequence that overlaps an A + T-rich region essential for transcription from the P-element promoter. We report here that transposase represses transcription from the P-element promoter in vitro. This transcriptional repression is blocked by prior formation of an RNA polymerase II transcription complex on the template DNA. Binding of transposase on the P-element promoter is blocked by prior binding of either the Drosophila RNA polymerase II complex or the yeast transcription factor TFIID. These data suggest that transposase represses transcription by preventing assembly of an RNA polymerase II complex at the P-element promoter.

Animals↗
PubMed1991

Regulation of Drosophila P element transposition.

Drosophila P transposable elements are the best-studied family of eukaryotic non-retroviral transposons. P element transposition is regulated in several different ways and has thus provided a unique system with which to study the control of DNA rearrangements and gene expression in metazoans. Recent genetic and biochemical experiments have begun to shed light on the mechanism of P element transposition and the mechanisms controlling the temporal and spatial patterns of transposition.

Animals↗
PubMed1990

Cytotype control of Drosophila P element transposition: the 66 kd protein is a repressor of transposase activity.

Drosophila P transposable elements encode two proteins, an 87 kd transposase protein and a 66 kd protein that has been hypothesized to repress transposition. We have made germline transformants carrying modified P element derivatives that encode only the 66 kd protein and shown that these elements repress transposase activity in both the germline and the soma. The position of these elements in the genome quantitatively affected their ability to negatively regulate transposase and to express the 66 kd protein. Single 66 kd element-containing strains did not exhibit the maternal inheritance of P cytotype characteristic of P strains. However, we demonstrated that a true P strain produced higher levels of the 66 kd protein during oogenesis than single 66 kd P elements. Thus, the expression of the 66 kd repressor during oogenesis may be a major determinant of the maternal effect of P cytotype.

Animals↗
PubMed1990

Regulated splicing of the Drosophila P transposable element third intron in vitro: somatic repression.

In eukaryotic cells alternative splicing of messenger RNA precursors (pre-mRNA's) is a means of regulating gene expression. Although a number of the components that participate in regulating some alternative splicing events have been identified by molecular genetic procedures, the elucidation of the biochemical mechanisms governing alternative splicing requires in vitro reaction systems. The tissue specificity of P element transposition in Drosophila depends on the germline restriction of pre-mRNA splicing of the P element third intron (IVS3). Drosophila P element IVS3 pre-mRNA substrates were spliced accurately in vitro in heterologous human cell extracts but not in Drosophila somatic cell splicing extracts. Components in Drosophila somatic cell extracts that specifically inhibited IVS3 splicing in vitro were detected by a complementation assay. Biochemical assays for Drosophila RNA binding proteins were then used to detect a 97-kilodalton protein that interacts specifically with 5' exon sequences previously implicated in the control of IVS3 splicing in vivo. Inhibition of IVS3 splicing in vitro could be correlated with binding of the 97-kD protein to 5' exon sequences, suggesting that one aspect of IVS3 tissue-specific splicing involves somatic repression by specific RNA-protein interactions.

Animals↗
PubMed1989

Drosophila P element transposase recognizes internal P element DNA sequences.

Drosophila P transposable elements encode an 87 kd trans-acting protein, transposase, that is required to catalyze P element transposition and excision. We show here that purified transposase is a site-specific DNA binding protein. P element transposase does not interact with the terminal 31 bp inverted repeats but instead interacts specifically with an internal 10 bp consensus sequence present at both the 5' and 3' ends of P element DNA. These binding sites lie within sequences known to be important for transposition in vivo. Transposase also displays an unusually high nonspecific affinity for DNA. The transposase binding site at the 5' and overlaps sequences we show to be essential for transcription from the P element promoter in vitro, which raises the possibility that either transposase or the related 66 kd P element protein may affect P element transcription. From these and other observations, we suggest that the P element transposition reaction probably requires the binding of additional Drosophila protein factors to the terminal DNA sequences.

Animals↗
PubMed1989

cis-acting DNA sequence requirements for P-element transposition.

The P transposable element of Drosophila melanogaster has a complex array of cis-acting DNA sequences necessary for efficient transposition. At the 3' end these sequences extend over more than 150 bp and include 11- and 31-bp sequences found repeated in inverted orientation at the 5' end. The P element's 5' end, however, cannot function as its 3' end. When two 3' P-element ends are present, the more proximal end is used preferentially. We found also that the duplication of the target site does not appear to play a role in forward transposition.

Animals↗
PubMed1988

Evidence for Drosophila P element transposase activity in mammalian cells and yeast.

Drosophila P element transposase expression is limited to the germline by tissue-specific splicing of one of its three introns. Removal of this intron by mutagenesis in vitro has allowed both P element excision and transposition to be detected in Drosophila somatic tissues. In order to determine if P element transposase can function in other organisms, we have expressed modified P elements either lacking one intron or lacking all three introns in mammalian cells and yeast, respectively. Using an assay for P element excision, we have detected apparent excision events in cultured monkey cells. Furthermore, expression of the complete P element cDNA is lethal to Saccharomyces cerevisiae cells carrying a mutation in the RAD52 gene, indicating that double-stranded DNA breaks are generated, presumably by transposase action.

Animals↗
PubMed1988

Identification and purification of a Drosophila protein that binds to the terminal 31-base-pair inverted repeats of the P transposable element.

We have used DNase I footprinting and partially fractionated nuclear extracts from Drosophila Kc tissue culture cells to identify DNA-binding proteins that interact with the terminal repeats of P transposable elements. We have identified a binding activity that interacts specifically with a region of the 31-base-pair terminal inverted repeats that is directly adjacent to the duplication of target site DNA. Binding occurs to both the 5' and 3' inverted terminal repeats irrespective of the sequence of the duplicated target DNA. UV photochemical crosslinking studies suggest that the binding activity resides in a polypeptide of 65-70 kDa. Biochemical fractionation and oligonucleotide affinity chromatography have been used to purify the binding activity to near homogeneity and identify a polypeptide of 66 kDa in the highly purified preparations. The site to which binding occurs is included in a region absolutely required for P element transposition, suggesting that this binding protein may be a cellular factor involved in P element transposition.

Animals↗
PubMed1988

Accurate and efficient pre-mRNA splicing in Drosophila cell-free extracts.

Synthetic mRNA precursors from the Drosophila fushi tarazu (ftz) gene were shown to be accurately and efficiently spliced in Drosophila nuclear extracts derived from Kc tissue culture cells or 0- to 12-hr embryos. Splicing the ftz pre-mRNA requires ATP and low levels of Mg2+. The reaction proceeds with a lag of 20-30 min prior to appearance of spliced mRNA and appears to proceed in two steps. The first step is cleavage at the 5' splice site to generate a 5' exon (E1) fragment and an intron-3' exon (IVS-E2) species. The second step involves cleavage at the 3' splice site, ligation of the two exons (E1-E2), and intron (IVS) release. The excised intron (IVS) and intron-3' exon (IVS-E2) exhibit anomalous electrophoretic mobility, suggesting that they contain branched structures. Nuclease analysis using two-dimensional thin-layer chromatography indicates that both the IVS and IVS-E2 species possess branched trinucleotides in which a guanosine residue at the 5' end of the intron is linked in a 2'-5' phosphodiester bond to the 2' hydroxyl group of an adenosine residue in the intron. The site of branchpoint formation was localized by debranching the Drosophila lariat with mammalian (HeLa) cell debranching enzyme and by P1 and T2 nuclease analysis. These findings indicate that nuclear extracts derived from Drosophila cultured cells or embryos can accurately splice mRNA precursors and that the reaction mechanism is the same as has been observed in yeast and mammalian cells. This system provides an initial step toward the biochemical analysis of developmentally regulated pre-mRNA splicing events in Drosophila.

Animals↗
PubMed1986

Tissue specificity of Drosophila P element transposition is regulated at the level of mRNA splicing.

We show that the germline specificity of P element transposition is controlled at the level of mRNA splicing and not at the level of transcription. In the major P element RNA transcript, isolated from somatic cells, the first three open reading frames are joined by the removal of two introns. Using in vitro mutagenesis and genetic analysis we demonstrate the existence of a third intron whose removal is required for transposase production. We propose that this intron is only removed in the germline and that its removal is the sole basis for the germline restriction of P element transposition.

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