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Biomedical subjects

P A Sharp

Publications and source records attributed to P A Sharp.

At least 199 records · Page 11Linked to original sources
PubMed1985

Effect of the tripartite leader on synthesis of a non-viral protein in an adenovirus 5 recombinant.

The EIa region of an Adenovirus 5 recombinant has been substituted by a modular gene encoding dihydrofolate reductase (DHFR). In this recombinant, the mouse DHFR cDNA was positioned behind sequences of the major late promoter and the complete tripartite leader. The leader sequences end in the normal 5' splice site (SS) of the third leader, so that RNA splicing joins the tripartite leader to a 3' splice site immediately upstream of the DHFR cDNA. At late stages of infection, high levels of DHFR mRNAs were synthesized. At early times in the late stage, this mRNA was efficiently translated; however, at later times translation of DHFR decreased probably due to poor competition with other late mRNAs. Synthesis of DHFR protein from an analogous Adenovirus 5 recombinant containing only the first late leader was studied in parallel. Equivalent levels of DHFR mRNA were expressed after infection with this recombinant virus; however, the efficiency of DHFR translation was at least 20 fold lower than that of the DHFR mRNA containing the tripartite leader. This suggests that the tripartite leader sequence is important for translation in the late stage of infection. As reported previously, the Ad5 recombinant containing only the first leader vastly overexpresses polypeptide IX from a novel mRNA, formed by the splicing of the first leader in the modular DHFR gene to the 3' splice site in the EIb region. Cells infected with this recombinant synthesize very little normal mRNA from the EIb region. Here, we demonstrated that coinfection of 293 cells with this recombinant and wild type Adenovirus 5 also results in decreased EIb mRNA synthesis. We propose that the overproduction of polypeptide IX suppresses mRNA expression from the EIb and IX promoter sites, probably by an autoregulation loop active during lytic growth.

Adenoviridae↗
PubMed1985

Amber, ochre and opal suppressor tRNA genes derived from a human serine tRNA gene.

Amber, ochre and opal suppressor tRNA genes have been generated by using oligonucleotide directed site-specific mutagenesis to change one or two nucleotides in a human serine tRNA gene. The amber and ochre suppressor (Su+) tRNA genes are efficiently expressed in CV-1 cells when introduced as part of a SV40 recombinant. The expressed amber and ochre Su+ tRNAs are functional as suppressors as demonstrated by readthrough of the amber codon which terminates the NS1 gene of an influenza virus or the ochre codon which terminates the hexon gene of adenovirus, respectively. Interestingly, several attempts to obtain the equivalent virus stock of an SV40 recombinant containing the opal suppressor tRNA gene yielded virus lacking the opal suppressor tRNA gene. This suggests that expression of an efficient opal suppressor derived from a human serine tRNA gene is highly detrimental to either cellular or viral processes.

Animals↗
PubMed1985

An RNA polymerase II transcription factor binds to an upstream element in the adenovirus major late promoter.

A gel electrophoresis DNA binding assay has been used to identify proteins in HeLa cell extracts that specifically bind to the major late promoter of adenovirus. A major late promoter transcription factor MLTF has been detected as a discrete protein-DNA complex. MLTF binds specifically and with high affinity to sequences upstream of the TATA box of the major late promoter. This factor protects a 17 bp (-50 to -66) region in a DNAase I footprinting assay. The same region has been shown to be important for efficient transcription from the major late promoter both in vivo and in vitro. MLTF stimulates in vitro transcription only from a template containing this upstream region. The binding, footprinting, and transcription-stimulatory activities of MLTF cofractionate through two chromatographic steps. These results suggest that direct binding of MLTF to an upstream element activates transcription from the major late promoter.

Adenoviruses, Human↗
PubMed1985

Accurate cleavage and polyadenylation of exogenous RNA substrate.

Purified precursor RNA containing the L3 polyadenylation site of late adenovirus 2 mRNA is accurately cleaved and polyadenylated when incubated with nuclear extract from HeLa cells. The reaction is very efficient; 75% of the precursor is correctly processed. Cleavage is rapidly followed by polymerization of an initial poly(A) tract of approximately 130 nucleotides. Additional adenosine residues are added during further incubation. In the presence of the ATP analog alpha-beta-methylene-adenosine 5' triphosphate, the precursor RNA is cleaved but not polyadenylated, suggesting that processing is not coupled to the synthesis of the initial poly(A) tract. In the absence of free Mg2+, a small RNA of approximately 46 nucleotides is stabilized against degradation. Fingerprint analysis suggests this RNA is produced by endonucleolytic cleavage at the L3 site. Like the in vitro splicing reaction, the in vitro polyadenylation reaction is inhibited by adding antiserum against the small nuclear ribonucleoprotein particle containing U1 RNA.

Adenosine Triphosphate↗
PubMed1985

Trans splicing of mRNA precursors in vitro.

Two exon segments from two separate RNA molecules can be joined in a trans splicing process. In trans splicing reactions, an RNA molecule containing an exon, a 5' splice site, and adjacent intron sequences was mixed with an RNA molecule containing an exon, a 3' splice site, and adjacent intron sequences. The efficiency of trans splicing of these two RNAs increased if the two termini of the intervening sequences were paired in a short RNA duplex. However, trans splicing of two RNA molecules with no significant complementarity was also observed. These results strongly suggest that significant secondary structures within intervening sequences could affect the splicing of flanking exons. Similarly, RNAs that are complementary to segments within the intervening sequences could potentially regulate the selection of splice sites. Finally, some organisms might use trans splicing to distribute a single exon to many different mRNAs.

Adenosine Triphosphate↗
PubMed1985

A multicomponent complex is involved in the splicing of messenger RNA precursors.

A multicomponent complex termed spliceosome (splicing body) is unique to the splicing of messenger RNA precursors in vitro. This 60S RNA-protein complex contains RNAs from the previously characterized bipartite splicing intermediate, the 5' exon RNA, and the lariat intervening sequence-3' exon RNA, as well as some intact 455 nucleotide precursor RNA. This complex contains snRNPs, particularly U1 RNP, as shown by immunoprecipitation with specific antisera. Formation of the 60S complex appears to be an early and essential step in splicing, because the 60S complex forms during the early stage, or lag time, of the reaction before the first covalent modification, cleavage at the 5' splice site of precursor RNA. The 60S complex forms only under conditions that permit splicing; both ATP and a precursor RNA containing authentic 5' and 3' splice sites are required for formation, while antiserum specific for U1 RNP inhibits its formation. RNA within the 60S complex, predominantly precursor RNA, was chased into products with accelerated kinetics and more complete conversion than purified precursor RNA.

Adenosine Triphosphate↗
PubMed1985

Nonconsensus branch-site sequences in the in vitro splicing of transcripts of mutant rabbit beta-globin genes.

Mutants of the rabbit beta-globin gene lacking the natural site of branch formation in the second intervening sequence have been analyzed for in vitro splicing activity. RNAs transcribed from these mutants were spliced, via lariat formation, at a reduced rate compared to wild-type RNA. The sites of branch formation were mapped by direct RNA analysis and primer-extension analysis. The sequences at the branch sites in the three mutants examined did not conform to the previously determined consensus sequence, nor were the 5' splice sites and branch sites complementary.

Animals↗
PubMed1984

Lariat RNA's as intermediates and products in the splicing of messenger RNA precursors.

The splicing of messenger RNA precursors in vitro proceeds through an intermediate that has the 5' end of the intervening sequence joined to a site near the 3' splice site. This lariat structure, which has been characterized for an adenovirus 2 major late transcript, has a branch point, with 2'-5' and 3'-5' phosphodiester bonds emanating from a single adenosine residue. The excised intervening sequence retains the branch site and terminates in a guanosine residue with a 3' hydroxyl group. The phosphate group at the splice junction between the two exons originates from the 3' splice site at the precursor.

Adenoviruses, Human↗
PubMed1984

Gas gangrene. An 11-year review of 73 cases managed with hyperbaric oxygen.

An 11-year review is presented of 73 patients treated for gas gangrene at The Prince Henry Hospital. The geographical collection area extended throughout the States of New South Wales and Queensland and to Papua New Guinea. The commonest aetiological factor was found to be motor-vehicle trauma, motor-cycle accidents producing compound limb fractures in particular. The infecting organisms in these cases were most commonly clostridia. The diagnosis of the disease remains a clinical one. Treatment consisted of three facets: conservative surgery, as much of the limb or viable tissue as possible being preserved; high-dose penicillin administration; and hyperbaric oxygen therapy. Death occurred in 15 cases, gas gangrene being the principal cause in seven. However the incidence of gas gangrene has diminished since 1971, and there are hopes that it will decrease further through adequate prophylaxis and prevention in civilian surgical practice.

Accidents, Traffic↗
PubMed1984

Dinucleotide priming of transcription mediated by RNA polymerase II.

Mammalian RNA polymerase II was shown to utilize dinucleoside monophosphates for priming of promoter specific RNAs. In a reconstituted system containing purified polymerase and HeLa cell fractions, dinucleotides were incorporated by complementarity with template sequences at the in vivo cap sites of the adenovirus major late and adenovirus early region IV promoters. Incorporation was shown by label transfer experiments and by determining the size of 5'-terminal RNase T1-resistant oligonucleotides. All 16 dinucleotides were tested for priming of RNA chains at the major late promoter. RNA polymerase II initiated with various primers over a contiguous region of 9 bases, centered around the in vivo initiation site. We suggest that the polymerase drifts or oscillates over this region. Using a dinucleotide challenge protocol, the rate of initiation at the major late promoter was measured following preincubation of the template DNA with RNA polymerase II and factors. Initiation with ATP was 90% complete within the 1st min after addition of nucleotide triphosphates. Stimulation of transcription by dinucleotides was not observed, due to this rapid initiation. The 5'-hydroxyl terminus of dinucleotide-primed RNAs remained unmodified. Although transcripts initiated with ATP were rapidly capped in whole cell extracts, ATP-primed RNA synthesized in the reconstituted system retained free 5'-terminal phosphates. Thus, capping was not essential for synthesis of long runoff RNAs.

Adenosine Triphosphate↗
PubMed1984

Interactions between RNA polymerase II, factors, and template leading to accurate transcription.

Accurate transcription by RNA polymerase II has been shown to require multiple factors in addition to the purified polymerase. In this study, we use a reconstituted transcription system, consisting of purified RNA polymerase II and three essential HeLa cell chromatographic fractions, to study events leading to transcription from the adenovirus major late promoter. A preincubation-pulse-chase protocol resolves the reaction into events occurring before and after nucleotide addition. Preincubation of template with a mixture of RNA polymerase II and factors allows formation of "activated" complexes, which are defined by the ability to rapidly commence accurate transcription when presented nucleotides. Maximal activation requires that polymerase, template, and each of the three HeLa fractions be present during preincubation. The activated complexes are template associated, as shown by their inability to exchange onto a second template added during further preincubation. Similar protocols are used to define functional intermediates leading to the activated complex. A template-associated functional complex is formed during the preincubation of template with just two of the HeLa fractions. Polymerase can associate with this intermediate complex in the absence of the third HeLa fraction. In the accompanying paper, we describe a direct analysis of initiation by "activated" complexes.

Animals↗
PubMed1984

Expression of dihydrofolate reductase, and of the adjacent EIb region, in an Ad5-dihydrofolate reductase recombinant virus.

A gene with the Ad2 MLP and first leader, and appropriate RNA processing signals (splicing, polyadenylation) positioned around a mouse DHFR cDNA clone was substituted for the EIa region of Ad5, and virus stocks of Ad5 (DHFR-I) were prepared on 293 cells. A DHFR RNA of the expected size and structure was expressed late after infection of 293 cells by Ad5 (DHFR-I), at levels comparable to that of other Ad5 late messages. Although this DHFR mRNA was translated as efficiently as other Ad late mRNAs in vitro, it was only poorly translated in vivo. The substitution of the DHFR gene for the Ad5 EIa region results in aberrant transcriptional activity in the adjacent EIb sequences. The transcriptional levels of the EIb 1kb message were down approximately 10-fold. In addition, a novel pIX-encoding mRNA was produced, generated by the splicing of the Ad first late leader onto sequences 14 bp upstream from the pIX initiation codon. This new mRNA was found to be potently efficient for translation both in vivo and in vitro.

Adenoviruses, Human↗
PubMed1984

Synthesis of an ochre suppressor tRNA gene and expression in mammalian cells.

We have used site-specific mutagenesis to change the anticodon of a Xenopus laevis tyrosine tRNA gene so that it would recognize ochre codons. This tRNA gene is expressed when amplified in monkey cells as part of a SV40 recombinant and efficiently suppresses termination at both the ochre codon separating the adenovirus 2 hexon gene from a 23-kd downstream gene and the ochre codon at the end of the NS1 gene of influenza virus A/Tex/1/68. Termination at an amber codon of a NS1 gene of another influenza virus strain was not suppressed by the (Su+) ochre gene suggesting that in mammalian cells amber codons are not recognized by ochre suppressor tRNAs. Finally, microinjection into mammalian cells of both (Su+) ochre tRNA genes and selectible genes containing ochre nonsense mutations gives rise to colonies under selective conditions. We conclude that it should be possible to isolate a wide assortment of mammalian cell lines with ochre suppressor activity.

Amino Acid Sequence↗
PubMed1984

Immunologic dysfunction during viral oncogenesis. II. Inhibition of cellular immunity to viral antigens by malignant rabbit fibroma virus.

The ability of two related viruses--Shope fibroma virus (SFV) and malignant rabbit fibroma virus (MV)--to induce virus-specific immune responses in lymphocytes of recipient animals was studied. SFV produces a benign local tumor which regresses in 12-14 days. Using an assay for virus-induced lymphocyte blastogenesis lymphocytes reactive to SFV were detected, both in rabbits bearing SFV-induced tumors and in rabbits whose SFV-induced tumor had regressed. These virus-reactive cells were detected in peripheral blood and spleen, and in lymph nodes draining the primary tumor. In contrast, MV produces a disseminated tumor and eventual death. MV does not induce detectable blastogenic responses in lymphocyte populations. SFV and MV are antigenically cross reactive: rabbits immune to SFV do not develop MV-induced tumors, and antisera to each virus neutralize both equally. Lymphocytes from SFV-infected rabbits proliferate in vitro in response to MV that has been inactivated by ultraviolet light (uv/MV) but not to infectious MV. In contrast, lymphocytes from rabbits infected with MV do not respond to uv-inactivated MV or to SFV. Thus, infectious MV inhibits the development of normal blastogenic responses in vivo and prevents the expression of those responses in lymphocytes from MV-resistant, SFV-immune rabbits in vitro. The relevance of this impairment to the differences in the clinical courses of SFV- and MV-induced tumors is discussed.

Animals↗
PubMed1984

Recognition of cap structure in splicing in vitro of mRNA precursors.

Substrate RNAs are only efficiently spliced in HeLa whole-cell extract when they possess capped 5' termini. This cap requirement is observed with substrate RNAs prepared by transcription with either mammalian RNA polymerase II or bacterial RNA polymerase. Addition of less than 10 microM of cap analogs such as m7G(5')ppp(5')N or m7GTP strongly inhibits splicing of capped RNAs. This observation, as well as experiments following the fate of substrate RNA, indicates that the dependence of splicing on a cap structure is not due to an effect on RNA stability. More interestingly, cap analogs inhibit splicing when added at the start of the reaction but not at later times of incubation. This suggests that the cap recognition might be an important step in the formation of a specific ribonucleoprotein complex required for splicing.

DNA-Directed RNA Polymerases↗
PubMed1984

Site-specific polyadenylation in a cell-free reaction.

A soluble HeLa cell extract accurately polyadenylates RNA transcribed from DNA templates containing the adenovirus L3 polyadenylation site. Regardless of the length of these DNA templates, the major polyadenylated species had 3' termini corresponding to the in vivo site. Polyadenylated RNA appears after an hour lag and only reaches maximum levels after 4 hr of incubation, a time course similar to that of splicing in this extract. Inhibitor studies suggest that the polyadenylation reaction is not coupled to active transcription. Unlike splicing in this extract where exogenous substrate is processed, addition of purified RNA precursor to the reaction does not yield product polyadenylated at L3 but rather results in addition of poly (A) to termini of the precursor. This suggests that part of the specificity of polyadenylation is established by in situ synthesis of RNA. Surprisingly, synthesis of accurately polyadenylated RNA may involve small nuclear ribonucleoprotein particles (snRNPs). The reaction is inhibited by antisera of Sm and U1 RNP specificities as well as antiserum to the nuclear antigen La, but is not inhibited by control serum and anti-(U2)RNP serum.

Cell-Free System↗