Showing papers by "Phillip A. Sharp published in 1984"
01 Jan 1984
890 citations
TL;DR: Cap analogs inhibit splicing when added at the start of the reaction but not at later times of incubation, suggesting that the cap recognition might be an important step in the formation of a specific ribonucleoprotein complex required for splicing.
500 citations
TL;DR: 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, which has been characterized for an adenovirus 2 major late transcript.
Abstract: 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.
404 citations
TL;DR: The gene product of a rearranged mouse c-myc gene is capable of stimulating expression of chimaeric genes containing a Drosophila hSP70 promoter region and 5′-flanking sequences, dependent on sequences located more than 200 bases 5′ of the normal start of hsp70 transcription.
Abstract: The myc gene seems to have a causal role in tumour formation in man, mouse and avian systems1–3. The myc gene product has been localized to the nucleus4,5, suggesting that it may be involved in the regulation of gene expression. The level of expression of the mammalian heat shock protein 70 (HSP70) gene is elevated in several tumour cell lines6, implying that a cellular function expressed in these tumour lines can stimulate HSP70 production. We report here that the gene product of a rearranged mouse c-myc gene is capable of stimulating expression of chimaeric genes containing a Drosophila hsp70 promoter region and 5′-flanking sequences. This stimulation is dependent on sequences located more than 200 bases 5′ of the normal start of hsp70 transcription.
234 citations
TL;DR: Results suggest that the splicing of mRNA precursors may involve sites in the intervening sequence, cleavage at the 5' splice site, cleaving at the 3' splicing site, and ligation of the two exons.
212 citations
TL;DR: A reconstituted transcription system, consisting of purified RNA polymerase II and three essential HeLa cell chromatographic fractions, is used to study events leading to transcription from the adenovirus major late promoter.
204 citations
TL;DR: Surprisingly, synthesis of accurately polyadenylated RNA may involve small nuclear ribonucleoprotein particles (snRNPs) and part of the specificity ofpolyadenylation is established by in situ synthesis of RNA.
173 citations
TL;DR: It is suggested that regulation of a promoter by the EIa region does not depend on the presence of a set of specific sequences, but instead reflects a characteristic of promoters that have been exogenously introduced into cells.
Abstract: During adenovirus infection, the EII promoter is positively regulated by products of the EIa region. The authors have studied this regulation by fusing a DNA segment containing the adenovirus EII promoter to a dihydrofolate reductase cDNA segment. Expression of this hybrid gene is stimulated in trans when cell lines containing an integrated copy are either transfected with plasmids carrying the EIa region or infected with adenovirus. This suggests that EIa activity regulates transcription of the EII promoter in the absence of other viral proteins and that this stimulation can occur when the EII promoter is organized in cellular chromatin. Transcription from the EII promoter is initiated at two sites in cell lines lacking EIa activity. Introduction of the EIa region preferentially stimulated transcription from one of these two sites. A sensitive, stable cotransfection assay was used to test for specific EII sequences required for stimulation. EIa activity stimulates all mutaant promoters; the most extensive deletion retained only 18 base pairs of sequences upstream of the initiation site. They suggest that regulation of a promoter by the EIa region does not depend on the presence of a set of specific sequences, but instead reflects a characteristic of promoters that have beenmore » exogenously introduced into cells. Insertion of the 72-base-pair repeat of simian-virus 40 in cis enhances transcription from the EII promoter. The stimulatory effects of EIa activity and of the simian virus 40 sequence are additive and appear to differ mechanistically.« less
85 citations
TL;DR: It is reported that an exogenous RNA substrate containing the first and second leaders of adenovirus 2 is accurately spliced when added to an extract of HeLa cells.
Abstract: The origin and functions of introns in protein coding genes is one of the enigmas of molecular biology. Splicing processes that remove intervening sequences from precursor RNAs must have either predated or co-evolved with introns. Inferences about the origin of introns and the possible modes of regulation of splicing should emerge from an understanding of the bio chemical mechanisms of splicing. The biochemistry of splicing of tRNA1,2 and rRNA3,4 precursors has rapidly advanced with the development of in vitro reactions containing soluble components that duplicate in vivo reactions. We have recently shown that accurate splicing of an adenovirus mRNA precursor occurs during a coupled transcription/splicing reaction in a soluble whole cell extract5. We now report that an exogenous RNA substrate containing the first and second leaders of adenovirus 2 is accurately spliced when added to an extract of HeLa cells. ATP and Mg2+ are essential cofactors for the reaction. The time course of splicing is unusual; a lag of 45 min is observed before the appearance of spliced product.
70 citations
TL;DR: 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, indicating that capping was not essential for synthesis of long runoff RNAs.
53 citations
TL;DR: A gene with the Ad2 MLP and first leader, and appropriate RNA processing signals positioned around a mouse DHFR cDNA clone was substituted for the EIa region of Ad5, and a novel pIX-encoding mRNA was produced, found to be potently efficient for translation both in vivo and in vitro.
Abstract: 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.
TL;DR: It is concluded that it should be possible to isolate a wide assortment of mammalian cell lines with ochre suppressor activity and microinjection into mammalian cells of both (Su+) o chre tRNA genes and selectible genes containing ochRE nonsense mutations gives rise to colonies under selective conditions.
Abstract: 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.
TL;DR: A series of deletion mutants spanning the adenovirus early region IV (EIV) promoter were tested for transcription activity in vitro and sequences residing upstream from -140 critically influence the level of EIV transcription.
Abstract: A series of deletion mutants spanning the adenovirus early region IV (EIV) promoter were tested for transcription activity in vitro. At least three elements were found to be important for maximal transcription in HeLa whole-cell extracts. Deletion of the TATA box drastically reduced the transcription activity from the EIV promoter. Sequences between nucleotides -58 and -44 are also important for efficient transcription since deletion of this region reduced activity by 50%. More importantly, sequences residing upstream from -140 critically influence the level of EIV transcription. Deletion of sequences between nucleotides -325 (the right terminus of adenovirus genome) and -140 reduced the level of transcription more than 10-fold. It is possible that a specific cellular factor stimulates EIV transcription by recognition of these upstream sequences. The dependence of transcription from the EIV promoter on a distal upstream element may explain some aspects of the regulation of this promoter.
01 Jan 1984
TL;DR: The enzyme of major interest is the eukaryotic DNA-dependent RNA polymerase (pol II), which is responsible for all cellular messenger RNA synthesis as well as Messenger RNA synthesis from many DNA viruses and proviral forms of RNA viruses.
Abstract: Gene expression in eukaryotic cells is often regulated at the level of transcription of the gene (see Darnell, 1982). In order to understand viral growth, the response of cells to external stimuli, and the processes of differentiation and development, it is of importance to understand what controls the initiation of transcription at a gene. The enzyme of major interest in this regard is the eukaryotic DNA-dependent RNA polymerase (pol II), which is responsible for all cellular messenger RNA synthesis as well as messenger RNA synthesis from many DNA viruses and proviral forms of RNA viruses.