Showing papers by "Phillip A. Sharp published in 1983"

In vitro transcription: whole-cell extract.

Abstract: Publisher Summary This chapter focuses on three classes of nuclear DNA-dependent RNA polymerases that have been identified in eukaryotic cells. Synthesis of mature RNA molecules requires additional enzymes and factors other than those needed to bring about accurate transcriptional initiation. For analysis by hybridization and SI nuclease digestion, it is important first to remove the template DNA. Titrations both of DNA and of extract yield nonlinear responses. At a constant extract concentration, measuring runoff transcription as a function of DNA concentration yields a threshold DNA concentration below which no transcription occurs and an inhibitory effect of high DNA concentration. For a given promoter, short runoff transcripts have a higher optimum DNA concentration than longer runoff transcripts. A number of inhibitory activities can be removed by fractionation on phosphocellulose, yielding a more efficient transcription extract.

Generation of adenovirus by transfection of plasmids

Abstract: Biologically active fragments of Adenovirus 5 (Ad5) DNA that span the entire genome have been cloned into plasmids. The covalently attached terminal protein was removed and Eco RI linkers added in a fashion that preserves the Ad5 terminal sequences. When plasmids containing overlapping fragments that represent the entire genome are cotransfected onto 293 cells, infectious virus is obtained. Generation of virus depends upon the release of the 0 or 100 mu Ad5 terminus from pBR322 DNA by Eco RI cleavage. During virus production the modified termini of the transfected fragments are corrected exactly to that of wt viral DNA. The above method for preparing adenovirus recombinants has been used to construct a mutant, Ad5 delta (78.9-84.3), lacking most of the non-essential EIII transcriptional unit. This mutant is phenotypically wild type with respect to burst size and kinetics of growth. Surprisingly, it inhibits wt viral growth upon mixed infections of HeLa or 293 cells, apparently at the level of DNA replication.

Conversion of RNA to DNA in mammals: Alu-like elements and pseudogenes

Abstract: Striking similarities between a subclass of mammalian pseudogenes and the Alu family of short repetitive DNA sequences which are dispersed throughout the mammalian genome, suggest that both are created by integration into the genome of DNA copies of RNA transcripts.

Splicing of adenovirus RNA in a cell-free transcription system

Abstract: A soluble whole-cell extract prepared accurately from HeLa cells splices 2-3% of the RNA transcribed from a DNA template containing the first and second leader exons of late adenovirus RNA. The spliced RNA was detected by a sensitive technique using hybridization to a single-stranded phage M13 cDNA clone, followed by binding to nitrocellulose filters. The identity of the spliced RNA was established by RNase T1 and pancreatic RNase two-dimensional peptide mapping. The bond formed during the in vitro splicing reaction appears to be a typical 3',5'-phosphodiester bond as judged by its sensitivity to RNase T1. The splicing reaction is specifically inhibited by KCl at concentrations greater than 50 mM and by the addition of cellular RNA. Three features of this system may account for the detection of splicing in a soluble extract: (i) the sensitive and unambiguous hybridization assay, (ii) the high transcriptional activity of the major late promoter of adenovirus, and (iii) the use of the first and second leader exon splice of adenovirus, which may be unusually rapid.

Sequences controlling in vitro transcription of SV40 promoters.

Abstract: A series of deletion mutants of SV40 were tested for early and late promoter activity in vitro in a transcription extract prepared from HeLa cells. These mutants had previously been characterized for expression in vivo. Transcription in vitro from both the SV40 early and late promoters was strongly dependent on an upstream region of DNA that contains six direct GC repeats. Sequences spanning two or more of these repeats stimulated transcription in a bidirectional fashion, at distances of 50-200 bp. These sequences may function by mediating the activity of a specific transcriptional factor. Little effect on transcription in vitro was observed upon deletion of the 72-bp enhancer elements. With this exception, the sequence dependence of early and late transcription in vitro was similar to that observed previously in vivo, both of the region including the GC repeats and of the early TATA sequence.

Evolution of chromosomal regions containing transfected and amplified dihydrofolate reductase sequences.

Abstract: A modular dihydrofolate reductase gene has been introduced into Chinese hamster ovary cells lacking dihydrofolate reductase. Clones capable of growth in the absence of added nucleosides contain one to five copies of the plasmid DNA integrated into the host genome. Upon stepwise selection to increasing methotrexate concentrations, cells are obtained which have amplified the transforming DNA over several hundredfold. A detailed analysis of the chromosomes in three clones indicated the appearance of cytologically distinct chromosomal regions containing the amplified plasmid DNA which differ in surrounding sequence composition, structure, and location. Two of the clones examined have extensive, homogeneously staining regions. The DNA in these homogeneously staining regions replicates in the early part of the S phase. The amplified plasmid DNA is found associated at or near the ends of chromosomes or on dicentric chromosomes. We propose that integration of DNA may disrupt telomeric structures and facilitate the formation of dicentric chromosomes, which may then undergo bridge breakage-fusion cycles. These phenomena are discussed in relation to DNA transfer experiments and modes of gene amplification and chromosome rearrangement.

Growth-dependent expression of dihydrofolate reductase mRNA from modular cDNA genes.

Abstract: Dihydrofolate reductase (DHFR) synthesis is regulated in a growth-dependent fashion. Dividing cells synthesize DHFR at a 10-fold-higher rate than do stationary cells. To study this growth-dependent synthesis. DHFR genes have been constructed from a DHFR cDNA segment, the adenovirus major late promoter, and fragments of simian virus 40 (SV40) which provide signals for polyadenylation. These genes have been introduced into Chinese hamster ovary cells. The DHFR mRNAs produced in different transformants are identical at their 5' ends, but differ in sequences in their 3' ends as different sites are utilized for polyadenylation. Three transformants that utilize either DHFR polyadenylation signals or the SV40 late polyadenylation signal exhibit growth-dependent DHFR synthesis. The level of DHFR mRNA in growing cells is approximately 10 times that in stationary cells for these transformants. This growth-dependent DHFR mRNA production probably results from posttranscriptional events. In contrast, three transformants that utilize the SV40 early polyadenylation signal and another transformant that utilizes a cellular polyadenylation signal do not exhibit growth-dependent DHFR synthesis. In these three cell lines, the fraction of mRNAs polyadenylated at different sites in a tandem array shifts between growing and stationary cells. These results suggest that the metabolic state of the cell is important in determining either the efficiency of polyadenylation at various sites or the stability of mRNA polyadenylated at various sites.

Inhibition of adenovirus early region IV transcription in vitro by a purified viral DNA binding protein

Abstract: Adenoviruses depend on cellular mechanisms for the decoding of their genetic information, and so provide a useful and simple model system for the investigation of mammalian gene expression. The five regions transcribed early in adenovirus infection are termed EIa, EIb, EII, EIII and EIV1,2. We report here that the primary product of the EII region, a 72,000 molecular weight DNA-binding protein3 (DBP), specifically represses transcription from the EIV promoter in an in vitro transcription system. Single-stranded DNA binds to the DBP with high affinity, and as a result inhibits its repressor activity. Our data extend previous genetic evidence that the DBP represses EIV transcription in vivo4–6, and suggest that it acts directly by suppressing transcription from the EIV promoter.

Adenovirus hexon monoclonal antibody that is group specific and potentially useful as a diagnostic reagent.

Abstract: A monoclonal antibody to the adenovirus 2 hexon protein was produced and characterized as a group-specific antibody. Positive reactivity in immunoprecipitation, indirect immunofluorescence, and radioimmunoassays was observed with human, canine, swine, bovine, murine, and simian adenoviruses. This monoclonal antibody should provide a specific and sensitive diagnostic reagent for detection of all mammalian adenoviruses. Images

Expression of chimeric genes in the early region of SV40.

Abstract: Chimeric genes have been constructed by inserting foreign gene sequences in the early region of SV40. The genes contained the first exon of the SV40 large T gene with 180 bp of its intron and either the third exon of the rat preproinsulin gene II with 488 bp of its large intron or the third exon of the mouse beta globin gene with 63 bp of its intron. The chimeric genes contained a 5' splicing site (SS) from SV40 and a 3' SS from the inserted gene. Both the preproinsulin and the globin insertions contained a polyadenylation signal. The SV40 early poly(A) addition signal was also retained. High-titer virus stocks were obtained when the recombinants, which contained SV40 origin of replication and the entire late region, were used to transfect a cloned line of COS cells (COS-M6). These stocks typically contained no detectable wild-type virus. RNA mapping demonstrated the following: (a) The SV40-rat preproinsulin chimeric RNA was initiated at the SV40 early promoter, spliced from the SV40 5' SS to the rat preproinsulin 3' SS, and polyadenylated solely at the SV40 poly(A) addition signal. (b) The SV40-mouse beta globin chimeric RNA was initiated at the SV40 early promoter, spliced from the 5' SS to the mouse beta globin 3' SS, and polyadenylated at the mouse beta globin poly(A) site. The chimeric RNAs were overproduced, owing to low levels of T antigen in the COS-M6 cells, which did not completely repress transcription from the early region. Fusion proteins of 15,500 molecular weight resulted from expression in vivo of the SV40-rat preproinsulin chimeric gene and of 11,500 molecular weight for the SV40-mouse beta globin chimeric gene. The molecular weights of the proteins suggested that they were initiated at the early SV40 AUG and that translation continued across the chimeric splice sites. The chimeric proteins were also overproduced.

Measurement of suppressor transfer RNA activity

Abstract: Transfer RNA (tRNA) suppression of nonsense mutations in prokaryotic systems has been widely used to study the structure and function of different prokaryotic genes. Through genetic engineering techniques, it is now possible to introduce suppressor (Su+) tRNA molecules into mammalian cells. A quantitative assay of the suppressor tRNA activity in these mammalian cells is described; it is based on the amount of tRNA-mediated readthrough of a terminating codon in the influenza virus NS1 gene after the cells are infected with virus. Suppressor activity in L cells continuously expressing Su+ (tRNAtyr) was 3.5 percent and that in CV-1 cells infected with an SV40- Su+ (tRNAtyr) recombinant was 22.5 percent.

Functional suppression in mammalian cells of nonsense mutations in the herpes simplex virus thymidine kinase gene by suppressor tRNA genes.

Abstract: A nonsense mutation (UAG) in the thymidine kinase gene of herpes simplex virus type 1 can be suppressed in vivo to produce active thymidine kinase by prior infection with a defective simian virus 40 stock which acts as a vector to introduce a functional suppressor tRNA gene into mammalian cells in culture. The suppression is specific for UAG, but not UGA or missense, mutants and restores thymidine kinase activity to 20 to 40% of the wild-type level. These results suggest that many cell lines susceptible to simian virus 40 infection may be transiently converted to a suppressor-positive phenotype for use in the genetic study of mammalian viruses.

Aberrant Distribution of Human Adenovirus Type 2 Late Proteins in Monkey Kidney Cells

Abstract: Monkey kidney cells (CV-C) infected with adenovirus type 2 displayed an aberrant distribution of 100K, 100K-hexon complex, hexon monomers, hexon trimers, penton base, and fiber proteins, relative to the patterns observed in adenovirus type 2-infected human cells. Human cell patterns were observed in CV-C cells when mutants selected for growth on monkey cells were used.