Biomedical subjects
S J Flint
Publications and source records attributed to S J Flint.
Splicing and the regulation of viral gene expression.
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A small nuclear ribonucleoprotein is required for splicing of adenoviral early RNA sequences.
The size and structure of viral RNA species synthesized in nuclei isolated during the early phase of productive infection by adenovirus type 2 have been examined by electrophoresis in denaturing polyacrylamide cells and the nuclease S1 assay. The major products of transcription in vitro of early regions 1 and 2 in the adenoviral genome are processed RNA molecules that appear to be correctly spliced in isolated nuclei. Splicing of adenoviral RNA molecules is inhibited when nuclei are preincubated with antibodies from systemic lupus erythematosus patients that immunoprecipitate small nuclear ribonucleoprotein particles. The specificity of these antibodies suggests that ribonucleoprotein particles containing U1 RNA are required for splicing of the adenoviral RNA sequences we have examined.
Transcription of adenoviral genetic information in isolated nuclei. Characterization of viral RNA sequences synthesized in vitro.
The virus-specific RNA sequences synthesized in nuclei isolated from adenovirus type 2-infected HeLa cells comprise a fraction of the total RNA similar to that observed with RNA made in vivo. By 16 h after infection, for example, some 25% of the RNA made in isolated nuclei is transcribed from adenoviral DNA. Only 10 to 15% of the adenoviral RNA sequences synthesized in nuclei isolated during the late phase of infection are transcribed by form III RNA polymerase. This RNA, whose synthesis is resistant to 0.5 microgram/ml, but sensitive to 200 microgram/ml of alpha-amanitin, sediments at about 5 S in denaturing sucrose gradients and must, therefore, represent the virus-associated RNAs. The remainder of the sequences are transcribed by form II RNA polymerase and sediment as several RNA species in denaturing sucrose gradients. The largest of these exhibits the size, 55 to 60 S, expected of a complete transcript of the major, adenoviral transcriptional unit expressed during the late phase. Hybridization of RNA 32P-labeled in nuclei isolated during the late phase of infection to restriction endonuclease fragments of adenoviral DNA immobilized on nitrocellulose filters suggests that sequences of this transcriptional unit are indeed transcribed in vitro. To make a detailed assessment of the fidelity of transcription in isolated nuclei, transcription reactions were performed in the presence of 5-mercuricytidine 5'-triphosphate and the RNA mercurated in vitro separated from endogenous RNA by chromatography on sulfhydryl-agarose columns by a stringent procedure. After demercuration, RNA made in nuclei isolated 20 h following adenovirus type 2 infection was hybridized to the separated strands of restriction endonuclease fragments of 32P-labeled adenovirus type 2 DNA. Such RNA is complementary to the r strand of adenoviral DNA from 16.6 units to a point to the right of 98.3 units. These sequences comprise the major transcriptional unit expressed during the late phase (Fig. 1). It is therefore clear that the fidelity of transcription of adenoviral DNA by form II RNA polymerase is preserved in isolated nuclei. Two 1-strand transcriptional units, those of the IVa2 and ts36 genes (see Fig. 1) are also active at 20 h after infection. The results of similar analysis of RNA made in nuclei isolated 4 and 12 h after infection are also presented and discussed in terms of the mapping of individual transcriptional units within the type 2 adenoviral genome and the temporal regulation of adenoviral gene expression during productive infection.
Identification of the adenovirus early proteins and their genomic map positions.
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Expression of transforming viral genes in semipermissive cells transformed by SV40 or adenovirus type 2 to type 5.
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Location of additional early regions within the left quarter of the adenoviral genome.
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Inhibition of HeLa cell protein synthesis during adenovirus infection. Restriction of cellular messenger RNA sequences to the nucleus.
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Location of additional early gene sequences in the adenoviral chromosome.
Genetic and biochemical analysis has revealed additional early gene sequences in the group C human adenovirus genome. Physical mapping by an improved marker rescue method showed that a group of "early function" mutations previously found by us to map within the left quarter of the genome do not map within the known early sequence (0-11.2) in that region, but instead lie within the coordinates 18.5-22.0. A search for early expression in this region of the genome using saturation hybridization techniques showed the presence of two discrete sets of viral cytoplasmic RNA sequences, present early in infection at low copy number and complementary to sequences on the I strand of the adenovirus genome lying between 11.2-14.5 and 19.8-23.5 untis.
Synthesis and processing of adenoviral RNA in isolated nuclei.
Adenoviral RNA sequences synthesized in nuclei isolated during the late phase of productive infection comprise, besides virus-associated, VA, RNA, five major species, ranging in size from approximately 13S to 55S. The latter RNA species is of the length predicted if the major transcriptional unit expressed during the late phase were completely copied in vitro. Some 30% of the RNA sequences labeled in vitro are polyadenylated, and about one-third of the polyadenylated RNA is virus specific. Hybridization analysis of the sequences immediately adjacent to polyadenylic acid in late RNA labeled in isolated nuclei suggests that polyadenylation in vitro occurs at the same sites recognized within the cell. The polyadenylic acid-containing viral RNA sequences made in isolated nuclei are found in three major species of RNA, sedimenting at approximately 28S, 18S, and 13S. These sizes are remarkably similar to those reported for late mRNA species, suggesting that additional processing steps can occur in isolated nuclei. Hybridization of RNA to XhoI fragments of adenovirus type 2 DNA transferred to nitrocellulose filters reveals that sequences complementary to the region from 22.0 to 26.5 units present in 55S RNA are absent from all smaller species, suggesting that the smaller RNA species labeled in isolated nuclei are generated by splicing. The splicing events necessary to generate the 5' leader segment common to the majority of late adenoviral mRNA species are shown to be performed correctly in isolated nuclei.
The generation and propagation of variegated chromosome structures.
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An altered subunit configuration associated with the actively transcribed DNA of integrated adenovirus genes.
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Two "early" mRNA species in adenovirus type 2-transformed rat cells.
mRNA isolated from adenovirus 2-infected HeLa cells at early times during the productive cycle and from two lines of adenovirus 2-transformed rat embryo cells (F17 and T2C4) was fractionated on sucrose gradients after disaggregation. Viral mRNA species were identified by hybridization across such gradients with the separated strands of restriction endonuclease fragments of 32P-labeled DNA known to be complementary to adeovirus 2 "early" and adenovirus 2-transformed cell mRNA. mRNA transcribed from the left-hand 14% of the adenovirus 2 genome was found to comprise two species, 16 to 17S and 20 to 21S: the same sized mRNA's were present both at early times during productive infection and in the two transformed rat cell lines. Direct comparison of the sequences present in these two mRNA species by additional saturation hybridizations suggests that they are not related to one another. Three additional regions of the adenovirus 2 genome, all of which are located in the right-hand 40% of the adenovirus 2 genome, are complementary to early mRNA sequences: each of these appears to specify one major mRNA species of about 22S. Thus, five major species of adenovirus type 2 early mRNA have been identified. Two of these, copied from the left-hand 14% of the viral genome, are also present in adenovirus 2-transformed rat cells.
Adenovirus transcription. V. Quantitation of viral RNA sequences in adenovirus 2-infected and transformed cells.
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Adenovirus transcription. III. Mapping of viral RNA sequences in cells productively infected by adenovirus type 5.
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Viral nucleic acid sequences in transformed cells. IV. A study of the sequences of adenovirus 5 DNA and RNA in four lines of adenovirus 5-transformed rodent cells using specific fragments of the viral genome.
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Adenovirus transcription.
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Characterization of single-stranded viral DNA sequences present during replication of adenovirus types 2 and 5.
Replication intermediates of adenovirus DNA apparently contain extensive stretches of single-stranded DNA. Such single-stranded viral DNA sequences homologous to different regions of the viral genome present in adenovirus-infected cells during viral DNA replication have therefore been characterized by hybridization to the separated strands of restriction endonuclease fragments of 32P-labeled adenovirus types 2 and 5 DNA. Saturation hybridization experiments with infected cell DNA extracted at late times suggest that all regions of the adenovirus genome are represented in the single-stranded fraction, but at unequal frequencies. This nonuniform representation has been characterized in more detail with self-annealed, total cell DNA extracted 18 hr after adenovirus type 2 infection: the concentration of single-stranded sequences homologous to different regions of the viral genome was determined by comparing the rates of hybridization of 32P-labeled, single-stranded DNA probes with such self-annealed 18 hr DNA to the rates of hybridization of the same probes with equal concentrations of their complements. This approach allows the concentration of single-stranded viral DNA sequences in excess of their complements to be determined. Such sequences can be represented by two concentration gradients across the viral genome: those homologous to the r strand increase in concentration from 27.8-40.9 units toward the right end, whereas sequences homologous to the 1 strand increase from an area 27.8-40.9 units toward the left end. The time course of synthesis of single-stranded viral DNA sequences relative to accumulation of total viral DNA during the productive cycle and their behavior following a shift of H5ts125-infected cells in which viral DNA replication has begun from a permissive to a nonpermissive temperature support the contention that these sequences are indeed generated as adenovirus DNA is replicated. These results are therefore discussed in terms of current models of adenovirus DNA replication.