Search PubMed⌕ Search

Biomedical subjects

S J Flint

Publications and source records attributed to S J Flint.

At least 55 records · Page 3Linked to original sources
PubMed1986

Identification of proteins and protein domains that contact DNA within adenovirus nucleoprotein cores by ultraviolet light crosslinking of oligonucleotides 32P-labelled in vivo.

A new approach to the identification of DNA binding proteins has been developed and used to study the DNA-protein interactions within the nucleoprotein core of subgroup C adenoviruses. Virions labelled in vivo with [32P]orthophosphate were exposed to ultraviolet light and the DNA digested by chemical or enzymatic methods. Labelled phosphoamino acids of the virion phosphoproteins were selectively hydrolysed by alkali, permitting proteins crosslinked to DNA to be identified by virtue of their covalently attached, 32P-labelled nucleotides. In parallel experiments, [3H]arginine-labelled virions were crosslinked by exposure to ultraviolet light and analysed by more conventional methods. The results indicate that proteins VII and V lie in close contact with viral DNA within the core. The compact arrangement of the nucleoprotein core appears to be capable of trapping protein VII molecules that are not covalently attached to DNA after exposure to ultraviolet light, suggesting that viral DNA might be wrapped around clusters of protein VII molecules. The domains of protein VII that lie in contact with DNA were identified by partial proteolytic mapping of the sites of covalent-attachment of the 32P-labelled oligonucleotides. The implications of these data for the nature of the interactions that mediate the packaging of viral DNA within the nucleoprotein core of adenovirions are discussed.

Adenoviridae↗
PubMed1986

Adenoviral protein VII packages intracellular viral DNA throughout the early phase of infection.

The proteins associated with parental, adenoviral DNA in productively-infected HeLa cells have been examined both directly and indirectly. HeLa cells infected with 32P-labelled Ad2 were irradiated with u.v. light at various points in the infectious cycle. Following degradation of the DNA, nuclear proteins carrying cross-linked nucleotides, or oligonucleotides, were distinguished from virion phosphoproteins by the resistance of their 32P radioactivity to 1 M NaOH. The major core protein of the virion, protein VII, was found to be associated with viral DNA throughout infection, even when cells were infected at a multiplicity of 0.14. Micrococcal nuclease digestion of intranuclear viral DNA 4 h after infection liberated two nucleoprotein particles containing viral DNA, neither of which co-migrated with HeLa cell mononucleosomes. These results indicate that core protein VII remains associated with parental adenoviral DNA during productive infections. The observation that protein VII can be cross-linked to DNA in cells infected at very low multiplicity, together with the results of a comparison of proteins cross-linkable to viral DNA in cells infected by wild-type virus and a non-infectious mutant containing the precursor to protein VII, suggest that nucleoproteins comprising viral DNA and protein VII must be the templates for expression of pre-early and early viral genes.

Adenoviruses, Human↗
PubMed1986

Partition of E1A proteins between soluble and structural fractions of adenovirus-infected and -transformed cells.

The partition of E1A proteins between soluble and structural framework fractions of human cells infected or transformed by subgroup C adenoviruses was investigated by using gentle cell fractionation conditions. A polyclonal antibody raised against a trpE-E1A fusion protein (K.R. Spindler, D.S.E. Rosser, and A. J. Berk, J. Virol. 132-141, 1984) synthesized in Escherichia coli was used to measure the steady-state levels of E1A proteins recovered in the various fractions by immunoblotting. The relative concentration of E1A proteins recovered in the soluble fraction of adenovirus type 2-infected cells was at least fivefold greater than the relative concentration in the corresponding fraction of transformed 293 cells. The observed distribution of E1A proteins was not altered by the sulfhydryl-blocking reagent N-ethylmaleimide. E1A proteins were recovered in nuclear matrix, chromatin, and cytoskeleton fractions after further fractionation of the structural framework fraction. However, the E1A protein species that could be identified by one-dimensional gel electrophoresis were not uniformly distributed among the subcellular fractions examined. The results obtained when fractionation was performed in the presence of the oxidation catalysts Cu2+ or (ortho-phenanthroline)2 Cu2+ indicate that E1A proteins can be efficiently cross-linked, via disulfide bonds, to the structural framework of both adenovirus-infected and adenovirus-transformed cells.

Adenoviridae Infections↗
PubMed1985

Infection and transformation of mouse cells by human adenovirus type 2.

The susceptibilities of C57Bl/6J mouse embryo fibroblasts (MEF) and baby mouse kidney (BMK) cells to infection or transformation by adenovirus type 2 have been compared to those of rat embryo fibroblasts (REF). Both MEF and BMK cells were some 10-fold less permissive to replication of the virus than were REF cells, even though similar fractions of all three cell types, a maximum of 50-60%, produced viral tumor and structural antigens. This observation suggests that a very late step in adenovirus production, such as assembly or maturation, occurs much less efficiently in mouse cells than it does in rat cells. No significant differences in the frequencies of transformation, as assayed by the appearance of foci of morphologically transformed cells, were observed following transfection of adenovirus type 2 or type 5 DNA into the three cell types. However, it proved extremely difficult to establish permanent lines of adenovirus-transformed mouse cells: only 2 of more than 100 attempts were successful, compared to a success rate of close to 100% with adenovirus type 2-transformed REF or SV40-transformed MEF or BMK cells. The two lines of type 2 adenovirus-transformed MEF that were established have been shown to retain and express viral genetic information.

Adenoviruses, Human↗
PubMed1985

The expression of the SV40 early region in the transformed human cell line SV80.

The one complete copy of the SV40 early region present in human cells of the transformed line SV80 carries a duplication of the 5' portion of the early region, including its transcriptional control region and splicing signals (W. Gish and M. Botchan, personal communication). Novel SV40-specific RNA species of sufficiently large size, 3.8 and 4.2 kb, to be expressed from the duplicated early transcriptional control region were detected in SV80 cytoplasmic and nuclear RNA preparations by blot hybridization. The results of transcription in a cell-free system of a plasmid, pSV80-04, representing this SV80 cell SV40 DNA integrate (W. Gish and M. Botchan, personal communication) and of nuclease protection experiments with end-labelled pSV80-04 DNA fragments support the conclusion that the duplicated early sequences are transcribed in SV80 cells. It has also been established that the duplicated early splicing signals are functional in SV80 cells. These results are discussed in relation to the large amounts of SV40 early mRNA and T-antigen synthesized in cells of the SV80 line.

Antigens, Viral, Tumor↗
PubMed1985

Interactions among the three adenovirus core proteins.

Interactions among the three adenovirus core polypeptides V, VII, and mu were examined, using the reversible chemical cross-linker dithiobis(succinimidyl propionate) and two-dimensional polyacrylamide gel electrophoresis. Cross-linked species obtained from gradient-purified adenovirus type 2 cores were well represented among the cross-linked products of pentonless virions and crude core preparations. The more efficiently formed cross-linked core species were also identified with the arginine-specific cross-linker, p-azidophenyl glyoxal. In addition to dimers of polypeptides V and VII, efficient cross-linking of V to VII, V to mu, and VII to V to mu was detected in adenovirus cores. Notably absent were cross-linked species corresponding to higher multimers of polypeptide VII. A major core-capsid interaction appeared to be via the association of polypeptide V with a dimer of polypeptide VI.

Adenoviruses, Human↗
PubMed1984

Specific transcription of an adenoviral gene that possesses no TATA sequence homology in extracts of HeLa cells.

Transcription of the adenovirus type 2 (Ad2) IVa2 gene, which contains no TATA-like sequence in the region immediately upstream of the IVa2 cap sites (Baker, C. C., and Ziff, E. B. (1981) J. Mol. Biol. 149, 189-221), has been examined in extracts of HeLa cells (Manley, J. L., Fire, A., Cano, A., Sharp, P. A., and Gefter, M.L. (1980) Proc. Natl. Acad. Sci. U.S.A. 77, 3855-3859). Run-off transcripts of the predicted length of those initiated at the IVa2 cap sites were synthesized from different Ad2 DNA templates, each of which also contained the major late transcriptional control region. Mapping of the 5' ends of the RNA made from one template by a nuclease protection assay established the fidelity of initiation of IVa2 transcription in vitro. The efficiency of IVa2 expression in whole HeLa extracts was influenced quite dramatically by monovalent and divalent metal ion concentrations and the concentration of extract protein present in the reaction mixture. Under certain conditions, IVa2 run-off transcripts were made almost as efficiently as those from the Ad2 major late transcriptional control region. However, conditions promoting optimal IVa2 transcription in vitro did not favor recognition of the major late transcriptional control region, and vice versa: the synthesis of IVa2 and major late run-off transcripts responded differently to all parameters tested.

Adenoviruses, Human↗
PubMed1984

Accumulation of early and intermediate mRNA species during subgroup C adenovirus productive infections.

The cytoplasmic, poly(A)-containing RNA species complementary to all regions of the adenovirus type 2 (Ad2) genome expressed before the onset of viral DNA synthesis have been examined by "Northern" blotting. HeLa cells infected with low multiplicities of Ad2 were harvested at 2-hr intervals until late in the infectious cycle to establish the temporal patterns of expression of each viral gene. Under these conditions, such late mRNA products as those of the L3 and L5 families were not detected until 14 hr after infection. Although individual mRNA species complementary to several genes showed different patterns of expression, the order of appearance in the cytoplasm of substantial concentrations of adenoviral mRNA species was E1A, E3, and E4 (4 to 6 hr), E2A and E2B (8 hr), 3.7- and 4.1-kb L1 mRNA species (10-12 hr), IX and IVa2 mRNAs (12 hr), and those encoded in the major late transcriptional unit, such as members of the L3 and L5 families (14 hr). The mRNA species encoding polypeptides IX and IVa2 were not produced when viral DNA synthesis was blocked, whereas the larger L1 mRNA species was made under these conditions. Two E2B mRNA species, some 5.0 and 7 kb, were observed at low concentrations at 8 hr after infection and their concentration increased until 24 hr after infection, as did that of the E2A mRNA species: the products of the E2 transcription unit appeared to be expressed coordinately and at a constant ratio throughout infection. Few of the early mRNA species decreased in concentration after the onset of the late phase of infection. Examination of the viral mRNA produced when protein synthesis was inhibited by 10 microM anisomycin added 3 hr after infection suggested that processing of certain viral, early transcripts was altered in the presence of the drug.

Adenoviruses, Human↗
PubMed1984

Transcription of adenovirus cores in vitro.

Transcription in whole HeLa cell extracts of the nucleoprotein core complexes released from adenovirus type 2 or type 5 virions has been examined. The average length of transcripts from deproteinized DNA templates increased steadily during a 90-min reaction in vitro, exhibiting an elongation rate of approximately 70 nucleotides per minute. On the other hand, transcripts made from viral core templates were restricted to a length of less than 2000 nucleotides. Accordingly, efficient transcription of cores (50 nucleotides elongated/min) ceased after 10-20 min of incubation in whole-cell extracts. Deproteinized viral DNA and viral nucleoprotein complexes appeared to support the initiation of a similar number of transcripts per template molecule, but the rate of initiation was faster when cores were provided as templates. Deproteinized viral DNA supported the synthesis of VA-RNA and of transcripts that hybridized to the region of the viral genome containing the 5' portion of the major late transcriptional. Viral cores also directed the synthesis of RNA products which hybridized to fragments of the viral genome containing E1A, E1B, and E4 regions. The results of nuclease protection experiments indicated that the presence of core proteins did not preclude accurate initiation of transcription from the E4 region.

Adenoviruses, Human↗
PubMed1984

Effect of adenovirus infection on expression of human histone genes.

The influence of adenovirus type 2 infection of HeLa cells upon expression of human histone genes was examined as a function of the period of infection. Histone RNA synthesis was assayed after run-off transcription in nuclei isolated from mock-infected cells and after various periods of adenovirus infection. Histone protein synthesis was measured by [3H]leucine labeling of intact cells and fluorography of electrophoretically fractionated nuclear and cytoplasmic proteins. The cellular representation of RNA species complementary to more than 13 different human histone genes was determined by RNA blot analysis of total cellular, nuclear or cytoplasmic RNA by using a series of 32P-labeled cloned human histone genes as hybridization probes and also by analysis of 3H-labeled histone mRNA species synthesized in intact cells. By 18 h after infection, HeLa cell DNA synthesis and all parameters of histone gene expression, including transcription and the nuclear and cytoplasmic concentrations of core and H1 mRNA species, were reduced to less than 5 to 10% of the control values. By contrast, transcription and processing of other cellular mRNA sequences have been shown to continue throughout this period of infection. The early period of adenovirus infection was marked by an inhibition of transcription of histone genes that accompanied the reduction in rate of HeLa cell DNA synthesis. These results suggest that the adenovirus-induced inhibition of histone gene expression is mediated in part at the transcriptional level. However, the persistence of histone mRNA species at concentrations comparable to those of mock-infected control cells during the early phase of the infection, despite a reduction in histone gene transcription and histone protein synthesis, implies that histone gene expression is also regulated post-transcriptionally in adenovirus-infected cells. These results suggest that the tight coupling between histone mRNA concentrations and the rate of cellular DNA synthesis, observed when DNA replication is inhibited by a variety of drugs, is not maintained after adenovirus infection.

Adenoviruses, Human↗
PubMed1983

Synthesis and processing of simian virus 40-specific RNA in adenovirus-infected, simian virus 40-transformed human cells.

Human simian virus 80 (SV80) cells transformed by simian virus 40 (SV40) synthesize substantial quantities of the SV40 large T-antigen (Henderson & Livingston, 1974; Tjian, 1978) and cytoplasmic, poly(A)-containing RNA species that exhibit spliced structures characteristic of the SV40, early messenger RNA species that encode both large and small T-antigens (Flint & Beltz, 1979). When SV80 cells were infected with type C adenovirus, both the synthesis of SV40 large T-antigen and the appearance in the cytoplasm of newly synthesized, SV40-specific RNA sequences were inhibited during the late phase of infection. The results of hybridization to SV40 DNA of SV80 nuclear RNA, prepared from mock- or adenovirus-infected cells after labeling for short periods in vivo or in vitro, indicated that transcription of integrated SV40 was, by contrast, not disrupted during the late phase of adenovirus infection. Poly(A)-containing, nuclear RNA species that hybridized to SV40 DNA sequences and exhibited the sizes of spliced, large and small T-antigen mRNA species were also synthesized in infected cells at a time when the corresponding mRNA sequences did not leave the nucleus. These results suggest that the failure of non-adenoviral mRNA sequences to enter the cytoplasm of adenovirus-infected cells does not reflect inhibition of either their transcription or the normal enzymatic processing reactions to which pre-mRNA species are subject. Several lines of evidence do, however, establish that nuclear, SV40-specific RNA sequences are less stable in adenovirus-infected compared to mock-infected SV80 cells.

Adenoviruses, Human↗
PubMed1983

The structure of nucleoprotein cores released from adenovirions.

The morphology, protein composition and DNA organization of nucleoprotein core complexes isolated from type 5 adenovirions have been examined by electron microscopy and biochemical techniques. The morphology of such core structures is in some ways strikingly similar to that exhibited by cellular chromatin. 'Native' core preparations contain compact and less highly-folded forms: the latter appear as thick fibres, 150-300A in diameter. Upon exposure to 0.4M NaCl, adenovirus cores undergo a transition to a beaded string form, reminiscent of nucleosomes. Of the three arginine-rich proteins, polypeptides V, VII and mu present in 'native' cores, only polypeptide VII remains associated with viral DNA in the presence of 0.4M NaCl. We therefore conclude that the nucleosome-like beads are constructed solely of polypeptide VII. The results of micrococcal nuclease digestion experiments suggest that polypeptide VII is sufficient to protect some 100-300bp of adenoviral DNA.

Adenoviruses, Human↗
PubMed1983

Effects of adenovirus infection on rRNA synthesis and maturation in HeLa cells.

The production of cytoplasmic and nucleolar rRNA species was examined in HeLa cells infected with high multiplicities of adenovirus type 5. Both 28S and 18S rRNA newly synthesized in infected cells ceased to enter the cytoplasm as reported previously (N. Ledinko, Virology 49: 79-89, 1972; H. J. Raskas, D. C. Thomas, and M. Green, Virology 40: 893-902, 1970). However, the effects on 28S cytoplasmic rRNA were observed considerably earlier in the infectious cycle than those on 18S rRNA. The inhibition of cellular protein synthesis and of the appearance in the cytoplasm of labeled cellular mRNA sequences (G. A. Beltz and S. J. Flint, J. Mol. Biol. 131: 353-373, 1979) were also monitored in infected cultures. During the later periods of an infectious cycle, from 18 h after infection, nucleolar rRNA synthesis and processing and exit of 18S rRNA from the nucleus were inhibited, probably reflecting the failure of infected cells to synthesize normal quantities of ribosomal proteins. The earliest responses of cellular RNA metabolism to adenovirus infection were, however, the rapid and apparently coordinate reductions in the levels of newly synthesized 28S rRNA and cellular mRNA sequences entering the cytoplasm.

Adenoviridae Infections↗
PubMed1982

Expression of the gene encoding the adenovirus DNA terminal protein precursor in productively infected and transformed cells.

The major product of in vitro translation of early RNA prepared from H5ts125-infected cells and selected by hybridization to adenoviral DNA fragments spanning the region from 14.7 to 31.5 map units had been shown to be identical to the 87-kilodalton terminal protein precursor. A 72- to 75-kilodalton polypeptide whose rRNA can be selected by DNA from this same region and made in the presence of anisomycin was indistinguishable from the 72-kilodalton single-stranded DNA-binding protein encoded by the region from 60.1 to 66.6 map units. The accumulation of cytoplasmic RNA sequences complementary to these l-strand genes under various conditions of infection and in certain lines of transformed cells has been investigated by solution hybridization of cytoplasmic RNA to the separated strands of restriction endonuclease fragments of adenoviral DNA. During the early phase, RNA sequences complementary to the region from 11.6 to 36.7 map units were present at a concentration of 10 to 60 copies per cell, regardless of the nature of the block used to inhibit viral DNA synthesis. By 24 h after infection in the absence of any such block, sequences complementary to the regions from 11.6 to 18.2 map units (IVa2) and from 18.6 to 36.7 map units (E2B) accumulated to concentrations of 4,800 and 280 copies per cell, respectively. The ratio of cytoplasmic E2A RNA sequences to E2B RNA sequences remained close to 10:1 throughout the time period investigated. Of the transformed cell lines which retained E2B DNA sequences that were examined, only the T2C4 line expressed these sequences in cytoplasmic RNA. The implications of these observations for regulation of expression of the adenoviral early l-strand genes are discussed.

Adenoviruses, Human↗