Simian virus 40 small-t antigen stimulates viral DNA replication in permissive monkey cells.

The simian virus 40 (SV40) large-T antigen is essential for SV40 DNA replication and for late viral gene expression, but the role of the SV40 small-t antigen in these processes is still unclear. We have previously demonstrated that small t inhibits SV40 DNA replication in vitro. In this study, we investigated the effect of small t on SV40 replication in cultured cells. CV1 monkey cell infection experiments indicated that mutant viruses that lack small t replicate less efficiently than the wild-type virus. We next microinjected CV1 cells with SV40 DNA with and without purified small-t protein and analyzed viral DNA replication efficiency by Southern blotting. Replication of either wild-type SV40 or small-t deletion mutant DNA was increased three- to fivefold in cells coinjected with purified small t. Thus, in contrast to our in vitro observation, small t stimulated viral DNA replication in vivo. This result suggests that small t has cellular effects that are not detectable in a reconstituted in vitro replication system. We also found that small t stimulated progression of permissive monkey cells--but not of nonpermissive rodent cells--from G0-G1 to the S phase of the cell cycle, possibly leading to an optimal intracellular environment for viral replication.     

Cloning of Multiple Forms of Goldfish Vimentin: Differential Expression in CNS

Abstract: In efforts to determine the primary structure of intermediate filament proteins in the goldfish visual pathway, we isolated clones from a retinal λgt11 cDNA expression library that represent goldfish vimentin. We show that there are at least two forms of goldfish vimentin, designated as vimentin α and vimentin β. RNase protection assays indicate that vimentin α mRNA is expressed in low amounts in retina, optic nerve, and brain and in higher amounts in spinal cord. In contrast, vimentin β mRNA is expressed in low amounts in retina, optic nerve, brain, and spinal cord and in very high amounts in eye lens. Immunohistochemical studies show that in the optic nerve, vimentin α is mainly restricted to blood vessels, meninges, and septa. Light staining is observed with this antibody in an astrocytic glial pattern throughout the optic nerve. Two-dimensional gel analysis shows that all of these goldfish vimentins are low abundant components of optic nerve cytoskeletal preparations.     

Polymorphism of the gene encoding the alpha subunit of the stimulatory G-protein of adenylyl cyclase (GNAS1)

A two-allele polymorphism of the human gene encoding for the alpha subunit of the guanine nucleotide-binding protein is described.     

ANTIVIRAL ACTIVITY OF CULTURED BLUE-GREEN ALGAE (CYANOPHYTA)1

Lipophilic and hydrophilic extracts from approximately 600 strains of cultured cyanophytes, representing some 300 species, were examined for antiviral activity against three pathogenic viruses. Approximately 10% of the cultures produced substances that caused significant reduction in cytopathic effect normally associated with viral infection. The screening program identified the order Chroococcales as commonly producing antiviral agents.     

Syncytium-inducing mutations localize to two discrete regions within the cytoplasmic domain of herpes simplex virus type 1 glycoprotein B.

Herpes simplex virus type 1 glycoprotein B (gB) is essential for virus entry, an event involving fusion of the virus envelope with the cell surface membrane, and virus-induced cell-cell fusion, resulting in polykaryocyte, or syncytium, formation. The experiments described in this report employed a random mutagenesis strategy to develop a more complete genetic map of mutations resulting in the syn mutant phenotype. The results indicate that syn mutations occur within two essential and highly conserved hydrophilic, alpha-helical regions of the gB cytoplasmic domain. Region I is immediately proximal to the transmembrane domain and includes residues R796 to E816/817. Region II is localized centrally in the cytoplasmic domain and includes residues A855 and R858. Positively charged residues were particularly affected in both regions, suggesting that charge interactions may be required to suppress the syn mutant phenotype. No syn mutations were identified within the transmembrane domain. A virus containing a rate of entry (roe) mutation at residue A851, either within or immediately proximal to syn region II, was isolated. Since roe mutations have also been discovered in the external domain of gB, it appears likely that the external and cytoplasmic domains cooperate in virus penetration. Moreover, the observation that both roe and syn mutations occur in the cytoplasmic domain further suggests that gB functions in an analogous manner in both membrane fusion events. It might be predicted from these observations that membrane fusion involves transduction of a fusion signal along the gB molecule through the transmembrane domain. Communication between the external and cytoplasmic domain may thus be required for gB-mediated membrane fusion events.     

The RR1 gene of herpes simplex virus type 1 is uniquely trans activated by ICP0 during infection.

As has been demonstrated for herpes simplex virus type 2, we show in this report that the herpes simplex virus type 1 ribonucleotide reductase large subunit (RR1) gene is trans activated in transient transfection assays by VP16 and ICP0 but not by ICP4. Deletion analysis demonstrated that responsiveness to induction to VP16 resides in an octamer/TAATGARAT sequence of the RR1 promoter and that the TATA box alone is sufficient to provide induction by ICP0. The induction of the RR1 gene by ICP0 but not by ICP4 suggested that it might be possible to identify the cis-acting element(s) responsive to ICP4 in an ICP4-inducible promoter. To this end, a series of chimeric promoters containing various portions of the regulatory sequences of the RR1 promoter and thymidine kinase (TK) promoter were constructed. The TK promoter is trans activated by both ICP0 and ICP4 in transient transfection assays and by ICP4 in infection. The data show that replacing the RR1 TATA region with the TK TATA region permits ICP4 inducibility even if the rest of the RR1 promoter elements remain intact. To test whether the RR1 gene is induced by ICP0 during infection, four mutant viruses were constructed. (i) TAATGARAT+ has the wild-type RR1 promoter driving chloramphenicol acetyltransferase (CAT) and the RR2 promoter driving the lacZ gene. The RR2 gene codes for the small subunit of the ribonucleotide reductase and is expressed as a beta gene. (ii) TAATGARAT- has a triple-base change in the octamer/TAATGARAT element which renders it unresponsive to VP16 trans activation, eliminating that portion of the activation of the RR1 gene. (iii) TAATGARAT- delta alpha 0 has a deletion of the alpha 0 gene. (iv) TAATGARAT- delta alpha 4 has a deletion of the alpha 4 gene. Infections were carried out in Vero cells at a multiplicity of infection of 10 per cell; cells were assayed for CAT and beta-galactosidase (beta-Gal) activities and for virus yields. The first two infections gave strong CAT and beta-Gal activities and high yields of progeny virus. Infection with the third virus showed no CAT activity but did produce high levels of beta-Gal activity and virus progeny. The fourth infection resulted in strong CAT activity but no beta-Gal activity or progeny virus. The data demonstrated that the RR1 promoter was activated in the absence of ICP4 but not in the absence of ICP0 in these infections.(ABSTRACT TRUNCATED AT 400 WORDS)     

Replication and mutagenesis of UV-damaged DNA templates in human and monkey cell extracts.

We have used in vitro DNA replication systems from human HeLa cells and monkey CV-1 cells to replicate a UV-damaged simian virus 40-based shuttle vector plasmid, pZ189. We found that replication of the plasmid was inhibited in a UV fluence-dependent manner, but even at UV fluences which caused damage to essentially all of the plasmid molecules some molecules became completely replicated. This replication was accompanied by an increase (up to 15-fold) in the frequency of mutations detected in the supF gene of the plasmid. These mutations were predominantly G:C-->A:T transitions similar to those observed in vivo. Treatment of the UV-irradiated plasmid DNA with Escherichia coli photolyase to reverse pyrimidine cyclobutane dimers (the predominant UV-induced photoproduct) before replication prevented the UV-induced inhibition of replication and reduced the frequency of mutations in supF to background levels. Therefore, the presence of pyrimidine cyclobutane dimers in the plasmid template appears to be responsible for both inhibition of replication and mutation induction. Further analysis of the replication of the UV-damaged plasmid revealed that closed circular replication products were sensitive to T4 endonuclease V (a pyrimidine cyclobutane dimer-specific endonuclease) and that this sensitivity was abolished by treatment of the replicated DNA with E. coli photolyase after replication but before T4 endonuclease treatment. These results demonstrate that these closed circular replication products contain pyrimidine cyclobutane dimers. Density labeling experiments revealed that the majority of plasmid DNA synthesized in vitro in the presence of bromodeoxyuridine triphosphate was hybrid density whether or not the plasmid was treated with UV radiation before replication; therefore, replication of UV-damaged templates appears to occur by the normal semiconservative mechanism. All of these data suggest that replication of UV-damaged templates occurs in vitro as it does in vivo and that this replication results in mutation fixation.     

INTERSPECIFIC TRANSFORMATION IN BACILLUS

Marmur, J. (Brandeis University, Waltham, Mass.), E. Seaman, and J. Levine. Interspecific transformation in Bacillus. J. Bacteriol. 85:461-467. 1963.-Deoxyribonucleic acids (DNA) from various species of the taxonomic group Bacillaceae were examined for base composition, ability to carry out interspecific transformation, and formation of molecular hybrids in vitro. The minimal requirement for genetic compatibility among different species and for DNA interaction (both reflecting base sequence homologies) is the similarity of the guanine plus cytosine contents of the DNA. The close correlation between the ability of DNA to be competent in interspecific transformation and to form hybrid molecules on denaturation and annealing provided a rational approach to the study of genetic relationship among organisms for which no genetic exchange has yet been demonstrated. Any or all of the criteria (base composition of DNA, transformation, molecular hybrid formation) can be used as tools in the taxonomic assessment of closely related microorganisms.     

Small-scale variation in growing season length affects size structure of scarlet monkeyflower

Growing season length can control plant size over altitudinal and biogeographic scales, but its effect at the scale of meters is largely unexplored. Within the riparian zone of a northern California river, scarlet monkeyflower, Mimulus cardinalis , grows significantly larger at sites high in the channel as compared to sites low in the channel, and even larger where tributaries meet the main stem of the river. We explored the hypothesis that markedly different growing season length controls this size variation. Due to the very gradual retreat of the water level following winter flooding, emergence time is three months longer for plants growing at tributary confluences than for plants growing at low elevations in the channel. Consistent with the growing season length hypothesis, we found no difference in transplant growth between river and tributary confluence sites in an experiment where we equalized growing season length at these locations. Moreover, a second experiment showed that individuals planted earlier in the year gain a distinct size advantage over those planted later, even though growing conditions are less ideal. These results suggest that emergence time may be a key determinant of plant size structure along rivers, an important result considering forecasted variation in water flows with climate change.