Search PubMed⌕ Search

Biomedical subjects

D R Dubbs

Publications and source records attributed to D R Dubbs.

At least 55 records · Page 3Linked to original sources
PubMed1970

Isolation of double lysogens from 3T3 cells transformed by plaque morphology mutants of SV40.

Simian virus 40 (SV40) rescued from 3T3 lines transformed independently by fuzzy plaque strain, SV40 (mKS-U4), and small-clear plaque strain, SV40 (mKS-U88), resembled the virus used to initiate the transformation. When a mixture of the two viruses was used for transformation, both plaque types could be rescued. Two clonal lines, after fusion with CV-1 cells, yielded both plaque types of infectious centers, and a third clonal line, 3T3(4-88)G-1, yielded fuzzy and small-clear plaque types of infectious centers plus large-clear infectious centers resembling "wild-type" SV40 clone 307L. Two secondary and 14 tertiary clones of 3T3(4-88)G-1 were isolated, all of which yielded fuzzy plaque type and clear infectious centers after fusion with CV-1 cells. When these infectious centers were picked and replated on CV-1 cells, the fuzzy and small-clear plaque types of SV40, as well as a large-clear plaque type resembling parental SV40, were isolated.

Animals↗
PubMed1970

Origin of thymidine kinase in adenovirus-infected human cell lines.

Human adenovirus type 5 enhances the thymidine kinase activity of KB cells but does not induce the enzyme in kinase-deficient HeLa (BU25) cells. Vaccinia induces thymidine kinase activity in both KB and HeLa (BU25) cells. Human adenovirus types 2, 4, 7, and 12 also fail to induce the enzyme in HeLa (BU25) cells. Vaccinia replicates equally well in the presence or absence of HATG (hypoxanthine-aminopterin-thymidine-glycine) in KB and HeLa (BU25) cells. Adenovirus type 5 replicates in KB and in HeLa (BU25) cells in the absence of HATG, and adenovirus type 5 replicates in kinase-positive KB cells in the presence of HATG. However, replication of adenovirus type 5 is grossly inhibited in HeLa (BU25) cells in the presence of HATG. These results suggest that human adenoviruses do not code for a new virus-specific thymidine kinase.

Adenoviridae↗
PubMed1970

Initial site of synthesis of virus during rescue of simian virus 40 from heterokaryons of simian virus 40-transformed and susceptible cells.

Simian virus 40 (SV40) can be rescued from certain SV40-transformed hamster cells by fusion with susceptible African green monkey kidney (CV-1) cells, in the presence of ultraviolet-irradiated Sendai virus. We have determined the sites in which SV40 is produced during rescue in these heterokaryons. To determine the sequence, nuclei were isolated from fused cells at various times after fusion, separated on sucrose-density gradients, and assayed for infectious center formation and virus content on CV-1 monolayers. Virus was first detected in the transformed nucleus (40 hr postfusion), and later associated with both transformed and susceptible nuclei (68 to 72 hr). Viral rescue apparently does not depend upon the transfer of SV40 deoxyribonucleic acid to a susceptible CV-1 nucleus, since the transformed nucleus is the primary site of virus production. The time course of certain cytological events in the rescue process and in productive infection was found to be similar.

Animals↗
PubMed1970

Identification of the simian virus 40 which replicates when simian virus 40-transformed human cells are fused with simian virus 40-transformed mouse cells or superinfected with simian virus 40 deoxyribonucleic acid.

Simian virus 40 (SV40) was rescued from heterokaryons of transformed mouse and transformed human cells. To determine whether the rescued SV40 was progeny of the SV40 genome resident in the transformed mouse cells, the transformed human cells, or both, rescue experiments were performed with mouse lines transformed by plaque morphology mutants of SV40. The transformed mouse lines that were used yielded fuzzy, small-clear, or large-clear plaques after fusion with CV-1 (African green monkey kidney) cells. The transformed human lines that were used did not release SV40 spontaneously or after fusion with CV-1 cells. From each mouse-human fusion mixture, only the SV40 resident in the transformed mouse cells was recovered. Fusion mixtures of CV-1 and transformed mouse cells yielded much more SV40 than those from transformed human and transformed mouse cells. The rate of SV40 formation was also greater from monkey-mouse than from human-mouse heterokaryons. Deoxyribonucleic acid (DNA) from SV40 strains which form fuzzy, largeclear, or small-clear plaques on CV-1 cells was also used to infect monkey (CV-1 and Vero), normal human, and transformed human cell lines. The rate of virion formation and the final SV40 yields were much higher from monkey than from normal or transformed human cells. Only virus with the plaque type of the infecting DNA was found in extracts from the infected cells. Two uncloned sublines of transformed human cells [W18 Va2(P363) and WI38 Va13A] released SV40 spontaneously. Virus yields were not appreciably enhanced by fusion with CV-1 cells. However, clonal lines of W18 Va2(P363) did not release SV40 spontaneously or after fusion with CV-1 cells. In contrast, several clonal lines of WI38 Va13A cells did continue to shed SV40 spontaneously.

Animals↗
PubMed1970

Temperature-sensitive simian virus 40 mutant defective in a late function.

A temperature-sensitive simian virus 40 (SV40) mutant, tsTNG-1, has been isolated from nitrosoguanidine-treated and SV40-infected African green monkey kidney (CV-1) cultures. Replication of virus at the nonpermissive temperature (38.7 C) was 3,000-fold less than at the permissive temperature (33.5 C). Plaque formation by SV40tsTNG-1 deoxyribonucleic acid (DNA) on CV-1 monolayers occurred normally at 33.5 C but was grossly inhibited at 38.7 C. The time at which virus replication was blocked at 38.7 C was determined by temperature-shift experiments. In shift-up experiments, cultures infected for various times at 33.5 C were shifted to 38.7 C. In shift-down experiments, cultures infected for various times at 38.7 C were shifted to 33.5 C. All cultures were harvested at 96 hr postinfection (PI). No virus growth occurred when the shift-up occurred before 40 hr PI. Maximum virus yields were obtained at 96 hr PI when the shift-down occurred at 66 hr, but only about 15% of the maximum yield was obtained when the shift-down occurred at 76 hr PI. These results indicate that SV40tsTNG-1 contains a conditional lethal mutation in a late viral gene function. Mutant SV40tsTNG-1 synthesized T antigen, viral capsid antigens, and viral DNA, and induced thymidine kinase activity at either 33.5 or 38.7 C. The properties of the SV40 DNA synthesized in mutant-infected CV-1 cells at 33.5 or 38.7 C were very similar to those of SV40 DNA made in parental virus-infected cells, as determined by nitrocellulose column chromatography, cesium-chloride-ethidium bromide equilibrium centrifugation, and by velocity centrifugation in neutral sucrose gradients. Mutant SV40tsTNG-1 enhanced cellular DNA synthesis in primary cultures of mouse kidney cells at 33.5 and 38.7 C and also transformed mouse kidney cultures at 36.5 C. SV40tsTNG-1 was recovered from clonal lines of transformed cells after fusion with susceptible CV-1 cells and incubation of heterokaryons at 33.5 C, but not at 38.7 C.

Animals↗
PubMed1969

Simian virus 40 deoxyribonucleic acid replication. I. Effect of cycloheximide on the replication of SV40 deoxyribonucleic acid in monkey kidney cells and in heterokaryons of SV40-transformed and susceptible cells.

Infectious deoxyribonucleic acid (DNA) was extracted from green monkey kidney (CV-1) cultures at various times after the cultures were infected with simian virus 40 (SV40) at input multiplicities of 0.01 and 0.1 plaque-forming unit (PFU) per cell. A pronounced decrease in infectious DNA was observed from 3 to 16 hr after virus infection, suggesting that structurally altered intracellular forms may have been generated early in infection. Evidence is also presented that SV40 DNA synthesis requires concurrent protein synthesis. DNA replication was studied in the presence and absence of cycloheximide in: (i) SV40-infected and uninfected cultures of CV-1 cells; (ii) cultures synchronized with 1-beta-d-arabinofuranosylcytosine (ara-C) for 24 to 30 hr prior to the addition of cycloheximide; and (iii) in heterokaryons of SV40-transformed hamster and susceptible monkey kidney cells. DNA synthesis was determined by pulse-labeling the cultures with (3)H-thymidine at various times from 24 to 46 hr after infection. In addition, the total infectious SV40 DNA was measured. Addition of cycloheximide, even after early proteins had been induced, grossly inhibited both SV40 and cellular DNA syntheses. The activities of thymidine kinase, DNA polymerase, deoxycytidylate deaminase, and thymidylate kinase were measured; these enzyme activities remained high for at least 9 hr in the presence of cycloheximide. SV40 DNA prelabeled with (3)H-thymidine before the addition of cycloheximide was also relatively stable during the time required for cycloheximide to inhibit further DNA replication.

Animals↗
PubMed1969

Properties of simian virus 40 rescued from cell lines transformed by ultraviolet-irradiated simian virus 40.

Simian virus 40 (SV40) strains have been rescued from various clonal lines of mouse kidney cells that had been transformed by ultraviolet (UV)-irradiated SV40. To learn whether some of the rescued SV40 strains were mutants, monkey kidney (CV-1) cells were infected with the rescued virus strains at 37 C and at 41 C. The SV40 strains studied included strains rescued from transformed cell lines classified as "good," "average," "poor," and "rare" yielders on the basis of total virus yield, frequency of induction, and incidence of successful rescue trials. Four small plaque mutants isolated from "poor" yielder lines and fuzzy and small plaque strains isolated from an "average" and a "good" yielder line, respectively, were among the SV40 strains tested. Virus strains rescued from all classes of transformed cells were capable of inducing the transplantation antigen, and they induced the intranuclear SV40-T-antigen, thymidine kinase, deoxyribonucleic acid (DNA) polymerase, and cellular DNA synthesis at 37 C and at 41 C. With the exception of four small plaque strains rescued from "poor" yielders, the rescued SV40 strains replicated their DNA and formed infectious virus with kinetics similar to parental SV40 at either 37 or 41 C. The four exceptional strains did replicate at 37 C, but replication was very poor at 41 C. Thus, only a few of the rescued virus strains exhibited defective SV40 functions in CV-1 cells. All of the virus strains rescued from the "rare" yielder lines were similar to parental SV40. Several hypotheses consistent with the properties of the rescued virus strains are discussed, which may account for the significant variations in virus yield and frequency of induction of the transformed cell lines.

Animals↗
PubMed1968

Isolation of defective lysogens from Simian virus 40-transformed mouse kidney cultures.

Rescue of simian virus 40 (SV40) from hamster and murine cell lines transformed by nonirradiated or by ultraviolet (UV)-irradiated SV40 (10(-3) to 10(-5) survival) was studied. A combination of tests was employed to detect induction of SV40 synthesis: (i) co-cultivation with susceptible monkey kidney (CV-1) cells; (ii) treating mixtures of transformed and CV-1 cells with UV-irradiated Sendai virus (UV-Sendai) prior to co-cultivation; and (iii) plating untreated or UV-Sendai-treated mixtures of transformed and CV-1 cells with freshly trypsinized CV-1 cells. The first and second tests provided a measure of the total infectious SV40 yield per culture, and the third test provided a measure of the frequency of induction (fraction of transformed cells giving rise to infectious centers). With the combination of tests, SV40 was rescued in all trials from TSV-5 hamster cells, mKS-BU100 mouse cells, and from several lines of mouse kidney cells transformed by UV-irradiated SV40 (mKS-U lines). The frequency of induction was about 7 x 10(-2) for TSV-5 cells, about 3 x 10(-3) for mKS-BU100 cells, greater than 10(-4) for the mKS-U lines which were "good" yielders, and about 10(-5) to 10(-4) for the mKS-U lines which were "average" yielders. SV40 of a plaque type different from parental virus was rescued from four of the mKS-U cell lines. Virus was also easily rescued from: (i) tumor cells produced from the mKS-A line of transformed mouse kidney cells; (ii) mouse kidney cells transformed by SV40 which had been rescued from mKS-BU100 cells; and (iii) tumor cells (HATS) which had been produced by inoculating newborn hamsters with SV40 rescued from mKS-BU100 cells. The frequency of induction of HATS cells was of the same order of magnitude as the frequency of induction of TSV-5 cells. In a study of the kinetics of virus induction, it was shown that SV40 could be detected 28, 40, and 48.5 hr after UV-Sendai treatment of mixtures of CV-1 and TSV-5, HATS, or mKS-BU100 cells, respectively. Although all of the mKS-U lines contained the SV40-specific tumor antigen, some were poor virus yielders (SV40 was recovered in less than 50% of the trials) and five lines were rare virus yielders (SV40 recovered only once in four or more trials). Forty-eight mKS-U lines were nonyielders; SV40 was never recovered by any test used thus far. UV-Sendai-treated mixtures of pairs of nonyielder mKS-U lines with CV-1 cells also did not yield infectious virus. Various factors affecting rescue have been discussed. The mKS-U lines which were poor virus yielders, rare yielders, or which never yielded virus have been classified tentatively as "defective lysogens" which contain mutational lesions at loci essential for detachment of SV40 from integration sites or for SV40 replication, or for both.

Animals↗
PubMed1967

Enzyme induction in green monkey kidney cultures infected with simian adenovirus.

Thymidine kinase was induced after infection of an established strain of green monkey kidney cells (CV-1) with simian adenovirus SV15. Increased levels of thymidine kinase were first observed 8 to 10 hr postinoculation (PI), and the levels increased four- to eightfold by 16 to 24 hr PI. A transient increase (1.5- to 3-fold) of deoxyribonucleic acid (DNA) polymerase activity was also observed about 18 hr PI, but the level of deoxycytidylic deaminase was not enhanced. The inductions of thymidine kinase and DNA polymerase were not obtained when protein synthesis was inhibited with 10(-5) M cycloheximide. However, the enzyme increases did take place when infected cultures were treated with 1-beta-D-arabinofuranosylcytosine (ara-C), an inhibitor of DNA synthesis and SV15 replication. The incorporation of tritium-labeled thymidine (H(3)-dT) into DNA was also stimulated 8 to 24 hr after infection with SV15.

Adenoviridae↗