Replication and Recombination of Gene 59 Mutant of Bacteriophage T4D

After infection of Escherichia coli B with phage T4D carrying an amber mutation in gene 59, recombination between two rII markers is reduced two- to three-fold. This level of recombination deficiency persists even when burst size similar to wild type is induced by the suppression of the mutant DNA-arrest phenotype. In the background of two other DNA-arrest mutants in genes 46 and 47, a 10- to 11-fold reduction in recombination is observed. The cumulative effect of gene 59 mutation on gene 46-47 mutant suggests that complicated interactions must occur in the production of genetic recombinants. The DNA-arrest phenotype of gene 59 mutant can be suppressed by inhibiting the synthesis of late phage proteins. Under these conditions, DNA replicative intermediates similar to those associated with wild-type infection are induced. Synthesis of late phage proteins, however, results in the degradation of mutant 200S replicative intermediate into 63S DNA molecules even in the absence of capsid assembly. Although these 63S molecules are associated with membrane, they do not replicate. These results suggest a role for gene 59 product, in addition to a possible requirement of concatemeric DNA in late replication of phage T4 DNA.     

The RecRO pathway of DNA recombinational repair in Helicobacter pylori and its role in bacterial survival in the host

Two pathways for DNA recombination, AddAB (RecBCD-like) and RecRO, were identified in Helicobacter pylori, a pathogenic bacterium that colonizes human stomachs resulting in a series of gastric diseases. In this study, we examined the physiological roles of H. pylori RecRO pathway in DNA recombinational repair. We characterized H. pylori single mutants in recR and in recO, genes in the putative gap repair recombination pathway, and an addA recO double mutant that is thus deficient in both pathways that initiate DNA recombinational repair. The recR or recO single mutants showed the same level of sensitivity to mitomycin C as the parent strain, suggesting that the RecRO pathway is not responsible for the repair of DNA double strand breaks. However, H. pylori recR and recO mutants are highly sensitive to oxidative stress and separately to acid stress, two major stress conditions that H. pylori encounters in its physiological niche. The complementation of the recR mutant restored the sensitivity to oxidative and acid stress to the wild type level. By measuring DNA transformation frequencies, the recR and recO single mutants were shown to have no effect on inter-genomic recombination, whereas the addA recO double mutant had a greatly (～12-fold) reduced transformation frequency. On the other hand, the RecRO pathway was shown to play a significant role in intra-genomic recombination with direct repeat sequences. Whereas the recA strain had a deletion frequency 35-fold lower than that of background level, inactivation of recR resulted in a 4-fold decrease in deletion frequency. In a mouse infection model, the three mutant strains displayed a greatly reduced ability to colonize the host stomachs. The geometric means of colonization number for the wild type, recR, recO, and addA recO strains were 6 x 10?, 1.6 x 10?, 1.4 x 10? and 4 x 103 CFU/g stomach, respectively. H. pylori RecRO-mediated DNA recombinational repair (intra-genomic recombination) is thus involved in repairing DNA damage induced by oxidative and acid stresses and plays an important role in bacterial survival and persistent colonization in the host.     

Incorporation of thymine-containing DNA precursors in wild-type and mutant T4-infected plasmolysed cells

Summary T4-infected cells, plasmolysed 15 min after infection, incorporate low concentrations (>20 M) of deoxythymidine (TdR) into DNA at a significantly greater rate than dTMP, dTTP or thymine. At higher concentrations (>40 M), dTMP incorporation rate is high, approaching that of TdR at 200 M. TdR is selectively incorporated at all concentrations tested, and is not inhibited by the other thymine containing DNA precursors. Incorporation of low concentrations of TdR requires the T4-induced thymidine kinase (tk) and is not significantly affected by the presence or absence of T4-induced thymidylate synthetase (td). We show that, in T4-infected plasmolysed cells, exogenously added TdR is preferentially incorporated into short DNA fragments during short pulse times. To explain these and other data a model is proposed in which thymidine plays a modulatory role between leading and lagging strand precursor feeds.Preliminary accounts of these data were presented at the West Coast Phage Meetings, Evergreen State College 1980, 1981     

Illegitimate and homologous recombination in mammalian cells: differential sensitivity to an inhibitor of poly(ADP-ribosylation).

We determined the effect of 3-methoxybenzamide (3-MB), a competitive inhibitor of poly(ADP-ribose)polymerase (E.C. 2.4.2.30), on illegitimate and extrachromosomal homologous recombination in mouse Ltk- cells. Cells were transfected with a wild type Herpes thymidine kinase (tk) gene or with two defective tk gene sequences followed by selection for tk-positive colonies. Using a wild type tk gene, colony formation required uptake, integration, and expression of the tk gene. Using defective tk genes, colony formation had the additional requirement for homologous recombination to reconstruct a functional tk gene. The presence of non-cytotoxic levels of 3-MB during and after transfection reduced the number of colonies recovered with a wild type tk gene in a dose-dependent manner, with 2 mM 3-MB causing a 10 to 20-fold reduction. 3-MB reduced the number of colonies recovered with defective tk genes only to the same extent as in transfections with a wild type gene. Treatment with 3-methoxybenzoic acid, a non-inhibitory analog of 3-MB, did not reduce the recovery of colonies in any experiment. Similar results were obtained using linear or supercoiled molecules and when defective tk genes were transfected into cells on one or two different DNA molecules. By assaying for transient expression of the tk gene, we found that 3-MB did not inhibit uptake or expression of the tk gene. We conclude that poly(ADP-ribosylation) plays a role in random integration (illegitimate recombination) of DNA but does not play an important role in extrachromosomal homologous recombination, demonstrating that these two recombination pathways in cultured mouse fibroblasts are biochemically distinct.     

Genetic analysis of 2',3'-dideoxycytidine incorporation into cultured human T lymphoblasts

In order to analyze the cellular determinants that mediate the action of 2',3'-dideoxycytidine, the growth inhibitory and cytotoxic effects and the metabolism of the dideoxynucleoside were examined in wild type human CEM T lymphoblasts and in mutant populations of CEM cells that were genetically deficient in either nucleoside transport or deoxycytidine kinase activity. Whereas 2',3'-dideoxycytidine at a concentration of 5 microM inhibited growth of the wild type CEM parental strain by 50%, two nucleoside transport-deficient clones were 4-fold resistant to the pyrimidine analog. The deoxycytidine kinase-deficient cell line was virtually completely resistant to growth inhibition by the dideoxynucleoside at a concentration of 1024 microM. An 80% diminished rate of 2',3'-[5,6-3H]dideoxycytidine influx into the two nucleoside transport-deficient lines could account for their resistance to the dideoxynucleoside, while the resistance of the deoxycytidine kinase-deficient cells to 2',3'-dideoxycytidine toxicity could be explained by a virtually complete failure to incorporate 2',3'-[5,6-3H]dideoxycytidine in situ. Two potent inhibitors of mammalian nucleoside transport, 4-nitrobenzylthioinosine and dipyridamole, mimicked the effects of a genetic deficiency in nucleoside transport with respect to 2',3'-dideoxycytidine toxicity and incorporation. These data indicate that the intracellular metabolism of 2',3'-dideoxycytidine in CEM cells is initiated by the nucleoside transport system and the cellular deoxycytidine kinase activity.     

Introduction of an active enzyme into permeable cells of Escherichia coli: acquisition of ultraviolet light resistance by uvr mutants on introduction of T4 endonuclease V.

Summary Plasmolysed cells of Escherichia coli N212 (uvrA recA) acquired ultraviolet resistance when the cells were exposed to high concentrations of T4 endonuclease V. With increasing concentrations of T4 enzyme, survivals of plasmolysed cells after ultraviolet irradiation increased while colony-forming ability of unirradiated plasmolysed cells was not significantly affected by the enzyme treatment. Under appropriate conditions more than 200 fold increase in survivals was observed. When plasmolysed cells were treated with a pre-heated enzyme preparation or enzyme fractions derived from T4v ₁(endonuclease V-deficient mutant)-infected cells, only little or no reactivation took place.Permeabilization of cells prior to the enzyme treatment was essential for the effective reactivation. Treatment of intact cells with the T4 enzyme did not cause any reactivation. Cells treated with 20 mM EGTA or 50 mM CaCl₂ in cold were reactivated to certain extents by the enzyme, but the extents of the reactivation were far less compared to those of plasmolysed cells.Plasmolysed cells of strains carrying a mutation in one of uvrA, uvrB and uvrC genes were reactivated by introduction of T4 endonuclease V, as was the uvrA recA double mutant. UvrD mutants were also reactivated, but rather slightly. However, wild type strain as well as strains having a mutation in recA or polA gene were not reactivated. From these results it was suggested that T4 endonuclease V, taken up into permeable cells, can function in vivo to replace defective functions, which are controlled by the uvr genes. The conditions established in the present study may be used for introduction of other proteins into viable bacterial cells.     

Mutational and pseudomutational effects of 5-bromodeoxyuridine in human lymphoblasts

We have studied the effects of 5-bromodeoxyuridine (BrdUrd) at two genetic loci in diploid human lymphoblast cells. In thymidine kinase heterozygotes (tk +/-), a 2-h dose of BrdUrd induced a transient, non-heritable resistance to the thymidine analogue, trifluorothymidine (F3TdR). We have called this phenomenon pseudomutation and have shown that affected cells acquire the ability to survive in the presence of F3TdR and then, after degradation of F3TdR in the medium, return to an apparently normal wild-type state. Our data suggest that BrdUrd incorporation into DNA as a thymidine analogue is responsible for the effect, which we interpret as a temporary loss of thymidine kinase activity. This effect is not seen in tk +/+ homozygotes. In contrast, at the hypoxanthine-guanine phosphoribosyl transferase locus in tk +/- heterozygotes, BrdUrd did not induce a permanent, heritable resistance to 6-thioguanine (gene locus mutation). We detected such mutations only in the tk +/+ homozygote and only at external BrdUrd concentrations considerably higher than those which saturate the uptake of BrdUrd into DNA as a thymidine analogue. We postulate that the reduced TK enzyme levels (30%) in the heterozygote prevent the build-up of a sufficiently high intracellular BrdUrd triphosphate pool to promote the misincorporations as deoxycytidine triphosphate which may be responsible for gene locus mutation.     

Properties of the nonlethal recombinational repair x and y mutants of bacteriophage T4. II. DNA synthesis.

The bacteriophage T4 recombination-deficient mutants x and y exhibited decreased rates of DNA synthesis as compared to wild-type T4. Mutant-induced DNA synthesis was more sensitive to mitomycin C than was wild-type synthesis. However, DNA synthesis in mutant- and wild-type-infected cells exhibited the same sensitivity to UV light and X-irradiation. When high-specific-activity label was administered at various times postinfection, mutant DNA synthesis resembled that of wild type for 12 min. after which time mutant-induced incorporation was greatly decreased and sensitive to mitomycin C as compared to that of the wild type. Rifampin and chloramphenicol studies indicated that the gene products necessary for synthesis measured at 15 min postinfection, including those of x+ and y+ were transcribed within 2 min and translated within 8 min postinfection. Administration of chloramphenicol to mutant x- or mutant y-infected cells exactly 8 min postinfection, however, allowed for increased synthesis at 15 min that was sensitive to mitomycin C. Cells coinfected with T4+ and T4x or T4x and T4y retained a reduced mutant-type synthesis, whereas cells coinfected with T4+ and T4y exhibited a synthesis more closely resembling that of wild type.     

Characterization of a cytochalasin D-resistant mutant of Entamoeba histolytica

Characterization of a cytochalasin D-resistant mutant of the human parasite Entamoeba histolytica capable of growing at 10 microM cytochalasin is described. The mutant cells also show resistance to 5 mM colchicine and 100 microM cytochalasin B, drugs proved deleterious for wild type trophozoites. The mutants show increased osmotic fragility and electric mobility but reduced phagocytic activity, and agglutination by Concanavalin A. On the other hand pinocytic activity remains unaltered when compared with the wild type cells. Polymerized actin, seen by staining with phalloidin, often appears polarized to one end of the trophozoites and forms few of the endocytic invaginations found in wild type amebas. An altered distribution of part of the actin could explain the differences in surface properties and motility observed in the mutant amebas.     

Selection of mutant Chinese hamster ovary cells altered glycoproteins by means of tritiated fucose suicide.

Mutant Chinese hamster ovary cells altered in glycoproteins have been isolated by selecting for ability to survive exposure to [6-3H]fucose. Mutagenized wild-type cells were permitted to incorporate [3H]fucose to approximately 1 cpm of trichloroacetic acid-insoluble radioactivity per cell and then frozen for several days to accumulate radiation damage. The overall viability of the population was reduced by 5- to 50-fold. Four consecutive selection cycles were carried out. The surviving cells were screened by replica plating-fluorography for clones showing decreased incorporation of fucose into trichloroacetic acid-insoluble macromolecules. Considerable enrichment for cells deficient in fucose uptake or incorporation into proteins (or both) was found in populations surviving the later selection cycles. Two mutant clones isolated after the fourth selection cycle had the same doubling time as the wild type, but contained only 30 to 40% as much fucose bound to proteins as the wild type. Sialic acid contents of the mutants and the wild type were similar. The mutants differed quantitatively and qualitatively from the wild type and from each other with respect to total glycoprotein profiles as visualized by sodium dodecyl sulfate gel electrophoresis. Differences were also found in resistances to cytotoxicity of lectins such as concanavalin A and wheat germ agglutinin.     

Cloning and characterization of DST2, the gene for DNA strand transfer protein beta from Saccharomyces cerevisiae.

The gene encoding the 180-kDa DNA strand transfer protein beta from the yeast Saccharomyces cerevisiae was identified and sequenced. This gene, DST2 (DNA strand transferase 2), was located on chromosome VII. dst2 gene disruption mutants exhibited temperature-sensitive sporulation and a 50% longer generation time during vegetative growth than did the wild type. Spontaneous mitotic recombination in the mutants was reduced severalfold for both intrachromosomal recombination and intragenic gene conversion. The mutants also had reduced levels of the intragenic recombination that is induced during meiosis. Meiotic recombinants were, however, somewhat unstable in the mutants, with a decrease in recombinants and survival upon prolonged incubation in sporulation media. spo13 or spo13 rad50 mutations did not relieve the sporulation defect of dst2 mutations. A dst1 dst2 double mutant has the same phenotype as a dst2 single mutant. All phenotypes associated with the dst2 mutations could be complemented by a plasmid containing DST2.     

Diverse herpes simplex virus type 1 thymidine kinase mutants in individual human neurons and Ganglia

Mutations in the thymidine kinase gene (tk) of herpes simplex virus type 1 (HSV-1) explain most cases of virus resistance to acyclovir (ACV) treatment. Mucocutaneous lesions of patients with ACV resistance contain mixed populations of tk mutant and wild-type virus. However, it is unknown whether human ganglia also contain mixed populations since the replication of HSV tk mutants in animal neurons is impaired. Here we report the detection of mutated HSV tk sequences in human ganglia. Trigeminal and dorsal root ganglia were obtained at autopsy from an immunocompromised woman with chronic mucocutaneous infection with ACV-resistant HSV-1. The HSV-1 tk open reading frames from ganglia were amplified by PCR, cloned, and sequenced. tk mutations were detected in a seven-G homopolymer region in 11 of 12 ganglia tested, with clonal frequencies ranging from 4.2 to 76% HSV-1 tk mutants per ganglion. In 8 of 11 ganglia, the mutations were heterogeneous, varying from a deletion of one G to an insertion of one to three G residues, with the two-G insertion being the most common. Each ganglion had its own pattern of mutant populations. When individual neurons from one ganglion were analyzed by laser capture microdissection and PCR, 6 of 14 HSV-1-positive neurons were coinfected with HSV tk mutants and wild-type virus, 4 of 14 were infected with wild-type virus alone, and 4 of 14 were infected with tk mutant virus alone. These data suggest that diverse tk mutants arise independently under drug selection and establish latency in human sensory ganglia alone or together with wild-type virus.     

Interaction of the yeast Pso5/Rad16 and Sgs1 proteins: influences on DNA repair and aging.

The interaction trap method was used to isolate putative binding partners of Rad16/Pso5, a protein responsible for repair of silent DNA. One of the interactors found was Sgs1, a DNA helicase influencing the life span of Saccharomyces cerevisiae, with homology to the human BLM, WRN and RECQL4 proteins. Using the same fusion proteins from the two-hybrid screening, we show evidence that both proteins also interact in vitro. We tested isogenic strains, containing mutant alleles of the two genes in single and double mutant combination, for phenotypic similarity. Life span in sgs1Delta single and sgs1Delta rad16Delta double mutants is about 40% of that of WT, and the rad16/pso5Delta single mutant also had its life span reduced to 75%. Sensitivity to different mutagens, whose lesions are poorly repaired in rad16/pso5Delta mutants, was tested in sgs1Delta mutants. The sgs1Delta conferred sensitivity to MMS, H2O2 and was moderately sensitive to UV(254nm) (UVC) and 4-NQO. An epistatic interaction between rad16 and sgs1 mutations after UVC, 4-NQO and H2O2 was observed. Moreover, we found that in a top3 background, functional Sgs1p and Rad16p apparently channel MMS, 4-NQO and H2O2 induced lesions into aberrant DNA repair. Our results demonstrate that Sgs1 is not only involved in genome stability, somatic recombination and aging, but is also implicated, together with Rad16/Pso5, in the repair of specific DNA damage.     

On the fidelity of DNA replication. The accuracy of T4 DNA polymerases in copying phi X174 DNA in vitro

The fidelity with which wild type T4 DNA polymerase copies phi X174 amber 3 plus strand DNA at position 587 in vitro has been measured. Synthesis is initiated by hybridizing to the template a HaeIII restriction fragment whose 3'-OH terminus is 83 nucleotides from the amber 3 site. Based on gel electrophoresis of product DNA molecules and genetic marker rescue data, T4 DNA polymerase copies significantly beyond the mutant site. Transfection analysis shows that the A X T leads to G X C mutation at position 587 occurs 10- to 100-fold less frequently with T4 DNA polymerase than with E. coli DNA polymerase I. The aberrant incorporation of cytosine opposite adenine at position 587 by the T4 polymerase alone is occurring at a frequency not greater than about 10(-7) which, for this particular locus, may be similar to the fidelity exhibited by the T4 accessory proteins plus the polymerase comprising the replication complex. A comparison of the accuracy of mutator L56 and antimutator L141 T4 DNA polymerases relative to wild type shows at most a 2- to 4-fold decrease and increase, respectively, in fidelity. When compared to 10- to 1000-fold effects on mutation frequencies that these same mutant alleles have in vivo, these results suggest that the wide range in expression of mutator and antimutator phenotypes in vivo may be dependent on an abnormal interaction of the aberrant DNA polymerases with other protein components of the replication complex.     

Altered nucleoside transporters in mammalian cells selected for resistance to the physiological effects of inhibitors of nucleoside transport

From a mutagenized population of wild type S49 T lymphoma cells, clones were generated that were resistant to the physiological effects of the potent inhibitor of nucleoside transport, 4-nitrobenzyl-6-thioinosine (NBMPR). These cells were selected for their ability to survive in semisolid medium containing 0.5 mM hypoxanthine, 0.4 microM methotrexate, 30 microM thymidine, 30 microM deoxycytidine, in the presence of 30 microM NBMPR. NBMPR protected wild type cells from the effects of a spectrum of cytotoxic nucleosides, whereas two mutant clones, KAB1 and KAB5, were still sensitive to nucleoside-mediated cytotoxicity in the presence of NBMPR. Comparisons of the abilities of wild type cells and mutant cells to incorporate exogenous nucleoside to the corresponding nucleoside triphosphate indicated that the KAB1 and KAB5 mutant cells were refractory to normal inhibition by NBMPR. Moreover, rapid transport studies indicated that mutant cells, unlike wild type parental cells, had acquired a substantial NBMPR-insensitive nucleoside transport component. Binding studies with [3H]NBMPR indicated that KAB5 cells were 70-75% deficient in the number of NBMPR binding sites, whereas KAB1 cells possessed a wild type complement of NBMPR binding sites. These data suggest that the NBMPR binding site in wild type S49 cells is genetically distinguishable from the nucleoside carrier site.     

Importance of the membrane-perturbing properties of the membrane-proximal external region of human immunodeficiency virus type 1 gp41 to viral fusion

The membrane-proximal external region (MPER; K(665)WASLWNWFNITNWLWYIK(683)) of the human immunodeficiency virus type 1 (HIV-1) gp41 ectodomain plays a critical role in envelope glycoprotein-mediated fusion. In addition, the epitopes of important neutralizing antibodies (2F5, Z13, and 4E10) and the sequence of the peptide fusion inhibitor T20 overlap this conserved region. The MPER has an unusually high percentage of tryptophan residues that likely contribute to the membrane-disrupting nature of the region, which is predicted to adopt an alpha-helical conformation on membrane contact. We have investigated the membrane-disruptive requirements for this region using a panel of mutants that replace most of the MPER with antibacterial, membrane-active peptides. The results demonstrate that the mutant Envs were processed, transported, and expressed on the cell surface similar to wild type. Some of the mutant Envs induced moderate levels of cell-cell fusion, demonstrating that the region can accommodate the substitution of proline-rich foreign peptides while retaining significant biological function. In contrast, the incorporation into and stability of the mutated Envs in virions was reduced, consistent with the severely impaired viral entry observed for all the mutants. These data suggest that both structural (for Env incorporation) and functional (membrane disruption) constraints may contribute to the highly conserved nature of this region.     

An anti-HLA class I monoclonal antibody alters the progression in the cell cycle of phytohemagglutinin-activated human T lymphocytes

The monomorphic anti-HLA Class I monoclonal antibody 01.65 inhibits the incorporation of tritiated thymidine ([3H]TdR) in Phytohemagglutinin (PHA)-activated human T lymphocytes. Our data indicate that 01.65 affects the average duration of the cell cycle by increasing the length of the early S subphase. As a consequence of the increase in the doubling time of the cell population, the absolute number of cells at harvesting time was reduced in 01.65-treated cultures compared to that of untreated cultures. The lengthening of the S-phase and the decrease in the cell number can together quantitatively account for the reduction of [3H]TdR incorporation observed in 01.65-treated cultures.     

Isolation and Characterization of Mutants of Thiophene Synthesis in Tagetes erecta

Two mutants of Tagetes erecta displaying aberrant thiophene composition were identified by screening more than 300 plants from a mutagenized M2 population using high-performance liquid chromatography analysis of root extracts. Both mutants, which may have originated from the same mutational event, contained high amounts of the C13 monothiophene 2-(but-3-en-1-ynyl)-5-(penta-1,3-diynyl)-thiophene that was previously not found in T. erecta and also high amounts of two C13 bithienyls that were absent or present at low concentrations in the wild type. The mutant phenotype was also expressed in 21 Agrobacterium rhizogenes transformed root clones derived from both mutants. Feeding experiments with root cultures derived from one mutant and from the wild type indicated that the monothiophene accumulating in the mutant is the common precursor for all bithienyl thiophenes in wild-type and mutant Tagetes erecta. These experiments also showed that one mutant is deficient in demethylation of the monothiophene.     

Ultrastructure and cell wall composition in cell division cycle mutants ofSchizosaccharomyces pombe deficient in septum formation

A number of temperature-sensitive cdc^- mutants ofSchizosaccharomyces pombe that are affected in septum formation were analyzed with respect to their ultrastructure and the composition of their cell wall polymers. One mutant strain, cdc 16–116, has a cell wall composition similar to the wild type (strain 972 h^-). However two other mutants, cdc 4 and cdc 7, show a higher galactomannan content and a lower -glucan content. In all the mutants tested, total glucose incorporation, protein, RNA and DNA synthesis increased similarly to wild type over 3 1/2 h. After 2–3 h of incubation at the non permissive temperature-35°C-, cell numbers remained constant although, increases in optical densities at 600 nm were observed. According to scanning electron microscopy, the mutants had aberrant shapes after 5h of incubation at 35°C. Transmission electron microscopy showed that cdc 3 is unable to complete septum formation. cdc 4 showed the most varied morphological shapes and aberrant depositions of cell wall material. cdc 8 exhibited a deranged plasma membrane and cell wall regions near of cell poles; an abnormal septum and several nuclei. cdc 7 showed elongated cells with several nuclei and with an apparently normal cell wall completely lacking in septum and septal material. cdc 16 showed more than one septum per cell.