Characterization Of Temperature-sensitive Mutants Research Articles

Role of vaccinia virus A20R protein in DNA replication: construction and characterization of temperature-sensitive mutants.

Previous analyses of randomly generated, temperature-sensitive vaccinia virus mutants led to the mapping of DNA synthesis negative complementation groups to the B1R, D4R, D5R, and E9L genes. Evidence from the yeast two-hybrid system that the D4R and D5R proteins can interact with the A20R protein suggested that A20R was also involved in DNA replication. We found that the A20R gene was transcribed early after infection, consistent with such a role. To investigate the function of the A20R protein, targeted mutations were made by substituting alanines for charged amino acids occurring in 11 different clusters. Four mutants were not isolated, suggesting that they were lethal, two mutants exhibited no temperature sensitivity, two mutants exhibited partial temperature sensitivity, and two mutants formed no plaques or infectious virus at 39 degrees C. The two mutants with stringent phenotypes were further characterized. Temperature shift-up experiments indicated that the crucial period was between 6 and 12 h after infection, making it unlikely that the defect was in virus entry, early gene expression, or a late stage of virus assembly. Similar patterns of metabolically labeled viral early proteins were detected at permissive and nonpermissive temperatures, but one mutant showed an absence of late proteins under the latter conditions. Moreover, no viral DNA synthesis was detected when cells were infected with either stringent mutant at 39 degrees C. The previous yeast two-hybrid analysis together with the present characterization of A20R temperature-sensitive mutants suggested that the A20R, D4R, and D5R proteins are components of a multiprotein DNA replication complex.

Isolation and characterization of temperature sensitive mutants of Bombyx mori nuclear polyhedrosis virus and their multiplication in a cell line and larvae of Bombyx mori

Isolation and characterization of temperature sensitive mutants of Bombyx mori nuclear polyhedrosis virus and their multiplication in a cell line and larvae of Bombyx mori

Isolation and Characterization of Temperature Sensitive Mutants of the F5 Deletion Mutant of Mycobacteriophage D29

Seven thermosensitive mutants of the F5 deletion mutant of the mycobacteriophage D29 were described. The mutants were obtained using N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) mutagenesis, and were characterized using temperature shift assays, complementation and recombination tests, electron microscopy of infected host bacteria at non-permissive temperature, and serum blocking power. Mutants deficient in tail assembly, and mutants deficient in head and tail assembly were described. Mutants deficient in head assembly but capable of assembling tails were not isolated during this study. From the data, 3 provisional linkage map of the phage F5 was proposed.

Isolation and Preliminary Characterization of Temperature-sensitive Mutants of Mouse Cytomegalovirus of Differing Virulence for 1-week-old Mice

To study the pathogenicity of murine cytomegalovirus (MCMV) and to identify virulence determinants, we have isolated and phenotypically characterized a set of temperature-sensitive mutants. One mutant, PP269/38, was avirulent for 1-week-old BALB/c mice and restricted in its plaque formation and replication at 39 degrees C. Mutants PP242/68 and PP268/38 were 100-fold less virulent than salivary gland-grown virus (SGV), even after two passages in the salivary glands of 1-week-old mice. The former mutant was unable to replicate or form plaques at 39 degrees C whereas the latter replicated poorly at 39 degrees C but not at all at 40 degrees C although it was able to form plaques at 40 degrees C with a reduced plaque size. PP31/15 exhibited a 40-fold reduction in virulence compared to SGV after two passages in vivo and was unable to form plaques or to replicate at 40 degrees C; at 39 degrees C it was able to, with reduced efficiency. The remaining two mutants, PP99/3 and PP392/31, were 10-fold less virulent than SGV and were restricted at 40 degrees C. The six mutants have been classified into at least four complementation groups. These mutants may be useful for studying various aspects of MCMV pathogenicity.

Isolation and characterization of temperature sensitive mutants of Broadhaven virus, a Kemerovo group orbivirus (family, Reoviridae).

Eighteen stable temperature sensitive (ts) mutants of Broadhaven virus were isolated without the aid of mutagens. Spontaneous mutants were detected using 41 degrees C as the nonpermissive temperature and 36 degrees C as the permissive temperature. High frequency pairwise recombination defined five recombination groups. Four mutants belonged to group I, three to group II, six to group III, two to group IV, and two to group V. ts 7 was a possible double mutant representing lesions corresponding to those of groups III and V mutants. This is the first reported isolation of temperature sensitive mutants of a tick-borne orbivirus.

Characterization of temperature sensitive mutants of Pichinde virus

Characterization of temperature sensitive mutants of Pichinde virus

Partial characterization of temperature-sensitive mutants of foot-and-mouth disease virus, O1 caseros strain.

The preliminary characterization of four temperature-sensitive (ts) mutants of the O1 Caseros strain of foot-and-mouth disease virus is described. Two mutants, ts 6 and ts 40, showed a very low RNA synthesis rate at nonpermissive temperature and were classified phenotypically as RNA(-). Shift-up experiments demonstrated an incapacity to organize the RNA replicative process at nonpermissive temperature. Another mutant (ts 139) behaved phenotypically as RNA(+) and its virions were more thermolabile than the wild type virus, so the defect in this mutant is likely to be in one of its structural proteins. Finally, mutant ts 5 was phenotypically RNA(+); its leak production at nonpermissive temperature was high, and the shift-up experiment showed a defect that was expressed either late in the replicative cycle or continuously.

Isolation and preliminary characterization of temperature-sensitive mutants of poliovirus type 1

Summary Following chemical mutagenesis, four temperature-sensitive mutants of the Mahoney strain of poliovirus type 1 were isolated. These mutants (clones 035, 203, 221 and 247) had a 4 × 102 to 4 × 103-fold lower plaquing efficiency on HeLa cells at 38.5° C than at 32.5° C. Standard growth curves showed that the viral mutants were restricted at 38.5° C but not at 37° C. The virions of clone 247 showed a greatly increased heat sensitivity: infectivity of the mutant stocks decreased by one log after a 1.5-min incubation at 45° C or after 15 min at 38.5° C. In contrast, equivalent heat inactivation of wild type virus or mutant ts 035 required about 6.5 min at 45° C and both viruses were completely stable for 30 min at 38.5° C. The virions of clones 203 and 221 showed partial heat sensitivity at 45° C but none at 38.5° C. Clones 035, 221 and 247 were unable to synthesize viral RNA at 38.5° C. Shift up of HeLa cells infected by any of these mutants from 32.5° C to 38.5° C, during the first 4 h of the virus growth cycle, resulted in prevention or arrest of viral RNA replication. Viral RNA synthesis continued unabated when the infected cells were shifted to non-permissive temperature at 6 h after infection. The RNA− phenotype at non-permissive temperature was therefore only manifest at early times after infection. Genetic recombination between the four mutants was investigated using both the conventional virus yield test and an infectious center assay. Recombinant plaques were found to occur with a significant frequency between ts 035 and ts 203, whereas no recombination was detected when ts 221 or ts 247 were one of the parents.

Genetics of Reovirus.

Genetic studies aimed at a better understanding of aspects of the structure and function of the genome of mammalian reoviruses were initiated in the late 1960s. These began with the isolation and characterization of temperature-sensitive mutants (Fields and Joklik 1969, Ikegami and Gomatos 1968). Much of the early research in this area, performed mainly before 1975, has been described in a recent, detailed review (Cross and Fields 1977).KeywordsGenome SegmentViral HemagglutinindsRNA SegmentReovirus TypeMammalian ReovirusThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Pathogenic murine coronaviruses III. Biological and biochemical characterization of temperature sensitive mutants of JHMV

JHMV is a neurotropic member of the hepatoencephalitis group of murine coronaviridae. The characteristics of the biology and intracellular viral RNA synthesis and the intracellular viral protein synthesis of JHMV are discussed in the two previous papers, respectively. This paper describes the neuropathogenesis of JHMV and the isolation and characterization of 34 temperature-sensitive mutants of JHMV. These mutants were selected for their inability to induce syncytia formation after low multiplicity infection (m.o.i. = 0.1 iU) in BALB/c 17CL-1 cells at 38.5° as compared to the induction of syncytia at 33°. N-Methyl-N′-nitrosoguanidine (14 mutants) and 5-fluorouridine (20 mutants) were used as mutagens at a concentration that reduced infectivity by 90–95%. Characterization of these mutants included: induction of syncytia; synthesis of JHMV-specific intracellular RNA; progeny yields at 33, 37, and 38.5°; synthesis of JHMV-specific antigens as determined by indirect immunofluorescence and sodium dodecyl sulfate-polyacrylamide gel electrophoresis; virion thermostability; neuropathogenesis including isolation of virus from infected brain, immunofluorescence of infected brain, and histopathology of brain and spinal cord by light and transmission electron microscopy; ability to protect mice from a lethal JHMV infection; and complementation. RNA-minus (1734), RNA-intermediate (1434), and RNA-plus (334) groups were defined. One mutant, N3, produces chronic meningitis and demyelination without typical JHMV encephalitis in spite of the fact that neurons are infected as detected by immunofluorescence. This altered neuropathogenesis cannot be explained by “leakiness” or reversion. In addition, non-temperature-sensitive variants of JHMV have been selected for altered neuropathogenesis and are described.

Isolation and Preliminary Characterization of Temperature-sensitive Mutants of the Murine Sarcoma Leukaemia Virus Complex

Five temperature-sensitive mutants (ts I to 5) were isolated from a stock of the Moloney strain of murine sarcoma leukaemia virus complex which had been mutagenized by ultraviolet irradiation or N-methyl-N'-nitro-N-nitrosoguanidine. In mouse cells at the non-permissive temperature the mutants formed fewer foci of transformed cells than at the permissive temperature. The ts mutants were characterized by testing: (I) murine leukaemia virus (MuLV) clones from the ts complex, (2) the effect of additional wild type MuLV on focus formation, (3) focus formation in rat cells and (4) focus formation with pseudotypes rescued from non-producer cells. Two mutants (ts 1 and ts 3) were found to be ts MuLVs which did not possess heat labile virion proteins and were not ts in post-penetration helper functions necessary for the fixation of sarcoma virus transformation. The remaining three mutants (ts 2, ts 4 and ts 5) were ts murine sarcoma viruses which, however, showed no temperature-sensitive effect on the maintenance of transformed cell morphology nor on colony forming efficiency in soft agar.

Isolation and characterization of temperature sensitive mutants of cyanophage LPP-1

Optimal conditions for the induction of temperature-sensitive (ts) mutants of cyanophage N-1 were established after mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). A treatment with MNNG (400 micrograms/ml) for 2 hrs at pH 8.0 induced ts-mutants at a maximum frequency of 1.46 x 10(-3). A characterization of 10 such ts-mutants with regard to adsorption, one-step growth and temperature-shift experiments with Nostoc muscorum as host bacterium led to the identification of temperature-sensitive steps in the phage multiplication at the restrictive temperature (37 degrees C). All the mutants were found to be conditionally lethal at 37 degrees C since they resumed growth upon shifting to 28 degrees C.

Isolation and characterization of temperature-sensitive mutants of Chinese hamster ovary cells after treatment with UV and x-irradiation.

The isolation of ten conditionally lethal temperature-sensitive mutants of the Chinese hamster ovary cell (CHO-Kl, pro-) by the BUdR-visible light selection procedure described. Treatment with radiation at doses known to cause single gene mutation in mammalian cells increases the mutation frequency by a factor of at least 14. These mutants will grow with normal plating efficiency at 34.5 degrees but will not grow at 39.5 degrees. Complementation analysis by two independent methods indicates that all mutants are recessive and allows the assignment of the mutants to six genetically independent complementation groups. Reversion analysis indicates that the TS-mutants are stable, spontaneous revertants arising at a frequency of less than 10(-6). Preliminary chromosome analysis revealed no systematic chromasomal abnormality in the mutants. Mitotic accumulation is used to study the generation time of the parental cells and representative mutants at 34.5 degrees and 39.5 degrees. The uses of these mutants for genetic analysis of mammalian cells in culture is discussed.

Isolation and partial characterization of temperature-sensitive mutants of Mengo virus

Abstract A total of 18 temperature-sensitive mutants of Mengo virus, produced by chemical mutagenesis, has been partially purified and characterized with respect to (1) single cycle growth at the permissive (33°) and nonpermissive (39°) temperatures, (2) ability to stimulate the synthesis of viral ribonucleates at the two temperatures, and (3) thermal stability. Of the 18, 11 and 7 were found to be RNA− and RNA+, respectively, and 5 were found to have significantly lower thermal stabilities than the wild-type.

Isolation and characterization of temperature-sensitive mutants in a Chinese hamster cell line

A replica plating method was used for the isolation of temperature-sensitive ( ts) mutants after treatment of Chinese hamster cells with ethyl methanesulfonate (EMS). No significant increase in ts mutants was found after this treatment. The limitations and advantages of the replicating procedure to detect such differences, as well as an alternative method, are discussed. Mutants isolated were classified into two general groups—density-dependent and clear-cut—as measured by survival at low and high cell densities at the restrictive temperature. The density-dependent mutants may be truly “leaky”, losing a metabolite to the medium at an excessive rate at the restrictive temperature. On the other hand, the one clear-cut mutant analyzed extensively dies at a rate determined by its ability to utilize one or more components from the medium. It shows an inverse density relationship in rate of death, as inferred from rates of macromolecular synthesis, as opposed to its growth rate at the permissive temperature.