Temperature-Sensitive Mutants of Bacteriophage Mu

Temperature-sensitive mutants of bacteriophage Mu, which grow at 32 C but not at 42 C, have been isolated. These mutants fall into two groups. Group 1 mutants fail to lyse host cells at nonpermissive temperatures, whereas lysis occurs normally with the group 2 mutants. All of the group 1 mutants apparently belong to the cistrons mapping to the left of gene C, whereas the group 2 mutants have lesions in various genes between D and S.     

A new gene of Escherichia coli K-12 whose product participates in T4 bacteriophage late gene expression: interaction of lit with the T4-induced polynucleotide 5''-kinase 3''-phosphatase.

We isolated five Escherichia coli mutants deficient in their ability to support the late (replication-coupled) gene expression of T4 bacteriophage at 30 degrees C. These mutants, which we call Lit mutants, define at least one novel gene at 25 min on the E. coli map. They were selected in an attempt to obtain mutants which restrict the growth of T4 mutants deficient in polynucleotide 5'-kinase 3'-phosphatase but not that of wild-type T4 at 37 degrees C. Some of the mutants do have these phenotypes under some conditions. Studies of the block in T4 development in some of the E. coli mutants suggest that Lit mutants are affected in a gene product involved in the metabolism of deoxyribonucleic acid nicks or single-strand gaps. None of the Lit mutants is deficient in the major, bacterial, 3'-phosphatase activity in crude extracts.     

Physiological characterization of temperature-sensitive mutants of mengovirus.

Twenty-four temperature-sensitive mutants of mengovirus were characterized physiologically with respect to phenotype. The mutants were separated into four classes on the basis of viral RNA synthesis. L-67-S cells infected with five of the mutants synthesized little viral RNA at 39.5 C. These mutants are designated RNA-. One mutant is designated RNA* since its RNA synthesis is altered at both 39.5 and 31.5 C. The other mutants were divided into two groups, RNA plus or minus (25 TO 49% of wild-type RNA synthesis) and RNA plus (50 to 100% of wild-type RNA synthesis). The time of expression of the mutation in the RNA- mutants was estimated from the results of reciprocal temperature-shift experiments. The mutatation in ts12 appears to be expressed at the time RNA synthesis normally begins. The defect in three of the mutants was expressed 1 to 2 h before RNA synthesis is normally detectable. Protein synthesis is required before RNA synthesis begins when the cells are shifted from 39.5 to 31.5 C. The RNA polymerase synthesized by cells infected with these RNA- mutants at 31.5 C was stable and fully active when assayed at 39.5 C in vitro. The sedimentation profiles of the viral RNA synthesized by cells infected with RNA plus and RNA plus or minus mutants are similar to wild-type profiles with the exception of ts148. Cells infected with this RNA plus or minus mutant synthesize RNA that sediments in a sucrose gradient like replicative-intermediate RNA, but little mature viral RNA is evident. The results of step-up experiments indicate that the temperature-sensitive period for the majority of the RNA plus and RNA plus and minus mutants extends through most of the replicative cycle. The temperature-sensitive defect of four of the mutants, however, was expressed in the first hour, suggesting that some undefined early function is required for the eventual maturation of mengovirus. The virions of three of the RNA- mutants were more thermolabile than wild-type virions. Five of the RNA plus and RNA plus or minus mutants were also thermolabile. Genetic complementation at a significant level was not detectable in mixed infections of the mutants described.     

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 Preliminary Characterization of Temperature-Sensitive Mutants of Influenza Virus

Isolation of temperature-sensitive (ts) mutants was attempted from the WSN strain of influenza A virus which was grown and assayed in MDBK cells. After growth of wild-type virus in the presence of 5-fluorouracil, 15 ts mutants were selected for which the ratio of plaquing efficiency at 39.5 C to that at 33 C was 10⁻³ or less. In pairwise crosses of ts mutants, recombination and complementation were either very efficient or undetectable. It is suggested, therefore, that the viral genome consists of physically discrete units and recombination occurs as an exchange of these units. All 15 mutants have been assigned with certainty into five recombination groups. Three mutants are suspected to be double mutants. Any two complementing mutants always recombined with each other, and noncomplementing mutants did not recombine. In physiological tests, mutants showed diverse patterns of functional defects at the nonpermissive temperature. However, it was not always possible to correlate these physiological defects with the results of genetic characterization.     

Mutants of Escherichia coli with High Minimal Temperatures of Growth.

O'Donovan, Gerard A. (University of California, Davis), Catherine L. Kearney, and John L. Ingraham. Mutants of Escherichia coli with high minimal temperatures of growth. J. Bacteriol. 90:611-616. 1965.-Three general classes of mutants showing increased minimal temperatures of growth have been isolated from Escherichia coli. These mutants do not grow at temperatures below 20 C, although their parents can grow at temperatures as low as 8 C. The first class of mutants (K-I) cannot grow below 20 C in either complex or minimal medium, but grows at nearly normal rates at 37 C on both types of media. Normal growth rate at 20 C can be conferred on these mutants by infection at a low multiplicity with a transducing phage grown on the parent. The second class of mutants (K-II) fails to grow only in minimal medium at 20 C. These mutants are characterized by their singular response to specific nutrients in minimal medium at 20 C. The third class of mutants (K-III) grows normally in minimal medium at all temperatures with either glucose or glycerol as the carbon source, but does not grow at 20 C with lactose as the carbon source.     

Isolation and characterization of temperature-sensitive mutants of measles virus.

Nine temperature-sensitive (ts) mutants of nonattenuated Edmonston strain measles virus were isolated from wild-type virus which was grown in the presence of 5-fluorouracil. Adsorption, temperature shift, and complementation experiments indicated that all these mutants were restricted at an intracellular stage of infection. However, all the mutants were more rapidly inactivated at 41 C than was wild-type virus, suggesting that the ts product of each mutant either influences or is a structural component of the virus. Three complementation groups were found to be represented among the mutants. Group A contained one mutant and it did not induce synthesis of detectable amounts of viral antigen at the nonpermissive temperature (39 C). Group B consisted of six mutants which did not induce viral antigen synthesis at 39 C and one mutant which did. Group C was represented by one mutant and it induced viral antigen synthesis at 39 C. The two mutants which induced sythesis of viral antigen also induced synthesis of relatively small amounts of virus-specific RNA at 39 C. These mutants, while producing cytoplasmic and nuclear accumulations of viral antigen at 39 C, were restricted in production of syncytia and hemadsorption. All the mutants were less neurovirulent than wild-type virus, as indicated by their inability to produce acute disease in newborn hamsters.     

The Escherichia coli dnaJ mutation affects biosynthesis of specific proteins, including those of the lac operon.

Temperature-sensitive dnaJ mutants of Escherichia coli showed a thermosensitive defect in the synthesis of beta-galactosidase. Synthesis of the lac mRNA was greatly reduced at the restrictive temperature. The mutants were also conditionally defective in the synthesis of a subset of membrane proteins such as succinate dehydrogenase, whereas the synthesis of anthranilate synthetase, encoded by trpED, as well as that of most cellular proteins, was unaffected at the restrictive temperature. The defect was specific for the dnaJ mutants among several dna mutants which are known to be involved in the initiation of DNA synthesis: dnaK, dnaA, and dnaB mutants synthesized each of these proteins normally even at the restrictive temperature. At the restrictive temperature, growth of the dnaJ mutants was arrested at a specific stage of the cell cycle.     

Virion functions of RNA+ temperature-sensitive mutants of Newcastle disease virus.

Virions from Newcastle disease virus mutants in four temperature-sensitive RNA+ groups were grown in embryonated hen eggs at the permissive temperature, purified, and then analyzed for biological properties at both the permissive and nonpermissive temperatures. At the permissive temperature, virions of mutants in groups B, C, and BC (11 mutants) were all lower in specific (per milligram of protein) hemagglutination, neuraminidase, and hemolysis activities compared with the wild type. These deficiencies were related to decreased amounts of hemagglutinin-neuraminidase glycoprotein in the virions. Activities of these mutant virions at both the permissive and nonpermissive temperatures were similar, indicating that hemagglutinin-neuraminidase synthesized at the permissive temperature was not temperature sensitive in function. The three group D mutants displayed a different pattern. At the permissive temperature, they had wild-type hemagglutination and neuraminidase activities but were deficient compared with the wild type in hemolysis. Again, functions were similar at both temperatures. Most of the B, C, and BC mutants had specific infectivities similar to that of the wild type despite lower hemagglutination, neuraminidase, and hemolysis functions. However, the D mutants were all less infectious. This evidence is consistent with a shared hemagglutinin-neuraminidase defect in the B, C, and BC mutants and a defect in either the F glycoprotein or the M protein in the D mutants.     

The effect of surface charge balance on thermodynamic stability and kinetics of refolding of firefly luciferase

Thermodynamic stability and refolding kinetics of firefly luciferase and three representative mutants with depletion of negative charge on a flexible loop via substitution of Glu by Arg (ER mutant) or Lys (EK mutant) as well as insertion of another Arg in ER mutants (ERR mutant) was investigated. According to thermodynamic studies, structural stability of ERR and ER mutants are enhanced compared to WT protein, whereas, these mutants become prone to aggregation at higher temperatures. Accordingly, it was concluded that enhanced structural stability of mutants depends on more compactness of folded state, whereas aggregation at higher temperatures in mutants is due to weakening of intermolecular repulsive electrostatic interactions and increase of intermolecular hydrophobic interactions. Kinetic results indicate that early events of protein folding are accelerated in mutants.     

Isolation and primary identification of methylotrophic yeast Hansenula polymorpha mutants for peroxisome biogenesis

After exposure of cells of the methylotrophic yeast Hansenula polymorpha HF246 leu1-1 to N-nitro-N-nitrosoguanidine, a collection of 227 mutants unable to grow on methanol at elevated temperature (45 degrees C) was obtained. Ninety four ts mutants (35% of the total number of mutants), which were unable to grow on methanol only at 45 degrees C but could grow at optimal temperature (37 degrees C), were isolated. Complementation analysis of mutants using 12 deletion mutants for genes of peroxisome biogenesis (PEX) (available in this yeast species by the beginning of our work) allowed to assign 51 mutants (including 16 ts) to the separate group of mutants unable to complement deletion mutants with defects in eight PEX genes. These mutants were classified into three groups: group 1 contained 10 pex10 mutants (4 ts mutants among them); group 2 included 19 mutants that failed to complement other pex testers: 1 pex1; 2 pex4 (1 ts); 6 pex5 (5 ts); 3 pex8; 6 (3ts)- pex19; group 3 contained 22 "multiple" mutants. In mutants of group 3, hybrids with several testers do not grow on methanol. All mutants (51) carried recessive mutations, except for mutant 108, in which the mutation was dominant only at 30 degrees C, which suggests that it is ts-dominant. Recombination analysis of mutants belonging to group 2 revealed that only five mutants (two pex5 and three pex8) carried mutations for the corresponding PEX genes. The remaining 14 mutants yielded methanol-utilizing segregants in an arbitrarily chosen sample of hybrids with the pex tester, which indicates mutation location in other genes. In 19 mutants, random analysis of ascospores from hybrids obtained upon crossing mutants of group 3 with a strain lacking peroxisomal disorders (ade11) revealed a single mutation causing the appearance of a multiple phenotype. A more detailed study of two mutants from this group allowed the localization of this mutation in the only PEX gene (PEX or PEX2). The revealed disorder of complementation interactions between nonallelic genes is under debate.     

Molecular biology of rotaviruses. III. Isolation and characterization of temperature-sensitive mutants of bovine rotavirus

Twenty-six temperature-sensitive (ts) mutants of United Kingdom tissue culture-adapted bovine rotavirus were isolated and characterized. Fourteen of these mutants were determined to be ts both by efficiency of plating and by virus yield at the nonpermissive temperature of 39.5 degrees C as compared with that at the permissive temperature of 32 degrees C. The remaining mutants were only ts by the criterion of efficiency of plating. High-frequency recombination (gene reassortment) was observed when some pairs of mutants were crossed, and this allowed the classification of the mutants into five separate recombination groups. Groups III and V have prototype ts mutants (ts34 and ts115, respectively) that do not synthesize RNA or polypeptides at 39.5 degrees C. The other groups, I, II, and IV, have prototype mutants (ts17, ts7, and ts6, respectively) that synthesize both RNA and polypeptides at 39.5 degrees C, although ts17 does so only at a reduced level.     

DNA synthesis and DNA polymerase activity of herpes simplex virus type 1 temperature-sensitive mutants.

Fifteen temperature-sensitive mutants of herpes simplex virus type 1 were studied with regard to the relationship between their ability to synthesize viral DNA and to induce viral DNA polymerase (DP) activity at permissive (34 C) and nonpermissive (39 C) temperatures. At 34 C, all mutants synthesized viral DNA, while at 39 C four mutants demonstrated a DNA+ phenotype, three were DNA+/-, and eight were DNA-. DNA+ mutants induced levels of DP activity similar to thhose of the wild-type virus at both temperatures, and DNA+/- mutants induced reduced levels of DP activity at 39 C but not at 34 C. Among the DNA- mutants three were DP+, two were DP+/-, and three showed reduced DP activity at 34 C with no DP activity at 39 C. DNA-, DP- mutants induced the synthesis of a temperature-sensitive DP as determined by in vivo studies.     

Isolation and Preliminary Characterization of Temperature-Sensitive Mutants of Rhizobiophage C

By using mutagenesis induced by N-methyl-N'-nitro-N-nitrosoguanidine, 150 temperature-sensitive mutants of rhizobiophage c were isolated. All were able to form plaques at 14 C but not at 29 C. They were classified into 21 complementation groups. Representative temperature-sensitive mutants from each complementation group were analyzed with regard to gene function. The approximate time of expression of the genes defined by the mutants was measured by temperature-shift experiments. Most genes began to be expressed at 4.0 to 7.5 h after infection at 14 C. Four genes were found which were expressed 2.5 to 3.5 h after infection. Some mutants showed no DNA synthesis at 29 C; some showed a delay in lysis. Some produced apparently normal particles after infection and lysis at 29 C; others produced various types of defective particles. Some mutants showing no DNA synthesis at 29 C nevertheless caused lysis at the normal time together with the production of phage structural components. Representative mutants from each complementation group were mapped by using two-factor crosses. A preliminary genetic map of phage c was constructed from the data.     

Repair of Ultraviolet Radiation Damage in Sensitive Mutants of Micrococcus radiodurans

Various aspects of the repair of ultraviolet (UV) radiation-induced damage were compared in wild-type Micrococcus radiodurans and two UV-sensitive mutants. Unlike the wild type, the mutants are more sensitive to radiation at 265 nm than at 280 nm. The delay in deoxyribonucleic acid (DNA) synthesis following exposure to UV is about seven times as long in the mutants as in the wild type. All three strains excise UV-induced pyrimidine dimers from their DNA, although the rate at which cytosine-thymine dimers are excised is slower in the mutants. The three strains also mend the single-strand breaks that appear in the irradiated DNA as a result of dimer excision, although the process is less efficient in the mutants. It is suggested that the increased sensitivity of the mutants to UV radiation may be caused by a partial defect in the second step of dimer excision.     

Physical mapping of herpes simplex virus type 1 mutations by marker rescue.

Thirty temperature-sensitive mutants of encephalomyocarditis virus have been isolated and partially characterized. Fifteen of these mutants are phenotypically RNA+ thirteen are RNA-, and two are RNA +/-. Six RNA + mutants, one RNA- mutants, and one RNA +/- mutant have virions which are more thermosensitive at 56 degree C than the wild-type virions. Hela cells infected at the nonpermissive temperature with any of the RNA+ mutants produced neither infective nor noninfective viral particles. The cleavage of the precursor polypeptides in cells infected with 11 of the RNA+ mutants was defective at the nonpermissive temperature. This defect in cleavage occurred only in those precursor polypeptides leading to capsid proteins.     

Temperature sensitive nitrogen fixation mutants of Azotobacter vinelandii

Summary Temperature-sensitive nitrogen fixation mutants of Azotobacter vinelandii were obtained by nitrosoguanidine mutagenesis and penicillin selection. The mutants were unable to grow on N₂ at 39° but grew normally at 30° on N₂ and at both temperatures in the presence of metabolizable nitrogen compounds. Growth experiments and assays of whole cells for nitrogenase activity separated the mutants into two classes: 1. mutants in which the nitrogenase activity present in cells grown at 30° was unaffected by a shift to 39°, and 2. mutants which lost their nitrogen fixation activity after such a temperature shift. Assays of cell-free extracts of the second class of mutants showed that in all cases tested the enzymatic activity of the nitrogenase complex itself was not affected by the mutation. These mutants might therefore contain some other temperature-sensitive proteins specifically involved in nitrogen fixation.     

Isolation of heat- and cold-sensitive mutants of chinese hamster lung cells affected in their ability to express the transformed state.

A clone of spontaneously transformed Chinese hamster lung cells was exposed to N-methyl-N'-nitro-N-nitroso-guanidine (MNNG), and six heat-sensitive and three cold-sensitive mutants were isolated after selection for inability to form colonies in soft agar at 39.5 degrees C and 34.5 degrees C, respectively. The heat-sensitive mutants had growth characteristics of transformed cells at 34.5 degrees C, but exhibited a normal phenotype at 39.5 degrees C. By contrast, cold-sensitive mutants displayed the characteristics of the normal cells at 34.5 degrees C and converted to a transformed phenotype at 39.5 degrees C. Transformed parent cells exhibited no obvious temperature-dependent properties. Temperature shift experiments showed that the colony-forming ability of both types of mutants was fully reversible. All of the mutants were able to grow well at both permissive and nonpermissive temperatures when grown on the surface of plastic dishes. Such mutants will be useful in analysis of factors involved in the expression of the transformed state or the maintenance of the nontransformed state.