An enrichment method for temperature-sensitive and auxotrophic mutants of yeast

Summary An enrichment procedure that exploits the difference in heat-sensitivity between exponentially growing and stationary phase cells has been developed for the isolation of yeast mutants. Enrichments of up to 12-fold for temperature-sensitive lethal mutants and of up to 15-fold for auxotrophs have been obtained with single cycles of selection. Still higher enrichments (to frequencies of greater than 90% and 80% for temperature-sensitive lethals and auxotrophs, respectively) have been obtained with multiple cycles of selection. The method requires no special parent strain, and seems adaptable to the selection of a wide variety of types of mutants.     

A screening procedure for the efficient recognition of Escherichia coli K-12 mutants

A method is described for the rapid screening of large numbers of E. coli colonies for detection of auxotrophs and mutants defective in sugar or succinate metabolism. The procedure permits recognition of forward mutations in a large number of functions and is applicable to the isolation of temperature-sensitive mutants in essential and nonessential genes.     

Temperature-sensitive revertants

About 50% of a series of non-temperature-sensitive, spontaneously revertable auxotrophs of Escherichia coli showed temperature-sensitive revertants when the selection was at 25° rather than at 37°. This procedure provides a simple device to select temperature-sensitive mutants.     

Isolation of Spontaneously Derived Mutants of CAULOBACTER CRESCENTUS

Caulobacter crescentus has a penicillinase which precludes the use of penicillin for mutant enrichment. However, two other antibiotics, fosfomycin and D-cycloserine, can be enrich for C. crescentus mutants. In enrichment procedures for C. crescentus auxotrophs, spontaneously derived mutants occur at a frequency of 5-10% among the survivors of an enrichment procedure. Consequently, large numbers of mutants are readily obtained without any need for mutagenesis. These mutants are heterogeneous both with regard to the type of mutation and to the nutritional requirement. A similar procedure has been used to isolate temperature-sensitive mutants.     

Comparative pathogenicity of auxotrophic mutants of Candida albicans

An induced mutant of Candida albicans with greatly decreased virulence for mice is described. The mutant was one of five auxotrophic mutants obtained by ultraviolet irradiation of a clinical isolate (strain MY 1044). The five mutants included two methionine auxotrophs, one methionine-cysteine auxotroph, one temperature-sensitive serine auxotroph, and one auxotroph with unknown growth requirements. Each of the mutants produced normal mycelium and had a normal profile of susceptibility to four antifungal drugs. The virulence of each mutant was compared with the parent strain by LD50 determination in mice. Four of the five auxotrophs exhibited LD50's that were not significantly different from the parent strain (mean LD50 = 7.5 x 10(5) cells). However, the temperature-sensitive serine auxotroph was significantly less virulent than the parent strain (LD50 greater than 10(7) cells), even though it grew well in vivo and in mouse serum at 37 degrees C in vitro. Use of this mutant in conjunction with its "isogenic" parent should help to elucidate true virulence factors in C. albicans.     

Temperature-sensitive glutamate dehydrogenase mutants of Salmonella typhimurium.

Mutants of Salmonella typhimurium defective in glutamate dehydrogenase activity were isolated in parent strains lacking glutamate synthase activity by localizcd mutagenesis or by a general mutagenesis combined with a cycloserine enrichment for glutamate auxotrophs. Two mutants with temperature-sensitive phenotypes had glutamate dehydrogenase activities that were more thermolabile than that of an isogenic control strain. Eight other mutants had less than 10% of the wild-type glutamate dehydrogenase activity. All the mutations were cotransducible with a Tn10 element (zed-2:Tn10) located at approximately 23 U on the S. typhimurium linkage map. These data strongly indicate that this region contains the structural gene (gdhA) for glutamate dehydrogenase.     

Action of temperature-sensitive mutants of myeloproliferative sarcoma virus suggests that fibroblast-transforming and hematopoietic transforming viral properties are related

The myeloproliferative sarcoma virus is molecularly related to the Moloney sarcoma virus (Pragnell et al., J. Virol. 38:952-957, 1981), but causes both fibroblast transformation in vitro and leukemic changes--including spleen focus formation--in adult mice. The fibroblast transforming properties of myeloproliferative sarcoma virus were used to select viral temperature-sensitive mutants at 39.5 degrees C, the nonpermissive temperature. These mutants are temperature sensitive in the maintenance of the transformed state. This was also shown by cytoskeletal changes of the infected cells at permissive and nonpermissive temperatures. Viruses released from cells maintained at both the permissive and nonpermissive temperature are temperature sensitive in fibroblast transformation functions. All temperature-sensitive mutants show only a low reversion rate to wild-type transforming function. The myeloproliferative sarcoma virus temperature-sensitive mutants are inefficient in causing leukemic transformation (spleen enlargement, focus formation) in mice at the normal temperature. A method to maintain a low body temperature (33 to 34 degrees C) in mice is described. One temperature-sensitive mutant was checked at low body temperature and did not induce leukemia. These data thus indicate that the same or related viral functions are responsible for hematopoietic and fibroblast transformation.     

Thymidine 5′-Monophosphate-Requiring Mutants of Saccharomyces cerevisiae Are Deficient in Thymidylate Synthetase

Thymidylate synthetase activity was measured in crude extracts of the yeast Saccharomyces cerevisiae by a sensitive radiochemical assay. Spontaneous non-conditional mutants auxotrophic for thymidine 5'-monophosphate (tmp1) lacked detectable thymidylate synthetase activity in cell-free extracts. In contrast, the parent strains (tup1, -2, or -4), which were permeable to thymidine 5'-monophosphate, contained levels of activity similar to those found in wild-type cells. Specific activity of thymidylate synthetase in crude extracts of normal cells or of cells carrying tup mutations was essentially unaffected by the ploidy or mating type of the cells, by the medium used for growth, by the respiratory capacity of the cells, by concentrations of exogenous thymidine 5'-monophosphate as high as 50 mug/ml, or by subsequent removal of thymidine 5'-monophosphate from the medium. Extracts of a strain bearing the temperature-sensitive cell division cycle mutation cdc21 lacked detectable thymidylate synthetase activity under all conditions tested. Its parent and another mutant (cdc8), which arrests with the same terminal phenotype under restrictive conditions, had normal levels of the enzyme. Cells of a temperature-sensitive thymidine 5'-monophosphate auxotroph arrested with a morphology identical to the cdc21 strain at the nonpermissive temperature and contained demonstrably thermolabile thymidylate synthetase activity. Tetrad analysis and the properties of revertants showed that the thymidylate synthetase defects were a consequence of the same mutation causing, in the auxotrophs, a requirement for thymidine 5'-monophosphate and, in the conditional mutants, temperature sensitivity. Complementation tests indicated that tmp1 and cdc21 are the same locus. These results identify tmp1 as the structural gene for yeast thymidylate synthetase.     

Effect of the Osmotic Pressure of the Growth Medium on fabB Mutants of Escherichia coli

Temperature-sensitive, unsaturated fatty acid (fabB) auxotrophs of Escherichia coli can grow at the restrictive temperature in the absence of unsaturated fatty acid in a medium with a high osmotic pressure. If a mutant culture was starved for unsaturated fatty acids and harvested just before the lysis started, the fatty acid composition of the cells was the same as that of cells grown until late log phase in a high-osmotic medium. Evidence is presented that the in vivo unsaturated fatty acid biosynthesis is significantly increased in a high osmotic medium. The increase is probably due to a partial activation of the temperature-sensitive fabB product. Besides the stimulation of the temperature-sensitive fabB product, a minimal osmotic pressure of the medium appeared to be necessary to allow growth of cells containing lipids with a changed fatty acid composition. fabA mutants are unable to grow in a high-osmotic medium in the absence of unsaturated fatty acids. No increase in the in vivo unsaturated fatty acid biosynthesis could be detected in the temperature-sensitive fabA mutants.     

Complementation analysis of measles virus mutants isolated from persistently infected lymphoblastoid cell lines.

Human lymphoblastoid cell lines persistently infected with measles virus release a heterogeneous population of virions. At least 80% of the infectious particles were temperature sensitive for plaque formation at 39 degrees C. Plaque-purified temperature-sensitive mutants from four persistently infected human lymphoblastoid cell lines were shown to be heterogeneous with respect to efficiency of plating at 31 and 39 degrees C, as well as to antigen and RNA production at 39 degrees C. The heterogeneity was confirmed by complementation analysis in which 21 temperature-sensitive isolates were found to represent at least four of the five previously described complementation groups of measles virus. Two isolates complemented four reference temperature-sensitive mutants. These isolates either represent new complementation groups or are members of the fifth complementation group, group E. The majority of isolates were found to have multiple mutations, and group B mutants (RNA-) predominated. Two temperature-sensitive isolates were able to interfere with production of parental measles virus at both permissive and nonpermissive temperatures.     

Distribution of Tn551 insertion sites responsible for auxotrophy on the Staphylococcus aureus chromosome

A method was devised to efficiently select isolates of Staphylococcus aureus 8325 in which Tn551, a transposon originating on the pI258 plasmid responsible for erythromycin resistance (Emr), had translocated to the host chromosome. This method consisted of selecting for Emr at 43 degrees C with a strain in which the pI258 plasmid was unable to replicate at 43 degrees C because of a temperature-sensitive plasmid mutation. By selecting isolates that were Emr at 43 degrees C and auxotrophic for nutrients not required by the parent strain. Tn551-induced auxotrophic mutants were readily isolated. The incidence of auxotrophic classes was not random; 80% of the isolates in one experiment were Trp-, whereas only a single example of each of some of the other classes was isolated. Among the Trp- mutants, the distribution of trp genes affected and the frequency of precise excision of Tn551 from individual sites varied. When analyzed by transformation, the Tn551-induced ala, his, ilv, lys, rib, thrA, thrB, and trp mutations were shown to occupy sites previously defined by nitrosoguanidine-induced mutations. Tn551-induced mutagenesis provided three previously unrecognized classes of auxotrophs (tyr, met, and thrC), and the Tn551 integration sites resulting in these mutations have been identified. In addition, a chromosomal region (uraB) was identified by Tn551 mutagenesis that is distinct from uraA (previously defined by chemical mutagenesis). Some Tn551-induced mutations (most notably pur) could not be linked to the known linkage groups of the chromosome by transformation. With the exception of two pur mutations, all of the Tn551-induced auxotrophic mutational sites cotransformed at unity with Tn551 and, in cases in which they were selected, prototrophic transformants were always Ems. Thus, the Tn551 and auxotrophic sites are identical.     

Selection by [3H] amino acids of CHO-cell mutants with altered leucyl- and asparagyl-transfer RNA synthetases.

Efficient selection procedures, using [3H]amino acids as the selecting agent, were developed for isolating temperature-sensitive (TS) mutations in CHO cells affecting protein synthesis. After chemical mutagenesis, leucyl-tRNA synthetase mutants were obtained when [3H]leucine was used as the selecting agent in two independent experiments. These mutations seem to involve the same genetic locus as the TSH1 mutant described previously (1). A selection with [3H]valine, in which all amino acids except leucine were at low concentration in the selective medium, resulted in a new class of mutants with reduced asparagyl-tRNA synthetase activity. These results were consistent with the finding that all mutants were phenotypically dependent on the concentration of amino acid, specific to the altered synthetase, in the medium. Our observations suggest that although leucyl synthetase mutations are a relatively common class of TS mutations in CHO cells, the spectrum of mutants obtained can be at least partially manipulated through concentrations of amino acids in selective media. The asparagyl-synthetase mutation was shown to be recessive and to complement the leucyl-synthetase mutation in cell-cell hybrids.     

Immunochemical characterization of glutamine synthetase from Neurospora crassa glutamine auxotrophs.

Glutamine synthetase derived from two Neurospora crassa glutamine auxotrophs was characterized. Previous genetic studies indicated that the mutations responsible for the glutamine auxotrophy are allelic and map in chromosome V. When measured in crude extracts, both mutant strains had lower glutamine synthetase specific activity than that found in the wild-type strain. The enzyme from both auxotrophs and the wild-type strain was partially purified from cultures grown on glutamine as the sole nitrogen source, and immunochemical studies were performed in crude extracts and purified fractions. Quantitative rocket immunoelectrophoresis indicated that the activity per enzyme molecule is lower in the mutants than in the wild-type strain; immunoelectrophoresis and immunochemical titration of enzyme activity demonstrated structural differences between the enzymes from both auxotrophs. On the other hand, the monomer of glutamine synthetase of both mutants was found to be of a molecular weight similar to that of the wild-type strain. These data indicate that the mutations are located in the structural gene of N. crassa glutamine synthetase.     

The effect of transformation-defective avian oncornavirus mutants on tumor antigen expression

The recent isolation of conditional (temperature sensitive) and nonconditional transformation-defective mutants of avian sarcoma virus strains has facilitated the investigation of the effect of virus transformation on the cell's phenotype, e.g., with respect to morphology, growth pattern, or cell surface antigenicity. Special emphasis was laid on elucidating the correlation between transformed phenotype and tumor antigen expression. All of the tested nontransforming deletion mutants and the majority of the temperature-sensitive mutants were unable to induce tumor antigens in phenotypically untransformed cells. However, 3 temperature-sensitive mutants were found which were able to support the expression of tumor specific surface antigens even at restrictive temperature, when cells otherwise exhibited a normal phenotype. The theoretical and practical implications of this association between normal phenotype and tumor antigen expression are discussed.     

Temperature-sensitive mutants of Saccharomyces cerevisiae variable in the methionine content of their protein.

Temperature-sensitive mutants were derived from Saccharomyces cerevisiae Y5alpha by ethyl methane sulfonate mutagenesis, in a search for mutants that would produce methionine-rich protein at the nonpermissive temperature. A total of 132 mutant strains were selected which showed adequate growth on minimal medium at 25 degrees C but little or no growth on the same medium supplemented with a high concentration (2 mg/ml) of l-methionine at 37 degrees C. Several of these mutants were found to increase the proportion of methionine in their protein to much higher levels than that of the wild-type parent after a temperature shift from 25 to 37 degrees C. Two strains, 476 and 438, which were temperature sensitive only in the presence of methionine, produced cellular protein with methionine contents as high as 3.6 and 4.3%, respectively, when incubated in the presence of methionine. The former strain contained 2.5% methionine even when incubated at 37 degrees C in the absence of methionine. Wild strain Y5alpha, on the other hand, had 1.75% methionine under all conditions tested. Most temperature-sensitive mutants isolated had the same methionine content as the wild strain. It is concluded that the proportion of a specific amino acid, such as methionine, in S. cerevisiae protein can be altered by culturing certain temperature-sensitive mutants at an elevated temperature.     

BHK cells expressing Sindbis virus-induced homologous interference allow the translation of nonstructural genes of superinfecting virus.

The process by which Sindbis virus excludes superinfecting homologous virus was investigated with the use of temperature-sensitive mutants. Mutants in two RNA-negative complementation groups were found to be defective in their ability to establish interference at the nonpermissive temperature. These mutants were unable to establish interference in a mixed infection (complementation), suggesting that both were defective in a common gene product. Homologous interference was found to block the replication of superinfecting virus after attachment, penetration, and translation of the nonstructural genes encoded in the virus RNA. The production of nonstructural gene products of superinfecting wild-type virus was found to enhance the replication of certain RNA- temperature-sensitive interfering viruses at the permissive and the nonpermissive temperature. The ability of certain RNA- mutants to establish homologous interference and to demonstrate enhanced growth after superinfection with wild-type virus was interpreted to produce a model implicating both virus and host components in the establishment of homologous interference and in the replication of Sindbis virus RNA.     

Analysis of Constructed E Gene Mutants of Mouse Hepatitis Virus Confirms a Pivotal Role for E Protein in Coronavirus Assembly

Expression studies have shown that the coronavirus small envelope protein E and the much more abundant membrane glycoprotein M are both necessary and sufficient for the assembly of virus-like particles in cells. As a step toward understanding the function of the mouse hepatitis virus (MHV) E protein, we carried out clustered charged-to-alanine mutagenesis on the E gene and incorporated the resulting mutations into the MHV genome by targeted recombination. Of the four possible clustered charged-to-alanine E gene mutants, one was apparently lethal and one had a wild-type phenotype. The two other mutants were partially temperature sensitive, forming small plaques at the nonpermissive temperature. Revertant analyses of these two mutants demonstrated that the created mutations were responsible for the temperature-sensitive phenotype of each and provided support for possible interactions among E protein monomers. Both temperature-sensitive mutants were also found to be markedly thermolabile when grown at the permissive temperature, suggesting that there was a flaw in their assembly. Most significantly, when virions of one of the mutants were examined by electron microscopy, they were found to have strikingly aberrant morphology in comparison to the wild type: most mutant virions had pinched and elongated shapes that were rarely seen among wild-type virions. These results demonstrate an important, probably essential, role for the E protein in coronavirus morphogenesis.     

Temperature-Sensitive Osmotic Remedial Mutants of Escherichia coli

A collection of temperature-sensitive mutants of Escherichia coli K-12 was examined for ability to grow at the restrictive temperature when the osmotic pressure of the medium was increased. Five of the fourteen mutants were found to be osmotic remedial. Four strains containing temperature-sensitive, osmotic-remedial mutations affecting aminoacyl-transfer ribonucleic acid synthetases were found to have altered permeability characteristics which may be attributable to changes in the lipopolysaccharide layer of the cell envelope at restrictive temperatures.     

Symbiotic characterization of isoleucine+valine and leucine auxotrophs of Sinorhizobium meliloti

Ten isoleucine+valine and three leucine auxotrophs of Sinorhizobium meliloti Rmd201 were obtained by random mutagenesis with transposon Tn5 followed by screening of Tn5 derivatives on minimal medium supplemented with modified Holliday pools. Based on intermediate feeding, intermediate accumulation and cross-feeding studies, isoleucine+valine and leucine auxotrophs were designated as ilvB/ilvG, ilvC and ilvD, and leuC/leuD and leuB mutants, respectively. Symbiotic properties of all ilvD mutants with alfalfa plants were similar to those of the parental strain. The ilvB/ilvG and ilvC mutants were Nod-. Inoculation of alfalfa plants with ilvB/ilvG mutant did not result in root hair curling and infection thread formation. The ilvC mutants were capable of curling root hairs but did not induce infection thread formation. All leucine auxotrophs were Nod+ Fix-. Supplementation of leucine to the plant nutrient medium did not restore symbiotic effectiveness to the auxotrophs. Histological studies revealed that the nodules induced by the leucine auxotrophs did not develop fully like those induced by the parental strain. The nodules induced by leuB mutants were structurally more advanced than the leuC/leuD mutant induced nodules. These results indicate that ilvB/ilvG, ilvC and one or two leu genes of S. meliloti may have a role in symbiosis. The position of ilv genes on the chromosomal map of S. meliloti was found to be near ade-15 marker.