Stimulation of growth rate of chondrocytes by Rous sarcoma virus is not coordinated with other expressions of the src gene phenotype

Infection and transformation of chondrocytes by Rous sarcoma viruses (RSVs) (Schmidt-Ruppin, Prague) stimulated the rate of cell growth. In contrast, several transformation-defective (td) mutants (tdPRA, tdNY105, tdNY106, tdNY107, and tdNY108) retaining various sizes of the src gene did not stimulate cell growth, indicating that the stimulation of growth of chondrocytes is due to the function of the src gene. With the use of various T (transformation)-class temperature-sensitive (ts) mutants of RSV, growth stimulation of chondrocytes by the src gene was examined. It was found that there are two types of T-class ts mutants with regard to the stimulatory effect on the growth of chondrocytes. One type (tsNY68) stimulates cell growth at both permissive (36 degrees C) and nonpermissive (41.5 degrees C) temperature, as does the wild type of RSV. Another type (ts GI201 [clone 9]) stimulates cell growth only at the permissive temperature. Chondrocytes infected with either of these two types of T-class ts mutants showed ts properties in other transformation markers, such as uptake of 2-deoxy-D-glucose, change of cell morphology, and focus formation. These data indicate that the effect of the src gene on cell growth does not occur coordinately with other transformation markers.     

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.     

Application of Arrhenius kinetic theory to viral eclipse: selection of bacteriophage phi X174 mutants.

Analysis of the bacteriophage phi X174 eclipse period in terms of Arrhenius kinetic theory suggests the following hypothesis: mutants should exist with two concomitant physiological characteristics as their phenotype. These are an eclipse rate lower than that of the wild type at permissive temperatures for plaque formation and an eclipse rate too low at lower temperatures to permit plaque development. Thus, enrichment of a mutagenized virus population for mutants that fail to eclipse during a short period at permissive temperatures should yield eclipse mutants with the cold-sensitive (cs; nonpermissive temperature, 25 degrees C), and not the temperature-sensitive (ts; nonpermissive temperature, 42 degrees C), plaque phenotype. In several trials, the frequency of the cs phenotype in the population increased from less than 0.2% to between 2 and 4% after the enrichment step, whereas the frequency of the ts phenotype remained unchanged (less than 0.2%). Moreover, 80% of these cs mutants have eclipse rates that are 3- to 40-fold lower than that of the wild type at both 37 degrees C and 25 degrees C. The successful application of the Arrhenius theory to phi X eclipse may provide insights into the molecular mechanism whereby the phi X174 genome is delivered into the host cell. Since the eclipse kinetics of other nonenveloped viruses are similar to those of phi X174, kinetic theory may be broadly applicable in the selection and characterization of viral eclipse mutants.     

A Highly Thermosensitive and Permeable Mutant of the Marine Yeast Cryptococcus aureus G7a Potentially Useful for Single-Cell Protein Production and its Nutritive Components

The highly thermosensitive and permeable mutants are the mutants from which intracellular contents including proteins can be released when they are incubated both in the low osmolarity water and at the nonpermissive temperature (usually 37 degrees C). After mutagenesis by using nitrosoguanidine, a highly thermosensitive and permeable mutant named Z114 was obtained from the marine yeast Cryptococcus aureus G7a. Of the total protein, 65.3% was released from the mutant cells suspended in distilled water after they were treated at 37 degrees C overnight. However, only 12.3% of the total protein was released from the mutant cells suspended in 1.0 M sorbitol solution after they were treated at 37 degrees C overnight. We found that intracellular density of the mutant treated at 37 degrees C was greatly decreased, and cell volume of the mutant treated at 37 degrees C was increased due to the increased protein release. However, no significant changes in the intracellular density and cell volume of the mutant were observed when its cells suspended in 1.0 M sorbitol solution were treated at 37 degrees C. It was found that no big changes in cell growth, protein content, vitamin C content, nucleic acid content, fatty acids, and amino acid compositions of both the mutant and its wild type were detected. Therefore, the highly thermosensitive and permeable mutant still can be a good candidate as single-cell protein. This means that the method used in this study is a simple and efficient way to release protein from the highly thermosensitive and permeable yeast mutant cells with high protein content.     

Sequential cold-sensitive mutations in Aspergillus fumigatus.

Mutants of thermotolerant fungus Aspergillus fumigatus I-21 (ATCC 32722) unable to grow at 37 degrees C were sought. Cold-sensitive mutants were enriched from progeny spores of gamma-irradiated conidia by two or more incubations at various nonpermissive temperatures alternating with filtrations through chessecloth. The approximate minimum, optimum, and maximum growth temperatures of the parent were 12, 40, and 50 degrees C, respectively. Mutants unable to grow at 37 degrees C were not successfully isolated directly from the wild type. A mutant unable to grow at 25 degrees C was isolated and mutations further increasing the cold sensitivity by increments of 3-5 degrees C were found to occur. Mutants completely unable to grow at 37 degrees C were obtained by five sequential mutations. All mutants grew as fast as the wild-type parent at 45 degrees C and higher. Each mutant produced revertants able to grow not only at the nonpermissive temperature used for its isolation but also at lower temperatures.     

Characterization of temperature-sensitive mutants of simian rotavirus SA11: protein synthesis and morphogenesis.

The synthesis of viral polypeptides, distribution of viral antigens, and morphogenesis of viral structures have been examined in cells infected with temperature-sensitive (ts) mutants of SA11 representing 10 recombination groups. At the permissive temperature (31 degrees C) the synthesis of viral polypeptides and the distribution of viral antigens did not differ significantly from those of the wild type. At the nonpermissive temperature (39 degrees C) some mutants (tsB, -C, -E, -F, and -G) synthesized significantly smaller amounts of viral polypeptides and had a very diffuse distribution of viral antigen. Several of the mutants synthesized one or more electrophoretically aberrant polypeptide species at both 31 and 39 degrees C. All of the mutants, except tsF, assembled morphogenetic intermediates at 39 degrees C. Aberrant intermediates were assembled in all mutants at 31 and 39 degrees C. No specific morphogenic defect could be associated with any of the ts mutants.     

Probing the Sinorhizobium meliloti-alfalfa symbiosis using temperature-sensitive and impaired-function citrate synthase mutants

To study the role of the decarboxylating leg of the bacterial TCA cycle in symbiotic nitrogen fixation, we used DNA shuffling and localized random polymerase chain reaction mutagenesis to construct a series of temperature-sensitive and impaired-function mutants in the Sinorhizobium meliloti Rm104A14 citrate synthase (gltA) gene. Reducing citrate synthase (CS) activity by mutation led to a corresponding decrease in the free-living growth rate; however, alfalfa plants formed fully effective nodules when infected with mutants having CS activities as low as 7% of the wild-type strain. Mutants with approximately 3% of normal CS activity formed nodules with lower nitrogenase activity and a mutant with less than 0.5% of normal CS activity formed Fix- nodules. Two temperature-sensitive (ts) mutants grew at a permissive temperature (25 degrees C) with 3% of wild-type CS activities but were unable to grow on minimal medium at 30 degrees C. Alfalfa plants that were inoculated with the ts mutants and grown with a root temperature of 20 degrees C formed functional nodules with nitrogenase activities approximately 20% of the wild type. When the roots of plants infected with the ts mutants were transferred to 30 degrees C, the nodules lost the ability to fix nitrogen over several days. Microscopic examination of these nodules revealed the loss of bacteroids and senescence, indicating that CS activity was essential for nodule maintenance.     

Thermophilic mutants of Pseudomonas fluorescens

Summary A series of heat tolerant mutants of Pseudomonas fluorescens were obtained which can grow at temperatures up to 54°C, in contrast to a maximum growth temperature of 37°C for the wild type. The minimum temperatures allowing growth of the mutant strains increased to the same extent as their maximum temperatures. Antibiotic sensitivity patterns suggested the mutants had altered ribosomes, but the purified mutant ribosomes showed no significant increase in thermostability. The virulence of the wild and mutant strains for mice correlated with their relative abilities to grow at the mouse body temperature of approximately 37°C.     

Respiratory syncytial virus ts mutants and nuclear immunofluorescence.

A replicated sector-plating procedure was used to isolate 35 induced temperature-sensitive (ts) mutants and one spontaneous ts mutant from a wild-type stock of respiratory syncytial (RS) virus cloned from recent clinical material. Seven of these mutants were ts for plaque formation at 37 degrees C as well as at the restrictive temperature of 39 degrees C. The wild-type strain did not differ markedly from standard laboratory strains of RS virus. It was dependent on exogenous arginine (84 mug/ml) for optimal growth, and was not significantly inhibited by mitomycin C (10 mug/ml). It was sensitive to actinomycin D (2.5 mug/ml) during the early part of the growth phase. A characteristic focal cytopathic effect was obtained in BS-C-1 cells. Staining of infected monolayers by an indirect immunofluorescence procedure revealed a profusion of filamentous processes extending from the plasma membrane, and a similar modification of the surface of infected cells could be visualized by scanning electron microscopy. Filament production was inhibited when certain ts mutants were incubated at 39 degrees C, confirming the virus-specific nature of the phenomenon. Thirty-four of the mutants were classified into three groups by immunofluorescence. Complementation was observed in mixed infection with a single mutant from each group. Nuclear, as well as cytoplasmic, immunofluorescence was detected in RS virus-infected cells using a high-titer bovine anti-bovine RS virus serum. Visualization of nuclear antigen was dependent on the inhibition of cytoplasmic fluorescence obtained when ts mutants in groups I and III were incubated at restrictive temperature.     

Distinguishable transformation-defective phenotypes among temperature-sensitive mutants of Rous sarcoma virus.

Eight transformation-defective, temperature-sensitive (ts) mutants of the Prague strain of Rous sarcoma virus, subgroup A, have been isolated after mutagenesis with 5-bromodeoxyuridine followed by selection on the basis of focus tests. Five of these mutants, ts GI201, GI202, GI203, GI204, and GI205, exhibit properties like most previously reported isolates in that they show a temperature-sensitive response to each of a variety of transformation-specific parameters tested. Interestingly, GI201, in addition to the temperature-sensitive defect, carries a lesion that was observed as a nonconditional loss of expression of plasminogen activator protease. Three mutants, ts GI251, GI252, and GI253 have been disignated partial transformation-defective (PTD) mutants since they behave as ts mutants according to some tests for transformation and as wild type according to others. These three mutants fail to form foci at the nonpermissive temperature (41 degrees C) and art nontumorigenic in 3-week-old chickens (body temperature, 42 degrees C). The agglutinability by concanavalin A of cells infected with these mutants shows a definite temperature sensitivity, as do the rate of 2-deoxyglucose uptake and the disappearance of the 250, 000-dalton normal cell glycoprotein (large, external, transformation sensitive [LETS]). Although the PTD mutant-infected cells, unlike cells infected with other transformation mutants, exhibit a cell-bound plasminogen activator protease at the nonpermissive temperature, this activator is not detectable as a free protease in the medium, as it is with wild-type, virus-infected cells. The PTD mutants behave like the wild-type parent in their ability to induce transformed growth properties in the infected cells, i.e., growth beyond normal cell saturation density with or without serum-supplemented medium and growth leading to colony formation in soft-agar- or methyl cellulose-containing suspension media.     

Isolation and characterization of a heat-inducible simian virus 40 mutant.

We have isolated a new type of temperature-sensitive mutant of simian virus 40 (SV40) that is capable of productive infection in permissive cells but not of maintenance of viral DNA integration in transformed cells at the conditional temperature. Virus development is induced when cells transformed by this mutant are shifted to temperatures above 39 degrees C, but is not induced below this temperature. The plaque-purified, temperature-sensitive mutant virus confers heat inducibility to new host cells, indicating that the conditional function is a property of the viral genome. Unlike previously described temperature-sensitive SV40 mutants, in (ts)-1501 is capable of productive infection in permissive cells at the conditional temperature. The morphology, growth, and oncogenicity of in (ts)-1501-transformed cells at 37 degrees C are similar to those of cell lines transformed by wild-type SV40. HK10-c2(in(ts)-1501), a cloned cell line, transformed at 37 degrees C by the mutant virus, exhibits a transient increase in DNA synthesis before cell death at the conditional temperature. Many properties of in(ts)-1501 are analogous to those of the heat-inducible mutants of bacteriophages in which a heat-inactivated protein is responsible for the stable integration of the prophage in the bacterial chromosome.     

Thermostability of temperature-sensitive folding mutants of the P22 tailspike protein

Temperature-sensitive folding mutations (tsf) of the thermostable P22 tailspike protein prevent the mutant polypeptide chain from reaching the native state at the higher end of the temperature range of bacterial growth (37-42 degrees C). At lower temperatures the mutant polypeptide chains fold and associate into native proteins. The melting temperatures of the purified native forms of seven different tsf mutant proteins have been determined by differential scanning calorimetry. Under conditions in which the wild type protein had a melting temperature of 88.4 degrees C, the melting temperatures of the mutant proteins were all above 82 degrees C, more than 40 degrees C higher than the temperature for expression of the folding defect. Because the folding defects were observed in vivo, the thermostability of the native protein was also examined with infected cells. Once matured at 28 degrees C, intracellular tsf mutant tailspikes remained native when the cells were transferred to 42 degrees C, a temperature that prevents newly synthesized tsf chains from folding correctly. These results confirm that the failure of tsf polypeptide chains to reach their native state is not due to a lowered stability of the native state. Such mutants differ from the class of ts mutations which render the native state thermolabile. The intracellular folding defects must reflect decreased stabilities of folding intermediates or alteration in the off-pathway steps leading to aggregation and inclusion body formation. These results indicate that the stability of a native protein within the cells is not sufficient to insure the successful folding of the newly synthesized chains into the native state.     

Two Delta9-stearic acid desaturases are required for Aspergillus nidulans growth and development

Unsaturated fatty acids are important constituents of all cell membranes and are required for normal growth. In the filamentous fungus Aspergillus nidulans, unsaturated fatty acids and their derivatives also influence asexual (conidial) and sexual (ascospore) sporulation processes. To investigate the relationship between fatty acid metabolism and fungal development, we disrupted the A. nidulans sdeA and sdeB genes, both encoding Delta9-stearic acid desaturases responsible for the conversion of palmitic acid (16:0) and stearic acid (18:0) to palmitoleic acid (16:1) and oleic acid (18:1). The effects of sdeA deletion on development were profound, such that growth, conidial and ascospore production were all reduced at 22 and 37 degrees C. Total fatty acid content was increased over 3-fold in the DeltasdeA strain, reflected in up-regulation of the expression of the fasA gene encoding the alpha chain of the fatty acid synthase, compared to wild type. Stearic acid accumulated approximately 3-fold compared to wild type in the DeltasdeA strain, while unsaturated fatty acid production was decreased. In contrast, disruption of sdeB reduced fungal growth and conidiation at 22 degrees C, but did not affect these processes at 37 degrees C compared to wild type. Interestingly, ascospore production was increased at 37 degrees C for DeltasdeB compared to wild type. Total fatty acid content was not increased in this strain, although stearic acid accumulated 2-fold compared to wild type, and unsaturated fatty acid production was decreased. Combining the DeltasdeA and DeltasdeB alleles created a synthetic lethal strain requiring the addition of oleic acid to the medium for a modicum of growth. Taken together, our results suggest a role for sdeA in growth and development at all temperatures, while sdeB is involved in growth and development at lower temperatures.     

Characterization of a temperature-sensitive, hexon transport mutant of type 5 adenovirus.

Infection of KB cells at 39.5 degrees C with H5ts147, a temperature-sensitive (ts) mutant of type 5 adenovirus, resulted in the cytoplasmic accumulation of hexon antigen; all other virion proteins measured, however, were normally transported into the nucleus. Immunofluorescence techniques were used to study the intracellular location of viral proteins. Genetic studies revealed that H5ts147 was the single member of a nonoverlapping complementation group and occupied a unique locus on the adenovirus genetic map, distinct from mutants that failed to produce immunologically reactive hexons at 39.5 degrees C ("hexon-minus" mutants). Sedimentation studies of extracts of H5ts147-infected cells cultured and labeled at 39.5 degrees C revealed the production of 12S hexon capsomers (the native, trimeric structures), which were immunoprecipitable to the same extent as hexons synthesized in wild type (WT)-infected cells. In contrast, only 3.4S polypeptide chains were found in extracts of cells infected with the class of mutants unable to produce immunologically reactive hexon protein at 39.5 degrees C. Hexons synthesized in H5ts147-infected cells at 39.5 degrees C were capable of being assembled into virions, to the same extent as hexons synthesized in WT-infected cells, when the temperature was shifted down to the permissive temperature, 32 degrees C. Infectious virus production was initiated within 2 to 6 h after shift-down to 32 degrees C; de novo protein synthesis was required to allow this increase in viral titer. If ts147-infected cells were shifted up to 39.5 degrees C late in the viral multiplication cycle, viral production was arrested within 1 to 2 h. The kinetics of shutoff was similar to that of a WT-infected culture treated with cycloheximide at the time of shift-up. The P-VI nonvirion polypeptide, the precursor to virion protein VI, was unstable at 39.5 degrees C, whereas the hexon polypeptide was not degraded during the chase. It appears that there is a structural requirement for the transport of hexons into the nucleus more stringent than the acquisition of immunological reactivity and folding into the 12S form.     

Peroxisomes in the methylotrophic yeast Hansenula polymorpha do not necessarily derive from pre-existing organelles.

We have identified two temperature-sensitive peroxisome-deficient mutants of Hansenula polymorpha (ts6 and ts44) within a collection of ts mutants which are impaired for growth on methanol at 43 degrees C but grow well at 35 degrees C. In both strains peroxisomes were completely absent in cells grown at 43 degrees C; the major peroxisomal matrix enzymes alcohol oxidase, dihydroxyacetone synthase and catalase were synthesized normally but assembled into the active enzyme protein in the cytosol. As in wild-type cells, these enzymes were present in peroxisomes under permissive growth conditions (< or = 37 degrees C). However, at intermediate temperatures (38-42 degrees C) they were partly peroxisome-bound and partly resided in the cytosol. Genetic analysis revealed that both mutant phenotypes were due to monogenic recessive mutations mapped in the same gene, designated PER13. After a shift of per13-6ts cells from restrictive to permissive temperature, new peroxisomes were formed within 1 h. Initially one--or infrequently a few--small organelles developed which subsequently increased in size and multiplied by fission during prolonged permissive growth. Neither mature peroxisomal matrix nor membrane proteins, which were present in the cytosol prior to the temperature shift, were incorporated into the newly formed organelles. Instead, these proteins remained unaffected (and active) in the cytosol concomitant with further peroxisome development. Thus in H.polymorpha alternative mechanisms of peroxisome biogenesis may be possible in addition to multiplication by fission upon induction of the organelles by certain growth substrates.