Three Genes Which Affect Founding of Aggregations in Polysphondylium Pallidum

PN6024 is an extraordinary mutant strain of the cellular slime mold Polysphondylium pallidum, characterized by having defects in many unlinked genes. New strains with altered development appeared spontaneously as aberrant clones of PN6024. Genetic crosses using the macrocyst sexual cycle were used to show that PN6030 (a clone like PN6024 in phenotype) carries mutations at two loci, emm and hge, whereas PN6031 (a clone of altered morphology) carries in addition a mutation at a third locus, mgt. hge and possibly mgt are linked to the mating type locus mat. The relatively high frequency of recombination between mat and hge is strong evidence that meiosis precedes macrocyst germination. The mutant genes themselves are of interest. A major effect of the emm-1 mutation is to remove the requirement for light to trigger aggregation. hge-1 greatly reduces the frequency of aggregation, whereas mgt-1 greatly increases it under standard conditions. None of these mutations interrupts later development leading to stalks and spore cells. It is hypothesized that all three genes act on steps immediately preceding the differentiation of the founder cells which initiate aggregation.     

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.     

Genetic characterization of Escherichia coli type 1 pilus adhesin mutants and identification of a novel binding phenotype

Five Escherichia coli type 1 pilus mutants that had point mutations in fimH, the gene encoding the type 1 pilus adhesin FimH, were characterized. FimH is a minor component of type 1 pili that is required for the pili to bind and agglutinate guinea pig erythrocytes in a mannose-inhibitable manner. Point mutations were located by DNA sequencing and deletion mapping. All mutations mapped within the signal sequence or in the first 28% of the predicted mature protein. All mutations were missense mutations except for one, a frameshift lesion that was predicted to cause the loss of approximately 60% of the mature FimH protein. Bacterial agglutination tests with polyclonal antiserum raised to a LacZ-FimH fusion protein failed to confirm that parental amounts of FimH cross-reacting material were expressed in four of the five mutants. The remaining mutant, a temperature-sensitive (ts) fimH mutant that agglutinated guinea pig erythrocytes after growth at 31 degrees C but not at 42 degrees C, reacted with antiserum at both temperatures in a manner similar to the parent. Consequently, this mutant was chosen for further study. Temperature shift experiments revealed that new FimH biosynthesis was required for the phenotypic change. Guinea pig erythrocyte and mouse macrophage binding experiments using the ts mutant grown at the restrictive and permissive temperatures revealed that whereas erythrocyte binding was reduced to a level comparable to that of a fimH insertion mutant at the restrictive temperature, mouse peritoneal macrophages were bound with parental efficiency at both the permissive and restrictive temperatures. Also, macrophage binding by the ts mutant was insensitive to mannose inhibition after growth at 42 degrees C but sensitive after growth at 31 degrees C. The ts mutant thus binds macrophages with one receptor specificity at 31 degrees C and another at 42 degrees C.     

Fluphenazine-resistant Saccharomyces cerevisiae mutants defective in the cell division cycle.

An fls1 mutant of Saccharomyces cerevisiae, which did not grow in the presence of 30 micrograms of fluphenazine per ml, was isolated. Mutants that were resistant to 90 micrograms of fluphenazine per ml and temperature sensitive for growth were obtained from the fls1 mutant. One fluphenazine-resistance mutation, fsr1, was located near the his7 locus on chromosome II. Growth of the fsr1 mutants at 35 degrees C was arrested after nuclear division. The other group of fluphenazine-resistant mutants, carrying fsr2 mutations, showed Ca2+-dependent growth at 35 degrees C. Growth of the fsr2 mutants at 35 degrees C was arrested at the G2 stage of the cell cycle in Ca2+-poor medium.     

Mutant chinese hamster cells with a thermosensitive hypoxanthine-guanine phosphoribosyltransferase.

By selecting variants of Chinese hamster cells that were resistant to 6-thioguanine at 39 degrees C, but which would continue to grow in HAT medium at 33 degrees C, we have isolated cell lines with thermosensitive phenotypes. These clones form colonies in HAT medium and incorporate 14-C-hypoxanthine much more efficiently at 33 degrees C than at 39 degrees C. The specific activity of hypoxanthine-guanine phosphoribo-syltransferase is at least 10 times higher in variant cells grown at 33 degrees C than in those grown in 39 degrees C, and the enzymes from the variant clones are inactivated in vitro at 39 degrees C 7-9 times more rapidly than is the enzyme from wild-type cells. The results are consistent with the conclusion that the selected clones have missense mutations in the structural gene for the enzyme.     

A temperature-sensitive mammalian cell mutant with thermolabile serine palmitoyltransferase for the sphingolipid biosynthesis

We devised an in situ assay method for the activity of serine palmitoyltransferase (SPT) that catalyzes the first step in sphingolipid biosynthesis and isolated a temperature-sensitive mutant of Chinese hamster ovary cells with thermolabile SPT. This mutant stopped growing at 40 degrees C after several generations, although the cells grew at 33 and 37 degrees C at rates similar to those of the parent. The SPT activity in cell homogenates of the mutant grown at low temperatures was 4-8% of that in the parent homogenates. When the cells were cultured for several generations at 40 degrees C, the activity in the mutant homogenate became negligible. When cell homogenates were incubated at 45 degrees C before enzyme assay, mutant SPT was more markedly inactivated than parental SPT, indicating that mutant SPT had become thermolabile. The rates of de novo synthesis of sphingolipids in the mutant were much slower at 40 degrees C than at lower temperatures, in contrast to those in the parent. The sphingomyelin content in the mutant cultivated at 40 degrees C for several generations was also less than that at low temperatures. These results indicate that SPT functions in the main pathway for sphingolipid biosynthesis. The temperature-sensitive growth of the mutant defective in sphingolipid synthesis suggests that sphingolipid(s) plays an essential role in cell growth.     

Tubulin composition and microtubule nucleation of a griseofulvin-resistant Chinese hamster ovary cell mutant with abnormal spindles

A griseofulvin-resistant Chinese hamster ovary (CHO) mutant (Grs-2) which has an altered beta-tubulin subunit as well as wild-type beta-tubulin is temperature-sensitive (ts) for growth at 40.5 degrees C. This growth defect appears to result from the formation of abnormal mitotic spindles at the non-permissive temperature (Abraham, I et al., J cell biol 97 (1983) 1055) [19]. Light microscopy of spindles isolated from mutant cells cultured at the permissive temperature showed a typical bipolar morphology, whereas spindles isolated at the non-permissive temperature were multipolar. In order to study the role of tubulin in spindle formation, we analyzed the tubulin composition of the multipolar spindles. Two-dimensional gels and immunoblotting analysis of one-dimensional electrophoretic gels stained with monoclonal anti-Chinese hamster brain beta-tubulin antibody revealed that both mutant and wild-type beta-tubulins were present in similar proportions in both bipolar spindles at 37 degrees C and multipolar spindles at 40.5 degrees C. The ratio between wild-type and mutant tubulin in spindles was also found to be the same as in the cytoplasmic microtubule network in interphase cells, providing evidence that the mutant beta-tubulin appeared to be incorporated in a similar manner into both interphase and mitotic microtubule structures. In vitro microtubule polymerization onto centrosomes prepared from mutant Grs-2 demonstrated that 80% of the sites for microtubule nucleation were without centrioles, suggesting fragmentation of pericentriolar material away from centrioles. This may be one of the causes of multipolar spindle formation in the mutant cells. These results, therefore, suggest that abnormal formation of spindles in mutant cells is due not to the presence of the mutant tubulin per se, but to the abnormal behavior of this mutant tubulin in the cellular environment during mitosis or abnormal interaction with other components in the spindle at 40.5 degrees C.     

Temporal sequence of the recovery of traits during phenotypic curing of a Cytophaga johnsonae motility mutant.

The lack of cell translocation and the resulting formation of nonspreading colonies of mutants of the gram-negative gliding bacterium Cytophaga johnsonae have been correlated with the loss of cell surface features of the organism. These cell surface traits include the ability to move polystyrene-latex beads over the cell surface and the ability to be infected by bacteriophages that infect the parent strain. In order to assess whether these traits reflect structures or functions that actually play a role in gliding, we studied a mutant (21A2I) selected for its inability to form spreading colonies; it is deficient in sulfonolipid, lacks bead movement ability, and is resistant to at least one bacteriophage. The provision of cysteate (a specific sulfonolipid precursor) restores lipid content and gliding to the mutant; hence, the lipids are necessary for motility. Growth with cysteate also restores bead movement and phage sensitivity. In order to determine the temporal relationship of these traits, we undertook a kinetic study of the appearance of them after addition of cysteate to the mutant. One predicts that appearance of a trait essential for cell translocation will either precede or accompany the appearance of this ability, while a nonessential trait need not do so. Sulfonolipid synthesis was the only trait that appeared before gliding; this is consistent with its established importance for motility. Bead movement and phage sensitivity first appeared only after gliding started, suggesting that the machinery involved in those processes is not necessary, at least for the initiation of gliding.     

Cysteine mutants of herpes simplex virus type 1 glycoprotein D exhibit temperature-sensitive properties in structure and function.

We previously constructed seven mutations in the gene for glycoprotein D (gD) of herpes simplex virus type 1 in which the codon for one of the cysteine residues was replaced by a serine codon. Each of the mutant genes was cloned into a eucaryotic expression vector, and the proteins were transiently expressed in mammalian cells. We found that alteration of any of the first six cysteine residues had profound effects on protein conformation and oligosaccharide processing. In this report, we show that five of the mutant proteins exhibit temperature-sensitive differences in such properties as aggregation, antigenic conformation, oligosaccharide processing, and transport to the cell surface. Using a complementation assay, we have now assessed the ability of the mutant proteins to function in virus infection. This assay tests the ability of the mutant proteins expressed from transfected plasmids to rescue production of infectious virions of a gD-minus virus, F-gD beta, in Vero cells. Two mutant proteins, Cys-2 (Cys-106 to Ser) and Cys-4 (Cys-127 to Ser), were able to complement F-gD beta at 31.5 degrees C but not at 37 degrees C. The rescued viruses, designated F-gD beta(Cys-2) and F-gD beta(Cys-4), were neutralized as efficiently as wild-type virus by anti-gD monoclonal antibodies, indicating that gD was present in the virion envelope in a functional form. Both F-gD beta(Cys-2) and F-gD beta(Cys-4) functioned normally in a penetration assay. However, the infectivity of these viruses was markedly reduced compared with that of the wild type when they were preincubated at temperatures above 37 degrees C. The results suggest that mutations involving Cys-106 or Cys-127 in gD-1 confer a temperature-sensitive phenotype on herpes simplex virus. These and other properties of the cysteine-to-serine mutants allowed us to predict a disulfide bonding pattern for gD.     

Nonlethal sec71-1 and sec72-1 mutations eliminate proteins associated with the Sec63p-BiP complex from S. cerevisiae.

The sec71-1 and sec72-1 mutations were identified by a genetic assay that monitored membrane protein integration into the endoplasmic reticulum (ER) membrane of the yeast Saccharomyces cerevisiae. The mutations inhibited integration of various chimeric membrane proteins and translocation of a subset of water soluble proteins. In this paper we show that SEC71 encodes the 31.5-kDa transmembrane glycoprotein (p31.5) and SEC72 encodes the 23-kDa protein (p23) of the Sec63p-BiP complex. SEC71 is therefore identical to SEC66 (HSS1), which was previously shown to encode p31.5. DNA sequence analyses reveal that sec71-1 cells contain a nonsense mutation that removes approximately two-thirds of the cytoplasmic C-terminal domain of p31.5. The sec72-1 mutation shifts the reading frame of the gene encoding p23. Unexpectedly, the sec71-1 mutant lacks p31.5 and p23. Neither mutation is lethal, although sec71-1 cells exhibit a growth defect at 37 degrees C. These results show that p31.5 and p23 are important for the trafficking of a subset of proteins to the ER membrane.     

A "CELL-CONTACT TEMPERATURE-SENSITIVE" MUTANT OF THE CELLULAR SLIME MOLD DICTYOSTELIUM MUCOROIDES

A mutant (TS-2) that is temperature-sensitive with respect to cell contact was isolated from the cellular slime mold, Dictyostelium mucoroides. The TS-2 were able to grow and develop normally at 21°C, but unable to grow at 31.5°C. When TS-2 were allowed to develop until the aggregation stage at 21°C and then shifted to 31.5°C, they instantly lost cell-to-cell contact, resulting in disintegration of the aggregation stream and flattening of the aggregation center. Although a slug transferred to 31.5°C retained its original shape, loss of cell-to-cell contact within the cell mass was evidenced by several facts. The TS-2 interphase amebas, at 31.5°C, also lost cell-to-substratum contact, and the loss of contact was followed by the production of cell-wall substance on their surface. The production of the same substance at 31.5°C was also observed in cells at aggregation and migration stages, but not in those at the vegetative stage. When TS-2 cells at various developmental stages were kept at 31.5°C for various periods of time and returned to 21°C they lost morphogenetic capacity in proportion to the production of the cell-wall substance.     

Direct selection of cybrids by streptomycin and valine resistance in tobacco

Summary Direct selection of cybrids by simultaneous selection for donor chloroplasts and for the recipient nuclei is described. Mesophyll protoplasts of two tobacco (Nicotiana tabacum) mutants, SR1 (streptomycin resistant) and Val^r-2 (valine resistant), were fused by polyethylene glycol treatment. Streptomycin resistance in the SR1 mutant is a maternally inherited chloroplast trait while valine resistance is a Mendelian (nuclear) digenic recessive character. The fused protoplast population was cultured and colonies were selected for resistance to valine (1 mM) and streptomycin (343 M). The efficiency of selection has been confirmed in three clones by demonstrating seed transmission of both streptomycin and valine resistances. In one subclone both streptomycin resistant and sensitive plants were obtained indicating that the streptomycin sensitive chloroplasts had not been totally eliminated by growth on the selective medium.     

Habituation to alkali and increased u.v.-resistance in DNA repair-proficient and -deficient strains of Escherichia coli grown at pH 9.0

Repair-deficient strains of Escherichia coli carrying polAI or recA mutations were more alkali-sensitive than was their repair-proficient parent but, like strain 1829 ColV, I-K94, they showed habituation to alkali (induction of increased resistance) when grown at pH 9.0. Occurrence of such increased alkali resistance in the recA mutant implies that habituation to alkali does not depend on induction of SOS-related repair mechanisms. Organisms of repair-proficient and repair-deficient strains also became more resistant to u.v.-irradiation after growth at pH 9.0; this increased u.v.-resistance also appeared to be RecA-independent.     

Effect of growth temperature on folding of carbamoylphosphate synthetases of Salmonella typhimurium and a cold-sensitive derivative.

The properties of homogeneous preparations of carbamoylphosphate synthetase (CPSase) from wild-type Salmonella typhimurium and a cold-sensitive derivative grown at different growth temperatures were examined. For the cold-sensitive mutant, the affinity for glutamine of the form of CPSase synthesized at 20 degrees C was lower than that of the form of the enzyme synthesized at 37 degrees C, regardless of the assay temperature. Thus, the cold sensitivity of the mutant reflects an effect of temperature on the synthesis of the enzyme rather than the activity of the folded enzyme. The two forms also differed in sensitivities to polyclonal antibodies as well as denaturational enthalpies. The combined results support the hypothesis that carAB mutations conferring cold sensitivity identify amino acid residues that are critical in the folding of CPSase. Quite unexpectedly, certain kinetic properties of cloned parent CPSase were also dependent on the growth temperature, although to a much lesser extent than those of the cold-sensitive mutant. The specific activity of wild-type CPSase synthesized at 15 degrees C was 60% of that synthesized at 37 degrees C. Further, CPSase synthesized at 15 degrees C was less thermostable than the enzyme synthesized at 37 degrees C; the difference in stability (delta G) is estimated to be 4,500 cal mol-1. Thus, variation of temperature within the physiological range for growth influences the folding and consequently the properties of CPSase from wild-type S. typhimurium.     

Nucleoside transport-deficient mutants of PK-15 pig kidney cell line.

Previous studies indicated that PK-15 pig kidney cells express solely a nitrobenzylthioinosine-sensitive, equilibrative nucleoside transporter. In the present study, PK-15 cells were mutagenized by treatment with ICR-170 and nucleoside transport-deficient mutants selected in a single step in growth medium containing tubercidin and cytosine arabinoside at a frequency of about 2 x 10(6). The mutants were simultaneously at least 100-times more resistant to tubercidin, cytosine arabinoside and 5-fluorodeoxyuridine than the wild-type parent cells. The mutants failed to transport thymidine and uridine and had lost all high affinity nitrobenzylthioinosine binding sites. Residual low level uptake of thymidine by the mutants was shown to be due to nonmediated permeation (passive diffusion), which explains the sensitivity of the mutants to growth inhibition by high concentrations of the nucleoside drugs. Passive diffusion of thymidine at a concentration of 16 microM was not rapid enough to support the growth of nucleoside transport-deficient mutant cells that had been made thymidine-dependent by treatment with methotrexate, whereas wild-type cells grew normally under these conditions. The nucleoside transport-deficient mutants exhibited about the same growth rate and plating efficiency (60-80%) as wild-type cells, but formed larger colonies than wild-type cells because of a more extensive spread of the cells on the surface of culture dishes. PK-15 cells adhere very strongly to the surface of culture dishes and have been transformed with high efficiency with plasmid DNA either via lipofection or electroporation.     

Increased ethanol production and tolerance by a pyruvate-negative mutant of Clostridium saccharolyticum

Summary To improve the conversion of hexoses and pentoses to ethanol, a pyruvate-negative (PN) mutant of Clostridium saccharolyticum, having lower acetate kinase activity, was obtained. The PN mutant used more substrate (glucose or xylose) and produced more biomass and ethanol, but less acetic acid. This shift in catabolism raised the ethanol/acetate ratio from 6.7 to 13. The PN mutant converted both glucose and xylose to ethanol at an efficiency of 80% of the theoretical yield as compared to 64% for C. saccharolyticum wild type. This improved production of ethanol was also accompanied by an increased tolerance to ethanol. The PN mutant showed 50% growth inhibition at an ethanol concentration of 6.5% (v/v) as compared to 3.5% for the parent strain.National Research Council of Canada No. 21316     

Genetic analysis of Escherichia coli K-12 chromosomal mutants defective in expression of F-plasmid functions: identification of genes cpxA and cpxB.

Two temperature-sensitive, chromosomal mutants of Escherichia coli were selected for their inability to express deoxyribonucleic acid donor activity and other activities associated with the conjugative plasmid F. These mutants were also auxotrophic for isoleucine and valine at 41 degrees C. Each mutant strain contained two altered genes: cpxA, located at 88 min on the E. coli K-12 genetic map, and cpxB, located at 41 min. Mutations in both genes were required for maximal expression of mutant phenotypes. The parent strain of mutants KN401 and KN312 already contained the cpxB mutation that is present in both mutants (cpxB1). This mutation by itself was cryptic. The cpxA mutations represent different mutant alleles since they are of independent origin. A cpxA mutation by itself significantly affected the expression of plasmid functions and growth at 41 degrees C in the absence of isoleucine and valine, but strains containing both a cpxA and cpxB mutation were more severely affected. Along with the observation that both cpxA mutations were revertable, the temperature sensitivity of cpxA cpxB+ cells suggests that both cpxA alleles contain point mutations that do not completely destroy the activity of the cpxA gene product.     

Virucidal activity of a GT-rich oligonucleotide against herpes simplex virus mediated by glycoprotein B

We have investigated the antiviral mechanism of a phosphorothioate oligonucleotide, ISIS 5652, which has activity against herpes simplex virus (HSV) in the low micromolar range in plaque reduction assays. We isolated a mutant that is resistant to this compound. Marker rescue and sequencing experiments showed that resistance was due to at least one of three mutations in the UL27 gene which result in amino acid changes in glycoprotein B (gB). Because gB has a role in attachment and entry of HSV, we tested the effects of ISIS 5652 at these stages of infection. The oligonucleotide potently inhibited attachment of virus to cells at 4 degrees C; however, the resistant mutant did not exhibit resistance at this stage. Moreover, a different oligonucleotide with little activity in plaque reduction assays was as potent as ISIS 5652 in inhibiting attachment. Similarly, ISIS 5652 was able to inhibit entry of pre-attached virions into cells at 37 degrees C, but the mutant did not exhibit resistance in this assay. The mutant did not attach to or enter cells more quickly than did wild-type virus. Strikingly, incubation of wild-type virus with 1 to 2 microM ISIS 5652 at 37 degrees C led to a time-dependent, irreversible loss of infectivity (virucidal activity). No virucidal activity was detected at 4 degrees C or with an unrelated oligonucleotide at 37 degrees C. The resistant mutant and a marker-rescued derivative containing its gB mutations exhibited substantial resistance to this virucidal activity of ISIS 5652. We hypothesize that the GT-rich oligonucleotide induces a conformational change in gB that results in inactivation of infectivity.     

Isolation in vitro of naphthaleneacetic acid-tolerant mutants of Nicotiana tabacum, which are impaired in root morphogenesis

Summary Mutants resistant to the toxic effect of the auxin analogue naphthaleneacetic acid have been selected in vitro in mutagenized populations of haploid mesophyll protoplasts of Nicotiana tabacum. Among the regenerated clones obtained, two clones impaired in root morphogenesis were further characterized. The parental mutant clones isolated were found to be heterozygotes for the mutations conferring the inability to root. These mutations were transmissible to the progeny as single nuclear dominant traits and cosegregated with resistance to naphthaleneacetic acid at the cellular level. Apart from the inability to root and a slightly modified leaf morphology, no obvious modification of stem structure, apical dominance, and flower fertility was detected in the development of homozygotes obtained in the progeny of one of the parental mutant clones when they were grafted onto normal plants.Abbreviations NAA 1-Naphthaleneacetic acid - PE plating efficiency - p-cells protoplast-derived dividing cells - MNNG N-methyl-N'-nitro-N-nitrosoguanidine - UV Ultraviolet light - 2,4-D 2,4-dichlorophenoxyacetic acid Dedicated to Professor Georg Melchers to celebrate his 50-year association with the journal     

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.