Respiration-deficient Chinese hamster cell mutants: biochemical characterization.

We have previously classified 35 of our respiration-deficient mutants into seven complementation groups and one "overlapping" mutant which does not complement mutants from groups I and II. In this paper we report on the biochemical characterization of representatives of complementation groups I, II, VII, and the "overlapping" mutant. We show that these mutants all have a defect in complex I of the electron-transport chain. The general features of these mutants are: (1) a low rate of O2 consumption in whole cells; (2) a low rate of release of 14CO2 from [2-14C] pyruvate, [1-14C] pyruvate, and [3-14C] beta-hydroxybutyrate; (3) a low rate of release of 14CO2 from [5-14C] glutamate and [1-14C] glutamate in mutants from groups II, VII, and the "overlapping" mutant, whereas a significant amount of 14CO2 is released in mutants from group I; (4) a substantial rate of release of 14CO2 from [U-14C] asparate; (5) in isolated mitochondria, succinate and alpha-glycerol phosphate stimulate O2 consumption whereas substrates which generate NADH, such as malate, do not; and (6) there is little or no rotenone-sensitive NADH oxidase activity in isolated mitochondria.     

Preliminary Physiological Characterization of Temperature-Sensitive Mutants of Vesicular Stomatitis Virus

Different temperature-sensitive mutants of vesicular stomatitis virus have been characterized in terms of their ability to induce synthesis of viral ribonucleic acid (RNA) in BHK-21 cells at 39 C (the restrictive temperature for these mutants). Mutants belonging to complementation groups I and IV (and probably II) did not induce actinomycin-resistant RNA synthesis in infected cells incubated at 39 C. All three mutants comprising complementation group III induced viral RNA synthesis at 39 C. The temperature sensitivity of the defective viral functions has also been studied by temperature-shift experiments. The functions associated with the mutants of groups I, II, and IV were required early, whereas the function associated with the group III mutants was not required until a late stage of the viral cycle. The heat sensitivity of extracellular virion was not correlated with complementation group.     

Mutant analysis of herpes simplex virus-induced cell surface antigens: resistance to complement-mediated immune cytolysis.

BHK-21 cells infected with temperature-sensitive mutants of herpes simplex virus type 1 strain KOS representing 16 complementation groups were tested for susceptibility to complement-mediated immune cytolysis at permissive (34 degrees C) and nonpermissive (39 degrees C) temperatures. Only cells infected by mutants in complementation group E were resistant to immune cytolysis in a temperature-sensitive manner compared with wild-type infections. The expression of group E mutant cell surface antigens during infections at 34 and 39 degrees C was characterized by a combination of cell surface radioiodination, specific immunoprecipitation, and gel electrophoretic analysis of immunoprecipitates. Resistance to immune lysis at 39 degrees C correlated with the absence of viral antigens exposed at the cell surface. Intrinsic radiolabeling of group E mutant infections with [14C]glucosamine revealed that normal glycoproteins were produced at 34 degrees C but none were synthesized at 39 degrees C. The effect of 2-deoxy-D-glucose on glycosylation of group E mutants at 39 degrees C suggested that the viral glycoprotein precursors were not synthesized. The complementation group E mutants failed to complement herpes simplex virus type 1 mutants isolated by other workers. These included the group B mutants of strain KOS, the temperature-sensitive group D mutants of strain 17, and the LB2 mutant of strain HFEM. These mutants should be considered members of herpes simplex virus type 1 complementation group 1.2, in keeping with the new herpes simplex virus type 1 nomenclature.     

The mitochondrial COB region in yeast codes for apocytochrome b and is mosaic.

Mitochondrial mutants of Saccharomyces cerevisiae defective in cytochrome b were analyzed genetically and biochemically in order to elucidate the role of the mitochondrial genetic system in the biosynthesis of this cytochrome. The mutants mapped between OLI1 and OLI2 on mitochondrial DNA in a region called COB. A fine structure map of the COB region was constructed by rho- deletion mapping and recombination analysis. The combined genetic and biochemical data indicate that the COB region is mosaic and contains at least five distinct clusters of mutants, A-E, with A being closest to OLI2 and E being closest to OLI1. Clusters A, C and E are probably coding regions for apocytochrome b, whereas clusters B and D seem to be involved in as yet unknown functions. These conclusions rest on the following evidence. 1. Most mutants in clusters A, C and E have specifically lost cytochrome b. Many of them accumulate smaller mitochondrial translation products; some of these were identified as fragments of apocytochrome b by proteolytic fingerprinting. The molecular weight of these fragments depends on the map position of the mutant, increasing in the direction OLI2 leads to OLI1. The mutant closest to OLI1 accumulates an apocytochrome b which is slightly larger than that of wild type. 2. A mutant in cluster C exhibits a spectral absorption band of cytochrome b that is shifted 1.5 nm to the red. 3. Mutants in clusters B and D are pleiotropic. A majority of them are conditional and lack the absorption bands of both cytochrome b and cytochrome aa3; these mutants also fail to accumulate apocytochrome b and subunit I of cytochrome c oxidase and instead form a large number of abnormal translation products whose nature is unknown. 4. Zygotic complementation tests reveal at least two complementation groups: The first group includes all mutants in cluster B and the second group includes mutants in clusters (A + C + D + E).     

Semaphorin 1a and semaphorin 1b are required for correct epidermal cell positioning and adhesion during morphogenesis in C. elegans

The semaphorin family comprises secreted and transmembrane proteins involved in axon guidance and cell migration. We have isolated and characterized deletion mutants of C. elegans semaphorin 1a (Ce-sema-1a or smp-1) and semaphorin 1b (Ce-sema-1b or smp-2) genes. Both mutants exhibit defects in epidermal functions. For example, the R1.a-derived ray precursor cells frequently fail to change anterior/posterior positions completely relative to their sister tail lateral epidermal precursor cell R1.p, causing ray 1 to be formed anterior to its normal position next to ray 2. The ray cells, which normally separate from the lateral tail seam cell (SET) at the end of L4 stage, remains connected to the SET cell even in adult mutant males. The ray 1 defects are partially penetrant in each single Ce-sema-1 mutant at 20 degrees C, but are greatly enhanced in Ce-sema-1 double mutants, suggesting that Ce-Sema-1a and Ce-Sema-1b function in parallel to regulate ray 1 position. Both mutants also have defects in other aspects of epidermal functions, including head and tail epidermal morphogenesis and touch cell axon migration, whereas, smp-1 mutants alone have defects in defecation and brood size. A feature of smp-1 mutants that is shared with mutants of mab-20 (which encodes Sema-2a) is the abnormal perdurance of contacts between epidermal cells.     

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.     

In vivo characterization of the nonessential budding yeast anaphase-promoting complex/cyclosome components Swm1p, Mnd2p and Apc9p

We have examined the in vivo requirement of two recently identified nonessential components of the budding yeast anaphase-promoting complex, Swm1p and Mnd2p, as well as that of the previously identified subunit Apc9p. swm1Delta mutants exhibit synthetic lethality or conditional synthetic lethality with other APC/C subunits and regulators, whereas mnd2Delta mutants are less sensitive to perturbation of the APC/C. swm1Delta mutants, but not mnd2Delta mutants, exhibit defects in APC/C substrate turnover, both during the mitotic cell cycle and in alpha-factor-arrested cells. In contrast, apc9Delta mutants exhibit only minor defects in substrate degradation in alpha-factor-arrested cells. In cycling cells, degradation of Clb2p, but not Pds1p or Clb5p, is delayed in apc9Delta. Our findings suggest that Swm1p is required for full catalytic activity of the APC/C, whereas the requirement of Mnd2p for APC/C function appears to be negligible under standard laboratory conditions. Furthermore, the role of Apc9p in APC/C-dependent ubiquitination may be limited to the proteolysis of a select number of substrates.     

P22-Mediated Transduction Analysis of the Rough A (rfa) Region of the Chromosome of Salmonella typhimurium

Seven temperature-sensitive rough mutants of Salmonella typhimurium were found to be sensitive to smooth-specific phages at low temperature (25 C, 30 C) and resistant or partially resistant to rough-specific phages, whereas at high temperatures (37 C, 45 C) they were resistant or partially resistant to smooth-specific phages but sensitive to rough-specific phages. These data indicate that at low temperature each strain makes lipopolysaccharide which is relatively normal, but at high temperatures O-specific side chains are not added to the lipopolysaccharide. At 45 C, these strains have the R-res-1 or R-res-2 phage sensitivity phenotype, and their genetic lesions map by P22-mediated transduction in the rfa gene cluster between cysE-pyrE, suggesting a mutation in genes with transferase functions. P22-mediated joint transduction with temperature-sensitive rfa mutants, leaky rfa mutants, and rfa P22 lysogens have shown the following order of genes in the S. typhimurium linkage map: xyl-mtlA-mtlB-cysE-rfaF-rfaG-pyrE. An rfaE allele was not jointly transduced in the cysE-pyrE segment.     

Two cyclophilin A homologs with shared and distinct functions important for growth and virulence of Cryptococcus neoformans

Cyclophilin A is the target of the immunosuppressant cyclosporin A (CsA) and is encoded by a single unique gene conserved from yeast to humans. In the pathogenic fungus Cryptococcus neoformans, two homologous linked genes, CPA1 and CPA2, were found to encode two conserved cyclophilin A proteins. In contrast to Saccharomyces cerevisiae, in which cyclophilin A mutations confer CsA resistance but few other phenotypes, cyclophilin A mutations conferred dramatic phenotypes in C. neoformans. The Cpa1 and Cpa2 cyclophilin A proteins play a shared role in cell growth, mating, virulence and CsA toxicity. The Cpa1 and Cpa2 proteins also have divergent functions. cpa1 mutants are inviable at 39°C and attenuated for virulence, whereas cpa2 mutants are viable at 39°C and fully virulent. cpa1 cpa2 double mutants exhibited synthetic defects in growth and virulence. Cyclophilin A active site mutants restored growth of cpa1 cpa2 mutants at ambient but not at higher temperatures, suggesting that the prolyl isomerase activity of cyclophilin A has an in vivo function.     

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.     

Characterization of a Phosphate-Accumulator Mutant of Arabidopsis thaliana

We have characterized a novel mutation of Arabidopsis thaliana at a locus designated pho2. pho2 mutants accumulated up to 3-fold more total P in leaves, mostly as inorganic phosphate (Pi), than wild-type seedlings. In addition, we isolated a mutant (locus designated pho1-2, an allelle of pho1-1 described by Y. Poirier, S. Thoma, C. Somerville, J. Schiefelbein [1991] Plant Physiol 97: 1087-1093) with low Pi concentrations in leaves. When grown under high transpiration conditions, leaves of pho2 seedlings became severely P intoxicated, whereas shoots of pho1-2 mutants were P deficient and wild-type seedlings were normal. A pho1/pho2 double mutant resulting from a cross between the single mutants was identified in the F2 generation and shown to have a pho1 phenotype. Prior to the development of P toxicity symptoms, P was the only mineral nutrient whose concentration was greater in pho2 mutants than wild-type seedlings. Compared to wild-type, pho2 mutants had greater Pi concentrations in stems, siliques, and seeds, but roots of pho2 mutants had similar or lower Pi concentrations than either pho1 mutants or wild-type seedlings. We suggest that the pho2 mutation affects a function normally involved in regulating the concentration of Pi in shoots of Arabidopsis.     

Effects of mutations of the bulged nucleotide in the conserved P7 pairing element of the phage T4 td intron on ribozyme function

The P7 element of group I introns contains a semiconserved "bulged" nucleotide, a C in group IA introns (nt 870 in the td intron) and an A in group IB introns [Cech, T.R. (1988) Gene 73, 259-271]. Variants U870, G870, and A870, isolated by a combination of in vitro and in vivo genetic strategies, indicate that C and A at position 870 are consistent with splicing whereas U and G are not. Although mutants G870 and U870 could be activated in vitro by increasing the Mg2+ concentration, their Km for GTP at pH 7 was 20-100-fold elevated, and they were unable to undergo site-specific hydrolysis. The dependence of the mutants on high guanosine concentrations could be substantially overcome by an increase in pH, suggesting that a tautomeric change, which makes U and G mimic C and A, is responsible for restoring function. In contrast to the striking Km effect, Vmax for the mutants differed by less than a factor of 2 from the wild type. Furthermore, streptomycin, an aminoglycoside antibiotic that competes with guanosine for its binding site, inhibited splicing of the U870 and G870 constructs at least as well as of the C870 and A870 variants, indicating that the guanosine-binding site of the mutants is proficient at interacting with a guanidino group. While our experiments argue against a hydrogen-bonding interaction between the C6-O of the cofactor and C4-NH2 of the bulged nucleotide, they are consistent with other models in which the C4-NH2 and/or N3 groups of the bulged C are involved in establishing an active ribozyme.     

Isolation and Classification of Temperature-Sensitive Mutants of Influenza B Virus

We isolated 25 temperature-sensitive mutants of B/Kanagawa/73 strain generated by mutagenesis with 5-fluorouracil and classified them into seven recombination groups by pair-wise crosses. All mutants showed a ratio of plaquing efficiency at the nonpermissive temperature (37.5 C) to the permissive temperature (32 C) of 10^–4 or less. At 37.5 C most of group I, II, and III mutants did not produce appreciable amounts of protein, but all other group mutants were protein synthesis-positive. A group VII mutant produced active hemagglutinin (HA) and neuraminidase (NA) at the nonpermissive temperature, but Group V mutants produced only active NA and were defective in the HA molecule. The other group mutants, including group IV mutants with mutation only in the NA gene (8, 10), lacked both activities at the nonpermissive temperature. One of nine influenza B virus isolates in 1989 had EOP 37.5/32 of 1/3 × 10^–2 and belonged to recombination group VII.     

A new class of rapidly developing mutants in Dictyostelium discoideum: implications for cyclic AMP metabolism and cell differentiation

Rapidly developing (rde) mutants of Dictyostelium discoideum, in which cells precociously differentiated into stalk and spore cells without normal morphogenesis, were investigated genetically and biochemically. Genetic complementation tests demonstrated that the 16 rde mutants isolated could be classified into at least two groups (groups A and C) and that the first described rde mutant FR17 (D. R. Sonneborn, G. J. White, and M. Sussman, 1963, Dev. Biol. 7, 79-93) belongs to group A. Morphological studies revealed several differences in development and final morphology between group A and group C mutants. In group A mutants, the time required for cell differentiation from vegetative cells to aggregation competent cells is reduced, whereas the time required for spore and stalk cell differentiation following the completion of aggregation is shortened in group C mutants. This suggests that group C mutants represent a new class of rde mutants and that there exist at least two mechanisms involved in regulating the timing of development in D. discoideum. Measurements of cell-associated and extracellular phosphodiesterase activities, and intracellular and total cAMP levels revealed that cAMP metabolism in both groups is significantly altered during development. Group A mutants showed precocious and excessive production of phosphodiesterase and cAMP during the entire course of development; intracellular cAMP levels in group C mutants were extremely low, and spore and stalk cell differentiation occurred without an apparent increase in these levels. Thus, while cAMP metabolism is abnormal in all the rde mutants studied, there exist several distinct types of derangement, not necessarily involving the overproduction of cAMP.     

Isolation and Primary Identification of Methylotrophic Yeast Hansenula polymorpha Mutants for Peroxisome Biogenesis

After exposure of cells of the methylotrophic yeast Hansenula polymorphaHF246leu1-1 to N-nitro-N-nitrosoguanidine, a collection of 227 mutants unable to grow on methanol at elevated temperature (45°C) was obtained. Ninety four ts mutants (35% of the total number of mutants), which were unable to grow on methanol only at 45°C but could grow at optimal temperature (37°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 10pex10 mutants (4ts mutants among them); group 2 included 19 mutants that failed to complement otherpex testers: 1 pex1; 2 pex4(1ts); 6 pex5(5ts); 3 pex8; 1 pex13; 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°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. Analysis of the spore population from the hybrids of remaining 14 mutants with the pex tester demonstrated the presence of methanol-utilizing segregants, which indicates mutation localization 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 us to localize this mutation in the only PEX gene (PEX1 or PEX2). The revealed disorder of complementation interactions between nonallelic genes is under debate.     

Genetic Characteristics of Conditional Lethal Mutants of Vesicular Stomatitis Virus Induced by 5-Fluorouracil, 5-Azacytidine, and Ethyl Methane Sulfonate

One hundred and seventy-five temperature-sensitive (ts) mutants of vesicular stomatitis virus (type Indiana-C) induced by 5-fluorouracil (FU), 5-azacytidine (ACR), and ethyl methane sulfonate (EMS) have been assigned to four complementation groups by a qualitative test. Group I contains 151 mutants; group II, 2 mutants; group III, 1 mutant; and group IV, 15 mutants; 6 are unclassified. FU was much more effective as a mutagen than either ACR or EMS. The proportion of the mutants belonging to groups I and IV, however, was similar in the case of all three mutagens. Fifteen mutants from groups I and IV have been used to obtain quantitative complementation data. Both groups appear to be homogeneous. Complementation yields increase with increasing multiplicity, but the number of particles per cell required to elicit maximal complementation is small. The pattern of genetic recombination parallels that of complementation. No recombination could be detected in crosses within group I (<0.001%) or group IV (<0.07%), whereas recombination (0.31 to 3.4%) was observed in crosses between groups I and IV. Recombination frequency did not increase with multiplicity above an input of 0.6 plaque-forming units per cell. Many group I mutants have very low reversion rates, and BHK 21 clone 13 cells infected with one of these mutants have been "cured" of infection by prolonged exposure at the restrictive temperature.