Characterization of a thermosensitive Escherichia coli aspartyl-tRNA synthetase mutant.

The Escherichia coli tls-1 strain carrying a mutated aspS gene (coding for aspartyl-tRNA synthetase), which causes a temperature-sensitive growth phenotype, was cloned by PCR, sequenced, and shown to contain a single mutation resulting in substitution by serine of the highly conserved proline 555, which is located in motif 3. When an aspS fragment spanning the codon for proline 555 was transformed into the tls-1 strain, it was shown to restore the wild-type phenotype via homologous recombination with the chromosomal tls-1 allele. The mutated AspRS purified from an overproducing strain displayed marked temperature sensitivity, with half-life values of 22 and 68 min (at 42 degrees C), respectively, for tRNA aminoacylation and ATP/PPi exchange activities. Km values for aspartic acid, ATP, and tRNA(Asp) did not significantly differ from those of the native enzyme; thus, mutation Pro555Ser lowers the stability of the functional configuration of both the acylation and the amino acid activation sites but has no significant effect on substrate binding. This decrease in stability appears to be related to a conformational change, as shown by gel filtration analysis. Structural data strongly suggest that the Pro555Ser mutation lowers the stability of the Lys556 and Thr557 positions, since these two residues, as shown by the crystallographic structure of the enzyme, are involved in the active site and in contacts with the tRNA acceptor arm, respectively.     

Calcium-sensitive cls4 mutant of Saccharomyces cerevisiae with a defect in bud formation.

A calcium-sensitive cls4 mutant of Saccharomyces cerevisiae ceased dividing in the presence of 100 mM CaCl2, producing large, round, unbudded cells. Since its DNA replication and nuclear division still continued after interruption of normal budding, the cls4 mutant had a defect in bud formation in Ca2+-rich medium. Its calcium content and calcium uptake activity were the same as those of the wild-type strain, suggesting that the primary defect of the mutation was not in a Ca2+ transport system. Genetic analysis showed that the cls4 mutation did not complement the cdc24-1 mutation, which is known to be a temperature-sensitive mutation affecting bud formation and localized cell surface growth at a restrictive temperature. Moreover, cls4 was tightly linked to cdc24, and a yeast 3.4-kilobase-pair DNA fragment carrying both the CLS4 and CDC24 genes was obtained. These results suggest that the cls4 mutation is allelic to the cdc24 mutation. Thus, Ca2+ ion seems to control bud formation and bud-localized cell surface growth.     

dnaT, dominant conditional-lethal mutation affecting DNA replication in Escherichia coli.

Normally, bacteria cease DNA replication in the absence of protein synthesis. A variety of treatments, such as thymine starvation or a shift-up to rich medium, lead to continued DNA replication in the absence of protein synthesis. Mutants are described which always terminate replication under these conditions. These conditional lethal mutants, dnaT1 and dnaT2, contransduce with serB and dnaC. The mutation also affects cell division. All aspects of the mutant phenotype (obligatory termination of replication, temperature sensitivity of DNA replication and growth, and aberrant cell division at permissive growth temperatures) were transdominant to the wild-type phenotype. Episomes carrying the dnaT mutation appeared to be unstable. The existence of such a dominant mutation was predicted by a model of chromosome termination proposed by Kogoma and Lark (J. Mol. Biol. 94:243-256, 1975).     

Characterization of a new simian virus 40 mutant, tsA3900, isolated from deletion mutant tsA1499.

The simian virus 40 (SV40) mutant tsA1499 contains an 81-base-pair deletion in the region of A gene encoding the C-terminal portion of the large T antigen. This mutant is particularly interesting, since it is a temperature-sensitive mutant that is apparently able to separate the lytic growth and transforming functions of the SV40 large T antigen at 38.5 degrees C. We report the isolation of a tsA1499 revertant (tsA1499-Rev) which is no longer temperature sensitive for lytic growth but still contains the 81-base-pair deletion of tsA1499. Marker rescue experiments with tsA1499-Rev or wild-type strain 830 (wt830) DNAs revealed that the original tsA1499 mutant contained a second mutation within the HindIII-Fnu4HI restriction fragment between 0.425 and 0.484 map units. Sequencing of this DNA fragment from the tsA1499, tsA1499-Rev, and wt830 viruses revealed that tsA1499 contained a single-base transversion (C to G) at 0.455 map units (nucleotide 4261). This transversion resulted in the creation of a new RsaI cleavage site in the tsA1499 DNA and predicts an arginine-to-threonine substitution at amino acid position 186 in the mutant large T antigen. The DNA sequence of the tsA1499-Rev HindIII-Fnu4HI fragment was identical to that of wt830. To determine whether tsA1499 was temperature sensitive for lytic growth solely as a result of the newly discovered point mutation or because of a combination of the point and deletion mutations, a series of viruses were constructed which contained the point mutation, the deletion mutation, both mutations, or neither. This was done by ligating the PstI A and B DNA fragments from either tsA1499 or wt830 and transfecting the ligated DNA into BSC-1H monkey kidney cells. This experiment revealed that all viruses containing the point mutation (the tsA1499 PstI A DNA fragment) were temperature sensitive for lytic growth, regardless of the presence of the 81-base-pair deletion (the tsA1499 PstI B DNA fragment). This newly discovered point mutation, at nucleotide 4261, is therefore unique, since to our knowledge it is the first tsA mutation to be described in the 0.455-map-unit region of the SV40 genome. We then tested the effect of this unique mutation on the ability of the SV40 virus to transform cultured rat cells to anchorage independence.(ABSTRACT TRUNCATED AT 400 WORDS)     

Characterization and cloning of the gerC locus of Bacillus subtilis 168

A Bacillus subtilis gerC spore germination mutant demonstrating a temperature-sensitive response to L-alanine as germinant has been characterized in detail. The gerC58 mutation is 50% cotransformed with aroB in the gene order gerC-aroB-trpC. The mutation is responsible for a severe growth defect which is manifest at all growth temperatures and is most extreme on rich media. A second, unlinked, mutation in the original strain suppressed this growth defect, but spores of the suppressed strain failed to germinate in alanine at 42 degrees C. As this germination defect is dependent on the presence of the gerC58 allele, it is likely to be the direct result of a mutant gerC protein. The gerC gene therefore appears to have a role in both spore germination and vegetative cell growth. A gene library of BclI-digested B. subtilis chromosomal DNA was constructed in phage vector phi 105J27. A derivative containing the gerC region was obtained by complementation of the growth defect of an unsuppressed gerC58 strain. This phage contained a 6.3 kb insert of bacterial DNA, which is above the reported packaging limit of the phage. It failed to form visible plaques, although it could be handled as a prophage and sufficient phage particles be isolated to allow characterization of the insert. A deletion derivative generated in vitro and carrying only 2.9 kb of insert DNA also complemented the gerC defect. This gerC locus is the second locus to be implicated in alanine-stimulated germination. The first, gerA, is a developmentally controlled operon whose gene products are present only in the spore. This study of gerC, in contrast, reveals a role in spore germination for a normally essential vegetative protein.     

Escherichia coli mutants deficient in deoxyuridine triphosphatase.

Mutants deficient in deoxyuridine triphosphatase (dUTPase) were identified by enzyme assays of randomly chosen heavily mutagenized clones. Five mutants of independent origin were obtained. One mutant produced a thermolabile enzyme, and it was presumed to have a mutation in the structural gene for dUTPase, designated dut. The most deficient mutant had the following associated phenotypes: less than 1% of parental dUTPase activity, prolonged generation time, increased sensitivity to 5'-fluorodeoxyuridine, increased rate of spontaneous mutation, increased rate of recombination (hyper-Rec), an inhibition of growth in the presence of 2 mM uracil, and a decreased ability to support the growth of phage P1 (but not T4 or lambda). This mutation also appeared to be incompatible with pyrE mutations. A revertant selected by its faster growth had regained dUTPase activity and lost its hyper-Rec phenotype. Many of the properties of the dut mutants are compatible with their presumed increased incorporation of uracil into DNA and the subsequent transient breakage of the DNA by excision repair.     

Suppressors of a host range mutation in the rabbitpox virus serpin SPI-1 map to proteins essential for viral DNA replication

The orthopoxvirus serpin SPI-1 is an intracellular serine protease inhibitor that is active against cathepsin G in vitro. Rabbitpox virus (RPV) mutants with deletions of the SPI-1 gene grow on monkey kidney cells (CV-1) but do not plaque on normally permissive human lung carcinoma cells (A549). This reduced-host-range (hr) phenotype suggests that SPI-1 may interact with cellular and/or other viral proteins. We devised a genetic screen for suppressors of SPI-1 hr mutations by first introducing a mutation into SPI-1 (T309R) at residue P14 of the serpin reactive center loop. The SPI-1 T309R serpin is inactive as a protease inhibitor in vitro. Introduction of the mutation into RPV leads to the same restricted hr phenotype as deletion of the SPI-1 gene. Second-site suppressors were selected by restoration of growth of the RPV SPI-1 T309R hr mutant on A549 cells. Both intragenic and extragenic suppressors of the T309R mutation were identified. One novel intragenic suppressor mutation, T309C, restored protease inhibition by SPI-1 in vitro. Extragenic suppressor mutations were mapped by a new procedure utilizing overlapping PCR products encompassing the entire genome in conjunction with marker rescue. One suppressor mutation, which also rendered the virus temperature sensitive for growth, mapped to the DNA polymerase gene (E9L). Several other suppressors mapped to gene D5R, an NTPase required for DNA replication. These results unexpectedly suggest that the host range function of SPI-1 may be associated with viral DNA replication by an as yet unknown mechanism.     

Involvement of host DNA gyrase in growth of bacteriophage T5.

Bacteriophage T5 did not grow at the nonpermissive temperature of 42 degrees C in Escherichia coli carrying a temperature-sensitive mutation in gyrB [gyrB(Ts)], but it did grow in gyrA(Ts) mutants at 42 degrees C. These findings indicate that the A subunit of host DNA gyrase is unnecessary, whereas the B subunit is necessary for growth of T5. The necessity for the B subunit was confirmed by a strong inhibition of T5 growth by novobiocin and coumermycin A1, which interfere specifically with the function of the B subunit of host DNA gyrase. However, T5 growth was also strongly inhibited by nalidixic acid, which interferes specifically with the function of the A subunit. This inhibition was due to the interaction of nalidixic acid with the A subunit and not just to its binding to DNA, because appropriate mutations in the gyrA gene of the host conferred nalidixic acid resistance to the host and resistance to T5 growth in such a host. The inhibition by nalidixic acid was also not due to a cell poison formed between nalidixic acid and the A subunit (K. N. Kreuzer and N. R. Cozzarelli, J. Bacteriol. 140:424-435, 1979) because nalidixic acid inhibited growth of T5 in a gyrA(Ts) mutant (KNK453) at 42 degrees C. We suggest that T5 grows in KNK453 at 42 degrees C because its gyrA(Ts) mutation is leaky for T5. Inhibition of T5 growth due to inactivation of host DNA gyrase was caused mainly by inhibition of T5 DNA replication. In addition, however, late T5 genes were barely expressed when host DNA gyrase was inactivated.     

Alteration of the largest subunit of RNA polymerase II and its effect on chromosome stability in Schizosaccharomyces pombe

A mutation in the RNA polymerase II largest subunit (RpII LS) that is related to abnormal induction of sister chromatid exchange has previously been described the CHO-K1 cell mutant tsTM4. To elucidate the molecular basis of this effect we introduced the mutation into the homologous site in the Schizosaccharomyces pombe rpb1 gene, which encodes RpII LS. Since the tsTM4 mutant exhibited a decrease in the rate of DNA synthesis in cells arrested in S phase at the nonpermissive temperature, we focussed on the study of growth, the cell cycle, and chromosome stability at various temperatures. First, we examined the effects of the mutation on haploid yeast cells. The mutant showed slower growth than the wild type, but cell growth was not arrested at the nonpermissive temperature. When growing cells were shifted to the nonpermissive temperature, an accumulation of cells in G1 and/or G0 was observed. Tetrad analysis suggested that these phenotypes were associated with the mutation. In diploid cells, chromosome instability was detected by loss of intragenic complementation between two alleles of the ade6 gene. An abnormal fraction of cells containing an intermediate DNA content was also observed by FACS analysis. The accumulation of this fraction may reflect the fact that a large number of cells are in S phase or have an abnormal DNA content as a result of chromosome instability. These observations demonstrate that the S. pomberpb1 mutant exhibits a phenotype very similar to that of the CHO-K1 cell mutant tsTM4.     

Characterization of a nalidixic-acid-resistant mutant of Escherichia coli as a strict aerobe

An Escherichia coli mutant, C18, which plates at an efficiency of 5.0 x 10(-4) under anaerobic condition, was isolated among spontaneous nalidixic-acid-resistant mutants. This strict aerobic mutation was mapped by P1 cotransduction with a gyrA linked transposon Tn10 and found to be at the gyrA gene. A low degree of superhelicity of pBR322 DNA isolated from C18 was demonstrated by agarose gel electrophoresis with various concentrations of ethidium bromide. The superhelical density of pBR322 isolated from C18 was 80% of the value of pBR322 isolated from wild-type bacteria cultured under aerobic condition, and 50% cultured under anaerobic condition. These results lead us to conclude that a certain mutation of the gyrA gene causes a decrease in DNA superhelicity and prevents anaerobic growth.     

The First Successful Prenatal Diagnosis on a Korean Family with Citrullinemia

DNA prenatal diagnosis was successfully performed on a family with citrullinemia. The father carried the G324S mutation and the mother carried the IVS6-2A > G mutation in the argininosuccinate synthase gene. They had a previous child with citrullinemia who died in the week after birth owing to complicated hyperammonemia. The lost child turned out to be a compound heterozygote. DNA was extracted from the cultured amniotic cells after amniocentesis done at 18-week gestation. For the detection of the G324S mutation, the PCR and restriction fragment length polymorphism method was used, and for the IVS6-2A > G mutation, allele-specific PCR was performed. The fetus was found to carry G324S but not IVS6-2A > G, suggesting a heterozygote carrier. Pregnancy was continued and a healthy boy was born. Plasma amino acid analysis performed on the third day after birth was normal and the serial ammonia level was in the normal range. A molecular study on his genomic DNA after birth also agreed with the previous fetal DNA analysis. He is now 2-months old with normal growth and development.     

Mutant of simian virus 40 large T-antigen that is defective for viral DNA synthesis, but competent for transformation of cultured rat cells.

A mutant was isolated which demonstrates that the transforming activity of simian virus 40 large T-antigen is separable from its function in viral DNA replication. The mutant, SVR9D, is nonconditionally defective for viral DNA synthesis, but competent at wild-type level for morphological transformation of cultured rat cells. The lytic growth defect in SVR9D is complemented by the simian virus 40 A gene product present in the transformed CV1 cell line, COS1. The lesion in SVR9D DNA was mapped genetically by marker rescue of plaque formation and localized to a 214-base-pair segment of the viral genome bounded by nucleotide numbers 4100 and 4314. DNA sequence analysis showed the mutation to be an adenine-to-guanine transition at nucleotide number 4178. This change predicts a lysine-to-glutamic acid amino acid change at residue number 214 of the mutant large T-antigen polypeptide.     

The First Successful Prenatal Diagnosis on a Korean Family with Citrullinemia

DNA prenatal diagnosis was successfully performed on a family with citrullinemia. The father carried the G324S mutation and the mother carried the IVS6–2A > G mutation in the argininosuccinate synthase gene. They had a previous child with citrullinemia who died in the week after birth owing to complicated hyperammonemia. The lost child turned out to be a compound heterozygote. DNA was extracted from the cultured amniotic cells after amniocentesis done at 18-week gestation. For the detection of the G324S mutation, the PCR and restriction fragment length polymorphism method was used, and for the IVS6–2A > G mutation, allele-specific PCR was performed. The fetus was found to carry G324S but not IVS6–2A > G, suggesting a heterozygote carrier. Pregnancy was continued and a healthy boy was born. Plasma amino acid analysis performed on the third day after birth was normal and the serial ammonia level was in the normal range. A molecular study on his genomic DNA after birth also agreed with the previous fetal DNA analysis. He is now 2-months old with normal growth and development.     

Alteration of the largest subunit of RNA polymerase II and its effect on chromosome stability in Schizosaccharomyces pombe

A mutation in the RNA polymerase II largest subunit (RpII LS) that is related to abnormal induction of sister chromatid exchange has previously been described the CHO-K1 cell mutant tsTM4. To elucidate the molecular basis of this effect we introduced the mutation into the homologous site in the Schizosaccharomyces pombe rpb1 gene, which encodes RpII LS. Since the tsTM4 mutant exhibited a decrease in the rate of DNA synthesis in cells arrested in S phase at the nonpermissive temperature, we focussed on the study of growth, the cell cycle, and chromosome stability at various temperatures. First, we examined the effects of the mutation on haploid yeast cells. The mutant showed slower growth than the wild type, but cell growth was not arrested at the nonpermissive temperature. When growing cells were shifted to the nonpermissive temperature, an accumulation of cells in G1 and/or G0 was observed. Tetrad analysis suggested that these phenotypes were associated with the mutation. In diploid cells, chromosome instability was detected by loss of intragenic complementation between two alleles of the ade6 gene. An abnormal fraction of cells containing an intermediate DNA content was also observed by FACS analysis. The accumulation of this fraction may reflect the fact that a large number of cells are in S phase or have an abnormal DNA content as a result of chromosome instability. These observations demonstrate that the S. pomberpb1 mutant exhibits a phenotype very similar to that of the CHO-K1 cell mutant tsTM4. Received: 1 October 1997 / Accepted: 29 December 1997     

Mutations in the gene encoding the adenovirus early region 1B 19,000-molecular-weight tumor antigen cause the degradation of chromosomal DNA

The adenovirus mutant Ad2ts111 has been previously shown to contain a mutation in the early region 2A gene encoding the single-stranded-DNA-binding protein that results in thermolabile replication of virus DNA and a mutation in early region 1 that causes degradation of intracellular DNA. A recombinant virus, Ad2cyt106, has been constructed which contains the Ad2ts111 early region 1 mutation and the wild-type early region 2A gene from adenovirus 5. This virus, like its parent Ad2ts111, has two temperature-independent phenotypes; first, it has the ability to cause an enhanced and unusual cytopathic effect on the host cell (cytocidal [cyt] phenotype) and second, it induces degradation of cell DNA (DNA degradation [deg] phenotype). The mutation responsible for these phenotypes is a single point mutation in the gene encoding the adenovirus early region 1B (E1B) 19,000-molecular-weight (19K) tumor antigen. This mutation causes a change from a serine to an asparagine in the 20th amino acid from the amino terminus of the protein. Three other mutants that affect the E1B 19K protein function have been examined. The mutants Ad2lp5 and Ad5dl337 have both the cytocidal and DNA degradation phenotypes (cyt deg), whereas Ad2lp3 has only the cytocidal phenotype and does not induce degradation of cell DNA (cyt deg+). Thus, the DNA degradation is not caused by the altered cell morphology. Furthermore, the mutant Ad5dl337 does not make any detectable E1B 19K protein product, suggesting that the absence of E1B 19K protein function is responsible for the mutant phenotypes. A fully functional E1B 19K protein is not absolutely required for lytic growth of adenovirus 2 in HeLa cells, and its involvement in transformation of nonpermissive cells to morphological variants is discussed.     

Deletion of DNA encoding the first five transmembrane domains of Epstein-Barr virus latent membrane proteins 2A and 2B.

A recombinant Epstein-Barr virus (EBV) was constructed, with a positive-selection marker inserted at the site of a deletion of a DNA segment which encodes the first five transmembrane domains of LMP2A and LMP2B. Despite the mutation, the mutant recombinant EBV was able to initiate and maintain primary B-lymphocyte growth transformation in vitro. Cells transformed with the mutant recombinant were not different from wild-type virus transformants in initial or long-term outgrowth, sensitivity to limiting cell dilution, or serum requirement. Expression of EBNA1, EBNA2, EBNA3A, EBNA3C, and LMP1 and permissivity for lytic EBV infection were also unaffected by the LMP2 deletion mutation. These results complete the molecular genetic studies proving LMP2 is dispensable for primary B-lymphocyte growth transformation, latent infection, and lytic virus replication in vitro.     

Mutational analysis of the mitochondrial Rieske iron-sulfur protein of Saccharomyces cerevisiae. I. Construction of a RIP1 deletion strain and isolation of temperature-sensitive mutants

A protocol has been devised to permit mutational analysis of the Rieske iron-sulfur protein of the mitochondrial cytochrome bc1 complex of Saccharomyces cerevisiae. The gene for this iron-sulfur protein (RIP1) has recently been cloned and sequenced (Beckmann, J. D., Ljungdahl, P. O., Lopez, J. L., and Trumpower, B. L. (1987) J. Biol. Chem. 262, 8901-8909). We have constructed a stable yeast deletion strain, JPJ1, in which the chromosomal copy of RIP1 was displaced by the yeast LEU2 gene by homologous recombination. A linear DNA fragment containing the LEU2 gene was inserted at the breakpoints of an 800-base pair deletion of the iron-sulfur protein gene and used to transform a leu- yeast strain. Leu+ transformants were obtained which were unable to grow on nonfermentable carbon sources. Southern analysis of the transformant, JPJ1, confirmed that the chromosomal copy of the RIP1 gene was deleted and replaced by the LEU2 gene. The genotype of JPJ1 was confirmed by genetic crosses. JPJ1 cannot grow on nonfermentable carbon sources but can be complemented to respiratory competence and transformed by yeast vectors containing the wild type RIP1 gene. The ability to complement strain JPJ1 with episomally encoded iron-sulfur protein provided the basis of a selection protocol by which mutagenized plasmids containing the RIP1 gene were assayed for mutations affecting respiratory growth. Five mutants of RIP1 were identified by their ability to complement JPJ1 to temperature-sensitive respiratory growth. DNA sequence analysis demonstrated that temperature-sensitive respiratory growth resulted from single point mutations within the protein coding region of RIP1. These mutations altered a single amino acid residue in each case. Mutations were dispersed throughout the terminal two-thirds of the protein. Each mutation was recessive and did not affect fermentative growth on dextrose. However, each mutation exerted unique temperature-sensitive growth characteristics on media containing the nonfermentable carbon source glycerol.