A new bacterial gene (groPC) which affects lambda DNA replication.

Summary A bacterial mutation affecting DNA replication, called groPC756, has been mapped between the thr and leu bacterial loci. Most of the parental DNA does not undergo even one round of replication in this host. Lambda mutants, called , which map in the P gene are able to overcome the inhibitory effect of the groPC756 mutation. It is shown that the mutation at the groPC locus also interferes with bacterial growth at 42°C. A -transducing phage, carrying the groPC⁺ allele, was isolated as a plaqueformer on groPC756 bacteria. Upon lysogenization, it restores both the gro ⁺ and temperature resistant phenotypes.     

Cloning and expression of the leucine gene from Thermus thermophilus in Escherichia coli

A pBR322-T.leu hybrid plasmid was constructed which contains a 3.75 Md HindIII-fragment derived from Thermus thermophilus HB27 chromosomal DNA. In the Escherichia coli host, this plasmid coded for the β-IPM dehy drogenase (product of leuB) activity, the optimal temperature of which was 80°C, suggesting that information on the thermostability of the enzyme lies in its structural gene. 10-day propagation of E. coli [pBR322-T.leu] at 37°C decreased the temperature optimum from 80°C to 75°C. This change, which was found to depend on the plasmid but not on the host cells, might be due to selection of some mutation at the non-restrictive temperature of 37°C. Our results suggest that the 3.75 Md HindIII-fragment of pBR322-T.leu carries a promoter of the thermophile, which could function in E. coli.     

The RAD3 gene of Saccharomyces cerevisiae: isolation and characterization of a temperature-sensitive mutant in the essential function and of extragenic suppressors of this mutant

Summary Mutations in the RAD3 gene of Saccharomyces cerevisiae were generated by integration of a mutagenized incomplete copy of the cloned gene into wild-type cells. Integrants were mass screened for colonies with abnormal growth characteristics at 37°C. A single temperature-sensitive mutant (rad3ts-1) was isolated and was shown to result from a missense mutation at codon 73 of the RAD3 gene. When shifted from 30° C to 37° C the strain undergoes only 2–4 cell doublings. This phenotype can be rescued by plasmids in which the essential function of the cloned RAD3 gene is intact, but not plasmids in which this function is inactivated. The mutant strain is weakly sensitive to ultraviolet (UV) radiation at restrictive temperatures. Measurement of RNA, DNA and protein synthesis at various times after shifting to restrictive temperatures does not show preferential inactivation of any one of these parameters and the temperature-sensitive mutation does not cause arrest at any specific phase of the cell cycle. The rad3ts-1 strain was transformed with multicopy plasmids from a normal yeast genomic library and two plasmids that partially suppress the temperature-sensitive phenotype were isolated. These suppressor genes (designated SRE1 and SRE2) are distinct from RAD3 and do not suppress the phenotype of several other temperature-sensitive mutants tested. Mutant strains carrying disruptions of the SRE1 gene are viable and are not sensitive to UV or radiation.     

Physiological and genetical studies on a mutant of Salmonella typhimurium which is temperature-sensitive for DNA synthesis

Summary The following evidence supports the view that a temperature-sensitive mutant of Salmonella typhimurium (11 G) is defective in DNA synthesis initiation: a) the increment in DNA synthesis at 38° is abolished by prior completion of rounds of replication at 25°. b) The extent of the increment at 38° is greatly increased by prior growth in the presence of a DNA synthesis inhibitor. c) Density gradient centrifugation demonstrates that the terminal region of the chromosomes is preferentially replicated at 38°. d) Preferential replication of the chromosome origins occurs at 25° after a period at 38°. The parental strain in the presence of a DNA synthesis inhibitor or the mutant at 38° (without inhibitor) show increased sensitivity to the detergent sodium deoxycholate, possibly due to a secondary effect of DNA synthesis inhibition on membrane composition. Strains of 11 G carrying episomes transfer the episomes very poorly at 38° suggesting a rôle for the chromosomal initiation apparatus in episome transfer. Continued cell division of 11 G with the production of DNA-less cells at 38° is not due to the presence of a rec mutation and no secondary mutation responsible for such division has been found. The lesion maps close to leu on the Salmonella chromosome.     

Growth and macromolecular synthesis phenotypes of a heat-sensitive mutant strain, rip-1, of Neurospora crassa

Summary A heat-sensitive mutant of Neurospora crassa, strain 4M(t), was isolated using ultraviolet-light mutagenesis followed by the inositol-less death enrichment technique. The heat-sensitivity is the result of a single gene mutation which maps to the distal end of the right arm of linkage group II. The mutation defines the rip-1 gene locus. Both conidial germination and mycelial extension are inhibited in the mutant at 35°C and above (the nonpermissive temperature) but prolonged incubation at that temperature is not lethal to either cell type. Analysis of the lateral mycelial growth rates of wild type and of the rip-1 mutant at a variety of temperatures between 10 and 40°C indicated that the maximal growth rate occurs at 35°C in the wild type, and at 25°C in the rip-1 strain. The rip-1 mutant grows 239-times slower at 35°C than at 25°C, whereas the wild type grows 1.4-times faster. Temperature shift-up experiments showed that even 3 h at 20°C is not sufficient to allow germination at 37°C, thereby showing that the mutant cannot accumulate enough heat-sensitive product at the permissive temperature to contribute to germination at 37°C. The reciprocal temperature shift-down experiments showed that the molecular events at 37°C may be qualitatively useful for germination after shifting to 20°C. Studies of macromolecular synthesis showed that the biochemical defect in the heat-sensitive strain appears to affect RNA synthesis before protein synthesis, although there were differences in the relative effects depending on the age of the germinating conidia and the inhibition of the two processes was never complete. Messenger RNA synthesis is normal in the mutant at 37°C. Previous work has shown that the rip-1 mutant strain has a conditional defect in the accumulation of 25S rRNA and, hence, in 60S ribosomal subunit production (Loo et al. 1981). There are also indications from those studies that the 60S ribosomal subunit may be functionally impaired at the higher temperature. Thus, the growth and macromolecular synthesis phenotypes may result as a consequence of a conditional, ribosome function defect and leads to the hypothesis that the mutation in the rip-1 strain may be in a gene for a 60S ribosomal subunit component, perhaps a ribosomal protein.     

Escherichia coli mutants with temperature-sensitive synthesis of DNA

Summary Seven mutants of E. coli with temperature-sensitive synthesis of DNA have been isolated. Synthesis of RNA, protein and DNA precursors does not appear to be directly affected. The mutants can be divided into at least two groups on the basis of their pattern of DNA synthesis, their ability to support phage growth at 41° and their genetic mapping.Mutants of the first group are heterogeneous in their pattern of DNA synthesis at 40°. Some mutants cease DNA synthesis abruptly upon transfer to 40° and any residual DNA synthesis is barely detectable. In others there is substantial residual synthesis at 40°. All these Group 1 mutants are alike, however, in that they support the growth of phage T4 but not Lambda at 41°. Two mutants with barely detectable residual DNA synthesis carry DNA mutations which have been mapped by P1 transduction and show about 72% linkage to the malB locus. It has not yet proved possible to map accurately the mutants showing substantial residual synthesis, and the possibility that these mutations are in a different gene(s) has not been excluded.A single mutant has been placed in a second group. Like some Group 1 mutants it synthesizes substantial amounts of DNA at 40° before synthesis stops. However, unlike them it supports the growth of T4 and Lambda at 41°. The DNA mutation maps near the leu locus. Certain properties of this mutant are consistent with the idea that initiation of DNA synthesis is temperature-sensitive in this strain.Adapted from a dissertation presented in partial fulfillment of the degree of Doctor of Philosophy. This investigation was supported in part by U.S. Public Health Services Grant 5-TO1-GM00829 from the National Institute of General Medical Sciences and in part by U.S.P.H.S. research grant GM12524.     

A missense mutation in the gene coding for ribosomal protein S17 (rpsQ) leading to ribosomal assembly defectivity in Escherichia coli.

Summary The conditionally lethal mutation, 286lmis, has been mapped inside the ribosomal protein gene cluster at 72 minutes on the Escherichia coli chromosome and was found to cotransduce at 97% with rpsE (S5). The 2861mis mutation leads to thermosensitivity and impaired assembly in vivo of 30S ribosomal particles at 42°C. The strain carrying the mutation has an altered S17 ribosomal protein; the mutational alteration involves a replacement of serine by phenylalanine in protein S17. Spontaneous reversion to temperature independence can restore the normal assembly in vivo of 30S ribosomal subunits at 42°C and the normal chromatographical sehaviour of the S17 ribosomal protein in vitro. We conclude therefore that the 2861mis mutation affects the structural gene for protein S17 (rpsQ).     

Mimicry of isozyme adaptive advantage by gene association II. Adh genotype frequency variations in Drosophila cage populations reared at different temperatures

The adaptive response of the Adh locus to different temperatures was determined in an experimental population with a stabilized gene pool in order to confirm the conclusions reached in a previous paper (Pieragostini et al., Genetica 56: 27–37, 1981) using as experimental material a laboratory population in which heterosis and strong interaction between loci were present.By sampling a stable lab population, three cage populations were constructed, heterozygous at the Adh locus and genetically identical but reared at different temperatures (P 18 °C, P 25 °C and P 28 °C). On these populations we studied the dynamics of Adh allele frequencies and of five metric traits up to the fifth generation of caging. The most interesting result is represented by the differences between P 28 °C and the other two populations (P 25 °C and P 18 °C). In particular P 25 °C and P 18 °C show a strong similarity with the original population both in Adh allele frequency and relationship between Adh genotype and the respective metric phenotype; this is not the case for P28 °C which differed from the original population and the other two newly founded ones not only in Adh allele frequencies but even in gene arrangements.Comparing these results with those obtained in the previous work it can be concluded that Adh frequency variations can be a side effect of the evolutionary pattern of the populations which in turn depends on the initial genetic composition.     

A new conditional lethal mutator (dnaQ49) in Escherichia coli K12.

Summary A conditional lethal mutator, dnaQ49, was found in Escherichia coli K12. The dnaQ49 mutation caused stimulation of rifampicin-, nalidixic acid- and streptomycin-resistant mutation frequencies 100 to 2000 fold at 30°C and the frequencies were further increased 50 to 100 fold at 35°C or higher temperatures. Cells carrying dnaQ49 were unable to grown in salt-free L-broth at 44.5°C, and DNA synthesis but not protein synthesis of the cells was suppressed under the restrictive conditions. The dnaQ gene was located at about 5 min on the E. coli linkage map and the order of the genes residing in this region was determined to be on A-dnaE-metD-dnaQ-proA.     

Effects on growth and sporulation of inactivation of a Bacillus subtilis gene (ctc) transcribed in vitro by minor vegetative cell RNA polymerases (E-sigma 37, E-sigma 32)

Summary The E-³⁷ gene ctc was inactivated by a site-specific insertion into the Bacillus subtilis chromosome. The resulting mutation inhibited sporulation by 95% at elevated temperatures (48° C). If the ctc ^- mutation is placed in a strain that carries a mutation in the closely linked but distinct spoVC gene, ctc now affects both growth and sporulation at elevated temperatures. Growth of the ctc ^- spoVC285 strain was transiently inhibited when exponentially growing cultures were shifted from 37° C to 48° C. A similar, but less pronounced growth lag, was also seen in a B. subtilis strain carrying only the spoVC-285 mutation. This finding suggests that both the ctc and spoVC products function in vegetatively growing B. subtilis.     

The temperature-sensitive mutation vir ts(virilizer) identifies a new gene involved in sex determination of Drosophila

Summary When XX animals homozygous for the temperature-sensitive mutation vir ^tsof virilizer (2–103.9) are raised at the restrictive temperature of 29° C, they are transformed into sterile intersexes with a morphology comparable to XX flies mutant at the sex-determining gene doublesex (dsx). The gonads of the vir ^tsintersexes are ovaries in which the germ cells undergo abortive oogenesis. At the permissive temperature of 25° C or below, XX vir ^tsanimals develop into marginally fertile females. The temperature-sensitive period of vir ^tsis within the third larval instar. XY males are not affected by the mutation. Animals that are homozygous for vir ^tsand either transformer (tra) or tra2 develop as pseudomales; on the other hand, constitutive expression of a female-specific tra product rescues XX animals from the effect of vir ^ts, but these females are sterile. The data show that tra and tra2 are epistatic to vir. Animals with only one wildtype copy of either tra or tra2 and mutant for vir ^tsare already transformed into intersexes at 25° C. Conversely, the presence of three copies of the tra ⁺ gene largely prevents the effect of vir ^tsat 29° C; such flies are practically female, but sterile. Animals homozygous for vir ^tsand heterozygous for dsx ^D/+, raised at 29° C, are transformed into severely masculinized intersexes or almost pseudomales. The observations suggest that vir acts above and via tra and tra2 to achieve proper female-specific expression of the dsx gene in XX zygotes. Offprint requests to: R. Nöthiger     

Cloning and sequencing of the leu C and npr M genes and a putative spo IV gene from Bacillus megaterium DSM319

The leuC gene, encoding 3-isopropylmalate dehydrogenase, the nprM gene (neutral protease) and a sporulation gene coding for a putative spoIV protein (spoIV) from Bacillus megaterium DSM319 were cloned and the nucleotide sequences were determined. The leuC gene is 1101 bp in length, preceded by a ribosome binding site; no promoter consensus sequence could be found. The nucleotide sequence from nprM when compared to the recently published gene from B. megaterium ATCC 14581 exhibited only a 17-base pair deviation. From a sporulation mutant isolated after transposonmutagenesis with transposon Tn917 the insertion site of the transposon was cloned and adjacent chromosomal fragments were characterized. An open reading frame that encodes for a putative spo protein of 247 amino-acid residues was identified.Sequence data presented in this contribution are part of doctoral theses of the Naturwissenschaftliche Fakultät Münster, Germany nprM (KDW); leuC and spoIV (MB)     

Mutations affecting growth of the Escherichia coli cell under a condition of DNA polymerase I-deficiency

Summary We constructed a strain of E. coli K12 carrying polA1 (an amber mutation of the DNA polymerase I gene; De Lucia and Cairns, 1969), and sup-126 (a temperature-sensitive amber suppressor; Nagata and Horiuchi, 1973). DNA polymerizing activity of the enzyme in this strain is virtually undetectable if the cells are grown at 42°C, but if grown at 30°C it is sufficiently present. By mutagenizing this strain, and after appropriate screening, we obtained mutants no longer able to grow at 42°, but able to do so when the normally functioning polA gene is present. One of them, called TS41, was most extensively studied. It acquired a mutation named pdeB41 which was found to be located between ilv and metE on the E. coli linkage map. Its phenotype is pleiotropic. The mutation by itself, i.e., if present in a polA ⁺ cell, does not kill the cell at 42°, but does so as in TS41 when it is reconstructed into a pdeB41 polA1 sup-126 triple mutant by P1 transduction. The mutation by itself renders the cell sensitive to UV, and tolerant to phage deficient in recombination. It is also a mutator.     

Conditionallethality of Escherichia coli strains carrying dnaE anddnaQ mutations

Summary A double mutant of Escherichia coli K12 which carries a conditional lethal mutator mutation, dnaQ49 (Horiuchi et al. 1978), and a DNA polymerase III-deficient mutation, dnaE486 (Wechsler and Gross 1971), was found to be more thermolabile than was either of the dnaQ49 or dnaE486 single mutants. The double mutant is able to grow at28° C but not at 30° C. Under the restrictive conditions DNA synthesis, but not protein synthesis, of the double mutant was suppressed. All the other combinations of dnaQ and dnaE mutation alleles tested so far rendered the cells thermolabile. a dnaZ mutation exerted a similar effect on the dnaQ strain. However, when non-specific temperaturesensitive graowth mutations were conbined with the dnaQ49 mutation, no such increase in thermosensitivity was observed. There is a possibility that the product of the dnaQ gene interacts directly with the DNA replicating enzyme complex.     

Involvement of DnaK protein in mini-F plasmid replication: temperature-sensitive seg mutations are located in the dnaK gene

Summary The seg mutants (seg-1 and seg-2) of Escherichia coli cannot support the replication of the F factor and mini-F plasmids at 42°C. We cloned the wild-type E. coli chromosomal DNA fragment complementing the seg-1 and seg-2 mutations and found that both mutations were complemented by the wild-type dnaK gene coding for a heat shock protein. Transduction with phage P1 indicated that the seg-2 mutation is located at about 0.3 min in the region containing the dnaK gene in the order trpR-thrA-seg-2-leuB, consistent with the locus of the dnaK gene. Cloning and sequencing of the dnaK gene of the seg mutants showed that there was one base substitution within the dnaK gene in each mutant causing an amino acid substitution. These results indicate that the seg gene in which the seg-1 and seg-2 mutations occurred is identical to the dnaK gene. The mini-F plasmid pXX325 did not transform a dnaK null mutant to ampicillin resistance at 30°C in contrast to plasmids pBR322, pACYC184 and pSC101, which did. The active dnaK (seg) gene product is therefore essential for replication of the mini-F plasmid at both 30° and 42°C.     

Control of stable RNA synthesis in a temperature-sensitive mutant of elongation factor G of Bacillus subtilis

Summary A temperature-sensitive EFG mutant of Bacillus subtilis was isolated and characterized. This mutant, ts32, synthesizes stable RNA at 48° C with or at 50° C without accompanied protein synthesis. The initial rate of the RNA synthesis at 48° C or 50° C was 1.5 to 2.0 times as much as that at 30° C.This mutant as well as its parent (both leu ^-) showed stringent response for the RNA synthesis upon deprivation of amino acids with an accumulation of the MS nucleotides (pp Gpp and pppGpp). On raising temperature to 48° C or 50° C, the ts-cells immediately began to synthesize the stable RNA with an initial increase of the MS nucleotides. No drastic decrease in amount of the MS was observed during the active RNA synthesis.These results suggest that EFG is somehow involved in repressing the stable RNA synthesis, and have broken the close relationship between the stable RNA synthesis and the MS nucleotides hitherto reported.     

Induction and repair of gene conversion in UV-sensitive mutants of yeast

Summary Photoreactivation effect on UV-induced allelic recombination has been examined using various combinations of leu 1 alleles in UV-sensitive and wild type diploid yeast, Saccharomyces cerevisiae. The frequencies of UV-induced heteroallelic reversion in UV-sensitive strains, presumably lacking dark-repair, are strikingly enhanced compared to those in wild type at the same doses under dark condition. However, these enhanced frequencies of reversion are diminished by photoreactivation almost to the level of those in wild type. The induced frequencies of homoallelic reversion (mutation) of relevant alleles are apparently lower than those of heteroallelic reversion. Phenotypic analysis for linked gene leu 1 on UV-induced heteroallelic revertants has shown that most of the revertants are of the nonreciprocal type recombination (mitotic gene conversion). These results would indicate that most of the dark-repairable damage leading to mitotic gene conversion after UV-light is due to pyrimidine dimers.On leave of absence from Radiation Center of Osaka Prefecture, Shinke-cho Sakai, Osaka, Japan.     

Regulatory mechanism of the tryptophan operon in Escherichia coli: possible interaction between trpR and trpS gene products

Summary The trpS5 mutation (a mutation in the structural gene for tryptophanyl-tRNA synthetase (TRSase) in E. coli), when present in the genetic background of strain KY913 (HfrH), results in the failure to grow at high temperature (42° C) in a complete medium. The rel (RC relaxed) marker present in this strain was found to be partly responsible for this temperature sensitivity. TRSase in such a strain was rapidly inactivated during growth at 42° C in rich media, but not in minimal media or in the presence of chloramphenicol. A partial derepression of anthranilate synthetase formation took place in the presence of excess tryptophan at growth-restricting temperatures. When some of the trpR mutations (including amber mutations) were combined with trpS5, the resulting double mutants (trpR trpS5) were temperature-insensitive, and TRSase was not inactivated at high temperature, in contrast to the trpR ⁺trpS5 strain. This effect of trpR mutations on temperature sensivity was shown not to be a secondary consequence of the constitutive expression of the trp operon. These findings suggest that the trpR ⁺ product interacts with the TRSase of the trpS5 mutant so as to bring about the growth-dependent inactivation of the enzyme. Furthermore, a special class of trpR mutants was obtained whose constitutivity with respect to the trp operon is manifested only in strains carrying trpS5 (but not trpS ⁺) grown at high temperatures. It is proposed that TRSase participates in repression trrough direct interaction with the product of the trpR gene.     

Three-factor reciprocal cross mapping of a gene that causes expression of feedback-resistant acetohydroxy acid synthase in Escherichia coli K-12

Summary The ilv-662 allele was previously identified as a mutation that caused acetohydroxy acid synthase activity to be resistant to feedback inhibition by valine (Davis et al. 1977). This allele was mapped between thr and leu by cotransduction analysis and labeled ilvJ. This report describes the mapping of ilvJ relative to genes that lie between thr and leu (ara, carA and pdxA) by three factor reciprocal cross analyses. We find that the probable gene order is thr-carA-pdxA-ilvJ-ara-leu. Although the phenotypic properties of ilvJ662 appear to be quite distinct from brnS, a gene reported to involve branched chain amino acid transport (Guardiola et al. 1974), we do not rule out possible allelism because of the uncertainty of the map position of brnS.