Isolation and initial characterization of a Schizosaccharomyces pombe mutant exhibiting temperature-dependent radiation sensitivity due to a mutation in a previously unidentified rad locus

Summary We have isolated a mutant of the yeast Schizosaccharomyces pombe which exhibits sensitivity to UV light when grown at either 30° or 37°C, as compared to the parental wild-type strain. This increased sensitivity is more pronounced when cells are grown at 37°C. The mutant is also sensitive to 18 MeV electrons at the high temperature. Tetrad analysis of spores generated by crossing the mutant and a Rad⁺ strain revealed that sensitivity to both types of radiation cosegregate 2:2, relative to wild-type resistance, indicating that a single altered chromosomal locus is responsible for the radiation sensitivities observed. In addition, analysis of spores resulting from crosses between the mutant and all other known S. pombe rad mutants indicates that the temperature-dependent sensitivity described in this report is mediated by a mutation in a previously unidentified rad locus.     

Evidence that heat and ultraviolet radiation activate a common stress-response program in plants that is altered in the uvh6 mutant of Arabidopsis thaliana.

The uvh6 mutant of Arabidopsis was previously isolated in a screen for increased sensitivity to ultraviolet (UV) radiation. uvh6 mutant plants were killed by incubation at 37 degrees C for 4 d, a treatment not lethal to wild-type plants. Furthermore, under permissive conditions, uvh6 plants were yellow-green with an approximately one-third lower chlorophyll content. Genetic analysis of the uvh6 mutant strongly suggested that all three mutant phenotypes were due to mutation at the same genetic locus. To understand UVH6 function more fully, the response of wild-type plants to growth at elevated temperatures and exposure to UV radiation was analyzed. Wild-type plants grown at 30 degrees C were as UV-hypersensitive and yellow-green as uvh6 mutant plants grown at 24 degrees C. Mutant uvh6 plants induced heat-shock protein HSP21 at a lower threshold temperature than wild-type plants, indicating that the uvh6 mutant was exhibiting signs of heat stress at a 4 to 5 degrees C lower temperature than wild-type plants. We propose the UV damage and heat induce a common stress response in plants that leads to tissue death and reduced chloroplast function, and that the UVH6 product is a negative regulator of this response.     

Characterization of Null Mutants of the RAD55 Gene of Saccharomyces cerevisiae: Effects of Temperature, Osmotic Strength and Mating Type

RAD55 belongs to a group of genes required for resistance to ionizing radiation, RAD50-RAD57, which are thought to define a pathway of recombinational repair. Since all four alleles of RAD55 are temperature conditional (cold sensitive) for their radiation phenotype, we investigated the phenotype produced by null mutations in the RAD55 gene, constructed in vitro and transplaced to the yeast chromosome. The X-ray sensitivity of these null mutant strains was surprisingly suppressed by increased temperature, osmotic strength of the growth medium and heterozygosity at the mating-type locus. These first two properties, temperature conditionality and osmotic remediability, are commonly associated with missense mutations; these rad55 null mutants are unique in that they exhibit these properties although the mutant gene cannot be expressed. X-ray-induced mitotic recombination was also cold sensitive in rad55 mutant diploids. Although mitotic growth was unaffected in these strains, meiosis was a lethal event at both high and low temperatures. Whereas the phenotype of rad55 null mutants is consistent with a role of RAD55 in recombination and recombinational repair, there is evidence for considerable RAD55-independent recombination, at least in mitotic cells, which is influenced by temperature and MAT. We discuss models for the role of RAD55 in recombination to explain the unusual properties of rad55 mutants.     

Radiation sensitivity of a mutant of Escherichia coli K-12 associated with DNA replication: Evidence for a new repair function

Summary The isolation and properties of a new radiation sensitive mutant of Escherichia coli K-12 are described which shows a correlation between radiation sensitivity and replication of irradiated DNA. The mutation, called rer, is located between argB and purD loci. The mutant, when grown in tryptone broth after irradiation, is sensitive to UV and -rays and incorporates little or no ³H-thymidine but in minimal glucose-salts medium both the radiation sensitivity and incorporation of ³H-thymidine remain identical to that of the parent strain. Studies with a temperature sensitive double mutant rer dnaC show that 1 hr incubation of irradiated cells at 42° C before their transfer to 30° C results in higher survival as compared to their incubation at 30° C only. It is suggested that rer controls the replication of irradiated DNA and thus regulates the coordination between replication and repair of DNA.     

Cloning and characterization of the rad4 gene of Schizosaccharomyces pombe; a gene showing short regions of sequence similarity to the human XRCC1 gene.

The rad4.116 mutant of the fission yeast Schizosaccharomyces pombe is temperature-sensitive for growth, as well as being sensitive to the killing actions of both ultraviolet light and ionizing radiation. We have cloned the rad4 gene by complementation of the temperature sensitive phenotype of the rad4.116 mutant with a S. pombe gene bank. The rad4 gene fully complemented the UV sensitivity of the rad4.116 mutant. The gene is predicted to encode a protein of 579 amino acids with a basic tail, a possible zinc finger and a nuclear location signal. The amino terminal part of the predicted rad4 ORF contains two short regions of similarity to the C-terminal part of the human XRCC1 gene. Codon usage suggests that the gene is very poorly expressed, and this was confirmed by RNA studies. Gene disruption showed that the rad4 gene was essential for the mitotic growth of S. pombe.     

Repair of DNA single-strand breaks in radiation-sensitive mutants of mouse cells

The radiation-sensitive mutant M10 of mouse lymphoma L5178Y cells was examined for its ability to rejoin DNA single-strand breaks induced by gamma-rays. The alkaline sucrose gradient sedimentation analysis revealed that M10 cells repaired single-strand breaks but simultaneously produced increasing amounts of small DNA fragments with time of postirradiation incubation, something which was not observed in L5178Y cells. Since small fragments did not appear in M10 cells irradiated at room temperature, DNA fragmentation may result from cold treatment during irradiation followed by incubation at 37 degrees C. This indicates that the cold susceptibility is characteristic of M10 cells and is not related to radiation sensitivity of this mutant. This conclusion is supported by the finding that no DNA degradation takes place after cold treatment with a subsequent incubation in the other radiosensitive mutant LX830 that belongs to the same complementation group as M10.     

Free radical scavenging and the expression of potentially lethal damage in X-irradiated repair-deficient Escherichia coli

When cells are exposed to ionizing radiation, they suffer lethal damage (LD), potentially lethal damage (PLD), and sublethal damage (SLD). All three forms of damage may be caused by direct or indirect radiation action or by the interaction of indirect radiation products with direct DNA damage. In this report I examine the expression of LD and PLD caused by the indirect action of X rays in isogenic, repair-deficient Escherichia coli. The radiosensitivity of a recA mutant, deficient both in pre- and post replication recombination repair and SOS induction (inducible error-prone repair), was compared to that of a recB mutant which is recombination deficient but SOS proficient and to a previously studied DNA polymerase 1-deficient mutant (polA) which lacks the excision repair pathway. Indirect damage by water radicals (primarily OH radicals) was circumvented by the presence of 2 M glycerol during irradiation. Indirect X-ray damage by water radicals accounts for at least 85% of the PLD found in exposed repair-deficient cells. The DNA polymerase 1-deficient mutant is most sensitive to indirect damage with the order of sensitivity polA1 greater than recB greater than or equal to recA greater than wild type. For the direct effects of X rays the order of sensitivity is recA greater than recB greater than polA1 greater than wild type. The significance of the various repair pathways in mitigating PLD by direct and indirect damage is discussed.     

Evidence for a presynaptic blockage of transmission in a temperature-sensitive mutant of Drosophila

In the temperature sensitive mutant of Drosophila, shibirets1 (shi), synaptic transmission in the dorsal longitudinal flight muscles (DLM) is normal at 19 degrees C, but is diminished progressively as the temperature is raised, and is blocked at 29 degrees C. The purpose of this paper is to determine whether this defect is located presynaptically, postsynaptically, or both. It is demonstrated here that the postsynaptic sensitivity to L-glutamate, the putative transmitter for this synapse, is not decreased at 29 degrees C. Furthermore, studies conducted with genetic mosaics of this mutant show that transmission is blocked when a mutant motor neuron synapses on a wild-type muscle fiber, but is not blocked when a wild-type motor neuron synapses on a mutant muscle fiber. Thus, the shi phenotype (temperature dependent transmission block) correlates with a shi motor neuron, not with a shi muscle fiber. The data, therefore, suggest that the defect is not postsynaptic, but presynaptic.     

Involvement of a protein kinase activity inducer in DNA double strand break repair and radioresistance of Deinococcus radiodurans

Transgenic bacteria producing pyrroloquinoline quinone, a known cofactor for dehydrogenases and an inducer of a periplasmic protein kinase activity, show resistance to both oxidative stress and protection from nonoxidative effects of radiation and DNA-damaging agents. Deinococcus radiodurans R1 encodes an active pyrroloquinoline quinone synthase, and constitutive synthesis of pyrroloquinoline quinone occurred in wild-type bacteria. Disruption of a genomic copy of pqqE resulted in cells that lacked this cofactor. The mutant showed a nearly 3-log decrease in gamma radiation resistance and a 2-log decrease in mitomycin C tolerance compared to wild-type cells. The mutant cells did not show sensitivity to UVC radiation. Expression of pyrroloquinoline quinone synthase in trans showed that there was functional complementation of gamma resistance and mitomycin C tolerance in the pqqE mutant. The sensitivity to gamma radiation was due to impairment or slow kinetics of DNA double strand break repair. Low levels of (32)P incorporation were observed in total soluble proteins of mutant cells compared to the wild type. The results suggest that pyrroloquinoline quinone has a regulatory role as a cofactor for dehydrogenases and an inducer of selected protein kinase activity in radiation resistance and DNA strand break repair in a radioresistant bacterium.     

Yeast cys3 and gsh1 mutant cells display overlapping but non-identical symptoms of oxidative stress with regard to subcellular protein localization and CDP-DAG metabolism

In a screen for temperature-sensitive (37 degrees C) mutants of Saccharomyces cerevisiae that are defective in the proper localization of the Golgi transmembrane protein Emp47p, we uncovered a constitutive loss-of-function mutation in CYS3/STR1, the gene coding for cystathionine-gamma-lyase. We showed by immunofluorescence, sucrose-gradient analysis and quantitative Western analysis that the mutant mislocalized Emp47p to the vacuole at high temperature, while Golgi structures were apparently normal and biosynthetic routing of the vacuolar carboxypeptidase Y (CPY) and the plasma membrane GPI-anchored protein Gas1p were unaffected. The effect of high temperature on Emp47p localization, as well as the temperature sensitivity of the mutant strain on rich medium, appear to be caused by oxidative stress and are correlated with severe reductions in the intracellular levels of low-molecular-weight thiols. In accordance with this conclusion, cys3-2 mutant cells were more sensitive to the oxidizing agent 1-chloro-2,4-dinitrobenzene, which also aggravated the mislocalization of Emp47p observed at high temperature. Furthermore, all the phenotypes of the mutant were completely complemented by exogenous supply of the main low-molecular-weight thiol, glutathione (GSH) and, importantly, the thiol beta-mercaptoethanol reversed the temperature sensitivity of the mutant. A comparison of our mutant with a mutant defective in GSH synthesis showed that gsh1Delta cells were similar to wild-type cells under the stress conditions tested, with the exception of one novel oxidative stress-related phenotype that is observed in both cys3-2 and gsh1Delta mutant cells - a defect in CDP-DAG metabolism upon shift to the non-permissive temperature. As most of the stress-related phenotypes of cys3-2 mutant cells are more severe than those seen in gsh1Delta cells, we conclude that cysteine as such is required and sufficient to confer some degree of protection from oxidative stress in yeast cells.     

Crystal structure of the temperature-sensitive and allosteric-defective chaperonin GroELE461K

The chaperonin GroEL adopts a double-ring structure with various modes of allosteric communication. The simultaneous positive intra-ring and negative inter-ring co-operativities alternate the functionality of the folding cavities in both protein rings. Negative inter-ring co-operativity is maintained through different inter-ring interactions, including a salt bridge involving Glu 461. Replacement of this residue by Lys modifies the temperature sensitivity of the substrate-folding activity of this protein, most likely as a result of the loss of inter-ring co-operativity. The crystal structure of the mutant chaperonin GroELE461K has been determined at 3.3A and compared with other structures: the wild-type GroEL, an allosteric defective GroEL double mutant and the GroEL-GroES-(ADP)7 complex. The inter-ring region of the mutant exhibits the following characteristics: (i) no salt-bridge stabilizes the inter-ring interface; (ii) the mutated residue plays a central role in defining the relative ring rotation (of about 22 degrees) around the 7-fold axis; (iii) an increase in the inter-ring distance and solvent accessibility of the inter-ring interface; and (iv) a 2-fold reduction in the stabilization energy of the inter-ring interface, due to the modification of inter-ring interactions. These characteristics explain how the thermal sensitivity of the protein's fundamental properties permits GroEL to distinguish physiological (37 degrees C) from stress (42 degrees C) temperatures.     

A bentazon and sulfonylurea sensitive mutant: breeding,genetics and potential application in seed production of hybrid rice

The use of a thermosensitive genic male sterility (TGMS) system in two-line hybrid rice breeding is affected greatly by the sterility instability of TGMS lines caused by temperature fluctuation beyond their critical temperatures for fertility reversion. To prevent seed production from self contamination, we have developed a system to secure seed purity using a herbicide-sensitive TGMS mutant, M8077S, obtained by radiation. Genetic analysis, using the F₁, F₂ and F₃ populations derived from this mutant and other normal varieties, revealed that bentazon lethality/sensitivity was controlled by a single recessive gene, which was named bel. The mutant can be killed at the seedling stage by bentazon at 300 mg/l or higher, a dosage that is safe for its F₁ hybrids and all other normal varieties. This mutant is also sensitive to all the tested sulfonylurea herbicides. Response of segregating plants to these two types of herbicide indicated that sulfonylurea sensitivity was also controlled by bel. By crossing this mutant with Pei-Ai 64S, an F₂ population was developed for genetic mapping. Surveying the two DNA pools from sensitive and non-sensitive F₂ plants identified four markers that were polymorphic between the pools. The putative linked markers were then confirmed with the F₂ population. The bel locus was located on chromosome 3, 7.1 cM from the closest microsatellite marker RM168. Phenotypic analysis indicated that the bel gene had no negative effect on agronomic traits in either a homozygous or heterozygous status. The mutant M8077S is valuable in the development of a TGMS breeding system for preventing impurity resulting from temperature fluctuation of the TGMS. Several two-line hybrid rice crosses using this system are under development.     

Effects of peroxide and catalase on near ultraviolet radiation sensitivity in Escherichia coli strains

The role of peroxide and catalase on NUV radiation sensitivity was examined in two repair competent E. coli strains, AB1157 and B/r. Exponential phase B/r is considerably more sensitive to NUV radiation than exponential phase AB1157. However, resistance to 5 mmol dm-3 H2O2 was induced in both AB1157 and B/r by pretreating growing cells with 30 mumol dm-3 H2O2. Pretreatment also induced resistance to broad-band NUV radiation in these strains. The addition of catalase to the post-irradiation plating medium increased survival to the same extent as that provided by pretreatment with 30 mumol dm-3 H2O2, in both strains. The NUV radiation sensitivity seen in B/r does not appear to be due to a deficiency in enzymes that scavenge H2O2, as a catalase deficient mutant, E. coli UM1, is more resistant to NUV radiation than B/r. Also, assays for H2O2 scavenging ability show little difference between AB1157 and B/r in this respect. Two hypotheses are put forward to account for the sensitivity of exponential phase B/r. Whilst it is apparent that peroxides and catalase do have a role in NUV radiation damage, it is clear that other factors also influence survival under certain conditions.     

Pleiotropic effects of ribosomal protein s4 studied in Escherichia coli mutants

Summary A temperature sensitive mutant of Escherichia coli was found to have two mutational alterations of its ribosomes: one of these was a streptomycin dependent mutation and the other was a suppressor alteration of S4, with a marked structural change. The altered form of S4 was studied in a strain that was constructed so that this alteration was the only one effecting the structure of the ribosome. Here, it was shown that the mutant form of S4 cause a temperature sensitive defect in the assembly of 30S subunits in vivo which is reflected in the inability of this mutant to properly process ribosomal RNA at the restrictive temperatures. An analysis of both transductants and revertants of this mutant show that the suppression of the streptomycin dependence phenotype, temperature sensitivity, and a defect in RNA processing all have their origin in a single mutational event effecting the structural gene for S4.     

On the molecular basis of the thermal sensitivity of an Escherichia coli topA mutant.

Studies of two temperature-sensitive Escherichia coli topA strains AS17 and BR83, both of which were supposed to carry a topA amber mutation and a temperature-sensitive supD43,74 amber-suppressor, led to conflicting results regarding the essentiality of DNA topoisomerase I in cells grown in media of low osmolarity. We have therefore reexamined the molecular basis of the temperature sensitivity of strain AS17. We find that the supD allele in this strain had lost its temperature sensitivity. The temperature sensitivity of the strain, in media of all osmolarity, results from the synthesis of a mutant DNA topoisomerase I that is itself temperature-sensitive. Nucleotide sequencing of the AS17 topA allele and studies of its expected cellular product show that the mutant enzyme is not as active as its wild-type parent even at 30 degrees C, a permissive temperature for the strain, and its activity relative to the wild-type enzyme is further reduced at 42 degrees C, a nonpermissive temperature. Our results thus implicate an indispensable role of DNA topoisomerase I in E. coli cells grown in media of any osmolarity.     

Mapping of a new genetic locus responsible for ampicillin resistance in Escherichia coli.

The ampicillin resistance locus of three different ampicillin-resistant, temperature-sensitive Escherichia coli mutants was mapped between proC and purE and does not correspond to any of the known genes in this region. The mutant gene responsible for the temperature sensitivity and consequent morphological changes in each mutant strain was not located in the same 5-min region, even though the two mutants characteristics co-reverted at a very high frequency.     

Lipid composition, lipid fluidity and radioresistance of Deinococcus radiodurans and two mutant strains

The lipid composition of D. radiodurans strain R1 and of two mutant strains has been studied in relation to membrane fluidity and sensitivity to X-ray radiation. No significant difference in the unsaturation degree of fatty acids was found between parental and mutant strains. An important decrease of carbohydrate-containing lipids was observed in the radiosensitive mutant strain. We also observed a higher fluidity in both mutant strains than in the parental one. Modification of membrane lipid fluidity by growing the parental strain at 39 degrees C did not lead to modified radioresistance. These results suggest that a particular chemical composition of the membrane leading to a special lipid phase may be an important parameter in controlling radiosensitivity.     

Effect of elevated temperatures on the radiation sensitivity of yeast cells of different species

Summary The influence of hyperthermia on the survival of irradiated yeast cells of different species has been studied. The experiments reported in the paper have shown: (1) simultaneous action of ionizing radiation and high temperatures appeared to increase the radiation response by a factor of approximately 2.7 for diploid and only by a factor of 1.5 for haploid cells of wild-type; (2) the combined action of high temperature and ionizing radiation had no synergistic effect for rad51 mutant diploid yeast cells; (3) heating before or after irradiation did not alter the radiation response of yeast cells; (4) enhancement of yeast cell sensitivity by simultaneous action of hyperthermia and²³⁹Pu--particles was negligible; (5) the magnitude and the rate of liquid holding recovery is lowered with increasing of irradiation temperature. On this basis, it was concluded that possible mechanism for thermal sensitization of yeast cells may involve the reduced capacity of cells to recover damages resulted from the combined action of both modalities.     

A nuclear mutation defective in mitochondrial recombination in yeast.

Homologous recombination (crossing over and gene conversion) is generally essential for heritage and DNA repair, and occasionally causes DNA aberrations, in nuclei of eukaryotes. However, little is known about the roles of homologous recombination in the inheritance and stability of mitochondrial DNA which is continuously damaged by reactive oxygen species, by-products of respiration. Here, we report the first example of a nuclear recessive mutation which suggests an essential role for homologous recombination in the stable inheritance of mitochondrial DNA. For the detection of this class of mutants, we devised a novel procedure, 'mitochondrial crossing in haploid', which has enabled us to examine many mutant clones. Using this procedure, we examined mutants of Saccharomyces cerevisiae that showed an elevated UV induction of respiration-deficient mutations. We obtained a mutant that was defective in both the omega-intron homing and Endo.SceI-induced homologous gene conversion. We found that the mutant cells are temperature sensitive in the maintenance of mitochondrial DNA. A tetrad analysis indicated that elevated UV induction of respiration-deficient mutations, recombination deficiency and temperature sensitivity are all caused by a single nuclear mutation (mhr1) on chromosome XII. The pleiotropic characteristics of the mutant suggest an essential role for the MHR1 gene in DNA repair, recombination and the maintenance of DNA in mitochondria.