Temperature-sensitive plant mutants isolated in vitro

Summary Conditional-lethal, temperature-sensitive plant mutants have been isolated using a simple protoplast cloning method. The leaf protoplasts used were obtained from sterile, haploid shoot cultures of Nicotiana plumbaginifolia. Recessive mutations are described at three loci: ts1, ts2 and ts3. The mutations are lethal when either tissue cultures or plants are incubated at 33°C but not at 26°C.     

Changes in regulation of ribosome synthesis during different stages of the life cycle of Saccharomyces cerevisiae.

Summary Diploid strains of Saccharomyces cerevisiae, each homozygous for one of the temperature sensitive mutations rna2, rna4, rna6 or rna8, are temperature sensitive for ribosome synthesis during vegetative growth, but are not inhibited for ribosomal synthesis at the restrictive temperature under sporulation conditions. The continued ribosome biosynthesis at the restrictive temperature (34° C) during sporulation includes de novo synthesis of both ribosomal RNA and ribosomal proteins. This lack of inhibition of ribosome biosynthesis is found even when cells committed to complete sporulation are returned to vegetative growth medium. The ribosomes synthesized at 34° C are apparently functional, as they are found in polyribosomes. Although the rna mutants do not regulate ribosome synthesis during sporulation, all of these diploid strains fail to complete sporulation at 34° C. The cells are arrested after the second meiotic nuclear division but before ascus formation. The failure to complete sporulation at the restrictive temperature and the inhibition of ribosome biosynthesis during growth are caused by the same mutation, because revertants selected for temperature independent growth were also able to sporulate at 34° C.     

Identification of ten genes that control ribosome formation in yeast

Summary Twenty-three temperature-sensitive mutants of Saccharomyces cerevisiae, all of which undergo a rapid cessation of net RNA accumulation following a shift from the permissive (23°) to the restrictive temperature (36°), have been characterized. Genetic studies demonstrate that these mutants belong to ten different complementation groups and that, in most cases, their properties are the result of a single, recessive mutation in a nuclear gene. Although the mutants were isolated for heat sensitivity, mutants from 2 of the complementation groups are cold sensitive (at 13°) as well. The mutants continue to synthesize protein, including an enzyme, alkaline phosphatase, for two to four hours following a shift from 23° to 36°, suggesting that they are capable of messenger RNA synthesis and the translation of messenger RNA with fidelity at the restrictive temperature. The small amount of RNA that is synthesized in these mutants at the restrictive temperature has been examined on sucrose gradients and by acrylmide gel electrophoresis; in addition, the RNA components in polyribosomes have been fractionated by a new technique that separates messenger RNA from ribosomal RNA. As a result of these analyses we conclude that these mutants are strongly inhibited in the accumulation of 5S, 7S, 17S, and 25S RNA components but are only slight if at all inhibited in the synthesis of messenger RNA and 4S RNA. The results reported here define ten genes, designated rna 2 through rna 11, that play an essential role in the formation or maturation of ribosomes in yeast.     

Mutants of Agrobacterium tumefaciens with temperature sensitivity in respect to their tumor inducing ability

Summary The isolation of six mutants of Agrobacterium tumefaciens which can induce tumors at low temperatures (22°C) but fail to do so at 28°C is described. At the nonpermissive temperature the following characteristics of the mutants are the same as those of the wild type: growth rates in vitro, growth rates in planta, and sensitivity towards agrocin 84, a marker for the presence of the virulence-plasmid. The tumors induced by the mutants at low temperature grow without addition of hormones at both 22°C and 28°C. The induction of the tumors but not the maintenance of the tumorous phenotypes are affected in the mutants isolated.     

Cold adaptation of the terrestrial isopod,Porcellio scaber,to subnivean environments

The terrestrial isopod, Porcellio scaber, was susceptible to subzero temperature: both freezing and chilling were injurious. The level of cold hardiness against chilling and freezing showed different patterns in their seasonal variation. The lower lethal temperature causing 50% mortality, an indicator of the tolerance to chilling, ranged from-1.37°C in August to-4.58°C in December. The whole body supercooling point, the absolute limit of freeze avoidance, was kept at about-7°C throughout the year. The winter decrease in lower lethal temperature was concomitant with an accumulation of low molecular weight carbohydrates which are possible protective reagents against chilling injury, whereas the less seasonally variable supercooling point seemed to be associated with the year-round presence of gut content. Food derivatives may act as efficient ice nucleators. The different trend in seasonal changes between lower lethal temperature and supercooling point may be related to the microclimate of the hibernacula in subnivean environments, where the winter temperature became lower than the lower lethal temperature in the summer active phase, but remained higher than the summer supercooling point.Abbreviations LLT₅₀ lower lethal temperature inducing 50% mortality - SCP supercooling point - T _a ambient air temperature - T _s soil surface temperature     

Genetic evidence for ribosomal antisuppressors inPodospora anserina

Summary Antisuppressors were screened for with the help of informational suppressors inPodospora anserina. Four mutations in the AS₁ locus and two in the AS₂ locus were isolated, using allele non specific suppressors supposed to be ribosomal ambiguity mutations. Four mutations in the AS₃ locus and 45 in the AS₄ locus were obtained, using a nonsense (tRNA like) suppressor. All antisuppressors are partially dominant. Most mutations in the AS₄ locus are lethal. The four mutants at the AS₃ locus and 6 out of the 8 viable mutants at the AS₄ locus are cold sensitive. Phenotypic properties and action spectra of the antisuppressors suggest that they are restrictive ribosomal mutations.     

Isolation and characterization of immunity temperature sensitive mutants of Enterobacter cloacae harbouring the cloacinogenic factor DF13

Summary Enterobacter cloacae cells, harbouring the cloacinogenic factor DF13 (Clo DF13) are immune to the cloacin they produce. We describe the isolation of eleven Enterobacter cloacae (Clo DF13) mutants, which are immune at 30°C, but lose their immunity at 42°C. The temperature sensitive immunity (Imm^ts) of these mutants appeared not to be transferable together with the Clo DF13 factor to non-cloacinogenic acceptor strains. Apparently host mutations are involved in the Imm^ts phenotype. Two different groups of Imm^ts mutants could be identified. Imm^tsC6 and Imm^tsC8, representatives of each group, have been compared with the parent strain. Imm^tsC6 as well as Imm^tsC8 is sensitive to crude cloacin at 42°C. Imm^ts mutants appeared to be also sensitive to cell components other than cloacin, indicating that the Imm^ts mutations may result in pleiotropic changes of cell properties.The Imm^tsC6 mutant is sensitive to deoxycholate and osmotic shock at 42°C. Spheroplasts of Imm^tsC6 cells incubated at 42°C are sensitive to DOC at 42°C and 30°C. The pleiotrophic changes of the Imm^tsC6 mutant may be attributed to a defect in the cell membrane.The Imm^tsC8, incubated at 42°C, is sensitive to deoxycholate, osmotic shock, ethylene-diaminetetraacetic acid, dyes, drugs and UV. Furthermore they form filaments. Imm^tsC8 spheroplasts are as sensitive to deoxycholate as the parent strain at 42°C. The pleiotropic changes in the phenotype of Imm^tsC8 are considered to be the result of a defect in the outer layers of the cell envelope, most likely the lipopolysaccharide layer.The possible relationship between the observed structural defects in the cell envelope of Imm^ts mutants and the phenomenon of immunity have been discussed.     

Temperature-sensitive yellow mutants of Chlamydomonas reinhardtii

Summary We have isolated and genetically characterized 10 mutants of Chlamydomonas reinhardtii carrying single, mendelian, temperature-sensitive yellow mutations. The mutants have a yellow phenotype at the restrictive temperature (33°C), but have a wildtype phenotype at the permissive temperature (25°C). Based on complementation and recombination tests, the ten mutations include alleles of two previously described yellow loci (y-1 and y-6) and three new yellow loci (y-8, y-9, and y-10). At the restrictive temperature, y-8, y-9, and y-10 are physiologically similar to other yellow mutants. They accumulate small amounts of protochlorophyllide when grown under dim light, but synthesize normal amounts of chlorophyll when grown in the light. Linkage tests indicate that the three new mutations are not linked to each other. y-8 is linked to y-7 on linkage group III, and y-10 is linked to y-5 and y-6 on linkage group I. y-9 is located on linkage group II. We conclude that the control of light-independent protochlorophyllide reduction is complex, involving several genetic loci which are scattered in the genome and which code for gene products able to complement in trans. Temperature-sensitive alleles at several of the yellow loci suggest that the gene products made by these loci are proteins.     

Temperature-sensitive,lipopolysaccharide-deficient mutants of Salmonella typhimurium

Two mutants of Salmonella typhimurium LT2, which were temperature-sensitive for lipopolysaccharide (LPS) synthesis, were isolated from a galE ^- strain based on their resistance to phage C21 and sensitivity to sodium deoxycholate at 42°C. They produced LPS of chemotype Rc at 30°C and deep-rough LPS at 42°C. P22-mediated transductional analysis showed that the mutations responsible for temperature sensitivity are located in the rfa cluster where several genes involved in the synthesis of the LPS core are mapped. A plasmid, carrying rfaC, D and F genes of Escherichia coli K-12, complemented these mutations. These genes are responsible for the synthesis of the inner-core region of the LPS molecule. This indicates that genetic defects in these temperature-sensitive mutants affect the inner-core region of LPS.     

Cold-sensitive mutations in the Z gene of prophage P2 that result in increased sensitivity of the lysogens to a low molecular weight product of the host bacteria

Summary Z mutants of bacteriophage P2 form clear plaques and are unable to give rise to stable lysogens in Escherichia coli C. To study the function of the Z gene in lysogenization by P2, temperature-sensitive mutants were isolated. Those that were classified as Z mutants by complementation were all cold-sensitive (cs); they were unable to form lysogens at 30° C, but had wild type phenotype at 42° C. When lysogens carrying such mutants, prepared at 42° C, were shifted to the lower temperature, the bacteria continued to multiply at the normal rate until they reached concentrations of about 5 × 10⁷ per ml, at which point the viable titer began to decrease. Inactivation of the bacteria at even lower concentrations occurred if they were transferred to medium taken from overnight cultures of the same strain, suggesting that they were sensitive to some material that had accumulated in the culture medium.The lethal material was produced not only by csZ lysogens, but by all derivatives of Escherichia coli C tested, including non-lysogens, and at both 30° C and 42° C. Only csZ lysogens were sensitive to it, however, and only at the lower temperature. A preliminary characterization of the material indicates that it is heat-stable, of low molecular weight and does not adsorb to activated charcoal.This work was supported by Research Grant 72 from the Swedish Medical Research Council     

CAENORHABDITIS ELEGANS Deficiency Mapping

Six schemes were used to identify 80 independent recessive lethal deficiencies of linkage group (LG) II following X-ray treatment of the nematode Caenorhabditis elegans. Complementation tests between the deficiencies and ethyl methanesulfonate-induced recessive visible, lethal and sterile mutations and between different deficiencies were used to characterize the extents of the deficiencies. Deficiency endpoints thus helped to order 36 sites within a region representing about half of the loci on LG II and extending over about 5 map units. New mutations occurring in this region can be assigned to particular segments of the map by complementation tests against a small number of deficiencies; this facilitates the assignment of single-site mutations to particular genes, as we illustrate. Five sperm-defective and five oocyte-defective LG II sterile mutants were identified and mapped. Certain deficiency-by-deficiency complementation tests allowed us to suggest that the phenotypes of null mutations at two loci represented by visible alleles are wild type and that null mutations at a third locus confer a visible phenotype. A segment of LG II that is about 12 map units long and largely devoid of identified loci seems to be greatly favored for crossing over.     

Lethal(1)aberrant immune response mutations leading to melanotic tumor formation in Drosophila melanogaster

Using P element-mediated mutagenesis we have isolated 20 X-linked lethal mutations, representing at least 14 complementation groups, which exhibit melanotic tumor phenotypes. We present the systematic analysis of this interesting group of lethal mutations that were selected for their visible melanotic or immune response. The lethal and melanotic tumor phenotypes of each lethal(1) aberrant immune response (air) mutation are pleiotropic effects of single genetic lesions. Lethality occurs throughout the larval and early pupal periods of development and larval development is extended in some air mutants. The air mutant lethal syndromes include abnormalities associated with the brain, haematopoietic organs, gut, salivary glands, ring glands, and imaginal discs. Additional characterization of the melanotic tumor mutations Tum^l and tu(1)Sz^ts have indicated that the melanotic tumor phenotype is similar to that observed in the air mutants. These studies have led to the proposal that two distinct classes of melanotic tumor mutations exist. Class 1 includes mutants in which melanotic tumors result from “autoimmune responses” or the response of an apparently normal immune system to the presence of abnormal target tissues. The Class 2 mutants display obvious defects in the haematopoietic organs or haemocytes, manifested as overgrowth, and the resulting aberrant immune system behavior may contribute to melanotic tumor formation.     

Drosophila melanogaster acetylcholinesterase: Identification and expression of two mutations responsible for cold- and heat-sensitive phenotypes

Ace ^IJ29 and Ac ^IJ40 are cold- and heat-sensitive variants of the gene coding for acetylcholinesterase in Drosophila melanogaster. In the homozygous condition, these mutations are lethal when animals are raised at restrictive temperatures, i.e., below 23° C for Ace ^IJ29 or above 25° C for Ace ^IJ40. The coding regions of the gene in these mutants were sequenced and mutations changing Ser³⁷⁴ to Phe in Ace ^IJ29 and Pro⁷⁵ to Leu in Ace ^IJ40 were found. Acetylcholinesterases bearing these mutations were expressed in Xenopus oocytes and we found that these mutations decrease the secretion rate of the protein most probably by affecting its folding. This phenomenon is exacerbated at restrictive temperatures decreasing the amount of secreted acetylcholinesterase below the lethality threshold. In parallel, the substitution of the conserved Asp²⁴⁸ by an Asn residue completely inhibits the activity of the enzyme and its secretion, preventing the correct folding of the protein in a non-conditional manner.     

Isolation and characterization ofgrandchildless-like mutants inDrosophila melanogaster

Summary Two temperature-sensitive sex-linkedgrandchildless (gs)-like mutations (gs(1)N26 andgs(1)N441) were induced by ethylmethane sulphonate inDrosophila melanogaster. They complemented each other and mapped at two different loci (1–33.8±0.7 forgs(1)N26 and 1–39.6±1.7 forgs(1)N441), which were not identical to those of any of thegs-like mutants reported in earlier work.Homozygous females of the newly isolated mutants produced eggs that were unable to form pole cells and developed into agametic adults. Competence of the embryos to form pole cells was not restored by wild-type sperm in either mutant; that is, the sterility caused by these mutations is controlled by a maternal effect.Fecundity and fertility ofgs(1)N26 females were low, and their male offspring showed a higher mortality than that of female offspring, causing an abnormal sex ratio. The frequency of agametic progeny was 93.1% and 55.8%, when the female parents were reared at 25° C and 18° C, respectively. In eggs produced by thegs(1)N26 females reared at 25° C, the migration of nuclei to the posterior pole was abnormal, and almost no pole cell formation occurred in these egg. Furthermore, half of these eggs failed to cellularize at the posterior pole. When the females were reared at 18° C, almost all of the eggs underwent complete blastoderm formation, and in half of these blastoderm embryos normal pole cells were formed.In the other mutant,gs(1)N441, the fecundity and fertility of the females were normal. The agametic frequency in the progeny was 70.8% and 18.6% when the female parents were reared at 25° C and 18° C, respectively. In the eggs laid by females reared either at 25° C or at 18° C, the migration of nuclei to the periphery and cellularization proceeded normally; nevertheless, in the majority of the embryos no pole cell formation occured at the stage when nuclei penetrated into the periplasm. When the females were reared at 18° C, some of the embryos from these females formed some round blastoderm cells with cytologically recognizable polar granules and nuclear bodies, which are attributes of pole cells. The temperature sensitive period ofgs(1)N441 was estimated to extend from stage 9 to 13 of King's stages of oogenesis.     

A maternal effect mutation leading to deficiencies of organs and homeotic transformations in the adults ofDrosophila

Summary The temperature sensitive mutationfs(l)h is characterized at the restrictive temperature of 29°C by both a maternal effect responsible for the early embryonic lethality and pupal zygotic lethality. The two phenotypes are inseparable and map at a short deletion in the X chromosome (7Dl, 7D5-6). At semipermissive temperatures, hemizygous mutant females produce adults with morphological defects, such as organ deficiencies and homeotic transformations of haltere to wing and third leg to second leg. These defects depend on the maternal genotype and are governed by an early temperature sensitive period, which covers the end of oogenesis and the first hours of embryogenesis. Furthermore, this maternal effect mutation interacts with some dominant mutations of the bithorax system. These properties suggest thatfs(l)h is somehow involved in segmental determination.     

Nuclear mutants of Neurospora crassa temperature-sensitive for the synthesis of cytochrome aa3. I. Isolation and preliminary characterization.

Summary Three nuclear mutants of Neurospora crassa, temperature-sensitive for the synthesis of cytochrome aa ₃ have been isolated. When grown at 41°C the mutants have large amounts of KCN-insensitive respiration, reduced amounts of cytochrome aa ₃ and cytochrome c oxidase activity, and grow more slowly than wild-type cultures grown at the same temperature. When the mutants are grown at 23°C, they are virtually indistinguishable from wild-type strains.The mutants were selected on the basis of their slow growth at 41°C in medium containing salicylhydroxamic acid, and by their inability to reduce 2,3,5-triphenyltetrazolium chloride at 41°C. The selection technique was designed to eliminate mutants that did not carry thermolabile electron transport chain components. However, studies on the thermolability of the cytochrome oxidase activity in isolated mitochondria indicate that the enzyme of the mutants is no more susceptible to heat denaturation than is the enzyme in wild-type mitochondria. This suggests that the synthesis or assembly of cytochrome aa ₃ may be altered in the mutants at the restrictive temperature.Supported by National Research Council of Canada Grant Number A-6351Recipient of a National Research Council of Canada Postgraduate Scholarship     

Rapid cold hardening increases cold and chilling tolerances more than acclimation in the adults of the sycamore lace bug, Corythucha ciliata (Say) (Hemiptera: Tingidae)

The sycamore lace bug, Corythucha ciliata is a new, invasive pest of Platanus trees in China. Although C. ciliata is often subjected to acute low temperatures in early winter and spring in northern and eastern China, the cold tolerance of C. ciliata has not been well studied. The objectives of this study were to determine whether adults of C. ciliata are capable of rapid cold hardening (RCH), and to compare the benefits of RCH vs. cold acclimation (ACC) in the laboratory. When the adult females incubated at 26 °C were transferred directly to the discriminating temperature (−12 °C) for 2 h, survival was only 22%. However, exposure to 0 °C for 4 h before transfer to −12 °C for 2 h induced RCH, i.e., increased survival to 68%. RCH could also be induced by gradual cooling of the insects at rates between 0.1 and 0.25 °C min⁻¹. The protection against cold shock obtained through RCH at 0 °C for 4 h was lost within 1 h if the adults were returned to 26 °C before exposure to −12 °C. Survival at both −12 and −5 °C was greater for RCH-treated than for ACC-treated adults (for ACC, adults were kept at 15 °C for 5 days), and the lethal temperature (2 h exposure) was lower for RCH-treated than for ACC-treated adults. The results suggest that RCH may help C. ciliata survive the acute low temperatures that often occur in early winter and early spring in northern and eastern China.     

Developmental genetics of a temperature-sensitive RNA polymerase II mutation in Drosophila melanogaster

Summary Purified RNA polymerase II (RNA nucleotidyl-transferase; EC 2.7.7.6) extracted from flies possessing lesions in the Ultrabithorax-like (Ubl) locus of Drosophila melanogaster has altered activity in vitro (Greenleaf et al. 1979, 1980; Coulter and Greenleaf 1982). This strongly suggests that the Ubl locus encodes a subunit of RNA polymerase II. Ethyl methanesulfonate was used to induce a temperature-sensitive mutation in this locus. Flies either homozygous or hemizygous for this new X–linked mutation (Ubl ^ts) display viability comparable to that of wild-type flies at 22° C but are lethal at 29° C. The temperature-sensitive period for Ubl ^ts flies is between gastrulation (6 h, 29° C) and pupation (9–10 days, 22° C). Zygotes shifted from 22° C to 29° C die at either the late embryonic or first larval instar stage while temperature shifts of second and third instar larvae result in the lethal phase occurring at the pupal stage. Most pupae shifted from 22° C to 29° C undergo metamorphosis and eclose as adults. Adults are viable if placed at 29° C; however, all females and some males become sterile if maintained at this temperature.Somatic recombination was used to induce clones homozygous for a null allele of Ubl at different stages of development. Clones of this null allele appear to be cell lethal indicating that the Ubl ⁺ gene product is required at all stages of development. The viability of Ubl ^ts pupae and adults at 29° C may result from only a partial reduction in activity caused by the mutation at this nonpermissive temperature.     

Regulation of polar surface structures in Caulobacter crescentus: Pleiotropic mutations affect the coordinate morphogenesis of flagella, pili and phage receptors

Summary A large number of Caulobacter mutants resistant to DNA or RNA phages were isolated. These phage-resistant mutants exhibited phenotypic variations with respect to cell motility and sensitivity to other phages.The majority of the mutants was resistant to both DNA and RNA phages tested. In addition, these mutants were either motile or non-motile. The analysis of spontaneous revertants from these mutants indicated that a single mutation is involved in these phenotypic variations. Other mutants were resistant to RNA phages and only to a certain DNA phage tested, and were also motile or non-motile.Several temperature-sensitive phage-resistant mutants were also isolated. One of them, CB13 ple-801, exhibited the wild type phenotype when grown at 25°C. However, at a higher temperature (35°C), the mutant cells became non-motile and resistant to both DNA and RNA phages. These phenotypes seem to be attributed to the concommitant loss of flagella, pili and phage receptors. In other respects (cell growth and morphology, and asymmetric stalk formation), CB13 ple-801 was normal at 35°C. The spontaneous revertants from CB13 ple-801 simultaneously regained the wild type phenotypes in all respects.It is suggested that a single mutation pleiotropically affects the formation of flagella, pili and phage receptors.     

An easy method for screening and isolating rod mutants of<Emphasis Type="Italic"> Bacillus subtilis</Emphasis>

A convenient and rapid method for screening and identifying rod mutants of Bacillus subtilis is described. At the restrictive temperature (45 °C), all rod mutants of B. subtilis screened lost their ability to sporulate. The morphology and colour of mutant colonies grown on sporulation agar plates differed from those of rod⁺ cells, which were able to sporulate even at elevated temperature. These characteristics provide an alternative approach for the identification of rod mutants in B. subtilis culture by streaking the cells onto a minimal glucose agar plate and incubating at the restrictive temperature. After 30 h of incubation at this temperature, rod mutants are easily identified. This method will facilitate the screening and isolation of rod mutants of B. subtilis.