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1
Baliga, Chetana, Sandipan Majhi, Kajari Mondal, Antara Bhattacharjee, K. VijayRaghavan, and Raghavan Varadarajan. "Rational elicitation of cold-sensitive phenotypes." Proceedings of the National Academy of Sciences 113, no. 18 (April 18, 2016): E2506—E2515. http://dx.doi.org/10.1073/pnas.1604190113.
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Cold-sensitive phenotypes have helped us understand macromolecular assembly and biological phenomena, yet few attempts have been made to understand the basis of cold sensitivity or to elicit it by design. We report a method for rational design of cold-sensitive phenotypes. The method involves generation of partial loss-of-function mutants, at either buried or functional sites, coupled with selective overexpression strategies. The only essential input is amino acid sequence, although available structural information can be used as well. The method has been used to elicit cold-sensitive mutants of a variety of proteins, both monomeric and dimeric, and in multiple organisms, namelyEscherichia coli,Saccharomyces cerevisiae, andDrosophila melanogaster. This simple, yet effective technique of inducing cold sensitivity eliminates the need for complex mutations and provides a plausible molecular mechanism for eliciting cold-sensitive phenotypes.
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Novick, P., B. C. Osmond, and D. Botstein. "Suppressors of yeast actin mutations." Genetics 121, no. 4 (April 1, 1989): 659–74. http://dx.doi.org/10.1093/genetics/121.4.659.
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Abstract Suppressors of a temperature-sensitive mutation (act1-1) in the single actin gene of Saccharomyces cerevisiae were selected that had simultaneously acquired a cold-sensitive growth phenotype. Five genes, called SAC (suppressor of actin) were defined by complementation tests; both suppression and cold-sensitive phenotypes were recessive. Three of the genes (SAC1, SAC2 and SAC3) were subjected to extensive genetic and phenotypic analysis, including molecular cloning. Suppression was found to be allele-specific with respect to actin alleles. The sac mutants, even in ACT1+ genetic backgrounds, displayed phenotypes similar to those of actin mutants, notably aberrant organization of intracellular actin and deposition of chitin at the cell surface. These results are interpreted as being consistent with the idea that the SAC genes encode proteins that interact with actin, presumably as components or controllers of the assembly or stability of the yeast actin cytoskeleton. Two unexpected properties of the SAC1 gene were noted. Disruptions of the gene indicated that its function is essential only at temperatures below about 17 degrees and all sac1 alleles are inviable when combined with act1-2. These properties are interpreted in the context of the evolution of the actin cytoskeleton of yeast.
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Flower, Ann M. "SecG Function and Phospholipid Metabolism inEscherichia coli." Journal of Bacteriology 183, no. 6 (March 15, 2001): 2006–12. http://dx.doi.org/10.1128/jb.183.6.2006-2012.2001.
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ABSTRACT SecG is an auxiliary protein in the Sec-dependent protein export pathway of Escherichia coli. Although the precise function of SecG is unknown, it stimulates translocation activity and has been postulated to enhance the membrane insertion-deinsertion cycle of SecA. Deletion of secG was initially reported to result in a severe export defect and cold sensitivity. Later results demonstrated that both of these phenotypes were strain dependent, and it was proposed that an additional mutation was required for manifestation of the cold-sensitive phenotype. The results presented here demonstrate that the cold-sensitive secG deletion strain also contains a mutation in glpR that causes constitutive expression of the glp regulon. Introduction of both the glpRmutation and the secG deletion into a wild-type strain background produced a cold-sensitive phenotype, confirming the hypothesis that a second mutation (glpR) contributes to the cold-sensitive phenotype of secG deletion strains. It was speculated that the glpR mutation causes an intracellular depletion of glycerol-3-phosphate due to constitutive synthesis of GlpD and subsequent channeling of glycerol-3-phosphate into metabolic pathways. In support of this hypothesis, it was demonstrated that addition of glycerol-3-phosphate to the growth medium ameliorated the cold sensitivity, as did introduction of a glpD mutation. This depletion of glycerol-3-phosphate is predicted to limit phospholipid biosynthesis, causing an imbalance in the levels of membrane phospholipids. It is hypothesized that this state of phospholipid imbalance imparts a dependence on SecG for proper function or stabilization of the translocation apparatus.
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Nonet, M. L., and R. A. Young. "Intragenic and extragenic suppressors of mutations in the heptapeptide repeat domain of Saccharomyces cerevisiae RNA polymerase II." Genetics 123, no. 4 (December 1, 1989): 715–24. http://dx.doi.org/10.1093/genetics/123.4.715.
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Abstract The largest subunit of RNA polymerase II contains a repeated heptapeptide sequence at its carboxy terminus. Yeast mutants with certain partial deletions of the carboxy-terminal repeat (CTR) domain are temperature-sensitive, cold-sensitive and are inositol auxotrophs. Intragenic and extragenic suppressors of the cold-sensitive phenotype of CTR domain deletion mutants were isolated and studied to investigate the function of this domain. Two types of intragenic suppressing mutations suppress the temperature-sensitivity, cold-sensitivity and inositol auxotrophy of CTR domain deletion mutants. Most intragenic mutations enlarge the repeat domain by duplicating various portions of the repeat coding sequence. Other intragenic suppressing mutations are point mutations in a conserved segment of the large subunit. An extragenic suppressing mutation (SRB2-1) was isolated that strongly suppresses the conditional and auxotrophic phenotypes of CTR domain mutations. The SRB2 gene was isolated and mapped, and an SRB2 partial deletion mutation (srb2 delta 10) was constructed. The srb2 delta 10 mutants are temperature-sensitive, cold-sensitive and are inositol auxotrophs. These phenotypes are characteristic of mutations in genes encoding components of the transcription apparatus. We propose that the SRB2 gene encodes a factor that is involved in RNA synthesis and may interact with the CTR domain of the large subunit of RNA polymerase II.
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Skiadopoulos, Mario H., Sonja Surman, Joanne M. Tatem, Maribel Paschalis, Shin-Lu Wu, Stephen A. Udem, Anna P. Durbin, Peter L. Collins, and Brian R. Murphy. "Identification of Mutations Contributing to the Temperature-Sensitive, Cold-Adapted, and Attenuation Phenotypes of the Live-Attenuated Cold-Passage 45 (cp45) Human Parainfluenza Virus 3 Candidate Vaccine." Journal of Virology 73, no. 2 (February 1, 1999): 1374–81. http://dx.doi.org/10.1128/jvi.73.2.1374-1381.1999.
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ABSTRACT The live-attenuated human parainfluenza virus 3 (PIV3) cold-passage 45 (cp45) candidate vaccine was shown previously to be safe, immunogenic, and phenotypically stable in seronegative human infants. Previous findings indicated that each of the three amino acid substitutions in the L polymerase protein of cp45 independently confers the temperature-sensitive (ts) and attenuation (att) phenotypes but not the cold-adaptation (ca) phenotype (29).cp45 contains 12 additional potentially important point mutations in other proteins (N, C, M, F, and hemagglutinin-neuraminidase [HN]) or in cis-acting sequences (the leader region and the transcription gene start [GS] signal of the N gene), and their contribution to these phenotypes was undefined. To further characterize the genetic basis for thets, ca, and att phenotypes of this promising vaccine candidate, we constructed, using a reverse genetics system, a recombinant cp45 virus that contained all 15 cp45-specific mutations mentioned above, and found that it was essentially indistinguishable from the biologically derived cp45 on the basis of plaque size, level of temperature sensitivity, cold adaptation, level of replication in the upper and lower respiratory tract of hamsters, and ability to protect hamsters from subsequent wild-type PIV3 challenge. We then constructed recombinant viruses containing the cp45 mutations in individual proteins as well as several combinations of mutations. Analysis of these recombinant viruses revealed that multiple cp45 mutations distributed throughout the genome contribute to the ts, ca, andatt phenotypes. In addition to the mutations in the L gene, at least one other mutation in the 3′ N region (i.e., including the leader, N GS, and N coding changes) contributes to thets phenotype. A recombinant virus containing all thecp45 mutations except those in L was more tsthan cp45, illustrating the complex nature of this phenotype. The ca phenotype of cp45 also is a complex composite phenotype, reflecting contributions of at least three separate genetic elements, namely, mutations within the 3′ N region, the L protein, and the C-M-F-HN region. The att phenotype is a composite of both ts and non-ts mutations. Attenuating ts mutations are located in the L protein, and non-ts attenuating mutations are located in the C and F proteins. The presence of multiple ts and non-ts attenuating mutations in cp45 likely contributes to the high level of attenuation and phenotypic stability of this promising vaccine candidate.
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Kuchka, Michael R., and Jonathan W. Jarvik. "Short-Flagella Mutants of Chlamydomonas reinhardtii." Genetics 115, no. 4 (April 1, 1987): 685–91. http://dx.doi.org/10.1093/genetics/115.4.685.
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ABSTRACT Six short-flagella mutants were isolated by screening clones of mutagenized Chlamydomonas for slow swimmers. The six mutants identify three unlinked Mendelian genes, with three mutations in gene shf-1, two in shf-2 and one in shf-3. shf-1 and shf-2 have been mapped to chromosomes VI and I, respectively. Two of the shf-1 mutations have temperature-sensitive flagellar-assembly phenotypes, and one shf-2 mutant has a cold-sensitive phenotype. shf shf double mutants were constructed; depending on the alleles present they showed either flagellaless or short-flagella phenotypes. Phenotypic revertants of shf-1 and shf-2 mutants were isolated, and certain of them were found to carry extragenic suppressors, some dominant and some recessive. We suspect that the shf mutations affect components of a specific flagellar size-control system, the existence of which has been suggested by a variety of physiological experiments.
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Puziss, J. W., T. A. Hardy, R. B. Johnson, P. J. Roach, and P. Hieter. "MDS1, a dosage suppressor of an mck1 mutant, encodes a putative yeast homolog of glycogen synthase kinase 3." Molecular and Cellular Biology 14, no. 1 (January 1994): 831–39. http://dx.doi.org/10.1128/mcb.14.1.831-839.1994.
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The yeast gene MCK1 encodes a serine/threonine protein kinase that is thought to function in regulating kinetochore activity and entry into meiosis. Disruption of MCK1 confers a cold-sensitive phenotype, a temperature-sensitive phenotype, and sensitivity to the microtubule-destabilizing drug benomyl and leads to loss of chromosomes during growth on benomyl. A dosage suppression selection was used to identify genes that, when present at high copy number, could suppress the cold-sensitive phenotype of mck1::HIS3 mutant cells. Several unique classes of clones were identified, and one of these, designated MDS1, has been characterized in some detail. Nucleotide sequence data reveal that MDS1 encodes a serine/threonine protein kinase that is highly homologous to the shaggy/zw3 kinase in Drosophila melanogaster and its functional homolog, glycogen synthase kinase 3, in rats. The presence of MDS1 in high copy number rescues both the cold-sensitive and the temperature-sensitive phenotypes, but not the benomyl-sensitive phenotype, associated with the disruption of MCK1. Analysis of strains harboring an mds1 null mutation demonstrates that MDS1 is not essential during normal vegetative growth but appears to be required for meiosis. Finally, in vitro experiments indicate that the proteins encoded by both MCK1 and MDS1 possess protein kinase activity with substrate specificity similar to that of mammalian glycogen synthase kinase 3.
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Puziss, J. W., T. A. Hardy, R. B. Johnson, P. J. Roach, and P. Hieter. "MDS1, a dosage suppressor of an mck1 mutant, encodes a putative yeast homolog of glycogen synthase kinase 3." Molecular and Cellular Biology 14, no. 1 (January 1994): 831–39. http://dx.doi.org/10.1128/mcb.14.1.831.
Full textAbstract:
The yeast gene MCK1 encodes a serine/threonine protein kinase that is thought to function in regulating kinetochore activity and entry into meiosis. Disruption of MCK1 confers a cold-sensitive phenotype, a temperature-sensitive phenotype, and sensitivity to the microtubule-destabilizing drug benomyl and leads to loss of chromosomes during growth on benomyl. A dosage suppression selection was used to identify genes that, when present at high copy number, could suppress the cold-sensitive phenotype of mck1::HIS3 mutant cells. Several unique classes of clones were identified, and one of these, designated MDS1, has been characterized in some detail. Nucleotide sequence data reveal that MDS1 encodes a serine/threonine protein kinase that is highly homologous to the shaggy/zw3 kinase in Drosophila melanogaster and its functional homolog, glycogen synthase kinase 3, in rats. The presence of MDS1 in high copy number rescues both the cold-sensitive and the temperature-sensitive phenotypes, but not the benomyl-sensitive phenotype, associated with the disruption of MCK1. Analysis of strains harboring an mds1 null mutation demonstrates that MDS1 is not essential during normal vegetative growth but appears to be required for meiosis. Finally, in vitro experiments indicate that the proteins encoded by both MCK1 and MDS1 possess protein kinase activity with substrate specificity similar to that of mammalian glycogen synthase kinase 3.
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Fane, B. A., and M. Hayashi. "Second-site suppressors of a cold-sensitive prohead accessory protein of bacteriophage phi X174." Genetics 128, no. 4 (August 1, 1991): 663–71. http://dx.doi.org/10.1093/genetics/128.4.663.
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Abstract This study describes the isolation of second-site suppressors which correct for the defects associated with cold-sensitive (cs) prohead accessory proteins of bacteriophage phi X174. Five phenotypically different suppressors were isolated. Three of these suppressors confer novel temperature-sensitive (ts) phenotypes. They were unable to complement a ts mutation in gene F which encodes the major coat protein of the phage. All five suppressor mutations confer nucleotide changes in the gene F DNA sequence. These changes define four amino acid sites in the gene F protein. Three suppressor mutations placed into an otherwise wild-type background display a cold resistant phenotype in liquid culture infections when compared to a wild-type phi X174 control.
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Hecht, Ralph M., Mary A. Norman, Tammy Vu, and William Jones. "A novel set of uncoordinated mutants inCaenorhabditis elegansuncovered by cold-sensitive mutations." Genome 39, no. 2 (April 1, 1996): 459–64. http://dx.doi.org/10.1139/g96-058.
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A set of uncoordinated (Unc) cold-sensitive (cs) mutants was isolated at a stringent condition of 11 °C. About half of the 13 independently isolated cs-Unc mutants were alleles of three X-linked Unc mutants that exhibited the "kinker" phenotype. The remaining four isolates identified new mutants that exhibited "kinker," "coiler," or severe paralytic phenotypes. The temperature-sensitive period (TSP) for each gene was determined. As a homozygous or heterozygous dominant, unc-125 exhibited a TSP throughout all stages of development. Its severe paralysis was immediately observed upon a shift down to 11 °C and reversed upon a shift up to 23 °C. The reversible thermolability of the unc-125 gene product indicated that it may function in a multicomponent process involved in neuro-excitation. Key words : Caenorhabditis elegans, cold-sensitive uncoordinated mutants, cs-Unc.
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Ramamurthy, Visvanathan, Vesna Dapíc, and Donald Oliver. "secG and Temperature Modulate Expression of Azide-Resistant and Signal Sequence Suppressor Phenotypes of Escherichia coli secA Mutants." Journal of Bacteriology 180, no. 23 (December 1, 1998): 6419–23. http://dx.doi.org/10.1128/jb.180.23.6419-6423.1998.
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ABSTRACT SecA is a dynamic protein that undergoes ATP-dependent membrane cycling to drive protein translocation across the Escherichia coli inner membrane. To understand more about this process, azide-resistant (azi) and signal sequence suppressor (prlD) alleles of secA were studied. We found that azide resistance is cold sensitive because of a direct effect on protein export, suggesting that SecA-membrane interaction is regulated by an endothermic step that is azide inhibitable. secGfunction is required for expression of azide-resistant and signal sequence suppressor activities of azi and prlDalleles, and in turn, these alleles suppress cold-sensitive and export-defective phenotypes of a secG null mutant. These remarkable genetic observations support biochemical data indicating that SecG promotes SecA membrane cycling and that this process is dependent on an endothermic change in SecA conformation.
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Krishnan, Karthik, and Ann M. Flower. "Suppression of ΔbipA Phenotypes in Escherichia coli by Abolishment of Pseudouridylation at Specific Sites on the 23S rRNA." Journal of Bacteriology 190, no. 23 (September 26, 2008): 7675–83. http://dx.doi.org/10.1128/jb.00835-08.
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ABSTRACT The BipA protein of Escherichia coli has intriguing similarities to the elongation factor subfamily of GTPases, including EF-Tu, EF-G, and LepA. In addition, phenotypes of a bipA deletion mutant suggest that BipA is involved in regulation of a variety of pathways. These two points have led to speculation that BipA may be a novel regulatory protein that affects efficient translation of target genes through direct interaction with the ribosome. We isolated and characterized suppressors of the cold-sensitive growth phenotype exhibited by ΔbipA strains and identified insertion mutations in rluC. The rluC gene encodes a pseudouridine synthase responsible for pseudouridine modification of 23S rRNA at three sites, all located near the peptidyl transferase center. Deletion of rluC not only suppressed cold sensitivity but also alleviated the decrease in capsule synthesis exhibited by bipA mutants, suggesting that the phenotypic effects of BipA are manifested through an effect on the ribosome. The suppressor effect is specific to rluC, as deletion of other rlu genes did not relieve cold sensitivity, and further, more than a single pseudouridine residue is involved, as alteration of single residues did not produce suppressors. These results are consistent with a role for BipA in either the structure or the function of the ribosome and imply that wild-type ribosomes are dependent on BipA for efficient expression of target mRNAs and that the lack of pseudouridylation at these three sites renders the ribosomes BipA independent.
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Martin, C., and R. A. Young. "KEX2 mutations suppress RNA polymerase II mutants and alter the temperature range of yeast cell growth." Molecular and Cellular Biology 9, no. 6 (June 1989): 2341–49. http://dx.doi.org/10.1128/mcb.9.6.2341-2349.1989.
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Suppressors of a temperature-sensitive RNA polymerase II mutation were isolated to identify proteins that interact with RNA polymerase II in yeast cells. Ten independently isolated extragenic mutations that suppressed the temperature-sensitive mutation rpb1-1 and produced a cold-sensitive phenotype were all found to be alleles of a single gene, SRB1. An SRB1 partial deletion mutant was further investigated and found to exhibit several pleiotropic phenotypes. These included suppression of numerous temperature-sensitive RNA polymerase II mutations, alteration of the temperature growth range of cells containing wild-type RNA polymerase, and sterility of cells of alpha mating type. The ability of SRB1 mutations to suppress the temperature-sensitive phenotype of RNA polymerase II mutants did not extend to other temperature-sensitive mutants investigated. Isolation of the SRB1 gene revealed that SRB1 is KEX2. These results indicate that the KEX2 protease, whose only known substrates are hormone precursors, can have an important influence on RNA polymerase II and the temperature-dependent growth properties of yeast cells.
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Martin, C., and R. A. Young. "KEX2 mutations suppress RNA polymerase II mutants and alter the temperature range of yeast cell growth." Molecular and Cellular Biology 9, no. 6 (June 1989): 2341–49. http://dx.doi.org/10.1128/mcb.9.6.2341.
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Suppressors of a temperature-sensitive RNA polymerase II mutation were isolated to identify proteins that interact with RNA polymerase II in yeast cells. Ten independently isolated extragenic mutations that suppressed the temperature-sensitive mutation rpb1-1 and produced a cold-sensitive phenotype were all found to be alleles of a single gene, SRB1. An SRB1 partial deletion mutant was further investigated and found to exhibit several pleiotropic phenotypes. These included suppression of numerous temperature-sensitive RNA polymerase II mutations, alteration of the temperature growth range of cells containing wild-type RNA polymerase, and sterility of cells of alpha mating type. The ability of SRB1 mutations to suppress the temperature-sensitive phenotype of RNA polymerase II mutants did not extend to other temperature-sensitive mutants investigated. Isolation of the SRB1 gene revealed that SRB1 is KEX2. These results indicate that the KEX2 protease, whose only known substrates are hormone precursors, can have an important influence on RNA polymerase II and the temperature-dependent growth properties of yeast cells.
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Pöntinen, Anna, Annukka Markkula, Miia Lindström, and Hannu Korkeala. "Two-Component-System Histidine Kinases Involved in Growth of Listeria monocytogenes EGD-e at Low Temperatures." Applied and Environmental Microbiology 81, no. 12 (April 3, 2015): 3994–4004. http://dx.doi.org/10.1128/aem.00626-15.
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ABSTRACTTwo-component systems (TCSs) aid bacteria in adapting to a wide variety of stress conditions. While the role of TCS response regulators in the cold tolerance of the psychrotrophic foodborne pathogenListeria monocytogeneshas been demonstrated previously, no comprehensive studies showing the role of TCS histidine kinases ofL. monocytogenesat low temperature have been performed. We compared the expression levels of each histidine kinase-encoding gene ofL. monocytogenesEGD-e in logarithmic growth phase at 3°C and 37°C, as well as the expression levels 30 min, 3 h, and 7 h after cold shock at 5°C and preceding cold shock (at 37°C). We constructed a deletion mutation in each TCS histidine kinase gene, monitored the growth of the EGD-e wild-type and mutant strains at 3°C and 37°C, and measured the minimum growth temperature of each strain. Two genes,yycGandlisK, proved significant in regard to induced relative expression levels under cold conditions and cold-sensitive mutant phenotypes. Moreover, the ΔresEmutant showed a lower growth rate than that of wild-type EGD-e at 3°C. Eleven other genes showed upregulated gene expression but revealed no cold-sensitive phenotypes. The results show that the histidine kinases encoded byyycGandlisKare important for the growth and adaptation ofL. monocytogenesEGD-e at low temperature.
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Loertscher, Jennifer, Lynnelle L. Larson, Clinton K. Matson, Mark L. Parrish, Alicia Felthauser, Aaron Sturm, Christine Tachibana, Martin Bard, and Robin Wright. "Endoplasmic Reticulum-Associated Degradation Is Required for Cold Adaptation and Regulation of Sterol Biosynthesis in the Yeast Saccharomyces cerevisiae." Eukaryotic Cell 5, no. 4 (April 2006): 712–22. http://dx.doi.org/10.1128/ec.5.4.712-722.2006.
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ABSTRACT Endoplasmic reticulum-associated degradation (ERAD) mediates the turnover of short-lived and misfolded proteins in the ER membrane or lumen. In spite of its important role, only subtle growth phenotypes have been associated with defects in ERAD. We have discovered that the ERAD proteins Ubc7 (Qri8), Cue1, and Doa10 (Ssm4) are required for growth of yeast that express high levels of the sterol biosynthetic enzyme, 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR). Interestingly, the observed growth defect was exacerbated at low temperatures, producing an HMGR-dependent cold sensitivity. Yeast strains lacking UBC7, CUE1, or DOA10 also assembled aberrant karmellae (ordered arrays of membranes surrounding the nucleus that assemble when HMGR is expressed at high levels). However, rather than reflecting the accumulation of abnormal karmellae, the cold sensitivity of these ERAD mutants was due to increased HMGR catalytic activity. Mutations that compromise proteasomal function also resulted in cold-sensitive growth of yeast with elevated HMGR, suggesting that improper degradation of ERAD targets might be responsible for the observed cold-sensitive phenotype. However, the essential ERAD targets were not the yeast HMGR enzymes themselves. The sterol metabolite profile of ubc7Δ cells was altered relative to that of wild-type cells. Since sterol levels are known to regulate membrane fluidity, the viability of ERAD mutants expressing normal levels of HMGR was examined at low temperatures. Cells lacking UBC7, CUE1, or DOA10 were cold sensitive, suggesting that these ERAD proteins have a role in cold adaptation, perhaps through effects on sterol biosynthesis.
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Zavanelli, M. I., J. S. Britton, A. H. Igel, and M. Ares. "Mutations in an essential U2 small nuclear RNA structure cause cold-sensitive U2 small nuclear ribonucleoprotein function by favoring competing alternative U2 RNA structures." Molecular and Cellular Biology 14, no. 3 (March 1994): 1689–97. http://dx.doi.org/10.1128/mcb.14.3.1689-1697.1994.
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Mutations in stem-loop IIa of yeast U2 RNA cause cold-sensitive growth and cold-sensitive U2 small nuclear ribonucleoprotein function in vitro. Cold-sensitive U2 small nuclear RNA adopts an alternative conformation that occludes the loop and disrupts the stem but does so at both restrictive and permissive temperatures. To determine whether alternative U2 RNA structure causes the defects, we tested second-site mutations in U2 predicted to disrupt the alternative conformation. We find that such mutations efficiently suppress the cold-sensitive phenotypes and partially restore correct U2 RNA folding. A genetic search for additional suppressors of cold sensitivity revealed two unexpected mutations in the base of an adjacent stem-loop. Direct probing of RNA structure in vivo indicates that the suppressors of cold sensitivity act to improve the stability of the essential stem relative to competing alternative structures by disrupting the alternative structures. We suggest that many of the numerous cold-sensitive mutations in a variety of RNAs and RNA-binding proteins could be a result of changes in the stability of a functional RNA conformation relative to a competing structure. The presence of an evolutionarily conserved U2 sequence positioned to form an alternative structure argues that this region of U2 is dynamic during the assembly or function of the U2 small nuclear ribonucleoprotein.
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Zavanelli, M. I., J. S. Britton, A. H. Igel, and M. Ares. "Mutations in an essential U2 small nuclear RNA structure cause cold-sensitive U2 small nuclear ribonucleoprotein function by favoring competing alternative U2 RNA structures." Molecular and Cellular Biology 14, no. 3 (March 1994): 1689–97. http://dx.doi.org/10.1128/mcb.14.3.1689.
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Mutations in stem-loop IIa of yeast U2 RNA cause cold-sensitive growth and cold-sensitive U2 small nuclear ribonucleoprotein function in vitro. Cold-sensitive U2 small nuclear RNA adopts an alternative conformation that occludes the loop and disrupts the stem but does so at both restrictive and permissive temperatures. To determine whether alternative U2 RNA structure causes the defects, we tested second-site mutations in U2 predicted to disrupt the alternative conformation. We find that such mutations efficiently suppress the cold-sensitive phenotypes and partially restore correct U2 RNA folding. A genetic search for additional suppressors of cold sensitivity revealed two unexpected mutations in the base of an adjacent stem-loop. Direct probing of RNA structure in vivo indicates that the suppressors of cold sensitivity act to improve the stability of the essential stem relative to competing alternative structures by disrupting the alternative structures. We suggest that many of the numerous cold-sensitive mutations in a variety of RNAs and RNA-binding proteins could be a result of changes in the stability of a functional RNA conformation relative to a competing structure. The presence of an evolutionarily conserved U2 sequence positioned to form an alternative structure argues that this region of U2 is dynamic during the assembly or function of the U2 small nuclear ribonucleoprotein.
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YANG, Yan, Jingxian ZHANG, Jun LIU, Xiaohui ZHOU, Shuqing LIANG, Songyu LIU, and Yong ZHUANG. "Global identification and functional prediction of cold-related lncRNAs in eggplant." Notulae Botanicae Horti Agrobotanici Cluj-Napoca 50, no. 4 (December 2, 2022): 12931. http://dx.doi.org/10.15835/nbha50312931.
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Long noncoding RNAs (lncRNAs) play critical roles in plant development and stress responses. So far, identification of lncRNA in eggplant response to stresses has been limited and the role in mediating response to cold stress is yet to be characterized in eggplant. In this study, there is reported the first dataset of lncRNAs responsive to cold stress in the cold tolerant and sensitive eggplants using RNA sequencing (RNA-seq). 227 and 225 differentially expressed (DE) lncRNAs were obtained in two genotypes with differential cold-tolerance. Functional characterization through gene ontology (GO) analysis indicated that target genes were particularly related to acyl-CoA dehydrogenase activity and pseudouridine synthase activity, which could result in the tolerant phenotypes. Kyoto Encyclopaedia of Genes and Genomes (KEGG) showed that target genes in both sensitive and tolerant eggplants were mainly involved in cold responsive pathways such as oxidative phosphorylation, peroxisome, protein processing in endoplasmic reticulum, ubiquitin mediated proteolysis and so on. However, the enriched pathways obtained by enrichment analysis in cold-tolerant eggplant were different from those in cold-sensitive eggplant, which further indicated the reason for different tolerances. Our findings highlight the potential contributions of lncRNAs in regulating eggplant response to cold stress and difference in cold tolerance.
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Kinzy, T. G., and J. L. Woolford. "Increased expression of Saccharomyces cerevisiae translation elongation factor 1 alpha bypasses the lethality of a TEF5 null allele encoding elongation factor 1 beta." Genetics 141, no. 2 (October 1, 1995): 481–89. http://dx.doi.org/10.1093/genetics/141.2.481.
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Abstract Translation elongation factor 1beta (EF-1beta) catalyzes the exchange of bound GDP for GTP on EF-1alpha. The lethality of a null allele of the TEF5 gene encoding EF-1beta in Saccharomyces cerevisiae was suppressed by extra copies of the TEF2 gene encoding EF-1alpha. The strains with tef5::TRP1 suppressed by extra copies of TEF were slow growing, cold sensitive, hypersensitive to inhibitors of translation elongation and showed increased phenotypic suppression of +1 frameshift and UAG nonsense mutations. Nine dominant mutant alleles of TEF2 that cause increased suppression of frameshift mutations also suppressed the lethality of tef5::TRP1. Most of the strains in which tef5::TRP1 is suppressed by dominant mutant alleles of TEF2 grew more slowly and were more antibiotic sensitive than strains with tef5::TRP1 is suppressed by wild-type TEF2. Two alleles, TEF2-4 and TEF2-10, interact with tef5::TRP1 to produce strains that showed doubling times similar to tef5::TRP1 strains containing extra copies of wild-type TEF2. These strains were less cold sensitive, drug sensitive and correspondingly less efficient suppressor of +1 frameshift mutations. These phenotypes indicate that translation and cell growth are highly sensitive to changes in EF-1alpha and EF-1beta activity.
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Thorsness, P. E., K. H. White, and T. D. Fox. "Inactivation of YME1, a member of the ftsH-SEC18-PAS1-CDC48 family of putative ATPase-encoding genes, causes increased escape of DNA from mitochondria in Saccharomyces cerevisiae." Molecular and Cellular Biology 13, no. 9 (September 1993): 5418–26. http://dx.doi.org/10.1128/mcb.13.9.5418-5426.1993.
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The yeast nuclear gene YME1 was one of six genes recently identified in a screen for mutations that elevate the rate at which DNA escapes from mitochondria and migrates to the nucleus. yme1 mutations, including a deletion, cause four known recessive phenotypes: an elevation in the rate at which copies of TRP1 and ARS1, integrated into the mitochondrial genome, escape to the nucleus; a heat-sensitive respiratory-growth defect; a cold-sensitive growth defect on rich glucose medium; and synthetic lethality in rho- (cytoplasmic petite) cells. The cloned YME1 gene complements all of these phenotypes. The gene product, Yme1p, is immunologically detectable as an 82-kDa protein present in mitochondria. Yme1p is a member of a family of homologous putative ATPases, including Sec18p, Pas1p, Cdc48p, TBP-1, and the FtsH protein. Yme1p is most similar to the Escherichia coli FtsH protein, an essential protein involved in septum formation during cell division. This observation suggests the hypothesis that Yme1p may play a role in mitochondrial fusion and/or division.
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Thorsness, P. E., K. H. White, and T. D. Fox. "Inactivation of YME1, a member of the ftsH-SEC18-PAS1-CDC48 family of putative ATPase-encoding genes, causes increased escape of DNA from mitochondria in Saccharomyces cerevisiae." Molecular and Cellular Biology 13, no. 9 (September 1993): 5418–26. http://dx.doi.org/10.1128/mcb.13.9.5418.
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The yeast nuclear gene YME1 was one of six genes recently identified in a screen for mutations that elevate the rate at which DNA escapes from mitochondria and migrates to the nucleus. yme1 mutations, including a deletion, cause four known recessive phenotypes: an elevation in the rate at which copies of TRP1 and ARS1, integrated into the mitochondrial genome, escape to the nucleus; a heat-sensitive respiratory-growth defect; a cold-sensitive growth defect on rich glucose medium; and synthetic lethality in rho- (cytoplasmic petite) cells. The cloned YME1 gene complements all of these phenotypes. The gene product, Yme1p, is immunologically detectable as an 82-kDa protein present in mitochondria. Yme1p is a member of a family of homologous putative ATPases, including Sec18p, Pas1p, Cdc48p, TBP-1, and the FtsH protein. Yme1p is most similar to the Escherichia coli FtsH protein, an essential protein involved in septum formation during cell division. This observation suggests the hypothesis that Yme1p may play a role in mitochondrial fusion and/or division.
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Shimajiri, Tomoyuki, and Joji M. Otaki. "Phenotypic Plasticity of the Mimetic Swallowtail Butterfly Papilio polytes: Color Pattern Modifications and Their Implications in Mimicry Evolution." Insects 13, no. 7 (July 19, 2022): 649. http://dx.doi.org/10.3390/insects13070649.
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Butterfly wing color patterns are sensitive to environmental stress, such as temperature shock, and this phenotypic plasticity plays an important role in color pattern evolution. However, the potential contributions of phenotypic plasticity to mimicry evolution have not been evaluated. Here, we focused on the swallowtail butterfly Papilio polytes, which has nonmimetic and mimetic forms in females, to examine its plastic phenotypes. In the nonmimetic form, medial white spots and submarginal reddish spots in the ventral hindwings were enlarged by cold shock but were mostly reduced in size by heat shock. These temperature-shock-induced color pattern modifications were partly similar to mimetic color patterns, and nonmimetic females were more sensitive than males and mimetic females. Unexpectedly, injection of tungstate, a known modification inducer in nymphalid and lycaenid butterflies, did not induce any modification, but fluorescent brightener 28, another inducer discovered recently, induced unique modifications. These results suggest that phenotypic plasticity in nonmimetic females might have provided a basis of natural selection for mimetic color patterns during evolution.
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Skiadopoulos, Mario H., Anna P. Durbin, Joanne M. Tatem, Shin-Lu Wu, Maribel Paschalis, Tao Tao, Peter L. Collins, and Brian R. Murphy. "Three Amino Acid Substitutions in the L Protein of the Human Parainfluenza Virus Type 3 cp45 Live Attenuated Vaccine Candidate Contribute to Its Temperature-Sensitive and Attenuation Phenotypes." Journal of Virology 72, no. 3 (March 1, 1998): 1762–68. http://dx.doi.org/10.1128/jvi.72.3.1762-1768.1998.
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ABSTRACT Studies were initiated to define the genetic basis of the temperature-sensitive (ts), cold adaptation (ca), and attenuation (att) phenotypes of the human parainfluenza virus type 3 (PIV3) cp45 live attenuated vaccine candidate. Genetic data had previously suggested that the L polymerase protein of cp45, which contains three amino acid substitutions at positions 942, 992, and 1558, contributed to its temperature sensitivity (R. Ray, M. S. Galinski, B. R. Heminway, K. Meyer, F. K. Newman, and R. B. Belshe, J. Virol. 70:580–584, 1996; A. Stokes, E. L. Tierney, C. M. Sarris, B. R. Murphy, and S. L. Hall, Virus Res. 30:43–52, 1993). To study the individual and aggregate contributions that these amino acid substitutions make to the ts, att, and ca phenotypes of cp45, seven PIV3 recombinant viruses (three single, three double, and one triple mutant) representing all possible combinations of the three amino acid substitutions were recovered from full-length antigenomic cDNA and analyzed for their ts, att, and caphenotypes. None of the seven mutant recombinant PIVs was cold adapted. The substitutions at L protein amino acid positions 992 and 1558 each specified a 105-fold reduction in plaque formation in cell culture at 40°C, whereas the substitution at position 942 specified a 300-fold reduction. Thus, each of the three mutations contributes individually to the ts phenotype. The triple recombinant which possesses an L protein with all three mutations was almost as temperature sensitive as cp45, indicating that these mutations are the major contributors to the ts phenotype ofcp45. The three individual mutations in the L protein each contributed to restricted replication in the upper or lower respiratory tract of hamsters, and this likely contributes to the observed stability of the ts and att phenotypes ofcp45 during replication in vivo. Importantly, the recombinant virus possessing L protein with all three mutations was as restricted in replication as was the cp45 mutant in both the upper and lower respiratory tracts of hamsters, indicating that the L gene of the cp45 virus is a major attenuating component of this candidate vaccine.
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Kiseleva, I., Q. Su, T. J. Toner, C. Szymkowiak, W. S. Kwan, L. Rudenko, A. R. Shaw, and R. Youil. "Cell-based assay for the determination of temperature sensitive and cold adapted phenotypes of influenza viruses." Journal of Virological Methods 116, no. 1 (March 2004): 71–78. http://dx.doi.org/10.1016/j.jviromet.2003.10.012.
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Mutero, Annick, Jean-Marc Bride, Madeleine Pralavorio, and Didier Fournier. "Drosophila melanogaster acetylcholinesterase: Identification and expression of two mutations responsible for cold- and heat-sensitive phenotypes." Molecular and General Genetics MGG 243, no. 6 (November 1994): 699–705. http://dx.doi.org/10.1007/bf00279580.
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Asare, Emmanuel, JoAnn Mugavero, Ping Jiang, Eckard Wimmer, and Aniko V. Paul. "A Single Amino Acid Substitution in Poliovirus Nonstructural Protein 2CATPaseCauses Conditional Defects in Encapsidation and Uncoating." Journal of Virology 90, no. 14 (April 13, 2016): 6174–86. http://dx.doi.org/10.1128/jvi.02877-15.
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ABSTRACTThe specificity of encapsidation of C-cluster enteroviruses depends on an interaction between capsid proteins and nonstructural protein 2CATPase. In particular, residue N252of poliovirus 2CATPaseinteracts with VP3 of coxsackievirus A20, in the context of a chimeric virus. Poliovirus 2CATPasehas important roles both in RNA replication and encapsidation. In this study, we searched for additional sites in 2CATPase, near N252, that are required for encapsidation. Accordingly, segments adjacent to N252were analyzed by combining triple and single alanine mutations to identify residues required for function. Two triple alanine mutants exhibited defects in RNA replication. The remaining two mutations, located in secondary structures in a predicted three-dimensional model of 2CATPase, caused lethal growth phenotypes. Most single alanine mutants, derived from the lethal variants, were either quasi-infectious and yielded variants with wild-type (wt) or temperature-sensitive (ts) growth phenotypes or had a lethal growth phenotype due to defective RNA replication. The K259A mutation, mapping to an α helix in the predicted structure of 2CATPase, resulted in a cold-sensitive virus.In vivoprotein synthesis and virus production were strikingly delayed at 33°C relative to the wt, suggesting a defect in uncoating. Studies with a reporter virus indicated that this mutant is also defective in encapsidation at 33°C. Cell imaging confirmed a much-reduced production of K259A mature virus at 33°C relative to the wt. In conclusion, we have for the first time linked a cold-sensitive encapsidation defect in 2CATPase(K259A) to a subsequent delay in uncoating of the virus particle at 33°C during the next cycle of infection.IMPORTANCEEnterovirus morphogenesis, which involves the encapsidation of newly made virion RNA, is a process still poorly understood. Elucidation of this process is important for future drug development for a large variety of diseases caused by these agents. We have previously shown that the specificity of encapsidation of poliovirus and of C-cluster coxsackieviruses, which are prototypes of enteroviruses, is dependent on an interaction of capsid proteins with the multifunctional nonstructural protein 2CATPase. In this study, we have searched for residues in poliovirus 2CATPase, near a presumed capsid-interacting site, important for encapsidation. An unusual cold-sensitive mutant of 2CATPasepossessed a defect in encapsidation at 37°C and subsequently in uncoating during the next cycle of infection at 33°C. These studies not only reveal a new site in 2CATPasethat is involved in encapsidation but also identify a link between encapsidation and uncoating.
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Okai, Haruka, Ryoko Ikema, Hiroki Nakamura, Mei Kato, Misako Araki, Ayumi Mizuno, Atsuko Ikeda, Paul Renbaum, Reeval Segel, and Kouichi Funato. "Cold‐sensitive phenotypes of a yeast null mutant of ARV1 support its role as a GPI flippase." FEBS Letters 594, no. 15 (June 11, 2020): 2431–39. http://dx.doi.org/10.1002/1873-3468.13843.
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Li, Dan, David Mahoudjro Bodjrenou, Shuting Zhang, Bin Wang, Hong Pan, Kai-Wun Yeh, Zhongxiong Lai, and Chunzhen Cheng. "The Endophytic Fungus Piriformospora indica Reprograms Banana to Cold Resistance." International Journal of Molecular Sciences 22, no. 9 (May 7, 2021): 4973. http://dx.doi.org/10.3390/ijms22094973.
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Banana (Musa spp.), one of the most important fruits worldwide, is generally cold sensitive. In this study, by using the cold-sensitive banana variety Tianbaojiao (Musa acuminate) as the study material, we investigated the effects of Piriformospora indica on banana cold resistance. Seedlings with and without fungus colonization were subjected to 4 °C cold treatment. The changes in plant phenotypes, some physiological and biochemical parameters, chlorophyll fluorescence parameters, and the expression of eight cold-responsive genes in banana leaves before and after cold treatment were measured. Results demonstrated that P. indica colonization reduced the contents of malondialdehyde (MDA) and hydrogen peroxide (H2O2) but increased the activities of superoxide dismutase (SOD) and catalase (CAT) and the contents of soluble sugar (SS) and proline. Noteworthily, the CAT activity and SS content in the leaves of P. indica-colonized banana were significant (p < 0.05). After 24 h cold treatment, the decline in maximum photochemistry efficiency of photosystem II (Fv/Fm), photochemical quenching coefficient (qP), efficient quantum yield [Y(II)], and photosynthetic electron transport rate (ETR) in the leaves of P. indica-colonized banana was found to be lower than in the non-inoculated controls (p < 0.05). Moreover, although the difference was not significant, P. indica colonization increased the photochemical conversion efficiency and electron transport rate and alleviated the damage to the photosynthetic reaction center of banana leaves under cold treatment to some extent. Additionally, the expression of the most cold-responsive genes in banana leaves was significantly induced by P. indica during cold stress (p < 0.05). It was concluded that P. indica confers banana with enhanced cold resistance by stimulating antioxidant capacity, SS accumulation, and the expression of cold-responsive genes in leaves. The results obtained from this study are helpful for understanding the P. indica-induced cold resistance in banana.
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Igoshin, Alexander, Nikolay Yudin, Ruslan Aitnazarov, Andrey A. Yurchenko, and Denis M. Larkin. "Whole-Genome Resequencing Points to Candidate DNA Loci Affecting Body Temperature under Cold Stress in Siberian Cattle Populations." Life 11, no. 9 (September 13, 2021): 959. http://dx.doi.org/10.3390/life11090959.
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Despite the economic importance of creating cold resilient cattle breeds, our knowledge of the genetic basis of adaptation to cold environments in cattle is still scarce compared to information on other economically important traits. Herein, using whole-genome resequencing of animals showing contrasting phenotypes on temperature maintenance under acute cold stress combined with the existing SNP (single nucleotide polymorphism) functional annotations, we report chromosomal regions and candidate SNPs controlling body temperature in the Siberian cattle populations. The SNP ranking procedure based on regional FST calculations, functional annotations, and the allele frequency difference between cold-tolerant and cold-sensitive groups of animals pointed to multiple candidate genes. Among these, GRIA4, COX17, MAATS1, UPK1B, IFNGR1, DDX23, PPT1, THBS1, CCL5, ATF1, PLA1A, PRKAG1, and NR1I2 were previously related to thermal adaptations in cattle. Other genes, for example KMT2D and SNRPA1, are known to be related to thermogenesis in mice and cold adaptation in common carp, respectively. This work could be useful for cattle breeding strategies in countries with harsh climates, including the Russian Federation.
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Ursic, D., J. C. Sedbrook, K. L. Himmel, and M. R. Culbertson. "The essential yeast Tcp1 protein affects actin and microtubules." Molecular Biology of the Cell 5, no. 10 (October 1994): 1065–80. http://dx.doi.org/10.1091/mbc.5.10.1065.
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Previously, we showed that the yeast Saccharomyces cerevisiae cold-sensitive mutation tcp1-1 confers growth arrest concomitant with cytoskeletal disorganization and disruption of microtubule-mediated processes. We have identified two new recessive mutations, tcp1-2 and tcp1-3, that confer heat- and cold-sensitive growth. Cells carrying tcp1 alleles were analyzed after exposure to the appropriate restrictive temperatures by cell viability tests, differential contrast microscopy, fluorescent, and immunofluorescent microscopy of DNA, tubulin, and actin and by determining the DNA content per cell. All three mutations conferred unique phenotypes indicative of cytoskeletal dysfunction. A causal relationship between loss of Tcp1p function and the development of cytoskeletal abnormalities was established by double mutant analyses. Novel phenotypes indicative of allele-specific genetic interactions were observed when tcp1-1 was combined in the same strain with tub1-1, tub2-402, act1-1, and act1-4, but not with other tubulin or actin mutations or with mutations in other genes affecting the cytoskeleton. Also, overproduction of wild-type Tcp1p partially suppressed growth defects conferred by act1-1 and act1-4. Furthermore, Tcp1p was localized to the cytoplasm and the cell cortex. Based on our results, we propose that Tcp1p is required for normal development and function of actin and microtubules either through direct or indirect interaction with the major cytoskeletal components.
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Patterson, Bruce, and James A. Spudich. "Cold-Sensitive Mutations of Dictyostelium Myosin Heavy Chain Highlight Functional Domains of the Myosin Motor." Genetics 143, no. 2 (June 1, 1996): 801–10. http://dx.doi.org/10.1093/genetics/143.2.801.
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Abstract Dictyostelium provides a powerful environment for characterization of myosin II function. It provides wellestablished biochemical methods for in vitro analysis of myosin's properties as well as an array of molecular genetic tools. The absence of myosin function results in an array of phenotypes that can be used to genetically manipulate myosin function. We have previously reported methods for the isolation and identification of rapideffect cold-sensitive myosin II mutations in Dictyostelium. Here, we report the development and utilization of a rapid method for localizing these point mutations. We have also sequenced 19 mutants. The mutations show distinct clustering with respect to three-dimensional location and biochemically characterized functional domains of the protein. We conclude that these mutants represent powerful tools for understanding the mechanisms driving this protein motor.
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Dutton, F. L., and A. Chovnick. "The l(3)S12 locus of Drosophila melanogaster: heterochromatic position effects and stage-specific misexpression of the gene in P element transposons." Genetics 128, no. 1 (May 1, 1991): 103–18. http://dx.doi.org/10.1093/genetics/128.1.103.
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Abstract l(3)S12 is a vital locus whose function is required in embryos, early larvae, late pupae and oogenesis. We have identified a cold-sensitive allele, l(3)S12(3), and characterized conditional misexpression of the gene associated with this mutation as well as with several euchromatic insertions of l(3)S12+ transposons. Surviving cold-sensitive mutants as well as underexpression variants generated by P element transformation display a phenotypic syndrome that can include delayed development, abnormal bristle morphology, and female sterility. Using these phenotypes, defects in putative "early" and "late" l(3)S12 expression can be identified. The sensitivity of certain l(3)S12+ insertions to site-specific euchromatic position effect appears to be due to separation of the gene from an endogenous enhancer element during cloning. This enhancerless construct can be used to identify and perhaps to select "permissive" euchromatic sites, presumably adjacent to enhancer elements, which in some cases permit elevated production not only of the l(3)S12 message, but also of a P element-l(3)S12 fusion transcript. Certain of these permissive sites appear to control stage-specific expression, and we propose that this system may be used to identify, clone, and characterize such loci. Heterochromatic position effect on this locus has been demonstrated. Available evidence suggests that the l(3)S12 gene may be involved in protein synthesis, perhaps encoding a ribosomal protein.
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Zhou, Bin, Victoria A. Meliopoulos, Wei Wang, Xudong Lin, Karla M. Stucker, Rebecca A. Halpin, Timothy B. Stockwell, Stacey Schultz-Cherry, and David E. Wentworth. "Reversion of Cold-Adapted Live Attenuated Influenza Vaccine into a Pathogenic Virus." Journal of Virology 90, no. 19 (July 20, 2016): 8454–63. http://dx.doi.org/10.1128/jvi.00163-16.
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ABSTRACTThe only licensed live attenuated influenza A virus vaccines (LAIVs) in the United States (FluMist) are created using internal protein-coding gene segments from the cold-adapted temperature-sensitive master donor virus A/Ann Arbor/6/1960 and HA/NA gene segments from circulating viruses. During serial passage of A/Ann Arbor/6/1960 at low temperatures to select the desired attenuating phenotypes, multiple cold-adaptive mutations and temperature-sensitive mutations arose. A substantial amount of scientific and clinical evidence has proven that FluMist is safe and effective. Nevertheless, no study has been conducted specifically to determine if the attenuating temperature-sensitive phenotype can revert and, if so, the types of substitutions that will emerge (i.e., compensatory substitutions versus reversion of existing attenuating mutations). Serial passage of the monovalent FluMist 2009 H1N1 pandemic vaccine at increasing temperaturesin vitrogenerated a variant that replicated efficiently at higher temperatures. Sequencing of the variant identified seven nonsynonymous mutations, PB1-E51K, PB1-I171V, PA-N350K, PA-L366I, NP-N125Y, NP-V186I, and NS2-G63E. None occurred at positions previously reported to affect the temperature sensitivity of influenza A viruses. Synthetic genomics technology was used to synthesize the whole genome of the virus, and the roles of individual mutations were characterized by assessing their effects on RNA polymerase activity and virus replication kinetics at various temperatures. The revertant also regained virulence and caused significant disease in mice, with severity comparable to that caused by a wild-type 2009 H1N1 pandemic virus.IMPORTANCEThe live attenuated influenza vaccine FluMist has been proven safe and effective and is widely used in the United States. The phenotype and genotype of the vaccine virus are believed to be very stable, and mutants that cause disease in animals or humans have never been reported. By propagating the virus under well-controlled laboratory conditions, we found that the FluMist vaccine backbone could regain virulence to cause severe disease in mice. The identification of the responsible substitutions and elucidation of the underlying mechanisms provide unique insights into the attenuation of influenza virus, which is important to basic research on vaccines, attenuation reversion, and replication. In addition, this study suggests that the safety of LAIVs should be closely monitored after mass vaccination and that novel strategies to continue to improve LAIV vaccine safety should be investigated.
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Miao, Mingjun, Huaqiang Tan, Le Liang, Haitao Huang, Wei Chang, Jianwei Zhang, Ju Li, et al. "Comparative transcriptome analysis of cold-tolerant and -sensitive asparagus bean under chilling stress and recovery." PeerJ 10 (March 22, 2022): e13167. http://dx.doi.org/10.7717/peerj.13167.
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Background Low temperature is a type of abiotic stress that threatens the growth and yield of asparagus bean. However, the key genes and regulatory pathways involved in low temperature response in this legume are still poorly understood. Methodology. The present study analyzed the transcriptome of seedlings from two asparagus bean cultivars—Dubai bean and Ningjiang 3—using Illumina RNA sequencing (RNA-seq). Correlations between samples were determined by calculating Pearson correlation coefficients (PCC) and principal component analysis (PCA). Differentially expressed genes (DEGs) between two samples were identified using the DESeq package. Transcription factors (TF) prediction, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of DEGs were also performed. Results Phenotypes and physiological indices indicated that Ningjiang 3 seedlings tolerated cold better than Dubai bean seedlings, in contrast to adult stage. The transcriptome dynamics of the two cultivars were closely compared using Illumina RNA-seq following 0, 3, 12, and 24 h of cold stress at 5 °C and recovery for 3 h at 25 °C room temperature. Global gene expression patterns displayed relatively high correlation between the two cultivars (>0.88), decreasing to 0.79 and 0.81, respectively, at 12 and 24 h of recovery, consistent with the results of principal component analysis. The major transcription factor families identified from differentially expressed genes between the two cultivars included bHLH, NAC, C2H2, MYB, WRKY, and AP2/ERF. The representative GO enrichment terms were protein phosphorylation, photosynthesis, oxidation-reduction process, and cellular glucan metabolic process. Moreover, KEGG analysis of DEGs within each cultivar revealed 36 transcription factors enriched in Dubai bean and Ningjiang 3 seedlings under cold stress. Conclusions These results reveal new information that will improve our understanding of the molecular mechanisms underlying the cold stress response of asparagus bean and provide genetic resources for breeding cold-tolerant asparagus bean cultivars.
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Campodonico, Eva, and Beate Schwer. "ATP-Dependent Remodeling of the Spliceosome: Intragenic Suppressors of Release-Defective Mutants of Saccharomyces cerevisiae Prp22." Genetics 160, no. 2 (February 1, 2002): 407–15. http://dx.doi.org/10.1093/genetics/160.2.407.
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Abstract The essential splicing factor Prp22 is a DEAH-box helicase that catalyzes the release of mRNA from the spliceosome. ATP hydrolysis by Prp22 is necessary but not sufficient for spliceosome disassembly. Previous work showed that mutations in motif III (635SAT637) of Prp22 that uncouple ATP hydrolysis from spliceosome disassembly lead to severe cold-sensitive (cs) growth defects and to impaired RNA unwinding activity in vitro. The cs phenotype of S635A (635AAT) can be suppressed by intragenic mutations that restore RNA unwinding. We now report the isolation and characterization of new intragenic mutations that suppress the cold-sensitive growth phenotypes of the T637A motif III mutation (SAA), the H606A mutation in the DEAH-box (DEAA), and the R805A mutation in motif VI (804QAKGRAGR811). Whereas the T637A and H606A proteins are deficient in releasing mRNA from the spliceosome at nonpermissive temperature in vitro, the suppressor proteins have recovered mRNA release activity. To address the mechanisms of suppression, we tested ATPase and helicase activities of Prp22 suppressor mutant proteins and found that the ability to unwind a 25-bp RNA duplex was not restored in every case. This finding suggests that release of mRNA from the spliceosome is less demanding than unwinding of a 25-bp duplex RNA; the latter reaction presumably reflects the result of several successive cycles of ATP binding, hydrolysis, and unwinding. Increasing the reaction temperature allows H606A and T637A to effect mRNA release in vitro, but does not restore RNA unwinding by T637A.
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Lux, F. G., and S. K. Dutcher. "Genetic interactions at the FLA10 locus: suppressors and synthetic phenotypes that affect the cell cycle and flagellar function in Chlamydomonas reinhardtii." Genetics 128, no. 3 (July 1, 1991): 549–61. http://dx.doi.org/10.1093/genetics/128.3.549.
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Abstract Through the isolation of suppressors of temperature-sensitive flagellar assembly mutations at the FLA10 locus of Chlamydomonas reinhardtii, we have identified six other genes involved in flagellar assembly. Mutations at these suppressor loci, termed SUF1-SUF6, display allele specificity with respect to which fla10- mutant alleles they suppress. An additional mutation, apm1-122, which confers resistance to the plant herbicides amiprophos-methyl and oryzalin, was also found to interact with mutations at the FLA10 locus. The apm1-122 mutation in combination with three fla10- mutant alleles results in synthetic cold-sensitive cell division defects, and in combination with an additional pseudo-wild-type fla10- allele yields a synthetic temperature-sensitive flagellar motility phenotype. Based upon the genetic interactions of these loci, we propose that the FLA10 gene product interacts with multiple components of the flagellar apparatus and plays a role both in flagellar assembly and in the cell cycle.
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Adachi, Y., and M. Yanagida. "Higher order chromosome structure is affected by cold-sensitive mutations in a Schizosaccharomyces pombe gene crm1+ which encodes a 115-kD protein preferentially localized in the nucleus and its periphery." Journal of Cell Biology 108, no. 4 (April 1, 1989): 1195–207. http://dx.doi.org/10.1083/jcb.108.4.1195.
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We isolated a novel class of Schizosaccharomyces pombe cold-sensitive mutants with deformed nuclear chromosome domains consisting of thread- or rodlike condensed segments at restrictive temperature. Their mutations were mapped in a novel, identical locus designated crm1 (chromosomal region maintenance). The crm1 mutants also show the following phenotypes. DNA, RNA, and protein syntheses diminish at restrictive temperature. At permissive temperature, the amount of one particular protein, p25, greatly increases. The mutant growth is hypersensitive to Ca2+ and resistant to protein kinase inhibitors. We cloned the 4.1-kb-long crm1+ gene that rescued the above phenotypes by transformation and determined its nucleotide sequence, which predicts a 1,077-residue protein. Affinity-purified antiserum raised against the crm1+ polypeptide expressed in Escherichia coli detected a 115-kD protein in S. pombe extracts. Genomic Southern hybridization and immunoblotting suggested that the crm1+ product might be highly conserved in distant organisms. Through immunofluorescence microscopy, the crm1+ protein appeared to be principally localized within the nucleus and also at its periphery. We speculate that the crm1+ protein might be one of those nuclear components that modify the chromosome structures or regulate the nuclear environment required for maintaining higher order chromosome structures.
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Habibu, Buhari, Lukuman Surakat Yaqub, Tavershima Dzenda, and Mohammed Umaru Kawu. "Sensitivity, Impact and Consequences of Changes in Respiratory Rate During Thermoregulation in Livestock – A Review." Annals of Animal Science 19, no. 2 (April 1, 2019): 291–304. http://dx.doi.org/10.2478/aoas-2019-0002.
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AbstractThis review discusses the thermal conservative and heat dissipating roles of one of the most sensitive thermoregulatory variables (respiratory rate) with the aim of enhancing its application in evaluating both cold and heat adaptation. During cold exposure, livestock enhance the economy of body heat through reduction in respiratory rate with the extent of reduction being greater and commencing at relatively higher ambient temperature in poorly adapted phenotypes. This is accompanied by an increase in tidal volume and alveolar oxygen uptake, but a decrease in partial pressure of oxygen. On the other hand, heat stress induces increase in respiratory rate to enhance evaporative heat loss with the magnitude of such increase being greater and commencing at relatively lower ambient temperature in phenotypes that are poorly-adapted to heat. This is accompanied by a decrease in tidal volume and the development of hypocapnia. The increase in respiratory rate is observed to be greater, moderate and lesser in livestock that are mainly (pigs, rabbits and poultry), moderately (sheep, goats and Bos taurus) and less (Zebu cattle) dependent on respiratory evaporative heat loss, respectively. The changes during chronic heat stress may cause acid-base crisis in all livestock, in addition to reduction in eggshell quality in birds; due to marked decrease in partial pressure of carbon dioxide and a compensatory increase in elimination of bicarbonate. Within and between breed variations in sensitivity of respiratory rhythm to both cold and heat stress has shown high applicability in identifying phenotypes that are more susceptible to thermal stress; with some cellular and metabolic changes occurring to protect the animal from the consequences of hypo- or hyper-thermia. The information in this review may provide basis for identification of genes that support or suppress thermoregulation and may also be of great use in animal breeding, genomics and selective thermal stress mitigation to provide maximum protection and comfort to poorly-adapted phenotypes.
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Voelker, R. A., S. M. Huang, G. B. Wisely, J. F. Sterling, S. P. Bainbridge, and K. Hiraizumi. "Molecular and genetic organization of the suppressor of sable and minute (1) 1B region in Drosophila melanogaster." Genetics 122, no. 3 (July 1, 1989): 625–42. http://dx.doi.org/10.1093/genetics/122.3.625.
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Abstract Recessive mutations at the suppressor of sable [su(s)] locus in Drosophila melanogaster result in suppression of second site mutations caused by insertions of the mobile element 412. In order to determine whether su(s) mutations might have other phenotypes, a saturation mapping of the su(s) region was carried out. The screen yielded 76 mutations that comprise ten genetic complementation groups ordered distal to proximal as follows: l(1)1Bh, l(1)1Bi, M(1)1B, su(s), l(1)1Bk, l(1)1Ca, mul, tw, l(1)lDa and brc. Twenty-three of the mutations are su(s) alleles, and all are suppressors of the 412-insertion-caused v1 allele. Although the screen could have detected su(s) mutations causing sex-specific dominant lethality or sterility as well as all types of recessive lethality or sterility, the only other phenotype observed was male sterility that is enhanced by cold temperature. This type of sterility is exhibited only by alleles induced by base-substitution-causing mutagens. Genetic functions of the poly(A+) messages transcribed from the su(s) microregion were identified by the reintroduction of cloned sequences into embryos by P element transformation. su(s) function has been attributed to a 5-kb message. The segment of DNA encoding only this 5-kb message rescues both the suppression and cold-sensitive male sterility phenotypes of su(s). Minute (1) 1B has been provisionally identified as encoding a 3.5-kb message; lethal (1)1Bi encodes a 1-kb message; and lethal (1)1Bk encodes a 4-kb message. The possible functions of su(s) and M(1)1B are discussed.
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Schatz, P. J., F. Solomon, and D. Botstein. "Isolation and characterization of conditional-lethal mutations in the TUB1 alpha-tubulin gene of the yeast Saccharomyces cerevisiae." Genetics 120, no. 3 (November 1, 1988): 681–95. http://dx.doi.org/10.1093/genetics/120.3.681.
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Abstract Microtubules in yeast are functional components of the mitotic and meiotic spindles and are essential for nuclear movement during cell division and mating. We have isolated 70 conditional-lethal mutations in the TUB1 alpha-tubulin gene of the yeast Saccharomyces cerevisiae using a plasmid replacement technique. Of the 70 mutations isolated, 67 resulted in cold-sensitivity, one resulted in temperature-sensitivity, and two resulted in both. Fine-structure mapping revealed that the mutations were located throughout the TUB1 gene. We characterized the phenotypes caused by 38 of the mutations after shifts of mutants to the nonpermissive temperature. Populations of temperature-shifted mutant cells contained an excess of large-budded cells with undivided nuclei, consistent with the previously determined role of microtubules in yeast mitosis. Several of the mutants arrested growth with a sufficiently uniform morphology to indicate that TUB1 has at least one specific role in the progression of the yeast cell cycle. A number of the mutants had gross defects in microtubule assembly at the restrictive temperature, some with no microtubules and some with excess microtubules. Other mutants contained disorganized microtubules and nuclei. There were no obvious correlations between these phenotypes and the map positions of the mutations. Greater than 90% of the mutants examined were hypersensitive to the antimicrotubule drug benomyl. Mutations that suppressed the cold-sensitive phenotypes of two of the TUB1 alleles occurred in TUB2, the single structural gene specifying beta-tubulin.
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Hirano, H., K. Tanaka, K. Ozaki, H. Imamura, H. Kohno, T. Hihara, T. Kameyama, et al. "ROM7/BEM4 encodes a novel protein that interacts with the Rho1p small GTP-binding protein in Saccharomyces cerevisiae." Molecular and Cellular Biology 16, no. 8 (August 1996): 4396–403. http://dx.doi.org/10.1128/mcb.16.8.4396.
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The RHO1 gene encodes a homolog of the mammalian RhoA small GTP-binding protein in the yeast Saccharomyces cerevisiae. Rho1p is localized at the growth site and is required for bud formation. The RHO1(G22S, D125N) mutation is a temperature-sensitive and dominant negative mutation of RHO1, and a multicopy suppressor of RHO1(G22S, D125N), ROM7, was isolated. Nucleotide sequencing of ROM7 revealed that it is identical to the BEM4 gene (GenBank accession number L27816), although its physiological function has not yet been reported. Disruption of BEM4 resulted in the cold- and temperature-sensitive growth phenotypes, and cells of the deltabem4 mutant showed abnormal morphology, suggesting that BEM4 is involved in the budding process. The temperature-sensitive growth phenotype was suppressed by overexpression of RHO1, ROM2, which encodes a Rho1p-specific GDP/GTP exchange factor, or PKC1, which encodes a target of Rho1p. Moreover, glucan synthase activity, which is activated by Rho1p, was significantly reduced in the deltabem4 mutant. Two-hybrid and biochemical experiments revealed that Bem4p directly interacts with the nucleotide-free form of Rho1p and, to lesser extents, with the GDP- and GTP-bound forms of Rho1p, although Bem4p showed neither GDP/GTP exchange factor, GDP dissociation inhibitor, nor GTPase-activating protein activity toward Rho1p. These results indicate that Bem4p is a novel protein directly interacting with Rho1p and is involved in the RHO1-mediated signaling pathway.
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Jin, Hong, Helen Zhou, Bin Lu, and George Kemble. "Imparting Temperature Sensitivity and Attenuation in Ferrets to A/Puerto Rico/8/34 Influenza Virus by Transferring the Genetic Signature for Temperature Sensitivity from Cold-Adapted A/Ann Arbor/6/60." Journal of Virology 78, no. 2 (January 15, 2004): 995–98. http://dx.doi.org/10.1128/jvi.78.2.995-998.2004.
Full textAbstract:
ABSTRACT The four temperature-sensitive (ts) loci identified in the PB1 and PB2 gene segments of cold-adapted A/Ann Arbor/6/60 influenza virus, the master donor virus for influenza A virus (MDV-A) FluMist vaccines, were introduced into a divergent A/Puerto Rico/8/34 influenza virus strain. Recombinant A/Puerto Rico/8/34 virus with these four introduced ts loci exhibited both ts and att phenotypes similar to those of MDV-A, which could be used as a donor virus for manufacturing large quantities of inactivated influenza virus vaccine against potential pandemic strains.
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Cyril, Jenith, R. R. Duncan, and W. V. Baird. "Changes in Membrane Fatty Acids in Cold-acclimated Turfgrass." HortScience 33, no. 3 (June 1998): 453c—453. http://dx.doi.org/10.21273/hortsci.33.3.453c.
Full textAbstract:
Three genotypes of seashore paspalum, `PI 299042', `Adalayd', and `PI 509018-1' considered to be cold-sensitive, intermediately cold-tolerant and cold-tolerant, respectively, were analyzed to investigate the biochemical basis of cold tolerance. The cultivars were acclimated to 8/4 °C day/night temperatures and rhizomes nodes and crowns were harvested at 7-day intervals over the 4-week experiment. Total lipid was extracted from these tissues, and the fatty acids present in the lipid fraction were identified by gas chromatography. Palmitic acid, stearic acid, linoleic acid and linolenic acid were the major fatty acids present. In cold acclimated tissues, the level of palmitic acid and stearic acid did not change significantly during the treatment period. There was a decrease in the level of linoleic acid by the second week of cold treatment. The amount of linolenic acid increased significantly during the second week of cold treatment corresponding to the decrease in linoleic acid. The change in the amount was significantly greater in `PI 509018-1' than in `Adalayd' or `PI 299042'. These results are similar to what was found for cultivars of bermudagrass that differ in their cold-tolerance phenotypes. Desaturases are enzymes involved in introducing the double bonds into the fatty acid chains. Research is underway to characterize and clone the genes encoding the Ω3, Ω6, and Ω9 desaturases, which may have an important role in affecting the cold tolerance by altering the degree of membrane lipid fatty acid saturation.
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Moritz, M., B. A. Pulaski, and J. L. Woolford. "Assembly of 60S ribosomal subunits is perturbed in temperature-sensitive yeast mutants defective in ribosomal protein L16." Molecular and Cellular Biology 11, no. 11 (November 1991): 5681–92. http://dx.doi.org/10.1128/mcb.11.11.5681-5692.1991.
Full textAbstract:
Temperature-sensitive mutants defective in 60S ribosomal subunit protein L16 of Saccharomyces cerevisiae were isolated through hydroxylamine mutagenesis of the RPL16B gene and plasmid shuffling. Two heat-sensitive and two cold-sensitive isolates were characterized. The growth of the four mutants is inhibited at their restrictive temperatures. However, many of the cells remain viable if returned to their permissive temperatures. All of the mutants are deficient in 60S ribosomal subunits and therefore accumulate translational preinitiation complexes. Three of the mutants exhibit a shortage of mature 25S rRNA, and one accumulates rRNA precursors. The accumulation of rRNA precursors suggests that ribosome assembly may be slowed in this mutant. These phenotypes lead us to propose that mutants containing the rpl16b alleles are defective for 60S subunit assembly rather than function. In the mutant carrying the rpl16b-1 allele, ribosomes initiate translation at the noncanonical codon AUA, at least on the rpl16b-1 mRNA, bringing to light a possible connection between the rate and the fidelity of translation initiation.
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Moritz, M., B. A. Pulaski, and J. L. Woolford. "Assembly of 60S ribosomal subunits is perturbed in temperature-sensitive yeast mutants defective in ribosomal protein L16." Molecular and Cellular Biology 11, no. 11 (November 1991): 5681–92. http://dx.doi.org/10.1128/mcb.11.11.5681.
Full textAbstract:
Temperature-sensitive mutants defective in 60S ribosomal subunit protein L16 of Saccharomyces cerevisiae were isolated through hydroxylamine mutagenesis of the RPL16B gene and plasmid shuffling. Two heat-sensitive and two cold-sensitive isolates were characterized. The growth of the four mutants is inhibited at their restrictive temperatures. However, many of the cells remain viable if returned to their permissive temperatures. All of the mutants are deficient in 60S ribosomal subunits and therefore accumulate translational preinitiation complexes. Three of the mutants exhibit a shortage of mature 25S rRNA, and one accumulates rRNA precursors. The accumulation of rRNA precursors suggests that ribosome assembly may be slowed in this mutant. These phenotypes lead us to propose that mutants containing the rpl16b alleles are defective for 60S subunit assembly rather than function. In the mutant carrying the rpl16b-1 allele, ribosomes initiate translation at the noncanonical codon AUA, at least on the rpl16b-1 mRNA, bringing to light a possible connection between the rate and the fidelity of translation initiation.
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Imai, Jun, Akio Toh-e, and Yasushi Matsui. "Genetic Analysis of the Saccharomyces cerevisiae RHO3 Gene, Encoding a Rho-Type Small GTPase, Provides Evidence for a Role in Bud Formation." Genetics 142, no. 2 (February 1, 1996): 359–69. http://dx.doi.org/10.1093/genetics/142.2.359.
Full textAbstract:
Abstract RHO3 encodes a Rho-type small GTPase of the yeast Saccharomyces cereuisiae. We isolated temperature-sensitive alleles and a dominant active allele of RHO3. Ts- rho3 cells lost cell polarity during bud formation and grew more isotropically than wild-type cells at nonpermissive temperatures. In contrast, cells carrying a dominant active mutant RHO3 displayed cold sensitivity, and the cells became elongated and bent, often at the position where actin patches were concentrated. These phenotypes of the rho3 mutants strongly suggest that RHO3 is involved in directing the growing points during bud formation. In addition, we found that SRO6, previously isolated as a multicopy suppressor of rho3, is the same as SEC4. The sec4-2 mutation was synthetic lethal with temperature-sensitive rho3 mutations and suppressed the cold sensitivity caused by a dominant active mutant RHO3. The genetic interactions between RHO3 and SEC4, taken together with the fact that the Rab-type GTPase Sec4p is required to fuse secretory vesicles together with plasma membrane for exocytosis, support a model in which the Rho3p pathway modulates morphogenesis during bud growth via directing organization of the actin cytoskeleton and the position of the secretory machinery for exocytosis.
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Phan, Huy, and Michael Schläppi. "Low Temperature Antioxidant Activity QTL Associate with Genomic Regions Involved in Physiological Cold Stress Tolerance Responses in Rice (Oryza sativa L.)." Genes 12, no. 11 (October 26, 2021): 1700. http://dx.doi.org/10.3390/genes12111700.
Full textAbstract:
Boosting cold stress tolerance in crop plants can minimize stress-mediated yield losses. Asian rice (Oryza sativa L.), one of the most consumed cereal crops, originated from subtropical regions and is generally sensitive to low temperature environments. Within the two subspecies of rice, JAPONICA, and INDICA, the cold tolerance potential of its accessions is highly variable and depends on their genetic background. Yet, cold stress tolerance response mechanisms are complex and not well understood. This study utilized 370 accessions from the Rice Diversity Panel 1 (RDP1) to investigate and correlate four cold stress tolerance response phenotypes: membrane damage, seedling survivability, and catalase and anthocyanin antioxidative activity. Most JAPONICA accessions, and admixed accessions within JAPONICA, had lower membrane damage, higher antioxidative activity, and overall, higher seedling survivability compared to INDICA accessions. Genome-wide association study (GWAS) mapping was done using the four traits to find novel quantitative trait loci (QTL), and to validate and fine-map previously identified QTL. A total of 20 QTL associated to two or more traits were uncovered by our study. Gene Ontology (GO) term enrichment analyses satisfying four layers of filtering retrieved three potential pathways: signal transduction, maintenance of plasma membrane and cell wall integrity, and nucleic acids metabolism as general mechanisms of cold stress tolerance responses involving antioxidant activity.
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Kirkpatrick, D., and F. Solomon. "Overexpression of yeast homologs of the mammalian checkpoint gene RCC1 suppresses the class of alpha-tubulin mutations that arrest with excess microtubules." Genetics 137, no. 2 (June 1, 1994): 381–92. http://dx.doi.org/10.1093/genetics/137.2.381.
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Abstract Microtubules in eukaryotic cells participate in a variety of nuclear and cytoplasmic structures, reflecting functional requirements and cell cycle position. We are studying the cellular regulation of microtubule assembly and organization in the yeast Saccharomyces cerevisiae. We screened for genes that when overexpressed suppress the growth phenotype of conditional mutants in alpha-tubulin that arrest with excess microtubules at the nonpermissive temperature (class 2 mutations). Here we describe one such suppressing element, called ATS1 (for Alpha Tubulin Suppressor). Overexpression of this gene rescues both the growth and microtubule phenotypes of all class 2 mutations, but not the cold-sensitive mutations that arrest with no microtubules (class 1 mutations). Deletion of ATS1 confers a modest slow growth phenotype which is slightly enhanced in strains containing both a deletion of ATS1 and a class 2 tub 1 mutation. The predicted ATS1 protein contains 333 amino acids and has considerable structural homology to the products of both the mammalian mitotic control gene RCC1 and the S. cerevisiae gene SRM1/PRP20. Overexpression of SRM1/PRP20 also suppresses class 2 mutants. The results suggest that this family of genes may participate in regulatory interactions between microtubules and the cell cycle.
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Budd, Martin E., Clara C. Reis, Stephanie Smith, Kyungjae Myung, and Judith L. Campbell. "Evidence Suggesting that Pif1 Helicase Functions in DNA Replication with the Dna2 Helicase/Nuclease and DNA Polymerase δ." Molecular and Cellular Biology 26, no. 7 (April 1, 2006): 2490–500. http://dx.doi.org/10.1128/mcb.26.7.2490-2500.2006.
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ABSTRACT The precise machineries required for two aspects of eukaryotic DNA replication, Okazaki fragment processing (OFP) and telomere maintenance, are poorly understood. In this work, we present evidence that Saccharomyces cerevisiae Pif1 helicase plays a wider role in DNA replication than previously appreciated and that it likely functions in conjunction with Dna2 helicase/nuclease as a component of the OFP machinery. In addition, we show that Dna2, which is known to associate with telomeres in a cell-cycle-specific manner, may be a new component of the telomere replication apparatus. Specifically, we show that deletion of PIF1 suppresses the lethality of a DNA2-null mutant. The pif1Δ dna2Δ strain remains methylmethane sulfonate sensitive and temperature sensitive; however, these phenotypes can be suppressed by further deletion of a subunit of pol δ, POL32. Deletion of PIF1 also suppresses the cold-sensitive lethality and hydroxyurea sensitivity of the pol32Δ strain. Dna2 is thought to function by cleaving long flaps that arise during OFP due to excessive strand displacement by pol δ and/or by an as yet unidentified helicase. Thus, suppression of dna2Δ can be rationalized if deletion of POL32 and/or PIF1 results in a reduction in long flaps that require Dna2 for processing. We further show that deletion of DNA2 suppresses the long-telomere phenotype and the high rate of formation of gross chromosomal rearrangements in pif1Δ mutants, suggesting a role for Dna2 in telomere elongation in the absence of Pif1.