Sequential cold-sensitive mutations in Aspergillus fumigatus. II. Analysis by the parasexual cycle

1981 ◽  
Vol 27 (3) ◽  
pp. 295-303 ◽  
Author(s):  
Wayne L. Levadoux ◽  
Kenneth F. Gregory ◽  
Ann Taylor
Keyword(s):  
Aspergillus Fumigatus ◽  
Minimal Medium ◽  
Cold Sensitivity ◽  
Nutritional Requirements ◽  
Parasexual Cycle ◽  
Multiple Genes ◽  
Ethylmethane Sulfonate ◽  
Cold Sensitive

From Aspergillus fumigatus I-21 (ATCC 32722), which grows at temperatures from 12 to 50 °C, three multistep, independently derived, cold-sensitive mutants unable to grow at 37 °C or below (Cs-37) were obtained by sequential exposure to ethylmethane sulfonate (strain AT2) or N-methyl-N′-nitro-N-nitrosoguanidine (AT1 and AT3). These mutants and ON5, a five-step Cs-37 mutant, were marked by mutations affecting spore color and nutritional requirements and crossed in four combinations by classical parasexual means.The heterokaryons demonstrated partial complementation with respect to auxotrophic requirements (suboptimal growth on minimal medium) and cold sensitivity (growth at 37 °C but not at 25 °C). Most presumed diploids, formed by exposure of the heterokaryons to d-camphor vapors, showed complete complementation but were unstable, as demonstrated by variations in spore sizes and markedly different ratios of segregant classes derived from different clones.Analysis of the segregants of the diploids or aneuploids, induced by Benomyl, indicated that multiple genes were responsible for cold sensitivity in each Cs-37 mutant, since segregants with various levels of cold sensitivity were obtained. The higher than predicted frequency of reversion to temperatures two or more steps back in the sequence of cold sensitivity mutations suggested that these genes or their products interacted. No Cs-37 segregant yielding a consistently lower frequency of revertants than the original mutants was obtained.

Sequential cold-sensitive mutations in Aspergillus fumigatus

1978 ◽  
Vol 24 (2) ◽  
pp. 84-88 ◽  
Author(s):  
Ole Nielsen ◽  
Kenneth F. Gregory
Keyword(s):  
Aspergillus Fumigatus ◽  
Type A ◽  
Maximum Growth ◽  
Cold Sensitivity ◽  
Nonpermissive Temperature ◽  
Wild Type ◽  
Cold Sensitive ◽  
Wild Type Parent ◽  
Thermotolerant Fungus

Mutants of the thermotolerant fungus Aspergillus fumigatus I-21 (ATCC 32722) unable to grow at 37 °C were sought. Cold-sensitive mutants were enriched from progeny spores of γ-irradiated conidia by two or more incubations at various nonpermissive temperatures alternating with filtrations through cheesecloth. The approximate minimum, optimum, and maximum growth temperatures of the parent were 12, 40, and 50 °C, respectively. Mutants unable to grow at 37 °C were not successfully isolated directly from the wild type. A mutant unable to grow at 25 °C was isolated and mutations further increasing the cold sensitivity by increments of 3–5 °C were found to occur. Mutants completely unable to grow at 37 °C were obtained by five sequential mutations. All mutants grew as fast as the wild-type parent at 45 °C and higher. Each mutant produced revenants able to grow not only at the nonpermissive temperature used for its isolation but also at lower temperatures.

Sequential cold-sensitive mutations in Aspergillus fumigatus. III. Mechanism of cold sensitivity

1981 ◽  
Vol 27 (3) ◽  
pp. 304-310 ◽  
Author(s):  
Anthony J. Arseneau ◽  
Kenneth F. Gregory
Keyword(s):  
Aspergillus Fumigatus ◽  
Rna Synthesis ◽  
Feedback Inhibition ◽  
Ribosomal Subunit ◽  
Cold Sensitivity ◽  
Nonpermissive Temperature ◽  
Cold Sensitive ◽  
Ribosomal Rna Processing

The mechanism of cold sensitivity of Aspergillus fumigatus ON5, a 37 °C-sensitive mutant derived from A. fumigatus I-21 (ATCC 32722) by five sequential mutations, was investigated. The rate of in vivo protein synthesis by ON5 was not affected for 2 h following a shift from 45 to 34 °C, but the rate of in vivo RNA synthesis dropped almost immediately. The RNA polymerases of ON5 possessed wild-type activity in vitro at a nonpermissive temperature (34 °C) indicating that the reduction in the rate of in vivo RNA synthesis did not result from cold sensitivity in transcription, but was possibly a result of rapid feedback inhibition of transcription. Mutant ON5 was not able to produce ribosomes at a nonpermissive temperature as evidenced by the fact that no 3H-labelled amino acids were incorporated into the monosome, large ribosomal subunit, or small ribosomal subunit at 34 °C. Ribosomal subunit assembly or ribosomal RNA processing appears, therefore, to be the cold-sensitive cellular function in ON5.

Suppressors of a Cold-Sensitive Mutation in Yeast U4 RNA Define Five Domains in the Splicing Factor Prp8 That Influence Spliceosome Activation

Genetics ◽  
2000 ◽  
Vol 155 (4) ◽  
pp. 1667-1682 ◽  
Author(s):  
Andreas N Kuhn ◽  
David A Brow
Keyword(s):  
Large Scale ◽  
Splicing Factor ◽  
Cold Sensitivity ◽  
Splicing Factors ◽  
Loss Of Function ◽  
U1 Snrnp ◽  
Snrnp Protein ◽  
Cold Sensitive ◽  
Spliceosome Activation ◽  
U4 Rna

AbstractThe highly conserved splicing factor Prp8 has been implicated in multiple stages of the splicing reaction. However, assignment of a specific function to any part of the 280-kD U5 snRNP protein has been difficult, in part because Prp8 lacks recognizable functional or structural motifs. We have used a large-scale screen for Saccharomyces cerevisiae PRP8 alleles that suppress the cold sensitivity caused by U4-cs1, a mutant U4 RNA that blocks U4/U6 unwinding, to identify with high resolution five distinct regions of PRP8 involved in the control of spliceosome activation. Genetic interactions between two of these regions reveal a potential long-range intramolecular fold. Identification of a yeast two-hybrid interaction, together with previously reported results, implicates two other regions in direct and indirect contacts to the U1 snRNP. In contrast to the suppressor mutations in PRP8, loss-of-function mutations in the genes for two other splicing factors implicated in U4/U6 unwinding, Prp44 (Brr2/Rss1/Slt22/Snu246) and Prp24, show synthetic enhancement with U4-cs1. On the basis of these results we propose a model in which allosteric changes in Prp8 initiate spliceosome activation by (1) disrupting contacts between the U1 snRNP and the U4/U6-U5 tri-snRNP and (2) orchestrating the activities of Prp44 and Prp24.

scholarly journals Suppression of ΔbipA Phenotypes in Escherichia coli by Abolishment of Pseudouridylation at Specific Sites on the 23S rRNA

2008 ◽  
Vol 190 (23) ◽  
pp. 7675-7683 ◽  
Author(s):  
Karthik Krishnan ◽  
Ann M. Flower
Keyword(s):  
Escherichia Coli ◽  
Target Genes ◽  
Regulatory Protein ◽  
Elongation Factor ◽  
Direct Interaction ◽  
Cold Sensitivity ◽  
23S Rrna ◽  
Pseudouridine Synthase ◽  
Suppressor Effect ◽  
Cold Sensitive

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.

Evidence for the involvement of peripheral cold-sensitive TRPM8 channels in human cutaneous hygrosensation

2020 ◽  
Vol 318 (3) ◽  
pp. R579-R589 ◽  
Author(s):  
Oliver Typolt ◽  
Davide Filingeri
Keyword(s):  
Human Skin ◽  
Chemical Activation ◽  
The Body ◽  
Cold Sensitivity ◽  
Trpm8 Channel ◽  
Body Regions ◽  
Skin Cooling ◽  
Cold Sensitive ◽  
Skin Wetness

In contrast to other species, humans are believed to lack hygroreceptors for sensing skin wetness. Yet, the molecular basis of human hygrosensation is currently unknown, and it remains unclear whether we possess a receptor-mediated sensing mechanism for skin wetness. The aim of this study was to assess the role of the cutaneous cold-sensitive transient receptor potential melastatin-8 (TRPM8) channel as a molecular mediator of human hygrosensation. To this end, we exploited both the thermal and chemical activation of TRPM8-expressing cutaneous Aδ cold thermoreceptors, and we assessed wetness sensing in healthy young men in response to 1) dry skin cooling in the TRPM8 range of thermosensitivity and 2) application of the TRPM8 agonist menthol. Our results indicate that 1) independently of contact with moisture, a cold-dry stimulus in the TRPM8 range of activation induced wetness perceptions across 12 different body regions and those wetness perceptions varied across the body following regional differences in cold sensitivity; and 2) independently of skin cooling, menthol-induced stimulation of TRPM8 triggered wetness perceptions that were greater than those induced by physical dry cooling and by contact with an aqueous cream containing actual moisture. For the first time, we show that the cutaneous cold-sensing TRPM8 channel plays the dual role of cold and wetness sensor in human skin and that this ion channel is a peripheral mediator of human skin wetness perception.

scholarly journals Chromatin-Level Differences Elucidate Potential Determinants of Contrasting Levels of Cold Sensitivity in Maize Lines

2020 ◽  
Author(s):  
Maciej Jończyk ◽  
Alicja Sobkowiak ◽  
Joanna Trzcinska-Danielewicz ◽  
Paweł Sowiński
Keyword(s):  
Low Temperature ◽  
Cold Stress ◽  
Photosynthetic Apparatus ◽  
Regulatory Elements ◽  
Cold Sensitivity ◽  
Regulatory Sequences ◽  
Physiological Level ◽  
Cold Tolerant ◽  
Tolerant Line ◽  
Cold Sensitive

Abstract Maize is a subtropical, cold-sensitive species. However, some varieties of this species have the potential to withstand long-term low temperatures, even at the seedling stage. The molecular basis of this phenomenon has not been determined. In a chromatin-level study, we compared the cold-stress reaction of seedlings of two maize inbred lines showing contrasting levels of cold sensitivity. The cold-tolerant line was selected based on field data and previous physiological and transcriptomic level studies. The first condition of gene expression—chromatin accessibility—was assessed by formaldehyde-aided isolation of regulatory elements method and DNA sequencing. Potentially expressed genes and cis-regulatory sequences open for interaction with transcription factors have been defined. The results of this study suggest that during cold stress, the tolerant maize line shifted resources from growth to defense. This shift was shown by potential hormone-level events—degradation of growth-promoting gibberellins and synthesis of jasmonic and abscisic acids. This finding is congruent with the xeromorphic morphology of seedlings of the cold-tolerant line and their ability to regrow when stress ceases. It is a common reaction of cold-tolerant maize lines. Moreover, in the cold-tolerant line, several genes from the low-temperature signaling pathways were potentially expressed. Additionally, numerous stress-response AP2/EREBP-binding cis-motifs were accessible in the cold-tolerant line. Differently in the cold-sensitive B73 line, MADS-binding cis-motifs were the most abundant. Development of the photosynthetic apparatus is crucial for the survival of maize seedlings at low temperature. Our results suggest efficient photosynthesis in seedlings of the cold-tolerant line, as was described earlier in physiological-level analyses.

scholarly journals Role of laeA in the Regulation of alb1, gliP, Conidial Morphology, and Virulence in Aspergillus fumigatus

2007 ◽  
Vol 6 (9) ◽  
pp. 1552-1561 ◽  
Author(s):  
Janyce A. Sugui ◽  
Julian Pardo ◽  
Yun C. Chang ◽  
Arno Müllbacher ◽  
Kol A. Zarember ◽  
...  
Keyword(s):  
Aspergillus Fumigatus ◽  
Survival Data ◽  
Mycelial Growth ◽  
Mrna Level ◽  
Nutritional Requirements ◽  
Wild Type ◽  
Conidial Morphology ◽  
Culture Filtrates ◽  
Data Culture

ABSTRACT The alb1 (pksP) gene has been reported as a virulence factor controlling the pigmentation and morphology of conidia in Aspergillus fumigatus. A recent report suggested that laeA regulates alb1 expression and conidial morphology but not pigmentation in the A. fumigatus strain AF293. laeA has also been reported to regulate the synthesis of secondary metabolites, such as gliotoxin. We compared the role of laeA in the regulation of conidial morphology and the expression of alb1 and gliP in strains B-5233 and AF293, which differ in colony morphology and nutritional requirements. Deletion of laeA did not affect conidial morphology or pigmentation in these strains, suggesting that laeA is not involved in alb1 regulation during conidial morphogenesis. Deletion of laeA, however, caused down-regulation of alb1 during mycelial growth in a liquid medium. Transcription of gliP, involved in the synthesis of gliotoxin, was drastically reduced in B-5233laeAΔ, and the gliotoxin level found in the culture filtrates was 20% of wild-type concentrations. While up-regulation of gliP in AF293 was comparable to that in B-5233, the relative mRNA level in AF293laeAΔ was about fourfold lower than that in B-5233laeAΔ. Strain B-5233laeAΔ caused slower onset of fatal infection in mice relative to that with B-5233. Histopathology of sections from lungs of infected mice corroborated the survival data. Culture filtrates from B-5233laeAΔ caused reduced death in thymoma cells and were less inhibitory to a respiratory burst of neutrophils than culture filtrates from B-5233. Our results suggest that while laeA is not involved in the regulation of alb1 function in conidial morphology, it regulates the synthesis of gliotoxin and the virulence of A. fumigatus.

scholarly journals Efficient Assembly of Ribosomes Is Inhibited by Deletion ofbipAin Escherichia coli

2015 ◽  
Vol 197 (10) ◽  
pp. 1819-1827 ◽  
Author(s):  
Promisree Choudhury ◽  
Ann M. Flower
Keyword(s):  
Low Temperature ◽  
Modified Nucleosides ◽  
Cold Sensitivity ◽  
23S Rrna ◽  
Ribosome Assembly ◽  
Rrna Processing ◽  
Pseudouridine Synthase ◽  
Assembly Factor ◽  
Cold Sensitive ◽  
And Function

ABSTRACTThe bacterial BipA protein belongs to the EF-G family of translational GTPases and has been postulated to be either a regulatory translation factor or a ribosome assembly factor. To distinguish between these hypotheses, we analyzed the effect ofbipAdeletion on three phenotypes associated with ribosome assembly factors: cold sensitivity, ribosome subunit distribution, and rRNA processing. We demonstrated that a ΔbipAstrain exhibits a cold-sensitive phenotype that is similar to, and synergistic with, that of a strain with a known ribosome assembly factor,deaD. Additionally, thebipAdeletion strain displayed a perturbed ribosome subunit distribution when grown at low temperature, similar to that of adeaDmutant, and again, the double mutant showed additive effects. The primary ribosomal deficiency noted was a decreased level of the 50S subunit and the appearance of a presumed pre-50S particle. Finally, deletion ofbipAresulted in accumulation of pre23S rRNA, as did deletion ofdeaD. We further found that deletion ofrluC, which encodes a pseudouridine synthase that modifies the 23S rRNA at three sites, suppressed all three phenotypes of thebipAmutant, supporting and extending previous findings. Together, these results suggest that BipA is important for the correct and efficient assembly of the 50S subunit of the ribosome at low temperature but when unmodified by RluC, the ribosomes become BipA independent for assembly.IMPORTANCEThe ribosome is the complex ribonucleoprotein machine responsible for protein synthesis in all cells. Although much has been learned about the structure and function of the ribosome, we do not fully understand how it is assembled or the accessory proteins that increase efficiency of biogenesis and function. This study examined one such protein, BipA. Our results indicate that BipA either directly or indirectly enhances the formation of the 50S subunit of the ribosome, particularly at low temperature. In addition, ribosomes contain a large number of modified nucleosides, including pseudouridines. This work demonstrates that the function of BipA is tied to the modification status of the ribosome and may help us understand why these modifications have been retained.

scholarly journals Genetic Interactions between the Escherichia coli umuDC Gene Products and the β Processivity Clamp of the Replicative DNA Polymerase

2001 ◽  
Vol 183 (9) ◽  
pp. 2897-2909 ◽  
Author(s):  
Mark D. Sutton ◽  
Mary F. Farrow ◽  
Briana M. Burton ◽  
Graham C. Walker
Keyword(s):  
Dna Damage ◽  
Dna Polymerase ◽  
Cold Sensitivity ◽  
Dna Damage Checkpoint ◽  
Gene Products ◽  
Missense Mutations ◽  
Dna Polymerase Iii ◽  
Checkpoint Control ◽  
Cold Sensitive ◽  
Growth Phenotype

ABSTRACT The Escherichia coli umuDC gene products encode DNA polymerase V, which participates in both translesion DNA synthesis (TLS) and a DNA damage checkpoint control. These two temporally distinct roles of the umuDC gene products are regulated by RecA–single-stranded DNA-facilitated self-cleavage of UmuD (which participates in the checkpoint control) to yield UmuD′ (which enables TLS). In addition, even modest overexpression of theumuDC gene products leads to a cold-sensitive growth phenotype, apparently due to the inappropriate expression of the DNA damage checkpoint control activity of UmuD2C. We have previously reported that overexpression of the ɛ proofreading subunit of DNA polymerase III suppresses umuDC-mediated cold sensitivity, suggesting that interaction of ɛ with UmuD2C is important for the DNA damage checkpoint control function of theumuDC gene products. Here, we report that overexpression of the β processivity clamp of the E. coli replicative DNA polymerase (encoded by the dnaN gene) not only exacerbates the cold sensitivity conferred by elevated levels of theumuDC gene products but, in addition, confers a severe cold-sensitive phenotype upon a strain expressing moderately elevated levels of the umuD′C gene products. Such a strain is not otherwise normally cold sensitive. To identify mutant β proteins possibly deficient for physical interactions with theumuDC gene products, we selected for noveldnaN alleles unable to confer a cold-sensitive growth phenotype upon a umuD′C-overexpressing strain. In all, we identified 75 dnaN alleles, 62 of which either reduced the expression of β or prematurely truncated its synthesis, while the remaining alleles defined eight unique missense mutations of dnaN. Each of the dnaNmissense mutations retained at least a partial ability to function in chromosomal DNA replication in vivo. In addition, these eightdnaN alleles were also unable to exacerbate the cold sensitivity conferred by modestly elevated levels of theumuDC gene products, suggesting that the interactions between UmuD′ and β are a subset of those between UmuD and β. Taken together, these findings suggest that interaction of β with UmuD2C is important for the DNA damage checkpoint function of the umuDC gene products. Four possible models for how interactions of UmuD2C with the ɛ and the β subunits of DNA polymerase III might help to regulate DNA replication in response to DNA damage are discussed.

scholarly journals Inflammatory and neuropathic cold allodynia are selectively mediated by the neurotrophic factor receptor GFRα3

2016 ◽  
Vol 113 (16) ◽  
pp. 4506-4511 ◽  
Author(s):  
Erika K. Lippoldt ◽  
Serra Ongun ◽  
Geoffrey K. Kusaka ◽  
David D. McKemy
Keyword(s):  
Chronic Pain ◽  
Neurotrophic Factor ◽  
Neutralizing Antibodies ◽  
Tissue Injury ◽  
Cold Sensitivity ◽  
Mechanical Stimuli ◽  
Cold Pain ◽  
Cold Allodynia ◽  
Cold Sensitive ◽  
Striking Contrast

Tissue injury prompts the release of a number of proalgesic molecules that induce acute and chronic pain by sensitizing pain-sensing neurons (nociceptors) to heat and mechanical stimuli. In contrast, many proalgesics have no effect on cold sensitivity or can inhibit cold-sensitive neurons and diminish cooling-mediated pain relief (analgesia). Nonetheless, cold pain (allodynia) is prevalent in many inflammatory and neuropathic pain settings, with little known of the mechanisms promoting pain vs. those dampening analgesia. Here, we show that cold allodynia induced by inflammation, nerve injury, and chemotherapeutics is abolished in mice lacking the neurotrophic factor receptor glial cell line-derived neurotrophic factor family of receptors-α3 (GFRα3). Furthermore, established cold allodynia is blocked in animals treated with neutralizing antibodies against the GFRα3 ligand, artemin. In contrast, heat and mechanical pain are unchanged, and results show that, in striking contrast to the redundant mechanisms sensitizing other modalities after an insult, cold allodynia is mediated exclusively by a single molecular pathway, suggesting that artemin–GFRα3 signaling can be targeted to selectively treat cold pain.

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