scholarly journals Mutant Kinesin-2 Motor Subunits Increase Chromosome Loss

2005 ◽  
Vol 16 (8) ◽  
pp. 3810-3820 ◽  
Author(s):  
Mark S. Miller ◽  
Jessica M. Esparza ◽  
Andrew M. Lippa ◽  
Fordyce G. Lux ◽  
Douglas G. Cole ◽  
...  
Keyword(s):  
Chromosome Segregation ◽  
Single Point ◽  
Intraflagellar Transport ◽  
Single Amino Acid ◽  
Single Point Mutation ◽  
Chromosome Loss ◽  
Temperature Sensitive ◽  
Dominant Effect ◽  
Flagellar Assembly ◽  
Gene Maps

The Chlamydomonas anterograde intraflagellar transport motor, kinesin-2, is isolated as a heterotrimeric complex containing two motor subunits and a nonmotor subunit known as kinesin-associated polypeptide or KAP. One of the two motor subunits is encoded by the FLA10 gene. The sequence of the second motor subunit was obtained by mass spectrometry and sequencing. It shows 46.9% identity with the Fla10 motor subunit and the gene maps to linkage group XII/XIII near RPL9. The temperature-sensitive flagellar assembly mutants fla1 and fla8 are linked to this kinesin-2 motor subunit. In each strain, a unique single point mutation gives rise to a unique single amino acid substitution within the motor domain. The fla8 strain is named fla8-1 and the fla1 strain is named fla8-2. The fla8 and fla10 alleles show a chromosome loss phenotype. To analyze this chromosome loss phenotype, intragenic revertants of fla8-1, fla8-2, and fla10-14 were generated. The analysis of the mutants and the revertants demonstrates the importance of a pocket in the amino terminus of these motor subunits for both motor activity and for a novel, dominant effect on the fidelity of chromosome segregation.

scholarly journals A Single Point Mutation, Asn16→Lys, Dictates the Temperature-Sensitivity of the Reovirus tsG453 Mutant

Viruses ◽  
2021 ◽  
Vol 13 (2) ◽  
pp. 289
Author(s):  
Kathleen K. M. Glover ◽  
Danica M. Sutherland ◽  
Terence S. Dermody ◽  
Kevin M. Coombs
Keyword(s):  
Amino Acid ◽  
Temperature Sensitivity ◽  
Reverse Genetics ◽  
Core Protein ◽  
Single Point ◽  
Single Amino Acid ◽  
Single Point Mutation ◽  
Temperature Sensitive ◽  
Amino Acid Substitutions ◽  
Gene Encoding

Studies of conditionally lethal mutants can help delineate the structure-function relationships of biomolecules. Temperature-sensitive (ts) mammalian reovirus (MRV) mutants were isolated and characterized many years ago. Two of the most well-defined MRV ts mutants are tsC447, which contains mutations in the S2 gene encoding viral core protein σ2, and tsG453, which contains mutations in the S4 gene encoding major outer-capsid protein σ3. Because many MRV ts mutants, including both tsC447 and tsG453, encode multiple amino acid substitutions, the specific amino acid substitutions responsible for the ts phenotype are unknown. We used reverse genetics to recover recombinant reoviruses containing the single amino acid polymorphisms present in ts mutants tsC447 and tsG453 and assessed the recombinant viruses for temperature-sensitivity by efficiency-of-plating assays. Of the three amino acid substitutions in the tsG453 S4 gene, Asn16-Lys was solely responsible for the tsG453ts phenotype. Additionally, the mutant tsC447 Ala188-Val mutation did not induce a temperature-sensitive phenotype. This study is the first to employ reverse genetics to identify the dominant amino acid substitutions responsible for the tsC447 and tsG453 mutations and relate these substitutions to respective phenotypes. Further studies of other MRV ts mutants are warranted to define the sequence polymorphisms responsible for temperature sensitivity.

scholarly journals A single point mutation converts a glutaryl-7-aminocephalosporanic acid acylase into an N-acyl-homoserine lactone acylase

2021 ◽  
Author(s):  
Shereen A. Murugayah ◽  
Gary B. Evans ◽  
Joel D. A. Tyndall ◽  
Monica L. Gerth
Keyword(s):  
Single Point ◽  
Quorum Quenching ◽  
Homoserine Lactone ◽  
Site Directed Mutagenesis ◽  
Saturation Mutagenesis ◽  
Single Amino Acid ◽  
Single Point Mutation ◽  
Acyl Homoserine Lactone ◽  
Signalling Molecules ◽  
Single Amino Acid Residue

Abstract Objective To change the specificity of a glutaryl-7-aminocephalosporanic acid acylase (GCA) towards N-acyl homoserine lactones (AHLs; quorum sensing signalling molecules) by site-directed mutagenesis. Results Seven residues were identified by analysis of existing crystal structures as potential determinants of substrate specificity. Site-saturation mutagenesis libraries were created for each of the seven selected positions. High-throughput activity screening of each library identified two variants—Arg255Ala, Arg255Gly—with new activities towards N-acyl homoserine lactone substrates. Structural modelling of the Arg255Gly mutation suggests that the smaller side-chain of glycine (as compared to arginine in the wild-type enzyme) avoids a key clash with the acyl group of the N-acyl homoserine lactone substrate. Conclusions Mutation of a single amino acid residue successfully converted a GCA (with no detectable activity against AHLs) into an AHL acylase. This approach may be useful for further engineering of ‘quorum quenching’ enzymes.

DBF8, an essential gene required for efficient chromosome segregation in Saccharomyces cerevisiae

1994 ◽  
Vol 14 (9) ◽  
pp. 6350-6360
Author(s):  
F Houman ◽  
C Holm
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Chromosome Segregation ◽  
Mutational Analysis ◽  
Essential Gene ◽  
Chromosome Loss ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Permissive Temperature ◽  
Nonsense Mutations ◽  
Carboxy Terminal

To investigate chromosome segregation in Saccharomyces cerevisiae, we examined a collection of temperature-sensitive mutants that arrest as large-budded cells at restrictive temperatures (L. H. Johnston and A. P. Thomas, Mol. Gen. Genet. 186:439-444, 1982). We characterized dbf8, a mutation that causes cells to arrest with a 2c DNA content and a short spindle. DBF8 maps to chromosome IX near the centromere, and it encodes a 36-kDa protein that is essential for viability at all temperatures. Mutational analysis reveals that three dbf8 alleles are nonsense mutations affecting the carboxy-terminal third of the encoded protein. Since all of these mutations confer temperature sensitivity, it appears that the carboxyl-terminal third of the protein is essential only at a restrictive temperature. In support of this conclusion, an insertion of URA3 at the same position also confers a temperature-sensitive phenotype. Although they show no evidence of DNA damage, dbf8 mutants exhibit increased rates of chromosome loss and nondisjunction even at a permissive temperature. Taken together, our data suggest that Dbf8p plays an essential role in chromosome segregation.

scholarly journals A single-point mutation in HCF causes temperature-sensitive cell-cycle arrest and disrupts VP16 function.

1997 ◽  
Vol 11 (6) ◽  
pp. 726-737 ◽  
Author(s):  
H Goto ◽  
S Motomura ◽  
A C Wilson ◽  
R N Freiman ◽  
Y Nakabeppu ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Arrest ◽  
Point Mutation ◽  
Single Point ◽  
Single Point Mutation ◽  
Temperature Sensitive ◽  
Sensitive Cell ◽  
Cycle Arrest

scholarly journals The role of retrograde intraflagellar transport in flagellar assembly, maintenance, and function

2012 ◽  
Vol 199 (1) ◽  
pp. 151-167 ◽  
Author(s):  
Benjamin D. Engel ◽  
Hiroaki Ishikawa ◽  
Kimberly A. Wemmer ◽  
Stefan Geimer ◽  
Ken-ichi Wakabayashi ◽  
...  
Keyword(s):  
Intraflagellar Transport ◽  
Retrograde Transport ◽  
Full Length ◽  
Temperature Sensitive ◽  
Nonpermissive Temperature ◽  
Flagellar Beat ◽  
Flagellar Assembly ◽  
Ph Shock ◽  
And Function

The maintenance of flagellar length is believed to require both anterograde and retrograde intraflagellar transport (IFT). However, it is difficult to uncouple the functions of retrograde transport from anterograde, as null mutants in dynein heavy chain 1b (DHC1b) have stumpy flagella, demonstrating solely that retrograde IFT is required for flagellar assembly. We isolated a Chlamydomonas reinhardtii mutant (dhc1b-3) with a temperature-sensitive defect in DHC1b, enabling inducible inhibition of retrograde IFT in full-length flagella. Although dhc1b-3 flagella at the nonpermissive temperature (34°C) showed a dramatic reduction of retrograde IFT, they remained nearly full-length for many hours. However, dhc1b-3 cells at 34°C had strong defects in flagellar assembly after cell division or pH shock. Furthermore, dhc1b-3 cells displayed altered phototaxis and flagellar beat. Thus, robust retrograde IFT is required for flagellar assembly and function but is dispensable for the maintenance of flagellar length. Proteomic analysis of dhc1b-3 flagella revealed distinct classes of proteins that change in abundance when retrograde IFT is inhibited.

scholarly journals A Single Point Mutation Controls the Cholesterol Dependence of Semliki Forest Virus Entry and Exit

1998 ◽  
Vol 140 (1) ◽  
pp. 91-99 ◽  
Author(s):  
Malini Vashishtha ◽  
Thomas Phalen ◽  
Marianne T. Marquardt ◽  
Jae S. Ryu ◽  
Alice C. Ng ◽  
...  
Keyword(s):  
Membrane Fusion ◽  
Semliki Forest Virus ◽  
Single Point ◽  
Cellular Membrane ◽  
Spike Protein ◽  
Single Amino Acid ◽  
Progeny Virus ◽  
Single Point Mutation ◽  
Protein Subunit ◽  
Single Amino Acid Change

Membrane fusion and budding are key steps in the life cycle of all enveloped viruses. Semliki Forest virus (SFV) is an enveloped alphavirus that requires cellular membrane cholesterol for both membrane fusion and efficient exit of progeny virus from infected cells. We selected an SFV mutant, srf-3, that was strikingly independent of cholesterol for growth. This phenotype was conferred by a single amino acid change in the E1 spike protein subunit, proline 226 to serine, that increased the cholesterol independence of both srf-3 fusion and exit. The srf-3 mutant emphasizes the relationship between the role of cholesterol in membrane fusion and virus exit, and most significantly, identifies a novel spike protein region involved in the virus cholesterol requirement.

scholarly journals Meiotic Segregation, Synapsis, and Recombination Checkpoint Functions Require Physical Interaction between the Chromosomal Proteins Red1p and Hop1p

2000 ◽  
Vol 20 (18) ◽  
pp. 6646-6658 ◽  
Author(s):  
Dana Woltering ◽  
Bridget Baumgartner ◽  
Sandipan Bagchi ◽  
Brittany Larkin ◽  
Josef Loidl ◽  
...  
Keyword(s):  
Chromosome Segregation ◽  
Single Point ◽  
Single Point Mutation ◽  
Physical Interaction ◽  
Spore Viability ◽  
Chromosomal Proteins ◽  
Meiotic Progression ◽  
Chromosome Synapsis ◽  
Recombination Checkpoint ◽  
Two Hybrid

ABSTRACT In yeast, HOP1 and RED1 are required during meiosis for proper chromosome segregation and the consequent formation of viable spores. Mutations in either HOP1 orRED1 create unique as well as overlapping phenotypes, indicating that the two proteins act alone as well as in concert with each other. To understand which meiotic processes specifically require Red1p-Hop1p hetero-oligomers, a novel genetic screen was used to identify a single-point mutation of RED1,red1-K348E, that separates Hop1p binding from Red1p homo-oligomerization. The Red1-K348E protein is stable, phosphorylated in a manner equivalent to Red1p, and undergoes efficient homo-oligomerization; however, its ability to interact with Hop1p both by two-hybrid and coimmunoprecipitation assays is greatly reduced. Overexpression of HOP1 specifically suppressesred1-K348E, supporting the idea that the only defect in the protein is a reduced affinity for Hop1p. red1-K348E mutants exhibit reduced levels of crossing over and spore viability and fail to undergo chromosome synapsis, thereby implicating a role for Red1p-Hop1p hetero-oligomers in these processes. Furthermore,red1-K348E suppresses the sae2/com1 defects in meiotic progression and sporulation, indicating a previously unknown role for HOP1 in the meiotic recombination checkpoint.

scholarly journals Mutations in Aspergillus fumigatus Resulting in Reduced Susceptibility to Posaconazole Appear To Be Restricted to a Single Amino Acid in the Cytochrome P450 14α-Demethylase

2003 ◽  
Vol 47 (2) ◽  
pp. 577-581 ◽  
Author(s):  
Paul A. Mann ◽  
Raulo M. Parmegiani ◽  
Shui-Qing Wei ◽  
Cara A. Mendrick ◽  
Xin Li ◽  
...  
Keyword(s):  
Cytochrome P450 ◽  
Aspergillus Fumigatus ◽  
Single Point ◽  
Point Mutations ◽  
Drug Accumulation ◽  
Resistant Isolate ◽  
Single Amino Acid ◽  
Single Point Mutation ◽  
Genes Encoding ◽  
Moderately Resistant

ABSTRACT To better understand the molecular basis of posaconazole (POS) resistance in Aspergillus fumigatus, resistant laboratory isolates were selected. Spontaneous mutants arose at a frequency of 1 in 108 and fell into two susceptibility groups, moderately resistant and highly resistant. Azole resistance in A. fumigatus was previously associated with decreased drug accumulation. We therefore analyzed the mutants for changes in levels of transcripts of genes encoding efflux pumps (mdr1 and mdr2) and/or alterations in accumulation of [14C]POS. No changes in either pump expression or drug accumulation were detected. Similarly, there was no change in expression of cyp51A or cyp51B, which encode the presumed target site for POS, cytochrome P450 14α-demethylase. DNA sequencing revealed that each resistant isolate carried a single point mutation in residue 54 of cyp51A. Mutations at the same locus were identified in three clinical A. fumigatus isolates exhibiting reduced POS susceptibility but not in susceptible clinical strains. To verify that these mutations were responsible for the resistance phenotype, we introduced them into the chromosome of a POS-susceptible A. fumigatus strain under the control of the glyceraldehyde phosphate dehydrogenase promoter. The transformants exhibited reductions in susceptibility to POS comparable to those exhibited by the original mutants, confirming that point mutations in the cyp51A gene in A. fumigatus can confer reduced susceptibility to POS.

scholarly journals A single-nucleotide change underlies the genetic assimilation of a plastic trait

2021 ◽  
Vol 7 (6) ◽  
pp. eabd9941
Author(s):  
Paul Vigne ◽  
Clotilde Gimond ◽  
Céline Ferrari ◽  
Anne Vielle ◽  
Johan Hallin ◽  
...  
Keyword(s):  
Evolutionary Biology ◽  
Single Point ◽  
Evolutionary Process ◽  
Resource Provisioning ◽  
Egg Laying ◽  
Genetic Assimilation ◽  
Single Amino Acid ◽  
Single Point Mutation ◽  
Single Amino Acid Change ◽  
Plastic Trait

Genetic assimilation—the evolutionary process by which an environmentally induced phenotype is made constitutive—represents a fundamental concept in evolutionary biology. Thought to reflect adaptive phenotypic plasticity, matricidal hatching in nematodes is triggered by maternal nutrient deprivation to allow for protection or resource provisioning of offspring. Here, we report natural Caenorhabditis elegans populations harboring genetic variants expressing a derived state of near-constitutive matricidal hatching. These variants exhibit a single amino acid change (V530L) in KCNL-1, a small-conductance calcium-activated potassium channel subunit. This gain-of-function mutation causes matricidal hatching by strongly reducing the sensitivity to environmental stimuli triggering egg-laying. We show that reestablishing the canonical KCNL-1 protein in matricidal isolates is sufficient to restore canonical egg-laying. While highly deleterious in constant food environments, KCNL-1 V530L is maintained under fluctuating resource availability. A single point mutation can therefore underlie the genetic assimilation—by either genetic drift or selection—of an ancestrally plastic trait.

Modulation of keratin intermediate filament assembly by single amino acid exchanges in the consensus sequence at the C-terminal end of the rod domain

1991 ◽  
Vol 99 (2) ◽  
pp. 351-362 ◽  
Author(s):  
M. Hatzfeld ◽  
K. Weber
Keyword(s):  
Intermediate Filament ◽  
Single Point ◽  
Consensus Sequence ◽  
Site Directed Mutagenesis ◽  
Single Amino Acid ◽  
Single Point Mutation ◽  
Wild Type ◽  
Filament Assembly ◽  
Elongation Process

All known intermediate filament (IF) proteins display -8 -4 -1 a consensus sequence TYRKLLEGE at the carboxyl end of the rod domain. To analyse the contribution of this sequence to the formation of IF we have changed two of the invariant positions of this motif by site-directed mutagenesis. We produced three mutant keratins, each containing a single point mutation. Tyrosine at position -8 was changed to alanine in keratin K8 (K8Y----A-8) and keratin K18 (K18Y----A-8) and leucine at position -4 was changed to glycine in keratin K18 (K18L----G-4). Mutant keratins were expressed in Escherichia coli, purified and analysed for their filament-forming capacity in vitro using either the complementary wild-type keratin or the corresponding mixture of mutant keratins. In standard filament buffer (50 mM Tris-HCl, pH7.5), assembly involving any of the mutants leads to large electron-dense aggregates instead of normal IF. In order to explain this effect, we studied the process of filament formation in more detail. Whereas the formation of tetramers in buffers containing 4M urea is unaffected, the elongation process seems slowed down. In buffer of lower ionic strength (10 mM Tris-HCl, pH7.5) mutant keratins K8Y----A-8 plus K18Y----A-8 become able to form long filaments, although short filaments and protofilamentous material are still detected. The filaments formed differ from normal keratin IF by their remarkable tendency to aggregate into thick cables. Assemblies involving K18L----G-4 can only form short IF lengths. The dense aggregates formed in standard filament buffer are able to dissociate into IF and their fragments upon dialysis into 10 mM Tris-HCl, pH7.5. The results show that the consensus sequence is needed for IF formation under normal conditions and that already one mutation per heterodimer affects the assembly.

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