scholarly journals Mutational fitness effects in RNA and single-stranded DNA viruses: common patterns revealed by site-directed mutagenesis studies

2010 ◽  
Vol 365 (1548) ◽  
pp. 1975-1982 ◽  
Author(s):  
Rafael Sanjuán
Keyword(s):  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Nucleotide Substitutions ◽  
Single Nucleotide ◽  
Single Stranded Dna ◽  
Dna Viruses ◽  
Fitness Effects ◽  
Lethal Mutations ◽  
Mean Fitness ◽  
The Mean

The fitness effects of mutations are central to evolution, yet have begun to be characterized in detail only recently. Site-directed mutagenesis is a powerful tool for achieving this goal, which is particularly suited for viruses because of their small genomes. Here, I discuss the evolutionary relevance of mutational fitness effects and critically review previous site-directed mutagenesis studies. The effects of single-nucleotide substitutions are standardized and compared for five RNA or single-stranded DNA viruses infecting bacteria, plants or animals. All viruses examined show very low tolerance to mutation when compared with cellular organisms. Moreover, for non-lethal mutations, the mean fitness reduction caused by single mutations is remarkably constant (0.10–0.13), whereas the fraction of lethals varies only modestly (0.20–0.41). Other summary statistics are provided. These generalizations about the distribution of mutational fitness effects can help us to better understand the evolution of RNA and single-stranded DNA viruses.

A novel shuttle vector for Streptomyces spp. and Escherichia coli as a tool in site-directed mutagenesis

1992 ◽  
Vol 38 (4) ◽  
pp. 350-353 ◽  
Author(s):  
A. Moreau ◽  
F. W. Paradis ◽  
R. Morosoli ◽  
F. Shareck ◽  
D. Kluepfel
Keyword(s):  
Escherichia Coli ◽  
Resistance Gene ◽  
Shuttle Vector ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Gene Encoding ◽  
Single Stranded Dna ◽  
E Coli ◽  
Streptomyces Spp ◽  
Ampicillin Resistance Gene

This paper describes the construction and utilization of a novel shuttle vector for Streptomyces spp. and Escherichia coli as a useful vector in site-directed mutagenesis. The shuttle vector pIAFS20 (6.7 kb) has the following features: a replicon for Streptomyces spp., isolated from plasmid pIJ702; the thiostrepton-resistance gene as a selective marker in Streptomyces; the ColE1 origin, allowing replication in E. coli; and the ampicillin-resistance gene as a selective markerin E. coli. Vector pIAFS20 also contains the phage fl intergenic region, which permits production of single-stranded DNA in E. coli after superinfection with helper phage M13K07. Moreover, the lac promoter is located in front of the multiple cloning sites cassette, allowing eventual expression of the cloned genes in E. coli. After mutagenesis and screeningof the mutants in E. coli, the plasmids can be readily used to transform Streptomyces spp. As a demonstration, a 3.2-kb DNA fragment containing the gene encoding the xylanase A from Streptomyces lividans 1326 was inserted into pIAFS20, and the promoter region of this gene served as a target for site-directed mutagenesis. The two deletions reported here confirm the efficiency of this new vector as a tool in mutagenesis. Key words: shuttle vector, single-stranded DNA, site-directed mutagenesis, Streptomyces spp., Escherichia coli.

scholarly journals Distribution of Fitness and Virulence Effects Caused by Single-Nucleotide Substitutions in Tobacco Etch Virus

2007 ◽  
Vol 81 (23) ◽  
pp. 12979-12984 ◽  
Author(s):  
Purificación Carrasco ◽  
Francisca de la Iglesia ◽  
Santiago F. Elena
Keyword(s):  
Single Case ◽  
Site Directed Mutagenesis ◽  
Relative Fitness ◽  
In Planta ◽  
Nucleotide Substitutions ◽  
Phenotypic Effect ◽  
Single Nucleotide ◽  
Mutant Genotype

ABSTRACT Little is known about the fitness and virulence consequences of single-nucleotide substitutions in RNA viral genomes, and most information comes from the analysis of nonrandom sets of mutations with strong phenotypic effect or which have been assessed in vitro, with their relevance in vivo being unclear. Here we used site-directed mutagenesis to create a collection of 66 clones of Tobacco etch potyvirus, each carrying a different, randomly chosen, single-nucleotide substitution. Competition experiments between each mutant and the ancestral nonmutated clone were performed in planta to quantitatively assess the relative fitness of each mutant genotype. Among all mutations, 40.9% were lethal, and among the viable ones, 36.4% were significantly deleterious and 22.7% neutral. Not a single case of beneficial effects was observed within the level of resolution of our measures. On average, the fitness of a genotype carrying a deleterious but viable mutation was 49% smaller than that for its unmutated progenitor. Deleterious mutational effects conformed to a beta probability distribution. The virulence of a subset of viable mutants was assessed as the reduction in the number of viable seeds produced by infected plants. Mutational effects on virulence ranged between 17% reductions and 24.4% increases. Interestingly, the only mutations showing a significant effect on virulence were hypervirulent. Competitive fitness and virulence were uncorrelated traits.

scholarly journals Distribution of Fitness Effects Caused by Single-Nucleotide Substitutions in Bacteriophage f1

Genetics ◽  
2010 ◽  
Vol 185 (2) ◽  
pp. 603-609 ◽  
Author(s):  
Joan B. Peris ◽  
Paulina Davis ◽  
José M. Cuevas ◽  
Miguel R. Nebot ◽  
Rafael Sanjuán
Keyword(s):  
Nucleotide Substitutions ◽  
Single Nucleotide ◽  
Fitness Effects

scholarly journals The distribution of fitness effects among synonymous mutations in a gene under selection

2019 ◽  
Author(s):  
E. Lebeuf-Taylor ◽  
N. McCloskey ◽  
S.F. Bailey ◽  
A. Hinz ◽  
R. Kassen
Keyword(s):  
Amino Acid ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Compelling Evidence ◽  
Competitive Fitness ◽  
Synonymous Substitutions ◽  
Synonymous Mutations ◽  
Fitness Effects ◽  
Growing Body ◽  
Direct Measures

AbstractThe fitness effects of synonymous mutations, nucleotide changes that do not alter the encoded amino acid, have often been assumed to be neutral, but a growing body of evidence suggests otherwise. We used site-directed mutagenesis coupled with direct measures of competitive fitness to estimate the distribution of fitness effects among synonymous mutations for a gene under selection. Synonymous mutations had highly variable fitness effects, both deleterious and beneficial, resembling those of nonsynonymous mutations in the same gene. This variation in fitness was underlain by changes in transcription linked to the creation of internal promoter sites. A positive correlation between fitness and the presence of synonymous substitutions across a phylogeny of related Pseudomonads suggests these mutations may be common in nature. Taken together, our results provide the most compelling evidence to date that synonymous mutations with non-neutral fitness effects may in fact be commonplace.

scholarly journals Nucleoside Analogue Mutagenesis of a Single-Stranded DNA Virus: Evolution and Resistance

2012 ◽  
Vol 86 (18) ◽  
pp. 9640-9646 ◽  
Author(s):  
Pilar Domingo-Calap ◽  
Marianoel Pereira-Gómez ◽  
Rafael Sanjuán
Keyword(s):  
Nucleoside Analogue ◽  
Rna Viruses ◽  
Spontaneous Mutation ◽  
Chemical Mutagenesis ◽  
Mutation Rates ◽  
Nucleotide Substitutions ◽  
Single Stranded Dna ◽  
Dna Viruses ◽  
Population Extinction ◽  
Nucleotide Mutation

It has been well established that chemical mutagenesis has adverse fitness effects in RNA viruses, often leading to population extinction. This is mainly a consequence of the high RNA virus spontaneous mutation rates, which situate them close to the extinction threshold. Single-stranded DNA viruses are the fastest-mutating DNA-based systems, with per-nucleotide mutation rates close to those of some RNA viruses, but chemical mutagenesis has been much less studied in this type of viruses. Here, we serially passaged bacteriophage ϕX174 in the presence of the nucleoside analogue 5-fluorouracil (5-FU). We found that 5-FU was unable to trigger population extinction for the range of concentrations tested, but it negatively affected viral adaptability. The phage evolved partial drug resistance, and parallel nucleotide substitutions appearing in independently evolved lines were identified as candidate resistance mutations. Using site-directed mutagenesis, two single-nucleotide substitutions in the lysis protein E (T572C and A781G) were shown to be selectively advantageous in the presence of 5-FU. In RNA viruses, base analogue resistance is often mediated by changes in the viral polymerase, but this mechanism is not possible for ϕX174 and other single-stranded DNA viruses because they do not encode their own polymerase. In addition to increasing mutation rates, 5-FU produces a wide variety of cytotoxic effects at the levels of replication, transcription, and translation. We found that substitutions T572C and A781G lost their ability to confer 5-FU resistance after cells were supplemented with deoxythymidine, suggesting that their mechanism of action is at the DNA level. We hypothesize that regulation of lysis time may allow the virus to optimize progeny size in cells showing defects in DNA synthesis.

scholarly journals A rapid, super-selective method for detection of single nucleotide variants in C. elegans

2020 ◽  
Author(s):  
Denis Touroutine ◽  
Jessica E. Tanis
Keyword(s):  
Low Cost ◽  
Primer Design ◽  
Site Directed Mutagenesis ◽  
Amplification Efficiency ◽  
Single Nucleotide Variants ◽  
Nucleotide Substitutions ◽  
Single Nucleotide ◽  
Colony Pcr ◽  
Sequence Composition ◽  
C Elegans

ABSTRACTWith the widespread use of single nucleotide variants generated through mutagenesis screens, the million mutation project, and genome editing technologies, there is pressing need for an efficient and low-cost strategy to genotype single nucleotide substitutions. We have developed a rapid and inexpensive method for detection of point mutants through optimization of SuperSelective (SS) primers for end point PCR in Caenorhabditis elegans. Each SS primer consists of a 5’ “anchor” that hybridizes to the template, followed by a non-complementary “bridge,” and a “foot” corresponding to the target allele. The foot sequence is short, such that a single mismatch at the terminal 3’ nucleotide destabilizes primer binding and prevents extension, enabling discrimination of different alleles. We explored how length, stability, and sequence composition of each SS primer segment affected selectivity and efficiency in order to develop simple rules for primer design that allow for distinction between any mismatches in various genetic contexts over a broad range of annealing temperatures. Manipulating bridge length affects amplification efficiency, while modifying the foot sequence can increase discriminatory power. Flexibility in the positioning of the anchor enables SS primers to be used for genotyping in regions with sequences that are challenging for standard primer design. In summary, we have demonstrated flexibility in design of SS primers and their utility for genotyping in C. elegans. Since SS primers reliably detect single nucleotide variants, we propose that this method could have broad application for SNP mapping, screening of CRISPR mutants, and colony PCR to identify successful site-directed mutagenesis constructs.

Estimation of Deleterious Genomic Mutation Parameters in Natural Populations by Accounting for Variable Mutation Effects Across Loci

Genetics ◽  
2002 ◽  
Vol 162 (3) ◽  
pp. 1487-1500 ◽  
Author(s):  
Hong-Wen Deng ◽  
Guimin Gao ◽  
Jin-Long Li
Keyword(s):  
Statistical Method ◽  
Mutation Rate ◽  
Genetic Variance ◽  
Natural Populations ◽  
Evolutionary Genetics ◽  
Simulation Studies ◽  
Fitness Effects ◽  
Mean Fitness ◽  
The Mean ◽  
Genomic Mutation

Abstract The genomes of all organisms are subject to continuous bombardment of deleterious genomic mutations (DGM). Our ability to accurately estimate various parameters of DGM has profound significance in population and evolutionary genetics. The Deng-Lynch method can estimate the parameters of DGM in natural selfing and outcrossing populations. This method assumes constant fitness effects of DGM and hence is biased under variable fitness effects of DGM. Here, we develop a statistical method to estimate DGM parameters by considering variable mutation effects across loci. Under variable mutation effects, the mean fitness and genetic variance for fitness of parental and progeny generations across selfing/outcrossing in outcrossing/selfing populations and the covariance between mean fitness of parents and that of their progeny are functions of DGM parameters: the genomic mutation rate U, average homozygous effect s, average dominance coefficient h, and covariance of selection and dominance coefficients cov(h, s). The DGM parameters can be estimated by the algorithms we developed herein, which may yield improved estimation of DGM parameters over the Deng-Lynch method as demonstrated by our simulation studies. Importantly, this method is the first one to characterize cov(h, s) for DGM.

Sign in / Sign up

Export Citation Format

Share Document