Designing multiple degenerate primers via consecutive pairwise alignments

<p>Abstract</p> <p>Background</p> <p>Different algorithms have been proposed to solve various versions of degenerate primer design problem. For one of the most general cases, multiple degenerate primer design problem, very few algorithms exist, none of them satisfying the criterion of designing low number of primers that cover high number of sequences. Besides, the present algorithms require high computation capacity and running time.</p> <p>Results</p> <p>PAMPS, the method presented in this work, usually results in a 30% reduction in the number of degenerate primers required to cover all sequences, compared to the previous algorithms. In addition, PAMPS runs up to 3500 times faster.</p> <p>Conclusion</p> <p>Due to small running time, using PAMPS allows designing degenerate primers for huge numbers of sequences. In addition, it results in fewer primers which reduces the synthesis costs and improves the amplification sensitivity.</p

Genetic Basis of Hidden Phenotypic Variation Revealed by Increased Translational Readthrough in Yeast

Eukaryotic release factors 1 and 3, encoded by SUP45 and SUP35, respectively, in Saccharomyces cerevisiae, are required for translation termination. Recent studies have shown that, besides these two key factors, several genetic and epigenetic mechanisms modulate the efficiency of translation termination. These mechanisms, through modifying translation termination fidelity, were shown to affect various cellular processes, such as mRNA degradation, and in some cases could confer a beneficial phenotype to the cell. The most studied example of such a mechanism is [PSI+], the prion conformation of Sup35p, which can have pleiotropic effects on growth that vary among different yeast strains. However, genetic loci underlying such readthrough-dependent, background-specific phenotypes have yet to be identified. Here, we used sup35C653R, a partial loss-of-function allele of the SUP35 previously shown to increase readthrough of stop codons and recapitulate some [PSI+]-dependent phenotypes, to study the genetic basis of phenotypes revealed by increased translational readthrough in two divergent yeast strains: BY4724 (a laboratory strain) and RM11_1a (a wine strain). We first identified growth conditions in which increased readthrough of stop codons by sup35C653R resulted in different growth responses between these two strains. We then used a recently developed linkage mapping technique, extreme QTL mapping (X-QTL), to identify readthrough-dependent loci for the observed growth differences. We further showed that variation in SKY1, an SR protein kinase, underlies a readthrough-dependent locus observed for growth on diamide and hydrogen peroxide. We found that the allelic state of SKY1 interacts with readthrough level and the genetic background to determine growth rate in these two conditions

Genetic Architecture of Highly Complex Chemical Resistance Traits across Four Yeast Strains

Many questions about the genetic basis of complex traits remain unanswered. This is in part due to the low statistical power of traditional genetic mapping studies. We used a statistically powerful approach, extreme QTL mapping (X-QTL), to identify the genetic basis of resistance to 13 chemicals in all 6 pairwise crosses of four ecologically and genetically diverse yeast strains, and we detected a total of more than 800 loci. We found that the number of loci detected in each experiment was primarily a function of the trait (explaining 46% of the variance) rather than the cross (11%), suggesting that the level of genetic complexity is a consistent property of a trait across different genetic backgrounds. Further, we observed that most loci had trait-specific effects, although a small number of loci with effects in many conditions were identified. We used the patterns of resistance and susceptibility alleles in the four parent strains to make inferences about the allele frequency spectrum of functional variants. We also observed evidence of more complex allelic series at a number of loci, as well as strain-specific signatures of selection. These results improve our understanding of complex traits in yeast and have implications for study design in other organisms

The genetic determinants and phenotypic consequences of variation in translation termination efficiency in Saccharomyces cerevisiae

Translation termination is a highly controlled process in the cell. In Saccharomyces cerevisiae, various regulatory factors employ genetic and epigenetic mechanisms to control this process. I used a quantitative dual luciferase reporter assay to demonstrate a difference in translation termination efficiency between two different yeast strains, BY4724 and RM11-1a. I then used a linkage mapping technique, X-QTL, to show that this difference is largely explained by a coding polymorphism in TRM10 (which encodes a tRNA-methylating enzyme) and a regulatory polymorphism in SUP45 (which encodes one of the translation termination factors). BY and RM carry variants of TRM10 and SUP45 with opposite effects on translation termination efficiency. These variants are common among 63 diverse S. cerevisiae strains and are in strong linkage disequilibrium with each other. This observation suggests that selection may have favored allelic combinations of the two genes that maintain an intermediate level of translation termination efficiency. [PSI+], the prion conformation of the S. cerevisiae translation termination factor Sup35p, is an epigenetic modifier of translation termination efficiency. It has been proposed that [PSI+] acts as a capacitor, releasing hidden genetic variation and generating heritable phenotypic variation with adaptive value. This hypothesis is based on observations that [PSI+] can create different growth phenotypes in strains with different genetic backgrounds by decreasing translation termination efficiency. However, genetic loci underlying such [PSI+]-induced, background-dependent phenotypes have yet to be identified. Here, I used sup35C653R, a partial loss-of-function allele of the SUP35 gene to model [PSI+] effect on translational termination efficiency in BY and RM. I used X-QTL to identify a number of readthrough-dependent loci for the growth conditions tested. I further showed that variation in SKY1, an SR protein kinase, underlies a readthrough-dependent locus observed for growth on diamide and hydrogen peroxide. I found that the allelic state of SKY1 interacts with readthrough level and the genetic background to determine growth rate in these two conditions. I believe this study is an effective step in understanding the mechanisms responsible for readthrough-dependent phenotypes. Extending this study to other strains, specifically strains shown to harbor [PSI+], would help in advancing our understandings of the genetic basis of the [PSI+]-dependent phenotypes

Variants in SUP45 and TRM10 underlie natural variation in translation termination efficiency in Saccharomyces cerevisiae.

Translation termination is a highly controlled process in the cell. In Saccharomyces cerevisiae, various regulatory factors employ genetic and epigenetic mechanisms to control this process. We used a quantitative dual luciferase reporter assay to demonstrate a difference in translation termination efficiency between two different yeast strains, BY4724 and RM11-1a. We then used a recently developed linkage mapping technique, extreme QTL mapping (X-QTL), to show that this difference is largely explained by a coding polymorphism in TRM10 (which encodes a tRNA-methylating enzyme) and a regulatory polymorphism in SUP45 (which encodes one of the yeast translation termination factors). BY and RM carry variants of TRM10 and SUP45 with opposite effects on translation termination efficiency. These variants are common among 63 diverse S. cerevisiae strains and are in strong linkage disequilibrium with each other. This observation suggests that selection may have favored allelic combinations of the two genes that maintain an intermediate level of translation termination efficiency. Our results also provide genetic evidence for a new role of Trm10p in translation termination efficiency

Genetic basis of hidden phenotypic variation revealed by increased translational readthrough in yeast.

Eukaryotic release factors 1 and 3, encoded by SUP45 and SUP35, respectively, in Saccharomyces cerevisiae, are required for translation termination. Recent studies have shown that, besides these two key factors, several genetic and epigenetic mechanisms modulate the efficiency of translation termination. These mechanisms, through modifying translation termination fidelity, were shown to affect various cellular processes, such as mRNA degradation, and in some cases could confer a beneficial phenotype to the cell. The most studied example of such a mechanism is [PSI+], the prion conformation of Sup35p, which can have pleiotropic effects on growth that vary among different yeast strains. However, genetic loci underlying such readthrough-dependent, background-specific phenotypes have yet to be identified. Here, we used sup35(C653R), a partial loss-of-function allele of the SUP35 previously shown to increase readthrough of stop codons and recapitulate some [PSI+]-dependent phenotypes, to study the genetic basis of phenotypes revealed by increased translational readthrough in two divergent yeast strains: BY4724 (a laboratory strain) and RM11_1a (a wine strain). We first identified growth conditions in which increased readthrough of stop codons by sup35(C653R) resulted in different growth responses between these two strains. We then used a recently developed linkage mapping technique, extreme QTL mapping (X-QTL), to identify readthrough-dependent loci for the observed growth differences. We further showed that variation in SKY1, an SR protein kinase, underlies a readthrough-dependent locus observed for growth on diamide and hydrogen peroxide. We found that the allelic state of SKY1 interacts with readthrough level and the genetic background to determine growth rate in these two conditions

Variants in SUP45 and TRM10 Underlie Natural Variation in Translation Termination Efficiency in Saccharomyces cerevisiae

Translation termination is a highly controlled process in the cell. In Saccharomyces cerevisiae, various regulatory factors employ genetic and epigenetic mechanisms to control this process. We used a quantitative dual luciferase reporter assay to demonstrate a difference in translation termination efficiency between two different yeast strains, BY4724 and RM11-1a. We then used a recently developed linkage mapping technique, extreme QTL mapping (X-QTL), to show that this difference is largely explained by a coding polymorphism in TRM10 (which encodes a tRNA–methylating enzyme) and a regulatory polymorphism in SUP45 (which encodes one of the yeast translation termination factors). BY and RM carry variants of TRM10 and SUP45 with opposite effects on translation termination efficiency. These variants are common among 63 diverse S. cerevisiae strains and are in strong linkage disequilibrium with each other. This observation suggests that selection may have favored allelic combinations of the two genes that maintain an intermediate level of translation termination efficiency

Readthrough-dependent strain-specific growth effects.

<p>The ratio of <i>sup35</i> strain growth rate and wildtype strain growth rate is plotted (mean ± SD) for BY (orange bars) and RM (purple bars) for the nine conditions in which the effect of <i>sup35^C653R</i>-mediated increase in readthrough on growth differed between the two strain backgrounds (uncorrected <i>p</i><0.05, FDR∼10%).</p