Dataset Information

Comprehensive Analysis of the SUL1 Promoter of Saccharomyces cerevisiae.

ABSTRACT: In the yeast Saccharomyces cerevisiae, beneficial mutations selected during sulfate-limited growth are typically amplifications of the SUL1 gene, which encodes the high-affinity sulfate transporter, resulting in fitness increases of >35% . Cis-regulatory mutations have not been observed at this locus; however, it is not clear whether this absence is due to a low mutation rate such that these mutations do not arise, or they arise but have limited fitness effects relative to those of amplification. To address this question directly, we assayed the fitness effects of nearly all possible point mutations in a 493-base segment of the gene's promoter through mutagenesis and selection. While most mutations were either neutral or detrimental during sulfate-limited growth, eight mutations increased fitness >5% and as much as 9.4%. Combinations of these beneficial mutations increased fitness only up to 11%. Thus, in the case of SUL1, promoter mutations could not induce a fitness increase similar to that of gene amplification. Using these data, we identified functionally important regions of the SUL1 promoter and analyzed three sites that correspond to potential binding sites for the transcription factors Met32 and Cbf1 Mutations that create new Met32- or Cbf1-binding sites also increased fitness. Some mutations in the untranslated region of the SUL1 transcript decreased fitness, likely due to the formation of inhibitory upstream open reading frames. Our methodology-saturation mutagenesis, chemostat selection, and DNA sequencing to track variants-should be a broadly applicable approach.

SUBMITTER: Rich MS

PROVIDER: S-EPMC4858773 | biostudies-literature | 2016 May

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Comprehensive Analysis of the SUL1 Promoter of Saccharomyces cerevisiae.

Rich Matthew S MS Payen Celia C Rubin Alan F AF Ong Giang T GT Sanchez Monica R MR Yachie Nozomu N Dunham Maitreya J MJ Fields Stanley S

Genetics 20160302 1

In the yeast Saccharomyces cerevisiae, beneficial mutations selected during sulfate-limited growth are typically amplifications of the SUL1 gene, which encodes the high-affinity sulfate transporter, resulting in fitness increases of >35% . Cis-regulatory mutations have not been observed at this locus; however, it is not clear whether this absence is due to a low mutation rate such that these mutations do not arise, or they arise but have limited fitness effects relative to those of amplification ...[more]

PMID: 26936925

Similar Datasets

Project description:DNA replication initiates from multiple replication origins (ORIs) in eukaryotes. Discovery and characterization of replication origins are essential for a better understanding of the molecular mechanism of DNA replication. In this study, the features of autonomously replicating sequences (ARSs) in Saccharomyces cerevisiae have been comprehensively analyzed as follows. Firstly, we carried out the analysis of the ARSs available in S. cerevisiae S288C. By evaluating the sequence similarity of experimentally established ARSs, we found that 94.32% of ARSs are unique across the whole genome of S. cerevisiae S288C and those with high sequence similarity are prone to locate in subtelomeres. Subsequently, we built a non-redundant dataset with a total of 520 ARSs, which are based on ARSs annotation of S. cerevisiae S288C from SGD and then supplemented with those from OriDB and DeOri databases. We conducted a large-scale comparison of ORIs among the diverse budding yeast strains from a population genomics perspective. We found that 82.7% of ARSs are not only conserved in genomic sequence but also relatively conserved in chromosomal position. The non-conserved ARSs tend to distribute in the subtelomeric regions. We also conducted a pan-genome analysis of ARSs among the S. cerevisiae strains, and a total of 183 core ARSs existing in all yeast strains were determined. We extracted the genes adjacent to replication origins among the 104 yeast strains to examine whether there are differences in their gene functions. The result showed that the genes involved in the initiation of DNA replication, such as orc3, mcm2, mcm4, mcm6, and cdc45, are conservatively located adjacent to the replication origins. Furthermore, we found the genes adjacent to conserved ARSs are significantly enriched in DNA binding, enzyme activity, transportation, and energy, whereas for the genes adjacent to non-conserved ARSs are significantly enriched in response to environmental stress, metabolites biosynthetic process and biosynthesis of antibiotics. In general, we characterized the replication origins from the genome-wide and population genomics perspectives, which would provide new insights into the replication mechanism of S. cerevisiae and facilitate the design of algorithms to identify genome-wide replication origins in yeast.

Project description:BackgroundSaccharomyces cerevisiae is widely studied for production of biofuels and biochemicals. To improve production efficiency under industrially relevant conditions, coordinated expression of multiple genes by manipulating promoter strengths is an efficient approach. It is known that gene expression is highly dependent on the practically used environmental conditions and is subject to dynamic changes. Therefore, investigating promoter activities of S. cerevisiae under different culture conditions in different time points, especially under stressful conditions is of great importance.ResultsIn this study, the activities of various promoters in S. cerevisiae under stressful conditions and in the presence of xylose were characterized using yeast enhanced green fluorescent protein (yEGFP) as a reporter. The stresses include toxic levels of acetic acid and furfural, and high temperature, which are related to fermentation of lignocellulosic hydrolysates. In addition to investigating eight native promoters, the synthetic hybrid promoter P3xC-TEF1 was also evaluated. The results revealed that P TDH3 and the synthetic promoter P3xC-TEF1 showed the highest strengths under almost all the conditions. Importantly, these two promoters also exhibited high stabilities throughout the cultivation. However, the strengths of P ADH1 and P PGK1 , which are generally regarded as 'constitutive' promoters, decreased significantly under certain conditions, suggesting that cautions should be taken to use such constitutive promoters to drive gene expression under stressful conditions. Interestingly, P HSP12 and P HSP26 were able to response to both high temperature and acetic acid stress. Moreover, P HSP12 also led to moderate yEGFP expression when xylose was used as the sole carbon source, indicating that this promoter could be used for inducing proper gene expression for xylose utilization.ConclusionThe results here revealed dynamic changes of promoter activities in S. cerevisiae throughout batch fermentation in the presence of inhibitors as well as using xylose. These results provide insights in selection of promoters to construct S. cerevisiae strains for efficient bioproduction under practical conditions. Our results also encouraged applications of synthetic promoters with high stability for yeast strain development.

Dataset Information

Comprehensive Analysis of the SUL1 Promoter of Saccharomyces cerevisiae.

Publications

Comprehensive Analysis of the SUL1 Promoter of Saccharomyces cerevisiae.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets