A new method for the construction of a mutant library with a predictable occurrence rate using Poisson distribution

Seong, Ki Moon; Park, Hweon; Kim, Seong-Jung; Ha, Hyo Nam; Lee, Jae Yung; Kim, Joon

doi:10.1016/j.mimet.2007.02.010

Cited by 6 publications

(11 citation statements)

References 28 publications

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“…This is plausible as Gcn4p is the best‐characterized transcription factor in yeasts and is known to have more than 500 trans ‐target genes (Hinnebusch, 2005). To investigate whether trans ‐activity has an indirect effect on RP transcription, Ser242, a highly conserved amino‐acid residue in the DNA binding domain of Gcn4p was mutated to leucine (Ellenberger et al , 1992; Kim and Struhl, 1995; Seong et al , 2007). Consequently, the transcriptional activity of this mutant was abolished (Figure 4B and E).…”

Section: Resultsmentioning

confidence: 99%

Gcn4p-mediated transcriptional repression of ribosomal protein genes under amino-acid starvation

et al. 2010

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Gcn4p is a well-characterized bZIP transcription factor that activates more than 500 genes encoding amino acids and purine biosynthesis enzymes, and many stressresponse genes under various stress conditions. Under these stresses, it had been shown that transcriptions of ribosomal protein (RP) genes were decreased. However, the detailed mechanism of this downregulation has not been elucidated. In this study, we present a novel mechanistic model for a repressive role of Gcn4p on RP transcription, especially under amino-acid starvation. It was found that Gcn4p bound directly to Rap1p, which in turn inhibited Esa1p-Rap1p binding. The inhibition of Esa1p recruitment to RP promoters ultimately reduced the level of histone H4 acetylation and RP transcription. These data revealed that Gcn4p has simultaneous dual roles as a repressor for RP genes as well as an activator for amino-acid biosynthesis genes. Moreover, our results showed evidence of a novel link between general control of amino-acid biosynthesis and ribosome biogenesis mediated by Gcn4p at an early stage of adaptation to amino-acid starvation.

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Section: Resultsmentioning

confidence: 99%

Gcn4p-mediated transcriptional repression of ribosomal protein genes under amino-acid starvation

et al. 2010

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“…Previously, Gcn4 proteins with a point mutation in their DNA‐binding domain were isolated using a probe containing a canonical AP‐1 binding site; these mutant Gcn4 proteins exhibited defective DNA‐binding activities compared to that of the wild‐type proteins (Seong, Park, et al , ). To identify a suppressor that can rescue the defective phenotypes of Gcn4 mutants undergoing amino acid depletion, we performed a multi‐copy suppression screening using the yeast genomic library cloned in a high copy plasmid (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…After a 30‐min incubation period, the reactions were resolved on 8% polyacrylamide gels in 0.5X Tris‐borate‐EDTA (TBE). The gel was then dried and autoradiographed using a phosphorImager (Seong, Park, et al , ). The probes were complementary, double stranded oligonucleotide sequences that were annealed in buffer (20 mM Tris‐Cl pH 8.0, 2 mM EDTA, and 100 mM NaCl).…”

Section: Methodsmentioning

confidence: 99%

Ssb2 is a novel factor in regulating synthesis and degradation of Gcn4 in Saccharomyces cerevisiae

Jung

Seong

Baek

et al. 2018

Molecular Microbiology

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Yeast cells respond to environmental stress by inducing the master regulator Gcn4 to control genes involved in biosynthesis of amino acids and purine pathways. Gcn4 is a member of the basic leucine Zipper family and binds directly as a homodimer to a conserved regulatory region of target genes. Ssb2 was discovered to rescue the mutant Gcn4 which has a point mutation that decreases DNA-binding affinity. Ssb2 is part of the Hsp70 protein family responsible for protein quality control and it is thought that Ssb2 assists the passage of nascent polypeptide chains from the ribosomes. To characterize the mechanism behind the rescue of the mutant gcn4 phenotype, transcriptional activity and protein levels of Gcn4 were analyzed. We found that Ssb2 improved the expression of Gcn4 target genes by increasing the DNA-binding affinity of gcn4 mutants to target gene promoters under conditions of amino acid starvation. Gcn4 levels increased at both translational and post-translational levels without regulating GCN4 steady-state mRNA levels. We also found that the nuclear export signal of Ssb2 is required for interaction with Gcn4 and rescue of the gcn4 mutant phenotype. These findings suggest that Ssb2 is a critical factor that modulates Gcn4 functions in the nucleus and cytosol.

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“…Application of Poisson distribution as a statistical tool for this purpose was suggested by Wang and Spadoro (47) for conventional PCR. In general, the analysis of positive and negative results in a cohort of samples was used by incidence of rare events such as frequency of alleles, mutations, levels of viruses, and clone frequencies in complex populations (1,6,11,22,34,35,41), and by several studies presenting new nucleic acid-based methods (5,9,12,14,19). Nevertheless, Poisson analysis was applied qualitatively, and the quantitative information was neglected.…”

Section: Discussionmentioning

confidence: 99%

A Novel Poisson Distribution-Based Approach for Testing Boundaries of Real-Time PCR Assays for Food Pathogen Quantification

Rossmanith

Wagner

2011

Journal of Food Protection

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The validation of quantitative real-time PCR systems and above all, proof of the detection limit of this method, is a frequently and intensively discussed topic in food pathogen detection. Among proper sample collection, assay design, careful experimental design, execution of real-time PCR, and data analysis, the validation of the method per se ensuring reliable quantification data is of prime importance. The purpose of this study was to evaluate a novel validation tool for real-time PCR assays, based on the theoretical possibility of the amplification of a single DNA target. The underlying mathematical basis for the work is Poisson distribution, which describes patterns of low particle numbers in a volume. In this context, we focused on the quantitative aspect of real-time PCR for the first time. This allowed for demonstration of the reliable amplification of a lone target DNA molecule and the demonstration of the distinct discrimination between integer molecular numbers when using low initial copy numbers. A real-time PCR assay amplifying a 274-bp fragment of the positive regulatory protein A locus of Listeria monocytogenes was used for this work. Evidence for a linear range of quantification from a single target copy to 10 ng of target DNA was experimentally demonstrated, and evidence for the significance of this novel validation approach is presented here.

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A new method for the construction of a mutant library with a predictable occurrence rate using Poisson distribution

Cited by 6 publications

References 28 publications

Gcn4p-mediated transcriptional repression of ribosomal protein genes under amino-acid starvation

Gcn4p-mediated transcriptional repression of ribosomal protein genes under amino-acid starvation

Ssb2 is a novel factor in regulating synthesis and degradation of Gcn4 in Saccharomyces cerevisiae

A Novel Poisson Distribution-Based Approach for Testing Boundaries of Real-Time PCR Assays for Food Pathogen Quantification

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