Systematic Mutational Analysis of the Intracellular Regions of Yeast Gap1 Permease

The yeast general amino acid permease Gap1 is a convenient model for studying the intracellular trafficking of membrane proteins. Present at the plasma membrane when the nitrogen source is poor, it undergoes ubiquitin-dependent endocytosis and degradation upon addition of a good nitrogen source, e.g. ammonium. It comprises 12 transmembrane domains (TM) flanked by cytosol-facing N- and C-terminal tails (NT, CT). The NT of Gap1 contains the acceptor lysines for ubiquitylation and its CT includes a sequence essential to exit from the endoplasmic reticulum (ER).Journal ArticleResearch Support, Non-U.S. Gov'tSCOPUS: ar.jinfo:eu-repo/semantics/publishe

Genome-Wide Analysis of Nucleotide-Level Variation in Commonly Used Saccharomyces cerevisiae Strains

Ten years have passed since the genome of Saccharomyces cerevisiae–more precisely, the S288c strain–was completely sequenced. However, experimental work in yeast is commonly performed using strains that are of unknown genetic relationship to S288c. Here, we characterized the nucleotide-level similarity between S288c and seven commonly used lab strains (A364A, W303, FL100, CEN.PK, ∑1278b, SK1 and BY4716) using 25mer oligonucleotide microarrays that provide complete and redundant coverage of the ∼12 Mb Saccharomyces cerevisiae genome. Using these data, we assessed the frequency and distribution of nucleotide variation in comparison to the sequenced reference genome. These data allow us to infer the relationships between experimentally important strains of yeast and provide insight for experimental designs that are sensitive to sequence variation. We propose a rational approach for near complete sequencing of strains related to the reference using these data and directed re-sequencing. These data and new visualization tools are accessible online in a new resource: the Yeast SNPs Browser (YSB; http://gbrowse.princeton.edu/cgi-bin/gbrowse/yeast_strains_snps) that is available to all researchers

α-Arrestins Aly1 and Aly2 Regulate Intracellular Trafficking in Response to Nutrient Signaling

Arrestins, known regulators of endocytosis, take on novel functions in nutrient-regulated endosomal recycling. Yeast α-arrestins, Aly1 and Aly2, redistribute the Gap1 permease from endosomes to the cell surface and interact with clathrin/AP-1. Aly2 is regulated by the Npr1 kinase and acts through mechanisms distinct from Aly1

Multicopy plasmid integration in Komagataella phaffii mediated by a defective auxotrophic marker

Background: A commonly used approach to improve recombinant protein production is to increase the levels of expression by providing extra-copies of a heterologous gene. In Komagataella phaffii (Pichia pastoris) this is usually accomplished by transforming cells with an expression vector carrying a drug resistance marker following a screening for multicopy clones on plates with increasingly higher concentrations of an antibiotic. Alternatively, defective auxotrophic markers can be used for the same purpose. These markers are generally transcriptionally impaired genes lacking most of the promoter region. Among the defective markers commonly used in Saccharomyces cerevisiae is leu2-d, an allele of LEU2 which is involved in leucine metabolism. Cells transformed with this marker can recover prototrophy when they carry multiple copies of leu2-d in order to compensate the poor transcription from this defective allele. Results: A K. phaffii strain auxotrophic for leucine (M12) was constructed by disrupting endogenous LEU2. The resulting strain was successfully transformed with a vector carrying leu2-d and an EGFP (enhanced green fluorescent protein) reporter gene. Vector copy numbers were determined from selected clones which grew to different colony sizes on transformation plates. A direct correlation was observed between colony size, number of integrated vectors and EGFP production. By using this approach we were able to isolate genetically stable clones bearing as many as 20 integrated copies of the vector and with no significant effects on cell growth. Conclusions: In this work we have successfully developed a genetic system based on a defective auxotrophic which can be applied to improve heterologous protein production in K. phaffii. The system comprises a K. phaffii leu2 strain and an expression vector carrying the defective leu2-d marker which allowed the isolation of multicopy clones after a single transformation step. Because a linear correlation was observed between copy number and heterologous protein production, this system may provide a simple approach to improve recombinant protein productivity in K. phaffii

Translocation of zeatin riboside and zeatin in soybean explants

Soybean explants consisting of a leaf, one or more young pods, and a subtending piece of stem were given a 1-h pulse of 3 H (ring-labeled)-zeatin riboside (ZR) or -zeatin (Z), via the base of the stem, followed by a 24-h incubation. At the end of the pulse, about 55% of the soluble 3 H was in the leaf blades, 11% in the petiole, 30% in the stem, 2% in the carpels, 0.1% in the seed coats, and 0.08% in the embryos. After 24 h, the percentages were 58, 7, 26, 6, 2, and 0.3, respectively. During this period, the total soluble 3 H decreased by 84%, the remainder being bound to “insoluble” material. The 3 H-cytokinin was rapidly converted to diverse metabolites including adenosine and adenine. At the end of the 1-h pulse, appreciable percentages (1–16%) of the total soluble 3 H in the seed coats chromatographed with ZR (or dihydro ZR) and with the 5′-phosphate of ZR, but these percentages declined markedly at 24 h. No distinct peaks of 3 H corresponded to known metabolites in the soluble extracts of embryos, and at 24 h, the 3 H equivalent to ZR must have been less than 0.0006% of the 3 H-ZR supplied. The bound 3 H corresponded to adenine and guanine in DNA and RNA. In contrast to cytokinin, 3 H-adenosine given as a pulse was readily translocated into the seed coats and embryos. Thus, cytokinin (ZR and Z) flowing up through the xylem from the root system does not readily enter the embryo (though metabolites such as adenosine could), and the seeds clearly do not compete with the leaves for this supply of cytokinin.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/45929/1/344_2005_Article_BF02042255.pd

Propagation of uncertainty in a rotating pipe mechanism to generate an impinging swirling jet flow for heat transfer from a flat plate

In Computational Fluid Dynamics (CFD) studies composed of the coupling of different simulations, the uncertainty in one stage may be propagated to the following stage and affect the accuracy of the prediction. In this paper, a framework for uncertainty quantification in the computational heat transfer by forced convection is applied to the two-step simulation of the mechanical design of a swirling jet flow generated by a rotating pipe (Simulation 1) impinging on a flat plate (Simulation 2). This is the first probabilistic uncertainty analysis on computational heat transfer by impinging jets in the literature. The conclusion drawn from the analysis of this frequent engineering application is that the simulated system does not exhibit a significant sensitivity to stochastic variations of model input parameters, over the tested uncertainty ranges. Additionally, a set of non-linear regression models for the stochastic velocity and turbulent profiles for the pipe nozzle are created and tested, since impinging jets for heat transfer at Reynolds number of Re = 23000 are very frequent in the literature, but stochastic inlet conditions have never been provided. Numerical results demonstrate a negligible difference in the predicted convective heat transfer with respect to the use of the profiles simulated via CFD. These suggested surrogate models can be directly embedded onto other engineering applications (e.g. arrays of jets, jet flows impinging on plates with different shapes, inlet piping in combustion, chemical mixing, etc.) in which a realistic swirling flow under uncertainty can be of interest

On the meaning of effects of substrate structure on biological transport

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/44802/1/10863_2005_Article_BF01516050.pd