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Candida guilliermondii (teleomorph Meyerozyma guilliermondii) is an ascomycetous species belonging to the fungal CTG clade. This yeast remains actively studied as a result of its moderate clinical importance and most of all for its potential uses in biotechnology. The aim of the present study was to establish a convenient transformation system for C. guilliermondii by developing both a methionine auxotroph recipient strain and a functional MET gene as selection marker. We first disrupted the MET2 and MET15 genes encoding homoserine-O-acetyltransferase and O-acetylserine O-acetylhomoserine sulphydrylase, respectively. The met2 mutant was shown to be a methionine auxotroph in contrast to met15 which was not. Interestingly, met2 and met15 mutants formed brown colonies when cultured on lead-containing medium, contrary to the wild-type strain, which develop as white colonies on this medium. The MET2 wild-type allele was successfully used to transfer a yellow fluorescent protein (YFP) gene-expressing vector into the met2 recipient strain. In addition, we showed that the loss of the MET2-containing YFP-expressing plasmid can be easily observed on lead-containing medium. The MET2 wild-type allele, flanked by two short repeated sequences, was then used to disrupt the LYS2 gene (encoding the α-aminoadipate reductase) in the C. guilliermondii met2 recipient strain. The resulting lys2 mutants displayed, as expected, auxotrophy for lysine. Unfortunately, all our attempts to pop-out the MET2 marker (following the recombination of the bordering repeat sequences) from a target lys2 locus were unsuccessful using white/brown colony colour screening. Nevertheless, this MET2 transformation/disruption system represents a new versatile genetic tool for C. guilliermondii., (Copyright © 2014 John Wiley & Sons, Ltd.)

We evaluated multiple effects of genetic variations of five candidate loci (LEP, LEPR, MC4R, PIK3C3 and VRTN) on four production traits (average daily weight gain (ADG); backfat thickness (BFT); loin eye muscle area (EMA); and intramuscular fat content (IMF)) in a closed nucleus herd of pure Duroc pigs. Polymorphisms in LEPR, MC4R and PIK3C3 had significant single gene effects on ADG and BFT. The additive genetic variance in ADG and BFT (16.99% and 22.51%, respectively) was explained by genetic effects of these three loci. No correlations were observed between the LEP genotype and production traits in this study. Although we detected marginally epistatic interactions between LEPR and PIK3C3 on the eye muscle area, there were no significant epistatic effects on any traits among all loci pairs. These results suggest that LEPR, MC4R, PIK3C3 and VRTN may independently influence growth rate and fat deposition. Furthermore, the statistical models for predicting the breeding values of each trait had the lowest Akaike's information criterion values when considering the effect of the MC4R, LEPR, PIK3C3 and VRTN genotype simultaneously. These results suggest that LEPR, MC4R, PIK3C3 and VRTN are useful markers for accurately predicting breeding values in Duroc pigs., (© 2013 Japanese Society of Animal Science.)

Equine breeding selection has been developed by applying quantitative genetic methods for calculating the heritability of the complex traits such as performance in racing or sport competitions. With the great development of biotechnologies, equine molecular genetics has come of age. The recent sequencing of the equine genome by an international consortium was a major advance that will impact equine genomics in the near future. With the rapid progress in equine genetics, new applications in early performance evaluation and the detection of disease markers become available. Many new biomolecular tools will change management of horse selection, disease diagnosis and treatment. The purpose of this review is to present new developments in equine genetics and genomics for performance evaluation and health markers after a short summary of the previous knowledge about the genetic components of the exercise performance traits., (© 2010 EVJ Ltd.)

Transcription factors of the ETS family are important regulators of endothelial and hematopoietic development. We have characterized the Xenopus orthologue of the ETS transcription factor, ETV2. Expression analysis shows that etv2 is highly expressed in hematopoietic and endothelial precursor cells in the Xenopus embryo. In gain-of-function experiments, ETV2 is sufficient to activate ectopic expression of vascular endothelial markers. In addition, ETV2 activated expression of hematopoietic genes representing the myeloid but not the erythroid lineage. Loss-of-function studies indicate that ETV2 is required for expression of all endothelial markers examined. However, knockdown of ETV2 has no detectable effects on expression of either myeloid or erythroid markers. This contrasts with studies in mouse and zebrafish where ETV2 is required for development of the myeloid lineage. Our studies confirm an essential role for ETV2 in endothelial development, but also reveal important differences in hematopoietic development between organisms.

Previous studies identified Inka1 as a gene regulated by AP-2alpha in the neural crest required for craniofacial morphogenesis in fish and frog. Here, we extend the analysis of Inka1 function and regulation to the mouse by generating a LacZ knock-in allele. Inka1-LacZ allele expression occurs in the cephalic mesenchyme, heart, and paraxial mesoderm prior to E8.5. Subsequently, expression is observed in the migratory neural crest cells and their derivatives. Consistent with expression of Inka1 in tissues of the developing head during neurulation, a low percentage of Inka1(-/-) mice show exencephaly while the remainder are viable and fertile. Further studies indicate that AP-2alpha is not required for Inka1 expression in the mouse, and suggest that there is no significant genetic interaction between these two factors during embryogenesis. Together, these data demonstrate that while the expression domain of Inka1 is conserved among vertebrates, its function and regulation are not.

Gene expression signatures can be developed as comprehensive pathway readouts and used as pharmacodynamic or patient-stratification biomarkers. While a consensus on the best practices for selecting gene expression signatures from microarray data is evolving, we have developed basic guidelines to ensure consistency and quality. Here we illustrate these guidelines through the identification of a growth factor gene expression signature that is responsive to phosphatidylinositol 3-kinase (PI3K) pathway perturbations in vitro and related to phosphatase and tensin homolog (PTEN) deregulation in vivo.

Recent studies have shown that resting-state functional networks as studied with fMRI, EEG, and MEG may be so-called small-world networks. We investigated to what extent the characteristic features of small-world networks are genetically determined. To represent functional connectivity between brain areas, we measured resting EEG in 574 twins and their siblings and calculated the synchronization likelihood between each pair of electrodes. We applied a threshold to obtain a binary graph from which we calculated the clustering coefficient C (describing local interconnectedness) and average path length L (describing global interconnectedness) for each individual. Modeling of MZ and DZ twin and sibling resemblance indicated that across various frequency bands 46-89% of the individual differences in C and 37-62% of the individual differences in L are heritable. It is asserted that C, L, and a small-world organization are viable markers of genetic differences in brain organization., (Copyright 2007 Wiley-Liss, Inc.)

The Pichia pastoris expression system is being used successfully for the production of various recombinant heterologous proteins. Recent developments with respect to the Pichia expression system have had an impact on not only the expression levels that can be achieved, but also the bioactivity of various heterologous proteins. We review here some of these recent developments, as well as strategies for reducing proteolytic degradation of the expressed recombinant protein at cultivation, cellular and protein levels. The problems associated with post-translational modifications performed on recombinant proteins by P. pastoris are discussed, including the effects on bioactivity and function of these proteins, and some engineering strategies for minimizing unwanted glycosylations. We pay particular attention to the importance of optimizing the physicochemical environment for efficient and maximal recombinant protein production in bioreactors and the role of process control in optimizing protein production is reviewed. Finally, future aspects of the use of the P. pastoris expression system are discussed with regard to the production of complex membrane proteins, such as G protein-coupled receptors, and the industrial and clinical importance of these proteins., (Copyright (c) 2005 John Wiley & Sons, Ltd.)

We describe the development of a new transformation system, using multiple auxotrophic marker genes, for the fission yeast Schizosaccharomyces pombe. We developed three new auxotrophic marker genes (arg12(+), tyr1(+) and ade7(+)) and generated a new host strain, YF043, by Cre-loxP-mediated gene disruption. YF043 possessed six mutated biosynthetic genes (leu1-32, ura4-M190T, arg12::loxP, tyr1::loxP, ade7::loxP and his2::loxP). The combination of this host strain and the new selectable markers can be used for gene disruption using the same preexisting transformation systems. In addition, Sz. pombe vectors were constructed, containing selectable marker genes that complement the auxotrophies of YF043. These new vectors are available for gene disruption and heterologous protein expression in strain YF043. The new Sz. pombe host strain will be a useful tool for molecular genetic studies of Sz. pombe where multiple recombinant modifications or multiple mutations are needed., (Copyright 2005 John Wiley & Sons, Ltd.)

Counter-selection is a useful gene manipulation technique for repeated gene disruptions, gene shufflings and gene replacements in yeasts. We developed a novel counter-selection system using a galactose-inducible growth inhibitory sequence (Kawahata et al.1999. Yeast 15: 1-10). This counter-selection marker, named GAL10p-GIN11, has several advantages over previous counter-selection markers, i.e. use of an inexpensive galactose medium for counter-selection, combined use with any transformation markers for gene introduction, and no requirement of specific mutations in the host strains. The GIN11 sequence, which is a part of an X-element of the subtelomeric regions, contained a conserved autonomously replicating sequence, causing the possibility of inefficient chromosomal integration. We isolated GIN11 mutants that lost the replication activity but retained the growth-inhibitory effect when overexpressed. A mutant GIN11M86 sequence was selected and fused to the CUP1 promoter for the counter-selection on a copper-containing medium. The GALp-GIN11M86 and the CUPp-GIN11M86 were used for constructing sets of integrating plasmids containing auxotrophic markers involving HIS3, TRP1, LEU2, URA3 or ADE2, or a drug-resistant marker PGKp-YAP1. In addition, a set of gene disruption cassettes that contained each of the auxotrophic markers and the GALp-GIN11M86, which were flanked by direct repeats of a hisG sequence, were constructed. The counter-selectable integrating plasmids and the gene disruption cassettes can allow the markers to be used repeatedly for yeast gene manipulations., (Copyright 2002 John Wiley & Sons, Ltd.)

A set of yeast strains based on Saccharomyces cerevisiae S288C in which commonly used selectable marker genes are deleted by design based on the yeast genome sequence has been constructed and analysed. These strains minimize or eliminate the homology to the corresponding marker genes in commonly used vectors without significantly affecting adjacent gene expression. Because the homology between commonly used auxotrophic marker gene segments and genomic sequences has been largely or completely abolished, these strains will also reduce plasmid integration events which can interfere with a wide variety of molecular genetic applications. We also report the construction of new members of the pRS400 series of vectors, containing the kanMX, ADE2 and MET15 genes.