Structural characterization of Saccharomyces cerevisiae prion-like protein Ure2

Sacchromyces cerevisiae prion-like protein Ure2 was expressed in Escherichia coli and was purified to homogeneity. We show here that Ure2p is a soluble protein that can assemble into fibers that are similar to the fibers observed in the case of PrP in its scrapie prion filaments form or that form on Sup35 self-assembly. Ure2p self-assembly is a cooperative process where one can distinguish a lag phase followed by an elongation phase preceding a plateau. A combination of size exclusion chromatography, sedimentation velocity, and electron microscopy demonstrates that the soluble form of Ure2p consists at least of three forms of the protein as follows: a monomeric, dimeric, and tetrameric form whose abundance is concentration-dependent. By the use of limited proteolysis, intrinsic fluorescence, and circular dichroism measurements, we bring strong evidence for the existence of at least two structural domains in Ure2p molecules. Indeed, Ure2p NH2-terminal region is found poorly structured, whereas its COOH-terminal domain appears to be compactly folded. Finally, we show that only slight conformational changes accompany Ure2p assembly into insoluble high molecular weight oligomers. These changes essentially affect the COOH-terminal part of the molecule. The properties of Ure2p are compared in the discussion to that of other prion-like proteins such as Sup35 and mammalian prion protein PrP.     

Molecular evolution and population genetics of duplicated accessory gland protein genes in Drosophila

To investigate the potential importance of gene duplication in D. melanogaster accessory gland protein (Acp) gene evolution we carried out a computational analysis comparing annotated D. melanogaster Acp genes to the entire D. melanogaster genome. We found that two known Acp genes are actually members of small multigene families. Polymorphism and divergence data from these duplicated genes suggest that in at least four cases, protein divergence between D. melanogaster and D. simulans is a result of directional selection. One putative Acp revealed by our computational analysis shows evidence of a recent selective sweep in a non-African population (but not in an African population). These data support the idea that selection on reproduction-related genes may drive divergence of populations within species, and strengthen the conclusion that Acps may often be under directional selection in Drosophila.     

Molecular population genetics and evolution of Drosophila meiosis genes

While many functional elements of the meiotic process are well characterized in model organisms, the genetic basis of most of the natural phenotypic variation observed in meiotic pathways has not been determined. To begin to address this issue, we characterized patterns of polymorphism and divergence in the protein-coding regions of 33 genes across 31 lines of Drosophila melanogaster and 6 lines of Drosophila simulans. We sequenced genes known to be involved in chromosome segregation, recombination, DNA repair, and related heterochromatin binding. As expected, we found several of the genes to be highly conserved, consistent with purifying selection. However, a subset of genes showed patterns of polymorphism and divergence typical of other types of natural selection. Moreover, several intriguing differences between the two Drosophila lineages were evident: along the D. simulans lineage we consistently found evidence of adaptive protein evolution, whereas along the D. melanogaster lineage several loci exhibited patterns consistent with the maintenance of protein variation.     

Molecular population genetics and the search for adaptive evolution in plants

The first papers on plant molecular population genetics were published approximately 10 years ago. Since that time, well over 50 additional studies of plant nucleotide polymorphism have been published, and many of these studies focused on detecting the signature of balancing or positive selection at a locus. In this review, we discuss some of the theoretical and statistical issues surrounding the detection of selection, with focus on plant populations, and we also summarize the empirical plant molecular population genetics literature. At face value, the literature suggests that a history of balancing or positive selection in plant genes is rampant. In two well-studied taxa (maize and Arabidopsis) over 20% of studied genes have been interpreted as containing the signature of selection. We argue that this is probably an overstatement of the prevalence of natural selection in plant genomes, for two reasons. First, demographic effects are difficult to incorporate and have generally not been well integrated into the plant population genetics literature. Second, the genes studied to date are not a random sample, so selected genes may be overrepresented. The next generation of studies in plant molecular population genetics requires additional sampling of local populations, explicit comparisons among loci, and improved theoretical methods to control for demography. Eventually, candidate loci should be confirmed by explicit consideration of phenotypic effects.     

Molecular population genetics of Xdh and the evolution of base composition in Drosophila

Few loci have been measured for DNA polymorphism and divergence in several species. Here we report such data from the protein-coding region of xanthine dehydrogenase (Xdh) in 22 species of Drosophila. Many of our samples were from closely related species, allowing us to confidently assign substitutions to individual lineages. Surprisingly, Xdh appears to be fixing more A/T mutations than G/C mutations in most lineages, leading to evolution of higher A/T content in the recent past. We found no compelling evidence for selection on protein variation, though some aspects of the data support the notion that a significant fraction of amino acid polymorphisms are slightly deleterious. Finally, we found no convincing evidence that levels of silent heterozygosity are associated with rates of protein evolution.     

Molecular population genetics of the gene encoding the human fertilization protein zonadhesin reveals rapid adaptive evolution

A hallmark of positive selection (adaptive evolution) in protein-coding regions is a d(N)/d(S) ratio >1, where d(N) is the number of nonsynonymous substitutions/nonsynonymous sites and d(S) is the number of synonymous substitutions/synonymous sites. Zonadhesin is a male reproductive protein localized on the sperm head, comprising many domains known to be involved in cell-cell interaction or cell adhesion. Previous studies have shown that VWD domains (homologous to the D domains of the von Willebrand factor) are involved directly in binding to the female zona pellucida (ZP) in a species-specific manner. In this study, we sequenced 47 coding exons in 12 primate species and, by using maximum-likelihood methods to determine sites under positive selection, we show that VWD2, membrane/A5 antigen mu receptor, and mucin-like domains in zonadhesin are rapidly evolving and, thus, may be involved in binding to the ZP in a species-specific manner in primates. In addition, polymorphism data from 48 human individuals revealed significant polymorphism-to-divergence heterogeneity and a significant departure from equilibrium-neutral expectations in the frequency spectrum, suggesting balancing selection and positive selection occurring in zonadhesin (ZAN) within human populations. Finally, we observe adaptive evolution in haplotypes segregating for a frameshift mutation that was previously thought to indicate that ZAN was a potential pseudogene.     

Specificity and genetics of S-adenosylmethionine transport in Saccharomyces cerevisiae.

The specificity of a transport system for S-adenosylmethionine was determined through the use of structurally related derivatives. Of the compounds tested, the analogues S-adenosylethionine and S-inosylmethionine and the naturally occurring compounds S-adenosyl-(5')-3-methylthiopropylamine and S-adenosylhomocysteine competitively inhibited uptake of the sulfonium compound. Ki values for these compounds indicate that the order of affinity for the transport protein is S-adenosylmethionine congruent to S-adenosyl-(5')-3-methyl-thiopropylamine greater than S-adenosylethionine greater than S-inosylmethionine greater than S-adenosylhomocysteins. S-adenosyl-(2-hydroxy-4-methylthio)butyric acid exerted inhibition of a mixed type. S-insoyl-(2-hydroxy-4-methylthio)butyric acid, S-inosylhomocysteine, and S-ribosylhomocysteine were without effect. On the basis of the inhibition data, the methionine-amino, adenine-amino, and methyl groups were identified as group important in the binding of S-adenosylmethionine to the transport protein. Comparison is made with the specificities of various transmethylating enzymes utilizing S-adenosylmethionine. In addition, a number of conventional and temperature-sensitive S-adenosylmethionine transport mutants were isolated and analyzed in an attempt to identify the structural character of the specific transport protein(s). The data obtained suggest that only a single gene (a single polypeptide) is involved in specific S-adenosylmethionine transport. Apparent interallelic complementation supports the assumption that the functional form of the protein is composed of two or more copies of a monomer.     

Induction and genetics of two alpha-galactosidase activities in Saccharomyces cerevisiae

Summary The induction of -galactosidase of Saccharomyces cerevisiae was investigated. We have demonstrated the existence of inducible internal and external -galactosidase activities and have studied the relationship between the two -galactosidases by examining a mutant strain which lacks both the internal and external activities. The mutant possesses a mutation in a single locus (mel 1-1) which does not affect the synthesis of the other galactose pathway enzymes or the ability of the yeast to grow on media containing only galactose as the carbon source. Genetic studies of the mutant indicate that mel 1-1 is recessive and allelic to the wild type allele for melibiose fermentation Mel 1.This investigation was supported in part by Grant No. AM15325 from the National Institutes of Arthritis, Metabolism and Digestive Diseases, and a grant from the Office of Research Administration, University of Hawaii. R.G. Buckholz was a National Science Foundation Pre-Doctoral Fellow during part of this study     

Molecular population genetics.

Molecular population genetics is entering a new era dominated by studies of genomic polymorphism. Some of the theory that will be needed to analyze data generated by such studies is already available, but much more work is needed. Furthermore, population genetics is becoming increasingly relevant to other fields of biology, for example to genetic epidemiology, because of disease gene mapping in general populations.     

Eliminating gene conversion improves high-throughput genetics in Saccharomyces cerevisiae

Synthetic genetic analysis was improved by eliminating leaky expression of the HIS3 reporter and gene conversion between the HIS3 reporter and his3Delta1. Leaky expression was eliminated using 3-aminotriazole and gene conversion was eliminated by using the Schizosaccharomyces pombe his5+ gene, resulting in a 5- to 10-fold improvement in the efficiency of SGA.     

Interactions of [NSI +] prion-like determinant with SUP35 and VTS1 genes in Saccharomyces cerevisiae

Previously we characterized [NSI ⁺], determinant, that possesses the features of a yeast prion. This determinant causes the nonsense suppression in strains that bear different N-substituted variants of Sup35p, which is a translation release factor eRF3. As a result of the genomic screen, we identified VTS1, the overexpression of which is a phenotypic copy of [NSI ⁺]. Here, we analyzed the influence of SUP35 and VTS1 on [NSI ⁺]. We demonstrated nonsense suppression in the [NSI ⁺] strains, which appears when SUP35 expression was decreased or against a background of general defects in the fidelity of translation termination. [NSI ⁺] has also been shown to increase VTS1 mRNA amounts. These findings facilitate the insight into the mechanisms of nonsense suppression in the [NSI ⁺] strains and narrow the range of candidates for [NSI ⁺] determinant.     

Molecular and physiological comparisons between Saccharomyces cerevisiae and Saccharomyces boulardii

In this paper, comparative molecular studies between authentic Saccharomyces cerevisiae strains, related species, and the strain described as Saccharomyces boulardii were performed. The response of a S. boulardii strain and a S. cerevisiae strain (W303) to different stress conditions was also evaluated. The results obtained in this study show that S. boulardii is genetically very close or nearly identical to S. cerevisiae. Metabolically and physiologically, however, it shows a very different behavior, particularly in relation to growth yield and resistance to temperature and acidic stresses, which are important characteristics for a microorganism to be used as a probiotic.     

Applications of the Saccharomyces cerevisiae Flp-FRT system in bacterial genetics

The Flp-FRT site-specific recombination system from Saccharomyces cerevisiae is a powerful and efficient tool for high-throughput genetic analysis of bacteria in the postgenomic era. This review highlights the features of the Flp-FRT system, describes current bacterial genetic methods incorporating this technology and, finally, suggests potential future uses of this system. In combination with improved allele replacement methods, recyclable FRT mutagenesis cassettes, whose antibiotic resistance markers can be excised from the chromosome in vivo, are useful for the rapid construction of multiple, unmarked mutations in the same chromosome, and thus aid in the generation of live vaccine strains or food-safe bacteria. The high-specificity of the Flp-FRT system makes it also applicable for manipulation of whole genomes, including in vivo cloning of large genomic segments. Integration-proficient vectors, from which antibiotic resistance markers and replication functions can be evicted after integration of the desired sequences into the chromosome, are useful for the construction of strains destined for environmental release, e.g. strains used as biosensors or for bioremediation. Although the Flp-FRT system is extremely efficient and easy to use, its true potential in bacterial genetics has not yet been fully exploited. On the contrary, in many instances this technology is probably greatly underutilized, especially in gram-positive bacteria.