Single cells (put a ring on it)

Proper spatial and temporal regulation of the small GTPase RhoA at the equatorial cortex represents a critical step in the specification of the division plane in eukaryotes. Despite increased understanding of the mechanisms whereby RhoA becomes active following chromosome segregation, far less is known about how RhoA is spatially regulated so that it concentrates precisely at the division site. In the April 1, 2009, issue of Genes & Development, Yoshida and colleagues (pp. 810–823) uncovered two genetically separable mechanisms whereby Rho1 is recruited to the bud neck in the budding yeast Saccharomyces cerevisiae to facilitate cytokinesis.     

Libraries against libraries for combinatorial selection of replicating antigen–antibody pairs

Antibodies are among the most highly selective tight-binding ligands for proteins. Because the human genome project has deciphered the proteome, there is an opportunity to use combinatorial antibody libraries to select high-affinity antibodies to every protein encoded by the genome. However, this is a large task because the selection formats used today for combinatorial antibody libraries are geared toward generating antibodies to one antigen at a time. Here, we describe a method that accelerates the identification of antibodies to a multitude of antigens simultaneously by matching combinatorial antibody libraries against eukaryotic antigen libraries so that replication-competent cognate antigen–antibody pairs can be directly selected. Phage and yeast display systems are used because they each link genotype to phenotype and can be replicated individually. When combined with cell sorting, the two libraries can be selected against each other for recovery of cognate antigen–antibody clones in a single experiment.     

Evaluation and management of vaginitis

OBJECTIVE: To evaluate recent advances in our understanding of the clinical relevance, diagnosis, and treatment of vaginal infections, and to determine an efficient and effective method of evaluating this clinical problem in the outpatient setting. DATA SOURCES: Relevant papers on vaginitis limited to the English language obtained through a MEDLINE search for the years 1985 to 1997 were reviewed. DATA SYNTHESIS: Techniques that enable the identification of the various strains of candida have helped lead to a better understanding of the mechanisms of recurrent candida infection. From this information a rationale for the treatment of recurrent disease can be developed. Bacterial vaginosis has been associated with complications, including upper genital tract infection, preterm delivery, and wound infection. Women undergoing pelvic surgery, procedures in pregnancy, or pregnant women at risk of preterm delivery should be evaluated for bacterial vaginosis to decrease the rate of complications associated with this condition. New, more standardized criteria for the diagnosis of bacterial vaginosis may improve diagnostic consistency among clinicians and comparability of study results. Use of topical therapies in the treatment of bacterial vaginosis are effective and associated with fewer side effects than systemic medication. Trichomonas vaginalis, although decreasing in incidence, has been associated with upper genital tract infection. Therapy of T. vaginalis infection has been complicated by an increasing incidence of resistance to metronidazole. CONCLUSIONS: Vaginitis is a common medical problem in women that is associated with significant morbidity and previously unrecognized complications. Research in recent years has improved diagnostic tools as well as treatment modalities for all forms of vaginitis.     

A 4-year longitudinal study of the oral prevalence of enteric gram–negative rods and yeasts in Chinese children

A 4-year longitudinal study of the oral prevalence of enteric gram–negative rods and yeasts in 116 Chinese primary school children in Hong Kong was conducted. The oral prevalence of enteric gram-negative rods for each consecutive year was 25.3%, 37.0%, 24.0%'and 25.8% respectively, with a weighted mean of 27.9%. Enterobacteriaceae , which comprised 57% of all enteric gram–negative rods, were more common in children with no caries experience. The oral prevalence of yeasts for each consecutive year was 7.7%, 12.0%, 14.4% and 15.5% respectively, with a weighted mean of 12.5%. Candida albicans comprised 84% of all yeasts isolated. Oral yeast carriage was significantly associated with caries prevalence. While the oral prevalence of enteric gram–negative rods in primary school children in Hong Kong may be higher than in other parts of the world, repeated isolation of either enteric gram–negative rods or Candida spp. from individual children over the 4–year study period was rare, suggesting that carnage of these organisms is transient.     

The genetics of ageing: insight from genome-wide approaches in invertebrate model organisms

Intense effort has been directed at understanding pathways modulating ageing in invertebrate model organisms. Prior to this decade, several longevity genes had been identified in flies, worms and yeast. More recently, with the development of RNAi libraries in worms and the yeast open reading frame (ORF) deletion collection, it has become routine to perform genome-wide screens for phenotypes of interest. A number of worm screens have now been performed to identify genes whose reduced expression leads to longer lifespan, and two ORF deletion longevity screens have been performed in yeast. Interestingly, these screens have linked previously unidentified cellular pathways to invertebrate ageing. More surprising, however, is the sheer number of longevity genes in worms and yeast. In this review, I discuss data from genome-wide screens in the context of evolutionary theories of ageing and raise issues regarding the increasing complexity associated with the genetics of longevity.     

Inhibition of the fungal fatty acid synthase type I multienzyme complex

Fatty acids are among the major building blocks of living cells, making lipid biosynthesis a potent target for compounds with antibiotic or antineoplastic properties. We present the crystal structure of the 2.6-MDa Saccharomyces cerevisiae fatty acid synthase (FAS) multienzyme in complex with the antibiotic cerulenin, representing, to our knowledge, the first structure of an inhibited fatty acid megasynthase. Cerulenin attacks the FAS ketoacyl synthase (KS) domain, forming a covalent bond to the active site cysteine C1305. The inhibitor binding causes two significant conformational changes of the enzyme. First, phenylalanine F1646, shielding the active site, flips and allows access to the nucleophilic cysteine. Second, methionine M1251, placed in the center of the acyl-binding tunnel, rotates and unlocks the inner part of the fatty acid binding cavity. The importance of the rotational movement of the gatekeeping M1251 side chain is reflected by the cerulenin resistance and the changed product spectrum reported for S. cerevisiae strains mutated in the adjacent glycine G1250. Platensimycin and thiolactomycin are two other potent inhibitors of KSs. However, in contrast to cerulenin, they show selectivity toward the prokaryotic FAS system. Because the flipped F1646 characterizes the catalytic state accessible for platensimycin and thiolactomycin binding, we superimposed structures of inhibited bacterial enzymes onto the S. cerevisiae FAS model. Although almost all side chains involved in inhibitor binding are conserved in the FAS multienzyme, a different conformation of the loop K1413–K1423 of the KS domain might explain the observed low antifungal properties of platensimycin and thiolactomycin.     

白念珠菌菌丝相与酵母相ERG11基因部分序列比较研究

目的通过对白念珠菌菌丝相与酵母相ERG11基因部分序列的比较，探讨两相细胞在DNA水平的差异。方法分别抽提7株来自同一亲本且对氟康唑敏感性不同的白念珠菌菌丝相与酵母相基因组DNA，根据ERG11基因编码序列设计一对引物，对ERG11基因第948位点至第1254位点307bp的碱基序列进行PCR扩增，以PCR产物直接测序比较两相在该片段碱基序列上的差异。结果7株白念珠菌菌丝相与酵母相在该片段的碱基序列无差异。结论白念珠菌菌丝相与酵母相ERG11基因的第948位点至第1254位点序列无差异。     

From the Cover: Vibrio effector protein VopQ inhibits fusion of V-ATPase–containing membranes

Vesicle fusion governs many important biological processes, and imbalances in the regulation of membrane fusion can lead to a variety of diseases such as diabetes and neurological disorders. Here we show that the Vibrio parahaemolyticus effector protein VopQ is a potent inhibitor of membrane fusion based on an in vitro yeast vacuole fusion model. Previously, we demonstrated that VopQ binds to the V_o domain of the conserved V-type H⁺-ATPase (V-ATPase) found on acidic compartments such as the yeast vacuole. VopQ forms a nonspecific, voltage-gated membrane channel of 18 Å resulting in neutralization of these compartments. We now present data showing that VopQ inhibits yeast vacuole fusion. Furthermore, we identified a unique mutation in VopQ that delineates its two functions, deacidification and inhibition of membrane fusion. The use of VopQ as a membrane fusion inhibitor in this manner now provides convincing evidence that vacuole fusion occurs independently of luminal acidification in vitro.Vesicle fusion governs many important physiological processes including neurotransmitter release and exocytosis. As such, many studies have focused on understanding this process and the proteins involved in fusion using various models such as yeast vacuoles and Drosophila synaptic vesicles (1, 2). Yeast vacuoles are an established and elegant model to study eukaryotic membrane fusion because of the ease of their isolation and the conserved nature of the fusion machinery required for their homotypic fusion (3). Although the core SNARE and Rab GTPase fusion machinery alone can drive the physiologically relevant fusion of liposomes in vitro (2), genetic and biochemical experiments have identified a number of additional regulators of vacuole fusion, including the membrane sector of the highly conserved V-type H⁺-ATPase (V-ATPase) (4, 5).The eukaryotic V-ATPase is the main electrogenic proton pump involved in the acidification of many intracellular organelles such as endosomes, lysosomes, and the yeast vacuole (6). The V-ATPase consists of two conserved, multisubunit domains: the cytoplasmic V₁ domain and the membrane bound V_o domain. The V₁ domain hydrolyzes ATP, providing the energy for proton translocation through the membrane-bound V_o proteolipid proton channel, thus acidifying the lumen of the vesicle. The loss of V-ATPase subunits is lethal in higher eukaryotes, highlighting the importance of this vital protein complex for normal eukaryotic physiology. However, yeast that lack subunits of the V-ATPase exhibit conditional lethality that is rescued by growth on acidic media, thus providing a unique and powerful system for the study of V-ATPase functions in vivo. In addition to its acidification function, the V-ATPase has been implicated in a broad range of biological processes, including the proper trafficking of secreted and endocytosed cargos (7), viral fusion (8), exocytosis (1, 9, 10), and the SNARE-dependent membrane fusion of yeast vacuoles (4, 5, 11, 12). Even though the role of V-ATPase in fusion has been demonstrated in various model organisms, its role in this process remains controversial (4, 13–16).Recent work has shown that a bacterial protein VopQ (also known as VP1680 or VepA) forms an outward rectifying, gated channel in membranes that contain the V-ATPase, resulting in the collapse of ion gradients and the disruption of autophagic flux (17). VopQ is a type III effector protein from the marine bacterium Vibrio parahaemolyticus that strongly associates with the V_o domain of the eukaryotic V-ATPase (17, 18). Given the proposed role of the V_o domain in fusion, we sought to examine the effect of VopQ on fusion using the well-defined biochemical model of eukaryotic membrane fusion from the budding yeast Saccharomyces cerevisiae. In this study, we demonstrate that expression of VopQ causes extensive yeast vacuolar fragmentation indicative of a defect in homotypic vacuole fusion. In vitro vacuole fusion assays confirmed VopQ is a potent inhibitor of a conserved Rab GTPase- and SNARE-dependent membrane fusion. Furthermore, we identify a mutant of VopQ, VopQ^S200P, that deacidifies the vacuole via its known channel-forming activity but no longer inhibits vacuole fusion in vitro because of its reduced affinity for the V-ATPase V_o domain. Therefore, by using a bacterial effector protein with yeast vacuoles containing wild-type V-ATPase machinery, we show that yeast vacuole fusion does not require luminal acidification in vitro.     

Molecular coevolution of a sex pheromone and its receptor triggers reproductive isolation in Schizosaccharomyces pombe

The diversification of sex pheromones is regarded as one of the causes of prezygotic isolation that results in speciation. In the fission yeast Schizosaccharomyces pombe, the molecular recognition of a peptide pheromone by its receptor plays an essential role in sexual reproduction. We considered that molecular coevolution of a peptide-mating pheromone, M factor, and its receptor, Map3, might be realized by experimentally diversifying these proteins. Here, we report the successful creation of novel mating-type pairs by searching for map3 suppressor mutations that rescued the sterility of M-factor mutants that were previously isolated. Several strong suppressors were found to also recognize WT M factor. The substituted residues of these Map3 suppressors were mapped to F204, F214, and E249, which are likely to be critical residues for M-factor recognition. These critical residues were systematically substituted with each of the other amino acids by in vitro mutagenesis. Ultimately, we successfully obtained three novel mating-type pairs constituting reproductive groups. These novel mating-type pairs could not conjugate with WT maters. Furthermore, no flow of chromosomally integrated drug-resistance genes occurred between the novel and the WT mating pairs, showing that each experimentally created reproductive group [e.g., M factor(V5H) and Map3(F214H)] was isolated from the WT group. In conclusion, we have succeeded in creating an artificial reproductive group that is isolated from the WT group. In keeping with the biological concept of species, the artificial reproductive group is a new species.Speciation is the most critical step in evolution (1). A new species branches off from an original species when a group of individuals is isolated reproductively (termed “reproductive isolation”) (2). Chemical communication between the two sexes is important in both attracting individuals of the opposite sex and the courtship reaction. Pheromone diversification may be a possible mechanism underlying reproductive isolation.Female-attracting peptide pheromones of newts are providing a promising means to explore this mechanism. A decapeptide called sodefrin was first identified during the analysis of a cDNA library of the abdominal gland of the red-bellied newt Cynops pyrrhogaster (3, 4). A closely related newt, the sword-tailed newt Cynops ensicauda, produces a similar peptide pheromone named Silefrin (5). Interestingly, a sodefrin variant, aonirin, was found in the Nara area of Japan; 1 of 10 amino acids in aonirin differs from those in sodefrin, the prototype peptide (6). This variant peptide was found to not be effective in attracting females in the Niigata and Chiba areas of Japan (7). It was, thus, speculated that altering the primary structure of the female-attracting peptide of the red-bellied newt and coevolution of the corresponding receptor protein may lead to reproductive isolation. To verify this speculation, we are interested in artificially altering a pheromone and its receptor, thereby mimicking coevolution in nature, by using a genetically amenable model organism, the fission yeast Schizosaccharomyces pombe.

Table 1.

Primary structure of peptide pheromones in some red-bellied newts (Cynops sp.) and fission yeasts (Schizosaccharomyces sp.)

Galactose metabolic genes in yeast respond to a ratio of galactose and glucose

Natural environments are filled with multiple, often competing, signals. In contrast, biological systems are often studied in “well-controlled” environments where only a single input is varied, potentially missing important interactions between signals. Catabolite repression of galactose by glucose is one of the best-studied eukaryotic signal integration systems. In this system, it is believed that galactose metabolic (GAL) genes are induced only when glucose levels drop below a threshold. In contrast, we show that GAL gene induction occurs at a constant external galactose:glucose ratio across a wide range of sugar concentrations. We systematically perturbed the components of the canonical galactose/glucose signaling pathways and found that these components do not account for ratio sensing. Instead we provide evidence that ratio sensing occurs upstream of the canonical signaling pathway and results from the competitive binding of the two sugars to hexose transporters. We show that a mutant that behaves as the classical model expects (i.e., cannot use galactose above a glucose threshold) has a fitness disadvantage compared with wild type. A number of common biological signaling motifs can give rise to ratio sensing, typically through negative interactions between opposing signaling molecules. We therefore suspect that this previously unidentified nutrient sensing paradigm may be common and overlooked in biology.The ability to integrate multiple cues about nutrient availability from the environment and coordinate uptake, metabolism, and regulatory networks is a major determinant of microbial cell fitness (1–3). The energy and building blocks needed for growth can come from many different sources, leading to a complex combinatorial signal integration problem. In an environment that contains a mixture of sugars, such as glucose and galactose, microbial cells regulate their response according to a carbon hierarchy mediated by catabolite repression. Galactose metabolic genes (GAL genes) are induced to a significant degree only after glucose-based catabolite repression is relieved, resulting in a lag in growth at the point of glucose exhaustion while GAL pathway proteins are produced (1–6). Recent studies of sugar integration in bacteria (7, 8) suggested that in these organisms the combinatorial response results from the multiplication of individual responses to different sugars.The response of Saccharomyces cerevisiae to galactose is one of the best-studied eukaryotic signaling pathways (1, 4–6, 9–12). The GAL response has become a canonical example for combinatorial signal integration based on a genetic switch (10–13). All GAL genes are induced by the activator Gal4p in response to galactose (14) but repressed by Mig1p when glucose is present (15). The inhibition of GAL genes by glucose is thought to occur at a threshold concentration, with signal integration occurring at promoters (11). These conclusions rest on a limited sampling of combinations of concentrations of glucose and galactose (SI Appendix, Fig. S6) (16–21). Our goal, therefore, was to use modern high-throughput techniques that allow us to characterize the GAL genes’ metabolic response in detail.