Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Mating Factor”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 235 records · Page 13Linked to original sources
PubMed1996

Expression of recombinant HLA-DR2 molecules. Replacement of the hydrophobic transmembrane region by a leucine zipper dimerization motif allows the assembly and secretion of soluble DR alpha beta heterodimers.

Major histocompatibility complex (MHC) class II molecules are membrane-anchored heterodimers that present peptides on the surface of antigen presenting cells to T cells. Soluble HLA-DR2 molecules were expressed for structural and functional characterization of the MHC/peptide/T cell receptor recognition unit. The alpha and beta chains of DR2 (encoded by the DRA, DRB1*1501 genes) did not assemble in mammalian or insect cell lines when the transmembrane regions of one or both chains were truncated. The hydrophobic transmembrane regions of DRalpha and DRbeta facilitate assembly of the heterodimer and were therefore replaced by the leucine zipper dimerization motifs from the transcription factors Fos and Jun, which assemble as a soluble, tightly packed coiled coil structure. The DRalpha-Fos and DRbeta-Jun constructs were expressed in a methyltrophic yeast, Pichia pastoris, using the alpha-mating factor secretion signal to direct expression to the secretory pathway. DR alphabeta heterodimers were purified from supernatants using an antibody specific for the DR alphabeta heterodimer. Kinetic and quantitative peptide binding experiments demonstrated that recombinant DR2 molecules were efficiently loaded with an antigenic peptide. Soluble DR2 molecules can be used to define structural aspects of the MHC/peptide/T cell receptor interaction and to study the signals induced by T cell receptor recognition of soluble DR2.peptide complexes.

Amino Acid Sequence↗
PubMed1989

Synthesis and secretion of human nerve growth factor by Saccharomyces cerevisiae.

The DNA coding for human nerve growth factor (hNGF) was chemically synthesized and introduced into Saccharomyces cerevisiae. Expression and secretion of hNGF was obtained by use of the yeast phosphoglycerate kinase-encoding gene promoter and the pre-pro sequence of the yeast alpha-mating factor. Immunoblotting with antiserum raised against a protein A-hNGF fusion protein, allowed the detection of an immunoreactive material secreted into the culture medium. A preparation from the culture medium, partially purified by ion-exchange column chromatography, stimulated neurite outgrowth from rat pheochromocytoma PC12h cells.

Amino Acid Sequence↗
PubMed2001

A filamentous growth response mediated by the yeast mating pathway.

Haploid cells of the budding yeast Saccharomyces cerevisiae respond to mating pheromones by arresting their cell-division cycle in G1 and differentiating into a cell type capable of locating and fusing with mating partners. Yeast cells undergo chemotactic cell surface growth when pheromones are present above a threshold level for morphogenesis; however, the morphogenetic responses of cells to levels of pheromone below this threshold have not been systematically explored. Here we show that MATa haploid cells exposed to low levels of the alpha-factor mating pheromone undergo a novel cellular response: cells modulate their division patterns and cell shape, forming colonies composed of filamentous chains of cells. Time-lapse analysis of filament formation shows that its dynamics are distinct from that of pseudohyphal growth; during pheromone-induced filament formation, daughter cells are delayed relative to mother cells with respect to the timing of bud emergence. Filament formation requires the RSR1(BUD1), BUD8, SLK1/BCK1, and SPA2 genes and many elements of the STE11/STE7 MAP kinase pathway; this response is also independent of FAR1, a gene involved in orienting cell polarization during the mating response. We suggest that mating yeast cells undergo a complex response to low levels of pheromone that may enhance the ability of cells to search for mating partners through the modification of cell shape and alteration of cell-division patterns.

Cell Division↗
PubMed1998

Characterization of a mitogen-activated protein kinase from Pneumocystis carinii.

The pathogenic fungus Pneumocystis carinii causes severe pneumonia in patients with impaired immunity, particularly patients with acquired immunodeficiency syndrome. The life cycle of P. carinii is poorly understood, and the inability to continuously culture P. carinii is a major limitation in understanding its cell biology. In fungi homologous to P. carinii, pheromone mating factors signal through a mitogen-activated protein kinase (MAPK) signal transduction cascade, resulting in mitotic cell cycle arrest and entry into a pathway of conjugation, cellular differentiation, and proliferation. Using degenerate PCR and library screening, we have identified a MAPK cDNA in P. carinii that is highly homologous to fungal MAPKs involved in the pheromone mating signal transduction cascade, and we demonstrate MAPK activity in P. carinii lysates with a specific antiserum derived from the translated P. carinii MAPK cDNA sequence.

Amino Acid Sequence↗
PubMed1995

The yeast homologue YTIS11, of the mammalian TIS11 gene family is a non-essential, glucose repressible gene.

The murine TIS11 primary response gene is rapidly and transiently induced in response to many extracellular signals. A CX8CX5CX3H sequence is present twice in the TIS11 protein, in two additional murine proteins, TIS11B and TIS11D, that share regions of strong sequence conservation with TIS11, and in a Drosophila homologue (DTIS11). Although immunolocalization of TIS11 protein to the nucleus and zinc binding have lead to the speculation that the TIS11 family proteins are transcription factors, no function for these proteins has yet been clearly determined. We have now identified a TIS11 homologue, YTIS11, from Saccharomyces cerevisiae. The Ytis11p protein conserves both the two putative zinc finger CX8CX5CX3H sequences and the spacing between them, as well as additional amino acids in this region. The amino terminal 169 amino acid portion of Ytis11p protein, which contains a large number of acidic amino acids, can serve as a transactivator when fused to the Gal4 DNA-binding domain. Expression of the YTIS11 gene is not induced in response to DNA damaging agents, heat shock, sporulation conditions, or mating factor. However, YTIS11 expression is subjected to rapid glucose repression. Disruption of the YTIS11 gene in the M12B strain of Saccharomyces cerevisiae does not effect viability, growth in rich or synthetic medium, mating, or spore formation. However, YTIS11 gene disruption causes an alteration in metabolism that is reflected by a pH color change when cells are grown on YP plates supplemented with 2% glucose. Overexpression of murine TIS11 or TIS11B proteins dramatically attenuates the growth of both ytis11 and wild-type yeast.

Amino Acid Sequence↗
PubMed1991

Role of alpha-factor and the MF alpha 1 alpha-factor precursor in mating in yeast.

The peptide pheromones secreted by a and alpha cells (called a-factor and alpha-factor, respectively) are each encoded by two structural genes. For strains of either mating type, addition of exogenous pheromone does not alleviate the mating defect of mutants with disruptions of both structural genes. In addition, a particular insertion mutation in the major alpha-factor structural gene (MF alpha 1) that should result in an altered product inhibits alpha mating. These results suggested that the pheromone precursors (the MF alpha 1 pro region in particular) might play a second role in mating separate from the role of pheromone production. To analyze the role of alpha-factor and the MF alpha 1 precursor in alpha mating, we have constructed two classes of mutants. The mating defects of mutants that should produce the MF alpha 1 pro region peptide but no alpha-factor could not be alleviated by addition of exogenous alpha-factor in crosses to a wild-type a strain, indicating that the previous results were not due to an inability of the disruption mutants to produce the pro region peptide. Mutants able to produce alpha-factor, but with a variety of alterations in MF alpha 1 precursor structure, mated at levels proportional to the levels of alpha-factor produced, suggesting that the only role of the alpha-factor precursor in mating is to produce alpha-factor. Both of these results argue against a role for the MF alpha 1 pro region separate from its role in alpha-factor production.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1995

Expression of a synthetic gene encoding the anticoagulant-antimetastatic protein ghilanten by the methylotropic yeast Pichia pastoris.

Ghilantens are a family of cysteine-rich inhibitors of the clotting enzyme, factor Xa, that are produced in the salivary glands of the South American leech Haementeria ghilianii. In this study, a gene, designed from the amino acid sequence of a specific ghilanten isoform, was assembled from eight double-stranded oligonucleotide fragments. A yeast expression plasmid, pPIC9HG-2, was constructed by making an inframe fusion of the ghilanten-coding sequences with the region encoding the pre-pro alpha-mating factor signal sequence for secretion. The expression of ghilanten in pPIC9HG-2 was under the control of the methanol-inducible, alcohol oxidase (AOX1) promoter. Pichia pastoris yeast strains KM 71 and SMD 1168 were transformed with linearized pPIC9HG-2 to target integration of the plasmid to the chromosomal 5'-AOX1 locus via homologous recombination. Both strains yielded His+ transformants that secreted a potent anticoagulant activity into the medium. Product yield was improved by using buffered media (pH 6.0) supplemented with either casamino acids or a mixture of yeast extract and peptone. The protease-deficient strain, SMD 1168, secreted about a twofold higher level of r-ghilanten than KM 71. Significant clonal variation in the expression of r-ghilanten was found among the His+ transformants. A high producing clone was selected for production at the 2-liter shake flask and 10-liter bioreactor scales. r-Ghilanten was recovered from the fermentation broths in a single step by heparin Sepharose affinity chromatography. Protein sequence analysis of the amino terminus showed that the correct processing to yield mature ghilanten varied with the fermentation conditions.

Amino Acid Sequence↗
PubMed1994

Identification of genes required for normal pheromone-induced cell polarization in Saccharomyces cerevisiae.

In response to mating pheromones, cells of the yeast Saccharomyces cerevisiae adopt a polarized "shmoo" morphology, in which the cytoskeleton and proteins involved in mating are localized to a cell-surface projection. This polarization is presumed to reflect the oriented morphogenesis that occurs between mating partners to facilitate cell and nuclear fusion. To identify genes involved in pheromone-induced cell polarization, we have isolated mutants defective in mating to an enfeebled partner and studied a subset of these mutants. The 34 mutants of interest are proficient for pheromone production, arrest in response to pheromone, mate to wild-type strains, and exhibit normal cell polarity during vegetative growth. The mutants were divided into classes based on their morphological responses to mating pheromone. One class is unable to localize cell-surface growth in response to mating factor and instead enlarges in a uniform manner. These mutants harbor special alleles of genes required for cell polarization during vegetative growth, BEM1 and CDC24. Another class of mutants forms bilobed, peanut-like shapes when treated with pheromone and defines two genes, PEA1 and PEA2. PEA1 is identical to SPA2. A third class forms normally shaped but tiny shmoos and defines the gene TNY1. A final group of mutants exhibits apparently normal shmoo morphology. The nature of their mating defect is yet to be determined. We discuss the possible roles of these gene products in establishing cell polarity during mating.

Alleles↗
PubMed2004

Population biology of the invasive freshwater snail Physa acuta approached through genetic markers, ecological characterization and demography.

Abstract The respective role of factors acting on population functioning can be inferred from a variety of approaches, including population genetics and demography. We here investigated the role of four of these factors (mating systems, population size, bottlenecks and migration) in the hermaphroditic freshwater snail Physa acuta. Twenty-four populations were sampled either around Montpellier (local scale), or at the scale of France (global scale). At local scale, eight populations were sampled twice, before and after summer drying out. The genetic structure of these populations was studied using microsatellite loci. Populations were classified according to openness (ponds vs. rivers) and water regime (permanent vs. temporary) allowing predictions on genetic patterns (e.g. diversity within populations and differentiation). At local scale, progeny-arrays analysis of the selfing rate was conducted, and size distributions of individuals were followed over two years. Results with regard to the four factors mentioned above were: (i) Estimates of population selfing rates derived from inbreeding coefficients were only slightly higher than those from progeny-arrays. (ii) More variation was detected in rivers than in ponds, but no influence of water regime was detected. One reason might be that permanent populations are not going less often through low densities than those from temporary habitats at the time scale studied. (iii) There was limited evidence for genetic bottlenecks which is compatible with the fact that even marked reduction in water availability was not necessarily associated with demographic bottlenecks. More generally, bottlenecks reducing genetic variation probably occur at population foundation. (iv) Lower genetic differentiation was detected among rivers than among ponds which might be related to limitations on gene flow. Demographic and temporal genetic data further indicates that flooding in rivers is unlikely to induce marked gene flow explaining the strong genetic differentiation at short geographical scale in such habitats. Finally, the demographic data suggest that some populations are transitory and subject to recurrent recolonization, a pattern that was also detected through genetic data.

Animals↗
PubMed1988

Evidence for a plasma membrane mating receptor in the myxomycete Didymium iridis.

Haploid isolates Panamanian 2-7A8 and Honduran 1-2A1 of the heterothallic myxomycete Didymium iridis were investigated in order to determine whether membrane mating factors or receptors were present and necessary in order that cell fusion could occur and zygote formation take place. Haploid and diploid controls grown on half strength cornmeal media, and experimental crosses grown on the same media plus concanavalin A, were analysed by quantitative cytophotometry employing the Feulgen nuclear reaction. Nuclear DNA analysis demonstrated that Con A treated cells when crossed did not produce zygotes or plasmodia, while those left untreated did so almost all of the time. On rare occasion plasmodia did develop in treated cultures (1.8% or two out of 110). These plasmodia demonstrated low viability and upon analysis were shown to be haploid. It was concluded that they had developed by apogamy, and therefore did not require plasma membrane bound mating receptors, cell fusion, or zygote formation.

Concanavalin A↗
PubMed1998

Endoplasmic reticulum membrane localization of Rce1p and Ste24p, yeast proteases involved in carboxyl-terminal CAAX protein processing and amino-terminal a-factor cleavage.

Proteins terminating in the CAAX motif, for example Ras and the yeast a-factor mating pheromone, are prenylated, trimmed of their last three amino acids, and carboxyl-methylated. The enzymes that mediate these activities, collectively referred to as CAAX processing components, have been identified genetically in Saccharomyces cerevisiae. Whereas the Ram1p/Ram2p prenyltransferase is a cytosolic soluble enzyme, sequence analysis predicts that the other CAAX processing components, the Rce1p and Ste24p proteases and the Ste14p methyltransferase, contain multiple membrane spans. To determine the intracellular site(s) at which CAAX processing occurs, we have examined the localization of the CAAX proteases Rce1p and Ste24p by subcellular fractionation and indirect immunofluorescence. We find that both of these proteases are associated with the endoplasmic reticulum (ER) membrane. In addition to having a role in CAAX processing, the Ste24p protease catalyzes the first of two cleavage steps that remove the amino-terminal extension from the a-factor precursor, suggesting that the first amino-terminal processing step of a-factor maturation also occurs at the ER membrane. The ER localization of Ste24p is consistent with the presence of a carboxyl-terminal dilysine ER retrieval motif, although we find that mutation of this motif does not result in mislocalization of Ste24p. Because the ER is not the ultimate destination for a-factor or most CAAX proteins, our results imply that a mechanism must exist for the intracellular routing of CAAX proteins from the ER membrane to other cellular sites.

Cell Fractionation↗
PubMed1994

Secretion, purification and characterization of a soluble form of the yeast KEX1-encoded protein from insect-cell cultures.

The Saccharomyces cerevisiae KEX1 gene encodes a carboxypeptidase involved in the C-terminal processing of the lysine and arginine residues from the precursors of K1 and K2 killer toxins and alpha-factor (mating pheromone). In order to produce large quantities of this unique carboxypeptidase for structural studies, a functional soluble form was obtained by deleting 224 amino acids from the C-terminus of the KEX1-encoded protein which includes a putative membrane-spanning domain. The resulting truncated KEX1 gene (KEX1 delta) has been expressed in the baculovirus/insect cell system. The protein (Kex1 delta p) is efficiently secreted into the culture medium and was purified to apparent homogeneity with a yield of approximately 4 mg/l culture. Kex1 delta p is a glycoprotein with a molecular mass of 56 kDa, its N-terminal sequence is identical to that of the full-length membrane-associated form of the enzyme [Latchinian-Sadek, L. & Thomas, D. Y. (1993) J. Biol. Chem. 268, 534-540], and like the full-length enzyme it is not made as a proenzyme. For the soluble enzyme form, the optimum pH for activity was 5.5-6.0, and the apparent pI value of the protein determined by isoelectric focusing was 4.2. The enzyme cleaves arginine from the C-terminus of the synthetic peptide benzoyl-Phe-Ala-Arg with Km 335 microM and Vmax 282 mumol.min-1 x mg protein-1. Insect-cell-derived Kex1 delta p processes alpha-factor-Lys-Arg, a known natural substrate, to mature active alpha-factor in a manner similar to the membrane-associated full-length enzyme. This secreted form of the enzyme is a convenient source for the isolation of substantial quantities of the pure enzyme for detailed kinetic and structural studies.

Amino Acid Sequence↗
PubMed1999

High-yield secretion of recombinant gelatins by Pichia pastoris.

Recombinant non-hydroxylated gelatins based on mouse type I and rat type III collagen sequences were secreted from the methylotrophic yeast Pichia pastoris, using the Saccharomyces cerevisiae alpha-mating factor prepro signal. Proteolytic degradation could be minimized to a large extent by performing fermentations at pH 3.0 and by adding casamino acids to the medium, even though gelatin is extremely susceptible to proteolysis due to its open, unfolded structure. Proteolytic cleavage at specific mono-arginylic sites, by a putative Kex2-like protease, could be successfully abolished by site-directed mutagenesis of these sites. Production levels as high as 14.8 g/l clarified both were obtained, using multicopy tranformants. To our knowledge, this represents the highest level of heterologous protein secretion reported to date for P. pastoris.

Amino Acid Sequence↗
PubMed1998

Simple approach to reducing proteolysis during secretory production of human parathyroid hormone in Saccharomyces cerevisiae.

A gene coding for human parathyroid hormone (hPTH) was synthesized and cloned into a yeast expression and secretion vector containing the mating factor alpha pre-pro leader sequence and the galactose-inducible promoter, GAL10. The intact hPTH(1-84) was found to be secreted into the culture medium. As observed in the previous reports on the secretory production of hPTH in yeast, however, the proteolytic cleavage occurred as the culture proceeded, resulting in a significant loss of the intact hPTH. Attempts were therefore made to reduce the extent of proteolysis by simply controlling the culture conditions. The proteolytic cleavage was significantly reduced by the addition of an excess amount of l-arginine (>/=0.2M) to the culture medium, which resulted in a marked improvement in the yield of intact hPTH. To examine whether l-arginine affects the activities of intracellular proteases such as KEX2 endoproteinase or extracellular proteases, the proteolysis experiments were performed by incubating the commercial intact hPTH in a yeast host culture supernatant. The results demonstrated that l-arginine at high concentrations reduced the rate of hPTH proteolysis by inhibiting extracellular proteases.

Arginine↗
PubMed2000

The 'SUN' family: yeast SUN4/SCW3 is involved in cell septation.

SUN4 is the fourth member of the SUN gene family from S. cerevisiae, whose products display high homology in their 258 amino acid C-terminal domain. SIM1, UTH1, NCA3 (the founding members) are involved in different cellular processes (DNA replication, ageing, mitochondrial biogenesis) and it is shown herein that SUN4 plays a role in the cell septation process. sun4 delta cells are larger than wild-type and begin a new cell cycle before they have separated from their mother cell. This phenotype is more pronounced in sun4Delta cells also deleted for UTH1. FACS analysis shows apparent polyploidy which disappears when the cell cycle is arrested by mating factor or nocodazole, indicating that cell septation is delayed without modification of the doubling time. Elutriated sun4 delta uth1 delta daughter cells are born larger, and therefore enter S phase sooner than their wild-type counterpart. S phase duration, as well as timing of Clb2 degradation, is normal, but cell septation is delayed. Sun4p/Scw3p was recently described as a cell wall protein (Cappellaro et al., 1998) and, consistent with this notion, electron micrographs of sun4 delta cells show defects in the final steps of cell wall septation. Our data suggest that Sun4p and Uth1p might contribute to the regulated process of cell wall morphogenesis and septation.

Basic Helix-Loop-Helix Proteins↗
PubMed1992

Is START a switch?

The cell cycle in Saccharomyces cerevisiae is controlled by regulation of START in late G1. The CLN1, CLN2 and CLN3 family of cyclin homologues is required for cells to pass START. They probably act by activating the CDC28 protein kinase. Expression of CLN1 or CLN3 under the control of an inducible promoter shows that transcription of either gene is sufficient for cyclin-deficient strains arrested in G1 to traverse START. A model of START regulation involves activation of CDC28 kinase by any CLN protein, leading to activation of CLN1 and CLN2 transcription in a positive feedback loop and passage through START. The cell cycle-dependent transcriptional regulators SWI4 and SWI6 may be components of the feedback loop. Cell cycle commitment entails resistance to the inhibitory action of mating factor, which correlates with peak levels of CLN1 and CLN2 mRNAs. FAR1 encodes an alpha-factor-dependent inhibitor of CLN function whose expression is markedly reduced at the time of START. The interplay of all these factors may sharpen the START transition such that it is close to an all-or-nothing switch event. This may be important for several START-dependent events to be activated at the same time, leading to coordinated cell cycle progression.

Cell Cycle↗
PubMed2004

Sexual conjugation in yeast: A paradigm to study G-protein-coupled receptor domain structure.

The yeast Saccharomyces cerevisiae undergoes cell fusion during sexual conjugation to form diploid cells. The haploids participating in this process signal each other through secreted peptide-mating factors (alpha-factor and a-factor) that are recognized by G-protein-coupled receptors. The receptor (Ste2p) recognizing the tridecapeptide alpha-factor is used as a model system in our laboratory to understand various aspects of peptide-receptor interactions and receptor structure. Using chemical procedures we have synthesized peptides corresponding to the seven transmembrane domains of Ste2p and studied their structures in membrane mimetic environments. Extension of these studies requires preparation of longer fragments of Ste2p. This article discusses strategies used in our laboratory to prepare peptides containing multiple domains of Ste2p. Data are presented on the use of chemical synthesis, biosynthesis, and native chemical ligation. Using biosynthetic approaches fusion proteins have been expressed that contain single receptor domains, two transmembrane domains connected by the contiguous loop, and the tail connected to the seventh transmembrane domain. Tens of milligrams of fusion protein were obtained per liter, and multimilligram quantities of the isotopically labeled target peptides were isolated using such biosynthetic approaches. Initial circular dichroism results on a chemically synthesized 64-residue peptide containing a portion of the cytosolic tail and the complete seventh transmembrane domain showed that the tail portion and the hydrophobic core of this peptide maintained individual conformational preferences. Moreover, this peptide could be studied at near millimolar concentrations in the presence of micelles and did not aggregate under these conditions. Thus, these constructs can be investigated using high-resolution nuclear magnetic resonance techniques, and the cytosolic tail of Ste2p can be used as a hydrophilic template to improve solubility of transmembrane peptides for structural analysis.

Amino Acid Sequence↗