Allelic divergence at Bα1 pheromone receptor genes of Schizophyllum commune

Abstract The multiallelic mating type locus Bal of Schizophyllum commune encodes a pheromone receptor and putative pheromone genes. A comparison of two alleles encoding receptors that share the same specificity Bα1 was performed using strains of different geographic origin. The amino acid sequence alignment revealed strong conservation of the largest part of the receptor. Only in the distal C-terminus major amino acid was divergence encountered. This C-terminal region of 117 of the total of 639 amino acids was shown to be unnecessary for function in vivo by transformation experiments.     

Two genes specifically expressed in fruiting dikaryons of Schizophyllum commune: homologies with a gene not regulated by mating-type genes

The nucleotide (nt) sequences of the Sc3 and Sc4 genes of the filamentous fungus Schizophyllum commune, and the deduced amino acid (aa) sequences, were determined; moreover, the previously published sequence for the ScI gene [Dons et al., EMBO J. 3 (1984) 2101–2106] was corrected. All three independently isolated genes were found to have similar structures and nt sequences of their coding regions. At the aa level the homology is 43–62% (63–69% in the C-terminal parts of the proteins), the hydrophobic aa predominate and the hydrophobicity patterns are similar. All three proteins contain leader sequences and eight cysteines among about 110 aa, conserved at the same positions. Yet these genes are differentially regulated: Sc1 and Sc4 are only expressed at high levels in fruiting dikaryons, whereas Sc3 is highly expressed in both monokaryons and dikaryons, independent from fruiting.     

The alpha-mating type locus of Cryptococcus neoformans contains a peptide pheromone gene.

The opportunistic fungal pathogen Cryptococcus neoformans has two mating types, MATa and MAT alpha. The MAT alpha strains are more virulent. Mating of opposite mating type haploid yeast cells results in the production of a filamentous hyphal phase. The MAT alpha locus has been isolated in this study in order to identify the genetic differences between mating types and their contribution to virulence. A 138-bp fragment of MAT alpha-specific DNA which cosegregates with alpha-mating type was isolated by using a difference cloning method. Overlapping phage and cosmid clones spanning the entire MAT alpha locus were isolated by using this MAT alpha-specific fragment as a probe. Mapping of these clones physically defined the MAT alpha locus to a 35- to 45-kb region which is present only in MAT alpha strains. Transformation studies with fragments of the MAT alpha locus identified a 2.1-kb XbaI-HindIII fragment that directs starvation-induced filament formation in MATa cells but not in MAT alpha cells. This 2.1-kb fragment contains a gene, MF alpha, with a small open reading frame encoding a pheromone precursor similar to the lipoprotein mating factors found in Saccharomyces cerevisiae, Ustilago maydis, and Schizosaccharomyces pombe. The ability of the MATa cells to express, process, and secrete the MAT alpha pheromone in response to starvation suggests similar mechanisms for these processes in both cell types. These results also suggest that the production of pheromone is under a type of nutritional control shared by the two cell types.     

Indole- and caffeine-resistant mutations of Schizophyllum commune are involved in the behavior of a class III B mating-type factor in trp1 cells

We have reported that a parental strain of Schizophyllun commune T11 (trp1) is fully compatible with a strain T37 (Bα1′β4, trp1), but indole- and caffeine-resistant mutants (In^R-13 and Caf^R-9 respectively) derived from the T11 are semicompatible with the same T37. The compound-resistant mutations, ind1 and cfn1, were linked to neither A nor B mating-type factors. In^R-13, Caf^R-9, and all of indole- and caffeine-resistant progenies (ind1, trp1; cfn1, trp1) were semicompatible with the T37, and compatible with a strain T40 (Bα1′β4, TRP1) although the T40 contained the same class III B mating-type factor as the T37. The In^R-13 and Caf^R-9 were also semicompatible with any tester strains (Bα1′β4, trp1) developed, and the resulting heterokaryons produced non-aggregated and semiaggregated masses of hyphae, respectively. In the mutant heterokaryons, nuclei were distributed irregularly especially in case of [trp1/trp1] background; anucleate, uninucleate, binucleate and multinucleate cells were found with modified hyphae which contained a different type of septation (pseudoclamps and simple septa other than true clamps). We concluded that the ind1 and cfn1 mutations alter the normal behavior of one of class III B mating-type factors in Trp⁻ cells.     

Structure of a yeast pheromone gene (MF alpha): a putative alpha-factor precursor contains four tandem copies of mature alpha-factor

We have cloned and sequenced a gene (MF alpha) coding for alpha-factor, a tridecapeptide mating factor secreted by yeast alpha cells. A plasmid carrying the MF alpha gene was identified by screening for production of alpha-factor by mat alpha 2 mutants, which fail to secrete alpha-factor because of simultaneous synthesis and degradation of the factor. The cloned segment codes for four mature alpha-factor within a putative precursor of 165 amino acids. The putative precursor begins as a signal sequence for secretion. The next segment, of approximately 60 amino acids, contains three potential glycosylation sites. The carboxy-terminal half of the precursor contains four tandem copies of mature alpha-factor, each preceded by spacer peptides of six or eight amino acids (variations of Lys-Arg-Glu-Ala-Asp-Ala-Glu-Ala), which are hypothesized to contain proteolytic processing signals.     

The bovine T cell receptor alpha/delta locus contains over 400 V genes and encodes V genes without CDR2

αβ T cells and γδ T cells perform nonoverlapping immune functions. In mammalian species with a high percentage of very diverse γδ T cells, like ruminants and pigs, it is often assumed that αβ T cells are less diverse than γδ T cells. Based on the bovine genome, we have created a map of the bovine TRA/TRD locus and show that, in cattle, in addition to the anticipated >100 TRDV genes, there are also >300 TRAV or TRAV/DV genes. Among the V genes in the TRA/TRD locus, there are several genes that lack a CDR2 and are functionally rearranged and transcribed and, in some cases, have an extended CDR1. The number of bovine V genes is a multiple of the number in mice and humans and may encode T cell receptors that use a novel way of interacting with antigen. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Analysis of the sexual development-promoting region of Schizophyllum commune TRP1 gene

This study aims to elucidate the mechanism of sexual development of basidiomycetous mushrooms from mating to fruit body formation. Sequencing analysis showed the TRP1 gene of basidiomycete Schizophyllum commune encoded an enzyme with three catalytic regions of GAT (glutamine amidotransferase), IGPS (indole-3-glycerol phosphate synthase), and PRAI (5-phosphoribosyl anthranilate isomerase); among these three regions, the trp1 mutant (Trp^?) had a missense mutation (L→F) of a 338th amino acid residue of the TRP1 protein within the IGPS region. To investigate the function of IGPS region related to sexual development, dikaryons with high, usual, and no expression of the IGPS region of TRP1 gene were made. The dikaryotic mycelia with high expression of the IGPS formed mature fruit bodies earlier than those with usual and no expression of the IGPS. These results showed that the IGPS region in TRP1 gene promoted sexual development of S. commune.     

Hemoglobin regulates expression of an activator of mating-type locus alpha genes in Candida albicans

Phenotypic switching from the white to the opaque phase is a necessary step for mating in the pathogenic fungus Candida albicans. Suppressing switching during vascular dissemination of the organism may be advantageous, because opaque cells are more susceptible to host defenses. A repressor of white-opaque switching, HBR1 (hemoglobin response gene 1), was identified based on its specific induction following growth in the presence of exogenous hemoglobin. Deletion of a single HBR1 allele allowed opaque phase switching and mating competence, accompanied by a lack of detectable MTL alpha1 and alpha2 gene expression and enhanced MTLa1 gene expression. Conversely, overexpression of Hbr1p or exposure to hemoglobin increased MTLalpha gene expression. The a1/alpha2 repressed target gene CAG1 was derepressed in the same mutant in a hemoglobin-sensitive manner. Regulation of CAG1 by hemoglobin required an intact MTLa1 gene. Several additional Mtlp targets were perturbed in HBR1 mutants in a manner consistent with commitment to an a mating phenotype, including YEL007w, MFalpha, HST6, and RAM2. Therefore, Hbr1 is part of a host factor-regulated signaling pathway that controls white-opaque switching and mating in the absence of allelic deletion at the MTL locus.     

RNA-mediated gene silencing in monokaryons and dikaryons of Schizophyllum commune

Disruption of genes by homologous recombination occurs at a low frequency in the basidiomycete Schizophyllum commune. For instance, the SC3 and SC15 genes were inactivated at frequencies of 1 and 5%, respectively. As an alternative to disruption, we used gene silencing through the introduction of a hairpin construct. The SC15 gene, which encodes an abundantly secreted structural protein, was silenced at a frequency of 80% in monokaryons of S. commune after introduction of a hairpin construct of the gene. Silencing also occurred in dikaryons in which one of the partners was not a silenced strain. The silencing mechanism resembles RNAi in other filamentous fungi and is a powerful tool for the functional analysis of genes expressed in monokaryons or dikaryons.     

Rational design and PCR-based synthesis of an artificial Schizophyllum commune xylanase gene.

A synthetic gene encoding the Schizophyllum commune xylanase XynA was constructed by a novel PCR-based procedure. Three long oligonucleotides were synthesized and used in combination with flanking PCR primers to generate a 607 base pair gene which contained 31 unique locations for restriction enzyme cleavage. The amino acid sequence was tailored for expression in Escherichia coli by using only those codons found in highly expressed E. coli genes. The availability of the gene will facilitate analysis of the structure and function of this and other beta-(1,4) xylanases.     

The interleukin 1 gene cluster contains a major susceptibility locus for ankylosing spondylitis

Ankylosing spondylitis (AS) is a common and highly heritable inflammatory arthropathy. Although the gene HLA-B27 is almost essential for the inheritance of the condition, it alone is not sufficient to explain the pattern of familial recurrence of the disease. We have previously demonstrated suggestive linkage of AS to chromosome 2q13, a region containing the interleukin 1 (IL-1) family gene cluster, which includes several strong candidates for involvement in the disease. In the current study, we describe strong association and transmission of IL-1 family gene cluster single-nucleotide polymorphisms and haplotypes with AS.     

Molecular cloning of a gene abundantly expressed during fruiting body initiation in Schizophyllum commune.

Complementary DNA was synthesized on polyadenylated RNA from a dikaryotic mycelium of the basidiomycete Schizophyllum commune bearing fruiting body initials. The complementary DNA was cloned into the PstI site of pBR327 by the deoxyguanidylate-deoxycytidylate tailing approach. After transformation into Escherichia coli cells, a differential screening was performed by colony hybridization with complementary [32P]DNA made on the RNAs of the monokaryon and dikaryon strains. Two clones were selected for further analysis by Northern blotting and hybrid release translation. Clone 1D10 hybridized with an mRNA of 775 nucleotides, coding for a polypeptide with an Mr of 15,000. Although this RNA was present in both monokaryotic and dikaryotic mycelia, its concentration appeared to change considerably over time and with different cultivation conditions. This mRNA is probably the most abundantly expressed sequence in S. commune. Clone 1G2 and its homologs hybridized with an mRNA of 650 nucleotides, coding for a polypeptide with an Mr of 13,000. This gene was exclusively expressed in the dikaryon strain. In liquid-grown cultures, the concentration of this mRNA was low but increased ca. 20-fold during the establishment of fruiting body primordia. A chromosomal fragment of 9 kilobase pairs which contained the 1G2 gene was cloned into pBR327 and used as a probe in Northern blot hybridization. It was found that surrounding sequences were not expressed at the same time or to the same extent as the 1G2 gene.