Expression of A mating type genes of Coprinus cinereus in a heterologous basidiomycete host

The A mating factor of Coprinus cinereus determines compatibility in mating by regulating part of a developmental sequence that leads to dikaryon formation. The A genes that trigger development encode two different classes of homeodomain proteins, and for a successful mating, a protein of one class, HD 1, must interact with a protein of the other class, HD 2. In this report we show that C. cinereus A genes that encode HD 2 proteins, a2-1 and b2-1, can elicit A-regulated development in the heterologous host C. bilanatus. Transformation rates were very low, suggesting that the genes were poorly transcribed. The fact that the HD 2 genes are functionally expressed implies successful heteromultimeric association of putative DNA-binding proteins coded by the two Coprinus species. This interaction was sufficient to satisfy the need for different A factors in the formation of a fertile C. bilanatus dikaryon, but fertile dikaryons were more readily produced in matings with the a2-1 gene transformants. The C. cinereus A genes, b1-1 and d1-1, which encode HD1 proteins, were either not expressed or their proteins were non-functional in C. bilanatus. These experiments raise some interesting questions regarding HD1–HD2 protein interactions.     

A mating-type factors of Coprinus cinereus have variable numbers of specificity genes encoding two classes of homeodomain proteins

We have identified the seven genes that constitute the A43 mating-type factor of Coprinus cinereus and compare the organisation of A43 with the previously characterised A42 factor. In both, the genes that trigger clamp cell development, the so-called specificity genes, are separated into and loci by 7 kb of noncoding sequence and are flanked by homologous genes -fg and -fg. The specificity genes are known to encode two classes of dissimilar homeodomain (HD1 and HD2) proteins and have different allelic forms which show little or no cross-hybridisation. By partial sequencing we identified a divergently transcribed HD1 (a1-2) and HD2 (a2-2) gene in the A43 locus. a2-2 failed to elicit clamp cell development in three different hosts, suggesting that it is non-functional. a1-2 elicited clamp cells in an A42 host that has only an HD2 gene (a2-1) in its locus, thus demonstrating that the compatible A mating interaction is between an HD1 and an HD2 protein. The A43 locus contains three specificity genes, the divergently transcribed HD1 and HD2 genes b1-2 and b2-2 and a third HD1 gene (d1-1) that was shown by hybridisation and transformation analyses to be functionally equivalent to d1-1 in A42. An untranscribed footprint of a third A42 HD1 gene, c1-1, was detected between the A43 b2-2 and d1-1 genes by Southern hybridisation.     

The MAT A locus of the dimorphic yeast Yarrowia lipolytica consists of two divergently oriented genes

The MAT A locus of Yarrowia lipolytica, which was on the basis of its ability to induce sporulation in a diploid B/B strain, represses the mating capacity of this strain. The gene functions required for induction of sporulation and repression of conjugation could be separated by subcloning. Sequence analysis revealed two ORFs in the MAT A locus. One of them (MAT A1) codes for a protein of 119 amino acids which is required to induce sporulation. The other (MAT A2) codes for a protein of 291 amino acids that is able to repress conjugation. Both genes are oriented divergently from a central promoter region, which possesses putative TATA and CAAT boxes for both genes. The product of MAT A1 shows no homology to any known protein and seems to represent a new class of mating-type genes. MAT A2 contains a HMG box with homology to other mating-type genes. Both MAT A1 and MAT A2 are mating-type specific. In cells of both mating types, the regions flanking the MAT A locus contain sequences with homology to either S. cerevisiae SLA2 and ORF YBB9, respectively. From hybridization and subcloning data we estimate that the MAT A region is approximately 2 kb long and is present only once in the genome. Received: 25 January 1999 / Accepted: 16 April 1999     

Isolation of Pheromone Precursor Genes of Magnaporthe grisea

In heterothallic ascomycetes one mating partner serves as the source of female tissue and is fertilized with spermatia from a partner of the opposite mating type. The role of pheromone signaling in mating is thought to involve recognition of cells of the opposite mating type. We have isolated two putative pheromone precursor genes of Magnaporthe grisea. The genes are present in both mating types of the fungus but they are expressed in a mating type-specific manner. The MF1-1 gene, expressed in Mat1-1 strains, is predicted to encode a 26-amino-acid polypeptide that is processed to produce a lipopeptide pheromone. The MF2-1 gene, expressed in Mat1-2 strains, is predicted to encode a precursor polypeptide that is processed by a Kex2-like protease to yield a pheromone with striking similarity to the predicted pheromone sequence of a close relative, Cryphonectria parasitica. Expression of the M. grisea putative pheromone precursor genes was observed under defined nutritional conditions and in field isolates. This suggests that the requirement for complex media for mating and the poor fertility of field isolates may not be due to limitation of pheromone precursor gene expression. Detection of putative pheromone precursor gene mRNA in conidia suggests that pheromones may be important for the fertility of conidia acting as spermatia.     

The trans action of HMRa in mating type interconversion

Summary HML and HMR are the sites of cryptic mating type genes in the yeast Saccharomyces cerevisiae. In the presence of the HO gene, the information from HML or HMR (an a or cassette) is transferred to the mating type locus (MAT). HML, HMR, and MAT are located on chromosome III, yet are widely separeted. Similarly, in other yeasts, at least some of the genes involved in mating type interconversion are linked to the mating type locus. We demonstrate here that a cassette donor (HMR) and the cassette target (MAT) need not be physically linked for successful mating type interconversion. In particular, we show that HMR a on one chromosome can donate an a cassette to the mating type locus on a homologous chromosome III.     

Regions in theZ5 mating gene ofSchizophyllum commune involved in Y-Z binding and recognition

The A mating locus of the woodrotting fungusSchizophyllum commune encodes two multiallelic genes,Y andZ, which regulate the A-pathway of development. TheY alleles contain a homeobox, suggesting that the Y proteins may be DNA-binding regulatory proteins. During mating, development is induced when Y from one mating partner interacts with Z from the other mating partner; self combinations of Y and Z are inactive. Two-hybrid analyses indicate that nonself combinations of Y and Z form heteromultimers and self combinations do not. To understand Y-Z binding and self- nonself recognition further we used mutagenesis and chimeras to identify regions in one allele ofZ(Z5) that are involved in these processes. Here we report the results, which broadly define regions in Z5 that are essential for activity, Y-Z binding and Z5 allelic specificity.The sequence reported in this paper has been deposited in the Genbank database under accession number U22049     

Molecular cloning and characterization of Drosophila genes encoding small GTPases of the rab and rho families

We have isolated eight genes from Drosophila, small GTPases. They can be classified into three rab family genes (Drab2, Drab5, Drab11) and five rho family genes (Drac1a, Drac1b, Drac3, Dcdc42, DrhoA). While Drac3 is a novel type of rac gene, others are homologues of known mammalian genes for small GTPases. Northern blot analyses showed that all the genes are expressed throughout all developmental stages from embryo to adult. In situ hybridization to embryos revealed that Drab2, Drac1b, and Drac3 are highly expressed in the nervous system, in the trunk mesoderm, and in the cephalic mesoderm, respectively. Since hemocytes are derived from the cephalic mesoderm, we carried out double stainings using a hemocyte marker – anti-peroxidasin antibody – and Drac3 in situ hybridization. We found that Drac3 is expressed in hemocyte precursor cells. In the Drac3 deficiency embryos, the hemocyte precursor cells start to differentiate normally, but never develop into mature hemocytes, indicating that Drac3 is essential for their maturation. The DrhoA and Dcdc42 genes complemented S. cerevisiae rho1 and cdc42 mutations in the same manner as human rhoA and CDC42, respectively. These results suggest functional similarity between Drosophila and mammalian small GTPase genes. Received: 7 May 1996 / Accepted: 6 January 1997     

绿豆VrWOX基因家族鉴定及表达分析北大核心CSCD

WOX(WUSCHEL-related homebox)基因家族是植物特有的一类转录因子,是同源盒(homeobox,HB)转录因子超家族中的重要成员。WOX基因在植物干细胞调节及生殖发育过程中具有重要作用,其功能已在多个植物物种中鉴定。然而绿豆(Vigna radiate)VrWOX基因家族信息尚不清楚。本研究通过同源比对和聚类分析,在绿豆基因组中鉴定了42个VrWOX基因。VrWOX基因在绿豆染色体中分布不均,其中7号染色体含有的VrWOX数量最多。VrWOX基因分为古老进化支(19个VrWOX)、中等进化支(12个VrWOX)和年轻进化支(WUSCHEL进化支,11个VrWOX)3个亚类。种内和种间共线性分析发现,VrWOX基因共有12个重复事件,与拟南芥(Arabidopsis thaliana)AtWOX有15个同源基因对,与菜豆(Phaseolus vulgaris)PvWOX有22个同源基因对。VrWOX基因在基因结构、保守基序等方面存在很大差异,因而可能存在功能差异。VrWOX基因启动子区域含有不同种类和不同数量的顺式作用元件,导致VrWOX基因在不同组织中表现出不同的基因表达模式。本研究对VrWOX基因家族信息和表达模式进行了分析,为绿豆VrWOX基因功能和调控网络的解析奠定了一定的理论依据。     

Double mutants of Phycomyces with abnormal phototropism

Summary Seven genes (madA to madG) are known which effect phototropism in Phycomyces. These genes have been partially ordered with respect to the associated stimulus-response pathway. Mutants affected in these genes serve as useful probes of photosensory transduction processes in this model system. To extend and deepen the analysis of the system, we have constructed a family of 21 double mutants in all combinations for the seven mad genes. A set of seven standard alleles was adopted for this work. The double mutants were isolated from crosses between isogenic single-mutant strains of opposite mating type. After a partial physiologic screening of the progeny, the double mutants were identified by complementation tests using single-mutant strains of known genotype. For all but three of the double mutants, the photogeotropism phenotypes were distinct from those of the respective single-mutant parentals. One triple mutant (madA madB madC) was constructed as part of this work. Various applications of the double mutants and the triple mutant are discussed. Recombination analyses were performed on the progeny from seven mad crosses to complete an earlier study. The results establish that all seven mad genes are unlinked.     

Mutants inducible for sexual agglutinability in Saccharomyces cerevisiae

Summary We have isolated the mutants, T55s-41(a) and T562s-161 () which have no sexual agglutinability when cultured at 28°C, but become sexually agglutinable by the action of the sex pheromone produced by respective opposite mating type. The sex-specific glycoproteins responsible for sexual agglutination were detected in the mutants treated with the opposite mating type pheromone, but not in those treated with the same mating type pheromone.The induction of sexual agglutinability by the pheromone required both nitrogen and carbon sources and was inhibited by cycloheximide. The induction by the pheromone of sexual agglutinability was much more sensitive to osmotic shock and Triton X-100 in T55s-41 than in H1-0, an inducible a strain found in our stock cultures. When cultured at 22°C both T55s-41 and T562s-161 produced respective agglutination substances without the sex pheromones.H1-0 carried more than one genes responsible for the inducibility (inducible genes). The inducible genes carried by T55s-41 and T562s-161 were recessive, possibly linked to none of the mating type locus, thr4 and his 4, and shown to be identical. The inducible gene in H22, an inducible a strain found in our stock cultures and at least one of the inducible genes in H1-0 were linked to the mating type locus. All the inducible genes observed so far were not specific to the mating type in their action.     

Revision of the genus Absidia (Mucorales, Zygomycetes) based on physiological, phylogenetic, and morphological characters; thermotolerant Absidia spp. form a coherent group, Mycocladiaceae fam. nov.

The genus Absidia comprises ubiquitously distributed soil fungi inhabiting different growth temperature optima ranging from 20–42 °C. Some of the mesophilic species are important biotechnologically in the biotransformation of steroids or as producers of rennin-like components, whereas species with higher growth temperature optima are of clinical relevance as opportunistic human pathogens. The aim of this study was to investigate the phylogenetic relationships between these species and to establish a revision of their systematics. For this purpose single and combined genealogies based on distance, MP, ML, and Bayesian analyses of aligned nucleotide sequences of the nuclear-encoded genes for actin (act) and for the 5.8S ribosomal RNA flanked by the ITS regions 1 and 2 (comprising 807 and 828 characters, respectively) of 16 Absidia species were reconstructed. The phylogenetic reconstructions suggest a trichotomy of the Absidia genus consisting of a mesophilic, a fast-growing thermotolerant, and a slowly-growing mycoparasitic Absidia group. The trichotomous phylogenetic grouping is concordant with the morphology of the zygospores, which are zygotes resulting from sexual conjugation between two compatible mating partners. Whereas the mesophilic group comprises the majority of absidiaceaeous species forming sterile hair-like, mycelial appendages on the suspensors of their zygospores, the thermotolerant group is characterised by the formation of smooth-walled zygospores, and the mycoparasitic group, namely Absidia parricida and A. zychae, by Mucor-like rough-walled zygospores. Based on the phylogenetic coherence of mesophilic and thermotolerant Absidia species, we propose that the two groups are separated into two distinct genera, Absidia for the mesophilic Absidia species resembling the Absidiaceae and Mycocladus for the thermotolerant species A. corymbifera, A. blakesleeana and A. hyalospora. Because Mycocladus is physiologically, phylogenetically, and morphologically distinct from the Absidiaceae s. str. we suggest that they are classified as a separate family, Mycocladiaceae fam. nov., which comprises the three species M. corymbifer, M. blakesleeanus and M. hyalospora.     

Cloning of the mating-type gene MATA of the yeast Yarrowia lipolytica

Summary The mating type gene MA TA of the dimorphic yeast Yarrowia lipolytica was cloned. The strategy used was based on the presumed function of this gene in the induction of sporulation. A diploid strain homozygous for the mating type B was transformed with an integrative gene bank from an A wild-type strain. A sporulating transformant was isolated, which contained a plasmid with an 11.6 kb insert. This sequence was rescued from the chromosomal DNA of the transformant and deletion mapping was performed to localize the MAT insert. The MAT gene conferred both sporulating and non-mating phenotypes on a B/B diploid. A LEU2 sequence targeted to this locus segregated like a mating type-linked gene. The A strain did not contain silent copies of the MAT gene.     

The conjugative transposon Tn925: enhancement of conjugal transfer by tetracycline in Enterococcus faecalis and mobilization of chromosomal genes in Bacillus subtilis and E. faecalis

Summary The effects of tetracycline on transfer of the conjugative, tetracycline-resistance transposon, Tn925, as well as the ability of the transposon to promote the transfer of chromosomal genes was examined in Enterococcus faecalis and Bacillus subtilis. To test for chromosomal transfer, multiply-marked strains of each organism, each carrying a single chromosomal copy of Tn925, were mated on filters with suitable recipient strains, under conditions where transformation and transduction were precluded. In both cases, transfer of a variety of chromosomal genes, at frequencies comparable to the frequency of Tn925 transfer, was detected readily. The presence of Tn925 in one of the members of the mating pair was absolutely required for chromosomal transfer, but transfer of Tn925 did not accompany every chromosomal transfer event. The results were consistent with a mating event resembling a type of cell fusion, allowing for extensive recombination between the genomes of the mating partners. Growth of Tn925-containing donor cells in the presence of tetracycline increased the transfer frequency of Tn925 by about tenfold in E. faecalis, but not in B. subtilis.Deceased, 7/89. O. Torres and R. Korman contributed equally to this work     

The relationship of flagellar length to sexual signalling in Chlamydomonas

The flagella of Chlamydomonas reinhardi are required for the initiation of mating between opposite mating type gametes. It has been suggested that flagellar length is a crucial factor in a cell's ability to transmit and receive the sexual signals necessary for fusion. Mating type + (mt⁺) cells of gam-5, a mutant which is characterized by variable length, paralyzed flagella, were mated with wild-type, mt⁻ cells. Activation of the mating structures of the gam-5 gametes, and therefore successful signalling, was demonstrated for cells with flagella as short as 1.5 μm (less than 1/6 normal length). Because this mutant displays aberrant axonemal structures, and because various mutants with other defects in axonemal structure are also able to mate, it seems likely that the flagellar membrane may provide the main conduit for gametic sexual signals.     

The biallelica mating type locus ofUstilago maydis: remnants of an additional pheromone gene indicate evolution from a multiallelic ancestor

Thea mating type locus ofUstilago maydis contains the structural genes for a pheromone-based cell recognition system that governs fusion of haploid cells. The locus exists in two alleles, termeda1 anda2. We have completed the analysis of the nucleotide sequences unique toa1 anda2. Within these dissimilar regions we find two short patches of DNA sequence similarity. Interestingly, one of these segments corresponds to the transcribed region of thea1 pheromone precursor. As a result of multiple nucleotide exchanges this sequence does not code for a functional product. The existence of a second pheromone gene in thea2 allele suggests that the present locus had a multiallelic ancestor. In addition, we describe the presence of two additional genes in thea2 allele. We have investigated the role of these genes during mating and pathogenic development and speculate that they might affect mitochondrial inheritance.     

Isolation of plasmids carrying the arginine repressor gene argR of Escherichia coli K12

Summary Twenty-two sexual crosses between strains of Phycomyces blakesleeanus carrying mutations affecting phototropism (madA, madD, madE), synthesis of carotenoids (carA), auxotrophy (leu-51, nicA, pur-51), and resistance to 5-fluorouracil (fur) were studied; mating type was also included as a marker. Recombination frequencies were obtained among the ten genes involved. Linkage was found between mating type and madE; leu-51 and madA; furA401, furB402 and madD. All other gene combinations tested are unliked.     

新疆野苹果AP2/ERF转录因子家族鉴定与响应腐烂病的表达分析北大核心CSCD

AP2/ERF转录因子家族参与了植物生长发育、抵抗胁迫以及植物激素响应等诸多生物过程,是植物中最重要的转录因子家族之一。该研究基于腐烂病菌侵染后的新疆野苹果(Malus sieversii)全长转录组,使用AP2保守结构域的隐马可夫模型PF00847,鉴定新疆野苹果的AP2/ERF家族成员。利用MEGA6、NCBI CDD-batch、MEME、EXPASY、BUSCA对MsAP2/ERF家族成员进行鉴定、分类和结构分析、理化性质和亚细胞定位分析。通过RNA-seq数据和qRT-PCR实验对差异表达的MsAP2/ERFs基因的表达水平进行分析和验证,旨在鉴定新疆野苹果中潜在具有腐烂病抗性的AP2/ERF家族基因资源。结果显示:(1)在新疆野苹果中共鉴定获得106个AP2/ERF基因,涵盖全部AP2(17个)、ERF(57个)、DREB(25个)、RAV(5个)和Soloist(2个)5个亚家族。(2)进一步的细化分类发现MsERF亚家族包括B1-B6 6个组,而MsDREB亚家族中只有A2、A4、A5、A6共4个组,缺少A1和A3组的基因成员。(3)RNA-seq表达模式分析结果表明,29个MsAP2/ERF基因在腐烂病感染过程中差异表达,其中MsERF亚家族中差异表达基因数量最多(19个)。(4)12个MsAP2/ERF代表基因的qRT-PCR结果表明:8个ERF亚家族基因均受腐烂病病菌诱导显著上调表达,其中B4类ERF成员基因(MsERF40)在腐烂病病菌侵染后5 d表达量上调表达倍数最高;4个MsDREB基因中,3个受到腐烂病病原菌诱导显著上调,1个下调表达;此外,含有TIR保守结构域的MsERF05在腐烂病病菌侵染1 d后上调表达69倍,表明ERF亚家族的MsERF40和MsERF05在新疆野苹果抗腐烂病过程中发挥重要作用。该研究结果为新疆野苹果AP2/ERF基因响应腐烂病的功能和机理研究奠定了基础。     

大豆GeBP转录因子基因家族的生物信息学分析

GeBP转录因子调控植物表皮毛的生长发育,并且参与控制植物叶片的发育。该文利用生物信息学方法,在大豆全基因组范围内搜索GeBP基因家族,并从氨基酸理化性质、基因结构、染色体的物理分布、系统进化、序列比对、功能结构域、组织表达情况等基本特征方面对GmGeBP基因家族进行分析。结果表明:(1)共获得9个GmGeBP转录因子基因家族成员,其中仅2个基因含有内含子,且都只有1个内含子,表明该家族成员基因构造比较简单但稳定。(2)GmGeBP编码的蛋白分子量为39.65~49.24 kD,理论等电点为4.65~9.08;这些成员基本上都是酸性氨基酸,属于亲水性、不稳定蛋白。(3)这9个基因不均匀的分布于7条染色体上,10和20号染色体上分别分布2个GeBP基因,3、5、13、15、19号染色体上各分布1个基因。(4)系统进化分析表明,大豆与拟南芥对应的GeBP成员亲缘关系较近,分别聚类到4个分支,而与水稻的距离较远。(5)结构域分析表明,9个GmGeBP成员都包含DUF573结构域,推测该部分在GeBP转录因子中很可能是与靶标基因顺式作用元件互作的结构域。(6)通过分析大豆GmGeBP转录因子基因家族的组织表达,发现不同基因在大豆不同组织的表达量不同,具有一定的特异性。该文对大豆GeBP转录因子基因家族的分析和鉴定为进一步研究大豆表皮毛发育的分子作用提供了理论基础。