Fuz1, a MYND domain protein, is required for cell morphogenesis in Ustilago maydis

Abstract: Ustilago maydis is a Basidiomycete fungus that exhibits a yeast-like nonpathogenic form and a dikaryotic filamentous pathogenic form. Generation of these two forms is controlled by two mating type loci, a and b. The fungus undergoes additional morphological transitions in the plant that result in formation of a third cell type, the teliospore. The fuz1 gene is necessary for this developmental program. Here we report cloning and sequencing of fuz1 and show that it contains an open reading frame with coding capacity for a protein of 1421 amino acids. The Fuz1 protein belongs to the family of MYND Zn finger domain proteins. We generate a null mutation in strains of opposite mating type and show that fuz1 is necessary for conjugation tube formation, a morphological transition that occurs in response to pheromones. We generate fuz1- diploid strains heterozygous at a and b and show that fuz1 is also necessary for postfusion events (maintenance of filamentous growth). We also demonstrate that fuz1 is necessary for cell morphogenesis of the yeast-like cell: normal cell length, location and number of septa, cell separation and constriction of the neck region. Fuz1 is also required for cell wall integrity and to prevent secretion of a dark pigment. We propose that the MYND domain may interact with different proteins to regulate cell morphogenesis.     

Identification of polypeptides encoded in open reading frame 1b of the putative polymerase gene of the murine coronavirus mouse hepatitis virus A59.

The polypeptides encoded in open reading frame (ORF) 1b of the mouse hepatitis virus A59 putative polymerase gene of RNA 1 were identified in the products of in vitro translation of genome RNA. Two antisera directed against fusion proteins containing sequences encoded in portions of the 3'-terminal 2.0 kb of ORF 1b were used to immunoprecipitate p90, p74, p53, p44, and p32 polypeptides. These polypeptides were clearly different in electrophoretic mobility, antiserum reactivity, and partial protease digestion pattern from viral structural proteins and from polypeptides encoded in the 5' end of ORF 1a, previously identified by in vitro translation. The largest of these polypeptides had partial protease digestion patterns similar to those of polypeptides generated by in vitro translation of a synthetic mRNA derived from the 3' end of ORF 1b. The polypeptides encoded in ORF 1b accumulated more slowly during in vitro translation than polypeptides encoded in ORF 1a. This is consistent with the hypothesis that translation of gene A initiates at the 5' end of ORF 1a and that translation of ORF 1b occurs following a frameshift at the ORF 1a-ORF 1b junction. The use of in vitro translation of genome RNA and immunoprecipitation with antisera directed against various regions of the polypeptides encoded in gene A should make it possible to study synthesis and processing of the putative coronavirus polymerase.     

Molecular cloning and characterization of acrA and acrE genes of Escherichia coli.

The DNA fragment containing the acrA locus of the Escherichia coli chromosome has been cloned by using a complementation test. The nucleotide sequence indicates the presence of two open reading frames (ORFs). Sequence analysis suggests that the first ORF encodes a 397-residue lipoprotein with a 24-amino-acid signal peptide at its N terminus. One inactive allele of acrA from strain N43 was shown to contain an IS2 element inserted into this ORF. Therefore, this ORF was designated acrA. The second downstream ORF is predicted to encode a transmembrane protein of 1,049 amino acids and is named acrE. Genes acrA and acrE are probably located on the same operon, and both of their products are likely to affect drug susceptibilities observed in wild-type cells. The cellular localizations of these polypeptides have been analyzed by making acrA::TnphoA and acrE::TnphoA fusion proteins. Interestingly, AcrA and AcrE share 65 and 77% amino acid identity with two other E. coli polypeptides, EnvC and EnvD, respectively. Drug susceptibilities in one acrA mutant (N43) and one envCD mutant (PM61) have been determined and compared. Finally, the possible functions of these proteins are discussed.     

The structural and accessory proteins M,ORF 4a,ORF 4b,and ORF 5 of Middle East respiratory syndrome coronavirus (MERS-CoV) are potent interferon antagonists

The newly emerged Middle East respiratory syndrome coronavirus (MERS-CoV) is a highly pathogenic respiratory virus with pathogenic mechanisms that may be driven by innate immune pathways. The goal of this study is to characterize the expression of the structural (S, E, M, N) and accessory (ORF 3, ORF 4a, ORF 4b, ORF 5) proteins of MERS-CoV and to determine whether any of these proteins acts as an interferon antagonist. Individual structural and accessory protein-coding plasmids with an N-terminal HA tag were constructed and transiently transfected into cells, and their native expression and subcellular localization were assessed using Wes tern blotting and indirect immunofluorescence. While ORF 4b demonstrated majorly nuclear localization, all of the other proteins demonstrated cytoplasmic localization. In addition, for the first time, our experiments revealed that the M, ORF 4a, ORF 4b, and ORF 5 proteins are potent interferon antagonists. Further examination revealed that the ORF 4a protein of MERS-CoV has the most potential to counteract the antiviral effects of IFN via the inhibition of both the interferon production (IFN-β promoter activity, IRF-3/7 and NF-κB activation) and ISRE promoter element signaling pathways. Together, our results provide new insights into the function and pathogenic role of the structural and accessory proteins of MERS-CoV.     

Conservation of the b mating-type gene complex among bipolar and tetrapolar smut fungi.

In the phytopathogenic fungus Ustilago hordei, one locus with two alternate alleles, MAT-1 and MAT-2, controls mating and the establishment of the infectious dikaryon (bipolar mating). In contrast, for U. maydis, these functions are associated with two different gene complexes, called a and b (tetrapolar mating); the a complex has two alternate specificities, and the b gene complex is multiallelic. We have found homologs for the b gene complex in U. hordei and have cloned one from each mating type using sequences from one bEast allele of U. maydis as a probe. Sequence analysis revealed two divergent open reading frames in each b complex, which we called bW (bWest) and bE (bEast) in analogy with the b gene complex of U. maydis. The predicted bW and bE gene products from the two different mating types showed approximately 75% identity when homologous polypeptides were compared. All of the characterized bW and bE gene products have variable amino-terminal regions, conserved carboxy-terminal regions, and similar homeodomain motifs. Sequence comparisons with the bW1 and bE1 genes of U. maydis showed conservation in organization and structure. Transformation of the U. hordei b gene complex into a U. hordei strain of opposite mating type showed that the b genes from the two mating types are functional alleles. The U. hordei b genes, when introduced into U. maydis, rendered the haploid transformants weakly pathogenic on maize. These results indicate that structurally and functionally conserved b genes are present in U. hordei.     

Severe acute respiratory syndrome coronavirus open reading frame (ORF) 3b, ORF 6, and nucleocapsid proteins function as interferon antagonists

The severe acute respiratory syndrome coronavirus (SARS-CoV) is highly pathogenic in humans, with a death rate near 10%. This high pathogenicity suggests that SARS-CoV has developed mechanisms to overcome the host innate immune response. It has now been determined that SARS-CoV open reading frame (ORF) 3b, ORF 6, and N proteins antagonize interferon, a key component of the innate immune response. All three proteins inhibit the expression of beta interferon (IFN-beta), and further examination revealed that these SARS-CoV proteins inhibit a key protein necessary for the expression of IFN-beta, IRF-3. N protein dramatically inhibited expression from an NF-kappaB-responsive promoter. All three proteins were able to inhibit expression from an interferon-stimulated response element (ISRE) promoter after infection with Sendai virus, while only ORF 3b and ORF 6 proteins were able to inhibit expression from the ISRE promoter after treatment with interferon. This indicates that N protein inhibits only the synthesis of interferon, while ORF 3b and ORF 6 proteins inhibit both interferon synthesis and signaling. ORF 6 protein, but not ORF 3b or N protein, inhibited nuclear translocation but not phosphorylation of STAT1. Thus, it appears that these three interferon antagonists of SARS-CoV inhibit the interferon response by different mechanisms.     

A carboxy-terminal region required by the adenovirus type 9 E4 ORF1 oncoprotein for transformation mediates direct binding to cellular polypeptides.

Human adenovirus type 9 (Ad9) is unique among oncogenic adenoviruses in that it elicits exclusively mammary tumors in rats and requires the viral E4 region open reading frame 1 (9ORF1) gene for tumorigenicity. The 9ORF1 oncogenic determinant codes for a 14-kDa transforming protein, and three separate regions of this polypeptide, including one at the extreme C terminus, are necessary for transforming activity. In this study, we investigated whether the 9ORF1 transforming protein interacts with cellular factors. Following incubation with cell extracts, a glutathione S-transferase (GST)-9ORF1 fusion protein associated with several cellular phosphoproteins (p220, p180, p160, p155), whereas GST fusion proteins of transformation-defective 9ORF1 C-terminal mutants did not. Similar interactions requiring the 9ORF1 C terminus were revealed with protein-blotting assays, in which a GST-9ORF1 protein probe reacted specifically with cellular polypeptides having gel mobilities resembling those of the 9ORF1-associated cellular phosphoproteins, as well as with additional cellular polypeptides designated p140/p130. In addition, GST fusion proteins containing 9ORF1 C-terminal fragments associated with some of the 9ORF1-associated cellular polypeptides, as did GST fusion proteins of full-length wild-type Ad5 and Ad12 E4 ORF1 transforming proteins. Significantly, the results of coimmunoprecipitation analyses suggested that the same cellular polypeptides also associate with wild-type but not C-terminal-mutant 9ORF1 proteins in vivo. Together, these findings suggest that the 9ORF1 C terminus, which is essential for transformation, participates in specific and direct binding of the 9ORF1 oncoprotein to multiple cellular polypeptides. We propose that interactions with these cellular factors may be responsible, at least in part, for the transforming activity of the 9ORF1 viral oncoprotein.     

Protein products of the open reading frames encoding nonstructural proteins of human astrovirus serotype 8

Human astroviruses have a positive-strand RNA genome, which contains three open reading frames (ORF1a, ORF1b, and ORF2). The genomic RNA is translated into two nonstructural polyproteins, nsp1a and nsp1ab, that contain sequences derived from ORF1a and from both ORF1a and ORF1b, respectively. Proteins nsp1a and nsp1ab are thought to be proteolytically processed to yield the viral proteins implicated in the replication of the virus genome; however, the intermediate and final products of this processing have been poorly characterized. To identify the cleavage products of the nonstructural polyproteins of a human astrovirus serotype 8 strain, antisera to selected recombinant proteins were produced and were used to analyze the viral proteins synthesized in astrovirus-infected Caco-2 cells and in cells transfected with recombinant plasmids expressing the ORF1a and ORF1b polyproteins. Pulse-chase experiments identified proteins of approximately 145, 88, 85, and 75 kDa as cleavage intermediates during the polyprotein processing. In addition, these experiments and kinetic analysis of the synthesis of the viral proteins identified polypeptides of 57, 20, and 19 kDa, as well as two products of around 27 kDa, as final cleavage products, with the 57-kDa polypeptide most probably being the virus RNA polymerase and the two approximately 27-kDa products being the viral protease. Based on the differential reactivities of the astrovirus proteins with the various antisera used, the individual polypeptides detected were mapped to the virus ORF1a and ORF1b regions.     

High level of divergence of male-reproductive-tract proteins, between Drosophila melanogaster and its sibling species, D. simulans

We compared male-reproductive-tract polypeptides of Drosophila melanogaster and D. simulans by using two-dimensional gel electrophoresis. Approximately 64% of male-reproductive-tract polypeptides were identical between two randomly chosen isofemale lines from these two species, compared with 83% identity for third-instar imaginal wing-disc polypeptides. Qualitatively similar differences were found between reproductive tracts and imaginal discs when D. sechellia was compared with D. melanogaster and with D. simulans. When genic polymorphism was taken into account, approximately 10% of male- reproductive-tract polypeptides were apparently fixed for different alleles between D. melanogaster and D. simulans; this proportion is the same as that found for soluble enzymes by one-dimensional gel electrophoresis. Strikingly, approximately 20% of male-reproductive- tract polypeptides of either D. melanogaster or D. simulans had no detectable homologue in the other species. We propose that proteins of the Drosophila male reproductive tract may have diverged more extensively between species than have other types of proteins and that much of this divergence may involve large changes in levels of polypeptide expression.      

Morphological Transitions in the Life Cycle of Ustilago maydis and Their Genetic Control by the a and b Loci

Banuett, F., and Herskowitz, I. 1994. Morphological transitions in the life cycle of Ustilago maydis and their genetic control by the a and b loci. Experimental Mycology 18: 247-266. Two forms characterize the life cycle of Ustilago maydis: a haploid yeast-like form and a filamentous dikaryotic form. Dimorphism and other aspects of the life cycle (including tumor induction) are governed by two mating type loci, a and b . Here we report characterization of two different morphological transitions in the life cycle of U. maydis. First, we describe an assay for conjugation tube formation in which cellular response is rapid and occurs synchronously and uniformly in the population. Using this assay, we demonstrate that different alleles of the a locus (but not the b locus) are necessary for conjugation tube formation. We also show that the b locus determines the type of filament formed after cell fusion: different b alleles lead to formation of true filaments, whereas identical b alleles result in production of pseudofilaments. Second, we analyze the role of a and b in postfusion events leading to filament formation in diploid strains. We show that diploid strains heterozygous for both a and b are capable of a dimorphic transition from yeast-like to filamentous growth when shifted from rich medium to low-nitrogen medium. This transition has two components: the first is dependent on the a locus and generates structures similar to conjugation tubes; the second is dependent on the b locus and produces true hyphal structures. We surmise that similar events take place in formation of the dikaryotic filament.     

The a locus governs cytoduction in Ustilago maydis.

We have developed a cytoduction assay to measure cell fusion quantitatively in the basidiomycete corn smut fungus Ustilago maydis. This assay employs a mutation conferring resistance to oligomycin that exhibits non-Mendelian inheritance and presumably affects the mitochondrial genome. After auxotrophic olir cells are mixed with prototrophic olis cells, prototrophic olir cells can be detected at a significant frequency after several hours of incubation, reaching a maximum of 10% of the total prototrophs in the mixture after 18 h. We demonstrate that this cell fusion event occurs only if the mating partners have different alleles of the a mating-type locus and is not influenced by the b locus. These studies support the view that the a locus but not the b locus controls establishment of the filamentous, pathogenic state.     

Conservation of the S10-spc-α Locus within Otherwise Highly Plastic Genomes Provides Phylogenetic Insight into the Genus Leptospira

S10-spc-α is a 17.5 kb cluster of 32 genes encoding ribosomal proteins. This locus has an unusual composition and organization in Leptospira interrogans. We demonstrate the highly conserved nature of this region among diverse Leptospira and show its utility as a phylogenetically informative region. Comparative analyses were performed by PCR using primer sets covering the whole locus. Correctly sized fragments were obtained by PCR from all L. interrogans strains tested for each primer set indicating that this locus is well conserved in this species. Few differences were detected in amplification profiles between different pathogenic species, indicating that the S10-spc-α locus is conserved among pathogenic Leptospira. In contrast, PCR analysis of this locus using DNA from saprophytic Leptospira species and species with an intermediate pathogenic capacity generated varied results. Sequence alignment of the S10-spc-α locus from two pathogenic species, L. interrogans and L. borgpetersenii, with the corresponding locus from the saprophyte L. biflexa serovar Patoc showed that genetic organization of this locus is well conserved within Leptospira. Multilocus sequence typing (MLST) of four conserved regions resulted in the construction of well-defined phylogenetic trees that help resolve questions about the interrelationships of pathogenic Leptospira. Based on the results of secY sequence analysis, we found that reliable species identification of pathogenic Leptospira is possible by comparative analysis of a 245 bp region commonly used as a target for diagnostic PCR for leptospirosis. Comparative analysis of Leptospira strains revealed that strain H6 previously classified as L. inadai actually belongs to the pathogenic species L. interrogans and that L. meyeri strain ICF phylogenetically co-localized with the pathogenic clusters. These findings demonstrate that the S10-spc-α locus is highly conserved throughout the genus and may be more useful in comparing evolution of the genus than loci studied previously.     

The divergence-homogenization duality in the evolution of the b1 mating type gene of Coprinus cinereus.

The A mating type locus of the fungus Coprinus cinereus is a complex, multigenic locus which regulates compatibility and subsequent sexual development. Genes within the A locus such as the b1 gene studied here exhibit extreme sequence variation. In this work, we asked how b1 alleles have evolved high levels of variation and, at the same time, conserved function. We compared sequence variation in 17 alleles characterized as belonging to seven different compatibility classes. Comparison of sequence variation between representatives of these seven classes shows that different regions of the b1 gene have been subject to varying levels of substitution, recombination, and structural/functional constraints. The N-terminal region of the encoded protein, which has been previously demonstrated to govern self/nonself recognition, exhibited hypervariability with levels of amino acid identity as low as 41%. We used a novel analysis of neutral mutations accumulating in this gene to rule out the possibility that the N-terminal region is hypermutable. In contrast, the C-terminal region displayed heterogeneous levels of variation, with functional motifs being better conserved. In fact, there is a duality in the b1 gene between variability and conservation; recombination events have homogenized the C-terminal region, while recombination events are undetectable in the N-terminal region. The ability to regulate sexual development is maintained in all of the mating compatibility alleles studied, and these data suggest that some functional motifs may tolerate high levels of substitution.     

The Drosophila erect wing gene, which is important for both neuronal and muscle development, encodes a protein which is similar to the sea urchin P3A2 DNA binding protein.

The erect wing (ewg) locus of Drosophila melanogaster encodes a vital function important for the development of the nervous system and the indirect flight muscles. In order to understand the ewg function at a molecular level, cDNA clones were isolated. Sequence analysis of cDNAs revealed a single open reading frame (ORF) encoding a protein of 733 residues. The translational start for this ORF is a CTG codon. A 225-amino-acid region of this protein is 71% identical to the DNA binding region of the Strongylocentrotus purpuratus P3A2 DNA binding protein. Additionally, the ORF contains large acidic and basic domains characteristic of those in proteins involved in nuclear regulatory functions. Immunoblot analysis using polyclonal anti-EWG antisera generated against a bacterial fusion protein reveals a single, 116-kDa protein present throughout development, beginning at approximately stage 12 of embryogenesis, which is enriched in adult heads and absent from embryos carrying certain ewg alleles. Additionally, we show that EWG is localized specifically to the nuclei of virtually all embryonic neurons. Finally, a minigene consisting of an ewg cDNA under control of the hsp70 promoter can provide the ewg function in transgenic ewg mutant flies.     

Biochemical genetics of some seed proteins of Pinus radiata

In a high-salt soluble fraction of the total protein from single seeds of Pinus radiata, up to 45 polypeptides were resolved on SDS-polyacrylamide gels. At least one-fifth of these polypeptides showed variation between seeds. In the 27,000–29,000 dalton region, two polypeptides were inherited as codominant alleles at a single locus and were shown to assort independently of another seed protein locus and three allozyme loci. A survey of 120 individuals from the five known native populations of P. radiata in California detected only the 27K and 29K alleles at the locus. In all populations, the 29K allele predominated, and the two island populations were monomorphic for the 29K allele. The 27 and 29 kdalton polypeptides were shown to have very similar amino acid sequences, and the allelic difference at this locus is most probably in the gene sequence for the polypeptide.     

The Rhizobium meliloti rhizopine mos locus is a mosaic structure facilitating its symbiotic regulation.

The Rhizobium meliloti L5-30 mos locus, encoding biosynthesis of the rhizopine 3-O-methyl-scyllo-inosamine, is shown to be a mosaic structure. The mos locus consists of four open reading frames (ORFs) (ORF1 and mosABC) arranged in an operon structure. Within this locus, several domains of homology with other prokaryotic symbiotic genes (nifH, fixA, fixU, and nifT) are present, suggesting that this locus may represent a hot spot for rearrangement of symbiotic genes. Unusually, these domains are present in the coding as well as noncoding regions of the mos locus. Proteins corresponding to those encoded by mosABC, but not ORF1, have been detected in nodule extracts by using antibodies. As ORF1 shows extensive homology with the 5' region of the nifH gene (P.J. Murphy, N. Heycke, S.P. Trenz, P. Ratet, F.J. de Bruijn, and J. Schell, Proc. Natl. Acad. Sci. USA 85:9133-9137, 1988) and a frameshift mutation indicates that expression of this ORF is not required for mos activity, we propose that the mos locus has acquired a duplicated copy of nifH, including the promoter region, in order to become symbiotically regulated. Surprisingly, since the functions are likely different, MosA has an amino acid sequence similar to that of the DapA protein of Escherichia coli. The central domain of MosB has extensive homology with a range of diverse proteins involved with carbohydrate metabolism in either antibiotic or outer-cell-wall biosynthesis. This region is also common to the regulatory proteins DegT and DnrJ, suggesting a regulatory role for MosB. The structure of MosC is consistent with its being a membrane transport protein.     

Conservation of the S10-spc-alpha locus within otherwise highly plastic genomes provides phylogenetic insight into the genus Leptospira

S10-spc-alpha is a 17.5 kb cluster of 32 genes encoding ribosomal proteins. This locus has an unusual composition and organization in Leptospira interrogans. We demonstrate the highly conserved nature of this region among diverse Leptospira and show its utility as a phylogenetically informative region. Comparative analyses were performed by PCR using primer sets covering the whole locus. Correctly sized fragments were obtained by PCR from all L. interrogans strains tested for each primer set indicating that this locus is well conserved in this species. Few differences were detected in amplification profiles between different pathogenic species, indicating that the S10-spc-alpha locus is conserved among pathogenic Leptospira. In contrast, PCR analysis of this locus using DNA from saprophytic Leptospira species and species with an intermediate pathogenic capacity generated varied results. Sequence alignment of the S10-spc-alpha locus from two pathogenic species, L. interrogans and L. borgpetersenii, with the corresponding locus from the saprophyte L. biflexa serovar Patoc showed that genetic organization of this locus is well conserved within Leptospira. Multilocus sequence typing (MLST) of four conserved regions resulted in the construction of well-defined phylogenetic trees that help resolve questions about the interrelationships of pathogenic Leptospira. Based on the results of secY sequence analysis, we found that reliable species identification of pathogenic Leptospira is possible by comparative analysis of a 245 bp region commonly used as a target for diagnostic PCR for leptospirosis. Comparative analysis of Leptospira strains revealed that strain H6 previously classified as L. inadai actually belongs to the pathogenic species L. interrogans and that L. meyeri strain ICF phylogenetically co-localized with the pathogenic clusters. These findings demonstrate that the S10-spc-alpha locus is highly conserved throughout the genus and may be more useful in comparing evolution of the genus than loci studied previously.