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C P Novotny

Publications and source records attributed to C P Novotny.

At least 19 recordsLinked to original sources
PubMed2001

The Aalpha6 locus: its relation to mating-type regulation of sexual development in Schizophyllum commune.

We have isolated and examined the Aalpha6 mating-type locus of Schizophyllum commune as a first step toward resolving a functional difference between this locus and the Aalpha loci previously studied. Our analyses show Aalpha6 to be remarkably similar to the Aalpha loci of known structure. The locus is composed of two, divergently transcribed genes similar in size to known Z and Y genes of the Aalpha loci. We have termed the two genes, Z6 and Y6, on the basis of their demonstrated mating activities and encoded protein motifs. The Z6 gene encodes a homeodomain-related sequence, two acidic regions and a predicted coiled-coil motif. The Y6 gene encodes a homeodomain, predicted coiled-coil motif, two regions with homology to the Abeta locus gene V6, a basic region encoding a putative nuclear localization sequence and a serine-rich region. The Z6 and Y6 proteins share these features with the other known Z and Y proteins, respectively. One of the two amino acid sequences with homology to the AbetaV6 protein has not previously been reported.

Amino Acid Sequence↗
PubMed1997

The specificity determinant of the Y mating-type proteins of Schizophyllum commune is also essential for Y-Z protein binding.

This paper concerns the manner in which combinatorial mating proteins of the fungus, Schizophyllum commune, recognize one another to form complexes that regulate target gene expression. In Schizophyllum, tightly linked Y and Z mating-type genes do not promote development in the combinations present in haploid strains (i.e., self combinations). When the Y and Z genes from two different mating types are brought together by the fusion of two haploid cells, the Y and Z proteins from different mating types recognize one another as nonself, form a complex and activate development. Several Y and Z alleles are present in the population and all nonself combinations of Y and Z alleles are equally functional. We have made chimeric genes among Y1, Y3, Y4 and Y5 and examined their mating-type specificities by transformation and mating tests. These studies show that the specificity of Y protein recognized by Z protein is encoded within a short region of N-terminal amino acids. The critical region is not precisely the same in each Y protein and in each Y-Z protein interaction. For Y3 protein compared with Y4 protein, the critical residues are in an N-terminal region of 56 amino acids (residues 17-72), with 40% identity and 65% similarity. Two-hybrid studies show that: the first 144 amino acids of Y4 protein are sufficient to bind Z3 and Z5 proteins, but not Z4 protein, and proteins deleted of the Y4 specificity region do not bind Z3, Z4 or Z5 protein. Thus the specificity determinant of the Y protein is essential for protein-protein recognition, Y-Z protein binding and mating activity.

Alleles↗
PubMed1997

Schizophyllum commune A alpha mating-type proteins, Y and Z, form complexes in all combinations in vitro.

Tha A alpha locus of the basidiomycete fungus, Schizophyllum commune, regulates sexual development via proteins Y and Z. Each A alpha mating type encodes unique Y and Z isoforms. We used two isoforms of Y (Y4 and Y5) and two isoforms of Z (Z4 and Z5) in affinity assays of protein binding. These assays identified two types of protein interactions. Each full-length Y or Z protein binds to itself and other Y or Z proteins regardless of the A alpha mating type from which they are encoded (i.e., mating-type independent binding). A second type of binding, detected with partial-length polypeptides, occurs only between N-terminal regions of Y and Z proteins encoded from different A alpha mating types (e.g., Y4Z5 or Y5Z4); we refer to this binding as mating-type dependent binding. Deletion analysis shows that the Y4 specificity domain (an N-terminal region conferring recognition uniqueness to the Y4 isoform) is essential for mating-type dependent binding. Other regions of Y and Z are involved in mating-type independent binding. These results, obtained in vitro, raise the possibility that either of several protein complexes composed of Y and/or Z proteins may occur in vivo.

Fungal Proteins↗
PubMed1996

Regions in the Z5 mating gene of Schizophyllum commune involved in Y-Z binding and recognition.

The A alpha mating locus of the woodrotting fungus Schizophyllum commune encodes two multiallelic genes, Y and Z, which regulate the A-pathway of development. The Y 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 of Z(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.

Alleles↗
PubMed1996

Cloning and characterization of a Schizophyllum gene with A beta 6 mating-type activity.

The A-pathway of development in the basidiomycete fungus Schizophyllum commune may be activated by either of two mating-type loci, A alpha and A beta. A alpha consists of two multiallelic genes, Y and Z. Y contains a putative homeodomain; Z contains a homeodomain-related region. Non-self combinations of Y and Z form heteromultimers which are thought to be transcription factors of developmental genes. To more completely understand A-regulated development it is necessary to address the issue of functional redundancy, i.e., how do two different mating loci, A alpha and A beta, both manage to regulate the same pathway. Here we report the structure of a gene with A beta 6 activity. This gene, denoted A beta V6, encodes a deduced polypeptide of 640 amino-acids with a homeodomain motif. V6 also contains a 20-amino acid sequence that is conserved in A alpha Y1, Y3 and Y4. Except for the homeodomain and the conserved sequence, the deduced V6 polypeptide shows no significant identity to A alpha Y, A alpha Z, or other known proteins. The presence of a homeodomain suggests that V, like Y and Z, may be a regulatory protein for genes in the A-pathway. Thus while A alpha and A beta encode different proteins, the general mechanism by which A alpha and A beta components signal A-regulated development may be similar.

Amino Acid Sequence↗
PubMed1996

The A alpha mating-type locus of Schizophyllum commune: structure and function of gene X.

Gene X maps immediately right of gene Y at the A alpha mating-type locus in Schizophyllum commune. Alleles X1, X3 and X4 were isolated, subcloned, and sequenced. The structure of the alleles and their relationship to the A alpha locus is described. The deduced X isoforms possess no recognized motifs common to other polypeptides and no significant similarity to any sequences in the protein data-bases. X alleles do not activate A-regulated development when transformed into recipient strains possessing different A alpha mating types or when these transformants are mated with tester strains. An X1-disrupted construction yielded a high frequency (33%) of homologous gene replacements. X-disrupted mutants have wild-type phenotypes and mate normally. Both the functional analyses and sequence data for X1, X3, and X4 suggest that the right boundary of the A alpha mating-type locus falls between A alpha Y and X. We propose that the Z and Y genes constitute the A alpha locus in its entirety.

Alleles↗
PubMed1996

Deletion of the Schizophyllum commune A alpha locus: the roles of A alpha Y and Z mating-type genes.

The A alpha locus is one of four master regulatory loci that determine mating type and regulate sexual development in Schizophyllum commune. We have made a plasmid containing a URA1 gene disruption of the A alpha Y1 gene. Y1 is the sole A alpha gene in A alpha 1 strains. We used the plasmid construction to produce an A alpha null (i.e., A alpha delta) strain by replacing the genomic Y1 gene with URA1 in an A alpha 1 strain. To characterize the role of the A alpha genes in the regulation of sexual development, we transformed various A alpha Y and Z alleles into A alpha delta strains and examined the acquired mating types and mating abilities of the transformants. These experiments demonstrate that the A alpha Y gene is not essential for fungal viability and growth, that a solitary Z A alpha mating-type gene does not itself activate development, that A beta proteins are sufficient to activate the A developmental pathway in the absence of A alpha proteins and confirm that Y and Z genes are the sole determinants of A alpha mating type. The data from these experiments support and refine our model of the regulation of A-pathway events by Y and Z proteins.

Gene Deletion↗
PubMed1995

Mammalian mitochondrial intermediate peptidase: structure/function analysis of a new homologue from Schizophyllum commune and relationship to thimet oligopeptidases.

Mitochondrial intermediate peptidase (MIP) is a component of the mitochondrial protein import machinery required for maturation of nuclear-encoded precursor proteins targeted to the mitochondrial matrix or inner membrane. We previously characterized this enzyme in rat (RMIP) and Saccharomyces cerevisiae (YMIP) and showed that MIP activity is essential for mitochondrial function in yeast. We have now defined the structure of a new MIP homologue (SMIP) from the basidiomycete fungus Schizophyllum commune. SMIP includes 4 exons of 523, 486, 660, and 629 bp separated by 3 short introns. The predicted SMIP, YMIP, and RMIP sequences share 31-37% identity and 54-57% similarity over 700 amino acids. When SMIP and RMIP were expressed in a yeast mip1 delta mutant, they were both able to rescue the respiratory-deficient phenotype caused by genetic inactivation of YMIP, indicating that the function of this enzyme is conserved in eukaryotes. Moreover, the MIP sequences show 20-24% identity and 40-47% similarity to a family of oligopeptidases from bacteria, yeast, and mammals. MIP and these proteins are characterized by a highly conserved motif, F-H-E-X-G-H-(X)2-H-(X)12-G-(X)5-D-(X)2-E-X-P-S-(X)3-E-X, centered around a zinc-binding site and appear to represent a new family of genes associated with proteolytic processing in the mitochondrial and cytosolic compartments.

Amino Acid Sequence↗
PubMed1995

The A alpha mating-type transcripts of Schizophyllum commune.

A alpha 1, A alpha 3, and A alpha 4 ds- and ss-DNA probes from the polymorphic A alpha mating-type locus of Schizophyllum commune were used to probe Northern blots of poly(A+) RNA extracted from strains of various A alpha mating types. The purpose of these experiments was to identify, map, and characterize the transcripts produced from the regions of the A alpha locus. The transcripts unique to A alpha mating type map colinear with the open reading frames identified from DNA sequence and are encoded within the fragments which activate the A developmental pathway in transformation. These data confirm the existence and structure of the previously hypothesized Y and Z A alpha mating-type genes. Transcripts from the Y and Z genes are present in vegetative cells of homokaryons and dikaryons and in cells of the fruiting bodies. The presence of the transcripts throughout the life cycle is consistent with the model of Y and Z proteins as "master switches" of A-regulated development.

Blotting, Northern↗
PubMed1994

Only one of the paired Schizophyllum commune A alpha mating-type, putative homeobox genes encodes a homeodomain essential for A alpha-regulated development.

The A alpha mating-type locus is one of four master regulatory loci controlling sexual development in Schizophyllum commune. The A alpha locus contains two homeobox genes, Y and Z, encoding two homeodomain-related proteins, Y and Z. Y and Z are each multiallelic genes. When haploid strains form fusion cells, only particular combinations of Y and Z alleles activate A alpha-regulated sexual development. The role of the putative homeodomain was examined in several Y and Z alleles by site-directed mutagenesis of regions critical to secondary structure and function of homeodomains. Mutations of the Z homeobox do not affect the function of Z proteins in A alpha-activated development, but mutations of Y homeoboxes destroy the ability of Y proteins to activate development. We conclude that only one of two A alpha homeodomain-related regulators relies upon the homeodomain motif to effect gene expression in sexual development. This conclusion affords a refinement of our working hypothesis for the mechanism by which A alpha proteins may regulate target gene expression. On the basis of our results with the Z protein, we speculate that the DNA-binding motifs of some transcriptional regulators may be lost or modified during evolution once these regulators have been recruited to participate in complexes with other DNA-binding proteins.

Amino Acid Sequence↗
PubMed1994

Mapping the heterogeneous DNA region that determines the nine A alpha mating-type specificities of Schizophyllum commune.

Classical genetic studies identified nine mating-type specificities at the A alpha locus of the Basidiomycete fungus Schizophyllum commune. We have used Southern blot hybridizations to generate EcoRI restriction maps of the A alpha locus for 18 strains, including all nine specificities. A alpha 1, A alpha 3 and A alpha 4 DNA was subcloned from three cosmids and used as probes. A unique region of DNA was found for each of the three cloned specificities. Hybridization was detected in this region only if the probe(s) and the blotted genomic DNAs were from strains with the same A alpha specificity. DNAs from strains with the same A alpha specificity hybridize regardless of geographic origin, but DNAs from strains with different A alpha specificities do not cross-hybridize. The results demonstrate two size classes of unique A alpha DNA. This unique DNA is about 4.5 kb in A alpha 1 strains and about 7.0-8.5 kb in other strains. Transcription regulators Z and Y, which were deduced previously from the DNA sequence of the A alpha 1, A alpha 3 and A alpha 4 loci, are probably encoded by all non-A alpha 1 loci. The smaller A alpha 1 loci appear to encode only Y and lack sequence for Z. No evidence was found for a locus that encodes only Z. The lack of hybridization detected between A alpha loci with different specificities suggests that the evolution of A alpha has resulted from extensive sequence divergence.

Blotting, Southern↗
PubMed1992

The A alpha mating locus of Schizophyllum commune encodes two dissimilar multiallelic homeodomain proteins.

The A alpha mating locus is one of four multiallelic loci that govern sexual development in the basidiomycete fungus Schizophyllum commune. We have determined the nucleotide sequence encoding three A alpha mating types, A alpha 1, A alpha 3, and A alpha 4. We have found that the locus for A alpha 3 and A alpha 4 consists of two genes: Y and Z. The locus for A alpha 1 encodes only one gene, Y. The Z polypeptides encoded by different alleles exhibit 42% identity. The Y polypeptides exhibit 49-54% identity. The finding that the deduced Z and Y polypeptides have homeodomain motifs suggests that these polypeptides may be DNA-binding regulatory proteins that control the expression of developmental genes. The deduced Z polypeptide also has acidic regions that might be functionally analogous to the acidic regions in yeast GAL4 and GCN4 that activate transcription. The Y polypeptide has a serine-rich region and a basic region that shows some identity to the lysine-rich region of H1 histones.

Alleles↗
PubMed1992

Functional analysis of the homeodomain-related proteins of the A alpha locus of Schizophyllum commune.

DNA-mediated transformation was used to correlate function with putative genes from three alternative A alpha mating-type loci (A alpha 1, A alpha 3, and A alpha 4) of Schizophyllum commune. Each DNA was tested in at least nine haploid strains, one for each of the nine A alpha mating types found in the world-wide population of S. commune. The Y and Z genes (tentatively identified by sequence analysis elsewhere) individually activate A alpha-regulated development when transformed into any strain with a different A alpha mating type. The only exceptions are when the Y alleles of A alpha 3 or A alpha 4 (i.e., Y3 or Y4, respectively) are introduced into an A alpha 1 strain (the A alpha 1 locus encodes Y1 but lacks a Z gene). These observations indicate that A alpha-regulated development is activated by the interaction (direct or indirect) of products from different genes (e.g., Z3 and Y1) rather than from different alleles of the same gene (e.g., Y1 and Y3). Therefore, the activating interaction is of the form ZiYj where i not equal to j and i and j are the A alpha mating types from which the Z and Y polypeptides, respectively, are derived. Transformations with truncated or mutagenized genes begin to define essential regions of the genes and their products. Activity is in some cases dependent upon the particular A alpha mating type of the recipient. A working hypothesis for the activation of A alpha-regulated development is proposed.

Crosses, Genetic↗
PubMed1992

Mitochondrial DNA of Schizophyllum commune: restriction map, genetic map, and mode of inheritance.

Mitochondrial DNA (mtDNA) found in the basidiomycete Schizophyllum commune (strain 4-40) is a circular molecule 49.75 kbp in length. A physical map containing 61 restriction sites revealed no repeat structures. Cloned genes from Neurospora crassa, Aspergillus nidulans, and Saccharomyces cerevisiae were used in Southern hybridizations to locate nine mitochondrial genes, including a possible pseudogene of ATPase 9, on the restriction map. A probe from a functional ATPase 9 gene identified homologous fragments only in the nuclear genome of S. commune. Restriction fragment length polymorphisms (RFLPs) between mtDNA isolated from different strains of S. commune were used to show that mitochondria do not migrate with nuclei during dikaryosis.

Adenosine Triphosphatases↗
PubMed1989

Sequence analysis of the URA1 gene encoding orotidine-5'-monophosphate decarboxylase of Schizophyllum commune.

The URA1 gene (encoding orotidine-5'-monophosphate decarboxylase) of the basidiomycete fungus Schizophyllum commune was mapped to a 1.4-kb BglI-BamHI fragment of two independent phage lambda clones previously isolated from a Schizophyllum genomic library. The fragment was identified by its ability to complement Schizophyllum ura1 mutants via transformation. The complete nucleotide sequence of the fragment containing the URA1 gene was determined. Sequence analysis revealed that the coding region of the URA1 gene encompasses a polypeptide of 279 amino acids (aa) interrupted by two small introns. The deduced aa sequence corresponds to 30.3 kDa and is substantially similar to the sequences of analogous polypeptides from other organisms. No canonical 5'-TATA sequence nor 3'-AATAAA polyadenylation signal are evident in the flanking regions of the URA1 gene.

Amino Acid Sequence↗
PubMed1989

Cloning and comparison of A alpha mating-type alleles of the Basidiomycete Schizophyllum commune.

An A alpha mating-type allele (A alpha 4) was isolated by walking the chromosome from the closely linked PAB1 gene. A cosmid clone containing the A alpha 4 allele isolated from the walk was used as a probe to recover the A alpha 1 allele from another cosmid library. Cosmids encoding mating-type activity were identified by transforming Schizophyllum cells and screening for activation of A-regulated development. Putative mating-type transformants were confirmed in mating tests and genetic analyses of progeny. The identity of the specific alleles isolated was demonstrated by showing that their effectiveness in transforming for mating type is limited to recipient strains possessing an A alpha allele different from the one encoded by the cloned sequences. Transforming DNA is active in trans, suggesting that A alpha encodes a diffusible product. Restriction mapping shows that A alpha 1 and A alpha 4 are coded in the same physical region of the genome, but within a subregion that contains extensive sequence divergence. In addition, Southern analyses show that there is only one copy of A alpha 1 or A alpha 4 per haploid genome, and that they do not cross-hybridize to one another or to any of the other A alpha alleles. A alpha 1 and A alpha 4 were subcloned as 2.8 and 1.2 kb fragments, respectively, retaining in transformation all the mating-type activity demonstrated of the original cosmids.

Alleles↗
PubMed1988

Organization of the ribosomal RNA genes of Schizophyllum commune.

The 18, 5.8, 25 and 5S ribosomal RNA (rRNA) cistrons have been mapped on the ribosomal DNA (rDNA) unit repeat of Schizophyllum commune strain 4-40. These genes are spatially ordered in the sequence given. The presence of a large primary precursor rRNA which is processed to form the mature 18, 5.8 and 25S rRNAs has been demonstrated. We have mapped the site of transcriptional initiation for this rRNA primary precursor. The sequence surrounding this site has been determined and shown to be highly conserved, with considerable identity to those in Neurospora crassa and Dictyostelium discoideum. The direction of transcription of the rRNA genes has been determined. The 5S rRNA cistron is transcribed in the same direction as the other rRNAs, however it is not transcribed as a part of the large primary precursor. The previously identified rDNA strain-specific length polymorphisms (Specht et al. 1984) are shown to be located within the transcribed region of the rDNA unit repeat.

Basidiomycota↗