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R L Metzenberg

Publications and source records attributed to R L Metzenberg.

At least 37 records · Page 2Linked to original sources
PubMed1994

Cloning and characterization of the pho-2+ gene encoding a repressible alkaline phosphatase in Neurospora crassa.

The Neurospora crassa phosphate-repressible alkaline phosphatase-encoding gene pho-2+ was cloned and its nucleotide sequence was determined. An open reading frame was found that contains four introns and encodes a putative protein of 555 amino acids. 'Activator-independent expression' of ectopically integrated pho-2+ was observed, as noted before for ectopically integrated pho-4+.

Alkaline Phosphatase↗
PubMed1994

Inactivation of the Neurospora crassa gene encoding the mitochondrial protein import receptor MOM19 by the technique of "sheltered RIP".

We have used a technique referred to as "sheltered RIP" (repeat induced point mutation) to create mutants of the mom-19 gene of Neurospora crassa, which encodes an import receptor for nuclear encoded mitochondrial precursor proteins. Sheltered RIP permits the isolation of a mutant gene in one nucleus, even if that gene is essential for the survival of the organism, by sheltering the nucleus carrying the mutant gene in a heterokaryon with an unaffected nucleus. Furthermore, the nucleus harboring the RIPed gene contains a selectable marker so that it is possible to shift nuclear ratios in the heterokaryons to a state in which the nucleus containing the RIPed gene predominates in cultures grown under selective conditions. This results in a condition where the target gene product should be present at very suboptimal levels and allows the study of the mutant phenotype. One allele of mom-19 generated by this method contains 44 transitions resulting in 18 amino acid substitutions. When the heterokaryon containing this allele was grown under conditions favoring the RIPed nucleus, no MOM19 protein was detectable in the mitochondria of the strain. Homokaryotic strains containing the RIPed allele exhibit a complex and extremely slow growth phenotype suggesting that the product of the mom-19 gene is important in N. crassa.

Amino Acid Sequence↗
PubMed1994

Analysis of the DNA-binding and dimerization activities of Neurospora crassa transcription factor NUC-1.

NUC-1, a positive regulatory protein of Neurospora crassa, controls the expression of several unlinked target genes involved in phosphorus acquisition. The carboxy-terminal end of the NUC-1 protein has sequence similarity to the helix-loop-helix family of transcription factors. Bacterially expressed and in vitro-synthesized proteins, which consist of the carboxy-terminal portion of NUC-1, bind specifically to upstream sequences of two of its target genes, pho2+ and pho-4+. These upstream sequences contain the core sequence, CACGTG, a target for many helix-loop-helix proteins. A large loop region (47 amino acids) separates the helix I and helix II domains. Mutations and deletion within the loop region did not interfere with the in vitro or in vivo functions of the protein. Immediately carboxy-proximal to the helix II domain, the NUC-1 protein contains an atypical zipper domain which is essential for function. This domain consists of a heptad repeat of alanine and methionine rather than leucine residues. Analysis of mutant NUC-1 proteins suggests that the helix II and the zipper domains are essential for the protein dimerization, whereas the basic and the helix I domains are involved in DNA binding. The helix I domain, even though likely to participate in dimer formation while NUC-1 is bound to DNA, is not essential for in vitro dimerization.

Base Sequence↗
PubMed1993

Amplification of the nucleolus organizer region during the sexual phase of Neurospora crassa.

Previously we have shown that the nucleolus organizer region (NOR) of Neurospora crassa displays frequent size changes during crosses. In these initial studies, we observed that decreases in NOR size are far more common than increases. Here, we have investigated the inheritance of NOR size in a strain with an unusually small NOR. We call this strain SNO for small nucleolus organizer. We found that progeny that inherit their rDNA from SNO receive either an NOR that is larger than that of SNO or, rarely, the same size, but never an NOR that is smaller than that of SNO. The number of progeny that inherit their NOR from SNO is not significantly different from the number that inherit their NOR from the other parent in the cross. This argues against the idea that the failure to find progeny with NORs smaller than that of SNO is due to inviability of spores carrying such an NOR, or that it is due to unconscious bias by the experimenter against isolating such spores. These results can most easily be explained by a combination of unequal sister chromatid exchanges in the rDNA, or sister chromatid conversion, coupled with selection against nuclei harboring small NORs during the premeiotic phase of the Neurospora life cycle. Other, less conventional, explanations are also possible, such as "directed" increase in the target NOR without corresponding loss at some other NOR.

DNA, Fungal↗
PubMed1993

Insertional mutagenesis in Neurospora crassa: cloning and molecular analysis of the preg+ gene controlling the activity of the transcriptional activator NUC-1.

The transcriptional activator NUC-1 controls the transcription of the genes for phosphorus acquisition enzymes, and its activity is regulated by the negative regulatory factors, PREG and PGOV In this report, we describe the cloning and molecular analysis of the preg+ gene. In Neurospora crassa, as in higher eukaryotes, transformation frequently results in nonhomologous integration of transforming DNA. Insertion of transforming DNA into host genes mutates the gene and provides a molecular tag for cloning it. We obtained two mutants that have an insertion in the preg+ and pgov+ genes, respectively, among 2 x 10(5) transformants. The preg+ gene was cloned by screening a Neurospora genomic DNA library with DNA sequences flanking the transforming DNA of the rescued plasmid. Northern analysis showed that the transcription of the preg+ gene is not regulated by phosphate. The carboxy-terminal half of PREG shows strong homology with Saccharomyces cerevisiae PHO80 whose function is analogous to that of PREG. The pregc mutations are located in the well conserved residues which may directly interact with the residues in the regulatory domain of NUC-1.

Alleles↗
PubMed1992

Sexual development genes of Neurospora crassa.

The filamentous fungus Neurospora crassa undergoes a complex program of sexual development to form a fruiting body composed of several kinds of specialized tissue. Subtractive hybridization was used to isolate genes that are expressed preferentially during this sexual phase. Many such sexual development (sdv) genes were identified in a cosmid library of Neurospora genomic DNA. Fourteen of the sdv genes were subcloned, and their expression in mutant strains and under crossing and vegetative growth conditions was examined. All of the regulated transcripts were less abundant (and in many cases not detectable) in strains grown under vegetative (high nitrogen) conditions, suggesting that nitrogen starvation is required for their synthesis. The expression of most of the sdv genes also required a functional A mating type product, even under crossing growth conditions, suggesting that this product functions as a master control in sexual development. To determine if the products of the sdv genes play essential roles in the sexual cycle, a reverse-genetic approach (based on RIP (repeat-induced point mutation)-mediated gene disruptions) was used to create mutations in the genes. A mutant strain (asd-1) with a recessive crossing defect (apparently caused by the RIP process) was isolated; in this strain, early development is normal and may asci are formed, but ascospores are never delineated. A second recessive mutant strain (asd-2) was apparently created by ectopic integration of the transforming DNA into a gene required for the sexual process; in this strain the sexual process was blocked at an early stage, and the ascogeneous tissue underwent little development.

Cell Differentiation↗
PubMed1990

Mating type and mating strategies in Neurospora.

In the heterothallic species Neurospora crassa, strains of opposite mating type, A and a, must interact to give the series of events resulting in fruiting body formation, meiosis, and the generation of dormant ascospores. The mating type of a strain is specified by the DNA sequence it carries in the mating type region; strains that are otherwise isogenic can mate and produce ascospores. The DNA of the A and a regions have completely dissimilar sequences. Probing DNA from strains of each mating type with labelled sequences from the A and the a regions has shown that, unlike in Saccharomyces cerevisiae, only a single copy of a mating type sequence is present in a haploid genome. The failure to switch is explainable by the physical absence of DNA sequences characteristic of the opposite mating type. While the mating type sequences must be of the opposite kind for mating to occur in the sexual cycle, two strains of opposite mating type cannot form a stable heterokaryon during vegetative growth; instead, they fuse abortively to give a heterokaryon incompatibility reaction, which results in death of the cells along the fusion line. The DNA sequences responsible for this reaction are coextensive with those sequences in the A and a regions which are necessary to initiate fruiting body formation. The genus Neurospora also includes homothallic species--ones in which a single haploid nucleus carries all the information necessary to form fruiting bodies, undergo meiosis, and produce new haploid spores. One such species, N. terricola, contains one copy each of the A and the a sequences within each haploid genome.(ABSTRACT TRUNCATED AT 250 WORDS)

Crosses, Genetic↗
PubMed1990

Neurospora crassa A mating-type region.

The mating-type locus of the haploid filamentous fungus Neurospora crassa is a regulatory region that controls entry into the sexual cycle and prevents formation of mixed mating-type heterokaryons in the vegetative phase. The locus consists of alternative sequences called A and a. The A mating-type DNA sequence of Neurospora crassa is composed of a region of 5301 base pairs that has little similarity to the sequence present at the mating-type locus in an a mating-type strain. However, the sequences flanking the mating-type locus in the A haploid and a haploid genome are essentially identical. The region of the A mating-type sequence required for expression of the heterokaryon incompatibility and sexual functions has been localized to a single open reading frame (ORF) encoding a polypeptide of 288 amino acids. Sequence analysis of sterile, heterokaryon-compatible mutants reveals frameshift mutations in this same ORF. The putative 288-amino acid product has a region of similarity to the MAT alpha 1 polypeptide of Saccharomyces cerevisiae.

Amino Acid Sequence↗
PubMed1990

Expansion and contraction of the nucleolus organizer region of Neurospora: changes originate in both proximal and distal segments.

Previously we have shown that the nucleolus organizer region (NOR) of Neurospora crassa changes size frequently during the premeiotic portion of the sexual phase. Here, we have investigated whether these changes in size originate only in specific regions of the NOR, or are distributed throughout the NOR. In two special strains of Neurospora, the NOR was divided into proximal and distal segments. In the first, the NOR was divided by a translocation breakpoint and, in the second, the NOR was divided by a meiotic crossover point. The two strains were crossed individually to normal sequence tester strains and the sizes of the proximal and distal segments were followed by pulsed-field gel electrophoresis. The analysis of progeny from both crosses indicates that the events affecting NOR size are not limited to a specific region of the NOR. Additionally, we have obtained evidence that the rDNA of N. crassa can undergo unequal sister chromatid exchange.

Crossing Over, Genetic↗
PubMed1990

Molecular analysis of nuc-1+, a gene controlling phosphorus acquisition in Neurospora crassa.

In response to phosphorus starvation, Neurospora crassa makes several enzymes that are undetectable or barely detectable in phosphate-sufficient cultures. The nuc-1+ gene, whose product regulates the synthesis of these enzymes, was cloned and sequenced. The nuc-1+ gene encodes a protein of 824 amino acids with a predicted molecular weight of 87,429. The amino acid sequence shows homology with two yeast proteins whose functions are analogous to that of the NUC-1 protein. Two nuc-1+ transcripts of 3.2 and 3.0 kilobases were detected; they were present in similar amounts during growth at low or high phosphate concentrations. The nuc-2+ gene encodes a product normally required for NUC-1 function, and yet a nuc-2 mutation can be complemented by overexpression of the nuc-1+ gene. This implies physical interactions between NUC-1 protein and the negative regulatory factor(s) PREG and/or PGOV. Analysis of nuc-2 and nuc-1; nuc-2 strains transformed by the nuc-1+ gene suggests that phosphate directly affects the level or activity of the negative regulatory factor(s) controlling phosphorus acquisition.

Alkaline Phosphatase↗
PubMed1989

Nucleotide sequence of pho-4+, encoding a phosphate-repressible phosphate permease of Neurospora crassa.

The nucleotide (nt) sequence of the Neurospora crassa pho-4+ gene, which encodes a phosphate-repressible phosphate permease, has been determined. The gene specifies a protein of 590 amino acids (aa) and contains two introns. Two RNA transcripts of 3.3 and 2.4 kb have been identified, and transcription start points (tsp) and termination sites and/or processing sites have been located. The 3.3-kb message is initiated about 890 nt upstream from the tsp for the 2.4-kb transcript. A hydropathy profile of the aa sequence suggests ten to twelve membrane-spanning helices with a large hydrophilic domain between the eighth and ninth helices. This model for the predicted secondary structure of the protein is very similar to models proposed for other sequenced integral membrane proteins from both prokaryotes and eukaryotes. Since very few permease-encoding genes of eukaryotes have been examined in molecular detail, it will be of interest to compare the sequence of pho-4+ with those encoding other anion transport proteins, as they become available.

Amino Acid Sequence↗
PubMed1989

Premeiotic change of nucleolus organizer size in Neurospora.

We have investigated the heritability of nucleolus organizer region (NOR) size in Neurospora crassa. By pulsed-field gel electrophoresis, we followed in genetic crosses the size of the normal or "terminal" NORs and the size of a small interstitial NOR. Tetrad analysis revealed that changes in NOR size occur frequently in the sexual phase. Moreover, most size changes occurred in the period between fertilization and meiosis, although some changes occurred during and after meiosis. Unexpectedly, increases and decreases in NOR size were not equally frequent: decreases were more common. The NOR size changes generated during meiosis were not the result of unequal crossing over between NORs on homologous chromosomes.

Autoradiography↗
PubMed1989

Nuclear gene for mitochondrial leucyl-tRNA synthetase of Neurospora crassa: isolation, sequence, chromosomal mapping, and evidence that the leu-5 locus specifies structural information.

We have isolated and characterized the nuclear gene for the mitochondrial leucyl-tRNA synthetase (LeuRS) of Neurospora crassa and have established that a defect in this structural gene is responsible for the leu-5 phenotype. We have purified mitochondrial LeuRS protein, determined its N-terminal sequence, and used this sequence information to identify and isolate a full-length genomic DNA clone. The 3.7-kilobase-pair region representing the structural gene and flanking regions has been sequenced. The 5' ends of the mRNA were mapped by S1 nuclease protection, and the 3' ends were determined from the sequence of cDNA clones. The gene contains a single short intron, 60 base pairs long. The methionine-initiated open reading frame specifies a 52-amino-acid mitochondrial targeting sequence followed by a 942-amino-acid protein. Restriction fragment length polymorphism analyses mapped the mitochondrial LeuRS structural gene to linkage group V, exactly where the leu-5 mutation had been mapped before. We show that the leu-5 strain has a defect in the structural gene for mitochondrial LeuRS by restoring growth under restrictive conditions for this strain after transformation with a wild-type copy of the mitochondrial LeuRS gene. We have cloned the mutant allele present in the leu-5 strain and identified the defect as being due to a Thr-to-Pro change in mitochondrial LeuRS. Finally, we have used immunoblotting to show that despite the apparent lack of mitochondrial LeuRS activity in leu-5 extracts, the leu-5 strain contains levels of mitochondrial LeuRS protein to similar to those of the wild-type strain.

Amino Acid Sequence↗
PubMed1989

Cytochrome oxidase subunit V gene of Neurospora crassa: DNA sequences, chromosomal mapping, and evidence that the cya-4 locus specifies the structural gene for subunit V.

The sequences of cDNA and genomic DNA clones for Neurospora cytochrome oxidase subunit V show that the protein is synthesized as a 171-amino-acid precursor containing a 27-amino-acid N-terminal extension. The subunit V protein sequence is 34% identical to that of Saccharomyces cerevisiae subunit V; these proteins, as well as the corresponding bovine subunit, subunit IV, contain a single hydrophobic domain which most likely spans the inner mitochondrial membrane. The Neurospora crassa subunit V gene (cox5) contains two introns, 398 and 68 nucleotides long, which share the conserved intron boundaries 5'GTRNGT...CAG3' and the internal consensus sequence ACTRACA. Two short sequences, YGCCAG and YCCGTTY, are repeated four times each in the cox5 gene upstream of the mRNA 5' termini. The cox5 mRNA 5' ends are heterogeneous, with the major mRNA 5' end located 144 to 147 nucleotides upstream from the translational start site. The mRNA contains a 3'-untranslated region of 186 to 187 nucleotides. Using restriction-fragment-length polymorphism, we mapped the cox5 gene to linkage group IIR, close to the arg-5 locus. Since one of the mutations causing cytochrome oxidase deficiency in N. crassa, cya-4-23, also maps there, we transformed the cya-4-23 strain with the wild-type cox5 gene. In contrast to cya-4-23 cells, which grow slowly, cox5 transformants grew quickly, contained cytochrome oxidase, and had 8- to 11-fold-higher levels of subunit V in their mitochondria. These data suggest (i) that the cya-4 locus in N. crassa specifies structural information for cytochrome oxidase subunit V and (ii) that, in N. crassa, as in S. cerevisiae, deficiencies in the production of nuclearly encoded cytochrome oxidase subunits result in deficiency in cytochrome oxidase activity. Finally, we show that the lower levels of subunit V in cya-4-23 cells are most likely due to substantially reduced levels of translatable subunit V mRNA.

Amino Acid Sequence↗
PubMed1988

DNAs of the two mating-type alleles of Neurospora crassa are highly dissimilar.

The mating-type alleles A and a of Neurospora crassa control mating in the sexual cycle and function in establishing heterokaryon incompatibility in the vegetative cycle. The A and a alleles were cloned, and they were shown to encode both the sexual functions and vegetative incompatibility. The mating-type clones contain nonhomologous DNA segments that are flanked by common DNA sequences. Neurospora crassa and all heterothallic and pseudohomothallic Neurospora species contain a single copy of one mating-type sequence or the other within each haploid genome. The six known self-fertile homothallic isolates contain an A homolog, but only one species also contains a homologous sequences. Homothallism in these species is not due to mating-type switching, as it is in Saccharomyces cerevisiae.

Chromosome Mapping↗
PubMed1988

The structural gene for a phosphorus-repressible phosphate permease in Neurospora crassa can complement a mutation in positive regulatory gene nuc-1.

van+, a gene encoding a phosphorus-repressible phosphate permease, was isolated by its ability to complement nuc-1, a positive regulatory locus that normally regulates van+ expression. This was unexpected because the nuc-1 host already contained a resident van+ gene. Plasmids carrying van+ complemented a nuc-2 mutation as well. Probing of RNA from untransformed wild-type (nuc-1+) and constitutive (nuc-1c) strains by van+ probes indicated that levels of the van+ transcript were subject to control by nuc-1+. Probing of the same RNAs with a cosmid clone, containing approximately 15 kilobases of upstream and downstream DNA, revealed no other detectable phosphorus-regulated transcripts within this 40-kilobase region of the chromosome.

Cloning, Molecular↗
PubMed1986

An upstream signal is required for in vitro transcription of Neurospora 5S RNA genes.

The DNA sequences upstream of the 5S RNA genes in Neurospora crassa are largely different from one another, but share a short consensus sequence located in the segment 29 to 26 nucleotides preceding the transcribed region. Differences among flanking sequences do not appear to affect transcription. Deletion analysis indicates, however, that a DNA segment including the conserved "TATA box" is required for in vitro transcription of Neurospora 5S RNA genes.

Base Sequence↗