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D J Ebbole

Publications and source records attributed to D J Ebbole.

26 records · Page 2Linked to original sources
PubMed1992

cot-1, a gene required for hyphal elongation in Neurospora crassa, encodes a protein kinase.

Neurospora crassa is a filamentous fungus that grows on semisolid media by forming spreading colonies. Mutations at several loci prevent this spreading growth. cot-1 is a temperature sensitive mutant of N.crassa that exhibits restricted colonial growth. At temperatures above 32 degrees C colonies are compact while at lower temperatures growth is indistinguishable from that of the wild type. Restricted colonial growth is due to a defect in hyphal tip elongation and a concomitant increase in hyphal branching. We have isolated a genomic cosmid clone containing the wild type allele of cot-1 by complementation. Sequence analyses suggested that cot-1 encodes a member of the cAMP-dependent protein kinase family. Strains in which we disrupted cot-1 are viable but display restricted colonial growth. Duplication, by ectopic integration of a promoter-containing fragment which includes the first one-third (209 codons) of the structural gene, unexpectedly resulted in restricted colonial growth. Our results suggest that an active COT1 kinase is required for one or more events essential for hyphal elongation.

Amino Acid Sequence↗
PubMed1991

cpc-1, the general regulatory gene for genes of amino acid biosynthesis in Neurospora crassa, is differentially expressed during the asexual life cycle.

CPCI, the principal regulatory protein required for cross-pathway control of amino acid biosynthetic genes in Neurospora crassa, contains a domain similar to the DNA-binding domain of GCN4, the corresponding general regulator in Saccharomyces cerevisiae. We examined binding by CPC1 synthesized in vitro and by CPC1 present in N. crassa whole-cell extracts. CPCI from both sources was shown to bind to the DNA sequence 5'-ATGACTCAT-3', which is also the preferred recognition sequence of GCN4, CPC1 was confirmed as the source of DNA-binding activity in extracts by immunoblotting. Slightly mobility differences between DNA complexes containing CPCI synthesized in vitro and CPC1 in mycelial extracts were observed. Analyses of N. crassa extracts from different stages of asexual development revealed that CPC1 was abundant immediately following spore germination and through early mycelial growth but was scarce subsequently. CPC1 levels could be increased at any time by imposing amino acid starvation. Copies of the CPC1 response element are located upstream of several genes regulated by cross-pathway control, including cpc-1 itself.

Amino Acids↗
PubMed1989

Interaction of a putative repressor protein with an extended control region of the Bacillus subtilis pur operon.

Transcription of the Bacillus subtilis pur operon is regulated independently by adenine and guanine levels in the growth medium. Transcription initiation is regulated by adenine, transcription attenuation by guanine (Ebbole, D. J., and Zalkin, H. (1987) J. Biol. Chem. 262, 8274-8287). A purC-lacZ fusion was constructed to monitor gene expression. Expression was reduced 11- and 18-fold by excess adenine and guanine, respectively. We have characterized the mechanism for the adenine-mediated control of transcription initiation. Deletions of DNA between nucleotides -193 and -37, relative to the start of transcription, resulted in high level constitutive expression and thus identified a control site for negative regulation. A candidate for a pur operon repressor protein was identified in a cell extract and was partially purified. This protein interacts specifically with an extended 5' flanking region of the pur operon that includes the control site identified by deletion analysis. DNase I footprinting defined the region of DNA-protein interaction between nucleotides -145 and -29. The protein-DNA interaction was not dependent on adenine, adenosine, or adenine nucleotides in vitro. These results suggest that a complex interaction of protein with an extended control region may repress transcription by blocking the -35 promoter site. This protein-DNA interaction is unlike that which regulates pur gene expression in Escherichia coli.

Adenine↗
PubMed1989

Bacillus subtilis pur operon expression and regulation.

The Bacillus subtilis pur operon is a 12-gene cluster, purEKB-purC(orf)QLF-purMNH(J)-purD, organized in groups of overlapping coding units separated by intercistronic gaps. Translational fusions of Escherichia coli lacZ were constructed to purE, purC, and purM, the first gene of each group. Analyses of gene fusions integrated into the chromosomal pur operon exclude the possibility of internal promoters in intercistronic regions and support the view that transcription is from the single sigma 43 promoter at the 5' end of the operon. Enzyme and mRNA measurements indicate that transcriptional regulation occurs solely at the 5' end of the operon. The relative levels of beta-galactosidase from purE-lacZ, purC-lacZ, and purM-lacZ were determined under repressing and nonrepressing conditions. These results indicate that expression of purC-lacZ was 3.0- to 6.8-fold higher than purE-lacZ because of enhanced translational efficiency. The enhanced translational efficiency of purC-lacZ was accompanied by a partial escape from regulation by purines. This anomalous effect on purC-lacZ was the only suggestion for posttranscriptional regulation.

Bacillus subtilis↗
PubMed1988

Detection of pur operon-attenuated mRNA and accumulated degradation intermediates in Bacillus subtilis.

Transcription of the Bacillus subtilis pur operon is regulated independently by adenine and guanine nucleotides (Ebbole, D. J., and Zalkin, H. (1987) J. Biol. Chem. 262, 8274-8287). Guanine nucleotides regulate transcription by a termination-antitermination mechanism in a 242-nucleotide, 5'-untranslated mRNA leader region. We have identified an apparently intact, terminated transcript of approximately 200 nucleotides in length, having a half-life of about 0.7 min. The terminated transcript is degraded in a series of discrete steps resulting in the accumulation of stable intermediates in vivo. We have used Northern blot analysis, primer extension, and nuclease S1 mapping to align the degradation intermediates with the nucleotide sequence and assign secondary structures that may contribute to the stability of the intermediates. Degradation is initiated by endonucleolytic cleavage of the approximately 200-nucleotide terminated transcript generating approximately 93- and approximately 97-nucleotide 5' and 3' moieties, respectively. The approximately 93-nucleotide 5' and approximately 97-nucleotide 3' intermediates are further degraded to approximately 88 and approximately 58 nucleotides, respectively. The 5'-end of pur operon mRNA and the attenuated transcript are degraded by different pathways.

Bacillus subtilis↗
PubMed1987

Cloning and characterization of a 12-gene cluster from Bacillus subtilis encoding nine enzymes for de novo purine nucleotide synthesis.

An approximately 16-kilobase pair region of the Bacillus subtilis chromosome at 55 degrees containing genes for de novo purine nucleotide synthesis (Piggot, P. J., and Hoch, J. A. (1985) Microbiol. Rev. 49, 158-179) was cloned. The nucleotide sequence of over 13 kilobase pairs indicates that this region contains a cluster of 12 genes, 11 of which encode enzymes that catalyze the 10 reactions for de novo purine nucleotide synthesis from 5-phosphoribosyl 1-pyrophosphate to IMP. The genes were identified by complementation of Escherichia coli pur mutants and by sequence comparisons with homologous enzymes. The cluster is likely an operon and is organized into three groups of overlapping genes followed by the last gene: purEKB-purC(orf)QLF-purMNH(J)-purD. Sequence comparisons provide evidence for homology of monofunctional purine nucleotide biosynthetic enzymes from B. subtilis with the corresponding multifunctional enzymes from yeast and Drosophila. Sequence alignment of the phosphoribosylaminoimidazole carboxylase heterodimer from B. subtilis with the monomeric enzyme from Methanobrevibacter smithii indicates an evolutionary relationship between these two enzymes. S1 nuclease analysis was used to map the mRNA 5' and 3' ends and to estimate levels of mRNA. These experiments indicate that synthesis of purine nucleotides is regulated independently by adenine and guanine nucleotides. Adenine nucleotides regulate transcription initiation. Guanine nucleotides regulate transcription by a termination-antitermination mechanism in a 242-nucleotide 5' untranslated mRNA leader region. Groups of overlapping genes, regulated at least in part by transcription termination-antitermination is likely to be a common theme for genetic organization and regulation of biosynthetic genes in this Gram-positive organism.

Amino Acid Sequence↗
PubMedDate not supplied

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.

Amino Acid Sequence↗