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C Yanofsky

Publications and source records attributed to C Yanofsky.

At least 91 records · Page 5Linked to original sources
PubMed1989

Nucleotide sequence of the Neurospora crassa trp-3 gene encoding tryptophan synthetase and comparison of the trp-3 polypeptide with its homologs in Saccharomyces cerevisiae and Escherichia coli.

The complete nucleotide sequence of the Neurospora crassa trp-3 gene-encoding tryptophan synthetase has been determined; we present an analysis of its structure. A comparison of the deduced amino acid sequence of the trp-3 polypeptide with its homologs in Saccharomyces cerevisiae (encoded by the TRP5 gene) and Escherichia coli (encoded by the trpA and trpB genes) shows that the A and B domains (amino acid segments homologous to the trpA and trpB polypeptides, respectively) of the N. crassa and yeast polypeptides are in the same order (NH2-A-B-COOH). This arrangement is the reverse of the gene order characteristic of all prokaryotes that have been examined. N. crassa tryptophan synthetase has strong homology to the yeast TRP5 polypeptide (A domains have 54% identity; B domains have 75% identity), and somewhat weaker homology to the E. coli trpA and trpB polypeptides (A domains have 31% identity; B domains have 50% identity). The two domains of the N. crassa polypeptide are linked by a connector of 54-amino acid residues that has less than 25% identity to the 45-residue connector of the yeast polypeptide, although secondary structure analysis predicts both connectors would be alpha-helical. In contrast to the yeast TRP5 gene, which has no introns, the trp-3 coding region is interrupted by two introns 77 and 71 nucleotides in length. Both introns are located near the 5'-end of the gene and therefore not near the segment encoding the connector.

Amino Acid Sequence↗
PubMed1989

Genes expressed during conidiation in Neurospora crassa: characterization of con-8.

The filamentous fungus Neurospora crassa, by a series of defined changes, differentiates from a mycelium composed of branching hyphae to form dormant spores, called conidia. Several genes of unknown function (con genes) that are preferentially expressed during this period have been cloned. Transcription of these genes has been examined in conidiation-defective mutants, and the results obtained revealed that con-6, con-8, con-10, con-11 and con-13 are most likely to play a unique role during conidiation, con-8 is expressed early during conidial differentiation. Genomic and cDNA sequence analyses with con-8 clones identified one open reading frame, interrupted by two introns, which encodes a weakly acidic 18.4 kDa polypeptide containing 176 amino acid residues, con-8 is unusual in that it is transcribed as two mRNA species, 1.0 and 1.25 kb in length. S1 nuclease mapping and primer extension analyses identify one major initiation site, one major polyadenylation site, and demonstrated the existence of heterogeneity at the messenger's 5' and 3' ends.

Amino Acid Sequence↗
PubMed1989

A morphological and genetic analysis of conidiophore development in Neurospora crassa.

The filamentous fungus Neurospora crassa responds to nutrient deprivation and dessication by producing asexual spores, or conidia. These conidia are derived from differentiated aerial structures called conidiophores. The process of conidiation was analyzed in wild-type and morphological mutants using scanning electron microscopy (SEM) and specific fluorescent probes. The first discernible morphological step of conidiation is the transition from growth by hyphal tip elongation to growth by repeated apical budding, resulting in the formation of chains of proconidia that resemble beads on a string. The initial proconidial chains are morphologically distinct from those that form later and are capable of reverting to hyphal growth, whereas the later chains are committed to conidiation. As the proconidial chains are formed, nuclei migrate into the conidiophore, and cross-walls arise between adjoining proconidia in a series of steps that have been defined by staining with Calcofluor, a fluorescent chitin-binding probe. The chains ultimately disarticulate in several discrete stages into free, morphologically mature conidia. Different conidiation-defective mutants were shown to be blocked at distinct stages in conidiation. Our observations permit us to derive a developmental timeline of conidiation relating the occurrence of morphological changes and the stage blocked in specific mutants.

Gene Expression Regulation↗
PubMed1988

Development of a trpE promoter-strength measuring system and its use in comparison of the trpEDCBA, trpR and aroH promoters.

An expression system was developed for measuring in vivo promoter strength at the single copy level and this system was used to compare the trp, aroH and trpR promoters. This system employs trpE enzyme activity as a measure of promoter strength and lacZ expression for internal copy number reference. Promoter-containing fragments are inserted into a cloning vector and subsequently recombined on to phage lambda by genetic exchange. Single lysogens are then prepared and used in promoter-strength analyses. The strength of several promoters was determined using this system. Among the promoters tested, the Escherichia coli trpEDCBA promoter was the strongest; it was four times more active than the lacUV5 promoter and about ten times stronger than the trpR and aroH promoters. To validate measurement of trpE enzyme activity as an indicator of promoter strength, trpE enzyme activity was compared with the level of trpE mRNA. There was excellent correspondence between the two, suggesting that with this system trpE enzyme activity accurately reflects promoter strength. We also examined a homologous promoter-strength measuring system in which the promoter-cloning plasmid lacked a 104 base-pair DNA spacer that was present immediately downstream from the promoter-cloning site in our preferred system. We found that the spacer was essential; the transcribed region accompanying a cloned promoter apparently affected trpE translational efficiency and/or trpE mRNA stability.

Anthranilate Synthase↗
PubMed1988

Sequence changes preceding a Shine-Dalgarno region influence trpE mRNA translation and decay.

In studies with a trpE promoter-strength measuring system we observed that constructs containing the Escherichia coli trp promoter and its adjacent transcribed region yielded lower levels of trpE protein than were expected. To analyze this observation we introduced mutational changes in the nucleotide sequence preceding the trpE Shine-Dalgarno region and examined their effects on trpE mRNA synthesis, translation and decay. We found that certain deletion, insertion and substitution mutations in the pre-Shine-Dalgarno region caused a two- to fivefold increase in trpE enzyme activity. These increases were accompanied by increases in steady-state levels of trpE mRNA. Pulse-chase analyses of trpE mRNA degradation revealed that the observed steady-state trpE mRNA levels correlated with changes in trpE mRNA stability. These findings are interpreted in terms of alternative models in which the primary effect of mutational changes that elevate trpE expression is to increase trpE mRNA translation, versus increasing trpE mRNA stability.

Anthranilate Synthase↗
PubMed1988

Ribosome release modulates basal level expression of the trp operon of Escherichia coli.

The leader peptide stop codon (UGA) of the Escherichia coli trp operon was replaced by UAA and UAG. The transcriptional behavior of the mutated leader regions in vitro and the extent of transcription termination observed with each in vivo were virtually identical to that of the wild type leader region. Introduction of a release factor 1 (UAA- and UAG-specific) mutation into strains with the different stop codons caused increased termination in strains with UAA and UAG, but not with UGA (in cells grown in the presence of tryptophan). This finding provides evidence for the view that ribosome release from the leader peptide stop codon is an important event in setting the basal level of transcription readthrough at the trp attenuator.

Amino Acid Sequence↗
PubMed1988

trp repressor interactions with the trp aroH and trpR operators. Comparison of repressor binding in vitro and repression in vivo.

Interaction of the Escherichia coli trp repressor with the promoter-operator regions of the trp, aroH and trpR operons was studied in vivo and in vitro. The three operators have similar, but non-identical, sequences; each operator is located in a different segment of its respective promoter. In vivo repression of the three operons was measured using single-copy gene fusions to lacZ. The extent of repression varied from 300-fold for the trp operon, to sixfold for the aroH operon and threefold for the trpR operon. To determine whether differential binding of repressor to the three operators was responsible for the differences in repression observed in vivo, three in vitro binding assays were employed. Restriction-site protection, gel retardation and DNase footprinting analyses revealed that repressor binds to the three operators with almost equal affinity. It was also shown in an in vivo competition assay that repressor binds approximately equally well to each of the three operators. It is proposed that the differential regulation observed in vivo may be due to the different relative locations of the three operators within their respective promoters.

Bacterial Proteins↗
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

Transcription attenuation.

Prokaryotic transcription attenuation mechanisms are described in which different metabolic signals and sensing events are used to regulate transcription termination at sites preceding structural genes. Suggestive eukaryotic examples also are mentioned.

Amino Acids↗
PubMed1988

Increased binding of operator DNA by trp superrepressor EK49.

The mechanism of superrepression by the mutant trp repressor EK49 was examined. This superrepressor has a glutamic acid-to-lysine change at residue 49. The purified EK49 trp repressor was found to have a 10-fold higher affinity than wild type repressor for trp operator DNA. This increased affinity was shown to be due to a decrease in dissociation rate. The binding of trp operator DNA by EK49 trp repressor in the filter binding assay was more sensitive to high salt concentrations than binding by wild type repressor.

Escherichia coli↗
PubMed1988

Formation of heterodimers between wild type and mutant trp aporepressor polypeptides of Escherichia coli.

Availability of the three-dimensional structure of the trp repressor of Escherichia coli and a large group of repressor mutants has permitted the identification and analysis of mutants with substitutions of the amino acid residues that form the tryptophan binding pocket. Mutant aporepressors selected for study were overproduced using a multicopy expression plasmid. Equilibrium dialysis with 14C-tryptophan and purified mutant and wild type aporepressors was employed to determine tryptophan binding constants. The results obtained indicate that replacement of threonine 44 by methionine (TM44) or arginine 84 by histidine (RH84) lowers the affinity for tryptophan approximately two- and four-fold, respectively. Replacement of arginine 54 by histidine (RH84) or glycine 85 by arginine (GR85) results in complete loss of tryptophan binding activity. Purified mutant and wild type aporepressors were used in in vitro heterodimer studies. The trp repressor of E. coli functions as a stable dimer. A large number of trp repressor mutants produces defective repressors that are transdominant to the wild type repressor in vivo. The transdominance presumably results from the formation of inactive or slightly active heterodimers between the mutant and wild type polypeptide subunits. An in vitro assay was developed to detect and measure heterodimer formation. Heterodimer formation was thermally induced, and heterodimers were separated on nondenaturing polyacrylamide gels. Aporepressors readily formed heterodimers upon treatment at 65 degrees C for 3 minutes. Heterodimer formation was significantly retarded by the presence of the corepressor, L-tryptophan. Indole-3-propionic acid, 5-methyl tryptophan, and other analogs of tryptophan, as well as indole, also inhibited heterodimer formation.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1988

The cross-pathway control gene of Neurospora crassa, cpc-1, encodes a protein similar to GCN4 of yeast and the DNA-binding domain of the oncogene v-jun-encoded protein.

Expression of the gene cpc-1 is required for cross-pathway-mediated regulation of amino acid-biosynthetic genes in Neurospora crassa. We have cloned cpc-1 and present an analysis of its structure and regulation. The cpc-1-encoded transcript contains three open reading frames, two of which are located in the 720-nucleotide leader segment preceding the cpc-1 coding region. The two leader open reading frames, if translated, would produce peptides 20 and 41 residues in length. The deduced amino acid sequence of the cpc-1 polypeptide, CPC1, contains segments similar to the DNA-binding and transcriptional activation domains of GCN4, the major cross-pathway regulatory protein of yeast. The structural and functional similarities of CPC1 and GCN4 proteins suggest that cpc-1 encodes the analogous transcriptional activator of N. crassa. Messenger RNA measurements indicate that cpc-1 is transcriptionally regulated in response to amino acid starvation. The segment of CPC1 similar to the DNA-binding domain of GCN4 also is similar to the DNA-binding domains of the avian sarcoma virus oncogene-encoded v-JUN protein and human c-JUN protein.

Amino Acid Sequence↗
PubMed1988

Second-site revertants of Escherichia coli trp repressor mutants.

Second-site reversion studies were performed with five missense mutants with defects in the trp repressor of Escherichia coli. These mutants were altered throughout the gene. The same unidirectional mutagen used in the isolation of these mutants, hydroxylamine, was used in reversion studies, to increase the likelihood that the revertants obtained would have second-site changes. Most of the second-site revertants were found to have the same amino acid substitutions detected previously as superrepressor changes. These second-site revertant repressors were more active in vivo than their parental mutant repressors, in the presence or absence of exogenous tryptophan. Apparently superrepressor changes at many locations in this protein can act globally to increase the activity of mutant repressors.

Bacterial Proteins↗
PubMed1988

Mutational analysis of the catalytic and feedback sites of the tryptophan-sensitive 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase of Escherichia coli.

The nucleotide sequence of aroH, the structural gene for the tryptophan-sensitive 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase [DAHPS(Trp)], is presented, and the deduced amino acid sequence of AroH is compared with that of the tyrosine-sensitive (AroF) and phenylalanine-sensitive (AroG) DAHPS isoenzymes. The high degree of sequence similarity among the three isoenzymes strongly indicates that they have a common evolutionary origin. In vitro chemical mutagenesis of the cloned aroH gene was used to identify residues and regions of the polypeptide essential for catalytic activity and for tryptophan feedback regulation. Missense mutations leading either to loss of catalytic activity or to feedback resistance were found interspersed throughout the polypeptide, suggesting overlapping catalytic and regulatory sites in DAHPS(Trp). We conclude that the specificity of feedback regulation of the isoenzymes was probably acquired by the duplication and divergent evolution of an ancestral gene, rather than by domain recruitment.

3-Deoxy-7-Phosphoheptulonate Synthase↗
PubMed1988

cis-acting sites in the transcript of the Bacillus subtilis trp operon regulate expression of the operon.

Transcription of the trp operon of Bacillus subtilis is regulated by attenuation. A trpE'-'lacZ gene fusion preceded by the wild-type trp promoter-leader region was used to analyze regulation. Overproduction of the trp leader transcript in trans from a multicopy plasmid caused constitutive expression of the chromosomal trpE'-'lacZ fusion, presumably by titrating a negative regulatory factor encoded by the mtr locus. Subsegments of the trp leader region cloned onto the multicopy plasmid were examined for their abilities to elevate beta-galactosidase activity. An RNA segment spanning the portion of the leader transcript that forms the promoter-proximal strand of the proposed antiterminator structure was most active in this trans test. The data suggest that the mtr gene product, when activated by tryptophan, binds to this RNA segment and prevents formation of the antiterminator. In this manner, the trans-acting factor promotes formation of the RNA structure that causes transcription termination. Secondary-structure predictions for the leader segment of the trp operon transcript suggest that if the mtr factor bound this RNA segment in a nonterminated transcript, the ribosome-binding site for the first structural gene, trpE, could be sequestered in a stable RNA structure. We tested this possibility by comparing transcriptional and translational fusions containing the initial segments of the trp operon. Our findings suggest that the mtr product causes both transcription attenuation and inhibition of translation of trpE mRNA. Inhibition of translation initiation would reduce ribosome density on trpE mRNA, perhaps making it more labile. Consistent with this interpretation, the addition of tryptophan to mtr+ cultures increased the rate of trpE'-'lacZ mRNA decay.

Bacillus subtilis↗
PubMed1988

An electrophoretic karyotype of Neurospora crassa.

A molecular karyotype of Neurospora crassa was obtained by using an alternating-field gel electrophoresis system which employs contour-clamped homogeneous electric fields. The migration of all seven N. crassa chromosomal DNAs was defined, and five of the seven molecules were separated from one another. The estimated sizes of these molecules, based on their migration relative to Schizosaccharomyces pombe chromosomal DNA molecules, are 4 to 12.6 megabases. The seven linkage groups were correlated with specific chromosomal DNA bands by hybridizing transfers of contour-clamped homogeneous electric field gels with radioactive probes specific to each linkage group. The mobilities of minichromosomal DNAs generated from translocation strains were also examined. The methods used for preparation of chromosomal DNA molecules and the conditions for their separation should be applicable to other filamentous fungi.

Chromosomes↗