共查询到20条相似文献,搜索用时 0 毫秒
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S. Ito Y. Matsui A. Toh-e T. Harashima H. Inoue 《Molecular genetics and genomics : MGG》1997,255(4):429-437
Genes belonging to the ras superfamily encode low-molecular-weight GTP/GDP-binding proteins that are highly conserved in wide variety of organisms. We used the polymerase chain reaction (PCR) to isolate a novel member of the ras superfamily from the filamentous fungus Neurospora crassa and obtained a mammalian Krev-1 homolog. We named the gene krev-1 and analyzed its structure and function. The krev-1 gene encodes a polypeptide of 225 amino acids, which is nearly 60% homologous to the mammalian Krev-1 p21. The krev-1 gene product (KREV1) is functionally analogous to mammalian Krev-1 p21 and Rsr1p/Bud1p, a Krev-1 homolog from the yeast Saccharomyces cerevisiae. GAL1-driven expression of KREV1 in a wild-type yeast strain resulted in a random budding pattern, as did its mammalian counterpart Krev-1 p21. We disrupted the krev-1 gene by RIP (repeat-induced point mutation), but the krev-1 disruptants showed no abnormalities. By in vitro mutagenesis, we constructed several mutant krev-1 genes (G21V, A68T, and D128A) which mimic constitutively active mutants of Ha-ras, and the krev-1 (K25N) mutant which is analogous to a dominant-negative mutant of Ha-ras. Each mutant gene was introduced into the wild-type strain and the phenotypes were analyzed. We could not observe any difference in vegetative growth between these transformants. When each strain was used as the female in mating tests, the development of perithecia from protoperithecia was inhibited in all cases. The results indicate that the krev-1 gene may be involved in sexual cycle progression. 相似文献
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G. Stauder H. Riesemann W.M. Joester K.E. Joester 《Biochimica et Biophysica Acta (BBA) - Gene Structure and Expression》1983,741(3)
A third DNA polymerase ‘C’ with low molecular weight was isolated and purified 3700-fold from ground hyphae of Neurospora crassa WT 74 A, which shows similarities to β- and γ-polymerases from higher eukaryotes: preference for poly(rA)(dT) as a template/primer, inhibition by p-chloromercuribenzoate, resistance against N-ethylmaleimide up to 10 mmol/l, and molecular weight of about 40 000. This polymerase elutes as a distinct peak from DEAE-cellulose at 0.60 mol/l KCl and has an optimum for K+ at 2–20 mmol/l, for Mn2+ at 0.8 mmol/l, for Mg2+ at 4.0 mmol/l, the pH optimum is 8.0. Its Km is 1.5 μmol/l using dTTP as substrate. The enzyme activity described here is free of endonuclease but contains detectable amounts of exonuclease. 相似文献
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Multiple GATA factors – regulatory proteins with consensus zinc finger motifs that bind to DNA elements containing a GATA
core sequence – exist in the filamentous fungus Neurospora crassa. One GATA factor, NIT2, controls nitrogen metabolism, whereas two others, WC-1 and WC-2, regulate genes responsive to blue
light induction. A gene encoding a new GATA factor, named SRE, was isolated from Neurospora using a PCR-mediated method. Sequence analysis of the new GATA factor gene revealed an ORF specifying 587 amino acids, which
is interrupted by two small introns. Unlike all previously known Neurospora GATA factors, which possess a single zinc-finger DNA-binding motif, SRE contains two GATA-type zinc fingers. The deduced
amino acid sequence of SRE shows significant similarity to URBS1 of Ustilago and SREP of Penicillium. A loss-of-function mutation was created by the RIP procedure. Analysis of sre
+ and sre
− strains revealed that SRE acts as a negative regulator of iron uptake in Neurospora by controlling the synthesis of siderophores. Siderophore biosynthesis is repressed by high iron concentrations in the wild-type
strain but not in sre
− mutant cells. The sre promoter contains a number of GATA sequences; however, expression of sre mRNA occurs in a constitutive fashion and is not regulated by the concentration of iron available to the cells.
Received: 20 January 1998 / Accepted: 23 April 1998 相似文献
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Mutants that show reduced DNA methylation were identified in a mutant screen based on the assumptions that (i) the nucleoside
analog 5-azacytidine (5-azaC) promotes the formation of potentially lethal DNA-methyltransferase adducts; (ii) reduction in
DNA methyltransferase will decrease the sensitivity of cells to 5-azaC; and (iii) this potential selective advantage will
be enhanced in mutants that are deficient in the repair of 5-azaC-induced DNA damage. Of fifteen potential repair mutants
screened for sensitivity to 5-azaC, five (mus-9, mus-10, mus-11, mus-18, and uvs-3) showed moderately increased sensitivity and two (mus-20, mei-3) showed highly increased sensitivity. A mus-20 mutation was used to isolate three non-complementing methylation mutants. The mutations, named dim-1 (defective in methylation), reduced female fertility, reduced methylation by 40–50%, and altered patterns of methylation. In wild-type strains
hypomethylation perse fails to alter methylation specificity. We demonstrate a growth-phase-dependent change in methylation patterns, detectable
only in hypomethylated DNA from dim
+ cultures. This may represent a growth-phase-dependent change in the relative amounts of distinct species of methyltransferase,
one of which may be encoded by the dim-1 gene.
Received: 3 January 1998 / Accepted: 26 March 1998 相似文献
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H. Prokisch O. Yarden M. Dieminger M. Tropschug I. B. Barthelmess 《Molecular & general genetics : MGG》1997,256(2):104-114
The function of Neurospora crassa calcineurin was investigated in N. crassa strains transformed with a construct that provides for the inducible expression of antisense RNA for the catalytic subunit
of calcineurin (cna-1). Induction of antisense RNA expression was associated with reduced levels of cna-1 mRNA and of immunodetectable CNA1 protein and decreased calcineurin enzyme activity, indicating that a conditional reduction
of the target function had been achieved in antisense transformants with multiple construct integrations. Induction conditions
caused growth arrest which indicated that the cna-1 gene is essential for growth of N. crassa. Growth arrest was preceded by an increase in hyphal branching, changes in hyphal morphology and concomitant loss of the
distinctive tip-high Ca2+ gradient typical for growing wild-type hyphae. This demonstrates a novel and specific role for calcineurin in the precise
regulation of apical growth, a common form of cellular proliferation. In vitro inhibition of N. crassa calcineurin by the complex of cyclosporin A (CsA) and cyclophilin20, and increased sensitivity of the induced transformants
to the calcineurin-specific drugs CsA and FK506 imply that the drugs act in N. crassa, as in T-cells and Saccharomyces cerevisiae, by inactivating calcineurin. The finding that exposure of growing wild-type mycelium to these drugs leads to a phenotype
very similar to that of the cna-1 antisense mutants is consistent with this idea.
Received: 18 February 1997 / Accepted: 20 April 1997 相似文献
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As a first step towards understanding the process of blue light perception, and the signal transduction mechanisms involved,
in Neurospora crassa we have used a pharmacological approach to screen a wide range of second messengers and chemical compounds known to interfere
with the activity of well-known signal transducing molecules in vivo. We tested the influence of these compounds on the induction
of the al-3 gene, a key step in light-induced carotenoid biosynthesis. This approach has implicated protein kinase C (PKC) as a component
of the light transduction machinery. The conclusion is based on the effects of specific inhibitors (calphostin C and chelerythrine
chloride) and activators of PKC (1,2-dihexanoyl-sn-glycerol). During vegetative growth PKC may be responsible for desensitization to light because inhibitors of the enzyme
cause an increase in the total amount of mRNA transcribed after illumination. PKC is therefore proposed here to be an important
regulator of transduction of the blue light signal, and may act through modification of the protein White Collar-1, which
we show to be a substrate for PKC in N. crassa.
Received: 4 December 1998 / Accepted: 21 May 1999 相似文献
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Summary Purrtins can be utilized as a secondary nitrogen source by Neurospora crassa during conditions of nitrogen limitation. The expression of purine catabolic enzymes is governed by the nitrogen regulatory circuit and requires induction by uric acid. The major positive-acting nitrogen regulatory gene, nit-2, turns on the expression of the purine catabolic enzymes, which may also be subject to negative regulation by a second control gene, nmr. We have cloned alc, the structural gene which encodes allantoicase, an inducible enzyme of the purine degradative pathway. The identity of the alc clone was confirmed by restriction fragment length polymorphism analysis and by repeat-induced mutation. The alc gene is transcribed to give a single messenger RNA, approximately 1.2 kb in length. The negative-acting nmr gene affects the expression of alc in the expected manner. Both the nit-2 and the nmr control genes affect alc mRNA levels and allantoicase enzyme activity in both the induced and nitrogen-repressed conditions. 相似文献
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The period length of the circadian conidiation rhythm was examined in a mutant strain of Neurospora crassa, un-18, that is temperature sensitive for mycelial growth. The un-18 mutant showed a temperature-sensitive phenotype with respect to both mycelial growth and the period length of the conidiation
rhythm. Below 22° C, the un-18 mutation did not affect the period length, but at temperatures between 22° C and 32° C, the period length of the un-18 mutant was ∼2 h longer than that of the wild-type strain. The un-18
+ gene was cloned and was found to encode the second-largest subunit of RNA polymerase I, which is involved in the synthesis
of rRNA. These results indicate that a defect in ribosome synthesis, which must result in a lower rate of protein synthesis,
lengthens the period of the circadian conidiation rhythm in Neurospora.
Received: 17 October 1997 / Accepted: 26 April 1998 相似文献
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A. Zsindely Kiss M. Schablik M. Szabolcs G. Szab 《Biochimica et Biophysica Acta (BBA) - Gene Structure and Expression》1983,741(3):273-278
The regulatory effect of inositol on inositol-1-phosphate synthase in Neurospora crassa strains was studied. Inositol represses enzyme production in the cultures of the wild type and that of the thermosensitive inositol-requiring mutant grown at 22°C. Enzyme activity as well as the quantity of enzyme protein decreased sharply in both strains by increasing concentrations of inositol in the medium. Inositol-requiring strains used in our experiments can be divided into two groups. The first group produces a protein related immunologically to inositol phosphate synthase, but which is enzymatically inactive. The synthesis of this defective enzyme was also repressed by inositol. In the second group, this protein was found to be completely lacking, in both the thermosensitive mutant grown at 37°C, and in a strain requiring inositol due to a reciprocal translocation. The thermostability and the cross immunoelectrophoresis of the enzyme suggest that in the case of the thermosensitive inositol-requiring mutant, the mutation did not occur in the structural gene of the enzyme, but its regulation was probably affected. 相似文献
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Patricia M. Okamoto Reginald H. Garrett George A. Marzluf 《Molecular & general genetics : MGG》1993,238(1-2):81-90
Nitrate reductase of Neurospora crassa is a dimeric protein composed of two identical subunits, each possessing three separate domains, with flavin, heme, and molybdenum-containing cofactors. A number of mutants of nit-3, the structural gene that encodes Neurospora nitrate reductase, have been characterized at the molecular level. Amber nonsense mutants of nit-3 were found to possess a truncated protein detected by a specific antibody, whereas Ssu-1-suppressed nonsense mutants showed restoration of the wild-type, full-length nitrate reductase monomer. The mutants show constitutive expression of the truncated nitrate reductase protein; however normal control, which requires nitrate induction, was restored in the suppressed mutant strains. Three conventional nit-3 mutants were isolated by the polymerase chain reaction and sequenced; two of these mutants were due to the deletion of a single base in the coding region for the flavin domain, the third mutant was a nonsense mutation within the amino-terminal molybdenum-containing domain. Homologous recombination was shown to occur when a deleted nit-3 gene was introduced by transformation into a host strain with a single point mutation in the resident nit-3 gene. New, severely damaged, null nit-3 mutants were created by repeat-induced point mutation and demonstrated to be useful as host strains for transformation experiments. 相似文献
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