共查询到20条相似文献,搜索用时 15 毫秒
1.
Ian F. Connerton 《Molecular & general genetics : MGG》1990,223(2):319-323
Summary Repeat-induced point mutation (RIP) has been used to generate new mutations in the previously uncharacterised gene for malate synthase in Neurospora crassa. Molecular clones carrying the am (NADP-glutamate dehydrogenase) gene and the malate synthase gene from either N. crassa or Aspergillus nidulans have been introduced into Neurospora as ectopic duplicate copies by transformation, selecting for the am function in a deletion host. A number of meiotic progeny derived from these transformants were unable to use acetate as sole carbon source, yielded no detectable malate synthase activity and demonstrated extensive cytosine methylation of their duplicated sequences. The new locus has been designated acu-9 and has been assigned to linkage group VII. 相似文献
2.
3.
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
4.
5.
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
6.
7.
8.
9.
10.
Celedonio González Nelida Brito George A. Marzluf 《Molecular & general genetics : MGG》1995,249(4):456-464
Nitrate reductase of Neurospora crassa is a complex multi-redox protein composed of two identical subunits, each of which contains three distinct domains, an amino-terminal domain that contains a molybdopterin cofactor, a central heme-containing domain, and a carboxy-terminal domain which binds a flavin and a pyridine nucleotide cofactor. The flavin domain of nitrate reductase appears to have structural and functional similarity to ferredoxin NADPH reductase (FNR). Using the crystal structure of FNR and amino acid identities in numerous nitrate reductases as guides, site-directed mutagenesis was used to replace specific amino acids suspected to be involved in the binding of the flavin or pyridine nucleotide cofactors and thus important for the catalytic function of the flavin domain. Each mutant flavin domain protein was expressed in Escherichia coli and analyzed for NADPH: ferricyanide reductase activity. The effect of each amino acid substitution upon the activity of the complete nitrate reductase reaction was also examined by transforming each manipulated gene into a nit-3
– null mutant of N. crassa. Our results identify amino acid residues which are critical for function of the flavin domain of nitrate reductase and appear to be important for the binding of the flavin or the pyridine nucleotide cofactors. 相似文献
11.
Patricia M. Okamoto Ying-Hui Fu George A. Marzluf 《Molecular & general genetics : MGG》1991,227(2):213-223
Summary The nit-3 gene of the filamentous fungus Neurospora crassa encodes the enzyme nitrate reductase, which catalyzes the first reductive step in the highly regulated nitrate assimilatory pathway. The nucleotide sequence of nit-3 was determined and translates to a protein of 982 amino acid residues with a molecular weight of approximately 108 kDa. Comparison of the deduced nit-3 protein sequence with the nitrate reductase protein sequences of other fungi and higher plants revealed that a significant amount of homology exists, particularly within the three cofactor-binding domains for molybdenum, heme and FAD. The synthesis and turnover of the nit-3 mRNA were also examined and found to occur rapidly and efficiently under changing metabolic conditions. 相似文献
12.
Summary A plasmid construct carrying the hygromycin phosphotransferase (hph) gene fused to the expression elements of the trpC gene of Aspergillus nidulans was used to obtain hygromycin B (Hyg)-resistant transformants of Neurospora crassa. The plasmid does not have any homology with the N. crassa genome. Here we demonstrate that most of the transformants arise from integration of the transforming DNA into only one of the nuclei present in the protoplasts. Furthermore, in most of the transformants the integrated transforming DNA is physically stable after growth of the transformants for about 25 nuclear divisions without Hyg selection, in spite of being present in multiple copies. In transformants carrying only a single insertion, phenotypic expression of the hph gene remains unaltered in conidial isolates obtained withoug Hyg selection. On the other hand, about 40% of transformants harbouring plasmid DNA integrated at more than one location yield conidial isolates showing reversible inactivation of the hph genes. Interestingly, the presence of methylated cytosine residues in the integrated DNA is strongly correlated with the number of plasmid copies. The hph genes are heavily methylated in transformants harbouring multiple copies but not in those harbouring only one copy of the plasmid. Phenotypic expression of the inactive hph genes can be restored by growing the transformants either under Hyg selection pressure or in the presence of 5-azacytidine. In the first case the hph genes are again inactivated when Hyg selection pressure is removed, while the activation of the hph gene by 5-azacytidine gives stable Hygr strains.Dedicated to Dr. T.A. Trautner on the occasion of his 60th birthday 相似文献
13.
The enzyme nitrate reductase, which catalyzes the reduction of nitrate to nitrite, is a multi-redox center homodimeric protein. Each polypeptide subunit is approximately 100 kDa in size and contains three separate domains, one each for a flavin, a heme-iron, and a molybdopterin cofactor. The heme-iron domain of nitrate reductase has homology with the simple redox protein, cytochrome b5, whose crystal structure was used to predict a three-dimensional structure for the heme domain. Two histidine residues have been identified that appear to coordinate the iron of the heme moiety, while other residues may be important in the folding or the function of the heme pocket. Site-directed mutagenesis was employed to obtain mutants that encode nitrate reductase derivatives with eight different single amino acid substitutions within the heme domain, including the two central histidine residues. Replacement of one of these histidines by alanine resulted in a completely nonfunctional enzyme whereas replacement of the other histidine resulted in a stable and functional enzyme with a lower affinity for heme. Certain amino acid substitutions appeared to cause a rapid turnover of the heme domain, whereas other substitutions were tolerated and yielded a stable and fully active enzyme. Three different single amino acid replacements within the heme domain led to a dramatic change in regulation of nitrate reductase synthesis, with significant expression of the enzyme even in the absence of nitrate induction. 相似文献
14.
Valerie-Ann Dumbrava Martin L. Pall 《Biochimica et Biophysica Acta (BBA)/General Subjects》1987,926(3)
UDPglucuronic acid and erythroascorbic acid were identified in extracts of the fungus Neurospora crassa. The concentrations of these two compounds are estimated, in growing wild type N. crassa, to be about 0.10 and 0.28 μmol/ml of cell water, respectively. The pools of these two compounds are regulated by cyclic AMP in Neurospora, both being elevated in the cr-1, adenylate cyclase deficient mutant and both being lowered by exogenous cyclic AMP. The pools of these two compounds are also elevated on nitrogen deprivation. The pools of a large number of other nucleotides are not influenced by cyclic AMP. Possible relationships between the metabolism of UDPglucuronic acid and erythroascorbic acid are discussed. It was found that exogenous cyclic AMP was much more effective in influencing cultures grown at 30–37°C than those grown at 25°C. We suggest that higher temperatures may render Neurospora more permeable to a variety of different compounds. 相似文献
15.
16.
Carl T. Yamashiro Oded Yarden Charles Yanofsky 《Molecular & general genetics : MGG》1992,236(1):121-124
Summary When Neurospora crassa is transformed using a Neurospora gene as the selectable marker, the vegetatively stable transformants obtained cannot be used successfully in a cross because the selectable marker will be inactivated by the process of RIP (repeat-induced point mutation). Introduction of the acetamidase-encoding gene amdS of Aspergillus nidulans into N. crassa by transformation yielded transformants that would grow in minimal medium containing acetamide as a sole nitrogen source. In mitotically stable transformants containing a single copy of the amdS gene, the capacity to utilize acetamide as a sole nitrogen source was maintained in the progeny of a sexual cross. Therefore, the A. nidulans amdS gene is an appropriate dominant selectable marker for use in transformation analyses with N. crassa in which sexual crosses will be subsequently performed. 相似文献
17.
A convenient assay to score repeat-induced point mutation (RIP) inNeurospora employs theerg-3 locus as a mutagenesis target. Using this assay we screened 132 wild-isolatedNeurospora crassa strains for ability to dominantly suppress RIP. RIP was exceptionally inefficient in crosses with the wild isolates Sugartown
(P0854) and Adiopodoume-7 (P4305), thereby suggesting the presence of dominant RIP suppressors in these strains. In other
experiments, we found no evidence for dominant RIP suppression by theSpore killer haplotypesSk-2 andSk-3. 相似文献
18.
19.
Prasanna Bhomkar Chandrama P. Upadhyay Mukesh Saxena Annamalai Muthusamy N. Shiva Prakash Mikhail Pooggin Thomas Hohn Neera B. Sarin 《Molecular breeding : new strategies in plant improvement》2008,22(2):169-181
A reproducible and efficient transformation system utilizing the nodal regions of embryonal axis of blackgram (Vigna mungo L. Hepper) has been established via Agrobacterium tumefaciens. This is a report of genetic transformation of Vigna mungo for value addition of an agronomic trait, wherein the gene of interest, the glyoxalase I driven by a novel constitutive Cestrum yellow leaf curling viral promoter has been transferred for alleviating salt stress. The overexpression of this gene under
the constitutive CaMV 35S promoter had earlier been shown to impart salt, heavy metal and drought stress tolerance in the
model plant, tobacco. Molecular analyses of four independent transgenic lines performed by PCR, Southern and western blot
revealed the stable integration of the transgene in the progeny. The transformation frequency was ca. 2.25% and the time required
for the generation of transgenic plants was 10–11 weeks. Exposure of T1 transgenic plants as well as untransformed control
plants to salt stress (100 mM NaCl) revealed that the transgenic plants survived under salt stress and set seed whereas the
untransformed control plants failed to survive. The higher level of Glyoxalase I activity in transgenic lines was directly
correlated with their ability to withstand salt stress. To the best of our knowledge this is the only report of engineering
abiotic stress tolerance in blackgram.
Prasanna Bhomkar, Chandrama P. Upadhyay are contributed equally.
An erratum to this article can be found at 相似文献
20.
To allow the regulated expression of cloned genes inCandida albicans, a plasmid was constructed using the inducible promoter of theC. albicans MAL2 gene. To demonstrate that theMAL2 promoter could regulate cloned genes placed under its control, a fusion construct was made with the coding sequence of theC. albicans URA3 gene. This plasmid was introduced into a Ura– strain ofC. albicans using the process of restriction enzyme-mediated integration (REMI). This procedure involves the transformation of theBamHI-linearized plasmid in the presence ofBamHI enzyme. The majority of transformants generated contained insertions of the plasmid at chromosomalBamHI sites. All transformants examined were inducible forURA3 expression, which was determined by growth analysis and by measuring the level ofURA3 gene product activity. The Ura+ phenotype of the transformants was stable during growth under nonselective conditions. This system offers the advantages of stable transformation, easy recovery of integrated DNA, and inducible expression of genes inC. albicans.Deceased, December 15, 1995 相似文献