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  • 1
    ISSN: 1432-0983
    Keywords: Neurospora transformation ; trp-1 gene
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary Neurospora trp-1 + transformants, obtained by transforming a trp-1 inl strain with plasmid DNA containing the wild type trp1 + gene, were characterized by genetic and Southern blot analyses. The transforming trp-1 gene integrated at or near the resident site in all of the trp-1 + transformants obtained with circular DNA or DNA cut within the trp-1 coding region. The frequency of homologous integration decreased substantially when the donor DNA was cleaved outside the trp-1 coding region. The transformants were very stable mitotically and, in general, also showed meiotic stability. Analysis of trp-1 + transformants obtained with another recipient strain, trp-1 + ga-2 aro-9 inl, showed that homologous integration of donor DNA occurred in only 20% of the transformants, whether circular or linear DNA was used. Thus, the host strain employed for transformation appears to be a major factor in determining the fate of transforming DNA. Southern blot analysis of transformants showed that integration of the transforming DNA at the homologous site occurred by double crossover or gene conversion events rather than by insertion of the entire plasmid DNA. Multiple and apparently non functional integration events were observed in some transformants.
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  • 2
    ISSN: 1432-0983
    Keywords: Neurospora ; Tomato ; Nitrate reductase ; Nitrate regulation ; GATA-binding factor
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary The nit-2 gene of Neurospora crassa encodes a trans-acting regulatory protein that activates the expression of a number of structural genes which code for nitrogen catabolic enzymes, including nitrate reductase. The NIT2 protein contains a Cys2/Cys2-type zinc-finger DNA-binding domain that recognizes promoter regions of the Neurospora nitrogen-related genes. The NIT2 zincfinger domain/β-Gal fusion protein was shown to recognize and bind in a specific manner to two upstream fragments of the nia gene of Lycopersicon esculentum (tomato) in vitro, whereas two mutant NIT2 proteins failed to bind to the same fragments. The dissociation kinetics of the complexes formed between the NIT2 protein and the Neurospora nit-3 and the tomato nia gene promoters were examined; NIT2 binds more strongly to the nit-3 promoter DNA fragment than it does to fragments derived from the plant nitrate reductase gene itself. The observed specificity of the binding suggests the existence of a NIT2-like homolog which regulates the expression of the nitrate assimilation pathway of higher plants.
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  • 3
    ISSN: 1432-0983
    Keywords: Neurospora crassa ; Nitrogen metabolism ; Regulation ; Heterokaryons
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary The nit-2 gene of Neurospora crassa is a major regulatory gene for control of nitrogen metabolism. Synthesis of the enzyme L-amino acid oxidase requires a functional nit-2 gene product and is also controlled by amino acid induction and nitrogen catabolite repression. Electrophoretic variants of L-amino acid oxidase have been employed to demonstrate that in heterokaryons, a nit-2 + gene product can turn on the expression of this enzyme in its own nucleus and also in nuclei that possess a nit-2 mutant. This trans-nuclear effect is only partial since the variant coded for in the nucleus containing the nit-2 mutant allele is always present in lower amounts than the alternative form. Two additional putative nitrogen control genes, MS5 and en(am)1, have been found to have clear effects upon the expression of L-amino acid oxidase. The en(am)1 mutant appears to result in an unusual case of reversal of the control present in wild-type: the enzyme is expressed in a constitutive fashion and inducers, required for enzyme synthesis in wild-type, actually reduce the level of L-amino acid oxidase in en(am)1. The MS5 mutant shows a substantial release from the usual nitrogen catabolite repression exerted by glutamine in wild-type.
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  • 4
    ISSN: 1432-0983
    Keywords: Neurospora ; Transformation ; Plasmids
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary A new, rapid, efficient and reliable method for transforming Neurospora crassa is described. In this procedure, germinated conidia are treated with lithium acetate, then incubated with DNA, followed by exposure to polyethylene glycol and then a brief heat shock, prior to plating on selective medium. Optimal conditions to achieve a high transformation rate are reported. Transformation can be obtained with both circular and linear plasmid DNA and also with genomic DNA. Although the rate is substantially decreased, transformation was also obtained with relatively impure DNA preparations, such as that made via rapid “miniprep” procedures. This transformation technique is simple and reliable and provides a considerable savings in time and materials.
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  • 5
    ISSN: 1432-0983
    Keywords: Plasmid recovery ; Liquid culture ; ars sequence ; Neurospora
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary The efficient recovery of plasmid DNA from Neurospora crassa transformants is described. Lithium acetate-treated spores were transformed with plasmid DNA and grown in mass in liquid culture. The resulting mycelial growth was harvested and plasmid DNA was extracted and used to transform E. coli to ampicillin resistance. Although at low frequency, routine recovery of plasmid pSD3 which carries the Neurospora qa-2 + gene and pBR322 sequences has been demonstrated. About 10% of the recovered plasmids carried deletions and transformed Neurospora at a higher frequency. The liquid culture procedure was also used in attempts to isolate autonomously replicating sequences (ars). In order to select for a stable vector which contains an ars sequence, a clone bank containing a selectable marker (qa-2 +) and Neurospora chromosomal BamHI fragments was constructed and used to transform Neurospora. Several plasmid isolates resulting from a screening of the clone bank showed an improvement in the efficiency of recovery from Neurospora transformants. The properties of one such isolated plasmid, pJP102, suggest that it may contain an ars sequence. Some potential applications of these results for cloning in Neurospora and other filamentous fungi are discussed.
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  • 6
    ISSN: 1432-0983
    Keywords: Amber nonsense mutations ; Regulatory and structural genes ; Neurospora crassa
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary Neurospora crassa possesses a set of nitrogen-regulated enzymes whose expression requires a lifting of nitrogen catabolite repression and specific induction. The nit-2 gene is a major regulatory locus which appears to act in a positive way to turn on the expression of these nitrogen-related enzymes whereas the nit-4 gene appears to mediate nitrate induction of nitrate and nitrite reductase. The nit-3 gene specifies nitrate reductase and is subject to control by both nit-2 and nit-4. Many new nit-2, nit-3, and nit-4 mutants were isolated in order to obtain amber nonsense mutations in these loci which were suppressible by the suppressor gene, Ssu-1. A nit-2 nonsense mutant was isolated which has altered regulatory properties for control of nitrate reductase, L-amino acid oxidase, and uricase, and which may encode a truncated regulatory protein. Four nit-3 nonsense mutations were isolated, each of which completely lacks nitrate reductase activity, which is restored to markedly different levels by suppression with Ssu-1. Studies of heat activation and thermal lability of nitrate reductase suggest a qualitative alteration of the enzyme occurs in two of the Ssu-1 nit-3 strains.
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  • 7
    ISSN: 1432-0983
    Keywords: Neurospora crassa ; RIP ; nmr ; Hygromycin B
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary The repeat induced point mutation (RIP) phenomenon has been used to generate new mutants of nmr, the negative nitrogen regulatory gene in Neurospora crassa. The wild-type nmr gene was cotransformed along with the hygromycin B resistance gene into wild-type cells by selecting for hygromycin B resistance. Following purification of primary transformants using microconidia, crosses to wild-type. Detailed analyses of some of the progeny revealed that we had generated authentic nmr mutants at high frequency. The polymerase chain reaction was used to amplify and clone a fragment of a mutagenized nmr copy from one of the mutants. The nucleotide sequence analysis showed that 14% of the guanine residues have been converted into adenines, resulting in numerous missense and nonsense mutations. The newly created nmr mutants were found suitable for use as host strains in transformation experiments.
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  • 8
    ISSN: 1432-0983
    Keywords: Neurospora crassa ; Nitrate assimilation ; Nitrogen regulation ; nit-4 gene ; Transformation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary nit-4 is a pathway-specific regulatory gene which controls nitrate assimilation in Neurospora crassa, and appears to mediate nitrate induction of nitrate and nitrite reductase. The NIT4 protein consists of 1090 amino-acid residues and possesses a single GAL4-like putative DNA-binding domain plus acidic, glutaminerich, and polyglutamine regions. Several mutants with amino-acid substitutions in the putative DNA-binding domain and a nit-4 deletion mutant, which encodes a truncated NIT4 protein lacking the polyglutamine region, are functional, i.e., they are capable of transforming a nit-4 mutant strain. However, transformants obtained with most of these nit-4 mutant genes possess a markedly reduced level of nitrate reductase and grow only slowly on nitrate, emphasizing the need to examine quantitatively the affects of in vitro-manipulated genes. The possibility that some mutant genes could yield transformants only if multiple copies were integrated was examined. The presence of multiple copies of wild-type or mutant nit-4 genes did not generally lead to increased enzyme activity or growth rate, but instead frequently appeared to be detrimental to nit-4 function. A hybrid nit-4-nirA gene transforms nit-4 mutants but only allows slow growth on nitrate and has a very low level of nitrate reductase.
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  • 9
    ISSN: 1432-0983
    Keywords: Neurospora ; Nitrogen regulation ; NIT2 ; L-amino acid oxidase ; DNA binding
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract NIT2, the major nitrogen regulatory protein of Neurospora crassa mediates nitrogen catabolite derepression of the structural genes which specify enzymes of nitrogen catabolism. The promoter of the structural gene for L-amino acid oxidase, a nitrogen-regulated enzyme, was found to contain two NIT2 binding sites, each with two copies of a GATA core consensus sequence. Site-directed mutagenesis was employed to create amino-acid substitutions within the single zinc-finger region of NIT2, which serves as the DNA-binding domain. The affect of those mutations upon NIT2 function in vivo in the activation of three separate structural genes was examined by transformation assays and relevant enzyme activities, and DNA-binding activity in vitro was determined by gel band mobility-shift assays. It was shown that specific amino-acid residues within the zinc-finger loop region of NIT2 are important for DNA-binding activity, whereas other residues influence the specificity of DNA binding. Mutant NIT2 proteins were obtained which retain DNA-binding activity and alter the specificity of DNA recognition, thus allowing a distinction between related DNA elements.
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  • 10
    ISSN: 1432-0983
    Keywords: Neurospora ; Nitrogen regulation ; NIT4 ; Transactivation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Expression ofnit-3 andnit-6, the structural genes which encode nitrate reductase and nitrite reductase inNeurospora crassa, requires the global-acting NIT2 and the pathway specific NIT4 regulatory proteins. NIT4, which consists of 1090 amino-acid residues, possesses a Cys6/Zn2 zinc cluster DNA-binding-domain. NIT4 was dissected to localize transactivation domains by fusion of various segments of NIT4 to the DNA-binding domain of GAL4 for in vivo analysis in yeast. Three separate activation subdomains, and one negative-acting region, which function in yeast were located in the carboxyl-terminal region of NIT4. The C-terminal tail of 28 amino-acid residues was identified as a minimal activation domain and consists of a novel leucine-rich, acidic region. Most deletions which removed even small segments of the NIT4 protein were found to lead to the loss of NIT4 function in vivo inN. crassa, implying that the central region of the protein which lies between the DNA-binding and activation domains is essential for function. The yeast two-hybrid system was employed to identify regions of NIT4 responsible for dimer formation. A short isoleucine-rich segment downstream from the zinc cluster, predicted to form a coiled coil, allowed dimerization in vivo; this same isoleucine-rich region also showed dimerization in vitro when examined via chemical cross linking. The enzyme nitrate reductase has been postulated to exert autogenous regulation by directly interacting with the NIT4 protein. This possible nitrate reductase-NIT4 interaction was investigated with the yeast two-hybrid system and by direct in vitro binding assays; both assays failed to identify such a protein-protein interaction.
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