Some unusual mutants of a locus related to the am locus in Neurospora

1968 ◽  
Vol 14 (6) ◽  
pp. 609-615 ◽  
Author(s):  
M. Kapoor ◽  
D. W. Smith
Keyword(s):  
Amino Acids ◽  
Neurospora Crassa ◽  
Glutamate Dehydrogenase ◽  
Growth Pattern ◽  
Specific Activity ◽  
Nutritional Requirement ◽  
Specific Enzyme ◽  
Mycelial Morphology ◽  
Appreciable Increase ◽  
Spontaneous Revertant

A mutation in a gene in Neurospora crassa leading to a semicolonial growth pattern and a deficiency of NADP-specific glutamate dehydrogenase is reported. The NAD-specific enzyme is not affected. The mutants (referred to as am-morphs) do not show any nutritional requirement nor is the growth pattern altered by addition of supplements in the medium. Addition of L-alanine, L-glutamate, and other α-amino acids to the medium does not result in an appreciable increase in specific activity of the NAD-specific glutamate dehydrogenase. This is due to the inability of amino acids to induce the NAD-specific enzyme, a phenomenon known to occur in the familiar am mutants. A spontaneous revertant of one of these mutants shows a mycelial morphology together with a normal glutamate dehydrogenase.The behavior of am-morphs is discussed in relation to other amination-deficient, mycelial mutants.

Catabolite-controlled regulation of glutamate dehydrogenases of Neurospora crassa

1970 ◽  
Vol 16 (1) ◽  
pp. 33-40 ◽  
Author(s):  
M. Kapoor ◽  
A. K. Grover
Keyword(s):  
Neurospora Crassa ◽  
Glutamate Dehydrogenase ◽  
Growth Medium ◽  
Nicotinamide Adenine Dinucleotide ◽  
Nicotinamide Adenine Dinucleotide Phosphate ◽  
Reciprocal Relationship ◽  
The Other ◽  
Nicotinamide Adenine ◽  
Specific Enzyme ◽  
Other Hand

The effect of the presence of catabolites in the growth medium on the synthesis of the two glutamate dehydrogenases of Neurospora crassa is reported. It has been demonstrated that the nicotinamide adenine dinucleotide (NAD) specific glutamate dehydrogenase is subject to repression by sucrose and glucose. Nicotinamide adenine dinucleotide phosphate (NADP) specific glutamate dehydrogenase, on the other hand, is induced by increasing concentrations of the catabolite. These data suggest that a reciprocal relationship exists between these two enzymes during synthesis in the presence of catabolites. Growth in higher concentrations of sucrose led to the formation of two isoenzymes of the NADP-specific enzyme; the second or the minor isozyme is not produced at very low catabolite concentrations. The catabolite effects produced by sucrose are overcome by glutamate, if the latter is incorporated into the growth medium. Glutamate represses both the isozymes of NADP-specific enzyme.

Disruption of the NAD+-specific glutamate dehydrogenase gene of Neurospora crassa by means of the RIP (repeat-induced point mutations) process

1996 ◽  
Vol 74 (1) ◽  
pp. 29-40 ◽  
Author(s):  
Y. Vijayaraghavan ◽  
M. Kapoor
Keyword(s):  
Neurospora Crassa ◽  
Glutamate Dehydrogenase ◽  
Blot Analysis ◽  
Specific Activity ◽  
Point Mutations ◽  
Constitutive Expression ◽  
Coding Sequence ◽  
Protein Levels ◽  
Hybridization Probes ◽  
Flanking Region

The structural gene for the catabolite-repressed, substrate-induced NAD+-specifïc glutamate dehydrogenase (gdh-1) of Neurospora crassa was disrupted using the process of repeat-induced point mutation (RIP). Plasmids containing incomplete copies of the gene, along with selectable markers, were introduced into germinated conidia by electroporation. The sexual progeny of a transformant containing an ectopically integrated copy of a plasmid, harbouring the 5′ flanking region and a part of the coding sequence of gdh-1 DNA, was examined for the occurrence of RIP by (i) Southern blot analysis of the genomic DNA digested with the isoschizomers MboI and Sau3A, (ii) Northern blot analysis of total RNA in cultures subjected to repression and induction conditions for NAD–GDH, (iii) direct assessment of enzymatic activity, and (iv) evaluation of protein levels by Western blot analysis using a polyclonal anti-GDH IgG preparation. Attempts were made at delineating different regions of the gene exhibiting RIP by using 32P-labelled DNA probes, corresponding to (i) the complete gene, (ii) a fragment containing the 5′ flanking region plus two-thirds of the coding sequence, and (iii) the 5′ flanking segment alone. The extent and relative location of RIP, as revealed by these hybridization probes, appeared to correlate with changes in specific activity under repression and derepression conditions. Mutant progeny, thus recovered, included isolates with altered regulatory features, such as constitutive expression, inability to elicit derepression, higher-than-wildtype GDH levels under derepression and inefficient repression.Key words: glutamate dehydrogenase, Neurospora, repeat-induced point mutations, RIP, regulatory mutants.

scholarly journals Isoenzyme pattern of glutamate dehydrogenase as a reflection of nitrogen metabolism in Lupinus albus

2015 ◽  
Vol 46 (2) ◽  
pp. 347-356 ◽  
Author(s):  
Lech Ratajczak ◽  
Wiktoria Ratajczak ◽  
Hanna Mazurowa
Keyword(s):  
Amino Acids ◽  
Glutamate Dehydrogenase ◽  
Nitrogen Metabolism ◽  
Specific Activity ◽  
Lupinus Albus ◽  
Ammonium Salts ◽  
Electrophoretic Separation ◽  
Trophic Conditions ◽  
Separation Pattern ◽  
Isoenzyme Patterns

Glutamate dehydrogenase (L-glutamate: NAD dehydrogenase, EC 1.4. 1.2; GDH) activity and electrophoretic separation pattern of the enzyme were studied. The enzyme was extracted from embryos of <i>Lupinus albus</i> decotyledonized and cultured for 24, 48 and 72 hours in media containing various combinations of saccharose, ammonium chloride, nitrate as well as amino acids: glutamate, aspartate, glutamine and asparagine. The absence of sugar in the medium resulted in an increase of specific activity of GDH, measured by the rate of NADH + H<sup>+</sup> oxidation, and induced formation of new isoenzymes of NAD<sup>+</sup> - dependent GDH. Most significant increase in GDH specific activity and most evident appearance of new isoenzymes in the embryos were noted when sugar was substituted in the medium by any of the mentioned amino acids. Induction of new isoenzymes could also be seen when ammonium salts were pre-sent in the medium. GHD isoenzymatic patterns were obtained in various trophic conditions. It is suggested that the GDH isoenzyme patterns may serve as a nitrogen metabolism index of a tissue from which the enzyme has been extracted.

scholarly journals A multi-enzyme cascade for efficient production of d-p-hydroxyphenylglycine from l-tyrosine

2021 ◽  
Vol 8 (1) ◽  
Author(s):  
Xu Tan ◽  
Sheng Zhang ◽  
Wei Song ◽  
Jia Liu ◽  
Cong Gao ◽  
...  
Keyword(s):  
Amino Acids ◽  
Specific Activity ◽  
Enantiomeric Excess ◽  
Efficient Production ◽  
Enzyme Cascade ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Rotation Strategy ◽  
Rate Limiting

AbstractIn this study, a four-enzyme cascade pathway was developed and reconstructed in vivo for the production of d-p-hydroxyphenylglycine (D-HPG), a valuable intermediate used to produce β-lactam antibiotics and in fine-chemical synthesis, from l-tyrosine. In this pathway, catalytic conversion of the intermediate 4-hydroxyphenylglyoxalate by meso-diaminopimelate dehydrogenase from Corynebacterium glutamicum (CgDAPDH) was identified as the rate-limiting step, followed by application of a mechanism-guided “conformation rotation” strategy to decrease the hydride-transfer distance d(C6HDAP−C4NNADP) and increase CgDAPDH activity. Introduction of the best variant generated by protein engineering (CgDAPDHBC621/D120S/W144S/I169P with 5.32 ± 0.85 U·mg−1 specific activity) into the designed pathway resulted in a D-HPG titer of 42.69 g/L from 50-g/L l-tyrosine in 24 h, with 92.5% conversion, 71.5% isolated yield, and > 99% enantiomeric excess in a 3-L fermenter. This four-enzyme cascade provides an efficient enzymatic approach for the industrial production of D-HPG from cheap amino acids.

scholarly journals Sequence and structure of mtr, an amino acid transport gene of Neurospora crassa

Genome ◽  
1991 ◽  
Vol 34 (4) ◽  
pp. 644-651 ◽  
Author(s):  
Kenneth Koo ◽  
W. Dorsey Stuart
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Neurospora Crassa ◽  
Rna Binding ◽  
Signal Sequence ◽  
Average Length ◽  
Open Reading Frame ◽  
Mrna Transcript ◽  
S1 Nuclease ◽  
Reading Frame

The gene product of the mtr locus of Neurospora crassa is required for the transport of neutral aliphatic and aromatic amino acids via the N system. We have previously cloned three cosmids containing Neurospora DNA that complement the mtr-6(r) mutant allele. The cloned DNAs were tightly linked to restriction fragment length polymorphisms that flank the mtr locus. A 2.9-kbp fragment from one cosmid was subcloned and found to complement the mtr-6(r) allele. Here we report the sequence of the fragment that hybridized to a poly(A)+ mRNA transcript of about 2300 nucleotides. We have identified an 845-bp open reading frame (ORF) having a 59-bp intron as the potential mtr ORF. S1 nuclease analysis of the transcript confirmed the transcript size and the presence of the intron. A second open reading frame was found upstream in the same reading frame as the mtr ORF and appears to be present in the mRNA transcript. The mtr ORF is predicted to encode a 261 amino acid polypeptide with a molecular mass of 28 613 Da. The proposed polypeptide exhibits six potential α-helical transmembrane domains with an average length of 23 amino acids, does not have a signal sequence, and contains amino acid sequence homologous to an RNA binding motif.Key words: sequence, membranes, ribonucleoprotein.

METHODS OF PROTEIN EXTRACTION FROM NEUROSPORA CRASSA

1964 ◽  
Vol 10 (1) ◽  
pp. 29-35 ◽  
Author(s):  
G. J. Stine ◽  
W. N. Strickland ◽  
R. W. Barratt
Keyword(s):  
Glutamic Acid ◽  
Neurospora Crassa ◽  
Total Protein ◽  
Extraction Method ◽  
Protein Extraction ◽  
Specific Activity ◽  
Dry Weight ◽  
Acid Dehydrogenase ◽  
Total Enzyme

Nine methods for disrupting the mycelium of Neurospora crassa have been compared. Protein percentages are calculated per gram dry weight of mycelium. A TPN-specific glutamic acid dehydrogenase was extracted and the efficiency of each extraction method is given as total enzyme extracted and specific activity. In terms of total protein, total enzyme, and practicality of the method, the Hughes Press, the French Press and the Raper–Hyatt Press were found to be the most efficient. The advantages and limitations of each method are considered.

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