Molecular cloning and expression in Saccharomyces cerevisiae and Neurospora crassa of the invertase gene from Neurospora crassa

A plasmid (named pCN2) carrying a 7.6 kb BamHI DNA insert was isolated from a Neurospora crassa genomic library raised in the yeast vector YRp7. Saccharomyces cerevisiae suco and N. crassa inv strains transformed with pNC2 were able to grow on sucrose-based media and expressed invertase activity. Saccharomyces cerevisiae suco (pNC2) expressed a product which immunoreacted with antibody raised against purified invertase from wild type N. crassa, although S. cerevisiae suc+ did not. The cloned DNA hybridized with a 7.6 kb DNA fragment from BamHI-restricted wild type N. crassa DNA. Plasmid pNC2 transformed N. crassa Inv- to Inv+ by integration either near to the endogenous inv locus (40% events) or at other genomic sites (60% events). It appears therefore that the cloned DNA piece encodes the N. crassa invertase enzyme. A 3.8 kb XhoI DNA fragment, derived from pNC2, inserted in YRp7, in both orientation, was able to express invertase activity in yeast, suggesting that it contains an intact invertase gene which is not expressed from a vector promoter.     

Molecular cloning and expression in Saccharomyces cerevisiae and Neurospora crassa of the invertase gene from Neurospora crassa

A plasmid (named pCN2) carrying a 7.6 kb BamHI DNA insert was isolated from a Neurospora crassa genomic library raised in the yeast vector YRp7. Saccharomyces cerevisiae suc ⁰ and N. crassa inv strains transformed with p NC2 were able to grow on sucrose-based media and expressed invertase activity. Saccharomyces cerevisiae suc ⁰ ( p NC2) expressed a product which immunoreacted with antibody raised against purified invertase from wild type N. crassa , although S. cerevisiae suc ⁺ did not. The cloned DNA hybridized with a 7.6 kb DNA fragment from BamHI -restricted wild type N. crassa DNA. Plasmid pNC2 transformed N. crassa Inv^- to Inv⁺ by integration either near to the endogenous inv locus (40% events) or at other genomic sites (60% events). It appears therefore that the cloned DNA piece encodes the N. crassa invertase enzyme. A 3.8 kb XhoI DNA fragment, derived from pNC2, inserted in YRp7, in both orientation, was able to express invertase activity in yeast, suggesting that it contains an intact invertase gene which is not expressed from a vector promoter.     

Molecular cloning and characterization of the cys-3 regulatory gene of Neurospora crassa.

The regulatory gene cys-3+ controls the synthesis of a number of enzymes involved in sulfur metabolism. cys-3 mutants show a multiple loss of enzymes in different pathways of sulfur metabolism. The cys-3+ gene was isolated by transformation of an aro-9 qa-2 cys-3 inl strain with a clone bank followed by screening with the "sib selection" method. The library used (pRAL1) contained inserts of Sau3a partial digest fragments of about 9 kilobases as well as the Neurospora qa-2+ gene. Double selection for qa-2+ and cys-3+ function was carried out. The transformants obtained with the isolated cys-3+ clone show recovery of the enzyme activities associated with the cys-3 mutation (e.g., arylsulfatase and sulfate permease). Restriction fragment length polymorphism experiments confirmed the identity of the clone, mRNA studies with Northern blots show that the expression of the cys-3+ gene is inducible. In contrast to cys-3+, the cys-3 (P22) mutant gene was not expressed at a higher level under sulfur-derepressed conditions.     

Molecular cloning of nit-2, a regulatory gene required for nitrogen metabolite repression in Neurospora crassa

We used an efficient sib-selection procedure to isolate a cosmid clone that complemented a mutated nit-2 gene of Neurospora crassa. Restriction fragment length polymorphism mapping indicated that the cosmid DNA insert was derived from linkage group IL, between 5S rDNA locus 12 and mt, the region of the N. crassa genome that contains nit-2. We conclude that the cosmid carries the nit-2 gene.     

Molecular cloning and characterization of the polyphenol oxidase gene from sweetpotato

Polyphenol oxidase is the enzyme responsible for enzymatic browning in sweetpotato that decreases the commercial value of sweetpotato products. Here we reported the cloning and characterization of a new cDNA encoding PPO from sweetpotato, designated as IbPPO (GeneBank accession number: AY822711). The full-length cDNA of IbPPO is 1984 bp with a 1767 bp open reading frame (ORF) encoding a 588 amino acid polypeptide with a calculated molecular weight of 65.7 kDa and theoretical pI of 6.28. The coding sequence of IbPPO was also directly amplified from the genomic DNA of sweetpotato that demonstrated that IbPPO was an intron-free gene. The computational comparative analysis revealed that IbPPO showed homology to other PPOs of plant origin and contained a 50 amino acid plastidial transit peptide at its N-terminal and the two conserved CuA and CuB copper-binding motifs in the catalytic region of IbPPO. A highly conserved serine-rich motif was firstly found in the transit peptides of plant PPO enzymes. Then the homology based structural modeling of IbPPO showed that IbPPO had the typical structure of PPO: the catalytic copper center was accommodated in a central four-helix bundle located in a hydrophobic pocket close to the surface. Finally, the results of the semiquantitative RT-PCR analysis of IbPPO in different tissues demonstrated that IbPPO could express in all the organs of sweetpotato including mature leaves, young leaves, the stems of mature leaves (petioles), the storage roots, and the veins but at different levels. The highest-level expression of IbPPO was found in the veins, followed by storage roots, young leaves and mature leaves; and the lowest-level expression of IbPPO was found in petioles. The present researches will facilitate the development of antibrown sweetpotato by genetic engineering. Published in Russian in Molekulyarnaya Biologiya, 2006, Vol. 40, No. 6, pp. 1006–1012. The article was submitted by the authors in English.