Isolation by genetic complementation of two differentially expressed genes for β-isopropylmalate dehydrogenase from Aspergillus niger

Abstract

We have constructed an Aspergillus niger cDNA library with a yeast expression vector. The library DNA complemented a leucine auxotroph of Saccharomyces cerevisiae (strain BWG1-7a) at a frequency of 4x10(-4). Plasmids rescued from the yeast prototrophs also complemented Escherichia coli (strain MC1066) deficient in leucine biosynthesis. Sequence determination of the rescued plasmids revealed two genes for beta-isopropylmalate dehydrogenase, which we called leu2A and leu2B. Genomic-blot analysis suggested that both leu2A and leu2B were derived from single-copy genes. Northern-blot hybridization showed that in nutrient-rich medium a leu2A transcript accumulated during germination and log-phase growth while the leu2B transcript appeared late in the growth phase. In minimal medium, only leu2A expression was greatly stimulated. We examined the codon preference of these two genes. Whereas leu2A shows a bias in codon usage typical of A. niger genes, leu2B does not. These results indicate the presence in A. niger of two highly divergent, differentially regulated, isozymes for beta-isopropylmalate dehydrogenase.

Analysis of the interaction between piD261/Bud32, an evolutionarily conserved protein kinase of Saccharomyces cerevisiae, and the Grx4 glutaredoxin

“…Next, these first-screening positive clones were tested for the activation of the second reporter gene (lacZ) by their ability to generate blue colonies in the presence of X-gal. Library plasmids were extracted from positive yeast clones and separated from bait plasmids by transformation of the MC1066 E. coli strain [17]. These candidate plasmids were reintroduced into the Y190 yeast strain together with the original bait plasmid pGBT9-Bud32, or the pGBT9 plasmid alone, in order to confirm the interaction.…”

Two FgLEU2 Genes with Different Roles in Leucine Biosynthesis and Infection-Related Morphogenesis in Fusarium graminearum

“…cerevisiae , functional analysis showed that disruption of LEU2 resulted in Leu-auxotroph and decreased resistance to acetic acid and to very high levels of ethanol stress of the mutants [7]. Very few documents characterized functions of LEU2 in filamentous fungi except for a relatively ancient and primary research in Aspergillus niger showed that this fungus elaborates two isoenzymes of IPMD and these two encoding genes were differentially expressed [8]. However, the detailed function of these two paralogous LEU2 genes in Leu biosynthesis or other cellular processes was not investigated in A .…”

Novel RING Finger Proteins, Air1p and Air2p, Interact with Hmt1p and Inhibit the Arginine Methylation of Npl3p

“…In total, ϳ7.0 ϫ 10 5 transformants were obtained, in which about 200 colonies grew on synthetic complete plates lacking tryptophan, leucine, and histidine (SC-Trp-Leu-His). As for the colonies, plasmids were recovered and transformed into an Escherichia coli strain MC1066 (29). Using the strain, the activation domain plasmids from the library were isolated by their ability to complement an E. coli leuB mutation as a result of the presence of the plasmid-borne LEU2 gene.…”