Aminoadipate reductase gene: a new fungal-specific gene for comparative evolutionary analyses

BACKGROUND: In fungi, aminoadipate reductase converts 2-aminoadipate to 2-aminoadipate 6-semialdehyde. However, other organisms have no homologue to the aminoadipate reductase gene and this pathway appears to be restricted to fungi. In this study, we designed degenerate primers for polymerase chain reaction (PCR) amplification of a large fragment of the aminoadipate reductase gene for divergent fungi. RESULTS: Using these primers, we amplified DNA fragments from the archiascomycetous yeast Saitoella complicata and the black-koji mold Aspergillus awamori. Based on an alignment of the deduced amino acid sequences, we constructed phylogenetic trees. These trees are consistent with current ascomycete systematics and demonstrate the potential utility of the aminoadipete reductase gene for phylogenetic analyses of fungi. CONCLUSIONS: We believe that the comparison of aminoadipate reductase among species will be useful for molecular ecological and evolutionary studies of fungi, because this enzyme-encoding gene is a fungal-specific gene and generally appears to be single copy

Molecular evolution of adenylating domain of aminoadipate reductase

BACKGROUND: Aminoadipate reductase (Lys2) is a fungal-specific protein. This enzyme contains an adenylating domain. A similar primary structure can be found in some bacterial antibiotic/peptide synthetases. In this study, we aimed to determine which bacterial adenylating domain is most closely related to Lys2. In addition, we analyzed the substitution rate of the adenylating domain-encoding region. RESULTS: Some bacterial proteins contain more than two similar sequences to that of the adenylating domain of Lys2. We compared 67 amino acid sequences from 37 bacterial and 10 fungal proteins. Phylogenetic trees revealed that the lys2 genes are monophyletic; on the other hand, bacterial antibiotic/peptide synthase genes were not found to be monophyletic. Comparative phylogenetic studies among closely related fungal lys2 genes showed that the rate of insertion/deletion in these genes was lower and the nucleotide substitution rate was higher than that in the internal transcribed spacer (ITS) regions. CONCLUSIONS: The lys2 gene is one of the most useful tools for revealing the phylogenetic relationships among fungi, due to its low insertion/deletion rate and its high substitution rate. Lys2 is most closely related to certain bacterial antibiotic/peptide synthetases, but a common ancestor of Lys2 and these synthetases evolutionarily branched off in the distant past

ジツヨウ コウボ SACCHAROMYCES オヨビ SACCHAROMYCES SENSU STRICTOニ オケル 2μ DNA ノ ブンプ

醸造酵母は実用面から見ると清酒酵母,ワイン酵母,ビール酵母などに分けられる。これらの酵母の多くはSaccharomyces cerevisiaeに属している。このS. cerevisiaeは近年遺伝子工学分野で頻繁に使用されている2μ DNAプラスミドを保持している。本研究では実用酵母としての観点から見た2μ DNAの保持について調べた。清酒酵母については,醸造協会保存の17株,ATCC保存の10株,長期間保存されている7株(IFO及びNI株),大手酒造工場から分離された22株,竹田らによって分離された93株,自然界から分離された43株の合計192株の全てが2μ DNAを保持していなかった。また,泡盛酵母も同様に15株全てが保持していなかった。ワイン酵母は55株中34株,パン酵母は20株中17株,ビール酵母は14株中13株が保持しており,高い保持率を示した。焼酎酵母は30株中10株で低い保持率であった。このように日本独自の酒類である清酒,泡盛,焼酎の製造に使用されている酵母は2μ DNAを保持していないか,または保持していても低率である結果であった。これに対し,パン酵母やビール酵母は高い保持率を示した。また,本研究では実験室酵母として使用されているS. cerevisiae, S. bayanus, S. pastorianus, S. paradoxus及び樹液酵母も使用した。その結果,興味あることにS. paradoxusと樹液酵母の保持は確認されなかった。Existence of 2μ DNA in industrial yeasts, those found in tree exudates, and Saccharomyces sensu stricto was examined. Regarding the yeast used for production of sake, none of the strains examined, i.e., the strains kept by Japanese Sake Association, ATCC strains, long-term-stored strains, strains isolated from the major sake factories, strains isolated from the natural soil and cultivated ornamental plants, retained 2μ DNA. Further, the proportion of 2μ DNA harboring strain was low in the yeast used for production of shochu, while it is fairly high in wine yeast and high in brewer\u27s yeast and baker\u27s yeast. Thus, the rate was characteristically low in the yeast used for production of liquor utilizing koji. Other than the industrial yeasts, neither the yeast found in tree exudates nor S. paradoxus carried 2μ DNA. Among Saccharomyces sensu stricto, all the reference strains of S. cerevisiae, S. bayanus and S. pastorianus retained 2μ DNA, distinguishing these three strains from the reference strain of S. paradoxus. As mentioned above, the group that did not retain 2μ DNA were sake yeast, awamori yeast, tree exudates yeast and S. paradoxus. In particular, it was extremely characteristic that none of the 192 strains of sake yeast retained 2μ DNA

Nuclear ribosomal internal transcribed spacer (ITS) region as a universal DNA barcode marker for Fungi

Six DNA regions were evaluated as potential DNA barcodes for Fungi, the second largest kingdom of eukaryotic life, by a multinational, multilaboratory consortium. The region of the mitochondrial cytochrome c oxidase subunit 1 used as the animal barcode was excluded as a potential marker, because it is difficult to amplify in fungi, often includes large introns, and can be insufficiently variable. Three subunits from the nuclear ribosomal RNA cistron were compared together with regions of three representative protein-coding genes (largest subunit of RNA polymerase II, second largest subunit of RNA polymerase II, and minichromosome maintenance protein). Although the protein-coding gene regions often had a higher percent of correct identification compared with ribosomal markers, low PCR amplification and sequencing success eliminated them as candidates for a universal fungal barcode. Among the regions of the ribosomal cistron, the internal transcribed spacer (ITS) region has the highest probability of successful identification for the broadest range of fungi, with the most clearly defined barcode gap between inter- and intraspecific variation. The nuclear ribosomal large subunit, a popular phylogenetic marker in certain groups, had superior species resolution in some taxonomic groups, such as the early diverging lineages and the ascomycete yeasts, but was otherwise slightly inferior to the ITS. The nuclear ribosomal small subunit has poor species-level resolution in fungi. ITS will be formally proposed for adoption as the primary fungal barcode marker to the Consortium for the Barcode of Life, with the possibility that supplementary barcodes may be developed for particular narrowly circumscribed taxonomic groups