Homologs of Aflatoxin Biosynthesis Genes and Sequence of
 aflR
 in
 Aspergillus oryzae
 and
 Aspergillus sojae

Watson, Adrian J.; Fuller, Linda J.; Jeenes, David J.; Archer, David B.

doi:10.1128/aem.65.1.307-310.1999

Cited by 64 publications

(25 citation statements)

References 18 publications

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“…Several molecular genetic techniques have been tested to classify Aspergillus section Flavi strains: random amplification of polymorphic DNA (RAPD) (Yuan et al, 1995), amplified fragment length polymorphism (Montiel et al, 2003), DNA restriction fragment polymorphism (Klich & Mullaney, 1987;Moody & Tyler, 1990a, b), and sequence analyses of (1) the mitochondrial cytochrome b gene (Wang et al, 2001), (2) the internal transcribed spacer (ITS) region (Kumeda & Asao, 1996;Henry et al, 2000;Kumeda & Asao, 2001;Rigo et al, 2002) and (3) the aflatoxin gene cluster (Chang et al, 1995;Watson et al, 1999;Tominaga et al, 2006). Although these studies provided important information about the phylogenetic relationships between species, none of them used singly was able to solve problems of identification.…”

Section: Introductionmentioning

confidence: 99%

Molecular strategy for identification inAspergillus sectionFlavi

Godet

Munaut

2010

FEMS Microbiology Letters

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Aspergillus flavus is one of the most common contaminants that produces aflatoxins in foodstuffs. It is also a human allergen and a pathogen of animals and plants. Aspergillus flavus is included in the Aspergillus section Flavi that comprises 11 closely related species producing different profiles of secondary metabolites. A six-step strategy has been developed that allows identification of nine of the 11 species. First, three real-time PCR reactions allowed us to discriminate four groups within the section: (1) A. flavus/Aspergillus oryzae/Aspergillus minisclerotigenes/Aspergillus parvisclerotigenus; (2) Aspergillus parasiticus/Aspergillus sojae/Aspergillus arachidicola; (3) Aspergillus tamarii/Aspergillus bombycis/Aspergillus pseudotamarii; and (4) Aspergillus nomius. Secondly, random amplification of polymorphic DNA (RAPD) amplifications or SmaI digestion allowed us to differentiate (1) A. flavus, A. oryzae and A. minisclerotigenes; (2) A. parasiticus, A. sojae and A. arachidicola; (3) A. tamarii, A. bombycis and A. pseudotamarii. Among the 11 species, only A. parvisclerotigenus cannot be differentiated from A. flavus. Using the results of real-time PCR, RAPD and SmaI digestion, a decision-making tree was drawn up to identify nine of the 11 species of section Flavi. In contrast to conventional morphological methods, which are often time-consuming, the molecular strategy proposed here is based mainly on real-time PCR, which is rapid and requires minimal handling.

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Section: Introductionmentioning

confidence: 99%

Molecular strategy for identification inAspergillus sectionFlavi

Godet

Munaut

2010

FEMS Microbiology Letters

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“…3). Recently, Watson et al (27) reported that sequences homologous to nor-1, ver-1, omtA, and aflR are also present in strains of A. oryzae and Aspergillus sojae, although no transcripts of these homologs have been found in any of the strains examined. Similar results have been reported by other researchers (15)(16)(17)(18).…”

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confidence: 99%

Cloning and Characterization of the O -Methyltransferase I Gene ( dmtA ) from Aspergillus parasiticus Associated with the Conversions of Demethylsterigmatocystin to Sterigmatocystin and Dihydrodemethylsterigmatocystin to Dihydrosterigmatocystin in Aflatoxin Biosynthesis

Motomura

Chihaya²,

Shinozawa

et al. 1999

Appl Environ Microbiol

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O-Methyltransferase I catalyzes both the conversion of demethylsterigmatocystin to sterigmatocystin and the conversion of dihydrodemethylsterigmatocystin to dihydrosterigmatocystin during aflatoxin biosynthesis. In this study, both genomic cloning and cDNA cloning of the gene encoding O-methyltransferase I were accomplished by using PCR strategies, such as conventional PCR based on the N-terminal amino acid sequence of the purified enzyme, 5′ and 3′ rapid amplification of cDNA ends PCR, and thermal asymmetric interlaced PCR (TAIL-PCR), and genes were sequenced by usingAspergillus parasiticus NIAH-26. A comparison of the genomic sequences with the cDNA of the dmtA region revealed that the coding region is interrupted by three short introns. The cDNA of the dmtA gene is 1,373 bp long and encodes a 386-amino-acid protein with a deduced molecular weight of 43,023, which is consistent with the molecular weight of the protein determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The C-terminal half of the deduced protein exhibits 76.3% identity with the coding region of the Aspergillus nidulans StcP protein, whereas the N-terminal half of dmtA exhibits 73.0% identity with the 5′ flanking region of the stcP gene, suggesting that translation of the stcP gene may start at a site upstream from methionine that is different from the site that has been suggested previously. Also, an examination of the 5′ and 3′ flanking regions of the dmtA gene in which TAIL-PCR was used demonstrated that the dmtA gene is located in the aflatoxin biosynthesis cluster between (and in the same orientation as) the omtA and ord-2 genes. Northern blotting revealed that expression of the dmtA gene is influenced by both medium composition and culture temperature and that the pattern correlates with the patterns observed for other genes in the aflatoxin gene cluster. Furthermore, Southern blotting and PCR analyses of thedmtA gene showed that a dmtA homolog is present in Aspergillus oryzae SYS-2.

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“…Two lines of evidence support such a notion. First, Watson et al (36) recently reported that A. sojae and A. oryzae, which are closely related to A. parasiticus and do not produce aflatoxins, contain an aflR homolog with a specific point mutation. This amber mutation at Arg383 of AFLR results in truncation of the last 62 amino acids in the carboxyl terminus.…”

Section: Discussionmentioning

confidence: 99%

The Carboxy-Terminal Portion of the Aflatoxin Pathway Regulatory Protein AFLR of Aspergillus parasiticus Activates GAL1 :: lacZ Gene Expression in Saccharomyces cerevisiae

Chang

Bhatnagar

et al. 1999

Appl Environ Microbiol

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AFLR, a DNA-binding protein of 444 amino acids, transactivates the expression of aflatoxin biosynthesis genes in Aspergillus parasiticus and Aspergillus flavus, as well as the sterigmatocystin synthesis genes in Aspergillus nidulans. We show here by fusion of various aflR coding regions to the GAL4 DNA-binding coding region that the AFLR carboxyl terminus contained a region that activatedGAL1::lacZ gene expression inSaccharomyces cerevisiae and that the AFLR internal region was required for the activation activity. Compared to the AFLR carboxy-terminal fusion protein (AFLRC), a mutant AFLRC retained approximately 75% of the activation activity after deletion of three acidic amino acids, Asp365, Glu366, and Glu367, in a previously identified acidic stretch. Removal of the carboxy-terminal amino acid, Glu444, did not affect the activation activity. Substitutions of acidic Glu423, Asp439, or Asp436/Asp439 with basic amino acids, Lys and His, resulted in 10- to 15-fold-lower activation activities. Strikingly, the Asp436His mutation abolished the activation activity. Substitutions of basic His428 and His442 with acidic Asp resulted in 20 and 40% decreases in the activation activities, respectively. Simultaneous substitutions of Arg427, Arg429, and Arg431 with Leu also significantly decreased the activation activity; the decrease was approximately 50-fold. Results suggest that the AFLR carboxy-terminal region is involved in transcription activation and that total acidity in this region is not a major determinant of AFLR’s activation ability inS. cerevisiae.

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Homologs of Aflatoxin Biosynthesis Genes and Sequence of aflR in Aspergillus oryzae and Aspergillus sojae

Cited by 64 publications

References 18 publications

Molecular strategy for identification inAspergillus sectionFlavi

Molecular strategy for identification inAspergillus sectionFlavi

Cloning and Characterization of the O -Methyltransferase I Gene ( dmtA ) from Aspergillus parasiticus Associated with the Conversions of Demethylsterigmatocystin to Sterigmatocystin and Dihydrodemethylsterigmatocystin to Dihydrosterigmatocystin in Aflatoxin Biosynthesis

The Carboxy-Terminal Portion of the Aflatoxin Pathway Regulatory Protein AFLR of Aspergillus parasiticus Activates GAL1 :: lacZ Gene Expression in Saccharomyces cerevisiae

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