Aspergillus nidulans verA is required for production of the mycotoxin sterigmatocystin.

Aspergillus nidulans produces the carcinogenic mycotoxin sterigmatocystin (ST), the next-to-last precursor in the aflatoxin (AF) biosynthetic pathway found in the closely related fungi Aspergillus flavus and Aspergillus parasiticus. We identified and characterized an A. nidulans gene, verA, that is required for converting the AF precursor versicolorin A to ST. verA is closely related to several polyketide biosynthetic genes involved in polyketide production in Streptomyces spp. and exhibits extended sequence similarity to A. parasiticus ver-1, a gene proposed to encode an enzyme involved in converting versicolorin A to ST. By performing a sequence analysis of the region 3' to verA, we identified two additional open reading frames, designated ORF1 and ORF2. ORF2 is closely related to a number of cytochrome P-450 monooxygenases, while ORF1 shares identity with the gamma subunit of translation elongation factor 1. Given that several steps in the ST-AF pathway may require monooxygenase activity and that AF biosynthetic genes are clustered in A. flavus and A. parasiticus, we suggest that verA may be part of a cluster of genes required for ST biosynthesis. We disrupted the verA coding region by inserting the A. nidulans argB gene into the center of the coding region and transformed an A. nidulans argB2 mutant to arginine prototrophy. Seven transformants that produced DNA patterns indicative of a verA disruption event were grown under ST-inducing conditions, and all of the transformants produced versicolorin A but negligible amounts of ST (200-fold to almost 1,000-fold less than the wild type), confirming the hypothesis that verA encodes an enzyme necessary for converting versicolorin A to ST.     

Cloning of the Aspergillus parasiticus apa-2 gene associated with the regulation of aflatoxin biosynthesis.

An Aspergillus parasiticus gene, designated apa-2, was identified as a regulatory gene associated with aflatoxin biosynthesis. The apa-2 gene was cloned on the basis of overproduction of pathway intermediates following transformation of fungal strains with cosmid DNA containing the aflatoxin biosynthetic genes nor-1 and ver-1. Transformation of an O-methylsterigmatocystin-accumulating strain, A. parasiticus SRRC 2043, with a 5.5-kb HindIII-XbaI DNA fragment containing apa-2 resulted in overproduction of all aflatoxin pathway intermediates analyzed. Specific enzyme activities associated with the conversion of norsolorinic acid and sterigmatocystin were increased approximately twofold. The apa-2 gene was found to complement an A. flavus afl-2 mutant strain for aflatoxin production, suggesting that apa-2 is functionally homologous to afl-2. Comparison of the A. parasiticus apa-2 gene DNA sequence with that of the A. flavus afl-2 gene (G. A. Payne, G. J. Nystorm, D. Bhatnagar, T. E. Cleveland, and C. P. Woloshuk, Appl. Environ. Microbiol. 59:156-162, 1993) showed that they shared > 95% DNA homology. Physical mapping of cosmid subclones placed apa-2 approximately 8 kb from ver-1.     

Structure and function of fas-1A, a gene encoding a putative fatty acid synthetase directly involved in aflatoxin biosynthesis in Aspergillus parasiticus.

A novel gene, fas-1A, directly involved in aflatoxin B1 (AFB1) biosynthesis, was cloned by genetic complementation of an Aspergillus parasiticus mutant strain, UVM8, blocked at two unique sites in the AFB1 biosynthetic pathway. Metabolite conversion studies localized the two genetic blocks to early steps in the AFB1 pathway (nor-1 and fas-1A) and confirmed that fas-1A is blocked prior to nor-1. Transformation of UVM8 with cosmids NorA and NorB restored function in nor-1 and fas-1A, resulting in synthesis of AFB1. An 8-kb SacI subclone of cosmid NorA complemented fas-1A only, resulting in accumulation of norsolorinic acid. Gene disruption of the fas-1A locus blocked norsolorinic acid accumulation in A. parasiticus B62 (nor-1), which normally accumulates this intermediate. These data confirmed that fas-1A is directly involved in AFB1 synthesis. The predicted amino acid sequence of fas-1A showed a high level of identity with extensive regions in the enoyl reductase and malonyl/palmityl transferase functional domains in the beta subunit of yeast fatty acid synthetase. Together, these data suggest that fas-1A encodes a novel fatty acid synthetase which synthesizes part of the polyketide backbone of AFB1. Additional data support an interaction between AFB1 synthesis and sclerotium development.     

The conversion of sterigmatocystin to O-methylsterigmatocystin and aflatoxin B1 by a cell-free preparation

A cell-free system derived from a versicolorin A-accumulating mutant of Aspergillus parasiticus was found to convert sterigmatocystin to both O-methylsterigmatocystin and aflatoxin B1. It is suggested that the similarity in the chromatographic properties of these two metabolites has caused erroneous conclusions to be made with regards to the biosynthesis of aflatoxin B1.     

Recombinational inactivation of the gene encoding nitrate reductase in Aspergillus parasiticus.

Functional disruption of the gene encoding nitrate reductase (niaD) in Aspergillus parasiticus was conducted by two strategies, one-step gene replacement and the integrative disruption. Plasmid pPN-1, in which an internal DNA fragment of the niaD gene was replaced by a functional gene encoding orotidine monophosphate decarboxylase (pyrG), was constructed. Plasmid pPN-1 was introduced in linear form into A. parasiticus CS10 (ver-1 wh-1 pyrG) by transformation. Approximately 25% of the uridine prototrophic transformants (pyrG+) were chlorate resistant (Chlr), demonstrating their inability to utilize nitrate as a sole nitrogen source. The genetic block in nitrate utilization was confirmed to occur in the niaD gene by the absence of growth of the A. parasiticus CS10 transformants on medium containing nitrate as the sole nitrogen source and the ability to grow on several alternative nitrogen sources. Southern hybridization analysis of Chlr transformants demonstrated that the resident niaD locus was replaced by the nonfunctional allele in pPN-1. To generate an integrative disruption vector (pSKPYRG), an internal fragment of the niaD gene was subcloned into a plasmid containing the pyrG gene as a selectable marker. Circular pSKPYRG was transformed into A. parasiticus CS10. Chlr pyrG+ transformants were screened for nitrate utilization and by Southern hybridization analysis. Integrative disruption of the genomic niaD gene occurred in less than 2% of the transformants. Three gene replacement disruption transformants and two integrative disruption transformants were tested for mitotic stability after growth under nonselective conditions. All five transformants were found to stably retain the Chlr phenotype after growth on nonselective medium.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cloning and nucleotide sequence of the murine interleukin-3 gene

Southern hybridization analysis using a probe derived from a murine interleukin-3 (IL-3) cDNA clone revealed the presence of a single IL-3 gene in the haploid murine genome. An 8600-base-pair (8.6-kb) murine genomic EcoRI fragment containing the IL-3 gene was isolated by screening a library of size-fractionated genomic EcoRI fragments cloned in lambda gtWES X lambda B. The nucleotide sequence of a 3.5-kb region of the cloned DNA encompassing the IL-3 gene was determined. The gene contains four introns of 96, 993, 135 and 122 base pairs (bp), located within the coding region. The large intron contains 12 copies of a 14-15-bp tandem repeating sequence which resembles a human cellular homologue of a BKV enhancer sequence. The nucleotide sequence of the exons agrees exactly with that of an IL-3 cDNA cloned from WEHI-3, a tumorigenic cell line which over-produces IL-3, establishing that the unprocessed primary structure of IL-3 is identical in WEHI-3 and in BALB/c mice. Southern hybridization has revealed genomic alteration in the vicinity of the IL-3 gene in WEHI-3 cells.     

Identification of averantin as an aflatoxin B1 precursor: placement in the biosynthetic pathway.

A new blocked mutant of Aspergillus parasiticus produces no detectable aflatoxin B1, but accumulates several polyhydroxyanthraquinones. One of these pigments was identified as averantin. This is the first report of its formation by A. parasiticus. Radiotracer studies with [14C]averantin showed that 15.3% of label from averantin was incorporated into aflatoxin B1. This incorporation was blocked by dichlorvos. With radiotracers and other mutants, averantin was placed after norsolorinic acid and before averufin in the biosynthetic pathway in which the general steps are norsolorinic acid leads to averantin leads to averufin leads to versiconal hemiacetal acetate leads to versicolorin A leads to sterigmatocystin leads to aflatoxin B1.     

Isolation of the gene for cytochrome P450L1A1 (lanosterol 14 alpha-demethylase) from Candida albicans

The Saccharomyces cerevisiae cytochrome P450 L1A1 (lanosterol 14 alpha-demethylase)-coding gene was used as a hybridization probe to isolate two HindIII fragments of 2.5 kb and 6.85 kb from a phage lambda library of Candida albicans nucleotide sequences. Restriction endonuclease mapping and Southern blot hybridization experiments indicated that these fragments represent two allelic forms of the same gene. This cloned sequence, when introduced into S. cerevisiae or C. albicans on a multiple copy vector, produced an increase in cytochrome P450 content and resistance to imidazole antifungal agents which are inhibitors of cytochrome P450 L1A1. In addition, the cloned sequence was able to complement a cytochrome P450 L1A1 gene disruption when introduced into S. cerevisiae. These data indicate that the cloned sequence codes for the lanosterol 14 alpha-demethylase cytochrome P450 L1A1 from C. albicans.     

Conversion of 11-hydroxy-O-methylsterigmatocystin to aflatoxin G1 in Aspergillus parasiticus

In aflatoxin biosynthesis, aflatoxins G(1) (AFG(1)) and B(1) (AFB(1)) are independently produced from a common precursor, O-methylsterigmatocystin (OMST). Recently, 11-hydroxy-O-methylsterigmatocystin (HOMST) was suggested to be a later precursor involved in the conversion of OMST to AFB(1), and conversion of HOMST to AFB(1) was catalyzed by OrdA enzyme. However, the involvement of HOMST in AFG(1) formation has not been determined. In this work, HOMST was prepared by incubating OrdA-expressing yeast with OMST. Feeding Aspergillus parasiticus with HOMST allowed production of AFG(1) as well as AFB(1). In cell-free systems, HOMST was converted to AFG(1) when the microsomal fraction, the cytosolic fraction from A. parasiticus, and yeast expressing A. parasiticus OrdA were added. These results demonstrated (1) HOMST is produced from OMST by OrdA, (2) HOMST is a precursor of AFG(1) as well as AFB(1), and (3) three enzymes, OrdA, CypA, and NadA, and possibly other unknown enzymes are involved in conversion of HOMST to AFG(1).     

Cloning and complete nucleotide sequence of mouse immunoglobulin gamma 1 chain gene.

The 6.6 kb DNA fragment coding for the immunoglobulin γ1 chain was cloned from newborn mouse DNA using λgtWES·λB as the EK2 vector. The complete nucleotide sequence (1823 bases) of the γ1 chain gene was determined. The cloned gene contained the entire constant region gene sequence as well as the poly(A) addition site, but not the variable region gene. The results indicate that the variable and constant region genes of immunoglobulin heavy chain are separated in newborn mouse DNA. The constant region genes of other gamma chains (that is, γ2a, γ2b and γ3) are not present in the cloned DNA fragment. The sequence demonstrates that the γ1 chain gene is interrupted by three intervening sequences at the junction of the domains and the hinge region, as previously shown in the γ2b and α chain genes and in the γ1 chain gene cloned from myeloma. The results suggest that the intervening sequence was introduced into the heavy chain gene before divergence of the heavy chain classes, and also support the hypothesis that the splicing mechanism has facilitated the evolution of eucaryotic genes by linking duplicated domains or prototype peptides not directly adjacent to one another. Comparison of the nucleotide sequence of the γ1 chain gene around the boundaries of the coding and intervening sequences with those of other mouse genes revealed extensive divergence, although short prevalent sequences of AG-GTCAG at the 5′ border of the intervening sequence and TCTGCAG-GC at the 3′ border were deduced. A limited homology of nucleotide sequences was found among domains and between the hinge region and the 5′ portion of the CH2 domain. Comparison of 3′ untranslated sequences from the γ1 and γ2b chain genes and the mouse major β-globin gene shows significant homology and a palindrome sequence surrounding the poly(A) addition site.     

Cloning and sequencing of beta toxin gene of Clostridium perfringens type C

A gene encoding beta toxin was amplified by polymerase chain reaction from C. perfringens type C isolate and cloned in pUC 19 vector. The nucleotide sequence was identical with C. perfringens type B beta toxin gene sequence. The Southern hybridization using labelled beta toxin gene probe revealed the presence of positive signals only in beta producing C. perfingens.     

Precursor recognition by kinetic pulse-labeling in a toxigenic aflatoxin B1-producing strain of Aspergillus.

Kinetic pulse-labeling of aflatoxin pathway compounds was carried out in Aspergillus parasiticus, beginning with radioactive acetate. Norsolorinic acid, averufin, versicolorin A, and sterigmatocystin (all known as compounds which can be incorporated into the aflatoxin molecule) were radiotraced to follow their order of appearance. Aflatoxin species B1, B2, G1, and G2 were included. Norsolorinic acid and averufin appeared as early transient intermediates followed in order by versicolorin A, aflatoxins, and sterigmatocystin. To date, a mutually confirming array of results has been obtained with established precursors in wild-type strains of A. parasiticus and A. versicolor (as well as with an aflatoxin pathway mutant of A. parasiticus), which together establish a practical methodology for recognition of new pathway intermediates. The kinetic of pulse-labeling for sterigmatocystin in relation to aflatoxins suggests that duel branchlets may exist to flatoxins; i.e., sterigmatocystin may not be an obligatory aflatoxin precursor.     

Peculiar feature of the organization of rRNA genes of the Chlorella chloroplast DNA.

The organization of a cloned rRNA gene cluster from Chlorella ellipsoidea chloroplast DNA (cpDNA) has been analyzed. Southern hybridization experiments with labelled chloroplast rRNAs as probes revealed an extraordinarily large size of the 16S-23S rRNA spacer region, ca. 4.8 kbp, almost twice as large as those of most higher plants. The nucleotide sequence determined on this region has shown that: (1) The tRNAIle gene locating in this region is similar to those of higher plant chloroplasts, blue-green algae and E. coli but does not contain any introns in contrast to higher plant chloroplasts. (2) The tRNAAla gene is absent from this region. (3) There are four open reading frames (ORFs) coding for 55, 102, 107 and 110 amino acids, respectively. (4) A few sets of unique sequence were found repeatedly in this region. (5) The 23S rRNA gene is coded on the opposite strand in the reverse order. This arrangement of the 16S-23S rRNA region of Chlorella cpDNA is quite different from any of those reported so far for various organisms.     

An aflatoxin biosynthesis cluster gene encodes a novel oxidase required for conversion of versicolorin a to sterigmatocystin

Disruption of the aflatoxin biosynthesis cluster gene aflY (hypA) gave Aspergillus parasiticus transformants that accumulated versicolorin A. This gene is predicted to encode the Baeyer-Villiger oxidase necessary for formation of the xanthone ring of the aflatoxin precursor demethylsterigmatocystin.     

Development of polyclonal antibodies for detection of aflatoxigenic molds involving culture filtrate and chimeric proteins expressed in Escherichia coli.

Polyclonal antibodies (PAb) were raised against an aflatoxigenic strain of Aspergillus parasiticus by using two different sources for antibody elicitation: (i) filtrate of a culture on which the fungus had been grown (ii) and two chimeric proteins, expressed in Escherichia coli as separate products, of the genes ver-1 and apa-2, which are involved in aflatoxin biosynthesis. The gene products were amplified by PCR, and each was cloned into the E. coli expression vector pGEX2T. Upon induction, the bacteria overexpressed 38- and 33-kDa chimeric proteins corresponding to the N-terminal domains of the genes ver-1 and apa-2, respectively. The chimeric proteins were isolated and affinity purified for use as antigens. The specificity of the raised antibodies was examined by enzyme-linked immunosorbent assay (ELISA). The PAbs raised against the culture filtrate reacted with all the species of Aspergillus and Penicillium tested but not with Fusarium species or corn gain. However, the PAbs elicited against the chimeric proteins were highly specific, showing significantly higher ELISA absorbance values (A405) against A. parasiticus and A. flavus than against the other fungi tested and the corn grain. The approach of utilizing gene products associated with aflatoxin biosynthesis for antibody production therefore appears to be feasible. Such a multiantibody system combined with the PCR technique, could provide a useful tool for the rapid, sensitive, and accurate detection of aflatoxin producers present in grains and foods.