Drug – bio-affecting and body treating compositions – Whole live micro-organism – cell – or virus containing – Fungus
Patent
1998-03-31
2000-08-15
Ketter, James
Drug, bio-affecting and body treating compositions
Whole live micro-organism, cell, or virus containing
Fungus
426627, 435 6, 536 2374, A01N 6304, C12Q 168
Patent
active
061032298
DESCRIPTION:
BRIEF SUMMARY
The present invention relates to a regulatory gene from the fungus Ustilago maydis, to the use of this gene, and to fungal mutants which harbor a mutation in this gene.
Ustilago maydis, the organism causing blister smut of corn, has a two-phase life cycle. The haploid stage grows like a yeast and is not pathogenic. The dikaryon shows filamentous growth and represents the pathogenic form. The fungus has two mating type loci. The a locus, of which two alleles exist, is responsible for the fusion of haploid cells and the formation of the dikaryon. The multiallelic b locus controls the pathogenicity and the sexual development of the fungus. It codes for two homeodomain proteins (bW and bE) which form functional heterodimers.
A b locus-regulated gene egl1 codes for an endoglucanase whose expression is induced in the filamentous phase. Expression of the glucanase egl1 can be detected reliably and unambiguously using indicator plates based on carboxymethylcellulose with congo red. Hence egl1 is suitable as reporter gene for searching for genes with regulatory functions for the expression of differentially expressed genes. Since the filamentous phase of Ustilago maydis is pathogenic for corn, the object was to identify genes or gene products linked to the regulation of the filamentous phase. Such genes or gene products ought to represent suitable possibilities for intervention to eliminate the pathogenicity.
We have now found a nucleic acid fragment from the fungus Ustilago maydis which contains a regulatory gene.
The invention relates to a nucleic acid fragment from the fungus Ustilago maydis which comprises the XbaI-BglII fragment depicted in FIG. 1 and which comprises the nucleic acid sequence indicated in FIG. 3 between the BglII and the XbaI cleavage site.
BRIEF DESCRIPTION OF THE FIGURES
FIG. 1 depicts a nucleic acid fragment from the fungus Ustilago maydis which comprises the XbaI-BglII fragment.
FIG. 2 depicts a section of the region of cosmid pUMcos7-H2 which harbors the XbaI-BglII nucleic acid fragment.
FIG. 3 depicts the sequence of the nucleic acid fragment from the fungus Ustilago maydis between the XbaI and BglII cleavage sites.
FIG. 4 depicts the amino acid sequence of the gene product of the nucleic acid fragment of FIG. 3.
The nucleic acid fragment was isolated in the following way:
A haploid Ustilago maydis strain FB1 (a1 b1) (Banuett, F. and Herskowitz, I. (1989), Different a alleles of Ustilago maydis are necessary for maintenance of filamentous growth but not for meiosis, Proc. Acad. Sci. U.S.A. 86: 5878-5882), which does not express the endoglucanase egl1 was subjected to UV mutagenesis. Screening of the resulting mutants for constitutive egl1 expression on carboxymethylcellulose plates followed by congo red staining revealed one mutant with the property which was sought, ie. filament-independent constitutive egl1 expression.
Detailed investigation of the resulting mutant revealed that other, normally differentially expressed, genes were expressed constitutively in this mutant. The gene affected by the mutation was further characterized by carrying out a complementation analysis (as described in detail in Example 3).
Analysis of the nucleic acid fragment revealed that the regulatory gene in this case presumably has a repressing action. This regulatory gene or the relevant gene product thus represents a possible specific site of action for fungicides. It is easily possible in a test method to test possible fungicidal compounds for interaction with the site of action which has been found, by bringing a haploid Ustilago strain which does not express endoglucinase egl1 in contact with the potential fungicide and determining whether egl1 expression takes place. In the positive case it is possible to assume interaction of the fungicide with the regulatory gene product.
A test method of this type can be carried out particularly straightforwardly if the screening for egl1 expression is done using carboxymethylcellulose plates, which are stained with congo red.
The invention furthermore relates to
REFERENCES:
Banuett et al., Proc. Natl. Acad. Sci., vol. 86, pp. 5878-5882, Aug. 1989.
Schauwecker et al., Biol. Chem. Hoppe-Seyler, vol. 376, pp. 617-625, Oct. 1995.
Barrett et al., Molecular Plant-Microbe Interactions, vol. 6, No. 3, pp. 274-283, 1993.
Schulz et al., Cell, vol. 60, pp. 295-306, Jan. 26, 1990.
Kahmann Regine
Quadbeck-Seeger Claudia
BASF - Aktiengesellschaft
Ketter James
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