Mutagenesis of aspergillus fungi and identification of genes essential for growth
OwnerMarie-Clair Grosjean-Cournoyer, Christ D'Enfert +3inventors
InventorsMarie-Clair Grosjean-Cournoyer, Christ D'Enfert, Arnaud Firon, Marc-Henri Lebrun, Roland Beffa
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Abstract
The present invention is directed to polynucleotides encoding proteins Essential For the Growth (EFG) of filaments fungi. The invention also deals with namely polypeptides encoded by said polynuctleotides, screening assays for identifying compounds capable of inhibiting said EFG proteins activities pharmaceutical or phytosanitary compositions comprising such compounds.
Claims29
A nucleic acid encoding an Essential For Growth (EFG) polypeptide selected from the group consisting of:
- (i) a nucleic acid molecule encoding a polypeptide comprising the amino acid sequence depicted in one of SEQ ID NO:
- 3, 6,9, 12, 15, 18, 21, 24, 27, 30, 33, 36, 39, 42, 45, 48, 51, 54, 57, 60, 94, 98, 102, 106, 110, 114, 118, 122, 126, 130, 134,138, 142, 146, 150,154, 158,162, 166, 170;
- (ii) a nucleic acid molecule comprising the nucleic acid sequence as depicted in one of SEQ ID NO:
- 1, 4, 7, 10, 13, 16, 19, 22, 25, 28, 31, 34, 37, 40, 43, 46, 49, 52, 55, 58, 92, 96, 100, 104, 108, 112, 116, 120, 124, 128, 132, 136, 140, 144, 148, 152, 156, 160, 164, 168;
- (iii) a nucleic sequence having at least 80, 85, 90, 95, 98, 99% identity with a sequence of SEQ ID NO:
- 1, 4, 7, 10, 13, 16, 19, 22, 25, 28, 31, 34, 37, 40, 43, 46, 49, 52, 55, 58, 92, 96, 100, 104, 108, 112, 116, 120, 124, 128, 132, 136, 140, 144, 148, 152, 156, 160, 164, 168;
- (iv) a nucleic acid molecule which hybridizes under stringent conditions to:
- (a) a nucleic acid as defined in (i), (ii) and (iii), or (b) a complementary strand of (a);
- (v) a nucleic acid the sequence of which is degenerate as a result of the genetic code to the sequence of a nucleic acid as defined in (i), (ii), (iii) and (iv).
An isolated nucleic acid, said nucleic acid comprising a nucleotide sequence encoding:
- (i) a EFG polypeptide comprising an amino acid sequence having at least 80% identity to a sequence of SEQ ID NO 3, 6, 9, 12, 15, 18, 21, 24, 27, 30, 33, 36, 39, 42, 45, 48, 51, 54, 57, 60, 94, 98, 102, 106, 110, 114, 118, 122, 126, 130, 134, 138, 142, 146, 150, 154, 158, 162, 166, 170;
- or
- (ii) a biologically active fragment of said polypeptide.
An isolated nucleic acid, said nucleic acid comprising a nucleotide sequence encoding:
- (i) a EFG polypeptide comprising an amino acid sequence which is orthologous to a sequence of SEQ ID NO:
- 3, 6, 9, 12, 15, 18, 21, 24, 27, 30, 33, 36, 39, 42, 45, 48, 51, 54, 57, 60, 94, 98, 102, 106, 110, 114, 118, 122, 126, 130, 134, 138, 142, 146, 150, 154, 158, 162, 166, 170;
- or
- (ii) a biologically active fragment of said polypeptide.
A method of identifying a candidate inhibitor of EFG polypeptide, said method comprising:
- (a) contacting said polypeptide with a test compound;
- (b) determining whether said compound selectively inhibits the activity of said polypeptide, said inhibition indicating that said compound is a candidate inhibitor.
A method for detecting the presence of a nucleic acid comprising a nucleotide sequence of SEQ ID NO 1, 4, 7, 10, 13, 16, 19, 22, 25, 28, 31, 34, 37, 40, 43, 46, 49, 52, 55, 58, 92, 96, 100, 104, 108, 112, 116, 120, 124, 128, 132, 136, 140, 144, 148, 152, 156, 160, 164, 168, a fragment or a variant thereof and a complementary sequence thereto in a sample, said method comprising the following steps of:
- (a) bringing into contact a nucleic acid probe or a plurality of nucleic acid probes which can hybridize with a nucleotide sequence included in a nucleic acid sequence of SEQ ID NO:
- 1, 4, 7, 10, 13, 16, 19, 22, 25, 28, 31, 34, 37, 40, 43, 46, 49, 52, 55, 58, 92, 96, 100, 104, 108, 112, 116, 120, 124, 128, 132, 136, 140, 144, 148, 152, 156, 160, 164, 168, a fragment or a variant thereof and a complementary sequence thereto and the sample to be assayed;
- and
- (b) detecting the hybrid complex formed between the probe and a nucleic acid in the sample.
A method for locating at least one gene essential for the growth of a haploid fungus, said method comprising the following successive steps:
- generation of diploid strain from fungal haploid strain
- mutagenesis of said diploid strain;
- haploidisation of the diploid transformant strain, in selection conditions such that the absence of haploid progeny is indicative of mutagenesis occurring in essential gene;
- wherein said mutagenesis is an in vivo transposon mutagenesis.
Plasmid pNIpyr (CNCM I-2815).
The diploid strain CEA 225 (CNCM I-2816).
The diploid strain CEA 226 (CNCM I-2817).
The diploid strain CEA 227 (CNCM I-2818).
A method for screening compounds that are active against A. fumigatus comprising:
- preparing an A. fumigatus strain that is heterozygous for an EFG gene (heterozygous EFGn/efgn);
- preparing an A. fumigatus strain that is homozygous for the EFG gene (homozygous EFGn/EFGn);
- comparing the effect of a candidate compound on the heterozygous EFGn/efgn and on the homozygous EFGn/EFGn,
- the higher inhibiting effect on the heterozygous EFGn/efgn than on the homozygous EFGn/EFGn indicating that the compound is an inhibitor.
Dependencies are inferred from the claim text.