Chemistry: molecular biology and microbiology – Process of mutation – cell fusion – or genetic modification – Introduction of a polynucleotide molecule into or...
Reexamination Certificate
2001-12-26
2003-09-09
Guzo, David (Department: 1636)
Chemistry: molecular biology and microbiology
Process of mutation, cell fusion, or genetic modification
Introduction of a polynucleotide molecule into or...
C435S440000, C435S471000, C435S476000, C435S477000, C435S480000, C435S481000, C435S243000, C435S254100, C435S254110, C435S254300, C435S254500, C435S254700, C435S320100, C536S023100, C536S024100
Reexamination Certificate
active
06617163
ABSTRACT:
The present invention relates to novel polynucleotides, and to the use of these polynucleotides, for insertional mutagenesis and gene tagging in fungi. The invention also relates to collections of fungus mutants obtained by random insertion of the Impala transposon from
Fusarium oxysporum
into the genome of these fungi. These collections of mutants represent an effective genetic tool for studying the function of genes in fungi.
Transposons may be defined as mobile genetic elements capable of moving between two DNA sequences. By virtue of their capacity to insert into genes (exons, introns, regulatory regions), they can be the cause of mutations. Because of this, they contribute to the evolution of the genomes in which they exist as a parasite. Transposons have been classified in two groups depending on their propagation method (Finnegan, 1989; Capy et al., 1998):
Class I elements (retroelements) transpose via an RNA intermediate which is reverse-transcribed into DNA by a reverse transcriptase. This class is subdivided into retroelements or retrotransposons which may or may not be bordered by LTRs (long terminal repeats). Among the LTR retroelements are the elements of the gypsy family and of the copia family. They have genes which are homologous to the gag and pol genes of retroviruses and differ in the organization of the various functional domains of the pol gene. In addition, the gypsy family has a gene which is homologous to env which, in retroviruses, contributes to their infectiousness. Among the non-LTR retroelements LINEs which have gag and pol genes and a poly-A sequence are distinguished, and also SINEs, which also have a poly-A tail but lack gag and pol sequences, are distinguished; they are presumed to derive from prior LINE elements (Eickbush, 1992; Okada and Hamada, 1997);
Class II elements transpose via a mechanism of excision and reinsertion of the transposon DNA sequence. Their general structure consists of two inverted repeat sequences (ITRs) bordering an open reading frame encoding a transposase required for the transposition of the element. These elements have been grouped together into superfamilies, according to the sequence homologies of their transposases and/or of the ITRs, including that of the Tc1/mariner elements (Doak et al., 1994), of the Fot1/Pogo elements (Capy et al., 1998; Smit and Riggs, 1996), of the hAT elements (Calvi et al., 1991), of the P elements (Kidwell, 1994) or of the CACTA elements (Gierl 1996).
The identification and study of fungus transposons is of very great value, in particular with a view to developing tools for insertional mutagenesis (Brown et al., Curr. Opin. Microbiol. 1:390-4, 1998) and also for studying the genome of these fungi (Dobinson et al., Trends in Microbiology, 1:348-3652, 1993).
Various strategies have therefore been implemented for identifying transposons in the genome of fungi. The first and second take advantage of the knowledge which derives from previously characterized elements. This involves the use of heterologous probes used in Southern hybridization experiments or amplifications using oligonucleotides derived from highly conserved domains, such as that of the LTR retroelement reverse transcriptase. The third strategy consists in characterizing repeat DNA sequences. In this case, differential hybridization between the genomic DNA and a ribosomal probe is required. Transposons of the Fot1 family have thus been identified in the
Magnaporthe grisea
genome (Kachroo et al., Current Genetics 31:361-369, 1997; Farman et al., Mol. Gen. Genetics 251:675-681 1996; Kachroo et al., Mol. Gen. Genetics 245:339-348, 1994). The final method, unlike the previous three, makes it possible to identify functional and active elements; this is the transposon trap. This strategy uses the inactivation of a marker gene in which the mutation engendered by the insertion of the element can be identified using a positive screen. Thus, the am (glutamate dehydrogenase) gene has made it possible to characterize the Tad retroelement, which is of the LINE type, in
Neurospora crassa
, following its reinsertion into this gene (Kinsey and Helber, 1989). The niaD (nitrate reductase) gene of
Aspergillus nidulans
has also been used for trapping transposons. Specifically, a mutation which inactivates this gene confers chlorate resistance. Various transposons have thus been identified in
Fusarium oxysporum
(Daboussi et al., Genetica 93:49-59, 1994) and in Aspergillus (U.S. Pat. No. 5,985,570). The class II element Fot1 from
Fusarium oxysporum
was the first transposon identified using inactivation of the niaD gene (Daboussi et al., 1992). In addition, the use of the niaD gene in
Fusarium oxysporum
has made it possible to trap the Impala transposon belonging to the superfamily of the Tc1/mariner-type elements (Langin et al., 1995). Various Impala transposon subfamilies have been identified in
Fusarium oxysporum
(Hua-Van et al., Mol. Gen. Genetics 259:354-362, 1998). The transposition of the Impala element has been studied in
Fusarium oxysporum
. When the Impala transposon is integrated into the promoter or the introns of a given gene, it may then inactivate the expression of this gene. On the other hand, after transposition of the Impala transposon, the gene is reactivated, which constitutes a positive control for the transposition event. Such a strategy for identifying the transposition has been used in Fusarium with a construct comprising the Impala transposon integrated into the promoter regulatory sequence of the nitrate reductase (niaD) gene from
Aspergillus nidulans
(Hua-Van, 1998).
It has not been possible to demonstrate the transposition of Impala, other than at an extremely low rate which is incompatible with the development of a tool for insertional mutagenesis, using the niaD/Impala gene construct of the pNi160 plasmid (Langin et al., 1995) in other fungi, and more particularly
Magnaporthe grisea
. These observations suggest that the niaD/Impala construct of the pNi160 plasmid (Langin et al., 1995), and more particularly that the Impala transposon itself, are not functional in other fungi, and in particular in
M. grisea.
Now, such a tool for creating a collection of insertion mutants in the fungus genome, and more particularly the genome of pathogenic filamentous fungi, is essential for studying their genome and for studying the function of their genes. Analyzing the functions of fungus genes is essential for discovering novel antifungal compounds which can be used for treating fungal conditions in human or animal health or for agriculture.
The present invention relates to novel polynucleotides comprising a marker gene which is functional in
Magnaporthe grisea
and which is inactivated by the insertion of an Impala transposon. These polynucleotides make it possible to demonstrate the transposition of the Impala element in fungi with a transposition rate which is compatible with the development of tools for insertional mutagenesis. A subject of the invention is also therefore methods for preparing fungus mutants by inserting an Impala transposon into their genome and methods for identifying a fungus gene associated with a phenotype of interest. Finally, the invention relates to collections of fungus insertion mutants and uses thereof.
DESCRIPTION OF THE INVENTION
Polynucleotides
The present invention therefore relates to a polynucleotide, in particular an isolated or purified polynucleotide, comprising a marker gene which is inactivated by the insertion of an Impala transposon, such that said marker gene comprises, in the direction of transcription, a promoter regulatory sequence which is functional in
Magnaporthe grisea
and which is functionally linked to the coding sequence of said marker gene.
The transformation of a fungus with a polynucleotide according to the invention and the excision of the transposon lead to the expression of the marker gene. The detection of the marker gene expression thus makes it possible to monitor the transposition events and to select the insertion mutants. The selection of the
Daboussi Marie-Josee
Grosjean-Cournoyer Marie-Claire
Lebrun Marc-Henri
Villalba Francois
Aventis Cropscience S.A.
Baker & Botts L.L.P.
Guzo David
Lambertson David A.
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