Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
1997-12-24
2001-05-29
Priebe, Scott D. (Department: 1632)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C435S320100, C536S024300, C536S024320
Reexamination Certificate
active
06239264
ABSTRACT:
Priority is claimed to Swiss App. No. 0016/97, filed Dec. 31, 1996, incorporated by reference.
FIELD OF THE INVENTION
The present invention relates to genomic DNA sequences obtained from terminal sequencing of random genomic fragments of the filamentous fungus
Ashbya gossypii
and uses thereof.
BACKGROUND OF THE INVENTION
The phytopathogenic fungus
Ashbya gossypii
is a filamentously growing ascomycete that was first isolated as a plant pathogen in tropical and subtropical regions. It infects the seed capsule of cotton plants (Ashby S. F. and Nowell W. (1926) Ann. Botany 40: 69-84) and has also been isolated from tomatoes and citrus fruits (Phaff H. J. and Starmer W. T. (1987) In “The Yeasts”, Vol. I Rose A. H., Harrison, J. S. (eds), Academic Press, London, 123 ff; Dammer K. H. and Ravelo H. G. (1990). Arch. Phytopathol. Pflanzenschutz, Berlin 26: 71-78Dammer and Ravelo, 1990). The infection of the seed capsule is caused by transmission of
A. gossypii
mycelium pieces or spores by stinging-sucking insects and causes a disease called stigmatomycosis.
Studies characterising the karyotype of
A. gossypii
have been performed (Wright, 1990; Wendland, 1993; Gaudenz, 1994, “The small genome of the filamentous fungus
Ashbya gossypii
: Assessment of the karyotype”, Diploma Thesis, Department of Applied Microbiology, Biocenter, University Basel). It has been found using yeast chromosomes of precisely known length as size markers that the genome of
A. gossypii
has a total nuclear genome size of 8.85 Mb.
A. gossypii
is systematically grouped to the endomycetales belonging to the family of spermophthoraceae (Lodder J (1970) General classification of the yeasts. In: “The Yeasts”, Lodder J. (edt.), North Holland Publishing Company, Amsterdam-London, 1ff Lodder, 1970). This classification is based on the observation that the spores that develop in hyphal compartments called sporangia look like ascospores, which are defined as endproducts of meiosis (Müller E. und Löffler W. (1971) Mykologie. Grundri&bgr; der Pilzkunde. DTV-Thieme, Stuttgart, 37 ff). However, in several respects,
A. gossypii
more closely resembles the budding yeast
Saccharomyces cerevisiae
than other filamentous fungi. For example, homologous recombination has been found to be the main mode of integration of transforming DNA (Steiner S. (1991). Diplomarbeit, Institut für Mikro- und Molekularbiologie der Justus Liebig Universität Gie&bgr;enSteiner et al., 1995), which is in contrast to findings made in many other filamentous fungi (reviewed by Fincham J. R. S (1989) Transformation in fungi. Microbiol. Rev. 53 (1): 148-170).
Additionally, sequence analysis of the
A gossypii
TEF, LEU2 and THR4 genes (Altmann-Jöhl and Philippsen, 1996; Mohr, May 1997; Steiner and Philippsen, 1994) has identified high sequence homology to their functional homologues in
S. cerevisiae
. In addition, for the latter genes, syntenic (positionally conserved) arrangement of adjacent homologous ORF's has been found. The growing number of completely sequenced reference genomes, such as for example
S. cerevisiae
, offers new prospects for rapid comparative gene and genome analysis of so far less characterized organisms, such as
A. gossypii
, in parallel or even before the application of genetic techniques.
SUMMARY OF THE INVENTION
In view of the above, the present invention provides genomic DNA sequences obtained from terminal sequencing of random genomic fragments of
Ashbya gossypii
. The present invention particularly relates to genomic
A. gossypii
DNA sequences that are obtainable from the series of clones listed in Table 1 and presented in the attached Sequence Listing. Some of these
A. gossypii
sequences are homologous to
S. cerevisiae
sequences and to sequences from other filamentous fungi, e.g. ORF's specifically required for growth in filamentous fungi. Others of these
A. gossypii
sequences, such as those set forth in Table 2, have no homology to
S. cerevisiae
sequences, including sequences which have no homology to known sequences from any other fungus. The sequences of the invention find particular use in forensic identification, chromosome mapping, chromosome identification, and tagging of genes of known and useful function. Procedures such as these can easily be carried out by those of ordinary skill in the art.
The present invention also concerns chimeric genes comprising the sequences of the invention, recombinant vectors comprising such chimeric genes, wherein the vectors are capable of being stably transformed into hosts, as well as hosts stably transformed with such vectors. Preferred hosts are fungi such as
A. gossypii
as well as bacteria.
Furthermore, the present invention relates to the identification and characterization of
A. gossypii
ORF's based on the high homology of primary structures in
A. gossypii
and
S. cerevisiae
and the sequences obtained therewith. The present invention also relates to the use of the
A. gossypii
sequences provided in the Sequence Listing to characterize genes and gene organization of this ascomycete by inter-genomic comparison, to identify biosynthetic genes that can be used as selection markers, to isolate promoters and terminators for application in a homologous as well as heterologous context, to find putative centromere containing clones, general information about genome organization and in addition to identify ORF's containing single read sequences (SRS) with no homology to
S. cerevisiae
or any other organism, which allows the identification of
A. gossypii
-specific genes.
The present invention also concerns a method for the detection of
Ashbya gossypii
, comprising the steps of isolating DNA from an organism to be characterized; subjecting said DNA to polymerase chain reaction amplification using a primer derived from one of the
A. gossypii
sequences provided in the Sequence Listing; and visualizing the product or products of said polymerase chain reaction amplification, whereby detection of the product or products of said polymerase chain reaction amplification indicates that the organism to be characterized is
Ashbya gossypii
.
ABBREVIATIONS
LIPS - Linked Pairs of Sequences
SRS - Single Read Sequence
MCS - Multi Cloning Site
RP - Reversed Primer
ORF - Open Reading Frame
UP - Universal Primer
DESCRIPTION OF THE INVENTION
Encompassed by the present invention is a method of sequencing the termini of randomly picked
A. gossypii
shotgun clones to obtain linked pairs of genomic sequences. Said linked pairs of genomic sequences can be used for identification of open reading frames (ORFs) showing or lacking homology to functionally characterized or uncharactezized genes from
S cerevisiae
, other fungi or other organisms. The sequence information provided herein in the attached Sequence Listing is sufficient to generate gene deletions in Ashbya by using, for example, by PCR-based gene targeting methods as described herein.
One of the main prerequisites for success in such an analysis is a relatively compact, organized genome. This is required to obtain a maximum of information from the limited length of single read sequence (SRS) analysis.
A. gossypii
represents such a compact genome. The presence within the Ashbya genome of short intergenic regions and rare occurrence of introns increases the probability of finding matches to open reading frames (ORF's) in the majority of SRS's.
Thus one embodiment of the present invention is a method to identify and characterize
A. gossypii
ORF's by sequence comparison of their
S. cerevisiae
homologues without the requirement of complete sequence information for the
A. gossypii
ORF'S.
Further encompassed by the invention is a method for characterization of an Ashbya gene, the knockout of which leads to a non-growth phenotype.
In a specific embodiment of the invention a method for characterization and validation of an Ashbya gene is provided comprising
(a) inserting into Ashbya sequences of genomic pAG clones as provided herein in the attached Sequence Listing a chimeric gene con
Knechtle Philipp
Mohr Christine
Philippsen Peter
Pöhlmann Rainer
Rebischung Corinne
Meigs J. Timothy
Priebe Scott D.
Syngenta Participations (AG)
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