Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
2001-07-23
2002-10-08
Wang, Andrew (Department: 1635)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S254300, C435S320100, C536S023100, C536S024500
Reexamination Certificate
active
06461826
ABSTRACT:
BACKGROUND OF THE INVENTION
The invention relates to essential fungal genes and their use in identifying antifungal agents.
Fungal infections (mycoses) may be cutaneous, subcutaneous, or systemic. Superficial mycoses include tinea capitis, tinea corporis, tinea pedis, perionychomycosis, pityriasis versicolor, oral thrush, and other candidoses such as vaginal, respiratory tract, biliary, eosophageal, and urinary tract candidoses. Systemic mycoses include systemic and mucocutaneous candidosis, cryptococcosis, aspergillosis, mucormycosis (phycomycosis), paracoccidioidomycosis, North American blastomycosis, histoplasmosis, coccidioidomycosis, and sporotrichosis. Fungal infections can also contribute to meningitis and pulmonary or respiratory tract diseases. Opportunistic fungal infections proliferate, especially in patients afflicted with AIDS or other diseases that compromise the immune system.
Examples of pathogenic fungi include dermatophytes (e.g.,
Microsporum canis
and other M. spp.; and Trichophyton spp. such as
T. rubrum
, and
T. mentagrophytes
), yeasts (e.g.,
Candida albicans, C. Tropicalis,
or other Candida species),
Torulopsis glabrata, Epidermophyton floccosum, Malassezia furfur
(
Pityropsporon orbiculare,
or
P. ovale
),
Cryptococcus neoformans, Aspergillus fumigatus,
and other Aspergillus sp., Zygomycetes (e.g., Rhizopus, Mucor),
Paracoccidioides brasiliensis, Blastomyces dermatitides, Histoplasma capsulatum, Coccidioides immitis,
and
Sporothrix schenckii.
Various strains of the fungus Aspergillus sp. cause aspergillosis, a potentially life-threatening disease in humans and other mammals. The clinical manifestations of aspergillosis in humans are very similar to those observed in rodents and cows. For example, necrosis, angioinvasion, and hematogenous dissemination are common features of aspergillosis in rodent and bovine model systems and in humans. In humans, aspergillosis typically is caused by inhalation of conidia (i.e., asexual spores produced by the fungus). In cattle, pathogenic Aspergillus typically enter the animal through the forestomach and then disseminate through the blood of the animal. Putative virulence factors produced by pathogenic species of Aspergillus include hydroxymate siderophores (i.e., compounds that compete with human iron-binding proteins to acquire iron to support fungal growth), lipids having the ability to inhibit complement and phagocytosis, and proteinases that can degrade elastin and other substrates.
SUMMARY OF THE INVENTION
The invention is based on the discovery of four new genes in the fungus
Aspergillus nidulans
that are essential for survival. These genes are referred to herein as AN97, AN80, AN17, and AN85; for convenience, the polypeptides encoded by these genes are referred to herein as “AN polypeptides.” The genes encoding the AN polypeptides are useful molecular tools for identifying similar genes in pathogenic microrganisms, such as pathogenic strains of Aspergillus (e.g.
Aspergillus fumigatus
and
Aspergillus flavus
). In addition, the AN polypeptides and the essential genes encoding them are useful targets for identifying compounds that are inhibitors of the pathogens in which the AN polypeptides are expressed. Such inhibitors inhibit fungal growth by being fungistatic (e.g., inhibiting reproduction or cell division) or by being fungicidal (i.e., by causing cell death).
The invention, therefore, features an isolated AN97 polypeptide having the amino acid sequence set forth as partial sequences in SEQ ID NOs 2 and 29, or conservative variations thereof. Nucleic acids encoding AN97 also are included within the invention. In particular, the invention includes an isolated nucleic acid of (a) SEQ ID NO: 1, as depicted in
FIG. 1
, or degenerate variants thereof; (b) SEQ ID NO:1, or degenerate variants thereof, wherein T is replaced by U; (c) nucleic acids complementary to (a) and (b); and (d) fragments of (a), (b), and (c) that are at least 15 base pairs in length and that hybridize under stringent conditions to genomic DNA encoding the polypeptide as partial sequences in SEQ ID NOs 2 and 29.
The invention also features an isolated AN80 polypeptide having the amino acid sequence set forth in SEQ ID NO:5, or conservative variations thereof. Nucleic acids encoding AN80 also are included. In particular, the invention includes an isolated nucleic acid of: (a) SEQ ID NO:4, as depicted in
FIG. 2
, or degenerate variants thereof; (b) SEQ ID NO:4, or degenerate variants thereof, wherein T is replaced by U; (c) nucleic acids complementary to (a) and (b); and (d) fragments of (a), (b), and (c) tat are at least 15 base pairs in length and which hybridize under stringent conditions to genomic DNA encoding the polypeptide of SEQ ID NO:5.
The invention also includes an isolated AN85 polypeptide having the amino acid sequence set forth as partial sequences in SEQ ID NOs:8, 30, 31, and 32, or conservative variations thereof. Nucleic acids encoding AN85 also are included. In particular, the invention includes an isolated nucleic acid of: (a) SEQ ID NO:7, as depicted in
FIG. 3
, or degenerate variants thereof; (b) SEQ ID NO:7, or degenerate variants thereof, wherein T is replaced by U; (c) nucleic acids complementary to (a) and (b); and (d) fragments of (a), (b), and (c) that are at least 15 base pairs in length and which hybridize under stringent conditions to genomic DNA encoding the polypeptide set forth as partial sequences in SEQ D NOs:8, 30, 31, and 32.
The invention also features an isolated AN17 polypeptide having the amino acid sequence set forth as partial sequences in SEQ ID NOs:l 1, 33, 34, and 35, or conservative variations thereof. Nucleic acids encoding AN17 also are included. In particular, the invention includes an isolated nucleic acid of: (a) SEQ ID NO:10, as depicted in
FIG. 4
, or degenerate variants thereof; (b) SEQ ID NO:10, or degenerate variants thereof, wherein T is replaced by U; (c) nucleic acids complementary to (a) and (b); and (d) fragments of (a), (b), and (c) tat are at least 15 base pairs in length and which hybridize under stringent conditions to genomic DNA encoding the polypeptide set forth as partial sequences in SEQ ID NOs:11, 33,34, and 35.
The invention also includes isolated nucleic acids that are at least 15 base pairs in length and which hybridize under stringent conditions to a nucleotide sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:7, and SEQ ID NO:10. In addition, the invention includes allelic variants (i.e., genes encoding isozymes) of the genes encoding AN97, AN17, AN80, and AN85. For example, the invention includes genes that encode an AN polypeptide but which gene includes point mutation, deletion, promoter variant, or splice site variant, provided that the resulting AN polypeptide functions as an AN polypeptide (e.g., as determined in a complementation assay, as described herein and elsewhere). Also included within the invention are isolated nucleic acid molecules containing the cDNA sequences contained with ATCC accession numbers 209473, 209472, 209484, and 209471 as well as polypeptides encoded by the cDNA sequences of these nucleic acid molecules.
Identification of the AN97, AN17, AN80, and AN85 genes and the determination that they are essential allows homologs of these genes to be found in other organisms (e.g., fungi, such as yeast like
S. cerevisiae
; mammalian cells, such as human or murine cells; or plant cells). Thus, the AN polypeptides used not only can be as a model for identifying similar essential genes in other Aspergillus strains, but also to identify homologous essential genes in other organisms, e.g.,
S. cerevisiae
. Because such genes are homologs, they can be expected to be essential for survival without the need for extensive characterization of the homologous gene or polypeptide. Even though some such homologous genes may have previously been identified, the invention allows one to determine that such genes are essential for survival. Having identified such homologous genes as essential, these genes and t
Gavrias Victoria
Koltin Yigal
Fish & Richardson P.C.
Millennium Pharmaceuticals Inc.
Wang Andrew
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