Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2000-01-20
2004-10-12
Ketter, James (Department: 1636)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S252300, C435S254220, C536S023100, C536S024100
Reexamination Certificate
active
06803191
ABSTRACT:
BACKGROUND OF THE INVENTION
Candida
is an opportunistic yeast that lives in the mouth, throat, intestines, and genitourinary tract of most humans. In a healthy human body, the population of
Candida
is kept in check by the immune system and by a competitive balance with other microorganisms. But when the body's immune system is compromised, as in AIDS patients and in patients undergoing immunosuppressive therapy,
Candida
will grow uncontrolled, leading to systemic infection called “
Candida mycosis
.” If left untreated, such systemic infections frequently lead to the death of the patients.
Candida albicans
is a species of particular interest to scientists and doctors because 90% of all cases of
Candida mycosis
are caused by this species.
At present, the therapy principally available for invasive infections is based on relatively few antimycotics, such as amphotericin B and flucytosine, or the azole derivatives fluconazole and itraconazole. These antimycotics cause serious side effects, such as renal insufficiency, hypocalcaemia and anaemia, as well as unpleasant constitutional symptoms such as fever, shivering and low blood pressure. Amphotericin B is toxic to the kidneys, for example, and yet the pharmaceutical is therapeutic only if administered at dose levels near to being toxic. A discussion of the pharmaceuticals used for treatment and their corresponding side effects can be found, for example, in Boyd, et al., BASIC MEDICAL MICROBIOLOGY (2d ed.), Little, Brown and Company, (1981).
Given the deficiencies of conventional therapies against
Candida
, a need exists for developing pharmaceuticals that are effective in this regard and also safe to use. One step in the development of such pharmaceuticals requires a method for screening compounds in order to identify pharmaceutical candidates.
SUMMARY OF THE INVENTION
It therefore is an object of the present invention to provide an isolated polynucleotide sequence coding for a protein that is linked to phenotypic switching in
Candida albicans.
It is a further object of the invention to provide a method for screening compounds to identify pharmaceutical candidates for effectively inhibiting the pathogenicity of
C. albicans.
In accomplishing these and other objects, there has been provided, according to one aspect of the present invention, an isolated polynucleotide that codes for such a protein and that hybridizes, under stringent conditions, to the polynucleotide sequence of SEQ ID NO:1, shown below in FIG.
1
. In a preferred embodiment, the polynucleotide has the sequence of SEQ ID NO:3 (FIG.
2
). In another preferred embodiment, the protein displays a kinase activity.
In accordance with another aspect of the present invention, a method is provided for screening compounds to identify pharmaceutical candidates. The inventive method comprises the steps of (A) providing a plurality of cells from yeast species that exhibit phenotypic switching, at least some of which contain (i) a polynucleotide coding for a CaNIK1 protein and (ii) a promoter that is operably linked to the polynucleotide, such that the plurality of cells produces the protein; then (B) bringing the plurality into contact with a test substance; and (C) assessing what effect, if any, the test substance has on the expression of the DNA segment. Assessment step (C) can comprise, for example, of monitoring the level either of the protein or the corresponding mRNA transcript produced by the plurality of cells. In another embodiment, step (C) comprises monitoring the level of kinase activity, within the plurality, that typifies the protein.
In yet another embodiment of the present invention, a promoter is operably linked to a reporter gene. In this context, step (C) comprises monitoring the level of transcription of the reporter gene, after contact between the plurality of cells and the test substance.
Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, only indicate preferred embodiments of the invention.
REFERENCES:
patent: 5939306 (1999-08-01), Alex et al.
patent: 96/40939 (1996-12-01), None
David R. Soll, “Gene regulation during high-frequency switching inCandida albicans” Microbiology, vol. 143, 1997, pp 279-288,XP002083237.
Database Swiss-Prot Accession No. p46588, Jun. 15, 1995 Ball T and Rosamond J: XP002083293 DNA Polymerase III gene (po13) fromCandida albicans.
Nagahashi et al., Isolation of CaSLNI and CaNIK1, the genes for osmosensing histidine kinase homologues, from the pathogenic fungusCandida albicans: Microbiology, vol. 144, 1998, pp 425-432, XP002083238.
Srikantha et al., The WH11 gene ofCandida albicansis regulated in two distinct developmental programs through the same transcription activation sequences: Journal Of Bacteriology, vol. 179, No. 12, 1997, pp 3837-3844, XP002083239.
Srikantha et al., “The sea pansyRenilla reniformisluciferas serves as a sensitive bioluminescent reporter, for differential gene expression inCandida albicans” Journal of Bacteriology, vol. 178, No. 1, 1996 pp 121-129, XP00208236.
Timerlake, W.E., “Cellular Reporters for Antifungal Drug Discovery”, PAP Conference Discovery Mode Action Antifungal Agent, 1995, pp 17-29 XP000603570.
Soll David R.
Srikantha Thyagarajan
Foley & Lardner LLP
Ketter James
Lambertson David
University of Iowa Research Foundation
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