Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Transferase other than ribonuclease
Reexamination Certificate
1998-06-25
2001-09-18
Prouty, Rebecca E. (Department: 1633)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Transferase other than ribonuclease
C536S023200, C536S023400
Reexamination Certificate
active
06291218
ABSTRACT:
FIELD OF THE INVENTION
This invention relates generally to the field of gene expression and specifically to genes essential for growth and to a vector and a method for the identification of such genes, as well as identification of eukaryotic promoters.
BACKGROUND OF THE INVENTION
Many eukaryotic genes are regulated in an inducible, cell type-specific or constitutive manner. There are several types of structural elements which are involved in the regulation of gene expression. There are cis-acting elements, located in the proximity of, or within, genes which serve to bind sequence-specific DNA binding proteins, as well as trans-acting factors. The binding of proteins to DNA is responsible for the initiation, maintenance, or down-regulation of transcription of genes.
The cis-acting elements which control genes are called promoters, enhancers or silencers. Promoters are positioned next to the start site of transcription and function in an orientation-dependent manner, while enhancer and silencer elements, which modulate the activity of promoters, are flexible with respect to their orientation and distance from the start site of transcription.
For many years, various drugs have been tested for their ability to alter the expression of genes or the translation of their messages into protein products. One problem with existing drug therapy is that it tends to act indiscriminately on genes and promoters and therefore affects healthy cells as well as neoplastic cells. Likewise, in the case of a pathogen-associated disease, it is critical to administer a pathogen-specific therapy to avoid any detrimental effect on the non-infected cells.
Chitin, a linear &bgr;-1,4 linked polymer of N-acetylglucosamine, is present in the cell walls of all true fungi, but is absent from mammalian cells. Studies in
S. cerevisiae
(reviewed in Bulawa, C.,
Mol. Cell. Biol
. 12:1764, 1992; Cabib et al.,
Arch. Med. Res
., 24:301, 1993) have shown that the synthesis of chitin is surprisingly complex, requiring at least three isozymes encoded by the CHS1, CHS2, and CSD2 genes. In cell-free extracts, all of the isozymes catalyze the formation of chitin using UDP-N-acetylglucosamine as the substrate. In cells, each isozyme makes chitin at a unique location in the cell during a specified interval of the cell cycle. Genetic analyses indicate that CHS2 is involved in the synthesis of the chitin-rich primary septum that separates mother and daughter cells, CSD2 is required for synthesis of the chitin rings, and CHS1 plays a role in cell wall repair. Thus, the three isozymes are not functionally redundant and do not substitute for one another.
Chitin synthase genes have been identified from a diverse group of fungi, and analysis of the deduced amino acid sequences of these genes has lead to the identification of two chitin synthase gene families (Bowen, et al.,
Proc. Natl. Acad. Sci., USA
, 89:519, 1992). Members of one family are related to the
S. cerevisiae
CHS genes (CHS family). Based on sequence analyses, the CHS family can be subdivided into classes I, II, and III. Members of the second family are related to the
S. cerevisiae
CSD2 gene.
The functions of class II CHS genes have been investigated in a number of fungi by gene disruption. In
S. cerevisiae
, the class II CHS mutant (designated chs2) is defective in cell separation (Bulawa and Osmond,
Proc. Natl. Acad. Sci., USA
, 87:7424, 1990; Shaw et al.,
J. Cell Biol
., 114(1):111, 1990). In
A. nidulans
(Yanai et al., Biosci. 58(10):1828, 1994) and
U. maydis
(Gold and Kronstad,
Molecular Microbiology
, 11(5):897, 1994), class II CHS mutants (designated chsA and chs1, respectively) have no obvious phenotype. Thus, all of the class II CHS genes studied to date are nonessential for growth. In addition, Young, et al. identified chitin synthase gene which encodes only part of the chitin synthase activity in
C. albicans
(
Molec. Micro
., 4(2):197, 1990).
There have been methods designed to identify virulence genes of microorganisms involved in pathogenesis. For example, Osbourn, et al. utilized a promoter-probe plasmid for use in identifying promoters that are induced in vivo in plants by
Xanthomonas campestris
(
EMBO, J
. 6:23, 1987). Random chromosomal DNA fragments were cloned into a site in front of a promoterless chloramphenicol acetyltransferase gene contained in the plasmid and the plasmids were transferred into Xanthomonas to form a library. Individual transconjugates were introduced into chloramphenicol-treated seedlings to determine whether the transconjugate displayed resistance to chloramphenicol in the plant.
Knapp, et al., disclosed a method for identifying virulence genes based on their coordinate expression with other known virulence genes under defined laboratory conditions (
J. Bacteriol
., 170:5059, 1988). Mahan, et al., (U.S. Pat. No. 5,434,065) described an in vivo genetic system to select for microbial genes that are specifically induced when microbes infect their host. The method depends on complementing the growth of an auxotrophic or antibiotic sensitive microorganism by integrating an expression vector by way of homologous recombination into the auxotrophic or antibiotic sensitive microorganism's chromosome and inducing the expression of a synthetic operon which encodes transcripts, the expression of which are easily monitored in vitro following in vivo complementation.
These systems all describe methods of identifying genes involved in pathogenesis in bacterial-host systems. There is a need to identify specific targets of eukaryotic pathogens, e.g., fungi, in an infected cell which are associated with the expression of genes whose expression products are implicated in disease, in order to increase eficacy of treatment of infected cells and to increase the efficiency of developing drugs effective against genes essential for survival of these pathogens.
The present invention provides a method for identifying targets essential for growth as well as specific targets identified by the method.
SUMMARY OF THE INVENTION
The present invention provides a yeast chitin synthase (CHS1) polypeptide and a polynucleotide encoding the polypeptide. In the present invention,the class II CHS gene of
C. albicans
(encoded by the CHS1 gene) is shown to be essential for growth under laboratory conditions and for colonization of tissues during infection in vivo. Thus, CHS1 is a target for the development of antifungal drugs.
CHS1 inhibitors are useful for inhibiting the growth of a yeast. Such CHS1 inhibitory reagents include, e.g., anti-CHS1 antibodies and CHS1 antisense molecules.
CHS1 can be used to determine whether a compound affects (e.g., inhibits) CHS1 activity, by incubating the compound with CHS1 polypeptide, or with a recombinant cell expressing CHS1, under conditions suficient to allow the components to interact, and then determining the effect of the compound on CHS1 activity or expression.
The invention also provides a vector for identifying a eukaryotic regulatory polynucleotide, including a selectable marker gene; a restriction endonuclease site located at the 5′ terminus of the selectable marker gene where a regulatory polynucleotide can be inserted to be operably linked to the selectable marker gene; and a polynucleotide for targeted integration of the vector into the chromosome of a susceptible host. Preferably, the eukaryotic regulatory polynucleotide is a promoter region, and most preferably, a promoter region of pathogenic yeast such as
Candida albicans
. The vector of the invention is preferably transferred to a library of host cells, wherein each host cell contains the vector.
The vector of the invention can be used to identify a eukaryotic regulatory polynucleotide. The method involves inserting genomic DNA of a eukaryotic organism into the vector, wherein the DNA is in operable linkage with the selectable marker gene; transforming a susceptible host with the vector; detecting expression of the selectable marker gene, wherein expression is indicative of operable linkage to a regulatory polynucleotide; and iden
Bulawa Chris
Gavrias Vicky
Koltin Yigal
Riggle Perry
Winter Kenneth R.
Fish & Richardson P.C.
Millennium Pharmaceuticals Inc.
Prouty Rebecca E.
LandOfFree
Identification of eukaryotic growth-related genes and... does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Identification of eukaryotic growth-related genes and..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Identification of eukaryotic growth-related genes and... will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-2501616