Human arsenite-resistance protein

Abstract

The invention provides a human arsenite-resistance protein (NITE) and polynucleotides which identify and encode NITE. The invention also provides expression vectors, host cells, antibodies, agonists, and antagonists. The invention also provides methods for diagnosing, treating or preventing disorders associated with expression of NITE.

Description

[0001] This application is a continuation application of U.S. application Ser. No. 09 / 300,172, filed Apr. 27, 1999, entitled HUMAN ARSENITE-RESISTANCE PROTEIN, which is a divisional application of U.S. application Ser. No. 09 / 074,512, filed May 7, 1998, issued Sep. 14, 1999 as U.S. Pat. No. 5,952,479, entitled HUMAN ARSENITE-RESISTANCE PROTEIN, all of which applications and patents are hereby expressly incorporated by reference.[0002] This invention relates to nucleic acid and amino acid sequences of a human arsenite-resistance protein and to the use of these sequences in the diagnosis, treatment, and prevention of cancer, developmental disorders, reproductive disorders, and autoimmune / inflammatory disorders.[0003] Arsenic is a toxin and carcinogen found in water, soil, food, and air. The natural source of arsenic is from weathering of arsenic-rich geologic formations. Arsenic also derives from human agricultural and industrial sources. Arsenic is used in pesticides, wood preservati...

AI Technical Summary

Benefits of technology

[0079] The nucleic acid sequences encoding NITE may be extended utilizing a partial nucleotide sequence and employing various PCR-based methods known in the art to detect upstream sequences, such as promoters and regulatory elements. For example, one method which may be employed, restriction-site PCR, uses universal and nested primers to amplify unknown sequence from genomic DNA within a cloning vector. (See, e.g., Sarkar, G. (1993) PCR Methods Applic. 2:318-322.) Another method, inverse PCR, uses primers that extend in divergent directions to amplify unknown sequence from a circularized template. The template is derived from restriction fragments comprising a known genomic locus and surrounding sequences. (See, e.g., Triglia, T. et al. (1988) Nucleic Acids Res. 16:8186.) A third method, capture PCR, involves PCR amplification of DNA fragments adjacent to known sequences in human and yeast artificial chromosome DNA. (See, e.g., Lagerstrom, M. et al. (1991) PCR Methods Applic. 1:111-119.) In this method, multiple restriction enzyme digestions and ligations may be used to insert an engineered double-stranded sequence into a region of unknown sequence before performing PCR. Other methods which may be used to retrieve unknown sequences are known in the art. (See, e.g., Parker, J. D. et al. (1991) Nucleic Acids Res. 19:3055-306). Additionally, one may use PCR, nested primers, and PromoterFinder.TM. libraries to walk genomic DNA (Clontech, Palo Alto, Calif.). This procedure avoids the need to screen libraries and is useful in finding intron / exon junctions. For all PCR-based methods, primers may be designed using commercially available software, such as OLIGO.TM. 4.06 Primer Analysis software (National Biosciences Inc., Plymouth, Minn.) or another appropriate program, to be about 22 to 30 nucleotides in length, to have a GC content of about 50% or more, and to anneal to the template at temperatures of about 68.degree. C. to 72.degree. C.

[0086] In order to express a biologically active NITE, the nucleotide sequences encoding NITE or derivatives thereof may be inserted into an appropriate expression vector, i.e., a vector which contains the necessary elements for transcriptional and translational control of the inserted coding sequence in a suitable host. These elements include regulatory sequences, such as enhancers, constitutive and inducible promoters, and 5' and 3' untranslated regions in the vector and in polynucleotide sequences encoding NITE. Such elements may vary in their strength and specificity. Specific initiation signals may also be used to achieve more efficient translation of sequences encoding NITE. Such signals include the ATG initiation codon and adjacent sequences, e.g. the Kozak sequence. In cases where sequences-encoding NITE and its initiation codon and upstream regulatory sequences are inserted into the appropriate expression vector, no additional transcriptional or translational control signals may be needed. However, in cases where only coding sequence, or a fragment thereof, is inserted, exogenous translational control signals including an in-frame ATG initiation codon should be provided by the vector. Exogenous translational elements and initiation codons may be of various origins, both natural and synthetic. The efficiency of expression may be enhanced by the inclusion of enhancers appropriate for the particular host cell system used. (See, e.g., Scharf, D. et al. (1994) Results Probl. Cell Differ. 20:125-162.)

[0094] For long term production of recombinant proteins in mammalian systems, stable expression of NITE in cell lines is preferred. For example, sequences encoding NITE can be transformed into cell lines using expression vectors which may contain viral origins of replication and / or endogenous expression elements and a selectable marker gene on the same or on a separate vector. Following the introduction of the vector, cells may be allowed to grow for about 1 to 2 days in enriched media before being switched to selective media. The purpose of the selectable marker is to confer resistance to a selective agent, and its presence allows growth and recovery of cells which successfully express the introduced sequences. Resistant clones of stably transformed cells may be propagated using tissue culture techniques appropriate to the cell type.

[0122] In other embodiments, any of the proteins, antagonists, antibodies, agonists, complementary sequences, or vectors of the invention may be administered in combination with other appropriate therapeutic agents. Selection of the appropriate agents for use in combination therapy may be made by one of ordinary skill in the art, according to conventional pharmaceutical principles. The combination of therapeutic agents may act synergistically to effect the treatment or prevention of the various-disorders described above. Using this approach, one may be able to achieve therapeutic efficacy with lower dosages of each agent, thus reducing the potential for adverse side effects.

[0135] Ribozymes, enzymatic RNA molecules, may also be used to catalyze the specific cleavage of RNA. The mechanism of ribozyme action involves sequence-specific hybridization of the ribozyme molecule to complementary target RNA, followed by endonucleolytic cleavage. For example, engineered hammerhead motif ribozyme molecules may specifically and efficiently catalyze endonucleolytic cleavage of sequences encoding NITE.

[0168] With respect to cancer, the presence of a relatively high amount of transcript in biopsied tissue from an individual may indicate a predisposition for the development of the disease, or may provide a means for detecting the disease prior to the appearance of actual clinical symptoms. A more definitive diagnosis of this type may allow health professionals to employ preventative measures or aggressive treatment earlier thereby preventing the development or further progression of the cancer.

Problems solved by technology

Acute arsenic poisoning can be fatal, killing by impairment of cellular respiration in the case of arsenite and arsenate, or by hemolysis in the case of arsine gas.

Glycolytic and .beta.-oxidation enzymes are inhibited by this reaction, which may cause increased and unregulated cell death.

This cell death may cause developmental defects because reduced cell proliferation is associated with neural tube defects in mice.

Inorganic arsenic impairs the assembly and disassembly of microtubules and may interfere with mitotic spindle formation, embryonic cell division, and neural tube formation.

Arsenite reaction with the thiols present in histones and nucleic acids may cause the chromosomal damage associated with arsenic exposure.

However, acyl arsenates are less stable than the corresponding phosphates and decompose rapidly, inhibiting glycolytic and oxidative phosphorylation enzymes.

The researchers concluded that humans may be unable to induce one or more protective proteins in response to arsenite.

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