Chemistry: molecular biology and microbiology – Vector – per se
Reexamination Certificate
1999-03-16
2002-04-23
Wortman, Donna C. (Department: 1645)
Chemistry: molecular biology and microbiology
Vector, per se
C435S070100, C435S071100, C536S023100, C536S023700
Reexamination Certificate
active
06376235
ABSTRACT:
This invention relates to newly identified polynucleotides, polypeptides encoded by such polynucleotides, the use of such polynucleotides and polypeptides, as well as the production and isolation of such polynucleotides and polypeptides. More particularly, the polynucleotides and polypeptides of the present invention have been putatively identified as transcriptional regulators.
Transcriptional regulators are factors that assist RNA polymerase to more accurately initiate and conduct transcription. Some transcription factors are general factors required for all promoters while others are gene-specific and are required only for certain promoters. Some general factors are also involved in the assembly of a multi-component protein complex of the promoter.
Transcriptional factors usually are found to have at least two functional domains, one for binding DNA and one for transcriptional activation. These functions are frequently found within circumscribed structural domains which frequently retain their function even when removed from the place of their natural occurrence.
Generally,
Enterococcus faecalis
is a pathogenic bacteria species which is known to cause infections, particularly endocarditis, in mammals. Therefore, suppression of a transcriptional regulator in such a bacteria would be detrimental to the growth of the bacteria and limit its ability to infect a host with endocarditis.
In accordance with one aspect of the present invention, there are provided novel transcriptional regulator polypeptides, as well as active fragments, analogs and derivatives thereof.
In accordance with another aspect of the present invention, there are provided isolated nucleic acid molecules encoding the transcriptional regulators of the present invention including mRNAs, cDNAs, genomic DNAs as well as active analogs and fragments of such transcriptional regulators.
In accordance with another aspect of the present invention there are provided isolated nucleic acid molecules encoding mature polypeptides expressed by the DNA contained in ATCC Deposit No. 98167.
In accordance with yet a further aspect of the present invention, there is provided a process for producing such polypeptides by recombinant techniques comprising culturing recombinant prokaryotic and/or eukaryotic host cells, containing a nucleic acid sequence of the present invention, under conditions promoting expression of said transcriptional regulators and subsequent recovery of said transcriptional regulators.
In accordance with yet a further aspect of the present invention, there is provided a process for utilizing such transcriptional regulators as reagents for testing pharmaceutical antibiotics for their activity in deactivating or controlling the activity of the transcriptional regulators as part of a screening process to identify pharmaceuticals for treating or controlling
Enterococcus faecalis
infections.
In accordance with another aspect of the present invention the polynucleotides or epitopic fragments thereof are useful as in vitro agents for producing monoclonal antibodies useful in screening procedures for diagnosing
Enterococcus faecalis
bacterial infections by identifying the presence of such bacteria in a specimen from a mammal suspected of having such an infection. Also, such polynucleotides or epitopic fragments are useful as reagents to test pharmaceutical chemicals for activity in suppressing the expression of such polynucleotides. Thus, the polynucleotides and polypeptides according to the invention are useful as in vitro agents for diagnostic and screening procedures for identifying and/or treating
Enterococcus faecalis
infections in mammals.
In another aspect of the present invention, an antisense construct prepared through the use of antisense technology, may be used to control gene expression through triple-helix formation or antisense DNA or RNA, both of which methods are based on binding of a polynucleotide to DNA or RNA. For example, the 5′ coding portion of the polynucleotide sequence, which encodes for the mature polypeptides of the present invention, is used to design an antisense RNA oligonucleotide of from about 10 to 40 base pairs in length. A DNA oligonucleotide is designed to be complementary to a region of the gene involved in transcription (triple helix -see Lee et al., Nucl. Acids Res., 6:3073 (1979); Cooney et al, Science, 241:456 (1988); and Dervan et al., Science, 251: 1360 (1991)), thereby preventing transcription and the production of the
E. faecalis
ivi locus genes. The antisense RNA oligonucleotide hybridizes to the mRNA in vivo and blocks translation of mRNA molecules into transcriptional regulator polypeptides (antisense—Okano, J. Neurochem., 56:560 (1991); Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, Fla. (1988)). The oligonucleotides described above can also be delivered to cells such that the antisense RNA or DNA may be expressed in vivo to inhibit production of the ivi locus transcriptional regulators.
In accordance with yet a further aspect of the present invention, there are also provided nucleic acid probes comprising nucleic acid molecules of sufficient length to specifically hybridize to a nucleic acid sequence of the present invention.
In accordance with yet a further aspect of the present invention, there is provided a process for utilizing such transcriptional regulator polypeptides, or polynucleotides encoding such polynucleotides, for in vitro purposes related to scientific research, for example, to generate probes for identifying similar sequences which might encode similar transcriptional regulators from other organisms by using certain regions, i.e., conserved sequence regions, of the nucleotide sequence.
These and other aspects of the present invention should be apparent to those skilled in the art from the teachings herein.
REFERENCES:
The Primer, random pd(N)6 kit (Boehringer Mannheim Biochemicals 1991 Catalog), 1991.*
Herzog et al., DNA and Cell Biology, 12(6):465-474, 1993.*
Rudinger et al., in “Peptide Hormones”, edited by Parsons, J.A., University Park Press, Jun. 1976, p. 6, 1976.*
Gold, et al., Database BIOSIS on Dialog, No.: 99494305, Gold, H.S. “Detection of Genes Involved in the Pathogenesis of Experimental Enterococcal Endocarditis Using in vivo Expression Technology (IVET),” abstract, Abstracts of the Interscience Conference on Antimicrobial Agents and Chemotheraphy, vol. 36, No. 0, 1996, p. 33.
Avant Immunotherapeutics, Inc.
Grant Alan J.
Olstein Elliot M.
Wortman Donna C.
Zeman Robert A.
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