Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
1999-04-16
2004-02-03
Wang, Andrew (Department: 1635)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S252300, C435S252330, C435S320100, C435S471000, C536S023100, C536S024100, C536S024500
Reexamination Certificate
active
06686174
ABSTRACT:
FIELD OF THE INVENTION
The invention relates to the field of biotechnology, and more particularly to the field of regulating the translation of mRNA and the production of proteins.
BACKGROUND OF THE INVENTION
Bacteria are the causative agents for a great many diseases in plants and animals, including humans. Before the advent of antibiotics, such as penicillin, bacterial infections were considered to be non-treatable. Since that time, additional antibiotics have been developed to control and kill bacteria and treat bacterial infections.
Unfortunately, however, many antibiotics have proven over time to be less and less effective at controlling bacterial populations due to the development of resistance of the bacteria to the antibiotics.
Science has responded by discovering newer and better antibiotics with which to treat resistant bacteria. As fast as new antibiotics can be produced, resistant strains of bacteria develop. Therefore, there is a clear and pressing need for new means of killing harmful bacteria.
The invention provides a novel mechanism for killing bacteria by disrupting bacterial protein production. The risk of development of bacterial resistance to the compounds and method of the invention is minimized, as compared to that encountered with traditional antibiotics, because the invention takes advantage of natural processes of the bacteria.
The invention further provides for the overproduction of the 5′ untranslated region of the mRNA of a target cold shock protein. Several other novel aspects are described further herein after.
SUMMARY OF EMBODIMENTS OF THE INVENTION
It has been unexpectedly discovered that protein synthesis in bacteria can be inhibited or even completely stopped by overexpressing in the bacteria an RNA comprising a sequence which is substantially complementary to a portion of the bacterial 16S rRNA adjacent to the decoding region, which portion is known as the anti-downstream box (ADB). The RNA sequence which is substantially complementary to the ADB is referred as a downstream box (DB) because, in a naturally occurring bacterial mRNA, the DB is positioned downstream from the initiation codon of the mRNA. The structure of the 3′ region of 16S rRNA and the function of the DB box as a translation initiation signal in bacteria is described in Sprengart, et al., EMBO Journal, 15(3):665-674 (1996), which is incorporated herein by reference.
The invention comprises several embodiments. In one embodiment, the invention is a method for arresting or inhibiting the production of bacterial proteins. The method of the invention comprises overexpressing in a bacterial cell an mRNA which comprises an initiation codon and a downstream box. The downstream box is preferably 3′ to the initiation codon, with an intervening nucleotide sequence of 0 to 30 nucleotides. Alternatively, the downstream box may overlap the initiation codon. In this latter situation, any or all of the three nucleotides of the initiation codon may constitute the 5′ end of the downstream box. The DB of the overexpressed mRNA is allowed to anneal to the ADB, thereby effectively binding the 16S rRNA and preventing translation of other mRNAs, ultimately preventing production of bacterial proteins.
In another embodiment, the invention is an oligonucleotide mRNA construct for the inhibition of protein synthesis in bacteria. The RNA construct has a nucleotide sequence which comprises an initiation codon and a DB sequence 3′ to, or overlapping, the initiation codon. Preferably, the RNA construct is free of a site for RNA endonucleases.
In another embodiment, the invention is an oligonucleotide DNA construct, which DNA construct codes for an mRNA which comprises an initiation codon and a DB sequence 3′ to, or overlapping, the initiation codon.
In a further embodiment, the invention is a vehicle for transforming a bacterial cell, which vehicle contains a DNA promoter sequence which is operably linked to a DNA sequence which codes for an mRNA which comprises an initiation codon and a DB sequence 3′ to, or overlapping, the initiation codon.
A further embodiment is a bacterial cell which has been transformed with a vehicle containing a DNA promoter sequence which is operably linked to a DNA sequence which codes for an mRNA which comprises an initiation codon and a DB sequence 3′ to or overlapping the initiation codon.
The invention is applicable to, and can be practiced in, all bacteria because of the existence of the 16S rRNA, which is a well-conserved sequence. Thus, the practice of the invention is not dependent on the bacteria species used, such as
E. coli
, which is used herein to illustrate the invention. See, Goodfellow and O'Donnell, Handbook of New Bacterial Systematics, Academic Press (1993); Stackebrandt and Goebel, International Journal of Systematic Bacteriology, 44(4):846-849 (1994); Durand and Gros, lFEMS Microbiology Letters, 140:193-198 (1996); and Olsen and Woese, FASEB Journal, 7:113123 (1993), each of which is incorporated herein by reference. The fact that bacteria in which the 16S rRNA is highly homologous with respect to that of
E. coli
includes mammalian pathogens such as Mycobacterium spp. and
Legionella pneumophila
, and even non-pathogen symbionts of marine animals, such as
Linga pensylvanica
and
Bathymodiolus thermophilus
, is indicative of the highly conserved nature of the 16S rRNA and the general applicability of the present invention. The conserved nature of the 16S rRNA permits identification of the ADB in a given bacteria from the nucleotide sequence of the 16S rRNA which can be found for bacteria in the GenBank database. Means of determining the nucleotide sequence of the 165 rRNA are known. See, for example, Lane et al., Proc. Natl. Acad. Sci., 82:6955-6959 (1985), and Bottger, FEMS Microbiology Letters, 65:171-176 (1989), each of which is incorporated herein by reference. The bacterial 16S rRNA contains, at its 3′ end, an anti-Shine-Dalgarno region (SD) and a decoding region. The ADB is a 12 to 14 nucleotide long region close to the decoding region of 16S rRNA. Once the ADB is identified and its sequence ascertained, the constructs of the invention may be readily constructed for any particular bacteria, as may the vehicle of the invention, and the method of the invention may likewise be practiced in any bacteria.
Moreover, because of the highly conserved nature of the sequence of the 3′ end region of the 16S rRNA, it is conceived that a DB which is substantially complementary to the ADB of the 16S rRNA of any one particular bacterial species will be sufficiently complementary to the ADB of the 16S rRNA of a second bacterial species to enable the method of the invention to be practiced in different species of bacteria using a DB of the same or similar sequence.
REFERENCES:
patent: 5654169 (1997-08-01), Oppenheim et al.
patent: 5714575 (1998-02-01), Inouye et al.
patent: 5981280 (1999-11-01), Fang et al.
Sprengart et al. The downstream box: an efficient and independent translation initiation signal inEscherichia coli. EMBO J. 15(3): 665-674 (1996).
Fang Li
Inouye Masayori
Jiang Weinning
Mitta Masanori
Larson Thomas G
Schnader Harrison Segal & Lewis LLP
The University of Medicine and Dentistry of New Jersey
Wang Andrew
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