Ribb

Details Ribb Ribb

1997-11-25

2001-06-26

Minnifield, Nita (Department: 1645)

Chemistry: natural resins or derivatives; peptides or proteins;

Proteins, i.e., more than 100 amino acid residues

C530S300000, C530S324000, C424S184100, C424S200100, C424S235100, C424S237100, C424S244100, C424S234100, C424S093430, C514S002600, C514S012200, C514S014800

Reexamination Certificate

active

06252044

ABSTRACT:

FIELD OF THE INVENTION
This invention relates to newly identified polynucleotides and polypeptides, and their production and uses, as well as their variants, agonists and antagonists, and their uses. In particular, the invention relates to novel polynucleotides and polypeptides of the Riboflavin synthase (&agr;-subunit) family, hereinafter referred to as “ribB”.
BACKGROUND OF THE INVENTION
The Streptococci make up a medically important genera of microbes known to cause several types of disease in humans, including, for example, otitis media, conjunctivitis, pneumonia, bacteremia, meningitis, sinusitis, pleural empyema and endocarditis, and most particularly meningitis, such as for example infection of cerebrospinal fluid. Since its isolation more than 100 years ago,
Streptococcus pneumoniae
has been one of the more intensively studied microbes. For example, much of our early understanding that DNA is, in fact, the genetic material was predicated on the work of Griffith and of Avery, Macleod and McCarty using this microbe. Despite the vast amount of research with
S. pneumoniae,
many questions concerning the virulence of this microbe remain. It is particularly preferred to employ Streptococcal genes and gene products as targets for the development of antibiotics.
Riboflavin (vitamin B2) is a member of the B complex of vitamins which function as coenzymes in metabolic reactions. Riboflavin has two coenzyme forms, flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) which act in oxidation-reduction reactions such as the cytochrome system of electron transport and the oxidative degradation of pyruvate, fatty acids and amino acids. The first committed step in the biosynthesis of riboflavin is the opening of the imidazole ring of GTP. In the presence of 3 H
2
O and Mg
++
, the C-8 of GTP is released as formate accompanied by the release of pyrophosphate by the action of GTP cyclohyrolase II (GCH2; EC 3.5.4.25). This enzyme function is encoded by ribA in bacteria and rib1 in yeast species. Through a series of steps, involving 3,4-dihydroxy-2-butanone 4 phosphate synthase (ribA), 6,7-dimethyl-8-ribityllumazine synthase (ribH), riboflavin synthase (ribB), pyrimidine deaminase and pyrimidine reductase (ribG), enzymes encoded by genes within the riboflavin biosynthesis operon, riboflavin is formed. Because the genes required for riboflavin biosynthesis are present in many pathogenic microorganisms, and since riboflavin biosynthesis has shown to be required for virulence in the swine pathogen
Actinobacillus pleuropneumoniae
(Fuller, T E, et al. (1996) A riboflavin auxotroph of
Actinobacillus pleuropneumoniae
is attenuated in swine. Infect. Immun. 64:4659-4664), these gene products represent broad spectrum antibacterial as well as antifungal targets.
The frequency of
Streptococcus pneumoniae
infections has risen dramatically in the past few decades. This has been attributed to the emergence of multiply antibiotic resistant strains and an increasing population of people with weakened immune systems. It is no longer uncommon to isolate
Streptococcus pneumoniae
strains which are resistant to some or all of the standard antibiotics. This phenomenon has created a demand for both new anti-microbial agents, vaccines, and diagnostic tests for this organism.
Clearly, there exists a need for factors, such as the ribB embodiments of the invention, that have a present benefit of being useful to screen compounds for antibiotic activity. Such factors are also useful to determine their role in pathogenesis of infection, dysfunction and disease. There is also a need for identification and characterization of such factors and their antagonists and agonists to find ways to prevent, ameliorate or correct such infection, dysfunction and disease.
Certain of the polypeptides of the invention possess amino acid sequence homology to a known
Actinobacillus pleuropnewnoniae
ribB protein. See Swiss Prot. Accession No. P50854; GenBank Accession No. U27202. Also see Perkins et al. In:
Bacillus subtilis and Other Gram-Positive Bacteria.
Eds: Sonenshein, A L, Hoch, J A and Losick, R. 1993. American Society for Microbiology.
SUMMARY OF THE INVENTION
It is an object of the invention to provide polypeptides that have been identified as novel ribB polypeptides by homology between the amino acid sequence set out in Table 1 [SEQ ID NO: 2 or 4] and a known amino acid sequence or sequences of other proteins such as
Actinobacillus pleuropneumoniae
ribB protein.
It is a further object of the invention to provide polynucleotides that encode ribB polypeptides, particularly polynucleotides that encode the polypeptide herein designated ribB.
In a particularly preferred embodiment of the invention the polynucleotide comprises a region encoding ribB polypeptides comprising a sequence set out in Table 1 [SEQ ID NO: 1 or 3] which includes a full length gene, or a variant thereof.
In another particularly preferred embodiment of the invention there is a novel ribB protein from
Streptococcus pneumoniae
comprising the amino acid sequence of Table 1 [SEQ ID NO:2 or 4], or a variant thereof.
A further aspect of the invention there are provided isolated nucleic acid molecules encoding ribB, particularly
Streptococcus pneumoniae
ribB, including mRNAs, cDNAs, genomic DNAs. Further embodiments of the invention include biologically, diagnostically, prophylactically, clinically or therapeutically useful variants thereof, and compositions comprising the same.
In accordance with another aspect of the invention, there is provided the use of a polynucleotide of the invention for therapeutic or prophylactic purposes, in particular genetic immunization. Among the particularly preferred embodiments of the invention are naturally occurring allelic variants of ribB and polypeptides encoded thereby.
Another aspect of the invention there are provided novel polypeptides of
Streptococcus pneumoniae
referred to herein as ribB as well as biologically, diagnostically, prophylactically, clinically or therapeutically useful variants thereof, and compositions comprising the same.
Among the particularly preferred embodiments of the invention are variants of ribB polypeptide encoded by naturally occurring alleles of the ribB gene.
In a preferred embodiment of the invention there are provided methods for producing the aforementioned ribB polypeptides.
In accordance with yet another aspect of the invention, there are provided inhibitors to such polypeptides, useful as antibacterial agents, including, for example, antibodies.
In accordance with certain preferred embodiments of the invention, there are provided products, compositions and methods for assessing ribB expression, treating disease, assaying genetic variation, and administering a ribB polypeptide or polynucleotide to an organism to raise an immunological response against a bacteria, especially a
Streptococcus pneumoniae
bacteria.
In accordance with certain preferred embodiments of this and other aspects of the invention there are provided polynucleotides that hybridize to ribB polynucleotide sequences, particularly under stringent conditions.
In certain preferred embodiments of the invention there are provided antibodies against ribB polypeptides.
In other embodiments of the invention there are provided methods for identifying compounds which bind to or otherwise interact with and inhibit or activate an activity of a polypeptide or polynucleotide of the invention comprising: contacting a polypeptide or polynucleotide of the invention with a compound to be screened under conditions to permit binding to or other interaction between the compound and the polypeptide or polynucleotide to assess the binding to or other interaction with the compound, such binding or interaction being associated with a second component capable of providing a detectable signal in response to the binding or interaction of the polypeptide or polynucleotide with the compound; and determining whether the compound binds to or otherwise interacts with and activates o

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