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
1998-08-31
2001-02-27
Priebe, Scott D. (Department: 1632)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S320100, C435S252300, C435S325000, C435S455000, C435S471000, C536S023100, C536S023200, C536S023400, C536S023700
Reexamination Certificate
active
06194170
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 polynucleotides and polypeptides of the MurF family, as well as their variants, hereinafter referred to as “MurF,” “MurF polynucleotide(s),” and “MurF polypeptide(s)” as the case may be.
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
Streptococcus 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.
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 an unmet medical need and demand for new anti-microbial agents, vaccines, drug screening methods, and diagnostic tests for this organism.
Moreover, the drug discovery process is currently undergoing a fundamental revolution as it embraces “functional genomics,” that is, high throughput genome- or gene-based biology. This approach is rapidly superseding earlier approaches based on “positional cloning” and other methods. Functional genomics relies heavily on the various tools of bioinformatics to identify gene sequences of potential interest from the many molecular biology databases now available as well as from other sources. There is a continuing and significant need to identify and characterize further genes and other polynucleotides sequences and their related polypeptides, as targets for drug discovery.
The enzyme UDP-N-acetylmuramoyl-L-alanyl-D-glutamyl-L-lysine:D-alanyl-D-alanine ligase, encoded by the gene MurF catalyses the addition of the last aminoacids (D-alanyl-D-alanine) of the peptide moiety in peptidoglycan biosynthesis to form UDP-N-acetylmuramate pentapeptide. The gene has been cloned and sequenced from
Escherichia coli
and the corresponding protein has been over-expressed, purified and characterised (Duncan, K., van Heijenoort, J. & Walsh, C. T. (1990) Biochemistry, 29, 2379-2386). MurF has also been found in such organisms as
Bacillus subtilis
and
Haemophilus influenzae.
The discovery of a MurF homologue in the human pathogen
Streptococcus pneumoniae
will allow us to produce UDP-N-acetylmuramoyl-L-alanyl-D-glutamyl-L-lysine:D-alanyl-D-alanine ligase enzyme which can then be used to screen for novel inhibitors. Inhibitors of this protein have utility in anti-bacterial therapy as they will prevent the construction of the bacterial cell wall.
Clearly, there exists a need for polynucleotides and polypeptides, such as the MurF embodiments of the invention, that have a present benefit of, among other things, being useful to screen compounds for antimicrobial 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.
SUMMARY OF THE INVENTION
The present invention relates to MurF, in particular MurF polypeptides and MurF polynucleotides, recombinant materials and methods for their production. In another aspect, the invention relates to methods for using such polypeptides and polynucleotides, including treatment of microbial diseases, amongst others. In a further aspect, the invention relates to methods for identifying agonists and antagonists using the materials provided by the invention, and for treating microbial infections and conditions associated with such infections with the identified agonist or antagonist compounds. In a still further aspect, the invention relates to diagnostic assays for detecting diseases associated with microbial infections and conditions associated with such infections, such as assays for detecting MurF expression or activity.
Various changes and modifications within the spirit and scope of the disclosed invention will become readily apparent to those skilled in the art from reading the following descriptions and from reading the other parts of the present disclosure.
DESCRIPTION OF THE INVENTION
The invention relates to MurF polypeptides and polynucleotides as described in greater detail below. In particular, the invention relates to polypeptides and polynucleotides of a MurF of
Streptococcus pneumoniae
, which is related by amino acid sequence homology to MurF from
B. subtilis
polypeptide. The invention relates especially to MurF having the nucleotide and amino acid sequences set out in Table 1 as SEQ ID NO:1 or 3 and SEQ ID NO:2 or 4 respectively. Note that sequences recited in the Sequence Listing below as “DNA” represent an exemplification of the invention, since those of ordinary skill will recognize that such sequences can be usefully employed in polynucleotides in general, including ribopolynucleotides.
TABLE 1
MurF Polynucleotide and Polypeptide Sequences
(A)
Streptococcus pneumoniae
MurF polynucleotide sequence [SEQ ID NO:1].
5′-ATGAAATTAACAATCCATGAAATTGCCCAAGTTGTTGGAGCCAAAAATGATATCAGTATC
TTTGAGGACACCCAGTTAGAAAAAGCTGAGTTTGATAGTCGTTTGATTGGAACTGGAGAT
TTATTTGTGCCACTTAAAGGTGCGCGTGATGGCCATGACTTTATTGAAACAGCCTTTGAA
AATGGTGCAGCAGTAACCTTGTCTGAGAAAGAGGTCTCAAATCATCCTTACATTCTAGTA
GATGATGTTTTGACAGCCTTTCAATCCCTAGCATCATACTATCTTGAAAAAACGACTGTT
GATGTCTTTGCTGTTACAGGTTCAAATGGCAAGACAACGACTAAGGATATGTTGGCGCAT
TTACTGTCAACGACCTACAAGACCTACAAAACACAAGGCAACTACAATAACGAGATTGGC
CTTCCCTACACAGTTCTACACATGCCTGAAGGAACAGAAAAGTTGGTTTTGGAGATGGGG
CAGGACCACTTGGGCGATATTCATCTCTTGTCTGAATTGGCTCGTCCAAAAACAGCCATC
GTGACCTTGGTTGGAGAAGCCCATTTGGCCTTTTTCAAAGACCGTTCAGAGATTGCTAAG
GGAAAAATGCAAATTGCAGACGGAATGGCTTCAGGTTCCTTGCTTTTAGTGCCGGCTGAC
CCTATCGTAGAGGACTATTTGCCAACTGATAAAAAGGTGGTCCGTTTTGGGCAAGGGGCA
GAGTTGGAAATCACAGACTTGGTTGAGCGTAAGGACAGTCTGACCTTTAAGGCTAATTTC
TTGGAACAAGCCCTTGATTTGCCAGTGACTGGTAAGTACAATGCTACCAATGCTATGATT
GCATCCTATGTTGCCCTACAAGAAGGAGTTTCAGAGGAGCAAATTCATCAGGCCTTCCAA
GGTCTTGAATTGACGCGTAATCGTACTGAGTGGAAGAAAGCAGCCAATGGAGCAGATATC
CTGTCAGACGTATATAATGCCAATCCAACTGCTATGAAGCTGATTTTAGAGACTTTCTCT
GCCATTCCAGCCAATGAAGGTGGCAAGAAAATTGCAGTGTTGGCGGATATGAAGGAGCTT
GGTGACCAGTCTATTCAACTCCATAACCAGATGATTTTGAGCCTTTCTCCAGATGTGCTT
GATACCGTTATTTTCTATGGAGAAGACATTGCTGAATTAGCCCAATTGGCCAGTCAAATG
TTCCCAATTGGCCACGTTTACTACTTCAAGAAAACAGAAGACCAAGACCAATTTGAAGAC
CTAGTCAAGCAGGTCAAGGAAAGCCTCAGTGCCAATGATCAAATCTTGCTCAAGGGCTCT
AACTCTATGAACCTAGCCATGTTGGTAGAAAGTTTAGAAAATGAAACCAAGTGATTTTGT
TAAGTATCTGCAAAGAATGATTGCCCTTACAGATACTGGCTTAACCTTTACAAAAGATCC
TTTTGACCGTGAGCGCTACGAGGACTTGCGAAGTCTGTTATCTGAAATGTTGAATCAGGG
ATCAGACCTGGATGCAGAAGAAGTAGCAGAAGTCTTGAAACCAACTTCAGCTTATGCGAC
TCCCTTAATGGACGTCCGTGCTTGGATTGTTGAGGATGAGAAAATTTGTCTGGTTAGGGG
ACAAGGAGAGGATAGTTGGGCTTTGCCANGTGGCTTTGGTGAAGTCGGCTATTCTCCAAC
TGAAAATATTCTCAAGGAAATT-3′
(B)
Streptococcus pneumoniae
MurF polypeptide sequence deduced from a
polynucleotide sequence in this table [SEQ ID NO:2].
NH
2
-MKLTIHEIAQVVGAKNDISIFEDTQLEKAEFDSRLIGTGDLFVPLKGARDGHDFIETAFE
NGAAVTLSEKE
Deibert Thomas S.
Gimmi Edward R.
King William T.
Priebe Scott D.
SmithKline Beecham Corporation
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