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
2000-04-25
2003-01-14
Brusca, John S. (Department: 1631)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S069300, C435S091410, C435S320100, C435S252300, C536S023700
Reexamination Certificate
active
06506581
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to the field of molecular biology. The present invention discloses compositions comprising the nucleotide sequence of
Haemophilus influenzae
, fragments thereof and usage in industrial fermentation and pharmaceutical development.
BACKGROUND OF THE INVENTION
The complete genome sequence from a free living cellular organism has never been determined. The first mycobacterium sequence should be completed by 1996, while
E. coli
and
S. cerevisae
are expected to be completed before 1998. These are being done by random and/or directed sequencing of overlapping cosmid clones. No one has attempted to determine sequences of the order of a megabase or more by a random shotgun approach.
H. influenzae
is a small (approximately 0.4×1 micron) non-motile, non-spore forming, germ-negative bacterium whose only natural host is human. It is a resident of the upper respiratory mucosa of children and adults and causes otitis media and respiratory tract infections mostly in children. The most serious complication is meningitis, which produces neurological sequelae in up to 50% of affected children. Six
H. influenzae
serotypes (a through f) have been identified based on immunologically distinct capsular polysaccharide antigens. A number of non-typeable strains are also known. Serotype b accounts for the majority of human disease.
Interest in the medically important aspects of
H. influenzae
biology has focused particularly on those genes which determine virulence characteristics of the organism. A number of the genes responsible for the capsular polysaccharide have been mapped and sequenced (Kroll et al.,
Mol. Microbiol.
5(6):1549-1560 (1991)). Several outer membrane protein (OMP) genes have been identified and sequenced (Langford et al,
J. Gen. Microbiol.
138:155-159 (1992)). The lipoligosaccharide (LOS) component of the outer membrane and the genes of its synthetic pathway are under intensive study (Weiser et al.,
J. Bacteriol.
172:3304-3309 (1990)). While a vaccine has been available since 1984, the study of outer membrane components is motivated to some extent by the need for improved vaccines. Recently, the catalase gene was characterized and sequenced as a possible virulence-related gene (Bishni et al., in press). Elucidation of the
H. influenzae
genome will enhance the understanding of how
H. influenzae
causes invasive disease and how best to combat infection.
H. influenzae
possesses a highly efficient natural DNA transformation system which has been intensively studied in the non-encapsulated (R), serotype d strain (Kahn and Smith,
J. Membrane Biology
81:89-103 (1984)). At least 16 transformation-specific genes have been identified and sequenced. Of these, four are regulatory (Redfield,
J Bacteriol.
173:5612-5618 (1991), and Chandler,
Proc. Natl. Acad. Sci. USA
89:1626-1630 (1992)), at least two are involved in recombination processes (Barouki and Smith,
J. Bacteriol.
163(2):629-634 (1985)), and at least seven are targeted to the membranes and periplasmic space (Tomb et al.,
Gene
104:1-10 (1991), and Tomb,
Proc. Natl. Acad. Sci. USA
89:10252-10256 (1992)), where they appear to function as structural components or in the assembly of the DNA transport machinery.
H. influenzae
Rd transformation shows a number of interesting features including sequence-specific DNA uptake, rapid uptake of several double-stranded DNA molecules per competent cell into a membrane compartment called the transformasome, linear translocation of a single strand of the donor DNA into the cytoplasm, and synapsis and recombination of the strand with the chromosome by a single-strand displacement mechanism. The
H. influenzae
Rd transformation system is the most thoroughly studied of the gram-negative systems and distinct in a number of ways from the gram-positive systems.
The size of
H. influenzae
Rd genome has been determined by pulsed-field agarose gel electrophoresis of restriction digests to be approximately 1.9 Mb, making its genome approximately 40% the size of
E. coli
(Lee and Smith,
J. Bacteriol.
170:4402-4405 (1988)). The restriction map of
H. influenzae
is circular (Lee et al.,
J. Bacteriol.
171:3016-3024 (1989), and Redfield and Lee, “
Haemophilus influenzae
Rd”, pp. 2110-2112, In O'Brien, S. J. (ed), Genetic Maps: Locus Maps of Complex Genomes, Cold Spring Harbor Press, N.Y.). Various genes have been mapped to restriction fragments by Southern hybridization probing of restriction digest DNA bands. This map will be valuable in verification of the assembly of a complete genome sequence from randomly sequenced fragments. GenBank currently contains about 100 kb of non-redundant
H. influenzae
DNA sequences. About half are from serotype b and half from Rd.
SUMMARY OF THE INVENTION
The present invention is based on the sequencing of the
Haemophilus influenzae
Rd genome. The primary nucleotide sequence which was generated is provided in SEQ ID NO:1.
The present invention provides the generated nucleotide sequence of the
Haemophilus influenzae
Rd genome, or a representative fragment thereof, in a form which can be readily used, analyzed, and interpreted by a skilled artisan. In one embodiment, present invention is provided as a contiguous string of primary sequence information corresponding to the nucleotide sequence depicted in SEQ ID NO:1.
The present invention further provides nucleotide sequences which are at least 99.9% identical to the nucleotide sequence of SEQ ID NO:1.
The nucleotide sequence of SEQ ID NO:1, a representative fragment thereof, or a nucleotide sequence which is at least 99.9% identical to the nucleotide sequence of SEQ ID NO:1 may be provided in a variety of mediums to facilitate its use. In one application of this embodiment, the sequences of the present invention are recorded on computer readable media. Such media includes, but is not limited to: magnetic storage media, such as floppy discs, hard disc storage medium, and magnetic tape; optical storage media such as CD-ROM; electrical storage media such as RAM and ROM; and hybrids of these categories such as magnetic/optical storage media.
The present invention further provides systems, particularly computer-based systems which contain the sequence information herein described stored in a data storage means. Such systems are designed to identify commercially important fragments of the
Haemophilus influenzae
Rd genome.
Another embodiment of the present invention is directed to isolated fragments of the
Haemophilus influenzae
Rd genome. The fragments of the
Haemophilus influenzae
Rd genome of the present invention include, but are not limited to, fragments which encode peptides, hereinafter open reading frames (ORFs), fragments which modulate the expression of an operably linked ORF, hereinafter expression modulating fragments (EMFs), fragments which mediate the uptake of a linked DNA fragment into a cell, hereinafter uptake modulating fragments (UMFs), and fragments which can be used to diagnose the presence of
Haemophilus influenzae
Rd in a sample, hereinafter, diagnostic fragments (DFs).
Each of the ORF fragments of the
Haemophilus influenzae
Rd genome disclosed in Tables 1(a) and 2, and the EMF found 5 to the ORF, can be used in numerous ways as polynucleotide reagents. The sequences can be used as diagnostic probes or diagnostic amplification primers for the presence of a specific microbe in a sample, for the production of commercially important pharmaceutical agents, and to selectively control gene expression.
The present invention further includes recombinant constructs comprising one or more fragments of the
Haemophilus influenzae
Rd genome of the present invention. The recombinant constructs of the present invention comprise vectors, such as a plasmid or viral vector, into which a fragment of the
Haemophilus influenzae
Rd has been inserted.
The present invention further provides host cells containing any one of the isolated fragments of the
Haemophilus influenzae
Rd genome of the present invention. The host ce
Adams Mark D.
Fleischmann Robert D.
Smith Hamilton O.
Venter J. Craig
White Owen
Brusca John S.
Human Genome Science, Inc.
Human Genome Sciences Inc.
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