Nucleotide sequence of the mycoplasma genitalium genome,...

Details Nucleotide sequence of the mycoplasma genitalium genome,... Nucleotide sequence of the mycoplasma genitalium genome,...

1995-10-19

2003-03-25

Ketter, James (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...

C536S023700, C536S024320, C435S252300, C435S320100

Reexamination Certificate

active

06537773

ABSTRACT:

BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to the field of molecular biology. The invention discloses compositions comprising the nucleotide sequence of
Mycoplasma genitalium
, fragments thereof, and its use in medical diagnostics, therapies and pharmaceutical development.
2. Related Background Art
Mycoplasmas are the smallest free-living bacterial organisms known (Colman, S. D. et al.,
Mol. Microbiol
. 4:683-687 (1990)). Mycoplasmas are thought to have evolved from higher gram-positive bacteria through the loss of genetic material (Bailey, C. C. et al.,
J. Bacteriol
. 176:5814-5819 (1994)).
Mycoplasma genitalium
(
M. genitalium
)is widely considered to be the smallest self-replicating biological system, as the molecular size of its genome has been shown to be only 570-600 kp (Pyle, L. E. et al.,
Nucleic Acids Res
. 16(13):6015-6025 (1988); Peterson, S. N. et al.,
J. Bacteriol
. 175:7918-7930 (1993)). All mycoplasmas lack a cell wall and have small genomes and a characteristically low G+C content (Razin, S.,
Microbiol. Rev
. 49(4):419-455 (1985); Peterson, S. N. et al.,
J. Bacteriol
. 175:7918-7930 (1993)). Some mycoplasmas, including
M genitalium
, have a specialized codon usage, whereby UGA encodes tryptophan rather than serving as a stop codon (Inamine, J. M. et al.,
J. Bacteriol
. 172:504-506 (1990); Tanaka, J. G. et al.,
Nucleic Acids Res
. 19:6787-6792 (1991); Yamao, F. A. et al.,
Proc. Natl. Acad. Sci. USA
82:2306-2309 (1985)).
Mycoplasmas are widely known to be significant pathogens of humans, animals, and plants (Bailey, C. C. et al.,
J. Bacteriol
. 176:5814-5819 (1994)). The metabolic systems of mycoplasmas indicate that they are generally biosynthetically deficient, and thus depend on the microenvironment of the host by characteristically adhering to host cells in order to obtain essential precursor molecules, i.e., amino acids, fatty acids and sterols etc. (Baseman, J. B., 1987
. Micoplasma Membranes
, Vol. 20. The Plenum Press, New York, N.Y.).
In particular,
M. genitalium
, a newly discovered species, is a pathogenic etiological agent first isolated in 1980 from the urethras of human males infected with non-gonococcal urethritis (Tully, J. G. et al., Lancet 1:1288-1291 (1981); Tully, J. G., et al.,
Int. J. Syst. Bacteriol
. 33:387-396 (1983)).
M. genitalium
has also been identified in specimens of pneumonia patients as a co-isolate of
Micoplasma pneumoniae
(Baseman, J. B. et al.,
J. Clin. Microbiol
. 26:2266-2269 (1988)).
M. genitalium
opportunistic infection has often been observed in individuals infected with human immunodeficiency virus type 1 (HIV-1) (Lo, S.-C. et al.,
Amer. J. Trop. Med. Hyg
. 41:601-616 (1989); Lo, S.-C. et al.,
Amer. J. Trop. Med. Hyg
. 41:601-616 (1989); Sasaki, Y. et al.,
AIDS Res. Hum. Retrov
. 9(8):775-780 (1993)). Mycoplasmas can also induce various cytokines, including tumor necrosis factor, which may enhance HIV replication (Chowdhury, I. H. et al.,
Biochem. Biophys. Res. Commun
. 170:1365-1370 (1990)).
A high amino acid homology exists between the attachment protein of
M. genitalium
and the aligned proteins of several human Class II major histocompatibility complex proteins (HLA), suggesting that
M. genitalium
infection may play an important role in triggering autoimmune mechanisms, thereby aggravating the immunodeficiency characteristics of acquired immune deficiency syndrome (AIDS) (Montagnier, L. et al.,
C.R. Acad. Sci. Paris
311(3):425-430 (1990); Root-Bernstein, R. S. et al.,
Res. Immunol
. 142:519-523 (1991); Bisset, L. R.
Autoimmunity
14:167-168 (1992)). A diagnostic immunoassay for detecting
M. genitalium
infection using monoclonal antibodies specific for some
M. genitalium
antigens has been developed. Baseman, J. B. et al., U.S. Pat. No. 5,158,870.
Due to its diminutive genomic size,
M. genitalium
provides a useful model for determining the minimum number of genes and protein products necessary for a host-independent existence.
M. genitalium
expresses a characteristically low number of base-pairs and low G+C content, which along with its UGA tryptophan codon, has hampered sequencing efforts by conventional techniques (Razin, A.,
Microbiol. Rev
. 49(4):419-455 (1985); Colman, S. D. et al.,
Gene
87:91-96 (1990); Dybvig, K. 1992
. Gene Transfer In
: Maniloff, J. (ed.)
Mycoplasmas: Molecular Biology and Pathogenesis., Am. Soc. Microbiol
. Washington, D.C., pp.355-362)).
M. genitalium
possesses a single circular chromosome (Colman, S. D. et al.,
Gene
87:91-96 (1990); Peterson, S. N. et al.,
J. Bacteriol
. 175:7918-7930 (1993)). The characterization of the genome of
M. genitalium
has also been hampered by the lack of auxotrophic mutants and by the lack of a system for genetic exchange, precluding reverse genetic approaches. Thus, the sequencing of the
M. genitalium
genome would enhance the understanding of how
M. genitalium
causes or promotes various invasive or immunodeficiency diseases and to how best to medically combat mycoplasma infection.
Prior attempts at characterizing the structure and gene arrangement of the chromosomes of mycoplasmas using pulsed-field gel electrophoretic methods (Pyle, L. E. et al.,
Nucleic Acids Res
. 16(13):6015-6025 (1988); Neimark, H. C. et al.,
Nucleic Acids Res
. 18(18):5443-5448 (1990)), indicated that mycoplasmas have genomes ranging widely in size. Southern blot hybridization of digested DNAs of
M. genitalium
compared to the well-known human pathogen,
M. pneumoniae
, indicated overall low homology values of approximately 6-8% (Yogev, D. et al.,
Int. J. Syst. Bacteriol
. 36(3):426-430 (1986)). However, high homologies have been reported between the adhesin genes of
M. genitalium
and
M pneumoniae
(Dallo, S. F. et al.,
Microbial Path
. 6:69-73 (1989)). Initial studies at characterizing the genome of
M. genitalium
by comparison to the well-known
M. pneumoniae
species, indicated that both species have three (3) rRNA genes clustered together in a chromosomal segment of about 5 kb and form a single operon organized in classical procaryotic fashion, but differences exist between their respective restriction sites (Yogev, D. et al.,
Int. J. Bacteriol
. 36(3):426-430 (1986)).
Restriction enzyme mapping of
M. genitalium
indicates that the genome is approximately 600 kb. Several genes have also been mapped, including the single ribosomal operon, and the gene encoding the MgPa cytadhesion protein (Su, C. J. et al.,
J. Bacteriol
. 172:4705-4707 (1990); Colman, S. D. et al.,
Mol. Microbiol
. 4(4):683-687 (1990)). The entire restriction map of the genome of
M. genitalium
has also been cloned in an ordered library of 20 overlapping cosmids and one &lgr; clone (Lucier, T. S. et al.,
Gene
150:27-34 (1994)).
An initial study using random sequencing techniques to characterize the
M. genitalium
genome resulted in forty-four (44) random clones being partially sequenced; several long open reading frames were also found (Peterson, S. N. et al.,
Nucleic Acids Rev
. 19:6027-6031 (1991)). Subsequent work using random sequencing of 508 random nonidentical clones has allowed sequence information to be compiled for approximately seventeen percent (17%) (100,993 nucleotides) of the
M. genitalium
genome (Peterson, S. N. et al.,
J. Bacteriol
. 175:7918-7930 (1993)). Sequence information indicates that the diminutive genome of
M. genitalium
contains numerous genes involved in various metabolic processes. The genome is estimated to encode approximately 390 proteins, indicating that
M. genitalium
makes very efficient use of its limited amount of DNA (Peterson, S. N. et al.,
J. Bacteriol
. 175:7918-7930 (1993)).
Several studies have been undertaken to sequence and characterize individual genes identified in
M. genitalium
. In particular, the medically important aspects of
M. genitalium
have helped to direct interest to those genes which determine the degree of infectivity and the virulence characteristics of the organism. The nucleotide sequence and deduced amino acid sequence for

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