Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1998-06-24
2001-09-25
Fredman, Jeffrey (Department: 1655)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C536S022100, C536S023100, C536S024100
Reexamination Certificate
active
06294328
ABSTRACT:
FIELD OF THE INVENTION
The present invention is directed to novel methodology, and DNA sequence libraries that result therefrom, whereby different strains of the tuberculosis bacterial pathogen,
Mycobacterium tuberculosis,
can be definitively identified, based upon the identification of differences in their respective DNA sequences. The invention has valuable application in the fields of tuberculosis genetics, epidemiology, patient treatment, and epidemic monitoring.
Reported Developments
Although certain chemotherapy and vaccine protocols have become available for he treatment of tuberculosis, the disease continues to claim more lives per year than any other infectious disease (see S. Cole et al.,
Nature,
393, pp.537-544, 1998). In fact, despite the widespread availablity of health measures in the industrialized world, the incidence of tuberculosis has been spreading in both the industrialized and developing nations. This increased incidence is of particular concern in view of the emergence of novel drug-resistant strains, and the strong presence of the disease in HIV-afflicted patients.
It has been the recognized understanding in the art (see S. Cole et al., and S. Sreevatsan et al.,
Proc. Natl. Acad. Sci, USA,
94, pp.9869-9874, 1997) that
M. tuberculosis
is a member of a complex of closely related species. The complex is understood to substantially lack interstrain genetic diversity, nucleotide changes being very rare. It has thus been the perception that both vaccine development and strain characterization would continue to be difficult, given that most proteins were expected to be identical between strains.
These difficulties are further compounded by the growth characteristics of
Mycobacterium tuberculosis
in patients and in culture. Cell growth is characterized by several unusual features including, for example, (1) very slow growth (a doubling time of circa 24 hours which is much slower that other bacteria such as
E. coli,
that have a doubling time of perhaps 30 minutes), (2) the capacity to become dormant in infected tissue for long periods of time, (3) the capacity to remain present at low density levels that probably avoids immune detection; and (4) the presence of unusual and complex cell wall components that probably contribute to pathogenicity and inflammation.
The present invention is directed to the discovery that, notwithstanding the above observations, very substantial differences in the DNA sequences between related Mycobacterium strains can be identified. Additionally, according to the practice of the present invention, it is not required that such DNA sequence differences be localized to protein encoding sequences.
SUMMARY OF THE INVENTION
According to the practice of the present invention, there is provided a nucleotide by nucleotide comparison between a well-recognized, but long ago characterized virulent strain, and a recent isolate correlated with a severe and persistent outbreak in the United States. Sequence differences bewtween the two strains are substantial, and point to loci in the DNA of Mycobacterium that can be used as markers for strain variation and characterization. Given that different strains have different susceptibilities to various therapeutic programs, providing proper identification of a strain responsible for a particular infection is of great importance to physicians.
Accordingly, there is provided a method of evaluating the virulence of a first strain of
Mycobacterium tuberculosis,
comprising the step of determining the nucleotide sequence of said strain at positions in the genome thereof, that correspond to positions where
M. tuberculosis
strains CDC 1551 and H37Rv differ as to sequence, and determining whether the nucleotide sequence of said first strain shows greater homology, at said positions, to the sequence of strain CDC 1551 or H37Rv.
REFERENCES:
Philipp et al. “Physical Mapping of Mycobacterium bovis BCG Pasteur reveals differences from the genome map of Mycobacterium tuberculosis H37Rv and from M. bovis”, Microbiology, vol. 142, pp. 3135-3145, 1996.*
Pevear et al, “Insights into Theiler's Virus Neurovirulence Based on a Genomic Comparison of the Neurovirulent GDVII and less virulent BeAn Strains”, Virology, pp. 1-12, 1988.*
NIAID Mycobacterial Genome Workshop Exploiting the TB Genome Executive Summary, Bethesda, MD, pp. 1-2, Sep. 15, 1997.*
Delcher et al., “Alignment of whole genomes”, Nucleic Acids Research, vol. 27(11), pp. 2639-2376, 1999.*
Behr et al., “Comparative Genomics of BCG Vaccines by Whole-Genome DNA Microarray”, Science, 284:15201523 (1999).
Cole et al., “Deciphering the Biology of Mycobacterium Tuberculosis from the Complete Genome Sequence”, Nature, 393:537-544 (1998).
Fleischmann et al “Whole-Genome Random Sequencing and Assembly of Haemophilus Influenzae Rd”, Science, 269:496-512 (1995).
Gordon et al., “Identification of Variable Regions in the Genomes of Tubercle Bacilli Using Bacterial Artificial Chromosome Arrays”, Molecular Microbiology, 32(3):643-655 (1999).
Mahairas et al., “Molecular Analysis of Genetic Differences Between Mycobacterium Bovis BCG and Virulent M. Bovis”, J. Bacteriology, 178(5):1274-1282 (1996).
Phillip et al. “Physical Mapping of Mycobacterium Bovis BCG Pasteur Reveals Differences from the Genome Map of Mycobacterium Tuberculosis H37Rv and from M. Bovis”, Microbiology, 142:3135-3145 (1997).
Sreevatsan et al., “Restricted Structural Gene Polymorphism in the Mycobacterium Tuberculosis Complex Indicates Evolutionarily Recent Global Dissemination”, Proc. Natl. Acad. Sci., USA, 94:9868-9874 (1997).
Valway et al., “An Outbreak Involving Extensive Transmission of a Virulent Strain of Mycobacterium Tuberculosis”, The New J. of Medicine, 338(10):633-639 (1998).
Wilson et al., “Exploring Drug-Induced Alterations in Gene Expression in Mycobacterium Tuberculosis by Microarray Hybridization”, Proc. Natl. Acad. Sci., 96(22):12833-12838 (1999).
S. Sreevatsan et al., “Restricted Structural Gene Polymorphism in the Mycobacterium Tuberculosis . . . ” Proc. Natl. Acad. Sci, USA, 94, pp. 9869-9874 (1997).
S. Cole et al., “Deciphering the Biology of Mycobacterium Tuberculosis from the Complete Genome Sequence”, Nature, 393, pp. 537-544 (1998).
S. Valway et al., “An Outbreak Involving Extensive Transmission of a Virulent Strain of Mycobacterium Tuberculosis”, New England Journal of Medicine, 338, pp. 633-639 (1988).
W. Philipp et al., “An Integrated Map of the Genome of the Tubercle Bacillus Mycobacterium Tuberculosis . . .” Proc. Natl. Acad. Sci. USA, 93, pp. 3132-3137 (1996).
Fleischmann Robert David
Fraser Claire Marie
Venter John Craig
White Owen Richardson
Chakrabarti Arun Kr.
Fredman Jeffrey
Morrison & Foerster / LLP
The Institute for Genomic Research
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