Chemistry: molecular biology and microbiology – Process of mutation – cell fusion – or genetic modification – Introduction of a polynucleotide molecule into or...
Reexamination Certificate
1999-07-08
2001-08-07
Guzo, David (Department: 1636)
Chemistry: molecular biology and microbiology
Process of mutation, cell fusion, or genetic modification
Introduction of a polynucleotide molecule into or...
C435S477000, C435S253100, C435S243000, C435S320100
Reexamination Certificate
active
06271034
ABSTRACT:
BACKGROUND OF THE INVENTION
In April 1993, tuberculosis was declared a global health emergency—the first such designation in the history of the World Health Organization. The distinction is regrettably justified because tuberculosis remains one of the largest causes of disease and death in the world (37), due in part to the increased susceptibility of HIV infected individuals and the ominous emergence of multi-drug resistant strains in both industrialized and developing countries. Effective new tuberculosis control and prevention strategies will require additional knowledge of the causative agent and its interaction with the human host.
In this regard, the determination of the genomic sequence of
Mycobacterium tuberculosis
(7) has provided many new opportunities for studying tuberculosis pathogenesis. Many of the genes in the genome of
M. tuberculosis
have no known function. A first step in establishing a function of an unknown gene lies in the generation of a mutation in that gene and characterization of the resulting mutant strain. Studying the behavior of these mutants in model systems of tuberculosis could reveal the mechanisms by which
M. tuberculosis
multiplies within the host cells and resists the immune effector functions of the host.
The availability of the
M. tuberculosis
genome sequences and the development of successful transformation protocols for the slow growing mycobacteria (32, 34) make the engineering of specific mutations readily achievable, but the introduction of these mutated alleles into their homologous sites in the chromosome, i.e. allelic exchange, has been notoriously difficult in this organism. Slow growing mycobacteria such as
M. bovis
BCG and
M. tuberculosis
can integrate exogenous DNA into their chromosome by both illegitimate and homologous recombination (13, 16). In recent years, numerous successful gene disruptions in various mycobacterial species were reported by using short (1, 13, 28) or long linear DNA fragments (2) as homologous DNA substrates. A “suicidal” vector approach, using recombinant plasmids unable to replicate in mycobacteria, was also extensively used to achieve allelic exchange in both fast- and slow-growing mycobacteria (5, 14) (22) (23) (24, 25, 31). Unfortunately, it is very difficult to estimate the real frequency of the allelic exchange events in these experiments due to the low number of transformants obtained, especially when using slow-growing mycobacteria. This led to the general conclusion that homologous recombination in the slow-growing mycobacteria is inefficient (16).
A two-step selection method using a selectable and counter-selectable marker positioned on either replicating or non-replicating plasmids has been successfully used in
M. smegmatis
(14, 24). Further, use of a conditionally replicating temperature sensitive plasmid as a delivery vector has greatly improved reproducibility of the allelic exchange in the slow growing mycobacteria (23).
However, the natural mechanisms of exchange of genetic information, such as conjugation or transduction, would be an alternative strategy to introduce homologous DNA into mycobacteria with high efficiency. Although conjugation has been described for
M. smegmatis
(17, 20, 33), it has not been demonstrated in the slow-growing mycobacteria. Similarly, transduction has been reported for
M. smegmatis
(26), but not for BCG or
M. tuberculosis.
Because the creation of mutants in
M. tuberculosis
and BCG is of essential importance in the analysis of gene function, it is desirable to develop effective means and methods for allelic exchange for
M. tuberculosis
, BCG and other slow-growing mycobacteria. Methods for efficient allelic exchange would facilitate the definition of wildtype and mutant genes of
M. tuberculosis
and BCG mycobacteria, and thereby provide the necessary tools for understanding the mechanisms by which these mycobacteria survive and replicate. In addition, it would further the development of vaccines and new drugs effective in the treatment of infection caused by
M. tuberculosis
, BCG, and other mycobacteria.
SUMMARY OF THE INVENTION
The present invention describes a novel one-step method for achieving high frequency allelic exchange in the fast- and slow-growing mycobacteria using in vitro generated conditional transducing phages. The present invention provides a method for producing a recombinant mutant mycobacterium, including a recombinant mutant slow-growing mycobacterium, comprising infecting a mycobacterium with a conditional transducing phage where the conditional transducing phage comprises a mutated DNA substrate for allelic exchange with a homologous wildtype nucleic acid sequence of the mycobacterium. The infected mycobacterium is cultured under conditions wherein the conditional transducing phage does not replicate. The mutated DNA substrate is incorporated into the chromosome of the mycobacterium by homologous recombination, thereby generating a recombinant mutant mycobacterium having the mycobacterial DNA substrate in lieu of the homologous wildtype nucleic acid sequence of the mycobacterium.
The present invention further provides a conditional transducing phage, comprising a conditional mycobacteriophage comprising an
E. coli
bacteriophage lambda cosmid inserted into a non-essential region of the mycobacteriophage, wherein said cosmid further comprises a mutated DNA substrate which is homologous to a wildtype nucleic acid sequence of a mycobacterium.
Also provided is a recombinant mutant mycobacterium, including recombinant mutants or auxotrophs resulting from the selective disruption of one or more genes in the biosynthetic pathway of a nutrient, structural component or amino acid, where the recombinant mutant mycobacterium is produced by a method comprising infecting a mycobacterium with the conditional transducing phage of the present invention, and culturing the infected mycobacterium under such conditions wherein the conditional transducing phage does not replicate and the mutated DNA substrate carried by the transducing phage is incorporated into the chromosome of the mycobacterium by homologous recombination. The present invention further provides for recombinant mutant slow- and fast-growing mycobacteria that are auxotrophic for lysine, as well as recombinant mutant slow-growing mycobacteria that are auxotrophic for leucine. Additional objects of the present invention will be apparent from the description which follows.
REFERENCES:
Bardarov, et al. Proceedings of the National Academy of Sciences, USA. vol. 94, pp. 10961-10966, Sep. 1997.*
Pelicic, et al. Proceedings of the National Academy of Sciences, USA vol. 94, pp. 10955-10960, Sep. 1997.
Bardarov Stoyan S.
Jacobs, Jr. William R.
Albert Einstein College of Medicine of Yeshiva University
Amster Rothstein & Ebenstein
Guzo David
Leffers, Jr. Gerald G.
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