Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1999-03-26
2001-03-27
Guzo, David (Department: 1636)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S473000, C435S029000, C435S468000, C435S455000, C435S091200, C536S023100
Reexamination Certificate
active
06207384
ABSTRACT:
BACKGROUND OF THE INVENTION
Nearly 40% of the
Haemophilus
(
H
.)
influenzae
genome is comprised of genes of unknown function, many of which have no recognizable functional orthologues in other species. Similar numbers of unidentified open reading frames (orfs) are present in other sequenced or partially sequenced genomes of infectious organisms. Comprehensive screens and selections for identifying functional classes of genes provide a crucial starting point for converting the vast body of growing sequence data into meaningful biological information that can be used for drug discovery.
One major and important class of genes consists of those bacterial genes that are essential for growth or viability of a bacterium. Because useful conventional antibiotics are known to act by interfering with the products of essential genes, it is likely that the discovery of new essential gene products will have a significant impact on efforts to develop novel antimicrobial drugs. Essential gene products have been traditionally identified through the isolation of conditional lethal mutants, or by transposon mutagenesis in the presence of a complementing wild type allele (balanced lethality). However, such approaches are laborious, as they require identification, purification, and study of individual mutant strains. These methods are also limited to species with well-developed systems for genetic manipulation and, therefore, cannot be readily applied to many of the potentially dangerous microorganisms whose genomes have recently been sequenced.
In order to facilitate the discovery of novel anti-microbial drugs, it would be desirable to have a rapid, generalized method of identifying essential growth/viability genes in pathogens. Such a method would be particularly useful for identifying essential genes in pathogens that are not genetically well-characterized. Such a method could also be used to identify essential genes in higher organisms, e.g., in animals and in plants.
SUMMARY OF THE INVENTION
We have developed a general system for the identification of essential genes in organisms. The system may be used to discover novel target genes for the development of therapeutic compounds, as well as for the discovery of genes that are involved in cell growth or viability. A related aspect of the invention allows for rapid construction of conditional mutations in essential genes.
In general, the invention features a method for locating an essential region in a portion of DNA from the genome of an organism. The method includes: a) mutagenizing DNA having the sequence of an essential portion of DNA, wherein the mutagenizing is performed using in vitro mutagenesis with a transposon; b) transforming cells of the organism with the mutagenized DNA of step a); c) identifying cells containing the mutagenized DNA; and d) locating the essential region of the DNA portion by detecting the absence of transposons in the essential region of DNA in cells containing the mutagenized DNA.
In various embodiments, the transposon may contain a selectable marker, the transposon may be mariner, and the method may further comprise the use of Himar 1 transposase.
In a preferred embodiment, the in vitro mutagenesis is high saturation mutagenesis. In further embodiments, the portion of DNA may be amplified using the polymerase chain reaction (PCR) prior to mutagenesis, or the portion of DNA may be cloned into a vector prior to mutagenesis. In another embodiment, prior to transforming the cells, the mutagenized DNA may be subjected to gap repair using DNA polymerase and DNA ligase. In still another embodiment, the transposon-mutagenized DNA may be recombined into the chromosome using an allelic replacement vector.
In another preferred embodiment, the locating of an essential region of DNA is done by performing PCR footprinting on a pool of transposon-mutagenized cells. The PCR footprinting is performed using a primer that hybridizes to the transposon, plus a primer that hybridizes to a specific location on the chromosome, after which the PCR products are separated on a footprinting gel. A PCR product on the gel represents a region of the chromosome that does not contain an essential gene, and the lack of a PCR product in an area of the gel, where a PCR product is expected, represents a region of the chromosome that contains an essential gene. Alternatively, a low level of the PCR product on the gel, relative to other PCR products on the gel, represents a region of the chromosome that contains an essential gene.
In still other embodiments, the cell may have a haploid growth phase, or be a single-cell microorganism, or be naturally competent for transformation, or be made competent for transformation, or be a fungus, such as a yeast (e.g.,
Saccharomyces cerevisiae
), or be a bacterium, including, but not limited to, a gram-positive bacterium. In a preferred embodiment, the bacterium is to be selected from the group consisting of:
Actinobacillus actinomycetemcomitans; Borrelia burgdorferi; Chlamydia trachomatis; Enterococcus faecalis; Escherichia coli; Haemophilus influenzae; Helicobacter pylori; Legionella pneumophila; Mycobacterium avium; Mycobacterium tuberculosis; Mycoplasma genitalium; Mycoplasma pneumonia; Neisseria gonorrhoeae; Neisseria meningitidis; Staphylococcus aureus; Streptococcus pneumoniae; Streptococcus pyogenes; Treponema pallidum
; and
Vibrio cholerae.
In another embodiment, the transposon may contain a selectable marker gene, and identifying the cells containing mutagenized DNA may be based upon the ability of the cells to grow on selective medium, wherein a cell containing a transposon can grow on selective medium, and a cell lacking a transposon cannot grow, or grows more slowly, on selective medium.
In still another embodiment, the transposon may contain a reporter gene, and identifying cells containing mutagenized DNA may be based on a reporter gene assay, wherein a cell confirming a transposon expresses the reporter gene and a cell lacking a transposon does not express the reporter gene.
In yet another embodiment, the method includes a step in which the cells are cultured in a medium that approximates a host environment for a pathogen.
In a second aspect, the invention provides a method for obtaining conditional mutations in essential genes. The method includes the steps of amplifying DNA containing a selective marker, as described herein, near an essential gene (e.g., a transposon) using mutagenic amplification (e.g., mutagenic PCR), transforming the DNA into a competent host under conditions allowing selection for those strains containing the selective marker, and screening for strains under permissive and non-permissive conditions such that conditional lethal mutations may be identified.
In a third aspect, the invention provides a method for isolating a compound that modulates the expression of a nucleic acid sequence operably linked to a gene promoter. The method includes a) providing a cell expressing a nucleic acid sequence operably linked to a gene promoter, wherein the gene promoter is the gene promoter for: HI0455; HI0456; HI0458; HI0599; HI0887; HI0904; HI0906; HI0907; HI0908; HI0909; HI1650; HI1651; HI1654; HI1655
; S. pneumoniae
rbfA;
S. pneumoniae
IF-2
; S. pneumoniae
L7AE; or
S. pneumoniae
nusA; b) contacting the cell with a candidate compound; and c) detecting or measuring expression of the gene following contact of the cell with the candidate compound.
In preferred embodiments of the third aspect, the nucleic acid sequence is a reporter gene (e.g., GFP, lacZ, or alkaline phosphatase) or is HI0455; HI0456; HI0458; HI0599; HI0887; HI0904; HI0906; HI0907; HI0908; HI0909; HI1650; HI1651; HI1654; HI1655
; S. pneumoniae
rbfA;
S. pneumoniae
IF-2
; S. pneumoniae
L7AE; or
S. pneumoniae
nusA.
In yet another preferred embodiment of the third aspect, the modulation in the expression of the nucleic acid sequence modulates cell growth or viability of the cell.
In a fourth aspect, the invention provides a method for identifying a nucleic acid sequence that is essential for cell growth or v
Akerley Brian J.
Camilli Andrew
Mekalanos John J.
Rubin Eric J.
Bieker-Brady Kristina
Clark & Elbing LLP
Guzo David
Leffers, Jr. Gerald G.
The General Hospital Corporation
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