Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2000-10-11
2003-08-19
Horlick, Kenneth R. (Department: 1655)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S005000, C435S091100, C435S091200, C435S252310, C435S252320, C435S252330, C435S252340, C435S252500, C536S023100, C536S024300, C536S024320, C536S024330
Reexamination Certificate
active
06607885
ABSTRACT:
FIELD OF THE INVENTION
This invention is in the field of bacterial gene expression. More specifically, this invention is a method for the high density, microarray-mediated gene expression profiling of
Escherichia coli
for comprehensive gene expression analysis.
BACKGROUND OF THE INVENTION
Escherichia coil
has been exhaustively studied for over 50 years. Early experiments measured the molecular fluxes from small compounds into macromolecular constituents. These studies were followed by others in which small molecule pools of central metabolic building blocks, nucleotides and amino acids were determined. The levels of several macromolecular components, including individual species of proteins, have been measured. Such measurements of the steady state provide a census of the cellular content while changes upon imposition of a stress catalogue the cell's fight for survival. This response to an insulting or adverse condition can take many forms from relieving end product inhibition to derepressing transcription.
In
E. coli
, experiments to define stress-related, global regulatory responses have often relied upon one of two approaches. In the first, operon fusions induced by a particular stress are isolated. In the second, proteomic measures in which the protein fractions from stressed and un-stressed cultures are separated by a two-dimensional method and then compared. Each method has an inherent technological hurdle; for the former, the map location of responsive gene fusions must be known precisely, and for the latter, induced or repressed proteins excised from the two-dimensional gels must be identified.
Another method uses a transposon-mediated mutagenesis (Spector et al.
J. Bacteriol.
170:345-351 (1988)). A reporter gene is inserted at a random location in the genome using a transposon. By assaying for the reporter gene before and after the treatment, genes affected by the treatment can be mapped and cloned by using the linked transposon as a marker. However, this method is limited to non-essential genes.
Alternatively, mRNA measurements utilizing techniques (such as hybridization to DNA and primer extension) have allowed the monitoring of individual gene's expression profiles. DeRisi et al. (
Science
278:680-686 (1997)) reported the expression profiling of most yeast genes. The measurements were facilitated by high-density arrays of individual genes and specific labeling of cDNA copies of eukaryotic mRNA using polyA tail-specific primers. The lack of a polyA tail and the extremely short bacterial mRNA half life represent hurdles for the application of DNA micro-array technology to prokaryotic research.
A comprehensive expression profiling has been performed previously with the yeast
Saccharomyces cerevisiae
. Adaptation of RNA isolation and labeling protocols from eukaryotes to prokaryotes is not straightforward since eukaryotic mRNA manipulations often exploit 3′-polyadenylation of this molecular species.
Chuang et al. (
J. Bacterol.
175:2026-2036 (1993)) reported an expression profiling using large DNA fragments from an ordered &lgr; library of
E. coli
genomic fragments as a capture reagent. It allowed the comparison of the expression patterns from large portions of DNA fragments by comparing mRNA levels from stressed and unstressed
E. coli
cultures. The resolution of this method, however, was unsatisfactory. Expression of groups of genes, as opposed to the expression of each individual gene was measured. Moreover, the method used radio-labeled DNA as a probe with the incumbent need for safety precautions. Furthermore, the use of radio-labeled probe prevents the simultaneous measurement of the expression level in a test sample and a control sample.
Richmond et al. (
Nucleic Acids Research,
19:3821-3835 (1999)) has recently reported genome-wide expression profiling of
E. coli
at a single ORF level of resolution. Changes in RNA levels after exposure to heat shock or IPTG were analyzed using comprehensive low density blots of individual ORFs on a nylon matrix and comprehensive high density arrays of individual ORFs spotted on glass slides. The results of the two methods were compared.
The methods recited above permit monitoring of the effect of environmental changes on gene expression by comparing expression levels of a limited number of genes. They, however, fail to monitor the comprehensive responses of a preponderance of individual genes in the genome of an organism in reliable, useful manner.
The problem to be solved, therefore, is to provide a way to measure the comprehensive gene expression profile analysis of the organism.
SUMMARY OF THE INVENTION
The invention provides a method for identifying gene expression changes within a bacterial species comprising:
(a) providing a comprehensive micro-array synthesized from DNA comprised in a bacterial species;
(b) generating a first set of labeled probes from bacterial RNA, the RNA isolated from the bacterial species of step (a);
(c) hybridizing the first:set of labeled probes of step (b) to the comprehensive micro-array of step (a), wherein hybridization results in a detectable signal generated from the labeled probe;
(d) measuring the signal generated by the hybridization of the first set of labeled probe to the comprehensive micro-array of step (c);
(e) subjecting the bacterial species of step (a) to a gene expression altering condition whereby the gene expression profile of the bacterial species is altered to produce a modified bacterial species;
(f) generating a second set of labeled probes from bacterial RNA, the RNA isolated from the modified bacterial species of step (e);
(g) hybridizing the second set of labeled probes of step (f) to the comprehensive micro-array of step (a), wherein hybridization results in a detectable signal generated from the labeled probe;
(h) measuring the signal generated by the hybridization of the second set of labeled probes to the comprehensive micro-array of step (g); and
(i) comparing signal generated from the first hybridization to the signal generated from the second hybridization to identify gene expression changes within a bacterial species.
Additionally the invention provides a method for identifying gene expression changes within a bacterial strain comprising:
(a) providing a comprehensive micro-array synthesized from DNA comprised in a bacterial species
(b) generating a first set of fluorescent cDNA from bacterial RNA, the RNA isolated from the bacterial species of step (a);
(c) hybridizing the first set of fluorescent cDNA of step (b) to the comprehensive micro-array of step (a), wherein hybridization results in a detectable signal generated from the fluorescent cDNA;
(d) measuring the signal generated by the hybridization of the first set of fluorescent cDNA to the comprehensive micro-array of step (c);
(e) subjecting the bacterial species of step (a) to a gene expression altering condition whereby the gene expression profile of the bacterial species is altered to produce a modified bacterial species;
(f) generating a second set of fluorescent cDNA from bacterial RNA, the RNA isolated from the modified bacterial species of step (e);
(g) hybridizing the second set of fluorescent cDNA of step (f) to the comprehensive micro-array of step (a), wherein hybridization results in a detectable signal generated from the fluorescent cDNA;
(h) measuring the signal generated by the hybridization of the second set of fluorescent cDNA to the comprehensive micro-array of step (g); and
(i) comparing signal generated from the first hybridization to the signal generated from the second hybridization to identify gene expression changes within a bacterial species.
In an alternate embodiment the invention provides a method for identifying gene expression changes within a genome comprising:
(a) providing a comprehensive micro-array synthesized from DNA comprised in a prokaryotic or eukaryotic species;
(b) generating a control set of fluorescent cDNA from total or polyadenylated RNA, the RNA isolated from the species of step (a), the fluorescent cDNA comprising at least on
Larossa Robert A.
Wei Yan
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