Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2001-04-11
2004-04-06
McKelvey, Terry (Department: 1636)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S029000
Reexamination Certificate
active
06716582
ABSTRACT:
FIELD OF THE INVENTION
This invention is in the field of bacterial gene expression. More specifically, this invention describes a method to monitor transcriptional changes on a genome-wide scale using a genome-registered gene fusion collection.
BACKGROUND OF THE INVENTION
DNA array analysis is a powerful method for comprehensive genome analysis of gene expression. Currently, this approach is the only available method for massively parallel analyses that allow the expression of each gene of a bacterial genome to be characterized simultaneously (Richmond et al., (1999)
Nucleic Acids Res.
27:3821-3835., 17, 25; Tao et al., (1999),
J. Bacteriol.
181:6425-6440; Wilson et al., (1999)
Proc. Natl. Acad. Sci. U.S.A.
96:12833-12838).
Richmond et al. ((1999)
Nucleic Acids Research,
27:3821-3835) has recently reported genome-wide expression profiling of
E. coli
at the 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. Richmond et al. states that radioactive probe/spot blots are inferior to fluorescent probe/micro-arrays. Moreover, the comparison of heat shock treatment between the two methods is fundamentally flawed since the RNA analyzed with spot blots were derived from broth grown cultures while those analyzed with micro-arrays were derived from cells grown in defined media. Despite the power of this new methodology, there are several problems that limit the reliability of results. For example, artifacts may arise during the isolation of microbial RNA (Tao et al., (1999)
J. Bacteriol.
181:6425-6440) or from cross hybridization to paralogous genes (Richmond et al., (1999)
Nucleic Acids Res.
27:3821-3835, 17, 25).
Another limitation of DNA array methodology is that RNA must be isolated, converted into DNA by reverse transcriptase with concomitant incorporation of fluorescent labels. These steps make it unlikely that facile high throughput screens could be developed based on DNA array technology. Thus, there exists a need for a method that adapts results from DNA array technology into high throughput screens. For the reasons mentioned above and others, alternative genome-wide expression profiling method as well as rapid methods to independently verify results from DNA array experiments are needed.
Gene fusion technology is an established method for gene expression monitoring. For example, the initial discovery of the SOS (DNA damage responsive) regulon of
E. coli
was done by Kenyon and Walker ((1980)
Proc. Natl. Acad. Sci. U.S.A.
77:2819-2823) by comparing the transcriptional responses of
Escherichia coli
to mitomycin C (MMC), a DNA damaging agent that intercalates into and forms a covalent attachment with double-stranded DNA. While these early experiments attempted to scan the bulk of the
E. coli
genome by using a transposon that put the lacZYA operon under the control of many promoter regions, it was not known if the entire genome had been surveyed because of the random nature of transposition and unknown location of the majority of transposition events. Accordingly, additional SOS regulon genes have been identified since these early experiments (Lomba et al., (1997)
Microbiol Lett
156:119-122; Walker, (1996) In
Escherichia coli and Salmonella
: Cellular and Molecular Biology. ASM Press, pp 1400-1416).
LaRossa et al. (U.S. Pat. No. 5,683,868) has transformed
E. coli
with at construct comprised of luxCDABE operably linked to a variety of stress promoters. They have used the microorganisms to detect a variety of environmental insults such as Ethanol, CdCl
2
and toluene. The presence of sublethal concentration of insults is indicated by an increase in bioluminescence. However, in order to generate the transformed host, the stress promoters has to be identified and characterized. Furthermore, this method is limited to the stress response only.
Ashby and Rine (U.S. Pat. No. 5,569,588) reported a method to measure the transcriptional responsiveness of an organism to a candidate drug by detecting reporter gene product signals from separately isolated cells of a target organism on genome-wide bases. Each cell contains a recombinant construct with the reporter gene operatively linked to a different endogenous transcriptional regulatory element of the target organism When cells were treated with a candidate drug, the transcriptional responsiveness of the organism to the candidate drug was measured by the detecting the reporter signal from each cells. However, this method is useful with the organism only after the majority of transcriptional regulatory elements of the target organism are known and mapped. Furthermore, the reporter signals are measured only after cells reached homeostasis in the presence of drug. The initial transcriptional responses to chemicals are not considered.
The Lux-A Collection of random
E. coli
genomic DNA fused to the luxCDABE had been used to screen for those gene fusions for which expression was induced by treatment with the herbicide sulfometuron methyl. The DNA sequence of 19 of these sulfometuron methyl inducible gene fusions (smi-lux) was determined and used to identify the promoter controlling expression of the luxCDABE reporter (Van Dyk et al., (1998)
J. Bacteriol.
180:785-792); the remaining 8047 gene fusions remained unidentified.
LaRossa and Van Dyk (U.S. Pat. No. 6,025,131) developed a method for the identification of gene regulatory regions, responsive to a particular cellular stress, such as that produced by herbicides or crop protection chemicals by randomly fusing regulatory regions to a bacterial luminescent gene complex where contacting the fusion in a suitable host with a cellular insult producing a cellular stress results in detection of that cellular stress by an increase in cellular luminescence. However, this method was limited to the perturbations in liquid media; luminescent responses were not detected on solid medium following overnight growth in the presence of a chemical stress. Furthermore, it did not allow regulatory region activity analysis in genome-wide scale.
The problem to be solved therefore is to provide a way to measure and follow the changes in gene expression using a genome-registered collection of reporter gene fusions in a manner that allows detection of initial transcriptional responses, and provide a way to cross-validate the results from other method (i.e., microarray) as well as to determine promoter and operon structure of genes, and further provide a way to test cellular responses to various environmental and genetic changes in high throughput manner.
SUMMARY OF THE INVENTION
A new method for the use of genome registered collection of reporter gene fusion is disclosed. Fragments of genomic DNA of host organism were fused to promoterless reporter gene. The reporter gene fusions were generated using restriction enzyme digestion, physical shearing of the genomic DNA, PCR, and transposition techniques. The reporter gene complexes were genome registered against the host genome on the basis of homology. Gene expression of each reporter gene complex is measure as reporter gene activity. The present invention provides a means to measure the changes in gene expression profiles in genome wide scale under various conditions in high throughput manner. In addition to being a stand-alone high throughput method, the present invention also provides a way to validate other genome-wide assays such as DNA microarray. The present invention also provides a method to confirm the response of several promoters to a particular insult (a condition or chemical of interest) as well as to identify a number of previously unknown operons responsive to that insult. Comparison of the gene expression patterns of two samples differing in one variable is also possible using this method. The present invention also provides the method to use an array of arrays by generating ge
Gonye Gregory E.
Hanafey Michael K.
LaRossa Robert A.
Rafalski J. Antoni
Van Dyk Tina K.
E. I. du Pont de Nemours and Company
McKelvey Terry
LandOfFree
Cellular arrays for the identification of altered gene... does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Cellular arrays for the identification of altered gene..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Cellular arrays for the identification of altered gene... will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-3240242