Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2001-09-19
2003-09-09
Whisenant, Ethan (Department: 1634)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C536S023100, C536S024300
Reexamination Certificate
active
06617112
ABSTRACT:
Current transcriptional profiling methods measure steady state mRNA levels in cells. Steady state message levels reflect the balance of gene expression (as transcription rate) to mRNA decay rate. Nuclear runoff assays measure transcription rates directly. I have discovered and disclose the utility of nuclear runoff assays to generate labeled transcript mRNA hybridization probes for use with transcription profiling arrays. This invention discloses and provides improvements in analyzing transcription of mRNA in eukaryotic cells using nuclear runoff transcription assays, including analysis of nuclear runoff transcription products by microarrays containing genes of interest, e.g. microarrays of nucleic acid molecules representing a genome of interest.
BACKGROUND OF THE INVENTION
Transcription profiling with labeled cDNA probes made by reverse transcription of oligo-dT-primed, whole-cell, total RNA measures steady state mRNA levels. Post transcriptional processes, for example, different mRNA decay rates, make steady state mRNA levels poor predictors of transcription rates. In nuclear runoff experiments isolated nuclei, in which transcription units are paused in the transcription process, are allowed to resume transcription in the presence of radiolabeled nucleotides, thereby labeling the nascent transcripts. Nuclear runoff labeled transcripts hybridized to immobilized DNA elements in the form of transcription profiling arrays containing most or all of the genes in an organism of interest have the potential to measure transcription rates on a whole genome basis. Such nuclear runoff transcription profiling has a variety of applications, e.g. in finding regulatory elements, in predicting mRNA stability from gene and mRNA structure and in constructing enhanced transgenic organisms.
Transcription profiling results from steady state mRNA measurements have been used in attempts to define genomic motifs that contribute to regulation of gene expression. See, for instance, Tavazoie et al., Nat Genet July 1999; 22(3):281-5. Post-transcriptional processes could confound such attempts. Nuclear runoff transcription profiling that is a predictor of transcription rate allows more accurate prediction of regulatory motifs. Knowledge of regulatory motifs that control gene expression can facilitate the design of recombinant organisms containing transgenes that are expressed only in specified cell types at specified times, e.g. in the light, in the dark, in drought or other stress.
The structural features that determine mRNA stability are not well known. See, for instance, Ohme-Takagi et al., Proc Natl Acad Sci USA, 90:24, pages 11811-5 (1993). Steady-state mRNA abundance equals the balance between message synthesis (that is, gene expression measured as transcription rate) and mRNA decay. See Hargrove, et al., Bioessays, 13(12), p. 667-674 (1991). Nuclear runoff transcription profiling together with steady state transcript profiling allows derivation of transcriptome estimates of mRNA decay rates. Such results combined with gene- and mRNA-structure predictions from large scale cDNA sequencing and whole genome sequencing allows correlation of mRNA structure with the derived mRNA decay rate.
Knowledge of structural aspects of mRNA that control mRNA stability can facilitate the design of a very stable mRNA message that can accumulate to high levels in a cell type for which only a weak cell-type-specific promoter was available. History and Methodology of Nuclear Runoff Assays
Nuclear runoff methods have found limited utility in prior practice as indicated by the following citations. For instance, newly transcribed mRNA can be identified using the nuclear runoff transcription assay, e.g. as described in Unit 4.10 contributed by Michael E. Greenberg and Timothy P. Bender to “Current Protocols in Molecular Biology” (1997) John Wiley & Sons, Inc., incorporated herein by reference. Nuclear runoff transcription is a procedure for measuring gene transcription as a function of cell state. In a typical method nuclei are first isolated from cultured cells or tissues so as to pause (but not kill) cellular processes including the extension of nascent mRNA transcripts. Isolated nuclei are incubated with labeled nucleoside triphosphate, e.g.
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P uridine triphosphate (
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P-UTP), and unlabelled nucleic acid triphosphates (NTPs) to label nascent mRNA transcripts. Labeled RNA is isolated and used to determine specific RNA transcript levels by hybridization to cDNA probes immobilized on nitrocellulose membrane.
The nuclear runoff transcription assay allows direct measurement and comparison of specific gene transcription in cells in various states of growth or differentiation in contrast to methods that measure steady state levels of mRNA. The nuclear runoff assay is often used with a steady state assay, e.g. northern blot, RNA gel blot, slot blot or dot blot, to assess whether changes in mRNA levels of a particular gene that occur as a function of cell state reflect a change in its synthesis as opposed to a change in mRNA degradation or transport from the nucleus to the cytoplasm. For reviews of nuclear runoff transcription methods, see Marzluff, W. F. Methods Cell Biol., 19:317-331, 1978; Marzluff, W. F. and Huang, R. C. C. Transcription and Translation: A Practical Approach (B. D. Hames and S. J. Higgins, eds.) pp 89-129 IRL Press, Oxford; Greenberg, M. E. and Ziff, E. B., Nature 311:433-438, 1984; and Groundine, M., et al. Mol. Cell. Biol. 1:281-288, 1981, the disclosures of all of which are incorporated herein by reference.
For instance, Berry-Lowe et al., Molecular and Cellular Biology, 5:8, 1910-1917, 1985, discloses the use of nuclear runoff analysis of ribulose-1,5-biphosphate carboxylase where nuclear RNA transcribed in vitro was labeled with [&agr;-
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P]UTP, isolated, and used to probe Southern blots and dot blots containing various DNA samples. Silverthorne et al., The Plant Cell, 2:1181-1190, 1990 disclose the use of nuclear runoff transcriptional analysis to study organ specific expression of the small subunit (SSU) of ribulose-1,5-biphosphate carboxylase/oxygenase. Slot blots of SSU DNA probes were hybridized to in vitro labeled transcripts from root and frond nuclei. And, Pilgrim et al., Plant Molecular Biology, 23:349-364, 1993 disclose the use of nuclear runoff transcriptional analysis to study circadian and light-regulated expression of nitrate reductase in Arabidopsis where plant leaf tissue was harvested at three hour intervals. Nuclei were then allowed to continue transcription in the presence of labeled nucleoside triphosphates to produce labeled RNA transcripts. The labeled RNA transcripts were hybridized to just three DNA targets on a slot blot. In these methods the number of genes that can be monitored is limited by the northern blot or slot blot size. Thus, the nuclear runoff transcription methods were limited in that significant trial and error, or a priori knowledge, was required to select genes which may hybridize to specific RNA transcripts.
OBJECTS OF THE INVENTION
An object of this invention is to provide improved methods for a more rapid, efficient and extensive analysis of RNA transcription rates.
Another object of this invention is to provide methods for determining genome-scale transcription rate of mRNA production in eukaryotic cells.
Another object of this invention is to provide data to allow more accurate prediction of regulatory motifs that influence gene expression.
Yet another object of this invention is to provide methods for constructing a recombinant organism containing a transgene that is specifically expressed in a cell type or in an environmental condition of interest.
Still another object of this invention is to provide methods for finding gene and mRNA structural elements that predict the structural basis and structural determinants of mRNA stability.
Still yet another object of this invention is to provide methods for constructing a recombinant organism with enhanced stability for mRNA transcribed from a gene of interest.
SUMMARY OF THE INVENTION
This inventi
Kelley Thomas E.
Monsanto Technology LLC
Whisenant Ethan
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