Methods for gene array analysis of nuclear runoff transcripts

#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.

#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.

#FIG. 1 is a histogram illustrating transcription frequency of a plurality of genes.

#FIG. 2 is a histogram illustrating bins of genes segregated by value of ratio of steady state mRNA level to nuclear runoff mRNA level which is useful in methods of this invention.

#The term “nascent mRNA transcript” as used herein means an mRNA precursor molecule that is partially transcribed.

#The term “nuclear runoff transcription assay” as used herein means methods for resuming a paused transcription of mRNA in the presence of labeled and unlabelled NTPs to produce completely transcribed, labeled mRNA molecules which can be hybridized to known DNA probes. More particularly, nuclear runoff transcription assay includes the methods which are described by Michael E. Greenberg and Timothy P. Bender in Unit 4.10 of “Current Protocols in Molecular Biology” (1997) John Wiley & Sons, Inc., incorporated herein by reference, and derivative methods specifically described herein.

#The term “nucleic acid molecule” as used herein means a DNA or RNA molecule ranging in length from oligonucleotides to full length genes or other large fragments of a genome.