Selective isolation of bacterial mRNA

Details Selective isolation of bacterial mRNA Selective isolation of bacterial mRNA

1999-10-01

2001-06-05

Zitomer, Stephanie W. (Department: 1655)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

C435S015000, C435S259000, C435S194000, C435S069200, C435S243000, C435S252330, C536S025400

Reexamination Certificate

active

06242189

ABSTRACT:

INTRODUCTION
1. Field of the Invention
The field of the invention is the selective isolation of bacterial mRNA.
2. Background of the Invention
RNA exists in three functionally different forms: ribosomal RNA (rRNA), transfer RNA (tRNA) and messenger RNA (mRNA). Whereas stable rRNA and tRNA are involved in catalytic processes in translation, mRNA molecules carry genetic information. Only about 1-5% of the total RNA consists of mRNA, about 15% of tRNA and about 80% of rRNA. In eukaryotic cells mRNA is polyadenylated (the 3′-terminal modification usually consists of about 200 adenosyl residues) and differs from rRNA and tRNA by this structural feature (Hereford & Roshbash (1977) Cell 10:453-462). Thus, eukaryotic mRNA can easily be purified by chromatography based on hybridization to oligo(dT)-nucleotides.
Bacterial mRNA, however, is not uniformly polyadenylated and only a few mRNA molecules have short 3′-terminal modifications (Nakazato et al. (1975) Nature 256:144-146; Sarkar (1997) Ann. Rev. Biochem. 66:173-197). Thus, in bacteria mRNA cannot be distinguished from rRNA or tRNA by a structural feature.
In the bacterial cell, rRNA is complexed with ribosomal proteins to high molecular weight structures: sub-ribosomal 30S and 50S particles, 70S ribosomes and polysomes. Polysomes represent a complex of an mRNA molecule with one or more ribosomes bound to it and polysomes can be purified. Amara & Vijaya showed that when purified polysomes are subjected to polyadenylation in vitro only the 3′-termini of mRNA, but not of rRNA are modified (Amara & Vijaya (1997) Nucl. Acid Res. 25:3465-3470). Their finding suggested that 3′-termini of rRNA in complexes with ribosomal proteins (at least in polysomes) are sterically blocked. However, mRNA molecules present in polysomes are only a subset of all cellular mRNA as those mRNA molecules are actively transcribed. To avoid biasing mRNA purification for actively transcribed mRNA molecules, we developed a method that allows isolation of mRNA representing all of the cellular mRNA population.
SUMMARY OF THE INVENTION
The invention provides methods and compositions for selectively isolating total bacterial mRNA. The general method comprises the steps of (a) contacting a bacterial lysate comprising total bacterial mRNA and nonisolated bacterial polysomes with an exogenous enzyme under conditions wherein the enzyme selectively modifies the mRNA to form modified mRNA, and (b) isolating the modified mRNA. In particular embodiments, the enzyme is selected from a poly(A) polymerase, a RNA ligase and a terminal deoxynucleotidyl transferase. Depending on the enzyme, the modified mRNA may have any of a variety of modifications, such as a 3′ tail, particularly a poly(A) tail, a specific sequence tag, a detectably labeled nucleotide, etc.
The conditions generally comprise an inhibitor of RNases, such as one or more chemical inhibitors and/or elevated temperature, used especially in conjunction with a thermostable enzyme. The subject compositions include products of the disclosed methods, particularly compositions comprising total bacterial mRNA, including species not of or from intact polysomes, wherein the mRNA is 3′ polyadenylated.


REFERENCES:
patent: 5525497 (1996-06-01), Keller et al.
patent: 5968767 (1999-10-01), Sheikh et al.
patent: 5972693 (1999-10-01), Rothberg et al.
patent: 5997913 (1999-12-01), Fowler et al.
Amara et al. Specific polyadenylation and purification of total messenger RNA fromEscherichia coli.Nucleic acids Reserch. vol. 25, No. 17, pp. 3465-3470, Dec. 1997.

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