Abstract
Bacterial messenger RNA (mRNA) is not coherently polyadenylated, whereas mRNA of Eukarya can be separated from stable RNAs by virtue of polyadenylated 3′-termini. We have developed a method to isolate Escherichia coli mRNA by polyadenylating it in crude cell extracts with E. coli poly(A) polymerase I and purifying it by oligo(dT) chromatography. Differences in lacZRNA levels were similar with purified mRNA and total RNA in dot blot hydridizations for cultures grown with or without gratuitous induction of the lactose operon. More broadly, changes in gene expression upon induction were similar when cDNAs primed from mRNA or total RNA with random hexanucleotides were hydridized to DNA microarrays for the E. coli genome. Comparable signal intensities were obtained with only 1% as much oligo(dT)-purified mRNA as total RNA, and hence in vitro poly(A) tailing appears to be selective for mRNA. These and additional studies of genome-wide expression with DNA microarrays provide evidence that in vitro poly(A) tailing works universally for E. coli mRNAs.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 205-213 |
| Number of pages | 9 |
| Journal | Analytical Biochemistry |
| Volume | 290 |
| Issue number | 2 |
| DOIs | |
| State | Published - Mar 15 2001 |
Bibliographical note
Funding Information:1This work was supported by a fellowship from the Deutsche Forschungsgemeinschaft to V.W., NIH Fellowship GM19862 to D.Z., NIH Grant GM31657 to N.C., and NIH Grant GM38361 and a grant from the Novartis Agricultural Discovery Institute to S.K. Work in the laboratory of Patrick O. Brown was supported by the Howard Hughes Medical Institute and NIH Grant HG00983.
Keywords
- Bacteria
- DNA microarrays
- Escherichia coli
- Messenger RNA
- Poly(A) polymerase
- Polyadenylation