Isolation of Escherichia coli mRNA and comparison of expression using mRNA and total RNA on DNA microarrays

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.     

Effects of an anti-beta monoclonal antibody on the interaction of the Escherichia coli RNA polymerase with the lac and TAC promoters

The effects of an inhibitory monoclonal antibody (mAb) raised against the beta subunit of the Escherichia coli RNA polymerase were determined on the kinetics and structural interactions during formation of the open promoter complex (RPo). Analysis of the kinetics of abortive initiation on linear and supercoiled templates of the lac and TAC16 promoters showed that abortive synthesis by mAb 210E8-RNA polymerase varied as a function of DNA topology. A kinetic analysis of RPl formation on the supercoiled lac UV5 promoter showed that mAb 210E8 effected a slight alteration in the isomerization rate and no effect on the initial rate of RNA polymerase binding to the promoter. The potent inhibition of initiation with linear promoters by mAb 210E8 was not apparent when the promoters were assayed in their supercoiled forms. Abortive synthesis with the TAC16 promoter was accompanied by an mAb 210E8 induced hindrance of ApUpU but not UpGpU synthesis. The data indicate that the inhibition by mAb 210E8 with the supercoiled TAC16 promoter is further alleviated when the spacer length is shifted from 16 base pairs (ApUpU formation) to 18 base pairs (UpGpU formation). When DNase I and dimethyl sulfate were used to probe DNA structure, mAb 210E8 was found to alter polymerase interactions with the lac promoter. DNase I footprinting indicated that the structural interactions for lac P+ promoter-RNA polymerase complexes were slightly altered in the presence of mAb 210E8. Treatment of the RNA polymerase-lac UV5 complex with dimethyl sulfate revealed an alternate mode of RNA polymerase interaction with essential guanine contacts which was intermediate between a fully protected and free promoter.(ABSTRACT TRUNCATED AT 250 WORDS)     

Isolation of a short, cytosine-rich repeating unit from the DNA of Escherichia coli

Hybridization of heterologous nucleic acids has provided the means for isolating a repeating sequence which is located next to template regions of DNA. Separated single strands of 32P-labelled DNA from Escherichia coli were to a limited extent able to anneal with DNA of Micrococcus lysodeikticus immobilized on nitrocellulose membrane filters. The resulting hybrid was resistant to enzymes specific for unpaired strands, nuclease S1 (Aspergillus oryzae) and exonuclease I (E. coli). The E. coli DNA so hybridized was isolated and characterized. It contained all four bases with cytosine predominating; strand length was about 50-60 nucleotides. Since these units occupied about 1-2% of the length of the E. coli chromosome, they would have to be repeated about 2000 times in a single cell. Formation of the unusual hybrid was not diminished by prior saturation of the E. coli DNA with homologous 3H-labelled RNA. In fact both RNA and additional increments of DNA were detected on the filters approximately in a 1:1 ratio, showing that some of the repeating sequences were physically continuous with transcribed regions of DNA.     

RNA biosensor for the rapid detection of viable Escherichia coli in drinking water

A highly sensitive and specific RNA biosensor was developed for the rapid detection of viable Escherichia coli as an indicator organism in water. The biosensor is coupled with protocols developed earlier for the extraction and amplification of mRNA molecules from E. coli [Anal. Biochem. 303 (2002) 186]. However, in contrast to earlier detection methods, the biosensor allows the rapid detection and quantification of E. coli mRNA in only 15-20 min. In addition, the biosensor is portable, inexpensive and very easy to use, which makes it an ideal detection system for field applications. Viable E. coli are identified and quantified via a 200 nt-long target sequence from mRNA (clpB) coding for a heat shock protein. For sample preparation, a heat shock is applied to the cells prior to disruption. Then, mRNA is extracted, purified and finally amplified using the isothermal amplification technique Nucleic acid sequence-based amplification (NASBA). The amplified RNA is then quantified with the biosensor. The biosensor is a membrane-based DNA/RNA hybridization system using liposome amplification. The various biosensor components such as DNA probe sequences and concentration, buffers, incubation times have been optimized, and using a synthetic target sequence, a detection limit of 5 fmol per sample was determined. An excellent correlation to a much more elaborate and expensive laboratory based detection system was demonstrated, which can detect as few as 40 E. coli cfu/ml. Finally, the assay was tested regarding its specificity; no false positive signals were obtained from other microorganisms or from nonviable E. coli cells.     

Synthesis of a Bacillus subtilis small, acid-soluble spore protein in Escherichia coli causes cell DNA to assume some characteristics of spore DNA.

Small, acid-soluble proteins (SASP) of the alpha/beta-type are associated with DNA in spores of Bacillus subtilis. Induction of synthesis of alpha/beta-type SASP in Escherichia coli resulted in rapid cessation of DNA synthesis, followed by a halt in RNA and then protein accumulation, although significant mRNA and protein synthesis continued. There was a significant loss in viability associated with SASP synthesis in E. coli: recA+ cells became extremely long filaments, whereas recA mutant cells became less filamentous. The nucleoids of cells with alpha/beta-type SASP were extremely condensed, as viewed in both light and electron microscopes, and immunoelectron microscopy showed that the alpha/beta-type SASP were associated with the cell DNA. Induction of alpha/beta-type SASP synthesis in E. coli increased the negative superhelical density of plasmid DNA by approximately 20%; UV irradiation of E. coli with alpha/beta-type SASP gave reduced yields of thymine dimers but significant amounts of the spore photoproduct. These changes in E. coli DNA topology and photochemistry due to alpha/beta-type SASP are similar to the effects of alpha/beta-type SASP on the DNA in Bacillus spores, further suggesting that alpha/beta-type SASP are a major factor determining DNA properties in bacterial spores.     

Studies on macronuclear DNA from Paramecium aurelia

Macronuclear DNA was isolated from purified macronuclei of Paramecium aurelia and the size distribution was determined with regard to growth phase and method of extraction. DNA molecules as long as 105 microns and as short as 0.2 microns were observed. It was concluded that the method of extraction affected the observed length of DNA extracted and that macronuclear DNA isolated from cells in balanced growth was less susceptible to nuclease degradation than was DNA isolated from cells in stationary phase. Renaturation studies were performed on macronuclear DNA and a kinetic complexity of 22-times E. coli DNA was determined. This value was similar to those values reported for Tetrahymena and Stylonychia macronuclear DNA. Correcting for GC base content yielded a kinetic complexity for Paramecium macronuclear DNA of 11-times E. coli DNA which corresponded to 3 X 10(10) daltons. There would be about 1400 copies of a unit genome of this complexity within each newly replicated macronucleus. Density gradient analysis indicated that the genes coding for ribosomal RNA had a greater density in CsCl than the bulk DNA. Molecular hybridization studies indicated that the genes coding for 25 S RNA represented 0.14 percent of the total macronuclear DNA. Correcting for GC base content, this corresponded to 30-35 25 S RNA genes per unit genome. These results on Paramecium are discussed in relationship to other ciliate macronuclear DNA.     

Design of a DNA-matrix with a specific structure for synthesizing RNA using the polymerase chain reaction]

Combination of chemical DNA synthesis and polymerase chain reactions has been used for obtaining DNA templates of preset structure coding for target mRNAs. The obtained DNA were 136 to 246 bp in length, contained promoters for RNA polymerases of phage T7 or E. coli and coded for 136-membered mRNA.     

End group of naturally terminated and UV lesion terminated T7 in vitro RNA.

The 3' terminal nucleosides of RNA transcribed in vitro by E. coli RNA polymerase from T7 DNA and UV irradiated TN DNA were determined. The 3' terminal nucleoside of naturally terminated (t1 termination site) RNA cytidine. In the case of RNA terminated at UV lesions, it is cytidine in 0 per cent of the molecules and adenosine in the remaining 30 per cent. Cytidine trialcohols are labile in high concentrations of KOH and at high temperature and appear to convert to uridine.     

Second-strand cDNA synthesis with E. coli DNA polymerase I and RNase H: the fate of information at the mRNA 5' terminus and the effect of E. coli DNA ligase

A simple method for generating cDNA libraries has been described (1) in which RNase H-DNA polymerase I-mediated second-strand cDNA synthesis primes from an RNA oligonucleotide derived from the 5' (capped) end of mRNA. The size of this oligonucleotide and the fate of the information corresponding to the RNA during subsequent cloning have not been established. We show here that the 5'-most RNA primer varies in length from 8 to 21 nucleotides, and that information corresponding to the length of the RNA primer is normally lost during cloning. A modification of the second-strand cDNA synthesis procedure is described which allows cloning of all, or almost all, of the primer sequence information. In addition, we show that the presence of E. coli DNA ligase during second-strand cDNA synthesis can increase the length of the cDNA clones obtained from long RNAs. Cloning by addition of linkers provides the greatest chance of obtaining near full-length cDNA clones from long mRNAs.