INTRODUCTION

Top Abstract Introduction Materials and Methods Results Discussion References

The first comprehensive studies of cellular response resulted from the development of two-dimensional gel electrophoresis and a complementary method for quantitatively measuring protein levels (15, 16). In parallel, Casadaban (2) created transcriptional fusion proteins to assist in the study of gene regulation, and these proteins have been particularly useful for analyzing genes whose products are difficult to characterize. These techniques have been refined (3, 12), and detection of transcriptional regulation has been simplified due to bioluminescent reporter proteins such as luciferase (18, 20) and green fluorescent protein (9).

Chuang et al. (4) demonstrated that global gene regulation in Escherichia coli could be analyzed by using single-stranded reverse-transcribed cDNA and a technique in which radiolabeled cDNA was hybridized with the Kohara set of overlapping  bacteriophage clones. These clones contain the entire E. coli genome and were used to map the locations of cDNA homologs. By performing follow-up Southern blotting (19), Chuang and Blattner identified 26 new heat shock genes (5). This technique was a significant improvement over the methods used previously (two-dimensional electrophoresis and transcriptional fusion) to analyze global genetic regulation; however, the ratio of mRNA signal to total RNA noise remained small (2, 15, 16). Wong and McClelland (23) used a random arbitrarily primed PCR amplification step, after reverse transcription (RT) of total RNA, to detect a stress-induced gene in Salmonella typhimurium. While this technique improved the mRNA signal level, the signal-to-noise ratio did not change due to random amplification of RNA templates. In addition, because sequencing gels were used for transcript identification, this technique did not allow quantification at the genomic level. An innovative refinement of the two methods mentioned above was described by de Saizieu et al. (6); in the refined technique nonradioactively labeled total prokaryotic RNA was hybridized directly to an oligonucleotide array that was synthesized on and bonded to a silicon chip. This method, which allowed quantification of a large subset of transcribed genes, required scanning confocal microscopy as RNA levels were detected as unamplified transcripts.

Conversely, there have been rapid advances in differential display techniques based on PCR amplification in eukaryotic systems due to mRNA polyadenlyation. The RT-PCR (14) and random arbitrarily primed PCR (RAP-PCR) (22) techniques were developed to enhance the signal-to-noise ratio in differential display experiments. While these techniques have provided methods for acquiring vast amounts of regulatory information concerning eukaryotic systems, differential display has not been widely utilized with prokaryotic systems due to the absence of polyadenylation in prokaryotic mRNA. Other developments in eukaryote-based RT-RAP-PCR include the use of random arbitrary primers or motif primers (7, 14, 22). Based on the results obtained, we suspected that an RT-RAP-PCR-based technique in which primers designed specifically for prokaryotic mRNA were used would improve differential display analysis of E. coli.

Fislage et al. (8) performed a detailed statistical evaluation of the coding regions extracted from bacterial genetic databases and designed 10 RT primers for the 3' end of prokaryotic mRNA and 10 PCR primers for the 5' end of prokaryotic mRNA. These primers exhibited increased specificity in the 3' and 5' regions surrounding E. coli genes and decreased specificity for rRNA or other abundant small RNA species, so that mRNA were preferentially transcribed. In their RT-PCR analysis, Fislage et al. used one RT primer in combination with a single PCR primer for an RT-PCR, and the process was subsequently repeated for each primer set so that 100 different amplification experiments were performed for every sample. Following amplification, each sample was analyzed by performing gel electrophoresis, reamplification, and sequencing.

In this paper, we describe an E. coli RT-PCR technique in which a novel combination of the primers of Fislage et al. (Fig. 1) is used in a degenerate fashion for mRNA-specific amplification. A single amplification experiment was required for each RNA sample, and repeated amplifications of the same RNA under identical conditions allowed us to analyze experimental error. This technique was made possible by the availability of an E. coli gene mapping membrane (4) which could be used for global analysis of amplified RNA. Specifically, by avoiding the use of sequencing gels, we were able to avoid performing multiple amplification experiments with the same RNA sample. The RT-PCR method described here was validated by comparing quantified Kohara clone intensities determined with amplified RNA from heat-shocked E. coli cells and non-heat-shocked E. coli cells. Since Kohara clones contain more than one E. coli gene, restriction enzyme-digested Kohara clones were hybridized with RT-PCR products to confirm that amplification of specific heat shock genes occurred. In addition, Northern blotting and total RNA dot blotting were performed to confirm the differentially displayed transcript levels.

View larger version (26K): [in this window] [in a new window]   FIG. 1.   Schematic diagram of the E. coli RT-PCR method used. Purified total RNA was treated with DNase I to remove chromosomal DNA and then repurified. RT and PCR were then performed sequentially in the presence of mRNA-specific primers. Amplified cDNA was end labeled and hybridized with an E. coli gene mapping membrane. Finally, the signal levels for repeated control and stressed samples were quantified, and the induction ratio and standard error were calculated as described in the text.

	MATERIALS AND METHODS

Top Abstract Introduction Materials and Methods Results Discussion References

Microorganisms. E. coli W3110(pIL2) [F IN(rrnD-rrnE)] was used exclusively in this work. W3110 was also utilized by Kohara et al. (13) to create the Kohara miniset.

Fermentation and media. Organisms were grown in Luria-Bertani media (17) supplemented with 0.1 mg of ampicillin per ml. The growth temperature was 37°C, and experiments were performed in 250-ml shake flasks. Shake flask experiments were performed by using either an air shaker (New Brunswick Scientific, Edison, N.J.) at 250 rpm or a reciprocating water bath shaker (New Brunswick Scientific) at 100 rpm.

Induction of stress response and sample preparation. Heat shock was induced in the mid-exponential phase of growth. All samples were taken immediately before the stress and 15 min after the stress. The heat shock was initiated by transferring shake flask cultures grown at 37°C to a 42°C water bath. Samples (25 ml) were removed from the shake flasks and immediately mixed with an equal volume of crushed ice in 50-ml centrifuge tubes. The samples were centrifuged at 5,000 × g for 15 min at 4°C, washed once in ice-cold 50 mM Tris buffer (pH 7.5), and then pelleted by centrifugation under the same conditions before total RNA was isolated.

RNA and DNA purification. RNA was purified by using an RNAqueous total RNA isolation kit (Ambion Inc., Austin, Tex.). This kit typically purifies 10 µg of total RNA from every 1 ml of E. coli culture at an optical density of 1.0. Purified RNA was incubated with 50 U of DNase I (Boehringer Mannheim, Indianapolis, Ind.) per ml at 37°C for 30 min and then was repurified by using an RNAqueous kit. This procedure has been shown to produce RNA that is pure enough to be used in RT-PCR mixtures (Ambion, Inc.). E. coli and RT-PCR product DNA were purified by ethanol-sodium acetate precipitation by using the protocol recommended by Boehringer Mannheim.  DNA was purified by using a  DNA purification kit (Promega, Madison, Wis.).

RT-PCR primers. The specific combinations of primers (based on the findings of Fislage et al. [8]) utilized for RT and PCR are shown in Table 1. Mixtures containing equimolar quantities of RT and PCR primers were utilized for the RT reactions and PCR, respectively. PCR primers PCR1, PCR3, and PCR5 were also added to the RT reaction mixtures at equimolar concentrations.

RT. The RT reaction was performed with a Peltier thermal cycler (MJ Research, Watertown, Mass.). Each reaction mixture contained avian myeoblastosis virus reverse transcriptase (AMVRT) reaction buffer (50 mM Tris HCl [pH 8.3], 30 mM KCl, 8 mM MgCl₂)(Boehringer Mannheim), each deoxynucleotide (Boehringer Mannheim) at a concentration of 0.5 mM, 0.8 U of RNase inhibitor (Boehringer Mannheim) per µl, 0.5 U of AMVRT (Boehringer Mannheim) per µl, each RT primer (Life Technologies, Gaithersburg, Md.) at a concentration of 0.5 µM, 15 µg of RNA sample, and enough autoclaved deionized water to bring the total volume to 60 µl. The RNA was denatured at 70°C for 10 min before it was mixed with the reaction solution. The RNA templates were allowed to anneal to the primers for 10 min at 25°C before the elongation reaction was started. The elongation reaction lasted for 3 h at 42°C. Final denaturation was carried out by heating the preparation at 95°C for 10 min, and cDNA was stored at 20°C.

PCR. The PCR was performed with a Peltier thermal cycler (MJ Research). Each reaction mixture contained Taq DNA polymerase reaction buffer (10 mM Tris HCl [pH 8.3], 50 mM KCl, 1.5 mM MgCl₂)(Boehringer Mannheim), 0.5 mM dATP, 0.5 mM dCTP, 0.5 mM dTTP, 0.5 mM dGTP (all deoxynucleoside triphosphates were obtained from Boehringer Mannheim), each RT primer (Life Technologies) at a concentration of 0.5 µM, each PCR primer (Life Technologies) at a concentration of 0.5 µM, 0.05 U of Taq DNA polymerase (Boehringer Mannheim) per µl, 5 µl of cDNA, and enough autoclaved deionized water to bring the total volume to 60 µl. The PCR cycle consisted of initial denaturation at 94°C for 4 min, followed by 40 cycles consisting of denaturation at 94°C for 1 min, annealing at 40°C for 1 min, and extension at 72°C for 1.5 min. A final extension step consisting of 72°C for 5 min was included. PCR products were quantified by determining absorbance at 260 nm.

3' end labeling of probe DNA with DIG-dUTP. The 3'-end-labeling reaction mixture contained 25 µg of probe DNA, 50 U of terminal transferase (Boehringer Mannheim), terminal transferase reaction preparation (Boehringer Mannheim), 20 µM dATP (Boehringer Mannheim), 3 µM digoxygenin-labeled dUTP (DIG-dUTP)(Boehringer Mannheim), and CoCl₂ (Boehringer Mannheim). This reaction mixture was placed in a 37°C water bath and incubated for 45 min. DIG-labeled probe was purified by ethanol-sodium acetate precipitation by using the protocol recommended by Boehringer Mannheim and was quantified by determining the absorbance at 260 nm.

Hybridization and detection of amplified mRNA. The E. coli gene mapping membranes used consisted of the Kohara set bound to nylon membranes in an ordered matrix (Panvera Inc., Milwaukee, Wis.). The E. coli gene-mapping membranes were prehybridized for 3 h in 15 ml of prehybridization-hybridization solution (Boehringer Mannheim). The prehybridization solution was decanted, and 5 ml of fresh prehybridization-hybridization solution was added. DNA was added to the hybridization mixtures at concentrations between 200 and 1,500 ng/ml. Hybridization occurred during incubation overnight at 40 to 65°C, and the membranes were developed by using the DIG development protocols recommended by Boehringer Mannheim, anti-DIG alkaline phosphatase (Boehringer Mannheim), and the CSPD chemiluminescent substrate (Boehringer Mannheim). Developed membranes were incubated at 37°C for 30 min and then exposed to X-ray film for 2 h. A schematic diagram of the overall RT-PCR and gene mapping technique is shown in Fig. 1.

Southern blot analysis. Kohara clones were amplified, and  DNA was purified by using a  DNA purification kit (Promega). The clones were restriction digested as described below before they were loaded onto an agarose gel. DNA that was separated by agarose gel electrophoresis was denatured by submersion in denaturation solution (0.5 M NaOH, 1.5 M NaCl) for 30 min. The gel was neutralized with neutralization solution (0.5 M Tris-HCl [pH 7.5], 3 M NaCl) for 30 min prior to blotting. The gel was blotted overnight onto a nylon membrane (Boehringer Mannheim) in 20× SSC (1× SSC is 0.15 M NaCl plus 0.015 M sodium citrate, pH 7). DNA was fixed to the membrane by using UV irradiation-induced cross-linking, and the membrane was stored at 20°C. The procedures used for hybridization with RT-PCR products and the DIG development procedures were identical to the procedures used for the gene mapping membrane.

Northern blotting. Samples of total RNA (10 µg/well) were separated by electrophoresing a 1% agarose denaturing gel (17% formaldehyde) at 75 V for approximately 2 h. Unused regions of the gel were removed, and blotting onto a nylon membrane (Boehringer Mannheim) was performed at 4°C overnight in 20× SSC. Total-RNA dot blots were prepared by pipetting 1 to 5 µg of total RNA (in 50% formamide-6.5% formaldehyde-1 × SSC) directly onto nylon membranes by using a microsample filtration manifold (Schleicher and Schuell, Keene, N.H.). All of the nylon membranes were fixed by UV-induced cross-linking. The procedures used for hybridization with DIG-labeled Northern probes and the DIG development procedures were identical to the procedures used for the gene-mapping membrane. The Northern probes were prepared by using a PCR DIG probe synthesis kit (Boehringer Mannheim) and were approximately 400 bp long. A detailed standard curve for serially diluted DIG-labeled  DNA was utilized to quantify Northern blots or total-RNA dot blots. The dilutions used spanned a 50-fold difference in DNA mass per dot, and the resulting standard curve had an r² of 0.9. Northern blotting was performed by using ibpA and groEL to correlate with dot blot data and to check for probe sensitivity. The following primer sequences were used to prepare probes: clpA, 5' ATGCTCAATCAAGAACT 3' and 5' AAAGTTCACCACATCGA 3'; ibpA, 5' ATGCGTAACTTTGATTT 3' and 5' TTAGTTGATTTCGATAC 3'; groEL, 5' ATGGCAGCTAAAGACGT 3' and 5' CTTTGTCCATCGCTTCA 3'; and degP, 5' TTTAATGACCGTCGCGT 3' and 5'CCACATTAGCACTGAGT 3'.

Quantification of RT-PCR product signal. Densitometric scanning was performed to analyze RT-PCR product signals. Images of exposed film were acquired with an Eagle Eye II image acquisition system (Stratagene, La Jolla, Calif.). Each image was quantified by using Scion Image analysis software (Scion, Inc., Frederick, Md.) to measure the area under a curve of the scanned image minus the background of the film surrounding the image. Images were analyzed by determining the densities of each Kohara clone that contained a known heat shock gene and the two adjacent Kohara clones. For example, clones 101, 201, and 301 were evaluated due to the presence of the dnaKJ gene on clone 101 (clones 201 and 301, which are clones that do not contain a heat shock gene, were directly adjacent). The three Kohara clones were scanned simultaneously to minimize the quantification error. Since the technique for integrating the density of a scanned image directly affected the value obtained, the method used for quantification was performed in the same way for all of the film samples analyzed.

	RESULTS

Top Abstract Introduction Materials and Methods Results Discussion References

Evaluation of RT-PCR primers. The ability of the RT-PCR primers described by Fislage et al. (8) (Table 1) to hybridize in a degenerate fashion was compared to the use of random hexamers for differential display analysis (data not shown). In these experiments, the RT reaction mixtures were incubated for 2 or 4 h, and this was followed by 10 or 30 PCR cycles. The best results, which yielded the greatest number of bands on agarose gels, were obtained when random hexamers were used for the RT reaction and an equimolar mixture of the 10 PCR primers of Fislage et al. (8) was used for the PCR. These primers, however, do not preferentially amplify mRNA so that a large amount of rRNA which confounds later analysis can be amplified (14). When RT primers were used in combination with the PCR primers, mRNA was preferentially targeted (8), but in our experiments the lowest number of bands was observed. Consequently, we sought a new combination of RT-PCR primers so that significant levels of mRNA would be amplified.

View larger version (39K): [in this window] [in a new window]   FIG. 2.   (A) Agarose (2%) gel containing RT-PCR samples from heat-shocked and control cells. Lanes 1 and 2 contained control and heat-shocked RNA, respectively. Lanes 3 and 4 contained the RT products in lanes 1 and 2, respectively. Lane 5 contained the products resulting from PCR amplification of the material in lane 3. Lanes 6 and 7 contained parallel PCR mixtures from lane 4. Lanes c4 through c7 contained controls. Lane c4 contained the product resulting from PCR amplification of purified RNA (lanes 1 and 2). Lane c5 contained the product resulting from a complete RT-PCR performed with no initial template RNA. Lane c6 contained the RT-PCR product obtained without AMVRT, and lane c7 contained the PCR product resulting from RNA obtained without DNA Taq polymerase. (B) Agarose (2%) gel containing new RNA samples loaded in the same manner as those in panel A. However, the purified RNA samples were contaminated with DNA, as shown by the multiple bands in control lanes c4 and c6.

Hybridization to the gene-mapping membrane. In order to quantify and map RT-PCR products, the end-labeled RT-PCR products were hybridized to gene-mapping membranes (Fig. 3A). All of the Kohara clones were clearly visible on all of the film samples analyzed; this indicated that amplified mRNA fragments representing the entire E. coli genome hybridized and were detected on the membranes. The signal-to-noise ratio was high enough to quantify the signal at each clone location. In fact, the signals for all 630 clones evaluated were the same within 1%. In addition, the signals for the clones containing rRNA genes were not the maximum signals for all of the clones analyzed, which reaffirmed the ability of the RT-PCR primers to preferentially amplify non-rRNA molecules.

View larger version (89K): [in this window] [in a new window]   FIG. 3.   (A) Hybridization of the RT-PCR product obtained from heat-shocked cells to an E. coli gene-mapping membrane. (B) Gene mapping membrane results obtained for control and heat-shocked films for the regions containing clones 131 and 265. Significantly induced clones 131 and 265 are visible in the heat shock films but are not visible in the control films. In addition, after quantification and analysis of repeated measurements, the induction ratios minus the standard errors were all greater than 1.

View larger version (66K): [in this window] [in a new window]   FIG. 4.   (A) Induction ratios for all of the clones analyzed. The clones identified on the figure were determined to be significantly induced. An induction ratio of 1 indicated that the clone signal did not change after the heat shock treatment. (B) Induction ratios and standard errors for all clones determined to be significantly induced. Note that the error bars for significantly induced clones do not go below 1.

Southern blotting and Northern blotting for identification of specific genes. To ensure that amplified Kohara clone signals were the result of heat shock genes and not other genes in each clone, Southern blotting with restriction enzyme-digested Kohara clones and Northern blot and total-RNA dot blot experiments were performed.

View larger version (34K): [in this window] [in a new window]   FIG. 5.   Southern blots obtained by using restriction enzyme-digested Kohara clones 117 and 212 and by probing with RT-PCR-amplified RNA from heat-shocked E. coli. Each clone was electrophoresed twice, in adjacent lanes of the same gel. Restriction maps for the clones are shown next to the Southern blots. Restriction sites are indicated by horizontal lines in the restriction maps. The restriction enzymes used are indicated below the clone numbers. The genes and locations of the genes on the restriction fragments are indicated. The vertical bars on the restriction maps indicate the fragments detected by Southern blotting. For clone 117 a single 2.5-kbp fragment was detected, and for clone 212 a smear of fragments corresponding to two 0.6-kbp fragments and two 1.0-kbp fragments was visible.

View larger version (49K): [in this window] [in a new window]   FIG. 6.   Total RNA dot blots for degP, clpA, ibpA, and groEL. The data in the bar graph are data obtained from dot blots of heat-shocked samples collected in triplicate under identical conditions. The signal intensity levels for each dot were quantified and averaged for each sample. The induction ratios were then calculated by dividing the average signal intensity for each heat-shocked sample by the corresponding average control signal intensity. For each dot blot, A was the control and B was the preparation obtained from cells that were heat shocked (15 min, 42°C). Note that the dark dot in each of the dot blots resulted from the apparatus used in the experiments.

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

To prove that the technique described here is an experimentally viable tool, we established that the RT-PCR product was the result of amplified mRNA (not contaminating DNA or total RNA) and that the amplified products indicated the relative mRNA amounts at the times that the samples were obtained. The first criterion was established by judiciously using experimental controls (Fig. 2). The second criterion, which was much more difficult to determine, was established by using a known stress response and by comparing our observed results to expected results. In addition, we utilized Southern blot-based mapping to ensure that the results observed corresponded to heat shock genes. Finally, Northern blotting and total-RNA dot blotting were performed to confirm the different levels of specific genes in our RNA samples.

RT-PCR primers and use of the gene-mapping membrane. The RT-PCR method, in which we used the combination of RT-PCR primers described here, allowed us to quantify induced and repressed Kohara clones containing differentially displayed genes in response to heat shock. Our results confirmed that using the primers of Fislage et al. (8), modified as described above, resulted in significant quantities of RT-PCR product cDNA that were representative of the relative amounts of mRNA before and after the heat shock.

RT-PCR as a screening method for differentially displayed genes. The RT-PCR technique used here to analyze differentially displayed genes in E. coli is useful as a screening technique for rapidly identifying Kohara clones containing genes that respond to certain environmental stresses. Since Kohara clones contain more than one E. coli gene, this technique must be followed by additional techniques for identifying specific genes that respond to previously uncharacterized stress responses. Specifically, Northern blotting with gene-specific probes or Southern blotting (19) with restriction enzyme-digested Kohara clones (5) can be used to identify differentially displayed genes. We found that Northern blotting with specific genes produced much better results given the amount of experimental work required. In addition, given the wealth of information about the E. coli genome, educated guesses can be made about differentially displayed genes in lieu of the somewhat lengthy Southern blotting steps.