Center for Pulmonary and Infectious Disease Control1, and Departments of Microbiology and Immunology2 and Medicine3, University of Texas Health Center, Tyler, TX 75708, USA
Center for Biomedical Inventions, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA4
Department of Pathology, University of Arkansas for Medical Sciences and the Central Arkansas Veterans Health Care System, Little Rock, AR 72205, USA5
Author for correspondence: Peter Barnes. Tel: +1 903 877 7790. Fax: +1 903 877 5516. e-mail: peter.barnes{at}uthct.edu
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ABSTRACT |
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Keywords: mycobacteria, gene expression, transcription
Abbreviations: gDNA, genomic DNA
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INTRODUCTION |
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The evaluation of gene expression by M. tuberculosis under different experimental conditions provides important insight into mycobacterial physiology, and these studies have been greatly facilitated by the availability of the genome sequences of M. tuberculosis (Cole et al., 1998 ; http://www.tigr.org/tigr-scripts/CMR2/CMRGenomes.spl). However, analysis of mRNA expression by M. tuberculosis is difficult, because prokaryotic mRNA is not extensively polyadenylylated, and cannot be separated from total cellular RNA and DNA by binding to an oligonucleotide containing multiple thymidine nucleotides (dT). To overcome this problem, several groups have developed complex methods to study M. tuberculosis gene expression, such as selective capture of transcribed sequences and customized amplification libraries (Alland et al., 1998
; Graham & Clark-Curtiss, 1999
).
In 1986, it was demonstrated that a poly(U) Sepharose column could isolate 0·8% of the total RNA isolated from Mycobacterium smegmatis, suggesting that a significant percentage of the mRNA produced by this organism was polyadenylylated (Katoch & Cox, 1986 ). More recently, two publications showed that, after total RNA from M. tuberculosis, Mycobacterium bovis BCG, M. smegmatis and Mycobacterium vaccae was reverse transcribed with an oligo(dT) primer, several mycobacterial genes could be amplified by PCR from cDNA, supporting the concept that some mRNAs of M. tuberculosis and other mycobacteria are polyadenylylated (Adilakshmi et al., 2000
; Rindi et al., 1998
).
If all mycobacterial mRNAs are polyadenylylated, oligo(dT) can be used to isolate and prime reverse transcription of mRNA, permitting accurate quantification of mycobacterial mRNA expression. On the other hand, if only a limited number of mycobacterial mRNAs are polyadenylylated or if the extent of polyadenylylation of different mRNAs is variable, this strategy will not provide an accurate picture of mycobacterial gene expression. To determine if priming reverse transcription with oligo(dT) yields representative samples of mycobacterial cDNA, we evaluated mRNA expression of multiple mycobacterial genes by competitive RT-PCR and by hybridization of mycobacterial cDNA to an M. tuberculosis microarray.
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METHODS |
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RNA isolation.
Mycobacteria were isolated by centrifugation and resuspended at 5x106 bacilli ml-1 in RNAzol B (Tel-Test), a chaotropic solution containing guanidine thiocyanate and phenol. The mycobacteria were disrupted by shaking with glass beads in an FP120 cell disrupter (Savant Instruments) at a setting of 6·5 for 23 s, and total RNA was obtained by phenol/chloroform extraction, followed by DNase treatment.
Quantification of mRNA expression by competitive RT-PCR.
We selected 12 M. tuberculosis genes that had the potential to contribute to virulence, based on published studies (Table 1). To quantify mRNA expression for these genes, we used the Omega 1.1 software program (Oxford Molecular) to design primers for each gene, based on the sequences available in the M. tuberculosis genome databases maintained by The Institute for Genomic Research and the Sanger Centre. The primer sequences are shown in Table 1
. The forward and reverse primers for 16S rRNA were 5'-GGACTGAGATACGGCCCAGACT-3' and 5'-CGCGACAAACCACCTACGA-3', respectively. Those for 23S rRNA were 5'-GAAACAGCCCAGATCGCC-3' and 5'-CCTACCCACACCCACCACA-3', respectively. In designing primers, the same criteria were used for each gene, thus minimizing differences in amplification efficiencies. Primers were 1921 bp in length, with a G+C content of 60 mol% and a Tm of 6065 °C, yielding amplicons of 150220 bp. For all genes, competitors were constructed that were amplified by the same set of primers to yield amplicons 100200 bp larger than those of the target sequences, using the MIMIIC system (Clontech Laboratory).
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Tenfold serial dilutions of known amounts of MIMIIC DNA were added to 1 µl of sample cDNA and amplified by PCR, using oligonucleotide primers specific for each gene. These preliminary experiments allowed a rough estimation of the amount of template cDNA. Serial twofold dilutions of MIMIIC DNA concentrations in the range of the estimated amount of template cDNA were then coamplified with sample cDNA by PCR. In this latter titration, the integrated density of the PCR product bands has a linear relationship to the amount of PCR product, and to the amount of template. PCR amplification was performed by running 2835 cycles, each cycle consisting of denaturation at 94 °C for 1 min and annealing/extension at 65 °C for 2 min. PCR products were subjected to electrophoresis and visualized by staining with ethidium bromide. To quantify PCR product, gels were photographed with a SPEEDLIGHT gel documentation system (B/T Scientific Technologies) and analysed with Quantity 1 software (Bio-Rad). This imaging and analysis system permits accurate comparison of the integrated density of the PCR product bands for target and MIMIIC DNA. By plotting the log of the ratio of integrated density of sample to MIMIIC PCR product against the log of the number of molecules of MIMIIC substrate DNA, the amount of substrate cDNA was determined, based on the point where the ratio of sample to MIMIIC PCR product was 1:1.
Microarray analysis.
Total mycobacterial RNA was isolated as described above. The RNA was reverse transcribed in the presence of Cy3-dCTP (Amersham Pharmacia Biotech), 0·1 M DTT, 10 µM dNTP except for 2·5 µM dCTP (Gibco-BRL), Superscript II (Gibco-BRL) and its buffer, and either the oligo(dT) or genome-directed primers, which were a mixture of 37 primers specifically designed to amplify all the ORFs in the M. tuberculosis genome (Talaat et al., 2000 ). Reverse transcription was performed at room temperature for 10 min, followed by 42 °C for 2 h. Unincorporated dye was removed by purifying the cDNA with QiaQuick purification kits (Qiagen) according to the manufacturers protocol. Mycobacterial genomic DNA was nick-translated to generate DNA fragments of approximately 500 bp (gDNA), and 2 µg DNA was labelled with Cy5 fluorescent dye, according to the manufacturers protocol (Promega). The Cy3-labelled cDNA was co-hybridized with Cy-5 labelled M. tuberculosis gDNA to an M. tuberculosis microarray that contained oligonucleotides (Operon Technologies) representing the 3924 ORFs present in the M. tuberculosis genome, as previously described (Kane et al., 2000
; Call et al., 2001
). Slides were allowed to hybridize overnight at 67 °C before washing in low-stringency buffer (1xSSC/0·1%SDS) for 5 min at room temperature, followed by another 5 min wash in a high-stringency buffer (0·1xSSC) and drying by centrifugation at 1000 r.p.m. for 5 min. Dry slides were scanned at 10 micropixel intensity (GenePix4000, Axon Instruments).
The signal and background Cy3 and Cy5 fluorescence intensities were calculated for each DNA spot, using image analysis software (GenePix 3.0 Pro, Axon Instrument), to average the intensities of every pixel inside the target region. The intensity of each spot was calculated as the difference between mean signal intensity and mean local background intensity (segmentation method). The ratio of intensity for Cy3- to Cy5-labelled probes was determined for each DNA spot, reflecting the abundance of Cy3-labelled cDNA relative to Cy5-labelled gDNA. A ratio of 1 or greater was considered to represent a positive signal where the mRNA of a particular gene was expressed above the hybridization signal generated from its counterpart gDNA. All hybridizations were repeated two to four times before inclusion in the analysis.
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RESULTS |
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RNA was extracted from two separate cultures of M. tuberculosis, and each RNA sample was divided into equal aliquots. Each aliquot was reverse transcribed to cDNA, using either the oligo(dT) primer or the arbitrary primer. The resultant cDNA was amplified by competitive PCR, using primers for the 12 M. tuberculosis genes in Table 1. Serial dilutions of known quantities of MIMIIC DNA were added to template cDNA. Representative results are shown in Fig. 1
for sigA and for the gene encoding antigen 85C.
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DISCUSSION |
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Although prokaryotic mRNA has traditionally been regarded as lacking 3' polyadenylylated sequences, more recent studies have demonstrated that polyadenylylation occurs in a variety of bacteria, including E. coli and Bacillus species (Sarkar, 1996 , 1997
). Bacterial polyadenylylated tracts are 1060 nucleotides long, significantly shorter than the 80200 nucleotides found in eukaryotic cells. In addition, for any single mRNA species, only 250% of mRNA molecules are polyadenylylated, compared to 100% in the case of eukaryotes (Sarkar, 1997
). Polyadenylylation is believed to be important for stability, maturation and translation of mRNA in eukaryotes. In contrast, a growing body of evidence suggests that polyadenylylation destabilizes certain bacterial mRNA species with stemloop structures, contributing to their degradation (Sarkar, 1997
).
Hybridization with an M. tuberculosis microarray revealed that oligo(dT) primed cDNA synthesis for only 550 genes, whereas priming with genome-directed primers yielded hybridization signals for 1847 genes. It is possible that the number of mRNAs primed by oligo(dT) was underestimated because the large amount of rRNA may depress cDNA synthesis from mRNA templates. In addition, we only considered a significant amount of cDNA to have been synthesized if hybridization of cDNA to the microarray equalled that of an arbitrary amount of gDNA. This method reduces the number of non-specific signals, but it may underestimate the number of mRNAs for which oligo(dT) can prime reverse transcription.
Although the 3' end of mycobacterial mRNA has not been sequenced, it has been hypothesized that polyadenylylation is present, because oligo(dT) can prime cDNA synthesis for several mycobacterial genes, which can then be amplified by non-competitive PCR (Adilakshmi et al., 2000 ; Rindi et al., 1998
). Although we confirmed these results, we found that the quantities of cDNA synthesized after priming with oligo(dT) or with an arbitrary primer were very similar, arguing against the presence of extensively polyadenylylated mycobacterial mRNA, which would be more efficiently reverse transcribed by oligo(dT).
The mechanism by which oligo(dT) primes reverse transcription of mycobacterial mRNA is uncertain. We speculate that the mRNA is reverse transcribed by non-specific binding of the oligo(dT) primer to the RNA template. When oligo(dT) is used to prime reverse transcription of eukaryotic mRNA, selective binding to the mRNA polyadenylylated tail minimizes random binding to other segments of mRNA and rRNA. However, even under these conditions, rRNA is occasionally reverse transcribed. When oligo(dT) is used to prime reverse transcription of mycobacterial mRNA, which is not extensively polyadenylylated, genes and rRNA with segments of several contiguous adenosines may function as the favoured binding sites for oligo(dT) at the relatively low temperature of 37 °C used for reverse transcription.
Our results do not exclude the possibility that oligo(dT) primes reverse transcription of mycobacterial mRNA by specific binding to the polyadenylylated 3' ends, and that a variable percentage of M. tuberculosis mRNA species are polyadenylylated. This could explain the variable priming efficiency of oligo(dT) for different mycobacterial mRNAs, compared to a random primer (Fig. 2). In addition, microarray analysis revealed that oligo(dT) primed reverse transcription of 114 genes more efficiently than genome-directed primers, suggesting that these genes may be polyadenylylated. However, if variable degrees of limited polyadenylylation are present, reverse transcription with oligo(dT) cannot be used to obtain a representative assessment of mRNA expression under different conditions or by different M. tuberculosis strains. Such studies are best pursued by using a mixture of primers that are specifically designed to amplify all the ORFs in the M. tuberculosis genome (Talaat et al., 2000
). Alternatively, because some mRNAs were amplified by oligo(dT) but not by the genome-directed primers, a combination of oligo(dT) and genome-directed primers may yield the most comprehensive collection of cDNAs. Others have used a combination of oligo(dT) and multiple arbitrary primers to perform mRNA differential display in two M. tuberculosis strains (Rindi et al., 1999
).
In summary, using competitive PCR and microarray analysis, we found that priming of reverse transcription of mycobacterial mRNA species with oligo(dT) amplifies a limited fraction of mycobacterial mRNAs, and does not yield representative samples of cDNA.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 9 October 2001;
revised 17 April 2002;
accepted 30 April 2002.
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