Department of Biological Sciences, University of Hull, Hull HU6 7RX, UK1
Author for correspondence: Colin Ratledge. Tel: +44 1482 465243. Fax: +44 1482 465458. e-mail: c.ratledge{at}biosci.hull.ac.uk
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ABSTRACT |
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Keywords: mycobacteria, polyadenylylation, mRNA, oligo d(T), fluorescein-11-dUTP
Abbreviations: DEPC, diethylpyrocarbonate
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INTRODUCTION |
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The entC and entD genes that have been used in this work were selected because of their possible involvement in iron utilization (Cole et al., 1998 ). We presume that these genes are involved in the biosynthesis of salicylic acid, which occurs as a response of mycobacteria to deprivation of iron (Ratledge, 1999
; Adilakshmi et al., 2000
). Another gene, mbtI (trpE2) was also included in this study as Quadri et al. (1998)
suggested that the protein product of this gene from Mycobacterium tuberculosis was homologous to salicylate- and anthranilate-forming enzymes from various other bacteria and therefore could be responsible for salicylate formation. In addition, hsp65, encoding a well-characterized 65 kDa antigen in mycobacteria (Shinnick, 1987
), was used.
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METHODS |
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Genomic DNA isolation.
Genomic DNA was isolated from mycobacteria according to the method of Gonzalez-y-Merchand et al. (1996) . Cells were collected from growth medium by centrifugation at 4000 g for 10 min and resuspended in 10 µl lysis buffer per mg cells (wet wt). Lysis buffer was 6 M guanidinium chloride, 1 mM 2-mercaptoethanol, 10 mM EDTA and 0·1% Tween 80. The cell suspension was subjected to two cycles of freezethawing by placing it in liquid N2 or ethanol/CO2 freezing mixture for 1 min and then transferred to a water bath at 65 °C for 10 min. The resulting suspension was chilled on ice for 5 min and extracted twice with 2 vols chloroform/isoamyl alcohol, (24:1, v/v). Two volumes of cold ethanol were added gradually and mixed gently by inverting the tube. This mixture was left at -20 °C for 30 min. The DNA precipitate was recovered by centrifugation and dissolved in TE buffer (10 mM Tris/HCl, 1 mM EDTA; pH 7·5) and stored at -20 °C for further use.
RNA extraction.
The method of Rajagopalan et al. (1995) was used to extract total RNA. One millilitre of the culture (OD600 0·5) was harvested, and centrifuged for 5 min at 4000 g at 4 °C; the supernatant was discarded and 1·5 ml Trizol reagent (phenol and guanidinium thiocyanate in a monophasic solution; Life Technologies) was added to the cell pellet. The suspension was ultrasonicated (Dawe Soniprobe) for 20 s followed by three cycles of freezing in liquid N2 and thawing at 4 °C. The cell debris was removed by centrifugation for 2 min at 4000 g. The supernatant was extracted with 0·75 ml chloroform. RNA was precipitated by adding 0·6 ml propan-2-ol and incubating for 10 min at room temperature. The RNA pellet was recovered by centrifugation at 12000 g for 15 min at 4 °C. It was then washed once with 70% (v/v) ethanol prepared in diethyl pyrocarbonate (DEPC)-treated water and resuspended in 40 µl RNase-free, DEPC-treated water using the method of Sambrook et al. (1989)
. RNA was quantified in a GeneQuant II analyser (Pharmacia Biotech) by measuring A260. The A260/A280 ratios for the RNA preparation were in the range 1·82·0.
DNase treatment.
RNA (1 µg) was treated with 5 units DNase I (Life Technologies) and incubated at 25 °C for 10 min to remove contaminating chromosomal DNA. EDTA (1 µl of 25 mM) was then added and the enzyme inactivated by heating to 65 °C for 15 min. The reaction mix was extracted with chloroform; the aqueous phase was precipitated with chilled propan-2-ol and centrifuged at 12000 g for 10 min at 4 °C. The supernatant was discarded and the pellet was once again washed with 70% (v/v) ethanol and recentrifuged at 12000 g for 10 min at 4 °C. The pellet was then suspended in 20 µl DEPC-treated water and requantified using the GeneQuant II analyser. The integrity of the RNA was checked on a 1·5% non-denaturing agarose gel as described by Mahenthiralingam (1998) .
cDNA synthesis.
Reverse transcription into cDNA was performed in 50 µl containing 1 µg RNA, 2 µg oligo(dT1218), 20 units RNAsin (both from Pharmacia), 10 mM DTT, 10 µl 5xnucleotide mix (dATP, dGTP, dCTP, dTTP and fluorescein-11-dUTP; Amersham), 10 units Superscript RNase H- reverse transcriptase (Life Technologies) and an appropriate volume of DEPC-treated water. The mixture was incubated for 1 h at 37 °C, chilled rapidly on ice and stored at -20 °C.
Southern blotting.
cDNA samples were electrophoresed at 100 V through a 0·8% agarose minigel in 1xTBE (45 mM Tris/borate, 1 mM EDTA). The cDNA could be seen directly under UV as a green fluorescent smear. However, resolution was better when it was stained with 0·5 µg ethidium bromide ml-1 for 30 min. The transfer of DNA from the gel to a nylon membrane was carried out overnight in 10xSSC (3 M NaCl, 0·3 M sodium citrate, pH 7·0) by capillary blotting and the DNA was fixed by UV cross-linking (UV Stratalinker, Stratagene).
Detection of cDNA.
The membrane was briefly rinsed with buffer A (100 mM Tris/HCl, 600 mM NaCl; pH 7·5) (2 ml cm-2) and incubated in anti-fluoresceinhorseradish peroxidase antibody conjugate (ECL; Amersham), diluted 1000-fold in freshly prepared 0·5% (w/v) bovine serum albumin fraction V in buffer A for 30 min. Unbound conjugate was removed by washing the membrane for 3x10 min periods in excess buffer A containing 0·1% (v/v) Tween 20. The washed membrane was treated with detection solution mixture according to the manufacturers instructions and exposed for 1 h to Hyperfilm (Amersham), which was then developed.
PCR.
The technique of message-amplification phenotyping (mRNA phenotyping), an application of the PCR technique, was used to detect the mRNA transcripts of hsp65, one of the well-characterized genes of mycobacterial metabolism (Shinnick, 1987 ), and entD, entC and trpE2 (see Introduction). Primers were designed using the Oligo 5.0 software and obtained from MWG-Biotech. All the other reagents used in the PCR reaction were from Life Technologies. The PCR mixture contained 2·5 µl nascent cDNA, 2·5 µl 10xPCR buffer, 1·5 mM MgCl2, 200 µM each of the dNTPs, 0·5 µg each of oligonucleotide primers and 2 units Taq DNA polymerase, and was made up to a final volume of 25 µl with sterile distilled water. Amplification was performed on a Peltier thermocycler (MJ Research) using the following thermocycle: (94 °C, 1 min; 55 °C, 1 min; 72 °C, 2 min)x30 cycles; (72 °C, 10 min)x1 cycle. DNA bands were visualized on agarose gel electrophoresis and photographed using the UVIPhoto V.97 software on Windows 95. The identity of the PCR products was confirmed by restriction enzyme analysis. All restriction enzymes were from Life Technologies.
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RESULTS |
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Specific mRNA transcripts of hsp65 were amplified with gene specific primers (Table 1) in three species of mycobacteria, M. bovis BCG, M. smegmatis and M. vaccae (Fig. 3
). As expected, a 441 bp sequence corresponding to the hsp65 product was observed. It is clear that the amplification of hsp65 was markedly pronounced in the cDNA prepared in presence of oligo(dT) alone (lanes 3, 7 and 11) and was equivalent to that obtained with genomic DNA amplification (lanes 4, 8 and 12).
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DISCUSSION |
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Little is known about gene expression patterns of mycobacteria; the definition of differentially expressed genes is central to understanding pathogenesis and virulence at a molecular level. The application of techniques such as differential display and subtractive hybridization, commonly used for the analysis of eukaryotic gene expression, has been limited in prokaryotes, owing to the generally accepted view that 3'-polyadenylylation of mRNA is a eukaryotic feature. Differential display in prokaryotes therefore requires the use of non-specific, random arbitrary primers (RAP) for cDNA synthesis, often involving extensive testing and optimization of primers (Kwaik & Pederson, 1996 ; Fislage et al., 1997
; Rivera-Marrero et al., 1998
). In addition, reamplification by PCR and subcloning of the isolated cDNA fragments is often difficult and results in failure. The main problem found with the RAP-PCR method is that, due to the abundance of rRNA and variations in the amount of rRNA in RNA preparations from different strains, amplification with arbitrary primers often results in a high number of false positives. Our finding therefore represents a significant advance in the study of gene expression in mycobacteria, as oligo(dT)-primed cDNA synthesis eliminates the pitfalls encountered with techniques such as RAP-PCR.
Sequential addition of adenylate residues to 3'-termini of RNA molecules is catalysed by the enzyme poly(A) polymerase (ATP:polyribonucleotide adenylyltransferase, EC 2 . 7 . 7 . 19). Multiple isoforms of poly(A) polymerases, probably derived from a single gene by alternate splicing or post-translational modification, occur in eukaryotes (Thuresson et al., 1994 ). In contrast, there is growing evidence for the occurrence of more than one such enzyme encoded by different genes (pcnA and pcnB) in Escherichia coli (Sarkar, 1997
). The deduced product of pcnB (PAP I), however, showed no homology to eukaryotic poly(A) polymerases and even moderate overexpression of this gene was lethal to E. coli (Cao & Sarkar, 1992
). On the other hand, disruption of this gene decreased the growth rate by two-thirds (Liu & Parkinson, 1989
). The protein product of pcnA of E. coli (PAP II) has no significant sequence homology either to PAP I or to the viral and eukaryotic poly(A) polymerases (Kalapos et al., 1994
), suggesting that the bacterial poly(A) polymerases have evolved independently. Sarkar (1997)
suggested that PAP I and PAP II possibly polyadenylate complementary classes of mRNA, or one enzyme initiates poly(A) chains and the other functions in their extension, and that the deletion of both the genes would be lethal to the organism. In Saccharomyces cerevisiae, there is a single gene for poly(A) polymerase whose inactivation leads to the loss of viability (Lingner et al., 1991
). The protein product of the pcnA gene (Rv3907c; Cole et al., 1998
) of M. tuberculosis has 80% similarity (BLASTP search) to the poly(A) polymerase-encoding gene pcnB of M. leprae (accession no. AAB53125; Fsihi et al., 1996
), suggesting that this gene is conserved in mycobacteria. In addition, this protein has a high similarity to poly(A) polymerases from various organisms including Bacillus subtilis, E. coli and Helicobacter pylori, supporting our finding that polyadenylylation does occur in mycobacteria. Understanding the importance of pcnA and pcnB to the survival of M. tuberculosis and M. leprae, respectively, might therefore open a new area for drug design to combat these pathogens.
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ACKNOWLEDGEMENTS |
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Received 28 June 1999;
revised 2 November 1999;
accepted 20 December 1999.