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Genetics and Molecular Biology |
Division of Mycobacterial Research, National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, UK1
Author for correspondence: K. G. Papavinasasundaram. Tel: +44 20 8959 3666. Fax: +44 20 8913 8528. e-mail: kpapavi{at}nimr.mrc.ac.uk
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Keywords: LexA, SOS induction, mycobacteria
a Present address: Trescowthick Research Laboratories, Peter MacCallum Cancer Institute, Locked Bag 1, A’Beckett Street, Melbourne VIC 8006, Australia.
b Present address: London School of Hygiene and Tropical Medicine, Keppel Street, London WC1E 7HT, UK.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). The system studied in most detail is that of Escherichia coli, where the so-called SOS response, the induction of around 30 genes in response to DNA damage (Fernandez de Henestrosa et al., 2000 ; Friedberg et al., 1995 ), is regulated by RecA in conjunction with LexA (Little & Mount, 1982 ). The recA and lexA genes are themselves part of the SOS regulon. LexA is a repressor protein which under normal conditions binds to a specific sequence, termed the SOS box, upstream of the SOS genes to restrict their expression (Brent & Ptashne, 1981 ; Little et al., 1981 ). When DNA damage occurs RecA protein binds to regions of single-stranded DNA resulting from processing of that damage or replication blockage (Sassanfar & Roberts, 1990 ) to form nucleoprotein filaments. In this form RecA stimulates the autocatalytic cleavage of LexA (Little, 1991 ). The resulting fragments of LexA do not bind to the SOS boxes (Bertrand-Burggraf et al., 1987 ); therefore, repression by LexA is alleviated and expression of the SOS genes is induced.
Although the details of this system have been worked out from studies of E. coli, the key elements appear to hold in other bacteria. Amongst Gram-positive bacteria the most information is available for Bacillus subtilis, where the functional homologue of LexA is called DinR (Haijema et al., 1996 ; Miller et al., 1996 ; Winterling et al., 1997 ). As with the LexA protein in E. coli, the DinR protein of B. subtilis binds to specific sites upstream of various DNA-damage-inducible genes including dinR and recA, although the sequence recognized is quite different from that bound by E. coli LexA (Cheo et al., 1991 , 1993 ; Winterling et al., 1998 ). In addition, DNA-damage induction is dependent on the presence of an intact recA gene (Gassel & Alonso, 1989 ; Lovett et al., 1988 ) and the cellular levels of DinR decrease following DNA damage (Lovett et al., 1993 ; Miller et al., 1996 ), indicating that the mechanism deduced in E. coli is also valid in B. subtilis.
In mycobacteria the recA and lexA genes have been identified and the LexA protein has been shown to bind to a specific site similar to the SOS box of B. subtilis upstream of each of these genes (Durbach et al., 1997 ; Movahedzadeh et al., 1997a , b ; Papavinasasundaram et al., 1997 ). In addition, the expression of recA has been shown to be inducible by DNA-damaging agents in both M. tuberculosis and M. smegmatis (Durbach et al., 1997 ; Movahedzadeh et al., 1997b ; Papavinasasundaram et al., 1997 ). Preliminary experiments had indicated that a longer period of induction was required for M. tuberculosis as compared with M. smegmatis to see similar levels of recA induction. In this study we have analysed the kinetics of recA induction in M. smegmatis and M. tuberculosis in detail.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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(Sambrook et al., 1989 ), and the mycobacterial strains Mycobacterium smegmatis mc2155 (Snapper et al., 1990 ) and Mycobacterium tuberculosis H37Rv have been described previously (Movahedzadeh et al., 1997b ). M. tuberculosis was grown in tissue culture flasks laid flat in a 37 °C incubator. Published protocols were followed for preparing electrocompetent cells of mycobacteria (Papavinasasundaram et al., 1998 ) and for electroporation (Jacobs et al., 1991 ). To induce DNA damage, mitomycin C (0·2 µg ml-1) or ofloxacin (1 µg ml-1) was added to growing cultures (at an OD600 of 0·6) and incubated for the time indicated. For pulse damage, the cultures were incubated with mitomycin C for a specified period and then the bacteria were harvested, washed and incubated in drug-free medium for expression. The total period of incubation (pulse treatment and expression) was 5 h for M. smegmatis and 24 h for M. tuberculosis. To compare the effects of the mitomycin C treatments on the viability of M. smegmatis and M. tuberculosis, colony-forming units were determined by serial dilution of mitomycin-C-treated cultures in 0·9 % NaCl/0·1 % Tween 80 and plating on 7H11 agar. The treatments assessed were exposure for 1 h or 5 h for M. smegmatis and for 1 h or 24 h for M. tuberculosis.
Recombinant DNA techniques and construction of plasmids.
Plasmid DNA was prepared using SNAP miniprep kits (Invitrogen). For other DNA manipulations, standard DNA protocols were followed (Sambrook et al., 1989 ). Through a series of plasmid manipulations, a lacZ transcriptional reporter plasmid (pEJ414) based on the mycobacterial integrating vector pMV306 (Stover et al., 1991 ) containing a promoterless E. coli lacZ gene from pMC1871 (Casadaban et al., 1983 ) was constructed. The plasmid pEJ414 (sequence available on request) had five copies of the trp terminator cloned at the beginning of a polylinker sequence to block readthrough from any vector promoters, as this number of copies had been shown to be effective in M. smegmatis. The M. tuberculosis recA promoter was cloned as a 0·35 kb PvuII–HindIII fragment from pFM6 (Movahedzadeh et al., 1997b ) into the NruI–HindIII sites of the polylinker in pEJ414 to make pEJ417. The same fragment had been shown previously to have promoter activity (Movahedzadeh et al., 1997b ). Nucleic acid sequences of the clones at the cloning junctions, and the promoter and the terminator sequences, were determined on an ABI PRISM 377 DNA sequencer using the ABI PRISM dRhodamine dye terminator cycle sequencing kit (PE Applied Biosystems). The promoter region was also recovered from the mycobacterial strains into which the clone was introduced by PCR of genomic DNA and the PCR products were sequenced to confirm no changes had occurred.
RNA extraction and real-time quantitative Taqman PCR assay.
Commercially available kits were used for the isolation of total RNA (Hybaid Ribolyser Blue kit) from bacterial cultures (100 ml), to digest contaminating DNA from the RNA preparations using RNase-free DNase (Roche), and subsequent cleanup procedures (RNeasy Mini Kit; Qiagen). First-strand cDNA synthesis was carried out using Superscript II (Life Technologies) following the published protocol (Papavinasasundaram et al., 1997 ). Real-time quantitative PCR was carried out on the ABI Prism 7700 Sequence Detection system using the Taqman Universal PCR Master Mix (PE Applied Biosystems). The primers and the Taqman probes (carrying both a fluorophore and a quencher) were designed using the Primer Express software and obtained from PE Applied Biosystems. The sequences of the primers and the probes are listed in Table 1.
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but using half-size reactions and reading the absorbance of 300 µl reaction mix in a flat-bottom microtitre plate reader. The specific activity in units (mg protein)-1 was calculated using the formula defined by Miller (1972) .
Western blotting.
Cell-free extracts corresponding to 20 µg protein were used in RecA Western blots and 5 µg in LexA Western blots. Following electrophoresis, the proteins were electroblotted onto a PVDF membrane using a semi-dry blotter (Hybaid) at 60 V for 1 h. Equal loading of the proteins was confirmed by Coomassie staining of an identical gel and the efficiency of transfer was verified by staining the blots with a solution of 0·1% Ponceau S in 1% acetic acid. Published protocols were followed for blocking non-specific sites and subsequent washing steps (Papavinasasundaram et al., 1998 ). The primary antisera, anti-RecA and anti-LexA raised in mice against recombinant M. tuberculosis RecA and LexA proteins respectively, were used at 1:1000 dilutions. LexA was purified as described previously (Movahedzadeh et al., 1997a ) and purified RecA was kindly provided by K. Muniyappa. Mouse antibody conjugated to horseradish peroxidase (Dako) was used as the second antibody. The blots were washed and developed with diaminobenzidine reagent solution as described previously (Davis et al., 1992 ).
Gel retardation analysis.
Oligonucleotides containing either the wild-type or mutated M. tuberculosis recA SOS box (Movahedzadeh et al., 1997b ) were designed such that following annealing, the double-stranded oligonucleotides had ‘AATT’ overhangs on both ends that were filled in with [-32P]dATP, dTTP and Klenow enzyme (Promega). This method of fill-in labelling helped to prevent non-specific binding of proteins such as single-strand-binding proteins in the cell-free extracts to single-stranded DNA. Approx. 0·4 pmol of the labelled oligonucleotide was incubated with cell-free extracts and 1 µg poly[d(I-C)] nonspecific competitor DNA in a 20 µl binding reaction [1xbinding buffer contained 20 mM HEPES (pH 7·6), 30 mM KCl, 10 mM (NH4)2SO4, 1 mM EDTA, 1 mM DTT and 0·2 % (w/v) Tween 20] for 15 min at room temperature. Protein–DNA complexes were resolved from free DNA on a 10% non-denaturing polyacrylamide gel by electrophoresis in 0·5xTBE buffer (Sambrook et al., 1989 ) at 180 V for 5 h at 4 °C. Gels were dried and the radioactive bands were visualized by autoradiography. Alternatively, gels were blotted onto a double layer of membranes, one being nitrocellulose and the other DE81 paper, following a published Shift-Western protocol (Demczuk et al., 1993 ). Proteins were retained on the nitrocellulose, which was developed using anti-LexA antibodies as described above, and DNA was retained on the DE81 paper and visualized by autoradiography.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Movahedzadeh et al., 1997b ; Papavinasasundaram et al., 1997 ). In this study we examined the kinetics of this response. Samples were taken at 1 h intervals following the addition of the DNA-damaging agent mitomycin C (0·2 µg ml-1) to an exponential phase (OD600 0·6) culture of M. smegmatis and analysed for RecA expression by Western blotting with an antibody raised to M. tuberculosis RecA protein (Fig. 1a). Induction of RecA expression was apparent from the earliest time point of 1 h, with maximal expression occurring between 3 and 6 h. However, when the equivalent experiment was done using M. tuberculosis very little if any induction was observed at time points up to 6 h (data not shown). Therefore, an extended time course was undertaken with M. tuberculosis, taking samples at intervals up to 36 h (Fig. 1b). Induction of RecA expression was clear from 12 h and peaked between 18 and 36 h. To test whether this difference in the kinetics of induction was specific to the particular DNA-damaging agent used, a similar set of experiments was undertaken but this time using ofloxacin (1 µg ml-1) to damage the DNA. Significantly, the kinetics of induction followed the same pattern with ofloxacin as had been found with mitomycin C (data not shown), although the mechanisms by which they damage DNA are different. These experiments demonstrated that whilst RecA expression was inducible in both species of mycobacteria there was a marked difference in the time required for this response, with essentially no induction occurring in M. tuberculosis at the very time when induction was maximal in M. smegmatis.
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) showed that although induction at the RNA level was detectable at 5 h, again there was a further increase in expression at 24 h, then the expression level remained constant to 36 h. As a control the expression of the gltS gene encoding glutamyl-tRNA synthase was measured in the same samples and as expected there was no induction in its expression following exposure to mitomycin C (Fig. 2). This analysis of the recA mRNA levels supports the results obtained by Western analysis of the RecA protein levels described above, and additionally indicates that the response is complete at 24 h.
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), was cloned into it to yield pEJ417. This clone was then introduced into both M. smegmatis mc2155 and M. tuberculosis H37Rv, and the expression of the lacZ reporter gene was determined by assaying ß-galactosidase activity at various time points following exposure to mitomycin C (0·2 µg ml-1) for both species. The time-course in M. tuberculosis (Fig. 3b) confirmed the earlier results obtained by Western analysis. There was no induction at 3 h, then a slight increase at 6 h followed by increasing levels of expression with time to 36 h, the last time point taken. The continued increase in expression at very late time points may reflect the stability of the reporter mRNA and protein, but again there was no clear response until 12 h. In contrast, the same clone containing the M. tuberculosis recA promoter exhibited a much more rapid response to DNA damage when present in M. smegmatis (Fig. 3a). Although there was no induction at 30 min, an increase in expression was detectable after 1 h and expression reached maximal levels between 4 and 7 h, mirroring the results obtained for expression of RecA protein from the M. smegmatis recA promoter described above. Thus, the M. tuberculosis recA promoter is capable of responding to an inducing signal quickly, and the slow induction of RecA expression in M. tuberculosis is not intrinsic to the M. tuberculosis recA promoter.
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). This was in marked contrast to cells harvested immediately after a 1 h exposure. Similarly, exposure of M. tuberculosis to mitomycin C for 1 h with a 23 h expression step in the absence of DNA-damaging agents was sufficient to induce an equivalent level of expression to that seen following continuous exposure for 24 h (Fig. 4b). Again, this result was in sharp contrast to those obtained when cells were harvested immediately after short time periods such as 3 h, when no induction was apparent. Whilst a 1 h exposure to mitomycin C resulted in a small decrease in viable counts the effect in the two species was comparable (82% for M. smegmatis and 83% for M. tuberculosis). These experiments established that DNA damage occurs within 1 h in both M. smegmatis and M. tuberculosis and thus that a delay in the appearance of damaged DNA cannot be the reason for the slow induction of recA expression.
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; Little & Mount, 1982 ). There is evidence for an analogous system in mycobacteria (Durbach et al., 1997 ; Movahedzadeh et al., 1997a , b ; Papavinasasundaram et al., 1997 ). Of particular significance to this work is the fact that the LexA homologue from M. tuberculosis has been shown to bind to a specific site upstream of the recA gene. It was possible that the slow induction of recA expression seen in M. tuberculosis was due to a slow rate of cleavage of LexA in this species.
To investigate this, the amount of LexA protein present in M. smegmatis and in M. tuberculosis after various times of DNA damage was assessed by Western blotting with an antibody raised to M. tuberculosis LexA protein, using the same samples as had been used above in the Western analysis of RecA expression levels. Under the conditions used the intensity of the signal from the antibody directly correlated with the amount of LexA protein applied to the blot as determined using varying amounts of purified LexA (data not shown). In M. smegmatis a gradual decline in LexA levels occurred with time up to about 3 h, after which there was no further change (Fig. 5a). At no time point examined did the LexA become undetectable, unlike the response of B. subtilis to mitomycin C (Miller et al., 1996 ). Surprisingly, in M. tuberculosis there was very little decrease in the amount of LexA present throughout the time course to 36 h (Fig. 5b).
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). In contrast the M. tuberculosis samples, both uninduced and induced, showed a relatively small variation in the degree of binding of LexA to the probe DNA (Fig. 6b). The use of different amounts of the uninduced extract in this assay established that it was sufficiently sensitive to detect a twofold reduction in the amount of LexA, and indicated that the amount of LexA in the induced samples changed by a maximum of a factor of two. This small decrease in the amount of LexA capable of DNA binding was not apparent until 12–18 h after treatment in accord with the timing of recA induction. These extracts had all been induced with 0·2 µg mitomycin C ml-1, so, in case the M. tuberculosis LexA needed higher levels of DNA damage for a clear response, we repeated the 24 h time point using 1 and 2 µg mitomycin C ml-1; the result was unchanged (data not shown).
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), confirming that LexA was required for the complex formation. In addition, when an oligonucleotide mutated at the LexA binding site was used no retardation was observed (data not shown). Thus, the reduction in repression by LexA following exposure to mitomycin C in M. tuberculosis is slower and to a lesser degree than that in M. smegmatis.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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, 1993 ). However, the activity of a recA–xylE reporter was greater 3 h following treatment than at earlier time points (Raymond-Denise & Guillen, 1992 ). Thus, the kinetics of RecA induction in M. smegmatis bear some similarity to the response in B. subtilis, another Gram-positive bacterium. In contrast, the induction of RecA by DNA damage in M. tuberculosis is markedly slower. This is despite the fact that the organism is capable of responding much more rapidly to other stresses. For example the heat-shock genes hsp60 and hsp70 are induced to maximum levels in less than 1 h following a shift to 45 °C (Patel et al., 1991 ), suggesting that the rate of mRNA synthesis is not limiting. The disparity in the response to DNA damage between the two mycobacterial species is reflected in their differential sensitivities to DNA damage, with M. tuberculosis being more sensitive to UV irradiation (David, 1973 ).
In each mycobacterial species there is a similar relationship between the induction time and the generation time of that species (about 3 h for M. smegmatis in the medium used here and about 20–24 h for M. tuberculosis). Thus, one possible explanation for the difference in kinetics between the species is that the time required for induction is related to the rate of replication of the chromosome, which has been reported to be 11 times slower in M. tuberculosis than in M. smegmatis (Hiriyanna & Ramakrishnan, 1986 ). In this scenario, the ssDNA-inducing signal would arise from replication blockage by the damaged DNA. Whilst this remains an attractive explanation it is perhaps noteworthy that neither mitomycin C nor ofloxacin depends on replication to generate the SOS-inducing signal in E. coli (Sassanfar & Roberts, 1990 ).
We established that the DNA-damaging agent is taken up and, therefore, as only a chemical reaction is then required between mitomycin C and DNA, presumably that the DNA is also damaged, within 1 h in both M. smegmatis and M. tuberculosis. Thus differential rates of damage cannot explain the different rates of RecA induction. In both species a period of further incubation, during which the damaging agent does not need to be present, is required for maximal levels of induction to be obtained. This time must be necessary for recognition and/or processing of the damage to generate the inducing signal and translation of this signal into increased expression. The mechanism responsible for this and the rate-limiting step in this pathway remain to be determined, but the process is evidently slower in M. tuberculosis than in M. smegmatis.
When using the reporter plasmid pEJ417 containing the M. tuberculosis recA promoter region we noticed that the basal level of expression we obtained from uninduced cultures was quite different in the two mycobacterial species. The ß-galactosidase activity in M. smegmatis was around a quarter to a third of that in M. tuberculosis. One potential explanation for this could be that there is a higher occupancy of the LexA binding site by repressor molecules in M. smegmatis, either because the LexA protein binds to the SOS box with higher affinity or because there is a higher intracellular concentration of LexA. Alternatively, it could be that the transcriptional machinery of M. smegmatis works less efficiently on the M. tuberculosis recA promoter than that of M. tuberculosis itself. It is to be noted that when the basal expression level of the native RecA protein in the two species is compared it is actually greater in M. smegmatis. This suggests that the primary reason for the lower ß-galactosidase activity from pEJ417 in M. smegmatis is reduced transcriptional efficiency of the heterologous promoter.
In E. coli, DNA damage is processed into regions of ssDNA by one of various mechanisms, depending on the nature of the damage. When RecA binds to such regions of ssDNA it becomes ‘activated’ and stimulates the auto-catalytic cleavage of LexA. The LexA cleavage products no longer bind to the SOS boxes upstream of the LexA-regulated genes, resulting in an increase in the expression of those genes (Friedberg et al., 1995 ; Little & Mount, 1982 ). In B. subtilis cleavage of the LexA homologue DinR following DNA damage by mitomycin C is clearly seen both by Western analysis (Miller et al., 1996 ) and by gel retardation assay (Lovett et al., 1993 ), with intact DinR declining to undetectable levels in less than 1 h. Whilst we detected a similar decrease in the ability of LexA to bind to a mycobacterial SOS box in induced extracts of M. smegmatis, we saw only a small change in this property in M. tuberculosis extracts following DNA damage and this only at extended time periods. The timing of this change in LexA binding coincides with that of recA induction in the two species of mycobacteria. Nevertheless, the relatively slight change seen in M. tuberculosis raises the possibility that other factors might be involved in recA induction in this species. A second mechanism for regulating gene expression in response to DNA damage might be beneficial to M. tuberculosis if it experienced conditions in which only a subset of the genes normally induced were required. The slow response to DNA damage in M. tuberculosis might be an adaptation permitting a more sustained response over a longer period of time, which could be advantageous for withstanding the defences of the macrophage on infection.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 27 April 2001;
revised 9 August 2001;
accepted 17 August 2001.
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K. K. Gopaul, P. C. Brooks, J.-F. Prost, and E. O. Davis Characterization of the Two Mycobacterium tuberculosis recA Promoters J. Bacteriol., October 15, 2003; 185(20): 6005 - 6015. [Abstract] [Full Text] [PDF]
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 S. Unniraman, M. Chatterji, and V. Nagaraja A hairpin near the 5' end stabilises the DNA gyrase mRNA in Mycobacterium smegmatis Nucleic Acids Res., December 15, 2002; 30(24): 5376 - 5381. [Abstract] [Full Text] [PDF]
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E. M. Dullaghan, P. C. Brooks, and E. O. Davis The role of multiple SOS boxes upstream of the Mycobacterium tuberculosis lexA gene - identification of a novel DNA-damage-inducible gene Microbiology, November 1, 2002; 148(11): 3609 - 3615. [Abstract] [Full Text] [PDF]
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S. Unniraman, M. Chatterji, and V. Nagaraja DNA Gyrase Genes in Mycobacterium tuberculosis: a Single Operon Driven by Multiple Promoters J. Bacteriol., October 1, 2002; 184(19): 5449 - 5456. [Abstract] [Full Text] [PDF]
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E. O. Davis, E. M. Dullaghan, and L. Rand Definition of the Mycobacterial SOS Box and Use To Identify LexA-Regulated Genes in Mycobacterium tuberculosis J. Bacteriol., June 15, 2002; 184(12): 3287 - 3295. [Abstract] [Full Text] [PDF]
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3') used in the real-time quantitative Taqman PCR assay
) and gltS (
) were normalized with gnd mRNA levels from the same samples assayed simultaneously. The values presented are the means of duplicate assays on two independent inductions; the error bars indicate standard deviation.
