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J. Biol. Chem., Vol. 275, Issue 42, 32611-32616, October 20, 2000

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Cadmium-inducible Expression of the Yeast GSH1 Gene Requires a Functional Sulfur-Amino Acid Regulatory Network^*

Ulla H. Dormer^§, John Westwater^§¶, Niall F. McLaren, Nicholas A. Kent, Jane Mellor, and Derek J. Jamieson^**

From the  Department of Biological Sciences, Heriot-Watt University, Riccarton, Edinburgh EH14 4AS, Scotland and the  Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, United Kingdom

Received for publication, May 16, 2000, and in revised form, July 25, 2000

	ABSTRACT

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Glutathione (-L-glutamyl-L-cysteinylglycine) is an important antioxidant molecule, helping to buffer the cell against free radicals and toxic electrophiles. Expression of the yeast GSH1 gene, encoding the first enzyme involved in glutathione biosynthesis, -glutamylcysteine synthetase, is regulated by oxidants and the heavy metal cadmium at the level of transcription. We present evidence that the transcription factors involved in controlling the network of sulfur amino acid metabolism genes are also responsible for regulating GSH1 expression in response to cadmium. In particular the transcription factors Met-4, Met-31, and Met-32 are essential for cadmium-mediated regulation of gene expression, whereas the DNA-binding protein Cbf1 appears to play a negative role in controlling GSH1 expression.

	INTRODUCTION

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Glutathione (-L-glutamyl-L-cysteinylglycine) is an important molecule that plays a major role in protecting cells against damage caused by oxidants, heavy metals, and pesticides (1). Glutathione can act as as a free radical scavenger, with the redox-active sulfhydryl group reacting with oxidants to produce oxidized glutathione (GSSG). In response to oxidative stress caused by compounds such as H₂O₂ and superoxide anion-generating agents (for example menadione), the yeast Saccharomyces cerevisiae induces stress responses that result in protection against subsequent toxic levels of oxidants (2-6). We and others (7-10) have shown that glutathione can protect S. cerevisiae against oxidative stress and is therefore an important antioxidant molecule in this organism. In addition to its role as an antioxidant, there is also evidence that glutathione can act as a storage compound for both sulfur and nitrogen (11, 12).

It is also apparent that much of the regulation of the oxidative adaptive stress responses in yeast appears to be at the level of transcription (4, 13-15). We have previously demonstrated (13) that exposure of yeast cells to oxidants results in an increase in the steady state level of GSH1 mRNA, which encodes -glutamylcysteine synthetase, the first enzyme in the GSH biosynthetic pathway. Furthermore, expression of the GSH1 gene has also been shown to be dependent upon the transcription factor Yap1 (13, 16). The expression of the gene encoding yeast glutathione reductase (GLR1) has also been shown to be regulated by oxidants and dependent upon the Yap1 protein (17). The pattern of gene expression controlled by the Yap1 protein has also been investigated using microarrays and high resolution two-dimensional gel electrophoresis (18, 19). We reported that GSH1 gene expression was also inducible by the heavy metal cadmium (20). Glutathione is known to be important in countering cadmium toxicity; indeed, gsh1 mutants are hypersensitive to cadmium (21, 22). Moreover, yap1 mutants are defective in regulation of GSH1 expression and are also sensitive to cadmium (16, 23). There is also clear evidence for a direct link between metal ions and oxidant resistance/sensitivity. Mutations in a number of genes give rise to either resistance or hypersensitivity toward toxic levels of both metal ions and oxidants, reviewed by Santoro and Thiel (6), although exposure of yeast cells to cadmium has been demonstrated to lead to the production of reactive oxidants (24). The link between oxidant stress and metal ion homeostasis has been reinforced by the observation that, by altering metal ion homeostasis, it is possible to suppress the oxidant sensitivity of sod1 mutants (25). Moreover, the close link between metal ions and oxidative stress seems to make biological sense, given the role of metal ions, such as Cu⁺ and Fe²⁺, in the production of oxidants, and as a result a coordinated response to both metals and oxidant stress would be desirable.

In view of the link between glutathione and cadmium, we wished to investigate the cadmium-induced regulation of GSH1 expression. We have identified sequences additional to the Yap1-binding sequence in the GSH1 promoter that regulate gene expression in response to cadmium. Moreover, we show that the genes involved in regulating sulfur amino acid biosynthesis also regulate GSH1 expression.

	MATERIALS AND METHODS

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Yeast Strains and Techniques-- Standard yeast methods and growth media were as described by Rose et al. (26). SD media, containing 2% glucose (w/v), 0.67% yeast nitrogen base without amino acids (Difco) was used throughout and supplemented where indicated with 0.1% (w/v) casamino acids (Difco) or amino acids at 40 µg/ml. Yeast cells were transformed with plasmid DNA using lithium acetate (27). -Galactosidase assays were performed as described by Rose et al. (26). Cadmium sulfate and H₂O₂ (30% w/v) were obtained from Sigma. Media components were obtained from Difco. All other chemicals were of analytical grade. Yeast strains used in this study are described in Table I. The CBF1 gene was deleted using PCR¹-mediated gene replacement, selecting for His⁺ transformants (28, 38).

Plasmid Construction-- The GSH1-lacZ gene fusion (pyDJ73) was described previously, and pyDJ95 is identical to pyDJ73 but contains the TRP1 gene instead of the URA3 gene as the selectable marker (20). The sequences of the oligonucleotide primers used to amplify the regions of the GSH1 promoter are shown in Table II. PCR-generated fragments of the GSH1 promoter were digested with BamHI and XhoI, ligated to plasmid pyDJ75 (20), and digested with BglII and XhoI, fusing the specified regions of the GSH1 promoter to the CYC1 basal promoter and lacZ gene. To fine map the cadmium-responsive promoter regions three overlapping complementary pairs of oligonucleotides were synthesized each containing BamHI and XhoI sticky ends at the 5' and 3' ends, respectively (oligonucleotides 11 and 12, 13 and 14, and 15 and 16), and these were ligated into BglII- and XhoI-digested pyDJ75 to fuse these regions of the GSH1 promoter to the basal CYC1 promoter. Integrative vectors containing various GSH1-lacZ gene fusions were digested with BcuI to excise the 2-µm origin of replication, gel-purified, and religated. Plasmids pDJ98, pDJ99, and pDJ100 were linearized by partial NcoI digestion and transformed into yeast, integrating at the ura3 locus.

Electrophoretic Mobility Shift Assays-- Protein extracts from S. cerevisiae were prepared by glass bead lysis. Yeast cells were grown aerobically at 30 °C in 500 ml of SD (plus appropriate supplements) to early exponential growth phase (A₆₀₀ = 0.1-0.2). Cells were harvested by centrifugation and washed once and resuspended in 200 µl of ice-cold breaking buffer (20 mM Tris/HCl, pH 8.0, 1 mM EDTA, 1 mM dithiothreitol, 10% glycerol (v/v), 1 mM phenylmethylsulfonyl fluoride, 1 mM benzamidine HCl). Cells were lysed by vortexing with 100 µl glass beads. Binding reactions (30 µl) were performed in binding buffer containing 20 mM Tris/HCl, pH 7.5, 100 mM NaCl, 2 mM EDTA, 5 mM MgCl₂, 10% glycerol, 2 µg poly(dI-dC)·(dI-dC), 1 µl of ³²P-labeled DNA probe (approximately 0.33 ng), and 30 µg of yeast protein extract. DNA probes were end-labeled using [-³²P]dATP and T4 polynucleotide kinase and purified on Sephadex G-50 spin columns (Amersham Pharmacia Biotech). In some cases probes were labeled by tailing with terminal transferase and digoxigenin-dUTP according to the manufacturer's instructions (Roche Molecular Biochemicals). Binding reactions were incubated at room temperature for 15 min. For competition reactions approximately 100 times excess of unlabeled DNA probe (33.3 ng) was added to the reaction mixture. Binding reactions were electrophoresed at 4 °C on pre-run (1 h) 4% polyacrylamide gels in 0.5× TBE buffer. After electrophoresis, gels were fixed, dried, and subjected to autoradiography. In the case of digoxigenin-labeled probes, the gels were electroblotted to nylon membranes, and protein complexes were identified following chemiluminescent detection on x-ray film.

Micrococcal Nuclease Digestion of Chromatin-- Chromatin was digested by micrococcal nuclease (MNase) in permeabilized yeast spheroplasts and analyzed by indirect end labeling as described previously (29). The indirect end label for GSH1 was derived from a 1283-bp Alw44I/PvuI fragment (spanning 584 to +699 bp) prepared by PCR using primers 5'-TGCACACGCCTGTTACTTCT-3' and 5'-ATCGTAGATGGAGTCATCCG-3'. The PCR product was cut with HincII to release the 339-bp PvuI/HincII probe.

	RESULTS

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Mapping the Cadmium-responsive Promoter Elements-- We had previously shown that the GSH1 gene was transcriptionally regulated by the heavy metal cadmium (20). GSH1 expression was also shown to be dependent upon the transcription factor Yap1 (16, 20). However, while characterizing the GSH1 promoter we observed that elements other than the Yap1-binding site were also required for cadmium regulation. We created a set of GSH1 promoter deletion mutations, which were fused to the non-regulated basal CYC1 promoter (to provide basal promoter function) and the reporter gene lacZ. Yeast harboring these plasmids were exposed to inducing levels of cadmium, and the level of expression was determined (Fig. 1). More detailed examination revealed that the promoter fragment in plasmid pyDJ81 (379 to 100) was capable of inducing gene expression in response to cadmium, whereas the sequence present in pyDJ80 (343 to 100) was not (Table III). The reduced levels of expression observed with pyDJ81 and 80, compared with pyDJ73, were found to be due to the absence of the Yap1-binding site in these constructs (384 to 379). Notwithstanding the lower level of expression, it is evident that there is an element in the GSH1 promoter, between nucleotides 379 and 343, which is responsible for cadmium-mediated gene expression. To characterize further this cadmium-responsive element, we synthesized three pairs of oligonucleotides whose sequences divide the region between nucleotides 379 and 343 into three portions. These were then fused to the basal CYC1 promoter and the lacZ gene. Yeast containing these three plasmids were exposed to inducing levels of cadmium to determine which contained the cadmium-responsive element. All of these plasmids possessed higher basal levels of expression compared with the larger promoter fragment in pyDJ81, suggesting that a repressor element may have been deleted. Nevertheless, two of the constructs displayed cadmium-regulated gene expression (Table IV). Given the high basal levels of gene expression observed, we were concerned that the presence of the cadmium-responsive element on episomal vectors could give misleading results. To counter this possibility we integrated the constructs into the yeast genome. Essentially identical results were obtained with the integrated constructs, indicating that the cadmium-responsive element lies between nucleotides 360 and 352 (Table IV).

View larger version (10K): [in this window] [in a new window]   Fig. 1.   Effect of promoter deletions on the cadmium induction of GSH1-CYC1-lacZ gene fusions. Cultures of S150-2B carrying the GSH1-lacZ gene fusions were grown aerobically in SD media to early exponential phase (A600 = 0.1-0.2) and exposed to cadmium (0.1 mM) for 2 h. After exposure to the stress, -galactosidase assays were performed. The results shown represent the degree of induction in response to cadmium.

View this table: [in this window] [in a new window]   Table III Cd-responsive element lies between nucleotides 379 and 343 The GSH1-lacZ gene fusions were transformed into wild type (S150-2B). Cultures of each transformant were grown in SD medium with appropriate supplements to early exponential phase A600 = 0.15 and exposed to cadmium (0.1 mM) for 2 h. After this time -galactosidase assays were performed as described under "Materials and Methods," and the results shown are the means of duplicate assays from three independent cultures.

View this table: [in this window] [in a new window]   Table IV Fine mapping of the cadmium-responsive element The GSH1-lacZ gene fusions were transformed into wild type (S150-2B). Cultures of each transformant were grown in SD medium with appropriate supplements to early exponential phase A600 = 0.15 and exposed to cadmium (0.1 mM) for 2 h. After this time -galactosidase assays were performed as described under "Materials and Methods," and the results shown are the means of duplicate assays from three independent cultures. Int, plasmids were integrated at the ura3 locus.

Protein Binding to the GSH1 Promoter in Vitro-- To complement the reporter gene assays we also examined protein-DNA interaction on the GSH1 promoter using an EMSA. A portion of the GSH1 promoter (from 497 to 198) covering the putative cadmium-regulatory element was used as a probe in an EMSA with protein extracts from yeast. We observed a specific complex binding to this region of the GSH1 promoter (Fig. 2A, lane 2).

View larger version (42K): [in this window] [in a new window]   Fig. 2.   Mapping DNA protein-binding sites on the GSH1 promoter. The GSH1 promoter from 497 to 198 was amplified by PCR and end-labeled. This was added to binding reactions containing protein extracts from wild type yeast (S150-2B) (A). Lane 1, free probe; lane 2, no competitor DNA, lane 3, competitor A; lane 4, competitor B; lane 5, competitor C; lane 6, competitor D. Complementary oligonucleotides containing the CDE1 site were annealed and added to binding reactions containing extracts from wild type yeast (S150-2B) (B). Lane 1, free probe; lane 2, no competitor; lane 3, competitor A; lane 4, competitor E. Binding reactions were electrophoresed on polyacrylamide gels as described under "Materials and Methods." Regions of the GSH1 promoter used in the competition experiments, to map the complex binding site, were as follows; A nucleotides 497 to 198, B 437 to 198, C 379 to 198. D343 to 198 and E 379 to 361, all relative to (+1) ATG.

In order to determine the specific region of the GSH1 promoter to which this protein complex was binding, various lengths of the GSH1 promoter were used in competition assays. Gel retardation experiments were performed with a labeled section of the GSH1 promoter (from nucleotides 497 to 198). Excess amounts of shorter unlabeled GSH1 promoter fragments generated by PCR were mixed in with the labeled probe in binding reactions (Fig. 2C). These experiments mapped the protein-binding site of the complex on the GSH1 promoter to nucleotides 379 to 341 (Fig. 2A, lanes 3-5). Further gel retardation experiments narrowed down the binding sequence yet further to the region between 379 and 361 (Fig. 2B, lanes 3 and 4).

The GSH1 Promoter Is Recognized by the Protein Cbf1-- The sequence identified by the EMSA experiments showed a perfect match to a sequence found in centromeric sequences (the CDEI motif) that occurs in the promoters of some methionine-regulated genes (Fig. 3) (30). The GSH1 promoter region contains two potential CDEI-binding sites (370 to 363) and (108 to 102). However the sequence between nucleotides 108 and 102 is not bound by the protein complex (data not shown). The CDE1 sequence element has been shown to be bound by the protein Cbf1. To determine whether the complex contained or at least required the Cbf1 protein for formation, gel retardation experiments were performed using extracts prepared from a strain containing a cbf1 null mutation. These extracts did not produce a specific protein-DNA complex in the EMSA experiments (Fig. 4, lane 4).

View larger version (22K): [in this window] [in a new window]   Fig. 3.   CDE1 flanking sequence in the GSH1 and MET genes. The CDE1 sequence of the GSH1 gene and flanking DNA sequences are aligned with those from some of the MET genes involved in methionine biosynthesis.

View larger version (81K): [in this window] [in a new window]   Fig. 4.   The cbf1 protein is important for complex formation. The GSH1 promoter from 497 to 198 was amplified by PCR and end-labeled. This was added to binding reactions containing extracts from either wild type (S150-2B) and a cbf1 null mutant (DJY169). Binding reactions were electrophoresed on polyacrylamide gels as described under "Materials and Methods." Competition experiments contained approximately 100-fold excess of unlabeled probe DNA. Lane 1, free probe; lane 2, S150-2B extract; lane 3, DJY169 extract; lane 4, S150-2B extract plus competitor DNA; lane 5, DJY169 extract plus competitor DNA.

The Cbf1 Protein Functions in Vivo to Regulate GSH1 Expression-- By having demonstrated that it is likely that the Cbf1 protein binds the GSH1 promoter, it was important to determine whether the Cbf1 protein was involved in regulating transcription in vivo. The expression of a GSH1-lacZ gene fusion was assayed in a cbf1 null mutant. The results of these experiments indicated that the Cbf1 protein does not appear to play a role in regulating the expression of the GSH1 gene in response to H₂O₂ (data not shown). However, the cbf1 null mutant did show elevated levels of basal GSH1 expression, compared with wild type (Table V). Indeed the elevated expression levels seen in the cbf1 mutant were similar to those seen in the wild type induced by cadmium, although no cadmium induction was observed in the cbf1 mutant.

View this table: [in this window] [in a new window]   Table V Methionine regulatory genes regulate GSH1 expression The GSH1-lacZ gene fusion pyDJ95 was transformed into wild type (S150-2B) as follows: a cbf1 mutant (DJY169), a met4 mutant (H313-10A), and a met31/met32 double mutant (CC845-1A). Cultures of each were grown in SD medium with appropriate supplements, including methionine, to early exponential phase A600 = 0.15 and exposed to cadmium (0.1 mM) for 2 h. After this time -galactosidase assays were performed as described under "Materials and Methods," and the results shown are the means of duplicate assays from three independent cultures.

A closer examination of the sequences required for cadmium induction revealed the presence of the following sequence CAACTGTGGC, corresponding to the binding site for the transcription factors Met-31 and Met-32 (31). Both Met-31 and Met-32 function as part of a large complex, which also includes Met-4 and Cbf1, to regulate expression of the methionine biosynthetic genes (32). We tested whether GSH1 expression and cadmium induction in particular also required these factors. A GSH1-lacZ gene fusion was transformed into a met4 and a met31/met32 double mutant, and GSH1 expression levels were measured. It was evident that there was no cadmium induction in either the met4 or the met31/met32 double mutant (Table V), supporting the hypothesis that these transcription factors play a role in regulating GSH1 gene expression in response to cadmium. This effect was specific to cadmium as induction of GSH1 by H₂O₂ still occurred in the met4 mutant (data not shown).

We next asked whether Cbf1 binds to the GSH1 promoter in vivo, by examining the accessibility of the GSH1 promoter to micrococcal nuclease digestion in spheroplasts from wild type and cbf1 mutant yeast (Fig. 5). In the absence of Cbf1 an additional nuclease hypersensitive site is evident in the region of the A-CDEI (Fig. 3) element of the GSH1 promoter, demonstrating that the promoter chromatin accessibility is altered by binding of the Cbf1 protein.

View larger version (103K): [in this window] [in a new window]   Fig. 5.   Indirect end label analysis of chromatin structure of the GSH1 promoter in wild type and cbf1 mutant yeast. Chromatin in yeast strains DBY745 (CBF1) and YAG93 (cbf1) was digested with 75, 150 and 300 units/ml MNase in permeabilized spheroplasts. Lanes 3-5, DBY745; lanes 6-8, YAG93; lane 1, marker digest consisting of DBY745 genomic DNA samples cut to completion with Alw 44I, HindIII and HincII; lane 2, Purified DBY745 genomic DNA digested with 10 units/ml MNase as a "naked DNA" control. All DNAs were cut to completion with PvuI, separated on a 1.5% agarose gel, blotted, and probed with the 339-bp PvuI/HincII end label marked on the gene map. Cbf1-binding motifs at 377bp and 115 bp are marked on the map as black boxes. A MNase cleavage site which occurs in the absence of Cbf1p is marked with an arrow to the left of the blot.

As glutathione is known to be important in countering cadmium toxicity, we examined the effect of the cbf1 mutation upon the cadmium sensitivity of yeast. We reasoned that as the levels of GSH1 gene expression in the cbf1 mutant were almost as high as those seen following cadmium induction that the cbf1 mutant would be more resistant to cadmium. This was indeed the case, with the cbf1 null mutant displaying considerably increased resistance to cadmium (Fig. 6).

View larger version (84K): [in this window] [in a new window]   Fig. 6.   A cbf1 mutant is hyper-resistant to cadmium. The strains S150-2B (wild type) and DJY169 (cbf1) were streaked out onto either (A) YPD plates or (B) YPD plates containing 0.025 µM CdSO4 and incubated for 3 days at 30 °C.

	DISCUSSION

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In view of the central importance of glutathione in countering environmental stress caused by reactive oxidants and heavy metals, we have studied the regulation of glutathione biosynthesis in S. cerevisiae. We have found that expression of the GSH1 gene, encoding the first and rate-limiting step in the GSH biosynthetic pathway, -glutamylcysteine synthetase, is regulated by reactive oxygen species, such as H₂O₂, superoxide anion generators, and the heavy metal cadmium (13, 20). Our results point to a direct link between sulfur amino acid metabolism and glutathione biosynthesis. We show that the genes regulating the methionine biosynthetic pathway are also responsible for the cadmium-mediated induction of GSH1 expression.

A series of promoter deletions enabled us to identify the region of the GSH1 promoter responsible for cadmium-mediated regulation of gene expression. In vitro experiments demonstrated that this sequence appeared to be bound by a specific protein complex, whereas subsequent sequence analysis of the promoter region bound by this complex revealed a sequence with a perfect match to the CDE1 box found in yeast centromeres and in the promoters of some genes involved in methionine biosynthesis (30, 33, 34). This sequence is known to bind a general transcription factor, the 39.4-kDa protein (Cbf1). A considerable amount of previous work, involving the methionine biosynthetic genes, has demonstrated that the Cbf1 protein can act as a recruitment factor for other transcription factors, such as Met-4 and Met-28 (35, 36). There is also evidence that suggests that Cbf1 can modulate chromatin structure to facilitate transcription factor binding (29). Our data strongly suggest that the Cbf1 protein binds to this sequence in the GSH1 promoter both in vivo and in vitro. Mutants lacking Cbf1 have considerably higher basal levels of GSH1 expression and also display enhanced resistance toward cadmium. Taken together these results support the idea that the Cbf1 protein has a repressive role on transcription from the GSH1 promoter.

The sequence responsible for mediating cadmium induction was found to be identical to that bound by the Met-31 and Met-32 proteins (31). The Met-31 and Met-32 proteins are zinc finger DNA-binding proteins that function to recruit the transcription factor Met-4 to the promoters of some of the methionine biosynthetic genes and are required for expression of these genes (32). Neither Met-31 nor Met-32, individually or together, are capable of activating transcription, and these proteins require the trans-activation function of the Met-4 protein to activate transcription (32). By using both met4 and met31/met32 mutants, it was evident that cadmium induction also required all three proteins. One likely scenario is that Met-31 and or Met-32 recruits the Met-4 protein to the GSH1 promoter; whether the Met-28 protein is also involved in this complex is not yet known. The role of the Cbf1 protein in the cadmium induction process is still poorly understood. The cbf1 mutant shows no significant cadmium induction of GSH1 expression; however, this may be because the rate of transcription is already maximal in this mutant.

The link between sulfur amino acid metabolism and GSH biosynthesis is particularly interesting as it has been suggested that GSH can act as a sulfur storage compound (11). This previous study showed that starvation for sulfur resulted in a depletion of the cellular GSH pool, largely through the action of -glutamyl transpeptidase. A more recent report also highlighted the role of GSH as a source of sulfur and suggested a direct link between GSH depletion and SO₄ accumulation in yeast (37). It seems reasonable to suppose that in response to GSH depletion through starvation for sulfur, cellular GSH levels will eventually need to be replenished. Equally, in the presence of excess methionine as a sulfur source, little GSH depletion would occur and therefore there would be little need for new synthesis.

The transcription factor Yap1 is clearly also very important in regulating GSH1 gene expression and in conferring cellular resistance toward cadmium (13, 16, 20, 34). Nevertheless our results show that other transcription factors are also involved. At present the relationship between the Yap1 protein and the Met-4·Met-31 (32) complex is unclear, nor is the physiological reason that this complex is involved in cadmium regulation of gene expression understood. Further work is required to understand the physiological relationship between the Yap1 and sulfur regulatory pathways.

	ACKNOWLEDGEMENTS

We are indebted to Dr. D. Thomas and Dr. H. Mountain for kindly providing strains. We are also grateful to Drs. E. Ellis and R. C. Schuurink for suggestions and critical reading of the manuscript. We also thank Raymond Wightman for help with preliminary experiments.

	FOOTNOTES

^* This work was supported in part by Grant 97/G08324 from the Biotechnology and Biological Science Research Council and the Society for General Microbiology.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^§ These authors contributed equally to the work.

^¶ Supported by a Biotechnology and Biological Science Research Council postgraduate studentship.

^** To whom correspondence should be addressed. Tel.: 44-131-451-3644; Fax: 44-131-451-3009; E-mail: d.j.jamieson@hw.ac.uk.

Published, JBC Papers in Press, July 31, 2000, DOI 10.1074/jbc.M004167200

	ABBREVIATIONS

The abbreviations used are: PCR, polymerase chain reaction; EMSA, electrophoretic mobility shift assay; Mnase, micrococcal nuclease; bp, base pair.

	REFERENCES

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