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Eukaryot Cell. 2006 Nov; 5(11): 1925–1933.
Published online 2006 Sep 15. doi:  10.1128/EC.00105-06
PMCID: PMC1694797

Effect of Sequence-Directed Nucleosome Disruption on Cell-Type-Specific Repression by α2/Mcm1 in the Yeast Genome


In Saccharomyces cerevisiae, a-cell-specific genes are repressed in MATα cells by α2/Mcm1, acting in concert with the Ssn6-Tup1 corepressors and the Isw2 chromatin remodeling complex, and nucleosome positioning has been proposed as one mechanism of repression. However, prior studies showed that nucleosome positioning is not essential for repression by α2/Mcm1 in artificial reporter plasmids, and the importance of the nucleosome positioning remains questionable. We have tested the function of positioned nucleosomes through alteration of genomic chromatin at the a-cell-specific gene BAR1. We report here that a positioned nucleosome in the BAR1 promoter is disrupted in cis by the insertion of diverse DNA sequences such as poly(dA) · poly(dT) and poly(dC-dG) · poly(dC-dG), leading to inappropriate partial derepression of BAR1. Also, we show that isw2 mutation causes loss of nucleosome positioning in BAR1 in MATα cells as well as partial disruption of repression. Thus, nucleosome positioning is required for full repression, but loss of nucleosome positioning is not sufficient to relieve repression completely. Even though disruption of nucleosome positioning by the cis- and trans-acting modulators of chromatin has a modest effect on the level of transcription, it causes significant degradation of the α-mating pheromone in MATα cells, thereby affecting its cell type identity. Our results illustrate a useful paradigm for analysis of chromatin structural effects at genomic loci.

In Saccharomyces cerevisiae, a-cell-specific genes are repressed in MATα cells by the α2/Mcm1 repressor, in concert with the corepressors Tup1-Ssn6, and several models for repression mechanisms have been proposed (46). Tup1-Ssn6 may interact with the general transcriptional machinery to inhibit transcription directly, or it may interfere with transcriptional activator function (12, 18, 20, 21, 29, 34). Nucleosomes are precisely positioned in the promoters of a-cell-specific genes in the repressed state in MATα cells but are not positioned in the activated state in MATa cells (9, 14, 37, 42), and the presence and absence of nucleosome positioning is not a consequence of transcription (9). Tup1 interacts with histones and histone deacetylases (5, 10, 11, 51, 52), and a tup1 mutation causes both disruption of nucleosome positioning and repression (9, 57). Also, the Isw2-Itc1 chromatin remodeling complex (17) is involved in regulation of Tup1-Ssn6-repressed genes (56), including a-cell-specific genes (16, 38). Thus, it has been proposed that positioned nucleosomes may modulate the accessibility of promoters to transcription factors to repress the genes (44).

However, some studies suggest that nucleosome positioning is not essential for repression by α2/Mcm1. When the a-cell-specific STE6 TATA box is placed at different locations in a positioned nucleosome and in the internucleosomal linker in STE6-lacZ reporter plasmids, no expression is detectable, even with the TATA box located in a linker region (31). Nucleosomes are not positioned in a test CYC1 promoter containing the α2 operator and Gal4 binding site and Gal4 can occupy its site, even though the test promoter is repressed by α2/Mcm1 (35). α2/Mcm1-dependent repression occurs in a naked DNA template in vitro (18). Also, the role of nucleosome positioning in repression of a-cell-specific genes has been examined by introducing mutations in histones and in other factors, such as Tup1, Ssn6, and histone deacetylases (37, 51). However, interpretation of these mutations is complicated by the fact that they have highly pleiotropic effects. Thus, although positioned nucleosomes have been observed in a number of promoters in yeast and mammalian cells, the importance of the positioning has remained questionable.

DNA can adopt several types of conformations as dictated by its sequence (45), and genomic analyses show that alternative DNA structure-forming sequences are represented in eukaryotic genomes (7, 39). Among such sequences, poly(dA) · poly(dT) and poly(dG) · poly(dC) as well as Z-DNA-forming sequences do not form nucleosomes reconstituted from purified histone octamer (2, 6, 15, 43), whereas CTG repeats preferentially bind to histone octamers in vitro (50). We have shown that the unusual B′ conformation, adopted by longer poly(dA) · poly(dT) sequences, disrupts an array of positioned nucleosomes in yeast cells (41). Poly(dA) · poly(dT) sequences in the yeast HIS3 promoter (19) and the Candida glabrata AMT1 gene (58) stimulate transcription by improving accessibility to the promoter in vivo. The nucleosome-free sequences were evolutionarily conserved and are enriched in poly(dA) · poly(dT) sequences as revealed by genome-scale analysis of yeast chromosome III (55). Poly(dA) · poly(dT) as well as (CCGNN)n, both of which do not favor nucleosome formation, can act as efficient boundaries of silent chromatin (4, 54). Z-DNA is required for the activation of the human CSF1 promoter by the SWI/SNF-like BAF complex, and it is thought that Z-DNA formation promoted by the BAF complex stabilizes the open chromatin structure at the promoter (23, 24). Thus, DNA structural properties may be used to modulate the positioning of nucleosomes in vivo to alter gene expression.

In this report, we have developed a systematic strategy to test critically the role of nucleosome positioning in repression of a-cell-specific gene BAR1 using nucleosome-disrupting sequences. We show here that longer poly(dA) · poly(dT) and poly(dC-dG) · poly(dC-dG) inserts block formation of a positioned nucleosome in the promoter to cause partial derepression of BAR1, while shorter inserts, CTG and GAGCTC repeats, are incorporated into a positioned nucleosome to maintain the repressed BAR1 state. These results indicate that nucleosome positioning contributes to full repression by α2/Mcm1, but it is solely responsible for repression.


Yeast strains and plasmids.

Yeast strains used were FY23 (MATa ura3-52 trp1Δ63 leu2Δ1) and FY24 (isogenic to FY23 except for MATα), which were obtained from the Yeast Genetics course at Cold Spring Harbor Laboratory. To construct strains with a modified BAR1 promoter, we cloned the −500 to +51 region of BAR1 into pRS306ΔKI, a pRS306 derivative in which the KpnI site in pRS306 was filled in, forming pYY1-2. Then, mutations in the sequence AATGT at a region −158 to −154 to GTACC were introduced to create a KpnI site in the BAR1 promoter by PCR, forming pAS1-8. A pair of oligonucleotides synthesized chemically was annealed and cloned into the KpnI site of pAS1-8. The portions of the BAR1 promoter sequence were replaced with (CTG)12 or (CG)7 by two-step PCR, and the modified promoter fragments were recombined with pAS1-8 in vivo in yeast. All the modified promoters were verified by DNA sequencing. Plasmids containing the modified BAR1 promoters were digested with XbaI and were integrated into the genomic BAR1 locus in FY23 and FY24, and the plasmid portions were looped out by two-step gene replacement. isw2Δ strains, MHS303 and MHS314, were constructed from wxy292 and wxy293, respectively, by one-step gene replacement using pFA6aMXHIS3 (53). Strains constructed in this study are listed in Table Table11.

Strains used in this study

Halo assay.

To assay for the generation of α-factor halo (27), MATa sst1 cells were grown for 24 h. Aliquots were then diluted to an optical density at 600 nm (OD600) of 0.5, and 100 μl of the diluted culture (∼106 cells) was spread on a YEPD (1% yeast extract, 2% peptone, 2% glucose) plate and allowed to dry. Spots of strains of interest were grown on YEPD plates overnight, the plates were replica plated to the sst1 spread plates, and the replica plates were incubated for 2 days.

Northern blot analysis.

Cells were grown to an OD600 of 0.5 to 1.0, harvested, and snap-frozen in a dry ice-ethanol bath. RNA was prepared by a hot phenol method (47). Northern blot analysis was performed as described previously (3). A BAR1 fragment (+1 to +500) was prepared by PCR using 5′-ATG TCT GCA ATT AAT CAT CTT TGT TTG AAA-3′ and 5′-ACG GGT GTC GTA GCA TAC TTG GCA ACT CCG-3′ as 5′-forward and 3′-reverse primers, respectively. A Northern probe for BAR1 was prepared by a random priming reaction with the BAR1 fragment or an ENO1 fragment as described elsewhere (3).

Analysis of chromatin structure.

Yeast cells were cultured in YEPD medium until the OD600 reached ∼1.0. Nuclei were isolated, and micrococcal nuclease (MNase) digestion proceeded as described previously (41, 42). Cleavage sites for MNase were analyzed by primer extension mapping using a primer with the BAR1 −391 to −357 sequence as described elsewhere (42).


Experimental design.

Previous studies indicate that the yeast α2/Mcm1 repressor positions nucleosomes adjacent to the α2 operator in a-cell-specific genes, such as BAR1, STE2, and STE6, in the genome (9, 14, 37, 42), as well as in yeast minichromosomes containing an α2 operator (36, 42). Here, we have implemented a strategy to test the functional significance of the positioned nucleosomes by introducing short, diverse nucleosome-disrupting sequences into the BAR1 genomic locus. The BAR1 promoter was modified by the insertion of poly(dA) · poly(dT), poly(dC-dG) · poly(dC-dG), CTG, or GAGCTC repeat sequences (Fig. (Fig.1;1; Table Table1)1) as follows. We cloned the −500 to +51 region of BAR1 in an integrative plasmid and introduced modification into the genomic BAR1 locus by two-step gene replacement. In strains constructed in this study, a KpnI site was created at −158 in the BAR1 promoter and An (n = 20, 25, 30, 34), A34GGTACCA34 (denoted as 2xA34), (CG)n (n = 4 to 7), (CG)7TATA(CG)7[denoted as (CG)14], (TGC)11T [denoted as (CTG)12, since this insert becomes (CTG)12, including the neighboring KpnI sequence], AC(GAGCTC)5GA (denoted as Sac5), or AC(GAGCTC)6GT (denoted as Sac6) were inserted into the KpnI site in the BAR1 promoter. Also, portions of the promoter sequence upstream of the KpnI site were replaced with (CTG)12 or (CG)7 [denoted as (CTG)12SB and (CG)7SB, respectively] to maintain the native distance in the BAR1 promoter. We predicted that longer poly(dA) · poly(dT) and poly(dC-dG) · poly(dC-dG) sequences would act as nucleosome-disrupting sequences (2, 6, 15, 41, 43, 48), whereas (CTG)12 and mixed sequences Sac5 and Sac6 would serve as control inserts, since CTG and GAGCTC repeats were shown previously to be incorporated into nucleosomes (41, 50).

FIG. 1.
Experimental design for this study. Nucleosomes (gray ellipses) are positioned at the BAR1 promoter in MATα cells from the α2 operator (α2 op) to the coding region, and the TATA box (T) is incorporated into the positioned nucleosome ...

Effect of introduced sequences on BAR1 expression.

We examined the effect of nucleosome-disrupting sequences, such as poly(dA) · poly(dT) and poly(dC-dG) · poly(dC-dG), on repression of BAR1 in MATα cells through a halo assay (27), which reflects degradation of the α mating pheromone (α-factor) by the BAR1 product, a protease. In this assay, strains to be tested for α-factor production were replica plated onto a lawn of the tester strain (MATa sst1), which is supersensitive to α-factor (8); a zone of growth inhibition (halo) in the sst1 cells surrounding a tested colony indicates that the colony secretes α-factor. If BAR1 were derepressed in MATα cells, α-factor would degraded and the size of the halo would be diminished.

A halo was observed around control wild-type MATα cells (α WT) but not wild-type MATa cells (a WT), as expected (Fig. (Fig.2A,2A, upper portion). Halo size was unaffected by introduction of the KpnI site into the BAR1 promoter (data not shown) or the insertion of control sequences (CTG)12, Sac5, or Sac6 (Fig. (Fig.2B2B and and3).3). Insertion of A20 also had no effect on halo formation, but increased length of An (n ≥ 25) decreased the halo size (Fig. 2C to E), indicating that these longer An tract insertions caused derepression. In addition to length, the orientation of poly(dA) · poly(dT) also affected derepression. Interestingly, Tn tracts (i.e., the T tract on the top strand of BAR1) were more effective than An tracts of identical length in causing BAR1 derepression (Fig. 2C and F, compare α T20 and α T25 with α A20 and α A25). Thus, in both orientations, poly(dA) · poly(dT) increased expression of BAR1. (CG)n also derepressed BAR1, as shown in Fig. 2G and H. The mating factor halo was undetectable after insertion of (CG)6 or (CG)7, and halo size was noticeably diminished by insertion of (CG)4 or (CG)5. Thus, (CG)n acts as a more powerful disruptor than An; the insertion of only ∼10 to 14 bp leads to derepression of BAR1.

Chromatin alteration by alternative DNA structure-forming sequences disrupts repression of the a-specific gene BAR1 in the yeast genome. Expression of BAR1, as analyzed by halo assay, is shown in photographs of plates. Increased expression of BAR1 in ...
FIG. 3.
Lack of effect of the distance of the α2 operator from the TATA box and of the helical orientation of the α2 operator with respect to the TATA box, as revealed by the halo assay. The designations Sac5 and Sac6 refer to CA(GAGCTC)5GA and ...

We evaluated the effect of spacing between the α2 operator and the TATA box that is altered by the insertions. First, three control inserts, (CTG)12 (34 bp), Sac5 (34 bp), and Sac6 (40 bp), did not affect repression, as revealed by the halo assay (Fig. (Fig.3).3). This result indicates that spacing alone does not relieve repression. Second, we replaced a portion of the promoter sequence with (CTG)12 or (CG)7 to maintain native distance between the α2 operator and the coding region (Fig. (Fig.1,1, bottom). Halo size was unaffected by the (CTG)12 substitution, whereas it was severely diminished by the (CG)7 substitution (Fig. (Fig.3),3), which agreed with the results for the insertion of (CTG)12 and (CG)7. Thus, the substitutions of these sequences caused the same effect as the insertions (Fig. 2B and H), indicating that changes in promoter distance of the α2 operator from the TATA box are not required to cause changes in repression. Furthermore, it should be noted that the derepression level of BAR1 increased as the length of (CG)n (insertions of 8, 10, 12, 14, and 32 bp) increased, as shown by Northern analysis (see Fig. Fig.4,4, below). Thus, changes in the helical orientation of the α2 operator and TATA box do not affect repression. Therefore, derepression must result from the nature of the inserted sequences, rather than their effects on overall promoter length.

FIG. 4.
Northern blot analysis of BAR1. Strains analyzed are indicated above each lane. The designations of strains are described in the legend of Fig. Fig.2,2, except for (CG)14, which refers to (CG)7TATA(CG)7 inserted into the BAR1 promoter. Northern ...

We examined BAR1 expression by Northern blot analysis to confirm our interpretation of the halo assays (Fig. (Fig.4).4). There was strong expression of BAR1 mRNA in the control MATa WT strain, whereas no signal was detected in MATα WT, MATα Kpn, MATα (CTG)12, MATα A20, MATα A25, MATα T20, and MATα (CG)4 strains. BAR1 mRNA was detectable to some extent (1 to 15% of full expression in MATa WT) in MATα A30, MATα A34, MATα 2xA34, MATα T25, and MATα (CG)n (n ≥ 5) strains. Even though mRNA levels are low in these strains, BAR1 expression is sufficient to cause substantial α-factor degradation. Thus, the halo assay seems more sensitive than Northern analysis for monitoring changes in BAR1 expression. Importantly, in keeping with the halo assays in Fig. Fig.2,2, the derepressed mRNA levels showed length dependence for poly(dA) · poly(dT) and poly(dC-dG) · poly(dC-dG), and (CG)n affected BAR1 expression more than An or Tn. We also examined expression from the modified BAR1 promoters in a set of MATa strains in order to determine whether the inserted sequences cause adventitious promoter activation (Fig. (Fig.4).4). The level of BAR1 mRNA in this series of MATa strains varied from 1.0- to 1.6-fold above the level in the MATa WT strain. Given that BAR1 mRNA is undetectable in the MATα WT strain, we infer that the derepression caused by active insertions is much greater than 1.6-fold. These results indicate that the insertion of nucleosome-disrupting sequences partially derepresses BAR1 in its native genomic context in MATα cells.

Chromatin alteration in the BAR1 promoter by introduced sequences.

We analyzed the chromatin structure of the genomic BAR1 promoter region by limited digestion of nuclei with MNase and subsequent high-resolution primer extension mapping (Fig. (Fig.2,2, lower portions). The BAR1 promoter region was cut with MNase in WT MATa cells, in which BAR1 is expressed, whereas a region of about 140 bp adjacent to the α2 operator was protected from MNase digestion in WT MATα cells, as indicated by a comparison of digested purified DNA (lanes marked “D”) and digested chromatin (lanes marked “C”) (Fig. (Fig.2A).2A). These results indicate that nucleosomes are positioned adjacent to the α2 operator in MATα cells but are not positioned in MATa cells, in good agreement with previous studies (9, 14, 37, 42, 44). The effects of promoter insertions on chromatin structure were monitored by the patterns of MNase cleavage between the α2 operator and TATA box (Fig. (Fig.2),2), the region in which the sequences were inserted. Insertion of control (CTG)12 or shorter inserts, A20 and (CG)4, did not significantly affect formation of positioned nucleosomes (Fig. 2B, C, and G). However, insertion of longer An · Tn (n ≥ 25) sequences in MATα cells led to increasing the MNase cleavage sites characteristic of MATa cell chromatin (Fig. 2C to F). Similarly, the MNase cleavage sites became stronger as the length of (CG)n (n ≥ 5) tracts increased (Fig. 2G and H).

The nucleosome positioning adjacent to the α2 operator was destabilized to roughly the same extent by these longer An, Tn, and (CG)n sequences, although (CG)n inserts caused greater derepression of BAR1 than An or Tn. At present, it is uncertain why the magnitude of derepression caused by poly(dA) · poly(dT) and poly(dC-dG) · poly(dC-dG) is different. It is possible that the types of alternative structures (B′ conformation or Z-DNA) or intrinsic structural properties (local distortion and stiffness) may cause this effect. Our studies indicate that loss of nucleosome positioning is usually accompanied by partial relief of cell-type-specific repression of BAR1, as shown by halo assay and Northern analysis, although there is no simple relationship between the magnitudes of the two effects.

Chromatin alteration and derepression of BAR1 in an isw2 mutant.

The Isw2 chromatin remodeling complex is required for nucleosome positioning by Crt1 and Tup1 at the DNA damage-inducible gene RNR3 (12, 16, 57) and for normal chromatin structure of the a-cell-specific gene STE6 in MATα cells (12, 16, 57). In addition, repression of a-cell-specific genes requires Itc1 (38), a subunit of the Isw2 complex (17). These prior studies suggest that an isw2Δ mutation might have an effect similar to nucleosome-disrupting sequences at BAR1.

Figure Figure2I2I shows the halo assay and mapping of MNase cleavage sites at BAR1 in isw2Δ isogenic strains. The halo assay indicates that BAR1 is derepressed in a MATα isw2Δ strain, in keeping with the report by Ruiz et al. (38) that an itc1 mutation causes derepression of a-cell-specific genes ASG7, BAR1, and STE2. Decrease in the halo size in the MATα isw2 strain (Fig. (Fig.2I)2I) was similar to that in MATα A30, MATα T25, and MATα (CG)5 strains (Fig. 2D, F, and G). In addition, BAR1 mRNA in the MATα isw2Δ strain was detectable to the same extent (2.3% of full expression in MATa WT) as in these strains (Fig. (Fig.4).4). As seen in Fig. Fig.2I,2I, the MNase cleavage pattern is nearly identical in MATa and MATα isw2Δ strains. These results reveal that the Isw2 chromatin remodeling complex is required for nucleosome positioning at the genomic BAR1 locus in MATα cells and that an isw2 mutation does not have a significant effect on the BAR1 transcription level. This result is consistent with a report by Zhang and Reese (57) that nucleosome positioning in the DNA damage-inducible gene RNR3 is disrupted by isw2 mutation, but the level of RNR3 mRNA was only slightly increased.


We have shown here that both the integrity of the positioned nucleosomes and MATα cell-type-dependent repression of BAR1 respond to the same cis- and trans-acting modulators of chromatin structure. The sequences we have used are diverse, yet both poly(dA) · poly(dT) and poly(dC-dG) · poly(dC-dG) share the ability to disrupt a positioned nucleosome and to cause inappropriate activation of BAR1. Even though chromatin alteration shows a modest effect on the level of BAR1 mRNA, it has a significant biological consequence, that is, it causes substantial degradation of the α-mating pheromone in MATα cells, thereby affecting its cell type identity. Thus, the most economical model to explain our data is that nucleosome positioning directly contributes to complete repression of the genomic BAR1 locus by α2/Mcm1.

However, loss of nucleosome positioning is not sufficient to relieve repression of BAR1 completely. One reason for this could be explained by the absence of activator function of Mcm1 in MATα cells; Mcm1 acts as an activator for a-cell-specific genes in MATa cells, whereas it acts as a repressor with α2 in MATα cells. Since Tup1 has high affinity for underacetylated histones and histone deacetylases (5, 10, 11, 52), complete relief of chromatin-mediated repression may require not only loss of nucleosome positioning but also other activities, such as action of a histone acetyltransferase.

The residual repression that persists despite the disruption of nucleosome positioning is also likely to be achieved by chromatin-independent mechanisms of Tup1-Ssn6 action (29, 56). Two additional mechanisms have been proposed for repression by α2/Mcm1: activator interference (20), in which Ssn6-Tup1 exerts repression while the activator still occupies its target DNA site (35), and general transcription machinery interference, in which Ssn6-Tup1 inhibits the transcription machinery directly and independently of chromatin or activators (18, 29). Our findings here do not rule out any repression mechanism. Rather, our results provide support for the contribution of nucleosome positioning to a-specific gene repression.

We note that insertions of longer An, Tn, or (CG)n sequences primarily disrupt one nucleosome in the promoter, while nucleosome positioning is preserved in the coding region. Interestingly, the coding region is separated from the α2 operator by the disrupted nucleosome. This may be explained by the fact that the Isw2 complex is associated with the entire region of the RNR3 gene (57) and that the Isw2 complex slides nucleosomes to remodel chromatin structure (12, 13). Thus, it is likely that the insertions disrupt only one nucleosome proximal to the site, and the preserved nucleosome positioning in the BAR1 coding region may be mediated by the Isw2 complex. This explanation is consistent with our results showing that the chromatin structure of BAR1 is nearly identical between MATa WT, MATa isw2, and MATα isw2 (no positioned nucleosomes).

It may seem possible that proteins that bind to poly(dA) · poly(dT) or poly(dC-dG) · poly(dC-dG) compete with binding of histone octamer; hence, the effects of these sequences might not be a consequence of intrinsic DNA structural properties. Alternatively, the absence of nucleosome positioning might be a consequence of affecting the ability of the α2/Mcm1 complex to recruit Ssn6/Tup1. However, the idea that DNA structural properties alter nucleosome positioning is founded on several lines of evidence. We and others previously demonstrated that longer An tracts exist as an unusual B′ conformation to create a nucleosome-free region in yeast cells (41, 48). Consistent with these reports, we found here that disruption of nucleosome positioning and derepression of BAR1 showed a length dependent of the An tract, indicating that the B′ conformation excludes histone octamers from the promoter. Interestingly, Tn disrupts BAR1 repression more effectively than An, as monitored in the halo assay. This difference in orientation can be explained by the fact that the unusual conformation of poly(dA) · poly(dT) is asymmetric; that is, the minor groove narrows asymmetrically from the 3′ end towards the 5′ end of a Tn stretch (1, 25, 28). Also, BAR1 expression was not affected by the dat1Δ mutation (data not shown), which lacks the only known poly(dA) · poly(dT) binding protein in S. cerevisiae (33). As for poly(dC-dG) · poly(dC-dG), its effects may be explained by the fact that (CG)n in the Z-form is not incorporated into nucleosomes in vitro (2, 6, 15). The length of (CG)n is critical for Z-DNA formation and stability in vivo (32), and the B-Z transition occurs from (CG)4 to (CG)5 at natural superhelical densities (22). Also, CG repeats longer than (CG)6 can form Z-DNA stably in vivo in yeast cells (30). These studies argue that it is the Z-DNA conformation of (CG)n (n ≥ 5) that disrupts nucleosome positioning at the genomic BAR1 promoter, though the existence of Z-DNA formation in the BAR1 promoter was uncertain in the present study. We cannot rule out a contribution of sequence-dependent general properties of the inserted DNA that may alter nucleosome organization (26, 40, 49). Whatever the structure of (CG)n in the BAR1 locus is, the key feature is that poly(dC-dG) · poly(dC-dG) as well as poly(dA) · poly(dT) sequences disrupt nucleosome positioning in a genomic context to alter gene expression. The intrinsic properties of these sequences make them useful tools for inquiring into local chromatin function in diverse cells and organisms as well as for artificial alteration of gene expression in vivo.


We thank Toshiki Kobayashi, Atsushi Sato, Yukino Mukai, and Mayu Yamaguchi for technical assistance in constructions of plasmids and strains in their undergraduate studies, Daichi Kurihara for construction of isw2Δ strains, and Karen Barwell and Clarissa Nobile for their help in Northern blot analysis. We thank Robert D. Wells of Texas A&M University for stimulating discussion and useful comments.

This work was supported, in part, by a JSPS research grant to M.S. and by NIH grants RO1 GM39531 and AI070272 to A.P.M.


Published ahead of print on 15 September 2006.


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