The NTPase/helicase activities of Drosophila maleless, an essential factor in dosage compensation
1997; Springer Nature; Volume: 16; Issue: 10 Linguagem: Inglês
10.1093/emboj/16.10.2671
ISSN1460-2075
Autores Tópico(s)DNA Repair Mechanisms
ResumoArticle15 May 1997free access The NTPase/helicase activities of Drosophila maleless, an essential factor in dosage compensation Chee-Gun Lee Chee-Gun Lee Graduate Program in Molecular Biology, Memorial Sloan-Kettering Cancer Center, Sloan-Kettering Institute, 1275 York Avenue, New York, NY, 10021 USA Search for more papers by this author Kimberly A. Chang Kimberly A. Chang Howard Hughes Medical Institute, Department of Cell Biology, Baylor College of Medicine, One Baylor Plaza, Houston, TX, 77030 USA Search for more papers by this author Mitzi I. Kuroda Mitzi I. Kuroda Howard Hughes Medical Institute, Department of Cell Biology, Baylor College of Medicine, One Baylor Plaza, Houston, TX, 77030 USA Search for more papers by this author Jerard Hurwitz Corresponding Author Jerard Hurwitz Graduate Program in Molecular Biology, Memorial Sloan-Kettering Cancer Center, Sloan-Kettering Institute, 1275 York Avenue, New York, NY, 10021 USA Search for more papers by this author Chee-Gun Lee Chee-Gun Lee Graduate Program in Molecular Biology, Memorial Sloan-Kettering Cancer Center, Sloan-Kettering Institute, 1275 York Avenue, New York, NY, 10021 USA Search for more papers by this author Kimberly A. Chang Kimberly A. Chang Howard Hughes Medical Institute, Department of Cell Biology, Baylor College of Medicine, One Baylor Plaza, Houston, TX, 77030 USA Search for more papers by this author Mitzi I. Kuroda Mitzi I. Kuroda Howard Hughes Medical Institute, Department of Cell Biology, Baylor College of Medicine, One Baylor Plaza, Houston, TX, 77030 USA Search for more papers by this author Jerard Hurwitz Corresponding Author Jerard Hurwitz Graduate Program in Molecular Biology, Memorial Sloan-Kettering Cancer Center, Sloan-Kettering Institute, 1275 York Avenue, New York, NY, 10021 USA Search for more papers by this author Author Information Chee-Gun Lee1, Kimberly A. Chang2, Mitzi I. Kuroda2 and Jerard Hurwitz 1 1Graduate Program in Molecular Biology, Memorial Sloan-Kettering Cancer Center, Sloan-Kettering Institute, 1275 York Avenue, New York, NY, 10021 USA 2Howard Hughes Medical Institute, Department of Cell Biology, Baylor College of Medicine, One Baylor Plaza, Houston, TX, 77030 USA The EMBO Journal (1997)16:2671-2681https://doi.org/10.1093/emboj/16.10.2671 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Drosophila maleless (mle) is required for X chromosome dosage compensation and is essential for male viability. Maleless protein (MLE) is highly homologous to human RNA helicase A and the bovine counterpart of RNA helicase A, nuclear helicase II. In this report, we demonstrate that MLE protein, overexpressed and purified from Sf9 cells infected with recombinant baculovirus, possesses RNA/DNA helicase, adenosine triphosphatase (ATPase) and single-stranded (ss) RNA/ssDNA binding activities, properties identical to RNA helicase A. Using site-directed mutagenesis, we created a mutant of MLE (mle-GET) that contains a glutamic acid in place of lysine in the conserved ATP binding site A. In vitro biochemical analysis showed that this mutation abolished both NTPase and helicase activities of MLE but affected the ability of MLE to bind to ssRNA, ssDNA and guanosine triphosphate (GTP) less severely. In vivo, mle-GET protein could still localize to the male X chromosome coincidentally with the male-specific lethal-1 protein, MSL-1, but failed to complement mle1 mutant males. These results indicate that the NTPase/helicase activities are essential functions of MLE for dosage compensation, perhaps utilized for chromatin remodeling of X-linked genes. Introduction Dosage compensation is the process by which the expression of X-linked genes is equalized between males and females. In Drosophila, dosage compensation is achieved by elevating the transcription of most genes on the single male X chromosome to a level equivalent to that of genes on the two female X chromosomes (Mukherjee and Beerman, 1965; Baker et al., 1994). In contrast, in mammals, dosage compensation is achieved by transcriptional inactivation of genes on one of the two female X chromosomes (Ballabio and Willard, 1992; Penny et al., 1996) and in nematodes, each X in the XX hermaphrodite is active but repressed relative to a single male X (Meyer and Casson, 1986; Kelley and Kuroda, 1995). The initial step in establishing dosage compensation in Drosophila occurs early in development in response to the ratio of X chromosomes to the haploid set of autosomes (the X:A ratio) (Maroni and Plaut, 1973). This process controls the expression of the developmental regulator, Sex lethal (Sxl). When the X:A ratio is 1.0, Sxl expression is directed by female-specific transcriptional activation of an early embyonic promoter; later, a positive autoregulatory loop is formed that maintains Sxl expression in females (Bell et al., 1991; Keyes et al., 1992). Active Sxl results in the female-specific splicing of late Sxl transcripts generated from a constitutive promoter (Bell et al., 1991). When the ratio of X:A is 0.5, Sxl protein is not expressed and male-specific regulators act to maintain equivalent levels of transcripts of most X-linked genes in males relative to females (Cline, 1978; Gorman et al., 1993). In Drosophila, there are four such male-specific regulatory genes, named for their mutant phenotypes: male-specific lethal-1 (msl-1), male-specific lethal-2 (msl-2), maleless (mle) and maleless on the third (mle3, also known as msl–3) (for references, see Baker et al., 1994). Sxl is proposed to negatively regulate dosage compensation in females through the post-transcriptional regulation of the msl-2 transcript, thereby repressing MSL-2 protein synthesis (Bashaw and Baker, 1995; Kelley et al., 1995; Zhou et al., 1995). As a result, MLE, MSL-1 and MSL–3 proteins are present in both sexes (Gorman et al., 1995), whereas MSL-2 protein is only found in males (Bashaw and Baker, 1995; Kelley et al., 1995; Zhou et al., 1995). These four positive regulators of dosage compensation (collectively called the MSL proteins) bind to hundreds of specific sites along the X chromosome only in males (Kuroda et al., 1991; Palmer et al., 1993; Hilfiker et al., 1994; Bashaw and Baker, 1995; Gorman et al., 1995; Kelley et al., 1995; Zhou et al., 1995). Histone H4 acetylated at lysine 16 is also found localized at numerous sites along the length of the male X chromosome, largely coincident with the MSL proteins (Turner et al., 1992; Bone et al., 1994). The concentration of this modified histone on the male X chromosome requires the wild-type products of the msl/mle genes. Thus, it has been postulated that dosage compensation in Drosophila involves complex formation of the four MSL proteins on the male X chromosome that alters the chromatin structure, resulting in increased transcription (Turner et al., 1992; Gorman et al., 1993; Bone et al., 1994). As the biochemical mechanism of dosage compensation has not been defined, it is of considerable interest to characterize potential activities of the MSL proteins that may contribute to in vivo function. MLE belongs to the DExH subfamily of nucleoside triphosphatase (NTPase) and/or helicase proteins (Kuroda et al., 1991). It displays more similarity to RNA- rather than DNA-dependent NTPases or helicases, and has an additional motif characteristic of double-stranded (ds) RNA binding proteins (Gibson and Thompson, 1994). Furthermore, the association of MLE with the X chromosome is RNase sensitive (Richter et al., 1996). However, previous analyses of mle mutants were inconclusive regarding the importance of helicase motifs in vivo (Richter et al., 1996) and no biochemical characterization of MLE protein has been documented. In this report we demonstrate that MLE has NTPase and both RNA and DNA helicase activities. Furthermore, we show that loss of the NTPase and helicase activities of MLE results in male lethality in Drosophila without eliminating the localization of the MLE–MSL complex to the male X chromosome. These results indicate that the NTPase/helicase activities of MLE are essential for dosage compensation. Results Both DNA and RNA helicase activities are associated with MLE Drosophila maleless shares extensive homology with human RNA helicase A (Lee and Hurwitz, 1993) and its bovine homolog nuclear DNA helicase II (Zhang et al., 1995), two enzymes that possess helicase and NTPase activities (Lee and Hurwitz, 1992; Zhang and Grosse, 1994). For this reason, we assayed recombinant histidine-tagged MLE protein for these activities in vitro. MLE protein was isolated from Sf9 cells infected with the recombinant baculovirus. After adsorption to and elution from a nickel column, followed by chromatographic purification through hydroxylapatite (see Materials and methods), the isolated recombinant protein was subjected to glycerol gradient sedimentation (Figure 1). MLE, which sedimented between bovine serum albumin (BSA) (4.3S) (fraction 12) and aldolase (7.4S) (fraction 19), peaked at fraction 16 with an estimated sedimentation coefficient of 6.2S (Figure 1A). Immunoblot analysis with polyclonal antibodies specific for MLE established that the major 140 kDa protein in the purified hydroxylapatite and peak glycerol fractions was full-length MLE (Figure 1B). Aliquots (0.1 μl) of each fraction were tested for both RNA and DNA helicase activities. Both activities, which sedimented coincidentally, were detected in fractions enriched in MLE (Figure 1C and D), demonstrating that these activities are intrinsically associated with MLE. Figure 1.Sedimentational analysis of MLE. An aliquot (53 μg) of the pooled hydroxylapatite fractions enriched in recombinant MLE was loaded onto a 20–40% glycerol gradient and centrifuged for 36 h at 45 000 r.p.m. in a Beckman SW50.1 rotor at 4°C. Subsequently, 30 fractions were collected from the bottom of the tube. (A) The distribution of protein in the glycerol gradient. Aliquots (12 μl) of each fraction were analyzed by electrophoresis on a 7.5% discontinuous SDS–polyacrylamide gel. Proteins were visualized by Coomassie blue staining. Lane M, molecular weight markers; lane L, 1 μg of the hydroxylapatite fraction. All other lanes represent the fraction number from the glycerol gradient. (B) An immunoblot analysis carried out with polyclonal anti-MLE 25 antibodies and aliquots (2 μl) of the indicated glycerol fractions. The distribution of helicase activities through the glycerol gradient is shown in (C) and (D). Unwinding reactions were performed with 50 fmol of partial duplex RNA or DNA substrate in the presence of the indicated glycerol fraction (0.1 μl) or 10 ng of MLE (lanes 3 and 4). After incubation for 30 min at 37°C, the substrate and displaced single-stranded products were analyzed by electrophoresis on an 8% polyacrylamide gel (30:1) containing 5% glycerol and 0.5× TBE. (C) An autoradiogram representing the distribution of DNA helicase activity in the glycerol gradient. Lane 1, substrate alone; lane 2, boiled substrate; lane 3, 10 ng of MLE without ATP; lanes 4–17, with ATP; lane 4, 10 ng of MLE; lanes 5–17, odd numbers of glycerol fractions 3–27. (D) The quantitation of results described in (C) and also of RNA helicase reactions performed with aliquots (0.1 μl) of the indicated glycerol fractions as described above. Download figure Download PowerPoint In order to determine the substrate specificity of MLE, four different substrates, containing identical ribonucleotide or deoxyribonucleotide sequences as shown in Figure 2 (RNA:RNA, RNA:DNA, DNA:RNA and DNA:DNA hybrids), were constructed and tested in the unwinding reaction with varying amounts of MLE (2.5–10 ng). MLE displaced substrates containing ssRNA regions, i.e. RNA:RNA and DNA:RNA hybrids, 2.5-fold more efficiently than substrates containing ssDNA regions (Figure 2). Figure 2.Substrate specificity of MLE helicase activity. The structures of the different substrates used are shown at the top of each panel. All substrates were constructed using mRNA transcripts synthesized in vitro and synthetic DNAs of the same size and sequence as described previously (Lee and Hurwitz, 1992). Unwinding reactions (20 μl) containing 50 fmol of the indicated substrate were incubated in the presence of increasing amounts of MLE. Lane 1, substrate alone; lane 2, boiled substrate; lane 3, 5 ng of MLE without ATP; lanes 4–6, 1.25, 2.5, 5 ng of MLE respectively, in the presence of ATP. Download figure Download PowerPoint The binding of MLE to ssRNA and ssDNA was examined (Figure 3). For this purpose, the longer strand of each duplex substrate described in Figure 2 (the 98mer ssRNA or ssDNA) was tested using a gel mobility shift assay in the presence of increasing levels of MLE (5–20 ng) (Figure 3). In keeping with the higher helicase activity observed with duplex substrates containing ssRNA, complexes with MLE were formed more efficiently (3- to 4–fold) with ssRNA than with ssDNA. Figure 3.The binding of MLE to ssRNA and ssDNA. The binding of MLE to ssRNA or ssDNA was measured in reaction mixtures (20 μl) containing 50 fmol of substrate and varying amounts of MLE, as described previously (Lee and Hurwitz, 1992). Complexes formed between MLE and ssRNA (left panel) or ssDNA (right panel) were quantitated following autoradiography after electrophoretic separation. The amounts of RNA or DNA complexed with MLE (bracketed regions) are presented at the bottom of each lane. Lane 1: substrate alone; lanes 2–4: 5, 10, 20 ng of MLE respectively. Download figure Download PowerPoint Characteristics of helicase activities associated with MLE The experiments described above demonstrated that MLE possesses RNA and DNA helicase activities. Previously, we reported that RNA helicase A, the human homolog of MLE, contained no detectable DNA helicase activity (Lee and Hurwitz, 1991). In contrast, Grosse's laboratory detected both RNA and DNA helicase activities with bovine nuclear helicase II, which is >90% identical to HeLa RNA helicase A (Zhang et al., 1995). In our earlier experiments with RNA helicase A, KCl or NaCl (50 mM) was added to unwinding reaction mixtures, which optimized the unwinding of partial duplex RNA substrates by RNA helicase A. Zhang and Grosse (1994) reported that DNA helicase activity of bovine nuclear helicase II was more salt sensitive than the RNA helicase activity. Indeed, as described by Zhang and Grosse (1994), RNA helicase A was found to contain DNA helicase activity that is more salt sensitive than RNA helicase activity (data not shown). In light of these findings, the influence of salt on RNA and DNA helicase activities of MLE was examined. As shown in Figure 4A, DNA helicase activity was markedly inhibited by 0.1 M NaCl, whereas RNA helicase activity was hardly affected by this salt concentration. At higher levels, however, both activities were inhibited. Figure 4.Properties of MLE helicase activity. (A) The influence of salt on the helicase activities of MLE. All reactions were carried out with 10 ng of MLE and 50 fmol of partial duplex RNA or DNA substrates in the presence of the indicated concentrations of NaCl. (B) The influence of various NTPs on MLE helicase activity. The unwinding reactions (20 μl) were carried out with 10 ng of MLE and 50 fmol of partial duplex RNA or DNA substrates in the presence of 1 mM of the indicated NTP. In the presence of 1 mM ATP, 37 fmol of ssRNA and 23 fmol of ssDNA were formed; there was no significant change in the efficiency of the unwinding reaction in the presence of other NTPs. Download figure Download PowerPoint The helicase activity of MLE required adenosine triphosphate (ATP) and in its absence, partial duplex substrates were not displaced (Figure 2, lanes 3 and 4). In addition to ATP, all other seven common nucleoside triphosphates (NTPs) supported helicase activity (Figure 4B). The Km for ATP in the helicase reaction was 10 μM and similar values were obtained for the other NTPs (data not shown), indicating that MLE utilizes all NTPs without preference. The partial duplex substrates described in Figure 2 contained single-stranded regions at both 3′- and 5′-ends. The directionality of a helicase is defined by the strand to which the enzyme binds and translocates. To determine the directionality of MLE helicase activity, three different substrates were prepared. The upper longer DNA of each substrate was the same but the lower shorter complementary DNA differed in each substrate. The duplex substrates B and C contained a single-stranded region exclusively at either the 3′- or 5′-end (Figure 5). The nucleotide sequence of substrate D which contained no single-stranded region was identical to the duplex region present in substrate B. These three substrates were examined in the unwinding reaction in the presence of various levels of MLE (2.5–10 ng). As shown in Figure 5, the 3′-tailed substrate was efficiently displaced whereas unwinding was not observed with the 5′-tailed substrate (Figure 5B and C). As expected, the unwinding reaction mediated by MLE was completely dependent on the presence of a single-stranded region; substrate D, devoid of any single-stranded region, was not utilized by MLE (Figure 5D). The same 3′ to 5′ directionality was observed with duplex RNA substrates (data not shown), indicating that MLE recognizes and binds both ssRNA and ssDNA and tranlocates in the 3′ to 5′ direction. These properties are identical to those observed with RNA helicase A (Lee and Hurwitz, 1992). Figure 5.Directionality of MLE helicase activity. In addition to the standard substrate (A), three different partial duplex substrates (B–D) were tested in the unwinding reaction. The structure of each substrate is shown at the top of each panel. Lane 1, substrate alone; lane 2: boiled substrate; lane 3, 10 ng of MLE without ATP; lanes 4–6 contained ATP and 2.5, 5, 10 ng of MLE respectively. Download figure Download PowerPoint Site-directed mutagenesis of ATP binding motif A of MLE The results described above demonstrated that MLE possesses RNA/DNA binding activity, NTPase activity, and DNA/RNA helicase activities. Next we determined whether these activities are essential for the role MLE plays in dosage compensation in Drosophila. If NTPase and helicase were essential functions of MLE, mutations affecting these activities should also affect dosage compensation. In earlier studies, the most highly conserved region of the ATPase site A within the helicase domain GKT, was changed to GNT. This mutation reduced male viability ∼50% (Richter et al., 1996). Reasoning that a lysine to asparagine change in the ATP binding motif GKT may have been inadequate to completely eliminate the function of MLE, a more substantial change (lysine to glutamic acid) was introduced into MLE, which reversed the ionic charge from positive to negative in the ATPase site A. In vitro analysis of the effect of the GET mutation in MLE The six-histidine-tagged mle-GET, possessing the lysine to glutamic acid mutation in the GKT conserved helicase motif, was expressed and purified from Sf9 cells infected with the recombinant virus. The expression level of mle-GET was low, and only 34 μg of mle-GET was isolated from 0.5 l of Sf9 cells after hydroxylapatite chromatography following Ni-affinity adsorption and elution (see Materials and methods). Hydroxylapatite fractions, highly enriched in mle-GET, were pooled (Figure 6, fractions 13–19) and used to characterize the biochemical properties of mle-GET. As shown in Figure 7, mle-GET contained no significant RNA-dependent NTPase activity or helicase activity with the DNA or RNA substrates. Since the unwinding reaction requires NTP hydrolysis for translocation and disruption of the duplex, the lack of helicase activity of mle-GET is most likely due to its inability to hydrolyze NTP. Figure 6.Purification of mle-GET by hydroxylapatite column chromatography. The pooled Ni-fraction (4 ml, 110 μg of protein), enriched in six-histidine tagged mle-GET, was directly loaded onto the hydroxylapatite column (0.5 ml) and bound proteins were eluted with a linear gradient (20–300 mM) of sodium phosphate, pH 6.0, containing 0.25 M NaCl, 2 mM DTT, 0.05% NP-40 and 10% glycerol. A total of 40 fractions (each 0.25 ml) was collected and aliquots (10 μl) were electrophoresed on a discontinuous 7.5% SDS–polyacrylamide gel. Fractions (13–19), enriched in mle–GET, were pooled and used in the present study following dialysis against 2 l of buffer containing 20 mM HEPES–NaOH, pH 7.4, 0.25 M NaCl, 0.1 mM EDTA, 0.5 mM PMSF, 2 mM DTT and 12.5% glycerol. Lane M, molecular weight marker; lane L, 0.3 μg of the pooled Ni-fraction; lane F, the flow-through fraction; lanes 1–31, odd numbered hydroxylapatite fractions. Download figure Download PowerPoint Figure 7.The influence of the GET mutation on the NTPase/helicase activities of MLE. (A) The ATPase activity of MLE and mle-GET was examined. Reaction mixtures (15 μl) containing 20 mM HEPES–NaOH, pH 7.4, 2 mM DTT, 3 mM MgCl2, 0.1 mM [γ-32P]ATP (4400 c.p.m./pmol) and 0.2 mg/ml BSA, were incubated for 30 min at either 25°C or 37°C in the presence of increasing levels of each protein. Aliquots (1 μl) of each reaction were analyzed on PEI plates and the extent of ATP hydrolysis was quantitated, as described previously (Lee and Hurwitz, 1992). (B) MLE and mle-GET were examined in unwinding reactions containing 50 fmol of either partial duplex RNA (left panel) or partial duplex DNA substrate (right panel). Lane 1, substrate alone; lane 2, boiled substrate; lanes 3 and 7, 10 ng of MLE and mle-GET respectively, without ATP; lanes 4–6 and 8–10 contained ATP and 2.5, 5, 10 ng of the indicated protein. In the presence of 10 ng of MLE, 39 fmol and 27 fmol of ssRNA and ssDNA were formed respectively. No substrate displacement was observed at any level of mle-GET protein added. Download figure Download PowerPoint Since the binding of MLE to single-stranded polynucleotides occurs in the absence of NTPs, we expected that the GET mutation would not affect the NTP-independent activities of MLE. As expected, the mle-GET bound both ssRNA (Figure 8A, lanes 5–7) and ssDNA (data not shown), though less efficiently (∼2.5-fold) than wild-type MLE (Figure 8A, lanes 2–4). In the presence of ATP, the complexes of MLE and RNA (and also DNA) displayed subtle changes in their migration and in the relative amount of each complex formed (compare Figure 8A, lanes 4 and 11). The fast-migrating complex decreased with the concomitant increase in the slower-migrating complexes. A similar result was obtained with complexes formed with mle-GET (compare Figure 8A, lanes 7 and 14). This result raised the possibility that although NTP hydrolysis activity was absent, mle-GET could still bind NTPs. Figure 8.The influence of the GET mutation on the binding of MLE to ssRNA and GTP. (A) The binding of MLE and mle-GET to ssRNA was examined in reactions containing 50 fmol of substrate in the absence (lanes 1–7) or presence (lanes 8–14) of ATP. After incubation for 30 min at 37°C, aliquots (10 μl) were loaded onto a 4.5% polyacrylamide gel (60:1) containing 5% glycerol in 0.5× TBE and electrophoresed at 15 mA for 1.5 h. Lanes 1 and 8: substrate alone; lanes 2–4 and 9–11: 5, 10, 20 ng of MLE respectively; lanes 5–7 and 12–14, 5, 10, 20 ng of mle-GET respectively. (B) The binding of MLE and mle-GET to GTP was examined in reactions (20 μl) containing [α-32P]GTP (10 μM, 5500 c.p.m./pmol) and increasing levels of protein (20–80 ng); reactions were incubated on ice for 10 min and then irradiated with UV-light (254 nm) for 10 min on ice. Formation of the GTP–protein complex was analyzed by SDS–PAGE and visualized by autoradiography. Lane 1: no protein; lanes 2 and 5: 20 ng of protein; lanes 3 and 6: 40 ng of protein; lanes 4 and 7: 80 ng of protein. Download figure Download PowerPoint UV-crosslinking (Pause et al., 1993) was used to measure the binding of nucleotides to MLE. Under the conditions used (Figure 8B), GTP was crosslinked to MLE more efficiently than ATP, and the efficiency of GTP crosslinking to MLE was maximal at a nucleotide concentration of 10 μM. The addition of RNA or DNA hardly affected the UV-crosslinking of GTP to MLE (data not shown). The binding of GTP to MLE and mle-GET was examined in the presence of [α-32P]GTP. Increasing levels of MLE (20–80 ng) resulted in an increased binding of GTP (Figure 8B, lanes 2–4). It was estimated that 20 fmol and 4.7 fmol of GTP were crosslinked to 80 ng (570 fmol) of MLE and mle-GET, respectively (lanes 4 and 7). The results presented in Figure 8B demonstrated that mle-GET bound GTP with an efficiency ∼5- to 10-fold lower than wild-type MLE. In vivo analysis of the GET mutation A genomic mle-GET P-element construct was injected into embryos, and 13 independent transgenic lines were established. Three independent insertions of the transgene were examined by Western blot analysis, and all three lines expressed a full-length transgenic MLE protein (Figure 9 and data not shown). The three insertion lines were tested for the ability of the mle-GET transgene to rescue mle1 males. Whereas a wild-type mle transgene fully complements the mle1 mutant phenotype (Richter, 1994; Richter et al., 1996), the percentage of mle1 males rescued by the mle-GET transgene was very low. One line rescued at 1.3% (n = 456), the second line rescued at 0.7% (n = 390) and the third line did not rescue at all (n = 223). The majority of the mle1 males with the mle-GET transgene died during mid to late larval stages, as do mle1 males lacking the transgene. The mle1 males that survived to adulthood were sterile, held out their wings and had disorganized abdominal bristles. Each of these phenotypes has been observed previously in rare surviving mle males carrying severe loss of function alleles (M.Kuroda, unpublished data). The low level of rescue suggests that the mle-GET mutation disrupts MLE function. MLE is thought to associate with the X chromosome in a complex with the other MSLs, and to act within that complex to facilitate dosage compensation. Figure 9.Western blot of MLE protein in flies carrying the mle-GET transgene. Lane 1, control nuclear extract from Schneider cells; lane 2, total protein from adult mle1 escaper males carrying one copy of the mle-GET transgene; lane 3, total protein from adult mle1 females carrying one copy of the mle-GET transgene. Anti-MLE antibody was used to detect the MLE protein. Download figure Download PowerPoint One copy of a wild-type mle transgene in mle1 mutant males is sufficient to produce co-localized MLE and MSL–1 immunostaining patterns that are indistinguishable from wild-type males (Richter, 1994). To determine if the mle-GET protein was able to assemble in an MSL complex on the X, the localization of MLE and MSL-1 was determined on polytene chromosomes obtained from w; mle1;[w+;mle-GET] male third instar larvae. The sole source of MLE in these larvae is from the mle-GET transgene, as mle1 mutants have no detectable MLE by Western blot or on polytene chromosomes (Richter et al., 1996). The staining results indicated that the mle-GET protein was able to bind the X chromosome (Figure 10A). The number of sites where the mle-GET protein associated with the X chromosome was reduced from the number seen in wild-type males, and the number varied from one male to the next. The MSL-1 pattern was also reduced, as is seen in mle mutants (Palmer et al., 1994). The mle-GET protein co-localized with all MSL-1 sites and also stained additional sites on the X and the autosomes (Figure 10B). We cannot determine whether the reduced number of the X chromosome sites and the increased staining of autosomal sites has functional significance, or reflects an indirect effect of the altered physiology of dying larvae. However, our results demonstrate that the mle-GET mutation does not dramatically alter MLE protein conformation, as mle-GET can still bind to the X chromosome coincident with MSL-1. That the mle-GET protein was able to form complexes on the X chromosome but unable to support a wild-type MSL pattern on the X or to rescue mle males, suggests that the NTPase/helicase activities are essential for the function of MLE in dosage compensation. Figure 10.Analysis of the ability of the mle-GET protein to bind to the male X chromosome. Indirect immunofluorescence was performed on polytene chromosomes from male salivary glands. Salivary glands were squashed from a wild-type male and a w; mle1;[mle-GET]/+ male. The chromosomes were stained with antibodies to MLE and MSL-1. The staining was visualized through the use of Texas Red and FITC conjugated secondary antibodies. The top row (A) shows the staining with the anti-MLE antibody. The bottom row (B) shows the double staining with anti-MLE and anti–MSL-1 antibodies. Where the red MLE staining overlaps with the green MSL-1 staining, a yellow color is observed. Download figure Download PowerPoint Discussion MLE and RNA helicase A are biochemically equivalent We have shown that both NTPase and helicase activities are intrinsically associated with MLE. Partial duplex substrates tested in the unwinding reaction, including those containing RNA:RNA, DNA:DNA and RNA:DNA duplexes, were efficiently displaced by MLE in an NTP-dependent manner. The requirement of a single-stranded region for the helicase activity of MLE is in keeping with its ability to bind ssRNA and ssD
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