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Address correspondence to Daniel St Johnston, Dept. of Genetics, Wellcome/CRC Institute, Tennis Court Rd., Cambridge CB2 1QR, UK. Tel.: 44-1223-334-127. Fax: 44-1223-334-089. E-mail: ds139{at}mole.bio.cam.ac.uk
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Abstract |
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Key Words: Drosophila; axis formation; pole plasm; microtubules; RNA transport
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Introduction |
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Although several mechanisms can target transcripts to a particular region of the cell, this is often thought to involve active transport along the cytoskeleton. This has been most clearly demonstrated in the case of Ash1 mRNA, which is transported to the bud tip along actin filaments (Bertrand et al., 1998; Beach et al., 1999). Ash1 mRNA is recognized by She2p, which then links it via the adaptor protein She3p to the myosin motor Myo4p, which moves this RNAprotein complex along actin cables (Bohl et al., 2000; Takizawa and Vale, 2000). Much less is known about mRNA localization in higher eukaryotes, but drug inhibitor studies have implicated the cytoskeleton in the localization of several mRNAs. For example, the localization of ß-actin RNA in fibroblasts is disrupted by actin-destabilizing drugs, whereas many other transcripts are localized in a microtubule-dependent manner including bicoid and oskar mRNAs in Drosophila (Clark et al., 1994; Pokrywka and Stephenson, 1995). However, it still remains to be proven that any of these RNAs are localized by active transport, and almost nothing is known about how they are coupled to the motors that are presumed to transport them.
One case which is very likely to involve active transport along microtubules is the localization of oskar mRNA to the posterior of the Drosophila oocyte. During stage 9 of oogenesis, oskar mRNA accumulates transiently at the anterior of the oocyte before moving to form a crescent at the posterior pole (Ephrussi et al., 1991; Kim-Ha et al., 1991). This anterior to posterior movement is disrupted by colchicine treatments, indicating that it requires an intact microtubule cytoskeleton (Clark et al., 1994). Furthermore, the site of oskar RNA localization correlates with the arrangement of the microtubules in the oocyte. During stages 79 of oogenesis, the oocyte microtubule cytoskeleton is reorganized in response to a polarizing signal from the posterior follicle cells, and a diffuse microtubule-organizing center at the anterior pole nucleates an anterior to posterior gradient of microtubules (Ruohola et al., 1991; Theurkauf et al., 1992). The polarity of these microtubules has been analyzed by expressing fusion proteins that contain microtubule motor domains: the plus enddirected motor kinesin fused to ß-galactosidase has been observed to localize to the posterior of the oocyte at exactly the same stage as oskar mRNA, whereas a nodßGal fusion localizes to the anterior (Clark et al., 1994, 1997). Thus, the minus ends of the microtubules seem to lie at the anterior of the oocyte with the plus ends extending towards the posterior pole. Furthermore, oskar mRNA still colocalizes with the plus ends of the microtubules in mutants that disrupt the polarity of the oocyte. For instance, oskar mRNA localizes to the center of the oocyte in gurken and PKA mutants, which led to the formation of a symmetric microtubule cytoskeleton with minus ends at both poles and the plus ends in the middle (González-Reyes et al., 1995; Lane and Kalderon, 1995; Roth et al., 1995). Finally, it has been shown recently that mutants in the heavy chain of kinesin I block the posterior localization of oskar mRNA and cause it to accumulate instead at the anterior of the oocyte (Brendza et al., 2000). Since these mutants do not appear to affect the organization of the microtubules, this motor seems to be directly involved in localizing oskar mRNA. It is therefore very attractive to propose that kinesin I transports oskar mRNA along microtubules from the anterior towards the plus ends at the posterior pole.
Besides the kinesin heavy chain, mutants in staufen, mago nashi, and tropomyosin II (TmII) have also been found to specifically block the posterior localization of oskar mRNA without affecting the organization of the microtubules (Ephrussi et al., 1991; Kim-Ha et al., 1991; Newmark and Boswell, 1994; Erdélyi et al., 1995; Tetzlaff et al., 1996). Staufen contains five copies of the dsRNA-binding domain and colocalizes with oskar mRNA throughout oogenesis in wild-type egg chambers and in all mutants that have been examined so far (St Johnston et al., 1991, 1992). Furthermore, the posterior localizations of Staufen and oskar mRNA are mutually dependent and require the RNA-binding activity of Staufen protein (Ferrandon et al., 1994; Ramos et al., 2000). Thus, it seems probable that Staufen binds directly to oskar mRNA and is required in some way to couple this complex to the translocation machinery. It is very important that Oskar protein is only produced at the posterior of the oocyte, and the translation of unlocalized oskar mRNA is therefore repressed through the binding of factors, such as Bruno protein, to sites within the 5' and 3' untranslated regions (UTRs)* (Kim-Ha et al., 1995; Gunkel et al., 1998). This repression must be relieved once oskar has been localized, and Staufen, which remains associated with the RNA at the posterior pole, performs a second function in the activation of oskar translation (Micklem et al., 2000).
In the case of Mago nashi and TmII, it has not yet been possible to establish a clear link between the molecular nature of the proteins and their effects on the localization of oskar mRNA. Mago is a mainly nuclear protein that has been extremely highly conserved during evolution and plays several roles during oogenesis (Newmark and Boswell, 1994; Micklem et al., 1997; Newmark et al., 1997). In the weakest mutant combinations, oskar mRNA remains at the anterior of the oocyte and never reaches the posterior pole. This phenotype is not due to the defect in microtubule organization, suggesting that Mago also has specific function in osk mRNA localization (Micklem et al., 1997). Consistent with this, small amounts of green fluorescent protein (GFP)-Mago colocalize with oskar mRNA at the posterior pole at stage 9 (Newmark et al., 1997). oskar mRNA also fails to localize to the posterior in TmIIgs homozygotes. This phenotype has been difficult to explain because TmII encodes a cytoplasmic tropomyosin that is known to function in the regulation of the actin rather than microtubule cytoskeleton. This analysis is further complicated by the fact that the TmIIgs mutation that disrupts oskar mRNA localization is not a null allele, but its exact effect on tropomyosin expression is not clear (Erdélyi et al., 1995).
Here, we report the phenotypic and molecular characterization of a novel locus called barentsz, which is specifically required for the movement of oskar mRNA from the anterior to the posterior of the oocyte and analyze the behavior of Barentz protein during oogenesis. These results suggest that Barentsz is an essential and specific component of the oskar mRNA localization complex.
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Results |
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The posterior localization of oskar mRNA and protein defines where the abdominal and germline determinants are localized, and staufen mutant females therefore give rise to embryos that lack both the abdomen and the pole cells. Surprisingly, the embryos laid by barentsz mutant females show much milder phenotypes. Embryos from btz2 homozygous females lack pole cells but develop an average of 5.4 ± 3.1 denticle belts. Furthermore, embryos from btz2/Df(3R)IR16 flies form a very similar number of 4.8 ± 3.1 denticle belts (n = 43), indicating that btz2 is a null mutation or a very strong hypomorph (Table I). The embryonic phenotype of btz1 is even weaker. At 18°C, almost all embryos lack pole cells (Fig. 2, C and E , and Table I) but have a normal number of denticle belts, but most embryos form a few pole cells and develop into viable and fertile adults if the mothers are kept at 25°C. As a consequence, the reduced number of pole cells, about a third of the adult female progeny, have only one gametic ovary, presumably because there were too few germ cells to populate both gonads.
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Given the role of barentsz in oskar mRNA localization, we wondered whether it might also be involved in the assembly of the pole plasm once Oskar protein is translated at the posterior. To test this possibility, we took advantage of an osk-bcd 3'UTR transgene in which the bicoid 3'UTR directs the anterior localization and translation of Oskar protein (Ephrussi and Lehmann, 1992). In a wild-type background, the ectopic Oskar produced from the transgene directs the formation of anterior pole plasm and pole cells, whereas normal pole cells develop at the posterior under the control of the endogenous oskar mRNA (Fig. 2, C and D). When this transgene is crossed into btz1 homozygotes at 18°C, the anterior pole cells still form, although the posterior ones are missing because oskar mRNA is not localized (Fig. 2, E and F). Although btz1 is not completely null at the restrictive temperature, this result strongly suggests that barentsz is also not required for any events in pole plasm assembly downstream of Oskar protein.
Cloning of barentsz
The analysis of the barentsz phenotype indicated that the gene has a unique and specific role in oskar mRNA localization, and we therefore decided to characterize it molecularly. Initial mapping placed barentsz at 98A-B on the right arm of the third chromosome in the region uncovered by the deficiency Df(3R)IR16 but not by other deletions in the area.
To identify the barentsz gene in this interval, we employed a novel positional cloning strategy that uses high resolution meiotic mapping between two nearby P element insertions. We first used P-mediated male recombination to map barentsz between PlacW 318-07 in 98A6 and PlacW 430-05 in 98B. We then generated btz1 PlacW430/PlacW318 females and used the miniwhite eye color phenotypes of the P elements to select for progeny in which recombination had occurred between them. Out of 16,000 flies, we recovered 203 lines in which recombination had occurred between the two P elements, corresponding to approximately one recombination event per 2 Kb (Fig. 3)
. At the same time, we extended two molecular walks into this region and identified restriction fragment length polymorphisms (RFLPs) between the two chromosomes. These enabled us to map btz1 between two RFLPs that define a 10.7-kb interval containing three genes, CG12876, CG12878, and carnitine octanyl transferase (Berkeley Drosophila Genome Project; http://www.fruitfly.org) (Fig. 3 B).
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The exact molecular nature of btz1 has not been determined: the coding sequence of barentsz is not affected, but PCR analysis of homozygous btz1 mutants suggests that an insertion has occurred in the 3'UTR or immediately downstream of the transcription unit. This leads to reduced expression of barentsz RNA and protein as determined by in situ and Western blot analysis (unpublished data). We also found that the P-element P{PZ}l(3)rL203 is inserted in the 5'UTR of btz but does not cause a phenotype, and we therefore used imprecise excision of this P element to generate new barentsz alleles. One of these, btz2, is very likely to be a null mutation in the gene, since the first 1,221 nt of the transcription unit are deleted, but the expression of the adjacent transcription unit CG12876 is unaffected as determined by whole-mount in situ analysis (Fig. 3 B). This deletion removes the NH2-terminal 332 amino acids of the barentsz-coding region, including the conserved domains described below. Homo- or hemizygous btz2 mutants are mostly pharate lethal, and the few adults that eclose die within a few days. Thus, barentsz seems to have a zygotic function in addition to its maternal role in oskar mRNA localization.
The sequence of barentsz was obtained from the Berkeley Drosophila Genome Project database (http://www.fruitfly.org) and was confirmed by direct sequencing of the longest EST. barentsz encodes a very hydrophilic protein with a predicted molecular weight of 83.7 kD and a pI of 5.2 but contains no known protein motifs. However, a region of 184 amino acids near its NH2 terminus shows homology to three other proteins: human MLN51 (sequence data available from Genbank/EMBL/DDBJ under accession no. 015234), which is overexpressed in breast cancer cell lines, a predicted protein from Caenorhabditis elegans (sequence data available from Genbank/EMBL/DDBJ under accession no. CAB07130) and partial sequence from two overlapping mouse ESTs (sequence data available from Genbank/EMBL/DDBJ under accession nos. AI876771 and AW496349) (Tomasetto et al., 1995). Apart from their homologies to each other, these proteins show no significant similarities to any other proteins in the databases, suggesting that they may represent a new but divergent protein family.
Barentsz localizes to the posterior of the oocyte with oskar mRNA
To investigate the function of Barentsz in oskar mRNA localization, we raised a polyclonal antiserum in rabbits against the NH2 terminus of the protein. On Western blots of wild-type ovary extracts, affinity purified antibody recognizes a single 125-kD band that is absent from btz2/Df(3R)IR16 extracts, confirming that it is Barentsz protein (Fig. 3 D). The antibody also specifically recognizes Barentsz in whole-mount stainings of ovaries, since all of the specific staining patterns described below are absent in btz2/Df(3R)IR16 ovaries and wild-type ovaries stained with preimmune serum.
Barentsz is expressed very early in oogenesis and localizes to the presumptive oocyte as soon as it can be identified in region 2B of the germarium (Fig. 4 A). The protein then concentrates at the posterior of the oocyte between the nucleus and the posterior follicle cells during stages 16 of oogenesis, before becoming dispersed throughout the oocyte cytoplasm at stage 8 (Fig. 4, BD). In addition, Barentsz shows a punctate staining of the nuclear membrane in all cells of the egg chamber except the oocyte itself, suggesting an association with nuclear pores (Fig. 4 F). The localization of Barentsz within the oocyte correlates with the behavior of the oocyte microtubule-organizing center, which moves to the posterior at around stage 1 and persists until stage 7 and is identical to that observed for oskar mRNA. However, these localizations appear to be independent of each other, since barentsz mutants have no effect on the early localization of oskar mRNA.
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To be able to examine the localization of Barentsz protein and Staufen simultaneously, we generated transformants that express a BtzGFP fusion protein under the control of a germ linespecific promoter. BtzGFP is functional, since it rescues the barentsz mutant phenotype and shows an identical localization in the germ line to that revealed by antibody staining. Labeling BtzGFP transgenic ovaries with anti-Staufen antibody revealed that BtzGFP and Staufen colocalize at the posterior from stage 9 onwards but that colocalization is lost after stage 10 (Fig. 5 , AF). Furthermore, the two proteins colocalize to the center of the oocyte in gurken mutants, suggesting that they are localized as part of the same complex (Fig. 5, GI). Since oskar mRNA is not translated in gurken mutants, this observation also indicates that Barentsz localization is independent of Oskar protein.
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Localization of Barentsz in other mutants that disrupt oskar mRNA transport
Barentsz protein shows an identical phenotype to oskar mRNA and Staufen protein in oskar protein-null mutants, such as osk54, in tropomyosin II and kinesin heavy chain mutants (Ephrussi et al., 1991; Kim-Ha et al., 1991; St Johnston et al., 1991; Erdélyi et al., 1995; Brendza et al., 2000). In the former, the protein forms a diffuse posterior cloud rather than a tight crescent because Oskar protein is required for the posterior anchoring of its own mRNA (Fig. 6, A and B)
(Markussen et al., 1995; Rongo et al., 1995). In tropomyosin II mutants and in kinesin heavy chain germ line clones, Barentsz accumulates at the anterior margin of the oocyte and fails to translocate to the posterior pole (Fig. 6, C and F).
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The final gene we examined is mago nashi, which in addition to its role in the polarization of the oocyte cytoskeleton has second and apparently independent function in the transport of oskar mRNA to the posterior pole (Newmark and Boswell, 1994; Micklem et al., 1997; Newmark et al., 1997). In mago1/Df or mago2/Df ovaries at permissive temperature, the microtubule organization appears normal, but oskar mRNA remains at the anterior of the oocyte. Furthermore, small amounts of myc- or GFP-tagged Mago protein can be detected at the posterior of the oocyte at stage 9, suggesting that Mago localizes with oskar mRNA (Newmark et al., 1997). Since both the phenotype and distribution of mago resembles that of barentsz, we generated flies expressing a GFP-Mago transgene to compare the localizations of two proteins in wild-type ovaries. As previously reported, GFP-Mago shows a weak localization to the posterior of the oocyte at stage 9 that can be detected by GFP fluorescence but not by anti-Mago antibodies (Fig. 7 A). Furthermore, Mago and Barentsz colocalize at the posterior pole, and both proteins accumulate only transiently at this site, unlike oskar mRNA and Staufen. However, the vast majority of Mago protein is nuclear, and it also appears to colocalize with Barentsz at the nurse cell nuclear membranes.
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The effect of barentsz mutations on GFP-Mago localization is less dramatic: the protein still accumulates normally in the nuclei but fails to localize to the posterior pole (Fig. 7 D). Thus, Mago is required for the posterior localization of Barentsz and vice versa.
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Discussion |
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barentsz is specifically required for oskar mRNA localization
The other genes involved in the posterior localization of oskar mRNA all have other functions: mago nashi is required for the polarization of the oocyte microtubule cytoskeleton (Micklem et al., 1997; Newmark et al., 1997), staufen is required for bicoid mRNA localization and for oskar and bicoid mRNA translation (Ferrandon et al., 1994; Micklem et al., 2000), kinesin I is required for the development of the fertilized egg and is also involved in fast axonal transport (Gho et al., 1992; Hurd et al., 1996), and tropomyosin II is an essential gene required for head development that seems to function in the polarization of the follicle cells (Erdélyi et al., 1995; Tetzlaff et al., 1996). In contrast, barentsz-null mutants block the transport of oskar mRNA from the anterior to the posterior of the oocyte but cause no other discernible phenotypes during oogenesis. In particular, Barentsz does not appear to be required for any other step in oskar mRNA biogenesis because btz mutants do not affect the transcription and export of the mRNA in the nurse cells, its accumulation in the early oocyte and at the anterior pole during stages 89, its colocalization with Staufen, or its translational activation. Furthermore, barentsz mutants have a stronger effect on oskar mRNA localization than either TmII or staufen. Very small amounts of oskar mRNA do reach the posterior pole in staufen-null mutant oocytes (Fig. 1 G), but the RNA is not maintained at the posterior for very long. On the other hand, in btz2 mutant oocytes no oskar mRNA or protein are ever detected at the posterior pole.
Given the severity of the oskar mRNA localization defect in barentsz mutants, it is surprising that the resulting embryos often form a normal abdomen, since this indicates that some Oskar protein must be produced at the posterior pole, although this cannot be detected by antibody staining. The translation of unlocalized oskar mRNA is repressed by factors such as Bruno protein, but this repression is specifically relieved at the posterior (Kim-Ha et al., 1995; Gunkel et al., 1998). Thus, any mRNA that diffuses to the posterior in a barentsz mutant should be translated there, leading to the production of trace amounts of Oskar protein at the posterior, which must be sufficient to induce a normal abdomen in the absence of RNA transport. Unlike Barentsz, Staufen protein is required for the translation of oskar mRNA, and this explains why staufen mutants produce a much stronger abdominal phenotype even though more oskar mRNA is localized to the posterior (Micklem et al., 2000). Thus, oskar mRNA localization is partially redundant with localized translational activation and is only necessary to produce the high levels of Oskar protein required for pole cell formation. To identify other mutants that specifically block oskar mRNA localization, it will therefore be important to design genetic screens that assay localization directly instead of the resulting embryonic phenotype.
Barentsz behaves like a component of the oskar RNA localization complex
Several lines of evidence indicate that Barentsz associates with oskar mRNA and Staufen protein during their movement from the anterior to the posterior of the oocyte. First, Barentsz localizes to the posterior pole at the same time as oskar mRNA and Staufen and colocalizes with Staufen in a posterior crescent at stage 9. However, unlike Staufen Barentsz does not remain at the posterior later in oogenesis and only colocalizes with oskar RNA during the stages when it is being transported to the posterior. Second, Staufen and Barentsz show an identical mislocalization to the center of the oocyte in gurken mutant egg chambers. Since oskar mRNA is not translated in these oocytes, this result argues against a role for Oskar protein in recruiting Barentsz to the complex. Third, like oskar mRNA and Staufen, Barentsz accumulates at the anterior of the oocyte in TmII and in kinesin heavy chain mutants. Thus, Barentsz colocalizes with oskar mRNA both before and after its transport to the posterior of the oocyte. Finally, the posterior localization of Barentsz seems to depend on oskar RNA. Although it is not possible to examine the localization of Barentsz in oskar RNA-null mutants, overexpression of oskar induces a corresponding increase in the amount of Barentsz that localizes to the posterior pole. In conjunction with the lack of oskar mRNA localization to the posterior in barentsz mutants, these results strongly suggest that Barentsz is an essential component of the oskar RNA transport complex.
Although it has been thought previously that Staufen is essential for oskar mRNA localization, our results show that a very small amount of the RNA can still reach the posterior pole at stage 9 in the complete absence of Staufen protein. Thus, Staufen cannot be the only RNA-binding protein that recognizes oskar mRNA and couples it to the transport machinery. In staufen mutants, Barentsz shows little if any accumulation at the anterior of the oocyte where the majority of oskar mRNA remains but colocalizes with the tiny fraction of RNA that reaches the posterior pole. Thus, Staufen seems to be required to promote or stabilize the efficient association of Barentsz with oskar mRNA. However, the Barentszoskar RNA complexes that do form in the absence of Staufen still localize to the posterior.
The sequence of Barentsz gives few clues as to its biochemical function, although it appears to have homologues in other species. Some insight into its role may be provided by the comparison of the oskar mRNA localization phenotype in btz mutants with that of mutants in the heavy chain of kinesin I (Brendza et al., 2000). In both cases, oskar mRNA does not localize to the posterior and accumulates instead at the anterior of the oocyte. Furthermore, khc mutants block the posterior localization of Barentsz protein, which remains with oskar mRNA at the anterior pole. Since kinesin I is a plus enddirected microtubule motor, a simple explanation for its role in oskar mRNA localization is that it actually transports oskar mRNA to the plus ends of the microtubules at the posterior pole. If this model is correct, the mutant phenotype and localization of Barentsz protein suggest that it acts somewhere between oskar mRNA and the kinesin. For example, Barentsz could play a role in coupling the RNA to the kinesin or in the activation of the motor once the complex has formed. On the other hand, we have been unable so far to detect an interaction between Barentsz and the kinesin heavy chain in Drosophila ovary extracts, although this may be due to the fact that only a fraction of the total Barentsz protein localizes with oskar mRNA, and this only occurs in stage 9 and 10 egg chambers, which represent a small proportion of the egg chambers in the ovary.
Most of our conclusions about Barentsz are also likely to apply to Mago nashi, which seems to serve a closely related function in oskar mRNA localization. mago nashi mutants cause a very similar failure in the translocation of oskar mRNA from the anterior to the posterior of the oocyte (Newmark and Boswell, 1994; Micklem et al., 1997). Furthermore, our results confirm that Mago protein also localizes transiently to the posterior pole, although the amounts are too low to detect by antibody staining (Newmark et al., 1997). Finally, Mago and Barentsz depend on each other for their localization to the posterior, since the localization of Mago is abolished in barentsz mutants and vice versa. Some clue to the relationship between the two may be provided by the fact that mago mutants disrupt the perinuclear localization of Barentsz in the nurse cells. This suggests that Mago may be required for the formation of functional Barentsz and that the two proteins are part of the same complex before they enter the oocyte. Consistent with this, Barentsz and Mago appear to colocalize at the periphery of the nurse cell nuclei and at the ring canals between the nurse cells and the oocyte, although we have been unable so far to detect a direct interaction between them.
Recent results have implicated hnRNP proteins that are predominantly nuclear in the cytoplasmic localization of several RNAs, suggesting that the nuclear history of a transcript may determine its fate in the cytoplasm (Hoek et al., 1998; Cote et al., 1999; Lall et al., 1999; Norvell et al., 1999). In this context, it is interesting to note that most Barentsz is associated with the nuclear membranes of the nurse cells, whereas almost all Mago nashi are found in the nuclei. Since oskar mRNA is transcribed in the nurse cell nuclei, this raises the possibility that Mago associates with the RNA in the nucleus and that Barentsz is then recruited to the complex as it exported into the cytoplasm. Consistent with this, the human homologue of Mago interacts with RBM8/Y14, a nucleocytoplasmic shuttling protein that binds to spliced mRNAs and remains associated with newly exported transcripts in the cytoplasm (Kataoka et al., 2000; Zhao et al., 2000). Thus, oskar mRNA may provide another example where factors loaded onto a transcript as it exits the nucleus determine its subsequent cytoplasmic localization. Neither Mago or Barentsz is required for oskar mRNA transport from the nurse cells into the oocyte, and they would therefore have to remain associated with the RNA during this phase of its localization before directing its subsequent transport to the posterior of the oocyte.
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Materials and methods |
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Creation of btz2
P{PZ}l(3)rL203 (Berkeley Drosophila Genome Project; http://www.fruitfly.org) was mobilized using TM3 2-3 (Robertson et al., 1988), and eight of the resulting ry- excision events were found to be new alleles of btz. These were crossed to Df(3R)IR16/TM6B flies, and hemizygous mutants were analyzed by PCR using several primers in the region. One of the alleles, btz2, has a deletion of the region distal to the P element that contains the NH2-terminal portion of Barentsz without affecting the expression of the adjacent proximal gene CG12876 as determined by whole-mount in situ analysis. The precise extent of the btz2 deficiency was determined by cloning and sequencing a PCR product spanning the breakpoints.
Whole-mount in situ hybridizations and antibody stainings
Antibody stainings were preformed as described in St Johnston et al. (1991). The following antibodies were used: affinity purified anti-Stau (1:1,000; St Johnston et al., 1991) anti-Osk (1:1,000; unpublished data and Grünert, S., personal communication), anti-Barentsz (1:5001:1,000; this paper); anti-ßGal (1:2,000; Cappel). In situ hybridizations were performed as described in Tautz and Pfeifle (1989).
Cloning of btz
Using male recombination, barentsz was mapped between two P elements in the 98A-B region, PlacW 318-07 (98A5-10) and PlacW 430-05 (98B) (Preston and Engels, 1996; Deak et al., 1997). To map barentsz further, w;e btz1 PlacW 430-05/e PlacW 318-07 females were created and crossed to w males. Meiotic recombination events in the interval between the P elements were selected on the basis of the red or white eye color of the F1 progeny. Flies with red eyes carry a third chromosome that contains both P elements and have therefore undergone a recombination event in the interval between the two P elements that juxtaposes the proximal part of e PlacW 318-07 chromosome and the distal part of e btz1 PlacW 430-05, whereas flies with white eyes carry neither P element and have undergone recombination in the opposite direction. Stocks of these recombinants were established and tested for the presence of btz. The position of barentsz was then refined by mapping it relative to RFLPs in two genomic walks in the region. Restriction fragments in these walks that were polymorphic between the e btz1 PlacW 430-05 and the PlacW 318-07 chromosomes were discovered by generating 4001,000-bp PCR products from each chromosome, digesting these with a battery of restriction enzymes with 4-bp recognition sequences and screening for differences between the two restriction digests on agarose gels. 75% of the PCR products that we tested showed a clear polymorphism between the two chromosomes, indicating that the ru st e ca background on which btz1 was induced is sufficiently different from that of the PlacW stocks to allow such an approach. The first walk contained cosmids 156B6 and 68H1, and the second started from the breakpoint of Df(3R)IR16. The distal end of the first walk was extended twice (27B9,105B7) using gridded cosmid filters from the United Kingdom Human Genome Mapping Project Resource Centre, and the last cosmid was found to extend beyond the barentsz locus. Mapping barentsz relative to further RFLPs in this cosmid placed the mutation in a 10.7-Kb interval that contains three predicted genes. Primer sequences and information on polymorphic enzymes are available on request (FvE).
A 7.3-Kb BamHI/Sca genomic fragment containing CG12876 and CG12878 was cloned in the BamHI/EcoRV sites of pBluescriptKS (Stratagene) and recloned as a KpnI fragment into the pWhite Rabbit transformation vector (pWRb) (Martin-Bermudo et al., 1997). The orientation of the KpnI fragment was such that CG12878 can use the polyA site encoded by pWRb. This construct was introduced into flies using standard methods and was able to rescue the maternal effect posterior group phenotype of osk nos btz1/btz1 females. Two mutant constructs were produced: one in which a frameshift was introduced after amino acid 25 in CG12876 using the Stratagene Quickchange kit and the other in which a NotI site in the CG12878 coding sequence was filled in, resulting in a frameshift after amino acid 181. In several independent transformant lines, only the first construct was able to rescue the barentsz mutant phenotype, indicating that CG12878 corresponds to btz.
Generation of Barentsz-GFP
The barentsz-coding region was amplified and cloned into BamHI/SpeI cut pD277-GFP6 to generate a transformation construct in which the 4-tubulin promoter drives germline-specific expression of Barentsz fused to GFP6. pD277-GFP6 was created by amplifying GFP6 (a gift from A. Brand, Welcome, CRC Institute, and J.V. Haseloff, University of Cambridge, Cambridge, UK) and cloning it between the NotI and XhoI sites of a modified version of pCaTub67MatpolyA (Micklem et al., 1997) in which the tubulin initiation codon had been mutated.
Antibody preparation
Amino acids 2383 of barentsz were amplified and cloned into pQE31 and expressed in M15[pRep4] cells (QIAGEN). The fusion protein was purified using the Amersham Pharmacia Biotech His-trap kit, and polyclonal antisera were produced in rabbits by Eurogentec.
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Footnotes |
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F.J.M. van Eeden's present address is The Hubrecht Laboratory, Uppsalalaan 8 3584CT, Utrecht, Netherlands.
M. Petronczki's present address is the Research Institute of Molecular Pathology, Dr. Bohr Gasse 7, A-1030 Vienna, Austria.
* Abbreviations used in this paper: GFP, green fluorescent protein; RFLP, restriction fragment length polymorphism; UTR, untranslated region.
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Acknowledgments |
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F.J.M. van Eeden was supported by an Human Frontier Science Program (HFSP) postdoctoral fellowship. I.M. Palacios was supported by an HFSP postdoctoral fellowship and the Royal Society Dorothy Hodgkin fellowship. M.J.D. Weston was a Wellcome Trust Prize student. M. Pertonczki and D. St Johnston were supported by Wellcome Trust Principal Research fellowship to D. St Johnston.
Submitted: 10 May 2001
Accepted: 27 June 2001
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