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Functioning of the Drosophila orb gene in gurken mRNA localization and translation

Jacqueline S. Chang*,{ddagger}, Lihua Tan*, Melisande R. Wolf* and Paul Schedl

Department of Molecular Biology, Princeton University, Princeton, NJ 08544, USA
* These authors contributed equally to this work
{ddagger} Present address: Department of Biology, DCMB Program, Duke University, Durham, NC 27708, USA



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  		Fig. 1. Defects in Orb and Grk expression in late stage orbmel egg chambers. Ovaries from wild-type and orbmel females were stained with antibodies against Orb protein (panels on left) and Grk protein (panels on right). The genotypes of the ovaries are as indicated. In wild-type stage 9 egg chambers (top panels), Orb protein accumulates at high levels in the cortical cytoplasm around the entire circumference of the oocyte. Grk protein is localized in a cap above the oocyte nucleus in the anterior corner of the oocyte. Note that the Grk antibody gives `nonspecific' background staining in nurse cells and follicle cells. This background staining is observed grk null chambers (not shown). Representative orbmel chambers are shown in the three lower panels. In the first of the group of orbmel chambers, the level of Orb protein appears reduced in most of the oocyte except at sites near the posterior pole. Little or no Grk protein is detected. In the panels in the middle, only low levels of Orb protein are observed, while there appears to be a small amount of Grk protein along anterior margin of the oocyte near the oocyte nucleus. The lower panel shows a stage 10 egg chamber with little or no Orb or Grk protein.

 


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  		Fig. 2. Orb and Grk are improperly localized in early stage egg chambers in orb303 and completely absent in orb343. Ovaries from wild-type and orb mutant females were stained with antibodies to Orb and Grk protein. Genotypes are as indicated. The two panels at the top show the pattern of Orb and Grk protein accumulation in the germarium, and early stages of a wild-type ovariole. Note that Orb protein can be detected in region 2 of the germarium, where it begins concentrating in the presumptive oocyte. In the stage 1 chamber in region 3 and older chambers, much of the Orb protein is in the oocyte at the posterior end. Grk protein can be detected in the stage 1 chamber that has not yet completely budded off from the germarium (left arrow) and in slightly older stages (right arrow) at the posterior of the oocyte. The two sets of panels in the middle show the Orb and Grk protein staining pattern in orb303 ovarioles. The Orb protein distribution in orb303 cysts and chambers is abnormal. Unusually high levels of Orb protein are observed in the cysts in region 2 and 3 of the germarium, and this protein appears to be distributed in all germ cells. High levels of uniformly distributed protein can be seen in pseudo-egg chambers just budded off from the germarium, but then the level of Orb protein begins to decline as the aberrant chambers age. Grk also shows an anomalous pattern of accumulation (panels on right). As indicated by the arrows on the left, Grk is expressed prematurely and abnormally high levels of Grk protein are observed in cysts in region 2 of the germarium. High levels of Grk protein are also visible in stage 1 pseudo-egg chambers that have just budded off from the germarium (arrows on right). When the level of Orb protein begins to decline, Grk protein is also reduced. The two panels at the bottom show that there is little or no Orb or Grk protein in orb343 ovarioles.

 


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  		Fig. 3. Defects in K(10) protein expression in orbmel ovaries. Wild-type and orbmel mutant ovaries were stained with Orb and K(10) antibodies. The two panels at the top show a stage 9 wild-type egg chamber stained with Orb (left) or K(10) antibody. Orb protein can be detected in the oocyte cortex, while K(10) protein is concentrated primarily (though not exclusively) in the oocyte nucleus. The four lower panels show Orb and K(10) proteins in stage 9 and early stage 10 orbmel egg chambers. Though the level of Orb protein in these two egg chambers is reduced compared with wild type, the extent of reduction is not great as seen in other mutant chambers (see, for example, Figs 1, 5). In the top orbmel chamber, some residual K(10) protein can be seen around the oocyte nucleus. In the bottom orbmel chamber there is little K(10) protein.

 


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  		Fig. 4. K(10) but not grk mRNA is in an immunoprecipitable complex with Orb protein. Anti-Orb or anti-Dorsal antibody was used for immunoprecipitation of wild-type ovary extracts. RNA isolated from the immunoprecipitates (IP) and from the total extracts was reverse transcribed with an anchored oligo(dT) primer (see Materials and Methods) and the resulting cDNAs were subjected to PCR amplification using the anchored primer and gene-specific primers for either K(10), grk or nos. The amplification products were displayed by electrophoresis and blotting to nitrocellulose and the filters were then hybridized with gene specific probes. K(10) sequences can be RT-PCR amplified from the Orb IP sample, as well as from the total RNA pool, but not from the Dorsal IP sample (middle panel). In both the Orb IP and the total samples, the K(10) probe hybridizes to a prominent band and an upward smear. The prominent band corresponds in size to a PCR amplification product extending from the K(10) primer in the 3' UTR to the beginning of the poly(A) tail. The smear arises from hybridization of the anchored oligo dT primer at different sites in the poly(A) tail. Neither nos PCR products (left panel) nor grk PCR products (right panel) could be detected in the Orb IP lanes, but could be amplified from the total RNA pool. As expected, no amplification products were observed when the RT step was omitted.

 


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  		Fig. 5. Orb and Gurken expression in K(10); orbmel double mutant ovaries. Ovaries from wild-type (w1), orbmel; K(10) and K(10); orbmel females were simultaneously stained with antibodies against Orb (red) and Grk (green). To make protein levels comparable between the different ovaries, the ovaries were stained in parallel and analyzed by confocal microscopy using identical conditions. Background staining by the Grk antibody is nonspecific and present in the same pattern in grk-null egg chambers (data not shown). The genotypes are as indicated in each panel. In the wild-type stage 10 egg chamber (top left panel) Grk protein is localized to the anterior corner of the oocyte above the nucleus, while Orb protein can be seen as a ring around the edges of the oocyte. In the orbmel stage 10 egg chamber (top middle), neither Grk nor Orb is observed. In the K(10) stage 8-9 egg chambers (top right), the level and localization of Orb resembles that seen in wild type, while Grk protein can be seen along the anterior margin of the oocyte (as yellow staining). In the K(10); orbmel egg chambers shown in the panels at the bottom, the level of Orb protein is greatly increased compared with that seen in the orbmel chamber, and the localization pattern appears to be quite similar to that seen in wild-type egg chambers. Note that red staining can be seen around the circumference of each of the double mutant oocytes. As in K(10) mutant chambers, Grk protein can be seen along the entire anterior margin of the double mutant oocytes.

 


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  		Fig. 6. Orb protein expression is increased in K(10); orbmel double mutants. Ovary extracts were prepared from wild-type w1, orbmel and K(10); orbmel females. The Orb protein level was then analyzed by western blotting. A representative blot is shown in this figure. The two bands seen in this blot correspond to the two Orb protein isoforms typically seen in wild-type and orbmel mutant ovaries. To control for the total protein in the extracts, the blots were reprobed with antibodies against the snRNP protein Snf or Armadillo (data not shown). Using NIH Image, the ratio of Orb protein to the loading control, Snf or Armadillo was calculated for each extract, and then normalized to that in wild-type ovaries. In the experiment shown here, the ratio is 1=w1; 0.3=K(10); orbmel and 0.05=orbmel. The ratios estimated for the double mutant ranged in different experiments from 0.3 to 0.5 and for orbmel from 0.05 to 0.1.

 

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