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poirot, a new regulatory gene of Drosophila oskar acts at the level of the short Oskar protein isoform

¹ Institute of Genetics, Biological Research Center of the Hungarian Academy of Sciences, H-6701 PO Box 521, Szeged, Hungary
² Institute of Biochemistry, Biological Research Center of the Hungarian Academy of Sciences, H-6701 PO Box 521, Szeged, Hungary

View larger version (82K): [in a new window]   Fig. 1. Embryonic germ cell-less (grandchildless) and abdomenless phenotypes of prtgs mutant embryos. (A) Wild type, early cellular blastoderm embryo with embryonic germ cells at the posterior at 25°C, as shown by staining with anti-Vasa antibody, and (B) embryo without embryonic germ cells from a prtgs/prtgs mother. (C,D) Cuticle preparation of wild-type and mutant, prtgs/prtgs female originated embryo at 18°C. (C) Wild-type embryo with normal abdominal segmentation, and (D) a mutant embryo from prtgs/ prtgs mother with a weak abdomenless phenotype. All embryos oriented anterior leftwards, dorsal upwards.

View larger version (55K): [in a new window]   Fig. 2. (A) The structure of the prt gene. prt contains four exons (1-4) and three introns. The Hobo element is inserted in the first intron of the prt gene that corresponds to 34569 bp in the DS04940 P1 phage. The hobo insertion is indicated by a triangle. The translation start site is in the first exon, marked by a star. Right-angled arrows show the orientation of the primers prt 1F, prt 4R and White 3R, which were used in RT-PCR reactions. (B) Multiple alignment of the amino acid sequences of PRT, human and mouse Sab-s, and C. elegans K03E6.7. Human and mouse Sab have been reported previously (Matsushita, 1998; Yamadori, 1999). The grey background indicates identical residues, and the black background indicates the minimum region required for binding to BTK in the human Sab sequence.

View larger version (37K): [in a new window]   Fig. 3. RT-PCR and western blot analysis of the prtgs mutation. (A) Amplification of the 1747 bp prt cDNA fragment with prt 1F and prt 4R (Fig. 4) primers. rp49 primers were used as a template amount control in the same reaction. (Lane a) molecular weight marker (1 kb ladder, Fermentas), (lane b) wild type female, (lane c) prtgs female, (lane d) wild-type embryo, (lane e) prtgs maternal null embryo originated from a cross of a prtgs homozygous female and a wild-type male, (lane f) maternal and zygotic prt null embryos, the progeny of prtgs homozygous females and males. (B) Demonstration of an aberrant splice variant in the prtgs mutant females by RT-PCR. Prt 1F and white 3R primers were used in the RT-PCR reaction. (Lane a) molecular weight marker. An 882 bp long RT-PCR product from the aberrant splice species was amplified in the mutant prtgs (lane c) but not in the wild type (lane b). (C) Western blot of wild-type (lane a) and prtgs/prtgs (lane b) ovary extracts. The blot was probed with an antibody against Drosophila PRT. A doublet of 53-55 kDa is recognised in ovary extracts from wild-type flies, but not from homozygotes for prtgs/prtgs. (D) PRT-GFP fusion protein distribution in different stages (st) of developing eggs in wild type.

View larger version (61K): [in a new window]   Fig. 4. (A-K) osk mRNA, STAU protein is normal in wild type and prtgs/prtgs mutant oocytes. OSK protein shows delocalisation or is missing from the posterior pole in the mutant oocyte and embryo. (L-N) PRT-GFP fusion protein and Osk protein colocalisation in oocyte. (A) osk mRNA is localised at the posterior pole in wild type and (B) prtgs/prtgs stage 10 oocytes. (C) STAU is localised at the posterior pole in wild-type and (D) prtgs/prtgs stage 10 oocyte. (E,G) Wild-type OSK localisation at the posterior pole of the stage 10 developing egg, (F) No OSK is detectable in the prtgs/prtgs egg chamber. (H,I) OSK protein is degraded from the posterior of prtgs/prtgs egg chambers, (J) OSK protein has posterior localisation in wild-type embryos, (K) there is no detectable OSK in prtgs/prtgs embryos. (L) PRT-GFP protein is subcortical in stage 10 wild type oocytes, (M) OSK protein is localised to the posterior pole and (N) in this stage they show colocalisation.

View larger version (51K): [in a new window]   Fig. 5. Western blot analysis of prtgs mutant ovaries. Western blot of ovary extracts probed with the anti-OSK antibody. Wild type, prtgs and Osk54/Df stands for an OSK protein null combination (osk54/Df(2)RPXT103). The bottom panel shows the same Western blot probed with anti -Tubulin antibody as a loading control. The molecular weight of the three OSK-specific bands long OSK (71 kDa), phosphorylated short OSK (57 kDa) and unphosphorylated short OSK (55 kDa) are indicated.