BDGP) were subcloned into pCaSpeR4 vectors for complementation tests (Fig. 1j).
5602LBsiWI and 5602LNheI transgenes were made by BsiWI and NheI enzyme digestion respectively followed by klenow treatment and self-ligation on the 5602L fragment.
The dDcp1R57A transgene with a R57A amino acid substitution on the 11183L fragment (Fig. 1j) was generated using a QuikChange site-directed mutagenesis kit (Strategene). P{UASp-HA-dDcp1} and P{UASp-HA-N180-dDcp1} were generated by introducing the N-terminal HA-tagged full length and N-terminal 180 amino acids of dDcp1 into the pUASp vector, respectively. The dDcp1 full length coding sequence was obtained from EST clone GH04763.
Antibody generation and immunochemistry
A peptide with sequence SAPQQPKQDSSQPAS, corresponding to amino acid residues 140 to 154 of dDcp1, was used to generate polyclonal rabbit antiserum. For Western blot analysis, a 1:2000 dilution of anti-dDcp1 rabbit antibody or 5 mg/ml of affinity purified anti-Yps rabbit antibody (a gift of James E. Wilhelm) or a 1:10000 dilution of anti-α-Tubulin mouse antibody (Sigma) were used as 1st antibodies.
Protein was detected by enhanced chemiluminescence using 1:15000 dilutions of HRP-conjugated goat anti–rabbit IgG (Jackson lab). For immunoflourescence staining, ovaries from 1 to 3 day-old females were dissected into PBS on ice and fixed for 20 minutes in fixative (600 ul of heptane, 200 ul of 2% paraformaldehyde in PBS,
and 1 µl of NP-40). After 3 washes with PBT (PBS plus 0.2% Tween20), the fixed ovaries were incubated in PBT containing 1% Triton-X 100 for 1 hour. Ovaries were then blocked for 3 to 5 hours in 5% normal goat serum in PBT, and incubated overnight at 4oC in primary antibody diluted in PBT (1:20 for anti-dDcp1, 1:20 for anti-Orb 6H4, 1:500 for anti-Yps, 1:3000 for anti-Osk, 1:4000 for anti-Stau, 1:200 for anti-Centrosomin, 1:500 for anti-Vasa, 1:100 for anti-GFP, 1:100 for anti-HA). The ovaries were then washed 3 times for 20 minutes each in PBT, and then incubated for 2 hours at room temperature in secondary antibody in PBT. Following final 3x 30 minute washes in PBT, the ovaries were mounted in DABCO anti-fading solution (PBS containing 50% glycerol and 2% DABCO). General procedures and ovary extract preparation for co-immunoprecipitation followed those described in (Wilhelm et al., 2000), except that 500 µl of ovary extract was immunoprecipitated with either 50 µl of anti-GFP agarose (Vector) or anti-HA agarose (Sigma) for 2 hours at 4oC with gentle shaking.
Northern blot analysis
Total RNAs were extracted from 1 hour interval staged embryo collections in different dDcp1 genotypes using Purescript RNA Isolation Kit (Gentra). DIG-labeled (Roche) osk (744 bp), bcd (766 bp), twe (711 bp), and rp49 (450 bp) RNA probes were made by in vitro transcription using DIG Northern Starter Kit (Roche). Northern blot procedures are based on “Dig Application Manual for Filter Hybridization”
(Roche).
Acknowledgements
We are in debt to everyone who contributed to the construction of the cFRT2L2R chromosome. We are grateful to Bob Boswell, Ira Clark, Ilan Davis, Anne Ephrussi, Peter Gergen, Tulle Hazelrigg, Daniel St. Johnston, Kaijun Li, Li-Mei Pai, Nobert Perrimon, Bill Saxton, Henry Sun, James E. Wilhelm, and the Bloomington stock center for their generous gifts of fly stocks and antibodies. We also thank Megerditch Kiledjian and Michael B. Melnick for critical comments on the manuscript. This project is supported by grants from National Sciences Council, Frontier in Genome Medicine Program (NSC89-2318-B002-011-M51; NSC90-2318-B002-001-M51), and Ministry of Education (89-B-FA01-1-4).
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Figure 1. Mutant phenotypes and molecular organization of the dDcp1 gene.
a-c, Reduction of maternal dDcp1 gives rise to an abdomen deletion phenotype.
Cuticle preparations of late embryos are shown. Embryos from b53 GLC (b) display a phenotype with deleted abdomen and deformed anterior region compared to wild type (a). Embryos from a dDcp1b53 female (c) display a pure abdominal deletion phenotype. d-i, The number of pole cells is reduced in a dDcp1 mutant. In dDcp1b53 cellular blastoderm stage embryos (f-i) the pole cells are fewer (f, g) or cannot be detected (h, i) compared to wild type (d, e). Embryos were viewed by DIG (d, f, h), and pole cells were marked by Vasa (red) and viewed by confocal (e, g, i). j, Organization of the dDcp1 locus at 60A8. The P{lArB}b53 insertion is 46 bp downstream of the transcription start site and 27 bp upstream of the translation start site of CG11183, and 273 bp upstream of the opposite-orientated CG5602.
P{GSV6}11684 is located 35 bp upstream of P{lArB}b53. The related deletion region in dDcp1442P is shown. The NcoI-BamHI 5K fragment, the SalI- SalI 11183L fragment, the EagI to BamHI 5602L fragment, the dDcp1R57A transgene, the 5602LNheI fragment and the 5602LBsiWI fragment used in complementation tests are shown. UASp-N180 transgene is also presented. k-l, Western blots of dDcp1 mutations. dDcp1 antibody specifically recognizes a 42kD band. In dDcp1b53 ovary extract, the 42 kD band is barely visible and a 22 kD band corresponding to the polypeptide produced by the N-terminal dDcp1 from the 5602L transgene is marked by asterisk (k). The 42 kD band cannot be detected in a dDcp1442P larval extract (l).
Figure 2. Sequence alignment of dDcp1, hDcp1a, hDcp1b and Dcp1p decapping proteins.
a, The N-terminal 140 amino acids of dDcp1, hDcp1a and hDcp1b are conserved with full length yeast Dcp1p. Conserved residues critical for decapping function (D18, R57 in dDcp1) in Dcp1 are marked with asterisks. Seven β sheets and an α helix that form the EVH1/WH1 domain in Dcp1p are presented (She et al., 2004). b, The carboxy-terminal regions of dDcp1, hDcp1a and hDcp1b are less well conserved.
Figure 3. dDcp1 is required for proper mRNA degradation.
a-c, Northern blot analyses of osk, bcd and twe mRNAs in different dDcp1 mutations.
osk mRNA diminishes after 2 hr AEL in wild type embryos. In contrast, osk mRNA remains stable until at least 5 hours AEL in b53 GLC embryos, and is partially rescued in the dDcp1b53 allele. osk mRNA is maintained until at least 5 hours AEL in the R57A mutated dDcp1, dDcp1442P/ dDcp1442P; dDcp1R57A/MKRS. This delayed mRNA degradation phenotype can be fully rescued by the 11183L transgene in b53/b53; 11183L/MKRS. (a). Similar patterns of mRNA degradation defects and results of 11183L transgenic rescue are observed using bcd or twe probes (b, c). The rp49 mRNA is used as an internal loading control for corresponding mutations. 1 µg of total RNA extracted from embryos collected at one hour intervals was applied in each lane.
Figure 4. Distribution of dDcp1 during oogenesis.
a-f, dDcp1 antibody staining pattern. dDcp1 accumulates in the posterior of the oocyte from stage 2 and 3 (a), stage 5 (b) and stage 6 (c). At stage 8, dDcp1 is transiently found at the anterior end of the oocyte (d). After stage 9, dDcp1 is once again found at the posterior pole of the oocyte (e) and remains until to stage 10 (f). g-i, HA antibody staining of the egg chambers of the nanos-Gal4VP16-driven UASp-HA-dDcp1 transgenic fly. The posterior accumulation in stage 2 (g), the transient anterior localization in stage 8 (h), and the posterior localization in stage 10A (i) are the same as the dDcp1 antibody staining.
Figure 5. Mutations in dDcp1 specifically affect the posterior deposition of maternal determinants.
a-d, in situ hybridization of osk and bcd mRNA in stage 10 b53 GLC egg chambers.
dDcp1 activity is required for the posterior localization of osk mRNA (a, b). By contrast, the localization of bcd mRNA is unaffected (c, d). e-l, Immunodetection of the Grk, Stau, Osk and Vasa proteins in dDcp1b53 egg chambers. The anterior-dorsal Grk distribution in the stage 9 egg chamber (e, f) is not affected. The posterior localization of Stau (g, h), Osk (i, j), and Vasa (k, l) is reduced or lost. In some cases, a faint and diffuse Stau can be recognized within the oocyte (h). The cell boundary was marked by Texas-red phalloidin (red).
Figure 6. The dDcp1b53 mutation does not disrupt microtubule organization in the oocyte.
a-d, The microtubule plus end marked by kinesin-lacZ (a, b) and minus end marked by Centrosomin (c, d) are not affected in stage 9 egg chambers. e, f, The overall microtubule organization examined by Tau-GFP is not affected in stage 9 egg chambers (c, f).
Figure 7. The posterior localization of dDcp1 is osk mRNA dosage- and position-dependent, and microtubule organization-dependent.
a-c, HA-dDcp1 (green, anti-HA) is co-localized with Stau (red, anti-Stau) in the posterior crescent of the oocyte. d, e, The posterior localization of dDcp1 (green) is lost and accumulated in the anterior and lateral cortex in khc27 GLC (d) and stauD3 (e) egg chambers. f, The posterior localization of dDcp1 (green) is increased in oocytes carring four copies of the osk gene. g, dDcp1 is ectopically localized to the anterior of the osk-bcd 3’UTR oocyte. h, i, dDcp1 (green) is mis-localized in the middle of grk2B6/HF48 oocytes. j-l, dDcp1 (red) is gone from the posterior poles in par-16821/6323; kin-lacZ/+ oocytes. The microtubule plus end was marked using Kinesin-lacZ (green, anti-lacZ). Stage 9 to 10 egg chambers are shown in all panels.
Figure 8. dDcp1 is required for the posterior localization of Exu, Yps and Orb and interacts with Exu and Yps.
a-f, dDcp1 (red) is colocalized with GFP-Exu (green) in the posterior end of oocytes (a-c) and the cytoplasm of nurse cells (d-f). g-i, dDcp1 is colocalized with Orb at the posterior end of the oocyte. Stage 5 (g), 9 (h) and 10 (i) egg chambers were doubly stained for dDcp1 (green) and Orb (red). j-l,The posterior localization of Exu (j, m, stage 9), Yps (k, n, stage 10), and Orb (l, o, stage 10) are disrupted in dDcp1b53 mutants (m, n and o). Genotype in (m): b53/b53; 5602L/GFP-Exu. p, GFP-Exu co-immunoprecipitates with dDcp1 in a RNase-sensitive manner. GFP-Exu ovary extract was immunoprecipitated using anti-GFP antibody. Western blot was performed using anti-dDcp1 antibody. q, HA-dDcp1 co-immunoprecipitates with Yps.
α4-tubulin Gal4VP16-driven HA-dDcp1 ovary extract was immunoprecipitated using anti-HA antibody. Western blot was performed using anti-Yps antibody. OR ovary extract was used as the control in (p) and (q). The input was 1/100 the volume of ovary extract used in the co-immunoprecipitation reaction.
參加第十屆 HUGO 國際會議心得報告
第十屆國際 HUGO( Human Genome Organization)大會於 94 年 4 月 18 日至 4 月 22 日在日本京都舉行。每年 The Human Genome Organisation 都會舉辦一個 國際性的人類基因體學年度大會，藉以提升全世界相關領域的學者對人類基因體 學的認識與了解。這個大會主要是提供一個激發和討論的環境，並以 plenary lectures, symposia, workshops, poster presentations 和 social events 的方式，來促使 有興趣的學者專家們共同分享個人或研究團隊的研究發現和經驗；同時也讓學界
由於它的出現頻度會因為不同族群而呈現差異性，因此族群遺傳學的研究也提供 了未來各族群間醫療資訊互通前一個非常重要的指標。針對單一核苷酸多型性的 應用，一個集鉅資與大量高承載、高產能的世界級研究組織團體已於 2003 年成 立，並如火如荼的展開各項測試，我們已經在本年度的人類基因體學大會中看到 一些成績了。另外，人類遺傳學家也注意到其他可供我們解釋很多生命現象的其 他基因體上的資源，它們可以用基因之表現度、基因之剪接和基因之標記來調控 一些特殊之生物功能。這些結果也都可以在一些人類疾病中得到驗證。再者，為 因應未來之醫學發展和個人化醫學之施行，英國和日本已經開始籌建屬於他們族 群特有之生物銀行，目標希望能夠繼續找出一些常見疾病的致病基因，尋求新的 藥物治療和診斷方法。
此次大會，因為日本為主辦國而且一直在人類基因體學研究各方面素有成 績，表現得很讓人另眼相看。台灣方面共來了十來位學者專家，大都有發表論文，
個人在此方面仍屬學習階段，但是收穫很多。