Truncated RanGAP encoded by the segregation distorter locus of Drosophila

Science

Volumn 283, Issue 5408, 1999, Pages 1742-1745

  Merrill, Cynthia ^a   Bayraktaroglu, Leyla ^a,b   Kusano, Ayumi ^a   Ganetzky, Barry ^a  

^a UNIVERSITY OF WISCONSIN MADISON   (United States)

^b HARVARD UNIVERSITY   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

CARRIER PROTEIN; COMPLEMENTARY DNA; GUANOSINE TRIPHOSPHATASE; PROTEIN RANGAP; UNCLASSIFIED DRUG;

ANIMAL EXPERIMENT; ANIMAL TISSUE; ARTICLE; CELL NUCLEUS; CELL TRANSFORMATION; CONTROLLED STUDY; DNA SEQUENCE; DROSOPHILA MELANOGASTER; GENE LOCUS; GENE SEGREGATION; INTRACELLULAR TRANSPORT; MALE; MEIOSIS; NONHUMAN; PHENOTYPE; PRIORITY JOURNAL; SEQUENCE HOMOLOGY; SPERMATID;

ANIMALS; CARRIER PROTEINS; CELL NUCLEUS; CROSSES, GENETIC; DNA, COMPLEMENTARY; DROSOPHILA MELANOGASTER; DROSOPHILA PROTEINS; FEMALE; GENE DUPLICATION; GENE EXPRESSION; GENES, INSECT; GTPASE-ACTIVATING PROTEINS; MALE; MEIOSIS; NUCLEAR PROTEINS; RNA, MESSENGER; SPERMATIDS; SULFOTRANSFERASES; TRANSCRIPTION, GENETIC; TRANSFORMATION, GENETIC;

EID: 0033548718     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.283.5408.1742     Document Type: Article

References (29)

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15. We used the 6.5-kb wild-type genomic fragment to screen libraries for wild-type and Sd cDNAs. Wildtype dHS2ST and dRanGAP cDNAs were isolated from a testes cDNA library provided by T. Hazelrigg. Sd cDNAs were isolated from an Sd-Mad male cDNA library that we constructed (Stratagene). We also constructed both wild-type and Sd-Mad Marathon cDNA libraries (Clontech) to extend incomplete cDNAs by polymerase chain reaction (PCR). Reverse transcription PCR was used to verify cDNA structure. cDNAs were mapped onto genomic DNA by direct sequence comparison.
16. Sequence alignments prepared with software (Wisconsin Package Version 9.0) from the Genetics Computer Group (Madison, WI) show that the wild-type dRanGAP polypeptide shares 34 and 36% amino acid identity with the Saccharomyces cerevisiae and mouse counterparts, respectively. The identities with the mouse protein are distributed throughout the entire sequence of 596 amino acids but are highest in the first 400 amino acids, with 40% identity and an additional 20% amino acid similarity. The top five matches identified by BLAST searches [S. F. Altschul et al., Nucleic Acids Res. 25, 3389 (1997)] of protein databases with the Drosophila sequence are RanGAP proteins from Xenopus, mouse, human, sea urchin, and nematode. The probabilities that the observed degree of similarity with these proteins occurs by chance range from 1.6e - 57 to 9.6e - 102.
17. Although the truncation of 234 amino acids from the distal version of dRanGAP is the most dramatic departure from the wild type protein, there are additional polymorphisms: an M-to-I substitution at amino acid position 9 in all SD strains examined, and a C-to-Y substitution at position 146 in only some SD strains. In addition, because the mutant cDNA is incomplete at the 5′ end, we have been unable to confirm that the distal dRanCAP transcript initiates at the equivalent point as the wild-type and proximal dRanGAP transcripts. If there is a difference here, it should affect only the 5′ untranslated leader and not the coding sequence.
18. A genomic fragment encoding amino acids 1 through 251 of the wild-type dRanGAP protein was cloned into the pQE30 expression vector (Qiagen) and expressed in Escherichia coli. The gel-purified polypeptide was injected into a rabbit, and subsequent bleeds were examined by protein immunoblot analysis for the ability to identify the wild-type and mutant dRanGAP proteins in Drosophila testes. The expressed protein was bound to nitrocellulose strips and incubated with crude antiserum to affinity purify the antibodies to RanGAP.
19. Ten independently isolated SD lines from the United States (SD-MAD, SD-5, SD-72, and SD-Weymouth). Italy (SD-Roma, SD-Oviedo, and SD-VO19], Spain (SD-Los Arenos), Japan (SD-NH2). and Australia (SD-Armindale] were examined by protein immunoblot analysis and found to express both the 66-kD and 40-kD proteins. Both proteins were present at consistent levels in testes, whole flies, carcasses (minus testes or ovaries), heads, larvae, and pupae. Because no somatic phenotypes are seen in SD heterozygotes or homozygotes, it is likely that some step in spermatogenesis, such as the high degree of chromatin compaction that takes place, is particularly sensitive to perturbations caused by expression of the truncated dRanGAP.
20. + Sd)12A] is remobilized to new insertion sites (C. Merrill and B. Ganetzky, unpublished results).
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22. Although how the truncated dRanGAP is functionally altered remains unknown, it is worth noting that the truncation eliminates the lysine residue at position 533, which is the presumed target site for covalent linkage to the small ubiquitin-related modifier SUMO-1 [S. Saitoh, R. T. Pu, M. Dasso, Trends Biochem. Sci. 22, 374 (1997); R. Mahajan, L. Gerace, F. Melchior, J. Cell Biol. 140, 259 (1998)]. Because this modification is essential for targeting dRanGAP to nuclear pore structures, subcellular mislocalization of the truncated dRanGAP is one potential consequence.
23. Although how the truncated dRanGAP is functionally altered remains unknown, it is worth noting that the truncation eliminates the lysine residue at position 533, which is the presumed target site for covalent linkage to the small ubiquitin-related modifier SUMO-1 [S. Saitoh, R. T. Pu, M. Dasso, Trends Biochem. Sci. 22, 374 (1997); R. Mahajan, L. Gerace, F. Melchior, J. Cell Biol. 140, 259 (1998)]. Because this modification is essential for targeting dRanGAP to nuclear pore structures, subcellular mislocalization of the truncated dRanGAP is one potential consequence.
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29. We thank R. Kreber and M. Schultz for technical assistance; M. Ashburner, A. Carpenter, T. Littleton, and R. Temin for helpful comments on the manuscript; and R. Temin for insightful discussion during the course of this work. Supported by NSF grant DMB-9014779 to B.G. This is paper number 3530 from the Laboratory of Genetics. 24 November 1998; accepted 5 February 1999