Sex-specific assembly of a dosage compensation complex on the nematode X chromosome

Science

Volumn 274, Issue 5293, 1996, Pages 1736-1739

  Chuang, Pao Tien ^a,b   Lieb, Jason D ^a   Meyer, Barbara J ^a  

^a UNIVERSITY OF CALIFORNIA   (United States)

^b HARVARD UNIVERSITY   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

ANIMAL CELL; ARTICLE; EMBRYO; GENE DOSAGE; GENE EXPRESSION REGULATION; GENE LOCATION; NEMATODE; NONHUMAN; PRIORITY JOURNAL; REGULATOR GENE; REGULATORY MECHANISM; X CHROMOSOME;

ANIMALS; CAENORHABDITIS ELEGANS; CAENORHABDITIS ELEGANS PROTEINS; CARRIER PROTEINS; DOSAGE COMPENSATION, GENETIC; ELECTROPHORESIS, POLYACRYLAMIDE GEL; FEMALE; GENES, HELMINTH; GENES, REGULATOR; HELMINTH PROTEINS; HERMAPHRODITISM; MALE; NUCLEAR PROTEINS; PRECIPITIN TESTS; RNA, HELMINTH; RNA, MESSENGER; SEX DETERMINATION (ANALYSIS); X CHROMOSOME;

ANIMALIA; CAENORHABDITIS; MAMMALIA; NEMATODA;

EID: 0029807745     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.274.5293.1736     Document Type: Article

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22. The sdc-2 (y74 or y82) and sdc-3(y126) null alleles cause such extensive XX-specific lethality and masculinization that mutants must be grown as her-1(hv1y101); xol-1(y9) set XO hermaphrodite strains. The XO-specific lethality caused by an xol-1 mutation is suppressed by an sdc mutation, and the rescued XO animals are transformed into fertile hermaphrodites by a her-1 mutation. The viable XO embryos and dying XX embryos were analyzed from this strain. Strains ot dpy-28(s939) and dpy-26(y65 or n199) null mutants did not require her-1 because dpy; xol-1 XO animals are hermaphrodites. We confirmed the results from the XO strains by examining anti-DPY-27 staining in a small sample of XX embryos from XX mutant mothers. The temperature-sensitive dpy-30(y228) and dpy-28(y1) XX mutants were grown as in (20). Because 30% of sdc-3(y129) XX mutants and all of the sdc-1 (n485) and dpy-21 (e428) mutants are viable, they were grown as XX strains.
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27. 2, 0.1 mM EDTA, 10% glycerol, 0.2 mM PMSF, and 0.1% NP-40] and incubated for 3 hours at 4°C with rocking. Embryonic nuclei (100 to 300 mg) in HEMK buffer were sonicated and microcentrifuged at 14,000 rpm for 30 min at 4°C. The supernatant was preincubated with protein A-beads, centrifuged as above, and added to the antibody-protein A complexes. After 4 hours at 4°C with rocking, the antibody-protein A complexes were washed twice (15 min each) in HEMK buffer and twice in HEMK buffer with 400 mM KCI. The bound proteins were analyzed by SDS-PAGE.
28. Partial purification of a dosage compensation complex: Nuclei (1 g) were resuspended in HEMK buffer, sonicated, and microcentrifuged at 14,000 rpm for 30 min at 4°C. The supernatant was added to SP-Sepharose resin (1 ml) and incubated for 1 hour at 4°C with rocking. The protein complex was washed with HEMK buffer and eluted from a column with HEMK buffer containing 400 mM KCI. The eluate was adjusted to 200 mM KCI, added to Q-Sepharose resin, and the mixture treated as above. The fractions containing DPY-27 were immunoprecipitated with anti-DPY-26 or anti-DPY-27 as described (17). Resins (Pharmacia) were washed with HEMK buffer containing 400 mM KCI and then HEMK buffer before use.
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31. dpy-30 probably exerts its effects on dosage compensation through regulation of sdc-3. sdc-3 XX null mutants do not exhibit a sex-determination defect, even though sdc-3 functions in sex determination (7). The sex determination defect is apparent in sdc-3(null); xol-1 XO animals, which are males. In contrast, xol-1 XO animals are hermaphrodites if also mutant in dpy-26, dpy-27, or dpy-28. dpy-30 mutations behave like sdc-3 null mutations in that extensive masculinization is apparent only in the dpy-30; xol-1 XO animals (3). For comparison, sdc-2 XX mutants are very masculinized.
32. We thank T. Cline, R. Tjian, G. Garriga, J. Rine, M. Botchan, I. Carmi, T. Davis, and H. Dawes for critical comments on the manuscript; C. Akerib for strain construction; and R. Kamakaka, H. Beckmann, and S. Lichtsteiner for advice. Supported by U.S. Public Health Service (USPHS) grant GM30702 and American Cancer Society grant DB-5B (to B.J.M.) and USPHS grant T32 GM07127 (to J.D.L.).