Dosage compensation proteins targeted to X chromosomes by a determinant of hermaphrodite fate

Science

Volumn 284, Issue 5421, 1999, Pages 1800-1804

  Dawes, Heather E ^a   Berlin, Dorit S ^a   Lapidus, Denise M ^a   Nusbaum, Chad ^a   Davis, Tamara L ^a   Meyer, Barbara J ^a  

^a UNIVERSITY OF CALIFORNIA   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

GENE PRODUCT; HELMINTH DNA; HELMINTH PROTEIN; DNA FRAGMENT;

ARTICLE; CAENORHABDITIS ELEGANS; CHROMOSOME MAP; CONTROLLED STUDY; GENE DOSAGE; GENE FUNCTION; GENE REPRESSION; GENE SWITCHING; GENE TARGETING; GENETIC ASSOCIATION; HERMAPHRODITISM; MOLECULAR DYNAMICS; NONHUMAN; PRIORITY JOURNAL; PROMOTER REGION; PROTEIN DNA INTERACTION; PROTEIN EXPRESSION; SEX DIFFERENTIATION; SPECIES DIFFERENCE; TRANSCRIPTION REGULATION; X CHROMOSOME; CHROMOSOME; GENE EXPRESSION REGULATION; GENETIC TRANSCRIPTION; MITOSIS; SEX DETERMINATION; SEXUAL DEVELOPMENT;

ANIMALS; CAENORHABDITIS ELEGANS; CAENORHABDITIS ELEGANS PROTEINS; DNA-BINDING PROTEINS; DOSAGE COMPENSATION, GENETIC; FEMALE; GENE EXPRESSION REGULATION, DEVELOPMENTAL; GENES, HELMINTH; HELMINTH PROTEINS; HERMAPHRODITISM; MALE; MOLECULAR SEQUENCE DATA; MUTATION; PROMOTER REGIONS (GENETICS); REPRESSOR PROTEINS; SEX DETERMINATION (GENETICS); TRANSGENES; X CHROMOSOME;

EID: 0033546359     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.284.5421.1800     Document Type: Article

References (32)

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20. We first detected the sdc-2(y74) deletion by Southern hybridization experiments in which genomic DNA from her-1(e1520); sdc-2(y74) XO animals was probed with sequences from pTY81, a 3.1-kb subclone of cosmid HHG9. We then detected the deletion by PCR with primers DML-3 (CTGTGAACACTCGGGAAATTAG) and DB-41 (GAACTCCCGATTCCATGTAATC) on DNA from single Unc male segregants of the strain sdc-2(y74) unc-3(e151)/szT1. We used DNA sequence analysis of PCR products to determine deletion breakpoints.
21. We raised rabbit antibodies to SDC-2 (anti-SDC-2) against a fusion protein composed of the first 455 amino acids of SDC-2 tagged with six tandem histidine residues. The plasmid used to express the antigen (pTY1068) was constructed by subcloning a RT-PCR product corresponding to base pairs (bp) 27 to 1391 of the sdc-2 transcript into the pRSET-A vector (Invitrogen). Fusion protein was expressed and purified as described (16). We used the pTY1068 antigen to affinity purify antibodies as described (16). Preimmune sera showed no immunoreactivity in C. elegans embryos. The affinity-purified antiserum contained cross-reacting contaminants that were removed by preabsorbing anti-SDC-2 antibodies to fixed sdc-2 (y74) mutant embryos. Affinity-purified antibodies were diluted to twice their working concentration and incubated with fixed her-1(hv1y101); xol-1(y9) sdc-2(y74) unc-9(e101) embryos overnight with nutation. Preabsorbed antibodies were separated from y74 embryos and cellular debris by centrifugation (three times for 15 min at 16,000g) and were used the same day. The SDC-2 specificity of the antibodies was demonstrated by the lack of staining in sdc-2(y74) embryos (Fig. 2, G to I). Embryos were collected from gravid hermaphrodites and fixed as described (16), except that the initial (4 °C) fixation was eliminated. Antibody staining of embryos was done as described (4), except that primary antibody incubations were for 1 to 4 hours. Laser confocal microscopy was performed on a Leica TCS-NT confocal microscope. To asses: the effect of dosage compensation mutations on the SDC-2 staining pattern, we used XO animals that had been converted to the XX mode of development by an xol-1 mutation and were rescued by the dpy or sdc mutation being characterized, as described (16). Dosage compensation mutations used in antibody staining experiments are molecular or genetic nulls. We viewed at least 1000 stained embryos of each genotype in multiple experiments.
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32. Supported by U.S. Public Health Service grants GM30702 (B.J.M.) and T32 GM07127 (H.E.D. and D.S.B.). B.J.M. is an investigator of the Howard Hughes Medical Institute. We thank A. Gonzalez-Serrichio and P. Sternberg for pPD49-78 and for the idea of GFP-tagged arrays, D. Lin for initial experiments, M. Kilgard for sequence analysis of cDNA clones, C. Akerib for strains, J. Lieb and I. Carmi for advice about array experiments, A. Chan for assistance with microscopy, J. Berger for protein sequence analysis, and T. Cline and C. Tsai for advice and comments on the manuscript.