DPY-26, a link between dosage compensation and meiotic chromosome segregation in the nematode

Science

Volumn 274, Issue 5293, 1996, Pages 1732-1736

  Lieb, Jason D ^a   Capowski, Elizabeth E ^b   Meneely, Philip ^b,c   Meyer, Barbara J ^a  

^a UNIVERSITY OF CALIFORNIA   (United States)

^b Fred Hutchinson Cancer Research Center   (United States)

^c HAVERFORD COLLEGE   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

PROTEIN;

ARTICLE; CAENORHABDITIS ELEGANS; GENE LOCATION; GENE SEGREGATION; GENETIC LINKAGE; GENETIC STABILITY; NEMATODE; PRIORITY JOURNAL; SEX DETERMINATION;

ANIMALS; CAENORHABDITIS ELEGANS; CAENORHABDITIS ELEGANS PROTEINS; CARRIER PROTEINS; CELL NUCLEUS; CHROMOSOMES; DOSAGE COMPENSATION, GENETIC; EMBRYONIC DEVELOPMENT; FEMALE; GENE EXPRESSION; GENES, HELMINTH; GERM CELLS; HELMINTH PROTEINS; HERMAPHRODITISM; MALE; MEIOSIS; MOLECULAR SEQUENCE DATA; MUTATION; NUCLEAR PROTEINS; X CHROMOSOME;

CAENORHABDITIS ELEGANS;

EID: 0029807952     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.274.5293.1732     Document Type: Article

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22. The dpy-26 gene had been mapped to linkage group IV, between unc-22 and unc-31 (9). To map dpy-26 more precisely, we used cosmids W03F2 and C03F9, which are flanked by unc-22 and unc-31, as probes to identity polymorphisms between Bristol N2 and Bergerac N62 strains. We mapped dpy-26 relative to these two polymorphisms by analyzing the DNA from Une non-Dpy recombinants picked from the strains dpy-26(n199) unc-31/++N62 and unc-22 dpy-26(n199)/++N62. Of the unc-31 recombinants, 20 had the N62 version of C03F9, and 5 had the N2 version. Of the unc-22 recombinants, 26 had the N62 version of W03F2, and 4 had the N2 version. Thus, dpy-26 mapped 0.18 map units (mu) to the left of C03F9 and 0.05 mu to the right of W03F2. Over-lapping cosmids that form a contiguous link between C03F9 and W03F2 (100 μg/ml) were injected into gonads of upy-26(n199) unc-31(e1B9)/unc-22 heterozygotes with the dominant injection marker rol-6 (100 μg/ml) [C. C. Mello, J. M. Kramer, D. Stinchcomb, V. Ambros, EMBO J. 10, 3959 (1991)]. Lines that stably transmitted the dominant rol-6 marker were isolated, and dpy-26 unc-31 mutant offspring were tested for rescue of lethality. Overlapping cosmids C25G4 (7 of 7 lines) and C01G3 (6 of 6 lines) rescued the XX-specific lethality. The dpy-26 rescuing region was narrowed to an 8.5-kb Sal l fragment of C25G4, pS2 (6 of 6 lines).
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24. A 3.5-kb cDNA clone (pJL2) was obtained from a mixed-stage cDNA library screened with the 8.5-kb pS2 fragment. To obtain the 5' end of the cDNA, we amplified the 5' 375 base pairs (bp) by reverse transcriptase polymerase chain reactions (RT-PCR) using primers specific for the SL1 (5'-TCTAGAATTCC- G GCGGTTTAATTACCCAAGTTTG-3') or SL2 (5'-T TCTAGAATTCCGCGGTTTTAACCCAGTTACTC-3 3') trans-splice leaders and a primer specific for dpy-26, jDL8 (5'-CCACTCGAGAGAGCTGAATCA-AT-3'). The dpy-26 transcript is trans-spliced to both SL1 and SL2 RNA leaders, suggesting that dpy-26 may be a member of an operon [T. Blumenthal, Trends Genet. 11, 132 (1995)]. Thedpy-26 genomic and cDNA clones were sequenced [M. Strathmann et al., Proc. Natl. Acad. Sci. U.S.A. 88, 1247 (1991)] and the dpy-26 cDNA sequence in GenBank (accession number U43562) and genomic sequence (accession number Z70680) were deposited.
25. Cloned Pfu polymerase was used with primers JDL18 (5'-TTCTCCTGCTTTCTATTATCTAA-3') and JDL19 (5'-CTTACTCATCCATCTGCTCAT-S') in a PCR to amplify the first 2200 bp of dpy-26 from single dpy-26(n199) homozygous mutant worms. PCR products from two independent reactions were cloned and sequenced on both strands.
26. Fragments of pJDL2 corresponding to amino acids 127 to 739 and 739 to 1263 of DPY-26 were sub-cloned into the T7 expression vector pRSET A (Invitrogen) fused to an epitope encoding six histidines. Expression of histidine-tagged DPY-26 fragments was induced in Escherichia coli BL21 pLys-S cells with 1 mM isopropyl-β-D-thiogalactoside, and the protein was purified by nickel-chelate chromatography and polyacrylamide gel electrophoresis. Mice received subcutaneous injections of 10 μ9 of protein mixed with Ribi adjuvant (RIBI ImmunoChem Research); rabbits received injections of 100 to 500 μg of protein mixed with Freund's adjuvant. All staining was performed with affinity-purified antibody. No staining was observed in wild-type embryos treated with preimmune sera.
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38. We thank D. Albertson for help with DPY-26 meiotic chromosome localization; P.-T. Chuang for DPY-27 germline experiments; A. Coulson for yeast artificial chromosome and cosmid clones; J. Sedat, W. Cande, and H. W. Bass for help with wide-field microscopy and deconvolution; A. Fire for a cDNA library; and T. Cline, G. Garriga, and P.-T. Chuang for critical comments. Supported by USPHS grant GM30702 and American Cancer Society grant DB-5 (B.J.M.), USPHS grant HD24324 and NSF grant DMB 9105708 (P.M.). and USPHS grant T32 GM07127 (J.D.L).