Worm Breeder's Gazette 14(1): 27 (October 1, 1995)
These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.
|1||Department of Molecular and Cell Biology, University of California, Berkeley, California 94720|
|2||FHCRC, Seattle, WA 98104|
|3||Haverford College, Haverford, PA 19041|
C. elegans equalizes X-linked gene expression between XX hermaphrodites and XO males by halving transcript levels from each of the hermaphrodite X chromosomes. Although the mechanism of dosage compensation is not known, molecular analysis of dpy-27 has yielded significant insight. DPY-27 is a member of the evolutionarily conserved SMC family of proteins, which are involved in chromosome condensation and segregation. Moreover, DPY-27 is specifically localized to the X chromosome in XX animals. The similarity of DPY-27 to SMC proteins and its X localization suggest that dosage compensation is achieved through changes in X chromosome structure. DPY-27, however, does not act alone. sdc-1, sdc-2, sdc-3, dpy-21, dpy-26, dpy-27, dpy-28, and dpy-30 are also required for the proper execution of dosage compensation. To further our understanding of dosage compensation we undertook a molecular analysis of dpy-26. Homozygous mutant daughters of dpy-26 heterozygotes are dumpy and produce 4% male progeny. The XX progeny of these homozygous mutants die as embryos or L1s, whereas XO animals develop into normal males. The eight alleles of dpy-26 have indistinguishable phenotypes. XX lethality is caused by the inability of these animals to implement the hermaphrodite mode of dosage compensation, leading to the overexpression of X-linked genes. Our cloning and DNA sequence analysis has revealed that the predicted dpy-26 gene product is an acidic 1263 aa (142.5 kD) protein with no similarity to any known motif, protein, or predicted protein sequence. The DNA change in dpy-26(n199) is a C to T transition that results in a premature termination of translation at codon 525. dpy-26 is trans-spliced to both SL1 and SL2 leaders. Preliminary analysis suggests that dpy-26 is the third gene in a three gene operon. Rescue of dpy-26 XX lethality is achieved with a subclone extending only 625 bp 5¹ of the first codon, indicating that the upstream portion of the operon is not required for rescue. Mouse polyclonal antibodies were raised against two DPY-26 polypeptides (aa 127-738 and aa 739-1263). Staining with either antibody revealed that DPY-26 co-localizes precisely with DPY-27 in older (>50 cells) wild-type XX embryos, indicating that DPY-26 is associated with the X chromosome. DPY-26 colocalizes with DPY-27 in all other embryos studied as well. DPY-26 is not localized to the X chromosome in XO male embryos or young (<50 cell) XX embryos, but is distributed diffusely throughout the nucleus. This diffuse nuclear staining is also observed in animals carrying either an sdc-2 or an sdc-3 mutation, revealing that these genes are required for the proper localization of DPY-26 to X. DPY-26 is mislocalized to the X chromosome of xol-1 XO embryos, which have inappropriately activated the XX mode of dosage compensation. DPY-26 X chromosome localization is unaffected by mutations in sdc-1 or dpy-21. dpy-26 and dpy-28 are necessary for the synthesis or stability of DPY-27, since there is abundant dpy-27 mRNA but no DPY-27 protein in dpy-26 or dpy-28 mutants. We find that DPY-27 and DPY-28 are probably required for the stability of DPY-26, since there is no detectable DPY-26 in either a dpy-27 or a dpy-28 mutant. Northern analysis to determine dpy-26 transcript levels in these mutants is underway. These data support the speculation that DPY-26, DPY-27, and DPY-28 are part of a large dosage compensation protein complex associated specifically with the X chromosome. There is one noteworthy difference between the distribution of DPY-26 and DPY-27. In wild-type XX adults, DPY-27 is excluded from the germline. As predicted by the Him phenotype of dpy-26 mutants, DPY-26 is expressed in the germline. We will continue our study of dpy-26-induced dosage compensation defects and meiotic non-disjunction in search of a molecular explanation of these phenotypes.