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Vol. 12, Issue 3, 761-776, March 2001
Department of Cell Biology and Molecular Genetics, University of
Maryland, College Park, Maryland 20742
During spermiogenesis in the water fern, Marsilea
vestita, basal bodies are synthesized de novo in cells that
lack preexisting centrioles, in a particle known as a blepharoplast. We
have focused on basal body assembly in this organism, asking what
components are required for blepharoplast formation. Spermiogenesis is
a rapid process that is activated by placing dry microspores into water. Dry microspores contain large quantities of stored protein and
stored mRNA, and inhibitors reveal that certain proteins are translated
from stored transcripts at specific times during development. Centrin
translation accompanies blepharoplast appearance, while 
-tubulin
translation occurs later, during axonemal formation. In asking whether
centrin is an essential component of the blepharoplast, we used
antisense, sense, and double-stranded RNA probes made from the
Marsilea centrin cDNA, MvCen1, to block centrin
translation. We employed a novel method to introduce these RNAs
directly into the cells. Antisense and sense both arrest spermiogenesis
when blepharoplasts should appear, and dsRNA made from the same cDNA is
an effective inhibitor at concentrations at least 10 times lower than
either of the single-stranded RNA used in these experiments. Blepharoplasts are undetectable and basal bodies fail to form. Antisense, sense, and dsRNA probes made from Marsilea
-tubulin permitted normal development until axonemes form. In
controls, antisense, sense, and dsRNA, made from a segment of HIV, had
no effect on spermiogenesis. Immunoblots suggest that
translational blocks induced by centrin-based RNA are gene specific and
concentration dependent, since neither -tubulin- nor HIV-derived
RNAs affects centrin translation. The disruption of centrin translation
affects microtubule distributions in spermatids, since centrin appears to control formation of the cytoskeleton and motile apparatus. These
results show that centrin plays an essential role in the formation of a
motile apparatus during spermiogenesis of M. vestita.
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