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Fig. 5. pal-1 mRNA and GLD-1 protein co-fractionate with ribosomes. (A)
RNase protection assays with pal-1 probe and 60 µg of yeast RNA or
RNA from wild-type and glp-4 (bn2ts) L4 larvae and adults.
Densitometry of two replicates indicates that approximately 75% of
pal-1 mRNA from wild-type L4s and 95% of pal-1 mRNA from
adults is associated with the germline. (B) A representative histogram
illustrating the total RNA in 25 0.5 ml fractions taken manually after worm
extracts were fractionated by sucrose density gradient sedimentation. Inset
shows the absorbance profile when 56 0.2 ml fractions were taken from a
gradient run under the same conditions. The fractions containing ribosomes are
indicated in red and bars below the graph indicate the fractions pooled for
RNase protection assays. Both pal-1 and mex-3 mRNA
co-fractionate with ribosomes. Y, control containing only yeast RNA. (C)
Metrizamide equilibrium sedimentation of L4 larvae. Curve denotes the
refractive index of the gradient fractions, while the bars denote the
percentage of total RNA recovered in the pooled fractions. RNase protection
assays on pooled fractions indicate that pal-1 mRNA is found at the
predicted refractive index for C. elegans ribosomes under these
conditions (1.412, indicated by dotted line and red bar). The mex-3
probe shown in the input lane was not included in the RNase protection assays.
(D) Distribution of GLD-1 protein from adult hermaphrodites following
metrizamide sedimentation. The index of refraction is indicated above each
lane. Western analysis with anti-GLD-1 antibody indicates that GLD-1 is
enriched in the predicted ribosome fraction (red).
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