Figure 3.
rnp-8 Transcripts and RNP-8L Protein. (A) Two
rnp-8 transcripts are detected in northern blots of mRNAs prepared from adults with wild-type,
rnp-8(
tm2435) homozygous, and
rnp-8(
q784) homozygous genotypes. cDNA probes are shown in Figure 1A.
eft-3 mRNA was the loading control; right, molecular weight markers (kb).(B) Staged expression of
rnp-8 mRNA. RT-PCR analysis of
rnp-8 mRNAs, with primers recognizing both
rnp-8L and
rnp-8S.
eft-3 mRNA was the control. Emb, embryo; L1-L4, first-fourth larval stage.(C) Germline expression of
rnp-8 mRNA. RT-PCR analysis of
rnp-8 mRNAs, with primers that recognize
rnp-8L; a similar experiment was performed with primers for the
rnp-8S transcript with equivalent results. RNA was prepared from wild-type adults, which possess normal germline tissue (+GL), or from
glp-1(
q175) mutant adults, which have essentially no germline (-GL) (Austin and Kimble, 1987).(D-F) Germline distribution of
rnp-8 mRNA in dissected adult hermaphrodite germlines. Arrowhead, distal end. All hybridizations and images were treated identically. (D) Wild-type (wt) germline probed with an antisense strand corresponding to exons 5 and 6 (3' probe; Figure 1A). (E) Wild-type (wt) germline probed with a sense strand of the same fragment as in (D) (
anti-3' probe). (F)
rnp-8(
q784) mutant germline probed with an antisense strand of the same fragment as in (D) (3' probe).(G) Western blot probed with rabbit anti-RNP-8 (top) or anti-actin (bottom). anti-RNP-8 recognizes the RNP-8L protein as a major band at 65 kD. On a longer gel, this single band resolves into several bands, which likely represent posttranslational modifications. anti-RNP-8 cannot detect RNP-8S. Emb, embryos; L1-L4, first-fourth larval stages; A, adult;
q784,
rnp-8(
q784) homozygotes;
tm2435,
rnp-8(
tm2435) homozygotes.
glp-1(
q224ts) hermaphrodites raised at the permissive temperature (15 C) have a nearly normal germline (+GL), but those raised at the restrictive temperature (25 C) have essentially no germline (-GL).(H-M) Immunocytochemistry of extruded adult hermaphrodite germlines. (H and I) (H) Wild-type and (I)
rnp-8(
tm2435) germlines were stained with rabbit anti-RNP-8 (green), and images were obtained on a fluorescence microscope. RNP-8 is abundant in meiotic pachytene germ cells and oocytes. Insets, RNP-8 is cytoplasmic and enriched in granules. RNP-8 is also detected, at a much lower level, in cytoplasmic granules in the distal germline. (J-M) Wild-type germline was double stained with (J) rat anti-RNP-8 (green) and (K) anti-PGL-1 (red), and images were obtained on a confocal microscope. (M) RNP-8 and PGL-1 overlap in all granules (yellow). Photos were taken in the pachytene region.