Fig. 4. K(10) but not grk mRNA is in an immunoprecipitable
complex with Orb protein. Anti-Orb or anti-Dorsal antibody was used for
immunoprecipitation of wild-type ovary extracts. RNA isolated from the
immunoprecipitates (IP) and from the total extracts was reverse transcribed
with an anchored oligo(dT) primer (see Materials and Methods) and the
resulting cDNAs were subjected to PCR amplification using the anchored primer
and gene-specific primers for either K(10), grk or nos. The
amplification products were displayed by electrophoresis and blotting to
nitrocellulose and the filters were then hybridized with gene specific probes.
K(10) sequences can be RT-PCR amplified from the Orb IP sample, as
well as from the total RNA pool, but not from the Dorsal IP sample (middle
panel). In both the Orb IP and the total samples, the K(10) probe
hybridizes to a prominent band and an upward smear. The prominent band
corresponds in size to a PCR amplification product extending from the K(10)
primer in the 3' UTR to the beginning of the poly(A) tail. The smear
arises from hybridization of the anchored oligo dT primer at different sites
in the poly(A) tail. Neither nos PCR products (left panel) nor
grk PCR products (right panel) could be detected in the Orb IP lanes,
but could be amplified from the total RNA pool. As expected, no amplification
products were observed when the RT step was omitted.
