The endogenous zebrafish cyclinB1 mRNA is polyadenylated during oocyte maturation and early embryogenesis. (A) Diagram of oligonucleotide/RNaseH treatment for analysis of mRNA poly (A) tails. i.) RNA samples were hybridized to a DNA oligonucleotide complementary to a region of the open reading frame close to the 3′UTR. Half of each sample was also hybridized to oligo/dT. ii.) Treatment with RNaseH cleaved the RNA in the region of the RNA/DNA hybrid. In the sample with oligo/dT RNaseH cleaved the poly (A) tail. iii.) Treated RNA samples were analyzed by high resolution RNA blot hybridization and the sizes of the 3′UTR fragments with and without poly (A) indicates the length of poly (A) tail present. (B) Analysis of the endogenous zebrafish cyclinB1 mRNA 3′UTR following oligonucleotide/RNaseH treatment. Total RNAs from zebrafish oocytes, mature oocytes and two-cell embryos were hybridized with a DNA oligonucleotide complimentary to the zebrafish cyclin B1 mRNA's 3′UTR nucleotides -393 to -372 relative to poly(A) addition site at +1. Half of each sample was also hybridized with oligo dT (lanes 1, 3, 5). All samples were then treated with RNaseH and analyzed by RNA blot hybridization using a radio labeled probe corresponding to the 3′UTR of zebrafish CyclinB1 mRNA (-199 to +1). The positions of RNA markers are indicated.
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