BECKHAM, Y.M.*; ELINSON, R.P.: Localization of mRNAs in Eleutherodactylus coqui oocytes and its implication for oocyte evolution

Eleutherodactylus coqui develops directly on land to a frog. The large 3.5 mm oocyte of E. coqui has enough yolk to allow development without an intermediate feeding tadpole. In the smaller Xenopus laevis oocyte, 1.3 mm in diameter, mRNAs involved in germ layer formation, such as VegT and Vg1, are localized to the vegetal cortex of the oocyte. We are interested in the localization of the E. coqui homologues of VegT and Vg1. We hypothesize that an animal shift has occurred in the localization of these mRNAs. Through a combination of degenerate RT-PCR and 5′ RACE we have cloned 474 bp of a T-box gene which NCBI blast results indicates as VegT type. Northern blot analysis suggests that there is one EcVegT transcript of approximately 2.5 kb. The cloned fragments of EcVegT show 74% and 86% identity with X. laevis VegT at the nucleotide and amino acid level, respectively. Through a combination of degenerate RT-PCR and 5′ and 3′ RACE we have cloned 1282 bp of EcVg1. Northern blot analysis suggests that EcVg1 is approximately 1.3 kb. The entire ORF of EcVg1 shows 60% nucleotide identity with X. laevis Vg1. Within the TGFb homology region the amino acid identity with X. laevis Vg1 is 72%. Radioactive RT-PCR demonstrated that both EcVegT and EcVg1 were predominately found in the animal third of the oocyte, with 35 times more of these transcripts in the animal third than in the vegetal third in a cDNA prep normalized for the amount of RNA. In situ hybridizations should provide more precise information on the localization of these mRNAs and yield insight into the evolution of the larger oocyte of E. coqui.