Localization of RNAs in oocytes of Eleutherodactylus coqui, a direct developing frog, differs from Xenopus laevis.
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 a 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 hypothesized that an animal shift has occurred in the localization of the E. coqui Orthologs of VegT and Vg1 due to the large egg size. Through a combination of degenerate reverse transcriptase polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE), we cloned 1634 bp of EcVegT and 1377 bp of EcVg1. Northern blot analysis shows that the lengths of these transcripts are 2.5 kb and 1.3 kb, respectively. This result suggests that we have obtained the complete Vg1 transcript, although this transcript has an extremely short 3' untranslated region compared with X. laevis, 256 bp and 1268 bp, respectively. Zygotic expression of EcVegT closely resembles that of VegT, supporting their orthology. Radioactive RT-PCR and in situ hybridization demonstrated the presence of EcVegT and EcVg1 predominantly near the animal pole of the oocyte. RT-PCR showed that the animal blastomeres, formed from the first horizontal cleavage, inherit half of the EcVegT and EcVg1 transcripts, although they contain only about 1% of the embryo volume. Our results indicate major differences between the molecular organization of the eggs of X. laevis and E. coqui.
Department of Biological Sciences, Duquesne University, 600 Forbes Avenue, Pittsburgh, PA 15282, USA.
Yvonne M
YM
Beckham
Kimberly
K
Nath
Richard P
RP
Elinson
D000818:Animals; D001001:Anura / Q000196:embryology / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D015152:Blotting, Northern; D017931:DNA Primers; D018507:Gene Expression Regulation, Developmental; D006023:Glycoproteins / Q000235:genetics / Q000378:metabolism*; D017403:In Situ Hybridization; D008969:Molecular Sequence Data; D021141:Nucleic Acid Amplification Techniques; D009865:Oocytes / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D020825:T-Box Domain Proteins / Q000235:genetics / Q000378:metabolism*; D016212:Transforming Growth Factor beta; D029867:Xenopus Proteins; D014982:Xenopus laevis; D015053:Zygote / Q000378:metabolism
D017931:DNA Primers; C513103:GDF1 protein, Xenopus; D006023:Glycoproteins; D012333:RNA, Messenger; D020825:T-Box Domain Proteins; D016212:Transforming Growth Factor beta; C103966:VegT protein, Xenopus; D029867:Xenopus Proteins