ABSTRACT
Xenopus laevis larvae with an elevated expression of c-src were generated by mating a transgenic X. laevis male frog carrying proviral Rous sarcoma virus (RSV) long terminal repeat (LTR) and most of the pol gene sequences in its sperm DNA and a normal X. laevis female frog. Offspring (15-20%) with a higher dosage of c-Src, detected in disorganized myotomal musculature and in cerebral and spinal neuronal cells by immunohistochemical analysis, developed abnormally, with edemas (in most cases), head deformities, and eye and axial system defects. In the remaining embryos, a small increase in c-src expression seemed to be compatible with normal embryogenesis. The dosage of c-Src correlated with the dosage of RSV LTR integrated in frog DNA as revealed by Southern and polymerase chain reaction (PCR) analyses. Authenticity of the integrated RSV LTR including enhancer sequence was proved by sequencing. Probing of total RNA from aberrant larvae demonstrated several times higher dosage of c-src mRNA in their tissues than in control tadpoles. We hypothesize that the integrated RSV regulatory sequences can stimulate the expression of c-src proto-oncogene of X. laevis above a threshold that interferes with the early developmental program of frog embryos.
Subject(s)
Proto-Oncogene Proteins pp60(c-src)/biosynthesis , Animals , Animals, Genetically Modified , Avian Sarcoma Viruses/genetics , DNA, Viral , Female , Gene Expression , Genome, Viral , Male , Proto-Oncogene Proteins pp60(c-src)/genetics , Proviruses/genetics , RNA, Messenger , Xenopus laevis/growth & developmentABSTRACT
Mature Xenopus laevis spermatozoa are capable of binding plasmid pAPrC carrying the complete Rous sarcoma virus (RSV) DNA. Each sperm cell associates, on an average, with 70-160 molecules of the plasmid DNA in a DNase resistant form, if the spermatozoa were exposed to the DNA at a concentration of 1.0-1.4 micrograms/10(7) sperm cells. Fertilization with pAPrC-treated spermatozoa induced developmental malformations in 25-30% of embryos. Immunohistochemical analysis of tissue sections from defective animals revealed aberrations in myotomal structures, and increased expression of pp60src protein in myoblasts, neuronal tube, and epidermis. The presence of characteristic v-src and RSV-long terminal repeat (LTR) sequences in X. laevis DNA was detected by PCR analysis. Embryonic RNA hybridized with a src-specific and an RSV-LTR specific probes indicating expression of the viral DNA. Plasmid DNAs without the v-src gene (pATV9) or completely free of any RSV sequences (pBR322) did not induce any changes in embryonic development. Our results provide evidence that the pBR322-cloned DNA form of the RSV genome associates with frog sperm cells in a DNase-resistant manner suggesting internalization and may be subsequently carried into eggs during the process of artificial fertilization. Correlation between the defective morphogenesis of X. laevis and increased expression of the src gene as well as an interference of RSV DNA with the developmental programs of frog embryos are discussed.
