Abstract
The relationship between the process of neural induction and the control of tyrosinase gene expression in the cells that derive from the neural crest of amphibians has been examined at the molecular level. [3H] Tyrosinase cDNA was utilized as a probe to measure the levels of tyrosinase RNA transcripts present in Rana pipiens embryos from the time of fertilization through Stage 25 of cleavage (operculum complete, 240 h) and to correlate these levels with those published for the rate of tyrosinase protein synthesis. R. pipiens [3H]tyrosinase cDNA was synthesized from a purified tyrosinase mRNA template using avian myeloblastosis virus reverse transcriptase and was enriched for full length copies by preparative polyacrylamide gel electrophoresis. This cDNA product was estimated to represent greater than 90% by length of tyrosinase mRNA and hybridized to tyrosinase mRNA to greater than 97% within two orders of magnitude of R0t values. The extent of hybridization of [3H]tyrosinase cDNA to total embryonic RNA throughout early development paralleled the rate of synthesis of tyrosinase protein. Tyrosinase RNA transcripts were first detected at Stage 12-13 (0.0032% of total RNA) and rose to maximal levels by Stage 19 (0.011%). This represents a 50-fold increase from preinduction levels. These results are consistent with a model which predicts that one of the early events in the development of neural crest derivatives is the transcriptionally dependent accumulation of functionally mature tyrosinase mRNA.
Highlights
The relationship between the process of neural in- the induction and accumulation of tyrosinase enzyme by 75
R. pipiens [‘Hltyrosinase cDNA was synthesized from a purified tyrosinasemRNA template using avian myeloblastosisvirus reverse transcriptase and was riched for full lengtchopies by preparative polyacrylamide gel electrophoresis
We describe the useof purified tyrosinase mRNA as a template for the synthesis of full length t[eo3nHq-]utaynrotisfiynathseecDacNcuAmaunldatiothneuosfe of this [3H]cDNAas a probe tyrosinase RNA transcripts during R. pipiens embryogenesis
Summary
Levels by Stage 19 (0.011%T)h.is representsa 60-fold Measurement of the Accumulation of Tyrosinase mRNA increase from preinduction levels. Values for the rate of tyrosinase protein synthesis were taken from the data of Benson and Triplett [8].The rateof tyrosinase synthesis was determined by comparing the amount of radiolabeled proteins present in antityrosinase immunoprecipitates to that intrichloroacetic acid precipitates of embryos pulsed 6 h priorwith a 3H-amino-acid mixture The coincidence of both the timing and amplitude of the tyrosinase RNA and tyrosinase protein inductive responses at theconclusion of gastrulation and through subsequent postinductive phases indicates that the translation of the tyrosinase zymogen is directly coupled to the accumulation of tyrosinase mRNA and that theaccumulation of tyrosinase mRNA is dependent upon active transcription of the tyrosinase gene ratherthanthe result of a processing of primary tyrosinase gene transcripts or an activation of masked tyrosinase mRNA
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