GENETIC EXPRESSION/DEVELOPMENT/CONTROL/MRNA UTILIZATION

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The objective of this research is to gain further understanding of the molecular mechanisms underlying development and differentiation. The fundamental theme will be the elucidation of the means operating in early embryogenesis for regulating the use of genetic information. Sea urchins will be used as a model embryonic system. Templates for protein synthesis present in the unfertilized egg are located primarily in postribosomal ribonucleoprotein complexes called free mRNP or informosomes, and a large portion of the mRNA synthesized by developing embryos enters the free RNP pool. Because of their cellular location it appears that the translation of these templates is controlled in the cytoplasm. We will systematically characterize both qualitatively and quantitatively specific species of mRNA present in the free RNPs in order to better understand the function of free RNPs and how the expression of the genetic information they contain is controlled. Our main approach will be to compare various properties of the free RNP mRNA to the mRNA actively engaged in protein synthesis, which is present in polysomes. A recombinant DNA library of expressed sequences corresponding to the low complexity-high abundance class of sea urchin poly(A) plus mRNA cloned in E.coli will be employed. We will select from this cDNA library a number of interesting cloned sequences for analysis. This selection involves appropriate DNA-RNA hybridization analyses and hybrid-arrest-translation experiments. The mRNAS isolated from embryos at various stages of development, will be characterized in terms of: concentration, cellular location (polysomes/free RNP distribution), time of synthesis in development, rate of synthesis, turnover kinetics (half-life), and efficiency to be translated in vitro. From these data it will be possible to gain considerable information concerning the metabolism and utilization of a number of specific messenger RNAs in a rapidly developing system and thereby the nature of translational control mechanisms operating in early development.