CHARACTERIZATION OF PRECURSOR TRANSFER-RNA IN SEA URCHIN EMBRYOS AND A STUDY OF THE KINETICS OF SYNTHESIS AND PROCESSING OF TRANSFER-RNA DURING EARLY EMBRYOGENESIS

DANIEL DAVID LOEWENSTEINER, Purdue University

Abstract

When RNA purified from sea urchin embryos is fractionated by the addition of LiCl to 2 molar, low molecular weight (LMW) RNA is partitioned into both the soluble (S) and insoluble (P) fractions. The S fraction contains most of the LMW RNA. S and P fraction RNA was isolated from embryos which had been incubated with radioactive nucleotide precursors for 30 min to 2 hours. Electrophoresis of this RNA on 15% acrylamide gels containing 7M urea results in most of the radioactivity being found in two peaks; one centered at 4S (peak I), and the other at approximately 4.5S (peak II). Four classes of 4- to 5S RNA are thus defined on the basis of salt solubility and molecular weight, and are designated SI, SII, PI, and PII. Chain length estimates of these RNA classes based on the mobility of E coli tRNA and 55 RNA are as follows: SI, 75 nucleotides; SII, 95 nucleotides; PI, 78 nucleotides, and PII, 100 nucleotides. On the basis of several criteria, SI is comprised of tRNA, and SII and PII appear to contain two classes of precursor tRNA; PI contains 'tRNA-like' molecules and may either represent precursor molecules in the final stages of maturation, or a class of tRNAs distinguishable from the major portion of tRNAs in a number of characteristics. All four classes of RNA are highly methylated, possess tRNA-like base compositions, and contain a spectrum of modified nucleotides characteristic of tRNA. The levels of methylation of RNA in the SII and PII fractions are lower than those in SI and PI, which are similar to each other. All four of these RNA fractions are represented in nuclei, although the fraction of total RNA which is nuclear may differ between the S and P fractions. A comparison of the modified nucleotide content of these RNA fractions isolated from nuclei and whole cells suggest that most of the nucleotide modifications occur within the nucleus. A detailed analysis of the kinetics of synthesis and turnover of these RNA fractions was made for cleavage, blastula, gastrula, and pluteus stage embryos. SII and PII RNAs were unstable and turned over with half lives of about 20- and 10 min, respectively. SI and PI molecules were stable, except in cleavage stage embryos where PI RNA was unstable. The rates of synthesis of all four RNAs was several fold lower in cleavage stage embryos compared to those in blastula stage embryos. The synthesis and processing of S and P fraction RNAs appeared to be regulated independently. While the rate of synthesis of SII RNA and the rate of accumulation of RNA in SI were similar in the three later stage embryos, those of PII and PI RNAs decreased several fold during development. There appeared to be quantitative processing of SII molecules to RNA found in fraction SI, with all of the RNA accumulating in SI derived from processing of SII molecules. This was not the case for the P fraction where there appeared to be a progressively increasing 'overproduction' of PII RNA with respect to the accumulation of PI molecules during development. Several additional kinetic properties of these RNAs are discussed. A comparison of the conformations of the four RNA fractions as probed by hydroxyapatite chromatography and SI nuclease digestion indicated that SII and PII molecules possessed conformations distinct from each other, whereas those of SI and PI molecules were quite similar.

Degree

Ph.D.

Subject Area

Biology

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