Distinct single-copy sequence sets in sea urchin nuclear RNAs.

Abstract

The purpose of this study was to determine whether nuclear RNAs (nRNA) of sea urchin embryos and adult tissues contain identical or partially distinct sets of single-copy sequence transcripts. A DNA tracer was prepared consisting mainly of sequences absent from gastrula nRNA; 3.6% of this tracer reacted with adult intestine nRNA but not with gastrula nRNA. The existence of a differentially transcribed DNA fraction was verified by its partial purification and rehybridization to intestine and gastrula nRNAs. About one-third of the genomic single-copy sequence is represented in both nRNAs, or about 2 x 108 nucleotides. The differentially transcribed portion of the single-copy genome identified in this work includes about 3.5 X 107 nucleotides. Recent measurements indicate that the nuclear RNAs (nRNAs) of several sea urchin embryo stages and adult tissues are of approximately equal sequence complexity. About a third of the single-copy DNA sequence is represented in the nRNAs of blastula (1), gastrula (2, 3), and pluteus (1) embryos and of previtellogenic ovarian oocytes (4) and adult intestine (5). Kleene and Humphreys (1) showed in addition that the single-copy sequence sets of blastula and pluteus nRNAs are largely overlapping. They concluded that with respect to single-copy sequence content, these nRNAs are in fact identical.Complete overlap in the single-copy sequence sets represented in diverse nRNAs would be paradoxical. The implication is that in the sea urchin differentiation need not be accompanied by alteration in the regions of the genome transcribed into nRNAs of typical prevalence. There are several reasons to question the conclusion that sea urchin nRNAs are identical. Scheller et al. (6) found recently that gastrula and adult intestine nRNAs differ strikingly in the representation of a number of repetitive sequence families. In the genome, these repetitive sequences are in general interspersed with single-copy sequence [ref. 7, and unpublished MATERIALS AND METHODS Preparation of Nuclear RNAs. Gastrula. Strongylocentrotus purpuratus were spawned, the eggs were fertilized, and the embryos were grown for 36 hr to the gastrula stage. nRNA was isolated as described earlier (6).Intestine. The intestine was removed from adult sea urchins, rinsed in seawater/EGTA [ethylene glycol-bis(,B-aminoethyl ether)-N,N,N',N'-tetraacetic acid] and lysed on ice in 50 mM Tris (pH 8.0), 5 mM magnesium chloride, 50 mM ammonium chloride, 5 mM EGTA, and 1% Triton X-100. Nuclei were pelleted through a 0.5 M sucrose cushion and the RNA was extracted as described earlier (5).Ovary. Nuclei were isolated from ovaries that contained previtellogenic oocytes and a high vitellogenic oocyte content, and the RNA was extracted by the methods of .Preparation of Labeled Single-Copy DNA. Single-copy DNA was renatured to 10-20 times the COtl/2 (moles of nucleotide liter-l sec) to form hyperpolymers (7,8). The DNA was labeled in vitro by the "gap translation" method with Escherichia coli polymerase 1 (8, 9). The single-copy tracer ...

Organ-specific nuclear RNAs in tobacco.

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