Organization of the ribosomal RNA gene cluster in Lytechinus variegatus. Restriction analysis and cloning of restriction fragments.

The DNA coding for ribosomal RNA from Lytechinus variegatus, an Atlantic sea urchin species, is arranged in repeating units. Each unit is cleaved into four fragments by the restriction endonuclease Eco RI. The same gene region is dissected by the enzyme Sal 1 to yield two fragments. The restriction nuclease Kpn I has one restriction site within each repeat unit, thus generating a single fragment of about 11.5 kilobase pairs. The Eco RI and Sal 1 fragments total 11,330 base pairs and 11,360 base pairs, respectively. The size of a repeat unit of sea urchin rDNA is therefore about 11.4 kilobase pairs or 7.5 x lo6 daltons. The four Eco RI rDNA fragments as well as the two Sal 1 fragments were incorporated into plasmid vectors and cloned in Escherichia coli. Using data from hybridization of different rDNA fragments to 18 or 26 S rRNA and from double digests of the cloned fragments with the enzymes Eco RI and Sal I, an arrangement of the fragments within the rDNA repeat unit was found and the rRNA coding regions were assigned. These data were confirmed by electron microscope length measurements of restriction fragments and RNA-DNA hybrids (R-loops). The Kpn I restriction site was mapped by the R-loop method. The spacer region located between transcriptional units was analyzed with the restriction enzyme Barn HI by cloning Barn HI restriction fragments and determining the restriction site within this region. The final arrangement of all investigated restriction fragments and the order of 18 and 26 S rRNA within the gene region is presented.