To investigate the precise structure of eucaryotic primer RNA made in vivo, short DNA chains isolated from nuclei of Drosophila melanogaster embryos were analyzed. Post-labeling of 5' ends of short DNA chains with polynucleotide kinase and [_Y-32P]ATP revealed that 7% of the DNA fragments were covalently linked with mono-to octaribonucleotide primers at their 5' ends. Octaribonucleotides, the major component (ca. 30%), formed the cap structure in the reaction with vaccinia guanylyltransferase and [o-32P]GTP, indicating that they were the intact primer RNA with tri-(or di-) phosphate termini, and the shorter ribooligomers were degradation intermediates. The intact primers started with purine (A/G ratio, 4:1), and the starting few ribonucleotide residues were rich in A.Primer RNA molecules for discontinuous DNA replication of eucaryotic genomes were extensively studied by using isolated nuclei of papovavirus-infected and noninfected mammalian cells (4,8,18,28,29). Chain length of the primer RNA was approximately decanucleotide, synthesis of the primer was started with pppA or pppG, and nucleotide sequence at the RNA-DNA junction was random (18). Recently, DNA primase activities associated with purified DNA polymerase a of various eucaryotic systems were found, and the size of primer RNA was approximately decanucleotide in most cases (1, 2,9,22,26). Primer RNA molecules in vivo were analyzed by using a human lymphoblastoid cell line (27) and a simian virus 40-infected monkey cell line (6). Their chain length varied from mono-up to approximately nonanucleotide, and it was suggested that the longest primer molecules probably corresponded to the primary products, whereas the shorter molecules were in various stages of excision process.The purposes of this work were to detect intact primer RNA molecules in eucaryotic cells, to determine their exact sizes, and to clarify whether any characteristic nucleotide sequences were present. Rigorous investigations on these points have not been done previously with eucaryotic primer RNA made in vivo. Early embryos of Drosophila inelanogaster are used for the analyses because their rapid rate of nuclear division suggests an abundance of nascent chains (10,17 A residues, though lesser so than the in vitro primer (32), and the 3'-terminal region is rich in A and U residues. These results raise the possibility that primer synthesis starts preferentially on a T-rich region on the template strands. MATERIALS AND METHODSReagents and enzymes. 32P, (carrier-free), G(5')pppA and G(5')pppG, nitrocellulose, nuclease P1, nuclease SW, and vaccinia guanylyltransferase (capping enzyme) have been described previously (32). Pancreatic DNase I, T4 polynucleotide kinase, Escherichia coli alkaline phosphatase, and T4 DNA polymerase have been described previously (15).[_-32PJATP and [a-3 PJGTP were prepared as described previously (32). Dihydroxyboryl Bio-Gel P-60 (boronate gel) was prepared by the method of Okayama et al. (16).Purification and terminal labeling of short DNA fragments. D. melanogaster...