Participation of deoxyribonucleic acid polymerase alpha in amplification of ribosomal deoxyribonucleic acid in Xenopus laevis.

Abstract

Aphidicolin, a known inhibitor of eucaryotic deoxyribonucleic acid (DNA) polymerase a, efficiently inhibited amplification of ribosomal DNA during oogenesis in Xenopus laevis. DNA polymerase a, but not DNA polymerase y, as isolated from ovaries, was sensitive to aphidicolin. DNA polymerase ,B was not detectable in Xenopus ovary extracts. Therefore, DNA polymerase a plays a major role in ribosomal ribonucleic acid gene amplification.During oogenesis in many different species, a large number of ribosomes are accumulated. These are needed for the extensive protein synthesis in the rapidly proliferating cells of the embryo. In some organisms, this process is facilitated by selective amplification of the genes that code for ribosomal ribonucleic acid (RNA) (9), thus providing additional templates for the massive ribosomal RNA synthesis which occurs during the development of the oocyte.In the South African clawed frog, Xenopus laevis, diploid somatic cells contain some 900 copies of the ribosomal RNA gene (2, 5) clustered together in the chromosomal nucleolus organizer regions (22). This repetitive deoxyribonucleic acid (DNA) contains, per repeat unit, the sequences for one copy each of 28S, 18S, and 5.8S ribosomal RNA and transcribed and nontranscribed spacers (2,5,7,17,26). During the early meiotic prophase in the oocyte (1, 6), the ribosomal RNA genes are amplified more than 1,000-fold and stored extrachromosomally in the nucleoplasn, forming hundreds of nucleoli (for review see reference 9).The mechanism of ribosomal DNA amplification in Xenopus proceeds by the selective replication of one of the repeat units of the ribosomal RNA genes, which exist in Xenopus as a multigene family whose members differ in the length of the nontranscribed DNA region (30,31). During amplification, a circularized copy of the ribosomal DNA is multiplied by a rollingcircle mechanism (13,23,31), producing multiple identical copies, tandemly arranged in circular and linear molecules (31). To date, it is not known which of the DNA polymerases is involved in ribosomal DNA amplification. We, therefore, investigated the role of DNA polymerase a in this process by using aphidicolin as a specific a polymerase inhibitor (14