161 Biochimica et Biophysica Acta, 349 (1974) 161--172 © Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands BBA 97995 REPLICATION OF THE KINETOPLAST DNA OF LEISHMANIA TARENTOLAE AND CRITHIDIA FASCICULA TA LARRY SIMPSON, AGDA M. SIMPSON and RONALD D. WESLEY* Department of Biology and the Molecular Biology Institute, University of California, Los Angeles, Calif. 90024 (U.S.A.) (Received December 10th, 1973) Summary 1. Replicating kinetoplast DNA networks from both Crithidia fasciculata and Leishmania tarentolae have an equilibrium density in ethidium bromide-- CsC1 which is less than that of covalently closed non-replicating networks. After a few hours of chase, these networks assume the covalently closed posi- tion in the gradient. 2. Pulse-labeled minicircles isolated from sonicated networks band in the upper position in ethidium bromide--CsC1 equilibrium gradients. Both "free" and network minicircles incorporate [3 H] thymidine at the same rate in a pulse. Labeled single stranded fragments of less than unit minicircle length are re- leased from pulse-labeled minicircles in alkali. 3. Intact networks of Leishmania and Crithidia can be isolated in the process of replication: replication involves a doubling of the surface area of the networks as visualized by spreading the kinetoplast DNA on glass slides, stain- ing and examining in the light microscope. 4. DNA replication within the kinetoplast DNA networks of both Leish- mania and Crithidia in all parts of the S phase is restricted to the periphery of the structures. In C. fasciculata the pulse-labeled DNA remains in position as the network enlarges by peripheral growth, and then becomes redistributed throughout the network sheet by an unknown mechanism after one cell genera- tion. 5. In the case of L. tarentolae it was demonstrated by density transfer experiments that all network minicircles replicate by an apparent semi-conser- vative pattern in one cell generation. This implies that there is some type of Abbreviation: SSC, 0.15 M NaCI--O.015 M sodium citrate, pH 7.0. * Present address: Department of Molecular, Cellular & Developmental Biology, University of Colorado, Boulder, Colo. 80302 (U.S.A.)