|
|
|
|
|
|
|
|
|||||
| Home Help Feedback Subscriptions Archive Search Table of Contents | |||||
Journal of Cell Science, Vol 103, Issue 4 907-918, Copyright © 1992 by Company of Biologists
JOURNAL ARTICLES |
JA Sanchez, D Marek and LJ Wangh
Department of Biology, Brandeis University, Waltham, MA 02254.
Injection of the circular plasmid FV1 (derived from type I bovine papilloma virus) into Xenopus eggs before the start of the first cell cycle dramatically increases the efficiency of plasmid replication once eggs are chemically activated. We call this the preloading effect and report kinetic and quantitative characterization of this phenomenon here. The timing and the amount of FV1 synthesis were measured by both BrdUTP density labelling and an optimized method of selective enzymatic digestion of replicated and unreplicated molecules using the three methyladenosine-sensitive isoschizomers, DpnI, MboI and Sau3a. DpnI in 100 mM NaCl proved particularly useful for distinguishing and quantitating unreplicated, once-replicated, and repeatedly replicated molecules accumulated over several cell cycles. Our results reveal that both the amount of DNA replicated and the timing of synthesis during the first S-phase correlate with the length of the preloading period. Longer preloading leads to larger amounts of DNA being replicated sooner. In fact, up to 30-50% of 1 ng injected plasmid can replicate in a semiconservative cell cycle-dependent manner during the first S-phase. But such high levels of synthesis during the first cell cycle appear to limit the egg's ability to rereplicate this material in subsequent cell cycles. The preloading effect does not depend on synthesis of either viral or egg proteins, but does appear to correlate with the extent of plasmid assembly into chromatin before the start of the cell cycle. We postulate that each plasmid molecule must achieve a critical degree of chromatin assembly before it can proceed along the replication pathway. These observations illuminate some of the difficulties inherent in building a vector for gene insertion into Xenopus embryos, but also suggest an experimental strategy toward this aim.
This article has been cited by other articles:
|
|
 |
|
  A.-Y. Xie, V. P. Bermudez, and W. R. Folk Stimulation of DNA Replication from the Polyomavirus Origin by PCAF and GCN5 Acetyltransferases: Acetylation of Large T Antigen Mol. Cell. Biol., November 15, 2002; 22(22): 7907 - 7918. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A.-Y. Xie and W. R. Folk Inhibition of Polyomavirus ori-Dependent DNA Replication by mSin3B J. Virol., October 25, 2002; 76(23): 11809 - 11818. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. Neuber, E. Havari, J. A. Sanchez, R. D. Powers, and L. J. Wangh Efficient Human Sperm Pronucleus Formation and Replication in Xenopus Egg Extracts Biol Reprod, October 1, 1999; 61(4): 912 - 920. [Abstract] [Full Text]
|
|
|
|
 |
|
 J. A. Sanchez, D. R. Wonsey, L. Harris, J. Morales, and L. J. Wangh Efficient Plasmid DNA Replication in Xenopus Egg Extracts Does Not Depend on Prior Chromatin Assembly J. Biol. Chem., December 15, 1995; 270(50): 29676 - 29681. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. M. Gilbert, A. Neilson, H. Miyazawa, M. L. DePamphilis, and W. C. Burhans Mimosine Arrests DNA Synthesis at Replication Forks by Inhibiting Deoxyribonucleotide Metabolism J. Biol. Chem., April 21, 1995; 270(16): 9597 - 9606. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. Wangh, D DeGrace, J. Sanchez, A Gold, Y Yeghiazarians, K Wiedemann, and S Daniels Efficient reactivation of Xenopus erythrocyte nuclei in Xenopus egg extracts J. Cell Sci., January 6, 1995; 108(6): 2187 - 2196. [Abstract] [PDF]
|
|
