Identification and characterization of a complex chromosomal replication origin in Schizosaccharomyces pombe.
In the budding yeast, S. cerevisiae, two-dimensional (2D) gel electrophoresis techniques permit mapping of DNA replication origins to short stretches of DNA (+/- 300 bp). In contrast, in mammalian cells and Drosophila, 2D gel techniques do not permit precise origin localization; the results have been interpreted to suggest that replication initiates in broad zones (several kbp or more). However, alternative techniques (replication timing, nascent strand polarity analysis, nascent strand size analysis) suggest that mammalian origins can be mapped to short DNA stretches, just like S. cerevisiae origins. Because the fission yeast, Schizosaccharomyces pombe, resembles higher organisms in several ways to a greater extent than does S. cerevisiae, we thought that S. pombe replication origins might prove to resemble--and thus be helpful models for--animal cell origins. An attempt to test this possibility using 2D gel techniques resulted in identification of a replication origin near the ura4 gene on chromosome III of S. pombe. The 2D gel patterns produced by this S. pombe origin indeed resemble the patterns produced by animal cell origins and show that the S. pombe origin cannot be precisely located. The data suggest an initiation zone of 3-5 kbp. Some aspects of the 2D gel patterns detected at the S. pombe origin cannot be explained by the rationale of initiation in broad zones, suggesting that future biochemical and genetic studies of this complex origin are likely to provide information useful in helping to understand the apparent conflict between the 2D gel mapping techniques and other mapping techniques at animal cell origins.
Zhu
J
J
Department of Molecular and Cellular Biology, Roswell Park Cancer Institute Buffalo, NY 14263.
Brun
C
C
Kurooka
H
H
Yanagida
M
M
Huberman
J A
JA
D015825:Chromosomes, Fungal; D015245:Deoxyribonuclease BamHI; D015247:Deoxyribonuclease HindIII; D015252:Deoxyribonucleases, Type II Site-Specific; D015180:Electrophoresis, Gel, Two-Dimensional; D012093:Replicon / Q000235:genetics*; D015183:Restriction Mapping; D012568:Schizosaccharomyces / Q000235:genetics*
D015245:Deoxyribonuclease BamHI; D015247:Deoxyribonuclease HindIII; D015252:Deoxyribonucleases, Type II Site-Specific; C027440:GATATC-specific type II deoxyribonucleases