Abstract
The nature of replication origins in eukaryotic chromosomes has been examined in some detail only in yeast, Drosophila, and mammalian cells. We have used highly synchronous cultures of plasmodia of the myxomycete Physarum and two-dimensional agarose gel electrophoresis to examine replication of two developmentally controlled, early replicated genes over time in S-phase. A single, discrete origin of replication was found within 4.8 kb of the LAV1-5 gene, which encodes a homolog of profilin. In contrast, the LAV1-2 gene appears to be surrounded by several origins. Two origins were identified within a 15 kb chromosomal domain and appear to be inefficiently used. Replication forks collide at preferred sites within this domain. These terminating structures are long lived, persisting for at least 2 h of the 3 h S-phase. Analysis of restriction fragment length polymorphisms (RFLPs) within the LAV1-2 domain indicates that replication of alleles on different parental chromosomes is a highly coordinated process. Our studies of the these two early replicated, plasmodium-specific genes indicate that both a fixed, narrow origin region and a broader zone containing two closely spaced origins of DNA replication occur in Physarum.
Similar content being viewed by others
References
Benard M (1990) Localisation d'origines de replication chromosomiques dans le genome de Physarum polycephalum. Dissertation. Universite de Paris-Sud, Centre d'Orsay
Benard M, Pierron G (1990) Physical relationship between a gene and its origin of replication in Physarum polycephalum. Exp Cell Res 186:299–305
Benard M, Pierron G (1992) Mapping of a Physarum chromosomal origin of replication tightly linked to a developmentally-regulated profilin gene. Nucleic Acids Res 20:3309–3315
Benbow RM, Zhao J, Larson DD (1992) On the nature of origins of DNA replication in eukaryotes. BioEssays 14:661–670
Binette F, Benard M, Laroche A, Pierron G, Lemieux G, Pallotta D (1990) Cell-specific expression of a profilin gene family. DNA Cell Biol 9:323–334
Brewer BJ (1988) When polymerases collide: replication and the transcriptional organization of the E. coli chromosome. Cell 53:679–686
Brewer BJ, Fangman WL (1987) The localization of replication origins on ARS plasmids in S. cerevisiae. Cell 51:463–471
Brewer BJ, Fangman WL (1988) A replication fork barrier at the 3′ end of yeast ribosomal RNA genes. Cell 55:637–643
Brewer BJ, Sena EP, Fangman WL (1988) Analysis of replication intermediates by two-dimensional agarose gel electrophoresis. Cancer Cells 6:229–234
Burhans WC, Vassilev LT, Caddle MS, Heintz NH, DePamphilis ML (1990) Identification of an origin of bidirectional DNA replication in mammalian chromosomes. Cell 62:955–965
Christensen ME, Moloo J, Swischuk JL, Schelling ME (1986) Characterization of the nucleolar protein, B-36, using monoclonal antibodies. Exp Cell Res 166:77–93
Cunningham DB, Dove WF (1992) Two alleles of a developmentally regulated α-tubulin locus in Physarum polycephalum replicate on different schedules. Mol Cell Biol 13:449–461
Daniel JW, Baldwin HH (1964) Methods of culture for plasmodial Myxomycetes. In: Prescott DM (ed) Methods in cell physiology. Academic Press, New York, pp 9–54
Daniel DC, Johnson EM (1989) Selective initiation of replication at origin sequences of the rDNA molecule of Physarum polycephalum using synchronous plasmodial extracts. Nucleic Acids Res 17: 8343–8362
Delidakis C, Kafatos FC (1989) Amplification enhancers and replication origins in the autosomal chorion gene cluster of Drosophila. EMBO J 8:891–901
Dijkwel PA, Vaughan JP, Hamlin JL (1991) Mapping of replication initiation sites in mammalian genomes by two-dimensional gel analysis: stabilization and enrichment of replication intermediates by isolation on the nuclear matrix. Mol Cell Biol 11:3850–3859
Dijkwel PA, Hamlin JL (1992) Initiation of DNA replication in the dihydrofolate reductase locus is confined to the early S period in CHO cells synchronized with the plant amino acid mimosine. Mol Cell Biol 12:3715–3722
Diller JD (1991) DNA replication chronology and replicon mapping of developmental genes in Physarum polycephalum. Dissertation, Texas A & M University
Fangman WL, Brewer BJ (1991) Activation of replication origins within yeast chromosomes. Annu Rev Cell Biol 7:375–402
Fangman WL, Brewer BJ (1992) A question of time: replication origins of eukaryotic chromosomes. Cell 71:363–366
Feinberg AP, Vogelstein B (1983) A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem 132: 6–13
Flanagan RR (1988) Studies in vitro and in vivo of cell-type specific gene expression in Physarum polycephalum. Masters Thesis, Texas A & M University
Funderud S, Andreassen R, Haugli F (1979) DNA replication in Physarum polycephalum: electron microscopic and autoradiographic analysis of replicating DNA from defined stages of the S-period. Nucleic Acids Res 6:1417–1431
Goldman MA, Holmquist GP, Gray MC, Caston LA, Nag A (1984) Replication timing of genes and middle repetitive sequences. Science 224:686–692
Hatton KS, Dhar V, Brown EH, Iqbal MA, Stuart S, Didamo VT, Schildkraut CL (1988) Replication program of active and inactive multigene families in mammalian cells. Mol Cell Biol 8:2149–2158
Heck MMS, Spradling AC (1990) Multiple replication origins are used during Drosophila chorion gene amplification. J Cell Biol 110:903–914
Huberman JA, Spotila LD, Nawotka KA, El-Assouli SM, Davis LR (1987) The in vivo replication origin of the yeast 2 μm plasmid. Cell 51:473–481
Iqbal MA, Chinsky J, Didamo V, Schildkraut CL (1987) Replication of proto-oncogenes early during the S phase in mammalian cell lines. Nucleic Acids Res 15:87–103
James CD, Leffak M (1986) Polarity of replication through the avian alpha-globin locus. Mol Cell Biol 6: 976–984
Kubbies M, Pierron G (1983) Mitotic cell cycle in Physarum. Unprecedented insights via flow-cytometry. Exp Cell Res 149:57–67
Laroche A, Lemieux G, Pallotta D (1989) The nucleotide sequence of a developmentally regulated cDNA from Physarum polycephalum. Nucleic Acids Res 17:10502
Linskens MHK, Huberman JA (1988) Organization of replication of ribosomal DNA in Saccharomyces cerevisiae. Mol Cell Biol 8:4927–4935
Linskens MHK, Huberman JA (1990) Ambiguities in results obtained with 2D gel replicon mapping techniques. Nucleic Acids Res 18:674–652
Martin-Parras L, Hernandez P, Martinez-Robles ML, Schvartzman JB (1991) Unidirectional replication as visualized by two-dimensional agarose gel electrophoresis. J Mol Biol 220:843–853
Murray MG, Thompson WF (1980) Rapid isolation of high molecular weight plant DNA. Nucleic Acids Res 8:4321–4325
Nader WF, Isenberg G, Sauer HW (1986) Structure of Physarum actin gene locus ardA: a nonpalindromic sequence causes inviability of phage lambda and recA-independent deletions. Gene 48:133–144
Nawotka KA, Huberman JA (1988) Two-dimensional gel electronphoresis method for mapping DNA replicons. Mol Cell Biol 8:1408–1413
Pallotta D, Laroche A, Tessier A, Schinnick T, Lemieux G (1986) Molecular cloning of stage-specific mRNAs from amoebae and plasmodia of Physarum polycephalum. Biochem Cell Biol 64:1294–1302
Pierron G, Durica DS, Sauer HW (1984) Invariant temporal order of replication of the four actin gene loci during the naturally synchronous mitotic cycles of Physarum polycephalum. Proc Natl Acad Sci USA 81:6393–6397
Pierron G, Benard M, Puvion E, Flanagan R, Sauer HW, Pallotta D (1989) Replication timing of 10 developmentally regulated genes in Physarum polycephalum. Nucleic Acids Res 17:553–566
Pierron G, Sauer HW, Toublan B, Jalouzot R (1982) Physical relationship between replicons and transcription units in Physarum polycephalum. Eur J Cell Biol 29:104–113
Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual, 2nd ed Cold Spring Harbor Laboratory Press. Cold Spring Harbor, New York
Smithies O (1982) The control of globin and other eukaryotic genes. J Cell Physiol 1:137–143
Stambrook PJ, Flickinger RA (1970) Changes in chromosomal DNA replication patterns in developing frog embryos. J Exp Zool 174:101–114
Taylor JH (1960) Asynchronous duplication of chromosomes in cultured cells of Chinese hamster. J Biophys Biochem Cytol 7:455–463
Trempe JP, Lindstrom YI, Leffak M (1988) Opposite replication polarities of transcribed and nontranscribed histone H5 genes. Mol Cell Biol 8:1657–1663
Vaughn JP, Dijkwel PA, Hamlin JL (1990) Replication initiates in a broad zone in the amplified CHO dihydrofolate reductase domain. Cell 61:1075–1087
Wilson K (1987) Preparation of genomic DNA from bacteria. In: Ausubel FM et al. (eds) Current protocols in molecular biology. Wiley, New York, pp 2.4.1–5
Zannis-Hadjopoulos M, Persico M, Martin RG (1981) The remarkable instability of replication loops provides a general method for the isolation of origins of DNA replication. Cell 27:155–163
Zhu J, Brun C, Kurooka H, Yanagida M, Huberman JA (1992) Identification and characterization of a complex chromosomal replication origin in Schizosaccharomyces pombe. Chromosoma 102:S7-S16
Author information
Authors and Affiliations
Additional information
Communicated by: J. Huberman
Rights and permissions
About this article
Cite this article
Diller, J.D., Sauer, H.W. Two early replicated, developmentally controlled genes of Physarum display different patterns of DNA replication by two-dimensional agarose gel electrophoresis. Chromosoma 102, 563–574 (1993). https://doi.org/10.1007/BF00368349
Received:
Revised:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF00368349