Summary
Hybrid plasmids containing the bacterial resistance-transfer factor pBR322 and the yeast leu2 +gene have been used to isolate DNA fragments of Physarum that are capable of initiating DNA replication in a yeast host. Five of forty hybrid plasmids containing Physarum sequences transform leu2 -yeast to Leu+ at high frequency. The resulting Leu+ transformants are characterized by phenotypic instability. Supercoiled plasmid molecules containing pBR322 sequences can be detected in the transformed yeast, indicating that the transforming DNA replicates autonomously. Plasmid DNA isolated from Leu+ yeast can transform leuB bacteria. The hybrid plasmid recovered from the Leu+ bacterial transformants is identical to the original plasmid, indicating structural integrity is maintained during passage through the yeast host. These hybrid plasmids containing Physarum sequences have the same characteristics as those containing autonomously replicating yeast chromosomal sequences. As the temporal sequence of DNA replication is particularly accessible to study in Physarum plasmodia, the functional significance of these segments should be amenable to study.
Similar content being viewed by others
References
Beach D, Piper M, Shall S (1980) Isolation of chromosomal origins of replication in yeast. Nature 284:185–187
Beggs JD (1978) Transformation of yeast by a replicating hybrid plasmid. Nature 275:104–109
Birnbohm HC, Doly J (1979) A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucl Acids Res 7:1513–1523
Blumenthal AB, Kriegstein HJ, Hogness DS (1973) The units of DNA replication in Drosophila melanogaster chromosomes. Cold Spring Harbor Symp Quant Biol 38:205–223
Bolivar F, Rodriguez RL, Greene PJ, Betlach MC, Heyneker HL, Boyer HW (1977) Construction and characterization of new cloning vehicles: A multipurpose cloning system. Gene 2:95–113
Braum R, Seebeck T (1979) Ribosomal DNA: Extrachromosomal genes of Physarum. In: Specific Eucaryotic Genes. Alfred Benzon Symposium 13. Munksgaard, Copenhagen, pp. 306–320
Braun R, Wili H (1969) Time sequence of DNA replication in Physarum. Biochim Biophys Acta 174:246–252
Broach JR, Hicks JB (1980) Replication and recombination functions associated with the yeast plasmid, 2 μ circle. Cell 21:501–508
Cameron JR, Panasenko SM, Lehman IR, Davis RW (1975) In vitro construction of bacteriophage λ carrying segments of the Escherichia coli chromosome: Selection of hybrids containing the gene for DNA ligase. Proc Natl Acad Sci USA 72:3416–3420
Campbell GR, Littau VC, Melera PW, Allfrey VG, Johnson EM (1979) Unique sequence arrangement of ribosomal genes in the palindromic rDNA molecule of Physarum polycephalum. Nucl Acids Res 6:1433–1447
Chan CSM, Type BK (1980) Autonomously replicating sequences in Sccharomyces cerevisiae. Proc Natl Acad Sci USA 77:6329–6333
Clarke L, Carbon J (1980) Isolation of a yeast centromere and construction of functional small circular chromosomes. Nature 287:504–509
Clewell DB (1972) Nature of Col El plasmid replication in the presence of chloramphenicol. J Bacteriol 110:667–676
Cooke DJ, Dee J (1975) Methods for the isolation and analysis of plasmodial mutants in Physarum polycephalum. Genet Res Camb 24:175–187
Dagert M, Ehrlich SD (1979) Prolonged incubation in calcium chloride improves the competence of Escherichia coli cells. Gene 6:23–28
Daniel JW, Baldwin HH (1964) Methods of culture of plasmodial myxomycetes. In: Prescott DM (ed) Methods in cell physiology, vol 1. Academic Press, New York, pp 9–41
Dugaiczyk A, Boyer HW, Goodman HM (1975) Ligation of EcoRI endonuclease-generated fragments into linear and circular structures. J Mol Biol 96:171–184
Funderud S, Andreassen R, Haugli F (1978a) Size distribution and maturation of newly replicated DNA through the S and G2 phases of Physarum polycephalum. Cell 15:1519–1526
Funderud S, Andreassen R, Haugli F (1978b) DNA replication in Physarum polycephalum: UV photolysis of maturing 5-bromode-oxyuridine substituted DNA. Nucl Acid Res 5:3303–3313
Guttes S, Guttes E (1968) Regulation of DNA replication in the nuclei of the slime mold Physarum polycephalum. J Cell Biol 37:761–772
Hicks JB, Hinnen A, Fink GR (1979) Properties of yeast transformation. Cold Spring Harbor Symp Quant Biol 43:1305–1314
Hinnen JB, Hicks JB, Fink GR (1978) Transformation of yeast. Proc Natl Acad Sci USA 75:1929–1933
Holt CE (1980) The nuclear replication cycle in Physarum polycephalum. In: Dove WF and Rusch HP (eds) Growth and differentiation in Physarum polycephalum. Princeton Univ Press, Princeton pp 9–63
Hsiao C-L, Carbon J (1979) High frequency transformation of yeast by plasmids containing the cloned yeast ARG4 gene. Proc Natl Acad Sci USA 76:3829–3833
Kingsman AJ, Clarke L, Mortimer RK, Carbon J (1979) Replication in Saccharomyces cerevisiae of plasmid pBR313 carrying DNA from the yeast trpl region. Gene 7:141–152
McDonell MW, Simon MN, Studier FW (1977) Analysis of restriction fragments of T7 DNA and determination of molecular weights by electrophoresis in neutral and alkaline gels. J Mol Biol 110:119–146
Miller JH (1972) Experiments in Molecular Genetics. Cold Spring Harbor Laboratory, New York, pp 431–433
Mohberg J, Rusch HP (1971) Isolation and DNA content of nuclei of Physarum polycephalum. Exptl Cell Res 66:305–316
Muldoon, JJ, Evans TE, Nygaard OF, Evans HH (1971) Control of DNA replication by protein synthesis at defined times during the S period in Physarum polycephalum. Biochim Biophys Acta 247:310–321
Nasmyth K, Reed SI (1980) Isolation of genes by complementation in yeast: Molecular cloning of a cell-cycle gene. Proc Natl Acad Sci USA 77:2119–2123
Ratzkin B, Carbon J (1977) Functional expression of cloned yeast DNA in Escherichia coli. Proc Natl Acad Sci USA 74:487–491
Rigby PWJ, Dieckmann M, Rhodes C, Berg P (1977) Labelling deoxyribonucleic acid to high specific activity in vitro by nick translation with DNA polymerase I. J Mol Biol 113:237–251
Southern EM (1975) Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol 98:503–517
Stinchcomb DT, Struhl K, Davis RW (1979) Isolation and characterization of a yeast chromosomal replicator. Nature 28:39–43
Stinchcomb DT, Thomas M, Kelly J, Selker E, Davis R (1980) Eucaryotic DNA segments capable of autonomous replication in yeast. Proc Natl Acad Sci USA 77:4559–4563
Struhl K, Cameron JR, Davis RW (1976) Functional expression of eukaryotic DNA in Escherichia coli. Proc Natl Acad Sci USA 73:1471–1475
Struhl K, Stinchcomb DT, Scherer S, Davis RW (1979) High-frequency transformation of yeast: Autonomous replication of hybrid DNA molecules. Proc Natl Acad Sci USA 76:1035–1039
Vogt VM, Braun R (1977) The replication of ribosomal DNA in Physarum polycephalum. Eur J Biochem 80:557–566
Wahl GM, Stern M, Stark GR (1979) Efficient transfer of large DNA fragments from agarose gels to diazobenzyloxymethyl paper and rapid hybridization by using dextran sulfate. Proc Natl Acad Sci USA 76:3683–3687
Zakian VA, Brewer BJ, Fangman W (1979) Replication of each copy of the yeast 2 micron DNA plasmid occurs during the S phase. Cell 17:923–934
Author information
Authors and Affiliations
Additional information
Communicated by G. Fink
Rights and permissions
About this article
Cite this article
Gorman, J.A., Dove, W.F. & Warren, N. Isolation of physarum DNA segments that support autonomous replication in yeast. Molec. Gen. Genet. 183, 306–313 (1981). https://doi.org/10.1007/BF00270633
Received:
Issue Date:
DOI: https://doi.org/10.1007/BF00270633