Summary
A set of artificial circular plasmids, named plasmoids, was constructed. They are about 1 kb in size and consist of a 178 bp λdv minimal DNA replication origin (ori) which has four direct repeats and the A+T-rich region conferring polarity to the ori fragment, a 775 bp DNA segment that codes for the CAT amino acid sequence and the 99 bp lac promoter (plac). They carry no other functional genes or genetic sites. The constructions involved various combinations of relative orientations of these components. These molecules do not replicate in vivo because they lack genes coding for initiation proteins, but they do replicate in an in vitro system (Fuller et al. 1981), and can be used for studies of interactions between transcription and replication. In these plasmoids, major transcription starts from the strong plac, and some weak unscheduled transcription starts from several other initiation sites. The major RNA synthesis was found to interfere with the unscheduled RNA synthesis, which was occurring on the opposite strand. The most active replication took place when the major RNA synthesis went through the λ origin region in the direction which occurs naturally in the λ genome. Under these conditions, DNA synthesis going against such transcription was less than that going along with the major transcription. When RNA synthesis through the λ origin region was in the opposite direction, DNA synthesis in the same direction was about half of that observed in the above case, whereas that going against transcription was very weak. Based on these observations, this paper discussed the interactions between the two transcription systems and between transcription and DNA synthesis.
Similar content being viewed by others
Abbreviations
- ccc:
-
covalently closed circular
- oc:
-
open circular
- CAT :
-
chloramphenicol acetyl transferase
- bp:
-
base pair
- plac :
-
lactose promoter
References
Alton NK, Vapnek D (1979) Nucleotide sequence analysis of the chloramphenicol resistance transposon Tn9. Nature 282:864–869
Backman K, Ptashne M, Gilbert W (1976) Construction of plasmids carrying the cI gene of bacteriophage λ. Proc Natl Acad Sci USA 73:4174–4178
Bähr W, Stender W, Scheit KH, Jovin TM (1976) Binding of rifampicin to Escherichia coli RNA polymerase: Thermodynamic and kinetic studies. In: Losick R, Chamberlin M (eds) RNA polymerase. Cold Spring Harbor Laboratory, New York, pp 369–396
Bolivar F, Rodriguez RL, Greene PJ, Betlach MC, Heyneker HL, Boyer HW, Grosa JH, Falkow S (1977) Construction and characterization of new cloning vehicles II. A multipurpose cloning system. Gene 2:95–113
Butler ET, Chamberlin MJ (1982) Bacteriophage SP6-specific RNA polymerase. J Biol Chem 257:5772–5778
Chang ACY, Cohen SN (1978) Construction and characterization of amplifiable multicopy DNA cloning vehicles derived from the P15A cryptic miniplasmid. J Bacteriol 134:1141–1156
Chen W, Yang H, Zubay G, Gellert M (1982) The effect of supercoiling on promoter function in prokaryotes. In: Rodriguez RL, Chamberlin MJ (eds) Promoters Structure and Function. Praeger Publishers, New York, pp 242–251
Chow LT, Davidson N, Berg D (1974) Electron microscope study of the structure of λdv DNAs. J Mol Biol 86:69–89
Christie GE, Farnham PJ, Platt T (1981) Synthetic sites for transcription terminaton and a functional comparison with tryptophan operon termination sites in vitro. Proc Natl Acad Sci USA 78:4180–4184
Denhardt DT (1966) A membrane-filter technique for the detection of complementary DNA. Biochem Biophys Res Commun 23:641–646
Dickson RC, Abelson J, Barnes WM, Reznikoff WS (1975) Genetic regulation: The lac control region. Science 187:27–35
Farnham PJ, Platt T (1980) A model for transcription termination suggested by studies on the trp attenuator in vitro using base analogs. Cell 20:739–748
Fuller RS, Kaguni JM, Kornberg A (1981) Enzymatic replication of the origin of the Escherichia coli chromosome. Proc Natl Acad Sci USA 78:7370–7374
Furth ME, Wickner SH (1983) Lambda DNA replication. In: Hendrix RW, Roberts JW, Stahl FW, Weisberg RA (eds) Lambda II. Cold Spring Harbor Laboratory, New York, pp 145–173
Hobom G, Grosschedl R, Lusky M, Scherer G, Schwarz E, Kossel H (1979) Functional analysis of the replicator structure of lambdoid bacteriophage DNAs. Cold Spring Harbor Symp Quant Biol 43:165–178
Kadesch TR, Williams RC, Chamberlin MJ (1980) Electron microscopic studies of the binding of Escherichia coli RNA polymerase to DNA. II. Formation of multiple promoter-like complexes at non-promoter sites. J Mol Biol 136:79–93
Kornberg A (1980) Replication mechanisms and operations. In: DNA replication. W.H. Freeman and Company, san Francisco, pp 347–413
Kourilsky P, Bourguignon MF, Gros F (1971) Kinetics of viral transcription after induction of prophage. In: Hershey AD (ed) The bacteriophage lambda. Cold Spring Harbor Laboratory, New York, pp 647–666
Matsubara K (1974) Preparation of plasmid λdv from bacteriophage λ: Role of promoter-operator in the plasmid replicon. J Virol 13:596–602
McKnight SL, Bustin M, Miller Jr, OL (1979) Electron microscopic analysis of chromosome metabolism in the Drosophila melanogaster embryo. Cold Spring Harbor Symp Quant Biol 42:741–754
Melançon P, Burgess RR, Record MT, Jr (1982) Nitrocellulose filter binding studies of the interactions of Escherichia coli RNA polymerase holoenzyme with deoxyribonucleic acid restriction fragments: Evidence for multiple classes of nonpromoter interactions, some of which display promoter-like properties. Biochemistry 21:4318–4331
Melton DA, Krieg PA, Rebagliati MR, Maniatis T, Zinn K, Green MR (1984) Efficient in vitro synthesis of biologically active RNA and RNA hybridization probes from plasmids containing a bacteriophage SP6 promoter. Nucleic Acids Res 12:7035–7056
Müller-Hill B, Crapo L, Gilbert W (1968) Mutants that make more lac repressor. Proc Natl Acad Sci USA 59:1259–1264
Müller-Hill B, Beyreuther K, Gilbert W (1971) Lac repressor from Escherichia coli. Methods Enzymol XXI:483–487
Schmeissner U, Court D, Shimatake H, Rosenberg M (1980) Promoter for the establishment of repressor synthesis in bacteriophage λ. Proc Natl Acad Sci USA 77:3191–3195
Schon E, Evans T, Welsh J, Efstratiadis A (1983) Conformation of promoter DNA: Fine mapping of S1-hypersensitive sites. Cell 35:837–848
Stahl SJ, Chamberlin MJ (1977) An expanded transcriptional map of T7 bacteriophage. Reading of minor T7 promoter sites in vitro by Escherichia coli RNA polymerase. J Mol Biol 112:577–601
Thomas R, Bertani LE (1964) On the control of the replication of temperate bacteriophages superinfecting immune hosts. Virology 24:241–253
Tsurimoto T, Matsubara K (1982) Replication of λdv plasmid in vitro promoted by purified λ O and P proteins. Proc Natl Acad Sci USA 79:7639–7643
Tsurimoto T, Matsubara K (1983) Replication of bacteriophage λ DNA. Cold Spring Harbor Symp Quant Biol XLVII: 681–691
Wang JC (1982) Unwinding at the promoter and the modulation of transcription by DNA supercoiling. In: Rodriguez RL, Chamberlin MJ (eds) Promoters Structure and Function. Praeger Publishers, New York, pp 229–241
Ward DF, Murray NE (1979) Convergent transcription in bacteriophage λ: Interference with gene expression. J Mol Biol 133:249–266
Author information
Authors and Affiliations
Additional information
Communicated by M. Takanami
Rights and permissions
About this article
Cite this article
Kouhara, H., Tsurimoto, T. & Matsubara, K. Direction of transcription affects the replication mode of λ in an in vitro system. Mol Gen Genet 208, 428–435 (1987). https://doi.org/10.1007/BF00328134
Received:
Issue Date:
DOI: https://doi.org/10.1007/BF00328134