Summary
A partially purified algal protein mixture which supports in vitro DNA replication consists of soluble proteins and proteins extracted from thylakoid membrane. The membrane extract is essential for the specific initiation of replication at a displacement loop (D-loop) site previously mapped by electron microscopy. D-loop site and its flanking sequences have been cloned and sequenced. In this study, fragment-retention assays using various subclones of the sequenced region indicate that some proteins in the membrane extract bind strongly and specifically with a 494 bp restriction fragment which partially overlaps the D-loop site. Protein gel analyses of the protein-DNA complex identify three DNA-binding polypeptides with apparent molecular weights of 18, 24 and 26 kDa, respectively. Treatment with chloramphenicol, an inhibitor of chloroplast protein synthesis, for 1 h has no obvious effect on the contents of the 24 or 26 kDa polypeptides but significantly reduces the content of the 18 kDa polypeptide in the membrane extract.
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References
Chua N-H, Gillham NW (1977) The sites of synthesis of the principle thylakoid membrane polypeptides in Chlamydomonas reinhardtii. J Cell Biol 74:441–452
Coleman AW (1979) Use of the fluorochrome 4′6-diamidino-2-phenylindole in genetic and developmental studies of chloroplast DNA. J Cell Biol 82:299
Douce R, Joyard J (1984) The regulatory role of the plastid envelope during development. In: Baker NR, Barber J (eds) Chloroplast Biogenesis, Elsevier, Amsterdam, pp 77–132
Gold B, Carrillo N, Tewari KK, Bogorad L (1987) Nucleotide sequence of a preferred maize chloroplast genome template for in vitro DNA synthesis. Proc Natl Acad Sci USA 84:194–198
Kuroiwa T, Kawano S, Nishibayashi S (1982) Epifluorescent microscopic evidence for maternal inheritance of chloroplast DNA. Nature 298:431–433
O'Farrell PH (1975) High resolution two-dimensional electrophoresis of proteins. J Biol Chem 200:4007–4021
Orozco EM Jr, Mullet JE, Chua N-H (1985) An in vitro system for accurate transcription initiation of chloroplast protein genes. Nucleic Acids Res 13:1283–1302
Rawlins DR, Rosenfeld PJ, Wides RJ, Challberg MD, Kelly TJ Jr (1984) Structure and function of the adenovirus origin of replication. Cell 37:309–319
Waddell J, Wang X-M, Wu M (1984) Electron microscopic localization of the chloroplast DNA replicative origins in Chlamydomonas reinhardtii. Nucleic Acids Res 12:3843–3856
Wang X-M, Chang CH, Waddell J, Wu M (1984) Cloning and delimiting one chloroplast DNA replicative origin of Chlamydomonas. Nucleic Acids Res 12:3857–3872
Wu M, Kong XF, Kung SD (1986a) Prokaryotic promoters in the chloroplast DNA replicative origin of Chlamydomonas reinhardtii. Curr Genet 10:819–822
Wu M, Lou JK, Chang DY, Chang CH, Nie ZQ (1986b) Structure and function of a chloroplast DNA replication origin of Chlamydomonas reinhardtii. Proc Natl Acad Sci USA 83:6761–6765
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Communicated by R. Herrmann
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Nie, Z.Q., Chang, D.Y. & Wu, M. Protein-DNA interaction within one cloned chloroplast DNA replication origin of Chlamydomonas . Molec Gen Genet 209, 265–269 (1987). https://doi.org/10.1007/BF00329652
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DOI: https://doi.org/10.1007/BF00329652