Abstract
Prior research suggested that the genes for large (L) and small (S) subunits of ribulose bisphosphate carboxylase/oxygenase (RuBisCO) are amplified in ampicillin-resistant pBR322-transformants of Anacystis nidulans 6301. We now report that chromosomal DNA from either untransformed or transformed A. nidulans cells hybridizes with nick-translated [32P]-pBR322 at moderately high stringency. Moreover, nick-translated [32-P]-pCS75, which is a pUC9 derivative containing a PstI insert with L and S subunit genes (for RuBisCO) from A. nidulans, hybridizes at very high stringency with restriction fragments from chromosomal DNA of untransformed and transformed cells as does the 32P-labeled PstI fragment itself. The hybridization patterns suggest the creation of two EcoRI sites in the transformant chromosome by recombination. In pBR322-transformants the RuBisCO activity is elevated 6- to 12-fold in comparison with that of untransformed cells. In spite of the difference in RuBisCO activity, pBR322-transformants grow in the presence of ampicillin at a similar initial rate to that for wild-type cells. Growth characteristics and RuBisCO content during culture in the presence or absence of ampicillin suggest that pBR322-transformants of A. nidulans 6301 are stable. The data also collectively suggest that a given plasmid in the transformed population replicates via a pathway involving recombination between the plasmid and the chromosome.
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References
Brooks A, Farquar GD (1985) Effect of temperature on the CO2/CO2 specificity of ribulose-1,5-bisphosphate carboxylase/oxygenase and the rate of respiration in the light. Planta 165:397–406
Buzby JS, Porter RD, Stevens SE (1985) Experssion of the Escherichia coli lac z gene on a plasmid vector in a cyanobacterium. Science 230:805–807
Daniell H, Sarojini G, McFadden BA (1986) Transformation of the cyanobacterium Anacystis nidulans 6301 with the Escherichia coli plasmid pBR 322. Proc Natl Acad Sci USA 83:2546–2550
Giles KW, Myers A (1965) An improved diphenylamine method for the estimation of deoxyribonuleic acid. Nature 206:93
Iglesias A, Bensi G, Canosi U, Trautner TA (1981) Plasmid transformation in Bacillus subtilis. Alterations introduced into the recipient-homologous DNA of hybrid plasmids can be corrected in transformation. Mol Gen Genet 184:405–409
Kolowsky KS, Williams JGK, Szalay AA (1984) Length of foreign DNA in chimeric plasmids determines the efficiency of its integration into the chromosome of the cyanobacterium Synechococcus R2. Gene 27:289–299
Kratz W, Myers J (1955) Nutrition and growth of several blue green algae. Am J Bot 42:282–287
Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685
Lopez P, Espinosa M, Stassi DL, Lacks SA (1982) Facilitation of plasmid transfer in Streptococcus pneumoniae by chromosomal homology. J Bacteriol 150:692–701
Maniatis T, Fritsch EF, Sambrook J (eds) (1982) Large-scale isolation of plasmid DNA, pp 86–94, in: Molecular Cloning a Laboratory Manual, Cold Spring Harbor, New York
Mannarelli BM, Lacks SA (1984) Ectopic intergation of chromosomal genes in Streptococcus pneumoniae. J Bacteriol 160:867–873
McFadden BA, Tabita FR, Kuehn GD (1975) Ribulose diphosphate carboxylase from the hydrogen bacteria and Rhodospirillum rubrum. In: Wood WA (ed) Methods in Enzymology, vol 42. Academic Press, New York, pp 461–470
Shinozaki K, Sugiura M (1985) Genes for the large and small subunits of ribulose-1,5-bisphosphate carboxylase/oxygenase constitute a single operon in a cyanobacterium Anacystis nidulans 6301. Mol Gen Genet 200:27–32
Shinozaki K, Sugiura M (1983) The gene for the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase is located close to the gene for the large subunit in the cyanobacterium Anacystis nidulans 6301. Nucl Ac Res 11:6957–6964
Smith GE, Summers MD (1980) The bidirectional transfer of DNA and RNA to nitrocellulose or diazobenzyloxymethyl paper. Anal Biochem 109:123–129
Tabita FR, Small CL (1985) Expression and assembly of active cyanobacterial ribulose-1,5-bisphsphate carboxylase/oxygenase in Escherichia coli containing stoichiometric amounts of large and small subunits. Proc Natl Acad Sci USA 82:6100–6103
Torres-Ruiz J, McFadden BA (1985) Isolation of L8 and L8S8 forms of ribulose bisphosphate carboxylase/oxygenase from Chromatium vinosum. Arch Microbiol 142:55–60
Towbin H, Staehelin T, Gordon J (1979) Electrophoretic transfer of proteins from polyacrylamide to nitrocellulose sheets. Procedure and some applications. Proc Natl Acad Sci USA 76:4350–4354
Vasseghi H, Claverys JP (1983) Amplification of a chimeric plasmid carrying an erythromycin-resistance determinant introduced into the genome of Streptococcus pneumoniae. Gene 21:285–292
Williams JGK, Szalay AA (1983) Stable integration of foreign DNA into the chromosome of the cyanobacterium Synechococcus R2. Gene 24:37–51
Wilamotte AMR, Stam WT (1984) Genetic relationships among cyanobacterial strains originally designated at Anacystis nidulans and some other Synechococcus strains. J Gen Microbiol 130:2737–2740
Wilson CR, Morgan AE (1985) Chromosomal DNA amplification in Bacillus subtilis. J Bacteriol 163:445–453
Young M (1984) Gene amplification in Bacillus subtilis. J Gen Microbiol 130:1613–1621
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Daniell, H., Torres-Ruiz, J.A., Inamdar, A. et al. Amplified expression of ribulose bisphosphate carboxylase/oxygenase in pBR322-transformants of Anacystis nidulans . Arch. Microbiol. 151, 59–64 (1988). https://doi.org/10.1007/BF00444670
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DOI: https://doi.org/10.1007/BF00444670