Summary
In vitro synthesized duplex DNA complementary to human foetal globin messenger RNA was integrated into bacterial plasmids and amplified by transformation of Escherichia coli. Recombinants carrying globin DNA were identified by hybridization of foetal globin messenger RNA to bacterial DNA in situ and by liquid hybridization of purified plasmids to specific globin complementary DNA probes. Heteroduplex mapping revealed either a simple insertion loop at the position of the EcoRI site of the parental plasmid or substitution loops due to insertion of globin DNA sequences combined with deletions of the parental plasmid DNA. We provide evidence to suggest that these deletions are the result of a site-specific nicking activity of the EcoRI preparations used in the formation of recombinant plasmids.
Similar content being viewed by others
References
Armstrong, K.A., Hershfield, V., Helinski, D.R.: Gene cloning and containment properties of plasmid ColEI and its derivatives. Science 196, 172–175 (1977)
Clegg, J.B., Naughton, M.A., Weatherall, D.J.: Abnormal human haemoglobins. Separation and characterisation of the α and β chains by chromatography, and the determination of two new variants, Hb Chesapeake and Hb J (Bangkok). J. molec. Biol. 19, 91–108 (1966)
Clegg, J.B., Weatherall, D.J.: Molecular basis of thalassaemia. Brit. med. Bull. 32, 262–269 (1976)
Godet, J., Verdier, G., Nigon, V., Belhani, M., Richard, F., Colonna, P., Mitchell, J., Williamson, R., Tolstoshev, P.: 70-1 from Algeria: Genetic and Molecular characterization. Blood 50, 463–470 (1977)
Higuchi, R., Paddock, G.V., Wall, R., Salser, W.: A general method for cloning eukaryotic structural gene sequences. Proc. nat. Acad. Sci. (Wash.), 73, 3146–3150 (1976)
Humphries, P., Cochet, M., Krust, A., Gerlinger, P., Kourilski, P., Chambon, P.: Molecular cloning of extensive sequences of the in vitro synthesized chicken ovalbumin structural gene. Nucleic Acids Research 4, 2389–2406 (1977)
Humphries, P., Old, R.W., Coggins, L.W., McShane, T., Watson, C., Paul, J.: Recombinant plasmids containing Xenopus laevis globin structural genes derived from complementary DNA. Nucleic Acids Research 5, 905–924 (1978)
Lang, A., Lorkin, P.A.: Genetics of human haemoglobins. Brit. med. J. 32, 239–245 (1976)
McReynolds, L.A., Monahan, J.J., Bendure, D.W., Sario, L.C., Woo, S.L.C., Paddock, G.V., Salser, W., Dorson, J., Moses R.E., O'Malley, B.: The ovalbumin gene: Insertion of ovalbumin gene sequences in chimeric bacterial plasmids. J. biol. Chem. 252, 1840–1843 (1977)
Maniatis, T., Kee S.G., Efstratiadis, A., Kafatos, F.C.: Amplification and characterization of β-globin gene synthesized in vitro. Cell 8, 163–182 (1976)
Maxam, A.M., Gilbert, W.: A new method for sequencing DNA. Proc. nat. Acad. Sci. (Wash.) 74, 560–564 (1977)
Mitchell, G.J., Williamson, R.: Genes for γ-globin in human adult erythroid DNA. Nucleic Acids Research 4, 3557–3562 (1977)
Ottolenghi, S., Lanyon, W.G., Williamson, R., Weatherall, D.J., Clegg, J.B., Pitcher, C.S.: Human globin gene analysis for a patient with 71-1. Proc. nat. Acad. Sci. (Wash.) 72, 2294–2299 (1975)
Paterson, B.M., Roberts, B.E., Kuff, E.L.: Structural gene identification and mapping by DNA mRNA hybrid-arrested cell-free translation. Proc. nat. Acad. Sci. (Wash.) 74, 4370–4374 (1978)
Paul, J.: Haemoglobin synthesis and cell differentiation. Brit. med. Bull. 32, 277–281 (1976)
Polisky, B., Greene, P., Garfin, D.E., McCarthy, B.J., Goodman, H.M., Boyer, H.W.: Specificity of substrate recognition by the EcoRI restriction endonuclease. Proc. nat. Acad. Sci. (Wash.) 72, 3310–3314 (1975)
Rabbitts, T.H.: Bacterial cloning of plasmids carrying copies of rabbit globin messenger RNA. Nature (Lond.) 260, 221–225 (1976)
Roberts, B.E., Paterson, B.M.: Efficient translation of tobacco mosaic virus RNA and rabbit globin 9S RNA in a cell-free system from commerical wheat germ. Proc. nat. Acad. Sci. (Wash.) 70, 2330–2334 (1973)
Rougeon, F., Kourilsky, P., Mach, B.: Insertion of a Rabbit β-globin gene sequence into on E. coli plasmid. Nucleic Acids Research 2, 2365–2378 (1975)
Schenk, T.E., Rhodes, C., Rigby, P.W.J., Berg, P.: Biochemical method for mapping mutational alterations in DNA with S1 nuclease: the location of deletions and temperature-sensitive mutations in Simion virus 40. Proc. Nat. Acad. Sci. (Wash.) 72, 989–993 (1975)
Wilson, J.T., Wilson, L.B., de Riel, J.K., Villa-Komaroff, L., Efstratiadis, A., Forget, B.G., Weissman, S.M.: Insertion of synthetic copies of human globin genes into bacterial plasmids. Nucleic Acid Research 5, 563–581 (1978)
Wood, W.G.: Haemoglobin synthesis during human fetal development. Brit. med. Bull. 32, 282–287 (1976)
Wood, K.O., Lee, J.: Integration of synthetic globin genes into an E. coli plasmid. Nucleic Acids Research 3, 1961–1971 (1976)
Author information
Authors and Affiliations
Additional information
Communicated by B.A. Bridges
Rights and permissions
About this article
Cite this article
Humphries, P., Coggins, L.W., Old, R.W. et al. Structure of recombinant plasmids containing synthetic human foetal globin gene sequences. Molec. Gen. Genet. 165, 65–71 (1978). https://doi.org/10.1007/BF00270377
Received:
Issue Date:
DOI: https://doi.org/10.1007/BF00270377