Abstract
A monoclonal antibody to chloramphenicol acetyl transferase (CAT) was used in an indirect competitive enzyme immunoassay (ELISA) for the quantitation of CAT in leaf extracts of eighteen transgenic tobacco plants containing the CAT gene fused to the cauliflower mosaic virus 35S promoter. The ELISA could be used to quantify CAT when present in extracts at 20 ng/ml. Enzymatic activity and electrophoretic mobility of CAT in these extracts was not different from CAT from Escherichia coli. Concentrations of CAT in these transgenic plants ranged from 79 to 732 ng CAT/mg protein. The average coefficient of variation among three replicate samples was 15%. All plants were sampled on two separate occasions. The CAT concentrations often varied between the two sampling dates. We determined the CAT gene copy number and the number of independently segregating loci in each plant by Southern blot analysis and progeny testing. We found no significant differences in CAT expression among all ten plants with a single CAT gene. We also found a significant correlation between CAT gene copy number and the level of CAT expressed in each plant, although plants with one gene copy sometimes had more CAT than plants with more than one gene copy. In this population, therefore, gene copy number contributed more to the variation in CAT expression than did position effects.
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References
An G: Development of plant promoter expression vectors and their use for analysis of differential activity of nopaline synthase promoter in transformed tobacco cells. Plant Physiol 81: 86–91 (1986).
Barker RF, Idler KB, Thompson DV, Kemp JD: Nucleotide sequence of the T-DNA region from the Agrobacterium tumefaciens octopine Ti plasmid pTi15955. Plant Mol Biol 2: 335–350 (1983).
Blake MS, Johnston KH, Russell-Jones GJ, Gotschlich EC: A rapid, sensitive method for detection of alkaline phosphatase-conjugated anti-antibody on western blots. Anal Biochem 136: 175–179 (1984).
Bradford MA: A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein dye-binding. Anal Biochem 72: 248–254 (1976).
Brugge JS, Erikson RL: Identification of a transformation-specific antigen induced by an avian sarcoma virus. Nature 269: 346–348 (1977).
Burns DK, Crowl RM: An immunological assay for chloramphenicol acetyltransferase. Anal Biochem 162: 399–404 (1987).
Clark MF, Lister RM, Bar-Joseph M: ELISA techniques. Meth Enzymol 118: 742–766 (1986).
Dean C, Favreau M, Tamaki S, Bond-Nutter D, Dunsmuir P, Bedbrook J: Expression of tandem gene fusions in transgenic tobacco plants. Nucl Acids Res 15: 7601–7617 (1988).
Dean C, Jones J, Favreau M, Dunsmuir P, Bedbrook J: Influence of flanking sequences on variability in expression levels of an introduced gene in transgenic tobacco plants. Nucl Acids res 16: 9269–9283 (1988).
Deroles SC, Gardner RC: Expression and inheritance of kanamycin resistance in a large number of transgenic petunias generated by Agrobacterium-mediated transformation. Plant Mol Biol 11: 355–364 (1988).
Deroles SC, Gardner RC: Analysis of the T-DNA structure in a large number of transgenic petunias generated by Agrobacterium-mediated transformation. Plant Mol Biol 11: 365–377 (1988).
Gidoni D, Bond-Nutter D, Brosio P, Jones J, Bedbrook J, Dunsmuir P: Co-ordinated expression between two photosynthetic petunia genes in transgenic plants. Mol Gen Genet 11: 507–514 (1988).
Gorman CM, Moffat LF, Howard BH: Recombinant genomes which express chloramphenicol acetyltransferase in mammalian cells. Mol Cell Biol 2: 1044–1051 (1982).
Gorman CM, Lane DP, Rigby PWJ: High efficiency gene transfer into mammalian cells. Phil Trans R Soc Lond B 307: 343–346 (1984).
Grosveld GF, vanAssendelft GB, Greaves DR, Kollias G: Position-independent, high-level expression of the human B-globin gene in transgenic mice. Cell 51: 975–985 (1987).
Hoffman LM, Donaldson DD, Herman EM: A modified storage protein is synthesized, processed, and degraded in the seeds of transgenic plants. Plant Mol Biol 11: 717–729 (1988).
Horsch RB, Fry JE, Hoffmann NL, Wallroth M, Eichholtz DA, Rogers SG, Fraley RT: A simple and general method for transferring genes into plants. Science 227: 1229–1231 (1985).
Jones JDG, Gilbert DE, Grady KL, Jorgensen RA: T-DNA structure and gene expression in petunia plants transformed by Agrobacterium tumefaciens C58 derivatives. Mol Gen Genet 207: 478–485 (1987).
Knight DM, Flomerfelt FA, Ghrayed J: Expression of the art/trs protein of HIV and study of its role in viral envelope synthesis. Science 236: 837–840 (1987).
Laemmli UK: Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680–685 (1970).
Maniatis T, Fritsch EF, Sambrook J: Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1982).
Marsh JL, Erfle M, Wykes EJ: The pIC plasmid and phage vectors with versatile cloning sites for recombinant selection by insertional inactivation. Gene 32: 481–485 (1984).
Murray MG, Kennard WC: Altered chromatin conformation of the higher plant gene phaseolin. Biochemistry 23: 4225–4232 (1984).
Murray MG, Paaren HE: Nucleic acid quantitation by continuous flow fluorometry. Anal Biochem 154: 638–642 (1986).
Muller AJ, Mendel RR, Schiemann J, Simoens C, Inze D: High meiotic stability of a foreign gene introduced into tobacco by Agrobacterium-mediated transformation. Mol Gen Genet 207: 171–175 (1987).
Odell JT, Nagy F, Chua N-H: Identification of DNA sequences required for the activity of the cauliflower mosaic virus 35S promoter. Nature 313: 810–812 (1985).
Odell JT, Nagy F, Chua N-H: Variability in 35S promoter expression between independent transformants. In: Key J, McIntosh L (eds) Plant Gene Systems and their Biology, pp. 289–299. AR Liss, New York (1987).
Ooms G, Hooykaas PJJ, vanVeen RJM, vanMeelen P, Regensburg-Tuink TJG, Schilperoort RA: Octopine Ti-plasmid deletion mutants of Agrobacterium tumefaciens with emphasis on the right side of the T-region. Plasmid 7: 15–29 (1982).
Shaw WV: Chloramphenicol acetyltransferase from chloramphenicol-resistant bacteria. Meth Enzymol 53: 737–755 (1985).
Simon R, Priefer U, Puhler A: Vector plasmids for in vivo and in vitro manipulations of gram-negative bacteria. In: APuhler (ed) Molecular Genetics of the Bacteria-Plant Interaction. pp. 98–106. Springer-Verlag, Berlin/Heidelberg (1983).
Sleigh MJ: A nonchromatographic assay for expression of chloramphenicol acetyltransferase gene in eukaryotic cells. Anal Biochem 156: 251–256 (1986).
Spielmann A, Simpson RB: T-DNA structure in transgenic tobacco with multiple integration sites. Mol Gen Genet 205: 34–41 (1986).
Towben H, Staehelin T, Gordon J: Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: Procedure and some applications. Proc Natl Acad Sci USA 76: 4350–4354 (1979).
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Gendloff, E.H., Bowen, B. & Buchholz, W.G. Quantitation of chloramphenicol acetyl transferase in transgenic tobacco plants by ELISA and correlation with gene copy number. Plant Mol Biol 14, 575–583 (1990). https://doi.org/10.1007/BF00027503
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DOI: https://doi.org/10.1007/BF00027503