Quantitation of chloramphenicol acetyl transferase in transgenic tobacco plants by ELISA and correlation with gene copy number

Elie H. Gendloff, Ben Bowen, Wallace G Buchholz

Research output: Contribution to journalArticle

36 Citations (Scopus)

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.

Original languageEnglish (US)
Pages (from-to)575-583
Number of pages9
JournalPlant Molecular Biology
Volume14
Issue number4
DOIs
StatePublished - Apr 1 1990

Fingerprint

chloramphenicol acetyltransferase
gene dosage
Gene Dosage
Tobacco
Genetically Modified Plants
Chloramphenicol
Transferases
tobacco
Genes
Enzyme-Linked Immunosorbent Assay
enzyme-linked immunosorbent assay
genetically modified organisms
genes
Caulimovirus
position effect (genetics)
progeny testing
Electrophoretic mobility
Cauliflower mosaic virus
enzyme immunoassays

Keywords

  • ELISA
  • chloramphenicol acetyl transferase
  • gene dosage
  • position effects
  • transgenic plants

ASJC Scopus subject areas

  • Agronomy and Crop Science
  • Genetics
  • Plant Science

Cite this

Quantitation of chloramphenicol acetyl transferase in transgenic tobacco plants by ELISA and correlation with gene copy number. / Gendloff, Elie H.; Bowen, Ben; Buchholz, Wallace G.

In: Plant Molecular Biology, Vol. 14, No. 4, 01.04.1990, p. 575-583.

Research output: Contribution to journalArticle

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