Maize line MON 863 was produced by biolistic transformation of the
inbred line A634 using linearized plasmid PV-ZMIR13 DNA purified
following Mlu I restriction endonuclease digestion. The introduced
DNA contained the modified cry3Bb1 gene from B. thuringiensis
subsp. kumamotoensis. The modified cry3Bb1 gene encodes a protein
of 653 amino acids whose amino acid sequence differs from that of
the wild-type protein by the addition of an alanine residue at
position 2 and by seven amino acid changes.
The introduced DNA also contained a copy of the neomycin
phosphotransferase II (NPTII) encoding gene (nptII) derived from
the Tn5 transposon of Escherichia coli. Due to the use of a unique
restriction site for the excision of nptII from Tn5, this gene
cassette also contains a 153 bp of the 378 bp bleomycin binding
protein gene (ble).This segment of ble is located 20 nucleotides
downstream of the nptII stop codon, and it is joined to the T-nos.
The mRNA that is transcribed from the nptII cassette contains
tandem open reading frames (ORF). The proximal ORF is the complete
nptII coding sequence while the distal ORF encodes approximately
40% of the bleomycin binding sequence. Due to differences in the
mechanism of initiation of translation between procaryotic and
eucaryotic organisms, it is highly unlikely that the partial ble
ORF will be translated into protein in Mon863. This means that
nptII will be expressed in Mon863, but the ble fragment will not.
According to the US FDA, if the partial ble gene were translated
into protein, the truncated peptide would not dimerize because it
lacks the necessary amino acids to dimerize, and also lacks
approximately 50% of the residues that are involved in bleomycin
Molecular analyses of the transformed plant show that one DNA
insert has been transferred to the genome of Mon863. This insert
contains one copy of the Mlu I plasmid fragment used in
transformation. Both cassettes are intact and no DNA from plasmid
backbone was detected.