Effect of point mutations in translation initiation context on the expression of recombinant human alpha(1)-proteinase inhibitor in transgenic tomato plants.
The functional and biological significance of translation initiation context sequence in determining high-level expression of modified synthetic human alpha(1)-proteinase inhibitor (alpha(1)-PI) gene was documented in stable transgenic tomato plants. Context sequence of initiator ATG codon derived from statistical analysis of databases was identified as taaA(A/C)aATGGCt in highly expressed dicot plant genes. Removal of initiator ATG context sequence reduced the expression of recombinant alpha(1)-PI protein to fourfolds. The mutation of consensus base at +4 position to a pyrimidine either alone or with substitution at -3 position eliminated most of the alpha(1)-PI expression, while mutation at -3 alone resulted in about sevenfold reduction. The presence of steady-state levels of alpha(1)-PI transcript in transgenic plants indicated that the variation in expression is entirely due to the point mutations incorporated in translation initiation context. These results indicated the significance of conserved nucleotide sequence around initiator ATG codon in augmenting post-transcriptional events and high-level expression of heterologous genes in transgenic plants.
Agarwal
Saurabh
S
Plant Transgenic Laboratory, National Botanical Research Institute, P.O. Box 436, Rana Pratap Marg, Lucknow 226001, India.
Jha
Shweta
S
Sanyal
Indraneel
I
Amla
D V
DV
D001483:Base Sequence; D015153:Blotting, Western; D018387:Codon, Initiator; D018506:Gene Expression Regulation, Plant; D017343:Genes, Plant / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D018551:Solanum lycopersicum / Q000235:genetics*; D008969:Molecular Sequence Data; D010442:Peptide Chain Initiation, Translational / Q000235:genetics*; D030821:Plants, Genetically Modified; D017354:Point Mutation / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism*; D000515:alpha 1-Antitrypsin / Q000378:metabolism*
D018387:Codon, Initiator; D012333:RNA, Messenger; D011994:Recombinant Proteins; D000515:alpha 1-Antitrypsin