I have made constructs where a coding sequence for a 78 amino acid
protein is fused upstream to the amino terminus of GFP. I deleted the
ATG start codon of GFP in order to reduce the chance of internal
translational initiation. Does anyone know if the GFP open reading
frame can initiate translation using, for example, the GTG codon found
immediately after the first ATG? I want GFP expression to be a
(obviously imperfect) measure of translation of my fusion protein, but
I need to know if GFP might start translation from other than the start
of the upstream protein. To complicate matters, my upstream sequence
(a putative gene) also lacks an ATG, but has only a GTG codon, which
can be used, according to the text books, but less efficiently than
ATG, but is rarely used in eukaryotic genes.
One more question: GFP fluoresces fine without the first methionine
(which I suppose would be cleaved anyway followoing translation), but
does GFP need the valine at position 2 for fluorescence?
Thanks.
David N. Levy
University of Alabama at Birmingham
Birmingham, AL 35294-0007
levy at uab.edu
