Interference 103,781
Q. And list - tell me all of those things - all of the
many things that you thought were potential problems as far
as Bt expression in plants in 1985 and 1986.
A. Yes. It occurred to me in 1985 that the Bt gene
was not expressing well in plants because it contained a
sequence which would lead to the RNA being cleaved [sic, at]
some individual site[-]specific recognition site that would
lead to RNA turnover. And it occurred to me that the Bt
gene would not express well in plants because it contained a
polymerase II termination sequence which had not been
described very well in terms of sequences in the literature,
but which I knew existed.
And that by virtue of the Bt gene not being a plant
gene, it inadvertently contained a sequence that polymerase
II would terminate at in eukaryotes. So that was another
possible explanation.
It occurred to me that there is a normal scanning
mechanism used in a plant for recognizing polyadenylation
and proceeding with that post transcriptional RNA
processing, and that there was some defect in the Bt gene as
expressed in plants. The Bt in the bacteria doesn’t
polyadenylate, as far as I know. And so, you know, it’s
evolved into being a very AT-rich sequence.
And when you move that AT-rich sequence, then into a
plant or some other eukaryote, that there is two steps - two
sequences that are recognized. One sequence is recognized
and you get cleavage, and then another sequence is
recognized and you get polyadenylation following the
cleavage. And there is a scanning mechanism for look for -
from the 3 prime end, the strongest sequence you might find,
and then in a plant you might pick from several and
polyadenylate.
So it occurred to me that the Bt gene could be not
being stably expressed because it was either cleaving
incorrectly, and then a kind of weak polyadenylation signal
was being used, and maybe you had to scan down from the
cleavage site, or that all these polyadenylation signals
that were in there might be a problem as well.
It also occurred to me in 1985 that the Bt gene,
because it was AT-rich, simply didn’t translate well in
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