A wide range of activity of Cry proteins against several orders of insects has resulted from a naturally occurring recombination and sequence diversity.125 Generally, Cry proteins have a defined spectrum of insecticidal activity within a particular insect order.
Cry proteins are composed of several functional domains that have highly conserved areas between the classes.126 For example, Cry1A proteins are highly conserved in domains I, II, and III. Sequence identity can indicate similarity in biological function, i.e., activity toward a similar spectrum of insects. These functional domains have been shown to determine the specificity of Cry proteins: domains I,
II, and III form the toxin portion (tryptic core), and a C-terminal protoxin domain is cleaved upon entry into the insect midgut.126 Domain I is involved in membrane insertion and pore formation and domain II is involved in specific receptor recognition and binding, as shown by mutagenesis studies. Domain III plays a role in receptor binding. The combination of domains I and II has been shown to determine insect specificity. The C-terminal protoxin domain plays a role in crystal formation. Domain swapping is a well-known mechanism for generating diversity. Mutagenesis and domain swapping is widely used in research in order to better understand function of each domain and have been described previously.125,127
The safety assessment of future Cry insecticidal proteins with enhanced insecticidal properties developed through domain swapping or other techniques can be confirmed using existing toxicological study designs. This would include the standard bioinformat-ics, in vitro digestibility, and high-dose rodent acute toxicity test required by the EPA for registration of PIPs. If indicated, confirmation of safety would also be possible through a 90-day rat feeding study with grain or seed containing the insecticidal protein. Other environmental toxicity tests, as outlined in Chapter 4, would also be needed to confirm selectivity toxicity against targeted insect pests and absence of toxicity to nontarget organisms, as exists for conventional Cry proteins. If the mode of action for the insec-ticidal protein is not well characterized, or raises questions about safety for consumers (such as the AFP example discussed earlier), then targeted toxicity tests designed to resolve safety questions may be needed based on a case-by-case assessment.
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