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[SANET-MG] Cry34Ab1 and Cry35Ab1 binary toxin genes



March 12, 2005
Prof. Joe Cummins
“Bacillus thuringiensis Cry34Ab1 and Cry35Ab1 binary toxin genes  to
control maize root worm”
    This year Dow AgroSciences Company  (Pioneer Seeds and Mycogen
Seeds) began the process leading to regulatory approval of maize
genetically modified (GM) with a novel binary toxin to control maize
rootworm. The maize plants have been modified with a pair of Bacillus
thuringiensis (Bt) toxin  genes (Cry 34Ab1 and Cry 35Ab1) that act
together to form channels on the membrane cells of rootworm larvae  that
promote water rushing into cells causing the cells to burst. All of the
previously employed Bt toxin genes and proteins have acted alone to
create pores in the larval gut cells but the novel binary toxins proved
most effective against the larvae of Diabrotica beetles one of the most
important pests of maize. In the United States the first major step in
regulatory approval of crops modified with pest control  agents is
consideration by an Environmental Protection Agency (EPA)  Scientific
Advisory Panel (SAP) and by the US Food and Drug Administration (FDA)
Office of Food additive Safety  . Following SAP and FDA  approval  the
United Sates Department of Agriculture (USDA) Animal and Plant Health
Inspection Service (APHIS) considers a petition for deregulation of the
GM crop. Dow  has completed the FDA evaluation  while decisions on EPA
approval is pending as is the decision  on the USDA/APHIS  petition for
deregulation. The final approval for commercial release of the  modified
maize  could  be approved by the end of this year unless there is
significant objection to the release.
    Bacillus thuringiensis Cry34Ab1/35Ab1 Corn Event DAS-59122-7 was
constructed  using a single plasmid to insert  a synthetic approximation
of the Cry 34Ab1 optimized for plant production of the toxin protein.
That gene was driven by a maize ubiquitin promoter and first intron
while transcription was terminated using the  potato protease inhibitor
terminator gene . A synthetic approximation of the  Cry35Ab1 gene was
driven by a root specific wheat peroxidase promoter and transcription
was terminated using the potato protease inhibitor terminator gene.  A
synthetic approximation of the bacterial pat (phosphoinothricin
acetyltransferase) gene was inserted  as a selectable marker and to
confer glufosinate herbicide tolerance. That gene was driven by the CaMV
promoter and transcription was terminated using the CaMV terminator gene
The bacterial plasmid used in the transformation contains antibiotic
resistance genes but those were not present in the  GM maize plants(1).
EPA reported that the   GM maize contained a single insert of the  Cry
34Ab1/Cry35Ab1 , pat gene sequences  and that the insert was stably
inherited (2).
The concentration of Cry34Ab1 was found to be 500 ppm in leaves while
Cry 35Ab1 was 120 ppm in leaves (2),  Cry 34Ab1 is 14kDa in size
Cry35Ab1 is 44kDa in size, for that reason about 13 times more 34 CryAb1
molecules than Cry35Ab1 was made  in maize leaves. Both  Cry 35Ab1 and
Cry 34Ab1 individually formed membrane channels in gut cells of the
rootworm but the two together were required for optimum  channel
formation and cell death. However,  the 44kDa toxin protein must be
shortened in the plant cell  by about  4kDa  (about 30 amino acid
molecules) to a 40kDa protein before it is optimally  active in
producing membrane channels when combined with the Cry34Ab1 toxin. The
optimum ratio  of  14kDa Cry 34Ab1 toxin to 40kDa  Cry35Ab1 is 3:1(3)
for lethal pore formation. About four times more of the 14kDa  toxin is
made than is needed for optimum  lethal pore formation.
 The toxin proteins produced in bacteria were used in the safety
evaluation of   the binary  toxins rather than the actual toxins
produced in GM maize. That risky procedure has been provided for the
corporate manufacturers because isolation and purification of the toxin
produced maize is a costly procedure while bacterial production is rapid
and inexpensive. Rather crude analytical procedures were used to
establish that the toxins produced from the bacterial genes were
equivalent to the toxins produced from the synthetic approximation of
those genes produced in GM maize. The evidence that the bacterial
products were identical was based primarily on gel electrophoresis
studies. The toxins produced from Cry35Ab1 bacterial and synthetic genes
provided equivocal evidence. The bacterial preparations produced a main
band at 44kDa and a minor band the activated 40KDa toxin while the plant
extracts  were either too faint or overloaded so that the equivalence of
bacterial and synthetic gene product was not clearly evident (4). The
toxin products were somewhat similar but that provides cold comfort  for
the millions who may soon consume the GM maize.
The EPA human Health Assessment of   the toxins in  Cry34Ab1/Cry35Ab1
maize focused almost exclusively on  allergenicity  of the toxin
Cry34Ab1. The reviewers believed that an allergen must  resist digestion
with stomach enzymes  and on that basis considered Cry35Ab1 unlikely to
be an allergen because it was rapidly partially degraded by stomach
enzymes. In contrast Cry34Ab1 was not rapidly degraded by stomach
enzymes. Nevertheless the EPA position was that neither toxins were
likely to be allergens (5). The Center for Food Safety  provided
extensive evidence that  the allergenicity of the Cry toxins had not
been adequately  studied to assure that the toxins were not allergenic,
The criticism went beyond the tests reported by EPA  to propose a fuller
test array to assure public safety(6).  It is worthwhile  pointing out
that the tests for allergenicity were not done, for the most part, with
the toxins produced in GM maize but on surrogate toxins produced in
bacteria. The evidence provided in the EPA  SAP review  certainly did
not provide convincing evidence that the toxins produced in  GM maize
were identical to the toxins produced in bacteria which were used in the
tests for allergenicity.
The safety tests on  Cry34Ab1/35Ab1 maize focused ,almost exclusively,
on  the allergenicity of the toxin proteins (and on toxin proteins
produced in bacteria from genes that  were different from the synthetic
genes in GM maize).. The data reviewed  by EPA was comparable to the
data sets used to approve  essentially all of the insect or herbicide
tolerant crops all of which focused on the toxin proteins or enzymes.
The GM  crops all contain  synthetic DNA and RNA which is copied from
the synthetic DNA. Synthetic DNA is employed in GM crops because  the
bacterial genes do not perform well in  plants because of an effect
called “codon bias”. The synthetic genes used to make GM crops have
never faced evolution and natural selection as have  the genes residing
in bacteria or plants (7). The synthetic DNA and RNA copied from it have
been assumed to be inactive as toxins in the cells of higher organisms
in the view of the   EPA SAPs and the FDA reviewers. However, that view
is unscientific  and ignores a large body of studies showing that naked
DNA and RNA alone profoundly effect animals and humans alike. For
example, vertebrate immune systems have learned to recognize bacterial
and viral DNA by way of unmethylated   CpG motifs DNA . Vertebrate  DNA
has low levels of the CpG motif and those motifs are methylated  for the
most part  prevent the genes from signaling a response to an invader
including activation the innate immune system and inflammatory
responses. Gene therapy vectors have been troubled by the inflammatory
responses associated with exposure vector DNA  (8) and that response can
be reduced by eliminating CpG motifs from the DNA (9). Microbial
infection in the human gastrointestinal tract is associated with
arthritis (10). Government regulators have simply ignored the impact of
 synthetic DNA in GM crops on humans and animals. Recently,  small RNA
molecules were  found to effect the immune system in a manner somewhat
similar to the DNA effect.(11).
    In conclusion,  the unique binary toxin  in Cry34Ab1/35Ab1 maize
requires much fuller study before it is released for commercial
application. In the review of that  genetic modification  as in the
previous reviews  the impact of synthetic DNA  and the RNA copied from
it has been ignored as in previous reviews of the commercial GM crops.
The commercial GM crops and the foods and feeds produced from them has
not been labeled  and for that reason deleterious impacts of the GM
crops have  escaped detection. The toxicity of synthetic DNA and the RNA
copied from it requires much fuller studies.
References
1.    US FDA CFSAN/Office of Food Additive Safety  Bacillus
thuringiensis Cry34Ab1/35Ab1 Corn Event DAS-59122-7 Biotechnology
Consultation Note to the File BNF No. 000081 2004
http://www.cfsan.fda.gov/~rdb/bnfm081.html
2.    United States Environment Protection Agency  Review of Product
Characterization and Expression Analysis for registration of Bt Cry
34Ab1/35Ab1 Construct PHP17662 corn 2004
3.    Masson,L,Schwab,G,Mazza,A,Brousseau,R,Potvin,L.and Schwartz,J. A
Novel Bacillus thuringiensis (PS149B1) Containing a Cry34Ab1/Cry35Ab1
Binary Toxin Specific for the Western Corn Rootworm Diabrotica Virgifera
 LeConte Forms Ion Channels in Lipid Membranes  2004 Biochemistry 43,
12349-57
4.    United States Environment Protection Agency  Review of Product
Characterization and Expression Analysis for registration of Bt Cry
34Ab1/35Ab1 Construct PHP17662 corn Appendix 1 (MRID46123906)2004
5.    United States Environment Protection Position Paper: Scientific
Issues Associated with the Human Health Assessment of the Cry34Ab1
Protein 2005
6.    Gurian-Sherman,D. Comments to the Scientific Advisory Panel
Evaluating Cry34Ab1/35Ab1, March 1-2, 2005, Docket No. OPP-2004-0395
2005 Center for Food Safety
7.    Cummins,J. Synthetic genes in food crops  2004 ISIS Press Release
http://www.i-sis.org.uk
8.    Krieg,A. Direct Immunologic Activity of CpG DNA and Implications
for Gene Therapy 1999 J.Gene Med. 1,56-63
9.    Yew,N,Zhao,H,Wu,I,Song,A,Tousignant,J,Przybylska,M. and Cheng,S.
Reduced Inflammatory Response to Plasmid DNA Vectors by Elimination and
Inhibition of Immunostimulatory CpG Motifs 2000 Molecular Therapy  1,255-63
10.    Zeuner,R,Ishii,K,Lizak,M,Gursel,I,Yamada,H,Klinman,D.and
Verthely,D. Reduction of CpG-Induced Arthritis by Suppressive
Oligodeoxynucleotides 2002 Arthritis and Rheumatism 46,2219-24
       11. Hornung V, Guenthner-Biller M, Bourquin C, Ablasser A,
Schlee M,   Uematsu S, Noronha A, Manoharan M, Akira S, de Fougerolles
A, Endres, S and Hartmann, G. . Sequence-specific potent induction of
IFN- by short interfering RNA in plasmacytoid dendritic cells through
TLR7 2005 Nature Medicine 11, 263-70

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