CBH-351 corn was obtained by transforming the backcrossed hybrid corn line (PA91 x H99) x H99, developed by Plant Genetic Systems, with two pUC19 based plasmids, pRVA9909 and pDE110.  These plasmids contain the modified cry9C gene and the bar gene, respectively, both of which have been engineered for expression in plants.  Both plasmids also contain the beta -lactamase (bla) gene derived from E. coli, but this gene is not expressed in CBH-351 corn plant tissues.  These genes and the encoded products are described below. <br>The modified cry9C gene (cry9C.PGS2a) was derived from the bacterium Bacillus thuringiensis subsp. tolworthi BTS02618A. The specific B.t. tolworthi strain used was isolated from grain dust collected in The Philippines.  The gene cry9C.PGS2a encodes a truncated protein which corresponds to the insecticidal, 68.7 kDa toxic fragment produced by initial trypsin-digestion of the Cry9C protoxin protein (previously named CryIH).  The modifications that have been introduced into the Cry9C protein expressed in CBH-351 corn, as compared to the wild type 129.8 kDa Cry9C protoxin include the following: <br>1) a C-terminal truncation that removes all the amino acids (aa) following<br>position 666 of the wild type protoxin just after the conserved sequence block 5<br>shared by certain Lepidoperta-active Bt Cry proteins;<br>2) a N-terminal truncation that removes the first 43 aa at the point corresponding to the N-terminus of the 68.7 kDa fragment of the wild type toxin produced by initial trypsin-digestion, and the subsequent N-terminal addition of the amino acids methionine and alanine; <br>3) a replacement of arginine by lysine at position 123 in the plant encoded<br>protein which reduces the susceptibility of the protein to trypsin cleavage to a<br>non-toxic 55 kDa fragment.  <br>These modifications do not appear to affect the insecticidal activity of the Cry9C protein against the primary targeted insect, European corn borer (ECB), Ostrinia nubilalis (Lambert et al., 1996).  The Cry9C toxin is also toxic against other Lepidoptera, including members of the families Pyralidae, Plutellidae, Sphingidae, and Noctuidae.  <br>The bar gene was cloned from the soil bacterium Streptomyces hygroscopicus.  It encodes the enzyme phosphinothricin-N-acetyltransferase (PAT) which inactivates phosphinothricin, the active component in glufosinate-ammonium herbicides. Acetylation of phosphinothricin by PAT results in detoxification of the herbicide. Expression of this enzyme allows for selection of transformed plant cells on selective medium, as well as whole-plant tolerance to glufosinate (DeBlock et al., 1987).<br>The bla gene was present on the plasmid vectors only as a selectable marker for plasmid DNA in the bacterial E. coli hosts.  When expressed in E. coli from a prokaryotic promoter, this gene encodes the enzyme  beta-lactamase which confers resistance to the antibiotic ampicillin (Yanisch-Perron et al., 1985).  <br>Noncoding DNA regulatory sequences were attached to the modified cry9C gene and the bar gene in the plasmids to facilitate their expression in plants.  These sequences include some which were derived from the plant pathogens cauliflower mosaic virus (CaMV) and A. tumefaciens.  Specifically,  the expression of the modified cry9C gene was directed by the 5' constitutive promoter and the 3' region containing the polyadenylation signal both from the 35S transcript of the CaMV, along with the leader sequence of the cab22L gene of petunia.  The expression of the bar gene was also directed by the 35S CaMV promoter along with the 3'  untranslated region from the nopaline synthase (nos) gene from A. tumefaciens which is involved in transcription termination and polyadenylation.  Although most of these regulatory regions were derived from plant pathogens, the regulatory sequences cannot cause plant disease by themselves or with the genes that they are designed to regulate.<br>The plasmids were introduced into the recipient corn via particle bombardment, a technique that results in direct introduction of DNA into the plant genome.  APHIS examined molecular and biochemical analyses which were presented to demonstrate the number of insertion sites, copy number, genetic stability, and expression level of the transgenes in CBH-351 corn.