Synthetic DNA Sequences Having Enhanced Expression in Monocotyledonous Plants and Method for Preparation Thereof

This invention generally relates to genetic engineering and more particularly to methods for enhancing the expression of a DNA sequence in a monocotyledonous plant and/or increasing the frequency of obtaining transgenic monocotyledonous plants which accumulate useful amounts of a transgenic protein.

One of the primary goals of plant genetic research is to provide transgenic plants which express a foreign gene in an amount sufficient to confer the desired phenotype to the plant. Significant advances have been made in pursuit of this goal, but the expression of some foreign genes in transgenic plants remains problematic. It is believed that numerous factors are involved in determining the ultimate level of expression of a foreign gene in a plant, and the level of mRNA produced in the plant cells is believed to be a major factor that limits the amount of a foreign protein that is expressed in a plant.

It has been suggested that the low levels of expression observed for some foreign proteins expressed in monocotyledonous plants (monocots) may be due to low steady state levels of mRNA in the plant as a result of the nature of the coding sequence of the structural gene. This could be the result of a low frequency of full-length RNA synthesis caused by the premature termination of RNA during transcription or due to unexpected mRNA processing during transcription. Alternatively, full-length RNA could be produced, but then processed by splicing or polyA addition in the nucleus in a fashion that creates a nonfunctional mRNA. It is also possible for the mRNA to be properly synthesized in the nucleus, yet not be suitable for sufficient or efficient translation in the plant cytoplasm.

Various nucleotide sequences affect the expression levels of a foreign DNA sequence introduced into a plant. These include the promoter sequence, intron sequences, the structural coding sequence that encodes the desired foreign protein, 3' untranslated sequences, and polyadenylation sites. Because the structural coding region introduced into the plant is often the only "non-plant" or "non-plant related" sequence introduced, it has been suggested that it could be a significant factor affecting the level of expression of the protein. In this regard, investigators have determined that typical plant structural coding sequences preferentially utilize certain codons to encode certain amino acids in a different frequency than the frequency of usage appearing in bacterial or non-plant coding sequences. Thus it has been suggested that the differences between the typical codon usage present in plant coding sequences as compared to the typical codon usage present in the foreign coding sequence is a factor contributing to the low levels of the foreign mRNA and foreign protein produced in transgenic monocot plants. These differences could contribute to the low levels of mRNA or protein of the foreign coding sequence in a transgenic plant by affecting the transcription or translation of the coding sequence or proper mRNA processing. Recently, attempts have been made to alter the structural coding sequence of a desired polypeptide or protein in an effort to enhance its expression in the plant. In particular, investigators have altered the codon usage of foreign coding sequences in an attempt to enhance its expression in a plant. Most notably, the sequence encoding insecticidal crystal proteins of B. thuringiensis (B.t.) has been modified in various ways to enhance its expression in a plant, particularly monocotyledonous plants, to produce commercially viable insect-tolerant plants.

In the European Patent Application No. 0359472 of Adang et al., a synthetic B.t. toxin gene was suggested which utilized codons preferred in highly expressed monocotyledonous or dicotyledonous proteins. In the Adang et al. gene design, the resulting synthetic gene closely resembles a typical plant gene. That is, the native codon usage in the B.t. toxin gene was altered such that the frequency of usage of the individual codons was made to be nearly identical to the frequency of usage of the respective codons in typical plant genes. Thus, the codon usage in a synthetic gene prepared by the Adang et al. design closely resembles the distribution frequency of codon usage found in highly expressed plant genes.

Another approach to altering the codon usage of a B.t. toxin gene to enhance its expression in plants was described in Fischhoff et al., European Patent Application No. 0385962. In Fischhoff et al., a synthetic plant gene was prepared by modifying the coding sequence to remove all ATTTA sequences and certain identified putative polyadenylation signals. Moreover, the gene sequence was preferably scanned to identify regions with greater than four consecutive adenine or thymine nucleotides and if there were more than one of the minor polyadenylation signals identified within ten nucleotides of each other, then the nucleotide sequence of this region was altered to remove these signals while maintaining the original encoded amino acid sequence. The overall G+C content was also adjusted to provide a final sequence having a G+C ratio of about 50%.

PCT Publication No W0 91/16432 of Cornelissen et al. discloses a method of modifying a DNA sequence encoding a B.t. crystal protein toxin wherein the gene was modified by reducing the A+T content by changing the adenine and thymine bases to cytosine and guanine while maintaining a coding sequence for the original protein toxin. The modified gene was expressed in tobacco and potato. No data was provided for maize or any other monocot.

Briefly, a method for modifying a nucleotide sequence for enhanced accumulation of its protein or polypeptide product in a monocotyledonous plant is provided. Surprisingly, it has been found that by reducing the frequency of usage of rare and semi-rare monocotyledonous codons in a foreign gene to be introduced into a monocotyledonous plant by substituting the rare and semi-rare codons with more preferred monocotyledonous codons, the accumulation of the protein in the monocot plant expressing the foreign gene and/or the frequency of obtaining a transformed monocotyledonous plant which accumulates the insecticidal B.t. crystal protein at levels greater than 0.005 wt % of total soluble protein is significantly improved. Thus, the present invention is drawn to a method for modifying a structural coding sequence encoding a polypeptide to enhance accumulation of the polypeptide in a monocotyledonous plant which comprises determining the amino acid sequence of the polypeptide encoded by the structural coding sequence and reducing the frequency of rare and semi-rare monocotyledonous codons in a coding sequence by substituting the rare and semi-rare monocotyledonous codons in the coding sequence with a more-preferred monocotyledonous codon which codes for the same amino acid.

The present invention is further directed to synthetic structural coding sequences produced by the method of this invention where the synthetic coding sequence expresses its protein product in monocotyledonous plants at levels significantly higher than corresponding wild-type coding sequences.

The present invention is also directed to a novel method comprising reducing the frequency of rare and semi-rare monocotyledonous codons in the nucleotide sequence by substituting the rare and semi-rare codons with a more-preferred monocotyledonous codon, reducing the occurrence of polyadenylation signals and intron splice sites in the nucleotide sequence, removing self-complementary sequences in the nucleotide sequence and replacing such sequences with nonself-complementary nucleotides while maintaining a structural gene encoding the polypeptide, and reducing the frequency of occurrence of 5'-CG-3' dinucleotide pairs in the nucleotide sequence, wherein these steps are performed sequentially and have a cumulative effect resulting in a nucleotide sequence containing a preferential utilization of the more-preferred monocotyledonous codons for monocotyledonous plants for a majority of the amino acids present in the polypeptide.

The present invention is also directed to a method which further includes analyzing the coding sequence in successive six nucleotide fragments (six-mers) and altering the sequence based on the frequency of appearance of the six-mers as compared to the frequency of appearance of the rarest 284, 484 and 664 six-mers in monocotyledonous plants. More particularly, the coding sequence to be introduced into a plant is analyzed and altered in a manner that (a) reduces the frequency of appearance of any of the rarest 284 monocotyledonous six-mers to produce a coding sequence with less than about 0.5% of the rarest 284 six-mers, (b) reduces the frequency of appearance of any of the rarest 484 monocotyledonous six-mers to produce a coding sequence with less than about 1.5% of the rarest 484 six-mers, and (c) reduces the frequency of appearance of any of the rarest 664 monocotyledonous six-mers to produce a coding sequence with less than about 3% of the rarest 664 six-mers.

The present invention is further directed to monocotyledonous plants and seeds containing synthetic DNA sequences prepared by the methods of this invention.

Therefore, it is an object of the present invention to provide synthetic DNA sequences that are capable of expressing their respective proteins at relatively higher levels that the corresponding wild-type DNA sequence and methods for the preparation of such sequences. It is a particular object of this invention to provide synthetic DNA sequences that express a crystal protein toxin gene of B.t. at such relatively high levels.

It is also an object of the present invention to provide a method for improving protein accumulation from a foreign gene transformed into a monocotyledonous plant (particularly maize) and/or improving the frequency of obtaining transformed monocotyledonous plants (particularly maize) which accumulate the insecticidal B.t. crystal protein at levels greater than 0.005 wt. % of total soluble protein, by altering the nucleotide sequence in the coding region of the foreign gene by reducing the frequency of codons that are infrequently utilized in monocotyledonous plant genes and substituting frequently utilized monocotyledonous plant codons therefor.

FIG. 1 is a table listing the frequency of abundance of each of the codons for each amino acid for typical monocotyledonous plant genes.

FIG. 2a is a list of the most rare 284 six-mers in typical monocotyledonous plant genes.

FIG. 2b and 2c are a list of the most rare 484 six-mers in typical monocotyledonous plant genes.

FIGS. 2d and 2e are a list of the most rare 664 six-mers in typical monocotyledonous plant genes.

FIGS. 3a, 3b and 3c are the DNA sequence of B.t. var. kurstaki (B.t. k.) CryIA(b) modified in accordance with the teachings of the present invention (SEQ ID NO:1).

FIGS. 4a and b are the DNA sequence of the CryIIB insecticidal protein modified in accordance with the teachings of the present invention (SEQ ID NO:2).

FIGS. 5a b and c are the DNA sequence of a synthetic DNA sequence encoding B.t. var. kurstaki CryIA(b)/CryIA(c) modified in accordance with one method of the prior art (SEQ ID NO:3).

FIG. 6 illustrates the construction of the intact CryIA(b) synthetic gene from subclones and the strategy involved;

FIG. 7 is a plasmid map of pMON19433.

FIG. 8 is a plasmid map of pMON10914.

FIGS. 9a, b and c are the DNA sequence of a B.t. var kurstaki insecticidal protein wherein the front half of the coding sequence is not modified and the back half is modified in accordance with the method of the present invention (SEQ ID NO:105).

FIG. 10 is a graphical representation of the range of expression of a B.t. DNA sequence modified in accordance with the method of the present invention in R0 corn plants as compared to a B.t. DNA sequence prepared by a method of the prior art.

FIG. 11 illustrates the method of construction of the CryIIB DNA sequence modified in accordance with a second embodiment of the present invention.

FIG. 12 is a plasmid map of pMON19470.

FIGS. 13a, b, c, d, e and f are a comparison of the wild-type bacterial B. t. k. CryIA(b) DNA coding sequence (SEQ ID NO:164) with the modified B. t. k. CryIA(b) DNA sequence as shown in FIG. 3 and identified as SEQ ID NO:1.

FIGS. 14a and b are the DNA sequence of the CryIIA synthetic DNA sequence which was used as the starting DNA sequence for the preparation of the CryIIB synthetic DNA according to one method of the present invention (SEQ ID NO:106).

The following definitions are provided for clarity of the terms used in the description of this invention.

"Rare monocotyledonous codons" refers to codons which have an average frequency of abundance in monocotyledonous plant genes of less than 10%. That is, for purposes of the present invention the rare monocotyledonous codons include GTA, AGA, CGG, CGA, AGT, TCA, ATA, TTA and CTA.

"Semi-rare monocotyledonous codons" refers to codons which have an average frequency of abundance in monocotyledonous plant genes of between 10%-20%. That is, for purposes of the present invention the semi-rare monocotyledonous codons include GGG, GGA, GAA, GCA, CGT, TCG, TCT, AAA, ACA, ACT, TGT, TAT, TTG, CTT and CCT.

An "average monocotyledonous codon" refers to codons which have an average frequency of greater than about 20%, but are not a "more-preferred monocotyledonous codon." That is, for purposes of the present invention the average monocotyledonous codons include GGT, GAT, GCT, AAT, ATT, ACG, TTT, CAT, CCG, CCA, GCG and CCC.

"More-preferred monocotyledonous codons" refers to the one or two most abundantly utilized monocotyledonous codons for each individual amino acid appearing in monocotyledonous plant genes as set forth in Table I below.

The determination of which codons are the more preferred monocotyledonous codons is done by compiling a list of mostly single copy monocotyledonous genes, where redundant members of multigene families have been removed. Codon analysis of the resulting sequences identifies the codons used most frequently in these genes. The monocot codon frequencies for each amino acid as determined by such an analysis is shown in FIG. 1 and is consistent with reported codon frequency determinations such as in Table 4 of E. E. Murray, et al. "Codon Usage in Plant Genes" NAR 17:477-498 (1989).

It has been discovered that a nucleotide sequence capable of enhanced expression in monocots can be obtained by reducing the frequency of usage of the rare and semi-rare monocotyledonous codons and preferentially utilizing the more-preferred monocotyledonous codons found in monocot plant genes. Therefore, the present invention provides a method for modifying a DNA sequence encoding a polypeptide to enhance accumulation of the polypeptide when expressed in a monocotyledonous plant. In another aspect, the present invention provides novel synthetic DNA sequences, encoding a polypeptide or protein that is not native to a monocotyledonous plant, that is expressed at greater levels in the plant than the native DNA sequence if expressed in the plant.

The invention will primarily be described with respect to the preparation of synthetic DNA sequences (also referred to as "nucleotide sequences, structural coding sequences or genes") which encode the crystal protein toxin of Bacillus thuringiensis (B.t.), but it should be understood that the method of the present invention is applicable to any DNA coding sequence which encodes a protein which is not natively expressed in a monocotyledonous plant that one desires to have expressed in the monocotyledonous plant.

DNA sequences modified by the method of the present invention are effectively expressed at a greater level in monocotyledonous plants than the corresponding non-modified DNA sequence. In accordance with the present invention, DNA sequences are modified to reduce the abundance of rare and semi-rare monocotyledonous codons in the sequence by substituting them with a more-preferred monocotyledonous codon. If the codon in the native sequence is neither rare, semi-rare nor a more-preferred codon, it is generally not changed. This results in a modified DNA sequence that has a significantly lower abundance of rare and semi-rare monocotyledonous codons and a greater abundance of the more-preferred monocotyledonous codons. In addition, the DNA sequence is further modified to reduce the frequency of CG dinucleotide pairs in the modified sequence. Preferably, the frequency of CG dinucleotides is reduced to a frequency of less than about 8% in the final modified sequence. The DNA sequence is also modified to reduce the occurrence of putative polyadenylation sites, intron splice sites and potential mRNA instability sites. As a result of the modifications, the modified DNA sequence will typically contain an abundance of between about 65%-90% of the more-preferred codons.

In order to construct a modified DNA sequence in accordance with the method of the present invention, the amino acid sequence of the desired protein must be determined and back-translated into all the available codon choices for each amino acid. It should be understood that an existing DNA sequence can be used as the starting material and modified by standard mutagenesis methods which are known to those skilled in the art or a synthetic DNA sequence having the desired codons can be produced by known oligonucleotide synthesis methods. For the purpose of brevity and clarity, the invention will be described in terms of a mutagenesis protocol. The amino acid sequence of the protein can be analyzed using commercially available computer software such as the BACKTRANSLATE.RTM. program of the GCG Sequence Analysis software Package.

Because most coding sequences of proteins of interest are of a substantial length, generally between 200-3500 nucleotides in length, the DNA sequence that encodes the protein is generally too large to facilitate mutagenesis or complete synthesis in one step. Therefore, it will typically be necessary to break the DNA sequence into smaller fragments of between about 300 bp to 1500 bp in length. To do this and to facilitate subsequent reassembly operations, desired restriction sites in the sequence are identified. The restriction site sequences will, therefore, determine the codon usage at those sites.

The sequence of the native DNA sequence is then compared to the frequency of codon usage for monocotyledonous plants as shown in FIG. 1. Those codons present in the native DNA sequence that are identified as being "rare monocotyledonous codons" are changed to the "more preferred monocotyledonous codon" such that the percentage of rare monocotyledonous codons in the modified DNA sequence is greater than about 0.1% and less than about 0.5% of the total codons in the resulting modified DNA sequence. Semi-rare monocotyledonous codons identified in the native DNA sequence are changed to the more-preferred monocotyledonous codon such that the percentage of semi-rare monocotyledonous codons is greater than about 2.5% and less than about 10% of the total codons in the resulting modified DNA sequence and, preferably less than about 5% of the total codons in the resulting modified DNA sequence. Codons identified in the native DNA sequence that are "average monocotyledonous codons" are not changed.

After the rare and semi-rare monocotyledonous codons have been changed to the more preferred monocotyledonous codon as described above, the DNA sequence is further analyzed to determine the frequency of occurrence of the dinucleotide 5'-CG-3'. This CG dinucleotide is a known DNA methylation site and it has been observed that methylated DNA sequences are often poorly expressed or not expressed at all. Therefore, if the codon changes as described above have introduced a significant number of CG dinucleotide pairs into the modified DNA sequence, the frequency of appearance of 5'-CG-3' dinucleotide pairs is reduced such that the modified DNA sequence has less than about 8% CG dinucleotide pairs, and preferably less than about 7.5% CG dinucleotides pairs. It is understood that any changes to the DNA sequence always preserve the amino acid sequence of the native protein.

The C+G composition of the modified DNA sequence is also important to the overall effect of the expression of the modified DNA sequence in a monocotyledonous plant. Preferably, the modified DNA sequence prepared by the method of this invention has a G+C composition greater than about 50%, and preferably greater than about 55%.

The modified DNA sequence is then analyzed for the presence of any destabilizing ATTTA sequences, putative polyadenylation signals or intron splice sites. If any such sequences are present, they are preferably removed. For purposes of the present invention, putative polyadenylation signals include, but are not necessarily limited to, AATAAA, AATAAT, AACCAA, ATATAA, AATCAA, ATACTA, ATAAA, ATGAAA, AAGCAT, ATTAAT, ATACAT, AAAATA, ATTAAA, AATTAA, AATACA and CATAAA. For purposes of the present invention, intron splice sites include, but are not necessarily limited to WGGTAA (5' intron splice site) and TRYAG (3' intron splice site), where W=A or T, R=A or G, and Y=C or T. When any of the ATTTA, putative polyadenylation signals or intron splice sites are changed, they are preferably replaced with one of the more preferred monocotyledonous codons or one of the average monocotyledonous codons. In essence, after the desired codon changes have been made to the native DNA sequence to produce the modified DNA sequence, the modified DNA sequence is analyzed according to the method described in commonly assigned U.S. patent application Ser. No. 07/476,661 filed Feb. 12, 1990, U.S. patent application Ser. No. 07/315,355 filed Feb. 24, 1989, and EPO 385 962 published Sep. 5, 1990. It is to be understood that while all of the putative polyadenylation signals and intron splice sites are preferably removed from the modified DNA sequence, a modified DNA sequence according to the present invention may include one or more of such sequences and still be capable of providing enhanced expression in monocotyledonous plants.

The resulting DNA sequence prepared according to the above description, whether by modifying an existing native DNA sequence by mutagenesis or by the de novo chemical synthesis of a structural gene, is the preferred modified DNA sequence to be introduced into a monocotyledonous plant for enhanced expression and accumulation of the protein product in the plant.

In a further embodiment of the present invention, an additional analysis is performed on the modified DNA sequence to further enhance its likelihood to provide enhanced expression and accumulation of the protein product in monocotyledonous plants. A list of rare monocotyledonous 6 mer nucleotide sequences is compiled from the same list of mostly single copy monocot genes as previously described for the compilation of the frequency of usage of monocotyledonous codons. A 6mer is six consecutive nucleotides in a sequence and proceeds in a successive fashion along the entire DNA sequence. That is, each adjacent 6mer overlaps the previous 6mer's terminal 5 nucleotides. Thus, the total number of six-mers in a DNA sequence is five less than the number of nucleotides in the DNA sequence. The frequency of occurrence of strings of six-mers was calculated from the list of monocotyledonous genes and the most rare 284, 484, and 664 monocotyledonous six-mers identified. The list of these most rare monocotyledonous six-mers is provided in FIG. 2. The modified DNA sequence is then compared to the lists of the most rare 284, 484, and 664 monocotyledonous six-mers and if one of the rare six-mers appears in the modified DNA sequence, it is removed by changing at least one of the nucleotides in the 6mer, but the amino acid sequence remains intact. Preferably, any such 6mer found in the modified DNA sequence is altered to produce a more preferred codon in the location of the 6mer. Preferably, the total number of the rarest 284 monocotyledonous six-mers in the modified DNA sequence will be less than about 1% of the total six-mers possible in the sequence, and more preferably less than about 0.5%, the total number of the rarest 484 monocotyledonous six-mers in the modified DNA sequence will be less than about 2% of the total six-mers possible in the sequence, and more preferably less than about 1.0%, and the total number of the rarest 664 monocotyledonous six-mers in the modified DNA sequence will be less than about 5% of the total six-mers possible in the sequence, and more preferably less than about 2.5%. It has been found that the removal of these 6mer sequences in this manner is beneficial for increased expression of the DNA sequence in monocotyledonous plants.

The method of the present invention has applicability to any DNA sequence that is desired to be introduced into a monocotyledonous plant to provide any desired characteristic in the plant, such as herbicide tolerance, virus tolerance, insect tolerance, drought tolerance, or enhanced or improved phenotypic characteristics such as improved nutritional or processing characteristics. Of particular importance is the provision of insect tolerance to a monocotyledonous plant by the introduction of a novel gene encoding a crystal protein toxin from B.t. into the plant. Especially preferred are the insecticidal proteins of B.t. that are effective against insects of the order Lepidoptera and Coleoptera, such as the crystal protein toxins of B.t. var. kurstaki CryIA(b) and CryIA(c) and the CryIIB protein.

The modified DNA sequences of the present invention are expressed in a plant in an amount sufficient to achieve the desired phenotype in the plant. That is, if the modified DNA sequence is introduced into the monocotyledonous plant to confer herbicide tolerance, it is designed to be expressed in herbicide tolerant amounts. It is understood that the amount of expression of a particular protein in a plant to provide a desired phenotype to the plant may vary depending upon the species of plant, the desired phenotype, environmental factors, and the like and that the particular amount of expression is determined in the particular situation by routine analysis of varying amounts or levels of expression.

A preferred modified DNA sequence for the control of insects, particularly Lepidopteran type insects, is provided as SEQ ID NO:1 and is shown in FIG. 3. A preferred modified DNA sequence expressing an effective B.t. CryIIB protein is provided as SEQ ID NO:2 and is shown in FIG. 4.

As will be described in more detail in the Examples to follow, the preferred modified DNA sequences were constructed by mutagenesis which required the use of numerous oligonucleotides. Generally, the method of Kunkel, T. A., Proc. Natl. Acad. Sci, USA (1985), Vol 82, pp. 488-492, for oligonucleotide mutagenesis of single stranded DNA to introduce the desired sequence changes into the starting DNA sequence was used. The oligonucleotides were designed to introduce the desired codon changes into the starting DNA sequence. The preferred size for the oligonucleotides is around 40-50 bases, but fragments ranging from 17 to 81 bases have been utilized. In most situations, a minimum of 5 to 8 base pairs of homology to the template DNA on both ends of the synthesized fragment are maintained to insure proper hybridization of the primer to the template. Multiple rounds of mutagenesis were sometimes required to introduce all of the desired changes and to correct any unintended sequence changes as commonly occurs in mutagenesis. It is to be understood that extensive sequencing analysis using standard and routine methodology on both the intermediate and final DNA sequences is necessary to assure that the precise DNA sequence as desired is obtained.

The expression of a foreign DNA sequence in a monocotyledonous plant requires proper transcriptional initiation regulatory regions, i.e. a promoter sequence, an intron, and a polyadenylation site region recognized in monocotyledonous plants, all linked in a manner which permits the transcription of the coding sequence and subsequent processing in the nucleus. A DNA sequence containing all of the necessary elements to permit the transcription and ultimate expression of the coding sequence in the monocotyledonous plant is referred to as a "DNA construct." The details of construction of such a DNA construct is well known to those in the art and the preparation of vectors carrying such constructs is also well-known.

Numerous promoters are known or are found to cause transcription of RNA in plant cells and can be used in the DNA construct of the present invention. Examples of suitable promoters include the nopaline synthase (NOS) and octopine synthase (OCS) promoters, the light-inducible promoter from the small subunit of ribulose bis-phosphate carboxylase promoters, the CaMV 35S and 19S promoters, the full-length transcript promoter from Figwort mosaic virus, ubiquitin promoters, actin promoters, histone promoters, tubulin promoters, or the mannopine synthase promoter (MAS). The promoter may also be one that causes preferential expression in a particular tissue, such as leaves, stems, roots, or meristematic tissue, or the promoter may be inducible, such as by light, heat stress, water stress or chemical application or production by the plant. Exemplary green tissue-specific promoters include the maize phosphoenol pyruvate carboxylase (PEPC) promoter, small submit ribulose bis-carboxylase promoters (ssRUBISCO) and the chlorophyll a/b binding protein promoters. The promoter may also be a pith-specific promoter, such as the promoter isolated from a plant TrpA gene as described in International Publication No. W093/07278, published Apr. 15, 1993. Other plant promoters may be obtained preferably from plants or plant viruses and can be utilized so long as it is capable of causing sufficient expression in a monocotyledonous plant to result in the production of an effective amount of the desired protein. Any promoter used in the present invention may be modified, if desired, to alter their control characteristics. For example, the CaMV 35S or 19S promoters may be enhanced by the method described in Kay et al. Science (1987) Vol. 236, pp.1299-1302.

The DNA construct prepared for introduction into the monocotyledonous plant also preferably contains an intron sequence which is functional in monocotyledonous plants, preferably immediately 3' of the promoter region and immediately 5' to the structural coding sequence. It has been observed that the inclusion of such a DNA sequence in the monocotyledonous DNA construct enhances the expression of the protein product. Preferably, the intron derived from the first intron of the maize alcohol dehydrogenase gene (MzADH1) as described in Callis et al. Genes and Devel. (1987) Vol. 1, pp1183-1200, or the maize hsp70 intron (as described in PCT Publication No. WO93/19189) is used. The HSP70 intron can be synthesized using the polymerase chain reaction from a genomic clone containing a maize HSP70 gene (pMON9502) Rochester et al. (1986) Embo J., 5:451-458.

The RNA produced by a DNA construct of the present invention also contains a 3' non-translated polyadenylation site region recognized in monocotyledonous plants. Various suitable 3' non-translated regions are known and can by obtained from vital RNA, suitable eukaryotic genes or from a synthetic gene sequence. Examples of suitable 3' regions are (a) the 3' transcribed, non-translated regions containing the polyadenylation signal of Agrobacterium tumefaciens (Ti) plasmid genes, such as the NOS gene, and (b) plant genes such as the soybean storage protein (7S) genes and the small subunit of the RuBP carboxylase (E9) gene.

The modified DNA sequence of the present invention may be linked to an appropriate amino-terminal chloroplast transit peptide or secretory signal sequence to transport the transcribed sequence to a desired location in the plant cell.

A DNA construct containing a structural coding sequence prepared in accordance with the method of the present invention can be inserted into the genome of a plant by any suitable method. Examples of suitable methods include Agrobacterium tumefaciens mediated transformation, direct gene transfer into protoplasts, microprojectile bombardment, injection into protoplasts, cultured cells and tissues or meristematic tissues, and electroporation. Preferably, the DNA construct is transferred to the monocot plant tissue through the use of the microprojectile bombardment process which is also referred to as "particle gun technology." In this method of transfer, the DNA construct is initially coated onto a suitable microprojectile and the microprojectile containing DNA is accelerated into the target tissue by a microprojectile gun device. The design of the accelerating device is not critical so long as it can produce a sufficient acceleration function. The accelerated microprojectiles impact upon the prepared target tissue to perform the gene transfer. The DNA construct used in a microprojectile bombardment method of DNA transfer is preferably prepared as a plasmid vector coated onto gold or tungsten microprojectiles. In this regard, the DNA construct will be associated with a selectable marker gene which allows transformed cells to grow in the presence of a metabolic inhibitor that slows the growth of non-transformed cells. This growth advantage of the transgenic cells permits them to be distinguished, over time, from the slower growing or non-growing cells. Preferred selectable marker genes for monocotyledonous plants include a mutant acetolactate synthase DNA sequence which confers tolerance to sulfonylurea herbicides such as chlorsulfuron, the NPTII gene which confers resistance to aminoglycosidic antibiotics such as kanamycin or G418, or a bar gene (DeBlock et al., 1987, EMBO J. 6:2513-2518; Thompson et al., 1987, EMBO J. 6:2519-2523) for resistance to phosphinothricin or bialaphos. Alternatively, or in conjunction with a selectable marker, a visual screenable marker such as the E. coli B-glucuronidase gene or a luciferase gene can be included in the DNA construct to facilitate identification and recovery of transformed cells.

Suitable plants for use in the practice of the present invention include the group of plants referred to as the monocotyledonous plants and include, but are not necessarily limited to, maize, rice and wheat.

The following examples are illustrative in nature and are provided to better elucidate the practice of the present invention and are not to be interpreted in a limiting sense. Those skilled in the art will recognize that various modifications, truncations, additions or deletions, etc. can be made to the methods and DNA sequences described herein without departing from the spirit and scope of the present invention.

This example is provided to illustrate the construction of a novel DNA sequence encoding the crystal toxin protein from B. thuringiensis var. kurstaki CryIA(b) according to the method of the present invention that exhibits enhanced accumulation of its protein product when expressed in maize.

As the starting DNA sequence to be modified in accordance with the method of the present invention, the synthetic CryIA(b)/CryIA(c) DNA sequence as described in European Patent Application Publication No. 0385962 was utilized. This DNA sequence encodes a fusion B.t. kurstaki protein with the insect specificity conferred by the amino-terminal CryIA(b) portion. This DNA has been modified to remove any ATTTA sites and any putative polyadenylation sites and intron splice sites. This sequence is identified as SEQ ID NO:3 and is shown in FIG. 5.

The amino acid sequence of this B.t. sequence was known and all of the available codon choices were determined by analyzing the amino acid sequence using the BACKTRANSLATE.RTM. program of the GCG Sequence Analysis Software Program. Because the B.t. gene is rather large (3569 bp in length) the mutagenesis process was conducted on a plurality of individual, smaller fragments of the starting DNA sequence as will be described below. The codon usage of the starting DNA sequence was then compared to the monocotyledonous codon frequency table as shown in FIG. 1 to determine which codons in the starting DNA sequence are rare or semi-rare monocotyledonous codons and are to be replaced with a more-preferred monocotyledonous codon. While keeping in mind the necessary restriction sites to facilitate religation of the DNA sequence after mutagenesis was complete, the modified DNA sequence design was determined. The modified DNA sequence design was then analyzed for any nucleotide strings of ATTTA or putative polyadenylation sites or intron splice sites. The modified DNA sequence design was then further modified to remove substantially all of such nucleotide strings, although one string of TTTTT, TRYAG, ATTTA, and AAGCAT remained in the design. The modified DNA sequence design was then analyzed for the occurrence of the dinucleotide 5'-CG-3' and, when possible, the modified DNA sequence was designed to remove such dinucleotide pairs, although all of such dinucleotide pairs were not removed. The resulting design is the preferred monocotyledonous CryIA(b) DNA sequence design and this sequence was compared to the starting DNA sequence by a sequence alignment program (Bestfit program of the GCG Sequence Analysis Software Package) to determine the number of mutagenesis primers needed to convert the starting DNA sequence into the modified DNA sequence.

The oligonucleotide mutagenesis primers were synthesized and purified by GENOSYS and the mutagenesis was carried out with the Bio-Rad Muta-Gene Enzyme Pack as described in the manufacturer's instruction manual. Following the mutagenesis reaction, a 10-30 .mu.l aliquot of the ligation mix was transformed into JM101 cells and selected on LBr Cb50. Individual transformed colonies were picked into 96-well microtiter plates containing 150 .mu.l 2XYT Cb50. After overnight growth at 37.degree. C., the cultures were replicated onto S&S Nytran filters on 2XYT-Cb50 plates and allowed to grow overnight at 37.degree. C. The filters were treated with denaturing solution (1.5M NaCl, 0.5M NaOH) for 5 minutes, neutralizing solution (3M NaOAc,pH5.0) for 5 minutes, air dried for 30 minutes, then baked for 1 hour at 80.degree. C.

The desired mutants were identified by differential primer melt-off at 65.degree. C. Mutagenesis oligonucleotides were end-labelled with either P.sup.32 or DIG-ddUTP. When P.sup.32 oligonucleotides were used, hybridizations were done overnight at 42.degree. C. in 50% formamide, 3.times. SSPE, 5.times. Denhardt's, 0.1%-20% SDS and 100 ug/ml tRNA. Filters were washed in 0.2.times. SSC, 0.1% SDS for 20 minutes at 65.degree. C. The filters were exposed to X-ray film for 1 hour. Colonies that contained the mutagenesis oligonucleotide retained the probe and gave a dark spot on the X-ray film. Parental colonies not subjected to mutagenesis were included in each screen as negative controls. For non-radioactive probes, the Genius DIG Oligonucleotide 3'-end labelling kit was used (Boehringer-Mannheim Biochemical, Indianapolis, Ind.) as per the manufacturer's instructions. Hybridization conditions were 50% formamide, 5.times. SSPE, 2% blocking solution, 0.1% N-laurylsarcosine, 0.02% SDS, and 100 .mu.g/ml tRNA. Temperatures for hybridization and filter washes were as previously stated for the radioactive method. Lumi-Phos 530 (Boehringer Mannheim) was used for detection of hybrids, following exposures of 1 hour to X-ray film. DNA from the positive colonies was sequenced to confirm the desired nucleotide sequences. If further changes were needed, a new round of mutagenesis using new oligonucleotides and the above described procedures were carried out.

Plasmids were transformed into the E. coli dut-, ung-, BW313 or CJ236 for use as templates for mutagenesis. Fifteen mls of 2XYT media containing 50 .mu.g/ml carbenicillin was inoculated with 300 .mu.l of overnight culture containing one of the plasmids. The culture was grown to an OD of 0.3 and 15 .mu.l of a stock of M13K07 helper phage was added. The shaking culture was harvested after 5 hours. Centrifugation at 10K for 15 minutes removed the bacteria and cell debris. The supernatant was passed through a 45 micron filter and 3.6 ml of 20% PEG/2.5M NaCl was added. The sample was mixed thoroughly and stored on ice for 30 minutes. The supernatant was centrifuged at 11K for 15 minutes. The phage pellet was resuspended in 400 .mu.l Tris-EDTA, pH8.0 (TE buffer) and extracted once with chloroform, twice with phenol:chloroform:isoamyl. Forty .mu.l of 7.5M NH.sub.4 OAc was added, then 1 ml ethanol. The DNA pellet was resuspended in 100 .mu.l TE.

The method employed in the construction of the modified CryIA(b) DNA sequence is illustrated in FIG. 6. The starting clones containing the starting CryIA(b)/CryIA(c) DNA sequence included pMON10922, which was derived from pMON19433 and is shown in FIG. 7, by replacement of the GUS coding region of pMON19433 with the NcoI-EcoRI restriction fragment from pMON10914, which is shown in FIG. 8 and which contains a pUC plasmid with a CaMV 35S promoter/NptII/NOS 3' cassette and an ECaMV 35S promoter(enhanced CaMV 35S promoter according to the method of Kay et al.)/Adh1 intron/(DNA sequence B. t. k. CryIA(b)/CryIA(c))/NOS 3' cassette, the only sequences used from pMON10914 are between the BglII site (nucleotide #1) at the 5' end of the CryIA(b)/CryIA(c) DNA sequence and the EcoRI site (nucleotide #3569) at the 3' end of the sequence; pMON19470 which consists of the ECaMV 35S promoter, the hsp70 intron and NOS 3' polyA region in a pUC vector containing a NPTII selectable marker; and pMON 19689 which is derived from pMON 10922, the 3' region of the CryIA(b)/CryIA(c) B.t. gene in pMON10922 was excised using XhoI (nucleotide #1839) and EcoRI (nucleotide #3569) and replaced with an oligonucleotide pair having the sequence

which creates XhoI and EcoRI cohesive ends when annealed that were ligated into pMON10922 to form pMON19689, which therefore contains a truncated CryIA(b) DNA sequence.

The five fragments of the starting CryIA(b)/CryIA(c) sequence from pMON10914 used for mutagenesis consisted of the following: pMON15740 which contained the 674 bp fragment from pMON10914 from the BglII to XbaI (nucleotide #675) restriction site cloned into the BamHI and XbaI sites of Bluescript SK+; pMON15741 which contains the sequence from the XbaI site to the SacI site (nucleotide #1354) cloned as a 679 bp XbaI-SacI fragment into the corresponding sites of Bluescript Sk+; pMON15742 which contains nucleotides between #1354-#1839 as a 485 bp SacI/XhoI fragment into the corresponding sites of Bluescript SK+; pMon 10928 which was derived from pMON10922 by excising the PvuII (nucleotide #2969) to EcoRI fragment and inserting it into the EcoRV to EcoRI site of Bluescript SK+; and pMON10927 which was derived from pMON10922 by excising the XhoI to PvuII fragment and inserting it into the XhoI to EcoRV site of pBS SK+.

The desired sequence changes were made to the section of the starting DNA sequence in pMON15741 by the use of oligonucleotide primers BTK15, BTK16, BTK17a and 17b (sequentially) and BTK18-BTK29 as shown in Table 2 below.

Plasmids with the desired changes were identified by colony hybridization with the mutagenesis oligonucleotides at temperatures that prevent hybridization with the original template, but allow hybridization with the plasmids that had incorporated the desired target sequence changes. In some cases unexpected sequence alterations were found. These were corrected by the use of oligonucleotides BTK44-BTK49 as shown in Table 3 below.

The final DNA sequence derived from pMON15741 was introduced into pMON15753 and contains the XbaI-SacI restriction fragment carrying nucleotides #669-1348 of the modified monocotyledonous CryIA(b) DNA sequence.

The desired sequence changes were made to the section of the starting DNA sequence in pMON15742 by the use of oligonucleotide primers BTK30-BTK41 as shown in Table 4 below

Plasmids with the desired changes were identified by colony hybridization with the mutagenesis oligonucleotides at temperatures that prevent hybridization with the original template, but allow hybridization with the plasmids that had incorporated the desired target sequence changes. In some cases unexpected sequence alterations were found. These were corrected by the use of oligonucleotides BTK42-BTK43 as shown in Table 5 below.

The final DNA sequence derived from pMON15742 was introduced into pMON15754 and contains the SacI-BstBI restriction fragment carrying nucleotides #1348-1833 of the modified monocotyledonous CryIA(b) DNA sequence.

The desired sequence changes were made to the section of the starting DNA sequence in pMON15740 by the use of oligonucleotide primers BTK0-BTK14 as shown in Table 6 below.

Plasmids with the desired changes were identified by colony hybridization with the mutagenesis oligonucleotides at temperatures that prevent hybridization with the original template, but allow hybridization with the plasmids that had incorporated the desired target sequence changes. In some cases unexpected sequence alterations were found. These were corrected by the use of oligonucleotides BTK50-BTK53 as shown in Table 7 below.

The final DNA sequence derived from pMON15740 was introduced into pMON15755 and contains the NcoI-XbaI restriction fragment carrying nucleotides #1-669 of the modified monocotyledonous CryIA(b) DNA sequence.

The desired sequence changes were made to the section of the starting DNA sequence in pMON10927 by the use of oligonucleotide primers BTK50D-BTK53D and BTK54-BTK61, and BTK63--BTK75 as shown in Table 8 below.

Plasmids with the desired changes were identified by colony hybridization with the mutagenesis oligonucleotides at temperatures that prevent hybridization with the original template, but allow hybridization with the plasmids that had incorporated the desired target sequence changes. In some cases unexpected sequence alterations were found. These were corrected by the use of oligonucleotides BTK91 and BTK94 as shown in Table 9 below.

The final DNA sequence derived from pMON10927 was introduced into pMON10947 and contains the BstBI-PvuI restriction fragment carrying nucleotides #1833-2888 of the modified monocotyledonous CryIA(b) DNA sequence.

The desired sequence changes were made to the section of the starting DNA sequence in pMON10928 by the use of oligonucleotide primers BTK76-BTK90 as shown in Table 10 below.

Plasmids with the desired changes were identified by colony hybridization with the mutagenesis oligonucleotides at temperatures that prevent hybridization with the original template, but allow hybridization with the plasmids that had incorporated the desired target sequence changes. In one case an unexpected sequence alteration was found. This was corrected by the use of oligonucleotide BTK92 as shown in Table 11 below.

The final DNA sequence derived from pMON10928 was introduced into pMON10944 and contains the PvuII-EcoRI restriction fragment carrying nucleotides #2888-3473 of the modified monocotyledonous CryIA(b) DNA sequence.

pMON15742 was subjected to oligonucleotide mutagenesis with oligonucleotide BTK41 (SEQ ID NO:38) to form pMON15767. The resulting B. t. k. CryIA DNA fragment of pMON15767 was excised with SacI and BstBI and inserted into the SacI and BstBI sites of pMON19689 to form pMON15768 which contains the NcoI-BstBI restriction fragment which contains nucleotides 7-1811 of the starting DNA sequence attached to nucleotides 1806-1833 of the modified DNA sequence.

Intermediate clones were prepared as follows: The SacI-BstBI fragment from pMON15754 was inserted into the SacI-BstBI sites of pMON19689 to form pMON15762 which contains nucleotides 7-1354 of the starting DNA sequence attached to nucleotides 1348-1833 of the modified DNA sequence; the XbaI to BstBI fragment of pMON19689 was excised and replaced with the XbaI to SacI fragment from pMON15753 and the SacI-BstBI fragment from pMON15762 resulting in pMON15765 which contains a truncated B.t. CryIA(b) DNA sequence where approximately the first third of the sequence from NcoI to XbaI of the starting DNA sequence is attached to XbaI-BstBI of the modified DNA sequence. Plasmid pMON15766 was prepared by excising the NcoI-XbaI fragment of pMON15765 and replaced by the NcoI-XbaI fragment from pMON15755 to yield pMON15766. pMON15766 thus encodes a truncated CryIA(b) sequence composed of nucleotides 1-1833 of the modified DNA sequence.

The final full length clones were prepared as follows: pMON10948 which encodes the full length CryIA(b) DNA sequence prepared in accordance with the method of this invention was made by inserting the BstBI to PvuII CryIA(b) fragment from pMON10947 and the PvuII-EcoRI fragment from pMON10944 into the BstBI-EcoRI site of pMON15766. The CryIA(b) B.t. DNA sequence of pMON10948 consists of the modified DNA sequence having nucleotides 1-3473; pMON10949, which encodes a full-length CryIA(b) DNA sequence where the first half of the gene consists of nucleotides 7-1811 of the starting DNA sequence attached to nucleotides 1806-3473 of the modified DNA sequence. pMON10949 was made by inserting the BstBI to EcoRI fragment from pMON10948 into the BstBI-EcoRI site of pMON15768. The sequence of the CryIA(b) DNA sequence in pMON10949 is identified as SEQ ID NO:105 and is shown in FIG. 9. pMON15722 was derived from pMON10948 by excising the entire CryIA(b) modified DNA sequence cassette, including the ECaMV promoter, hsp70 intron and NOS3' polyadenylation site region, as a NotI fragment and inserting it between the NotI sites of pMON19470 (this does not change any of the modified B. t. k. CryIA DNA sequence). pMON15774 was derived from pMON10948 by excising the entire CryIA(b) DNA sequence including the promoter, intron, CryIA(b) coding sequence, and NOS 3' polyadenylation site region as a NotI fragment and inserted between the NotI sites of pMON19470 (this does not change any of the B.t. DNA sequences).

The resulting modified CryIA(b) DNA sequence (SEQ ID NO:1) has a total abundance of 0.25% rare monocotyledonous codons and 3.8% semi-rare monocotyledonous codons. The total abundance of more preferred monocotyledonous codons is 86%. The CG dinucleotide frequency in the resulting modified DNA sequence was 7.5%. The modified CryIA(b) DNA sequence is compared to the wild-type bacterial CryIA(b) DNA sequence in FIG. 13.

This example illustrates the transient gene expression of the modified B. t. k. CryIA DNA sequence described in Example 1 and the CryIA(b)/CryIA(c) B.t. DNA sequence modified by the Fischoff et al. method in corn leaf protoplasts.

The level of expression of the modified CryIA(b) B.t. DNA sequence in pMON10948 and pMON15772 which contains the B. t. k. CryIA DNA sequence modified in accordance with the method of the present invention, the dicot/modified CryIA(b) B.t. DNA sequence in pMON10949 which has the 5' half of the DNA sequence modified in accordance with the method of Fischoff et al. and the 3' half modified in accordance with the method of the present invention, and the CryIA(b)/CryIA(c) DNA sequence modified by the Fischoff et al. method in pMON19493, were compared in a transient gene expression system in corn leaf protoplasts. The protoplasts were isolated from young corn seedlings. The DNA sequences were transferred into the protoplasts by electroporation and, after allowing time for gene expression, the electroporated samples were harvested and analyzed for gene expression. Samples were performed in duplicate and the ELISA values (performed in triplicate) were averaged for each experiment. The protein levels were measured by ELISA and the values indicated that 9-fold more CryIA(b) protein was produced from the modified B. t. k. CryIA DNA sequence in pMON10948 or pMON15772 than from pMON19493 containing the prior art CryIA(b)/CryIA(c) DNA sequence. The mixed B.t. DNA sequence in pMON10949 was expressed at 7 fold higher levels than pMON19493 indicating that most of the benefit of the modified B.t. DNA sequence of this invention is in the 3' portion of the CryIA(b) DNA sequence. This data is presented in Table 12.

This Example illustrates the expression of a modified B.t. DNA sequence modified by the method of the present invention in stably transformed corn cells.

Black Mexican Sweet (BMS) suspension cells were stably transformed using the microprojectile bombardment method and the chlorsulfuron EC9 selectable marker. Transgenic calli expressing the DNA sequence were initially identified by their insecticidal activity against tobacco hornworm larvae in a diet assay containing the calli. B.t. protein levels from individual insecticidal transgenic BMS calli were measured by ELISA from 48 calli expressing pMON15772 DNA and 45 calli expressing pMON19493. This comparison found that the average B.t. protein levels produced in pMON15772 calli was 6.5 fold higher than the average B.t. protein levels produced in pMON19493 calli. Western blot analysis confirmed the ELISA results and that the shorter processed forms of the proteins, predominantly the CryIA(b) portion, were in the extracts. These results demonstrate that the B. t. k. CryIA DNA sequence modified according to the method of the present invention functions better than the dicot CryIA(b)/CryIA(c) DNA sequence in stably transformed corn cells.

The ELISA assay used herein is a direct double antibody sandwich that utilizes a single polyclonal rabbit antibody against CryIA(b) as antigen (F137) for the capture and detection of the B. t. k. CryIA protein. Unconjugated antibody is used to coat 96 well polystyrene dishes. Alkaline phosphatase conjugated F137 antibody is added to the antibody coated dishes along with the test extracts or purified standard and allowed to incubate. The amount of B. t. k. CryIA protein present in the sample is directly proportional to the amount of alkaline phosphate-antibody bound. Color development with the p-nitrophenyl phosphate allows for quantitation of the CryIA(b) concentration in the samples using linear regression of the calibration curve prepared with the purified CryIA(b) protein standard.

Because the CryIA(b)/CryIA(c) protein differs from the CryIA(b) protein in the carboxyl terminus region, it needed to be confirmed that the ELISA measurements were accurately quantitating the CryIA(b)/CryIA(c) and CryIA(b) proteins produced from the full length synthetic DNA sequences. A trypsin treatment was used to produce identical amino terminal truncated CryIA(b) proteins in each extract. Bovine pancreatic trypsin (Calbiochem) was prepared as a 5 mg/ml solution in 50 mM sodium carbonate, pH8.5-9 and 3.5 .mu.l of the trypsin solution was added per 100 .mu.l tissue extract, mixed and incubated at 23.degree. C. for 1.5 hours. The reaction was stopped by the addition of 2.5 .mu.l of a 50 mM solution of PMSF in isopropanol, per 100 .mu.l extract.

A Western blot of the trypsin treated and untreated samples demonstrated that adding trypsin did convert the CryIA(b)/CryIA(c) and CryIA(b) proteins into a truncated size identical to the amino terminal portion of trypsin treated bacterial CryIA(b). The abundance of the B.t. proteins of either the untreated or trypsin treated samples was comparable to those found by the ELISA measurements of the protoplast extracts. This confirms that the ELISA assays accurately measure the amount of B.t. protein present, regardless of whether it is CryIA(b)/CryIA(c) or CryIA(b). The Western blot independently confirmed that the CryIA(b) DNA sequence prepared in accordance with the method of the present invention and the mixed prior art/modified CryIA(b) DNA sequence expressed at considerably greater levels than the B.t. full length synthetic DNA sequence of the prior art in pMON19493.

Additionally, the Western blot revealed that in protoplast extracts a considerable portion of the B.t. protein, either CryIA(b)/CryIA(c) or CryIA(b), was present as shorter, processed form of the full-length B.t. protein. Similar processed B.t. protein forms are present in extracts from both transgenic callus and plant tissue. This further explains why the ELISA assay provides accurate results against both the CryIA(b)/CryIA(c) and CryIA(b) proteins from the full-length DNA sequences, as it is effectively measuring the same amino terminal portions of the proteins.

This Example illustrates the expression of pMON15772 and pMON19493 in transgenic corn plants.

A highly embryogenic, friable Type II callus culture is the preferred tissue for obtaining transgenic, whole corn plants. The age of the embryogenic culture can be from the initial callus formation on the immature embryos, approximately one week after embryo isolation, to older established cultures of 6 months to 2 years old, however, it is preferred to use younger cultures to enhance the potential for recovery of fertile transgenic plants. Type II cultures were initiated from immature HiII embryos on N6 2-100-25 medium containing 10 .mu.M silver nitrate and solidified with 0.2% Phytagel. The most friable Type II calli were picked after about two weeks growth, and transferred onto fresh N6 2-100-25 medium containing 10 uM silver nitrate, in the center of the plate, in preparation for bombardment.

Four days after the calli were picked and transferred, the corn cell were bombarded 2 or 3 times with M10 tungsten particles coated with pMON15772 or pMON19493 mixed with pMON19574 as the selectable marker plasmid, using the particle preparation protocol described below. M10 particles at 100 mg/ml in 50% glycerol are sonicated to resuspend the particles. An aliquot of 12.5 .mu.l is placed into a small microfuge tube and 2.5 .mu.l of the desired DNA at 1 .mu.g/l is added and mixed well by pipetting up and down rapidly several times. A freshly prepared CaCl.sub.2 /spermidine pre-mix is added in an amount of 17.5 .mu.l and again mixed thoroughly. The particles are allowed to settle undisturbed for about 20 minutes and then 12.5 .mu.l of the supernatant was removed. The particles are ready for use and are used in microprojectile bombardment within one hour of their preparation.

After bombardment, the cells were transferred to fresh N6 2-100-25 medium containing 10 .mu.M silver nitrate for seven days without any selective pressure. The cells were then transferred to N6 1-0-25 media containing 3 mM glyphosate. Two weeks later, the cells were transferred to fresh selective media of the same composition. After a total of 6 or 7 weeks post-bombardment, glyphosate-tolerant calli could be observed growing on the selection media. Occasionally, the cell population would be transferred to fresh selective plates at this time to carry on the selection for 10-12 weeks total time. Glyphosate resistant calli were picked onto fresh N6 1-0-25 media containing 3 mM glyphosate for increasing the amount of callus tissue prior to initiating plant regeneration.

Plant regeneration was initiated by placing the transgenic callus tissue on MS 0.1 D media for two weeks. At two weeks, the tissue was transferred to N6 6% OD media for another two week period. The regenerating tissues are then transferred to MS 0 D media and transferred into lighted growth chambers. After another two weeks in the same media in larger containers, the young plants are hardened off, followed by transfer to the greenhouse where they were maintained in the same manner as normal corn plants. In most instances, the regeneration process was performed with 0.01 mM glyphosate in the regeneration media.

The corn plants were allowed to grow and the level of B. t. k. CryIA protein expressed in the leaves of the plant were measured by ELISA. As is commonly observed in transgenic plants, a large range of expression values were observed and, therefore, a large number of independently derived transgenic plants were examined. The B.t. levels in 44 pMON19493 plants and 86 pMON15722 plants were measured by ELISA assays of leaf material. Each line of plants were derived from embryogenic callus expressing the B.t. DNA sequence as determined by insecticidal activity against tobacco hornworm. Thus, the percentage of transformants that do not express the B.t. DNA sequence, as occurs in the transformation process, are not included in the data set. Western blots demonstrated that the majority of B.t. protein in the leaf extracts was processed to the predominantly CryIA(b) form of the protein, which has been shown to be recognized equivalently by the ELISA antibody assay. These results illustrate that the average level of B.t. expression with pMON15722 plants is at least 5 fold higher than the average level of B.t. expression from pMON19493 plants as shown in Table 13.

This example illustrates the preparation of another form of a crystal toxin protein from B.t., namely the CryIIB DNA sequence (Widner et al., J Bact., 171: 965-974), according to the method of the present invention and also utilizing the 6mer analysis of the DNA sequence to construct a modified DNA sequence that exhibits enhanced expression in a monocotyledonous plant.

The starting DNA sequence for this Example was the CryIIA synthetic DNA sequence identified by SEQ ID NO:106. The CryIIB synthetic DNA sequence was constructed from SEQ ID NO:106 by a new gene construction process. The CryIIA gene was used as a template for annealing oligonucleotides. These oligonucleotides fit precisely adjacent to each other such that DNA ligase could close the gap to form a covalent linkage. After the ligation reaction, the linked oligonucleotides were amplified by PCR and subcloned. Thus, this process is a form of oligonucleotide mutagenesis that ligates the oligonucleotides into one contiguous fragment of the desired new sequence. Because of the large size of the CryIIA gene, the process was carried out on five smaller fragments designated A, B, C, D and E. A representation of the steps by which the CryIIB synthetic DNA sequence of the present invention was prepared is presented in FIG. 11.

A double stranded plasmid containing the CryIIA synthetic DNA sequence (SEQ ID NO:106) in pBSKS+, referred to hereinafter as the P2syn DNA sequence, was digested and used as an annealing template for the different oligonucleotide combinations. For the A fragment, oligonucleotides A1 through A4, as shown in Table 14, were annealed to linearized pP2syn and ligated with T4 DNA ligase. The new strand of the contiguous oligonucleotides was amplified using primers AP5 and AP3, as shown in Table 14, under standard PCR conditions. The amplified double stranded fragment was digested with the restriction enzymes XbaI and BamHI and cloned into similarly digested pBSKS+ to form pMON19694.

For the B fragment, oligonucleotides B1 through B6, were annealed to pP2syn and ligated with T4 DNA ligase. The new strand of the contiguous oligonucleotides was amplified using primers BP5 and BP3, as shown in Table 15, under standard PCR conditions. The amplified double stranded fragment was digested with the restriction enzymes BglII and PstI and cloned into similarly digested pMON19694 to form pMON19700.

For the C fragment, oligonucleotides C1 through C7, as shown in Table 16, were annealed to pP2syn and ligated with T4 DNA ligase. The new strand of the contiguous oligonucleotides was amplified using primers CP5 and CP3, as shown in Table 16, under standard PCR conditions. The amplified double stranded fragment was digested with the restriction enzymes PstI and XhoI and cloned into similarly digested pBSKS+ to form pMON19697.

For the D fragment, oligonucleotides D1 through D7, as shown in Table 17, were annealed to pP2syn and ligated with T4 DNA ligase. The new strand of the contiguous oligonucleotides was amplified using primers DP5 and DP3, as shown in Table 17, under standard PCR conditions. The amplified double stranded fragment was digested with the restriction enzymes XhoI and KpnI and cloned into similarly digested pBSKS+ to form pMON19702.

For the E fragment, oligonucleotides E1 through E8, as shown in Table 18, were annealed to pP2syn and ligated with T4 DNA ligase. The new strand of the contiguous oligonucleotides was amplified using primers EP5 and EP3, as shown in Table 18, under standard PCR conditions. The amplified double stranded fragment was digested with the restriction enzymes BamHI and KpnI and cloned into similarly digested pBSKS+ to form pMON19698.

Some sequence errors occurred during the construction process. The repair oligonucleotides A5 and A6 were used to repair fragment A, and oligonucleotides B7-B10, C8-C10, D8-D10, and E9-E11, were used to repair fragments B-E, respectively, using the single stranded oligonucleotide mutagenesis described in Example 1. These oligonucleotides are shown in Table 19.

pMON19694 was then restricted at the PstI and XhoI sites in the pBSKS+ polylinker, removing a small oligonucleotide region. The insert from pMON19697 was excised with PstI and XhoI and ligated into the PstI and XhoI digested pMON19694 to form pMON19703. pMON19703 was digested with BclI and PstI, removing a small oligonucleotide region, and the BglII and PstI digested insert from pMON19700 was ligated into pMON19703 to form pMON19705. pMON19705 was digested with XhoI and KpnI and the XhoI to KpnI excised insert of pMON19702 was ligated into pMON19705 to form pMON19706. pMON19706 was digested with BclI and KpnI and the BamHI to KpnI excised insert of pMON19701 was ligated into pMON19706 to form pMON19709. This comprises the final CryIIB sequence and contains the DNA sequence identified as SEQ ID NO: 2. This sequence contains 0.15% rare monocotyledonous codons, 9.7% semi-rare monocotyledonous codons, and has a CG dinucleotide composition of 6.7% The resulting modified CryIIB DNA sequence also has 0.05% of the rarest 284 six-mers, 0.37% of the rarest 484 six-mers, and 0.94% of the rarest 664 six-mers. The bacterial CryIA(b) DNA sequence has 9.13% of the rarest 284 six-mers, 15.5% of the rarest 484 six-mers, and 20.13% of the rarest 664 six-mers. The modified DNA sequence as described in Example 1, the monocotyledonous modified B.t. CryIA(b) contains 0.35% of the rarest 284 six-mers, 1.12% of the rarest 484 six-mers, and 2.1% of the rarest 664 six-mers.

pMON19709 was digested with BglII and BclI and inserted into pMON19470, a plasmid map of which is provided in FIG. 12, to form pMON15785. The starting DNA sequence comprising a synthetic CryIIA DNA sequence prepared by the method of Fischoff et al. was inserted into pMON19470 for use as a control for expression studies in corn.

Corn leaf protoplasts were electroporated with CryIIB plasmid DNA or CryIIA plasmid DNA using the protocol described above. The CryIIB DNA sequence, pMON15785, was compared to the CryIIA DNA sequence, pMON19486, in the same corn gene expression cassette. The protoplast electroporation samples were done in duplicate for Western blot analysis and in triplicate for insect bioactivity assays. The protoplast extracts were assayed by diet incorporation into insect feeding assays for tobacco hornworm (THW) and European corn borer (ECB). The protein produced by the CryIIB DNA sequence in pMON15785 showed excellent insecticidal activity that was superior to the insecticidal activity of the CryIIA DNA sequence in the same vector in pMON19486. This data is presented in Table 20 below.

Western blots also demonstrated that more protein was detected from pMON15785 than from pMON19486. The antibody used in the Western was raised against CryIIA, so the detection of more CryIIB is significant. Initial transgenic corn plant studies with the CryIIB DNA sequence modified by the method of the present invention have demonstrated insecticidal activity against the European corn borer when the insect was feeding on leaf discs from the transgenic plant. One of fourteen independent transgenic plants containing the modified CryIIB killed the insect. This confirms the initial transient data that the CryIIB DNA sequence is expressed in the plant and is insecticidal to European corn borer and other Lepidopteran pests.