Chemical pesticides have provided an effective method of pest control; however, the public has become concerned about the amount of residual chemicals which might be found in food, ground water, and the environment. Because of the problems associated with the use of synthetic chemical pesticides, there exists a clear need to limit the use of these agents and a need to identify alternative control agents. The replacement of synthetic chemical pesticides, or combination of these agents with biological pesticides, could reduce the levels of toxic chemicals in the environment.

A biological pesticidal agent that is being used with increasing popularity is the soil microbe Bacillus thuringiensis (B. t.). The soil microbe Bacillus thuringiensis (B. t.) is a Gram-positive, spore-forming bacterium. Most strains of B. t. do not exhibit pesticidal activity. Some B. t. strains produce, and can be characterized by, parasporal crystalline protein inclusions. These"o-endotoxins,"which typically have specific pesticidal activity, are different from exotoxins, which have a non-specific host range.

These inclusions often appear microscopically as distinctively shaped crystals. The proteins can be highly toxic to pests and are specific in their toxic activity.

Preparations of the spores and crystals of B. thuringiensis subsp. kurstaki have been used for many years as commercial insecticides for lepidopteran pests. For example, B. thuringiensis var. kurstaki HD-1 produces a crystalline 8-endotoxin which is toxic to the larvae of a number of lepidopteran insects.

The cloning and expression of a B. t. crystal protein gene in Escherichia coli was described in the published literature more than 15 years ago (Schnepf, H. E., H. R.

Whiteley [1981] Proc. Natl. Acad. Sci. USA 78: 2893-2897.). U. S. Patent No. 4,448,885

and U. S. Patent No. 4,467,036 both disclose the expression of B. t. crystal protein in E. coli. Recombinant DNA-based B. t. products have been produced and approved for use.

With the use of genetic engineering techniques, new approaches for delivering B. t. toxins to agricultural environments are under development, including the use of plants genetically engineered with B. t. toxin genes for insect resistance and the use of stabilized, microbial cells as delivery vehicles of B. t. toxin (Gaertner, F. H., L. Kim [1988] TIBTECH 6: S4-S7). Thus, isolated B. t. endotoxin genes are becoming commercially valuable. As a result of extensive research and resource investment, patents continue to issue for new B. t. isolates, toxins, and genes, and for new uses of B. t. isolates. See Feitelson et al., supra, for a review. Various improvements have been achieved by modifying B. t. toxins and/or their genes. For example, U. S. Patent Nos.

5,380,831 and 5,567,862 relate to the production of synthetic insecticidal crystal protein genes having improved expression in plants.

Marker genes are useful tools for selecting cells that have been transformed with a gene of interest. Techniques for screening for transformants are well known.

Typically, a certain transformation technique is used in an attempt to introduce a gene of interest, such as a toxin gene, and a marker gene into one or more target cells. These two genes can be present on the same vector so that successfully transformed cells would receive both genes. The target cells that have undergone the attempted transformation can then be screened for successful transformants.

Marker genes encode proteins that are easily detectable using techniques known in the art. For example, some marker genes encode proteins, such as fluorescent proteins, the presence of which yields a detectable color change in transformed cells. Cells successfully expressing the detectable fluorescent protein, for example, can be distinguished from cells that do not exhibit the fluorescent color under selective conditions. One such protein that is known in the art is the green-fluorescent protein from Aequorea victoria.

Another marker scheme is to use genes that convey a detectable trait to successfully transformed cells. One example of this type of marker gene is a gene encoding a protein that conveys resistance to a certain herbicide. Successfully transformed cells would be expected to grow in the presence of the herbicide while non-

transformants would not. An example of a protein expressed by this type of marker gene include the phosphinothricin acetyl transferase (PAT) protein which confers phosphinothricin resistance. Another example is the cyanamide hydratase (cat) protein.

A chemical called cyanamide can be broken down by this enzyme into urea, which plants can use as a fertilizer. Cyanamide, itself, can be herbicidal.

There remains a need for marker genes that can be successfully expressed at adequate levels, in order to facilitate easy detection, in plant cells and in plants.

Brief Summary of the Invention The subject invention concerns materials and methods useful for identifying transformed cells. More specifically, the subject invention provides polynucleotide marker sequences that are optimized for expression in plants. Plant cells (and plants) can be transformed, using techniques known to those skilled in the art, in order to confer an easily detectable trait upon the transformed cells. The polynucleotide sequences of the subject invention have certain modifications, compared to wild-type sequences, that make them particularly well-suited for optimized expression in plants. In preferred embodiments, the subject invention provides polynucleotide referred to herein as GFPAV1-PO, PATV4-PO, CAH-M-PO, and CAH-C-PO.

Description of the Sequences SEQ ID NO. 1 is a plant-optimized polynucleotide sequence for a synthetic gene designated GFPAV1-PO, which encodes an Aequorea victoria green-fluorescent protein.

This gene can be used as a transformation marker.

SEQ ID NO. 2 is a plant-optimized polynucleotide sequence for a synthetic gene designated PATV4-PO. This gene encodes a phosphinothricin acetyl transferase protein.

This gene can be used to confer phosphinothricin herbicide resistance to a transformed host. This gene can be used as a transformation marker.

SEQ ID NO. 3 is a maize-optimized polynucleotide sequence designated CAH- M-PO encoding a cyanamide hydratase (cah) protein. This gene can be used to confer cyanamide herbicide resistance to a transformed host. This gene can be used as a transformation marker.

SEQ ID NO. 4 is a cotton-optimized polynucleotide sequence designated CAH- C-PO encoding a cyanamide hydratase (cah) protein. This gene can be used to confer

cyanamide herbicide resistance to a transformed host. This gene can be used as a transformation marker.

Detailed Disclosure of the Invention The subject invention concerns materials and methods useful for identifying transformed cells. More specifically, the subject invention provides polynucleotide marker sequences that are optimized for expression in plants. Plant cells (and plants) can be transformed, using techniques known to those skilled in the art, in order to confer an easily detectable trait upon the transformed cells. The polynucleotide sequences of the subject invention have certain modifications, compared to wild-type sequences, that make them particularly well-suited for optimized expression in plants. The subject polynucleotides can be used alone or in combination to"mark"the transformation of a host cell with one or more genes of interest. In preferred embodiments, the subject invention provides polynucleotides referred to herein as GFPAV1-PO, PATV4-PO, CAH-M-PO, and CAH-C-PO.

Using techniques such as computer-or software-assisted sequence alignments, differences can be noted in the nucleotide sequence of the subject plant-optimized genes as compared to the wild-type genes or to previously known genes.

It should be apparent to a person skilled in this art that, given the sequences of the genes as set forth herein, the genes of the subject invention can be obtained through several means. In preferred embodiments, the subject genes may be constructed synthetically by using a gene synthesizer, for example. The specific genes exemplified herein can also be obtained by modifying, according to the teachings of the subject invention, certain wild-type genes (for example, by point-mutation techniques).

The polynucleotides of the subject invention can be used to form complete "genes"to encode proteins or peptides in a desired host cell. For example, as the skilled artisan would readily recognize, the sequences exemplified herein are shown without stop codons. These sequences can be appropriately placed under the control of a promoter in a host of interest, as is readily known in the art.

As the skilled artisan would readily recognize, DNA can exist in a double- stranded form. In this arrangement, one strand is complementary to the other strand and vice versa. The"coding strand"is often used in the art to refer to the strand having a

series of codons (a codon is three nucleotides that can be read three-at-a-time to yield a particular amino acid) that can be read as an open reading frame (ORF) to form a protein or peptide of interest. In order to express a protein in vivo, a strand of DNA is typically translated into a complementary strand of RNA which is used as the template for the protein. As DNA is replicated in a plant (for example) additional, complementary strands of DNA are produced. Thus, the subject invention includes the use of either the exemplified polynucleotides shown in the attached sequence listing or the complementary strands. RNA and PNA (peptide nucleic acids) that are functionally equivalent to the exemplified DNA are included in the subject invention.

The subject invention includes, in preferred embodiments, a polynucleotide sequence optimized for expression in a plant, wherein said sequence is selected from the group consisting of SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, and SEQ ID NO. 4.

Certain DNA sequences of the subject invention have been specifically exemplified herein. These sequences are exemplary of the subject invention. It should be readily apparent that the subject invention includes not only the genes and sequences specifically exemplified herein but also equivalents and variants thereof (such as mutants, fusions, chimerics, truncations, fragments, and smaller genes) that exhibit the same or similar characteristics relating to expression in plants, as compared to those specifically disclosed herein. As used herein,"variants"and"equivalents"refer to sequences which have nucleotide (or amino acid) substitutions, deletions (internal and/or terminal), additions, or insertions which do not materially affect the expression of the subject genes, and the resultant activity, in plants.

Genes can be modified, and variations of genes may be readily constructed, using standard techniques. For example, techniques for making point mutations are well known in the art. In addition, commercially available exonucleases or endonucleases can be used according to standard procedures, and enzymes such as Sa/31 or site-directed mutagenesis can be used to systematically cut off nucleotides from the ends of these genes. Useful genes can also be obtained using a variety of restriction enzymes.

It should be noted that equivalent genes will encode proteins having high amino acid identity or homology with the toxins encoded by the subject genes. The amino acid homology will be highest in critical regions of the protein which account for biological

activity or are involved in the determination of three-dimensional configuration which ultimately is responsible for the activity. In this regard, certain substitutions are acceptable and can be expected if these substitutions are in regions which are not critical to activity or are conservative amino acid substitutions which do not affect the three- dimensional configuration of the molecule. For example, amino acids may be placed in the following classes: non-polar, uncharged polar, basic, and acidic. Conservative substitutions whereby an amino acid of one class is replaced with another amino acid of the same type fall within the scope of the subject invention so long as the substitution does not materially alter the biological activity of the compound. Table 1 provides a listing of examples of amino acids belonging to each class.

Table 1.

Class of Amino Acid Examples of Amino Acids Nonpolar Ala, Val, Leu, Ile, Pro, Met, Phe, Trp Uncharged Polar Gly, Ser, Thr, Cys, Tyr, Asn, Gln Acidic Asp, Glu Basic Lys, Arg, His In some instances, non-conservative substitutions can also be made. The critical factor is that these substitutions must not significantly detract from the ability of plants to express the subject DNA sequences or from the activity of the protein.

As used herein, reference to"isolated"polynucleotides and/or"purified"toxins refers to these molecules when they are not associated with the other molecules with which they would be found in nature and would include their use in plants. Thus, reference to"isolated"or"purified"signifies the involvement of the"hand of man"as described herein.

Recombinant hosts. The genes of the subject invention can be introduced into a wide variety of microbial or plant hosts. In some embodiments of the subject invention, transformed microbial hosts can be used in preliminary steps for preparing precursors,

for example, that will eventually be used to transform, in preferred embodiments, plant cells and plants so that they express the proteins encoded by the genes of the subject invention. Microbes transformed and used in this manner are within the scope of the subject invention. Recombinant microbes may be, for example, B. t., E. coli, or Pseudomonas. Transformations can be made by those skilled in the art using standard techniques. Materials necessary for these transformations are disclosed herein or are otherwise readily available to the skilled artisan. Thus, in preferred embodiments, expression of the subject gene results, directly or indirectly, in the intracellular production and maintenance of the protein.

The subject gene is introduced via a suitable vector into a host, preferably a plant cell or cells. There are many crops of interest, such as corn, wheat, rice, cotton, soybeans, and sunflowers. The genes of the subject invention are particularly well suited for providing stable maintenance and expression, in the transformed plant cells, of the gene expressing the polypeptide.

While the subject invention provides specific embodiments of synthetic genes, other genes that are functionally equivalent to the genes exemplified herein can also be used to transform hosts, preferably plant hosts. Additional guidance for the production of synthetic genes can be found in, for example, U. S. Patent No. 5,380, 831.

All of the publications and patent references cited herein are hereby incorporated by reference in their entirety to the extent that they are not inconsistent with the explicit teachings of this specification.

Following is an example which illustrates procedures for practicing the invention.

This example should not be construed as limiting.

Example 1-Insertion of Marker Genes Into Plants One aspect of the subject invention is the transformation of plants with the subject polynucleotide sequences. The transformed plant cells express the protein encoded by the polynucleotide sequence of interest. The genes of the subject invention are optimized for use in plants.

Obviously, a promoter region capable of expressing the gene in a plant is needed.

Thus, for in planta expression, the DNA of the subject invention is under the control of an appropriate promoter region. Techniques for obtaining in planta expression by using

such constructs is known in the art. Once the inserted DNA has been integrated in the genome, it is relatively stable there and, as a rule, does not come out again.

The genes of the subject invention can be inserted into plant cells using a variety of techniques which are well known in the art. For example, a large number of cloning vectors comprising a replication system in E. coli and a marker that permits selection of the transformed cells are available for preparation for the insertion of foreign genes into higher plants. The vectors comprise, for example, pBR322, pUC series, M13mp series, pACYC 184, etc. Accordingly, the sequence encoding the B. t. toxin can be inserted into the vector at a suitable restriction site. The resulting plasmid is used for transformation into E. coli. The E. coli cells are cultivated in a suitable nutrient medium, then harvested and lysed. The plasmid is recovered. Sequence analysis, restriction analysis, electrophoresis, and other biochemical-molecular biological methods are generally carried out as methods of analysis. After each manipulation, the DNA sequence used can be cleaved and joined to the next DNA sequence. Each plasmid sequence can be cloned in the same or other plasmids.

Depending on the method of inserting desired genes into the plant, other DNA sequences may be necessary. If, for example, the Ti or Ri plasmid is used for the transformation of the plant cell, then at least the right border, but often the right and the left border of the Ti or Ri plasmid T-DNA, has to be joined as the flanking region of the genes to be inserted. The use of T-DNA for the transformation of plant cells has been intensively researched and sufficiently described in EP 120 516; Hoekema (1985) In: The Binary Plant Vector System, Offset-durkkerij Kanters B. V., Alblasserdam, Chapter 5; Fraley et al., Crit. Rev. Plant Sci. 4: 1-46; and An et al. (1985) EMBO J. 4: 277-287.

A large number of techniques are available for inserting DNA into a plant host cell. Those techniques include transformation with T-DNA using Agrobacterium tumefaciens or Agrobacterium rhizogenes as transformation agent, fusion, injection, biolistics (microparticle bombardment), or electroporation as well as other possible methods. If Agrobacteria are used for the transformation, the DNA to be inserted has to be cloned into special plasmids, namely either into an intermediate vector or into a binary vector. The intermediate vectors can be integrated into the Ti or Ri plasmid by homologous recombination owing to sequences that are homologous to sequences in the

T-DNA. The Ti or Ri plasmid also comprises the vir region necessary for the transfer of the T-DNA. Intermediate vectors cannot replicate themselves in Agrobacteria. The intermediate vector can be transferred into Agrobacterium tumefaciens by means of a helper plasmid (conjugation). Binary vectors can replicate themselves both in E. coli and in Agrobacteria. They comprise a selection marker gene and a linker or polylinker which are framed by the right and left T-DNA border regions. They can be transformed directly into Agrobacteria (Holsters et al. [1978] Mol. Gen. Genet. 163: 181-187). The Agrobacterium used as host cell is to comprise a plasmid carrying a vir region. The vir region is necessary for the transfer of the T-DNA into the plant cell. Additional T-DNA may be contained. The bacterium so transformed is used for the transformation of plant cells. Plant explants can advantageously be cultivated with Agrobacterium tumefaciens or Agrobacterium rhizogenes for the transfer of the DNA into the plant cell. Whole plants can then be regenerated from the infected plant material (for example, pieces of leaf, segments of stalk, roots, but also protoplasts or suspension-cultivated cells) in a suitable medium, which may contain antibiotics or biocides for selection. The plants so obtained can then be tested for the presence of the inserted DNA. No special demands are made of the plasmids in the case of injection and electroporation. It is possible to use ordinary plasmids, such as, for example, pUC derivatives.

The transformed cells grow inside the plants in the usual manner. They can form germ cells and transmit the transformed trait (s) to progeny plants. Such plants can be grown in the normal manner and crossed with plants that have the same transformed hereditary factors or other hereditary factors. The resulting hybrid individuals have the corresponding phenotypic properties.

It should be understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and the following claims.