WO1998010734A9

WO1998010734A9 - MsENOD40 PROMOTER COMPOSITIONS AND METHODS OF USE

Info

Publication number: WO1998010734A9
Application number: PCT/US1997/016102
Authority: WO
Filing date: 1997-09-10
Publication date: 1998-06-18

Abstract

Disclosed are compositions comprising MsENOD promoters and methods of use in the preparation of transformed host cells, and transgenic plant and animal cells. Also disclosed are methods for expressing nucleic acid segments (such as heterologous genes, antisense constructs, and ribozymes) in an inducible, and/or tissue-specific manner in a transformed host cell. In certain embodiments, the invention provides methods of introducing and controlling the expression of transgenic DNA segments in plants and animals, and in particular, methods for increasing desirable characteristics in a plant cell, such as increased herbicide resistance, increased insect resistance, increased fungal resistance, and the like, and methods for introducing genes of interest under the control of an inducible promoter for use in gene therapies.

Description

DESCRIPTION

MsENOD40 PROMOTER COMPOSITIONS AND METHODS OF USE

1. BACKGROUND OF THE INVENTION

The present application is a continuing application based on U. S. Provisional

Patent Serial Number 60/025730, filed September 10, 1996, the entire contents of which is specifically incorporated herein by reference in its entirety. The United States Government has rights to the present invention pursuant to Grant NSF 90-23888 from the National Science Foundation.

1.1 FIELD OF THE INVENTION

The present invention relates generally to the field of molecular biology. Certain embodiments concern methods of expressing nucleic acid segements (such as heterologous genes, antisense constructs, and ribozymes) in an inducible, and/or tissue- specific manner in a transformed host cell. More specifically, the invention relates to a method of expressing such nucleic acid segments in transgenic plant and animal ceils. Furthermore, the invention provides methods of introducing and controlling the expression of transgenic DNA segments in plants and animals, and in particular, methods for increasing desirable characteristics in a plant cell, such as increased herbicide resistance, increased insect resistance, increased fungal resistance, and the like.

1.2 DESCRIPTION OF THE RELATED ART

The symbiotic interaction between leguminous plants and rhizobia results in the development on plant roots of a novel organ, the nodule, in which rhizobia provide fixed nitrogen to the host in exchange for carbon and protection. To establish this mutualistic relationship, the host plant and rhizobia must continually exchange molecular signals. Rhizobial nodulation (nod) gene expression is induced in response to different fiavonoids that are excreted by plant roots or seeds (McKhann and Hirsch. 1994; Long. 1996). In turn, nod gene products synthesize and secrete nodulation signal molecules called Nod factors, which have been identified as modified lipo- chitooligosaccharide molecules (Lerouge et al, 1990; Truchet et al, 1991 ; van Rhijn and Vanderleyden, 1995; Long, 1996; Denarie et al, 1996). Nod factor is considered a primary morphogenetic signal for nodulation because it triggers on a compatible host the earliest stages of nodule development, including root hair deformation and curling as well as cortical cell divisions (Lerouge et al, 1990; Truchet et al, 1991 ; Spaink et al, 1991). Furthermore, Nod factor elicits the expression of several early nodulin (ENOD) genes, such as ENOD12 and ENOD40 (Vijn et al, 1993, 1995a; Horvath et al, 1993; Bauer et al, 1994; Crespi et al, 1994; Journet et al, 1994; Hirsch et al, 1997). Nodulins are plant-encoded proteins that are expressed during nodule development. ENOD40 is one of the earliest nodulins to be expressed upon Rhizobium inoculation, and has been cloned from a number of legumes (Kouchi and Hata, 1993; Matvienko et al, 1994; Asad et al, 1994; Crespi et al, 1994; Vijn et al, 1995b; Papadopoulou et al, 1996) as well as one non-legume, tobacco (van de Sande et al, 1996). In alfalfa, ENOD40 transcripts have been detected in dividing cells and cells that are competent to divide as well as some partially differentiated cells (Asad et al. 1994; Crespi et al. 1994).

1.3 DEFICIENCIES IN THE PRIOR ART The lack of general availability of a variety of inducible promoters to express nucleic acid segments has confounded the area of molecular biology for some time. Particularly, it has been difficult to utilize native inducible promoters in animal systems, because of the generalized nature in which native activators induce not only the promoter of interest, but other systems as well. The availability of an inducible promoter which is regulated by non-animal activators (such as plant cytokinins, and other small molecules) would facilitate the use of gene constructs in animal systems which could be specifically activated using promoter compositions which respond to non-animal activators.

Likewise, it has been difficult to obtain inducible promoters for use in preparing transformed plant cells to express particularl heterologous genes, antisense constructs, ribozymes and other nucleic acid segments of interest and the like in a repeatable and desired fashion. The availability of promoters which are functional in plants to drive expression of such nucleic acid segments would be particular desirable.

What is lacking in the prior art are inducible promoter sequences which may be used to promote the expression of antisense constructs, ribozymes, and heterologous genes of interest in a transformed cell, and in particular, transformed and transgenic plant and animal cells.

2.0 SUMMARY

The present invention seeks to overcome these and other limitations in the prior art by providing methods and compositions related to expressing a nucleic acid segment in a cell. In particular, the invention discloses and claims compositions and methods of use for inducible promoters which are regulated by cytokinins and other compounds such as chitin, chitin fragments, and the like. U. S. Patent 5, 1 18,610, specifically incorporated herein by reference, describes assays for chitin, and methods for the detection and quantitation of chitin in a sample. Such assays are desirable when the use of chitin is desired for induction of the promoters described herein.

In an important embodiment, the present invention relates to an isolated MsENOD40 promoter comprising a contiguous nucleic acid sequence of at least about 17 nucleic acids from SEQ ID NO:l or SEQ ID NO:2. Alternatively the MsENOD40 promoter may comprise a nucleic acid sequence having from about 60% to about 65%, to about 70%. to about 75%, to about 80%, to about 85%, to about 90%, to about 95%, even up to and including about 96%, about 97%, about 98%, or about 99% or greater sequence identity with either the sequence of SEQ ID NOT or SEQ ID NO:2. Of course, the percent identity to the sequence of either SEQ ID NOT or SEQ ID NO:2 need not be limited to the specific percentages given, but is also meant to include all integers between about 60%o and about 99% identity, such as percentage identities of about 86%, 87%, 88%, and 89%, or even about 91% or 92% or 93% or 94%, etc. identity with the sequence of either SEQ ID NOT or SEQ ID NO:2. In fact, all such sequences are contemplated to fall within the scope of the present invention, so long as the particular sequence retains an ability to promote transcription of a nucleic acid segment operatively linked to the DNA sequence comprising the MsENOD40 promoter. In preferred embodiments, the MsENOD40-l and MsENOD40-2 promoters described in Example 1 have been shown by the inventors to be sufficient for promoting transcription of one or more nucleic acid segments operatively linked to either of the DNA sequences. Particularly preferred DNA sequences include, but are not limited to, the DNA sequences disclosed in SEQ ID NOT and SEQ ID NO:2, and all such DNA sequences which have from about 60% to about 99% sequence identity with such sequences.

In an exemplary embodiment, the nucleic acid segment to be transcriptionally controlled (or promoted) by one or more of these promoters include, but are not limited to, heterologous genes, ribozymes, and antisense constructs.

Another important aspect of the present invention is the ability to modulate, alter, or regulate, either positively, or negatively, the amount, extent, or efficiency of one or more of the disclosed promoters through the use of transcriptional enhancers, and particularly, transcriptional activators. In an exemplary embodiment, the inventors have demonstrated that compounds such as cytokinins and chitin fragments can alter the effectiveness of these promoters. Particularly preferred activators include the plant hormones such as BAP. Also preferred are compounds isolated from chitin and/or chitin fragments, which may also serve to regulate the activity of the MsENOD40 promoters.

When it is desirable to negatively, or "down-regulate" the expression of a particular gene or nucleic acid segment in a particular cell, the inventors contemplate the preparation of antisense constructs using the promoters of the present invention either with, or without, the addition of such activators as cytokinin or chitin fragments. Antisense constructs are well-known in the art, and in their simplest terms, relate to the use of antisense mRNA to reduce or lessen the transcription or translation or otherwise impair the net production of the encoded polypeptide. The use of antisense constructs are described herein. Alternatively, the inventors contemplate the use of the promoters disclosed herein in the preparation of ribozymes, which are also well-known in the art.

In important aspects of the present invention, there are provided DNA constructs comprising one or more MsENOD40 promoters operably linked to or operatively positioned with respect to one or more heterologous genes. Exemplary heterologous genes which are contemplated to be useful include, but are not limited to, reporter genes such as GFP. GUS, lac, lux. β-lactamase. xylE, α-amylase. a tyrosinase gene, and 6^" aequorin; cell cycle control genes such as Rb, p53, a cell cycle dependent kinase, a CDK kinase or a cyclin gene; insecticidal resistance genes such as the crystal protein encoding genes obtained from Bacillus thuringiensis; microbial resistance genes encode proteins such as b-1 , 3-glucanases, chitinases . osmotin, UDA, and hevein; and herbicide resistance genes such as bar, pat, glyphosate tolerant EPSP synthase genes, the glyphosate degradative enzyme gene gox encoding glyphosate oxidoreductase, deh (encoding a dehalogenase enzyme that inactivates dalapon), acetolactate synthase, and bxn genes (encoding a nitrilase enzyme that degrades bromoxynil). Alternatively, the heterologous genes could be one or more microbial resistance genes, drought tolerance genes, or a gene encoding a pheromone, hormone, antihormones, hormone inhibitors.storage proteins (e.g.. lectin), an enzyme, or a structural protein.

A further aspect of the present invention provides a method of expressing a heterologous nucleic acid segment in a cell. The method generally involves transforming said cell with a vector comprising a heterologous nucleic acid segment operatively linked to an MsENOD40 promoter and culturing the cell under conditions effective to express the heterologous nucleic acid segment from the promoter.

Preferably, the MsENOD40 promoter comprises an MsENOD40-l or an MsENOD40-2 promoter, and has substantially the sequence of SEQ ID NOT or SEQ ID NO:2. Preferably, the cell is a plant, bacterial, fungal, or animal cell, and in particular, plant cells such as those from Arahidopsis, corn, cotton, rye, flax, rice, canola, wheat, alfalfa, tobacco, tomato, potato, soybean, sunflower, citrus, nuts, fruits, berries, shrubs, cacti, succulents, grasses, and other ornamental plants, or alternatively, animal cells such as those from humans, monkeys, hamsters, caprines, such as goats, felines such as cats, canines such as dogs, horses and other equines, pigs and other porcines, rabbits and other lupines, and murines such as mice and rats. Of course, in certain embodiments, particularly in the preparation of recombinant vectors and the like, it may be desirable to prepare the constructs of the present invention for use in bacterial cells such as E. coli. A tumejaciens. and R. meliloti cells or in yeast.

In another embodiment the present invention provides a method of changing the characteristics of a cell. Characteristics include, but are not limited to, herbicide resistance, insect resistance, and fungal resistance. The method generally involves the expression of a gene operatively linked to an MsENOD40 promoter in the cell. Genes ( providing herbicide resistance include, but are not limited to, bar, pat, glyphosate tolerant EPSP synthase genes, the glyphosate degradative enzyme gene gox encoding glyphosate oxidoreductase, deh (encoding a dehalogenase enzyme that inactivates dalapon), acetolactate synthase, and bxn genes (encoding a nitrilase enzyme that degrades bromoxynil). Genes providing insect resistance include, but are not limited to, the crystal protein encoding genes obtained from Bacillus thuringiensis. Genes providing microbial resistance include, but are not limited to, genes encoding proteins such as b-1, 3- glucanases, chitinases , osmotin, UDA, and hevein.

In a preferred embodiment, the present invention relates to a recombinant vector comprising an MsENOD40 promoter operatively linked to a heterologous nucleic acid segment, in such an orientation as to control expression of said segment. The recombinant vector may be a plasmid, a cosmid, a YAC, a BAC, or a viral vector. Viral vectors include, but are not limited to, a bacteriophage vector, a Raus sarcoma virus vector, a p21 virus vector, an adeno-associated virus vector, and adenoviral vectors. Adenovirus vectors may be replication deficient of replication competent. In certain embodiments, the recombinant vector may be dispersed in a pharmaceutically acceptable solution.

In an important embodiment, the present invention relates to a nucleic acid segment comprising an MsENOD40 promoter having substantially the nucleotide sequence of SEQ ID NOT or SEQ ID NO:2 and operatively linked to a heterologous gene. Alternatively the nucleic acid segment may comprise a sequence having about 60% to about 65%, to about 70%, to about 75%. to about 80%, to about 85%, to about 90%. to about 95%o. even up to and including about 96%, about 97%, about 98%, or about 99% or greater sequence identity with either the sequence of SEQ ID NO: 1 or SEQ ID NO:2. Of course, the percent identity to the sequence of either SEQ ID NOT or SEQ ID NO:2 need not be limited to the specific percentages given, but is also meant to include all integers between about 60% and about 99% identity, such as percentage identities of about 86%, 87%. 88%. and 89%, or even about 91% or 92% or 93% or 94%, etc identity with the sequence of either SEQ ID NOT or SEQ ID NO:2. In fact, all such sequences are contemplated to fall within the scope of the present invention, so long as the particular sequence retains an ability to promote transcription of a nucleic acid segment operatively linked to the DNA sequence comprising the MsENOD40-l or MsENOD40-

2 promoter.

3.0 BRIEF DESCRIPTION OF THE DRAWINGS The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

FIG. IA, FIG. IB, FIG. 1C, FIG. ID, FIG. IE, and FIG. IF. Presented on six panels, the comparison of the promoter sequences of MsENOD40-l (top sequence) (SEQ ID NOT) and MsENOD40-2 (bottom sequence) (SEQ ID NO:2) is shown. The lines between the two sequences represent the identical nucleotides between them. Sequence analyses were performed on a VAX/VMS computer using program GAP in the UWGCG software package. Note that the first 1431 bp (1 -1431) of the 3' end were identical between the two promoters, and they are at the proximal end of the promoters (the unshaded region in FIG. 2). The remaining 5' distal sequences are only 40%o similar to each other. Approximately 200 bp of sequence of the 5' end of MsENOD40-

1 is not shown here, but the promoter sequence of MsENOD40-2 is complete. The bold letters indicate the Hinάlll restriction site. The single-underlined letters are the Bell site; the double-underlined letters indicate the putative TATA box in the promoter. The boxed regions are the putative "nodule-specific motifs".

FIG. 2. A diagram shows the restriction map of the constructs used for alfalfa transformation. The vector pBI101.3 is a promoterless construct, and is not shown here. The black box represent the reporter gene, uidA (gus). The unshaded box is the region shared by both promoters. The hatched box is the upstream region in the

MsENOD40-l promoter that is only 40% similar to the lined region in the MsENOD40-

2 promoter. Hind: Hindlll; Spe: Spel; Cla: Clal; Eco: EcoRV; Bel: Bell: Ace: Accl; Bam: BamHI.

FIG. 3A. The localization of Gus protein in the transgenic alfalfa plants in the absence of R meliloti. Transverse section of a root of plant line A27 (MsENOD40- 2). Blue color was detected in the stele, in the pericycle, endodermis, and inner cortex. Bar = 55 μm. FIG. 3B. The localization of Gus protein in the transgenic alfalfa plants in the absence of R. meliloti. A cross section of a stem of plant line A17 (MsENOD40-2). Blue color is localized to the stem procambium and the protophloem. Bar = 1 10 μm.

FIG. 3C. The localization of Gus protein in the transgenic alfalfa plants in the absence of R. meliloti. A longitudinal section of a root tip of plant line A 17 (MsENOD40-2). Note that the blue staining is present even in the root cap. Same magnification as FIG. 3B.

FIG. 3D. The localization of Gus protein in the transgenic alfalfa plants in the absence of R. meliloti. A lateral root primordium on transgenic plant line A27 (MsENOD40-2). Bar = 1 10 μm.

FIG. 3E. The localization of Gus protein in the transgenic alfalfa plants in the absence of R. meliloti. Elongated lateral roots on the same plant shown in FIG. 3D. Same magnification as FIG. 3D.

FIG. 3F. The localization of Gus protein in the transgenic alfalfa plants in the absence of R. meliloti. Elongated lateral roots on the same plant shown in FIG. 3D. Same magnification as FIG. 3D.

FIG. 3G. The localization of Gus protein in the transgenic alfalfa plants in the absence of R. meliloti. A root of plant line al8 (MsENOD40-l) without any treatment. Bar = 1 10 μm. FIG. 3H. The localization of Gus protein in the transgenic alfalfa plants in the absence of R. meliloti. A root of line al 8 4 d after treatment with 10^"6 M BAP. The arrowhead points to the root hairs. FIG. 3H is the same magnification as FIG. 3G.

FIG. 31. The localization of Gus protein in the transgenic alfalfa plants in the absence of R. meliloti. A root of line al8 4 d post-treatment with 10^" M purified Nod factor (PNF). FIG. 31 is the same magnification as FIG. 3G.

FIG. 3J. The localization of Gus protein in the transgenic alfalfa plants in the absence of R. meliloti. A root of plant line A27 (MsENOD40-2) without any treatment. FIG. 3.1 is the same magnification as FIG. 3G.

FIG. 3K. The localization of Gus protein in the transgenic alfalfa plants in the absence of R. meliloti. A 10^"6 Ivl BAP-treated root of line A27 after 4 d. The arrowhead points to the root hairs. Bar = 1 10 μm. FIG. 3L. The localization of Gus protein in the transgenic alfalfa plants in the absence of R. meliloti. A root of line A27 treated with 10^"8 M PNF for 4 d. FIG. 3L is the same magnification as FIG. 3K.

FIG. 3M. The localization of Gus protein in the transgenic alfalfa plants in the absence of R. meliloti. A root of line a23 treated with 10^"6 M 2,4-D for 4 d; no blue color is present. FIG. 3M is the same magnification as FIG. 3K.

FIG. 3N. The localization of Gus protein in the transgenic alfalfa plants in the absence of R. meliloti. A nodule primordium formed on plant line al 8

(MsENOD40-l) after BAP-treatment for 4 d. Bar = 55 μm. FIG. 3O. The localization of Gus protein in the transgenic alfalfa plants in the absence of R. meliloti. A cross-section of a primordium in FIG. 3N that was induced by BAP.

FIG. 3P. The localization of Gus protein in the transgenic alfalfa plants in the absence of R. meliloti. A primordium formed on plant line A27 (MsENOD40-2) after BAP treatment for 4 d. Same magnification as FIG. 3N.

FIG. 3Q. The localization of Gus protein in the transgenic alfalfa plants in the absence of R. meliloti. A nodule primordium formed on plant line A27

(MsENOD40-2) following 4 d of PNF-treatment. Bar = 55 μm. Arrows indicate starch grains and the arrowhead points to a root hair that is out of the plane of section. FIG. 4A. The localization of Gus protein during wild-type nodule development on roots of plants containing MsENOD40-l and MsENOD40-2 promoter- gus constructs. The roots illustrated here were flood-inoculated, but each developmental stage is comparable to that found by spot-inoculation. Plants shown here contained the MsENOD40-l promoter construct. Two areas showing the blue color on a root of plant line al 8. The area pointed by the small arrowheads shows the blue color mainly in the outer cortical cells and the epidermis. The area indicated by the large arrowhead shows the blue color in the dividing inner cortical cells, including the pericycle. Bar = 1 14 μm.

FIG. 4B. The localization of Gus protein during wild-type nodule development on roots of plants containing MsENOD40-l and MsENOD40-2 promoter- gus constructs. Shown here is an enlargement of the area indicated by the large arrowhead in FIG. 4A. Bar = 57 μm. FIG. 4C. The localization of Gus protein during wild-type nodule development on roots of plants containing MsENOD40-l and MsENOD40-2 promoter- gus constructs. Two adjacent areas show the blue color on a root of plant line a33 at two stages similar to FIG. 4A. Bar = 55 μm. FIG. 4D. The localization of Gus protein during wild-type nodule development on roots of plants containing MsENOD40-l and MsENOD40-2 promoter- gus constructs. A small nodule primordium on a root of plant line al 8 (cell divisions in the inner cortical cells). The blue staining of Gus protein is displayed mainly in the inner cortical cells and its derivatives as well as in the root pericycle. Bar = 1 10 μm. FIG. 4E. The localization of Gus protein during wild-type nodule development on roots of plants containing MsENOD40-l and MsENOD40-2 promoter- gus constructs. A well-developed nodule primordium on a root of plant line a33. Same magnification as FIG. 4D.

FIG. 4F. The localization of Gus protein during wild-type nodule development on roots of plants containing MsENOD40-l and MsENOD40-2 promoter- gus constructs. Nodule primordia/nodules at different developmental stages for MsENOD40-2 transgenic plants. The blue staining for Gus protein is present in the cortex as well as in the root stele. FIG. 4F was from plant line A4. Bar = 55 μm.

FIG. 4G. The localization of Gus protein during wild-type nodule development on roots of plants containing MsENOD40-l and MsENOD40-2 promoter- gus constructs. Nodule primordia/nodules at different developmental stages for MsENOD40-2 transgenic plants. The blue staining for Gus protein is present in the cortex as well as in the root stele. FIG. 4G was from plant line A27. Same magnification as FIG. 4B. FIG. 4H. The localization of Gus protein during wild-type nodule development on roots of plants containing MsENOD40-l and MsENOD40-2 promoter- gus constructs. Nodule primordia/nodules at different developmental stages for MsENOD40-2 transgenic plants. The blue staining for Gus protein is present in the cortex as well as in the root stele. FIG. 4H was from plant line A27. Same magnification as FIG. 4F.

FIG. 41. The localization of Gus protein during wild-type nodule development on roots of plants containing MsENOD40-l and MsENOD40-2 promoter- gus constructs. Nodule primordia/nodules at different developmental stages for MsENOD40-2 transgenic plants. The blue staining for Gus protein is present in the cortex as well as in the root stele. FIG. 41 was from plant line A27. Bar = 110 μm.

FIG. 4J. The localization of Gus protein during wild-type nodule development on roots of plants containing MsENOD40-l and MsENOD40-2 promoter- gus constructs. Nodules at different stages (post-primordium) on plant line al 8 or a33. FIG. 4J is the same magnification as FIG. 41.

FIG. 4K. The localization of Gus protein during wild-type nodule development on roots of plants containing MsENOD40-l and MsENOD40-2 promoter- gus constructs. Nodules at different stages (post-primordium) on plant line al 8 or a33. Bar = HO μm.

FIG. 4L. The localization of Gus protein during wild-type nodule development on roots of plants containing MsENOD40-l and MsENOD40-2 promoter- gus constructs. Nodules at different stages (post-primordium) on plant line al 8 or a33. FIG.4L is the same magnification as FIG. 4K.

FIG. 4M. The localization of Gus protein during wild-type nodule development on roots of plants containing MsENOD40-l and MsENOD40-2 promoter- gus constructs. A longitudinal section of a mature nodule on plant line b5-2 (containing construct pBIlal-2). FIG. 4M is the same magnification as FIG. 4K. Note that the blue color is present in the nodule meristem, in peripheral infected cells, and in cells associated with the nodule vascular bundles.

FIG. 5A. Gus localization during the development of ineffective nodules.

FIG. 5C represents different stages of nodules induced by 10^"5 M NPA on plants containing the MsENOD40-2 promoter construct (7 different lines were examined). The blue color is not only found in the nodule primordium, but also in the root cortex. Shown is a very young nodule primordium on plant line A27. Arrowheads indicate starch grains.

FIG. 5B. Gus localization during the development of ineffective nodules.

FIG. 5B represents different stages of nodules induced by 10° M NPA on plants containing the MsENOD40-2 promoter construct (7 different lines were examined).

The blue color is not only found in the nodule primordium, but also in the root cortex. Shown is an NPA-induced nodule on plant line A32. FIG. 5B is the same magnification as FIG. 5A. Bar = 1 10 μm.

FIG. 5C. Gus localization during the development of ineffective nodules.

FIG. 5C represents different stages of nodules induced by 10^"3 M NPA on plants containing the MsENOD40-2 promoter construct (7 different lines were examined). The blue color is not only found in the nodule primordium, but also in the root cortex. Dark-field picture of FIG. 5B. FIG. C is the same magnification as FIG. 5A. Bar = 1 10 μm.

FIG. 5D. Gus localization during the development of ineffective nodules. The blue color is not only found in the nodule primordium, but also in the root cortex. FIG. 5D shows the different stages of Rhizobium exo (strain 7094) mutant-induced nodule development (8 different lines were examined). A nodule primordium on plant line al 8 (MsENOD40-l). Bar = 55 μm.

FIG. 5E. Gus localization during the development of ineffective nodules. The blue color is not only found in the nodule primordium, but also in the root cortex. FIG. 5E shows the different stages of Rhizobium exo (strain 7094) mutant-induced nodule development (8 different lines were examined). A well-formed nodule primordium on plant line al 8 (MsENOD40-l). Arrowheads indicate starch grains. FIG. 5E is the same magnification as FIG. 5A. Bar = 1 10 μm. FIG. 5F. Gus localization during the development of ineffective nodules.

The blue color is not only found in the nodule primordium, but also in the root cortex. FIG. 5F shows the different stages of Rhizobium exo (strain 7094) mutant-induced nodule development (8 different lines were examined). A mature exo nodule on plant line A27. The blue color is mainly located in the apical and peripheral area of the nodule. FIG. 5F is the same magnification as FIG. 5A. Bar = 1 10 μm.

FIG. 5G. Gus localization during nodule formation on plants containing the composite promoter in pBIlal-7 (see FIG. 2H). The blue color is not only found in the nodule primordium. but also in the root cortex. An infection thread formed in a curled root hair on plant line H2. The picture was taken with Nomarski optics. Bar = 92 μm.

FIG. 5H. Gus localization during nodule formation on plants containing the composite promoter in pBIlal -7 (see FIG. 2H). The blue color is not only found in 16 the nodule primordium, but also in the root cortex. Bright-field picture of FIG. 5G, showing the blue color in the cortical cells where the infection thread has penetrated.

The arrow indicates the curled root hair.

FIG. 51. Gus localization during nodule formation on plants containing the composite promoter in pBI lal-7 (see FIG. 2H). The blue color is not only found in the nodule primordium, but also in the root cortex. A very young nodule primordium on plant line H35. Same magnification as FIG. 5 A.

FIG. 5J. Gus localization during nodule formation on plants containing the composite promoter in pBIlal-7 (see FIG. 2H). The blue color is not only found in the nodule primordium, but also in the root cortex. A nodule primordium on plant line

H2. Bar = 55 μm.

FIG. 5K. Gus localization during nodule formation on plants containing the composite promoter in pBI lal -7 (see FIG. 2FI). The blue color is not only found in the nodule primordium, but also in the root cortex. A young nodule on plant line HI 5. Bar = 460 μm.

FIG. 5L. Gus localization during nodule formation on plants containing the composite promoter in pBIlal -7 (see FIG. 2H). The blue color is not only found in the nodule primordium, but also in the root cortex. A section of a mature nodule formed on plant line H25. Bar = 1 10 μm. FIG. 5M. Gus localization during nodule formation on plants containing the composite promoter in pBI lal -7 (see FIG. 2H). The blue color is not only found in the nodule primordium. but also in the root cortex. Dark-field picture of FIG. 5L.

FIG. 6A. Plants containing plasmid pBI6cl -l (the full-length

MsENOD40-2 promoter). The results of Gus colorimetric assays in the transgenic alfalfa roots. Plants with construct pBI6cl-l . Gus activity is expressed as per mg per h per mL. Plants containing the same construct are represented with the same letter, e.g.. letter "A" for construct pBI6cl -l , "a" for pBIlal-1. "b" for pBIlal-2. "c" for pBI101.3.

"e" for pBIlal-4, and "f for pBI lal-5. The number following the letter represents an individual plant line; a different number represents a different transgenic plant. Plant roots were harvested after a 4 d-treatment with the following: FIG. 6C, Jensen's medium only; 10^"6 M BAP: 10^"8 M PNF; Rm, wild-type R. meliloti strain 1021. The line above and below the average values re apresent the standard deviation. If absent, the standard deviations were too small to be displayed.

FIG. 6B. Plants containing plasmid pBIlal-1 (the full-length MsENOD-1 promoter). The results of Gus colorimetric assays in the transgenic alfalfa roots. Plants with construct pBIlal-1. vGus activity is expressed as per mg per h per mL. Plants containing the same construct are represented with the same letter, e.g., letter "A" for construct pBI6cl-l, "a" for pBIlal-1, "b" for pBIlal-2, ^*'c" for pBI101.3, "e" for pBIlal-4, and "f for pBIlal-5. The number following the letter represents an individual plant line; a different number represents a different transgenic plant. Plant roots were harvested after a 4 d-treatment with the following: FIG. 6C, Jensen's medium only; 10^"6 M BAP; 10^"8 M PNF; Rm. wild-type R. meliloti strain 1021. The line above and below the average values represent the standard deviation. If absent, the standard deviations were too small to be displayed.

FIG. 6C. The results of Gus colorimetric assays in the transgenic alfalfa roots. Plants containing construct pBIlal -2. Gus activity is expressed as per mg per h per mL. Plants containing the same construct are represented with the same letter, e.g., letter "A" for construct pBI6cl-l , "a" for pBIlal-1, "b" for pBIlal-2, "c" for pBI101.3, "e" for pBIlal-4, and "f for pBIlal-5. The number following the letter represents an individual plant line; a different number represents a different transgenic plant. Plant roots were harvested after a 4 d-treatment with the following: FIG. 6C, Jensen's medium only; 10^"6 M BAP; 10^"8 M PNF; Rm, wild-type R. meliloti strain 1021. The line above and below the average values represent the standard deviation. If absent, the standard deviations were too small to be displayed.

FIG. 6D. The results of Gus colorimetric assays in the transgenic alfalfa roots. Plants containing construct pBIlal -4. Gus activity is expressed as per mg per h per mL. Plants containing the same construct are represented with the same letter, e.g., letter "A" for construct pBI6cl-l . "a" for pBIlal -1 , "b" for pBIl al -2, "c" for pBI101.3. "e" for pBIlal-4, and "f for pBIlal-5. The number following the letter represents an individual plant line; a different number represents a different transgenic plant. Plant roots were harvested after a 4 d-treatment with the following: FIG. 6C, Jensen's medium only; 10^"6 M BAP; 10^"8 M PNF; Rm. wild-type R. meliloti strain 1021. The IS line above and below the average values represent the standard deviation. If absent, the standard deviations were too small to be displayed.

FIG. 6E. The results of Gus colorimetric assays in the transgenic alfalfa roots. Plants containing construct pBIlal -5, the minimal promoter. Gus activity is expressed as per mg per h per mL. Plants containing the same construct are represented with the same letter, e.g., letter "A" for construct pBI6cl-l, "a" for pBIlal-1 , "b" for pBIlal -2, "c" for pBI101.3, "e" for pBIlal -4, and "f* for pBI lal -5. The number following the letter represents an individual plant line; a different number represents a different transgenic plant. Plant roots were harvested after a 4 d-treatment with the following: FIG. 6C, Jensen's medium only; 10^"6 M BAP; 10^"8 M PNF; Rm, wild-type R. meliloti strain 1021. The line above and below the average values represent the standard deviation. If absent, the standard deviations were too small to be displayed.

FIG. 6F. The results of Gus colorimetric assays in the transgenic alfalfa roots. Plants containing vector pBI101.3, the promoterless vector. Gus activity is expressed as per mg per h per mL. Plants containing the same construct are represented with the same letter, e.g., letter "A" for construct pBI6cl-l , "a" for pBIlal-1 , "b" for pBIlal-2, "c" for pBI101.3, "e" for pBIlal-4, and "f^* for pBIlal-5. The number following the letter represents an individual plant line; a different number represents a different transgenic plant. Plant roots were harvested after a 4 d-treatment with the following: FIG 6C, Jensen's medium only; 10^"6 M BAP; 10^"8 M PNF; Rm, wild-type R. meliloti strain 1021. The line above and below the average values represent the standard deviation. If absent, the standard deviations were too small to be displayed.

FIG. 7. Eppendorf tubes photographed under UV light. The tubes with arrows contain mycorrhizal root extracts (myc) that fluoresce more brightly than the control (con) root extracts after 4 h (top two rows) and 5 h (bottom two rows) of incubation. Tubes arranged in vertical rows represent individual MsENOD40 promoter-gw constructs. The plants for each construct are clonal. The tubes on the right (c) are positive controls; the roots had been treated with the cytokinin BAP, which also up-regulates the expression of MsENOD40. lb

4.0 DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

The effects of Nod factor on legume plants can be phenocopied by modulating the balance of endogenous plant hormones. For example, applying 2,3,5-triiodobenzoic acid (TIB A) or Λ -(naphthyl)phthalamic acid (NPA), which presumably block auxin polar transport in the root, elicits the formation of nodule-like structures (pseudonodules) on alfalfa, Afghanistan pea roots, or sweetclover sym mutants (Hirsch et al, 1989; Scheres et al, 1992; Wu et al, 1996a). Transcripts for the early nodulin genes, ENOD2, ENOD8 and ENOD12, were detected in these pseudonodules. Similarly, when alfalfa roots were inoculated with Rhizobium meliloti nodulation mutants carrying the Agrobacterium tumefaciens tzs gene (encoding DMA transferase, an enzyme which is associated with tπ s-zeatin biosynthesis; Beaty et al. 1986), small, uninfected nodules developed. MsENOD2 (Cooper and Long, 1994) and MsENOD40 (Hirsch et al, 1997) transcripts were detected in these nodules. Likewise, cytokinin application triggered ENOD2 expression in Sesbania roots (Dehio and deBruijn, 1992) and ENOD2 and ENOD12A expression in alfalfa roots (Bauer et al, 1996). Furthermore, exogenous cytokinin can bypass the block of a non-nodulating alfalfa mutant, M NN-1008, such that both MsENOD2 and MsENOD40 were expressed in the mutant roots, although at a reduced level when compared to the wild- type alfalfa level (Hirsch et al, 1997). These results suggest that a change in endogenous hormone level and/or sensitivities may be the consequence of Nod factor perception in a responsive host plant (Hirsch and Fang, 1994).

How the change of hormone (presumably auxin/cytokinin) balance is brought about remains unknown. However, the fact that some early nodulin (ENOD) genes are hormone-responsive may lead to the identification of specific transcription factors or cz^'s-acting elements that are involved in regulating these genes upon Nod factor application. Thus, studying the promoter elements of MsENOD40 should help us elucidate the c/s-acting region(s) responsible for its expression as well as those necessary for induction by Nod factors and/or cytokinin. In this way, the inventors can achieve a better understanding of the signal transduction pathway in the nitrogen-fixing symbiosis as well as plant hormone (especially cytokinin)-regulation of plant genes. » 7 4.1 SYMBIOSIS

Fungi colonize roots of most plants forming symbiotic associations called mycorrhizae. The arbuscular mycorrhizal symbiosis is one of the oldest and most widespread mutualistic associations on Earth. About 80% of modern plant species benefit from improved mineral uptake mediated by arbuscular mycorrhizal (AM) fungi

(Smith and Read, 1997). Based on fossils from the Devonian period, AM fungi are believed to have played an instrumental role in early land plant establishment and survival under the nutrient-limiting conditions of the terrestrial environment

(Pirozynski and Malloch, 1975; Simon et al, 1993; Remy et al, 1994). AM fungi colonize roots of most land plants and improve their uptake of phosphorous; most soils are phosphate-deficient. Absence of AM fungi from sites disturbed or created by human activities such as mining, construction or industrial waste disposal, severely hinders their revegetation (Pawlowska, 1991). Introduction of AM fungi during reclamation practices is therefore of great importance for the establishment of stable vegetation cover at these sites. AM fungi provide a substitute for chemical fertilizers in nutrient-poor soils. They also protect plants from drought and numerous pathogenic fungi (Smith and Read, 1997). None of these applications can be overlooked, especially when considering revegetation or agricultural practices in developing countries. Despite of all these potential benefits, the arbuscular mycorrhizal symbiosis is poorly understood mostly because of the inability to culture AM fungi in the absence of the host. Without the knowledge of the principles governing symbiont recognition and root colonization, this symbiotic association cannot be fully exploited.

Nevertheless, even though AM fungi (Glomales, Zygomycetes) are obligate biotrophs, they reproduce by asexual spores formed mostly outside of the host roots, and each new host generation is colonized de novo (Becard and Fortin, 1988).

Based on several lines of evidence from legume plants, whose roots can harbor both phosphate-acquiring AM fungi and nitrogen-fixing symbiotic bacteria, e.g. , Rhizobium and Bradyrhizobium, it has been postulated that these two symbiotic associations share the same host-microorganism recognition pathway (LaRue and Weeden, 1994). Arbuscular mycorrhizae and the Rhizobium-legume symbiosis differ vastly in their biology and temporal origin based on geological time. The Rhizobium- legume symbiosis involves the formation of a new organ, the nodule in which the bacteria fix atmospheric nitrogen into ammonia. It also has been in existence only since the evolution of the angiosperms. The biology of the Rhizobium-legume symbiosis has long attracted attention due to its commercial importance in agriculture. It has been estimated that biological organisms fix 255 million tons of nitrogen each year, and that some 175 million tons (69% of all the nitrogen gas reduced) are produced by the Rhizobium-legume symbiosis.

The Rhizobium-legume symbiosis in contrast to that between plants and AM fungi is highly specific. The recognition specificity is brought about by Nod factors synthesized by the action of rhizobial nod genes (Fisher and Long, 1992). Nod factor is a complex lipochitooligosaccharide; various R-groups on the reducing and non- reducing ends of the chitin backbone impart specificity. For example, R. meliloti, which nodulates alfalfa and not pea, produces a sulfated (at the R, position) Nod factor. If the sulfate is removed, then this Nod factor is no longer active on alfalfa, but it is active on pea. Consequently, each legume host must have a specific receptor that recognizes a particular Nod factor.

When bacterial cells that produce Nod factor or even if the Nod factor molecule alone comes in contact with susceptible legume root hair cells, a cascade of physiological and morphological changes leading to root colonization, nodule formation and, if Rhizobium bacteria are present, nitrogen fixation itself, are induced. Nod factor receptors are presumed to be located on the root hairs which can be invaded by rhizobia. When these Nod factor receptor are no longer expressed or if the gene encoding the Nod factor receptor is mutated, then the root hairs are no longer responsive to rhizobia.

Despite its ubiquity and importance for plants, the molecular aspects of arbuscular mycorrhizal symbiosis are much less investigated than those of the Rhizobium-legume symbiosis. Elucidation of additional similarities between recognition cascades in arbuscular mycorrhizal and Rhizobium-legume symbioses may have enormous implications for the breeding of crop species. Arbuscular mycorrhizae are found in many important crop plants such as rice or corn, whereas the Rhizobium- legume symbiosis is restricted to legumes. If the signal transduction pathways are indeed conserved between these two plant-microbe interactions, it may be possible to utilize the recognition pathway already existing in AM plants for the purpose of N genetically engineering the roots of non-legume plants so that they support rhizobial colonization and ultimately, nitrogen fixation.

Transformed rice roots containing a construct consisting of the MsENOD12 promoter (ENOD12 is an early nodulin gene which is expressed in legumes upon Rhizobium inoculation; see below) and the reporter gene gusA turn blue upon treatment with Rhizobium Nod factor. It can be inferred from these studies that plants that are mycorrhizal may have receptor proteins that recognize Nod factor, and perhaps these receptors can be manipulated in non-nodulating plants to interact effectively with

Rhizobium. Some of the strongest evidence indicating that signal transduction pathways for these two interactions may be conserved comes from genetics. Studies of legume mutants unable to form nodules upon inoculation with rhizobia demonstrated that AM fungi do not colonize their roots either (Due et al, 1989; Gianinazzi-Pearson et al, 1991 ; Bradbury et al, 1991). Genetic analysis of non-nodulating and non-mycorrhizal plant phenotypes has shown that they are caused by mutations in the same gene (Bradbury et al, 1993). This implies that a receptor for Nod factor is also a receptor for a putative Myc factor.

The fact that both symbiotic associations are affected by flavonoids also suggests that there are other shared elements. Plant-derived flavonoids induce expression of rhizobial nod genes, described above as being responsible for the biosynthesis of Nod factor, the molecule necessary for recognition between symbionts (Hirsch, 1992). However, a chitin-like molecule such as Nod factor is quite an unusual product for Gram-negative bacteria. Typically, fungi, including AM fungi, produce a chitin-based cell wall. Nod factor's role in symbiont recognition suggests that it is a product of convergent evolution and provides rhizobia with a tool to mimic AM fungi in the early stages of root colonization (LaRue and Weeden, 1994). Similar to rhizobia, AM fungi are significantly affected by flavonoids which induce spore germination and hyphal proliferation resulting in greater physical contact between the symbiotic partners (Gianinazzi-Pearson et al, 1989; Becard et al, 1992). Another parallel between the two symbioses is host behavior upon penetration of root cells by rhizobia or AM fungi. In both cases, an initial transient burst of defense responses does not prevent root colonization (Harrison and Dixon, 1993, 1994; Volpin 2. et al, 1994, 1995). Thus, symbiotic microbes, in contrast to pathogenic bacteria and fungi, suppress a host defense response. Nod factor may have evolved to converge with the structure of an AM-fungal wall component for this very purpose.

4.2 NOMENCLATURE OF THE NOVEL PROMOTERS

The inventors have arbitrarily assigned the designations MsENOD40-l and MsENOD40-2 to the novel promoters of the invention, any re-designations of the compositions of the present invention are also contemplated to be fully within the scope of the present disclosure.

4.3 CHARACTERISTICS OF THE MsENOD40 PROMOTERS

The present invention provides promoter regions allowing expression of functional RNA.

In a preferred embodiment, the invention discloses and claims the MsENOD40- 1 promoter region. The MsENOD40-l promoter region comprises a 2531 bp nucleic acid sequence, which is given in SEQ ID NOT, and shown in FIG. IA, FIG. IB, FIG. 1C, FIG. ID, FIG. IE, and FIG. IF.

In a second embodiment, the invention discloses and claims the MsENOD40-2 promoter region. The MsENOD40-2 promoter region comprises a 2520 bp nucleic acid sequence, which is given in SEQ ID NO:2, and shown in FIG. IA, FIG. IB, FIG. 1C, FIG. ID, FIG. IE, and FIG. IF.

4.4 INDUCERS

The promoters described by this invention provide for inducible expression of sequences that are operatively linked to said promoters. In a preferred embodiment, induction of gene expression is provided by plant hormones. In the most preferred embodiment, the plant hormone is a cytokinin. Exemplary cytokinins include for example, 6-benzylaminopurine (BAP), which the inventors have shown to be particularly useful in increasing the activity of the disclosed promoters. Other compounds related to BAP as well as other cytokinin molecules are also contemplated to be particularly useful in the methods disclosed herein. In fact, the use of any molecule or inducer which regulates the activity of the disclosed promoters are contemplated to be useful. The inventors contemplate that one could either directly contact a transformed cell or a transgenic plant or animal comprising the promoter constructs disclosed herein with such an inducer, or alternatively, in the case of plant cell transformation, contact the plant with one or more substances which increase the production of the particular cytokinin. such as BAP, and thus cause an increase in trascription from the promoter constructs. Particularly, the inventors believe that the presence of compounds such as nod factors, chitin, chitin fragments, chitin extracts, cytokinin regulatory proteins, or even infection or symbiosis with bacterial species such as Rhizobium may affect the production of cytokinins in the cell, or even may directly or indirectly affect the transcriptional promoting activity of the particular MsENOD promoter constructs).

4.5 RECOMBINANT HOST CELLS

The nucleotide sequences of the subject invention can be introduced a gene of interest into a wide variety of microbial hosts. Suitable host cells may include either prokaryotes or eukaryotes, normally being limited to those cells which do not produce substances toxic to higher organisms, such as mammals. However, organisms which produce substances toxic to higher organisms could be used, where the toxin is unstable or the level of application sufficiently low as to avoid any possibility or toxicity to a mammalian host. As hosts, of particular interest will be the prokaryotes and the lower eukaryotes. such as fungi. Illustrative prokaryotes, both Gram-negative and Gram- positive, include Enterobacteriaceae, such as Escherichia, Erwinia, Shigella, Salmonella, and Proteus; Bacillaceae; Rhizobiceae, such as Rhizobium; Spirillaceae, such as photobacterium, Zymomonas, Serratia, Aeromonas, Vibrio, Desulfυvibrio, Spirillum; Lactobacillaceae; Pseudomonadaceae, such as Pseudomonas and Acetobacter; Azotobacteraceae, Actinomycetales, and Nitrobacteraceae. Among eukaryotes are fungi, such as Phycomycetes and Ascomycetes, which includes yeast, such as Saccharomyces and Schizosaccharomyces; and Basidiomycetes yeast, such as Rhodotorula, Aureobasidium, Sporobolomyces, and the like. Characteristics of particular interest in selecting a host cell for purposes of production include ease of introducing the promoters of the present invention and the gene of interest into the host, availability of expression systems, efficiency of expression. stability of the gene of interest in the host, and the presence of auxiliary genetic capabilities. Characteristics of interest include protective qualities for the gene product of interest, such as thick cell walls, pigmentation, and intracellular packaging or formation of inclusion bodies; leaf affinity; lack of mammalian toxicity; ease of killing and fixing without damage to the gene product of interest; and the like. Other considerations include ease of formulation and handling, economics, storage stability, and the like.

Host organisms of particular interest include yeast, such as Rhodotorula sp., Aureobasidium sp., Saccharomyces sp., and Sporobolomyces sp.; phylloplane organisms such as Pseudomonas sp., Erwinia sp. and Flavohacterium sp. ; or such other organisms as Escherichia, Lactobacillus sp., Bacillus sp., Streptomyces sp., and the like. Specific organisms include Pseudomonas aeruginosa. Pseudomonas fluorescens. Saccharomyces cerevisiae, Bacillus thuringiensis, Escherichia coli, Bacillus subtilis, Bacillus megaterium, Bacillus cereus. Streptomyces lividans and the like.

Treatment of the microbial cell, e.g., a microbe containing promoters of the present invention and the gene of interest, can be by chemical or physical means, or by a combination of chemical and/or physical means, so long as the technique does not deleteriously affect the properties of the product of the gene of interest, nor diminish the cellular capability in protecting the product. Examples of chemical reagents are halogenating agents, particularly halogens of atomic no. 17-80. More particularly, iodine can be used under mild conditions and for sufficient time to achieve the desired results. Other suitable techniques include treatment with aldehydes, such as formaldehyde and glutaraldehye: anti-infectives, such as zephiran chloride and cetylpyridinium chloride; alcohols, such as isopropyl and ethanol; various histologic fixatives, such as Lugol's iodine, Bouin's fixative, and Helly's fixatives, ( e.g., Humason, 1967); or a combination of physical (heat) and chemical agents that preserve and prolong the activity of the toxin produced in the cell when the cell is administered to the host animal. Examples of physical means are short wavelength radiation such as γ-radiation and X-radiation, freezing. UV irradiation, lyophilization. and the like. The cells employed will usually be intact and be substantially in the proliferative form when 33 treated, rather than in a spore form, although in some instances spores may be employed.

Where a promoter of the current invention and gene of interest are introduced into a microbial host, and said host is applied to the environment in a living state, it is essential that certain host microbes be used. Microorganism hosts are selected which are known to occupy the "phytosphere" (phylloplane, phyllosphere, rhizosphere, and/or rhizoplane) of one or more crops of interest. These microorganisms are selected so as to be capable of successfully competing in the particular environment (crop and other insect habitats) with the wild-type microorganisms, provide for stable maintenance and expression of the gene of interest, and, desirably, provide for improved protection of the gene product from environmental degradation and inactivation.

A large number of microorganisms are known to inhabit the phylloplane (the surface of the plant leaves) and/or the rhizosphere (the soil surrounding plant roots) of a wide variety of important crops. These microorganisms include bacteria, algae, and fungi. Of particular interest are microorganisms, such as bacteria, e.g., genera Bacillus, Pseudomonas, Erwinia, Serratia, Klebsiella, Zanthomonas, Streptomyces, Rhizobium, Rhodopseudomonas, Methylophilius, Agrobacterium, Acetobacter, Lactobacillus, Arthrobacter, Azotobacter, Leuconostoc, and Alcaligenes; fungi, particularly yeast, e.g. , genera Saccharomyces, Cryptococcus, Kluyveromyces, Sporobolomyces, Rhodotorula, and Aureobasidium. Of particular interest are such phytosphere bacterial species as Pseudomonas syringae, Pseudomonas fluorescens, Serratia marcescens, Acetobacter xylinum, Agrobacterium tumefaciens, Rhodobacter sphaeroides, Xanthomonas campestris, Rhizobium melioti, Alcaligenes eutrophus, and Azotobacter vinlandii; and phytosphere yeast species such as Rhodotorula rubra, R. glutinis, R. marina, R. aurantiaca. Cryptococcus alhidus, C. diffluens, C. laurentii, Saccharomyces rosei, S. pretoriensis, S. cerevisiae, Sporobolomyces roseus, S. odorus, Kluyveromyces veronae, and Aureobasidium pollulans.

4.6 DNA SEQUENCES Virtually any DNA composition may be used for delivery to recipient plant cells to ultimately produce fertile transgenic plants in accordance with the present invention. For example, DNA segments in the form of vectors and plasmids, or linear DNA fragments, in some instances containing only the DNA element to be expressed in the plant, and the like, may be employed.

In certain embodiments, it is contemplated that one may wish to employ replication-competent viral vectors in plant transformation. Such vectors for monocots include, for example, wheat dwarf virus (WDV) and "shuttle" vectors, such as pWl-1 1 and PW1-GUS (Ugaki et al, 1991 ). Likewise any virus which infects a plant for which introduction of the promoter-nucleic acid constructs described herein is desired, is also considered to fall within the scope of the present invention. Such viruses are well- known to those of skill in the art in the area of plant transformation, and plant virology, and any such suitable construct is contemplated to be useful for delivery of the nucleic acid segments disclosed herein. For example, viruses which infect cotton would be optimal hosts for introduction of the constructs into cotton plants. Likewise, viruses which attack alfalfa plants would be useful for introduction into alfalfa. Tobacco mosaic virus, for example, may also prove to be useful in introduction of constructs into those plant species such as tobacco, for which this virus is a pathogen.

As an example of the use of the promoters of the present invention in a plant species, such as corn, the WDV "shuttle" vectors are capable of autonomous replication in maize cells as well as E. coli, and as such may provide increased sensitivity for detecting DNA delivered to transgenic cells. A replicating vector may also be useful for delivery of genes flanked by DNA sequences from transposable elements such as Ac. Ds, or Mu. It has been proposed (Laufs et al, 1990) that transposition of these elements within the plant genome requires DNA replication. It is also contemplated that transposable elements would be useful for introducing DNA fragments lacking elements necessary for selection and maintenance of the plasmid vector in bacteria, e.g., antibiotic resistance genes and origins of DNA replication. It is also proposed that use of a transposable element such as Ac, Ds, or Mu would actively promote integration of the desired DNA and hence increase the frequency of stably transformed cells.

Vectors, plasmids. phagemids, cosmids, viral vectors, shuttle vectors, baculovirus vectors, BACs (bacterial artificial chromosomes), HACs (human artificial chromosomes), YACs (yeast artificial chromosomes) and DNA segments for use in transforming cells with a nucleic acid construct of interest, will, of course, generally comprise at least one MsΕNOD40 promoter region, and in particular, at least one or more MsENOD40-l or MsENOD40-2 promoters, such as those in SEQ ID NOT or SEQ ID NO:2, or alternatively, will comprise at least one or more promoters which have at least about 80% or 85% or 90%> or 95% sequence identity to the promoters disclosed in SEQ ID NOT or SEQ ID NO:2. These DNA constructs may contain a cDNA, or one or more genes which one desires to introduce into the cells. These DNA constructs can include structures such as promoters, enhancers, polylinkers, or even regulatory genes as desired. The DNA segment or gene chosen for cellular introduction will often encode a protein which will be expressed in the resultant recombinant cells, such as will result in a screenable or selectable trait and/or which will impart an improved phenotype to the regenerated plant. However, this may not always be the case, and the present invention also encompasses transgenic plants incorporating non- expressed transgenes. Alternatively, the nucleic acid constructs may contain antisense constructs, or ribozyme-encoding regions when delivery or introduction of such nucleic acid constructs are desirable.

4.6.1 METHODS FOR PREPARING MUTAGENIZED MsENOD40 PROMOTERS

In certain circumstances, it may be desirable to modify or alter one or more nucleotides in one or more of the promoter sequences disclosed herein for the purpose of altering or changing the trasncriptional activity or other property of the promoter region. In general, the means and methods for mutagenizing a DNA segment such as one comprising an MsENOD40 promoter region are well-known to those of skill in the art. Modifications to such promoter regions may be made by random, or site-specific mutagenesis procedures. The promoter region may be modified by altering its structure through the addition or deletion of one or more nucleotides from the sequence which encodes the corresponding un-modified promoter region.

Mutagenesis may be performed in accordance with any of the techniques known in the art such as and not limited to synthesizing an oligonucleotide having one or more mutations within the sequence of a particular promoter region. In particular, site- specific mutagenesis is a technique useful in the preparation of promoter mutants, through specific mutagenesis of the underlying DNA. The technique further provides a ready ability to prepare and test sequence variants, for example, incorporating one or more of the foregoing considerations, by introducing one or more nucleotide sequence changes into the DNA. Site-specific mutagenesis allows the production of mutants through the use of specific oligonucleotide sequences which encode the DNA sequence of the desired mutation, as well as a sufficient number of adjacent nucleotides, to provide a primer sequence of sufficient size and sequence complexity to form a stable duplex on both sides of the deletion junction being traversed. Typically, a primer of about 17 to about 75 nucleotides or more in length is preferred, with about 10 to about 25 or more residues on both sides of the junction of the sequence being altered.

In general, the technique of site-specific mutagenesis is well known in the art, as exemplified by various publications. As will be appreciated, the technique typically employs a phage vector which exists in both a single stranded and double stranded form. Typical vectors useful in site-directed mutagenesis include vectors such as the Ml 3 phage. These phage are readily commercially available and their use is generally well known to those skilled in the art. Double stranded plasmids are also routinely employed in site directed mutagenesis which eliminates the step of transferring the gene of interest from a plasmid to a phage.

In general, site-directed mutagenesis in accordance herewith is performed by first obtaining a single-stranded vector or melting apart of two strands of a double stranded vector which includes within its sequence a DNA sequence which encodes the desired promoter region or peptide. An oligonucleotide primer bearing the desired mutated sequence is prepared, generally synthetically. This primer is then annealed with the single-stranded vector, and subjected to DNA polymerizing enzymes such as E. coli polymerase I Klenow fragment, in order to complete the synthesis of the mutation-bearing strand. Thus, a heteroduplex is formed wherein one strand encodes the original non-mutated sequence and the second strand bears the desired mutation. This heteroduplex vector is then used to transform or transfect appropriate cells, such as E. coli cells, and clones are selected which include recombinant vectors bearing the mutated sequence arrangement. A genetic selection scheme was devised by Kunkel et al. (1987) to enrich for clones incorporating the mutagenic oligonucleotide. Alternatively, the use of PCR™ with commercially available thermostable enzymes such as Tag polymerase may be used to incorporate a mutagenic oligonucleotide primer into an amplified DNA fragment that can then be cloned into an appropriate cloning or expression vector. The PCR™-mediated mutagenesis procedures of Tomic et al. an

(1990) and Upender et al. (1995) provide two examples of such protocols. A PCR™ employing a thermostable ligase in addition to a thermostable polymerase may also be used to incorporate a phosphorylated mutagenic oligonucleotide into an amplified DNA fragment that may then be cloned into an appropriate cloning or expression vector. The mutagenesis procedure described by Michael (1994) provides an example of one such protocol.

The preparation of sequence variants of the selected promoter-encoding DNA segments using site-directed mutagenesis is provided as a means of producing potentially useful species and is not meant to be limiting as there are other ways in which sequence variants of DNA sequences may be obtained. For example, recombinant vectors encoding the desired promoter sequence may be treated with mutagenic agents, such as hydroxylamine, to obtain sequence variants.

As used herein, the term "oligonucleotide directed mutagenesis procedure" refers to template-dependent processes and vector-mediated propagation which result in an increase in the concentration of a specific nucleic acid molecule relative to its initial concentration, or in an increase in the concentration of a detectable signal, such as amplification. As used herein, the term "oligonucleotide directed mutagenesis procedure" also is intended to refer to a process that involves the template-dependent extension of a primer molecule. The term template-dependent process refers to nucleic acid synthesis of an RNA or a DNA molecule wherein the sequence of the newly synthesized strand of nucleic acid is dictated by the well-known rules of complementary base pairing (Watson, 1987). Typically, vector mediated methodologies involve the introduction of the nucleic acid fragment into a DNA or RNA vector, the clonal amplification of the vector, and the recovery of the amplified nucleic acid fragment. Examples of such methodologies are provided by U. S. Patent No. 4,237,224, specifically incorporated herein by reference in its entirety.

A number of template dependent processes are available to amplify the target sequences of interest present in a sample. One of the best known amplification methods is the polymerase chain reaction (PCR™) which is described in detail in U. S. Patent Nos. 4,683,195, 4,683,202 and 4,800,159, each of which is incorporated herein by reference in its entirety. Briefly, in PCR™, two primer sequences are prepared which are complementary to regions on opposite complementary strands of the target sequence. An excess of deoxynucleoside triphosphates are added to a reaction mixture along with a DNA polymerase (e.g., Taq polymerase). If the target sequence is present in a sample, the primers will bind to the target and the polymerase will cause the primers to be extended along the target sequence by adding on nucleotides. By raising and lowering the temperature of the reaction mixture, the extended primers will dissociate from the target to form reaction products, excess primers will bind to the target and to the reaction products and the process is repeated. Preferably a reverse transcriptase PCR™ amplification procedure may be performed in order to quantify the amount of mRNA amplified. Polymerase chain reaction methodologies are well known in the art.

Another method for amplification is the Iigase chain reaction (referred to as LCR), disclosed in Eur. Pat. Appl. Publ. No. 320,308, incorporated herein by reference in its entirety. In LCR, two complementary probe pairs are prepared, and in the presence of the target sequence, each pair will bind to opposite complementary strands of the target such that they abut. In the presence of a Iigase, the two probe pairs will link to form a single unit. By temperature cycling, as in PCR™, bound ligated units dissociate from the target and then serve as "target sequences" for ligation of excess probe pairs. U. S. Patent No. 4,883,750, incorporated herein by reference in its entirety, describes an alternative method of amplification similar to LCR for binding probe pairs to a target sequence.

Qbeta Replicase, described in Intl. Pat. Appl. Publ. No. PCT/US87/00880. incorporated herein by reference in its entirety, may also be used as still another amplification method in the present invention. In this method, a replicative sequence of RNA which has a region complementary to that of a target is added to a sample in the presence of an RNA polymerase. The polymerase will copy the replicative sequence which can then be detected.

An isothermal amplification method, in which restriction endonucleases and ligases are used to achieve the amplification of target molecules that contain nucleotide 5'-[α-thio]triphosphates in one strand of a restriction site (Walker et al, 1992. incorporated herein by reference in its entirety), may also be useful in the amplification of nucleic acids in the present invention. A

Strand Displacement Amplification (SDA) is another method of carrying out isothermal amplification of nucleic acids which involves multiple rounds of strand displacement and synthesis, i.e. nick translation. A similar method, called Repair Chain Reaction (RCR) is another method of amplification which may be useful in the present invention and is involves annealing several probes throughout a region targeted for amplification, followed by a repair reaction in which only two of the four bases are present. The other two bases can be added as biotinylated derivatives for easy detection. A similar approach is used in SDA.

Still other amplification methods described in Great Britain Pat. Appl. No. 2 202 328, and in Intl. Pat. Appl. Publ. No. PCT/US89/01025, each of which is incorporated herein by reference in its entirety, may be used in accordance with the present invention. In the former application, "modified" primers are used in a PCR™ like, template and enzyme dependent synthesis. The primers may be modified by labeling with a capture moiety (e.g., biotin) and/or a detector moiety (e.g., enzyme). In the latter application, an excess of labeled probes are added to a sample. In the presence of the target sequence, the probe binds and is cleaved catalytically. After cleavage, the target sequence is released intact to be bound by excess probe. Cleavage of the labeled probe signals the presence of the target sequence.

Other nucleic acid amplification procedures include transcription-based amplification systems (TAS) (Kwoh et al, 1989; Intl. Pat. Appl. Publ. No. WO 88/10315, incorporated herein by reference in its entirety), including nucleic acid sequence based amplification (NASBA) and 3SR. In NASBA, the nucleic acids can be prepared for amplification by standard phenol/chloroform extraction, heat denaturation of a sample, treatment with lysis buffer and minispin columns for isolation of DNA and RNA or guanidinium chloride extraction of RNA. These amplification techniques involve annealing a primer which has crystal protein-specific sequences. Following polymerization, DNA/RNA hybrids are digested with RNase H while double stranded DNA molecules are heat denatured again. In either case the single stranded DNA is made fully double stranded by addition of second crystal protein-specific primer, followed by polymerization. The double stranded DNA molecules are then multiply transcribed by a polymerase such as T7 or SP6. In an isothermal cyclic reaction, the RNAs are reverse transcribed into double stranded DNA. and transcribed once against with a polymerase such as T7 or SP6. The resulting products, whether truncated or complete, indicate crystal protein-specific sequences.

Eur. Pat. Appl. Publ. No. 329,822, incorporated herein by reference in its entirety, disclose a nucleic acid amplification process involving cyclically synthesizing single-stranded RNA ("ssRNA"), ssDNA, and double-stranded DNA (dsDNA), which may be used in accordance with the present invention. The ssRNA is a first template for a first primer oligonucleotide, which is elongated by reverse transcriptase

(RNA-dependent DNA polymerase). The RNA is then removed from resulting

DNA:RNA duplex by the action of ribonuclease H (RNase H, an RNase specific for RNA in a duplex with either DNA or RNA). The resultant ssDNA is a second template for a second primer, which also includes the sequences of an RNA polymerase promoter (exemplified by T7 RNA polymerase) 5' to its homology to its template. This primer is then extended by DNA polymerase (exemplified by the large "Klenow" fragment of E. coli DNA polymerase I), resulting as a double-stranded DNA ("dsDNA") molecule, having a sequence identical to that of the original RNA between the primers and having additionally, at one end, a promoter sequence. This promoter sequence can be used by the appropriate RNA polymerase to make many RNA copies of the DNA. These copies can then re-enter the cycle leading to very swift amplification. With proper choice of enzymes, this amplification can be done isothermallv without addition of enzymes at each cycle. Because of the cyclical nature of this process, the starting sequence can be chosen to be in the form of either DNA or RNA.

PCT Intl. Pat. Appl. Publ. No. WO 89/06700, incorporated herein by reference in its entirety, disclose a nucleic acid sequence amplification scheme based on the hybridization of a promoter/primer sequence to a target single-stranded DNA ("ssDNA") followed by transcription of many RNA copies of the sequence. This scheme is not cyclic; i.e. new templates are not produced from the resultant RNA transcripts. Other amplification methods include "RACE" (Frohman, 1990), and "one-sided PCR™" (Ohara. 1989) which are well-known to those of skill in the art. Methods based on ligation of two (or more) oligonucleotides in the presence of nucleic acid having the sequence of the resulting "di-oligonucleotide", thereby amplifying the di-oligonucleotide (Wu and Dean, 1996. incorporated herein by reference in its entirety), may also be used in the amplification of DNA sequences of the present invention.

4.6.2 REGULATORY ELEMENTS Constructs will include the promoters of the present invention functionally linked to a gene of interest, and optionally including a 3' end DNA sequence that acts as a signal to terminate transcription and allow for the poly-adenylation of the resultant mRNA. The most preferred 3' elements are contemplated to be those from the nopaline synthase gene of Agrobacterium tumefasciens (Bevan et al, 1983), the terminator for the T7 transcript from the octopine synthase gene of Agrobacterium tumefasciens, and the 3' end of the protease inhibitor I or II genes from potato or tomato. Regulatory elements such as Adh intron 1 (Callis et al, 1987), sucrose synthase intron (Vasil et al , 1989) or TMV omega element (Gallie, et al, 1989), may further be included where desired. As the DNA sequence between the transcription initiation site and the start of the coding sequence, i.e. the untranslated leader sequence, can influence gene expression, one may also wish to employ a particular leader sequence. Preferred leader sequences are contemplated to include those which include sequences predicted to direct optimum expression of the attached gene, i.e. to include a preferred consensus leader sequence which may increase or maintain mRNA stability and prevent inappropriate initiation of translation. The choice of such sequences will be known to those of skill in the art in light of the present disclosure. Sequences that are derived from genes that are highly expressed in plants, and in alfalfa in particular, will be most preferred. In one embodiment, the inventors contemplate that vectors comprising the promoters of the present invention may be constructed to include an enhancer element such as an ocs element. This element was first identified as a 16 bp palindromic enhancer from the octopine synthase (ocs) gene of Agrobacterium (Ellis et al. 1987), and is present in at least 10 other promoters (Bouchez et al. 1989). It is proposed that the use of an enhancer element, such as the ocs element and particularly multiple copies of the element, will act to increase the level of transcription from adjacent promoters when applied in the context of plant transformation. 3^ 4.6.3 GENES OF INTEREST

Ultimately, the most desirable DNA segments for introduction into a plant genome may be homologous genes or gene families which encode a desired trait (e.g., increased yield per acre) in said plant. Indeed, it is envisioned that a particular use of the promoters of the present invention will be the targeting of a gene in a tissue-specific manner using the promoter of SEQ ID NOT . For example, genes encoding proteins with particular activity against rootworm may be targeted directly to root tissues.

With respect to tissue specific expression, it is contemplated that an antisense gene may be introduced that is expressed only in those tissues where the gene product is not desired. Furthermore, it is contemplated that the tissue specific expression of an antisense gene could be used in conjunction with a ubiquitously expressed transgene to allow expression only in those tissues which lack antisense gene expression.

Additionally, vectors may be constructed and employed in the intracellular targeting of a specific gene product within the cells of a transgenic plant or in directing a protein to the extracellular environment. This will generally be achieved by joining a DNA sequence encoding a transit or signal peptide sequence to the coding sequence of a particular gene. The resultant transit, or signal, peptide will transport the protein to a particular intracellular, or extracellular destination, respectively, and will then be post- translationally removed. Transit or signal peptides act by facilitating the transport of proteins through intracellular membranes, e.g., vacuole, vesicle, plastid and mitochondrial membranes, whereas signal peptides direct proteins through the extracellular membrane.

A particular example of such a use concerns the direction of a herbicide resistance gene, such as the EPSPS gene, to a particular organelle such as the chloroplast rather than to the cytoplasm. This is exemplified by the use of the rbcS transit peptide which confers plastid-specific targeting of proteins. In addition, it is proposed that it may be desirable to target certain genes responsible for male sterility to the mitochondria, or to target certain genes for resistance to phytopathogenic organisms to the extracellular spaces, or to target proteins to the vacuole.

It is also contemplated that it may be useful to target DNA itself within a cell. For example, it may be useful to target introduced DNA to the nucleus as this may increase the frequency of transformation. Within the nucleus itself it would be useful to target a gene in order to achieve site specific integration. For example, it would be useful to have a gene introduced through transformation replace an existing gene in the cell.

4.6.4 MARKER GENES

In order to improve the ability to identify transformants, one may desire to employ a selectable or screenable marker gene as, or in addition to, the expressible gene of interest. "Marker genes" are genes that impart a distinct phenotype to cells expressing the marker gene and thus allow such transformed cells to be distinguished from cells that do not have the marker. Such genes may encode either a selectable or screenable marker, depending on whether the marker confers a trait which one can ^"select' for by chemical means, i.e., through the use of a selective agent (e.g., a herbicide, antibiotic, or the like), or whether it is simply a trait that one can identify through observation or testing, i.e., by ^'screening' (e.g., the R-locus trait). Of course, many examples of suitable marker genes are known to the art and can be employed in the practice of the invention.

Included within the terms selectable or screenable marker genes also are genes which encode a "secretable marker" whose secretion can be detected as a means of identifying or selecting for transformed cells. Examples include markers which encode a secretable antigen that can be identified by antibody interaction, or even secretable enzymes which can be detected by their catalytic activity. Secretable proteins fall into a number of classes, including small, diffusible proteins detectable, e.g., by ELISA; small active enzymes detectable in extracellular solution (e.g., -amylase, β-lactamase, phosphinothricin acetyltransferase); and proteins that are inserted or trapped in the cell wall (e.g., proteins that include a leader sequence such as that found in the expression unit of extensin or tobacco PR-S).

With regard to selectable secretable markers, the use of a gene that encodes a protein that becomes sequestered in the cell wall, and which protein includes a unique epitope is considered to be particularly advantageous. Such a secreted antigen marker would ideally employ an epitope sequence that would provide low background in plant tissue, a promoter-leader sequence that would impart efficient expression and targeting across the plasma membrane, and would produce protein that is bound in the cell wall and yet accessible to antibodies. A normally secreted wall protein modified to include a unique epitope would satisfy all such requirements.

One example of a protein suitable for modification in this manner is extensin, or hydroxyproline rich glycoprotein (HPRG). The use of the maize HPRG (Steifel et al, 1990) which is preferred as this molecule is well characterized in terms of molecular biology, expression and protein structure. However, any one of a variety of extensins and/or glycine-rich wall proteins (Keller et al, 1989) could be modified by the addition of an antigenic site to create a screenable marker. The exemplary selectable and/or screenable marker is the GUS gene. Of course, in light of this disclosure, numerous other possible selectable and/or screenable marker genes will be apparent to those of skill in the art in addition to the one set forth hereinbelow. Therefore, it will be understood that the following discussion is exemplary rather than exhaustive. In light of the techniques disclosed herein and the general recombinant techniques which are known in the art, the present invention renders possible the introduction of any gene, including marker genes, into a recipient cell to generate a transformed plant.

4.6.4.1 SELECTABLE MARKERS Possible selectable markers for use in connection with the present invention include, but are not limited to, a neo gene (Potrykus et al , 1985) which codes for kanamycin resistance and can be selected for using kanamycin, G418, etc.; a bar gene which codes for bialaphos resistance; a mutant aroA gene which encodes an altered EPSP synthase protein (Hinchee et al, 1988) thus conferring glyphosate resistance; a nitrilase gene such as hxn from Klebsiella ozaenae which confers resistance to bromoxynil (Stalker et al, 1988); a mutant acetolactate synthase gene (ALS) which confers resistance to imidazolinone. sulfonylurea or other ALS inhibiting chemicals (European Patent Application 154,204, 1985); a methotrexate resistant DHFR gene (Thillet et al , 1988), or a dalapon dehalogenase gene that confers resistance to the herbicide dalapon; or a mutated anthranilate synthase gene that confers resistance to 5- methyl tryptophan. Where a mutant EPSP synthase gene is employed, additional benefit may be realized through the incorporation of a suitable chloroplast transit peptide, CTP (European Patent Application 0,218,571, 1987)

An illustrative embodiment of a selectable marker gene capable of being used in systems to select transformants is the genes that encode the enzyme phosphinothricin acetyltransferase, such as the bar gene from Streptomyces hygroscopicus or the pat gene from Streptomyces viridochromogene The enzyme phosphinothricin acetyl transferase (PAT) inactivates the active ingredient in the herbicide bialaphos, phosphinothricin (PPT) PPT inhibits glutamine synthetase, (Murakami et al , 1986, Twell et al , 1989) causing rapid accumulation of ammonia and cell death Where one desires to employ a bialaphos resistance gene in the practice of the invention, a particularly useful gene for this purpose is the bar or pat genes obtainable from species of Streptomyces (e g ATCC No 21,705) The cloning of the bar gene has been descπbed (Murakami et al , 1986, Thompson et al , 1987) as has the use of the bar gene in the context of plants (De Block et al , 1987, De Block et al , 1989)

4.6.4.2 SCREENABLE MARKERS

Screenable markers that may be employed include a β-glucuronidase or utdA gene (GUS) which encodes an enzyme for which various chromogenic substrates are known, an R-locus gene, which encodes a product that regulates the production of anthocyanm pigments (red color) in plant tissues (Dellaporta et al , 1988), a β-lactamase gene (Sutchffe, 1978), which encodes an enzyme for which various chromogenic substrates are known (e g PADAC, a chromogenic cephalospoπn), a xylE gene (Zukowsky et al , 1983) which encodes a catechol dioxygenase that can convert chromogenic catechols, an α-amylase gene (Ikuta et al , 1990), a tyrosinase gene (Katz et al , 1983) which encodes an enzyme capable of oxidizing tyrosine to DOPA and dopaquinone which in turn condenses to form the easily-detectable compound melanin, a β-galactosidase gene, which encodes an enzyme for which there are chromogenic substrates, a luciferase (lux) gene (Ow et al , 1986) or green fluorescent protein (GFP) and the derivatives thereof (U S Patent No 5,625,048), which allow for bioluminescence detection, or even an aequoπn gene (Prasher et al , 1985) which may be employed in calcium-sensitive bioluminescence detection A further screenable marker contemplated for use in the present invention is firefly luciferase, encoded by the lux gene. The presence of the lux gene in transformed cells may be detected using, for example, X-ray film, scintillation counting, fluorescent spectrophotometry, low-light video cameras, photon counting cameras or multiwell luminometry. It is also envisioned that this system may be developed for populational screening for bioluminescence, such as on tissue culture plates, or even for whole plant screening.

4.6.5 PLANT TRANSGENE COMPOSITIONS A particularly important advance of the present invention is that it provides methods and compositions for the transformation of plant cells with genes in addition to, or other than, marker genes. Such transgenes will often be genes that direct the expression of a particular protein or polypeptide product, but they may also be non- expressible DNA segments, e.g., transposons such as Ds that do no direct their own transposition. As used herein, an "expressible gene" is any gene that is capable of being transcribed into RNA (e.g., mRNA. antisense RNA, etc.) or translated into a protein, expressed as a trait of interest, or the like, etc., and is not limited to selectable, screenable or non-selectable marker genes. The invention also contemplates that, where both an expressible gene that is not necessarily a marker gene is employed in combination with a marker gene, one may employ the separate genes on either the same or different DNA segments for transformation. In the latter case, the different vectors are delivered concurrently to recipient cells to maximize cotransformation.

The choice of the particular DNA segments to be delivered to the recipient cells will often depend on the purpose of the transformation. One of the major purposes of transformation of crop plants is to add some commercially desirable, agronomically important traits to the plant. Such traits include, but are not limited to, herbicide resistance or tolerance; insect resistance or tolerance; disease resistance or tolerance (viral, bacterial, fungal, nematode); stress tolerance and/or resistance, as exemplified by resistance or tolerance to drought, heat, chilling, freezing, excessive moisture, salt stress; oxidative stress; increased yields; food content and makeup; physical appearance; male sterility; drydown; standability; prolificacy; starch properties; oil quantity and quality; and the like. One may desire to incorporate one or more genes conferring any such desirable trait or traits, such as, for example, a gene or genes encoding herbicide resistance.

In certain embodiments, the present invention contemplates the transformation of a recipient cell with more than one advantageous transgene. Two or more transgenes can be supplied in a single transformation event using either distinct transgene-encoding vectors, or using a single vector incorporating two or more gene coding sequences. For example, plasmids bearing the bar and aroA expression units in either convergent, divergent, or colinear orientation, are considered to be particularly useful. Further preferred combinations are those of an insect resistance gene, such as a Bt gene, along with a protease inhibitor gene such as pinll, or the use of bar in combination with either of the above genes. Of course, any two or more transgenes of any description, such as those conferring herbicide, insect, disease (viral, bacterial, fungal, nematode) or drought resistance, male sterility, drydown, standability, prolificacy, starch properties, oil quantity and quality, or those increasing yield or nutritional quality may be employed as desired.

4.6.5.1 HERBICIDE RESISTANCE

The genes encoding phosphinothricin acetyltransferase (bar and pat), glyphosate tolerant EPSP synthase genes, the glyphosate degradative enzyme gene gox encoding glyphosate oxidoreductase, deh (encoding a dehalogenase enzyme that inactivates dalapon), herbicide resistant (e.g., sulfonylurea and imidazolinone) acetolactate synthase, and hxn genes (encoding a nitrilase enzyme that degrades bromoxynil) are good examples of herbicide resistant genes for use in transformation. The bar and pat genes code for an enzyme, phosphinothricin acetyltransferase (PAT), which inactivates the herbicide phosphinothricin and prevents this compound from inhibiting glutamine synthetase enzymes. The enzyme 5-enolpyruvylshikimate 3-phosphate synthase (EPSP Synthase), is normally inhibited by the herbicide N-(phosphonomethyl)glycine (glyphosate). Flowever, genes are known that encode glyphosate-resistant EPSP Synthase enzymes. The deh gene encodes the enzyme dalapon dehalogenase and confers resistance to the herbicide dalapon. The bxn gene codes for a specific nitrilase enzyme that converts bromoxynil to a non-herbicidal degradation product. 2>t 4.6.5.2 INSECT RESISTANCE

An important aspect of the present invention concerns the introduction of insect resistance-conferring genes into a plant cell. Exemplary insect resistance genes which may be introduced into a plant cell, or used for generating a transgenic plant itself, include, but are not limited to, those insect resistance genes known to those of skill in the art. For example, Bacillus thuringiensis crystal toxin, or Bt, genes (Watrud et al , 1985), or endotoxin genes from other species of B. thuringiensis which affect insect growth or development may also be employed in this regard. A representative list of B. thuringiensis endotoxin genes which may be placed under the control of one or more of the disclosed promoters for expression in a transformed cell includes, those genes listed in Table 1 :

TABLE 1

B. THURINGIENSIS Δ-ENDOTOXINS CONTEMPLATED TO BE USEFUL IN THE

PREPARATION OF INSECT-RESISTANT TRANSFORMED CELLS^A

Current Nomenclature Former Name GenBank Accession #

CrylAa CryΙA(a) M1 1250 CrylAb CryΙA(b) M13898 Cry 1 Ac CryΙA(c) Ml 1068 Cry 1 Ad CryΙA(d) M73250 CrylAe CryΙA(e) M65252 CrylBa CrylB X0671 1 CrylBb ET5 L32020 Cry 1 Be PEG5 Z46442 Cryl Ca CrylC X07518 Cryl Cb CryΙC(b) M97880 Cry 1 Da CrylD X54160 CrylDb PrtB Z2251 1 Cry 1 Ea CrylE X53985 CrylEb CryΙE(b) M73253 Cry 1 Fa CrvIF M63897 3q

Current Nomenclature Former Name GenBank Accession #

CrylFb PrtD Z22512

CrylG PrtA Z22510

CrylH PrtC Z22513

CrylHb U35780

Cry 1 la CryV X62821

Cry lib CryV U07642

CrylJa ET4 L32019

CrylJb ET1 U31527

Cry IK U28801

Cry2Aa CryllA M31738

Cry2Ab CryllB M23724

Cry2Ac CryllC X57252

Cry3A CrylllA M22472

Cry3Ba CrylllB X17123

Cry3Bb CryIIIB2 M89794

Cry3C CrylllD X59797

Cry4A CrylVA Y00423

Cry4B CrylVB X07423

Cry5Aa CryVA(a) L07025

Cry5Ab CryVA(b) L07026

Cry5B U 19725

CryόA Cry VIA L07022

CryόB CryVIB L07024

Cry7Aa CrylllC M64478

Cry7Ab CrvIIICb U04367

Cry8A CrylllE U04364

Cry8B CrylllG U04365

Cry8C CrylllF U04366

Cry9A CrylG X58120

Cry9B CrylX X75019 HO

Current Nomenclature Former Name GenBank Accession #

Cry9C CrylH Z37527

CrylOA CrylVC Ml 2662

Cryl lA CrylVD M31737

Cry 1 IB Jeg80 X86902

Cryl2A CryVB L07027

Cryl3A CryVC L07023

Cryl4A CryVD U13955

Cryl5A 34kDa M76442

CrylόA cbm71 X94146

CytlA CytA X03182

Cyt2A CytB Z 14147 Adapted from: http://epunix.biols.susx.ac.uk/Home Neil_Crickmore/Bt/index.html

While some non-native genes are known to be poorly expressed in plants (such as Bt toxin genes, antifungal genes, herbicide resistance genes, etc.) in their native state, the inventors contemplate that such genes may readily be modified prior to their introduction into the cell of interest. In fact, introduction of heterologous genes in plants is a well- documented phenomenon (Vaeck et al, 1987; Barton et al, 1987), and methodologies for improving the expression of heterologous genes in a particular cell line have been described in the literature for genes such as those encoding Bt endotoxins (for example, see Perlak et al, 1991). In such embodiments, therefore, it may be desirable to alter the coding sequence (or "plantize") the transgene of interest prior to transformation to improve expression of the gene in the plant host cell.

In other embodiments, when expression of genes under the control of one or more MsENOD40 promoters of the present invention are contemplated in animal cells, it may be desirable to alter the coding sequence prior to transformation to improve expression. For example, it is often desirable to "humanize" coding sequences to incorporate codons which are more common for expression in human cells. The correlation between the abundance of tRNAs and the occurrence of the respective codons in protein-expressing genes has been described for E. coli, yeast and other organisms (Bennetzen and Hall. 1982; Grantham et al. 1980; Grantham et al, 1981 ; HI Ikemura, 1981a; 1981b; 1982; Wada et al, 1990). For example, using the information in Table 2 and Table 3, a person of skill in the art would be able to determine the appropriate changes to the nucleic acid sequence that would allow efficient expression in human cells. Methods for mutagenizing and altering DNA segments are known to those of skill in the art, and discussed in detail, herein. Generally, the mutagenesis of a transgene will involve random mutagenesis, transposon mutagenesis, site-directed mutagenesis, nucleotide addition or deletion, truncation, or gene fusion techniques.

For example, with regards to increasing insect resistance in a transformed cell, protease inhibitors have been shown to enhance insect resistance (Johnson et al, 1989), and may thus have utility in plant transformation. The use of a protease inhibitor II gene, pinll, from tomato or potato is envisioned to be particularly useful. Even more advantageous is the use of a pinll gene in combination with a Bt toxin gene, the combined effect of which has been discovered by the present inventors to produce synergistic insecticidal activity. Other genes which encode inhibitors of the insects' digestive system, or those that encode enzymes or co-factors that facilitate the production of inhibitors, may also be useful. This group may be exemplified by oryzacy statin and amylase inhibitors such as those from wheat and barley.

Also, genes encoding lectins may confer additional or alternative insecticide properties. Lectins (originally termed phytohemagglutinins) are multivalent carbohydrate-binding proteins which have the ability to agglutinate red blood cells from a range of species. Lectins have been identified recently as insecticidal agents with activity against weevils, ECB and rootworm (Murdock et al, 1990; Czapla & Lang, 1990). Lectin genes contemplated to be useful include, for example, barley and wheat germ agglutinin (WGA) and rice lectins (Gatehouse et al, 1984), with WGA being preferred.

TABLE 2 PREFERRED DNA CODONS FOR HUMAN USE

Amino Acids Codons Preferred in Human G< 2nes

Alanine Ala A GCC GCT GCA GCG

Cysteine Cys C TGC TGT

Aspartic acid Asp D GAC GAT

Glutamic acid Glu E GAG GAA

Phenylalanine Phe F TTC TTT

Glycine Gly G GGC GGG GGA GGT

Histidine His H CAC CAT

Isoleucine He 1 ATC ATT ATA 5

Lysine Lys K AAG AAA

Leucine Leu L CTG CTC TTG CTT CTA² TTA

Methionine Met M ATG

Asparagine Asn N AAC AAT

Proline Pro P CCC CCT CCA CCG

Glutamine Gin Q CAG CAA

Arginine Arg R CGC AGG CGG AGA CGA CGT

Serine Ser S AGC TCC TCT AGT TCA TCG

Threonine Thr T ACC ACA ACT ACG Valine Val V GTG GTC GTT GTA Tryptophan Trp W TGG Tyrosine Tyr Y TAC TAT

The codons at the left represent those most preferred for use in human genes, with human usage decreasing towards the right.

Double underlined codons represent those which are almost never used in human genes.

TABLE 3 PREFERRED RNA CODONS FOR HUMAN USE §

Amino Acids Codons Preferred in Human Genes

Alanine Ala A GCC GCU GCA GCG Cysteine Cys C UGC UGU Λspartic acid Asp D GAC GAU Glutamic acid Glu E GAG GAA Phenylalanine Phe F UUC UUU Glycine Gly G GGC GGG GGA GGU l listidine His H CAC CAU Isoleucine He I AUC AUU AUA

Lysine Lys K AAG AAA

Leucine Leu L CUG cue UUG CUU CUA² UUA

Mcthionine Met M AUG

Asparagine Asn N AAC AAU

Proline Pro P CCC ecu CCA CCG

Glutamine Gin Q CAG CAA

Λrginine Arg R CGC AGG CGG AGA CGA CGU

Serine Ser S AGC UCC ucu AGU UCA UCG

Threonine Thr T ACC ACA ACU ACG

Valine Val V GUG GUC GUU GUA

Tryptophan Trp w UGG

Tyrosine Tyr Y UAC UAU

The codons at the left represent those most preferred for use in human genes, with human usage decreasing towards the right. ^"Double underlined codons represent those which are almost never used in human genes.

I . Genes controlling the production of large or small polypeptides active against insects when introduced into the insect pests, such as, e.g., lytic peptides, peptide hormones and toxins and venoms, form another aspect of the invention. For example, it is contemplated that the expression of juvenile hormone esterase, directed towards specific insect pests, may also result in insecticidal activity, or perhaps cause cessation of metamorphosis (Hammock et al, 1990).

Transgenic plants expressing genes which encode enzymes that affect the integrity of the insect cuticle form yet another aspect of the invention. Such genes include those encoding, e.g., chitinase, proteases, lipases and also genes for the production of nikkomycin, a compound that inhibits chitin synthesis, the introduction of any of which is contemplated to produce insect resistant plants. Genes that code for activities that affect insect molting, such those affecting the production of ecdysteroid UDP-glucosyl transferase, also fall within the scope of the useful transgenes of the present invention. Genes that code for enzymes that facilitate the production of compounds that reduce the nutritional quality of the host plant to insect pests are also encompassed by the present invention. It may be possible, for instance, to confer insecticidal activity on a plant by altering its sterol composition. Sterols are obtained by insects from their diet and are used for hormone synthesis and membrane stability. Therefore alterations in plant sterol composition by expression of novel genes, e.g., those that directly promote the production of undesirable sterols or those that convert desirable sterols into undesirable forms, could have a negative effect on insect growth and/or development and hence endow the plant with insecticidal activity. Lipoxygenases are naturally occurring plant enzymes that have been shown to exhibit anti-nutritional effects on insects and to reduce the nutritional quality of their diet. Therefore, further embodiments of the invention concern transgenic plants with enhanced lipoxygenase activity which may be resistant to insect feeding.

The present invention also provides methods and compositions by which to achieve qualitative or quantitative changes in plant secondary metabolites. One example concerns transforming maize to produce DIMBOA which, it is contemplated, will confer resistance to European corn borer, rootworm and several other maize insect pests. Candidate genes that are particularly considered for use in this regard include those genes tjto at the bx locus known to be involved in the synthetic DIMBOA pathway (Dunn et al. ,

1981). The introduction of genes that can regulate the production of maysin, and genes involved in the production of dhurrin in sorghum, is also contemplated to be of use in facilitating resistance to earworm and rootworm, respectively. Tripsacum dactyloides is a species of grass that is resistant to certain insects, including corn root worm. It is anticipated that genes encoding proteins that are toxic to insects or are involved in the biosynthesis of compounds toxic to insects will be isolated from Tripsacum and that these novel genes will be useful in conferring resistance to insects. It is known that the basis of insect resistance in Tripsacum is genetic, because said resistance has been transferred to Zea mays via sexual crosses (Branson and Guss, 1972).

Further genes encoding proteins characterized as having potential insecticidal activity may also be used as transgenes in accordance herewith. Such genes include, for example, the cowpea trypsin inhibitor (CpTI; Hilder et al, 1987) which may be used as a rootworm deterrent; genes encoding avermectin (Avermectin and Abamectin. , Campbell, W.C., Ed.. 1989; Ikeda et al, 1987) which may prove particularly useful as a corn rootworm deterrent; ribosome inactivating protein genes; and even genes that regulate plant structures. Transgenic plants including anti-insect antibody genes and genes that code for enzymes that can covert a non-toxic insecticide (pro-insecticide) applied to the outside of the plant into an insecticide inside the plant are also contemplated.

4.6.5.3 ENVIRONMENT OR STRESS RESISTANCE

Improvement of a plant's ability to tolerate various environmental stresses such as, but not limited to, drought, excess moisture, chilling, freezing, high temperature, salt, and oxidative stress, can also be effected through expression of novel genes. It is proposed that benefits may be realized in terms of increased resistance to freezing temperatures through the introduction of an "antifreeze" protein such as that of the Winter Flounder (Cutler et al, 1989) or synthetic gene derivatives thereof. Improved chilling tolerance may also be conferred through increased expression of glycerol-3- phosphate acetyltransferase in chloroplasts (Wolter et al, 1992). Resistance to oxidative stress (often exacerbated by conditions such as chilling temperatures in combination with high light intensities) can be conferred by expression of superoxide V7 dismutase (Gupta et al, 1993), and may be improved by glutathione reductase (Bowler et al, 1992). Such strategies may allow for tolerance to freezing in newly emerged fields as well as extending later maturity higher yielding varieties to earlier relative maturity zones. It is contemplated that the expression of novel genes that favorably effect plant water content, total water potential, osmotic potential, and turgor will enhance the ability of the plant to tolerate drought. As used herein, the terms "drought resistance" and "drought tolerance" are used to refer to a plants increased resistance or tolerance to stress induced by a reduction in water availability, as compared to normal circumstances, and the ability of the plant to function and survive in lower- water environments. In this aspect of the invention it is proposed, for example, that the expression of genes encoding for the biosynthesis of osmotically-active solutes, such as polyol compounds, may impart protection against drought. Within this class are genes encoding for mannitol dehydrogenase (Lee and Saier, 1983) and trehalose-6-phosphate synthase (Kaasen et al, 1992). Through the subsequent action of native phosphatases in the cell or by the introduction and coexpression of a specific phosphatase, these introduced genes will result in the accumulation of either mannitol or trehalose, respectively, both of which have been well documented as protective compounds able to mitigate the effects of stress. Mannitol accumulation in transgenic tobacco has been verified and preliminary results indicate that plants expressing high levels of this metabolite are able to tolerate an applied osmotic stress (Tarczynski et al, 1992, 1993).

Similarly, the efficacy of other metabolites in protecting either enzyme function

(e.g., alanopine or propionic acid) or membrane integrity (e.g., alanopine) has been documented (Loomis et al, 1989), and therefore expression of genes encoding for the biosynthesis of these compounds might confer drought resistance in a manner similar to or complimentary to mannitol. Other examples of naturally occurring metabolites that are osmotically active and/or provide some direct protective effect during drought and/or desiccation include fructose, erythritol (Coxson et al, 1992), sorbitol, dulcitol (Karsten et al, 1992), glucosylglycerol (Reed et al, 1984; ErdMann et al, 1992), sucrose, stachyose (Koster and Leopold, 1988; Blackman et al, 1992). raffinose (Bernal-Lugo and Leopold, 1992), proline (van Rensburg et al, 1993) and glycinebetaine (Wyn-Jones and Storey, 1982), ononitol and pinitol (Vernon and Bohnert, 1992). Continued canopy growth and increased reproductive fitness during times of stress will be augmented by introduction and expression of genes such as those controlling the osmotically active compounds discussed above and other such compounds. Currently preferred genes which promote the synthesis of an osmotically active polyol compound are genes which encode the enzymes mannitol- 1 -phosphate dehydrogenase, trehalose-6-phosphate synthase and myoinositol 0-mefhyltransferase.

It is contemplated that the expression of specific proteins may also increase drought tolerance. Three classes of Late Embryogenic Proteins have been assigned based on structural similarities ( Dure et al , 1989). All three classes of LEAs have been demonstrated in maturing (i.e desiccating) seeds. Within these 3 types of LEA proteins, the Type-II (dehydrin-type) have generally been implicated in drought and/or desiccation tolerance in vegetative plant parts (i.e Mundy and Chua, 1988; Piatkowski et al, 1990; Yamaguchi-Shinozaki et al, 1992). Recently, expression of a Type-Ill LEA (HVA-1) in tobacco was found to influence plant height, maturity and drought tolerance (Fitzpatrick, 1993). Expression of structural genes from all three LEA groups may therefore confer drought tolerance. Other types of proteins induced during water stress include thiol proteases, aldolases and transmembrane transporters (Guerrero et al , 1990), which may confer various protective and/or repair-type functions during drought stress. It is also contemplated that genes that effect lipid biosynthesis and hence membrane composition might also be useful in conferring drought resistance on the plant.

Many of these genes for improving drought resistance have complementary modes of action. Thus, it is envisaged that combinations of these genes might have additive and/or synergistic effects in improving drought resistance in plants. Many of these genes also improve freezing tolerance (or resistance); the physical stresses incurred during freezing and drought are similar in nature and may be mitigated in similar fashion. Benefit may be conferred via constitutive expression of these genes, but the preferred means of expressing these novel genes may be through the use of a turgor- induced promoter (such as the promoters for the turgor- induced genes described in Guerrero et al , 1987 and Shagan et al , 1993 which are incorporated herein by reference). Spatial and temporal expression patterns of these genes may enable plants to better withstand stress. It is proposed that expression of genes that are involved with specific morphological traits that allow for increased water extractions from drying soil would be of benefit. For example, introduction and expression of genes that alter root characteristics may enhance water uptake. It is also contemplated that expression of genes that enhance reproductive fitness during times of stress would be of significant value. For example, expression of genes that improve the synchrony of pollen shed and receptiveness of the female flower parts, i.e., silks, would be of benefit. In addition it is proposed that expression of genes that minimize kernel abortion during times of stress would increase the amount of grain to be harvested and hence be of value. Given the overall role of water in determining yield, it is contemplated that enabling plants to utilize water more efficiently, through the introduction and expression of novel genes, will improve overall performance even when soil water availability is not limiting. By introducing genes that improve the ability of plants to maximize water usage across a full range of stresses relating to water availability, yield stability or consistency of yield performance may be realized.

4.6.5.4 DISEASE RESISTANCE

It is proposed that increased resistance to diseases may be realized through introduction of genes into plants. It is possible to produce resistance to diseases caused by viruses, bacteria, fungi and nematodes. It is also contemplated that control of mycotoxin producing organisms may be realized through expression of introduced genes.

Resistance to viruses may be produced through expression of novel genes. For example, it has been demonstrated that expression of a viral coat protein in a transgenic plant can impart resistance to infection of the plant by that virus and perhaps other closely related viruses (Cuozzo et al. 1988, Hemenway et al, 1988, Abel et al, 1986). It is contemplated that expression of antisense genes targeted at essential viral functions may impart resistance to said virus. For example, an antisense gene targeted at the gene responsible for replication of viral nucleic acid may inhibit said replication and lead to resistance to the virus. It is believed that interference with other viral functions through the use of antisense genes may also increase resistance to viruses. Further it is So proposed that it may be possible to achieve resistance to viruses through other approaches, including, but not limited to the use of satellite viruses.

It is proposed that increased resistance to diseases caused by bacteria and fungi may be realized through introduction of novel genes. It is contemplated that genes encoding so-called "peptide antibiotics," pathogenesis related (PR) proteins, toxin resistance, and proteins affecting host-pathogen interactions such as morphological characteristics will be useful. Peptide antibiotics are polypeptide sequences which are inhibitory to growth of bacteria and other microorganisms. For example, the classes of peptides referred to as cecropins and magainins inhibit growth of many species of bacteria and fungi. It is proposed that expression of PR proteins in monocotyledonous plants such as maize may be useful in conferring "resistance to bacterial disease. These genes are induced following pathogen attack on a host plant and have been divided into at least five classes of proteins (Bol, Linthorst, and Cornelissen, 1990). Included amongst the PR proteins are b-1 , 3-glucanases, chitinases, and osmotin and other proteins that are believed to function in plant resistance to disease organisms. Other genes have been identified that have antifungal properties, e.g., UDA (stinging nettle lectin) and hevein (Broakaert et al, 1989; Barkai-Golan et al, 1978). It is known that certain plant diseases are caused by the production of phytotoxins. It is proposed that resistance to these diseases would be achieved through expression of a novel gene that encodes an enzyme capable of degrading or otherwise inactivating the phytotoxin. It is also contemplated that expression novel genes that alter the interactions between the host plant and pathogen may be useful in reducing the ability the disease organism to invade the tissues of the host plant, e.g., an increase in the waxiness of the leaf cuticle or other morphological characteristics. Plant parasitic nematodes are a cause of disease in many plants, including maize. It is proposed that it would be possible to make the corn plant resistant to these organisms through the expression of novel genes. It is anticipated that control of nematode infestations would be accomplished by altering the ability of the nematode to recognize or attach to a host plant and/or enabling the plant to produce nematicidal compounds, including but not limited to proteins. St

4.6.5.5 MYCOTOXIN REDUCTION/ELIMINATION

Production of mycotoxins, including aflatoxin and fumonisin, by fungi associated with monocotyledonous plants such as maize is a significant factor in rendering the grain not useful. These fungal organisms do not cause disease symptoms and/or interfere with the growth of the plant, but they produce chemicals (mycotoxins) that are toxic to animals. It is contemplated that inhibition of the growth of these fungi would be reduce the synthesis of these toxic substances and therefore reduce grain losses due to mycotoxin contamination. It is also proposed that it may be possible to introduce novel genes into monocotyledonous plants such as maize that would inhibit synthesis of the mycotoxin without interfering with fungal growth. Further, it is contemplated that expression of a novel gene which encodes an enzyme capable of rendering the mycotoxin nontoxic would be useful in order to achieve reduced mycotoxin contamination of grain. The result of any of the above mechanisms would be a reduced presence of mycotoxins on grain.

4.6.5.6 CROP COMPOSITION OR QUALITY

One mechanism for increasing the biosynthesis of the amino acids is to introduce genes that deregulate the amino acid biosynthetic pathways such that the plant can no longer adequately control the levels that are produced. This may be done by deregulating or bypassing steps in the amino acid biosynthetic pathway which are normally regulated by levels of the amino acid end product of the pathway. Examples include the introduction of genes that encode deregulated versions of the enzymes aspartokinase or dihydrodipicolinic acid (DHDP)-synthase for increasing lysine and threonine production, and anthranilate synthase for increasing tryptophan production. Reduction of the catabolism of the amino acids may be accomplished by introduction of DNA sequences that reduce or eliminate the expression of genes encoding enzymes that catalyze steps in the catabolic pathways such as the enzyme lysine-ketoglutarate reductase.

The protein composition of the plant may be altered to improve the balance of amino acids in a variety of ways including elevating expression of native proteins, decreasing expression of those with poor composition, changing the composition of native proteins, or introducing genes encoding entirely new proteins possessing . S3 superior composition. Examples may include the introduction of DNA that decreases the expression of members of the zein family of storage proteins. This DNA may encode ribozymes or antisense sequences directed to impairing expression of zein proteins or expression of regulators of zein expression such as the opaque-2 gene product. It is also proposed that the protein composition of the plant may be modified through the phenomenon of cosupression, i.e., inhibition of expression of an endogenous gene through the expression of an identical structural gene or gene fragment introduced through transformation (Goring et al, 1991 ). Additionally, the introduced DNA may encode enzymes which degrade zeins. The decreases in zein expression that are achieved may be accompanied by increases in proteins with more desirable amino acid composition or increases in other major seed constituents such as starch. Alternatively, a chimeric gene may be introduced that comprises a coding sequence for a native protein of adequate amino acid composition such as for one of the globulin proteins or 10 kD zein of maize and a promoter or other regulatory sequence designed to elevate expression of said protein. The coding sequence of said gene may include additional or replacement codons for essential amino acids. Further, a coding sequence obtained from another species, or, a partially or completely synthetic sequence encoding a completely unique peptide sequence designed to enhance the amino acid composition of the seed may be employed. The introduction of genes that alter the oil content of the plants may be of value.

Increases in oil content may result in increases in metabolizable-energy-content and - density of the seeds for uses in feed and food. The introduced genes may encode enzymes that remove or reduce rate-limitations or regulated steps in fatty acid or lipid biosynthesis. Such genes may include, but are not limited to, those that encode acetyl- CoA carboxylase, ACP-acyltransferase, b-ketoacyl-ACP synthase, plus other well known fatty acid biosynthetic activities. Other possibilities are genes that encode proteins that do not possess enzymatic activity such as acyl carrier protein. Genes may be introduced that alter the balance of fatty acids present in the oil providing a more healthful or nutritive feedstuff. The introduced DNA may also encode sequences that block expression of enzymes involved in fatty acid biosynthesis, altering the proportions of fatty acids present in the grain such as described below. S3 Genes may be introduced that enhance the nutritive value of the starch component of the plant, for example by increasing the degree of branching, resulting in improved utilization of the starch in cows by delaying its metabolism.

In addition to direct improvements in feed or food value, genes may also be introduced which improve the processing of plants and improve the value of the products resulting from the processing. Plants may be improved though the expression of novel genes that increase the efficiency and reduce the cost of processing such as by decreasing steeping time.

Improving the value of processed products may include altering the quantity or quality of starch, or oil of the plant. Elevation of starch may be achieved through the identification and elimination of rate limiting steps in starch biosynthesis or by decreasing levels of the other components of the plant resulting in proportional increases in starch. An example of the former may be the introduction of genes encoding ADP-glucose pyrophosphorylase enzymes with altered regulatory activity or which are expressed at higher level. Examples of the latter may include selective inhibitors of protein or oil biosynthesis.

The properties of starch may be beneficially altered by changing the ratio of amylose to amylopectin, the size of the starch molecules, or their branching pattern. Through these changes a broad range of properties may be modified which include, but are not limited to, changes in gelatinization temperature, heat of gelatinization, clarity of films and pastes, rheological properties, and the like. To accomplish these changes in properties, genes that encode granule-bound or soluble starch synthase activity or branching enzyme activity may be introduced alone or combination. DNA such as antisense constructs may also be used to decrease levels of endogenous activity of these enzymes. The introduced genes or constructs may possess regulatory sequences that time their expression to specific intervals in starch biosynthesis and starch granule development. Furthermore, it may be worthwhile to introduce and express genes that result in the in vivo derivatization, or other modification, of the glucose moieties of the starch molecule. The covalent attachment of any molecule may be envisioned, limited only by the existence of enzymes that catalyze the derivatizations and the accessibility of appropriate substrates in the starch granule. Examples of important derivations may include the addition of functional groups such as amines, carboxyls, or phosphate Si groups which provide sites for subsequent in vitro derivatizations or affect starch properties through the introduction of ionic charges. Examples of other modifications may include direct changes of the glucose units such as loss of hydroxyl groups or their oxidation to aldehyde or carboxyl groups. Oil is another product which may be improved by introduction and expression of genes. Oil properties may be altered to improve its performance in the production and use of cooking oil, shortenings, lubricants or other oil-derived products or improvement of its health attributes when used in the food-related applications. Novel fatty acids also may be synthesized which upon extraction can serve as starting materials for chemical syntheses. The changes in oil properties may be achieved by altering the type, level, or lipid arrangement of the fatty acids present in the oil. This in turn may be accomplished by the addition of genes that encode enzymes that catalyze the synthesis of novel fatty acids and the lipids possessing them or by increasing levels of native fatty acids while possibly reducing levels of precursors. Alternatively DNA sequences may be introduced which slow or block steps in fatty acid biosynthesis resulting in the increase in precursor fatty acid intermediates. Genes that might be added include desaturases, epoxidases, hydratases, dehydratases, and other enzymes that catalyze reactions involving fatty acid intermediates. Representative examples of catalytic steps that might be blocked include the desaturations from stearic to oleic acid and oleic to linolenic acid resulting in the respective accumulations of stearic and oleic acids. Another example is the blockage of elongation steps resulting in the accumulation of C₈ to C₁₂ saturated fatty acids.

In addition it may further be considered that the plant be used for the production or manufacturing of useful biological compounds that were either not produced at all, or not produced at the same level, in the plant previously. The novel plants producing these compounds are made possible by the introduction and expression of genes by plant transformation methods. The vast array of possibilities include but are not limited to any biological compound which is presently produced by any organism such as proteins, nucleic acids, primary and intermediary metabolites, carbohydrate polymers, etc. The compounds may be produced by the plant, extracted upon harvest and/or processing, and used for any presently recognized useful purpose such as pharmaceuticals, fragrances, industrial enzymes to name a few. So

4.6.5.7 PLANT AGRONOMIC CHARACTERISTICS

Two of the factors determining where plants can be grown are the average daily temperature during the growing season and the length of time between frosts. Within the areas where it is possible to grow alfalfa, there are varying limitations on the maximal time it is allowed to grow to maturity and be harvested. The plants to be grown in a particular area is selected for its ability to mature and dry down to harvestable moisture content within the required period of time with maximum possible yield. Therefore, com of varying maturities is developed for different growing locations. Apart from the need to dry down sufficiently to permit harvest is the desirability of having maximal drying take place in the field to minimize the amount of energy required for additional drying post-harvest. Also the more readily the grain can dry down, the more time there is available for growth and kernel fill. It is considered that genes that influence maturity and/or dry down can be identified and introduced into com lines using transformation techniques to create new com varieties adapted to different growing locations or the same growing location but having improved yield to moisture ratio at harvest. Expression of genes that are involved in regulation of plant development may be especially useful, e.g., the liguleless and rough sheath genes that have been identified in corn. It is contemplated that genes may be introduced into plants that would improve standability and other plant growth characteristics, such as stronger stalks or improved root systems. The expression of genes that increase the efficiency of photosynthesis and/or the leaf canopy would further increase gains in productivity. Such approaches would allow for increased plant populations in the field.

4.6.5.8 NUTRIENT UTILIZATION

The ability to utilize available nutrients may be a limiting factor in growth of plants such as alfalfa. It is proposed that it would be possible to alter nutrient uptake, tolerate pH extremes, mobilization through the plant, storage pools, and availability for metabolic activities by the introduction of novel genes. These modifications would allow a plant such as alfalfa to more efficiently utilize available nutrients. It is contemplated that an increase in the activity of, for example, an enzyme that is normally present in the plant and involved in nutrient utilization would increase the availability of a nutrient. An example of such an enzyme would be phytase. It is also contemplated that expression of a novel gene may make a nutrient source available that was previously not accessible, e.g., an enzyme that releases a component of nutrient value from a more complex molecule, perhaps a macromolecule.

4.6.5.9 MALE STERILITY

Male sterility is useful in the production of hybrid seed. It is proposed that male sterility may be produced through expression of novel genes. For example, it has been shown that expression of genes that encode proteins that interfere with development of the male inflorescence and/or gametophyte result in male sterility. In an exemplary embodiment, the inventors contemplate the use of chimeric ribonuclease genes that express in the anthers of transgenic tobacco and oilseed rape have been demonstrated to lead to male sterility (Mariani et al, 1990). A number of mutations were discovered in maize that confer cytoplasmic male sterility. One mutation in particular, referred to as T cytoplasm, also correlates with sensitivity to Southern corn leaf blight. A DNA sequence, designated TURF- 13 (Levings, 1 90), was identified that correlates with T cytoplasm. It is proposed that it would be possible through the introduction of TURF- 13 via transformation to separate male sterility from disease sensitivity. As it is necessary to be able to restore male fertility for breeding purposes and for grain production it is proposed that genes encoding restoration of male fertility may also be introduced.

4.6.5.10 NEGATIVE SELECTABLE MARKERS Introduction of genes encoding traits that can be selected against may be useful for eliminating undesirable linked genes. It is contemplated that when two or more genes are introduced together by cotransformation that the genes will be linked together on the host chromosome. For example, a gene encoding a Bt gene that confers insect resistance on the plant may be introduced into a plant together with a bar gene that is useful as a selectable marker and confers resistance to the herbicide Ignite® on the plant. However, it may not be desirable to have an insect resistant plant that is also resistant to the herbicide Ignite®. It is proposed that one could also introduce an . 7 antisense bar gene that is expressed in those tissues where one does not want expression of the bar gene, e.g.. in whole plant parts. Hence, although the bar gene is expressed and is useful as a selectable marker, it is not useful to confer herbicide resistance on the whole plant. The bar antisense gene is a negative selectable marker. It is also contemplated that a negative selection is necessary in order to screen a population of transformants for rare homologous recombinants generated through gene targeting. For example, a homologous recombinant may be identified through the inactivation of a gene that was previously expressed in that cell. The antisense gene to neomycin phosphotransferase II (nptll) has been investigated as a negative selectable marker in tobacco (Nicotiana tabacum) and Arahidopsis thaliana (Xiang, C. and Guerra, D.J. 1993). In this example both sense and antisense npt II genes are introduced into a plant through transformation and the resultant plants are sensitive to the antibiotic kanamycin. An introduced gene that integrates into the host cell chromosome at the site of the antisense nptll gene, and inactivates the antisense gene, will make the plant resistant to kanamycin and other aminoglycoside antibiotics. Therefore, rare site specific recombinants may be identified by screening for antibiotic resistance. Similarly, any gene, native to the plant or introduced through transformation, that when inactivated confers resistance to a compound, may be useful as a negative selectable marker. It is contemplated that negative selectable markers may also be useful in other ways. One application is to construct transgenic lines in which one could select for transposition to unlinked sites. In the process of tagging it is most common for the transposable element to move to a genetically linked site on the same chromosome. A selectable marker for recovery of rare plants in which transposition has occurred to an unlinked locus would be useful. For example, the enzyme cytosine deaminase may be useful for this purpose (Stouggard, J., 1993). In the presence of this enzyme the compound 5-fluorocytosine is converted to 5-fluorouracil which is toxic to plant and animal cells. If a transposable element is linked to the gene for the enzyme cytosine deaminase, one may select for transposition to unlinked sites by selecting for transposition events in which the resultant plant is now resistant to 5-fluorocytosine. The parental plants and plants containing transpositions to linked sites will remain sensitive to 5-fluorocytosine. Resistance to 5-fluorocytosine is due to loss of the 6Υ cytosine deaminase gene through genetic segregation of the transposable element and the cytosine deaminase gene. Other genes that encode proteins that render the plant sensitive to a certain compound will also be useful in this context. For example, T-

DNA gene 2 from Agrobacterium tumefaciens encodes a protein that catalyzes the conversion of a-naphthalene acetamide (NAM) to a-naphthalene acetic acid (NAA) renders plant cells sensitive to high concentrations of NAM (Depicker et al, 1988).

It is also contemplated that negative selectable markers may be useful in the construction of transposon tagging lines. For example, by marking an autonomous transposable element such as Ac, Master Mu, or En/Spn with a negative selectable marker, one could select for transformants in which the autonomous element is not stably integrated into the genome. It is proposed that this would be desirable, for example, when transient expression of the autonomous element is desired to activate in trans the transposition of a defective transposable element, such as Ds, but stable integration of the autonomous element is not desired. The presence of the autonomous element may not be desired in order to stabilize the defective element, i.e., prevent it from further transposing. However, it is proposed that if stable integration of an autonomous transposable element is desired in a plant the presence of a negative selectable marker may make it possible to eliminate the autonomous element during the breeding process.

4.6.6 NON-PROTEIN-EXPRESSING SEQUENCES 4.6.6.1 RNA-Expressing

DNA may be introduced into alfalfa and other plants for the purpose of expressing RNA transcripts that function to affect plant phenotype yet are not translated into protein. Two examples are antisense RNA and RNA with ribozyme activity. Both may serve possible functions in reducing or eliminating expression of native or introduced plant genes.

Genes may be constructed or isolated, which when transcribed, produce antisense RNA that is complementary to all or part(s) of a targeted messenger RNA(s). The antisense RNA reduces production of the polypeptide product of the messenger RNA. The polypeptide product may be any protein encoded by the plant genome. The aforementioned genes will be referred to as antisense genes. An antisense gene may thus be introduced into a plant by transformation methods to produce a novel transgenic plant with reduced expression of a selected protein of interest. For example, the protein may be an enzyme that catalyzes a reaction in the plant. Reduction of the enzyme activity may reduce or eliminate products of the reaction which include any enzymatically synthesized compound in the plant such as fatty acids, amino acids, carbohydrates, nucleic acids and the like. Alternatively, the protein may be a storage protein, such as a zein, or a structural protein, the decreased expression of which may lead to changes in seed amino acid composition or plant morphological changes respectively. The possibilities cited above are provided only by way of example and do not represent the full range of applications.

Genes may also be constructed or isolated, which when transcribed produce RNA enzymes, or ribozymes, which can act as endoribonucleases and catalyze the cleavage of RNA molecules with selected sequences. The cleavage of selected messenger RNA's can result in the reduced production of their encoded polypeptide products. These genes may be used to prepare novel transgenic plants which possess them. The transgenic plants may possess reduced levels of polypeptides including but not limited to the polypeptides cited above that may be affected by antisense RNA.

It is also possible that genes may be introduced to produce novel transgenic plants which have reduced expression of a native gene product by a mechanism of cosuppression. It has been demonstrated in tobacco, tomato, and petunia (Goring et al, 1991 ; Smith et al, 1990; Napoli, C. et al, 1990; van der Krol et al, 1990) that expression of the sense transcript of a native gene will reduce or eliminate expression of the native gene in a manner similar to that observed for antisense genes. The introduced gene may encode all or part of the targeted native protein but its translation may not be required for reduction of levels of that native protein.

4.6.6.2 NON-RNA-EXPRESSING

For example, DNA elements including those of transposable elements such as

Ds. Ac, or Mu, may be inserted into a gene and cause mutations. These DNA elements may be inserted in order to inactivate (or activate) a gene and thereby "tag" a particular trait. In this instance the transposable element does not cause instability of the tagged mutation, because the utility of the element does not depend on its ability to move in (p ύ the genome. Once a desired trait is tagged, the introduced DNA sequence may be used to clone the corresponding gene, e.g., using the introduced DNA sequence as a PCR primer together with PCR gene cloning techniques (Shapiro, 1983; Dellaporta et al, 1988). Once identified, the entire gene(s) for the particular trait, including control or regulatory regions where desired may be isolated, cloned and manipulated as desired. The utility of DNA elements introduced into an organism for purposed of gene tagging is independent of the DNA sequence and does not depend on any biological activity of the DNA sequence, i.e. , transcription into RNA or translation into protein. The sole function of the DNA element is to disrupt the DNA sequence of a gene. It is contemplated that unexpressed DNA sequences, including novel synthetic sequences could be introduced into cells as proprietary "labels" of those cells and plants and seeds thereof. It would not be necessary for a label DNA element to disrupt the function of a gene endogenous to the host organism, as the sole function of this DNA would be to identify the origin of the organism. For example, one could introduce a unique DNA sequence into a plant and this DNA element would identify all cells, plants, and progeny of these cells as having arisen from that labelled source. It is proposed that inclusion of label DNAs would enable one to distinguish proprietary germplasm or germplasm derived from such, from unlabelled germplasm.

Another possible element which may be introduced is a matrix attachment region element (MAR), such as the chicken lysozyme A element (Stief, 1989), which can be positioned around an expressible gene of interest to effect an increase in overall expression of the gene and diminish position dependant effects upon incorporation into the plant genome (Stief et al, 1989; Phi-Van et al, 1990).

4.7 IMMUNOCHEMICAL METHODS

One means of identifying expression of the heterologous protein promoted by a promoter composition of the present invention involves detection of protein products using immunochemical methods.

4.7.1 METHODS FOR PREPARING ANTIBODY COMPOSITIONS

One of the uses for the promoters of the present invention may be to generate antibodies. Reference to antibodies throughout the specification includes whole polyclonal and monoclonal antibodies (mAbs), and parts thereof, either alone or conjugated with other moieties. Antibody parts include Fab and F(ab)₂ fragments and single chain antibodies. The antibodies may be made in vivo in suitable laboratory animals, by immunizing donors (preferably humans), or in vitro using recombinant DNA techniques. In a preferred embodiment, an antibody is a polyclonal antibody. Means for preparing and characterizing antibodies are well known in the art (See, e.g., Harlow and Lane, 1988).

Briefly, a polyclonal antibody is prepared by immunizing an animal with an immunogen comprising a polypeptide expressed by a promoter of the present invention and collecting antisera from that immunized animal. A wide range of animal species can be used for the production of antisera. Typically an animal used for production of anti- antisera is a rabbit, a mouse, a rat, a hamster, a goat, or a guinea pig. Because of the relatively large blood volume of rabbits, a rabbit is a preferred choice for production of polyclonal antibodies. Antibodies, both polyclonal and monoclonal, specific for the polypeptide of interest may be prepared using conventional immunization techniques, as will be generally known to those of skill in the art. A composition containing antigenic epitopes of the polypeptide of interest can be used to immunize one or more experimental animals, such as a rabbit or mouse, which will then proceed to produce specific antibodies against the polypeptide. Polyclonal antisera may be obtained, after allowing time for antibody generation, simply by bleeding the animal and preparing serum samples from the whole blood.

The amount of immunogen composition used in the production of polyclonal antibodies varies upon the nature of the immunogen, as well as the animal used for immunization. A variety of routes can be used to administer the immunogen (subcutaneous, intramuscular, intradermal. intravenous and intraperitoneal). The production of polyclonal antibodies may be monitored by sampling blood of the immunized animal at various points following immunization. A second, booster injection, also may be given. The process of boosting and titering is repeated until a suitable titer is achieved. When a desired level of immunogenicity is obtained, the immunized animal can be bled and the serum isolated and stored, and/or the animal can be used to generate mAbs (below). One of the important features obtained by the present invention is a polyclonal sera that is relatively homogenous with respect to the specificity of the antibodies therein.

Typically, polyclonal antisera is derived from a variety of different "clones," i.e., B-cells of different lineage. mAbs, by contrast, are defined as coming from antibody-producing cells with a common B-cell ancestor, hence their "mono" clonality.

When polypeptides are used as antigens to raise polyclonal sera, one would expect considerably less variation in the clonal nature of the sera than if a whole antigen were employed. Unfortunately, if incomplete fragments of an epitope are presented, the polypeptide may very well assume multiple (and probably non-native) conformations. As a result, even short polypeptides can produce polyclonal antisera with relatively plural specificities and, unfortunately, an antisera that does not react or reacts poorly with the native molecule.

Polyclonal antisera according to present invention is produced against polypeptides that are predicted to comprise whole, intact epitopes. It is believed that these epitopes are, therefore, more stable in an immunologic sense and thus express a more consistent immunologic target for the immune system. Under this model, the number of potential B-cell clones that will respond to this peptide is considerably smaller and, hence, the homogeneity of the resulting sera will be higher. In various embodiments, the present invention provides for polyclonal antisera where the clonality, i.e., the percentage of clone reacting with the same molecular determinant, is at least 80%. Even higher clonality - 90%, 95% or greater - is contemplated.

To obtain mAbs, one would also initially immunize an experimental animal, often preferably a mouse, with a polypeptide of interest composition. One would then, after a period of time sufficient to allow antibody generation, obtain a population of spleen or lymph cells from the animal. The spleen or lymph cells can then be fused with cell lines, such as human or mouse myeloma strains, to produce antibody-secreting hybridomas. These hybridomas may be isolated to obtain individual clones which can then be screened for production of antibody to the desired polypeptide.

Following immunization, spleen cells are removed and fused, using a standard fusion protocol with plasmacytoma cells to produce hybridomas secreting mAbs against the peptide of interest. Flybridomas which produce mAbs to the selected antigens are identified using standard techniques, such as ELISA and Western blot methods. Hybridoma clones can then be cultured in liquid media and the culture supernatants purified to provide the desired mAbs.

It is proposed that the mAbs anticipated by the present invention will also find useful application in immunochemical procedures, such as ELISA and Western blot methods, as well as other procedures such as immunoprecipitation, immunocytological methods, etc. which may utilize antibodies specific to the polypeptide of interest. In particular, antibodies may be used in immunoabsorbent protocols to purify native or recombinant polypeptide species or synthetic or natural variants thereof.

The antibodies disclosed herein may be employed in antibody cloning protocols to obtain cDNAs or genes encoding a polypeptide from other species or organisms, or to identify proteins having significant homology to the polypeptide of interest. They also may be used in inhibition studies to analyze the effects of polypeptide of interest in cells, tissues, or whole animals. The operation of all such immunological techniques will be known to those of skill in the art in light of the present disclosure.

4.7.2 ANTIBODY COMPOSITIONS AND FORMULATIONS THEREOF

Means for preparing and characterizing antibodies are well known in the art (See, e.g.. Harlow and Lane (1988); incorporated herein by reference). The methods for generating mAbs generally begin along the same lines as those for preparing polyclonal antibodies. Briefly, a polyclonal antibody is prepared by immunizing an animal with an immunogenic composition in accordance with the present invention and collecting antisera from that immunized animal. A wide range of animal species can be used for the production of antisera. Typically the animal used for production of anti-antisera is a rabbit, a mouse, a rat, a hamster, a guinea pig or a goat. Because of the relatively large blood volume of rabbits, a rabbit is a preferred choice for production of polyclonal antibodies.

As is well known in the art, a given composition may vary in its immunogenicity. It is often necessary therefore to boost the host immune system, as may be achieved by coupling a peptide or polypeptide immunogen to a carrier. Exemplary and preferred carriers are keyhole limpet hemocyanin (KLH) and bovine serum albumin (BSA). Other albumins such as ovalbumin, mouse serum albumin or rabbit serum albumin can also be used as carriers. Means for conjugating a polypeptide to a carrier b protein are well known in the art and include glutaraldehyde, m-maleimidobenzoyl-V- hydroxysuccinimide ester, carbodiimide and bis-biazotized benzidine. mAbs may be readily prepared through use of well-known techniques, such as those exemplified in U. S. Patent 4,196,265, incorporated herein by reference. Typically, this technique involves immunizing a suitable animal with a selected immunogen composition, e.g., a purified or partially purified protein, polypeptide or peptide. The immunizing composition is administered in a manner effective to stimulate antibody producing cells. Rodents such as mice and rats are preferred animals, however, the use of rabbit, sheep or frog cells is also possible. The use of rats may provide certain advantages (Goding, 1986), but mice are preferred, with the BALB/c mouse being most preferred as this is most routinely used and generally gives a higher percentage of stable fusions.

Following immunization, somatic cells with the potential for producing antibodies, specifically B-lymphocytes (B-cells), are selected for use in the mAb generating protocol. These cells may be obtained from biopsied spleens, tonsils or lymph nodes, or from a peripheral blood sample. Spleen cells and peripheral blood cells are preferred, the former because they are a rich source of antibody-producing cells that are in the dividing plasmablast stage, and the latter because peripheral blood is easily accessible. Often, a panel of animals will have been immunized and the spleen of animal with the highest antibody titer will be removed and the spleen lymphocytes obtained by homogenizing the spleen with a syringe. Typically, a spleen from an immunized mouse

7 8 contains approximately about 5 x 10 to about 2 x 10 lymphocytes.

The antibody-producing B lymphocytes from the immunized animal are then fused with cells of an immortal myeloma cell, generally one of the same species as the animal that was immunized. Myeloma cell lines suited for use in hybridoma-producing fusion procedures preferably are non-antibody-producing, have high fusion efficiency, and enzyme deficiencies that render them incapable of growing in certain selective media which support the growth of only the desired fused cells (hybridomas).

Any one of a number of myeloma cells may be used, as are known to those of skill in the art (Goding, 1986; Campbell, 1984). For example, where the immunized animal is a mouse, one may use P3-X63/Ag8, X63-Ag8.653, NSl/l .Ag 4 1 , Sp210-Agl4,

FO. NSO/U. MPC-1 1 , MPC1 1-X45-GTG 1.7 and S194/5XX0 Bui; for rats, one may use lob^' R210.RCY3, Y3-Ag 1.2.3, IR983F and 4B210; and U-266, GM1500-GRG2,

LICR-LON-HMy2 and UC729-6 are all useful in connection with human cell fusions.

One preferred murine myeloma cell is the NS-1 myeloma cell line (also termed

P3-NS-l-Ag4-l), which is readily available from the NIGMS Human Genetic Mutant Cell Repository by requesting cell line repository number GM3573. Another mouse myeloma cell line that may be used is the 8-azaguanine-resistant mouse murine myeloma

SP2/0 non-producer cell line.

Methods for generating hybrids of antibody-producing spleen or lymph node cells and myeloma cells usually comprise mixing somatic cells with myeloma cells in a 2:1 ratio, though the ratio may vary from about 20: 1 to about 1 :1, respectively, in the presence of an agent or agents (chemical or electrical) that promote the fusion of cell membranes. Fusion methods using Sendai virus have been described (Kohler and Milstein, 1975; 1976), and those using polyethylene glycol (PEG), such as 37% (v/v) PEG, by Gefter et al. (1977). The use of electrically induced fusion methods is also appropriate (Goding, 1986).

Fusion procedures usually produce viable hybrids at low frequencies, about 1 x 10^" to about 1 x 10^" . However, this does not pose a problem, as the viable, fused hybrids are differentiated from the parental, unfused cells (particularly the unfused myeloma cells that would normally continue to divide indefinitely) by culturing in a selective medium. The selective medium is generally one that contains an agent that blocks the de novo synthesis of nucleotides in the tissue culture media. Exemplary and preferred agents are aminopterin, methotrexate, and azaserine. Aminopterin and methotrexate block de novo synthesis of both purines and pyrimidines, whereas azaserine blocks only purine synthesis. Where aminopterin or methotrexate is used, the media is supplemented with hypoxanthine and thymidine as a source of nucleotides (HAT medium). Where azaserine is used, the media is supplemented with hypoxanthine.

The preferred selection medium is HAT. Only cells capable of operating nucleotide salvage pathways are able to survive in HAT medium. The myeloma cells are defective in key enzymes of the salvage pathway, e.g.. hypoxanthine phosphoribosyl transferase (HPRT), and they cannot survive. The B-cells can operate this pathway, but they have a limited life span in culture and generally die within about two weeks. Therefore, the only cells that can survive in the selective media are those hybrids formed from myeloma and B-cells.

This culturing provides a population of hybridomas from which specific hybridomas are selected. Typically, selection of hybridomas is performed by culturing the cells by single-clone dilution in microtiter plates, followed by testing the individual clonal supernatants (after about two to three weeks) for the desired reactivity. The assay should be sensitive, simple and rapid, such as radioimmunoassays, enzyme immunoassays, cytotoxicity assays, plaque assays, dot immunobinding assays, and the like. The selected hybridomas would then be serially diluted and cloned into individual antibody-producing cell lines, which clones can then be propagated indefinitely to provide mAbs. The cell lines may be exploited for mAb production in two basic ways. A sample of the hybridoma can be injected (often into the peritoneal cavity) into a histocompatible animal of the type that was used to provide the somatic and myeloma cells for the original fusion. The injected animal develops tumors secreting the specific mAb produced by the fused cell hybrid. The body fluids of the animal, such as serum or ascites fluid, can then be tapped to provide mAbs in high concentration. The individual cell lines could also be cultured in vitro, where the mAbs are naturally secreted into the culture medium from which they can be readily obtained in high concentrations. mAbs produced by either means may be further purified, if desired, using filtration, centrifugation and various chromatographic methods such as HPLC or affinity chromatography.

4.7.3 IMMUNOASSAYS Turning first to immunoassays, in their most simple and direct sense, preferred immunoassays of the invention include the various types of enzyme linked immunosorbent assays (ELISAs), as are known to those of skill in the art. However, it will be readily appreciated that other useful embodiments include RIAs and other non- enzyme linked antibody binding assays and procedures. In preferred ELISA assays, proteins or peptides incorporating polypeptides expressed by a promoter of the present invention are immobilized onto a selected surface, preferably a surface exhibiting a protein affinity, such as the wells of a polystyrene (e l microtiter plate. After washing to remove incompletely adsorbed material, one would then generally desire to bind or coat a nonspecific protein that is known to be antigenically neutral with regard to the test antisera, such as bovine serum albumin (BSA) or casein, onto the well. This allows for blocking of nonspecific adsorption sites on the immobilizing surface and thus reduces the background caused by nonspecific binding of antisera onto the surface.

After binding of antigenic material to the well, coating with a non-reactive material to reduce background, and washing to remove unbound material, the immobilizing surface is contacted with the antisera or clinical or biological extract to be tested in a manner conducive to immune complex (antigen/antibody) formation. Such conditions preferably include diluting the antisera with diluents such as BSA, bovine gamma globulin (BGG) and phosphate buffered saline (PBS)/Tween™. These added agents also tend to assist in the reduction of nonspecific background. The layered antisera is then allowed to incubate for, e.g., from 2 to 4 h, at temperatures preferably on the order of about 25° to about 27°C. Following incubation, the antisera-contacted surface is washed so as to remove non-immunocomplexed material. A preferred washing procedure includes washing with a solution such as PBS/Tween™, or borate buffer.

Following formation of specific immunocomplexes between the test sample and the bound antigen, and subsequent washing, the occurrence and the amount of immunocomplex formation may be determined by subjecting the complex to a second antibody having specificity for the first. Of course, in that the test sample will typically be of human origin, the second antibody will preferably be an antibody having specificity for human antibodies. To provide a detecting means, the second antibody will preferably have an associated detectable label, such as an enzyme label, that will generate a signal, such as color development upon incubating with an appropriate chromogenic substrate. Thus, for example, one will desire to contact and incubate the antisera-bound surface with a urease or peroxidase-conjugated anti-human IgG for a period of time and under conditions that favor the development of immunocomplex formation (e.g., incubation for 2 h at room temperature in a PBS-containing solution such as PBS-Tween™). After incubation with the second enzyme-tagged antibody, and subsequent to washing to remove unbound material, the amount of label is quantified by incubation with a chromogenic substrate such as urea and bromocresol purple or 2,2'-azino-di-(3-ethyl- benzthiazoline)-6-sulfonic acid (ABTS) and H₂O₂, in the case of peroxidase as the enzyme label. Quantitation is then achieved by measuring the degree of color generation, e.g.. using a visible spectrum spectrophotometer.

4.7.4 IMMUNOPRECIPITATION

The antibodies of the present invention are particularly useful for the isolation of antigens by immunoprecipitation. Immunoprecipitation involves the separation of the target antigen component from a complex mixture, and is used to discriminate or isolate minute amounts of protein. For the isolation of cell-surface localized proteins, nonionic salts are preferred, since other agents such as bile salts, precipitate at acid pH or in the presence of bivalent cations.

In an alternative embodiment the antibodies of the present invention are useful for the close juxtaposition of two antigens. This is particularly useful for increasing the localized concentration of antigens, e.g., enzyme-substrate pairs.

4.7.5 WESTERN BLOTS

The compositions of the present invention will find great use in immunoblot or western blot analysis. The antibodies may be used as high-affinity primary reagents for the identification of proteins immobilized onto a solid support matrix, such as nitrocellulose, nylon or combinations thereof. In conjunction with immunoprecipitation, followed by gel electrophoresis, these may be used as a single step reagent for use in detecting antigens against which secondary reagents used in the detection of the antigen cause an adverse background. This is especially useful when the antigens studied are immunoglobulins (precluding the use of immunoglobulins binding bacterial cell wall components), the antigens studied cross-react with the detecting agent, or they migrate at the same relative molecular weight as a cross-reacting signal. Immunologically-based detection methods in conjunction with Western blotting (including enzymatically-. radiolabel-. or fluorescently-tagged secondary antibodies against the toxin moiety) are considered to be of particular use in this regard. 4.8 PRODUCTION AND CHARACTERIZATION OF STABLE TRANSGENIC PLANTS 4.8.1 SELECTION

An example of a screenable marker trait is the red pigment produced under the control of the R-locus in maize. This pigment may be detected by culturing cells on a solid support containing nutrient media capable of supporting growth at this stage and selecting cells from colonies (visible aggregates of cells) that are pigmented. These cells may be cultured further, either in suspension or on solid media. The R-locus is useful for selection of transformants from bombarded immature embryos. In a similar fashion, the introduction of the C 1 and B genes will result in pigmented cells and/or tissues.

The enzyme luciferase is also useful as a screenable marker in the context of the present invention. In the presence of the substrate luciferin, cells expressing luciferase emit light which can be detected on photographic or x-ray film, in a luminometer (or liquid scintillation counter), by devices that enhance night vision, or by a highly light sensitive video camera, such as a photon counting camera. All of these assays are nondestructive and transformed cells may be cultured further following identification. The photon counting camera is especially valuable as it allows one to identify specific cells or groups of cells which are expressing luciferase and manipulate those in real time. The above techniques also could be utilized if the screenable marker is the green fluorescent protein.

It is further contemplated that combinations of screenable and selectable markers will be useful for identification of transformed cells. In some cell or tissue types a selection agent, such as bialaphos or glyphosate, may either not provide enough killing activity to clearly recognize transformed cells or may cause substantial nonselective inhibition of transformants and nontransformants alike, thus causing the selection technique to not be effective. It is proposed that selection with a growth inhibiting compound, such as bialaphos or glyphosate at concentrations below those that cause 100% inhibition followed by screening of growing tissue for expression of a screenable marker gene such as luciferase would allow one to recover transformants from cell or tissue types that are not amenable to selection alone. Therefore it is proposed that combinations of selection and screening will enable one to identify transformants in a wider variety of cell and tissue types. 4.8.2 CHARACTERIZATION

To confirm the presence of the exogenous DNA or "transgene (s)" in the regenerating plants, a variety of assays may be performed. Such assays include, for example, "molecular biological" assays, such as Southern and Northern blotting and

PCR; "biochemical" assays, such as detecting the presence of a protein product, e.g., by immunological means (ELISAs and Western blots) or by enzymatic function; plant part assays, such as leaf or root assays; and also, by analyzing the phenotype of the whole regenerated plant.

4.8.2.1 DNA INTEGRATION, RNA EXPRESSION AND INHERITANCE

Genomic DNA may be isolated from callus cell lines or any plant parts to determine the presence of the exogenous gene through the use of techniques well known to those skilled in the art. Note, that intact sequences will not always be present, presumably due to rearrangement or deletion of sequences in the cell.

The presence of DNA elements introduced through the methods of this invention may be determined by polymerase chain reaction (PCR™). Using this technique discreet fragments of DNA are amplified and detected by gel electrophoresis. This type of analysis permits one to determine whether a gene is present in a stable transformant. but does not prove integration of the introduced gene into the host cell genome. It is the experience of the inventors, however, that DNA has been integrated into the genome of all transformants that demonstrate the presence of the gene through PCR™ analysis. In addition, it is not possible using PCR™ techniques to determine whether transformants have exogenous genes introduced into different sites in the genome, i.e.. whether transformants are of independent origin. It is contemplated that using PCR™ techniques it would be possible to clone fragments of the host genomic DNA adjacent to an introduced gene.

Positive proof of DNA integration into the host genome and the independent identities of transformants may be determined using the technique of Southern hybridization. Using this technique specific DNA sequences that were introduced into the host genome and flanking host DNA sequences can be identified. Hence the Southern hybridization pattern of a given transformant serves as an identifying characteristic of that transformant. In addition it is possible through Southern hybridization to demonstrate the presence of introduced genes in high molecular weight DNA, i.e.. confirm that the introduced gene has been integrated into the host cell genome. The technique of Southern hybridization provides information that is obtained using PCR™ e.g., the presence of a gene, but also demonstrates integration into the genome and characterizes each individual transformant.

It is contemplated that using the techniques of dot or slot blot hybridization which are modifications of Southern hybridization techniques one could obtain the same information that is derived from PCR™, e.g., the presence of a gene. Whereas DNA analysis techniques may be conducted using DNA isolated from any part of a plant, RNA will only be expressed in particular cells or tissue types and hence it will be necessary to prepare RNA for analysis from these tissues. PCR™ techniques may also be used for detection and quantitation of RNA produced from introduced genes. In this application of PCR™ it is first necessary to reverse transcribe RNA into DNA, using enzymes such as reverse transcriptase, and then through the use of conventional PCR™ techniques amplify the DNA. In most instances PCR™ techniques, while useful, will not demonstrate integrity of the RNA product. Further information about the nature of the RNA product may be obtained by Northern blotting. This technique will demonstrate the presence of an RNA species and give information about the integrity of that RNA. The presence or absence of an RNA species can also be determined using dot or slot blot Northern hybridizations. These techniques are modifications of Northern blotting and will only demonstrate the presence or absence of an RNA species.

4.8.2.2 GENE EXPRESSION

While Southern blotting and PCR™ may be used to detect the gene(s) in question, they do not provide information as to whether the gene is being expressed.

Expression may be evaluated by specifically identifying the protein products of the introduced genes or evaluating the phenotypic changes brought about by their expression.

Assays for the production and identification of specific proteins may make use of physical-chemical, structural, functional, or other properties of the proteins. Unique physical-chemical or structural properties allow the proteins to be separated and identified by electrophoretic procedures, such as native or denaturing gel electrophoresis or isoelectric focussing, or by chromatographic techniques such as ion exchange or gel exclusion chromatography. The unique structures of individual proteins offer opportunities for use of specific antibodies to detect their presence in formats such as an ELISA assay. Combinations of approaches may be employed with even greater specificity such as western blotting in which antibodies are used to locate individual gene products that have been separated by electrophoretic techniques. Additional techniques may be employed to absolutely confirm the identity of the product of interest such as evaluation by amino acid sequencing following purification. Although these are among the most commonly employed, other procedures may be additionally used.

Assay procedures may also be used to identify the expression of proteins by their functionality, especially the ability of enzymes to catalyze specific chemical reactions involving specific substrates and products. These reactions may be followed by providing and quantifying the loss of substrates or the generation of products of the reactions by physical or chemical procedures. Examples are as varied as the enzyme to be analyzed and may include assays for PAT enzymatic activity by following production of radiolabeled acetylated phosphinothricin from phosphinothricin and C- acetyl CoA or for anthranilate synthase activity by following loss of fluorescence of anthranilate, to name two.

Very frequently the expression of a gene product is determined by evaluating the phenotypic results of its expression. These assays also may take many forms including but not limited to analyzing changes in the chemical composition, moφhology, or physiological properties of the plant. Chemical composition may be altered by expression of genes encoding enzymes or storage proteins which change amino acid composition and may be detected by amino acid analysis, or by enzymes which change starch quantity which may be analyzed by near infrared reflectance spectrometry. Moφhological changes may include greater stature or thicker stalks. Most often changes in response of plants or plant parts to imposed treatments are evaluated under carefully controlled conditions termed bioassays. An example is to evaluate resistance to insect feeding. 4.9 EXPRESSION VECTORS

The present invention contemplates an expression vector comprising a polynucleotide of the present invention. Thus, in one embodiment an expression vector is an isolated and purified DNA molecule comprising a promoter of the present invention operatively linked to a coding region that encodes a polypeptide, which coding region is operatively linked to a transcription-terminating region, whereby the promoter drives the transcription of the coding region.

In another embodiment, the promoter of the present invention is operatively linked to a coding region that encodes a functional RNA. A functional RNA may encode for a polypeptide(mRNA), be a tRNA, have ribozyme activity, or be an antisense RNA.

As used herein, the term "operatively linked" means that a promoter is connected to a functional RNA in such a way that the transcription of that functional RNA is controlled and regulated by that promoter. Means for operatively linking a promoter to a functional RNA are well known in the art.

A transgenic plant of the present invention produced from a plant cell transformed with a tissue specific promoter can be crossed with a second transgenic plant developed from a plant cell transformed with a different tissue specific promoter to produce a hybrid transgenic plant that shows the effects of transformation in more than one specific tissue.

Exemplary plant tissue-specific promoters are corn sucrose synthetase 1 (Yang et al. , 1990), corn alcohol dehydrogenase 1 (Vogel et al. , 1989), corn light harvesting complex (Simpson, 1986), corn heat shock protein (Odell et al, 1985), pea small subunit RuBP carboxylase (Poulsen et al, 1986; Cashmore et al, 1983), petunia chalcone isomerase (Van Tunen et al, 1988), bean glycine rich protein 1 (Keller et al, 1989), CaMV 35s transcript (Odell et al , 1985) and Potato patatin (Wenzler et al, 1989). Exemplary plant promoters include the promoters of the curent invention, along with the cauliflower mosaic virus (CaMV 35S) promoter and the S-E9 small subunit RuBP carboxylase promoter.

The choice of which expression vector and ultimately to which promoter a polypeptide coding region is operatively linked depends directly on the functional properties desired, e.g., the location and ti πmiing of protein expression, and the host cell to be transformed. These are well known limitations inherent in the art of constructing recombinant DNA molecules. However, a vector useful in practicing the present invention is capable of directing the expression of the functional RNA to which it is operatively linked.

Typical vectors useful for expression of genes in higher plants are well known in the art and include vectors derived from the tumor-inducing (Ti) plasmid of Agrobacterium tumefaciens described (Rogers et al, 1987). However, several other plant integrating vector systems are known to function in plants including pCaMVCN transfer control vector described (Fromm et al , 1985). Plasmid pCaMVCN (available from Pharmacia, Piscataway, NJ) includes the cauliflower mosaic virus CaMV 35S promoter.

In preferred embodiments, the vector used to express the coding region includes a selection marker that is effective in a plant cell, preferably a drug resistance selection marker. One preferred drug resistance marker is the gene whose expression results in kanamycin resistance; i.e. , the chimeric gene containing the nopaline synthase promoter, Tn5 neomycin phosphotransferase II (nptll) and nopaline synthase 3' non- translated region described (Rogers et al, 1988).

RNA polymerase transcribes a coding DNA sequence through a site where polyadenylation occurs. Typically, DNA sequences located a few hundred base pairs downstream of the polyadenylation site serve to terminate transcription. Those DNA sequences are referred to herein as transcription-termination regions. Those regions are required for efficient polyadenylation of transcribed messenger RNA (mRNA).

Means for preparing expression vectors are well known in the art. For example, expression (transformation) vectors used to transform plants and methods of making those vectors are described in U. S. Patent Nos. 4,971 ,908, 4,940,835, 4,769,061 and

4.757,01 1. the disclosures of which are incorporated herein by reference. Those vectors can be modified to include a coding sequence in accordance with the present invention.

A variety of methods has been developed to operatively link DNA to vectors via complementary cohesive termini or blunt ends. For instance, complementary homopolymer tracts can be added to the DNA segment to be inserted and to the vector ηS DNA. The vector and DNA segment are then joined by hydrogen bonding between the complementary homopolymeric tails to form recombinant DNA molecules.

4.10 DNA SEGMENTS AS HYBRIDIZATION PROBES AND PRIMERS In another aspect, DNA sequence information provided by the invention allows for the preparation of relatively short DNA (or RNA) sequences having the ability to specifically hybridize to gene sequences of the selected polynucleotides disclosed herein. In these aspects, nucleic acid probes of an appropriate length are prepared based on a consideration of a selected MsENOD40 promoter sequence, e.g., a sequence such as that shown in SEQ ID NOT and SEQ ID NO:2. The ability of such nucleic acid probes to specifically hybridize to a MsENOD40 promoter sequence lends them particular utility in a variety of embodiments. Most importantly, the probes may be used in a variety of assays for detecting the presence of complementary sequences in a given sample. In certain embodiments, it is advantageous to use oligonucleotide primers. The sequence of such primers is designed using a polynucleotide of the present invention for use in detecting, amplifying or mutating a defined segment of a MsENOD40 promoter from alfalfa using PCR™ technology. Segments of related MsENOD40 promoters from other species may also be amplified by PCR™ using such primers. To provide certain of the advantages in accordance with the present invention, a preferred nucleic acid sequence employed for hybridization studies or assays includes sequences that are complementary to at least a 14 to 30 or so long nucleotide stretch of a MsENOD40 promoter sequence, such as that shown in SEQ ID NOT and SEQ ID NO:2. A size of at least 14 nucleotides in length helps to ensure that the fragment will be of sufficient length to form a duplex molecule that is both stable and selective. Molecules having complementary sequences over stretches greater than 14 bases in length are generally preferred, though, in order to increase stability and selectivity of the hybrid, and thereby improve the quality and degree of specific hybrid molecules obtained. One will generally prefer to design nucleic acid molecules having gene- complementary stretches of 14 to 20 nucleotides, or even longer where desired. Such fragments may be readily prepared by, for example, directly synthesizing the fragment by chemical means, by application of nucleic acid reproduction technology, such as the 7to PCR™ technology of U. S. Patent Nos. 4,683,195, and 4,683,202, herein incoφorated by reference, or by excising selected DNA fragments from recombinant plasmids containing appropriate inserts and suitable restriction sites.

Of course, for some applications, for example, where one desires to prepare mutants employing a mutant primer strand hybridized to an underlying template or where one seeks to isolate a MsENOD40 promoter sequence for related species, functional equivalents, or the like, less stringent hybridization conditions will typically be needed in order to allow formation of the heteroduplex. In these circumstances, one may desire to employ conditions such as about 0.15 M to about 0.9 M salt, at temperatures ranging from about 20°C to about 55°C. Cross-hybridizing species can thereby be readily identified as positively hybridizing signals with respect to control hybridizations. In any case, it is generally appreciated that conditions can be rendered more stringent by the addition of increasing amounts of formamide, which serves to destabilize the hybrid duplex in the same manner as increased temperature. Thus, hybridization conditions can be readily manipulated, and thus will generally be a method of choice depending on the desired results.

In addition to the use in directing the expression of functional RNA of the present invention, the nucleic acid sequences contemplated herein also have a variety of other uses. For example, they also have utility as probes or primers in nucleic acid hybridization embodiments. As such, it is contemplated that nucleic acid segments that comprise a sequence region that consists of at least a 14 nucleotide long contiguous sequence that has the same sequence as, or is complementary to, a 14 nucleotide long contiguous DNA segment of SEQ ID NOT and SEQ ID NO:2 will find particular utility. Longer contiguous identical or complementary sequences, e.g., those of about 20, 30, 40, 50, 100, 200, 500, 1000, 2000, 5000, 10000 etc. (including all intermediate lengths and up to and including full-length sequences will also be of use in certain embodiments.

The ability of such nucleic acid probes to specifically hybridize to MsENOD40 promoter sequence will enable them to be of use in detecting the presence of complementary sequences in a given sample. However, other uses are envisioned, including the use of the sequence information for the preparation of mutant species primers, or primers for use in preparing other genetic constructions. ?? Nucleic acid molecules having sequence regions consisting of contiguous nucleotide stretches of 10-14, 15-20, 30, 50, or even of 100-200 nucleotides or so, identical or complementary to DNA sequences of SEQ ID NOT and SEQ ID NO:2 are particularly contemplated as hybridization probes for use in, e.g., Southern and Northern blotting. Smaller fragments will generally find use in hybridization embodiments, wherein the length of the contiguous complementary region may be varied, such as between about 10-14 and about 100 or 200 nucleotides, but larger contiguous complementarity stretches may be used, according to the length complementary sequences one wishes to detect. The use of a hybridization probe of about 14 nucleotides in length allows the formation of a duplex molecule that is both stable and selective. Molecules having contiguous complementary sequences over stretches greater than 14 bases in length are generally preferred, though, in order to increase stability and selectivity of the hybrid, and thereby improve the quality and degree of specific hybrid molecules obtained. One will generally prefer to design nucleic acid molecules having gene-complementary stretches of 15 to 20 contiguous nucleotides, or even longer where desired.

Of course, fragments may also be obtained by other techniques such as, e.g., by mechanical shearing or by restriction enzyme digestion. Small nucleic acid segments or fragments may be readily prepared by, for example, directly synthesizing the fragment by chemical means, as is commonly practiced using an automated oligonucleotide synthesizer. Also, fragments may be obtained by application of nucleic acid reproduction technology, such as the PCR™ technology of U. S. Patent Nos. 4,683,195 and 4,683,202 (each incoφorated herein by reference), by introducing selected sequences into recombinant vectors for recombinant production, and by other recombinant DNA techniques generally known to those of skill in the art of molecular biology.

Accordingly, the nucleotide sequences of the invention may be used for their ability to selectively form duplex molecules with complementary stretches of DNA fragments. Depending on the application envisioned, one will desire to employ varying conditions of hybridization to achieve varying degrees of selectivity of probe towards target sequence. For applications requiring high selectivity, one will typically desire to employ relatively stringent conditions to form the hybrids, e.g. , one will select 7S relatively low salt and/or high temperature conditions, such as provided by about 0.02

M to about 0.15 M NaCl at temperatures of about 50°C to about 70°C. Such selective conditions tolerate little, if any, mismatch between the probe and the template or target strand, and would be particularly suitable for isolating MsENOD40 promoter DNA segments. Detection of DNA segments via hybridization is well-known to those of skill in the art, and the teachings of U. S. Patent Nos. 4,965,188 and 5,176,995 (each incorporated herein by reference) are exemplary of the methods of hybridization analyses. Teachings such as those found in the texts of Maloy et al, 1994; Segal 1976;

Prokop, 1991 ; and Kuby, 1994, are particularly relevant. Of course, for some applications, for example, where one desires to prepare mutants employing a mutant primer strand hybridized to an underlying template or where one seeks to isolate an MsENOD40 promoter from related species, functional equivalents, or the like, less stringent hybridization conditions will typically be needed in order to allow formation of the heteroduplex. In these circumstances, one may desire to employ conditions such as about 0.15 M to about 0.9 M salt, at temperatures ranging from about 20°C to about 55°C. Cross-hybridizing species can thereby be readily identified as positively hybridizing signals with respect to control hybridizations. In any case, it is generally appreciated that conditions can be rendered more stringent by the addition of increasing amounts of formamide, which serves to destabilize the hybrid duplex in the same manner as increased temperature. Thus, hybridization conditions can be readily manipulated, and thus will generally be a method of choice depending on the desired results.

In certain embodiments, it will be advantageous to employ nucleic acid sequences of the present invention in combination with an appropriate means, such as a label, for determining hybridization. A wide variety of appropriate indicator means are known in the art, including fluorescent, radioactive, enzymatic or other ligands, such as avidin/biotin, which are capable of giving a detectable signal. In preferred embodiments, one will likely desire to employ a fluorescent label or an enzyme tag, such as urease, alkaline phosphatase or peroxidase, instead of radioactive or other environmental undesirable reagents. In the case of enzyme tags, colorimetric indicator substrates are known that can be employed to provide a means visible to the human eye or spectrophotometrically, to identify specific hybridization with complementary nucleic acid-containing samples.

In general, it is envisioned that the hybridization probes described herein will be useful both as reagents in solution hybridization as well as in embodiments employing a solid phase. In embodiments involving a solid phase, the test DNA (or RNA) is adsorbed or otherwise affixed to a selected matrix or surface. This fixed, single- stranded nucleic acid is then subjected to specific hybridization with selected probes under desired conditions. The selected conditions will depend on the particular circumstances based on the particular criteria required (depending, for example, on the G+C content, type of target nucleic acid, source of nucleic acid, size of hybridization probe, etc.). Following washing of the hybridized surface so as to remove nonspecifically bound probe molecules, specific hybridization is detected, or even quantitated, by means of the label.

4.11 TRANSFORMED OR TRANSGENIC PLANT CELLS

A bacterium, a yeast cell, or a plant cell or a plant transformed with an expression vector of the present invention is also contemplated. A transgenic bacterium, yeast cell, plant cell or plant derived from such a transformed or transgenic cell is also contemplated. Means for transforming bacteria and yeast cells are well known in the art. Typically, means of transformation are similar to those well known means used to transform other bacteria or yeast such as E. coli or Saccharomyces cerevisiae.

Methods for DNA transformation of plant cells include Agrobacterium- mediated plant transformation, protoplast transformation, gene transfer into pollen, injection into reproductive organs, injection into immature embryos and particle bombardment. Each of these methods has distinct advantages and disadvantages. Thus, one particular method of introducing genes into a particular plant strain may not necessarily be the most effective for another plant strain, but it is well known which methods are useful for a particular plant strain. There are many methods for introducing transforming DNA segments into cells, but not all are suitable for delivering DNA to plant cells. Suitable methods are believed to include virtually any method by which DNA can be introduced into a cell, such as by Agrobacterium infection, direct delivery of DNA such as, for example, by PEG- mediated transformation of protoplasts (Omirulleh et al, 1993), by desiccation inhibition-mediated DNA uptake, by electroporation, by agitation with silicon carbide fibers, by acceleration of DNA coated particles, etc. In certain embodiments, acceleration methods are preferred and include, for example, microprojectile bombardment and the like.

Technology for introduction of DNA into cells is well-known to those of skill in the art. Four general methods for delivering a gene into cells have been described: (1) chemical methods (Graham and van der Eb, 1973; Zatloukal et al, 1992); (2) physical methods such as microinjection (Capecchi, 1980), electroporation (Wong and Neumann, 1982; Frornm et al , 1985) and the gene gun (Johnston and Tang, 1994; Fynan et al, 1993); (3) viral vectors (Clapp, 1993; Lu et al , 1993; Eglitis and Anderson, 1988a; 1988b); and (4) receptor-mediated mechanisms (Curiel et al, 1991 ; 1992; Wagner et al, 1992).

4.11.1 ELECTROPORATION

The application of brief, high-voltage electric pulses to a variety of animal and plant cells leads to the formation of nanometer-sized pores in the plasma membrane. DNA is taken directly into the cell cytoplasm either through these pores or as a consequence of the redistribution of membrane components that accompanies closure of the pores. Electroporation can be extremely efficient and can be used both for transient expression of clones genes and for establishment of cell lines that carry integrated copies of the gene of interest. Electroporation, in contrast to calcium phosphate-mediated transfection and protoplast fusion, frequently gives rise to cell lines that carry one, or at most a few, integrated copies of the foreign DNA.

The introduction of DNA by means of electroporation, is well-known to those of skill in the art. In this method, certain cell wall-degrading enzymes, such as pectin- degrading enzymes, are employed to render the target recipient cells more susceptible to transformation by electroporation than untreated cells. Alternatively, recipient cells are made more susceptible to transformation, by mechanical wounding. To effect transformation by electroporation one may employ either friable tissues such as a suspension culture of cells, or embryogenic callus, or alternatively, one may transform immature embryos or other organized tissues directly. One would partially degrade the cell walls of the chosen cells by exposing them to pectin-degrading enzymes (pectolyases) or mechanically wounding in a controlled manner. Such cells would then be recipient to DNA transfer by electroporation. which may be carried out at this stage, and transformed cells then identified by a suitable selection or screening protocol dependent on the nature of the newly incoφorated DNA.

4.11.2 MICROPROJECTILE BOMBARDMENT

A further advantageous method for delivering transforming DNA segments to plant cells is microprojectile bombardment. In this method, particles may be coated with nucleic acids and delivered into cells by a propelling force. Exemplary particles include those comprised of tungsten, gold, platinum, and the like.

An advantage of microprojectile bombardment, in addition to it being an effective means of reproducibly stably transforming monocots, is that neither the isolation of protoplasts (Cristou et al, 1988) nor the susceptibility to Agrobacterium infection is required. An illustrative embodiment of a method for delivering DNA into maize cells by acceleration is a Biolistics Particle Delivery System, which can be used to propel particles coated with DNA or cells through a screen, such as a stainless steel or Nytex screen, onto a filter surface covered with corn cells cultured in suspension. The screen disperses the particles so that they are not delivered to the recipient cells in large aggregates. It is believed that a screen intervening between the projectile apparatus and the cells to be bombarded reduces the size of projectiles aggregate and may contribute to a higher frequency of transformation by reducing damage inflicted on the recipient cells by projectiles that are too large. For the bombardment, cells in suspension are preferably concentrated on filters or solid culture medium. Alternatively, immature embryos or other target cells may be arranged on solid culture medium. The cells to be bombarded are positioned at an appropriate distance below the macroprojectile stopping plate. If desired, one or more screens are also positioned between the acceleration device and the cells to be bombarded. Through the use of techniques set forth herein one may obtain up to 1000 or more foci of cells transiently expressing a marker gene. The number of cells in a focus which express the exogenous gene product 48 hours post-bombardment often range from 1 to 10 and average 1 to 3.

In bombardment transformation, one may optimize the prebombardment culturing conditions and the bombardment parameters to yield the maximum numbers of stable transformants. Both the physical and biological parameters for bombardment are important in this technology. Physical factors are those that involve manipulating the DNA/microprojectile precipitate or those that affect the flight and velocity of either the macro- or microprojectiles. Biological factors include all steps involved in manipulation of cells before and immediately after bombardment, the osmotic adjustment of target cells to help alleviate the trauma associated with bombardment, and also the nature of the transforming DNA, such as linearized DNA or intact supercoiled plasmids. It is believed that pre-bombardment manipulations are especially important for successful transformation of immature embryos.

Accordingly, it is contemplated that one may wish to adjust various of the bombardment parameters in small scale studies to fully optimize the conditions. One may particularly wish to adjust physical parameters such as gap distance, flight distance, tissue distance, and helium pressure. One may also minimize the trauma reduction factors (TRFs) by modifying conditions which influence the physiological state of the recipient cells and which may therefore influence transformation and integration efficiencies. For example, the osmotic state, tissue hydration and the subculture stage or cell cycle of the recipient cells may be adjusted for optimum transformation. The execution of other routine adjustments will be known to those of skill in the art in light of the present disclosure.

4.11.3 AGROBACTERI UM-MEDIATED TRANSFER

Agrobacterium-mediated transfer is a widely applicable system for introducing genes into plant cells because the DNA can be introduced into whole plant tissues, thereby bypassing the need for regeneration of an intact plant from a protoplast. The use of Agrobacterium-mediated plant integrating vectors to introduce DNA into plant cells is well known in the art. See. for example, the methods described (Fraley et α , 1985; Rogers et αl . 1987). Further, the integration of the Ti-DNA is a relatively precise process resulting in few rearrangements. The region of DNA to be transferred 3 is defined by the border sequences, and intervening DNA is usually inserted into the plant genome as described (Spielmann et al, 1986; Jorgensen et al, 1987).

Modern Agrobacterium transformation vectors are capable of replication in E. coli as well as Agrobacterium. allowing for convenient manipulations as described (Klee et al, 1985). Moreover, recent technological advances in vectors for

Agrobacterium-mediated gene transfer have improved the arrangement of genes and restriction sites in the vectors to facilitate construction of vectors capable of expressing various polypeptide coding genes. The vectors described (Rogers et αl., 1987), have convenient multi-linker regions flanked by a promoter and a polyadenylation site for direct expression of inserted polypeptide coding genes and are suitable for present puφoses. In addition, Agrobacterium containing both armed and disarmed Ti genes can be used for the transformations. In those plant strains where Agrobacterium- mediated transformation is efficient, it is the method of choice because of the facile and defined nature of the gene transfer. Agrobacterium-mediated transformation of leaf disks and other tissues such as cotyledons and hypocotyls appears to be limited to plants that Agrobacterium naturally infects. Agrobacterium-mediated transformation is most efficient in dicotyledonous plants. Few monocots appear to be natural hosts for Agrobacterium, although transgenic plants have been produced in asparagus using Agrobacterium vectors. Therefore, commercially important cereal grains such as rice, corn, and wheat must usually be transformed using alternative methods. However, as mentioned above, the transformation of asparagus using Agrobacterium can also be achieved.

A transgenic plant formed using Agrobacterium transformation methods typically contains a single gene on one chromosome. Such transgenic plants can be referred to as being heterozygous for the added gene. However, inasmuch as use of the word "heterozygous" usually implies the presence of a complementary gene at the same locus of the second chromosome of a pair of chromosomes, and there is no such gene in a plant containing one added gene as here, it is believed that a more accurate name for such a plant is an independent segregant, because the added, exogenous gene segregates independently during mitosis and meiosis.

More preferred is a transgenic plant that is homozygous for the added structural gene; i.e., a transgenic plant that contains two added genes, one gene at the same locus fry on each chromosome of a chromosome pair. A homozygous transgenic plant can be obtained by sexually mating (selfing) an independent segregant transgenic plant that contains a single added gene, germinating some of the seed produced and analyzing the resulting plants produced for enhanced carboxylase activity relative to a control (native, non-transgenic) or an independent segregant transgenic plant.

It is to be understood that two different transgenic plants can also be mated to produce offspring that contain two independently segregating added, exogenous genes. Selfing of appropriate progeny can produce plants that are homozygous for both added, exogenous genes that encode a polypeptide of interest. Back-crossing to a parental plant and out-crossing with a non-transgenic plant are also contemplated.

Transformation of plant protoplasts can be achieved using methods based on calcium phosphate precipitation, polyethylene glycol treatment, electroporation, and combinations of these treatments (, e.g., Potrykus et al. 1985; Lorz et al, 1985; Fromm et al, 1985; Uchimiya et α/., 1986; Callis et al, 1987; Marcotte et al, 1988). Application of these systems to different plant strains depends upon the ability to regenerate that particular plant strain from protoplasts. Illustrative methods for the regeneration of cereals from protoplasts are described (Fujimura et al, 1985; Toriyama et al, 1986; Yamada et al, 1986; Abdullah et al, 1986).

To transform plant strains that cannot be successfully regenerated from protoplasts, other ways to introduce DNA into intact cells or tissues can be utilized. For example, regeneration of cereals from immature embryos or explants can be effected as described (Vasil, 1988). In addition, "particle gun" or high-velocity microprojectile technology can be utilized (Vasil, 1992).

Using that latter technology, DNA is carried through the cell wall and into the cytoplasm on the surface of small metal particles as described (Klein et al, 1987; Klein et al, 1988; McCabe et al. 1988). The metal particles penetrate through several layers of cells and thus allow the transformation of cells within tissue explants.

4.12 EXPRESSION IN ANIMAL CELLS The inventors contemplate that the MsENOD40 promoters may also be utilized to allow inducible expression of genes in animal cells. Induction of the gene of interest would comprise of contacting a cell containing an MsENOD40 expression construct with an inducer of the promoter. The animal cells may be mammalian cells such as human or other primate,murine, canine, bovine, equine, epine, or porcine. The cells may be in a normal or transformed state.

4.12.1 PROTEINS

A variety of different proteins can be expressed according to the present invention. Proteins can be grouped generally into two categories - secreted and non- secreted - discussions of each are detailed below.

First, it is contemplated that many proteins will not have a single sequence but, rather, will exists in many forms. These forms may represent allelic variation or, rather, mutant forms of a given protein. Second, it is contemplated that various proteins may be expressed advantageously as "fusion" proteins. Fusions are generated by linking together the coding regions for two proteins, or parts of two proteins. This generates a new, single coding region that gives rise to the fusion protein. Fusions may be useful in producing secreted forms of proteins that are not normally secreted or producing molecules that are immunologically tagged. Tagged proteins may be more easily purified or monitored using antibodies to the tag. A third variation contemplated by the present invention involves the expression of protein fragments. It may not be necessary to express an entire protein and, in some cases, it may be desirable to express a particular functional domain, for example, where the protein fragment remains functional but is more stable, or less antigenic, or both.

4.12.1.1 SECRETED PROTEINS

Expression of several proteins that are normally secreted can be engineered into animal cells. The cDNA's encoding a number of useful human proteins are available.

Examples would include soluble CD-4, Factor VIII. Factor IX, von Willebrand Factor,

TPA. urokinase, hirudin, interferons, TNF, interleukins. hematopoietic growth factors, antibodies, albumin, leptin, transferin and nerve growth factors.

Peptide hormones are grouped into three classes with specific examples given for each. These classes are defined by the complexity of their post-translational processing. Class I proteins generally are considered to include growth hormone, prolactin, placental lactogen. luteinizing hormone, follicle-stimulating hormone. chorionic gonadotropin, and thyroid-stimulating hormone. These require relatively limited proteolytic processing followed by storage and stimulated release from secretory granules.

Class II is represented human peptide hormones such as adrenocorticotropin (ACTFI), angiotensin I and II, β-endoφhin, β-melanocyte stimulating hormone (β- MSH), cholecystokinin, endothelin I, galanin, gastric inhibitory peptide (GIP), glucagon, insulin, lipotropins, neurophysins, and somatostatin. Further proteolytic processing is required, with both endoproteases and carboxypeptidases processing of larger precursor molecules occurring in the secretory granules. Class III includes, for example, Calcium Metabolism Peptides such as calcitonin. calcitonin gene related peptide (CGRP), β-calcitonin gene related peptide, hypercalcemia of malignancy factor (1-40) (PTH-rP), parathyroid hormone-related protein (107-139) (PTH-rP), and parathyroid hormone-related protein (107-1 1 1) (PTH- rP); Gastrointestinal Peptides, such as cholecystokinin (27-33) (CCK), galanin message associated peptide, preprogalanin (65-105), gastrin I, gastrin releasing peptide, glucagon-like peptide (GLP-1), pancreastatin, pancreatic peptide, peptide YY, PHM, secretin, and vasoactive intestinal peptide (VIP); and Pituitary Peptides, such as oxytocin, vasopressin (AVP), and vasotocin; Enkephalins, such as enkephalinamide, and metoφhinamide (adrenoφhin). Also included in Class III are peptides such as alpha melanocyte stimulating hormone (α-MSH), atrial natriuretic factor (5-28) (ANF), amylin, amyloid P component (SAP-1), corticotropin releasing hormone (CRH), growth hormone releasing factor (GHRH), luteinizing Hormone-releasing hormone (LHRH). neuropeptide Y, substance K (Neurokinin A ), substance P, and thyrotropin releasing hormone (TRH). In addition to the proteolytic processing found in the Class II peptides. amidation of the C-terminus is required for proper biological function.

4.12.1.2 NON-SECRETED PROTEINS

Expression of non-secreted proteins can be engineered into animal cells. Two general classes of such proteins can be defined. The first are proteins that, once expressed in cells, stay associated with the cells in a variety of destinations. These destinations include the cytoplasm, nucleus, mitochondria, endoplasmic reticulum, golgi, membrane of secretory granules and plasma membrane. Non-secreted proteins are both soluble and membrane associated. The second class of proteins are ones that are normally associated with the cell, but have been modified such that they are now secreted by the cell. Modifications would include site-directed mutagenesis or expression of truncations of engineered proteins resulting in their secretion as well as creating novel fusion proteins that result in secretion of a normally non-secreted protein.

Cells engineered to produce such proteins could be used for either in vitro production of the protein or for in vivo, cell-based therapies. In vitro production would entail purification of the expressed protein from either the cell pellet for proteins remaining associated with the cell or from the conditioned media from cells secreting the engineered protein. In vivo, cell-based therapies would either be based on secretion of the engineered protein or beneficial effects of the cells expressing a non-secreted protein.

The cDNA's encoding a number of therapeutically useful human proteins are available. These include cell surface receptors, transporters and channels such as GLUT2, CFTR, leptin receptor, sulfonylurea receptor, β-cell inward rectifying channels, etc. Other proteins include protein processing enzymes such as PC2 and PC3, and PAM, transcription factors such as IPF1, and metabolic enzymes such as adenosine deaminase, phenylalanine hydroxylase, glucocerebrosidase.

4.12.2 GENETIC CONSTRUCTS

Also claimed in this patent are examples of DNA expression plasmids designed to optimize production of the heterologous proteins. These include a number of enhancers/promoters from both viral and mammalian sources that drive expression of the genes of interest in aniamal cells. Elements designed to optimize messenger RNA stability and translatability in animal cells are defined

4.12.2.1 VECTOR BACKBONE

Throughout this application, the term "expression construct" is meant to include any type of genetic construct containing a nucleic acid coding for a gene product in which part or all of the nucleic acid encoding sequence is capable of being transcribed.

The transcript may be translated into a protein, but it need not be. In certain embodiments, expression includes both transcription of a gene and translation of mRNA into a gene product. In other embodiments, expression only includes transcription of the nucleic acid encoding a gene of interest.

In preferred embodiments, the nucleic acid encoding a gene product is under transcriptional control of an MsENOD40 promoter. A "promoter" refers to a DNA sequence recognized by the synthetic machinery of the cell, or introduced synthetic machinery, required to initiate the specific transcription of a gene. The phrase "under transcriptional control" means that the promoter is in the correct location and orientation in relation to the nucleic acid to control RNA polymerase initiation and expression of the gene.

The term promoter will be used here to refer to a group of transcriptional control modules that are clustered around the initiation site for RNA polymerase II. Much of the thinking about how promoters are organized derives from analyses of several viral promoters, including those for the HSV thymidine kinase (tk) and SV40 early transcription units. These studies, augmented by more recent work, have shown that promoters are composed of discrete functional modules, each consisting of approximately 7-20 bp of DNA, and containing one or more recognition sites for transcriptional activator or repressor proteins.

At least one module in each promoter functions to position the start site for RNA synthesis. The best known example of this is the TATA box, but in some promoters lacking a TATA box, such as the promoter for the mammalian terminal deoxynucleotidyl transferase gene and the promoter for the SV40 late genes, a discrete element overlying the start site itself helps to fix the place of initiation.

Additional promoter elements regulate the frequency of transcriptional initiation. Typically, these are located in the region 30-1 10 bp upstream of the start site, although a number of promoters have recently been shown to contain functional elements downstream of the start site as well. The spacing between promoter elements frequently is flexible, so that promoter function is preserved when elements are inverted or moved relative to one another. In the tk promoter, the spacing between promoter elements can be increased to 50 bp apart before activity begins to decline. Depending on the promoter, it appears that individual elements can function either co-operatively or independently to activate transcription. Enhancers were originally detected as genetic elements that increased transcription from a promoter located at a distant position on the same molecule of DNA. This ability to act over a large distance had little precedent in classic studies of prokaryotic transcriptional regulation. Subsequent work showed that regions of DNA with enhancer activity are organized much like promoters. That is, they are composed of many individual elements, each of which binds to one or more transcriptional proteins.

The basic distinction between enhancers and promoters is operational. An enhancer region as a whole must be able to stimulate transcription at a distance; this need not be true of a promoter region or its component elements. On the other hand, a promoter must have one or more elements that direct initiation of RNA synthesis at a particular site and in a particular orientation, whereas enhancers lack these specificities. Promoters and enhancers are often overlapping and contiguous, often seeming to have a very similar modular organization. In preferred embodiments of the invention, the expression construct comprises a virus or engineered construct derived from a viral genome. The ability of certain viruses to enter cells via receptor-mediated endocytosis and to integrate into host cell genome and express viral genes stably and efficiently have made them attractive candidates for the transfer of foreign genes into mammalian cells (Ridgeway, 1988; Nicolas and Rubenstein, 1988; Baichwal and Sugden, 1986; Temin. 1986). The first viruses used as gene vectors were DNA viruses including the papovaviruses (simian virus 40, bovine papilloma vims, and polyoma) (Ridgeway, 1988; Baichwal and Sugden, 1986) and adenoviruses (Ridgeway, 1988; Baichwal and Sugden, 1986). These have a relatively low capacity for foreign DNA sequences and have a restricted host spectrum. Furthermore, their oncogenic potential and cytopathic effects in permissive cells raise safety concerns. They can accommodate only up to 8 kB of foreign genetic material but can be readily introduced in a variety of cell lines and laboratory animals (Nicolas and Rubenstein, 1988; Temin, 1986).

4.12.2.2 REGULATORY ELEMENTS

Where a cDNA insert is employed, one will typically desire to include a polyadenylation signal to effect proper polyadenylation of the gene transcript. The qo nature of the polyadenylation signal is not believed to be crucial to the successful practice of the invention, and any such sequence may be employed. Also contemplated as an element of the expression cassette is a terminator. These elements can serve to enhance message levels and to minimize read through from the cassette into other sequences.

4.12.2.3 SELECTABLE MARKERS

In certain embodiments of the invention, the delivery of a nucleic acid in a cell may be identified in vitro or in vivo by including a marker in the expression construct. The marker would result in an identifiable change to the transfected cell permitting easy identification of expression. Usually the inclusion of a drug selection marker aids in cloning and in the selection of transformants, for example, neomycin, puromycin, hygromycin. DHFR, GPT, zeocin and histidinol. Alternatively, enzymes such as heφes simplex virus thymidine kinase (tk) (eukaryotic) or chloramphenicol acetyltransferase (CAT) (prokaryotic) may be employed. Immunologic markers also can be employed. The selectable marker employed is not believed to be important, so long as it is capable of being expressed simultaneously with the nucleic acid encoding a gene product. Further examples of selectable markers are well known to one of skill in the art.

4.12.2.4 MULTIGENE CONSTRUCTS AND IRES

In certain embodiments of the invention, the use of internal ribosome binding sites (IRES) elements are used to create multigene, or polycistronic, messages. IRES elements are able to bypass the ribosome scanning model of 5' methylated Cap dependent translation and begin translation at internal sites (Pelletier and Sonenberg, 1988; Yang et al , 1988). IRES elements from two members of the picanovirus family (polio and encephalomyocarditis) have been described (Pelletier and Sonenberg, 1988), as well an IRES from a mammalian message (Macejak and Sarnow, 1991). IRES elements can be linked to heterologous open reading frames. Multiple open reading frames can be transcribed together, each separated by an IRES, creating polycistronic messages. By virtue of the IRES element, each open reading frame is accessible to 9 / ribosomes for efficient translation. Multiple genes can be efficiently expressed using a single promoter/enhancer to transcribe a single message.

Any heterologous open reading frame can be linked to IRES elements. This includes genes for secreted proteins, multi-subunit proteins, encoded by independent genes, intracellular or membrane-bound proteins and selectable markers. In this way, expression of several proteins can be simultaneously engineered into a cell with a single construct and a single selectable marker.

4.12.3 IN VIVO DELIVERY AND TREATMENT PROTOCOLS It may be desirable to introduce genetic constructs to cells in vivo. There are a number of ways in which nucleic acids may be introduced into cells. Several methods are outlined below.

4.12.3.1 ADENOVIRUS One of the preferred methods for in vivo delivery involves the use of an adenovirus expression vector. "Adenovirus expression vector" is meant to include those constructs containing adenovirus sequences sufficient to (a) support packaging of the construct and (b) to express an antisense polynucleotide that has been cloned therein. In this context, expression does not require that the gene product be synthesized.

The expression vector comprises a genetically engineered form of adenovirus. Knowledge of the genetic organization or adenovirus, a 36 kB, linear, double-stranded DNA vims, allows substitution of large pieces of adenoviral DNA with foreign sequences up to 7 kB (Grunhaus and Horwitz, 1992). In contrast to retrovirus. the adenoviral infection of host cells does not result in chromosomal integration because adenoviral DNA can replicate in an episomal manner without potential genotoxicity. Also, adenoviruses are structurally stable, and no genome rearrangement has been detected after extensive amplification. Adenovirus can infect virtually all epithelial cells regardless of their cell cycle stage. So far, adenoviral infection appears to be linked only to mild disease such as acute respiratory disease in humans.

Adenovirus is particularly suitable for use as a gene transfer vector because of its mid-sized genome, ease of manipulation, high titer, wide target-cell range and high infectivity. Both ends of the viral genome contain 100-200 base pair inverted repeats (ITRs), which are cis elements necessary for viral DNA replication and packaging. The early (E) and late (L) regions of the genome contain different transcription units that are divided by the onset of viral DNA replication. The El region (El A and E1B) encodes proteins responsible for the regulation of transcription of the viral genome and a few cellular genes. The expression of the E2 region (E2A and E2B) results in the synthesis of the proteins for viral DNA replication. These proteins are involved in DNA replication, late gene expression and host cell shut-off (Renan, 1990). The products of the late genes, including the majority of the viral capsid proteins, are expressed only after significant processing of a single primary transcript issued by the major late promoter (MLP). The MLP, (located at 16.8 m.u.) is particularly efficient during the late phase of infection, and all the mRNA's issued from this promoter possess a 5'- tripartite leader (TPL) sequence which makes them preferred mRNA's for translation.

In a current system, recombinant adenovirus is generated from homologous recombination between shuttle vector and pro virus vector. Due to the possible recombination between two proviral vectors, wild-type adenovirus may be generated from this process. Therefore, it is critical to isolate a single clone of vims from an individual plaque and examine its genomic structure.

Generation and propagation of the current adenovirus vectors, which are replication deficient, depend on a unique helper cell line, designated 293, which was transformed from human embryonic kidney cells by Ad5 DNA fragments and constitutively expresses El proteins (Graham et al , 1977). Since the E3 region is dispensable from the adenovirus genome (Jones and Shenk, 1978), the current adenovirus vectors, with the help of 293 cells, carry foreign DNA in either the El. the D3 or both regions (Graham and Prevec, 1991). In nature, adenovirus can package approximately 105% of the wild-type genome (Ghosh-Choudhury et al, 1987), providing capacity for about 2 extra kB of DNA. Combined with the approximately 5.5 kB of DNA that is replaceable in the El and E3 regions, the maximum capacity of the current adenovirus vector is under 7.5 kB, or about 15% of the total length of the vector. More than 80% of the adenovirus viral genome remains in the vector backbone and is the source of vector-borne cytotoxicity. Also, the replication deficiency of the El -deleted virus is incomplete. For example, leakage of viral gene expression has been observed with the currently available vectors at high multiplicities of infection (MOI) (Mulligan, 1993).

Helper cell lines may be derived from human cells such as human embryonic kidney cells, muscle cells, hematopoietic cells or other human embryonic mesenchymal or epithelial cells. Alternatively, the helper cells may be derived from the cells of other mammalian species that are permissive for human adenovirus. Such cells include, e.g., Vero cells or other monkey embryonic mesenchymal or epithelial cells. As stated above, the preferred helper cell line is 293.

Recently, Racher et al. (1995) disclosed improved methods for culturing 293 cells and propagating adenovirus. In one format, natural cell aggregates are grown by inoculating individual cells into 1 liter siliconized spinner flasks (Techne, Cambridge, UK) containing 100-200 ml of medium. Following stirring at 40 φm, the cell viability is estimated with trypan blue. In another format, Fibra-Cel microcarriers (Bibby Sterlin, Stone, UK) (5 g/1) is employed as follows. A cell inoculum, resuspended in 5 ml of medium, is added to the carrier (50 ml) in a 250 ml Erlenmeyer flask and left stationary, with occasional agitation, for 1 to 4 h. The medium is then replaced with 50 ml of fresh medium and shaking initiated. For virus production, cells are allowed to grow to about 80% confluence, after which time the medium is replaced (to 25% of the final volume) and adenovirus added at an MOI of 0.05. Cultures are left stationary overnight, following which the volume is increased to 100% and shaking commenced for another 72 h.

Other than the requirement that the adenovirus vector be replication defective, or at least conditionally defective, the nature of the adenovirus vector is not believed to be crucial to the successful practice of the invention. The adenovirus may be of any of the 42 different known serotypes or subgroups A-F. Adenovirus type 5 of subgroup C is the preferred starting material in order to obtain the conditional replication-defective adenovirus vector for use in the present invention. This is because Adenovirus type 5 is a human adenovirus about which a great deal of biochemical and genetic information is known, and it has historically been used for most constructions employing adenovirus as a vector.

As stated above, the typical vector according to the present invention is replication defective and will not have an adenovims El region. Thus, it will be most convenient to introduce the polynucleotide encoding the gene of interest at the position from which the El -coding sequences have been removed. However, the position of insertion of the construct within the adenovirus sequences is not critical to the invention. The polynucleotide encoding the gene of interest may also be inserted in lieu of the deleted E3 region in E3 replacement vectors as described by Karlsson et al. (1986) or in the E4 region where a helper eel line or helper virus complements the E4 defect.

Adenovirus is easy to grow and manipulate and exhibits broad host range in vitro and in vivo. This group of viruses can be obtained in high titers, e.g., 10⁹-10^π plaque-forming units per ml, and they are highly infective. The life cycle of adenovims does not require integration into the host cell genome. The foreign genes delivered by adenovirus vectors are episomal and, therefore, have low genotoxicity to host cells. No side effects have been reported in studies of vaccination with wild-type adenovims (Couch et al , 1963; Top et al, 1971), demonstrating their safety and therapeutic potential as in vivo gene transfer vectors.

Adenovirus vectors have been used in eukaryotic gene expression (Levrero et al, 1991 ; Gomez-Foix et al , 1992) and vaccine development (Grunhaus and Horwitz, 1992; Graham and Prevec. 1992). Recently, animal studies suggested that recombinant adenovims could be used for gene therapy (Stratford-Perricaudet and Perricaudet, 1991 ; Stratford-Perricaudet et al, 1990; Rich et al, 1993). Studies in administering recombinant adenovirus to different tissues include trachea instillation (Rosenfeld et al, 1991 ; Rosenfeld et al , 1992), muscle injection (Ragot et al, 1993). peripheral intravenous injections (Herz and Gerard, 1993) and stereotactic inoculation into the brain (Le Gal La Salle et al, 1993).

4.12.3.2 RETROVIRUSES

The retroviruses are a group of single-stranded RNA viruses characterized by an ability to convert their RNA to double-stranded DNA in infected cells by a process of reverse-transcription (Coffin, 1990). The resulting DNA then stably integrates into cellular chromosomes as a provirus and directs synthesis of viral proteins. The integration results in the retention of the viral gene sequences in the recipient cell and its descendants. The retroviral genome contains three genes, gag, pol, and env that code for capsid proteins, polymerase enzyme, and envelope components, respectively. A sequence found upstream from the gag gene contains a signal for packaging of the genome into virions. Two long terminal repeat (LTR) sequences are present at the 5' and 3' ends of the viral genome. These contain strong promoter and enhancer sequences and are also required for integration in the host cell genome (Coffin, 1990).

In order to constmct a retroviral vector, a nucleic acid encoding a gene of interest is inserted into the viral genome in the place of certain viral sequences to produce a vims that is replication-defective. In order to produce virions, a packaging cell line containing the gag, pol, and env genes but without the LTR and packaging components is constructed (Mann et al , 1983). When a recombinant plasmid containing a cDNA, together with the retroviral LTR and packaging sequences is introduced into this cell line (by calcium phosphate precipitation for example), the packaging sequence allows the RNA transcript of the recombinant plasmid to be packaged into viral particles, which are then secreted into the culture media (Nicolas and Rubenstein, 1988; Temin, 1986; Mann et al, 1983). The media containing the recombinant retroviruses is then collected, optionally concentrated, and used for gene transfer. Retroviral vectors are able to infect a broad variety of cell types. However, integration and stable expression require the division of host cells (Paskind et al, 1975). A novel approach designed to allow specific targeting of retrovirus vectors was recently developed based on the chemical modification of a retrovirus by the chemical addition of lactose residues to the viral envelope. This modification could permit the specific infection of hepatocytes via sialoglycoprotein receptors.

A different approach to targeting of recombinant retroviruses was designed in which biotinylated antibodies against a retroviral envelope protein and against a specific cell receptor were used. The antibodies were coupled via the biotin components by using streptavidin (Roux et al . 1989). Using antibodies against major histocompatibility complex class I and class II antigens, they demonstrated the infection of a variety of human cells that bore those surface antigens with an ecotropic virus in vitro (Roux et al, 1989).

There are certain limitations to the use of retrovirus vectors in all aspects of the present invention. For example, retrovirus vectors usually integrate into random sites in the cell genome. This can lead to insertional mutagenesis through the interruption of host genes or through the insertion of viral regulatory sequences that can interfere with the function of flanking genes (Varmus et al, 1981). Another concern with the use of defective retrovirus vectors is the potential appearance of wild-type replication- competent virus in the packaging cells. This can result from recombination events in which the intact_ sequence from the recombinant virus inserts upstream from the gag, pol, env sequence integrated in the host cell genome. However, new packaging cell lines are now available that should greatly decrease the likelihood of recombination (Markowitz et al, 1988; Hersdorffer et al, 1990).

4.12.3.3 OTHER VIRAL VECTORS AS EXPRESSION CONSTRUCTS

Other viral vectors may be employed as expression constructs in the present invention. Vectors derived from viruses such as vaccinia virus (Ridgeway, 1988; Baichwal and Sugden, 1986; Coupar et al, 1988) adeno-associated virus (AAV) (Ridgeway, 1988; Baichwal and Sugden, 1986; Hermonat and Muzycska, 1984) and heφesviruses may be employed. They offer several attractive features for various mammalian cells (Friedmann, 1989; Ridgeway, 1988; Baichwal and Sugden, 1986; Coupar et al , 1988; Horwich et al, 1990).

With the recent recognition of defective hepatitis B viruses, new insight was gained into the structure-function relationship of different viral sequences. In vitro studies showed that the virus could retain the ability for helper-dependent packaging and reverse transcription despite the deletion of up to 80% of its genome (Horwich et al.. 1990). This suggested that large portions of the genome could be replaced with foreign genetic material. The hepatotropism and persistence (integration) were particularly attractive properties for liver-directed gene transfer. Chang et al. recently introduced the chloramphenicol acetyltransferase (CAT) gene into duck hepatitis B virus genome in the place of the polymerase, surface, and pre-surface coding sequences. It was cotransfected with wild-type virus into an avian hepatoma cell line. Culture media containing high titers of the recombinant virus were used to infect primary duckling hepatocytes. Stable CAT gene expression was detected for at least 24 days after transfection (Chang et al, 1991 ). 4.12.3.4 NON-VIRAL VECTORS °l 7

In order to effect expression of sense or antisense gene constmcts, the expression construct must be delivered into a cell. This delivery may be accomplished in vitro, as in laboratory procedures for transforming cells lines, or in vivo or ex vivo, as in the treatment of certain disease states. As described above, the preferred mechanism for delivery is via viral infection where the expression construct is encapsidated in an infectious viral particle.

Several non-viral methods for the transfer of expression constructs into cultured mammalian cells also are contemplated by the present invention. These include calcium phosphate precipitation (Graham and Van Der Eb, 1973; Chen and Okayama, 1987; Rippe et al , 1990) DEAE-dextran (Gopal, 1985), electroporation (Tur-Kaspa et al, 1986; Potter et al , 1984), direct microinjection (Harland and Weintraub, 1985), DNA-loaded liposomes (Nicolau and Sene, 1982; Fraley et al , 1979) and lipofectamine-DNA complexes, cell sonication (Fechheimer et al, 1987), gene bombardment using high velocity microprojectiles (Yang et al, 1990), and receptor- mediated transfection (Wu and Wu, 1987; Wu and Wu, 1988). Some of these techniques may be successfully adapted for in vivo or ex vivo use.

Once the expression construct has been delivered into the cell the nucleic acid encoding the gene of interest may be positioned and expressed at different sites. In certain embodiments, the nucleic acid encoding the gene may be stably integrated into the genome of the cell. This integration may be in the cognate location and orientation via homologous recombination (gene replacement) or it may be integrated in a random, non-specific location (gene augmentation). In yet further embodiments, the nucleic acid may be stably maintained in the cell as a separate, episomal segment of DNA. Such nucleic acid segments or "episomes" encode sequences sufficient to permit maintenance and replication independent of or in synchronization with the host cell cycle. How the expression construct is delivered to a cell and where in the cell the nucleic acid remains is dependent on the type of expression construct employed.

In one embodiment of the invention, the expression construct may simply consist of naked recombinant DNA or plasmids. Transfer of the construct may be performed by any of the methods mentioned above which physically or chemically permeabilize the cell membrane. This is particularly applicable for transfer in vitro but it may be applied to in vivo use as well. Dubensky et al. (1984) successfully injected polyomavirus DNA in the form of calcium phosphate precipitates into liver and spleen of adult and newborn mice demonstrating active viral replication and acute infection. Benvenisty and Neshif (1986) also demonstrated that direct intraperitoneal injection of calcium phosphate-precipitated plasmids results in expression of the transfected genes. It is envisioned that DNA encoding a gene of interest may also be transferred in a similar manner in vivo and express the gene product.

Another embodiment of the invention for transferring a naked DNA expression construct into cells may involve particle bombardment. This method depends on the ability to accelerate DNA-coated microprojectiles to a high velocity allowing them to pierce cell membranes and enter cells without killing them (Klein et al , 1987). Several devices for accelerating small particles have been developed. One such device relies on a high voltage discharge to generate an electrical current, which in turn provides the motive force (Yang et al, 1990). The microprojectiles used have consisted of biologically inert substances such as tungsten or gold beads.

Selected organs including the liver, skin, and muscle tissue of rats and mice have been bombarded in vivo (Yang et al, 1990; Zelenin et al, 1991). This may require surgical exposure of the tissue or cells, to eliminate any intervening tissue between the gun and the target organ, i.e. , ex vivo treatment. Again, DNA encoding a particular gene may be delivered via this method and still be incorporated by the present invention.

In a further embodiment of the invention, the expression construct may be entrapped in a liposome. Liposomes are vesicular structures characterized by a phospholipid bilayer membrane and an inner aqueous medium. Multilamellar liposomes have multiple lipid layers separated by aqueous medium. They form spontaneously when phospholipids are suspended in an excess of aqueous solution. The lipid components undergo self-rearrangement before the formation of closed structures and entrap water and dissolved solutes between the lipid bilayers (Ghosh and Bachhawat. 1991 ). Also contemplated are lipofectamine-DNA complexes. Liposome-mediated nucleic acid delivery and expression of foreign DNA in vitro has been very successful. Wong et al. (1980) demonstrated the feasibility of liposome-mediated delivery and expression of foreign DNA in cultured chick embryo, HeLa and hepatoma cells. Nicolau et al. (1987) accomplished successful liposome- mediated gene transfer in rats after intravenous injection.

In certain embodiments of the invention, the liposome may be complexed with a hemagglutinating virus (HVJ). This has been shown to facilitate fusion with the cell membrane and promote cell entry of liposome-encapsulated DNA (Kaneda et al, 1989). In other embodiments, the liposome may be complexed or employed in conjunction with nuclear non-histone chromosomal proteins (HMG-1) (Kato et al, 1991). In yet further embodiments, the liposome may be complexed or employed in conjunction with both HVJ and HMG-1. In that such expression constmcts have been successfully employed in transfer and expression of nucleic acid in vitro and in vivo, then they are applicable for the present invention. Where a bacterial promoter is employed in the DNA constmct, it also will be desirable to include within the liposome an appropriate bacterial polymerase.

Other expression constructs which can be employed to deliver a nucleic acid encoding a particular gene into cells are receptor-mediated delivery vehicles. These take advantage of the selective uptake of macromolecules by receptor-mediated endocytosis in almost all eukaryotic cells. Because of the cell type-specific distribution of various receptors, the delivery can be highly specific (Wu and Wu, 1993).

Receptor-mediated gene targeting vehicles generally consist of two components: a cell receptor-specific ligand and a DNA-binding agent. Several ligands have been used for receptor-mediated gene transfer. The most extensively characterized ligands are asialoorosomucoid (ASOR) (Wu and Wu, 1987) and transferrin (Wagner et al. 1990). Recently, a synthetic neoglycoprotein, which recognizes the same receptor as ASOR, has been used as a gene delivery vehicle (Ferkol et al, 1993; Perales et al, 1994) and epidermal growth factor (EGF) has also been used to deliver genes to squamous carcinoma cells (Myers, EPO 0273085).

In other embodiments, the delivery vehicle may comprise a ligand and a liposome. For example, Nicolau et al. (1987) employed lactosyl-ceramide, a galactose- terminal asialganglioside. incoφorated into liposomes and observed an increase in the uptake of the insulin gene by hepatocytes. Thus, it is feasible that a nucleic acid encoding a particular gene also may be specifically delivered into a cell type such as lung, epithelial or tumor cells, by any number of receptor-ligand systems with or too without liposomes. For example, epidermal growth factor (EGF) may be used as the receptor for mediated delivery of a nucleic acid encoding a gene in many tumor cells that exhibit upregulation of EGF receptor. Mannose can be used to target the mannose receptor on liver cells. Also, antibodies to CD5 (CLL), CD22 (lymphoma), CD25 (T- cell leukemia) and MAA (melanoma) can similarly be used as targeting moieties.

In certain embodiments, gene transfer may more easily be performed under ex vivo conditions. Ex vivo gene therapy refers to the isolation of cells from an animal, the delivery of a nucleic acid into the cells in vitro, and then the return of the modified cells back into an animal. This may involve the surgical removal of tissue/organs from an animal or the primary culture of cells and tissues. Anderson et al, U.S. Patent

5,399,346, and incorporated herein in its entirety, disclose ex vivo therapeutic methods.

4.12.3.5 PHARMACEUTICAL COMPOSITIONS

Where clinical applications are contemplated, it will be necessary to prepare pharmaceutical compositions - either gene delivery vectors or engineered cells - in a form appropriate for the intended application. Generally, this will entail preparing compositions that are essentially free of pyrogens, as well as other impurities that could be harmful to humans or animals.

One will generally desire to employ appropriate salts and buffers to render delivery vectors stable and allow for uptake by target cells. Buffers also will be employed when recombinant cells are introduced into a patient. Aqueous compositions of the present invention comprise an effective amount of the vector to cells, dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium. Such compositions also are referred to as inocula. The phrase "pharmaceutically or pharmacologically acceptable" refer to molecular entities and compositions that do not produce adverse, allergic, or other untoward reactions when administered to an animal or a human. As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like. The use of such media and agents for pharmaceutically active substances is well know in the art. Except insofar as any- conventional media or agent is incompatible with the vectors or cells of the present to t invention, its use in therapeutic compositions is contemplated. Supplementary active ingredients also can be incoφorated into the compositions.

Solutions of the active ingredients as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with surfactant, such as hydroxypropylcellulose. Dispersions also can be prepared in glycerol, liquid polyethylene glycols, mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent growth of microorganisms.

The expression vectors and delivery vehicles of the present invention may include classic pharmaceutical preparations. Administration of these compositions according to the present invention will be via any common route so long as the target tissue is available via that route. This includes oral, nasal, buccal, rectal, vaginal or topical. Alternatively, administration may be by orthotopic, intradermal, subcutaneous, intramuscular, intraperitoneal or intravenous injection. Such compositions would normally be administered as pharmaceutically acceptable compositions, described supra.

The vectors and cells of the present invention are advantageously administered in the form of injectable compositions either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid prior to injection also may be prepared. These preparations also may be emulsified. A typical composition for such purposes comprises a 50 mg or up to about 100 mg of human serum albumin per milliliter of phosphate-buffered saline. Other pharmaceutically acceptable carriers include aqueous solutions, non-toxic excipients, including salts, preservatives, buffers and the like. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oil and injectable organic esters, such as ethyloleate. Aqueous carriers include water, alcoholic/aqueous solutions, saline solutions, parenteral vehicles such as sodium chloride. Ringer's dextrose, etc. Intravenous vehicles include fluid and nutrient replenishers. Preservatives include antimicrobial agents, anti-oxidants, chelating agents and inert gases. The pH and exact concentration of the various components in the pharmaceutical are adjusted according to well-known parameters.

Additional formulations are suitable for oral administration. Oral formulations include such typical excipients as. for example, pharmaceutical grades of mannitol, tθ 2. lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate and the like. The compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders. When the route is topical, the form may be a cream, ointment, salve or spray. An effective amount of the therapeutic agent is determined based on the intended goal. The term "unit dose" refers to a physically discrete unit suitable for use in a subject, each unit containing a predetermined quantity of the therapeutic composition calculated to produce the desired response in association with its administration, i.e., the appropriate route and treatment regimen. The quantity to be administered, both according to number of treatments and unit dose, depends on the subject to be treated, the state of the subject, and the protection desired. Precise amounts of the therapeutic composition also depend on the judgment of the practitioner and are peculiar to each individual.

4.13 BIOLOGICAL FUNCTIONAL EQUIVALENTS

Modification and changes may be made in the structure of the peptides of the present invention and DNA segments which encode them and still obtain a functional molecule that encodes a protein or peptide with desirable characteristics. The following is a discussion based upon changing the amino acids of a protein to create an equivalent, or even an improved, second-generation molecule. In particular embodiments of the invention, mutated crystal proteins are contemplated to be useful for increasing the insecticidal activity of the protein, and consequently increasing the insecticidal activity and/or expression of the recombinant transgene in a plant cell. The amino acid changes may be achieved by changing the codons of the DNA sequence, according to the codons given in TABLE 4.

TABLE 4

Amino Acids Codons

Alanine Ala A GCA GCC GCG GCU

Cysteine Cys C UGC UGU

Aspartic acid Asp D GAC GAU

Glutamic acid Glu E GAA GAG

Phenylalanine Phe F UUC UUU

Glycine Gly G GGA GGC GGG GGU tO-3

Histidine His H CAC CAU

Isoleucine He I AUA AUC AUU ysine Lys K AAA AAG eucine Leu L UUA UUG CUA cue CUG CUU

Methionine Met M AUG

Asparagine Asn N AAC AAU

Proline Pro P CCA CCC CCG ecu

Glutamine Gin Q CAA CAG

Arginine Arg R AGA AGG CGA CGC CGG CGU

Serine Ser S AGC AGU UCA UCC UCG UCU

Threonine Thr T ACA ACC ACG ACU

Valine Val V GUA GUC GUG GUU

Tryptophan Trp UGG

Tyrosine Tyr Y UAC UAU

For example, certain amino acids may be substituted for other amino acids in a protein structure without appreciable loss of interactive binding capacity with structures such as, for example, antigen-binding regions of antibodies or binding sites on substrate molecules. Since it is the interactive capacity and nature of a protein that defines that protein's biological functional activity, certain amino acid sequence substitutions can be made in a protein sequence, and, of course, its underlying DNA coding sequence, and nevertheless obtain a protein with like properties. It is thus contemplated by the inventors that various changes may be made in the peptide sequences of the disclosed compositions, or corresponding DNA sequences which encode said peptides without appreciable loss of their biological utility or activity.

In making such changes, the hydropathic index of amino acids may be considered. The importance of the hydropathic amino acid index in conferring interactive biologic function on a protein is generally understood in the art (Kyte and Doolittle, 1982, incorporate herein by reference). It is accepted that the relative hydropathic character of the amino acid contributes to the secondary structure of the resultant protein, which in turn defines the interaction of the protein with other molecules, for example, enzymes, substrates, receptors, DNA. antibodies, antigens, and the like. Each amino acid has been assigned a hydropathic index on the basis of their hydrophobicity and charge characteristics (Kyte and Doolittle, 1982), these are: tό f isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cystine

(+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-

0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamate (-

3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (- 4.5).

It is known in the art that certain amino acids may be substituted by other amino acids having a similar hydropathic index or score and still result in a protein with similar biological activity, i.e., still obtain a biological functionally equivalent protein.

In making such changes, the substitution of amino acids whose hydropathic indices are within ±2 is preferred, those which are within ±1 are particularly preferred, and those within ±0.5 are even more particularly preferred.

It is also understood in the art that the substitution of like amino acids can be made effectively on the basis of hydrophilicity. U. S. Patent No. 4,554,101 , incoφorated herein by reference, states that the greatest local average hydrophilicity of a protein, as governed by the hydrophilicity of its adjacent amino acids, correlates with a biological property of the protein.

As detailed in U. S. Patent No. 4,554,101, the following hydrophilicity values have been assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartate

(+3.0 ± 1); glutamate (+3.0 ± 1); serine (+0.3); asparagine (+0.2); glutamine (+0.2) glycine (0); threonine (-0.4); proline (-0.5 ± 1); alanine (-0.5): histidine (-0.5) cysteine (-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8) tyrosine (-2.3); phenylalanine (-2.5); tryptophan (-3.4).

It is understood that an amino acid can be substituted for another having a similar hydrophilicity value and still obtain a biologically equivalent, and in particular, an immunologically equivalent protein. In such changes, the substitution of amino acids whose hydrophilicity values are within ±2 is preferred, those which are within ±1 are particularly preferred, and those within ±0.5 are even more particularly preferred.

As outlined above, amino acid substitutions are generally therefore based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity. charge, size, and the like. Exemplary substitutions which take various of the foregoing characteristics into consideration are well known to /Ob^" those of skill in the art and include: arginine and lysine; glutamate and aspartate; serine and threonine; glutamine and asparagine; and valine, leucine and isoleucine.

5.0 EXAMPLES The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

5.1 EXAMPLE 1 -- ISOLATION AND CHARACTERIZATION OF MsENOD40-l AND MsENOD40-2 PROMOTERS

The present example relates to the isolation and characterization of the promoter regions from two independent genomic clones (la and 6c of MsENOD40) from an alfalfa genomic library. The two discrete promoters were individually fused to a reporter gene uidA (gus), and introduced into alfalfa (Medicago sativa cv. Regen) via Agrobacterium tumefaciens-mediated transformation. The inventors followed the spatial and temporal expression patterns of the two ENOD40 genes under both non- symbiotic and symbiotic conditions. The expression patterns of both promoter constructs were also examined in NPA- or Rhizobium meliloti exopolysaccharide mutant-induced nodules, both of which are bacteria-free. To define the c/ acting region of the MsENOD40 promoter required for its induction by Nod factor or cytokinin. a series of nested truncated promoters of the MsENOD40-l promoter was constructed and introduced into alfalfa plants. The activities of these truncated promoters were studied in the transgenic plants. f ό o

5.1.1 MATERIALS AND METHODS

5.1.1.1 ISOLATION OF MSENOD40 GENOMIC CLONES AND SEQUENCING ANALYSIS

One million plaques from an alfalfa (M. sativa cv. Chief) genomic library (Clontech. Palo Alto, CA.) were screened with a ³²P-radiolabeled MsENOD40 cDNA (Asad et al, 1994). Five individual plaques, la, 2b, 4B, 6c, 9B', which hybridized to the probe were isolated and the phage DNAs were purified. Two clones, la and 6c, were selected and the putative promoter regions located within the Hindlll fragments were subcloned into pBluescript II KS vector (Stratagene, La Jolla, CA.) to generate lal (~2.8 kb) and 6cl (-2.6 kb), respectively. Fragment lal was then designated as the MsENOD40-l promoter, and 6c 1 as the MsENOD40-2 promoter. A set of nested deletions from either the 5' or 3' end of both clones was generated using the method described by Henikoff (1984).

Both DNA strands were sequenced using the double-stranded dideoxy chain termination method according to the manufacturer's protocol (Sequenase® v 2, USB, Cleveland, OFF). Sequence analyses were performed on a VAX/VMS computer using the UWGCG (University of Wisconsin) software package.

5.1.1.2 SOUTHERN ANALYSIS

Phage DNAs were isolated according to the method described by Kernodle et al. (1993). DNA was digested with different enzymes and blotted for Southern analysis (Asad et α/., 1994).

5.1.1.3 CONSTRUCTION OF 5' TRUNCATED MsENOD40-l PROMOTERS

AND OTHER PLASMIDS The Hindlll-Bc L fragments of lal and 6cl were individually ligated into the

Hindlll-BamHl site of the binary vector pBI101.3 (Clontech) to create pBIlal-1 and pBI6cl-l . respectively. The Bell site is about 35 bp downstream of the putative TATA box of MsENOD40 gene (Asad et al. 1994). A Spel-BcR (~ 2.3 kb) and a Clal-Bc l (~ 1.6 kb) fragment from lal were cloned into pBI 101.3 to produce pBIlal -2 and pBIlal - 4, respectively. The EcoRY-Bcll (231 bp) fragment was used to create pBIlal-5, which is considered to be the minimal promoter in this paper. The Spel-Clal fragment of lal was fused at the 5' end of pBI lal-5 to produce pBIlal -7, a composite promoter or? for MsENOD40-l, consisting of the minimal promoter and the 616 bp Spel-Clal fragment (see FIG. 2). All pBI plasmids were electroporated into A. tumefaciens strain LBA4404 and then transformed into alfalfa (Medicago sativa cv. Regen) using the alfalfa transformation and regeneration procedure described in Hirsch et al (1995).

5.1.1.4 TRANSGENIC PLANT PROPAGATION AND TREATMENTS

All the transgenic plants used for the studies were primary transformants which were propagated vegetatively. Healthy stems were cut and rooted in a vermiculite/perlite mixture soaked with 1/4 strength complete Hoagland's plant medium. After 12 to 14 d, the rooted plants were transferred into sterile 50-mL Falcon tubes containing nitrogen-free Jensen's medium and placed in Conviron growth chamber. The tubes were wrapped with aluminum foil to exclude light from the roots. After 6 to 7 d, the plants were transferred to fresh Jensen's medium without any additives (control) or inoculated with R. meliloti strain 1021 , or treated with 10^" M PNF (NodRmIV(C16:l, S)), or 10^"6 M BAP. These concentrations were determined previously to give an optimal response (see Hirsch et al, 1997). Roots were harvested 4 d post-treatment for Gus histochemical staining or for total protein extraction. To obtain nodules at different stages, the rooted plants were either spot-inoculated (see Asad et al. 1994) or flood-inoculated with R. meliloti strain 1021 (wild-type), strain 7094 (exoB::Tn5) or treated with 10° M NPA. Nodules or other tissues (e.g., roots) were harvested approximately 2 to 5 weeks post-inoculation.

5.1.1.5 Gus HISTOCHEMICAL AND COLORIMETRIC ASSAY

The inventors used the method developed by Jefferson (1987) for assaying Gus activity histochemically. Briefly, plant tissues were placed in the fixing buffer (Jefferson, 1987) for 45 min., rinsed three times with 50 mM phosphate buffer and incubated with 1 mM X-Gluc substrate at 37°C from 1 h to overnight. After staining, the tissues were rinsed in phosphate buffer, and kept in 50%> ethanol. Some tissues were further cleared using a 20% bleach solution under vacuum for 15 to 30 min. The blue-stained tissues were either used directly for observation after washing in distilled water or embedded in paraffin via the TBA series (McKhann and Hirsch, 1993). If fό paraffin embedded, the tissues were sectioned at 15 to 25 μm, and mounted on slides for observation and photography using a Zeiss Axiophot microscope.

To assay the enzymatic activity of Gus protein, total protein was extracted from roots 4 d following the various treatments (see above). Twenty ng of protein were used for the Gus assays in microtiter plates using N-nitrophenyl glucuronide as a substrate.

The assay was adapted from the method described by Breyne et al. (1993). The plates were read in a Titertek plate reader at wavelength 405 nm after a 6 h-incubation at

37°C. Ten independent transgenic plants generated from each construct were tested, and each plant was assayed three times with three individual sets of plant cuttings per treatment. The colorimetric Gus assay was repeated, and the readings were averaged and plotted using Microsoft Excel and Cricket Graph programs.

5.1.2 RESULTS

5.1.2.1 THERE ARE AT LEAST Two DIFFERENT MsENOD40 GENES IN ALFALFA After three rounds of screening one million plaques, five independent phage clones were isolated from an alfalfa genomic library using the full-length MsENOD40 cDNA as a probe. Two phage clones were found to be identical to each other; this was confirmed later by sequence analysis. The putative promoter region, which was located within the Hindlll fragment in each phage clone, varied in size. The inventors knew that the Hindlll fragment contained the potential promoter region based on the inventors' previous finding that there is a Hindlll site at the very 5' end of the MsENOD40 cDNA sequence (Asad et al, 1994). This was verified by sequencing analysis (see later section). Genomic Southern analysis had suggested that one or two MsENOD40 genes were present in the alfalfa genome (Asad et al, 1994). Preliminary analysis showed that the putative promoters in phages la and 6c were the largest fragments, and differed in their restriction patterns. On this basis, phages la and 6c were selected for further characterization.

After sequencing analysis, the inventors confirmed that the promoter of MsENOD40 gene was located in either a 2.8 kb ( lal) or a 2.6 kb (6c 1 ) Hindlll fragment for clone l a or 6c, respectively. These H dIII fragments also included the first 75 bp sequence found at the 5' end of the MsENOD40 cDNA, including the TATA box. Sequence comparison revealed that the proximal sequences of these two to y promoters, which span ca. 1.4 kb long, were identical. However, the upstream sequences shared only 40% similarity (FIG. 1). The different restriction sites in these regions also supported this conclusion (FIG. 1 , FIG. 2). This finding suggested that these two clones (la and 6c) were likely to represent promoters of two different MsENOD40 genes in alfalfa (Asad et al, 1994). Thus, the designation MsENOD40-l was assigned to clone lal , and MsENOD40-2 was used for clone 6cl .

5.1.2.2 THE NON-SYMBIOTIC EXPRESSION OF THE MsENOD40 PROMOTERS IN TRANSGENIC ALFALFA To investigate the expression patterns of the two different MsENOD40 genes, each promoter was fused to the reporter gene, uidA (gus), to produce plasmid pBIlal-1 and pBI6cl-l , respectively (FIG. 2). The putative transcriptional start site in the promoter was determined by primer-extension studies; thus, these constmcts were transcriptional fusions. Both constructs were subsequently introduced into alfalfa (Medicago sativa cv. Regen) via Agrobacterium tumefaciens-mediated transformation to generate transgenic plants (Hirsch et αl, 1995). More than 30 individual transgenic plants generated for each construct were examined by the histochemical assay, and approximately 70%) of them expressed the gus gene in nodules at locations determined previously by in situ hybridization analysis (Asad et αl, 1994). On this basis, it seemed unlikely that sequences other than those located within the MsENOD40 promoter were affecting the spatial patterns of gene expression. Twenty of the 30 independent transgenic lines from each construct were selected for more detailed studies. Using Southern analysis, the inventors found that most lines contained multiple inserts of the T-DNA. However, the level of gus gene expression did not correlate with the copy number of the inserts.

By performing histochemical staining for Gus protein (Jefferson, 1987) in various plant organs, the expression patterns of the two MsENOD40 genes were shown to differ under non-symbiotic conditions. During the process of somatic embryogenesis, the inventors could not detect any gus expression in callus cells or somatic embryos for either construct. In mature transgenic plants, the MsENOD40-l promoter was usually not active in uninoculated roots. For example, 16 of 20 plants showed no blue staining in roots whereas the remaining 4 plants expressed the gus gene 1 16 in the vascular tissues of the root and the stem. Of the 20 plants, 9 of them (45%) showed the blue staining indicative of Gus protein in emerging lateral root primordia. When the lateral root emerged from the parent root, however, the blue color was no longer apparent. In contrast, 17 of 21 (81%) of the transgenic plants containing the MsENOD40-

2 promoter-gus constmct expressed gus in the root stele and in the stem procambium/phloem region even in the absence of Rhizobium (FIG. 3A and FIG. 3B). Furthermore, gus expression was detected at the root tip (sometimes in the root cap) in 40%) of the plants examined (FIG. 3C). About 80% of the plants studied also expressed gus in developing lateral roots (FIG. 3D, FIG. 3E and FIG. 3F). The blue stain indicating Gus protein was seen in the central cells of the lateral root primordium (FIG. 3D). Upon lateral root elongation and emergence from the parent root, gus expression became restricted to the central vasculature at the proximal end of the lateral root, at the point where it was attached to the parent root. It remained high in the root tip, however (FIG. 3E and FIG. 3F).

Despite the differences between the two promoters, their combined expression patterns in transgenic alfalfa correlated with the inventors' previous findings of MsENOD40 transcript localization using in situ hybridization (Asad et al, 1994). Two new locations were detected, however: the root tip and the node where the leaf is attached. Only the MsENOD40-2 promoter was active in the root tip, and occasionally in the root cap of some transgenic plants (FIG. 3C). The MsENOD40-l promoter construct was occasionally expressed in the node but not in other parts of the stem.

5.1.2.3 THE EXPRESSION OF THE MsENOD40 PROMOTERS DURING WILD-TYPE NODULE DEVELOPMENT

The inventors then followed gus gene expression driven by the MsENOD40 promoter constructs in the plants inoculated with wild-type R. meliloti strain 1021. The two promoters were found to respond to Rhizobium inoculation to different degrees. For the transgenic plants bearing the MsENOD40-l promoter-gws fusion construct, less than 50% of them expressed gus in the stele upon inoculation (FIG. 4A, FIG. 4B, FIG 4C, FIG. 4D, and FIG. 4E), whereas nearly 90% of the transgenic alfalfas containing the MsENOD40-2 promoter responded in this way (FIG. 4F, FIG. 4G, FIG. 4H. and tn

FIG. 41). However, both constructs demonstrated similar expression patterns as the nodule developed. Before root cortical cell divisions were apparent, gus expression was detected in the root outer cortex as well as in the epidermal cells (small arrowheads in FIG. 4C). When the inner cortical cells were activated to divide, the blue color indicating Gus protein was found in the dividing inner cortical cells as well as in the pericycle (large arrowhead in FIG. 4A, FIG. 4C; and FIG. 4F). Blue color was subsequently detected in all cells of the nodule primordium (FIG. 4D, FIG. 4E; FIG. 4G, and FIG. 4H). Following initiation of a nodule meristem and the further development of the nodule, gus expression became localized to the nodule meristem (including the prefixing zone), to cells on the periphery of the central region, and to cells associated with the nodule vascular bundles (FIG. 41, FIG. 4J, FIG. 4K, and FIG. 4L). This pattern persisted in the mature nodule (FIG. 4M). For plants bearing the MsENOD40-2 promoter construct, gus expression was much stronger. The time required to detect the appearance of the blue color was 1 to 6 h depending on plant line compared to 6 to 24 h for the MsENOD40-l construct. In addition, blue staining indicating Gus protein was detected in the root cortex following R. meliloti inoculation for the MsENOD40-2 construct (FIG. 4F, FIG. 4G, FIG. 4H, and FIG. 41). The localization of the reporter gene driven by the MsENOD40 promoters in nodules formed on the transgenic plants is consistent with the in situ hybridization results found earlier (Asad et al, 1994).

For the transgenic plants containing the vector control pBI101.3, no gus expression was detected in any tissue or organ, even after incubation in the solution with the X-Gluc substrate for 2 to 3 d (more than 30 plants examined). Thus, gus expression directed by the MsENOD40 promoters in the transgenic alfalfas was specific.

5.1.2.4 THE EXPRESSION PATTERN OF THE MsENOD40 PROMOTERS IN BACTERIA- FREE NODULES

Earlier, the inventors found using northern blot analysis that MsENOD40 is expressed in ineffective nodules elicited by NPA and R. meliloti exo mutants (Asad et al, 1994). To examine whether both promoter constructs are expressed in these bacteria-free nodules, 16 individual plants which contained either the MsENOD40-l- or the MsENOD40-2-gus construct were tested. The NPA-induced nodule-like structures gave the largest difference with respect to the expression patterns of the two promoter constructs. No gus expression was detected in the NPA-induced pseudonodules that were formed on the plants carrying the MsENOD40-l promoter construct, unless these plants expressed the gus gene in the root stele constitutively (20% of the plants). In comparison, the majority (80%) of the plants containing the MsENOD40-2 promoter expressed gus in NPA-induced nodules. Following treatment with 10^"5 M NPA, gus expression was detected in the dividing cortical cells and pericycle (FIG. 5A). In an older NPA-induced pseudonodule, Gus activity were detected in the area around the central vascular bundle as well as in the peripheral area consisting of pericycle and cortical cell derivatives (FIG. 5B and FIG. 5C).

In contrast, when roots of the transgenic plants containing either of the promoter-g ' constructs were inoculated with R. meliloti strain 7094 (exoB::Tn5), the blue staining for gus protein was detected in the uninfected nodules that developed on the roots. Both promoters, including the composite MsENOD40-l promoter (pBIlal -7, see later section), expressed the gus gene in a pattern similar to that of wild-type nodules, i.e., in dividing cortical cells (FIG. 5D), in the nodule primordium (FIG. 5E), and in the peripheral region that is comparable to the meristem of the wild-type nodule (Yang et al, 1992) in mature, bacteria- free nodules (FIG. 5F). In addition, the inventors observed amyloplast deposition in roots (arrowheads in FIG. 5A and FIG. 5E) following treatment with NPA or inoculation with R. meliloti strain 7094.

5.1.2.5 THE EXPRESSION PATTERNS OF THE TWO DIFFERENT MsENOD40 PROMOTER CONSTRUCTS IN CYTOKININ- OR NOD FACTOR-TREATED ROOTS Earlier the inventors found using northern blot analysis that MsENOD40 was induced by BAP application (van Rhijn et al, 1997). Treatment with 2,4-D or other plant hormones did not elicit MsENOD40 expression (Hirsch et al. 1997; FIG. 3M). The expression patterns of the two MsENOD40 promoter constructs were observed after treating the transgenic plant roots with BAP or Nod factor (six independent plant lines for each construct were examined). For both constructs, Nod factor or cytokinin triggered similar responses. In roots without any treatment (Jensen's medium alone), Gus protein was not detected in roots of the MsENOD40-l transgenic plants (FIG. 3G) i t3 or was detected in the root stele and root tip of the MsENOD40-2 transgenic plants

(FIG. 3J). In roots treated with BAP or PNF for 4 d, blue staining indicating Gus protein was present in the root cortex and epidermal cells for both promoter constructs (FIG. 3H, FIG. 31, FIG. 3K, and FIG. 3L). In some cases, gus expression was also found in root hairs (arrowheads in FIG. 3H and FIG. 3K). Gus expression in the root cortex and epidermis was seen mainly in the root elongation zone, where the root expanded laterally following BAP or PNF treatment (FIG. 3H, FIG. 31, FIG. 3K, and FIG. 3L). This change in moφhology and induction of gus expression took place as early as 2 d post-treatment. In the root treated with either BAP or PNF, small nodule primordia resulting from inner cortical cell divisions developed; blue staining indicating Gus protein was present in all the cells of the nodule primordium (FIG. 3N, FIG. 30, FIG. 3P, and FIG. 3Q). Starch grain accumulation in BAP- or PNF-treated roots was also observed (arrows in FIG. 3Q)

5.1.2.6 A 616 BP REGION IN THE MsENOD40-l PROMOTER IS ESSENTIAL AND SUFFICIENT FOR MsENOD40-l EXPRESSION IN NODULES

To define the cis-ac mg region in the MsENOD40 promoter which is important for its activity, a series of 5' truncated promoters of MsENOD40-l was constmcted, fused to uidA (FIG. 2), and subsequently transformed into alfalfa. The MsENOD40-l promoter was chosen because its expression levels in roots and nodules correlated well with a previous study (Asad et αl. 1994) and also because it was expressed almost exclusively under symbiotic conditions in contrast to the MsENOD40-2 promoter construct. By performing the Gus histochemical assay on 30 independent lines, the promoter in pBIlal -4 (FIG. 2) was found to be completely inactive in any tissues or organs of the transgenic plants, including nodules. This promoter contained the region common to both promoters. If the promoter contained the upstream region up to Spel site (pBIlal -2; FIG. 2), the same gus expression patterns as the full-length MsENOD40-l promoter was detected in 30 independent lines. The minimal promoter construct of MsENOD40-l (231 bp. pBIlal -5 in FIG. 2) behaved exactly the same as the promoterless vector pBI101.3 (30 independent lines examined for each construct). These results suggested that the region between Spel and Clαl site was required for the MsENOD40-l promoter activity. A composite promoter was created by directly linking the Spel-Clal fragment (616 bp) to the minimal promoter in pBIlal -5 (pBI lal -7, see FIG. 2). This constmct was introduced into alfalfa plants to produce transgenic lines. In 40%> of these transgenic plants, gus expression was detected in the inner and outer root cortical cells at early stages of Rhizobium infection, but was absent or below detection levels in the root stele (including the pericycle) (see FIG. 5G and FIG. 5H). When the nodule primordium was formed, gus expression was found within the cells of the primordium (FIG. 51 and FIG. 5K). In the nodule, gus expression was detected at locations similar to those of the full-length MsENOD40-l promoter, e.g., in the nodule meristem and in cells associated with the nodule vascular bundle (FIG. 5J, FIG. 5L and FIG. 5M).

Compared to the full-length promoter, only about 40% (versus >70%) of the transgenic plants examined (1 1 out of 25 plants) demonstrated the expected expression patterns in nodules. Like the full-length promoter construct, the composite promoter construct was active in nodules induced by R. meliloti exo mutants, but not by NPA. In addition, the composite promoter in pBIlal -7 was exclusively expressed in nodules. It completely lost its activity under non-symbiotic conditions, even in young lateral root primordia.

5.1.2.7 REGION RESPONSIBLE FOR INDUCING MsENOD40-l EXPRESSION BY NOD FACTORS AND CYTOKININ IS OVERLAPPING

Having the truncated promoters of the MsENOD40-l, the inventors were able to study the -acting region required for BAP and/or PNF induction. Ten independent transgenic alfalfa plants from each transformation with each construct, including that of the vector control, were tested. Rooted plants generated from the stem cuttings were treated with wild-type R. meliloti strain 1021 , 10^"6 M BAP, or 10^"8 M PNF. Total protein was extracted from roots 4 d following each treatment, and Gus activity was assayed with a microtiter plate assay using /V-nitrophenyl glucuronide as a colorimetric substrate. Both promoters (MsENOD40-l and MsENOD40-2) were enhanced 2 to 3- fold on average in roots by exogenous BAP as well as by R. meliloti inoculation or addition of PNF (FIG. 6A, FIG. 6B, FIG. 6C, FIG. 6D, FIG. 6E, and FIG. 6F). Furthermore, the average basal level of the MsENOD40-2 promoter activity was measured at approximately 3 -fold higher than that of the MsENOD40-l promoter t/S

(compare the different scales in FIG. 6A and FIG. 6B). When the MsENOD40-l promoter was deleted from the 5' end to the Spel site (pBIlal -2 in FIG. 2), it acted like the full-length promoter (FIG. 6C). However, for the transgenic plants containing the construct pBI lal -4, gus expression could no longer be induced by BAP, by PNF, or by Rhizobium inoculation (FIG. 6D). The same result was obtained for the minimal promoter (pBIlal -5) and the vector control (pBI101.3) (FIG. 6E and FIG. 6F). These results suggest the 616 bp region in the MsENOD40-l promoter is involved in regulating expression induced by either Nod factors or cytokinin.

5.1.3 DISCUSSION

Here, the inventors have extended their investigations on ENOD40 gene expression in alfalfa to studying promoter-gw.v fusions in transgenic plants. Five independent genomic clones were isolated from an alfalfa genomic library using the MsENOD40 cDNA as a probe, and two of them were found to be identical. The promoter sequences from two distinct clones, la and 6c, were cloned for detailed characterizations. Sequence analyses revealed that the two promoters differed at their 5' distal end (40% similarity), but were identical at the proximal region (1431 bp long) (FIG. 1 ). Based on these results and the inventor's previous Southern analysis (Asad et al 1994), these two clones (la and 6c) are proposed to represent two distinct ENOD40 genes in the alfalfa genome. The inventors have designated them MsENOD40-l and MsENOD40-2. respectively.

More than one ENOD40 gene has also been isolated from other legumes, such as soybean and French bean (Kouchi and Hata, 1993; Papadopoulou et al, 1995). A soybean GmENOD40-2 promoter-gws fusion has been introduced into a heterologous plant, vetch, and found to be expressed only in the pericycle of the nodule vascular bundle and at the region of the root where the nodule was attached (Roussis et al, 1995). However, the spatial and temporal expression patterns of the second soybean ENOD40 gene in a transgenic plant have not yet been investigated. The inventors fused the promoters of the two MsENOD40 genes individually to gus, and subsequently transformed these constructs into alfalfa plants. By doing so, the inventors were able to investigate the expression patterns of these two genes in a homologous transgenic plant system with or without Rhizobium inoculation. tb

During the process of alfalfa transformation and regeneration, no gus expression was detected in the callus cells or somatic embryos using the gus histochemical assay. When using northern analysis, the inventors found that MsENOD40 was not expressed in alfalfa cell cultures, even after cytokinin treatment. Under non-symbiotic conditions, MsENOD40-l was usually not expressed in the uninoculated primary root of the transgenic alfalfa plants, but was activated when lateral roots or nodule primordia are initiated. In contrast, the expression of MsENOD40-2 is likely to be constitutive, because the promoter construct was expressed in the vascular tissues of the root and the stem even in the absence of Rhizobium (see FIG. 3). In addition, the expression of the MsENOD40-2 promoter construct was also detected at the root tip and throughout lateral root development (FIG. 3). The expression pattern of MsENOD40-2 in lateral roots is very similar to that found for PvENOD40 in bean (Papadopoulou et al, 1996), suggesting that PvENOD40 may be a MsENOD40-2 homolog. Using in situ hybridization analysis (Asad et al, 1994), the inventors detected the combined expression patterns of MsENOD40-l and MsENOD40-2. Transcripts were localized in the stem procambium, in the root stele, in the margins of the young leaf primordia, and in emerging lateral roots (see Table 5). The inventors found two new sites of expression by using the Gus histochemical assay. Both promoter constructs were expressed in the stem node at the point of petiole attachment in some transgenic plants, which has not been examined previously by in situ hybridizations. Moreover, MsENOD40-2 was expressed in the root tip, including the root cap in some transgenic plants. Comparing the inventors' previous findings (Asad et al, 1994) and the results from studying the transgenic plants, the expression of the two MsENOD40 genes in alfalfa appears to be differentially regulated under non-symbiotic conditions. Transcripts detected in the stem and in lateral roots by the in situ method are likely to be derived from the expression of MsENOD40-2. TABLE 5 SUMMARY OF THE EXPRESSION PATTERNS OF THE Two MsENOD40 GENES IN TRANSGENIC PLANTS

in situ results pBI6cl -l pBI lal-1 pBIlal-2 pBIlal-4 pBIlal-5 pBIlal-7 pBI101.3

In the root stele - +(80%) +(20%) +(20%)

(uninoculated)

In the stem + +(80%) +(20%) +(20%) . . . . procambium i

In the lateral root + +(90%) +(45%) +(45%) . . . . primordium

In the root tip +/- +(~50%) -

In the nodule ± + + + - - + - primodium

In the nodule + + + meristem and vascular bundles

in situ results pBI6cl-l pBIlal -1 pBIlal -2 pBIlal -4 pBIlal-5 pBIlal-7 pBI101.3

In NPA-induced + +(80%) +(10%) +(10%) - - - - nodules In Rhizobium exo - + + + + . . . mutant- induced nodules Plasmids represent the different promoter constructs shown in FIG. 2. The percentage is the number of plants that demonstrated the indicate Gus localization over the total number of the plants examined.

ι

When transgenic plants were inoculated with wild-type R. meliloti strain 1021, both MsENOD40-l and MsENOD40-2 constmcts were expressed in a similar fashion throughout all stages of nodule development. Both promoter constmcts were activated in the cortical cells/pericycle in the pre-infection and infection stages. They were subsequently expressed in the nodule primordium, and later in the nodule meristem and peripheral cells around the central tissues as well as in the nodule vascular bundles of the mature nodule (see FIG. 4). Although MsENOD40-2 expression appears to be stronger than MsENOD40-l (3-fold higher), the inventors cannot distinguish between the two promoters in terms of Gus localization as the nodule develops. Also, because Gus localization correlates with the inventors' previous findings obtained by in situ hybridization (Asad et al, 1994), the inventors could not determine whether only one or both genes are expressed during the alfalfa- Rhizobium interaction.

On the other hand, even though the two promoter constmcts behaved similarly during nodule development, they are regulated differently in the NPA-induced pseudonodules. Only the MsENOD40-2 promoter constmct was expressed in the nodule-like stmctures elicited by NPA treatment (FIG. 5A, FIG. 5B, and FIG. 5C). In contrast, both promoter constructs were expressed in nodules induced by R. meliloti exo mutants (FIG. 5D, FIG. 5E, and FIG. 5F). Part of the explanation for the differential expression may be in the fact that NPA-elicited nodules moφhologically and physiologically resemble lateral roots more closely than nodules, whereas Rhizobium exo mutant-induced nodules follow the wild-type nodule-developmental pathway (Yang et αl., 1992; Asad et αl., 1994). Thus the differential expression patterns of the two MsENOD40s may reflect two distinct pathways for these different types of ineffective nodules. MsENOD40-l is not usually active in the root stele or in the lateral root, and consequently it is not expressed in NPA-induced nodules. In contrast, the MsENOD40- 2 gene is active in the main root or lateral roots; thus it is expressed in NPA-elicited nodules (FIG. 5A, FIG. 5B, and FIG. 5C). Both genes show similar expression patterns during the formation of wild-type nodules, and consequently both genes are expressed in Rhizobium exo mutant-induced nodules (FIG. 5D. FIG. 5E, and FIG. 5F). These results suggest that these two MsENOD40 genes have different but complementary roles in the development of different organs. A

The inventors have found that MsENOD40 gene expression was enhanced in roots treated with either cytokinin or purified Nod factors (van Rhijn et al, 1997; Hirsch et al, 1997). Here the inventors confirm this observation by testing gus gene expression in roots of transgenic plants carrying either of the MsENOD40 promoters. The expression of MsENOD40-l as well as MsENOD40-2 was induced by BAP or Nod factor treatment, and the extent of induction is similar for both genes (2 to 3-fold), although the basal level of MsENOD40-2 expression is generally higher (FIG. 6). In addition, both genes were also expressed in a similar pattern in roots following treatment with cytokinin or Nod factor for 4 d (see FIG. 3G, FIG. 3H, FIG. 31, FIG. 3J, FIG. 3K, FIG. 3L, FIG. 3M, FIG. 3N, FIG. 30, FIG 3P, and FIG. 3Q).

The differential expression patterns of MsENOD40-l and MsENOD40-2 may reflect their different sensitivities to hormone changes or other signals. This is based on several observations. Under non-symbiotic conditions, 80% of plants (compared to 20%) of the MsENOD40-l transgenic plants) containing the MsENOD40-2-gus construct expressed gus in the root stele as well as in lateral roots. Following inoculation with R. meliloti strain 1021, MsENOD40-2 was expressed not only in nodules, but also in the root cortex; this latter location was not observed for the MsENOD40-l constmct. MsENOD40-2 expression was also detected in the root cortex after addition of either NPA or R. meliloti exo mutants. This difference between two genes may be dependent on the 5' upstream regions which are only 40% similar between the two promoters.

To define the c s-acting region involved in cytokinin or Nod factor induction, a series of 5' deletions of the MsENOD40-J promoter was constructed and introduced into plants to study their activities. By assaying Gus enzymatic activities in roots after various treatments, a 616 bp Spel-Clal region in the MsENOD40-l promoter was found to be absolutely required for its expression. This region plus the minimal promoter (the composite promoter) is sufficient to drive reporter gene expression at similar locations in the nodule as the full-length promoter (see FIG. 5). Furthermore, this region is also involved in the induction of MsENOD40-l gene by cytokinin or by Nod factors. However, this region does not contain any known consensus sequences needed for hormonal regulation, such as auxin-responsive elements (ARE), ethylene-responsive /*/ elements, G boxes etc., confirming the inventors' finding that MsENOD40 is not induced by other plant hormones (Hirsch et al, 1997).

Similar to the GmENOD40-2 promoter, the two MsENOD40 promoters also contain two conserved sequence motifs AAAGAT and CTCTT (see boxed regions in FIG. 1) that have been identified in several nodulin gene promoters (Sandal et al, 1987; Bak Ramlov et al, 1993; Miao and Verma, 1993; Roussis et al, 1995). Using deletion analysis and site-specific mutagenesis, these sequences have been found to be required for nodule-specific expression for certain genes, such as lbc3 and nodulin-26 (Stougaard et al, 1990; Miao and Verma, 1993; Bak Ramlov et al, 1993; Szczyglowski et al, 1994). Whether these motifs determine or are responsible for nodule-specific expression of MsENOD40 will be investigated in future work.

The inventors' studies do not clarify whether the ENOD40 gene product functions as a small peptide (van de Sande et al, 1996) or as a riboregulator (Crespi et al, 1994). However, it is very likely that the induction of expression of ENOD40 by cytokinin or other endogenous molecules upon Rhizobium inoculation could serve as an amplification mechanism thereby triggering a localized hormone imbalance, a state which initiates cell divisions in the root cortex. Also, the inventors should caution that they have analyzed the expression patterns of trangenes in this study. Most of the inventors' results, with few exceptions, corresponded very well with the inventors' previous findings from in situ hybridization studies. The exceptions may reflect the difference between detecting transcripts from the endogenous genes versus detecting the reporter gene, i.e. gus, from the introduced trangenes.

Abbreviations: BAP (6-benzylaminopurine); ENOD, early nodulin gene; NPA, N- - (naphthyl)phthalamic acid; PNF, purified Nod factor.

5.2 EXAMPLE 2

5.2.1 ASSESSMENT OF AM FUNGAL MYCELIUM AND SPORE COMPONENTS

The inventors have produced transgenic plants carrying MsENOD40-Gus constmcts, and based on these assays, the arbuscular-mycorrhizal fungi induce several of the MsENOD40 promoter-Gus constructs (FIG. 7). Axenically grown transgenic plants carrying ENOD40-Gus constructs will be incubated with spore and/or hyphal fractions of axenically grown Glomus intraradices. Glomus intraradices will be separated into spore and hyphal fractions according to Horn et al. (1992). Roots will be examined for Gus activity using histochemical staining procedures and fluorimetry for quantitative evaluations (Gallager, 1992).

If activity is found to be associated with the spore wall, the wall material will be prepared according to the procedure of Ren and West (1992). Their strategy will be utilized to identify the active component from the AM fungi. Isolated AM fungal walls will be pretreated with wall-degrading enzymes to see if such treatment influences Gus- inducing activity. Such an approach led Ren and West to conclude that the most potent wall fraction in their bioassay consisted of chitin fragments. If chitin fragments are implicated, then an acid hydrolysis of insoluble chitin using the method of Barber et al. (1989) will be employed. However, it is possible that chitin or chitin fragments are not active inducers of the sE /D40-promoter Gus constructs even though chitin is one of the principle components of the AM-fungal cell walls (Bago et al, 1996). The inventors then will treat the most potent wall fraction inducer with other cell-wall degrading enzymes to learn which is the most active.

If the wall component is determined to be chitin-based, a strategy similar to that utilized by Lerouge et al. (1990) for extracting R. meliloti Nod factor may be employed. Wall fractions may be extracted with butanol and successively purified by preparative reverse C₁₈ HPLC, by gel permeation on a Sephadex LFI20 column, and by ion exchange chromatography on a DEAE column. Alternatively, one may will utilize an affinity-purification procedure developed for Nod factor extraction by E.M. Atkinson et al., . Ultraviolet light absorbance may be monitored for each fraction, and each fraction tested for its ability to induce MsENOD40-Gus expression.

5.2.2 EXPRESSION OF ENOD GENES IN NON-MYCORRHIZAL ALFALFA MUTANTS

Roots of two alfalfa genotypes, non-nodulating (MN NN-1008) and nodulating but ineffective in nitrogen fixation (MN IN-381 1). do not become colonized by AM fungi (Bradbury et al, 1991 , 1993). Although appressoria are developed on the surface of root epidermis, AM colonization was slight for MN IN-381 1 and essentially non- existent for MN NN-1008. These alfalfa mutants provide a great opportunity to understand the importance of appressoria formation for triggering plant responses to AM fungal colonization. Mutant alfalfa plants will be inoculated with Glomus intraradices. Expression of ENOD genes will be assessed using northern-blot analysis according to standard procedures with radioactively labeled probes from ENOD2 and ENOD40 clones. In situ hybridization will be performed to detect spatial localization of ENOD transcripts in relation to distribution of appressoria formed by AM fungi attempting to colonize the roots.

5.2.3 ENOD40 EXPRESSION IN NON-LEGUME AM HOSTS

A homolog of ENOD40 has been detected in and cloned from tobacco (van de Sande et al, 1996). To evaluate whether host responses to AM-fungal colonization are conserved between legume and non-legume plants, tobacco plants will be grown axenically and inoculated with Glomus intraradices spores. Expression of ENOD40 in tobacco AM-fungal-colonized roots may be assessed using northern-blot analysis. In situ hybridization may be performed to detect spatial localization of ENOD40 transcripts in relation to distribution of fungal stmctures within AM-fungal-colonized tobacco roots.

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All of the compositions and methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims. . SEQUENCE LISTING ^{/ V}

(1) GENERAL INFORMATION:

(l) APPLICANT:

(A) NAME: Regents of the University of California

(B) STREET: 10945 Le Cont Avenue

(C) CITY: Los Angeles

(D) STATE: California

(E) COUNTRY: US

(F) POSTAL CODE (ZIP) : 90095

(G) TELEPHONE: (512) 418-3000 (H) TELEFAX: (713) 789-2679

(A) NAME: Yiwen Fang

(B) STREET: Department of Molecular, Cell, and Developmental Biology, 405 Hilgard Avenue

(C) CITY: Los Angeles

(D) STATE: California

(E) COUNTRY: US

(F) POSTAL CODE (ZIP) : 90095-1606

(G) TELEPHONE: (512) 418-3000 (H) TELEFAX: (713) 789-2679

(A) NAME: Ann M. Hirsch

(C) CITY: Los Angeles

(D) STATE: California

(E) COUNTRY: US

(F) POSTAL CODE (ZIP) : 90095-1606

(G) TELEPHONE: (512) 418-3000 (H) TELEFAX: (713) 789-2679

(n) TITLE OF INVENTION: MsENOD40 Promoter Compositions and Methods of Use

(m) NUMBER OF SEQUENCES: 2

( v) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS/MS-DOS

(D) SOFTWARE: Patentln Release #1.0, Version #1.30 (EPO)

(vi) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER: US 60/025,730

(B) FILING DATE: 10-AUG-1996

(2) INFORMATION FOR SEQ ID NO : 1: (l) SEQUENCE CHARACTERISTICS: (A) LENGTH: 2531 base pairs °

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 1:

GGCACCTAAA TTGCAGCGGA AAGAATCAAA CTTGAGACCT CAAGGAGAAG TTCAATCCTA 60

AAATTGTTAG TTAAGTGTAA AGAGATTTAA ACGATTTCCT CGAAATGCAG TTAATTAACT 120

TTCGATTACT CCCCGTTCTA TTATGAATGT ACATTTGAAG AAAAAAAAAG TTCTACAATA 180

AGTGTAATTT TTAATGTATT TTTTTTTTAT GAAACTAGTT TTCATTGTAA TTAATGTTAA 240

TTTTTTTTCT CTTAATGATT TATTTAATAA TATGTGCAGA AAAGGTTAAT TTGGTAAAAA 300

AAAATCTTTC ATTTATTATT TCTCCCTAAT TTGTACGAAA TAATTAATCA AAGATTCCTT 360

AAAAAAAAAA ATTAATCAAA GATAACACTC AATAGAAAAG TGTGTATTAA TATTACTTTT 420

GTAGCCTCCT CATATTAATC AGTTCTATTT TTTTTTTTTT CCTTCACTTC TTTGTGGCTA 480

TTGTTTATTT TTAGTAAAAC CAGCTCACCG AAATTGGTAT CATTGGAAAC AAACCTGAAA 540

ACTTAAGACG AGCATGCTCT AAAGTCGCAA ATCATCACCA TCCAACTAAC ACAAGTGGGT 600

TCTTTGTGGC GATTGTTGTT ATTAAATAAC AAAGCCATTA AATTAACTTG GTCAGGGATG 660

GGAAAATTAA TAAATCAAAT AAGACTCTAA GAAGAACTAA AGAGTTAGGA TAAGTGAATG 720

GAAAAGTCAA CTCTAGTTTT TCTGAAAAGA ACCTTTGATT CCTCTTGTTG CAAAAGTTTT 780

TAAATGTTTA TTAAAACCTT GGCTTATTGC CTTTTATAAA TCAAATTAGA ATCGATCATT 840

GTTGTCCACC ACTTTCAATT GGACAAAGGA GGATCAACGG AAAATGTTGT GGTCCTAAGT 900

TGATTCCAAT TTGCAATCAA GTTTTGGGTC CCTTATAATC CAAAATTTCC ACATTTTACT 960

GTTAATTCAG TTTAATTTAC ATTCAAACGT GGTTAATTTT CTGATTAGTC ATCATCTTAG 1020

TAATAATTAT AAATATGGTC CCTCTACTTT CCACGTGCCA TTTGAAGCCA TAGGAAATTT 1080

GAATTTTCCA TGATGTGGTC TTTAGAGTCT TGCTATATAG ACCGAAAGAA TTTTCGCGTG 1140

GATGGCACGA ATATGTTATT TAATGCACAG TATATCTCGC TTAGTGCCTT TAATTGTTTT 1200

TTTTAAGCAA GTAGCTTTAA TTGTTATTGC TATAGTTTAT AGGGTTGTGC TACATATTGT 1260

TCTTAATGAG ATTGTTCTTC AATGCTAACT ATAGTTTATG GGGTTAATTA AGTTTTTAGT 1320

CCTTATAAAT ATAGCGAATT TTATTTTTAG TTCTCATTAA AAAAAAGTGA TAGGTTTTGG 1380

TTCCTATAAA TATTTTCACC ACGTAATTTT AGTCCTTGTT AAATTAAAAT TTGTTTAATT 1440 t *j

ATGCATAAAT TTCTAAATTT TTGAATGATT TTTTACAGAC ATGTTAATAT CATCATAAGA 1500

AACTCTTTTA TAAAATATTT TAGTTTTTTA ACATGTCACA AACTAAATGT GGATTTTTCT 1560

AAAAGCCATA TTTTCAAGAT TAAATTAATG AAAATTTGGA CCAAATAATC AAATTTTAAT 1620

TTAACAGTGA CTAAAAACAA ACTTACTTAA AAACAAAAGA TTACATGAAA ATTGACTATA 1680

ATAATAGTGA CCAAAAACTT ATTTCCCAAT TTTTGTTTAA TTACAATGAT GACATGAGCT 1740

AAATATGAGG GATGAGTTAA AAAAAGAGAG AGGTGATGAG GTAAT ATAA GGGAAATTTG 1800

AAAGAAATCT GATGGCTATG CAAGTAACAG CTCATTCGAG GGTATAGCAC GAAATTTTAT 1860

CTCGTGCAAT TTAGCATTTT TCATAAAATT AGTTCATCTT TTCTTGACAA ATAAAATGAG 1920

TTCATCTTAA ATGGTAGAAA AAATAGTTAA GGTAACAAAT TAGAAATTAG ATTATAGATC 1980

ACCAATATTA TGTTAAAATT TTGTGGATAA AAAAATATTG AAAGAGAAGA CCCTATTTAA 2040

GATAGTTAGC GAAATTTCTT ACCAAAAATT AATAATCAAC AAAACCGATA GAAGCTCGTA 2100

TCTAAACTTT ATTCCCATGT AAACCAGTTT GAAGGAAAAT AAAAGAGTAA CAAATCCAGC 2160

TAGGTGAGGT ATATCTTGGC TATGATGAGA TTGATATCAA CAAAAGGTTA GGCCATTAGG 2220

TTAAGATTGA GATTGAATTA ATCAAACAAA CATCTTTATC CATTATTTTA ATAGACACAC 2280

AGTAGTACTA TTTTCTTTTA TGTGAGTGCC TATACGTTGC AACTTTTGAT GGTGTTAAAT 2340

AGTCATTTTA GATAATGAAA AAGAATGATA CACACACCAC TTTCATATAT ACTTCTCAAC 2400

CATCACAAGT ATTTAAAACA ATGATCAGAG ACACCAACTT CCCCACTACC TTTCTATGTG 2460

GAGCCCTTTA AGCATCCTCT AAACCAATCC ATCAAGACTT GAATCTTGTT TGTAATAAGG 2520

ATGAAGCTTC T 2531

(2) INFORMATION FOR SEQ ID NO : 2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 2520 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 2:

AAGCTTGATT GTTATACATT TTCAATGTAA AAAGTTTTGC TAAAATTTCT GATTGGAGAG 60

AGTAAAATTA GTGGATAATT AGGAATTGAA AAATGAGTTG TTGTATTAAC AATTTCTCTT 120

GACATCTAAT CATTTTGAAA CAGGAAGAGT TGTTATTGTA GTAACCTGAA TTTCGTGCCA 180 HS

TGAAGGAAAT CAAGCAGCGA AAAATCTCGA TCATTGGATG AAGATCAAAC GATTCGTATT 240

CTAACTTCAT TAAATTGACT TCAAAACATC CTAAAATGGA AGTCATGCAA TATTTATTTA 300

ACAGTCAAGA TCCAACGGTA AATCCGTGTT TAATATTTCT TATCTAGTTC CTAGTTGTGA 360

AGAAGTATTA TGAATGAGTT CTATAATTCA ATTTTTGCTT CTAACAACTT CTATCTAAAC 420

AAATTAAACA ATAAATTTTG TGTATCAAAA ATAAAAGAAA AAATTTCTTA TGAATAAATA 480

GGTTTTTCTT TTTTGTAATG GAAATCTAAT AAAGAGGTTT ATTAAATAGA TTCAACAAGA 540

TATATAATTA TTGCAATTTA GGATATAAAA TGAATTAGTG AATCGTGCCA ATTTTCCATG 600

GAAGAGACAT CTAATTTAAA TTTTGTATCA TGACTCATAA AAGGAAATTT ATATCAAGGT 660

CAACATAAAC TTTAAGCATG GGTTCCCACA CTTTACTTTT TCATCTATGC TTGACCTTTG 720

ATAATCGGAG GCTTTTATTG CATAGTGGAT CCACTCACTA TAATAAGACT CTATCCCCAC 780

CACCTCTCTT CCAACAATAA TTATAGGGGA AATTAGTCAC TTTAGTCCCT GAATGGGTAA 840

CGATTCGTCA ATTTAGTCCC TTGAATAAAG ATAATATGCA AAACAGTAAT TGCATGGTCA 900

ATTTCAGGGA CTAAAGTGAC TAACGAGTCT ACATTCAGTG ACTAAATTGA CTAACGGAGT 960

CTGCATTCAG GGACTAAATT GACTAACAAA GTCTACATTC AGGGACTATT TTGCAATTTA 1020

TCTGCATTCA GGGACTAAAT TGACGAAACG TTCCCCATTC AGGGACTAAA GTGACTAATT 1080

CCCCAATAAT TTAGAGTCTT GCTATATAGA CCGAAAGAAT TTTCGCGTGG ATGGCACGAA 1140

TATGTTATTT AATGCACAGT ATATCTCGCT TAGTGCCTTT AATTGTTTTT TTTAAGCAAG 1200

TAGCTTTAAT TGTTATTGCT ATAGTTTATA GGGTTGTGCT ACATATTGTT CTTAATGAGA 1260

TTGTTCTTCA ATGCTAACTA TAGTTTATGG GGTTAATTAA GTTTTTAGTC CTTATAAATA 1320

TAGCGAATTT TATTTTTAGT TCTCATTAAA AAAAAGTGAT AGGTTTTGGT TCCTATAAAT 1380

ATTTTCACCA CGTAATTTTA GTCCTTGTTA AATTAAAATT TGTTTAATTA TGCATAAATT 1440

TCTAAATTTT TGAATGATTT TTTACAGACA TGTTAATATC ATCATAAGAA ACTCTTTTAT 1500

AAAATATTTT AGTTTTTTAA CATGTCACAA ACTAAATGTG GATTTTTCTA AAAGCCATAT 1560

TTTCAAGATT AAATTAATGA AAATTTGGAC CAAATAATCA AATTTTAATT TAACAGTGAC 1620

TAAAAACAAA CTTACTTAAA AACAAAAGAT TACATGAAAA TTGACTATAA TAATAGTGAC 1680

CAAAAACTTA TTTCCCAATT TTTGTTTAAT TACAATGATG ACATGAGCTA AATATGAGGG 1740

ATGAGTTAAA AAAAGAGAGA GGTGATGAGG TAATAATAAG GGAAATTTGA AAGAAATCTG 1800

ATGGCTATGC AAGTAACAGC TCATTCGAGG GTATAGCACG AAATTTTATC TCGTGCAATT 1860 TAGCATTTTT CATAAAATTA GTTCATCTTT TCTTGACAAA TAAAATGAGT TCATCTTAAA 1920

TGGTAGAAAA AATAGTTAAG GTAACAAATT AGAAATTAGA TTATAGATCA CCAATATTAT 1980

GTTAAAATTT TGTGGATAAA AAAATATTGA AAGAGAAGAC CCTATTTAAG ATAGTTAGCG 2040

AAATTTCTTA CCAAAAATTA ATAATCAACA AAACCGATAG AAGCTCGTAT CTAAACTTTA 2100

TTCCCATGTA AACCAGTTTG AAGGAAAATA AAAGAGTAAC AAATCCAGCT AGGTGAGGTA 2160

TATCTTGGCT ATGATGAGAT TGATATCAAC AAAAGGTTAG GCCATTAGGT TAAGATTGAG 2220

ATTGAATTAA TCAAACAAAC ATCTTTATCC ATTATTTTAA TAGACACACA GTAGTACTAT 2280

TTTCTTTTAT GTGAGTGCCT ATACGTTGCA ACTTTTGATG GTGTTAAATA GTCATTTTAG 2340

ATAATGAAAA AGAATGATAC ACACACCACT TTCATATATA CTTCTCAACC ATCACAAGTA 2400

TTTAAAACAA TGATCAGAGA CACCAACTTC CCCACTACCT TTCTATGTGG AGCCCTTTAA 2460

GCATCCTCTA AACCAATCCA TCAAGACTTG AATCTTGTTT GTAATAAGGA TGAAGCTTCT 2520

Claims

CLAIMS:

l . An isolated MsENOD40 promoter comprising a contiguous nucleic acid sequence of at least about 17 nucleic acids from SEQ ID NOT or SEQ ID NO:2, or a nucleic acid sequence having from about 80% to about 95% sequence identity with the sequence of SEQ ID NOT or SEQ ID NO:2, wherein said sequence is effective to promote transcription of a heterologous nucleic acid segment operatively linked to said promoter in a cell.

The promoter of claim 1 , wherein said nucleic acid sequence has at least about 80% sequence identity to the sequence of SEQ ID NOT or SEQ ID NO:2.

3. The promoter of claim 2, wherein said nucleic acid sequence has at least about

85% sequence identity to the sequence of SEQ ID NOT or SEQ ID NO:2.

4. The promoter of claim 3, wherein said nucleic acid sequence has at least about

90% sequence identity to the sequence of SEQ ID NOT or SEQ ID NO:2.

5. The promoter of claim 4, wherein said nucleic acid sequence has at least about

95% sequence identity to the sequence of SEQ ID NOT or SEQ ID NO:2.

6. The promoter of claim 1 , wherein said nucleic acid sequence comprises the sequence of SEQ ID NOT or SEQ ID NO:2.

7. The promoter of claim 1. further defined as an MsENOD40-l or an MsENOD40-

2 promoter. H%

8. The promoter of claim 1, wherein said promoter is regulated by a cytokinin, chitin, or chitin fragment.

9. The promoter of claim 8, wherein said cytokinin is BAP.

10. The promoter of claim 8, wherein said cytokinin or chitin increases transcription from said promoter.

11. The promoter of claim 1 , wherein said nucleic acid segment is a ribozyme, antisense construct or a heterologous gene.

12. The promoter of claim 1 1, wherein said nucleic acid segment comprises a ribozyme or an antisense constmct.

13. The promoter of claim 1 1 , wherein said nucleic acid segment comprises a heterologous gene.

14. The promoter of claim 13, wherein said heterologous gene is a reporter gene, a cell cycle control gene, an insecticidal resistance gene, a microbial resistance gene, a herbicide resistance gene, a drought tolerance gene, or a gene encoding a pheromone, hormone, antihormones, hormone inhibitor, storage protein, enzyme, or a stmctural protein. H 15. The promoter of claim 14, wherein said insecticidal gene encodes a Bacillus thuringiensis crystal protein.

16. The promoter of claim 14, wherein said reporter gene is a GUS gene, a lac gene, a GFP gene, a lux gene, a β-lactamase gene, a xylE, a α-amylase gene, a tyrosinase gene, or a aequorin gene.

17. The promoter of claim 14, wherein said cell cycle control gene is selected from the group consisting of Rb, p53, a cell cycle dependent kinase, a CDK kinase and a cyclin gene.

18. The promoter of claim 14, wherein said herbicide resistance gene is a bar gene, a a pat gene, a glyphosate tolerant EPSP synthase gene, a gox gene, a deh gene, a acetolactate synthase gene, or a bxn gene.

19. The promoter of claim 14, wherein said microbial resistance gene is a b-1 gene, a lac gene, a 3-glucanase gene, a chitinase, a osmotin gene, a UDA gene, or a hevein gene.

20. A method of expressing a heterologous nucleic acid segment in a cell comprising transforming said cell with a vector comprising a heterologous nucleic acid segment operatively linked to an MsENOD40 promoter and culturing said cell under conditions effective to express said heterologous nucleic acid segment from said promoter. I SO

21. The method of claim 20, wherein said MsENOD40 promoter comprises an MsENOD40-l or an MsENOD40-2 promoter.

22. The method of claim 21, said promoter having substantially the sequence of SEQ ID NOT or SEQ ID NO:2.

23. The method of claim 22, said promoter comprising the sequence of SEQ ID NO: 1 or SEQ ID NO:2.

24. The method of claim 20, wherein said cell is a plant, bacterial, fungal, or animal cell.

25. The method of claim 24, wherein said plant cell is a corn, cotton, Arahidopsis, flax, rye, rice, canola, wheat, alfalfa, tobacco, tomato, potato, soybean, sunflower, citrus, nut, fruit, berry, shrub, cactus, succulent, or ornamental plant cell.

26. The method of claim 24, wherein said bacterial cell is an E. coli, A. tumefaciens, or a R. meliloti cell, and said fungal cell is a yeast or a mycorrhizal cell.

27. The method of claim 24, wherein said animal cell is a human, dog, cat, rat, mouse, rabbit, goat, horse, pig, monkey, or hamster cell.

28. The method of claim 20, wherein said heterologous nucleic acid segment comprises a ribozyme, an antisense constmct, or a heterologous gene.

29. The method of claim 28. wherein said gene comprises a cell cycle control gene, an insecticidal resistance gene, a fungal resistance gene, a bacterial resistance gene, a viral resistance gene, a herbicide resistance gene, a drought tolerance gene, or a gene encoding a pheromone, hormone, antihormones, hormone inhibitor, storage protein, enzyme, or a stmctural protein.

30. A method of increasing herbicide resistance in a cell comprising expressing in said cell a herbicide resistance gene operatively linked to an MsENOD40 promoter.

31. A method of increasing insect resistance in a cell comprising expressing in said cell an insect resistance gene operatively linked to an MsENOD40 promoter.

32. A method of increasing microbial resistance in a cell comprising expressing in said cell a microbial resistance gene operatively linked to an MsENOD40 promoter.

A recombinant vector comprising an MsENOD40 promoter operatively linked to a heterologous nucleic acid segment, in a position to control expression of said segment.

34. The recombinant vector of claim 33, selected from the group consisting of a plasmid, a phagemid, a cosmid. a YAC, a BAC, and a viral vector. lb^~X

35. The recombinant vector of claim 34, wherein said viral vector is a bacteriophage vector, a raus sarcoma vims vector, a p21 virus vector an adeno-associated vims vector or an adenoviral vector.

36. The recombinant vector of claim 35, wherein said vector is further defined as a replication deficient adenovims.

37. The recombinant vector of claim 33, dispersed in a pharmaceutically acceptable solution.

38. A nucleic acid segment comprising a promoter sequence region having substantially the nucleotide sequence of SEQ ID NOT or SEQ ID NO:2.

39. A nucleic acid segment comprising an MsENOD40 promoter having substantially the sequence of SEQ ID NOT or SEQ ID NO:2 operatively linked to a heterologous gene, said promoter having transcriptional promoting activity for said gene.

40. The nucleic acid segment of claim 39, comprising an MsENOD40 promoter sequence region having about 80% to about 95% identity with the sequence of SEQ ID NOT or SEQ ID NO:2.

41. The nucleic acid segment of claim 40, wherein said sequence region has at least about 80% identity with the sequence of SEQ ID NOT or SEQ ID NO:2.

42. The nucleic acid segment of claim 41 , wherein said sequence region has at least about 85% identity with the sequence of SEQ ID NO: 1 or SEQ ID NO:2.

43. The nucleic acid segment of claim 42, wherein said sequence region has at least about 90% identity with the sequence of SEQ ID NO: 1 or SEQ ID NO:2.

44. The nucleic acid segment of claim 43, wherein said sequence region has at least about 95% identity with the sequence of SEQ ID NOT or SEQ ID NO:2.

45. The nucleic acid segment of claim 44, wherein said sequence region comprises the sequence of SEQ ID NOT or SEQ ID NO:2.