CA2389813A1

CA2389813A1 - Genes encoding enzymes for lignin biosynthesis and uses thereof

Info

Publication number: CA2389813A1
Application number: CA002389813A
Authority: CA
Inventors: Timothy G. Helentjaris
Original assignee: Individual
Current assignee: Pioneer Hi Bred International Inc
Priority date: 1999-11-05
Filing date: 2000-11-02
Publication date: 2001-05-17
Also published as: WO2001034817A3; HUP0203221A2; AU1465201A; EP1226260A2; MXPA02004482A; WO2001034817A2

Abstract

The present invention provides methods and compositions relating to altering lignin biosynthesis content and/or composition of plants. The invention provides isolated nucleic acids and their encoded proteins that are involved in lignin biosynthesis. The invention further provides recombinant expression cassettes, host cells, transgenic plants, and antibody compositions.

Description

GENES ENCODING ENZYMES FOR LIGNIN BIOSYNTHESIS
AND USES THEREOF
TECHNICAL FIELD
The present invention relates generally to plant molecular biology. More specifically, it relates to nucleic acids and methods for modifying the lignin content in plants.
BACKGROUND OF THE INVENTION
Differences in plant cell wall composition account for much of the variation in chemical reactivity, mechanical strength, and energy content of plant material. In turn, differences in chemical and mechanical properties of plant material greatly impact the utilization of plant biomass by agriculture and industry. One abundant component of many types of plant cells, and one which has garnered increasing attention because of its importance in plant utilization, are lignins.
Lignins are a class of complex heterpolymers associated with the polysaccharide components of the wall in specific plant cells. Lignins play an essential role in providing rigidity, compressive strength, and structural support to plant tissues. They also render cell walls hydrophobic allowing the conduction of water and solutes. Reflecting their importance, lignins represent the second most abundant organic compound on Earth after cellulose accounting for approximately 25% of plant biomass. Lignins result from the oxidative coupling of three monomers: coumaryl, coniferyl, and sinapyl alcohols.
Variability in lignin structure is dependent, in part, upon the relative proportion of the three constitutive monomers.
The biosynthesis of lignins proceeds from phenylalanine through the phenylpropanoid pathway to the cinnamoyl CoAs which are the general precursors of a wide range of phenolic compounds. The enzymes involved in this pathway are phenylalanine ammonia-lyase (PAL), cinnamate-4-hydroxylase (C4H), 4-coumarate-hydroxylase (C3H), O-methyltransferase (OMT), ferulate-5-hydroxylase (FSH), caffeoyl-CoA 3-O-methyltransferase (CCoA-OMT), and 4-coumarate:CoA ligase (4CL).
Whetten and Sederoff, The Plant Cell, 7: 1001-1013 (1995); Boudet and Grima-Pettenati, Molecular Breeding, 2:25-39 (1996).
The lignin specific pathway channels cinnamoyl CoAs towards the synthesis of monolignols and lignins. This pathway involves two reductive enzymes that convert the hydroxycinnamoyl-CoA esters into monolignols: cinnamoyl-CoA reductase (CCR), and cinnamyl alcohol dehydrogenase (CAD).
While lignins are a vital component in terrestrial vascular plants, they often pose an obstacle to the utilization of plant biomass. For example, in the pulp and paper industry lignins have to be separated from cellulose by an expensive and polluting process. Lignin content also limits the digestibility of crops consumed by livestock. While reduction of lignin content for such applications is generally desirable, increasing lignin content in plant material intended as a chemical feedstock for production of phenolics, for use as a fuel source, or for improvement in agronomically desirable properties (e.g., standability) is also advantageous. Accordingly, what is needed in the art is the ability to modulate lignin content in plants. The present invention addresses these and other needs.
SUMMARY OF THE INVENTION
Generally, it is the object of the present invention to provide nucleic acids and proteins relating to lignin biosynthesis; transgenic plants comprising the nucleic acids of the present invention; and methods for modulating, in a transgenic plant, the expression of the nucleic acids of the present invention.
Therefore, in one aspect, the present invention relates to an isolated nucleic acid comprising a member selected from the group consisting of (a) a polynucleotide having specified sequence identity to a polynucleotide encoding a polypeptide of the present invention; (b) a polynucleotide which is complementary to the polynucleotide of (a); and (c) a polynucleotide comprising a specified number of contiguous nucleotides from a polynucleotide of (a) or (b). The isolated nucleic acid can be DNA.
In another aspect, the present invention relates to recombinant expression cassettes, comprising a nucleic acid of the present invention operably linked to a promoter.
In some embodiments, the nucleic acid is operably linked in antisense orientation to the promoter.
In another aspect, the present invention is directed to a host cell into which has been introduced the recombinant expression cassette.

In a further aspect, the present invention relates to an isolated protein comprising a polypeptide having a specified number of contiguous amino acids encoded by an isolated nucleic acid of the present invention.
In another aspect, the present invention relates to an isolated nucleic acid comprising a polynucleotide of a specified length which selectively hybridizes under stringent conditions to a polynucleotide of the present invention. In some embodiments, the isolated nucleic acid is operably linked to a promoter.
In another aspect, the present invention relates to a recombinant expression cassette comprising a nucleic acid, wherein the nucleic acid is operably linked to a promoter. In some embodiments, the present invention relates to a host cell transfected with this recombinant expression cassette. In some embodiments, the present invention relates to a protein of the present invention which is produced from this host cell.
In yet another aspect, the present invention relates to a transgenic plant comprising a recombinant expression cassette comprising a plant promoter operably linked to any of the isolated nucleic acids of the present invention. The present invention also provides transgenic seed from the transgenic plant.
In a further aspect, the present invention relates to a method of modulating expression of the genes encoding the proteins of the present invention in a plant cell capable of plant regeneration.

Definitions Units, prefixes, and symbols may be denoted in their SI accepted form. Unless otherwise indicated, nucleic acids are written left to right in 5' to 3' orientation; amino acid sequences are written left to right in amino to carboxy orientation, respectively. Numeric S ranges are inclusive of the numbers defining the range and include each integer within the defined range. Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-ICIB
Biochemical Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes. Unless otherwise provided for, software, electrical, and electronics terms as used herein are as defined in The New IEEE Standard Dictionary of Electrical and Electronics Terms (5th edition, 1993). The terms defined below are more fully defined by reference to the specification as a whole.
By "amplified" is meant the construction of multiple copies of a nucleic acid sequence or multiple copies complementary to the nucleic acid sequence using at least one 1 S of the nucleic acid sequences as a template. Amplification systems include the polymerise chain reaction (PCR) system, ligase chain reaction (LCR) system, nucleic acid sequence based amplification (NASBA, Cangene, Mississauga, Ontario), Q-Beta Replicase systems, transcription-based amplification system (TAS), and strand displacement amplification (SDA). See, e.g., Diagnostic Molecular Microbiology: Principles and Applications, D. H.
Persing et al., Ed., American Society for Microbiology, Washington, D.C.
(1993). The product of amplification is termed an amplicon.
As used herein, "antisense orientation" includes reference to a duplex polynucleotide sequence which is operably linked to a promoter in an orientation where the antisense strand is transcribed. The antisense strand is sufficiently complementary to an endogenous transcription product such that translation of the endogenous transcription product is often inhibited.
As used herein, "chromosomal region" includes reference to a length of chromosome which may be measured by reference to the linear segment of DNA
which it comprises. The chromosomal region can be defined by reference to two unique DNA
sequences, i.e., markers.
The term "conservatively modified variants" applies to both amino acid and nucleic acid sequences. With respect to particular nucleic acid sequences, conservatively modified variants refers to those nucleic acids which encode identical or conservatively modified variants of the amino acid sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein.
For instance, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine.
Thus, at every position where an alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide. Such nucleic acid variations are "silent variations" and represent one species of conservatively modified variation. Every nucleic acid sequence herein which encodes a polypeptide also describes every possible silent variation of the nucleic acid. One of ordinary skill will recognize that each codon in a nucleic acid (except AUG, which is ordinarily the only codon for methionine) can be modified to yield a functionally identical molecule.
Accordingly, each silent variation of a nucleic acid which encodes a polypeptide of the present invention is implicit in each described polypeptide sequence and is within the scope of the present invention.
As to amino acid sequences, one of skill will recognize that individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide, or protein sequence which alters, adds or deletes a single amino acid or a small percentage of amino acids in the encoded sequence is a "conservatively modified variant" where the alteration results in the substitution of an amino acid with a chemically similar amino acid. Thus, any number of amino acid residues selected from the group of integers consisting of from 1 to 15 can be so altered. Thus, for example, l, 2, 3, 4, 5, 7, or 10 alterations can be made.
Conservatively modified variants typically provide similar biological activity as the unmodified polypeptide sequence from which they are derived. For example, substrate specificity, enzyme activity, or ligand/receptor binding is generally at least 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the native protein for it's native substrate.
Conservative substitution tables providing functionally similar amino acids are well known in the art.
The following six groups each contain amino acids that are conservative substitutions for one another:
1) Alanine (A), Serine (S), Threonine (T);
2) Aspartic acid (D), Glutamic acid (E);
3) Asparagine (N), Glutamine (Q);
4) Arginine (R), Lysine (K);

5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).
See also, Creighton (1984) Proteins W.H. Freeman and Company.
By "encoding" or "encoded", with respect to a specified nucleic acid, is meant comprising the information for translation into the specified protein. A
nucleic acid encoding a protein may comprise non-translated sequences (e.g., introns) within translated regions of the nucleic acid, or may lack such intervening non-translated.
sequences (e.g., as in cDNA). The information by which a protein is encoded is specified by the use of codons. Typically, the amino acid sequence is encoded by the nucleic acid using the "universal" genetic code. However, variants of the universal code, such as is present in some plant, animal, and fungal mitochondria, the bacterium Mycoplasma capricolum, or the ciliate Macronucleus, may be used when the nucleic acid is expressed therein.
When the nucleic acid is prepared or altered synthetically, advantage can be taken of known codon preferences of the intended host where the nucleic acid is to be expressed.
For example, although nucleic acid sequences of the present invention may be expressed in both monocotyledonous and dicotyledonous plant species, sequences can be modified to account for the specific codon preferences and GC content preferences of monocotyledons or dicotyledons as these preferences have been shown to differ (Murray et al.
Nucl. Acids Res. 17: 477-498 (1989)). Thus, the maize preferred codon for a particular amino acid may be derived from known gene sequences from maize. Maize codon usage for 28 genes from maize plants are listed in Table 4 of Murray et al., supra.
As used herein "full-length sequence" in reference to a specified polynucleotide or its encoded protein means having the entire amino acid sequence of, a native (non-synthetic), endogenous, catalytically active form of the specified protein. A
full-length sequence can be determined by size comparison relative to a control which is a native (non-synthetic) endogenous cellular form of the specified nucleic acid or protein. Methods to determine whether a sequence is full-length are well known in the art including such exemplary techniques as northern or western blots, primer extension, S 1 protection, and ribonuclease protection. See, e.g., Plant Molecular Biology: A Laboratory Manual, Clark, Ed., Springer-Verlag, Berlin (1997). Comparison to known full-length homologous (orthologous and/or paralogous) sequences can also be used to identify full-length sequences of the present invention. Additionally, consensus sequences typically present at the 5' and 3' untranslated regions of mRNA aid in the identification of a polynucleotide as full-length. For example, the consensus sequence ANNNNAUGG, where the underlined codon represents the N-terminal methionine, aids in determining whether the polynucleotide has a complete 5' end. Consensus sequences at the 3' end, such as polyadenylation sequences, aid in determining whether the polynucleotide has a complete 3' end.
As used herein, "heterologous" in reference to a nucleic acid is a nucleic acid that originates from a foreign species, or, if from the same species, is substantially modified from its native form in composition and/or genomic locus by deliberate human intervention. For example, a promoter operably linked to a heterologous structural gene is from a species different from that from which the structural gene was derived, or, if from the same species, one or both are substantially modified from their original form. A
heterologous protein may originate from a foreign species or, if from the same species, is substantially modified from its original form by deliberate human intervention.
By "host cell" is meant a cell which contains a vector and supports the replication and/or expression of the expression vector. Host cells may be prokaryotic cells such as E. coli, or eukaryotic cells such as yeast, insect, amphibian, or mammalian cells.
Preferably, host cells are monocotyledonous or dicotyledonous plant cells. A
particularly preferred monocotyledonous host cell is a maize host cell.
The term "hybridization complex" includes reference to a duplex nucleic acid structure formed by two single-stranded nucleic acid sequences selectively hybridized with each other.
The term "introduced" in the context of inserting a nucleic acid into a cell, means "transfection" or "transformation" or "transduction" and includes reference to the incorporation of a nucleic acid into a eukaryotic or prokaryotic cell where the nucleic acid may be incorporated into the genome of the cell (e.g., chromosome, plasmid, plastid or mitochondria) DNA), converted into an autonomous replicon, or transiently expressed (e.g., transfected mRNA).
The terms "isolated" refers to material, such as a nucleic acid or a protein, which is:
(1) substantially or essentially free from components which normally accompany or interact with it as found in its naturally occurnng environment. The isolated material optionally comprises material not found with the material in its natural environment; or (2) _ g _ if the material is in its natural environment, the material has been synthetically (non-naturally) altered by deliberate human intervention to a composition and/or placed at a locus in the cell (e.g., genome or subcellular organelle) not native to a material found in that environment. The alteration to yield the synthetic material can be performed on the material within or removed from its natural state. For example, a naturally occurring nucleic acid becomes an isolated nucleic acid if it is altered, or if it is transcribed from DNA which has been altered, by non-natural, synthetic (i.e., "man-made") methods performed within the cell from which it originates. See, e.g., Compounds and Methods for Site Directed Mutagenesis in Eukaryotic Cells, Kmiec, U.S. Patent No.
5,565,350; In Vivo Homologous Sequence Targeting in Eukaryotic Cells; Zarling et al., PCT/US93/03868.
Likewise, a naturally occurnng nucleic acid (e.g., a promoter) becomes isolated if it is introduced by non-naturally occurring means to a locus of the genome not native to that nucleic acid. Nucleic acids which are "isolated" as defined herein, are also referred to as "heterologous" nucleic acids.
Unless otherwise stated, the term "lignin biosynthesis nucleic acid" means a nucleic acid comprising a polynucleotide ("lignin biosynthesis polynucleotide") encoding a lignin biosynthesis polypeptide. A "lignin biosynthesis gene" refers to a non-heterologous genomic form of a full-length lignin biosynthesis polynucleotide.
As used herein, "localized within the chromosomal region defined by and including" with respect to particular markers includes reference to a contiguous length of a chromosome delimited by and including the stated markers.
As used herein, "marker" includes reference to a locus on a chromosome that serves to identify a unique position on the chromosome. A "polymorphic marker"
includes reference to a marker which appears in multiple forms (alleles) such that different forms of the marker, when they are present in a homologous pair, allow transmission of each of the chromosomes in that pair to be followed. A genotype may be defined by use of one or a plurality of markers.
As used herein, "nucleic acid" includes reference to a deoxyribonucleotide or ribonucleotide polymer in either single- or double-stranded form, and unless otherwise limited, encompasses known analogues having the essential nature of natural nucleotides in that they hybridize to single-stranded nucleic acids in a manner similar to naturally occurring nucleotides (e.g., peptide nucleic acids).

- g _ By "nucleic acid library" is meant a collection of isolated DNA or RNA
molecules which comprise and substantially represent the entire transcribed fraction of a genome of a specified organism. Construction of exemplary nucleic acid libraries, such as genomic and cDNA libraries, is taught in standard molecular biology references such as Berger and Kimmel, Guide to Molecular Cloning Techniques, Methods in Enzymologv, Vol.
152, Academic Press, Inc., San Diego, CA (Berger); Sambrook et al., Molecular Cloning - A
Laboratory Manual, 2nd ed., Vol. 1-3 (1989); and Current Protocols in Molecular Biology, F.M. Ausubel et al., Eds., Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc. (1994).
As used herein "operably linked" includes reference to a functional linkage between a promoter and a second sequence, wherein the promoter sequence initiates and mediates transcription of the DNA sequence corresponding to the second sequence.
Generally, operably linked means that the nucleic acid sequences being linked are contiguous and, where necessary to join two protein coding regions, contiguous and in the same reading frame.
As used herein, the term "plant" includes reference to whole plants, plant parts or organs (e.g., leaves, stems, roots, etc.), plant cells, seed and progeny of same. Plant cell, as used herein includes, without limitation, cells obtained from or found in:
seeds, suspension cultures, embryos, meristematic regions, callus tissue, leaves, roots, shoots, gametophytes, sporophytes, pollen, and microspores. Plant cells can also be understood to include modified cells, such as protoplasts, obtained from the aforementioned tissues. The class of plants which can be used in the methods of the invention is generally as broad as the class of higher plants amenable to transformation techniques, including both monocotyledonous and dicotyledonous plants. Particularly preferred plants include maize, soybean, sunflower, sorghum, canola, wheat, alfalfa, cotton, rice, barley, and millet.
As used herein, "polynucleotide" includes reference to a deoxyribopolynucleotide, ribopolynucleotide, or analogs thereof that have the essential nature of a natural ribonucleotide in that they hybridize, under stringent hybridization conditions, to substantially the same nucleotide sequence as naturally occurring nucleotides and/or allow translation into the same amino acids) as the naturally occurnng nucleotide(s). A
polynucleotide can be full-length or a subsequence of a native or heterologous structural or regulatory gene. Unless otherwise indicated, the term includes reference to the specified sequence as well as the complementary sequence thereof. Thus, DNAs or RNAs with backbones modified for stability or for other reasons are "polynucleotides" as that term is intended herein. Moreover, DNAs or RNAs comprising unusual bases, such as inosine, or modified bases, such as tritylated bases, to name just two examples, are polynucleotides as the term is used herein. It will be appreciated that a great variety of modifications have been made to DNA and RNA that serve many useful purposes known to those of skill in the art.
The term polynucleotide as it is employed herein embraces such chemically, enzymatically or metabolically modified forms of polynucleotides, as well as the chemical forms of DNA and RNA characteristic of viruses and cells, including among other things, simple and complex cells.
The terms "polypeptide", "peptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical analogue of a corresponding naturally occurnng amino acid, as well as to naturally occurring amino acid polymers. The essential nature of such analogues of naturally occurring amino acids is that, when incorporated into a protein, that protein is specifically reactive to antibodies elicited to the same protein but consisting entirely of naturally occurring amino acids. The terms "polypeptide", "peptide" and "protein" are also inclusive of modifications including, but not limited to, glycosylation, lipid attachment, sulfation, gamma-carboxylation of glutamic acid residues, hydroxylation and ADP-ribosylation. Exemplary modifications are described in most basic texts, such as, Proteins - Structure and Molecular Properties, 2nd ed., T. E.
Creighton, W. H. Freeman and Company, New York (1993). Many detailed reviews are available on this subject, such as, for example, those provided by Wold, F., Posttranslational Protein Modifications: Perspectives and Prospects, pp. 1-12 in Posttranslational Covalent Modification of Proteins, B. C. Johnson, Ed., Academic Press, New York (1983);
Seifter et al., Meth. Enzymol. 182: 626-646 (1990) and Rattan et al., Protein Synthesis:
Posttranslational Modifications and Aging, Ann. N. Y. Acad. Sci. 663: 48-62 (1992). It will be appreciated, as is well known and as noted above, that polypeptides are not always entirely linear. For instance, polypeptides may be branched as a result of ubiquitination, and they may be circular, with or without branching, generally as a result of posttranslation events, including natural processing event and events brought about by human manipulation which do not occur naturally. Circular, branched and branched circular polypeptides may be synthesized by non-translation natural process and by entirely synthetic methods, as well.
Modifications can occur anywhere in a polypeptide, including the peptide backbone, the amino acid side-chains and the amino or carboxyl termini. In fact, blockage of the amino or carboxyl group in a polypeptide, or both, by a covalent modification, is common in naturally occurring and synthetic polypeptides and such modifications may be present in polypeptides of the present invention, as well. For instance, the amino terminal residue of polypeptides made in E. coli or other cells, prior to proteolytic processing, almost invariably will be N-formylmethionine. During post-translational modification of the peptide, a methionine residue at the NHZ-terminus may be deleted. Accordingly, this invention contemplates the use of both the methionine-containing and the methionineless amino terminal variants of the protein of the invention.
As used herein "promoter" includes reference to a region of DNA upstream from the start of transcription and involved in recognition and binding of RNA
polymerise and other proteins to initiate transcription. A "plant promoter" is a promoter capable of initiating transcription in plant cells whether or not its origin is a plant cell. Exemplary plant promoters include, but are not limited to, those that are obtained from plants, plant viruses, and bacteria which comprise genes expressed in plant cells such Agrobacterium or Rhizobium. Examples of promoters under developmental control include promoters that preferentially initiate transcription in certain tissues, such as leaves, roots, seeds, fibers, xylem vessels, tracheids, or sclerenchyma. Such promoters are referred to as "tissue preferred". Promoters which initiate transcription only in certain tissue are referred to as "tissue specific". A "cell type" specific promoter primarily drives expression in certain cell types in one or more organs, for example, vascular cells in roots or leaves.
An "inducible"
or "repressible" promoter is a promoter which is under environmental control.
Examples of environmental conditions that may effect transcription by inducible promoters include anaerobic conditions or the presence of light. Tissue specific, tissue preferred, cell type specific, and inducible promoters constitute the class of "non-constitutive"
promoters. A
"constitutive" promoter is a promoter which is active under most environmental conditions.
The term "lignin biosynthesis polypeptide" is a peptide of the present invention and refers to one or more amino acid sequences, in glycosylated or non-glycosylated form. The term is also inclusive of fragments, variants, homologs, alleles or precursors (e.g., preproproteins or proproteins) thereof. A "lignin biosynthesis protein" is a protein of the present invention and comprises a lignin biosynthesis polypeptide.
As used herein "recombinant" includes reference to a cell or vector, that has been modified by the introduction of a heterologous nucleic acid or that the cell is derived from a cell so modified. Thus, for example, recombinant cells express genes that are not found in identical form within the native (non-recombinant) form of the cell or express native genes that are otherwise abnormally expressed, under-expressed or not expressed at all as a result of deliberate human intervention. The term "recombinant" as used herein does not encompass the alteration of the cell or vector by naturally occurring events (e.g., spontaneous mutation, natural transformation/transduction/transposition) such as those occurring without deliberate human intervention.
As used herein, a "recombinant expression cassette" is a nucleic acid construct, generated recombinantly or synthetically, with a series of specified nucleic acid elements which permit transcription of a particular nucleic acid in a target cell. The recombinant expression cassette can be incorporated into a plasmid, chromosome, mitochondrial DNA, plastid DNA, virus, or nucleic acid fragment. Typically, the recombinant expression cassette portion of an expression vector includes, among other sequences, a nucleic acid to be transcribed, and a promoter.
The term "residue" or "amino acid residue" or "amino acid" are used interchangeably herein to refer to an amino acid that is incorporated into a protein, polypeptide, or peptide (collectively "protein"). The amino acid may be a naturally occurnng amino acid and, unless otherwise limited, may encompass known analogs of natural amino acids that can function in a similar manner as naturally occurnng amino acids.
The term "selectively hybridizes" includes reference to hybridization, under stringent hybridization conditions, of a nucleic acid sequence to a specified nucleic acid target sequence to a detectably greater degree (e.g., at least 2-fold over background) than its hybridization to non-target nucleic acid sequences and to the substantial exclusion of non-target nucleic acids. Selectively hybridizing sequences typically have about at least 80% sequence identity, preferably 90% sequence identity, and most preferably 100%
sequence identity (i.e., complementary) with each other.

The terms "stringent conditions" or "stringent hybridization conditions"
includes reference to conditions under which a probe will hybridize to its target sequence, to a detectably greater degree than other sequences (e.g., at least 2-fold over background).
Stringent conditions are sequence-dependent and will be different in different circumstances. By controlling the stringency of the hybridization and/or washing conditions, target sequences can be identified which are 100% complementary to the probe (homologous probing). Alternatively, stringency conditions can be adjusted to allow some mismatching in sequences so that lower degrees of similarity are detected (heterologous probing). Generally, a probe is less than about 1000 nucleotides in length, preferably less than 500 nucleotides in length.
Typically, stringent conditions will be those in which the salt concentration is less than about 1.5 M Na ion, typically about 0.01 to 1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30°C for short probes (e.g., 10 to 50 nucleotides) and at least about 60°C for long probes (e.g., greater than 50 nucleotides). Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide. Exemplary low stringency conditions include hybridization with a buffer solution of 30 to 35% formamide, 1 M NaCI, 1% SDS (sodium dodecyl sulphate) at 37°C, and a wash in 1X to 2X SSC (20X SSC = 3.0 M NaCI/0.3 M trisodium citrate) at 50 to 55°C. Exemplary moderate stringency conditions include hybridization in 40 to 45% formamide, 1 M NaCI, 1% SDS at 37°C, and a wash in 0.5X to 1X
SSC at 55 to 60°C. Exemplary high stringency conditions include hybridization in 50%
formamide, 1 M NaCI, 1% SDS at 37°C, and a wash in O.1X SSC at 60 to 65°C.
Specificity is typically the function of post-hybridization washes, the critical factors being the ionic strength and temperature of the final wash solution. For DNA-DNA
hybrids, the Tm can be approximated from the equation of Meinkoth and Wahl, Anal.
Biochem., 138:267-284 (1984): Tm = 81.5 °C + 16.6 (log M) + 0.41 (%GC) -0.61 (%
form) - 500/L; where M is the molarity of monovalent cations, %GC is the percentage of guanosine and cytosine nucleotides in the DNA, % form is the percentage of formamide in the hybridization solution, and L is the length of the hybrid in base pairs.
The Tm is the temperature (under defined ionic strength and pH) at which 50% of a complementary target sequence hybridizes to a perfectly matched probe. Tm is reduced by about 1 °C for each 1 % of mismatching; thus, Tm, hybridization and/or wash conditions can be adjusted to hybridize to sequences of the desired identity. For example, if sequences with >90%
identity are sought, the Tm can be decreased 10 °C. Generally, stringent conditions are selected to be about 5 °C lower than the thermal melting point (Tm) for the specific sequence and its complement at a defined ionic strength and pH. However, severely S stringent conditions can utilize a hybridization and/or wash at 1, 2, 3, or 4 °C lower than the thermal melting point (Tm); moderately stringent conditions can utilize a hybridization and/or wash at 6, 7, 8, 9, or 10 °C lower than the thermal melting point (Tm); low stringency conditions can utilize a hybridization and/or wash at 11, 12, 13, 14, 15, or 20 °C
lower than the thermal melting point (Tm). Using the equation, hybridization and wash compositions, and desired Tm, those of ordinary skill will understand that variations in the stringency of hybridization and/or wash solutions are inherently described. If the desired degree of mismatching results in a Tm of less than 45 °C (aqueous solution) or 32 °C
(formamide solution) it is preferred to increase the SSC concentration so that a higher temperature can be used. An extensive guide to the hybridization of nucleic acids is found in Tijssen, Laboratory Techniques in Biochemistry and Molecular Biology--Hybridization with Nucleic Acid Probes, Part I, Chapter 2 "Overview of principles of hybridization and the strategy of nucleic acid probe assays", Elsevier, New York (1993); and Current Protocols in Molecular Biology, Chapter 2, Ausubel, et al., Eds., Greene Publishing and Wiley-Interscience, New York (1995).
As used herein, "transgenic plant" includes reference to a plant which comprises within its genome a heterologous polynucleotide. Generally, the heterologous polynucleotide is stably integrated within the genome such that the polynucleotide is passed on to successive generations. The heterologous polynucleotide may be integrated into the genome alone or as part of a recombinant expression cassette.
"Transgenic" is used herein to include any cell, cell line, callus, tissue, plant part or plant, the genotype of which has been altered by the presence of heterologous nucleic acid including those transgenics initially so altered as well as those created by sexual crosses or asexual propagation from the initial transgenic. The term "transgenic" as used herein does not encompass the alteration of the genome (chromosomal or extra-chromosomal) by conventional plant breeding methods or by naturally occurring events such as random cross-fertilization, non-recombinant viral infection, non-recombinant bacterial transformation, non-recombinant transposition, or spontaneous mutation.

As used herein, "vector" includes reference to a nucleic acid used in transfection of a host cell and into which can be inserted a polynucleotide. Vectors are often replicons.
Expression vectors permit transcription of a nucleic acid inserted therein.
The following terms are used to describe the sequence relationships between two or more nucleic acids or polynucleotides: (a) "reference sequence", (b) "comparison window", (c) "sequence identity", (d) "percentage of sequence identity", and (e) "substantial identity".
(a) As used herein, "reference sequence" is a defined sequence used as a basis for sequence comparison. A reference sequence may be a subset or the entirety of a specified sequence; for example, as a segment of a full-length cDNA or gene sequence, or the complete cDNA or gene sequence.
(b) As used herein, "comparison window" means includes reference to a contiguous and specified segment of a polynucleotide sequence, wherein the polynucleotide sequence may be compared to a reference sequence and wherein the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. Generally, the comparison window is at least 20 contiguous nucleotides in length, and optionally can be 30, 40, 50, 100, or longer. Those of skill in the art understand that to avoid a high similarity to a reference sequence due to inclusion of gaps in the polynucleotide sequence a gap penalty is typically introduced and is subtracted from the number of matches.
Methods of alignment of sequences for comparison are well-known in the art.
Optimal alignment of sequences for comparison may be conducted by the local homology algorithm of Smith and Waterman, Adv. Appl. Math. 2: 482 (1981); by the homology alignment algorithm of Needleman and Wunsch, J. Mol. Biol. 48: 443 ( 1970); by the search for similarity method of Pearson and Lipman, Proc. Natl. Acad. Sci. 85:

(1988); by computerized implementations of these algorithms, including, but not limited to: CLUSTAL in the PC/Gene program by Intelligenetics, Mountain View, California, GAP, BESTFIT, BLAST, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group (GCG), 575 Science Dr., Madison, Wisconsin, USA;
the CLUSTAL program is well described by Higgins and Sharp, Gene 73: 237-244 (1988);
Higgins and Sharp, CABIOS 5: 151-153 (1989); Corpet, et al., Nucleic Acids Research 16: 10881-90 (1988); Huang, et al., Computer Applications in the Biosciences 8: 155-65 (1992), and Pearson, et al., Methods in Molecular Biology 24: 307-331 (1994).
The BLAST family of programs which can be used for database similarity searches includes:
BLASTN for nucleotide query sequences against nucleotide database sequences;
BLASTX
for nucleotide query sequences against protein database sequences; BLASTP for protein query sequences against protein database sequences; TBLASTN for protein query sequences against nucleotide database sequences; and TBLASTX for nucleotide query sequences against nucleotide database sequences. See, Current Protocols in Molecular Biology, Chapter 19, Ausubel, et al., Eds., Greene Publishing and Wiley-Interscience, New York (1995).
Unless otherwise stated, sequence identity/similarity values provided herein refer to the value obtained using the BLAST 2Ø1 suite of programs using default parameters.
Altschul et al., Nucleic Acids Res. 25:3389-3402 (1997).
Software for performing BLAST analyses is publicly available, e.g., through the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/).
This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold. These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues;
always > 0) and N (penalty score for mismatching residues; always < 0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score.
Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X
determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 1 l, an expectation (E) of 10, a cutoff of 100, M=5, N=-4, and a comparison of both strands. For amino acid sequences, the BLASTP
program uses as defaults a wordlength (W) of 3, an expectation (E) of 10, and the scoring matrix (see Henikoff & Henikoff (1989) Proc. Natl. Acad. Sci. USA
89:10915).
In addition to calculating percent sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin &
Altschul, Proc. Nat'l. Acad. Sci. USA 90:5873-5877 (1993)). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance.
As those of ordinary skill in the art will understand, BLAST searches assume that proteins can be modeled as random sequences. However, many real proteins comprise regions of nonrandom sequences which may be homopolymeric tracts, short-period repeats, or regions enriched in one or more amino acids. Such low-complexity regions may be aligned between unrelated proteins even though other regions of the protein are entirely dissimilar. A number of low-complexity filter programs can be employed to reduce such low-complexity alignments. For example, the SEG (Wooten and Federhen, Comput. Chem., 17:149-163 (1993)) and XNU (Claverie and States, Comput. Chem., 17:191-201 (1993)) low-complexity filters can be employed alone or in combination.
GAP can also be used to compare a polynucleotide or polypeptide of the present invention with a reference sequence. GAP uses the algorithm of Needleman and Wunsch (J. Mol. Biol. 48: 443-453, 1970) to find the alignment of two complete sequences that maximizes the number of matches and minimizes the number of gaps. GAP
considers all possible alignments and gap positions and creates the alignment with the largest number of matched bases and the fewest gaps. It allows for the provision of a gap creation penalty and a gap extension penalty in units of matched bases. GAP must make a profit of gap creation penalty number of matches for each gap it inserts. If a gap extension penalty greater than zero is chosen, GAP must, in addition, make a profit for each gap inserted of the length of the gap times the gap extension penalty. Default gap creation penalty values and gap extension penalty values in Version 10 of the Wisconsin Genetics Software Package for protein sequences are 8 and 2, respectively. For nucleotide sequences the default gap creation penalty is SO while the default gap extension penalty is 3. The gap creation and gap extension penalties can be expressed as an integer selected from the group of integers consisting of from 0 to 100. Thus, for example, the gap creation and gap extension penalties can each independently be: 0, l, 2, 3, 4, S, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60 or greater.
GAP presents one member of the family of best alignments. There may be many members of this family, but no other member has a better quality. GAP displays four figures of merit for alignments: Quality, Ratio, Identity, and Similarity. The Quality is the metric maximized in order to align the sequences. Ratio is the quality divided by the number of bases in the shorter segment. Percent Identity is the percent of the symbols that actually match. Percent Similarity is the percent of the symbols that are similar. Symbols that are across from gaps are ignored. A similarity is scored when the scoring matrix value for a pair of symbols is greater than or equal to 0.50, the similarity threshold. The scoring matrix used in Version 10 of the Wisconsin Genetics Software Package is (see Henikoff & Henikoff (1989) Proc. Natl. Acad. Sci. USA 89:10915).
(c) As used herein, "sequence identity" or "identity" in the context of two nucleic acid or polypeptide sequences includes reference to the residues in the two sequences which are the same when aligned for maximum correspondence over a specified comparison window. When percentage of sequence identity is used in reference to proteins it is recognized that residue positions which are not identical often differ by conservative amino acid substitutions, where amino acid residues are substituted for other amino acid residues with similar chemical properties (e.g. charge or hydrophobicity) and therefore do not change the functional properties of the molecule. Where sequences differ in conservative substitutions, the percent sequence identity may be adjusted upwards to correct for the conservative nature of the substitution. Sequences which differ by such conservative substitutions are said to have "sequence similarity" or "similarity". Means for making this adjustment are well-known to those of skill in the art. Typically this involves scoring a conservative substitution as a partial rather than a full mismatch, thereby increasing the percentage sequence identity. Thus, for example, where an identical amino acid is given a score of 1 and a non-conservative substitution is given a score of zero, a conservative substitution is given a score between zero and 1. The scoring of conservative substitutions is calculated, e.g., according to the algorithm of Meyers and Miller, Computer Applic. Biol. Sci., 4: 11-17 (1988) e.g., as implemented in the program PC/GENE
(Intelligenetics, Mountain View, California, USA).
(d) As used herein, "percentage of sequence identity" means the value determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity.
(e) (i) The term "substantial identity" of polynucleotide sequences means that a polynucleotide comprises a sequence that has at least 70% sequence identity, preferably at least 80%, more preferably at least 90% and most preferably at least 95%, compared to a reference sequence using one of the alignment programs described using standard parameters. One of skill will recognize that these values can be appropriately adjusted to determine corresponding identity of proteins encoded by two nucleotide sequences by taking into account codon degeneracy, amino acid similarity, reading frame positioning and the like. Substantial identity of amino acid sequences for these purposes normally means sequence identity of at least 60%, more preferably at least 70%, 80%, 90%, and most preferably at least 95%.
Another indication that nucleotide sequences are substantially identical is if two molecules hybridize to each other under stringent conditions. However, nucleic acids which do not hybridize to each other under stringent conditions are still substantially identical if the polypeptides which they encode are substantially identical.
This may occur, e.g., when a copy of a nucleic acid is created using the maximum codon degeneracy permitted by the genetic code. One indication that two nucleic acid sequences are substantially identical is that the polypeptide which the first nucleic acid encodes is immunologically cross reactive with the polypeptide encoded by the second nucleic acid.
(e) (ii) The terms "substantial identity" in the context of a peptide indicates that a peptide comprises a sequence with at least 70% sequence identity to a reference sequence, preferably 80%, more preferably 85%, most preferably at least 90% or 95%
sequence identity to the reference sequence over a specified comparison window.
Preferably, optimal alignment is conducted using the homology alignment algorithm of Needleman and Wunsch, J. Mol. Biol. 48: 443 (1970). An indication that two peptide sequences are substantially identical is that one peptide is immunologically reactive with antibodies raised against the second peptide. Thus, a peptide is substantially identical to a second peptide, for example, where the two peptides differ only by a conservative substitution. Peptides which are "substantially similar" share sequences as noted above except that residue positions which are not identical may differ by conservative amino acid changes.
DETAILED DESCRIPTION OF THE INVENTION
Overview The present invention provides, among other things, compositions and methods for modulating (i.e., increasing or decreasing) the total levels of proteins of the present invention and/or altering their ratios in plants. In particular, the polypeptides of the present invention can be expressed temporally or spatially, e.g., at developmental stages, in tissues, and/or in quantities, which are uncharacteristic of non-recombinantly engineered plants.
Thus, the present invention provides utility in such exemplary applications as improving the digestibility of fodder crops, increasing the value of plant material for pulp and paper production, improving the standability of crops, as well as for improving the utility of plant material where lignin content or composition is important, such as the use of plant lignins as a chemical feedstock, or the use of hyperlignified plant material for use as a fuel source.
The present invention also provides isolated nucleic acid comprising polynucleotides of sufficient length and complementarity to a lignin biosynthesis gene to use as probes or amplification primers in the detection, quantitation, or isolation of gene transcripts. For example, isolated nucleic acids of the present invention can be used as probes in detecting deficiencies in the level of mRNA in screenings for desired transgenic plants, for detecting mutations in the gene (e.g., substitutions, deletions, or additions), for monitoring upregulation of expression or changes in enzyme activity in screening assays of compounds, for detection of any number of allelic variants (polymorphisms), orthologs, or paralogs of the gene, for use as molecular markers in plant breeding programs, or for site directed mutagenesis in eukaryotic cells (see, e.g., U.S. Patent No.
5,565,350). The isolated nucleic acids of the present invention can also be used for recombinant expression of lignin biosynthesis polypeptides, or for use as immunogens in the preparation and/or screening of antibodies. The isolated nucleic acids of the present invention can also be employed for use in sense or antisense suppression of one or more lignin biosynthesis genes in a host cell, tissue, or plant. Attachment of chemical agents which bind, intercalate, cleave and/or crosslink to the isolated nucleic acids of the present invention can also be used to modulate transcription or translation. Further, using a primer specific to an insertion sequence (e.g., transposon) and a primer which specifically hybridizes to an isolated nucleic acid of the present invention, one can use nucleic acid amplification to identity insertion sequence inactivated lignin biosynthesis genes from a cDNA
library prepared from insertion sequence mutagenized plants. Progeny seed from the plants comprising the desired inactivated gene can be grown to a plant to study the phenotypic changes characteristic of that inactivation. See, Tools to Determine the Function of Genes, 1995 Proceedings of the Fiftieth Annual Corn and Sorghum Industry Research Conference, American Seed Trade Association, Washington, D.C., 1995. Additionally, non-translated 5' or 3' regions of the polynucleotides of the present invention can be used to modulate turnover of heterologous mRNAs and/or protein synthesis. Further, the codon preference characteristic of the polynucleotides of the present invention can be employed in heterologous sequences, or altered in homologous or heterologous sequences, to modulate translational level and/or rates.
The present invention also provides isolated proteins comprising polypeptides including an amino acid sequence from the lignin biosynthesis polypeptides (e.g., preproenzyme, proenzyme, or enzymes) as disclosed herein. The present invention also provides proteins comprising at least one epitope from a lignin biosynthesis polypeptide.
The proteins of the present invention can be employed in assays for enzyme agonists or antagonists of enzyme function, or for use as immunogens or antigens to obtain antibodies specifically immunoreactive with a protein of the present invention. Such antibodies can be used in assays for expression levels, for identifying and/or isolating nucleic acids of the present invention from expression libraries, or for purification of lignin biosynthesis polypeptides.
The isolated nucleic acids and polypeptides of the present invention can be used over a broad range of plant types, particularly monocots such as the species of the family Gramineae including Hordeunz, Secale, Triticum, Sorghum (e.g., S. bicolor) and Zea (e.g., Z. mays). The isolated nucleic acid and proteins of the present invention can also be used in species from the genera: Cucurbita, Rosa, Vitis, Juglans, Fragaria, Lotus, Medicago, Onobrychis, Trifolium, Trigonella, Vigna, Citrus, Linum, Geranium, Manihot, Daucus, Arabidopsis, Brassica, Raphanus, Sinapis, Atropa, Capsicum, Datura, Hyoscyamus, Lycopersicon, Nicotiana, Solanum, Petunia, Digitalis, Majorana, Ciahorium, Helianthus, S Lactuca, Bromus, Asparagus, Antirrhinum, Heterocallis, Nemesis, Pelargonium, Panieum, Pennisetum, Ranunculus, Senecio, Salpiglossis, Cucumis, Browallia, Glycine, Pisum, Phaseolus, Lolium, Oryza, and Avena.
Nucleic Acids The present invention provides, among other things, isolated nucleic acids of RNA, DNA, and analogs and/or chimeras thereof, comprising a lignin biosynthesis polynucleotide encoding such enzymes as: ferulate-5-hydroxylase (FSH), caffeoyl-CoA 3-O-methyltransferase (CCoA-OMT), and cinnamyl alcohol dehydrogenase (CAD).
The lignin biosynthesis nucleic acids of the present invention comprise isolated lignin biosynthesis polynucleotides which, are inclusive of:
(a) a polynucleotide encoding a lignin biosynthesis polypeptide of SEQ m NOS: 2, 6, 10 and 14 and conservatively modified and polymorphic variants thereof, including exemplary polynucleotides of SEQ ID NOS: 1, 5, 9 and 13;
(b) a polynucleotide which is the product of amplification from a Zea mays nucleic acid library using primer pairs from amongst the consecutive pairs from SEQ ~
NOS: 3, 7, 11, 15 and 4, 8, 12, 16, which amplify polynucleotides having substantial identity to polynucleotides from amongst those having SEQ >D NOS: 1, 5, 9, and 13 or using primer pairs which selectively hybridize under stringent conditions to loci within a polynucleotide selected from the group consisting of SEQ ID NOS: l, 5, 9, and 13;
(c) a polynucleotide which selectively hybridizes to a polynucleotide of (a) or (b);
(d) a polynucleotide having a specified sequence identity with polynucleotides of (a), (b), or (c);
(e) a polynucleotide encoding a protein having a specified number of contiguous amino acids from a prototype polypeptide, wherein the protein is specifically recognized by antisera elicited by presentation of the protein and wherein the protein does not detectably immunoreact to antisera which has been fully immunosorbed with the protein;
(f) complementary sequences of polynucleotides of (a), (b), (c), (d), or (e);
and (g) a polynucleotide comprising at least a specific number of contiguous nucleotides from a polynucleotide of (a), (b), (c), (d), (e), or (f).
A. Polynucleotides Encoding A Polypeptide of the Present Invention or Conservatively Modified or Polymorphic Variants Thereof As indicated in (a), above, the present invention provides isolated nucleic acids comprising a polynucleotide of the present invention, wherein the polynucleotide encodes a polypeptide of the present invention, or conservatively modified or polymorphic variants thereof. Those of skill in the art will recognize that the degeneracy of the genetic code allows for a plurality of polynucleotides to encode for the identical amino acid sequence.
Such "silent variations" can be used, for example, to selectively hybridize and detect allelic variants of polynucleotides of the present invention. Accordingly, the present invention includes, for example, polynucleotides of SEQ ID NOS: 1, 5, 9, and 13, and silent variations of polynucleotides encoding a polypeptide of SEQ >D NOS: 2, 6, 10, and 14.
Thus, each silent variation of a nucleic acid which encodes a polypeptide of the present invention is implicit in each described polypeptide sequence and is within the scope of the present invention. Accordingly, the present invention includes polynucleotides of SEQ >D
NOS: 1, 5, 9, and 13, and polynucleotides encoding a polypeptide of SEQ ID
NOS: 2, 6, 10, and 14.
Cinnamyl alcohol dehydrogenase (CAD) is coded for by the polypeptide of SEQ >D
NO: 10 which is encoded for by the polynucleotide of SEQ >D NO: 9.
Caffeoyl-CoA 3-O-methyltransferase (CCoA-OMT) is coded for by the polypeptide of SEQ >D NO: 14 which is encoded for by the polynucleotide of SEQ >D NO: 13.
Ferulate-5-hydroxylase (FSH) is coded for by the polypeptides of SEQ >D NOS: 2 and 6 which are encoded for by the polynucleotides of SEQ m NOS: 1 and 5, respectively.
The present invention further provides isolated nucleic acids comprising polynucleotides encoding conservatively modified variants of a polypeptide of SEQ >D
NOS: 2, 6, 10, and 14. Additionally, the present invention further provides isolated nucleic acids comprising polynucleotides encoding one or more allelic (polymorphic) variants of polypeptides/polynucleotides. Polymorphisms are frequently used to follow segregation of chromosomal regions in, for example, marker assisted selection methods for crop improvement.
B. Polynucleotides Amplified from a Zea mays Nucleic Acid Library As indicated in (b), above, the present invention provides an isolated nucleic acid comprising a lignin biosynthesis polynucleotide of the present invention, wherein the polynucleotides are amplified from a Zea mays nucleic acid library. Zea mays lines B73, PHRE1, A632, BMS-P2#10, W23, and Moll are known and publicly available. Other publicly known and available maize lines can be obtained from the Maize Genetics Cooperation (Urbana, IL). The nucleic acid library may be a cDNA library, a genomic library, or a library generally constructed from nuclear transcripts at any stage of intron processing. cDNA libraries can be normalized to increase the representation of relatively rare cDNAs. In preferred embodiments, the cDNA library is constructed mature lignified tissue such as root, leaf, or tassel tissue. The cDNA library can be constructed using a full-length cDNA synthesis method. Examples of such methods include Oligo-Capping (Maruyama, K. and Sugano, S. Gene 138: 171-174, 1994), Biotinylated CAP
Trapper (Carninci, P., Kvan, C., et al. Genomics 37: 327-336, 1996), and CAP Retention Procedure (Edery, E., Chu, L.L., et al. Molecular and Cellular Biology 15: 3363-3371, 1995). cDNA
synthesis is preferably catalyzed at 50-55°C to prevent formation of RNA secondary structure. Examples of reverse transcriptases that are relatively stable at these temperatures are Superscript II Reverse Transcriptase (Life Technologies, Inc.), AMV
Reverse Transcriptase (Boehringer Mannheim) and RetroAmp Reverse Transcriptase (Epicentre).
Rapidly growing tissues, or rapidly dividing cells are preferably used as mRNA
sources.
The polynucleotides of the present invention include those amplified using the following primer pairs:
SEQ ID NOS: 3 and 4 which yield an amplicon comprising a sequence having substantial identity to SEQ ID NO: l;
SEQ ID NOS: 7 and 8 which yield an amplicon comprising a sequence having substantial identity to SEQ ID NO: 5;
SEQ ID NOS: 11 and 12 which yield an amplicon comprising a sequence having substantial identity to SEQ ID NO: 9; and SEQ >D NOS: 15 and 16 which yield an amplicon comprising a sequence having substantial identity to SEQ >D NO: 13.
The present invention also provides subsequences of the polynucleotides of the present invention. A variety of subsequences can be obtained using primers which selectively hybridize under stringent conditions to at least two sites within a polynucleotide of the present invention, or to two sites within the nucleic acid which flank and comprise a polynucleotide of the present invention, or to a site within a polynucleotide of the present invention and a site within the nucleic acid which comprises it. Primers are chosen to selectively hybridize, under stringent hybridization conditions, to a polynucleotide of the present invention. Generally, the primers are complementary to a subsequence of the target nucleic acid which they amplify but may have a sequence identity ranging from about 85%
to 99% relative to the polynucleotide sequence which they are designed to anneal to. As those skilled in the art will appreciate, the sites to which the primer pairs will selectively hybridize are chosen such that a single contiguous nucleic acid can be formed under the desired amplification conditions.
In optional embodiments, the primers will be constructed so that they selectively hybridize under stringent conditions to a sequence (or its complement) within the target nucleic acid which comprises the codon encoding the carboxy or amino terminal amino acid residue (i.e., the 3' terminal coding region and 5' terminal coding region, respectively) of the polynucleotides of the present invention. Optionally within these embodiments, the primers will be constructed to selectively hybridize entirely within the coding region of the target polynucleotide of the present invention such that the product of amplification of a cDNA target will consist of the coding region of that cDNA. The primer length in nucleotides is selected from the group of integers consisting of from at least 15 to 50.
Thus, the primers can be at least 15, 18, 20, 25, 30, 40, or 50 nucleotides in length. Those of skill will recognize that a lengthened primer sequence can be employed to increase specificity of binding (i.e., annealing) to a target sequence. A non-annealing sequence at the 5'end of a primer (a "tail") can be added, for example, to introduce a cloning site at the terminal ends of the amplicon.
The amplification products can be translated using expression systems well known to those of skill in the art and as discussed, infra. The resulting translation products can be confirmed as polypeptides of the present invention by, for example, assaying for the appropriate catalytic activity (e.g., specific activity and/or substrate specificity), or verifying the presence of one or more linear epitopes which are specific to a polypeptide of the present invention. Methods for protein synthesis from PCR derived templates are known in the art and available commercially. See, e.g., Amersham Life Sciences, Inc, Catalog '97, p.354.
Methods for obtaining 5' and/or 3' ends of a vector insert are well known in the art.
See, e.g., RACE (Rapid Amplification of Complementary Ends) as described in Frohman, M. A., in PCR Protocols: A Guide to Methods and Applications, M. A. Innis, D.
H.
Gelfand, J. J. Sninsky, T. J. White, Eds. (Academic Press, Inc., San Diego), pp. 28-38 (1990)); see also, U.S. Pat. No. 5,470,722, and Current Protocols in Molecular Biology, Unit 15.6, Ausubel, et al., Eds., Greene Publishing and Wiley-Interscience, New York (1995); Frohman and Martin, Techniques 1:165 (1989).
C. Polynucleotides Which Selectively Hybridize to a Polynucleotide of (A) or (B) As indicated in (c), above, the present invention provides isolated nucleic acids comprising polynucleotides of the present invention, wherein the polynucleotides selectively hybridize, under selective hybridization conditions, to a polynucleotide of paragraphs (A) or (B) as discussed above. Thus, the polynucleotides of this embodiment can be used for isolating, detecting, and/or quantifying nucleic acids comprising the polynucleotides of (A) or (B). For example, polynucleotides of the present invention can be used to identify, isolate, or amplify partial or full-length clones in a deposited library. In some embodiments, the polynucleotides are genomic or cDNA sequences isolated or otherwise complementary to a cDNA from a dicot or monocot nucleic acid library.
Exemplary species of monocots and dicots include, but are not limited to:
maize, canola, soybean, cotton, wheat, sorghum, sunflower, alfalfa, oats, sugar cane, millet, barley, and rice. Optionally, the cDNA library comprises at least 30% to 95% full-length sequences (for example, at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% full-length sequences). The cDNA libraries can be normalized to increase the representation of rare sequences. Low stringency hybridization conditions are typically, but not exclusively, employed with sequences having a reduced sequence identity relative to complementary sequences. Moderate and high stringency conditions can optionally be employed for sequences of greater identity. Low stringency conditions allow selective hybridization of sequences having about 70% to 80% sequence identity and can be employed to identify orthologous or paralogous sequences.
D. Polynucleotides Having a Specifac Sequence Identity with the Polynucleotides of (A), (B) or (C) As indicated in (d), above, the present invention provides isolated nucleic acids comprising polynucleotides of the present invention, wherein the polynucleotides have a specified identity at the nucleotide level to a polynucleotide as disclosed above in paragraphs (A), (B), or (C). Identity can be calculated using, for example, the BLAST of GAP algorithms under default conditions. The percentage of identity to a reference sequence is at least 60% and, rounded upwards to the nearest integer, can be expressed as an integer selected from the group of integers consisting of from 60 to 99.
Thus, for example, the percentage of identity to a reference sequence can be at least 70%, 75%, 80%, 85%, 90%, or 95%.
Optionally, the polynucleotides of this embodiment will encode a polypeptide that will share an epitope with a polypeptideencoded by the polynucleotides of sections (A), (B), or (C). Thus, these polynucleotides encode a first polypeptide which elicits production of antisera comprising antibodies which are specifically reactive to a second polypeptide encoded by a polynucleotide of (A), (B), or (C). However, the first polypeptide does not bind to antisera raised against itself when the antisera has been fully immunosorbed with the first polypeptide. Hence, the polynucleotides of this embodiment can be used to generate antibodies for use in, for example, the screening of expression libraries for nucleic acids comprising polynucleotides of (A), (B), or (C), or for purification of, or in immunoassays for, polypeptides encoded by the polynucleotides of (A), (B), or (C). The polynucleotides of this embodiment embrace nucleic acid sequences which can be employed for selective hybridization to a polynucleotide encoding a polypeptide of the present invention.
Screening polypeptides for specific binding to antisera can be conveniently achieved using peptide display libraries. This method involves the screening of large collections of peptides for individual members having the desired function or structure.
Antibody screening of peptide display libraries is well known in the art. The displayed peptide sequences can be from 3 to 5000 or more amino acids in length, frequently from 5-100 amino acids long, and often from about 8 to 15 amino acids long. In addition to direct chemical synthetic methods for generating peptide libraries, several recombinant DNA
methods have been described. One type involves the display of a peptide sequence on the surface of a bacteriophage or cell. Each bacteriophage or cell contains the nucleotide sequence encoding the particular displayed peptide sequence. Such methods are described in PCT patent publication Nos. 91/17271, 91/18980, 91/19818, and 93/08278.
Other systems for generating libraries of peptides have aspects of both in vitro chemical synthesis and recombinant methods. See, PCT Patent publication Nos. 92/05258, 92/14843, and 96/19256. See also, U.S. Patent Nos. 5,658,754; and 5,643,768. Peptide display libraries, vectors, and screening kits are commercially available from such suppliers as Invitrogen (Carlsbad, CA).
E. Polynucleotides Encoding a Protein Having a Subsequence from a Prototype Polypeptide and is Cross-Reactive to the Prototype Polypeptide As indicated in (e), above, the present invention provides isolated nucleic acids comprising polynucleotides of the present invention, wherein the polynucleotides encode a protein having a subsequence of contiguous amino acids from a prototype lignin biosynthesis polypeptide of the present invention such as are provided in (a), above. The length of contiguous amino acids from the prototype polypeptide is selected from the group of integers consisting of from at least 10 to the number of amino acids within the prototype sequence. Thus, for example, the polynucleotide can encode a polypeptide having a subsequence having at least 10, 15, 20, 25, 30, 35, 40, 45, or 50, contiguous amino acids from the prototype polypeptide. Further, the number of such subsequences encoded by a polynucleotide of the instant embodiment can be any integer selected from the group consisting of from 1 to 20, such as 2, 3, 4, or 5. The subsequences can be separated by any integer of nucleotides from 1 to the number of nucleotides in the sequence such as at least 5, 10, 15, 25, 50, 100, or 200 nucleotides.
The proteins encoded by polynucleotides of this embodiment, when presented as an immunogen, elicit the production of polyclonal antibodies which specifically bind to a prototype polypeptide such as, but not limited to, a polypeptide encoded by the polynucleotide of (a) or (b), above. Generally, however, a protein encoded by a polynucleotide of this embodiment does not bind to antisera raised against the prototype polypeptide when the antisera has been fully immunosorbed with the prototype polypeptide. Methods of making and assaying for antibody binding specificity/affinity are well known in the art. Exemplary immunoassay formats include ELISA, competitive immunoassays, radioimmunoassays, Western blots, indirect immunofluorescent assays and the like.
In a preferred assay method, fully immunosorbed and pooled antisera which is elicited to the prototype polypeptide can be used in a competitive binding assay to test the protein. The concentration of the prototype polypeptide required to inhibit 50% of the binding of the antisera to the prototype polypeptide is determined. If the amount of the protein required to inhibit binding is less than twice the amount of the prototype protein, then the protein is said to specifically bind to the antisera elicited to the immunogen.
Accordingly, the proteins of the present invention embrace allelic variants, conservatively modified variants, and minor recombinant modifications to a prototype polypeptide.
A polynucleotide of the present invention optionally encodes a protein having a molecular weight as the non-glycosylated protein within 20% of the molecular weight of the full-length non-glycosylated polypeptides of the present invention..
Molecular weight can be readily determined by SDS-PAGE under reducing conditions. Preferably, the molecular weight is within 15% of a full length lignin biosynthesis polypeptide, more preferably within 10% or S%, and most preferably within 3%, 2%, or 1 % of a full length polypeptide of the present invention.
Optionally, the polynucleotides of this embodiment will encode a protein having a specific enzymatic activity at least 50%, 60%, 80%, or 90% of a celluler extract comprising the native, endogenous, full-length polypeptide of the present invention.
Further, the proteins encoded by polynucleotides of this embodiment will optionally have a substantially similar affinity constant (Km ) and/or catalytic activity (i.e., the microscopic rate constant, k~at) as the native endogenous, full-length protein. Those of skill in the art will recognize that k°at/Kr" value determines the specificity for competing substrates and is often referred to as the specificity constant. Proteins of this embodiment can have a k~at/Kr" value at least 10% of a full-length lignin biosynthesis polypeptide of the present invention as determined using the endogenous substrate of that polypeptide.
Optionally, the k~at/K", value will be at least 20%, 30%, 40%, 50%, and most preferably at least 60%, 70%, 80%, 90%, or 95% the k~a,/Km value of the full-length polypeptide of the present invention. Determination of k~at, Km , and k~at/K,T, can be determined by any number of means well known to those of skill in the art. For example, the initial rates (i.e., the first 5% or less of the reaction) can be determined using rapid mixing and sampling techniques (e.g., continuous-flow, stopped-flow, or rapid quenching techniques), flash photolysis, or relaxation methods (e.g., temperature jumps) in conjunction with such exemplary methods of measuring as spectrophotometry, spectrofluorimetry, nuclear magnetic resonance, or radioactive procedures. Kinetic values are conveniently obtained using a Lineweaver-Burk or Eadie-Hofstee plot.
F. Polynucleotides Complementary to the Polynucleotides of (A)-(E) As indicated in (f), above, the present invention provides isolated nucleic acids comprising polynucleotides complementary to the polynucleotides of paragraphs A-E, above. As those of skill in the art will recognize, complementary sequences base-pair throughout the entirety of their length with the polynucleotides of sections (A)-(E) (i.e., have 100% sequence identity over their entire length). Complementary bases associate through hydrogen bonding in double stranded nucleic acids. For example, the following base pairs are complementary: guanine and cytosine; adenine and thymine; and adenine and uracil.
G. Polynucleotides Which are Subsequences of the Polynucleotides of (A)-(F) As indicated in (g), above, the present invention provides isolated nucleic acids comprising lignin biosynthesis polynucleotides which comprise at least 15 contiguous bases from the polynucleotides of sections (A) through (F) as discussed above.
The length of the polynucleotide is given as an integer selected from the group consisting of from at least 15 to the length of the nucleic acid sequence from which the polynucleotide is a subsequence of. Thus, for example, polynucleotides of the present invention are inclusive of polynucleotides comprising at least 15, 20, 25, 30, 40, 50, 60, 75, or 100 contiguous nucleotides in length from the polynucleotides of (A)-(F). Optionally, the number of such subsequences encoded by a polynucleotide of the instant embodiment can be any integer selected from the group consisting of from 1 to 20, such as 2, 3, 4, or 5. The subsequences can be separated by any integer of nucleotides from 1 to the number of nucleotides in the sequence such as at least 5, 10, 15, 25, 50, 100, or 200 nucleotides.

The subsequences of the present invention can comprise structural characteristics of the sequence from which it is derived. Alternatively, the subsequences can lack certain structural characteristics of the larger sequence from which it is derived.
For example, a subsequence from a polynucleotide encoding a polypeptide having at least one linear epitope in common with a prototype sequence as provided in (a) above, may encode an epitope in common with the prototype sequence. Alternatively, the subsequence may not encode an epitope in common with the prototype sequence but can be used to isolate the larger sequence by, for example, nucleic acid hybridization with the sequence from which it is derived. Subsequences can be used to modulate or detect gene expression by introducing into the subsequences compounds which bind, intercalate, cleave and/or crosslink to nucleic acids. Exemplary compounds include acridine, psoralen, phenanthroline, naphthoquinone, daunomycin or chloroethylaminoaryl conjugates.
Construction of Nucleic Acids The isolated nucleic acids of the present invention can be made using (a) standard recombinant methods, (b) synthetic techniques, or combinations thereof. In some embodiments, the polynucleotides of the present invention will be cloned, amplified, or otherwise constructed from a monocot. In preferred embodiments the monocot is Zea mays. Particularly preferred is the use of Zea mays tissue from root, leaf, or tassel.
The nucleic acids may conveniently comprise sequences in addition to a polynucleotide of the present invention. For example, a multi-cloning site comprising one or more endonuclease restriction sites may be inserted into the nucleic acid to aid in isolation of the polynucleotide. Also, translatable sequences may be inserted to aid in the isolation of the translated polynucleotide of the present invention. For example, a hexa-histidine marker sequence provides a convenient means to purify the proteins of the present invention. A polynucleotide of the present invention can be attached to a vector, adapter, or linker for cloning and/or expression of a polynucleotide of the present invention. Additional sequences may be added to such cloning and/or expression sequences to optimize their function in cloning and/or expression, to aid in isolation of the polynucleotide, or to improve the introduction of the polynucleotide into a cell. Typically, the length of a nucleic acid of the present invention less the length of its polynucleotide of the present invention is less than 20 kilobase pairs, often less than 15 kb, and frequently less than 10 kb. Use of cloning vectors, expression vectors, adaptors, and linkers is well known in the art. Exemplary nucleic acids include such vectors as: M13, lambda ZAP
Express, lambda ZAP II, lambda gtl0, lambda gtl l, pBK-CMV, pBK-RSV, pBluescript II, lambda DASH II, lambda EMBL 3, lambda EMBL 4, pWElS, SuperCos 1, SurfLap, Uni-ZAP, pBC, pBS+/-, pSGS, pBK, pCR-Script, pET, pSPUTK, p3'SS, pOPRSVI CAT, pOPI3 CAT, pXTl, pSGS, pPbac, pMbac, pMClneo, pOG44, pOG45, pFRT(3GAL, pNEO/3GAL, pRS403, pRS404, pRS405, pRS406, pRS413, pRS414, pRS415, pRS416, lambda MOSSIox, and lambda MOSEIox. For a description of various nucleic acids see, for example, Stratagene Cloning Systems, Catalogs 1995, 1996, 1997 (La Jolla, CA); and, Amersham Life Sciences, Inc, Catalog '97 (Arlington Heights, IL).
A. Recombinant Methods for Constructing Nucleic Acids The isolated nucleic acid compositions of this invention, such as RNA, cDNA, genomic DNA, or a hybrid thereof, can be obtained from plant biological sources using any number of cloning methodologies known to those of skill in the art. In some embodiments, oligonucleotide probes which selectively hybridize, under stringent conditions, to the polynucleotides of the present invention are used to identify the desired sequence in a cDNA or genomic DNA library. Isolation of RNA, and construction of cDNA and genomic libraries is well known to those of ordinary skill in the art. See, e.g., Plant Molecular Biology: A Laboratory Manual, Clark, Ed., Springer-Verlag, Berlin (1997);
and, Current Protocols in Molecular Biology, Ausubel, et al., Eds., Greene Publishing and Wiley-Interscience, New York (1995).
The following highlights some of the methods employed.
Al. mRNA Isolation and Purification Total RNA from plant cells comprises such nucleic acids as mitochondria) RNA, chloroplastic RNA, rRNA, tRNA, hnRNA and mRNA. Total RNA preparation typically involves lysis of cells and removal of proteins, followed by precipitation of nucleic acids.
Extraction of total RNA from plant cells can be accomplished by a variety of means.
Frequently, extraction buffers include a strong detergent such as SDS and an organic denaturant such as guanidinium isothiocyanate, guanidine hydrochloride or phenol.
Following total RNA isolation, poly(A)+ mRNA is typically purified from the remainder RNA using oligo(dT) cellulose. Exemplary total RNA and mRNA isolation protocols are described in Plant Molecular Biology: A Laboratory Manual, Clark, Ed., Springer-Verlag, Berlin (1997); and, Current Protocols in Molecular Biology, Ausubel, et al., Eds., Greene Publishing and Wiley-Interscience, New York (1995). Total RNA and mRNA
isolation kits are commercially available from vendors such as Stratagene (La Jolla, CA), Clonetech (Palo Alto, CA), Pharmacia (Piscataway, NJ), and 5'-3' (Paoli, PA). See also, U.S. Patent Nos. 5,614,391; and, 5,459,253. The mRNA can be fractionated into populations with size ranges of about 0.5, 1.0, 1.5, 2.0, 2.5 or 3.0 kb. The cDNA synthesized for each of these fractions can be size selected to the same size range as its mRNA prior to vector insertion.
This method helps eliminate truncated cDNA formed by incompletely reverse transcribed mRNA.
A2. Construction of a cDNA Library Construction of a cDNA library generally entails five steps. First, first strand cDNA synthesis is initiated from a poly(A)+ mRNA template using a poly(dT) primer or random hexanucleotides. Second, the resultant RNA-DNA hybrid is converted into double stranded cDNA, typically by a combination of RNAse H and DNA polymerase I (or Klenow fragment). Third, the termini of the double stranded cDNA are ligated to adaptors.
Ligation of the adaptors will produce cohesive ends for cloning. Fourth, size selection of the double stranded cDNA eliminates excess adaptors and primer fragments, and eliminates partial cDNA molecules due to degradation of mRNAs or the failure of reverse transcriptase to synthesize complete first strands. Fifth, the cDNAs are ligated into cloning vectors and packaged. cDNA synthesis protocols are well known to the skilled artisan and are described in such standard references as: Plant Molecular Biology: A
Laboratory Manual, Clark, Ed., Springer-Verlag, Berlin (1997); and, Current Protocols in Molecular Biology, Ausubel, et al., Eds., Greene Publishing and Wiley-Interscience, New York (1995). cDNA synthesis kits are available from a variety of commercial vendors such as:
Stratagene, and Pharmacia.
A number of cDNA synthesis protocols have been described which provide substantially pure full-length cDNA libraries. Substantially pure full-length cDNA
libraries are constructed to comprise at least 90%, and more preferably at least 93% or 95%
full-length inserts amongst clones containing inserts. The length of insert in such libraries can be from 0 to 8, 9, 10, 11, 12, 13, or more kilobase pairs. Vectors to accommodate inserts of these sizes are known in the art and available commercially. See, e.g., Stratagene's lambda ZAP Express (cDNA cloning vector with 0 to 12 kb cloning capacity).
An exemplary method of constructing a greater than 95% pure full-length cDNA
library is described by Carninci et al., Genomics, 37:327-336 (1996). Other methods for producing full-length libraries are known in the art. See, e.g., Edery et al., Mol. Cell Biol.,l5(6):3363-3371 (1995); and, PCT Application WO 96/34981.
A3. Normalized or Subtracted cDNA Libraries A non-normalized cDNA library represents the mRNA population of the tissue it was made from. Since unique clones are out-numbered by clones derived from highly expressed genes their isolation can be laborious. Normalization of a cDNA
library is the process of creating a library in which each clone is more equally represented.
A number of approaches to normalize cDNA libraries are known in the art. One approach is based on hybridization to genomic DNA. The frequency of each hybridized cDNA in the resulting normalized library would be proportional to that of each corresponding gene in the genomic DNA. Another approach is based on kinetics.
If cDNA reannealing follows second-order kinetics, rarer species anneal less rapidly and the remaining single-stranded fraction of cDNA becomes progressively more normalized during the course of the hybridization. Specific loss of any species of cDNA, regardless of its abundance, does not occur at any Cot value. Construction of normalized libraries is described in Ko, Nucl. Acids. Res., 18(19):5705-5711 (1990); Patanjali et al., Proc. Natl.
Acad. U.S.A., 88:1943-1947 (1991); U.S. Patents 5,482,685, and 5,637,685. In an exemplary method described by Soares et al., normalization resulted in reduction of the abundance of clones from a range of four orders of magnitude to a narrow range of only 1 order of magnitude. Proc. Natl. Acad. Sci. USA, 91:9228-9232 (1994).
Subtracted cDNA libraries are another means to increase the proportion of less abundant cDNA species. In this procedure, cDNA prepared from one pool of mRNA
is depleted of sequences present in a second pool of mRNA by hybridization. The cDNA:mRNA hybrids are removed and the remaining un-hybridized cDNA pool is enriched for sequences unique to that pool. See, Foote et al. in, Plant Molecular Biology:
A Laboratofy Manual, Clark, Ed., Springer-Verlag, Berlin (1997); Kho and Zarbl, Technique, 3(2):58-63 (1991); Sive and St. John, Nucl. Acids Res., 16(22):10937 (1988);
Current Protocols in Molecular Biology, Ausubel, et al., Eds., Greene Publishing and Wiley-Interscience, New York (1995); and, Swaroop et al., Nucl. Acids Res., 19)8):1954 (1991). cDNA subtraction kits are commercially available. See, e.g., PCR-Select (Clontech, Palo Alto, CA).
A4. Construction of a Genomic Library To construct genomic libraries, large segments of genomic DNA are generated by random fragmentation, e.g. using restriction endonucleases, and are ligated with vector DNA to form concatemers that can be packaged into the appropriate vector.
Methodologies to accomplish these ends, and sequencing methods to verify the sequence of nucleic acids are well known in the art. Examples of appropriate molecular biological techniques and instructions sufficient to direct persons of skill through many construction, cloning, and screening methodologies are found in Sambrook, et al., Molecular Cloning: A
Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Vols. 1-3 (1989), Methods in Enzymology, Vol. 152: Guide to Molecular Cloning Techniques, Berger and Kimmel, Eds., San Diego: Academic Press, Inc. (1987), Current Protocols in Molecular Biology, Ausubel, et al., Eds., Greene Publishing and Wiley-Interscience, New York (1995); Plant Molecular Biology: A Laboratory Manual, Clark, Ed., Springer-Verlag, Berlin (1997).
Kits for construction of genomic libraries are also commercially available.
A5. Nucleic Acid Screening and Isolation Methods The cDNA or genomic library can be screened using a probe based upon the sequence of a polynucleotide of the present invention such as those disclosed herein.
Probes may be used to hybridize with genomic DNA or cDNA sequences to isolate homologous genes in the same or different plant species. Those of skill in the art will appreciate that various degrees of stringency of hybridization can be employed in the assay; and either the hybridization or the wash medium can be stringent. As the conditions for hybridization become more stringent, there must be a greater degree of complementarity between the probe and the target for duplex formation to occur. The degree of stringency can be controlled by temperature, ionic strength, pH and the presence of a partially denaturing solvent such as formamide. For example, the stringency of hybridization is conveniently varied by changing the polarity of the reactant solution through manipulation of the concentration of formamide within the range of 0%
to 50%.
The degree of complementarity (sequence identity) required for detectable binding will vary in accordance with the stringency of the hybridization medium and/or wash medium.
The degree of complementarity will optimally be 100 percent; however, it should be understood that minor sequence variations in the probes and primers may be compensated for by reducing the stringency of the hybridization and/or wash medium.
The nucleic acids of interest can also be amplified from nucleic acid samples using amplification techniques. For instance, polymerase chain reaction (PCR) technology can be used to amplify the sequences of polynucleotides of the present invention and related genes directly from genomic DNA or cDNA libraries. PCR and other in vitro amplification methods may also be useful, for example, to clone nucleic acid sequences that code for proteins to be expressed, to make nucleic acids to use as probes for detecting the presence of the desired mRNA in samples, for nucleic acid sequencing, or for other purposes. Examples of techniques sufficient to direct persons of skill through in vitro amplification methods are found in Bergen Sambrook, and Ausubel, as well as Mullis et al., U.S. Patent No. 4,683,202 (1987); and, PCR Protocols A Guide to Methods and Applications, Innis et al., Eds., Academic Press Inc., San Diego, CA (1990).
Commercially available kits for genomic PCR amplification are known in the art. See, e.g., Advantage-GC Genomic PCR Kit (Clontech). The T4 gene 32 protein (Boehringer Mannheim) can be used to improve yield of long PCR products.
PCR-based screening methods have also been described. Wilfmger et al. describe a PCR-based method in which the longest cDNA is identified in the first step so that incomplete clones can be eliminated from study. BioTechniques, 22(3): 481-486 (1997).
In that method, a primer pair is synthesized with one primer annealing to the 5' end of the sense strand of the desired cDNA and the other primer to the vector. Clones are pooled to allow large-scale screening. By this procedure, the longest possible clone is identified amongst candidate clones. Further, the PCR product is used solely as a diagnostic for the presence of the desired cDNA and does not utilize the PCR product itself. Such methods are particularly effective in combination with a full-length cDNA construction methodology, above.

B. Synthetic Methods for Constructing Nucleic Acids The isolated nucleic acids of the present invention can also be prepared by direct chemical synthesis by methods such as the phosphotriester method of Narang et al., Meth.
Enzymol. 68: 90-99 (1979); the phosphodiester method of Brown et al., Meth.
Enzymol.
68: 109-151 (1979); the diethylphosphoramidite method of Beaucage et al., Tetra. Lett. 22:
1859-1862 (1981); the solid phase phosphoramidite triester method described by Beaucage and Caruthers, Tetra. Letts. 22(20): 1859-1862 (1981), e.g., using an automated synthesizer, e.g., as described in Needham-VanDevanter et al., Nucleic Acids Res., 12:
6159-6168 (1984); and, the solid support method of U.S. Patent No. 4,458,066.
Chemical synthesis generally produces a single stranded oligonucleotide. This may be converted into double stranded DNA by hybridization with a complementary sequence, or by polymerization with a DNA polymerise using the single strand as a template.
One of skill will recognize that while chemical synthesis of DNA is limited to sequences of about 100 bases, longer sequences may be obtained by the ligation of shorter sequences.
Recombinant Expression Cassettes The present invention further provides recombinant expression cassettes comprising a nucleic acid of the present invention. A nucleic acid sequence coding for the desired polynucleotide of the present invention, for example a cDNA or a genomic sequence encoding a full length polypeptide of the present invention, can be used to construct a recombinant expression cassette which can be introduced into the desired host cell. A recombinant expression cassette will typically comprise a polynucleotide of the present invention operably linked to transcriptional initiation regulatory sequences which will direct the transcription of the polynucleotide in the intended host cell, such as tissues of a transformed plant.
For example, plant expression vectors may include (1) a cloned plant gene under the transcriptional control of 5' and 3' regulatory sequences and (2) a dominant selectable marker. Such plant expression vectors may also contain, if desired, a promoter regulatory region (e.g., one confernng inducible or constitutive, environmentally- or developmentally-regulated, or cell- or tissue-specific/selective expression), a transcription initiation start site, a ribosome binding site, an RNA processing signal, a transcription termination site, andlor a polyadenylation signal.
A plant promoter fragment can be employed which will direct expression of a polynucleotide of the present invention in all tissues of a regenerated plant.
Such promoters are referred to herein as "constitutive" promoters and are active under most environmental conditions and states of development or cell differentiation.
Examples of constitutive promoters include the cauliflower mosaic virus (CaMV) 35S
transcription initiation region, the 1'- or 2'- promoter derived from T-DNA of Agrobacterium tumefaciens, the ubiquitin 1 promoter, the Smas promoter, the cinnamyl alcohol dehydrogenase promoter (U.5. Patent No. 5,683,439), the Nos promoter, the pEmu promoter, the rubisco promoter, the GRPl-8 promoter, and other transcription initiation regions from various plant genes known to those of skill.
Alternatively, the plant promoter can direct expression of a polynucleotide of the present invention in a specific tissue or may be otherwise under more precise environmental or developmental control. Such promoters are referred to here as "inducible" promoters. Environmental conditions that may effect transcription by inducible promoters include pathogen attack, anaerobic conditions, or the presence of light.
Examples of inducible promoters are the Adhl promoter which is inducible by hypoxia or cold stress, the Hsp70 promoter which is inducible by heat stress, and the PPDK promoter which is inducible by light.
Examples of promoters under developmental control include promoters that initiate transcription only, or preferentially, in certain tissues, such as leaves, roots, fruit, seeds, or flowers. Exemplary promoters include the anther specific promoter S 126 (U.5.
Patent Nos.
5,689,049 and 5,689,051), glob-1 promoter, and gamma-zero promoter. The operation of a promoter may also vary depending on its location in the genome. Thus, an inducible promoter may become fully or partially constitutive in certain locations.
Both heterologous and non-heterologous (i.e., endogenous) promoters can be employed to direct expression of the nucleic acids of the present invention.
These promoters can also be used, for example, in recombinant expression cassettes to drive expression of antisense nucleic acids to reduce, increase, or alter lignin biosynthesis content and/or composition in a desired tissue. Thus, in some embodiments, the nucleic acid construct will comprise a promoter functional in a plant cell, such as in Zea mays, operably linked to a polynucleotide of the present invention. Promoters useful in these embodiments include the endogenous promoters driving expression of a polypeptide of the present invention.
In some embodiments, isolated nucleic acids which serve as promoter or enhancer elements can be introduced in the appropriate position (generally upstream) of a non-heterologous form of a polynucleotide of the present invention so as to up or down regulate expression of a polynucleotide of the present invention. For example, endogenous promoters can be altered in vivo by mutation, deletion, and/or substitution (see, Kmiec, U.S. Patent 5,565,350; Zarling et al., PCT/LJS93/03868), or isolated promoters can be introduced into a plant cell in the proper orientation and distance from a lignin biosynthesis gene so as to control the expression of the gene. Gene expression can be modulated under conditions suitable for plant growth so as to alter lignin biosynthesis content and/or composition. Thus, the present invention provides compositions, and methods for making, heterologous promoters and/or enhancers operably linked to a native, endogenous (i.e., non-heterologous) form of a polynucleotide of the present invention.
Methods for identifying promoters with a particular expression pattern, in terms of, e.g., tissue type, cell type, stage of development, and/or environmental conditions, are well known in the art. See, e.g., The Maize Handbook, Chapters 114-115, Freeling and Walbot, Eds., Springer, New York (1994); Corn and Corn Improvement, 3rd edition, Chapter 6, Sprague and Dudley, Eds., American Society of Agronomy, Madison, Wisconsin (1988).
A typical step in promoter isolation methods is identification of gene products that are expressed with some degree of specificity in the target tissue. Amongst the range of methodologies are: differential hybridization to cDNA libraries; subtractive hybridization;
differential display; differential 2-D gel electrophoresis; DNA probe arrays;
and isolation of proteins known to be expressed with some specificity in the target tissue.
Such methods are well known to those of skill in the art. Commercially available products for identifying promoters are known in the art such as Clontech's (Palo Alto, CA) Universal GenomeWalker Kit.
For the protein-based methods, it is helpful to obtain the amino acid sequence for at least a portion of the identified protein, and then to use the protein sequence as the basis for preparing a nucleic acid that can be used as a probe to identify either genomic DNA
directly, or preferably, to identify a cDNA clone from a library prepared from the target tissue. Once such a cDNA clone has been identified, that sequence can be used to identify the sequence at the 5' end of the transcript of the indicated gene. For differential hybridization, subtractive hybridization and differential display, the nucleic acid sequence identified as enriched in the target tissue is used to identify the sequence at the 5' end of the transcript of the indicated gene. Once such sequences are identified, starting either from protein sequences or nucleic acid sequences, any of these sequences identified as being from the gene transcript can be used to screen a genomic library prepared from the target organism. Methods for identifying and confirming the transcriptional start site are well known in the art.
In the process of isolating promoters expressed under particular environmental conditions or stresses, or in specific tissues, or at particular developmental stages, a number of genes are identified that are expressed under the desired circumstances, in the desired tissue, or at the desired stage. Further analysis will reveal expression of each particular gene in one or more other tissues of the plant. One can identify a promoter with activity in the desired tissue or condition but that do not have activity in any other common tissue.
To identify the promoter sequence, the 5' portions of the clones described here are analyzed for sequences characteristic of promoter sequences. For instance, promoter sequence elements include the TATA box consensus sequence (TATAAT), which is usually an AT-rich stretch of 5-10 by located approximately 20 to 40 base pairs upstream of the transcription start site. Identification of the TATA box is well known in the art. For example, one way to predict the location of this element is to identify the transcription start site using standard RNA-mapping techniques such as primer extension, S 1 analysis, and/or RNase protection. To confirm the presence of the AT-rich sequence, a structure-function analysis can be performed involving mutagenesis of the putative region and quantification of the mutation's effect on expression of a linked downstream reporter gene.
See, e.g., The Maize Handbook, Chapter 114, Freeling and Walbot, Eds., Springer, New York, (1994).
In plants, further upstream from the TATA box, at positions -80 to -100, there is typically a promoter element (i.e., the CAAT box) with a series of adenines surrounding the trinucleotide G (or T) N G. J. Messing et al., in Genetic Engineering in Plants, Kosage, Meredith and Hollaender, Eds., pp. 221-227 1983. In maize, there is no well conserved CART box but there are several short, conserved protein-binding motifs upstream of the TATA box. These include motifs for the trans-acting transcription factors involved in light regulation, anaerobic induction, hormonal regulation, or anthocyanin biosynthesis, as appropriate for each gene.
Once promoter and/or gene sequences are known, a region of suitable size is selected from the genomic DNA that is 5' to the transcriptional start, or the translational S start site, and such sequences are then linked to a coding sequence. If the transcriptional start site is used as the point of fusion, any of a number of possible 5' untranslated regions can be used in between the transcriptional start site and the partial coding sequence. If the translational start site at the 3' end of the specific promoter is used, then it is linked directly to the methionine start codon of a coding sequence.
If polypeptide expression is desired, it is generally desirable to include a polyadenylation region at the 3'-end of a polynucleotide coding region. The polyadenylation region can be derived from the natural gene, from a variety of other plant genes, or from T-DNA. The 3' end sequence to be added can be derived from, for example, the nopaline synthase or octopine synthase genes, or alternatively from another plant gene, or less preferably from any other eukaryotic gene.
An intron sequence can be added to the 5' untranslated region or the coding sequence of the partial coding sequence to increase the amount of the mature message that accumulates in the cytosol. Inclusion of a spliceable intron in the transcription unit in both plant and animal expression constructs has been shown to increase gene expression at both the mRNA and protein levels up to 1000-fold. Buchman and Berg, Mol. Cell Biol.
8: 4395-4405 (1988); Callis et al., Genes Dev. 1: 1183-1200 (1987). Such intron enhancement of gene expression is typically greatest when placed near the 5' end of the transcription unit.
Use of maize introns Adhl-S intron 1, 2, and 6, the Bronze-1 intron are known in the art.
See generally, The Maize Handbook, Chapter 116, Freeling and Walbot, Eds., Springer, New York (1994).
The vector comprising the sequences from a polynucleotide of the present invention will typically comprise a marker gene which confers a selectable phenotype on plant cells. Usually, the selectable marker gene will encode antibiotic resistance, with suitable genes including genes coding for resistance to the antibiotic spectinomycin (e.g., the aada gene), the streptomycin phosphotransferase (SPT) gene coding for streptomycin resistance, the neomycin phosphotransferase (NPTII) gene encoding kanamycin or geneticin resistance, the hygromycin phosphotransferase (HPT) gene coding for hygrornycin resistance, genes coding for resistance to herbicides which act to inhibit the action of acetolactate synthase (ALS), in particular the sulfonylurea-type herbicides (e.g., the acetolactate synthase (ALS) gene containing mutations leading to such resistance in particular the S4 and/or Hra mutations), genes coding for resistance to herbicides which act to inhibit action of glutamine synthase, such as phosphinothricin or basta (e.g., the bar gene), or other such genes known in the art. The bar gene encodes resistance to the herbicide basta, the nptll gene encodes resistance to the antibiotics kanamycin and geneticin, and the ALS gene encodes resistance to the herbicide chlorsulfuron.
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 by Rogers et al., Meth. in Enzymol., 153:253-277 (1987). These vectors are plant integrating vectors in that on transformation, the vectors integrate a portion of vector DNA into the genome of the host plant. Exemplary A. tumefaciens vectors useful herein are plasmids pKYLX6 and pKYLX7 of Schardl et al., Gene, 61:1-11 (1987) and Berger et al., Proc.
Natl. Acad. Sci. U.S.A., 86:8402-8406 (1989). Another useful vector herein is plasmid pBI101.2 that is available from Clontech Laboratories, Inc. (Palo Alto, CA).
A polynucleotide of the present invention can be expressed in either sense or anti-sense orientation as desired. It will be appreciated that control of gene expression in either sense or anti-sense orientation can have a direct impact on the observable plant characteristics. Antisense technology can be conveniently used to gene expression in plants. To accomplish this, a nucleic acid segment from the desired gene is cloned and operably linked to a promoter such that the anti-sense strand of RNA will be transcribed.
The construct is then transformed into plants and the antisense strand of RNA
is produced.
In plant cells, it has been shown that antisense RNA inhibits gene expression by preventing the accumulation of mRNA which encodes the enzyme of interest, see, e.g., Sheehy et al., Proc. Nat'l. Acad. Sci. (USA) 85: 8805-8809 (1988); and Hiatt et al., U.S.
Patent No.
4,801,340.
Another method of suppression is sense suppression. Introduction of nucleic acid configured in the sense orientation has been shown to be an effective means by which to block the transcription of target genes. For an example of the use of this method to modulate expression of endogenous genes see, Napoli et al., The Plant Cell 2:

(1990) and U.S. Patent No. 5,034,323.
Catalytic RNA molecules or ribozymes can also be used to inhibit expression of plant genes. It is possible to design ribozymes that specifically pair with virtually any S target RNA and cleave the phosphodiester backbone at a specific location, thereby functionally inactivating the target RNA. In carrying out this cleavage, the ribozyme is not itself altered, and is thus capable of recycling and cleaving other molecules, making it a true enzyme. The inclusion of ribozyme sequences within antisense RNAs confers RNA-cleaving activity upon them, thereby increasing the activity of the constructs. The design and use of target RNA-specific ribozymes is described in Haseloff et al., Nature 334: 585-591 (1988).
A variety of cross-linking agents, alkylating agents and radical generating species as pendant groups on polynucleotides of the present invention can be used to bind, label, detect, and/or cleave nucleic acids. For example, Vlassov, V. V., et al., Nucleic Acids Res (1986) 14:4065-4076, describe covalent bonding of a single-stranded DNA
fragment with alkylating derivatives of nucleotides complementary to target sequences. A
report of similar work by the same group is that by Knorre, D. G., et al., Biochimie ( 1985) 67:785-789. Iverson and Dervan also showed sequence-specific cleavage of single-stranded DNA
mediated by incorporation of a modified nucleotide which was capable of activating cleavage (JAm Chem Soc (1987) 109:1241-1243). Meyer, R. B., et al., JAm Chem Soc (1989) 111:8517-8519, effect covalent crosslinking to a target nucleotide using an alkylating agent complementary to the single-stranded target nucleotide sequence. A
photoactivated crosslinking to single-stranded oligonucleotides mediated by psoralen was disclosed by Lee, B. L., et al., Biochemistry (1988) 27:3197-3203. Use of crosslinking in triple-helix forming probes was also disclosed by Home, et al., JAm Chern Soc (1990) 112:2435-2437. Use of N4, N4-ethanocytosine as an alkylating agent to crosslink to single-stranded oligonucleotides has also been described by Webb and Matteucci, JAm Chem Soc (1986) 108:2764-2765; Nucleic Acids Res (1986) 14:7661-7674; Feteritz et al., J. Am. Chem. Soc. 113:4000 (1991). Various compounds to bind, detect, label, and/or cleave nucleic acids are known in the art. See, for example, U.S. Patent Nos.
5,543,507;
5,672,593; 5,484,908; 5,256,648; and, 5,681941.

Proteins The isolated proteins of the present invention comprise a polypeptide having at least 10 amino acids encoded by any one of the polynucleotides of the present invention as discussed more fully, supra, or polypeptides which are conservatively modified variants thereof. The proteins of the present invention or variants thereof can comprise any number of contiguous amino acid residues from a polypeptide of the present invention, wherein that number is selected from the group of integers consisting of from 10 to the number of residues in a full-length lignin biosynthesis polypeptide. Optionally, this subsequence of contiguous amino acids is at least 15, 20, 25, 30, 35, or 40 amino acids in length, often at least 50, 60, 70, 80, or 90 amino acids in length. Further, the number of such subsequences can be any integer selected from the group consisting of from 1 to 20, such as 2, 3, 4, or 5.
The present invention further provides a protein comprising a polypeptide having a specified sequence identity with a polypeptide of the present invention. The percentage of sequence identity is an integer selected from the group consisting of from 50 to 99.
Exemplary sequence identity values include 60%, 65%, 70%, 75%, 80%, 85%, 90%, and 95%. Sequence identity can be determined using, for example, the GAP or BLAST
algorithms.
As those of skill will appreciate, the present invention includes catalytically active polypeptides of the present invention (i.e., enzymes). Catalytically active polypeptides have a specific activity at least 20%, 30%, or 40%, and preferably at least 50%, 60%, or 70%, and most preferably at least 80%, 90%, or 95% that of the native (non-synthetic), endogenous polypeptide. Further, the substrate specificity (k~at/K",) is optionally substantially similar to the native (non-synthetic), endogenous polypeptide.
Typically, the Kr" will be at least 30%, 40%, or 50%, that of the native (non-synthetic), endogenous polypeptide; and more preferably at least 60%, 70%, 80%, or 90%. Methods of assaying and quantifying measures of enzymatic activity and substrate specificity (k~at/Km), are well known to those of skill in the art.
Generally, the proteins of the present invention will, when presented as an immunogen, elicit production of an antibody specifically reactive to a polypeptide of the present invention encoded by a polynucleotide of the present invention as described, supra.
Exemplary polypeptides include those which are full-length, such as those disclosed in SEQ ID NOS: 1-18 and 73-75. Further, the proteins of the present invention will not bind to antisera raised against a polypeptide of the present invention which has been fully immunosorbed with the same polypeptide. Immunoassays for determining binding are well known to those of skill in the art. A preferred immunoassay is a competitive immunoassay as discussed, infra. Thus, the proteins of the present invention can be employed as immunogens for constructing antibodies immunoreactive to a protein of the present invention for such exemplary utilities as immunoassays or protein purification techniques.
Expression of Proteins in Host Cells Using the nucleic acids of the present invention, one may express a protein of the present invention in a recombinantly engineered cell such as bacteria, yeast, insect, mammalian, or preferably plant cells. The cells produce the protein in a non-natural condition (e.g., in quantity, composition, location, and/or time), because they have been genetically altered through human intervention to do so.
It is expected that those of skill in the art are knowledgeable in the numerous expression systems available for expression of a nucleic acid encoding a protein of the present invention. No attempt to describe in detail the various methods known for the expression of proteins in prokaryotes or eukaryotes will be made.
In brief summary, the expression of isolated nucleic acids encoding a protein of the present invention will typically be achieved by operably linking, for example, the DNA or cDNA to a promoter (which is either constitutive or regulatable), followed by incorporation into an expression vector. The vectors can be suitable for replication and integration in either prokaryotes or eukaryotes. Typical expression vectors contain transcription and translation terminators, initiation sequences, and promoters useful for regulation of the expression of the DNA encoding a protein of the present invention. To obtain high level expression of a cloned gene, it is desirable to construct expression vectors which contain, at the minimum, a strong promoter to direct transcription, a ribosome binding site for translational initiation, and a transcription/translation terminator. One of skill would recognize that modifications can be made to a protein of the present invention without diminishing its biological activity. Some modifications may be made to facilitate the cloning, expression, or incorporation of the targeting molecule into a fusion protein.
Such modifications are well known to those of skill in the art and include, for example, a methionine added at the amino terminus to provide an initiation site, or additional amino acids (e.g., poly His) placed on either terminus to create conveniently located purification sequences. Restriction sites or termination codons can also be introduced.
A. Expression in Prokaryotes Prokaryotic cells may be used as hosts for expression. Prokaryotes most frequently are represented by various strains of E. coli; however, other microbial strains may also be used. Commonly used prokaryotic control sequences which are defined herein to include promoters for transcription initiation, optionally with an operator, along with ribosome binding site sequences, include such commonly used promoters as the beta lactamase (penicillinase) and lactose (lac) promoter systems (Chang et al., Nature 198:1056 (1977)), the tryptophan (trp) promoter system (Goeddel et al., Nucleic Acids Res.
8:4057 (1980)) and the lambda derived P L promoter and N-gene ribosome binding site (Shimatake et al., Nature 292:128 (1981)). The inclusion of selection markers in DNA vectors transfected in E. coli is also useful. Examples of such markers include genes specifying resistance to ampicillin, tetracycline, or chloramphenicol.
The vector is selected to allow introduction into the appropriate host cell.
Bacterial vectors are typically of plasmid or phage origin. Appropriate bacterial cells are infected with phage vector particles or transfected with naked phage vector DNA. If a plasmid vector is used, the bacterial cells are transfected with the plasmid vector DNA. Expression systems for expressing a protein of the present invention are available using Bacillus sp.
and Salmonella (Palva, et al., Gene 22: 229-235 (1983); Mosbach, et al., Nature 302: 543-545 (1983)).
B. Expression in Eukaryotes A variety of eukaryotic expression systems such as yeast, insect cell lines, plant and mammalian cells, are known to those of skill in the art. As explained briefly below, a of the present invention can be expressed in these eukaryotic systems. In some embodiments, transformed/transfected plant cells, as discussed infra, are employed as expression systems for production of the proteins of the instant invention.
Synthesis of heterologous proteins in yeast is well known. Sherman, F., et al., Methods in Yeast Genetics, Cold Spring Harbor Laboratory (1982) is a well recognized work describing the various methods available to produce the protein in yeast.
Suitable vectors usually have expression control sequences, such as promoters, including 3-phosphoglycerate kinase or other glycolytic enzymes, and an origin of replication, termination sequences and the like as desired. For instance, suitable vectors are described in the literature (Botstein, et al., Gene 8: 17-24 (1979); Broach, et al., Gene 8: 121-133 ( 1979)).
A protein of the present invention, once expressed, can be isolated from yeast by lysing the cells and applying standard protein isolation techniques to the lysates. The monitoring of the purification process can be accomplished by using Western blot techniques or radioimmunoassay of other standard immunoassay techniques.
The sequences encoding proteins of the present invention can also be ligated to various expression vectors for use in transfecting cell cultures of, for instance, mammalian, insect, or plant origin. Illustrative of cell cultures useful for the production of the peptides are mammalian cells. Mammalian cell systems often will be in the form of monolayers of cells although mammalian cell suspensions may also be used. A number of suitable host cell lines capable of expressing intact proteins have been developed in the art, and include the HEK293, BHK21, and CHO cell lines. Expression vectors for these cells can include expression control sequences, such as an origin of replication, a promoter (e.g., the CMV
promoter, a HSV tk promoter or pgk (phosphoglycerate kinase) promoter), an enhancer (Queen et al., Immunol. Rev. 89: 49 (1986)), and necessary processing information sites, such as ribosome binding sites, RNA splice sites, polyadenylation sites (e.g., an SV40 large T Ag poly A addition site), and transcriptional terminator sequences.
Other animal cells useful for production of proteins of the present invention are available, for instance, from the American Type Culture Collection Catalogue of Cell Lines and Hybridomas (7th edition, 1992).
Appropriate vectors for expressing proteins of the present invention in insect cells are usually derived from the SF9 baculovirus. Suitable insect cell lines include mosquito larvae, silkworm, armyworm, moth and Drosophila cell lines such as a Schneider cell line (See Schneider, J. EmbYyol. Exp. Morphol. 27: 353-365 (1987).
As with yeast, when higher animal or plant host cells are employed, polyadenlyation or transcription terminator sequences are typically incorporated into the vector. An example of a terminator sequence is the polyadenlyation sequence from the bovine growth hormone gene. Sequences for accurate splicing of the transcript may also be included. An example of a splicing sequence is the VP1 intron from SV40 (Sprague, et al., J. Virol. 45: 773-781 (1983)). Additionally, gene sequences to control replication in the host cell may be incorporated into the vector such as those found in bovine papilloma virus type-vectors. Saveria-Campo, M., Bovine Papilloma Virus DNA a Eukaryotic Cloning Vector in DNA Cloning 101. II a Practical Approach, D.M. Glover, Ed., IRL
Press, Arlington, Virginia pp. 213-238 (1985).
Transfection/Transformation of Cells The method of transformation/transfection is not critical to the instant invention;
various methods of transformation or transfection are currently available. As newer methods are available to transform crops or other host cells they may be directly applied.
Accordingly, a wide variety of methods have been developed to insert a DNA
sequence into the genome of a host cell to obtain the transcription and/or translation of the sequence to effect phenotypic changes in the organism. Thus, any method which provides for efficient transformation/transfection may be employed.
A. Plant Transformation A DNA sequence coding for the desired polynucleotide of the present invention, for example a cDNA or a genomic sequence encoding a full length protein, will be used to construct a recombinant expression cassette which can be introduced into the desired plant.
Isolated nucleic acid acids of the present invention can be introduced into plants according to techniques known in the art. Generally, recombinant expression cassettes as described above and suitable for transformation of plant cells are prepared.
Techniques for transforming a wide variety of higher plant species are well known and described in the technical, scientific, and patent literature. See, for example, Weising et al., Ann. Rev.
Genet. 22: 421-477 (1988). For example, the DNA construct may be introduced directly into the genomic DNA of the plant cell using techniques such as electroporation, polyethylene glycol (PEG), poration, particle bombardment, silicon fiber delivery, or microinjection of plant cell protoplasts or embryogenic callus. See, e.g., Tomes, et al., Direct DNA Transfer into Intact Plant Cells Via Microprojectile Bombardment.
pp.197-213 in Plant Cell, Tissue and Organ Culture, Fundamental Methods. eds. O. L.
Gamborg and G.C. Phillips. Springer-Verlag Berlin Heidelberg New York, 1995.
Alternatively, the DNA constructs may be combined with suitable T-DNA flanking regions and introduced into a conventional Agrobacterium tumefaciens host vector. The virulence functions of the Agrobacterium tumefaciens host will direct the insertion of the construct and adjacent marker into the plant cell DNA when the cell is infected by the bacteria. See, U.S. Patent No. 5,591,616.
The introduction of DNA constructs using polyethylene glycol precipitation is described in Paszkowski et al., Embo J. 3: 2717-2722 (1984). Electroporation techniques are described in Fromm et al., Proc. Natl. Acad. Sci. 82: 5824 (1985).
Ballistic transformation techniques are described in Klein et al., Nature 327: 70-73 (1987).
Agrobacterium tumefaciens-meditated transformation techniques are well described in the scientific literature. See, for example Horsch et al., Science 233: 496-498 (1984), and Fraley et al., Proc. Natl. Acad. Sci. 80: 4803 (1983). Although Agrobacterium is useful primarily in dicots, certain monocots can be transformed byAgrobacterium. For instance, Agrobacterium transformation of maize is described in U.S. Patent No.
5,550,318.
Other methods of transfection or transformation include (1) Agrobacterium rhizogenes-mediated transformation (see, e.g., Lichtenstein and Fuller In:
Genetic Engineering, vol. 6, PWJ Rigby, Ed., London, Academic Press, 1987; and Lichtenstein, C.
P., and Draper, J,. In: DNA Cloning, Vol. II, D. M. Glover, Ed., Oxford, IRI
Press, 1985),Application PCT/LTS87/02512 (WO 88/02405 published Apr. 7, 1988) describes the use of A.rhizogenes strain A4 and its Ri plasmid along with A. tumefaciens vectors pARC8 or pARCl6 (2) liposome-mediated DNA uptake (see, e.g., Freeman et al., Plant Cell Physiol. 25: 1353, 1984), (3) the vortexing method (see, e.g., Kindle, Proc.
Natl. Acad.
Sci., USA 87: 1228, (1990).
DNA can also be introduced into plants by direct DNA transfer into pollen as described by Zhou et al., Methods in Enzymology, 101:433 (1983); D. Hess, Intern Rev. Cytol., 107:367 (1987); Luo et al., Plane Mol. Biol. Reporter, 6:165 (1988). Expression of polypeptide coding genes can be obtained by injection of the DNA into reproductive organs of a plant as described by Pena et al., Nature, 325.:274 (1987). DNA can also be injected directly into the cells of immature embryos and the rehydration of desiccated embryos as described by Neuhaus et al., Theor. Appl. Genet., 75:30 (1987); and Benbrook et al., in Proceedings Bio Expo 1986, Butterworth, Stoneham, Mass., pp. 27-54 (1986). A variety of plant viruses that can be employed as vectors are known in the art and include cauliflower mosaic virus (CaMV), geminivirus, brome mosaic virus, and tobacco mosaic virus.
B. Transfection of Prokaryotes, dower Eukaryotes, and Animal Cells Animal and lower eukaryotic (e.g., yeast) host cells are competent or rendered competent for transfection by various means. There are several well-known methods of introducing DNA into animal cells. These include: calcium phosphate precipitation, fusion of the recipient cells with bacterial protoplasts containing the DNA, treatment of the recipient cells with liposomes containing the DNA, DEAF dextran, electroporation, biolistics, and micro-injection of the DNA directly into the cells. The transfected cells are cultured by means well known in the art. Kuchler, R.J., Biochemical Methods in Cell Culture and Virology, Dowden, Hutchinson and Ross, Inc. (1977).
Synthesis of Proteins The proteins of the present invention can be constructed using non-cellular synthetic methods. Solid phase synthesis of proteins of less than about 50 amino acids in length may be accomplished by attaching the C-terminal amino acid of the sequence to an insoluble support followed by sequential addition of the remaining amino acids in the sequence. Techniques for solid phase synthesis are described by Barany and Mernfield, Solid-Phase Peptide Synthesis, pp. 3-284 in The Peptides: Analysis, Synthesis, Biology.
Vol. 2: Special Methods in Peptide Synthesis, Part A.; Mernfield, et al., J.
Am. Chem. Soc.
85: 2149-2156 (1963), and Stewart et al., Solid Phase Peptide Synthesis, 2nd ed., Pierce Chem. Co., Rockford, Ill. (1984). Proteins of greater length may be synthesized by condensation of the amino and carboxy termini of shorter fragments. Methods of forming peptide bonds by activation of a carboxy terminal end (e.g., by the use of the coupling reagent N,N'-dicycylohexylcarbodiimide)) is known to those of skill.
Purification of Proteins The proteins of the present invention may be purified by standard techniques well known to those of skill in the art. Recombinantly produced proteins of the present invention can be directly expressed or expressed as a fusion protein. The recombinant protein is purified by a combination of cell lysis (e.g., sonication, French press) and affinity chromatography. For fusion products, subsequent digestion of the fusion protein with an appropriate proteolytic enzyme releases the desired recombinant protein.
The proteins of this invention, recombinant or synthetic, may be purified to substantial purity by standard techniques well known in the art, including selective precipitation with such substances as ammonium sulfate, column chromatography, immunopurification methods, and others. See, for instance, R. Scopes, Protein Purification: Principles and Practice, Springer-Verlag: New York (1982);
Deutscher, Guide to Protein Purification, Academic Press (1990). For example, antibodies may be raised to the proteins as described herein. Purification from E. coli can be achieved following procedures described in U.S. Patent No. 4,511,503. The protein may then be isolated from cells expressing the protein and further purified by standard protein chemistry techniques as described herein. Detection of the expressed protein is achieved by methods known in the art and include, for example, radioimmunoassays, Western blotting techniques or immunoprecipitation.
Trans~enic Plant Regeneration Transformed plant cells which are derived by any of the above transformation techniques can be cultured to regenerate a whole plant which possesses the transformed genotype. Such regeneration techniques often rely on manipulation of certain phytohormones in a tissue culture growth medium. For transformation and regeneration of maize see, Gordon-Kamm et al., The Plant Cell, 2:603-618 (1990).
Plants cells transformed with a plant expression vector can be regenerated, e.g., from single cells, callus tissue or leaf discs according to standard plant tissue culture techniques. It is well known in the art that various cells, tissues, and organs from almost any plant can be successfully cultured to regenerate an entire plant. Plant regeneration from cultured protoplasts is described in Evans et al., Protoplasts Isolation and Culture, Handbook ofPlant Cell Culture, Macmillilan Publishing Company, New York, pp. 124-176 (1983); and Binding, Regeneration ofPlants, Plant Protoplasts, CRC Press, Boca Raton, pp. 21-73 (1985).
The regeneration of plants containing the foreign gene introduced by Agrobacterium from leaf explants can be achieved as described by Horsch et al., Science, 227:1229-1231 (1985). In this procedure, transformants are grown in the presence of a selection agent and in a medium that induces the regeneration of shoots in the plant species being transformed as described by Fraley et al., Proc. Natl. Acad. Sci. U.S.A., 80:4803 (1983). This procedure typically produces shoots within two to four weeks and these transformant shoots are then transferred to an appropriate root-inducing medium containing the selective agent and an antibiotic to prevent bacterial growth. Transgenic plants of the present invention may be fertile or sterile.
Regeneration can also be obtained from plant callus, explants, organs, or parts thereof. Such regeneration techniques are described generally in Klee et al., Ann. Rev. of Plant Phys. 38: 467-486 (1987). The regeneration of plants from either single plant protoplasts or various explants is well known in the art. See, for example, Methods for Plant Molecular Biology, A. Weissbach and H. Weissbach, eds., Academic Press, Inc., San Diego, Calif. (1988). This regeneration and growth process includes the steps of selection of transformant cells and shoots, rooting the transformant shoots and growth of the plantlets in soil. For maize cell culture and regeneration see generally, The Maize Handbook, Freeling and Walbot, Eds., Springer, New York (1994); Corn and Corn Improvement, 3rd edition, Sprague and Dudley Eds., American Society of Agronomy, Madison, Wisconsin (1988).
One of skill will recognize that after the recombinant expression cassette is stably incorporated in transgenic plants and confirmed to be operable, it can be introduced into other plants by sexual crossing. Any of a number of standard breeding techniques can be used, depending upon the species to be crossed.
In vegetatively propagated crops, mature transgenic plants can be propagated by the taking of cuttings or by tissue culture techniques to produce multiple identical plants.
Selection of desirable transgenics is made and new varieties are obtained and propagated vegetatively for commercial use. In seed propagated crops, mature transgenic plants can be self crossed to produce a homozygous inbred plant. The inbred plant produces seed containing the newly introduced heterologous nucleic acid. These seeds can be grown to produce plants that would produce the selected phenotype, (e.g., altered lignin biosynthesis content or composition).
Parts obtained from the regenerated plant, such as flowers, seeds, leaves, branches, fruit, and the like are included in the invention, provided that these parts comprise cells comprising the isolated nucleic acid of the present invention. Progeny and variants, and mutants of the regenerated plants are also included within the scope of the invention, provided that these parts comprise the introduced nucleic acid sequences.
Transgenic plants expressing the selectable marker can be screened for transmission of the nucleic acid of the present invention by, for example, standard immunoblot and DNA detection techniques. Transgenic lines are also typically evaluated on levels of expression of the heterologous nucleic acid. Expression at the RNA level can be determined initially to identify and quantitate expression-positive plants.
Standard techniques for RNA analysis can be employed and include PCR amplification assays using oligonucleotide primers designed to amplify only the heterologous RNA
templates and solution hybridization assays using heterologous nucleic acid-specific probes.
The RNA-positive plants can then analyzed for protein expression by Western immunoblot analysis using the specifically reactive antibodies of the present invention. In addition, in situ hybridization and immunocytochemistry according to standard protocols can be done using heterologous nucleic acid specific polynucleotide probes and antibodies, respectively, to localize sites of expression within transgenic tissue. Generally, a number of transgenic lines are usually screened for the incorporated nucleic acid to identify and select plants with the most appropriate expression profiles.
A preferred embodiment is a transgenic plant that is homozygous for the added heterologous nucleic acid; i.e., a transgenic plant that contains two added nucleic acid sequences, one gene at the same locus on each chromosome of a chromosome pair.
A
homozygous transgenic plant can be obtained by sexually mating (selfing) a heterozygous transgenic plant that contains a single added heterologous nucleic acid, germinating some of the seed produced and analyzing the resulting plants produced for altered lignification relative to a control plant (i.e., native, non-transgenic). Back-crossing to a parental plant and out-crossing with a non- transgenic plant are also contemplated.
Modulating Polypeptide Content and/or Com osition The present invention further provides a method for modulating (i.e., increasing or decreasing) the concentration or ratio of the lignin biosynthesis polypeptide in a plant or part thereof. Modulation can be effected by increasing or decreasing the concentration and/or the ratio of the polypeptides of the present invention in a plant. The method comprises transforming a plant cell with a recombinant expression cassette comprising a polynucleotide of the present invention as described above to obtain a transformed plant cell, growing the transformed plant cell under plant forming conditions, and inducing expression of a polynucleotide of the present invention in the plant for a time sufficient to modulate lignin biosynthesis polypeptide concentration and/or the ratios of the polypeptides in the plant or plant part.
In some embodiments, lignification in a plant may be modulated by altering, in vivo or in vitro, the promoter of a non-isolated lignin biosynthesis gene to up- or down-regulate gene expression. In some embodiments, the coding regions of native lignin biosynthesis genes can be altered via substitution, addition, insertion, or deletion to decrease activity of the encoded enzyme. See, e.g., Kmiec, U.S. Patent 5,565,350; Zarling et al., PCT/LJS93/03868. And in some embodiments, an isolated nucleic acid (e.g., a vector) comprising a promoter sequence is transfected into a plant cell. Subsequently, a plant cell comprising the promoter operably linked to a polynucleotide of the present invention is selected for by means known to those of skill in the art such as, but not limited to, Southern blot, DNA sequencing, or PCR analysis using primers specific to the promoter and to the gene and detecting amplicons produced therefrom. A plant or plant part altered or modified by the foregoing embodiments is grown under plant forming conditions for a time sufficient to modulate lignin biosynthesis content and/or composition in the plant.
Plant forming conditions are well known in the art and discussed briefly, supra.
In general, content or composition is increased or decreased by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% relative to a native control plant, plant part, or cell lacking the aforementioned recombinant expression cassette.
Modulation in the present invention may occur during and/or subsequent to growth of the plant to the desired stage of development. Modulating nucleic acid expression temporally and/or in particular tissues can be controlled by employing the appropriate promoter operably linked to a polynucleotide of the present invention in, for example, sense or antisense orientation as discussed in greater detail, supra. Induction of expression of a polynucleotide of the present invention can also be controlled by exogenous administration of an effective amount of inducing compound. Inducible promoters and inducing compounds which activate expression from these promoters are well known in the art. In preferred embodiments, lignification is modulated in monocots, particularly maize.
Molecular Markers The present invention provides a method of genotyping a plant comprising a polynucleotide of the present invention. Optionally, the plant is a monocot, such as maize or sorghum. Genotyping provides a means of distinguishing homologs of a chromosome pair and can be used to differentiate segregants in a plant population.
Molecular marker methods can be used for phylogenetic studies, characterizing genetic relationships among crop varieties, identifying crosses or somatic hybrids, localizing chromosomal segments affecting monogenic traits, map based cloning, and the study of quantitative inheritance.
See, e.g., Plant Molecular Biology: A Laboratory Manual, Chapter 7, Clark, Ed., Springer-Verlag, Berlin (1997). For molecular marker methods, see generally, The DNA
Revolution by Andrew H. Paterson 1996 (Chapter 2) in: Genome Mapping in Plants (ed.
Andrew H.
Paterson) by Academic Press/R. G. Landis Company, Austin, Texas, pp.7-21.
The particular method of genotyping in the present invention may employ any number of molecular marker analytic techniques such as, but not limited to, restriction fragment length polymorphisms (RFLPs). RFLPs are the product of allelic differences between DNA restriction fragments caused by nucleotide sequence variability.
As is well known to those of skill in the art, RFLPs are typically detected by extraction of genomic DNA and digestion with a restriction enzyme. Generally, the resulting fragments are separated according to size and hybridized with a probe; single copy probes are preferred.
Restriction fragments from homologous chromosomes are revealed. Differences in fragment size among alleles represent an RFLP. Thus, the present invention further provides a means to follow segregation of a lignin biosynthesis gene or nucleic acid of the present invention as well as chromosomal sequences genetically linked to these genes or nucleic acids using such techniques as RFLP analysis. Linked chromosomal sequences are within 50 centiMorgans (cM), often within 40 or 30 cM, preferably within 20 or 10 cM, more preferably within 5, 3, 2, or 1 cM of a lignin biosynthesis gene.
In the present invention, the nucleic acid probes employed for molecular marker mapping of plant nuclear genomes selectively hybridize, under selective hybridization conditions, to a gene encoding a polynucleotide of the present invention. In preferred embodiments, the probes are selected from polynucleotides of the present invention.
Typically, these probes are cDNA probes or Pst I genomic clones. The length of the probes is discussed in greater detail, supra, but are typically at least 15 bases in length, more preferably at least 20, 25, 30, 35, 40, or 50 bases in length. Generally, however, the probes are less than about 1 kilobase in length. Preferably, the probes are single copy probes that hybridize to a unique locus in a haploid chromosome complement.
Some exemplary restriction enzymes employed in RFLP mapping are EcoRI, EcoRv, and SstI.
As used herein the term "restriction enzyme" includes reference to a composition that recognizes and, alone or in conjunction with another composition, cleaves at a specific nucleotide sequence.
The method of detecting an RFLP comprises the steps of (a) digesting genomic DNA of a plant with a restriction enzyme; (b) hybridizing a nucleic acid probe, under selective hybridization conditions, to a sequence of a polynucleotide of the present of said genomic DNA; (c) detecting therefrom a RFLP. Other methods of differentiating polymorphic (allelic) variants of polynucleotides of the present invention can be had by utilizing molecular marker techniques well known to those of skill in the art including such techniques as: 1) single stranded conformation analysis (SSCP); 2) denaturing gradient gel electrophoresis (DGGE); 3) RNase protection assays; 4) allele-specific oligonucleotides (ASOs); 5) the use of proteins which recognize nucleotide mismatches, such as the E. coli mutS protein; and 6) allele-specific PCR. Other approaches based on the detection of mismatches between the two complementary DNA strands include clamped denaturing gel electrophoresis (CDGE); heteroduplex analysis (HA); and chemical mismatch cleavage (CMC). Exemplary polymorphic variants are provided in Table I, supra. Thus, the present invention further provides a method of genotyping comprising the steps of contacting, under stringent hybridization conditions, a sample suspected of comprising a polynucleotide of the present invention with a nucleic acid probe. Generally, the sample is a plant sample; preferably, a sample suspected of comprising a maize polynucleotide of the present invention (e.g., gene, mRNA). The nucleic acid probe selectively hybridizes, under stringent conditions, to a subsequence of a polynucleotide of the present invention comprising a polymorphic marker. Selective hybridization of the nucleic acid probe to the polymorphic marker nucleic acid sequence yields a hybridization complex.
Detection of the hybridization complex indicates the presence of that polymorphic marker in the sample. In preferred embodiments, the nucleic acid probe comprises a polynucleotide of the present invention.
UTR's and Codon Preference In general, translational efficiency has been found to be regulated by specific sequence elements in the 5' non-coding or untranslated region (5' UTR) of the RNA. Positive sequence motifs include translational initiation consensus sequences (Kozak, Nucleic Acids Res.15:8125 (1987)) and the 7 methylguanosine cap structure (Drummond et al., Nucleic Acids Res.
13:7375 (1985)). Negative elements include stable intramolecular 5' UTR
stem-loop structures (Muesing et al., Cell 48:691 (1987)) and AUG sequences or short open reading frames preceded by an appropriate AUG in the 5' UTR (Kozak, supra, Rao et al., Mol. and Cell. Biol. 8:284 (1988)). Accordingly, the present invention provides S' and/or 3' untranslated regions for modulation of translation of heterologous coding sequences.
Further, the polypeptide-encoding segments of the polynucleotides of the present invention can be modified to alter codon usage. Altered codon usage can be employed to alter translational efficiency and/or to optimize the coding sequence for expression in a desired host or to optimize the codon usage in a heterologous sequence for expression in maize. Codon usage in the coding regions of the polynucleotides of the present invention can be analyzed statistically using commercially available software packages such as "Codon Preference" available from the University of Wisconsin Genetics Computer Group (see Devereaux et al., Nucleic Acids Res. 12: 387-395 (1984)) or MacVector 4.1 (Eastman Kodak Co., New Haven, Conn.). Thus, the present invention provides a codon usage frequency characteristic of the coding region of at least one of the polynucleotides of the present invention. The number of polynucleotides that can be used to determine a codon usage frequency can be any integer from 1 to the number of polynucleotides of the present invention as provided herein. Optionally, the polynucleotides will be full-length sequences. An exemplary number of sequences for statistical analysis can be at least 1, 5, 10, 20, 50, or 100.
Sequence Shuffling The present invention provides methods for sequence shuffling using polynucleotides of the present invention, and compositions resulting therefrom. Sequence shuffling is described in PCT publication No. 96/19256. See also, Zhang, J.-H., et al.
Proc. Natl. Acad. Sci. USA 94:4504-4509 (1997). Generally, sequence shuffling provides a means for generating libraries of polynucleotides having a desired characteristic which can be selected or screened for. Libraries of recombinant polynucleotides are generated from a population of related sequence polynucleotides which comprise sequence regions which have substantial sequence identity and can be homologously recombined in vitro or in vivo. The population of sequence-recombined polynucleotides comprises a subpopulation of polynucleotides which possess desired or advantageous characteristics and which can be selected by a suitable selection or screening method. The characteristics can be any property or attribute capable of being selected for or detected in a screening system, and may include properties of: an encoded protein, a transcriptional element, a sequence controlling transcription, RNA processing, RNA stability, chromatin conformation, translation, or other expression property of a gene or transgene, a replicative element, a protein-binding element, or the like, such as any feature which confers a selectable or detectable property. In some embodiments, the selected characteristic will be an increased Km andlor K~at over the wild-type protein as provided herein. In other embodiments, a protein or polynculeotide generated from sequence shuffling will have a ligand binding affinity greater than the non-shuffled wild-type polynucleotide. The increase in such properties can be at least 110%, 120%, 130%, 140% or at least 150% of the wild-type value.
Generic and Consensus Se ug ences Polynucleotides and polypeptides of the present invention further include those having: (a) a generic sequence of at least two homologous polynucleotides or polypeptides, respectively, of the present invention; and, (b) a consensus sequence of at least three homologous polynucleotides or polypeptides, respectively, of the present invention. The generic sequence of the present invention comprises each species of polypeptide or polynucleotide embraced by the generic polypeptide or polynucleotide sequence, respectively. The individual species encompassed by a polynucleotide having an amino acid or nucleic acid consensus sequence can be used to generate antibodies or produce nucleic acid probes or primers to screen for homologs in other species, genera, families, orders, classes, phyla, or kingdoms. For example, a polynucleotide having a consensus sequence from a gene family of Zea mays can be used to generate antibody or nucleic acid probes or primers to other Gramineae species such as wheat, rice, or sorghum.
Alternatively, a polynucleotide having a consensus sequence generated from orthologous genes can be used to identify or isolate orthologs of other taxa. Typically, a polynucleotide having a consensus sequence will be at least 9, 10, 15, 20, 25, 30, or 40 amino acids in length, or 20, 30, 40, 50, 100, or 150 nucleotides in length. As those of skill in the art are aware, a conservative amino acid substitution can be used for amino acids which differ amongst aligned sequence but are from the same conservative substitution group as discussed above. Optionally, no more than 1 or 2 conservative amino acids are substituted for each 10 amino acid length of consensus sequence.
Similar sequences used for generation of a consensus or generic sequence include any number and combination of allelic variants of the same gene, orthologous, or paralogous sequences as provided herein. Optionally, similar sequences used in generating a consensus or generic sequence are identified using the BLAST algorithm's smallest sum probability (P(N)). Various suppliers of sequence-analysis software are listed in chapter 7 of Current Protocols in Molecular Biology, F.M. Ausubel et al., Eds., Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley &
Sons, Inc.
(Supplement 30). A polynucleotide sequence is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.1, more preferably less than about 0.01, or 0.001, and most preferably less than about 0.0001, or 0.00001. Similar polynucleotides can be aligned and a consensus or generic sequence generated using multiple sequence alignment software available from a number of commercial suppliers such as the Genetics Computer Group's (Madison, WI) PILEUP software, Vector NTI's (North Bethesda, MD) ALIGNX, or Genecode's (Ann Arbor, MI) SEQUENCHER. Conveniently, default parameters of such software can be used to generate consensus or generic sequences.
Computer Applications The present invention provides machines, data structures, and processes for modeling or analyzing the polynucleotides and polypeptides of the present invention.

WO 01/34817 PCT/US00t30456 A. Machines and Data Structures The present invention provides a machine having a memory comprising data representing a sequence of a polynucleotide or polypeptide of the present invention. The machine of the present invention is typically a digital computer. The memory of such a machine includes, but is not limited to, ROM, or RAM, or computer readable media such as, but not limited to, magnetic media such as computer disks or hard drives, or media such as CD-ROM. Thus, the present invention also provides a data structure comprising a sequence of a polynucleotide of the present invention embodied in a computer readable medium. As those of skill in the art will be aware, the form of memory of a machine of the present invention or the particular embodiment of the computer readable medium is not a critical element of the invention and can take a variety of forms.
B. Homology Searches The present invention provides a process for identifying a candidate homologue (i.e., an ortholog or paralog) of a polynucleotide or polypeptide of the present invention. A
candidate homologue has statistically significant probability of having the same biological function (e.g., catalyzes the same reaction, binds to homologous proteins/nucleic acids) as the reference sequence to which it's compared. Accordingly, the polynucleotides and polypeptides of the present invention have utility in identifying homologs in animals or other plant species, particularly those in the family Gramineae such as, but not limited to, sorghum, wheat, or rice.
The process of the present invention comprises obtaining data representing a polynucleotide or polypeptide test sequence. Test sequences are generally at least 25 amino acids in length or at least 50 nucleotides in length. Optionally, the test sequence can be at least 50, 100, 150, 200, 250, 300, or 400 amino acids in length. A test polynucleotide can be at least 50, 100, 200, 300, 400, or 500 nucleotides in length. Often the test sequence will be a full-length sequence. Test sequences can be obtained from a nucleic acid of an animal or plant. Optionally, the test sequence is obtained from a plant species other than maize whose function is uncertain but will be compared to the test sequence to determine sequence similarity or sequence identity; for example, such plant species can be of the family Gramineae, such as wheat, rice, or sorghum. The test sequence data are entered into a machine, typically a computer, having a memory that contains data representing a reference sequence. The reference sequence can be the sequence of a polypeptide or a polynucleotide of the present invention and is often at least 25 amino acids or 100 nucleotides in length. As those of skill in the art are aware, the greater the sequence identity/similarity between a reference sequence of known function and a test sequence, the greater the probability that the test sequence will have the same or similar function as the reference sequence.
The machine further comprises a sequence comparison means for determining the sequence identity or similarity between the test sequence and the reference sequence.
Exemplary sequence comparison means are provided for in sequence analysis software discussed previously. Optionally, sequence comparison is established using the BLAST or GAP suite of programs.
The results of the comparison between the test and reference sequences can be displayed. Generally, a smallest sum probability value (P(N)) of less than 0.1, or alternatively, less than 0.01, 0.001, 0.0001, or 0.00001 using the BLAST 2.0 suite of algorithms under default parameters identifies the test sequence as a candidate homologue (i.e., an allele, ortholog, or paralog) of the reference sequence. A nucleic acid comprising a polynucleotide having the sequence of the candidate homologue can be constructed using well known library isolation, cloning, or in vitro synthetic chemistry techniques (e.g., phosphoramidite) such as those described herein. In additional embodiments, a nucleic acid comprising a polynucleotide having a sequence represented by the candidate homologue is introduced into a plant; typically, these polynucleotides are operably linked to a promoter. Confirmation of the function of the candidate homologue can be established by operably linking the candidate homolog nucleic acid to, for example, an inducible promoter, or by expressing the antisense transcript, and analyzing the plant for changes in phenotype consistent with the presumed function of the candidate homolog.
Optionally, the plant into which these nucleic acids are introduced is a monocot such as from the family Gramineae. Exemplary plants include maize, sorghum, wheat, rice, canola, alfalfa, cotton, and soybean.
C. Computer Modeling The present invention provides a process of modeling/analyzing data representative of the sequence a polynucleotide or polypeptide of the present invention. The process comprises entering sequence data of a polynucleotide or polypeptide of the present invention into a machine, manipulating the data to model or analyze the structure or activity of the polynucleotide or polypeptide, and displaying the results of the modeling or analysis. A variety of modeling and analytic tools are well known in the art and available from such commercial vendors as Genetics Computer Group (Version 10, Madison, WI).
Included amongst the modeling/analysis tools are methods to: 1) recognize overlapping sequences (e.g., from a sequencing project) with a polynucleotide of the present invention and create an alignment called a "contig"; 2) identify restriction enzyme sites of a polynucleotide of the present invention; 3) identify the products of a T1 ribonuclease digestion of a polynucleotide of the present invention; 4) identify PCR
primers with minimal self complementarity; 5) compare two protein or nucleic acid sequences and identifying points of similarity or dissimilarity between them; 6) compute pairwise 1 S distances between sequences in an alignment, reconstruct phylogentic trees using distance methods, and calculate the degree of divergence of two protein coding regions;
7) identify patterns such as coding regions, terminators, repeats, and other consensus patterns in polynucleotides of the present invention; 8) identify RNA secondary structure;

9) identify sequence motifs, isoelectric point, secondary structure, hydrophobicity, and antigenicity in polypeptides of the present invention; and, 10) translate polynucleotides of the present invention and backtranslate polypeptides of the present invention.
Detection of Nucleic Acids The present invention further provides methods for detecting a polynucleotide of the present invention in a nucleic acid sample suspected of comprising a polynucleotide of the present invention, such as a plant cell lysate, particularly a lysate of corn. In some embodiments, a lignin biosynthesis gene or portion thereof can be amplified prior to the step of contacting the nucleic acid sample with a polynucleotide of the present invention.
The nucleic acid sample is contacted with the polynucleotide to form a hybridization complex. The polynucleotide hybridizes under stringent conditions to a gene encoding a polypeptide of the present invention. Formation of the hybridization complex is used to detect a gene encoding a polypeptide of the present invention in the nucleic acid sample.

Those of skill will appreciate that an isolated nucleic acid comprising a polynucleotide of the present invention should lack cross-hybridizing sequences in common with non-lignin biosynthesis genes that would yield a false positive result.
Detection of the hybridization complex can be achieved using any number of well known methods. For example, the nucleic acid sample, or a portion thereof, may be assayed by hybridization formats including but not limited to, solution phase, solid phase, mixed phase, or in situ hybridization assays.
Detectable labels suitable for use in the present invention include any composition detectable by spectroscopic, radioisotopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means. Useful labels in the present invention include biotin for staining with labeled streptavidin conjugate, magnetic beads, fluorescent dyes, radiolabels, enzymes, and colorimetric labels. Other labels include ligands which bind to antibodies labeled with fluorophores, chemiluminescent agents, and enzymes.
Labeling the nucleic acids of the present invention is readily achieved such as by the use of labeled PCR primers.
Although the present invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be obvious that certain changes and modifications may be practiced within the scope of the appended claims.

Assays for Compounds that Modulate Enzymatic Activity or Expression The present invention also provides means for identifying compounds that bind to (e.g., substrates), and/or increase or decrease (i.e., modulate) the enzymatic activity of, catalytically active polypeptides of the present invention. The method comprises contacting a polypeptide of the present invention with a compound whose ability to bind to or modulate enzyme activity is to be determined. The polypeptide employed will have at least 20%, preferably at least 30% or 40%, more preferably at least 50% or 60%, and most preferably at least 70% or 80% of the specific activity of the native, full-length lignin biosynthesis polypeptide (e.g., enzyme). Generally, the polypeptide will be present in a range sufficient to determine the effect of the compound, typically about 1 nM
to 10 ~M.
Likewise, the compound will be present in a concentration of from about 1 nM
to 10 p.M.
Those of skill will understand that such factors as enzyme concentration, ligand concentrations (i.e., substrates, products, inhibitors, activators), pH, ionic strength, and temperature will be controlled so as to obtain useful kinetic data and determine the presence of absence of a compound that binds or modulates polypeptide activity. Methods of measuring enzyme kinetics is well known in the art. See, e.g., Segel, Biochemical Calculations, 2°d ed., John Wiley and Sons, New York (1976).
Although the present invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be obvious that certain changes and modifications may be practiced within the scope of the appended claims.

This example describes the construction cDNA libraries.
Total RNA Isolation Total RNA was isolated from corn tissues with TRIzoI Reagent (Life Technology Inc. Gaithersburg, MD) using a modification of the guanidine isothiocyanate/acid-phenol procedure described by Chomczynski and Sacchi (Chomczynski, P., and Sacchi, N.
Anal.
Biochem. 162, 156 (1987)). In brief, plant tissue samples were pulverized in liquid nitrogen before the addition of the TRIzoI Reagent, and then were further homogenized with a mortar and pestle. Addition of chloroform followed by centrifugation was conducted for separation of an aqueous phase and an organic phase. The total RNA was recovered by precipitation with isopropyl alcohol from the aqueous phase.
PolvlAl+ RNA Isolation The selection of poly(A)+ RNA from total RNA was performed using PolyATact system (Promega Corporation. Madison, WI). In brief, biotinylated oligo(dT) primers were used to hybridize to the 3' poly(A) tails on mRNA. The hybrids were captured using streptavidin coupled to paramagnetic particles and a magnetic separation stand. The mRNA was washed at high stringent condition and eluted by RNase-free deionized water.
cDNA Library Construction cDNA synthesis was performed and unidirectional cDNA libraries were constructed using the Superscript Plasmid System (Life Technology Inc.
Gaithersburg, MD). The first stand of cDNA was synthesized by priming an oligo(dT) primer containing a Not I site. The reaction was catalyzed by Superscript Reverse Transcriptase II at 45°C.
The second strand of cDNA was labeled with alpha-32P-dCTP and a portion of the reaction was analyzed by agarose gel electrophoresis to determine cDNA sizes. cDNA
molecules smaller than 500 base pairs and unligated adaptors were removed by Sephacryl-chromatography. The selected cDNA molecules were ligated into pSPORTl vector in between of NotI and SaII sites.

This example describes cDNA sequencing and library subtraction.
Seduencing Template Preparation Individual colonies were picked and DNA was prepared either by PCR with M13 forward primers and M13 reverse primers, or by plasmid isolation. All the cDNA
clones were sequenced using M 13 reverse primers.
(~-bot Subtraction Procedure cDNA libraries subjected to the subtraction procedure were plated out on 22 x cm2 agar plate at density of about 3,000 colonies per plate. The plates were incubated in a 37°C incubator for 12-24 hours. Colonies were picked into 384-well plates by a robot colony picker, Q-bot (GENETIX Limited). These plates were incubated overnight at 37°C.
Once sufficient colonies were picked, they were pinned onto 22 x 22 cm2 nylon membranes using Q-bot. Each membrane contained 9,216 colonies or 36,864 colonies.
These membranes were placed onto agar plate with appropriate antibiotic. The plates were incubated at 37°C for overnight. After colonies were recovered on the second day, these filters were placed on filter paper prewetted with denaturing solution for four minutes, then were incubated on top of a boiling water bath for additional four minutes. The filters were then placed on filter paper prewetted with neutralizing solution for four minutes. After excess solution was removed by placing the filters on dry filter papers for one minute, the colony side of the filters were place into Proteinase K solution, incubated at 37°C for 40-50 minutes. The filters were placed on dry filter papers to dry overnight. DNA
was then cross-linked to nylon membrane by UV light treatment.
Colony hybridization was conducted as described by Sambrook,J., Fritsch, E.F.
and Maniatis, T., (in Molecular Cloning: A laboratory Manual, 2°d Edition).
The following probes were used in colony hybridization:
1. First strand cDNA from the same tissue as the library was made from to remove the most redundant clones.
2. 48-192 most redundant cDNA clones from the same library based on previous sequencing data.
3. 192 most redundant cDNA clones in the entire maize sequence database.
4. A Sal-A20 oligo nucleotide: TCG ACC CAC GCG TCC GAA AAA AAA
AAA AAA AAA AAA, removes clones containing a poly A tail but no cDNA.
S. cDNA clones derived from rRNA.
The image of the autoradiography was scanned into computer and the signal intensity and cold colony addresses of each colony was analyzed. Re-arraying of cold-colonies from 384 well plates to 96 well plates was conducted using Q-bot.

This example describes identification of the gene from a computer homology search.

Gene identities were determined by conducting BLAST (Basic Local Alignment Search Tool; Altschul, S. F., et al., (1993) J. Mol. Biol. 215:403-410; see also www.ncbi.nlm.nih.gov/BLAST/) searches under default parameters for similarity to sequences contained in the BLAST "nr" database (comprising all non-redundant GenBank CDS translations, sequences derived from the 3-dimensional structure Brookhaven Protein Data Bank, the last major release of the SWISS-PROT protein sequence database, EMBL, and DDBJ databases). The cDNA sequences were analyzed for similarity to all publicly available DNA sequences contained in the "nr" database using the BLASTN
algorithm.
The DNA sequences were translated in all reading frames and compared for similarity to all publicly available protein sequences contained in the "nr" database using the BLASTX
algorithm (Gish, W. and States, D. J. Nature Genetics 3:266-272 (1993)) provided by the NCBI. In some cases, the sequencing data from two or more clones containing overlapping segments of DNA were used to construct contiguous DNA sequences.
The above examples are provided to illustrate the invention but not to limit its scope. Other variants of the invention will be readily apparent to one of ordinary skill in the art and are encompassed by the appended claims. All publications, patents, patent applications, and computer programs cited herein are hereby incorporated by reference.

SEQUENCE LISTING
<110> Pioneer Hi-Bred International, Inc.
<120> Genes Encoding Enzymes for Lignin Biosynthesis and Uses Thereof <130> 07098-PCT
<150> US 60/057,082 <151> 1997-08-27 <150> US 09/076,851 <151> 1998-05-12 <150> US 09/434,229 <151> 1999-11-05 <160> 16 <170> FastSEQ for Windows Version 3.0 <210> 1 <211> 2086 <212> DNA
<213> Zea mays <220>
<221> CDS
<222> (107)...(1702) <400> 1 gtcgacccac gcgtccgaag agcgctctac cagtacaact acaaccacac taaaacccaa 60 acaaaaaaaa ggaagcaggt gctgccgata tatcgattga tcgatc atg gcg gcg 115 Met Ala Ala gcc gtg gcc aat atc tgc atg gag tgg ctc caa gac cct ctg agc tgg 163 Ala Val Ala Asn Ile Cys Met Glu Trp Leu Gln Asp Pro Leu Ser Trp gtg ttc ctg ggc acg gtg tgc ttg gtg gtc ctg cag cag ctg cga cgt 211 Val Phe Leu Gly Thr Val Cys Leu Val Val Leu Gln Gln Leu Arg Arg cgg cgg ggc aaa gcg ccg ctt ccg cct ggg ccg aag ccg ctg ccg atc 259 Arg Arg Gly Lys Ala Pro Leu Pro Pro Gly Pro Lys Pro Leu Pro Ile gta ggc aac atg ggg atg atg gac cag ctg acg cac cgc ggg ctg gcg 307 Val Gly Asn Met Gly Met Met Asp Gln Leu Thr His Arg Gly Leu Ala gcg ctg gcg gag acg tac ggc ggg ctg ctg cac ctc cgg ctg ggg cgg 355 Ala Leu Ala Glu Thr Tyr Gly Gly Leu Leu His Leu Arg Leu Gly Arg ctg cac gcg ttc gcg gtg tcg acg ccc gag tac gcg cgc gag gtg ctg 403 Leu His Ala Phe Ala Val Ser Thr Pro Glu Tyr Ala Arg Glu Val Leu cag gcg cag gac ggc gcg ttc tcg aac cgg cct gcg acc gcg gcc atc 451 Gln Ala Gln Asp Gly Ala Phe Ser Asn Arg Pro Ala Thr Ala Ala Ile gcg tac ctg acg tac gac cgc gcg gac atg gcg ttc gcg cac tac ggg 499 Ala Tyr Leu Thr Tyr Asp Arg Ala Asp Met Ala Phe Ala His Tyr Gly ccc ttc tgg cgg cag atg cgc aag ctg tgc gtg atg aag ctg ttc agc 547 Pro Phe Trp Arg Gln Met Arg Lys Leu Cys Val Met Lys Leu Phe Ser cggcggcgc gccgagacg tgggcg gccgtg cgcgacgag tgcgcggcg 595 ArgArgArg AlaGluThr TrpAla AlaVal ArgAspGlu CysAlaAla ctggtccgg gccgtggcc gtgggc ggcggg agcgggggc gaagccgtg 643 LeuValArg AlaValAla ValGly GlyGly SerGlyGly GluAlaVal aacctgggc gagctcatc ttcagc ctgacg aagaacgtg acgttccgc 691 AsnLeuGly GluLeuIle PheSer LeuThr LysAsnVal ThrPheArg gcggcgttc ggcacccgc gacggc gagggc caggaggag ttcatcgcc 739 AlaAlaPhe GlyThrArg AspGly GluGly GlnGluGlu PheIleAla atcctgcag gagttctcc aagctg ttcggcgcc ttcaac gtgggcgac 787 IleLeuGln GluPheSer LysLeu PheGlyAla PheAsn ValGlyAsp ttcctaccg tggctgggt tggatg gacctgcag ggcatc aaccggcgc 835 PheLeuPro TrpLeuGly TrpMet AspLeuGln GlyIle AsnArgArg ttgcgcgcc gcccgctcc gcgctg gaccggttc atcgac aagatcatc 883 LeuArgAla AlaArgSer AlaLeu AspArgPhe IleAsp LysIleIle gacgagcac gtgaggcgc gggaag agccccgac gacgcc gacgccgac 931 AspGluHis ValArgArg GlyLys SerProAsp AspAla AspAlaAsp atggtcgac gacatgctc gcgttc ttcgtcgag gccacg cccggcaag 979 MetValAsp AspMetLeu AlaPhe PheValGlu AlaThr ProGlyLys gcgacgggc gccgccgcc getget gacggcggc gacgac ctgcacaac 1027 AlaThrGly AlaAlaAla AlaAla AspGlyGly AspAsp LeuHisAsn accctccgg ctcacgcgc gacaac atcaaggcc atcatc atggacgtg 1075 ThrLeuArg LeuThrArg AspAsn IleLysAla IleIle MetAspVal atgttcggc gggacggag acggtg gcgtcggcg atcgag tgggccatg 1123 MetPheGly GlyThrGlu ThrVal AlaSerAla IleGlu TrpAlaMet gcg gag atg atg cac agc ccc gac gac atg cgc cgg gtg cag cag gag 1171 Ala Glu Met Met His Ser Pro Asp Asp Met Arg Arg Val Gln Gln Glu ctc gcc gac gtc gtg ggc ctc gac cgc aac gtg agc gag tcg gac ctg 1219 Leu Ala Asp Val Val Gly Leu Asp Arg Asn Val Ser Glu Ser Asp Leu gac agg ctc ccc ttc ctc agg tgc gtc atc aag gag acg ctc cgg ctg 1267 Asp Arg Leu Pro Phe Leu Arg Cys Val Ile Lys Glu Thr Leu Arg Leu cac ccg ccc atc ccg ctg ctc ctc cac gag acc gcc gac gac tgc gtc 1315 His Pro Pro Ile Pro Leu Leu Leu His Glu Thr Ala Asp Asp Cys Val gtg gcc ggg tac tcc gtg ccc agg ggc tcc cgc gtc atg gtc aac gtc 1363 Val Ala Gly Tyr Ser Val Pro Arg Gly Ser Arg Val Met Val Asn Val tgg gcc atc ggc cgc cac cgc gcc tcg tgg aag gac gcc gac gcg ttc 1411 Trp Ala Ile Gly Arg His Arg Ala Ser Trp Lys Asp Ala Asp Ala Phe cgc ccg tcg cgg ttc gcg gcg ccc gag ggg gag gcc gcg ggg ctc gac 1459 Arg Pro Ser Arg Phe Ala Ala Pro Glu Gly Glu Ala Ala Gly Leu Asp ttc aag ggc ggg tgc ttc gag ttc ctg ccg ttc ggg tcg ggc cgc cgg 1507 Phe Lys Gly Gly Cys Phe Glu Phe Leu Pro Phe Gly Ser Gly Arg Arg tcc tgc ccc ggg atg gcg ctc ggc ctg tac gcg ctg gag ctc gcc gtc 1555 Ser Cys Pro Gly Met Ala Leu Gly Leu Tyr Ala Leu Glu Leu Ala Val gcc cag ctc gcg cac gcc ttc aac tgg tcg ctg ccc gac gga atg aag 1603 Ala Gln Leu Ala His Ala Phe Asn Trp Ser Leu Pro Asp Gly Met Lys ccc tcg gag atg gac atg ggc gac atc ttc ggc ctt acc gcg ccg cgc 1651 Pro Ser Glu Met Asp Met Gly Asp Ile Phe Gly Leu Thr Ala Pro Arg gcc acg cgg ctc tac gcc gtg cct acg ccc cgg ctc aac tgc ccc ttg 1699 Ala Thr Arg Leu Tyr Ala Val Pro Thr Pro Arg Leu Asn Cys Pro Leu tac tgacgccctg cacgtggcgc gcggggactg ccattacgca tgcatgcgtt 1752 Tyr tggactttggtgttcatccctggggtggggccgccgtgggggaagttaggagtttggtgg1812 ctttctagctctgtcttcttgtattctgtttattataaattttcccaacccttccatgcc1872 tgatcgatgttgcggtaataattgttagaaaatgtgacattttgtatgtaatcaatctat1932 ggggtgcaattgttatctcgtcaaaggacacaccactcgacttgcaccccttcatgtata1992 tatatacatacacgaacatcccctgcaataaagaaatcgctgtcactttctttcggaaaa2052 aaaaaaaaaaaaaaaaaaaaaaaagggcggccgc 2086 <210> 2 <211> 532 <212> PRT
<213> Zea mays <400> 2 Met Ala Ala Ala Val Ala Asn Ile Cys Met Glu Trp Leu Gln Asp Pro Leu Ser Trp Val Phe Leu Gly Thr Val Cys Leu Val Val Leu Gln Gln Leu Arg Arg Arg Arg Gly Lys Ala Pro Leu Pro Pro Gly Pro Lys Pro Leu Pro Ile Val Gly Asn Met Gly Met Met Asp Gln Leu Thr His Arg Gly Leu Ala Ala Leu Ala Glu Thr Tyr Gly Gly Leu Leu His Leu Arg Leu Gly Arg Leu His Ala Phe Ala Val Ser Thr Pro Glu Tyr Ala Arg Glu Val Leu Gln Ala Gln Asp Gly Ala Phe Ser Asn Arg Pro Ala Thr Ala Ala Ile Ala Tyr Leu Thr Tyr Asp Arg Ala Asp Met Ala Phe Ala His Tyr Gly Pro Phe Trp Arg Gln Met Arg Lys Leu Cys Val Met Lys Leu Phe Ser Arg Arg Arg Ala Glu Thr Trp Ala Ala Val Arg Asp Glu Cys Ala Ala Leu Val Arg Ala Val Ala Val Gly Gly Gly Ser Gly Gly Glu Ala Val Asn Leu Gly Glu Leu Ile Phe Ser Leu Thr Lys Asn Val Thr Phe Arg Ala Ala Phe Gly Thr Arg Asp Gly Glu Gly Gln Glu Glu Phe Ile Ala Ile Leu Gln Glu Phe Ser Lys Leu Phe Gly Ala Phe Asn Val Gly Asp Phe Leu Pro Trp Leu Gly Trp Met Asp Leu Gln Gly Ile Asn Arg Arg Leu Arg Ala Ala Arg Ser Ala Leu Asp Arg Phe Ile Asp Lys Ile Ile Asp Glu His Val Arg Arg Gly Lys Ser Pro Asp Asp Ala Asp Ala Asp Met Val Asp Asp Met Leu Ala Phe Phe Val Glu Ala Thr Pro Gly Lys Ala Thr Gly Ala Ala Ala Ala Ala Asp Gly Gly Asp Asp Leu His Asn Thr Leu Arg Leu Thr Arg Asp Asn Ile Lys Ala Ile Ile Met Asp Val Met Phe Gly Gly Thr Glu Thr Val Ala Ser Ala Ile Glu Trp Ala Met Ala Glu Met Met His Ser Pro Asp Asp Met Arg Arg Val Gln Gln Glu Leu Ala Asp Val Val Gly Leu Asp Arg Asn Val Ser Glu Ser Asp Leu Asp Arg Leu Pro Phe Leu Arg Cys Val Ile Lys Glu Thr Leu Arg Leu His Pro Pro Ile Pro Leu Leu Leu His Glu Thr Ala Asp Asp Cys Val Val Ala Gly Tyr Ser Val Pro Arg Gly Ser Arg Val Met Val Asn Val Trp Ala Ile Gly Arg His Arg Ala Ser Trp Lys Asp Ala Asp Ala Phe Arg Pro Ser Arg Phe Ala Ala Pro Glu Gly Glu Ala Ala Gly Leu Asp Phe Lys Gly Gly Cys Phe Glu Phe Leu Pro Phe Gly Ser Gly Arg Arg Ser Cys Pro Gly Met Ala Leu Gly Leu Tyr Ala Leu Glu Leu Ala Val Ala Gln Leu Ala His Ala Phe Asn Trp Ser Leu Pro Asp Gly Met Lys Pro Ser Glu Met Asp Met Gly Asp Ile Phe Gly Leu Thr Ala Pro Arg Ala Thr Arg Leu Tyr Ala Val Pro Thr Pro Arg Leu Asn Cys Pro Leu Tyr <210> 3 <211> 25 <212> DNA
<213> Zea mays <400> 3 atggcggcgg ccgtggccaa tatct 25 <210> 4 <211> 25 <212> DNA
<213> Zea mays <400> 4 tcagtacaag gggcagttga gccgg 25 <210> 5 <211> 1859 <212> DNA
<213> Zea mays <220>
<221> CDS
<222> (79)...(1656) <400> 5 gtcgacccac gcgtccgcta aaacccaaac gaaataaaag gaagaggtca aaaaataaaa 60 ggtgttgtgc aatcgatc atg gtg acc gtg gcc aag atc gcc atg gag tgg 111 Met Val Thr Val Ala Lys Ile Ala Met Glu Trp ctc caa gac cct ctg agc tgg gtg ttc ctg ggc acg ctg gcc ttg gtg 159 Leu Gln Asp Pro Leu Ser Trp Val Phe Leu Gly Thr Leu Ala Leu Val gtc ctg cag ctg cga cga cgg ggc aaa gcg ccg ctg ccg ccc ggg ccg 207 Val Leu Gln Leu Arg Arg Arg Gly Lys Ala Pro Leu Pro Pro Gly Pro aag ccg ctg ccg atc gtg ggc aac atg gcg atg atg gac cag ctg acc 255 Lys Pro Leu Pro Ile Val Gly Asn Met Ala Met Met Asp Gln Leu Thr cac cgc ggg ctg gcg gcg ctg gcc gag agg tac ggc ggg ctg ctg cac 303 His Arg Gly Leu Ala Ala Leu Ala Glu Arg Tyr Gly Gly Leu Leu His ctc cgc ctg ggc cgg ctg cac gcg ttc gcg gtg tcg acg ccc gag tac 351 Leu Arg Leu Gly Arg Leu His Ala Phe Ala Val Ser Thr Pro Glu Tyr gcg cgc gag gtg ctg cag gcg cag gac ggc gcg ttc tcg aac cgg ccg 399 Ala Arg Glu Val Leu Gln Ala Gln Asp Gly Ala Phe Ser Asn Arg Pro gcc act atc gcc atc gcg tac ctg acg tac gac cgc gcc gac atg gcg 447 Ala Thr Ile Ala Ile Ala Tyr Leu Thr Tyr Asp Arg Ala Asp Met Ala ttc gcg cac tac ggg ccc ttc tgg cgc cag atg cgc aag ctg tgc gtg 495 Phe Ala His Tyr Gly Pro Phe Trp Arg Gln Met Arg Lys Leu Cys Val atg aag ctg ttc agc cgg cgc cgc gcc gag acg tgg gtg gcc gtg cgc 543 Met Lys Leu Phe Ser Arg Arg Arg Ala Glu Thr Trp Val Ala Val Arg gac gag tgc gcg gcg ctg gtc cgc gcc gtg gcg tcc ggc ggc ggc ggc 591 Asp Glu Cys Ala Ala Leu Val Arg Ala Val Ala Ser Gly Gly Gly Gly ggc ggc gag gcc gtg aac ctg ggc gag ctc atc ttc aac ctg acc aag 639 Gly Gly Glu Ala Val Asn Leu Gly Glu Leu Ile Phe Asn Leu Thr Lys aac gtg acg ttc cgc gcc gcc ttc ggc acc cgc gac ggc gag gac cag 687 Asn Val Thr Phe Arg Ala Ala Phe Gly Thr Arg Asp Gly Glu Asp Gln gag gag ttc atc gcc atc ctg cag gag ttc tcg aag ctg ttc ggc gcc 735 Glu Glu Phe Ile Ala Ile Leu Gln Glu Phe Ser Lys Leu Phe Gly Ala ttc aac gtc gtc gac ttc ctg ccg tgg ctg agc tgg atg gac ctg cag 783 Phe Asn Val Val Asp Phe Leu Pro Trp Leu Ser Trp Met Asp Leu Gln ggc atc aac cgc cgc ctc cgc gcc gca cga tcc gcg ctg gac cgg ttc 831 Gly Ile Asn Arg Arg Leu Arg Ala Ala Arg Ser Ala Leu Asp Arg Phe atc gac aag atc atc gac gag cac gtg agg cgg ggg aag aac ccc gac 879 Ile Asp Lys Ile Ile Asp Glu His Val Arg Arg Gly Lys Asn Pro Asp gac gcc gac gcc gac atg gtc gac gac atg ctc gcc ttc ttc gcc gag 927 Asp Ala Asp Ala Asp Met Val Asp Asp Met Leu Ala Phe Phe Ala Glu gcc aag ccg ccc aag aag ggg ccc gcc gcc gcc gcg gac ggt gac gac 975 Ala Lys Pro Pro Lys Lys Gly Pro Ala Ala Ala Ala Asp Gly Asp Asp ctg cac aac acc ctc cgg ctc acg cgc gac aat atc aag get atc atc 1023 Leu His Asn Thr Leu Arg Leu Thr Arg Asp Asn Ile Lys Ala Ile Ile atg gac gtg atg ttt ggc ggg acg gag acg gtg gcg tcg gcg atc gag 1071 Met Asp Val Met Phe Gly Gly Thr Glu Thr Val Ala Ser Ala Ile Glu tgg gcg atg gcg gag atg atg cac agc ccc gac gac ctg cgc cgg ctg 1119 Trp Ala Met Ala Glu Met Met His Ser Pro Asp Asp Leu Arg Arg Leu cag cag gag ctc gcc gac gtc gtg ggc ctg gac cgg aac gtg aac gag 1167 Gln Gln Glu Leu Ala Asp Val Val Gly Leu Asp Arg Asn Val Asn Glu tcg gac ctg gac aag ctc ccc ttc ctc aag tgc gtc atc aag gag acg 1215 Ser Asp Leu Asp Lys Leu Pro Phe Leu Lys Cys Val Ile Lys Glu Thr ctc cgg ctg cac ccg ccg atc ccg ctg ctc ctg cac gag acc gcc ggc 1263 Leu Arg Leu His Pro Pro Ile Pro Leu Leu Leu His Glu Thr Ala Gly gac tgc gtc gtg ggc ggc tac tcc gtg ccc agg ggc tcc cgc gtc atg 1311 Asp Cys Val Val Gly Gly Tyr Ser Val Pro Arg Gly Ser Arg Val Met gtc aac gtg tgg gcc atc ggc cgc cac cgc gcc tcg tgg aag gac gcc 1359 Val Asn Val Trp Ala Ile Gly Arg His Arg Ala Ser Trp Lys Asp Ala gac gcg ttc cgg ccg tcg cgc ttc acg ccc gag ggc gag gcc gcg ggg 1407 Asp Ala Phe Arg Pro Ser Arg Phe Thr Pro Glu Gly Glu Ala Ala Gly ctc gac ttc aag ggc ggc tgc ttc gag ttc ctg ccc ttc ggc tcc ggc 1455 Leu Asp Phe Lys Gly Gly Cys Phe Glu Phe Leu Pro Phe Gly Ser Gly cgc cgc tcg tgc ccc ggc acg gcg ctg ggc ctg tac gcg ctg gag ctc 1503 Arg Arg Ser Cys Pro Gly Thr Ala Leu Gly Leu Tyr Ala Leu Glu Leu gcc gtc gcc cag ctc gcg cac ggc ttc aac tgg tcg ctg ccc gac ggc 1551 Ala Val Ala Gln Leu Ala His Gly Phe Asn Trp Ser Leu Pro Asp Gly atg aag ccc tcg gag ctg gac atg ggc gac gtc ttc ggc ctc acc gcg 1599 Met Lys Pro Ser Glu Leu Asp Met Gly Asp Val Phe Gly Leu Thr Ala ccg cgc gcc acg agg ctc tac gcc gtg cct acg ccc cgg ctc aac tgc 1647 Pro Arg Ala Thr Arg Leu Tyr Ala Val Pro Thr Pro Arg Leu Asn Cys ccc ttg tac tgacgccatg cgcgggcgac tgccattacc atcgtcccct 1696 Pro Leu Tyr cgggtgggtg tggggtacgg gggtaggagt ttggtgcctt tctctgtcgt cttttttccc 1756 tttaaaaaac atgcctggtc gatgttgtag ggtgtgttgt agacagccat tatcaatttt 1816 ttttattctc aaaaaaaaaa aaaaaaaaaa aaagggcggc cgc 1859 <210> 6 <211> 526 <212> PRT
<213> Zea mays <400> 6 Met Val Thr Val Ala Lys Ile Ala Met Glu Trp Leu Gln Asp Pro Leu Ser Trp Val Phe Leu Gly Thr Leu Ala Leu Val Val Leu Gln Leu Arg Arg Arg Gly Lys Ala Pro Leu Pro Pro Gly Pro Lys Pro Leu Pro Ile Val Gly Asn Met Ala Met Met Asp Gln Leu Thr His Arg Gly Leu Ala Ala Leu Ala Glu Arg Tyr Gly Gly Leu Leu His Leu Arg Leu Gly Arg Leu His Ala Phe Ala Val Ser Thr Pro Glu Tyr Ala Arg Glu Val Leu Gln Ala Gln Asp Gly Ala Phe Ser Asn Arg Pro Ala Thr Ile Ala Ile Ala Tyr Leu Thr Tyr Asp Arg Ala Asp Met Ala Phe Ala His Tyr Gly Pro Phe Trp Arg Gln Met Arg Lys Leu Cys Val Met Lys Leu Phe Ser Arg Arg Arg Ala Glu Thr Trp Val Ala Val Arg Asp Glu Cys Ala Ala Leu Val Arg Ala Val Ala Ser Gly Gly Gly Gly Gly Gly Glu Ala Val Asn Leu Gly Glu Leu Ile Phe Asn Leu Thr Lys Asn Val Thr Phe Arg Ala Ala Phe Gly Thr Arg Asp Gly Glu Asp Gln Glu Glu Phe Ile Ala Ile Leu Gln Glu Phe Ser Lys Leu Phe Gly Ala Phe Asn Val Val Asp Phe Leu Pro Trp Leu Ser Trp Met Asp Leu Gln Gly Ile Asn Arg Arg Leu Arg Ala Ala Arg Ser Ala Leu Asp Arg Phe Ile Asp Lys Ile Ile Asp Glu His Val Arg Arg Gly Lys Asn Pro Asp Asp Ala Asp Ala Asp Met Val Asp Asp Met Leu Ala Phe Phe Ala Glu Ala Lys Pro Pro Lys Lys Gly Pro Ala Ala Ala Ala Asp Gly Asp Asp Leu His Asn Thr Leu Arg Leu Thr Arg Asp Asn Ile Lys Ala Ile Ile Met Asp Val Met Phe Gly Gly Thr Glu Thr Val Ala Ser Ala Ile Glu Trp Ala Met Ala Glu Met Met His Ser Pro Asp Asp Leu Arg Arg Leu Gln Gln Glu Leu Ala Asp Val Val Gly Leu Asp Arg Asn Val Asn Glu Ser Asp Leu Asp Lys Leu Pro Phe Leu Lys Cys Val Ile Lys Glu Thr Leu Arg Leu His Pro Pro Ile Pro Leu Leu Leu His Glu Thr Ala Gly Asp Cys Val Val Gly Gly Tyr Ser Val Pro Arg Gly Ser Arg Val Met Val Asn Val Trp Ala Ile Gly Arg His Arg Ala Ser Trp Lys Asp Ala Asp Ala Phe Arg Pro Ser Arg Phe Thr Pro Glu Gly Glu Ala Ala Gly Leu Asp Phe Lys Gly Gly Cys Phe Glu Phe Leu Pro Phe Gly Ser Gly Arg Arg Ser Cys Pro Gly Thr Ala Leu Gly Leu Tyr Ala Leu Glu Leu Ala Val Ala Gln Leu Ala His Gly Phe Asn Trp Ser Leu Pro Asp Gly Met Lys Pro Ser Glu Leu Asp Met Gly Asp Val Phe Gly Leu Thr Ala Pro Arg Ala Thr Arg Leu Tyr Ala Val Pro Thr Pro Arg Leu Asn Cys Pro Leu Tyr <210> 7 <211> 25 <212> DNA
<213> Zea mays <400> 7 atggtgaccg tggccaagat cgcca 25 <210> 8 <211> 25 <212> DNA
<213> Zea mays <400> 8 tcagtacaag gggcagttga gccgg 25 <210> 9 <211> 1294 <212> DNA
<213> Zea mays <220>
<221> CDS
<222> (108)...(1103) <400> 9 gtcgacccac gcgtccgcgg acgcgtgggc ggacgcgtgg gcggacgcgt gggcaggtct 60 ccagcgaagc aggctcaggc gcaacgcaac aacacaaggc ggcggtt atg gcc tct 116 Met Ala Ser get get gcg acg get acg atg ggg acg ggg aag gtg gtt tgc gtc acc 164 Ala Ala Ala Thr Ala Thr Met Gly Thr Gly Lys Val Val Cys Val Thr ggc gca tca ggg tac atc gcg tcc tgg att gtc agg ctc cta ctc gac 212 Gly Ala Ser Gly Tyr Ile Ala Ser Trp Ile Val Arg Leu Leu Leu Asp cgc ggc tac acc gtc cgc gcc acc gtg cga gac acc get gac cca aag 260 Arg Gly Tyr Thr Val Arg Ala Thr Val Arg Asp Thr Ala Asp Pro Lys aaa aca ttg cac ctg act gcg ttg gat ggt get aag gat agg ctg cac 308 Lys Thr Leu His Leu Thr Ala Leu Asp Gly Ala Lys Asp Arg Leu His tta ttt aaa get agc ctg cta gaa gag ggt tct ttc gat get gca gtt 356 Leu Phe Lys Ala Ser Leu Leu Glu Glu Gly Ser Phe Asp Ala Ala Val cat gga tgc gac act gta ttt cat act gcc tct ccc ttt tat cac aat 404 His Gly Cys Asp Thr Val Phe His Thr Ala Ser Pro Phe Tyr His Asn gtc aag gat get aag gca gag tta ctt gac cca gca gtt aag gga aca 452 Val Lys Asp Ala Lys Ala Glu Leu Leu Asp Pro Ala Val Lys Gly Thr ctc aat gtt ctc ggt tca tgc aag aaa get tct att aaa aag gtg gtt 500 Leu Asn Val Leu Gly Ser Cys Lys Lys Ala Ser Ile Lys Lys Val Val gta aca tca tct atg get get gta gcc tac aac agg agg cca agg act 548 Val Thr Ser Ser Met Ala Ala Val Ala Tyr Asn Arg Arg Pro Arg Thr cct gag gtc aca gtt gac gag aca tgg ttt tct gat cca caa att tgt 596 Pro Glu Val Thr Val Asp Glu Thr Trp Phe Ser Asp Pro Gln Ile Cys gaa aca aat cag caa tgg tat att ttg tcc aag acg ctg gca gag gag 644 Glu Thr Asn Gln Gln Trp Tyr Ile Leu Ser Lys Thr Leu Ala Glu Glu get get tgg aag ttc tca agg gat aat gga ctt gaa atc gtt act ata 692 Ala Ala Trp Lys Phe Ser Arg Asp Asn Gly Leu Glu Ile Val Thr Ile aac cca gcg atg gtt att ggt ccc ctg ctg cag cct aca cta aat acc 740 Asn Pro Ala Met Val Ile Gly Pro Leu Leu Gln Pro Thr Leu Asn Thr agt get gaa gca atc ctg aag tta atc aat ggg tca tcg tct aca tat 788 Ser Ala Glu Ala Ile Leu Lys Leu Ile Asn Gly Ser Ser Ser Thr Tyr ccc aat ttt tgc ttt gga tgg gtt aat gtc aag gat gtt gcc ctg gcc 836 Pro Asn Phe Cys Phe Gly Trp Val Asn Val Lys Asp Val Ala Leu Ala cat atc ctt gca tac gag gtt ccc tca tca aat gga agg tac tgc atg 884 His Ile Leu Ala Tyr Glu Val Pro Ser Ser Asn Gly Arg Tyr Cys Met gtg gaa aga gtt gtt cac tac tca gaa ctg gtg aac att atc cgc aac 932 Val Glu Arg Val Val His Tyr Ser Glu Leu Val Asn Ile Ile Arg Asn atg tat cct acc ctt cct ctt ccg gac aag tgt gca gat gac aag ccg 980 Met Tyr Pro Thr Leu Pro Leu Pro Asp Lys Cys Ala Asp Asp Lys Pro ttt gtc cca ccc tac caa gta tca aag gag aag ata aaa agc ata ggc 1028 Phe Val Pro Pro Tyr Gln Val Ser Lys Glu Lys Ile Lys Ser Ile Gly atc gag ttg att cca ctg gag acg agc gtc aag gag acc atc gaa agc 1076 Ile Glu Leu Ile Pro Leu Glu Thr Ser Val Lys Glu Thr Ile Glu Ser ttg aaa gag aag ggg ttc get agt ttt tgactcgagc aagctgtgaa 1123 Leu Lys Glu Lys Gly Phe Ala Ser Phe acataagtgt tactgtacta cgtgatgttg cattgtatac atgcatcgga cttcgcagtt 1183 cccaagctat tttggtagct ttggtctgta agattaccta aaataaactg agccacgttg 1243 ccacgctgta gcttttggaa aaaaaaaaaa aaaaaaaaag ggcggccgct c 1294 <210> 10 <211> 332 <212> PRT
<213> Zea mays <400> 10 Met Ala Ser Ala Ala Ala Thr Ala Thr Met Gly Thr Gly Lys Val Val Cys Val Thr Gly Ala Ser Gly Tyr Ile Ala Ser Trp Ile Val Arg Leu Leu Leu Asp Arg Gly Tyr Thr Val Arg Ala Thr Val Arg Asp Thr Ala Asp Pro Lys Lys Thr Leu His Leu Thr Ala Leu Asp Gly Ala Lys Asp Arg Leu His Leu Phe Lys Ala Ser Leu Leu Glu Glu Gly Ser Phe Asp Ala Ala Val His Gly Cys Asp Thr Val Phe His Thr Ala Ser Pro Phe Tyr His Asn Val Lys Asp Ala Lys Ala Glu Leu Leu Asp Pro Ala Val Lys Gly Thr Leu Asn Val Leu Gly Ser Cys Lys Lys Ala Ser Ile Lys Lys Val Val Val Thr Ser Ser Met Ala Ala Val Ala Tyr Asn Arg Arg Pro Arg Thr Pro Glu Val Thr Val Asp Glu Thr Trp Phe Ser Asp Pro Gln Ile Cys Glu Thr Asn Gln Gln Trp Tyr Ile Leu Ser Lys Thr Leu Ala Glu Glu Ala Ala Trp Lys Phe Ser Arg Asp Asn Gly Leu Glu Ile Val Thr Ile Asn Pro Ala Met Val Ile Gly Pro Leu Leu Gln Pro Thr Leu Asn Thr Ser Ala Glu Ala Ile Leu Lys Leu Ile Asn Gly Ser Ser Ser Thr Tyr Pro Asn Phe Cys Phe Gly Trp Val Asn Val Lys Asp Val Ala Leu Ala His Ile Leu Ala Tyr Glu Val Pro Ser Ser Asn Gly Arg Tyr Cys Met Val Glu Arg Val Val His Tyr Ser Glu Leu Val Asn Ile Ile Arg Asn Met Tyr Pro Thr Leu Pro Leu Pro Asp Lys Cys Ala Asp Asp Lys Pro Phe Val Pro Pro Tyr Gln Val Ser Lys Glu Lys Ile Lys Ser Ile Gly Ile Glu Leu Ile Pro Leu Glu Thr Ser Val Lys Glu Thr Ile Glu Ser Leu Lys Glu Lys Gly Phe Ala Ser Phe <210> 11 <211> 25 <212> DNA
<213> Zea mays <400> 11 atggcctctg ctgctgcgac ggcta 25 <210> 12 <211> 25 <212> DNA
<213> Zea mays <400> 12 tcaaaaacta gcgaacccct tctct 25 <210> 13 <211> 1218 <212> DNA
<213> Zea mays <220>
<221> CDS
<222> (112)...(900) <400> 13 gtcgacccac gcgtccgata cccgacgcgc aaccagtgcc gcacccagac cagatctccg 60 cgacatatca gtcgttcgtc cagctaactg cactgcactg cactgcacgc a atg gcc 117 Met Ala acc acg gcg acc gag gcg gcc aag get gca ccg gcg cag gag cag cag 165 Thr Thr Ala Thr Glu Ala Ala Lys Ala Ala Pro Ala Gln Glu Gln Gln gcc aac ggc aac ggc aac ggc gag cag aag acg cgc cac tcc gag gtc 213 Ala Asn Gly Asn Gly Asn Gly Glu Gln Lys Thr Arg His Ser Glu Val ggc cac aag agc ctg ctc aag agc gac gac ctg tac cag tac atc ctg 261 Gly His Lys Ser Leu Leu Lys Ser Asp Asp Leu Tyr Gln Tyr Ile Leu gac acg agc gtg tac ccg cgg gag ccg gag agc atg aag gag ctg cgc 309 Asp Thr Ser Val Tyr Pro Arg Glu Pro Glu Ser Met Lys Glu Leu Arg gagatcaccgcc aagcaccca tggaacctg atgaccacc tccgcc gac 357 GluIleThrAla LysHisPro TrpAsnLeu MetThrThr SerAla Asp gagggccagttc ctcaacatg ctcatcaag ctcatcggc gccaag aag 405 GluGlyGlnPhe LeuAsnMet LeuIleLys LeuIleGly AlaLys Lys accatggagatc ggcgtctac accggctac tcgctcctc gccacc gcg 453 ThrMetGluIle GlyValTyr ThrG1yTyr SerLeuLeu AlaThr Ala ctcgcactcccg gaggacggc acgatcttg gccatggac atcaac cgc 501 LeuAlaLeuPro GluAspGly ThrIleLeu AlaMetAsp IleAsn Arg gagaactacgag ctaggcctt ccctgcatc aacaaggcc ggcgtg ggc 549 GluAsnTyrGlu LeuGlyLeu ProCysIle AsnLysAla GlyVal Gly cacaagatcgac ttccgcgag ggccccgcg ctccccgtc ctggac gac 597 HisLysIleAsp PheArgGlu GlyProAla LeuProVal LeuAsp Asp ctc gtg gcg gac aag gag cag cac ggg tcg ttc gac ttc gcc ttc gtg 645 Leu Val Ala Asp Lys Glu Gln His Gly Ser Phe Asp Phe Ala Phe Val gac gcc gac aag gac aac tac ctc agc tac cac gag cgg ctc ctg aag 693 Asp Ala Asp Lys Asp Asn Tyr Leu Ser Tyr His Glu Arg Leu Leu Lys ctg gtg agg ccc ggc ggc ctc atc ggc tac gac aac acg ctg tgg aac 741 Leu Val Arg Pro Gly Gly Leu Ile Gly Tyr Asp Asn Thr Leu Trp Asn ggc tcc gtc gtg ctc ccc gac gac gcg ccc atg cgc aag tac atc cgc 789 Gly Ser Val Val Leu Pro Asp Asp Ala Pro Met Arg Lys Tyr Ile Arg ttc tac cgc gac ttc gtc ctc gcc ctc aac agc gcg ctc gcc gcc gac 837 Phe Tyr Arg Asp Phe Val Leu Ala Leu Asn Ser Ala Leu Ala Ala Asp gac cgc gtc gag atc tgc cag ctc ccc gtc ggc gac ggc gtc acg ctc 885 Asp Arg Val Glu Ile Cys Gln Leu Pro Val Gly Asp Gly Val Thr Leu tgc cgc cgc gtc aag tgaaaaaaag aagaagaaga aaaaaaacat aataccctgc 940 Cys Arg Arg Val Lys gttcctgctgccccggctgtctggcccccactactgccaccgacggcggc gccgaacccc1000 cgttccaatcatcatatcgtagacgacgcgcagcattaaactatcaatca ccggatctgg1060 ctctttcttggccctgtactgtactattaatgttccgttcttgttttttt attcggaatt1120 gtcgccgtttcagtatacgtaaatctcgaggtcgataatacagtaatact accaatttaa1180 ctgtataaaaaaaaaaaaaaaaaaaaaagggcggccgc 1218 <210> 14 <211> 263 <212> PRT
<213> Zea mays <400> 14 Met Ala Thr Thr Ala Thr Glu Ala Ala Lys Ala Ala Pro Ala Gln Glu Gln Gln Ala Asn Gly Asn Gly Asn Gly Glu Gln Lys Thr Arg His Ser Glu Val Gly His Lys Ser Leu Leu Lys Ser Asp Asp Leu Tyr Gln Tyr Ile Leu Asp Thr Ser Val Tyr Pro Arg Glu Pro Glu Ser Met Lys Glu Leu Arg Glu Ile Thr Ala Lys His Pro Trp Asn Leu Met Thr Thr Ser Ala Asp Glu Gly Gln Phe Leu Asn Met Leu Ile Lys Leu Ile Gly Ala Lys Lys Thr Met Glu Ile Gly Val Tyr Thr Gly Tyr Ser Leu Leu Ala Thr Ala Leu Ala Leu Pro Glu Asp Gly Thr Ile Leu Ala Met Asp Ile Asn Arg Glu Asn Tyr Glu Leu Gly Leu Pro Cys Ile Asn Lys Ala Gly Val Gly His Lys Ile Asp Phe Arg Glu Gly Pro Ala Leu Pro Val Leu Asp Asp Leu Val Ala Asp Lys Glu Gln His Gly Ser Phe Asp Phe Ala Phe Val Asp Ala Asp Lys Asp Asn Tyr Leu Ser Tyr His Glu Arg Leu Leu Lys Leu Val Arg Pro Gly Gly Leu Ile Gly Tyr Asp Asn Thr Leu Trp Asn Gly Ser Val Val Leu Pro Asp Asp Ala Pro Met Arg Lys Tyr Ile Arg Phe Tyr Arg Asp Phe Val Leu Ala Leu Asn Ser Ala Leu Ala Ala Asp Asp Arg Val Glu Ile Cys Gln Leu Pro Val Gly Asp Gly Val Thr Leu Cys Arg Arg Val Lys <210> 15 <211> 25 <212> DNA
<213> Zea mays <400> 15 atggccacca cggcgaccga ggcgg 25 <210> 16 <211> 25 <212> DNA
<213> Zea mays <400> 16 tcacttgacg cggcggcaga gcgtg 25

Claims

WHAT IS CLAIMED IS:

1. An isolated nucleic acid comprising a member selected from the group consisting of:
(a) a polynucleotide having at least 80% sequence identity, as determined by the BLAST 2.0 algorithm under default parameters, to a polynucleotide encoding a polypeptide selected from the group consisting of SEQ ID NOS: 2, 6, 10, and 14;
(b) a polynucleotide encoding a polypeptide selected from the group consisting of SEQ ID NOS: 2, 6, 10, and 14;
(c) a polynucleotide amplified from a Zea mays nucleic acid library using primers which selectively hybridize, under stringent hybridization conditions, to loci within a polynucleotide selected from the group consisting of SEQ ID
NOS: 1, 5, 9, and 13;
(d) a polynucleotide which selectively hybridizes, under stringent hybridization conditions and a wash in 2X SSC at 50°C, to a polynucleotide selected from the group consisting of SEQ ID NOS: 1, 5, 9, and 13;
(e) a polynucleotide selected from the group consisting of SEQ ID NOS: 1, 5, 9, and 13;
(f) a polynucleotide which is complementary to a polynucleotide of (a), (b), (c), (d), or (e); and (g) a polynucleotide comprising at least 25 contiguous nucleotides from a polynucleotide of (a), (b), (c), (d), (e), or (f).

2. A recombinant expression cassette, comprising a member of claim 1 operably linked, in sense or anti-sense orientation, to a promoter.

3. A host cell comprising the recombinant expression cassette of claim 2.

4. A transgenic plant comprising a recombinant expression cassette of claim 2.

5. The transgenic plant of claim 4, wherein said plant is a monocot.

6. The transgenic plant of claim 4, wherein said plant is selected from the group consisting of: maize, soybean, sunflower, sorghum, canola, wheat, alfalfa, cotton, rice, barley, and millet.

7. A transgenic seed from the transgenic plant of claim 4.

8. A method of modulating the level of lignin biosynthesis in a plant, comprising:
(a) introducing into a plant cell a recombinant expression cassette comprising a lignin biosynthesis polynucleotide of claim 1 operably linked to a promoter;
(b) culturing the plant cell under plant cell growing conditions; and (c) inducing expression of said polynucleotide for a time sufficient to modulate the level of lignin biosynthesis in said plant.

9. The method of claim 8, wherein the plant is selected from the group consisting of: maize, soybean, sunflower, sorghum, canola, wheat, alfalfa, cotton, rice, barley, and millet.

10. An isolated protein comprising a member selected from the group consisting of:
(a) polypeptide of at least 20 contiguous amino acids from a polypeptide selected from the group consisting of SEQ ID NOS: 2, 6, 10, and 14;
(b) a polypeptide selected from the group consisting of SEQ ID NOS: 2, 6, 10, and 14;
(c) a polypeptide having at least 80% sequence identity to, and having at least one linear epitope in common with, a polypeptide selected from the group consisting of SEQ ID NOS: 2, 6, 10, and 14, wherein said sequence identity is determined using BLAST 2.0 under default parameters; and, at least one polypeptide encoded by a member of claim 1.