WO1999064562A2 - Expression control elements from genes encoding starch branching enzymes - Google Patents

Abstract

Nucleic acid molecules for directing and modulating gene expression in transformed cells are disclosed. The molecules comprise 5' and 3' regulatory sequences isolated from genomic clones having homology in their coding regions with maize genes encoding Type I and Type IIB starch branching enzymes. Chimeric genes comprising these expression controlling nucleic acid molecules, and cells transformed with such chimeric genes, are also disclosed.

Description

EXPRESSION CONTROL ELEMENTS FROM GENES ENCODING STARCH BRANCHING ENZYMES

Pursuant to 35 U.S.C. §202 (c) , it is acknowledged that the U.S. Government has certain rights in the invention described herein, which was made in part with funds from the Department of Energy, Grant No. DE- FG02-96ER20234.

This application claims priority to U.S. Provisional Application Serial Nos. 60/089,049 and 60/089,050, filed June 12, 1998, the entireties of which are incorporated by reference herein.

FIELD OF THE INVENTION

The present invention relates to the field of genetic manipulation in plants. In particular, the invention provides a novel transcription and translation control elements isolated from genes encoding starch branching enzymes .

BACKGROUND OF THE INVENTION

Various publications or patents are referenced in this application to describe the state of the art to which the invention pertains. Each of these publications or patents is incorporated by reference herein. Starch provides carbon and energy for vegetative and reproductive development of most higher plants. It is found as a water-insoluble granule which is mainly composed of two different polysaccharides, amylose and amylopectin. Amylose is considered to consist of linear α- 1,4 -linked glucose chains of about 1,000 residues long. However, amylopectin is a more highly branched macromolecule consisting of linear α-1,4- glucose chains with α-1 , 6-glucosidic bonds at branch points .

Starch is synthesized in higher plants through the action of four classes of enzymes, ADP-glucose pyrophosphorylase (EC 2.7.7.23), starch synthase (EC 2.4.1.21), starch branching enzyme (EC 2.4.1.28) and starch debranching enzyme (EC 3.2.1.41). Starch branching enzymes (SBE) catalyze the formation of α-1, 6 glucan linkages, thereby playing an important role in the synthesis of the amylopectin fraction of starch.

Multiple forms of starch branching enzymes (SBE) differing in enzymatic and biochemical properties have been identified and characterized in various plants. Three SBE isoforms, SBEI, Ila and lib, have been isolated and characterized in maize. Isolation of the maize cDNAs encoding the SBE isoforms ( Sbel , 2a and 2b) enabled the investigation of the Sjbe genes at molecular level (Fisher et al., Plant Physiol . 102: 1045-1046, 1993; Fisher et al., Plant Physiol. 108: 1313-1314, 1995; Gao et al . , Plant Mol. Biol. 30: 1223-1232, 1996; Plant Physiol. 114 : 69-78, 1997). Fisher et al . (Plant Physiol. 110: 611- 619, 1996) determined that SBEIIa and lib are the product of separate genes, and Gao et al . (1996, 1997, supra) demonstrated that the Sbe genes are differentially expressed during kernel development and in various tissues, suggesting that they play distinct roles in starch biosynthesis. For example, while SJbel and 2a are expressed in vegetative tissues, Ae is not. Moreover, unlike Sbel and 2b, Sbe2a is more highly expressed in embryos than in endosperm. However, all three genes are highly expressed in developing maize seed from mid to late development in both embryo and endosperm tissue. Developmental and tissue specificity of gene expression is governed by the expression regulatory regions of a gene. Typically, these include promoters, enhancers, 5' untranslated leaders, translation terminators and polyadenylation signals, among others. These cis-acting sequences control gene expression by regulating the timing, location and amount of transcription of the gene and/or stability or efficiency of translation of the encoded mRNA .

As maize seed is a major global food source, the ability to engineer protein production in seed will have many applications in agriculture and industry. High-level and tissue-specific expression regulatory sequences will be needed for the development of such genetically engineered plants. The expression regulatory sequences of the above-described Sjbe and Ae genes from maize would be well suited for such applications, but heretofore have been unavailable.

SUMMARY OF THE INVENTION According to one aspect of the present invention, an isolated nucleic acid molecule for controlling expression of genes in transformed plant cells is provided, which comprises a segment of an Sjbel gene from Zea mays or related species . In a preferred embodiment, the nucleic acid molecule is isolated from a gene having a coding sequence at least 60% homologous with the coding sequence defined by the exons of SEQ ID NO:l. In one embodiment, the segment begins at a location about 3,000 bases upstream from the transcription initiation site of the gene, and ends at a location about 250 bases downstream from the transcription initiation site. The molecule contains one or more specific regions for effecting high level expression or sugar regulation. In another embodiment, the segment comprises a 3 ' untranslated region commencing at a stop codon for the gene's coding sequence, and ending at a location about 5900 bases downstream from the gene's transcription initiation site.

According to another aspect of the invention, a DNA segment for effecting expression of coding sequences operably linked to the segment is provided, which is isolated from a gene whose coding region hybridizes under stringent conditions with a coding region defined by exons of SEQ ID NO:l. Preferably, the gene is a maize Sjbel gene. In one embodiment, the segment comprises a promoter and a transcription initiation site, and may further comprise (1) an element included in the first exon of SEQ ID NO:l, which is capable of increasing promoter activity of homologous or heterologous promoters operably linked thereto, or (2) an element that confers sugar-regulatability on expression of the coding sequence. In another embodiment, the segment comprises a 3' untranslated region of the gene.

According to another aspect of the invention, another isolated nucleic acid molecule for controlling expression of genes in transformed plant cells is provided, which comprises a segment of a plant Ae gene. In a preferred embodiment, the nucleic acid molecule is isolated from a gene having a coding sequence at least 60% homologous with the coding sequence defined by the exons of SEQ ID NO : 2 , and most preferably it is a maize gene. In one embodiment, the segment begins at a location about 3,000 bases upstream from a transcription initiation site of the gene, and ends at a location about 100 bases downstream from the transcription initiation site. In another embodiment, the segment comprises a 3' untranslated region commencing at a stop codon for gene ' s coding sequence, and ending at a location about 20,500 bases downstream from the gene's transcription initiation site . In another aspect of the invention, a DNA segment for effecting expression of coding sequences operably linked to the segment is provided, which is isolated from a gene whose coding region hybridizes under stringent conditions with a coding region defined by exons of SEQ ID NO : 2. Preferably, the gene is a maize Ae gene. In one embodiment, the segment comprises a promoter and a transcription initiation site. In another embodiment, the segment comprises a 3' untranslated region. According to another aspect of the invention, a chimeric gene comprising a coding sequence operably linked to one or more of the aforementioned expression- regulatory sequences is provided. The chimeric gene preferably is inserted into a vector for transforming cells. Cells transformed with the vector are provided. In a preferred embodiment, they are plant cells, and are regenerated into fertile transgenic plants.

These and other features and advantages of the present invention will be described in greater detail in the description and examples set forth below.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1. Structure of the lambda clone 5-1-1 containing the Sjbel gene. Fig. 1A. Restriction map of the 5-1-1 clone. pBI5-l and pBI5-2 indicate subclones in plasmid pBluescript' SK^" . Fig. IB. Genomic structure of the SJbel gene. The thin black lines indicate the 5'- or 3 '-flanking sequences of the Sbel gene. The solid black boxes indicate exons and the open boxes denote introns . The numbers represent positions relative to the transcription initiation site (+1) .

Figure 2. Nucleotide sequence of the Sjbel gene and 5'- and 3 ' -flanking regions. Flanking regions and introns are shown in lowercase letters, while exons are presented in uppercase letters. The deduced amino acid sequences are shown below the string of exon sequences. Numbers indicate the distance relative to the transciption start site (+1) which is indicated by the arrowhead. The consensus TATA and G-box sequences as well as putative polyadenylation signal are underlined. The regions containing at least 82% sequence homology with the rice Sjbel 5' flanking region is also underlined. The asterisk and dot indicate the stop codon and putative polyadeylation site, respectively.

Figure 3. Genomic structure of the Ae gene. The complete structure of the Ae gene was constructed using two overlapping genomic clones: λ 3-2-1 and 7-2-1. The thick black lines indicate the 5 ' - or 3 '-flanking sequences of the Sjbel gene. The solid —black boxes indicate exons and the open boxes denote introns . The position of a putative TATA-box relative to the transcription initiation site (+1) is indicated. Figure 4. Nucleotide sequence of the Ae gene and 5 '-and 3 '-flanking regions. Numbers indicate the distance relative to the transciption start site (+1) which is indicated by the arrow. Intron sequences are omitted. Putative cis-elements are indicated below the sequence and boxed. The end points of the 5' deletion constructs were indicated by arrowheads below the first nucleotides of the deletions. Direct repeat sequences are underlined. The translation initiation codon (ATG) and the translation termination codon (TGA) are double underlined. The dot indicates polyadenylation site. The region exhibiting 80% sequence similarity with the PREM-2 internal and 3' LTR region is boxed. The wavy line represents polypurine tract.

Figure 5. Schematic diagram of the 5' deletion chimeric constructs. Fig. 5A. The thick black lines denote the Ae promoter sequences . The numbers at left indicate deletion-end points relative to the transcription initiation site (+1) of the Ae gene. The open and stripped boxes indicate luciferase gene and nopaline synthase 3' end sequences, respectively. Fig. 5B. The junction sequences between the Ae gene and LUC. The BamHI site used to join the two genes is underlined. The translation start site of LUC is indicated by boldface letters. Fig. 5C. Effect of 5' deletions on Ae promoter activity. The relative activity values of the constructs are percentages of pKL201 level. Each value represents the average of three independent shootings. Error bars indicate standard errors of the means.

Figure 6. Effect of Sjbel gene exon/introns and 3 ' end on the level of LUC expression driven by the Sjbel promoter. Fig. 6A. Schematic diagram of chimeric Sjbel promoter-luciferase constructs. Numbers indicate distance relative to the SJbel transcription start site. Translation initiation starts at a position, + 28. The light grey (stippled) boxes indicate the Sbel promoter region. Angled lines indicate exons and introns in the Sjbel gene. Open and solid black boxes indicate luciferase (LUC) reporter gene and nopaline synthase 3' end sequences, respectively. The striped box indicates the Sjbel 3' flanking sequence. Fig. 6B. The junction sequences between the Sjbel gene and LUC. The BamHI sites used to join the two genes are underlined. The translation start site of LUC is indicated by boldface letters. Fig. 6C. Expression levels of the construct shown in (A) . LUC/GUS ratios were calculated as described in Methods. Each value represents the average of four independent shootings. Error bars indicate standard errors of the mean.

Figure 7. Effect of 5' deletions on Sjbel promoter activity. Fig. 7A. Schematic diagram of the 5' deletion chimeric constructs. The thick black lines denote the Sjbel promoter sequences . The numbers at left indicate deletion-end points relative to the transcription initiation site (+1) of the SJbel gene. The light grey (stippled) boxes and the thin black angled line represent the first exon and intron in the Sjbel gene, respectively. The open boxes indicate the luciferase gene. The solid black boxes denote nopaline synthase 3' end sequences. Fig. 7B. The relative activity levels of the constructs shown in (A) . The relative activity values are percentages of pKLNlOl level . Each value represents the average of six to eight independent experiments. Error bars indicate standard errors of the means .

Figure 8. Linker-scan analyses of the 60 -bp region in the SJbel promoter. Fig. 8A. Schematic diagram of the linker-scan constructs. DNA sequence of the 60-bp region in the Sjbel promoter is shown to the right of the wild-type construct pKLN105. The mutated bases in the linker-scan constructs are shown in lowercase letters. Dashes represent the unaltered nucleotides. For an explanation of the other symbols, refer to the legend to Figure 13. Fig. 8B. Relative LUC activity levels of the constructs shown in (A) . The relative activity values are percentages of construct -314 level. Each value represents the average of four independent experiments. Error bars indicate standard errors of the means.

Figure 9. Sucrose responsiveness of the Sjbel promoter. Each construct was bombarded onto the maize endosperm suspension cell cultures supplemented with 0% (-) sucrose or 9% (+) sucrose and incubated for 48 hr at 25°C in the dark. The relative activity values are percentages of pKLNlOl level in 0% sucrose. Each value represents the average of three independent experiments. Error bars indicate standard errors of the means .

Figure 10. Effect of mEmBP-1 overexpression on Sjbel promoter activity. 4 μg each of reporter plasmid (Sjbel promoter-LUC; pKLNlOl, or Ubiquitin-LUC; pACH18) and reference plasmid (CaMV 35S-GUS; pBI221) were co- precipitated onto gold particles with or without 4 μg of an effector plasmid (CaMV 35S-mEmBP-l) . Maize endosperm suspension cells were bombarded wiht the gold particles and incubated at 25°C for 24 hr in the dark. The relative acvtivity values are percentages of the pKLNlOl or pACH18 levels without mEmBP-1 overexpression. Each value represents the average of two independent shootings. Error bars indicate standard errors of the means .

DETAILED DESCRIPTION OF THE INVENTION I. Definitions

Various terms used throughout the specification and claims to describe the invention. Unless otherwise specified, these terms are defined as set forth below. With reference to nucleic acid molecules, the term "isolated nucleic acid" is sometimes used. This term, when applied to DNA, refers to a DNA molecule that is separated from sequences with which it is immediately contiguous (in the 5' and 3' directions) in the naturally occurring genome of the organism from which it was derived. For example, the "isolated nucleic acid" may comprise a DNA molecule inserted into a vector, such as a plasmid or virus vector, or integrated into the genomic DNA of a procaryote or eucaryote . An "isolated nucleic acid molecule" may also comprise a cDNA molecule. With respect to RNA molecules, the term "isolated nucleic acid" primarily refers to an RNA molecule encoded by an isolated DNA molecule as defined above. Alternatively, the term may refer to an RNA molecule that has been sufficiently separated from RNA molecules with which it would be associated in its natural state (i.e., in cells or tissues), such that it exists in a "substantially pure" form (the term "substantially pure" is defined below) .

The term "substantially pure" refers to a preparation comprising at least 50-60% by weight the compound of interest (e.g., nucleic acid, oligonucleotide, protein, etc.) . More preferably, the preparation comprises at least 75% by weight, and most preferably 90-99% by weight, the compound of interest. Purity is measured by methods appropriate for the compound of interest (e.g. chromatographic methods, agarose or polyacrylamide gel electrophoresis, HPLC analysis, and the like) .

Nucleic acid sequences and amino acid sequences can be compared using computer programs that align the similar sequences of the nucleic or amino acids thus define the differences. For purposes of this invention, the GCG Wisconsin Package version 9.1, available from the Genetics Computer Group in Madison, Wisconsin, and the default parameters used (gap creation penalty=12 , gap extension penalty=4) by that program are the parameters intended to be used herein to compare sequence identity and similarity. The term "substantially the same" refers to nucleic acid or amino acid sequences having sequence variation that do not materially affect the functionality of cis acting regulatory sequences (e.g, promoters, transcriptional response elements, etc.) or the nature of the encoded gene product (i.e. the structure, stability characteristics, substrate specificity and/or biological activity of the protein) . With particular reference to nucleic acid sequences, the term "substantially the same" is intended to refer to conserved sequences governing expression and to the coding region (referring primarily to degenerate codons encoding the same amino acid, or alternate codons encoding conservative substitute amino acids in the encoded polypeptide) .

The terms "percent identical" and "percent similar" are also used herein in comparisons among nucleic acid sequences. When referring to nucleic acid molecules, "percent identical" refers to the percent of the nucleotides of the subject nucleic acid sequence that have been matched to identical nucleotides by a sequence analysis program. When referring to amino acid sequences, "percent identical" refers to the percent of the amino acids of the subject amino acid sequence that have been matched to identical amino acids in the compared amino acid sequence by a sequence analysis program. "Percent similar" refers to the percent of the amino acids of the subject amino acid sequence that have been matched to identical or conserved amino acids. Conserved amino acids are those which differ in structure but are similar in physical properties such that the exchange of one for another would not appreciably change the tertiary structure of the resulting protein. Conservative substitutions are defined in Taylor (1986, J. Theor. Biol. 119:205) .

With respect to oligonucleotides or other single-stranded nucleic acid molecules, the term "specifically hybridizing" refers to the association between two single-stranded nucleic acid molecules of sufficiently complementary sequence to permit such hybridization under pre-determined conditions generally used in the art (sometimes termed "substantially complementary"). In particular, the term refers to hybridization of an oligonucleotide with a substantially complementary sequence contained within a single-stranded DNA or RNA molecule, to the substantial exclusion of hybridization of the oligonucleotide with single-stranded nucleic acids of non-complementary sequence.

A "coding sequence" or "coding region" refers to a nucleic acid molecule having sequence information necessary to produce a gene product, when the sequence is expressed.

The term "operably linked" or "operably inserted" means that the regulatory sequences necessary for expression of the coding sequence are placed in a nucleic acid molecule in the appropriate positions relative to the coding sequence so as to enable expression of the coding sequence. This same definition is sometimes applied to the arrangement other transcription control elements (e.g. enhancers) in an expression vector.

When describing the organization of a nucleic acid molecule, the term "upstream" refers to the 5' direction and the term "downstream" refers to the 3 ' direction.

Transcriptional and translational control sequences, sometimes referred to herein as "expression control" sequences or elements, or "expression regulating" sequences or elements, are DNA regulatory elements such as promoters, enhancers, ribosome binding sites, polyadenylation signals, terminators, and the like, that provide for the expression of a coding sequence in a host cell. The term "expression" is intended to include transcription of DNA and translation of the mRNA transcript .

The terms "promoter" , "promoter region" or "promoter sequence" refer generally to transcriptional regulatory regions of a gene, which may be found at the 5 ' or 3 ' side of the coding region, or within the coding region, or within introns. Typically, a promoter is a DNA regulatory region capable of binding RNA polymerase in a cell and initiating transcription of a downstream (3' direction) coding sequence. The typical 5' promoter sequence is bounded at its 3' terminus by the transcription initiation site and extends upstream (5' direction) to include the minimum number of bases or elements necessary to initiate transcription at levels detectable above background. Within the promoter sequence is a transcription initiation site (conveniently defined by mapping with nuclease SI) , as well as protein binding domains (consensus sequences) responsible for the binding of RNA polymerase .

A "vector" is a replicon, such as plasmid, phage, cosmid, or virus to which another nucleic acid segment may be operably inserted so as to bring about the replication or expression of the segment.

The term "nucleic acid construct" or "DNA construct" is sometimes used to refer to a coding sequence or sequences operably linked to appropriate regulatory sequences and inserted into a vector for transforming a cell. This term may be used interchangeably with the term "transforming DNA" . Such a nucleic acid construct may contain a coding sequence for a gene product of interest, along with a selectable marker gene and/or a reporter gene .

A "heterologous" region of a nucleic acid construct is an identifiable segment (or segments) of the nucleic acid molecule within a larger molecule that is not found in association with the larger molecule in nature. Thus, when the heterologous region encodes a mammalian gene, the gene will usually be flanked by DNA that does not flank the mammalian genomic DNA in the genome of the source organism. In another example, coding sequence is a construct where the coding sequence itself is not found in nature (e.g., a cDNA where the genomic coding sequence contains introns, or synthetic sequences having codons different than the native gene) . Allelic variations or naturally-occurring mutational events do not give rise to a heterologous region of DNA as defined herein.

A cell has been "transformed" or "transfected" by exogenous or heterologous DNA when such DNA has been introduced inside the cell. The transforming DNA may or may not be integrated (covalently linked) into the genome of the cell. For example, the transforming DNA may be maintained on an episomal element such as a plasmid. With respect to eukaryotic cells, a stably transformed cell is one in which the transforming DNA has become integrated into a chromosome so that it is inherited by daughter cells through chromosome replication. This stability is demonstrated by the ability of the eukaryotic cell to establish cell lines or clones comprised of a population of daughter cells containing the transforming DNA. A "clone" is a population of cells derived from a single cell or common ancestor by mitosis. A "cell line" is a clone of a primary cell that is capable of stable growth in vi tro for many generations.

II . Description

The genes encoding starch branching enzymes (SBEI, Ila and lib) in maize are differentially regulated in tissue specificity and during kernel development. The expression-controlling sequences governing the differential regulation of genes encoding SBEI and SBEIIb are the subject matter of the present invention. These sequences are of great utility and value in genetic modification of plants to produce other gene products in the same tissue- and developmentally specific manner as are the two aforementioned starch branching enzymes. This is accomplished by placing the coding sequence of a gene of interest under the control of one or more of these expression-controlling sequences. cDNAs for the starch branching enzymes have been disclosed, but such sequences, for the most part, do not contain the regulatory elements that control expression of a gene. To obtain these expression- controlling sequences, the actual genes must be isolated. In accordance with the present invention, the isolation and charcterization of two starch branching genes from maize, SJbel and Ae, has enabled the identification of elements that regulate the expression of these genes.

The inventors have isolated and sequenced a maize genomic DNA (-2190 to +5929) which contains the entire coding region of SBEI ( Sbel) as well as 5 '-and 3'- flanking sequences. Sequence analysis of this gene (SEQ ID N0:1) is shown in Figure 2. Using this clone, the complete genomic organization of the maize SJbel gene was established. The transcribed region consists of 14 exons and 13 introns, distributed over 5.7 kb . A consensus TATA-box and a G-box containing a perfect palindromic sequence, CCACGTGG, were found in the 5 '-flanking region. Genomic Southern blot analysis indicated that two Sjbel genes with divergent 5 '-flanking sequences exist in the maize genome, indicating that they may be differentially regulated. A chimeric construct containing the 5'- flanking region of Sbel (-2190 to +27) fused to the β- glucuronidase gene (pKGlOl) showed promoter activity after it was introduced into maize endosperm suspension cells by particle bombardment. Although the 2.2-kb 5'- flanking sequence between -2190 and +27 relative to the transcription initiation site was sufficient to promote transcription, addition of the transcribed region between +28 and +228 containing the first exon and intron resulted in high-level expression in maize endosperm suspension cells. A series of 5' deletion and linker- substitution mutants identified two critical positive cis-elements, -314 to -295 and -284 to -255. Electrophoretic mobility shift assay showed that nuclear proteins prepared from maize kernels interact with the

60-bp fragment containing these two elements. Expression of the Sjbel gene is regulated by sugar concentration in cultured maize endosperm suspension cells, and the region -314 to -145 is essential for this effect. Expression of mEmBP-1, a bZIP transcription activator, in maize endosperm suspension cells resulted in a 5-fold decrease in the Sjbel promoter activity, suggesting a possible regulatory role of the G-box present in the SJbel promoter from -227 to -220 .

A genomic clone of a SJbel gene from rice has been described (Kawasaki et al . , 1993, supra) , as has an Sjbel promoter from wheat (U.S. Patent No. 5,866,793 to Baga, et al . ) However, the presently described expression regulatory elements have several distinguishing features, described in greater detail below, which have not been reported for the rice or wheat promoter elements. The amylose -extender (Ae) gene encoding SBEIIb in maize is predominantly expressed in endosperm and embryos during kernel development . To obtain the expression-controlling sequences of this gene, a maize genomic DNA fragment (-2,964 to +20,485) containing the Ae gene was isolated and sequenced. Sequence analysis of this gene (SEQ ID NO : 2) is shown in Figure 9. The maize Ae mRNA is derived from 22 exons distributed over 16,914 bp . Twenty one introns, differing in length from 76 bp to 4,020 bp, all have conserved junction sequences (GT . . AG) . Sequence analysis of the 5'- and 3' -flanking regions revealed a consensus TATA-box sequence located 28 bp upstream of the transcription initiation site as determined by primer extension analysis, and a putative polyadenylation signal observed 29 bp upstream of the polyadenylation site based on cDNA sequence. Genomic

Southern blot analysis revealed that a single Ae gene is present in the maize genome. Promoter activity was confirmed by testing a transcriptional fusion of the Ae 5 '-flanking region between -2,964 and +100 to a luciferase reporter gene in a transient expression assay using maize endosperm suspension cultured cells. 5' deletion analysis revealed that the 111-bp region from - 160 to -50 is essential for high-level promoter activity. The present invention is drawn to all expression controlling elements of genes encoding SBEI and SBEIIB, particularly as exemplified by the maize SJbel and Ae genes described in detail herein. With respect to the the maize Sjbel gene, the following sequences are considered particularly preferred for use in the present invention:

1. The 5' untranslated region, nucleotides - 2190 to +27 relative to the transcription initiation site, which contains sequences sufficient to promote transcription;

2. The region containing the first exon and intron, nucleotides +28 to +228 relative to the transcription initiation site, which, when added to the promoter region, results in high level gene expression in the endosperm;

3. The region between -314 and -145 relative to the transcription initiation site, which is needed for sugar regulation of gene expression; and within this region

4. two critical positive cis-elements, -314 to -295 and -284 to -255; nuclear proteins prepared from maize kernels interact with the 60-bp fragment containing these two elements; 5. The 3' untranslated region, nucleotides

+5390 to +5910, containing the polyadenylation signal and other sequences that may contribute to the stability or translation efficiency of the encoded mRNA.

With respect to the the maize Ae gene, the following sequences are considered particularly preferred for use in the present invention:

1. the 5' untranslated region, nucleotides - 2,964 to +100 relative to the transcription initiation site, which contains sequences sufficient to promote transcription, and within this region;

2. the 111-bp region from -160 to -50, which is essential for high-level promoter activity; and 3. The 3' untranslated region, nucleotides

+16,695 to +20,485, containing the polyadenylation signal and other sequences that may contribute to the stability or translation efficiency of the encoded mRNA .

Although the genomic clones of the maize Sjbel and Ae are exemplified herein, this invention is intended to encompass nucleic acid sequences from other organisms, particularly higher plants, that are sufficiently similar to be used instead of the maize Sjbel and Ae nucleic acid moleucles for the purposes described below. These include, but are not limited to, allelic variants and natural mutants of SEQ ID ΝOS : 1 and 2 and the particular portions thereof listed above, which are likely to be found in different maize cultivars, as well as in different plant species. Of particular relevance to this invention are monocotyledenous plant species, and most especially those of agronomic importance, such as wheat, rice, barley and sorghum, for example. Also of particular relevance to this invention are taxonomic relatives of Zea mays (which includes cultivated maize and teosinte) , including other members of the genus Zea, such as Zea diploperennis (diploperennial teosinte) , Zea luxurians and Zea perennis (perennial teosinte) , as well as Tripsacum spp. and Sorghum spp .

Variants from other cultivars and species are expected to possess certain differences in nucleotide sequence. Moreover, it is known that 5' and 3' regulatory regions of genes, while often sharing overall functional similarites, do not share a high degree of sequence homology. Accordingly, this invention provides nucleic acid molecules comprising one or more the above- described expression regulatory sequences, which are isolated from genes with coding sequences (exons) having at least about 60% (preferably 70%, more preferably over 80% and most preferably over 90%) sequence homology with the respective coding sequences (exons) of the maize Sjbel or Ae genes, as exemplified by SEQ ID NO:l or SEQ ID NO: 2. The isolation of genes with exons having these levels of homology is accomplished by nucleic acid hybridization at a selected stringency, which is described in greater detail below.

It will also be understood by persons skilled in the art that the Sjbel and Ae expression-regulating elements of the invention include single- or double- stranded DNA or RNA.

The following description sets forth the general procedures involved in practicing the present invention. To the extent that specific materials are mentioned, it is merely for purposes of illustration and is not intended to limit the invention. Unless otherwise specified, general cloning procedures, such as those set forth in Sambrook et al . , Molecular Cloning, Cold Spring Harbor Laboratory (1989) (hereinafter "Sambrook et al.") or Ausubel et al . (eds) Current Protocols in Molecular Biology, John Wiley & Sons (1999) (hereinafter "Ausubel et al . " ) are used.

Nucleic acid molecules comprising the promoters and/or other expression regulatory sequences of the invention may be prepared by two general methods: (1) they may be synthesized from appropriate nucleotide triphosphates, or (2) they may be isolated from biological sources. Both methods utilize protocols well known in the art .

The availability of nucleotide sequence information, such as the aforementioned expression- controlling regions of SEQ ID NO : 1 and SEQ ID NO : 2 , enables preparation of isolated nucleic acid molecules of the invention by oligonucleotide synthesis. Synthetic oligonucleotides may be prepared by the phosphoramadite method employed in the Applied Biosysterns 38A DNA Synthesizer or similar devices. Variants of the aforementioned sequences also may be synthesized as described above .

Sjbel and Ae genes and their expression- regulatory elements also may be isolated from appropriate biological sources using methods known in the art . In the exemplary embodiment of the invention, the genomic clones having SEQ ID NO:l and SEQ ID NO : 2 were isolated from a genomic library of maize inbred line B73. Genomic libraries of other maize cultivars or plant species, as discussed above, are also suitable sources for isolating these genes. A preferred means for isolating clones from a genomic library is PCR amplification using genomic templates and Sjbel or Ae-specific primers derived from exons of SEQ ID NO:l or SEQ ID NO:2.

In accordance with the present invention, genomic clones having the appropriate level sequence homology with the coding regions of SEQ ID NO : 1 or 2 may be identified by using hybridization and washing conditions of appropriate stringency. For example, hybridizations may be performed, according to the method of Sambrook et al . , using a hybridization solution comprising: 5X SSC, 5X Denhardt ' s reagent, 1.0% SDS, 100 μg/ml denatured, fragmented salmon sperm DNA, 0.05% sodium pyrophosphate and up to 50% formamide. Hybridization is carried out at 37-42°c for at least six hours. Following hybridization, filters are washed as follows: (1) 5 minutes at room temperature in 2X SSC and 1% SDS; (2) 15 minutes at room temperature in 2X SSC and 0.1% SDS; (3) 30 minutes-1 hour at 37oc in 2X SSC and 0.1% SDS; (4) 2 hours at 45-55oin 2X SSC and 0.1% SDS, changing the solution every 30 minutes.

One common formula for calculating the stringency conditions required to achieve hybridization between nucleic acid molecules of a specified sequence homology (Sambrook et al . , 1989) :

T_m = 81.5°C + 16.6Log [Na+] + 0.41(% G+C) - 0.63 (% formamide) - 600/#bp in duplex

As an illustration of the above formula, using [N+] =

[0.368] and 50% formamide, with GC content of 42% and an average probe size of 200 bases, the T_ra is 57°C. The T_ra of a DNA duplex decreases by 1 - 1.5°C with every 1% decrease in homology. Thus, targets with greater than about 75% sequence identity would be observed using a hybridization temperature of 42 °C. Such a sequence would be considered substantially homologous to the sequences of the present invention. In a preferred embodiment, the hybridization is at 37°C and the final wash is at 42°C, in a more preferred embodiment the hybridization is at

42° and the final wash is at 50°, and in a most preferred embodiment the hybridization is at 42 °C and final wash is at 65°C, with the above hybridization and wash solutions. Conditions of high stringency include hybridization at 42 °C in the above hybridization solution and a final wash at 65°C in 0. IX SSC and 0.1% SDS for 10 minutes.

Nucleic acids of the present invention may be maintained as DNA in any convenient cloning vector. In a preferred embodiment, clones are maintained in plasmid cloning/expression vector, such as pBluescript SK- (Stratagene, La Jolla CA) which is propagated in a suitable E. coli host cell. The expression-controlling elements of the invention, as mentioned above, include transcriptional control elements such as promoters and enhancers that occur in flanking regions, in introns and sometimes in the coding region of the gene, as well as translational control elements (5' and 3') that affect the translation efficiency and/or stability of the mRNA. These elements are contemplated for use in a variety of applications in accordance with the present invention.

A preferred use of the expression-controlling elements is to drive transcription and translation of native and heterologous genes in transgenic plants, for the purpose of increasing the production of the endogenous gene products, or for producing new gene products in the tissue- and developmentally-specific manner governed by the expression-controlling sequences. Examples of useful gene products that can be expressed in seeds and other starch storage locations in plants include, but are not limited to: (1) gene products conferring herbicide tolerance; (2) gene products involved in starch synthesis, such as starch synthases or ADP-glucose pyrophosphorylase; (3) gene products involved in fatty acid biosynthesis, such as fatty acid desaturases; (4) gene products which are seed storage proteins, such as zeins; (5) gene products conferring resistance to insect pests, such as the crystal BT toxin; (6) gene products conferring resistance to microbial plant pathogens, including, e.g., viral coat proteins for virus resistance, or fungal cell wall lytic enzymes or phytoalexins for fungal resistance; and (7) gene products that comprise or produce pharmaceutical or biological agents, such as antibiotics, secondary metabolites, antibodies, peptides or vaccines. To use the expression-controlling sequences for introducing native or foreign genes into plants, the selected codings sequences and expression-controlling sequences are operably linked to one another, then inserted into vectors (or used directly, in some transformation protocols) for transforming plant cells.

Methods for manipulating DNA sequences to accomplish this are well known in the art, as exemplified by Sambrook et al . and Ausubel et al . The design of such DNA constructs will depend on the expression functions desired for the gene of interest. As one example, all of the 5' and 3' expression-controlling sequences, as well as the +28 - +228 region, of the maize SJbel gene may be operably linked to a coding sequence, such as a cDNA encoding a zein protein. A similar construct can be made using all of the 5' and 3' expression controlling elements of the Ae gene. In transgenic plants containing such chimeric genes, the zein protein would be produced in the same tissues under the same physiological conditions as the SBEI or SBEIIb proteins are produced. In another embodiment, if it is desired to produce a sugar- inducible gene, the segments of the 5' region of Sbel controlling sugar regulation (-314 to -145) could be utilized independently .

In addition, certain of the expression regulatory elements (e.g., the -314 to -145 element or the +28 to +228 element of Sbel) may be combined with constitutive or inducible promoters from other sources. Examples of constitutive promoters suitable for this purpose are the rice actin promoter, the maize ubiquitin promoter and the Cauliflower Mosaic Virus (CaMV) 35S promoter. An example of a suitable inducible promoter is the tetracycline repressor/operator controlled promoter. Other examples of ways to combine the various

5 ' , 3 ' and internal expression-controlling elements of the SJbel and Ae genes will be apparent to the person of skill in the art.

Transgenic plants expressing the native or heterologous chimeric genes described above can be generated using standard plant transformation methods known to those skilled in the art. These include, but are not limited to, Agrobacterium vectors, PEG treatment of protoplasts, biolistic DNA delivery, UV laser microbeam, gemini virus vectors, calcium phosphate treatment of protoplasts, electroporation of isolated protoplasts, agitation of cell suspensions with microbeads coated with the transforming DNA, direct DNA uptake, liposome-mediated DNA uptake, and the like. Such methods have been published in the art. See, e.g., Methods for Plant Molecular Biology (Weissbach & Weissbach, eds . , 1988); Methods in Plant Molecular Biology (Schuler & Zielinski, eds., 1989); Plant Molecular Biology Manual (Gelvin, Schilperoort , Verma, eds. , 1993) ; and Methods in Plant Molecular Biology - A Laboratory Manual (Maliga, Klessig, Cashmore, Gruissem & Varner, eds., 1994).

The method of transformation depends upon the plant to be transformed. The biolistic DNA delivery method is useful for nuclear transformation, especially of monocotyledenous plants. Methods for performing transformation using particle bombardment are described in detail in the Examples.

In another embodiment of the invention, Agrobacterium vectors are used to advantage for efficient transformation of plant nuclei. In this embodiment, Agrobacterium "superbinary" vectors have been used successfully for the transformation of maize (Ishida et al . , Nature Biotechnology 14: 745-750, 1996) and rice (Hiei et al . , Plant Journal 6: 271-282, 1994).

Using an Agrobacterium superbinary vector system for transformation, the selected coding sequence under control of the expression-controlling elements of the invention, as described above, is linked to a nuclear drug resistance marker, such as kanamycin resistance or phosphothricin herbicide resistance. Agrobacterium- mediated transformation of plant nuclei is accomplished according to the following procedure: (1) the gene is inserted into the selected

Agrobacterium binary vector;

(2) transformation is accomplished by co- cultivation of plant tissue (e.g., leaf discs) with a suspension of recombinant Agrobacterium, followed by incubation (e.g., two days) on growth medium in the absence of the drug used as the selective medium;

(3) plant tissue is then transferred onto the selective medium to identify transformed tissue; and

(4) identified transformants are regenerated to intact plants.

Regardless of the transformation system used, it should be recognized that the amount of expression, as well as the tissue specificity of expression of the gene in transformed plants can vary depending on the position of their insertion into the nuclear genome. Such position effects are well known in the art. For this reason, several nuclear transformants should be regenerated and tested for expression of the transgene. Another use of the expression-controlling elements is to inhibit or prevent transcription and translation of native SJbel or Ae genes in plants. For this use, the elements are used as antisense molecules to block gene expression at critical points. Antisense molecules can be transiently expressed in plants by introduction as single-stranded molecules (DNA or RNA) , according to known methods. Alternatively, vectors encoding the antisense molecules can be introduced into plants and the antisense molecules thereafter produced by transcription of the encoding sequences on the vectors, using the above-described methods. In another embodiment, overexpression of Sjbel or Ae is induced to generate a co-suppression effect. This excess expression serves to promote down-regulation of both endogenous and exogenous Sjbel or Ae genes .

The following specific examples are provided to illustrate embodiments of the invention. They are not intended to limit the scope of the invention in any way.

EXAMPLE 1 Genomic Organization and Promoter Activity of Maize Starch Branching Enzyme I Gene

This example describes the isolation of a full- length maize genomic DNA fragment containing the entire SJbelgene . Structural and functional analysis of this gene revealed a complete genomic organization and demonstrated the transcriptional activity of its promoter region. Materials and methods

PCR amplification. Maize ( Zea mays L., inbred W64A) genomic DNA prepared from 22 -DAP kernels using the method of Rogers and Bendich (1985) was amplified in a 50 μL reaction mixture containing 1 μg of target DNA, 1 μM of each primer, 200 μM of each dNTP, 10 mM Tris-HCl (pH 8.3), 50 mM KCl , 1.5 mM MgCl2, 0.01% gelatin (w/v) , and 2.5 U of Tag DNA polymerase (Boehringer Mannheim, Indianapolis, IN) . The mixture without dNTP was overlaid with 50 μL of mineral oil (Sigma, St. Louis, MO) and incubated for 5 min at 94 °C. The dNTPs were added to the mixture at the end of the incubation and then the mixture was cycled 35 times in a thermal cycler (ERICOMP, San Diego, CA) as follows: 94°C for 30 s; 55°C for 1 min; and 72°C for 2 min; with a final 72°C extension of 7 min. The primers were designed according to the published sequence data of the maize partial SJbel cDNA (Baba et al . , Biochem. Biophys . Res. Commun. 181 : 87-94, 1991). The 5' primer, 5 ' -GACTGAATTCCTGCGCAGGAGGCAGAGCTT-3 ' (SEQ ID NO: 3), and the 3' primer, 5 ' -GATCGAATTCCATAGATACG

TGGAGCAGCA-3 ' (SEQ ID NO: 4), are homologous and complementary to DNA sequences of the maize Sjbel cDNA from 438 bp to 457 bp and 745 bp to 764 bp, respectively. Each primer contains an BcoR I restriction enzyme site and 4 extra nucleotides (underlined) at their 5' ends for convenience of subsequent cloning of the PCR product . After amplification, 15 μL of the reaction sample was run on an agarose gel (1.5 %,w/v) in IX TAE buffer containing 0.04 M Tris-acetate and 1 mM EDTA. A single PCR product of about 0.5 kb was detected on an ethidium bromide- stained agarose gel^', digested with EcoR I restriction enzyme and cloned into the corresponding site of pBluescript SK^~ (Stratagene, La Jolla, CA) creating plasmid pBl .

Maize genomic library screening and DNA sequencing. An EMBL-3 genomic library (Clonetech, Palo Alto, CA) prepared from maize seedlings (2-leaf stage, B73) was screened essentially according to Sambrook et al . (1989) . Approximately 3 x 10⁵ pfu were transferred onto nylon membranes (Hybond-N+, Amersham, UK) and hybridized with the ³²P-labeled Sjbel genomic PCR product excised from pBl . Hybridization was performed at 55°C for 20 h in 0.5 M Na₂HP0₄ (pH 7.2) and 7% SDS with gentle agitation at 40 cycles per minute on a rotary shaker. Following the hybridization, filters were washed twice in 5% SEN (5% SDS (w/v) , 1 mM EDTA, 0.04 M Na₂HP0₄ , pH 7.2) and once in 1% SEN (1% SDS, 1 mM EDTA, 0.04 M Na₂HP0₄, pH 7.2) for 15 min at 65°C. Plaques strongly hybridizing to the probe were selected and purified through three rounds of screening. Phage DNAs were isolated from the positive plaques according to Chisholm' s method (1988) and were digested with Pst I to release the inserts from the EMBL- 3 vector. The restriction fragments were separated on a

0.8% agarose gel and blotted onto nylon membranes. The blots were probed with ³²P-labeled full-length SJbel cDNA (Fisher et al . , 1995, supra) as described above and hybridizing DNA fragments were identified and subcloned into pBluescript SK- . DNA sequences were determined by the dideoxynucleotide chain termination method with Sequenase Version 2.0 (United States Biochemical Co., Cleveland, OH) . Sequence analyses were performed using programs from DNASTAR Inc. (Madison, WI) . Primer extension analysis. To locate the transcription initiation site of the Sjbel gene, an oligonucleotide, 5 ' -GGCGACACGAGGCACAGCAT-3 ' (SEQ ID NO:5), which is complementary to the sense strand sequence of the Sbel cDNA from +1 to +20 relative to the translation start site (ATG) was radiolabeled at its 5 ' terminus with T4 polynucleotide kinase and γ³²P-ATP. Approximately 10⁵ cpm of the labeled primer was hybridized at 35°C with 10 μg of total RNA, which was isolated from 30 -DAP maize kernels (B73) according to the protocol of Vries et al . (Plant. Mol. Biol. Manual B6: 1- 13, 1988) . After hybridization for 8 h, complementary DΝA was synthesized from the annealed primer by the addition of reverse transcriptase and dΝTP . Following the addition of EDTA and RΝAase A into the reaction, the nucleic acid was precipitated with ethanol. The reaction products were resuspended in sequencing gel loading buffer, denatured at 95°C, electrophoresed through a 5% polyacrylamide sequencing gel (w/v) , and visualized by autoradiography . In order to provide size markers, part of the Sbel gene was sequenced with the same primer used in the primer extension experiment .

3 ' -Rapid amplification of cDΝA ends (3 '-RACE). To isolate the 3' end of the Sjbel transcript, the 3 ' -RACE method was used (Frohman et al . , 1988) . First-strand cDΝA synthesis reaction was performed as follows: 14 μL of a mixture containing 5 μg of total RΝA from 30-DAP maize kernels (B73) and 50 pmol of a 39-bp oligonucleotide with 17 dT residues and an adaptor sequence, 5 ' -GGTCGACTCGAGTCGACATCGATTTTTTTTTTTTTTTTT-3 ' (SEQ ID NO: 6), was heated at 70°C for 10 min and quickly chilled on ice. To the chilled sample, 2 μL of 10X synthesis buffer ( 200 mM Tris-HCl, pH 8.4, 500 mM KC1 , 25 mM MgCl₂, and 1 mg/mL BSA) , 1 μL of 10 mM dNTP mix, 2 μL of 0.1 M DTT and 1 μL (200U) of SUPERSCRIPT reverse transcriptase (GIBCO BRL, Grand Island, NY) were added and incubated at room temperature for 10 min. The reaction mixture was then placed in a 42 °C water bath for 50 min and transferred to a 90 °C water bath. After 5 min incubation, 1 μL of RNase H (2U/μL) was added and incubation at 37°C was continued for 20 min. Next, the first -strand cDNA obtained was amplified directly by the PCR method using a gene-specific primer (5 ' -GACTGAGCTCA TACCAAATGAAGCCAGGAG-3 ' (SEQ ID N0:7)), which is a homologous to sequence of the Sjbel gene from +5382 to +5401, and an adaptor primer (5 ' -GGTCGACTCGAGTCGACATCGA- 3' (SEQ ID N0:8)). After amplification, a single DNA band detected on an agarose gel (1.5%, w/v) was isolated and digested with Sac I and Xho I (underlined within the primers) . The resulting fragment, approximately 350 bp in length, was cloned into a pBlusScript SK^" (pB13R) and sequenced.

Genomic DNA blot analysis. Maize genomic DNA was prepared from 7-day-old etiolated seedlings (inbred B73) according to the method described by Junghans and Metzlaff (Junghans and Metzlaff, Biotechniques , 8_.: 176, 1990) . 10 μg of genomic DNA was digested with restriction enzymes BamΑ I, EcoR I, Bgl II, and Hind III, separated on 0.8% agarose gels, and transferred onto nylon membranes (Hybond-N, Amersham, UK) in 20 X SSC containing 3 M NaCI and 0.3 M sodium citrate (pH 7.0) according to Sambrook et al . (1989) . The DNA was crosslinked to the membrane by 3.5 min of UV irradiation on a transilluminator (312 nm) . The genomic blots were prehybridized at 65°C for 1 h in 0.5 M Na₂HP0₄ (pH 7.2), 7% SDS, and 100 μg/mL denatured salmon sperm DNA. Using the random primed DNA labeling kit (Boehringer Mannheim,

Indianapolis, IN) , 25 ng of a full-length Sbel cDNA (Fisher et al . , 1995, supra) was labeled with [α-³²P] - dCTP. This labeled probe was added to the prehybridization solution and incubated at 65 °C for 18 h. Blots were washed twice in 5% SEN and once in 1% SEN for 15 min at 65°C and were exposed to Kodak X-AR film at - 80°C for 48 h using two intensifying screens. Construction of Sbel promoter-UidA expression plasmids. To make a transcriptional chimeric construct consisting of the Sjbel promoter (-2190 to +27) fused to a β-glucuronidase (GUS) reporter gene, UidA, a BamΑ I restriction enzyme site was created just before the translation initiation site of the Sjbel gene as follows: the DNA sequence between -253 and +27 of the Sbel gene was amplified via polymerase chain reaction using Pfu DNA polymerase (Stratagene, La Jolla, CA) to enhance the fidelity of PCR amplification. The 5' primer, 5'- CCAGCTCCACGGTTGTT CGTGT-3 ' (SEQ ID NO: 9), is homologous to sequence of the Sjbel gene from -253 to -232. An Apa I restriction enzyme site (GGGCCC) is located immediately downstream of the 5 ' -primer-binding region of the Sjbel promoter, -203 to -198. The 3' primer, 5 ' -CGATGGATCC TGTGACGGCGTGTGAGT CCC-3 ' (SEQ ID NO:10), consists of a

DNA sequence complementary to that of the Sjbel gene from +8 to +27 and a BairLU. I restriction enzyme site (GGATCC) flanked with four random nucleotides (underlined) . The PCR product was digested with Apa I and BamR I , and the resulting 236-bp fragment was cloned into pBluescript SK^" and sequenced in order to verify that no misincorporation had occurred in the DNA sequence during the PCR amplification. To incorporate this mutation into the context of a longer promoter fragment, the SJbel genomic clone (λ 5-1-1) was digested with Sal I to isolate the approximately 3.0-kb fragment, which was then blunt-ended with Klenow and ligated with Pst I linkers (New England Biolabs) . After complete digestion with Pst I and Apa I, the resulting 2-kb Sjbel promoter fragment was ligated to the 236-bp Apa I -BairiH. I fragment and cloned into plasmid pBI221 cut with Pst I and BamH I, thereby creating plasmid pKGlOl. Transient gene expression assay. Suspension culture cells of maize endosperm (inbred A636) , kindly provided by J. L. Anthony (DEKALB Genetics Corporation, Mystic, CT) were grown in 250-mL large-mouth Erlenmeyer flasks containing 80 mL of Murashige and Skoog basal salt medium (Murashige and Skoog, 1962) supplemented with 0.4 mg/L thiamine, 2 g/L asparagine, and 30 g/L sucrose (Shannon and Liu, 1977) . The culture was maintained in the dark at 29°C on a rotary shaker (120 rpm) and was subcultured every 7 days by transferring a portion of the cell suspension into fresh medium. For particle bombardment, the growing cells (3 days after subculture) were evenly distributed over the surface of three layers of filter paper (Whatmann #4, 55 mm in diameter) moistened with 3 mL of the liquid medium and positioned in the middle of a 10-cm petri dish. Three milligrams of 1.6-μm gold particles were coated with 10 μg of pKGlOl according to the method described by Xu et al . (Xu et al., Plant Mol. Biol., 31: 1117-1127, 1996) and introduced into the cells using a Bio-Rad PDS-1000/He Biolistic Particle Delivery system. Bombardments were performed at 650 psi under a vacuum of 26 inches of Hg with a distance of 10 cm between the cells and the microprojectile launch site of the particle gun. Following the bombardments, the petri dishes were sealed with parafilm and then incubated in the dark at 25°C for 24 h.

Histochemical GUS staining. Histochemical assays of GUS activity were performed according to the method described by Jefferson et al . (Jefferson et al . , Plant Mol. Biol. Rep., 5: 387-405, 1987) with minor modifications. Briefly, after 24 h incubation, cells on the filter paper were transferred into petri dishes containing 2 mL of GUS staining solution (50 mM sodium phosphate buffer, pH 8.0 , 0.05 mM potassium ferricyanide, 0.05 mM potassium ferrocyanide, 0.2% Triton X-100 (w/v) , 20% methanol (v/v) , and 1 mg/mL 5-bromo-4-chloro-3- indolyl-β-D-glucuronide) . The petri dishes were then sealed and incubated at 37°C overnight. The cells were examined and photographed under a Nikon SMZ-U dissecting microscope .

Results PCR amplification of maize genomic DNA. In order to obtain a DNA probe for genomic library screening, maize genomic DNA prepared from 22 -DAP kernels (W64A) was amplified by polymerase chain reaction (PCR) using upper and lower primers designed to anneal the SJbel cDNA (Baba et al . , Biochem. Biophys . Res. Commun. , 181 : 87-94, 1991) from 438 to 457 and 745 to 764, respectively. A single amplified DNA band, approximately 450 bp in length, was observed on an ethidium bromide- stained agarose gel . The PCR product containing EcoR I sites at both 5'- and 3 ' -ends was digested with EcoR I and the resulting fragment was cloned and sequenced.

Alignment of sequences between the maize PCR genomic fragment and the published Sbel cDNA (Baba et al . , 1991, supra) shows that the PCR fragment contained the predicted Sjbel cDNA sequence with only 3 nucleotide differences and a 103 -bp intron. The differences could be explained by cultivar polymorphisms or misincorporation of bases during PCR amplification by Tag DNA polymerase which does not have proofreading activity. This PCR fragment was ³²P-labeled and used as a hybridization probe for screening a maize genomic library to isolate Sbel genomic clones. Isolation and analysis of a maize Sbel genomic clone. In a screen of approximately 3 x 10⁵ plaque- forming units from a genomic library prepared from maize seedlings (inbred B73), 8 positive lambda clones were isolated which strongly hybridized to the probe. Restriction mapping and partial DNA sequencing of these clones indicated they all probably originated from the same genetic locus. A full-length clone (λ 5-1-1), containing the entire coding region of the Sjbel gene, as well as 5 ' - and 3'- flanking sequences, was selected for further analyses. Figure 1A shows a restriction map of the genomic clone. The 3.0-kb Sal I fragment and the 6.2-kb Pst I fragment from the clone were subcloned into pBluescript SK^" producing plasmids pBI5-l and pBI5-2, and their nucleotide sequences were completely determined. A consensus TATA-box as well as a G-box containing a perfect palindromic sequence known as a G- box, CCACGTGG, were found in the 5' flanking region of the gene (Fig. 2) . Primer extension analysis displayed a single extended product of 44 nucleotides, which co- migrates with an A residue in the sequencing ladder generated with the Sbel 5 '-flanking region. This indicates that consistant with many eukaryotic genes, the transcription initiates at a position which is located 25 bp downstream from the putative TATA box, suggesting that the TATA box may be a functional element of the Sjbel promoter.

To determine the polyadenylation site of the Sbel gene, 3' RACE was conducted. It demonstrated that a poly (A) tail occurs at an adenine nucleotide located 29 bp downstream from a putative polyadenlyation signal (AATAAA) in the Sjbel gene . Along with the primer extension result, this indicated that the transcribed region in the SJbel gene is 5,690 bp in length.

Structure of the Sbel genomic clone. Alignment of the genomic sequence with the published maize Sjbel cDNA sequence (Fisher et al . , 1995, supra) revealed that the gene is composed of 14 exons and 13 introns distributed over 5.7 Kb. Figure IB summarizes the organization of the maize Sjbel gene. The cDNA sequence is identical to the corresponding genomic sequence except for a 1-bp mismatch in exon 14. Table 1 shows the sequences around the exon/intron junctions and a list of putative branch point consensus sequences, which were derived as described by Brown (Brown, J.W.S., Nucl. Acids Res., 14: 9549-9559, 1986).

Table 1. List of introns and sequences of exon/intron borders in the Sbel gene

Intron Intron GC content number Exon / Putative intron branch point* /Exon size(bp) ^(0/-

1 TCGCG GTAAG CTGAT 17^γ CGCAG GGTGG 82 46. 3 2 GGAAG GTAGA CTGAA 24 GGAAG GTCAA 377 36 9

3 TAAAG GTTAG ATCAT 30 TTCAG GCTAT 120 34 2

4 GCGCA GTAAG TTGAC 25 TGTAG GGAGG 319 34 .2

5 GAAAG GTCTC ATGAC 33 TGCAG GTACA 103 35 9

6 ATCAG GTACC ATGAC 43 TTCAG TCTAT 262 32 1

7 AAAAG GTTCC CTCAA 33 TCCAG ATGAT 97 39 .2

8 ATGAG GTGAA TTGAT 17 TCTAG TTTGG 488 35 .2

9 ACAAG GTTAT CTAAC 37 AACAG TACAT 565 37 .3

10 AAAAG GTAAG GTCAG 25 TTCAG GTTAT 160 39 .4

11 GAGGG GTAAG CTTAC 31 TGCAG CTACA 107 41 .1

12 GAAGA GTAAG CTCAT 16 CGCAG GTTGG 140 49 .3

13 GTGTG GTAAT CTGAC 18 GCCAG GCTTA 73 49 .3

* Consensus sequences between introns are underlined. ^γ Numbers indicate number of nucleotides between adjacent sequences . The introns, relatively AT-rich (61%) compared to the exons (52%) , vary in length from 73 bp to 565 bp, and all of which have the conserved sequences at their 5 ' and 3 ' ends, following the ^VGT . . AG' rule of plant introns (Brown, 1986, supra) . Exon 1, containing 27 bp of 5'- untranslated DNA sequence, and exon 2 occur in the transit peptide region which may be essential for transporting the gene product into the amyloplast . Exon 14 contains the translation stop codon (TGA) and 3 ' - untranslated region, as well as the putative polyadenylation signal (AATAAA) . The exons vary in length from 63 to 907 bp.

Comparison of the maize and rice Sjbel genomic DNA sequences (Kawasaki et al . , Mol. Gen. Genet., 237 : 10-16, 1993b) revealed two large highly conserved regions in the 5 '-flanking sequences (Fig. 2) . One region, 161 bp in length, was present between -2190 and -1890 in the maize Sbel 5'- flanking sequence, and has 82% similarity with the corresponding rice region. The other, 342 bp in length, is located from -1804 to -1611, and shows 85% similarity with the corresponding rice region. These sequences conserved between the species suggest that the regions may play an important role in gene expression. Interestingly, a part of the latter region from -1804 to -1611 (194 bp in length) shares 83% similarity with the 5 ' -untranslated sequence of a Ca²⁺-dependent protein kinase gene in rice (Kawasaki et al . , Gene., 129 : 183- 189, 1993a), suggesting that a maize version of protein kinase gene may be located immediately upstream of the Sbel gene. In the rice genome, the two genes are separated by approximately 1.4 kb, and transcribed divergently from each other.

5 '-flanking sequences downstream from the two conserved regions did not show sequence similarity between the two genes (less than 25%) except for a G-box motif and several small segments adj cent to the G-box. This may indicate that the G-box plays a role in regulation of Sjbel gene expression. The G-box is found in other plant genes which respond to diverse environmental or physiological stimuli and are often associated with additional regions which possibly act as coupling elements determining signal response specificity.

Another notable feature derived from the sequence comparison is that the maize and rice Sbel genomic structures are quite similar. Both genes consist of 14 exons and 13 introns, the positions of which are conserved between the two species. The sizes of exons (exon 3 to exon 13) constituting most of the mature proteins are identical except exon 5, in which the rice Sbel gene has one more codon compared to the maize gene. They share more than 86% and almost 90% similarity in nucleotide and amino acid sequences, respectively.

However, exons not encoding the mature proteins (exon 1, 2 and most of exon 14) do not display any significant sequence similarity and vary in size. The carboxyl- terminal 64 (67 in rice) amino acids encoded by exon 14 in the maize gene is the only region which is not conserved in the mature protein. Unlike the exons, homology was not found in any of the introns other than the splice junction sequences. The large intron (2212 bp) present in the rice Sbel gene is not found in the maize gene.

Genomic Southern blot analysis . To determine the number of Sbel genes in the maize genome, Southern blot analysis was performed. When blots were hybridized with the full-length maize Sbel cDNA (Fisher et al . , 1995, supra) under high-stringency conditions, at least three bands were detected in each lane. Comparison of the hybridization patterns with the restriction map of the Sjbel genomic clone (Fig. 1A) revealed that not all the bands in the Southern blot corresponded to the genomic map, suggesting more than one Sbel gene is present in the maize genome.

To confirm this, a 0.6-kb genomic DNA probe which did not have any restriction enzyme sites used in the genomic blot was prepared from the genomic clone λ 5- 1-1 by BamH I -Hind III digestion. The genomic probe will produce only one hybridizing band in every lane if there is a single copy of the Sbel gene in the maize genome. However, the genomic probe (probe 2) containing the central region of the Sjbel cDNA detected in each lane one or two additional bands apart from the bands predicted by the genomic map. This indicates that along with the isolated SJbel gene, another Sbel gene or a gene very closely related to SJbel exists in the maize genome.

These will be referred to as Sbela (identified) and Sbelb (unidentified) to distinguish them when appropriate. Interestingly, when a 1.7-kb BamH I-Pst I genomic fragment consisting of the Sbela promoter and transit peptide-coding region was used as a probe (probe 1) , only the bands predicted from the identified Sbela genomic DNA sequence were detected. Taken together, these results suggest that although two SJbel genes are present in the maize genome, their 5' flanking sequences and the 5 '-end of the coding regions (at least in DNA level) are quite divergent from each other.

Genetic mapping of Sbe genes . In order to map Sbel loci onto the existing framework map (Gardiner et al., Genetics, 134: 917-930, 1993), the full length Sjbel cDNA was used to probe a population of 54 immortalized F₂ individuals from the cross Tx303 X C0159 as part of the mapping efforts of the University of Missouri-Columbia Maize RFLP Laboratory. Two loci were identified on chromosome 6, bin 6.01 and chromosome 10, 10.04. This supports the conclusion made above based on genomic Southern analysis that two SJbel genes exist in the maize genome. However, at present the data do not allow us to distinguish which gene is located on which chromosome. Promoter activity. A transient expression system was used to test whether the 5 '-flanking sequence of the cloned Sbel gene (λ 5-1-1) is sufficient to support transcriptional activity. A chimeric gene containing a 2.2-kb 5' Sbel fragment (-2191 to +27) fused to UidA reporter gene in pUC19 was first constructed and designated pKGlOl. The chimeric plasmid was introduced into maize endosperm suspension cells via particle bombardment. Iodine staining and northern blot analysis showed that the maize endosperm suspension cells actually produce starch and the genes involved in starch biosynthesis are expressed. The bombarded cells were incubated for 24 h at 25°C in the dark and histochemical GUS assays were performed to visualize GUS expression. Blue spots were observed, indicating that the 5 '-flanking sequence has the ability to drive gene expression in the maize endosperm cells. Control cells bombarded with gold particles coated with promoterless UidA construct did not show any blue spots. EXAMPLE 2 Molecular Cloning and Characterization of the Amylose -Extender Gene Encoding Starch Branching Enzyme IIB in Maize

This example describes the complete genomic organization of the Ae gene and the promoter regions critical for its expression in maize endosperm cells.

Materials and Methods

Maize Genomic Library Screening and DNA Sequencing. Using ³P-labeled full-length Ae cDNA (Fisher et al . , 1993, supra) as probe, an EMBL-3 genomic library (Clonetech, Palo Alto, CA) prepared from maize seedlings (B73, 2 leaf stage) was screened essentially according to Sambrook et al . Approximately 3 x 10⁵ plaque forming units were transferred onto nylon membranes (Hybond-N+, Amersham, UK) . Hybridization was performed at 55°C for 20 h in a solution containing 0.5 M Na₂HP0₄, pH 7.2, and 7% SDS with gentle agitation at 40 cycles per minute on a rotary shaker. Following the hybridization, filters were washed twice in 5% SEN (5% SDS (w/v) , 1 mM EDTA, 0.04 M Na₂HP0₄, pH 7.2) and once in 1% SEN (1% SDS, 1 mM EDTA, 0.04 M Na₂HP0₄, pH 7.2) for 15 min at 65°C. Plaques strongly hybridizing to the probe were selected and purified through three rounds of screening. Phage DNAs were isolated from the positive plaques according to Chisholm' s method and Sail -digested inserts were subcloned into pBluescript SK^" . DNA sequences were determined by the dideoxynucleotide chain termination method with Sequenase Version 2.0 (United States Biochemical Co., Cleveland, OH). Sequence analyses were performed using programs from DNASTAR Inc. (Madison, WI) . Primer Extension Analysis. To locate the transcription initiation site of the Ae gene, an oligonucleotide, 5 ' -GATCGGATCGAACTGATCAG-3 ' (SEQ ID N0:11), which is complementary to the sense strand sequence of the Ae cDNA from -35 to -16 relative to the translation start site (ATG) was radiolabeled at its 5' terminus with T4 polynucleotide kinase and γ³²P-ATP. Approximately 10⁵ cpm of the labeled primer was hybridized at 35°C with 10 μg of total RNA, which was isolated from 30 DAP maize kernels (B73) according to the protocol of Vries et al . (In: Gelvin, SB, Schilperoot, RA (eds) Plant Molecular Biology Manual B6 : 1-13, Kluwer Academic Publishers, Dordrecht, Netherlands, 1988). After hybridization for 8 h, complementary DNA was synthesized from the annealed primer by the addition of reverse transcriptase and dNTP. Following the addition of EDTA and RNAase A into the reaction, the nucleic acid was precipitated with ethanol. The reaction products were resuspended in sequencing gel loading buffer, denatured at 95°C, electrophoresed through a 5% polyacrylamide sequencing gel (w/v) , and visualized by autoradiography. In order to provide size markers, part of the Ae gene was sequenced with the same primer used in the primer extension experiment . Genomic Southern Blot Analysis. Maize genomic

DNA was prepared from 7-day-old etiolated seedlings (inbred B73) according to the method described by Junghans et al (1990, supra) . 10 μg of genomic DNA was digested with restriction enzymes, separated on 0.8% agarose gels, and transferred onto nylon membranes

(Hybond-N, Amersham, UK) in 20 X SSC containing 3 M NaCI and 0.3 M sodium citrate, pH 7.0 according to Sambrook et al . DNA was crosslinked to the membrane by 3.5 min of UV irradiation on a transilluminator (312 nm) . Genomic blots were prehybridized at 65°C for 1 h in 0.5 M Na₂HP0₄, pH 7.2, 7% SDS, and 100 μg/mL denatured salmon sperm DNA. 25 ng of a full-length Ae cDNA (Fisher et al . , 1993, supra) was labeled with [α-³²P] -dCTP using a random primed DNA labeling kit (Boehringer Mannheim, Indianapolis, IN) . Labeled probe was added to the prehybridization solution and incubated at 65°C for 18 h. Blots were washed twice in 5% SEN and once in 1% SEN for 15 min at 65 °C and were exposed to Kodak X-AR film at -80°C for 48 h using two intensifying screens.

Construction of Plasmids. For a transcriptional fusion of the Ae promoter to a luciferase (LUC) reporter gene, a BamHI restriction enzyme site was created just before the translation initiation site (ATG) of the Ae gene as follows. The DNA sequence between -14 and +100 of the Ae gene was first amplified via polymerase chain reaction. The primer (PII-2) , 5 ' -CCTAATTGTAGCCCTGCAGTCA-3 ' (SEQ ID N0:12), is homologous to sequence of the Ae gene from -10 to +12. A Pstl site (CTGCAG) is located immediately downstream of the primer binding region of the Ae promoter, +4 to +9. The 3' primer (PII-3), 5'- GACTGGATCCTCGCCTTCGCAGCCGGATCG-3 ' (SEQ ID NO: 13), consists of a DNA sequence complementary to that of the Ae gene from +80 to +100 and a BamHI restriction enzyme site (GGATCC) flanked with four random nucleotides (underlined) . The PCR product was digested with Pstl and BamHI, and the resulting 100 -bp fragment was ligated to the 2,977-bp Sall-Pstl Ae promoter fragment and cloned into plasmid pLN cut with Sail and BamHI ( promoterless LUC-NOS gene in pUC119) , thereby creating plasmid pKL201. This construct as well as all the following constructs were verified by DNA sequencing.

To construct a translational fusion of the Ae promoter containing the first exon and intron to a LUC reporter plasmid, the Ae genomic clone, 3-2-1, was digested with Xhol and the resulting 866-bp fragment was gel purified. The fragment was blunt ended by Klenow fill-in DNA synthesis and ligated with BamHI linkers (CGGGATCCCG) . After complete digestion with Pstl and BamHI, the 325-bp DNA fragment was isolated and used to replace the 100-bp Pstl-BamHI region in pKL201. This construct was designated pKLN201.

A series of 5 ' deletion mutants were derived from pKL201 using available restriction enzyme sites and PCR techniques. To create pKL202, pKL203, pKL205 and pKL206, pKL201 was first digested with Accl (-1719 to - 1714), Spel (-1128 to -1123), Xhol (-537 to -532) and Apal (-348 to -343) , respectively. Then, each linearlized plasmid was separately gel purified and blunt ended by Klenow fragment. After Sail linker ligation, the modified plasmids were digested with Sail, and the larger DNA fragments from each reaction were isolated and self-ligated to produce the relevant plasmids carrying different deletion end points.

For a construction of pKL204, the Ae promoter region between -755 and -500 was amplified with two primers. The 5' primer containing a Sail (GTCGAC) flanked with four extra nucleotides (underlined), 5'- GAAAGTCGACGAAGAGAGAATGAAAGCGAA-3 ' (SEQ ID NO: 14) , and the 3' primer, 5 ' -GCGCGGGTCCGTCGTGCCTTTT-3 ' (SEQ ID NO:15), were designed to anneal to DNA sequences of the Ae gene from -755 to -736 and from -521 to 500, respectively. The amplified 266-bp product was digested with Sail and Xhol which was located 10 bp upstream of the 3 ' primer binding region, and the resulting 230-bp fragment was used to substitute the 2.4-kb SalI -Xhol fragment in pKL201.

To make pKL207, the Ae promoter region between -160 and +100 was first amplified with 5' primer, 5'-

GATAGTCGACCGACGCGCAACGGCCTGCCT-3 ' (SEQ ID NO: 16), and 3' primer, 5 ' -GACTGGATCCTCGCCTTCGCAGCCGGATCG-3 ' (SEQ ID NO:17), which contain Sail and BamHI sites, respectively, along with four arbitrary extra bases (underlined) . Next, the PCR product was digested with Sail and BamHI and the resulting 267-bp DNA fragment was then used to replace the 3.1-kb Sail -BamHI fragment in pKL201. The same 3 ' primer and method were used for a construction of pKL208 except for the 5' primer, 5'- GATCGTCGACCGCTCGTCTCCGTCCTATAT-3 ' (SEQ ID NO: 18), homologous to the DNA sequence of the Ae gene from -49 to -32.

Particle Bombardment. Suspension culture cells of maize (inbred A636) endosperm provided by J. L. Anthony (DEKALB Genetics Corporation, Mystic, CT) were grown in 250-ml large-mouth Erlenmeyer flasks containing 80 ml of Murashige and Skoog basal salt medium supplemented with 0.4 mg/1 thiamine 2 g/1 asparagine and 30 g/1 sucrose. The culture was maintained in the dark at 29°C on a rotary shaker (120 rpm) and was subcultured every 7 days by transferring a spoonful of the cell suspension into 80 ml of fresh medium.

For particle bombardment, about 600 mg (fresh weight) of actively growing cells 3 days after subculture was evenly distributed over the surface of filter paper (Whatmann #4, 55 mm in diameter) by vacuum filtration of 8 ml of suspension culture. The filter paper bearing the cells was then placed over three layers of filter paper (Whatmann #4, 70 mm in diameter) moistened with 5 ml of the liquid medium containing 12% sucrose and positioned in the middle of a 10 cm petri dish.

60 mg of gold microcarriers (1.6 μm particle size) were washed three times with 1 ml of 100% ethanol and twice with 1 ml of sterile deionized H₂0, resuspended in 1 ml of sterile deionized H₂0, and dispensed in 50-μl aliquots (3 mg/50 μl) . The Sjbel Promoter-LUC constructs and a GUS reference plasmid (pBl 221; Clontech, Palo Alto, CA) were co-precipitated onto gold particles as follows: under continuous vortexing, the following were added in order to each 50-μl aliquot of gold particles: 5 μl of DNA (8 μg of LUC reporter plasmid, 4 μg of GUS reference plasmid), 50 μl of 2.5 M CaCl₂, and 20 μl of 0.1 M spermidine (free base, tissue culture grade) . The gold particles coated with DNA were pelleted in an Eppendorf centrifuge at 10,000 rpm for 10 sec, rinsed with 250 μl of 100% ethanol, and resuspended in 60 μl of 100% ethanol. Immediately after sonication, 8 μl of the DNA-coated gold particles were pipetted onto the center of macrocarriers (Bio-Rad, Hercules, CA) and dried in a low humidity environment.

A Bio-Rad PDS-1000/He Biolistic Particle Delivery system was used for particle bombardment. Bombardment parameters which were optimized include He pressure, gap distance (distance from power source to macroprojectile) , and target distance (distance from microprojectile launch site to sample target) . After optimization, all bombardments were performed in a dim room at 650 psi under a vacuum of 26 inches of Hg with a distance of 10 cm between the cells and the barrel of the particle gun. Following the bombardments, the petri dishes were sealed with Parafilm and then incubated in the dark at 25°C for 24 hr.

GUS and LUC Assays. The bombarded cells were harvested from the plates by vacuum filtration, frozen in liquid nitrogen, and ground with a pestle and mortal to a fine powder. The powder was then transferred into a microfuge tube and extracted with cell culture lysis buffer containing 300 mM Tris-phosphate, pH 7.8 , 2 mM DTT, 2 mM 1,2-diaminocyclohexane-N, N, N' , N' -tetraacetic acid, 10% glycerol and 1% Triton X-100 (0.3 ml/g of tissue) . Cell debris was pelleted in an Eppendorf centrifuge at 14,000 rpm for 10 min at 4°C and the supernatant was split into two aliquots for assays of GUS and LUC activity.

For fluorometric GUS assays, 30 μl of the crude extract was incubated at 37°C with 2 mM 4-methyl umbelliferyl glucuronide in 0.3 ml of GUS assay buffer (50 mM NaP0₄, pH 7 , 10 mM EDTA, 0.1% Triton X-100, 0.1% Sarkosyl, 10 mM β-mercaptoethanol , 20% methanol) . After 0, 1, and 2 hr of incubation, 0.1 ml aliquots were removed and added to 0.9 ml of 0.2 M Na₂C0₃ to terminate the reaction. A TKO 100 fluorometer (Hoeffer, San Francisco, CA) calibrated by setting a 100-nM MU to 1,000 fluorescence units was used to measure fluorescence of the product, 4-methyl umbel1iferone (4-MU) . For each sample, results of GUS assay were plotted in a graph of

OD₄₀₅ (Y-axis) versus time in minutes and the GUS activity was expressed simply as the slope of the line. GUS activity from the maize endosperm suspension cells that had been bombarded with the naked gold particles (no DNA) was used as a control.

Using a luminometer (Monolight 1500; Analytical Luminescence Laboratory, San Diego, CA) , luciferase activity was determined by measuring luminescence for 10 sec after mixing 20 μl of cell extract with 100 μl of luciferase assay reagent containing 20 mM tricine, pH 7.8, 1.07 mM (MgC0₃) ₄Mg (OH) ₂ • 5H₂0 , 2.67 mM MgS0₄, 0.1 mM EDTA, 33.3 mM DTT, 270 μM coenzyme A, 470 μM luciferin and 530 μM ATP. LUC activity from the maize endosperm suspension cells that had been bombarded with the pLN (promoterless LUC plasmid) was used as a control . To correct differences in sample variability and transfection efficiency, the luciferase activity (in light units) was normalized with GUS activity, yielding the LUC/GUS ratio of each sample.

Results

Cloning and Characterization of the Ae Gene. After screening approximately 3 x 10⁵ plaque-forming units of a genomic library prepared from maize seedlings (inbred B73) , 12 lambda clones that strongly hybridized to the full-length Ae cDNA probe were isolated. These clones were hybridized to 5' or 3 ' cDNA fragment probes revealing that none of them contained both ends of the Ae gene. Based on restriction endonuclease maps, two clones, λ 3-2-1 and λ 7-2-1, were selected, subcloned, and sequenced. DNA sequences of the two clones revealed that the λ 3-2-1 clone containing the 5 ' -end of the Ae gene had approximately 1.5-kb of overlap with the λ 7-2-1 clone containing the 3 ' -end of the gene. A complete restriction map of the Ae gene was constructed by combining the two overlapping genomic clones which encompasses the entire coding region of the gene as well as large regions of 5'- and 3'- flanking sequences (Fig. 3) .

Genomic Organization of the Ae Gene. Primer extension analysis was conducted to determine the transcription initiation site of the Ae gene. A single major reverse transcription product was observed which co-migrated with a G residue in the sequencing ladder, indicating the transcription initiates mainly at a position which is located 28 bp downstream from a putative TATA box. The transcription initiation site was numbered +1 in the sequence shown in Figure 4. In addition to the TATA box, a number of potential cis- regulatory elements were found in the 5 '-flanking region of the Ae gene (Fig. 4) . The proximal promoter region from -300 to -87 contains two MRE boxes (TGCRCNC, R = purine, Y = pyrimidine) , motifs essential for metal ion- dependent induction of both mouse and human metallothionein genes. In addition, this region contains four GC boxes (CCGCCC) , sequences recognized by the mammalian transcription factor Spl . In a region further upstream, sequences identical to a Hex (ACGTCA) , a conserved element found in plant histone gene promoters; an I box (GATAGG) , an element conserved in various RBCS genes encoding the small subunit of ribulose 1,5- bisphosphate carboxylase/oxygenase; and a RY repeat (ATGCCATG) , a distal regulatory element which comprises a portion of the 28-bp legumin box were present at positions -999, -647, and -1491, respectively. Interestingly, DNA sequences from -505 to +463 are extremely high in G+C content (66.5%) and CpG dinucleotide frequency (10.1 per 100 bp) compared to an average of 40% G+C and 3.2 CpG per 100 bp for the rest of the genomic fragment. This region includes the proximal promoter region, two exons, and one intron. These characteristics are typical of CpG islands found in the mammalian genome, which are usually nonmethylated and flanked by methylated regions. The genomic structure of the Ae gene was established by alignment with the published sequence of Ae cDNA. The transcribed region of the gene consists of 22 exons and 21 introns distributed over 16,914 bp in length. Figure 3 summarizes the organization of the Ae gene. The published cDNA sequence is identical to the corresponding genomic sequence except for three nucleotides present in exon 4 and exon 6. This may be due to the different genetic stocks used in the two studies . Table 2 shows the sequences around the exon/intron junctions and a list of putative branch point consensus sequences . Table 2. List of introns and sequences of exon/intron

Dorders in the Ae gene .

Intron Intron GC content number Exon/ Putative intron branch point* /Exon size(bp) (%)

1 ACTC GTAA.... .GTGAA. .24^γ GCAG GGGG 106 51 9

2 GGAG GTTC .... . CTGAA . .29. .CCAG GTAC 244 41 4

3 ATCG GTAT .... . TTCAA . .21. ■ACAG GTAC 1086 43 9

4 GCAG GTAT .... .ATAAC. .24. • GTAG CGCG 76 25 0

5 ATTT GTAT .... . TTCAG . .25. ■ TTAG TCTG 196 31 1

6 CAAA GTAT .... .CTAAA. .22. .GCAG AATG 499 33 5

7 AAAG GTAG .... . TTAAC . .23. .ATAG GTGA 81 40 7

8 AAGA GTCT .... . TTAAG . .21. ■ GCAG GTAA 567 30 3

9 CCCG GTAT .... .ATAAT. .23. .TTAG GAAC 774 32 0

10 TTGG GTAA .... . TTAAT . .21. .GCAG ATAC 751 33 2

11 ATAG GTAA.... . TTAAC . .22. .GCAG TCAT 4020 44 0

12 GGAA GTAC .... . TTTAT . .49. ■ GCAG GTTT 86 34 9

13 ACAA GTAA.... .CTAAA. .19. .TTAG GTAA 148 31 1

14 GTAA GTGC .... .TTCAA. .20. ■ TCAG GTTA 3051 41 8

15 TCAA GTAA.... . TTCAA . .19. .ACAG GCAA 872 31 8

16 CAAG GTTA.... .ATGAG. .25. • GCAG GATA 457 32 6

17 CCTG GTGA.... . GTCAT . .21. .GCAG AATG 144 30 6

18 CCTG GTAA.... .ATTAT . .28. ■ TCAG GGTG 226 30 1

19 TGAA GTAT .... . ATGAA . .19. ■ GCAG TTCA 266 31 2

20 TAAG GTAT .... . CTGAC . .21. ■ CCAG GTGG 448 40 .0

21 CGCC GTAA.... . CTAAC . .24. ■ GCAG GACT 96 45 .8

* Consensus sequences between introns are underlined. ^γ Numbers indicate number of nucleotides between adjacent sequences . The introns are relatively AT-rich (61%) compared to the exons (54%) , and all have the conserved splice site sequences at their 5' and 3' ends, following the ^VGT...AG' rule of plant introns. The introns vary in length from 76 bp (intron 4) to 4,020 bp (intron 11), and the exons vary in length from 43 bp to 303 bp . Exon 1 contains 100 bp of 5' untranslated DNA sequence, and exon 22 contains the translation stop codon (TGA) and 3 ' untranslated region. Although the canonical polyadenylation signal, AATAAA, was not found in the 3'- end of the gene, a similar sequence (AATTAAA) was observed 29 bp upstream of the polyadenylation site.

In addition, sequence analysis revealed that the 3 '-flanking region of the Ae gene contains many direct repeat sequences and has a high degree of similarity to the pollen retroelement maize-2 (PREM-2) , a copia-type retroelemt in maize which is expressed in a tissue-specific manner (Fig. 4). A 13-bp polypurine tract (AAAAAGGGGGAGA) which is present in PREM-2 and necessary for retroelement replication was also found upstream of the region which is very similar to the 3 ' long terminal repeat (LTR) of the PREM-2. Thus, the 3'- flanking region is likely to possess part of a PREM-2 - type retroelement . Genomic Southern Blot Analysis. Southern blot analyses were performed to determine the number of genes in the maize genome that are similar to Ae . When blots were probed with the full-length Ae cDNA under high- stringency conditions, at least two strongly hybridizing bands were observed in each lane. The band patterns agreed with the restriction map of the Ae genomic DNA, suggesting all the bands were derived from a single genetic locus. To confirm this, the blots were probed with a small fragment of the Ae genomic DNA which does not have any restriction enzyme sites for BamHI, EcoRI , Bglll and Hindlll. As expected, only a single band was observed in every lane, supporting the conclusion that a single copy of Ae is present in the maize genome.

Analysis of the 5' -flanking Region of the Ae Gene. To identify the 5' -flanking regions necessary for Ae gene expression, we utilized a transient expression assay system in maize endosperm suspension cells. Iodine staining and northern blot analysis showed that the maize endosperm suspension cells actually produce starch and the genes involved in starch biosynthesis are expressed. In an initial experiment, a transcriptional chimeric construct containing the Ae gene fragment between -2964 and +100 linked to a luciferase (LUC) reporter gene in pUC119 was created and called pKL201. Since there are several examples of plant genes which are regulated by the first exon and/or intron sequences, a translational fusion construct (pKLN201) containing the corresponding region of the Ae gene was also created to test its effect on gene expression. pKLN201 was created by including an additional Ae DNA sequence from +101 to +329 in the plasmid pKL201. These plasmids were then tested by assaying LUC activity after introduction of DNA into maize endosperm suspension cells by particle bombardment. Plasmid pBI221 containing the CaMV 35S promoter linked to a GUS gene was used as an internal control to correct for transfection efficiency. The results showed that levels of LUC expression driven by the two constructs were almost the same , suggesting that the first exon and intron region of Ae is not necessary for high level gene expression in maize endosperm cells.

To define the promoter sequences important for Ae gene expression in maize endosperm cells via transient expression assays, a series of 5' deletion mutants were derived from the plasmid pKL201 using available restriction enzyme sites and PCR techniques (Fig. 5A,B) . The activity of each 5 ' deletion construct is presented in Fig. 5C. Two consecutive deletions of the Ae 5'- flanking sequence down to -1128 decreased the level of the LUC expression to approximately 40% of the full- length promoter level. However, the removal of an additional 353 bp, to -775, restored LUC activity (Fig. 5C) . This suggests that potent positive and negative distal cis-regulatory elements may be located in the regions from -2964 to -1129 and from -1128 to -776, respectively. Although further deletions down to -160 did not significantly change the levels of LUC expression, a dramatic reduction in the promoter strength was observed when an additional 111 bp, to -49, was deleted. This indicates that very strong positive regulatory element (s) are located at the 111-bp region from -160 to -50. The presence of two GC boxes and one MRE motif in the region suggests the possibility that these conserved motifs may actually act as cis-regulatory elements essential for gene expression in maize endosperm cells .

EXAMPLE 3 Functional Analysis of SJbel Promoter

We report in this example a functional analysis of the Sjbel promoter which reveals DNA sequence elements important for the high level and sugar responsive expression of the Sbel gene in maize endosperm cells. Materials and Methods ;

Construction of Chimeric Plasmids. A transcriptional fusion of the Sbel promoter to a luciferase (LUC) reporter gene was made as follows. A BamHI restriction enzyme site was first created just before the translation initiation site of the Sbel gene by polymerase chain reaction (PCR) : The DNA sequence between -253 and +27 of the Sbel gene was PCR-amplified with PI-1 and PI -2 primers (Table 3) .

Table 3. Oligonucleotides used in PCR to create SJbel LUC chimeric constructs .

Primer³ Sequence⁰

Annealing Region⁰

PI- -1 U CCAGCTCCACGGTTGTTCGTGT -253 to -232

PI- -2 L cgatggatccTGTGACGGCGTGTGAGTCCC +8 to +27

PI- -3 L agtcggatccTCAGGCGCACATTGCCGCCA +209 to +228

PI- -4 U gactgagctcATACCAAATGAAGCCAGGAG +5382 to +5401

PI- -5 L actggaattcGGAACAAGGAACGAAGAAAC +5761 to +5780

PI- -6 U gatcaagcttACCAGCTCCACGGTTGTTCG -254 to -235

PI- -7 U attcaagcttCAGATCCGGCTCAGGGTCAT -196 to -177

PI- -8 L TGCGACAAGGAGGGGGCCAT -165 to -146 ^a U and L indicate upper (sense) and lower (antisense) primers relative to the Sbel , respectively. ^b The lowercase letters designate restriction sites used for cloning. ^c Numbers represent distance relative to the transcription start site (+1) of the Sbel .

Four additional bases were included at 5 ' -end of the primers to provide for restriction enzyme sites at the ends of the PCR products for subsequent cloning. The bases were chosen randomly considering their effect on Tm value, dimer and stem-loop formation of the primers. —Pfu

DNA polymerase (Stratagene, La Jolla, CA) , which has proofreading activity, was used to enhance the fidelity of PCR amplification ( Pfu DNA polymerase was used for all the following PCRs) . Since an Apal restriction enzyme site (GGGCCC) is located immediately downstream of the 5' primer (PI-1) binding region of the Sjbel promoter, -203 to -198, the PCR product was digested with Apal and BamHI. The resulting 236-bp fragment was then cloned into pBluescript SK^" and sequenced to verify that no misincorporation had occurred in the DNA sequence during the PCR amplification (all the following PCR products were sequenced) . Next, the 236-bp fragment was ligated to the 1991-bp Sall-Apal Sbel promoter fragment and cloned into plasmid pLN cut with Sail and BamHI

(promoterless LUC-NOS gene in pUC119) (Montgomery et al . , Proc. Natl. Acad. Sci. USA, 90.: 5939-5943, 1993), thereby creating plasmid pKLlOl.

To construct a translational fusion of the Sjbel promoter containing the first exon and intron to a LUC reporter plasmid, the DNA sequence between -253 and +228 was amplified with the PI-1 primer and a 3' primer (PI-3) designed to anneal to the region just downstream of the first intron of the Sbel gene. The 493-bp PCR product was digested with Apal and BamHI, and the resulting 436- bp fragment was used to replace the Apal -BamHI fragment in pKLlOl. This construct was called pKLNlOl. To make pKLMlOl which contains the Sbel promoter with four exons and introns, the 236-bp Apal and BamHI fragment in pKLlOl was replaced with the 1816-bp Sbel genomic DNA fragment. The plasmid pKLNSlOl was derived from pKLNlOl by replacing the nopaline synthase (NOS) 3 ' sequence with the native Sbel 3' -flanking sequence. To accomplish this, two primers PI -4 and PI -5 were designed to amplify SJbel DNA sequences containing the transcription stop signal and the polyadenylation site (from + 5382 to + 5780) . A 419-bp PCR product was digested with Sad and EcoRI, and the resulting fragment was then used for substituting a 255-bp SacI-EcoRI NOS 3' sequence in the pKLNlOl.

To create a series of 5 ' deletions in the Sbel promoter, pKLNlOl was first modified as follows: pKLNlOl DNA was digested with Hindlll and the resulting 7190-bp fragment lacking the 452 -bp HindiII fragment was gel purified. The fragment was blunt ended by Klenow fill-in DNA synthesis and ligated with Sail linkers. After complete digestion with Sail, the DNA fragment was partially digested with BamHI to isolate the 1993 -bp Sail -BamHI fragment, which was then gel purified and cloned into pLN cut with Sail and BamHI to produce pKLNlOl-l.

A series of 5 ' deletion mutants were made from the plasmid pKLNlOl-l using the SI nuclease based Erase- a-Base system (Promega, Madison, WI) to produce the 5' deletion series plasmids, pKLN102 to pKLN107. All constructs were sequenced with pUC/M13 reverse primer to verify deletion end points. For the -254 and -196 deletion constructs, two regions of the Sbel promoter, - 254 to -146 and -196 to -146, were PCR-amplified by primer PI-6 and PI-8, PI-7 and PI-8, respectively. Primers used in PCR to create the Sbel-LUC constructs are shown in Table 2. Since each 5' primer, PI-7 and PI-8, contains a Hindlll restriction enzyme site and a BstXI restriction enzyme site is located between -173 and -162, the PCR products were digested with Hindlll and BstXI and the resulting fragments were used to replace the 2,047-bp Hindlll-BstXI fragment of pKLNlOl. Linker-Scanning Mutagenesis. A series of linker-scan mutations were introduced into the 60-bp DNA region from -314 to -255 as described by Kunkel et al (Kunkel et al . , Methods Enzymol., 154: 367-382, 1987) . Briefly, the Hindlll-BamHI (-314 to +235) fragment from pKLN105, containing the DNA region to be altered, was subcloned into the corresponding sites of a M13mpl9 vector to produce a single-stranded template. In order to increase mutant recovery efficiencies, the template was prepared from an E. coli dut^" ung^' strain (CJ236) which allows the incorporation of uracil into the newly synthesized DNA. Next, a set of oligonucleotides with 10 -bp mismatches shown in Table 4 were annealed to the template and extended with T7 DNA polymerase. After addition of T4 DNA ligase, the resulting heteroduplexes were introduced into a wild-type E.coli strain (MV1190) to generate mutated double-stranded DNAs . DNA sequencing was performed to verify that the desired mutations were correctly introduced and no unintended mutations had occurred.

To create the mutated Sjbe 1 promoter-LUC constructs (pLSl-1 to pLSl-6) , the Hindlll-BamHI fragment in pKLN105 was replaced by each mutated DNA sequence.

Table 4. Oligonucleotides used in linker-scanning mutagenesis .

Constructs Oligonucleotides* pLS1-1 CCCGGTTTGCCTTTTTTgcTgcaggacAAGCTTGGCGTAATCAT pLS1-2 TTGCACGCTTCCCGGTTgaCtgcaggcTATTTTATGTAAGCTTG pLSl-3 GCCTTTGGGCTTGCACGtcTagatagcTGCCTTTTTTTATTTTA pLS1-4 GGGCCGATTGGCCTTTGactgcaggtaCTTCCCGGTTTGCCTTT pLS1-5 AGCTGGTTCTGGGCCGAcgactgcagaGGCTTGCACGCTTCCCG pLSl-6 ACAACCGTGGAGCTGGTgtcGactatcTTGGCCTTTGGGCTTGC

* The mutated bases are shown in lowercase letters, and restriction sites used for convenience of screening are underlined. Particle Bombardment. Suspension culture cells of maize ( Zea mays) endosperm (inbred A636) provided by J. L. Anthony (DEKALB Genetics Corporation, Mystic, CT) were grown in 250 -mL large-mouth Erlenmeyer flasks containing 80 mL of Murashige and Skoog basal salt medium supplemented with 0.4 mg/L thiamine 2 g/L asparagine and 30 g/L sucrose (Shannon and Liu, Physiol. Plant, 40_.: 285- 291, 1977) . The culture was maintained in the dark at 29°C on a rotary shaker (120 rpm) and was subcultured every 7 days by transferring a portion of the cell suspension into fresh medium.

For particle bombardment, about 600 mg (fresh weight) of actively growing cells 3 days after subculture was evenly distributed over the surface of a piece of filter paper (Whatmann #4, 55 mm in diameter) by vacuum filtration of 8 mL of suspension culture. The filter paper bearing the cells was then placed over three layers of filter paper (Whatmann #4, 70 mm in diameter) moistened with 5 mL of the liquid medium containing 12% sucrose and positioned in the middle of a 10 cm petri dish.

60 mg of gold microcarriers (1.6 μm particle size) were washed three times with 1 ■ mL of 100% ethanol and twice with 1 mL of sterile deionized H₂0, resuspended in 1 mL of sterile deionized H₂0, and dispensed in 50-μL aliquots (3 mg/50 μL) . The SJbel Promoter-LUC constructs and a GUS reference plasmid (pBI221, Jefferson, Plant Mol. Biol. Rep., 5: 387-405, 1987) were co-precipitated onto the gold particles as follows: under continuous vortexing, the following were added in order to each 50- μL aliquot of gold particles: 5 μL of DNA (8 μg of LUC reporter plasmid, 4 μg of GUS reference plasmid) , 50 μL of 2.5 M CaCl₂, and 20 μL of 0.1 M spermidine (free base, tissue culture grade) . The gold particles coated with DNA were pelleted in an Eppendorf centrifuge at 10,000 rpm for 10 sec, rinsed with 250 μL of 100% ethanol, and resuspended in 60 μL of 100% ethanol. Immediately after sonication, 8 μL of the DNA-coated gold particles were pipetted onto the center of macrocarriers (Bio-Rad, Hercules, CA) and dried in a low humidity environment.

A Bio-Rad PDS-1000/He Biolistic Particle Delivery system was used for particle bombardment. Bombardment parameters which were optimized include He pressure, gap distance (distance from power source to macroprojectile) , and target distance (distance from microprojectile launch site to sample target) . After optimization, all bombardments were performed in a dimly light room at 650 psi under a vacuum of 26 inches of Hg with a distance of 10 cm between the cells and the barrel of the particle gun. Following bombardment, the petri dishes were sealed with Parafilm and then incubated in the dark at 25°C for 24 hr. GUS and LUC Assays. The bombarded cells were harvested from the plates by vacuum filtration, frozen in liquid nitrogen, and ground with a pestle and mortal to a fine powder. The powder was then transferred into a microfuge tube and extracted with cell culture lysis buffer containing 300 mM Tris-phosphate, pH 7.8 , 2 mM

DTT, 2 mM 1, 2-diaminocyclohexane-N, N, N' , N' -tetraacetic acid, 10% glycerol and 1% Triton X-100 (0.3 mL/g of tissue) . Cell debris was pelleted in an Eppendorf centrifuge at 14,000 rpm for 10 min at 4°C and the supernatant was split into two aliquots for assays of GUS and LUC activity.

For fluorometric GUS assays (Jefferson, Plant Mol. Biol. Rep., 5: 387-405, 1987), 30 μL of the crude extract was incubated at 37°C with 2 mM 4-methyl umbelliferyl glucuronide in 0.3 mL of GUS assay buffer (50 mM NaP0₄, pH 7.0 , 10 mM EDTA, 0.1% Triton X-100, 0.1% Sarkosyl, 10 mM β-mercaptoethanol , 20% methanol) . After 0, 1, and 2 hr of incubation, 0.1 mL aliquots were removed and added to 0.9 mL of 0.2 M Na₂C0₃ to terminate the reaction. A TKO 100 fluorometer (Hoeffer, San Francisco, CA) calibrated by setting a 100-nM MU to 1,000 fluorescence units was used to measure fluorescence of the product, 4-methyl umbelliferone (4-MU) . For each sample, results of GUS assay were plotted in a graph of OD₄₀₅ (Y-axis) versus time in minutes and the GUS activity was expressed simply as the slope of the line. GUS activity from the maize endosperm suspension cells that had been bombarded with the naked gold particles (no DNA) was used as a control .

Using a luminometer (Monolight 1500; Analytical Luminescence Laboratory, San Diego, CA) , luciferase activity was determined by measuring luminescence for 10 sec after mixing 20 μL of cell extract with 100 μL of luciferase assay reagent containing 20 mM tricine, pH 7.8, 1.07 mM (MgC0₃) ₄Mg (OH) ₂ • 5H₂0 , 2.67 mM MgS0₄, 0.1 mM EDTA, 33.3 mM DTT, 270 μM coenzyme A, 470 μM luciferin and 530 μM ATP. LUC activity from the maize endosperm suspension cells that had been bombarded with the pLN (promoterless LUC plasmid) was used as a control . To correct differences in sample variability and transfection efficiency, the luciferase activity (in light unit) was normalized with GUS activity, yielding the LUC/GUS ratio of each sample.

Nuclear Extract Preparation. Maize kernels (inbred B73) were harvested 30 days after pollination and frozen in liquid nitrogen. Nuclear extract was prepared essentially according to the method described by Jensen et al. (Jensen et al . , EMBO J. , 7: 1265-1271, 1988). Protein concentration was determined using a BCA protein assay kit (Pierce, Rockford, IL) , according to the manufacturer's instructions.

DNA Probe Preparation. The Sjbel promoter region from -314 to -255 was PCR-amplified with a forward primer (5 ' -GGACTTACATAAAATAAAAAAAGG CA) and a reverse primer (5 ' -TGCTAAGCTTTCTGGGCCGATTGGCCTTTG) which contain BamHI and Hindlll restriction enzyme sites, respectively, at their 5' ends (underlined) . The PCR product was digested with BamHI and Hindlll, and the resulting fragment was cloned into pBlueScript SK^" cut with BamHI and Hindlll to create plasmid pRb4-l. The plasmid construct was verified with DNA sequencing. For electrophoretic mobility shift assays, the DNA fragment was cut out from the plasmid pRb4-l with Hindlll and BamHI, purified from agarose gels, and end-labeled with [α-³²P]-dCTP using the Klenow fragment. Electrophoretic Mobility Shift Assay. The DNA- protein binding reaction was performed in 20 μL of solution containing 0.5 ng of labeled probe, 10 μg of nuclear protein, 1 μg of poly (dl-dC)-poly (dl-dC), 12% glycerol, 12 mM HEPES-NaOH (pH 7.9), 4 mM Tris-Cl (pH 7.9), 60 mM KCl , 1 mM EDTA and 1 mM DTT. After a 20-min incubation at room temperature, the samples were loaded into a 4% native polyacrylamide gel which was pre-run at 4°C for 1 hr at 150 V and electrophoresed for 2.5 hr at 150 V in Tris-glycine buffer at 4°C. Following electrophoresis, the gel was dried with a gel dryer (Bio-

Rad) and exposed to Kodak X-ray film with two intensifying screens for 24 hr. Northern Blot Analysis. Total RNA was isolated according to Vries et al . (Vries et al . , Plant Mol . Biol . Manual, B6: 1-13, 1988) from maize endosperm suspension cells which had been incubated for 24 hr in the MS basal salt media supplemented with 0.4 mg/L thiamine, 2 g/L asparagine and various amounts of sucrose from 0% to 15%. Northern blot analysis was performed as described in Gao et al . (1996, supra) . Radioactivity was detected with a Phosphorlmager and quantified with the ImageQuant software program (Molecular Dynamics) . To correct for minor loading errors between the lanes, the blot was wash at 95°C in a 0.1% (w/v) SDS solution to remove the ³²P- labeled Sbel cDNA probe and rehybridized with a ³²P- labeled tomato cDNA for 26S rRNA.

Results ;

Transcribed Regions of the Sbel Gene are involved in gene expression. To determine whether the 5' flanking sequence of the SJbel gene has all of the DNA elements necessary to initiate transcription, a 2217-bp fragment upstream of the translation start site (-2191 to +27) was fused to the luciferase (LUC) reporter gene in pUC119 (pKLlOl) as shown in Figure 6A. The chimeric plasmid was then introduced into maize endosperm cells via particle bombardment along with a reference plasmid containing the cauliflower mosaic virus (CaMV) 35S promoter linked to a GUS gene (pBI221, Jefferson, 1987, supra) to correct for transfection efficiency. However, only very low levels of LUC activity were detected relative to other promoters previously described.

Since many reports indicated that DNA sequences within transcribed regions such as exons, introns and 3' flanking regions are involved in the expression of genes in either qualitative or quantitative manners, three different types of translational fusion constructs were created to test the effect of downstream elements on Sbel gene expression. First, the 5 '-flanking sequence as well as the first exon and intron of the Sbel gene (-2190 to +228) were fused in-frame to the LUC reporter gene to make pKLNlOl (Figure 6A, B) . Second, the nopaline synthase (NOS) 3 ' sequence in the pKLNlOl was replaced with the SJbel 3' flanking sequence (399 bp in length), which contains the translation stop codon and polyadenlylation signal to create pKLSlOl. Finally, to determine whether an increase in the number of exon/introns enhances the gene expression, three more exons and introns from the Sbel gene were added to the pKLNlOl to make pKLMlOl (-2190 to +1617) .

The results of transient expression assays using the chimeric constructs are shown in Figure 6C. Inclusion of the DNA sequence from +28 to +228 containing the first exon and intron increased the level of LUC expression by 14-fold, suggesting that the first exon and intron region is required for high level expression of the SJbel gene in maize endosperm cells. Since pKLNlQl produce a fusion protein, however, we cannot completely rule out the possibility that the increase may be due to changes in enzyme activity and/or turnover rate caused by the added amino acid sequences. If the additional amino acids have a negative effect, the enhancement of LUC activity observed would be greater than 14 fold.

Replacement of the NOS 3' end in pKLNlOl with the SJbel 3 ' region did not have a significant effect on the level of LUC expression, implying that the Sbel 3 ' untranslated region does not have indispensable control elements. However, it is still possible that the region may be important for Sbel gene expression in other cell types or inductive conditions.

Construct pKLMlOl showed a slight reduction in LUC activity compared to pKLNlOl, indicating that additional exons and introns had an adverse effect on LUC expression in maize endosperm suspension cells. The adverse effect could be explained by inefficient splicing, resulting from the introduction of multiple copies of the plasmid into a single cell, or by formation of fusion protein consisting of the 5 ' -end of SBEI and luciferase, thus lowering LUC activity. Alternatively, it could also be actually due to the presence of negative cis-elements in this region.

5¹ deletion down to -314 did not significantly affect the Sbel promoter activity. To identify promoter sequences critical for Sbel expression in maize endosperm cells, a series of 5' deletion mutants were derived from pKLNlOl as shown in Figure 7A. The activity of each 5' deletion construct is presented in Figure 7B . Removal of the sequences to -1332 caused a decrease in the level of the LUC expression, while deletion of an additional 422 bp, to -910, resulted in an increase in the activity of the construct. This suggests that potential positive and negative distal cis-regulatory elements may be located in the regions from -2190 to -1332 and from -1332 to -910, respectively. Further deletions down to -315 did not significantly affect the promoter activity, but a severe reduction in the activity was observed when an additional 169 bp, to -145, was deleted. The -72 deletion construct produced a level of the LUC activity slightly over background, showing that the minimal promoter is functional . A 60-bp region is critical for the promoter activity. To further delimit sequences essential for high level expression of the promoter, two additional 5' deletions with about 60-bp intervals were created between -315 and -145. As shown in Figure 7, a deletion to -255 (pKLN108) severely reduced the expression of the LUC reporter gene, while a further deletion to -196 (pKLN109) did not further reduce promoter strength. This indicates that a strong positive regulatory element (s) is present in the 60-bp region between -315 and -255.

Linker-Scan Analysis reveals two cis-elements within the 60-bp region. Since the 5' deletion analyses indicated that the region of the Sbel promoter from -314 to -255 is critical for the promoter activity, the 60-bp DNA fragment was further dissected by oligonucleotide- directed in vi tro mutagenesis, as described by Kunkel et al . (1987, supra) . A series of six different substitution mutants, designated pLSl to pLS6, were created by altering the wild-type DNA sequence of the SJbel promoter at 10-bp intervals. The mutations were made by creating transversion substitutions where possible, while at the same time introducing restriction enzyme sites for simplifying identification of the mutant forms. The mutated constructs were tested for their promoter activity using the transient assay system, and the results of the experiments are shown in Figure 8. Mutations in the regions from -314 to -305 and -304 to - 295, corresponding to pLS-1 and pLS-2, caused a decrease in the SJbel promoter activity to 60% and 72% of wild-type (pKLN105) expression, respectively. The pLS3 construct showed almost the same level of the LUC expression as the wild-type promoter, suggesting the nucleotides from -294 to -285 are not important for the promoter activity in maize endosperm cells. However, mutation in the pLS-4 region (-284 to -275) decreased promoter activity to 40% of the wild-type level. Also, other two mutants, pLS-5 and pLS-6, resulted in a reduction of the promoter activity to 55% and 50% of the wild-type promoter, respectively .

The 60-bp fragment interacts with DNA-binding proteins. In order to investigate the possibility that a nuclear protein (s) might interact with the 60-bp Sbel promoter fragment from -314 to -255, electrophoretic mobility shift assays were performed. The 60-bp fragment was ³²P end-labeled with Klenow fill-in reaction and then incubated with nuclear extract prepared from 30 days after pollination (DAP) maize kernels (B73) , which have been demonstrated to highly express Sbel gene (Gao et al . , 1996, supra) . Two major shifted bands were observed in the lane containing nuclear extract (lane 2) compared to the control (lane 1) . The bands were not detected after inclusion of proteinase K in the binding reaction (lane 7) , indicating the shifted bands represent DNA- protein complexes .

Competition assays were conducted to determine whether or not the complexes are due to the binding of sequence-specific proteins. Inclusion of 10-fold and 100-fold excess of the unlabeled 60-bp fragment in the binding reaction significantly reduced formation of the complexes (lane 3 and 4) , while the same amount of nonspecific competitor DNA failed to compete for binding (lane 5 and 6) . Thus, the complexes appear to be the results of sequence-specific interactions between a nuclear protein (s) and the DNA fragment, consistent with the functional identification of this region as an important regulatory element. Using six 60-bp fragments of linker scan mutants (LS-1 to 6) as competitors, we have found that LS-1 did not affect the intensity of the lower band, although the rest of linker scan mutants abolished its formation. This suggests that the lower band may be the result of interactions between a transacting factor (s) and the sequence ACATAAAATA, located within LS-1. All of the linker scan mutants reduced the intensity of the slower migrating complex to varying degrees. Since LS-4, 5 and 6 were less effective competitors, wild type sequences spanning these regions (-284 to -255) may be involved in formation of this complex, however, binding may involve several overlapping regions in this fragment.

Expression of the Sbel gene is sugar-regulated. The SBEs are expressed in a coordinate fashion with the granule-bound starch synthase (GBSS) and ADP-glucose pyrophosphorylase during maize endosperm development (Gao et al . , 1996, supra) . The ADP-glucose pyrophosphorylase gene (AGPase S) from potato and the genes encoding GBSS and SBE in cassava plants have been shown to be induced by an exogenous supply of sugars (Muller-Rober et al . , Mol. Gen. Genet., 224 : 136-146, 1990; Giroux et al . , Plant Physiol., 106 : 713-722, 1994; Salehuzzaman et al . , Plant Sci., 98_.: 53-62, 1994). This led us to speculate that the Sbel gene in maize may also be regulated by external sugar concentration.

To test this, maize endosperm suspension cells were incubated in MS media containing different concentrations of sucrose and their total endogenous RNAs were analyzed by northern blot hybridization. Sucrose was used in preference to other metabolizable sugars, because it is known to be the major sugar unloading from the pedicel tissue of maize kernels. Increase in sucrose concentration from 0% to 9% elevated the Sbel mRNA level by two- fold, and at higher concentrations the increase was reduced. Hexoses such as glucose, fructose and myo- inositol also increased the level of the transcript in a similar fashion. However, L-glucose and PEG 200 at concentrations calculated to have the same osmotic potential as a 9% sucrose solution (263 mM) did not exhibit any effect, indicating that the response is not an osmotic effect but a sugar-specific phenomenon. These results suggest that expression of the Sbel gene in maize endosperm cells is regulated by sugar availability like other starch biosynthetic genes (Giroux et al . , 1994, supra) . This metabolic feedback mechanism may serve as a system to fine tune the expression levels of Sbe genes relative to physiological status of a plant. In fact,

Shannon et al . (Shannon et al . , Plant Physiol., 110 : 835- 843, 1996) showed that non-allelic starch mutants of maize accumulating high levels of sucrose in endosperm contain increased SBE activities compared to normal. Since we determined that two SJbel genes (SJbela and Sbelb) with divergent 5 '-flanking regions exist in the maize genome (Example 1) , it was necessary to determine whether or not expression of the isolated SJbel gene ( Sbela) promoter is responding to external sucrose concentrations. To test this, a gene which is not regulated by sugar concentration was required for an internal control for the transient assay system. Since a CaMV 35S promoter has been used as a control in other studies investigating sucrose responsiveness of plant genes, the effect of sucrose on expression of the CaMV

35S promoter-GUS chimeric gene (pBI221) in maize endosperm cells was first investigated.

The plasmid pBI221 was bombarded into maize endosperm suspension cells supplemented with 0% sucrose or 9% sucrose media and incubated at 25°C in the dark. After 48 hr incubation, GUS activity and protein concentration were measured from each sample to calculate specific GUS activity. The results showed that specific GUS activities of 9% sucrose samples were almost 2.5-fold higher than those of 0% sucrose samples, which is consistent with other reports. Since similar results were obtained from a ubiquitin promoter (pACHlδ) and -64 CaMV 35S minimal promoter which does not have an activation sequence (as)-l, a binding site for the transcription factor TGA-la) , it appeared that the elevated levels of expression by the CaMV 35S and ubiquitin promoters in 9% sucrose may be a general phenomenon simply due to an increase in energy source rather than a sugar-specific effect. Therefore, we reasoned that if the chimeric construct pKLNlOl (the SJbel promoter-LUC) is sugar-modulated, it will further enhance the level of LUC expression beyond the general increase at the higher sucrose concentration.

As shown in Figure 9, after normalization to GUS activity driven by the CaMV 35S promoter the plasmid pKLNlOl still showed approximately two-fold greater LUC activity in 9% sucrose media than in 0% sucrose media. This is consistent with the result of the endogenous RNA analysis, indicating that the identified Sbel gene is regulated by sugar availability. It also suggests that the nucleotide sequence containing a 2.2 kb 5 '-flanking region and the first exon/intron of the SJbel gene is sufficient to confer sugar responsiveness in maize endosperm cells.

Next, to delimit a region (s) necessary for the response, two deletion constructs, pKLN105 and pKLN106 were also tested in the transient expression system (Figure 9) . Like pKLNlOl, pKLN105 (deletion end point - 314) responded to a high sucrose concentration (9%) and increased LUC expression by approximately two-fold. However, pKLN106 (deletion end point -145) showed similar levels of LUC expression in both low and high sucrose conditions. These results suggest-s- that the region between -314 and -145 contains a cis-regulatory element (s) necessary for the sugar response in maize endosperm cells. In addition, because the expression level was reduced in both + and - sucrose treated cells, other regulatory elements may also reside in this region. Overexpression of mEmBP-1 protein represses the Sbel gene expression. The canonical G-box sequence, CCACGTGG, was found in the 5 '-flanking sequence of the maize Sbel (-228 to -221) as well as the rice Sbel gene (-170 to -163) . This evolutionary conservation suggests a possible role of the G-box motif in the regulation of gene expression, although our 5 ' deletion analysis did not show it as an important regulatory element. It is known that the G-box motif resides in the promoters of many plant genes responding to a variety of different environmental and physiological stimuli and is often associated with additional regions which act as coupling elements determining signal response specificity.

To test whether or not the G-box in the maize SJbel promoter is interacting with a G-box binding protein in maize, mEmBP-1, which is a homologue of the wheat EmBP-1 (Guiltinan et al . , Science, 250: 267-271, 1990) and expressed during endosperm development, EMSA and

DNase I footprint analyses were performed. As expected, the analyses clearly showed that EmBP-1 interacts with the G-box sequence in vi tro . Since EmBP-1, a member of the basic leucine zipper (bZIP) transcription factors, is implicated in ABA-induced Em gene expression in wheat. The data prompted us to ask two questions. First, is the SJbel gene expression regulated by ABA concentration? Second, can mEmBP-1 protein transactivate Sbel gene expression? Transient expression assays failed to show a relationship between exogenous ABA concentrations (1 to 100 μM) and the Sbel promoter activity in the maize endosperm suspension cells, suggesting the G-box in the Sbel promoter is not ABA-responsive .

To address the second question, a chimeric construct containing the cauliflower mosic virus (CaMV) 35S promoter fused to the full-length mEMBP-1 cDNA (35S- mEmBP-1) was created and co-introduced with the plasmid pKLNlOl (a full-length Sbel promoter-LUC) into the maize endosperm suspension cells. We predicted overexpression of mEmBP-1 protein would enhance the LUC expression driven by the Sbel promoter, since mEmBP-1 is known as a bZIP transcription activator. Contrary to the prediction, overexpression of mEmBP-1 protein actually resulted in a significant reduction (5-fold) of the SJbel promoter activity as shown in Figure 10. The effect was apparently selective for the Sbel promoter, since mEmBP-1 had little effect on expression of a LUC reporter gene linked to the ubiquitin promoter (pACHlδ) .

Interestingly, substitution of the G-box sequence (CCACGTGG) in pKLN105 with TTGAACTA did not cause a reduction in promoter activity, suggesting that the G-box sequence is not required for high level expression of the Sbel gene in maize endospem cells. Discussion

The expression pattern of the maize Sbel gene has been investigated in almost all maize tissues (Gao et al . , 1996) . The Sbel gene is constitutively expressed at a low level in vegetative tissues, while it is modulated during kernel development. Especially in the endosperm, SJbel mRNA began to accumulate to high levels at the onset of rapid starch deposition. These findings suggest that the expression of Sbel is regulated by certain factors which vary in concentration or activity during kernel development .

As a step toward understanding regulatory mechanisms controlling Sbel gene expression, we analyzed the Sbel promoter regions using a transient gene expression system. Transient expression assays showed that expression driven by the maize Sbel promoter greatly depends on the presence of the DΝA region spanning the first exon and intron of the maize Sbel . Addition of the DΝA sequence (+28 to +228) containing the first exon and intron of the Sbel gene into the transcriptional chimeric construct (pKLlOl) increased reporter gene expression in maize endosperm suspension cells up to 14-fold. Since such DΝA sequences containing transcriptional stimulating effects are useful in investigations of gene expression in plant cells and for plant genetic engineering, it will be necessary to determine whether or not the DΝA sequence has the ability to increase gene expression under the control of other promoters . There are several examples of plant genes which are regulated by DΝA sequences within the transcribed region. Among them, the first exon and intron sequences of the maize Shi gene are one of the best examples studied so far (Vasil et al . , Plant Physiol., 91: 1575-1579, 1985; Maas et al . , Plant Mol. Biol., 16: 199-207, 1991; Clancy et al . , Plant Sci., 98: 151-161, 1994). The Shi exon appears to have two separate cis-elements which act independently to increase gene expression via different mechanisms. One of the elements may contain a novel promoter element which has the ability to interact with transcription factors binding upstream. The other acts possibly at the level of translation efficiency or mRNA stability. The enhancing effect of the Shi intron is likely the result of an increase in the level of mature cytoplasmatic mRNA level like the maize Adhl first intron.

5' deletion analysis of the maize Sbel promoter revealed several cis-regulatory elements affecting the promoter activity in maize endosperm cells. Of special interest was the identification of the 60-bp positive element located in the region from -314 to -255 relative to the transcription initiation site. Further investigation of the region using linker-scan analysis identified at least two separate regions, -314 to -295 and -284 to -255, which are critical for gene expression in maize endosperm cells.

Interestingly, the -314/-295 region has striking similarity with the sucrose-responsive element (SURE-1) of the potato patatin-1 promoter (Grierson et al., Plant J. , 5: 815-826, 1994), which has been shown to interact with a sucrose-inducible nuclear protein(s). Grierson et al . demonstrated that a 100-bp patatin-1 promoter fragment encompassing SURE-1 is sufficient to confer sucrose responsiveness. DΝA sequences similar to the -314/-295 region are also found in the promoter regions of other sugar-inducible genes, such as maize sucrose synthase (Shaw et al . , Plant Physiol., 106 : 1659- 1665, 1994), Arabidopsis β-amylase (Mita et al . , Plant Physiol., 107 : 895-904, 1995), and potato sporamin (Ohta et al., Mol. Gen. Genet., 225: 360-378, 1991). This finding along with the sugar enhanced expression of the Sbel demonstrated by northern blot analysis and transient expression assay strongly suggest that the conserved sequences may be implicated in mediating sugar responsiveness of the Sbel gene. This is further supported by our recent finding showing that the -314/- 196 region of the Sbel promoter is sufficient to confer the sucrose responsiveness to -64 CaMV 35S minimal promoter. Since high sucrose concentration media were used for the transient expression assays to maximize gene expression, it is understandable that mutation of this region decreased the level of LUC expression. It remains to be tested whether or not other Sbe genes are also sugar-modulated. To date, we have not detected sugar- dependent DNA binding activity associated with the SJbel promoter.

In potato and cassava plants, sugars have been shown to regulate expression of genes involved in starch biosynthesis. Our results demonstrated that the maize Sbel is also modulated by sugar concentration. Such sugar effect was not due to change in osmotic potential, because L-glucose and PEG which are osmotically active did not affect SJbel gene expression. Recently, it has been reported that hexokinase is involved in sensing sugar concentration in higher plants, and sugar signaling mediated through hexokinase is uncoupled from sugar metabolism. Sequence comparison between the rice (Kawasaki et al . , 1993, supra) and maize SJbel genomic DNAs (Example 1) revealed that the 5 '-flanking sequences proximal to the protein-coding regions are highly divergent except for the canonical G-box sequences (CCACTGG) which are located in similar positions relative to the corresponding transcription initiation sites. This evolutionary conservation between the species led us to postulate that the G-box may be involved in regulation of the Sbel gene expression possibly in response to one of the environmental or physiological stimuli, even though we failed to show the importance of the G-box in the Sbel promoter activity using the 5' deletion analysis. It is possible that G-box dependent mechanism controlling Sbel promoter activity could not be appraised in our endosperm suspension cultured cells. This hypothesis is supported by the results showing interaction of the G-box with mEmBP-1 protein in vi tro and repression of the Sbel promoter activity by over-expression of mEmBP-1. Along with these, the finding that disruption of the G-box sequence (CCACGTGG) in pKLN105 did not cause a reduction in promoter activity led us to speculate that the G-box and its binding proteins are involved in down-regulation of the Sjbel gene expression rather than up-regulation.

Although a specific role for the G-box motif in Sbel gene expression has not been identified, there is a possibility that the G-box in the SJbel promoter may play a critical role under different environmental conditions or in different tissues.

It has been noted that mutations decreasing starch accumulation in maize endosperm also reduce storage protein synthesis, implying possible interactions between these pathways. It has been shown that mutations affecting synthetic events in one biosynthetic pathway affect expression of genes in both pathway, and demonstrated that expression of genes involved in starch and storage protein synthesis of the maize endosperm are coordinately regulated. Elevation in sugar concentration or alteration of the osmotic potential of the endosperm was proposed to be a possible candidate for the primary signal triggering this coordinate expression. In this context, knowledge of the Sbe promoter elements and their associated regulatory proteins may eventually lead to a better understanding of the regulatory mechanisms controlling all of the starch biosynthetic genes and the genes encoding storage proteins in maize endosperm.

While certain of the preferred embodiments of the present invention have been described and specifically exemplified above, it is not intended that the invention be limited to such embodiments. Various modifications may be made thereto without departing from the scope and spirit of the present invention, as set forth in the following claims.