Transgenic barley expressing a protein-engineered ... - PNAS

Proc. Natl. Acad. Sci. USA Vol. 93, pp. 3487-3491, April 1996 Applied Biological Sciences

Transgenic barley expressing a protein-engineered, thermostable (1,3-1,4)-(3-glucanase during germination (Hordeum vulgare/codon usage/malt production/animal feed)

LISBETH GATH JENSEN*, OLE OLSEN*, OLIVER KOPSt, NORBERT WOLFt, KARL KRISTIAN THOMSEN*, DITER VON WETTSTEIN*t§ *Carlsberg Laboratory, Department of Physiology, Gamle Carlsberg Vej 10, DK-2500 Copenhagen, Valby, Denmark; tWeissheimer Research Laboratory, Department of Biotechnology, Schaarstrasse 1, D-56626 Andernach, Germany; and *Departments of Crop and Soil Sciences and Genetics and Cell Biology, Washington State University, Pullmann, WA 99164

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Contributed by Diter von Wettstein, December 14, 1995

(14) between the genes from Bacillus amyloliquefaciens (15) and Bacillus macerans (16)-e.g., H(A12-M)AY13 (H, hybrid; A, amino acid from B. amyloliquefaciens; M, amino acid from B. macerans), which exhibits a half-life of >4 h at 70°C (pH 5.0) (17). Computer modeling, using the coordinates of H(A16-M) (1,3-1,4)-p3-glucanase (18) (Fig. 1 Left), suggested that hydrogen bond formation between the spatially close Nand C-terminal B-sheets forms the structural basis for the increased enzymic stability. The promoter of the barley (1,3-1,4)-f3-glucanase isoenzyme EII gene-directed transient expression of the chloramphenicol acetyltransferase gene upon transfection of barley aleurone protoplasts and showed a gibberellin A3 response (20), but this promoter did not direct detectable expression of a hybrid bacterial (1,3-1,4)-,3-glucanase gene in barley aleurone protoplasts. However, transient expression and secretion was obtained with the same 13-glucanase gene controlled by the barley low-pI a-amylase gene promoter and the low-pI a-amylase signal peptide (21). The codon usage for barley (1,3-1,4),3-glucanase isoenzyme EII exhibits strong preference for G or C in the third position (22, 23), resulting in a G+C content of 65.9% in the coding region, while such codon bias is not observed in Bacillus (1,3-1,4)-/3-glucanase genes (15, 16). Modification of a bacterial gene, crylA(b), toward plant gene codon usage increased its expression in planta (24, 25). To obtain value-added barley lines synthesizing thermostable (1,3-1,4)-p3-glucanase during germination, the codons for hybrid H(A12-M)AY13 were modified to match those of the gene encoding barley (1,3-1,4)-p1-glucanase isoenzyme EII, and the modified gene was tested for expression with the barley (1,3-1,4)-B1-glucanase isoenzyme EII gene promoter in aleurone protoplasts. The codon adapted hybrid gene, cloned behind the barley high-pI a-amylase promoter and signal peptide encoding sequences, was introduced into immature embryo cells and germinating grains from regenerated plants were analyzed for thermostable (1,3-1,4)-13-glucanase.

The codon usage of a hybrid bacterial gene ABSTRACT encoding a thermostable (1,3-1,4)-13-glucanase was modified to match that of the barley (1,3-1,4)-p-glucanase isoenzyme El gene. Both the modified and unmodified bacterial genes were fused to a DNA segment encoding the barley high-pI a-amylase signal peptide downstream of the barley (1,3-1,4),3-glucanase isoenzyme EII gene promoter. When introduced into barley aleurone protoplasts, the bacterial gene with adapted codon usage directed synthesis of heat stable (1,31,4)-p8-glucanase, whereas activity of the heterologous enzyme was not detectable when protoplasts were transfected with the unmodified gene. In a different expression plasmid, the codon modified bacterial gene was cloned downstream of the barley high-pI a-amylase gene promoter and signal peptide coding region. This expression cassette was introduced into immature barley embryos together with plasmids carrying the bar and the uidA genes. Green, fertile plants were regenerated and -75% of grains harvested from primary transformants synthesized thermostable (1,3-1,4)-p3-glucanase during germination. All three trans genes were detected in 17 progenies from a homozygous T1 plant.

vitro

The (1,3-1,4)-13-glucans from barley (Hordeum vulgare L.) are linear polysaccharides consisting of glucose units joined by (1,3)-43 and (1,4)-,B glycosidic linkages (1-3). These polymers are the major constituents of barley endosperm cell walls (4) and their degradation is a prerequisite for the enzymatic mobilization of endosperm storage components, which serve as nutrients for the growing embryo. Efficient degradation of endosperm cell walls is also important for utilization of barley as a monogastric animal feed (5, 6) and in industrial processes such as malting and brewing (7). Furthermore, extraction of non-food products deposited in the endosperm of transgenic barley would be facilitated by the action of highly efficient, heat stable cell wall-degrading enzymes. The (1,3-1,4)-3glucanases (EC 3.2.1.73) synthesized by the aleurone and scutellum tissues during barley grain germination (8) are susceptible to irreversible thermoinactivation at temperatures above 55°C (9, 10), which may result in incomplete degradation of cell wall 13-glucans and limit the utility of barley for industrial processes unless a thermostable (1,3-1,4)-43glucanase is present during extraction at elevated temperatures. Bacillus species synthesize and secrete (1,3-1,4)-,3glucanases with the same specificity as the barley enzymesi.e., hydrolysis of (1-4)-j3-glycosidic linkages joining 3-0 substituted glucose units (1, 11, 12) but the bacterial enzymes are more thermotolerant than their barley counterparts (13). Hybrid (1,3-1,4)-3-glucanases with improved thermostability at pH 5.0 have been obtained by intragenic recombination in

EXPERIMENTAL PROCEDURES

Organisms and Materials. Grains of Hordeum vulgare L., cv. Himalaya (1985 harvest at Washington State University, Pullman, WA) were used for preparation of protoplasts, and immature embryos were isolated from cv. Golden Promise. Genomic DNA from cultivars Golden Promise and Carlsberg II was purified according to the procedure by Edwards et al. (26). Escherichia coli cells of strain DH5a (27) (Life Technologies, Grand Island, NY) were used for propagation of plasmids, which were purified using the Wizard System (Promega). Nucleotide sequence analysis was on an Applied Biosystems

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Abbreviations: A, amino acid from Bacillus amyloliquefaciens; M, amino acid from Bacillus macerans; H, hybrid. §To whom reprint requests should be addressed.

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Proc. Natl. Acad. Sci. USA 93 (1996)

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FIG. 1. Hybrid (1,3-1,4)-p3-glucanases. (Left) Three-dimensional structure of hybrid enzyme H(A16-M) derived from the structure amplitudes and coordinates deposited in the Protein Data Bank (reference 1AYH) by Keitel et al. (18). The peptide backbone of residues originating from the B. amyloliquefaciens and B. macerans parental enzymes are shown in blue and red, respectively. Side chains of active site residues Glu-105, Asp-107, and Glu-109 (Glu-129, Asp-131, and Glu-133 in the preenzyme listed on the right) are illustrated with white sticks. Since the parental B. macerans enzyme adopts a similar conformation (19), only minor differences are expected for H(A12-M)AY13. (Right) Synthetic DNA sequence used to direct synthesis and secretion of H(A12-M)AY13 in barley. Amino acid sequences of the barley a-amylase signal sequence, B. amyloliquefaciens (1,3-1,4)-j3-glucanase and B. macerans (1,3-1,4)-p3-glucanase are shown on green, blue, and red backgrounds, respectively. Nucleotides that have been changed to those preferred for barley (1,3-1,4)-13-glucanase isoenzyme EII are shown in capital letters. Of 215 codons specifying the reading frame for the mature enzyme, 141 (corresponding to 66%) have been changed.

model 373A nucleotide sequencer. Oligonucleotides were synthesized on a model 380B synthesizer (Applied Biosystems). For PCRs Taq enzyme (Perkin-Elmer/Cetus) was used according to the supplier's recommendations. Genomic DNA was digested with BamHI when used as template in PCR. Other recombinant DNA techniques were as described (28). Polyclonal antibodies were raised in rabbits against purified H(A12-M)AY13 (1,3-1,4)-f3-glucanase produced in E. coli. Plasmid Constructions. The plasmid pEmuGN contains the uidA (29) gene encoding the ,3-glucuronidase and has been described (30). The plasmid pUBARN carries the bar gene (31). The plasmids pEII-aH(A12-M)AY13-N and pAMYaH(A12-M)AY13-N carry the unmodified bacterial (1,3-1,4)f3-glucanase gene fused to the high-pI a-amylase signal peptide coding sequence under control of the promoters from barley (1,3-1,4)-j3-glucanase isoenzyme EII and high-pI a-amylase genes, respectively. These and the plasmids carrying the modified gene [pEII-aH(A12-M)AY13-GC-N and pAMYaH(A12-M)AY13-GC-N] will be described elsewhere. Transfection of Aleurone Protoplasts. Aleurone protoplasts were prepared (32) and transfected with 50 ,ug of plasmid DNA by PEG-mediated DNA uptake (33). Gibberellin A3 at a final concentration of 1 ,tM was included in all experiments and incubation was for 65 or 110 h at room temperature.

Protoplasts were removed by centrifugation at 1000 x g for 5 min and the supernatant was assayed for (1,3-1,4)-,3-glucanase activity. Plant Transformation, Selection, and Regeneration. Media used for tissue culture and plants were callus induction medium (CIM); plant growth medium (PGM), which is CIM without hormones added as described (34); as well as the hormone-free FHG medium (35). Plantlet induction medium (PIM) is CIM in which the auxin 3,6-dichloro-o-anisic acid (Dicamba) is replaced by the cytokinin 6-benzylaminopurine

(1 mg/ml). The selective agent bialaphos (Meiji Seika Kaisha Ltd., Tokyo) was used in the concentrations suggested by Wan

and Lemaux (34). A 1:1:1 mixture of linearized pEmuGN, pUBARN, and pAMY-aH(A12-M)AY13-GC-N was introduced into immature embryos by particle bombardment using a DuPont 1000 He device. After passage through the selection procedure (34) embryogenic clusters from uniformly growing callus lines were transferred to PIM, and after 10-14 d in darkness pieces of callus were transferred to hormone-free FHG medium and exposed to light. When plantlets reached a size of 1-1.5 cm, they were transferred to PGM in culture cylinders for further development and finally transferred to soil and grown to maturity. For rapid propagation, immature embryos from mother plants (To) and their offspring (Ti and T2) were allowed to germinate in darkness with the scutella facing downward on FHG medium without the selective agent. Green seedlings with well developed roots were transferred to soil and grown to maturity in the greenhouse. Mature grains from putative transgenic plants were surface sterilized and left to germinate in darkness on sterile, humidified filter paper at 10°C. When coleoptiles were '10 mm, the germinating grains were transferred to room temperature (low light conditions) for further development for 1-2 d before separation of the seedlings from the grains. Residual grain material was ground in liquid nitrogen and extracted with 50 mM 2-(N-morpholino)ethanesulfonic acid/5 mM CaCl2 using 500 ,ll per grain. Extracts were kept on ice for 30 min with occasional mixing followed by centrifugation at 15,000 x g for 10 min. Supernatants were stored at -20°C. Enzyme Assays. (1,3-1,4)-/-Glucanase activity was determined by the method of McCleary (36) using 200 ,ul of azobarley f3-glucan substrate. Analysis of protoplast supernatants was in 50 mM sodium acetate, pH 6.0/5 mM CaCl2 and

Applied Biological Sciences: Jensen et al. incubation was at 56°C. Determination of bacterial (1,3-1,4)P3-glucanase activity in extracts from germinating grains was in 50 mM BisTris (pH 7.4) at 65°C, and homologous barley (1,3-1,4)-43-glucanase activity was determined in 50 mM sodium acetate (pH 4.5) at 30°C. Samples to be analyzed were incubated for 10 min in 200 ,il of buffer before addition of substrate and then further incubated for 30 min. Reactions were stopped by addition of 1 ml of precipitation solution (36). Samples were centrifuged at 5000 x g for 3 min and the absorbance at 590 nm was measured. In this report, 1 unit of (1,3-1,4)-/3-glucanase is defined as the amount of enzyme that in the described assay results in an A590 of 1.0. One unit of H(A12-M)AY13 corresponds to 8 ng of protein. Analysis of Proteins from Transfected Protoplasts and Germinating Grains. Proteins were separated by SDS/PAGE, transferred to nitrocellulose membranes, and probed with antibodies (37-39). Grain extracts were analyzed by isoelectric focusing using IsoGel agarose plates (FMC) with a separation range from pH 3 to 10. Focused proteins were transferred to nitrocellulose filters and probed with the antibodies. Alternatively the focusing gel was overlaid with a 1% agarose gel containing 0.5% lichenin and incubated at 55°C for 1 h. Undigested lichenin was stained with Congo Red (40) and clear zones of ,3-glucanase activity were revealed.

RESULTS AND DISCUSSION Effect of Codon Modification. Three different protoplast preparations were transfected with the (1,3-1,4)-j3-glucanase encoding plasmids pEII-aH(A12-M)AY13-N and pEIIaH(A12-M)AY13-GC-N. In experiment B (Fig. 2), seven wells containing protoplasts transfected with the codon adapted, G+C-rich construct gave an average production of 40 ng (1,3-1,4)-p3-glucanase per 2 x 105 protoplasts (s = ± 18 ng) after incubation for 110 h, while no (1,3-1,4)-13-glucanase activity was detectable in any of the six wells containing protoplasts transfected with pEII-aH(A12-M)AY13-N. Two different protoplast preparations were incubated for 65 h upon transfection with (1,3-1,4)-j3-glucanase encoding plasmids. In 12 wells, protoplasts were transfected with the high G+C construct, giving an average production of 30 ng per 2 x 105 protoplasts (s = ± 12.8 ng), whereas no (1,3-1,4)-p3-glucanase activity was detected when the nonmodified gene was used for transfection (Fig. 2B). Furthermore, in all experiments per-

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formed, essentially all (1,3-1,4)-j3-glucanase activity was in the supernatant and only negligible amounts were associated with the pelleted protoplast fraction. Proteins from aleurone protoplasts, transfected with pEIIaH(A12-M)AY13-GC-N were separated by SDS/PAGE, transferred to nitrocellulose membranes, and probed with antibodies. Fig. 2A shows the reaction with proteins from transfected protoplasts (lanes 4 and 5). Three bands of different Mr react with the antibodies, indicating that the (1,3-1,4),3-glucanase has been exported via the secretory system resulting in an array of glycoforms as encountered in yeast expression studies (41, 42) (lane 2). The protein band with the lowest Mr (24,000) comigrates with (1,3-1,4)-f3-glucanase from E. coli (lane 1). Plant Transformation and Regeneration. The plasmid pAMY-aH(A12-M)AY13-GC-N encoding heat stable (1,31,4)-,3-glucanase was introduced into immature barley embryos together with plasmids carrying the bar and uidA genes. In 10 experiments, 293 bisected and 45 whole embryos were bombarded. Twenty-two lines survived the selection procedure and the callus obtained grew well on bialaphoscontaining medium. Long-term resistance was never observed with wild-type tissue. Two of the transformation experiments gave a total of 14 green plants. Four were from experiment 6 (37 half embryos) and 10 were from experiment 8 (17 whole embryos). These plants were morphologically indistinguishable from wild-type Golden Promise plants grown under identical conditions in the green house (Fig. 3). Analysis of Primary Transformants and Their Offspring. Analysis of the 14 primary transformants by PCR showed that all plants carried all three genes. An example is shown in Fig. 4A. Eight T, plants were obtained by germination of immature embryos from transgenic mother plant 6.2.2. Six of the offspring had inherited all three heterologous genes, while the other two did not carry any of them (examples shown in Fig. 4B), indicating Mendelian segregation and linkage of the introduced genes. Immature embryos from one spike of each of four TI plants were germinated to produce T2 plants. From TI plant 6.2.2.2, 17 offspring plants were obtained and ana-

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plasts. (A) Proteins secreted by two independent sets of transfected aleurone protoplasts were subjected to SDS/PAGE and Western blot analysis (lanes 4 and 5). H(A12-M)AY13 (1,3-1,4)-p3-glucanase from E. coli (lane 1), an N-glycosylated preparation (lane 2), and a deglycosylated preparation from yeast (lane 3) were used as standards. (B) Quantitation of heat stable (1,3-1,4)-,3-glucanase synthesized by transfected aleurone protoplasts. 1, Protoplasts transfected with pEIIaH(A12-M)AY13-GC-N incubated for 110 h; 2, as in 1, but incubation was for 65 h; 3, protoplasts transfected with pEII-aH(A12-M)AY13-N.

FIG. 3. Spikes of regenerated plants. A spike from transformant 8.2.1 (red tag) is compared with a spike from a regenerated wild-type plant (white tag). Spikes are fully fertile and grains are morphologically

indistinguishable.

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Applied Biological Sciences: Jensen et al. 29

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FIG. 4. PCR analysis of transgenic barley plants. Genomic DNA of To plant 6.2.2 (A), four T1 plants (B), and two T2 plants (C) were used as templates for amplification of homologous barley sequences [a 509-bp fragment spanning bases 3036-3545 of the barley (1,3-1,4)-3glucanase isoenzyme EII gene (20) (A, lane 1) and bases -679 to + 190 of the gene encoding high-pI a-amylase (43) (B, lanes 7-10). DNA sequence analysis has shown that the minor band is due to a deletion

corresponding to -610 to -531]. A heterologous 845-bp fragment spanning the entire bar and nos sequences (31) was amplified from To (A, lane 4), while a 429-bp fragment, covering the coding region was obtained from T2 plants (C, lanes 9 and 10). A 539-bp fragment of the uidA gene (29) was identified in To (A, lane 5) and in T2 (C, lanes 6 and 7). The gene encoding H(A12-M)AY13 was detected with primers spanning the signal peptide code to the stop codon in To (A, lane 3) and with primers spanning the signal peptide code to the terminator region in T1 (B, lanes 3-6) and T2 (C, lanes 3 and 4) giving rise to fragments of 705 and 972 bp, respectively. The H(A12-M)AY13 gene could not be detected in a sister plant segregating as wild type (B, lane 1). Lane 2 in B and lanes 2, 5, and 8 in C are plasmid control reactions for H(A12-M)AY13 uidA and bar, respectively.

lyzed by PCR, which demonstrated the presence of all three heterologous genes in all 17 T2 plants (examples shown in Fig. 4C), indicating that T, plant 6.2.2.2 was homozygous. Aqueous extracts of germinating grains from primary transformants 6.2.1, 6.2.2, and 8.2.1 were assayed for heat stable (1,3-1,4)f3-glucanase activity. Plants originating from grains producing heat stable (1,3-1,4)-j3-glucanase were positive for the pAMYaH(A12-M)AY13-GC-N gene. The numbers obtained are in agreement with a Mendelian 3:1 segregation for all three mother plants (Table 1). Protein extracted from leaf, root, and stem tissues of transgenic plants showed no heat stable (1,31,4)-f3-glucanase activity. Plants obtained from grains without heat stable (1,3-1,4)-,3-glucanase were subsequently analyzed by PCR and the absence of the corresponding genes was confirmed in all cases. PCR analysis of T2 seedlings generated by germination of immature embryos from a single spike of the homozygous T, plant 6.2.2.2 confirmed the presence of the gene encoding heat stable (1,3-1,4)-j3-glucanase. Aqueous extracts were prepared from 10 germinating grains of this plant and all 10 extracts contained heat stable (1,3-1,4)-,3-glucanase, demonstrating activity of the introduced gene in the second generation after transformation. Extracts of randomly chosen transformed and untransformed grains were analyzed for endogenous barley (1,3-1,4)-/3-glucanase. This enzyme was Table 1. Segregation analysis No. tested To 30 6.2.1 74 6.2.2 25 8.2.1

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7

present in all grain extracts, irrespective of expression of the heterologous (1,3-1,4)-3-glucanase gene. Characterization of Heterologous (1,3-1,4)-f3-Glucanase. Aliquots of single grain extracts were analysed by isoelectric focusing. Fig. 5A shows that heat stable (1,3-1,4)-p3-glucanase from a germinating grain of To plant 6.2.1 (lane 2) has the same pI value as a control sample of H(A12-M)AY13 (1,3-1,4)-/3glucanase produced in E. coli (lane 1). Extract from a wild-type grain gives no activity zone. Fig. SB shows the reaction of focused proteins with antibodies. Soluble proteins from germinating grains of transgenic barley were separated by SDS/ PAGE, transferred to nitrocellulose filters, and probed with antibodies. Fig. 5C shows four samples reacting with different intensities. This difference is in agreement with the activities measured using azobarley glucan as substrate. The mobility of the transgenic (1,3-1,4)-,3-glucanase is slightly decreased in comparison with the enzyme from E. coli. This is most likely due to N-glycosylation, since there are three sites for Nglycosylation in H(A12-M)AY13. The enzyme from germinating grains appeared as a single band, whereas three major forms are released from aleurone protoplasts. When H(A16-M) is secreted from Saccharomyces cerevisiae two major molecular weight classes appear, each representing an array of different glycoforms (Fig. 2A) (41, 42). Analysis of barley (1,3-1,4)-,3-glucanase isoenzyme EII showed that a seemingly uniform preparation contained four major glycoforms (44). It is therefore to be-expected that the bacterial enzyme with three sites for N-glycosylation will appear in different glycoforms when synthesized in barley. Conclusion. In this plant breeding project, we endeavoured to produce barley plants that during steeping and germination express a (1,3-1,4)-f3-glucanase that survives the high temperatures used for kiln drying of green malt. This would allow the enzyme to act in the mash tun as do the thermostable a-amylases from barley, wheat, and rice. Such a heat stable

(1,3-1,4)-f3-glucanase synthesized during germination might

eliminate the requirement of complete endosperm wall depolymerization in the malting schedule and thereby provide new opportunities for the application of malting and mashing in production of conventional and novel biotechnological commodities. The design of the enzyme with dramatically improved thermostability rested on the discovery that hybrid (1,3-1,4)-,3-glucanases obtained by intragenic recombination can exhibit molecular heterosis or hybrid vigor (45). The heat stability of the bacterial enzyme was further optimized with reference to the three-dimensional structure of H(A16-M) (18). The improved enzyme has been produced in transgenic E. coli and successfully tested in pilot mashing (14) and feed pellet production (K.K.T., unpublished data). In the present 2

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4 5 6 3 FIG. 5. Analysis of heat stable (1,3-1,4)-p-glucanase from transgenic barley. (A) Zones of activity after isoelectric focusing and staining with Congo Red. Lanes: 1, (1,3-1,4)-p3-glucanase from E. coli; 2 and 3, extracts from germinating sister grains. (B) Same three samples were blotted onto nitrocellulose and probed with antibodies. (C) SDS/PAGE and Western blot analysis of extracts from germinating grains. Lanes: 1, (1,3-1,4)-/3-glucanase from E. coli; 2-5, extracts with different amounts of heat stable (1,3-1,4)-/3-glucanase; 6, extract from a germinating wild-type grain.

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Applied Biological Sciences: Jensen et al. communication, we report the synthesis of the engineered thermotolerant (1,3-1,4)-p3-glucanase during germination and the fidelity of its inheritance in fully fertile transgenic barley. Dr. S0ren Knudsen is thanked for valuable advice regarding plant transformation and regeneration. This work was supported by a grant to D.v.W. from the Danish Programme for Food Technology, Projekt 6028 and by a Ph.D. fellowship to L.G.J. from the Danish Agricultural Science Research Council.

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