Discovery of a protein required for photosynthetic membrane assembly.
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Bacteria↗
Biomedical subjects
D von Wettstein
Publications and source records attributed to D von Wettstein.
Improved barley broiler feed with transgenic malt containing heat-stable (1,3-1,4)-beta-glucanase.
The low nutritional value of barley for poultry is because of the absence of an intestinal enzyme for efficient depolymerization of (1, 3-1,4)-beta-glucan, the major polysaccharide of the endosperm cell walls. This leads to high viscosity in the intestine, limited nutrient uptake, decreased growth rate, and unhygienic sticky droppings adhering to chickens and floors of the production cages. Consequently, the 7.5 billion broiler chickens produced annually in the United States are primarily raised on corn-soybean diets. Here we show that addition to normal barley of 6.2% transgenic malt containing a thermotolerant (1,3-1,4)-beta-glucanase (4.28 microg.g(-1) soluble protein) provides a weight gain equivalent to corn diets. The number of birds with adhering sticky droppings is drastically reduced. Intestines and excrements of chickens fed the barley control diet contained large amounts of soluble (1,3-1,4)-beta-glucan, which was reduced by 75 and 50%, respectively, by adding transgenic malt to the diet. The amount of active recombinant enzyme in the small intestine corresponded to that present in the feed, whereas an 11-fold concentration of the enzyme was observed in the ceca, and a 7.5-fold concentration occurred in the excrement. Glycosylation of the beta-glucanase isolated from the ceca testified to its origin from the transgenic barley. Analysis of the data from this trial demonstrates the possibility of introducing individual recombinant enzymes into various parts of the gastrointestinal tract of chickens with transgenic malt and thereby the possibility of evaluating their effect on the metabolism of a given ingredient targeted by the enzyme.
Animal Feed↗
The production of recombinant proteins in transgenic barley grains.
The grain of the self-pollinating diploid barley species offers two modes of producing recombinant enzymes or other proteins. One uses the promoters of genes with aleurone-specific expression during germination and the signal peptide code for export of the protein into the endosperm. The other uses promoters of the structural genes for storage proteins deposited in the developing endosperm. Production of a protein-engineered thermotolerant (1, 3-1, 4)-beta-glucanase with the D hordein gene (Hor3-1) promoter during endosperm development was analyzed in transgenic plants with four different constructs. High expression of the enzyme and its activity in the endosperm of the mature grain required codon optimization to a C+G content of 63% and synthesis as a precursor with a signal peptide for transport through the endoplasmic reticulum and targeting into the storage vacuoles. Synthesis of the recombinant enzyme in the aleurone of germinating transgenic grain with an alpha-amylase promoter and the code for the export signal peptide yielded approximately 1 microgram small middle dotmg(-1) soluble protein, whereas 54 microgram small middle dotmg(-1) soluble protein was produced on average in the maturing grain of 10 transgenic lines with the vector containing the gene for the (1, 3-1, 4)-beta-glucanase under the control of the Hor3-1 promoter.
Blotting, Southern↗
Molecular basis for semidominance of missense mutations in the XANTHA-H (42-kDa) subunit of magnesium chelatase.
During biosynthesis of bacteriochlorophyll or chlorophyll, three protein subunits of 140, 70, and 42 kDa interact to insert Mg2+ into protoporphyrin IX. The semidominant Chlorina-125, -157, and -161 mutants in barley are deficient in this step and accumulate protoporphyrin IX after feeding on 5-aminolevulinate. Chlorina-125, -157, and -161 are allelic to the recessive xantha-h mutants and contain G559A, G806A, and C271T mutations, respectively. These mutations cause single amino acid substitutions in residues that are conserved in all known primary structures of the 42-kDa subunit. In vitro complementation and reconstitution of Mg-chelatase activity show that the 42-kDa subunits are defective in the semidominant Chlorina mutants. A mutated protein is maintained in the Chlorina plastids, unlike in the xantha-h plastids. Heterozygous Chlorina seedlings have 25-50% of the Mg-chelatase activity of wild-type seedlings. Codominant expression of active and inactive 42-kDa subunits in heterozygous Chlorina seedlings is likely to produce two types of heterodimers between the strongly interacting 42-kDa and 70-kDa subunits. Reduced Mg-chelatase activity is explained by the capacity of heterodimers consisting of mutated 42-kDa and wild-type 70-kDa protein to bind to the 140-kDa subunit. The 42-kDa subunit is similar to chaperones that refold denatured polypeptides with respect to its ATP-to-ADP exchange activity and its ability to generate ATPase activity with the 70-kDa subunit. We hypothesize that the association of the 42-kDa subunit with the 70-kDa subunit allows them to form a specific complex with the 140-kDa subunit and that this complex inserts Mg2+ into protoporphyrin IX.
Amino Acid Sequence↗
Barley glutamyl tRNAGlu reductase: mutations affecting haem inhibition and enzyme activity.
Glutamyl tRNA(Glu) reductase converts glutamate molecules that are ligated at their alpha-carboxyl groups to tRNA(Glu) into glutamate 1-semialdehyde, an intermediate in the synthesis of 5-aminolevulinate, chlorophyll and haem. The mature plant enzymes contain a highly conserved extension of 31-34 amino acids at the N-terminus not present in bacterial enzymes. It is shown that barley glutamyl tRNAGlu reductases with a deletion of the 30 N-terminal amino acids have the same high specific activity as the untruncated enzymes, but are highly resistant to feed-back inhibition by haem. This peptide domain thus interacts directly or indirectly with haem and the toxicity of the 30 amino acid peptide for Escherichia coli experienced in mutant rescue and overexpression experiments can be explained by extensive haem removal from the metabolic pools that cannot be tolerated by the cell. Induced missense mutations identify nine amino acids in the 451 residue long C-terminal part of the barley glutamyl tRNA(Glu) reductase which upon substitution curtail drastically, but do not eliminate entirely the catalytic activity of the enzyme. These amino acids are thus important for the catalytic reaction or tRNA binding.
Aldehyde Oxidoreductases↗
Magnesium chelatase: association with ribosomes and mutant complementation studies identify barley subunit Xantha-G as a functional counterpart of Rhodobacter subunit BchD.
Magnesium chelatase catalyses the insertion of Mg2+ into protoporphyrin and is found exclusively in organisms which synthesise chlorophyll or bacteriochlorophyll. Soluble protein preparations containing >10 mg protein/ml, obtained by gentle lysis of barley plastids and Rhodobacter sphaeroplasts, inserted Mg2+ into deuteroporphyrin IX in the presence of ATP at rates of 40 and 8 pmoles/mg protein per min, respectively. With barley extracts optimal activity was observed with 40 mM Mg2+. The activity was inhibited by micromolar concentrations of chloramphenicol. Mutations in each of three genetic loci, Xantha-f, -g and -h, in barley destroyed the activity. However, Mg-chelatase activity was reconstituted in vitro by combining pairwise the plastid stroma protein preparations from non-leaky xantha-f -g and -h mutants. This establishes that, as in Rhodobacter, three proteins are required for the insertion of magnesium into protoporphyrin IX in barley. These three proteins, Xantha-F, -G and -H, are referred to as Mg-chelatase subunits and they appear to exist separate from each other in vivo. Active preparations from barley and Rhodobacter yielded pellet and supernatant fractions upon centrifugation for 90 min at 272,000 x g. The pellet and the supernatant were inactive when assayed separately, but when they were combined activity was restored. Differential distribution of the Mg-chelatase subunits in the fractions was established by in vitro complementation assays using stroma protein from the xantha-f, -g, and -h mutants. Xantha-G protein was confined to the pellet fraction, while Xantha-H was confined to the supernatant. Reconstitution assays using purified recombinant BchH, BchI and partially purified BchD revealed that the pellet fraction from Rhodobacter contained the BchD subunit. The pellet fractions from both barley and Rhodobacter contained ribosomes and had an A260:A280 ratio of 1.8. On sucrose density gradients both Xantha-G and BchD subunits migrated with the plastid and bacterial ribosomal RNA, respectively.
Chlorophyll↗
Expression of catalytically active barley glutamyl tRNAGlu reductase in Escherichia coli as a fusion protein with glutathione S-transferase.
delta-Aminolevulinate in plants, algae, cyanobacteria, and several other bacteria such as Escherichia coli and Bacillus subtilis is synthesized from glutamate by means of a tRNA(Glu) mediated pathway. The enzyme glutamyl tRNA(Glu) reductase catalyzes the second step in this pathway, the reduction of tRNA bound glutamate to give glutamate 1-semialdehyde. The hemA gene from barley encoding the glutamyl tRNA(Glu) reductase was expressed in E. coli cells joined at its amino terminal end to Schistosoma japonicum glutathione S-transferase (GST). GST-glutamyl tRNA(Glu) reductase fusion protein and the reductase released from it by thrombin digestion catalyzed the reduction of glutamyl tRNA(Glu) to glutamate 1-semialdehyde. The specific activity of the fusion protein was 120 pmol.micrograms-1.min-1. The fusion protein used tRNA(Glu) from barley chloroplasts preferentially to E. coli tRNA(Glu) and its activity was inhibited by hemin. It migrated as an 82-kDa polypeptide with SDS/PAGE and eluted with an apparent molecular mass of 450 kDa from Superose 12. After removal of the GST by thrombin, the protein migrated as an approximately equal to 60-kDa polypeptide with SDS/PAGE, whereas gel filtration on Superose 12 yielded an apparent molecule mass of 250 kDa. Isolated fusion protein contained heme, which could be reduced by NADPH and oxidized by air.
Aldehyde Oxidoreductases↗
Transgenic barley expressing a protein-engineered, thermostable (1,3-1,4)-beta-glucanase during germination.
The codon usage of a hybrid bacterial gene encoding a thermostable (1,3-1,4)-beta-glucanase was modified to match that of the barley (1,3-1,4)-beta-glucanase isoenzyme EII gene. Both the modified and unmodified bacterial genes were fused to a DNA segment encoding the barley high-pI alpha-amylase signal peptide downstream of the barley (1,3-1,4)-beta-glucanase isoenzyme EII gene promoter. When introduced into barley aleurone protoplasts, the bacterial gene with adapted codon usage directed synthesis of heat stable (1,3-1,4)-beta-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 alpha-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 approximately 75% of grains harvested from primary transformants synthesized thermostable (1,3-1,4)-beta-glucanase during germination. All three trans genes were detected in 17 progenies from a homozygous T1 plant.
Amino Acid Sequence↗
Structural genes for Mg-chelatase subunits in barley: Xantha-f, -g and -h.
Barley mutants in the loci Xantha-f, Xantha-g and Xantha-h, when fed with 5-aminolevulinate in the dark, accumulate protoporphyrin IX. Mutant alleles at these loci that are completely blocked in protochlorophyllide synthesis are also blocked in development of prolamellar bodies in etioplasts. In contrast to wild type, the xan-f, -g and -h mutants had no detectable Mg-chelatase activity, whereas they all had methyltransferase activity for synthesis of Mg-protoporphyrin monomethyl ester. Antibodies recognising the CH42 protein of Arabidopsis thaliana and the OLIVE (OLI) protein of Antirrhinum majus immunoreacted in wild-type barley with 42 and 150 kDa proteins, respectively. The xan-h mutants lacked the protein reacting with antibodies raised against the CH42 protein. Two xan-f mutants lacked the 150 kDa protein recognised by the anti-OLI antibody. Barley genes homologous to the A. majus olive and the A. thaliana Ch-42 genes were cloned using PCR and screening of cDNA and genomic libraries. Probes for these genes were applied to Northern blots of RNA from the xantha mutants and confirmed the results of the Western analysis. The mutants xan-f27, -f40, -h56 and -h57 are defective in transcript accumulation while -h38 is defective in translation. Southern blot analysis established that h38 has a deletion of part of the gene. Mutants xan-f10 and -f41 produce both transcript and protein and it is suggested that these mutations are in the catalytic sites of the protein. It is concluded that X an-f -h genes encode two subunits of the barley Mg-chelatase and that X an-g is likely to encode a third subunit. The XAN-F protein displays 82% amino acid sequence identity to the OLI protein of Antirrhinum, 66% to the Synechocystis homologue and 34% identity to the Rhodobacter BchH subunit of Mg-chelatase. The XAN-H protein has 85% amino acid sequence identity to the Arabidopsis CH42 protein, 69% identity to the Euglena CCS protein, 70% identity to the Cryptomonas BchA and Olisthodiscus CssA proteins, as well as 49% identity to the Rhodobacter BchI subunit of Mg-chelatase. Identification of the barley X an-f and X an-h encoded proteins as subunits required for Mg-chelatase activity supports the notion that the Antirrhinum OLI protein and the Arabidopsis Ch42 protein are subunits of Mg-chelatase in these plants. The expression of both thet X an-f and -h genes in wild-type barley is light induced in leaves of greening seedlings, and in green tissue the genes are under the control of a circadian clock.
Amino Acid Sequence↗
Three separate proteins constitute the magnesium chelatase of Rhodobacter sphaeroides.
The insertion of magnesium into protoporphyrin IX is the first step unique to chlorophyll production and is catalyzed by magnesium chelatase. The Rhodobacter sphaeroides genes, bchI and bchD together, and bchH alone, were cloned and expressed with the pET3a vector in Escherichia coli strain BL21 (DE3). The 40-kDa BchI protein was synthesized in greater abundance compared to the 70-kDa BchD protein when both were expressed together from the same plasmid. The production of large amounts of the 140-kDa BchH protein in E. coli was accompanied by an accumulation of protoporphyrin IX. The accumulated protoporphyrin IX was bound specifically to BchH in an approximate molar ratio of 1:1. All three recombinant proteins were soluble; BchH was monomeric, Bchl was dimeric, while BchD appeared to be polymeric with a molecular mass of approximately 550 kDa. The BchH and BchI proteins were purified to apparent homogeneity while BchD was separated from BchI and partially purified. Magnesium was inserted into protoporphyrin IX and deuteroporphyrin by combining these three proteins in the presence of ATP. One monomer of BchH to one dimer of BchI gave the optimal magnesium chelatase activity and the activity was dependent on the amount of partially purified BchD added to the assay at the optimum BchH:BchI ratio. The reaction was dissected into two parts with an activation step requiring BchI, BchD, and Mg2+-ATP, and a metal-insertion step which in addition requires Mg2+, protoporphyrin IX, and BchH. The stoichiometric binding of protoporphyrin IX to BchH in vitro is direct evidence for BchH carrying out such a role in vivo whereas the other two proteins are involved in ATP activation and magnesium insertion.
Amino Acid Sequence↗
Transplanting two unique beta-glucanase catalytic activities into one multienzyme, which forms glucose.
Endo cellulases of plant pathogenic erwinias degrade cellulose as well as the cellulosic domains of barley (1-3,1-4)-beta-glucan. Depolymerization of the latter substrate is mainly caused by (1-3,1-4)-beta-glucanases, which hydrolyze (1-4)-beta glycosidic linkages adjacent to (1-3)-beta linkages. To construct an enzyme for efficient degradation of barley (1-3,1-4)-beta-glucan, the sequence encoding the catalytic domain and interdomain linker of the cellulase from Erwinia carotovora subspecies atroseptica was fused to that for the heat stable Bacillus hybrid, H(A12-M) delta Y13 (1-3,1-4)-beta glucanase. The chimeric enzyme secreted from Escherichia coli cells did not remain covalently assembled as judged by SDS-PAGE. However, the glycosylated and intact enzyme (denoted CELGLU) is secreted from the yeast Pichia pastoris. CELGLU exhibits both cellulase and (1-3,1-4)-beta-glucanase catalytic activities, and was accordingly classified a true multienzyme. HPLC and NMR analyses revealed that among the products from CELGLU, di- and trimeric oligosaccharides were identical to those produced by the parental cellulase. Tetrameric oligosaccharides, derived from the (1-3,1-4)-beta-glucanase activity of CELGLU, were further degraded by the cellulase moiety to yield glucose and trimers. Compared with the parental enzymes, CELGLU exhibits substantially higher Vmax for degradation of both soluble cellulose and barley (1-3,1-4)-beta-glucan. These findings point to construction of multienzymes as an effective approach for engineering enzymes with novel characteristics.
Artificial Gene Fusion↗
Expression of an Erwinia pectate lyase in three species of Aspergillus.
Transgenic filamentous fungi of the species Aspergillus niger, A. nidulans and A. awamori expressing and secreting Erwinia carotovora subsp. atroseptica pectate lyase 3 (PL3) were generated. Correct processing of the pre-enzyme was achieved using the A. niger pectin lyase A (PEL A) signal peptide. With the prepro-peptide of A. niger polygalacturonase II, secreted enzymes still possessed the 6- aa pro-sequence, indicating the importance of the conformation of the precursor protein for correct cleavage of the signal sequence. PL3 expression was markedly increased in media optimized for limited protease activity, and reached 0.4, 0.8 and 2.0 mg/l for expression in A. niger, A. awamori and A. nidulans, respectively. Glycans attached to the PL3 enzymes exhibited species-specific differences, and an increase of molecular mass coincided with reduced specific activities of the enzymes.
Amino Acid Sequence↗
Characterization of the different spectral forms of glutamate 1-semialdehyde aminotransferase by mass spectrometry.
Glutamate 1-semialdehyde aminotransferase produces delta-aminolevulinate for the synthesis of chlorophyll, heme, and other tetrapyrrole pigments. The native enzyme from Synechococcus is pale yellow and has absorption maxima at 338 and 418 nm from vitamin B6. Yellow, colorless, and pink forms of the protein are obtained by treatment with 4,5-dioxovalerate, 4,5-diaminovalerate, and acetylenic GABA, respectively. Compared to the native enzyme, the 418 nm absorption maximum in the yellow enzyme is enhanced and the 338 nm maximum reduced while the colorless enzyme has a heightened maximum at 338 nm and a barely detectable peak at 418 nm. The pink enzyme has an absorption maximum at 560 nm. When the native and colorless enzymes are repeatedly diluted in 0.5 M Na2HPO4, pH 7.0, and reconcentrated, pyridoxamine 5'-phosphate is released and the 338 nm maximum lost. Thus the 338 nm absorption maximum is associated with noncovalently bound pyridoxamine 5'-phosphate. NaBH4 reduction proved that the absorbance at 418 nm is from pyridoxal 5'-phosphate cofactor bound by a Schiff base to the protein. When the native, colorless, and yellow enzymes were subjected to electrospray ionization mass spectrometry, the B6 cofactor dissociated from the protein and gave a molecular weight of 46,401-46,418. Acetylenic GABA and NaBH4 were used for protein modification, and they reacted with the native and yellow enzymes but had no effect on the colorless enzyme. Pyridoxal 5'-phosphate bound covalently to the protein after NaBH4 reduction. Acetylenic GABA attached covalently to the enzyme produced an additional mass peak, 123-126 mass units higher, in the electrospray ionization spectrum.(ABSTRACT TRUNCATED AT 250 WORDS)
Amino Acid Sequence↗
Magnesium-protoporphyrin chelatase of Rhodobacter sphaeroides: reconstitution of activity by combining the products of the bchH, -I, and -D genes expressed in Escherichia coli.
Magnesium-protoporphyrin chelatase lies at the branch point of the heme and (bacterio)chlorophyll biosynthetic pathways. In this work, the photosynthetic bacterium Rhodobacter sphaeroides has been used as a model system for the study of this reaction. The bchH and the bchI and -D genes from R. sphaeroides were expressed in Escherichia coli. When cell-free extracts from strains expressing BchH, BchI, and BchD were combined, the mixture was able to catalyze the insertion of Mg into protoporphyrin IX in an ATP-dependent manner. This was possible only when all three genes were expressed. The bchH, -I, and -D gene products are therefore assigned to the Mg chelatase step in bacteriochlorophyll biosynthesis. The mechanism of the Mg chelation reaction and the implications for chlorophyll biosynthesis in plants are discussed.
Adenosine Triphosphate↗
The expression of serine carboxypeptidases during maturation and germination of the barley grain.
cDNA clones encoding three additional serine carboxypeptidases (Ser-CPs) have been isolated from a gibberellic acid-induced barley aleurone cDNA library. The three deduced Ser-CPs belong to the two-chain subfamily of Ser-CPs; they are synthesized as precursors with a putative signal peptide, propeptide, and linker peptide between the A and B chains. Their identification provides the proof for the existence of more than three Ser-CPs in cereal grains, and, based on their sequences, they may exhibit new substrate specificities. The expression of these and of the three previously isolated Ser-CPs from barley grains (CP-MI, CP-MII, and CP-MIII) has been investigated by Northern and Western analysis and RNA PCR. CP-MII is the only Ser-CP to be expressed and accumulate in the developing grain and is stored in its active form in the mature grain. All six Ser-CPs are expressed de novo in the germinating grain, in the scutellum, and/or in the aleurone. Furthermore, at least CP-MI, CP-MII, and CP-MIII are secreted into the endosperm. In addition, all Ser-CPs (except CP-MI) are also expressed in the roots and shoots of the growing seedling. This enzyme family thus appears to be ubiquitous in the barley plant, which suggests that Ser-CPs play additional roles besides their participation in the mobilization of storage proteins.
Amino Acid Sequence↗
A visible marker for antisense mRNA expression in plants: inhibition of chlorophyll synthesis with a glutamate-1-semialdehyde aminotransferase antisense gene.
Glutamate 1-semialdehyde aminotransferase [(S)-4-amino-5-oxopentanoate 4,5-aminomutase, EC 5.4.3.8] catalyzes the last step in the conversion of glutamate to delta-aminolevulinate of which eight molecules are needed to synthesize a chlorophyll molecule. Two full-length cDNA clones that probably represent the homeologous Gsa genes of the two tobacco (Nicotiana tabacum) genomes have been isolated. The deduced amino acid sequences of the 468-residue-long precursor polypeptides differ by 10 amino acids. The cDNA sequence of isoenzyme 2 was inserted in reverse orientation under the control of a cauliflower mosaic virus 35S promoter derivative in an expression vector and was introduced by Agrobacterium-mediated transformation into tobacco plants. Antisense gene expression decreased the steady-state mRNA level of glutamate 1-semialdehyde aminotransferase, the translation of the enzyme, and chlorophyll synthesis. Remarkably, partial or complete suppression of the aminotransferase mimics in tobacco a wide variety of chlorophyll variegation patterns caused by nuclear or organelle gene mutations in different higher plants. The antisense gene is inherited as a dominant marker.
Amino Acid Sequence↗
Enzymic and mechanistic studies on the conversion of glutamate to 5-aminolaevulinate.
Higher plants, algae, cyanobacteria and several other photosynthetic and non-photosynthetic bacteria synthesize 5-aminolaevulinate by a tRNA(Glu)-mediated pathway. Glutamate is activated at the alpha-carboxyl by ligation to tRNA(Glu) with an aminoacyl-tRNA synthetase. An NADPH-dependent reductase converts glutamyl-tRNA(Glu) to glutamate 1-semialdehyde, which is finally converted to 5-aminolaevulinate by an aminotransferase. These components are soluble and in plants and algae are located in the chloroplast stroma. In plants and algae the tRNA(Glu) is encoded in chloroplast DNA whereas the enzymes are encoded in nuclear DNA. The tRNA(Glu) has a hypermodified 5-methylaminomethyl-2-thiouridine-pseudouridine-C anticodon and probably plays a role in the light-dark regulation of 5-aminolaevulinate synthesis. Ligation of glutamate to tRNA(Glu) requires ATP and Mg2+ and proceeds via a ternary intermediate. Glutamyl-tRNA(Glu) reduction appears to involve formation of a complex. Glutamate 1-semialdehyde non-enzymically synthesized by reductive ozonolysis from gamma-vinyl GABA is used as substrate by the last enzyme. Glutamate-1-semialdehyde aminotransferase contains pyridoxal phosphate as a prosthetic group. The enzyme is converted to spectrally different forms by treatment with 4,5-diaminovalerate or 4,5-dioxovalerate. The pyridoxamine 5'-phosphate form of the enzyme converts (S)-glutamate 1-semialdehyde to 5-aminolaevulinate via 4,5-diaminovalerate through a bi-bi ping-pong mechanism.
Aminolevulinic Acid↗
Sodium azide mutagenesis: preferential generation of A.T-->G.C transitions in the barley Ant18 gene.
The molecular basis for the absence of anthocyanins and proanthocyanidins in four independent sodium azide-induced ant18 mutants of barley was examined by sequencing the gene encoding dihydroflavonol 4-reductase in these mutants. Sodium azide generated 21 base substitutions, which corresponds to 0.17% of the 12,704 nucleotides sequenced. Of the substitutions, 86% were nucleotide transitions, and 14% were transversions. A.T-->G.C base pair transitions were about 3 times more frequent than G.C-->A.T transitions. No deletions or mutation hot spots were found. The absence of dihydroflavonol 4-reductase activity in ant18-159, ant18-162, and ant18-164 plants is caused by missense mutations in the respective genes. By using microprojectile bombardment, a plasmid harboring the wild-type Ant18 gene was introduced into ant18-161 mutant cells and resulted in the development of anthocyanin pigmentation, which demonstrates that the mutation is corrected by expression of the introduced gene. On the other hand, a plasmid derivative with the two ant18-161-specific base transitions at the 5' splice site of intron 3 prevented complementation. It is concluded that the absence of detectable mRNA for dihydroflavonol 4-reductase in ant18-161 cells is due to the mutations in the pre-mRNA splice donor site.
Alcohol Oxidoreductases↗