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PubMed1995

Characterization of the progesterone 21-hydroxylase activity of canine cytochrome P450 PBD-2/P450 2B11 through reconstitution, heterologous expression, and site-directed mutagenesis.

Canine hepatic cytochrome P450 PBD-2 metabolizes 2,2',4,4',5,5'-hexachlorobiphenyl and catalyzes the 21-hydroxylation of progesterone, thereby distinguishing PBD-2 as unique among 2B P450s. Heterologous expression of the PBD-2 cDNA, P450 2B11, in COS and yeast systems produced a protein capable of androstenedione metabolism; however, this P450 did not metabolize progesterone in a manner consistent with PBD-2. Modification of PBD-2 reconstitution parameters resulted in significantly increased catalytic activities and further emphasized differences between PBD-2 and the heterologously expressed enzyme. Subsequent Escherichia coli expression of 2B11 generated a protein that possessed substrate specificities indistinguishable from those of PBD-2 and provided a system in which the determinants of 2B11 progesterone 21-hydroxylation could be examined via site-directed mutagenesis. Site-directed mutants of 2B11 expressed in E. coli revealed that substitution of Ile with Val at position 363 converted 2B11 into a highly active and specific progesterone 16 alpha-hydroxylase. Mutants Val-114 --> Ile, Asp-290 --> Ile, and Ile-365 --> Phe exhibited decreased progesterone 21- and 16 alpha-hydroxylase activities, in accordance with decreases in androstenedione hydroxylase activities. In contrast, replacement of Ile-365 with Val or Leu resulted in much greater changes in progesterone than androstenedione hydroxylation. Thus, the combination of P450 reconstitution techniques, heterologous expression, and site-directed mutagenesis has revealed PBD-2 to be an important progesterone 21-hydroxylase in canine liver and has identified several amino acid residues that alter progesterone metabolism by 2B11.

Animals↗
PubMed2002

Dbeta3, an atypical nicotinic acetylcholine receptor subunit from Drosophila : molecular cloning, heterologous expression and coassembly.

Insect nicotinic acetylcholine receptors (nAChRs) play a central role in mediating neuronal synaptic transmission and are the target sites for the increasingly important group of neonicotinoid insecticides. Six nicotinic acetylcholine receptor (nAChR) subunits (four alpha-type and two beta-type) have been cloned previously from the model insect species Drosophila melanogaster. Despite extensive efforts, it has not been possible to generate functional recombinant nAChRs by heterologous expression of any combination of these six subunits. It has, however, been possible to express functional hybrid receptors when Drosophila alpha subunits are co-expressed with vertebrate beta subunits. This has led to the assumption that successful heterologous expression might require an, as yet, uncloned beta-type insect subunit. Examination of the recently completed Drosophila genomic sequence data has identified a novel putative nAChR beta-type subunit. Here we report the molecular cloning, heterologous expression and characterization of this putative Drosophila nAChR subunit (Dbeta3). Phylogenetic comparisons with other ligand-gated ion channel subunit sequences support its classification as a nAChR subunit but show it to be a distantly related member of this neurotransmitter receptor subunit family. Evidence that the Dbeta3 subunit is able to coassemble with other Drosophila nAChR subunits and contribute to recombinant nAChRs has been obtained by both radioligand binding and coimmunoprecipitation studies in transfected Drosophila S2 cells.

Animals↗
PubMed2006

Rare codon clusters at 5'-end influence heterologous expression of archaeal gene in Escherichia coli.

Proteins from hyperthermophilic microorganisms are attractive candidates for novel biocatalysts because of their high resistance to temperature extremes. However, archaeal genes are usually poorly expressed in Escherichia coli because of differences in codon usage. Genes from the thermoacidophilic archaea Sulfolobus solfataricus and Thermoplasma acidophilum contain high proportions of rare codons for arginine, isoleucine, and leucine, which are recognized by the tRNAs encoded by the argU, ileY, and leuW genes, respectively, and which are rarely used in E. coli. To examine the effects of these rare codons on heterologous expression, we expressed the Sso_gnaD and Tac_gnaD genes from S. solfataricus and T. acidophilum, respectively, in E. coli. The Sso_gnaD product was expressed at very low levels when the open reading frame (ORF) was cloned in pRSET and expressed in E. coli BL21(DE3), and was expressed at much higher levels in the E. coli BL21(DE3)-CodonPlus RIL strain, which contains extra copies of the argU, ileY, and leuW tRNA genes. In contrast, Tac_gnaD was expressed at similar levels in both E. coli strains. Comparison of the Sso_gnaD and Tac_gnaD gene sequences revealed that the 5'-end of the Sso_gnaD sequence was rich in AGA(arg) and ATA(Ile) codons. These codons were replaced with the codons commonly used in E. coli by polymerase chain reaction-mediated site-directed mutagenesis. The results of expression studies showed that a non-tandem repeat of rare codons is critical in the observed interference in heterologous expression of this gene. We concluded that the level of heterologous expression of Sso_gnaD in E. coli was limited by the clustering of the rare codons in the ORF, rather than on the rare codon frequency.

Codon↗
PubMed2004

beta-Amyloid directly inhibits human alpha4beta2-nicotinic acetylcholine receptors heterologously expressed in human SH-EP1 cells.

Amyloid-beta (Abeta) accumulation and aggregation are thought to contribute to the pathogenesis of Alzheimer's disease (AD). In AD, there is a selective decrease in the numbers of radioligand binding sites corresponding to the most abundant nicotinic acetylcholine receptor (nAChR) subtype, which contains human alpha4 and beta2 subunits (halpha4beta2-nAChR). However, the relationships between these phenomena are uncertain, and effects of Abeta on halpha4beta2-nAChR function have not been investigated in detail. We first confirmed expression of halpha4 and hbeta2 subunits as messenger RNA in transfected, human SHEP1 cells by reverse transcription-polymerase chain reaction and mRNA fluorescence in situ hybridization analyses. Immunoprecipitation Western analyses confirmed alpha4 and beta2 subunit protein expression and co-assembly. Whole cell current recording demonstrated heterologous expression in SH-EP1-halpha4beta2 cells of functional halpha4beta2-nAChRs with characteristic responses to nicotinic agonists or antagonists. Nicotine-induced whole cell currents were suppressed by Abeta(1-42) in a dose-dependent manner. Functional inhibition was selective for Abeta(1-42) compared with the functionally inactive, control peptide Abeta(40-1).Abeta(1-42)-mediated inhibition of halpha4beta2-nAChR function was non-competitive, voltage-independent, and use-independent. Pre-loading of cells with guanyl-5'-yl thiophosphate failed to prevent Abeta(1-42)-induced inhibition, suggesting that down-regulation of halpha4beta2-nAChR function by Abeta(1-42) is not mediated by nAChR internalization. Sensitivity to Abeta(1-42) antagonism at 1 nm was evident for halpha4beta2-nAChRs, but not for heterologously expressed human alpha7-nAChRs, although both nAChR subtypes were functionally inhibited by 100 nm Abeta(1-42), with the magnitude of functional block being higher for 100 nm Abeta(1-42) acting on halpha7-nAChRs. These findings suggest that halpha4beta2-nAChRs are sensitive and perhaps pathophysiologically relevant targets for Abeta neurotoxicity in AD.

Amyloid↗
PubMed2004

Overproduction of trehalose: heterologous expression of Escherichia coli trehalose-6-phosphate synthase and trehalose-6-phosphate phosphatase in Corynebacterium glutamicum.

Trehalose is a disaccharide with potential applications in the biotechnology and food industries. We propose a method for industrial production of trehalose, based on improved strains of Corynebacterium glutamicum. This paper describes the heterologous expression of Escherichia coli trehalose-synthesizing enzymes trehalose-6-phosphate synthase (OtsA) and trehalose-6-phosphate phosphatase (OtsB) in C. glutamicum, as well as its impact on the trehalose biosynthetic rate and metabolic-flux distributions, during growth in a defined culture medium. The new recombinant strain showed a five- to sixfold increase in the activity of OtsAB pathway enzymes, compared to a control strain, as well as an almost fourfold increase in the trehalose excretion rate during the exponential growth phase and a twofold increase in the final titer of trehalose. The heterologous expression described resulted in a reduced specific glucose uptake rate and Krebs cycle flux, as well as reduced pentose pathway flux, a consequence of downregulated glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase. The results proved the suitability of using the heterologous expression of Ots proteins in C. glutamicum to increase the trehalose biosynthetic rate and yield and suggest critical points for further improvement of trehalose overproduction in C. glutamicum.

Corynebacterium↗
PubMed1999

Heterologous expression of correctly assembled methylamine dehydrogenase in Rhodobacter sphaeroides.

The biosynthesis of methylamine dehydrogenase (MADH) from Paracoccus denitrificans requires four genes in addition to those that encode the two structural protein subunits, mauB and mauA. The accessory gene products appear to be required for proper export of the protein to the periplasm, synthesis of the tryptophan tryptophylquinone (TTQ) prosthetic group, and formation of several structural disulfide bonds. To accomplish the heterologous expression of correctly assembled MADH, eight genes from the methylamine utilization gene cluster of P. denitrificans, mauFBEDACJG, were placed under the regulatory control of the coxII promoter of Rhodobacter sphaeroides and introduced into R. sphaeroides by using a broad-host-range vector. The heterologous expression of MADH was constitutive with respect to carbon source, whereas the native mau promoter allows induction only when cells are grown in the presence of methylamine as a sole carbon source and is repressed by other carbon sources. The recombinant MADH was localized exclusively in the periplasm, and its physical, spectroscopic, kinetic and redox properties were indistinguishable from those of the enzyme isolated from P. denitrificans. These results indicate that mauM and mauN are not required for MADH or TTQ biosynthesis and that mauFBEDACJG are sufficient for TTQ biosynthesis, since R. sphaeroides cannot synthesize TTQ. A similar construct introduced into Escherichia coli did not produce detectable MADH activity or accumulation of the mauB and mauA gene products but did lead to synthesizes of amicyanin, the mauC gene product. This finding suggests that active recombinant MADH is not expressed in E. coli because one of the accessory gene products is not functionally expressed. This study illustrates the potential utility of R. sphaeroides and the coxII promoter for heterologous expression of complex enzymes such as MADH which cannot be expressed in E. coli. These results also provide the foundation for future studies on the molecular mechanisms of MADH and TTQ biosynthesis, as well as a system for performing site-directed mutagenesis of the MADH gene and other mau genes.

Bacterial Proteins↗
PubMed2000

Cloning, characterization and heterologous expression of a polyketide synthase and P-450 oxidase involved in the biosynthesis of the antibiotic oleandomycin.

The gene cluster encoding the deoxyoleandolide polyketide synthase (OlePKS) was isolated from the oleandomycin producing strain Streptomnyces antibioticus. Sequencing of the first two genes encoding OlePKS, together with the previously identified third gene revealed an overall genetic and protein architecture similar to that of the erythromycin gene cluster encoding the 6-deoxyerythronolide B synthase (DEBS) from Saccharopolyspora erythraea. When the entire OlePKS (10,487 amino acids) was expressed in the heterologous host Streptomyces lividans, it produced 8,8a-deoxyoleandolide, an aglycone precursor of oleandomycin. The role of the P-450 monooxygenase, OleP, in oleandomycin biosynthesis was also examined in vivo by co-expression with DEBS in S. lividans. The production of 8,8a-dihydroxy-6-deoxyerythronolide B and other derivatives indicates that OleP is involved in the epoxidation pathway of oleandomycin biosynthesis. Since there are currently no genetic systems available for manipulation of the natural oleandomycin producing strain, the heterologous expression system reported here provides a useful tool for studying this important macrolide antibiotic.

Anti-Bacterial Agents↗
PubMed2003

Functional properties of homomeric, human alpha 7-nicotinic acetylcholine receptors heterologously expressed in the SH-EP1 human epithelial cell line.

alpha 7-Nicotinic acetylcholine receptors (alpha 7-nAChRs) are broadly distributed in the central nervous system, where they play important roles in chemical and electrical signaling, and perhaps in neurite outgrowth, synaptic plasticity, and neuronal death/survival. To help elucidate their normal and pathophysiological roles, we have heterologously expressed human alpha 7-nAChR in transfected SH-EP1 human epithelial cells. Reverse transcription-polymerase chain reaction and mRNA fluorescence in situ hybridization analyses demonstrate expression of human alpha 7 subunits as messenger RNA. Patch-clamp recordings exploiting a novel strategy to prevent functional rundown of whole-cell peak current responses to repeated acute challenges with nicotinic agonists show successful expression of functional alpha 7-nAChR that mediate inward currents characterized by rapid phases of activation and inactivation. Concentration-response curves show that nicotine, acetylcholine, and choline are efficacious agonists at human alpha 7-nAChRs. Current-voltage relationships show inward rectification for agonist-induced currents. Human alpha 7-nAChRs exhibit some sensitivity to alpha 7-nAChR antagonists alpha-bungarotoxin (Bgt) or methyllycaconitine (MLA) when applied coincidentally with agonist, but much higher affinity block occurs when cells and alpha 7-nAChRs are pre-exposed to antagonists for 2 min before challenge with agonist. Both Bgt and MLA are competitive inhibitors of alpha 7-nAChR function. Whole-cell current peak amplitudes and half-times for inactivation of alpha 7-nAChR functional responses to nicotine are dramatically reduced in the absence of extracellular Ca2+, suggestive of high Ca2+ permeability of the alpha 7-nAChR channel. Thus, heterologously expressed human alpha 7-nAChR in mammalian cells have properties of native alpha 7-nAChR or of alpha 7-nAChR heterologously expressed in other systems and serve as excellent models for studies of molecular bases of alpha 7-nAChR function.

Acetylcholine↗
PubMed2001

Heterologous expression of lacticin 3147 in Enterococcus faecalis: comparison of biological activity with cytolysin.

Lacticin 3147 is a broad-spectrum, two-component, lanthionine-containing bacteriocin produced by Lactococcus lactis DPC3147 which has widespread food and biomedical applications as a natural antimicrobial. Other two-component lantibiotics described to date include cytolysin and staphylococcin C55. Interestingly, cytolysin, produced by Enterococcus faecalis, has an associated haemolytic activity. The objective of this study was to compare the biological activity of lacticin 3147 with cytolysin. The lacticin 3147-encoding determinants were heterologously expressed in Ent. faecalis FA2-2, a plasmid-free strain, to generate Ent. faecalis pOM02, thereby facilitating a direct comparison with Ent. faecalis FA2-2.pAD1, a cytolysin producer. Both heterologously expressed lacticin 3147 and cytolysin exhibited a broad spectrum of activity against bacterial targets. Furthermore, enterococci expressing active lacticin 3147 did not exhibit a haemolytic activity against equine blood cells. The results thus indicate that the lacticin 3147 biosynthetic machinery can be heterologously expressed in an enterococcal background resulting in the production of the bacteriocin with no detectable haemolytic activity.

Amino Acid Sequence↗
PubMed2003

Heterologous expression of G-protein-coupled receptors: comparison of expression systems from the standpoint of large-scale production and purification.

G-protein-coupled receptors (GPCRs) are of prime importance for cell signal transduction mechanisms and are the target of many current and potential drugs. However, structural data on these membrane proteins is still scarce because of their low natural abundance and the low efficiency of most of the expression systems currently available. This review presents the most important expression systems currently employed for heterologous expression of GPCRs; Escherichia coli, yeast, insect cells and mammalian cells. After briefly recalling the specificity, advantages and limitations of each system, particular emphasis is put on the quantitative comparison of these expression systems in terms of overall expression yield, and on the influence of various factors (primary sequence, origin, cell type, N- and C-terminal tags) on the results.

Animals↗
PubMed1999

Purification, cloning, and heterologous expression of a catalytically efficient flavonol 3-O-galactosyltransferase expressed in the male gametophyte of Petunia hybrida.

Flavonols are plant-specific molecules that are required for pollen germination in maize and petunia. They exist in planta as both the aglycone and glycosyl conjugates. We identified a flavonol 3-O-galactosyltransferase (F3GalTase) that is expressed exclusively in the male gametophyte and controls the formation of a pollen-specific class of glycosylated flavonols. Thus an essential step to understanding flavonol-induced germination is the characterization of F3GalTase. Amino acid sequences of three peptide fragments of F3GalTase purified from petunia pollen were used to isolate a full-length cDNA clone. RNA gel blot analysis and enzyme assays confirmed that F3GalTase expression is restricted to pollen. Heterologous expression of the F3GalTase cDNA in Escherichia coli yielded active recombinant enzyme (rF3GalTase) which had the identical substrate specificity as the native enzyme. Unlike the relatively nonspecific substrate usage of flavonoid glycosyltransferases from sporophytic tissues, F3GalTase uses only UDP-galactose and flavonols to catalyze the formation of flavonol 3-O-galactosides. Kinetic analysis showed that the k(cat)/K(m) values of rF3GalTase, using kaempferol and quercetin as substrates, approaches that of a catalytically perfect enzyme. rF3GalTase catalyzes the reverse reaction, generation of flavonols from UDP and flavonol 3-O-galactosides, almost as efficiently as the forward reaction. The biochemical characteristics of F3GalTase are discussed in the context of a role in flavonol-induced pollen germination.

Amino Acid Sequence↗
PubMed2006

Heterologous expression of proteins from Plasmodium falciparum: results from 1000 genes.

As part of a structural genomics initiative, 1000 open reading frames from Plasmodium falciparum, the causative agent of the most deadly form of malaria, were tested in an E. coli protein expression system. Three hundred and thirty-seven of these targets were observed to express, although typically the protein was insoluble. Sixty-three of the targets provided soluble protein in yields ranging from 0.9 to 406.6 mg from one liter of rich media. Higher molecular weight, greater protein disorder (segmental analysis, SEG), more basic isoelectric point (pI), and a lack of homology to E. coli proteins were all highly and independently correlated with difficulties in expression. Surprisingly, codon usage and the percentage of adenosines and thymidines (%AT) did not appear to play a significant role. Of those proteins which expressed, high pI and a hypothetical annotation were both strongly and independently correlated with insolubility. The overwhelmingly important role of pI in both expression and solubility appears to be a surprising and fundamental issue in the heterologous expression of P. falciparum proteins in E. coli. Twelve targets which did not express in E. coli from the native gene sequence were codon-optimized through whole gene synthesis, resulting in the (insoluble) expression of three of these proteins. Seventeen targets which were expressed insolubly in E. coli were moved into a baculovirus/Sf-21 system, resulting in the soluble expression of one protein at a high level and six others at a low level. A variety of factors conspire to make the heterologous expression of P. falciparum proteins challenging, and these observations lay the groundwork for a rational approach to prioritizing and, ultimately, eliminating these impediments.

Animals↗
PubMed1999

The use of heterologously expressed drug metabolizing enzymes--state of the art and prospects for the future.

The first report of the functional, heterologous expression of a mammalian cytochrome P450 (CYP) enzyme occurred more than a decade ago. In the intervening years, these expression systems have been optimized with regard to the specific requirements for production of catalytically active enzymes. In this review, we discuss the strengths and limitations of heterologously expressed enzymes as they affect in vitro drug metabolism studies. Emphasis is given to new applications (screens for CYP inhibition and novel enzyme mixtures) that have been enabled by high level, functional expression of CYP enzymes.

Animals↗
PubMed1999

A heterologous expression system for bovine lens transmembrane main intrinsic protein (MIP) in Nicotiana tabacum plants.

We have developed a heterologous expression system for transmembrane lens main intrinsic protein (MIP) in Nicotiana tabacum plant tissue. A native bovine MIP26 amplicon was subcloned into an expression cassette under the control of a constitutive Cauliflower Mosaic Virus promoter, also containing a neomycin phosphotransferase operon. This cassette was transformed into Agrobacterium tumefaciens by triparental mating and used to infect plant tissue grown in culture. Recombinant plants were selected by their ability to grow and root on kanamycin-containing media. The presence of MIP in the plant tissues was confirmed by PCR, RT-PCR and immunohistochemistry. A number of benefits of this system for the study of MIP will be discussed, and also its application as a tool for the study of heterologously expressed proteins in general.

Animals↗
PubMed1995

Heterologous expression of metabotropic glutamate receptors in adult rat sympathetic neurons: subtype-specific coupling to ion channels.

A novel heterologous expression system was used to examine the coupling of metabotropic glutamate receptors (mGluRs) to neuronal voltage-gated ion channels. Cytoplasmic injection of mGluR2 cRNA into adult rat sympathetic neurons resulted in the expression of receptors that negatively coupled to N-type Ca2+ channels through a pertussis toxin-sensitive pathway. Injection of mGluR1 alpha cRNA resulted in the expression of receptors that inhibited M-type K+ channels via a pertussis toxin-insensitive pathway. Coupling was restricted to specific transduction elements and effectors, since mGluR2 did not inhibit M channels and mGluR1 alpha had minimal effects on Ca2+ channels. These findings demonstrate that heterologously expressed, and thus unambiguously identified, mGluR subtypes modulate specific neuronal ion channels through discrete signal transduction pathways.

Animals↗
PubMed1997

Prolactin-like protein-B: heterologous expression and characterization of placental and decidual species.

Prolactin-like protein-B (PLP-B) is a member of a family of proteins expressed by the rat placenta and/or decidua with characteristics similar to prolactin (PRL). In this report, we present the heterologous expression and characterization of PLP-B. Recombinant PLP-B heterologously expressed in Chinese hamster ovary cells exhibited similar immunoreactive and electrophoretic characteristics with PLP-B produced by rat placental and decidual tissues. N-terminal sequencing verified the identity and purity of the recombinant PLP-B species and the site of cleavage of the signal peptide from the mature secreted PLP-B species. Polyclonal antibodies were generated to the recombinant PLP-B and used for Western blot and immunocytochemical analyses. Recombinant and native PLP-B migrated as a doublet at 30-31 kDa in SDS-PAGE under reducing conditions. Treatment of recombinant and native PLP-B with N-glycanase accelerated their electrophoretic mobility, indicative of their glycoprotein nature. PLP-B was localized exclusively to decidual cells in the developing deciduum and spongiotrophoblast cells in the placental junctional zone. The level of PLP-B protein expression dramatically declined prior to parturition. Potential PRL-like biological actions of PLP-B were also investigated. PLP-B bound weakly to ovarian and liver PRL receptors and did not stimulate the proliferation of lactogen-dependent Nb2 lymphoma cells. In conclusion, recombinant PLP-B possesses characteristics similar to native decidual and placental PLP-B and may represent a hormone/cytokine that has important modulatory actions during the establishment of pregnancy and the initiation of parturition.

Animals↗
PubMed2005

[Heterologous expression and functional identification of a chitinase gene from wheat].

Chitinases are functional in plant defense against pathogens and also involved in plant development as well as human disease responses. In this study a chitinase gene, Wch2, from wheat was expressed transiently in tobacco leaves and stably in transgenic Arabidopsis thaliana plants to evaluate its heterologous expression and potential application. Western blot analysis revealed that the expressed wheat chitinase was about 30 kD as was expected (Fig.1) and had hydrolytic activity towards chitin polymers (Fig.2). Integration and expression of Wch2 gene in transgenic Arabidopsis were confirmed by PCR (Fig.3) and Western blot (Fig.4) analyses. Wch2 accumulated to a high level in transgenic plants without any detectable degradation. Transgenic plants expressing Wch2 displayed a much higher resistance to Fusarium oxysporum f. sp. matthiolae, a fungus infectious to Arabidopsis thaliana than the plants containing green fluorescence protein (GFP) (Fig.5). These results indicated that Wch2 was suitable for heterologous expression in different organisms and could be used for enhancement of plant resistance in genetic engineering. The Arabidopsis-Fusarium interaction may be one of the ideal plant-fungus interaction systems for the evaluation of antifungal proteins in planta.

Arabidopsis↗
PubMed1999

A constitutively expressed Myc-like gene involved in anthocyanin biosynthesis from Perilla frutescens: molecular characterization, heterologous expression in transgenic plants and transactivation in yeast cells.

The coordinate expression of anthocyanin biosynthetic genes in leaves and stems of a red forma of Perilla frutescens is presumably controlled by regulatory gene(s). A Myc-like gene (Myc-rp) was isolated from a cDNA library prepared from the leaves of red P. frutescens, and its deduced amino acid sequence shows 64% identity with that of delila from snapdragon. The Myc-rp gene was expressed in leaves and roots of both red and green P. frutescens equally. Comparison of deduced amino acid sequence of Myc-rp with that of Myc-gp, the second allele isolated from a green forma of P. frutescens, indicates that the 132nd amino acid, alanine, existing in MYC-RP was changed to serine in MYC-GP. The heterologous expression of these two alleles of Myc-like gene in tobacco and tomato resulted in an increase of the anthocyanin contents in flowers of tobacco and vegetative tissues and flowers of tomato. However, the flowers of transgenic tobacco expressing the fragment with a partial deletion (encoding 1-115 amino acids deleted) of Myc(-gp gave no change in anthocyanin accumulation, but some morphological changes of the flower were observed. In yeast, the MYC-RP/GP and Delila protein exhibited transactivation activity on the GAL-1 promoter from yeast and the promoter of dihydroflavonol 4-reductase (DFR) gene from P. frutescens. A transactivation domain of MYC-RP/GP and Delila could be located in the region between the 193rd and the 420th amino acid of MYC-RP/GP proteins. Our data indicate that this Myc-like gene presumably functions in the regulation of anthocyanin biosynthesis similarly in different tissues of dicot plants.

Alcohol Oxidoreductases↗