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Query: UNIPROT:P06889 (Mol)
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Three polyketide synthase genes (PKS1, PKS2, PKS3) from cell suspension cultures of raspberry (Rubus idaeus L. cv. Royalty) were characterized. They showed high similarity in both their nucleotide and deduced amino acid sequences. All three proteins contain the amino acid residues identified in previous work as essential for chalcone synthase (CHS) function. Enzyme activities were investigated after heterologous expression in Escherichia coli. RiPKS1 is a typical naringenin CHS that synthesizes the chalcone as the main reaction product, and p-coumaryltriacetic acid lactone (CTAL) as a minor by-product. RiPKS3 differed from RiPKS1 in four positions (K49R, M64R, P120L, V188A), and the products in vitro were predominantly CTAL and low levels of chalcone. RiPKS2 had the same four differences from RiPKS1 as RiPKS3, but in addition two further exchanges (R259H, F344L), and the protein had no detectable enzyme activity. Experiments with RiPKS1 containing either 259H or 344L showed that each of the exchanges was sufficient to completely eliminate enzyme activity. These experiments identify amino acid residues in CHS which are important for folding of the tetraketide intermediate to the chalcone (PKS3) and which are in general essential for CHS activity (PKS2). The possible functions of these residues are discussed.
Plant Mol Biol 2001 May
PMID:Molecular and biochemical characterization of three aromatic polyketide synthase genes from Rubus idaeus. 1143 45

DNA sequence variations of chalcone synthase (Chs) and Apetala3 gene promoters from 22 cruciferous plant species were analyzed to identify putative conserved regulatory elements. Our comparative approach confirmed the existence of numerous conserved sequences which may act as regulatory elements in both investigated promoters. To confirm the correct identification of a well-conserved UV-light-responsive promoter region, a subset of Chs promoter fragments were tested in Arabidopsis thaliana protoplasts. All promoters displayed similar light responsivenesses, indicating the general functional relevance of the conserved regulatory element. In addition to known regulatory elements, other highly conserved regions were detected which are likely to be of functional importance. Phylogenetic trees based on DNA sequences from both promoters (gene trees) were compared with the hypothesized phylogenetic relationships (species trees) of these taxa. The data derived from both promoter sequences were congruent with the phylogenies obtained from coding regions of other nuclear genes and from chloroplast DNA sequences. This indicates that promoter sequence evolution generally is reflective of species phylogeny. Our study also demonstrates the great value of comparative genomics and phylogenetics as a basis for functional analysis of promoter action and gene regulation.
Mol Biol Evol 2001 Oct
PMID:Comparative genomics and regulatory evolution: conservation and function of the Chs and Apetala3 promoters. 1155 94

The mutable flaked or a (flaked) (a(f)) line of the common morning glory (Ipomoea purpurea) displays white flowers with colored flakes, and the a(f) mutation is caused by the insertion of a transposable element named Tip100 into the CHS-D gene for anthocyanin biosynthesis. The 3.9-kb Tip100 element belongs to the Ac/Ds family and contains an ORF encoding a polypeptide of 808 amino acids. The frequency and timing of flower variegation vary in different a(f) lines, and a genetic element termed Modulator has been postulated to affect the variegation pattern. Since the pattern of flower variegation is determined by the frequency and timing of excision of Tip100 from the CHS-D gene, we wished to determine whether Tip100 is an autonomous element that is itself capable of transposition in a heterologous host. To do this, we introduced the element into the genome of tobacco plants by Agrobacterium-mediated transformation. The intact Tip100 element was able to excise from its original position in the chromosome and reinsert into new sites in the tobacco genome, whereas an internal deletion derivative was not. Based on these results, we conclude that Tip100 is an autonomous element. We also discuss the nature of the putative Modulator element affecting flower and leaf variegation in various mutable lines of the morning glory.
Mol Genet Genomics 2002 Jan
PMID:The transposon Tip100 from the common morning glory is an autonomous element that can transpose in tobacco plants. 1181 Feb 46

Screening of a tobacco (Nicotiana tabacum) cDNA library resulted in the isolation of a clone encoding the bZIP transcription factor BZI-1. With respect to amino acid sequence, conservation of protein domains, genomic exon-intron structure and expression pattern, BZI-1 is closely related to CPRF2, OHP1/2, BLZ1 and REB, a group of bZIP proteins which have been described in a number of dicot and monocot species. BZI-1 exhibits the characteristics of a transcription factor. It binds to G-box and C-box cis-elements in vitro, it is localised in the nucleus, and the N-terminal region of BZI-1 functions as an activation domain in both yeast and plant cells. Since BZI-1-related transcription factors have been isolated from dicots by in vitro binding to G-box elements in the chalcone synthase ( CHS) promoter, it has been suggested that phenylpropanoid pathway genes, such as CHS and PAL (phenylalanine ammonia-lyase), are targets of these proteins in vivo. However, after infection with Pseudomonas syringae or Tobacco Mosaic Virus, no changes in pathogen-induced PAL expression were observed in transgenic plants expressing increased levels of BZI-1 or a dominant negative form of the protein, BZI-1-DeltaN. In contrast to the tissue-specific expression of CHS and PAL, BZI-1 was found to be ubiquitously expressed in tobacco plants. Furthermore, no changes in the tissue-specific expression of PAL or CHS were observed in plants that were transgenic for BZI-1-DeltaN. Expression of a VP16-BZI-1 fusion protein would be expected to result in constitutive activation of the BZI-1 target genes. However, tetracycline-dependent expression of a VP16-BZI-1 protein in tobacco plants did not result in activation of CHS or PAL. On the basis of these data, we conclude that the phenylpropanoid pathway genes analysed are not targets of BZI-1 in vivo. Thus, the pattern of in vitro DNA binding of transcription factors need not always reflect their in vivo function.
Mol Genet Genomics 2002 Mar
PMID:The tobacco bZIP transcription factor BZI-1 binds to G-box elements in the promoters of phenylpropanoid pathway genes in vitro, but it is not involved in their regulation in vivo. 1191 11

The soluble, diffusible red-brown pigment produced by a Saccharopolyspora erythraea "red variant" has been shown to contain glycosylated and polymerized derivatives of 2,5,7-trihydroxy-1,4-naphthoquinone (flaviolin). Flaviolin is a spontaneous oxidation product of 1,3,6,8-tetrahydroxynaphthalene (THN), which is biosynthesized in bacteria by a chalcone synthase-like (CS-like) type III polyketide synthase (PKS). A fragment of the gene responsible for THN biosynthesis in S. erythraea E_8-7 was amplified by polymerase chain reaction (PCR) using degenerate primers based on conserved regions of known plant CS and bacterial CS-like genes. From the isolated fragment, a suicide vector was prepared, which was subsequently used to disrupt the red-brown pigment-producing (rpp) locus in S. erythraea, generating a mutant that displayed an albino phenotype. Chromosomal DNA from the albino mutant was subsequently used in a vector-recapture protocol to isolate a plasmid that contained an insert spanning the entire rpp locus. Sequencing of the insert revealed that the disrupted open reading frame (ORF) encodes a CS-like protein displaying 69% sequence identity to the rppA gene of Streptomyces griseus. The S. griseus rppA gene encodes RppA, the first characterized bacterial CS-like protein, which is sufficient in vitro for the synthesis of THN from malonyl-CoA. The rppA disruption mutant and rppA sequence provided a means by which to address the mechanism of diffusible pigment biosynthesis, as well as to investigate any link between this and the modulation of erythromycin A titre, which has been observed for S. erythraea variants.
Mol Microbiol 2002 Jun
PMID:Identification and cloning of a type III polyketide synthase required for diffusible pigment biosynthesis in Saccharopolyspora erythraea. 1202 78

Markers corresponding to 27 plant defense genes were tested for linkage disequilibrium with quantitative resistance to late blight in a diploid potato population that had been used for mapping quantitative trait loci (QTLs) for late blight resistance. Markers were detected by using (i) hybridization probes for plant defense genes, (ii) primer pairs amplifying conserved domains of resistance (R) genes, (iii) primers for defense genes and genes encoding transcriptional regulatory factors, and (iv) primers allowing amplification of sequences flanking plant defense genes by the ligation-mediated polymerase chain reaction. Markers were initially screened by using the most resistant and susceptible individuals of the population, and those markers showing different allele frequencies between the two groups were mapped. Among the 308 segregating bands detected, 24 loci (8%) corresponding to six defense gene families were associated with resistance at chi2 > or = 13, the threshold established using the permutation test at P = 0.05. Loci corresponding to genes related to the phenylpropanoid pathway (phenylalanine ammonium lyase [PAL], chalcone isomerase [CHI], and chalcone synthase [CHS]), loci related to WRKY regulatory genes, and other -defense genes (osmotin and a Phytophthora infestans-induced cytochrome P450) were significantly associated with quantitative disease resistance. A subset of markers was tested on the mapping population of 94 individuals. Ten defense-related markers were clustered at a QTL on chromosome III, and three defense-related markers were located at a broad QTL on chromosome XII. The association of candidate genes with QTLs is a step toward understanding the molecular basis of quantitative resistance to an important plant disease.
Mol Plant Microbe Interact 2002 Jun
PMID:Plant defense genes associated with quantitative resistance to potato late blight in Solanum phureja x dihaploid S. tuberosum hybrids. 1205 7

The KAP-2 protein that binds to the H-box (CCTACC) element in the bean CHS15 chalcone synthase promoter was purified, and internal peptide sequence used to design primers leading to the cloning of KAP-2 from bean (Phaseolus vulgaris) and barrel medic (Medicago truncatula). KAP-2 shares sequence similarity to the large subunit of mammalian Ku autoantigen, a protein proposed to be involved in control of DNA recombination and transcription. KAP-2 sequences were present in genomic DNA from a range of legumes, and a related protein is found in Arabidopsis thaliana. Recombinant KAP-2 expressed in insect cells showed the same binding specificity for the CHS15 H-box as the protein purified from bean cell extracts. In vitro transcription assays confirmed that KAP-2 stimulates transcription from a promoter harboring the H-box cis element.
Plant Mol Biol 2002 Jul
PMID:KAP-2, a protein that binds to the H-box in a bean chalcone synthase promoter, is a novel plant transcription factor with sequence identity to the large subunit of human Ku autoantigen. 1209 Jun 26

The cholesterol sulphate sulphohydrolase (CHS-ase) exhibiting molecular weight of 30 kDa was purified from human placenta microsomes. The microsomal proteins were extracted with 0.5% Triton X-100. The DEAE-cellulose chromatography of the solubilized microsomal proteins, performed at pH 7.6 allowed to separate two enzymatically active fractions. One of them was associated with the protein fraction unbound by DEAE-cellulose, the other was tightly bound by ion exchanger. The 30 kDa cholesterol sulphate sulphohydrolase was purified to homogenity from the protein fraction tightly bound by DEAE-cellulose. The highly purified enzyme preparation (specific activity 385 nmol min(-1)mg(-1) of protein) exhibited optimal activity at pH 6.4, the K(m) was established to be 6.7 x 10(-6)M, the pI value was 7.4. The 30 kDa cholesterol sulphate sulphohydrolase, in contrast to the CHS-ase form originated from the protein fraction unbound by DEAE-cellulose, was not sensitive to alkaline phosphatase treatment and phosphohydrolase inhibitors. The effects of steroids, -SH reacting agents and sulphohydrolase inhibitors on the enzyme activity were tested.
J Steroid Biochem Mol Biol 2002 Jul
PMID:Sterolsulphate sulphohydrolase from human placenta microsomes--30 kDa molecular weight form of cholesterol sulphate sulphohydrolase. 1216 38

Flavonoids are thought to function in the plant stress response and male fertility in some, but not all, species. We examined the effects of a self-fertile chalcone synthase null allele, a, for the effects of heat and light stress on fertilization success and flower production in Ipomoea purpurea. Pollen recipients and pollen donors of both homozygous genotypes exhibit reduced fertilization success at high temperatures, indicating that high temperature acts as a stress-lowering fertilization success. Homozygous aa individuals exhibit reduced male and female fertilization success, compared to AA individuals, at high temperatures but not at low temperatures. In addition, aa individuals produce fewer flowers than AA individuals at low temperatures, but not at high temperatures. These results suggest that flavonoids alleviate heat stress on fertilization success. They also suggest that pleiotropic effects at the A locus may explain the low frequency of the a allele in natural populations.
Mol Ecol 2003 May
PMID:Analysis of a chalcone synthase mutant in Ipomoea purpurea reveals a novel function for flavonoids: amelioration of heat stress. 1269 76

Over a broad taxonomic range that spans monocots and dicots, upstream enzymes of the anthocyanin pigment pathway have evolved less rapidly than downstream enzymes. In this article we show that this pattern is also evident within the genus Ipomoea. Specifically, the most upstream enzyme, chalcone synthase (CHS-D), evolves more slowly than the two most downstream enzymes, ancyocyanidin synthase (ANS) and UDP glucose flavonoid 3-oxy-glucosyltransferase (UFGT). This pattern appears not to be due to variation in mutation rates, because the CHS-D gene exhibits higher synonymous substitution rates than the genes for the other two enzymes. Codon-based tests for positive selection suggest that it has been negligible or absent in all three genes. In addition, the mean number of indel-creating events is four times as high in the downstream genes as in CHS-D. Unlike the downstream genes, CHS-D also exhibits evidence of codon bias. Together, the evidence suggests that the difference in nonsynonymous substitution rates between upstream and downstream genes is due to relaxed constraint on the downstream genes rather than a greater frequency of positively selected substitutions.
Mol Biol Evol 2003 Nov
PMID:Evolutionary rate variation in anthocyanin pathway genes. 1288 63


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