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Query: UNIPROT:P06889 (Mol)
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Analysis of Agrobacterium-transferred DNA (T-DNA) revealed strong correlations between transgene structures and floral pigmentation patterns from chalcone synthase (chs) co-suppression among 47 Petunia transformants. Presented here are the full details of T-DNA structural organization in that population. Sixteen transformants (34%) carried one T-DNA copy while 31 (66%) carried 106 complete and partial T-DNA elements in 54 linkage groups. Thirty linkage groups contained multiple T-DNA copies; 15 of these contained only contiguously repeated copies, 8 contained only dispersed copies and 7 contained both. Right-border inverted repeats were three times more frequent than left-border inverted or direct repeats. Large fragments of binary-vector sequences were linked to the T-DNA in seven plants.
Plant Mol Biol 1996 Dec
PMID:Details of T-DNA structural organization from a transgenic Petunia population exhibiting co-suppression. 900 21

To investigate further the signal transduction pathway that mediates the cold-stress response in maize, we isolated a low temperature-inducible cDNA clone (ZmCDPK1) that encodes a calcium-dependent protein kinase. Time-course experiments revealed that the low-temperature induction of ZmCDPK1 precedes that of mlip15, another cold-inducible gene that codes for a DNA-binding protein of the basic region/leucine zipper (bZIP) type, indicating that ZmCDPK1 might be located upstream of mlip15 in the cold-stress signal transduction pathway. We observed that the steady-state mRNA level of mlip15 drastically increased after cycloheximide treatment. In addition to mlip15, cycloheximide elevates the transcript levels of two other low temperature-induced genes, ZmCDPK1, and Adh1, which encodes alcohol dehydrogenase 1. In contrast, the chalcone synthase gene was only inducible by low temperature. The accumulation of the mlip15 transcript at low temperatures and in response to cycloheximide was significantly reduced by pretreatment with a calcium chelator, suggesting that calcium is involved in both cases of mlip15 induction. The signal transduction pathways triggered by low temperature and cycloheximide are discussed in relation to these observations.
Mol Gen Genet 1997 Apr 16
PMID:Cycloheximide induces a subset of low temperature-inducible genes in maize. 915 Feb 61

Chediak-Higashi syndrome is an autosomal recessive, immune deficiency disorder of human (CHS) and mouse (beige, bg) that is characterized by abnormal intracellular protein transport to, and from, the lysosome. Recent reports have described the identification of homologous genes that are mutated in human CHS and bg mice. Here we report the sequences of two major mRNA isoforms of the CHS gene in human and mouse. These isoforms differ both in size and in sequence at the 3' end of their coding domains, with the smaller isoform (approximately 5.8 kb) arising from incomplete splicing and reading through an intron. These mRNAs also differ in tissue distribution of transcription and in predicted biological properties. Novel mutations were identified within the region of the coding domain common to both isoforms in three CHS patients: C-->T transitions that generated stop codons (R50X and Q1029X) were found in two patients, and a novel frameshift mutation (deletion of nucleotides 3073 and 3074 of the coding domain) was found in a third. Northern blots of lymphoblastoid mRNA from CHS patients revealed loss of the largest transcript (approximately 13.5 kb) in two of seven CHS patients, while the small mRNA was undiminished in abundance. These results suggest that the small isoform alone cannot complement Chediak-Higashi syndrome.
Hum Mol Genet 1997 Jul
PMID:Identification of mutations in two major mRNA isoforms of the Chediak-Higashi syndrome gene in human and mouse. 921 80

We have isolated seven genomic chalcone synthase (CHS) genes and six classes of CHS cDNA from elicitor-treated pea tissues. Comparison of the nucleotide sequences of the coding regions revealed the existence of eight members of the CHS gene family in pea. These can essentially be divided into three groups (PSCHS1, 2 and 8; PSCHS3, 4 and 5; and PSCHS6 and 7) on the basis of nucleotide and or amino acid sequence comparisons of the coding regions, introns and promoter regions. We previously reported that the accumulation of CHS mRNAs is induced by elicitor treatment. Accumulation of CHS mRNA was observed mainly in roots and very little was found in floral organs. To specifically detect expression of each CHS gene in various types of pea cells. S1 nuclease protection assays were performed. Interestingly, the classification of the eight members of the CHS gene family based on the sequence identity was found to reflect their expression patterns as determined by the S1 nuclease protection assay. The first group of CHS genes, PSCHS1, 2 and 8, was strongly induced not only by elicitor treatment and UV irradiation but is also constitutively expressed in root and flower tissues. The second group, PSCHS3, 4 and 5, was also strongly induced by elicitor treatment and UV irradiation but is constitutively expressed only in root. Expression of the third group, PSCHS6 and 7 was barely detectable in any of the organs tested and was not influenced by environmental stimuli such as elicitor or UV. Furthermore, sequence analysis of the promoter region of each member of the CHS gene family revealed that putative cis-regulatory elements, such as Box-I. Box-II and G-Box, were conserved only in PSCHS1, 2, 3, 4 and 5. From these results we propose that an ancestral CHS gene might have given rise to defense response-related (UV irradiation- and elicitor-responsive) and -unrelated (unresponsive) genes at an early stage of evolution, followed by divergence within these subclasses based upon the developmental program in pea.
Mol Gen Genet 1997 Jun
PMID:Molecular evolution and functional relevance of the chalcone synthase genes of pea. 923 Aug 96

The expression, during flower development, of the gene encoding the anthocyanin pathway key enzyme dihydroflavonol 4-reductase (DFR) was investigated in floral organs of Forsythia x intermedia cv. 'Spring Glory'. Full-length DFR and partial chalcone synthase (CHS) cDNAs, the gene of interest and a flavonoid pathway control gene respectively, were obtained from petal RNA by reverse transcription PCR. Whereas for CHS northern blot analysis enabled the study of its expression pattern, competitive PCR assays were necessary to quantify DFR mRNA levels in wild-type plants and in petals of 2 transgenic clones containing a CaMV 35S promoter-driven DFR gene of Antirrhinum majus. Results indicated a peak of CHS and DFR transcript levels in petals at the very early stages of anthesis, and different expression patterns in anthers and sepals. In comparison to wild-type plants, transformants showed a more intense anthocyanin pigmentation of some vegetative organs, and a dramatic increase in DFR transcript concentration and enzymatic activity in petals. However, petals of transformed plants did not accumulate any anthocyanins. These results indicate that other genes and/or regulatory factors should be considered responsible for the lack of anthocyanin production in Forsythia petals.
Plant Mol Biol 1997 Oct
PMID:Molecular cloning and expression analysis of dihydroflavonol 4-reductase gene in flower organs of Forsythia x intermedia. 934 54

Three clonal genotypes of Lotus corniculatus L. (bird's foot trefoil) were transformed with an antisense chalcone synthase (CHS) gene construct made using a stress induced CHS17 cDNA from Phaseolus vulgaris under the control of the constitutive CaMV 35S promoter and Nos terminator via Agrobacterium rhizogenes. After initial screening, ten antisense and five control co-transformation events from each recipient clonal genotype were analysed. After elicitation with glutathione, the level of tannin accumulation was found to be increased in a number of antisense root cultures derived from the low (S33) and moderate (S50) tannin recipient genotypes. Six antisense and four control transformed lines from genotype S50 were selected for more detailed study. The antisense CHS construct was found to be integrated into the genome, with a copy number ranging from 1 to 5 and antisense orientation was confirmed by PCR. In transformed root cultures, increased CHS transcript levels were noted in a number of antisense lines. Biochemical analyses of glutathione-elicited-root cultures indicated a significant increase in tannin accumulation in antisense CHS lines and mean vestitol levels were reduced. These results show that the introduction of a heterologous antisense chalcone synthase construct into L. corniculatus resulted in an unpredicted molecular and biochemical phenotype. Such findings are discussed in relation to manipulation of this complex multigene family.
Plant Mol Biol 1997 Nov
PMID:Differential modification of flavonoid and isoflavonoid biosynthesis with an antisense chalcone synthase construct in transgenic Lotus corniculatus. 934 73

Two cultivars of Perilla frutescens, red and green formas are known to differ in anthocyanin accumulation in leaves and stems. cDNA clones encoding the enzymes involved in anthocyanin biosynthesis, chalcone synthase (CHS), flavanone 3-hydroxylase (F3H), dihydroflavonol 4-reductase (DFR), and UDP glucose: flavonoid 3-O-glucosyltransferase (3GT), were isolated from cDNA libraries derived from the leaves of a red forma of P. frutescens by screening with partial fragments amplified by means of polymerase chain reaction (PCR) and heterologous cDNAs as probes. The deduced amino acid sequences of these four genes exhibited 40-90% identity with those reported for the corresponding gene from other unrelated species. Southern blot analysis for these genes and two other structural genes, the leucoanthocyanidin dioxygenase (LDOX, anthocyanidin synthase) and anthocyanin acyltransferase (AAT) genes, indicated that each gene comprises a small multi-gene family. More than three copies of the CHS gene are present, two copies of the other genes being present. The expression of five genes, the exception being the CHS gene, was detected only in red leaves of the red forma of P. frutescens, i.e. not in green leaves of the green forma plant. The CHS gene was expressed in both red and green leaves, but 10-fold more in red leaves than in green leaves. These results suggest that the expression of all structural genes examined is coordinately regulated in a forma-specific manner. Under weak-light conditions, the accumulation of both anthocyanin and mRNAs of biosynthetic enzymes was lower in leaves of the red forma. High-intensity white light coordinately induced the accumulation of transcripts of all six genes examined in the mature leaves of red P. frutescens.
Plant Mol Biol 1997 Dec
PMID:Cloning and molecular analysis of structural genes involved in anthocyanin biosynthesis and expressed in a forma-specific manner in Perilla frutescens. 942 10

Protoplasts isolated from the primary leaves of Phaseolus vulgaris L. were used in transient expression experiments to identify promoter sequences of the P. vulgaris rbcS2 gene, encoding ribulose 1,5-bisphosphate carboxylase/oxygenase small subunit, concerned with sucrose repression. The protoplasts supported high rates of expression of the chloramphenicol acetyl transferase reporter gene fused to 1433 bp of the rbcS2 5' flanking sequences. Expression was repressed by 50 mM sucrose whereas that driven by control promoters was not. Assays of promoter deletions revealed that 203 bp 5' to the transcription start site were sufficient for high rates of sucrose-repressible expression. A -187 bp deletion supported much lower rates of expression and was not subject to sucrose repression. The -203 to -187 bp region contains sequences resembling elements involved in the sugar stimulation of transcription of other genes: the SURE (sucrose response element) of plant genes and the ChoRE (carbohydrate response element) of mammalian genes. A G-box (CACGTG) located at -200 to -205 was important for high levels of sucrose-repressible expression, since deletion of a nucleotide from this element in the context of the 1433 bp promoter gave much reduced expression. However, a modified G-box (CcCGTG) in the -203 bp fusion and adjacent vector sequences remained functional. Measurements of rbcS and chalcone synthase (CHS) transcript levels in the protoplasts indicated that 4 mM sucrose was sufficient to repress or stimulate the respective genes. Further experiments suggested that metabolism of 6-carbon sugars is the signal for rbcS repression and CHS stimulation.
Plant Mol Biol 1997 Dec
PMID:A sucrose repression element in the Phaseolus vulgaris rbcS2 gene promoter resembles elements responsible for sugar stimulation of plant and mammalian genes. 942 11

Anthocyanin production in higher plants is a function of the tissue considered and its developmental stage, and is modulated by environmental factors. In maize, the best characterized system, regulation of the pathway is achieved largely through the action of proteins with homology to the transcriptional factors encoded by myc and myb proto-oncogenes of animals; these homologues control the expression of structural genes and thus regulate the availability of anthocyanin biosynthetic enzymes. We have studied anthocyanin biosynthesis and its regulation in flowers of pea (Pisum sativum). Our results demonstrate a correlation between anthocyanin accumulation and steady-state mRNA levels for genes encoding chalcone synthase, flavanone 3 beta-hydroxylase, and dihydroflavonol 4-reductase in developing flowers. Patterns of expression for these biosynthetic genes in both a and a2 mutants confirm the regulatory roles of the two a loci. The reduced expression of all three biosynthetic genes in mutant lines suggests that genes acting both early and late in the anthocyanin biosynthetic pathway are controlled by a and a2. Particle bombardment of floral tissue demonstrates the ability of two maize R-like genes, Lc and R-S, but neither myb-like genes nor R-like genes from snapdragon or petunia, functionally to complement a and a2 mutations. We cannot distinguish whether a and a2 act coordinately or sequentially in anthocyanin regulation, but the epistatic action of maize R-like genes suggests that they mimic the action of a gene that normally functions downstream of both a and a2 in the regulatory cascade.
Mol Gen Genet 1998 Jan
PMID:Anthocyanin regulatory mutations in pea: effects on gene expression and complementation by R-like genes of maize. 949 Oct 78

To identify DNA sequences of the Arabidopsis thaliana chalcone synthase gene (CHS) concerned with induction by UV-B and UV-A/blue light, AtCHS promoter constructions were assayed by transient expression in protoplasts prepared from two different lines of cultured A. thaliana cells. The protoplasts responded similarly to A. thaliana leaf tissue in light-dependent CHS transcript accumulation. The reporter enzyme beta-glucuronidase (GUS) was used to monitor light-responsive promoter activity. A 1972 bp promoter conferred UV-B and UV-A/blue light induction of GUS activity. Deletion to 164 bp resulted in reduced promoter strength but retention of responsiveness to UV-B and UV-A/blue light. Further deletion abolished transcriptional activity. The 164 bp promoter contains sequences closely resembling LRUPcCHS, (light-responsive unit of the Petroselinum crispum CHS promoter). This A. thaliana CHS promoter region, designated LRUAtCHS, was sufficient to confer UV-B and UV-A/blue light responsiveness to a heterologous core promoter. Mutation of sequences in LRUAtCHS corresponding to the ACGT element and the MYB recognition element of LRUPcCHS resulted in inactivation of the 164 bp and 335 bp promoter deletions. However, the mutant 668 bp promoter retained residual UV-B and UV-A/blue light-induced expression, indicating the presence of additional functional sequences upstream of -335. Mutation of a single G-box-like sequence around -442 had no effect on light responsiveness, indicating that it does not function in light regulation of this promoter. Since no difference in responsiveness to UV-B and UV-A/blue light was observed with any promoter variant, we conclude that the two phototransduction pathways regulate transcription factors which interact with common promoter elements. The results from-our analysis of a A. thaliana light-responsive promoter will facilitate the study of light-dependent gene regulation by genetic means in Arabidopsis thaliana.
Plant Mol Biol 1998 Mar
PMID:Identification of UV/blue light-response elements in the Arabidopsis thaliana chalcone synthase promoter using a homologous protoplast transient expression system. 952 7


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