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Query: UNIPROT:P06889 (Mol)
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The sequence of the S-adenosyl-L-methionine:trans-caffeoyl-CoA O-methyltransferase (CCoAOMT, EC2.1.1.104) gene, including the 5'-flanking region of 5 kb, was determined from parsley (Petroselinum crispum) plants. The enzyme appears to be encoded by one or two genes, and the ORF is arranged in five exons spaced by introns from 107 to 263 bp in length. The genomic sequence matches the ORF of the cDNA previously reported from elicited parsley cell cultures, showing only three base changes that do not affect the enzyme polypeptide sequence. S1 nuclease protection assays and primer extension analyses with genomic and cDNA templates revealed the transcription start site 67 bp upstream of the translation start codon, indicating a shorter 5'-UTR than reported previously for the transcript. Promoter regulatory consensus elements such as two 'CAAT' boxes and one 'TATA' box were identified at -196, -127 and -31, respectively, relative to the transcription start site, and an SV 40-like enhancer element is located 347 bp upstream. Most notably, three putative cis-regulatory elements were recognized by sequence alignments, which represent motifs recurring in the promoters of several genes of the stress-inducible phenylpropanoid pathway (boxes P, A and L). Transient expression assays with a set of 5'-truncated promoter-GUS fusions show that significant promoter activity is retained in a 354 bp promoter fragment. In vitro DNase 1 footprint experiments and electrophoretic mobilty shift assays (EMSA) identified in this fragment a unique sequence motif with elicitor-inducible trans-factor binding activity, which was unrelated to boxes P, A, or L. This novel cis-regulatory element, designated box E, appears to be conserved in the TATA-proximal regions of other stress-inducible phenylpropanoid genes, and in vitro binding of nuclear protein was confirmed in EMSA assays for such an element from the PAL-1 promoter (-54 to -45). Moreover, the deletion of box E reduced the activity and erased the elicitor-responsiveness of the CCoAOMT promoter in transient expression assays. The results corroborate the proposed physiological function of CCoAOMT in elicited plant cells and may shed new light on the sequential action of trans-active factors in the regulation of phenylpropanoid genes.
Plant Mol Biol 1997 Jan
PMID:Structure of the parsley caffeoyl-CoA O-methyltransferase gene, harbouring a novel elicitor responsive cis-acting element. 903 50

We have identified a new En/Spm-like transposable element, Tdc1, in the 5' flanking region of a phenylalanine ammonia-lyase gene (gDcPAL1) that is normally induced by transferring cells of carrot suspension cultures to fresh liquid medium (transfer or dilution effect). The initial integration into gDcPAL1 occurred more than 4 years after culture initiation. Tdc1 was first detected in gDcPAL1 genomic clones of a genomic library made from cells of the same cultured cell line 7 years after its initiation and thus following repeated subculturing. Twelve years after initiation, about 5-10% of the cells had Tdc1 inserted into the gDcPAL1 gene, indicating that Tdc1 insertion into gDcPAL1 occurred in one (or more) cell(s) during the first 4-7 years of subculturing. These mutant cells did not disappear during numerous passages; instead the proportion of cells having this Tdc1 inserted into gDcPAL1 has been increasing over the last 5 years. The promoter activity and the inducibility by transfer/dilution of the gDcPAL1 gene harboring Tdc1 is reduced relative to wild type. Finally, we show that insertion of a transposable element is one of the mechanisms that can cause variation of plant cell cultures during repeated subculture.
Mol Gen Genet 1997 Apr 28
PMID:Somatic variation during long-term subculturing of plant cells caused by insertion of a transposable element in a phenylalanine ammonia-lyase (PAL) gene. 918 Jun 94

Depending on the pH of the growth medium, the yeast Yarrowia lipolytica secretes both an acidic proteinase and an alkaline proteinase, the synthesis of which is also controlled by carbon, nitrogen, and sulfur availability, as well as by the presence of extracellular proteins. Recessive mutations at four unlinked loci, named PAL1 to PAL4, were isolated which prevent alkaline proteinase derepression under conditions of carbon and nitrogen limitation at pH 6.8. These mutations markedly affect mating and sporulation. A dominant suppressor of all four PAL mutations was isolated from a wild-type genomic library, which turned out to be a C-terminally truncated form of a 585-residue transcriptional factor of the His2Cys2 zinc finger family, which we propose to call YlRim101p. Another C-terminally truncated version of YlRim101p (419 residues) is encoded by the dominant RPH2 mutation previously isolated as expressing alkaline protease independently of the pH. YlRim101p is homologous to the transcriptional activators Rim101p of Saccharomyces cerevisiae, required for entry into meiosis, and PacC of Aspergillus nidulans and Penicillium chrysogenum, which were recently shown to mediate regulation by ambient pH. YlRim101p appears essential for mating and sporulation and for alkaline proteinase derepression. YlRIM101 expression is autoregulated, maximal at alkaline pH, and strongly impaired by PAL mutations.
Mol Cell Biol 1997 Jul
PMID:Genetic analysis of regulatory mutants affecting synthesis of extracellular proteinases in the yeast Yarrowia lipolytica: identification of a RIM101/pacC homolog. 919 31

Genomic methylation patterns of mammals can vary among individuals and are subject to dynamic changes during development. In order to gain a better understanding of this variation, we have analyzed patterns of cytosine methylation within a 200 bp region at the CpG island of the human FMR1 gene from leukocyte DNA. FMR1 is normally methylated during inactivation of the X chromosome in females and it is also methylated and inactivated upon expansion of CGG repeats in fragile-X syndrome. Patterns of methylation (epigenotypes) were determined by the sequencing of bisulfite-treated alleles from normal males and females and alleles from a family of five brothers who are methylation mosaics and are affected to various degrees by the fragile-X syndrome. Our data indicate that: (i) methylation of individual CpG cytosines is strikingly variable in hypermethylated epigenotypes obtained from a single individual, suggesting that maintenance of cytosine methylation is a dynamic process; (ii) methylation of non-CpG cytosines in the region studied may occur but is rare; (iii) mosaicism of methylation in the analyzed fragile-X males is remarkably similar to that found for the active X and inactive X alleles in normal females, suggesting that the methylation mosaicism of some fragile-X males reflects similar on and off states of FMR1 expression that exist in normal females; (iv) hypermethylation is slightly more pronounced on fragile-X alleles than on normal inactive X alleles of females; (v) the general dichotomy of hypo- and hypermethylated alleles persisted over the 5 year period that separated samplings of the fragile-X males; (vi) methylation variability was most pronounced at a consensus binding sequence for the alpha-PAL transcription factor, a sequence that may play a role in regulating expression of FMR1.
Hum Mol Genet 1997 Oct
PMID:Epigenetic variation illustrated by DNA methylation patterns of the fragile-X gene FMR1. 930 55

Fragile X mental retardation syndrome is associated with an expansion of a CGG repeat within the 5'UTR of the first exon of the FMR1 gene, abnormal methylation of the CpG island in the promoter region, and a transcriptional silencing of this gene. We studied transcriptional regulation of the FMR1 gene using protein footprint analysis of the active and inactive gene in vivo . We identified four footprints within the FMR1 promoter region which correspond to consensus binding sites of known transcription factors, alpha-PAL/NRF1, Sp1, H4TF1/Sp1-like and c-myc. These footprints were present in normal cells with a transcriptionally active FMR1 gene. The same footprints were present in different cell types: primary fibroblasts, lymphoblastoid cells and peripheral lymphocytes. However, for the 1.1 kb region analyzed, no footprints were detected in a variety of cell types derived from patients with fragile X syndrome which have a transcriptionally inactive FMR1 gene. A BLAST nucleotide search identified sequence similarities between the region of the FMR1 gene containing the footprints and an analogous region within the promoter region of the gene for the heterogeneous nuclear ribonucleoprotein (hnRNP) A2, a member of a family of ribonucleoproteins implicated in mRNA processing and nuclear-cytoplasm transport. The nucleotide sequences identified in the hnRNP-A2 promoter region correspond to the same consensus binding sites showing DNA-protein interactions in the FMR1 gene. Our previous functional studies and the studies of others demonstrate that FMR proteins, like hnRNP-A2, are also ribonucleoproteins which appear to be involved in mRNA transport. The results from our footprint studies suggest that the expression of the FMR1 gene is regulated by the binding of specific transcription factors to sequence elements in the 5' region of the gene and that this expression may be regulated by elements in common with the hnRNP-A2 gene. Common regulation of these two genes might play an important role in the cooperative processing and transport of mRNA from the nucleus to the translation machinery.
Hum Mol Genet 1997 Nov
PMID:Structural and functional characterization of the human FMR1 promoter reveals similarities with the hnRNP-A2 promoter region. 932 68

A study of the expression of a bean phenylalanine ammonia-lyase (PAL) promoter/beta-glucuronidase gene fusion in transgenic tobacco has shown that the PAL2 promoter has a modular organization. Expression of the PAL2 promoter in the vascular system involves positive and negative regulatory cis elements. Among these elements is an AC-rich motif implicated in xylem expression and a suppressing cis element for phloem expression. Using radiolabelled complementary oligonucleotides bearing the AC-rich motif, a cDNA clone encoding a DNA-binding protein has been isolated from a tobacco lambda gt11 expression library. This factor, named AC-rich binding factor (ACBF), showed binding specificity to the AC-rich region. The specificity of ACBF for the AC-rich region was also shown using a gel retardation assay with an ACBF recombinant protein extract. The deduced amino acid sequence from ACBF contains a long repeat of glutamine residues as found in well characterized transcription factors. Interestingly, ACBF shared sequence similarity to conserved amino acid motifs found in RNA-binding proteins. Genomic gel blot analysis indicated the presence of a small gene family of sequences related to ACBF within the tobacco nuclear genome. Analysis of tobacco mRNA using the ACBF cDNA as probe showed that while ACBF mRNA was present in all tissues examined, the highest transcript accumulation occurred in stem tissues. The functional characteristics of the AC-rich sequence were examined in transgenic tobacco. A heptamer of the AC-rich sequence, in front of a minimal 35S promoter from cauliflower mosaic virus (-46 to +4), conferred specific expression in xylem.
Plant Mol Biol 1997 Oct
PMID:Characterization of a gene encoding a DNA-binding protein that interacts in vitro with vascular specific cis elements of the phenylalanine ammonia-lyase promoter. 934 52

Phenylalanine ammonia-lyase (PAL, EC 4.3.1.5), the first enzyme in phenylpropanoid biosynthesis, catalyzes the elimination of ammonium ion from L-phenylalanine. In the present study, PAL was purified through ammonium sulfate fractionation, DEAE-cellulose chromatography, Sephadex G-200 chromatography, and Q-Sepharose chromatography from the cytosolic fraction of leaf mustard (Brassica juncea var. integrifolia). It consists of 4 subunits, each having an estimated molecular weight of about 40,000 on SDS-polyacrylamide gel electrophoresis (SDS-PAGE). The optimal pH and temperature of the purified enzyme are 9.0 and 45 degrees C, respectively. Its activity is inhibited by Zn2+ ion, and it is strongly activated by caffeic acid. The purified PAL seems to have some characteristics different from those obtained with other PALs.
Mol Cells 1997 Dec 31
PMID:Purification and properties of phenylalanine ammonia-lyase from leaf mustard. 950 10

To determine the regulatory mechanism of gene expression in the early stages of tracheary element (TE) differentiation, we isolated and characterized a genomic fragment of TED3 whose mRNA is expressed preferentially in differentiating TEs 12-24 h before morphological changes in the in vitro Zinnia system. Transgenic Arabidopsis plants with a chimeric gene of the 537 bp TED3 promoter and the beta-glucuronidase (GUS) reporter gene indicated the strong expression of the GUS gene by the TED3 promoter in TEs, in particular in immature TEs as well as stipules and trichomes. GUS expression driven by the promoter was also induced in callus, in which GUS activity was localized in immature TEs and slender cells around TEs that may be TE precursor cells. The TED3 promoter was not significantly activated by wounding. This pattern of expression differed clearly from that of other vascular tissue-related genes such as PAL, 4CL, and GRP1.8. The nature of TED3 promoter enables us to use it to monitor TE differentiation in tissue and to introduce foreign genes preferentially into immature TE.
Plant Mol Biol 1998 Apr
PMID:Expression of the Zinnia TED3 promoter in developing tracheary elements of transgenic Arabidopsis. 952 Feb 82

Throughout the plant kingdom expression of the flavonoid biosynthetic pathway is precisely regulated in response to developmental signals, nutrient status, and environmental stimuli such as light, heat and pathogen attack. Previously we showed that, in developing Arabidopsis seedlings, flavonoid genes are transiently expressed during germination in a light-dependent manner, with maximal mRNA levels occurring in 3-day-old seedlings. Here we describe the relationship between developmental and environmental regulation of flavonoid biosynthesis by examining phenylalanine ammonia-lyase (PAL), chalcone synthase (CHS), chalcone isomerase (CHI), and dihydroflavonol reductase (DFR) mRNA levels in germinating Arabidopsis seedlings as a function of light, developmental stage and temperature. We show that seedlings exhibit a transient potential for induction of these four genes, which is distinct from that observed for chlorophyll a/b-binding protein(CAB). The potential for flavonoid gene induction was similar in seedlings grown in darkness and red light, indicating that induction potential is not linked to cotyledon expansion or the development of photosynthetic capacity. The evidence for metabolic regulation of flavonoid genes during seedling development is discussed.
Plant Mol Biol 1998 May
PMID:A light-independent developmental mechanism potentiates flavonoid gene expression in Arabidopsis seedlings. 961 95

A mutant strain of the cyanobacterium Synechocystis PCC 6803, called PAL, (PC-, delta apcAB, delta apcE), lacking phycocyanin, allophycocyanin and the core-membrane linker (Lcm), was constructed. The strain was characterized by absorption and fluorescence spectroscopy. The mutant compensates for the absence of the major PS II antenna by increasing its PS II/PS I ratio. It is stable and grows well albeit more slowly than wild type.
Plant Mol Biol 1998 Jun
PMID:Construction and characterization of a phycobiliprotein-less mutant of Synechocystis sp. PCC 6803. 961 24


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