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At least 721 records · Page 40Linked to original sources
PubMed1996

Identification of an Arabidopsis thaliana ribulose-1,5-bisphosphate carboxylase/oxygenase activase (RCA) minimal promoter regulated by light and the circadian clock.

Transcription of the Arabidopsis thaliana gene encoding ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) activase (RCA) is organ-specific, light-responsive, and regulated by the circadian clock. RCA is transcribed throughout the green parts of the plant, but not in roots and petals. Responses elicited by short pulses of light indicate that the light response is mediated, at least in part, by phytochrome. Analysis of transgenic tobacco and Arabidopsis carrying RCA 5' untranscribed regions fused to reporter genes (uidA, encoding beta-glucuronidase, or cat, encoding chloramphenicol acetyltransferase) indicate that elements sufficient to confer organ-specific, light-responsive, and clock-regulated transcription are localized within 317 base pairs upstream of the site of transcription initiation. A clock-responsive element sufficient to confer a low-amplitude (approximately 2-fold) circadian oscillation lies within 317 base pairs of the trascription start, but other elements necessary for high-amplitude (approximately 10-fold) circadian oscillation lie upstream of -317 and are removed by deletion from -970 to -317.

Arabidopsis↗
PubMed1997

Sequencing, genomic organization, and preliminary promoter analysis of a black cherry (R)-(+)-mandelonitrile lyase gene.

The flavoprotein (R)-(+)-mandelonitrile lyase (MDL; EC 4.1.2.10) plays a key role in cyanogenesis in rosaceous stone fruits. An MDL gene (mdl3) and its corresponding cDNA (MDL3) were isolated from black cherry (Prunus serotina) and characterized. The mdl3 gene contains 2292 bp of the 5' flanking region, the entire coding region, and 300 bp of the 3' flanking region. The coding region is interrupted by three short introns, of which one possesses the usual GC-AG splice junction dinucleotides. This gene encodes a polypeptide of 573 amino acids that includes a putative signal sequence, 13 potential N-glycosylation sites, and a presumptive flavin adenine dinucleotide-binding site. To determine whether the 5' flanking region of the mdl3 gene is capable of driving MDL expression, it was fused to the beta-glucuronidase reporter gene for Agrobacterium-mediated transformation into tobacco. Matching endogenous MDL expression patterns, beta-glucuronidase staining was observed in maturing embryos and seeds; it also occurred in postembryonic tissues, especially in association with vascular tissues. After developing a homologous transient transformation system to facilitate identification of putative regulatory sequences, we demonstrated that 125 bp (-107 to +18) of the 5' flanking sequence of the mdl3 gene is sufficient for MDL expression in protoplasts derived from immature black cherry embryos.

Aldehyde-Lyases↗
PubMed1989

Constitutive expression of pathogenesis-related proteins PR-1, GRP, and PR-S in tobacco has no effect on virus infection.

Samsun NN tobacco cells were transformed with chimeric genes for pathogenesis-related (PR) proteins derived from genomic (PR-1a, GRP) or cDNA (PR-S) clones under the transcriptional control of the cauliflower mosaic virus 35S promoter. Regenerated plants were assayed by RNA and protein gel blotting, and plants showing high specific expression of the inserted genes were selected for self-pollination and seed formation. Inspection of second generation transformants showed that constitutive expression of PR-1a, GRP, and PR-S in tobacco in general does not have an effect on the phenotypic appearance of the plants or the expression of other endogenous PR genes. Furthermore, constitutive expression of the above genes does not affect the susceptibility of the plants to infection with tobacco mosaic virus or alfalfa mosaic virus.

Base Sequence↗
PubMed2000

Silencing of retrotransposons in arabidopsis and reactivation by the ddm1 mutation.

Gene silencing associated with repeated DNA sequences has been reported for many eukaryotes, including plants. However, its biological significance remains to be determined. One important function that has been proposed is the suppression of transposons. Here, we address transposon suppression by examining the behavior of the tobacco retrotransposon Tto1 and endogenous retrotransposons in Arabidopsis. After an initial increase in copy number because of active transposition in the Arabidopsis genome, Tto1 became silent. The amount of transcript was reduced, and the inactivated Tto1 became methylated. This silencing correlated with an increase in copy number. These phenomena mimic repeat-induced gene silencing. The homozygous ddm1 (for decrease in DNA methylation) mutation of Arabidopsis results in genomic DNA hypomethylation and the release of silencing in repeated genes. To investigate the role of DNA methylation and the gene-silencing machinery in the suppression of Tto1, we introduced the ddm1 mutation into an Arabidopsis line carrying inactivated Tto1 copies. In the homozygous ddm1 background, Tto1 became hypomethylated and transcriptionally and transpositionally active. In addition, one of the newly isolated endogenous Arabidopsis retrotransposon families, named Tar17, also became hypomethylated and transcriptionally active in the ddm1 mutant background. Our results suggest that the inactivation of retrotransposons and the silencing of repeated genes have mechanisms in common.

Arabidopsis↗
PubMed1991

Developmental analysis of elongation factor-1 alpha expression in transgenic tobacco.

The developmental regulation of the translational elongation factor EF-1 alpha has been analyzed in tobacco. A gene fusion was constructed consisting of the 5' and 3' regions of the tomato genomic clone LeEF-A from the EF-1 alpha gene family and the beta-glucuronidase coding region. Analysis of the transgenic plants containing this chimeric gene demonstrated that the tomato LeEF-A flanking sequences were sufficient to confer expression patterns similar to those of the endogenous tobacco EF-1 alpha gene. The patterns of beta-glucuronidase activity in this system indicated that during plant growth and development EF-1 alpha is regulated with increased expression corresponding to regions of high protein synthesis, including meristems, rapidly growing tissues, and developing gametophytes. In addition, EF-1 alpha expression responds rapidly to changes in growth patterns induced by hormone treatment. Our results are in agreement with studies in animals indicating that EF-1 alpha expression may be rate limiting for protein synthesis and demonstrate that the analysis of EF-1 alpha is of value for studying interrelationships between protein synthesis and developmental control.

2,4-Dichlorophenoxyacetic Acid↗
PubMed1994

Soybean GH3 promoter contains multiple auxin-inducible elements.

The soybean GH3 gene is transcriptionally induced in a wide variety of tissues and organs within minutes after auxin application. To determine the sequence elements that confer auxin inducibility to the GH3 promoter, we used gel mobility shift assays, methylation interference, deletion analysis, linker scanning, site-directed mutagenesis, and gain-of-function analysis with a minimal cauliflower mosaic virus 35S promoter. We identified at least three sequence elements within the GH3 promoter that are auxin inducible and can function independently of one another. Two of these elements are found in a 76-bp fragment, and these consist of two independent 25- and 32-bp auxin-inducible elements. Both of these 25- and 32-bp auxin-inducible elements contain the sequence TGTCTC just upstream of an AATAAG. An additional auxin-inducible element was found upstream of the 76-bp auxin-inducible fragment; this can function independently of the 76-bp fragment. Two TGA-box or Hex-like elements (TGACGTAA and TGACGTGGC) in the promoter, which are strong binding sites for proteins in plant nuclear extracts, may also elevate the level of auxin inducibility of the GH3 promoter. The multiple auxin-inducible elements within the GH3 promoter contribute incrementally to the overall level of auxin induction observed with this promoter.

Agrobacterium tumefaciens↗
PubMed1995

Impaired photoassimilate partitioning caused by phloem-specific removal of pyrophosphate can be complemented by a phloem-specific cytosolic yeast-derived invertase in transgenic plants.

Constitutive expression of the Escherichia coli ppa gene encoding inorganic pyrophosphatase resulted in sugar accumulation in source leaves and stunted growth of transgenic tobacco plants. The reason for this phenotype was hypothesized to be reduced sucrose utilization and loading into the phloem. To study the role of PPi in phloem cells, a chimeric gene was constructed using the phloem-specific rolC promoter of Agrobacterium rhizogenes to drive the expression of the ppa gene. Removal of cytosolic PPi in those cells resulted in photoassimilate accumulation in source leaves, chlorophyll loss, and reduced plant growth. From these data, it was postulated that sucrose hydrolysis via sucrose synthase is essential for assimilate partitioning. To bypass the PPi-dependent sucrose synthase step, transgenic plants were produced that express various levels of the yeast suc2 gene, which encodes cytosolic invertase, in their phloem cells. To combine the phloem-specific expression of the ppa gene and the suc2 gene, crosses between invertase- and pyrophosphatase-containing transgenic plants were performed. Analysis of their offspring revealed that invertase can complement the phenotypic effects caused by the removal of PPi in phloem cells.

Base Sequence↗
PubMed1997

The dominant developmental mutants of tomato, Mouse-ear and Curl, are associated with distinct modes of abnormal transcriptional regulation of a Knotted gene.

The Curl (Cu) and Mouse-ear (Me) mutations of tomato cause two seemingly unrelated developmental syndromes with a wide range of pleiotropic phenotypes. Yet, the distinct morphogenic alterations in shoots, leaves, and inflorescences conferred by the two mutations appear to be caused by unchecked meristematic activity that characterizes dominant mutations in Knotted1 (Kn1)-like genes of monocot plants. We have been unable to separate the two closely linked Cu and Me mutations, and they may lie in the same gene. A homeobox-containing class I Kn1-like gene, TKn2, also maps to the same location. Significantly, the dominant mutations are associated with two aberrant modes of TKn2 transcription. Overexpression of the two in-frame wild-type transcripts of TKn2 is associated with the Cu mutation, whereas misexpression of an abundant and oversized fusion mRNA is associated with the Me mutation. Available molecular evidence strongly suggests that the defective Me-TKn2 transcript is generated via a novel splicing event that merges transcripts of two closely linked genes. The translated fusion product is comprised of most of the 5' end of the adjacent PPi-dependent fructose 6-phosphate phosphotransferase (PFP) transcript spliced in-frame to coding position 64 of the TKn2 transcript, leaving the TKn2 homeobox intact. We suggest that class I Kn1-like genes were selected early during evolution to regulate basic programs of aerial meristems and that subtle alterations in their function may be the basis for the wide diversity in growth parameters of shoot systems, leaves, and inflorescences among plant species.

Amino Acid Sequence↗
PubMed1995

Structure and expression analysis of the gdcsPA and gdcsPB genes encoding two P-isoproteins of the glycine-cleavage system from Flaveria pringlei.

In Flaveria pringlei, a C3 plant, P protein of the glycine-cleavage system is encoded by a small gene family consisting of at least five transcriptionally active genes. We have cloned and sequenced two of these genes, gdcsPA and gdcsPB, and provide the first detailed report on the complete structure of eukaryotic gdcsP genes. Based on the lengths of exons and intervening sequences, the P-protein genes can be subdivided into two parts. In both cases the N-terminal region consists of one very long exon followed by a long intron. In contrast, the C-terminal parts show a complex mosaic structure of relatively small exons and introns. A highly conserved leucine-zipper motif was identified, which is supposed to participate in the assembly of the glycine decarboxylase multienzyme complex. The transcript derived from the gdcsPA sequence corresponds perfectly to a leaf cDNA isolated earlier. Reverse-transcriptase PCR experiments show that both genes are preferentially active in leaves. Stems contain distinctly less P protein mRNA and the relative level in roots is very low but still clearly detectable. In all three organs, but most significantly in roots, the gdcsPA transcript level is distinctly higher than that of gdcsPB. Analysis of promoter-beta-glucuronidase fusions in transgenic tobacco suggests that far-upstream elements enhance the transcriptional activity of both genes in leaves relative to stems. The analysis of distal gdcsPA promoter deletions reveals the presence of regulatory elements acting with a distinct organ preference and indicates their approximate location.

Amino Acid Oxidoreductases↗
PubMed1994

Recombination between viral RNA and transgenic plant transcripts.

Transformed plants expressing the 3' two-thirds of the cowpea chlorotic mottle virus (CCMV) capsid gene were inoculated with a CCMV deletion mutant lacking the 3' one-third of the capsid gene. Although the deletion inoculum replicates in inoculated cells, systemic infections occur only if recombination restores a functional capsid gene. Four of 125 inoculated transgenic plants, representing three different transgenic lines, became systemically infected. Analysis of viral RNA confirmed that RNA recombination had united the transgenic messenger RNA and the challenging virus through aberrant homologous recombination.

Amino Acid Sequence↗
PubMed1997

A heterologous maize rpoB editing site is recognized by transgenic tobacco chloroplasts.

Single nucleotides in plant chloroplast transcripts are edited from the genomically encoded C to U, often resulting in changes of the encoded protein sequence. Site-specific trans-acting factors are postulated to direct the selection of edited residues. In order to further define cis sequences required for RNA editing, we investigated whether two editing sites present in maize rpoB mRNA would be recognized by the editing machinery of transformed tobacco chloroplasts. A 93-nucleotide (nt) segment surrounding site I is sufficient to direct editing of the maize sequence in tobacco chloroplasts. However, an 86-nt segment surrounding maize site IV (which is genomically encoded as a T in tobacco) does not confer editing of this site, suggesting that trans-acting factors necessary for recognition of site IV are not present in tobacco. The maize sequences surrounding site I were found to compete with the endogenous rpoB for a depletable trans factor and to reduce editing of endogenous site I. The presence of exogenous maize site I was also found to decrease editing of endogenous tobacco site II, indicating that there is a shared aspect of editing for some closely spaced editing sites.

Base Sequence↗
PubMed2001

The transposition pattern of the Ac element in tobacco cultured cells.

We investigated physical distances and directions of transposition of the maize transposable element Ac in tobacco cultured cells. We introduced a T-DNA construct that carried a non-autonomous derivative of Ac (designated dAc-I-RS) that included sites for cleavage by restriction endonuclease MluI. Another cleavage site was also introduced into the T-DNA region outside of the dAc-I-RS transposable element. The tobacco cultured cell line BY-2 was transformed with the T-DNA and several transformed lines that had a single copy of the T-DNA at a different chromosomal location were isolated. These lines were co-cultured with Agrobacterium tumefaciens cells that carried a cDNA for the Ac transposase gene under the control of various promoters. Sublines of cultured cells in which dAc-I-RS had been transposed, were isolated. The genomic DNAs of these sublines were isolated and digested with MluI. Sizes of DNA segments generated by digestion were determined by pulse-field gel electrophoresis. Our results showed that 20 to 70% of transposition events had occurred within several hundreds kilo-base pairs (kb) on the same chromosome. These results demonstrate that the Ac-Ds element preferentially transposed to regions near the original site in a tobacco chromosome. In addition, the present results are an example of asymmetric transposition as demonstrated by the distance of transposition on the chromosome.

Agrobacterium tumefaciens↗
PubMed2000

[Phenotypic changes in transgenic tobacco plants with an antisense form of the hmg1 gene].

Tobacco plants were genetically transformed with the Arabidopsis thaliana heterologous hmg1 gene encoding 3-hydroxy-3-methylglutaryl-CoA reductase, a key enzyme involved in the metabolism of terpenoid compounds. The hmg1 gene was inserted under the control of the 35S RNA double promoter from the cauliflower mosaic virus (CaMV 35S) both in direct and reverse orientation relative to the promoter. DNA analysis by polymerase chain reaction (PCR) and Southern blotting confirmed the transgenic nature of the tobacco plants obtained. DNA-RNA hybridization revealed expression of the hmg1 gene in these tobacco plants. The plants transformed with the antisense copy of the hmg1 gene differed from the control plants in delayed development and in flower color and shape.

Antisense Elements (Genetics)↗
PubMed2001

Targeting a nuclear anthranilate synthase alpha-subunit gene to the tobacco plastid genome results in enhanced tryptophan biosynthesis. Return of a gene to its pre-endosymbiotic origin.

Anthranilate synthase (AS), the control enzyme of the tryptophan (Trp) biosynthetic pathway, is encoded by nuclear genes, but is transported into the plastids. A tobacco (Nicotiana tabacum) cDNA (ASA2) encoding a feedback-insensitive tobacco AS alpha-subunit was transformed into two different sites of the tobacco plastid genome through site-specific insertion to obtain transplastomic plants with normal phenotype and fertility. A high and uniform level of ASA2 mRNA was observed in the transplastomic plants but not in the wild type. Although the plants with the transgene insertion at ndhF-trnL only expressed one size of the ASA2 mRNA, the plants with the transgene incorporated into the region between accD and open reading frame (ORF) 184 exhibited two species of mRNA, apparently due to readthrough. The transplastomic plants exhibited a higher level of AS alpha-subunit protein and AS enzyme activity that was less sensitive to Trp-feedback inhibition, leading to greatly increased free Trp levels in leaves and total Trp levels in seeds. Resistance to an AS inhibitor, 5-methyl-Trp, was found during seed germination and in suspension cultures of the transplastomic plants. The resistance to the selection agent spectinomycin and to 5-methyl-Trp was transmitted maternally. These results demonstrate the feasibility of modifying the biosynthetic pathways of important metabolites through transformation of the plastid genome by relocating a native gene from the nucleus to the plastid genome. Very high and uniform levels of gene expression can be observed in different lines, probably due to the identical insertion sites, in contrast to nuclear transformation where random insertions occur.

Anthranilate Synthase↗
PubMed2000

Plant research. Can genetically modified crops go 'greener'?

A flood of new information from projects such as the sequencing of the genome of the mustard plant Arabidopsis has pinpointed genes involved in key processes such as speeding up flowering, changing a plant's basic architecture, or improving pest resistance. One example appears on page 344 of this issue; researchers report the cloning of an Arabidopsis gene called FRIGIDA and show that natural mutations leading to loss of FRIGIDA function are associated with early flowering, a helpful adaptation in some cold climates. Such work could allow researchers to enhance the traits they want by introducing one or a few genes from another plant, or by modifying the regulation of genes in their original settings.

Arabidopsis↗
PubMed1996

Modification of fatty acid composition by over- and antisense-expression of a microsomal omega-3 fatty acid desaturase gene in transgenic tobacco.

omega-3 fatty acid desaturases, which catalyse the conversion of linoleic acid (18:2) to linolenic acid (18:3) in lipids, are located in the microsomes and plastid membranes. Transgenic tobacco plants were produced that express the transcripts of a tobacco microsomal omega-3 fatty acid desaturase gene (NtFad3) in antisense and sense orientations under the control of the cauliflower mosaic virus 35S promoter. The antisense construct has the 0.5-kb fragment of the NtFad3 cDNA containing a 3'-flanking region and a part of the coding region in antisense orientation. The antisense-transformant lines showed decreases of the steady-state NtFad3 mRNA level to 30% of the control plants. In these lines, the 18:3 content decreased to about 80% in root tissues and to about 70-80% in leaf tissues when compared with the control plants. The sense construct has the 1.4-kb full-length cDNA of NtFad3. In one of the sense-transformant lines, the NtFad3 mRNA level increased 8 times when compared with that of the control plants. In this line, the 18:3 content increased by about 1.5-fold in root tissues and by about 1.1-fold in leaf tissues. These results indicate that the up- and down-regulation of the transcript level in the microsomal omega-3 fatty acid desaturase gene is useful to modify the 18:3 content in the vegetative tissues of higher plants.

Antisense Elements (Genetics)↗
PubMed1992

Control of gene expression in tobacco cells using a bacterial operator-repressor system.

We have investigated the efficacy of using the Escherichia coli lac operator-repressor system to control plant gene expression. The lacI gene was modified to allow optimal expression in plant cells and then placed downstream of the cauliflower mosaic virus (CaMV) 35S RNA promoter. This construct was introduced into tobacco plants by leaf disc transformation. Transgenic tobacco plants synthesized significant quantities of LacI protein (up to 0.06% of total soluble protein). We have used the E.coli beta-glucuronidase gene (gus) as the reporter gene by placing it downstream of the maize chlorophyll a/b binding protein (CAB) gene promoter. Lac operators were introduced into several positions within the CAB promoter and operator-free plasmid was used as control. Repression was assessed by comparing the transient expression from CAB-operator-gus reporter constructs in protoplasts expressing lac protein, with that in control cells not expressing the repressor. Repression varied between 10 and 90% with different operator positions. Transient assays were also performed in the presence of the inducer, isopropyl-beta-D-thiogalactoside (IPTG). In lacI protoplasts the presence of IPTG manifested itself in a 4.2-fold relief of repression. The study was extended to show regulation of expression in stable transformants. Tobacco transformants harbouring a CAB-operator-gus reporter construct and the lacI gene were shown to have repressed GUS levels, but in the presence of IPTG, repression was relieved 15-fold. We conclude that the lac repressor can enter the plant cell nucleus, find its cognate operator sequence in the chromatin to form a repressor--operator complex and effectively block transcription of a downstream gene.

Bacterial Proteins↗