Gene fusions to lacZ reveal new expression patterns of chimeric genes in transgenic plants.

Teeri, Teemu H.; Lehväslaiho, Heikki; Franck, M.; Uotila, Jussi; Heino, Pekka; Palva, E. Tapio; Montagu, Marc Van; Herrera‐Estrella, Luis

doi:10.1002/j.1460-2075.1989.tb03383.x

Cited by 117 publications

(69 citation statements)

References 40 publications

(29 reference statements)

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“…The TR2' promoter is stimulated by wounding and exhibits a marked tissue specificity (34,46). The NPTII level in plants transformed with the dicistronic constructs is expected to follow the same pattern.…”

Section: Resultsmentioning

confidence: 98%

Expression of dicistronic transcriptional units in transgenic tobacco.

Angenon¹,

Uotila²,

Kurkela³

et al. 1989

Mol. Cell. Biol.

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We investigated whether the two cistrons of a dicistronic mRNA can be translated in plants to yield both gene products. The coding sequences of various reporter genes were combined in dicistronic units, and their expression was analyzed in stably transformed tobacco plants at the RNA and protein levels. The presence of an upstream cistron resulted in all cases in a drastically reduced expression of the downstream cistron. The translational efficiency of the gene located downstream in the dicistronic units was 500-to 1,500-fold lower than that in a monocistronic control; a 500-fold lower value was obtained with a dicistronic unit in which both cistrons were separated by 30 nucleotides, whereas a 1,500-fold lower value was obtained with a dicistronic unit in which the stop codon of the upstream cistron and the start codon of the downstream cistron overlapped. As a strategy to select indirectly for transformants with enhanced levels of expression of a gene which is by itself nonselectable, the gene of interest can be cloned upstream from a selectable marker in a dicistronic configuration. This strategy can be used provided that the amount of dicistronic mRNA is high. If, on the other hand, the expression of the dicistronic unit is too low, selection of the downstream cistron will primarily give clones with rearranged dicistronic units.The most consistent model to describe initiation of translation in eucaryotes is the modified scanning model (27), which states that 40S ribosomal subunits bind at or near the 5' cap structure of the mRNA and migrate downstream along the mRNA until the first AUG codon is reached. If this AUG codon occurs in a favorable context for initiation, the 40S ribosomal subunits are halted, the 60S subunits associate with them, and translation is started; if the context around the first AUG codon is less favorable, some or most of the 40S subunits proceed to the next AUG. The favorable contexts for initiation, as established by nucleotide sequence analysis and site-directed mutagenesis, are AACAAIUGGC for plant mRNAs (23,25,36,45) and (GCC)GCC'CCAUG for vertebrate mRNAs (29-31). The scanning model is in accordance with the finding that nucleus-encoded genes in eucaryotes are normally organized in monocistronic transcriptional units.The concept of reinitiation has been included in the scanning model to account for the observation that an upstream AUG codon, even in an optimal context, still allows initiation of translation at a downstream AUG codon provided that a termination codon in-frame with the upstream AUG codon precedes the downstream AUG codon. Indeed, in several reports it has been demonstrated that in mammalian cells a "minicistron" inserted upstream from the main cistron does not abolish translation of the main cistron (22,28,35). After translation of the minicistron, reinitiation of translation seems to occur at the start codon of the main cistron on the same mRNA. Similar processes have been described in barley protoplasts (16). Kozak (32) showed that the efficiency of reinitiation...

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Section: Resultsmentioning

confidence: 98%

Expression of dicistronic transcriptional units in transgenic tobacco.

Angenon¹,

Uotila²,

Kurkela³

et al. 1989

Mol. Cell. Biol.

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“…Leaves from the bombarded plants were vacuum-infiltrated with 1% glutaraldehyde in ZЈ buffer at room temperature for 1 h to inhibit endogenous ␤-galactosidase activity (18,19). The leaves were rinsed three times in ZЈ buffer and then vacuum-infiltrated with staining solution as described previously (19) with 0.1% 5-bromo-4-chloro-3-indolyl ␤-D-galactopyranoside (X-gal) at room temperature for 1 h followed by incubation at 37°C for 14 h. Finally, the leaves were treated with ZЈ buffer and incubated with 70 and 100% ethanol to remove chlorophyll.…”

Section: Methodsmentioning

confidence: 99%

Mutation of the Matrix Metalloproteinase At2-MMP Inhibits Growth and Causes Late Flowering and Early Senescence in Arabidopsis

Golldack

Popova

Dietz

2002

Journal of Biological Chemistry

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This study characterizes the expression and functional significance of the member of the matrix metalloproteinase (MMP) family At2-MMP from Arabidopsis. By transcript analysis, expression of At2-MMP was found in leaves and roots of juvenile Arabidopsis and leaves, roots, and inflorescences of mature flowering plants showing strong increase of transcript abundance with aging. Cell specificity of expression of At2-MMP was studied by in situ hybridizations in leaves and flowers of Arabidopsis. In leaves, the gene was expressed in the phloem, in developing xylem elements, epidermal cells, and neighboring mesophyll cell layers. In flowers, signals were localized in pistils, ovules, and receptacles. In an Arabidopsis mutant (at2-mmp-1) carrying a tDNA insertion in At2-MMP, neither germination nor development of plants was modified in comparison to the wild type in the juvenile rosette stage. Starting with the onset of shoots, growth of roots, leaves, and shoots was inhibited compared with the wild type, and the plants were characterized by late flowering. Besides the flowering, at2-mmp-1 plants showed fast degradation of chlorophyll in leaves and early senescence. These results demonstrate the involvement of At2-MMP in plant growth, morphogenesis, and development with particular relevance for flowering and senescence.The family of zinc-dependent endopeptidases that has been particularly characterized in vertebrates is divided into four subfamilies based on structural and functional characteristics: matrix metalloproteinases (matrixins), adamalysins, serralysins, and astacins. All members of these zinc metalloproteinases have a similar structure with the conserved consensus sequence HEXXHXXGXXH in their catalytic site and a conserved methionine residue that forms the "Met-turn" structure (1). Members of the subfamily of matrix metalloproteinases are gelatinases, collagenases, and stromelysins (2). Matrix metalloproteinases are synthesized as prepro-enzymes with a signaling peptide targeting the enzyme to the extracellular space. The pre-domain is followed by a pro-peptide with a conserved PRCG(V/N)PD motif that contains the so-called "cysteine switch." This Cys residue ligates the catalytic zinc thus maintaining the latency of the inactive pro-enzymes (3). In vitro, pro-matrix metalloproteinases can be activated proteolytically by proteinases as well as by mercurial compounds and reactive oxygen, for example, whereas in vivo activation by proteinases is most likely (3). The function of matrix metalloproteinases is the degradation and remodeling of the extracellular matrix (2). The enzymes play a role in development, embryogenesis, and organ morphogenesis but also in wound healing in vertebrates (4). They also participate in pathological processes such as cancer and arthritis (5). In vertebrates, the enzyme activity of matrix metalloproteinases is transcriptionally regulated as well by proteolytic activation of the mature enzyme from the inactive pro-enzyme and by co-secretion with endogenous inhibitors (4).In addition ...

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“…We expressed the LPVC marker RFP-Rha1 under the transcriptional control of the weak pNOS promoter (Teeri et al, 1989) to avoid merging of PVC and LPVC markers (Bottanelli et al, 2012). Upon coexpression with either GFP-VSR2D19 or GFP-VSR2D15, we tested potential colocalization with the LPVC ( Figure 8B).…”

Section: Evidence For Yxxɸ-independent Vsr Targeting To the Pvcmentioning

confidence: 99%

Golgi-Dependent Transport of Vacuolar Sorting Receptors Is Regulated by COPII, AP1, and AP4 Protein Complexes in Tobacco

et al. 2014

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The cycling of vacuolar sorting receptors (VSRs) between early and late secretory pathway compartments is regulated by signals in the cytosolic tail, but the exact pathway is controversial. Here, we show that receptor targeting in tobacco (Nicotiana tabacum) initially involves a canonical coat protein complex II-dependent endoplasmic reticulum-to-Golgi bulk flow route and that VSRligand interactions in the cis-Golgi play an important role in vacuolar sorting. We also show that a conserved Glu is required but not sufficient for rate-limiting YXXɸ-mediated receptor trafficking. Protein-protein interaction studies show that the VSR tail interacts with the m-subunits of plant or mammalian clathrin adaptor complex AP1 and plant AP4 but not that of plant and mammalian AP2. Mutants causing a detour of full-length receptors via the cell surface invariantly cause the secretion of VSR ligands. Therefore, we propose that cycling via the plasma membrane is unlikely to play a role in biosynthetic vacuolar sorting under normal physiological conditions and that the conserved Ile-Met motif is mainly used to recover mistargeted receptors. This occurs via a fundamentally different pathway from the prevacuolar compartment that does not mediate recycling. The role of clathrin and clathrin-independent pathways in vacuolar targeting is discussed.

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Gene fusions to lacZ reveal new expression patterns of chimeric genes in transgenic plants.

Cited by 117 publications

References 40 publications

Expression of dicistronic transcriptional units in transgenic tobacco.

Expression of dicistronic transcriptional units in transgenic tobacco.

Mutation of the Matrix Metalloproteinase At2-MMP Inhibits Growth and Causes Late Flowering and Early Senescence in Arabidopsis

Golgi-Dependent Transport of Vacuolar Sorting Receptors Is Regulated by COPII, AP1, and AP4 Protein Complexes in Tobacco

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