Removal of a cryptic intron and subcellular localization of green fluorescent protein are required to mark transgenic Arabidopsis plants brightly
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The green fluorescent protein (GFP) from the jellyfish Aequorea victoria is finding wide use as a genetic marker that can be directly visualized in the living cells of many heterologous organisms as discussed by the authors.Abstract:
The green fluorescent protein (GFP) from the jellyfish Aequorea victoria is finding wide use as a genetic marker that can be directly visualized in the living cells of many heterologous organisms. We have sought to express GFP in the model plant Arabidopsis thaliana, but have found that proper expression of GFP is curtailed due to aberrant mRNA processing. An 84-nt cryptic intron is efficiently recognized and excised from transcripts of the GFP coding sequence. The cryptic intron contains sequences similar to those required for recognition of normal plant introns. We have modified the codon usage of the gfp gene to mutate the intron and to restore proper expression in Arabidopsis. GFP is mainly localized within the nucleoplasm and cytoplasm of transformed Arabidopsis cells and can give rise to high levels of fluorescence, but it proved difficult to efficiently regenerate transgenic plants from such highly fluorescent cells. However, when GFP is targeted to the endoplasmic reticulum, transformed cells regenerate routinely to give highly fluorescent plants. These modified forms of the gfp gene are useful for directly monitoring gene expression and protein localization and dynamics at high resolution, and as a simply scored genetic marker in living plants.read more
Citations
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Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana
Steven J. Clough,Andrew F. Bent +1 more
TL;DR: The modified method should facilitate high-throughput transformation of Arabidopsis for efforts such as T-DNA gene tagging, positional cloning, or attempts at targeted gene replacement.
Journal ArticleDOI
The green fluorescent protein
TL;DR: In just three years, the green fluorescent protein from the jellyfish Aequorea victoria has vaulted from obscurity to become one of the most widely studied and exploited proteins in biochemistry and cell biology.
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An enhanced transient expression system in plants based on suppression of gene silencing by the p19 protein of tomato bushy stunt virus.
TL;DR: A system based on co-expression of a viral-encoded suppressor of gene silencing, the p19 protein of tomato bushy stunt virus, that prevents the onset of PTGS in the infiltrated tissues and allows high level of transient expression is described.
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pGreen: a versatile and flexible binary Ti vector for Agrobacterium-mediated plant transformation.
TL;DR: The pGreen plasmid system allows any arrangement of selectable marker and reporter gene at the right and left T-DNA borders without compromising the choice of restriction sites for cloning, since the pGreen cloning sites are based on the well-known pBluescript general vector plasmids.
Journal ArticleDOI
The SHORT-ROOT Gene Controls Radial Patterning of the Arabidopsis Root through Radial Signaling
Yrjö Helariutta,Hidehiro Fukaki,Joanna Wysocka-Diller,Keiji Nakajima,Jee Jung,Giovanni Sena,Marie-Theres Hauser,Philip N. Benfey +7 more
TL;DR: It is concluded that SHR functions upstream of SCR and participates in a radial signaling pathway, and ectopic expression of SHR results in supernumerary cell divisions and abnormal cell specification in the root meristem.
References
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GUS fusions: beta‐glucuronidase as a sensitive and versatile gene fusion marker in higher plants.
TL;DR: GUS is very stable, and tissue extracts continue to show high levels of GUS activity after prolonged storage, and Histochemical analysis has been used to demonstrate the localization of gene activity in cells and tissues of transformed plants.
Journal ArticleDOI
Green fluorescent protein as a marker for gene expression
TL;DR: A complementary DNA for the Aequorea victoria green fluorescent protein produces a fluorescent product when expressed in prokaryotic or eukaryotic cells, which can be used to monitor gene expression and protein localization in living organisms.
Journal ArticleDOI
Primary structure of the Aequorea victoria green-fluorescent protein.
Douglas Prasher,Virginia K. Eckenrode,William W. Ward,Frank G. Prendergast,Milton J. Cormier +4 more
TL;DR: The cloning and sequencing of both cDNA and genomic clones of GFP from the cnidarian, Aequorea victoria, show three different restriction enzyme patterns which suggests that at least three different genes are present in the A. victoria population.
Journal ArticleDOI
Crystal structure of the Aequorea victoria green fluorescent protein.
TL;DR: The green fluorescent protein (GFP) from the Pacific Northwest jellyfish Aequorea victoria has generated intense interest as a marker for gene expression and localization of gene products.
Journal ArticleDOI
Wavelength mutations and posttranslational autoxidation of green fluorescent protein
TL;DR: The availability of two visibly distinct colors should significantly extend the usefulness of GFP in molecular and cell biology by enabling in vivo visualization of differential gene expression and protein localization and measurement of protein association by fluorescence resonance energy transfer.
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