Green‐fluorescent protein fusions for efficient characterization of nuclear targeting

Abstract

SummaryThe green-fluorescent protein (GFP) from Aequorea victoria has been shown to be a convenient and flexible reporter molecule within a variety of eukaryotic systems, including higher plants. It is particularly suited for applications in vivo, since the mechanism of fluorophore formation involves an intramolecular autoxidation and does not require exogenous co-factors. Unlike standard histochemical procedures of fixation and staining required for analysis of the cellular or tissue-specific expression of other popular reporter molecules, such as the I]-glucuronidase (GUS) marker, analysis of GFP can be done in living cells with no specific pretreatments. This implies that GFP might also be particularly suited for studies of intracellular protein targeting. In this paper, the use of GUS is compared with that of GFP for the analysis of nuclear targeting in tobacco. A novel oligopeptide motif from a tobacco protein is described which confers nuclear localization of GUS. The use of this oligopeptide and two from potyviral proteins to target GFP to the nucleus is examined. An essential modification of GFP is described, which specifically increases its molecular weight to eliminate its passive penetration into the nucleus. Three examples of the targeting of these enlarged GFP molecules to the nucleus are illustrated. GFP, in combination with confocal microscopy, offers significant advantages over traditional methods of studying nuclear targeting.

Cellulose Binding Protein from the Parasitic Nematode Heterodera schachtii Interacts with Arabidopsis Pectin Methylesterase: Cooperative Cell Wall Modification during Parasitism

“…For PME3, primer sequences used were PME3(SP) forward (59-TATAAAGCTTATGGCACCATCAATGAAA-GAAATTTTTTC-39) or PME3 forward (59-TATAAAGCTTATGATCTCTG-CCGGAGCTTCAAAAGCC-39) and PME3 reverse (59-ATGATTCTAGA-TAAGACCGAGCGAGAAGGGGAAACCG-39). The resulting amplified fragments were cloned into the respective sites in the modified pRJG23 vector (Grebenok et al, 1997) before the start codon of GFP fused into the GUS reporter gene and under the control of double CaMV 35S promoter. All constructs were confirmed by DNA sequencing.…”

A Novel Endo-β-Mannanase Gene in Tomato LeMAN5 Is Associated with Anther and Pollen Development

“…The PCR products were digested with SacI and SalI and were cloned to the SacI and SalI sites in a shuttle vector pRJG23 (Grebenok et al, 1997) that contained the uidA (GUS) gene. The promoter-GUS construct (3.9 kb) in pRJG23 was cleaved out with SacI and SpeI and was subcloned into SacI and XbaI sites in pGPTV-KAN binary vector (Becker et al, 1992).…”

Matrix Attachment Region Binding Protein MFP1 Is Localized in Discrete Domains at the Nuclear Envelope

“…Although original problems with expressing the GFP at suffi- MAR Binding Protein at the Nuclear Envelope 1123 ciently high levels in plants have been overcome (Haseloff et al, 1997;Davis and Vierstra, 1998), its application for highresolution subcellular localization is still less widespread than it is in the animal field. This is due in part to the fact that its excitation wavelength also causes strong red fluorescence of chlorophyll, which can partially obscure the green fluorescence signal (Grebenok et al, 1997a). In addition, the fact that the protein diffuses passively into the nucleus due to its small size has discouraged researchers from using the GFP routinely for nuclear localization experiments (Grebenok et al, 1997a(Grebenok et al, , 1997b.…”

“…This is due in part to the fact that its excitation wavelength also causes strong red fluorescence of chlorophyll, which can partially obscure the green fluorescence signal (Grebenok et al, 1997a). In addition, the fact that the protein diffuses passively into the nucleus due to its small size has discouraged researchers from using the GFP routinely for nuclear localization experiments (Grebenok et al, 1997a(Grebenok et al, , 1997b. Here, we demonstrate that the GFP can be used in combination with confocal laser scanning microscopy to obtain high-resolution information about subcellular protein localization in plants.…”