Characterization of the targeted nuclear accumulation of GFP within the cells of transgenic plants

Robert J. Grebenok, Georgina M. Lambert, David W Galbraith

Research output: Contribution to journalArticle

41 Citations (Scopus)

Abstract

The soluble proteins of the nucleoplasm are synthesized on cytoplasmic ribosomes. Proteins larger than about 40 kDa are post-translationally targeted to the nucleus via energy-dependent processes, passing through the nuclear pore complex into the nucleoplasm. Targeting involves nuclear localization signals (NLSs) found within the primary sequences of the imported proteins. In higher plants, information has come primarily from study of proteins carrying 'classical' NLSs, comprising stretches of basic amino acids, and has required assays to measure nuclear uptake both in vitro and in vivo. In general, these assays are not entirely satisfactory; they are either technically demanding, are of limited accuracy and statistical rigor, or are unsuitable for in vivo applications. The green-fluorescent protein (GFP) of Aequorea victoria has recently emerged as a versatile marker for transgenic expression in vivo. Conditions under which GFP gene fusions can be employed for the analysis of nuclear targeting in plant protoplasts have been described. This study demonstrates for the first time the nuclear targeting of chimeric GFP molecules in transgenic tobacco. This is accompanied by a description and evaluation of novel analytical methods, involving flow and image cytometry, for the quantitative temporal and spatial analysis of nuclear targeting, and these unique methods are used to provide information concerning the targeting process. Finally, the way in which the chimeric GFP molecules might be employed for the study of various important problems in plant cell and developmental biology is discussed.

Original languageEnglish (US)
Pages (from-to)685-696
Number of pages12
JournalPlant Journal
Volume12
Issue number3
StatePublished - 1997

Fingerprint

Genetically Modified Plants
Green Fluorescent Proteins
green fluorescent protein
transgenic plants
Nuclear Localization Signals
nuclear localization signals
Proteins
proteins
Aequorea victoria
Image Cytometry
cells
nucleoporins
Spatio-Temporal Analysis
Nuclear Pore
Developmental Biology
Basic Amino Acids
gene fusion
Protoplasts
Gene Fusion
Plant Cells

ASJC Scopus subject areas

  • Plant Science

Cite this

Characterization of the targeted nuclear accumulation of GFP within the cells of transgenic plants. / Grebenok, Robert J.; Lambert, Georgina M.; Galbraith, David W.

In: Plant Journal, Vol. 12, No. 3, 1997, p. 685-696.

Research output: Contribution to journalArticle

Grebenok, Robert J. ; Lambert, Georgina M. ; Galbraith, David W. / Characterization of the targeted nuclear accumulation of GFP within the cells of transgenic plants. In: Plant Journal. 1997 ; Vol. 12, No. 3. pp. 685-696.
@article{4f0ca96d79884df8ab94c661e0630b0d,
title = "Characterization of the targeted nuclear accumulation of GFP within the cells of transgenic plants",
abstract = "The soluble proteins of the nucleoplasm are synthesized on cytoplasmic ribosomes. Proteins larger than about 40 kDa are post-translationally targeted to the nucleus via energy-dependent processes, passing through the nuclear pore complex into the nucleoplasm. Targeting involves nuclear localization signals (NLSs) found within the primary sequences of the imported proteins. In higher plants, information has come primarily from study of proteins carrying 'classical' NLSs, comprising stretches of basic amino acids, and has required assays to measure nuclear uptake both in vitro and in vivo. In general, these assays are not entirely satisfactory; they are either technically demanding, are of limited accuracy and statistical rigor, or are unsuitable for in vivo applications. The green-fluorescent protein (GFP) of Aequorea victoria has recently emerged as a versatile marker for transgenic expression in vivo. Conditions under which GFP gene fusions can be employed for the analysis of nuclear targeting in plant protoplasts have been described. This study demonstrates for the first time the nuclear targeting of chimeric GFP molecules in transgenic tobacco. This is accompanied by a description and evaluation of novel analytical methods, involving flow and image cytometry, for the quantitative temporal and spatial analysis of nuclear targeting, and these unique methods are used to provide information concerning the targeting process. Finally, the way in which the chimeric GFP molecules might be employed for the study of various important problems in plant cell and developmental biology is discussed.",
author = "Grebenok, {Robert J.} and Lambert, {Georgina M.} and Galbraith, {David W}",
year = "1997",
language = "English (US)",
volume = "12",
pages = "685--696",
journal = "Plant Journal",
issn = "0960-7412",
publisher = "Wiley-Blackwell",
number = "3",

}

TY - JOUR

T1 - Characterization of the targeted nuclear accumulation of GFP within the cells of transgenic plants

AU - Grebenok, Robert J.

AU - Lambert, Georgina M.

AU - Galbraith, David W

PY - 1997

Y1 - 1997

N2 - The soluble proteins of the nucleoplasm are synthesized on cytoplasmic ribosomes. Proteins larger than about 40 kDa are post-translationally targeted to the nucleus via energy-dependent processes, passing through the nuclear pore complex into the nucleoplasm. Targeting involves nuclear localization signals (NLSs) found within the primary sequences of the imported proteins. In higher plants, information has come primarily from study of proteins carrying 'classical' NLSs, comprising stretches of basic amino acids, and has required assays to measure nuclear uptake both in vitro and in vivo. In general, these assays are not entirely satisfactory; they are either technically demanding, are of limited accuracy and statistical rigor, or are unsuitable for in vivo applications. The green-fluorescent protein (GFP) of Aequorea victoria has recently emerged as a versatile marker for transgenic expression in vivo. Conditions under which GFP gene fusions can be employed for the analysis of nuclear targeting in plant protoplasts have been described. This study demonstrates for the first time the nuclear targeting of chimeric GFP molecules in transgenic tobacco. This is accompanied by a description and evaluation of novel analytical methods, involving flow and image cytometry, for the quantitative temporal and spatial analysis of nuclear targeting, and these unique methods are used to provide information concerning the targeting process. Finally, the way in which the chimeric GFP molecules might be employed for the study of various important problems in plant cell and developmental biology is discussed.

AB - The soluble proteins of the nucleoplasm are synthesized on cytoplasmic ribosomes. Proteins larger than about 40 kDa are post-translationally targeted to the nucleus via energy-dependent processes, passing through the nuclear pore complex into the nucleoplasm. Targeting involves nuclear localization signals (NLSs) found within the primary sequences of the imported proteins. In higher plants, information has come primarily from study of proteins carrying 'classical' NLSs, comprising stretches of basic amino acids, and has required assays to measure nuclear uptake both in vitro and in vivo. In general, these assays are not entirely satisfactory; they are either technically demanding, are of limited accuracy and statistical rigor, or are unsuitable for in vivo applications. The green-fluorescent protein (GFP) of Aequorea victoria has recently emerged as a versatile marker for transgenic expression in vivo. Conditions under which GFP gene fusions can be employed for the analysis of nuclear targeting in plant protoplasts have been described. This study demonstrates for the first time the nuclear targeting of chimeric GFP molecules in transgenic tobacco. This is accompanied by a description and evaluation of novel analytical methods, involving flow and image cytometry, for the quantitative temporal and spatial analysis of nuclear targeting, and these unique methods are used to provide information concerning the targeting process. Finally, the way in which the chimeric GFP molecules might be employed for the study of various important problems in plant cell and developmental biology is discussed.

UR - http://www.scopus.com/inward/record.url?scp=0030804143&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0030804143&partnerID=8YFLogxK

M3 - Article

AN - SCOPUS:0030804143

VL - 12

SP - 685

EP - 696

JO - Plant Journal

JF - Plant Journal

SN - 0960-7412

IS - 3

ER -