An internal standard improves the reliability of transient expression studies in plant protoplasts

1991 ◽  
Vol 10 (8) ◽  
Author(s):  
Marc Lepetit ◽  
Martine Ehling ◽  
Claude Gigot ◽  
G�nther Hahne
Keyword(s):  
Transient Expression ◽  
Internal Standard ◽  
Plant Protoplasts ◽  
Expression Studies

Influence of bacterial strain genotype on transient expression of plasmid DNA in plant protoplasts

1993 ◽  
Vol 4 (3) ◽  
pp. 587-592 ◽  
Author(s):  
Jorge Tovar Torres ◽  
Annette Block ◽  
Klaus Hahlbrock ◽  
Imre E. Somssich
Keyword(s):  
Transient Expression ◽  
Plasmid Dna ◽  
Bacterial Strain ◽  
Plant Protoplasts

scholarly journals Use of Fluorescent Protein Tags to Study Nuclear Organization of the Spliceosomal Machinery in Transiently Transformed Living Plant Cells

2004 ◽  
Vol 15 (7) ◽  
pp. 3233-3243 ◽  
Author(s):  
Zdravko J. Lorković ◽  
Julia Hilscher ◽  
Andrea Barta
Keyword(s):  
Transient Expression ◽  
Rna Processing ◽  
Fluorescent Protein ◽  
Nuclear Organization ◽  
Plant Cells ◽  
Cajal Bodies ◽  
Plant Protoplasts ◽  
Living Plant ◽  
Small Nuclear Ribonucleoprotein ◽  
Specific Proteins

Although early studies suggested that little compartmentalization exists within the nucleus, more recent studies on metazoan systems have identified a still increasing number of specific subnuclear compartments. Some of these compartments are dynamic structures; indeed, protein and RNA-protein components can cycle between different domains. This is particularly evident for RNA processing components. In plants, lack of tools has hampered studies on nuclear compartmentalization and dynamics of RNA processing components. Here, we show that transient expression of fluorescent protein fusions of U1 and U2 small nuclear ribonucleoprotein particle (snRNP)-specific proteins U1-70K, U2B″, and U2A ′, nucleolar proteins Nop10 and PRH75, and serine-arginine-rich proteins in plant protoplasts results in their correct localization. Furthermore, snRNP-specific proteins also were correctly assembled into mature snRNPs. This system allowed a systematic analysis of the cellular localization of Arabidopsis serine-arginine-rich proteins, which, like their animal counterparts, localize to speckles but not to nucleoli and Cajal bodies. Finally, markers for three different nuclear compartments, namely, nucleoli, Cajal bodies, and speckles, have been established and were shown to be applicable for colocalization studies in living plant protoplasts. Thus, transient expression of proteins tagged with four different fluorescent proteins is a suitable system for studying the nuclear organization of spliceosomal proteins in living plant cells and should therefore allow studies of their dynamics as well.

scholarly journals Molecular basis of a hereditary type I protein S deficiency caused by a substitution of Cys for Arg474

Blood ◽  
1996 ◽  
Vol 87 (11) ◽  
pp. 4643-4650 ◽  
Author(s):  
T Yamazaki ◽  
A Katsumi ◽  
K Kagami ◽  
Y Okamoto ◽  
I Sugiura ◽  
...  
Keyword(s):  
Amino Acid ◽  
Missense Mutation ◽  
Transient Expression ◽  
Molecular Basis ◽  
Protein S ◽  
Basic Amino Acid ◽  
Sex Hormone Binding Globulin ◽  
Type I ◽  
Intracellular Degradation ◽  
Expression Studies

The molecular basis for a hereditary type I protein S (PS) deficiency was investigated. DNA sequence analysis in the proband showed a novel missense mutation substituting Cys (TGT) for Arg474 (CGT) that is a highly conserved amino acid residue among the related proteins. This missense mutation cosegregated with the type I PS deficiency in this family. Transient expression studies showed that the secretion of the recombinant Cys-mutant PS was markedly decreased compared with that of the recombinant wild-type PS, reproducing the observed phenotype of type I deficiency. Stable expression and pulse-chase experiments demonstrated an intracellular degradation and an impaired secretion of the recombinant Cys-mutant PS. Furthermore, the substitution of Arg474 by Ala or Glu, but not by Lys, markedly reduced the secretion of the recombinant PS mutants in transient expression studies, suggesting that a positively charged basic amino acid might be needed at residue 474 and might play a key role in the protein structure and conformation of the sex hormone binding globulin-homology domain of the PS molecule. We postulate that the loss of the highly conserved Arg474 might be responsible for the type I PS deficiency inherited in this family.

Characterization of frozen hydrated biological specimens by low-loss EELS

1992 ◽  
Vol 50 (2) ◽  
pp. 1572-1573
Author(s):  
Songquan Sun ◽  
Richard D. Leapman
Keyword(s):  
Water Content ◽  
Direct Method ◽  
Internal Standard ◽  
Dry Weight ◽  
Dark Field ◽  
Protein Solutions ◽  
Subcellular Compartment ◽  
Differential Shrinkage ◽  
Electron Energy Loss

Analyses of ultrathin cryosections are generally performed after freeze-drying because the presence of water renders the specimens highly susceptible to radiation damage. The water content of a subcellular compartment is an important quantity that must be known, for example, to convert the dry weight concentrations of ions to the physiologically more relevant molar concentrations. Water content can be determined indirectly from dark-field mass measurements provided that there is no differential shrinkage between compartments and that there exists a suitable internal standard. The potential advantage of a more direct method for measuring water has led us to explore the use of electron energy loss spectroscopy (EELS) for characterizing biological specimens in their frozen hydrated state.We have obtained preliminary EELS measurements from pure amorphous ice and from cryosectioned frozen protein solutions. The specimens were cryotransfered into a VG-HB501 field-emission STEM equipped with a 666 Gatan parallel-detection spectrometer and analyzed at approximately −160 C.

STEM measurement of subcellular water distributions

1993 ◽  
Vol 51 ◽  
pp. 568-569
Author(s):  
R.D. Leapman ◽  
S.Q. Sun ◽  
S-L. Shi ◽  
R.A. Buchanan ◽  
S.B. Andrews
Keyword(s):  
Water Content ◽  
Internal Standard ◽  
Dry Weight ◽  
Dry Mass ◽  
Scanning Transmission Electron Microscope ◽  
Dark Field ◽  
Bulk Water ◽  
Inelastic Electron ◽  
Scanning Transmission ◽  
Annular Dark Field

Recent advances in rapid-freezing and cryosectioning techniques coupled with use of the quantitative signals available in the scanning transmission electron microscope (STEM) can provide us with new methods for determining the water distributions of subcellular compartments. The water content is an important physiological quantity that reflects how fluid and electrolytes are regulated in the cell; it is also required to convert dry weight concentrations of ions obtained from x-ray microanalysis into the more relevant molar ionic concentrations. Here we compare the information about water concentrations from both elastic (annular dark-field) and inelastic (electron energy loss) scattering measurements.In order to utilize the elastic signal it is first necessary to increase contrast by removing the water from the cryosection. After dehydration the tissue can be digitally imaged under low-dose conditions, in the same way that STEM mass mapping of macromolecules is performed. The resulting pixel intensities are then converted into dry mass fractions by using an internal standard, e.g., the mean intensity of the whole image may be taken as representative of the bulk water content of the tissue.

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