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Table 1

Relative Intensities of Fluorescence in Three Different Compartments of 3T3 Fibroblasts Transfected with the GFP Fusion Constructs

ConstructCellular localization
CytoplasmNucleoplasmNucleoli
pEGFP ++ ++ − 
pEGFP-Rpp38 −+ ++ +++ 
pEGFP-Rpp38(1-245) −+ +++ − 
pEGFP-Rpp38(246-283) −+ ++ +++ 
pNS38 −+ ++ +++ 
pNS38KN +++ ++ − 
pNS38KN23 ++ ++ ++ 
pNS38KN45 ++ ++ ++ 
pNS38KN78 +++ − 
pNS38KN59 ++ ++ 
pNS38R13A ++ ++ 
pNS38S18A −+ ++ ++ 
pNS38T22A −+ ++ ++ 
pNS38P23A −+ +++ 
pNS38ATDPP ++ ++ 
pEGFP-Rpp29 − +++ 
pEGFP-Rpp29(52-85) −+ +++ 
pEGFP-Rpp29(63-85) −+ +++ 
pNS29RN4 −+ +++ 
pNS29KN4 ++ ++ ++ 
pNS29HN − +++ 
pNS29RHKRK ++ +++ 
ConstructCellular localization
CytoplasmNucleoplasmNucleoli
pEGFP ++ ++ − 
pEGFP-Rpp38 −+ ++ +++ 
pEGFP-Rpp38(1-245) −+ +++ − 
pEGFP-Rpp38(246-283) −+ ++ +++ 
pNS38 −+ ++ +++ 
pNS38KN +++ ++ − 
pNS38KN23 ++ ++ ++ 
pNS38KN45 ++ ++ ++ 
pNS38KN78 +++ − 
pNS38KN59 ++ ++ 
pNS38R13A ++ ++ 
pNS38S18A −+ ++ ++ 
pNS38T22A −+ ++ ++ 
pNS38P23A −+ +++ 
pNS38ATDPP ++ ++ 
pEGFP-Rpp29 − +++ 
pEGFP-Rpp29(52-85) −+ +++ 
pEGFP-Rpp29(63-85) −+ +++ 
pNS29RN4 −+ +++ 
pNS29KN4 ++ ++ ++ 
pNS29HN − +++ 
pNS29RHKRK ++ +++ 

Constructs indicated above are shown in Fig. 1 A and their construction is described in Materials and Methods. The relative fluorescence intensities distributed in different cell compartments, cytoplasm, nucleoplasm, and nucleoli, are presented from + to +++ ranking, and are based on 3–7 transfection experiments with each GFP fusion construct using the same set of parameters for measuring fluorescence intensity by confocal microscopy. Background autofluorescence of 3T3 fibroblasts is indicated by minus (−), whereas a slightly higher signal that may result from the expression of the fusion protein is indicated by minus and plus (−+).

The frequency of transfection, as measured by cells exhibiting fluorescence, was ∼2–10% with all constructs except for the pEGFP-Rpp38. This latter construct yielded transfectants at about one tenth the frequency of the other plasmids, presumably because RNase P incorporating this fusion protein is less active than in other cases. We note that for each construct tested, all transfected cells (i.e., cells exhibiting fluorescence) yielded the same phenotype as shown in Table.

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