Fig. 1. A synthetic NoLS targets GFP to nucleoli. (A) Schematic representation of the generation of NoLS. The upper diagram corresponds to the DNA sequence encoding human sirtuin-1, the middle one to the PCR product after misreading of the DNA polymerase and the bottom one to the resulting mutant protein. A partial nucleotide sequence of sirtuin-1 is indicated in the upper diagram, and the corresponding misread sequence is indicated in the middle diagram. The resulting mutant protein corresponds to the first 87 amino acids of sirtuin-1 (sirtuin-1) followed by a synthetic sequence of 93 amino acids (NoLS). The numbers are nucleotide or amino acid positions. (B) NoLS with the stretch of basic residues in red. (C) Constructs of NoLS containing the entire or partial basic stretch in red. (D) Fluorescence microscopy observations of the NoLS constructs fused to GFP and expressed in HeLa cells. Only the constructs possessing the stretch of basic residues were predominantly (NoLS/31-63, i,j and NoLS/1-50, e,f), if not exclusively (NoLS, a,b and NoLS/33-93, c,d) localized in nucleoli, whereas the other constructs were not (NoLS/51-93, k,l) or only slightly (NoLS/1-32, g,h) enriched in nucleoli. Arrowheads in panel b represent nucleoli visible by phase-contrast image. Bar, 10 µm.