title: [Coding region of far-red fluorescent protein katushka contains a strong donor splice site].
abstract: Computer analysis predicted a strong donor splice site within the 3'-part of the far-red fluorescent protein Katushka coding region. To test the functional activity of this site a model vector has been constructed. This vector encoded Katushka and green fluorescent protein TagGFP2 with a gene fragment of tafazzin in between. Normal splicing of this pre-mRNA should result in a frameshift between Katushka and TagGFP2. Alternatively, after splicing at internal katushka donor splice site appearance of Katushka-TagGFP2 fusion protein was expected. Expression of this construct in a mammalian cell culture led to bright red and green fluorescence. Therefore, katushka-specific donor splice site is functional. Disruption of this splice site by several silent substitutions resulted in red-only fluorescent cells that corresponded to normal splicing. The mutant katushka can be used for visualization of pre-mRNA splicing at single cell level by fluorescence microscopy and flow cytometry.
authors: N G
authors: NG
authors: Gurskaia
authors: D B
authors: DB
authors: Staroverov
authors: A F
authors: AF
authors: Fradkov
authors: K A
authors: KA
authors: Luk'ianov
mesh_terms: D000217:Acyltransferases; D005091:Exons; D049452:Green Fluorescent Proteins / Q000235:genetics; D057809:HEK293 Cells; D006801:Humans; D008164:Luminescent Proteins / Q000737:chemistry / Q000235:genetics*; D012322:RNA Precursors / Q000235:genetics; D022821:RNA Splice Sites / Q000235:genetics*; D012326:RNA Splicing; D014157:Transcription Factors / Q000235:genetics; D000097573:Red Fluorescent Protein
chemical_list: D000217:Acyltransferases; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins; D012322:RNA Precursors; D022821:RNA Splice Sites; D014157:Transcription Factors; D000097573:Red Fluorescent Protein; C108675:TAFAZZIN protein, human