title: Heterologous expression from the human D-Loop in organello.
abstract: We report the expression of a linear reporter construct in isolated human mitochondria. The reporter construct contained the entire human D-Loop with adjacent tRNA (MTT) genes (mt.15956-647), the human ND1 gene with an in frame GFP gene and adjacent endogenous MTT genes and heterologous rat MTT genes. Natural competence of isolated human mitochondria of HepG2 cells was used to import reporter constructs. The import efficiency of various fluorescently labelled PCR-generated import substrates in the range of 250bp up to 3.5kb was assessed by quantitative PCR and evaluated by confocal microscopy. Heterologous expression of the imported construct was confirmed at RNA level by a circular RNA (cRNA)-RT-PCR assay for the expression of tRNAs and by in organello [α-(32)P]-UTP labelling and subsequent hybridisation to reporter-specific sequences for monitoring mRNA expression. Heterologous expression of rat mitochondrial tRNA(Leu(UUR)) (rMT-TL1) was confirmed by co-/post-transcriptional trinucleotide (CCA) addition. Interestingly, the rat-specific MT-TL1 was correctly processed in isolated human mitochondria at the 3' end, but showed an aberrant 5' end processing. Correct 3' end processing of the heterologous expressed mitochondrial rat tRNA(Ser2) (MT-TS2) was detected. These findings demonstrate the feasibility of genetic manipulation of human mitochondria, providing a tool for characterisation of cis-acting elements of the human mitochondrial genome and for the study of human mitochondrial tRNA processing in organello.
authors.affiliation: Division of Human Genetics, Departments of Paediatrics Clinical Research, Inselspital, University of Berne, Switzerland; Graduate School for Cellular and Biomedical Sciences, University of Berne, Switzerland. Electronic address: christopher.jackson@insel.ch.
authors.forename: C B
authors.initials: CB
authors.lastname: Jackson
authors.affiliation: Division of Human Genetics, Departments of Paediatrics Clinical Research, Inselspital, University of Berne, Switzerland. Electronic address: christina.zbinden@vetsuisse.unibe.ch.
authors.forename: C
authors.initials: C
authors.lastname: Zbinden
authors.affiliation: Division of Human Genetics, Departments of Paediatrics Clinical Research, Inselspital, University of Berne, Switzerland. Electronic address: sabina.gallati@insel.ch.
authors.forename: S
authors.initials: S
authors.lastname: Gallati
authors.affiliation: Division of Human Genetics, Departments of Paediatrics Clinical Research, Inselspital, University of Berne, Switzerland. Electronic address: andre.schaller@insel.ch.
authors.forename: A
authors.initials: A
authors.lastname: Schaller
mesh_terms: D000818:Animals; D019677:Artificial Gene Fusion; D004272:DNA, Mitochondrial / Q000235:genetics*; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation; D050259:Genes, Mitochondrial; D017930:Genes, Reporter; D005818:Genetic Engineering / Q000379:methods; D056945:Hep G2 Cells; D006801:Humans; D051381:Rats
chemical_list: D004272:DNA, Mitochondrial
keywords: Heterologous expression; In organello; Mitochondria; Mitochondrial gene expression; Mitochondrial transfection; Natural competence