کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
---|---|---|---|---|
8399388 | 1544426 | 2014 | 31 صفحه PDF | دانلود رایگان |
عنوان انگلیسی مقاله ISI
Heterologous expression from the human D-Loop in organello
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کلمات کلیدی
موضوعات مرتبط
علوم زیستی و بیوفناوری
بیوشیمی، ژنتیک و زیست شناسی مولکولی
بیوفیزیک
پیش نمایش صفحه اول مقاله
![عکس صفحه اول مقاله: Heterologous expression from the human D-Loop in organello Heterologous expression from the human D-Loop in organello](/preview/png/8399388.png)
چکیده انگلیسی
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 250 bp up to 3.5 kb 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-32P]-UTP labelling and subsequent hybridisation to reporter-specific sequences for monitoring mRNA expression. Heterologous expression of rat mitochondrial tRNALeu(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 3Ⲡend, but showed an aberrant 5Ⲡend processing. Correct 3Ⲡend processing of the heterologous expressed mitochondrial rat tRNASer2 (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.
ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Mitochondrion - Volume 17, July 2014, Pages 67-75
Journal: Mitochondrion - Volume 17, July 2014, Pages 67-75
نویسندگان
C.B. Jackson, C. Zbinden, S. Gallati, A. Schaller,