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Cosegregation of novel mitochondrial 16S rRNA gene mutations with the age-associated T414G variant in human cybrids.

Seibel P, Di Nunno C, Kukat C, Schäfer I, Del Bo R, Bordoni A, Comi GP, Schön A, Capuano F, Latorre D, Villani G - Nucleic Acids Res. (2008)

Bottom Line: In the present work, we have analyzed the bioenergetic properties associated with the age-related T414G mutation of the mtDNA control region in transmitochondrial cybrids.The results show that the T414G mutation does not cause per se any detectable bioenergetic change.The results are discussed in the more general context of the complex heterogeneity and the dramatic instability of the mitochondrial genome during cell culture of transmitochondrial cybrids.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Cell Therapy, Center for Biotechnology and Biomedicine, Universität Leipzig, Deutscher Platz 5, 04103 Leipzig, Germany. peter.seibel@bbz.uni-leipzig.de

ABSTRACT
Ever increasing evidence has been provided on the accumulation of mutations in the mitochondrial DNA (mtDNA) during the aging process. However, the lack of direct functional consequences of the mutant mtDNA load on the mitochondria-dependent cell metabolism has raised many questions on the physiological importance of the age-related mtDNA variations. In the present work, we have analyzed the bioenergetic properties associated with the age-related T414G mutation of the mtDNA control region in transmitochondrial cybrids. The results show that the T414G mutation does not cause per se any detectable bioenergetic change. Moreover, three mtDNA mutations clustered in the 16S ribosomal RNA gene cosegregated together with the T414G in the same cybrid cell line. Two of them, namely T1843C and A1940G, are novel and associate with a negative bioenergetic phenotype. The results are discussed in the more general context of the complex heterogeneity and the dramatic instability of the mitochondrial genome during cell culture of transmitochondrial cybrids.

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Mispairing PCR and RFLP analyses of heteroplasmic point mutations in the mitochondrial 16S rRNA gene. The 1843, 1940 and 2623 positions were analyzed by RFLP analyses as described under Materials and methods section. XmiI, SacI and BshTI restriction enzymes were used to cleave the mispairing PCR products carrying the wild-type genotype at positions 1843, 1940 and 2623, respectively (upper panels). RsaI and Bsp120I restriction enzymes were used to cleave the mispairing PCR products carrying the mutant genotype at positions 1843 and 1940 or 2623, respectively (lower panels). The displayed ethidium bromide stained gels (upper and lower panels) refer to representative experiments.
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Figure 2: Mispairing PCR and RFLP analyses of heteroplasmic point mutations in the mitochondrial 16S rRNA gene. The 1843, 1940 and 2623 positions were analyzed by RFLP analyses as described under Materials and methods section. XmiI, SacI and BshTI restriction enzymes were used to cleave the mispairing PCR products carrying the wild-type genotype at positions 1843, 1940 and 2623, respectively (upper panels). RsaI and Bsp120I restriction enzymes were used to cleave the mispairing PCR products carrying the mutant genotype at positions 1843 and 1940 or 2623, respectively (lower panels). The displayed ethidium bromide stained gels (upper and lower panels) refer to representative experiments.

Mentions: To quantify the heteroplasmic states of the 16S rRNA mutations, we decided to develop a mispairing PCR test. In this context, a partial region of the 16S rRNA gene was amplified that encompassed the positions of all three point mutations (see Materials and methods section for details). The amplified product served as target for nested PCRs, so that upon RFLP analysis, mutated and wild-type mtDNA could be distinguished by gel electrophoresis (Figure 2) and quantified accordingly. The heteroplasmic mutation levels obtained as the mean value of the quantification of the cleaved fragments were of 54, 64 and 54% for the T1843C, A1940G and A2623G, respectively. These values were in agreement with the relative values of the wild-type and mutant peaks of the electropherograms displayed in Figure 1.Figure 2.


Cosegregation of novel mitochondrial 16S rRNA gene mutations with the age-associated T414G variant in human cybrids.

Seibel P, Di Nunno C, Kukat C, Schäfer I, Del Bo R, Bordoni A, Comi GP, Schön A, Capuano F, Latorre D, Villani G - Nucleic Acids Res. (2008)

Mispairing PCR and RFLP analyses of heteroplasmic point mutations in the mitochondrial 16S rRNA gene. The 1843, 1940 and 2623 positions were analyzed by RFLP analyses as described under Materials and methods section. XmiI, SacI and BshTI restriction enzymes were used to cleave the mispairing PCR products carrying the wild-type genotype at positions 1843, 1940 and 2623, respectively (upper panels). RsaI and Bsp120I restriction enzymes were used to cleave the mispairing PCR products carrying the mutant genotype at positions 1843 and 1940 or 2623, respectively (lower panels). The displayed ethidium bromide stained gels (upper and lower panels) refer to representative experiments.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2566888&req=5

Figure 2: Mispairing PCR and RFLP analyses of heteroplasmic point mutations in the mitochondrial 16S rRNA gene. The 1843, 1940 and 2623 positions were analyzed by RFLP analyses as described under Materials and methods section. XmiI, SacI and BshTI restriction enzymes were used to cleave the mispairing PCR products carrying the wild-type genotype at positions 1843, 1940 and 2623, respectively (upper panels). RsaI and Bsp120I restriction enzymes were used to cleave the mispairing PCR products carrying the mutant genotype at positions 1843 and 1940 or 2623, respectively (lower panels). The displayed ethidium bromide stained gels (upper and lower panels) refer to representative experiments.
Mentions: To quantify the heteroplasmic states of the 16S rRNA mutations, we decided to develop a mispairing PCR test. In this context, a partial region of the 16S rRNA gene was amplified that encompassed the positions of all three point mutations (see Materials and methods section for details). The amplified product served as target for nested PCRs, so that upon RFLP analysis, mutated and wild-type mtDNA could be distinguished by gel electrophoresis (Figure 2) and quantified accordingly. The heteroplasmic mutation levels obtained as the mean value of the quantification of the cleaved fragments were of 54, 64 and 54% for the T1843C, A1940G and A2623G, respectively. These values were in agreement with the relative values of the wild-type and mutant peaks of the electropherograms displayed in Figure 1.Figure 2.

Bottom Line: In the present work, we have analyzed the bioenergetic properties associated with the age-related T414G mutation of the mtDNA control region in transmitochondrial cybrids.The results show that the T414G mutation does not cause per se any detectable bioenergetic change.The results are discussed in the more general context of the complex heterogeneity and the dramatic instability of the mitochondrial genome during cell culture of transmitochondrial cybrids.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Cell Therapy, Center for Biotechnology and Biomedicine, Universität Leipzig, Deutscher Platz 5, 04103 Leipzig, Germany. peter.seibel@bbz.uni-leipzig.de

ABSTRACT
Ever increasing evidence has been provided on the accumulation of mutations in the mitochondrial DNA (mtDNA) during the aging process. However, the lack of direct functional consequences of the mutant mtDNA load on the mitochondria-dependent cell metabolism has raised many questions on the physiological importance of the age-related mtDNA variations. In the present work, we have analyzed the bioenergetic properties associated with the age-related T414G mutation of the mtDNA control region in transmitochondrial cybrids. The results show that the T414G mutation does not cause per se any detectable bioenergetic change. Moreover, three mtDNA mutations clustered in the 16S ribosomal RNA gene cosegregated together with the T414G in the same cybrid cell line. Two of them, namely T1843C and A1940G, are novel and associate with a negative bioenergetic phenotype. The results are discussed in the more general context of the complex heterogeneity and the dramatic instability of the mitochondrial genome during cell culture of transmitochondrial cybrids.

Show MeSH