Non-random mtDNA segregation patterns indicate a metastable heteroplasmic segregation unit in m.3243A>G cybrid cells.

Many pathogenic mitochondrial DNA mutations are heteroplasmic, with a mixture of mutated and wild-type mtDNA present within individual cells. The severity and extent of the clinical phenotype is largely due to the distribution of mutated molecules between cells in different tissues, but mechanisms underpinning segregation are not fully understood. To facilitate mtDNA segregation studies we developed assays that measure m.

Transient and constitutive repression of cytoplasmic translation signaling in cells with mtDNA mutation.

Cytoplasmic translation is under sophisticated control but how cells adapt its rate to constitutive loss of mitochondrial oxidative phosphorylation is unknown. Here we show that translation is repressed in cells with the pathogenic A3243G mtDNA mutation or in mtDNA-less rho(0) cells by at least two distinct pathways, one transiently targeting elongation factor eEF-2 and the other initiation factor eIF-2alpha constitutively. Under conditions of exponential cell growth and mammalian target of rapamycin (mTOR) activation, eEF-2 becomes transiently phosphorylated by an AMP-activated protein kinase (AMPK)-dependent pathway, especially high in mutant cells.

Probe labeling and fluorescence in situ hybridization.

This unit describes in detail basic protocols for probe labeling, denaturing of in situ target DNA, in situ hybridization, and post-hybridization washes. Support protocols for probe labeling cover probe purification and quality control.

Basic preparative techniques for fluorescence in situ hybridization.

This unit presents protocols for preparing human metaphase chromosome slides from peripheral blood lymphocytes, isolating interphase nuclei from lymphocytes and paraffin-embedded tissues, and preparing DNA fibers. The protocols are designed so that the resulting preparations are amenable to FISH. The methods correspond to a selection of the specimens that can be analyzed with FISH techniques, and the choice of sample preparation method is highly dependent on the molecular cytogenetics question being addressed.

Overview of fluorescence in situ hybridization techniques for molecular cytogenetics.

This unit presents an overview of the FISH methodology. It covers such topics as direct versus indirect methods, sensitivity, multiplicity, resolution, and applications.