Abstract

In the studied mutant strain of Drosophila subobscura, 78% of the mitochondrial genomes lost >30% of the coding region by deletion. The mutations was genetically stable. Despite this massive loss of mitochondrial genes, the mutant did not seem to be affected. Distribution of the two genome types, cell levels of mitochondrial DNA, steady-state concentrations of the mitochondrial gene transcripts, mitochondrial enzymatic activities, and ATP synthesis capacities were measured in the head, thorax, and abdomen fractions of the mutant strain in comparison with a wild type strain. Results indicate that the deleted genomes are detected in all fractions but to a lesser extent in the male and female abdomen. In all fractions, there is a 50% increase in cellular mitochondrial DNA content. Although there is a decrease in steady-state concentrations of mitochondrial transcripts of genes affected by deletion, this is smaller than expected. The variations in mitochondrial biochemical activities in the different fractions of the wild strain are upheld in the mutant strain. Activity of complex I (involved in mutation) nevertheless shows a decrease in all fractions; activity of complex III (likewise involved) shows little or no change; finally, mitochondrial ATP synthesis capacity is identical to that observed in the wild strain. This latter finding possibly accounts for the lack of phenotype. This mutant is a good model for studying mitochondrial genome alterations and the role of the nuclear genome in these phenomena.

Highlights

  • Various alterations in the mitochondrial genome have been correlated with severe human pathology [1,2,3]

  • The first investigations, performed in the whole adult fly [13], showed a strong heteroplasmic tendency in the deleted molecules; this affected 78% of the mitochondrial genomes. mtDNA cell content showed a 50% increase in the mutant strain compared with the D. subobscura wild strain

  • Measurement of mtDNA Content—Cellular mtDNA content extracted from the various fractions of the wild and mutant strains is compared with nuclear DNA content as described previously [13] using two types of probes: a mitochondrial probe (12 S) and a nuclear probe (18 S)

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Summary

EXPERIMENTAL PROCEDURES Strains

The studied flies belong to the D. subobscura strain and were raised on medium standard cornmeal as described [12] at 19 °C. The wild strain (W) acts as a control to study the effects of deletion on the mutant strain (H)

Preparation of the Different Tissues
Total RNA Extraction
Measurement of Heteroplasmy
Measurement of Mitochondrial DNA Content
Measurement of RNA Content
Isolation of Mitochondria
Measurement of Biochemical Activities
The Different Fractions
Measurement of Heteroplasmy and mtDNA Content
Measurement of Relative Concentrations of Mitochondrial Transcripts
Mitochondrial Biochemistry
ATP Synthesis Capacity
DISCUSSION
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