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Deletions and rearrangements in Kluyveromyces lactis mitochondrial DNA
C M Hardy1, C L Galeotti, G D Clark-Walker
1Molecular and Population Genetics Group, Research School of Biological Sciences, Australian National University, Canberra, A.C.T.
Abstract:
Three classes of respiratory deficient mutants have been isolated from a fusant between Kluyveromyces lactis and Saccharomyces cerevisiae that contains only K. lactis mtDNA. One class (15 isolates), resemble rho 0 mutants of S. cerevisiae as they lack detectable mtDNA. A second class (16 isolates), resemble point mutations (mit-) or nuclear lesions (pet-) of S. cerevisiae as no detectable change is found in their mtDNA. The third class (five isolates), with deletions and rearrangements in their mtDNA are comparable to S. cerevisiae petite (rho-) mutants. Surprisingly, three of the five deletion mutants have lost the same 8.0 kb sector of the mtDNA that encompasses the entire cytochrome oxidase subunit 2 gene and the majority of the adjacent cytochrome oxidase subunit 1 gene. In the other strains, deletions are accompanied by complex rearrangements together with substoiciometric bands and in one instance an amplified sector of 800 bp. By contrast to G + C rich short direct repeats forming deletion sites in S. cerevisiae mtDNA, excision of the 8.0 kb sector in K. lactis mtDNA occurs at an 11 bp A + T rich direct repeat CTAATATATAT. The recovery of three strains manifesting this deletion suggests there are limited sites for intramolecular recombination leading to excision in K. lactis mtDNA.
Insights
Researchers identified three types of respiratory-deficient mutants in yeast, including those with mtDNA deletions and rearrangements. A specific 8.0 kb deletion in Kluyveromyces lactis mtDNA occurred at an A+T-rich repeat, differing from S. cerevisiae.
Area of Science:
- Mitochondrial genetics
- Yeast molecular biology
Background:
- Respiratory deficient mutants are crucial for understanding mitochondrial DNA (mtDNA) stability and function.
- Kluyveromyces lactis and Saccharomyces cerevisiae are model organisms for studying yeast genetics and mitochondrial inheritance.
Purpose of the Study:
- To isolate and characterize respiratory deficient mutants from a Kluyveromyces lactis-Saccharomyces cerevisiae fusant containing only K. lactis mtDNA.
- To investigate the nature of mtDNA alterations, including deletions and rearrangements, in these mutants.
- To compare the mechanisms of mtDNA deletion in K. lactis with those in S. cerevisiae.
Main Methods:
- Isolation of respiratory deficient mutants from yeast fusants.
- Analysis of mitochondrial DNA (mtDNA) integrity using techniques to detect deletions and rearrangements.
- Sequencing and characterization of mtDNA deletion sites.
Main Results:
- Three classes of mutants were identified: mtDNA-lacking (rho 0-like), mtDNA with no detectable change (mit-/pet-like), and mtDNA with deletions/rearrangements (rho-like).
- A conserved 8.0 kb deletion, encompassing cytochrome oxidase subunit 2 and part of subunit 1 genes, was found in three mutants.
- mtDNA deletions in K. lactis occurred at an 11 bp A+T-rich direct repeat (CTAATATATAT), contrasting with G+C-rich repeats in S. cerevisiae.
Conclusions:
- The study identified distinct classes of respiratory deficient mutants in K. lactis mtDNA.
- The findings reveal a specific mechanism for large-scale mtDNA deletion in K. lactis, mediated by A+T-rich repeats.
- The limited sites for intramolecular recombination suggest specific hotspots for deletions in K. lactis mtDNA.