Mitochondrial Genome Instability in W303-SK1 Yeast Cytoplasmic Hybrids

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The well-written manuscript describes a descriptive but elegant genetic and biochemical study of determinants of "suppressivity" in mitochondrial inheritance in yeast. The authors concluded that the primary determinant is mitochondrial DNA by itself, not nuclear DNA. Two strains, W303 and SK1, are genetically distant (e.g., PMID:37175502, PMID:37244009). However, mitochondrial genomes retain their properties, though foreign mitochondria decreased respiratory ability. Interestingly, the mitochondrial DNA of the SK1 strain was not very stable in the W303 background, while the mitochondrial DNA of W303 was stable in SK1. The work's only weakness is that the discussion of evolutionary aspects is based on comparing only two strains.

Minor comments

We do not see the analysis of genomic differences of W303 and SK1 in the nuclear genes CCE1, RPO41, PET127, or other yet unidentified nuclear-encoded genes regulating suppressivity that are discussed in the Introduction (lines 80-81).

The authors should mention (e.g., in the Methods section or legends to figures) what dots, rectangles, and lines represent in all figures.

 

 

Author Response

First of all, we would like to thank the referee for useful comments and advises and are ready to answer the questions raised.

Reviewer 1

The well-written manuscript describes a descriptive but elegant genetic and biochemical study of determinants of "suppressivity" in mitochondrial inheritance in yeast. The authors concluded that the primary determinant is mitochondrial DNA by itself, not nuclear DNA. Two strains, W303 and SK1, are genetically distant (e.g., PMID:37175502, PMID:37244009). However, mitochondrial genomes retain their properties, though foreign mitochondria decreased respiratory ability. Interestingly, the mitochondrial DNA of the SK1 strain was not very stable in the W303 background, while the mitochondrial DNA of W303 was stable in SK1. The work's only weakness is that the discussion of evolutionary aspects is based on comparing only two strains.

 

Minor comments:

Сomments 1: “We do not see the analysis of genomic differences of W303 and SK1 in the nuclear genes CCE1, RPO41, PET127, or other yet unidentified nuclear-encoded genes regulating suppressivity that are discussed in the Introduction (lines 80-81).”

Response 1: We have added two sentences to the Results section and Supplementary Text 1 with the analysis of these genes.  There are very few conservative amino acid replacements which are unlikely to affect the protein function.

 

Сomments 2: “The authors should mention (e.g., in the Methods section or legends to figures) what dots, rectangles, and lines represent in all figures.”

Response 2: Done, We added the following text to the Statistice, Data Visualisation and Analysis section.

“The figures show boxplots where the lower and upper borders correspond to the the 25th and 75th percentiles (InterQuartile Range, IQR). The upper whisker extends from the box border to the largest value within 1.5 * IQR. The lower whisker reaches the smallest value within 1.5 * IQR. The result of individual data points are shown as circles, points beyond whiskers are outliers.”

 

Author Response File: Author Response.docx

Reviewer 2 Report

Comments and Suggestions for Authors

The paper by Epremyan et al. presents new data on suppressivity of eukaryotic cells using W303-SK1 yeast cytoplasmic hybrids. The manuscript is well written, organized and adds new understanding to mitochondrial genome instability.

Minor comments:

-   It is useful to divide Figure 1 into several separate parts.

-   In paragraph 2.4 of the Methods, the pH of the medium is missing. The value was probably 5 or 5.5.

-   Paragraph 2.7 does not specify the programme that was used to statistically process the data.

-   Stylistically, the expression ‘On the one hand’ is repeated in lines 49, 56, 74, 78-79; it should be paraphrased in some cases.

-   The Conclusions part of the manuscript is missing. If the authors have thoughts about, for example, the application of the fundamental data obtained in practical biology, this section makes sense to add.

I do not have any major concerns, and recommend consideration for inclusion in BIOLOGY.

Author Response

First of all, we would like to thank the referee for useful comments and advises and are ready to answer the questions raised.

Reviewer 2

The paper by Epremyan et al. presents new data on suppressivity of eukaryotic cells using W303-SK1 yeast cytoplasmic hybrids. The manuscript is well written, organized and adds new understanding to mitochondrial genome instability.

 

Minor comments:

Сomments 1: “It is useful to divide Figure 1 into several separate parts.”

Response 1: Done.

 

Сomments 2: “In paragraph 2.4 of the Methods, the pH of the medium is missing. The value was probably 5 or 5.5.”

Response 2: Done. We updated the 2.4 section.

 

Сomments 3: “Paragraph 2.7 does not specify the programme that was used to statistically process the data.”

Response 3: All analyses were performed using the base R programming language library.  We updated the material and methods section to mention this.

 

Сomments 4: “Stylistically, the expression ‘On the one hand’ is repeated in lines 49, 56, 74, 78-79; it should be paraphrased in some cases.”

Response 4: Done. We paraphrased the ‘on the one hand’ expression at the second occasion.

 

Сomments 5: “The Conclusions part of the manuscript is missing. If the authors have thoughts about, for example, the application of the fundamental data obtained in practical biology, this section makes sense to add.”

Response 5: We edited the last paragraph of the discussion to fulfill the role of the conclusion. Given that there are some speculations, we prefer not to make a separate conclusions section. Now it looks like as follow

“To summarise, our study demonstrated that swapping mtDNA between laboratory yeast strains increases the rate of mtDNA loss while decreasing respiration and growth rates. Although expected, this observation suggests that mutual adaptation of mitochondrial and nuclear genomes occurs at the intraspecific level in yeast. At the same time, our data shows that mutant mtDNA variant's ability to outcompete another variant in a heteroplasmic cell can be primarily determined by mtDNA sequences rather than the nuclear genome background.

In the state of mitochondrial heteroplasmy, over the course of generations, only one variant of mitochondrial DNA usually remains in the cells. This outcome is determined by both random genetic drift and the relative fitness levels of the different mtDNA variants [37]. The relative fitness of different variants can depend on the context (e.g., tissue type of the multicellular organism), which is created in the mitochondrial matrix mainly by proteins encoded in the nuclear genome [38,39]. However, the results of suppressivity tests in yeast SK1-W303 cybrids demonstrate that, in some cases, the relative competition of mtDNA in yeast is determined by differences in the mtDNA itself. These results demonstrate that different mtDNAs from standard laboratory strains can be subject to varying degrees of displacement by selfish mitochondrial DNA elements. We propose that mtDNA sequences evolved under constant pressure from such selfish elements and may contain adaptations to counteract them.“

Author Response File: Author Response.docx