Inheritance and organisation of the mitochondrial genome differ between two Saccharomyces yeasts

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Abstract

Petite-positive Saccharomyces yeasts can be roughly divided into the sensu stricto, including Saccharomyces cerevisiae, and sensu lato group, including Saccharomyces castellii; the latter was recently studied for transmission and the organisation of its mitochondrial genome. S. castellii mitochondrial molecules (mtDNA) carrying point mutations, which confer antibiotic resistance, behaved in genetic crosses as the corresponding point mutants of S. cerevisiae. While S. castellii generated spontaneous petite mutants in a similar way as S. cerevisiae, the petites exhibited a different inheritance pattern. In crosses with the wild type strains a majority of S. castellii petites was neutral, and the suppressivity in suppressive petites was never over 50%. The two yeasts also differ in organisation of their mtDNA molecules. The 25,753 bp sequence of S. castellii mtDNA was determined and the coding potential of both yeasts is similar. However, the S. castellii intergenic sequences are much shorter and do not contain sequences homologous to the S. cerevisiae biologically active intergenic sequences, as ori/rep/tra, which are responsible for the hyper-suppressive petite phenotype found in S. cerevisiae. The structure of one suppressive S. castellii mutant, CA38, was also determined. Apparently, a short direct intergenic repeat was involved in the generation of this petite mtDNA molecule.

Introduction

The genetic material of Saccharomyces cerevisiae consists of 16 nuclear chromosomes and a separate circular mitochondrial genome. The multi-copy mitochondrial DNA (mtDNA) has the size of 85.8 kilobases (kb), a low gene density and extensive intergenic regions, which cover a majority of the mitochondrial genome.1 These regions are composed of long adenosine and thymine (A+T) stretches, short guanosine and cytosine (G+C) clusters, and a special class of intergenic sequences, ori/rep/tra, which are present in eight copies.2., 3. A number of short repeats, direct or indirect, can be found within the S. cerevisiae mitochondrial sequence, especially within the intergenic regions.1., 3.

S. cerevisiae is a facultative anaerobe and can obtain energy purely by fermentation.4 Several respiratory deficient mutants, which can grow only on fermentable carbon sources, can be isolated.5 The largest class of these mutants is called petites, and is characterised by grossly rearranged and deleted mtDNA molecules (for review, see Bernardi6). Crosses of wild type cells with petite mutants exhibit a non-Mendelian segregation of the mutation, yielding only wild type progeny or both wild type and petite mutants present in different proportions. In the first case the petites entering the cross are called neutral and in the second one suppressive.7 Among spontaneously arising petites, suppressive petites are the most commonly found class.8 The degree of suppressiveness of petite clones is measured by the percentage of zygotes giving rise to petite clones. A special subclass, hyper-suppressive petites, is defined by the criterion that close to 100% of cells resulting from crosses are petites. mtDNA molecules of hyper-suppressive petites reveal a common organisation: they consist of only a short multiplied intergenic segment carrying an ori/rep/tra sequence.9., 10. Upon a cross the petite mtDNA molecule out-competes the wild type mtDNA molecule in the zygote and gets almost exclusively transmitted to the progeny (reviews5., 11., 12.). Similarly, respiratory competent, mitochondrial mutants lacking some of the ori/rep/tra sequences are out-competed by the wild type mtDNA molecules.13 Apparently, the ori/rep/tra sequence embedded in a proper environment of neighbouring sequences, provides a structural basis for interactions between mtDNA and other elements involved in the process of selective transmission.14 However, while ori/rep/tra sequences play a crucial role in transmission of the S. cerevisiae mtDNA molecule, these sequences seem to be present only in a limited number of yeast species.15

A majority of yeasts are petite-negative, including Schizosaccharomyces pombe, and only their mutator strains can yield mitochondrial respiratory mutants.16 The genus Saccharomyces contains several species,17 which can be divided into a group of petite-positive, including S. cerevisiae, and a group of petite-negative yeasts; the latter includes Saccharomyces kluyveri.18 Apparently, the common progenitor of these two groups was a petite-negative yeast. Upon separation of the S. kluyveri and S. cerevisiae lineages the S. kluyveri lineage has remained petite negative, while the other lineage has developed the petite-positive characteristics.18 These petite-positive Saccharomyces yeasts can be divided into sensu stricto yeasts, including S. cerevisiae, and sensu lato yeasts. The sensu stricto mtDNA molecules are phylogenetically very closely related, are larger than 60 kb in size and show similar gene order configurations.19 On the other hand, the sensu lato mtDNA molecules are smaller in size and show a higher degree of phylogenetic and gene order diversity.19 So far, sensu lato yeasts have not been studied for the inheritance of their mitochondrial genome.

In this project, the sensu lato yeast, Saccharomyces castellii, was studied for transmission and organisation of the mitochondrial genome and compared to S. cerevisiae.

Section snippets

Heterothallic strains

S. cerevisiae mitochondrial inheritance has been studied using crosses between different mitochondrial mutants. Therefore, we intended to develop similar mutants in S. castellii. Upon mutagenesis a haploid heterothallic strain, Y239, was isolated from a diploid, well sporulating homothallic strain of S. castellii (Y188). Further on, haploid strains of the opposite mating type, carrying different auxotrophic markers, as Y252, Y257, Y258, Y319, Y320, and Y321, were developed using mutagenesis,

Discussion

While S. cerevisiae is one of the best studied organisms, so far very little molecular biology research has been done on its closest relatives belonging to the genus Saccharomyces. For example, S. cerevisiae mitochondrial genetics has been studied for over five decades by a number of laboratories and has been presented in hundreds of publications (reviews5., 11., 14., 22.). It is still only poorly understood if the molecular mechanisms operating during transmission of the S. cerevisiae

Yeast strains

The parental strains of S. cerevisiae and S. castellii used in this study are listed in Table 1. Previously, the diploid S. castellii, Y188, was sporulated and the mutagenised spores gave several haploid ho mutants.25 One of these strains, Y239, was further mutagenised and screened for auxotrophic mutants,25 thus giving Y252, Y257 and Y258. Haploid strains, with the opposite mating type, were obtained in the following way. Y258 was crossed with spores obtained from the diploid, HO+, strain Y235,

Supplementary Files

Acknowledgements

This work has been partially supported by grants from the Danish Research Council and the Novo Nordisk Foundation to J.P., and a grant from the Danish Research Foundation to Søren Brunak. The authors thank G.I. Naumov for his advice and help during development of the heterothallic strains.

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