Microbiol. Res.

(2002) 157, 197–200 (795)

http://www.urbanfischer.de/journals/microbiolres

Isolation and characterisation of nuclear mutants

with enhanced mitochondrial mutability in the fission yeast
Schizosaccharomyces pombe
Domenica Rita Massardo1, Luigi Del Giudice1, Angelica Del Giudice2, Klaus Wolf2
1
International Institute of Genetics and Biophysics, CNR, via G. Marconi 10, I-80125 Naples, Italy
2
Department of Biology IV (Microbiology), Aachen University of Technology, Worringer Weg, D-52056 Aachen, Germany

Accepted: April 4, 2002

Abstract
In this paper we report the isolation and preliminary charac-
terisation of nuclear mutants with increased mitochondrial
mutability in fission yeast. Screening of about 2000 clones
after nitrosoguanidine mutagenesis led to the isolation of ten
mutator mutants. For one of them (mut-1) we show that the
mutation is chromosomally encoded. The activity of the muta-
tor is restricted to the mitochondrial genome, since it increases
the mutation rate to mitochondrially encoded drug resistance
considerably, whereas the mutability of nuclear genes is not
altered.
and Fox 1992). Mitochondrial mutants completely
devoid of mitochondrial DNA can only be achieved
after a mutational event in one or more nuclear gene(s)
(Haffter and Fox 1992). In order to identify nuclear
genes involved in mitochondrial genome stability, we
started the isolation of nuclear mutants with an elevated
mutation rate of the mitochondrial genome, without
changing the mutability of nuclear genes.

Key words: mutator – mitochondria – fission yeast – drug
resistance
Introduction
The fission yeast Schizosaccharomyces pombe harbors a
small mitochondrial genome of only 15 to 20 kilobase
pairs (depending on the strain used ; Merlos-Lange
et al. 1987), which has been sequenced entirely (Sankoff
et al. 1992). Unlike the mitochondrial genome of Sac-
charomyces cerevisiae it is very stable. Mitochondrial
mutations are only obtained either in mutator strains
carrying a mutation in the mitochondrially encoded
rps3 gene (ribosomal protein of the small subunit of the
ribosome; formerly urf a; Seitz-Mayr and Wolf 1982;
Neu et al. 1998 ; Schäfer and Wolf in press) or after a
mutation in a hitherto unknown nuclear gene (Haffter
Corresponding author: L. Del Giudice
e-mail: Delgiudi@iigbna.iigb.na.cnr.it
Materials and methods
Mutants. Mutant strains ade6-424 and ura4-D18h– were
kindly provided by J. Kohli, University of Berne, Swit-
zerland.
Methods. Standard genetic methods were used accord-
ing to the fission yeast handbook (http://bio.uva.n1/
pombe/handbook/).
Media. Standard yeast media were were used as de-
scribed in the fission yeast handbook.
Results and discussion
For measuring mitochondrial mutation rates we used the
resistance to the inhibitor diuron (diur) which acts by
binding to cytochrome b and thus blocking electron
transport. Mutants resistant to this drug carry mutations
in the gene encoding apocytochrome b (cob) (Wolf and
Del Giudice 1988). In addition we used an inhibitor of
protein synthesis, erythromycin (Wolf 1995). Mutations

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 Fig. 1

198 Microbiol. Res. 157 (2002) 3


leading to resistance to this drug (eryr) are located in the
gene encoding the large ribosomal RNA (rns). Both
genes are located distantly on the mitochondrial genome
(Wolf and Del Giudice 1988). After mutagenesis with
nitrosoguanidine (survival rate 5%) (Del Giudice and
Puglisi 1974) cells were diluted and plated out onto glu-
cose complete medium at 28 °C to form single colonies.
After four days of incubation a small portion of the cell
material of several hundred colonies was picked and
each sample transferred to a well of microtiter plates
filled with 0.2 ml of glucose complete medium. After
incubation at 28 °C for four days the cultures reached a
titer of approximately 107 cells/ml. Aliquots of 0.1 ml
were spread an the surface of petri dishes containing
glycerol complete medium supplemented with 0.05%
glucose and 5 µg/ml of diuron (Fig. 1 A) or 400 µg/ml
of erythromycin (Fig. 1 C). More than 400 colonies per
plate, varying in size, were obtained after 14 days of
incubation on diuron medium, whereas 15 – 40 colonies
appeared an diuron plates when a wild type strain was
plated out (Fig. 1 B). From the mutator strain also more
than 400 colonies grew on erythromycin containing
medium, but no colonies were observed from the wild
type on erythromycin containing medium (Fig. 1D).
Fig. 1. Demonstration of mutator activity by plating. A. Colonies from a mutator mutant on diuron. B. Colonies from a
wild-type strain on diuron. C. Colonies from a mutator strain on erythromycin. D. No visible colonies from the wild type on
erythromycin. See text for further details.
Fig. 2. Demonstration of mutator
activity by drop-out. Dropping out
of mutator mutant and wild type
onto media containing diuron (A)
and to erythromycin (B) The drop
of wild type cells, which does not
contain papillae, is indicated by
the arrowheads. For further expla-
nation see text.
Genetic analysis revealed that the diuron resistant
mutants originating from the wild type were of nuclear
inheritance (K. Wolf, unpublished results) and could be
deficient in the uptake of diuron.
Since the next goal in this work is the identification
of the nuclear mutator gene(s) and since the only select-
able phenotype is the amount of colonies growing an
drug media, we had to scale down the selection process.
This was done by dropping out up to 100 spots per petri
dish.
An example that scaling down is possible, is given in
Fig. 2A for diuron and Fig. 2B for erythromycin. Only
for demonstration purposes the drop-out of only one
mutator and one non-mutator is shown on the plate. The
arrowheads mark the position of the non-mutator strain,
where no papillae were visible. Drop-out of mutator
strains gave between 20 and 50 papillae both, for diuron
and erythromycin.
The method also works with a metal device to direct-
ly replicate cells from a microtiter plate onto drug
medium (about 100 spots per plate).
The mitochondrial mutator encoded by the rps3 gene
gives not only rise to drug resistant mutants, but also
produces up to several percent of mitochondrial respira-
Microbiol. Res. 157 (2002) 3 199
tory deficient mutants. In order to test whether the
nuclearly encoded mutator also produces respiratory
deficient mutants, cells were plated onto petri dishes
with glucose complete medium (about 10,000 cells al-
together) and replica plated onto glycerol complete
medium plus 0.05% of glucose without drugs. No res-
piratory deficient colony was identified among 10,000
colonies.
Ten mutator mutants identified with the help of drop-
out technique (mut1–mut10) were purified three times
and again tested for their mutator property by dropping
out on drug plates. The percentage of drug resistant
papillae was the same as in the preliminary studies. This
demonstrates that the mutator is a stable heritable trait.
In order to test the possible influence of the mutator
an nuclear genes, we used a combined system to analyse
both, forward and reverse mutation. The ade6 mutation
leads to the accumulation of a red pigment on adenine
limiting medium. By plating out 6 × 106 cells an plates
containing complete medium with low amounts of yeast
extract (0.5%), cells form a lawn which is red due to the
adenine limitation. On top of this background white
colonies appear which can either originate from a back-
mutation of the ade6 mutation to the wild genotype or
from the mutation of any other gene which is epistatic
to ade6. Both in the wild type and in the mutant, about
6 – 20 white colonies appeared among 4 × 107 cells
plated altogether. In both cases half of them proved to
be prototrophic (due to reversion of ade6 to ADE6 or
to a suppressor mutation), the other half was still ade-
nine requiring (very likely to a mutation in another gene
in the adenine pathway). From these results it can be
concluded that the mutator exclusively acts on mito-
chondrial DNA but not on nuclear genes.
In order to prove that the mutator phenotype is due to
a single nuclear mutation, we crossed the mutant mut1
(ade6 -424 ura4-D18 h–) with the strain leu2-120 h+.
Analysis of 800 random spores showed a nearly 1:1
segregation of auxotrophic markers and the mating type
(data not shown). Concerning the mutator property,
47.5% of the progeny was wild type and 52.5% showed
a mutator phenotype which is identical to that observed
in the parental mutant strain. This demonstrates that the
mutator phenotype is due to a single nuclear mutation.
In order to isolate the wild type allele(s) for the muta-
tor trait, work is in progress to complement the mutant
with a genomic library of the wild type. The trans-
formants have to be analysed for the restoration of
normal (low) mitochondrial mutation frequency by the
technique outlined in this paper.
The role of the gene product needed for maintaining
the integrity of the mitochondrial genome could be
direct or indirect. A direct role could be imagined for a
subunit or a cofactor of the mitochondrial DNA poly-
merase. Alternatively the protein could also be engaged
200 Microbiol. Res. 157 (2002) 3
in repair processes of mitochondrial DNA, which so far
have not been studied in fission yeast. The mutants de-
scribed in this paper provide the basis for analysis of
genes involved in the stable maintenance of mitochon-
drial DNA. Since all eukaryotic organisms are faced
with the problem to conserve the integrity of their mit-
ochondrial genomes, these genes were probably con-
served during evolution. This would then allow the
isolation of homologous genes in humans by functional
complementation. This approach has already proved to
be successful in the characterisation of genes involved
in the regulation of cell cycle.
Acknowledgements
The technical assistance of Mr. G. De Simone (Naples) is
acknowledged. Research was supported by the Ministero delle
Politiche Agricole e Forestali (MIPAF), Italy, special grant
‘Piano Nazionale Biotecnologie Vegetali’ to L. D. G. and by
the Deutsche Forschungsgemeinschaft to K. W.
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