FEMS Yeast Research, 16, 2016, fow004
doi: 10.1093/femsyr/fow004
Advance Access Publication Date: 28 January 2016
Retrospective
RETROSPECTIVE
Piotr P. Slonimski – The Warrior Pope:
The discovery of mitochondrial (petite)
mutants and split genes
Piotr P. Slonimski† , Terrance G. Cooper∗ and Robert C. (Jack) von Borstel†
∗
Corresponding author: Department of Microbiology, Immunology & Biochemistry, University of Tennessee, Memphis, TN 38163, USA.
Tel: +1-901-448-6179; Fax: +1-901-448-3244; E-mail: [email protected]
†
This retrospective derives from an interview that Jack von Borstel conducted over a period of two weeks in 1996 with Piotr Slonimski in his laboratory in
Gif-sur-Yvette. Piotr was asked to write a chapter for the Hall and Linder book ‘The Early Days of Yeast’. After one page of writing he quit, feeling that he
was writing an obituary. Fortunately, Jack convinced him to share his story verbally, and Piotr and Jack collaboratively wrote it down. It remained buried
in Jack’s papers until he sent it to Terry Cooper in August, 2010 after the Vancouver Yeast meeting and asked if he would finish the bibliography and
publish it. This is Slonimski and von Borstel’s manuscript, to whose title was added ‘The Warrior Pope’ and which was edited only to shorten it. Piotr
Slonimski and Jack von Borstel are both deceased. Therefore, Terrance G. Cooper will serve as the corresponding author.
Editor: Terrance Cooper
Keywords: retrospective; Slonimski; mitochondrial genetics; petite mutants; split genes; introns; exons; polish underground
army; world war II
PRE-PRO-HISTORY OF MITOCHONDRIAL
GENETICS
Memoirs are more often written by generals than by scientists.
Although the highest rank I received during my five years in the
Polish Underground Army, Armia Krajowa, did not exceed that of
a corporal-chief (not a very brilliant accomplishment!), it would
have been much easier, and possibly even more interesting for
the readers, to write about this period. At least there was action,
blood, fantastic friends and beautiful girls: a more-or-less classical potpourri of clichés that a survivor (who, in the language we
are accustomed to, is nothing more than a random colony that
survives on a petri plate after a drastic treatment with a highly
efficient inhibitor) might use. But this is not what I’m asked to
do. I have to write about yeast genetics.
Strangely enough, there is a link between my interest in genetics and my ancient terrorist activities. It is an indirect, devious link, but an essential link, nevertheless.
As a child, I was interested in natural sciences, and certainly, this was because of a long-standing family tradition. My
father was an embryologist and histologist investigating blood
formation at the University of Warsaw. Grandfathers, Chaim
Zelig Slonimski and Abraham Stern, were mathematicians and
astronomers. I collected beetles, grew tadpoles and parame-
cia in stinking water, and read ‘Microbe Hunters’ and Claude
Bernard.
AWARENESS OF THE FATALISTIC KIND
In 1943, von Bertalanffy led me to Franz Moewus, and Franz
Moewus inspired me to work on the chemical nature of the gene.
And here is how that came about.
Joining the Army at the age of 16 1/2 is not a good idea in
peace-time, but the notion is even less romantic in war-time
when there were two opposing fronts with a German blitzkrieg
force at one of them and the other populated by Russian forces
ready to pounce, and I as a young soldier was in between. Hitler
and Stalin had decided to divide Poland between them, and I
was in the Polish army facing the USSR troops. That lasted but a
few days, and then I was in a prisoner-of-war camp deep in the
Soviet Union at a place called Starobielsk.
There were many thousands of prisoners there, and by accident I ran into my father who was housed in another barrack.
One night there was lots of shouting and shooting at my father’s barrack, where a prisoner had just escaped. I went to stay
in the barrack with my father, and everyone was called outside to count off. Although there was one man missing, the
Received: 16 January 2016; Accepted: 22 January 2016
C FEMS 2016. All rights reserved. For permissions, please e-mail: [email protected]
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FEMS Yeast Research, 2016, Vol. 16, No. 2
less, but my friend, who feared nothing, replied sarcastically, ‘A
disassembled machine gun’. The soldiers laughed harshly, and
one of them hit him hard on the side of the face for making such
a stupid joke, and said, ‘Get on with you!’ We trudged away, pretending our suitcases were full of air.
After the uprising by the Polish underground and the levelling of Warsaw, I went to Cracow and continued my medical studies. I worked at the Institute for Embryology. After the
war ended in 1945, I was arrested and sent to prison by the Polish communists for being a member of the Polish underground
army. Fortunately, my uncle, the poet, was being wooed by the
Polish communist government to return to Poland. He returned,
and courageously observed that his nephew, the only surviving
member of his family, had disappeared, and he demanded that I
be found within 24 h, or he would return to England. I was found
within 48 h and released. My uncle decided to remain in Poland.
So I returned to Cracow to finish my thesis on the origin of the
pineal gland in the axolotl (Slonimski 1947). I succeeded in solving the old puzzle of whether the single organ was derived from
one anlagen or from two (it was from two).
PURSUIT OF THE GENE
Figure 1. Piotr and his famous pipe in a plenary session at the International Conference on Yeast. Genetics and Molecular Biology, Prague, Czech Republic, 2005.
head-count showed that the correct number of prisoners were
there. The commanding officer soon discovered that I was the
replacement for the missing head. When called in front of him,
he saw a young, small, sick and hungry waif in a uniform much
too large. The officer said, ‘We are not swine; we don’t keep children as prisoners of war’, and offered to let me return home.
I refused, saying, ‘I don’t want to go home; I want to stay with
my father’. So then my father was brought forward, and he suggested to the officer that we both go home. He was a physician,
and he convinced the officer that physicians should be in Warsaw where there was plenty of work for them to do. He also gave
the officer a Waterman gold fountain pen.
So beginning in late November 1939, or early December, my
father and I walked back to Warsaw. We arrived just before
Christmas. In Warsaw, masses of people were ordered to go to
the ghetto, but my father simply refused. He just wanted to stay
with my mother who had been injured by shrapnel at the time
of the dual invasion of Poland by Germany and the USSR.
From 1940 to 1943, I studied medicine with the underground
faculty of medicine. There were very few students, and the training by the faculty was excellent because it was almost one-onone. All students and teachers were volunteers. Discovery of one
or the other of them was punishable by death, and this happened more than once. The black market kept me alive; everything was for sale. I was paid 4 British pounds a month to be
in the underground army. There I also finished the underground
training needed to become an officer in the underground. During this time my uncle, the poet Antoni Slonimski, was a refugee
in England.
One day my friend and I were carrying, in two suitcases, a disassembled old-fashioned machine gun, the kind that was placed
on a tripod. Two members of the German occupation forces confronted us, and asked us what we were carrying. I was speech-
But to return to Warsaw in 1943, one evening, a small force was
sent to attack the military police to obtain arms and ammunition; mostly, the ammunition was needed. We killed the two military police, who were on duty, and I found two volumes by Ludwig von Bertalanffy, which I took with me. These books excited
my interest in genetics, because one chapter was on the amazing work of Franz Moewus, who had found that the gene was
crocetin. At the end of the chapter, there was a short discussion of the work of George Beadle and Boris Ephrussi, but von
Bertalanffy made it clear that their chemistry was not as good
as the chemistry of Franz Moewus. This chapter excited me immensely, and I started to consider how to find out more about
the chemical nature of the gene. So, in 1947, after completing
my thesis, I decided to follow the dictates of my 1943 discovery
of the gene. Moewus was in Germany, and I didn’t want to go to
Germany. Beadle was at the other end of the United States, so I
decided to work with Boris Ephrussi, who, for some obscure reason, I believed to be in Brussels. I went to Brussels. Jean Brachet,
who knew my supervisor in Cracow, very kindly put me in touch
with Ephrussi. I went to Paris, and told Ephrussi that I wanted
to work on the chemical nature of the gene in relation to development. Ephrussi told me he would take me on the condition
that I would learn to speak French in one month. So I found a
French-speaking Swiss woman. We went together to live in Lausanne for a month bolstered by the universally held philosophy
that in order to learn a foreign language properly, one had to live
with the language 24 h a day. She was willing. So was I.
LIFE NEED NOT BE DEFINED BY THE PRESENCE
OF CYTOCHROMES
One month later I returned to Paris. In March 1947, Ephrussi
found funding for me and put me on the problem of transplantation of the nucleus of a sea urchin from one egg to another.
I accomplished the transplant, but to my chagrin, the injected
nuclei always were spat out of the eggs.
Then Ephrussi, who had anticipated that nuclear transplantation might be a failure, suggested a problem associated
with the petite mutants in yeast he had discovered earlier.
Specifically, he believed that the petite yeast must be deficient in
Slonimski et al.
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Figure 2. Poet Antoni (left) and nephew Piotr Slonimski. Scanned from Antoni Slonimski:Alfabet Wspomnień, PWN, Warsaw, Poland, 1975. Open source photograph.
Figure 3. Piotr Slonimski with his mentor, Boris Ephrussi (right). Paryz, 1949–1950. From Genetk I historia (Genetist and History) by R. Jarocki, Rosner and Wspolnicy,
2003.
melibiose metabolism, because Jacques Monod had found a mutant strain of Escherichia coli that had a small colony phenotype.
This E. coli mutant strain was deficient in maltose metabolism.
Also, Sol Spiegelman and Carl Lindegren had found that yeast
mutants deficient in galactose grew into small colonies.
I began using a Warburg apparatus to check the fermentation rate of all of the sugars, and found myself in the worst of
all scientific worlds. There was a 5%–10% difference from wildtype (grande) cells with each of the sugars, and every experiment
was reproducible. No conclusion could be drawn. So then, in order to try out another approach, I checked the respiration rate,
and found a 100% difference. The petite mutants did not respire.
There must be a leak in the manometer. There was no leak. I
found a hand spectroscope, and there were no cytochromes a or
b in the mutant yeast. This was in July or August of 1947.
Whenever one finds himself at odds with the ‘conventional
wisdom’, as we have called it for a long time, or a ‘paradigm’,
as it was renamed by Thomas Kuhn [The Structure of Scien-
tific Revolutions], there is nothing to expect other than hell from
the great unknown presences, and averse and chilling responses
from the people you thought were your friends. There is something profane about upsetting the conventional wisdom. It does
not fit into the category of an unexpected result, where, to paraphrase Louis Agassiz, the first response is, ‘We don’t believe it’,
the second response is, ‘Someone else discovered it first’, and
the third response is, ‘We’ve always known it’. The dispelling of
a paradigm is tantamount to being asked to be ostracized, and
one can expect to be flayed publicly.
At the time I found that petite yeast did not contain cytochromes, the biochemists had certified that cytochromes were
necessary in every living organism, and that the presence of cytochromes was truly a definition of a living organism. There was
only one thing to do. Confront the arbiters of the conventional
wisdom. David Keilin was in the Multino Institute at Cambridge
University where he had found a rich source of cytochromes
in the flight muscles of bees. He recognized the richness of
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or more of petite cells packed into the tip of a conical centrifuge
tube. Only cytochromes a and b were deficient. It is clear now
that cytochromes a and b are encoded by the mitochondrial DNA
and cytochrome c is encoded by the nuclear DNA. So simple to
say in a few words—so difficult to have worked out in the laboratory.
BIOCHEMICAL GENETICS OF MITOCHONDRIA
The 1960s were good years for yeast genetics. Yeast geneticists
everywhere were just beginning to understand what was similar and what was dissimilar about eukaryotes and prokaryotes.
Also the biochemistry and genetics of yeast were beginning to fit
perfectly together. I remember finding a bright red petite colony,
just one, after treating yeast with a mutagen; that mutant appeared to have some interesting properties. The mutant was nuclearly inherited (it segregated 2:2 in a cross), and it appeared
from its anaerobic and aerobic growth habits on different carbon sources, to be a mutant in the oxidative phosphorylation
pathway. When we published our paper (Kovác, Lachowicz and
Slonimski 1967), it was extremely difficult in the world of biochemistry to separate the steps of oxidative phosphorylation.
Our paper was published as a model way to explore difficult
biochemical pathways by a genetic method, using biochemistry
as a way to describe the phenotype. What pleases me most
was that we predicted, from genetic reasoning, that the mutant,
op1, was likely to be the last step of the oxidative phosphorylation pathway, and it most likely encoded the adenine nucleotide
translocation step in the pathway. This turned out to be true. I
gave the mutant to Fred Sherman, who confirmed our results,
but he changed the name of my mutant gene from op1 to pet9.
The nomenclature rho- and rho+ came to us from Marquardt in
Freiburg. Petites inherited by the nuclear genome are known as
‘pet’ mutants, and mitochondrially inherited petites are known
as ‘rho-’ or ‘mit-’ mutants.
Figure 4. Piotr at Princeton University, 1952.
cytochromes probably had to do with the tremendous energy
required to keep bees in the air. He was the reigning king of cytochromes at the time.
Therefore, I went to England in 1948. I remember going to Professor Keilin’s office, and this friendly little man stepped out into
the hall. I told him that I had a yeast mutant that grew perfectly
well and did not have cytochromes. He asked me to give him a
sample. I did. He walked back into his office, and I began to pace
outside of his door. I paced and paced. Not such a long time after,
although it seemed forever at the time, Professor Keilin stepped
out of his office, where he had examined the cells with a spectroscope, and said, ‘You are right’. It was like being knighted by
the king.
In 1949, Ephrussi’s group published seven papers in the Annals of the Pasteur Institute, and in one that I co-authored with
him (Slonimski and Ephrussi 1949) we dared to say, in as careful
a way as we could say it, that these ‘petites’ might be mitochondrial mutants. But proving this took time. But in time, we and
others demonstrated it to be true. The petite yeast cells were
not completely deficient in cytochromes. Colonies on plates are
bone white in colouration. However, cytochrome c is present in
abundance, as can be seen from the pink colouration of a gram
THE GENETIC BASIS OF THE PETITE
PHENOMENON
During the decade of the 1950s into the mid-1960s, Ephrussi’s
group developed the genetics and biochemistry for handling
every aspect of petite yeast that we could imagine. Chen,
Ephrussi and Hottinguer found that some of the petites segregated during meiosis and therefore existed in the nuclear
genome. When Herschel Roman came to Paris on his first
sabbatical leave, he worked together with Boris Ephrussi and
Helene de Margerie-Hottinguer, and discovered suppressive
petites (rhos ). This was a breakthrough, because partial petites, although we didn’t understand them, opened the way
for us to make genetic crosses with organelles in the cellular cytoplasm, and to show that these organelles were indeed
mitochondria.
We also found a way to construct petite yeast that contained
no mitochondrial DNA whatsoever (Slonimski, Perrodon and
Croft 1968). Ethidium bromide exhibits a long binding time to
mitochondria, and to practically everything else as well, including the glass walls of growth tubes. We showed that cells lost
their mitochondrial DNA in the presence of ethidium bromide,
even when treated before growth was initiated. These are now
known as ‘rho0 ’ mutants. This is an excellent way to make a perfect control for eliminating all interference from mitochondria
when studying a cellular reaction.
Slonimski et al.
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Figure 5. Piotr receiving the Honoris Causa degree in the Collegium Anthrologicum at the University of Wroclaw in 1976. Courtesy of Library of Wroclaw University.
Figure 6. Piotr showing the sequence of his split BOX3 gene.
MAPPING THE GENES IN THE YEAST
MITOCHONDRION
Although we struggled mightily with the suppressive mutants,
the omega initiation point (polarity point), and the presence of
cytochromes a and b and cytochrome oxidase in the mitochondrial genome, it was Thomas and Wilkie who opened the door to
mapping mitochondrial genes by their discovery that mitochondrial antibiotic resistance mutants could be easily induced, and
recombination between the mutations was detectable. We now
had enough knowledge of mitochondrial interactions within the
cell to know that we could map genes and develop rules for recombination and segregation of the mitochondrial gene. So we
began. A summary of our work appears in Coen et al. (1969).
One of the obvious properties of mitochondria is that they
exist in the cell as multiple copies. We used this property of ‘amplification’ with suppressive mutants to build multiple copies of
short pieces of DNA to produce a selective purification of different mitochondrial tRNAs, rRNAs and mRNAs (Faye et al. 1973)
This was a large piece of work involving our laboratory and that
of Murray Rabinowitz for several years.
Finally, we accomplished a cytogenetic analysis that demonstrated that the suppressive petite mutants, those retaining
some DNA, always retained the remaining DNA in a circular configuration.
INTERVENING SEQUENCES WITHIN THE
CODING REGIONS OF MITOCHONDRIAL GENES
From a sporadic pile of inconsistencies in studies of mutants of
BOX3, we were on the trail of introns (Slonimski et al. 1978a,b) at
about the same time that Phillip Sharp and Richard Roberts were
developing their findings for which they received the Nobel Prize
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Figure 7. Piotr in his office at Centre de Genetique Moleculaire in
Gif-sur-Yvette. Website of the city of Gif-sur-Yvette, http://www.villegif.fr/mairie/democratie-locale/concertation-sur-les-noms-du-moulon/.
Figure 8. Piotr at Cold Spring Harbor Mitochondrial Mtg, 1981.
in 1993. What we found was that an intron for the BOX3 gene
that encoded cytochrome b contained a splicing protein that in
fact removed the intron itself from the BOX3 mRNA (Lazowska,
Jacq and Slonimski 1980). This turned out not to be the usual
way that the cell carries out splicing reactions, but it certainly
was a clear demonstration of the way that at least one mechanism was selected to solve evolutionary problems. [Slonimski
was nominated for the Nobel Prize for this work.]
THE IMPORTANCE OF INTERNATIONAL
MEETINGS
From 25 June to 3 July, 1948, a very important meeting took place
in Paris. It was arranged by Andre Lwoff. It was entitled ‘Unités
Biologique Douées de Continuité Génétique’ [Endowed Organic
Units of Genetic Continuity] and was published soon thereafter.
All of the important microbial geneticists were present. It was
there that Boris Ephrussi announced the work of his laboratory,
‘Action de l’acriflavine sur les levures’. His talk was a big hit. It
was really the launching pad for yeast to be taken seriously and
put in its rightful place as a serious contender in the world of
microbial genetics. Also, it was my first scientific meeting, and
first scientific meetings are remembered very clearly.
In those days international meetings about current research
were small, rare and exciting events. News did not travel by way
of fax or the internet; it travelled via seminars and small meetings, with pre-publication findings being disseminated back to
the research groups.
It was at one of those meetings that I became known as
Mrs Magni. There was to be a small meeting of microorganism
Figure 9. Piotr and the International Community of Yeast Genetics and Molecular
Biology’s first Pope, Hershel Roman.
Slonimski et al.
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geneticists in Spallanza, and we still had difficulties in traveling
from country to country. As a Pole residing in France without a
passport, there was no possible way for me to get permission
from the police to travel to Italy. Moreover, wherever one stayed,
the hotel records, and often our passports, were given to the local police daily to make certain that everything was in order. I
wanted to go to that meeting in Spallanza. So I decided to climb
over the Alps.
The hike was arduous. I actually succeeded in blazing a new
trail through a pass in the Alps that was both unused and unguarded. Eventually, I arrived in Spallanza, and had to find a hotel, which of course, I could not do because of not wanting my
whereabouts to be known by the authorities. Giovanni Magni
came up with the solution, saying ‘I have an extra bed in my
room, and you will be called Mrs Magni!’ That was a perfect solution because wives who accompanied husbands did not have
to submit passports. After that hike I needed a bath and a good
night’s sleep. I got both. Except that Giovanni snored.
INTERNATIONALIZATION
OF YEAST GENETICS
Figure 10. Piotr Slonimski.
Figure 11. Cover of Robert Jarocki’s biography of Piotr, ‘Genetist and History’.
The International Conferences on Yeast Genetics had begun in
1961, although not everyone in the yeast community was aware
of it. We held the first really scientific Conference on Yeast Genetics in 1963 at Gif-sur-Yvette just before the International
Congress of Genetics in The Hague. About 30 people attended
from all over the world, and we had a joyful time together; good
food and good wine are great liberators of the spirit and the
tongue. The first yeast genetics meeting had been held in Carbondale, Illinois, two years earlier, gathering as many people
from as many labs everywhere (all 11 of them) to tackle the terrible problem of nomenclature which was consuming everyone’s
energy. Giovanni Magni called the Carbondale meeting ‘Conference Number Zero’ because it was mostly a tugging match
among laboratories to determine which gene should be called
what. Fortunately for both of us, the problems were worked out
with neither Herschel Roman nor I being present, although he
and I attended all Conferences thereafter.
In 1982, Herschel Roman had decided to resign as the ‘Pope’
of the yeast community, as Giovanni Magni had so aptly named
him. Urs Leupold managed the election by sending letters to everyone in the yeast community asking them to suggest names.
Urs counted the ballots, and I had received the most nominations. That was it—no run-offs among those receiving the most
nominations. Jack von Borstel announced my election to the assembled participants at the 1984 Conference in Edinburgh by
saying, ‘The Catholic Church is not the only international organization with a Polish Pope!’
The International Conference on Yeast Genetics and Molecular Biology, as it subsequently became known, is a haphazardly
managed organization, fully suited to my inclination never to
answer a letter. The Finance and Policy Committee (one member
from every nation present at the Conference) gathers together
every two years at the time of the Conferences, and we all try to
convince someone to manage the Conferences during the next
2-, 4- and 6-year periods. We also try to deal with little crises like
having to skip one year while the US Yeast Cell Biology meetings
and the US Yeast Genetics and Molecular Biology meetings were
put in different years.
[Piotr preferred not to talk about the many awards he
received. They are enumerated along with a description of
his contributions to sequencing of the yeast genome in the
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FEMS Yeast Research, 2016, Vol. 16, No. 2
C.J. Herbert article in Yeast. Additional material may be found
at http://www.normalesup.org/∼adanchin/lectures/slonimski
.html.]
ACKNOWLEDGEMENTS
Life was infused into the text of this retrospective through the
generosity of Drs Laura Frontali, Joanna Rytka and Nicola Altamura who provided the photographs for it. Their effort and generosity is gratefully acknowledged.
Conflict of interest. None declared.
REFERENCES
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methodology and phenomenology. In: Symposium 24 of the
Society for Exprimental Biology, Control of Organelle Development.
Cambridge University Press, 1969, 449–96.
Faye G, Fukuhara H, Grndchamp C et al. Mitochondrial nucleic
acids in the petite colonie mutants: deletions and repetitions
of genes. Biochimie 1973;55:779–92.
Kovác L, Lachowicz TM, Slonimski PP. Biochemical genetics of
oxidative phosphorylation. Science 1967;158:1564–7.
Lazowska J, Jacq C, Slonimski PP. Sequence of introns and flanking exons in wild-type and box3 mutants of cytochrome b
reveals an interlaced splicing protein encoded by an intron.
Cell 1980;22:333–48.
Slonimski PP, Claisse ML, Foucher M et al. Mosaic organization
and expression of the mitochondrial DNA region controlling
cytochrome c reductase and oxidase III. A model of structure
and function. In: Bacila M, Horecker BL, Stoppani AWN (eds).
Biochemistry and Genetics of Yeast. New York: Academic Press,
1978b, 391–401.
Slonimski PP, Ephrussi B. Action de l’acriflavin sur les levures. V.
Le système des cytochromes des mutants ‘petite colonie’ de
la levure. Ann Institute Pasteur 1949;77:47–64.
Slonimski PP, Pajot P, Jacq C et al. Mosaic organization and
expression of the mitochondrial DNA region controlling
cytochrome c reductase and oxidase. I. Genetic, physical, and complementation maps of the box region. In:
Bacila M, Horecker BL, Stoppani AWN (eds). Biochemistry
and Genetics of Yeast. New York: Academic Press, 1978a,
339–68.
Slonimski PP, Perrodon G, Croft JH. Ethidium bromide induced
mutation: Complete transformation of cells into repiratory
deficient non-chromosomal petites. Biochem Bioph Res Co
1968;30:232–9.