Селен і дріжджі. Генетичні механізми толерантності дріжджів до сполук селену та їхніх аналогів
Селен (Se) і його сполуки проявляють токсичну і канцерогенну дію на організми людини і тварин, а в малих кількостях цей мікроелемент відіграє суттєву роль для живих істот. Тому важливо встановити молекулярні основи токсичності Se і резистентності клітин до нього. Значну кількість досліджень зазначен...
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| Published in: | Біополімери і клітина |
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| Date: | 2006 |
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Інститут молекулярної біології і генетики НАН України
2006
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| Cite this: | Селен и дрожжи. Генетические механизмы толерантности дрожжей к соединениям селена и их аналогам / Н.Н. Стенчук, Л.Б. Чабан, М.В. Гончар // Біополімери і клітина. — 2006. — Т. 22, № 1. — С. 3-17. — Бібліогр.: 70 назв. — укр. |
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Digital Library of Periodicals of National Academy of Sciences of Ukraine| _version_ | 1859998146381217792 |
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| author | Стенчук, Н.Н. Чабан, Л.Б. Гончар, М.В. |
| author_facet | Стенчук, Н.Н. Чабан, Л.Б. Гончар, М.В. |
| citation_txt | Селен и дрожжи. Генетические механизмы толерантности дрожжей к соединениям селена и их аналогам / Н.Н. Стенчук, Л.Б. Чабан, М.В. Гончар // Біополімери і клітина. — 2006. — Т. 22, № 1. — С. 3-17. — Бібліогр.: 70 назв. — укр. |
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| description | Селен (Se) і його сполуки проявляють токсичну і канцерогенну дію на організми людини і тварин, а в малих кількостях цей мікроелемент відіграє суттєву роль для живих істот. Тому важливо встановити молекулярні основи токсичності Se і резистентності клітин до нього. Значну кількість досліджень зазначеної проблеми здійснено на бактеріях. Можливість використання молекулярно-генетичних методів зробила дріжджі (в основному Saccharomyces cerevisiae) зручною модельною системою для вивчення механізмів резистентності еукаріотних клітин до селену на молекулярному рівні. Виходячи з цього в огляді підсумовано дані, особливо генетичні, щодо механізмів чутливості/резистентності дріжджів до селенових сполук.
Selenium (Se) and its compounds have toxic and carcinogenic effect on animal and human beings, but in small concentrations this microelement is essential for life. Therefore, it is important to establish the molecular basis of Se toxicity and cell resistance to this metaloid. Many investigations of this problem have been carried out on bacteria. Genetic approaches, available for the yeasts, made these eukaryotic microorganisms, especially S. cerevisiae, a convenient model for the investigation of molecular mechanisms of Se tolerance. This review summarizes the knowledge of genetic mechanisms involved in Se tolerance in yeasts.
Селен (Se) и его соединения оказывают токсическое и канцерогенное действие на организмы человека и животных, а в небольших количествах он является существенным микроэлементом для живых существ. Поэтому важно установить молекулярные основы токсичности Se и резистентности к нему клеток. Значительное количество исследований этой проблемы проведено на бактериях. Возможность использования молекулярно-генетических методов сделала дрожжи (в основном Sacccharomyces cerevisiae) удобной модельной системой для изучения механизмов резистентности эукариотных клеток к селену на молекулярном уровне. Исходя из этого в данном обзоре суммированы данные, особенно генетические, полученные при исследовании механизмов чувствительности/резистентности дрожжей к селеновым соединениям.
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| first_indexed | 2025-12-07T16:35:05Z |
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Se le nium and yeast: Ge netic mech a nisms of the yeast
tol er ance to se le nium com pounds and their analogs
M. M. Stenchuk1, L. B. Chaban1, 2, M. V. Gonchar1, 2
1Institute of Cell Biology, NAS of Ukrain
14/16, Dragomanov Str., 79005 Lviv, Ukraine
2Ivan Franko Na tional Uni ver sity of Lviv
4, Hrushevskyi Str., 79005 Lviv, Ukraine
gonchar@biochem.lviv.ua
Se le nium (Se) and its com pounds have toxic and car ci no genic ef fect on an i mal and hu man be ings, but in small con cen tra -
tions this microelement is es sen tial for life. There fore, it is im por tant to es tab lish the mo lec u lar ba sis of Se tox ic ity and cell
re sis tance to this el e ment. Many in ves ti ga tions of this prob lem have been car ried out on bac te ria. Ge netic ap proaches,
avail able for the yeasts, made these eukaryotic mi cro or gan isms, es pe cially S. cerevisiae, a con ve nient model for the in ves ti -
ga tion of mo lec u lar mech a nisms of Se tol er ance. This re view sum ma rizes the knowl edge of ge netic mech a nisms in volved in
Se tol er ance in yeasts.
Key words: yeast, se le nium, tol er ance.
In tro duc tion. Se le nium is a vi tally im por tant microelement
for most or gan isms – from bac te ria and al gae to mam mals.
Though it was dis cov ered by J.J. Bercelius in 1817, it was
rec og nized as an im por tant microelement only in 1957,
and be fore the tox ic ity of this el e ment was the main sub ject
of re searches. The study of se le nium role in bi ol ogy, med i -
cine and vet er i nary may be a vivid ex am ple of tight con nec -
tion of ba sic and ap plied sci ence. Se le nium is an el e ment of
the 4th group of Mendeleev’s pe ri odic sys tem, there fore, it
re veals non-me tal lic as well as me tal lic prop er ties. It com -
poses chem i cal com pounds (in or ganic and or ganic) anal o -
gous to sul fur com pounds, namely sel e nite (SeO3
2-),
selenate (SeO4
2-), selenides, selenomethionine,
selenocysteine, etc. The main source of se le nium for hu -
mans and an i mals is the plants that as sim i late this el e ment
de pend ing on its con cen tra tion, avail abil ity, forms of se le -
nium in the ground, and plant va ri ety. The most sta ble
forms of se le nium in the ground are sel e nite and selenate.
The to tal amount of se le nium in the ground has a broad
range from 0.000005 to 1.2 g/kg. Such a broad range re sults
in the neg a tive con se quences for or gan isms which are in
the ar eas of ul ti mate con cen tra tions – suf fer ing from the
lack of se le nium as a microelement or from its ex ces sive
amount. Mean while the range of se le nium con cen tra tions
from min i mal re quire ments to le thal dose is very nar row.
Thus, min i mum fod der level for an i mals is about 0.05-0.10
mg/kg of dry fod der, while 2-5 mg/kg is toxic al ready [1].
Ad mis si ble con cen tra tions of se le nium in food prod ucts
for a hu man are con sid ered to be 0.1-1.0 mg/kg, and rec -
om mended se le nium dose for pre ven tive treat ment is 5
мg/kg of hu man weight [2]. A nar row range be tween use ful
and harm ful lev els com pli cates pre ven tive treat ment and
ther a peu tic us age of selenocompounds for hu mans and an -
i mals, so fur ther re searches of sen si tiv ity/re sis tance mech -
a nisms of or gan isms to se le nium com pounds are worth
con sid er ing.
3
ISSN 0233-7657. BIOPOLYMERS AND CELL, 2006, VOL. 22, ISS. 1. TRANS LATED FROM UKRAI NIAN
ã M. M. STENCHUK, L. B. CHABAN, M. V. GONCHAR
REVIEWS
Se le nium and its com pounds are used in elec tronic in -
dus try at pro duc tion of copy ing equip ment and
photoelements, and in build ing in dus try at pro duc tion of
glass ar chi tec ture blocks, col or ing agents within plas tic and
paints, so there is an ur gent ques tion of bioremediation of
en vi ron ment pol luted by se le nium, us ing yeast and other
mi cro or gan isms in par tic u lar, which ob vi ously should have
max i mum re sis tance to the toxic ac tion of se le nium. The
prob lem of tol er ance arises also while us ing such met al -
loid-con tain ing poi sons as fun gi cides and in sec ti cides, be -
cause pests and par a sites of ten show un de sired high re sis -
tance to metalloids which pro hib its ef fec tive pro cess ing [3,
4].
In or ganic se le nium can be as sim i lated by all kinds of
or gan isms though the ef fec tive ness of com pounds
bioutilization de pends on their chem i cal na ture and kind
of or gan isms. Thus, as sim i la tion of selenate which is the
main source of se le nium in the ground oc curs in plants as
well as in yeasts through the path way of sul fate re duc tion,
as cor re spond ing en zymes do not dis crim i nate sul fur and
se le nium: selenate ч adenosyl-phosphoselenate ч
adenosine-3-phospho-5-phosphoselenate ч sel e nite ч
selenide ч selenocysteine (SeCys) ч selenomethionine
(SeMet). In Archea and Eubacteria (prob a bly, not in all of
them) and in an i mals (in ver te brate and ver te brate) se le -
nium can be in cluded into polypeptide chains as so called
twenty first amino acid SeCys [5]. Thus, the syn the sis of
these pro teins which are usu ally called selenoproteins
should in clude selenospecific mech a nisms which would
dif fer en ti ate be tween se le nium and sul fur. UGA is the
codon which pro vides SeCys bind ing, and which func tions
as non sense-codon in other con di tions (at the end of a
gene). This way of selenocysteine in clu sion is spe cific – Se
be comes the com po nent of ac tive cen ters of a num ber of
selenoproteins, for in stance, bac te rial formate
dehydrogenases, eukaryotic glutathione peroxidases and
thioredoxin re duc tases func tion ing as cat a lyt i cally ac tive
heteroatom. Dur ing sev eral de cades af ter the dis cov ery of
se le nium as an im por tant microelement, more than 20
eukaryotic and 15 prokaryotic selenoproteins con tain ing
twenty first amino acid selenocysteine were iden ti fied. The
ma jor ity of these pro teins par tic i pate in re dox-re ac tions
through selenocysteine which acts as an im por tant com po -
nent of the cat a lytic cy cle. They play an im por tant role in
the me tab o lism of ma lig nant tu mors, in the con trol of cell
di vi sion, ox y gen me tab o lism, de tox i fi ca tion pro cesses,
apoptosis in duc tion, im mune sys tem func tion ing etc.
Selenoproteins have not been dis cov ered in yeasts and
higher plants [6, 7].
The sec ond way of se le nium in cor po ra tion in pro teins
is not spe cific – it can sub sti tute sul fur with the for ma tion
of free amino ac ids of selenocysteine and
selenomethionine which are in cluded non-spe cif i cally
into pro teins in stead of cysteine and methionine, re spec -
tively. Such pro teins are not called selenoproteins. With
the us age of bac te ria as model sys tems it was shown that the
ef fect of sub sti tut ing methionine and cysteine for their se -
le nium de riv a tives de pended on the num ber of sub sti tu -
tions and their lo ca tion in polypeptide – they can ei ther in -
crease or de crease its func tion even to zero, or not in flu -
ence it [8-10]. This way func tions at all the lev els of liv ing
sys tems, in yeasts, in par tic u lar, ir re spec tive of the pres -
ence of syn the sis mech a nisms of selenoproteins in them.
Due to its an ti ox i dant prop er ties se le nium sup ple ment
to the diet in cer tain con cen tra tion pro tects hu man or gan -
ism from car dio vas cu lar dis eases, vi ral in fec tions, rheu ma -
toid ar thri tis, liver dis eases and some forms of can cer. The
pres ence of se le nium in fod der in op ti mum con cen tra tions
re sults in de crease of tu mors oc cur rence fre quency in ex -
per i men tal an i mals. Many years of the re search in the USA
on 1300 pa tients showed that con sump tion of se le nium for
6 years in the amount of 200 мg per day in the form of
Se-en riched yeast bio mass de creased to tal fre quency of
ma lig nant tu mors by 50% [11]. Its de fi ciency in food prod -
ucts and fod der for an i mals re sults in a num ber of dis eases
(in hu mans – Keshan syn drome (cardiomiopathy),
Kashin-Beck (chondronecrosis)) etc. Epidemiologic study
in the USA showed the cor re la tion be tween low amount of
se le nium in food prod ucts and in creased fre quency of
some kinds of tu mors in hu mans [11].
Yeast as a model or gan ism in the study of as sim i la tion
and tox ic ity of se le nium com pounds. The role of se le nium in
bi ol ogy, med i cine and vet er i nary is de scribed more sub -
stan tially in the re views [1, 5, 6, 11, 12], where it mainly
goes about bac te ria, plants and higher eukaryotes. The aim
of this re view is to sum ma rize the re sults of the study of se -
le nium com pounds in flu ence on the yeast cells. These mi -
cro or gan isms are one of the most stud ied eukaryotic cells
which have a num ber of fun da men tal prop er ties com mon
with the mam ma lian cells. This al lows us ing them to study
met a bolic pro cesses which are not stud ied yet or enough in
lower, as well as in higher eukaryotes. In this re view we
con cen trated on the main con cepts of phys i ol ogy and bio -
chem is try of se le nium in yeasts, mainly in Saccharomyces
cerevisiae, in clud ing se le nium con sump tion and trans port
as well as mech a nisms of se le nium tox ic ity and yeast tol er -
ance to it.
As it was al ready stated, that se le nium me tab o lism in
yeast does not in clude selenoproteins syn the sis. Full se -
quenc ing of the yeast ge nome did not re veal intragenic opal
codons UGA, lo cated in an open frame se quences with
AUG [5]. It should be noted that it con cerns only
4
STENCHUK M. M. ET ALL.
Saccharomyces. Se le nium me tab o lism in non-con ven -
tional yeasts has not been stud ied un til pres ent, so it is pos -
si ble that yeast selenoproteins would be re vealed at fur ther
study of nu mer ous gen era and spe cies of these mi cro or gan -
isms. Even re cently, it was con sid ered that plants do not
have selenoproteins ei ther. This fact would mean that ei -
ther they lost the cor re spond ing mech a nism in the course
of evo lu tion or in suf fi cient amount of ma te rial was stud ied.
Ev i dently, the lat ter is true, as re cently it has been proved
that in a rep re sen ta tive of the veg e ta ble king dom
Chlamidomonas reinhardtii glutathione peroxidase con -
tains selenocysteine, the in clu sion of which into
polypeptide oc curs due to UGA codon [13].
The study of 404 yeast strains which be long to 40 gen -
era showed that their sen si tiv ity/re sis tance to selenate var -
ies widely. The growth of some strains can be sup pressed by
se le nium even at the con cen tra tion of 0.1 mM, while some
oth ers grow in the pres ence of 100 mM. Gen er ally,
ascomycetes are more tol er ant to se le nium, than
basidiomycetic fungi. The chem i cal com po si tion of the
me dia, in par tic u lar, the con tent of sul fate and sul fur-con -
tain ing amino ac ids, in flu ences the sen si tiv ity to this el e -
ment [14].
A con sid er able part of ex per i men tal works, ded i cated
to the study of se le nium in flu ence on yeast, was per formed
us ing selenate as its source. It was shown that this an a logue
of sul fate in flu ences the growth of S. cerevisiae de pend ing
on its con cen tra tion. At the pres ence of 1-5 mM selenate in
a me dium, the ex po nen tial growth of a cul ture slows down,
and its tran si tion to the sta tion ary phase oc curs at a smaller
cells’ con cen tra tion. Life cy cle of 60 % of cells treated with
selenate (5 mM) is stopped at the bud ding stage, though
the smaller part fin ishes their di vi sion with the sep a ra tion
of doughter cells, which may tes tify the ad ap ta tion of a part
of the cul ture to the toxic ac tion of sel e nite. Sel e nite has
also le thal ac tion on cells of S. cerevisiae de pend ing on its
con cen tra tion in a me dium. Ev i dently, a neg a tive in flu -
ence of sel e nite on the growth of yeast cul ture oc curs
mainly due to sup pres sion of mi to sis as well as its le thal ac -
tion on cells [15]. Non-con ven tional flavinogenic yeast
Pichia guilliermondii is more sen si tive to sel e nite than S.
cerevisiae – its growth in a syn thetic me dium is al most
com pletely blocked at its con cen tra tion of 0.5-1.0 mM
[16]. Mam ma lian cells are even more sen si tive – sel e nite is
ex tremely toxic for them even in micromolar con cen tra -
tions [17]. Prob a bly, one of the rea sons of dif fer ent sen si -
tiv ity of cells to sel e nite is their dif fer ent ca pac ity to
detoxicate it. The cells of Esch e richia coli neu tral ize toxic
sel e nite by its re duc tion to el e men tary se le nium (Se°) [18].
The pro cess of re duc tion leads to the ap pear ance of spe -
cific red color of cul ture and to the for ma tion of H2O2 and
O2
-, which are con sid ered [19] to cause the tox ic ity of sel e -
nite in Sal mo nella typhimurium. The cul ture of S. cerevisiae
also red dens dur ing cul ti va tion in a me dium with sel e nite
be cause of the ac cu mu la tion of red sub stance in vac u oles,
which, in the au thors’ opin ion [15], is el e men tary se le nium
(Se°). It was shown that at cul ti vat ing yeast Candida
tropicalis in a me dium with se le nium (SeO2) the gran ules of
free se le nium ac cu mu late in their vac u oles as well [20, 21].
The study of mu tants, avoid of vac u oles as well as de -
fec tive in vacuolar H+-ATPase, also ev i dences an im por -
tant role of S. cerevisiae vac u ole in se le nium detoxication.
All three tested strains re vealed an in creased sen si tiv ity to
the tellurite and chromate, be cause of oxyanions ac cu mu -
la tion in the cytosol. Ev i dently, their de tox i fi ca tion needs
in tact vac u ole which par tic i pates in met al loid
compartmentalization, as well as in reg u la tion of cytosolic
de tox i fi ca tion pro cesses, i.e. re duc tion. Vice versa, the mu -
tants ac cu mu lated less se le nium and were more tol er ant to
sel e nite than par ent strain. Ac cord ing to the au thors of [22]
this re sult shows that se le nium ac cu mu la tion oc curs
mainly in vac u ole and the ac tiv ity of V-H+-ATPase may be
in volved in this pro cess.
The col lec tion of sel e nite-re sis tant (Sitr) mu tants of
the yeast P. guilliermondii was iso lated, and the char ac ter is -
tic fea ture of them, con trary to the par ent strain, was quick
change of their bio mass color from white to red dur ing the
growth in a me dium sup ple mented with com par a tively low
con cen tra tions of sel e nite [23]. The cells of the wild strain
can also change its color, but only to pink and only af ter
5
SE LE NIUM AND YEAST: GE NETIC MECH A NISMS OF THE YEAST TOL ER ANCE
Ta ble 1
Con tent (%) of in or ganic and or ganic selenocompounds in the yeast bio -
mass (to tal se le nium con tent – 1922 mg/kg of dry bio mass) [28].
Compound %
Selenate Not de tected
Sel e nite 1
Selenocystine 0.5
Selenocystathionine 1
Se-methylselenocysteine 0.5
?-Glutamyl-Se-methylcysteine 0.5
Selenomethionine 85
Se-adenosylselenohomocysteine 3
Selenolanthionine 1.5
To tal se le nium 93
long-term (>7 days) cul ti va tion in the pres ence of
sublethal con cen tra tion of sel e nite. The pre vi ous char ac -
ter iza tion of the sel e nite-in duced stained sub stance iso -
lated from the mu tant cells of P. guilliermondii ev i dences
the for ma tion of a red form of Se° in them. These data in di -
rectly ap prove the ex is tence of the reductive path way of
sel e nite de tox i fi ca tion in yeast [23], but for ma tion of el e -
men tal Se° in the cells should be con firmed also by di rect
chem i cal iden ti fi ca tion.
Se le nium and sul fur have very sim i lar physico-chem i -
cal prop er ties, which al low con sump tion and as sim i la tion
of se le nium through the met a bolic way for sul fur. Or ganic
com pounds of se le nium and se le nium-con tain ing amino
ac ids (bioselenium prep a ra tions) are con sid ered to be the
most fa vor able sources of Se for hu mans and an i mals. At
cor re spond ing con di tions yeast can ac cu mu late se le nium
and in clude it into these com pounds. At cul ti vat ing in a
me dium with sel e nite at cer tain con di tions about 60% of
or ganic se le nium is lo cated in struc tural com part ments of
the yeast, namely, in the frac tion of microsome mem -
branes, endoplasmic re tic u lum, Goldgi-vac u oles and
other organelles. Mi to chon dria also con tain a con sid er able
amount of or ganic se le nium. “Org-se le nium” was found in
pep tides, where se le nium sub sti tutes for sul fur, in lipid
frac tions (mem bra nous and non-mem bra nous),
glycoprotein frac tions of cel lu lar walls and in struc tural
com po nents of the yeast sur face. There is still no def i nite
an swer to the ques tion, whether the ef fi cacy of this com pli -
cated bi o log i cal sys tem de pends on the sub sti tu tion of sul -
fur with se le nium. Prob a bly, as in the case of en zymes in
bac te ria, the ef fect will de pend on the amount and lo ca tion
of sub sti tu tions.
As for quan ti ta tive ra tio of dif fer ent se le nium com -
pounds in the bio mass of “selenized” yeast, it de pends on
the con di tions of the yeast cul ti va tion. It is pos si ble to pro -
duce the baker’s yeast of a high qual ity en riched with se le -
nium at con di tions pro vid ing a spe cific rate of its
as sim i la tion near 40-50 mg·g-1·hour-1. The con tent of un -
de sired in or ganic se le nium in cells can be de creased to
5-6%, though the bio mass yield will be 20% less [24, 25]. At
cor re spond ing con di tions (pH, t°, airation, se le nium con -
tent in a me dium, etc) the S. cerevisiae cells can ac cu mu -
late se le nium up to 1000-2000 мg/g of dry bio mass, and
more than 80% of se le nium can be in the form of SeMet
(Ta ble 1). For the op ti mi za tion of the yeast cul ti va tion it is
prof it able to use pro cesses of con tin u ous fer men ta tion [26,
27].
SeMet is con sid ered less toxic for or gan isms than in or -
ganic com pounds of se le nium [1]. How ever, this con clu -
sion seems to con cern not all the kinds of or gan isms. Thus,
a com par a tive study of the in flu ence of two se le nium com -
pounds, or ganic (selenomethionine) and in or ganic (sel e -
nite), on the growth, vi a bil ity and an ti ox i dant sta tus of S.
cerevisiae cells showed that both forms of se le nium at in -
creas ing con cen tra tion in a me dium de creased pro por tion -
ally the cells vi a bil ity and their quan tity in a cul ture, though
se le nium con tent was in creased in cells, and the ef fect of
selenomethionine was stron ger [29].
Yeasts as the source of se le nium for an i mals and hu mans
Selenized yeasts are more as sim i lated and there fore they
are a better source of se le nium not only for hu mans but also
for an i mals. There fore, to es ti mate the avail abil ity of dif -
fer ent forms of se le nium the male rats (4 weeks age, ex per i -
men tal group) were fed for 4 weeks with fod der sup ple -
mented by se le nium (0.04, 0.08, 0.16, and 0.32 мg/g) ei -
ther in the form of sel e nite or as en riched yeast bio mass
(“SeY”). The con trol group was kept on non-se le nium
diet. Se le nium did not in flu ence the growth and bio chem i -
cal tests of blood and se rum. Its con tent and the ac tiv ity of
glutathione peroxidase in liver, se rum, and eryth ro cytes in -
creased grad u ally ac cord ing to se le nium added. At low se -
le nium doses (0.04 and 0.08 мg/g), sel e nite pro vided a
greater con tent of se le nium and a higher ac tiv ity of
glutathione peroxidase when com pared to SeY, though at
higher se le nium level (0.32 мg/g) these val ues were greater
for SeY. It was shown that bioavailability of se le nium as
SeY in com par i son with sel e nite (100%) was higher in tis -
sues - 135-165 %, as well as for glutathione peroxidase ac -
tiv ity - 105-197 %, i.e. SeY is a better source of se le nium for
rats than sel e nite [30].
The con cen tra tion of se le nium in liver of pigs,
weighted about 24 kg, which were fed for 9 weeks with
selenized yeasts (0.3 mg se le nium per kg) was con sid er ably
higher than in the liver of those fed with the ad di tion of sel -
e nite (0.3 mg of se le nium per kg) [31].
6
STENCHUK M. M. ET ALL.
Ta ble 2
Anticancerogenic ef fi ciency of dif fer ent se le nium com pounds for sup pres -
sion of mam mary tu mors in rats [39]
Component
Selenium dose (ppm) for
50% suppression
Seleno-methyl-selenocysteine 2
Selenobethaine 2
Methyl ether of selenobethaine 2-3
Sel e nite 3
Selenomethionine 4-5
Selenocysteine 4-5
The in ves ti ga tion of abil ity of sel e nite, selenate, and
SeY to in crease the Se con cen tra tion in milk, se rum and
blood of cows showed that the en rich ment of fod der with
sel e nite and selenate had no sig nif i cant in flu ence on Se
amount in milk, and there was no dif fer ence be tween the
ef fects of these two se le nium com pounds. In stead, the ac -
tion of or ganic se le nium (SeY) (com par ing to in or ganic se -
le nium) was 2 or 3 times more ef fec tive in the in crease of
se le nium level in milk, blood, and se rum [32-34].
New born lambs, the moth ers of which were fed with
SeY, had a higher con tent of se le nium and the ac tiv ity of
glutathione peroxidase in blood, than those the moth ers of
which were fed by sel e nite. The ewes fed with SeY had a
higher quan tity of se le nium in foremilk in com par i son with
nour ish ing with sel e nite. Thus, se le nium of SeY passed
eas ier into foremilk and foe tuses of lamb ing ewes than in
form of sel e nite.
Though the need of hu mans and an i mals for se le nium
is well-founded, still the ques tion about its form for con -
sump tion is un der con sid er ation. Pref er a bly, the se le nium
should be con sumed in the form which is found in nat u ral
prod ucts. As the nat u ral prod ucts con tain se le nium mainly
as L-iso mer of selenomethionine (SeMet), the syn thetic
L-SeMet or food en riched by it can be used as ad di tional
forms of se le nium for hu mans. It is im por tant not only for
adults but also for chil dren. It is shown, for ex am ple, that
bioavailability of se le nium in the form of SeY for pre ma -
ture in fants is higher than other se le nium com pounds.
Chil dren whose par ents lived in the area of Hun gary, de fi -
cient in se le nium, re ceived 4.8 mg SeY (5 мg of Se) ev ery
day dur ing first 14 days af ter birth. The au thors did not
found any com pli ca tions or side ef fects af ter giv ing chil -
dren the prep a ra tion of SeY [36, 37]. Still the ques tion
arises which has no a grounded an swer yet – how bi o log i -
cally ac tive are other nu mer ous com pounds of se le nium
(or ganic and in or ganic)? Prob a bly it is ad van ta geous to use
not only SeMet, but also other se le nium com pounds for
dif fer ent aethiologies. Se le nium is known to re veal a num -
ber of antitumor ef fects but only some of them de pend on
glutathione peroxidase, which pro tects DNA from the ac -
tions of muta gens, and on reg u la tory pro tein p53 (tu mor
sup pres sor) which con trols DNA rep a ra tion and the ac tiv -
ity of which can be in creased by SeMet as an
anticancerogenic fac tor in this case [38].
It is still not known whether there are any other mech a -
nisms of se le nium antitumor ac tion. Ev i dently, they ex ist,
as it was shown in the ex per i ments on higher eukaryotes
that selenocompounds can sig nif i cantly dif fer by their
mode of in flu enc ing a cell [39-41]. For ex am ple, it was
shown that not selenomethionine and selenocysteine, but
other se le nium com pounds (in or ganic sel e nite, in par tic u -
lar) can be more ef fec tive in pre vent ing tu mors in rats (Ta -
ble 2). It is worth men tion ing that
selenomethylselenocysteine which was one of the ef fec tive
com po nents in this ex per i ment, as well as SeMet, is syn -
the sized by yeast cells and maybe it is pos si ble to se lect
yeast strains, the bio mass of which would be en riched with
this com po nent and not SeMet (and, prob a bly with both).
This prob lem is im por tant also be cause there are data
about the pres ence of some fac tors (de riv a tives of se le nium
me tab o lism?) in food and fod der, en riched by se le nium,
which me di ate se le nium ac tion in can cer aethiology (prob -
a bly, in other aethiologies as well). There are an tag o nists of
se le nium among them which sup press the antitumor ac -
tion not only of se le nium but also of other an ti ox i dants –
ascor bic acid, retinol, в-carotin, б-tocopherol, that syn er -
gis ti cally in ter act as antitumor prep a ra tions [42].
Genotoxic and mutagenic po ten tial of se le nium. It is
known that se le nium re veals two ef fects in the cells of
mam ma lian cells: in the nanomolar range it stim u lates
cells growth, while micromolar con cen tra tions are ex -
tremely toxic [17]. It also has a dual ef fect on yeast: at
ten-fold higher con cen tra tions (in the micromolar range)
sup presses spon ta ne ous mu ta gen e sis, but be comes toxic,
genotoxic in par tic u lar, at milimolar con cen tra tions. Sel e -
nite in the con cen tra tion of 1-15 мmoles/plate sup pressed
spon ta ne ous mu ta gen e sis com pletely in two in de pend ent
loci his1-7 (missense mu ta tion) and lys1-1 (am ber mu ta -
tion) of S. cerevisiae [43]. The de gree of sup pres sion de -
pended on sel e nite con cen tra tion, strain and lo cus.
30-times higher sel e nite con cen tra tion was nec es sary to
sup press the fre quency of spon ta ne ous re ver sions in the
histidine lo cus, in com par i son with the lysine lo cus. Both
loci re acted dif fer ently to two other in or ganic se le nium de -
riv a tives. Spon ta ne ous mu ta gen e sis in the lysine lo cus was
sup pressed com pletely by sel e nite in the con cen tra tion of 3
мmoles/plate while the histidine re ver sions were sup -
pressed only at 30 мmoles. Selenate sup pressed re ver sions
in the lysine but not in the histidine lo cus. These re sults
show that ex og e nous ad di tives (in this case se le nium com -
pounds) can in flu ence con sid er ably ge net i cally con trolled
readi ness of the or gan ism to re spond to mu ta gen e sis and
they prove the com plex ity of such in ter ac tions. The mech -
a nisms of sup press ing the fre quency of spon ta ne ous mu ta -
tions by se le nium in yeast are not known. Prob a bly, the se -
le nium com pounds act as an ti ox i dants, de creas ing a pool
of mutagenic free rad i cals.
At higher con cen tra tion (1-10 mM) se le nium acts as a
mutagen, re veal ing tox ic ity as well. In S. cerevisiae it in -
duces di rect mu ta tions in the CAN1 lo cus, which codes
arginine permease. The mu tants canr are re sis tant to
canavanine and their reg is tra tion can be eas ily per formed
7
SE LE NIUM AND YEAST: GE NETIC MECH A NISMS OF THE YEAST TOL ER ANCE
in the me dium with this toxic an a logue of arginine. Their
fre quency un der the treat ment of cells with a high con cen -
tra tion of sel e nite (10 mM) in creased 6 times and
amounted to about 80·10-6 [15]. The fre quency of these di -
rect mu ta tions was close to the fre quency of back mu ta -
tions, in duced by sel e nite [44].
In dip loid strains of S. cerevisiae sel e nite, be sides tox -
ic ity man i fes ta tion, re vealed recombinogenic and
mutagenic ef fects: it in duced gene con ver sion, mi totic
cross ing-over, back mu ta tions and ap pear ance of ab er rant
col o nies [44]. In ter est ingly that selenomethionine, as well
as sel e nite, has mutagenic and antimutagenic ef fects in
hap loid strains of S. cerevisiae – de pend ing on the con cen -
tra tion, and the lat ter is ob served in both sta tion ary and
log a rith mic phases of the growth. The au thors used hy dro -
gen per ox ide as a mutagen. The ac tiv ity of catalase,
superoxide dismutase and glutathione peroxidase was
much higher in the pres ence of both se le nium com pounds
[45].
The mech a nisms of sel e nite ac tion as a mutagenic fac -
tor. As sel e nite in flu ences neg a tively the growth of S.
cerevisiae by block ing mi to sis, it was sup posed [15] that this
ac tion was caused by DNA dam age, the rep a ra tion of
which de mands prior de lay in mi to sis and which is known
to be con trolled by the RAD9 gene. In deed, a mu tant rad9
was shown to be more sen si tive to sel e nite than the wild
type strain. The con clu sion was made that de lay in mi to sis
un der the RAD9 con trol is nec es sary for the rep a ra tion of
DNA dam age, in duced by sel e nite. To un der stand the na -
ture of these dam ages, the sen si tiv ity to sel e nite was de ter -
mined for the mu tants, de fec tive in dif fer ent ways of DNA
rep a ra tion. Tak ing into con sid er ation a pos si ble ox i diz ing
ca pac ity of sel e nite, the sen si tiv ity of strains de fec tive in
the rep a ra tion of DNA ox i da tive dam ages (the rep a ra tion
by ex ci sion of bases) was first to study. None of the de fec -
tive genes ogg1, ntg1, ntg2 and apn1 in flu enced re sis tance to
sel e nite con sid er ably. Prob a bly, the DNA dam ages caused
by sel e nite are not re paired by the prod ucts of wild type al -
leles of these genes.
Mu ta tions in the genes RAD51 and RAD52 which code
for the com po nents of rep a ra tion of the DNA dou -
ble-strand breaks did not in crease sig nif i cantly the sen si tiv -
ity to sel e nite, which, then, does not cause such DNA
dam age. A sim i lar con clu sion was made con cern ing RAD1,
which par tic i pates in the DNA rep a ra tion by ex ci sion of
nu cleo tides. At last, it was re vealed that only one of four
known ways of the DNA rep a ra tion par tic i pates in the
elim i na tion of DNA dam ages, in duced by sel e nite – the
one where DNA-poly mer ase ж takes place [15]. Mu ta tions
in the REV3 gene which codes for this en zyme, in creased
con sid er ably the sen si tiv ity of cells to sel e nite. This rep a ra -
tion type is mutagenic, as the en zyme di rects rep li ca tion of
DNA on a dam aged (prob a bly by sel e nite) cod ing strand of
DNA dur ing which a prob a bil ity of in clu sion into newly
syn the sized strand of non-com ple men tary nu cle o tide is
in creased, which may be the cause of mu ta tions in this site.
As the rep a ra tion of DNA, dam aged by sel e nite, oc curs not
by ex ci sion of bases but with the par tic i pa tion of the REV3
gene, the au thors came to a con clu sion that sel e nite gen er -
ates in DNA not ox i dized bases, but large ad ducts, which
can be avoided only by DNA-poly mer ase ж [15].
It is not im prob a ble that some of genotoxic sel e nite ef -
fects can be caused by hy dro gen per ox ide, which is pro -
duced due to the re ac tion of sel e nite with glutathione [19].
Sel e nite in duces one-strand splits of DNA in the cells of
mam mary tu mors of mice [40].
Mu tants re sis tant to selenate and chromate. The ef fec -
tive ap proach for re veal ing genes which de ter mine sen si -
tiv ity/re sis tance of cells to some fac tors is the iso la tion of
mu tant cells with a changed tol er ance to these fac tors, the
ge netic anal y sis of which in the com bi na tion with other
meth ods al lows re veal ing spe cific rea sons of their re sis -
tance to the fac tors un der study. In the work [46] the au -
thors used selenate and chromate as se lec tive fac tors to ob -
tain cells, re sis tant to these toxic an a logues of sul phur. It
was known [47], that the se lec tion of strains, re sis tant to
chromate and selenate, mainly re sults in the iso la tion of
mu tants, which are de fec tive in the MET3 gene (which
codes for ATP-sulfurylase). To avoid this, the strain which
had ad di tional cop ies of the MET3 gene on a multy-cop ies
plasmid, was used as the ini tial one. The cells of S.
cerevisiae, treated with ethylmethansulfonate, were plated
on syn thetic me dium which con tained selenate (2 mM) or
chromate (0.1 mM) or both com pounds si mul ta neously at
the same con cen tra tions.
10 out of 39 se lected re sis tant mu tants were
methinine-in de pend ent (phe no type Met+) and 29 –
methinine auxotrophes (Met-). The re sults of the com ple -
men tary anal y sis showed that each of 24 Met--strains con -
tained one of al ready known al leles met1, met4, met14 or
met16. One of the strains con tained two mu ta tions – met22
(al ready known) and sul3 (al lele of a new gene SUL3). 4
other Met--strains con tained mu tant al leles of a new gene
MET28 which codes for an ac ti va tor of tran scrip tion.
Four most re sis tant strains were se lected out of 10
methionine-in de pend ent strains for fur ther anal y sis and it
was shown that two of them car ried one al lele of new genes
SUL3 or SUL2, and two oth ers were dou ble mu tants: one –
genes SUL2 and SUL1 (al ready known), and the other –
genes SUL1 and MET14. Mu ta tions in gene SUL3 are
dom i nant. It is note wor thy that there was no re la tion ship
found be tween the quan tity and the classes of above men -
8
STENCHUK M. M. ET ALL.
tioned mu tants and the types of se lec tive me dia – with
selenate, chromate or their mix ture.
The rate of sul fate con sump tion by cells of the tested
strains (nmoles·min-1·mg-1 of dry bio mass) was for: wild
type – 5.9; sul1 – 4.4; sul2 – 3.4; sul3 – 2.9; sul1 sul2 - <
0.1; sul1 sul3 - < 0.1; sul2 sul3 – 2.3. Thus, though in the
sin gle mu tants the rate of sul fate con sump tion was not
much lower than in the wild type, it was suf fi cient to iden -
tify them by re sis tance to sul phur an a logues. Only the
com bi na tion of two mu ta tions – sul1 sul2 or sul1 sul3 in
one ge nome re sulted in con sid er able de crease in the ef fi -
cacy of sul fate con sump tion by the cells of such strains –
they were not able to grow in a me dium with 1 mM sul fate
like a wild type, but de manded it in the con cen tra tion of 30
mM.
These dou ble mu tants were shown to be con ve nient for
the SUL genes clon ing, the re sults of which dem on strated
that the gene SUL1 is lo cated on II chro mo some and codes
for a pro tein which con tains 859 amino acid res i dues and
has 11 po ten tial transmembrane do mains. Its struc ture is
iden ti cal to the pro tein re vealed by Smith [48]. The gene
SUL2 is lo cated on XII chro mo some and codes for a pro -
tein which con tains 893 amino ac ids and has 10
transmembrane do mains. Both pro teins are highly ho mol -
o gous (62%).
The above men tioned re sults showed that yeast cells
have two high-af fin ity sul fate trans port ers which are coded
by the genes SUL1 and SUL2. The func tion of the gene
SUL3 re mained still un de fined. The in abil ity of dou ble
mu tants sul1 sul2 and sul1 sul3 to grow in a me dium with 1
mM sul fate could mean that Sul3p reg u lates the ac tiv ity of
Sul2p or the ex pres sion of the gene SUL2. The ki netic study
of the genes SUL1 and SUL2 derepression in the wild type
strain and the sul3 showed that Sul3p ac ti vates the tran -
scrip tion of the gene SUL2. A low level of the gene SUL2
tran scrip tion in the mu tant sul3 ex plains why dou ble mu -
tants sul1 sul2 and sul1 sul3 have the same phe no type. It is
still not known whether S. cerevisiae have a low-af fin ity
sys tem of sul fate trans port into the cell. The fact that the
mu tant sul1 sul2 grows in the me dium with a high con tent
of sul fate (30 mM) in di cates the prob a bil ity of its ex is tence.
It is also not known why the mu tant sul1, iso lated in the
work [45], does not grow in a me dium which con tains less
than 5 mM sul fate. Prob a bly, the ini tial strain was al ready
the mu tant sul2? One more prob lem is that the mu tant sul3
has a higher re sis tance to selenate than the strain sul2, so it
is not im prob a ble that the gene SUL3 par tic i pates in the
reg u la tion of other genes which take part in the sul phur
me tab o lism.
Selenate-an ion turned out to be toxic for
Schizosaccharomyces pombe as well, which gave a pos si bil -
ity to iso late from this yeast selenate-re sis tant mu tants,
which could not uti lise sul fate, so they de manded for their
nor mal growth such sources of sul phur as sul fite,
thiosulfate, cysteine or glutathione, but not methionine
[49, 50].The mu tants were trans formed us ing the ge nome
li brary of S. pombe and the gene, which com ple mented
selenate re sis tance, was iden ti fied as the one which codes
for the en zyme ATP-sulfurylase, which in S.cerevisiae is
coded by the gene MET3. On the au thors’ opin ion, the in -
abil ity of the mu tants to use methionine as the source of
sul phur is caused by the ab sence of re verse way of
transsulfurylatiton in this or gan ism. The wild type strains
can use methionine as a source of sul phur af ter its deg ra da -
tion with the for ma tion of sul fate. The au thors did not use
the above men tioned method [46], which al lows iso lat ing
other mu tants, be sides MET3, which are de fec tive in sul -
fate as sim i la tion.
Re sis tance/sen si tiv ity to sul fite. The re search of mech -
a nisms of cells tol er ance, yeast in par tic u lar, to sul fites is of
the o ret i cal and prac ti cal im por tant. Sul fites are widely
used for food con ser va tion, so it is im por tant to know the
con di tions un der which their ac tion on the mi cro or gan -
isms (in hibit – do not in hibit) will be ef fec tive. Sul fite-re -
sis tance is an im por tant fea ture of the wine strains of S.
cerevisiae, and un der stand ing its ba sis and the fac tors in flu -
enc ing it, can pro vide better man age ment of sul fite us age.
Sul fite is po ten tially toxic but at the same time it is a nor mal
me tab o lite of many an i mals, plants and mi cro or gan isms,
so they should have de tox i fi ca tion mech a nisms se lec tive
for this an ion. It is im por tant to know what are these mech -
a nisms, and whether they are spe cific only for sul fite, or are
more gen eral and pre vent the neg a tive in flu ence of other
re lated me tab o lites as well (for ex am ple, se le nium com -
pounds, sel e nite first of all). Fi nally, there is not much
known about the rea sons of hy per sen si tiv ity of some peo -
ple to sul fites added to food prod ucts and drinks. It can be
ex pected that the study on the ways of sul fites me tab o lism
in yeast will be come an im por tant con tri bu tion into solv ing
this im por tant prob lem as well. Though the sul fite me tab o -
lism in hu mans and yeast oc curs in dif fer ent ways, it is not
im prob a ble that cel lu lar tar gets for sul fite (pro tein or other
mol e cules) may be sim i lar in these or gan isms. The re sults
of com plete se quenc ing of the hu man and S. cerevisiae
genomes showed that much can be learned about the hu -
man fea tures, study ing anal o gous pro cesses in the yeast.
At the be gin ning of the study of the yeasts the au thors
con sid ered the fol low ing mech a nisms of sul fite de tox i fi ca -
tion as pos si ble though not ex cep tional: 1) the as sim i la tion
of sul fite trough in creased for ma tion of methionine and
cysteine – the main fi nal prod ucts of a nor mal sul fite-gen -
er at ing met a bolic path way of the yeast; 2) in creased syn -
9
SE LE NIUM AND YEAST: GE NETIC MECH A NISMS OF THE YEAST TOL ER ANCE
the sis of sul fite-bind ing agents – for ex am ple, ace tic al de -
hyde; 3) de creased con sump tion of sul fite. At the first stage
of in ves ti ga tions, the sul fite-re sis tant and sul fite-sen si tive
vari ants or mu tants were iso lated, the growth of which
would not be in flu enced by sul fite in the con cen tra tion
which is toxic to a par ent strain or vice versa is more toxic
for the mu tant than for a wild strain.
Gene FZF1. The col lec tion of sul fite-re sis tant mu tants
of S .cerevisiae was first ob tained and char ac ter ized by a
group of Ital ian sci en tists [51-53]. The char ac ter is tic fea -
tures of mu tants were in creased intracellular con tent of
glutathione, higher glutathione reductase ac tiv ity, smaller
con cen tra tion of extracellular glutathione and el e vated
amount of extracellular ace tic al de hyde in the pres ence as
well as in the ab sence of sul fite. The re sis tant strains had
con sid er ably smaller abil ity to ac cu mu late sul fite. No dif -
fer ence has been found be tween re sis tant and sen si tive
strains in the sen si tiv ity of their glyceraldehyde-3-phos -
phate dehydrogenase to sul fite or glutathione. It was shown
that re sis tance of seven mu tants was in her ited as one dom i -
nant mu ta tion and that all seven mu ta tions are allelic, i.e.
they are lo cated in one gene, which in the au thors’ opin ion
is vi tal in de fin ing sen si tiv ity/re sis tance of S. cerevisiae to
sul fite. The re sults of clon ing and se quenc ing a gene
showed that it is iden ti cal to al ready de scribed gene FZF1
[54], which codes for a pro tein with 5 zinc fin gers, three of
which are lo cated in tan dem in N-ter mi nal part of the pro -
tein. It in di cates the pro tein be longs to the class of
transcriptional fac tors but the ques tion of its role in the sul -
fite me tab o lism re mains un known.
Gene SSU1. New light on the mech a nisms of sen si tiv -
ity/re sis tance of yeast to sul fite was shed by the re search on
one more mu tants’ col lec tion of S. cerevisiae, which in -
cluded the strains of two types – sen si tive and re sis tant to
sul fite (1-2 mM) [55]. The ge netic anal y sis showed that
mu ta tions in creas ing cells sen si tiv ity to sul fite are re ces sive
and lo cated in 4 genes (SSU1, SSU2, SSU3, SSU4). None
of the sen si tive mu tants was de fec tive in biosynthesis of
methionine or cysteine which ex cluded the loss of sul -
fite-reductase ac tiv ity as a pos si ble rea son of sen si tiv ity. All
the mu tants of the group did not re veal sen si tiv ity or re sis -
tance to other tested an ti ox i dants (re duc ing agents), be -
sides sul fite: cysteine, ascorbate, dithiothreitole, ni trite,
glutathione and thiosulfate.
Fur ther re search showed that SSU1 gene, the mu tant
al leles of which in crease cells’ sen si tiv ity to sul fite, codes
for pro tein of 458 amino ac ids, lo cal ized in the mem brane
with 10 po ten tial transmembrane do mains. The con clu -
sion was made that SSU1 codes for the trans porter which
par tic i pates in ex trud ing sul fite out of the cell, but not in its
as sim i la tion. The fact that zero-mu tants on SSU1 gene ac -
cu mu lated more sul fite in cells, tes ti fied in fa vour of this
con clu sion, and vice versa gene overexpression on
multi-cop ies plasmid re sulted in con sid er able de creas ing
its quan tity in the cell in com par i son with its wild coun ter -
part. The rate of ex trud ing sul fite out of cells was more in -
tense in the case of SSU1 ex pres sion on multi-cop ied
plasmid [56].
It was dis cov ered that some dom i nant mu ta tions in
ear lier iden ti fied gene FZF1 as com pared to the wild type
al lele in crease the tran scrip tion of SSU1 gene 8-fold, and
SSU1 be ing multi-cop ied in creases sul fite-re sis tance of
cells 3-8-fold. The rate of ex trud ing bound sul fite from
cells of wild type, ssul1 and fzf1 mu tants or cells with ac tive
multi-cop ied SSU1 did not dif fer no tice ably. It is in ac cor -
dance with the con clu sion that the pro tein Ssu1p keeps a
low, non-toxic level of sul fite by re mov ing solely its free
form out of the cells. A site of the gene SSU1 pro moter was
iden ti fied to which the pro tein Fzf1p binds in vi tro and reg -
u lates pos i tively its tran scrip tion [57]. The role of Fzf1p as
an ac ti va tor of SSU1 is proved by the fact that Fzf1p sup -
presses sul fite sen si tiv ity of dif fer ent mu tant classes but not
ssu1. Thus, the overexpression of genes FZF1 or/and SSU1
is one of the ef fec tive ways to in crease the yeast tol er ance to
sul fite due to the in crease of the Ssu1p ac tion as a “sul fite
pump” [58].
Sim i lar trans port ers, known as Mdr (multidrug
resistance) or Pdr (pleiotropic drug resistance) are widely
spread in liv ing na ture – from bac te ria to hu mans [59], but
Ssu1p dif fers from them in struc ture and mode of ac tion.
Trans port ers Mdr, on the con trary to Ssu1p, need ATP en -
ergy to take a wide spec trum of toxic sub stances out of a
cell.
Can Ssu1p re move some other toxic com pounds, be -
sides sul fite, out of a cell? Polyfunctional abil i ties of this
pro tein as a pump were dem on strated by the in crease of the
ex pres sion of the genes SSU1 and FZF1 18-fold and 5-fold,
re spec tively, af ter treat ing the cells with alkylating com -
pound – methyl methanesulphonate, which is known to
dam age nu cleic ac ids and pro teins [60]. It is still not known
whether Ssu1p can iden tify and take this super-mutagen
out of the cell, but the very fact of ac ti va tion of both genes
ex pres sion by it de serves spe cial at ten tion. Ev i dently,
Ssu1p rec og nizes a toxic an a logue of sul fite, sel e nite, as
well, as the in crease of yeast re sis tance to the lat ter un der
the con di tion of SSU1 overexpression in cells was dis cov -
ered [15, 57].
The mu tant ssu2 was shown to be an al lele to al ready
known gene GRR1 [56], in volved in glu cose me tab o lism
and other func tions [61]. The mu ta tion grr1 is pleiotropic:
it de ter mines chang ing cells mor phol ogy, in creased sen si -
tiv ity to re duc ing agents dithiothreitole, ni trites and
10
STENCHUK M. M. ET ALL.
thiosulfate, de creases ex cre tion of ace tic al de hyde and de -
creases the con tent of re duced glutathione in cells. Two
lat ter me tab o lites in ter act with sul fite and this could be the
way of its de tox i fi ca tion, but the sen si tiv ity to sul fite of in -
de pend ently ob tained mu tant with con sid er ably de creased
glutathione con tent was not de creased [55]. Prob a bly, the
grr1 mu tants grow slowly on glu cose be cause of the de fect
in high-af fin ity sys tem of its trans port. Still the sen si tiv ity
of the mu tant grr1 to sul fite is prob a bly caused by this de -
fect only par tially, since it showed an in creased sen si tiv ity
on other car bon sources as well (galactose, malt ose, ac e -
tate, eth a nol and glyc erol). Be sides, a sup pres sor rgl1 of the
de fect of glu cose me tab o lism in the mu tant grr1 did not
sup press its sen si tiv ity to sul fite com pletely, though FZF1
gene on multi-cop ies plasmid sup pressed the sul fite sen si -
tiv ity com pletely [55, 56]. There upon, the main rea son of
sul fite sen si tiv ity of mu tant cells rgl1 (ssu2) is con sid ered a
de creased pool of ace tic al de hyde, the re ac tion of which
with sul fite gen er ates a non-toxic prod uct –
hydroxyethanesulfonate. Thus, the for ma tion of ace tic al -
de hyde by the yeast is an im por tant way of sul fite de tox i fi -
ca tion. In fa vour of this as sump tion is the above men tioned
fact of the ex cre tion by sul fite-re sis tant mu tants fzf1 (all the
mu ta tions are dom i nant) of much higher amounts of ace tic
al de hyde in com par i son with the wild type cells [52]. It is
also known that ex og e nous sul fite in duces ex cre tion of this
me tab o lite by the cells of the wild type S. cerevisiae and
Saccharomycodes ludwigii [62].
The re sis tance mu ta tions are re vealed to be dom i nant
and lo cated in the same gene, la belled as RSU [55], shown
to be iden ti cal to the gene FZF1 [53, 58]. The dif fer ence
be tween the mu tants’ tol er ance to sul fite ap peared to be
small. Thus, the re sis tant mu tant RSU1 en dures 3 times
higher con cen tra tion of sul fite than a wild type strain
which is twice more tol er ant to sul fite than the most sen si -
tive mu tants. The dom i nance of mu ta tion FZF1-4 makes it
con ve nient for the se lec tion of in dus trial and lab o ra tory
strains of S. cerevisiae [58].
While study ing mo lec u lar mech a nisms of sel e nite de -
tox i fi ca tion by the yeast cells, the au thors [15] searched for
genes which be ing overexpressed would in crease the re sis -
tance to sel e nite. The wild strain S. cerevisiae was trans -
formed us ing a ge nome li brary on multi-cop ies plasmid.
About 60000 transformants were transfered to a min i mum
me dium, which con tained 20 mM sel e nite. Two plasmids
were se lected which pro vided in creased re sis tance of cells
to sel e nite, each of them car ried a sin gle gene – al ready
known SSU1, en sur ing cells re sis tance due to the mu ta tion
[56], and GLR1 which codes for NADPH-de pend ent
glutathione reductase. Overexpression of the lat ter, as well
as the for mer, in creased the sel e nite re sis tance of cells.
Overexpression of both genes (SSU1 and GLR1) si mul ta -
neously in the same cell caused a higher re sis tance to sel e -
nite than overexpression of only one of two genes.
There fore, both genes par tic i pate in the sel e nite de tox i fi -
ca tion through an ad di tive mech a nism. Overexpression of
SSU1 was also found to in crease the re sis tance of both wild
type strain and ycf1 mu tant, while overexpression of GLR1
in flu enced the sen si tiv ity of only wild type strain, not ycf1.
Un ex pect edly, the ycf1 mu tant was shown to be more re sis -
tant to sel e nite in com par i son with the wild type strain. The
rea son seems to be an ac tive trans port of
selenodiglutathione into a vac u ole in the wild-type cells
con trary to the ycf1 mu tants, that re sults in the ex haus tion
of a pool of re duced glutathione in cytosol, which in its turn
causes a less ef fec tive re duc tion of toxic sel e nite.
As overexpression of glutathione reductase re sults in a
higher sul fite-re sis tance, it was stud ied, whether sel e nite
in flu ences the ac tiv ity of genes, which con trol the re sponse
to the ox i da tive stress. It was shown that the ex pres sion of
TRR1 (which codes for cytosol thioredoxin reductase),
GLR1, and YCF1 is in creased by sel e nite 14, 4 and 2-fold,
re spec tively, and this pro cess is Yap1p-de pend ent. Sel e -
nite does not in flu ence the ac tiv ity of SSU1 [15]. Thus, sel -
e nite acts to yeast as an ac tive ox i dant, in duc ing genes
which de fend the cell from ox i da tive stress (GLR1 and
TRR1).
Wine yeast and SSU1. The study of mech a nisms of sul -
fite sen si tiv ity/re sis tance is be ing per formed also with the
us age of wine yeast as a model ob ject. In the course of thou -
sands of years this yeast was un der se lec tion to ac quire such
prop er ties as the abil ity to fer ment must with a high con tent
of sugar quickly and ef fec tively, be re sis tant to high con -
cen tra tions of eth a nol and sul phur di ox ide, and high tem -
per a ture. Be cause of this they have unique ge netic and bio -
chem i cal char ac ter is tics which dif fer them con sid er ably
from the other yeasts, baker’s and brewer’s, in par tic u lar.
Un like the lab o ra tory strains S. cerevisiae, which are hap -
loids or dip loids, the wine yeasts are mainly dip loids,
aneuploids or polyploids, homotallic and highly het ero zy -
gous [63, 64]. They re veal high poly mor phism along the
chro mo somes [65, 66] and the in creased fre quency of the
mi totic re com bi na tion [67]. It strength ens their abil ity to
re or ga nize its ge nome and pro vides a quick ad ap ta tion to
the changes of a me dium.
Con se quently an im por tant ques tion arose about a role
of sul fite in the ge netic poly mor phism of the wine yeast,
since it is used as an an ti bac te rial agent at the pro duc tion of
wine. The gene SSU1 was the one be ing in ves ti gated be -
cause of its abil ity to mod u late yeast sen si tiv ity to sel e nite
as was stated ear lier. It was shown to be ex pressed much
higher in the cells of wine strains as com pared with the lab -
11
SE LE NIUM AND YEAST: GE NETIC MECH A NISMS OF THE YEAST TOL ER ANCE
12
STENCHUK M. M. ET ALL.
Ta ble 3.
Тhe list of genes which mod u late the yeast tol er ance to se le nium com pounds and sul fite.
Genes Protein function Phenotype/Genotype of mutants Source
S. cerevisiae:
MET1
MET3
MET4
MET14
MET16
MET22
MET28
SUL1
SUL2
SUL3
Uroporphyrinogene-3-methylase
АТP-sulfurylase
Reg u la tory gene
Adenosine-5`- phosphosulfite kinase
3`-phospho-5`-adenylylsulfate
reductase
Biphosphonucleoside
phosphohydralase
Tran scrip tion ac ti va tor
Sul fate trans porter
Sul fate trans porter
Gene SUL2 tran scrip tion ac ti va tor
Satr Сhrr Met-
Satr Сhrr Met-
Satr Сhrr Met-
Satr Сhrr Met
Satr Сhrr Met-
Satr Сhrr Met-
Satr Сhrr Met-
Satr Сhrr
Satr Сhrr
Satr, Сhrr; all mu tants are dom i nant.
46
46, 47
46
46
46
46
46
46
46
46
FZF1 (RSU1)
SSU1
Gene SUL1 tran scrip tion ac ti va tor
Trans porter (“sul fate pump”):
Takes out sul fite and sel e nite out of the
cell;
Dom i nant mu tants Sulr; the in creased
glutathione con tent, the in creased glutathione
reductase ac tiv ity; the de creased quan tity of
extracellular glutathione; the in creased con -
cen tra tion of extracellular ace tic al de hyde.
Re ces sive mu tants: Suls. The in creased ex pres sion
in wine yeast cells. Am pli fy ing SSU1: > Sulr, Sitr (3
mM); ssu1 > Suls.
51-54
14, 55-58
SSU2 (GRR1)
SSU3
SSU4
Un known
Un known
Un known
Mu tant grr1 pleiotropic Suls, the change of cell
mor phol ogy, the in creased sen si tiv ity to re -
duc ing agents, the de creased ex cre tion of ace -
tic al de hyde, the de creased level of re newed
glutathione in cells, the de creased growth on
glu cose.
Suls
Suls
55, 56, 61
55
55
ECM34
GLR1
REV3
RAD9
Un known
NADPH-de pend ent
glutathionreductase
DNA-poly mer ase z
The mi to sis de lay be fore the com ple tion
of DNA repara tion.
Du pli ca tion of pro moter of this gene and its
translocation in SSU1 pro moter leads to high
ex pres sion of the lat ter and there fore to
Sulr-phe no type.
Sitr
Sits
Sits
68, 69
15
15
15
o ra tory ones, be sides the in creased level of cor re spond ing
mRNA cor re lated with a high tol er ance of the strains tested
in re spect to sul fite [68]. The re sults of clon ing with sub se -
quent se quenc ing of gene SSU1 showed that a high sul fite
re sis tance of the strains was pro vided by a new al lele of the
gene SSU1, called SSU1-R. It arises due to the re cip ro cal
translocation be tween VIII and XVI chro mo somes as a re -
sult of un equal cross ing-over be tween microhomologous
pro moter re gions of the genes ECM34 and SSU1, which are
lo cated on VIII and XIV chro mo somes, re spec tively. Each
of such short se quences of 76 bp (so called “upper
activating sequence” – UAS) is the site for bind ing the
tran scrip tion ac ti va tor, namely Fzf1p. On the first stage of
the re or ga ni za tion of two chro mo somes the amount of
UAS in the ECM34 pro moter in creases by du pli ca tion(-s)
from one to sev eral, af ter which as a re sult of translocation
they are re lo cated within the SSU1 pro moter, while the
sin gle UAS is re mained within the ECM34 pro moter. It is
note wor thy that the func tion of the lat ter is not known yet
[69].
First, such re or ga ni za tion leads to a high poly mor -
phism of two pairs of ho mol o gous chro mo somes of the
cor re spond ing dip loid: if be fore the translocation each of
the chro mo somes of VIII pair had the length of 562 kbp,
af ter the translocation, one of them was ex tended till 921
kbp. Anal o gously, af ter the re or ga ni za tion, one of the ho -
mol o gous chro mo somes of XVI pair had only 599 kbp in -
stead of 948 kbp. Sec ond, by means of the de scribed
translocation the pro moter of the gene SSU1 can con tain
from 2 to 6 UAS, due to this the level of ex pres sion of the
gene SSU1 in creases rap idly in the com par i son with the
wild type strain. A high pos i tive cor re la tion was shown be -
tween the amount of UAS and the level of sul fite re sis tance
of the cor re spond ing yeast cell [69].
Non-ho mol o gous re com bi na tion, de scribed above, is
a par tic u larly rare phe nom e non in the wild type strains. Its
fre quency is con sid ered not to ex ceed 3.5 x 10-10. In spite of
this all the stud ied wine strains (more than 10) con tain al -
lele SSU1-R which ap pears as a re sult of such ex tremely
rare event, while the wild type strains do not have it [68,
69]. Ev i dently, sul fite in must is one of the im por tant se lec -
tive fac tors, in the pres ence of which the cells with rare mu -
ta tions of sul fite re sis tance have an ad van tage in the growth
rate over the par ent cells, which con trib utes to their better
sur vival, and, thus, to the evo lu tion of cor re spond ing
strains of wine yeasts in the di rec tion of the sul fite re sis -
tance. It should be noted that poly mor phism of a cer tain
strain in the length of chro mo somes, which ap peared as a
re sult of above men tioned translocation, can pro mote fur -
ther struc tural re or ga ni za tion of chro mo somes and, thus,
con trib ute to ad ap ta tion of the strain to other un fa vour able
en vi ron men tal con di tions.
To tally, at pres ent there are re vealed 20 genes which
mod u late the yeast tol er ance to se le nium com pounds and
sul fite (Ta ble 3). How com plete is this list? It is very prob a -
ble that it will be sup ple mented by other yet un known
genes. The facts ob tained in the study of ar senic in flu ence
on yeast sup port this as sump tion. Ar senic and se le nium
13
SE LE NIUM AND YEAST: GE NETIC MECH A NISMS OF THE YEAST TOL ER ANCE
Genes Protein function Phenotype/Genotype of mutants Source
YCF1
TRR1
YAP1
Schizosaccha-ro
myces pombe:
MET3
Pichia
guilliermondii
SIT1, SIT2
Ycf1p – vacuolar pro tein which per forms the
role of pump, be sides of cad mium de tox i fi ca -
tion
Cytosolic tioredoxin reductase
Tran scrip tion ac ti va tor TRR1, GLR1, YCF1
АТP-sulfurylase
Un known
Sitr
Phe no type is un known. Sel e nite de -
presses pro tein syn the sis.
Sits
Satr
Sitr
15
15
15
49, 50
16, 23
R e m a r k : Phe no type mark ing: Met
-
- methionine-de pend ence; Sat
r
, Chr
r
, selenate- or chromate-re sis tance, re spec tively; Sul
s
, Sul
r
– sen si tiv ity or
re sis tance to sul fite, re spec tively; Sit
s
,
Sit
r
– sen si tiv ity or re sis tance to sel e nite re spec tively.
have a num ber of sim i lar fea tures, neigh bour ing in the
fourth pe riod of the pe ri odic sys tem of el e ments. Both are
nec es sary in trace amounts for me tab o lism and growth as
microelements, but they are toxic at higher con cen tra tions.
It was shown [70], that the treat ment of S. cerevisiae cells
dur ing 2 hours with 0.1-1.0 mM so lu tion of NaAsO2 had
in sig nif i cant in flu ence on the growth of the yeast, but the
ex pres sion of 829 genes out of 6240 stud ied was changed
for a rather long pe riod. Three of them, which ac ti vate
tran scrip tion (MET4, MET28, and YAP1) are in duced by
se le nium as well. Prob a bly, these three fac tors de ter mine
cross-sen si tiv ity/re sis tance of the cells to both metalloids.
One can not also ex clude, that se le nium it self mod u lates
the ex pres sion of not doz ens but hun dreds of genes which
con trol the yeast tol er ance to it.
There fore, the yeasts ac quired cer tain mech a nisms of
de tox i fi ca tion in or der to sur vive at non- fa vor able con di -
tions, due to the pres ence of se le nium oxianions and their
an a logues, namely:
a con trol of their trans port into and ex cre tion out of the
cell;
the oxianions may be re duced in side the cell and me -
tab o lized into dif fer ent forms which are less toxic for the
cell (se le nium-con tain ing or ganic com pounds, the el e -
men tal form of se le nium);
compartmentalization (se ques tra tion) of their de riv a -
tives in organelles;
complexation with cell me tab o lites to form less toxic
com pounds;
oxianions’ ac ti vated tran scrip tion of genes which code
for the prod ucts pro tect ing the cell at these con di tions (an -
ti ox i dant sys tem).
М. М. Стен чук, Л. Б. Ча бан, М. В. Гон чар
Селен і дріжджі. генетичні механізми толерантності
дріжджів до сполук селену та їх аналогів
Резюме
Се лен (Se) і його спо лу ки про яв ля ють ток сич ну і кан це ро ген ну дію на
організми лю ди ни і тва рин, а у ма лих кількос тях він є важ ли вим мікро -
е ле мен том для жи вих істот. Тому важ ли во вста но ви ти мо ле ку лярні
осно ви ток сич ності Se та ре зис тен тності клітин до ньо го. Знач на
кількість досліджень цієї про бле ми про ве де на на бак теріях і висвітле -
на у ряді оглядів, Мож ливість ви ко рис тан ня мо ле ку ляр но-ге не тич них
ме тодів зро би ла дріжджі, в основ но му Sacccharomyces cerevisiae, зруч -
ною мо дель ною сис те мою для досліджен ня ме ханізмів ре зис тен тності
еу каріот ич них клітин до се ле ну на мо ле ку ляр но му рівні. Тому у цьо му
огляді підсу мо ва но дані, особ ли во ге не тичні, про ме ханізми чут ли -
вості/ре зис тен тності дріжджів до се ле но вих спо лук.
Клю чові сло ва: дріжджі, се лен, то ле рантність; yeast, selenium,
tolerance.
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УДК (579.25+579.22):546.23
17
Название статьи
|
| id | nasplib_isofts_kiev_ua-123456789-154190 |
| institution | Digital Library of Periodicals of National Academy of Sciences of Ukraine |
| language | Ukrainian |
| last_indexed | 2025-12-07T16:35:05Z |
| publishDate | 2006 |
| publisher | Інститут молекулярної біології і генетики НАН України |
| record_format | dspace |
| spelling | Стенчук, Н.Н. Чабан, Л.Б. Гончар, М.В. 2019-06-15T09:50:31Z 2019-06-15T09:50:31Z 2006 Селен и дрожжи. Генетические механизмы толерантности дрожжей к соединениям селена и их аналогам / Н.Н. Стенчук, Л.Б. Чабан, М.В. Гончар // Біополімери і клітина. — 2006. — Т. 22, № 1. — С. 3-17. — Бібліогр.: 70 назв. — укр. DOI: http://dx.doi.org/10.7124/bc.000715 https://nasplib.isofts.kiev.ua/handle/123456789/154190 (579.25 +579.22 ) :546.23 Селен (Se) і його сполуки проявляють токсичну і канцерогенну дію на організми людини і тварин, а в малих кількостях цей мікроелемент відіграє суттєву роль для живих істот. Тому важливо встановити молекулярні основи токсичності Se і резистентності клітин до нього. Значну кількість досліджень зазначеної проблеми здійснено на бактеріях. Можливість використання молекулярно-генетичних методів зробила дріжджі (в основному Saccharomyces cerevisiae) зручною модельною системою для вивчення механізмів резистентності еукаріотних клітин до селену на молекулярному рівні. Виходячи з цього в огляді підсумовано дані, особливо генетичні, щодо механізмів чутливості/резистентності дріжджів до селенових сполук. Selenium (Se) and its compounds have toxic and carcinogenic effect on animal and human beings, but in small concentrations this microelement is essential for life. Therefore, it is important to establish the molecular basis of Se toxicity and cell resistance to this metaloid. Many investigations of this problem have been carried out on bacteria. Genetic approaches, available for the yeasts, made these eukaryotic microorganisms, especially S. cerevisiae, a convenient model for the investigation of molecular mechanisms of Se tolerance. This review summarizes the knowledge of genetic mechanisms involved in Se tolerance in yeasts. Селен (Se) и его соединения оказывают токсическое и канцерогенное действие на организмы человека и животных, а в небольших количествах он является существенным микроэлементом для живых существ. Поэтому важно установить молекулярные основы токсичности Se и резистентности к нему клеток. Значительное количество исследований этой проблемы проведено на бактериях. Возможность использования молекулярно-генетических методов сделала дрожжи (в основном Sacccharomyces cerevisiae) удобной модельной системой для изучения механизмов резистентности эукариотных клеток к селену на молекулярном уровне. Исходя из этого в данном обзоре суммированы данные, особенно генетические, полученные при исследовании механизмов чувствительности/резистентности дрожжей к селеновым соединениям. uk Інститут молекулярної біології і генетики НАН України Біополімери і клітина Огляди Селен і дріжджі. Генетичні механізми толерантності дріжджів до сполук селену та їхніх аналогів Selenium and yeast: genetic mechanisms of the yeast tolerance to selenium compounds and their analogs Селен и дрожжи. Генетические механизмы толерантности дрожжей к соединениям селена и их аналогам Article published earlier |
| spellingShingle | Селен і дріжджі. Генетичні механізми толерантності дріжджів до сполук селену та їхніх аналогів Стенчук, Н.Н. Чабан, Л.Б. Гончар, М.В. Огляди |
| title | Селен і дріжджі. Генетичні механізми толерантності дріжджів до сполук селену та їхніх аналогів |
| title_alt | Selenium and yeast: genetic mechanisms of the yeast tolerance to selenium compounds and their analogs Селен и дрожжи. Генетические механизмы толерантности дрожжей к соединениям селена и их аналогам |
| title_full | Селен і дріжджі. Генетичні механізми толерантності дріжджів до сполук селену та їхніх аналогів |
| title_fullStr | Селен і дріжджі. Генетичні механізми толерантності дріжджів до сполук селену та їхніх аналогів |
| title_full_unstemmed | Селен і дріжджі. Генетичні механізми толерантності дріжджів до сполук селену та їхніх аналогів |
| title_short | Селен і дріжджі. Генетичні механізми толерантності дріжджів до сполук селену та їхніх аналогів |
| title_sort | селен і дріжджі. генетичні механізми толерантності дріжджів до сполук селену та їхніх аналогів |
| topic | Огляди |
| topic_facet | Огляди |
| url | https://nasplib.isofts.kiev.ua/handle/123456789/154190 |
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