Genetic mechanisms of Escherichia coli resistance to target inactivation. Genes governing purine metabolism in enterobacteria: an unexpected sequence found via complementation selection
Using enierobacterial strains having block in 3 different genes required for GMP synthesis, 3 groups of inserts with different restriction patterns were expected. But the fragments cloned represented 5 such, groups. One consisted of Salmonella typhimurium DNA fragments with 2 SacI sites available. S...
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| Date: | 1997 |
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| Language: | English |
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Інститут молекулярної біології і генетики НАН України
1997
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| Cite this: | Genetic mechanisms of Escherichia coli resistance to target inactivation. Genes governing purine metabolism in enterobacteria: an unexpected sequence found via complementation selection / E. Cherepenko, S. Craig // Биополимеры и клетка. — 1997. — Т. 13, № 5. — С. 403-407. — Бібліогр.: 16 назв. — англ. |
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| author | Cherepenko, E. Craig, S. |
| author_facet | Cherepenko, E. Craig, S. |
| citation_txt | Genetic mechanisms of Escherichia coli resistance to target inactivation. Genes governing purine metabolism in enterobacteria: an unexpected sequence found via complementation selection / E. Cherepenko, S. Craig // Биополимеры и клетка. — 1997. — Т. 13, № 5. — С. 403-407. — Бібліогр.: 16 назв. — англ. |
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| description | Using enierobacterial strains having block in 3 different genes required for GMP synthesis, 3 groups of inserts with different restriction patterns were expected. But the fragments cloned represented 5 such, groups. One consisted of Salmonella typhimurium DNA fragments with 2 SacI sites available. Sequencing revealed 100 % homology of the cloned insert to the N-tenn of Y protein of the hemC-hemD linkage group of E. coli chromosome (85 min locus). It is suggested that Y may represent the gpp gene, coding for guanosinepentaphosphatase. It was also shown that a Salmonella DNA fragment resulting from PCR amplification with 20-mer primers complementary to the N- and C-terms of the hpt gene of E. coli did not encode an hypoxanthine phosplioribosyltransferase (HPRTase)and some other gene complemented GMP synthesis block in tie novo and salvage pathways in E. coli cells.
Використання штамів ентеробактерій, які дозволяють клону вати три різних гени шляхів синтезу GMP, надавало мож ливість одержання трьох груп рестрикційних фрагментів. Однак було клоновано п'ять групп таких фрагментів. Фрагменти однієї з цих груп вміщували 2 SacI-сайти, Секвенування фрагмента геному Salmonella typhimurium показало 100 % гомологію з N-кінцем гена Y, який належить оперону hemC-hemD Е. coli. Зроблено припущення, що цей фрагмент вміщує ген, подібний гену gpp Е. coli. Також показано, що фрагмент геному S. typhimurium, який комплементує блок синтезу GMP, ампліфікований у PCR-реакції за. допомогою 20-членних прайме рів до N- та С-кінців гена hpt Е. coli, не кодує HPRT.
Использование штаммов энтеробактерий, позволяющих клонировать три различных гена путей синтеза GMP, предполагало получение трех групп рестрикционных фрагментов. Однако были клонированы пять групп таких фрагментов. Фрагменты одной из этих групп содержали 2 SacI-сайта. Секвенирование фрагмента генома. Salmonella typhimurium показало 100 % гомологии с N-концом гена У, принадлежащим оперону hemC-hemD Е. coli Сделано предположение, что этот фрагмент содержит ген, подобный гену gpp Е. coli Также показано, что фрагмент генома S. typhimurium, комплементирующий блок синтеза GMP, амплифицируемый в PCR-реакции с помощью 20-членных прайме ров к N- и С-концам гена hpt Е. coli, не кодирует HPRT.
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ISSN 0233-7657 . Б и о п о л и м е р ы и клетка . 1997. Т. 13 . № 5
Genetic mechanisms of Escherichia coli resistance
to target inactivation* Genes governing purine
metabolism in enterobacteria: an unexpected
sequence found via complementation selection
E. Cherepenko, S. Craig1
Institute of Molecular Biology and Genetics , National Academy of Sciences of Ukraine
150 Zabolotnogo str., Kyiv, 252143 , Ukraine
School of Pha rmacy , University of North Carolina
Chapel Hill, NC 27510 , USA.
Using enterobacterial strains having block in 3 different genes required for GMP synthesis, 3 groups of
inserts with different restriction patterns were expected. But the fragments cloned represented 5 such
groups. One consisted of Salmonella typhimurium DNA fragments with 2 SacI sites available. Sequencing
revealed 100 % homology of the cloned insert to the hi-term of Y protein of the hemC-hemD linkage group
of E. coli chromosome (85 min locus). It is suggested that Y may represent the gpp gene, coding for
guanosinepentaphosphatase. It was also shown that a Salmonella DNA fragment resulting from PGR
amplification with 20~mer primers complementary to the N- and С-terms of the hpt gene of E. coli did
not encode an hypoxanthine phosphoribosyltransferase (HPRTase)and some other gene complemented
GMP synthesis block in de novo and salvage pathways in E. coli cells.
Introduction. If a chemical compound selectively inac
tivates only a certain type of molecules, a target , and
if the function of this target is pivotal for cell survival,
then an effective control of cell viability could be
possible. To discover such compounds as drugs or
pesticides an innovative approach has recently been
developed known as s t ructure-based drug design (for
rev. see [1 , 2 ] ) . This approach utilizes three-dimen
sional structural data to design a selective inhibitor to
the active site of an enzyme and aliosteric sites as
well. Thus , if mutat ions r ende r a target insensitive to
an inhibitor to the active site, inhibitors to the
ailosteric sites could be developed to overcome the
problem of drug resis tance.
But alongside with this target approach others are
needed as well because non- target specific cell resis
tance may also occur. Such resis tance mechanisms
include target site overproduction due to gene ampli
fication or promoter changes, reduced inhibitor upta
ke, induction of efflux processes and inhibited de to-
© E. C H E R E P B N K . O , S. C R A I G , 1 9 9 7
xification or sequestrat ion. Moreover, cells can also
exploit some other means to survive with a pivotal
metabolic target blocked. For example, Escherichia
coli thermosensit ive phenyla lan ine- tR N A synthetase
could be protected at non-permissive temperatures
when the level of its cognate tRNA gene expression
was elevated (for rev. see [3 ]). But the enzyme could
also be protected, when cells were transformed with
multicopy colEl p lasmids which use RNA1 resem
bling the s t ructure of t R N A F h e for controlling plasmid
DNA replication [4 J.
May protection of D N A replication targets inac-
tivation with some unknown genes also occur? To
answer this question the s tudy of pur ine metabolism
is of special interest . In enterobacter ia , there are de
novo and salvage pa thways for G M P synthesis . Both
have been very well s tudied [5, 6 ] . Different bacterial
s t rains are available which allow the cloning via
complementation selection of genes belonging to both
pathways. This makes it possible to determine if
genes of only these pathways a re capable of com
plementing the block of GMP synthes is , or if some
403
CHfckEPKNKO Е., С К А К ; S.
other genes also exist which help a cell to overcome
this block.
Materials and Methods Materials. Guan ine was
obtained from «Sigma (USA). Bacto-tryptone and
vitamin-free Casamino acids were purchased from
«Difco» (USA). All other reagents used in this work
were from «Sigma» and of the highest purity avai
lable.
Bacterial strains and growth media. For comple
mentation experiments E. coli S06O9 strain {F-Dpro-
gpt-lac, hpt, purH, J, thi, pup, ara, str A) [6 J unable
to grow on selective medium containing guanine was
used as a recipient in transformation experiments
with the gene library.
Salmonella typhimurium G P 3 6 (DproAB-gpt,
purE66, sug) [7 ] with a changed subs t ra te specificity
of the hpt gene coding for hypoxanth ine phospho-
ribosyltransferase and allowing cells to grow not only
Division of S. typhimurium DNA inserts complementing GMP
synthesis block in the de novo and salvage pathway into groups
on the basis of differences in restriction pattern
Group number Restriction endonuclease
site in the insert cloned
Number of sites available
Sad
EcoRI
EcoRV
SmaJ
HindJIJ
Sail
HindJIJ
EcoRV
Sail
1 2 3
5 6 7 8 9 10 11
Fig. 1. Examples of restriction pat
terns of some Salmonella typhi
murium DNA inserts cloned via
complementat ion of cells with a
block of G M P synthesis in de novo
and salvage pathways in E. coli, a:
4 clones studied with: lanes 1 — 4:
EcoRV (clones 3 , 4 contain this
si te); lanes 5 — 8: Hindi 11 (clones
1—3 contain this si te); lanes 9 —
12: Sail (no clone contains this
si te); lanes 13 — 16: Smal (clone 4
contains this si te); b: 11 clones
studied with Hindll 1 (clones 7, 9,
11 contain this s i te) ; c: lanes 1 and
9 are Bluescript S K + vector DNA
only, other lanes show top and
bottom clones study with EcoRI,
EcoRV, Kpnl, Pstl, Sail, Xbal,
Xhol appropriately (bottom clone of
lane 10 contains EcoRI site)
on hypoxanthine but on guanine as well. This strain
was used for the gene l ibrary construction.
As a basal medium in transformation experiments
of S06O9 made competent after H a n a h a n [8} a
defined MOPS medium [9] containing 0.2 % glucose,
0.2 % vitamin-free casaminoacids, 1.5 mM thiamine,
20 jAg/rnl guanine, 50 ^ g / m l ampicillin and 40 mM
NaP0 4 -bu f f e r was used.
Generation of a genomic library of S. typhimurium
DNA and complementation. High-molecular weight
DNA obtained from strain GP36 was partially diges
ted with restriction endonuclease Sau3A and frag
ments obtained were resolved in an agarose gel.
Fragments of about 3 kb identified by reference to 1
Hindlll size markers were recovered from the gel
using the glassmilk procedure (Bio 101 Inc). The
fragments thus obtained were ligated in 1:1 ratio with
BamHI digested, dephosphoryla ted Bluescript SK"
vector (Statagene) at 4 °С overnight [10] . With this
ligation products , maximal efficiency competent DH5a
cells (from Bethesda Res . Lab) were transformed
giving rise to 80 % of white colonies on indicator LB
medium containing X-gal and I P T G [10] a 10000-
member gene l ibrary was thus constructed with every
gene present .
Alternative methods to clone genes capable of
complementing a block in GMP synthesis. Numerous
clones obtained via complementat ion selection of E.
coli S06O9 recipient cells with the GP36 S. typhi
murium DNA gene l ibrary were divided into groups
on the basis of different availability of restriction sites
in the insertions cloned. T h e clones from different
groups were studied routinely as in [11 J in the PGR
404
reaction with 20-mer primers to the N - and C-terms
of E. coli hpt gene using the sequence of the gene
published in [12 ]. T h e sequence of the PCR amplified
DNAs of the clones studied were confirmed with
3 5 S-iabelied dATP by the dideoxy chain termination
method as described previously [13] .
Results and Discussion. Using E. coli S06O9
competent cells and a GP36 S. typhimurium gene
library (in both these strains the gpt gene is deleted)
3 groups of insertions could be cloned via com
plementation selection. These groups should represent
the hpt sug allele of GP36 conferring on the salvage
pathway the ability to the recipient to grow on
guanine, the purH, J gene of Salmonella which
belongs to the de novo pathway and Salmonella pup
gene making conversion of aden ine , adenosine and
deoxyadenosine to guanine nucleotides possible [6 ].
Plating of transformed and washed S06O9 cells onto
selective MOPS medium supplemented with 20 /*g/ml
of guanine and 50 /-tg/ml of ampicillin produced more
than 50 colonies after 2 days of incubation, while the
negative control (Bluescript vector alone) produced
none. The clones obtained were picked, plasmid DNA
minipreps were isolated as in 110J and studied in
transformation experiments and with restriction en-
donucleases. High efficiency of retransformation was
shown for every insert cloned.
An example of the restriction pat terns of some
inserts complementing G M P synthesis block in E, coli
1 2 3 4
Fig. 2. Study of the Salmonella typhymurium DNA insert comp
lementing GMP synthesis block on de novo and salvage pathway in
E. coli cells with Sad restriction endonuclease (lanes I and 4: size
markers X HindiII and a ladder appropriately; lanes 2 and 3:
concentration of DNA loaded differs by the factor of 2)
G E N E T I C M E C H A N I S M O F E S C H E R I C H I A COLI R E S I S T A N C E
C A C C T G G T G G AAAAACCAAA
GCCGAAAACC C G C C A T C A C G T
Fig. 3. A small par t of the sequence obtained with the forward primer
in sequencing S. typhimurium. DNA insert containing 2 Sad sites
and complementing G M P synthesis block on the de novo and salvage
pathways of E. coli
* 4 4 ** - ***** <mm *8*w ^ * лота, « А ^ІЙИМ ^ у і - і ^ Т ^ Ж
Fig. 4. PCR-araplification with 20-mer primers to the N~ and
C-terms of E. coli hpt gene of Salmonella typhimurium DNA inserts
complementing G M P synthesis block on de novo and salvage
pathways in E. coli: lanes / and 13 are 1 Hindi If size markers; lanes
I—8: different inserts; lane 9: E. coli genomic DNA; lane 10: G P 6 6 0
Salmonella DNA; lane II: G P 3 6 Salmonella. DNA; lane 12: vector
DNA only
S06O9 strain is shown in Fig. 1. As seen from this
figure, inserts cloned could be divided into groups on
the basis of different restriction sites available. Table
demonstra tes that all these inser ts could be divided
into 5 such groups whereas only 3 groups were
anticipated with cloning system used.
Because it was known that pur Я , J of E, coli
contained an EcoRl site [141 and the hpt gene of E.
coli contained both EcoRV and Sail sites [12] , and
even though the genes studied belong to different
enterobacteria, we paid a t tent ion to the fragment
containing 2 Sad sites (Fig. 2) . Double-s t randed
DNA of this fragment was sequenced using the
Sequenase 2.0 kit (U. S. Biochemical Corp. Cleveland,
OH) and universal and reverse pr imers . A small part
of this sequence obtained with the forward primer is
shown in Fig. 3.
405
CHfckt/PBNKO E., CRAIG S.
Analysis of the sequence shown in Fig. 3 by
Simlran and dfastp programs did not reveal any
similarity to genes involved in purine metabolism, but
showed strong homology to the hypothetical protein У
of E. coli [15] . The sequnce coding for this protein
was found in 4260 bp E. coli DNA fragment mapped
at 85 min of the E. coli chromosome map and includes
three genes (hemC, hemD, X) which were suggested
to be a part of Uro operon. The function of У remains
unknown. Because the «2 Sacl» containing fragment
of Salmonella DNA is strongly homologous to У and
conferred the ability to S06O9 to grow on exogenous
guanine, we suggested on the basis of map position
that У could be identified as the gpp gene coding for
guanosinepentaphosphatase. This enzyme is respon
sible for a conversion of pppGpp into ppGpp which is
an important regulator molecule in bacterial cell (for
rev. see [16]) . In the conditions of overexpression
could this enzyme be capable to the resynthesis with
exogenous guanine and phosphates providing for
DNA synthesis precursor? Because due to this en
zyme activity also ppGp compound is formed in cells
[16 | , degradation of this molecule also could supply
cells with a precursor needed for DNA replication.
This suggestion remains to be verified and the gene
identified.
Besides «2 Sacl» insert we also studied if a
fragment containing an EcoRI site and fragments
containing EcoRV and Sail sites could be amplified in
the PCR reaction with 20-mer primers complementary
to the N- and C-terms of the hpt gene of E. coli. Fig.
4 shows results of this s tudy. The positive PCR
amplification with the primers used is observed with
E. coli DNA containing the wild type hpt allele, with
S. typhimurium GP660 DNA containing also the wild
type allele of the gene and with Salmonella GP36
DNA containing the sug mutat ion in the hpt gene
(ability to grow on hypoxanth ine and guanine) . As
seen from Fig. 4, lane 8, the fragment containing an
EcoRI could not be amplfied by PCR with primers
used.
This shows that there is no cell DNA conta
mination in the plasmid DNA preparat ions used. T h e
inserts of S. typhimurium. DNA containing EcoRV,
Sail, Smal and HindiII sites were all positive in the
PCR reaction with generic 20-mer primers comple
mentary to the N - and C-terms of E. coli hpt gene.
At present these fragments are under s tudy, but
sequencing of one of the positive fragments (data not
shown) demonstrated that it did not encode an
HPRTase . If this insert does not include the pup
gene, then some new gene capable to complement a
block in GMP synthesis might be identified.
О. Й. Черспенко, С. Крег
Генетичні механізми стійкості клітин Escherichia coli
до інактивації мішеней. Гени пуринового метаболізму:
клонування за допомогою комплементаці ї
невідомої послідовності
Резюме
Використання штамів ентеробактерій, які дозволяють кло
ну вати три різних гени шляхів синтезу GMP, надавало мож
ливість одержання трьох груп рестрикційних фрагментів.
Однак було клоновано п'ять групп таких фрагментів. Фраг
менти однієї з цих груп вміщували 2 Sad-сайти, Секвенуваяня
фрагмента геному Salmonella typhimurium показало 100 %
гомологію з N-кінцем гена Y, який належить оперону ІіетС-
hemD Е. coli. Зроблено припущення, що цей фрагмент вміщує
ген, подібний гену gpp Е. coli. Також показано, що фрагмент
геному S. typhimurium, який комплементує блок синтезу GMP,
ампліфікований у PCR-реакції за. допомогою 20-членних прай
ме рів до N- та С-кінців гена hpt Е. coli, не кодує HPRT.
Е. И. Черспенко, С. Крэг
Генетические механизмы устойчивости клеток Escherichia coli
к инактивации мишени . Гены пуринового метаболизма:
клонирование с помощью комплементации неизвестной
последовательности
Резюме
Использование штаммов энтеробактерий, позволяющих кло
нировать три различных гена путей синтеза GMP, предпола
гало получение трех групп рестрикционных фрагментов. Од
нако были клонированы пять групп таких фрагментов. Фраг
менты одной из этих групп содержали 2 Sad-сайта. Секвени-
рование фрагмента генома. Salmonella typhimurium показало
100 % гомологии с N-концом гена У, принадлежащим оперону
hemC-hemD Е. coli Сделано предположение, что этот фраг
мент содержит ген, подобный гену gpp Е. coli Также показано,
что фрагмент генома S. typhimurium, комплементирующий
блок синтеза GMP, амплифицируемый в PCR-реакции с по
мощью 20-членных прайме ров к N- и С-концам гена hpt Е. coli,
не кодирует HPRT.
R E F E R E N C E S
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Received 24.02.97
407
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| id | nasplib_isofts_kiev_ua-123456789-155647 |
| institution | Digital Library of Periodicals of National Academy of Sciences of Ukraine |
| issn | 0233-7657 |
| language | English |
| last_indexed | 2025-12-07T17:41:52Z |
| publishDate | 1997 |
| publisher | Інститут молекулярної біології і генетики НАН України |
| record_format | dspace |
| spelling | Cherepenko, E. Craig, S. 2019-06-17T09:43:09Z 2019-06-17T09:43:09Z 1997 Genetic mechanisms of Escherichia coli resistance to target inactivation. Genes governing purine metabolism in enterobacteria: an unexpected sequence found via complementation selection / E. Cherepenko, S. Craig // Биополимеры и клетка. — 1997. — Т. 13, № 5. — С. 403-407. — Бібліогр.: 16 назв. — англ. 0233-7657 DOI: http://dx.doi.org/10.7124/bc.00049E https://nasplib.isofts.kiev.ua/handle/123456789/155647 Using enierobacterial strains having block in 3 different genes required for GMP synthesis, 3 groups of inserts with different restriction patterns were expected. But the fragments cloned represented 5 such, groups. One consisted of Salmonella typhimurium DNA fragments with 2 SacI sites available. Sequencing revealed 100 % homology of the cloned insert to the N-tenn of Y protein of the hemC-hemD linkage group of E. coli chromosome (85 min locus). It is suggested that Y may represent the gpp gene, coding for guanosinepentaphosphatase. It was also shown that a Salmonella DNA fragment resulting from PCR amplification with 20-mer primers complementary to the N- and C-terms of the hpt gene of E. coli did not encode an hypoxanthine phosplioribosyltransferase (HPRTase)and some other gene complemented GMP synthesis block in tie novo and salvage pathways in E. coli cells. Використання штамів ентеробактерій, які дозволяють клону вати три різних гени шляхів синтезу GMP, надавало мож ливість одержання трьох груп рестрикційних фрагментів. Однак було клоновано п'ять групп таких фрагментів. Фрагменти однієї з цих груп вміщували 2 SacI-сайти, Секвенування фрагмента геному Salmonella typhimurium показало 100 % гомологію з N-кінцем гена Y, який належить оперону hemC-hemD Е. coli. Зроблено припущення, що цей фрагмент вміщує ген, подібний гену gpp Е. coli. Також показано, що фрагмент геному S. typhimurium, який комплементує блок синтезу GMP, ампліфікований у PCR-реакції за. допомогою 20-членних прайме рів до N- та С-кінців гена hpt Е. coli, не кодує HPRT. Использование штаммов энтеробактерий, позволяющих клонировать три различных гена путей синтеза GMP, предполагало получение трех групп рестрикционных фрагментов. Однако были клонированы пять групп таких фрагментов. Фрагменты одной из этих групп содержали 2 SacI-сайта. Секвенирование фрагмента генома. Salmonella typhimurium показало 100 % гомологии с N-концом гена У, принадлежащим оперону hemC-hemD Е. coli Сделано предположение, что этот фрагмент содержит ген, подобный гену gpp Е. coli Также показано, что фрагмент генома S. typhimurium, комплементирующий блок синтеза GMP, амплифицируемый в PCR-реакции с помощью 20-членных прайме ров к N- и С-концам гена hpt Е. coli, не кодирует HPRT. en Інститут молекулярної біології і генетики НАН України Биополимеры и клетка Геном и его регуляция Genetic mechanisms of Escherichia coli resistance to target inactivation. Genes governing purine metabolism in enterobacteria: an unexpected sequence found via complementation selection Генетичні механізми стійкості клітин Escherichia coli до інактивації мішеней. Гени пуринового метаболізму: клонування за допомогою комплементації невідомої послідовності Генетические механизмы устойчивости клеток Escherichia coli к инактивации мишени. Гены пуринового метаболизма: клонирование с помощью комплементации неизвестной последовательности Article published earlier |
| spellingShingle | Genetic mechanisms of Escherichia coli resistance to target inactivation. Genes governing purine metabolism in enterobacteria: an unexpected sequence found via complementation selection Cherepenko, E. Craig, S. Геном и его регуляция |
| title | Genetic mechanisms of Escherichia coli resistance to target inactivation. Genes governing purine metabolism in enterobacteria: an unexpected sequence found via complementation selection |
| title_alt | Генетичні механізми стійкості клітин Escherichia coli до інактивації мішеней. Гени пуринового метаболізму: клонування за допомогою комплементації невідомої послідовності Генетические механизмы устойчивости клеток Escherichia coli к инактивации мишени. Гены пуринового метаболизма: клонирование с помощью комплементации неизвестной последовательности |
| title_full | Genetic mechanisms of Escherichia coli resistance to target inactivation. Genes governing purine metabolism in enterobacteria: an unexpected sequence found via complementation selection |
| title_fullStr | Genetic mechanisms of Escherichia coli resistance to target inactivation. Genes governing purine metabolism in enterobacteria: an unexpected sequence found via complementation selection |
| title_full_unstemmed | Genetic mechanisms of Escherichia coli resistance to target inactivation. Genes governing purine metabolism in enterobacteria: an unexpected sequence found via complementation selection |
| title_short | Genetic mechanisms of Escherichia coli resistance to target inactivation. Genes governing purine metabolism in enterobacteria: an unexpected sequence found via complementation selection |
| title_sort | genetic mechanisms of escherichia coli resistance to target inactivation. genes governing purine metabolism in enterobacteria: an unexpected sequence found via complementation selection |
| topic | Геном и его регуляция |
| topic_facet | Геном и его регуляция |
| url | https://nasplib.isofts.kiev.ua/handle/123456789/155647 |
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