Lysogeny by MS2 phage. Analysis of a recombinant plasmid containing MS2 RNA-like sequence
pL34 recombinant plasmid coding MS2-llke RNA synthesis in plasmid-containing cells has been studied using physical mapping and blot-hybridisation with P32-labelled pMS27 plasmid fragments including a DNA copy of MS2 phage RNA. The authors have determined the size of a fragment containing MS2-like se...
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Інститут молекулярної біології і генетики НАН України
1995
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| Цитувати: | Lysogeny by MS2 phage. Analysis of a recombinant plasmid containing MS2 RNA-like sequence / Т.P. Pererva, N.Yu. Mirjuta, A.Yu. Mirjuta, M.I. Woodmaska, E.N. Zherebtsova // Биополимеры и клетка. — 1995. — Т. 11, № 1. — С. 61-65. — Бібліогр.: 10 назв. — англ. |
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irk-123456789-1556512019-06-18T01:27:16Z Lysogeny by MS2 phage. Analysis of a recombinant plasmid containing MS2 RNA-like sequence Pererva, T.P. Mirjuta, N.Yu. Mirjuta, A.Yu. Woodmaska, M.I. Zherebtsova, E.N. pL34 recombinant plasmid coding MS2-llke RNA synthesis in plasmid-containing cells has been studied using physical mapping and blot-hybridisation with P32-labelled pMS27 plasmid fragments including a DNA copy of MS2 phage RNA. The authors have determined the size of a fragment containing MS2-like sequence and its localisation inside the hybrid plasmid. Рекомбінантну плазміду pL34, що кодує синтез MS2-подібної РНК у плазмідовмісних клітинах, вивчали методом фізичного картування та блот-гібридизації. Як зонд використовували фрагмент плазміди pMS27, що містить ДНК-копію РНК фага MS2. Було визначено розміри фрагменту, який включає MS2 РНК-подібну послідовність, та його локалізацію в гібридній плазміді. Рекомбинантная плазмида pL34, кодирующая синтез MS2-подобной РНК в плазмидосодержащих клетках, изучена методами физического картирования и блот-гибридизации с использованием в качестве зонда фрагмента плазмиды pMS27, содержащей ДНК-копию плазмиды фага MS2. Оп ределены размеры фрагмента, включающего MS2 РНК-подобную последовательность, и его локализацию в составе гибридной плазмиды. 1995 Article Lysogeny by MS2 phage. Analysis of a recombinant plasmid containing MS2 RNA-like sequence / Т.P. Pererva, N.Yu. Mirjuta, A.Yu. Mirjuta, M.I. Woodmaska, E.N. Zherebtsova // Биополимеры и клетка. — 1995. — Т. 11, № 1. — С. 61-65. — Бібліогр.: 10 назв. — англ. 0233-7657 DOI: http://dx.doi.org/10.7124/bc.0003D4 http://dspace.nbuv.gov.ua/handle/123456789/155651 577.21 en Биополимеры и клетка Інститут молекулярної біології і генетики НАН України |
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English |
| description |
pL34 recombinant plasmid coding MS2-llke RNA synthesis in plasmid-containing cells has been studied using physical mapping and blot-hybridisation with P32-labelled pMS27 plasmid fragments including a DNA copy of MS2 phage RNA. The authors have determined the size of a fragment containing MS2-like sequence and its localisation inside the hybrid plasmid. |
| format |
Article |
| author |
Pererva, T.P. Mirjuta, N.Yu. Mirjuta, A.Yu. Woodmaska, M.I. Zherebtsova, E.N. |
| spellingShingle |
Pererva, T.P. Mirjuta, N.Yu. Mirjuta, A.Yu. Woodmaska, M.I. Zherebtsova, E.N. Lysogeny by MS2 phage. Analysis of a recombinant plasmid containing MS2 RNA-like sequence Биополимеры и клетка |
| author_facet |
Pererva, T.P. Mirjuta, N.Yu. Mirjuta, A.Yu. Woodmaska, M.I. Zherebtsova, E.N. |
| author_sort |
Pererva, T.P. |
| title |
Lysogeny by MS2 phage. Analysis of a recombinant plasmid containing MS2 RNA-like sequence |
| title_short |
Lysogeny by MS2 phage. Analysis of a recombinant plasmid containing MS2 RNA-like sequence |
| title_full |
Lysogeny by MS2 phage. Analysis of a recombinant plasmid containing MS2 RNA-like sequence |
| title_fullStr |
Lysogeny by MS2 phage. Analysis of a recombinant plasmid containing MS2 RNA-like sequence |
| title_full_unstemmed |
Lysogeny by MS2 phage. Analysis of a recombinant plasmid containing MS2 RNA-like sequence |
| title_sort |
lysogeny by ms2 phage. analysis of a recombinant plasmid containing ms2 rna-like sequence |
| publisher |
Інститут молекулярної біології і генетики НАН України |
| publishDate |
1995 |
| url |
http://dspace.nbuv.gov.ua/handle/123456789/155651 |
| citation_txt |
Lysogeny by MS2 phage. Analysis of a recombinant plasmid containing MS2 RNA-like sequence / Т.P. Pererva, N.Yu. Mirjuta, A.Yu. Mirjuta, M.I. Woodmaska, E.N. Zherebtsova // Биополимеры и клетка. — 1995. — Т. 11, № 1. — С. 61-65. — Бібліогр.: 10 назв. — англ. |
| series |
Биополимеры и клетка |
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2025-07-14T07:50:47Z |
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2025-07-14T07:50:47Z |
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| fulltext |
УДК 577.21
Т. Pererva, N. Mirjuta, A. Mirjuta, M. Woodmaska, E. Zherebtsova
LYSOGENY BY MS2 PHAGE. ANALYSIS OF A RECOMBINANT
PLASMID CONTAINING MS2 RNA-LIKE SEQUENCE
pL34 recombinant plasmid coding MS2-like RNA synthesis in plasmid-containing cells
has been studied using physical mapping and blot-hybridisation with P32-labelled pMS27
plasmid Iragmenis including a DNA copy of MS2 phage RNA. The authors have deter
mined the size of a fragment containing MS2-like sequence and its localisation inside
the hybrid plasmid.
Introduction. Dispite of RNA-containing phages being among the best
studied biosystems, some of their properties, especially phage-host inter
actions, have not been completely investigated. One of the problems is a
cause of multiple resistant cells development in E. coli cultures infected
by RNA-containing phages in the conditions not favorable for quick cell
division. In some previous papers concerning RNA-containing phages bio
logy this phenomenon has been discussed from the point of view of pos
sible preexisting mutants selection. Nevertheless, such an interpretation
does not seem to be completely convincing because of large scale phage
resistance development. In our previous paper [1] we have shown that
more than 1 % of infected bacterium offspring receives MS2-re-
sistance marker which may not be due to pre-existing mutants selection,
We interpret such a fact as a non-direct proof of direct interactions bet
ween phage genome and cell host DNA; out interpretation has been con
firmed by non-stability of primary MS2-induced mutations and derivative
forms segregation from these ones [2]. In order to prove that these mu
tants properties are caused not by a persistent MS2 infection but due to
phage genome integration into the host cell chromosome we have created
a genetic library of EcoRI fragments of a segregant obtained by us — a
lysing E. coli AB 259 Hfr 3000 mutant [3]. A total RNA preparation iso
lated from plasmid-forming cells has been hybridised both with pMS27
DNA containing MS2 phage cDNA and with a part of this cDNA without
vector sequence. The results obtained prove phage specific RNA to be
transcripted from chromosomal DNA of the MS2-induced E. coli mutant;
so we have really demonstrated that MS2 phage is able to establish true
lysogeny as a state based on physical integration of phage and cellular
DNA's. In the present paper we present our first data on our study of
cloned host cell DNA fragment having the goal to determine how large
is MS2-specific region and which is its localisation on the genetic map
of our recombinant plasmid; we aim also to find there some recognition
sites for several popular restrictive enzymes.
Materials and methods. P 1 a s m і d s. Recombinant plasmid pL34 has
been constructed by us using non-replicative Лр-fragment of pCVl6 plas
mid and described in the previous paper [3] as pL26 plasmid. A strain
containing pCV16 plasmid [4}' has been received from the Institute of
Biochemistry and Physiology of Micro-organisms (Russian Academy of
Sciences, Pushtchino-na-Okie, Moscow district, Russian Federation).
A strain carrying pMS27 plasmid [5, 6] is a friendly present of Dr R. De-
vos (Gent University, Belgium).
© T. Pererva, N. Mirjuta, A. Mirjuta, M. Woodmaska, E. Zherebtsova, 1995
ISSN 0233-7657. БИОПОЛИМЕРЫ И КЛЕТКА. 1995. Т. И. № I 61
N u t r i e n t m e d i a . We have mostly used 0.6% and 1.2% ami
no peptide (AP) containing agar and AP-containing broth. Plasmid iso
lation, restriction analysis, and blot-hybridisation have been perfected
according to [7]; physical mapping of plasmid DNA sequences has been
made with mutual and single digestion approaches [8].
Results and discussion. To determine the size of a recombinant DNA
plasmid fragment carrying MS2-\\ke sequence and its localisation in the
f 2 3 4 5 6'7 8 9 1 0 f Z 3 4 5 6 7 8 9 10
Fig. 1. Electrophoregram of
pCV16 and pL34 plasmids DNA
(a) and radio autograph obtai
ned during hybridisation of the
corresponding filter with r e
labelled pMS27 (b) and pMS27
fragment (c) DNA's; lanes. 1 —
pMS27 DNA; 2 — pCV16 DNA;
3 — pCV16 DNA+EcoRI; 4 —
pCV16 T>NA+PstI; 5 — pCV16
DNA+PslI+EcoRI; 6 — pL34
DNA; 7 — pL34 DNA+EcoRI;
8 — PL34 DNA+PstI; 9 — pL34
DNA+EcoRI+PstI; W —
lambda phage DNA+EcoRI+
-\-HindIlI (marker lane)
recombinant plasmid we have used physical mapping and blot-hybridi
sation. Our probes were both the whole pMS27 plasmid containing MS2
cDNA and a fragment of this DNA copy without vector sequence. The
results of an experiment with EcoRI and PstI treatment of pL34 and
pCV16 plasmids are shown in the Fig. 1, a.
It is clear from the Fig. \,b that the labelled pMS27 having been
used as a probe gives hybridisation with all the fragments of both plas
mids (pCV16 and pL34). It may be due to pCV16 origin, i. e. to the pre
sence of some homologies with pBR322 sequences in the vector part of
pMS27. In the next blot-hybridisation experiment (see Fig. l,c) where
62 ISSN 0233-7657. БИОПОЛИМЕРЫ И КЛЕТКА. 1995. Т. И . № 1
file://-/-HindIlI
cDNA copies of phage MS2 RNA have been used hybridisation lines cor
respond to pCV16 fragments of the size 5.1 kbs and to pL34 fragments-
as large as 5.1 and 4.8 kbs in single and double treatments. The smaller
pCVJ6 fragments (their sizes are about 1.1 and 0.7 kbs) corresponding
to Ар-fragment of the total size 1.8 kbs do not hybridise with pMS27
fragment, i. e. a region of the recombinant plasmid able to hybridise with
pMS27 fragment is not a part of pCV16 plasmid.
Fig. 2. Eleclrophoregram of pL34 plasmid DNA (a) and radio autograph obtained du
ring hybridisation of the corresponding filter with P32-labelled pMS27 fragment DNA (6)-
lanes: l — pL34 DNA+Pstl; 2 — pL34 DNA+Pstl+BamHI; 3 — pL34 VNA+ВатШ;
4 — pL34 ША+ВатНІ+НШІП; 5 — pL34. DNA+HindIII; 6 — pL34 DNA +
+Hindlll+Pstl; 7 — pL34 VNA+Pstl+EcoRI; 8 — pL34 ША+EcoRI; 9 — pL34 DNA+
+EcoRI+BamHI; 10 — pL34 ША+EcoRI+HindlII; 11 — lambda phage DNA+ '
+EcoRI+HindIII (marker lane)
Fig. 3. Physical maps of pCV16 and pL34 plasmids. A shaded part corresponds to the
fragment including ampicilline resistance gene, an unshaded and wider one corresponds
to a fragment containing MS2-like sequence
Our data obtained in the experiments of recombinant and vector plas
mids hybridisation with pMS27 plasmid carrying MS2 cDNA and with
this cDNA copy permit us to conclude that the sequence homoloo-ical to
MS2 RNA is situated in the large pL34 fragment (5.1 kbs) limited by
Pstl sites. The hybridisation observed with pCV16 DNA is thought to be
most probably due to random short sequences, pCV16 DNA beino- non-
hybrisidable both with MS2 RNA and also total RNA isolated from a
strain containing a recombinant plasmid and being able to hybridise both
with labelled pMS27 plasmid and with its fragment [3] These data sug
gest that on the contrary to recombinant plasmid pCV16 plasmid con-
ISSN 0233-7657. БИОПОЛИМЕРЫ И КЛЕТКА. 1995. Т. 11. № 1
63
tains no sequence coding MS2-like RNA synthesis and forming hybrids
with such RNA. So the hybridisation observed in pCV16 experiments may
be explained by a presence of a random sequence or of a sequence with
the biological function analogue to lysogenic phage integration site in
host cell chromosome. Electrophoregrams of the recombinant plasmid af
ter PstI, BamHI, Hindlll, and EcoRI restrictive digests as well as blot-
hybridisation of fragments obtained with pMS27 fragment are demonstra
ted in the Fig. 2.
Our results lead to the conclusion that ЛІ52-1іке sequence in most
probably localised inside the fragment as large as 3.9 kbs limited by PstI
and BamHI cleavage sites. The localisation of this
fragment is shown on the recombinant plasmid map
given in the Fig. 3.
The undoubtful hybridisation of this fragment
with a fragment of MS2 cDNA fragment and almost
total coincidence of its size (3.9 kbs) with MS2 RNA
size (3.569 kbs) [9] permit to think that MS2-like
sequence integrated into pL34 recombinant plasmid
is localised inside this fragment. It should be noted,
however, that this region contains no EcoRI and
Sail sites detected inside MS2 cDNA [10]. A Sail
site situated inside of Pstl-BamHI fragment (see
Fig. 4) is localised not far from one of its termini
corresponding in its localisation to none of two Sail
sites detected inside DNA copy of the phage RNA
Fig. 4. Electrophoregram of pL34 plasmid with corresponding
lanes: l — pL34 DNA+Pstl+BamHI; 2 — pL34 DN A+Pstl+
+BamHI+PvuII; 3 — pL34 DNA+Pstl+BamHI+BgUI; 4 —
pL34 DNA+Pstl+BamHI+Sall; 5 —lambda phage DNA-f
+EcoRI+HindIII (marker lane)
[10]. So the region of recombinant plasmid being correspondent to MS2
sequence according to hybridisation experiments is probably a fragment
of the structure more complex than a double-stranded one. Such a con
clusion is not contradictory to our previous results demonstrating that
labelled recombinant plasmid DNA fails to hybridise with MS2 RNA [3] .
The absence of recombinant plasmid DNA and MS2 RNA hybridisation
accompanied by MS2-like RNA synthesis in plasmid-containing cells may
be most probably explained by a hypothesis that this plasmid contains
a plus-chain of MS2 RNA-like DNA or even MS2 RNA and has a three-
stranded structure in the fragment of the size about 3.9 kbs. Our point
of view is the last version is also probable because of being able to ex
plain our negative results obtained when we have attempted to detect
some restrictive sites inside pL34 plasmid Pstl-BamHI fragment with si
ze of 3.9 kb, these ones being undoubtfully present in the DNA copy of
MS2 RNA.
This work was supported by Fundamental Investigation Fund of Sta
te Committee on Science and Technology under Parliament of Ukraine.
Т. П. Перерва, Н. Ю. Мірюта, Г. Ю. Мірюта, М. І. Вудмаска, Е. М. Жеребцева
ЛІЗОГЕШЯ У ФАГА MS2. АНАЛІЗ РЕКОМБІНАНТНОІ ПЛАЗМІДИ,
ЩО МІСТИТЬ MS2 РНК-ПОДІБНУ ПОСЛІДОВНІСТЬ
Р е з ю м е
Рекомбінантну плазміду pL34, що кодує синтез ЛІ52-подібної РНК у шіазмідовмісних
клітинах, вивчали методом фізичного картування та блот-гібридизації. Як зонд ви
користовували фрагмент плазміди pMS27, що містить ДНК-копію РНК фага MS2.
Було визначено розміри фрагменту, який включає MS2 РНК-подібну послідовність,
та його локалізацію в гібридній плазміді.
<64 ISSN 0233-7667. БИОПОЛИМЕРЫ И КЛЕТКА. 1995. Т. 11. № 1
REFERENCES
1. Перерва Т. П. Устойчивость к фагу MS2, индуцированная у Ё, coll при заражении
этим фагом // Цитология и генетика.—19*77.—11, № 1.— С. 3—9.
2. Перерва Т. П., Малюта С. С. Система Л152-индуцированных мутантов Е. coll по
F-фактору//Молекуляр. 'биология,— L984.— 38.— С. 81—90.
3. Перерва Т. П., Мирюта Н. Ю., Мирюта А. Ю,. іЛизогения у фага MS2. Синтез
фагоспецифичной РНК на клеточной ДНК // Биополимеры и клетка.— 1993.—
9, № 1.—С. 45—50.
4. Копылова-Свиридова Т. Н., Фодор И., Баев А. А. Новые векторные плазмиды для
селекции рекомбинантных клонов Е. coli // Докл. АН СССР.— 1979.— 245, № 5.—
С. 1250—1253.
5. Devos R., Van Emmelo I., Contreras R., Fiers W. Construction and characterisation
of a plasmid containing a nearly full-size DNA copy of bacteriophage MS2 RNA //
J. Мої. Biol.—1979.—128, N 4,—P. 595—619.
6. Devos R., Contreras R., Van Emmelo /., Fiers W. Identification of the transloca-
table element IS1 in a molecular chimera constructed with plasmid pBR322 DNA
into which a bacteriophage MS2 copy was inserted by the poly(dA)poly(dT; linker
method // Ibid.—P. 621—632.
7. Маниатис Т., Фрич Э., Сэмбрук Д. Молекулярное клонирование.— М. : Мир, 19в4.—
479 с.
8. Fitch W. М., Smith Т. F., Ralph W. W. Mapping the order of DNA restriction frag
ments // Gene,—1983,—22, N 1.—P. 19—29.
9. Fiers W., Contreras R., Duerinck F. et al. Complete nucleotide sequence of bacte
riophage MS2 RNA // Nature,—1976.—260.—P. 500—507.
10. Berkhout B,, Van Duin J. Mechanism of translational coupling between coat protein
and replicase gene of RNA bacteriophage MS2 // Nucl. Acids Res.— 1985,— 13,
N 19.—P. 6955—6967.
Inst. Мої. Biol, and Genet. 25.07.94
the Nat. Acad. Sci. of Ukraine, Kiev
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