Physical mapping of Malacosoma neustria nuclear polyhedrosis virus genome and its modification in Antheraea pernyi cell culture
A physical map of the M. neustria nuclear polyhedrosis virus (ManeNPV) genome was constructed, the complete order of BamHl, Kpnl and PstI restriction enzyme sites was determined, a polyhedrin gene was localized on the map. The viral DNA size was calculated to be about 139 kbp. Restriction endonuclea...
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| Zitieren: | Physical mapping of Malacosoma neustria nuclear polyhedrosis virus genome and its modification in Antheraea pernyi cell culture / I.М. Kіhno, L.I. Strokovskaya, R.A. Meleshko, J. Michalic, A.P. Solomko // Вiopolymers and Cell. — 2002. — Т. 18, № 6. — С. 522-528. — Бібліогр.: 16 назв. — англ. |
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Kikhno, I.M Strokovskaya, L.I. Meleshko, R.A. Michalic, J. Solomko, A.P. 2019-06-18T11:21:15Z 2019-06-18T11:21:15Z 2002 Physical mapping of Malacosoma neustria nuclear polyhedrosis virus genome and its modification in Antheraea pernyi cell culture / I.М. Kіhno, L.I. Strokovskaya, R.A. Meleshko, J. Michalic, A.P. Solomko // Вiopolymers and Cell. — 2002. — Т. 18, № 6. — С. 522-528. — Бібліогр.: 16 назв. — англ. 0233-7657 DOI:http://dx.doi.org/10.7124/bc.000630 https://nasplib.isofts.kiev.ua/handle/123456789/156308 577.29 A physical map of the M. neustria nuclear polyhedrosis virus (ManeNPV) genome was constructed, the complete order of BamHl, Kpnl and PstI restriction enzyme sites was determined, a polyhedrin gene was localized on the map. The viral DNA size was calculated to be about 139 kbp. Restriction endonuclease profiles of the DNA of ManeNPV plaques isolate propagated in A. pernyi cells demonstrated "persistent heterogeneity", submolar bands were shown to appear in a digestion pattern of DNA of the first passage virus. These bands were proved to be due to the DNA molecules presence in the non-homogeneous virus DNA pool, their chains having been shown to carry a putative break in a definite site. Such a "break site" was localized on the physical, map of ManeNPV genome. Складено фізичну карту геному вірусу ядерного поліедрозу М. neustria (ManeNPV), визначено розташування сайтів для рестриктаз BamHl, Kpnl та PstI, на карті локалізовано ген поліедрину. Розмір вірусної ДНК визначено приблизно в 139 тис. п. н. Профілі рестриктів ДНК ManeNPV, ізольованих з бляшок, отриманих у клітинах A. pernyi, демонструють «тривку гетерогенність», субмолярні фрагменти виявлено в паттерні рестрикції після першого пасажу вірусу. Встановлено, що ці фрагменти присутні в пулі гетерогенної вірусної ДНК, а її нитки мають розриви в певному місці. Таке місце розриву було локалізовано на фізичній карті геному ManeNPV. Составлена физическая карта генома вируса ядерного полиэдроза М. neustria (ManeNPV), определено расположение сайтов для рестриктаз BamHl, Kpnl и PstI, на карте локализован ген полиэдрина. Размер вирусной ДНК определен приблизительно в 139 тыс п. к. Профили рестриктов ДНК ManeNPV, изолироанных из бляшек, полученных в клетках A. pernyi, демонстрируют «устойчивую гетерогенность», субмолярные фрагменты обнаружены в паттерне рестрикции после первого пассажа вируса. Установлено, что эти фрагменты присутствуют в пуле гетерогенной вирусной ДНК, а ее нити имеют разрыв в определенном месте. Такое место разрыва было локализовано на физической карте генома ManeNPV. en Інститут молекулярної біології і генетики НАН України Біополімери і клітина Віруси та клітина Physical mapping of Malacosoma neustria nuclear polyhedrosis virus genome and its modification in Antheraea pernyi cell culture Фізичне картування геному вірусу ядерного поліедрозу Malacosoma neustria і його модифікація в клітинній культурі Antheraea pernyi Физическое картирование генома вируса ядерного полиэдроза Malacosoma neustria и его модификация в клеточной структуре Antheraea pernyi Article published earlier |
| institution |
Digital Library of Periodicals of National Academy of Sciences of Ukraine |
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DSpace DC |
| title |
Physical mapping of Malacosoma neustria nuclear polyhedrosis virus genome and its modification in Antheraea pernyi cell culture |
| spellingShingle |
Physical mapping of Malacosoma neustria nuclear polyhedrosis virus genome and its modification in Antheraea pernyi cell culture Kikhno, I.M Strokovskaya, L.I. Meleshko, R.A. Michalic, J. Solomko, A.P. Віруси та клітина |
| title_short |
Physical mapping of Malacosoma neustria nuclear polyhedrosis virus genome and its modification in Antheraea pernyi cell culture |
| title_full |
Physical mapping of Malacosoma neustria nuclear polyhedrosis virus genome and its modification in Antheraea pernyi cell culture |
| title_fullStr |
Physical mapping of Malacosoma neustria nuclear polyhedrosis virus genome and its modification in Antheraea pernyi cell culture |
| title_full_unstemmed |
Physical mapping of Malacosoma neustria nuclear polyhedrosis virus genome and its modification in Antheraea pernyi cell culture |
| title_sort |
physical mapping of malacosoma neustria nuclear polyhedrosis virus genome and its modification in antheraea pernyi cell culture |
| author |
Kikhno, I.M Strokovskaya, L.I. Meleshko, R.A. Michalic, J. Solomko, A.P. |
| author_facet |
Kikhno, I.M Strokovskaya, L.I. Meleshko, R.A. Michalic, J. Solomko, A.P. |
| topic |
Віруси та клітина |
| topic_facet |
Віруси та клітина |
| publishDate |
2002 |
| language |
English |
| container_title |
Біополімери і клітина |
| publisher |
Інститут молекулярної біології і генетики НАН України |
| format |
Article |
| title_alt |
Фізичне картування геному вірусу ядерного поліедрозу Malacosoma neustria і його модифікація в клітинній культурі Antheraea pernyi Физическое картирование генома вируса ядерного полиэдроза Malacosoma neustria и его модификация в клеточной структуре Antheraea pernyi |
| description |
A physical map of the M. neustria nuclear polyhedrosis virus (ManeNPV) genome was constructed, the complete order of BamHl, Kpnl and PstI restriction enzyme sites was determined, a polyhedrin gene was localized on the map. The viral DNA size was calculated to be about 139 kbp. Restriction endonuclease profiles of the DNA of ManeNPV plaques isolate propagated in A. pernyi cells demonstrated "persistent heterogeneity", submolar bands were shown to appear in a digestion pattern of DNA of the first passage virus. These bands were proved to be due to the DNA molecules presence in the non-homogeneous virus DNA pool, their chains having been shown to carry a putative break in a definite site. Such a "break site" was localized on the physical, map of ManeNPV genome.
Складено фізичну карту геному вірусу ядерного поліедрозу М. neustria (ManeNPV), визначено розташування сайтів для рестриктаз BamHl, Kpnl та PstI, на карті локалізовано ген поліедрину. Розмір вірусної ДНК визначено приблизно в 139 тис. п. н. Профілі рестриктів ДНК ManeNPV, ізольованих з бляшок, отриманих у клітинах A. pernyi, демонструють «тривку гетерогенність», субмолярні фрагменти виявлено в паттерні рестрикції після першого пасажу вірусу. Встановлено, що ці фрагменти присутні в пулі гетерогенної вірусної ДНК, а її нитки мають розриви в певному місці. Таке місце розриву було локалізовано на фізичній карті геному ManeNPV.
Составлена физическая карта генома вируса ядерного полиэдроза М. neustria (ManeNPV), определено расположение сайтов для рестриктаз BamHl, Kpnl и PstI, на карте локализован ген полиэдрина. Размер вирусной ДНК определен приблизительно в 139 тыс п. к. Профили рестриктов ДНК ManeNPV, изолироанных из бляшек, полученных в клетках A. pernyi, демонстрируют «устойчивую гетерогенность», субмолярные фрагменты обнаружены в паттерне рестрикции после первого пассажа вируса. Установлено, что эти фрагменты присутствуют в пуле гетерогенной вирусной ДНК, а ее нити имеют разрыв в определенном месте. Такое место разрыва было локализовано на физической карте генома ManeNPV.
|
| issn |
0233-7657 |
| url |
https://nasplib.isofts.kiev.ua/handle/123456789/156308 |
| citation_txt |
Physical mapping of Malacosoma neustria nuclear polyhedrosis virus genome and its modification in Antheraea pernyi cell culture / I.М. Kіhno, L.I. Strokovskaya, R.A. Meleshko, J. Michalic, A.P. Solomko // Вiopolymers and Cell. — 2002. — Т. 18, № 6. — С. 522-528. — Бібліогр.: 16 назв. — англ. |
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ISSN 0233-7657. Біополімери і клітина. 2002. Т. 18. № 6
ВІРУСИ І КЛІТИНА
Physical mapping of Malacosoma neustria nuclear
polyhidrosis virus genome and its modification in
Antheraea pernyi cell culture
I. M. Kikhno, L. L Strokovskaya, R. A. Meleshko, J. Michalic1, A. P. Solomko
Institute of Molecular Biology and Genetics, National Academy of Science of Ukraine
Vul. Zabolotnoho, 150, Kyiv, 03143 , Ukraine
department of grotein Biosynthesis, Institute of Biochemistry and Biophysics NAS of Poland
A. Pawinskega 5 , 02 -106 , Warsawa, Poland
A physical map of the M. neustria nuclear polyidrosis virus (ManeNPV) genome was constructed, the
complete order of BamHJ, Kpnl and Pstl restriction enzyme sites was determinedt a polyhedrin gene was
localized on the map. The viral DNA size was calculated to be about 139 kbp. Restriction endonuclease
profiles of the DNA of ManeNPV plaques isolate propagated in A. pernyi cells demonstrated ^persistent
heterogeneity*, submolar bands were shown to appear in a digestion pattern of DNA of the first passage
virus. These bands were proved to be due to the DNA molecules presence in the non-homogeneous virus
DNA pool, their chains having been shown to carry a putative break in a definite site. Such a *break site»
was localized on the physical, map of ManeNPV genome.
The Baculoviridae contain double-stranded circular
DNA viruses infecting a lot of species belonging
mainly to the Lepidoptera> Diptera> and Hymenoptera
orders. Baculoviruses with their large genomes and
complex reproduction cycle accompanied by the cas
cade gene expression regulation have become an
interesting and well-made topic of molecular biology
studies. The interest to the representatives of this
family is also caused by three aspects of their
practical use. First of them is an application of the
baculoviruses as vectors for gene therapy, the problem
being investigated during last decade [1, 2 ] . Two
other aspects are more traditional: baculoviruses are
considered as viral insecticides for pest insect control
and are widely used as vectors for recombinant
protein synthesis in both in vivo and in vitro cul
tivated insect cells [3 J.
M. neustria {Mane) larvae (Lepidoptera, Lasio-
campidae) is a well known garden fruit pest insect,
especially on apple trees. However, outbreaks of the
pest mass reproduction occur often also in woods
(first of all, in oak woods) of the forest-steppe zone.
© I. M. KIKHNO, L. I. STROKOVSKAYA, R. A. MELESHKO,
J. M I C H A U C , A. P . SOLOMKO, 2 0 0 2
For example, during such an outbreak in the middle
of the XX century many thousands of hectares of
Eastern Europe woods were completely deprived of
leafage. Currently, Mane populations are controlled
by the application of chemical insecticides.
In our laboratory a virus strain has been isolated
from a diseased Mane larvae [4 ]. M. neustria nuclear
polyhedrosis virus (ManeNPV) is a species in the
genus Nucleopolyhedravirus, family Baculoviridae. It
has been adapted to the cell line MCAp-1 derived
from A. pernyi larvae [5 J. Later the ManeNPV
polyhedrin gene has been localized in an EcoRI-A
fragment of virus genome [6 ] and complete sequence
of this gene has been determined. High productivity
of ManeNPV in cultivated MCAp-1 cells as well as a
high level of polyhedrin expression in the late infec
tion stage have made this cell-virus system a good
candidate for use as an expression system for recom
binant protein production under the polyhedrin gene
promoter control [7, 8 ] . Working with ManeNPV we
have detected its interesting feature. Submolar bands
have been observed in the REN profiles of virus DNA
indicating that the virus isolate used in this study is
not homogenous and contains minor genotypic va
riants. We have failed to select out a homogenous
522
PHYSICAL MAPPING O F MALACOSOMA NEUSTRIA
virus clone using routine multiple plaque-forming
approach, minor submolar bands have been detected
in the REN profiles of the DNA of all plaque isolates.
Aiming at usage of ManeNPV as an insecticide
and a vector for recombinant protein expression as
well as understanding the nature of its unusual
«persistent heterogeneity* we have begun to study its
genetic organization.
Materials and Methods. MCAp-1 cells were main
tained in the Grace's medium supplemented with
10 % heat-inactivated fetal bovine serum. Some de
tails of the cell cultivation, virus propagation, and
plaque assay were described previously [9 ] . Plaque
assays were incubated for 4 days at 27 ° С Infected
cells and extracellular virus (ECV) fluids for viral
DNA extraction, virus stock obtaining and virus titer
estimation were made 7 days p. i.
Viral DNA from infected cells was isolated ac
cording to the method previously described [10] with
some modifications. This method permits to obtain
supercoiled forms of viral DNA with minimal cellular
DNA contamination. DNA was isolated directly from
virus-infected cells without preliminary virus puri
fication. To isolate each preparation, (5—8) • 10 6 cells
were suspended in 100 pi of 0.05 M EDTA solution.
An equal volume of 2 * buffer containing 50 mM
EDTA, 0.5 mg/ml spermidine, 0.4 mg/ml ethidium
bromide was then added. The pH of this mixture was
adjusted to 11.5 with 0.25 M NaOH and then
neutralized with 0.1 M HC1, 10 pi of RNAse A
(10 mg/ml) was added. After incubation (37 °С,
30 min) sodium laurylsulphate was added to the
lysate (1 %) as well as proteinase К (1 / ig /ml) . The
mixture was incubated overnight at 37 °С. DNA was
extracted three times by phenol, one time by chloro-
phorm and then ethanol precipitated. Alternatively,
viral DNA was isolated from infected cells and ECV
according to the standard procedures described by
O'Reily [9 ] .
Viral DNA was digested with different restriction
endonucleases and then run in 0.7 % agarose gel
electrophoresis using Tris-borate buffer system [11] .
Molecular sizes of the ManeNPV DNA fragments
obtained after digestion have been estimated by
comparison with phage DNA Hindi IIIEcoRI/BamHI
markers. For high molecular weight fragments the
second enzyme was used to generate smaller frag
ments and to provide better estimation. For this
purpose viral REN fragments were separated in 0.7 %
low melting agarose gel, gel slices including large
fragments were excised, heated to 68 °С, two-fold
diluted with ТЕ buffer and digested using appropriate
buffer.
DNA probes were labelled with [ 3 2 P JdCTP using
random primer labelling kit («Fermentas», Lithua
nia)) according to the manufacturer's protocol. Elec-
trophoretically separated the restriction fragments
were transferred to nylon filter (Hybond-N, «Amer-
sham», Great Britain)) and hybridized with labelled
probes according to Maniatis [11 ] .
Results and Discussion. Our first step was the
construction of the ManeNPV physical map and
locating on this map the site the changes of which
cause the appearance of submolar bands in electro-
phoregrams of the digested virus DNA.
One of two ManeNPV strains isolated from Mane
larvae — the isolate K2 [4 ] — was selected for fur
ther investigations. The REN digestion pattern of this
isolate did not contain submolar bands, therefore we
have supposed K2 to have a single genotype. How
ever, the further REN digestion followed by the
hybridization of blotted REN fragments with the
3 2 P-labelled DNA of the same virus demonstrated low
intensity of submolar bands in the digested DNA
patterns. As a result of more attentive study of the
electrophoregrams obtained we have revealed such
submolar bands also in the REN profiles formed after
the excessive virus DNA introduction into sample
wells. These data are against homogeneity of the
clone obtained; so we purified it additionally, three
plaque passages were made. Fifteen plaques were
selected out during the third passage; each plaque
variant was reproduced to obtain virus stocks. These
stocks were used for the cell culture infection, the
MOI being 5 pfu/cell. The viral DNAs obtained from
the reproduced virus isolates were REN digested. We
attempted to select out in such a way any virus isolate
presenting either a single major or a single minor
fraction of the K2 isolate. Among 15 REN digested
virus DNA preparations 13 ones were shown to
possess submolar fractions. Two virus isolates pre
sented single major fraction following such a treat
ment. However, after the second in vitro passage of
the both isolates their DNAs were not homogenous. It
should be noted that «homogeneity» or «hetero-
geneity» are not strict terms, so-called homogenous
DNA samples may contain microquantities of the
molecules of minor fraction undetectable by the
following REN analysis. All 15 isolates demonstrated
the identical heterogenous REN DNA profiles, the
only difference concerned the submolar bands inten
sity. An isolate, Wl (referred then as ManeNPV),
was chosen among 15 ones for further investigations
because of the higher intensity of its minor bands
comparing to the minor bands of other isolates. Our
investigations have shown the constant quantity and
size of the ManeNPV submolar fragments for each
restriction endonuclease used; they do not depend on
523
KIKHNO I M. E T AJL
Fig. 1. Electrophoregram
(A) and hybridization
(Я, C) of the ManeNPV
DNA digested with Eco-
RI ( 7 ) , BamHI (2) ,
Kpnl (J) , Hindi 11 (4),
BgUl (5) , PstI (6) and
EcoRI-Hindlll-clewed
(phage DNA standards).
Arrows point to submolar
bands. Blots were hybri
dized with the ^ - l a b e l
led BamHl-ml (B) and
BamHI-m2 (C) submo
lar fragments
passage number (the virus after 1—10 passages was
taken) and on MOI (0.1, 1.0, 10.0). Two bands
appeared after the EcoRI, BamHI, Kpnl, BgUI treat
ment; one band after Hindlll treatment and no
submolar bands were found after the PstI action (see
fig. 1, A).
The virus population heterogeneity is not a
unique phenomenon. Natural baculoviral populations
contain different genotype variants which can be
cloned out using the cell culture methods. However,
such a cloned and «originally» homogenous virus
strains are shown to be changed after their passages
in vitro, new patterns having been detected in the
REN profiles of genome DNAs of virus plaque isolates
originated from such strains. Previous studies with
the serial passage of cloned virus have shown that
DNA insertions and deletions are relatively common
in the baculovirus genome. The REN analysis of
baculovirus genomes has revealed several types of
genomic alterations occuring upon serial passages in
vitro. In some cases viruses isolated from a serially
passaged stock virus carry a host-derived transposable
element insertion [12, 13]. In other cases the viral
plaque isolates selected from the serial passaged stock
contain viral DNA insertions at reiterated regions of
the viral genome [14] . Some baculovirus deletion
mutants have been recorded to appear also after the
in vitro passaging of undiluted virus stocks [15] .
Accumulation of defective interfering particles in
the serially passaged virus stock is detected in the
REN profiles of DNA prepared from this stock. The
restriction fragments from deleted regions are present
in submolar quantities in the REN profiles of he
terogenous virus DNA preparations. In this case
submolar bands are seen in the REN patterns of virus
DNA after serial passages, usually with high МОЇ. A
striking property of the ManeNPV is its «persistent
heterogeneity* detected already after the first passage
by virus materials of low MOI.
Comparing our data with the results of other
authors would not be adequate because of the special
method of cellular virus (CV) DNA isolation used.
Besides, other data concerning the baculovirus he
terogeneity are usually based on the analysis of DNA
isolated from the ECV. To rule out any artifact due
to the specificity of the DNA extraction method, we
524
PHYSICAL MAPPING O F MALACOSOMA NEUSTRIA
have compared the REN profiles of the DNAs isolated
from CV using both our protocol and the standard
common one. All of the DNA samples always pos
sessed minor submolar bands regardless of the pro
tocol used. We have also analyzed the baculovirus
DNA isolated by the alkali-ethidium method from the
Spodoptera frugiperda (Sf9) cells infected by the
Autographa californica NPV. The electrophoresis of
the REN-digested DNA of this virus has not revealed
any new bands (data not shown). We have also
compared the digested ManeNPV DNAs isolated from
the cells and from ECV. The REN analysis has
demonstrated the identity of samples obtained and
the presence of minor bands in both of them (data not
shown). All these data, having been taken into
consideration, suggest our results to be independent
of the specificity of the alkali-ethidium DNA isolation
protocol, although any artifact cannot be ruled out.
To continue the study of the factors causing this
phenomenon first of all we had to answer the question
what kind of alterations contains the minor genotypic
variant comparing to the major genotypic one. The
refore, the ManeNPV physical map had to be cons
tructed. In our following investigations concerning the
physical mapping of the ManeNPV major fraction
genome we used heterogenous ManeNPV DNA. The
DNA samples were treated with the BamHl, Kpnl,
and PstI restriction endonucleases. The electrophore-
tic patterns obtained are shown in fig. 1, Л (lanes 7,
2, 6 correspondingly) and fig. 2. We obtained 10
2?am#/-fragments including also two submolar frag
ments, 11 /f/w/-fragments including two submolar
ones, and 9 /^ / - fragments . The fragment sizes are
given in the table. Some highly molecular weight
fragments obtained (BamHI-A, В, С; KpnI-A, С, D)
were treated in addition with PstI, Kpnl, BamHl, or
EcoRI. Their sizes were calculated as a sum of the
fragments obtained.
We also performed a double ManeNPV digestion
using the BamHI-Kpnl, BamHI-PstI, KpnI-PstI
combinations (data not shown). As a result the full
genome size was determined to be of about 139 kbp.
The DNA-DNA hybridization technique was used to
construct a physical ManeNPV genome map. The
2?am#/-fragments extracted from the gel or cloned
into pUC 18 (F- and G-fragments) were labelled and
hybridized to Southern blots of viral DNA digested by
Kpnl and PstL The order of respective restriction
sites was confirmed by the direct comparison of single
and double digests on agarose gels and by comparison
of the sizes of individual single-digest fragments with
the sizes of double-digest fragments. According to the
proposal [161, the smallest fragment containing a
putative polyhedrin gene should be taken as a zero
Size of the restriction endonuclease fragments of Malacosoma
neustria NPV
The size of the genome as estimated by the sum of the restriction
fragments (submolar fragments, indicated as ml and m2 were not
included in the sum) is shown at the bottom of each column.
•Restriction endonuclease fragment has been cleaved.
point on the physical map of baculovirus DNA. We
have earlier cloned the EcoRI-A-fragmtnt of the
ManeNPV genome containing the polyhedrin gene
[6 ] , a physical map of the given plasmid was cons
tructed. The comparing of physical maps of the
ManeNPV genome and its f?co/*/-fragment has per
mitted to locate the full polyhedrin gene in the
/W-C- fragment This fragment was taken as a zero
point of the linearized circular map. The linear
BamHl, Kpnl, and PstI physical maps of the genome
of the major fraction of ManeNPV are shown in fig.
3.
The BamHl and Kpnl minor fragments were also
located on this map. Our results of blot-hybridization
have shown the BamHl-ml and m2 minor fragments
(5.5 and 3.4 kbp correspondingly) to be components
of the BamHI-D-fragmmt of 8.9 kbp size. The
Kpnl-ml and m2 minor fragments (4.1 and 0.5 kbp
correspondingly) were included into the Kpnl-G-trag-
ment (4.6 kbp), in other words an additional site for
every restriction endonuclease was present on the
physical map of the minor variant of ManeNPV
genome. The localization of submolar components on
the physical map of the ManeNPV DNA has proved
the additional sites for BamHl and Kpnl to be
situated in the same locus of this map (indicated by
525
KJKHNO I. M. ET AL.
Fig. 3. Physical BamHI, КрпІ and maps of the ManeNPV
DNA. Letters correspond to the fragments in fig. 2 and the table.
The arrows indicate the position and direction of the polyhedrin gene
and position of the «break point*
m2
Fig. 2. Diagram of the REN profiles of ManeNPV digested with
BamHI (2 ) , КрпІ (J) , PstI (4) and Hindi11-cleaved (phage DNA
standards (7))
arrow in fig. 3) which coincids with the PstI recog
nition site. Co-location of the sites for three casually
chosen restriction endonucleases has permitted us to
suppose this point to be not a specific recognition site
for each one, but a site of non-specific or specific
virus DNA break. To confirm this idea, we had to
locate in the virus genome some submolar fragments
obtained after digestions with other restriction en
donucleases.
The results of the blot-hybridization experiments
(fig. 1, В, C) demonstrated without any doubt that
the sums of minor fragments sizes obtained after the
EcoRI and BgUI digestions corresponded to the sizes
of higher molecular fragments hybridizing with 3 2 P -
labelled BamHI minor fragments (10 kbp + 14 k b p e
= 24 kbp for EcoRI; 2.0 kbp + 0.9 kbp - 2.9 kbp for
BgUI). Earlier we have mentioned the lack of the
second submolar band in the Яшс/Ш-digested viral
DNA electrophoregrams (fig. 1, Л). The submolar
band of 4.8 kbp has been shown by hybridization (fig.
1, В, О to be present in the HindiII^REN pattern in
major fraction fragments of the same size as hidden.
T h e s e Hindlll submolar fragments s izes sum
(4.8 kbp + 1 . 3 kbp) corresponded to the size of the
#mrf///-fragment hybridizing with the probes (6.3
kbp). We could not locate the EcoRI, Hindi11 and
^/ / / - fragments directly on the ManeNPV physical
map as the recognition sites for these restriction
enzymes were not ordered in the full ManeNPV
genome. However, the comparison of the full-size
physical map and the mentioned £?cojR/-^-fragment
map has shown that both BamHI-D and Kpnl-G-
fragments are included in the £coff/-.4-fragment
giving us a possibility to locate the break point on the
£coK/-^-fragment map. To confirm our hypothesis
concerning the existence of this point, we have
analyzed the order of BgUI and Hindi I I sites inside
the 2?caR/-;4-fragment.
Our results have demonstrated that a possible
break point is localized inside the 2.9 kpb-fragment
dividing it into two parts of 2.0 and 0.9 kpb. The
Hindi 11-fragment (6.1 kpb) is also a part of the
£caR/-A-fragment and is divided by the break point
into two fragments — 1.3 and 4.8 kpb. Two minor
£cojR/-fragments of 14.0 kpb and 10.0 kpb may be a
result of the jEcoKZ-fragment (24.0 kpb) damage at
the same point. All these results confirm the break
point in the site located at a distance of approximately
21.0 kbp from the zero point on the ManeNPV
physical map. To locate this site more precisely and
to determine the distance from the PstI site, we have
made the additional double REN digestion of the
virus DNA using BamHI, Kpnl, Hindi 11, BgUI com
bined with PstI, the REN products being hybridized
with the labelled BamHI fragments (3.4 and 5.5 kpb)
(data not shown). According to these results, the
526
PHYSICAL MAPPING O F M A L A C O S 0 M A NEUSTRIA
break point is situated at a distance less than 0.1 kbp
downstream the PstI site. This fact explains the
«absence» of submolar bands on the PstI REN pattern
in fig. 1, A. The 2 L 0 kpb-fragment and the submolar
fragment are almost of the same size, the difference
being less than 0.1 kpb; so it is beyond the dif
ferential ability of the REN analysis. The second
band corresponding to the second minor component
(less than 0Л kpb) is of a very low intensity and
cannot be detected even by blot-hybridization ap
proach.
Having detected the break in the minor DNA
fraction we are now to answer the question if such
break is «naturally» present in a part of virus DNA
molecules (before DNA isolation) or it appears as a
result of the DNA molecule damage during its extrac
tion and purification since some DNA molecules are
damage-sensitive in a determined specific point. Our
similar results with the virus DNA isolation using
different protocols do not rule out this last possibility
because each of them includes a phenol-chloroform
extraction stage able to damage the DNA molecules.
We have found that the use of more «unfavorable»
conditions of DNA purification (longer phenol-chloro
form extraction procedure and more intensive mixing)
causes the increasing of total quantity of damaged
DNA molecules and, as a result, the smearing of the
REN DNA profiles, the intensity of minor bands
having, however, no tendency to increase. According
to these data the most probable situation is the
existence of a minor virus variant carrying break-
containing genomes. Any two-stranded DNA break is
known to disclose a supercoiled DNA and form linear
DNA molecules. However, the circular DNA form is
well known to be a natural feature of any baculovirus
life cycle, no undamaged «naturab linear forms
having been found for any baculovirus studied. Some
facts suggest ManeNPV is a typical baculovirus with
a typical genome. First, any virus DNA isolation
using the alkali-ethidium approach permits to obtain
only supercoiled molecules, all the linear ones being
destroyed by the alkali treatment. The presence of
the linear molecules in such samples is proved to be
a result of the following chloroform-phenol deproteini-
zation. Further, such break-containing viral genomes
have been shown to be presented not only in the
infected cells, which can contain any forms of viral
DNA but in the ECV too. The linear viral genomes
seem incapable to be packed into maturing particles
and to form virions. These data and considerations
develop a clear contradiction: it goes that the viral
genome integrity is damaged but this genome exists
simultaneously as a supercoiled structure. This con
tradiction may be explained by a hypothesis that any
DNA molecule of the minor genotypic virus variant
really contains a break. However, the molecule is
stabilized by a protein or proteins, hence a protein
«Іоскег» ties both ends of the DNA molecules. Such
protein (s) are destroyed during the DNA deproteini-
zation and phenol-chloroform treatment causing the
DNA linearization.
As we have already discussed usual accumulation
of modified virus genomes in a virus clone seeming
originally homogenous is a stepwise process following
virus passages. The fact that the heterogeneity of the
plaque ManeNPV isolates is seen in the first passage
virus with low MOI testifies that the term «mo-
dification» is hardly applied to this phenomenon. The
nature of changes detected here — a break at a
determined point of the DNA sequence-permits to
make an assumption, that it is not the modification in
a strict sense of this word, which means some changes
comparing with the natural state, but a phenomenon,
which can reflect a natural step of the ManeNPV
intracellular reproduction. Our data permit only to
make some speculations and to suppose an important
functional role of the break point — for example,
during replication and/or recombination process or
other processes requiring DNA strands break. Some
reparation processes are possibly realized with low
effectiveness in the virus-cell system studied here, so
the break-containing DNA molecules are easily detec
ted in the virus DNA pool.
Either the DNA molecule break is a result of high
damage sensitivity of its locus during purification or
it is masked by protective protein «naturally» present
in baculovirus genome — in any case, this unusual
phenomenon is of great interest and to be studied in
future.
/. M. Кіхно, Л. I. Строковська, P. А. Мелешко, Ж. Михайлик,
О. П. Соломко
Ф і з и ч н е картування геному вірусу ядерного поліедрозу
Malacosoma neustria і його модифікація в клітинній культурі
Antheraea pernyi
Резюме
Складено фізичну карту геному вірусу ядерного поліедрозу М.
neustria (ManeNPV), визначено розташування сайтів для ре-
стриктаз BamHl, Kpnl та PstI, на карті локалізовано ген
поліедрину. Розмір вірусної ДНК визначено приблизно в 139
тис. п. н. Профілі рестриктів ДЯК ManeNPV, ізольованих з
бляшок, отриманих у клітинах A. pernyi, демонструють
«тривку гетерогенність», субмолярні фрагменти виявлено в
паттерні рестрикції після першого пасажу вірусу. Встановле
но, що ці фрагменти присутні в пулі гетерогенної вірусної
ДНК, а її нитки мають розриви в певному місці. Таке місце
розриву було локалізовано на фізичній карті геному ManeNPV.
527
KJKHNO I. M. В Т AL.
И. M. KUXHO, Л. И. Строковская, P. А. Мелеиіко,
Ж. Михайлик, А. П. Соломко
Физическое картирование генома вируса ядерного полиэдроза
Malacosoma neustria и его модификация в клеточной структуре
Antheraea pernyi
Резюме
Составлена физическая карта генома вируса ядерного полиэд
роза М. neustria (ManeNPV), определено расположение сайтов
для рестриктаз BamHl, Kpnl и PstI, на карте локализован ген
полиэдрина. Размер вирусной ДНК определен приблизительно в
139 тыс п. к. Профили рестриктов ДНК ManeNPV, изолиро
ванных из бляшек, полученных в клетках A. pernyi, демонст
рируют «устойчивую гетерогенность», субмолярные фрагмен
ты обнаружены в паттерне рестрикции после первого пассажа
вируса. Установлено, что эти фрагменты присутствуют в
пуле гетерогенной вирусной ДНК, а ее нити имеют разрыв в
определенном месте. Такое место разрыва было локализовано
на физической карте генома ManeNPV.
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УДК 577.29
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