DNA recombinant molecules carrying different allels of human preproinsulin gene as mutagens in Chinese hamster cells
In our earlier investigations it was discovered the nuttagenic activity of two recombinant plasmids (pBR322ins and pAins) containing the human preproinsulin gene in cultivated somatic mammalian cells. The preproinsulin gene (allel-1) was able to function under its own promoter in different cell type...
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| Zitieren: | DNA recombinant molecules carrying different allels of human preproinsulin gene as mutagens in Chinese hamster cells / E.L. Rubashevsky, Y.V. Patskovsky, S.D. Kirilenko, I.S. Varzanova, O.M. Bogolyubova, O.F. Lysenko, L.L. Lukash // Биополимеры и клетка. — 1996. — Т. 12, № 5. — С. 83-92. — Бібліогр.: 30 назв. — англ. |
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Rubashevsky, E.L. Patskovsky, Y.V. Kirilenko, S.D. Varzanova, I.S. Bogolyubova, O.M. Lysenko, O.F. Lukash, L.L. 2019-06-15T11:17:38Z 2019-06-15T11:17:38Z 1996 DNA recombinant molecules carrying different allels of human preproinsulin gene as mutagens in Chinese hamster cells / E.L. Rubashevsky, Y.V. Patskovsky, S.D. Kirilenko, I.S. Varzanova, O.M. Bogolyubova, O.F. Lysenko, L.L. Lukash // Биополимеры и клетка. — 1996. — Т. 12, № 5. — С. 83-92. — Бібліогр.: 30 назв. — англ. 0233-7657 DOI:http://dx.doi.org/10.7124/bc.00044E https://nasplib.isofts.kiev.ua/handle/123456789/154234 575.155:575.224.46 In our earlier investigations it was discovered the nuttagenic activity of two recombinant plasmids (pBR322ins and pAins) containing the human preproinsulin gene in cultivated somatic mammalian cells. The preproinsulin gene (allel-1) was able to function under its own promoter in different cell types since it lacked a regulatory element providing tissue-specific expression. Introduction of a frameshift mutation into the region of initiation of the translation of the transgene (allel-2) diminished the mutagenic effect of a corresponding recombinant plasmid on gene level. In this work the mutagenic activity of the recombinant plasmids carrying allel-1 (pBR322ins) andallel-2 (pBR322insN) of the preproinsulin gene was studied using cytogenetic test. It was shown that the presence of allel-2 (that probably had altered expression of the transgene) in the recombinant plasmid structure decreased the level of induced chromosome breaks per cell. It was made a conclusion that not only viruses and their genes but also recombinant DNAs of nonviral origin are able to induce chromosome and gene mutations in mammalian cells. The mutagenic activity of studied recombinant plasmids depends on the level of expression of the transgenes in the cells. У наших попередніх дослідженнях було відкрито мутагенну активність двох рекомбінантних плазмід (pBR322ins та pAins), які несуть ген препроінсуліну людини, у соматичних клітинах ссавців, що культивуються. Ген препроінсуліну (алель-1) має здатністьфункціонувати під своїм власним промотором у різних типах клітин, оскільки втратив регуляторний елемент, який забезпечує тканинну специфічність експресії. Введення мутації зсуву рамки зчитування в район ініціації трансляції трансгена (алель-2) призводило до зменшення мутагенного ефекту відповідної рекомбінантної плазміди на генному рівні. У цій роботі вивчалась мутагенна активність рекомбінантних плазмід, що несуть алель-1 (pBR322ins) та алель-2 (pBR322insN) гена препроінсуліну, з використанням цитогенетичного тесту. Показано, що присутність алеля-2 (який, можливо, має пошкодження експресії трансгена) у структурі рекомбінантної плазміди призводить до зниження рівня хромосомних розривів на клітину. Зроблено висновок стосовно того, що не тільки віруси та їх гени, а й рекомбінантні ДНК невірусної природи здатні індукувати хромосомні та генні мутації у клітинах ссавців. Мутагенна активність досліджуваних рекомбінантних плазмід залежить від експресії трансгенів у клітинах. В наших предыдущих исследованиях была обнаружена мутагенная активность двух рекомбинантных плазмид (pBR322ins и pAins), содержащих ген инсулина человека, в культивируемых соматических клетках млекопитающих. Ген инсулина (аллель-1) способен функционировать под своим собственным промотором в различных типах клеток, поскольку утратил регуляторний элемент, обеспечивающий тканеспецифичность экспрессии. Введение мутации сдвига рамки считывания в район инициации трансляции трансгена (аллель-2) приводило к снижению мутагенного эффекта соответствующей рекомбинантной плазмиды на генном уровне. В этой работе изучена мутагенная активность рекомбинантных плазмид, несущих аллель-1 (pBR322ins) и аллель-2 (pBR322insN) гена препроинсулина, с использованием цитогенетического теста. Показано, что присутствие аллеля-2 (имеющего, вероятно, поврежденную экспрессию трансгена) в структуре рекомбинантной плазмиды, вызывало снижение уровня хромосомных разрывов на клетку. Сделан вывод о том, что не только вирусы и их гены, а также рекомбинантные ДНК невирусной природы способны индуцировать хромосомные и генные мутации в клетках млекопитающих. Мутагенная активность исследуемых рекомбинантных плазмид зависит от экспрессии трансгенов в клетках. We thank Dr. Nina B. Varshaver (Institute of Molecular Genetics, Russian Academy of Sciences, Moscow) for useful consultations and for critical reading of the manuscript of this paper. This work was supported by the Grant of the National Academy of Sciences of Ukraine and the Grant of the National Programme «Gene Fund Protection of the Population of Ukraine». en Інститут молекулярної біології і генетики НАН України Биополимеры и клетка DNA recombinant molecules carrying different allels of human preproinsulin gene as mutagens in Chinese hamster cells Рекомбінантні ДНК, що містять різні алелі гена препроінсуліну людини, як мутагени в клітинах китайського хом'ячка Рекомбинантные ДНК, несущие разные аллели гена препроинсулина человека, как мутагены в клетках китайского хомячка Article published earlier |
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Digital Library of Periodicals of National Academy of Sciences of Ukraine |
| collection |
DSpace DC |
| title |
DNA recombinant molecules carrying different allels of human preproinsulin gene as mutagens in Chinese hamster cells |
| spellingShingle |
DNA recombinant molecules carrying different allels of human preproinsulin gene as mutagens in Chinese hamster cells Rubashevsky, E.L. Patskovsky, Y.V. Kirilenko, S.D. Varzanova, I.S. Bogolyubova, O.M. Lysenko, O.F. Lukash, L.L. |
| title_short |
DNA recombinant molecules carrying different allels of human preproinsulin gene as mutagens in Chinese hamster cells |
| title_full |
DNA recombinant molecules carrying different allels of human preproinsulin gene as mutagens in Chinese hamster cells |
| title_fullStr |
DNA recombinant molecules carrying different allels of human preproinsulin gene as mutagens in Chinese hamster cells |
| title_full_unstemmed |
DNA recombinant molecules carrying different allels of human preproinsulin gene as mutagens in Chinese hamster cells |
| title_sort |
dna recombinant molecules carrying different allels of human preproinsulin gene as mutagens in chinese hamster cells |
| author |
Rubashevsky, E.L. Patskovsky, Y.V. Kirilenko, S.D. Varzanova, I.S. Bogolyubova, O.M. Lysenko, O.F. Lukash, L.L. |
| author_facet |
Rubashevsky, E.L. Patskovsky, Y.V. Kirilenko, S.D. Varzanova, I.S. Bogolyubova, O.M. Lysenko, O.F. Lukash, L.L. |
| publishDate |
1996 |
| language |
English |
| container_title |
Биополимеры и клетка |
| publisher |
Інститут молекулярної біології і генетики НАН України |
| format |
Article |
| title_alt |
Рекомбінантні ДНК, що містять різні алелі гена препроінсуліну людини, як мутагени в клітинах китайського хом'ячка Рекомбинантные ДНК, несущие разные аллели гена препроинсулина человека, как мутагены в клетках китайского хомячка |
| description |
In our earlier investigations it was discovered the nuttagenic activity of two recombinant plasmids (pBR322ins and pAins) containing the human preproinsulin gene in cultivated somatic mammalian cells. The preproinsulin gene (allel-1) was able to function under its own promoter in different cell types since it lacked a regulatory element providing tissue-specific expression. Introduction of a frameshift mutation into the region of initiation of the translation of the transgene (allel-2) diminished the mutagenic effect of a corresponding recombinant plasmid on gene level. In this work the mutagenic activity of the recombinant plasmids carrying allel-1 (pBR322ins) andallel-2 (pBR322insN) of the preproinsulin gene was studied using cytogenetic test. It was shown that the presence of allel-2 (that probably had altered expression of the transgene) in the recombinant plasmid structure decreased the level of induced chromosome breaks per cell. It was made a conclusion that not only viruses and their genes but also recombinant DNAs of nonviral origin are able to induce chromosome and gene mutations in mammalian cells. The mutagenic activity of studied recombinant plasmids depends on the level of expression of the transgenes in the cells.
У наших попередніх дослідженнях було відкрито мутагенну активність двох рекомбінантних плазмід (pBR322ins та pAins), які несуть ген препроінсуліну людини, у соматичних клітинах ссавців, що культивуються. Ген препроінсуліну (алель-1) має здатністьфункціонувати під своїм власним промотором у різних типах клітин, оскільки втратив регуляторний елемент, який забезпечує тканинну специфічність експресії. Введення мутації зсуву рамки зчитування в район ініціації трансляції трансгена (алель-2) призводило до зменшення мутагенного ефекту відповідної рекомбінантної плазміди на генному рівні. У цій роботі вивчалась мутагенна активність рекомбінантних плазмід, що несуть алель-1 (pBR322ins) та алель-2 (pBR322insN) гена препроінсуліну, з використанням цитогенетичного тесту. Показано, що присутність алеля-2 (який, можливо, має пошкодження експресії трансгена) у структурі рекомбінантної плазміди призводить до зниження рівня хромосомних розривів на клітину. Зроблено висновок стосовно того, що не тільки віруси та їх гени, а й рекомбінантні ДНК невірусної природи здатні індукувати хромосомні та генні мутації у клітинах ссавців. Мутагенна активність досліджуваних рекомбінантних плазмід залежить від експресії трансгенів у клітинах.
В наших предыдущих исследованиях была обнаружена мутагенная активность двух рекомбинантных плазмид (pBR322ins и pAins), содержащих ген инсулина человека, в культивируемых соматических клетках млекопитающих. Ген инсулина (аллель-1) способен функционировать под своим собственным промотором в различных типах клеток, поскольку утратил регуляторний элемент, обеспечивающий тканеспецифичность экспрессии. Введение мутации сдвига рамки считывания в район инициации трансляции трансгена (аллель-2) приводило к снижению мутагенного эффекта соответствующей рекомбинантной плазмиды на генном уровне. В этой работе изучена мутагенная активность рекомбинантных плазмид, несущих аллель-1 (pBR322ins) и аллель-2 (pBR322insN) гена препроинсулина, с использованием цитогенетического теста. Показано, что присутствие аллеля-2 (имеющего, вероятно, поврежденную экспрессию трансгена) в структуре рекомбинантной плазмиды, вызывало снижение уровня хромосомных разрывов на клетку. Сделан вывод о том, что не только вирусы и их гены, а также рекомбинантные ДНК невирусной природы способны индуцировать хромосомные и генные мутации в клетках млекопитающих. Мутагенная активность исследуемых рекомбинантных плазмид зависит от экспрессии трансгенов в клетках.
|
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0233-7657 |
| url |
https://nasplib.isofts.kiev.ua/handle/123456789/154234 |
| citation_txt |
DNA recombinant molecules carrying different allels of human preproinsulin gene as mutagens in Chinese hamster cells / E.L. Rubashevsky, Y.V. Patskovsky, S.D. Kirilenko, I.S. Varzanova, O.M. Bogolyubova, O.F. Lysenko, L.L. Lukash // Биополимеры и клетка. — 1996. — Т. 12, № 5. — С. 83-92. — Бібліогр.: 30 назв. — англ. |
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ISSN 0233-7657. Биополимеры и клетка. 1996. Т. 12. № 5
DNA recombinant molecules carrying different allels of human
preproinsulin gene as mutagens in Chinese hamster cells
Eugeny L. Rubashevsky, Yuriy V. Patskovsky, Sergei D. Kirilenko, Irina S.
Varzanova, Oksana M. Bogolyubova, Olena F. Lysenko, Lubov L. Lukash
Institute of Molecular Biology and Genetics, National Academy of Sciences of the Ukraine
150 Zabolotnogo str., Kyiv, 252143, Ukraine
In our earlier investigations it was discovered the mutagenic activity of two recombinant
plasmids (pBR322ins and pAins) containing the human preproinsulin gene in cultivated
somatic mammalian cells. The preproinsulin gene (allel-I) was able to function under its
own promoter in different cell types since it lacked a regulatory element providing tissue-
specific expression. Introduction of aframeshift mutation into the region of initiation of
the translation of the transgene (allel-2) diminished the mutagenic effect of a
corresponding recombinant plasmid on gene level In this work the mutagenic activity of
the recombinant plasmids carrying allel-1 (pBR322ins) and allel-2 (pBR322insN) of the
preproinsulin gene was studied using cytogenetic test. It was shown that the presence of
allel-2 (that probably had altered expression of the transgene) in the recombinant plasmid
structure decreased the level of induced chromosome breaks per cell. It was made a
conclusion that not only viruses and their genes but also recombinant DNAs of nonviral
origin are able to induce chromosome and gene mutations in mammalian cells. The
mutagenic activity of studied recombinant plasmids depends on the level of expression of
the transgenes in the cells.
Introduction.The mutagenicity of some viruses and recombinant DNAs carrying
viral genes in the somatic mammalian cells in culture was demonstrated by us
earlier [1—7]. Recombinant plasmids containing human preproinsulin gene
(pBR322ins and pAins) has been shown to induce gene mutations and
chromosomal aberrations in the same cells as well [8—11]. The preproinsulin
gene (allel-1) was able to function in different cell types under its own promoter
since it lacked a regulatory element providing a tissue-specific expression [12,
13].Introduction of a frameshift mutation into the region of initiation of the
translation of the transgene (allel-2) diminished the mutagenic effect of the
corresponding recombinant plasmid (pBR322insN) on gene level [10].
Here we present the results of our experiments which aimed: 1) to study
the ability of the recombinant plasmids pBR322ins, pAins (allel-1 of the
preproinsulin gene codes a protein of normal structure) and pBR322insN
(allel-2 probably codes a changed protein) to induce chromosome aberrations
in subsequent generations of transfected cells and 2) using chromosome
mutation assay to study the role of the expression of the transgene in genome
instability induced in the Chinese hamster cells.
Materials and methods Cell line The Blld-ii-FAF 28 Chinese hamster cell
line was described in detail previously [1, 2, 4, 8]. It contains 18 chromosomes
•Correspondence address.
© E. L. RUBASHEVSKY, Yu. V- PATSKOVSKY, S. D. KIRILENKO, I. S. VARZANOVA,
О. M. BOGOLYUBOVA. О. F. LYSENKO. L. L. LUKASH. 1996
83
RUBASHEVSKY E. L. ET AL.
in the modal class (65—80 % of cells examined). Cells were grown in standard
Eagle's medium supplemented with 10 % fetal bovine serum and 50 jig/ml
kanamycin at 37 °С in an atmosphere of 5 % C02 and 95 % air. Stocks of cells
were stored in liquid nitrogene in medium containing 10 % DMSO.
Recombinant plasmid DNAs. The human preproinsulin gene containing its
own promoter but lacking a regulatory element providing tissue specificity of the
expression (Bglll-Tagl-fragmQnX) was cloned into the recombinant plasmids
pBR322ins and pAins in our institute [12]. (Fig. 1 and 2). Intracellular
expression of the human preproinsulin gene included into the plasmid vectors
was demonstrated after introduction into fibroblasts of different origin in our
earlier investigations [13]. The pBR322insN plasmid carrying the frameshift
mutation in the region of initiation of the translation of the human preproinsulin
gene was also constructed in our Institute (Fig. 3 and 4).
Transfection with the recombinant plassmid DNAs. Chinese hamster cell
populations were treated by DEAE-dextrane (2-106 mm) at the final
concentration of 100 ^g/ml with and without DNA as previously described [4,
8]. The intracellular presence of the 'plasmid DNAs was proved by a
dot-hybridization [14]. Plasmid DNAs in the decreasing concentrations were
observed up to the 3rd recultivation of the cells transfected (Fig. 5).
Chromosome aberrations assay. The method was described previously [1,
4, 8]. Chromosome aberrations were scored for 100 and more cells for each
variant. We repeated all experiments three times (the average data of three
Fig. 2. Map of the pBR322ins recombinant plasmid DNA
Fig. 3. Map of the pBR322insN recombinant plasmid DNA
84
DNA RECOMBINANT MOLECULES CARRYING DIFFERENT ALLELS
Fig. 5. Intracellular presence of the pBR322
bacterial DNA and the pBR322ins and
pBR322insN recombinant plasmid DNAs:
III — pBR322\ VII — pBR322ins\ IX —
pBR322insN\ X — positive control pBR322
experiments have been performed in the tables). Observed aberrations were
classified into breaks (chromatid and chromosome breaks, acentric fragments)
and exchanges (translocations, rings, dicentrics) according to the criteria of
ISCN [15]. We used two indices: percentage of cells with aberration and
number of breaks per cell.
Criteria for judgement whether the recombinant plasmid DNAs induced
chromosome aberrations and whether the frameshift mutation within human
preproinsulin gene effected an ability of the corresponding recombinant plasmid
to induce chromosome mutations in the transfected cells were as follows: 1)
significant increase in the portion of the cells with chromosome aberrations and
2) significant increase in the number of breaks per cell.
85
RUBASHEVSKY E. L. ET AL.
The following scheme of experiments was used (Fig. 6). The Chinese
hamster cell populations were transfected by plasmid DNAs (pAins, pBR322ins
and pBR322insN). Every cell population was divided into three parts. One part
of the cell population was reactivated in 24 hrs post-transfection procedure.
Another part of the cell population was used for karyotype analysis. The third
part was used in dot-hybridization procedure in order 1) to estimate the
effectivity of the plasmid DNAs penetration into the cells, 2) to study the
presence of introduced DNAs in succeeding cell generations and 3) to establish
the relationship between mutation events and presence of the exogeneous DNAs
in succeeded generations of transfected cells.
Statistical analysis. Statistical comparisons were made with the help of
Fisher's criterium. The confidential intervals were determined as it was
described [16].
Results and discussion Induction of chromosome aberrations by plasmid
DNAs. This part of our investigations was devoted to the study of the mutagenic
activity of recombinant DNAs (pAins, pBR322ins and pBR322insN) in different
time after transfection (Table 1). A total number of 6653 metaphases has been
examined.
All recombinant DNAs studied at both concentrations 5 and 10 ̂ g/ml have
been found to induce chromosome aberrations in Chinese hamster cells. There
was no difference in maximum mutagenic effect on chromosome level induced
by these agents. Chromosome aberrations including chromosome and chromatid
breaks as well as pair-acentric fragments were observed in 24 hrs and 72 hrs
post-transfection time. It was shown that the level of breaks sharply declined
at 120 hrs post-transfection.
The chromosome exchanges appeared in 24 hrs (for pBR322insN it is
evident) and mostly in 72 hrs (all other plasmids) after transfection.
Thus, the recombinant DNAs carrying allel-1 and allel-2 of human
preproinsulin gene and pBR322 bacterial plasmid were shown to induce both
types of chromosome aberrations in transfected Chinese hamster cells. The
breaks of chromosome are the dominant type of aberrations.
The effect of structure of the recombinant DNAs on the occurance of
chromosome aberrations. There are two criteria being used in cytogenetic
Fig. 6. The design of the experiments
86
DNA RECOMBINANT MOLECULES CARRYING DIFFERENT ALLELS
Table 1
Chromosome aberration induced by different plasmids carrying human preproinsulin gene in Chinese
hamster cells
The average data of three experiments. The confidential intervals are given in brackets
Relationship to the control level.
research. The first of them is the per cent of metaphase plates with chromosome
aberrations, it reflects the efficiency of treatment. The second one is the
number of breaks per cell, it describes the degree of alteration of the genetic
apparatus of treated cells. We used the last one for comparing the mutagenic
effect of two recombinant plasmids pBR322ins and pBR322insN. The data
obtained are presented in Table 2.
Two experimental variants (72 and 120 hrs post-transfection) out of the
three ones showed the decreased aberration level for pBR322insN as compared
to the pBR322ins. The statistical analysis of these data by the Fisher's test
illustrated that the level of chromosome aberrations in Chinese hamster cells
transfected with the pBR322insN recombinant plasmid was significantly lower
(P < 0.01, P<0.001). Thus, we demonstrated a specific frameshift mutation
effect on the ability of recombinant DNA to induce mutations at gene [10] and
chromosome level as well.
87
RUBASHEVSKY E. L. ET AL.
Table 2
Effect of the frameshift mutation in the human preproinsulin gene inserted into the pBR322insN
plasmid on an ability of the recombinant DNA to induce chromosome aberrations
The average data of three experiments. The confidential intervals are given in brackets.
***The significant differences between the pBR322ins level and the pBR322insN level (P < 0.01
and P < 0.001 respectively)
These data are in agreement with the results of our earlier investigations
demonstrating the mutagenic activity of the DNA-containing viruses [1—3, 7]
and the recombinant DNAs in the same cell system [2, 4, 7—11 ]. Inactivation
of exogeneous DNAs of different origin with a help of UV-light [6, 17] and
methylation [11] decreased their mutagenic effect.
We studied the ability of the recombinant molecules constructed on the
basis of pBR325 plasmid and carrying the oncogene of the Bovine Adenovirus
of the 3rd type (pBR325-D) and the tk-gene of the Herpes Simplex Virus of
the 1st type (pBR325tk) to induce chromosome and gene mutations in Chinese
hamster cells [2—5]. The mutagenic activity of those recombinant plasmids
might be due to the presence of the early viral genes since parental plasmid
pBR325 was not mutagenic. It was confirmed by the results that demonstrated
a dependence of mutagenic effect on the functional activity of SV40 A gene [19,
20] and the dNTP content as the result of the tk-gzne activity in the cells
transfected by HSV-2 virus [21, 22].
It was shown that bacterial plasmid pBR322 behaves as week mutagen by
itself [11]. This property was probably connected with a toxic nucleotide
sequence in the plasmid DNA. The presence of the allel-1 of the preproinsulin
gene in the structure of the recombinant plasmid pBR322ins showed just a
tendency of increasing a mutagenic activity [10, 11 ]. And the presence of the
allel-2 in the same vector decreased the mutagenic activity. So we have shown
a possibility to modulate the mutagenic effect of the bacterial plasmid with a
help of different allels of the transgene. It is opening a way to influence on the
mutagenic process in the system exogeneous recombinant DNA — cell if
changing the structure of the transgene. Presumably an alteration in the
expression of the gene-mutator prevents the induction of a part of the
premutagenic lesions or provides better conditions for repair of these lesions in
cultivated mammalian cells.
Similar results were obtained when studying the mutagenic and the
88
DNA RECOMBINANT MOLECULES CARRYING DIFFERENT ALLELS
transforming effect of the recombinant plasmid pEJ6.6 [23]. This one also
derives from pBR322 plasmid and contains the ras oncogene from human
bladder carcinoma cells. The recombinant plasmid pEJ6.6 induced gene
mutations (hprt locus) only if c-ras gene was activated. And it lacked
transforming and mutagenic properties if c-ras gene was inactivated by deletion.
These results confirm the idea that the functional activity of recombinant DNAs
and particularly transgenes plays an important role in induction of mutagenesis.
The expression of exogenous preproinsulin gene (allel-1) included into the
pBR322ins recombinant plasmid in cultivated fibroblasts of different origin have
been shown [12, 13]. At the present time we have no comparative data on the
expression of two studied allels of the human preproinsulin gene in Chinese
hamster cells. However, a decrease of frequences of chromosome breaks and
gene mutations [10] if using the recombinant plasmid pBR322insN (allel-2) і»
evident.
The results presented here permit us to suggest a dependence of mutagenic
effect of the recombinant DNAs in transfected cells on the expression of the
transgene. The alteration in the transgene structure would lead to a changing
of a propriate protein (if transcription and translation are realized in the cell
system). Comparison of the molecular structures of normal preproinsulin and
the speculated protein may reveal that the last one consists of 20 amino acid
residues (a stop-codon is located in the position 21), and only two of them are
homologous to the preproinsulin. It is not ruled out that the translation system
«pays no attention» to the CATG insertion which is introduced into the
start-codon region. In this case a normal protein would be synthesized. Then
there is a possibility that the insertion of four nucleotides upsets the translation
and the level of synthesized protein is reduced.
The differences in the functioning between two allels of preproinsulin gene
may be determined by immunologic methods using antibodies to the human
preproinsulin and/or to the hypothetical oligopeptide. Only special studies will
permit us to estimate the effect of the insertion into the region of initiation of
the translation on the expression of the mutant preproinsulin gene. Such an
approach may allow to study a regulation of translation and mutagenesis on the
level of the primary structure of the transgene.
In [9, 11] we have described the pALl and pAins recombinant plasmids
that had induced the gene mutations with high efficiency. Those plasmids
carried Alu-repeat from human genome (Fig. 1). It was found that Л/w-repeat
enhanced the mutagenic activity of the recombinant plasmids on gene level. But
we did not obtain significant difference between pBR322ins and pAins (both
contain allel-1 of the preproinsulin gene) in their mutagenic action on
chromosomes. Probably percentage of aberrant cells reflects the portion of cells
in the population which are the most sensitive to mutagenic action of different
exogeneous DNAs.
It is obvious that transgenesis may be used as a good approach for
investigation of mutator properties of different genes and estimation of the risk
of the gene therapy manipulation with recombinant viruses and DNA molecules.
We focus our attention on a mutagenic potential of recombinant DNAs and
influence of introduced genes on a variety of methabolic pathways involving
activation of proto-oncogenes as well [2]. This concerns especially anti-diabet
strategy using recombinant adenovirus [24, 25] and plasmids containing the
preproinsulin gene [13].
Insulin exerts profound effects on human physiology by controlling the
activities of many enzymes, especially those involved in energy metabolism [26,
27 ]. The list of insulin-regulated genesis is extensive and rapidly growing [28 ].
It contains the following groups of enzytoes: 1) integral membrane proteins
(insulin and growth hormone receptors, etc.); 2) proteins involved in energy
89
RUBASHEVSKY E. L. ET AL.
methabolism (pyruvate kinase, ATP cytrate lyase, etc.); 3) proteins involved in
reproduction (casein, ovalbumin); 4) secreted proteins/hormones (IGF-1 and
IGF-2, prolactin, glucagon, etc.); 5).miscellaneous proteins (p53, thyroglobulin)
and 6) transcription factors (proteins coded by c-fos and c-jun, etc.). Insulin
may have either positive or negative effects on expression of many cellular genes
[28—30 ] depending on the tissue specificity and cause the cascade of reactions.
So changing of the expression of the normal preproinsulin gene or
functioning of a mutant one would lead to different consequenses. The most
dangerous are destabilization of cellular genome and malignant transformation.
And we have shown that the recombinant plasmids carrying the human
preproinsulin gene reveal mutagenic properties depending on the presence of
different regulatory elements and mutations in the structure of the transgene.
That is why we emphasize the gene therapy experiments should be followed by
testing of recombinant molecules on mutagenic and transforming abilities.
The main conclusion of this investigation is that not only viruses and their
genes but also recombinant DNAs of nonviral origin are able to induce
chromosome and gene mutations in somatic mammalian cells. The mutagenic
activity of studied recombinant DNAs depends on the expression of the
transgenes in transfected cells.
Acknowledgements. We thank Dr. Nina B. Varshaver (Institute of
Molecular Genetics, Russian Academy of Sciences, Moscow) for useful consult-
ations and for critical reading of the manuscript of this paper.
This work was supported by the Grant of the National Academy of Sciences
of Ukraine and the Grant of the National Programme «Gene Fund Protection
of the Population of Ukraine».
Є. Л. Рубашевський, Ю. В. Пацковський, С. Д. Кириленко, I. С. Варзанова, О. М. Боголюбова,
О. Ф. Лисенко, Л. Л. Лукаш
Рекомбінантні ДНК, що містять різні алелі гена препроінсуліну людини, як мутагени в клітинах
китайського хом'ячка
Резюме
У наших попередніх дослідженнях було відкрито мутагенну активність двох рекомбінантних
плазмід (pBR322ins та pAins), які несуть ген препроінсуліну людини, у соматичних клітинах
ссавців, що культивуються. Ген препроінсуліну (алель-1) має здатністьфункціонувати під своїм
власним промотором у різних типах клітин, оскільки втратив регуляторний елемент, який
забезпечує тканинну специфічність експресії. Введення мутації зсуву рамки зчитування в район
ініціації трансляції трансгена (алель-2) призводило до зменшення мутагенного ефекту відповід-
ної рекомбінантної плазміди на генному рівні. У цій роботі вивчалась мутагенна активність
рекомбінантних плазмід, що несуть алель-1 (pBR322ins) та алель-2 (pBR322insN) гена препро-
інсуліну, з використанням цитогенетичного тесту. Показано, що присутність алеля-2 (який,
можливо, має пошкодження експресії трансгена) у структурі рекомбінантної плазміди призво-
дить до зниження рівня хромосомних розривів на клітину. Зроблено висновок стосовно того, що
не тільки віруси та їх гени, а й рекомбінантні ДНК невірусної природи здатні індукувати хромо-
сомні та генні мутації у клітинах ссавців. Мутагенна активність досліджуваних рекомбінан-
тних плазмід залежить від експресії трансгенів у клітинах.
Е. Л. Рубашевский, Ю. В. Пацковский, С. Д. Кириленко, И. С. Варзанова, О. Н. Боголюбова,
Е. Ф. Лысенко, Л. Л. Лукаш
Рекомбинантные ДНК, несущие разные аллели гена препроинсулина человека, как мутагены
в клетках китайского хомячка
Резюме
В наших предыдущих исследованиях была обнаружена мутагенная активность двух рекомби-
нантных плазмид (pBR322ins и pAins), содержащих ген инсулина человека, в культивируемых
соматических клетках млекопитающих. Ген инсулина (аллель-1) способен функционировать под
своим собственным промотором в различных типах клеток, поскольку утратил регуляторний
90
DNA RECOMBINANT MOLECULES CARRYING DIFFERENT ALLELS
элемент, обеспечивающий тканеспецифичность экспрессии. Введение мутации сдвига рамки счи-
тывания в район инициации трансляции трансгена (аллель-2) приводило к снижению мутаген-
ного эффекта соответствующей рекомбинантной плазмиды на генном уровне. В этой работе
изучена мутагенная активность рекомбинантных плазмид, несущих аллель-1 (pBR322ins) и ал-
лель-2 (pBR322insN) гена препроинсулина, с использованием цитогенетического теста. Пока-
зано, что присутствие аллеля-2 (имеющего, вероятно, поврежденную экспрессию трансгена) в
структуре рекомбинантной плазмиды, вызывало снижение уровня хромосомных разрывов на
клетку. Сделан вывод о том, что не только вирусы и их гены, а также рекомбинантные ДНК
невирусной природы способны индуцировать хромосомные и генные мутации в клетках мле-
копитающих. Мутагенная активность исследуемых рекомбинантных плазмид зависит от экс-
прессии трансгенов в клетках.
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УДК 575.155:575.224.46 Received 25.01.96
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