Biopolym. Cell. 1996; 12(5):83-92.
DNA recombinant molecules carrying different allels of human preproinsulin gene as mutagens in Chinese hamster cells
1Rubashevsky E. L., 1Patskovsky Yu. V., 1Kirilenko S. D., 1Varzanova I. S., 1Bogolyubova O. M., 1Lysenko O. F., 1Lukash L. L.
  1. Institute of Molecular Biology and Genetics, NAS of Ukraine
    150, Akademika Zabolotnoho Str., Kyiv, Ukraine, 03680

Abstract

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.

References

[1] Lukash LL, Varshaver NB, Buzhiyevskaya TI, Shapiro NI. The oncogene of BAV-3 as a mutagen. J Cell Sci. 1985;78:97-103.
[2] Lukash LL. Mutation process and malignant transformation induced by adenoviruses in mammalian cells. Tsitol Genet. 1986;20(1):55-8, 72.
[3] Buzhievskaia TI. Virus-induced mutagenesis and various aspects of genetic engineering in humans. Tsitol Genet. 1986;20(1):66-72.
[4] Rubashevski? EL. Plasmid pBR325tk as a mutagen. Tsitol Genet. 1986;20(1):76-8,
[5] Rubashevsky EL, Lukash LL, Buzhievskaya TI et al. Mutagenicity of the Herpes Simplex Virus Type 1 DNA and pBR322tk plasmid DNA in the permanent mammalian cells. Dokl. Acad. Nauk SSSR. 1984; 279(3): 252-4.
[6] Rubashevsky EL, Lykash LL, Shved AD et al. The mutagenic activity of the native and inactivated samples of Herpes Simple Virus. Biol Sci. 1988; (9): 83-7.
[7] Buzhievskaya TI, Lukash LL, Rubashevsky EL. Mutagenic and transforming action of some viruses, genes and recombinant molecules. Biotechnology. Current Progress. Eds P. N. Cheremisinoff, L. M. Ferrante-Lancaster (USA). New York: Technomic Publ. Comp., 1991: 133-156.
[8] Rubashevsky EL, Lukash LL, Lysenko EF, Neborachko LN, Kirilenko SI, Patskovsky YuV, Buzhievskaya TI. The chromosome mutations in established chinese hamster fibroblasts, transfected by pBR322 recombinant plasmid carrying human insulin gene. Biopolym Cell. 1990; 6(6):96-9.
[9] Rubashevski? EL, Patskovski? IuV, Podol'skaia SV, Sukhorada EM, Lysenko EF, Kostetskaia EV, Kostetski? IE, Varzanova IS, Lukash LL. The induction of mutations in 6-mercaptopurine resistance in Chinese hamster cells under the action of the recombinant plasmid pAins containing the human insulin gene. Tsitol Genet. 1993;27(3):63-8.
[10] Rubashevski? EL, Patskovski? IuV, Lysenko EF, Kirilenko SD, Lukash LL. The mutagenic effect of the recombinant plasmid pBR322ins carrying a native and a modified human insulin gene. Tsitol Genet. 1993;27(4):44-51.
[11] Lukash LL, Podolskaya SV, Suhorada HM, Kostetskaya KV, Kostetsky IE, Varzanova IS, Patskovsky YuV, Vavilina IV, Deis SV. The influence of alkylating agent MNNG on mutagenic effect of exogeneous recombinant DNA. Biopolym Cell. 1995; 11(1):87-91.
[12] Neborachko LN, Lukash LL, Troyanovsky BM, Varzanova IS, Buzhievskaya TI, Kordium VA. Studies in possibility of expression of exogenic human insulin gene in cultured mammalian fibroblasts. Biopolym Cell. 1989; 5(2):58-61.
[13] Kordyum VA, Frolkis VV, Lukash LL, Shulzhenko VN, Shpilevaya SP, Kostetsky IE, Titok TG, Varsanova IS, Likhacheva LI, Irodov DM. Gene therapy of mass pathologies. Biopolym Cell. 1993; 9(4):63-104.
[14] Maniatis T, Fritch EF, Sambrook J. Molecular Cloning. A Laboratory Manual. New York: Cold Spring Harbor Lab., 1982. 545 p.
[15] An International System for Human Cytogenetic Nomenclature (1985) ISCN 1985. Report of the Standing Committee on Human Cytogenetic Nomenclature. Birth Defects Orig Artic Ser. 1985;21(1):1-117.
[16] Urbakh UU. Biometrics Methods. Moscow: Nauka, 1964. 415 p.
[17] Rubashevsky EL, Buzhievskaya TI, Dyachenko NS, Nosach LN. Mutagenic activity of the human adenovirus type 1 on the Chinese hamster fibroblasts. IV Meet, of the Ukr. Soc. of Geneticists and Selectionists. Kiyv: Nauk. Dumka, 1981. Pt 6: 152-153 (Ukraine).
[18] Popov LS, Gorbunova LV, Varshaver NB, Shapiro NI. Integration of SV40 DNA into the cell genome and viral mutagenesis. Genetika. 1986;22(9):2213-9.
[19] Gorbunova LV, Varshaver NB, Marshak MI, Shapiro NI. The role of the transforming A gene of SV40 in the mutagenic activity of the virus. Mol Gen Genet. 1982;187(3):473-6.
[20] Drize OB, Sokova OI, Nikashina EB, Shliankevich MA, Shapot VS. Possible role of the T antigen in inducing chromosome aberrations in SV40 virus-transformed cells. Tsitologiia. 1985;27(1):76-82.
[21] Pilon L, Royal A, Langelier Y. Increased mutation frequency after herpes simplex virus type 2 infection in non-permissive XC cells. J Gen Virol. 1985;66 ( Pt 2):259-65.
[22] Pilon L, Langelier Y, Royal A. Herpes simplex virus type 2 mutagenesis: characterization of mutants induced at the hprt locus of nonpermissive XC cells. Mol Cell Biol. 1986;6(8):2977-83.
[23] Baron EM, Bobrysheva IV, Varshaver NB. The plasmid pEJ6.6 carrying the activated c-Ha-ras-1 oncogene increases the mutation frequency in Chinese hamster cells. Genetika. 1992;28(6):35-40.
[24] Becker TC, Noel RJ, Johnson JH. et al Use of recombinant adenovirus for high-efficiency gene transfer into the islets of Langerhans (Abstract) Diabetes. 1993; 42, Suppl. 1:11 A.
[25] Becker TC, Noel RJ, Coats WS, G?mez-Foix AM, Alam T, Gerard RD, Newgard CB. Use of recombinant adenovirus for metabolic engineering of mammalian cells. Methods Cell Biol. 1994;43 Pt A:161-89.
[26] Lawrence JC Jr. Signal transduction and protein phosphorylation in the regulation of cellular metabolism by insulin. Annu Rev Physiol. 1992;54:177-93.
[27] Makino H, Manganiello VC, Kono T. Roles of ATP in insulin actions. Annu Rev Physiol. 1994;56:273-95.
[28] O'Brien RM, Granner DK. Regulation of gene expression by insulin. Biochem J. 1991;278 ( Pt 3):609-19.
[29] Taub R, Roy A, Dieter R, Koontz J. Insulin as a growth factor in rat hepatoma cells. Stimulation of proto-oncogene expression. J Biol Chem. 1987;262(22):10893-7.
[30] Stumpo DJ, Stewart TN, Gilman MZ, Blackshear PJ. Identification of c-fos sequences involved in induction by insulin and phorbol esters. J Biol Chem. 1988;263(4):1611-4.