Zbornik - Društvo genetičara Srbije

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64 ZBORNIK ABSTRAKATA III KONGRESA GENETIÈARA SRBIJE III-Uvo-1 Subotica, 30. novembar - 4. decembar 2004. GENETIÈKO INENJERSTVO - OSNOVA MOLEKULARNE BIOTEHNOLOGIJE Lj. Topisiroviæ Institut za molekularnu genetiku i genetièko inenjerstvo, Beograd Istraivanja u proteklih dvadeset godina nedvojbeno su pokazala da je genetièko inenjerstvo otvorilo perspektivu razvoja molekularne biotehnologije. Genetièko inenjerstvo omoguæava (a) izuèavanje strukture i funkcije gena bilo kog organizma, (b). izazivanje mutacija na specifiènom mestu u genu (dirigovana mutageza) koja za posledicu moe imati poveæanje sinteze produkta za koga taj gen nosi informaciju, (c) prevazilaenje uskih grla u biosintetskim putevima razlièitih metabolita od koristi, kao i (d) dobijanje proizvoda koje je na drugi naèin teško, skupo ili nemoguæe dobiti. Svakako da je najveæi doprinos genetièkog inenjerstva molekularnoj biotehnologiji upravo u ovom domenu. Naime, genetièko inenjerstvo omoguæava manipulaciju genima i prebacivanjem tih gena iz jedne biološke vrste u drugu, što se u prirodi ne moe spontano dešavati. S druge strane, moguæe je konstruisati himerne gene in vitro) i tako konstruisane gene ubaciti u ivi organizam. Sve svetske prognoze oko osnova tehnologije 21. veka se slau u tome da æe upravo molekularna biotehnologija biti pokretaèka snaga razvoja savremene civilizacije. Uvoðenjem genetièkog inenjerstva u cilju unapreðivanja biotehnoloških procesa, dobio se jedan sasvim novi kvalitet. Sada je moguæe od mikroorganizama, biljnih ili ivotinjskih æelija napraviti «biološke fabrike» koje æe proizvoditi veliku kolièinu ekonomski vrednih jedinjenja, odnosno moguæe je konstruisati transgene organizme koji mogu biti osnova za proširenje palete biotehnoloških proizvoda. GENETIC ENGINEERING – THE BASIS OF MOLECULAR BIOTECHNOLOGY Results obtained by using genetic engineering in the research of living systems in the past twenty years indisputably showed that genetic engineering opened real perspective for the development of molecular biotechnology. The application of genetic engineering in the research allowed (a) investigation of structure and function of genes of any organism, (b) direct introduction of mutations into the gene of interest, (c) overcoming the bottle-necks in biosynthetic pathways as well as (d) production of products that are very hard or at all impossible to get in traditional processes. By all means, the greatest contribution of genetic engineering in molecular biotechnology lies in this domain. This is because, genetic engineering enable the gene manipulation and transfer of gene(s) from one into another species, what is impossible to happen in nature. On the other hand, it is possible to construct chimerical genes in vitro and introduce such genes into chosen host organism. According to general prognosis, the molecular biotechnology will be a basis for 21st century technologies and that it would be a moving force for development of modern civilisation. Introduction of genetic engineering for an improvement of biotechnology processes opens qualitatively new approaches. Molecular biotechnology facilitate the using of microorganisms, plant and animal cells as «biological factories» that are able to produce higher quantities of high value-added compounds, that is, it would be possible to construct transgenic organisms that could be a basis for broadening of biotechnology products.
III-Uvo-2 ZBORNIK ABSTRAKATA III KONGRESA GENETIÈARA SRBIJE 65 Subotica, 30. novembar - 4. decembar 2004. POREÐENJE AKTIVNOSTI RAZLIÈITIH kgmB::lacZ GENSKIH I OPERONSKIH FUZIJA Sandra Vojnoviæ, Nataša Milojeviæ i Branka Vasiljeviæ Institut za molekularnu genetiku i genetièko inenjerstvo, Beograd KgmB (rezistencija na kanamicin-gentamicin) metilaza iz soja Streptomyces tenebrarius modifikuje G-1405 u okviru16S rRNK unutar sekvence CGUCA i na taj naèin štiti bakteriju od sopstvenog toksiènog proizvoda. Pentanukleotid CGCCC je prisutan u okviru istog regiona 16S rRNK pri èemu se i preklapa sa CGUCA sekvencom. Pokazano je da su obe sekvence prisutne i u uzvodnom regionu kgmB gena, ispred RBS-a, ali su ovde razdvojene. Predloili smo regulatorni model po kome KgmB metilaza moe da se vee za sopstvenu iRNK, u okviru ove dve potencijalne regulatorne sekvence, i na taj naèin spreèi dalju translaciju, nakon što je modifikovala sve ribozome. Sa ciljem izuèavanja translacione autoregulacije kgmB gena konstruisali smo razlièite kgmB::lacZ genske i operonske fuzije i pratili uticaj KgmB metilaze na aktivnost tih fuzija kako in-cis tako i in-trans. Pokazali smo da èak i kada se PCR-om mutira CGTCA pentanukleotid, aktivnost fuzionog proteina opada u prisustvu funkcionalne KgmB metilaze. Paralelno smo uradili i eksperimente sa KgmB proteinom inaktiviranim u okviru BglII restrikcionog mesta. Uprkos èinjenici da inaktivirani KgmB protein ima samo 66 originalnih amino kiselina od ukupno 135, uticaji skraæenog KgmB proteina i originalne KgmB metilaze na aktivnost razlièitih kgmB::lacZ fuzija su vrlo slièni. Iz ovih rezultata proizilazi da bi KgmB protein mogao da ima dva odvojena domena, obzirom da je regulatorna funkcija skraæenog KgmB proteina oèuvana, a rezistencija na kanamicin i gentamicin narušena. Eksperimenti sa kgmB::lacZ operonskim fuzijama su pokazali da KgmB protein, kompletan ili skraæen, nema uticaja na ekspresiju lacZ gena. COMPARATIVE ANALYSIS OF DIFFERENT kgmB::lacZ GENE AND OPERON FUSIONS The KgmB (kanamycin-gentamicin resistance) methylase from Streptomyces tenebrarius acts at G-1405 of 16S rRNA within the sequence CGUCA and thus protects bacteria from its own toxic product. The pentanucleotide CGCCC is present in the same region of 16S rRNA overleaping with the CGUCA sequence. It was shown that both of these sequences are present in the upstream region of the kgmB gene, in front of RBS but, they are separated. We proposed a regulatory model in which the KgmB methylase can bind to its own mRNA, within these two potentional regulatory sequences, preventing further translation once all the ribosomes are modified. To study translational autoregulation of the kgmB gene different kgmB::lacZ gene and operon fusions were constructed and the effect of overexpression of kgmB gene to these fusions was analyzed in-cis as well as in-trans. We showed that even if CGTCA pentanucleotide is mutated by PCR, there is still decrease in the activity of the fusion protein. We performed analogous experiments with the kgmB gene inactivated in BglII restriction site. Despite the fact that inactivated KgmB protein has only 66 sense amino acids out of 135, the effect of truncated KgmB protein to different kgmB::lacZ fusions was similar. These results suggested that the KgmB protein might have two separate domains since regulatory function of the truncated KgmB protein is preserved in spite of the fact that resistance to kanamycin and gentamicin was lost. The experiments with kgmB::lacZ operon fusions showed that the KgmB protein, wild type or truncated, had no effect on the expression of the lacZ fusions.
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DRUŠTVO GENETIČARA SRBIJE SERBIAN
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III KONGRES GENETIÈARA SRBIJE Subo
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Uvodno predavanje Opening lecture
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I tematska oblast / I topic: Geneti
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ZAVRŠNO PREDAVANJE CLOSING LECTURE
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A Aksentijeviæ I. 194 Aleksiæ J.
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Kovaèev L. 73, 90, 125, 140, 147 K
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Stanojeviæ B. 223 Stanojeviæ J. 2
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Zbornik - Društvo genetičara Srbije

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