Zbornik - Društvo genetičara Srbije

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32 ZBORNIK ABSTRAKATA III KONGRESA GENETIÈARA SRBIJE I-Pos-11 Subotica, 30. novembar - 4. decembar 2004. ANTIMUTAGENA SVOJSTVA PRIRODNIH ANTIOKSIDANATA DETEKTOVANA Escherichia coli K12 TEST SISTEMOM B. Nikoliæ, J. Kneeviæ-Vukèeviæ, B. Vukoviæ-Gaèiæ, D. Mitiæ-Æulafiæ, T. Beriæ-Bjedov, J. Stanojeviæ, S. Stankoviæ i D. Simiæ Katedra za mikrobiologiju, Biološki fakultet, Univerzitet u Beogradu, Beograd Prirodni antioksidanti, prisutni u mnogim biljnim ekstraktima, mogu zaštiti genom od reaktivnih kiseoniènih vrsti (ROS) i time spreèiti nastajanje i fiksiranje ošteæenja koja su najznaèajniji izvor endogene mutageneze. Ispitivanje njihovog antimutagenog potencijala je znaèajno za prevenciju kancerogeneze, imunodeficijencije, neurodegeneracije i starenja. U cilju detekcije antimutagenih svojstava antioksidanata koristili smo E.coli K12 test sistem za praæenje arg - Arg + reverzija. Test sistem je sastavljen iz dva testa – Testa A i B. U Testu A se prati oksidativna mutageneza indukovana t-butilhidroperoksidom (t-BOOH) na reparativno sposobnom soju. Test B se izvodi na mutator sojevima: mutS (defektan u ‘mismatch’ reparaciji) i mutT (defektan u uklanjanju 8-oxo-G); on je dizajniran u cilju praæenja efekta antioksidanta na spontanu mutagenezu, ali i blieg odreðivanja molekularnog mehanizma njegovoga dejstva. Ispitivan je potencijal vitamina E (á-tokoferola), etarskog ulja (EO) bosiljka (Ocimum basilicum L.) i njegovog konstituenta linaloola (èini oko 70% EO) da redukuju oksidativnu mutagenezu. Protektivni efekat model antioksidanta vitamina E je detektovan u oba testa, ali je najveæa inhibicija mutageneze ostvarena u mutS soju (76%). EO bosiljka, kao i linalool, su pokazali umeren inhibitorni potencijal u Testu A (~35%), ali je snaan efekat ostvaren u mutS soju (inhibicija preko 60%). Ispitivani derivati bosiljka nisu imali uticaja na mutagenezu posredovanu 8-oxo-G. ANTIMUTAGENIC PROPERTIES OF NATURAL ANTIOXIDANTS IN THE Escherichia coli K12 REVERSION ASSAY Natural antioxidants, widely distributed in plant extracts, might protect genomic DNA from lesions induced by reactive oxygen species (ROS), a significant source of endogenous mutagenesis. The study of their antimutagenic potential can promote the prevention of carcinogenesis, immunodeficiency, neurodegeneration and aging. In order to detect antimutagenic potential of antioxidants, we used E.coli K12 arg - Arg + reversion assay consists of two tests, named Test A and Test B. Test A is created to evaluate the antimutagenic potential against t-butyl hydroperoxide (t-BOOH)-induced oxidative mutagenesis and is performed on repair proficient strain. Test B is performed on mutator strains mutS (deficient in mismatch repair) and mutT (deficient in removing 8-oxo-G); it is designed to elucidate the effect of antioxidants on spontaneous mutagenesis and to determine the molecular mechanism of its action. The potential of vitamin E (-tocopherol), essential oil (EO) of basil (Ocimum basilicum L.) and its major constituent linalool (~70% of EO) to reduce the oxidative mutagenesis, was studied.. The protective effect of model antioxidant vitamin E against oxidative mutagenesis was detected in both tests, but the strongest inhibition was obtained in mutS strain (76%). EO of basil, as well as linalool, exhibited moderate inhibitory potential in Test A (~35%), while powerful inhibition was detected with both test substances in mutS strain (over 60%). EO of basil as well as linalool showed no effect on mutagenesis mediated by 8-oxo-G lesions.
I-Pos-12 ZBORNIK ABSTRAKATA III KONGRESA GENETIÈARA SRBIJE 33 Subotica, 30. novembar - 4. decembar 2004. MOLEKULARNA KARAKTERIZACIJA BAKTERIJA MLEÈNE KISELINE (BMK) IZ KOZIJEG SIRA Milica Nikoliæ, Amarela Terziæ-Vidojeviæ, B. Jovèiæ, Lj. Topisiroviæ i Nataša Goliæ Institut za molekularnu genetiku i genetièko inenjerstvo, Beograd Klasiène metode za identifikaciju i determinaciju mikroorganizama, zasnovane na kultivaciji i biohemijskim metodama, nisu uvek bile pogodne za dobijanje informacija o mikrobiološkom sastavu razlièitih uzoraka. Analiza mikroflore klasiènim metodama je ogranièena na mikroorganizme koji se lako kultivišu, tako da se ne dobija informacija o kompletnoj populaciji iz uzorka. U ovom radu su korišæene molekularne tehnike kojima je moguæe okarakterisati razlièite bakteije mleène kiseline (BMK) iz kozijeg sira. Totalna DNK iz sira je izolovana i korišæena za PCR reakciju, gde su kao prajmeri korišæeni U968-GC i L1401, komplementarni V6 i V8 regionima eubakterijske 16s rDNK. Sintetisani DNK fragmenati velièine 450 bp predstavljaju zbir umnoenih DNK fragmenata koji su poreklom od razlièitih vrsta bakterija iz sira. Fragmenti su uklonirani u plazmid pUCEry19, a ligacionom smešom je transformisan soj E. coli DH5. Transformanti su analizirani na prisustvo plazmida sa ukloniranim PCR fragmentom. U daljem radu biæe uraðena DGGE analiza kloniranih fragmenata. Fingerprint tehnikom, kao što je DGGE, se utvrðuje genetièki diverzitet mikroflore sira. Meðusobno razlièiti fragmenti æe biti sekvenciirani u cilju precizne identifikacije mikroorganizama prisutnih u siru. Dobijeni rezultati biæe poreðeni sa rezultatima klasiène mikrobiološke analize. MOLECULAR CHARACTERIZATION OF LACTIC ACID BACTERIA IN THE GOAT CHEESE Conventional methods for the identification and determination of microorganisms, based on cultivation and biochemical methods, usually were suitable enough for getting the information about the composition of the microorganisms present in variable environmental samples. However, the analysis of microflora by conventional methods is limited to easy cultivable microorganisms and does not give the information about the complete population in the sample. In this study molecular techniques were used to characterize different lactic acid bacteria (LAB) from a goat cheese. The first step was the isolation of total DNA from the cheese that has been used as a template for PCR reaction. PCR, by using primers U968-GC and L1401 complementary to the V6 and V8 region of eubacterial 16s rDNA was performed. Obtained DNA products of 450 bp represent the sum of amplified DNA fragments originated from different species of bacteria from the cheese. These fragments were cloned into a plasmid pUCEry19, and ligation mixture was transformed into a competent E. coli strain DH5. Transformants were screened for the presence of plasmids containing cloned PCR fragments. The aim of further research will be the DGGE analysis of the cloned fragments. DGGE, as a fingerprinting technique is suitable to describe the genetic diversity of the cheese composition. Different fragments will be sequenced and the obtained data would be used for the identification of the microorganisms present in the goat cheese. These results will be compared with results obtained by classical microbiological methods.
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Zbornik - Društvo genetičara Srbije

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