Zbornik - Društvo genetičara Srbije

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56 ZBORNIK ABSTRAKATA III KONGRESA GENETIÈARA SRBIJE II-Pos-1 Subotica, 30. novembar - 4. decembar 2004. ã-OZRAÈENJE SUVOG BAKTERIOFAGA ë Desanka Boin Institut za nuklearne nauke «Vinèa», Beograd Cilj istraivanja je efekat ã-ozraèivanja suvog ë1390 spi- bakteriofaga. Bakteriofag je proizveden u P2 lizogenom soju E. coli i preèišæen diferencijalnim sedimentacijom (centrifugiranje na 10 000 rpm, 2 puta po 10 min). Bakteriofag je sušen 170 sati na sobnoj temperaturi na vazduhu ili u visokom vakumu (10 -5 mbar). U poreðenju sa uzorkom sušenim u vakumu, dehidracija na vazduhu uzrokuje 11 puta veæu inaktivaciju bakteriofaga, veoma slabi stabilnost proteina kapsida i 3 puta poveæava frekvencu rekombinacije. Mutacije u DNK nisu registrovane ni u uzorku sušenom na vazduhu, niti u uzorku sušenom u visokom vakumu. Dehidrirani ë1390 spi- bakteriofagi su ozraèeni ã zracima iz 60Co izvora. Istu dozu ã zraka preivljava 1x10-1 bakteriofaga osušenih na vazduhu i 1.3x10 -2 bakteriofaga osušenih u vakumu. U oba uzorka suvih faga, frekvenca rekombinacije je 2-3 puta veæa nego u kontrolnom uzorku a DNK mutacije nisu registrovane. ã-IRRADIATED DRY BACTERIOPHAGE ë The effects of ã-irradiation on dry ë1390 spi- bacteriophages were investigated. Bacteriophages were preparing by confluent lysis in P2 lysogene strain of E. coli and subsequently purified by differential sedimentation (centrifugation at 10 000 rpm, 2 times per 10 min). Dehydration of the bacteriophage for 170 hours was performed at room temperature on air or in high vacuum (10 -5 mbar). Dehydration on air caused 11 times higher inactivation of bacteriophage than dehydration in high vacuum, resulting in very weakened stability of the capside proteins and 3 times higher frequency of recombination. DNA mutations were not detected neither in the sample dried on air nor in the sample dried in vacuum. Dehydrated ë1390 spi- bacteriophages were ã-irradiated from a 60 Co source. For the same dose of ã-rays, survival was 1x10 -1 for bacteriophages dried on air and 1.3x10-2 for bacteriophages dried in vacuum. In both dried samples, the frequency of recombination was 2-3 times higher than in the control sample and DNA mutations were not detected.
II-Pos-2 ZBORNIK ABSTRAKATA III KONGRESA GENETIÈARA SRBIJE 57 Subotica, 30. novembar - 4. decembar 2004. ULOGA OPŠTEG TRANSKRIPCIONOG FAKTORA NFY U REGULACIJI EKSPRESIJE HUMANOG SOX3 GENA A. Krstiæ, Marija Mojsin, Nataša Kovaèeviæ-Grujièiæ i Milena Stevanoviæ Institut za molekularnu genetiku i genetièko inenjerstvo, Beograd Rani stadijumi neurogeneze i neuronalne diferencijacije su precizno kontrolisani serijom gena. Sox3 gen je eksprimiran u mozgu veæ u najranijim fazama razviæa. Smatra se da je ovaj gen jedan od najranijih neuralnih markera kod kièmenjaka, odreðujuæi sudbinu neurona. Da bi se rasvetlili molekularni mehanizmi odgovorni za ekspresiju humanog SOX3 gena korišæen je kompjuterski program ( MatInspector) koji pretrauje baze podataka vezivnih matriksa, a sa ciljem identifikacije potencijalnih mesta vezivanja transkripcionih faktora u promotoru humanog SOX3 gena. Niz potencijalnih vezivnih mesta za poznate transkripcione faktore je identifikovan u 5’ nekodirajuæem regionu humanog SOX3 gena. Prisustvo tri evolutivno konzervisana CCAAT boksa, koji predstavlja potencijalno vezivno mesto za opšti transkripcioni faktor NFY, sugeriše moguæi znaèaj ovog faktora u regulaciji ekspresije humanog SOX3 gena. Eksperimenti smanjene elktroforetske pokretljivosti i modifikacija ove metode uz upotrebu specifiènih antitela dokazuju specifiènost vezivanja transkripcionog faktora NFY za sva tri identifikovana CCAAT kontrolna elementa. Da bi se ispitalo da li je potencijalni CCAAT boks funkcionalan, uraðena je poziciono usmerena mutageneza. Sposobnost da utièe na ekspresiju cat gena u NT2/D1 æelijskoj liniji, poreðena je izmeðu mutiranog i nemutiranog konstrukta. Pokazano je da je mutageneza CCAAT boksa (-101 bp do -105 bp) umanjila ekspresiju reporter gena više od 5 puta u poreðenju sa ekspresijom nemutiranog konstrukta. Ovaj rezultat ukazuje da CCAAT boks vezivni domen prisutan u promotoru SOX3 gena ima funkcionalnu ulogu u transkripciji ovog gena. THE ROLE OF THE GENERAL TRANSCRIPTION FACTOR NFY IN THE REGULATION OF THE EXPRESSION OF HUMAN SOX3 GENE Early stadiums of the neurogenesis and neuronal differentiation are precisely controlled by a series of genes. Sox3 is expressed in the brain from the earliest stages of development. It is considered to be one of the earliest neural markers in vertebrates playing the role in specifying neuronal fate. In order to elucidate molecular mechanisms underlying the regulation of the human SOX3 gene expression, computer prediction software (MatInspector) was used to search the matrix family library database and to identify potential transcription binding sites in the human SOX3 promoter. The number of putative consensus binding sites for known transcription factors was identified in the 5’ noncoding region of the human SOX3 gene. The presence of three evolutionary conserved CCAAT boxes, representing the putative binding sites for the general transcription factor NFY, suggests the potential importance of this factor in the regulation of the human SOX3 gene expression. EMSA and «supershift» experiments are performed to prove the specificity of the NFY binding to all three identified CCAAT control elements. To examine whether the putative CCAAT box is functional, site directed mutagenesis was done. The ability of the mutant and its wild-type counterpart to drive expression of the cat reporter gene was compared in NT2/D1 cell line. We have shown that mutagenesis of the CCAAT box (-101 bp to -105 bp) reduced the expression of the reporter gene more than 5-fold when compared to the wild-type expression. This result indicates that the CCAAT box motif present in the SOX3 promoter plays a functional role in the transcription of this gene.
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DRUŠTVO GENETIČARA SRBIJE SERBIAN
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III KONGRES GENETIÈARA SRBIJE Subo
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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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Zbornik - Društvo genetičara Srbije

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