Zbornik - Društvo genetičara Srbije
Zbornik - Društvo genetičara Srbije
Zbornik - Društvo genetičara Srbije
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III-Uvo-2 ZBORNIK ABSTRAKATA III KONGRESA GENETIÈARA SRBIJE 65<br />
Subotica, 30. novembar - 4. decembar 2004.<br />
POREÐENJE AKTIVNOSTI RAZLIÈITIH kgmB::lacZ GENSKIH I<br />
OPERONSKIH FUZIJA<br />
Sandra Vojnoviæ, Nataša Milojeviæ i Branka Vasiljeviæ<br />
Institut za molekularnu genetiku i genetièko inenjerstvo, Beograd<br />
KgmB (rezistencija na kanamicin-gentamicin) metilaza iz soja Streptomyces tenebrarius<br />
modifikuje G-1405 u okviru16S rRNK unutar sekvence CGUCA i na taj naèin štiti<br />
bakteriju od sopstvenog toksiènog proizvoda. Pentanukleotid CGCCC je prisutan u<br />
okviru istog regiona 16S rRNK pri èemu se i preklapa sa CGUCA sekvencom. Pokazano<br />
je da su obe sekvence prisutne i u uzvodnom regionu kgmB gena, ispred RBS-a, ali su<br />
ovde razdvojene. Predloili smo regulatorni model po kome KgmB metilaza moe da se<br />
vee za sopstvenu iRNK, u okviru ove dve potencijalne regulatorne sekvence, i na taj<br />
naèin spreèi dalju translaciju, nakon što je modifikovala sve ribozome.<br />
Sa ciljem izuèavanja translacione autoregulacije kgmB gena konstruisali smo razlièite<br />
kgmB::lacZ genske i operonske fuzije i pratili uticaj KgmB metilaze na aktivnost tih<br />
fuzija kako in-cis tako i in-trans. Pokazali smo da èak i kada se PCR-om mutira CGTCA<br />
pentanukleotid, aktivnost fuzionog proteina opada u prisustvu funkcionalne KgmB<br />
metilaze. Paralelno smo uradili i eksperimente sa KgmB proteinom inaktiviranim u<br />
okviru BglII restrikcionog mesta. Uprkos èinjenici da inaktivirani KgmB protein ima<br />
samo 66 originalnih amino kiselina od ukupno 135, uticaji skraæenog KgmB proteina i<br />
originalne KgmB metilaze na aktivnost razlièitih kgmB::lacZ fuzija su vrlo slièni. Iz ovih<br />
rezultata proizilazi da bi KgmB protein mogao da ima dva odvojena domena, obzirom da<br />
je regulatorna funkcija skraæenog KgmB proteina oèuvana, a rezistencija na kanamicin i<br />
gentamicin narušena. Eksperimenti sa kgmB::lacZ operonskim fuzijama su pokazali da<br />
KgmB protein, kompletan ili skraæen, nema uticaja na ekspresiju lacZ gena.<br />
COMPARATIVE ANALYSIS OF DIFFERENT kgmB::lacZ GENE AND<br />
OPERON FUSIONS<br />
The KgmB (kanamycin-gentamicin resistance) methylase from Streptomyces tenebrarius<br />
acts at G-1405 of 16S rRNA within the sequence CGUCA and thus protects bacteria<br />
from its own toxic product. The pentanucleotide CGCCC is present in the same region of<br />
16S rRNA overleaping with the CGUCA sequence. It was shown that both of these sequences<br />
are present in the upstream region of the kgmB gene, in front of RBS but, they<br />
are separated. We proposed a regulatory model in which the KgmB methylase can bind to<br />
its own mRNA, within these two potentional regulatory sequences, preventing further<br />
translation once all the ribosomes are modified.<br />
To study translational autoregulation of the kgmB gene different kgmB::lacZ gene and<br />
operon fusions were constructed and the effect of overexpression of kgmB gene to these<br />
fusions was analyzed in-cis as well as in-trans. We showed that even if CGTCA<br />
pentanucleotide is mutated by PCR, there is still decrease in the activity of the fusion protein.<br />
We performed analogous experiments with the kgmB gene inactivated in BglII restriction<br />
site. Despite the fact that inactivated KgmB protein has only 66 sense amino acids<br />
out of 135, the effect of truncated KgmB protein to different kgmB::lacZ fusions was<br />
similar. These results suggested that the KgmB protein might have two separate domains<br />
since regulatory function of the truncated KgmB protein is preserved in spite of the fact<br />
that resistance to kanamycin and gentamicin was lost. The experiments with kgmB::lacZ<br />
operon fusions showed that the KgmB protein, wild type or truncated, had no effect on<br />
the expression of the lacZ fusions.