Zbornik - Društvo genetičara Srbije

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.