Effect of the kanamycin resistance marker on stability of 2μ-based expression plasmids

U ovom radu opisan je uticaj gena za kanamicinsku rezistenciju (Kmr) na održavanje 2μm plazmida u Saccharomyces cerevisiae. Prisustvo ovog marker gena dovodi do gubitka stabilnog model-vektora konstantnom stopom nezavisnom od izvora ugljenika i stope rasta kulture. Kod sojeva za sintezu heterolognih proteina sa galaktoznog promotora (GALUAS) uvođenje Kmr rezultira "čišćenjem" od kvaščevih epizomalnih plazmida (YEp) u svega nekoliko generacija. Primena selektivnog pritiska na sojeve koji proizvode rekombinantnu penicilin G amidazu (rPGA) nije dovela do očekivanog povećanja prinosa proteina. Ispitivanjem uticaja samih genetičkih elemenata za proizvodnju heterolognih proteina na stabilnost vektora pokazano je da najjače destabilišuće dejstvo ima prisustvo i eksprecija stranog gena.In this paper we describe the effect of the kanamycin resistance gene (Kmr) on 2μm-based plasmid maintenance in Saccharomyces cerevisiae. The influence of this marker gene on the loss of the stable model-vectors proved to be constant, as well as independent of carbon source and culture growth rates. In strains for GALUAS - driven heterologous protein production introduction of Kmr resulted in curing of the yeast episomal plasmid (YEp) from the population in a small number of generations. Application of selective pressure on the strain producing recombinant penicillin G amidase (rPGA) did not provide the expected increase of protein yield. The influence of genetic elements for heterologous protein production on vector stability was examined, and the most destabilizing factors prove to be the presence and expression of the foreign gene

Краду накит и иконе

Разговор о књизи "Уметност и криминал" аутора др Ренате Самарџић (Нови Сад : Платонеум, 2017

Хитлер је хтео Вермера, Рембранта и Рубенса : најновији подаци о крађи уметнина током Другог светског рата

Разговор о књизи "Уметност и криминал" аутора др Ренате Самарџић (Нови Сад : Платонем, 2017

Metagenomic analysis of soil microbial communities

Ramonda serbica i Ramonda nathaliae, retke biljke 'vaskrsnice' koje rastu na Balkanskom poluostrvu, u odgovoru na stres produkuju velike količine fenola. Bakterijske zajednice poreklom iz rizosfere ovih biljaka analizirane su metagenomskim pristupom. Fluorescentna 'in situ' hibridizacija (FISH) i DAPI bojenje pokazali su da u analiziranim zemljištima ima svega 5% metabolički aktivnih bakterija. Upotrebom prajmera specifičnih za bakterijsku DNK umnoženi su geni za 16S rDNK i konstruisane su dve genske biblioteke. Biblioteke su pretraživane uz pomoć RFLP metode. Od ukupno 192 klona dobijena iz uzorka rizosfere R. nathaliae identifikovano je 35 operativnih taksonomskih jedinica (OTJ), dok je iz uzorka rizosfere R. serbica dobijeno 13 OTJ od ukupno 80 klonova. Predstavnici svake OTJ su sekvencirani. Analizirane zajednice odlikuje veoma mali diverzitet i većina dobijenih sekvenci je pokazala malu sličnost sa DNK sekvencama do sada kultivisanih bakterija.Ramonda serbica and Ramonda nathaliae, rare resurrection plants growing in the Balkan Peninsula, produce a high amount of phenolic compounds as a response to stress. The composition and size of bacterial communities in two rhizosphere soil samples of these plants were analyzed using a metagenomic approach. Fluorescent in situ hybridization (FISH) experiments together with DAPI staining showed that the metabolically active bacteria represent only a small fraction, approximately 5%, of total soil bacteria. Using universal bacteria - specific primers 16S rDNA genes were amplified directly from metagenomic DNAs and two libraries were constructed. The Restriction Fragment Length Polymorphism (RLFP) method was used in library screening. Amongst 192 clones, 35 unique operational taxonomic units (OTUs) were determined from the rhizosphere of R. nathaliae, and 13 OTUs out of 80 clones in total from the library of R. serbica. Representative clones from each OTU were sequenced. The majority of sequences from metagenomes showed very little similarity to any cultured bacteria. In conclusion, the bacterial communities in the studied soil samples showed quite poor diversity

Overexpression of sgm 5’ UTR mRNA reduces gentamicin resistance in both Escherichia coli and Micromonospora melanosporea cells

16S rRNK metilaze su eksprimirane u većini bakterija koje proizvode antibiotike da bi se zaštitile od dejstva antibiotika putem metilacije 16S rRNK na pozicijama koje su bitne za njihovo dejstvo. Gen sgm koji je odgovoran za rezistenciju na sisomicin i gentamicin u soju Micromonospora zionensis, metilu je G1405 u okviru A mesta 16S rRNA gde se nalazi i CCGCCC heksanukleotid. Isti heksanukleotid se nalazi i 14 nukleotida ispred mesta vezivanja ribozoma na sgm informacionoj RNK. Predloženi model translacione regulacije sgm gena pretpostavlja da se Sgm protein vezuje za ovaj motiv kako na 16S rRNK, tako i na 5’ netranslirajućem regionu (UTR) sopstvene informacione RNK. 5’ UTR sekvenca je overeksprimirana na sgm informacionoj RNK sa skraćenim 3’ krajem i testiran je efekat na gentamicinsku rezistenciju u ćelijama E. coli i Micromonospora melanosporea. Overekspresija ove regulatorne sekvence dovodi do smanjenja rezistencije u oba testirana soja najverovatnije zbog titracije Sgm molekula od strane 5’ UTR-a.The 16S rRNA methylases are expressed by most of the antibiotic producing bacteria in order to protect themselves against antibiotics by methylation of 16S rRNA at positions which are crucial for their action. The sgm sisomicin-gentamicin resistance gene from Micromonospora zionensis methylates G1405 positioned in the A site of 16S rRNA, which includes a CCGCCC hexanucleotide. The same hexanucleotide is also present 14 nucleotides in front of the ribosome binding site of sgm mRNA. The model proposed for translational regulation of sgm assumes that Sgm binds to this motif, both on 16S rRNA and on the 5’ untranslated region (UTR) of its own mRNA. The 5’ UTR mRNA sequence was overexpressed on 3’-truncated sgm mRNA, and the effect on gentamicin resistance conferred by Sgm was tested in Escherichia coli and in Micromonospora melanosporea. Overexpression of the sgm mRNA regulatory region decreases the resistance to gentamicin in both E. coli and M. melanosporea. This effect is likely to be due to titration of Sgm molecules by the overexpressed 5’ UTR

Пикасо и Сезан као таоци мафије

Разговор о књизи "Уметност и криминал" аутора др Ренате Самарџић (Нови Сад : Платонеум, 2017

Краду накит и иконе

Разговор о књизи "Уметност и криминал" аутора др Ренате Самарџић (Нови Сад : Платонеум, 2017

Glycosylation and pH stability of penicillin G acylase from providencia rettgeri produced in Pichia pastoris

Penicilin G acilaza (PAC) je jedan od najšire korišćenih enzima u industrijskoj sintezi polusintetskih antibiotika. U ovom radu dobijeni nivo ekspresije PAC gena iz Providencia rettgeri u ekspresionom sistemu Pichia pastoris iznosio je 2.7 U/ml. Rekombinantni enzim je prečišćen i određen je njegov glikozilacioni status. Nađeno je da osim što su obe subjedinice enzima (α i β) N-glikozilovane, β subjedinica sadrži još i O-glikane. Takođe je ustanovljeno da je rekombinantna PACP. rett. stabilna u širokom pH opsegu što ju je, zajedno sa predhodno ustanovljenom visokom termostabilnošću, učinilo izuzetno privlačnim biokatalizatorom sa industrijske tačke gledišta.Penicillin G acylase (PAC) is one of the most widely used enzymes in industrial synthesis of semi-synthetic antibiotics. The Providencia rettgeri pac gene was expressed to a level of 2.7 U/ml using the Pichia pastoris expression system. The recombinant enzyme was purified and its glycosylation status was determined. It was found that both subunits (α and β) of the enzyme were N-glycosylated, while the β-subunit also contained O-glycans. It was also observed that rPACP.rett. was stable in a wide range of pH, which, in addition to the previously proved high thermostability, makes it an attractive biocatalyst from an industrial point of view

Analysis of secondary structure within sgm and kgmB mRNA

Sgm methyltransferase from Micromonospora zionensis and KgmB methyltransferase from Streptoalloteichus tenebrarius are resistant to aminoglycoside antibiotics as a result of their ability to specifically methylate G1405 within the bacterial 16S rRNA A-site. The (C)CGCCC motif, assumed to be a regulatory sequence responsible for the autoregulation of the sgm gene, could most likely also be responsible for the autoregulation of the kgmB gene. This sequence, found within the 5' untranslated region of both sgm and kgmB mRNAs, as indicated by in silico prediction, may be involved in the formation of a specific stem-loop structure. Sgm and KgmB are mutually down-regulated and it is likely that they share the same cis-acting elements. Structure probing experiments confirmed the existence of a stable secondary structure within the 5' UTR of the sgm mRNA, while the analysis of kgmB mRNA failed to confirm the predicted structure.

Analysis of secondary structure within sgm and kgmB mRNA

Sgm metiltransferaza iz soja Micromonospora zionensis i KgmB metiltransferaza iz soja Streptoalloteichus tenebrarius ostvaruju rezistenciju na aminoglikozidne antibiotike metilacijom nukleotida na poziciji G1405 u okviru A mesta na 16S rRNK. Smatra se da je za autoregulaciju sgm gena odgovoran (C)CCGCCC motiv. Najverovatnije je ista sekvenca odgovorna i za autoregulaciju kgmB gena. Po kompjuterskoj predikciji, ovaj motiv, lociran u 5' netranslatirajućem regionu iRNK molekula oba gena, bi mogao učestvovati u formiranju sekundarne strukture tipa ukosnice. Kako Sgm i KgmB metiltransferaze jedna drugu autoregulišu, moguće je da prepoznaju iste cis elemente u iRNK molekulima. Eksperimenti ispitivanja strukture su, s jedne strane potvrdili prisustvo stabilne sekundarne strukture u okviru 5' netranslatirajućeg regiona iRNK molekula sgm gena, a sa druge, nisu dokazali postojanje modelovane sekundarne strukture u iRNK molekulu kgmB gena.Sgm methyltransferase from Micromonospora zionensis and KgmB methyltransferase from Streptoalloteichus tenebrarius are resistant to aminoglycoside antibiotics as a result of their ability to specifically methylate G1405 within the bacterial 16S rRNA A-site. The (C)CGCCC motif, assumed to be a regulatory sequence responsible for the autoregulation of the sgm gene, could most likely also be responsible for the autoregulation of the kgmB gene. This sequence, found within the 5' untranslated region of both sgm and kgmB mRNAs, as indicated by in silico prediction, may be involved in the formation of a specific stem-loop structure. Sgm and KgmB are mutually down-regulated and it is likely that they share the same cis-acting elements. Structure probing experiments confirmed the existence of a stable secondary structure within the 5' UTR of the sgm mRNA, while the analysis of kgmB mRNA failed to confirm the predicted structure