Procjena zdravstvene ispravnosti poljoprivrednih kultura oplemenjenih genetičkim inženjerstvom - kako umanjiti nenamjerne učinke?

Scientists started applying genetic engineering techniques to improve crops two decades ago; about 70 varieties obtained via genetic engineering have been approved to date. Although genetic engineering offers the most precise and controllable genetic modification of crops in entire history of plant improvement, the site of insertion of a desirable gene cannot be predicted during the application of this technology. As a consequence, unintended effects might occur due to activation or silencing of genes, giving rise to allergic reactions or toxicity. Therefore, extensive chemical, biochemical and nutritional analyses are performed on each new genetically engineered variety. Since the unintended effects may be predictable on the basis of what is known about the insertion place of the transgenic DNA, an important aim of plant biotechnology is to define techniques for the insertion of transgene into the predetermined chromosomal position (gene targeting). Although gene targeting cannot be applied routinely in crop plants, given the recent advances, that goal may be reached in the near future.Genetičko inženjerstvo primjenjuje se u oplemenjivanju poljoprivrednih kultura u posljednjih dvadeset godina. Temelji se uglavnom na ugradnji jednog ili dvaju novih gena u genom biljaka. Do danas je odobreno za uzgoj oko 70 sorta oplemenjenih tom tehnologijom. U usporedbi sa svim tehnologijama koje se primjenjuju u oplemenjivanju bilja, genetičkim inženjerstvom postižu se najpreciznije promjene u genetičkome materijalu. Međutim, tijekom primjene te tehnologije ne može se predvidjeti mjesto u genomu u koje će se ugraditi željeni gen. Zbog toga može doći do nenamjerne aktivacije ili inaktivacije određenih gena, a to može uzrokovati nepoželjne promjene, primjerice u alergološkim ili toksikološkim značajkama. Zato se prije komercijalizacije provode iscrpne kemijske, biokemijske i nutricionističke analize svake nove sorte oplemenjene tom tehnologijom. Budući da se nenamjerni učinci mogu predvidjeti u određenoj mjeri na temelju spoznaja o mjestu u genomu u koje se željeni gen ugradio, jedan od najvažnijih ciljeva moderne biljne biotehnologije svakako je razvoj tehnika koje će omogućiti ugradnju željenih gena u unaprijed odabrano mjesto u genomu. Ta se metoda naziva "gene targeting". Za razliku od svih ostalih skupina organizama, "gene targeting" još nije metoda koja se može rutinski primijeniti u biljaka. Međutim, uzimajući u obzir nedavna postignuća na tom području, taj će se cilj vjerojatno ostvariti u bliskoj budućnosti

Controversy Associated With the Common Component of Most Transgenic Plants – Kanamycin Resistance Marker Gene

Plant genetic engineering is a powerful tool for producing crops resistant to pests, diseases and abiotic stress or crops with improved nutritional value or better quality products. Currently over 70 genetically modified (GM) crops have been approved for use in different countries. These cover a wide range of plant species with significant number of different modified traits. However, beside the technology used for their improvement, the common component of most GM crops is the neomycin phosphotransferase II gene (nptII), which confers resistance to the antibiotics kanamycin and neomycin. The nptII gene is present in GM crops as a marker gene to select transformed plant cells during the first steps of the transformation process. The use of antibiotic-resistance genes is subject to controversy and intense debate, because of the likelihood that clinical therapy could be compromised due to inactivation of the oral dose of the antibiotic from consumption of food derived from the transgenic plant, and because of the risk of gene transfer from plants to gut and soil microorganisms or to consumer’s cells. The present article discusses these possibilities in the light of current scientific knowledge

Usporedba intraplazmidne rekombinacije u bakterijama Agrobacterium tumefaciens i Escherichia coli

In this work we have constructed a plasmid to compare intraplasmid recombination efficiency in Agrobacterium tumefaciens and Escherichia coli. The plasmid contains two directly repeated copies of spectinomycin resistance gene, one lacking 5’ and the other lacking 3’ end. These two copies share a 570-bp region of homology and are separated by the ampicillin resistance gene. Homologous recombination between repeated copies of incomplete spectinomycin resistance genes results in the restoration of spectinomycin resistance. During this process, ampicillin resistance gene is either deleted or incomplete spectinomycin genes are amplified along with the ampicillin resistance gene. This experimental system enabled us to follow for the first time the generation of deletions and amplifications during intraplasmid recombination in A. tumefaciens. We show here that predominantly RecA-independent mechanism contributes to the formation of deletion and amplification products in both, A. tumefaciens and E. coli. Additionally, deletion and amplification products were detected at similar frequencies, suggesting that amplifications and deletions probably occur by a similar mechanism.U ovom smo radu konstruirali plazmid koji nam je omogućio da usporedimo intraplazmidnu rekombinaciju u bakterijama Agrobacterium tumefaciens i Escherichia coli. Plazmid sadržava dvije istosmjerno ponovljene kopije gena odgovornog za rezistenciju na spektinomicin, pri čemu jednoj kopiji nedostaje 5\u27, a drugoj 3\u27 kraj gena, a međusobno su homologne u duljini od 570 pb. Osim toga, DNA koja se nalazi između ove dvije istosmjerno ponovljene sekvencije sadržava gen koji daje rezistenciju na antibiotik ampicilin. Homolognom rekombinacijom između nepotpunih gena za rezistenciju na spektinomicin nastaje funkcionalni gen, odgovoran za pojavu rezistencije. Pritom može doći do delecije gena za rezistenciju na ampicilin ili njegovog umnožavanja, zajedno s nepotpunim genima za otpornost na spektinomicin. Ovaj eksperimentalni sustav omogućio nam je da po prvi put pratimo pojavu delecija i amplifikacija tijekom intraplazmidne rekombinacije u bakteriji A. tumefaciens. Pokazali smo da delecije i amplifikacije u bakterijama Agrobacterium tumefaciens i Escherichia coli nastaju prvenstveno RecA-neovisnim mehanizmom. Osim toga, ustanovili smo da se delecije i amplifikacije pojavljuju s podjednakom učestalošću, što upućuje na to da je mehanizam oba rekombinacijska događaja sličan

Comparison of Intraplasmid Rearrangements in Agrobacterium tumefaciens and Escherichia coli

In this work we have constructed a plasmid to compare intraplasmid recombination efficiency in Agrobacterium tumefaciens and Escherichia coli. The plasmid contains two directly repeated copies of spectinomycin resistance gene, one lacking 5’ and the other lacking 3’ end. These two copies share a 570-bp region of homology and are separated by the ampicillin resistance gene. Homologous recombination between repeated copies of incomplete spectinomycin resistance genes results in the restoration of spectinomycin resistance. During this process, ampicillin resistance gene is either deleted or incomplete spectinomycin genes are amplified along with the ampicillin resistance gene. This experimental system enabled us to follow for the first time the generation of deletions and amplifications during intraplasmid recombination in A. tumefaciens. We show here that predominantly RecA-independent mechanism contributes to the formation of deletion and amplification products in both, A. tumefaciens and E. coli. Additionally, deletion and amplification products were detected at similar frequencies, suggesting that amplifications and deletions probably occur by a similar mechanism