Expression of two pfk-l genes coding human 6-phosphofructo-1-kinases in Saccharomyces cerevisiae

Encim 6-fosfofrukto-1-kinaza je pomemben regulatorni encim glikolize. Obstajajo trije tipi encima in jetrni tip tega encima, Pfk-L, je bil v rakastih celicah zaznan v višjih koncentracijah. Značilnost encima Pfk-L je tudi postrtanslacijska modifikacija, pri kateri nastane tudi krajša, bolj aktivna oblika encima. V našem delu smo hoteli primerjati aktivnost nativne in skrajšane oblike encima v različnih pogojih. Tako smo gena za obe obliki encima vnesli v plazmid in nato plazmid vnesli v sev kvasovke, ki nima lastne 6-fosfofrukto-1-kinaze. Z gojenjem transformiranih sevov v osnovnem gojišču brez uracila smo potrdili prisotnost samega plazmida v celicah kvasovk. Nato smo seve gojili še na trdnih in tekočih gojiščih, ki so kot vir ogljika vsebovali glukozo, fruktozo ali maltozo. Transformirana seva sta dobr rasla le na trdnem gojišču z maltozo, v tekočem gojišču pa sta rasla le ob dodatku etanola. Vse to je nakazovalo na dejstvo, da vnesena gena ne omogočata nastanka aktivne oblike encima. Pri merjenju encimske aktivnosti pa smo to še potrdili, saj je bila njuna aktivnost komaj zaznavna. Glede na to, da tudi nativna oblika ni rasla na enostavnih sladkorjih, lahko sklepamo, da že izbrani gen verjetno ni bil optimalen za uporabo v takšnem poskusu. Uporabljena oblika gena je namreč nastala z alternativnim izrezovanjem in ni kanonična oblika gena.Enzyme 6-phosphofructo-1-kinase is an important regulatory enzyme of glycolysis. There are three types of this enzyme and the liver type, Pfk-L, was found in higher concentrations in human cancer cells. A known characteristic of Pfk-L is a posttranslational modification which results in a shorter, but a more active isoform of the enzyme. We inserted genes for both isoforms of the enzyme in a plasmid, and the plasmids were inserted in a Pfk-null yeast strain. We grew both strains in a medium without uracil, to confirm the presence of the plasmid in the cells. Then, we grew those two strains with other control strains on solid and in liquid media. These media had as a source of carbon different sugars: glucose, fructose or maltose. Transformed strains grew best on solid media with maltose and in liquid media with maltose and ethanol. This was probably due to fact that the inserted genes could not be translated and/or folded into an active form of the enzyme. Minimal activity was also confirmed with an experiment where we measured only PFK-L enzyme activity. Since even the native form of the enzyme could not enable growth on glucose or fructose, we concluded that the selected gene was not the best option, because it was not the canonical one