Identification, cloning and expression of sponge Eunapius subterraneus myc gene

Proučavanje gena povezanih s nastankom raka na nivou najjednostavnijih višestaničnih životinja (spužve) može dati bolji uvid u njihovu biokemijsku i biološku funkciju te pomoći u razjašnjavanju složenijih interakcija njihovih homologa kod ljudi i objasniti moguće razloge njihovog onkogenog potencijala. Od posebnog interesa su protoonkogeni koji su uključeni u regulaciju staničnog rasta i diferencijacije. U ovom je radu identificiran i analiziran protoonkogen myc iz spužve Eunapius subterraneus, koja je jedini poznati slatkovodni predstavnik spužvi (porodica Spongillidae) među stigobiontima na svijetu. Usporedbom spužvinog proteina Myc s homolozima iz drugih organizama uočeno je da su gotovo sve domene, osim MB IV, sačuvane kod svih uspoređenih organizama. Utvrđeno je postojanje jednog introna u sredini gena myc dužine 60 nukleotida. Usporedbom gena myc iz spužve s homolozima iz drugih analiziranih organizama zaključili smo da su pozicija i faza tog introna sačuvane od čovjeka do spužve. cDNA koja kodira za protein Myc je uspješno uklonirana u ekspresijski vektor pET28b te je napravljena prekomjerna ekspresija koja je i potvrđena western analizom. U drugom dijelu diplomskog rada cilj je bio osmisliti 90-minutni nastavni sat u kojem će se obraditi tema elektroforeze s materijalima koji su svima dostupni i jeftini. Pregledom nastavnih programa za osnovnu i srednju školu iz kemije, fizike i biologije uočeno je da nema pokusa koji se bave elektroforezom, a učenici stječu potrebna znanja koja mogu primijeniti i utvrditi tumačeći elektroforetski pokus.Studying genes, associated with the emergence of tumors at the level of simpler organisms like Sponges, provides a better insight into their biochemical and biological function. This approach to cancer research will help clarify the complex interactions of their homologues with humans and thus explain possible reasons for their oncogenic potential. Of particular interest are the protooncogenes involved in regulation of cell growth and differentiation. In this Diploma Thesis protooncogene myc from Eunapius subterraneus (Spongillidae), which is the only freshwater stygobitic sponge, has been indentified and analyzed. Comparison of sponge protein Myc with homologs from other organisms showed that almost all domains, except MB IV, were preserved in all comparing organisms. The existence of an intron in the middle of the myc gene (60 nucleotides) was established. By comparing the myc gene from the sponge with the homologs from other analyzed organisms, we concluded that the intron's position and phase were preserved from the human to the sponge. The cDNA coding for Myc protein was successfully cloned in the expression vector pET28b and excessive expression was made, which was confirmed by western analysis. The second part of this diploma thesis deals with electrophoresis and its potential implications in chemistry teaching. Examining the curriculum for elementary and high school chemistry, physics and biology indicates that they lack electrophretic or similar experiments. Furthermore, students should easily understand this technique because they have acquired educational achievements needed to understand and explain electrophoretic experiment.A 90-minute lesson was devised that uses simple and cheap electrophoretic experiment available to everyone