Introduction of deletion in gene Scw4 using 'megaprimer' PCR method

Proteini stanične stijenke kvasca Sacharomyces cerevisiae se u staničnu stijenku mogu vezati putem dva mehanizma: kovalentno i nekovalentno. Nekovalentno vezani proteini se iz stanične stijenke izoliraju tretmanom sa vrućim SDS-om, a kovalentno vezani se mogu izolirati pomoću enzima glukanaza (tzv. GPI-proteini) ili ekstrakcijom pomoću 30 mM NaOH (tzv. Pir-proteini). Protein Scw4p je specifičan po tome što se u staničnu stijenku veže putem oba mehanizma tj. kovalentno i nekovalentno. Kovalentno vezani Scw4p izolira se iz stijenki prekonoćnom inkubacijom u 30 mM NaOH na 40C kao i proteini Pir porodice. Pir proteini su vezani na β-1,3-glukan esterskom vezom između hidroksilne grupe glukoze i γ-karboksilne grupe određenog ostatka glutaminske kiseline smještenog unutar ponavljajuće sekvence specifične za ovu porodicu proteina. Budući da unutar gena SCW4 takva sekvenca nije prisutna, mehanizam vezanja Scw4p u stijenku je nepoznat. Unutar gena SCW4 je međutim pronađena sekvenca slična PIR ponavljajućem motivu za koju se stoga pretpostavlja da bi mogla biti zaslužna za kovalentno vezanje Scw4p u staničnu stijenku kvasca. Kako bi se ta pretpostavka provjerila u ovom radu je koristeći PCR metodu „megapočetnice“ ta sekvenca deletirana iz gena SCW4. Yeast cell wall proteins can be linked to the Saccharomyces cerevisiae cell wall by two different mechanisms: covalently and non-covalently. Non-covalently linked proteins can be isolated from cell wall by treatment with hot SDS, and covalently linked proteins can be isolated with glucanase (GPI-proteins) or with 30mM NaOH (Pir-proteins). Protein Scw4p is specific because it can be linked to the cell wall both covalently and non-covalently. Covalently linked Scw4p is isolated by overnight extraction in 30mM NaOH on +40C like proteins of Pir family. Pir proteins are linked to β-1,3-glukan by ester bound between hydroxyl group of glucose and γ-carbonyl group of specific glutamic acid residue, placed in repetitive sequence specific for proteins of this family. Since that sequence is not present in SCW4 gene the mechanism of Scw4p covalent linkage to the cell wall is unknown. However, in the SCW4 gene sequence quite similar to PIR repetitive motive was found, that might be involved in covalently bounding of Scw4p in yeast cell wall. In order to check this assumption deletion of that sequence from the SCW4 gene was performed using „megaprimer“ PCR method.