Ekspresijski profili gena rac1A, rac1B i rac1C u jednostrukim i dvostrukim rac1 mutantima amebe Dictyostelium discoideum

master's thesis

• Title (english): Expression profiles of rac1A, rac1B and rac1C genes in single and double rac1 mutants in Dictyostelium discoideum cells
• Author: Marija Šimić
• Mentor: Vedrana Filić Mileta (mentor)
• Mentor: Petra Korać (komentor)
• Committee member: Petra Korać (predsjednik povjerenstva)
• Committee member: Biljana Balen (član povjerenstva)
• Committee member: Inga Urlić (član povjerenstva)
• Committee member: Petra Peharec Štefanić (član povjerenstva)
• Granter: University of Zagreb; Faculty of Science; (Department of Biology); Zagreb
• Defense date and country: 2024-02-27, Croatia
• Scientific / art field, discipline and subdiscipline: NATURAL SCIENCES; Biology

Abstract

Proteini Rac1A, Rac1B i Rac1C iz amebe Dictyostelium discoideum pripadaju porodici GTPaza Rho i reguliraju aktinski citoskelet. Zbog velike homologije među njima i nepostojanja značajnih promjena u fenotipu jednostrukih mutanata, u literaturi se uvriježilo mišljenje da ovi proteini imaju preklapajuće funkcije. Međutim, karakterizacija mutanata pojedinačno deficijentnih za gene rac1 konstruiranih u Laboratoriju za staničnu dinamiku Instituta Ruđer Bošković u Zagrebu pokazala je da stanice rac1A- imaju defekte tijekom vegetativnog rasta. Nadalje, u odsutnosti proteina Rac1A opaženo je veliko statistički značajno povećanje ekspresije gena rac1B i rac1C. Kako bih ispitala dolazi li do povećanja ekspresije preostalog gena rac1 u dvostrukim mutantima deficijentnima za gen rac1A, u stanicama rac1A- uklonila sam gene rac1B i rac1C. Uklanjanje gena potvrdila sam metodom PCR i Southernovom metodom otiska. Potom sam metodom qPCR analizirala ekspresiju sva tri gena rac1 u stanicama divljeg tipa i mutanata. U stanicama rac1A-/rac1B- i rac1A-/rac1C- ekspresija preostalog gena rac1 ostala je značajno viša u odnosu na stanice divljeg tipa. Također opazila sam promjene fenotipa specifične za stanice rac1A-/rac1B-. Ovi rezultati pokazuju da povećana ekspresija gena rac1B i/ili rac1C ne može u potpunosti nadomijestiti funkciju proteina Rac1A, što ukazuje na to da proteini Rac1, osim preklapajućih, imaju i specifične uloge u stanici.

Abstract (english)

The D. discoideum proteins Rac1A, Rac1B and Rac1C are GTPases of the Rho family that are involved in the regulation of the actin cytoskeleton. Due to the high homology between them, it is assumed that they have overlapping functions. Furthermore, cells lacking the single Rac1 protein showed no significant phenotypic changes. However, analyzes of rac1A-, rac1B- and rac1C- mutants produced in the Laboratory of cell dynamics (RBI Zagreb) revealed that rac1A- cells are defective during vegetative growth. Moreover, rac1B and rac1C expression was significantly increased in the absence of Rac1A. To analyze the expression profile of double mutants lacking Rac1A, I generated rac1A-/rac1B- and rac1A-/rac1C- cells. After confirming the deletion of rac1B and rac1C genes in rac1A- cells by PCR and Southern blot, I analyzed the expression of the three rac1 genes by qPCR. Relative quantification of expression showed that in both rac1A-/rac1B- and rac1A-/rac1C- double mutants, the expression of the third rac1 gene remains strikingly elevated. In addition, preliminary observations indicate phenotypic changes specific to the rac1A-/rac1B- genotype. Therefore, overexpression of rac1B and/or rac1C cannot fully rescue the phenotype of mutants lacking Rac1A and Rac1 GTPases not only have overlapping functions but also perform specific roles in the cell.

• Keywords: Rac1; Rho; GTPaza; ekspresija gena; Dictyostelium
• Keywords (english): Rac1; Rho; GTPase; gene expression; Dictyostelium
• Language: croatian
• URN:NBN: urn:nbn:hr:217:146055
• Study programme: Title: Molecular Biology-University graduate programme; Study programme type: university; Study level: graduate; Academic / professional title: sveučilišni/a magistar/magistra molekularne biologije (sveučilišni/a magistar/magistra molekularne biologije)
• Type of resource: Text
• File origin: Born digital
• Access conditions: Embargoed access; Embargo expiration date: 2026-02-27
• Created on: 2024-03-13 10:57:18