Učinak promijenjene ekspresije gena BPM na de novo metilaciju DNA uročnjaka (Arabidopsis thaliana L.)

master's thesis

• Title (english): Effect of altered BPM gene expression on de novo DNA methylation in Arabidopsis thaliana L.
• Author: Petar Mohorović
• Mentor: Nataša Bauer (mentor)
• Committee member: Nataša Bauer (predsjednik povjerenstva)
• Committee member: Željka Vidaković-Cifrek (član povjerenstva)
• Committee member: Vesna Benković (član povjerenstva)
• Committee member: Dunja Leljak-Levanić (član povjerenstva)
• Granter: University of Zagreb; Faculty of Science; (Department of Biology); Zagreb
• Defense date and country: 2017-01-30, Croatia
• Scientific / art field, discipline and subdiscipline: NATURAL SCIENCES; Biology

Abstract

Proteini BPM, kojima je dosada opisana uloga u degradaciji proteina putem ubikvitin proteasoma, ulaze u interakciju s proteinima koji su uključeni u de novo metilaciju DNA uročnjaka Arabidopsis thaliana. Nije poznato utječu li proteini BPM na metiliranost molekule DNA, a kako bi se provjerila potencijalna uloga proteina BPM u metilaciji DNA, koristile su se linije biljaka u kojima je promijenjena ekspresija gena BPM. Analizirala se metiliranost lokusa molekule DNA koji bi mogli biti pod utjecajem de novo metilacije. Analiza metiliranosti DNA provela se enzimatskom digestijom restrikcijskim enzimima koji su ovisni ili osjetljivi na metilaciju DNA, nakon koje je uslijedilo umnažanje istraživanih lokusa lančanom reakcijom polimerazom. Nakon gel elektroforeze i denzitometrijske obrade, te usporedbe rezultata dobivenih za nepocijepanu DNA i DNA pocijepanu metilacijski ovisnim enzimima MspJI i metilacijski osjetljivim enzimima HaeIII ili AluI, utvrdile su se promjene u metiliranosti u biljaka divljeg tipa kao i u biljaka koje različito eksprimiraju gene BPM. Rezultati pokazuju da promijenjene ekspresije gena BPM dovode do promjene u stupnju metiliranosti ponavljajućih sekvenci molekule DNA dok rezultati istraživanja promotorskih regija određenih gena ne daju jasnu sliku promjene obrasca metiliranosti te su potrebna dodatna istraživanja kako bi se to utvrdilo.

Abstract (english)

BPM proteins, which have a known role in protein degradation via the ubiquitin proteasome pathway, interact with proteins involved in de novo DNA methylation in Arabidopsis thaliana. It is still unknown how BPMs effect DNA methylation. To assess the role of BPMs in DNA methylation, different plant mutants with altered expression of BPM genes were used. DNA methylation analysis was performed using the methylation dependent and methylation sensitive restriction enzymes after which the polymerase chain reaction was used to amplify specific, targeted regions of DNA which may be under the influence of de novo methylation. After the gel electrophoresis and densitometric analysis of the amplified undigested DNA and DNA digested with methylation dependant MspJI enzyme and with methylation sensitive HaeIII or AluI enzymes, changes of DNA methylation in wild type, as well in plants with altered BPM gene expression,were determined. Results show that there is a change in methylation pattern in repeating DNA sequences while the results of examined promotor regions of specific genes don't give clear image on how the methylation pattern changes so further research is needed.

• Keywords: BPM; ubikvitin-proteasomni put; de novo metilacija DNA; RdDM; metilacijski ovisni restrikcijski enzimi; metilacijski osjetljivi restrikcijski enzimi; MspJI; Arabidopsis thaliana
• Keywords (english): BPM; ubiquitin proteasome pathway; de novo DNA methylation; RdDM; methylation dependant restriction enzymes; methylation sensitive restriction enzymes; MspJI; Arabidopsis thaliana
• Language: croatian
• URN:NBN: urn:nbn:hr:217:216748
• Study programme: Title: Molecular Biology; Study programme type: university; Study level: graduate; Academic / professional title: magistar/magistra molekularne biologije (magistar/magistra molekularne biologije)
• Type of resource: Text
• File origin: Born digital
• Access conditions: Open access
• Created on: 2017-05-22 09:37:30