Istraživanje preferencije vezanja proteina HB6, RDM1 i DMS3 na BPM1

master's thesis

• Title (english): Preferential protein-protein binding of HB6, RDM1 and DMS3 to BPM1
• Author: Mirta Tokić
• Mentor: Nataša Bauer (mentor)
• Committee member: Nataša Bauer (predsjednik povjerenstva)
• Committee member: Željka Vidaković-Cifrek (član povjerenstva)
• Committee member: Martina Šeruga Musić (član povjerenstva)
• Committee member: Dunja Leljak-Levanić (član povjerenstva)
• Granter: University of Zagreb; Faculty of Science; (Department of Biology); Zagreb
• Defense date and country: 2019-02-15, Croatia
• Scientific / art field, discipline and subdiscipline: NATURAL SCIENCES; Biology

Abstract

BPM proteini vrste A. thaliana usmjeravaju nekoliko transkripcijskih faktora u 26S
proteasomsku razgradnju ovisnu o ubikvitinu. U preliminarnom istraživanju, nakon
kopurifikacije proteina TAP-tag metodom iz klijanaca uročnjaka, pokazana je interakcija
između proteina BPM1 i DMS3, odnosno RDM1, članova puta DNA metilacije usmjerene
RNA molekulom (RdDM). U ovom diplomskom radu, interakcije između BPM1,
transkripcijskog faktora HB6 te DMS3, odnosno RDM1 proteina, analizirane su metodom
kopurifikacije (pull-down), jedan na jedan i jedan na dva. U tu su svrhu prikladni ekspresijski
plazmidi konstruirani pomoću ligacije ili In-Fusion metode. Plazmidi su kemijski
transformirani u E. coli soj RosettaTM. Nakon indukcije ekspresije gena, fuzijski su proteini
pročišćeni afinitetnom kromatografijom i vizualizirani SDS-PAGE metodom, bojanjem
proteina u gelu i imunodetekcijom. U ovom diplomskom radu, potvrđena je interakcija
između RDM1 i BPM1, te DMS3 i BPM1. Interakcija između HB6 i BPM1-GST nije
potvrđena, budući da se protein HB6 vezao i na GST (negativna kontrola). Ovi eksperimenti
predstavljaju osnovu za daljnja istraživanja koja bi mogla razriješiti potencijalnu ulogu
proteina BPM1 u putu RdDM.

Abstract (english)

BPM proteins in A. thaliana are known to direct several transcription factors to ubiquitinmediated
26S proteasome degradation. Preliminary investigation, after protein copurification
by TAP-tag from Arabidopsis seedlings, has shown that BPM1 also potentially interacts with
members of the RNA-directed DNA methylation (RdDM) pathway, DMS3 and RDM1. Here,
interactions of BPM1 with transcription factor HB6 and proteins DMS3 and RDM1 were
analyzed in one to one and one to two pull-down assays. For this purpose, suitable expression
plasmids were generated using ligation or In-Fusion cloning methods. RosettaTM E. coli strain
was chemically transformed with generated constructs. Protein expression was induced, and
fusion proteins were purified. Finally, pull-down assays were performed and proteins
visualized by SDS-PAGE, staining and immunodetection. In the conditions carried out here,
RDM1 and DMS3 interacted with BPM1. Since HB6 interacted with both BPM1-GST and
GST alone (negative control), a direct HB6 interaction could not be shown. These
experiments provide a basis for future analyses which could elucidate the potential function of
BPM1 in the RdDM pathway.

• Keywords: interakcija proteina; kopurifikacija; RdDM; degradacija proteina; BPM1; Arabidopsis thaliana
• Keywords (english): protein interaction; pull-down; RdDM; protein degradation; BPM1; Arabidopsis thaliana
• Language: croatian
• URN:NBN: urn:nbn:hr:217:554796
• 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; Embargo expiration date: 2020-02-15
• Created on: 2019-03-26 09:43:49