Universität zu Köln

Hu, Zhoubo, Ghosh, Ajit ORCID: 0000-0001-9045-3916, Stolze, Sara C., Horvath, Mihaly, Bai, Bing, Schaefer, Sabine, Zuendorf, Simone, Liu, Shanda, Harzen, Anne ORCID: 0000-0001-7370-4939, Hajheidari, Mohsen, Sarnowski, Tomasz J., Nakagami, Hirofumi ORCID: 0000-0003-2569-7062, Koncz, Zsuzsa and Koncz, Csaba (2019). Gene modification by fast-track recombineering for cellular localization and isolation of components of plant protein complexes. Plant J., 100 (2). S. 411 - 430. HOBOKEN: WILEY. ISSN 1365-313X

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Abstract

To accelerate the isolation of plant protein complexes and study cellular localization and interaction of their components, an improved recombineering protocol is described for simple and fast site-directed modification of plant genes in bacterial artificial chromosomes (BACs). Coding sequences of fluorescent and affinity tags were inserted into genes and transferred together with flanking genomic sequences of desired size by recombination into Agrobacterium plant transformation vectors using three steps of E. coli transformation with PCR-amplified DNA fragments. Application of fast-track recombineering is illustrated by the simultaneous labelling of CYCLIN-DEPENDENT KINASE D (CDKD) and CYCLIN H (CYCH) subunits of kinase module of TFIIH general transcription factor and the CDKD-activating CDKF;1 kinase with green fluorescent protein (GFP) and mCherry (green and red fluorescent protein) tags, and a PIPL (His(18)-StrepII-HA) epitope. Functionality of modified CDKF;1 gene constructs is verified by complementation of corresponding T-DNA insertion mutation. Interaction of CYCH with all three known CDKD homologues is confirmed by their co-localization and co-immunoprecipitation. Affinity purification and mass spectrometry analyses of CDKD;2, CYCH, and DNA-replication-coupled HISTONE H3.1 validate their association with conserved TFIIH subunits and components of CHROMATIN ASSEMBLY FACTOR 1, respectively. The results document that simple modification of plant gene products with suitable tags by fast-track recombineering is well suited to promote a wide range of protein interaction and proteomics studies.

Item Type:	Journal Article
Creators:	Creators Email ORCID ORCID Put Code Hu, Zhoubo UNSPECIFIED UNSPECIFIED UNSPECIFIED Ghosh, Ajit UNSPECIFIED orcid.org/0000-0001-9045-3916 UNSPECIFIED Stolze, Sara C. UNSPECIFIED UNSPECIFIED UNSPECIFIED Horvath, Mihaly UNSPECIFIED UNSPECIFIED UNSPECIFIED Bai, Bing UNSPECIFIED UNSPECIFIED UNSPECIFIED Schaefer, Sabine UNSPECIFIED UNSPECIFIED UNSPECIFIED Zuendorf, Simone UNSPECIFIED UNSPECIFIED UNSPECIFIED Liu, Shanda UNSPECIFIED UNSPECIFIED UNSPECIFIED Harzen, Anne UNSPECIFIED orcid.org/0000-0001-7370-4939 UNSPECIFIED Hajheidari, Mohsen UNSPECIFIED UNSPECIFIED UNSPECIFIED Sarnowski, Tomasz J. UNSPECIFIED UNSPECIFIED UNSPECIFIED Nakagami, Hirofumi UNSPECIFIED orcid.org/0000-0003-2569-7062 UNSPECIFIED Koncz, Zsuzsa UNSPECIFIED UNSPECIFIED UNSPECIFIED Koncz, Csaba UNSPECIFIED UNSPECIFIED UNSPECIFIED
URN:	urn:nbn:de:hbz:38-132392
DOI:	10.1111/tpj.14450
Journal or Publication Title:	Plant J.
Volume:	100
Number:	2
Page Range:	S. 411 - 430
Date:	2019
Publisher:	WILEY
Place of Publication:	HOBOKEN
ISSN:	1365-313X
Language:	English
Faculty:	Unspecified
Divisions:	Unspecified
Subjects:	no entry
Uncontrolled Keywords:	Keywords Language KINASE-ACTIVATING KINASES; C-TERMINAL DOMAIN; NUCLEOTIDE EXCISION-REPAIR; T-LOOP; ESCHERICHIA-COLI; EMERGING ROLES; TAGGING SYSTEM; HISTONE H3.1; IN-VIVO; DNA Multiple languages Plant Sciences Multiple languages
Refereed:	Yes
URI:	http://kups.ub.uni-koeln.de/id/eprint/13239