Please use this identifier to cite or link to this item: http://dx.doi.org/10.25673/486
Title: Structural requirements for CENH3 targeting to centromeric chromatin
Author(s): Moraes, Izabel C. R.
Referee(s): Reuter, Gunter, Prof. Dr.
Diekmann, Stephan, Prof. Dr.
Schubert, Ingo, Prof. Dr.
Granting Institution: Martin-Luther-Universität Halle-Wittenberg
Issue Date: 2011
Extent: Online-Ressource (85 Bl. = 1,47 mb)
Type: Hochschulschrift
Type: PhDThesis
Exam Date: 2011-05-24
Language: English
Publisher: Universitäts- und Landesbibliothek Sachsen-Anhalt
URN: urn:nbn:de:gbv:3:4-5475
Subjects: Online-Publikation
Hochschulschrift
Abstract: Eukaryotic chromosomes need centromeres to ensure their faithful transmission to daughter nuclei during nuclear division. The centromere is the site where the kinetochore assembles for chromosome attachment to the spindle microtubules, directing the chromosome segregation during nuclear division. Kinetochore assembly requires deposition of the centromeric histone H3 variant (CENH3) into centromeric nucleosomes. CENH3 has a variable N-terminal and a more conserved C-terminal part, including the loop1 region of the histone fold domain, which is considered to be critical for centromere targeting. To investigate the structural requirements for centromere targeting, constructs for tagged CENH3 of A. lyrata, A. arenosa, Capsella bursa-pastoris, Zea mays and Luzula nivea (the latter with holocentric chromosomes) were transformed into A. thaliana. Except for LnCENH3, all recombinant CENH3 proteins targeted A. thaliana centromeres, but the more distantly related the alien protein is, the lower is the efficiency of targeting. Alignment of CENH3 sequences revealed that the tested species share only three amino acids at loop1 region: threonine (T/2), arginine (R/12) and alanine (A/15). These three amino acids were substituted by asparagine, proline and valine encoding sequences within a recombinant EYFP-AtCENH3 construct via PCR mutagenesis prior to transformation of A. thaliana. After transformation, immunostaining of root tip nuclei with anti-GFP antibodies yielded only diffuse signals, indicating that the original three amino acids are necessary but not sufficient for targeting A. thaliana centromeres.
URI: https://opendata.uni-halle.de//handle/1981185920/7125
http://dx.doi.org/10.25673/486
Open Access: Open access publication
License: In CopyrightIn Copyright
Appears in Collections:Biochemie

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