Recognition of <Emphasis Type="Italic">A. thaliana</Emphasis> centromeres by heterologous CENH3 requires high similarity to the endogenous protein |
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Authors: | Izabel C R Moraes Inna Lermontova Ingo Schubert |
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Institution: | (1) Leibniz Institute of Plant Genetics and Crop Plant Research (IPK), Corrensstrasse 3, 06466 Gatersleben, Germany |
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Abstract: | The centromere is an essential chromosomal component assembling the kinetochore for chromosome attachment to the spindle microtubules
and for 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 EYFP-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 heterologous 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: threonine2, arginine12 and
alanine15. 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. |
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