Supplementary Material and Method 1
Binary vector construction
The N-terminal part of A. thaliana cenH3 sequence (N-cenH3) was amplified from
the p35S-BAM/cenH3-EYFP intermediate vector (Lermontova et al. 2006) with
primer pair CENPA-SpeI-F and NCENPA-XmaI-R (Supplementary Table 1), and cut
with SpeI and SmaI restriction enzymes. To generate the construct with N-cenH3
sequence fused with EYFP, the full-length cenH3 sequence of p35S-BAM/cenH3EYFP (Lermontova et al. 2006) was replaced with N-cenH3 sequence. First, the
p35S-BAM/cenH3-EYFP plasmid DNA was linearized with SpeI and SmaI restriction
enzymes to release the full-length cenH3 sequence from this plasmid. The
SpeI/SmaI-cut N-cenH3 fragment was then cloned into the linearized p35SBAM/cenH3-EYFP. This construct was designated as p35S-BAM/N-cenH3-EYFP
(Supplementary Fig. 1a).
For the construction of the pCLacI or pNCLacI intermediate vectors
(Supplementary Fig. 1b, c), the GFP-LacI-NLS sequence was amplified from A.
thaliana transgenic line EL702C (Kato and Lam 2001) using forward primer GFP5HindIII-F and reverse primer NLS-SalI-R with with a HindIII and a SaII site at the 5’
end of these primers, respectively (Supplementary Table 1) and the PCR amplified
fragments were cut with HindIII and SalI restriction enzymes. The EYFP sequence
was released from the p35S-BAM/cenH3-EYFP and pNCENH3-7 constructs using
HindIII and SalI restrictions enzymes and replaced with HindIII/SalI-cut GFP-LacINLS fragments. Both intermediate constructs contained Pro35S promoter sequence
and NOSTer terminator sequence located upstream or downstream of the fusion
protein coding sequence.
To generate the pcenH3 and pN-cenH3 T-DNA binary vector constructs
(Supplementary Fig. 1d, e), the Pro35S:cenH3- or N-cenH3-GFP-LacI-NLS:NOSter
sequences were amplified from intermediate vectors (pCLacI or pNCLacI) using
primer pair p35S-XhoI-F and tNOS-SmaI-R and inserted in the SmaI site of the
pCMBarL binary vector (kindly provided by Andreas Finke). The pCMBarL vector
sequence consists of a pCAMBIA1300 backbone sequence, an expression cassette
for the phosphinotricine resistance marker including the ProMAS promoter and
35STer terminator sequence, and a LacZ coding sequence plus multiple cloning
sites for the screening for insertions.
For the construction of the pC-CENPC-N-cenH3 and pMIS12-N-cenH3 binary
vector constructs (Supplementary Fig. 1f, g), the C-terminal part of CENP-C and fulllength MIS12 of A. thaliana accession Columbia (Col-0), was amplified from cDNA
obtained by reverse transcription of RNA isolated from flower buds with the primer
pairs AtCENPC-SpeI-F and AtCENPC-SpeI-R or AtMIS12-SpeI-F and AtMIS12SpeI-R, respectively (Supplementary Table 1). The PCR fragments were cut with
SpeI restriction enzyme prior to clone into upstream of the N-cenH3 sequence in the
unique SpeI site of the pN-cenH3 T-DNA vector.