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Supplemental Methods and Figure Legends
Supplemental methods. Plasmids for expressing P. angusta H3 and H4 in S. cerevisiae: The S.
cerevisiae HHT2 and HHF2 genes (respectively, chr. XIV coordinates 575,265-576,092 and
576,046-577,238) were amplified by PCR and cloned separately into pGEM-T (Promega). An
XhoI site was incorporated into the downstream primer for HHT2. The resulting plasmids
(pRB633, HHT2; pRB591, HHF2) were used as templates for site-directed mutagenesis using
the QuikChange Multi system (Stratagene). P. angusta H3 and H4 each differ from the
respective S. cerevisiae proteins at three amino acid positions. The H3 replacements are S32A,
A111C, and K126Q. H4 replacements are G49A, S65N, and S70A. Mutagenic primers were
designed using Stratagene’s online QuikChange Primer Design Program, and mutagenesis was
carried out following the manufacturer’s instructions. HHT2 mutagenesis was accomplished in a
single step; HHF2 mutagenesis required two steps due to overlap in S65N and S70A primer
annealing sites. The coding regions of all resulting plasmids were sequenced to verify the
presence of intended mutations and the absence of mutations at other sites. We refer to the “P.
angusta-ized” HHT2 as HHT2Pang and the P. angusta-ized HHF2 as HHF2Pang. A complete S.
cerevisiae HHT2-HHF2 locus was regenerated in plasmid pRB642 by stepwise ligation of the
1145-bp BamHI-SpeI fragment containing HHF2 and the 800-bp BamHI/XhoI fragment
containing HHT2 into the URA3-CEN vector pRS316 (SIKORSKI and HIETER 1989). An
analogous CEN-LEU2 plasmid, pRB648, was constructed by excising the ApaI-XbaI fragment of
pRB642 containing the HHT2-HHF2 locus and ligating it into ApaI-XbaI-cut pRS315 (SIKORSKI
and HIETER 1989). Plasmids pRB652, pRB653, and pRB654 were obtained by exchanging,
respectively, the HHF2, HHT2, and HHT2-HHF2 segments of pRB648 with their P. angusta
counterparts. Plasmid pRB666 contains the P. angusta HHT2Pang-HHF2Pang insert from pRB654
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transferred to the URA3 integrating vector pRS306 (SIKORSKI and HIETER 1989). The BstBI site
located in the URA3 coding region of pRB666 was destroyed by QuikChange site-directed
mutagenesis, making a synonymous codon change (TTC GAA changed to TTT GAA).
Strain construction: All strains are isogenic to S288C strains BY4741 and BY4742
(BRACHMANN et al. 1998). The strain used for testing H3-H4 function was R400-3B (MAT
his31 leu20 ura30 met150 hht1-hhf1::HIS3 hht2-hhf2::HIS3 [pRB642]). The
hht1-hhf1::HIS3 and hht2-hhf2::HIS3 alleles were obtained by one-step PCR mediated gene
deletion (LAFONTAINE and TOLLERVEY 1996) in parental strains 13144 (hhf1::kanMX4) and
5356 (hhf2::kanMX4) obtained through the systematic yeast gene deletion project (Open
Biosystems). The deletion strains were then crossed to obtain a diploid which was transformed
with pRB642 and sporulated to yield the meiotic segregant R400-3B.
Strain R411-11B (MAT his31 leu20 ura30 trp163 hht1-hhf1::HIS3
HHT2-HHF2Pang cse4::kanMX4 [pRB163]) was used to test P. angusta Cse4 function in the
presence of P. angusta H4. Plasmid pRB163 carries wild-type CSE4 on a CEN-URA3 backbone.
The HHT2- HHF2Pang allele was obtained by pop-in/pop-out gene replacement (ROTHSTEIN
1991) in strain 5356 (HHF2::kanMX4), screening for loss of kanamycin resistance after the
pop-out event. The integrative plasmid was BstBI-cut pRB666. During the integration event,
the HHT2Pang allele of pRB666 was converted to wild-type S. cerevisiae HHT2. The
hht1-hhf1::HIS3 and cse4::kanMX4 alleles were introduced by standard genetic crosses,
scoring HHF2Pang in segregants of the final cross by performing colony PCR of the HHF2 locus
and sequencing the products to identify the HHF2Pang allele.
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Legend to Supplemental Figure S1. Plasmid shuffle test for complementation of S. cerevisiae
H3 and H4 function by P. angusta orthologs. Plasmids expressing P. angusta H3, P. angusta
H4, or both P. angusta H3 and H4 were transformed into tester strain R400-3B and tested for
their ability to provide H3/H4 function, as observed by growth on FOA medium. Sc, S.
cerevisiae; Pa, P. angusta.
Supplemental References.
BRACHMANN, C. B., A. DAVIES, G. J. COST, E. CAPUTO, J. LI et al., 1998 Designer deletion
strains derived from Saccharomyces cerevisiae S288C: a useful set of strains and
plasmids for PCR-mediated gene disruption and other applications. Yeast 14: 115-132.
KISHINO, H., and M. HASEGAWA, 1989 Evaluation of the maximum likelihood estimate of the
evolutionary tree topologies from DNA sequence data, and the branching order in
hominoidea. J Mol Evol 29: 170-179.
LAFONTAINE, D., and D. TOLLERVEY, 1996 One-step PCR mediated strategy for the construction
of conditionally expressed and epitope tagged yeast proteins. Nucleic Acids Res 24:
3469-3471.
ROTHSTEIN, R., 1991 Targeting, disruption, replacement, and allele rescue: integrative DNA
transformation in yeast. Methods Enzymol 194: 281-301.
SIKORSKI, R. S., and P. HIETER, 1989 A system of shuttle vectors and yeast host strains designed
for efficient manipulation of DNA in Saccharomyces cerevisiae. Genetics 122: 19-27.
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