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 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 (MATa his3D1 leu2D0 ura3D0 met15D0 hht1-hhf1D::HIS3 hht2-hhf2D::HIS3 [pRB642]). The hht1hhf1D::HIS3 and hht2-hhf2D::HIS3 alleles were obtained by one-step PCR mediated gene deletion (Lafontaine and Tollervey 1996) in parental strains 13144 (hhf1D::kanMX4) and 5356 (hhf2D::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 (MATa his3D1 leu2D0 ura3D0 trp1D63 hht1-hhf1D::HIS3 HHT2HHF2Pang cse4D::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 (HHF2D::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 hht1hhf1D::HIS3 and cse4D::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.

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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