CELL COUNTING DATA

Cell Counting Data

Cell Counting Data (Saccharomyces cerevisiae)

Yeast strains and YACs

Yeast strains used in this study were all of SK1 background and are presented in Table 1. The zip1::LYS2 deletion/disruption mutation was originally constructed by Mary Sym (Sym and Roeder 1994). This mutation consists of an insertion of an EcoRI-XbaI fragment containing the LYS2 promoter and ORF into the ORF of ZIP1, deleting most of the latter. The LYS2 ORF is inserted in the opposite direction to ZIP1. After insertion of LYS2, a residual ORF consisting of 40 ZIP1 amino acids fused to 14 amino acids from the inserted fragment remain (the sequence of the junction of the insertion mutation with the ZIP1 ORF is shown in Supplementary Figure S1). To construct a full deletion mutant of ZIP1, we amplified by PCR the region surrounding the ZIP1 ORF in the zip1? mutant strain from the EUROSCARF deletion library (http://web.uni-frankfurt.de/fb15/mikro/euroscarf/). This PCR fragment was then inserted by transformation into strain RD6-X to create a heterozygous zip1? SK1 strain.

Table 1 Strains used in this study

Strain

Genotype

NS1a

MATa/MATa, ura3/ura3, lys2/lys2, trp1/trp1, his3/his3, ade2/ade2, leu2/leu2, can1/can1, YAC (18ED5)::URA3,ADE2, YAC(18ED5)::LEU2,TRP1

NS2

MATa/MATa, ura3/ura3, lys2/lys2, trp1/trp1, his3/his3, ade2/ade2, leu2/leu2, can1/can1, YAC (yWXD4932)::URA3,ADE2, YAC(yWXD4932)::LEU2,TRP1

Mk21X65

MATa/MATa, ade2/ade2, ura3/ura3, his4/his4, leu2/leu2, trp1/trp1, lys2/lys2, can1/CAN1, zip1::LYS2/ZIP1, YAC (18ED5)::URA3,ADE2, YAC(18ED5)::LEU2,TRP1

Mk26X81

MATa/MATa, ade2/ade2, ura3/ura3, his4/his4, leu2/leu2, trp1/trp1, lys2/lys2, can1/CAN1, zip1::LYS2/ZIP1, YAC (yWXD4932)::URA3,ADE2, YAC(yWXD4932)::LEU2,TRP1

Mk9X23

MATa/MATa, ade2/ade2, ura3/ura3, his4/his4, leu2/leu2, trp1/trp1, lys2/lys2, zip1::LYS2/zip1::LYS2, YAC (18ED5)::URA3,ADE2, YAC(18ED5)::LEU2,TRP1

Mk29X25

MATa/MATa, ade2/ade2, ura3/ura3, his4/his4, leu2/leu2, trp1/trp1, lys2/lys2, zip1::LYS2/zip1::LYS2, YAC (yWXD4932)::URA3,ADE2, YAC(yWXD4932)::LEU2,TRP1

Mk65AX61

MATa/MATa, ade2/ade2, ura3/ura3, leu2/leu2, trp1/trp1, lys2/lys2,HIS4/his4, CYH2/cyh2, YAC (18ED5)::URA3,ADE2, YAC(18ED5)::LEU2,TRP1

Mk81AX83

MATa/MATa, ade2/ade2, ura3/ura3, leu2/leu2, trp1/trp1, lys2/lys2,HIS4/his4, CYH2/cyh2, YAC (yWXD4932)::URA3,ADE2, YAC(yWXD4932)::LEU2,TRP1

Mk30X9

MATa/MATa, ade2/ade2, ura3/ura3, his4/his4, leu2/leu2, trp1/trp1, lys2/lys2, zip1::LYS2/zip1::LYS2, CAN1/can1, YAC (18ED5)::URA3,ADE2, YAC(18ED5)::LEU2,TRP1

Mk25X34

MATa/MATa, ade2/ade2, ura3/ura3, his4/his4, leu2/leu2, trp1/trp1, lys2/lys2, zip1::LYS2/zip1::LYS2, CAN1/can1, YAC (yWXD4932)::URA3,ADE2, YAC(yWXD4932)::LEU2,TRP1

RD6-X

MATa/MATa, ura3/ura3, LEU1/leu1::HIS4, lys2/lys2, his4/his4, TRP5/trp5, CYH2/cyh2, CAN1/can1, arg4/ARG4

RD6-XH

MATa/MATa, ura3/ura3, LEU1/leu1::HIS4, lys2/lys2, his4/his4, TRP5/trp5, ZIP1/zip1::LYS2, CAN1/can1, CYH2/cyh2, arg4/ARG4

RD6-Y

MATa/MATa, ura3/ura3, LEU1/leu1::HIS4, his4/his4, trp5/TRP5, lys2/lys2, CYH2/cyh2, CAN1/can1, arg4/ARG4, zip1::LYS2/zip1::LYS2

4022

MATa/MATa, ura3/ura3, leu2::hisG/leu2::hisG, trp1::hisG/trp1::hisG, lys2/lys2, ho::LYS2/ho::lys2, ZIP1/zip1::LYS2, his4X::LEU2::URA3/HIS4

aYAC end markers are listed, telomeric marker first

To mark chromosomes VII for the study of non-disjunction, the LEU1 locus near the centromere of a haploid his4 strain was disrupted using HIS4 (Arbel 1992). This haploid strain was then mated to a his4 strain with unperturbed LEU1.

YACs yWXD4932 (100 Kb) and 18ED5 (190 Kb) contain DNA from the pseudoautosomal region of the human X chromosome. Their construction is presented elsewhere (Ried et al. 1995), following another study (Burke et al. 1987). YACs were transferred into haploid strains of SK1 genetic background by Kar1- matings, using the standard set of intermediate strains, as described (Hugerat et al. 1994). To test recombination and non-disjunction of the YACs, a pair of such elements needs to be present in the same diploid strain, with different genetic markers at their ends and the same human DNA backbone. To achieve this situation, the markers were replaced by one-step transformation (Rothstein 1991) as described previously (Hugerat and Simchen 1993).

Media and growth conditions

Yeast cultures were grown on standard rich (YPD) or defined (Comp) medium ...