Supplemental Table 1 This table lists the masses of predicted tryptic peptides from SMC-1,
SMC-3 and TIM-1 which match the measured tryptic peptides obtained from the ≈150 kDa
proteins precipitated by SMC-1 antibodies. Deviations () between predicted and measured
peptide masses are provided along with the location of the peptide within the full length protein.
For the peptide mass fingerprinting, proteins in excised gel slices were subjected to in-gel
digestion as described 1, except the DTT and iodoacetamide incubation steps were omitted, and
the tryptic digestion included 10% (v/v) 1-propanol. The masses of the extracted peptides were
determined by MALDI-MS and matched against predicted open reading frames in the C. elegans
genome database using the search programs MS-Fit and ProFound. Searches were conducted
with a mass tolerance of 0.150 dalton.
Supplemental Table 2 RNAi treatments against tim-1 and each of the four cohesin subunit
genes result in embryonic lethality of F1 progeny laid 24-48 hours post-injection. The arrested
embryos contain 150-200 cells (data not shown).
Supplemental Table 3 This table lists the percentage distribution of FISH signals per nucleus in
the pre-meiotic and transition zones of wild-type and mutant gonads. The number of nuclei (n)
scored is given. The probability (p) values are shown, where p ≤ 0.001 indicates significant
deviation.
Supplemental Table 4 This table lists the gene-specific primers used to amplify exon-rich
regions of him-1, scc-1, scc-3, rec-8 and tim-1 from genomic DNA. The T3 and T7 promoter
sequences are underlined.
Supplemental Figure 1 a Confocal photomicrographs of DAPI-stained gonads from maternally
rescued (m+/z-) him-1(h55 or h134) homozygotes show the range of undersized gonads that
result from somatic and germline mitotic defects. b Confocal photomicrographs show similar
cell-cycle dependent patterns of accumulation and subcellular localization between cohesin
(represented by SMC-1) and TIM-1. In embryos (upper panels) SMC-1 and TIM-1 have diffuse
nuclear localization during interphase (white arrowheads), and the proteins are excluded from
metaphase (yellow arrowheads) and anaphase (yellow asterisks) chromosomes. In pre-meiotic
germline nuclei (lower panels), TIM-1 and cohesin (as represented by SMC-1) have a diffuse
nuclear appearance and are excluded from metaphase (yellow arrowhead) and anaphase (yellow
asterisks) chromosomes. c Chromosome aberrations in the pre-meiotic germline of a
him-1(h134) mutant were confirmed by 5S rDNA FISH analysis, which revealed more than the
two FISH signals expected for diploid nuclei. This defect is also observed following RNAi of
cohesin subunits and is most pronounced in the pre-meiotic germline of F1 escapers (data not
shown). d Similar to tim-1(ts) and cohesin(RNAi) mutants, him-1(e879ts) homozygotes raised at
the 25˚C non-permissive temperature contained univalents, demonstrating the absence of
chiasmata. e Localization of SMC-3 to pachytene and diakinesis chromosomes is unaffected in
rec-8(RNAi) animals and tim-1(ts); rec-8(RNAi) animals, unlike the localization of SCC-3. In
tim-1(ts) mutants, the TIM-1 protein level was reduced but the residual protein was correctly
restricted to pre-meiotic nuclei (data not shown), indicating that SCC-3 mislocalization was not
due to ectopic distribution of TIM-1 mutant protein. Scale bars, 10 µm (a) and 5 µm (b-e)
Supplemental Figure 2 Schematic representations of the Drosophila, C. elegans and mouse
Timeless protein homologs. HEAT/Armadillo (Arm) repeats (E ≤ 0.2) are colored in blue and the
PERIOD interaction regions in TIMELESS are represented by red bars. Homology searches
were conducted on worm TIM-1 (accession no. 6467560), mouse mTim1 (accession no.
4115716) and fly TIMEOUT (accession no. 8133124) using 3D-PSSM 2. The HEAT/Arm
repeats predicted in mTim1 and TIMEOUT agree closely with those previously described 3.
Positions of the TIMELESS HEAT/Arm repeats and PERIOD interaction motifs were described
by Vodovar et al. 3
SUPPLEMENTAL REFERENCES
1.
Shevchenko, A., Wilm, M. & Mann, M. Peptide sequencing by mass spectrometry for
homology searches and cloning of genes. J. Protein Chem. 16, 481-90 (1997).
2.
Kelley, L. A., MacCallum, R. M. & Sternberg, M. J. Enhanced genome annotation using
structural profiles in the program 3D-PSSM. J. Mol. Biol. 299, 499-520 (2000).
3.
Vodovar, N., Clayton, J. D., Costa, R., Odell, M. & Kyriacou, C. P. The Drosophila
clock protein Timeless is a member of the Arm/HEAT family. Curr. Biol. 12, R610-R611
(2002).