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THE JOURNAL OF CELL BIOLOGY
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Yan et al., http://www.jcb.org/cgi/content/full/jcb.200904040/DC1
Figure S1. Cytological analysis of meiosis in WT and solo spermatocytes. Testes were stained with anti–-tubulin to visualize spindles and with DAPI to
visualize DNA. (A) Meiosis I. Both WT and soloZ2-0198/Df(2L)A267 spermatocytes exhibit three compact and separate chromatin masses representing the
three large bivalents at prometaphase I (PMI). Chromosomes successfully align at metaphase I (MI) and segregate equally to opposite poles at anaphase I
(AI) in both genotypes. (B) Quantification of cytological phenotypes during meiosis I in solo mutants. Abnormal cells were defined as follows: prometaphase
I, cells with more than three large DNA clumps; metaphase I, cells with more than one DNA clump; anaphase I, cells with unequal poles or one or more
laggards. (C) Meiosis II. Sister chromatids separate precociously at metaphase II (MII) in soloZ2-0198/Df(2L)A267 spermatocytes and segregate unequally at
anaphase II (AII). (D) Quantification of cytological phenotypes at MII and AII in solo mutants. Abnormal cells were defined as follows: metaphase II, cells
with more than one DNA clump; anaphase II, cells with unequal poles or one or more laggards. The number of nuclei scored is shown in parentheses.
Bars, 5 µm.
SOLO: a novel meiotic cohesion protein • Yan et al.
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Figure S2. Premature loss of third chromosome cohesion in solo mutants. The dodeca repeats adjacent to the third chromosome centromere were visualized by FISH using a labeled dodeca probe, and DNA was stained by DAPI. (A) dodeca cohesion in WT primary spermatocytes. Two dodeca foci,
each representing the paired sister centromeres of homologous third chromosomes, are visible within one bivalent at prometaphase I (PMI). The two foci
segregate to opposite spindle poles at anaphase I (AI). Sister foci remain fused, indicating that sister centromeres are held together during prometaphase
I and anaphase I. (B) Dodeca cohesion in soloZ2-0198/Df(2L)A267 primary spermatocytes. Four dodeca foci are evident at prometaphase I within the chromosome 3 bivalent, indicating that sister centromeres have prematurely separated but sister chromatids are still held together within the bivalent. Note,
however, that the bivalents appear loosely packed, with chromatin strands extending out of the bivalent. At anaphase I, fully separated sister chromatids
are evident. Two dodeca foci segregate to each pole. Based on genetic data (Table I and Table S1), the cosegregating third chromatids could be either
sister or homologous chromatids. Bars, 5 µm.
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Figure S3. SOLO is not required for arm cohesion or mitotic chromatid segregation. Arm cohesion was assayed by counting GFP spots in spermatogonia
and spermatocytes from males hemizygous for a chromosome 2 transgene carrying a 256-mer tandem array of lacO repeats and heterozygous for a
transgene (also on chromosome 2) expressing a GFP-LacI chimeric protein under control of the hsp83 promoter. The genotype of the tested males was
w1118/Y; Df(2L)A267, {GFP-LacI} {lacO}/soloZ2-0198. Testes were dissected from young adults, and GFP-LacI foci were imaged by native fluorescence. DNA
was stained with DAPI. (A) GFP::LacI foci in early prophase I spermatocytes. Only one spot is evident in each nucleus, although there are two copies of the
lacO array on sister chromatids of one of the second chromosomes, which is indicative of arm cohesion. Bar, 5 µm. (B) Quantification of GFP::LacI foci in
spermatogonia (gonia) and early prophase I spermatocytes (S1 and S2).
SOLO: a novel meiotic cohesion protein • Yan et al.
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Figure S4. Venus::SOLO in WT and mei-S332 spermatocytes. (A and B) Expression of Venus::SOLO was induced by nos-GAL4::VP16. Venus foci were detected by native fluorescence. Besides bright foci, diffuse Venus::SOLO foci can be seen on one bivalent (arrow). This is probably the X-Y bivalent because
it contains more heterochromatin than the second or third chromosome bivalents, but this conjecture has not yet been directly tested. Mutant spermatocytes
are from mei-S3324/mei-S3328; {nos-GAL4::VP16}/{UASp-Venus::SOLO} males. All images are sum or maximum projections of 3D-deconvolved z series
planes. S1, early prophase I; S6, late prophase I; PMI, prometaphase I; AI, anaphase I; MI, metaphase I; MII, metaphase II. Bar, 5 µm.
Figure S5. SOLO colocalizes with CID in germarium. Venus::SOLO (green) was induced by nos:GAL4-VP16 and detected by native fluorescence. CID is
shown in red. Arrows indicate meiotic cells (nurse cells and oocytes). Arrowheads indicate follicle cells. Bar, 5 µm.
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Table S1. Second chromosome NDJ in solo mutant males
Sperm genotype
b pr/cn bw
b pr/b pr
cn bw/cn bw
O
NDJ type
Egg genotype
Progeny phenotype
No. of progeny
Homologue
Sister
Sister
Both
O
O
O
2^2, bw sp
WT
b pr
cn bw
bw sp
414
60
71
637
Parameters: number of males tested, 75; total progeny, 1,182; progeny/male, 15.8; %sis, 22. %sis = 100 x (cn bw + b pr)/(cn bw + b pr +WT). soloZ2-0198, cn bw/b vas7
pr males were crossed singly with three C(2)EN, bw sp females. vas7 is null for both vas and solo function. C(2)EN females carry two copies of each arm of chromosome 2 attached to a single centromere and produce only diplo-2 (2^2) and nullo-2 (O) eggs. Because zygotes that are trisomic or monosomic for chromosome 2
are inviable, the only viable progeny result from fertilization by sperm resulting from paternal chromosome 2 NDJ (2/2 and O sperm). Thus, the frequency of second
chromosome NDJ is proportional to progeny per male. Because the paternal second chromosomes carry different recessive markers (cn bw and b pr, respectively),
the percentage of sister chromatid NDJ out of total NDJ (%sis) can be estimated from the proportions of NDJ progeny that are homozygous or heterozygous for the
markers. cn bw progeny and b pr progeny result from sister chromatid NDJ, whereas WT (cn bw/b pr) progeny result from homologue NDJ. 54 control solo/+ sibling
males were also tested, but only one male produced one progeny due to spontaneous NDJ; the control data are not shown in the table.
SOLO: a novel meiotic cohesion protein • Yan et al.
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