Supplementary data contents:
Supplementary Note 1. Search for Platypus SRY
Supplementary Figure 2
Supplementary Figure 3
Supplementary Table 1
Supplementary Table 2
Supplementary Note 1. Search for Platypus SRY.
Strenuous attempts to identify and isolate a male-specific platypus (or echidna)
homologue of the mammal sex determining gene SRY have been made by many members
of, and visitors to, the Graves laboratory over more than ten years (principally J. Watson,
J. Foster, P. Western, A. Pask, P. Kirby, K. Nagai). The uniformly negative results have
been reportedS1-S5 but the details never published. These experiments have included PCR
based approaches using a range of primers derived from human, mouse and marsupial
SRY or SOX3 sequences, screening platypus and echidna libraries with a variety of
probes, and Southern blotting.
PCR using several SRY-specific primer pairs that specifically amplify a male-specific
band in human, mouse or marsupial (tammar wallaby and Sminthopsis maculata)
produced no product on platypus or echidna DNA. Nor did RT-PCR using the same
primers on platypus RNA or a platypus cDNA library. To circumvent problems arising
from the poor conservation of SRY, the same templates were also PCR amplified by
primers designed from sequence of the much more conserved SOX3 gene, which, because
it lies on the X chromosome and is the closest relative of SRY, is thought to be the gene
from which SRY evolvedS6-S8. Products were obtained by this strategy, but none were
male-specific.
A number of monotreme (platypus and echidna) genomic and cDNA libraries have
been screened with a variety of probes in order to isolate a clone containing a monotreme
SRY orthologue. Platypus and echidna lambda genomic libraries and a platypus testis
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cDNA library were screened with human, mouse, tammar and Sminthopsis SRY. Many
weak positives were recovered, but none proved to contain a relative of SRY. Screening
the same libraries with tammar SOX3 yielded many positive clones, but these proved to
contain either unrelated sequences, or SOX genes that were present in both sexes. Two
platypus SOXB genes (SOX2 and SOX14) were characterized and mappedS9.
The strategy that was ultimately accepted for demonstrating the absence of SRY in the
mole vole Ellobius lutescens, was to probe Southern blots containing male and female
DNA with SRY isolated from a closely related species Ellobius fuscocapillus that retains
a Y chromosome and an SRY genesS10. This strategy is unavailable for monotremes, since
they diverged from therian mammals (marsupials and eutherians) about 210 million years
ago and are equally distantly related to human, mouse, tammar and Sminthopsis.
Southern blotting, using DNA cut with a barrage of restriction enzymes from male and
female platypus and echidna has been carried out many times. However, probing with
human, mouse, tammar or Sminthopsis SRY produced only weak SOX bands present in
both sexes in both species. Again, the absence of an SRY-homologous male-specific band
might be just the result of the rapid sequence divergence of this gene.
The most successful strategy for identifying and isolating a Y-borne gene across
species has proved to be Southern blotting, using as probe the X-borne homologue, rather
than the Y-borne gene itself. Whereas the Y-borne genes (e.g. RBMY, UBE1Y, TSPY,
ATRY) have diverged beyond recognition such that they do not detect orthologues in
distantly related species, their X-borne homologues (RBMX, UBE1X, TSPX and ATRX)
are all highly conserved, and detect both X and Y homologues on Southern blots, even
across distantly related speciesS6,S11-S14. SOX3 has diverged much less than SRYS15.
Southern blots of male and female DNA from platypus and/or echidna, cut with different
restriction enzymes, were therefore probed with human and tammar SOX3. Several strong
bands were identified, but all were present in both sexes in both species. Supplementary
Fig 1 shows a representative Southern blot, in which a male-specific band is absent.
Similar experiments have been published to show the absence of a male specific
SRY/SOX genes in chickenS16,S17.
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Our conclusion, therefore, is that platypus lacks a male-specific SRY orthologue.
Supplementary Figure 1 Male and female platypus DNA cut with EcoRI and probed
with tammar wallaby SOX3. Several bands representing platypus SOX genes can be seen
in both sexes, but there is no trace of a male-specific band. Photograph kindly supplied
by Dr Andrew Pask, present address Zoology Department,University of Melbourne.
S1. Graves, J.A.M. & Shetty, S. Sex from W to Z: evolution of vertebrate sex
chromosomes and sex determining genes. J. Exp. Zool. 290, 449-462 (2001)
S2. Graves, J.A.M. The rise and fall of SRY. Trends in Genetics 18, 259-264 (2002)
S3. Pask, A., Renfree, M.B. & Graves, J.A.M. The human sex-reversing ATRX gene has
a homologue on the marsupial Y chromosome, ATRY: implications for the
evolution of mammalian sex determination. Proc. Natl. Acad. Sci. U S A. 97,1319813202 (2000)
S4. O'Neill, R.J., Brennan, F.E., Delbridge, M.L., Crozier, R.H. & Graves, J.A.M. De
novo insertion of an intron into the mammalian sex determining gene, SRY. Proc.
Natl. Acad. Sci. U S A. 95, 1653-1657 (1998)
S5. Grützner, F., Deakin, J., Rens, W., El-Mogharbel, N. & Graves, J.A.M. The
monotreme genome: a patchwork of reptile, mammal and unique features? Comp.
Biochem. Physiol. A 136, 867-881 (2003)
S6. Foster, J.W. & Graves, J.A.M. An SRY-related sequence on the marsupial X
chromosome: implications for the evolution of the mammalian testis-determining
gene. Proc. Natl. Acad. Sci. U.S.A. 91, 1927-1931 (1994)
S7. Katoh, K., & Miyata, T. A heuristic approach of maximum likelihood method for
inferring phylogenetic ree and an application to the mammalian SOX-3 origin of
the testis-detrmining gene SRY. FEBS Letts. 463, 129-132 (1999)
S8. Bowles, J., Schepers, G. & Koopman, P. Phylogeny of the SOX family of
developmentall transcription factors based on sequence and structural indicators.
Dev. Biol. 15, 239-255 (2000)
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S9. Kirby, P.J., Waters, P.D, Delbridge, M.L., Svartman, M., Stewart, A.N., Nagai, K. &
Graves, J.A.M. Cloning and mapping of platypus SOX2 and SOX14 – insights into
SOX group B evolution. Cytogenet. & Genome Res. 98, 96 –100 (2002)
S10. Just, W., Rau, W., Akhverdian, M., Fredga, K., Graves, J.A.M., Lyapunova, E. &
Vogel, W. Sex determination in Ellobius lutescens and E. tancrei in the absence of
the Y chromosome and the Sry gene. Nature Genetics, 11, 117-118 (1995)
S11. Mitchell, M.J., Woods, D.R., Wilcox, S.A. Graves, J.A.M. and Bishop, C.E. The
marsupial Y chromosome encodes a homologue of the mouse Y-linked candidate
spermatogenesis gene Ube1y. Nature 359, 528-531 (1992)
S12. Mitchell, M.J., Wilcox, S.A., Watson, J.M., Lerner, J., Woods, D., Scheffler, J.,
Hearn, J., Bishop, C. and Graves, J.A.M. The origin and loss of the ubiquitin
activating enzyme gene on the mammalian Y chromosome. Hum. Mol. Genet. 7,
429-434 (1998)
S13. Delbridge, M.L., Lingenfelter, P.A., Disteche, C.M. and Graves, J.A.M. The
candidate spermatogenesis gene RBMY has a homologue on the human X
chromosome. Nat. Genet. 22, 223–224 (1999)
S14. Delbridge, M.L., Longepied, G., Depetris, D., Mattei, M-G., Disteche, C.M.,
Graves, J.A.M. & Mitchell, M.J. TSPY, the cadidate gonadoblastoma gene on the
human Y chromosome, has a widely expressed homologue on the X; implications
for Y chromosome evolution. Chrom. Res., in press.
S15. Nagai, K. Molecular evolution of Sry and Sox genes. Gene 270, 161-169 (2001)
S16. Griffiths, R. The isolation of conserved DNA sequences related to the human sexdetermining region Y gene from the lesser black-backed gull (Larus fuscus). Proc.
R. Soc. Lond. B Biol. Sci. 244, 123-128 (1991)
S17. Mizuno, S., Kunita, R., Nakabayashi, O., Kuroda, Y., Arai, N., Harata, M., Ogawa,
A., Itoh, Y., Teranishi, M. & Hori, T. Z and W chromosomes of chickens: studies
on their gene functions in sex determination and sex differentiation. Cytogenet.
Genome Res. 99, 236-244 (2002)
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Supplementary Figure 2 DAPI inverted karyotype of male platypus taken from a male
cell. The unpaired chromosomes are assembled at the bottom of the karyotype.
Supplementary Figure 3 Amino-acid sequence alignment of platypus DMRT1 with
DMRT1 from different species (accession #: medaka AF319994, chicken AF123456, pig
NM_214111, mouse NM_015826, human NM_021951). a, shows partial exon 1
(accession # AJ744848) and in b, partial exon3 (accession # AJ744847).
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