Silencing of the Y-chromosomal gene tspy during murine evolution
Institut fu¨r Humangenetik, Medizinische Hochschule, Hannover, D-30625 Hannover, Germany
Institut fu¨r Biologie, Medizinische Universita¨t Lu¨beck, Germany
Institut fu¨t Wildtierbiologie, Tiera¨rztliche Hochschule, Hannover, Germany
Max-Planck-Institut fu¨r molekulare Genetik (MPIMG), Berlin-Dahlem, Germany
Department of Zoology, Banaras Hindu University, India
Received: 23 April 1999 / Accepted: 13 December 1999
Abstract. We have studied the process of tspy gene silencing in
murine evolution. We have isolated functional tspy sequences from
Apodemus agrarius, A. sylvaticus, A. flavicollis, and Mus platythrix
(subgenus Pyromys) and nonfunctional tspy sequences from spe-
cies of the subgenus Mus. We present two alternative models as to
how tspy may have lost its function in the murine lineage.
The testis-specific gene family TSPY (testis-specific protein, Y-
encoded) was first discovered in man [Arnemann et al. 1987].
TSPY-homologous gene families have subsequently been detected
in other primates including the great apes, owl monkey, gibbon,
lemurs [Zhang et al. 1992; Kim et al. 1996] and macaques [Jaku-
biczka et al. 1993], artiodactyles including cattle, sheep, goat, and
pig [Vogel et al. 1997; Vogel and Schmidtke 1998], perissodac-
tyles such as the horse [Manz et al. 1998] and rodents [Vogel et al.
1998; Mazeyrat and Mitchell 1998; Dechend et al. 1999]. In hu-
man, TSPY is moderately repetitive with 30–60 copies per chro-
mosome, whereas in the bull the copy number ranges from 50 to
200 [Arnemann et al. 1987; Jakubiczka et al. 1993; Manz et al.
1993]. Human TSPY members show up to 10% nucleotide diver-
sity at the genomic level, if apparently non-transcribed pseudo-
genes are included, while functional members are only up to 3%
divergent [Vogel and Schmidtke 1998]. Each TSPY transcription
unit is 2.8 kb in length and consists of six exons and five introns.
TSPY is expressed almost exclusively in spermatogonia and only
to very small extent in primary spermatocytes, suggesting a role in
spermatogonial proliferation [Schnieders et al. 1996] and thus
serving as one of several male-specific “fertility factors” [Vogt
1997]. Bovine TSPY resembles its human homolog in all aspects
of organization and expression [Vogel et al. 1998].
In contrast to all other species investigated so far, tspy is
single- or low-copy in Murinae. While tspy is single-copy and
apparently nonfunctional in the house mouse (Mus musculus)ow-
ing to several mutational events [Vogel et al. 1998; Mazeyrat and
Mitchell 1998], the rat tspy locus apparently consists of one com-
plete functional and one truncated, probably nonfunctional copy
[Dechend et al. 1998]. This indicated that the loss of function of
tspy may have taken place after the mouse lineage had separated
from the rat lineage. The aim of this study was to investigate tspy
silencing in rodent evolution, a model for the general decay of the
Y chromosome [Graves 1996; Jegalian and Page 1998], in greater
detail. To this end, we searched for functional tspy homologs in
other species of the genera Apodemus and Mus. We were able to
isolate functional tspy genes from A. agrarius, A. sylvaticus, A.
flavicollis, and from M. platythrix (subgenus Pyromys), but not
from different lineages of the subgenus Mus.
Materials and methods
The three Apodemus species were trapped in Lower
Saxony, Germany. M. spretus originates from Southern France, M. mace-
donicus from Greece, M. booduga from Mysore (South India) and Varanasi
(North India), M. platythrix from Mysore (South India), and M. cervicolor
from Kathmandu (Nepal).
PCR, RT-PCR, and product cloning.
Amplification of DNA or cDNA
was achieved by PCR with 10 pmol primer and 1 unit of Taq or Pfu
polymerase (Qiagen or Stratagene) in a 30-l reaction volume. Standard
conditions were 37 cycles of 1 min at 94°C, 1 min annealing and 1–5 min
at 72°C, and subsequently 10 min at 72°C. First-strand cDNA syntheses
were performed with DNase-digested RNA with poly dT-primer and the
First Strand Synthesis Kit (Pharmacia, Freiburg, Germany), according to
the manufacturer’s protocol. Amplification products were cloned with the
Zero Background/Kab Cloning Kit (Invitrogen, Groningen, Netherlands)
and the Eucaryotic Dual Promotor TA Cloning Kit (Invitrogen).
Sequencing of plasmid DNA and PCR fragments was
done by using the non-radioactive DNA-sequencing kit-Big Dye Termi-
nator Cycle Sequencing Ready Reaction (Applied Biosystems, Weiterstadt,
Germany) and the ABI 310 Genetic Analyzer (Applied Biosystems). Se-
quence analyses were carried out with “GAP”, using the Needleman and
Wunsch algorithm, which is included in the HUSAR software (EMBL,
Heidelberg). Standard parameters were a gap weight of 5.0 and a gap
length of 0.3. In rare cases, computer-aided alignments were corrected
Ten microgram of male and female DNA of different
murine species were digested with EcoRI, electrophoresed on 0.8% aga-
rose gels in TAE buffer, blotted onto Nytran 13 N membrane from Schlei-
cher and Schuell, hybridized with ␣
-P-dCTP-labeled pRat3Ј (rat TSPY
cDNA clone, covering exons 2–6), under low stringent conditions
[Church’s hybridization at 55°C, buffer 0.5
and washed twice with 200 m
, pH 7.2, at 55°C for 30 min, once
in 0.5 m
, pH 7.2 at 40°C for 15 min, and 0.5 m
at 53°C for 15 min.
In order to time the evolutionary loss of function of tspy in the
murine lineage more precisely, we have attempted to isolate tspy
The nucleotide sequence data reported in this paper have been submitted to
GenBank and have been assigned the accession numbers: AF205111,
AF205112, AF205113, AF205114, AF152846, AF152847.
* Both authors have contributed equally to this work
Correspondence to: e-mail: email@example.com
Mammalian Genome 11, 288–291 (2000).
© Springer-Verlag New York Inc. 2000