Differential expression of the zinc finger gene Zfp105
The Jackson Laboratory, Bar Harbor, Maine 04609, USA
Department of Biochemistry and Cellular and Molecular Biology, University of Tennessee, Knoxville, Tennessee 37996-0840, USA
Received: 11 February 1998 / Accepted: 1 May 1998
Abstract. We report the isolation of Zfp105, the mouse homolog
of the human ZNF35 zinc finger gene. Zfp105 and ZNF35 are
highly conserved at the protein and nucleotide level, and Zfp105
maps to a region of mouse Chromosome (Chr) 9 that is homolo-
gous to the human region containing ZNF35. Zpf105 is highly
expressed in the testis, especially in pachytene spermatocytes and
round spermatids. The possible role of this gene product in main-
taining an ordered germ cell differentiation process is discussed.
Spermatogenesis is a complex process initiated by the formation of
type-A spermatogonia from neonatal primordial germ cells. These
cells either cycle as pluripotent stem cells or further differentiate
into type-B spermatogonia that give rise to primary spermatocytes.
Meiotic division of spermatocytes yields haploid spermatids that
undergo radical morphological and functional differentiation to
produce spermatozoa. It is likely that the complex pattern of gene
expression during spermatogenesis is at least partially controlled at
the transcriptional level.
Several genes encoding transcription factors are expressed at
various stages of spermatogenesis (reviewed by Wolgemuth and
Watrin 1991). Among these are several C
zinc finger DNA-
binding proteins, including Zfp38 (Noce et al. 1992), TSGA (Hoog
et al. 1991), Zfp29 (Denny and Ashworth 1991), Zfp35 (Cunliffe et
al. 1990), Zfp37 (Hosseini et al. 1994), Zfy-related genes (Koop-
man et al. 1989; Nagamine et al. 1990; Ashworth et al. 1990), and
members of the Gli family (Persengiev et al. 1997). Support for the
importance of expression of such genes comes from the demon-
stration of decreased fertility, owing to a reduction in germ cells,
in mice homozygous or hemizygous for a targeted mutation of the
X-linked zinc finger gene, Zfx (Luoh et al. 1997).
We now describe the isolation of the mouse homolog (desig-
nated Zfp105) of the human ZNF35 zinc finger gene, a gene regu-
lated during terminal differentiation of myeloid cells. Zfp105 is
highly expressed in spermatogenesis, during the progression of
spermatocytes to round spermatids in the mouse adult testis. We
suggest that this gene product may play a role in coordinate stage-
specific gene expression.
Materials and methods
A mouse testis cDNA library (Stratagene, La Jolla, CA)
was screened with a
P-labeled probe at 60°C by conventional methods
(Sambrook et al. 1989). Final washing conditions were 1 × SSC, 0.5% SDS
for 20 min at 60°C. cDNA clones were sequenced with di-deoxyterminator
chemistry on an ABI 373 Stretch DNA sequencer.
Mapping of Zfp105 was performed by restriction frag-
ment length polymorphism (RFLP) analysis of 94 animals from the inter-
specific backcross panel (C57BL/6J × SPRET/Ei)F
× SPRET/Ei from The
Jackson Laboratory, Bar Harbor, Me. (Rowe et al. 1994). Genomic DNAs
were digested with BamHI, Southern blotted, and hybridized with2×10
cpm/ml of denatured
P-random-labeled Zfp105 full-length cDNA. This
analysis detected 5.0- and 1.1-kb bands from C57BL/6J DNA and 5.0- and
2.3-kb bands from M. spretus DNA (Sambrook et al. 1989). DNAs were
scored for the presence or absence of the C57BL/6J-specific 1.1-kb band.
Map locations were determined by the Map Maker program.
Northern blot analyses.
Total RNA was prepared from various adult
mouse tissues (B6D2F
/J; The Jackson Laboratory) with guanidinium
isothiocyanate (Chomczynski and Sacchi 1987), and poly[A
] RNA was
isolated by oligo (dT) cellulose chromatography. Northern blotting was
carried out by standard methods (Sambrook et al. 1989); 10 g of poly[A
RNA and 15 g of total RNA were loaded per lane for the multiple tissue
and germ cell fractionation blots, respectively. Hybridizations were per-
P-random-labeled probe in 50% form-
amide,5×SSC,5×Denhardt’s solution, 50 m
buffer, pH 7.4, 10% dextran sulfate, and 150 g/ml denatured salmon
sperm DNA at 42°C. Final washing conditions were 65°C in 0.1 × SSC,
Germ cell fractionation.
Testes of adult CD-1 mice were suspended in
EKRB buffer [120.1 m
NaCl, 4.8 m
KCl, 25.2 m
, 1.2 m
, 1.2 m
O, 1.3 m
glucose, 1 ×
essential amino acids (Sigma, St. Louis, Mo.), 1 × non-essential amino
acids (Sigma)] and digested with collagenase, trypsin, and DNase as de-
scribed (Romrell et al. 1976; Bellve` et al. 1977). After being washed in
EKRB containing 0.5% bovine serum albumin (BSA), the single-cell sus-
pension was sedimented at unit gravity througha2to4%BSAgradient
generated in a STAPUT chamber (Johns Scientific, Ontario). Leptotene
and zygotene (L/Z) spermatocytes were isolated in a similar manner from
testes of 17-day-old animals. The average purity of the pachytene sper-
matocyte population and the leptotene and zygotene (L/Z) spermatocyte
population was 85–90%. Major contaminants in the pachytene spermato-
cyte fraction were Sertoli cells and symplasts composed of round sperma-
tids and spermatogonia, and Sertoli cells were the main contaminants in the
(L/Z) spermatocyte fraction. Total RNA was isolated (Cathala et al. 1983)
from pooled germ cell fractions and also from the testes of wild-type mice
and from germ-cell-deficient testes of adult XXSxr mice (Cattanach et al.
In situ hybridization.
Adult mouse testes were immersion fixed in 4%
paraformaldehyde in phosphate-buffered saline (PBS, pH 7.4) overnight,
dehydrated, cleared in xylene, and embedded in paraffin wax prior to
sectioning (6 m) and mounting on Plus slides (Fisher).
-antisense riboprobes corresponding to the 5Ј region of the isolated clone(s)
were prepared by in vitro transcription according to the manufacturer’s
protocol (Stratagene). Tissue pretreatment, hybridization, and post-hybrid-
Correspondence to: S.L. Ackerman
The nucleotide sequence data reported in this paper have been submitted to
GenBank and have been assigned the accession number AF045565.
Mammalian Genome 9, 758–762 (1998).
© Springer-Verlag New York Inc. 1998