Identification
of a Novel Human Zinc Finger Protein Gene ZNF313

MA
Yong-Xin, ZHANG Si-Zhong*,
HOU Yi-Ping, HUANG Xin-Li¹, WU Qia-Qing, SUN Yan

( The Key Laboratory of Biotherapy, Ministry of
Education; Department of Medical Genetics, West China Hospital, Sichuan
University, Chengdu 610041, China; ¹Center for Human
Genetics, Boston University, Boston, MA 02118, USA )

Abstract       A
novel human zinc finger protein gene that contains both ring finger and C2H2
domain was first isolated by mRNA differential display between the testes of
fertile adults and azoospermic patients followed by rapid amplification of cDNA
ends (RACE). Total 6 exons of the human gene span a 17 484 bp genomic DNA
sequence that was mapped to chromosome 20q13 by fluorescence in situ
hybridization. The mature processed mRNA encodes a 228-amino acid protein with
a C3HC4 ring finger and three C2H2
domains. Genomic analysis of the human gene identified two polyadenylation
signals in exon 6 resulting in alternative 3′-untranslated regions. Results of
Northern blot and RT-PCR of RNAs extracted from multiple tissues revealed that
the gene has two transcripts of which the shorter transcript was expressed
abundantly in fertile adult testes, but much less in testes of azoospermic
patient, fetus as well as other human tissues. These data suggest that the gene
may play a role in human spermatogenesis and male fertility.

Key words   human ZNF313 gene; spermatogenesis; male
infertility; alternative polyadenylation signals

Idiopathic azoospermia or severe
oligozoospermia is observed in about 20% of all infertile men[1]. In these
cases, systematic analysis of molecular deletions of Y chromosome indicates
that three non-overlapping regions, AZFa, AZFb and AZFc, in band Yq11 are
involved in male spematogenesis[2]. A series of genes including DAZ and RBM
genes have been identified in these AZF regions as candidate genes for
azoospermia. They are categorized as a testis-specific type and a
multiple-tissue expression type[3–5]. However, gene-specific mutations causing
the azoospermia phenotype have only been found in the USP9Y gene[3,6]. Some
autosomal genes are also associated with spermatogenesis in mouse and man
including DAZLA (the autosomal DAZ gene homologue), CREM, and HSP70-2 with its
human homologue HSPA2[7–10].

The products of many genes are essential for spermatogenesis,
but a small number affect spermatogenesis exclusively. Identification of these
genes and their role is important in understanding the biology of
spermatogenesis[11]. Using mRNA differential display (mRNA-DD) between the mRNA
transcription pool of the testicular tissues of two healthy fertile adults and
two patients with meiotic spermatogenic disruptions at the spermatocyte phase,
we have identified a series of known and novel transcripts expressed
specifically or in high concentrations in normal testicular tissues. Among
these, a transcription product has proved to be a part of the previously
uncharacterized zinc finger protein gene, ZNF313, which is expressed highly in
testicular tissues of fertile adults.

More than 20 different zinc finger protein encoding
genes located both on sex chromosomes and on autosomes have been proposed to
play a regulatory role in mammalian spermatogenesis including the genes ZNF76,
Sperizin, ZFY and ZFX[12–14], which are usually expressed ubiquitously,
function primarily during meiosis and early spermatid development. In contrast,
zinc finger protein genes encoding both ring finger and C2H2 domains are rare.
Cloning and characterization of ZNF313 may provide an important clue.

1   Materials
and Methods

1.1  Subjects and RNA preparation

Fresh testicular tissues of two normal adults who
died in accidents and two six-month human fetal testicular tissues were
obtained from the West China Hospital, Sichuan University with the consent of
their relatives. Testis bioptic material of azoospermic patients came from the
Department of Urology, Sichuan College of Genital Health. The diagnosis of
azoospermia was made by sperm counting and confirmed by pathohistological
examination of testis. The spermatogenesis was arrested at spermatocyte stage.
Total RNA of testicular tissues were prepared by using RNeasy mini Kit (Qiagen)
and treated with DNase I(RNase-free, Boehringer-Mannheim) to eliminate DNA
contamination. Pellets of the total RNA were resuspended in DEPC-treated water
and stored at -80 ℃ until use.

1.2  mRNA differential display

Isolated RNA from the testicular tissues of two
fertile adults and two azoospermic patients were used for the mRNA differential
display performed according to the protocol of Liang and Pardee[15] using the
RNAimage Kit (Gene Hunter). The amplified fragments were separated on a 6%
polyacrylamide sequencing gel for differential analysis. Bands displayed in two
control lanes (N1,N2 ) but at a reduced or undetectable level in patients
(P1,P2) were cut out from the gel and the DNA fragments were recovered by QIAEX
II Gel Extraction Kit (Qiagen, Victoria, Australia) according to the
manufacturer’s instructions. The eluted fragments were reamplified with the
same set of primers used in the mRNA-DD experiment. The reamplified fragments
were cloned into the pGEM-T Easy Vector (Promega, Madison, WI, USA) as
described by the manufacturer, and sequenced with Thermo Sequenase Cycle
Sequencing Kit (Amersham Pharmacia) using an ALF express Automatic DNA
Sequencer (Pharmacia, Ontaria, Canada).

1.3  Rapid amplification of cDNA ends
(5′-RACE)

5′-RACE experiments were performed using the SMART
RACE cDNA Construction Kit (Clontech). Briefly, 5′-RACE-Ready cDNA was obtained
by reverse transcription using total RNA from testicular tissues of healthy
fertile adults as templates. Universal primer mix (UPM) provided by the kit and
gene specific primers based on the sequence of the fragments obtained from
mRNA-DD were used for the 5′-RACE experiments. The touch-down PCR amplification
profile was 94℃ for 2 min; 94 ℃ for 30 s, 68–0.5 ℃/cycle for 30 s, 72 ℃ for 3
min,10 cycles; 94 ℃ for 30 s, 63 ℃ for 30 s, 72 ℃ for 3 min, 22 cycles;
followed by a final extension at 72 ℃ for 10 min. The PCR products were
recovered using QIAEX II Gel Extraction Kit (Qiagen) and cloned as described
above. Clones with the inserts of the expected size were identified by EcoRI
digestion and sequenced as described above.

1.4  Northern blot

Multiple Tissue Northern (MTN) Blot Membranes
(Clontech cat#7759-1 and 7760-1) with mRNA from 16 tissues were used to
determine the tissue expression pattern of ZNF313 gene. A clone contained
ZNF313 gene insert was digested with EcoRI, separated on a 1.5% agarose gel and
the insert was recovered as a cDNA probe. The probe was labeled with [³²P]dCTP
with Random Primed DNA Labeling Kit (Roche, Indianapolis, IN,USA) according to
the manual’s protocol and purified with NucleoTrap PCR Purification Kit
(Clontech). Northern hybridization was then performed as described by the
manual and human β-actin gene as a control.

1.5  Cloning of 2.4 kb transcript of ZNF313
gene

Based on the result of Northern blot, the ZNF313 gene
has a 2.4 kb transcript in addition to the expected 0.75 kb transcript. During
the homology analysis of the sequence of the 0.75 kb transcript, we found a
1852 bp sequence AF131742 without a detectable open reading frame. Because this
sequence overlaps the 0.75 kb fragment by 143 bp, five primers were designed to
confirm the existence of the 2.4 kb transcript in testis. The sequences of the
three pair of primers were(Table 1): P4F3: 5′-TCCGTGCTTGC-TATCTGTCTCATG-3′ and
P4R3: 5′-GCATTTAAGA-CTGAACCAGTGAACTC-3′; P4F4:
5′-GAGTTCACT-GGTTCAGTCTTAAATGC-3′ and P4R4: 5′-CAGATT-AGGCTGACAGCTCTTGGAC-3′;
P4F5: 5′-GTCCAA-GAGCTGTCAGCCTAATCTG-3′ and CDSIII/3′PCR primer delivered by the
SMART RACE cDNA Construction Kit. The PCR products were then cloned into pGEM-T
easy vector and sequenced.

Table 1 The sequences of PCR primers used for ZNF313
amplification

1.6  Chromosomal mapping

ZNF313 gene was mapped by fluorescence in situ
hybridization (FISH) of an 8.1 kb PCR amplified genomic DNA fragment to
metaphase human cell separated by standard protocols. The primers for the PCR
were G4F1 and G4R2: 5′-ATCCGGTCCCAC-GTGGCTACTTGTTCC-3′ and 5′-GCGCGAGGTAG-CACTTGCAGCCCCATC-3′.
The amplified genomic products was labeled with digoxigenin-11-dUTP (Enzo
Diagnosis) by nick-translation (Intergen, Purchase, NY, USA) and hybridized to
the denaturedchro-mosomes at a final concentration of 20 μg/L in 50% formamide,
10% dextran sulfate, 2 × SSC, 0.2 g/L Cot-1 DNA (Gibco BRL, Gathersburg, MD,
USA), 2 g/L salmon sperm DNA, and 2 g/L E.coli tRNA. The hybridized signals
were detected with anti-digoxigen-rhodamine (Boehringer Mannheim).Chromosomal
DNA were counterstained with DAPI II (4,6-diamidino-2-phenylindole) and the
slides were examined through a Zeiss fluorescent microscope with a integrating
CCD camera (Photometrics). These images were captured by a Cyto Vision-Ultra
Workstation (Applied Imaging, Santa Clara) using the Probe Software.

1.7  Genomic structure analysis

Exon-intron boundaries of the gene were identified by
aligning the cDNA sequences with corresponding genomic sequence (GenBank
accession No.AL 031685).

1.8  RT-PCR analysis

Total RNAs from the normal human tissues including
testis, skeletal muscle, lung, liver, brain, spleen, kidney and stomach were
extracted for RT-PCR analysis. A 175 bp fragment of ZNF313 was amplified by PCR
with primers G4F1 and P4. The sequence of primer P4 was:
5′-TGCAGTGTTCCAC-AAGTCCTTCCT-3′. As an internal control, the 401 bp β-actin
gene fragment was amplified with the following primers:
5′-GACCTGACTGACTACCTCATGA-3′ and 5′-TGATCTCCTTCTGCATCCTGTC-3′. The PCR reaction
profile was 94 ℃ for 2 min; 94 ℃ for 30 s, 63 ℃ for 30 s, 72 ℃ for 90 s,
totally 32 cycles, with final extension at 72 ℃ for 10 min. The PCR products
were then separated on 1.5% agarose gel and analyzed for RNA expression.

For comparison study of ZNF313 expression, RT-PCR
with RNAs extracted from the testicular tissues of two normal fertile adults,
two fetus and two azoospermic patients was carried out under the same condition
as described above.

2   Results

2.1
Transcription products displayed differen-tially in fertile and infertile men
include known and novel genes

Thirty-six fragments from the mRNA differential
display cDNA pool were obtained by polyacrylamide gel electrophoresis. 12 of
these between 200 bp and 600 bp in length were reamplified, cloned and
sequenced. The ESTs used for 5′-RACE reactions were renamed as EST DD1 to DD6,
and the other 6 ESTs were renamed as EST DD7 to DD12. BLAST analysis of
nucleotide sequences in the GenBank database revealed that these 12 ESTs could
be grouped into four categories: novel ESTs, ESTs homologous to those in the
testis library, known gene sequences and ubiquitously expressed ESTs. For
example, EST DD-7 and DD-8 is identical to the downstream region of a human
TCP1 gene and HsMCAK gene, respectively, and DD-9 is part of TnP1 gene. Finding
of these genes which are involved in or essential for spermatogenesis suggests
that our experimental approach for the isolation of novel human spermatogenesis
genes by mRNA-DD is successful.

For cloning of novel full length
cDNAs with a potential role in human spermatogenesis, 5 ESTs (DD-1, DD-2, DD-3,
DD-4 and DD-6) that have homologous ESTs from testis but not in full length and
1 novel ESTs (DD-5) were used as starting points to isolate the related full
length cDNA sequences by RACE technique. The DD-1-DD-6 differential display
products are displayed (Fig.1).

Fig.1 mRNA-differential display between patients with
idiopathic azoospermia and two normal fertile adults

Arrows indicate the bands expressed in
both normal fertile adults (N1 and N2) but not or at significantly reduced
level in the patients (P). DD-5 is a novel EST, while DD-1, DD-2, DD-3, DD-4
and DD-6 are found in the database as testicular ESTs but not as full length
cDNAs.

2.2 Isolation
of the human ZNF313 gene shorter transcript

The results of 5′-RACE of DD-1 to DD-6 with a SMART
cDNA Construction kit are shown in Fig.2. As from the figure, 5′-RACE fragments
were obtained for DD-2 (R2, about 1.3 kb) and DD-4 (R4, about 0.5 kb). These
fragments were then cloned into pGEM-T easy vector and sequenced. The segments
that overlap with the corresponding mRNA-DD sequences including the poly(A)
tail were assembled to obtain two full-length cDNAs.

Fig.2 Results of 5′-RACE

R2 and R4
are 1.3 kb and 0.5 kb RACE products respectively. M is 1 kb DNA ladder.

The complete sequence of R4+DD4 represents a novel
zinc finger protein gene. Its complete sequence was deposited in the DDBJ, EMBL
and GenBank databases under the accession No.AF265215, and received official
approval from HGNC. Excluding poly(A) the ZNF313 cDNA is 753 bp in length and
contains a 684 bp open reading frame which starts at position 7 and ends at
position 693. It encodes a polypeptide of 228 amino acid residues with a C3HC4
zinc finger domain from 29 to 67 amino acid residue and three non-standard C2H2
zinc finger domains, the first one from 89 to 110, the second one from 141 to
164 and the last one from 171 to 199 amino acid residue. As expected, the
non-canonical polyadenylation signal ATTAAA was found 5 bp upstream of the
polyA tail (Fig.3).

Fig.3 Nucleotide and predicted amino acid sequence of ZNF313 gene

The translation initiation codon is
underlined. The stop codon at the 3′-end of the sequence is underlined and
shaded. The predicted C3HC4 zinc finger motif is boxed and the C2H2 zinc finger
motifs is boxed and shaded. The proximal polyadenylation signal sequence is
shaded and the distal polyadenylation signal sequence is underlined and bolded.
The shorter transcript is from 1 to 753 bp. The sequences of the primers are
double underlined.

The second full-length cDNA (R2+DD2) obtained by RACE
is hBACH gene. It was cloned and reported previously by Yamada et al.[16] and
encodes human brain long-chain acyl-CoA hydrolase. Our results from mRNA-DD and
RACE suggest that this gene is also expressed in testis and may play a role in
spermatogenesis. Recently, the WDC146 gene whose 3′-region is identical to EST
DD6 was reported to be a novel WD40 repeat protein that is highly expressed
during spermatogenesis in a stage-specific manner[17]. The highest expression
of the mouse gene was observed in testis while in situ hybridization to
rat tissues preferentially in the pachytene stage[17]. These results further
confirm the validity of the mRNA DD approach.

2.3 The
shorter ZNF313 gene message is highly expressed in testis

Northern blot analysis of human
sixteen tissues was used to characterize the expression profiles of the ZNF313
gene in various tissues (Fig.4). Of the two different length transcripts
detected, the expected 750 bp ZNF313 gene transcript was expressed most highly
in testis, and at reduced levels in skeletal muscle, kidney, pancreas, liver,
spleen, heart, colon and placenta, but was undetectable in other tissues. In
contrast, a 2.4 kb ZNF313 gene transcript was detected in all 16 tissues with
relatively more quantities in skeletal muscle, kidney, pancreas, liver,
placenta and spleen, but quite less quantities in brain, lung and peripheral
leucocytes. Multiple tissue RT-PCR was performed to confirm these Northern blot
analysis results. As expected, 175 bp fragment of ZNF313 gene was detected most
highly in testis [Fig.5(A)]. The RNA samples used for this experiment had been
treated with RNase-free DNase I and the primer pairs for the reaction were
located in different exons, so that false positive amplification from minute
DNA contaminations could be excluded.

Fig.4      Expression
analysis of ZNF313 in multiple human tissues by Northern blot

From lane 1 to lane 16 are RNAs from
heart, brain, placenta, lung, liver, skeletal muscle, kidney, pancreas, spleen,
thymus, prostate, testis, ovary, small intestine, colon, and peripheral blood
leukocyte. There are two transcripts detected: the 0.75 kb transcript is
expressed most highly in testis, but at a significantly reduced level in
skeletal muscle, kidney, pancreas, liver, spleen, heart, colon and placenta;
the 2.4 kb transcript is observed in all 16 tissues, although the bands in
lanes of brain, lung and peripheral blood leukocyte are very faint. Human
β-actin gene as a control.

When we searched against UniGene database with cDNA
of ZNF313 gene, a UniGene cluster (Hs.10590) was obtained. The cluster included
434 ESTs which came from testis, heart, brain, lung, prostate, kidney as well
as some other tissues used or not used in our expression studies. This result
was consistent with the results of our Northern and RT-PCR analyses.

2.4
Determination of the sequence of the ZNF313 longer transcript

These sequences of the amplified fragments by PCR
with four pair primers(P4F3 and P4R3; P4F4 and P4R4; P4F5 and CDSIII/3′PCR
primer; P4F3 and P4R4) were 100% identical with AF131742. Thus these sequences
and shorter transcript AF265215 were assembled into a 2462 bp contig. Its
sequence is consistent with the 2.4 kb transcript observed in Fig.4 and shown
in Fig.3. The PCR products are shown in Fig.5.

Fig.5 Amplication of 3′-untranslated region of the
2.4 kb transcript of ZNF313

1, the 624 bp product of amplification
with primers P4F3 and P4R3; 2, the 629 bp product of amplification with primers
P4F4 and P4R4; 3, the 585 bp product of amplification with primers P4F5 and
CDSIII/3′PCR primer; 4, 1237 bp product of amplification with primers P4F3 and
P4R4; M, the 100 bp DNA ladder.

2.5  ZNF313 gene is localized at 20q13

The chromosome location of the
human ZNF313 gene was determined by fluorescence in situ hybridization(Fig.6).
One hundred metaphases were analyzed by recording fluorescent signals on
DAPI-banded chromosomes. The identity of these chromosomes were determined by
comparison to Q-banded chromosome images. The figure shows typical dual
hybridization signal localized at 20q13. This assignment is in full accordance
with the result of mapping by bioinformatic approaches using the UniGene
database.

Fig.6 Chromosome localization of ZNF313
gene by FISH

Arrows
indicate that the gene is mapped to the 20q13.

2.6  Genomic structures of human ZNF313

The genomic structure of human ZNF313 gene was
obtained from a 115 935 bp sequence submission (GenBank accession No.AL031685)
from Clone RP5-963K23 on chromosome 20q13, which included the entire ZNF313
gene sequence. The exon-intron boundaries and intron sizes are shown in Table
2. All boundaries conform to the GT/AG rule[18].

Table 2 Genomic structure of the human ZNF313
gene Exon

The splice acceptor/donor columns show
sequences that span splicing junctions. The exonic sequences are in upper case,
and the intron sequences in lower case. Canonical nucleotides (gt/ag) are in
bold type.

2.7  Two polyadenylation signals of human
ZNF313 gene result in two different 3′-untranslated region

Based on the sequences of the two transcripts and the
genomic structure analysis of human ZNF313 gene, we found that the two
different transcripts were resulted from two polyadenylation signal: ATTAAA
from 744–749 bp and AATAAA from 2414-2418 bp. Even though both signals are
located in the same exon, the two 3′-untranslated regions are very different in
length: 60 bp and 1742 bp. In addition, there are other two polyadenylation
signal: AATAAA from 1828-1833 bp and AATAAA from 1901-1905 bp. However, there
is no transcripts of 2.0 kb fragment detected in Northern blot, the two
polyadenylation signals may not be used.

2.8  The expression pattern of the ZNF313 in
ontogenesis and in fertile and azoospermic patients suggests its role in
spermatogenesis

RT-PCR experiments with testicular tissues were
performed to examine the expression of the ZNF313 gene in testis during development
and to compare this expression in fertile adults and infertile patients
[Fig.7(B)]. As from the figure, the expected amplified products are present in
two fertile adults, but significantly reduced level in the two fetuses and in
the azoospermic patients. This means that the gene is expressed only at a low
level in early stage of individual development when the spermatogenesis does
not occur.

Fig.7 Expression analysis of ZNF313 by RT-PCR

The lanes 1-8 are RNAs from testis,
skeletal muscle, lung, liver, brain, spleen, kidney and stomach, respectively.
M is the 100 bp ladder. The expected 175 bp product is expressed most highly in
testis but at a markedly reduced level in other tissues. (B) RNA from normal
adults (1,2), the patients with azoospermia (3, 4) and fetuses (5, 6) and the
175 bp products is highly expressed in normal adults but much less in patients
and fetuses. In both A and B, the human β-actin gene (401 bp) is co-amplified
as an internal control.

3   Discussion

The complex process of sperm formation requires
coordinated regulation of numerous genes. Among the genes shown to be expressed
exclusively or predominantly in the testis, many display different isoforms
resulting from alternative splicing or polyadenylation[19].Poly(A) site
regulation produces not only alternative protein products but also mRNAs with
different 3′-UTRs, which may alter the stability, translation, or localization
of the mRNA, although the same protein will be produced[20]. The formation of
mature mRNAs in vertebrates involves the cleavage and polyadenylation of the
pre-mRNA, 10-30 nt downstream of an AAUAAA or AUUAAA signal sequence. Many mRNAs
displayed two or more polyadenylation sites. Of them, the poly(A) sites
proximal tends to use variant signals more often, while the 3′-most site tends
to use a canonical signal. Besides, the position of the site in the
untranslated region may also play a role in polyadenylation rate[21]. So, it
would be intriguing to study the transcription rates and the function
relationship of the two distinct transcripts of the ZNF313 gene.

The well known zinc finger protein gene family, one
of the largest human gene families, plays an important role in the regulation
of transcription[22,23]. This family can be divided into many subfamilies
including C2H2, glucocorticoid receptor, ring finger, GATA-1, GAL4 and LIM
family genes[24–26]. The C2H2 domain is composed of 25 to 30 amino-acid
residues including 2 conserved Cys and 2 conserved His residues in a
C-2-C-12-H-3(4)-H type motif and bind an atom of Zn. The 12 residues separating
the second Cys and the first His are mainly polar and basic, implicating that
this region in particular may involve in nucleic acid binding[22]. The C3HC4
zinc finger domains resemble a ring finger, a more specialized zinc finger
structure containing about 40 to 60 amino acid residues that binds two atoms of
zinc with the consensus sequence C-x (2)-C-x (9 to 39)-C-x (1 to 3)-H-x (2 to
3)-C-x (2)-C-x (4 to 48)-C-x (2)-C. The 3D structure of the zinc complex
structure is unique to the ring domain and referred to as the ‘cross-brace’
motif [23, 24, 27]. Proteins with such structure generally are involved in both
protein-DNA and protein-protein interactions [26–28]. It is noteworthy that
although the C2H2 and the ring finger are common zinc finger domains, genes
with both two types of domains are scarce. Thus, it will be intriguing to fully
characterize the function of the C3HC4 domain and C2H2 domains in the ZNF313
protein.

In addition, when the open reading frame sequence of
human ZNF313 gene was used for Blast analysis, we also obtained some ESTs from
chicken, rat, dog, pig and cow that share identity more than 80% with human
ZNF313 gene. Being consistent with their evolutional relationship, mammal’s
ESTs display more identity than chicken’s. The presence of these homolgues and
their conservation in evolution suggest that ZNF313 may be important for these
organisms.

This paper describes the novel
spermatogenesis-related zinc finger protein gene, ZNF313, on chromosome band
20q13 with two different 3′-UTRs that has a ring finger and three C2H2 domains.
Results of multiple tissue Northern blot and RT-PCR analysis of testicular
tissues of fertile and infertile men suggest that the gene may play a role in
normal spermatogenesis and male fertility.

References

1     Tiepolo L, Zuffardi O.
Localization of factors controlling spermatogenesis in the nonfluorescent portion
of the human Y chromosome long arm. Hum Genet, 1976, 34: 119-124

2     Vogt PH, Edelmann A,
Kirsch S, Henegariu O, Hirschmann P, Kiesewetter F, Kohn FM et al. Human Y
chromosome azoospermia factors (AZF) mapped to different subregions in Yq11.
Hum Mol Genet, 1996, 5: 933-943

3     Vogt PH. Human
chromosome deletions in Yq11, AZF candidate genes and male infertility: history
and update. Mol Hum Reprod, 1998, 4: 739-744

4     Ma K, Inglis JD,
Sharkey A, Bickmore WA, Hill RE, Prosser EJ, Speed RM et al. A Y chromosome
gene family with RNA-binding protein homology: Candidates for the azoospermia
factor AZF controlling human spermatogenesis. Cell, 1993, 75: 1287-1295

5     Reijo R, Lee TY, Salo
P, Alagappan R, Brown LG, Rosenberg M, Rozen S et al. Diverse spermatogenic defects
in humans caused by Y chromosome deletions encompassing a novel RNA-binding
protein gene. Nat Genet, 1995, 10: 383-393

6     Sun C, Skaletsky H,
Birren B, Devon K, Tang Z, Silber S, Oates R et al. An azoospermic man with a
de novo point mutation in the Y-chromosomal gene USP9Y. Nat Genet, 1999, 23(4):
429-432

7     Bonnycastle LL, Yu CF,
Hunt CR, Trask BJ, Clancy KP, Weber JL, Patterson D et al. Cloning, sequencing
and mapping of the human chromosome 14 heat shock protein gene (HSPA2).
Genomics, 1994, 23(1): 85-93

8     Blendy JA, Kaestner KH,
Weinbauer GF, Nieschlag E, Schutz G. Severe impairment of spermatogenesis in
mice lacking the CREM gene. Nature, 1996, 380: 162-165

9     Dix DJ, Allen JW, Collins BW,
Mori C, Nakamura N, Poorman-Allen P, Goulding EH et al. Targeted gene
disruption of Hsp70-2 results in failed meiosis, germ cell apoptosis, and male
infertility. Proc Natl Acad Sci USA, 1996, 93: 3264-3268

10   Ruggiu M, Speed R, Taggart M, Mckay SJ,
Kilanowski F, Saunders P, Dorin J et al. The mouse Dazla gene encodes a
cytoplasmic protein essential for gametogenesis. Nature, 1997,389(6646):73-77

11   Cooke HJ, Hargreave T, Elliott DJ.
Understanding the genes involved in spermatogenesis: A progress report. Ferti
Steril, 1998,69(6):989-995

12   Palmer MS, Berta P, Sinclair AH, Pym B,
Goodfellow PN. Comparison of human ZFY and ZFX transcripts. Proc Natl Acad Sci
USA, 1990, 87: 1681-1685

13   Ragoussis J， Senger G，
Mockridge I， Sanseau P， Ruddy S，
Dudley K， Sheer D et al. A testis- expressed Zn
finger gene (ZNF76) in human 6p21.3 centromeric to the MHC is closely linked to
the human homolog of the t-complex gene tcp-11. Genomics, 1992, 14(3): 673-679

14   Fujii T, Tamura K, Copeland NG, Gilbert
DJ, Jenkins NA, Yomogida K, Tanaka H et al. Sperizin is a murine ring zinc-finger
protein specifically expressed in haploid germ cells. Genomics,1999, 57: 94-101

15   Liang P, Pardee AB. Differential display
of eukaryotic messenger RNA by means of the polymerase chain reaction.
Science,1992, 257: 967-971

16   Yamada J, Kurata A, Hirata M, Taniguchi
T, Takama H, Furihata T, Shiratori K et al. Purification, molecular cloning,
and genomic organization of human brain long-chain acyl-CoA hydrolase. J
Biochem (Tokyo), 1999, 126(6): 1013-1019

17   Ito S, Sakai A, Nomura T, Miki Y, Ouchida
M, Sasaki J, Shimizu K. A novel WD40 repeat protein, WDC146, highly expressed
during spermatogenesis in a stage-specific manner. Biochem Biophys Res
Commun,2001, 280(3): 656-663

18   Mount SM. A catalogue of splice junction
sequences. Nucleic Acids Res, 1982, 10: 459-472

19   Venables JP, Eperon I. The roles of
RNA-binding proteins in spermatogenesis and male infertility. Curr Opin Genet
Dev, 1999, 9(3): 346-354

20   Proudfoot N. Ending the message is not so
simple. Cell, 1996, 87:779-781

21   Beaudoing E, Freier S, Wyatt JR, Claverie
JM, Gautheret D. Patterns of variant polyadenylation signal usage in human
genes. Genome Res, 2000, 10(7): 1001-100

22   Klug A, Schwabe JW. Protein motifs 5.
Zinc fingers. FASEB J, 1995, 9: 597-604

23   Freemont PS. The ring finger. A novel
protein sequence motif related to the zinc finger. Ann N Y Acad Sci,1993, 684:
174-192

24   Borden KL, Freemont PS. The ring finger
domain: A recent example of a sequence-structure family. Curr Opin Struct Biol,
1996, 6: 395-401

25   Hammarstrom A, Berndt KD, Sillard R,
Adermann K, Otting G. Solution structure of a naturally-occurring zinc-peptide
complex demonstrates that the N-terminal zinc-binding module of the Lasp-1 LIM
domain is an independent folding unit. Biochemistry, 1996, 35: 12723-12732

26   Barlow PN, Luisi B, Milner A, Elliott M,
Everett R. Structure of the C3HC4 domain by 1H-nuclear magnetic resonance
spectroscopy. A new structural class of zinc-finger. J Mol Biol, 1994, 23:
201-211

27   Freemont PS, Hanson IM, Trowsdale J. A
novel cystein-rich sequence motif. Cell, 1991, 64: 483-484

28   Borden KL, Boddy MN, Lally J, O’Reilly
NJ, Martin S, Howe K, Solomon E et al. The solution structure of the ring
finger domain from the acute promyelocytic leukaemia proto-oncoprotein PML.
EMBO J, 1995, 14: 1532-1541

Received: September 23, 2002       Accepted:
November 22, 2002

This work was supported by grants from the National Natural Science
Foundation of China (No. 30200153, No. 30170359, No. 39993420, No. 30170525 and
No. 39970404) and the National High Technology Research and Development Program
of China (863 Program) (No. 2001AA216091)

*Corresponding author: Tel, 86-28-85422749; Fax, 86-28-5501518; e-mail, [email protected]