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Copyright © 2000, The American Society for Cell Biology Wpkci, Encoding an Altered Form of PKCI,
Is Conserved Widely on the Avian W Chromosome and Expressed in Early
Female Embryos: Implication of Its Role in Female Sex Determination Judith Kimble, Monitoring Editor ‡Corresponding author. E-mail address:
s-mizuno/at/brs.nihon-u.ac.jp. Received June 5, 2000; Revised July 10, 2000; Accepted July 27, 2000.  This article has been cited by other articles in PMC. | |||||||||||||||||||
Abstract Two W chromosome–linked cDNA clones, p5fm2 and p5fm3, were
obtained from a subtracted (female minus male) cDNA library prepared
from a mixture of undifferentiated gonads and mesonephroi of male or
female 5-d (stages 26–28) chicken embryos. These two clones were
demonstrated to be derived from the mRNA encoding an altered form of
PKC inhibitor/interacting protein (PKCI), and its gene was named
Wpkci. The Wpkci gene reiterated ~40
times tandemly and located at the nonheterochromatic end of the chicken
W chromosome. The W linkage and the moderate reiteration of
Wpkci were conserved widely in Carinatae birds. The
chicken PKCI gene, chPKCI, was shown to
be a single-copy gene located near the centromere on the long arm of
the Z chromosome. Deduced amino acid sequences of Wpkci and chPKCI
showed ~65% identity. In the deduced sequence of Wpkci, the HIT
motif, which is essential for PKCI function, was absent, but the
α-helix region, which was conserved among the PKCI family, and a
unique Leu- and Arg-rich region, were present. Transcripts from both
Wpkci and chPKCI genes were present at
significantly higher levels in 3- to 6-d (stages 20–29) embryos. These
transcripts were detected in several embryonic tissues, including
undifferentiated left and right gonads. When the green fluorescent
protein–fused form of Wpkci was expressed in male chicken embryonic
fibroblast, it was located almost exclusively in the nucleus. A model
is presented suggesting that Wpkci may be involved in
triggering the differentiation of ovary by interfering with PKCI
function or by exhibiting its unique function in the nuclei of early
female embryos. | |||||||||||||||||||
INTRODUCTION The constitution of sex chromosomes in vertebrates is either XX
(female)/XY (male), as in mammals, or ZW (female)/ZZ (male), as in
birds and some reptiles. The gene SRY/Sry, which triggers
male sex determination, is present on the Y chromosome of mammals
(Gubbay et al., 1990 In the present study, we prepared a subtracted (female minus male) cDNA
library from the pooled tissues of undifferentiated gonads and
mesonephroi of 5-d male or female chicken embryos (corresponding to
stages 26–28) (Hamburger and Hamilton, 1951 | |||||||||||||||||||
MATERIALS AND METHODS DNA Preparations High-molecular-weight DNAs were prepared according to Ogawa
et al. (1997)  from blood samples of the following male and
female species of birds: ostrich and emu belonging to the order
Struthioniformes of Ratitae, and 24 species belonging to the following
12 orders of Carinatae: Galliformes (white leghorn chicken, red jungle
fowl, chukar partridge, green pheasant, common turkey, Japanese quail,
Guinea fowl, common peafowl), Anseriformes (domestic duck),
Psittaciformes (scarlet macaw), Musophagiformes (violet
plantain-eater), Strigiformes (snowy owl), Columbiformes (rock dove),
Gruiformes (Japanese crane), Falconiformes (secretary bird),
Ciconiformes (Oriental white stork), Sphenisciformes (king penguin,
bearded penguin, Humboldt penguin, macaroni penguin), Procellariiformes
(streaked shearwater), and Passeriformes (common finch, white Java
sparrow, Rothschild's starling).Identification of the Sexes of Chicken Embryos Fertilized eggs of white leghorn chickens were incubated at
38°C. The sexes of 2- to 7-d embryos (stages 12–31) were determined
by Southern blot hybridization with the W chromosome–specific,
300-base pair (bp) EE0.6 genomic probe (Ogawa et al., 1997 )
and the Z chromosome–specific ZOV3 probe (a 2-kilobase
[kb] HindIII–SacI fragment of the cDNA clone)
(Saitoh et al., 1993 ; Kunita et al., 1997 ) to the
EcoRI-digested genomic DNA prepared from the extraembryonic
membrane of an individual embryo. The sexes of later-stage embryos were
identified by the morphology of gonads.cDNA Cloning of Female-specific Transcripts by the PCR-Select cDNA
Subtraction Method Total RNA was prepared with Trizol reagent (GIBCO-BRL,
Gaithersburg, MD) from a mixture of undifferentiated gonads and
mesonephroi isolated from 37 male and 37 female embryos at about d 5 of
incubation (stages 26–28), and poly(A)+ RNA was
obtained with the use of Oligotex-dT30 “Super” (Roche Diagnostics,
Mannheim, Germany). The following procedure was adopted
essentially according to the PCR-Select cDNA Subtraction Kit user
manual (Clontech, Palo Alto, CA). Double-stranded and blunt-ended cDNA
was synthesized, digested with RsaI, and ligated with
RsaI adaptors (Gurskaya et al., 1996 ). The
adaptor-ligated cDNA was amplified by PCR and redigested with
RsaI. The RsaI-digested cDNA from the female
(tester) was ligated with either adaptor-1 or adaptor-2R. The first
hybridization was performed between the excess of
RsaI-digested cDNA from the male (driver) and each one of
the adaptor-ligated tester cDNAs. The two reaction mixtures were then
mixed together, and the second hybridization was performed by adding
freshly denatured driver cDNA. The hybrids between the
adaptor-1–ligated and the adaptor-2R–ligated tester cDNAs were
amplified by PCR, digested with RsaI, and cloned into the
SmaI site of pBluescriptII KS(+) (Stratagene, La Jolla, CA).The subtracted cDNA library was screened by reverse Northern blot
hybridization (von Stein et al., 1997 Isolation of cDNA Clones for Wpkci and PKCI from Chicken, Quail,
and Duck Poly(A)+ RNAs were prepared from the mixed
tissues of undifferentiated gonads and mesonephroi of female chicken
embryos at stages 26–28 and from the sex-undetermined 3-d whole
embryos of Japanese quail and domestic duck. The double-stranded and
blunt-ended cDNA was synthesized as described above, except that the
first-strand cDNA was synthesized with the use of pd(T)12-18 (Amersham
Pharmacia Biotech, Uppsala, Sweden) as a primer. The cDNA was ligated
with EcoRI–NotI–BamHI adaptor
(Takara), and its 5′ ends were phosphorylated and size-selected for
0.3- to 2-kb fragments by 1.2% agarose gel electrophoresis. The cDNA
library was constructed by ligating the cDNA fragments with Lambda
ZAPII/EcoRI, CIAP (Stratagene), followed by in vitro
packaging with the use of Max Plax Packaging Extract (Epicentre
Technologies, Madison, WI). The chicken cDNA library was
screened with the insert of p5fm2, which had been
32P-labeled by the random priming method
described above, and pWpkci-7, pWpkci-8, and pfst5.2-5 (carrying a
sequence region containing exon III of Wpkci) clones were
obtained. The N-terminal 214-bp cDNA fragments of human and mouse PKCI
were obtained by reverse transcription PCR from the total RNAs of HeLa
cells and murine C127 cells, respectively, with the use of the
following primers: 5′-TGGC(A/T)GA(T/C)GAGATTGCCAAGG-3′ (forward) and
5′-CTTTCATCATCATC(T/A)TCTGC-3′ (reverse). This chicken cDNA library was
screened with a mixture of the PCR-amplified human and mouse PKCI cDNA
probes, which were 32P-labeled as described
above, and the pchPKCI-3 clone was obtained. The Japanese quail and the
domestic duck cDNA libraries were screened with the insert of pWpkci-8
or the chPKCI cDNA fragment (nucleotide positions 30–410)
as a 32P-labeled probe, and pquWpkci-16,
pquPKCI-2, pduWpkci-20, and pduPKCI-8 clones were obtained.Construction and Screening of a Bacterial Artificial Chromosome
Library from Female Chicken Genomic DNA Cultured fibroblasts established from an 8-d female embryo of
white leghorn chicken were suspended in PBS (8.1 mM
NaH2PO4, 1.47 mM
KH2PO4, 137 mM NaCl, 2.7 mM
KCl) at a concentration of 1 × 108
cells/ml. The cell suspension and an equal volume of 1.2% solution of
SeaPlaque GTG agarose (FMC Bioproducts, Rockland, ME) in PBS were
incubated at 42°C, mixed, and poured into a sample mold of GeneLine
System (Beckman, Fullerton, CA) and solidified at 4°C. The gel plug
was soaked successively in ES solution (0.5 M EDTA, 1%
N-lauroylsarcosine Na salt) at room temperature for 30 min,
twice in ESP solution (1 mg/ml proteinase K in ES solution) at 50°C
for 24 h with gentle shaking, and five times in TE50 (10 mM
Tris-HCl, pH 8, 50 mM EDTA) at room temperature for 30 min. The
extremely high-molecular-weight genomic DNA in the gel plug was
digested partially with HindIII and subjected to
pulsed-field gel electrophoresis in CHEF mapper (Bio-Rad, Richmond,
CA), and DNA fragments of 150–200 kb were recovered. These DNA
fragments were ligated with pBAC-Lac vector (Asakawa et al.,
1997 ), which had been digested with HindIII and treated with
alkaline phosphatase (Roche Diagnostics), and subjected to
electroporation into Escherichia coli DH10B
electrocompetent cells (Hanahan et al., 1991 ) by Gene Pulser
(Bio-Rad) according to Asakawa et al. (1997) . Individual
recombinant clones were picked up and transferred into individual wells
of 96-well microtiter plates, each well containing 150 μl of LB (1%
bacto-tryptone, 0.5% bacto-yeast extract, 1% NaCl) containing 7.5%
glycerol and 12.5 μg/ml chloramphenicol. Altogether, 512 plates
containing 4.9 × 104 clones were made, and
those plates were incubated at 37°C for 12 h. High-density
replica filters (eight 22 × 7 cm filters per set) were prepared
from these plates with the use of Bio-Grid (BioRobotics, Cambridge,
UK), spotting 6144 clones per filter. The mean insert size of
these clones was ~150 kb. The whole bacterial artificial chromosome
(BAC) library contained ~3.2 times as much chicken genomic DNA as the
diploid genome equivalent, assuming that the diploid genome
corresponded to ~2300 megabase (Mizuno et al., 1978 ).Two sets of filters (16 high-density replica filters) were hybridized with the 32P-labeled insert of p5fm2 or p5fm3 (Wpkci probes), and 10 BAC clones that gave hybridization signals on both filters were picked up. Similarly, three BAC clones were picked up after hybridization with the 32P-labeled insert of pchPKCI-3 as a probe. DNA Sequencing and Data Analysis Sequencing was carried out with the use of the BigDye
Terminator Cycle Sequencing Kit (Applied Biosystems, Foster City, CA)
and the ABI 377 DNA sequencer. Sequence data were analyzed by
DNASIS-Mac version 3.0 (Hitachi Software Engineering, Tokyo, Japan).
Deduced amino acid sequences were aligned with the use of Clustal W1.7
(http://dot.imgen.bcm.tmc.edu:9331/multi-align/multi-align.html),
and common residues were shown with the use of Boxshade 3.21
(http://www.isrec.isb-sib.ch:8080/software/BOX_form.html).Southern Blot Hybridization and Determination of Reiteration
Frequencies of the Wpkci and chPKCI Genes EcoRI-digested genomic DNAs from various species of
birds were electrophoresed (5 μg/lane) on a 1% agarose gel,
transferred to a Biodyne B nylon membrane (Pall Specialty
Materials), hybridized with the 32P-labeled
StHi-0.37 genomic fragment (exon III probe for Wpkci; see
Figure 2A) in CG buffer at 63°C for 12 h, and washed in 0.3×
SSC, 0.1% SDS at 63°C for 30 min.
The BAC clone 216G1, selected with the p5fm2 cDNA probe for Wpkci, was first digested with NotI, followed by partial digestion with PstI with the use of six different enzyme concentrations. Each digest was separated by electrophoresis on a 0.4% Seakem Gold agarose (FMC) gel in 0.5× TBE (1× TBE is 0.089 M Tris-borate, 0.089 M boric acid, 0.002 M EDTA), treated with 0.25 M HCl for 10 min, and transferred onto a Biodyne B membrane in 0.4 N NaOH. The blot was hybridized with the 32P-labeled insert of the cDNA clone pfst5.2-5 (for Wpkci) in CG buffer at 65°C for 12 h, followed by washing in 0.3× SSC, 0.1% SDS at 65°C for 30 min, and subjected to autoradiography. The BAC clone 224D8 was digested with NotI, followed by partial digestion with BamHI, with the use of eight different enzyme concentrations. Each digest was separated by pulsed-field gel electrophoresis in GeneLine System (Beckman) and subjected to Southern blot hybridization as described above, but with 32P-labeled fragment (nucleotide positions 30–410) of the chPKCI-3 cDNA sequence as a probe. Different amounts (0.25–3.0 μg) of EcoRV-digested genomic DNA of female white leghorn chicken and 3.0 μg each of EcoRV-digested genomic DNA of male white leghorn chicken mixed with different amounts (10–90 pg) of EcoRI-cut p5fm2 DNA (3.83 kb) were electrophoresed on a 1% LO3 agarose (Takara) gel in 0.5× TBE and transferred onto a Biodyne B membrane in 0.4 N NaOH. The DNA blot was hybridized with the 32P-labeled insert of p5fm2 and washed under the same conditions described above. Hybrids were detected and intensities of signals were quantified with a FLA-2000 image analyzer (Fuji Film, Tokyo, Japan). The reiteration frequency of the sequence detected with the p5fm2 probe in the diploid genome was calculated with the use of the following equations: Ng × X/Nc = Sg/Sc, and X = Nc × Sg/Ng × Sc, where X = reiteration frequency, Ng = genomic DNA (pg) fixed on the membrane/diploid genome size of chicken (2.54 pg), Nc = molecular numbers of p5fm2 clone fixed on the membrane, calculated from the size of the clone (3.83 kb) with 650 as the mean molecular weight of a base pair, Sg = intensity value of the hybridization signal to the genomic DNA, and Sc = intensity value of the hybridization signal to p5fm2 DNA. A fixed amount (3 μg) of the BamHI-digested genomic DNA from male chicken and different amounts (5–100 pg) of HindIII-digested DNA of the cDNA clone pchPKCI-3 (3.589 kb) were subjected to Southern blot hybridization as described above, with a 32P-labeled subfragment (nucleotide positions 30–410, generated by PCR) of pchPKCI-3 as a probe. By comparing the former signal intensity with the slope of the latter signal intensities, the reiteration frequency of the chPKCI sequence in the haploid male genome was calculated. Northern Blot Hybridization Poly(A)+ RNAs were prepared as described
above from sex-identified whole embryos: 6 each for 3-d (stage 20), 4
each for 4-d (stage 24), and one each for 5- to 14.5-d (stages 27–40)
embryos; from undifferentiated gonads plus mesonephroi of
sex-identified 5- to 16-d (stages 27–42) embryos: 37 each for 5-d, 30
each for 6-d, 29 male and 18 female for 7-d, 22 male and 18 female for
8-d, and 3 each for 16-d embryos; and also from different tissues of an
80-d male or female chicken. Poly(A)+ RNA (1 μg
each) was electrophoresed on an agarose/formaldehyde gel (Sambrook
et al., 1989 ) and capillary transferred onto a Biodyne B
membrane in 7.5 mM NaOH. The RNA blot was hybridized with a
32P-labeled probe in CG buffer at 65°C for
12 h, followed by washing in 2× SSC, 0.1% SDS at 65°C for 15
min and autoradiography. The 63-bp Wpkci-specific probe
(nucleotide positions 365–427 in Figure 1A) was prepared by PCR with
the use of 5′-ATGGCTGTGAGATACC-3′ (forward) and
5′-ACCCAGAATACAGAATATGG-3′ (reverse) primers, and similarly, the 72-bp
chPKCI-specific probe (nucleotide positions 312–383 in
Figure 1B) was prepared with the use of 5′-CGGATGGTTTTGAATG-3′
(forward) and 5′-ACCTCCCAGAATATGG-3′ (reverse) primers. The mRNA of
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was probed with the
cloned chicken GAPDH cDNA sequence (Dugaiczyk et
al., 1983 ). Intensities of hybridization signals with the
Wpkci or chPKCI probe were divided by the
intensities of GAPDH signals, and their relative levels were
compared through embryonic development.
When the mRNA molar ratio of Wpkci and chPKCI was determined, different amounts (0.25–2 μg) of poly(A)+ RNA preparations from female 6-d (stage 29) whole embryo or from a mixture of undifferentiated gonads and mesonephroi of female 6-d embryo were electrophoresed and transferred to the membrane as described above. The cDNA clones pWpkci-8 and pchPKCI-3 were linearized by digestion with BamHI, and different amounts (10–200 pg) were electrophoresed on an agarose gel and transferred to the membrane as for the RNA samples. The RNA and the DNA blots were hybridized together with the Wpkci or chPKCI-specific probe described above, washed as described above, and the radioactivity of the hybrids was converted to the fluorescence intensity values by FLA-2000 image analyzer. The intensity of the Northern blot signal was corrected with the signal intensity for GAPDH mRNA and also with the ratio of signal intensity (pWpkci-8/pchPKCI-3) by Southern blot hybridization. In Situ Hybridization to Mitotic Chromosome Preparations Fluorescence in situ hybridization (FISH) to mitotic chromosomes
prepared from chicken embryonic fibroblasts was carried out as
described (Hori et al., 1996 ). Probes used were pCZTH5–8
(pFN-1–type macrosatellite sequence on the chicken Z chromosome; Hori
et al., 1996 ), pUGD1201 (EcoRI family sequence on
the chicken W chromosome; Saitoh et al., 1991 ), pGP-3
(5.6-kb PstI genomic fragment containing a Wpkci
gene; this study), and 224D8 (BAC clone containing the
chPKCI gene; this study), which were labeled by nick
translation with either digoxigenin (DIG)-11-dUTP (Boehringer Mannheim,
Indianapolis, IN) or biotin-16-dUTP (Boehringer Mannheim) according to
Saitoh and Mizuno (1992) .In Situ Detection of Wpkci and chPKCI Transcripts in the Whole
Mount Preparation or Paraffin Sections of Early Chicken Embryos For the whole mount hybridization, a part of the 4.5-d (stage
25) embryo containing undifferentiated gonads and mesonephroi was
dissected out and fixed in 4% paraformaldehyde in PBT (0.1% Tween 20
in PBS) at 4°C overnight, and the preparation was hybridized with
DIG-labeled antisense or sense riboprobe for Wpkci or
chPKCI, as described below, according to Wilkinson and Nieto
(1993) . For hybridization to paraffin sections, stages 26 to 27 chicken
embryos were fixed in 4% paraformaldehyde and 0.2% glutaraldehyde in
PBS at 4°C for 8 h and embedded in paraffin. Sections 4 μm
thick were cut with a microtome (Sledge Microtome IVS-400, Sakura,
Tokyo, Japan) and attached to the surface of
3-aminopropyltriethoxysidane-coated slide glasses. DIG-labeled
antisense or sense riboprobe was prepared by transcribing
XbaI- or HindIII-digested pBluescriptII KS(+)
containing the Wpkci cDNA fragment (nucleotide positions
63–534) or the chPKCI cDNA fragment (nucleotide positions
30–410) with T3- or T7-RNA polymerase, respectively, with the use of
DIG-RNA labeling mix (Boehringer Mannheim). The DIG-labeled hybrids
were detected with the use of anti-DIG antibody–coupled alkaline
phosphatase (Boehringer Mannheim) and the AP-Conjugate Substrate Kit
(Bio-Rad).Construction and Expression of GFP-fused Forms of Wpkci and chPKCI The sequence of the Wpkci-encoding region was amplified by PCR
with the use of the cDNA clone pWpkci-8 as a template, the following
primers: 5′-GAAGATCTATGGCCGGCGGGATCGT-3′ (forward) and
5′-GGCTGCAGCTAGGCTGACGGGCAAC-3′ (reverse), and Pfu DNA
polymerase (Toyobo, Osaka, Japan). The sequence of the
chPKCI-encoding region was amplified as described above but with the
use of pchPKCI-3 and the following primers:
5′-GAAGATCTATGGCTGACGAGATCC-3′ (forward) and
5′-TTCTGCAGTTAGCCAGGAGGCCAGCCCA-3′ (reverse). The amplified fragments
were digested with BglII (the site is present in the forward
primer) and PstI (the site is present in the reverse primer)
and ligated to pS65T-C1 vector (Clontech), which had been digested with
BglII and PstI. The recombinant plasmids and the
plasmid vector were purified with the use of the Concert High-Purity
Plasmid Purification System (GIBCO-BRL). Fibroblasts established from
an 8-d (stage 34) male chicken embryo (2 ×
105 cells/24 × 24 mm coverglass) were
cultured in DMEM (Sigma Chemical, St. Louis, MO) containing 0.03%
l-glutamine, 0.16% Na-bicarbonate, 8% FBS (JRH
Biosciences, Lenexa, KS), 2% chicken serum (JRH Biosciences),
50 U/ml penicillin G, and 50 μg/ml streptomycin at 37°C in 5%
CO2/95% air and subjected to lipofection with 1
μg of the recombinant plasmid, or the plasmid vector as a control,
and 5 μl of Lipofectin Reagent (GIBCO-BRL), according to the
manufacturer's protocol. After incubation in the serum-free medium for
6 h, the medium was changed to one containing sera, and the
culture was continued for 12 h at 37°C for cells transfected
with Wpkci-containing vector or for 24 h for cells
transfected with chPKCI-containing vector or the control
vector. The temperature of each culture was then shifted to 30°C for
8 h. Cells were washed in PBS, fixed in PBS containing 4%
paraformaldehyde for 30 min, stained for 1 min with 1 μg/ml DAPI
(Sigma) in PBS, covered with Vectashield antifade (Vector Laboratories,
Burlingame, CA), and observed under a Leica (Bensheim, Germany)
DMRB fluorescence microscope with the Cytovision (Applied Imaging,
Santa Clara, CA) image-processing system. | |||||||||||||||||||
RESULTS cDNA and Deduced Amino Acid Sequences of Wpkci and chPKCI Poly(A)+ RNA was prepared from pooled
tissues of undifferentiated gonads and mesonephroi of male or female
chicken embryos at about d 5 of incubation (stages 26–28), and those
RNA preparations were subjected to cDNA synthesis and PCR-Select cDNA
subtraction. In this process, the female-derived tester cDNA molecules
ligated with one of the two different adaptors were hybridized with the
excess male-derived driver cDNA without those adaptors, the cDNA
duplexes formed between the two different adaptor-ligated tester cDNA
molecules were amplified by PCR with the use of primers complementary
to a part of each adaptor sequence, and the PCR products were cloned
with the use of pBluescriptII KS(+). Two hundred colonies were picked
up randomly, and their inserts were amplified by PCR and subjected to
reverse Northern blot hybridization (von Stein et al., 1997 )
with 32P-labeled PCR-amplified cDNA fragments
from male or female embryos that had been used for the PCR-Select cDNA
subtraction. Twenty-nine clones were confirmed to be derived from mRNA
species expressed in a female-specific manner. Of these clones, two
(5fm2 and 5fm3) were suggested to be derived from genes on the W
chromosome because of their female-specific patterns in Southern blot
hybridization. A cDNA library, which was prepared from mixed tissues of
undifferentiated gonads and mesonephroi of 5-d (stages 26–28) female
embryos and size-selected for 0.3- to 2-kb cDNA molecules, was screened
with 5fm2 or 5fm3 as a probe. A clone selected with the 5fm2 probe and
containing an ~660-bp insert was sequenced (Figure
1A) and designated pWpkci-8, because the
130 amino acid residues deduced from the sequence of its ORF showed
61% identity to PKCI of human (Brzoska et al., 1995 ; Lima
et al., 1996 ). The 5fm3 clone was later found to be derived
from the same gene.
Next, a cDNA clone for the chicken PKCI was obtained from the same cDNA
library described above but by probing with the mixed sequences of
human and mouse PKCI cDNAs (Lima et al.,
1996 Molecular mass values of Wpkci and chPKCI, calculated from the deduced
amino acid sequences, were almost identical, 13.9 and 13.8 kDa,
respectively. On the other hand, the deduced isoelectric point values
were significantly different: 10.93 for Wpkci and 6.33 for chPKCI. When
the deduced sequences of Wpkci and chPKCI were compared with respect to
their different regions (Figure 1C), the HIT motif, a characteristic
motif of PKCI containing the conserved triad of histidine residues
(Seraphin, 1992 Structure of Wpkci and chPKCI Genes The BAC genomic library of the female chicken was screened with
the 5fm2 or 5fm3 cDNA clone (Figure 2A)
as a probe. One clone, 216G1, which was hybridized with both probes,
contained tandem repeats of a 5-kb BamHI fragment and a
5.6-kb PstI fragment, both of which contained the
Wpkci sequence. These two repeating units were then
subcloned to yield pGB-1 (5-kb BamHI fragment) and pGP-3
(5.6-kb PstI fragment) (Figure 2A). Sequencing of the pGB-1
and pGP-3 clones revealed that the Wpkci gene consisted of
three exons and suggested that the gene was reiterated tandemly (Figure
2A).
A BAC clone, 224D8, containing the chPKCI gene sequence was obtained by screening the female chicken BAC library with the pchPKCI cDNA clone. Subregions (1.3- and 3.3-kb HindIII fragments) of this BAC clone, each containing a part of the chPKCI sequence, were subcloned (pGH1.3-1 and pGH3.3-3 in Figure 2C). Sequences of these subcloned regions and a part of the 224D8 BAC clone, flanked with these subcloned sequences, were determined, which indicated that the chPKCI gene also consisted of three exons (Figure 2C). Intron-1 of the chPKCI gene was 2682 bp long and that of the Wpkci gene was 985 bp long, and no significant homology was found between these sequences. On the other hand, intron-2 of the chPKCI gene was 762 bp long and that of the Wpkci gene was 831 bp long, and their sequence identity was 57%. The relatively high similarity of exon sequences (Figure 1C) and the intron-2 sequence between chPKCI and Wpkci genes suggested that these two genes had evolved from the same origin. Sequencing of the pGB-1 and pGP-3 genomic clones disclosed that
part of the CR1 repeat, corresponding to the 3′ half of ORF2 of the CR1
consensus sequence (Haas et al., 1997 Chromosomal Locations of Wpkci and chPKCI Genes Chromosomal locations of Wpkci and chPKCI
genes were examined by FISH to mitotic chromosome sets from the female
chicken embryonic fibroblasts with the use of the following probes: the
5.6-kb insert of pGP-3 (Figure 2A) for the Wpkci gene and
the BAC clone 224D8, which contains the chPKCI gene sequence
(Figure 2C). W and Z chromosomes were identified by cohybridization
with the chromosome-specific repetitive sequence probes: pUGD1201 for
the EcoRI family sequence on the long arm of the chicken W
chromosome (Saitoh et al., 1991 ; Saitoh and Mizuno, 1992 )
and pCZTH5-8 for the pFN-1 macrosatellite sequence at the terminal
region of the long arm of the chicken Z chromosome (Hori et
al., 1996 ). The results are shown in Figure
3A and summarized in Figure 3B. The
reiterated Wpkci genes were located close to the terminus of
the nonheterochromatic end on the short arm of the W chromosome, and
the chPKCI gene was located near the centromere on the long
arm of the Z chromosome, i.e., at 53.4% of the entire length of the Z
chromosome, measured from the end of the short arm.
Reiteration Frequencies of Wpkci and chPKCI Genes Tandem reiteration of the Wpkci gene, as suggested from
genomic structural analysis (Figure 2A), was confirmed by Southern blot
hybridization. The 140-kb insert of the BAC clone 216G1 was digested
partially with PstI and electrophoresed, and the blot was
hybridized with the 0.58-kb insert of the cDNA clone pfst5.2-5, which
had been derived from the pre-mRNA and consisted of sequences
of a part of intron-2 and exon III of Wpkci (Figure 2A) as a
probe. The probe hybridized to bands corresponding to multiples of the
5.6-kb PstI fragment (Figure
4A). When the BAC clone 224D8, which
contains the chPKCI sequence, was digested with
NotI, further digested with BamHI at eight
different concentrations, and subjected to pulsed-field gel
electrophoresis and Southern blot hybridization with the
32P-labeled chPKCI cDNA fragment
(nucleotide positions 30–410; Figure 1B), 9.4-kb and three
higher-molecular-mass bands were detected, but the sizes of the latter
bands were not multiples of 9.4 kb (Figure 4B).
To estimate the reiteration frequency of the Wpkci gene, Southern blot hybridization was carried out with the 32P-labeled insert of the p5fm2 cDNA clone, which had been derived from the pre-mRNA (Figure 2A), as a probe to different amounts (0.25–3 μg) of EcoRV-digested genomic DNA of the female chicken (Figure 4C, upper panel) and to a fixed amount (3 μg) of EcoRV-digested genomic DNA of the male chicken mixed with different amounts (10–90 pg) of the linearized p5fm2 DNA (Figure 4C, lower panel). Hybridization signals were quantified by the image analyzer, which converted levels of radioactivity to fluorescence intensity values. The reiteration frequency of the Wpkci gene was calculated to be 44 times per diploid genome of the female, or 44 copies on the W chromosome, by comparing slopes of fluorescence intensity values obtained from the two sets of reactions described above. To estimate the number of chPKCI genes per genome, a fixed amount (3 μg) of the BamHI-digested genomic DNA of the male chicken and different amounts (5–100 pg) of the HindIII-digested pchPKCI-3 DNA were subjected to Southern blot hybridization with the 32P-labeled subfragment (nucleotide positions 30–410) of pchPKCI-3 (Figure 4D). By comparing the intensity of hybridization to the genomic DNA with the slope of signal intensities to the cloned DNA, the copy number of chPKCI gene per haploid male genome was calculated to be 1.3. These results indicated that a single copy of the chPKCI gene existed on the Z chromosome. Conservation of the Wpkci Gene in the Female Genomes of Carinatae
Birds Southern blot hybridization was carried out with the cDNA
probe for the exon III region (StHi-0.37; Figure 2A) of the
Wpkci gene, under stringent conditions, to the
EcoRI-digested genomic DNA preparations from 26 pairs of
male and female species belonging to 12 different orders of Carinatae
birds and 1 order of Ratitae birds (ostrich and emu). The results
demonstrated that the Wpkci gene was present and reiterated
in all of the genomes of the female species of Carinatae birds tested
(Figure 5). The extent of reiteration
seemed to be similar among the species belonging to the order
Galliformes (chicken, red jungle fowl, chukar partridge, green
pheasant, common turkey, Japanese quail, Guinea fowl, common peafowl)
but somewhat less so in the species belonging to other orders (Figure
5). The band(s) hybridized in each sample from the male was interpreted
to represent the sequence of chPKCI gene on the Z
chromosome, because overall similarity of the exon III sequence between
Wpkci and chPKCI was ~73%. The reiteration of
Wpkci-related sequence in the female genome was not observed
for the two Ratitae species; thus, we were unable to conclude from
these results if the Wpkci gene was present in the genomes
of these species.
Comparison of Deduced Amino Acid Sequences of PKCI from Mammals,
Birds, and a Plant with Those of Wpkci from Birds The deduced sequences of PKCI from human (GenBank accession number
[GB] U51004), mouse (GB U60001), rabbit (GB Y11175), bovine (GB
U09405), maize (GB Z29643), chicken, quail, and duck (this study) and
of Wpkci from chicken, quail, and duck (this study) were aligned and
compared (Figure 6, A and B). For the
chicken Wpkci, sequences deduced from two different cDNA clones were
compared in which only a single amino acid change was noticed: His-81
(Wpkci-7; minor type) or Arg-81 (Wpkci-8; major type). The deduced
sequence of chicken PKCI was almost identical (98% identity) to the
sequences of quail and duck and was highly similar (87–90% identity)
to those of mammals. On the other hand, the deduced sequences of Wpkci
of birds and those of PKCI were much less similar, except for the
α-helix region and the C-terminal region (Figures 1C and 6A),
although the duck PKCI lacked the C-terminal 14 residues (Figure 6, A
and B).
The highly conserved α-helix region has been suggested to be the site
of intermolecular contact in the formation of a homodimer of PKCI (Lima
et al., 1996 Transcriptional Expression of Both Wpkci and chPKCI Genes Is Higher
during Early Stages of Embryonic Development The levels of expression of Wpkci and chPKCI
genes were examined by Northern blot hybridization to
poly(A)+ RNA preparations from whole embryos at 3
to 14.5 d of incubation (stages 20–40) or from different tissues
of 80-d chickens with the use of the PCR-generated probe for a part of
the exon III sequence of each gene, which detected Wpkci
mRNA or chPKCI mRNA specifically. The results shown in
Figure 7, A (Wpkci mRNA) and B
(chPKCI mRNA), demonstrate that both genes were expressed as
~0.65-kb mRNAs and that the levels of both mRNAs were higher during
the 3- to 6-d stages (stages 20–29) than in later stages of embryonic
development or in different tissues of 80-d chickens. The higher-level
expression of both Wpkci and chPKCI genes in
stage 20–29 embryos was shown more clearly when the ratios of
Wpkci mRNA, or chPKCI mRNA, to GAPDH
mRNA were plotted as in Figure 7D (a and b).
The higher-level expression of both Wpkci and
chPKCI genes in the 5- to 6-d (stages 27–29) embryos was
also demonstrated for the poly(A)+ RNA
preparations from mixed tissues of undifferentiated gonads plus
mesonephroi (Figure 7, C and D, c and d). It was of interest to note
that both Wpkci and chPKCI genes were expressed
at higher levels during stages 20–29 of embryonic development, which
were before the onset of gonadal differentiation in the female embryo;
a thickening of the germinal epithelium in the left gonad was seen
after 7 d of incubation (stage 31 or later) (Romanoff, 1960 In the Northern blot patterns for the transcripts of the Wpkci gene (Figure 7, A and C), transcripts of higher molecular mass (1.4 kb, ~2.4 kb, and a band at the top of the gel) were detected as minor components. The 1.4- and 2.4-kb components were likely mRNA species having longer 3′ untranslated regions, because the downstream cDNA probe, 5fm3 (Figure 2A), hybridized to the 1.4- and 2.4-kb bands but not to the 0.65-kb band, and sequencing of the 3′ untranslated region of the 1.4-kb component suggested that an alternative polyadenylation signal in the downstream region was used. The Abundance of Wpkci Transcripts Exceeds That of chPKCI
Transcripts in Early Embryos It was suggested that the Wpkci mRNA was more abundant
than the chPKCI mRNA in the early-stage embryos, because the
intensity of the 0.65-kb bands shown in Figure 7A was obtained after
24 h of exposure of the x-ray film but the intensity of the bands
shown in Figure 7B was obtained after 8 h of exposure of the
imaging plate for the bioimage analyzer, which was ~30 times
as sensitive as autoradiography. To confirm this notion, molar ratios
of both mRNA species in the whole 6-d (stage 29) female embryos and in
the undifferentiated gonads plus mesonephroi from the 6-d female
embryos were determined (Figure 8). In
this determination, the intensity values of Wpkci and
chPKCI mRNA bands in the Northern blot hybridization were
corrected by the intensity values of GAPDH mRNA and further
by the ratio of signal intensity values of pWpkci-8 and pchPKCI-3 cDNA
clones in Southern blot hybridization (Figure 8, A and B). The
corrected molar ratio (Wpkci mRNA/chPKCI mRNA)
was 10.3 for the whole embryo and 6.7 for the undifferentiated gonads
plus mesonephroi.
In Situ Detection of Wpkci and chPKCI Transcripts in the
Undifferentiated Gonads of Chicken Embryos To examine the presence of Wpkci and chPKCI
transcripts in undifferentiated gonads, the part of an embryo
containing undifferentiated gonads and mesonephroi was dissected out
from the 4.5-d (stage 25) female or male embryo and subjected to whole
mount in situ hybridization with the use of the DIG-labeled antisense
or sense riboprobe for Wpkci or chPKCI (Figure
9). The antisense riboprobe for
Wpkci detected Wpkci transcripts in the
undifferentiated left and right gonads and mesonephroi in the female
embryo, but not in the male embryo (Figure 9A). The antisense riboprobe
for chPKCI detected chPKCI transcripts as for the
Wpkci transcripts but in both female and male embryos
(Figure 9B). The level of expression of chPKCI in
undifferentiated gonads seemed to be higher in the male than in the
female, which was consistent with the results of Northern blot
hybridization (Figure 7C). The sense riboprobe did not give signals of
hybridization in each case (Figure 9, A and B).
To confirm that Wpkci and chPKCI genes were expressed in undifferentiated gonads in the female embryo, paraffin sections of embryonic tissues containing the undifferentiated gonads were prepared from the 5-d (stages 26 to 27) female embryo and subjected to in situ hybridization with the DIG-labeled antisense riboprobe for Wpkci or chPKCI (Figure 10). It was evident that transcripts of both Wpkci and chPKCI genes were detectable in the left and right undifferentiated gonads, mesonephric tubules, spinal cord, spinal ganglion, and myotome. In the undifferentiated gonads of the female embryo, transcripts of both Wpkci and chPKCI genes were detected widely in the tissue, except for some large cells (Figure 10, b, c, e, and f). However, we could not conclude in this study that the latter cells were primordial germ cells.
Nuclear Localization of GFP-fused Forms of Wpkci and chPKCI
Expressed in Chicken Embryonic Fibroblasts The intracellular localization of Wpkci and chPKCI was estimated
by expressing their full-length cDNA sequences, ligated in frame to the
3′ end of the GFP-encoding sequence, in the male chicken embryonic
fibroblasts. The GFP-fused form of Wpkci was found exclusively in the
nucleus (Figure 11, A–C). The
GFP-fused form of chPKCI was distributed in both nucleus and cytoplasm
(Figure 11, D–F), but its nuclear distribution seemed to be more
conspicuous compared with the distribution of GFP alone (Figure 11,
G–I).
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DISCUSSION Sex Chromosome Linkage of Wpkci and chPKCI In the present study, two cDNA clones, p5fm2 and p5fm3, were
isolated from the subtracted (i.e., female minus male) cDNA library
prepared from the mixed tissues of undifferentiated gonads and
mesonephroi of 5-d (stages 26–28) chicken embryos. The full-length
cDNA clone was then obtained with the use of p5fm2 as a probe and
identified as that for a gene encoding an altered form of PKCI because
of its substantial homology with the cDNA sequences of mammalian
PKCI. This gene was designated Wpkci because of
its location near the terminus of the nonheterochromatic end of the W
chromosome, the female-specific sex chromosome, of chickens and its
encoding a related but not identical protein with PKCI.The female-specific Wpkci gene was conserved among all the
Carinatae species of birds examined. Subsequently, a cDNA clone for the
chicken homologue of PKCI (chPKCI) was identified
because its deduced sequence was nearly 90% identical with those of
mammalian PKCI and it contained the conserved HIT motif HVHLH (residues
110–114) near the C terminus. The chPKCI also contained the conserved
His-51, which was suggested to participate in the binding of zinc with
His-112 and His-114 based on analysis of the three-dimensional
structure of hPKCI-1 by x-ray crystallography (Lima et al.,
1996 The chPKCI gene was located near the centromere of the long
arm of the Z chromosome, the sex chromosome common to the female and
the male. The human hPKCI-1 gene was mapped to 5q31.2
(Brzoska et al., 1996 When the deduced sequences of Wpkci and chPKCI are compared,
there are two substantially different regions (the second and fifth
regions from the N terminus; Figure 1C). The second region of Wpkci is
characterized by the relatively high content of Leu and Arg (4 residues
each out of 20 residues), which may give a unique property to Wpkci
and/or cause the distinct nuclear localization of Wpkci, as suggested
from the localization of the GFP-fused form of Wpkci (Figure 11). The
fifth region contains the HIT motif in chPKCI, but this motif is absent
in Wpkci. Wpkci also does not contain His-51. Thus, Wpkci should not
have zinc-binding ability. Overall, Wpkci does not display the same
biological function as chPKCI. However, the presence of highly
conserved regions (the fourth region from the N terminus and the
C-terminal region; Figure 1C) is notable, because it has been suggested
from three-dimensional analysis of hPKCI that it forms a homodimer by
means of close contact between the α-helix formed by the fourth
region and the C-terminal region of each protomer (Lima et
al., 1996 These structural features of Wpkci suggest that, although it does not
have a function comparable to that of chPKCI, it may form a homodimer
and/or a heterodimer with chPKCI. Our finding that the region
corresponding to the C-terminal region of chicken Wpkci was absent but
the fourth region, which was predicted to form an α-helix (Figure
6C), was conserved in duck Wpkci suggests that the α-helix region
might be sufficient for intermolecular association. The facts that
hPKCI, lacking most of the N-terminal region, was isolated originally
by its association with the regulatory domain (N-terminal 317-residue
polypeptide) of PKC-β (Lima et al., 1996 A Model of the Role of Wpkci in Female Sex Determination Molecular mechanisms of sex determination in birds have not been
elucidated. Considering the female heterogametic sex chromosomes, the
following two possibilities may be postulated: 1) the W chromosome
contains a gene whose expression in the early embryonic stage triggers
the cascade of gene expression toward female sex differentiation; and
2) the double dosage of a gene on the Z chromosome (ZZ in the male)
causes male determination, whereas the single dosage (ZW in the female)
of the gene causes female sex determination. In the latter case, the W
chromosome may be required only for the proper meiotic segregation of
the sex chromosomes. At present, there is no strong evidence favoring
either of these possibilities.The W chromosome of chicken is largely heterochromatic, and genes
involved in female sex determination, if present, are expected to be
located in the terminal nonheterochromatic region on the short arm
(Mizuno and Macgregor, 1998 The Z chromosome of chicken contains genes that seem to be required for
the function of the female gonad: VLDLR (Barber et
al., 1991 However, a positive function of the W chromosome may be inferred from
studies on triploid chickens. In ZZZ chickens, gonadal and excurrent
duct development was normal as in ZZ male chickens, although meiosis
and spermiogenesis were somewhat abnormal. On the other hand, in ZZW
chickens, the right gonad developed into a testis but the left gonad
developed into an ovotestis at hatching, although no excurrent ducts
were associated with it and it started to degenerate by 1 wk of age
(Lin et al., 1995 In the present study, we found the Wpkci gene on the W chromosome, and the following facts, revealed in this study, suggest that Wpkci may function in the process of female sex determination in Carinatae birds. 1) Wpkci is located on the female-specific W chromosome, enabling its female-specific expression, and its locus is in the nonheterochromatic end region of this largely heterochromatic sex chromosome, facilitating its gene expression. 2) Wpkci is moderately reiterated on the W chromosome, whereas chPKCI is a single-copy gene on the Z chromosome. The higher dosage of Wpkci may contribute to the presence of larger amounts of Wpkci mRNA over chPKCI mRNA in early female chicken embryos. 3) Wpkci is present and moderately reiterated in the female genomes of all of the Carinatae species examined (12 orders, 24 species), indicating that its W linkage and reiteration have been maintained during the evolution of modern birds. 4) Wpkci is transcribed and its translation products are likely localized in the nucleus, suggesting its possible function within the nucleus. 5) The level of expression of Wpkci is significantly greater during the early stages of development of female embryos, which correspond to the period before the onset of gonadal differentiation, and its timing of active expression is about the same as that of the chPKCI gene. 6) The deduced sequence of Wpkci lacks the HIT motif and His-51, indicating that Wpkci should have no zinc-binding function, whereas Wpkci has a unique Leu- and Arg-rich region, and the central α-helix region is well conserved, suggesting its ability to form a homodimer or a heterodimer with chPKCI through contact between the α-helix region of each molecule. However, when a possible role of Wpkci in female sex determination is considered, the fact that its expression is not limited to the undifferentiated gonads needs to be reconciled. In the model presented in Figure 12, it is proposed that the excess Wpkci forms the heterodimer with chPKCI efficiently and thereby interferes with the association of the chPKCI homodimer with a specific target protein (factor X) in undifferentiated gonads. If the association of the chPKCI homodimer with factor X is a key event in triggering the cascade of gene expression toward the differentiation of testis, this interference may, by itself, switch the pathway toward the differentiation of ovary. Another possibility is that the homodimer of Wpkci, the Wpkci monomer, or the Wpkci–chPKCI heterodimer, may play a positive role by interacting with another target protein (factor Y) in undifferentiated gonads, triggering the cascade of gene expression toward the differentiation of ovary.
To assess this model, it is essential to identify the Wpkci homodimer, the Wpkci–chPKCI heterodimer, and/or the target factors X and Y at the level of protein in undifferentiated gonads. It also would be desirable to transfer the Wpkci gene into the male embryonic stem cells and examine its effect on the fate of gonadal differentiation during embryogenesis. | |||||||||||||||||||
ACKNOWLEDGMENTS We thank Dr. K. Murata (Kobe Municipal Oji Zoo) for his help in collecting blood samples from various species of birds, and Drs. E. Satoh and T. Yamaguchi (Tohoku University) for their help in the preparation of paraffin sections of embryonic tissues. This work was supported by a Grant-in-Aid for Scientific Research (B) 09556072 from the Ministry of Education, Science, Sports, and Culture, Japan, and by the Nippon Life Insurance Foundation. | |||||||||||||||||||
REFERENCES
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