 1997 Oxford University Press
3428–3432 Nucleic Acids Research, 1997, Vol. 25, No. 17
Peg5/Neuronatin is an imprinted gene located on
sub-distal chromosome 2 in the mouse
Fusako Kagitani1, Yoshimi Kuroiwa1, Shigeharu Wakana2, Toshihiko Shiroishi3,
Naoki Miyoshi1, Shin Kobayashi1, Michiru Nishida1, Takashi Kohda1,
Tomoko Kaneko-Ishino4 and Fumitoshi Ishino1,5,*
1Gene
Research Center, Tokyo Institute of Technology, 4259 Nagatsuta-cho, Midori-ku, Yokohama-226, Japan,
Institute for Experimental Animals, 1430 Nogawa, Miyamae-ku, Kawasaki-216, Japan, 3Department of
Mammalian Genetics, National Institute of Genetics, 1-1111 Yata, Mishima, Shizuoka-411, Japan, 4Tokai
University, School of Health Sciences, Bohseidai, Isehara-shi, Kanagawa 259-11, Japan and 5PRESTO, Japan
Science and Technology Corporation (JST), 4-1-8 Hon-machi, Kawaguchi-shi, Saitama 332, Japan
2Central
Received May 30, 1997; Revised and Accepted July 14, 1997
ABSTRACT
We have established a systematic screen for imprinted
genes using a subtraction–hybridization method with
day 8.5 fertilized and parthenogenetic embryos. Two
novel imprinted genes, Peg1/Mest and Peg3, were
identified previously by this method, along with the
two known imprinted genes, Igf2 and Snrpn. Recently
three additional candidate imprinted genes, Peg5–7,
were detected and Peg5 is analyzed further in this
study. The cDNA sequence of Peg5 is identical to
Neuronatin, a gene recently reported to be expressed
mainly in the brain. Two novel spliced forms were
detected with some additional sequence in the middle
of the known Neuronatin sequences. All alternatively
spliced forms of Peg5 were expressed only from the
paternal allele, confirmed using DNA polymorphism in
a subinterspecific cross. Peg5/Neuronatin maps to
sub-distal Chr 2, proximal to the previously established imprinted region where imprinted genes cause
abnormal shape and behavior in neonates.
DDBJ/EMBL/GenBank accession no. AB004048
morphological abnormalities. Therefore, each of these regions
must contain imprinted gene(s). Seventeen imprinted genes have
been identified in the mouse (5–17), most of which map to these
imprinted regions. The majority of these genes are also imprinted
in humans (18). However, the biological significance and molecular
mechanism of genomic imprinting in mammals remains to be
elucidated. To clarify common features of imprinted genes, such
as biochemical function or expression specificity, it is necessary
to identify and characterize additional imprinted genes. We have
established a genome wide systematic screen for new imprinted
genes using parthenogenetic and normal mouse embryos at 8.5
days postcoitum (d.p.c.) as the source of material (11). Two novel
imprinted genes, Peg1/Mest (11) and Peg3 (12) have been
isolated previously with our cDNA subtractive hybridization and
differential screening method. Here we demonstrate that Peg5,
which is identical to neuronatin, is also imprinted. Neuronatin is
expressed in the nervous system especially in the hindbrain and
pituitary gland during development and is highly conserved among
the three mammalian species, rat, mouse and human (19,20). We
have mapped this gene to sub-distal mouse Chr 2, proximal to the
previously established imprinted region (distal Chr 2) affecting
development, behavior and viability in neonates (21).
INTRODUCTION
Proper development in mammals requires both a maternal and a
paternal genome because of the presence of imprinted genes
whose expression is dependent on their parental origin (1–3).
From genetic studies, chromosomes 2, 6, 7, 11, 12 and 17 have
been identified in the mouse as chromosomes whose influence on
development is dictated by their parental origin (4) and the
responsible regions have been narrowed down to nine established
imprinted regions on these chromosomes. Several imprinting
effects were observed following maternal or paternal duplication
of these established imprinted regions, such as early embryonic
lethality, mid fetal lethality, neonatal lethality, excessive or
deficient prenatal growth, excessive or deficient postnatal growth
and hyperkinetics or hypokinetics in neonates with different
MATERIALS AND METHODS
Peg5/Neuronatin
The DNA sequence of Peg5/Neuronatin-5 was registered on
DDBJ with accession number AB004048.
Expression analysis using RT–PCR
Total RNA was extracted from day 9.5 fertilized embryos (males
and females) and parthenogenetic embryos of (C57BL/6 × C3H)
F1 (Fig. 1a) using ISOGEN (Nippon Gene) as described previously
(11,12). Reverse transcription was carried out using 100 ng of
total RNA isolated from each embryo. One hundredth of the cDNA
sample was used for amplification of Peg5/Neuronatin-2, 3, 4, 5
*To whom correspondence should be addressed. Tel: +81 45 924 5812; Fax: +81 45 924 5814; Email: [email protected]
3429
Nucleic Acids
Acids Research,
Research,1994,
1997,Vol.
Vol.22,
25,No.
No.117
Nucleic
3429
Figure 1. (a) Predicted structures of Peg5/Neuronatin transcripts. Each transcript is shown as a solid line and open reading frames are shown as open boxes. The position
of the two introns are indicated by arrows. The number of nucleotides is according to the data in GenBank (X83569 and X83570) (20). In our analysis of
Peg5/Neuronatin-5, the sequence was almost the same as the published data, except in four sites (deletions, addition and base change) resulting in a 964 bp region
instead of a 962 bp region, and an extra 13 bp sequence at the 3′ end that is also reported in the rat Neuronatins (19,30). The total number of nucleotides in
Peg5/Neuronatin-4 and 5 is based on our results (indicated by ). (b) Primer sets to detect Peg5/Neuronatin-2 and 3 (F4–R1) and Peg5/Neuronatin-4 and 5 (F2–R1).
(c) Theoretical amino acid sequences of Peg5/Neuronatin-2, 3, 4 and 5. Asterisks indicate putative protein kinase C phosphorylation sites (Peg5/Neuronatin-2 and
3) and a putative sulfatation site (Peg5/Neuronatin-4 and 5). The gaps in the sequences shown in the broken lines correspond to exons that are spliced out in each
transcript.
and one thousandth for β-actin. Reaction mixtures contained 1×
ExTaq buffer, 2.5 mM dNTP mixture, 80 pmol primers and 2.5 U
ExTaq (Takara) in 100 µl, and the amplification cycle was set at
95C for 30 s, 65C for 30 s and 72C for 60 s in a Perkin-Elmer
GeneAmp PCR system 9600. Primer sets used were 5-CAGAACTGCTCATCATCGGC-3 (F4) and 5-TGCGTGAGACCAGGGATAAG-3 (R1), 5-ACTTGCCAAGGTCAGTGAGG-3 (F5)
and 5-TCATGGTAGGATCTTGTG CG-3 (R3) for Peg5/Neuronatin-2 and 3 and 5-AGCCGGGAACAAAGACTCAG-3 (F2)
and R1 for Peg5/Neuronatin-4 and 5. β-actin (222 bp) was
amplified as a control using primers 5-AAGTGTGACGTTGACATCCG-3 and 5-GATCCACATCTGCTGGAAGG-3. For
analysis of neonatal organs and tissues (Fig. 3), one hundredth of
the cDNA samples was amplified for 30 cycles for Peg5/Neuronatin-2, 3 and 35 cycles for Peg5/Neuronatin-4, 5, respectively.
RFLP analysis
Total RNA from adult brains (C3H, C57BL/6 and MSM), neonatal
brains (C57BL/6 × MSM) F1 and reciprocal F1 of [(MSM ×
C57BL/6) F1] and day 13 whole embryos [(C3H × MSM) F1]
were used for RT–PCR. For Peg5/Neuronatin-2 and 3, the first 25
cycles of amplification were carried out using F4 and R1. After
confirming that only Peg5/Neuronatin-2 and 3 had been amplified,
one hundredth of the samples was reamplified for another 13
cycles with primers F5 and R3. For Peg5/Neuronatin-4 and 5, the
first amplification was for 25 cycles. Then, one hundredth of the
samples was reamplified for another 13 cycles with the same
primers. PCR products were digested with TaqI restriction enzyme
at 65C for 4 h.
RESULTS
Identity of Peg5 with Neuronatin and analysis of alternatively
spliced forms
DNA sequence analysis showed that Peg5 cDNA is identical to
the previously described mouse neuronatin (20). It also shares
significant homology to rat and human Neuronatin (20). Peg5
cDNA has an additional 275 bp in the coding region and this
transcript seems to be the largest of the alternatively spliced forms
of Neuronatin (Fig. 1a). We detected four transcripts of
Peg5/Neuronatin by RT–PCR, designated Peg5/Neuronatin-2, 3,
4 and 5 in ascending order of the size of the transcript.
Peg5/Neuronatin-2 and 3 were named after the previously
isolated mouse neuronatin-2 and 3 (20) and correspond to the rat
Neuronatin-β and α (19). Peg5/Neuronatin-4 and 5 are novel
transcripts that have an additional 275 bp of sequence; the original
cDNA identified as Peg5 corresponds to Peg5/Neuronatin-5.
Comparing several RT–PCR products with corresponding PCR
products of genomic DNA, there are apparently two introns
between 275 and 81 bp and between 81 and 964 bp regions.
However, no intron seems to exist between the 138 and 275 bp
sequences. DNA sequence of the genomic DNA confirmed this
result (Fig. 1a). This indicates two possible usages of exon 1; the
275 bp sequence in the 3 part of exon 1 is spliced out to produce
Peg5/Neuronatin-2 and 3, while it is read through from the
preceding 138 bp sequence to produce Peg5/Neuronatin-4 and 5.
Peg5/Neuronatin-2 and 3 encode peptides that consist of 54 and
81 amino acids, respectively (19,20), and both Peg5/Neuronatin-4
and 5 have the same open reading frame of 44 amino acids
because a stop codon appears within the 275 bp sequence. All
peptides have a common N-terminal amino acid sequence of 24
residues, most of which are hydrophobic residues that may form
3430 Nucleic Acids Research, 1997, Vol. 25, No. 17
Figure 2. (a) Peg5/Neuronatin expression analysis by RT–PCR in normal and parthenogenetic embryos. Expression was examined in day 9.5 normal (C57B/6 × C3H)
F1 male (lanes 1 and 2) and female (lanes 3 and 4), and in day 9.5 parthenogenetic (C57B/6 × C3H) F1 embryos (lanes 5–7). PCR amplification of Peg5/Neuronatin-2
and 3 (upper) consisted of 30 cycles using primers F4–R1. Peg5/Neuronatin-4 and 5 (middle) were amplified for 30 cycles using primers F2–R1 and reamplification
was carried out for 10 more cycles using one tenth of the aliquot. β-actin (lower) was amplified to serve as a control. (b–e) Verification of Peg5/Neuronatin imprinting
in subinterspecific hybrid embryos by RT–PCR and RFLP analysis (b and c). Analysis of whole day 13.5 embryos. RT–PCR products of adult brains of C3H and MSM
were examined (lanes 1 and 2). RT–PCR products of (C3H × MSM) F1 embryos (lanes 3 and 4) show that paternal MSM Peg5/Neuronatin-2 and 3 (b) and
Peg5/Neuronatin-4 and 5 (c) which gave 40 and 42 bp bands are expressed. (d and e) Analysis of Peg5/Neuronatin in neonatal brains. RT–PCR products of adult brains
of B6 and MSM (lanes 1 and 2) were examined. Neonatal brain of the (B6 × MSM) F1 (lane 3) which gave 40 and 42 bp bands corresponding to the MSM allele and
in the reciprocal cross the (MSM × B6) F1 (lane 4) which gave a 82 bp band corresponding to the B6 allele shows that only paternal alleles are expressed in all cases.
The 40, 42 and 82 bp bands are indicated by asterisks. (f and g) Primers used in RFLP analysis. (f) For Peg5/Neuronatin-2 and 3, F4–R1 primers were used in the
first round of PCR. After confirming that only Peg5/Neuronatin-2 and 3 were amplified, a second round of PCR was carried out using internal primers F5–R3, because
one TaqI fragment from the F4–R1 region was similar to the 82 bp in question. (g) For Peg5/Neuronatin-4 and 5, amplification was carried out using the F2–R1 primers,
followed by TaqI digestion.
a transmembrane region (19,20). Peg5/Neuronatin-2 and 3 have
common C-terminal sequences of 30 amino acids; the latter has
an additional 27 amino acid sequence in the middle (Fig. 1c; 19,20).
Peg5/Neuronatin-2 and 3 peptides have four and five putative
phosphorylation sites for protein kinase C (22,23) in the
C-terminal region, respectively (Thr and Ser; asterisks in Fig. 1c).
Peptides encoded by both of Peg5/Neuronatin-4 and 5 have
C-terminal sequences consisting of 20 amino acids that differ
entirely from the Peg5/Neuronatin-2 and 3 C-terminal amino acid
sequences. There is also one candidate for a sulfatation site (24)
(Tyr; asterisks in Fig. 1c), although its significance is unknown.
Expression and imprinting of Peg5/Neuronatin
We examined the expression and imprinting status of all of the
Peg5 transcripts. Only the expression of Peg5/Neuronatin-2 and
3 could be detected by RT–PCR using the F4–R1 primer set
(Fig. 1b), indicating that expression levels of Peg5/Neuronatin-4
and 5 are very low compared with those of Peg5/Neuronatin-2
and 3 (see below and Fig. 2 legend). Peg5/Neuronatin-4 and 5
were amplified specifically using the primer set, F2–R1 (Fig. 1b),
because the F2 primer was designed within the 275 bp region. As
shown in Figure 2a, all transcripts were expressed only in normal
embryos and not in parthenogenetic embryos. All four transcripts
are expressed from the paternal allele only. This was determined
by analyzing subinterspecific hybrid embryos using a DNA
polymorphism in the 3 UTR (shown in Fig. 2f and g) in Mus
musculus molossinus (MSM) and Mus musculus musculus strains
of C57BL/6 and C3H mice (Fig. 2b–e). A MSM-specific TaqI site
is contained within the 82 bp TaqI fragment from B6 or C3H
cDNA and gives 40 and 42 bp fragments from MSM cDNA when
digested with TaqI together with the other TaqI fragments
indicated in Figure 2f and g. In day 13.5 embryos of (C3H ×
MSM) F1, only the MSM (paternal) alleles of Peg5/Neuronatin-2
3431
Nucleic Acids
Acids Research,
Research,1994,
1997,Vol.
Vol.22,
25,No.
No.117
Nucleic
3431
Figure 3. Analysis of Peg5 expression in neonatal tissues. Primers were used
as described in Figure 1b and conditions of RT–PCR were the same as described
in the legend to Figure 2a. Except for the lung, no expression was detected in
other tissues and organs, including stomach, liver, spleen, thymus, heart, muscle
and tongue.
and 3 (Fig. 2b) or Peg5/Neuronatin-4 and 5 (Fig. 2c) were
detected. In neonatal brain of (B6 × MSM) F1 and the reciprocal
(MSM × B6) F1, only paternal alleles (MSM for the former and
B6 for the latter) were detected for both Peg5/Neuronatin-2 and 3
(Fig. 2d) and Peg5/Neuronatin-4 and 5 (Fig. 2e)
Brain specific expression of Neuronatin during development
was examined in detail using in situ hybridization by Wijnholds
et al. (20). We confirmed their results by carrying out the same
experiment using our Peg5/Neuronatin-5 probe that has an
additional 275 bp of sequence. RT–PCR analysis on neonates also
showed that all four transcripts were expressed mainly in the brain
and eyes; very low expression was detected in the bladder, colon
(Fig. 3e) and lung (data not shown). The expression levels of
Peg5/Neuronatin-2, 3 were always higher than those of
Peg5/Neuronatin-4 and 5 (estimated at ∼50–100-fold by RT–PCR).
There were subtle differences in the levels of expression of
various transcripts, but the sites of their expression appear to be
similar. However, it is possible that only certain transcripts may
be expressed in embryos prior to day 8.5 in the mouse because
Joseph et al. reported that rat Neuronatin-α, corresponding to our
Peg5/Neuronatin-3, was only expressed in day 7–10 embryos (19).
Mapping of Peg5/Neuronatin to sub-distal chromosome 2
Mapping of Peg5/Neuronatin was carried out using linkage
analysis as shown in Figure 4. The gene maps to distal Chr 2. An
imprinted region was previously detected here based on genetic
studies using the T2Wa translocation breakpoint to generate
embryos of different genotypes. Neonatal mice with duplication
of maternal Chr 2 and paternal deficiency (MatDp.dist2. T2Wa)
have thin bodies with arched backs and are hypokinetic, while the
PatDp.dist2. T2Wa have square bodies and show hyperkinetic
movement (25). Both of these conditions are neonatal lethal and
this provides evidence for the existence of imprinted gene(s) on
distal Chr 2. Indeed, Gnas which maps to this region has
previously been reported to be imprinted (26). Peg5/Neuronatin
must be proximal to Gnas (see Fig. 4 legend). As shown in
Figure 4, Peg5/Neuronatin is also proximal to Ada. This mapping
data may have important implications for defining the imprinted
region on distal Chr 2, as Ada is proximal to the T2Wa breakpoint
(Peters, J., personal communication). Thus, Peg5/Neuronatin
Figure 4. Mapping of Peg5. Linkage analysis was carried out with a total of 254
mice of the subinterspecific backcross (DBA × MSM)F1 × MSM using the TaqI
polymorphism in the 3 UTR as described in the legend to Figure 2. The ratios
of the total number of mice exhibiting recombinant chromosomes to total
number of mice analyzed for each pair of loci is –D2Mit22–6/255–D2Mit48–
5/254–Peg5/Neuronatin–2/254–D2Mit411–1/254–D2Mit453–2/254– D2Mit197–
7/254–Ada–0/254–D2Mit29–1/254–D2Mit49–16/255–D2Mit25. The calculated
genetic distances in centiMorgans (cM) are shown. Gnas is located distal to
D2Mit25 (31), indicating that the location of Peg5/Neuronatin is very different
from that of Gnas. Moreover, the Peg5/Neuronatin locus is proximal to Ada
which is proximal to the T2Wa breakpoint (Peters, J., personal communication).
An imprinted region that affects neonatal viability and behavior is located distal
to T2Wa, suggesting that Peg5/Neuronatin maps to a novel imprinted region.
may be proximal to the T2Wa breakpoint and also proximal to the
previously identified imprinted region affecting neonatal behavior.
DISCUSSION
Systematic screening for paternally expressed genes (Peg) has
been carried out using a subtraction–hybridization method with
day 8.5 fertilized and parthenogenetic embryos. So far, two novel
imprinted genes, Peg1/Mest (11) and Peg3 (12), have been
identified along with the two known imprinted genes, Igf2 (27)
and Snrpn (13). These four imprinted genes map to four
independent imprinted regions, proximal Chr 6 (Peg1/Mest),
proximal Chr 7 (Peg3), distal Chr 7 (Igf2) and central Chr 7 (Snrpn).
In this study, we analyzed Peg5/Neuronatin and mapped it to
sub-distal Chr 2. It is noteworthy that five imprinted genes that are
located in five different imprinted regions were obtained with our
subtraction–hybridization method and these results indicate that
this subtraction method is a very powerful tool for the isolation
of differentially expressed genes from very small amounts of
biological tissues.
3432 Nucleic Acids Research, 1997, Vol. 25, No. 17
Peg5/Neuronatin is highly expressed in the brain during
development and also in neonates and adults. Our initial mapping
data suggest that Peg5/Neuronatin is located on distal Chr 2, a
region that has been implicated in neonatal behavior and viability
(21) and it seems that Peg5/Neuronatin might be a good candidate
to explain these phenotypes. However, more precise mapping
data has suggested that it is located proximal to and outside the
established imprinted region. There have been no reports suggesting
the existence of an imprinted region at this locus by previous
genetic studies. However, recently, a novel imprinted gene, Grf1,
was isolated by the RLGS (restriction landmark genome scanning)
method and was mapped to Chr 9, where no imprinted region had
previously been detected by genetic studies (16). It is noteworthy
that the majority of the imprinted genes so far identified are
located within genetically defined imprinted regions. However,
since not all imprinted genes will necessarily produce a distinct
phenotype, it is possible that other novel imprinted genes may
exist in regions not identified by genetic studies. Thus, systematic
screening for paternally expressed genes has led to the discovery of
a novel imprinted region in the mouse.
In this study, we identified four alternatively spliced forms of
Neuronatin. Three different forms of Neuronatin, neuronatin-1,
2 and 3 have been reported previously (20). Neuronatin-2 and 3
are identical to our Peg5/Neuronatin-2 and 3. Two novel
alternatively spliced forms, Peg5/Neuronatin-4 and 5, have some
additional sequence. The expression pattern of these two
transcripts appeared to be the same as that of Peg5/Neuronatin-2
and 3. The first exon which includes both the 138 bp and the 275 bp
sequence is used in two different ways. Firstly, it is used as an
exon of 138 bp for Peg5/Neuronatin-2 and 3. Secondly, it is used
as a continuous 403 bp exon for Peg5/Neuronatin-4 and 5 by
reading through from the 138 bp sequence to the 275 bp segment.
A similar splicing pattern is seen in three transcripts of polyoma
T antigen genes (28).
Because there have been no reports of genetic studies using
mice showing uniparental disomy of the sub-distal region of
chromosome 2 where Peg5/Neuronatin is located, the function of
Peg5/Neuronatin is currently unclear. It will be very interesting
to identify the function of Peg5/Neuronatin in neural cells during
development. All of the alternatively spliced transcripts encode
short polypeptides that have a common N-terminal putative
transmembrane region consisting of 24 hydrophobic amino acids.
Peg5/Neuronatin-2, 3 peptides consist of a total of 54 and 81
amino acids, respectively (19,20), while both Peg5/Neuronatin-4
and 5 have the same open reading frame of a total of 44 amino
acids. Peg5/Neuronatin-2, 3 peptides have four and five putative
phosphorylation sites for protein kinase C, respectively (22,23)
in the C-terminal region, and peptides encoded by both of
Peg5/Neuronatin-4, 5 have one candidate sulfatation site (24) in
their C-terminal sequence. These structural features suggest that the
C-terminal region may extend into the cytoplasm and that the
Peg5/Neuronatin peptides might function as membrane channels
or membrane receptors (19,20) for signal transduction, perhaps
by forming multimers or a complex with other protein(s).
Recently, it has been reported that the amino acid sequence of
human neuronatin shows 50% homology with two ion channel
proteins, PMP1 and phospholamban, that function as subunits of
an H+-ATPase and a Ca2+-ATPase, respectively (29). It seems
likely that Peg5/Neuronatin has an essential role in brain
development or neuronal function as it is highly conserved among
mammalian species (19,20,30). Detailed investigations from
genetic, molecular and cellular viewpoints will be required to
elucidate the role of the Peg5/Neuronatin gene.
ACKNOWLEDGEMENTS
We thank Dr Kuwano for helpful advice on the relationship
between Calbindin and Neuronatin and Dr Hayashizaki for
providing the linkage data for the CSC panel. This work was
supported by grants from the Ciba-Geigy Foundation (Japan) for
the Promotion of Science, the Kato Memorial Bioscience
Foundation to T.K.-I., the Asahi Glass Foundation and Precursory
Research for Embryonic Science and Technology from JST to F.I.
REFERENCES
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
Cattanach, B. M. and Beechey, C. V. (1990) Development (Suppl.), 63–72.
McGrath, J. and Solter, D. (1984) Cell, 37, 179–183.
Surani, M. A., Barton, S. C. and Norris, M. L. (1986) Cell, 45, 127–136.
Cattanach, B. M. and Beechey, C. V. (1996) Mouse Genome, 94, 96–99.
Hayashizaki, Y., Shibata, H., Hirotsune, S., Sugino, H., Okazaki, Y.,
Sasaki, N., Hirose, K., Imoto, H., Okuizumi, H., Muramatsu, M. et al.
(1994) Nature Genet., 6, 33–40.
Hatada, I. and Mukai, T. (1995) Nature Genet., 11, 204–206.
Guillemot, F., Caspary, T., Tilghman, S. M., Copeland, N. G., Gilbert, D. J.,
Jenkins, N. A., Anderson, D. J., Joyner, A. L., Rossant, J. and Nagy, A.
(1995) Nature Genet., 9, 235–242.
Giddings, S. J., King, C. D., Harman, K. W., Flood, J. F. and Carnaghi, L. R.
(1994) Nature Genet., 6, 310–313.
Bartolomei, M. S., Zemel, S. and Tilghman, S. M. (1991) Nature, 351,
153–155.
Barlow, D. P., Stoger, R., Herrmann, B. G., Saito, K. and Schweifer, N.
(1991) Nature, 349, 84–87.
Kaneko-Ishino, T., Kuroiwa, Y., Miyoshi, N., Kohda, T., Suzuki, R.,
Yokoyama, M., Viville, S., Barton, S. C., Ishino, F. and Surani, M. A.
(1995) Nature Genet., 11, 52–59.
Kuroiwa, Y., Kaneko-Ishino, T., Kagitani, F., Kohda, T., Li, L. L., Tada, M.,
Suzuki, R., Yokoyama, M., Shiroishi, T., Wakana, S., et al. (1996) Nature
Genet., 12, 186–190.
Leff, S. E., Brannan, C. I., Reed, M. L., Ozcelik, T., Francke, U.,
Copeland, N. G. and Jenkins, N. A. (1992) Nature Genet., 2, 259–264.
Matsuoka, S., Thompson, J. S., Edwards, M. C., Bartletta, J. M., Grundy, P.,
Kalikin, L. M., Harper, J. W., Elledge, S. J. and Feinberg, A. P. (1996)
Proc. Natl. Acad. Sci. USA, 93, 3026–3030.
Lee, M. P., Hu, R. J., Johnson, L. A. and Feinberg, A. P. (1997) Nature
Genet., 15, 181–185.
Plass, C., Shibata, H., Kalcheva, I., Mullins, L., Kotelevtsevva, N.,
Mullins, J., Kato, R., Sasaki, H., Hirotsune, S., Okazaki, Y., et al. (1996)
Nature Genet., 14, 106–109.
Villar, A. J. and Pedersen, R. A. (1994) Nature Genet., 8, 373–379.
Ledbetter, D. H. and Engel, E. (1995) Hum. Mol. Genet., 4, 1757–1764.
Joseph, R., Dou, D. and Tsang, W. (1995) Brain Res., 690, 92–98.
Wijnholds, J., Chowdhury, K., Wehr, R. and Gruss, P. (1995) Dev. Biol.,
171, 73–84.
Cattanach, B. M. and Kirk, M. (1985) Nature, 315, 496–498.
Woodgett, J. R., Gould, K. L. and Hunter, T. (1986) Eur. J. Biochem., 161,
177–184.
Kishimoto, A., Nishiyama, K., Nakanishi, H., Uratsuji, Y., Nomura, H.,
Takeyama, Y. and Nishizuka, Y. (1985) J. Biol. Chem., 260, 12492–12499.
Hunter, W. B. (1987) Trends Biochem. Sci., 12, 361–363.
Cattanach, B. M., Evans, E. P., Burtenshaw, M. and Beechey, C. V. (1992)
Mouse Genome, 90, 82.
Williamson, C. M., Schofield, J., Dutton, E. R., Seymoor, A., Beechey, C. V.,
Edwards, V. H. and Peters, J. (1996) Genomics, 36, 280–287.
DeChiara, T. M., Robertson, E. J. and Efstratiadis, A. (1991) Cell, 64,
849–859.
Soeda, E., Arrand, J. R., Smolar, N. and Griffin, B. E. (1979) Cell, 17,
357–370.
Dou, D. and Joseph, R. (1996) Brain Res., 723, 8–22.
Joseph, R., Dou, D. and Tsang, W. (1994) Biochem. Biophys. Res.
Commun., 201, 1227–1234.
Cattanach, B. M. (1986) J. Embryol. Exp. Morphol., 97, 137–150.