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Developmental patterns of zygotes from transgenic female mice with elevated tissue glutathione

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JOURNAL OF EXPERIMENTAL ZOOLOGY 286:149–156 (2000)
Heterogeneity of Chicken Slow Skeletal Muscle
Troponin T mRNA
I. YONEMURA, T. HIRABAYASHI, AND J.-I. MIYAZAKI*
Institute of Biological Sciences, University of Tsukuba, Tsukuba, Ibaraki
305-8572, Japan
ABSTRACT
The troponin T (TnT) transcripts in chicken slow skeletal muscle were characterized by S1 nuclease mapping and nucleotide sequencing of cDNA produced by RT-PCR and 5′RACE. We found two kinds of transcripts in the 5′-region, one having the codon for alanine (position
135–137), C (258), and A (262) and the other lacking the codon and having T (258) and G (262)
instead of C and A. In the 3′-region, we found four single base substitutions at 703 (T or C), 774 (C
or T), 797 (C or T), and 827 (G or A). Four of the six substitutions lead to amino acid changes in
chicken sTnT isoforms. We determined the genomic structure of the 3′-region of the chicken sTnT
gene. The region includes 7 exons corresponding to position 249–891 of the chicken sTnT cDNA
and no alternative exon, showing that the 3′-heterogeneity in sTnT transcripts was due to allelic
variation. J. Exp. Zool. 286:149–156, 2000. © 2000 Wiley-Liss, Inc.
Skeletal muscle cells are characterized by precise organization of the contractile proteins into
the repeating units of overlapping thick and thin
filaments, i.e., sarcomeres, that organize in turn
myofibrils. The thick filaments are composed primarily of myosin, and major components of the
thin filaments are actin, tropomyosin and troponin. These contractile proteins have multiple
isoforms that are produced by multigene families
and by respective genes through alternative splicing (for review, see Andreadis et al., ’87) and/or
use of alternative promoters (Robert et al., ’84;
Concordet et al., ’93). Functional significance of
these multiple isoforms is not fully clarified so far
in spite of much effort devoted for it.
Troponin, the key protein of Ca2+-sensitive molecular switching for contraction in vertebrate
striated muscle, consists of three subunits, troponin T (TnT), troponin I, and troponin C (for reviews, see Zot and Potter, ’87; Schiaffino and
Reggiani, ’96). TnT isoforms are encoded by three
genes characteristic of slow skeletal muscle
(sTnT), fast skeletal muscle (fTnT), and cardiac
muscle (cTnT) (Breitbart et al., ’85; Cooper and
Ordahl, ’85; Gahlmann et al., ’87; Smillie et al.,
’88; Mesnard et al., ’93). Each gene can generate
a variety of transcripts by alternative splicing
(Breitbart et al., ’85; Cooper and Ordahl, ’85;
Gahlmann et al., ’87; Schachat et al., ’95). Molecular organization of the rat fTnT gene has revealed its capacity to produce 128 different fTnT
mRNAs by differential alternative splicing (Med© 2000 WILEY-LISS, INC.
ford et al., ’84; Breitbart et al., ’85; Breitbart and
Nadal-Ginard, ’87; Morgan et al., ’93). Smiillie et
al. (’88) have found four variants of chicken fTnT
cDNA, and Schachat et al. (’95) 16 variants of
chicken fTnT 5′-cDNA. The chicken cTnT gene
generates one embryonic and one adult transcripts, derived from inclusion and exclusion of
exon 5, respectively (Cooper and Ordahl, ’85). A
more complex alternative splicing pattern involving exons 4 and 12 has been found in the rat cTnT
gene (Jin and Lin, ’89; Jin et al., ’92).
On the other hand, very poor information is
available on the mode of alternative splicing of
the sTnT gene. The human sTnT gene can generate three or possibly four transcripts, which differ in the presence or absence of two short inserts
(33 and 48 nucleotides) in the 5′- and 3′-end regions (Gahlmann et al., ’87; Samson et al., ’94).
Chicken slow skeletal muscle, such as anterior
latissimus dorsi (ALD), has been shown so far to
contain two kinds of sTnT transcripts differing in
inclusion or exclusion of one codon encoding an
alanine residue in the 5′-end region (Yonemura
et al., ’96). To elucidate the alternative splicing
Abbreviations used: ALD, anterior latissimus dorsi; bp, base pair(s);
cTnT, cardiac TnT; fTnT, fast muscle TnT; nt, nucleotide(s); PCR,
polymerase chain reaction; 5′-RACE, 5′-rapid amplification of cDNA
ends; RT-PCR, reverse transcription-PCR; sTnT, slow muscle TnT;
TnT, troponin T.
*Correspondence to: Jun-Ichi Miyazaki, Institute of Biological Sciences, University of Tsukuba, Tsukuba, Ibaraki 305-8572, Japan. Email: junichi@sakura.cc.tsukuba.ac.jp
Received 22 October 1998; Accepted 11 May 1999
150
I. YONEMURA ET AL.
pattern of the sTnT transcripts and to deduce the
gene structure it is very important to investigate
heterogeneity of sTnT transcripts.
In the present work, we perform S1 nuclease
mapping and nucleotide sequencing of cDNA produced by RT-PCR and 5′-RACE to search for heterogeneity of chicken sTnT mRNA. We show that
5′ and 3′ heterogeneities in sTnT transcripts are
caused by single base substitutions at six positions and by inclusion or exclusion of the codon
coding for alanine. We also show the partial genomic structure of the chicken sTnT gene.
MATERIALS AND METHODS
Materials
Adult and one-day-old (H1) white leghorn chickens (Gallus domesticus [L]) were obtained from
commercial sources. Slow muscle, anterior latissimus dorsi (ALD), mixed muscle, complexus, and
fast muscle, pectoralis major, were dissected out
and used for RNA preparation.
RNA preparation and reverse transcriptionpolymerase chain reaction (RT-PCR)
Total RNA was prepared by using Isogen (Nippon
gene) following its protocol. Reverse transcription
was performed with 10 µg of total RNA, 0.5 µg of
oligo(dT)12–18 and Superscript II (Gibco BRL) at
42°C for 60 min. For PCR of the 5′-region of
chicken sTnT, the forward primer, 5′-GGCTCGAGGAGCCAACAGGACCG-3′ (position 1 to 15
of the cDNA + Xho I site), was synthesized based
on the chicken sTnT cDNA sequence. The 3′-mixed
primer (position 522 to 541) was used as the reverse primer (Yonemura et al., ’96). PCR was performed by 30 cycles of 1 min denaturation at 95°C,
1 min annealing at 60°C and 1 min extension with
Taq DNA polymerase (Gibco BRL) at 72°C, followed by final 10 min extension at 72°C. For PCR
of the 3′-region of chicken sTnT, the reverse
primer, 5′-GGGAATTCGACGAGCAGAGCTTTATTGG-3′ (Primer R, position 871 to 891 of the cDNA
+ EcoR I site), was also synthesized based on the
chicken sTnT cDNA sequence. The 5′-mixed
primer (position 248 to 269) was used as the forward primer (Yonemura et al., ’96). PCR was performed by 35 cycles of 1 min denaturation at 94°C,
1 min annealing at 65°C and 1 min extension with
Taq DNA polymerase at 72°C, followed by final
10 min extension at 72°C. The 5′-rapid amplification of cDNA ends (5′-RACE) was performed as
in Frohman et al. (’88) and Yonemura et al. (’96).
Products were cloned in the pBluescript II KS+
phagemid (Stratagene) and sequenced.
Antisense DNA probes and
S1 nuclease mapping
Two clones, stnt-5′R1 and sTnT-a3, were obtained by cloning chicken sTnT cDNA (Yonemura
et al., ’96). The former includes 1 to 541, and the
latter 78 to 898 of sTnT cDNA. The stnt-5′R1 clone
was digested with Xho I and Pst I, and the resulting fragment was subcloned into the Xho IPst I double-digested sTnT-a3 to obtain the
full-length cDNA, which did not have three bases
(135–137) encoding alanine. This full-length cDNA
was cut with Xho I and BamH I and a 690 nt
fragment, probe A, was obtained. The probe A was
labeled with [32P] ATP using T4 polynucleotide kinase (Nippon gene). Another probe, probe B, was
generated by PCR with sTnT-a3 as a template using the 5′-mixed primer as the forward primer
(Yonemura et al., ’96) and Primer R as the reverse primer. PCR was performed by 30 cycles of
30 sec denaturation at 95°C, 10 sec annealing at
60°C and 30 sec extension with KOD Dash
(TOYOBO) at 74°C, followed by final 10 min extension at 72°C. The probe B was labeled with
[32P] dATP using KOD Dash (TOYOBO). Total
RNA (5 or 10 µg) was precipitated with 105 cpm
of each probe. The pellet was dissolved in 20 µl of
the hybridization buffer. Hybridization was performed overnight at 55°C before digestion with
S1 nuclease (Gibco BRL). Protected fragments
were separated on 6% acrylamide/8 M urea gels,
which were exposed to X-ray film (Kodak) with
intensifying screen.
Cloning of the chicken sTnT gene
For PCR of the chicken sTnT gene, the forward
primer, 5′-GCAGCCATGTCCGAAGCTGAG-3′
(position 39–62 of the cDNA sequence), was synthesized based on the chicken sTnT cDNA sequence. Primer R was used as the reverse primer.
PCR was performed by 30 cycles of 30 sec denaturation at 96°C and 10 min annealing and extension at 74°C with 130 ng of chicken liver
genomic DNA using KOD Dash (TOYOBO). Amplified DNA was cloned in the pBluescript II KS+
phagemid (Stratagene) and sequenced.
RESULTS
Heterogeneity in the 5¢-region of
chicken sTnT mRNA
We described previously two truncated cDNA
clones, stnt-5′R1 and sTnT-a3 (Yonemura et al.,
’96). Using a 690 nt antisense probe (probe A) derived from the full-length cDNA, we performed
CHICKEN SLOW TROPONIN T mRNA
S1 nuclease mapping of total RNA from adult and
one-day-old chick (H1) ALD to characterize the
5′-region of chicken sTnT mRNA (Fig. 1). The 690
nt fragment free from S1 nuclease digestion
showed that the transcript completely matching
with the probe was expressed in both adult and
H1 ALD. In addition, two other fragments of 558
nt and about 440 nt were also generated, suggesting that adult and H1 chicken ALD had possibly three kinds of transcripts which differed in
at least two positions in the 5′-region. The consistent patterns of S1 nuclease mapping were obtained with several individuals (data not shown).
As expected, no protected band was found in adult
fast muscle, pectoralis major. The result is consistent with our previous data by Northern blotting (Yonemura et al., ’96).
To collect precise information on the sequences
which caused the variation in the 5′-region, we
performed nucleotide sequencing of RT-PCR and
5′-RACE products from total RNA of adult chicken
ALD and neck muscle, complexus, which is known
to express sTnT isoforms (Yao et al., ’92). We obtained two RT-PCR products from ALD, A1-1R and
Aall-1R, and one 5′-RACE product from complexus, 20-2R (Fig. 2). The 20-2R had GCA coding for alanine at 135 to 137 of chicken sTnT
cDNA just as stnt-5′R20b, but A1-1R, Aall-1R and
stnt-5′R1 did not (Fig. 2). Furthermore, two single
base substitutions were found at 258 and 262. The
clones, stnt-5′R20b and 20-2R, had C and A at
those positions, respectively, while the remaining
clones, stnt-5′R1, A1-1R and Aall-1R, had T and
G instead of C and A. Those differences lead to
amino acid substitutions of arginine (258) and
lysine (262) in the former to tryptophan and arginine in the latter, respectively. Such nucleotide
substitutions are also seen in the 5′-region of human sTnT cDNA. Two human sTnT cDNA clones,
M1 and MSL-2-27, have G and one clone, H22h,
has C at the position 117, resulting in the amino
acid changes from glutamic acid in the M1 and
MSL-2-27 to aspartic acid in H22h (Gahlmann et
al., ’87; Samson et al., ’94). However, we could not
find the sequence corresponding to the 33 nt insert reported in the 5′-region of human sTnT
cDNA (Gahlmann et al., ’87).
Therefore, the determination of sequences
showed that the two kinds of transcripts, one having the codon for alanine and C (258) and A (262)
and the other lacking the codon and having T (258)
and G (262), were expressed in chicken slow skeletal muscle. The results are consistent with the
above data from S1 nuclease mapping, showing
151
Fig. 1. S1 nuclease mapping of sTnT mRNA with an
antisense probe from the 5′-region of sTnT cDNA. The upper panel shows schematically the structure of sTnT mRNA
and the position of the 690 nt antisense probe (probe A) derived from the cDNA clones, stnt-5′R1 and sTnT-a3, with
the 32P-labeled 3′-end (closed circle). Total RNA from adult
(Ad) chicken ALD, 1-day-old chick (H1) ALD or adult pectoralis major was hybridized to the probe A and digested with
S1 nuclease. Protected fragments were separated on the 6%
acrylamide/8 M urea gel. No band was detected in fast skeletal muscle, pectoralis major (PM), but three bands of 690
nt, 558 nt and about 440 nt were found in slow skeletal
muscle, ALD (A).
152
I. YONEMURA ET AL.
Fig. 2. Nucleotide and deduced amino acid sequences of the
5′-region of chicken sTnT cDNA. The cDNA was generated by
RT-PCR and 5′-RACE with total RNA from chicken adult ALD
and complexus. Two RT-PCR products from ALD, A1-1R and
Aall-1R, and one 5′-RACE product from complexus, 20-2R, were
sequenced. The remaining stnt-5′R1 and stnt-5′R20b were described previously (Yonemura et al., ’96). The bracket and dashes
(-) indicate the absence of GCA coding for alanine and gaps,
respectively. The amino acid sequences are indicated in the
single-letter code above the nucleotide sequences. Two substitutions at 258 and 262 are marked with asterisks (*) with the
corresponding amino acid residues. Only single sequence is
shown in 45 to 104, 165 to 224, and 285 to 344 (designated as
com.), because the sequence was identical among the clones.
that the ca. 440 nt fragment was generated by digestion at 258 and/or 262 and the other fragment
of 558 nt by digestion at 135–137. Therefore, incomplete digestion at 258 and 262 might have occurred to produce the 558 nt fragment, because
S1 nuclease recognizes normally more than duplex
substitutions. Those results showed the heterogeneity in the 5′-region of chicken sTnT mRNA.
completely with the probe B (Fig. 3). In addition,
minor and smear bands of faster mobilities were
found. Similar pattern was detected in gastrocnemius of the 12-day-old embryo (data not shown).
In order to characterize those minor protected
fragments, we sequenced RT-PCR products of total RNA from adult ALD and complexus (Fig. 4).
We found four single base substitutions in the 3′region of sTnT cDNA at 703 (T or C), 774 (C or
T), 797 (C or T), and 827 (G or A). The substitutions at 703 and 774 cause the amino acid changes
from leucine in the five clones (b20-10, bA3, bA1,
b20-4, and b20-3) to proline in the other two clones
and from arginine in the two clones (b20-1- and
b20-3) to cysteine in the five remaining clones,
respectively. Two single base substitutions at 797
and 827 do not cause amino acid changes. The
results showed that chicken slow skeletal muscle
had at least four kinds of transcripts in the 3′-
Heterogeneity in the 3¢-region of
chicken sTnT mRNA
To search for heterogeneity in the 3′-region of
chicken sTnT mRNA, we performed S1 nuclease
mapping of total RNA from adult ALD with a 658
nt PCR-produced antisense probe (probe B) which
included the 6 nt restriction enzyme site (Xho I
or EcoR I) and 2 nt optional sequence at each end.
The major protected fragment was 642 nt long,
showing that the majority of transcripts matched
CHICKEN SLOW TROPONIN T mRNA
Fig. 3. S1 nuclease mapping of sTnT mRNA with an
antisense probe from the 3′-region of sTnT cDNA. The upper
panel shows schematically the structure of sTnT mRNA and
the position of the 658 nt antisense probe (probe B) generated by PCR. The probe included the 6 nt restriction enzyme
site and 2 nt optional sequence at each end and was 32Plabeled internally. Total RNA from adult chicken ALD (A) was
hybridized to the probe B and digested with S1 nuclease, and
protected fragments were separated on the 6% acrylamide/8
M urea gel. The major (642 nt) and minor protected fragments (bracket) were found.
region. In the 3′-region of human sTnT cDNA,
single base substitutions are also seen at positions
648 (G or C) and 649 (C or G) (Novelli et al., ’92).
These substitutions cause the amino acid changes
from lysine to asparagine (648) and from leucine
to valine (649), respectively. However, we could
not find the sequence corresponding to the 48 nt
insert reported in the 3′-region of human sTnT
cDNA (Gahlmann et al., ’87).
Structure of the 3¢-region of the
chicken sTnT gene
To investigate how the heterogeneity in the
chicken sTnT transcripts is generated, we deter-
153
mined the partial sequence of the chicken sTnT
gene. The size of the chicken sTnT gene was only
about 3.5 kb (data not shown), but we encountered serious difficulties in sequencing its 5′-region possibly due to extensive G-C tracts in
introns. The 3′-region of the gene was composed
of seven exons with the last exon including the
stop codon and polyadenylation signal (Fig. 5A).
We tentatively designated the exons as exon A to
G. There was no alternative exon. Each one of
the variable nucleotides at the four positions in
the transcripts was found in exon E (T at 703),
exon F (C at 774) and exon G (C and G at 797
and 827), suggesting that the 3′-heterogeneity in
the transcripts might be derived from allelic variation. We confirmed the absence of the sequence
corresponding to the 48 nt insert reported in the
3′-region of human sTnT cDNA (Gahlmann et al.,
’87), showing one major difference in the genomic
structure between chicken and human sTnT
genes. On comparison of the chicken sTnT gene
with the quail fTnT gene (Bucher et al., ’89), exons A, B, C, D, E, F, and G in the former had
sequence homology to exons 11, 12, 13, 14, 15, 17
and 18 in the latter, respectively (Fig. 5B). However, the length from exons A to G (1547 bp) was
considerably shorter than that from exons 11 to
17 (about 7.5 kb) due to highly small introns (84
to 329 bp) in the chicken sTnT gene.
DISCUSSION
We found the two kinds of transcripts in the
5′-region, one having the codon for alanine (135–
137) and C (258) and A (262) and the other lacking the codon and having T and G instead of C
and A. In the 3′-region, we found four single
base substitutions at 703 (T or C), 774 (C or T),
797 (C or T), and 827 (G or A). Four out of six
substitutions in the 5′- and 3′-regions lead to
amino acid changes in chicken sTnT isoforms.
Such nucleotide substitutions are also seen in
human sTnT (Gahlmann et al., ’87; Samson et
al., ’90; Novelli et al., ’92; Samson et al., ’94).
However, no single substitution has been reported in fTnT and cTnT cDNAs so far (Garfinkel et al., ’82; Medford et al., ’84; Breitbart
et al., ’85; Cooper and Ordahl, ’85; Smillie et
al., ’88; Bucher et al., ’89; Jin and Lin, ’89; Jin
et al., ’92; Morgan et al., ’93; Wu et al., ’94;
Briggs et al., ’94; Schachat et al., ’95; Wang and
Jin, ’97) with two exceptions in human fetal and
pathological cTnT cDNA (Mesnard et al., ’93, ’95;
Townsend et al., ’95).
How is the heterogeneity in chicken sTnT then
154
I. YONEMURA ET AL.
Fig. 4. Nucleotide and deduced amino acid sequences of
the 3′-region of chicken sTnT cDNA. The cDNA was generated by RT-PCR with total RNA from adult chicken ALD and
complexus. Two RT-PCR products from ALD, bA1 and bA3,
and five RT-PCR products from complexus, b20-1, b20-3, b204, b20-9, and b20-10, were sequenced. Four single base substitutions (marked with asterisks, *) are seen at 703 (T or
C), 774 (C or T), 797 (C or T), and 827 (G or A). The substitutions at 703 and 774 cause amino acid changes from leucine
to proline and from arginine to cysteine, respectively, but those
at 797 and 827 do not. The amino acid sequences are indicated above the nucleotide sequences and amino acid residues are also shown below the sequences at the positions
where amino acid changes are found.
generated? In the 5′-region, we can consider three
possible mechanisms. First, two exons covering
135 to 262 may exist, one having the codon for
alanine (135–137) and C (258) and A (262) and
the other lacking the codon and having T and G.
Two kinds of sTnT transcripts can be generated
by mutually exclusive alternative splicing of the
two exons. Secondly, when the variable nucleotides
in the 5′-region are included in more than two
exons, inclusion of one exon may be tightly linked
to that of the other(s) resulting in production of
only two transcripts. Therefore, alternative exons
of each set can be synchronously spliced. Thirdly,
we can not rule out completely that two kinds of
transcripts may be derived from different alleles
of this gene.
From the cDNA sequences and sTnT gene structure, we showed that four single base substitu-
tions in the 3′-region might be derived from allelic variation. Since human sTnT cDNAs have
also several single base substitutions and deletions in the 3′-region (Samson et al., ’90; Novelli
et al., ’92), it seems likely that the 3′-region of
the sTnT gene could accumulate mutations, possibly suggesting the moderate structural constraints on the COOH region of the sTnT protein.
Our data also showed the structural difference
between chicken and human sTnT genes, because
the chicken sTnT gene did not have the sequence
corresponding to the 48 nt insert reported in the
3′-region of human sTnT cDNA (Gahlmann et al.,
’87). The structural differences between avian and
mammalian genes have also been reported in fTnT
(Miyazaki et al., unpublished data; Breitbart and
Nadal-Ginard, ’86) and cTnT (Cooper and Ordahl,
’85; Jin et al., ’92).
CHICKEN SLOW TROPONIN T mRNA
155
Fig. 5. Structure of the 3′-region of the chicken sTnT
gene. (A) The sequence of the 3′-region of the chicken
sTnT gene (1547 bp) was determined. It included seven
exons, tentatively designated as exons A to G. Capital
and lower-case letters indicate exon and intron sequences, respectively. The amino acid sequences are indicated below the nucleotide sequences. Outlined letters
in exons E to G indicate the positions of four single base
substitutions. The asterisk (*) indicates the stop codon.
The bold underline indicates the putative polyadenylation signal. Italic letters within thin underlines indicate the 5′ and 3′ splice sites. (B) Comparison of the
3′-regions between chicken sTnT and quail fTnT genes
(Bucher et al., ‘89). The size of the 3′-region of the former
was considerably smaller than that of the latter. The
asterisks (*) indicate the positions of four single base
substitutions.
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development, patterns, transgenic, mice, female, glutathione, elevated, tissue, zygotes
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