.n) 1990 Oxford University Press
Nucleotide
Nucleic Acids Research, Vol. 18, No. 10 3053
sequence
of
cofilin cDNA
mouse
Kenji Moriyama, Seiji Matsumoto1, Eisuke Nishida, Hikoichi Sakai and Ichiro Yahara1 Department of Biophysics and Biochemistry, Faculty of Science, University of Tokyo, Hongo, Tokyo 113 and 1Department of Cell Biology, The Tokyo Metropolitan Institute of Medical Science, Honkomagome, Tokyo 113, Japan EMBL accession no. D00472
Submitted April 12, 1990
combination of cDNAs thus obtained has covered the overall sequence of the mouse brain cofilin message. The deduced amino acid sequence of mouse brain cofilin is 99% identical with that of porcine brain cofilin. Thereare only two different amino acid residues (boxed) out of 166 residues between them. Thus, mouse brain cofflin also contains a sequence (marked with *) similar to the nuclear transport signal sequence of SV40 large T antigen, a hexapeptide (overlined) identical with the amino-terminal sequence of tropomyosin, and series of portions (dotted) homologous to other actin-binding proteins. The sequence conservation of cofilin between these two species may suggest stringent requirements of the entire amino acid sequence for the functions of cofilin.
Cofflin is a widely distributed, pH-sensitive actin-modulating protein, which forms intranuclear and/or cytoplasmic actin-cofilin rods in cultured cells exposed to various stimuli (1, 2). Previously we isolated porcine brain cofilin cDNAs, and found that cofilin contains a putative nuclear location signal sequence and sequences homologous to other actin-binding proteins (3). Using these cDNAs as probes, here, we screened a mouse brain cDNA library (Clontech Lab., Inc.) and isolated 5 clones with a completely identical protein-coding sequence. To determine the 5'-noncoding sequence of the mouse brain cofilin mRNA, we constructed a cDNA library by primer extension method using a 17 base oligonucleotide complementary to nucleotide sequence 116-132 shown below. From this library we obtained 34 independent clones with cDNA inserts almost equal in size of 130 bp. We determined DNA sequence of 4 clones and found 3 clones extended up to 94 nucleotides and one clone up to 92 nucleotides upstream of the translation initiation codon. This suggests that 5' ends of these cDNAs are at or in the vicinity of the transcriptional start sites. We further isolated a cDNA clone containing a polyadenylation signal sequence (underlined) from another cDNA library we made by standard protocol. The
REFERENCES 1. Nishida,E., Maekawa,S. and Sakai,H. (1984) Biochemistry 23, 5307-5313. 2. Nishida,E., Iida,K., Yonezawa,N., Koyasu,S., Yahara,I. and Sakai,H. (1987) Proc. Natl. Acad. Sci. USA 84, 5262-5266. 3. Matsuzaki,F., Matsumoto,S., Yahara,I., Yonezawa,N., Nishida,E. and Sakai,H. (1988) J. Biol. Chem. 263, 11564-11568.
GCCGGGAGAGGCCGCGTTCAGTCGGATCCCGGCAGCAGCTGCGGCGGCTCAGGTCTTCAGTTGCTCTTTCTGT M A S G V A V S D G V I K V F N D M K V CTCCCTTTCGTTTCCGGAAACATGGCCTCTGGTGTGGCTGTCTCTGATGGTGTCATCAAGGTGTTCAATGACATGAAGGTT R
K
S
S
T
*
*
*
*
*
*
*
*
*
*
*
P
E
E
V
K
K
R
K K A
V
L
F
C
L
S
E
D
K K
N
I
73
154
CGCAAGTCTTCAACACCAGAAGAAGTGAAGAAMCGCAAGAAGGCGGTGCTCTTTTGCCTGAGTGAGGACAAGAMGAACATC
235
GGQ ATCCTGGAGGAGGGCAGGAGATCCTGGTAGGAGATGTGGGGCAGACTGTGGACGACCCCTACACCACTTTTGTCAGATG
316
I
L
L P
E
E
G K E
D
K
D
C
R
I
L
Y A
V
G
L Y
D
V
D A
T
Y
T
V
D
D
P
Y
T
F
V
K M
E
T
K
E
S
K K E
D
L
V
F
CTGCCAGACAAGGACTGCCGCTATGCACTCTATGATGCAACCTATGAGACCAAGGAGAGCAAGAAGGAGGACCTGGTGTTC 397 I F W A P E A P L K S K M I Y A S S K D A I K K K L ATCTTCTGGGCCCCCGAGATGCACCCCTCAAGAGCAAAATGATCTATGCCAGCTCCAAGGATGCCATCAAGAAGAAGCTG 478 T GIT K H E"L Q. A.NOC Y E E V K D R C T L A E K L G G ACAGGAATCAAGCATGAATTACAGCTAACTGCTACGAGGAGGTCAAGGACCGCTGCACCCTGGCAGAGAAACTAGGTGGC 559
SAV. .....E P AGCGCCGTCATTTCCCTGGAGGGCAAGCCTTTGTGAGCCACCTCCAGCCCCCTGCCTGGAGCATCTTAACAGCCCCAGACC 640 TGCTCTTGGGTGTTGCAGGCTGCCCCTTTCCTGCCAGACCGGAGGGGCTGGGGGGATCCCAGCAGGGGGAGGGCTATCCCT 721 TCACCCCAGTTGCCAAACATCCCTCCCACCCCCTGGACCGTCCTTCTCCCTCCATCCCTGACGGTTCTGGCCTTCCCAAAC 802 TGCTTTTGATCTTCTGATTCCTCTTGGGTTGACGCAGACCAAGTCCCGTCCTAGGCACCCAGTTTGGGGGGAGCCTGTATT 883 TTTTTTTTTAACGACACCCCTACTCCGTATCCCTCCCCATCCCATGCTGCCAACTTCTAACCACAATAGTGACTCTGTGCT 964 TGTCTGTTTAGTTCTGTGTGTAAATGAAATGTGGAAATGACCCTCCCTGCCCCAGCTGGCTGCCCTCCCCTTTCCTTTGAT 1045 CTTGACCACTCATGGAAGCAGGACCAGTAAGGGACCTTCAATTTAAAACAAAACAAAACAAAAAAACAATAAAAAGGCTAA 1126 1134 TTAACAAC G
K
L
Nucleotide
Nucleic Acids Research, Vol. 18, No. 10 3053
sequence
of
cofilin cDNA
mouse
Kenji Moriyama, Seiji Matsumoto1, Eisuke Nishida, Hikoichi Sakai and Ichiro Yahara1 Department of Biophysics and Biochemistry, Faculty of Science, University of Tokyo, Hongo, Tokyo 113 and 1Department of Cell Biology, The Tokyo Metropolitan Institute of Medical Science, Honkomagome, Tokyo 113, Japan EMBL accession no. D00472
Submitted April 12, 1990
combination of cDNAs thus obtained has covered the overall sequence of the mouse brain cofilin message. The deduced amino acid sequence of mouse brain cofilin is 99% identical with that of porcine brain cofilin. Thereare only two different amino acid residues (boxed) out of 166 residues between them. Thus, mouse brain cofflin also contains a sequence (marked with *) similar to the nuclear transport signal sequence of SV40 large T antigen, a hexapeptide (overlined) identical with the amino-terminal sequence of tropomyosin, and series of portions (dotted) homologous to other actin-binding proteins. The sequence conservation of cofilin between these two species may suggest stringent requirements of the entire amino acid sequence for the functions of cofilin.
Cofflin is a widely distributed, pH-sensitive actin-modulating protein, which forms intranuclear and/or cytoplasmic actin-cofilin rods in cultured cells exposed to various stimuli (1, 2). Previously we isolated porcine brain cofilin cDNAs, and found that cofilin contains a putative nuclear location signal sequence and sequences homologous to other actin-binding proteins (3). Using these cDNAs as probes, here, we screened a mouse brain cDNA library (Clontech Lab., Inc.) and isolated 5 clones with a completely identical protein-coding sequence. To determine the 5'-noncoding sequence of the mouse brain cofilin mRNA, we constructed a cDNA library by primer extension method using a 17 base oligonucleotide complementary to nucleotide sequence 116-132 shown below. From this library we obtained 34 independent clones with cDNA inserts almost equal in size of 130 bp. We determined DNA sequence of 4 clones and found 3 clones extended up to 94 nucleotides and one clone up to 92 nucleotides upstream of the translation initiation codon. This suggests that 5' ends of these cDNAs are at or in the vicinity of the transcriptional start sites. We further isolated a cDNA clone containing a polyadenylation signal sequence (underlined) from another cDNA library we made by standard protocol. The
REFERENCES 1. Nishida,E., Maekawa,S. and Sakai,H. (1984) Biochemistry 23, 5307-5313. 2. Nishida,E., Iida,K., Yonezawa,N., Koyasu,S., Yahara,I. and Sakai,H. (1987) Proc. Natl. Acad. Sci. USA 84, 5262-5266. 3. Matsuzaki,F., Matsumoto,S., Yahara,I., Yonezawa,N., Nishida,E. and Sakai,H. (1988) J. Biol. Chem. 263, 11564-11568.
GCCGGGAGAGGCCGCGTTCAGTCGGATCCCGGCAGCAGCTGCGGCGGCTCAGGTCTTCAGTTGCTCTTTCTGT M A S G V A V S D G V I K V F N D M K V CTCCCTTTCGTTTCCGGAAACATGGCCTCTGGTGTGGCTGTCTCTGATGGTGTCATCAAGGTGTTCAATGACATGAAGGTT R
K
S
S
T
*
*
*
*
*
*
*
*
*
*
*
P
E
E
V
K
K
R
K K A
V
L
F
C
L
S
E
D
K K
N
I
73
154
CGCAAGTCTTCAACACCAGAAGAAGTGAAGAAMCGCAAGAAGGCGGTGCTCTTTTGCCTGAGTGAGGACAAGAMGAACATC
235
GGQ ATCCTGGAGGAGGGCAGGAGATCCTGGTAGGAGATGTGGGGCAGACTGTGGACGACCCCTACACCACTTTTGTCAGATG
316
I
L
L P
E
E
G K E
D
K
D
C
R
I
L
Y A
V
G
L Y
D
V
D A
T
Y
T
V
D
D
P
Y
T
F
V
K M
E
T
K
E
S
K K E
D
L
V
F
CTGCCAGACAAGGACTGCCGCTATGCACTCTATGATGCAACCTATGAGACCAAGGAGAGCAAGAAGGAGGACCTGGTGTTC 397 I F W A P E A P L K S K M I Y A S S K D A I K K K L ATCTTCTGGGCCCCCGAGATGCACCCCTCAAGAGCAAAATGATCTATGCCAGCTCCAAGGATGCCATCAAGAAGAAGCTG 478 T GIT K H E"L Q. A.NOC Y E E V K D R C T L A E K L G G ACAGGAATCAAGCATGAATTACAGCTAACTGCTACGAGGAGGTCAAGGACCGCTGCACCCTGGCAGAGAAACTAGGTGGC 559
SAV. .....E P AGCGCCGTCATTTCCCTGGAGGGCAAGCCTTTGTGAGCCACCTCCAGCCCCCTGCCTGGAGCATCTTAACAGCCCCAGACC 640 TGCTCTTGGGTGTTGCAGGCTGCCCCTTTCCTGCCAGACCGGAGGGGCTGGGGGGATCCCAGCAGGGGGAGGGCTATCCCT 721 TCACCCCAGTTGCCAAACATCCCTCCCACCCCCTGGACCGTCCTTCTCCCTCCATCCCTGACGGTTCTGGCCTTCCCAAAC 802 TGCTTTTGATCTTCTGATTCCTCTTGGGTTGACGCAGACCAAGTCCCGTCCTAGGCACCCAGTTTGGGGGGAGCCTGTATT 883 TTTTTTTTTAACGACACCCCTACTCCGTATCCCTCCCCATCCCATGCTGCCAACTTCTAACCACAATAGTGACTCTGTGCT 964 TGTCTGTTTAGTTCTGTGTGTAAATGAAATGTGGAAATGACCCTCCCTGCCCCAGCTGGCTGCCCTCCCCTTTCCTTTGAT 1045 CTTGACCACTCATGGAAGCAGGACCAGTAAGGGACCTTCAATTTAAAACAAAACAAAACAAAAAAACAATAAAAAGGCTAA 1126 1134 TTAACAAC G
K
L
