Sequence of the cDNA encoding an actin homolog in the crayfish Procumbarus clarkii
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Gene, 133(1993) 303-304 0 1993 Elsevier Science Publishers B.V. All rights reserved. 0378-l 119/93/$06.00 303 GENE 07355 Sequence of the cDNA encoding an actin homolog in the crayfish Procumbarus clarkii (Nucleotide sequencing; cDNA library; open reading frame; poly(A)+ mRNA; recombinant DNA) W.-K. Kang” and Yoko Nayab “Life Sciences Division, King’s College London, Campden Hill Road, London W7AH, UK; and bSUNBOR (Suntory Institutefor Bioorganic Research), Shimamoto-cho, Mishima-gun, Osaka 618, Japan Received by J.A. Engler: 18 March 1993; Accepted 21 May 1993; Received at publishers: 21 June 1993 SUMMARY A cDNA library was constructed by using mRNAs purified from crayfish (Procambarus clarkii) muscle. Using a homology search of the nucleotide (nt) sequences, a clone of the library was found to encode a protein homologous to actin (Act). The insert fragment of this cDNA clone was 1072 nt in length. The amino acid sequence deduced from the nt sequence showed significant similarity to Act of various organisms as follows: 88.1% to Drosophila melanogaster, 88.2% to silk worm, 87.3% to brine shrimp, 86.3% to rat, and 86.3% to human (% identity). A cDNA library was constructed from crayfish muscle. One gram of muscle yielded approximately 1.5 mg of total RNA by acid guanidium thiocyanate-phenol- chloroform extraction (Chomczynski and Sacchi, 1987). Then using oligo(dT) cellulose (Invitrogen, San Diego, CA, USA), 10 pg of poly(A)+mRNA was obtained from 500 pg of total RNA, and 5 ug of it served as template to synthesize cDNA with cDNA synthesis kit (Stratagene, La Jolla, CA, USA). The cDNA library containing ap- proximately lo6 clones was then constructed by ligating the cDNAs to hZAPI1 vectors and packaging. Twelve clones were selected at random in order to investigate randomness of the resultant cDNA library; the sizes of their insert DNAs were determined by 1% agarose gel electrophoresis and their partial nt sequences were deter- mined by the method of Sanger et al. (1977) with non- RIDNA sequence kit (Millipore, Tokyo, Japan) and BcaBest DNA polymerase (Takara, Kyoto, Japan). By homology search against GenBank and PIR data bases, a clone was found to encode the protein similar to Act proteins. Therefore, we determined the complete nt se- quence of the insert fragment of the cDNA clone. As the result, the cDNA clone was found to contain the insert DNA of 1072 nt (Fig. 1) with poly(A) sequence of 27 nt [poly(A).sequence not shown]. This clone was, however, found to lack 150 nt at the N-terminus of the open read- ing frame of the actin gene judging from the similarity to other Act-encoding genes. The deduced aa sequence showed significant similarity to other actins: 88.1% to Drosophila, 88.2% to silk worm, 87.3% to brine shrimp, 86.3% to rat and 86.3% to human (% identity). This agrees with the notion that Act proteins are well conserved. Correspondence to: Dr. Y. Naya, SUNBOR, Wakayamadai, Shimamoto-cho, Mishima-gun, Osaka 618, Japan. Tel. (81-75) 962- 1660; Fax (81-75) 962-2115 Abbreviations: aa, amino acid(s); Act, actin( bp, base pair(s); kb, kilo- base(s) or 1000 bp; nt, nucleotide(s). ACKNOWLEDGEMENT We are grateful to Prof. K. Isono (Kobe University, Japan) for accession to the DDBJ data base nt sequences.
Transcript of Sequence of the cDNA encoding an actin homolog in the crayfish Procumbarus clarkii
Gene, 133(1993) 303-304 0 1993 Elsevier Science Publishers B.V. All rights reserved. 0378-l 119/93/$06.00 303
GENE 07355
Sequence of the cDNA encoding an actin homolog in the crayfish Procumbarus clarkii
(Nucleotide sequencing; cDNA library; open reading frame; poly(A)+ mRNA; recombinant DNA)
W.-K. Kang” and Yoko Nayab
“Life Sciences Division, King’s College London, Campden Hill Road, London W7AH, UK; and bSUNBOR (Suntory Institutefor Bioorganic Research), Shimamoto-cho, Mishima-gun, Osaka 618, Japan
Received by J.A. Engler: 18 March 1993; Accepted 21 May 1993; Received at publishers: 21 June 1993
SUMMARY
A cDNA library was constructed by using mRNAs purified from crayfish (Procambarus clarkii) muscle. Using a homology search of the nucleotide (nt) sequences, a clone of the library was found to encode a protein homologous to actin (Act). The insert fragment of this cDNA clone was 1072 nt in length. The amino acid sequence deduced from the nt sequence showed significant similarity to Act of various organisms as follows: 88.1% to Drosophila melanogaster, 88.2% to silk worm, 87.3% to brine shrimp, 86.3% to rat, and 86.3% to human (% identity).
A cDNA library was constructed from crayfish muscle. One gram of muscle yielded approximately 1.5 mg of total RNA by acid guanidium thiocyanate-phenol- chloroform extraction (Chomczynski and Sacchi, 1987). Then using oligo(dT) cellulose (Invitrogen, San Diego, CA, USA), 10 pg of poly(A)+mRNA was obtained from 500 pg of total RNA, and 5 ug of it served as template to synthesize cDNA with cDNA synthesis kit (Stratagene, La Jolla, CA, USA). The cDNA library containing ap- proximately lo6 clones was then constructed by ligating the cDNAs to hZAPI1 vectors and packaging. Twelve clones were selected at random in order to investigate randomness of the resultant cDNA library; the sizes of their insert DNAs were determined by 1% agarose gel electrophoresis and their partial nt sequences were deter- mined by the method of Sanger et al. (1977) with non- RIDNA sequence kit (Millipore, Tokyo, Japan) and
BcaBest DNA polymerase (Takara, Kyoto, Japan). By homology search against GenBank and PIR data bases, a clone was found to encode the protein similar to Act proteins. Therefore, we determined the complete nt se- quence of the insert fragment of the cDNA clone. As the result, the cDNA clone was found to contain the insert DNA of 1072 nt (Fig. 1) with poly(A) sequence of 27 nt [poly(A).sequence not shown]. This clone was, however, found to lack 150 nt at the N-terminus of the open read- ing frame of the actin gene judging from the similarity to other Act-encoding genes. The deduced aa sequence showed significant similarity to other actins: 88.1% to Drosophila, 88.2% to silk worm, 87.3% to brine shrimp, 86.3% to rat and 86.3% to human (% identity). This agrees with the notion that Act proteins are well conserved.
Correspondence to: Dr. Y. Naya, SUNBOR, Wakayamadai, Shimamoto-cho, Mishima-gun, Osaka 618, Japan. Tel. (81-75) 962- 1660; Fax (81-75) 962-2115
Abbreviations: aa, amino acid(s); Act, actin( bp, base pair(s); kb, kilo- base(s) or 1000 bp; nt, nucleotide(s).
ACKNOWLEDGEMENT
We are grateful to Prof. K. Isono (Kobe University, Japan) for accession to the DDBJ data base nt sequences.
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GGCACGAGCGTCGGTGATGAGGCTCAGAGCAAGAGAGGTATCCT~CTCT~GTACCCCATCGAGCACGGTAT~TCACC~CT~GACGACATffiAG~GATCT~TACCACACCTTC 120
GTSVGDEAQSKRGILTLKYP IEHGIITNWDDMEKIWYHTF 40
TACAATGAGCTCCGTATCGCCCCTGAGGAGTCCCC~C~TGCT~CTG~GCTCCCCTC~CCCC~~CC~CCGTGAG~GATGACT~TCATGTTT~~CTTT~CTT~CC 240
YNELRIAPEESPTMLTEAPLNPKANREKMTQIMFEAFNLP SO
GCCACTTACGTTACCATCCAGGCTGTGCTGTCCCTGTCCCTGTACGCCTCTGGTCGTACCACTGGTCT~TGTGCGACTCTGGT~T~TGTCTCT~~TGGTCCCCGTCTATG~GGTTTCGCC 360
ATYVTIQAVLSLYASGRTTGLVCDSGDGVSHMVPVYEGFA 120
CTTCCTCATGCCATCCTTCGTCTTGACCTTGCTT~TGGTCGTGACCT~CC~CTACCT~TG~GATCATGACTGA~GT~CTATTCCTTCACCACCACC~T~~GTGA~TTGTCCGT 480
LPHAILRLDLAGRDLTNYLMKI MTERGYSFTTTAEREIVR 160
GACATCAAGGAGAAGCTTTGCTACATCGCCCTTGACTTCGAG~TGAGAT~TGTATCT~AGCGTCTTCGTCTATTGA~GTCCTACGA~TTCCCGAC~TCAGGT~TCACCATT 600
DIKEXLCYIALDFENEMNVSAASSSIDKSYELPDGQVITI 200
GGCAATGAACGTTTCCGTTGTCCCGAGGCTCTCTCTTCCA~CTTCCTTCCTTGGTAT~~TCTGTT~TGTTCATG~CCGTCCA~~TCCATCATGA~TGCGACATTGACATCA~ 720
GNERFRCPEALFQPSFLGMESVGVHETVHSSIMRCDIDIR 240
AAGGACCTGTTCGCCIlACATTGTCGTATCTGGTGGTACCACCATGTACCCTGGTATTGCT~TC~TT~AG~~~T~CT~~TG~ACCT~TACCATC~GATC~GATCATT 840
KDLFANIVVSGGTTMYPGIADRLQKEITALAPATIKIKII 280
GCTCCTCCCGAGCGTAAGTACTCCGTCTGGATCGGTGGT~CTCCATCCTG~CTCTCTGTCCACTTTC~GACCATGT~AT~CC~ffiAGGAGTAC~CGAGTCCG~CCAG~ATCGTC 960
APPERKYSVWIGGSILASLSTFQTMWITKEEYDESGPGIV 320
CACCGCAAGTGCTTCTA GTTATATCTAACCAAGTTGGCATTATACCGTGTGCAAGAACCACT~TGTTTTTAATT 1045
HRKCF* 325
Fig. 1. Nucleotide sequence of the cDNA encoding actin protein of P. &&ii , and the deduced aa sequence. Two underlined AATAAA sequences
show the putative polyadenylation signals (Proudfoot, 1991). Asterisk marks the stop codon. The start codon is not shown, and the total Act is
probably 375 aa long Every tenth nt is marked with a colon.
REFERENCES
Chomczynski, P. and Sacchi, N.: Single-step method of RNA isolation
by acid guanidium thiocyanate phenol chloroform extraction. Anal.
Biochem. 162 (1987) 156-159.
Proudfoot, N.: Poly(A) signals. Cell 64 (1991) 671-674.
Sanger, F., Nicklen, S. and Coulson, A.R.: DNA sequencing with chain-
terminating inhibitors. Proc. Natl. Acad. Sci. USA 74 (1977)
5463-5467.
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