- Email: [email protected]
Structure of the mouse calpain small subunit gene
Biochimica et Biophysica Acta 1388 (1998) 247^252
Short sequence-paper
Structure of the mouse calpain small subunit gene J. Simon C. Arthur a , Peter A. Greer b , John S. Elce
a;
*
a
b
Department of Biochemistry, Queen's University, Kingston, Ontario K7L 3N6, Canada Departments of Biochemistry and Pathology, and the Cancer Research Laboratories, Queen's University, Kingston, Ontario K7L 3N6, Canada Received 5 May 1998; revised 13 July 1998; accepted 28 July 1998
Abstract The calpains comprise a family of heterodimeric (80+28 kDa) Ca2 -dependent cysteine proteases, probably having roles in signal transduction and cytoskeletal remodelling. We describe cloning and sequencing of the 28 kDa calpain subunit cDNA from mouse (coding for 268 amino acids), and characterization of its gene. The gene spans 7 kb and contains 11 exons. The promoter region, like those of other calpain genes, lacks an obvious TATA box, but contains several Sp1 binding sites. ß 1998 Elsevier Science B.V. All rights reserved. Keywords: Calpain; Small subunit; cDNA; Gene; (Mouse)
The calpains are cytosolic Ca2 -dependent cysteine proteases, and are essentially ubiquitous in mammalian tissue. Their physiological roles are unclear, but roles in integrin-mediated signal transduction, cytoskeletal remodelling and cell cycle control have been proposed [1,2]. The enzymes also appear to be involved in the tissue damage associated with ischemia in the brain and heart, and Alzheimer's disease [3]. In mammals there are two groups of calpains, referred to as ubiquitous and tissue-speci¢c [2]. The two ubiquitous calpains, W- and m-calpain, which have been extensively studied both at the DNA and protein levels, contain two subunits, a large (catalytic) 80 kDa subunit and a small (regulatory) 28 kDa subunit. The 80 kDa subunit contains 4 domains (I^IV), and its amino acid sequence di¡ers between W- and
* Corresponding author. Fax: +1-613-545-2497; E-mail: [email protected]
m-calpain. The 28 kDa subunit is identical in both Wand m-calpain, and consists of two domains; an Nterminal glycine-rich domain (V), and a C-terminal Ca2 -binding domain (VI). The small subunit is always present in W- and mcalpain isolated from natural sources, suggesting that it has an essential role, although the details of this role are presently subject to some debate. It has long been assumed that the small subunit is involved in biochemical regulation of enzyme activity [1], and also in regulating subcellular localization, since calpain binds via domain V to phospholipid membranes in the presence of Ca2 [4,5]. Alternatively it has been suggested that the small subunit serves only to assist in the correct folding of the large subunit, and that it dissociates from the large subunit in the presence of Ca2 [6,7]. In disagreement with the latter hypothesis, immunoprecipitation experiments showed that both subunits were still present in active calpain [8]; it has also been found that the two subunits remain associated on ion-exchange chromatog-
0167-4838 / 98 / $ ^ see front matter ß 1998 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 7 - 4 8 3 8 ( 9 8 ) 0 0 1 6 6 - 6
BBAPRO 30412 6-10-98
248
J.S.C. Arthur et al. / Biochimica et Biophysica Acta 1388 (1998) 247^252
Fig. 1. DNA sequence of the mouse calpain 28 kDa subunit. The sequence shown was assembled from sequences in several genomic DNA subclones, and includes all of exons 1 to 11. The longest cDNA clone obtained by PCR started at position 42 of exon 1 (V). Nucleotides in the PCR-derived cDNA clone which di¡ered from the genomic sequence are shown above the main sequence. The encoded amino acid sequence is shown below. The ¢rst coding nt of each exon is marked ( s ) and the polyadenylation sequence is underlined.
BBAPRO 30412 6-10-98
J.S.C. Arthur et al. / Biochimica et Biophysica Acta 1388 (1998) 247^252
249
Fig. 2. Amino acid sequence comparison of the calpain small subunits of six mammals. (1) human; (2) cow; (3) pig; (4) rabbit; (5) rat; (6): mouse. A dash indicates an identical residue, and an asterisk indicates a gap introduced to maximize the sequence identities.
raphy in the presence of Ca2 [9]. Further work is required to resolve these di¡erences. Sequences of the cDNA for the 28 kDa subunit have been reported for several species [9^14], and the structure of the human gene has been described [15]. Here we report the isolation and analysis both of cDNA and of genomic sequences for the murine 28 kDa subunit of calpain. The cDNA was obtained by screening a mouse leukemia cell line library in Vgt11 [16], using cDNA for the C-terminal region of the rat 28 kDa protein as a probe. The longest clone was subcloned by PCR into pUC19 and sequenced. The cDNA contained 33bp of the 5P untranslated region and the complete coding and 3P untranslated regions. A 129Sv mouse genomic library in V-Dash was then probed with cDNA for the mouse 28 kDa protein. Two overlapping genomic clones were isolated and used to map the intron^exon structure of the gene. The entire gene was sequenced except for some sections of the two longest introns (introns 6 and 9). The DNA coding sequence as derived from the genomic clones is shown in Fig. 1, together with some di¡erences which were observed between the cDNA and genomic sequences. Most of these were
in the third codon position and do not a¡ect the deduced amino acid sequence. They probably arise from di¡erences between the two mouse strains used for the production of the cDNA and genomic libraries, although PCR-generated errors in the cDNA clone cannot be excluded. An extra glycine codon was found at amino acid residue 37 in the cDNA, when compared with the genomic sequence: this difference was found in independent PCR clones of this section of the cDNA and therefore appears to represent a real di¡erence between mouse strains. It is noteworthy that the calpain small subunit sequences from various species di¡er within domain V by one to four glycine residues (Fig. 2). The gene in the 129Sv mouse encodes a 268-amino acid protein with a theoretical molecular mass of 28 389 Da and a high degree of amino acid sequence identity (93^ 97%) with calpain small subunits of other mammalian species. The amino acid sequences of six mammalian calpain small subunits are compared in Fig. 2. The sequence of the presumed promoter region containing exon 1 is shown in Fig. 3. The longest cDNA isolated contained 33 bp of 5P untranslated
BBAPRO 30412 6-10-98
250
J.S.C. Arthur et al. / Biochimica et Biophysica Acta 1388 (1998) 247^252
Fig. 3. Features of the presumed promoter region. The sequence of exon 1 is shown in upper-case letters and the 5P-terminal nucleotide of the longest cDNA clone is marked (V). The potential transcription initiation sequence is single-underlined and potential Sp1 sites are double-underlined. Sequences from this work are available from the authors and from GenBank under accession nos. AF058297 and AF058298.
sequence, of which the ¢rst 18 nt are assumed to be transcribed from the identical sequence found in genomic DNA, and located in a proposed exon 1 approximately 900 bp upstream from the start of exon 2. The transcription start site has not been determined experimentally, but analysis of the sequence shown in Fig. 3 revealed a potential promoter region upstream of the proposed exon 1, consisting of a transcription initiation sequence (CCCGAACCG) and several Sp1 binding sites (positions 316, 364 and 3248) (programs TSSW, TSSG, and TESS transcription start recognition, at http://dot.imgen.bcm.tmc.edu:9331/gene¢nder/gene-search.html). No TATA boxes were observed, but there are AT-
rich regions upstream of the start site which may increase the activity of the promoter [17]. Comparison of the putative promoter sequence shown in Fig. 3 with the corresponding region in the promoter sequence of the human 28 kDa subunit gene [15] revealed a stretch of 122 bp (from position 383 to +39) with 83% sequence identity; this region includes the Sp1 site at position 316 with 100% identity between mouse and human genes. No signi¢cant sequence identity was observed further upstream of this region. The promoter region of the human mcalpain large subunit gene is similar in that it lacks a TATA box and contains several Sp1 sites [18]. The genomic structure of the gene for the 28 kDa
Fig. 4. Structure of the gene for the 28 kDa calpain subunit. Exons are shown in grey and EcoRI (E), HindIII (H) and PstI (P) sites are arrowed. To optimise the scale of the diagram, two restriction sites outside the diagram and relevant to the Southern blot in Fig. 5 are marked by an asterisk.
BBAPRO 30412 6-10-98
J.S.C. Arthur et al. / Biochimica et Biophysica Acta 1388 (1998) 247^252
protein is summarized in Fig. 4. The gene spans approximately 7 kb and contains 11 exons. The 5P untranslated region is contained in exon 1 and the start of exon 2, and the N-terminal glycine-rich domain V of the subunit is encoded by exons 2 and 3. The Cterminal domain VI contains 5 EF-hand motifs [19] of which both EF1 and EF2 are encoded by 2 exons (exons 4^5 and 6^7, respectively), while EF3, EF4, and EF5 are each encoded by single exons (exons 8^ 10). Sequence for the last 8 amino acid residues and the whole 3P untranslated region is found in exon 11. The intron^exon boundaries (Table 1) all correspond well to the consensus sequences for splice sites. The positions of the intron^exon break points within the coding sequence are identical to those of the human gene, and the intron lengths are also similar between the two species. A Southern blot was performed on genomic DNA from 129Sv mice digested with PstI, HindIII, and EcoRI (Fig. 5). The blot was probed with a digoxigenin-labelled cDNA fragment (Boehringer-Mannheim, in accordance with the manufacturer's instructions) extending from the middle of exon 4 to the 3Pend of exon 11. Only the bands predicted from the genomic map were observed, suggesting that this is a single copy gene. The predicted fragments at 7.5 kb (HindIII) and 2.5 kb (EcoRI) are faint, which is thought to be due to weak hybridization of the probe to the short exons 4^6. These bands were con¢rmed by their strong hybridization to a 600 bp probe consisting of a fragment of genomic DNA spanning
251
Fig. 5. Southern blot of 129Sv mouse genomic DNA. The DNA was digested with PstI, HindIII, and EcoRI, and probed with a DIG-labelled cDNA probe extending from the middle of exon 4 to the 3P end of exon 11. Tracks: P, PstI digest; H, HindIII digest; E, EcoRI digest. Some V DNA size markers are indicated by arrows on the right.
exons 4^6 (data not shown). The genomic clones are currently being used for construction of a targeting vector to generate mice lacking a 28 kDa calpain subunit gene, in order to investigate the physiological functions of calpain. The 129Sv mouse genomic library was kindly provided by A. Reaume, R. Zirngibl and J. Rossant. This work was supported by the Medical Research Council of Canada and the Protein Engineering Network of Centres of Excellence (PENCE). P.A.G. is an MRC Scholar, and J.S.C.A. and J.S.E. are members of PENCE. We are grateful to Carol Hegadorn for expert technical assistance.
Table 1 Intron and exon lengths, in bp, and junction sequences Exon
5P Exon boundary
3P Exon boundary
Exon length
Intron classa
Intron length
1 2 3 4 5 6 7 8 9 10 11
^ ttacagGACTCA cctcagCGAGGC ctgcagCCTCCA cgtcagGACATG nntcagACCCGG tgttagAGCGAC ccttagGCTATA tcccagGATTCC ccgcagGCGCTT ttccagTGGCTG
GGATCCgtaagt CATCAGgtaagg CCTCCGgtaagc GGAGACgtaagt CCCGACgtgagt ATGGATgtatcc TGGCAGgtaagt CAGCAGgtgtgc TGTTCCgtgagt CAGGAGgtgagc ^
59 224 34 90 58 65 69 79 117 59 567
0 2 0 0 1 0 0 1 1 0
917 1063 274 125 93 V1300 99 310 V2000 121
a
Class 0 indicates a splice site between two codons, class 1 after the ¢rst nucleotide of a codon and class 2 after the second nucleotide.
BBAPRO 30412 6-10-98
252
J.S.C. Arthur et al. / Biochimica et Biophysica Acta 1388 (1998) 247^252
References [1] D.E. Croall, G.N. DeMartino, Calcium activated neutral protease (calpain) system: structure^function and regulation, Physiol. Rev. 71 (1991) 813^847. [2] K. Suzuki, H. Sorimachi, T. Yoshizawa, K. Kinbara, S. Ishiura, Calpain: novel family members, activation and physiological function, Biol. Chem. Hoppe-Seyler 376 (1995) 523^529. [3] K.K.W. Wang, P.W. Yuen, Calpain inhibition: an overview of its therapeutic potential, Trends Pharmacol. Sci. 15 (1994) 412^419. [4] J.S.C. Arthur, C. Crawford, Investigation of the interaction of calpain with phospholipids, Biochim. Biophys. Acta 1293 (1996) 201^206. [5] S. Imajoh, H. Kawasaki, K. Suzuki, The amino terminal hydrophobic region of the calcium-activated neutral protease (CANP) is essential for its activation by phosphatidylinositol, J. Biochem. 99 (1986) 1281^1284. [6] E.M. Vilei, S. Calderara, J. Anagli, S. Berardi, K. Hitomi, M. Maki, E. Carafoli, Functional properties of recombinant calpain I and mutants lacking domains III and IV of the catalytic subunit, J. Biol. Chem. 272 (1997) 25802^25808. [7] T. Yoshizawa, H. Sorimachi, S. Tomioka, S. Ishiura, K. Suzuki, A catalytic subunit of calpain possesses full catalytic activity, FEBS Lett. 358 (1995) 101^103. [8] W. Zhang, R.L. Mellgren, Calpain subunits remain associated during catalysis, Biochem. Biophys. Res. Commun. 227 (1996) 891^896. [9] J.S. Elce, P.L. Davies, C. Hegadorn, D.H. Maurice, J.S.C. Arthur, The e¡ects of truncation of the small subunit on mcalpain activity and heterodimer formation, Biochem. J. 326 (1997) 31^38. [10] Y. Emori, H. Kawasaki, S. Imajoh, S. Kawashima, K. Suzuki, Isolation and sequence analysis of cDNA clones for the small subunit of rabbit calcium-dependent protease, J. Biol. Chem. 261 (1986) 9472^9476.
[11] P. McCelland, J.A. Lash, D.R. Hathaway, Identi¢cation of major autolytic cleavage sites in the regulatory subunit of vascular calpain; a comparison of partial amino-terminal sequences to deduced sequence from complementary DNA, J. Biol. Chem. 264 (1989) 17428^17431. [12] S. Ohno, Y. Emori, K. Suzuki, Nucleotide sequence of a cDNA coding for the small subunit of human calcium-dependent protease, Nucleic Acids Res. 14 (1986) 5559. [13] T. Sakihama, H. Kakidani, K. Zenita, N. Yumoto, T. Kikuchi, T. Sasaki, R. Kannagi, S. Nakanishi, M. Ohmori, K. Takio, K. Titani, T. Murachi, A putative Ca2 binding protein: structure of the light subunit of porcine calpain elucidated by molecular cloning and protein sequence analysis, Proc. Natl. Acad. Sci. U.S.A. 82 (1985) 6075^6079. [14] H. Sorimachi, S. Amano, S. Ishiura, K. Suzuki, Primary sequences of rat W-calpain large and small subunits are respectively, moderately and highly similar to those of human, Biochim. Biophys. Acta 1309 (1996) 37^41. [15] M. Miyake, Y. Emori, K. Suzuki, Gene organisation of the small subunit of human calcium-activated neutral protease, Nucleic Acids Res. 14 (1986) 8805^8817. [16] Y. Ben-David, K. Letwin, L. Tannock, A. Bernstein, T. Pawson, A mammalian protein kinase with potential for serine/threonine and tyrosine phosphorylation is related to cell cycle regulators, EMBO J. 10 (1991) 317^325. [17] L. Weis, D. Reinberg, Accurate positioning of RNA polymerase II on a natural TATA-less promoter is independent of TATA-binding-protein-associated factors and initiatorbinding proteins, Mol. Cell. Biol. 17 (1997) 2973^2984. [18] A. Hata, S. Ohno, Y. Akita, K. Suzuki, Tandomly reiterated negative enhancer-like elements regulate transcription of a human gene for the large subunit of calpain-dependent protease, J. Biol. Chem. 264 (1989) 6404^6411. [19] H. Blanchard, P. Grochulski, L. Yunge, J.S.C. Arthur, P.L. Davies, J.S. Elce, M. Cygler, Structure of a calpain Ca2 binding domain reveals a novel EF-hand and Ca2 -induced conformational changes, Nat. Struct. Biol. 4 (1997) 532^538.
BBAPRO 30412 6-10-98
Short sequence-paper
Structure of the mouse calpain small subunit gene J. Simon C. Arthur a , Peter A. Greer b , John S. Elce
a;
*
a
b
Department of Biochemistry, Queen's University, Kingston, Ontario K7L 3N6, Canada Departments of Biochemistry and Pathology, and the Cancer Research Laboratories, Queen's University, Kingston, Ontario K7L 3N6, Canada Received 5 May 1998; revised 13 July 1998; accepted 28 July 1998
Abstract The calpains comprise a family of heterodimeric (80+28 kDa) Ca2 -dependent cysteine proteases, probably having roles in signal transduction and cytoskeletal remodelling. We describe cloning and sequencing of the 28 kDa calpain subunit cDNA from mouse (coding for 268 amino acids), and characterization of its gene. The gene spans 7 kb and contains 11 exons. The promoter region, like those of other calpain genes, lacks an obvious TATA box, but contains several Sp1 binding sites. ß 1998 Elsevier Science B.V. All rights reserved. Keywords: Calpain; Small subunit; cDNA; Gene; (Mouse)
The calpains are cytosolic Ca2 -dependent cysteine proteases, and are essentially ubiquitous in mammalian tissue. Their physiological roles are unclear, but roles in integrin-mediated signal transduction, cytoskeletal remodelling and cell cycle control have been proposed [1,2]. The enzymes also appear to be involved in the tissue damage associated with ischemia in the brain and heart, and Alzheimer's disease [3]. In mammals there are two groups of calpains, referred to as ubiquitous and tissue-speci¢c [2]. The two ubiquitous calpains, W- and m-calpain, which have been extensively studied both at the DNA and protein levels, contain two subunits, a large (catalytic) 80 kDa subunit and a small (regulatory) 28 kDa subunit. The 80 kDa subunit contains 4 domains (I^IV), and its amino acid sequence di¡ers between W- and
* Corresponding author. Fax: +1-613-545-2497; E-mail: [email protected]
m-calpain. The 28 kDa subunit is identical in both Wand m-calpain, and consists of two domains; an Nterminal glycine-rich domain (V), and a C-terminal Ca2 -binding domain (VI). The small subunit is always present in W- and mcalpain isolated from natural sources, suggesting that it has an essential role, although the details of this role are presently subject to some debate. It has long been assumed that the small subunit is involved in biochemical regulation of enzyme activity [1], and also in regulating subcellular localization, since calpain binds via domain V to phospholipid membranes in the presence of Ca2 [4,5]. Alternatively it has been suggested that the small subunit serves only to assist in the correct folding of the large subunit, and that it dissociates from the large subunit in the presence of Ca2 [6,7]. In disagreement with the latter hypothesis, immunoprecipitation experiments showed that both subunits were still present in active calpain [8]; it has also been found that the two subunits remain associated on ion-exchange chromatog-
0167-4838 / 98 / $ ^ see front matter ß 1998 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 7 - 4 8 3 8 ( 9 8 ) 0 0 1 6 6 - 6
BBAPRO 30412 6-10-98
248
J.S.C. Arthur et al. / Biochimica et Biophysica Acta 1388 (1998) 247^252
Fig. 1. DNA sequence of the mouse calpain 28 kDa subunit. The sequence shown was assembled from sequences in several genomic DNA subclones, and includes all of exons 1 to 11. The longest cDNA clone obtained by PCR started at position 42 of exon 1 (V). Nucleotides in the PCR-derived cDNA clone which di¡ered from the genomic sequence are shown above the main sequence. The encoded amino acid sequence is shown below. The ¢rst coding nt of each exon is marked ( s ) and the polyadenylation sequence is underlined.
BBAPRO 30412 6-10-98
J.S.C. Arthur et al. / Biochimica et Biophysica Acta 1388 (1998) 247^252
249
Fig. 2. Amino acid sequence comparison of the calpain small subunits of six mammals. (1) human; (2) cow; (3) pig; (4) rabbit; (5) rat; (6): mouse. A dash indicates an identical residue, and an asterisk indicates a gap introduced to maximize the sequence identities.
raphy in the presence of Ca2 [9]. Further work is required to resolve these di¡erences. Sequences of the cDNA for the 28 kDa subunit have been reported for several species [9^14], and the structure of the human gene has been described [15]. Here we report the isolation and analysis both of cDNA and of genomic sequences for the murine 28 kDa subunit of calpain. The cDNA was obtained by screening a mouse leukemia cell line library in Vgt11 [16], using cDNA for the C-terminal region of the rat 28 kDa protein as a probe. The longest clone was subcloned by PCR into pUC19 and sequenced. The cDNA contained 33bp of the 5P untranslated region and the complete coding and 3P untranslated regions. A 129Sv mouse genomic library in V-Dash was then probed with cDNA for the mouse 28 kDa protein. Two overlapping genomic clones were isolated and used to map the intron^exon structure of the gene. The entire gene was sequenced except for some sections of the two longest introns (introns 6 and 9). The DNA coding sequence as derived from the genomic clones is shown in Fig. 1, together with some di¡erences which were observed between the cDNA and genomic sequences. Most of these were
in the third codon position and do not a¡ect the deduced amino acid sequence. They probably arise from di¡erences between the two mouse strains used for the production of the cDNA and genomic libraries, although PCR-generated errors in the cDNA clone cannot be excluded. An extra glycine codon was found at amino acid residue 37 in the cDNA, when compared with the genomic sequence: this difference was found in independent PCR clones of this section of the cDNA and therefore appears to represent a real di¡erence between mouse strains. It is noteworthy that the calpain small subunit sequences from various species di¡er within domain V by one to four glycine residues (Fig. 2). The gene in the 129Sv mouse encodes a 268-amino acid protein with a theoretical molecular mass of 28 389 Da and a high degree of amino acid sequence identity (93^ 97%) with calpain small subunits of other mammalian species. The amino acid sequences of six mammalian calpain small subunits are compared in Fig. 2. The sequence of the presumed promoter region containing exon 1 is shown in Fig. 3. The longest cDNA isolated contained 33 bp of 5P untranslated
BBAPRO 30412 6-10-98
250
J.S.C. Arthur et al. / Biochimica et Biophysica Acta 1388 (1998) 247^252
Fig. 3. Features of the presumed promoter region. The sequence of exon 1 is shown in upper-case letters and the 5P-terminal nucleotide of the longest cDNA clone is marked (V). The potential transcription initiation sequence is single-underlined and potential Sp1 sites are double-underlined. Sequences from this work are available from the authors and from GenBank under accession nos. AF058297 and AF058298.
sequence, of which the ¢rst 18 nt are assumed to be transcribed from the identical sequence found in genomic DNA, and located in a proposed exon 1 approximately 900 bp upstream from the start of exon 2. The transcription start site has not been determined experimentally, but analysis of the sequence shown in Fig. 3 revealed a potential promoter region upstream of the proposed exon 1, consisting of a transcription initiation sequence (CCCGAACCG) and several Sp1 binding sites (positions 316, 364 and 3248) (programs TSSW, TSSG, and TESS transcription start recognition, at http://dot.imgen.bcm.tmc.edu:9331/gene¢nder/gene-search.html). No TATA boxes were observed, but there are AT-
rich regions upstream of the start site which may increase the activity of the promoter [17]. Comparison of the putative promoter sequence shown in Fig. 3 with the corresponding region in the promoter sequence of the human 28 kDa subunit gene [15] revealed a stretch of 122 bp (from position 383 to +39) with 83% sequence identity; this region includes the Sp1 site at position 316 with 100% identity between mouse and human genes. No signi¢cant sequence identity was observed further upstream of this region. The promoter region of the human mcalpain large subunit gene is similar in that it lacks a TATA box and contains several Sp1 sites [18]. The genomic structure of the gene for the 28 kDa
Fig. 4. Structure of the gene for the 28 kDa calpain subunit. Exons are shown in grey and EcoRI (E), HindIII (H) and PstI (P) sites are arrowed. To optimise the scale of the diagram, two restriction sites outside the diagram and relevant to the Southern blot in Fig. 5 are marked by an asterisk.
BBAPRO 30412 6-10-98
J.S.C. Arthur et al. / Biochimica et Biophysica Acta 1388 (1998) 247^252
protein is summarized in Fig. 4. The gene spans approximately 7 kb and contains 11 exons. The 5P untranslated region is contained in exon 1 and the start of exon 2, and the N-terminal glycine-rich domain V of the subunit is encoded by exons 2 and 3. The Cterminal domain VI contains 5 EF-hand motifs [19] of which both EF1 and EF2 are encoded by 2 exons (exons 4^5 and 6^7, respectively), while EF3, EF4, and EF5 are each encoded by single exons (exons 8^ 10). Sequence for the last 8 amino acid residues and the whole 3P untranslated region is found in exon 11. The intron^exon boundaries (Table 1) all correspond well to the consensus sequences for splice sites. The positions of the intron^exon break points within the coding sequence are identical to those of the human gene, and the intron lengths are also similar between the two species. A Southern blot was performed on genomic DNA from 129Sv mice digested with PstI, HindIII, and EcoRI (Fig. 5). The blot was probed with a digoxigenin-labelled cDNA fragment (Boehringer-Mannheim, in accordance with the manufacturer's instructions) extending from the middle of exon 4 to the 3Pend of exon 11. Only the bands predicted from the genomic map were observed, suggesting that this is a single copy gene. The predicted fragments at 7.5 kb (HindIII) and 2.5 kb (EcoRI) are faint, which is thought to be due to weak hybridization of the probe to the short exons 4^6. These bands were con¢rmed by their strong hybridization to a 600 bp probe consisting of a fragment of genomic DNA spanning
251
Fig. 5. Southern blot of 129Sv mouse genomic DNA. The DNA was digested with PstI, HindIII, and EcoRI, and probed with a DIG-labelled cDNA probe extending from the middle of exon 4 to the 3P end of exon 11. Tracks: P, PstI digest; H, HindIII digest; E, EcoRI digest. Some V DNA size markers are indicated by arrows on the right.
exons 4^6 (data not shown). The genomic clones are currently being used for construction of a targeting vector to generate mice lacking a 28 kDa calpain subunit gene, in order to investigate the physiological functions of calpain. The 129Sv mouse genomic library was kindly provided by A. Reaume, R. Zirngibl and J. Rossant. This work was supported by the Medical Research Council of Canada and the Protein Engineering Network of Centres of Excellence (PENCE). P.A.G. is an MRC Scholar, and J.S.C.A. and J.S.E. are members of PENCE. We are grateful to Carol Hegadorn for expert technical assistance.
Table 1 Intron and exon lengths, in bp, and junction sequences Exon
5P Exon boundary
3P Exon boundary
Exon length
Intron classa
Intron length
1 2 3 4 5 6 7 8 9 10 11
^ ttacagGACTCA cctcagCGAGGC ctgcagCCTCCA cgtcagGACATG nntcagACCCGG tgttagAGCGAC ccttagGCTATA tcccagGATTCC ccgcagGCGCTT ttccagTGGCTG
GGATCCgtaagt CATCAGgtaagg CCTCCGgtaagc GGAGACgtaagt CCCGACgtgagt ATGGATgtatcc TGGCAGgtaagt CAGCAGgtgtgc TGTTCCgtgagt CAGGAGgtgagc ^
59 224 34 90 58 65 69 79 117 59 567
0 2 0 0 1 0 0 1 1 0
917 1063 274 125 93 V1300 99 310 V2000 121
a
Class 0 indicates a splice site between two codons, class 1 after the ¢rst nucleotide of a codon and class 2 after the second nucleotide.
BBAPRO 30412 6-10-98
252
J.S.C. Arthur et al. / Biochimica et Biophysica Acta 1388 (1998) 247^252
References [1] D.E. Croall, G.N. DeMartino, Calcium activated neutral protease (calpain) system: structure^function and regulation, Physiol. Rev. 71 (1991) 813^847. [2] K. Suzuki, H. Sorimachi, T. Yoshizawa, K. Kinbara, S. Ishiura, Calpain: novel family members, activation and physiological function, Biol. Chem. Hoppe-Seyler 376 (1995) 523^529. [3] K.K.W. Wang, P.W. Yuen, Calpain inhibition: an overview of its therapeutic potential, Trends Pharmacol. Sci. 15 (1994) 412^419. [4] J.S.C. Arthur, C. Crawford, Investigation of the interaction of calpain with phospholipids, Biochim. Biophys. Acta 1293 (1996) 201^206. [5] S. Imajoh, H. Kawasaki, K. Suzuki, The amino terminal hydrophobic region of the calcium-activated neutral protease (CANP) is essential for its activation by phosphatidylinositol, J. Biochem. 99 (1986) 1281^1284. [6] E.M. Vilei, S. Calderara, J. Anagli, S. Berardi, K. Hitomi, M. Maki, E. Carafoli, Functional properties of recombinant calpain I and mutants lacking domains III and IV of the catalytic subunit, J. Biol. Chem. 272 (1997) 25802^25808. [7] T. Yoshizawa, H. Sorimachi, S. Tomioka, S. Ishiura, K. Suzuki, A catalytic subunit of calpain possesses full catalytic activity, FEBS Lett. 358 (1995) 101^103. [8] W. Zhang, R.L. Mellgren, Calpain subunits remain associated during catalysis, Biochem. Biophys. Res. Commun. 227 (1996) 891^896. [9] J.S. Elce, P.L. Davies, C. Hegadorn, D.H. Maurice, J.S.C. Arthur, The e¡ects of truncation of the small subunit on mcalpain activity and heterodimer formation, Biochem. J. 326 (1997) 31^38. [10] Y. Emori, H. Kawasaki, S. Imajoh, S. Kawashima, K. Suzuki, Isolation and sequence analysis of cDNA clones for the small subunit of rabbit calcium-dependent protease, J. Biol. Chem. 261 (1986) 9472^9476.
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