Cloning and characterization of alternatively spliced

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clone and characterize four alternatively spliced Dp71 transcripts from cultured ... cDNAs encoding these Dp71 transcripts were shown to be alternatively spliced ...
7995 Oxford University Press

Human Molecular Genetics, 1995, Vol. 4, No. 9

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Cloning and characterization of alternatively spliced isoforms of Dp71 Richard C.Austin*, Perry L.Howard1, Vinita N.D'Souza1, Henry J.KIamut2 and Peter N.Ray1 Department of Pathology, McMaster University and the Hamilton Civic Hospitals Research Centre, Hamilton, Ontario, L8V 1C3, 1 Genetics Department and Research Institute, The Hospital for Sick Children, 555 University Avenue, Toronto, Ontario, M5G 1X8 and ^ h e Ontario Cancer Institute, 500 Sherbourne Street, Toronto, Ontario, Canada, M4X 1K9 Received March 16, 1995; Revised and Accepted May 29, 1995

INTRODUCTION Duchenne muscular dystrophy (DMD) is a lethal X-linked inherited disease affecting primarily skeletal and cardiac muscle (1,2), as well as some nonmuscle tissues including brain and retina (3-6). The DMD gene consists of at least 79 exons spanning over 2.4 Mb of DNA on the human X chromosome (7,8). Its gene product, dystrophin, is transcribed in muscle from a 14 kb transcript encoding a 3685 amino acid protein of 427 kDa (9). Immunohistochemical analysis using dystrophinspecific antibodies showed that dystrophin is localized to the sarcolemma of skeletal muscle (10,11), suggesting a structural role in maintaining membrane stability (12-14). Recently, a 70-75 kDa C-terminal dystrophin isoform has been identified as the major DMD gene product in many nonmuscle tissues, including brain and liver (15-20), but is absent or barely detectable in normal skeletal muscle (16,21,22). This C-terminal dystrophin isoform, termed Dp71 or apo-dystrophin-1, is generated from a 6.5 kb (16,19) and/

*To whom correspondence should be addressed

or a 4.8 kb mRNA transcript (18) arising from a C-terminal promoter situated between exons 62 and 63 of the human DMD gene (15). Dp71 was initially identified and cloned from human amniotic fluid cells (16) and: (i) encodes the cysteinerich and C-terminal domains of dystrophin; (ii) has a unique exon one encoding seven novel amino acids at the N-terminus; and (iii) has both exons 71 and 78 spliced from the transcript. While the loss of exon 71 does not change the reading frame of the transcript, the loss of exon 78 does, resulting in the 13 C-terminal amino acids of dystrophin being replaced by 31 new amino acids in the Dp71 protein product (16). At present, the function of Dp71 and its significance in the pathogenesis of DMD is unknown. DMD patients lacking Dp71 due to C-terminal deletions in the dystrophin gene appear clinically similar to DMD patients retaining Dp71 (2). There has been speculation that Dp71, in conjunction with Dpi 16 (23) and Dpl40 (24) may be causally involved in DMD-

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Dp71, a C-terminal isoform of dystrophin, has been identified as the major DMD gene product in many nonmuscle tissues. In this report, reverse transcriptase-polymerase chain reaction (RT-PCR) was used to clone and characterize four alternatively spliced Dp71 transcripts from cultured human amniocytes. The cDNAs encoding these Dp71 transcripts were shown to be alternatively spliced for exons 71 and/or 78. RT-PCR analysis also revealed that Dp71 transcripts alternatively spliced for exons 71 and/or 78 were expressed at varying levels in a number of adult human tissues, including muscle, heart, brain, kidney, lung, testis and liver. To investigate size heterogeneity at the translational level, Dp71 cDNAs isolated from amniocytes were expressed in E.coli to generate recombinant Dp71 fusion proteins. These fusion proteins were identified on immunoblots using antibodies specific for the C-terminal sequences of dystrophin that either included (antibody 1461) or excluded exon 78 (antibody 462B). The molecular masses of the Dp71 fusion proteins ranged from 71-75 kDa on SDS-PAGE, consistent with their predicted values. Immunoblot analysis using antibodies 1461 and 462B identified multiple Dp71 isoforms of approximately 70-75 kDa on SDS-PAGE in total protein lysates from amniocytes and various adult human tissues. This variation in molecular mass is consistent with the expression of Dp71 isoforms derived from transcripts alternatively spliced for exons 71 and/or 78. Total protein lysates from normal skeletal muscle, DMD muscle, amniocytes and brain were shown to contain p-dystroglycan, a component of the dystrophin-associated glycoprotein complex (DGC). Taken together, these results indicate that Dp71 is alternatively spliced for exons 71 and/or 78, and encodes multiple protein isoforms in a wide range of human tissues. The co-expression of these isoforms with p-dystroglycan suggests that they may form part of a 'nonmuscle' DGC somewhat similar to that observed in muscle.

1476 Human Molecular Genetics, 1995, Vol. 4, No. 9

RESULTS Cloning of alternatively spliced Dp71 cDNAs from cultured human amniocytes Previous studies have shown that human amniotic fluid cells express Dp71 (16) and to a lesser extent full-length dystrophin (30). In order to identify and clone additional Dp71 isoforms, Dp71 cDNA was synthesized from total human amniocyte RNA using a reverse primer (513; nucleotides 11519-11541 of human dystrophin cDNA) complementary to a sequence present in the 3'-untranslated region of dystrophin mRNA (Fig. 1A). PCR analysis, using the same reverse primer and a forward primer (512) corresponding to a sequence in the 5'untranslated region of the Dp71 transcript (Fig. 1 A), revealed a PCR product of approximately 2.1 kb (Fig. IB, lane 3). This product was purified from agarose and re-amplified with nested primers 741 and 2296 (Fig. 1A). This second amplification included the open-reading frame of the Dp71 cDNA and enabled it to be cloned into the £coRI site (in the correct reading frame and orientation) of the bacterial expression vector pFL-1 (see Materials and Methods). Initially, the 1.9 kb nested PCR product (Fig. IB, lane 4) was ligated into pBluescript(KS) and used to transform E.coli cells. Analysis of £coRI digested plasmids derived from four representative independent recombinant clones revealed subtle differences in insert size ranging from approximately 1.8 to 1.9 kb (Fig. 2). To investigate the possibility that the size variation in the clones was due to alternative splicing of the primary gene transcript, one of the clones, pBS-S2 (+/-) (Fig. 2), was sequenced and found to differ from the sequence previously reported by Lederfein et al. (16), in that exon 71 (nucleotides 10432-10470 of dystrophin cDNA) was present in the cDNA. PCR amplification with primers flanking exons 71 (695 and 926) and 78 (837 and 2296) were used to determine if the other representative clones Ell (+/+), S3 (-/+) and S8 (-/-) were alternatively spliced (Fig. 3A). Exon 78 (nucleotides 11223-11254 of the dystrophin gene) was also examined since

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Figure 1. Identification of Dp71 transcripts in human amniocytes. (A) Schematic diagram of Dp71 and the location of the oligonucleotide primers used for RT-PCR and nested PCR. The boxed area represents the open reading frame for the Dp71 transcript. The arrowhead indicates the splice-junction site between the unique exon 1 of Dp71 and the sequence common to dystrophin mRNA. The location and orientation of the primers used for the RT-PCR (512 and 513) and nested PCR (741 and 2296) are shown. (B) Cloning of the Dp71 transcripts using RT-PCR. Primer 513 was used initially to reverse transcribe RNA isolated from cultured human amniocytes. The cDNA was amplified by PCR with the same 513 primer and a primer (512) located within the unique exon 1 of the C-terminal transcript. Lane 1, negative control RT-PCR in the absence of primers 512 and 513; lane 2, negative control RT-PCR in the absence of amniocyte cDNA; lane 3, amniocyte cDNA amplified with primers 512 and 513 (dark arrow); lane 4, PCR product using nested primers 741 and 2296 and gel-purified DNA from lane 3 as a template (light arrow). M, 1 kb DNA ladder. Primer 741 is positioned at the initiator ATG while primer 2296 is positioned at the unique stop codon of Dp7l cDNA, as previously described (16).

this exon has also been shown to be alternatively spliced in full-length dystrophin and Dp71 (16,31,32). PCR analysis showed that clone Ell (+/+) contained both exons 71 and 78, clone S2 (+/-) contained exon 71 but not 78 (confirming sequencing data), clone S3 (-/+) contained exon 78 but not 71 and clone S8 (-/-) contained neither exons 71 nor 78 (Fig. 3B). Double-stranded sequencing of both strands of each Dp71 cDNA showed that the exon boundaries, with or without exons 71 and 78, were identical to those previously described by Feener et al. (32) (data not shown). RT-PCR analysis of alternative splicing of Dp71 transcripts in adult human tissues In order to detect individual Dp71 mRNA populations and also to determine whether there were tissue-specific differences in alternative splicing of exons 71 and/or 78, randomly primed cDNA from a variety of adult human tissues was analyzed by PCR. Amplification with primers 512 and 513, followed by nested primers 741 and 2296 (see Fig. 1), resulted in a Dp7I cDNA band of approximately 1.9 kb present in each tissue except muscle and kidney (Fig. 4A). However, 10 additional cycles of PCR on the muscle and kidney samples resulted in the appearance of a 1.9 kb band (data not shown). In brain, in addition to the 1.9 kb band, there was a smaller band of

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associated cognitive impairment (25). However, no abnormal neuropathy has been reported in either DMD patients lacking Dp71 (2) or mdxCv3 mice deficient in both dystrophin and Dp71 (20). Unlike dystrophin, Dp71 does not contain the spectrinlike repeat domain nor the N-terminal actin-binding domain, suggesting that its cellular function is different from fulllength dystrophin. Based on its ability to associate with the plasma membrane (22,26,27), Dp71 could be involved in membrane anchorage and/or organization via dystrophinassociated proteins (DAPs) such as fj-dystroglycan (28) or with proteins distinct from the DAPs described in muscle (12,13). In fact, Dp71 has been shown to associate with syntrophin in a variety of tissues (29). In an analysis of rodent tissues, Kramarcy et al. (29) demonstrated the existence of multiple Dp71 isoforms and suggested that they result from alternative splicing in the Dp71 transcript. In this report, we show, at both the transcript and protein level, that Dp71 is alternatively spliced for exons 71 and/or 78 to generate multiple protein products in a number of adult human tissues. The coexpression of these Dp71 isoforms with (3-dystroglycan in these tissues suggests that these proteins may form part of a 'nonmuscle' DGC similar to that characterized in muscle.

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Figure 2. Size heterogeneity of the different Dp71 cDNAs. Miniprep plasmid DNA isolated from four representative independent bacterial clones Ell (+/ +), S2 (+/-), S3 (-/+) and S8 (-/-) was digested with EcoRl and separated on a 1% agarose-TBE gel containing ethidium bromide. pBS, pBluescript(KS); insert, represents the cDNAs encoding the Dp71 cDNAs. The + and symbols in brackets represent the presence or absence of exons 71 and/or 78, respectively (see Fig. 3).

approximately 1.6 kb which may be the result of additional splicing. To determine if exons 71 and/or 78 were alternatively spliced, Dp71 cDNAs shown in Figure 4A were gel-purified and used as DNA templates for subsequent PCR analysis with primers flanking either exon 71 (Fig. 4B) or 78 (Fig. 4C). In Figure 4B, brain, muscle, kidney, lung, testis and liver were shown to contain Dp71 transcripts with exon 71 present or spliced out. Heart contained only Dp71 transcripts spliced for exon 71. In Figure 4C, brain, muscle, kidney, lung and testis contained Dp71 transcripts with exon 78 either present or spliced out while heart and liver had only Dp71 transcripts spliced for exon 78. These results suggest that there is tissuespecific expression of Dp71 with multiple forms in most tissues, including brain, muscle, kidney, liver and testis. These data do not, however, rule out the possibility that other Dp71 transcripts alternatively spliced for exons other than 71 or 78 may exist in some tissues. Expression of the different Dp71 cDNAs in E.coli To determine size heterogeneity of the Dp71 isoforms at the protein level, cDNAs encoding the different Dp71 isoforms cloned from amniocytes were expressed as fusion proteins in E.coli using the bacterial expression vector pFL-1 (33) and examined by immunoblotting. To distinguish between Dp71 fusion proteins differing in their C-terminal sequence (due to alternative splicing of exon 78) two C-terminal dystrophinspecific antibodies were used: (i) antibody 1461, which recognizes the last 17 amino acids of dystrophin; and (ii) antibody 462B, which recognizes the alternative 31 amino acid Cterminus due to the splicing of exon 78 (Drs L.Kunkel and F.M.Boyce, personal comm.). An anti-FLAG Ml monoclonal antibody, which recognizes the N-terminal 8 amino acid FLAG sequence, was used to identify the FLAG-Dp71 fusion proteins. Based on alternative splicing within the different Dp71 cDNAs,

Figure 3. Alternative splicing within'the different Dp71 cDNAs. (A) Schematic of the cDNA for Dp71 and the primer sets used for amplification of the regions flanking exons 71 and 78. Location and orientation of the primers used are shown by arrows. e71, exon 71. e78, exon 78. ATG, initiator methionine. (B) PCR analysis of the Dp71 cDNAs with primers flanking exons 71 and 78. Dp71 cDNAs described in Figure 2 were gel-purified and amplified by PCR using primer sets which flank either exons 71 or 78. Amplified products were combined and separated on a 1.5% agarose-TBE gel containing ethidium bromide. Two specific PCR products were evident in lanes containing cDNA as a template. The 266 bp product represents Dp71 cDNAs having an intact exon 71 while the 227 bp product represents cDNAs spliced for exon 71. The 160 bp product represents Dp71 cDNAs having an intact exon 78 while the 128 bp product represents cDNAs spliced for exon 78. The + and - symbols in brackets represent the presence or absence of exons 71 and/or 78, respectively, -primer, PCR reaction in the absence of primers, -template, PCR reaction in the absence of DNA template. 123 bp, 123 bp DNA ladder.

the translated products of Ell (+/+), S2 (+/-), S3 (-/+) and S8 (-/-) would have molecular masses of approximately 70.4, 72.2, 68.9 and 70.8 kDa, respectively. The additional 17 amino acids encoded by the N-terminal fusion would increase the molecular mass of each recombinant protein by 2.1 kDa (see Materials and Methods). Size heterogeneity for the four FLAGDp71 fusion proteins shown using the anti-FLAG Ml monoclonal antibody and their migration positions were consistent with their predicted molecular masses on SDS-PAGE (Fig. 5). The FLAG-Dp71 fusion proteins Ell ( + / + ) and S3 (-/+) containing an intact exon 78 stained intensely with the 1461 antibody (Fig. 5). In addition, a bacterial fusion protein containing maltose-binding protein (mbp) fused to the last 330 amino acids of dystrophin stained intensely with this antibody (Fig. 5, mbp-C33O). This observation was expected since the 1461 antibody is directed primarily to exon 78 with only part

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Figure 4. RT-PCR analysis of alternatively spliced transcripts of Dp71 from adult human tissues. Randomly primed cDNAs derived from adult human brain (B), muscle (M), heart (H), kidney (K), lung (L), testis (T) and liver (Li) were PCR-amplified as described in Materials and Methods and Figure 1. RT-PCR products were separated on 2% agarose gels and stained with ethidium bromide. (A) PCR analysis using primers 741 and 2296 which flank the open reading frame of Dp71. +/-, PCR reactions in the presence or absence of DNA template. Cl, PCR reaction in the absence of primers. C2, PCR reaction in the absence of template. Dp71 cDNAs from (A) were gelpurified and amplified by PCR using primer sets which flank either exons 71 or 78 described in Figure 3. (B) PCR analysis using primers 695 and 926 which flank exon 71. The upper band (266 bp) corresponds to the presence of exon 71 while the lower band (227 bp) corresponds to the splicing of exon 71. (C) PCR analysis using primers 837 and 2296 which flank exon 78. The upper band (160 bp) corresponds to the presence of exon 78 while the lower band (128 bp) corresponds to the splicing of exon 78. S2, Dp71 cDNA clone containing exon 71 but not 78. S3, Dp71 cDNA clone containing exon 78 but not 71. 1 kb, 1 kb DNA ladder. 100 bp, 100 bp DNA ladder.

of the antigen (five amino acids) being derived from exon 77 (11). These fusion proteins did not stain with the 462B antibody. Conversely, the FLAG-Dp71 fusion proteins S2 (+/-) and S8 (-/-) containing the alternative 31 amino acid C-terminal end due to the removal of exon 78 stained intensely with the 462B antibody but did not stain well with the 1461 antibody. These data indicate that antibodies 1461 and 462B were able to recognize different Dp71 fusion proteins alternatively spliced for exon 78. Expression and tissue distribution of the Dp71 isoforms To study the expression and tissue distribution of the Dp71 isoforms, total protein lysates from human amniocytes and various adult human tissues were examined by immunoblotting using the C-terminal antibodies 1461 and 462B (Fig. 6). Using antibody 1461, Dp71 isoforms which retained the C-terminal sequence of dystrophin were detected at varying levels in the 70-75 kDa range in all tissues studied (Fig. 6A). This antibody

Figure 5. Immunoblot analysis of recombinant Dp71 fusion proteins expressed in E.coli. Total cell lysates from IPTG-induced bacterial clones Ell (+/+), S2 (+/-), S3 (-/+) and S8 (-/-) were separated on 10% SDS-polyacrylamide gels, under reducing conditions and transferred to nitrocellulose. Fusion proteins containing Dp71 isoforms fused to an N-terminal FLAG extension (FLAG-Dp71) were detected using the anti-FLAG Ml monoclonal antibody (anti-FLAG). FLAG-Dp71 fusion proteins containing the dystrophin Cterminal sequences were detected using the 1461 antibody while fusion proteins containing the alternative 31 amino acid C-terminus (due to the splicing of exon 78) were detected using the 462B antibody. mbp-C330 represents a fusion protein consisting of maltose-binding protein (mbp) fused to the last 330 amino acids of skeletal muscle dystrophin. The + and symbols represent the presence or absence of exons 71 or 78 within the cDNA. kDa, molecular mass markers.

also detected full-length dystrophin in skeletal muscle lysates. Amniocytes, and to a lesser degree DMD muscle, were shown to contain at least three Dp71 isoforms immunoreactive for antibody 1461. In brain lysates, the isoforms appeared to migrate as a doublet of 71-73 kDa with an intensity of staining similar to that observed for muscle dystrophin, and is consistent with previous observations (16,18). In kidney lysates, a single reactive band corresponding to the lower band in brain was observed. Consistent with the RT-PCR data suggesting that liver should not contain Dp71 isoforms immunoreactive for antibody 1461, only a very weak signal was observed at 73 kDa (Fig. 6A). With antibody 462B, human amniocytes and all tissues examined were shown to contain varying levels of Dp71 isoforms having the alternative 31 amino acid C-terminal tail (Fig. 6B). In most tissues, these isoforms appeared as doublets in the range of 72-75 kDa. Normal and DMD muscle were shown to contain only a single band corresponding to the lower band observed in the other tissues. The difference in molecular mass of Dp71 isoforms, detected using either antibodies 1461 or 462B, is consistent with Dp71 isoforms derived from transcripts alternatively spliced for exons 71 and/ or 78. The observation that DMD muscle contained higher levels of Dp71 isoforms, compared to normal skeletal muscle, suggests that, like utrophin, Dp71 may be upregulated in response to the loss of dystrophin or that a larger proportion of nonmuscle cells expressing Dp71 are present in DMD muscle compared to normal muscle (34). Antibodies directed against the spectrin-like repeat domain of full-length dystrophin (MANDYS1) were shown to react only with full-length dystrophin in normal skeletal muscle and, to a lesser extent, in brain

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462B Figure 6. Immunoblot analysis of Dp71 isoforms expressed in human amniocytes and various adult human tissues. Total protein lysates (40 ng) were separated on 7.5% SDS-polyacrylamide mini-gels, transferred to nitrocellulose and immunostained with the appropriate dystrophin-specific antibody. (A) Detection of dystrophin and Dp71 isoforms retaining the Cterminal sequence of dystrophin using the 1461 antibody. (B) Detection of Dp7l isoforms containing the alternative 31 amino acid C-terminus (due to the loss of exon 78) using the 462B antibody. (C) Detection of full-length dystrophin using a monoclonal antibody (MANDYS1) directed to the spectrinrepeat rod domain. The migration positions of the Dp71 isoforms (70-75 kDa) are shown by the asterisk while the migration position of full-length dystrophin (427 kDa) is shown by the arrowhead. kDa, molecular mass markers.

(Fig. 6C). These results indicate that the immunoreactive bands detected with antibodies 1461 and 462B were indeed isoforms of Dp71 and were not the result of degradation or cleavage products of full-length dystrophin. Expression and tissue distribution of P-dystroglycan Recent studies in mdx mice transgenic for Dp71 have shown that Dp71 can localize to the sarcolemma membrane and restore normal expression and localization of all members of the muscle DGC (26,27). Although the normal function of Dp71 in nonmuscle tissues is unknown it is possible that it may associate with members of the DGC that are not musclespecific, such as p-dystroglycan (28). Immunoblot analysis using a monoclonal antibody (43DAG/8D5) specific for Pdystroglycan (35) showed the presence of this DGC component in amniocytes, brain and muscle (Fig. 7).

DISCUSSION Previous studies have shown that Dp71 is the major DMD gene product expressed in many nonmuscle tissues, including brain and liver (15-21,29). At present, neither the number of expressed isoforms nor the function of Dp71 in human tissues is known. In this report, we have identified and characterized alternatively spliced Dp71 transcripts and their protein products from cultured human amniocytes and various human tissues. Specifically, we have shown that: (i) Dp71 transcripts are

Figure 7. Immunoblot analysis of P-dystroglycan in human amniocytes, brain and muscle. Total protein lysates (40 jig) were separated on a 10% SDSpolyacrylamide mini-gel, transferred to nitrocellulose and immunostained with a monoclonal antibody to P-dystroglycan (43DAG/8D5). The antibody shows the presence of P-dystroglycan in all samples examined, with the lowest amount in amniocytes. P-dystroglycan is present at considerably lower levels in DMD muscle then in normal skeletal muscle. The migration position of Pdystroglycan is shown by the arrowhead. kDa, molecular mass markers.

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Figure 8. Amino acid sequence alignment of the extreme C-terminus of dystrophin, Dp71 (with and without exon 78) and utrophin. The amino acids encoding exon 78 of dystrophin are underlined. The boxed sequence in dystrophin represents a p34 c d " consensus site. The arrow represents the point of divergence in the amino acid sequence of Dp71 due to the splicing of exon 78 in the transcript.

alternatively spliced for exons 71 and/or 78 in a variety of adult human tissues; and (ii) Dp71 is expressed as multiple protein isoforms in a tissue-specific manner. Four alternatively spliced Dp71 cDNAs were initially cloned from cultured human amniocytes using RT-PCR followed by nested PCR and their translational products characterized by expression in E.coli. These alternatively spliced Dp71 isoforms result exclusively from the presence or absence of exons 71 and/or 78 within the transcript. One of these cDNA clones, S8 (-/-), has been cloned previously by RT-PCR using RNA from human amniotic fluid cells (16). The fact that we have cloned three additional, alternatively spliced transcripts from the same cultured cells may reflect differences in cell growth and differentiation (30). In an effort to characterize the protein products of these cloned Dp71 cDNAs, we expressed them in E.coli to generate recombinant Dp71 fusion proteins. The

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Human Molecular Genetics, 1995, Vol. 4, No. 9 reports have shown that Dp71 is phosphorylated at serine and threonine residues of the C-terminus of dystrophin in myoblast cell cultures and in skeletal muscle from mdx mice transgenic for Dp71 (26). This is consistent with reports showing that the C-terminal domain of full-length dystrophin can be phosphorylated by endogenous protein kinases (39-42). In particular, Milner et al. (40) showed that the C-terminal region of dystrophin is a substrate for in vitro phosphorylation by p34cdc2 protein kinase. Amino acid sequence analysis shows that two consensus sites for p34cdc2 protein kinase lie within the Cterminal region of dystrophin, with one being located in exon 78 (Fig. 8). These phosphorylation sites are not conserved in utrophin (43,44), suggesting that phosphorylation may be specific for dystrophin and/or Dp71 isoforms which retain the C-terminal amino acid sequence of muscle dystrophin. This would imply that the phosphorylation of these Dp71 isoforms by endogenous protein kinases may modulate their interaction with other cytoskeletal proteins in vivo, a phenomena observed with erythrocyte proteins 4.1 and 4.9 and ankyrin (45). Thus, alternative splicing may mediate the phosphorylation of Dp71, resulting in functional variation due to differential proteinprotein interactions. At present, however, the specific protein kinases responsible for this post-translational modification in vivo on dystrophin and Dp71 have not been determined. In skeletal muscle, the C-terminus of dystrophin is associated with a complex of sarcolemmal glycoproteins, including (3dystroglycan and syntrophin (12,13,29,46). Recent reports have suggested that this C-terminal region of dystrophin, as well as Dp71, can be subdivided into a 'dystroglycan-binding domain' (amino acids 3080-3408) and a 'syntrophin-binding domain' (amino acids 3409-3685) (47,48). In addition, alternative splicing of exons 71 through 74 was shown to mediate dystrophin-syntrophin and Dp71 -syntrophin interactions. The observation that Dp71 isoforms and P-dystroglycan are coexpressed in brain and amniocytes suggests that these proteins may form part of a 'nonmuscle' DGC similar to that observed in muscle. Thus, alternative splicing of Dp71 may be important either for the interaction with components of the DGC, or with proteins distinct from those originally identified in muscle. Whether all of the Dp71 isoforms interact with the same cellular components has not been determined. However, based on the relative abundance of Dp71 isoforms in nonmuscle tissues and their association with the plasma membrane (R.Austin, unpublished observations; 22,26,27,49) the possibility exists that they play a significant role in membrane-cytoskeletal interactions.

MATERIALS AND METHODS Cell culture Approximately 4 ml of amniotic fluid was mixed with an equal volume of cc-MEM media containing 15% fetal calf serum and grown until confluent at 37°C in 5% CO2. To harvest cells, the media was replaced with PBS and the cells resuspended using a sterile rubber policeman. The cells were pelleted and washed twice in PBS before protein and RNA extraction. RT-PCR Total RNA was extracted from cultured human amniocytes using the method of Chomczynski and Sacchi (50). The reverse transcriptase reaction was done as follows. Briefly, 1 \lg of total RNA and 50 ng of reverse primer 513, in a total volume of 10 u.1, was heated to 70°C for 10 min. The mixture was then immediately chilled on ice and made up to 20 |il with a premix that contained 4 ul 5X reverse transcription buffer (BRL), 2 (ll 0.1 M dithiothreitol, 0.5 mM

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predicted molecular masses of the Dp71 fusion proteins fit well with their mobility observed on SDS-PAGE. The observation that recombinant Dp71 fusion proteins containing exon 78 stained intensely with the 1461 antibody while recombinant Dp71 fusion proteins lacking exon 78 stained intensely with the 462B antibody confirms our PCR data and indicates that antibodies directed against specific epitopes of the dystrophin protein can be used to detect the protein products of Dp71 transcripts alternatively spliced for exon 78. The identification of alternatively spliced Dp71 transcripts in cultured human amniocytes suggested that Dp71 may also be differentially spliced for exons 71 and/or 78 in other human tissues expressing Dp71. As demonstrated by RT-PCR analysis, Dp71 transcripts were present in a variety of human tissues. Although this transcript is expressed predominantly in nonmuscle tissues, we could detect it at varying levels in all tissues examined. In addition, various combinations of Dp71 transcripts alternatively spliced for exons 71 and/or 78 were identified in the majority of tissues examined. It has been suggested that different dystrophin mRNAs may arise in a developmentally regulated fashion either through selection of alternative promoters or differential RNA processing pathways resulting in alternative splicing (36). Alternative splicing is a common feature of many developmentally regulated proteins and has been documented for many contractile and cytoskeletal proteins including troponin T (37) and p-tropomyosin (38). Feener et al. (32) showed that the 3'-end of the dystrophin transcript can be alternatively spliced to create multiple isoforms differing at their C-terminus in fetal muscle and brain. Interestingly, alternative splicing of dystrophin in muscle and brain is identical to splicing observed in Dp71 transcripts expressed in cultured human amniocytes and various human tissues described in our studies. These observations suggest that either some of the original cDNA clones, which were attributed to the dystrophin transcript, were in fact Dp71 cDNAs, or that processing pathways resulting in alternative spliced transcripts are functionally similar for both muscle and nonmuscle tissues. Size heterogeneity of Dp71 was originally observed in studies using antibodies to the C-terminus of dystrophin (15,16,18). However, it was not known whether this heterogeneity was due to degradation, alternative splicing or posttranslational modification of the protein since the antibodies used in previous studies were unable to distinguish between alternatively spliced isoforms of Dp71. Recently, Kramarcy et al. (29), using antibodies which recognized Dp71 isoforms alternatively spliced for exon 78, showed multiple Dp71 isoforms of 70-80 kDa in a wide range of rodent tissues. We have used similar antibodies, 1461 and 462B, to show that multiple protein isoforms of Dp71 observed on SDS-PAGE are expressed in a variety of adult human tissues. This is consistent with the hypothesis that alternative splicing of Dp71 transcripts results in the tissue-specific expression of multiple protein isoforms of Dp71. At present, the function of alternative splicing in Dp71 is unknown. Recent transgenic mouse experiments have shown that Dp71, either with or without exons 71 or 78, cannot replace the function of full-length dystrophin and correct the muscle defect, although it does restore the DGC (26,27). This result suggests that although Dp71 and dystrophin may interact with the same proteins they have distinct functions. Recent

Human Molecular Genetics, 1995, Vol. 4, No. 9 1481 buffer (52). Total protein samples from adult liver, testis and kidney were purchased from Clontech (Mississauga, ON). Samples were heated to 100°C for 2 min and separated on 7.5% SDS-polyacrylamide gels under reducing conditions using the Mini-Protean II gel apparatus (Bio-Rad, Toronto, ON). Gels were either stained with Coomassie brilliant blue or electroblotted on to nitrocellulose (Bio-Rad) (53). The efficiency of protein transfer was determined by staining the blots with Ponceau S (Sigma, St. Louis, MO). The nitrocellulose membranes were washed briefly in TBST [10 mM Tris-HCl (pH 8.0); 150 mM NaCl; 0.05% (v/v) Tween-20] to remove the Ponceau S stain and then blocked overnight at 4°C in TBST containing 5% non-fat milk and 1% fetal calf serum. Membranes were incubated at room temperature for 4 h with the appropriate polyclonal or monoclonal antibody diluted in TBST containing 1% non-fat milk. Following a wash step with TBST (3X10 min at room temperature) the membranes were incubated with the appropriate alkaline phosphatase- or horse radish peroxidase-conjugated secondary antibody, diluted in TBST containing 1% non-fat milk for 2-4 h at room temperature. Following another wash step with TBST (4X10 min) the membranes were developed with either nitroblue tetrazolium and bromo-chloro-indoylphophate (Promega-Biotec, Toronto, ON) or with the Renaissance chemiluminescence reagent kit (DuPont NEN, Mississauga, ON). Production, purification and characterization of the 1461 antibody has been described previously (11,54). The MANDYS1 (55) and 43DAG/8D5 (35) monoclonal antibodies have been previously described in detail. Molecular mass marker proteins (Helixx Technologies, Scarborough, ON) were rabbit muscle myosin (200 kDa), E.coli P-galactosidase (116 kDa), rabbit muscle phosphorylase b (97 kDa), bovine serum albumin (66 kDa), bovine glutamate dehydrogenase (55 kDa), porcine lactate dehydrogenase (36 kDa), bovine erythrocyte carbonic anhydrase (31 kDa), soybean trypsin inhibitor (21 kDa) and chicken egg white lysozyme (14 kDa).

cDNA screening using PCR Bacterial miniprep isolation using the alkaline lysis method was carried out according to Sambrook et al. (51). All samples were resuspended in TE (pH 8.0) containing 20 |ig/ml RNase A. Plasmid DNA (100 pg) from each miniprep was used as template for PCR amplification. All samples were subjected to 30 cycles of amplification (94°C for 30 s, 58°C for 30 s, 72°C for I min) in a final volume of 50 Hi containing 100 ng of each primer, 2.5 U Taq polymerase (Cetus) and a buffer containing 1.5 mM MgCl2, 50 mM KG, 10 mM Tris-HCl (pH 8.8) and 0.25 mM each dNTP. The following PCR oligonucleotide primers were used to amplify DNA segments flanking exons 71 and 78, respectively: 695, 5'-TCTAGAATTCATGGTGGAATATTGCACTCCG-3' (position 10273-10295); 926, 5'-ATTGCGTGAATGAGTATCATCGT-3' (position 10519-10496); 837, 5'-CCTTCCCTAGTAGTTCAAGAGG3' (position 11205-11223); 2296 (Fig. 3A). Primer 695 contained a terminal EcoRI site. PCR products were combined and separated on 1.5% agaroseTBE gels containing ethidium bromide. PCR analysis using randomly primed cDNA from normal adult human brain, muscle, heart, kidney, lung, testis and liver was performed as described above. Amplified PCR products were separated on 2% agarose-TBE gels containing ethidium bromide.

ACKNOWLEDGEMENTS

Cloning and expression of the Dp71 cDNAs in E.coli To generate Dp71 fusion proteins in E.coli, the cDNAs in pBluescript (KS) were digested with EcoRl, gel-purified and ligated in the correct reading frame into the EcoRl site of the bacterial expression vector pFL-1 (33). pFL-1 is a modified version of the commercially available expression vector pFLAG1 (InterScience, Markham, ON) and was kindly provided by Dr M.A.Blanar (University of California, San Francisco). It contains a 17 amino acid Nterminal extension with recognition sites for a Ca2+-dependent monoclonal antibody (anti-FLAG), a specific enterokinase cleavage site and a recognition sequence for the catalytic subunit of bovine heart muscle kinase. DH5a cells harbouring the pFL-l-Dp7l cDNA constructs, were grown at 37°C in terrific broth containing ampicillin (100 |ig/ml) to an OD^OQ of 0.5. Cell cultures were induced by the addition of IPTG to a final concentration of 1.5 mM. After 3 h, 1.5 ml of the culture was centrifuged in a microcentrifuge and the pelleted cells were washed and resuspended in PBS at room temperature. The cells were again pelleted and resuspended in cracking buffer [0.01 M NaPO4 (pH 7.2); 1% P-mercaptoethanol; 1% SDS; 6 M urea] prior to SDSPAGE analysis. Immunoblot analysis Adult human tissues and amniocytes were solubilized in cracking buffer for 30 min on ice and resuspended in an equal volume of 2X SDS-PAGE sample

We thank Ko Gyi for his expert technical assistance in isolating and culturing the human amniocytes. We also thank Drs L.Kunkel and F.M.Boyce for the affinity-purified 462B polyclonal antibody directed against the alternative 31 amino acid C-terminal end of dystrophin, Dr L.V.B.Anderson for the monoclonal antibody 43DAG/8D5 directed against p-dystroglycan, and Drs Nguyen thi Man and Glen Morris for the monoclonal antibody MANDYS 1 directed against the spectrin-like repeat domain of muscle dystrophin. We also thank Dr Johanna Rommens for the randomly primed human tissue cDNAs. This research was supported by the Muscular Dystrophy Association of Canada (P.N.R.) and The Medical Research Council of Canada (P.N.R.). R.C.A. was a recipient of a post-doctoral fellowship of the Medical Research Council of Canada.

ABBREVIATIONS DMD, Duchenne muscular dystrophy; RT-PCR, reverse transcriptasepolymerase chain reaction; DGC, dystrophin-associated glycoprotein complex; DAPs, dystrophin-associated proteins; SDS-PAGE, sodium dodecyl sulfatepolyacrylamide gel electrophoresis.

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(final) of each dNTP (Pharmacia), 37.5 U RNAguard (Pharmacia) and 200 U Superscript reverse transcriptase (BRL). cDNA synthesis was performed at 37°C for 60 min. The reaction was then heated to 95°C for 10 min and quick chilled on ice. Two |il of the cDNA reaction was used as a template for PCR amplification. Reactions took place in a final volume of 50 (il containing 100 ng of primers 512 and 513, 2.5 U Taq polymerase (Cetus) and a buffer containing 1.5 mM MgCl,, 50 mM KC1, 10 mM Tris-HCl (pH 8.8) and 0.5 mM of each dNTP. AH samples were subjected to amplification in a PerkinElmer Cetus Thermal Cycler with a step program consisting of 30 cycles of 94°C for 1 min, 58°C for 1 min, and 72°C for 2 min. The following PCR oligonucleotide primers (Fig. 1A) were used to amplify the Dp71 cDNA from human amniocytes: 512, 5'-GAAGCTCACTCCTCCACTCGTACC-3' (positioned 28 bp 5' of the initiator ATG); 513,5'-TGCATAGACGTGTAAAACCTGCC-3' (position 11541-11519 of the human dystrophin cDNA). For nested PCR, the previously amplified products were gel-purified using the Qiagen gel purification kit (Qiagen, Chatsworth, CA) and used as a template for an additional 30 cycles of PCR using the primers 741 (5'TCTAGAATTCATGAGGGAACAGCTCAAAGG-3'; positioned at the initiator ATG of Dp71) and 2296 (5 '-TCTAGAATTCTTATTCTGCTCCTTCTTC3'; positioned 11352-11335 of the dystrophin cDNA) (see Fig. 1). Both primers contained a terminal extension EcoRl restriction site for subsequent subcloning into pBluescript (KS) (Stratagene, La Jolla, CA) and the bacterial expression vector pFL-1. Amplified products using the nested primers 741 and 2296 were gelpurified, blunt-ended with T4 DNA polymerase (BRL) and initially subcloned into either the £coRV or Smal sites of pBluescript (KS). Authenticity of the cDNA inserts encoding the Dp71 isoforms was confirmed by double-stranded DNA sequencing of both strands using a modified T7 polymerase system (Sequenase, USB).

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