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Human Molecular Genetics Pages 801-808
The Emery-Dreifuss muscular dystrophy protein, emerin, is a nuclear membrane protein
Introduction
Results
Discussion
Materials And Methods
   PCR amplification of STA cDNA
   Cloning of cDNA into pMW172 and expression of fusion protein
   Sequencing of cloned inserts
   Production of monoclonal antibodies
   Clinical details
   Immunofluorescence microscopy
   SDS-PAGE and Western blotting
   Subcellular fractionation
Acknowledgements
References

The Emery-Dreifuss muscular dystrophy protein, emerin, is a nuclear membrane protein

The Emery-Dreifuss muscular dystrophy protein, emerin, is a nuclear membrane protein S. Manilal1, , Nguyen thi Man1, C. A. Sewry2 and G. E. Morris1,*

1MRIC Biotechnology Group, N.E. Wales Institute, Deeside, Clwyd CH5 4BR, UK and 2Neuromuscular Unit, Department of Paediatrics and Neonatal Medicine and Muscle Cell Biology Group, MRC Clinical Sciences Centre, Royal Postgraduate Medical School, Du Cane Road, London W12 ONN, UK

Received February 7, 1996; Revised and Accepted March 21, 1996

A large fragment of emerin cDNA was prepared by PCR and expressed as a recombinant protein in Escherichia coli. Using this as immunogen, we prepared a panel of 12 monoclonal antibodies which recognise at least four different epitopes on emerin in order to ensure that emerin can be distinguished from non-specific cross-reacting proteins. All the mAbs recognised a 34 kDa protein in all tissues tested, though minor emerin-related bands were also detected in some tissues. Immunofluorescence microscopy showed that emerin is located at the nuclear rim in all tissues examined. A muscle biopsy from an Emery-Dreifuss muscular dystrophy (EMDM) patient showed complete absence of emerin by both Western blotting and immunohisto- chemistry, suggesting a simple diagnostic antibody test for EDMD families. Biochemical fractionation of brain and liver tissues showed that emerin was present in nuclei purified by centrifugation through 65% sucrose and was absent from soluble fractions (post-100 000 g). From these results, together with sequence and structural homologies between emerin, thymopoietins and the nuclear lamina-associated protein, LAP2, we suggest that emerin will prove to be one member of a family of inner nuclear membrane proteins.

INTRODUCTION

Emery-Dreifuss muscular dystrophy (EDMD) is a neuromuscular disease characterised by the following features: slowly progressive skeletal muscle wasting of the shoulder girdle and distal leg muscles, early contractures of the elbows and Achilles tendons and cardiomyopathy which often presents as atrial ventricular block and eventually results in death unless a cardiac pacemaker is inserted immediately after diagnosis. EDMD is inherited either as an autosomal dominant form or an X-linked recessive disorder. The two forms are clinically very similar with a preponderance of the X-linked form (1 ).

Linkage analysis of the X-linked disorder has shown this gene to lie between the loci DXS52/DXS15 and the gene for factor eight in the telomeric region of the Xq28 (2 ). This region has been further refined with the aid of physical mapping studies. Of the eight genes in this region which were highly expressed in brain and muscle, one in particular, STA, was a strong candidate for EDMD as mutations in this gene were identified in affected individuals (3 -5 ).

Although the gene is highly expressed in skeletal muscle and heart, it has been shown that STA is ubiquitously expressed at high levels in most tissues. STA encodes a 254 amino acid serine-rich protein, identified as emerin. The emerin sequence is highly hydrophilic except for a hydrophobic carboxy-terminal tail. No homology to other proteins with known functions was apparent, although two regions of homology with thymopoietins were observed (3 ). Thymopoietin (TMPO) was originally isolated as a 5 kDa, 49 amino acid protein from bovine thymus in studies of the effects of thymic extracts on neuromuscular transmission, though these effects were later found to be due to a contaminant in the preparation. Three proteins produced by alternative splicing with this sequence at their amino-termini were later identified and named as [alpha] (75 kDa), [beta] (51 kDa) and [gamma] (39 kDa) thymopoietins (6 ), but there were few clues to their function until the cDNA for a rat nuclear lamina-associated protein, LAP2, was cloned and found to be the rat homologue of [beta]-thymopoietin (7 ,8 ).

We now describe the PCR cloning and expression of the human emerin hydrophilic region and its use for the production of monoclonal antibodies. We show that emerin is a nuclear membrane protein and suggest that it will prove to be a member of a family of nuclear lamina-associated proteins (LAPs).

RESULTS

A fragment of STA cDNA encoding the first 188 amino acids of emerin (out of 254) was amplified from total human skeletal muscle cDNA and cloned into the expression plasmid, pMW172. The C-terminal hydrophobic domain of emerin was avoided because it might have adverse effects on recombinant protein expression (9 ). After electroporation into E.coli BL21(DE3), good expression of a protein of the expected size (21 kDa) was obtained. A panel of 12 mAbs was generated by standard hybridoma methods using partially purified protein as immunogen.

The mAbs were shown to recognise at least four different epitopes on emerin as shown in Table 1 . Only three mAbs (MANEM1-3) cross-reacted with rabbit emerin on Western blots and only one of these (MANEM3) failed to bind when 41 amino acids were removed from the recombinant emerin by chemical cleavage at Cys147 using NTCB. This establishes two distinct epitopes for MANEM1-2 and MANEM3. For seven mAbs (MANEM4-10) which do not recognise rabbit emerin, Western blotting of chymotryptic fragments produced two quite distinct fragmentation patterns (Table 1 ). This establishes two more epitopes, making four distinct epitopes in all. We can be confident that any antigen recognised by mAbs from all four epitope groups, whether by Western blotting or immunohistochemistry, will be authentic emerin and not a cross-reacting protein; the latter is always a possibility when a single antibody or a polyclonal antiserum is used. None of the 12 mAbs cross-react with mouse emerin.

Figure 1 shows that the emerin mAbs detect a protein band of ~34 kDa in normal human muscle. This band is completely absent from the muscle of an EDMD patient, confirming its identity with emerin. The muscle membrane dystrophin-associated protein, 43DAG or [beta]-dystroglycan, is used as a control to show that equal amounts of protein were loaded on the gel [43DAG and dystrophin itself are unchanged in EDMD (10 )]. On SDS-PAGE, emerin migrates more slowly than expected from its predicted size (29 kDa). This could be due to post-translational modification, since the emerin sequence has a glycosylation site and several potential sites for phosphorylation. This might also explain a faint additional band at ~36 kDa in Figure 1 . The estimated size for emerin on SDS-PAGE of 34 kDa was established by careful comparison with several different pre-stained and unstained marker sets (see Materials and Methods).


Figure 1. Western blots of normal muscle and EDMD biopsy muscle extracts. MANDAG1 mAb to the muscle membrane protein, 43DAG, was used as control to show that equal amounts of normal and EDMD protein were loaded onto the gel. A single major band at 34 kDa observed in normal muscle is completely absent in EDMD muscle following staining with the emerin mAb, MANEM1.

Figure 2 shows that emerin is localised almost exclusively at the periphery of nuclei in frozen sections of human skeletal muscle. The same result was obtained with all the mAbs, and double labelling with ethidium bromide and anti-emerin mAbs (not shown) confirmed the nuclear localisation. The nuclear membrane staining is seen in normal muscle and in a biopsy from a patient with an uncharacterised myopathy, but EDMD biopsy showed no staining with any of the 12 anti-emerin mAbs, consistent with the Western blot results in Figure 1 . EDMD sections in Figure 2 were also stained with anti-dystrophin mAbs (confirming that dystrophin is unaffected in EDMD) and ethidium bromide (to show that nuclei are present in the EDMD biopsy sections). Careful examination of EDMD sections showed that all sections tested contained many ethidium bromide-stained nuclei, but no nuclear staining by anti-emerin mAbs was ever detected. Staining of major blood vessels in the biopsy sections (Fig. 3 ) shows that the presence of emerin in normal nuclei and its absence from EDMD nuclei is not restricted to myofibre nuclei. Most of the nuclei are in the smooth muscle layer of the blood vessels.

Table 1 Characterization of 12 emerin mAbs
mAb

 Clone

 NTCBa

 ChymoTb

 Rabbitc

 IMFd

 Epitope groupe
MANEM1

 5D10

 1

 1

 ++

 ++

 1
MANEM2

 9B2

 1

 1

 ++

 ++

 1
MANEM3

 6D2

 2

 2

 +

 ++

 2
MANEM4

 6C4

 1

 2

 -

 ++

 3
MANEM5

 8A1

 1

 2

 -

 +

 3
MANEM6

 8E9

 1

 2

 -

 ++

 3
MANEM7

 7A9

 1

 3

 -

 ++

 4
MANEM8

 7B9

 1

 3

 -

 ++

 4
MANEM9

 9F8

 1

 3

 -

 +

 4
MANEM10

 1H9

 1

 3

 -

 +

 4
MANEM11

 7D9

 nd

 nd

 -

 ++

 nd
MANEM12

 5B11

 nd

 nd

 -

 weak

 nd
aNTCB (nitrothiocyanobenzoic acid) cleaves the recombinant emerin at Cys-147: group 1 mAbs bind to the large fragment, while the group 2 mAb does not.bChymotrypsin digests of the recombinant emerin give three different patterns when different mAbs are used on Western blots.cOnly three mAbs cross-react with emerin in rabbit muscle extracts.dmAbs were tested on frozen sections of human muscle by immunofluorescence microscopy.emAbs were placed in four epitope groups on the basis of a, b and c. All mAbs belong to the IgG1 subclass.nd, not determined.


Figure 4 a shows that emerin levels are similar in all foetal and adult human tissues tested. This is consistent with mRNA data (3 ) and with a nuclear location. The slightly higher Mr band seen in Figure 1 is also present in all the human tissues (with a third band visible in foetal liver). Figure 4 b shows that emerin is present at similar concentrations in all rabbit tissues tested. The main `34 kDa' emerin band migrates slightly faster in the heart, kidney and skeletal muscle extracts. This is quite reproducible, but its significance remains to be determined. It is important to examine carefully any stained protein bands other than the 34 kDa emerin because of the possible existence of isoforms or modified forms. The lower Mr bands could be degradation products, but they are noticeably absent from skeletal muscle and sciatic nerve (see also the human skeletal muscle extract in Fig. 1 ) and all the extracts were freshly prepared. These bands are also present in foetal and adult human tissues, except that they are much less prominent in adult skeletal muscle (Fig. 4 a).


Figure 2.Immunohistochemical localisation of emerin at the nuclear membrane in normal muscle and its absence in EDMD muscle. Frozen sections of normal muscle, muscle from a patient with an undiagnosed myopathy (control) and muscle from an EDMD patient were stained with emerin mAb, MANEM1. EDMD sections from the same biopsy were also stained with anti-dystrophin mAb, MANDYS1, to show the sarcolemma and with ethidium bromide to show that nuclei are present in the sections. The different nuclear shapes in different sections are due to differences in orientation (see Figure 3, where various intermediates between transverse and longitudinal nuclei are visible in the same blood vessel).


Figure 3. Immunofluorescence of blood vessels in frozen muscle sections stained with emerin mAb, MANEM1. Staining of nuclear membranes is observed in normal blood vessel sections but was absent in EDMD biopsy sections. Most of the nuclei are in the smooth muscle layer of the vessel and can be seen both in circular section (transverse; right) and in elongated form (top left). The non-nuclear, fibrous staining in both normal and EDMD sections is due mainly to autofluorescence from elastic fibres on each side of the smooth muscle layer. This autofluorescence is red and easily distinguished from the apple-green anti-emerin staining. In the upper panel, the empty lumen of the blood vessel is clearly visible in the lower right hand quadrant; in the lower panel, the blood vessel has broken open in the upper left hand quadrant during sectioning.


Figure 4.Western blots of emerin in adult and foetal human tissues and in rabbit tissues. (a) Adult and foetal muscle, brain and lung extracts from post-mortem tissues were screened with the emerin mAb, MANEM1, after SDS-PAGE on 12% polyacrylamide gels. The emerin band is present in all the extracts, together with a fainter second band at slightly higher Mr of 36 kDa (foetal lung has a third band). Lower Mr bands may be degradation products, though the most prominent of them corresponds in size (17 kDa) to the labelled rabbit band in (b). (b) Rabbit tissue samples were resolved by SDS-PAGE on a 3-12.5% gradient polyacrylamide gel. The 34 kDa emerin band is present in all tissue extracts examined, but the lower Mr band indicated is absent from muscle (MU) and sciatic nerve (SN). Other lanes are Mr: prestained molecular weight markers, LI: liver, HT: heart, KI: kidney, BR: brain and SP: spleen.

The nuclear rim localisation was also observed in tissues other than skeletal muscle, including kidney, heart and spleen (results not shown). In rabbit sciatic nerve, as in skeletal muscle, emerin was also found almost exclusively at the nuclear periphery (Fig. 5 ). Most of the nuclei are found in the perineurium, connective tissue which surrounds the fibre, at the bottom of the figure and appear elongated in this section. The few nuclei visible in circular section among the axons are likely to be from Schwann cells or blood vessels. The control section without primary antibody shows that the faint non-nuclear staining is non-specific.


Figure 5.Localisation of emerin in a transverse section of rabbit sciatic nerve. Nuclear membranes stained with emerin mAb, MANEM1, are visible in circular section, sparsely distributed amongst the packed myelinated and unmyelinated axons (these few nuclei are probably derived from Schwann cells or blood vessels) and, more abundantly, in elongated form in the connective tissue which surrounds the nerve fibre (perineurium). Faint staining of the axons is non-specific, since it is also seen in the control section without primary Ab.

To confirm the immunohistochemical results, purified nuclei were prepared from rabbit brain by centrifugation through 65% sucrose and shown to contain emerin by Western blotting (Fig. 6 ). A simple subcellular fractionation by differential centrifugation was performed as outlined in Figure 7 and the fractions were tested for emerin by Western blotting (Fig. 7 ). About half the emerin was found in the 1000 g pellet containing nuclei and unbroken cells (lane 1), but none of this was extracted by Triton X-100 (cf. lanes 2 and 3). This implies that emerin is retained in nuclei not only via its transmembrane domain but also by non-membrane-dependent interactions. The nuclear membrane protein LAP2 is also Triton-insoluble because of its interaction with nuclear lamins (7 ). Most of the remaining emerin was found in the `mitochondrial' pellet (cf. lanes 4 and 5), though this may also contain some nuclei or nuclear fragments. None of the emerin remained in the supernatant after high-speed centrifugation (lane 7). Similar results were obtained with rabbit liver (results not shown). The results cannot be used as evidence for non-nuclear emerin because we cannot rule out the possibility that nuclear membrane fragments are responsible for the emerin content of the `mitochondrial' and `microsomal' pellets obtained by this very simple fractionation procedure.


Figure 6.Isolation of rabbit brain nuclei and emerin analysis by Western blot. Rabbit brain (0.5 g) was homogenised in 10 ml of RSB buffer (10 mM NaCl; 1.5 mM MgCl2; 10 mM Tris-HCl, pH 7.5) using a Dounce hand homogeniser. The homogenate was centrifuged at 1000 g for 10 min at 4oC. The pellet was resuspended in 10 ml of RSB solution, homogenised and sedimented as described above. The crude nuclear pellet was resuspended in 65% sucrose/5 mM MgCl2 and centrifuged at 100 000 g for 80 min in a 3*5 ml swing-out rotor. The nuclear pellet was washed with RSB to remove residual sucrose and resuspended in RSB. Phase-contrast microscopy (upper panel) shows that the nuclear preparation is reasonably pure, though some have `cytoplasmic tags' (cytoskeletal material) attached. The Western blot (12% polyacrylamide gel) with MANEM1 mAb (lower panel) shows that emerin is present in the purified nuclei (Mr: prestained markers, lane 1: rabbit muscle extract, lane 2: purified nuclei).


Figure 7. Flow diagram depicting the subcellular fractionation of rabbit brain by differential centrifugation and Western blot analysis of the subcellular fractions. The crude nuclear pellet (1) will also contain unbroken cells. The 6600 g pellet (4) will contain mainly mitochondria, `heavy' membranes and some nuclei. The 100 000 g pellet (6) is the microsomal fraction containing mainly ER and `light' membranes. Aliquots of each fraction were removed and analysed on 12% SDS-polyacrylamide gels for Western blotting with MANEM1 mAb. Lanes 1-7 correspond to the fractions in the flow diagram above. **Non-specific staining by second antibodies.

DISCUSSION

We have shown that emerin is a nuclear membrane protein. Several striking sequence and structural homologies with LAP2 ([beta]-thymopoietin) suggest, as a working hypothesis at least, that emerin belongs to a family of type II integral membrane proteins which are anchored to the inner nuclear membrane near their carboxy-termini, with the remainder of the molecule projecting into the nucleoplasm, as illustrated in Figure 8 . A sequence of 39 amino acids at the human emerin amino-terminus (Asp6-Gln44) shows 41% identity (70% conserved residues) with a sequence in all three human thymopoietins (Glu114-Gln152). The last 35 amino acids of emerin show the same degree of identity with a sequence very close to the carboxy-terminus of [beta]- and [gamma]-thymopoietins, including a hydrophobic transmembrane domain of ~20 amino acids (Fig. 8 ). [alpha]-Thymopoietin lacks this transmembrane domain and localises to the nucleoplasm rather than the nuclear membrane (8 ). In between the two regions of homology at each end of the molecule, emerin is about the same length as [gamma]-thymopoietin, but there is little obvious sequence homology, apart from the presence of numerous potential phosphorylation sites.


Figure 8.Schematic representation of predicted organisation of emerin and thymopoietin in the nuclear membrane. Based on the model for LAP2 (7).

We have not yet confirmed the orientation of emerin in the nuclear membrane by electron microscopy, partly because the mAb epitopes are destroyed by most common fixation procedures, making EM technically difficult. The arrangement of positively and negatively charged amino acids on each side of the transmembrane sequence argues strongly against the amino- terminal region being in the lumen between inner and outer membranes (11 ). A cytoplasmic orientation in the outer nuclear membrane is unlikely because it is not clear what could target emerin to nuclei rather than to the rest of endoplasmic reticulum (ER), of which the outer nuclear membrane forms a part. In contrast, LAP2 and other inner nuclear membrane proteins appear to be inserted first into ER membranes where they diffuse laterally throughout the ER system until they are fixed in the inner nuclear membrane only by specific interactions with the nuclear lamina (12 ).

LAP2 is known to interact with lamin B polymers below the inner nuclear membrane, a property shared with another type II integral membrane protein, LBR (lamin B receptor or p58). The interaction between LAP2 and lamin B is regulated during mitosis by phosphorylation of LAP2 which prevents lamin B binding in vitro (12 ). This involves cdc2 kinase and emerin does not appear to have any potential cdc2 kinase sites, suggesting that emerin does not interact with lamin B in the same way as LAP2, if at all. The amino-terminal sequence of emerin which is shared with LAP2 does have three predicted phosphorylation sites which are also conserved in thymopoietins, and it most likely has a common function in all four proteins. Neither this sequence nor the transmembrane sequence appear to be essential for nuclear membrane localisation, however, since deletion mutants lacking either or both will still target to the nuclear rim in a Triton-stable manner in transfected HeLa cells (7 ). A 73 amino acid sequence in the middle of LAP2 was identified as particularly important for targetting to the nuclear rim and the nucleoplasmic [alpha]-thymopoietin lacks this, as well as the transmembrane domain (8 ). Emerin also lacks this sequence, so proteins other than lamin B could be responsible for Triton-stable targetting of emerin to the nuclear rim. Indeed, it is not yet known whether interaction with lamin B is solely responsible for LAP2 targetting, since the deletion mutant results suggested that multiple interactions are involved. In a somewhat similar way, both the amino-terminal domain (13 ) and the carboxy-terminal transmembrane domain (14 ) of the lamin B receptor can independently target proteins to the nuclear envelope. We are currently investigating possible interactions between our recombinant emerin and other proteins.

Since EDMD is a variable, but generally rather mild, genetic disease, the complete absence of emerin in all the nuclei of at least some patients suggests that emerin function must be redundant in most situations. This is consistent with emerin being a member of a family of related proteins, with other members capable of performing some of its functions. The tissue-specific effects of EDMD could therefore reflect the tissue distribution of emerin-related proteins rather than emerin itself. Alternatively, tissue-specific alternative splicing or modification might generate isoforms of emerin with different functions or localisations [cf. LAP2 phosphorylation and deletion mutants (7 ,12 )]. Bands other than 34 kDa emerin are detected by anti-emerin mAbs on Western blots in many tissues, but they are weak or absent in adult skeletal muscle and sciatic nerve (Fig. 4 ). The possibility cannot yet be excluded that some of these protein bands are different forms of emerin, generated by post-translational modification, alternative splicing or proteolysis in vivo, although degradation during experimental manipulation has not been ruled out as an explanation for the lower Mr bands. The gene for the autosomal form of EDMD has not yet been identified, but it may well shed further light on emerin function. Although the severity of EDMD can vary, there is concordance of severity in affected members within a family. The clinical variability between families might therefore relate to the effect of different gene mutations on protein function, since it is not yet known whether emerin is completely absent in all EDMD cases.

The discovery that emerin is primarily a nuclear protein was unexpected. Many other genetic muscle-wasting diseases are caused by defects in genes encoding sarcolemmal proteins: Duchenne and Becker MDs by defects in the dystrophin gene (15 ) and three of the five limb-girdle MDs by defects in genes for each of the three sarcoglycan components of the transmembrane complex which attaches dystrophin to the muscle membrane (16 -18 ). A calcium-activated protease, calpain, is defective in a fourth form of limb-girdle MD (19 ). Myotonic dystrophy is associated with changes in a protein kinase gene (20 ,21 ), which may also show some homology with thymopoietins (22 ). The homology is at the amino-terminus of both proteins and is different from the homology shown by emerin towards these proteins. It does not target the kinase to the nucleus, since the expressed protein is cytoplasmic in transfected COS-1 cells (22 ). The diseases themselves have in common progressive wasting and/or weakness of skeletal muscles, but also have important distinguishing features. In EDMD, for example, a striking feature is a failure of impulse conduction in the heart, which can be corrected with a cardiac pacemaker. There is little that is common to all the gene products, other than a possible association with cell signalling and signal transduction. Thus, although it is appealing to look for links between the functions of the various gene products, such links may prove to be obscure and rather remote.

While this paper was under revision, a paper by Nagano et al. (27 ) appeared in which a nuclear membrane localisation for emerin was demonstrated using two different polyclonal antisera raised against synthetic peptides derived from the C-terminal half of the emerin sequence. We concur with this conclusion and have taken it further by drawing attention to the important relationship between emerin and LAP2. Most of the specificity problems encountered by Nagano et al. [non-specific staining of the plasma membrane and non-specific protein bands on Western blots (27 )] are avoided using our panel of 12 mAbs which recognise the N-terminal region of the emerin molecule. We did not observe in rabbit tissues the tissue-specific differences in human emerin distribution reported by Nagano et al. We found emerin in nuclear membranes in all tissues studied, although this does not rule out the possibility that the proportion of emerin-positive nuclei, or the intensity of nuclear rim staining, varies between tissues. More detailed studies of emerin distribution in both rabbit and human tissues and with as wide a range of anti-emerin antibodies as possible are clearly required.

MATERIALS AND METHODS

PCR amplification of STA cDNA

Human skeletal muscle cDNA (10 ng; Clontech Laboratories) was subjected to PCR amplification using forward (5'-gcggatccgacaactacgcagatctttcg) and reverse (5'-gcaggcctaagaggtggaggaggaagtagg) primers designed to the cDNA of the STA gene. Restriction sites were engineered into the 5' ends of these primers to facilitate cloning of the amplified products into BamHI and StuI sites of the pMW172 expression vector. PCR was carried out in sterile microfuge tubes in a final reaction volume of 50 [mu]l consisting of 1.5 mM MgCl2, 50 mM KCl, 10 mM Tris-HCl (pH 8.4), 200 [mu]M dNTP (Pharmacia) and 1 [mu]M forward and reverse primers. Following a 5 min denaturation step at 94oC, 0.5 U of Taq polymerase was added to the reaction tube. PCR was performed for 25 cycles (56oC, 30 s/72oC, 30 s/95oC, 30 s) followed by a final 5 min extension step at 72oC. The PCR product was purified from a 1% agarose gel using Qiaex resin (Diagen, Dusseldorf, Germany).

Cloning of cDNA into pMW172 and expression of fusion protein

The PCR product was subcloned into the Novagen pT7Blue vector (50 ng; Novagen) containing compatible single T-nucleotide overhangs. Ligation was carried out in a final reaction volume of 10 [mu]l in the presence of 100 mM dithiothreitol (DTT), 10 mM ATP and 2 U of T4 DNA ligase (Boehringer Mannheim). Following incubation at 16oC for 5 h, 1 [mu]l of the ligation mixture was directly transformed into chemically competent pT7 NovaBlue cells (20 [mu]l; available with the kit). Colony PCR was performed on recombinant clones to confirm the presence of the cloned PCR product using pT7 forward (5'-taatacgactcactataggg) and reverse primers (5'-ggttttcccagtcacgacgt). The pT7 construct (100 ng) was digested with 1 U of BamHI and StuI restriction enzymes (Boehringer Mannheim) for 1 h at 37oC in a final reaction volume of 20 [mu]l. The digested fragment was purified using the Qiaex DNA purification kit, cloned into BamHI-StuI-digested, alkaline phosphatase-treated, pMW172 vector and electrotransformed into 20 [mu]l of electrocompetent BL21(DE3) cells in ice-cold electroporation cuvettes (0.2 cm). Recombinant clones were subjected to colony PCR to confirm the presence of the insert using pMW172 forward (5'-gactcactatagggagaccacaac) and reverse (5'-gttattgctcagcggtggcagcag) primers.

Luria Bertani media (100 ml) was inoculated with 1 ml of overnight culture of pMW172 recombinant clone. The media was cultured by shaking at 37oC until the O.D.600nm was 0.8. Following induction (0.8 mM IPTG) at 37oC for 3 h, the culture was centrifuged at 6600 g for 10 min at 4oC. The pellet was resuspended in 30 ml of ice-cold TNE buffer, pH 8.0 (50 mM NaCl, 1.27 mM EDTA, 50 mM Tris-HCl) and sedimented as described above. This procedure was repeated twice. The resulting pellet was extracted twice by sonication in 2 M urea.

Sequencing of cloned inserts

DNA sequencing of the pT7 and pMW172 constructs containing the EDMD cDNA was performed by the dideoxy chain termination protocol (23 ). DNA was sequenced in both orientations using vector-specific oligonucleotides and the Sequenase kit version 2.0 (Amersham Life Science).

Production of monoclonal antibodies

mAbs were produced by immunisation of BALB/c mice and fusion of spleen cells with Sp2/0 myeloma cells as described elsewhere (24 ). Hybridoma culture supernatants were tested by ELISA (24 ), and 12 cell lines were successfully established after two rounds of cloning by limiting dilution.

Clinical details

A needle biopsy from the quadriceps was taken from a boy aged 16 years with features of EDMD. He had contractures of the elbows and Achilles tendon, weakness of the tibialis anterior, a stiff spine, progressive muscle weakness and junctional bradiacardia. There was a strong suggestion of X-linked inheritance with an affected maternal cousin with progressive muscle weakness, contractures and an arrhythmogenic cardiomyopathy requiring a pacemaker. This sample was compared with four controls with no evidence of X-linked inheritance.

Immunofluorescence microscopy

Unfixed, frozen sections of human and rabbit tissues (6 mm) were mounted on glass slides and stored at -70oC. FITC-labelled anti-mouse IgG (DAKO) was used to detect bound antibody as described previously (25 ).

SDS-PAGE and Western blotting

SDS-PAGE and Western blotting were carried out essentially as described elsewhere (26 ). Antibody reacting bands were visualised following development with a biotin-avidin detection system for mouse immunoglobulin (Vectastain kit; Vector Laboratories) and diaminobenzidine (Sigma, Dorset, UK).

In order to get an accurate estimate of molecular weight for the emerin protein, a number of different sets of prestained markers (Novex, CA) were checked and re-calibrated against unstained Sigma markers (Sigma, SDS-7).

Subcellular fractionation

Rabbit liver and brain tissues (0.5 g) were homogenised in ice-cold RSB buffer (10 mM NaCl/1.5 mM MgCl2/10 mM Tris-HCl, pH 7.5) using a Dounce hand homogeniser. All steps were carried at 4oC. The homogenate was centrifuged at 1000 g for 10 min. The pellet was extracted with 1% Triton X-100/10 mM Tris-HCl, pH 7.5 and centrifuged at 100 000 g for 30 min whilst the supernatant fraction was centrifuged at 6600 g for 10 min. The supernatant resulting from this fraction was further subjected to a high-speed spin (100 000 g) for 30 min.

ACKNOWLEDGEMENTS

We thank Francesco Muntoni (RPMS) for valuable comments on the manuscript and Fiona Wilkinson and Abosede Salako (NEWI) for technical assistance. This work was supported by a research development award (DevR) from the Higher Education Funding Council (Wales).

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12 Foisner, R. and Gerace, L. (1993) Integral membrane proteins of the nuclear envelope interact with lamins and chromosomes, and binding is modulated by mitotic phosphorylation. Cell, 73, 1267-1279 MEDLINE Abstract

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20 Brook, J. D., McCurrach, M. E., Harley, H. G., Buckler, A. J., Church, D., Aburatani, H., Hunter, K., Stanton, V. P., Thirion, J. P., Hudson, T., Sohn, R., Zemelman, B., Snell, R. G., Rundel, S. A., Crow, S., Davies, J., Shelbourne, P., Buxton, J., Jones, C., Juvonen, V., Johnson, K., Harper, P. S., Shaw, D. J. and Houseman, D. E. (1992) Molecular-basis of myotonic-dystrophy-expansion of a trinucleotide (CTG) repeat at the 3' end of a transcript encoding a protein kinase family member. Cell, 68, 799-808. MEDLINE Abstract

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22 Sasagawa, N., Sorimachi, H., Maruyama, K., Arahata, K., Ishiura, S. and Suzuki, K. (1994) Expression of a novel human myotonin protein kinase (MtPK) cDNA clone which encodes a protein with a thymopoietin-like domain in COS cells. FEBS Lett., 351, 22-26. MEDLINE Abstract

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*To whom correspondence should be addressed

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