* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Download Regulation of transcript encoding the 43K
Artificial gene synthesis wikipedia , lookup
Messenger RNA wikipedia , lookup
Biochemistry wikipedia , lookup
Paracrine signalling wikipedia , lookup
Ribosomally synthesized and post-translationally modified peptides wikipedia , lookup
Biosynthesis wikipedia , lookup
Silencer (genetics) wikipedia , lookup
Expression vector wikipedia , lookup
Epitranscriptome wikipedia , lookup
Magnesium transporter wikipedia , lookup
Genetic code wikipedia , lookup
G protein–coupled receptor wikipedia , lookup
Point mutation wikipedia , lookup
Interactome wikipedia , lookup
Homology modeling wikipedia , lookup
Ancestral sequence reconstruction wikipedia , lookup
Bimolecular fluorescence complementation wikipedia , lookup
Metalloprotein wikipedia , lookup
Gene expression wikipedia , lookup
Western blot wikipedia , lookup
Nuclear magnetic resonance spectroscopy of proteins wikipedia , lookup
Protein structure prediction wikipedia , lookup
Protein purification wikipedia , lookup
Protein–protein interaction wikipedia , lookup
557 Development 104, 00-00 (1988) Printed in Great Britain © The Company of Biologists Limited 1988 Regulation of transcript encoding the 43K subsynaptic protein during development and after denervation TIMOTHY J. BALDWIN, JULIE A. THERIOT, CORINNE M. YOSHIHARA and STEVEN J. BURDEN Biology Department, Massachusetts Institute of Technology, Cambridge, MA 02139, USA Summary The postsynaptic membrane of vertebrate neuromuscular synapses is enriched in the four subunits of the acetylcholine receptor (AChR) and in a peripheral membrane protein of Mr = 4 3 x l 0 3 (43K). Although AChRs are virtually restricted to the postsynaptic membrane of innervated adult muscle, developing and denervated adult muscle contain AChRs at nonsynaptic regions. These nonsynaptic AChRs accumulate because the level of mRNA encoding AChR subunits increases in response to a loss of muscle cell electrical activity. We have determined the level of mRNA encoding the 43K subsynaptic protein in developing muscle and in innervated and denervated adult muscle. We isolated a cDNA that encodes the entire protein-coding region of the 43K subsynaptic protein from Torpedo electric organ and used this cDNA to isolate a cDNA that encodes the 43K subsynaptic protein from Xenopus laevis. We used the Xenopus cDNA to measure the level of transcript encoding the 43K protein in embryonic muscle and in innervated and denervated adult muscle by RNase protection. The level of transcript encoding the 43K protein is low in innervated adult muscle and increases 25- to 30-fold after denervation. The level of transcript encoding the alpha subunit of the AChR increases to a similar extent after denervation. Moreover, during development, transcripts encoding the 43K protein and the alpha subunit are expressed initially at late gastrula and are present in similar quantities in embryonic muscle. These results demonstrate that transcripts encoding the 43K protein and AChR subunits appear coordinately during embryonic development and that the level of mRNA encoding the 43K protein is regulated by denervation. Introduction 43K protein is not required for ligand-gated AChR channel function, since AChR channels retain their ligand-gated channel characteristics in the absence of the 43K protein (Neubig et al. 1979; Mishina et al. 1984). The correspondence between the location of AChR and 43K protein at synaptic sites and the 1:1 stoichiometry of AChR and 43K protein at these sites has led to the suggestion that the 43K protein is involved in the formation and/or maintenance of high-density packing of AChRs at synaptic sites. Although AChRs are virtually restricted to the postsynaptic membrane in innervated adult muscle, developing muscle and denervated adult muscle contain AChRs at their nonsynaptic surface (for reviews, see Fambrough, 1979; Salpeter, 1987). These AChRs The 43X103 (43K) protein is a peripheral membrane protein that is highly concentrated at vertebrate neuromuscular synapses (for reviews, see Froehner, 1986; Burden, 1987). The 43K protein is a major protein of the postsynaptic apparatus, since it is present at 1:1 stoichiometry with the acetylcholine receptor (AChR) (Burden et al. 1983; LaRochelle & Froehner, 1986). Moreover, chemical cross-linking experiments demonstrate that the 43K protein is in close physical proximity to the cytoplasmic domain(s) of the beta subunit of the AChR and raise the possibility that the 43K protein directly interacts with the AChR (Burden et al. 1983). Nevertheless, the Key words: neuromuscular synapse, denervation, peripheral membrane protein, postsynaptic membrane, Xenopus laevis, Torpedo. 558 T. J. Baldwin are synthesized and accumulate at nonsynaptic regions in response to a lack of muscle cell electrical activity. Denervation causes a 10- to 100-fold increase in AChR mRNA and AChR protein (for review, see Anderson, 1987). These nonsynaptic AChRs are not clustered at high density, but rather are present at 20to 100-fold lower concentration than at the synapse. Although fluorescence and histochemical methods have been used to detect AChRs that are clustered at synapses, these methods are not sufficiently sensitive to detect readily the lower density of nonsynaptic AChRs in developing and denervated muscle. The lower density of nonsynaptic AChRs can be detected, however, by electrophysiological methods and by autoradiography. Moreover, the high affinity and specificity of alpha-bungarotoxin binding allows measurement of AChR protein in detergent extracts of denervated muscle (for review, see Fambrough, 1979). Similarly, antibodies against the 43K protein have been used to detect the 43K protein at synapses, but these immunochemical methods are not sufficiently sensitive to determine whether the 43K protein is present at nonsynaptic regions of denervated and developing muscle (Froehner et al. 1981; Burden, 1985; Peng & Froehner, 1985). Thus, it is not clear whether the 43K protein is associated with both synaptic and nonsynaptic AChRs or whether the 43K protein is associated with only clustered AChRs. Moreover, it is not clear whether muscle cell electrical activity regulates the level of 43K protein as electrical activity regulates the level of AChR. Since methods for measuring low levels of 43K protein are not presently available, we have used a nucleic acid probe to measure the level of transcript encoding the 43K protein in developing muscle, and in innervated and denervated adult muscle. We demonstrate that the level of transcript encoding the 43K protein corresponds to the level of transcript encoding the AChR in developing muscle, and in innervated and denervated adult muscle: denervation results in a 25- to 30-fold increase in the level of transcript encoding the 43K protein and a 30- to 35fold increase in the level of transcript encoding the alpha subunit of the AChR. Moreover, during development, AChR mRNA and 43K protein mRNA are expressed initially at late gastrula and are present at similar amounts in developing muscle. These results demonstrate that transcripts encoding the major proteins of the postsynaptic membrane appear coordinately during embryonic development. Moreover, the level of mRNA encoding the 43K protein is regulated by denervation and suggests that the level of mRNA encoding the 43K protein is regulated by myofibre electrical activity. Materials and methods Isolation of cDNA encoding the 43K subsynaptic protein from Torpedo electric organ A lambda gtll cDNA library was prepared from Torpedo electric organ poly(A)+ RNA. The RNA was copied into cDNA as described (Gubler & Hoffman, 1983; Baldwin et al. 1988), except that first-strand synthesis was primed with random primers. 200000 recombinant phage were screened with a nicktranslated (Rigby et al. 1977) cDNA 32P-probe encoding a truncated form of the Torpedo 43K subsynaptic protein. This cDNA was generously provided by Drs Frail and Merlie (Washington University School of Medicine, St Louis, MO) and has been designated T43k.l (Frail et al. 1987). Filters were hybridized with 32P-probe in 5xSSPE, 5xDenhardt's, 100^gmP 1 calf thymus DNA, and 0-1% SDS at 68°C and washed in 0-lxSSC, 0 4 % SDS at 68°C (Benton & Davis, 1977; Maniatis et al. 1982). Using these procedures, we detected 90 positive plaques, purified phage from 24 plaques and analysed DNA from these phage by Southern blots. Phage DNA was digested separately with EcoRl and Stul and Southern blots were probed with 32P-T43k.l cDNA. Based upon the restriction maps of cDNA clones that encode an incorrect C-terminal region (T43k.l) and a second cDNA clone, which is incomplete (T43k.7), but encodes the correct C-terminal region of the 43K subsynaptic protein (Frail et al. 1987), a 1051 bp fragment was predicted to span from a Stul site at amino acid 80 to a Stul site 68bp 3' to the C-terminus. We detected two phage that harboured cDNA inserts that contained a 1051 bp Stul fragment; one cDNA insert is 2-5 kb in length and the other is 1-8 kb in length. The 1-8 kb cDNA was mapped with restriction endonucleases and approximately 300 bp from the 5' and 3' ends were sequenced. The restriction map of the cDNA (Tor 43K) was equivalent to a spliced product of T43k.l and T43k.7. Isolation of cDNA encoding the 43K subsynaptic protein from Xenopus laevis 200000 phage from a lambda gtlO cDNA library prepared from Xenopus embryo poly(A)+ RNA isolated from stage22 to -24 embryos (Kintner & Melton, 1987; Baldwin et al. 1988) were screened with a nick-translated cDNA 32Plabelled probe encoding the Torpedo 43K protein (see above). Filters were hybridized with 32P-probe in 5xSSPE, 5xDenhardt's, 100^gml"1 calf thymus DNA, and 0-1% SDS at 52°C and washed in 0-5xSSC, 0-1 % SDS at 52°C (Maniatis et al. 1982). These hybridization and wash conditions were established with Northern blots of Xenopus embryo RNA. Using these procedures, we detected one positive plaque and this phage was purified (Xen 43.1). The cDNA insert was subcloned into plasmid vectors, mapped with restriction endonucleases and sequenced (Baldwin et al. 1988). Northern blots and RNase protection Total RNA was isolated from Xenopus embryos using proteinase K/SDS as previously described (Baldwin et al. 1988). Total RNA was isolated from adult muscle using Regulation of 43K subsynaptic protein guanidinium isothiocyanate as described (Chirgwin et al. 1979). The triceps femoris muscle was denervated as described previously (Baldwin et al. 1988). Poly(A)+ RNA was isolated as described and was used for Northern blots (Baldwin et al. 1988). Hybridization with nick-translated full-length cDNA ^P-labelled probes was in 5xSSPE, 5xDenhardt's, 100/igmr 1 calf thymus DNA, 0-1% SDS at 65 °C. Hybridization with 32P-labelled RNA probe was in 50% formamide, 5xSSPE, 5% SDS at 56°C. Filters were washed in 0-lxSSC, 0-1% SDS at 68°C (Maniatis et al. 1982). Total cellular RNA was used in RNase protection assays (Baldwin et al. 1988). The 32P-43K RNA probe was synthesized with SP6 polymerase from SP65-Xen 43.1 cDNA (3' EcoBJ to 5' EcoRl) linearized with HindlU (nucleotide 791; Fig. 1). The probe was 448 nucleotides 559 long; 403 nucleotides were protected by cellular and synthetic sense RNA. The 32P-alpha subunit probe was 449 nucleotides long; 429 nucleotides were protected by cellular and synthetic sense RNA (Baldwin et al. 1988). Purification of probes, hybridization, digestion and analysis of protected labelled products was as described (Baldwin et al. 1988). Results Isolation and characterization of cDNA encoding the Torpedo 43K subsynaptic protein Two cDNA clones that encode a portion of the 43K subsynaptic protein were isolated from a Torpedo Xenopus laevis 43K subsynaptic protein 5' CCCTTCCCTGTTCAATTCAATTACTGCCCTAACTCCTGAGCTGCCCA -159 TTTCCCAAACAGGACCACCAGTCATCCTGTCTCTCATGCCAAGCCTTGAGTGAAACCCTCCATTCTTTGGACGCTGAGC "80 TTCCTAATGTTTTGTAAGCCAGACGGGCTCTGCAGTGGCTCTGCATACCCAGGATTATATGAGACTTTCTGGTGCCGCG "' 1 20 HET GLY GLN ASP GLN THR LYS GLN GLN ILE GLN LYS GLY LEU GLN MET TYR GLN SER ASN ATG GGT CAG GAC CAA ACC AAA CAG CAG ATC CAA AAG GGC CTT CAG ATG TAT CAG TCC AAC 60 40 GLN THR GLU LYS ALA LEU GLN ILE TRP THR LYS VAL LEU GLU LYS THR THR ASP ALA ALA CAG ACA GAG AAG GCT TTG CAG ATC TGG ACT AAA GTC TTG GAG AAG ACC ACT GAT GCG GCC '20 60 GLY ARG PHE ARG VAL LEU GLY CYS LEU ILE THR ALA HIS SER GLU HET GLY ARG TYR LYS GGG AGG TTC CGG GTT CTT GGC TGC CTG ATC ACG GCC CAC TCG GAG ATG GGA AGA TAC AAG '80 80 ASP MET LEU LYS PHE ALA VAL ILE GLN ILE ASP THR ALA ARG GLU LEU GLU GLU PRO ASP GAT ATG TTA AAG TTT GCA GTG ATC CAA ATC GAC ACG GCT CGG GAG CTG GAG GAG CCA GAC 240 100 PHE LEU THR GLU SER TYR LEU ASN LEU ALA ARG SER ASN GLU LYS LEU CYS GLU PHE GLN TTT TTG ACC GAG AGT TAC CTC AAC CTG GCC CGT AGC AAC GAG AAG CTC TGC GAG TTC CAG 300 120 LYS THR ILE SER TYR CYS LYS THR CYS LEU ASN MET GLN GLY THR SER VAL SER LEU GLN AAA ACC ATT TCC TAC TGC AAG ACC TGC CTC AAT ATG CAG GGA ACC TCG GTC AGC CTC CAG 360 HO LEU ASN GLY GLN VAL CYS LEU SER LEU GLY ASN ALA TYR LEU GLY LEU SER VAL PHE GLN CTA AAC GGA CAG GTG TGC CTG AGT CTG GGC AAT GCC TAC CTG GGC CTT AGC GTC TTC CAG 420 160 LYS ALA LEU GLU CYS PHE GLU LYS ALA LEU ARG TYR ALA HIS ASN ASN ASP ASP LYS HET AAA GCC CTG GAA TGC TTC GAG AAG GCC CTG CGC TAC GCC CAC AAC AAC GAT GAC AAG ATG 480 180 LEU GLU CYS ARG VAL CYS CYS SER LEU GLY GLY LEU TYR THR GLN LEU LYS ASP LEU GLU CTG GAG TGC AGG GTC TGC TGC AGC CTG GGA GGT CTC TAC ACT CAA CTT AAG GAT CTG GAG 540 200 LYS ALA LEU PHE PHE PRO CYS LYS ALA ALA GLU LEU VAL ASN ASP TYR GLY LYS GLY TRP AAA GCG CTC TTC TTC CCA TGC AAG GCG GCA GAG CTG GTG AAT GAC TAC GGG AAA GGC TGG 600 220 SER LEU LYS TYR ARG ALA MET SER GLN TYR HIS MET ALA VAL ALA TYR ARG LYS LEU GLY AGC CTC AAA TAC AGA GCG ATG AGT CAG TAC CAC ATG GCG GTC GCT TAC CGC AAG TTG GGC 660 240 ARG LEU ALA ASP ALA MET GLU CYS CYS GLU GLU SER HET LYS ILE ALA LEU GLN HIS GLY CGT TTA GCC GAC GCA ATG GAG TGT TGT GAG GAG TCA ATG AAG ATC GCC CTT CAG CAT GGA 720 260 ASP ARG PRO LEU GLN ALA LEU CYS LEU LEU ASN PHE ALA ASP ILE HIS ARG SER HIS GLY GAC CGA CCG CTT CAA GCC CTT TGT CTG CTC AAC TTT GCC GAT ATC CAC AGA AGT CAC GGT 780 280 ASP ILE GLU LYS ALA PHE PRO ARG TYR ASP SER SER HET SER ILE MET THR ASP ILE GLY GAC ATT GAG AAA GCT TTT CCC CGC TAC GAC TCC TCC ATG AGT ATC ATG ACT GAC ATC GGT 840 300 ASN ARG LEU GLY GLN THR HIS VAL MET ILE GLY VAL ALA LYS CYS TRP LEU HIS GLN LYS AAC CGC CTG GGT CAG ACC CAT GTA ATG ATA GGA GTG GCG AAA TGT TGG CTC CAT CAG AAG 900 320 GLU MET ASP LYS ALA LEU ASP CYS LEU GLN LYS THR GLN GLU LEU ALA GLU ASP ILE GLY GAG ATG GAC AAG GCT CTG GAT TGT CTC CAA AAG ACC CAA GAG CTG GCG GAA GAT ATT GGA 960 340 TYR LYS HIS CYS LEU LEU LYS VAL HIS CYS LEU SER GLU ILE ILE PHE ARG THR LYS GLN TAT AAG CAC TGC CTG CTG AAA GTT CAC TGC CTG AGT GAG ATT ATA TTC CGG ACA AAG CAG 1020 360 GLN GLN ARG GLU LEU ARG ALA HIS VAL VAL ARG PHE HIS GLU CYS VAL GLU GLU MET GLU CAG CAA CGC GAG CTC CGC GCC CAT GTG GTG CGA TTT CAT GAA TGT GTG GAG GAG ATG GAG 1080 380 LEU TYR CYS GLY HET CYS GLY GLU SER ILE GLY GLU LYS ASN CYS GLN LEU GLN ALA LEU TTA TAC TGT GGA ATG TGT GGG GAG TCC ATT GGG GAG AAG AAC TGC CAA CTT CAG GCA CTT 1 140 399 PRO CYS SER HIS VAL PHE HIS LEU ARG CYS LEU GLN THR ASN GLY THR ARG GLY CYS CCG TGC TCC CAT GTC TTT CAT CTG CGG TGT CTT CAG ACC AAT GGA ACC CGA GGC TGC G 1198 Fig. 1. Nucleotide sequence and deduced amino acid sequence of the 43K subsynaptic protein of Xenopus laevis. Nucleotide 1 indicates the first nucleotide of the codon encoding the amino terminal residue and nucleotides to the 5' side of this amino terminal residue are indicated with negative numbers. The number of the nucleotide residue at the end of each line is provided. The predicted amino acid sequence is shown above the nucleotide sequence. Amino acid residues are numbered beginning with the amino terminal residue. The 5' and 3' ends of the Xen 43.1 cDNA is bordered by EcoRI linkers which were introduced during cloning. 560 T. J. Baldwin electric organ cDNA library (Frail et al. 1987). One cDNA clone encodes a protein whose amino acid sequence corresponds to the sequence of the 43K subsynaptic protein from Torpedo electric organ, except that the cDNA encodes a different C-terminus and does not encode the last 23 amino acids of the 43K subsynaptic protein (Carr et al. 1987). A second cDNA clone encodes 42 amino acid residues which correspond to the C-terminal region and C-terminal residue of the 43K protein; this cDNA clone, however, is incomplete and 370 of the 412 amino acid residues are not encoded by the cDNA (Frail et al. 1987). We sought a cDNA that encodes the entire 43K subsynaptic protein from Torpedo electric organ. Our strategy for isolating this cDNA is described in Materials and methods. We isolated a cDNA (Tor 43K) that encodes the entire protein-coding region of the 43K subsynaptic protein from Torpedo electric organ. The cDNA is 1-8 kb in length, contains 38 bp of the 5' untranslated region, the entire protein-coding region that corresponds to the protein sequence of the Torpedo 43K subsynaptic protein (Carr et al. 1987) and approximately 520 bp of the 3' untranslated region (see Materials and methods; Fig. 2). The Tor 43K cDNA was used as a probe to screen a Xenopus embryo cDNA library. Isolation and characterization of cDNA encoding the Xenopus 43K subsynaptic protein We isolated a cDNA clone encoding the Xenopus 43K protein by screening a Xenopus laevis embryo cDNA library with a cDNA clone encoding the Torpedo electric organ 43K protein (see above and Materials and methods). The sequence of the cDNA was determined and both the nucleic acid and deduced amino acid sequences were compared to that for the Torpedo 43K protein (Figs 1 and 2). Xen 43.1 cDNA is 1403 bp long, contains 205 bp of the 5' untranslated region and 1198 bp of protein-coding region. Comparison of the deduced amino acid sequence with the sequence of the Torpedo 43K protein indicates that Xen 43.1 cDNA ends prior to a termination codon and that the last 13 amino acids of the Xenopus 43K protein are not encoded by Xen 43.1 cDNA (Figs 1 and 2). Xen 43.1 cDNA does, however, encode 10 amino acids beyond the Cterminus encoded in a truncated Torpedo 43K clone (Frail et al. 1987). Since Northern blots of Xenopus embryo RNA demonstrate that the cDNA clone hybridizes to transcripts of 4-0kb and 2-0 kb (Fig. 3; see below), the Xenopus cDNA clone is not fulllength. The Xenopus 43K protein is 71 % homologous to the Torpedo 43K protein (Fig. 2). The DNA sequences within the protein-coding region are 72% homologous. Moreover, the similarity in amino acid sequence is rather evenly distributed over the length of the protein (Fig. 2). It is noteworthy that the homology between the Xenopus and Torpedo sequences does not extend 5' to the codon encoding the methionine that has been designated as the /V-terminus; since the TV-terminal sequence of the Torpedo 43K protein has not been established (Carr et al. 1987), the initiator methionine was assigned on the basis of other criteria (Frail et al. 1987). The dissimi- Alignment of the amino acid sequences of the 43K protein from Xenopus and Torpedo XN43 TOR43 MGQDQTKQQIQKGLQMYQSNQTEKALQIWTKVLEKTTDAAGRFRVLGCLITAHSEMGRYKDMLKFAVIQIDTARELEEPD E L—A-E-G E—QQ-V-RS-ELP A K-E R A-SEA—QMGD-E 10' 20' 30* 40* 50" 607080- XN43 TOR4 3 FLTESYLNLARSNEKLCEFQKTISYCKTCIJmQGTSVSLQLNGQVCLSLGNAYLGLSVFQKALECFEKALRYAHNNDDKM RV—A GH SEAVA—R GAE-GPLR—F M F A G 90100110120130140150160- XN43 TOR43 LECRVCCSLGGLYTQLKDLEKALFFPCKAAELVNDYGKGWSLKYRAMSQYHMAVAYRKLGRLADAMECCEESMKIAIiQHG AF-V Y S A—R K—R A MD Q 170180' 190200210220230' 240 A XN43 TOR43 DRPLQALCLLNFADIHRSHGDIEKAFPRYDSSHSIHTDIGNRLGQTHVMIGVAKCWLHQKEMDKALDCLQKTQELAEDIG C HRS—G--L E--LN E A--LLNI MTE-KL--T-GW--AE DAV250" 260270280290" 300310320" XN43 TOR43 YKHCLLKVHCLSEI1FRTKQQQRELRAHWRFHECVEEMELYCGMCGESIGEKNCQLQALPCSHVFHLRCLQTNGTRGC N-LV A Y-T-Y-EMGSDQL—D K M-D L DQ-S L K N P 330340350360370380390400- TOR43 NCKRSSVKPGYV 410- Fig. 2. Alignment of the amino acid sequences of the Xenopus laevis and Torpedo californica 43K proteins. The Xenopus (XN 43) sequence is shown by the one-letter amino acid notation. Identical residues in the Torpedo (TOR 43) sequence are indicated with a dash (—) and amino acid substitutions are shown by the one-letter amino acid notation. 71 % (282/399) of the amino acid residues in the two sequences are identical. Regulation of 43K subsynaptic protein larity in sequence between the Xenopus and Torpedo clones in the region 5' to this methionine codon and the striking similarity in sequence thereafter provides support for the correct identification of the initiator methionine. In addition, a glycine residue that is a putative yV-terminal myristylation addition site (amino acid residue number two) is conserved in Xenopus. cDNA encoding the 43K protein hybridizes to 4-0 kb and 2-0 kb transcripts in Northern blots of Xenopus embryo RNA Northern blots of poly(A)+ RNA from Xenopus embryos and denervated adult muscle were probed with either nick-translated Xen 43.1 cDNA 32Plabelled probe or a 32P-labelled RNA probe encoding the C-terminal region of the 43K protein (Materials and methods). Northern blots of embryo RNA and denervated adult muscle RNA are identical: strong hybridization is detected to a 4-0 kb transcript and less intense hybridization to a 2'0kb transcript (Fig. 3). We do not know whether the 2-0kb transcript is less abundant than the 4-0 kb transcript and/or whether the 2-0 kb transcript is less homologous to the Xen 43.1 cDNA. Moreover, we cannot exclude the possibility that the 2-0 kb transcript is a degradation product of the 4-0 kb transcript. Fig. 3. cDNA encoding the 43K protein hybridizes to transcripts of 4-0 kb and 2-0 kb in Northern blots of Xenopus embryo RNA. 1 n% of poly(A)+ RNA from stage-41 Xenopus embryos was fractionated by electrophoresis in a formaldehyde agarose (1%) gel, transferred to Zetabind, and hybridized to 32Plabelled Xen 43.1 RNA probe (Materials and methods). The RNAs that hybridize migrate at 40 and 2-0kb (arrowheads). The filter was exposed to X-ray film with an intensifying screen at —70°C for lday. 43K INN DEN 561 AChR INN DEN Fig. 4. Transcript encoding the 43K protein and transcript encoding the AChR alpha subunit are 25- to 35-fold more abundant in denervated than in innervated adult muscle. The triceps femoris muscle of adult Xenopus was denervated for 10 days and AChR alpha subunit and 43K protein transcript levels were measured by RNase protection. 5 fig of RNA from innervated (INN) and denervated (DEN) muscle were included in hybridizations with 32P-labelled probes. The amount of alpha subunit and 43K protein transcript is low in innervated muscle and increases 25- to 35-fold in denervated muscle. Denervation results in no change in either the amount of total RNA or the level of transcript encoding the translation elongation factor Ef-1-alpha (Baldwin et al. 1988). The arrowheads mark the positions of the protected fragments at 403 nucleotides (43K protein) and 429 nucleotides (AChR alpha subunit). Transcript encoding 43K protein is 25- to 30-fold more abundant in denervated than in innervated adult muscle We isolated total cellular RNA from innervated adult Xenopus muscle and denervated adult Xenopus muscle and measured the level of transcripts encoding the 43K protein and the alpha subunit of the AChR. We had demonstrated previously that transcript encoding the alpha subunit increases 50- to 100-fold after denervation of Xenopus muscle, whereas denervation results in no change in either the amount of total RNA or transcript encoding the translation elongation factor Ef-1-alpha (Baldwin et al. 1988). Fig. 4 demonstrates that alpha subunit transcript level increases 30- to 35-fold and 43K protein transcript level increases 25- to 30-fold after denervation. Since both the 2-0 and 4-0 kb RNAs hybridize to the 562 T. J. Baldwin 12 14 20 41 Fig. 5. Transcript encoding the 43K protein is first expressed at late gastrula. 43K protein transcript levels were measured in Xenopus embryos by RNase protection. RNA from 30 eggs (E) and 30 embryos at stages 10, 12 and 14 were included in hybridizations with 32P-labelled Xen 43 probe. Hybridization reactions with RNA from embryos at later stages (20 and 41) included RNA from 10 embryos. The gel was exposed to X-ray film with an intensifying screen at —70°C for 4 days to analyse expression at early stages (egg, 10, 12 and 14) and for 3 days to analyse later stages (20 and 41). The stages are indicated at the top of each lane; the arrowhead marks the position of the protected fragment at 403 nucleotides. encoding the subsynaptic 43K protein increases 25- to 30-fold after denervation of adult skeletal muscle. Denervation of adult muscle produces a similar increase in mRNA encoding AChR subunits (Merlie et al. 1984; Evans et al. 1987; Moss et al. 1987; Baldwin et al. 1988). Thus, loss of myofibre electrical activity results in a 25- to 100-fold increase in the level Transcript encoding 43K protein is expressed initially of transcripts encoding the major proteins of the at late gastrula during embryonic development postsynaptic membrane. To determine when transcript encoding the 43K Although denervation results in a similar increase protein is first expressed during embryonic developin AChR mRNA and AChR protein (Merlie et al. ment, we isolated total cellular RNA from Xenopus 1984), we have not measured the level of 43K protein embryos and measured the level of transcript enin innervated or denervated muscle. Nevertheless, coding the 43K protein. Fig. 5 demonstrates that the results presented here suggest that the level of transcript encoding the 43K protein isfirstdetected in 43K protein increases following denervation and raise Xenopus embryos at late gastrula (stage 12); tranthe possibility that the 43K protein is associated with script is not detected in eggs or in stage-10 embryos. AChRs that are neither clustered nor present at At later stages of development transcript encoding synaptic sites. the 43K subsynaptic protein is more abundant Transcript encoding the 43K protein is readily (Fig. 5). Initial expression of AChR subunit trandetected in Xenopus embryos at stage 14 (16-25 h of scripts also occurs at stage 12 and similar increases in development) and is first detected at stage 12 AChR subunit transcript levels occur during later (13-75 h). Transcripts encoding AChR subunits are stages of development (Baldwin et al. 1988). Morealso initially expressed at stage 12 of development over, the absolute quantity of transcripts encoding (Baldwin et al. 1988). Moreover, the amount of AChR subunits and transcript encoding 43K protein transcript encoding the 43K protein is similar to that is similar at all stages. Thus, expression of transcripts encoding AChR subunits throughout development encoding AChR subunits and the 43K protein are (Baldwin et al. 1988). Thus, transcript encoding the regulated in a similar manner during development as 43K protein is present before synapses form (stage 21, well as in innervated and denervated adult muscle. 22-5 h) (Kullberg et al. 1977) and before AChRs cluster at synapses (stage 22, 24 h) (Chow & Cohen, 1983). Thus, synapse formation and clustering of Discussion AChRs are not required to initiate transcription of the gene encoding the 43K protein. This study demonstrates that the level of transcript 43K RNA probe at high stringency (Fig. 3), the RNase protection analysis measures the sum total of each transcript. Thus, the level of transcripts encoding both the alpha subunit of the AChR and the 43K protein are low in innervated adult muscle and increase to a similar extent following denervation. Regulation of 43K subsynaptic protein Previous immunocytochemical studies have demonstrated that the 43K protein is restricted to the postsynaptic membrane of vertebrate skeletal muscle cells. The 43K protein has not been detected by indirect immunofluorescence at synaptic sites on parasympathetic neurones in the frog cardiac ganglion or in either plexiform layer of the frog retina (unpublished data). The availability of a probe that provides a sensitive assay for the transcript encoding the 43K protein should provide a different and more sensitive assay for the expression of the 43K protein in the nervous system. We would like to thank Drs Frail and Merlie for providing us with Tor43k.l cDNA. This work was supported by a grant from the National Institutes of Health (NS 21579). References D. J. (1987). Molecular biology of the acetylcholine receptor: structure and regulation of biogenesis. In The Vertebrate Neuromuscular Junction (ed. M. M. Salpeter), pp. 285-315. New York: Alan R. Liss. ANDERSON, BALDWIN, T. J., YOSHIHARA, C. M., BLACKMER, K., KINTNER, C. R. & BURDEN, S. J. (1988). Regulation of acetylcholine receptor transcript expression during development in Xenopus laevis. J. Cell Biol. 106, 469-478. BENTON, W. D. & DAVIS, R. W. (1977). Screening lambda gt recombinant clones by hybridization to single plaques in situ. Science 196, 180-182. BURDEN, S. J. (1985). The subsynaptic 43 kd protein is concentrated at developing nerve-muscle synapses in vitro. Proc. natn. Acad. Sci. U.S.A. 82, 8270-8273. BURDEN, S. J. (1987). The extracellular matrix and subsynaptic sarcoplasm at nerve-muscle synapses. In The Vertebrate Neuromuscular Junction (ed. M. M. Salpeter), pp. 163-186. New York: Alan R. Liss. BURDEN, S. J., DEPALMA, R. L. & GOTTESMAN, G. S. (1983). Crosslinking of proteins in acetylcholine receptor-rich membranes: associatipn between the beta-subunit and the 43 kd subsynaptic protein. Cell 35, 687-692. CARR, C , MCCOURT, D. & COHEN, J. B. (1987). The 43kilodalton protein of Torpedo nicotinic postsynaptic membranes: purification and determination of primary structure. Biochemistry 26, 7090-7102. CHIRGWIN, J. M., PRZYBYLA, A. E., MACDONALD, R. J. & RUTTER, W. J. (1979). Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry 18, 5294-5299. CHOW, I. & COHEN, M. W. (1983). Developmental changes in the distribution of acetylcholine receptors in the myotomes of Xenopus laevis. J. Physiol. 339, 553-571. EVANS, S., GOLDMAN, D., HEINEMANN, S. & PATRICK, J. (1987). Muscle acetylcholine receptor biosynthesis. /. biol. Chem. 262, 4911-4916. 563 FAMBROUGH, D. M. (1979). Control of acetylcholine receptors in skeletal muscle. Physiol. Rev. 59, 165-227. FRAIL, D. E., MUDD, J., SHAH, V., CARR, C , COHEN, J. B. & MERLIE, J. P. (1987). cDNAs for the postsynaptic 43-kDa protein of Torpedo electric organ encode two proteins with different carboxyl termini. Proc. natn. Acad. Sci. U.S.A. 84, 6302-6306. FROEHNER, S. C. (1986). The role of the postsynaptic cytoskeleton in AChR organization. Trends Neurosci. 9, 37-41. FROEHNER, S. C , GULBRANDSEN, V., HYMAN, C , JENG, A. Y., NEUBIG, R. R. & COHEN, J. B. (1981). Immunofluorescence localization at the mammalian neuromuscular junction of the Mr 43,000 protein of Torpedo postsynaptic membranes. Proc. natn. Acad. Sci. U.S.A. 78, 5230-5234. GUBLER, U. & HOFFMAN, B. J. (1983). A simple and very efficient method for generating cDNA libraries. Gene 25, 263-269. KINTNER, C. R. & MELTON, D. A. (1987). Expression of Xenopus N-CAM RNA in ectoderm is an early response to neural induction. Development 99, 311-325. KULLBERG, R. W., LENTZ, T. L. & COHEN, M. W. (1977). Development of the myotomal neuromuscular junction in Xenopus laevis: an electrophysiological and finestructural study. Devi Biol. 60, 101-129. LAROCHELLE, W. J. & FROEHNER, S. C. (1986). Determination of the tissue distribution and relative concentrations of the postsynaptic 43-kDa protein and AChR in Torpedo. J. biol. Chem. 261, 5270-5274. MANIATIS, T., FRTTSCH, E. F. & SAMBROOK, J. (1982). Molecular Cloning. Cold Spring Harbor, New York: Cold Spring Harbor Laboratory. MERLIE, J. P., ISENBERG, I. E., RUSSELL, S. D. & SANES, J. R. (1984). Denervation supersensitivity in skeletal muscle: analysis with a cloned cDNA probe. /. Cell Biol. 99, 332-335. MlSHINA, M . , KUROSAKI, T . , TOBIMATSU, T . , MORIMOTO, Y., NODA, M., YAMAMOTO, T., TERAO, M., LINDSTROM, J., TAKAHASHI, T., KUNO, M. & NUMA, S. (1984). Expression of functional acetylcholine receptor from cloned cDNAs. Nature, Lond. 307, 604-608. Moss, S. J., BEESON, D. M. W., JACKSON, J. F., DARLISON, M. G. & BARNARD, E. A. (1987). Differential expression of nicotinic acetylcholine receptor genes in innervated and denervated chicken muscle. EMBO J. 6, 3917-3921. NEUBIG, R. R., KRODEL, E. K., BOYD, N. D. & COHEN, J. B. (1979). Acetylcholine and local anesthetic binding to Torpedo nicotinic postsynaptic membranes after removal of nonreceptor peptides. Proc. natn. Acad. Sci. U.S.A. 76, 690-694. PENG, H. B. & FROEHNER, S. C. (1985). Association of the postsynaptic 43 k protein with newly formed acetylcholine receptor clusters in cultured muscle cells. 564 T. J. Baldwin J. Cell Biol. 100, 1698-1705. of the neuromuscular junction and of the junctional RIGBY, P. W. J., DIECKMANN, M., RHODES, C. & BERG, acetylcholine receptor. In The Vertebrate P. (1977). Labeling deoxyribonucleic acid to high specific activity in vitro by nick translation with DNA polymerase I. J. molec. Biol. 113, 237-251. SALPETER, M. M. (1987). Development and neural control Neuromuscular Junction (ed. M. M. Salpeter), pp. 55-115. New York: Alan R. Liss. {Accepted 30 July 1988)