Proc. Nat/. A cad. Sci. USA Vol. 81. pp. 7970-7974. December 1984 Neurobaology
Isolation and characterization of a eDNA clone for the complete protein coding region of the 5 subunit of the mouse
acetylcholine receptor
(bacteriophage ~gUO/sequence homology profiles/BCJH-1 cells)
ROBERT J. LAPOLLA*. KATHARINE MIXTER MAYNEt. AND NORMAN DAVIDSON*
•Depanment of Chem1s1ry and tDiv•sion of Biolo8)'. Cahfom1a lns111u!e of Technology. Pasadena. CA 911.:!5 Contributed by Nurman Dal·idson. AURliSI 20. 1984
ABSTRACT A mouse cD:'oiA clone has been isolated that contains the complete codin~ re~ion of a protein high!~· ho- mologous to the ii subunit of the Torpedo acet~·lcholine recep- tor I AcChoRI. The cD:"A librarv was constructed in the vector
>.gtiO from membrane-associat~d poly! AI• R:"iA from BC3H- I mouse cells. Surprisingly. the ii clone was selected by hybrid- ization with eDNA encoding the y subunit of the Torpedo Ac- ChoR. The nucleotide sequence of the mouse cON A clone con- tains an open reading frame of 520 amino acids. This amino acid sequence exhibits 59% and 50% sequence homology to the Torpedo AcChoR 6 and ysubunits, respectivelv. However.
the mouse nucleotide sequence has several stretches of high homology with the Torpedo y subunit cD:"A. but not with 6.
The mouse protein bas the same general structural features as do the Torpedo subunits. It is encoded by a 3.3-kilobase mR:'o/A. There is probably only one. hut at most two, chromo- somal genes coding for this or closely related sequences.
The nicotinic acetylcholine receptor IAcChoRl is a postsyn- aptic integral membrane protein complex composed of four subunits. denoted a. {3. y. and li. with gel electrophoretic molecular weights ranging from 40.000 to 65.000. The recep- tor is very abundant in the electric organ of the ray Torpedo and it has been extensively characterized at the biochemical.
functional. and sequence levels Ill. The amino acid se- quences of the four Torpedo subunits have been determined from nucleotide sequences of full-length eDNA clones 12-7).
Nicotinic AcChoRs occur in lower overall abundances per unit mass of tissue at the neuromuscular junctions of vene- brate striated muscle and in various muscle-like cell systems in culture. Venebrate systems are of greater interest than Torpedo for most electrophysiological studies and for cell biological studies of assembly I see ref. 81. The subunits of the venebrate receptor are similar in general propenies to those of Torpedo. but they are clearly somewhat divergent at the amino acid sequence level 191.
Full-length eDNA clones for the a subunit of a bovine re- ceptor and chromosomal genes for the a subunit of humans and chickens have been characterized 110. 111. as has a par- tial eDNA clone for the a subunit of the mouse AcChoR t 121.
As a fun her step toward the complete characterization of the venebrate genes. we repon here the isolation and character- ization of a eDNA clone containing the complete protein- encoding region of the li subunit of the mouse AcChoR.
MATERIALS AND METHODS
Preparation of Membrane-Associaled Polysomal PolyiAI•
RNA. BCJH-1 mouse cells were grown and induced to un- dergo differentiation on reaching connuency so as to express The publicatiOn costs of thiS an1cle were defrayed m part by pa~e charge payment. This artacle must therefore be hereby marked "adn•mum~nt"
in accordance w1th 1~ U.S.C. §1734 'iolely to indicate this fact.
7970
AcChoR as described I 13 l. A membrane pellet enriched in membrane-bound polyribosomes was prepared by a modifi- cation of the method of Merlie er a/. 1141. Our procedure differed in some details. includino omission of the nuclear separation step and use of emetin~·HCI in all isolation butf- ers to prevent ribosome run-off. The final membrane pellet was dissolved in 6 M ouanidine·HCI/20 m~l sodium acetate.
pH 5/1 mM dithioth,:e1tol 1151. RNA was purified by four cycles of precipitation with ethanoli0.55 voll and redissolu- tion in the same buffer. PolviAI- RNA was isolated bv two cycles of oligoldTi-cellulos~ chromatography. ·
eDNA Cloning in >.gtlO. A eDNA library was prepared in the bacteriophage vector l.gtlO by a method devised by Charles Rice of this institution I personal commumcationl.
based in pan on the procedure of Oka~·ama and Berg 1!61.
First strand synthesis was carried out with 10 1'-g oft he mem- brane-bound poly! AI· RNA essentially as described t 161 ex- cept that dT,,_,., was used for primmg. Four micrograms of actinomycin D and 40 units of human placental ribonuclease inhibitor were included in the 100-JJ.I reaction volume. Sec- ond strand synlhesis by replacement of RNA from 4 1'-g of the RNA·DNA hybrid was carried out without addition of primer by adding Escherichia coli DNA ligase and polymer- ase I. ribonuclease H. dNTPs. and other components as de- scribed I 161. Internal EcoRI sites in the resulting duplex eDNA were then protected by methylation with EcoRl methylase. The duplex eDNA was blunt-ended with T4 DNA polymerase and all four dNTPs. Two micrograms of phosphorylated £cvRllinkers 11~-mersl was added with T4 DNA ligase. After digestion with EcoRl. free linkers and their fragments were removed by four cycles of precipitation from 2 M ammonium acetate with 0.6 vol of isopropyl alco- hol at 2YC. Approximately 2.4 1'-g of the final eDNA prepa- ration was then ligated to 4.6 1'-g of £coRl-digested l.gtlO DNA in 25 JJ.I. using T-1 DNA ligase. The reaction mixture was incubated at 15"C for 15 hr. Recombinant l.gtiO DNA was packaged in 1·itro 1 ref. 17. pp. 264-2681. Packaged phage were plated on£. coli strains C600 I ref. 17. p. 5041 to assess the proponion of recombinants in the library or on C600 t!.Hfl !provided by Carol Nottenburg of the University of California. San Francisco) for screening purposes. An unam- plified library of approximately 1 x to• recombinants was obtained.
Library Screening. We screened 1 x 10' recombinant plaques in duplicate by plaque filter hybridization tref. 17.
pp. 324-3281 at a density of 2 x to• recombinant phage per 150-mm !diameter! plate. Full length Torpedo eDNA clones.
kindly provided by D. Noonan Ia subunitl. T. Claudio 1{3 and
/i). and S. Heinemann I y: see ref. 51. were nick-translated to
specific activities of approximately 1.0 x 10• cpm/ ~-The hybridization solution contained 50% (vol/voll formamide.
Abbreviations: AcChoR. acetylcholine receptor: kb. kilobasetsl:
bp, base pairt sl.
Neurobiology: LaPolla eta/.
0.9 M NaCI/~0 mM sodium phosphate. pH 7.4/5 mM EDTA t~x SSPE11ref. 17. p. 4471. 0.1'7< Ficoll. 0.1'7r polyvinylpyr- rolidone. 0.1'/f bovine ~crum albumin. O.l'h sarcosine. dena- tured salmon sperm DNA at 100 1-'g/ml. polylrAI. polylrCl.
and polyirGI at 2 !'g/ml each. and I l'g of labeled plasmid probe. Hybridization was carried out at 42'C for 48 hr. Fil- ters were washed in 30 mM NaCI/3 mM sodium ci- trate/0.05'7r sarcosine at 50'C. Positive plaques were puri- fied and rescreened at least three times.
RESULTS AND DISCUSSION
Source or mR:"'A. Differentiation of the myogenic nonfus- ing mouse cell line BC3H-I results in the biosvnthesis of rel- atively large amounts of AcChoR 1131. An additional enrich- ment of AcChoR mRNA by a factor of about 5 is achieved by isolation of membrane-bound poly somes 1141.
Screening the eDNA Library. The eDNA library was screened at moderate stringency with hybridization probes prepared from full-length eDNA clones for each of the four subunits of the Torpedo AcChoR. The a. /3. and y probes each identified between 10 and 25 apparent positive plaques.
No positives were obtained with the Torpedo li probe. even when screened under less stnngent conditions [33'7r lvol/
voll formamide/5 x SSPE. 42'C[. We chose to characterize first the mouse eDNA clones selected by using the Torpedo y probe.
Restriction Enl\'me Analvsis or the Mouse eDNA Clones.
Five clones that ·gave strong signals with the Torpedo y probe in gel blots and contained inserts of length greater than 1.0 kilobase lkbi were analvzed in detail. Their restriction endonuclease maps are shown in Fig. I. Indicated on the figure for reference purposes are the sites of the initiator me- thionine and the terminator codon as determined bv nucleo- tide sequence analvsis I see Fig. 31. Several features'bear not- ing. First. all five clones possess identical restriction maps in their coding regions. indicating they were derived from the same or closely related messages. Second. the inserts have widely different 3' ends. Nucleotide sequence determination revealed A-rich regions in the coding strand of the >-58 insert corresponding to the 3' ends of >--16 and >-53. OligoidTI prim- ing from A-rich regions in the mRNA could be responsible for thiS phenomenon. Third. the s· ends of all five inserts are within 33 nucleotides of the initiator A TG codon !determined bv sequence analysis: data not shown!. >-60 extends the fur- thest upstream from the initiator ATG 133 nucleotidesl. The sequence of >-58. the clone subjected to detailed sequence analysis. extends five nucleotides upstream of the ATG. A53 begins at position 7 on Fig. 3 and >-61 begins at position 14 on Fig. 3. I None of these values includes the £coR I linker se- quence.! Fourth. the inserts from >-58 and '-60 have different restriction maps near their 3' ends. RNA gel blot experi- ments suggest that this difference is due to a cloning artifact in the 3' untranslated region of >-58 !see Fig. 6 below).
. s~ ·"-"- -- ---=
FtG. I. Restriction maps of insens from recombinant A eDNA clones. Po~ it ions of the: inillator mc:thtonine ( ~1ell and the termina- tion codon tTrmJ v.ere determtned from nucleotide sequence analy-
~ts !see Fig. 31. bp. H::tse pairs.
Proc. Nat/. Acad. Sci. USA 81 11984! 7971
"" e
0 0
6 •566
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'"'' SlaNt enrEn No< I """' EroRI
. .
420 666 •n 25• ,,., '9t2FtG. ~- Sequencing 'itrategy for the protein-encoding region EcoRI fragment from >..58 subcloned in pUC9. £. cvli strain TBl
!provided bv T. 0. Baldwin. Texas A & M Uni·tersitvJ was used for transformation. Plasmtd DNA was isolated by thf" alkaline ly- sts/CsCI gradient method (ref. 17. pp. 90-911. The small closed ar- rows show the directton of sequencing. Protruding 5' ends were la- beled by using T4 DNA kinase and fy-·"PidATP 1>7000 Ci/mmol.
ICN: 1 Ci = 37 GBqJ. Recessed 3' ends were labeled by using£. cvli DNA polymerase I Klenow fragment (Bethesda Research Labora- tonesl and the appropriate a:-·':P-Iabeled dNTPs I ==JOOO Ci/mmol.
Amershaml. The venicallines on the ends of the arrows indicate the position oft he: radioactive label. The nucleotide ~equence was deter- mined by the method of Maxam and Gilbert as modified bv Smith and Calvo I 191. The numbers indicate the position in nucieotides tfrom Fig. 31 of the restriction site'i. The large open arrows show the locauons of the initiator methiontne tMeo and the termination !Trm) codons.
:"ucleotide Sequence Determination. The EcoRI fragments in the five eDNA clones shown in Fig. I were subcloned separately in the plasmid vector pUC9il81. Fig. 2 shows the strategy used to determine the nucleotide sequence of the
£coR I fragment from A58 that had been characterized as hy- bridizing to the coding region of the Torpedo AcChoR y sub- unit eDNA. Both strands of thiS fragment were completely sequenced.
Fig. 3 gives the nucleotide sequence of the coding 1 mRN A sensei strand of this fragment and the deduced amino acid sequence of the complete polypeptide chain. The first base of mouse eDNA from A58 is at position I of the nucleotide sequence in Fig. 3. The initiator ATG codon occurs at posi- tion 6. A signal peptide of 24 amino acid residues is encoded by nucleotides 6-77. The first amino acid of the mature pro- tein begins at position 78 of the nucleotide sequence. as judged by comparison with the protein sequences of Ac- ChoR subumts from fetal calf 191. There is an open reading frame of 1560 nucleotides !beginning at position 61. coding for a polypeptide chain of 520 amino acids and terminated by a TAG codon at position 1566. There are two additional ter- mination codons in phase with the reading frame at positions 1581 and 1605. There is no sequence corresponding to the
£cuRl linker at the 3' end of this fragment. indicating that this £wRI site is a natural internal £wRI site in the mouse sequence. The calculated molecular weights of the mature polypeptide chain and its precursor are S7.104 and 59.393.
respectively.
Sequence Comparisons. In Fig. 4 we have aligned the de- duced amino acid sequence of the cloned mouse polypeptide chain with the amino acid sequences of the T urpedu AcChoR y and li subunits. The similarity of the mouse amino acid sequence to the sequences of both of the Torpedo subunits is striking. The surprising result is that the mouse amino acid sequence is actually more similar overall to the Torpedo Ac- ChoR li subunit !59'/C homology! than it is to the Torpedo y subunit i50'7r homology!. lin these calculations. gaps insert- ed into the sequences for alignment purposes are 1gnored by the computer. 1 When the individual amino acids were re- placed by their functional groups !nonpolar. polar. acidic.
and basic! and the same comparison made. the mouse poly-
7972 Neurobiology: LaPolla er a/. Proc. Nat/. AcaJ. Sci. USA 8/1/9/3./i
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Ftc. 3. Nucleotide sequence oft he £coR I fragment from mouse eDNA clone A. 58 containing the protein coding region. The deduced am1no acid sequence tstandard one-leiter code! of the open reading frame IS g:tven above the nucleotide sequence. Sudeoude no. l 1s th~ fir,t nucleotide of the eDNA. Eight nucleotidcs df A·A· T· T-C-G·G·GI present at the 5' end of the s~quence ~Acre Jcrived from th~ £c oRIIinkc!r u'cd in the cloning procedure and have been deleted from the figure. Amino actd no. 1 (•) ts thJt asstgned as the first ammo a~o:td of th~ mature protein. The arrows indicate potential sites of asparagme N-glycosylallon.
peptide chain was again more homologous overall to the Tor- pedo AcChoR o subunit 177"<) than it was to the Torpedo y subunit 17~'7c). When the nucleic acid sequences were aligned as in Fig. 4. the mouse sequence was also more simi- lar overall to the Torpedo o subunit sequence i60?i-l than it
was to the Torpedo y subunit sequence i55'7c) !data not shown). It is important to reiterate at this point that the Tor- pedo AcChoR y subunit eDNA was used to 1solate this puta- tive mouse AcChoR eDNA clone and that no positives were obtained when the Torpedo o subunit cON A was used as hybridization probe.
A clue to the cause for this paradox is provided by the percent homology profiles for both the nucleotide and ammo acid sequences 1 Fig. 5). This analysis displays areas of high and low homology between two sequences as peaks and troughs. respectively. From the nucleic acid sequence com- parisons it is apparent that the Torpedo y subunit eDNA se- quence contains longer reg1ons of high homology with the mouse eDNA <equence I Fig. 5Al than does the Torpedo o
subunit eDNA I Fig. 5CJ. We estimate that only sequences in the peaks of length ca. 75 nucleotiues and homology greater than 65-70'7c in Fig. 5A for the Torpedo y eDNA probe
would hvbridize with the mouse sequence under the condi- tions used i50?i- formam1de/S x SSPE. ~~·o. whereas no such rcgtons occur in the Torpedo S cD~·"'·
In contrast 10 the nucleic acid homologv profiles just de- scribed. the amino ac1d homology profiles show longer stretches of high homology for mouse vs. Torpedo 6 than for mouse vs. Torpedo yiFig. 5 D and 81. in agreement \\lth the overall percent homology calculations. These data ind1cate that the subunit of the mouse AcChoR that we have cloned is generally more similar to the Torpedo AcChoR 6 subunit than to the y subunit. The nucleic acid sequence homology profiles explain the technical result of selecting a mou>e 6 eDNA clone with a Torpedo y probe: however. the biologi- cal and evolutionary significance of these homology relation- ships is unclear.
Conti-Tronconi era/. 19) have reported ~H,-terminal ami- no acid sequence data for all four subunits of the fetal calf AcChoR. The data clearly showed sequence homology among the four subunits of the fetal calf AcChoR. Similar to that observed in Torpedo and Electrophorus. and they estab- lished the subunit stoichiometrY in fetal calf 10 be 2:1:1:1.
However. the data diu not perm.it subunit molecular weights
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