Jul 22, 1985 - John A. Hammer 111, Edward D. Korn, and Bruce M. PatersonS ...... Hammer, J. A., 111, Korn, E. D., and Paterson, B. M. (1985) J. Biol. Chem.
Vol. 261,No.4,Issue of February 5,pp. 1949-1956,1986 Printed in U. S. A.
THEJOURNALOF BIOLOGICAL CHEMISTRY
Isolation of a Non-muscle Myosin Heavy Chain Gene from Acanthamoeba” (Received for publication, July 22, 1985)
John A. Hammer 111, Edward D. Korn, and Bruce M. PatersonS From the Laboratory of Cell Biology, National Heart, Lung, and Blood Institute and the$Laboratory of Biochemistry, National Cancer Institute, National Institutesof Health, Bethesda, Maryland 20205
We have isolated a non-muscle myosin heavy chain gene from Acanthamoeba castellanii using as a heterologous probe a sarcomeric myosin heavy chain gene from Caenorhabditiselegans. The amoeba genomic clone has been tentatively identified as containing a myosin I1 heavy chain gene based on hybridization to a 5300-nucleotide RNA species, hybrid selection of a mRNA encoding a 185-kDa polypeptide, specific immunoprecipitation of this polypeptide with antiserum to myosin 11, and an exact match between the DNA sequence and a carboxyl-terminal myosin I1 peptide previously sequenced by protein chemical methods (Cote, G. P., Robinson, E. A., Appella, E., and Korn, E. D. (1984) J.Biol. Chem. 259, 12781-12787). We also sequenced a region of the gene whose deduced amino acid sequence shows stronghomology with that region of muscle myosins which is thought to be involved in nucleotide binding. These results indicate that the amoeba genomic clone contains at least 90%of the coding information for the 185-kDa heavy chain polypeptide and that the bulk of the gene contains very little intron DNA.Genomic blots of amoebaDNA probed with a portion of this myosin gene indicate the presence of additional highly related sequences within the amoeba genome.
chain of myosin 11. This cytoplasmic myosin clone was identified using a sarcomeric myosin heavy chain gene from the nematode as aheterologousprobe (3). While a number of sarcomeric myosin heavy chain genes have been isolated from both vertebrates (4-11) and invertebrates (12-14), this is the first report of the isolation of a non-muscle myosin heavy chain gene. Just as we used the nematodegene to identify the amoeba gene, the amoebagene may be useful as a heterologous probe to isolate other cytoplasmic myosin genes from both vertebrates and invertebrates. EXPERIMENTAL PROCEDURES
Materials-Acanthamoeba polyadenylated RNA was prepared from mid-log phase cells as describedpreviously (15). High-molecularweight amoeba DNA was isolatedfromamoebanuclei as follows. Saline-washed cells (4 “C) were Dounce homogenized in a solution containing 300 mM sucrose, 20 mM Tris (pH 7.5), 20 mM KCl, and 5 mM EDTA (40 ml of buffer/5 ml of packed cell pellet) and centrifuged at 1800 rpm for 8 min (IEC Clinical). To the crude nuclear pellet was added 15 ml of lysis buffer, which contained 0.2 M EDTA (pH 9.75), 2% SDS,’ and 0.5 mg/ml proteinase K. The mixture was vortexed and incubated for 90 min a t 50 “C. DNA was purified by phenol/ chloroform extraction, treatment with DNase-free RNase, andspooling through 2-propanol (16). The recombinant X1059 phage containing the nematode unc-54 myosin heavy chain gene (3) was a generous gift of J. Karn and L. Barnett (Medical Research Council, Cambridge, United Kingdom). Myosin I1 was a gift of M. A. L. Atkinson (Laboratory of Cell Biology, National Heart, Lung, and Blood Institute). Myosin I1 polyclonal antiserum was prepared as described previously Three non-muscle myosins have been purified from the soil (15). All enzymes and cloning vectors were purchased from Bethesda amoeba, Acanthamoeba (for review, see Refs. 1 and 2). Myo- ResearchLaboratories.Formamide from Fluka was deionized as sins IA and IB are structurally unusual myosins, each con- described by Maniatis et al. (16). All chemicals were reagent grade. General Methods-General procedures including bacterial transtaining asinglelow-molecular-mass heavy chain (130 kDa and 125 kDa,respectively) and a single light chain(17 and 25 formation, subcloning, restriction enzyme digestion, and gel electrowere performed using standard methods (16). Plasmid DNA kDa, respectively). These molecules are roughly globular, lack phoresis was prepared by two bandings in cesium cbloride/ethidium bromide an extended rod-like tail, and appear incapable of self-assem- gradients as described elsewhere (17). Nick translation of DNA fragbly into bipolar filaments. Acanthamoeba myosin I1 is, on the ments prepared by electroelution from agaroseor acrylamide gels was other hand, structurally similar to vertebrate muscle myosins, performed using the kit from Bethesda Research Laboratories sup(Amersham Corp., 3000 Ci/ being composed of a pair of 185-kDa heavy chains and two plemented with 100 pCi of [(u-~’P]~CTP pairs of light chains (17 and17.5 kDa) arranged in a typical, mmol) and 2 pM dCTP. Blots-DNA digests were fractionated on agarose gels highly asymmetric molecule possessingtwoglobular heads andSouthern transferred tonitrocellulose (Schleicher & Schuell) by published and a longa-helical coiled-coil tail. In our effort to understand procedures (18, 19). Prior to blotting, the top half of t.he gel was the structure and function of these cytoplasmic myosins at incubated for 15 min in 0.25 M HCI to facilitate transfer of highthe molecular level, we are isolating thegenes encoding these molecular-weight fragments (20). Exceptwhen oligonucleotide probes proteins. Theavailability of cloned myosin heavychain genes were used (see below),blots were prehybridized in a solution containprovidesa way toobtain sequence information relatively ing 6 X SSPE ( 1 X SSPE is 0.15 M NaC1, 10 mM sodium phosphate, 1 mM EDTA,pH 7.0), 5 X Denhardt’s solution ( 1 X Denhardt’s rapidly and eventually to study the relationships of protein solution is 0.02% Ficoll 400, 0.02% polyvinylpyrrolidone, 0.02% bostructure and functionvia mutagenesis of the gene. We report vine serum albumin), and0.2 mg/ml sheared salmon sperm DNA for here the isolation and initial characterization of an amoeba 6 h at 68 ”C and then hybridized with radioactive probes in a solution myosin heavy chain gene which apparently encodes the heavy containing 30% formamide, 5 X SSPE, 2 X Denhardt’s solution, and 0.1 mg/ml salmon sperm DNA for 18 h at 42 “C. For screening of * The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby The abbreviations used are: SDS, sodium dodecyl sulfate; DBM, marked “aduertisement” in accordance with 18 U.S.C. Section 1734 diazobenzyloxymethyl; PIPES, piperazine-N,Nf-bis-(2-ethanesulsolely to indicate this fact. fonic acid); bp, base pair; kb, kilobase pair.
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library liftoffs, PUC9 DNA was alsoaddedtothe hybridization SDS. The hybridization solution (35pl final volume/disc),which solution (1 pg/ml). When the heterologous nematode probes were contained the same components plus 50 pg/ml tRNA (calf liver; used (Fig. 1 and screening of the genomic library), the blots were Boehringer Mannheim) and600 pg/ml amoeba polyadenylated RNA, washed twice with 2 X SSPE, 0.1% SDS (55 "C), andtwice with 0.5 was heated for 3min a t 70 "C and added to the disc following removal X SSPE, 0.1% SDS (55 "C). When amoeba DNA probes were used, of the prehybridization solution. Following an 18-h hybridization at the blots were washed as described in the legend to Fig. 7. 42 "C, the filters were washed 10 times with 1 ml each of a solution Northern Blots-Amoeba polyadenylated RNA was fractionated containing 1 X SSC, 1 mM EDTA, 0.1% SDS (60 "C), washed once on a 1% agarose gel after denaturation with glyoxal and dimethyl with 2 mM EDTA (pH 7.5) (20 "C), and the bound RNA eluted by sulfoxide (16,21) and blotted on to DBM paper (Schleicher & Schuell) incubation for 5 min at 65 "C in 200 p1 of 99% formamide, 10 mM by published procedures (20). Blots were prehybridized for 8 h a t Tris (pH7.5). The eluted RNA was recovered as described previously 42 "cin a solution containing 50% formamide, 0.9 M NaC1, 20 mM (16), dissolvedin 5 pl of H20, and translated using rabbit reticulocyte sodium phosphate (pH 7.0), 5 mM EDTA, 0.4 mg/ml salmon sperm lysates as previouslydescribed(15). The [35S]methionine-labeled DNA, and 0.1% SDS and were then hybridized with radioactive myosin I1 heavy chain polypeptide synthesized in vitro was immuprobes in a solution containing the same components for 40 h a t noprecipitated usinga myosin I1 polyclonal antiserum and the results 42 "C. When nematode DNA probes were used, the blotswere washed visualized by SDS-polyacrylamide gel electrophoresis and fluorogratwice with 2 X SSC (1 X SSC is 0.15 M NaC1, 15 mM sodium citrate), phy as described previously (15). 0.1% SDS (50 "C), and twice with 0.8 X SSC, 0.1% SDS (50 "C). DNA Sequencing-DNA fragments were suhcloned in phage m13 When amoeba DNA probes were used, the blots were washed twice mp18 or mp19 (usinghost Escherichia coli strain J M 101) and with 1 X SSC, 0.1% SDS (55 "C) and twice with 0.2 X SSC, 0.1% sequenced by the dideoxynucleotide chain termination method of SDS (55 "C). The DBM blots were regenerated as described previSanger etal. (27) on8%sequencing gels. The sequences were analyzed ously (22). with the DEC-10 DNA sequence analysis program (Division of ComOligonucleotides-The mixed 20-bp oligonucleotide probe was syn- puter Research andTechnology, National Institutes of Health). thesized by the phosphotriester method (23)by the Nucleic Acid and Peptide Synthesis Laboratory (Frederick Cancer Research Center, RESULTS Frederick, MD). The mixed 20-bp oligomers were purified on a 16% To determine thefeasibility of using the nematode myosin sequencing gel, end-labeled using T4 polynucleotide kinase and [y "PIATP (Amersham) to a final specific activity of 1 X lo9 dpm/pg gene as a heterologous probe to identify amoebamyosin genes, (16), and separatedfrom unincorporated radioactivity by chromatog- we tested the ability of the nematode gene to recognize both raphyonSephadexG-50superfine.Blots to beprobedwiththe amoeba DNA sequences and amoeba RNA transcripts. We oligomer were prehybridized for 4 h at 68 "C ina solution containing used as a heterologous probe a 2830-bp BamHI fragmentfrom 6 X SSC, 10X Denhardt's solution, and0.5% SDS. For hybridization, the probe was added directlyto the prehybridization solution and thethe nematode unc-54 myosin heavy chain gene, which conblot incubated at 50 "C for 40 h. The blots were washed twice with 1 tains 2181 bp of coding sequence interrupted by four interX SSC, 0.1% SDS (50 "C), and twice with 0.25 X SSC, 0.1% SDS vening sequences of 479,79,53, and 38 bp(3). This fragment (50 "C). encodes amino acid residues 35 through 760, or approximately Construction of the Acanthamoeba Genomic Library-High-molec90% of the nematode myosin head including the actin and ular-weight amoeba DNA was partially digested with MboI under ATP binding sites and thereactive thiols. We found that this conditions which maximize the sequence representation of molecules in the 15-20-kb size range (16, 24). The 15-20-kb MboI fragments nematode probe hybridized to eight amoeba DNA restriction were isolated by electroelution following fractionation through a 0.5% fragments (10.5, 9.6, 8.6, 7.7, 7.0, 5.6,3.9, and 3.2 kb) in a Southern blot of BamHI-digested amoebagenomic DNA (Fig. agarose gelby published procedures (16). The phage vectorused, phage X2001, is a modified form of the SPI phenotype vector X1059 1, lune 1 ) . Furthermore, this probe hybridized to two high(14), and was a generous gift of J. Karn and L. Barnett (Medical molecular-weight amoeba mRNA species (5300 and 4250 nuResearch Council).The conditions of the ligation reaction, including cleotides) in a Northern blotof amoeba polyadenylated RNA the concentrations and ratios of BamHI-digested X2001 DNA and (Fig. 1, lane 3 ) . Therefore, this heterologous probe recognized 15-20-kb amoeba MboI DNA fragments, were as described by Karn et al. (14). In vitro packaging was performed asdescribed by Maniatis both discrete amoeba DNA sequences and RNA transripts, and one of these transcripts was large enough to encode the et al. (16), using the kit from Promega Biotec. The yield was -5 X IO5recombinant phage/pg of amoeba DNA. Unamplified recombinant amoeba myosin I1 heavy chain (the 185-kDa heavy chain phage (80,000 plaque-formingunits,orapproximately 40 genome would requirea minimum of approximately 5000 nucleoequivalents(25)) were grown onbacterialstrain Q359 (14) and tides'). These results strongly suggested that the nematode screened by the plaque hybridization technique of Benton and Davis probe might identify amoebamyosin I1 gene sequences within (26) with the isolated, nick-translated 2.8-kb BamHI fragment from the nematode unc-54myosin heavy chain gene (3). The hybridization a genomic library of amoeba DNA. We constructed a genomic library of Acanthamoeba DNA and washing conditions were those described above. Phage clones which were positive on replicate filters(-4/genome equivalent) were in phage X2001 with 15-20-kb DNA fragments generated by chosen randomly, plaque purified, and amplified by the plate lysate partial digestion of amoeba genomic DNA with MboI (see techniqueas describedby Maniatiset al. (16).Phage DNA was "Methods"). We screened approximately 40 genome equivaprepared from large scale liquid lysates by polyethylene glycol 6000 lents (80,000 plaques) with the nematode probe and obtained precipitation, glycerol step gradient centrifugation, and DNA extracapproximately 4 positivephage/genome equivalent. This yield tion (16). For long term storage, the phage library was amplified by is in good agreement with the results inFig. 1 (lane I ) , where plate lysate and storeda t 4 "C over chloroform (16). HybridSelectionAnalysis-Recombinantphage DNA was sub- eight BamHI restriction fragments totaling nearly 60 kb of DNA were recognized by the nematode probe. Forty positive jected tohybrid selection analysisby a modification of the method of Miller et al. (22). Phage clone DNA (10 pg) was digested with Xba, phage clones were picked at random and plaque purified. purified, dissolved in 20 p1 of HzO,boiled for 3 min, quick-frozen, and These were rescreened with a subfragment of the nematode applied immediately on thawing to 7-mm diameter discs of DBM probe, a 490-bp AvaI/BamHI piece which encodes amino acid paper (Schleicher & Schuell) treated as previouslydescribed (20). residues 598 through 760. This region contains the 2 reactive The DBM discs containing bound DNA were washed 3 times with cysteines at residue positions 705 and 715. This "reactive H,O, cut into -1 mm2 pieces,washed 3 times with 0.4 N NaOH (37 "C),washed 4 times with H20, and incubatedfor 1 h (50 "C) ina thiol" region is highly conserved in myosins (3, 28). Restricsolution containing 65% formamide, 10 mM PIPES (pH 6.4), 0.4 M This estimate is based on the averagemolecular mass of the NaCl, and 1 mM EDTA. The DBM discs were subjected to two mock myosin I1 heavy chain (111.74 Da,basedon elutions: (i) 1 h (65 "C) in99% formamide, 10 mM Tris (pH 7.51, and aminoacidsinthe quantitative aminoacid composition of the purified heavy chain) and (ii) 2 min (100 "C) in 1 mM EDTA (pH 7.5). The discs were prehyon the molecular mass of the heavy chain (185 kDa, as estimated by bridized for 30 min (42 "C) in 100 pl of a solution containing 50% formamide, 80 mM Tris (pH 7.8), 0.6 M NaCI, 2 mM EDTA, and0.1% SDS-polyacrylamide gel electrophoresis).
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proteins translated in uitro from mRNA hybrid-selected by the control DNA (vector DNA only; lane 2) and the X4.13 DNA (lane 3) revealed a 185-kDa protein which was unique to the X4 13 DNA selection. This radioactive protein comigrated exactly with the authentic, purified myosin I 1 heavy chain. Furthermore, this 185-kDa radioactive protein was 10.5, quantitatively and specifically immunoprecipitated by a 9.6polyclonal antiserum to myosin I 1 (lane 4 ) . The protein was 8.6 - w w not precipitated by non-immune serum (lane 5 ) , nor was a 7.7 185-kDa protein precipitated from the control DNA selection 7.0using the myosin I1 antiserum (lane 6). This preliminary identification of the myosin I 1 gene was further supported by 5.6the fact that the 5’ 11-kb Xba fragment in X4.13 hybridized 5300 to a 5300-nucleotide amoeba mRNA (Fig. 1, lane 4 ) . This 4626 mRNA co-migrated exactly with the 5300-nucleotide mRNA 4250recognized by the nematode probe (Fig. 1; compared lanes 3 and 4 ) . The coding information for the myosin I 1 heavy chain appeared to be localized to this 11-kb Xba fragment, as the 3.92400 remainder of the insert DNA in X4.13 and X4.8 to the right of this 11-kb Xba fragment did not hybridize to the 5300*Oo0 1800 nucleotide mRNA (data notshown). Therefore, we subcloned 3.2this 11-kb Xba fragment in the plasmid PUC12 and determined a more detailed restriction enzyme map (Fig. 2, part B). We found an exact match between a region of DNA sequence in phage clone X4 13 and a 58-amino acid residue sequence previously determined by protein chemical sequencing of a carboxyl-terminal, chymotryptic peptide of the myosin I 1 heavy chain (see Ref. 29 and Fig. 4). To identify this region of the gene, we prepared a 20-bp mixed oligonucleotide probe which contained all the possible codon permutations for a portion of this peptide sequence (His-GluLys-Asn-Lys-Gln-Leu). The probe was a mixture of 128 different sequences (CA(T/C)GA(A/G)AA(A/G)AA(T/C)AAFIG. 1. Hybridization of the nematode myosin gene probe (A/G)CA(A/G)(T/C)T) and encoded the first 6 amino acids to Acanthamoeba DNA and RNA. Lune 1, Southern blot of and the first 2 base pairs of the Leu codon. To locate this BarnHI-digested amoeba genomic DNA (2 pg, fractionated on 0.85% sequence within the gene, the oligomers were end-labeled and agarose) probed with the 2.8-kb nematode probe (see “Methods”). The numbers indicate the sizes of the hybridizing amoeba DNA hybridized to restriction enzyme digests of the 11-kb Xba fragments in kilobases, which were determined from the migration of fragment subcloned in PUC12. Two small overlapping restricHindIII-digested phage X (not shown). The 3.9- and 3.2-kb bands tion enzyme fragments, a 1.9-kb KpnlSstII fragment and a were clearly visible in longer autoradiographic exposures. Lanes 2A 1.3-kb SstI fragment, hybridized with the mixedoligomer and 2B, Southern blots of BarnHI-digested amoeba genomic DNA (2 (data notshown). The position of these two fragments within p g ) (2A) and BarnHI-digested genomic clone X4.13 (25 ng) (2B) probed with the 2.8-kb nematode probe. Lune 3, Northern blot of genomic clone X4-13 is shown in Fig. 2, part B. Fig. 4 shows amoeba polyadenylated RNA (6 pg) probed with the 2.8-kb nematode the DNA sequence obtained from the 5‘ end of the 1.3-kb probe. The four marker RNA species (arrows) were electrophoresed SstI fragment (277 bp). Written below the nucleic acid sein an adjacent lane and are chicken 28 S RNA (4626 nucleotides), quence is the amino acid sequence deduced from one of the the 2400 and 2000 nucleotide pieces of amoeba 26 S RNA, and a fused three possible reading frames (starting a t nucleotide 57). doublet of chicken and amoeba 18 S RNA (1800 nucleotides). Lane Written below this deduced amino acid sequence, starting a t 4, Northern blot of amoeba polyadenylated RNA (same DBM strip as lane 3, after regeneration) probed with the 11-kb Xba fragment nucleotide 60, is the 58-amino acid residue sequence from the myosin I 1 carboxyl-terminal peptide (29). As can be seen, the from amoeba genomic clone X4.13. peptide sequence and the sequence deduced from the DNA tion mapping of the seven phage clones which were positive match exactly. We find, as suspected from the peptide sefor this reactive thiol probe yielded 2 groups of overlapping quencing work (29), an additional 8 amino acid residues at phage (three inone group, two in the other) andtwo individ- the carboxyl terminus followed by a terminationcodon (TAA). ual, non-overlapping phage. The group containing three mem- These results strongly support the conclusion that X4.13 bers and spanningroughly 19 kb of DNA has been identified contains a myosin I 1 heavy chain gene, identify the 3’ end of as containing an amoeba myosin I 1 heavy chain gene based the heavy chain polypeptide coding sequence, and provide on the observations described below. Fig. 2 (part A ) shows the transcriptional orientation. Approximately 7 kb of DNA exrestriction enzyme maps of these three overlapping phage ists in the5’ direction in the phage clone group to encode the clones (X2.12, X4.8, and X4 13). Fig. 1 (lanes 2A and 2B) balance of the 5300-nucleotide myosin I 1 mRNA (see Fig. 2, shows that phage clone X4 13 and amoeba genomic DNA parts A and B ) . To gain insight into the structureof the gene, we sought to share two co-migrating BamHI fragments (8.6 and 3.9 kb) identify within the genomic clone a DNA sequence encoding which hybridize with the nematode probe. Amoeba genomic clone X4.13 was tentatively identified as a recognizable region of the myosin I 1 globular head. Again, containing a myosin I 1 heavy chain gene by hybrid selection we made use of a portion of the nematode myosin gene as a analysis (Fig. 3). Comparison of the [35S]methionine-labeled heterologous probe to identify such as a sequence. The probe
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FIG. 2. Restriction enzyme maps of Acanthamoeba genomic clones. Part A , amoebagenomic phage clones X2.12, h4.8, and h4.13 were mapped with BanHI, EcoRI, and Xba. The left and right arms of the phage vector are designated LA and RA, respectively, and both contain anXba site adjacent to the insert.Part B, the 5' 11-kb Xba fragment of h4.13 was subcloned in PUC12 and mapped with BglII, Cla, Kpn, Pst, and SstII. The SstI sites (in parentheses)were not mapped throughout the sequence. The 1.9-kb KpnlSstII and 1.3-kb SstI fragments which hybridized with the mixed oligonucleotide probe are bracketed. The 5' end of the 1.3-kb SstI fragment yielded the sequence described in Fig. 4. The 2.6-kb SstII fragment described in the legend to Fig. 7 overlaps the 1.3-kb SstI fragment in the 5' direction. The thick arrow indicates theposition of the termination codon. Part C, the 5' 3.9-kb BamHI fragment of the Puc 12 subclone in part B was subcloned in Puc 12 and mapped with Nco, Sal, and SstI. The MboI sites (in parentheses)were not mapped throughout the sequence. The two contiguous SstI fragments (450 and 391 bp) which hybridized with the354-bp PstlAuaII nematode probe are bracketed. The arrows in parts B and C indicate the direction and extent of the areaswhich were sequenced. The restrictionenzyme maps above are written left to right in the 5' to 3' direction relative to the orientation of the myosin I1 heavy chain gene.
was a 354-bp PstlAvaII fragment which encodes amino acid binding site andobserved two contiguous SstI fragments (450 and 391 bp) which hybridized strongly with the probe (data residues 115 through 218 (3). This portion of the primary sequence falls within the amino-terminal 11%of the nema- not shown).Fig. 2 (part C) indicates the position of these two contiguous SstI fragments within the gene. Fig. 5 shows the tode heavy chain polypeptide. This nematode sequence also shows 60% exact homology witha rabbitskeletal muscle nucleotide sequence of these fragments and the myosin I1 amino acid sequence encoded by this region, which was demyosin sequence which is similarly positioned close to the amino terminus(30). Furthermore, thispolypeptide region in duced by homology with the nematode myosin sequence (Fig. boththenematodeandrabbit myosins is thoughtto be 6). The deduced myosin I1 amino acid sequence shows a 58% involved in the bindingof ATP, as determinedby conforma- exact match with residues 91 through 300 of the nematode tional analysis of the sequence (3, 30), sequence homology myosin and a 58% exact match with residues 92 through 301 of the rabbit skeletal muscle myosin (Fig. 6). Taking into with otherATP-bindingproteins(3),and chemicalcrosslinking with nucleotide analogs (31-33). We probed an SstI consideration conservative amino acid changes (34), the hodigest of a PUC12 subclone containing the 5' 3.9-kb BamHI mology is 74% with the nematode myosin and 70% with the fragment derived from genomic clone X4.13 (see Fig. 2, part rabbit muscle myosin. Thealignment of these sequences requires only a single correction, which is a gap of 11 residues C) with this nematode fragment containing the putative ATP
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6 AACTCGGCCCTCGAGTCCGACAAGCAGATCCTCGAGGACGAGATCGGCGACCTC~~ N S A L E S D K O I L E D E I G D L H N S A L E S D K O I L E D E I G D L H
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FIG. 4. The nucleotide sequence encoding the carboxyl terminus of the myosin I1 heavy chain. Top line, the nucleotide sequence determined from the 5' end of the 1.3-kb SstI fragment (see Fig. 2, part B, for the orientation of this sequence within genomic clone X4.13). The sequencing of both strands was accomplished by 67 + directed sequencing from the 5' end SstI site (1-310 bases), synthesizing a 17-bp oligonucleotide which is the compliment of nucleotides 294-310, and using this oligonucleotide as the primer for sequencing ".. the template with the opposite orientation. Middle line, the amino acid sequence deduced from one of the three possible reading frames "(starting at nucleotide 57). Bottom line, the amino acid sequence of the myosin I1 carboxyl-terminal peptide,sequenced previously by protein chemical methods (startinga t nucleotide 60). The numbering to theleft refers to nucleotides. The numbers beneath the bottom line FIG. 3. Hybrid selection analysis of amoeba genomic clone denote the 58-amino acid residue sequence of the carboxyl-terminal X4.13. Lanes 1-3, autoradiogram of an SDS-polyacrylamide gel (7%) myosin I1 peptide. The standard single letter amino acid code is of the ["S]methionine-labeled proteins synthesizedin vitro from total positioned in the middle of the corresponding triplet codon. The 3 amoeba polyadenylated RNA (lune I ) , mRNA selected by X2001 phosphorylatable serines are a t positions 46, 51, and 56 (29). Also vector DNA (lune 2), and mRNA selected by phage cloneX4.13 DNA shown are the position of the SstII site in the overlapping 1.9-kb (lune3 ) .Lunes 4 and 5,autoradiograms of the [35S]methionine-labeled Kpn/SstII fragment (see text), the termination codon (boxed),and proteins immunoprecipitated from the X4.13 DNA-selected material the genesequencewhich is complimentary to the oligonucleotide using a polyclonal antiserum to myosin I1 (lane 4 ) or non-immune probe (underlined).We have tentatively identified by nuclease S-1 serum (lune 5).Lane 6, autoradiogram of the [35S]methionine-labeled mapping a splice site a t nucleotide 56 (data notshown). The assignproteins immunoprecipitated from the X2001 vector DNA-selected ment of this putative intronsequence as well as the3' nontranslated material using the polyclonal antiserum to myosin 11. Lanes 2 and 3 sequences is preliminary. are 6-h exposures while lunes 4-6 are 48-h exposures. The position where the Coomassie Blue-stained,authentic 185-kDamyosin I1 ( M I I ) heavy chain polypeptide migrated in an adjacent lane is indi- this region of the gene, which contains the bulk of the heavy cated. The M, standards are: skeletal muscle myosin heavy chain, chain coding information, is certainly less than 10% intron DNA. The 841-bp myosin I1 sequence shown in Fig. 5 is 200 kDa; @ galactosidase, 116 kDa; phosphorylase b, 94 kDa; bovine serum albumin, 67 kDa; and actin, 42 kDa. interrupted, nevertheless, by two small apparent intronsof 70
94-
in the myosin I1 sequence relative to both the nematode and rabbit sequences (Fig. 6). This gap falls at the site where trypsin cleaves the rabbit myosin head into the23-kDa aminoterminal peptide and the50-kDa central peptide (30). Assuming that the amoeba myosin I1 sequence shown in Fig. 6 is positioned within the myosin I1 heavy chain polypeptide in an analogous fashion to the position of the homologous sequences in nematode and rabbit myosins (ie. within the amino-terminal 5-15% of the polypeptide), then these results, in combination with our previous identification of the gene region encoding the myosin I1 carboxyl-terminus, indicate that themyosin I1 genomic clone contains at least 90% of the coding information for the 185-kDa heavy chain polypeptide. This conclusion is supported by the recent finding that the glutamate a t position 95 in the myosin I1 sequence in Fig. 6 is cross-linked to a UTP analog by photoaffinity labeling of an enzymatically active, amino-terminal 68-kDa proteolytic fragment of the myosin I1 heavy chain (35). The position within the gene of the 841-bp nucleotide sequence shown in Fig. 5 indicatesthat thebulk of the myosin I1 heavy chain gene must containvery little intron DNA. The 5' end of the nucleotide sequence in Fig. 5 falls within genomic clone X4.13 approximately 4800 bp 5' of the identified termination codon (see Fig. 2). This 4800-bp region should contain approximately 90% of the myosin I1 heavy chain coding sequence, which would require -4400 bp.' Therefore,
bp (nucleotides 73 through 142) and 177 bp (nucleotides 327 through 504). These introns were identified because (i) they interrupt a contiguous stretch of myosin I1 amino acid sequence which is highly homologous to the nematode and rabbit myosin sequences, (ii)they have 5' donor and 3' acceptor splice sites which conform to the GT . . . AG rule (36), and (iii) they containstrong runsof pyrimidines just 5' of the splice acceptor site, which are seen in typical introns within higher eukaryotic genes (36). We probed restriction enzyme digests of Acanthamoeba genomic DNA with a portion of phage clone X4.13 to look for evidence of additional highly related sequences within the amoeba genome. The probe used was the 1.3-kb SstI fragment (see Fig. 2, part B ) , which contains the coding information for the carboxyl-terminal -5% of the myosin I1 heavy chain and about 1 kb of 3' flanking DNA. Fig. 7 shows Southern blots of co-electrophoresed amoeba genomic DNA (A) and 14.13 DNA ( B ) which were digested with BamHI (l), Kpn (2), Xba (3), BglII (4), and Pst (5), probed with the 1.3-kb SstI fragment, and washed to reveal related sequences. In each case the genomic DNA contains, in addition to a single band co-migrating with X4.13 DNA, either one (BamHI,Xba, and Pst) or two (Kpn and BglII) additional bands not present in the X4.13 digest. The signal intensities of these additional bands are equivalent to the bands shared by the genomic DNA and X4.13 DNA samples. Qualitatively similar results were obtained using as a probe the 2.6-kb SstII fragment (see
Nan-muscle Myosin
1954
Acanthamoeba Gene from
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TATTGAGCTCAAATTGCTTGCATGGTAACTGACGAATTTTTTCTTATATTTTTTTAACCCAC~TGGTCAGTCGGGAGCTGGT~AGACGGAG~AC~CGA~GAAGGTC~TC55o G E S G A G K T E N T K K V I CAGTACCTGACGGCCATCGCCGGTCGCGCTGAGGGCGGTCTGCTGGAGCAGCAGCTGCTCGAGTTC~ACCCGATCCTCG~GGCCTTCGGTAACGCC~AGACGACC~~G~A66o O V L T A I A G R A E C G L L E O O L L E F N P I L E A F G N ~ K T T K N CAACAACTCGTCGCCTTTCGGTAAGTTCATCGAGCTGCAGTTCA~CGCCGGCGGTC~GATCACGGGCGCCAACACGTTCATCT~CCTGCTCGAGAAGTCGCGCGTGACCG11o N N S S R F G K F I E L O F N A G G O I T G A N T F I V L L E K S R V T s a lI CCCAGGCTGCCGGCGAGCGTAACTTCCACATCTTCTACCAGATCCTGTCCAAGGCCATGCCCGAGGAGCTC A O C A G E R N F H I F Y O I L S K A M P E E L
FIG. 5. Nucleotide sequence and deduced amino acid sequence of the putative ATP binding site region of amoeba myosin 11. The two contiguous SstI fragments (450 and 391 bp) which hybridized with the nematode ATP binding site probe were sequenced as described under "Methods" and in Fig. 2 (part C). The amino acid sequence was deduced by homology with the aminoacid sequence of the nematode unc-54myosin (see Fig. 6). The three SstI sites and the positions of the 2 apparent introns are shown. The 5' donor (GT) and 3' acceptor (AG) splice sites are boxed and the characteristic runof pyrimidines 5' of the acceptor sites areunderlined.
NLSFLNDASWL~NLRSRVAAMLIVTYSGLFCWVINPYKRLPIVTDSCARMFMGKRKTEMPP~LFAVSDEAYRNMLODHENOSMLITGESGAGKTENTKKVIC
FIG. 6. Comparison of the putative ATP binding site regions in amoeba myosin 11, nematode unc-54 myosin, and rabbit skeletal muscle myosin. Shown is a comparison of the myosin I1 amino acid sequence (deduced from thenucleotide sequence in Fig. 5 ) with the nematode (3) and rabbit myosin sequences (Ref. 30 and Footnote 3). The numbering for the nematode and rabbit myosins refers to residue 1 as the amino terminusof the proteins. The numbering of the myosin I1 sequence is arbitrary. Alignment of the three sequences required only one correction, an 11-residue gap in the myosin I1 sequence (dashes). Bars indicate an exact match and colons indicate a conservative amino acid change. The per cent homology presented in the text was calculated based on the total numberof possible matches, andwas therefore correctedfor the 11-residue gap in themyosin I1 sequence.
Fig. 2, part B ) from X4.13, which probably encodes most of the myosin I1 coiled-coil tail sequence (data not shown). At present we do not know if these additional sequences represent an additional amoeba myosin heavy chain gene(s), althoughthesignalstrengths of theseadditional sequences indicate that they are highly related to theX4.13 probes used. It shouldbe possible toisolatethese sequences from the amoeba genomic library and determine if they contain additional myosin heavy chain genes. DISCUSSION
The results reported here indicate that, a t least in the case from asarcomeric of Acanthamoeba, aheterologousprobe myosin gene (nematode) will identify a non-muscle myosin M. Elzinga, personal communication.
gene. Nguyen et al. (6) found, on the other hand, that a rat embryonic skeletal muscle myosin cDNA clone, which contains coding sequencefor the carboxyl terminus of the myosin coiled-coil tail, hybridized to all sarcomericmyosin heavy chain mRNAs tested, butfailed to detect non-muscle myosin heavy chainmRNAsand genes. Similarly, we found that fragments of the nematode gene encoding portions of the coiled-coil tail sequence failed to detect discrete amoeba DNA sequences (data not shown). While our results and those of Nguyen et al. (6) cannot be compareddirectly, the results taken together suggest that myosin gene fragments encoding portions of the globular head where the enzymatic active sites reside (like those fragments used in the present study) may be moreuseful as heterologous myosin probes than gene fragments encoding portions of the coiled-coil tail. This interpretation is supportedby a comparison of the nematode unc-
Non-muscle MyosinGene from Acanthamoeba
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FIG. 7. Acantharnoeba genomic DNA probed with a portion of phage clone X4.13 reveals additional highly related sequences. Amoeba genomic DNA (1.25 pg) ( A ) and X4.13 DNA (25 ng) ( B ) were digested to completion with BarnHI ( l ) , Kpn (2), Xba (3), BglII (4), and Pst (5),co-electrophoresed on 0.85% agarose, and a Southern blot probed with the 1.3-kb SstI fragment from X4.13 (see Fig. 2, part B). The blots were washed once with 1 X SSPE, 0.1% SDS (55 "C),twice with 0.25 X SSPE, 0.1% SDS (55 "C), and twice with 0.1 X SSPE, 0.1% SDS (60 "C). Autoradiograms of blots following the washes with 0.25 X SSPE, 0.1% SDS (55 "C) did not reveal any significant bands in the genomic DNAlanes in addition to those shown. The autoradiogram exposures were 4 h for X4.13 DNA lanes and 16 h for genomic DNA lanes. The 6-kb Pst fragment in lane 5B arises from a Pst site -1.2 kb 3' of the 11-kb Xba fragment shown in Fig. 2, part B (data not shown). All the remaining bands in the X4.13 DNA digests can be accounted for by the restriction enzyme sites provided in Fig. 2.
54 myosin heavy chain protein sequence with that of a second nematode sarcomeric myosin and with rabbit skeletal muscle myosin (3), which indicates that thecoiled-coil tail sequences are much less conserved than are the globular head sequences. This may reflect (i) a strong conservation of sequences which fold to form the geometrically correct binding and catalytic sites in the myosin globular head and (ii) a greater tolerance within the coiled-coil tail sequence for amino acid changes, so long as the chemical nature of the rod sequence is preserved (ie. the position of hydrophobic, charged, and skip residues) (3,37). However, because nucleotide sequence conservation is
not required for protein sequence conservation, the feasibility of using a particular heterologous myosin gene probe should be tested first in experiments similar to those described in Fig. 1. A clear understanding of the relatedness of sarcomeric and non-sarcomeric myosin heavy chain sequences must await the complete sequencing of at least several non-muscle myosin genes. Only 7 of the 40 purified phage selected initially from the amoeba genomic library using the 2.8-kb nematode probe were positive with the 490-bp reactive thiol site nematode probe. This region of the myosin primary sequence is highly
1956
Non-muscle Myosin
Gene from Acanthamoeba
conserved between rabbit and nematode myosins (3, 28) and may represent a "myosin-specific" sequence. The three overlapping amoebagenomic clones containing a myosin I1 heavy chain gene were all thiol-positive. In contrast, 10 randomly chosen phageclones selectedusing the2.8-kb nematode probe, but negative for the reactive thiol site nematode probe, overlapped and hybrid selected an amoeba mRNA encoding a 107kDa protein (data not shown). While the identity of this protein is unknown, it is clearly smaller than any known amoeba myosinheavy chain polypeptide. Apparently, this gene was selected by homology with a portion of the 2.8-kb nematode probe other than the reactive thiolsite region. These results again illustrate the advantage carefully in choosing the heterologous probe. The hybrid selection results essentially rule out thepossibility that X4- 13 encodes a myosin IA or IB heavy chain, since thesemolecules possess much smallerheavy chains than myosin I1 and since antibodies tomyosin I1 and myosin I do not cross-react (15). We still cannot be absolutely certain, however, that phage clone X4.13 encodes the specific myosin I1 heavy chain polypeptide previously isolated and sequenced (29). While the exact match between the DNA and peptide sequences in Fig. 4 makes this quite probable, the fact that the amoeba maycontain more than onemyosin I1 heavy chain gene (as suggested by the results in Fig. 7) raises the possibility that X4.13 might encodea closely related isoform of the purified, sequenced myosin I1 polypeptide. We also cannot be certain that themyosin I1 gene contained in phageclone X4. 13 isa transcriptionally activegene. The open reading frames so far sequenced, the normal splice signals in thetwo identified introns, and the exact match between the nucleotide sequence and the myosin I1 peptide sequence allstrongly suggest, however, that this is a functional gene. We estimate that theregion of the gene encoding approximately 90% of the heavy chain polypeptide contains less than 10% intron DNA (ie. less than -500 bp). This observation contrasts sharply with the structureof vertebrate sarcomeric myosingenes,where the analogous region of the genes is interrupted by numerous large introns which in most cases total in excess of 10-15 kb of DNA (38-40). In general, the genes of simple eukaryotes like Acanthmoeba (whose genome complexity is -1/100 that of humans (25)) containfewer and smaller introns than their counterparts in vertebrates (41). Nevertheless, we did identify two small apparent introns of 70 and 177 bp within the myosin I1 DNA sequence in Fig. 5. These introns are similar in structure to typical introns of higher eukaryotes (36) and to the single intron in an Acanthamoeba actin gene (42). The 70-bp intron which splits the myosin I1 sequence in Fig. 5 interrupts the coding sequence in exactly the same location that the nematode unc-54, rat a cardiac, and rat embryonic myosin heavy chain genes are split by an intron (3, 38). Recently, Strehleret al. (38) reported that the intron positions in the 5' end region of these latter 3 myosin heavy chain genes are conserved. Those authorsconcluded that this structural feature may be important in myosin gene expression orregulation and that theconserved interruptions of the myosin heavy chain genes suggest the existence of a highly split ancestral myosin gene from which difference lineages
removed and/or added specific sets of introns (38).The data reported heresuggest that this conservation of intron position inthe 5' end region of the gene mayalso existinthis cytoplasmic myosin gene from amoeba. Acknowledgments-J. H. thanks Rob Horlick, Rudi Billeter, and Anne Seiler-Tuyns for their helpful advice during this work. J. H. also thanks in particular Carl Schmidt for his excellent advice on technical matters and his many helpful discussions during the course of this work. J. H. thanks GrahamC6t6formaking available his protein sequence data prior to publication. REFERENCES 1. Korn, E. D. (1978) Proc. Nutl. Acud. Sci. U. S. A. 75,588-599 2. Pollard, T. D. (1982) Methods Cell Biol. 24, 333-371 3. Karn, J., Brenner, S., and Barnett, L. (1983) Proc. Natl. Acad. Sci. U. S. A. 80, 4253-4257 4. Mahdavi, V., Periasamy, M., and Nadal-Ginard, B. (1982) Nature 2 9 7 , 659-664 5. Leinwand, L. A., Saez, L., McNally, E., and Nadal-Gindard,B. (1983) Proc. Natl. Acud. Sci. U. S. A . 80,3716-3720 6. Nguyen H. T. Gubits, R.M., Wydro R. M., and Nadal-Ginard, B. (1982) Proc. Natl. &ad. Sei. ti.S. A . 79,5230-5234 7. Sinha, A. M., Umeda, P. K., Kavinsky, C. J., Rajamanickam, C., Hsu, H. J., Jakovcic, S., and Rabinowitz,M. (1982) Proc. Natl. Acad. Sci. U, S. A. 79,5847-5851 8. Kavinsky, C. J., Umeda, P. K., Sinha, A. M., Elzinga, M., Tong, S. W., Zak, R., Jakovcic, S., and Rabinowitz, M. (1983) J. B i d . Chem. 2 5 8 , 51~ ~ - 5 7 n 5 """ " l "
9. Freyer, G. A., and Robbins, J. (1983) J. Biol. Chem. 258, 7149-7154 A. Q. (1982) Arch. Biochem. Bwphys. 2 1 7 , 710-720 11. Weydert, A,, Daubas, P., Caravatti, M., Minty, A,, Bugaisky, G., Cohen, A,, Robert, B., and Buckingham, M. (1983) J. Bid. Chem. 258,13867-13874 12. Bernstein, S. I., Mogami, K., Donady, J. J., and Emerson, C. P., Jr. (1983) Nature 302,393-397 13. Rosek, C. E., and Davidson, N. (1983) Cell 32,23-24 14. Karn, J., Brenner, S., Barnett, L., and Cesareni,G. (1980) Proc. Natl.Acud. Sci. U. S. A . 77,5172-5176 15. Hammer, J. A., 111, Korn, E. D., and Paterson, B. M. (1985) J. Biol. Chem. 259, 11157-11159 16. Maniatis, T., Fritsch, E. F., and Sambrook, J. (1982) Molecular Clonin A Laboratory Manual,Cold Spring Harbor Laboratory, Cold Spring Harkor, 10. Jakowlew, S. B., and Siddiqui, M.
NV
17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42.
Pateison, B. M., and Eldridge, J. D. (1984) Science 224,1436-1438 Southern, E. M. (1975) J. Mol. Biol. 98,503-517 Southern, E. (1979) Methods Enzymol. 68, 152-176 Wahl, G. M., Stern, M., and Stark, C. R. (1979) Proc. Natl. Acad.Sei. U. S. A. 76, 3683-3687 McMaster. G. K.. and Carmichael. G. G. (1977) Proc. Natl. Acud.Sei. U. S. A. '74,4835-4838 Miller, J. S., Roberts, B. E., and Paterson, B. M. (1982) in Genetic En i neering-Principles and Methods (Setlow, J. K., and Hollaender, A,,e b i Vol. 4,103-117, PlenumPress, New York Miyoshi, Miyake, T., Hozumi, T., and Itakura, K. (1980) Nueleic Acids Res. 8,5461-5471 Seed, B., Parker, R. C., and Davidson, N. (1982) Gene (Amst.) 19,201-209 Bohnert, H. J., and Herrman, R. G. (1974) Eur. J. Biochem. 50,83-90 Benton, W. D., and Davis, R. W. (1977) Science 196,180-182 Sanger, F., Nicklen, S., and Coulson, A. R. (1977) Proc. Nutl. Acud. Sci. U. S. A . 74,5463-5467 Elzinga, M., and Collins, J. H. (1977) Proc. Natl. Acad. Sci. U. S. A . 7 4 , 4281-4284 C6t6, G . P., Robinson, E. A,, Appella, E., and Korn, E. D. (1984) J . Biol. Chem. 259, 12781-12787 Tong, S. W., and Elzinga, M. (1983) J. Biol. Chem. 2 5 8 , 13100-13110 Szilagyi, L., Balint, M., Streter, F. A,, and Gergely, J. (1979) Biochem. Biophys. Res. Commun. 87,936-945 Okamoto, Y.,and Yount, R. G. (1985) Proc. Nutl. Acad. Sci. U. S. A. 82, 1575-1579 Mahmood, R., and Yount, R. G . (1984) J. Biol. Chem. 259, 12956-12959 Dayhoff, M. 0. (1972) Atlas of Protein Sequence und Structure, National Biomedical Research Foundation, Washington, D. C. E., and Korn, E. D. (1985) J . Atkinson. M. A. L.. Robinson, E.A,.. Amella, .. Biol. Chem. in press Mount, S. M. '(1982) Nucleic Acids Res..10,459~472 McLachlan, A. D. (1984) Annu. Reo. Bcophys. Baoeng. 13,167-189 Strehler. E. E., Mahdavi. V., Periasamv, M., and Nadal-Ginard, B. (1985) J. Bid. Chem. 260,468-471 Friedman, D. J., Umeda, P. K., Sinha, A. M.,,Hsu, H. J., Jakovcic,S., and Rabinowitz, M. (1984) Proc. Nutl. Acud. Sa: U. S. A. 8 1 , 3044-3048 Mahdavi, V., Chambers, A. P., and Nadal-Glnard, B. (1984) Proc. Natl. U. S. A . 8 1 , 2626-2630 Acud. SCL. Gilbert, W. (1985) Science 228,823-824 Nellen, W., and Gallwitz, D. (1982) J. Mol. Biol. 159, 1-18
