Aug 5, 2016 - Jeffrey Boone Miller$, Stephanie B. Teal, and Frank E. StockdaleQ. From the ...... Namsawa, M., Fitzsimmons, R. B., Izumo, S., Nadal-Ginard, B.,.
THEJOURNAL OF BIOLOGICAL CHEMISTRY
Vol. 264, No. 22, Issue of August 5, pp. 13122-13130 1989 Printed in d.S.A.
0 1989 by The American Society for Biochemistry and Molecular Biology, Inc.
Evolutionarily Conserved Sequencesof Striated Muscle Myosin Heavy Chain Isoforms EPITOPE MAPPING BY cDNA EXPRESSION* (Received for publication, September 12, 1988, and in revised form April 21, 1989)
Jeffrey Boone Miller$, Stephanie B. Teal, and Frank E. StockdaleQ From the Department of Medicine, Stanford University School of Medicine, Stanford, California94305-5306
A cDNA expression strategy was used to localize amino acid sequences which were specific for fast, as opposed to slow,isoforms of the chicken skeletal muscle myosin heavy chain (MHC) and which were conserved in vertebrate evolution. Five monoclonal antibodies (mAbs), termed FlS, F27, F30, F47, and F59, were prepared that reacted with all of the known chicken fast MHC isoforms but did not react with any of the known chicken slow nor with smooth muscle MHC isoforms. The epitopes recognized by mAbsFlS, F30, F47, and F59 were on the globular head fragment of the MHC, whereas the epitope recognized by mAb F27 was on the helical tail orrod fragment. Reactivity of all fivemAbs also was confined to fast MHCs in the rat, with the exception of mAb F59, which also reacted with the@-cardiacMHC, the single slow MHC isoform common to both the rat heart and skeletal muscle. None of the five epitopes was expressed on amphioxus, nematode, or Dictyostelium MHC. The F27 and F59 epitopes were found on shark, electric ray, goldfish, newt,frog,turtle, chicken, quail, rabbit,and rat MHCs. The epitopes recognized by these mAbs were conserved, therefore, to varying degrees through vertebrate evolution and differed in sequence from homologous regions of a number of invertebrate MHCs and myosin-like proteins. The sequence of those epitopes on the head were mapped using a two-part cDNA expression strategy. First, Bd3 1 exonuclease digesof a tion was used to rapidlygeneratefragments chicken embryonic fast MHC cDNAthat were progressively deleted from the 3’ end. These cDNA fragments were expressed as @-galactosidase/MHCfusion proteins using the pUR290 vector; the fusion proteins were tested by immunoblotting for reactivity with the mAbs; and the approximate locations of the epitopes were determined from the sizes of the cDNA fragments that encoded a particular epitope. The epitopes were then precisely mapped by expression of overlapping cDNA fragments of known sequence that covered the approximate location of the epitopes. With this method, the epitope recognized by mAb F59 was mapped to amino acids 2 11-23 1 of the chicken embryonic fast * This work was supported by grants from the National Institutes of Health and theMuscular Dystrophy Association of America. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. This paper is dedicated to thememory of Dr. James A. Magnuson of the Washington State University. $ Present address: Day Neuromuscular Research Center, Massachusetts General Hospital-East, Charlestown, MA02129, and Program in Neuroscience, Harvard Medical School, Boston, MA 02115. 5 To whom correspondence should be addressed.
MHC and the three distinct epitopes recognized by mAbs F18, F30, and F47 weremapped to amino acids -65-92. Each of these epitope sequences is at or near the ATPase active site.
The myosin heavy chain (MHC)’ is a large (-220 kDa), evolutionarily conserved protein that is required for many types of cell motility, ranging from slime mold cytokinesis to skeletal muscle contraction (reviewed by Emerson and Bernstein, 1987; Warrick and Spudich, 1987). Vertebrate organisms generally have multiple genes encoding distinct MHC isoforms that areexpressed in cell-specific and developmental stage-specific patterns (Fallon and Nachmias, 1980; Bader et al., 1982; Kavinsky et al., 1983; Bandman, 1985; Izumo et al., 1986; Robbins et al., 1986; Emerson and Bernstein, 1987; Stockdale and Miller, 1987). In birds and mammals, for example, multiple genes encode the cytoskeletal, smooth muscle, and multiple fast andslow, skeletal muscle and cardiac MHC isoforms. These MHCshave several structural andfunctional domains which have been evolutionarily conserved. The two major structural domains of MHC arethe globular head (Subfragment 1or Sl),and thehelical tail orrod. The ATPase active site, the light chain-binding sites,and theactin-binding site are functional domains found on S1, whereas the sites for dimerization and thick filament assembly are found on the rod. Many structural aspects of MHC functional domains are poorly understood. Though much work, including the determination of MHC amino acid sequences, has been directed toward understanding structural features involved in the different functions carried out by the MHC protein (Bernstein and Emerson, 1987; Warrick and Spudich, 1987), comparatively little attention has been given to structural features that distinguish among the different MHC isoforms and that may be responsible for their distinctfunctional properties. Structural differences among MHC isoforms may account for the differences in thick filament assembly, sarcomere structure, regulatory mechanisms, and ATPase activities found in myosins from differentanimals and different muscle cell types(Squire, 1986). Our interest has focused on the enzymatic and structural differences that distinguish fastand slow MHC isoforms expressed in chicken skeletal muscle. For skeletal muscle myosins, the ATPase active site is on the S1 (head) of the MHC (Wagner and Giniger, 1981; Sivramakrishnan and Burke, 1982), the ATPase activities of fast muscle isoforms
* The abbreviations used are: MHC, myosin heavy chain; mAbs, monoclonal antibodies; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; kb, kilobase; IPTG, isopropyl-l-thio@-D-galactopyranoside.
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Structure Myosin Isoform
13123
control of the laCZ operator and promoter, and a multiple cloning site (each vector has one of the three possible reading frames) at the 3’ end of the lncZ coding sequence (Fig. 1).A cDNA encoding most of the S1 of the chicken embryonic fast MHC (Fig. 1) was a gift from Dr. Jeffrey Robbins (University of Cincinnati). The 2.94-kb MHC cDNA wascloned in the PstIsite of pUC18 and encoded amino acids 34-1014 out of the 1940 amino acids that compose the entire chicken embryonic fast MHC (Molina et al., 1987). Escherichia coli strain DGlOl (thi-I, hsdRl7, endA44, supE44, h Z q , lncZAM15) was used as host. Generation and Subcloning of MHC cDNA Fragments-The 2.94kb PstI-PstI fragment of MHC cDNA and a 2.13-kb PstI-Hind111 subfragment of the MHC cDNA (encoding amino acids 34-745, Fig. 1) were prepared by digestion of the parent plasmid and separated from the pUC18 in a 0.9%, a low-gelling temperature agarose gel (Seaplaque, FMC Bioproducts, Rockland, ME). The band containing the MHC cDNA was excised, the agarose was melted, and thecDNA was ligated into PstIdigested, or PstI andHindIII digested, pUR290, pUR291, or pUR292 using the method of Struhl (1985). Expression of a /3-galactosidaselMHC fusion protein carrying the F59 epitope was obtained only with the cDNA fragments ligated into pUR290. A pUR290 plasmid carrying a 1.4-kb PstI-BglII fragment of the MHC cDNA (encoding amino acids 34-468, Fig. 1)was prepared by digesting at the unique BglII site (nucleotide 1407 of the coding region of the cDNA, Molina et al., 1987) found in the recombinant pUR290 plasmid carrying the PstI-HindIII cDNA fragment, treated with the Klenow fragment of DNA polymerase in the presence of NTPs to prepare blunt-ends, and recircularizing by ligation with T4 DNA ligase. To generate progressively smaller MHC cDNA fragments, the pUR290 plasmid carrying the 2.13-kb PstI-Hind111 MHC cDNA insert was linearized by digesting a t the unique BglII site, treated with Ba131 exonuclease for different amounts of time (Silhavy et al., 1981) to progressively delete portions of the cDNA by digestion in both directions from the BglII site, treated with HindIII to remove the remaining 3’ ends of the cDNA in the cloning site, made bluntended by treatment with the Klenow fragment of DNA polymerase and NTPs, andrecircularized by ligation with T4 DNA ligase (ManEXPERIMENTALPROCEDURES iatis et al., 1981). This protocol resulted in a family of plasmids Monoclonal Antibodiesand Zmmunoblotting-The preparation and carrying progressively shorter pieces (depending on duration of BaZ31 properties ofmAb F59 have been described in detail (Crow and treatment) of the 5’ end of the MHC cDNA fused in the proper Stockdale, 1984; Miller et al., 1985; Miller and Stockdale, 1986a, reading frame to the 3’ end of the @-galactosidasegene of pUR290. 1986b; Crowand Stockdale, 1986a, 1986b;Schafer et al., 1987; Stock- The plasmids were usedto transform DGlOl and transformants were dale and Miller, 1987). Briefly, the epitope with whichmAb F59 selected by ampicillin resistance (Maniatis et al., 1981). Individual reacts isfound on all chicken skeletal muscle fast MHC isoforms; i.e. transformant colonies were replicate plated, grown in LB supplemAb F59 reacts with the chicken embryonic fast, neonatal fast, and mented with ampicillin, and induced with IPTG to synthesize fusion adultfast MHC isoforms as these isoforms have been defined proteins which were analyzed by immunoblotting as described below. (Whalen et al., 1981; Bandman et al., 1982; Bader et al., 1982; Lowey The inserted cDNA fragments were released for size analysis on et al., 1983), but this epitope is not found oneither of the two agarose gels by digestion of the recombinant plasmids with EcoRI. (Matsuda et al., 1982) chicken skeletal muscleslow MHCs or on One EcoRI site is -60 base pairs to the5’ side of the multiple cloning chicken smooth muscle MHC; i.e.mAbF59 does not react with site of the PUR plasmids, and thesecond EcoRI site is -30 base pairs chicken slow MHC 1, chicken slow MHC 2, chicken vascular MHC to the 3’ side of the multiple cloning site (Ruther and Muller-Hill, (Evans et al., 1988),or chicken gizzard MHC (Table I). The properties 1983, Fig. 1). of four additional mAbs, F18, F27, F30, and F47, that react with As described under Results and Table 11, other restriction fragchicken fast MHCs, but do not react with chicken slow MHCs or ments of the MHC cDNAwere prepared and made blunt-ended, smooth muscle MHCs, are described here. Hybridomas producing purified bygel electrophoresis, and sub-cloned into the EcoRI or monoclonal antibodies F18, F30, and F47 were generated as described HindIII sites (also made blunt-ended) of the PUR plasmid with the (Crow and Stockdale, 1984; Miller et al., 1985) using MHC from adult proper reading frame using the same methods as above, except that, chicken pectoral muscle as the immunogen, and mAb F27 was gen- in some cases, the cDNA fragment was purified from the agarose slice erated using MHC from the adult chicken medial adductor muscle. by silica gel adsorption as described by the manufacturer (Geneclean To test the reactivities of the mAbs against different MHCs, crude kit, Bio 101, San Diego, CA). Restriction analysis with appropriate myosin preparations were prepared by high salt extraction of muscle enzymes was used to confirm the structure of each plasmid contissues (Miller et al., 1985; Evans et al., 1988), and the S1 and rod structed for fusion protein expression. All enzymes were from New chymotryptic fragments of chicken adult fast MHC were prepared England Biohbs. (Weeds and Taylor, 1975; Crow and Stockdale, 1986a). The myosin Analysis of Transformant Plasmids and Detection of Epitope preparations were subjected to SDS-PAGE in 5% gels to separate Expression-Individual transformant colonies were inoculated into 5 isoforms (Rushbrook and Stracher, 1979; Matsuda et al., 1983; Miller ml of LB supplemented with ampicillin and 5 mM IPTG, and grown et al., 1985;Miller and Stockdale, 1986a, 1986b;Stockdale and Miller, at 37 “C for 4-6 h until the logarithmic phase of growth was reached. 1987), and the separated proteins were transferred to nitrocellulose Plasmids were prepared from 1 ml of culture using a rapid procedure paper. The transfers were incubated for 1 h with a 1:10 dilution of (Riggs and McLachlan, 19851, digested with appropriate restriction the hybridoma supernatant tobe tested, and mAb binding was visu- enzymes, and analyzed for insert size by electrophoresis in agarose alized using an alkaline phosphatase-conjugated goat anti-mouse gels. A 1-3-ml culture sample was used to prepare P-galactosidasesecondary antibody (immunoblotting grade, Bio-Rad) (Miller et al., MHC fusion proteins for analysis by SDS-PAGE in 5% gels and by 1985). immunoblotting. Bacteria incubated with IPTG were harvested by Plasmtds, cDNA, and Bacteria-The plasmids pUR290, pUR291, centrifugation, and protoplasts were prepared by the method of Weiss and pUR292 (Ruther andMuller-Hill, 1983) wereused as vectors for (1976) as modified (Miller and Amy, 1983). Protoplasts were lysed by expression of @-galactosidase/MHC fusion proteins (Miller et al., resuspension and incubation for 20 min a t 37 ‘C in 100 pl of 1 mM 1986). These -5.2-kb plasmids contain an ampicillin resistance gene, MgCL containing 1pg/ml RNase A and 1Fg/ml DNase (Sigma). The almost the entire lncZ coding sequence under the transcriptional suspensions were mixed with an equal volume of 2 X SDS sample
are several times greaterthan those of slow muscle or smooth muscle isoforms (Barany et al., 1967), and theATPase activities of the MHCs in a muscle fiber largely determine the contraction velocity of a fiber (Reiser et al., 1985; Sweeney et al., 1988). The differential expression of MHC isoforms with distinct ATPase activities and structural properties thus underlies the formation of diverse types of fast andslow skeletal muscle fibers (reviewed by Jolesz and Srkter, 1981; Kelly, 1983; Bandman, 1985; Pette andVrbovi, 1985; Whalen, 1985; Miller and Stockdale, 1987; Stockdale and Miller, 1987). Because the most highly conserved structural regions of large proteins appear to be those that are most critical for function, our approach to analysis of structural features that may determine the distinct functional properties of fast and slow MHCs was to identify MHC regions that were conserved, but were restricted to particular functional groups of MHC isoforms. To identify such regions on skeletal muscle MHCs, we prepared monoclonal antibodies (mAbs) that (i) reacted with all chicken fast MHCs, (ii) did not react with chicken slow or smooth muscle MHCs, and (iii) recognized epitopes conserved through vertebrate evolution. We determined the location of the chicken fast isoform-specific epitopes on the MHC by generating cDNA fragments encoding different regions of a chicken fast MHC isoform and by determining which of the cDNA fragments encoded the epitope when expressed in bacteria as p-galactosidaselMHC fusion proteins. The MHC structural regions identified by this analysis are likely to be important in determining the distinct functional properties of the chicken fast, as opposed to slow, MHC isoforms.
Myosin Isoform Structure
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TABLEI Specificities of monoclonal antibodies and evolutionary conseruationof epitopes Species
Reaction of MHC isoform with mAbb
MHC isoform or source'
F18
Embryonic fast Neonatal fast Adult fast Slow MHC 1 Slow MHC 2 Gizzard (smooth)
+++ +++ +++ +++ -
Quail
Adult fast
Rat
Embryonic fast Neonatal fast Type IIA fast Type IIB fast a-Cardiac P-Cardiaclslow
+++ + ++++ -
Chicken
Rabbit
Back muscle
Turtle
Biceps brachii
Newt
Thigh muscle
Frog
Pectoral muscle
Goldfish
Body muscle
Electric ray
Tail muscle
Shark
Red body wall muscle
Nematode
Body muscle
-
F21
F30
+++ +++ +++ -
-
+++
+++
+++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++
F41
F59
+++ +++ -
+ +++ +++
-
-
+++ +++ +++ -
+++ + ++++ -
+++ + +++ +++ +++ -
-
+++ +++
+++ +++ +++ +++ +
-
+++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++
Slime mold Large MHC Amphioxus Body wall muscle Individual chicken MHC isoforms were distinguished as described previously (Stockdale and Miller, 1987). For individual rat MHC isoforms, the embryonic fast isoform was prepared from embryonic day 15 rat limb buds, the neonatal isoform was from 1-week-old rat thighs, the Type IIA fast isoform was from adult soleus and adult diaphragm muscles, the Type IIB fast isoform was from adult quadriceps muscle, the a-cardiac isoform was from embryonic day 15 rat hearts and adult rat ventricles, and the p-cardiaclslow skeletal isoform was from embryonic day 15 hearts and adult rat soleus muscle. The rat 0-cardiaclslow skeletal isoform is electrophoretically separable from the a-cardiac and other rat fastMHCs allowing an unambiguous demonstration of mAb specificity in mixtures of the two isoforms such as found in the embryonic rat heart (Lompre et al., 1984). Individual MHC isoforms or MHCs from whole muscle were subjected to SDS-PAGE in 5% gels, transferred to immunoblots, and tested for reaction with each mAb. Equal amounts of myosin from each sample were analyzed and the intensity of mAb reaction with each sample was compared; +++, strong reaction; +, weak reaction; -, no reaction. buffer, boiled, and approximately one-fourth of the suspension was analyzed in each gel lane. Duplicate gels were prepared. One was stained with Coomassie Blue to detect fusion protein expression. Proteins separated in the second gel weretransferred to nitrocellulose and fusion proteins that reacted with the different mAbs were detected by immunoblotting as above. One-dimensional immunopeptide maps were performed as previously described (Schafer et al., 1987). RESULTS
rnAbs Specific for Chicken Fast Myosin Heavy Chins-A group of mAbs specific for myosin heavy chains was prepared, and immunoblotting was used to identify those mAbs that reacted with chicken skeletal muscle fast MHC isoforms but did not react with chicken skeletal muscle slow MHC isoforms. For immunoblotting, fast MHC isoforms were prepared from the adult chicken pectoral muscle, and slow MHC isoforms were prepared from the adult chicken anterior latissimus dorsi, subjected to SDS-PAGE in 5% gels, and transferred to nitrocellulose. The reaction of each mAb with fast
and slow MHC isoforms was determined by Western blotting and mAbs that reacted with fast MHC isoforms but not with the slow MHC isoforms were identified. Those mAbs that reacted with the adult chicken fast MHC but not with theadult chicken slow MHCs were further analyzed to identify the mAbs that reacted with other known fast MHCs from chicken and to determine if the epitopes recognized by the mAbs had been conserved in vertebrate evolution. Five mAbs (termed F18, F27, F30, F47, and F59) were found that reacted with the embryonic, neonatal, and adult fast MHC isoforms of chicken skeletal muscle but did not react with the chicken slow MHC 1, slow MHC 2, or smooth muscle (gizzard) MHC isoforms (Table I). Each mAb reacted strongly with each of the chicken fast MHCs, except mAb F47 which reacted more weakly with the embryonic than with the neonatal and adult fastisoforms. The specificity of mAb F59 for the different chicken skeletal muscle fast MHC isoforms was as found previously (Miller and Stockdale,
Structure Myosin Isoform
13125
frog, or newt MHCs. Though F30 did not react with the two amphibian MHCs, it did with the MHC of a reptile, the turtle. None of the five mAbs reacted with chicken smooth muscle MHC or invertebrate MHCsfrom amphioxus (Brunchiostornu californiensis), nematodes (Cuenorhabditis elegans), or slime molds (Dictyostelium). When the reactivities of the mAbs with individual rat MHC isoforms were tested, mAbs F27 and F59 reacted strongly with all known rat skeletal muscle fast MHCs (the embryonic, neonatal, adult Type IIA, adult Type IIB, and a-cardiac isoforms); mAb F47 did not react with the a-cardiac MHC but did react with varying intensities with the other rat fast MHCs; and mAbs F18 and F30 did not react with the adult Type IIA or a-cardiac isoforms but did react with varying intensities with the embryonic, neonatal, and adult Type IIB isoforms (Tables I).To determine if the epitopes were also restricted to fast MHCs in rats, themAbs were tested for reaction with the only known slow MHC in the rat, the rat @-cardiaclslowskeletal MHC isoform, which is encoded by a single gene that is expressed in both cardiac and skeletal muscle (Lompri. et al., 1984; Narusawa et al., 1987). As expected, mAbs F18, F27, F30, and F47 did not react with the rat slow isoform. Contrary to what was expected, mAb F59 did react with the rat@-cardiaclslowskeletal MHC as well as with the rat fast MHCs. The epitope recogaal aa 1940 nized by mAb F59, therefore, was restricted to fast MHCs in N- I s1 I Rod CC 1 I I chickens but was found on both the fast MHC and the one MHC protein aa 34 aa 1014 slow MHC inrats, whereas the F18, F27, F30, and F47 epitopes were found only on fast MHCs in bothchickens and I I nt 100 nt 3043 rats. Epitope Localizationto MHC Proteolytic Fragments-As MHC cDNA the first step inmapping, the epitopes recognized by the five mAbs were localized to either the S1 or rod portion of the Pst I Bgl I1 Hind In Pst I nt 1 0 0 1407 2235 3043 MHC. The reactions of the different mAbs with the -90-kDa S1 and -110-kDa rod chymotryptic fragments were determined by immunoblotting. Four of the mAbs, including F18, F30, F47, and, as shown previously (Crow and Stockdale, 1986a), F59 reacted with the S1 (Fig. 2, lanes 2 and 4-43), whereas mAb F27 reacted withthe rod (Fig. 2, lune 3). Because the head region of the S1 contains the ATPase active site, the actin-bindingsite, and other known regions of functional importance, the epitopes on the S1 recognized by mAbs F18, F30, F47, and F59 were further localized using a cDNA FIG. 1. Plasmid and MHC cDNA used for epitope localization. Amino acids (aa) 34-1014, comprising most of the S1 and a expression strategy. Epitope Mapping by cDNA Expression-The epitopes recportion of the S2 region of the MHC rod, were encoded by the 2.94kDa PstI fragment including nucleotides (nt) 100-3043 of embryonic ognized by mAbs F18, F30, F47, and F59 were encoded by the chicken MHC cDNA. Fragments of the MHC cDNA were prepared 2.94-kb PstI-PstI MHC cDNA fragment (Molinaet al., 1987) using the indicated PstI, BglII, HindIII, and other siteslisted in Table which encodes amino acids 34-1014 of the chicken embryonic 11, and were ligated in reading frame into the3’ end of the Lac2 gene carried by pUR290. The PstI ( P ) ,HindIII (H), and two EcoRI ( R ) fast MHC (Fig. 1). When grown in the presence of IPTG, sitesin the multiple cloning site of pUR290 are shown. The transformants carrying pUR290 with the PstIfragment of the PUR 290 plasmid, including the ampicillin resistant ( A p ) and Lac2 MHC gene inserted expressed a full-length @-galactosidase/ genes, is not drawn to scale. MHC fusion protein with a M, of -230 kDa (Fig. 3, lunes 1
1986a). The classification of chicken MHC isoforms as fast or slow wasbased on electrophoretic mobility in 5% gels, fiber type (ATPase histochemistry) invivo,reactivity with specific mAbs, and, when known, therate of ATPase enzymatic activity (Stockdale and Miller, 1987). The epitopes recognized by the fivemAbs specific for chicken fast MHCs were conserved to different degrees during vertebrate evolution. Myosin was prepared from different classes of vertebrates, and the reactivities of the five mAbs with the MHCs were determined by immunoblotting. As shown in Table I, mAbs F27 and F59 reacted strongly with MHCs prepared from a shark red body wall muscle, electric ray (Discopyge ommata, an elasmobranch) tail muscle, goldfish body muscle, newt thigh muscle, frog pectoral muscle, turtle biceps brachii muscle, quail pectoral muscle, rat thigh muscle, and rabbit back muscle. In contrast,mAb F47 reacted strongly withamphibian (frog and newt),reptile (turtle), avian (chicken and quail), and mammalian (rat and rabbit) MHCs, weakly with goldfish MHC, and did not react with the MHCs of two elasmobranchs (shark and electric ray); whereas mAbs F18 and F30 reacted with the avian and mammalian MHCs but not with the shark, electric ray, goldfish,
TABLE I1 Expression of MHC cDNA fragments and reaction of mAbs with @-ghhctosidase/myosinheavy chain fusion Droteins Restriction fragment
MHC nucleotides in fragment
MHC amino acids encoded
F18
Reaction of fusion protein with mAb” F30 F47 F59
PstI-StuI 100-694 34-231 +++ +++ + +++ AluI 601-837 202-279 +++ Fnu4HI 629-910 211-303 +++ PstI-BalI 100-274 34-92 +++ +++ + PstI-HDhI 34-71 100-212 - __ a Restriction fragments of embryonic chicken MHC cDNA were made by digestion with the indicated restriction endonucleases; the cDNA fragment was purified and ligated to the appropriate PUR vector; and @-galactosidase/ MHC fusion protein expression was induced as described under “ExperimentalProcedures.” Reaction of the fusion proteins with mAbs was determined by immunoblotting as in TableI and Figs. 3 and 4.
Myosin Isoform Structure
13126
1 w
2
3
4
5
6
1
2
3
4
5
6
+IPTG F18 CB
F27
F30
F47
7
r t
Rod U
i ' S1
# a n
"4
116-
CB F18 F27 F30 F47 F59 FIG.2. Location of fast MFIC mAb epitopes on S1 (head) or tail (rod) regions of the chicken myosin heavy chain.The adult fast MHC isoform was purified from the pectoral muscle of adult chickens and cleaved with chymotrypsin to give the -90-kDa S1 (head) and -110-kDa tail fragments. The MHCfragments were analyzed by SDS-PAGE in 5% gels and by immunoblotting. Lane I, Coomassie Blue-stained gel of chymotrypsin-cleaved MHC, lanes 26, immunoblots of duplicate gels showing reaction of chymotryptic fragments with mAbs F18 (lane 2 ) , F27 (lane 3 ) , F30 (lane 4 ) , F47 (lane5),and F59 (lane 6).
and 2). Proteins smaller than 230 kDa werealso induced, but these appeared to be fragments of the full length fusion protein (see below). This size of fusion protein was expected as the &galactosidase accounted for 116 kDa and the 981 amino acids encoded by the inserted MHC cDNA accounted for the remaining mass. That the epitopes recognized bymAbs F18, F30, F47,and F59 were encoded bythis fragment of the cDNA was shown bythe reaction of these mAbs with the full length -230-kDa fusion protein and with the shorter, immunoreactive fragments of the full length fusion protein (Fig. 3, lanes 3 and 5-7). The gel lanes shown in Figs. 3 and 4 were overloaded to allow visualization of the fusion protein fragments (see "Discussion"). The reaction of mAb F47 with the 8-galactosidaselembryonic fast MHC fusion protein was weak, as was the reaction of this mAb with the chicken embryonic fast MHC prepared from muscle (Table I). The epitope on the MHC rod that was recognized by mAb F27 was not found on the fusion protein (Fig. 3, lane 4 ) , thus the epitope recognized by mAb F27 wasnot on the S1 nor on the first 200 amino acids (808-1014) of the S2 region of the rod. To localize the epitopes on the S1 of the fast MHC, cDNA fragments were generated by deleting progressively from the 3' end of the MHC cDNA inserted in pUR290, and each fragment was tested for epitope expression. The 2.13-kb PstIHind111 fragment (encoding amino acids 34-745) and a 1.4kb PstI-BglI fragment (encoding amino acids 34-468) of the MHC cDNA wereprepared, expressed as fusion proteins, and found to encode the F18, F30, F47, and F59 epitopes. Transformants carrying the 2.1-kb PstI-HindIIIMHC cDNA fragment inserted into pUR290 produced a fusion protein with M , 190 kDa upon induction with IPTG, and mAbs F18, F30, F47, and F59 reacted with this fusion protein in immunoblots (Fig. 4, lane 1). Similarly, transformants carrying the inserted 1.4-kb PstI-BgZII fragment produced a fusion protein with M , 165 kDa which also reacted with these four mAbs (Fig. 4, lane 2). To more closely determine the regions of the MHC cDNA that encoded the different epitopes, a series of progressively
-
-
-ImG
FIG. 3. A &galactosidase/MHC S1 fusion protein expressed epitopes recognized by mAbs specific to chicken fast MHC isoforms. A 2.94-kb PstI cDNA fragment encoding amino acids 341014 (out of 1940 total) comprising the S1 (head) and a portion of the S2 regions of the chicken embryonic fast MHC was subcloned into the PstI site of pUR290. Proteins expressed by transformants carrying this recombinant plasmid were analyzed by SDS-PAGE in 5% gels and by immunoblotting before (lane I ) and after (lanes 2-7) IPTG induction of 8-galactosidaselMHC fusion protein synthesis. Lane I, Coomassie Blue (CB)-stained gelof bacterial proteins expressed before IPTG induction. Lane 2, Coomassie Blue-stained gel of bacterial proteins expressed after IPTGinduction of fusion protein synthesis. Note the appearance of the full length fusion protein a t -230 kDa in the IPTG-induced bacteria. Lanes 3-7, immunoblot analysis of j3-galactosidaselMHC fusion protein reaction with mAbs F18 (lane3),F27 (lane 4 ) , F30 (lane 5),F47 (lane 6), and F59 (lane 7). The -230-kDa full length fusion protein and thesmaller fragments of the fusion protein reacted strongly with mAbs F18, F30, and F59, and weakly with mAB F47, but did not react with mAb F27. Gellanes in Figs. 3 and 4 were overloaded to allow visualization of fusion protein fragments (see "Discussion").
smaller fragments of the MHC cDNAwere produced by deleting from the 3' end of the MHC cDNA with BaZ31 exonuclease treatment (see "Experimental Procedures"); and the fusion proteins expressed from these fragments were tested for reaction with the mAbs. Three MHC cDNA fragments of-1.0,-0.7, and -0.5 kb that were inserted into pUR290 were among those produced with this method, and the p-galactosidase-MHC fusion proteins encoded by these plasmids were of-155,-145, and -135 kDa, respectively (Fig. 4, lanes 3-5). In immunoblots, mAbs F18, F30, and F47 reacted with each of these differently sized fusion proteins, whereas mAb F59 reacted with the fusion proteins of -155 and -145 kDa but did not react with the smallest fusion protein of -135 kDa (Fig. 4). From the sizes of the inserted cDNAs and the pattern of mAb reactivities with the fusion proteins, it appeared that the epitopes for mAbs F18, F30, and F47 were located between amino acid 34 (the first amino acid encoded by the cDNA) and approximately amino acid 200, and that theepitope recognized bymAb F59 waslocated between approximately amino acid 200 and amino acid 250. Utilization ofBa131 exonuclease to generate a family of progressively smaller cDNAs thus allowed a very rapid low resolution mapping of the MHC epitopes. The method was limited in two ways. First, the gels used to size the fusion proteins were of limited resolution. Second, the Ba131 exonuclease digested in both directions from the opened BglI site; thus the recovery of very small cDNA fragments (c0.5
Myosin Isoform Structure 1 ‘
Z
2
3
4
5
1 r-
!
2
3
7-
’ ”
5
4
1 .
-1
1
- ‘
m
2
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4
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190-
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2
3
4
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i
1651551451 ll6-
._,
-
. J
I L
-
F18
,
”
4
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C
F47
FIG.4. Localization of epitopes by expression ofMHCcDNA
F59
fragments as @-galactosidase/MHC
fusion proteins. A family of progressively shorter MHC cDNAs was generated by progressive deletion from the 3’ end of the 2.94-kb PstI fragment of the chicken embryonic fast cDNA, and each cDNA fragment was ligated in reading frame into pUR290 (see “Experimental Procedures”).Transformants carrying each recombinant plasmid were induced by IPTG to synthesize 8-galactosidaselMHC fusion proteins which were analyzed by SDS-PAGE in 5% gels and by immunoblotting. The cDNA fragments expressed as fusion proteins were the 2.13-kb PstI-Hind111 fragment (lane I), the 1.4-kb PstI-BglII fragment (lane 2 ) , and fragments of -1.0 kb (lane 3), -0.7 kb (lane 4 ) , and -0.5 kb (lane 5) generated by Bal31 deletion from the 3’ end of the PstI-BglII fragment as described under “Experimental Procedures.” The fusion proteins synthesized upon IPTG induction of bacteria carrying these recombinant plasmids were analyzed by immunoblotting with mAbs F18, F30, F47, and F59 as indicated.
pressed as fusion proteins and theepitopes they encoded are listed in Table 11. Final assignments of epitopes to particular amino acid sequences were determined from the smallest overlapping regions of cDNA fragments that encoded the amino acid sequences reactive with a particular mAb.As shown in Table 11, mAb F59 reacted with two @-galactosidase/ MHC fusion proteins which included MHC amino acids 34231 and amino acids 211-303. The F59 epitope was, therefore, assigned to amino acids 211-231. Similarly, mAbs F18, F30, and F47 all reacted with a fusion protein that included MHC amino acids 34-92 but did not react with a fusion protein that included amino acids 34-71. This result suggested that these three epitopes were located either entirely between amino acids 72 and 92 or that one or more epitopes spanned the region including amino acids 71 and 72. Because continuous epitopes usually include five to seven amino acids, the epitopes recognizedbymABs F18,F30, and F47 were assigned to amino acids -65-92. One-dimensional immunopeptide mapping was usedto determine if the epitopes recognized by mAbs F18, F30, and F47 were identical or three distinct epitopes. F18 F30 F47 Partial proteolytic fragments of the chicken adult fast MHC FIG.5. Distinct epitopes were recognized by mAbs F18, isoform were prepared, separated by SDS-PAGE, and tested F30,and F47. Chicken adult fast MHC from the pectoralis muscle for reactivity with mAbs (Fig. 5). The three mAbs did not was partially cleaved with Staphylococcus aureus V8 protease, the react with a single set of MHC peptides as would have been peptide fragments were separated by SDS-PAGE in 15% gels and expected if each mAb recognized the same epitope. Rather, transferred to nitrocellulose. The transfers were incubated with mAb each of the three mAbs reacted with a distinct setof the MHC F18, F30, or F47 as indicated, and mAb binding was visualized with an alkaline phosphatase-linked secondary antibody. Symbols indicate peptides (Fig. 5), thus showing that mAbs F18, F30, and F47 differences in the partial MHC peptides that reacted with the differ- reacted with three distinctepitopes within MHC amino acids ent mAbs. Because each of the three mAbs reacted with a distinct -65-92. pattern of MHC fragments, the F18, F30, and F47 epitopes were in three distinct amino acid sequences.
DISCUSSION
kb) was prevented when the Ba131 digested beyondthe 3’ end of the initial 2.13-kb insert into the ampicillin resistance gene. With the approximate locations of the epitopes known, however, the resolution of mapping was increased by expressing as fusion proteins cDNA restriction fragments that encoded known amino acid sequencesin the epitope regions. The expression of known cDNA restriction fragments allowed the assignment of the F59 epitope to amino acids 211231 and theF18, F30, and F47 epitopes to amino acids -6592. The most informative cDNA fragments that were ex-
The epitopes recognized by mAbsF18, F27, F30, F47, and F59 were characteristic of all fastmyosin heavychain isoforms in chicken skeletal musclebecause each mAb reacted on immunoblots with known chicken fast MHC isoforms, but did not react with known chicken slow or smooth muscle MHC isoforms. The epitopes characteristic of fast MHCs in the chicken were, in most cases, also restricted to fast MHCs in therat. The exception was the epitope recognized by mAb F59 whichwas present on the rat @-cardiaclslowskeletal MHC as well as on the chicken and rat fast MHCs. The unexpected finding that the F59 epitope was shared by the
13128
Myosin Isoform Structure
rat a-cardiac and @-cardiaclslowskeletal MHCssuggests that the cardiac and skeletal muscle MHCs may have distinct evolutionary histories; an idea supported by the finding that cardiac MHCs, at leastin the mouse, areonaseparate chromosome from the cluster of genes encoding the skeletal muscle MHCs (Barton and Buckingham, 1985). Each of the five mAbs reacted with skeletal muscle MHCs prepared from species of two or more classes of vertebrates (mammalian, avian, reptilian, amphibian, fish, and elasmobranch species), but none of the mAbs reacted with the invertebrate MHCs tested (TableI). Thus, each of the epitopes has been conserved in vertebrate evolution, with the F27 and F59 epitopes first appearing on vertebrate skeletal muscle MHCs at least 350 million years ago. A comparison was made of the amino acid sequences of four vertebrate skeletalmuscle fast MHCs that did react with mAbs F18, F30, F47,and F59: embryonic chicken fast (Molina et al., 1987),adult chicken fast (Maita et al., 1987),embryonic rat fast(Strehler et al., 1986),and adultrabbit fast (Tong and Elzinga, 1983), and of three MHCs that did not react with these mAbs: two invertebrate MHCs (Dictyostelium, Warrick et al., 1986; and nematode, Karn etal., 1983),and thesmooth muscle MHC of the chicken (Yanagisawa, et al., 1987) (Fig. 6). The four vertebrate skeletal muscle MHCs (Fig. 6 A ) had F18,F30,
F41 EpitOpe Ragion 1a.a.s 6 5
-
f59 Epitope 1a.a.s211-2311
92)
E G G E T L T V K E D Q I F S ~ P P K Y D K I E D ~ PAGKMQGTLEDQIISANPLLE " . """"S Q ""y s p ""m 5 " K """" -DNR"V"pEDWA""-F"""" TP""""""T""" -A-ASV"""v-p"""""". VHLfPY-E--K-LLO---I--N.K~N-LSK-D-QK""-fs-v"" OpN-KKV-~----VQT--V-VTArtEn-L-KEL~QE-----fE-~"-S K ~ M Q - - K - D ~ Q R - - I - F - ~ - - - ~NQANGSGV-Q--LO--NI"
". 0
100
200
300
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500
Emb. c h i c k e n f a s t Ad. c h i c k e nf a s t r a tf a s t Ad. r a b b i tf a s t C h i c k e ng i z z a r d Nematode Dictyostelium
~mb.
600
700
800
Amino Acid Numbsr
FIG. 6. Comparison of MHC amino acid sequences. A, MHC amino acid sequences were compared in the F18, F30, and F47 epitope region and in the F59 epitope region. Epitope region sequences are shown from four vertebrateMHCs that reacted with each mAb (chicken embryonic fast, Molina et al., 1987;chicken adult fast, Maita et at., 1987; rat embryonic fast, Strehler et at., 1986; and rabbit adult fast, Tong and Elzinga, 1983), and from three MHCs that did not react with the mAbs (chicken gizzard, Yanagisawa, et al., 1987; nematode, Karn et al., 1983; and Dictyostelium, Warrick et al., 1986). Amino acids were aligned according to Warrick and Spudich (1987) with gaps removed and amino acids numbered to correspond to the chicken embryonic fast MHC (Molina et al., 1987). Dashes represent amino acids that are identical to the corresponding amino acids in the chicken embryonic fast MHC sequence. B, the amino acid sequence of the S1 (head) region of the chicken embryonic fast MHC wasused as the reference sequence, and the percentages of mismatched amino acids in the corresponding sequences of the three vertebrate MHCs (darkly stippled area under the lower line) and the two invertebrate MHCs (lightly stippled area under the upper line) were determined as described in the text. Highly conserved sequences thus appear as valleys and unconserved sequences appear as peaks. Indicated sequences include the consensus ATP-binding site, the 2550-kDa subfragment border, the 50-20-kDa subfragment border, the actin-binding site, and the conserved thiols (Warrick and Spudich, 1987), as well as the regions comprising the F59 epitope and theF18, F30, and F47 epitopes.
nearly identical sequences in the F59 epitope region between amino acids 214 and 231 as only two substitutions occurred (the ratMHC has Lys instead of Gln at position 216 and the rabbit MHC has Thr instead of Ser at position 225). Amino acids 211, 212, and 213 were not conserved among the four vertebrate skeletalmuscle MHCs that reacted withmAb F59, suggesting that theF59 epitope was between amino acids 214 and 231. As predicted from the lack of reaction of mAb F59 with chicken smooth muscle MHC and invertebrate MHCs, there were multiple amino acid differences between the F59 epitope region of the vertebrate skeletal muscle MHCs and the corresponding region of the chicken smooth muscle and invertebrate MHCs. The region in which the F18, F30, and F47 epitopes were localized also had stretches of sequence identity (particularly within amino acids 80-92) among the four vertebrate skeletalmuscle MHCs. Invertebrate sequences (Acanthnmoeba myosin I and 11, yeast, nematode, and Dictyostelium myosin) were also compared with those of chicken embryonic fast MHC and all fell in a range of 65-85% sequence mismatch with the F59 epitope region and 61-82% sequence mismatch with the F18,F30,F47 epitope region (Junget al., 1987; Hammer et al., 1987; Hoshimaru and Nakanishi, 1987). Several ATPases and nucleotide-binding proteins were found with up to 30% sequence identity in the F59 epitope region (6 amino acids out of 20, but therewas no pattern of conserved amino acids in this region found among these non-myosin proteins, such as ninaC (85% mismatch) because each of the amino acids in theF59 epitope region was about equally likely to be conserved. In thecase of Acanthamoeba myosin I and ninuC myosin-like protein, the homologous region for the F18, F30, and F47 epitopes is beyond the NH2 terminus of these proteins and lacks 15 of the 28 amino acids of the epitope region (Hammer et al., 1987; Jung et al., 1987; Monte11 and Rubin, 1988). The F59 epitope region was as highly conserved as any of the known functional regions of the S1 in vertebrateskeletal muscle MHCs and was relatively more conserved than much of the S1. A diagram of sequence conservation is shown in Fig. 6B in which the chicken embryonic fast MHC sequence was used as the reference to which other sequences were compared. The percentage of amino acid mismatches between the chicken embryonic MHC sequence and either the three other vertebrate skeletalmuscle MHC sequences (darkly stippled area under the lower line) or the two invertebrate sequences (lightly stippled area under the upper line) was determined with a sliding 10-amino acid window. This stringent test of conservation was used because of the relatively high degree of conservation among different skeletalmuscle MHCs and thelikely sensitivity of mAb binding to small changesin sequence. Therefore, conserved regions appear as ''valleys" and unconserved regions as "peaks" in Fig. 6B. MHC regions known to be required for function of the S1, including the ATP-binding site, actin-binding site, and thiol region, were highly conserved and tended to be highly conserved even when comparisons were made between vertebrates and invertebrates (Fig. 6B; cf. Warrick and Spudich, 1987). There are, however, additional MHC regions which are conserved on specific groups of vertebrate skeletal muscle MHC isoforms, including the epitopes mapped here, to which no functionhas been assigned; and theseregions could account for the distinct enzymatic and structural features of different isoforms. Poorly conserved regions included the NH2-terminalof the protein and the trypsin-sensitive sites that define the boundaries of the 25-,50-, and 20-kDa tryptic subfragments of the S1 (Molina et al., 1987). The epitopes recognized by mAbs F18, F30, F47, and F59
Myosin Isoform Structure
13129
may be part of or near the ATPase site. The two-partcDNA the epitopes could be localized on the three-dimensional strucexpression strategy allowed assignment of the epitopes rec- ture of the MHC using rotary shadowing and electron microscopy of mAbs bound to native myosin (Winkelmann et ognized by mAbs F18, F30, and F47 to amino acids -65-92 and the epitope recognized by mAb F59 to amino acids 211- al., 1983; Winkelmann and Lowey, 1986). It seems likely that 231. Modification of Lys-84, in the region of the F18, F30, these mAbs will bind near the ATP-binding site which has and F47 epitopes, causes a large decrease in ATPase activity been localized (Tokunaga et al., 1987). The functions of amino acid sequences in the epitope regions can be probed by in (Mornet et al., 1980; Hozumi and Muhlrad, 1981), and an ATP derivative photoaffinity labels Trp-131 indicating that vitro mutagenesis of MHCs expressed from cDNAs (Bauer it is near the ATP-binding site (Okamato and Yount, 1985). and Robbins, 1988). The F59 epitope is approximately 40 amino acids toward the Acknowledgments-We thank Dr. Jeffrey Robbins (University of COOH terminus from the highly conserved ATP-binding Cincinnati) for providing the MHC cDNA with sequence and restricconsensus sequence of G-X-X-G-X-G-K-T beginning at Gly- tion map, Dr. Kathleen M.Buckley for invaluable instruction on 179. If MHCs are similar to other ATP- or GTP-binding cDNA manipulations and for providing the electric ray muscle, Drs. proteins (Fry et al., 1986; Weinmaster et al., 1986; Dever et David Shelton and Gary Radeke for help with the protein database al., 1987; deVoset al., 1988), then amino acids on the 50-kDa search, Drs. James Spudich and William Sharrock for samples of fragment COOH terminal to the nucleotide-binding site (such Dictyosteliurn and nematode myosins, and Gloria Garcia for secreas the F59 epitope region) may loop back and perhaps com- tarial assistance. prise part of the ATPase enzymatic site. Mahmoud and Yount REFERENCES (1984) have shown that another ATPderivative reacts with a D., Masaki, T., and Fischman, D. A. (1982) J. Cell Bwl. 95, site on the 50-kDa fragment that must be within 0.7 nm of Bader, 763-770 the nucleotide-binding site. Bandman, E., Matsuda, R., and Strohman, R.C. (1982) Deu. Biol. Epitope mapping by cDNA expression using the pUR290 93,508-518 series of plasmids was simple, rapid, and efficient. Unlike Bandman, E. R. (1985) Znt. Reu. Cytol. 97,97-131 other methods, the method used here does not require gener- Barany, M. (1967) J. Gen. Physiol. 50, 197-218 ation, identification,and analysis of multiple proteolytic frag- Barton, P. J. R., and Buckingham, M. E. (1985) Biochern. J. 2 3 1 , 249-261 ments, synthesis of multiple overlapping peptides, in vitro Bauer, B. J., and Robbins, J. (1988) J. Cell Biol. 1 0 7 , 3 6 (abstr.) mutagenesis, or sequencing of multiple overlapping cDNAs Crisanti, A,, Mueller, H.-M., Hilbich, C., Sinigaglia, F., Matile, H., (Reinach and Fischman, 1985; Mehra et al., 1986; Sigal et al., McKay, M., Scaife, J., Beyreuther, K., and Bujard, H. (1988) 1986; Van Regenmortel, 1987; Sheshberadaran and Payne, Science 240,1324-1326 1988; Doorbar et al., 1988). As our workwas in progress, Crow, M. T., and Stockdale, F.E. (1984) Exp. Biol. Med. 9, 165-174 Doorbar et al. (1988) used Ba131 to generate multiple overlap- Crow, M. T., and Stockdale, F. E. (1986a) Deu. Biol. 113, 238-254 ping fragments of a short viral cDNA which were sequenced Crow, M. T., and Stockdale, F. E. (198613) Deu. Biol. 118, 333-342 Dever, T. E., Glynias, M. J., and Merrick, W. C. (1987) Proc. Natl. and expressed to localize epitopes on 100-amino a acid protein, Acad. Sci. U. S. A. 84, 1814-1818 and Crisanti et al. (1988) used exonuclease 111 to generate deVos, A. M., Tong, L., Milburn, M. V., Matias, R. M., Jancarik, J., shortened pieces of cDNA for expression as fusion proteins Noguchi, S., Nishimura, S., Miura, K., Ohtsuka, E., and Kim, S.H. (1988) Science 239,888-893 to localize a glycoprotein epitope. An unexpected advantage of the strategy we used here came from the immunoreactive Doorbar, J., Evans, H. S., Coneron, I., Crawford, L. V., and Gallimore, P. H.(1988) EMBO J. 7,825-833 fragments of the @-galactosidaselMHCfusion proteins. The Doolittle, R. F. (1979) in The Proteins, 3rd ed., Vol. 4 (Neurath, H., sizes of the smallest abundant fragmentsappeared to be and Hill R. L., eds) Academic Press, New York predictors of epitope locations. In the case of the epitope Emerson, C. P., Jr., and Bernstein, S.I. (1987)Annu. Reu. Biochern. recognized by mAb F59, for instance, the smallest immuno56,695-726 reactive product had M, 140 kDa (Fig. 4). Assuming that Evans, D., Miller, J. B., and Stockdale, F. E. (1988) Deu. Biol. 127, 376-383 the fusion protein fragments were missing amino acids from only the COOH-terminal (MHC) endof the full length fusion Fallon, J. R., and Nachmias, V. T. (1980) J. Cell Biol. 8 7 , 237-247 Fry, D. C., Kuby, S. A., and Mildvan, A. S.(1986) Proc. Natl. Acad. protein (perhaps due to bacterial protease digestion or transSci. U. S. A. 83,907-911 lation of incompletely transcribed mRNAs), then the amino Hammer, J. A., 111, Bower,B., Paterson, B.M., and Korn, E. D. acids comprising the F59 epitope would have been predicted (1987) J. Cell Biol. 105, 913-925 to be about 24 kDa (-140-kDa fusion protein less 116 kDa Hozumi, T., and Muhlrad, A. (1981) Biochemistry 20,2945-2950 due to @-galactosidase)or about 200 amino acids from the Izumo, S., Nadal-Ginard, B., and Mahdavi, V. (1986) Science 2 3 1 , 597-600 most NHz-terminal MHC amino acid encoded by the cDNA Jolesz, F., and Sreter, F. A. (1981) Annu. Reu. Physwl. 44, 531-552 (which was amino acid 34), and this was the region in which Jung, G., Korn, E. D., and Hammer, J. A. I11 (1987)Proc. Natl. Acad. the F59 epitope was found. If such immunoreactive fragments Sei. U. S. A. 84,6720-6724 of full length fusion proteins result when cDNAs of other Karn, J., Brenner, S., and Barnett, L. (1983) Proc. Natl. Acad. Sci. U. S. A. 80, 4253-4257 large proteins are expressed, this system will provide a very Kavinsky, C. J., Umeda, P. K., Sinha, A. 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Myosin Isoform Structure
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