Proc. Natl. Acad. Sci. USA Vol. 89, pp. 12078-12082, December 1992 Genetics Organization of the human skeletal heavy chain cluster (musde /yeast ardtfcial /physcal map) SUNG-JOO YOON*, STEPHANIE H. SEILERt, RAJu KUCHERLAPATI*, AND LESLIE LEINWAND*t Departments of *Molecular Genetics and tMicrobiology and Immunology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461 Communicated by Frank Lilly, October 5, 1992

ABSTRACT Myosin is an important structural and enzy- The clustering of MYH genes is in distinction to the matic component of skeletal muscle. Multiple myosin isoforms dispersed location of members of other contractile are encoded by a multigene family and are expressed in multigene families such as those encoding and myosin different developmental stages and fiber types. In humans and light chains. These differences in gene organization of the mice, skeletal myosin heavy chain (MYH) genes are clustered different families have led to the proposal that clustering of on a single (17p and 11, respectively). Since the MYH genes has a regulatory significance (39). Although structural organization of the gene duster may affect its characterization of the regulatory elements of cardiac MYH expression as well as shed light on MYH genetic alterations, a genes has begun (20, 21), little is known about the regulatory physical map ofthe human MYH gene cluster was constructed. sequences within the skeletal MYH genes. If the globin gene Nine yeast artificial chromosomes containing MYH genes were cluster serves as a model, it seems likely that the organization isolated and used to construct a contiguous set (contig) of of the skeletal MYH genes will be important in their regula- overlapping yeast ar l chromosomes. This contig encom- tion. In addition to identifying domains or elements regulating passes a genetic marker mapped to 17pl3.1. Six MYH genes expression ofthe skeletal MYH genes, a physical map will be were located within a 500-kilobase sgent of human DNA. essential in identifying gross structural mutations in skeletal The order of the genes within this duster does not correspond MYH genes responsible for muscle disease. Mutations in the to the developmental pattern of expression of individual mem- human (3 cardiac MYH gene have recently been demon- bers. strated in several kindreds with the autosomal dominant disease familial hypertrophic cardiomyopathy (22), lending Myosin is the molecular motor of muscle and is composed of support to the hypothesis that mutations in the structural a pair of myosin heavy chains (MYHs) and two pairs of components of muscle may be responsible for some forms of nonidentical light chains. At least seven sarcomeric MYH muscle genetic disease. isoforms are expressed in mammalian skeletal muscle, in- To understand the organization of the human MYH gene cluding embryonic, perinatal, fast Ila, fast HIb, fast lId, cluster, yeast artificial chromosome (YAC) clones containing extraocular, and slow (J3 cardiac) forms (1-3). MYH genes MYH genes were isolated and analyzed, revealing that six are expressed at different times during development and in distinct genes are located within a 500-kb interval. Five different fiber types (4-8). The MYH isoform content of any correspond to previously identified MYH genes and/or given muscle can also vary in response to physiological cDNAs: embryonic; perinatal; two fast II MYH genes; and changes such as innervation or hormonal perturbation (for a an adult skeletal MYH gene designated MYH2. The identity review, see ref. 9), and quantitative differences in myosin of the sixth gene has not yet been determined. The physical ATPase activity have been correlated with the contractile order of these genes does not correspond to their develop- velocity of the muscle (10). mental program and may represent evolutionary expansion of The cardiac and skeletal MYH genes are clustered on two the gene family to accommodate the physiological needs of different chromosomes in mice and humans. The a and (3 mammalian muscle. cardiac MYH genes are separated by only 4 or 5 kilobases (kb) of DNA on human and mouse chromosomes 14 (11-13). With the exception of the slow/,B cardiac MYH gene, the MATERIALS AND METHODS skeletal MYH genes are clustered on human Nomencature. MYH genes have been given the numerical and mouse chromosome 11 (12, 14, 40). Linkage analysis with designations as follows: embryonic MYHCE, MYH3; peri- a polymorphic probe proximal to an adult skeletal MYH gene natal MYHpn, MYH8; adult fast skeletal MYH (not subtype (MYH2) localized this gene to human 17p13 (15). In addition, defined, but described as a A genomic clone, AMHC10, in ref. two other human skeletal MYH genes (embryonic, MYH3; 3), MYH2. perinatal, MYH8) have been mapped to 17p (16, 17). Several YAC Screang. PCR-based screening (23) was used to lines of evidence suggest that all or most of the vertebrate isolate clones containing the human MYH locus from the skeletal MYH genes are in close genetic and physical prox- Washington University human DNA YAC library (24) using imity to each other. Interspecific backcrosses and pulsed- MYHem- and MYHp.-specific primers (MYHEM and field gel electrophoresis (PFGE) analysis in mice demon- MYHPM, respectively; see Table 1). strated that three MYH genes (embryonic, perinatal, and fast DNA Analyi. DNAs for PFGE were prepared by estab- IIB) are clustered on mouse chromosome 11 within 370 kb of lished procedures (25). Restriction fragments or intact yeast each other (12, 18). Two additional mouse MYH genes, fast chromosomes in agarose plugs were separated in a 1% Ila and a gene expressed abundantly in several adult skeletal agarose gel using a contour-clamped, homogeneous electric muscles (termed MdMS), are separated by about 5 kb of field apparatus (Bio-Rad) as described (26). DNA from yeast DNA (19). The order and organization ofthe human skeletal for conventional electrophoretic analysis was prepared by MYH genes are not known. established procedures (27), and restriction enzyme-digested

The publication costs ofthis article were defrayed in part by page charge Abbreviations: MYH, myosin heavy chain; YAC, yeast artificial payment. This article must therefore be hereby marked "advertisement" chromosome; PFGE, pulsed-field gel electrophoresis; STS, se- in accordance with 18 U.S.C. §1734 solely to indicate this fact. quence-tagged site. 12078 Downloaded by guest on September 26, 2021 Genetics: Yoon et al. Proc. Natl. Acad. Sci. USA 89 (1992) 12079

yeast DNA was separated in a 0.8% agarose gel in lx TAE The VL primer used for Alu-vector PCR is a 29-mer of the (28). DNAs were transferred to GeneScreenPlus (DuPont/ composition 5'-CACCCGTTCTCGGAGCACTGTCCGAC- NEN) and the filters were hybridized with appropriate probes CGC-3' and is designated RK152. The VL primer used for in a solution containing 1 M NaCl, 1% SDS, and 10o dextran vectorette PCR, designated LL384, is a 30-mer of the com- sulfate at 65°C. Fifty micrograms of sheared salmon sperm position 5'-TTAAGGCGCAAGACTTTAATTTATCAC- DNA per ml was added during prehybridization and with the TAC-3'. The VR primer used for both types ofPCR reactions denatured probe. was designated RK151 and had the composition 5'-ATAT- Rescue of DNA Sequences from the Ends of YACs. Three AGGCGCCAGCAACCGCACCTGTGGCG-3'. The se- methods for end sequence rescue were used. In the first, quences that constituted the vectorette are RK154 and 155. referred to as vectorette or bubble PCR (29), yeast DNA was RK154 has the sequence 5'-GA/TCAAGGAGAGGACGC- digested with Ava II, ligated with a synthetic vectorette (see TGTCTGTCGAAGGTAAGGAACGGACGAGAGAAGG- below), and amplified with one primer corresponding to part GAGAG-3'. RK155 has the, partially complementary se- of the vectorette and the other corresponding to the left (VL) quence 5'-CTCTCCCTTCTCGAATCGTAACCGTTCG- or right (VR) end part of the YAC vector. DNA ligation TACGAGAATCGCTGTCCTCTCCTT-3'. To generate the reactions were carried out under conditions described by vectorette, oligonucleotides RK154 and RK155 were an- Maniatis et al. (28). Amplification was conducted in a total nealed and used for ligation with Ava 11-digested yeast DNA. volume of 50 Il containing 10 mM Tris HCl (pH 8.3), 50 mM The vectorette primer designated RK153 is 5'-CGAATCG- KCl, 1.5 mM MgCl2, 250 ,LM of each ofthe four dNTPs, and TAACCGTTCGTACGAGAATCGCT-3' (29). The following 1 ,uM of each primer in a Perkin-Elmer thermal cycler under PCR primers were used in the inverse PCR reactions. For the conditions of denaturation at 940C for 4 min, 38 cycles of left end, we used the primers RK156 (5'-GTTGGTTTAAG- denaturation (920C for 1 min), annealing (660C for 30 sec), and GCGCAAGA-3') and RK204 (5 '-GCGATGCTGTCG- extension (720C for 2 min). The second method for end rescue GAATGGAC-3'). For the right end, we used RK205 (5'- is referred to as vector-Alu PCR, using the VL and VR primers GTCGAACGCCCGATCTCAAG-3') and LL385 (5'-TAT- and the Alu primer 517 or 559 (30). These reactions were GTCTCCATTCACTTCCCA-3'). carried out in 50-,lJ volumes containing 5 ng of yeast DNA in PCR Conditions for Sequence-Tagged Sites (STSs). PCR the reaction mixture described above. Amplification condi- with gene-specific primers (Table 1) was carried out in 10 ,ul tions were denaturation at 940C for 4 min, followed by 35 with 0.5 AM primers using 5 ng of yeast DNA or 100 ng of cycles ofdenaturation (940C for 1 min), annealing (650C for 2 human DNA as controls in the conditions described above. min), and extension (72°C for 5 min), followed by a final Initial denaturation was at 940C for 4 min, followed by 30 extension period of 10 min. The third method to generate end cycles of 940C denaturation (1 min), 57°C (for MYHpn), or sequences is referred to as inverse PCR (31). Yeast DNA was 62°C (for MYHEM, MYHPM) annealing (1 min), and 720C digested with EcoRV (for left end) and Xmn I (for right end), extension. PCR reactions with other STS primers were and the products were ligated under conditions that enhance carried out in 50 ,l with 1 AM of each primer using 25 ng of intramolecular ligation and amplified using primers within the yeast or 500 ng of human DNA. The PCR conditions for all vector that are oriented in opposite directions. STS pairs are initial denaturation at 94°C for 4 min, followed Table 1. STSs for the MYH gene cluster contig Primer Product PCR STS name Origin name Primer sequence size, bp condition* A213C41 A213C4 RK207 5'-CTTTTTGAGTCTCAGTGCCTCA-3' 116 55t RK206 5'-ACCCGGCTGCCTTTATTATT-3' MYHE5 MYHem LL347 5'-GGTGGTCAAACCAGAGGATGTG-3' 1200 55/2 LL290 5'-GTCAACATGAACTGATAGGCA-3' MYHEM MYHem LL137 5'-GTGTGCAGAGGGTTCCTCATGCGTG-3' 360 62/1 LL60 5'-CTCTTGGACCAGAGTCACCAG-3' MYHE3 MYHem LL182 5'-GGATGCAAGGAACGCTGAG-3' 3500 58/2 LL181 5'-GTCCTGCTCCAGAAGGGC-3' B120C11L B120C11 LL438 5'-CTTCCCATGCGAGATGATCG-3' 124 65t LL437 5'-GATAGAGCTGAAAAGAGAGC-3' MYHAS8 MYH&S8 LL175 5'-AGATGGAGGACATTCTCCAG-3' 3000 55/2 LL176 5'-TTGCAACAGGGTAGAATACAC-3' A288E1L A288E1 LL446 5'-TAGCCATTGCCTATATCAAG-3' 108 55t LL447 5'-GCATCGAGCTCATCGAGAAG-3' MYHIIa MYHa LL174 5'-TGCAAGCAAAGGTGAAATCCT-3' 301 57/1 LL155 5'-TGGCAGATAAATTTTTATCTCC-3' MYHP5 MYHpn LL289 5'-TTTATCTGGAACTCCAGAAGC-3' 600 58/1 LL290 5'-GTCAACATGAACTGATAGGCA-3' MYHPM MYHpn LL77 5'-TTAAGCCCCTCCTCAAGAGTGCA-3' 900 62/1 LL120 5'-TCAAGCTTCTTCCTCCTCCTCAGCTCTTTC-3' MYHP3 MYHpn LL179 5'-GGTTGAAAATGAACAGAAACGT-3' 2600 54/2 LL180 5'-TATTCAGCTTTAACAGGAAAATAA-3' A56B1OR A56B10 LL444 5'-ATCTACACAAAATAGCCTCCTGC-3' 68 55t LL445 5'-TCATCCCCAGAAAAGATTTAGG-3' B12OC11R B12OC11 LL443 5'-CTATCTACTCTCATCTCACG-3' 147 55t LL442 5'-GCAGCAACAGTAACGGAACTGG-3' bp, Base pairs. *Annealing temperature/time of extension (°C/min). tThis reaction did not have a designated extension period. Downloaded by guest on September 26, 2021 12080 Genetics: Yoon et al. Proc. Natl. Acad. Sci. USA 89 (1992) by 30 cycles of denaturation at 940C for 1 min and annealing for 30 sec at temperatures shown in Table 1. Probes. Total human DNA was used as a probe for PFGE analysis and for DNA fingerprint assays. Gene-specific probes, MYHm, MYHpN, MYHas, and MYHa, were gel- i! purified fragments from the 3' untranslated regions of the corresponding genes and have been described (refs. 17, 32, and 33; I. Karsch-Mizrachi and L.L., unpublished data). The consensus 3' probe designated LL1000 was prepared by PCR -_ ',i with the perinatal cDNA as a template using primers from W @4_ exons 37 and 39, which correspond to sequences identical in (HiI all published mammalian skeletal MYH genes. These primers w _ - in! were 5'-CAGGACACCAGCGCCCA-3' (sense) and 5'- (antisense). End frag- TCCTCGGCCTCCTCCAGCTC-3' FIG. 1. Sizes of YACs from the MYH gene cluster. Electropho- ments rescued by PCR were digested with EcoRI to remove retic karyotypes of yeast carrying DNA from the MYH gene cluster vector sequence, followed by gel purification. All probes were transferred to nylon membranes and hybridized with total were radiolabeled using the random primer method of Fein- human DNA. Lanes: a, A213C4, 275 kb; b, A24D2, 140 kb; c, berg and Vogelstein (34). A258D4, 140 kb; d, A23Dq7, 330 kb; e, A288E1, 200 kb; f, B72A12, Cell Lines. Two somatic cell hybrid cell lines, kindly 300 kb; g, A51D4, 280 kb; h, A56B10, 340 kb; and i, B120C11, 410 provided by R. E. K. Fournier, were 7AE-13 and 7AC-2. kb. Hybrids 7AE-13 and 7AC-2 contain intact human chromo- somes 17 and 17q, respectively, in a rat background. include MYH gene-specific markers, internal markers gen- DNA Sequencing. Plasmid DNA was prepared and se- erated from Alu-PCR products from individual YACs, and quenced essentially as described (35). Sequence data were markers from the ends of individual YACs. YAC end se- analyzed using the PRIMER program (Genome Center, White- quences were obtained and used to probe each of the YACs head Institute, Cambridge, MA) to generate oligomers foruse by hybridization and/or PCR. If end sequences failed to as primers in the PCR. hybridize to any YACs other than the one from which it was derived, it could represent one of two ends of the contig or be derived from a chimeric YAC. The latter feature was RESULTS ascertained by determining whether the probe could hybrid- Isolation of YACs. To understand the structural organiza- ize to DNA from a somatic cell hybrid containing chromo- tion of the human MYH genes, clones were isolated from a some 17 as its only human component. human YAC library by B. Brownstein (Washington Univer- Three different methods were used to isolate end-specific sity) by PCR using primers (MYHEM; Table 1) that amplify sequences (see Materials and Methods). PCR products cor- a unique product from the embryonic skeletal MYH gene. responding to the 18 ends of the nine YACs were generated. This screen yielded nine YACs. A second screening of this During an initial analysis, 14 of these products were hybrid- library was conducted at the Genome Center of the Baylor ized to the nine YACs. Six of the products hybridized to all College ofMedicine using a pair ofprimers from the perinatal YACs, indicating that they contain repetitive sequences. Of MYH gene (MYHPN). This screen yielded five YACs. The the remaining eight end-specific probes tested, four electrophoretic karyotype of each clone was obtained by (A288E1L, A288E1R, A56B1OR, B12OC11L) hybridized to PFGE and the sizes of the YACs containing human inserts subsets ofthe family of YACs, enabling us to infer that these were determined by hybridization to total human DNA and probes are chromosome 17 specific and overlapping. These to MYH gene-specific probes. From both screenings, 3 PCR products were cloned and partially sequenced to gen- clones had YACs containing human genomic DNA but failed erate primer pairs that define STSs for this part of chromo- to hybridize to the gene-specific MYHem or MYHpn probes. some 17 (Table 1). The others (A213C4L, B120C11R, These clones (A258E1, A201F4, A120F5) were not subjected A23D7L, B72A12L) did not hybridize to any YACs other to further analysis. The sizes of YACs from the remaining 11 than the one from which they were derived. Primers that clones ranged from 135 to 410 kb. Results of hybridization of define B12OC11R amplify the appropriate size product from the YACs to human DNA are shown in Fig. 1. the DNA of the somatic cell hybrid containing chromosome Characterization ofthe YAC Clones. To begin to determine 17, suggesting that it constitutes one ofthe ends ofthe contig. the degree ofoverlap between the clones, repetitive sequence Probe B72A12L was derived from a YAC that contained fingerprints were determined by digesting total yeast DNA markers internal to the contig and did not hybridize to other with EcoRI, blotting, and hybridization with total human members ofthe contig. We conclude that this probe is derived DNA. In the case of YACs that were either indistinguishable from a chromosome other than 17 and the YAC is chimeric. or very close in size and in their fingerprint patterns, only one The other two end products, A213C4L and A23D7L, hybrid- member of these pairs was used for further analysis. This ized to the YACs from which they were derived but not to any eliminated A256D4, which was identical to A253D4, and others, suggesting that one was chimeric and one represents eliminated A288F8, which was 20 kb smaller but entirely the true end of the contig. Since this determination had no overlapping with A288E1. This process of elimination left effect on ascertaining the organization of the MYH gene five YACs that hybridized to the MYHm probe and four cluster (see below), it was not investigated further. These YACs that hybridized to the MYHpn probe. data established the contig encompassing the MYH gene Construction of the YAC Contig. Although the fingerprint cluster. The level ofchimerism observed (30%o) was similar to patterns provided clues to the degree of overlap between that described in an earlier report about this library (36). We different YACs, they were insufficient to construct a YAC confirmed the nature of the contig by PFGE analysis of contig. A simple method to construct YAC contigs is to individual YACs (data not shown). The YAC contig estab- generate a series of DNA markers internal to the YACs and lished by all of these analyses is shown in Fig. 2. The from their ends followed by ascertainment of their presence estimated size of the contig is 650 kb. in each ofthe YACs. Such an analysis permits ordering ofthe Organization of the MYH Genes. To understand the orga- markers as well as of the YACs with respect to each other. nization of the individual members of the MYH gene family The three types of markers we used to construct this contig within the contig, DNA from each YAC was hybridized with Downloaded by guest on September 26, 2021 Genetics: Yoon et al. Proc. Natl. Acad. Sci. USA 89 (1992) 12081 I-,.-v",- + ¶e995f0q i i.oo 'b

A213C4

A24D2 -- -a-.-.-

A258D4 . __

A23D7

A288E1 i_

B72A12 _ __ A51D4

A56B10 O_* B12OC11 ,

FIG. 2. Physical map ofthe skeletal MYH gene cluster. The top line shows the different probes that are used to establish the physical map. The relative distances between all markers are not known. Markers MYH2, p10-5, and A56B1OR are within a 15-kb region. Open circles, location of individual MYH genes; closed circles, STSs derived from YACs; open boxes, DNA marker; closed boxes, ends that have not been analyzed; dashed lines, uncertainty about the length of that region; wavy lines, sequences from a chromosome other than 17.

MYH gene-specific DNA probes or PCR was carried out with contig and MYH2 was determined by hybridizing a single- gene-specific primers (see Table 1). These probes correspond copy sequence from MYH2 (p10-s in ref. 3) to DNA from the to five distinct MYH genes and include the 3' untranslated nine YACs. Clones A56B10 and B12OC11 showed positive regions of the embryonic and perinatal skeletal MYH DNAs hybridization and yielded an EcoRI band of appropriate size (16, 32), an adult fast skeletal MYH cDNA (33), and a cDNA (data not shown). In addition, the STS A56B1OR was de- from an MYH gene expressed primarily in adult skeletal tected to be present in a A genomic clone containing the muscle (I. Karsch-Mizrachi and L.L., unpublished data). The MYH2, thus defining the end ofthe YAC to occur within the fifth probe corresponds to an adult skeletal MYH gene MYH2 gene. The 10-kb EcoRI band corresponds to a sixth (MYH2) that was used earlier in linkage analysis and permit- MYH gene whose identity is not yet known. Based on these ted its assignment to 17p13.1 (15). Because of the known data, we conclude that the order ofthe six MYH genes in this genetic location, this probe allowed us to integrate the cluster is MYHem (MYH3)-MYHas-MYHa-MYHpn (MYH8)- physical map of the MYH cluster into the genetic map of MYH?-MYH2. To establish that the organization ofthe MYH chromosome 17. These analyses permitted us to conclude genes in the YACs represents the pattern in the human that these five MYH genes are located within the contig. Two genome, human genomic DNA was digested with EcoRI and ofthe YACs, B72A12 and A51D4, contained the 5' end ofthe hybridized to LL1000. Several bands in the 10- to 12-kb size MYHp. but did not contain the 3' end of this gene. A56B10 and B12OC11 contained the 5' and 3' ends ofthe gene. Based on these data, the transcriptional orientation ofMYHp. gene, with respect to the contig, can be unambiguously established (left to right in Fig. 2). To determine whether there were additional MYH genes in the YAC contig and to determine the order ofthe genes within the cluster, DNA from each of the YACs was digested with EcoRI and blot-hybridized with a probe derived from a highly conserved region corresponding to the myosin rod (LL1000). Results of this analysis are shown in Fig. 3. YACs A213C4, A24D2, A258D4, and A23D7, containing only the MYHFm gene, yielded an 11-kb EcoRI fragment (Fig. 3, lanes b-e), consistent with the known organization of the MYH[I. gene (3). YAC A288E1, which is known to contain the MYH.m and the MYHa8 genes (Fig. 3, lane f), shows the 11-kb band and another slightly larger (412 kb) band that must correspond to the MYHaa gene. YAC B72A12 contains the MYH and MYHa as well as the 5' end of the MYHpn gene. This YAC (Fig. 3, lane g) has the MYH,,8 band and, in addition, has two other bands 1.7 kb and 0.9 kb in length. YACs A56B10 and B12OC11 contain bands corresponding to the MYHasg, MYHa, and MYHIp, genes. Thus, the 1.7-kb band must correspond to MYHa and the 0.9-kb band must correspond to YACs A56B10 and B120C11 two MYHPn. contain other FIG. 3. Location of MYH genes in different YACs. Total yeast bands (2.1 and 10 kb) that represent two additional MYH DNA was digested with EcoRI and blot-hybridized with a MYH 3' genes. The 2.1-kb EcoRI band corresponds to the adult consensus sequence probe (LL1000). Lanes: a, YPH252 (no YAC); skeletal MYH gene (MYH2) that had previously been used in b, A213C4; c, A24D2; d, A258D4; e, A23D7; f, A288E1; g, B72A12; linkage analysis (15). The relationship between the YAC h, A51D4; i, A56B10; and j, B12OC11.

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range and all other bands observed in individual YACs were 4. Whalen, R. G., Sell, S. M., Butler-Browne, G., Schwartz, K., observed (data not shown). These results indicate that the Bouveret, P. & Pinset-Harstrom, 1. (1981) Nature (London) 292, organization ofthe MYH genes in the YAC contig represents 803-809. the 5. Rubinstein, N. & Kelly, A. (1981) J. Cell Biol. 90, 128-144. organization of these genes in the genome. 6. Nadal-Ginard, B., Medford, R., Nguyen, H., Periasamy, M., Wy- dro, R., Hornig, D., Gubits, R., Garfinkel, L., Weiczorek, D., DISCUSSION Bekesi, E. &Mahdavi, V. (1982) in Muscle Development:Molecular and Cellular Control, eds. Pearson, M. L. & Epstein, H. F. (Cold Nine YAC clones containing six distinct human skeletal Spring Harbor Lab., Cold Spring Harbor, NY), pp. 43-168. MYH genes on chromosome 17 have been isolated and 7. Weydert, A., Daubas, P., Caravatti, M., Minty, A., Bugaisky, G., characterized. The physical map encompasses 650kb and has Cohen, A., Robert, B. & Buckingham, M. (1983)J. Biol. Chem. 258, been defined with 13 STSs. Two of the YACs, A288E1 and 13867-13874. B120C11, which together comprise 500 kb of DNA, contain 8. Lyons, G., Ontell, M., Cox, R., Sasson, D. & Buckingham, M. (1990) J. Cell Biol. 111, 1465-1476. all of the MYH genes known to be located in this region. 9. Pette, D. & Staron, R. S. (1990) Rev. Physiol. Biochem. Pharmacol. MYH genes have been mapped to 17p by a number of 116, 1-76. different methods. Of the genes mapped, MYH2 is mapped 10. Barany, M. (1967) J. Gen. Physiol. 50, 197-218. with the greatest precision to 17p13.1 (15). Because this gene 11. Saez, L. J., Gianola, K. M., McNally, E., Feghali, R., Eddy, R., is contained in the YAC contig, it is possible to assign all six Shows, T. & Leinwand, L. (1987) NucleicAcidsRes. 15,5443-5459. MYH genes to 17p13.1. 12. Weydert, A., Daubas, P., Lazaridis, I., Barton, P., Garner, I., In the current study, the six MYH genes that have been Leader, D., Bonhomme, F., Catalan, J., Simon, D., Guenet, J., Gros, F. & Bucki gham, M. (1985) Proc. Natl. Acad. Sci. USA 82, found in the 650-kb YAC contig include embryonic, perinatal, 7183-7187. three adult skeletal fast forms (either Ila, Ilb, or lId), and an 13. Gulick, J., Subramaniam, A., Neumann, J. & Robbins, J. (1991) J. unidentified gene. Four have been identified by comparison Biol. Chem. 26, 9180-9185. with cloned cDNAs. Two YAC fiagments that hybridize to a 14. Edwards, J., Parkar, M., Povey, S., West, L. F., Parrington, J. M. consensus MYH probe are evident at the rightward end ofthe & Solomon, E. (1985) Ann. Hum. Genet. 49, 101-109. locus (YACs A56B10 and B120C11). One of these additional 15. Schwartz, C. E., McNally, E., Leinwand, L. & Skolnick, M. H. bands (2.1-kb corresponds to an adult skeletal MYH (1986) Cytogenet. Cell Genet. 43, 117-120. EcoRI) 16. Karsch-Mizrachi, I., Travis, M., Blau, H. & Leinwand, L. A. (1989) locus from which arestriction fiagment length polymorphism Nucleic Acids Res. 17, 6167-6179. (plo05) has been described and used in linkage analysis (3, 17. Karsch-Mizrachi, I., Feghali, R., Shows, T. B. & Leinwand, L. A. 15). Its precise idetjfity has not yet been determined and will (1990) Gene 89, 289-294. require DNA sequence and RNA hybridization analysis, but 18. Cox, R., Weydert, A., Barlow, D. & Buckingham, M. (1991) Dev. preliminary data suggest it is an adult fast MYH form. The Biol. 143, 36-43. sixth MYH gene in the contig does not correspond to any of 19. Tbornburg, J., Bauer, B., Palermo, J. & Robbins, J. (1992) Dev. the available human genes or cDNA sequences and is a Biol. 159, 99-107. likely 20. Tsika, R. W., Bahl, J. J., Leinwand, L. A. & Morkin, E. (1990) candidate for the extraocular MYH gene. Proc. Natd. Acad. Sci. USA 87, 379-383. Several examples of related gene clusters include- globin, 21. Subramaniam, A., Jones, W., Gulick, J., Wert, S., Neumann, J. & proline-rich salivary , genes, homeobox, and Robbins, J. (1991) J. Biol. Chem. 266, 24613-24620. the major histocompatibility complex. In the globin gene 22. Geisteafer-Lawrance, A. A., Kass, S., Tanigawa, G., Vosberg, complex and the homeobox clusters, the organization of the H.-P., McKenna, W., Sediman, C. E. & Seidman, J. G. (1990) Cell 62, 999-1006. genes reflects the developmental pattern of expression (37, 23. Green, E. D. & Olson, M. V. (1990) Proc. Natl. Acad. Sci. USA 87, 38). It has been suggested that structural organization within 1213-1217. a cluster might reflect the functional aspects of individual 24. Burke, D. T., Carle, G. F. & Olson, M. V. (1987) Science 236, members. The organization of the skeletal MYH genes does 806-812. not follow their developmental expression order. For exam- 25. Schwartz, D. C. & Cantor, C. R. (1984) Cell 37, 67-75. 26. Chu, G., Vollrath, D. & Davis, R. W. (1986) Science 234, 1582- ple, the embryonic and perinatal MYH genes are expressed 1585. sequentially during development but are not located adjacent 27. Davis, R. W., Thomas, M., Cameron, J. R., St. John, T. P., to each other. The functional significance of the organization Scherer, S. & Padgett, R. A. (1980) Methods Enzymol. 65,404-411. of MYH genes, if any, has yet to be elucidated. 28. Maniatis, T., Fritsch, E. F. & Sambrook, J. (1982) Molecular Understanding the organization of the MYH genes may Cloning: A Laboratory Manual (Cold Spring Harbor Lab., Cold permit us to initiate studies ofpathological aspects associated Spring Harbor, NY), p. 156. with defects of these genes. 29. Riley, J., Butler, R., Ogilvie, D., Finniear, R., Jenner, D., Powell, Examination of the organization S., Anand, R., Smith, J. C. & Markham, A. F. (1990) Nucleic Acids of the MYH genes in patients who have dystrophic muscle Res. 18, 2887-2890. may indicate that, in some of these patients, abnormalities in 30. Ledbetter, S. A., Nelson, D. L., Warren, S. T. & Ledbetter, D. H. the individual members of the MYH gene family cause the (1990) Genomics 6, 475-481. pathology. 31. Silverman, G. A., Ye, R. D., Pollock, K. M., Sader, J. E. & Korsmeyer, S. J. (1989) Proc. Natd. Acad. Sci. USA 86, 7485-7489. The manuscript was prepared by V. Gradus. We also acknowledge 32. Feghali, R. & Leinwand, L. A. (1989) J. Cell 8iol. 10, 1791-1797. the contribution of somatic cell hybrid DNA from R. E. K. Fournier 33. Saez, L. J. & Leinwand, L. A. (1986) Nucleic Acids Res. 14, (Fred Hutchinson Cancer Center, Seattle) and the contribution of 2951-2969. Ilene Karsch-Mizrachi in initiating the analysis of MYH sequences in 34. Feinberg, A. P. & Vogelstein, B. (1983) Anal. Biochem. 132, 6-13. the YAC clones. 35. Kraft, R., Tardiff, J., Krauter, K. S. & Leinwand, L. A. (1988) This work is supported by grants from the National Biotechniques 6, 544-547. Institutes of Health (GM33943 and HG00380 to R.K. and GM29090 to 36. Green, E. D. & Olson, M. V. (1990) Science 250, 94-98. L.L.), a Wills Foundation grant to R.K., and a Cancer Center grant 37. Efstraiatis, A., Posakony, J. W., Maniatis, T., Lawn, R. M., to the Albert Einstein College of Medicine (CA13330). L.L. is the O'Connell, C., Spritz, R. A., DeRiel, J. K., Forget, B. G., Weiss- recipient ofan IrnaT. Hirschl Award. The YAC libraries were kindly man, S. M., Slightom, J. L., Blechl, A. E., Smithies, O., Baralle, screened by Dr. B. Brownstein (Washington University) and by the F. E., Shoulders, C. C. & Proudfoot, N. J. (1980) Cell 21, 653-668. Genome Center of the Baylor College of Medicine (Houston). 38. Hart, C. P., Fainsod, A. & Ruddle, F. H. (1987) Genomics 1, 182-195. 1. Weydert, A. (1988) Bull. Inst. Pasteur 86, 157-210. 39. 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