GENOMICS

7,%9-293

(1990)

SHORT COMMUNICATION Human Embryonic/Atria1 Myosin Alkali Light Chain Gene: Characterization, Sequence, and Chromosomal Location JEGATHEESAN SEHARASEYON,* EVA Bom,t CHIH-LIN HSIEH,$ WILLIAM L. FODOR,~ UTA FRANCKE,* HANS-HENNING ARNOLD,t AND ELIO F. VANIN*” *Department of Neurology, Ohio State University, Columbus, Ohio 432 10; tDepartment of Toxicology, Medical School, University of Hamburg, Hamburg 13, Federal Republic of Germany; *Howard Hughes Medical Institute Research Laboratories, Beckman Center for Molecular Genetics, Stanford University Medical Center, Stanford, California 943055428; and §Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, Connecticut 06510 Received

November

22. 1989;

revised

February

7, 1990

muscle tissues express an embryonic isoform, MLC1emb.The MLC-l,,b protein has been found to be transiently expressed during the development of skeletal muscle in chicken (Katoh and Kubo, 1978; TakanoOhmuro et aZ., 1985), rat (Whalen et al., 1978; Whalen and Sell, 1980), and man (Cummins et aZ., 1980; Strohman et at., 1983). Interestingly, the MLC-l,,b isoform from both fetal ventricular (Cummins et al., 1980) and fetal skeletal muscle (Whalen et al., 1978,1982; Whalen and Sell, 1980) has been found to corn&rate with the atria1 myosin alkali light chain isoform MLC-lA . The isolation of cDNA clones corresponding to the chicken isoforms (Kawashima et al., and human MLC-l,,,, 1987; Arnold et al., 1988), the mouse and human MLC1~ isoforms (Barton et al., 1985; Kurabayashi et al., 1988), and the mouse MLC-l,,, gene (Barton et al., 1988) has been reported. Kawashima et al. (1987) found that MLC-1 embmRNA (L23 mRNA) was present in chicken embryonic skeletal, cardiac, and smooth muscle, confirming the results obtained previously by protein analysis. Arnold et al. (1988) found that mRNA species that hybridized to their human MLC-&, isoform cDNA clone were present in human fetal skeletal and cardiac muscle as well as in adult atria. Kurabayashi et al. (1988), using a human MLC-1A isoform cDNA clone, found that hybridizing mRNAs were present in fetal ventricle, atria, and iliopsoas muscle as well as in adult atria. Barton et al. (1985) found that the mouse MLC-1~ mRNA was present in adult atria and in the Purkinje fibers of the heart. Thus, only in humans have cDNA clones corresponding to both the MLC-l,,b and the MLC-lA mRNAs been isolated. In this communi-

We have isolated and sequenced the gene encoding the human embryoniciatrial myosin alkali light chain isoform (MLC-1 emb,A). The gene is split into seven exons by six introns; the last exon, as in all MLC isoform genes sequenced to date, is completely 3’ untranslated sequence. Comparison of the MLC-l,,,,A isoform gene with the other MLC-1 genes showed that the exonintron arrangement of the human MLC- lemb,* isoform gene is analogous to that of the other MLC- 1 type isoform genes. We have also mapped the human MLCl,,,,* isoform gene to the long arm of chromosome 17; the corresponding mouse gene has been mapped to chromosome 11. This gene, together with a number of others such as the collagen(I) arl, galactokinase, and thymidine kinase genes, is part of the largest syntenic group between mouse and man. o IBSOAcademic P~BPS, IIIC.

Myosin, which is the major structural and functional component of muscle myofibrils, is composed of two heavy chains (MHC) and two pairs of light chains (MLC) forming a hexamer. The light chains can be divided into phosphorylatable or regulatory light chains (MLC-2 type) and nonphosphorylatable or “alkali” light chains (MLC-1 and MLC-3 type). Analysis of the myosin alkali light chains that are expressed during development has shown that some ’ To whom correspondence should be addressed. Present address: Promega Corporation, 2800 Woods Hollow Road, Madison, WI 53711.

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osss-7543/90 $3.00 Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.

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aggaggaagaagaggaggagg-----

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(6.8kbp)-----aggctgggtcttctctccacagAGTTC~QA~~~CA~Q~T~c 1uPhGLysQluAlaPhoGGrLGuPhGAsp

QCCCM~CT(ULaOgtcagtgcggtcgcatggcagacctctc~cagggtccagtgtgcacgcccttggcttcc LGuArgV81LGuQlyLysProLy8ProQlaQ CTQCQTQTQCPQQQCM

tccttgtccag-----(1.7kbp)

-----ccttgagcacctgcctacattgatttctctttctttaccctgcctgcctgaagA~T~T lull.tAan

(0.5kbp)-----accacctcccaaacccaccgcaqQAQAQMQATQACT

cagagatgaagaccaagtgggaggaatggagggtgg-----

gggcc-----(0.6kbp)

gccctagaaggcatctgtc-----(0.3kbp)

1yQluLyGBlGtThr

---cctagaggcgctgagccacatggtggctgattttcacttttttcctagCC~TQTCM~ACA~TQ

laPhGValLysHi8I1GHGt

---tgggtctaggagggtgaaatgccctttcctcctagcacttctttcagQ~C~~

FIG. 1. Nucleotide sequence of the human MLC-1 e,,,b,~ isoform gene. The nucleotide sequences of exons 1 through flanking sequences are presented in capital letters, while the intron sequences are in lowercase. The numbers within the approximate distance between the sequences shown. The predicted amino acid sequence, in the three-letter terminology, is nucleotide sequence. The transcriptional start site and the site of polyadenylation are indicated by vertical arrow heads. The at -30 to -26 and the “AATAAA” hexanucleotide are all indicated by asterisks.

cation we report the isolation, characterization, and chromosomal localization of the human MLC-l,,,, isoform gene. Initially, a human partial Sau3AI library in Charon 28 was screened using a 431-bp EcoRI fragment from

7 and the 5’ and 3’ introns indicate the indicated “TATA"

below

the

homology

the rat MLC-lr isoform cDNA (Periasamy et aZ., 1984). This approach was previously used to isolate the human MLC-lv/sb isoform gene (Fodor et aZ., 1989). Three clones were isolated and each contained a 6.0-kb EcoRI fragment that hybridized to the probe. The same size

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TABLE Summary

291

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1

of Rodent-Human

Somatic

Cell Hybrid

Human Hybridization/chromosome

+/+ -/+/-/+ Discordant Informative

hybrids hybrids

1

2

3

4

5

10001102001011102121121 9 10 4 5 10 12321110220211120101001 4 4 8 6 3 5 15

6 16

11 15

8 13

4 15

Data

chromosome

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

X

4

10

9

10

12

8

8

7

2

6

6

14

I

7

10

4

5

3

9

3

4

3

2

5

6

7

10

8

6

0

6

I

4

8

8

3

10 15

4 14

4 15

5 15

4 16

5 14

8 16

8 16

11 14

9 16

8 14

0 16

7 15

7 16

5 16

8 13

8 15

4 8

Note. The results of both EcoRI and HindI digests of DNAs from various rodent-human somatic cell hybrid lines are summarized. The numbers under each human chromosome indicate the total number of hybrids that gave results marked as either +/+, -/-, +/-, or -/+. The +/+ indicates that hybridization to the human-specific fragment was observed and that the respective chromosome was present. For example, only one somatic cell hybrid that contained chromosome 1 was found to contain the human-specific fragment upon genomic Southern analysis. Furthermore, the +/indicates that hybridization to the human-specific fragment was seen yet the somatic cell hybrid analyzed did not contain that particular chromosome, and -/+ that hybridization to the specific fragment was not seen yet that particular chromosome was present. The number of discordant hybrids under a particular human chromosome indicates the total number of hybrids that were either +/- or -/+. The number of informative hybrids under each human chromosome indicates the total number of hybrids analyzed that contained that particular chromosome intact and at a frequency of greater than 0.1 copy/cell.

EcoRI fragment was observed when an EcoRI digest of human genomic DNA was probed with both the rat cDNA probe used above and a human MLC-l,,b isoform cDNA clone (GT14), which has subsequently become available (Arnold et al., 1988). Upon further analysis it was found that these clones did not contain exons 1 and 2 of the human MLC-&,,A gene. Therefore a different human partial Sau3AI library in EMBL3 was screened with the insert from the human MLC-l,,,, cDNA clone and two clones were isolated, one of which contained the entire gene and the other only exons 1 and 2. Exons 3,4, and 5 of the gene were localized using fragments of the rat MLC-lr isoform cDNA. The locations of exons 1, 6, and 7 were determined using the appropriate fragments from the human MLC- lembisoform cDNA (Arnold et al., 1988), while exon 2 was localized using an oligonucleotide probe. The complete sequence of the human MLC-l,,,,~ isoform gene together with 5’ and 3’ flanking regions is shown in Fig. 1. Comparison of this gene sequence with that of the human MLC-l,,b cDNA allowed us to define precisely the exon-intron boundaries of this gene. The gene is divided into seven exons of 208,28, 150,174,78,44, and 171 bp. Six of these exons, exons 1 through 6, code for a polypeptide of 197 amino acids with each exon coding for 45,9,49,57,25, and 8 amino acids, respectively; four amino acids are encoded by codons that are formed as a result of exon splicing. Exon 7 is completely 3’ untranslated sequence. Upon comparing the cDNA clone corresponding to the human MLC-l,,,,b isoform mRNA (Arnold et al., 1988) with the cDNA corresponding to the human MLC-1~ isoform mRNA (Kurabayashi et al., 1988), we found a

total of seven differences, consisting of six base differences and a deletion (in the 3’ untranslated region). At each of these positions the genomic sequence that we report above is identical to the sequence of Arnold et al. (1988). We also determined the chromosomal location of the human MLC-l,,,,,,, gene (MYLH) using a panel of 16 rodent X human somatic cell hybrids (Yang-Feng et aZ., 1986). The probe used in these studies was a 0.3kb SmaI-KpnI fragment which contains exon 4 of the human MLC-l,,b,* isoform gene as well as flanking intron sequences.Hybridization of this probe to EcoRIdigested human genomic DNA detected a single 6.0kb fragment and a 5.0-kb EcoRI fragment in mouse genomic DNA, but no band was detected in Chinese hamster genomic DNA. The human-specific 6.0-kb EcoRI was observed in every hybrid in which human chromosome 17 was present, whereas it was not observed when human chromosome 17 was absent. Similar results were obtained when Hind111 was used instead of EcoRI (data not shown). In other words, the presence or absence of the human MYL4 sequence was in perfect concordance with human chromosome 17 in hybrid cell lines (Table 1). All other human chromosomes were excluded by at least 4 discordant hybrids. We also found that the 6.0-kb EcoRI human-specific fragment was present in the mouse-human hybrid containing only the 17q21-qter chromosomal region. These results are in agreement with the assignment of MYL4 to human chromosome 17 reported by CohenHaguenauer et al. (1989). Using the same SmuI-KpltI exon 4 containing fragment, we have also localized the IIlOUSe MLC- lem,,/Aisoform gene. A total of 12 Chinese

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hamster-mouse somatic cell hybrids having reduced numbers of mouse chromosomes (Francke et al., 1977; Francke and Taggart, 1979) were analyzed and the mouse-specific fragment was found to be absent in all of the hybrid DNAs. Each of the mouse chromosomes, except chromosome 11, is present in at least one of the hybrids tested. Therefore these results indicate that the mouse MLC-l,,,,,~ gene must be on mouse chromosome 11. This is consistent with the results of Robert et al. (19&S), who were able to localize the mouse MLC-led/A isoform gene to chromosome 11 by following the pattern of segregation of RFLPs through an F1 backcross of MUS musculus and Mus spretus. It is interesting to note that a large conserved syntenic group of genesexists on human chromosome 17 and the distal portion of mouse chromosome 11 (Munke and Francke, 1987). In fact, all loci on human chromosome 17 that have been mapped in mouse are on mouse chromosome 11 (Lalley et al., 1988). This conserved region represents one of the largest conserved regions identified between mouse and man to date (Nadeau and Taylor, 1984; Nadeau and Reiner, 1989).

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9. ACKNOWLEDGMENTS J.S. and E.F.V. thank Arthur and Susan Burghes as well as the members of Dr. Arthur Burghes’ laboratory, in the Department of Neurology at Ohio State University, for their encouragement and support during this difficult year. Their timely intervention has helped in many ways and will continue to be of influence in the following years. We also thank Drs. J. Mendell and Rammohan, also of the Department of Neurology, for their encouragement; Louis DeLuca and Yana Kogan for help in the isolation of the phage clones; and Dr. Caroline Breitenberger, Dr. Vidya Rao, and Barbara Kemp Duerr for helpful discussions. E.B. and H.H.A. were supported by the Deutsche Sorschungsgen., BSG Grant 2HHA, and the Deutsche Muskelschwundbilfe. C.-L.H. and U.F. were supported by NIH Grant GM26105. U.F. is an Investigator at the Howard Hughes Medical Institute, and C.-L.H. is an Associate Investigator at the Howard Hughes Medical Institute.

REFERENCES ARNOLD, H.-H., LOHSE, P., SEIDEL, U., AND BOBER, E. (1988). A novel human myosin alkali light chain is developmentally regulated: Expression in fetal cardiac and skeletal muscle and in adult atria. Eur. J. Biochem. 178: 53-60. BARON, P. J. R., ROBERT, B., COHEN, A., GARNER, I., SASSOON, D., WEYDERT, A., AND BUCKINGHAM, M. E. (1988). Structure and sequence of the myosin alkali light chain gene: Expressed in adult cardiac atria and fetal striated muscle. J. Bid. Chem. 269: 12,669-12,676. BARTON, P. J. R., ROBERT, B., FIZMAN, M. Y., LJZADER,D. P., AND BUCKINGHAM, M. E. (1985). The same myosin alkali light

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chain gene is expressed in adult cardiac atria and fetal skeletal muscle. J. Muscle Res. Cell Motil. 6: 461-475. COHEN-HAGUENAUER, O., BARTON, P. J. R., VAN CONG, N., COHEN, A., MASSET, M., BUCKINGHAM, M., AND FREZAL, J. (1989). Chromosomal assignment of two myosin alkali light chain genes encoding the ventricular/slow twitch skeletal muscle isoform and the atrial/fetal muscle isoform (MYL3, MYL4). Hum. Genet. 81: 278-282. CUMMINS, P., PRICE, K. M., AND LI’ITLER, W. A. (1980). Foetal myosin light chain in human ventricle. J. Muscle Res. Cell. Motil. 1: 357-366. FODOR, W. L., DARRAS, B., SEHARASEYON, J., FALKENTHAL, S., FRANCKE, U., AND VANIN, E. F. (1989). Human ventricular/ slow twitch myosin alkali light chain gene: Characterization, sequence and chromosomal location. J. Bid. C&em. 264: 21432149. FRANCKE, U., LALLEY, P. A., Moss, W., Iw, J., AND MINNA, J. D. (1977). Gene mapping in Mus musculus by interspecies cell hybridization: Assignment of the genes for tripeptidase-1 to chromosome 10, dipeptidsse-2 to chromosome 12, and adenylate kinase-1 to chromosome 2. Cytogenet. Cell Genet. 19: 57-84. FRANCKE, U., AND TAGGART, R. T. (1979). Assignment of gene for cytoplasmic superoxidase dismutase (Sod-l) to a region of chromosome 16 and of Hprt to a region of the X chromosome in the mouse. Proc. Natl. Acad. Sci. USA 76: 5230-5233. KATOH, N., AND KUBO, S. (1978). Light chains of chicken embryonic gizzard myosin. Biochim. Biophys. Acta 635: 401-411. KAWASHIMA, M., NABESHIMA, Y.-I., OBINATA, T., AND FUJIIKURIYAMA, Y. (1987). A common myosin light chain is expressed in chicken embryonic skeletal, cardiac and smooth muscle and in brain continuously from embryo to adult. J. Bid. Chcm. 262: 14,408-14,414. KURABAYASHI, M., KOMURO, I., TSUCHIMOCHI, H., TAKAKU, F., AND YAZAKI, Y. (1988). Molecular cloning and characterization of human atria1 and ventricular myosin slksli light chain cDNA clones. J. Biol. C&m. 263: 13,930-13,936. LALLEY, P. A., DAVISSON, M. T., GRAVES, J. A. M., O’BRIEN, S. J., RODERICK, H., DOOLITTLE, D. P., AND HILLYARD, A. L. (1988). Report of the committee on comparative mapping. Cytogenet.

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M., AND FRANCKE, U. (1987). The physical map of Mus musculus chromosome 11 reveals evolutionary relationships with different syntenic groups of gene in Homo sapiens. J. Mol. Euol. 25: 134-140. 14. NADEAU, J. H., AND REINER, A. (1989). A chromosomal map of linkage homologies in mouse and man. In “Genetic Variants and Strains of the Laboratory Mouse” (M. F. Lyon and A. G. Searle, Eds.), Oxford Univ. Press, Oxford/London. 15. NADEAU, J. H., AND TAYLOR, B. A. (1984). Lengths of chromosomal segments conserved since divergence of man and mouse. Proc. Natl. Acad. Sci. USA 81: 814-818. MUNKE,

16. PERIASAMY, M., STREHLER, E. E., GARFINKEL, L. I., GUBITS, R. M., RUIZ-OPAZO, N., AND NADAL-GINARD, B. (1984). Fast skeletal muscle myosin light chains are produced from a single gene by a combined process of differential RNA transcription and splicing. J. Bid. Chem. 269: 13,595-13,604. 17. ROBERT, B., BARTON, P., MINTY, A., DAUBAS, P., WEYDERT, A., BONHOMME, F., CATALAN, J., CHAZO~ES, D., GUENET, J.-L., AND BUCKINGHAM, M. (1985). Investigation of genetic

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atrial myosin alkali light chain gene: characterization, sequence, and chromosomal location.

We have isolated and sequenced the gene encoding the human embryonic/atrial myosin alkali light chain isoform (MLC-1emb/A). The gene is split into sev...
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