Abstract
We have isolated a non-muscle myosin heavy chain gene from Acanthamoeba castellanii using as a heterologous probe a sarcomeric myosin heavy chain gene from Caenorhabditis elegans. The amoeba genomic clone has been tentatively identified as containing a myosin II heavy chain gene based on hybridization to a 5300-nucleotide RNA species, hybrid selection of a mRNA encoding a 185-kDa polypeptide, specific immunoprecipitation of this polypeptide with antiserum to myosin II, and an exact match between the DNA sequence and a carboxyl-terminal myosin II peptide previously sequenced by protein chemical methods (Côté, G.P., Robinson, E.A., Appella, E., and Korn, E. D. (1984) J. Biol. Chem. 259, 12781-12787). We also sequenced a region of the gene whose deduced amino acid sequence shows strong homology with that region of muscle myosins which is thought to be involved in nucleotide binding. These results indicate that the amoeba genomic clone contains at least 90% of the coding information for the 185-kDa heavy chain polypeptide and that the bulk of the gene contains very little intron DNA. Genomic blots of amoeba DNA probed with a portion of this myosin gene indicate the presence of additional highly related sequences within the amoeba genome.
Highlights
We have isolated a non-muscle myosin heavy chain chain of myosin 11
We found an exact match between a region of DNA sequence in phage clone X4 e 13 and a 58-amino acid residue sequence previously determined by protein chemical sequencing of a carboxyl-terminal, chymotryptic peptide of the myosin I 1 heavy chain
Vector DNA,and mRNAselected by phage cloneX4.13 DNA shown are the position of the SstII site in the overlapping 1.9-kb .Lunes 4 and 5,autoradiograms of the [35S]methionine-labeled Kpn/SstII fragment, the termination codon,and proteins immunoprecipitated from the X4.13 DNA-selected material the genesequencewhich is complimentary to the oligonucleotide using a polyclonal antiserum to myosin I1 or non-immune probe.We have tentatively identified by nuclease S-1 serum.Lane 6, autoradiogram of the [35S]methionine-labeled mapping a splice site a t nucleotide 56
Summary
Vol 261,No.4,Issue of February 5,pp. 1949-1956,1986 Printed in U.S.A. From the Laboratory of Cell Biology,National Heart, Lung, and Blood Institute and the$Laboratory of Biochemistry, National Cancer Institute, National Instituteosf Health, Bethesda, Maryland 20205. The amoeba genomic clone has been tentatively identified as containing a myosin I1 heavy chain gene based on hybridization to a 5300-nucleotide RNA species, hybrid selection of a mRNA encoding a 185-kDa polypeptide, specific immunoprecipitation of this polypeptide with antiserum to myosin 11, and an exact match between the DNA sequence and a carboxyl-terminal myosin I1 peptide sarcomeric myosin heavy chain genes have been isolated from both vertebrates [4,5,6,7,8,9,10,11] and invertebrates [12,13,14], this is the first report of the isolation of a non-muscle myosin heavy chain gene.
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