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Myosins from angiosperms, ferns, and algae amplification of gene fragments with versatile PCR primers and detection of protein products with a monoclonal antibody to a conserved head epitope

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Summary

Myosins providing the motors for the actin-based motility that occurs in diverse plants have proved difficult to study. To facilitate those studies, we describe polymerase chain reaction primers that reliably amplify part of the myosin head from diverse plants, consensus sequences that characterise the amplified product as encoding a class V or class VIII myosin, and a monoclonal antibody that recognises an epitope conserved in the head of most plant, fungal, and animal myosins. A pair of stringent oligonucleotide primers was designed that, when used in the polymerase chain reaction, amplified at least eleven different myosins from five species of angiosperms and one sequence from each of the fernAzolla and the algaeNitella andPhaeodactylum. The amplified products, comprising 126 to 135 nucleotides encoding part of the myosin head domain, can be used as myosin-specific probes to screen genomic and cDNA libraries. To identify the products of plant myosin genes, we raised a monoclonal antibody (anti-CHE) to a nine amino acid peptide matching a conserved head epitope showing not more than single amino acid substitutions in most published myosin genes. This antibody recognises rabbit skeletal myosin and multiple polypeptides of >100 kDa in four angiosperms and in the algaNitella. Relating the Mr values of immunoreactive bands inArabidopsis extracts to the predicted Mr values of the products of five myosin genes supports the view that the antibody recognises both myosins V and VIII together with the products of some as yet unsequenced genes. The previously described MB170 antibodies may, in contrast, be specific for one or more type V myosins. Together, the polymerase chain reaction primers and the antibody represent versatile tools for identifying and categorising myosins in diverse plants.

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References

  • Baskin TI, Betzner AS, Hoggart R, Cork A, Williamson RE (1992) Root morphology mutants inArabidopsis thaliana. Aust J Plant Physiol 19: 427–437

    Google Scholar 

  • Cheney RE, Riley MA, Mooseker MS (1993) A phylogenetic analysis of the myosin superfamily. Cell Motil Cytoskeleton 24: 215–223

    Google Scholar 

  • Espreafico EM, Cheney RE, Matteoli M, Nascimento AAC, De Camilli PV, Larson RE, Mooseker MS (1992) Primary structure and cellular localization of chicken brain myosin V (p190), an unconventional myosin with calmodulin light chains. J Cell Biol 119: 1541–1557

    Google Scholar 

  • Feng D-F, Doolittle RF (1987) Progressive sequence alignment as a prerequisite to correct phylogenetic trees. J Mol Evol 25: 351–360

    Google Scholar 

  • Goodson HV, Spudich JA (1993) Molecular evolution of the myosin family: relationships derived from comparisons of amino acid sequences. Proc Natl Acad Sci USA 90: 659–663

    Google Scholar 

  • Grolig F, Williamson RE, Parke J, Miller C, Anderton BH (1988) Myosin and Ca2+-sensitive streaming in the algaChara: detection of two polypeptides reacting with a monoclonal anti-myosin and their localization in the streaming endoplasm. Eur J Cell Biol 47: 22–31

    Google Scholar 

  • Haarer BK, Petzold A, Lillie SH, Brown SS (1994) Identification ofMY04, a second class V myosin gene in yeast, J Cell Sci 107: 1055–1064

    Google Scholar 

  • Higashi-Fujime S, Ishikawa R, Iwasawa H, Kagami O, Kurimoto E, Kohama K, Hozumi T (1995) The fastest actin-based motor protein from the green algae,Chara, and its distinct mode of interaction with actin. FEBS Lett 375: 151–154

    Google Scholar 

  • Jablonsky PP, Elliott J, Williamson RE (1993) Purification of a mung bean protein binding to microtubules through two defined sites in the carboxyl-terminal domain of β-tubulin. Plant Sci 94: 35–45

    Google Scholar 

  • Johnston GC, Prendergast JA, Singer RA (1991) TheSaccharomyces cerevisiae MYO2 gene encodes an essential myosin for vectorial transport of vesicles. J Cell Biol 113: 539–551

    Google Scholar 

  • Joshi HC, Palacios MJ, McNamara L, Cleveland DW (1992) γ-Tubulin is a centrosomal protein required for cell cycle-dependent microtubule nucleation. Nature 356: 80–83

    Google Scholar 

  • Ketchum AS, Stewart CT, Stewart M, Kiehart DP (1990) Complete sequence of theDrosophila nonmuscle myosin heavy-chain transcript: conserved sequences in the myosin tail and differential splicing in the 5′ untranslated sequence. Proc Natl Acad Sci USA 87: 6316–6320

    Google Scholar 

  • Kinkema M, Schiefelbein J (1994) A myosin from a higher plant has structural similarities to class V myosins. J Mol Biol 239: 591–597

    Google Scholar 

  • —, Wang H, Schiefelbein J (1994) Molecular analysis of the myosin gene family inArabidopsis thaliana. Plant Mol Biol 26: 1139–1153

    Google Scholar 

  • Knight AE, Kendrick-Jones J (1993) A myosin-like protein from a higher plant. J Mol Biol 231: 148–154

    Google Scholar 

  • Korn ED, Hammer JA (1988) Myosins of nonmuscle cells. Annu Rev Biophys Biophys Chem 17: 23–45

    Google Scholar 

  • La Claire JW, Chen R, Herrin DL (1995) Identification of a myosinlike protein inChlamydomonas reinhardtii (Chlorophyta). J Phycol 31: 302–306

    Google Scholar 

  • Lin Q, Grolig F, Jablonsky PP, Williamson RE (1989) Myosin heavy chains: detection by immunoblotting in higher plants and localization by immunofluorescence in the algaChara. Cell Biol Int Rep 13: 107–117

    Google Scholar 

  • —, Jablonsky PP, Elliott J, Williamson RE (1994) A 170 kDa poly-peptide from mung bean shares multiple epitopes with rabbit skeletal myosin and binds ADP-agarose. Cell Biol Int 18: 1035–1047

    Google Scholar 

  • Margossian RB, Lowey S (1982) Preparation of myosin and its subfragments from rabbit skeletal muscle. Methods Enzymol 85: 55–71

    Google Scholar 

  • Menzel D (ed) (1992) The cytoskeleton of the algae. CRC Press, Boca Raton

    Google Scholar 

  • Mercer JA, Seperack PK, Strobel MC, Copeland NG, Jenkins NA (1991) Novel myosin heavy chain encoded by murine dilute coat colour locus. Nature 349: 709–713

    Google Scholar 

  • Miller DD, Scordilis SP, Hepler PK (1995) Identification and localization of three classes of myosins in pollen tubes ofLilium longiflorum andNicotiana alata. J Cell Sci 108: 2549–2563

    Google Scholar 

  • Moepps B, Conrad S, Schraudolf H (1993) PCR-dependent amplification and sequence characterization of partial cDNAs encoding myosin-like proteins inAnemia phyllitidis (L.) Sw. andArabidopsis thaliana (L.) Heynh. Plant Mol Biol 21: 1077–1083

    Google Scholar 

  • Mornet D, Bonet A, Audemard E, Bonicel J (1989) Functional sequences of the myosin head. J Muscle Res Cell Motil 10: 10–24

    Google Scholar 

  • Murray MG, Thompson WF (1980) Rapid isolation of high-molecular weight plant DNA. Nucleic Acids Res 8: 4321–4324

    Google Scholar 

  • Parke J, Miller C, Anderton BH (1986) Higher plant myosin heavychain identified using a monoclonal antibody. Eur J Cell Biol 41: 9–13

    Google Scholar 

  • Plazinski J, Zheng Q, Taylor R, Rolfe BG, Gunning BES (1989) Use of DNA/DNA hybridization techniques to authenticate the production of newAzolla-Anabaena symbiotic associations. FEMS Microbiol Lett 65: 199–204

    Google Scholar 

  • Rayment I, Rypniewski WR, Schmidt-Base K, Smith R, Tomchick DR, Benning MM, Winkelmann DA, Wesenberg G, Holden HM (1993) Three-dimensional structure of myosin subfragment-1: a molecular motor. Science 261: 50–58

    Google Scholar 

  • Reichlin M (1980) Use of glutaraldehyde as a coupling agent for proteins and peptides. Methods Enzymol 70: 159–165

    Google Scholar 

  • Reyrat JM, David M, De Philip P, Garnerone AM, Batut J, Boistard P (1993) In vivo and in vitro oxygen control of nitrogen fixation gene expression inRhizobium leguminosarum. In: Palacios R, Mora J, Newton WE (eds) New horizons in nitrogen fixation. Kluwer, Dordrecht, pp 399–403

    Google Scholar 

  • Warrick HM, Spudich J (1987) Myosin structure and function in cell motility. Annu Rev Cell Biol 3: 379–421

    Google Scholar 

  • Williamson RE (1993) Organelle movements. Annu Rev Plant Physiol Plant Mol Biol 44: 181–202

    Google Scholar 

  • —, Hurley UA (1986) Growth and regrowth of actin bundles inChara: bundle assembly by mechanisms differing in sensitivity to cytochalasin. J Cell Sci 85: 21–32

    Google Scholar 

  • —, Perkin JL, McCurdy DW, Craig S, Hurley UA (1986) Production and use of monoclonal antibodies to study the cytoskeleton and other components of the cortical cytoplasm ofChara. Eur J Cell Sci 41: 1–8

    Google Scholar 

  • Yamamoto K, Kikuyama M, Sutoh-Yamamoto N, Kamitsubo E (1994) Purification of actin based motor protein fromChara corallina. Proc Jap Acad Ser B 70: 175–180

    Google Scholar 

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Authors and Affiliations

  1. Plant Cell Biology Group, Research School of Biological Sciences, Institute of Advanced Studies, Australian National University, GPO Box 475, 2601, Canberra, ACT, Australia

    J. Plazinski, J. Elliott, U. A. Hurley, J. Burch & R. E. Williamson

  2. Cooperative Research Centre for Plant Science, Research School of Biological Sciences, Institute of Advanced Studies, Australian National University, Canberra, ACT

    T. Arioli & R. E. Williamson

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  1. J. Plazinski
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  2. J. Elliott
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  3. U. A. Hurley
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  4. J. Burch
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  5. T. Arioli
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  6. R. E. Williamson
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Correspondence to J. Plazinski.

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Plazinski, J., Elliott, J., Hurley, U.A. et al. Myosins from angiosperms, ferns, and algae amplification of gene fragments with versatile PCR primers and detection of protein products with a monoclonal antibody to a conserved head epitope. Protoplasma 196, 78–86 (1997). https://doi.org/10.1007/BF01281061

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  • DOI: https://doi.org/10.1007/BF01281061

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