Abstract
A useful approach to the study of differentiation consists of isolating and characterizing the genes specifically expressed in the various stages and then studying their regulation. This chapter describes the methodology used in our laboratory to clone sequences from stage-specific mRNAs of Physarum polycephalum. It involved the preparation of intact poly (A)+ RNA and the construction of cDNA libraries for four developmental stages (amoebae, plasmodia, spores, and spherules). These libraries were then screened by differential hybridization, and the specificity of the positive clones was confirmed by Northern blot hybridization. The quality of the results obtained at each step was controlled, and the methodology was found to be of general use in the identification of Physarum stage-specific mRNAs.
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References
Bernier, F., Seligy, V. L., Pallotta, D., and Lemieux, G., 1985, Changes in gene expression during spherulation in Physarumpolycephalum, Can. J. Biochem. Cell Biol., in press.
Birnboim H. C., and Doly, J., 1979, A rapid alkaline extraction procedure for screening recombinant plasmid DNA, Nucl. Acids Res ., 7: 1513.
Burland, T. G., Gull, K., Boston, A. S., and Dove, W. F., 1983, Cell type-dependent expression of tubulins in Physarum, J. Cell Biol ., 97: 1852.
Chirgwin, J. M., Przybyla, A. E., MacDonald, R. J., and Rutter, W. J., 1979, Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease, Biochemistry, 18: 5294.
Cooke, D. J., and Dee, J., 1975, Methods for the isolation and analysis of plasmodial mutants in Physarum polycephalum, Genet. Res ., 24: 175.
Cox, R. A., and Smulian, N. J., 1983, A single step procedure for the isolation of individual mRNA species from crude lysates of Physarum polycephalum, FEBS Lett ., 155: 73.
Daniel, J. W., and Baldwin, H. H., 1964, Methods of culture for plasmodial myxomycetes, in: “Methods in Cell Physiology”, D. M. Prescott, ed., p. 9, Academic Press, New York.
Fyrberg, E. A., Kindle, K. L., Davidson, N., and Sodja, A., (1980), The actin genes of Drosophila: a dispersed multigene family, Cell, 19: 365.
Gubler, U., and Hoffman, B. J., 1983, A simple and very efficient method for generating cDNA libraries, Gene, 25: 263.
Guttes, E. S., Guttes, S., andRusch, H. P., 1961, Morphological observations on growth and differentiation of Physarum polycephalum in pure culture, Dev. Biol ., 3: 588.
Hanahan, D., and Meselson, M., 1983, Plasmid screening at high colony density, Methods Enzymol ., 100: 333.
Holmes, D. S., and Quigley, M., 1981, A rapid boiling method for the preparation of bacterial plasmids, Anal. Biochem ., 114: 193.
Hoopes, B. C., and McClure, W. R., 1981, Studies on the selectivity of DNA precipitation by spermine, Nucl. Acids Res ., 9: 5493.
Maizels, N., 1976, Dictyostelium 17S, 25S and 5S rDNAs lie within a 38,000 base pair repeated unit, Cell, 9:431.
Mandel, H., and Higa, A., 1970, Calcium dependent bacteriophage DNA infection, J. Mol. Biol ., 53: 154.
Maniatis, T., Fritsch, E. F., and Sambrook, J., 1982a, Electrophoresis of RNA through gels containing formaldehyde, in: “Molecular Cloning, A Laboratory Manual”, p. 202, Cold Spring Harbor Laboratory, Cold Spring Harbor.
Maniatis, T., Fritsch, E. F., and Sambrook J., 1982b, Synthesis of double stranded DNA, In: “Molecular Cloning, A Laboratory Manual”, p. 230, Cold Spring Harbor Laboratory, Cold Spring Harbor.
McCullough, C. H. R., Dee, J., and Foxon, J. L., 1978, Genetic factors determining the growth of Physarum polycephalum amoebae in axenic medium, J. Gen. Microbiol ., 106: 297.
Melera, P. W., and Rusch, H. P., 1973, A characterization of ribonucleic acid in the myxomycete Physarum polycephalum, Exp. Cell Res ., 82: 197.
Michelson, A. M., and Orkin, S. H., 1982, Characterization of the homopolymer tailing reaction catalyzed by terminal deoxynucleotidyl transferase. Implication for the cloning of cDNA, J. Biol. Chem ., 257: 14773.
Okayama, H., and Berg, P., 1982, High efficiency cloning of full length cDNA, Mol. Cell. Biol ., 2: 161.
Putzer, H., Werenskiold, K., Verfuerth, C., and Schreckenbach, T., 1983, Blue light inhibits slime mold differentiation at the mRNA level, EMBO J ., 2: 261.
Rigby, P. W. J., Dieckmann, M., Rhodes, C., and Berg, P., 1977, Labeling deoxyribonucleic acid to high specific activity in vitro by nick translation with DNA polymerase, J. Mol. Biol ., 113: 237.
Thomas, P. S., 1983, Hybridization of denatured RNA transferred or dotted to nitrocellulose paper, Methods Enzymol ., 100: 255.
Youngman, P. J., Pallotta, D., Hosler, B., Struhl, G., and Holt, C. E., 1979, A new mating compatibility locus in Physarum polycephalum, Genetics, 91: 683.
Zellweger, A., and Braun, R., 1971, RNA of Physarum. I. Preparation and properties, Exp. Cell Res ., 65: 413.
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© 1986 Plenum Press, New York
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Pallotta, D., Bernier, F., Hamelin, M., Martel, R., Lemieux, G. (1986). cDNA Cloning Of Physarum polycephalum Stage-Specific mRNAs. In: Dove, W.F., Dee, J., Hatano, S., Haugli, F.B., Wohlfarth-Bottermann, KE. (eds) The Molecular Biology of Physarum polycephalum . NATO ASI Series, vol 106. Springer, Boston, MA. https://doi.org/10.1007/978-1-4613-2203-0_24
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DOI: https://doi.org/10.1007/978-1-4613-2203-0_24
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