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Characterization of yeast ribosomal DNA fragments generated by EcoR1 restriction endonuclease

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Summary

The action of Escherichia coli restriction endonuclease R1 (EcoR1) on DNA isolated from Saccharomyces cerevisiae (strain MAR-33) generates three predominent homogenously sized DNA fragments (species of 1.8, 2.2 and 2.5 kilo nucleotide base pairs (KB). Many DNA species of molecular weight greater than 2 million daltons can be recognized upon incomplete EcoR1 digestion of yeast DNA. Four additional DNA species ranging from 0.3–0.9 KB can be identified as the second major class of EcoR1-yeast DNA products.

Hybridization with radioactive ribosomal RNA (rRNA) and competition with nonradioactive rRNA show that of the three predominent EcoR1-yeast DNA species, the 2.5 KB species hybridizes only with the 25S rRNA while the lighter 1.8 KB species hybridizes with the 18S rRNA. The intermediate DNA species of 2.2 KB hybridizes to a small extent with the 25S rRNA and could be a result of the presence of the 2.5 KB DNA species. The mass proportions and hybridization values of these 3 DNA species account for about 60% of the total ribosomal DNA (rDNA).

The 5EcoR1-yeast DNA species of less than 0.9 KB (4 major and 1 minor species) hybridize to varying degrees with the 2 rRNA and can be grouped in two classes. In one class there are 3 DNA species that hybridize exclusively with the 18S rRNA. In the second class there are 2 DNA species that besides hybridizing predominently with the 25S rRNA also hybridize with the 18S rRNA. The 7 EcoR1-yeast DNA species (excluding the 2.2 KB DNA species) that hybridize with the two rRNA account for nearly a 5 million dalton DNA segment, which is very close to the anticipated gene size of rRNA precursor molecule. If the 2.2 KB DNA species is a part of the rDNA that is not transcribed or 5 sRNA then the cistron encoding the rRNA in S. cerevisiae has at least 8 EcoR1 recognition sites resulting in 8 DNA fragments upon digestion with the EcoR1. Consideration is given to the relationship of the rRNA species generated by EcoR1 digestion and the chromosomes containing ribosomal cistrons.

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References

  • Allet, B., Jeppesen, P.G.N., Katagiri, K.J., Delius, H.: Mapping of DNA fragments produced by cleavage of λDNA with endonuclease R1. Nature (Lond.) 241, 120–123 (1973)

    Google Scholar 

  • Betz, J.L., Hereford, L.M., Magee, P.T.: Threonine deaminase from S. cerevisiae mutationally altered in regulatory properties. Biochemistry (Wash.) 10, 1818–1824 (1971)

    Google Scholar 

  • Bollon, A.P.: Fine structure analysis of a eucaryotic multifunctional gene. Nature (Lond.) 250, 630–634 (1974)

    Google Scholar 

  • Bollon, A.P.: Regulation of the ilv1 multifunctional gene in Saccharomyces cerevisiae. Molec. gen. Genet. 142, 1–12 (1975)

    Google Scholar 

  • Bollon, A.P., Magee, P.T.: Involvement of threonine deaminase in multivalent repression of the isoleucine-valine pathway in Saccharomyces cerevisiae. Proc. nat. Acad. Sci. (Wash.) 68, 2169–2172 (1971)

    Google Scholar 

  • Bonner, J., Kung, G., Bekhor, I.: A method for the hybridization of nucleic acid molecules at low temperature. Biochemistry (Wash.) 6, 3650–3653 (1967)

    Google Scholar 

  • Botchan, M.R.: Bovine satellite I DNA consists of repetitive units 1,400 base pairs in length. Nature (Lond.) 251, 288–292 (1974)

    Google Scholar 

  • Cohen, S.N., Chang, A.C.Y.: Recircularization and autonomous replication of a sheared R-factor DNA segment in Escherichia coli transformants. Proc. nat. Acad. Sci. (Wash.) 70, 1293–1297 (1973)

    Google Scholar 

  • Cohen, S.N., Chang, A.C.Y.: A method for selective clonging of eucaryotic DNA fragments on Escherichia coli by repeated transformation. Molec. gen. Genet. 134, 133–141 (1974)

    Google Scholar 

  • Cohen, S.N., Chang, A.C.Y., Hsu, L.: Nonchromosomal antibiotic resistance in bacteria: Genetic information of Escherichia coli by R-factor DNA. Proc. nat. Acad. Sci. (Wash) 69, 2110–2116 (1972)

    Google Scholar 

  • Cramer, J.H., Bhargava, M.M., Halvorson, H.O.: Isolation and characterization of rDNA of Saccharomyces cerevisiae. J. molec. Biol. 71, 11–20 (1972)

    Google Scholar 

  • Finkelstein, D.B., Blamire, J., Marmur, J.: Location of ribosomal RNA cistrons in yeast. Nature (Lond.) 240, 279–281 (1972)

    Google Scholar 

  • Gilliespie, D., Spiegelman, S.: A quantitative assay for DNA-RNA hybrids with DNA immobilized on a membrane. J. molec. Biol. 12, 829–842 (1965)

    Google Scholar 

  • Glover, D.M., White, R.L., Finnegan, D.J., Hogness, D.S.: Characterization of six cloned DNAs from Drosophila melanogaster, including one that contains the genes for rRNA. Cell 5, 149–157 (1975)

    Google Scholar 

  • Goldberg, S., Øyen, T., Idriss, J.M., Jalvorson, H.O.: Use of disomic strains to study the arrangement of ribosomal cistrons in Saccharomyces cerevisiae. Molec. gen. Genet. 116, 139–157 (1972)

    Google Scholar 

  • Hedgpeth, J., Goodman, H.M., Boyer, H.W.: DNA nucleotide sequence restricted by the R1 endonuclease. Proc. nat. Acad. Sci. (Wash.) 69, 3448–3452 (1972)

    Google Scholar 

  • Helser, T.L., McLaughlin, C.S.: Small ribonucleic acid molecules produced during ribosome biosynthesis in Saccharomyces cerevisiae. J. biol. Chem. 250, 2003–2007 (1975)

    Google Scholar 

  • Hershfield, V., Boyer, H.W., Yanofsky, C., Lovett, M.A., Helinski, D.R.: Plasmid Col El as a molecular vehicle for cloning and amplification of DNA. Proc. nat. Acad. Sci. (Wash.) 71, 3455–3459 (1974)

    Google Scholar 

  • Kedes, L.H., Chang, A.C.Y., Houseman, D., Cohen, S.N.: Isolation of histone genes from unfractionated sea urchin DNA by subculture cloning in E. coli. Nature (Lond.) 255, 533–538 (1975)

    Google Scholar 

  • Kaback, D.B., Bhargava, M.M., Halvorson, H.O.: Location and arrangement of genes coding for ribosomal RNA in Saccharomyces cerevisiae. J. molec. Biol. 79, 735–739 (1973)

    Google Scholar 

  • Mertz, J.E., Davis, R.W.: Cleavage of DNA by R1 restriction endonuclease generates cohesive ends. Proc. nat. Acad. Sci. (Wash.) 69, 3370–3874 (1972)

    Google Scholar 

  • Morrow, J.F., Cohen, S.N., Chang, A.C.Y., Boyer, H.W., Goodman, H.M., Helling, R.B.: Replication and transcription of eukaryotic DNA in Escherichia coli. Proc. nat. Acad. Sci. (Wash.) 71, 1743–1747 (1974)

    Google Scholar 

  • Nath, K., Bollon, A.P.: Generation of discrete yeast DNA fragments by endonuclease R1. Nature (Lond.) 257, 155–157 (1975)

    Google Scholar 

  • Philippsen, P., Streeck, R.E., Zachau, H.G., Muller, W.: Investigation of the repetitive sequences in calf DNA by cleavage with restriction nucleases. Europ. J. Biochem. 57, 55–68 (1975)

    Google Scholar 

  • Retel, J., Keulen H. van: Characterization of Yeast ribosomal DNA. Europ. J. Biochem. 58, 51–57 (1975)

    Google Scholar 

  • Retel, J., Planta, R.J.: The investigation of the ribosomal RNA sites in yeast DNA by the hybridization technique. Biochim. biophys. Acta (Amst.) 169, 416–429 (1968)

    Google Scholar 

  • Retel, J., Planta, R.J.: Non-conservative processing of ribosomal precursor RNA in yeast. Biochim. biophys. Acta (Amst.) 199, 286–288 (1970)

    Google Scholar 

  • Retel, J., Planta, R.J.: On the mechanism of the biosynthesis of ribosomal RNA in yeast. Biochim. biophys. Acta (Amst.) 224, 458–469 (1970)

    Google Scholar 

  • Rubin, G.M., Sulston, J.E.: Physical linkage of the 5S cistrons to the 18S and 28S ribosomal RNA cistrons in Saccharomyces cerevisiae. J. molec. Biol. 79, 521–530 (1973)

    Google Scholar 

  • Schweizer, E., Mackenzie, C., Halvorson, H.O.: The redundancy of ribosomal and transfer RNA genes in Saccharomyces cerevisiae. J. molec. Biol. 40, 261–277 (1969)

    Google Scholar 

  • Sgaramella, V.: Enzymatic oligomerization of bacteriophage P22 DNA and of linear simian virus 40 DNA. Proc. nat. Acad. Sci. (Wash.) 69, 3389–3393 (1972)

    Google Scholar 

  • Studier, F.W.: Sedimentation studies of the size and shape of DNA. J. molec. Biol. 11, 373–390 (1965)

    Google Scholar 

  • Taber, R.L. Jr., Vincent, W.S.: The synthesis and processing of ribosomal RNA precursor molecules in yeast. Biochim. biophys. Acta (Amst.) 186, 317–325 (1969)

    Google Scholar 

  • Tanaka, T., Weisblum, B.: Construction of a colicin E1-R factor composite plasmid in vitro: means for amplification of deoxyribonucleic acid. J. Bact. 121, 354–362 (1975)

    Google Scholar 

  • Thomas, M., Davis, R.W.: Studies on the cleavage of bacteriophage lambda DNA with EcoR1 restriction endonuclease. J. molec. Biol. 91, 315–328 (1975)

    Google Scholar 

  • Udem, S.A., Warner, J.R.: Ribosomal RNA synthesis in Saccharomyces cerevisiae. J. molec. Biol. 65, 227–242 (1972)

    Google Scholar 

  • Udem, S.A., Warner, J.R.: The cytoplasmic maturation of a ribosomal precursor ribonucleic acid in yeast. J. biol. Chem. 248, 1412–1416 (1973)

    Google Scholar 

  • Umbarger, H.E.: The regulation of enzyme levels in the pathways to the branch-chain amino acids. In: Metabolic regulation. Metabolic pathways (H.J. Vogel, ed.), pp. 447–462. New York: Academic Press 1971

    Google Scholar 

  • Wensink, P.C., Finnegan, D.J., Donelson, J.E., Hogness, D.S.: A system for mapping DNA sequences in the chromosomes of Drosophila melanogaster. Cell 3, 315–325 (1974)

    Google Scholar 

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  1. Biochemistry Department, The University of Texas, Health Science Center, 75235, Dallas, Texas, USA

    Kamalendu Nath & Arthur P. Bollon

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  1. Kamalendu Nath
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  2. Arthur P. Bollon
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Communicated by G. O'Donovan

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Nath, K., Bollon, A.P. Characterization of yeast ribosomal DNA fragments generated by EcoR1 restriction endonuclease. Molec. Gen. Genet. 147, 153–168 (1976). https://doi.org/10.1007/BF00267567

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  • DOI: https://doi.org/10.1007/BF00267567

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