Abstract
A rapid, convenient and economical method for the hybridization of electrophoretically resolved RNA to DNA restriction fragments immobilized on nitrocellulose filters is described. DNA was digested, electrophoresed on agarose gels in a wide band and transferred to a nitrocellulose filter. The filter was then placed on the surface of a second gel containing radioactively labeled RNA electrophoresed under denaturing conditions in a similar way. The filter and gel were oriented so that the DNA and RNA bands were perpendicular to one another and the RNA was transferred from the gel through the filter under conditions which promote RNA-DNA hybridization. Following washing, the filter was autoradiographed. RNA-DNA sequence relationships could be conveniently determined from the spots produced at regions of intersection of homologous nucleic acids. The two dimensional array formed in this procedure fascilitates the rapid ordering of DNA restriction fragments. An example of its use for this purpose is presented.
Full text
PDF











Images in this article
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Alwine J. C., Kemp D. J., Stark G. R. Method for detection of specific RNAs in agarose gels by transfer to diazobenzyloxymethyl-paper and hybridization with DNA probes. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5350–5354. doi: 10.1073/pnas.74.12.5350. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Bailey J. M., Davidson N. Methylmercury as a reversible denaturing agent for agarose gel electrophoresis. Anal Biochem. 1976 Jan;70(1):75–85. doi: 10.1016/s0003-2697(76)80049-8. [DOI] [PubMed] [Google Scholar]
- Bell G. I., DeGennaro L. J., Gelfand D. H., Bishop R. J., Valenzuela P., Rutter W. J. Ribosomal RNA genes of Saccharomyces cerevisiae. I. Physical map of the repeating unit and location of the regions coding for 5 S, 5.8 S, 18 S, and 25 S ribosomal RNAs. J Biol Chem. 1977 Nov 25;252(22):8118–8125. [PubMed] [Google Scholar]
- Bicknell J. N., Douglas H. C. Nucleic acid homologies among species of Saccharomyces. J Bacteriol. 1970 Feb;101(2):505–512. doi: 10.1128/jb.101.2.505-512.1970. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Burckhardt J., Birnstiel M. L. Analysis of histone messenger RNA of Drosophila melanogaster by two-dimensional gel electrophoresis. J Mol Biol. 1978 Jan 5;118(1):61–79. doi: 10.1016/0022-2836(78)90244-9. [DOI] [PubMed] [Google Scholar]
- Burckhardt J., Telford J., Birnstiel M. L. Detection of labelled RNA species by contact hybridization. Nucleic Acids Res. 1979 Jul 11;6(9):2963–2971. doi: 10.1093/nar/6.9.2963. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Christiansen C., Bak A. L., Stenderup A., Christiansen G. Repetitive DNA in yeasts. Nat New Biol. 1971 Jun 9;231(23):176–177. doi: 10.1038/newbio231176a0. [DOI] [PubMed] [Google Scholar]
- Cockburn A. F., Newkirk M. J., Firtel R. A. Organization of the ribosomal RNA genes of Dictyostelium discoideum: mapping of the nontranscribed spacer regions. Cell. 1976 Dec;9(4 Pt 1):605–613. doi: 10.1016/0092-8674(76)90043-x. [DOI] [PubMed] [Google Scholar]
- Cox R. A., Peden K. A study of the organisation of the ribosomal ribonucleic acid gene cluster of Neurospora crassa by means of restriction endonuclease analysis and cloning in bacteriophage lambda. Mol Gen Genet. 1979 Jul 2;174(1):17–24. doi: 10.1007/BF00433300. [DOI] [PubMed] [Google Scholar]
- Dons J. J., de Vries O. M., Wessels J. G. Characterization of the genome of the basidiomycete Schizophyllum commune. Biochim Biophys Acta. 1979 Jun 20;563(1):100–112. doi: 10.1016/0005-2787(79)90011-x. [DOI] [PubMed] [Google Scholar]
- Free S. J., Rice P. W., Metzenberg R. L. Arrangement of the genes coding for ribosomal ribonucleic acids in Neurospora crassa. J Bacteriol. 1979 Mar;137(3):1219–1226. doi: 10.1128/jb.137.3.1219-1226.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Gall J. G. Free ribosomal RNA genes in the macronucleus of Tetrahymena. Proc Natl Acad Sci U S A. 1974 Aug;71(8):3078–3081. doi: 10.1073/pnas.71.8.3078. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hall L., Braun R. The organisation of genes for transfer RNA and ribosomal RNA in amoebae and plasmodia of Physarum polycephalum. Eur J Biochem. 1977 Jun 1;76(1):165–174. doi: 10.1111/j.1432-1033.1977.tb11582.x. [DOI] [PubMed] [Google Scholar]
- Hereford L. M., Rosbash M. Number and distribution of polyadenylated RNA sequences in yeast. Cell. 1977 Mar;10(3):453–462. doi: 10.1016/0092-8674(77)90032-0. [DOI] [PubMed] [Google Scholar]
- Hudspeth M. E., Timberlake W. E., Goldberg R. B. DNA sequence organization in the water mold Achlya. Proc Natl Acad Sci U S A. 1977 Oct;74(10):4332–4336. doi: 10.1073/pnas.74.10.4332. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Johnson P. H., Grossman L. I. Electrophoresis of DNA in agarose gels. Optimizing separations of conformational isomers of double- and single-stranded DNAs. Biochemistry. 1977 Sep 20;16(19):4217–4225. doi: 10.1021/bi00638a014. [DOI] [PubMed] [Google Scholar]
- Lauer G. D., Roberts T. M., Klotz L. C. Determination of the nuclear DNA content of Saccharomyces cerevisiae and implications for the organization of DNA in yeast chromosomes. J Mol Biol. 1977 Aug 25;114(4):507–526. doi: 10.1016/0022-2836(77)90175-9. [DOI] [PubMed] [Google Scholar]
- LéJohn H. B. Enzyme regulation, lysine pathways and cell wall structures as indicators of major lines of evolution in fungi. Nature. 1971 May 21;231(5299):164–168. doi: 10.1038/231164a0. [DOI] [PubMed] [Google Scholar]
- Maizels N. Dictyostelium 17S, 25S, and 5S rDNAs lie within a 38,000 base pair repeated unit. Cell. 1976 Nov;9(3):431–438. doi: 10.1016/0092-8674(76)90088-x. [DOI] [PubMed] [Google Scholar]
- McConaughy B. L., Laird C. D., McCarthy B. J. Nucleic acid reassociation in formamide. Biochemistry. 1969 Aug;8(8):3289–3295. doi: 10.1021/bi00836a024. [DOI] [PubMed] [Google Scholar]
- Molgaard H. V., Matthews H. R., Bradbury E. M. Organisation of genes for ribosomal RNA in Physarum polycephalum. Eur J Biochem. 1976 Sep 15;68(2):541–549. doi: 10.1111/j.1432-1033.1976.tb10842.x. [DOI] [PubMed] [Google Scholar]
- Sato S., Hutchinson C. A., 3rd, Harris J. I. A thermostable sequence-specific endonuclease from Thermus aquaticus. Proc Natl Acad Sci U S A. 1977 Feb;74(2):542–546. doi: 10.1073/pnas.74.2.542. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Schweizer E., MacKechnie C., Halvorson H. O. The redundancy of ribosomal and transfer RNA genes in Saccharomyces cerevisiae. J Mol Biol. 1969 Mar 14;40(2):261–277. doi: 10.1016/0022-2836(69)90474-4. [DOI] [PubMed] [Google Scholar]
- Southern E. M. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol. 1975 Nov 5;98(3):503–517. doi: 10.1016/s0022-2836(75)80083-0. [DOI] [PubMed] [Google Scholar]
- Timberlake W. E. Alterations in RNA and protein synthesis associated with steroid hormone-induced sexual morphogenesis in the water mold Achlya. Dev Biol. 1976 Jul 15;51(2):202–214. doi: 10.1016/0012-1606(76)90138-x. [DOI] [PubMed] [Google Scholar]
- Timberlake W. E., Griffin D. H. Differential effects of cycloheximide and other inhibitors of protein synthesis on in vivo ribosomal RNA synthesis in Achlya bisexualis. Biochim Biophys Acta. 1974 Jun 27;353(2):248–252. doi: 10.1016/0005-2787(74)90189-0. [DOI] [PubMed] [Google Scholar]
- Timberlake W. E., Shumard D. S., Goldberg R. B. Relationship between nuclear and polysomal RNA populations of Achlya: a simple eucaryotic system. Cell. 1977 Apr;10(4):623–632. doi: 10.1016/0092-8674(77)90095-2. [DOI] [PubMed] [Google Scholar]
- Vogt V. M., Braun R. Structure of ribosomal DNA in Physarum polycephalum. J Mol Biol. 1976 Sep 25;106(3):567–587. doi: 10.1016/0022-2836(76)90252-7. [DOI] [PubMed] [Google Scholar]


