Use of gene 32 protein staining of single-strand polynucleotides for gene mapping by electron microscopy: application to the phi80d3ilvsu+7 system.
A method for visualizing RNA-DNA duplex regions along a single strand of DNA in the electron microscope is described. A preparation of RNA molecules is hybridized to a long DNA strand containing the coding sequences (genes) for some of the RNAs. T4 gene 32 protein, which binds selectively and cooperatively only to the single-strand regions, is added, followed by glutaraldehyde. The resulting nucleic acid-gene 32 complex is adsorbed to the surface of an electron microscope grid in the presence of ethidium bromide. The single-strand regions are relatively thick (8.5 nm) compared to the duplex (RNA-DNA hybrid) regions (3.5 nm), so that the two kinds of regions are readily recognized by electron microscopy. In favorable cases, tRNA-DNA hybrids of length about 80 nucleotide pairs can be recognized (although with difficulty). The positions of a number of interesting genetic sequences on the DNA of the transducing phage phi80d3ilvsu+7 have been mapped. The r strand contains 16S, 23S, and 5S rRNA coding sequences in that order. The spacer between 16S and 23S genes has a length of 500 nucleotides and contains the coding sequence for a tRNA2Glu gene in agreement with previous biochemical observations. The spacer between the 23S and 5S genes has a length of 180 nucleotides. The su+7 tRNATrp coding sequence has been mapped on the l strand at a position just to the left of the ilv genes. Secondary structure loops due to short inverted repeat sequences flanking the 16S, 23S, tRNATrp, and F sequences in the DNA have been observed.
D002874:Chromosome Mapping; D003090:Coliphages; D004269:DNA, Bacterial / Q000032:analysis*; D004277:DNA, Single-Stranded / Q000032:analysis*; D004279:DNA, Viral / Q000032:analysis; D004996:Ethidium; D005796:Genes; D008854:Microscopy, Electron / Q000379:methods; D009693:Nucleic Acid Hybridization; D012329:RNA, Bacterial / Q000378:metabolism; D012335:RNA, Ribosomal / Q000378:metabolism; D012343:RNA, Transfer / Q000378:metabolism; D014161:Transduction, Genetic; D014764:Viral Proteins
D004269:DNA, Bacterial; D004277:DNA, Single-Stranded; D004279:DNA, Viral; D012329:RNA, Bacterial; D012335:RNA, Ribosomal; D014764:Viral Proteins; D012343:RNA, Transfer; D004996:Ethidium