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Use of gene 32 protein staining of single-strand polynucleotides for gene mapping by electron microscopy: Application to the phi-80d3ilvsu+7 system

Wu, Madeline and Davidson, Norman (1975) Use of gene 32 protein staining of single-strand polynucleotides for gene mapping by electron microscopy: Application to the phi-80d3ilvsu+7 system. Proceedings of the National Academy of Sciences of the United States of America, 72 (11). pp. 4506-4510. ISSN 0027-8424.

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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 tRNA2{}Glu 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.

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Additional Information:© 1975 by the National Academy of Sciences. Contributed by Norman Davidson, September 2, 1975. Drs. Bruce Alberts and Peter von Hippel have made generous gifts of gene 32 protein and have contributed advice and information about its properties. We are indebted to Drs. Steve Eisenberg and Mike Yarus for gifts of purified tRNAGlu and tRNATrp. We have profited from consultations with Drs. Larry Soll, M. Yarus, and Harumi Kasamatsu. We had been informed prior to our studies of the general results of the mapping experiments by Elsebet Lund, James Dahlberg, and Fred Blattner as to the positions of the several short RNA species on phi80d3ilv as described in the text. We have profited from the previous preliminary studies of this system, including the discovery of the F loop, by Drs. Louise Chow and Thomas Broker. This research has been supported by Grant NIGMS 20927 from the USPHS.
Subject Keywords:visualization of RNA-DNA duplex regions; rRNA genes; tRNA genes; secondary structure mapping
Issue or Number:11
Record Number:CaltechAUTHORS:WUMpnas75
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Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:10509
Deposited By: Tony Diaz
Deposited On:13 May 2008
Last Modified:03 Oct 2019 00:10

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