RNA can hybridize to double-stranded DNA in the presence of 70% formamide by displacing the identical DNA strand. The resulting structure, called an R-oop, is fonned in formamide probably because of the greater thermodynamic stability of the RNA-DNA hybrid when it is near the denaturation temperature of duplex DNA. The rate of R-loop formation is maximal at the temperature at which half of the dIlex DNA is irreversibly converted to single-stranded DNA (the strand separation temperature or ts) of the duplex DNA and falls precipitously a few degrees above or below that temperature. This maximal rate is similar to the rate of hybridization of RNA to single-stranded DNA under the same conditions. At temperatures above the t5s the rate is proportional to the RNA concentration. However, at temperatures below t55 the rate of Rloop formation is less dependent upon the RNA concentration.Once formed, the R-loops display considerable stability; the formamide can be removed and the DNA can be cleaved with restriction endonucleases without loss of R-loop structures. It has recently been observed by R. L. White and D. Hogness (manuscript in preparation) that rRNA can hybridize to duplex DNA that codes for rRNA (rDNA) of Drosophila melanogaster. As observed in the electron microscope, these R-loop structures appear similar to the D-loop structures reported by Robberson et al. (1). However, the duplex portion of the loop is an RNA-DNA hybrid rather than a DNA-DNA hybrid and has thus been termed an R-loop. In order to optimize the formation and utilization of R-loops observed by White and Hogness, we have studied their rate of formation and kinetic stability under a variety of conditions. The ability to form R-loops at high efficiency under controlled conditions may facilitate the mapping and isolation of DNA DNA cleaved with EcoRI endonuclease was placed in the Rloop formation buffer as described above. Samples of 4 AI each were taken at 10 intervals starting at 45°. Five minutes were allowed for equilibration between intervals. Each sample was diluted 20-fold into 0.35 M ammonium acetate at pH 8, 0.01 M Na3EDTA, and 75 Mg/ml of cytochrome c at 00. Samples were mounted for electron microscopy by the aqueous drop method. Drops of 25 Ml of the above DNA plus cytochrome solutions were placed on a polished teflon bar. A parlodioncoated microscope grid was touched to the side of each drop and was stained with uranyl acetate (2). At the strand separation temperature (tss) of a specific EcoRI DNA fragment, the duplex strands are converted into collapsed single-strand bushes. The tss of the RNA-DNA hybrid was determined in an identical manner, except that RNA was first hybridized to its EcoRIcleaved complementary DNA strand at 470 in the R-loop formation buffer.Our initial work on R-loop formation was complicated by DNA degradation and nonreproducible reaction rates. We believe this was the consequence of changes in the reaction constituents and their concentrations during the course of the reaction. The following revisions...

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A selective scheme is presented for isolating sequences capable of replicating autonomously in the yeast Saccharomyces cerevisiae. YIp5, a vector Table 1. YNN27 is a ura3-52 strain that is transformed by YRp12 (see Fig. 2) at a particularly high frequency (2000-10,000 colonies per gg of DNA). It was obtained by crossing YNN6 and YNN34 and assessing the transformation ability of strains grown from individual spores. Growth and storage conditions used for all strains have been described (27).DNA. Bacterial plasmid DNA was purified by repeated isopycnic centrifugation in CsCl (27). Chromosomal yeast DNA was prepared by the method of Cameron (28). N. crassa DNA was purified from conidia (unpublished method). E. coil, D. melanogaster, D. discoideum, C. elegans, and Z. mays DNAs were generous gifts of Lee Rowan, Louise Prestidge, Alan Jacobsen, David Hirsh, and Irwin Rubenstein, respectively. pSY317, a kanamycin-resistant plasmid carrying the E. coli origin of replication, was provided by Seiichi Yasuda.Enzymes and Reagents. EcoRI endonuclease was purified by the published procedure (29). T4 DNA ligase and DNA polymerase I were generously provided by Stewart Scherer. All other enzymes and reagents were purchased from commercial suppliers and were used as described (27).Construction of Hybrid DNA Molecules. Random DNA fragments were inserted into YIp5 to produce pools of hybrid molecules. After digestion with the appropriate restriction endonuclease(s) (EcoRI, HindIII, BamHI, or codigestion with EcoRI and HindIII), the YIp5 and chromosomal DNAs (each at 15-20 ,ug of DNA per ml) were mixed and ligated with 0.1 ,g of T4 DNA ligase in 100 mM NaCl/50 mM Tris-HCl, pH 7.4/10 mM MgSO4/1 mM ATP/10 mM dithiothreitol at 40C for hr. This ligation mixture was directly used to transform yeast cells.Hybrids were constructed between YIp5 and the E. coil origin of replication, oriC, by mixing and ligating EcoRI-digested pSY317 and YIp5 DNAs (as described above). The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.

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A bacteriophage X strain has been constructed and a method developed by which DNA from potentially any source can be covalently inserted through EcoRI cohesive ends into the middle of the X DNA. These hybrid DNAs can infect nonrestricting Escherichia coli cells and can then propagate as plaque-forming phage. A unique feature of this X strain is that extra DNA in the middle of its genome is required for plaque formation. A large number of such phages have been produced with E. coli DNA and Drosophila melanogaster DNA.Hybrid DNA molecules can be constructed in vitro by joining any two or more DNA molecules through the cohesive ends that are generated by cleavage with EcoRI endonuclease (1). This discovery has made possible the formation of hybrid DNA molecules that can be propagated in Escherichia coli. Such hybrids may be formed between a vector molecule, which can be any molecule cleaved by EcoRI endonuclease capable of self propagation in E. coli, and potentially any other DNA molecule with cohesive ends generated by EcoRI endonuclease.There already exist in bacteriophage X (Fig. la) viable deletion mutants that lack most of the center two EcoRI-endonuclease-generated B and C fragments (2, 3). It was established that fragments B and C are in fact not required for lytic growth by cleaving X DNA with EcoRI endonuclease, randomly rejoining the fragments with DNA ligase, and, after DNA infection, selecting viable phage that lack the B and C fragments (Abraham, Thomas, and Davis, in preparation). Therefore, it should be possible to construct viable hybrid DNA molecules by inserting foreign DNA into X DNA in place of the B and C fragments. To facilitate such an insertion, the two rightmost EcoRI restriction sites were eliminated by mutation.An advantage of X DNA as a vector molecule is that an insertion of foreign DNA can be made to be essential for plaque formation. The shortest X DNA molecules that produce plaques of nearly normal size are 25% deleted (J. S. Parkinson, unpublished observation). Apparently, if too much of even nonessential DNA is deleted from the X genome, it cannot be packaged into virus particles. The EcoRI-B and -C fragments represent 21.1% of the X DNA (Thomas and Davis, submitted to J. Mol. Biol.). Deletion of the nonessential nin5 region (6.1%o of ? DNA) (2) as well as the EcoRI-B and -C fragments yields a DNA that would not be expected to produce plaque-forming phage even though no essential genes have been deleted. However, this deleted X DNA can be made to produce plaques if a new DNA segment is inserted into the remaining central EcoRI restriction site. This constitutes a positive selection for X phage carrying a DNA insertion. The X strain used for the construction of these hybrid phages, hereafter referred to as Xgt-XC, contains the X EcoRI-C fragment in this central region in order to retain sufficient DNA that it may be propagated. This segment is removed after EcoRI endonuclease cleavage, and EcoRI-endonuclease-cleaved foreign DNA is inserted. MATERIALS AND METHODSTerminology....