Molecular mechanisms of genetic recombination in bacteriophage

Tomizawa, Jun-ichi; Anraku, Naoyo; Iwama, Yukiko

doi:10.1016/0022-2836(66)90095-7

Cited by 132 publications

(20 citation statements)

References 8 publications

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“…The HDPs of bacteriophage T4 (6)(7)(8) and of Escherichia col1 (9,10) have been implicated directly in DNA replication, recombination, and repair. Similar functions in vivo for eukaryotic HDPs have been suggested only indirectly (see Results and Discussion).…”

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confidence: 99%

Immunoreactive helix-destabilizing protein localized in transcriptionally active regions of Drosophila polytene chromosomes.

Patel¹,

Thompson²

1980

Proc. Natl. Acad. Sci. U.S.A.

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A highly purified helix-destabilizing protein (HDP) obtained from rat liver has been used to elicit specific, high-titer anti-HDP sera in rabbits. These antisera show immunological crossreaction with single-stranded DNA binding proteins from several very diverse eukaryotic sources, including Drosophila embryos. The use of such antisera in the labeling of Drosophila salivary gland chromosomes by indirect immunofluorescence shows concentrations of immunoreactive HDP in many regions, but especially in chromosome puffs. There is a striking localization of HDP in heat shock puffs known to be sites of new transcription. The pattern of HDP distribution seems to implicate a transcriptional role, with some specificities independent of puffing itself.Every aspect of the processing of chromosomal information requires the concerted action of several proteins. For example, the DNA replication complex of bacteriophage T4 may comprise seven distinct proteins induced by the phage (1). One of these proteins, which is coded by phage gene 32, was purified on the basis of its preferential binding to single-stranded DNA and was shown to destabilize and unwind DNA from its duplex helical conformation (2). During the past 10 years, proteins with similar properties have been identified in various organisms (for reviews see refs. 3 and 4). Proteins of this class are referred to as helix-destabilizing proteins (HDP) (5).The HDPs of bacteriophage T4 (6-8) and of Escherichia col1 (9, 10) have been implicated directly in DNA replication, recombination, and repair. Similar functions in vivo for eukaryotic HDPs have been suggested only indirectly (see Results and Discussion). Our goal has been to provide detailed information concerning the role(s) and functional regulation of eukaryotic HDPs, with particular focus on rat liver HDP. The purification and characterization of a rat liver HDP (unpublished data) provided the basis for undertaking a broad spectrum of immunological approaches to this end.In this report we show that an antiserum raised against rat HDP crossreacts with single-stranded DNA binding proteins isolated from heterologous species, including Drosophila, and that the localization of immunoreactive material on polytene chromosomes of Drosophila larvae by the method of indirect immunofluorescence is similar to that reported for RNA polymerase (11). Consequently, we propose that some eukaryotic HDPs may play a role in gene transcription.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. 6749 MATERIALS AND METHODSPurification of Rat Liver HDP and Preparation of Anti-HDP Serum. A rat liver HDP was purified to homogeneity by procedures to be published elsewhere. In brief, a nucleic acid-free extract of Sprague-Dawley rat livers was fractionated by sequential affinity chromatography on native and denatured DNA-cellulose columns (12) to obtain proteins preferentially bi...

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confidence: 99%

Immunoreactive helix-destabilizing protein localized in transcriptionally active regions of Drosophila polytene chromosomes.

Patel¹,

Thompson²

1980

Proc. Natl. Acad. Sci. U.S.A.

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“…Recombinationdeficient mutants of bacteriophage and bacteria have been useful in the study of recombination processes (1)(2)(3)(4)(5)(6)(7)(8)(9)(10). Recombination deficient, radiation-sensitive mutants of Bacillus subtilis have been isolated and described by several investigators (11)(12)(13)(14).…”

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Fate of Transforming DNA After Uptake by Competent Bacillus subtilis : Failure of Donor DNA to Replicate in a Recombination-Deficient Recipient

Davidoff-Abelson¹,

Dubnau²

1971

Proc. Natl. Acad. Sci. U.S.A.

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The fate of radioactively-labeled transforming DNA was studied in a recombination-deficient strain of Bacillus subtilis that carried the recB2 mutation (Rec-) Bacterial transformation is an advantageous system for the study of recombination, since the fate of purified donor DNA can be studied in the course of the different steps before, during, and after integration. Recombinationdeficient mutants of bacteriophage and bacteria have been useful in the study of recombination processes (1-10). Recombination deficient, radiation-sensitive mutants of Bacillus subtilis have been isolated and described by several investigators (11)(12)(13)(14). This paper reports the results of experiments in which the fate of transforming DNA was examined in one of these mutants (recB2). Our results indicate that in recB2, the donor-recipient complex is formed to the same extent as in an isogenic Rec+ strain, but that donor DNA fails to replicate in the recipient after formation of the complex. MATERIALS AND METHODS Bacterial strainsThe bacterial strains used were derivatives of Bacillus subtilis 168. Strains BD170 (thr-5 trp-2) and BD191 (thr-5 trp-2 recB2) were derived from the same parent and were isogenic for all markers except the recB2 marker, which was introduced by transformation as was described (15). The recB2 marker is derived from the mitomycin-sensitive strain mc-1 (12). RecB2 has been shown to be radiation sensitive and deficient in both transformability and transducibility (12)(13)(14). Strain BD204 (hisB2 thy) was used to prepare radioactively-labeled transforming DNA.BD55 (trp-2 hisBW), known also as SB25, was used to assay the biological activity of DNA fractions isolated from the transformed cells. The trp-2 and hisB2 markers are cotransformed at a frequency of about 60%. Media and solutionsThe media for plating and for the development of competence were prepared as described (16). Transformed cells were washed in SP buffer (0.15 M NaCl-0.02 M K2HPO4, pH 7.5). The washed samples were resuspended in lysis medium (0.05 M NaCl-0.1 M EDTA, pH 6.9).SPII medium contained Spizizen salts (17), 0.02% casamino acids, 0.1% Difco yeast extract, 0.5% glucose, 0.5 mM CaC12, 2.5 mM MgCl2, and 50,gg/ml of each required amino acid. Re-extraction experimentsCompetent cells were prepared and frozen as was described (16). Frozen competent Rec+ and Rec-cells were quickly thawed and incubated for 5 min at 370C. To begin the reaction, [3H]DNA, isolated from BD204 as described (16), was then added to a final concentration of 1-2 gg/ml. The specific activity of the DNA preparations ranged from 4 to 6 X 105 cpm/htg. The mixture was incubated at 370C for 7 min or 9 min, at which time an excess (100 ,Ag/ml) of salmonsperm DNA (Calbiochem) was added to stop further DNA uptake. In some experiments the reaction mixture was then transferred to 300C. At different times after addition of the salmon-sperm DNA, samples of the transformed cells were chilled rapidly, washed three times by centrifugation in icecold SP, and resuspended in lysis ...

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“…In bacteriophage T4, the roles ofgene 32 protein have also been well characterized both in vitro and in vivo. Genetic studies have demonstrated that the product of gene 32 is essential for DNA replication, recombination, and repair (38)(39)(40)(41)(42). It binds cooperatively to single-stranded DNA, affects denaturation and renaturation of DNA, and interacts specifically with T4 DNA polymerase and the recombination proteins uvsX and uvsY (43)(44)(45)(46) Why is gene 2.5 protein essential for T7 growth and replication?…”

Section: Discussionmentioning

confidence: 99%