Heteroduplex structures of bacteriophage Mu DNA

Daniell, Ellen; Abelson, John; Kim, J.S.; Davidson, Norman

doi:10.1016/0042-6822(73)90385-1

Cited by 93 publications

(40 citation statements)

References 7 publications

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“…The mean length of the split ends in wild type is 1450 + 500 base pairs. This large deviation in mean length is in agreement with the deviation reported by Daniell et al (7). As shown in the histogram in Fig.…”

Section: Analysis Of Mu Dnas With Restriction Endonucleasessupporting

confidence: 82%

“…All the molecules show single-stranded tails or split ends, measuring about 4% of the molecular length. The split ends, which apparently represent sequences picked up randomly from the, E. coli chromosome, are always found at the S gene end, called here the SE end, of Mu DNA (6)(7)(8). Some molecules show another nonrenaturable region, termed the G bubble, near the SE end (9).…”

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confidence: 99%

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Influence of insertions on packaging of host sequences covalently linked to bacteriophage Mu DNA.

Bukhari¹,

Taylor²

1975

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

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Insertions in bacteriophage Mu DNA have been identified. These insertions are responsible for at least seven X mutations, all of which eliminate essential Mu functions. The insertions are about 800 base pairs long and are located to the left of the cleavage site of restriction endonuclease EcoRI, near the immunity end of Mu DNA. We have found that such-insertions cause a reduction in the length of nonhomologous terminal sequences which are seen as split ends in denatured and renatured Mu DNA molecules. These heterogeneous sequences apparently arise from packaging of host DNA from maturation precursors in which Mu and host DNA are covalently linked. We infer that a single Mu genome length is too short to be cut during morphogenesis, and thus some host DNA is packaged into mature virions. Since the insertions increase the length of Mu DNA, they decrease the amount of host DNA needed for packaging.The temperate bacteriophage Mu is characterized by the highly promiscuous integration of its DNA into the genome of its host bacterium Escherichia coli (1-3). The prophage Mu DNA can be excised precisely from the different integration sites, and as a result wild-type function of the gene into which Mu was inserted can be restored (4). This reversal of Mu integration can be seen if the prophages contain the X mutations, which block the lethal and essential phage functions.Mu X mutants revert to wild type at a frequency of about 10-8. However, no X amber mutants have been found. Thus, we proposed that the X mutations are insertions (4). We have investigated the nature of the X mutations by agarose-gel electrophoresis of Mu DNA fragments produced by specific endonucleases, and by electron microscopic examination of Mu DNA heteroduplexes. These studies have shown that the X mutations are insertions near the immunity (c) end of Mu DNA. In one X mutant, the insertion is demonstrated to be of about 720 base pairs, located 4 kilobases (kb) away from the c end.The isolation of insertions in Mu DNA has allowed us to analyze further some aspects of Mu DNA structure. Mu DNA, a double-stranded linear duplex of about 37 kb (5), has some interesting features. When it is denatured and reannealed, two types of homoduplex molecules are obtained (Fig. 1). All the molecules show single-stranded tails or split ends, measuring about 4% of the molecular length. The split ends, which apparently represent sequences picked up randomly from the, E. coli chromosome, are always found at the S gene end, called here the SE end, of Mu DNA (6-8). Some molecules show another nonrenaturable region, termed the G bubble, near the SE end (9). Recently, we observed that the c end of Mu DNA is also not fixed, and probably varies in length by about 100 base pairs (10, 11).Abbreviation: kb, kilobase. t To whom correspondence should be directed.One plausible model for the origin of the heterogeneity of the SE end is that Mu DNA is packaged into phage heads from maturation precursors which contain both Mu DNA and host sequences. This hypothesis implies that...

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Section: Analysis Of Mu Dnas With Restriction Endonucleasessupporting

confidence: 82%

mentioning

confidence: 99%

Influence of insertions on packaging of host sequences covalently linked to bacteriophage Mu DNA.

Bukhari¹,

Taylor²

1975

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

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“…Phage were purified by banding on cesium chloride equilibrium gradients. * This is contrary to our report (1) (7), depending on the orientation of Mu in the Mu-induced chlD mutant from which the hybrid arose (8) (Fig. 2).…”

Section: Methodscontrasting

confidence: 56%

“…When a temperature-inducible mutant of bacteriophage Mu-1 cIts is induced from a lysogen, the DNA of the phage in the burst is inhomogeneous in two respects (1). When the DNA is denatured, reannealed, and observed in an electron microscope, the inhomogeneities show up as single-stranded split ends in virtually all of the duplexes* and in the form of a nonhomology loop in 30-50% of the duplexes (Fig.…”

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confidence: 99%

Genetic Mapping of the Inversion Loop in Bacteriophage Mu DNA

Daniell

Boram

Abelson

1973

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

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An inversion loop seen in heteroduplex mapping of the DNA of mature Mu phage induced from a lysogen is observed also in defective X phage carrying one end of Mu. 14% of the DNA of Mu, including this region, designated the G loop, is shown to be to the right of all known genes in the prophage map. The inhomogeneous ends of Mu are observed as a separate phenomenon and appear in all mutants investigated. The recA and recBC functions of the host are not needed for the inversion responsible for the G loop to take place. Deletions of Mu DNA in the G-loop region have been isolated and are under study.When a temperature-inducible mutant of bacteriophage Mu-1 cIts is induced from a lysogen, the DNA of the phage in the burst is inhomogeneous in two respects (1). When the DNA is denatured, reannealed, and observed in an electron microscope, the inhomogeneities show up as single-stranded split ends in virtually all of the duplexes* and in the form of a nonhomology loop in 30-50% of the duplexes (Fig. 1A).This loop, about 3000 base-pairs long (8% the length of Mu) has been shown to be due to an inversion (2).We took advantage of a set of hybrids between X and Mu we had constructed (3) to determine the relation between the physical position of the G loop and the genetic map Survivors were picked and again treated with pyrophosphate. After four cycles, the surviving phage were centrifuged to equilibrium on cesium chloride. Fractions were collected. Plaques from the least-dense fractions were purified. Hightiter stocks were prepared and banded in CsCl. They banded at a lower density than wild-type Mu.Electron Microscopy. DNA was extracted and prepared for microscopy as in Davis et al. (6). Photographs were taken on glass plates, and measurements were made from tracings of negatives enlarged on a photographic enlarger.Measurements of DNA. All measurements are reported as equivalent single-stranded molecular weight of a DNA segment measured. The calculations are based on length measurements of the DNA in question and of RF 4X174 and single-stranded 4X174 DNA in the same microscope preparation, taking 1.7 X 106 as the single-stranded molecular weight of qX174 DNA (6). RESULTS Mapping of the G loopThe G loop observed in self-annealed mature Mu DNA extends 15% in from one end of the Mu molecule. XdpglMu particles contain varying amounts of the Mu genome from one end or the other of the Mu prophage map (7), depending on the orientation of Mu in the Mu-induced chlD mutant from which the hybrid arose (8) (Fig. 2).Prophage Mu has the same gene order as vegetative Mu (9), so we supposed that the genes carried on XdpglMu phages are near the physical ends of the mature phage genome. If the region of the G loop is within the piece of Mu DNA contained in a particular hybrid phage, and if the inversion responsible for the loop can occur with only the piece of Mu present in the hybrid, we could hope to see G loops upon selfrenaturation of the denatured hybrid.Phage in a given series of XdpglMus result from a single

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“…The finding that ColV,I-K94 + strains were also resistant to phage Plcmclrl00 produced by induction of a lysogen indicates that ColV,I-K94 + strains are not sensitive to the C(-) form either [induction of lysogens produces a mixture of C(+) and C(-) forms; Daniell et al, 1973;Van de Putte et al, 1980;Iida et aL, 1982].…”

Section: Discussionmentioning

confidence: 92%