Cleavage map of bacteriophage T4 cytosine-containing DNA by sequence-specific endonucleases SalI and KpnI

Carlson, Karin; Nicolaisen, Berit

doi:10.1128/jvi.31.1.112-123.1979

Cited by 11 publications

(13 citation statements)

References 32 publications

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“…Since the isolation of bacteriophage T4 mutants which allow the growth and packaging of DNA containing cytosine instead of the glucosylated hydroxymethylcytosine residues normally present (15), T4 DNA has been amenable to analysis with restriction endonucleases. Circular cleavage maps of the 166-kilobase pair T4 genome have been published for restriction enzymes BamHI, BglII, KpnI, SalI, SmaI, and XhoI (3,6,14), and maps for BglI, EcoRI, HindIII, and PstI have been completed recently (2,12). In the accompanying report, the sites of cleavage by PvuI and XbaI were added to a computer-fitted, unified map of the BamHI, Bglll, KpnI, and SailI cleavage sites (9).…”

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confidence: 99%

Alignment of a restriction map with the genetic map of bacteriophage T4

Yee¹,

Marsh²

1981

J Virol

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A restriction map of the bacteriophage T4 genome was aligned with the T4 genetic map. Included were the cleavage sites for BamHI, BglII, KpnI, PvuI, Sall, and XbaI. The alignment utilized the fact that the T4 genetic map had been oriented previously with respect to a T2/T4 heteroduplex map. DNA fragments from a BgII digestion of cytosine-containing DNA from a T4 dCTPase-denA denB(rIIH23B) alc mutant were hybridized with full-length chromosomal strands of bacteriophage T2, and the heteroduplexes were examined by electron microscopy. From their lengths and patterns of substitution and deletion loops, the heteroduplexes formed with 6 of the 13 BglIl fragments could be unambiguously identified and positioned on the T2/T4 heteroduplex map. The ends of the T4 DNA strands in the heteroduplexes directly identified the location of 10 BglII cleavage sites. The remaining three BglII cleavage sites could be assigned to the T2/T4 heteroduplex map based on their relative locations on the restriction map. It was also possible to identify the source of the DNA strands (i.e., T2 or T4) in four previously unassigned deletion loops on the T2/T4 heteroduplex. Among the BglII fragments identified in heteroduplexes was the fragment containing the rIIH23B deletion; this deletion was used as the primary point of reference for alignment of the T4 restriction map with the T2/T4 heteroduplex map and, hence, with the T4 genetic map. Snyder (15). Originally, this T4 alc mutant was thought to carry the 4.2-kb rIIH23 deletion, but instead it has been found to carry a longer rII deletion, defined genetically as extending from or near the end 115 on July 6, 2020 by guest

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confidence: 99%

Alignment of a restriction map with the genetic map of bacteriophage T4

Yee¹,

Marsh²

1981

J Virol

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“…As shown in Fig. 2C, fragment K-G was adjacent to K-E. Having accidentally overlooked K-G, we previously assigned K-F to this position to account for a partially cleaved KpnI fragment (4). K-F, however, was obtained from Cyt-9,12 and, therefore, must be located elsewhere (see below).…”

Section: Fig 1cmentioning

confidence: 94%

“…The order of the SalI fragments I and G within K-C was derived by assignment of a partially cleaved SK fragment (4). Neither S-I nor S-G was cleaved by BglI (or BamHI).…”

Section: Fig 1cmentioning

confidence: 99%

“…Enzymes. Endonucleases Sall, KpnI, and BamHI were obtained and assayed as described previously (4). BglI was purchased from New England Biolabs and assayed in the same manner as Sall.…”

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confidence: 99%

“…Electrophoresis. Agarose gel electrophoresis was carried out as described previously (4). Acrylamide gels contained 8% acrylamide and 0.27% bisacrylamide in 50% glycerol-45 mM Tris-borate (pH 8.3)-1.2 mM Na2EDTA (20).…”

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confidence: 99%

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Correlation between genetic map and map of cleavage sites for sequence-specific endonucleases SalI, KpnI, BglI, and BamHI in bacteriophage T4 cytosine-containing DNA

Carlson¹

1980

J Virol

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Cleavage sites for SalI, KpnI, BglI, and BamHI in cytosine-containing DNA from T4 alclO(alc) nd28(denA) D2a2(denB) amE5lx5(56) amN55x5(42) have been mapped relative to each other, and the positions of deletions saa9(Dl-stp), r1589(rII), del(39-56)12, and tk2(rI-tk) relative to these cleavage sites have been determined. Based on these analyses, a physical map of the T4 genome containing 166 kilobase pairs has been constructed. Recent studies from this (4) and other (14, 21, 30) laboratories have yielded cleavage maps of cytosine-containing DNA from bacteriophage T4 for several sequence-specific endonucleases (restriction enzymes). The potential value of these maps, of course, hinges on the accuracy with which the genetic content of each fragment is known. Deletion mutants of T4 may be utilized to establish directly the correlation between genetic and physical maps, since their genetic deficiencies can be compared with the altered cleavage patterns of their DNA. This paper presents a cleavage map for T4 DNA constructed with the enzymes SalI, KpnI, BglI, and BamHI and its correlation to the genetic map of the phage genome based on an analysis of deletion mutants. Correlations between restriction cleavage maps and the genetic map of T4 have been derived independently by O'Farreil et al. (P. O'Farreil, E. Kutter, and M. Nakanishi, manuscript in preparation) and by R. Marsh (manuscript in preparation) by different methods. MATERIALS AND METHODS Strains. (i) Phage. The phage strains employed in this study are listed in Table 1, and the construction steps leading up to strains Cyt-9,1589, Cyt-9,12, and Cyt-9,tk2 are summarized in Table 2. Relevant genotypes were verified as follows. (a) akc-denA-denBam dCTPase-("Cyt phenotype"). Phage stocks prepared in Escherichia coli B834, which is supo hsd, will plate on E. coli B834, but not on E. coli B23, which is supo hsd+ (23). (b) "rH23" (rII). There is no growth in E. coli K803(X) and no complementation or marker rescue in K803(X) of rII point mutants; it is resistant to acriflavine (10 i.g/ml in LB). "rH23" is not identical to the

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