Physical map of the bacteriophage T5 genome based on the cleavage products of the restriction endonucleases SalI, SmaI, BamI, and HpaI

Abstract

A physical map of the bacteriophage T5 genome was constructed by ordering the fragments produced by cleavage of T5 DNA with the restriction endonucleases SalI (4 fragments), SmaI (4 fragments), BamI (5 fragments), and HpaI (28 fragments). The following techniques were used to order the fragments. (i) Digestion of DNA from T5 heat-stable deletion mutants was used to identify fragments located in the deletable region. (ii) Fragments near the ends of the T5 DNA molecule were located by treating T5 DNA with X exonuclease before restriction endonuclease cleavage. (iii) Fragments spanning other restriction endonuclease cleavage sites were identified by combined digestion of T5 DNA with two restriction endonucleases. (iv) The general location of some fragments was determined by isolating individual restriction fragments from agarose gels and redigesting the isolated fragments with a second restriction enzyme. (v) Treatment of restriction digests with X exonuclease before digestion with a second restriction enzyme was used to identify fragments near, but not spanning, restriction cleavage sites. (vi) Exonuclease III treatment of T5 DNA before restriction endonuclease cleavage was used to locate fragments spanning or near the natural T5 single-chain interruptions. (vii) Analysis of the products of incomplete restriction endonuclease cleavage was used to identify adjacent fragments. MATERIALS AND METHODS Bacteriophage. Wild-type T5 and the heat-stable deletion mutants T5st(0) and T5st(102) (13, 15) were grown on Escherichia coli B23. EcoRI endonuclease. Purification and assay conditions for EcoRI have been described (11). Sally endonuclease. Streptomyces albus G (from G. Hayward) was grown at 30°C with shaking in a medium containing, in 1 liter: 0.33 g of yeast extract (Difco), 0.33 g of beef extract (Difco), 0.67 g of tryptone (Difco), trace FeSO4, and 3.33 g of glucose. After 2 days of growth, the mycelia were harvested by centrifugation and suspended in 0.01 M Tris, pH 7.9, containing 0.01 M 2-mercaptoethanol (approximately 10 ml per 15 g [wet weight] of mycelia). The cells were disrupted by sonication. Cell debris was removed from the extract by low-speed (8,000 rpm for 15 min in a Sorvall SS34 rotor) and high-speed centrifugation (33,000 rpm for 90 min in a Beckman SW50.1 rotor). The high-speed supernatant was made 1.0 M in NaCl and applied to a 200-ml column 249

Restriction cleavage map of SP01 DNA: general location of early, middle, and late genes

“…(A simple analysis of the data presented in Table 3 directly confirmed much of this map order. For example, HaeIl fragments 5 and 3 and HaeIll fragments 2 and 3 overlapped EcoRI* fragments 7 and 22, respectively. Thus, these HaeIl fragments had to be in the order 2-3-5.…”

Physical map of bacteriophage BF23 DNA: terminal redundancy and localization of single-chain interruptions

“…BF23 DNA was treated with T4 ligase, E. coli exonuclease III, and SI endonuclease from Aspergillus oryzae, as described previously for T5 DNA (17,18). Digestion of T5st(+) DNA with HpaI was carried out as described by Hamlett et al (5). RESULTS The sites where single-chain interruptions occur in BF23 DNA were determined, as reported previously for T5 DNA (17), by an electrophoretic analysis of two analogous sets of fragments.…”

“…2. The molecular weights of the duplex fragments of BF23 DNA were determined by coelectrophoresis in the presence of EcoRI and HpaI digests of T5 DNA (5,16). The results of these measurements are given in Table 1.…”

Physical map of bacteriophage BF23 DNA: restriction enzyme analysis

“…These studies have been aided by construction ofrestriction endonuclease cleavage maps of both genomes. A number of cleavage maps of T5 are available (2,4,8,10). This paper presents cleavage maps of BF23 DNA for the Sall, BamHI, EcoRI, BalI, and HpaI restriction endonucleases.…”

“…The locations of the st deletions were determined from heteroduplex analysis (unpublished data). (2). Fragments were isolated by the procedure of Blin et al (1).…”