Diaz L A, Hardisson C, Rodicio M R
Departamento de Biología Funcional, Area de Microbiología, Universidad de Oviedo, Spain.
J Gen Microbiol. 1991 Feb;137(2):293-8. doi: 10.1099/00221287-137-2-293.
A restriction map of phi A7 DNA (46.7 kb) was established for nine endonucleases (BclI, ClaI, EcoRI, EcoRV, HpaI, PvuI, SacII, SphI and XbaI) which cut the phage genome up to 11 times. There was no sites for BamHI, BglII, HindIII, PstI, PvuII, SacI or SalI. phi A7 DNA, circularized through its cohesive ends, could integrate into the genome of several Streptomyces hosts, to form stable lysogens. Integration occurred by recombination between unique attachment sites on the phage (attP) and the host (attB) genomes. The attP site has been located on the phi A7 restriction map. Deletion mutants of phi A7 DNA were obtained by selecting for pyrophosphate- or EDTA-resistant clones. The deletions occurred either near the left-hand end of the conventional restriction map, or about 18 kb from the right-hand end, close to, but not affecting the unique SacII site. Together, the deletions defined at least 7.9 kb of DNA (16.9% of the phage genome) non-essential for plaque formation. phi A7 DNA was introduced into S. lividans protoplasts by liposome-assisted transfection. Since the phage does not adsorb to intact cells of this strain, and therefore does not form plaques, an overlay of S. antibioticus spores was used to detect the infectious progeny released by the protoplasts. Using this technique, phi A7 could be introduced into S. antibioticus with an efficiency of about 6 x 10(6) p.f.u. per micrograms DNA (equivalent to 3 x 10(-4) p.f.u. per DNA molecule).
构建了噬菌体φA7 DNA（46.7 kb）的限制性酶切图谱，该图谱针对9种核酸内切酶（BclI、ClaI、EcoRI、EcoRV、HpaI、PvuI、SacII、SphI和XbaI），这些酶可将噬菌体基因组切割多达11次。不存在BamHI、BglII、HindIII、PstI、PvuII、SacI或SalI的酶切位点。通过其粘性末端环化的φA7 DNA可整合到几种链霉菌宿主的基因组中，形成稳定的溶原菌。整合通过噬菌体（attP）和宿主（attB）基因组上独特的附着位点之间的重组发生。attP位点已定位在φA7限制性酶切图谱上。通过选择对焦磷酸或EDTA抗性的克隆获得了φA7 DNA的缺失突变体。缺失发生在传统限制性酶切图谱的左端附近，或距右端约18 kb处，靠近但不影响独特的SacII位点。这些缺失共同定义了至少7.9 kb的DNA（占噬菌体基因组的16.9%），其对于噬菌斑形成是非必需的。通过脂质体辅助转染将φA7 DNA引入变铅青链霉菌原生质体。由于该噬菌体不吸附于该菌株的完整细胞，因此不形成噬菌斑，所以使用抗生链霉菌孢子覆盖物来检测原生质体释放的感染性后代。使用该技术，可将φA7以约每微克DNA 6×10⁶噬菌斑形成单位（p.f.u.）的效率引入抗生链霉菌（相当于每个DNA分子3×10⁻⁴ p.f.u.）。
