Bacteriophage P22, like other double-stranded DNA bacteriophages, packages DNA in a preassembled, DNA-free procapsid. The P22 procapsid and P22 bacteriophage have been electrophoretically characterized; the procapsid has a negative average electrical surface charge density (uf) higher in magnitude than the negative uf of the mature bacteriophage. Dextrans, sucrose, and maltose were shown to have a dramatic stimulatory effect on the in vitro packaging of DNA by the P22 procapsid. However,

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... tulose, smaller sugars, and smaller polyols did not stimulate in vitro P22 DNA packaging. These and other data suggest that an osmotic pressure difference across some particle, probably a capsid, stimulates P22 DNA packaging. After in vitro packaging was optimized by including dextran 40 in extracts, the entry kinetics of DNA into P22 capsids were measured. Packaged DNA was detected by: (i) DNA-specific staining of intact capsids after fractionation by agarose gel electrophoresis and (ii) agarose gel electrophoresis of DNase-resistant DNA after release of DNase-resistant DNA from capsids. It was found that the first DNA was packaged by 1.5 min after the start of incubation. The data further suggest that either P22 capsids with DNA partially packaged in vitro are too unstable to be detected by the above procedures or entry of DNA into the capsid occurs in less than 0.25 min. All double-stranded DNA bacteriophages that have been studied package DNA in vivo in a preformed, DNA-free procapsid. For all sufficiently studied bacteriophages, the external surface of the procapsid differs from the external surface of the mature bacteriophage capsid in having a rounder shape and smaller radius (reviewed in references 8, 9, 22, and 39). In the case of bacteriophage T7, the procapsid was also shown to have a negative average electrical surface charge density (cr) higher in magnitude than the negative cr of the mature bacteriophage capsid (37, 38). For analysis of DNA packaging, it is desirable to have packaging as synchronous as possible and to maximize control of the compounds present during packaging. To help accomplish these goals, DNA is packaged in extracts of bacteriophage-infected cells (in vitro). DNA packaging in vitro has been achieved with bacteriophages 4129 (5). In all of these studies, production of an infective particle was used as the assay for DNA packaging, and it was found that production of an infective particle required either ATP or some other ribonucleoside triphosphate. In the case of T7, a 10to 50-fold stimulation of in vitro infectious particle assembly was observed in the presence of dextrans and some smaller, related compounds (36). To determine the rate at which DNA enters bacteriophage capsids in vitro and to determine the effects of added compounds on this rate, it is necessary to have an assay for DNA entry into capsids, independent of other assembly events needed for an infective particle. Thus far, only bacteriophages T3 and 4)29 have been made to package DNA efficiently enough to assay physically for packaged DNA. In these studies (T3 [21] ; 4)29 [3, 4] ), velocity sedimentation in sucrose gradients, sometimes after DNase treatment, was used to detect capsids with packaged DNA. However, no attempt was made to measure the rate of DNA entry. In addition, velocity sedimentation in sucrose gradients has the following limitations as an assay for DNA entry. (i) The state of the capsid (procapsid or its larger, more angular conversion product) is not reliably determined (4). (ii) DNA may empty from capsids during the assay. (iii) The cost (in time and materials) can become excessive during studies of entry rates. A possible alternative DNA packaging assay for overcoming limitations (i) and (iii) above is DNA-specific staining of DNase-resistant DNA comigrating during agarose gel electrophoresis 96 on May 9, 2020 by guest