Abstract
Effects of the size of template DNA on the DNA packaging reaction of bacteriophage phi X174 were studied using plasmids of various sizes which contain the phi X174 origin of DNA replication and the in vitro phage synthesizing system (Aoyama, A., Hamatake, R. K., and Hayashi, M. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 4195-4199). DNA between 78.5% and 101% of the length of phi X174 DNA produced infectious particles efficiently. Packaging of DNA smaller or larger than this range produced uninfectious defective particles. Although these particles contained circular single-stranded DNA, they suffered structural changes which altered the sedimentation properties or the ability to adsorb to the cells. Mutant phage were found from the packaging reaction of DNA larger than 101% of phi X174 DNA. These mutants deleted the termination region of DNA, suggesting that they were produced by early termination of the phage synthesizing reaction.
Highlights
Stage I11 is the synthesis of the circular viral In this report, the packaginogf DNA smaller orlarger than DNA using R F DNA as template
Packagingof viral DNA is the limit was studied using the in vitro phage synthesizing coupled to DNA synthesis during stage I11 (for review, see system developed in our laboratory [19]
The synthesisof the viral were constructed by cloning 4x174 HincII-3DNA fragment, DNA starts from this point which contains theorigin of 4x174 viralDNA synthesis, into and proceeds 5‘ to 3’ by a rolling circle mechanism [8].The plasmid pBR322, pBR325, or their derivatives
Summary
The PuuII sites (nucleotides 375-2069 of pBR322), pAS976,a pBR322 derivativewith a deletion bet,ween the EcoRI and the BamHI Stage 111 Reactions Using Recombinant Plasmid DNA as sites, pBR322, or pBR325 DNA. Deletion Mutants Made in Group C System-To study the nature of infectious phage made inlow efficiency in group A TheDNAisolated from the cellsinfectedwithgroupA or C system, the DNAwere isolated from cells infected with products co-migrated with the respective template DNA as these products and analyzed by gel electrophoresis (Fig. 6). 6 , l t o7 in c and d).the DNA from the cells infected vitro stage II(+) system is capable of synthesizing circular ssDNA with the group C products lost one of the EcoRI sites, which when 6x174 RF I DNA [25]or the recombinant plasmid DNA which resulted in the loss of the 4x174 HincII-3 DNA fragments carries 4x174 HincII-3 DNA fragment was added astemplate?. Only group B DNA could produce infectious phage efficiently, group A, whose genome sizes are between 3.25 kb and 3.65 kb, or group C , whose genome sizes are between 5.65
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