Bacteriophage T7 DNA packaging. I. Plasmids containing a T7 replication origin and the T7 concatemer junction are packaged into transducing particles during phage infection.
Bacteriophage T7 DNA is a linear duplex molecule with a 160 base-pair direct repeat (terminal redundancy) at its ends. During replication, large DNA concatemers are formed, which are multimers of the T7 genome linked head to tail through recombination at the terminal redundancy. We define the sequence that results from this recombination, a mature right end joined to the left end of T7 DNA, as the concatemer junction. To study the processing and packaging of T7 concatemers into phage particles, we have cloned the T7 concatemer junction into a plasmid vector. This plasmid is efficiently (at least 15 particles/infected cell) packaged into transducing particles during a T7 infection. These transducing particles can be separated from T7 phage by sedimentation to equilibrium in CsCl. The packaged plasmid DNA is a linear concatemer of about 40 x 10(3) base-pairs with ends at the expected T7 DNA sequences. Thus, the T7 concatemer junction sequence on the plasmid is recognized for processing and packaging by the phage system. We have identified a T7 DNA replication origin near the right end of the T7 genome that is necessary for efficient plasmid packaging. The origin, which is associated with a T7 RNA polymerase promoter, causes amplification of the plasmid DNA during T7 infection. The amplified plasmid DNA sediments very rapidly and contains large concatemers, which are expected to be good substrates for the packaging reaction. When cloned in pBR322, a sequence containing only the mature right end of T7 DNA is sufficient for efficient packaging. Since this sequence does not contain DNA to the right of the site where a mature T7 right end is formed, it was expected that right ends would not form on this DNA. In fact, with this plasmid the right end does not form at the normal T7 sequence but is instead formed within the vector. Apparently, the T7 packaging system can also recognize a site in pBR322 DNA to produce an end for packaging. This site is not recognized solely by a "headful" mechanism, since there can be considerable variation in the amount of DNA packaged (32 x 10(3) to 42 x 10(3) base-pairs). Furthermore, deletion of this region from the vector DNA prevents packaging of the plasmid. The end that is formed in vector DNA is somewhat heterogeneous. About one-third of the ends are at a unique site (nucleotide 1712 of pBR322), which is followed by the sequence 5'-ATCTGT-3'. This sequence is also found adjacent to the cut made in a T7 DNA concatemer to produce a normal T7 right end.
Chung
Y B
YB
Department of Biology, University of Rochester, NY 14627.
Hinkle
D C
DC
D001483:Base Sequence; D002499:Centrifugation, Density Gradient; D003001:Cloning, Molecular / Q000379:methods; D004261:DNA Replication; D004279:DNA, Viral / Q000302:isolation & purification / Q000502:physiology*; D004587:Electrophoresis, Agar Gel; D005822:Genetic Vectors; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D009024:Morphogenesis; D010957:Plasmids; D012045:Regulatory Sequences, Nucleic Acid; D015183:Restriction Mapping; D013604:T-Phages / Q000235:genetics* / Q000302:isolation & purification / Q000648:ultrastructure; D014161:Transduction, Genetic; D014779:Virus Replication
D004279:DNA, Viral