Bacteriophage replication

Other experiments also pointed to the conclusion that DNA was the genetic material. DNA was found localized in the nuclei of eukaryotic cells. The absolute amount per cell was constant for a given species. Studies of bacteriophage replication pointed strongly to DNA as the genetic material.11 In 1952 Hershey and Chase showed that when a phage particle infects a cell the viral DNA enters the bacterium, but the protein "coat" remains outside.13 This was demonstrated by... 

Sinha NK, Morris CF, Alberts BM. Efficient in vitro replication of double-stranded DNA templates by a purified T4 bacteriophage replication system. J. Biol. Chem. 1980 255 4290-4293. 

Figure 16.1 Viruses vary in size and shape from the simplest satellite viruses (a) that need another virus for their replication to the T-even bacteriophages (d) that have developed sophisticated mechanisms for injecting DNA into bacteria. Four different virus particles are shown to scale.

PAC A high capacity (70-95 kb) cloning vector based upon the lytic E. colt bacteriophage PI that replicates in bacteria as an extrachromosomal element. 

An alternative to repeated cloning of PCR products is a recombination-based approach developed by Liu et al. (1998) to permit the cloning of a PCR product into a plasmid and the rapid conversion of the plasmid to a number of different expression systems without the necessity of cloning the PCR product multiple, independent times. The method, termed the univector plasmid-fusion system (UPS), involves the insertion of the PCR product into a particular type of plasmid, called the univector, which can then be placed under the control of a variety of promoters or fused in-frame to various tag sequences. The system is based upon plasmid fusion using the Cre-lox site-specific recombination system of bacteriophage PI (Sternberg et al., 1981). The Cre enzyme is a site-specific recombinase that catalyzes recombination between two 34 base pair (bp) loxP sequences and is involved in the resolution of dimers formed during replication of the... 

The basic problem of virus replication can be simply put the virus must somehow induce a living host cell to synthesize all of the essential components needed to make more virus particles. These components must then be assembled into the proper structure and the new virus particles must escape from the cell and infect other cells. The various phases of this replication process in a bacteriophage can be categorized in seven steps ... 

Figure 5.21 Consequences of infection by a temperate bacteriophage. The alternatives upon infection are integration of the virus DNA into the host DNA (lysogenization) or replication and release of mature virus (lysis). The lysogenic cell can also be induced to produce mature virus and lyse.

In E. coli cells, DNA replication starts at a specific site called oriC. The oriC locus contains only 245 base pairs. Similar sequences are responsible for initiating the synthesis of plasmid and bacteriophage DNA. The oriC nucleotide sequence binds several units of the tetrameric form of the dnaA protein. This protein is named for the gene that encodes it. The dnaB and dnaC proteins then bind to the complex. As a result of binding these proteins, a portion of the helical DNA is unwound. This forces the rest of the DNA into a left-handed double helix that wraps around the proteins to give a structure... 

DNA very efficiently into their host bacteria. A single bacteriophage DNA can be replicated approximately 100-fold or more per cell depending on the type of phage and its host. 

An essential feature of the cloning vector used is that it must be capable of self-replication in the cell into which it is introduced, which is usually E. coli. Two of the most commonly used types of vector in conjunction with E. coli are plasmids and bacteriophage X. Plasmids are circular extra-chromosomal DNA molecules, generally between 5000 and 350 0000 bp in length, that are found naturally in a wide range of bacteria. They generally house several... 

In lysogenic cytlos. replication of the bacteriophage lambda occurs without lysis of the infected tell. 

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