Phage cycle

Replication Replication of lambda DNA occurs in two distinct fashions during different parts of the phage production cycle. Initially, liberation of lambda DNA from the host results in replication of a circular DNA, but subsequently linear concatamers are formed, which replicate in a different way. Replication is initiated at a site close to gene 0 and from there proceeds in opposite directions (bidirectional... 

Temperate bacteriophage, the best known being phage X, have a very different life cycle. Their DNA usually becomes integrated at a specific point into the genome of the bacterium (Chapter 27). Only rarely is an infected cell lysed. The retroviruses that attack mammals and birds have a similar characteristic. 

Of the 50 genes present in native X, only about half are necessary for replication in the lytic cycle. Thus, it is possible to delete about 1 / 3 of the genome to make room for more passenger DNA. However, to form mature phage particles the length of the DNA must be at least 75% of the native length. No more than 110% of the native amount may be present. The total DNA must fall between 38 and 53 kb in length. 

Particularly relevant are studies on the replication of RNA-con-taining viruses, all of which have a double-stranded stage in their life cycles. Additionally, it may yield specific limited cleavages of such single-stranded RNA molecules as tRNA, ribosomal RNA, and phage RNA. Finally its ability to digest the RNA of DNA-RNA hybrids should provide a further measure of specificity in DNA-RNA hybridization experiments. ... 

As the lytic cycle progresses, the phage DNA replicates. The increase in the number of gene copies can result in overexpression of the cl, which could shut down the lytic cycle. This effect is overcome by the regulatory protein cro, which is specifically designed to inhibit cl synthesis during late infection. 

Figure 22.2 Cellular activation by CpG DNA. CpG DNA directly activates dendritic cells (DCs), monocytes and macrophages, to express increased levels of co-stimu-latory molecules, to increase antigen presentation, and to secrete high levels of chemokines and cytokines, such as interleukin 12 (IL-12), interferon-a(IFN-a), and tumor necrosis factor-a (TNF-a), and monocytes and macro-phages have increased antibody-dependent cellular cytotoxicity (ADCC) activity. NK cells are induced to express IFN-7 by these cytokines acting in concert with CpG, and have increased lytic activity. B cells rapidly produce IL-b and IL-10 and express increased levels of costimulatory molecules. B cells rapidly enter the cell cycle and become resistant to some forms of activation-induced cell death. T cells are not directly activated by CpG, but because of the T helper 1 (Thl)-like cytokine environment, and the increased antigen presenting cell (APC) activity, antigen-specific Thl cells and cytotoxic T lymphocytes (CTL) are generated.

A non-contact heating method called infrared-mediated temperature control has been employed for PCR. Because the chip material (polyimide) does not absorb IR, only the solution absorbs IR. Therefore, the low thermal mass of the solution allows for fast thermal cycling, and 15 cycles have been achieved in 240 s Amplification of X phage DNA (500 bp) was first conducted at 94°C for 10 s, followed by 15 cycles of 94°C (2 s), 68°C (2 s), 72°C (2 s), then finally stopped at 72°C for 10 s [192],... 

In another report, PCR and subsequent CGE separation were integrated on a glass microchip (see Figure 9.1). PCR of X phage DNA was conducted in a sample reservoir with thermal cycling by a Peltier heater/cooler. Subsequent CGE separation was conducted immediately after PCR because the PCR reservoir led to the CE channel. In addition, an on-chip DNA pre-concentration device was included. This reduced the analysis time to 20 min (by decreasing the number of thermal cycles required to 10 cycles), and the starting DNA copy number to 15 (0.3 pM) [925],... 

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