Bacteriophages synthesis

Figure 8.1 A region of DNA in the related bacteriophages lambda, 434, and P22 that controls the switch for synthesis of new phage particles. Two structural genes are involved in this switch one coding for a repressor protein and one coding for the Cro protein. Between these genes there is an operator region (OR) that contains three protein binding sites—ORl, OR2, and OR3.

Tomato Agritrope/1996 5-Adenosylmethionine hydrolase Escherichia coli bacteriophage T3 Delayed fruit ripening due to reduced ethylene synthesis... 

Figure 5.15 The filamentous single-stranded DNA bacteriophage fd. Orientation of the proteins and genes in the virion. Note the intergenic space which contains the origin of DNA synthesis. Gene cloning is done in this intergenic space.

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... 

Indicators of microbial activity in soil represent measurements at the ecosystem level (e.g., processes regulating decomposition of organic residues and nutrient cycling, especially nitrogen, sulfur and phosphorus). Measurements at the community level include bacterial DNA and protein synthesis. Frequency of bacteriophages is a measurement at the population level. 

A prerequisite for cRNA transcript synthesis is the presence of the cDNA of interest within a suitable expression vector containing a bacteriophage polymerase promoter (e.g. T3, T7 or SP6). The vector must be linearized downstream of the cDNA insert prior to its use as the template in the in vitro reaction. 

Prokaryotic Bacterial cells infected with bacteriophage Transient expression Infected cells Viral (bacterial phage) promoter and other elements required to support viral protein synthesis... 

Some E. coli bacteriophages, including f2, MS2, R17, and Qj8, as well as some eukaryotic viruses (including influenza and Sindbis viruses, the latter associated with a form of encephalitis) have RNA genomes. The single-stranded RNA chromosomes of these viruses, which also function as mRNAs for the synthesis of viral proteins, are replicated in the host cell by an RNA-dependent RNA polymerase (RNA replicase). All RNA viruses—with the exception of retroviruses—must encode a protein with RNA-dependent RNA polymerase activity because the host cells do not possess this enzyme. 

Figure 5-13 Electron micrograph of a DNA molecule (from a bacterial virus bacteriophage T7) undergoing replication. The viral DNA is a long ( 14 pm) duplex rod containing about 40,000 base pairs. In this view of a replicating molecule an internal "eye" in which DNA has been duplicated is present. The DNA synthesis was initiated at a special site (origin) about 17% of the total length from one end of the duplex. The DNA was stained with uranyl acetate and viewed by dark field electron microscopy. Micrograph courtesy J. Wolfson and D. Dressier.

A sample of double-stranded DNA is denatured. One of the resulting single strands is used as a template to direct the synthesis of a complementary strand of radioactive DNA using a suitable DNA polymerase. The "Klenow fragment" of E. coli, DNA polymerase I, reverse transcriptase from a retrovirus, bacteriophage T7 DNA polymerase, Taq polymerase, and specially engineered enzymes produced from cloned genes have all been used. 

In 1961, Jacob and Monod postulated messenger RNA (mRNA) as a short-lived polynucleotide.30 32 33 An abundance of additional evidence supported the proposal. For example, RNA molecules produced after infection of E. coli by bacteriophage T4 underwent hybridization (Chapter 5) with denatured DNA of the bacteriophage. Furthermore, this virus-specific mRNA became associated with preexisting bacterial ribosomes and provided the template for synthesis of phage proteins.34 The experiment provided direct evidence for transcription of mRNA from genes of the viral DNA. 

In addition, they assist in replication of RNA viruses, another process that requires RNA synthesis. Viruses sometimes make use of host RNA polymerases but often synthesize their own catalytic subunits. Bacteriophage T4 uses the E. coli RNA polymerase and o factors but modifies their action through the binding of several phage-encoded proteins.248 In contrast, phage T7 encodes its own relatively simple RNAP whose initiation complex (Section A,2)29 and elongation complexes have been studied 249-249b... 

Most translation is terminated at this stop codon, but frameshifting, which is induced by a high polyamine concentration, allows read-through and synthesis of the antizyme protein. In rare cases frameshifting may lead to translational bypass of some codons on the mRNA. Such a case is found in a bacteriophage T4 mRNA for which the E. coli ribosomes bypass 50 nucleotides in order to complete the synthesis of a... 

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