T4 infected E. coli

T4-DNA kinase. Enzyme isolated from phage T4 infected E. coli which catalyses the transfer of the y-phosphate group of rATP to the 5 -hydroxyl terminus of DNA and RNA T4-DNA ligase. Enzyme isolated from phage T4 infected E. coli which catalyses its formation of a phosphodiester bond between adjacent 5 -phosphorylated and 3 -hydroxy terminated DNA strands... 

The thioredoxin reductases are highly specific for their thioredoxin substrate. For instance, yeast thioredoxin reductase is specific for the reduction of the homologous thioredoxins and does not interact with thioredoxins from E. coli or from T4-infected E. coli. Furthermore E. coli thioredoxin reductase does not use the thioredoxins from Novikoff hepatoma, yeast, and L. leichmannii. 

DNA fragments can be joined (G3) to create artificially recombinant molecules. There are currently three methods for joining DNA fragments in vitro. The first of these capitalizes on the ability of DNA ligase to join covalently the annealed cohesive ends produced by certain restriction enzymes. The second depends upon the ability of DNA ligase from phage T4-infected E. coli to catalyze the formation... 

Soon after T4 phage has infected E. coli, host protein synthesis is inhibited and the bacterium cannot be superinfected with RNA phage. This is reflected by the inability of ribosomes from T4 infected E. coli to translate MS2, f2, or R17 RNA in vitro or to bind to the initiation sites at the beginning of each cistron (Dube and Rudland, 1970 Steitz et al., 1970 Hsu and Weiss, 1969 Klem et al, 1970). If ribosomes from infected cells are washed in 2.0 M NH,C1 and then supplemented with initiation factors from normal ribosomes they are once more able to translate phage RNA. On the other hand, the wash fraction from infected ribosomes prevents, and even reverses, the formation of initiation complexes between normal ribosomes and MS2 RNA. These observations suggest that after T4 infection E. coli initiation factors are selectively... 

The a subunit, the gene product (329 amino acids, 36,512) of rpoA, is required for the assembly of the core enzyme and plays a role in promoter recognition. When phage T4 infects E. coli, an Arg residue of the a subunit is modified by ADP-ribosylation which results in a reduction of the affinity of the promoter to the holoenzyme. The carboxyl-terminal domain (CTD, 99 amino acids) is regarded as the contact site for transcription activators, e.g.. 

Pollack, Y., Groner, Y., Aviv (Greenshpan), H., Revel, M. Role of initiation factor B (F3) in the preferential translation of T4 late messenger RNA in T4 infected E. coli. FEBS Letters 9, 218-221 (1970). 

To make blunt ends out of DNA fragments having 3 overhangs, T4 DNA polymerase is recommended. T4 DNA polymerase is an enzyme isolated from bacteriophage T4-infected E. coli. Like the Klenow fragment, it also has 5 to 3 polymerization and 3 to 5 exonuclease activities (Sambrook and Russell, 2001). However, the activity of 3 to 5 exonuclease is 200-fold greater than that of the Klenow fragment. 

Penetration The means by which the vims penetrates into the cell depends on the nature of the host cell, especially on its surface stmctures. Cells with cell walls, such as bacteria, are infected in a different manner from animal cells, which lack a cell wall. The most complicated penetration mechanisms have been found in viruses that infect bacteria. The bacteriophage T4, which infects E. coli, can be used as an example. 

Escherichia coli (strain B [28] overproducing strain JM83(pKT8P3) [31] infected and uninfected [32] infected by bacteriophage T4, host-coded enzyme from infected E. coli is part of bacteriophage T4 dNTP-synthesiz-ing multi-enzyme complex [25]) [25, 28, 31, 32]... 

Sadowski and Hurwitz have described two endonucleases synthesized in T4 phage-infected E. coli which they have named T4 endonucleases II and IV (66). 

Thioredoxin systems have also been demonstrated in L. leichmannii (39), rat Novikoff hepatoma (40), T4 phage infected E. coli (41, 42), yeast (43), rat liver (44), calf liver (45) and germinating wheat embryo (32). 

Bacteriophages T 5 and T 6, but not T 7 or a 2 phage, also induce new ribonucleotide reductase activities in infected E. coli cells. The enzymes of T4 and T6 were immunologically related but did not cross-react with E. coli enzyme. The unstable reductase of phage T5 differed again because of its hydroxyurea sensitivity it is probably an iron protein, too. 

The 3 -phosphatase-free T4 PNK has also been prepared from mutant phage T4 amN81 pseTl-infected E. coli BB cells (23,26,50). The mutant enzyme has almost intact 5 -kinase activity but none of the 3 -phosphatase activity. The 3 -phosphatase-free T4 PNK is commercially available from BMB. 

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. 

T4 DNA polymerase E. coli infected with bacteriophage T4 5 —> 3 chain growth 3 —> 5 exonuclease... 

A DNA polymerase isolated from phage T4 infected coli. Possesses a 3 - 5 exonuclease activity but lacks the 5 - 3 activity found in E. coli DNA poll. Used for synthesis of DNA on a single-stranded template (Section 1.3.4.)... 

B. Ribonucleoside Diphosphate Reductase Induced in E. coli by Infection with Bacteriophage T4... 

Both E. coli and T4 thioredoxin have recently been purified by immu-noabsorbent affinity chromatography. The specific immunoadsorbents were prepared by coupling the a-globular fractions of rabbit anti-thio-redoxin antisera to Sepharose. This technique makes it possible to isolate both thioredoxins from phage-infected cells by two consecutive chromatography steps on the specific adsorbents (125, 126). 

The life cycle of T4 (Figure 17L) begins with adsorbing the virion to the surface of an E. coli cell. Because the bacterial cell wall is rigid, the entire virion cannot penetrate into the cell s interior. Instead, the DNA is injected by flexing and constricting the tail apparatus. Once the DNA has entered the cell, the infective process is complete, and the next phase (replication) begins. 

Within 2 minutes after the injection of T4 phage DNA into an E. coli cell, synthesis of host DNA, RNA, and protein stops and phage mRNA synthesis begins. Phage mRNA codes for the synthesis of capsid proteins and some of the enzymes required for the replication of the viral genome and the assembly of virion components. In addition, other enzymes are synthesized that weaken the host cell s cell wall, so that new phage can be released for new rounds of infection. Approximately 22 minutes after viral DNA (vDNA) is injected, the host cell, now filled with several hundred new virions, lyses. Upon release, the virions attach to nearby bacteria, thus initiating new infections. 

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