RNA Polymerase primed DNA synthesis in T7 Bacteriophage system

Enzymatic RNA Synthesis using Bacteriophage T7 RNA Polymerase

Bacteriophage T7 DNA polymerase – sequenase

An ideal DNA polymerase for chain-terminating DNA sequencing should possess the following features: (1) incorporate dideoxy- and other modified nucleotides at an efficiency similar to that of the cognate deoxynucleotides; (2) high processivity; (3) high fidelity in the absence of proofreading/exonuclease activity; and (4) production of clear and uniform signals for detection. The DNA polymerase...

Function of DNA polymerase I in RNA-primed synthesis of bacteriophage M-13 duplex DNA.

Cell-free extracts from Escherichia coli contain a DNA polymerase activity resistant to SH-blocking agents, which is capable of synthesizing complementary strand DNA on a circular M-13 DNA template by extension of RNA primers. This activity is considered to be identical with DNA polymerase I (or some altered form of this enzyme) since it is missing in extracts from po1A- cells. DNA synthesis in...

Cytoplasmic expression system based on constitutive synthesis of bacteriophage T7 RNA polymerase in mammalian cells.

A mouse cell line that constitutively synthesizes the bacteriophage T7 RNA polymerase was constructed. Fluorescence microscopy indicated that the T7 RNA polymerase was present in the cytoplasmic compartment. The system provided, therefore, a unique opportunity to study structural elements of mRNA that affect stability and translation. The in vivo activity of the bacteriophage polymerase was dem...

Escherichia coli thioredoxin stabilizes complexes of bacteriophage T7 DNA polymerase and primed templates.

The DNA polymerase activity induced after bacteriophage T7 infection of Escherichia coli is found in a complex of two proteins, the T7 gene 5 protein and a host protein, thioredoxin. Gene 5 protein is a DNA polymerase and a 3' to 5' exonuclease. Thioredoxin binds tightly to the gene 5 protein and increases the processivity of polymerization some 1000-fold. Gene 5 protein forms a short-lived com...