Sequencing Strategy for a Protein-Linked Genome: Spontaneous Reversions of Ochre Triplet in the Phage ϕ29 Gene 17

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Title:Sequencing Strategy for a Protein-Linked Genome: Spontaneous Reversions of Ochre Triplet in the Phage ϕ29 Gene 17
Creators:
Fučík, Vladimír
Chaloupecký, Vladimír
Beran, Jaroslav
Jonák, Jiří
Journal or Publication Title:
Collection of Czechoslovak Chemical Communications, 62, 7, pp. 1136-1142
Uncontrolled Keywords:Terminal protein, Proteinase K, T7 gene 6 exonuclease, Bacteriophage fi29, Spontaneous mutations

Abstract

A direct method using T7 gene 6 exonuclease to prepare single-stranded templates was adapted for the sequencing of the phage ϕ29 genome carrying a covalently linked terminal protein at the 5' ends of both DNA chains. The terminal protein was found to block completely the action of the exonuclease. The treatment of the phage DNA by proteinase K did not restore the accessibility of the 5' ends to the exonuclease to the full extent but only partially. The digestion of proteinase K-treated terminal fragments of the genome by 5' exonuclease could proceed now from both 5' ends, however, at different rates. The delay of exonucleolytic digestion apparently due to residual amino acid(s) was calculated to be about 800 nucleotides. The relative rates of exonucleolytic splitting of both chains were of decisive significance for the choice of suitable primers for the sequencing of desired regions. In all Sus<sup>+</sup> revertants sequenced by the described method the TAA(ochre) codon in the gene 17 was found changed either to the original CAA(Gln) or to TAT(Tyr) with the same frequencies.

Title:Sequencing Strategy for a Protein-Linked Genome: Spontaneous Reversions of Ochre Triplet in the Phage ϕ29 Gene 17
Creators:
Fučík, Vladimír
Chaloupecký, Vladimír
Beran, Jaroslav
Jonák, Jiří
Uncontrolled Keywords:Terminal protein, Proteinase K, T7 gene 6 exonuclease, Bacteriophage fi29, Spontaneous mutations
Divisions:Life and Chemical Sciences > Institute of Organic Chemistry and Biochemistry > Collection of Czechoslovak Chemical Communications
Journal or Publication Title:Collection of Czechoslovak Chemical Communications
Volume:62
Number:7
Page Range:pp. 1136-1142
ISSN:0010-0765
E-ISSN:1212-6950
Publisher:Institute of Organic Chemistry and Biochemistry
Related URLs:
URLURL Type
http://dx.doi.org/10.1135/cccc19971136UNSPECIFIED
ID Code:1193
Item Type:Article
Deposited On:06 Feb 2009 17:07
Last Modified:06 Feb 2009 16:07

Citation

Fučík, Vladimír; Chaloupecký, Vladimír; Beran, Jaroslav; Jonák, Jiří (1997) Sequencing Strategy for a Protein-Linked Genome: Spontaneous Reversions of Ochre Triplet in the Phage ϕ29 Gene 17. Collection of Czechoslovak Chemical Communications, 62 (7). pp. 1136-1142. ISSN 0010-0765

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