Concatemerization and packaging of bacteriophage T7 DNA in vitro

determination of the concatemers' length and appearance kinetics by use of rotating gel electrophoresis

Marjatta Son, Shirley J. Hayes, Philip Serwer

Research output: Contribution to journalArticle

21 Citations (Scopus)

Abstract

During its morphogenesis both in intact infected cells (in vivo) and in lysates of infected cells (in vitro), bacteriophage T7 forms end-to-end concatemers of its mature DNA, a linear, nonpermuted, terminally repetitious DNA. During morphogenesis, in vivo T7 concatemers are packaged in preformed capsids and cut to mature size. In the present study the lengths and appearance kinetics of concatemers formed in vitro from mature T7 DNA have been determined. The following procedures are used here for the first time: (a) 20-35% efficient in vitro concatemerization and packaging of T7 DNA; the mixture used for packaging contained two lysates that together had all T7 gene products, and (b) fractionation of concatemers by rotating gel electrophorsis (RGE), which improves the resolution by length of concatemer-length DNA. Concatemerization at 30° was so fast that some other process must be rate limiting for packaging. The concatemers formed were linear and joined left-end to right-end by complementary base pairing, not by blunt-end ligation. Concatemers formed at 30° were reconverted to mature DNA by packaging in vitro. Reducing the temperature to 0° both slowed concatemerization to the time scale (minutes) needed for control of the extent of concatemerization and reduced packaging to insignificant levels, thereby also uncoupling packaging from concatemerization. At both 30° and 0° bands of discrete-length concatemers were observed by RGE. The lengths were n times the length of mature T7 DNA; n was found to be any integer from 2 to 15. The bands were stronger at 0° than they were at 30° in comparison to a background of heterogeneous DNA. No evidence for the favoring of any value of n was found. In addition, it was found by two-dimensional agarose gel electrophoresis that a comparatively small amount of circular DNA was produced in vitro.

Original languageEnglish (US)
Pages (from-to)38-46
Number of pages9
JournalVirology
Volume162
Issue number1
DOIs
StatePublished - 1988

Fingerprint

Bacteriophage T7
Product Packaging
Electrophoresis
Gels
DNA Packaging
DNA
Morphogenesis
Concatenated DNA
Circular DNA
Agar Gel Electrophoresis
Capsid
Electrophoresis, Gel, Two-Dimensional
Base Pairing
Ligation
In Vitro Techniques
Temperature
Genes

ASJC Scopus subject areas

  • Virology
  • Infectious Diseases

Cite this

Concatemerization and packaging of bacteriophage T7 DNA in vitro : determination of the concatemers' length and appearance kinetics by use of rotating gel electrophoresis. / Son, Marjatta; Hayes, Shirley J.; Serwer, Philip.

In: Virology, Vol. 162, No. 1, 1988, p. 38-46.

Research output: Contribution to journalArticle

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abstract = "During its morphogenesis both in intact infected cells (in vivo) and in lysates of infected cells (in vitro), bacteriophage T7 forms end-to-end concatemers of its mature DNA, a linear, nonpermuted, terminally repetitious DNA. During morphogenesis, in vivo T7 concatemers are packaged in preformed capsids and cut to mature size. In the present study the lengths and appearance kinetics of concatemers formed in vitro from mature T7 DNA have been determined. The following procedures are used here for the first time: (a) 20-35{\%} efficient in vitro concatemerization and packaging of T7 DNA; the mixture used for packaging contained two lysates that together had all T7 gene products, and (b) fractionation of concatemers by rotating gel electrophorsis (RGE), which improves the resolution by length of concatemer-length DNA. Concatemerization at 30° was so fast that some other process must be rate limiting for packaging. The concatemers formed were linear and joined left-end to right-end by complementary base pairing, not by blunt-end ligation. Concatemers formed at 30° were reconverted to mature DNA by packaging in vitro. Reducing the temperature to 0° both slowed concatemerization to the time scale (minutes) needed for control of the extent of concatemerization and reduced packaging to insignificant levels, thereby also uncoupling packaging from concatemerization. At both 30° and 0° bands of discrete-length concatemers were observed by RGE. The lengths were n times the length of mature T7 DNA; n was found to be any integer from 2 to 15. The bands were stronger at 0° than they were at 30° in comparison to a background of heterogeneous DNA. No evidence for the favoring of any value of n was found. In addition, it was found by two-dimensional agarose gel electrophoresis that a comparatively small amount of circular DNA was produced in vitro.",
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