Concatemerization and packaging of bacteriophage T7 DNA in vitro: determination of the concatemers' length and appearance kinetics by use of rotating gel electrophoresis.
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 degrees 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 degrees were reconverted to mature DNA by packaging in vitro. Reducing the temperature to 0 degrees 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 degrees and 0 degrees 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 degrees than they were at 30 degrees 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.
Son
M
M
Department of Biochemistry, University of Texas Health Science Center, San Antonio 78284-7760.
Hayes
S J
SJ
Serwer
P
P
D003080:Cold Temperature; D004270:DNA, Circular; D004279:DNA, Viral; D004587:Electrophoresis, Agar Gel; D066298:In Vitro Techniques; D007700:Kinetics; D009024:Morphogenesis; D009690:Nucleic Acid Conformation; D013604:T-Phages / Q000235:genetics / Q000648:ultrastructure; D014779:Virus Replication
D004270:DNA, Circular; D004279:DNA, Viral