A small (58-nm) attached sphere perturbs the sieving of 40-80-kilobase DNA in 0.2-2.5% agarose gels: analysis of bacteriophage T7 capsid-DNA complexes by use of pulsed field electrophoresis.
Although the icosahedral bacteriophage T7 capsid has a diameter (58 nm) that is 234-fold smaller than the length of the linear, double-stranded T7 DNA, binding of a T7 capsid to T7 DNA is found here to have dramatic effects on the migration of the DNA during both pulsed field agarose gel electrophoresis (PFGE; the field inversion mode is used) and constant field agarose gel electrophoresis (CFGE). For these studies, capsid-DNA complexes were obtained by expelling DNA from mature bacteriophage T7; this procedure yields DNA with capsids bound at a variable position on the DNA. When subjected to CFGE at 2-6 V/cm in 0.20-2.5% agarose gels, capsid-DNA complexes arrest at the electrophoretic origin. Progressively lowering the electrical potential gradient to 0.5 V/cm results in migration; most complexes form a single band. The elevated electrical potential gradient (3 V/cm) induced arrest of capsid-DNA complexes is reversed when PFGE is used instead of CFGE. For some conditions of PFGE, the mobility of capsid-DNA complexes is a function of the position of the capsid on the DNA. During either CFGE (0.5 V/cm) or PFGE, capsid-DNA complexes increasingly separate from capsid-free DNA as the percentage of agarose increases. During these studies, capsid-DNA complexes are identified by electron microscopy of enzymatically-digested pieces of agarose gel; this is apparently the first successful electron microscopy of DNA from an agarose gel.(ABSTRACT TRUNCATED AT 250 WORDS)
Serwer
P
P
Department of Biochemistry, University of Texas Health Science Center, San Antonio 78284-7760.
Hayes
S J
SJ
Moreno
E T
ET
Park
C Y
CY
D002213:Capsid / Q000737:chemistry* / Q000648:ultrastructure; D004279:DNA, Viral / Q000737:chemistry* / Q000648:ultrastructure; D016521:Electrophoresis, Gel, Pulsed-Field; D004706:Endodeoxyribonucleases / Q000378:metabolism; D046911:Macromolecular Substances; D013604:T-Phages / Q000737:chemistry* / Q000648:ultrastructure
D004279:DNA, Viral; D046911:Macromolecular Substances; D004706:Endodeoxyribonucleases