Transient orientation of linear DNA molecules during pulsed-fleld gel electrophoresis

Cytoskeletal crowding plays a key role in the diffusion of DNA molecules through the cell, acting as a barrier to effective intracellular transport and conformational stability required for processes such as transfection, viral infection, and gene therapy. Here, we elucidate the transport properties and conformational dynamics of linear and ring DNA molecules diffusing through entangled and crosslinked composite networks of actin and microtubules. We couple single-molecule conformational tracking with differential dynamic microscopy to reveal that ring and linear DNA exhibit unexpectedly distinct transport properties that are influenced differently by cytoskeleton crosslinking. Ring DNA coils are swollen and undergo heterogeneous and biphasic subdiffusion that is hindered by crosslinking. Conversely, crosslinking actually facilitates the single-mode subdiffusion that compacted linear chains exhibit. Our collective results demonstrate that transient threading by cytoskeleton filaments plays a key role in the dynamics of ring DNA, whereas the mobility of the cytoskeleton dictates transport of linear DNA.

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Replication forms of the gene-sized DNA molecules of hypotrichous ciliates.

Molecular and Cellular Biology ◽

10.1128/mcb.3.9.1562 ◽

1983 ◽

Vol 3 (9) ◽

pp. 1562-1566 ◽

Cited By ~ 16

Author(s):

K G Murti ◽

D M Prescott

Keyword(s):

Electron Microscopy ◽

S Phase ◽

Origin Of Replication ◽

Dna Molecules ◽

Linear Dna ◽

Dna Molecule ◽

Independent Unit

Using a method for obtaining DNA from 10 to 40 macronuclei for electron microscopy, we analyzed the structure of gene-sized, linear DNA molecules from S-phase macronuclei of two hypotrichous ciliates, Euplotes eurystomus and Styx sp. Three types of putative replicating intermediates were observed: (i) molecules with a bubble close to one end, (ii) molecules with single forks, and (iii) molecules with two forks. We conclude that: (i) each macronuclear DNA molecule replicates as an independent unit, (ii) the molecules contain an origin of replication close to one or both ends, and (iii) the mode of replication is bidirectional.

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Restriction mapping of recombinant λ DNA molecules using pulsed field gel electrophoresis

Analytical Biochemistry ◽

10.1016/0003-2697(90)90389-q ◽

1990 ◽

Vol 191 (1) ◽

pp. 70-74 ◽

Cited By ~ 2

Author(s):

Bruce L. Goode ◽

Stuart C. Feinstein

Keyword(s):

Gel Electrophoresis ◽

Pulsed Field Gel Electrophoresis ◽

Restriction Mapping ◽

Dna Molecules ◽

Pulsed Field

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Looping Dynamics of Linear DNA Molecules and the Effect of DNA Curvature: A Study by Brownian Dynamics Simulation

Biophysical Journal ◽

10.1016/s0006-3495(98)74002-0 ◽

1998 ◽

Vol 74 (2) ◽

pp. 773-779 ◽

Cited By ~ 55

Author(s):

Holger Merlitz ◽

Karsten Rippe ◽

Konstantin V. Klenin ◽

Jörg Langowski

Keyword(s):

Brownian Dynamics ◽

Dynamics Simulation ◽

Dna Curvature ◽

Dna Molecules ◽

Brownian Dynamics Simulation ◽

Linear Dna

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An electrophoretic karyotype of Neurospora crassa.

Molecular and Cellular Biology ◽

10.1128/mcb.8.4.1469 ◽

1988 ◽

Vol 8 (4) ◽

pp. 1469-1473 ◽

Cited By ~ 176

Author(s):

M J Orbach ◽

D Vollrath ◽

R W Davis ◽

C Yanofsky

Keyword(s):

Neurospora Crassa ◽

Electric Fields ◽

Gel Electrophoresis ◽

Electrophoretic Karyotype ◽

Dna Molecules ◽

Linkage Groups ◽

Homogeneous Electric Field ◽

Chromosomal Dna ◽

Molecular Karyotype ◽

Electrophoresis System

A molecular karyotype of Neurospora crassa was obtained by using an alternating-field gel electrophoresis system which employs contour-clamped homogeneous electric fields. The migration of all seven N. crassa chromosomal DNAs was defined, and five of the seven molecules were separated from one another. The estimated sizes of these molecules, based on their migration relative to Schizosaccharomyces pombe chromosomal DNA molecules, are 4 to 12.6 megabases. The seven linkage groups were correlated with specific chromosomal DNA bands by hybridizing transfers of contour-clamped homogeneous electric field gels with radioactive probes specific to each linkage group. The mobilities of minichromosomal DNAs generated from translocation strains were also examined. The methods used for preparation of chromosomal DNA molecules and the conditions for their separation should be applicable to other filamentous fungi.

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