scholarly journals Two-dimensional gel electrophoretic method for mapping DNA replicons.

1988 ◽  
Vol 8 (4) ◽  
pp. 1408-1413 ◽  
Author(s):  
K A Nawotka ◽  
J A Huberman
Keyword(s):  
Gel Electrophoresis ◽  
Replication Origin ◽  
Replication Fork ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Two Dimensional ◽  
Agarose Gel Electrophoresis ◽  
Electrophoretic Method ◽  
Short Probe

We describe in detail a method which allows determination of the directions of replication fork movement through segments of DNA for which cloned probes are available. The method uses two-dimensional neutral-alkaline agarose gel electrophoresis followed by hybridization with short probe sequences. The nascent strands of replicating molecules form an arc separated from parental and nonreplicating strands. The closer a probe is to its replication origin or to the origin-proximal end of its restriction fragment, the shorter the nascent strands that are detected by the probe. The use of multiple probes allows determination of directions of replication fork movement, as well as locations of origins and termini. In this study, we used simian virus 40 as a model to demonstrate the feasibility of the method, and we discuss its applicability to other systems.

Two-dimensional gel electrophoretic method for mapping DNA replicons

1988 ◽  
Vol 8 (4) ◽  
pp. 1408-1413 ◽  
Author(s):  
K A Nawotka ◽  
J A Huberman
Keyword(s):  
Gel Electrophoresis ◽  
Replication Origin ◽  
Replication Fork ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Two Dimensional ◽  
Agarose Gel Electrophoresis ◽  
Electrophoretic Method ◽  
Short Probe

We describe in detail a method which allows determination of the directions of replication fork movement through segments of DNA for which cloned probes are available. The method uses two-dimensional neutral-alkaline agarose gel electrophoresis followed by hybridization with short probe sequences. The nascent strands of replicating molecules form an arc separated from parental and nonreplicating strands. The closer a probe is to its replication origin or to the origin-proximal end of its restriction fragment, the shorter the nascent strands that are detected by the probe. The use of multiple probes allows determination of directions of replication fork movement, as well as locations of origins and termini. In this study, we used simian virus 40 as a model to demonstrate the feasibility of the method, and we discuss its applicability to other systems.

scholarly journals Composite patterns in neutral/neutral two-dimensional gels demonstrate inefficient replication origin usage.

1996 ◽  
Vol 16 (9) ◽  
pp. 4915-4922 ◽  
Author(s):  
R F Kalejta ◽  
J L Hamlin
Keyword(s):  
Replication Origin ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Yeast Cells ◽  
Eukaryotic Cells ◽  
Mapping Technique ◽  
Two Dimensional ◽  
Composite Pattern ◽  
Composite Gel ◽  
Bona Fide

The neutral/neutral two-dimensional (2-D) gel replicon mapping technique has been used to great advantage to localize and characterize origins of replication. Interestingly, many yeast origins display a composite pattern consisting of both a bubble arc and a single-fork arc. Moreover, in every instance in which neutral/neutral 2-D gels have been used to analyze origins in higher eukaryotic cells, two or more adjacent fragments display these composite patterns. We believe that composite patterns signal inefficient origin usage in yeast cells because the replicators in question are not active in every cell cycle and in higher eukaryotic replicons because initiation sites are chosen from among many potential sites lying within a zone. However, others have suggested that the single-fork arcs in these composite gel patterns arise from nicking activity that converts replication bubbles to branched structures that comigrate with bona fide single forks. Here, we have used three different replicon mapping strategies to show that broken simian virus 40 replication bubbles trace unique arcs that are clearly distinguishable from classic, intact single forks. Thus, it is likely that composite 2-D gel patterns represent origins that are inefficiently utilized.

Size determination of multienzyme complexes using two-dimensional agarose gel electrophoresis

1989 ◽  
Vol 5 (3) ◽  
pp. 224-232 ◽  
Author(s):  
Richard A. Easom ◽  
Michael S. Debuysere ◽  
Merle S. Olson ◽  
Philip Serwer
Keyword(s):  
Gel Electrophoresis ◽  
Agarose Gel ◽  
Two Dimensional ◽  
Size Determination ◽  
Agarose Gel Electrophoresis ◽  
Multienzyme Complexes

The distribution of large T antigen in simian virus 40 nucleoprotein complexes

1984 ◽  
Vol 30 (5) ◽  
pp. 622-631 ◽  
Author(s):  
Peter Milthorp ◽  
Ernst Baumann ◽  
Roger Hand
Keyword(s):  
Gel Electrophoresis ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Absolute Amount ◽  
Agarose Gel Electrophoresis ◽  
Large T Antigen ◽  
Dna Molecules ◽  
Infected Cells ◽  
Nucleoprotein Complexes

Simian virus 40 large T antigen binds to two types of nucleoprotein complexes from lytically infected cells: those containing replicating virus DNA (100S complexes) and those containing nonreplicating virus DNA (70S complexes). Analysis by agarose gel electrophoresis showed that replicating DNA was found exclusively in 100S complexes, although these complexes also contained large amounts of form I and form II DNA. In contrast, no replicating DNA was found in 70S complexes, and pulse-labeled DNA in these complexes migrated as form I and form II DNA that presumably had recently completed replication. Immunoprecipitation and gel electrophoresis showed that large T antigen was associated with both types of complexes. From 21 to 62% of replicating DNA in 100S complexes was bound to T antigen. Our estimates indicated, however, that more than three-fourths of the DNA molecules in 100S complexes were nonreplicating and unassociated with T antigen. In 70S complexes, 12 to 31% of pulse-labeled DNA was bound to T antigen, but because there were more DNA molecules in the 70S complexes, they contained a greater absolute amount of T antigen.

scholarly journals Mapping of replication initiation sites in human ribosomal DNA by nascent-strand abundance analysis.

1995 ◽  
Vol 15 (5) ◽  
pp. 2482-2489 ◽  
Author(s):  
Y Yoon ◽  
J A Sanchez ◽  
C Brun ◽  
J A Huberman
Keyword(s):  
Ribosomal Dna ◽  
Gel Electrophoresis ◽  
Repeat Unit ◽  
Low Frequency ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Replication Initiation ◽  
Size Analysis ◽  
Two Dimensional ◽  
Two Dimensional Gel Electrophoresis

New techniques for mapping mammalian DNA replication origins are needed. We have modified the existing nascent-strand size analysis technique (L. Vassilev and E.M. Johnson, Nucleic Acids Res. 17:7693-7705, 1989) to provide an independent means of studying replication initiation sites. We call the new method nascent-strand abundance analysis. We confirmed the validity of this method with replicating simian virus 40 DNA as a model. We then applied nascent-strand abundance and nascent-strand size analyses to mapping of initiation sites in human (HeLa) ribosomal DNA (rDNA), a region previously examined exclusively by two-dimensional gel electrophoresis methods (R.D. Little, T.H.K. Platt, and C.L. Schildkraut, Mol. Cell. Biol. 13:6600-6613, 1993). Our results partly confirm those obtained by two-dimensional gel electrophoresis techniques. Both studies suggest that replication initiates at relatively high frequency a few kilobase pairs upstream of the transcribed region and that many additional low-frequency initiation sites are distributed through most of the remainder of the ribosomal DNA repeat unit.

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