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.

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 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.

Binding properties of replication protein A from human and yeast cells

1992 ◽  
Vol 12 (7) ◽  
pp. 3050-3059 ◽  
Author(s):  
C Kim ◽  
R O Snyder ◽  
M S Wold
Keyword(s):  
Dna Replication ◽  
Dna Binding ◽  
Protein A ◽  
Simian Virus 40 ◽  
Replication Protein A ◽  
Simian Virus ◽  
Yeast Cells ◽  
Origin Of Replication ◽  
Binding Properties ◽  
Single Stranded Dna

Replication protein A (RP-A; also known as replication factor A and human SSB), is a single-stranded DNA-binding protein that is required for simian virus 40 DNA replication in vitro. RP-A isolated from both human and yeast cells is a very stable complex composed of 3 subunits (70, 32, and 14 kDa). We have analyzed the DNA-binding properties of both human and yeast RP-A in order to gain a better understanding of their role(s) in DNA replication. Human RP-A has high affinity for single-stranded DNA and low affinity for RNA and double-stranded DNA. The apparent affinity constant of RP-A for single-stranded DNA is in the range of 10(9) M-1. RP-A has a binding site size of approximately 30 nucleotides and does not bind cooperatively. The binding of RP-A to single-stranded DNA is partially sequence dependent. The affinity of human RP-A for pyrimidines is approximately 50-fold higher than its affinity for purines. The binding properties of yeast RP-A are similar to those of the human protein. Both yeast and human RP-A bind preferentially to the pyrimidine-rich strand of a homologous origin of replication: the ARS307 or the simian virus 40 origin of replication, respectively. This asymmetric binding suggests that RP-A could play a direct role in the process of initiation of DNA replication.

scholarly journals Enhancer and promoter elements from simian virus 40 and polyomavirus can substitute for an upstream activation sequence in Saccharomyces cerevisiae.

1990 ◽  
Vol 10 (3) ◽  
pp. 947-957 ◽  
Author(s):  
N J Axelrod ◽  
G G Carmichael ◽  
P J Farabaugh
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Sequences ◽  
Evolutionary Relationship ◽  
Simian Virus 40 ◽  
Regulatory Elements ◽  
Simian Virus ◽  
Yeast Cells ◽  
Upstream Activation Sequence ◽  
Beta Galactosidase ◽  
Activation Sequence

Ten fragments of higher eucaryotic DNA were tested for upstream activation sequence activity in Saccharomyces cerevisiae by inserting them upstream of a CYC1::lacZ promoter lacking an upstream activation sequence. Fragments containing the 21-base-pair repeat region, the enhancer of simian virus 40 or both strongly stimulated beta-galactosidase synthesis, and three fragments from the polyomavirus enhancer region stimulated moderate levels. Three of the four controls of random DNA sequences failed to stimulate significant levels, and the fourth stimulated moderate levels. The stimulation in all cases was independent of the orientation of the inserted fragment. Two series of clones were examined in which between one and six tandemly arranged copies of a fragment were inserted into the XhoI site of the vector. Very interestingly, we detected an apparent exponential relationship between the number of copies of a fragment and the amount of beta-galactosidase produced. Southern analysis showed that increases in enzyme activity were not a result of increased plasmid copy number. Rather, quantitative S1 nuclease analysis demonstrated that the increases were correlated with steady-state levels of lacZ-specific mRNA. We suggest that there may be an evolutionary relationship between some transcriptional activation sequences in yeast cells and the higher eucaryotic regulatory elements that we tested.

scholarly journals Evolutionarily selected replication origins: functional aspects and structural organization.

1986 ◽  
Vol 6 (9) ◽  
pp. 3077-3085 ◽  
Author(s):  
G J Lee-Chen ◽  
M Woodworth-Gutai
Keyword(s):  
Inverted Repeat ◽  
Replication Origin ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Inverted Repeats ◽  
Regulatory Sequences ◽  
Functional Aspects ◽  
Cellular Sequence ◽  
Transfection Mixture

A selective replicative pressure occurs during the evolution of simian virus 40 variants. When the replication origin is duplicated as an inverted repeat, there is a dramatic enhancement of replication. Having regulatory sequences located between the inverted repeat of ori magnifies their enhancing effect on replication. A passage 20 variant and a passage 45 variant containing three pairs of an inverted repeat of ori replicated more efficiently than a passage 13 variant containing nine copies of ori arranged in tandem. A 69-base-pair cellular sequence inserted between inverted repeats of ori of both passage 40 and 45 variants enhanced simian virus 40 DNA replication. Differences in replication efficiencies became greater as the total number of replicating species was increased in the transfection mixture, under conditions where T antigen is limiting. In a competitive environment, sequences flanking the replication origin may be inhibitory to replication.

scholarly journals Transcription factor Oct1 binds to the AT-rich segment of the simian virus 40 replication origin.

1995 ◽  
Vol 69 (1) ◽  
pp. 575-578 ◽  
Author(s):  
J Kilwinski ◽  
M Baack ◽  
S Heiland ◽  
R Knippers
Keyword(s):  
Transcription Factor ◽  
Replication Origin ◽  
Simian Virus 40 ◽  
Simian Virus

Signal-mediated import of bacteriophage T7 RNA polymerase into the Saccharomyces cerevisiae nucleus and specific transcription of target genes

1990 ◽  
Vol 10 (1) ◽  
pp. 353-360
Author(s):  
B M Benton ◽  
W K Eng ◽  
J J Dunn ◽  
F W Studier ◽  
R Sternglanz ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Rna Polymerase ◽  
Target Genes ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Yeast Cells ◽  
T7 Rna Polymerase ◽  
Bacteriophage T7 ◽  
T7 Promoter

Bacteriophage T7 RNA polymerase and derivatives that contain the nuclear localization signal (NLS) from simian virus 40 T antigen (J. J. Dunn, B. Krippl, K. Bernstein, H. Westphal, and F. W. Studier, Gene 68:259-266, 1988) were expressed in Saccharomyces cerevisiae under the control of the inducible GAL1 promoter. As determined by indirect immunofluorescence, T7 RNA polymerase lacking the NLS remained mostly in the cytoplasm, whereas the protein containing the NLS localized to the nucleus. T7 RNA polymerase containing a mutated NLS remained mostly cytoplasmic. Hybrid proteins containing the NLS near the amino terminus were enzymatically active in the yeast cell, initiating transcription selectively at a T7 promoter placed in yeast chromosomal or plasmid DNA and stopping at a specific T7 terminator. At limiting enzyme concentrations, 5 to 10 times as much target RNA was produced when the polymerase contained the NLS, presumably because more enzyme reached the nucleus. Although substantial amounts of intact mRNA accumulated, no translation of target mRNAs in yeast cells was detected.

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