scholarly journals DNA thermodynamics shape chromosome organization and topology

2013 ◽  
Vol 41 (2) ◽  
pp. 548-553 ◽  
Author(s):  
Andrew A. Travers ◽  
Georgi Muskhelishvili
Keyword(s):  
Dna Sequence ◽  
Dna Sequences ◽  
Chromosome Organization ◽  
Topological Properties ◽  
Genetic Organization ◽  
And Topology ◽  
Dna Translocases

How much information is encoded in the DNA sequence of an organism? We argue that the informational, mechanical and topological properties of DNA are interdependent and act together to specify the primary characteristics of genetic organization and chromatin structures. Superhelicity generated in vivo, in part by the action of DNA translocases, can be transmitted to topologically sensitive regions encoded by less stable DNA sequences.

Simian virus 40 major late promoter: an upstream DNA sequence required for efficient in vitro transcription

1984 ◽  
Vol 4 (1) ◽  
pp. 133-141
Author(s):  
J Brady ◽  
M Radonovich ◽  
M Thoren ◽  
G Das ◽  
N P Salzman
Keyword(s):  
Dna Sequence ◽  
Dna Sequences ◽  
Simian Virus 40 ◽  
Promoter Sequence ◽  
In Vitro Transcription ◽  
Simian Virus ◽  
Upstream Promoter ◽  
Upstream Promoter Sequence

We have previously identified an 11-base DNA sequence, 5'-G-G-T-A-C-C-T-A-A-C-C-3' (simian virus 40 [SV40] map position 294 to 304), which is important in the control of SV40 late RNA expression in vitro and in vivo (Brady et al., Cell 31:625-633, 1982). We report here the identification of another domain of the SV40 late promoter. A series of mutants with deletions extending from SV40 map position 0 to 300 was prepared by nuclease BAL 31 treatment. The cloned templates were then analyzed for efficiency and accuracy of late SV40 RNA expression in the Manley in vitro transcription system. Our studies showed that, in addition to the promoter domain near map position 300, there are essential DNA sequences between nucleotide positions 74 and 95 that are required for efficient expression of late SV40 RNA. Included in this SV40 DNA sequence were two of the six GGGCGG SV40 repeat sequences and an 11-nucleotide segment which showed strong homology with the upstream sequences required for the efficient in vitro and in vivo expression of the histone H2A gene. This upstream promoter sequence supported transcription with the same efficiency even when it was moved 72 nucleotides closer to the major late cap site. In vitro promoter competition analysis demonstrated that the upstream promoter sequence, independent of the 294 to 304 promoter element, is capable of binding polymerase-transcription factors required for SV40 late gene transcription. Finally, we show that DNA sequences which control the specificity of RNA initiation at nucleotide 325 lie downstream of map position 294.

scholarly journals Nucleosome positioning on large tandem DNA repeats of the '601' sequence engineered in Saccharomyces cerevisiae

2021 ◽  
Author(s):  
Astrid Lancrey ◽  
Alexandra Joubert ◽  
Evelyne Duvernois-Berthet ◽  
Etienne Routhier ◽  
Saurabh Raj ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Sequence ◽  
Dna Sequences ◽  
Nucleosome Occupancy ◽  
Nucleosome Positioning ◽  
Dna Repeats ◽  
Repeat Array ◽  
Synthetic Dna

The so-called 601 DNA sequence is often used to constrain the position of nucleosomes on a DNA molecule in vitro. Although the ability of the 147 base pair sequence to precisely position a nucleosome in vitro is well documented, in vivo application of this property has been explored only in a few studies and yielded contradictory conclusions. Our goal in the present study was to test the ability of the 601 sequence to dictate nucleosome positioning in Saccharomyces cerevisiae in the context of a long tandem repeat array inserted in a yeast chromosome. We engineered such arrays with three different repeat size, namely 167, 197 and 237 base pairs. Although our arrays are able to position nucleosomes in vitro as expected, analysis of nucleosome occupancy on these arrays in vivo revealed that nucleosomes are not preferentially positioned as expected on the 601-core sequence along the repeats and that the measured nucleosome repeat length does not correspond to the one expected by design. Altogether our results demonstrate that the rules defining nucleosome positions on this DNA sequence in vitro are not valid in vivo, at least in this chromosomal context, questioning the relevance of using the 601 sequence in vivo to achieve precise nucleosome positioning on designer synthetic DNA sequences.

scholarly journals DNA Gap Repair-Mediated Site-Directed Mutagenesis is Different from Mandecki and Recombineering Approaches

2018 ◽  
Author(s):  
George T. Lyozin ◽  
Luca Brunelli
Keyword(s):  
Dna Sequence ◽  
Dna Sequences ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Gene Products ◽  
E Coli ◽  
Health And Disease ◽  
First Time ◽  
Disease Understanding

AbstractSite-directed mutagenesis allows the generation of mutant DNA sequences for downstream functional analysis of genetic variants involved in human health and disease. Understanding the mechanisms of different mutagenesis methods can help select the best approach for specific needs. We compared three different approaches for in vivo site-directed DNA mutagenesis that utilize a mutant single-stranded DNA oligonucleotide (ssODN) to target a wild type DNA sequence in the host Escherichia coli (E. coli). The first method, Mandecki, uses restriction nucleases to introduce a double stranded break (DSB) into a DNA sequence which needs to be denatured prior to co-transformation. The second method, recombineering (recombination-mediated genetic engineering), requires lambda red gene products and a mutant ssODN with homology arms of at least 20 nucleotides. In a third method described here for the first time, DNA gap repair, a mutant ssODN targets a DNA sequence containing a gap introduced by PCR. Unlike recombineering, both DNA gap repair and Mandecki can utilize homology arms as short as 10 nucleotides. DNA gap repair requires neither red gene products as recombineering nor DNA denaturation or nucleases as Mandecki, and unlike other methods is background-free. We conclude that Mandecki, recombineering, and DNA gap repair have at least partly different mechanisms, and that DNA gap repair provides a new, straightforward approach for effective site-directed mutagenesis.

scholarly journals Modulating the properties of DNA-SWCNT sensors using chemically modified DNA

2021 ◽  
Author(s):  
Alice J. Gillen ◽  
Benjamin P. Lambert ◽  
Alessandra Antonucci ◽  
Daniel Molina-Romero ◽  
Ardemis A. Boghossian
Keyword(s):  
Dna Sequence ◽  
Fluorescence Intensity ◽  
Dna Sequences ◽  
Attractive Alternative ◽  
Laser Exposure ◽  
Chemically Modified ◽  
Modified Dna ◽  
Penetration Depths

AbstractProperties of SWCNT-based sensors such as brightness and detection capabilities strongly depend on the characteristics of the wrapping used to suspend the nanotubes. In this study, we explore ways to modify the properties of DNA-SWCNT sensors by using chemically modified DNA sequences, with the aim of creating sensors more suitable for use in in vivo and in vitro applications. We show that both the fluorescence intensity and sensor reactivity are strongly impacted not only by the chemical modification of the DNA but also by the method of preparation. In the absence of modifications, the sensors prepared using MeOH-assisted surfactant exchange exhibited higher overall fluorescence compared to those prepared by direct sonication. However, we demonstrate that the incorporation of chemical modifications in the DNA sequence could be used to enhance the fluorescence intensity of sonicated samples. We attribute these improvements to both a change in dispersion efficiency as well as to a change in SWCNT chirality distribution.Furthermore, despite their higher intensities, the response capabilities of sensors prepared by MeOH-assisted surfactant exchange were shown to be significantly reduced compared to their sonicated counterparts. Sonicated sensors exhibited a globally higher turn-on response towards dopamine compared to the exchanged samples, with modified samples retaining their relative intensity enhancement. As the increases in fluorescence intensity were achieved without needing to alter the base sequence of the DNA wrapping or to add any exogenous compounds, these modifications can - in theory - be applied to nearly any DNA sequence to increase the brightness and penetration depths of a variety of DNA-SWCNT sensors without affecting biocompatibility or reducing the near-limitless sequence space available. This makes these sensors an attractive alternative for dopamine sensing in vitro and in vivo by enabling significantly higher penetration depths and shorter laser exposure times.

scholarly journals Absence of MeCP2 binding to non-methylated GT-rich sequences in vivo

2020 ◽  
Vol 48 (7) ◽  
pp. 3542-3552 ◽  
Author(s):  
John C Connelly ◽  
Justyna Cholewa-Waclaw ◽  
Shaun Webb ◽  
Verdiana Steccanella ◽  
Bartlomiej Waclaw ◽  
...  
Keyword(s):  
Dna Sequence ◽  
Dna Sequences ◽  
Cytosine Methylation ◽  
Epigenetic Mark ◽  
Primary Role ◽  
Flanking Sequence ◽  
Primary Determinant ◽  
Human Neuronal Cells

Abstract MeCP2 is a nuclear protein that binds to sites of cytosine methylation in the genome. While most evidence confirms this epigenetic mark as the primary determinant of DNA binding, MeCP2 is also reported to have an affinity for non-methylated DNA sequences. Here we investigated the molecular basis and in vivo significance of its reported affinity for non-methylated GT-rich sequences. We confirmed this interaction with isolated domains of MeCP2 in vitro and defined a minimal target DNA sequence. Binding depends on pyrimidine 5′ methyl groups provided by thymine and requires adjacent guanines and a correctly orientated A/T-rich flanking sequence. Unexpectedly, full-length MeCP2 protein failed to bind GT-rich sequences in vitro. To test for MeCP2 binding to these motifs in vivo, we analysed human neuronal cells using ChIP-seq and ATAC-seq technologies. While both methods robustly detected DNA methylation-dependent binding of MeCP2 to mCG and mCAC, neither showed evidence of MeCP2 binding to GT-rich motifs. The data suggest that GT binding is an in vitro phenomenon without in vivo relevance. Our findings argue that MeCP2 does not read unadorned DNA sequence and therefore support the notion that its primary role is to interpret epigenetic modifications of DNA.

Structure of the Schizosaccharomyces pombe cytochrome c gene

1982 ◽  
Vol 2 (2) ◽  
pp. 106-116
Author(s):  
P R Russell ◽  
B D Hall
Keyword(s):  
Cytochrome C ◽  
Schizosaccharomyces Pombe ◽  
Dna Sequence ◽  
Dna Sequences ◽  
Protein Function ◽  
Hybridization Probe ◽  
Yeast Plasmid ◽  
Nonfermentable Carbon Source ◽  
Drastic Effect

The cytochrome c gene of the fission yeast Schizosaccharomyces pombe has been cloned by using the Saccharomyces cerevisiae iso-1-cytochrome c gene as a molecular hybridization probe. The DNA sequence and the 5' termini of the mRNA transcripts of the gene have been determined. The DNA sequence has confirmed, with two exceptions, the previously determined protein sequence. The nonrandom distribution of silent third base differences which was observed between the two cytochrome c genes of S. cerevisiae does not extend to the S. pombe cytochrome c gene, suggesting that there are no constraints other than protein function and codon usage which have acted to conserve the cytochrome DNA sequences of the two yeasts. Introduction of the S. pombe cytochrome c gene on a yeast plasmid into a S. cerevisiae mutant which lacked functional cytochrome c transformed that recipient strain for the ability to grow on a nonfermentable carbon source. This implies that the S. pombe cytochrome c gene has all the regulatory signals which are required for its expression in S. cerevisiae, and that none of the amino acid differences between the cytochrome c proteins of the two yeasts has a drastic effect on the function of the protein in vivo.

scholarly journals Discovering epistatic feature interactions from neural network models of regulatory DNA sequences

2018 ◽  
Author(s):  
Peyton Greenside ◽  
Tyler Shimko ◽  
Polly Fordyce ◽  
Anshul Kundaje
Keyword(s):  
Dna Sequence ◽  
Dna Sequences ◽  
Chromatin Accessibility ◽  
Feature Interaction ◽  
Core Motif ◽  
Feature Interactions ◽  
Binding Models ◽  
Regulatory Dna Sequences ◽  
Regulatory Dna

AbstractMotivationTranscription factors bind regulatory DNA sequences in a combinatorial manner to modulate gene expression. Deep neural networks (DNNs) can learn the cis-regulatory grammars encoded in regulatory DNA sequences associated with transcription factor binding and chromatin accessibility. Several feature attribution methods have been developed for estimating the predictive importance of individual features (nucleotides or motifs) in any input DNA sequence to its associated output prediction from a DNN model. However, these methods do not reveal higher-order feature interactions encoded by the models.ResultsWe present a new method called Deep Feature Interaction Maps (DFIM) to efficiently estimate interactions between all pairs of features in any input DNA sequence. DFIM accurately identifies ground truth motif interactions embedded in simulated regulatory DNA sequences. DFIM identifies synergistic interactions between GATA1 and TAL1 motifs from in vivo TF binding models. DFIM reveals epistatic interactions involving nucleotides flanking the core motif of the Cbf1 TF in yeast from in vitro TF binding models. We also apply DFIM to regulatory sequence models of in vivo chromatin accessibility to reveal interactions between regulatory genetic variants and proximal motifs of target TFs as validated by TF binding quantitative trait loci. Our approach makes significant strides in improving the interpretability of deep learning models for genomics.AvailabilityCode is available at: https://github.com/kundajelab/dfim.Contact: [email protected]

scholarly journals Half-Site DNA Sequence and Spacing Length Contributions to PrrA Binding to PrrA Site 2 of RSP3361 in Rhodobacter sphaeroides 2.4.1

2009 ◽  
Vol 191 (13) ◽  
pp. 4353-4364 ◽  
Author(s):  
Jesus M. Eraso ◽  
Samuel Kaplan
Keyword(s):  
Dna Binding ◽  
Dna Sequence ◽  
Rhodobacter Sphaeroides ◽  
Dna Sequences ◽  
Mutational Analysis ◽  
Consensus Sequence ◽  
Binding Affinities ◽  
Transcriptional Fusions

ABSTRACT The consensus DNA binding sequence for PrrA, a global regulator in Rhodobacter sphaeroides 2.4.1, is poorly defined. We have performed mutational analysis of PrrA site 2, of the RSP3361 gene, to which PrrA binds in vitro (J. M. Eraso and S. Kaplan, J. Bacteriol. 191:4341-4352, 2009), to further define the consensus sequence for DNA binding. Two half-sites of equal length, containing 6 nucleotides each, were required for PrrA binding to this DNA sequence. Systematic nucleotide substitutions in both inverted half-sites led to a decrease in binding affinity of phosphorylated PrrA in vitro, the level of which was dependent on the substitution. The reduced binding affinities were confirmed by competition experiments and led to proportional decreases in the expression of lacZ transcriptional fusions to the RSP3361 gene in vivo. The 5-nucleotide spacer region between the half-sites was found to be optimal for PrrA binding to the wild-type half-sites, as shown by decreased PrrA DNA binding affinities to synthetic DNA sequences without spacer regions or with spacer regions ranging from 1 to 10 nucleotides. The synthetic spacer region alleles also showed decreased gene expression in vivo when analyzed using lacZ transcriptional fusions. We have studied three additional DNA sequences to which PrrA binds in vitro. They are located in the regulatory regions of genes positively regulated by PrrA and contain spacer regions with 5 or 8 nucleotides. We demonstrate that PrrA can bind in vitro to DNA sequences with different lengths in the spacer regions between the half-sites.

scholarly journals Structure of the Schizosaccharomyces pombe cytochrome c gene.

1982 ◽  
Vol 2 (2) ◽  
pp. 106-116 ◽  
Author(s):  
P R Russell ◽  
B D Hall
Keyword(s):  
Cytochrome C ◽  
Schizosaccharomyces Pombe ◽  
Dna Sequence ◽  
Dna Sequences ◽  
Protein Function ◽  
Hybridization Probe ◽  
Yeast Plasmid ◽  
Nonfermentable Carbon Source ◽  
Drastic Effect

The cytochrome c gene of the fission yeast Schizosaccharomyces pombe has been cloned by using the Saccharomyces cerevisiae iso-1-cytochrome c gene as a molecular hybridization probe. The DNA sequence and the 5' termini of the mRNA transcripts of the gene have been determined. The DNA sequence has confirmed, with two exceptions, the previously determined protein sequence. The nonrandom distribution of silent third base differences which was observed between the two cytochrome c genes of S. cerevisiae does not extend to the S. pombe cytochrome c gene, suggesting that there are no constraints other than protein function and codon usage which have acted to conserve the cytochrome DNA sequences of the two yeasts. Introduction of the S. pombe cytochrome c gene on a yeast plasmid into a S. cerevisiae mutant which lacked functional cytochrome c transformed that recipient strain for the ability to grow on a nonfermentable carbon source. This implies that the S. pombe cytochrome c gene has all the regulatory signals which are required for its expression in S. cerevisiae, and that none of the amino acid differences between the cytochrome c proteins of the two yeasts has a drastic effect on the function of the protein in vivo.

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