scholarly journals Ramified rolling circle amplification for efficient and flexible synthesis of nucleosomal DNA sequences

2019 ◽  
Author(s):  
Clara L. van Emmerik ◽  
Ivana Gachulincova ◽  
Vincenzo R. Lobbia ◽  
Mark A. Daniëls ◽  
Hans A. Heus ◽  
...  
Keyword(s):  
Dna Sequence ◽  
Dna Sequences ◽  
Protein Complexes ◽  
Rolling Circle Amplification ◽  
Fold Increase ◽  
One Pot ◽  
Efficient Manner ◽  
Rolling Circle ◽  
Nucleosomal Dna

ABSTRACTNucleosomes are a crucial platform for the recruitment and assembly of protein complexes that process the DNA. Mechanistic and structuralin vitrostudies typically rely on recombinant nucleosomes that are reconstituted using artificial, strong-positioning DNA sequences. To facilitate such studies on native, genomic nucleosomes, there is a need for methods to produce any desired DNA sequence in an efficient manner. The current methods either do not offer much flexibility in choice of sequence or are less efficient in yield and labor. Here, we show that using ramified rolling circle amplification (RCA) milligram amounts of a genomic nucleosomal DNA fragment can be produced in a scalable, one-pot reaction overnight. The ramified RCA reaction is more efficient than the existing methods, is flexible in DNA sequence and shows a 10-fold increase in yield compared to PCR, rivalling the production using plasmids. We demonstrate the method by producing the genomic DNA from the human LIN28B locus and show that it forms functional nucleosomes capable of binding pioneer transcription factor Oct4.

scholarly journals Ramified rolling circle amplification for synthesis of nucleosomal DNA sequences

2020 ◽  
Vol 588 ◽  
pp. 113469 ◽  
Author(s):  
Clara L. van Emmerik ◽  
Ivana Gachulincova ◽  
Vincenzo R. Lobbia ◽  
Mark A. Daniëls ◽  
Hans A. Heus ◽  
...  
Keyword(s):  
Dna Sequences ◽  
Rolling Circle Amplification ◽  
Rolling Circle ◽  
Nucleosomal Dna

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.

DNA flower-encapsulated horseradish peroxidase with enhanced biocatalytic activity synthesized by an isothermal one-pot method based on rolling circle amplification

Nanoscale ◽  
2018 ◽  
Vol 10 (47) ◽  
pp. 22456-22465 ◽  
Author(s):  
Yongcun Yan ◽  
Juan Li ◽  
Wenhui Li ◽  
Ye Wang ◽  
Weiling Song ◽  
...  
Keyword(s):  
Horseradish Peroxidase ◽  
Visual Analysis ◽  
Rolling Circle Amplification ◽  
One Pot ◽  
Rolling Circle

A one-pot method was developed to directly encapsulate horseradish peroxidase in DNA flowers during rolling circle amplification, which demonstrated enhanced biocatalytic activity and was applied to colorimetric and visual analysis.

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 The use of enzymopathic human red cells in the study of malarial parasite glucose metabolism

Blood ◽  
1988 ◽  
Vol 71 (5) ◽  
pp. 1408-1413 ◽  
Author(s):  
E Jr Roth ◽  
V Joulin ◽  
S Miwa ◽  
A Yoshida ◽  
J Akatsuka ◽  
...  
Keyword(s):  
Dna Sequences ◽  
Cdna Probe ◽  
Phosphoglycerate Kinase ◽  
Fold Increase ◽  
Plasmodium Falciparum Malaria ◽  
Red Cells ◽  
Host Protein ◽  
Malarial Parasite ◽  
Parasite Genome

Abstract The in vitro growth of Plasmodium falciparum malaria parasites was assayed in mutant red cells deficient in either diphosphoglycerate mutase (DPGM) or phosphoglycerate kinase (PGK). In addition, cDNA probes developed for human DNA sequences coding for these enzymes were used to examine the parasite genome by means of restriction endonuclease digestion and Southern blot analysis of parasite DNA. In both types of enzymopathic red cells, parasite growth was normal. In infected DPGM deficient red cells, no DPGM activity could be detected, and in normal red cells, DPGM activity declined slightly in a manner suggestive of parasite catabolism of host protein. However, in infected PGK deficient red cells, there was a 100-fold increase in PGK activity, and in normal red cells, a threefold increase in PGK activity was observed. Parasite PGK could be recovered from isolated parasites, and a marked increase in heat instability of parasite PGK as compared with the host cell enzyme was noted. Neither cDNA probe was found to cross- react with DNA sequences in the parasite genome. It is concluded that the parasite has no requirement for DPGM, and probably has no gene for this enzyme. On the other hand, the parasite does require PGK, (an adenosine triphosphate [ATP] generating enzyme) and synthesizes its own enzyme, which must have been encoded in the parasite genome. The parasite PGK gene most likely lacks sufficient homology to be detected by a human cDNA probe. Enzymopathic red cells are useful tools for elucidating the glycolytic enzymology of parasites and their co- evolution with their human hosts.

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 DNA sequence preferences of GAL4 and PPR1: how a subset of Zn2 Cys6 binuclear cluster proteins recognizes DNA.

1996 ◽  
Vol 16 (7) ◽  
pp. 3773-3780 ◽  
Author(s):  
S D Liang ◽  
R Marmorstein ◽  
S C Harrison ◽  
M Ptashne
Keyword(s):  
Crystal Structure ◽  
Dna Sequence ◽  
Dna Sequences ◽  
Tight Binding ◽  
Recognition Sequence ◽  
Sequence Dependence ◽  
Network Of Hydrogen Bonds ◽  
Dna Binding Assay ◽  
Related Proteins

Biophysical and genetic experiments have defined how the Saccharomyces cerevisiae protein GAL4 and a subset of related proteins recognize specific DNA sequences. We assessed DNA sequence preferences of GAL4 and a related protein, PPR1, in an in vitro DNA binding assay. For GAL4, the palindromic CGG triplets at the ends of the 17-bp recognition site are essential for tight binding, whereas the identities of the internal 11 bp are much less important, results consistent with the GAL4-DNA crystal structure. Small reductions in affinity due to mutations at the center-most 5 bp are consistent with the idea that an observed constriction in the minor groove in the crystalline GAL4-DNA complex is sequence dependent. The crystal structure suggests that this sequence dependence is due to phosphate contacts mediated by arginine 51, as part of a network of hydrogen bonds. Here we show that the mutant protein GAL4(1-100)R51A fails to discriminate sites with alterations in the center of the site from the wild-type site. PPR1, a relative of GAL4, also recognizes palindromic CGG triplets at the ends of its 12-bp recognition sequence. The identities of the internal 6 bp do not influence the binding of PPR1. We also show that the PPR1 site consists of a 12-bp duplex rather than 16 bp as reported previously: the two T residues immediately 5' to the CGG sequence in each half site, although highly conserved, are not important for binding by PPR1. Thus, GAL4 and PPR1 share common CGG half sites, but they prefer DNA sequences with the palindromic CGG separated by the appropriate number of base pairs, 11 for GAL4 and 6 for PPR1.

Sign in / Sign up

Export Citation Format

Share Document