The Role of DNA Concentration

1969 ◽  
Vol 24 (5) ◽  
pp. 511-514 ◽  
Author(s):  
Samarendra Basu
Keyword(s):  
Thermal Denaturation ◽  
Melting Profile ◽  
Dna Concentration ◽  
Melting Temperatures

Melting temperatures (Tm) of several DNA’s do not vary with the change of DNA concentration. However, at any temperature above Tm, the fractional hyperchromism was always greater at the lower concentration. This result was also evident in the presence of formaldehyde.Reversibility on renaturation decreased with the progress of thermal denaturation along the melting profile. The effect was much enhanced at low DNA concentration and for a DNA of low G—C content.The occurrence of some permanent changes on the dissociated molecules has been suspected to be the only cause for the observed effects.

scholarly journals The Phenanthridine-modified Tyrosine Dipeptide

2019 ◽  
Vol 92 (2) ◽  
pp. 249-258
Author(s):  
Antonija Erben ◽  
Josipa Matić ◽  
Nikola Basarić ◽  
Ivo Piantanida
Keyword(s):  
Unnatural Amino Acids ◽  
Thermal Denaturation ◽  
Specific Binding ◽  
Quinone Methide ◽  
Melting Temperatures ◽  
Double Helices ◽  
Fluorescence Titrations ◽  
Alkylate Dna ◽  
Ct Dna ◽  
Phenanthridine Derivative

Dipeptide 4 containing two unnatural amino acids, a modified tyrosine and a phenanthridine derivative, was synthesized. Binding of the dipeptide to a series of polynucleotides including ct-DNA, poly A - poly U, poly (dAdT)2, poly dG - poly dC and poly (dGdC)2 was investigated by thermal denaturation experiments, fluorescence spectroscopy and circular dichroism. Thermal denaturation experiments indicated that dipeptide 4 at pH 5.0, when phenanthridine is protonated, stabilizes ds-DNA, whereas it destabilizes ds-RNA. At pH 7.0, when the phenanthridine is not protonated, effects of 4 to the polynucleotide melting temperatures are negligible. At pH 5.0, dipeptide 4 stabilized DNA double helices, and the changes in the CD spectra suggest different modes of binding to ds-DNA, most likely the intercalation to poly dG- poly dC and non-specific binding in grooves of other DNA polynucleotides. At variance to ds-DNA, addition of 4 destabilized ds-RNA against thermal denaturation and CD results suggest that addition of 4 probably induced dissociation of ds-RNA into ss-RNA strands due to preferred binding to ss-RNA. Thus, 4 is among very rare small molecules that stabilize ds-DNA but destabilize ds-RNA. However, fluorescence titrations with all polynucleotides at both pH values gave similar binding affinity (log Ka ≈ 5), indicating nonselective binding. Preliminary photochemical experiments suggest that dipeptide 4 reacts in the photochemical reaction, which affects polynucleotides chirality, presumably via quinone methide intermediates that alkylate DNA.

Studies on the Melting of DNA

1974 ◽  
Vol 29 (3-4) ◽  
pp. 130-132
Author(s):  
Gokul Chandra Das
Keyword(s):  
Ionic Strength ◽  
Melting Temperature ◽  
Thermal Denaturation ◽  
Spectrophotometric Methods ◽  
Melting Temperatures

Abstract The thermal denaturation of the native DNA in solvents of varying salt concentrations was studied by viscometric and spectrophotometric methods. It was observed that within the molarity range of 0.02 ᴍ to 0.3 ᴍ, the melting temperatures obtained by the two independent methods agreed well, but that at lower ionic strength the agreement was not satisfactory. Both the visco­metric and the spectrophotometric measurements showed an increase of the melting temperature with increasing counterion concentration and a levelling off effect in the neighbourhood of 0.3 ᴍ.

Nucleic acid content of turkey heart with a note on hemodynamic findings

1964 ◽  
Vol 207 (5) ◽  
pp. 1102-1106
Author(s):  
Robert G. Sumner ◽  
Thomas C. Hoyle ◽  
Henry D. McIntosh ◽  
Robert E. Whalen
Keyword(s):  
Protein Concentration ◽  
Systemic Blood Pressure ◽  
Laboratory Animals ◽  
Nucleic Acid Content ◽  
Dna Concentration ◽  
Cellular Change ◽  
Marked Disparity ◽  
Left And Right ◽  
Rna Concentration

In order to study the marked disparity in thickness of the left and right ventricular (LV and RV) walls of the turkey heart, DNA, RNA, and protein content of LV and RV myocardium were determined in fowl aged 4, 6, and 11 weeks and at maturity. DNA concentration in RV tissue was significantly greater than that in LV tissue in each bird and at each age. DNA concentration in both ventricles, moreover, decreased with increasing age. Protein concentration in both ventricles increased as did protein-to-DNA ratios. Although RNA concentration showed less consistent changes with age, the RNA-to-DNA ratio increased. It was concluded that the predominant cellular change with age was hypertrophy. In addition, electrocardiograms and hemodynamic data were obtained on birds in each group. Although pulmonary artery pressure is comparable to that in other laboratory animals, a markedly elevated systemic blood pressure develops between the ages of 6 and 11 weeks. The role of hypertension as a determinant of cellular size in the LV was not elucidated by this study.

scholarly journals Single Residue Substitution at N-Terminal Affects Temperature Stability and Activity of L2 Lipase

Molecules ◽  
2020 ◽  
Vol 25 (15) ◽  
pp. 3433
Author(s):  
Noramirah Bukhari ◽  
Adam Thean Chor Leow ◽  
Raja Noor Zaliha Raja Abd Rahman ◽  
Fairolniza Mohd Shariff
Keyword(s):  
Rational Design ◽  
Thermal Denaturation ◽  
Temperature Stability ◽  
Industrial Applications ◽  
Terminal Region ◽  
Wild Type ◽  
Functional Region ◽  
Target Region ◽  
Residue Substitution

Rational design is widely employed in protein engineering to tailor wild-type enzymes for industrial applications. The typical target region for mutation is a functional region like the catalytic site to improve stability and activity. However, few have explored the role of other regions which, in principle, have no evident functionality such as the N-terminal region. In this study, stability prediction software was used to identify the critical point in the non-functional N-terminal region of L2 lipase and the effects of the substitution towards temperature stability and activity were determined. The results showed 3 mutant lipases: A8V, A8P and A8E with 29% better thermostability, 4 h increase in half-life and 6.6 °C higher thermal denaturation point, respectively. A8V showed 1.6-fold enhancement in activity compared to wild-type. To conclude, the improvement in temperature stability upon substitution showed that the N-terminal region plays a role in temperature stability and activity of L2 lipase.

scholarly journals Optimization of the experimental parameters of the ligase cycling reaction

2019 ◽  
Vol 4 (1) ◽  
Author(s):  
Niels Schlichting ◽  
Felix Reinhardt ◽  
Sven Jager ◽  
Michael Schmidt ◽  
Johannes Kabisch
Keyword(s):  
Secondary Structure ◽  
Thermal Denaturation ◽  
Screening System ◽  
Experimental Procedure ◽  
Melting Temperatures ◽  
Assembly Method ◽  
High Efficient ◽  
Experimental Parameters ◽  
Molecular Crosstalk ◽  
Validation Experiments

Abstract The ligase cycling reaction (LCR) is a scarless and efficient method to assemble plasmids from fragments of DNA. This assembly method is based on the hybridization of DNA fragments with complementary oligonucleotides, so-called bridging oligos (BOs), and an experimental procedure of thermal denaturation, annealing and ligation. In this study, we explore the effect of molecular crosstalk of BOs and various experimental parameters on the LCR by utilizing a fluorescence-based screening system. The results indicate an impact of the melting temperatures of BOs on the overall success of the LCR assembly. Secondary structure inhibitors, such as dimethyl sulfoxide and betaine, are shown to negatively impact the number of correctly assembled plasmids. Adjustments of the annealing, ligation and BO-melting temperature further improved the LCR. The optimized LCR was confirmed by validation experiments. Based on these findings, a step-by-step protocol is offered within this study to ensure a routine for high efficient LCR assemblies.

scholarly journals Influence of Magnesium Ions on the Preparation and Storage of DNA Tetrahedrons in Micromolar Ranges

Molecules ◽  
2019 ◽  
Vol 24 (11) ◽  
pp. 2091 ◽  
Author(s):  
Yue Hu ◽  
Zhou Chen ◽  
Zheng Hou ◽  
Mingkai Li ◽  
Bo Ma ◽  
...  
Keyword(s):  
The Novel ◽  
Magnesium Ions ◽  
Dna Nanostructures ◽  
Structure And Stability ◽  
Dna Concentration ◽  
Factors Influencing ◽  
Dna Tetrahedron ◽  
And Storage

The DNA tetrahedron (Td), as one of the novel DNA-based nanoscale biomaterials, has been extensively studied because of its excellent biocompatibility and increased possibilities for decorating precisely. Although the use of Td in laboratories is well established, knowledge surrounding the factors influencing its preparation and storage is lacking. In this research, we investigated the role of the magnesium ions, which greatly affect the structure and stability of DNA. We assembled 1, 2, 5, 10 and 20 μM Td in buffers containing different Mg2+ concentrations, demonstrating that 2 and 5 mM Mg2+ is optimal in these conditions, and that yields decrease dramatically once the DNA concentration reaches 20 μM or the Mg2+ concentration is lower than 0.5 mM. We also verified that the Td structure is retained better through freeze-thawing than lyophilization. Furthermore, a lower initial Mg2+ (≤2 mM) benefited the maintenance of Td structure in the process of lyophilization. Hence, our research sheds light on the influence of Mg2+ in the process of preparing and storing Td, and also provides some enlightenment on improving yields of other DNA nanostructures.

scholarly journals Interaction between Chromosomal Protein HMGB1 and DNA Studied by DNA-Melting Analysis

2014 ◽  
Vol 2014 ◽  
pp. 1-5 ◽  
Author(s):  
Elena V. Chikhirzhina ◽  
Starkova J. Tatiana ◽  
Alexander M. Polyanichko
Keyword(s):  
Circular Dichroism ◽  
Thermal Denaturation ◽  
Circular Dichroism Spectroscopy ◽  
Dna Melting ◽  
Chromosomal Protein ◽  
Melting Temperatures ◽  
Melting Analysis ◽  
Circular Dichroïsm ◽  
Nonhistone Chromosomal Protein ◽  
Biphasic Process

Interaction of HMGB1 nonhistone chromosomal protein with DNA was studied using circular dichroism spectroscopy and thermal denaturation of DNA. Melting DNA in the complex was shown to be a biphasic process. The characteristic melting temperatures of unbound DNA and the DNA bound to HMGB1 in 0.25 mM EDTA solutions were found to beTmI=44.0±0.5°C andTmII=62.0±1°C, respectively. It was shown that the binding of the HMGB1 molecule affects the melting of the DNA region approximately 30 b.p. long.

scholarly journals Role of non-compatible osmolytes in the stabilization of proteins during heat stress

1998 ◽  
Vol 329 (1) ◽  
pp. 137-143 ◽  
Author(s):  
Vikas RISHI ◽  
Farah ANJUM ◽  
Faizan AHMAD ◽  
Wolfgang PFEIL
Keyword(s):  
Thermal Denaturation ◽  
Rnase A ◽  
Transition Curve ◽  
Evolutionary Perspective ◽  
Inhibitory Effects ◽  
Absorption Coefficients ◽  
Ph Values ◽  
Stabilizing Effect ◽  
Compatible Osmolytes

This study is a systematic attempt to understand the roles of non-compatible osmolytes, i.e. solutes that have inhibitory effects on enzymes, in the stabilization of proteins against denaturing stress. Thermal denaturation of RNase A, holo-α-lactalbumin, apo-α-lactalbumin, lysozyme and metmyoglobin in the absence and presence of various concentrations of free basic amino acids was studied by observing changes in the absorption coefficients of these proteins. It has been observed that arginine and histidine destabilize all proteins in terms of the midpoint of the transition curve and Gibbs energy change on denaturation. Study of the heat-induced denaturation of the proteins in the presence of various concentrations of arginine at different pH values demonstrated that arginine binds to the denatured molecules. In contrast with the effect of arginine and histidine on protein stability, it was observed that the effect of lysine on proteins stability is unpredictable, i.e. it may have a stabilizing effect, no effect or a destabilizing effect on proteins during denaturing stress. The results of this study are considered from an evolutionary perspective.

Conformation and Thermal Denaturation of Apocalmodulin:  Role of Electrostatic Mutations†

Biochemistry ◽  
1997 ◽  
Vol 36 (8) ◽  
pp. 2017-2024 ◽  
Author(s):  
Irina Protasevich ◽  
Bijan Ranjbar ◽  
Vladimir Lobachov ◽  
Alexander Makarov ◽  
Robert Gilli ◽  
...  
Keyword(s):  
Thermal Denaturation

Histological evidence for the role of mechanical stress in modulating thermal denaturation of collagen

2005 ◽  
Vol 4 (4) ◽  
pp. 201-210 ◽  
Author(s):  
P. B. Wells ◽  
S. Thomsen ◽  
M. A. Jones ◽  
S. Baek ◽  
J. D. Humphrey
Keyword(s):  
Mechanical Stress ◽  
Thermal Denaturation ◽  
Histological Evidence
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