Comparison of N-terminal affinity fusion domains: effect on expression level and product heterogeneity of recombinant restriction endonuclease EcoRV

1994 ◽  
Vol 42 (1) ◽  
pp. 73-77 ◽  
Author(s):  
T. Oswald ◽  
W. Wende ◽  
A. Pingoud ◽  
U. Rinas
Keyword(s):  
Restriction Endonuclease ◽  
Expression Level ◽  
Product Heterogeneity

Electron microscopic assays of restriction endonuclease cutting, exonuclease digestion, and hybridization

1984 ◽  
Vol 42 ◽  
pp. 686-687
Author(s):  
Mark Hannibal ◽  
Jacob Varkey ◽  
Michael Beer
Keyword(s):  
Electron Microscope ◽  
Low Temperature ◽  
Restriction Endonuclease ◽  
Double Helix ◽  
Test Material ◽  
Electron Microscopic ◽  
Exonuclease Iii ◽  
Dna Molecule ◽  
Linear Molecules ◽  
Exonuclease Digestion

Workman and Langmore have recently proposed a procedure for isolating particular chromatin fragments. The method requires restriction endonuclease cutting of the chromatin and a probe, their digestion with two exonucleases which leave complimentary single strand termini and low temperature hybridization of these. We here report simple electron microscopic monitoring of the four reactions involved.Our test material was ϕX-174 RF DNA which is cut once by restriction endonuclease Xho I. The conversion of circles to linear molecules was followed in Kleinschmidt spreads. Plate I shows a circular and a linear DNA molecule. The rate of cutting is shown in Figure 1.After completion of the endonuclease cutting, one portion of the DNA was treated with exonuclease III, an enzyme known to digest the 3' terminals of double helical DNA. Aliquots when examined in the electron microscope reveal a decreasing length of double helix and increasing bushes at the ends.

Use of Antibody to aid Visualization of Protein at the Termini of Bacteriophage Ø29 DNA

1980 ◽  
Vol 38 ◽  
pp. 540-541
Author(s):  
Dwight Anderson ◽  
Charlene Peterson ◽  
Gursaran Notani ◽  
Bernard Reilly
Keyword(s):  
Electron Microscopy ◽  
Bacillus Subtilis ◽  
Dna Synthesis ◽  
Restriction Endonuclease ◽  
Protein Complex ◽  
Protein Product ◽  
Viral Dna ◽  
Small Proteins ◽  
Antibody Labeling ◽  
Subtilis Bacteriophage

The protein product of cistron 3 of Bacillus subtilis bacteriophage Ø29 is essential for viral DNA synthesis and is covalently bound to the 5’-termini of the Ø29 DNA. When the DNA-protein complex is cleaved with a restriction endonuclease, the protein is bound to the two terminal fragments. The 28,000 dalton protein can be visualized by electron microscopy as a small dot and often is seen only when two ends are in apposition as in multimers or in glutaraldehyde-fixed aggregates. We sought to improve the visibility of these small proteins by use of antibody labeling.

Potential predictive marker of treatment effectiveness in patients with osteopenic syndrome after surgical menopause

GYNECOLOGY ◽  
2020 ◽  
Vol 22 (4) ◽  
pp. 39-42
Author(s):  
Yansiiat Z. Zaydieva ◽  
Elena V. Kruchinina ◽  
Olga S. Gorenkova ◽  
Elena Yu. Polyakova ◽  
Elena N. Kareva ◽  
...  
Keyword(s):  
Vitamin D ◽  
Combination Therapy ◽  
Vitamin D Receptor ◽  
Surrogate Marker ◽  
Predictive Marker ◽  
Expression Level ◽  
Mineral Density ◽  
Vitamin D Receptors ◽  
Surgical Menopause

Introduction. Patients with surgical menopause have a risk for osteopenic syndrome (OS). Menopausal hormone therapy (MHT) in combination with calcium and vitamin D promotes increase in bone mineral density (BMD). The expression level of vitamin D receptor in mononuclear fraction cells (MNFC) of blood can be considered as a predictive marker of effectiveness of OS therapy. Aim. To search a molecular predictive marker of the effectiveness of OS treatment. Materials and methods. The study included 100 women aged 4055 years with a duration of surgical menopause from 12 months to 6 years. The criterion for including patients in the study was the absence of contraindications to the use of MHT. The subject of the study was the determination of BMD by dual-energy X-ray absorptiometry, polymerase chain reaction diagnostics of the level of expression of vitamin D genes, estradiol and progesterone receptors, determination of 25-OH vitamin D in the blood. Results. Analysis of 12-month OS therapy effectiveness evaluated with a surrogate marker BMD. The increase in BMD up to 34% per year was treated as absence of negative dynamics, more than 4% per year as positive one. Significant effect of combination therapy compared with MHT on BMD in patients with surgical menopause with a low baseline level of BMD (due to hypovitaminosis D) is associated with the anti-inflammatory, bone-protective effect of vitamin D. In both groups of patients not responding; to the prescribed therapy we were able to conduct a comparative analysis of expression level of the target molecules in the MNFC before the start of treatment. The efficacy of MHT and combination therapy for BMD disorders is positively associated with the expression level of vitamin D receptors in MNFC before treatment. Therefore, the vitDR mRNA level is a potential predictive marker of the effectiveness of OS treatment. The expression levels of nuclear estradiol beta receptor and membrane receptor for progesterone in MNFC before treatment showed an upward trend in women responding to therapy. Conclusion. The expression level of the vitamin D receptor in MNFC of blood is significantly lower in the group of women with no/insufficient effect on 12-month combined therapy. This indicator can be considered as a predictive marker of the effectiveness of OS therapy.

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