scholarly journals Visualization of miniSOG Tagged DNA Repair Proteins in Combination with Electron Spectroscopic Imaging (ESI)

10.3791/52893 ◽  
2015 ◽  
Author(s):  
Hilmar Strickfaden ◽  
Zhi Zhong Xu ◽  
Michael J. Hendzel
Keyword(s):  
Dna Repair ◽  
Spectroscopic Imaging ◽  
Electron Spectroscopic Imaging ◽  
Dna Repair Proteins

Visualisation of E. coli ribosomal RNA in situ by electron spectroscopic imaging and image averaging

1992 ◽  
Vol 50 (1) ◽  
pp. 466-467
Author(s):  
Daniel Beniac ◽  
George Harauz
Keyword(s):  
Ribosomal Rna ◽  
Three Dimensional ◽  
Spectroscopic Imaging ◽  
Electron Spectroscopic Imaging ◽  
E Coli ◽  
Microscopical Technique ◽  
Quantitative Image ◽  
Dimensional Reconstruction ◽  
Image Averaging ◽  
Protein Components

The structures of E. coli ribosomes have been extensively probed by electron microscopy of negatively stained and frozen hydrated preparations. Coupled with quantitative image analysis and three dimensional reconstruction, such approaches are worthwhile in defining size, shape, and quaternary organisation. The important question of how the nucleic acid and protein components are arranged with respect to each other remains difficult to answer, however. A microscopical technique that has been proposed to answer this query is electron spectroscopic imaging (ESI), in which scattered electrons with energy losses characteristic of inner shell ionisations are used to form specific elemental maps. Here, we report the use of image sorting and averaging techniques to determine the extent to which a phosphorus map of isolated ribosomal subunits can define the ribosomal RNA (rRNA) distribution within them.

Transcription factor/DNA interactions visualized by electron spectroscopic imaging

1992 ◽  
Vol 50 (1) ◽  
pp. 450-451
Author(s):  
David P. Bazett-Jones ◽  
Mark L. Brown
Keyword(s):  
Transcription Factor ◽  
Dna Sequences ◽  
Transcription Initiation ◽  
Molecular Mechanisms ◽  
Phosphorus Content ◽  
Spectroscopic Imaging ◽  
Electron Spectroscopic Imaging ◽  
Dna Backbone ◽  
Two Parameters ◽  
High Level

A multisubunit RNA polymerase enzyme is ultimately responsible for transcription initiation and elongation of RNA, but recognition of the proper start site by the enzyme is regulated by general, temporal and gene-specific trans-factors interacting at promoter and enhancer DNA sequences. To understand the molecular mechanisms which precisely regulate the transcription initiation event, it is crucial to elucidate the structure of the transcription factor/DNA complexes involved. Electron spectroscopic imaging (ESI) provides the opportunity to visualize individual DNA molecules. Enhancement of DNA contrast with ESI is accomplished by imaging with electrons that have interacted with inner shell electrons of phosphorus in the DNA backbone. Phosphorus detection at this intermediately high level of resolution (≈lnm) permits selective imaging of the DNA, to determine whether the protein factors compact, bend or wrap the DNA. Simultaneously, mass analysis and phosphorus content can be measured quantitatively, using adjacent DNA or tobacco mosaic virus (TMV) as mass and phosphorus standards. These two parameters provide stoichiometric information relating the ratios of protein:DNA content.

Localization of DNA in chromatin using ESI and osmium ammine staining

1990 ◽  
Vol 48 (3) ◽  
pp. 116-117
Author(s):  
C.L. Woodcock ◽  
R.A. Horowitz ◽  
D. P. Bazett-Jones ◽  
A.L. Olins
Keyword(s):  
Spectroscopic Imaging ◽  
Energy Losses ◽  
Electron Spectroscopic Imaging ◽  
Feulgen Reaction ◽  
Specific Location ◽  
Eukaryotic Nucleus ◽  
Chromatin Fibers ◽  
Patiria Miniata ◽  
Model Structures

In the eukaryotic nucleus, DNA is packaged into nucleosomes, and the nucleosome chain folded into ‘30nm’ chromatin fibers. A number of different model structures, each with a specific location of nucleosomal and linker DNA have been proposed for the arrangment of nucleosomes within the fiber. We are exploring two strategies for testing the models by localizing DNA within chromatin: electron spectroscopic imaging (ESI) of phosphorus atoms, and osmium ammine (OSAM) staining, a method based on the DNA-specific Feulgen reaction.Sperm were obtained from Patiria miniata (starfish), fixed in 2% GA in 150mM NaCl, 15mM HEPES pH 8.0, and embedded In Lowiciyl K11M at -55C. For OSAM staining, sections 100nm to 150nm thick were treated as described, and stereo pairs recorded at 40,000x and 100KV using a Philips CM10 TEM. (The new osmium ammine-B stain is available from Polysciences Inc). Uranyl-lead (U-Pb) staining was as described. ESI was carried out on unstained, very thin (<30 nm) beveled sections at 80KV using a Zeiss EM902. Images were recorded at 20,000x and 30,000x with median energy losses of 110eV, 120eV and 160eV, and a window of 20eV.

scholarly journals DNA repair proteins may differentiate papillary thyroid cancer from chronic lymphocytic thyroiditis and nodular colloidal goiter

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Bahri Evren ◽  
Sami Yılmaz ◽  
Neşe Karadağ ◽  
Ayşe Çıkım Sertkaya ◽  
Ömercan Topaloğlu ◽  
...  
Keyword(s):  
Dna Repair ◽  
Thyroid Cancer ◽  
Papillary Carcinoma ◽  
Staining Intensity ◽  
Follicular Cell ◽  
Papillary Thyroid ◽  
Chronic Thyroiditis ◽  
Dna Repair Proteins ◽  
The Difference ◽  
Repair Genes

AbstractMalignant thyroid lesions are the most common malignancy of the endocrine glands with increasing rates in the last two decades. Papillary thyroid cancer is the most common thyroid malignancy. In our study, we aimed to quantitatively evaluate the levels of DNA repair proteins MSH2, MLH1, MGMT, which are representative blocks of patients diagnosed with papillary carcinoma, chronic thyroiditis, or colloidal goiter. Total or subtotal thyroidectomy material of 90 patients diagnosed with papillary carcinoma, nodular colloidal goiter, or chronic thyroiditis between 2009 and 2012 were retrospectively evaluated. Tissue samples obtained from paraffin blocks were stained with MGMT, MSH2, MLH1 proteins and their immunohistochemistry was evaluated. Prepared sections were examined qualitatively by an impartial pathologist and a clinician, taking into account the staining method under the trinocular light microscope. Although there was no statistically significant difference in MGMT, MSH2, MLH1, follicular cell positivity, staining intensity, and immunoreactivity values, papillary carcinoma cases showed a higher rate of follicular cell positivity, and this difference was more pronounced between papillary carcinoma and colloidal goiter. In the MSH2 follicular cell positivity evaluation, the difference between chronic thyroiditis and colloidal goiter was significant (p = 0.023). The difference between chronic thyroiditis and colloidal goiter was significant in the MSH2 staining intensity evaluation (p = 0.001). The difference between chronic thyroiditis and colloidal goiter was significant in MLH1 immunoreactivity evaluation (p = 0.012). Papillary carcinoma cases were demonstrated by nuclear staining only for MSH2 and MLH1 proteins as opposed to hyperplastic nodules. The higher levels of expression of DNA repair genes in malignant tumors compared to benign tumors are attributed to the functional activation of DNA repair genes. Further studies are needed for DNA repair proteins to be a potential test in the development and progression of thyroid cancer.

scholarly journals Host DNA Repair Proteins in Response toPseudomonas aeruginosain Lung Epithelial Cells and in Mice

2010 ◽  
Vol 79 (1) ◽  
pp. 75-87 ◽  
Author(s):  
Min Wu ◽  
Huang Huang ◽  
Weidong Zhang ◽  
Shibichakravarthy Kannan ◽  
Andrew Weaver ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dna Repair ◽  
Epithelial Cells ◽  
Critical Role ◽  
Lung Epithelial Cells ◽  
Host Cells ◽  
Myeloperoxidase Activity ◽  
Gram Negative ◽  
Lung Epithelial ◽  
Dna Repair Proteins

ABSTRACTAlthough DNA repair proteins in bacteria are critical for pathogens' genome stability and for subverting the host defense, the role of host DNA repair proteins in response to bacterial infection is poorly defined. Here, we demonstrate, for the first time, that infection with the Gram-negative bacteriumPseudomonas aeruginosasignificantly altered the expression and enzymatic activity of 8-oxoguanine DNA glycosylase (OGG1) in lung epithelial cells. Downregulation of OGG1 by a small interfering RNA strategy resulted in severe DNA damage and cell death. In addition, acetylation of OGG1 is required for host responses to bacterial genotoxicity, as mutations of OGG1 acetylation sites increased Cockayne syndrome group B (CSB) protein expression. These results also indicate that CSB may be involved in DNA repair activity during infection. Furthermore, OGG1 knockout mice exhibited increased lung injury after infection withP. aeruginosa, as demonstrated by higher myeloperoxidase activity and lipid peroxidation. Together, our studies indicate thatP. aeruginosainfection induces significant DNA damage in host cells and that DNA repair proteins play a critical role in the host response toP. aeruginosainfection, serving as promising targets for the treatment of this condition and perhaps more broadly Gram-negative bacterial infections.

Imaging techniques used for the detection of 8-oxoguanine adducts and DNA repair proteins in cells and tissues

2001 ◽  
Vol 36 (9) ◽  
pp. 1483-1494 ◽  
Author(s):  
Rebecca L. Persinger ◽  
Robert Melamede ◽  
Ivan Bespalov ◽  
Susan Wallace ◽  
Douglas J. Taatjes ◽  
...  
Keyword(s):  
Dna Repair ◽  
Imaging Techniques ◽  
Dna Repair Proteins ◽  
In Cells
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