Successful deoxyribonuclease I (DNase I) phenotyping from a small piece of used sock

Abstract The Leydig insulin-like gene (Ley I-L), a member of the insulin-related gene family, is specifically expressed in pre- and postnatal Leydig cells of the testis and in postnatal theca cells of the ovary. To determine the functional region of the mouse Ley I-L promoter and factors controlling the Ley I-L gene expression, we used 2.1 kb of the 5′-flanking region of the mouse Ley I-L gene to generate chimeric constructs with the chloramphenicol acetyltransferase gene (CAT). Transient transfections of MA10 Leydig cells, LTK− fibroblasts, and F9 embryonic cells by a series of 5′-deleted mouse Ley I-L promoter-CAT constructs revealed that the sequence between nucleotides −157 to +4 directs the transcription of the reporter gene in MA10 but not in LTK− and F9 cells, indicating that the determinants of Leydig cell-specific expression reside within this region. Deoxyribonuclease I (DNase I) footprint analysis revealed that the sequences designated SF-1/1, SF-1/2, and SF-1/3 within three DNase I-protected regions are homologous to the consensus binding site of the steroidogenic factor-1 (SF-1). Competition and antibody studies showed that the three SF-1-binding sites in the Ley I-L promoter have similar binding affinities for SF-1. Furthermore, transient transfections of MA10 cells with mutant reporter constructs, in which SF-1/1 or both SF-1/2 and SF-1/3 were deleted, demonstrated that all three SF-1- binding sites are required for SF-1-mediated stimulation of Ley I-L transcription. Cotransfection of an SF-1-containing expression vector together with a Ley I-L promoter-CAT construct into HeLa cells, which lack the endogenous SF-1 protein, resulted in CAT gene transcription, which indicated that SF-1 can transactivate the Ley I-L promoter. These data demonstrate an essential role of SF-1 in transcriptional activation of the Ley I-L promoter.

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Usefulness of deoxyribonuclease I (DNase I) polymorphism for individualization from small aged urine stains

Legal Medicine ◽

10.1016/s1344-6223(03)00049-x ◽

2003 ◽

Vol 5 (2) ◽

pp. 105-107 ◽

Cited By ~ 3

Author(s):

Toshihiro Yasuda ◽

Haruo Takeshita ◽

Misuzu Ueki ◽

Tamiko Nakajima ◽

Koichi Mogi ◽

...

Keyword(s):

Dnase I ◽

Deoxyribonuclease I

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Genetic Control of Urinary Deoxyribonuclease I (DNase I) Activity Levels in Mice.

Experimental Animals ◽

10.1538/expanim.45.245 ◽

1996 ◽

Vol 45 (3) ◽

pp. 245-250 ◽

Cited By ~ 7

Author(s):

Tsutomu KOIZUMI

Keyword(s):

Genetic Control ◽

Dnase I ◽

Activity Levels ◽

Deoxyribonuclease I

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Enhanced eradication of bacterial biofilms with DNase I-loaded silver-doped mesoporous silica nanoparticles

Nanoscale ◽

10.1039/c9nr08467c ◽

2020 ◽

Vol 12 (4) ◽

pp. 2328-2332 ◽

Cited By ~ 8

Author(s):

Winda Tasia ◽

Chang Lei ◽

Yuxue Cao ◽

Qingsong Ye ◽

Yan He ◽

...

Keyword(s):

Mesoporous Silica ◽

Silica Nanoparticles ◽

Mesoporous Silica Nanoparticles ◽

Dnase I ◽

Bacterial Biofilms ◽

Deoxyribonuclease I

Deoxyribonuclease I (DNase I) was loaded and delivered by silver-doped mesoporous silica nanoparticles (MSN-Ag) for biofilm treatments.

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A New Individualization Marker of Sweat: Deoxyribonuclease I (DNase I) Polymorphism

Journal of Forensic Sciences ◽

10.1520/jfs14012j ◽

1996 ◽

Vol 41 (5) ◽

pp. 14012J ◽

Cited By ~ 8

Author(s):

Toshihiro Yasuda ◽

Haruo Takeshita ◽

Kazumi Sawazaki ◽

Daita Nadano ◽

Reiko Iida ◽

...

Keyword(s):

Dnase I ◽

Deoxyribonuclease I

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Purification of multiple functional leaf-actin isoforms from Phaseolus vulgaris L

Biochemical Journal ◽

10.1042/bj3430597 ◽

1999 ◽

Vol 343 (3) ◽

pp. 597-602

Author(s):

Claudia DÍAZ-CAMINO ◽

Marco A. VILLANUEVA

Keyword(s):

Phaseolus Vulgaris ◽

Affinity Purification ◽

Dnase I ◽

Rabbit Muscle ◽

Fresh Tissue ◽

Western Blots ◽

Actin Isoforms ◽

Deoxyribonuclease I ◽

A Cell ◽

Actin Concentration

Plant actins show diversity in their gene sequences, protein isovariants and tissue distribution in eukaryotes. Besides general difficulties with the isolation of proteins from plant material (i.e. the presence of a cell wall and high proteolytic activity), the actin concentration in any vegetative plant tissue is much lower than in cytoplasmic animal tissues. In this study, we adapted a deoxyribonuclease I-Sepharose affinity purification scheme and we were able to enrich and isolate multiple functional plant actin isovariants from common bean leaves (Phaseolus vulgaris). Urea (4 M) elution proved that the DNase I column was able to bind at least eight actin isoforms with pI values ranging from 5.5 to 5.9, as observed by two-dimensional Western blots. Three of the most acidic actin isoforms, with pI values of ≈ 5.6-5.7, were eluted partially with 0.75 M urea. The purified actin was also able to bind leaf and rabbit muscle profilin, phalloidin and DNase I. Moreover, the protein could polymerize into filaments that contained the main isoforms eluted from the column. The average actin recovery using this procedure was ≈ 4-8 μg from 20 g of fresh tissue, of which at least 80% was able to form filaments. This is the first report of the purification of multiple plant-actin isoforms that are functional by the criteria of both binding to other ligands and polymerization.

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Novel High-Throughput Deoxyribonuclease 1 Assay

CrossRef Listing of Deleted DOIs ◽

10.1177/1087057114555828 ◽

2014 ◽

Vol 20 (2) ◽

pp. 202-211 ◽

Cited By ~ 3

Author(s):

Dae Song Jang ◽

Narsimha R. Penthala ◽

Eugene O. Apostolov ◽

Xiaoying Wang ◽

Tariq Fahmi ◽

...

Keyword(s):

Cell Death ◽

High Throughput ◽

High Volume ◽

Dnase I ◽

Radiation Injuries ◽

Chemical Library ◽

Chemical Libraries ◽

Deoxyribonuclease I ◽

Highly Sensitive

Deoxyribonuclease I (DNase I), the most active and abundant apoptotic endonuclease in mammals, is known to mediate toxic, hypoxic, and radiation injuries to the cell. Neither inhibitors of DNase I nor high-throughput methods for screening of high-volume chemical libraries in search of DNase I inhibitors are, however, available. To overcome this problem, we developed a high-throughput DNase I assay. The assay is optimized for a 96-well plate format and based on the increase of fluorescence intensity when fluorophore-labeled oligonucleotide is degraded by the DNase. The assay is highly sensitive to DNase I compared to other endonucleases, reliable (Z’ ≥ 0.5), and operationally simple, and it has low operator, intraassay, and interassay variability. The assay was used to screen a chemical library, and several potential DNase I inhibitors were identified. After comparison, 2 hit compounds were selected and shown to protect against cisplatin-induced kidney cell death in vitro. This assay will be suitable for identifying inhibitors of DNase I and, potentially, other endonucleases.

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