High frequency of constitutive alkali-labile sites in mouse major satellite DNA, detected by DNA breakage detection-fluorescence in situ hybridization

Rivero, María Teresa; Vázquez-Gundı́n, Fernando; Goyanes, Vicente; Campos, Ana Carolina da Cruz; Blasco, Marı́a A.; Gosálvez, Jaime; Fernández, José Luis Fernández

doi:10.1016/s0027-5107(01)00218-4

Cited by 20 publications

(11 citation statements)

References 20 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The latter are DNA modifications that are transformed into SBs by alkaline treatment. ALS are particularly common in genomes with tightly-packaged DNA (i.e., human and mouse sperm, chicken erythrocytes, mouse kidney) or are concentrated within specific heterochromatic noncoding regions and are considered a functional feature of condensed chromatin [Singh et al, 1989;Fairbain et al, 1994;Haines et al, 1998;Fernandez et al, 2001;Rivero et al, 2001]. The elevated levels of SBs detected by alkaline SCGE in several marine invertebrates were interpreted as the result of high levels of ALS rather than to SBs generated by the SCGE procedure.…”

Section: Discussionmentioning

confidence: 99%

Influence of the SCGE protocol on the amount of basal DNA damage detected in the Mediterranean mussel, Mytilus galloprovincialis

Machella

Battino

Pisanelli

et al. 2006

Environ and Mol Mutagen

View full text Add to dashboard Cite

Genotoxicity studies using the single cell gel electrophoresis (SCGE) assay indicate that basal levels of DNA strand breaks (SBs) in marine invertebrates are higher and more variable than those in marine vertebrates. This elevated level of DNA damage was attributed to a large number of alkali-labile sites, which are characteristic of the tightly-packaged DNA in invertebrate cells. To investigate if altering the SCGE protocol can artificially modulate high levels of SBs, SCGE experiments were performed on haemocytes from the Mediterranean mussel (Mytilus galloprovincialis) using proteinase K (PK) digestion in combination with assay buffers containing various concentrations of EDTA. In addition, the effects of Trolox (soluble antioxidant) and aurintricarboxylic acid (ATA; inhibitor of Ca(2+)/Mg(2+)-dependent nucleases) also were tested. The levels of SBs in M. galloprovincialis cells were compared with SBs in cells from a terrestrial mollusk (the snail Helix aspersa), and a teleost fish (the seabass Dicentrarchus labrax). The integrity of M. galloprovincialis DNA isolated with phenol extractions using EDTA, Trolox, and ATA was further assayed by gel electrophoresis. High SBs in mussel cells were reduced by combining EDTA with PK digestion, or using Trolox or ATA during cell processing for the SCGE assay. Snails and seabass had lower levels of SBs in the SCGE assay, and the levels were not affected by the protocol modifications. Adding EDTA, Trolox, or ATA to phenol extractions of M. galloprovincialis genomic DNA also reduced the extent of DNA fragmentation. These results suggest that the internal fluids of M. galloprovincialis may increase the basal levels of DNA SBs through oxidative and/or enzyme-mediated pathways. M. galloprovincialis is used extensively as a sentinel species for assessing the genotoxic hazard of marine pollutants. Our data suggest that the SCGE protocol should be carefully considered when assessing DNA damage in these species.

show abstract

Section: Discussionmentioning

confidence: 99%

Influence of the SCGE protocol on the amount of basal DNA damage detected in the Mediterranean mussel, Mytilus galloprovincialis

Machella

Battino

Pisanelli

et al. 2006

Environ and Mol Mutagen

View full text Add to dashboard Cite

show abstract

“…It is noteworthy that when a whole-genome DNA probe is hybridized to somatic cells, the background DBD-FISH signal is not homogeneous and certain chromatin regions are selectively and strongly labeled; this is especially evident when high levels of alkali denaturation are used on the native sperm chromatin (Figure 3, DBD-FISH High). It is of interest to highlight that the DNA sequences related with constitutive ALS mostly correspond with certain specific highly repetitive DNA sequences (Fernández et al, 2001; Rivero et al, 2001, 2004). In human leukocytes, the more intense background DBD-FISH areas within the genome correspond to DNA domains containing 5-bp satellite DNA sequences (Fernández and Gosálvez, 2002).…”

Section: Sperm Dna Comet Under Alkaline Denaturing Conditionsmentioning

confidence: 99%

“…In human leukocytes, the more intense background DBD-FISH areas within the genome correspond to DNA domains containing 5-bp satellite DNA sequences (Fernández and Gosálvez, 2002). In mouse splenocytes, the background labeled areas correspond with highly repetitive major DNA satellite sequences located in pericentromeric regions (Rivero et al, 2001), and in Chinese hamster cells, they match to pericentromeric interstitial telomeric-like DNA sequence blocks (Rivero et al, 2004). As indicated, all these native highly alkali-sensitive regions correspond to strongly compacted chromatin domains present in somatic nuclei.…”

Section: Sperm Dna Comet Under Alkaline Denaturing Conditionsmentioning

confidence: 99%

Interpreting sperm DNA damage in a diverse range of mammalian sperm by means of the two-tailed comet assay

et al. 2014

View full text Add to dashboard Cite

Key Concepts The two-dimensional Two-Tailed Comet assay (TT-comet) protocol is a valuable technique to differentiate between single-stranded (SSBs) and double-stranded DNA breaks (DSBs) on the same sperm cell.Protein lysis inherent with the TT-comet protocol accounts for differences in sperm protamine composition at a species-specific level to produce reliable visualization of sperm DNA damage.Alkaline treatment may break the sugar–phosphate backbone in abasic sites or at sites with deoxyribose damage, transforming these lesions into DNA breaks that are also converted into ssDNA. These lesions are known as Alkali Labile Sites “ALSs.”DBD–FISH permits the in situ visualization of DNA breaks, abasic sites or alkaline-sensitive DNA regions.The alkaline comet single assay reveals that all mammalian species display constitutive ALS related with the requirement of the sperm to undergo transient changes in DNA structure linked with chromatin packing.Sperm DNA damage is associated with fertilization failure, impaired pre-and post- embryo implantation and poor pregnancy outcome.The TT is a valuable tool for identifying SSBs or DSBs in sperm cells with DNA fragmentation and can be therefore used for the purposes of fertility assessment.Sperm DNA damage is associated with fertilization failure, impaired pre-and post- embryo implantation and poor pregnancy outcome. A series of methodologies to assess DNA damage in spermatozoa have been developed but most are unable to differentiate between single-stranded DNA breaks (SSBs) and double-stranded DNA breaks (DSBs) on the same sperm cell. The two-dimensional Two-Tailed Comet assay (TT-comet) protocol highlighted in this review overcomes this limitation and emphasizes the importance in accounting for the difference in sperm protamine composition at a species-specific level for the appropriate preparation of the assay. The TT-comet is a modification of the original comet assay that uses a two dimensional electrophoresis to allow for the simultaneous evaluation of DSBs and SSBs in mammalian spermatozoa. Here we have compiled a retrospective overview of how the TT-comet assay has been used to investigate the structure and function of sperm DNA across a diverse range of mammalian species (eutheria, metatheria, and prototheria). When conducted as part of the TT-comet assay, we illustrate (a) how the alkaline comet single assay has been used to help understand the constitutive and transient changes in DNA structure associated with chromatin packing, (b) the capacity of the TT-comet to differentiate between the presence of SSBs and DSBs (c) and the possible implications of SSBs or DSBs for the assessment of infertility.

show abstract

“…The comet formation pattern is determined by the size of the DNA fragments and the number of broken ends. [10] As the percent of damage increases, the free DNA fragments move more in the tail. To perform this test, a suspension of the separated cells should be prepared.…”

Section: Introductionmentioning

confidence: 99%

Genotoxicity evaluation of hydroalcoholic and aqueous extracts of Dorema aucheri by the comet assay

et al. 2016

View full text Add to dashboard Cite

Background:Dorema aucheri is a plant of Apiaceae family which is used widely in some states of Iran. Different extracts and essential oil of Dorema species contain flavonoids and cumarin compounds which have anti-hypertensive, cholesterol- and triglycerides-lowering properties. This study was undertaken to evaluate the genotoxic properties of hydroalcoholic and aqueous extracts of D. aucheri on human hepatoma cells using the comet assay method for safety evaluation.Materials and Methods:In this method, after incubation of cells with different concentrations of extracts, cell suspensions were added to pre-coated normal agarose slides. After lysis, electrophoresis and neutralization process, staining was done by ethidium bromide and comets were observed using a fluorescence microscope. Tail length, percentage of DNA in tail and tail moment parameters were measured.Results:Statistical analysis of the results demonstrated that concentrations more than 500 μg/ml of hydroalcoholic and aqueous extract of D. aucheri were genotoxic.Conclusion:It can be concluded from the results that taking the concentrations less than these dosages of extracts are safe but more studies are required to determine genotoxic mechanisms of this plant.

show abstract

High frequency of constitutive alkali-labile sites in mouse major satellite DNA, detected by DNA breakage detection-fluorescence in situ hybridization

Cited by 20 publications

References 20 publications

Influence of the SCGE protocol on the amount of basal DNA damage detected in the Mediterranean mussel, Mytilus galloprovincialis

Influence of the SCGE protocol on the amount of basal DNA damage detected in the Mediterranean mussel, Mytilus galloprovincialis

Interpreting sperm DNA damage in a diverse range of mammalian sperm by means of the two-tailed comet assay

Genotoxicity evaluation of hydroalcoholic and aqueous extracts of Dorema aucheri by the comet assay

Contact Info

Product

Resources

About