A fraction of mouse sperm chromatin is organized in nucleosomal hypersensitive domains enriched in retroposon DNA

1999 ◽  
Vol 112 (20) ◽  
pp. 3537-3548 ◽  
Author(s):  
C. Pittoggi ◽  
L. Renzi ◽  
G. Zaccagnini ◽  
D. Cimini ◽  
F. Degrassi ◽  
...  
Keyword(s):  
Nuclease Activity ◽  
Sperm Chromatin ◽  
Dorsal Margin ◽  
Chromosomal Dna ◽  
Active Chromatin ◽  
Immunofluorescence Analysis ◽  
Major Satellite ◽  
Chromatin Fraction ◽  
Sperm Nuclei ◽  
Endogenous Nuclease

We have characterized a nuclease hypersensitive chromatin fraction from murine spermatozoa. Endogenous nuclease activity can be induced in mouse epididymal spermatozoa by appropriate stimuli and cause the localized degradation of chromosomal DNA. Based on these observations, we have isolated nuclease hypersensitive chromatin regions released from spermatozoa in the supernatant of pelleted sperm cells, and have cloned and characterized the DNA. Gel electrophoresis of end-labelled released DNA fragments showed a typical nucleosomal distribution. Peripherally distributed nucleohistones were visualized by immunofluorescence in sperm nuclei, and histones were identified by western blot in sperm chromatin. Moreover, the released DNA is enriched in retroposon DNA from a variety of families. FISH and immunofluorescence analysis showed that retroposon DNA and nucleohistone chromatin co-localize and are both peripherically distributed in nuclei of spermatozoa. In contrast, a major satellite DNA probe, used for control, co-localizes with highly condensed chromatin in the central region of sperm nuclei. The nuclear Ran and RCC1 proteins were also visualized in the dorsal margin of sperm nuclei, and were abundantly released with the hypersensitive chromatin fraction. Together, these results indicate that nucleohistone chromatin fraction(s) with typical features of ‘active’ chromatin are present in murine spermatozoa, are hypersensitive to nuclease cleavage, enriched in retroposon DNA and organized in nucleosomal domains. These observations suggest that nucleohistone domains identify a fraction of the sperm genome which may be functional during early embryogenesis.

The assessment of sperm DNA fragmentation in the saltwater crocodile (Crocodylus porosus)

2017 ◽  
Vol 29 (3) ◽  
pp. 630 ◽  
Author(s):  
S. D. Johnston ◽  
C. López-Fernández ◽  
F. Arroyo ◽  
J. L. Fernández ◽  
J. Gosálvez
Keyword(s):  
Dna Damage ◽  
Dna Fragmentation ◽  
Sperm Chromatin ◽  
Sperm Dna Fragmentation ◽  
Fragmented Dna ◽  
Sperm Dna ◽  
Saltwater Crocodile ◽  
Crocodylus Porosus ◽  
Dispersion Test ◽  
Sperm Nuclei

Herein we report a method of assessing DNA fragmentation in the saltwater crocodile using the sperm chromatin dispersion test (SCDt) after including frozen–thawed spermatozoa in a microgel (Halomax; Halotech DNA, Madrid, Spain). Following controlled protein depletion, which included a reducing agent, sperm nuclei with fragmented DNA showed a homogeneous and larger halo of chromatin dispersion with a corresponding reduced nucleoid core compared with sperm with non-fragmented DNA. The presence of DNA damage was confirmed directly by incorporation of modified nucleotides using in situ nick translation (ISNT) and indirectly by studying the correlation of the SCDt with the results of DNA damage visualisation using a two-tailed comet assay (r = 0.90; P = 0.037). Results of the SCDt immediately following thawing and after 5 h incubation at 37°C in order to induce a range of DNA damage revealed individual crocodile differences in both the baseline level of DNA damage and DNA longevity.

Chromatin organization in the homogeneously staining regions of a methotrexate-resistant mouse cell line: interspersion of inactive and active chromatin domains distinguished by acetylation of histone H4

1996 ◽  
Vol 109 (9) ◽  
pp. 2221-2228 ◽  
Author(s):  
L. Nicol ◽  
P. Jeppesen
Keyword(s):  
Cell Line ◽  
Dna Sequences ◽  
Satellite Dna ◽  
Mouse Cell ◽  
Histone H4 ◽  
Active Chromatin ◽  
Chromatin Domains ◽  
Resistant Mouse ◽  
Major Satellite ◽  
Drosophila Heterochromatin

We have analyzed the organization of the homogeneously staining regions (HSRs) in chromosomes from a methotrexate-resistant mouse melanoma cell line. Fluorescence in situ hybridization techniques were used to localize satellite DNA sequences and the amplified copies of the dihydrofolate reductase (DHFR) gene that confer drug-resistance, in combination with immunofluorescence using antibody probes to differentiate chromatin structure. We show that the major DNA species contained in the HSRs is mouse major satellite, confirming previous reports, and that this is interspersed with DHFR DNA in an alternating tandem array that can be resolved at the cytological level. Mouse minor satellite DNA, which is normally located at centromeres, is also distributed along the HSRs, but does not appear to interfere with centromere function. The blocks of major satellite DNA are coincident with chromatin domains that are labelled by an autoantibody that recognizes a mammalian homologue of Drosophila heterochromatin-associated protein 1, shown previously to be confined to centric heterochromatin in mouse. An antiserum that specifically recognizes acetylated histone H4, a marker for active chromatin, fails to bind to the satellite DNA domains, but labels the intervening segments containing DHFR DNA. We can find no evidence for the spreading of the inactive chromatin domains into adjacent active chromatin, even after extended passaging of cells in the absence of methotrexate selection.

scholarly journals Histone composition of a chromatin fraction containing ribosomal deoxyribonucleic acid isolated from the macronucleus of Tetrahymena pyriformis

1978 ◽  
Vol 173 (1) ◽  
pp. 155-164 ◽  
Author(s):  
R W Jones
Keyword(s):  
Ribosomal Dna ◽  
Sodium Dodecyl Sulphate ◽  
Gel Electrophoresis ◽  
Deoxyribonucleic Acid ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Tetrahymena Pyriformis ◽  
Active Chromatin ◽  
Chromatin Fraction ◽  
Inactive Chromatin

The histone compositions of a chromatin fraction containing ribosomal DNA and of the remaining macronuclear chromatin of Tetrahymena pyriformis was analysed by gel electrophoresis. These chromatin fractions were used as models for transcriptionally active and inactive chromatin respectively. The extent of histone modification, as indicated by the distribution of histone between differently charged subspecies in acid-urea gels, is not grossly different in the two chromatin fractions. However, histone H1 is present, but may be differently modified in the two chromatin fractions. The histone/DNA ratio in ribosomal chromatin, measured after sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of samples of chromatin, was found to be the same whether chromatin was extracted from growing or stationary organisms, and to be approx. 40% of this ratio in the remaining macronuclear chromatin. The implications of these results for the possible structure of transcriptionally active chromatin are discussed.

Can alcohol retain the reproductive and genetic potential of sperm nuclei? Chromosome analysis of mouse spermatozoa stored in alcohol

Zygote ◽  
1998 ◽  
Vol 6 (3) ◽  
pp. 233-238 ◽  
Author(s):  
Hiroyuki Tateno ◽  
Teruhiko Wakayama ◽  
W. Steven Ward ◽  
R. Yanagimachi
Keyword(s):  
Primary Structure ◽  
Reproductive Potential ◽  
Chromosome Analysis ◽  
Sperm Chromatin ◽  
Small Molecular Weight ◽  
Simple Method ◽  
Sperm Nuclei ◽  
High Concentrations ◽  
Dna Strand ◽  
Normal Offspring

Alcohol is known to preserve genomic DNA and the primary structure of sperm protamines. To determine whether alcohol can retain the genetic and reproductive potential of mammalian sperm nuclei, mature mouse spermatozoa were stored in 70% ethanol or propanol for up to 2 months before injection into oocytes. Live offspring were obtained after injection of spermatozoa stored in 70% ethanol for 1 day at -20 °C. About 20% of the spermatozoa stored under this condition had normal chromosomes. The remaining 80% of spermatozoa and all the spermatozoa stored in 70% ethanol for 2 months had structurally aberrant chromosomes, and none could support the development of normal embryos. High concentrations of alcohol do not alter the primary structure of either DNA or small-molecular-weight protamines. However, alcohol may modify protamine—protamine or protamine—DNA interactions in a manner that results in the induction of DNA strand breaks during sperm chromatin decondensation within the oocyte. The limited success in obtaining normal offspring with ethanol-stored spermatozoa is encouraging. It may be possible to overcome these problems and develop a simple method for preserving mammalian spermatozoa without freezing.

scholarly journals Structure of active chromatin: isolation and characterization of transcriptionally active chromatin from rat liver

1997 ◽  
Vol 322 (1) ◽  
pp. 273-279 ◽  
Author(s):  
Kulbhushan TIKOO ◽  
Sunita GUPTA ◽  
Q. Anwar HAMID ◽  
Vanya SHAH ◽  
Bishwanath CHATTERJEE ◽  
...  
Keyword(s):  
Rat Liver ◽  
Blot Hybridization ◽  
Southern Blot Hybridization ◽  
Micrococcal Nuclease ◽  
Active Chromatin ◽  
Chromatin Fraction ◽  
Isolation And Characterization ◽  
Bivalent Cations ◽  
Composite Gel ◽  
A Minor

Rat liver nuclei were isolated in low-ionic-strength buffer in the absence of bi- and multi-valent cations. Digestion of these nuclei by endogenous nuclease, micrococcal nuclease and DNase I revealed that a minor chromatin fraction was preferentially digested into poly- and oligo-nucleosomes. Southern blot hybridization with various active gene probes confirmed that these chromatin fragments represent coding and 5ƀ upstream regions of transcriptionally active chromatin. Active chromatin fragments were released selectively into the medium, with inactive chromatin remaining inside the nuclei, under the above ionic conditions. The inclusion of bivalent cations during the digestion of nuclei reversed the solubility behaviour of active chromatin. Rearrangement and exchange of histone H1 between chromatin fragments was prevented by using low-salt conditions in all steps in the absence of bivalent cations. All histones, including H1, were present in stoichiometric amounts in this active chromatin fraction. Active nucleosomes showed a lower electrophoretic mobility than bulk nucleosomes in an acrylamide/agarose composite gel in the absence of Mg2+, but were selectively bound to the gel in the presence of this ion.

scholarly journals Purification and Characterization of aBacillus subtilis168 Nuclease, YokF, Involved in Chromosomal DNA Degradation and Cell Death Caused by Thermal Shock Treatments

2001 ◽  
Vol 276 (50) ◽  
pp. 47046-47051 ◽  
Author(s):  
Jin J. Sakamoto ◽  
Miho Sasaki ◽  
Tetsuaki Tsuchido
Keyword(s):  
Thermal Shock ◽  
Nuclease Activity ◽  
Dna Degradation ◽  
Wild Type ◽  
Chromosomal Dna ◽  
Purification And Characterization ◽  
Membrane Perturbation

We purified and characterized a 39-kDaBacillus subtilis168 nuclease that has been suggested in this laboratory to be involved in chromosomal DNA degradation induced by lethal heat and cold shock treatmentsin vivo. The nuclease activity was inhibitedin vitroby aurintricalboxylic acid but not by Zn2+. By the mutant analysis, we identified the 39-kDa nuclease as a product ofyokFgene. TheyokFgene contained a putative lipoprotein signal peptide motif. Afterin vivoexposure to lethal heat and cold stresses, the chromosomal DNA fragmentation was reduced in theyokFmutant, which demonstrated about a 2–10-fold higher survival rate than the wild type. TheyokFmutant was found to be more sensitive to mitomycin C than the wild type. The transformation efficiency of theyokFmutant was about 10 times higher than that of the wild type. It is suggested that whenB. subtiliscells are exposed to a stressful thermal shock resulting in membrane perturbation, YokF nuclease consequently dislocates into the cytoplasm and then attacks DNA.

Electron Microscopy of EDTA-Treated Ribosomal Subunits from Escherichia coli

1971 ◽  
Vol 29 ◽  
pp. 432-433
Author(s):  
John H. Nisbet ◽  
Henry S. Slayter
Keyword(s):  
Escherichia Coli ◽  
Ribosomal Rna ◽  
Gradient Analysis ◽  
Ribosomal Subunit ◽  
Nuclease Activity ◽  
Final Concentration ◽  
Ribosomal Subunits ◽  
Magnesium Removal ◽  
Low Ionic Strength ◽  
Endogenous Nuclease

Several studies have indicated that treatment of ribosomes and ribosomal subunits with EDTA at low ionic strength results in the formation of slower-sedimenting species which have not lost any significant amount of protein (see, for example, reference 1). Here we attempt to follow morphologically the process of magnesium removal from the 50S ribosomal subunit of Escherichia coli, using strain Q13 which exhibits a low RNase I activity. 50S particles, initially in 0.01M Tris, 0.0001M MgCl2, pH 7.4, were treated with EDTA either by dialysis against 0.002M Tris, 0.001M EDTA, pH 7.4, or by direct addition of a 0.1M solution of EDTA to a final concentration of 0.001M. On sucrose gradients made up in the Tris-EDTA buffer, the particles sedimented as a sharp peak in the 10-12S region, with no detectable slower - sedimenting species. Sucrose gradient analysis of the ribosomal RNA following EDTA treatment of 50S subunits showed that 23S RNA were largely converted to 16S, reflecting a low level of endogenous nuclease activity in the subunit preparation.

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