Patterns of DNase I sensitivity in the holocentric chromosomes of the aphid Megoura viciae

Genome ◽  
1998 ◽  
Vol 41 (2) ◽  
pp. 169-172 ◽  
Author(s):  
Gian Carlo Manicardi ◽  
Mauro Mandrioli ◽  
Davide Bizzaro ◽  
Umberto Bianchi
Keyword(s):  
Dnase I ◽  
Holocentric Chromosomes ◽  
Telomeric Region ◽  
Nick Translation ◽  
In Situ Nick Translation ◽  
Embryo Cells ◽  
Dnase I Sensitivity ◽  
Megoura Viciae ◽  
Active Genes

Using the in situ nick translation technique, we looked for the presence of DNase I sensitive sites in Megoura viciae chromosomes, to study the distribution of active or potentially active genes in aphids, a group of insects possessing holocentric chromosomes. Cytological preparations obtained by the spreading of embryo cells were treated in situ with increasing concentrations (ranging from 5 to 200 ng/mL) of DNase I. At DNase I concentrations below 50 ng/mL, only one hypersensitive site was observed, and this was located on a telomeric region of the X chromosome that contains transcriptionally active nucleolar organizing regions, as assayed by silver staining. Interestingly, at intermediate concentrations of DNase, the incorporation of biotinylated nucleotide occurred uniformly throughout all chromosomes, whereas at concentrations above 100 ng/mL, a C-like banding pattern was produced. Our data differ from results obtained with mammalian, frog, and grasshopper chromosomes, where it was found that DNase I nicking is concentrated at the distal regions of all chromosomes.Key words: aphids, holocentric chromosomes, DNase I sensitivity, nick translation.

The distribution of genes on human chromosomes as studied by in situ nick translation

Genome ◽  
1992 ◽  
Vol 35 (5) ◽  
pp. 890-894 ◽  
Author(s):  
J. de la Torre ◽  
A. T. Sumner ◽  
J. Gosalvez ◽  
L. Stuppia
Keyword(s):  
Cpg Islands ◽  
Dnase I ◽  
Human Chromosomes ◽  
Nick Translation ◽  
X Chromosomes ◽  
Dnase I Hypersensitivity ◽  
In Situ Nick Translation ◽  
Inactive X Chromosome ◽  
Active Genes

We have studied the distribution of potentially active genes on human chromosomes, using two methods: DNAse I hypersensitivity and restriction enzyme – nick translation with enzymes sensitive to methylation of CpG doublets. DNAse hypersensitivity is known to be associated with potentially active genes, and, when the reaction is detected by "in situ" nick translation, produces an R-banding pattern. Digestion of chromosomes with HpaII or CfoI, both of which should preferentially cut unmethylated sequences in the CpG islands associated with the majority of genes, also produces R-banding patterns. Deviations are attributable to overdigestion of the chromosomes, leading to extraction of DNA and loss of the specific sites that were to be detected. Contrary to the results of a number of previous workers, we have failed to demonstrate any differences between the DNAse I hypersensitivity or the degree of methylation of the active and inactive X chromosomes in metaphases from females.Key words: human chromosomes, gene distribution, DNAse I hypersensitivity, in situ nick translation, R-banding, CpG islands, DNA methylation, inactive X chromosome.

Patterns of DNase I sensitivity in the holocentric chromosomes of the aphid Megoura viciae

Genome ◽  
1998 ◽  
Vol 41 (2) ◽  
pp. 169-172 ◽  
Author(s):  
Gian Carlo Manicardi ◽  
Mauro Mandrioli ◽  
Davide Bizzaro ◽  
Umberto Bianchi
Keyword(s):  
Dnase I ◽  
Holocentric Chromosomes ◽  
Dnase I Sensitivity ◽  
Megoura Viciae

scholarly journals In situ nick translation at the electron microscopic level: a tool for studying the location of DNAse I-sensitive regions within the cell.

1991 ◽  
Vol 39 (6) ◽  
pp. 871-874 ◽  
Author(s):  
M Thiry
Keyword(s):  
Ultrastructural Level ◽  
Dnase I ◽  
Electron Microscopic Level ◽  
Nick Translation ◽  
Dna Polymerase I ◽  
Electron Microscopic ◽  
Thin Sections ◽  
In Situ Nick Translation ◽  
Polymerase I

The in situ nick translation method was adapted to the ultrastructural level, to study the location of DNAse I-sensitive sequences within the cell. Ultra-thin sections of Lowicryl-embedded cells were incubated in a medium containing DNAse I, DNA polymerase I, and all four deoxyribonucleotides, some being biotinylated. The nick-translated sites were then visualized by an indirect immunogold labeling technique. The resulting labeling pattern is closely dependent on the DNAse I concentration in the nick-translation medium. The method reveals with great precision the specific DNAse I-sensitive regions within the nucleus. This technique can be used to discriminate between active and inactive regions of interphase chromatin.

Heterochromatin heterogeneity in the holocentric X chromatin of Megoura viciae (Homoptera, Aphididae)

Genome ◽  
1996 ◽  
Vol 39 (2) ◽  
pp. 465-470 ◽  
Author(s):  
G. C. Manicardi ◽  
D. Bizzaro ◽  
E. Galli ◽  
U. Bianchi
Keyword(s):  
X Chromosome ◽  
Holocentric Chromosomes ◽  
Dna Base Composition ◽  
Banding Patterns ◽  
Dna Base ◽  
Embryo Cells ◽  
Chromatin Compactness ◽  
Megoura Viciae ◽  
Endonuclease Digestion

Holocentric chromosomes, prepared by spreading embryo cells obtained from Megoura viciae parthenogenetic females, have been C-banded, enzymatically digested in situ using the specific endonucleases DdeI (C↓TNAG), DraI (TTT↓AAA), Tru9I (TT↓AA), and CfoI (GCG↓C), and subsequently stained with Giemsa, DAPI, CMA3, and AgNO3. We observed that the X chromosome had the best defined banding patterns. In the M. viciae X chromosome there is a certain amount of heterogeneity in heterochromatic DNA composition. In fact, the GC-rich NOR-associated heterochromatin differs from other heterochromatic bands that are characterized by AT-rich DNAs. Our data also indicate that, in M. viciae holocentric chromosomes, all heterochromatic blocks are accessible to in situ enzyme attack, the only limit to the digestion being the presence or absence of recognition targets. This is an interesting point, since, in monocentric chromosomes, it is well known that in situ endonuclease digestion is heavily affected not only by DNA base composition but also by chromatin compactness that may limit enzyme accessibility to their specific targets. Key words : heterochromatin, holocentric chromosomes, aphids.

Analysis of DNase 1 sensitivity and methylation of active and inactive X chromosomes of kangaroos (Macropus robustus) by in situ nick translation

Chromosoma ◽  
1993 ◽  
Vol 102 (2) ◽  
pp. 81-87 ◽  
Author(s):  
David A. Loebel ◽  
Peter G. Johnston
Keyword(s):  
Nick Translation ◽  
X Chromosomes ◽  
In Situ Nick Translation ◽  
Dnase 1

scholarly journals Detection of DNA fragmentation in apoptosis: application of in situ nick translation to cell culture systems and tissue sections.

1993 ◽  
Vol 41 (7) ◽  
pp. 1023-1030 ◽  
Author(s):  
R Gold ◽  
M Schmied ◽  
G Rothe ◽  
H Zischler ◽  
H Breitschopf ◽  
...  
Keyword(s):  
Cell Death ◽  
Dna Fragmentation ◽  
Specific Cell ◽  
Nick Translation ◽  
Proliferating Cells ◽  
Cell Surface Antigens ◽  
Simultaneous Application ◽  
Tissue Sections ◽  
In Situ Nick Translation

Since DNA fragmentation is a key feature of programmed cell death (PCD) and also occurs in certain stages of necrosis, we have adapted the methodology of in situ nick-translation (ISNT) to detect DNA fragmentation on a single-cell level. We first established the technique for cell preparations. Apoptosis was induced by gamma-irradiation on freshly isolated rat thymocytes. After fixation procedures, ISNT was performed by overnight incubation either with fluorescein-12-dUTP or with digoxigenin-labeled 11-dUTP and DNA polymerase I. The enzymatic incorporation of labeled nucleotides at sites of DNA fragmentation was detected by flow cytometry either directly or indirectly with fluorescein-conjugated anti-digoxigenin. The quantitative results demonstrated close correlation with morphological essays for apoptosis, DNA gel electrophoresis, and ISNT. Proliferating cells determined by bromodeoxyuridine immunofluorescence were not labeled by ISNT. Immunocytochemistry for cell surface antigens in combination with ISNT allowed the identification of specific cell types undergoing PCD. Furthermore, the simultaneous application of photolabeling techniques with ethidium monoazide and ISNT led to the identification of DNA fragmentation in cells with still intact membranes. Extending ISNT to tissue sections of paraformaldehyde-fixed, paraffin-embedded material reliably revealed labeling of cells with typical morphological features of apoptosis. However, this technique was not useful in detecting early stages of necrotic cell death.

Microprocedure for in situ nick translation of chromosomes

1992 ◽  
Vol 62 (2) ◽  
pp. 150-153
Author(s):  
Li Zhengang ◽  
Howard L. Hosick ◽  
Kang Fan
Keyword(s):  
Nick Translation ◽  
In Situ Nick Translation

In situ DNAse I sensitivity assay indicates DNA conformation differences between CHO cells and the radiation-sensitive CHO mutant IRS-20

2004 ◽  
Vol 104 (1-4) ◽  
pp. 100-103 ◽  
Author(s):  
D.G. Marañon ◽  
A.O. Laudicina ◽  
M. Muhlmann
Keyword(s):  
Cho Cells ◽  
Dnase I ◽  
Dna Conformation ◽  
Dnase I Sensitivity

DNA single-strand breaks in postischemic gerbil brain detected by in situ nick translation procedure

1995 ◽  
Vol 200 (2) ◽  
pp. 129-132 ◽  
Author(s):  
Muneshige Tobita ◽  
Isao Nagano ◽  
Shozo Nakamura ◽  
Yasuto Itoyama ◽  
Kyuya Kogure
Keyword(s):  
Single Strand ◽  
Nick Translation ◽  
Strand Breaks ◽  
In Situ Nick Translation ◽  
Gerbil Brain ◽  
Single Strand Breaks ◽  
Translation Procedure

scholarly journals Gamma rays and bleomycin nick DNA and reverse the DNase I sensitivity of beta-globin gene chromatin in vivo.

1987 ◽  
Vol 7 (5) ◽  
pp. 1917-1924 ◽  
Author(s):  
B Villeponteau ◽  
H G Martinson
Keyword(s):  
Globin Gene ◽  
Dnase I ◽  
Globin Genes ◽  
Torsional Stress ◽  
Beta Globin ◽  
Cell Penetration ◽  
Dnase I Sensitivity ◽  
Chicken Erythrocytes ◽  
Active Genes

The active beta-globin genes in chicken erythrocytes, like all active genes, reside in large chromatin domains which are preferentially sensitive to digestion by DNase I. We have recently proposed that the special structure of chromatin in active domains is maintained by torsional stress in the DNA (Villeponteau et al., Cell 39:469-478, 1984). This hypothesis predicts that nicking of the DNA within any such chromosomal domain in vivo will relax the DNA and lead to loss of the special DNase I-sensitive state. Here we have tested this prediction by using gamma irradiation and bleomycin treatment to cleave DNA within intact chicken embryo erythrocytes. Both treatments cause reversal of DNase I sensitivity. Moreover, reversal occurs at approximately one nick per 150 kilobase pairs for both agents despite their entirely unrelated modes of cell penetration and DNA attack. These results suggest that the domain of DNase I sensitivity surrounding the beta-globin genes comprises 150 kilobase pairs of chromatin under torsional stress and that a single DNA nick in this region is sufficient to reverse the DNase I sensitivity throughout the entire domain.

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