Distribution of TaqI sites along human chromosomes revealed by in situ enzyme-nick translation

We have studied the relative richness of TaqI sites along human chromosomes by means of a nonradioactive in situ enzyme-nick translation procedure. Regions with a higher content of these sequences are shown to be the noncentromeric heterochromatin blocks, whereas within euchromatin, terminal R-bands are the domains more enriched in these sites. Results obtained suggest that the method of performing enzyme digestions using time as a variable, and then in situ nick translation, provides much more complete information about the distribution of enzyme sequences along chromosomes than standard enzyme digestions.Key words: TaqI, nick translation, biotin, T-bands, heterochromatin.

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

Neuroscience Letters ◽

10.1016/0304-3940(95)12097-n ◽

1995 ◽

Vol 200 (2) ◽

pp. 129-132 ◽

Cited By ~ 35

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

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In situ nick translation of meiotic chromosomes to demonstrate homologous heterochromatin heterogeneity

Genome ◽

10.1139/g93-037 ◽

1993 ◽

Vol 36 (2) ◽

pp. 268-270 ◽

Cited By ~ 3

Author(s):

J. de la Torre ◽

C. López-Fernández ◽

P. Herrero ◽

J. Gosálvez

Keyword(s):

Tandem Duplication ◽

Dna Recombination ◽

Nick Translation ◽

Meiotic Chromosomes ◽

In Situ Nick Translation ◽

Living Species ◽

Longitudinal Differentiation ◽

Translation Procedure ◽

Restriction Endonuclease Cleavage

The in situ nick translation procedure performed on fixed meiotic chromosomes partially cleaved with restriction endonucleases shows a different staining of homologous heterochromatic regions, which could be explained through a differential restriction endonuclease cleavage. Mutations occurring before massive tandem duplication and involving those DNA motifs that produce these heterochromatic blocks, together with the absence of DNA recombination that characterizes these particular regions, could explain the observed results. This method for chromosome labelling is most useful to demonstrate a certain level of heterochromatin heterogeneity that is present in the genome of living species but remained cryptic to other techniques that are also able to induce longitudinal differentiation of the chromosomes.Key words: cytogenetics, nick translation, meiosis, heterochromatin.

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A new banding pattern of human chromosomes by in situ nick translation using ECO RI and biotin-dUTP

Clinical Genetics ◽

10.1111/j.1399-0004.1985.tb00379.x ◽

2008 ◽

Vol 28 (2) ◽

pp. 173-176 ◽

Cited By ~ 9

Author(s):

Jörn Bullerdiek ◽

Jürgen Dittmer ◽

Angelika Faehre ◽

Sabine Bartnitzke ◽

Volker Kasche ◽

...

Keyword(s):

Banding Pattern ◽

Human Chromosomes ◽

Nick Translation ◽

In Situ Nick Translation ◽

Eco Ri

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

Genome ◽

10.1139/g92-135 ◽

1992 ◽

Vol 35 (5) ◽

pp. 890-894 ◽

Cited By ~ 17

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.

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Ultrastructural banding induced by DraI or HaeIII progressive digestion and in situ nick translation on human chromosomes

Genome ◽

10.1139/g94-135 ◽

1994 ◽

Vol 37 (6) ◽

pp. 950-956

Author(s):

Rosalba Del Coco ◽

Liborio Stuppia ◽

Caterina Cinti ◽

Rita Peila ◽

Nadir Mario Maraldi

Keyword(s):

Electron Microscopy ◽

Conformational Changes ◽

Restriction Enzymes ◽

Human Chromosomes ◽

Nick Translation ◽

Metaphase Chromosomes ◽

In Situ Nick Translation ◽

Transmission Electron ◽

Dna Loss

Fixed human metaphase chromosomes were progressively digested with DraI or HaeIII restriction enzymes, submitted to in situ nick translation, and observed by transmission electron microscopy to obtain further information on the localization of the endonuclease target sequences and on the conformational changes in chromosomal bands. This approach allows us to detect specific nick translation patterns, namely, G-banding or R-like banding after short DraI and HaeIII endonuclease digestion, respectively. Intermediate banding recognizable as C-negative banding and G + C banding are induced by longer HaeIII digestion, before the C-positive banding. These patterns appear to depend both on different target sites of the employed endonucleases and on the DNA loss at different digestion times.Key words: human chromosomes, in situ nick translation, DraI banding, HaeIII banding, electron microscopy.

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

Chromosoma ◽

10.1007/bf00356024 ◽

1993 ◽

Vol 102 (2) ◽

pp. 81-87 ◽

Cited By ~ 16

Author(s):

David A. Loebel ◽

Peter G. Johnston

Keyword(s):

Nick Translation ◽

X Chromosomes ◽

In Situ Nick Translation ◽

Dnase 1

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

Journal of Histochemistry & Cytochemistry ◽

10.1177/41.7.8515045 ◽

1993 ◽

Vol 41 (7) ◽

pp. 1023-1030 ◽

Cited By ~ 188

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.

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