Nick Translation Research Articles

Structure of human sperm DNA and background damage, analysed by in situ enzymatic treatment and digital image analysis.

DNA breakage detection-fluorescence in situ hybridization (DBD-FISH) is a procedure to detect and quantify DNA breaks in situ, on a cell-by-cell basis. A comparison between sperm nuclei versus peripheral blood leukocytes using this method demonstrated that the nucleoids from mature human sperm are 12.7 times more sensitive to alkaline denaturation than those from human peripheral blood leukocytes. To investigate the origin of this alkali sensitivity, different approaches were employed. First, free 3'-OH ends of background DNA breaks were labelled by Klenow polymerase, or by DNA polymerase I following the in situ nick translation assay. Second, the presence of abasic sites, the other recognized DNA lesions that lends to constitutive alkali sensitivity, and DNA breaks with blocked 3' ends, were determined by in situ exonuclease III digestion prior to the polymerase labelling. The results demonstrated that the sperm nucleoid contains approximately 2.5-fold higher density of background DNA breaks with 3'-OH ends, and also approximately 2.8-fold higher density of basal abasic sites and DNA breaks with blocked 3' termini, than leukocytes. These differences only partially explain the significant alkali sensitivity of sperm DNA. However, in situ digestion with mung bean nuclease before DNA break labelling showed that sperm DNA is 9-fold more enriched in segments of ssDNA than DNA from leukocytes. The high frequency of partially denatured regions may result from a greater torsional stress of DNA loops in sperm chromatin due to its higher degree of compaction. Moreover, these short unpaired ssDNA stretches should be included in the category of alkali-labile sites detected by all techniques that measure DNA breaks through an alkaline unwinding step. These results provide new insights into the nature of DNA packaging in sperm nuclei.

PHOTOPROBER® Biotin: An Alternative Method for Labeling Archival DNA for Comparative Genomic Hybridization

Comparative genomic hybridization (CGH) represents a powerful method for screening the entire genome of solid tumors for chromosomal imbalances. Particularly it enabled the molecular cytogenetic analysis of archival, formalin‐fixed, paraffin‐embedded (FFPE) tissue. A well‐known dilemma, however, is the poor DNA quality of this material with fragment sizes below 1000 bp. Nick translation, the conventionally used enzymatic DNA labeling method in CGH, leads to even shorter fragments often below a critical limit for successful analysis. In this study we report the alternative application of non‐enzymatic, PHOTOPROBE® biotin labeling for conjugation of the hapten to the DNA prior to in situ hybridization and fluorescence detection. We analyzed 51 FFPE tumor samples mainly from the upper respiratory tract by both labeling methods. In 19 cases, both approaches were successful. The comparison of hybridized metaphases showed a distinct higher fluorescence signal of the PHOTOPROBE® samples sometimes with a discrete cytoplasm background which however did not interfere with specificity and sensitivity of the detected chromosomal imbalances. For further 32 cases characterized by an average DNA fragment size below 1000 bp, PHOTOPROBE® biotin was the only successful labeling technique thus offering a new option for CGH analysis of highly degraded DNA from archival material.

Transcriptional repression mechanisms of nucleolus organizer regions (NORs) in humans and chimpanzees.

Polymorphisms related to transcriptional inactivation of nucleolus organizer regions (NORs) have long been described in many animals, particularly humans. However, the precise aetiology of such variations is not always clear. We conducted analyses to investigate the repression mechanisms in humans and chimpanzees using FISH (fluorescence in situ hybridisation) with 18S rDNA, Ag-NOR (silver nitrate) staining, C-banding, and the in situ nick translation technique with the HpaII restriction enzyme. Examination of 48 humans and 46 chimpanzees suggested that there are at least three different mechanisms that produce inactivation of NORs. These include: (1) elimination of rDNA; (2) DNA methylation: (3) gene silencing due to position effects induced by heterochromatin (C-bands) and/or telomeres.

Neuronal apoptosis following human brain injury

Neuronal apoptosis has been investigated in paraffin-embedded brain tissue from 103 individuals who had sustained blunt head injury by use of the in situ nick translation (ISNT) technique. In order to provide reliable data for a forensic wound age estimation, a quantitative morphometric analysis was performed. Apoptotic neuronal cells could be detected in a cortical contusion with a wound age of 45 min at the earliest and in the majority of the cases with postinfliction intervals up to 2 weeks, numerous ISNT-positive cells were found adjacent to the traumatically injured area. The presented data indicate that neuronal apoptosis peaks at about 1 day and persists for at least 22 weeks after blunt head injury. The time-dependent occurrence of apoptotic cells can contribute to a forensic timing of cortical contusions and complements other immunohistochemical parameters, especially in the early postinfliction interval.

Vitamin E affects cell death in adult rat dentate gyrus.

We have previously reported the presence of dying cells in the granule cell layer (GCL) of adult rat dentate gyrus (DG), where neurogenesis occurs. In particular, we found that cell death in the GCL increased in vitamin E deficiency and decreased in vitamin E supplementation. These findings were regarded as related to changes in neurogenesis rate, which in turn was influenced by vitamin E availability; a neuroprotective effect of vitamin E on cell death was also proposed. In order to verify this latter hypothesis, we have studied cell death in all layers of DG in vitamin E-deficient and vitamin E-supplemented rats and in control rats at different ages, using TUNEL and nick translation techniques. The phenotype of TUNEL-positive cells was characterized and the existence of dying BrdU-positive cells was investigated. Dying cells with neuronal phenotype were observed throughout the DG in all experimental groups. The number of TUNEL-positive cells decreased from juvenile to adult age. A higher number of TUNEL-positive cells in vitamin E-deficient rats and a lower number in vitamin E-supplemented rats, with respect to age-matched controls, were found; moreover, in these groups, TUNEL-positive cells had a different percentage distribution in the different layers of the DG. Our results confirm the occurrence of cell death in DG, demonstrate that cell death affects neuronal cells and support the hypothesis that the effect of vitamin E on cell death is not related to neurogenesis.

Fluorescence in situ hybridization in diluted and flow cytometrically sorted boar spermatozoa using specific DNA direct probes labelled by nick translation.

Successful evaluation of X- and Y-chromosome-bearing sperm separation technology using flow cytometry-cell sorter is of great importance. Fluorescence in situ hybridization (FISH), which allows for the detection of specific nucleic acid sequences on morphologically preserved spermatozoa, is an ideal method for quantitatively and qualitatively assessing the purity of sorted sperm samples. In this study specific pig DNA direct probes for small regions of chromosomes 1 and Y were used. Chromosome 1 was labelled in green and used as internal control to detect a lack of hybridization, whereas chromosome Y was labelled in red. Nick translation was used as the labelling method for the preparation of these probes. Spermatozoa, unsorted and sorted for high and low Y-chromosome purity from ejaculates of five boars, were fixed on slides and two-colour direct FISH was performed for chromosomes 1 and Y. About 500 non-sorted and 200 sorted spermatozoa per sample were scored. The proportion of Y-chromosome-bearing spermatozoa was determined by the presence of a red fluorescent signal on the sperm head and the proportion of X-chromosome-bearing spermatozoa was determined by subtraction. The efficiency of the hybridization procedure was established as near 98% on sorted and unsorted samples. The results of this study confirm that direct FISH using specific pig DNA probes labelled by nick translation provides a useful tool for laboratory validation of sperm separation by flow sorting technology. Moreover, the ease of nick translation and the quality of the fluorescent signal obtained using this method makes this procedure the most appropriate method for labelling pig DNA probes to be used for direct FISH on pig spermatozoa.

Fluorescence in situ hybridization in diluted and flow cytometrically sorted boar spermatozoa using specific DNA direct probes labelled by nick translation

Successful evaluation of X- and Y-chromosome-bearing sperm separation technology using flow cytometry-cell sorter is of great importance. Fluorescence in situ hybridization (FISH), which allows for the detection of specific nucleic acid sequences on morphologically preserved spermatozoa, is an ideal method for quantitatively and qualitatively assessing the purity of sorted sperm samples. In this study specific pig DNA direct probes for small regions of chromosomes 1 and Y were used. Chromosome 1 was labelled in green and used as internal control to detect a lack of hybridization, whereas chromosome Y was labelled in red. Nick translation was used as the labelling method for the preparation of these probes. Spermatozoa, unsorted and sorted for high and low Y-chromosome purity from ejaculates of five boars, were fixed on slides and two-colour direct FISH was performed for chromosomes 1 and Y. About 500 non-sorted and 200 sorted spermatozoa per sample were scored. The proportion of Y-chromosome-bearing spermatozoa was determined by the presence of a red fluorescent signal on the sperm head and the proportion of X-chromosome-bearing spermatozoa was determined by subtraction. The efficiency of the hybridization procedure was established as near 98% on sorted and unsorted samples. The results of this study confirm that direct FISH using specific pig DNA probes labelled by nick translation provides a useful tool for laboratory validation of sperm separation by flow sorting technology. Moreover, the ease of nick translation and the quality of the fluorescent signal obtained using this method makes this procedure the most appropriate method for labelling pig DNA probes to be used for direct FISH on pig spermatozoa.

Residualizing Indium-111-Radiolabel for Plasmid DNA and Its Application to Tissue Distribution Study

To develop a suitable vector and an administration technique for in vivo gene transfer, the tissue distribution of plasmid DNA (pDNA) needs to be understood. In this study, a novel residualizing radiolabel for pDNA was developed. 4-[p-Azidosalicylamido]butylamine (ASBA) was coupled with diethylenetriaminepentaacetic acid (DTPA) anhydride, then the conjugate was reacted with pDNA by photoactivation, followed by labeling with [(111)In]InCl(3) to obtain (111)In-pDNA. The overall structure of pDNA was well preserved, and the retention of its transcriptional activity was 40-98%. After intravenous injection of (111)In-pDNA into mice, about 50% of the radioactivity was recovered in the liver within 3 min. The level remained stable for at least 2 h, followed by a very slow decrease to 45% at 24 h. This contrasted with the results obtained with (32)P-pDNA by nick translation, in which a rapid decrease in hepatic radioactivity was observed. The amount of radioactivity in the lung following the administration of polyethyleneimine/(111)In-pDNA complexes correlates well with the transgene expression. These results indicate that the novel residualizing radiolabel clearly demonstrates the cells that have taken up pDNA and, therefore, gives us useful information about how to design a better approach for nonviral in vivo gene delivery.

HU Protein of Escherichia coli Has a Role in the Repair of Closely Opposed Lesions in DNA

Closely opposed lesions form a unique class of DNA damage that is generated by ionizing radiation. Improper repair of closely opposed lesions could lead to the formation of double strand breaks that can result in increased lethality and mutagenesis. In vitro processing of closely opposed lesions was studied using double-stranded DNA containing a nick in close proximity opposite to a dihydrouracil. In this study we showed that HU protein, an Escherichia coli DNA-binding protein, has a role in the repair of closely opposed lesions. The repair of dihydrouracil is initiated by E. coli endonuclease III and processed via the base excision repair pathway. HU protein was shown to inhibit the rate of removal of dihydrouracil by endonuclease III only when the DNA substrate contained a nick in close proximity opposite to the dihydrouracil. In contrast, HU protein did not inhibit the subsequent steps of the base excision repair pathway, namely the DNA synthesis and ligation reactions catalyzed by E. coli DNA polymerase and E. coli DNA ligase, respectively. The nick-dependent selective inhibition of endonuclease III activity by HU protein suggests that HU could play a role in reducing the formation of double strand breaks in E. coli.

Expression of Tumor-Associated Membrane Antigen, RCAS1, in Human Colorectal Carcinomas and Possible Role in Apoptosis of Tumor-Infiltrating Lymphocytes

RCAS1, a novel tumor-associated antigen, is expressed in advanced human neoplasias including uterine and ovarian carcinomas. RCAS1 protein was indicated to induce cell cycle arrest and apoptosis of cultured human lymphoid and myeloid cell lines and normal lymphocytes. In the present study, we investigated the expression and prognostic value of RCAS1 in 58 patients with colorectal carcinomas. RCAS1 protein was detected by immunoperoxidase staining using a mouse monoclonal anti-RCAS1 antibody (22-1-1 antibody). Immunohistochemical examination showed expression of RCAS1 in 75% of colorectal carcinomas with lymph node metastases (n = 24), whereas it was present in only 41% of tumors without metastases (n = 34, P < .05). Patients with RCAS1-positive tumors showed a significantly poorer prognosis than those negative for RCAS1 (P < .05). Multivariate analysis using the Cox regression model indicated that RCAS1 positivity was an independent negative predictor for survival (P = .0300; risk ratio, 0.496). In addition, apoptotic cells of tumor-infiltrating lymphocytes were examined using nonradioactive in situ nick translation in paraffin-embedded sections. The proportion of apoptotic tumor-infiltrating lymphocytes was significantly higher in RCAS1-positive colorectal carcinomas (11.2 ± 1.0) than in RCAS1-negative tumors (7.9 ± 1.0, P < .05). Our results suggest that overexpression of RCAS1 may negatively affect the prognosis of human colorectal carcinomas and that RCAS1 may play a role in tumor immune privilege in vivo.

Okazaki Fragment Maturation in Yeast: I. DISTRIBUTION OF FUNCTIONS BETWEEN FEN1 AND DNA2

In the presence of proliferating cell nuclear antigen, yeast DNA polymerase delta (Pol delta) replicated DNA at a rate of 40-60 nt/s. When downstream double-stranded DNA was encountered, Pol delta paused, but most replication complexes proceeded to carry out strand-displacement synthesis at a rate of 1.5 nt/s. In the presence of the flap endonuclease FEN1 (Rad27), the complex carried out nick translation (1.7 nt/s). The Dna2 nuclease/helicase alone did not efficiently promote nick translation, nor did it affect nick translation with FEN1. Maturation in the presence of DNA ligase was studied with various downstream primers. Downstream DNA primers, RNA primers, and small 5'-flaps were efficiently matured by Pol delta and FEN1, and Dna2 did not stimulate maturation. However, maturation of long 5'-flaps to which replication protein A can bind required both DNA2 and FEN1. The maturation kinetics were optimal with a slight molar excess over DNA of Pol delta, FEN1, and proliferating cell nuclear antigen. A large molar excess of DNA ligase substantially enhanced the rate of maturation and shortened the nick-translation patch (nucleotides excised past the RNA/DNA junction before ligation) to 4-6 nt from 8-12 nt with equimolar ligase. These results suggest that FEN1, but not DNA ligase, is a stable component of the maturation complex.

Okazaki Fragment Maturation in Yeast: II. COOPERATION BETWEEN THE POLYMERASE AND 3′–5′-EXONUCLEASE ACTIVITIES OF POL δ IN THE CREATION OF A LIGATABLE NICK

To address the different functions of Pol delta and FEN1 (Rad27) in Okazaki fragment maturation, exonuclease-deficient polymerase Pol delta-01 and Pol delta-5DV (corresponding to alleles pol3-01-(D321A, E323A) and pol3-5DV-(D520V), respectively) were purified and characterized in this process. In the presence of the replication clamp PCNA, both wild-type and exo(-) Pol delta carried out strand displacement synthesis with similar rates; however, initiation of strand displacement synthesis was much more efficient with Pol delta-exo(-). When Pol delta-exo(-) encountered a downstream primer, it paused with 3-5 nucleotides of the primer displaced, whereas the wild type carried out precise gap filling. Consequently, in the absence of FEN1, Pol delta exonuclease activity was essential for closure of simple gaps by DNA ligase. Compared with wild type, Okazaki fragment maturation with Pol delta-exo(-) proceeded with an increased duration of nick translation prior to ligation. Maturation was efficient in the absence of Dna2 and required Dna2 only when FEN1 activity was compromised. In agreement with these results, the proposed generation of double strand breaks in pol3-exo(-) rad27 mutants was suppressed by the overexpression of DNA2. Further genetic studies showed that pol3-exo(-) rad27 double mutants were sensitive to alkylation damage consistent with an in vivo defect in gap filling by exonuclease-deficient Pol delta.

Pitfalls and Caveats in Histochemically Demonstrating Apoptosis

For investigating the biological significance of apoptosis, the exact and sensitive histochemical identification of apoptosis and apoptotic cells is essential. However, we need to recognize both the pitfalls and caveats in performing histochemical staining and in the findings obtained. DNA fragmentation-based approaches, such as TdT- mediated dUTP-biotin nick end-labeling (TUNEL), in situ nick translation (ISNT) and immunostaining for single-stranded DNA, represent DNA alterations in the apoptotic cell, but they are technically unstable and occasionally give false- positive and false negative findings. In contrast, immunostaining for intracellular proteins cleaved and activated by caspases, including cleaved caspase 3, cleaved poly(ADP-ribose) polymerase, cleaved cytokeratin 18 and cleaved actin interpreting (fractin), is technically reproducible, but the intracellular accumulation of the activated proteins is not necessarily sychronized. The present review focuses on the pretreatments for enhancing the nsensitivity of these techniques, as well as their limitations and comparisons inhistochemically demonstrating apopto sis and apoptotic cells.

Produção de sonda de DNA diretamente biotinilada com N-hidroxisuccinimida biotina ester (BNHS).

&lt;span class="texto"&gt;A utilização de sondas de DNA para análise genômica em fase sólida tem se expandido e ultrapassado as áreas da pesquisa, estendendo-se ao terreno do diagnóstico de rotina nas áreas de medicina humana e animal, bem como nas ciências forenses. Estas sondas têm sido utilizadas desde o surgimento da primeira técnica de hibridização descrita com o southern blotting, que envolve a imobilização, em filtros de papel, de fragmentos de DNA separados eletroforeticamente. Outra técnica descrita como o dot-hibridização é menos complexa do que o southern convencional por não requerer o passo prévio de eletroforese e por ser menos exigente quanto à qualidade do DNA, já que dispensa a digestão com enzima de restrição. A marcação mais comum para estas sondas é o fósforo radioisótopo (32P), que, apesar de sua alta sensibilidade, apresenta os inconvenientes de risco físico, necessidade de autorização para manipulação de radioisótopos, alto custo e uma vida média muito curta. Uma série de alternativas para o 32P têm sido utilizadas, com marcas diretas ou secundárias, entre elas a biotina, que tem mostrado grandes vantagens e tem sido usada também em laboratórios especializados e bem equipados. Este trabalho descreve uma modificação do método clássico descrito para sondas de DNA acopladas por biotina hidrazida. Utilizamos a N-hidroxisuccinimida biotina (BNHS) em dimetilformamida, para marcar resíduos de citosina disponíveis em segmentos de fita simples de DNA extraído de Corynebacterium pseudotuberculosis. A variação da proporção de biotina e de DNA, bem como sua especificidade e sensibilidade, foram cuidadosamente testadas e criteriosamente analisadas através de ensaios de hibridização, adsorvendo-se DNA cromossômico do C. pseudotuberculosis e outros DNAs em papel de nitrocelulose, seguindo-se a incubação com as sondas biotiniladas descritas,avidina-peroxidase e revelação com 4-cloro-1-naftol. Os resultados mostraram que a sonda de DNA produzida tem as características favoráveis esperadas. Desde que não é necessário o nick translation nem a utilização de material radioativo ou mesmo de hidrazida, sondas deste tipo podem ser utilizadas no auxílio do diagnóstico de infecções por C. pseudotuberculosis e outros microorganismos.&lt;/span&gt;

IDENTIFICATION OF PARENTAL CHROMOSOMES AND DETECTION OF RIBOSOMAL DNA SEQUENCES IN INTERSPECIFIC HYBRIDS OF LILIUM REVEALED BY MULTICOLOR IN SITU HYBRIDIZATION

IDENTIFICATION OF PARENTAL CHROMOSOMES AND DETECTION OF RIBOSOMAL DNA SEQUENCES IN INTERSPECIFIC HYBRIDS OF LILIUM REVEALED BY MULTICOLOR IN SITU HYBRIDIZATION

In situ fluorescent nick translation procedure for plant chromosomes

Pectinase and cellulase, which are used to macerate plant material, always show traces of DNase activities that result in DNA nicking. Moreover, the DNA polymerase I usually applied in the in situ nick translation techniques shows both 5′ to 3′ and 3′ to 5′ exonuclease activities. As a result, significant nonspecific labeling appears in control preparations that are not digested by a restriction endonuclease. Our procedure includes blocking nonspecific nick labeling before incubation with restriction enzymes (HpaII and HaeIII). This is achieved by incorporation of ddGTP into DNA by the Taq polymerase which lacks 3′ to 5′ exonuclease activity. This method gives satisfactory results because it eliminates nonspecific nick translation signals that are present after applying the methods described for animal material.

Internal labeling of DNA probes.

AbstractOne of the most common precursors to undertaking a protocol for mutation detection is the production of a suitably labeled DNA probe (1). Labeled nucleotides (radioactive or fluorescent) can be incorporated efficiently into doublestranded DNA by a number of methods. One of the most common is by a process termed nick translation. Nick translation works by using DNase and DNA polymerase I enzymes. DNase cuts one strand of the DNA, exposing 5′-phosphoryl and 3′-hydroxyl (OH) termini. Polymerase I adds dNTPs, including labeled dNTPs to the exposed 3′-OH strand, and at the same time, the polymerase exonuclease activity digests from the exposed 5′ end. In this way, a new complementary strand, including labeled dNTPs, is produced (2). It is also possible to incorporate radioactive nucleotides into a DNA using a enzymatic primer extension technique, usually termed random primer labeling (3). In this method, random hexanucleotides are annealed to denatured DNA to be used as the probe. These are used as a primer for enzymatic extension in the presence of the four deoxyribonucleotides, one of which is radiolabeled. Alternative probes may be prepared where the label occurs on one of the termini of the DNA, either the 3′ or the 5′ end. The protocol for this type of labeling is found in Chapter 3.KeywordsAmersham Pharmacia BiotechNick TranslationRandom Primer LabelAlternative ProbeRandom HexanucleotidesThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Construction of a cDNA fragment library from SH-SY5Y cells using restriction display PCR

A complementary DNA (cDNA) fragment library from SH-SY5Y cells is constructed using a restriction display polymerase chain reaction (RD-PCR) technique. Messenger RNA (mRNA) is extracted from SH-SY5Y cells and single-strand cDNA synthesised using an anchored oligo primer (dT18). The second strand is produced by nick translation. The double strands are cleaved with the restriction enzyme Sau3AI and the fragments ligated with universal linker. The products are amplified with universal primers and selected primers, ligated into the pMDI8-T vector, and then sequenced. The library constructed contained 136 subgroups, each comprising seven to 12 cDNA fragments. RD-PCR proved a simple, effective way to construct a cDNA library, and this will contribute to the investigation of gene expression in the neuron in future microarray studies.

A modified nick translation method used with FISH that produces reliable results with archival tissue sections.

Nick translation is used to label DNA and RNA to produce probes for in situ hybridization and Northern and Southern blotting. Fluorescence in situ hybridization (FISH) is a widely applied technique used to determine chromosomal and genetic anomalies in many biological samples. Initially the technique was applied to metaphase preparations, but the usefulness of detecting genetic anomalies in solid tumors in situ has resulted in the development of modified protocols. Formalin fixed paraffin processed tissue sections present novel challenges when applying FISH; the probes must be small (between 200 and 600 base pairs) and pretreatment is necessary before the probes can be applied to tissue sections, to promote probe access to target DNA. Here we report on a modification of a nick translation method to produce a probe that can reliably be used with FISH in paraffin processed tissue sections.

In situ nick translation at the electron microscopic level.

The nick translation procedure has been introduced in 1977 by Righy et al. (). It is commonly used to label purified DNA in vitro. This technique relies on combined 5′→3′ polymerase and 5′→3′ exonuclease activities of Escherichia coli DNA polymerase I. If a nick or single strand break with a 3′ hydroxyl terminus is present in a duplex DNA molecule, the enzyme will translocate it, removing nucleotides ahead of it by 5′→3′ exonuclease activity and synthesizing the new DNA strand behind it using 5′→3′ polymerase activity (). When labeled deoxyribonucleotides are used as substrates, the original unlabeled nucleotides in the DNA template are replaced by the labeled ones.