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R Drouin

Publications and source records attributed to R Drouin.

At least 55 records · Page 3Linked to original sources
PubMed1995

Mapping of copper/hydrogen peroxide-induced DNA damage at nucleotide resolution in human genomic DNA by ligation-mediated polymerase chain reaction.

The ligation-mediated polymerase chain reaction was used to map the frequency of reactive oxygen species-induced DNA damage at nucleotide resolution in genomic DNA purified from cultured human male fibroblasts. Damaged pyrimidine and purine bases were recognized and cleaved by the Nth and Fpg proteins from Escherichia coli, respectively. Strand breaks and modified bases were induced in vitro by copper ion-mediated reduction of hydrogen peroxide in the presence of ascorbate; reactant concentrations were adjusted to induce lesions at a frequency of 1 per 2-3 kilobases in purified genomic DNA. Glyoxal gel analysis demonstrated that the ratio of induced strand breaks to induced base damage was 0.8/2.7 in DNA dialyzed extensively to remove adventitious transition metal ions. Ligation-mediated polymerase chain reaction analysis of the damage frequency in the promoter region of the transcriptionally active phosphoglycerate kinase (PGK 1) gene revealed that (Cu(II)/ascorbate/H2O2 caused DNA base damage by a sequence-dependent mechanism, with the 5' bases of d(pGn) and d(pCn) being damage hot spots, as were the most internal guanines of d(pGGGCCC) and d(pCCCGGG). Since base damage occurs after formation of a DNA-Cu(I)-H2O2 complex, these data suggest that the local DNA sequence affects formation of DNA-Cu(I)-H2O2 complexes and/or the efficiency of base oxidation during resolution of this complex.

Base Sequence↗
PubMed1994

DNA repair rates mapped along the human PGK1 gene at nucleotide resolution.

The repair of cyclobutane pyrimidine dimers (CPDs), DNA lesions induced by ultraviolet light, was studied at nucleotide resolution. Human fibroblasts were irradiated with ultraviolet light and allowed to repair. The DNA was enzymatically cleaved at the CPDs, and the induced breaks along the promoter and exon 1 of the PGK1 gene were mapped by ligation-mediated polymerase chain reaction. Repair rates within the nontranscribed strand varied as much as 15-fold, depending on nucleotide position. Preferential repair of the transcribed strand began just downstream of the transcription start site but was most pronounced beginning at nucleotide +140 in exon 1. The promoter contained two slowly repaired regions that coincided with two transcription factor binding sites.

Binding Sites↗
PubMed1993

Detection of DNA adducts at the DNA sequence level by ligation-mediated PCR.

Many carcinogens and mutagens interact with DNA to form specific adducts. Base-specificity and sequence-specificity of adduct formation has been analyzed previously with cloned, end-labelled DNA fragments. However, the distribution of adducts along a mammalian chromosome may be modulated by chromatin structure and could be different from that in naked plasmid DNA. Recently, a method has been developed that utilizes the sensitivity of the polymerase chain reaction (PCR) to detect DNA adducts at the DNA sequence level in mammalian cells. The sequence position of adducts can be mapped whenever it is possible to convert the adduct, either chemically or enzymatically, into a DNA strand break with a 5'-phosphate group. Fragments containing these ligatable breaks are amplified in a single-sided, ligation-mediated PCR reaction. We have used ligation-mediated PCR for detection of alkylguanine adducts and UV-induced cyclobutane pyrimidine dimers and (6-4) photoproducts. We discuss the sensitivity of the method, its limitations, and its potential for mapping other DNA adducts at the DNA sequence level in mammalian cells.

Animals↗
PubMed1992

Binding of transcription factors creates hot spots for UV photoproducts in vivo.

Cyclobutane dipyrimidines and less than mean value of 6-4 dipyrimidines are the two major classes of mutagenic DNA photoproducts produced by UV irradiation of cells. We developed a method to map cyclobutane dipyrimidines at the DNA sequence level in mammalian cells. The frequency of this class of photoproducts was determined at every dipyrimidine along the human phosphoglycerate kinase-1 (PGK1) promoter sequence and was compared to the UV-induced frequency distribution of mean value of 6-4 dipyrimidines. After irradiation of living cells containing active or inactive PGK1 genes, enzymatic or chemical cleavage at UV photoproducts, and amplification by ligation-mediated polymerase chain reaction, photofootprints were seen in all regions which bind transcription factors and appear as DNase I footprints. Photoproduct frequency within transcription factor binding sites was suppressed or enhanced relative to inactive genes or naked DNA with enhancements of up to 30-fold. Since photoproducts are mutagenic, this indicates that photoproduct (mutation?) hot spots may be tissue specific in mammals.

Animals↗
PubMed1991

Simultaneous visualization of chromosome bands and hybridization signal using colloidal-gold labeling in electron microscopy.

Electron microscopy (EM) is seldom used with in situ hybridization to localize DNA sequences because banding methods for chromosome identification could not be coupled to EM techniques. We have applied an immunochemical replication-banding method specific for EM to solve this problem. A thymidine synchronization/BrdUrd release protocol allows BrdUrd incorporation only into late replicating bands. A biotinylated DNA probe is hybridized in situ to its complementary sequence. The biotinylated probe and the BrdUrd-substituted DNA are simultaneously localized by different reporter/detection systems using different-sized colloidal gold particles as electron-dense tags. We demonstrate the high precision of this mapping procedure by localizing on long prophase chromosomes (greater than 1000 bands per haploid set) the pXBR-1 sequence to a small subregion of the centromeric subband Xp11.1-Xq11.1. This localization to a part of an individual prophase subband is the most precise localization ever reported on human banded mitotic chromosomes.

Cells, Cultured↗
PubMed1991

In vivo mapping of a DNA adduct at nucleotide resolution: detection of pyrimidine (6-4) pyrimidone photoproducts by ligation-mediated polymerase chain reaction.

DNA adducts in unique sequences along the mammalian genome are mapped in vivo at single-nucleotide resolution. Pyrimidine (6-4) pyrimidone photoproducts [(6-4) photoproducts] represent one of the two major adduct classes found after UV irradiation of DNA and were shown to play an important role in UV-induced mutagenesis. After UV light treatment of cells, DNA is prepared and chemically cleaved at (6-4) photoproducts with piperidine. Gene-specific fragments are then amplified from total genomic DNA by use of a ligation-mediated polymerase chain reaction. Analysis of the human chromosome X-linked phosphoglycerate kinase (PGK1) gene's promoter has shown that the frequency of (6-4) photoproducts expressed as piperidine-labile sites is (i) high at TpC and CpC dinucleotides, (ii) dependent on the nearest-neighbor bases, (iii) inhibited by the binding of a transcription factor, and (iv) different for DNA derived from the active and inactive X chromosome. This latter difference is mainly a consequence of the presence of 5-methylcytosine (m5C) in CpG dinucleotides on the inactive X chromosome. 5-Methylcytosine in the sequences Tm5CG and Cm5CG inhibits the formation of (6-4) photoproducts. Thus, in addition to in vivo mapping of a DNA adduct at nucleotide resolution, we also report another method for methylation analysis and photofootprinting.

Animals↗
PubMed1991

High-resolution R-banding at the 1250-band level. III. Comparative analysis of morphologic and dynamic R-band patterns (RHG and RBG).

High-resolution human chromosomes were obtained from lymphocytes after thymidine synchronization. The block was released either with thymidine to produce GTG (G-bands by trypsin using Giemsa) and RHG (R-bands by heating using Giemsa) banding or with BrdU (5-bromo-2'-deoxyuridine) for RBG (R-bands by BrdU using Giemsa) banding. RHG and RBG band patterns are only 75 to 85% congruent. The dissimilarities increase with the band number per genome and vary from one chromosome region to another. After high-resolution RBG banding, the BrdU-substituted bands show an unequal condensation delay, which can be, according to the bands involved, very important, minimal, or even absent. The bands showing the highest degree of condensation delay are the bands replicating the latest. The GTG- and RHG-band patterns show complementary matching for about 90% of the bands. It was found that two third of the chromosome surface appears positively stained after R-banding. This suggests that more DNA is replicated during early S-phase than during late S-phase. To obtain a fully developed RBG-band pattern in 90 to 95% of harvested mitoses, a period of 4.5 hours after the removal of the blocking agent is optimal. Such a brief release period also implies that late S-phase is much shorter than early S-phase.

Adult↗
PubMed1991

Chromosome condensation from prophase to late metaphase: relationship to chromosome bands and their replication time.

As chromosomes condense during early mitosis, their subbands fuse in a highly coordinated fashion. Subband fusion occurs when two large subbands flanking one minor subband come together to form one band, which takes on the cytological characteristics of the original flanking subbands. Using four different banding techniques--GTG (G-bands obtained with trypsin and Giemsa), GBG (G-bands obtained with BrdU and Giemsa), RHG (R-bands obtained by heating and Giemsa), and RBG (R-bands obtained with BrdU and Giemsa)--we studied subband fusion from prophase (1,250 bands per haploid set) to late metaphase (300 bands). To quantify the condensation process, a fusion index was established. We found that chromosomes contain preferential zones of condensation. From prophase to late metaphase, the early replicating subbands (R-subbands) fuse more readily with each other than do the late-replicating subbands (G-subbands). R-bands usually replicate early and condense late independently of the adjacent G-bands, which replicate late but condense early. Therefore, chromosome bands can undergo DNA replication and chromatin condensation relatively autonomously. Our data suggest that (1) chromosome replication and condensation are closely connected in time, (2) the metaphase bands represent independent units of chromatin condensation, and (3) the condensation process is an important feature of chromosome organization.

Cells, Cultured↗
PubMed1990

High-resolution dynamic and morphological G-bandings (GBG and GTG): a comparative study.

A high-resolution replication banding technique, dynamic GBG banding (G-bands after 5'-bromodeoxyuridine [BrdUrd] and Giemsa), showed that, at a resolution of 850 bands/genome, GBG banding and GTG banding (G-bands after trypsin and Giemsa) produce almost identical patterns. RBG band (R-bands after BrdUrd and Giemsa) and RHG band (R-bands after heat denaturation and Giemsa) patterns were previously shown to be only 75%-85% coincident; thus GTG banding more accurately reflects replication patterns than does RHG banding. BrdUrd synchronization uses high concentrations of BrdUrd both to substitute early replicating DNA and to arrest cells before the late bands replicate. Release from the block is via a low thymidine concentration. The banding is revealed by the fluorochrome-photolysis-Giemsa (FPG) technique and produces the GBG banding that includes concomitant staining of constitutive heterochromatin. As opposed to other replication G-banding procedures, BrdUrd synchronization and GBG banding produces a reproducible replication band pattern. The discordance between homologs after GBG banding is similar to that after GTG banding and no lateral asymmetry of the constitutive heterochromatin has been observed. Also, BrdUrd synchronization neither significantly depresses the mitotic index, nor induces chromosome breaks. Thus, GBG banding seems as clinically useful as GTG banding and provides important information regarding replication time.

Adult↗
PubMed1990

Analysis of DNA replication during S-phase by means of dynamic chromosome banding at high resolution.

The characteristic patterns of dynamic banding (replication banding) were analysed. Extremely high resolution (850 to 1,250 bands per genome) G- and R-band patterns were obtained after 5-bromo-2'-deoxyuridine (BrdUrd) incorporation either during the early or the late S-phase. We synchronized human lymphocytes with high concentrations of thymidine or BrdUrd as blocking agents, followed by low concentrations of BrdUrd or thymidine respectively as releasing agents, and obtained R- or G-band patterns respectively. The dynamic R- and G-band patterns were complementary for all chromosomes, even for the late-replicating X chromosome. There was no overlapping and every part of each chromosome was positively stained by one of the two banding procedures. The complementarity of the two patterns shows that both high thymidine and high BrdUrd concentrations blocked S-phase progression near the R-band to G-band replication transition in the middle of S-phase. Some bands of the inactive X chromosome replicate before this transition concurrently with R-band replication. The 48 different telomeric regions could be classified into 5 distinct morphotypes based upon the distribution of early and late-replicating DNA in each telomeric region. The dynamic band patterns are particularly useful for the study of the structural and physiological organization of chromosomes at high resolution and should prove invaluable for assessing the replication behavior of rearranged chromosomes.

Adult↗
PubMed1990

Detection by electron microscopy of a small subband 13q14.11 deletion in an hereditary retinoblastoma.

High-resolution banding, specific for electron microscopy, was applied to chromosomes of synchronized blood lymphocytes obtained from a child with bilateral retinoblastoma. Ultrastructural analysis of the subbands in region q14.1, after synchronization and immunochemical banding, showed that the deletion in the abnormal chromosome 13 corresponds to subband 14.11, thus evidencing that the retinoblastoma gene is located within subband q14.11. This first application to a diagnostic problem of immunochemical banding suggests that, coupled with electron microscopy, this banding provides a higher resolution than that obtained with light microscopy and should be useful to pinpoint important localizations.

Child, Preschool↗
PubMed1990

Ultrastructural, immunohistochemical, and cytogenetic study of a malignant peripheral neuroectodermal tumor in a patient seropositive for human immunodeficiency virus.

A case of malignant peripheral neuroectodermal tumor occurring during the course of a human immunodeficiency virus (HIV) infection is reported. The patient was a male homosexual who presented with a rapidly enlarging tumor of the posterior lower thoracic wall. By light microscopic examination the tumor was a small cell tumor showing occasional structures suggestive of Homer-Wright rosettes. The strong positivity for neuron-specific enolase and the neurosecretory granules indicated the neural differentiation of the tumor. Its precise nature was shown cytogenetically by the presence of the t(11;22) translocation, which distinguished it from the classical neuroblastoma.

Adult↗
PubMed1989

Synchronization of amniotic fluid cells for high resolution cytogenetics.

A high resolution technique was applied to amniotic fluid cells by synchronization. After inoculation, the cells were incubated for 30 h in the presence of either thymidine or 5-bromodeoxyuridine (BrdU). After removal of the blocking agent and addition of a low concentration of thymidine, the cells were incubated for another 6 1/2 7 h, then harvested in prometaphase without colcemid. This technique gives a mitotic index of 3.7 per cent after thymidine synchronization and of 3.2 per cent after BrdU synchronization, and more than half of the mitoses were in the earlier phases with the chromosomes showing more than 550 bands per haploid set. GBG, GTG, and RHG prometaphases are presented. Precise high-resolution banding of chromosomes of amniotic fluid cells can increase diagnostic accuracy.

Amniotic Fluid↗
PubMed1989

Dynamic G- and R-banding of human chromosomes for electron microscopy.

Synchronized human lymphocytes were exposed to 5-bromo-2'-deoxyuridine (BrdUrd) for incorporation in either G- or R-bands. The substituted bands were revealed by monoclonal anti-BrdUrd antibodies disclosed with either gold-labeled antibodies or with the protein A-gold complex. Sharp G- or R-banding, specific for electron microscopy (EM), was obtained. These banding patterns, referred to as GB-AAu (G-bands by BrdUrd using Antibodies and gold [Au]) and RB-AAu (R-bands by BrdUrd using Antibodies and gold [Au]), resemble dynamic band patterns (GBG and RBG) much more than they do morphologic band patterns (GTG and RHG). The G- and R-band patterns allow accurate chromosome identification and karyotyping. An actual karyotype of human GB-AAu-banded chromosomes at the 750 band level, photographed in the EM, is presented. The method produces excellent band separation and band contrast. Variations in band staining intensities were noted and correlated with BrdUrd enrichment. The C-band regions were positively stained after GB-AAu banding while they were negatively stained after RB-AAu banding. Telomeres appeared heterogeneous after GB-AAu banding suggesting that part of the telomeric bands might be late replicating.

Antibodies, Monoclonal↗
PubMed1989

DNA denaturation for ultrastructural banding and the mechanism underlying the fluorochrome-photolysis-Giemsa technique studied with anti-5-bromodeoxyuridine antibodies.

G- and R-bands produced by an immunochemical approach were studied by electron microscopy (EM) to evaluate the role of DNA denaturation on banding quality. Excellent banding was observed only after adequate denaturation by HCl, NaOH and formamide, used in appropriate concentrations to provide uniform 5-bromodeoxyuridine (BrdUrd) exposure by generating single-stranded DNA. Formamide treatment resulted in less intercellular variability. High temperature and high concentrations of NaOH and HCl altered chromosomal morphology. Besides formamide, Hoechst 33258 prestaining which does not interfere with the binding of the anti-BrdUrd antibody and UV irradiation associated with formamide also produced high quality banding. On the other hand, consecutive Hoechst and UV treatment completely inhibited the immunochemical banding. The data indicate that Hoechst and UV act synergistically to disintegrate BrdUrd-substituted chromatin from which DNA is then extracted, leaving only the unsubstituted DNA stainable with Giemsa.

Animals↗
PubMed1989

High-resolution R-banding at the 1250-band level. II. Schematic representation and nomenclature of human RBG-banded chromosomes.

Detailed characterization of the RBG-banding pattern at the 1250-band level has been done after thymidine synchronization and block release with 5-bromo-2'-deoxyuridine (BrdU), which induces chromosome elongation and improves definition of chromosomal bands. Optimal conditions for the incorporation of BrdU and the use of the FPG (fluorochrome-photolysis-Giemsa) technique produced excellent band separation and band contrast even in highly elongated prophase chromosomes. Moreover, we did not observe lateral asymmetry in C-banded regions. The schematic representation of these elongated chromosomes in the 1250-band range per haploid set was prepared showing the relative position, the specific size, and the characteristic staining intensity for each band. To this idiogram was extended the International Standard Cytogenetic Nomenclature. This realistic idiogram should help in the preparation of R-banded prophase karyotypes and in the identification and localization of chromosomal rearrangements. Because differences exist between RBG and RHG bands, a brief comparative description of each RBG-banded chromosome is included. Moreover, a minute analysis of the banding pattern revealed that various parts of chromosomes contract differently. We also observed the presence of R-positive bands in heterochromatic regions of the short arms of the acrocentrics, and of chromosomes 1, 9, 16, and Y.

Adult↗
PubMed1989

Electron microscopy of gold-labeled human and equine chromosomes.

We present an immunochemical technique for the detection of 5-bromo-2'-deoxyuridine (BrdU) incorporated discontinuously into the chromosomal DNA. A monoclonal anti-BrdU antibody and a protein A-gold complex were used to produce chromosome banding of human and equine chromosomes, specific for electron microscopy (EM). Well-defined bands, symmetry of sister chromatids, concordance between homologues, and band patterns similar to those observed by light microscopy facilitate chromosome identification and karyotyping. From prophase to late metaphase, chromosomes condense and bands appear to fuse. The fusion appears to be owing to chromatin reorganization. Our results underline the value of using immunogold reagents, which are ideal probes for antigen localization on chromosomes.

Animals↗