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B Malfoy

Publications and source records attributed to B Malfoy.

At least 55 records · Page 3Linked to original sources
PubMed1994

Detection of methylcytosine-rich heterochromatin on banded chromosomes. Application to cells with various status of DNA methylation.

A procedure including incorporation of 5-bromodeoxyuridine (BrdU) in DNA and a thermal denaturation step was developed to obtain both R-banding and efficient binding of anti-5-methylcytosine antibodies on metaphase chromosomes. BrdU incorporation improved the efficiency of antibody binding disclosed by immunofluorescence staining. This method allowed semiquantitative analysis of the antibody binding sites on straightforward characterized metaphase chromosomes and was applied to normal human lymphocytes and lymphoblastoid cell lines for which DNA methylation status had been previously analyzed. A correlation was established between level of DNA methylation and the semiquantitative estimate of antibody fixation. This procedure can be used to study DNA methylation on metaphase chromosomes in transformed and cancerous cell lines.

Chromosome Banding↗
PubMed1994

Increased FISH efficiency using APC probes generated by direct incorporation of labeled nucleotides by PCR.

Probes of various sizes from the adenomatous polyposis coli gene (APC) were directly biotinylated by polymerase chain reaction (PCR) from genomic DNA. PCR labeling gave high efficiency in detection of fluorescence in situ hybridization (FISH) signals. Probes as small as 250 base pairs could be visualized through a fluorescence microscope without any image processing.

Adenomatous Polyposis Coli↗
PubMed1994

In situ hybridization approach at infragenic level on metaphase chromosomes.

Analysis of the retinoblastoma locus (RB1) by in situ hybridization at the infragenic level was done using human chromosomes. A high level of resolution was attained with nonisotopic in situ hybridization on banded chromosomes in both fluorescence and electron microscopy. DNA sequences less than 100 kb apart could be positioned on band 13q14 in the order expected from molecular mapping. These observations suggest that the DNA target of hybridized probes on metaphase chromosomes may correspond to nucleoprotein loops giving a resolution comparable to that obtained in interphase nuclei.

Genes, Retinoblastoma↗
PubMed1994

[Characterization of chromosomal rearrangements by in situ hybridization in glioblastoma].

In a case of glioblastoma, the following karyotype was determined: 47, X, - Y, + der(1) t(1;9)(p21;p23), t(1;9)(p21;p23), + 3, + 7, der(9) t(Y;9)(q11;p21), - 13, t(13;16)(p13,p11), del(14)(q11q22). Classical satellite DNAs are mainly located in chromosomes 1, 9, 15, 16 and Y. Because, most of these chromosomes were implicated in the rearrangements, a detailed cytogenetic study was undertaken. This study included in situ hybridization of the satellite and alphoid DNAs of chromosomes 1, 9, 16 and Y combined with various chromosome banding methods (DA-DAPI, quinacrine mustard and R-banding). The data obtained, demonstrated that the breakpoints were always located outside the areas containing the satellite and alphoid DNAs. The situation observed here differs from that reported in breast cancers for which a high proportion of the breakpoints occur within these areas. These findings suggest that in glioblastoma, chromosome rearrangements result from different mechanisms than those implicated in breast cancers. Thus, in cancers, chromosomal instabilities may result from several mechanisms.

Azacitidine↗
PubMed1994

Oncogene amplification in human gliomas: a molecular cytogenetic analysis.

Nine cases of malignant gliomas were selected for the presence of double minutes (dmin) or homogeneously staining regions (hsr) detected by conventional cytogenetics. Analyses were performed on fresh (2 cases) or xenografted (5 cases) tumors or both (2 cases). A modified comparative genomic hybridization technique (mCGH) was applied exhibiting a single amplified locus in 8 tumors and 4 amplified loci in one tumor. Recurrent sites of amplification were detected in 7p11-p12 (5 cases) and 1q32.1 (2 cases). Signals were also observed in 4q11-q12, 5p15.1, 7q31, 8q24.1 and 9p2 in one tumor each. Southern blotting demonstrated that the genes for EGFR (epidermal growth factor receptor), PDGFRA (platelet derived growth factor receptor alpha), MET and MYC oncogenes were involved in 7p11-p12, 4q11-q12, 7q31 and 8q24.1 amplifications, respectively. These amplifications were found by in situ hybridization on tumor spreads, in dmin or episomes for EGFR, dmin for PDGFRA and MET, and hsr and dmin for MYC genes. Other mCGH signals, for which no target genes could be proposed, were confirmed by chromosome paintings on tumor metaphases. In one of the tumors, the coamplification of DNA from 5p15.1 and 9p2 bands in the same dmin was demonstrated.

Adult↗
PubMed1993

Two-color FISH characterization of i(1q) and der(1;16) in human breast cancer cells.

Two-color fluorescent in situ hybridizations using probes for alphoid (alpha) and classical satellite (CS) DNAs from chromosomes 1 and 16 were performed to characterize i(1q), der(1;16), and complex rearrangements observed in breast cancer cells from fresh tumors and established cell lines. Six of seven i(1q) occurred after breakage in the alpha 1 containing region and one of seven was dicentric, with breakage in 1p11.2. The five der(1;16)(q10;p10) studied appeared to result from a variety of breakpoints involving alpha 1, alpha 16, CS1, and CS16 DNAs. All had conserved alpha 16 DNA, suggesting a segregation of the der(1;16) leading to a loss of 16q and a gain of 1q in most cases. One complex rearrangement of chromosome 1 also appeared to involve chromosome 16, suggesting that a der(1;16) occurred first, followed by another rearrangement. Both the apparent preferential involvement of constitutive heterochromatin harboring alpha and CS DNAs and the variety of breakpoints spanning along heterochromatin suggest that the important consequence of the rearrangement is not the breakage per se but the resulting imbalance.

Adenocarcinoma↗
PubMed1993

Human carbonyl reductase (CBR) localized to band 21q22.1 by high-resolution fluorescence in situ hybridization displays gene dosage effects in trisomy 21 cells.

Human carbonyl reductase (CBR) belongs to a group of NADPH-dependent enzymes called aldo-keto reductases. The enzyme can function as an aldo-keto reductase or as a quinone reductase with potential for modulating quinone-mediated oxygen free radicals. The CBR gene was mapped by high-resolution fluorescence in situ hybridization to band 21q22.12, very close to the SOD1 locus at position 21q22.11. CBR displayed gene dosage effects in trisomy 21 human lymphoblasts at the DNA and mRNA levels. Lymphoblasts with increasing chromosome 21 ploidy also showed increased aldo-keto reductase activity and increased quinone reductase activity. Both aldo-keto reductase activity and quinone reductase activity have been shown to be associated with carbonyl reductase. The location of CBR near SOD1 and the increased enzyme activity and potential for free radical modulation in trisomy 21 cells implicate CBR as a candidate for contributing to the pathology of certain diseases such as Down syndrome and Alzheimer disease.

Alcohol Oxidoreductases↗
PubMed1993

Hypomethylation of classical satellite DNA and chromosome instability in lymphoblastoid cell lines.

To determine possible relationships between DNA hypomethylation and chromosome instability, human lymphoblastoid cell lines from different genetic constitutions were studied with regard to 1) uncoiling and rearrangements, which preferentially affect the heterochromatic segments of chromosomes 1 and 16; 2) the methylation status of the tandemly repetitive sequences (classical satellite and alphoid DNAs) from chromosomes 1 and 16, and of the L1Hs interspersed repetitive sequences. The methylation status largely varied from cell line to cell line, but for a given cell line, the degree of methylation was similar for all the repetitive DNAs studied. Two cell lines, one obtained from a Fanconi anemia patient and the other from an ataxia telangiectasia patient were found to be heavily hypomethylated. The heterochromatic segments of their chromosomes 1 and 16 were more frequently elongated and rearranged than those from other cell lines, which were found to be less hypomethylated. Thus, in these lymphoblastoid cell lines, alterations characterized by uncoiling and rearrangements of heterochromatic segments from chromosomes 1 and 16 seem to correlate with the hypomethylation of their repetitive DNAs. Two-color in situ hybridizations demonstrated that these elongations and rearrangements involved only classical satellite-DNA-containing heterochromatin. This specificity may be related to the excess of breakages affecting the chromosomes carrying these structures in a variety of pathological conditions.

Ataxia Telangiectasia↗
PubMed1993

Specific induction of uncoiling and recombination by azacytidine in classical satellite-containing constitutive heterochromatin.

Azacytidine (ACR) is known to induce uncoiling and somatic association involving the constitutive heterochromatin of human chromosomes 1, 9, 15, and 16 and the Y. These regions are composed of alphoid and classical satellite DNA sequences. Using specific probes for chromosomes 1 and 16, we have performed two-color fluorescence in situ hybridization on human lymphocytes cultured in the presence of ACR. We demonstrate that for these two chromosomes (1) uncoiling and association specifically occur in classical satellite-containing regions at the first cell generation, (2) breakages also affect these regions, and (3) somatic recombinations occur between these regions and lead to translocations at the next cell generation. These results suggest that changes in methylation of repetitive DNA sequences are related to chromosomal instability occurring during cell transformation and tumorigenesis.

Azacitidine↗
PubMed1992

Detection of small, single-copy genes on protein-G-banded chromosomes by electron microscopy.

A method for the detection by electron microscopy of chromosome banding after in situ hybridization of small, nonradioactive DNA sequences is described. Typical high-resolution G-banding is produced by adding 5-bromodeoxyuridine (BrdU) during the last part of the S-phase and by applying a monoclonal antibody against the BrdU-substituted chromosome segments, followed by the addition of protein G, but no further treatment. A protocol for in situ hybridization of small, single-copy biotinylated DNA sequences and their detection by immunogold tagging on banded chromosomes is also described. This combined approach permits high-resolution mapping of small DNA sequences and should be useful in discriminating between neighboring DNA fragments.

Antibodies, Monoclonal↗
PubMed1991

Recurrent homogeneously staining regions in 8p1 in breast cancer and lack of amplification of POLB, LHRH, and PLAT genes.

In a cytogenetic study of 125 primary and untreated breast cancers, 107 were selected for the quality of their metaphases permitting detection of amplifications:homogeneously staining regions (HSRs), abnormally banded region (ABRs), and double minutes (dmins). HSRs and ABRs were detected in 62 cases (58%), but no cases of dmins were observed. The localizations of HSRs and ABRs were not random because they were observed in the 8p1 position in 14 cases. The possible amplifications of five sequences, MOS (8q1), LHRH (8p21.1), POLB (8p11.2), PLAT (8p12), and D8Z2 (8c) were investigated in three tumors with HSR on the short arm of chromosome 8. Because these sequences were not amplified, two interpretations can be proposed: 1) there is a frequent amplification of a sequence from the 8p1 region, located between the investigated sequences; and 2) the amplifications do not occur in 8p1, but HSRs or ABRs of undetermined origin have a strong tendency to be translocated onto 8p. Because cases with HSR(8p) have less complex karyotypes than with HSRs in other locations, the first interpretation is the most likely: HSRs may be formed in 8p and further translocated on other chromosomes in the course of tumor progression.

Breast Neoplasms↗
PubMed1991

A t(X;15)(q23;q25) with Xq reactivation in a lymphoblastoid cell line from Fanconi anemia.

A t(X:15)(q23;q25) was detected during cytogenetic investigation of a lymphoblastoid cell line established from a female patient with Fanconi anemia. The translocation was apparently balanced at passage 300 and unbalanced at passage 13. A chromatid exchange between both the normal and the der(15), between the centromere and band 15q25, may explain these results. Replication studies, following BrdU incorporation, indicate that the segment Xq23----qter from the der(15) is early replicating whereas segment Xpter----q23 from the der(X) is late replicating. Since the normal X was early replicating, it is concluded that the segment of the long arm of chromosome X, separated from its inactivation center by the translocation, was reactivated. This interpretation is confirmed by the methylation patterns of the hypoxanthine phosphoribosyltransferase gene (HPRT), mapped on Xq26, which corresponds to that of an active gene, whereas that of phosphoglycerate kinase (PGK1), which remained on the der(X), corresponds to that of an inactive gene. This is the first example of reactivation of a segment of the X chromosome following a structural rearrangement in somatic cells.

Cell Line↗
PubMed1991

GST pi gene is frequently coamplified with INT2 and HSTF1 proto-oncogenes in human breast cancers.

The glutathione S-transferase gene (GST pi) is located on the same chromosome band (11q13) as proto-oncogenes INT2 and HSTF1 which are frequently amplified in breast cancer. Using the Southern blot technique, we looked for the amplification of the GST pi gene in 17 fresh tumors from human mammary carcinoma. The tumors were preselected because either they had an amplification of the INT2 proto-oncogene detected by dot blot, or their karyotypes exhibited or did not exhibit homogeneously staining regions, a cytogenetic character indicating amplification. Coamplification of GST pi, HSTF1 and INT2 was observed in five tumors, and coamplification of GST pi and HSTF1 without amplification of INT2 in another tumor. We also observed coamplification of GST pi, INT2, HSTF1 in the mammary carcinoma cell line MDA/MB134, whereas GST pi alone was amplified in the mammary epithelial cell line HBL100. These results indicate that INT2, HSTF1 and GST pi belong to the same large amplicon. Since GST pi is involved in intracellular detoxication and since chemotherapeutic drugs are among its substrates, it will be of interest to study GST pi gene expression as well as the response to chemotherapy in patients presenting this amplicon.

Blotting, Southern↗
PubMed1989

Theoretical prediction of base sequence effects in DNA. Experimental reactivity of Z-DNA and B-Z transition enthalpies.

Molecular modeling is used to study the sequence dependence of conformation and stability within helically regular duplex Z-DNA. The variations of conformation that are found are sufficiently important to be classified as a new type of polymorphism within the Z family. It is also demonstrated that certain sequences can adopt more than one of these polymorphic forms. Comparison with experimental studies of chemical reactivity within a natural DNA fragment, forced into a left-handed conformation, suggests that the results of our modeling may be used to explain the chemical reactivity observed. Comparison of the Z results with similar studies of the B form allow enthalpies of transition to be calculated as a function of base sequence.

Algorithms↗
PubMed1988

A study of the B-Z transition of the AC-rich region of the repeat unit of a satellite DNA from Cebus by means of chemical probes.

The conformational changes induced by negative supercoiling in the AC-rich region of the repeat unit of a Cebus satellite DNA has been studied by chemical probes sensitive to alterations in DNA conformation. This region is constituted of a (GT/CA)n stretch (15 less than or equal to n less than or equal to 18) associated to a sequence rich in GT/CA. At high superhelical density, at least 100 base pairs in the AC-rich region adopt the Z conformation as judged by diethyl pyrocarbonate reactivity. This is confirmed by diethyl pyrocarbonate footprinting of the complex between antibodies to Z-DNA and the AC-rich region. Osmium tetroxide and hydroxylamine reveal some distortions of the Z double helix in the (GT/CA)n stretch also. The terminal T residues of the stretch are hyperreactive with osmium tetroxide; the terminal left C residues but not the terminal right C residues are hyperreactive with hydroxylamine. Substitution of a few base pairs in the middle of the (GT/CA)n stretch induces also some distortions of the Z double helix. In the GT/CA-rich sequence, distortion of the Z double helix is also supported by the hyperreactivity of osmium tetroxide with several T and C residues.

Animals↗
PubMed1988

Studies of a novel repetitive sequence family in the genome of mice.

A new middle repetitive sequence is described in the mouse genome. It has been revealed with a recombinant clone isolated from a Mus musculus BamHI gene library constructed in pBR322 and containing an insertion of 1.73 kb. When digests of genomic DNA were subjected to Southern blot hybridization, using the 1.73-kb insert as probe, we obtained a light smear and discrete bands, indicating a dispersion in the mouse genome of this sequence. This 1.73-kb sequence seems to be a part of a greater repetitive sequence at least 6 kb in length. The sizes of the bands hybridizing with the 1.73-kb insert are similar when compared between different laboratory strains but differ remarkably between the two species M. musculus and Mus caroli. We have shown also a great variation in the copy number of the sequence studied between these two species. When rat DNA is probed with the 1.73-kb insert, no hybridization is observed. Subcloning of the 1.73-kb sequence in three fragments has pointed out that the reiteration was not homogeneous along the 1.73-kb sequence. The 1.73-kb clone was sequenced and compared with other interspersed repetitive sequences, previously described in the rodent genome, and no homology was found.

Animals↗
PubMed1988

Chloroacetaldehyde reacts with Z-DNA.

We show that chloroacetaldehyde, a chemical compound known to be reactive with unpaired adenine and cytosine residues, reacts with adenine residues (syn conformation) but not with cytosine residues (anti conformation) within Z-DNA. These modified residues are sensitive to cleavage by piperidine, which allows mapping at the single nucleotide level.

Acetaldehyde↗