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T Cremer

Publications and source records attributed to T Cremer.

At least 73 records · Page 4Linked to original sources
PubMed1994

Multicolor fluorescence in situ hybridization on metaphase chromosomes and interphase Halo-preparations using cosmid and YAC clones for the simultaneous high resolution mapping of deletions in the dystrophin gene.

We report on multicolor fluorescence in situ hybridization protocols for the simultaneous visualization of deletion-prone regions for carrier detection of Duchenne/Becker (DMD/BMD) muscular dystrophy. Cosmid and yeast artificial chromosome (YAC) clones specific for preferentially deleted subregions of the dystrophin gene were labeled differentially and detected with three different fluorochromes using digital imaging microscopy. This approach allows for an assessment of the carrier status of female relatives even in families where no index patient is available. Cosmid and YAC clones, and different probe-generation protocols are compared with respect to their feasibility for carrier detection. The use of histone-depleted interphase nuclei (Halo-preparations) for deletion mapping is demonstrated and shown to have a resolution power of 5 kb.

Cell Line↗
PubMed1994

The origin of human chromosome 2 analyzed by comparative chromosome mapping with a DNA microlibrary.

Fluorescence in situ hybridization (FISH) of microlibraries established from distinct chromosome subregions can test the evolutionary conservation of chromosome bands as well as chromosomal rearrangements that occurred during primate evolution and will help to clarify phylogenetic relationships. We used a DNA library established by microdissection and microcloning from the entire long arm of human chromosome 2 for fluorescence in situ hybridization and comparative mapping of the chromosomes of human, great apes (Pan troglodytes, Pan paniscus, Gorilla gorilla, Pongo pygmaeus) and Old World monkeys (Macaca fuscata and Cercopithecus aethiops). Inversions were found in the pericentric region of the primate chromosome 2p homologs in great apes, and the hybridization pattern demonstrates the known phylogenetically derived telomere fusion in the line that leads to human chromosome 2. The hybridization of the 2q microlibrary to chromosomes of Old World monkeys gave a different pattern from that in the gorilla and the orang-utan, but a pattern similar to that of chimpanzees. This suggests convergence of chromosomal rearrangements in different phylogenetic lines.

Animals↗
PubMed1994

Comparative chromosome painting discloses homologous segments in distantly related mammals.

Comparative chromosome painting, termed ZOO-FISH, using DNA libraries from flow sorted human chromosomes 1, 16, 17 and X, and mouse chromosome 11 discloses the presence of syntenic groups in distantly related mammalian orders ranging from primates (Homo sapiens), rodents (Mus musculus), even-toed ungulates (Muntiacus muntjak vaginalis and Muntiacus reevesi) and whales (Balaenoptera physalus). These mammalian orders have evolved separately for 55-80 million years (Myr). We conclude that ZOO-FISH can be used to generate comparative chromosome maps of a large number of mammalian species.

Animals↗
PubMed1994

Specific loss of chromosomes 1, 2, 6, 10, 13, 17, and 21 in chromophobe renal cell carcinomas revealed by comparative genomic hybridization.

We analyzed 19 chromophobe renal cell carcinomas by means of comparative genomic hybridization. Two tumors revealed no numerical abnormalities. In the remaining 17 cases we found loss of entire chromosomes with underrepresentation of chromosome 1 occurring in all 17 cases; loss of chromosomes 2, 10, and 13 in 16 cases; loss of chromosomes 6 and 21 in 15 tumors; and loss of chromosome 17 in 13 cases. The loss of the Y chromosome was observed in 6 of 13 tumors from male patients, whereas 1 X chromosome was lost in 3 of 4 tumors obtained from females. Comparative genomic hybridization results were verified by interphase cytogenetics. We conclude that a specific combination of multiple chromosomal losses characterizes chromophobe renal cell carcinomas and may help to differentiate them unequivocally from other types of kidney cancer.

Adult↗
PubMed1994

[Detection of amplified DNA sequences by comparative genomic in situ hybridization with human glioma tumor DNA as probe].

Comparative genomic hybridization (CGH) provides a new possibility for the investigation of genetic alterations in tumour genomes. In our experiments CGH was carried out using genomic DNA from human glioblastoma multiforme (GBM) as a probe for chromosomal in situ suppression hybridization. Amplified DNA sequences contained in the tumour DNA showed specific signals, revealing the chromosomal positions of these sequences. Using this approach we detected amplifications of different chromosomal segments in individual GBM specimens. In accordance with the results from Southern analysis demonstrating amplification of the EGFR gene in 45% of human GBM, CGH signals in different GBM mapped to the region of this gene on chromosome 7p. Other signals detected by CGH involved chromosome 12q and 8q. Our data demonstrate CGH as a novel comprehensive and rapid approach for the analysis of complex genomic alterations in glial tumours.

Base Sequence↗
PubMed1993

Characterization of two marker chromosomes in a patient with acute nonlymphocytic leukemia by two-color fluorescence in situ hybridization.

A patient with acute nonlymphocytic leukemia (ANLL), M5b according to French-American-British (FAB) classification, showed monosomy 16, an extra 1p-, and a 21q+. These derivative chromosomes could not be defined by GTG-banding. For better characterization, we performed two-color fluorescence in situ hybridization (FISH) experiments applying DNA libraries from sorted human chromosomes, chromosome-specific repetitive probes, and a band-specific YAC-clone. With these FISH studies the karyotype could be characterized as 46,XY, +der(1)t(1;21)(p11;?), -16,der(21)t(16;21) (p11.1;q22).

Adult↗
PubMed1993

Differences of size and shape of active and inactive X-chromosome domains in human amniotic fluid cell nuclei.

It is a widely held belief that the inactive X-chromosome (Xi) in female cell nuclei is strongly condensed as compared to the largely decondensed active X-chromosome (Xa). We have reconsidered this problem and painted X-chromosome domains in nuclei of subconfluent, female and male human amniotic fluid cell cultures (46,XX and 46,XY) by chromosomal in situ suppression (CISS) hybridization with biotinylated human X-chromosome specific library DNA. FITC-conjugated avidin was used for probe detection and nuclei were counterstained with propidium iodide (PI). The shape of these nuclei resembling flat ellipsoids or elliptical cylinders makes them suitable for both two-dimensional (2D) and three-dimensional (3D) analyses. 2D analyses of Xi- and Xa-domains were performed in 34 female cell nuclei by outlining of the painted domains using a camera lucida. Identification of the sex chromatin body in DAPI-stained nuclei prior to CISS-hybridization was confirmed by its colocalization with one of the two painted X-domains. In 31 of the 34 nuclei the area AXi for the inactive X-domain was smaller than the area AXa for the active domain (mean ratio AXa/AXi = 1.9 +/- 0.8 SD, range 1.0-4.3). The signed rank test showed a highly significant (P < .0001) difference both between AXa and AXi and between the ratios r(Xa) and r(Xi), calculated by dividing the maximum length L of each X-domain by its maximum width W. In most nuclei (26/34) we found r(Xa) > r(Xi) demonstrating a generally more elongated structure of Xa. For 3D analysis a confocal scanning laser fluorescence microscope (CSLFM) was used. Ten to 20 light optical sections (PI-image, FITC-image) were registered with equal spacings (approx. 0.4 microns). A thresholding procedure was applied to determine the PI-labeled nuclear and FITC-labeled X-domain areas in each section. Estimated slice volumes were used to compute total nuclear and X-domain volumes. In a series of 35 female nuclei most domains extended from the top to the bottom nuclear sections. The larger of the two X-chromosome domains comprised (3.7 +/- 1.7 S.D.)% of the nuclear volume. A mean ratio of 1.2 +/- 0.2 SD (range 1.1-2.3) was found for the volumes of the larger and the smaller X-domains in these female nuclei. In a series of 27 male amniotic fluid cell nuclei the relative X-chromosome domain volume comprised (4.0 +/- 2.6 S.D.)%.(ABSTRACT TRUNCATED AT 400 WORDS)

Amniotic Fluid↗
PubMed1993

Detection of amplified DNA sequences by reverse chromosome painting using genomic tumor DNA as probe.

A modification of "reverse chromosome painting" was carried out using genomic DNA from tumor cells as a complex probe for chromosomal in situ suppression hybridization to normal metaphase chromosome spreads. Amplified DNA sequences contained in such probes showed specific signals, revealing the normal chromosome positions from which these sequences were derived. As a model system, genomic DNAs were analyzed from three tumor cell lines with amplification units including the proto-oncogene c-myc. The smallest amplification unit was about 90 kb and was present in 16-24 copies; the largest unit was bigger than 600 kb and was present in 16-32 copies. Specific signals that co-localized with a differently labeled c-myc probe on chromosome band 8q24 were obtained with genomic DNA from each cell line. In further experiments, genomic DNA derived from primary tumor material was used in the case of a male patient with glioblastoma multiforme (GBM). Southern blot analysis using an epidermal growth factor receptor gene (EGFR) probe that maps to 7p13 indicated the amplification of sequences from this gene. Using reverse chromosome painting, signals were found both on band 7p13 and bands 12q13-q15. Notably, the signal on 12q13-q15 was consistently stronger. The weaker 7p13 signal showed co-localization with the major signal of the differently labeled EGFR probe. A minor signal of this probe was seen on 12q13, suggesting cross-hybridization to ERB3 sequences homologous to EGFR. The results indicate co-amplification of sequences from bands 12q13-q15, in addition to sequences from band 7p13. Several oncogenes map to 12q13-q15 providing candidate genes for a tumor-associated proto-oncogene amplification. Although the nature of the amplified sequences needs to be clarified, this experiment demonstrates the potential of reverse chromosome painting with genomic tumor DNA for rapidly mapping the normal chromosomal localization of the DNA from which the amplified sequences were derived. In addition, a weaker staining of chromosomes 10 and X was consistently observed indicating that these chromosomes were present in only one copy in the GBM genome. This rapid approach can be used to analyze cases where no metaphase spreads from the tumor material are available. It does not require any preknowledge of amplified sequences and can be applied to screen large numbers of tumors.

Chromosome Aberrations↗
PubMed1993

Detection of complete and partial chromosome gains and losses by comparative genomic in situ hybridization.

Comparative genomic in situ hybridization (CGH) provides a new possibility for searching genomes for imbalanced genetic material. Labeled genomic test DNA, prepared from clinical or tumor specimens, is mixed with differently labeled control DNA prepared from cells with normal chromosome complements. The mixed probe is used for chromosomal in situ suppression (CISS) hybridization to normal metaphase spreads (CGH-metaphase spreads). Hybridized test and control DNA sequences are detected via different fluorochromes, e.g., fluorescein isothiocyanate (FITC) and tetraethylrhodamine isothiocyanate (TRITC). The ratios of FITC/TRITC fluorescence intensities for each chromosome or chromosome segment should then reflect its relative copy number in the test genome compared with the control genome, e.g., 0.5 for monosomies, 1 for disomies, 1.5 for trisomies, etc. Initially, model experiments were designed to test the accuracy of fluorescence ratio measurements on single chromosomes. DNAs from up to five human chromosome-specific plasmid libraries were labeled with biotin and digoxigenin in different hapten proportions. Probe mixtures were used for CISS hybridization to normal human metaphase spreads and detected with FITC and TRITC. An epifluorescence microscope equipped with a cooled charge coupled device (CCD) camera was used for image acquisition. Procedures for fluorescence ratio measurements were developed on the basis of commercial image analysis software. For hapten ratios 4/1, 1/1 and 1/4, fluorescence ratio values measured for individual chromosomes could be used as a single reliable parameter for chromosome identification. Our findings indicate (1) a tight correlation of fluorescence ratio values with hapten ratios, and (2) the potential of fluorescence ratio measurements for multiple color chromosome painting. Subsequently, genomic test DNAs, prepared from a patient with Down syndrome, from blood of a patient with T-cell prolymphocytic leukemia, and from cultured cells of a renal papillary carcinoma cell line, were applied in CGH experiments. As expected, significant differences in the fluorescence ratios could be measured for chromosome types present in different copy numbers in these test genomes, including a trisomy of chromosome 21, the smallest autosome of the human complement. In addition, chromosome material involved in partial gains and losses of the different tumors could be mapped to their normal chromosome counterparts in CGH-metaphase spreads. An alternative and simpler evaluation procedure based on visual inspection of CCD images of CGH-metaphase spreads also yielded consistent results from several independent observers. Pitfalls, methodological improvements, and potential applications of CGH analyses are discussed.

Aneuploidy↗
PubMed1993

A strategy for the characterization of minute chromosome rearrangements using multiple color fluorescence in situ hybridization with chromosome-specific DNA libraries and YAC clones.

The identification of marker chromosomes in clinical and tumor cytogenetics by chromosome banding analysis can create problems. In this study, we present a strategy to define minute chromosomal rearrangements by multicolor fluorescence in situ hybridization (FISH) with "whole chromosome painting" probes derived from chromosome-specific DNA libraries and Alu-polymerase chain reaction (PCR) products of various region-specific yeast artificial chromosome (YAC) clones. To demonstrate the usefulness of this strategy for the characterization of chromosome rearrangements unidentifiable by banding techniques, an 8p+ marker chromosome with two extra bands present in the karyotype of a child with multiple anomalies, malformations, and severe mental retardation was investigated. A series of seven-color FISH experiments with sets of fluorochrome-labeled DNA library probes from flow-sorted chromosomes demonstrated that the additional segment on 8p+ was derived from chromosome 6. For a more detailed characterization of the marker chromosome, three-color FISH experiments with library probes specific to chromosomes 6 and 8 were performed in combination with newly established telomeric and subtelomeric YAC clones from 6q25, 6p23, and 8p23. These experiments demonstrated a trisomy 6pter-->6p22 and a monosomy 8pter-->8p23 in the patient. The present limitations for a broad application of this strategy and its possible improvements are discussed.

Abnormalities, Multiple↗
PubMed1993

Evidence for a nuclear compartment of transcription and splicing located at chromosome domain boundaries.

The nuclear topography of splicing snRNPs, mRNA transcripts and chromosome domains in various mammalian cell types are described. The visualization of splicing snRNPs, defined by the Sm antigen, and coiled bodies, revealed distinctly different distribution patterns in these cell types. Heat shock experiments confirmed that the distribution patterns also depend on physiological parameters. Using a combination of fluorescence in situ hybridization and immunodetection protocols, individual chromosome domains were visualized simultaneously with the Sm antigen or the transcript of an integrated human papilloma virus genome. Three-dimensional analysis of fluorescence-stained target regions was performed by confocal laser scanning microscopy. RNA transcripts and components of the splicing machinery were found to be generally excluded from the interior of the territories occupied by the individual chromosomes. Based on these findings we present a model for the functional compartmentalization of the cell nucleus. According to this model the space between chromosome domains, including the surface areas of these domains, defines a three-dimensional network-like compartment, termed the interchromosome domain (ICD) compartment, in which transcription and splicing of mRNA occurs.

Amniotic Fluid↗
PubMed1993

Evaluation of the utility of interphase cytogenetics to detect residual cells with a malignant genotype in mixed cell populations: a Burkitt lymphoma model.

Interphase cytogenetics has been used to detect tumor cells in the presence of a large excess of normal cells. Probes for fluorescence in situ hybridization were chosen to reveal a specific hybridization pattern in tumor cell nuclei as well as to provide an internal control for the assessment of the hybridization results. By enumerating mixtures of cytogenetically normal cells and tumor cells from a Burkitt lymphoma cell line, we were able to detect tumor cells at a frequency of one in 500. Normal cells could be differentiated from Burkitt lymphoma cells with a specificity of approximately 99.9%.

Burkitt Lymphoma↗
PubMed1993

Molecular cytogenetic analysis of formalin-fixed, paraffin-embedded solid tumors by comparative genomic hybridization after universal DNA-amplification.

We present a technique which allows the detection and chromosomal localization of DNA sequence copy number changes in solid tumor genomes from frozen sections and paraffin embedded, formalin fixed specimens. Based on comparative genomic hybridization and on universal DNA amplification procedures this technique is possible even if only a few tumor cells are available. We demonstrate the feasibility of this method to visualize complete and partial chromosome gains and losses and gene amplifications in archived solid tumor samples.

Base Sequence↗
PubMed1993

Detection of residual host cells after bone marrow transplantation using non-isotopic in situ hybridization and karyotype analysis.

Karyotype analysis and interphase cytogenetics by means of non-radioactive in situ hybridization (NISH) with Y and X-specific probes were used to detect residual host cells in BM of 18 male patients who had received a BMT from their sisters. All patients but one had a malignant blood disease; 17 patients were clinically in continuous remission at the time of the investigations and throughout the follow-up period. No host cells were detected by karyotype analysis in patients who were in clinical remission. NISH with a biotinylated Y-specific probe showed residual host cells in 16 of the 17 patients in clinical remission. In samples from three patients the existence of host cells was further confirmed by revealing the XY-containing cells with two-color fluorescence in situ hybridization (FISH) using X- and Y-specific probes on direct BM smears showed > 3% residual host cells in 50% of the samples studied at or later than 2 months post-BMT. On conventional cytogenetic preparations < 1% Y-specific cells were detected in all but two samples at > or = 2 months post-BMT. There was no difference in the proportion of host cells between patients conditioned with total body irradiation and CY and those who received busulphan and CY. The recipient's stromal or epithelial cells in the aspirates probably account for most of the host cells detected. In conclusion, small numbers of residual host cells detected by interphase cytogenetics with a Y-specific probe do not indicate an imminent relapse.(ABSTRACT TRUNCATED AT 250 WORDS)

Adolescent↗
PubMed1992

Reconstruction of genomic rearrangements in great apes and gibbons by chromosome painting.

The homology between hylobatid chromosomes and other primates has long remained elusive. We used chromosomal in situ suppression hybridization of all human chromosome-specific DNA libraries to "paint" the chromosomes of primates and establish homologies between the human, great ape (chimpanzee, gorilla, and orangutan), and gibbon karyotypes (Hylobates lar species group, 2n = 44). The hybridization patterns unequivocally demonstrate the high degree of chromosomal homology and synteny of great ape and human chromosomes. Relative to human, no translocations were detected in great apes, except for the well-known fusion-origin of human chromosome 2 and a 5;17 translocation in the gorilla. In contrast, numerous translocations were detected that have led to the massive reorganization of the gibbon karyotype: the 22 autosomal human chromosomes have been divided into 51 elements to compose the 21 gibbon autosomes. Molecular cytogenetics promises to finally allow hylobatids to be integrated into the overall picture of chromosomal evolution in the primates.

Animals↗