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T Cremer

Publications and source records attributed to T Cremer.

At least 91 records · Page 5Linked to original sources
PubMed1992

Metaphase and interphase cytogenetics with Alu-PCR-amplified yeast artificial chromosome clones containing the BCR gene and the protooncogenes c-raf-1, c-fms, and c-erbB-2.

A human yeast artificial chromosome (YAC) library was screened by polymerase chain reaction with oligonucleotide primers defined for DNA sequences of the BCR gene and the protooncogenes c-raf-1, c-fms, and c-erbB-2. Alu-PCR-generated human DNA sequences were obtained from the respective YAC clones and used for fluorescence in situ hybridization experiments under suppression conditions. After chromosomal in situ suppression hybridization to GTG-banded human prometaphase chromosomes, seven of nine initially isolated YAC clones yielded strong signals exclusively in the chromosome bands containing the respective genes. Two clones yielded additional signals on other chromosomes and were excluded from further tests. The band-specific YACs were successfully applied to visualize specific structural chromosome aberrations in peripheral blood cells from patients with myelodysplasia exhibiting del(5)(q13q34), chronic myeloid leukemia and acute lymphocytic leukemia with t(9;22)(q34;q11), acute promyelocytic leukemia (M3) with t(15;17)(q22;q21), and in a cell line established from a proband with the constitutional translocation t(3;8)(p14.2;q24). In addition to the analysis of metaphase spreads, we demonstrate the particular usefulness of these YAC clones in combination with whole chromosome painting to analyze specific chromosome aberrations directly in the interphase nucleus.

Base Sequence↗
PubMed1992

Molecular and classical cytogenetic analyses demonstrate an apomorphic reciprocal chromosomal translocation in Gorilla gorilla.

The existence of an apomorphic reciprocal chromosomal translocation in the gorilla lineage has been asserted or denied by various cytogeneticists. We employed a new molecular cytogenetic strategy (chromosomal in situ suppression hybridization) combined with high-resolution banding, replication sequence analysis, and fluorochrome staining to demonstrate that a reciprocal translocation between ancestral chromosomes homologous to human chromosome 5 and 17 has indeed occurred.

Animals↗
PubMed1992

Specific metaphase and interphase detection of the breakpoint region in 8q24 of Burkitt lymphoma cells by triple-color fluorescence in situ hybridization.

Triple fluorescence in situ hybridization with a plasmid DNA library from sorted human chromosomes 8 in combination with bacteriophage clones flanking the breakpoint in 8q24 of the Burkitt lymphoma cell line J1 was used for the specific delineation of this breakpoint in individual tumor cells. With this approach, tumor-specific breakpoints in translocation chromosomes can be detected at all stages of the cell cycle with high specificity.

Burkitt Lymphoma↗
PubMed1992

Chromosomal in situ suppression hybridization of immunologically classified mitotic cells in hematologic malignancies.

Chromosomal in situ suppression (CISS) hybridization was performed with library DNA from sorted human chromosomes 8, 9, 15, 17, 21, and 22 on immunologically stained bone marrow cells of four patients with a hematologic neoplasm, including two patients with myelodysplastic syndrome and trisomy 8, one patient with promyelocytic leukemia bearing the translocation t(15;17)(q22;q11-12), and one patient with chronic myeloid leukemia and the translocation t(9;22)(q34;q11). In all patients, the results of conventional karyotype analysis could be confirmed by one- or two-color CISS hybridization using the appropriate chromosome-specific libraries. Our results show that CISS hybridization can detect both numerical and structural chromosome changes in immunologically classified cells with high specificity and reliability. The fact that chromosome spreads of very poor quality can now be included in such analyses is a decisive advantage of this approach. In addition, the suitability of this approach for interphase cytogenetics is discussed.

Chromosome Aberrations↗
PubMed1992

Rapid generation of chromosome-specific alphoid DNA probes using the polymerase chain reaction.

Non-isotopic in situ hybridization of chromosome-specific alphoid DNA probes has become a potent tool in the study of numerical aberrations of specific human chromosomes at all stages of the cell cycle. In this paper, we describe approaches for the rapid generation of such probes using the polymerase chain reaction (PCR), and demonstrate their chromosome specificity by fluorescence in situ hybridization to normal human metaphase spreads and interphase nuclei. Oligonucleotide primers for conserved regions of the alpha satellite monomer were used to generate chromosome-specific DNA probes from somatic hybrid cells containing various human chromosomes, and from DNA libraries from sorted human chromosomes. Oligonucleotide primers for chromosome-specific regions of the alpha satellite monomer were used to generate specific DNA probes for the pericentromeric heterochromatin of human chromosomes 1, 6, 7, 17 and X directly from human genomic DNA.

Animals↗
PubMed1992

Homologies in human and Macaca fuscata chromosomes revealed by in situ suppression hybridization with human chromosome specific DNA libraries.

We established chromosomal homologies between all chromosomes of the human karyotype and that of an old world monkey (Macaca fuscata) by chromosomal in situ suppression (CISS) hybridization with human chromosome specific DNA libraries. Except for the human chromosome 2 library and limited cross-hybridization of X and Y chromosome libraries all human DNA libraries hybridized to single GTG-banded macaque chromosomes. Only three macaque chromosomes (2, 7, 13) were each hybridized by two separate human libraries (7 and 21, 14 and 15, 20 and 22 respectively). Thus, an unequivocally high degree of synteny between human and macaque chromosomes has been maintained for more than 20 million years. As previously suggested, both Papionini (macaques, baboons, mandrills and cercocebus monkeys, all of which have nearly identical karyotypes) and humans are chromosomally conservative. The results suggest, that CISS hybridization can be expected to become an indispensable tool in comparative chromosome and gene mapping and will help clarify chromosomal phylogenies with speed and accuracy.

Animals↗
PubMed1992

Fluorescence in situ hybridization of YAC clones after Alu-PCR amplification.

Alu-PCR protocols were optimized for the generation of human DNA probes from yeast strains containing yeast artificial chromosomes (YACs) with human inserts between 100 and 800 kb in size. The resulting DNA probes were used in chromosome in situ suppression (CISS) hybridization experiments. Strong fluorescent signals on both chromatids indicated the localization of specific YAC clones, while two clearly distinguishable signals were observed in greater than or equal to 90% of diploid nuclei. Signal intensities were generally comparable to those observed using chromosome-specific alphoid DNA probes. This approach will facilitate the rapid mapping of YAC clones and their use in chromosome analysis at all stages of the cell cycle.

Base Sequence↗
PubMed1992

Development of a biological dosimeter for translocation scoring based on two-color fluorescence in situ hybridization of chromosome subsets.

Recently fluorescence in situ hybridization protocols have been developed which allow the painting of individual chromosomes using DNA-libraries from sorted human chromosomes. This approach has the particular advantage that radiation induced chromosome translocations can be easily detected, if chromosomes of distinctly different colors take part in the translocation event. To enhance the sensitivity of this approach two metaphase chromosome subsets A and B (A: chromosomes 1, 2, 4, 8, 16; B: 3, 5, 9, 10, 13) were simultaneously painted in green and red color. Counterstaining of the chromosomes with DAPI resulted in a third subset which exhibited blue fluorescence only. Green-red, green-blue and red-blue translocation chromosomes could be easily detected after irradiation of lymphocyte cultures with 137Cs-gamma-rays. Analyses of painted chromosomes can be combined with conventional GTG-banding analyses. This new biological dosimeter should become useful to monitor both long term effects of single irradiation events and the cumulative effects of multiple or chronic irradiation exposures. In contrast to translocation scoring based on the analysis of banded chromosomes, this new approach has the particular advantage that a rapid, automated scoring of translocations can now be envisaged.

Chromosomes↗
PubMed1991

Translocation (8;21) in acute nonlymphocytic leukemia delineated by chromosomal in situ suppression hybridization.

In situ suppression hybridization with recombinant bacteriophage DNA libraries for chromosomes 8 and 21 was performed in two cases of acute nonlymphocytic leukemia, type FAB M2. In both cases, cytogenetic analysis by conventional G-banding revealed t(8;21)(q22;q22). In situ suppression hybridization was able to prove the reciprocal nature of the translocation in both cases by identifying the terminal end of chromosome 21 translocated to the derivative chromosome 8q-.

Chromosome Aberrations↗
PubMed1991

Analysis of genes and chromosomes by nonisotopic in situ hybridization.

Nonisotopic in situ hybridization is a powerful tool to analyze the organization of complex genomes. Current approaches utilizing this technique for the analysis of linear and spatial genome organizations are presented. Clinical applications of these approaches, which open new avenues for diagnosis of disease-related chromosomal changes, are also discussed.

Chromosome Mapping↗
PubMed1991

Painting of defined chromosomal regions by in situ suppression hybridization of libraries from laser-microdissected chromosomes.

"Painting" of defined chromosomal regions provides a powerful tool for cytogenetic analyses. Here, we demonstrate that chromosomal in situ suppression (CISS)-hybridization of DNA libraries derived by microcloning laser-microdissected chromosomal regions can be applied to achieve this goal. As an example, we used unbanded metaphase spreads from a female patient carrying a balanced translocation. t(1;7)(1qter----1p36::7q11----7qter). Fragments from the long arms of 130 translocation chromosomes were microdissected. After microcloning, human inserts with an average size of about 3 kb were pooled from 400 recombinant bacteriophage DNA clones and used as a complex probe set in CISS-hybridization experiments. This resulted in painting of the translocation chromosome along the region 7q35 to 1p31. Painted chromosomal subregions in normal chromosomes 1 and 7 were consistent with this finding. This approach may be used to perform painting of any chromosome regions for which microlibraries can be established. Possible applications include the definition of marker chromosomes in clinical and tumor cytogenetics and studies of chromosomal evolution, as well as studies of nuclear chromosome topography in animal and plant species.

Cells, Cultured↗
PubMed1990

Detection of monosomy 7 in interphase cells of patients with myeloid disorders.

Six patients, five with acute myeloid leukemia (AML) and one with a myelodysplastic syndrome (MDS), were found to have monosomy 7 by conventional cytogenetics at diagnosis. Repetitive DNA sequences from the heterochromatic region of human chromosomes 1 and 7 were used as probes for in situ hybridization experiments on interphase cells of these patients. A double hybridization protocol was used to reveal the particular chromosomes as distinct spots or clusters of signals within interphase nuclei. The chromosome 1 sequence served as an internal control. Simultaneous detection of the sequences showed the presence of two normal number 1 chromosomes and a missing 7 chromosome from individual cells. While cytogenetic preparations showed only -7 metaphases in 3 AML and 1 MDS patients, in situ hybridization of interphase cells showed many normal cells as well as the presence of -7 in fully mature granulocytes. One AML patient studied in remission showed only normal metaphases yet had 9% interphase cells with a missing 7 and relapsed within 3 months. We conclude that examination of interphase cells by in situ hybridization provides clinically useful data since every cell including mature granulocytes can be examined, the lineage of a cell can be determined, and efficacy of differentiation therapy can be evaluated.

Chromosome Deletion↗
PubMed1990

Rapid metaphase and interphase detection of radiation-induced chromosome aberrations in human lymphocytes by chromosomal suppression in situ hybridization.

Chromosomal in situ suppression (CISS)-hybridization of biotinylated phage DNA-library inserts from sorted human chromosomes was used to decorate chromosomes 1 and 7 specifically from pter to qter and to detect structural aberrations of these chromosomes in irradiated human peripheral lymphocytes. In addition, probe pUC1.77 was used to mark the 1q12 subregion in normal and aberrant chromosomes 1. Low LET radiation (60Co-gamma-rays; 1.17 and 1.33 MeV) of lymphocyte cultures was performed with various doses (D = 0, 2, 4, 8 Gy) 5 h after stimulation with phytohaemagglutinin. Irradiated cells were cultivated for an additional 67 h before Colcemid arrested metaphase spreads were obtained. Aberrations of the specifically stained chromosomes, such as deletions, dicentrics, and rings, were readily scored after in situ hybridization with either the 1q12 specific probe or DNA-library inserts. By the latter approach, translocations of the specifically stained chromosomes could also be reliably assessed. A linear increase of the percentage of specifically stained aberrant chromosomes was observed when plotted as a function of the square of the dose D. A particular advantage of this new approach is provided by the possibility to delineate numerical and structural chromosome aberrations directly in interphase nuclei. These results indicate that cytogenetic monitoring of ionizing radiation may be considerably facilitated by CISS-hybridization.

Chromosome Aberrations↗
PubMed1990

Chromosomal in situ suppression hybridization of human gonosomes and autosomes and its use in clinical cytogenetics.

DNA libraries from sorted human gonosomes were used selectively to stain the X and Y chromosomes in normal and aberrant cultured human cells by chromosomal in situ suppression (CISS-) hybridization. The entire X chromosome was stained in metaphase spreads. Interphase chromosome domains of both the active and inactive X were clearly delineated. CISS-hybridization of the Y chromosome resulted in the specific decoration of the euchromatic part (Ypter-q11), whereas the heterochromatic part (Yq12) remained unlabeled. The stained part of the Y chromosome formed a compact domain in interphase nuclei. This approach was applied to amniotic fluid cells containing a ring chromosome of unknown origin (47,XY: +r). The ring chromosome was not stained by library probes from the gonosomes, thereby suggesting its autosomal origin. The sensitivity of CISS-hybridization was demonstrated by the detection of small translocations and fragments in human lymphocyte metaphase spreads after irradiation with 60Co-gamma-rays. Lymphocyte cultures from two XX-males were investigated by CISS-hybridization with Y-library probes. In both cases, metaphase spreads demonstrated a translocation of Yp-material to the short arm of an X chromosome. The translocated Y-material could also be demonstrated directly in interphase nuclei. CISS-hybridization of autosomes 7 and 13 was used for prenatal diagnosis in a case with a known balanced translocation t(7:13) in the father. The same translocation was observed in amniotic fluid cells from the fetus. Specific staining of the chromosomes involved in such translocations will be particularly important, in the future, in cases that cannot be solved reliably by conventional chromosome banding alone.

Chromosome Aberrations↗
PubMed1990

Direct carrier detection by in situ suppression hybridization with cosmid clones of the Duchenne/Becker muscular dystrophy locus.

A basic problem in genetic counseling of families with Duchenne/Becker muscular dystrophy (DMD/BMD) concerns the carrier status of female relatives of an affected male. In about 60% of these patients, deletions of one or more exons of the dystrophin gene can be identified. These deletions preferentially include exon 45, which can be detected by multiplex polymerase chain reaction (PCR) and Southern blot analysis of genomic cosmid clones that map to this critical region. As a new approach for definitive carrier detection, we have performed chromosomal in situ suppression (CISS) hybridization with these cosmid clones in female relatives of four unrelated patients. In normal females, most metaphases showed signals on both X chromosomes, whereas only one X chromosome was labeled in carriers. Our results demonstrate that CISS hybridization can define the carrier status in female relatives of DMD patients exhibiting a deletion in the dystrophin gene.

Blotting, Southern↗
PubMed1990

Painting of human chromosomes with probes generated from hybrid cell lines by PCR with Alu and L1 primers.

Specific amplification of human sequences of up to several kb length has recently been accomplished in man-hamster and man-mouse somatic hybrid cell DNA by IRS-PCR (interspersed repetitive sequence - polymerase chain reaction). This approach is based on oligonucleotide primers that anneal specifically to human Alu- or L1-sequences and allows the amplification of any human sequences located between adequately spaced, inverted Alu- or L1-blocks. Here, we demonstrate that probe pools generated from two somatic hybrid cell lines by Alu- and L1-PCR can be used for chromosome painting in normal human lymphocyte metaphase spreads by chromosomal in situ suppression (CISS-) hybridization. The painted chromosomes and chromosome subregions directly represent the content of normal and deleted human chromosomes in the two somatic hybrid cell lines. The combination of IRS-PCR and CISS-hybridization will facilitate and improve the cytogenetic analysis of somatic hybrid cell panels, in particular, in cases where structurally aberrant human chromosomes or human chromosome segments involved in interspecies translocations cannot be unequivocally identified by classical banding techniques. Moreover, this new approach will help to generate probe pools for the specific delineation of human chromosome subregions for use in cytogenetic diagnostics and research without the necessity of cloning.

Animals↗
PubMed1990

Distribution of chromosome 18 and X centric heterochromatin in the interphase nucleus of cultured human cells.

In situ hybridization of human chromosome 18 and X-specific alphoid DNA-probes was performed in combination with three dimensional (3D) and two dimensional (2D) image analysis to study the interphase distribution of the centric heterochromatin (18c and Xc) of these chromosomes in cultured human cells. 3D analyses of 18c targets using confocal laser scanning microscopy indicated a nonrandom disposition in 73 amniotic fluid cell nuclei. The shape of these nuclei resembled rather flat cylinders or ellipsoids and targets were preferentially arranged in a domain around the nuclear center, but close to or associated with the nuclear envelope. Within this domain, however, positionings of the two targets occurred independently from each other, i.e., the two targets were observed with similar frequencies at the same (upper or lower) side of the nuclear envelope as those on opposite sides. This result strongly argues against any permanent homologous association of 18c. A 2D analytical approach was used for the rapid evaluation of 18c positions in over 4000 interphase nuclei from normal male and female individuals, as well as individuals with trisomy 18 and Bloom's syndrome. In addition to epithelially derived amniotic fluid cells, investigated cell types included in vitro cultivated fibroblastoid cells established from fetal lung tissue and skin-derived fibroblasts. In agreement with the above 3D observations 18c targets were found significantly closer (P less than 0.01) to the center of the 2D nuclear image (CNI) and to each other in all these cultures compared to a random distribution derived from corresponding ellipsoid or cylinder model nuclei. For comparison, a chromosome X-specific alphoid DNA probe was used to investigate the 2D distribution of chromosome X centric heterochromatin in the same cell types. Two dimensional Xc-Xc and Xc-CNI distances fit a random distribution in diploid normal and Bloom's syndrome nuclei, as well as in nuclei with trisomy X. The different distributions of 18c and Xc targets were confirmed by the simultaneous staining of these targets in different colors within individual nuclei using a double in situ hybridization approach.

Amniotic Fluid↗