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T Cremer

Publications and source records attributed to T Cremer.

At least 109 records · Page 6Linked to original sources
PubMed1990

Molecular cytotaxonomy of primates by chromosomal in situ suppression hybridization.

A new strategy for analyzing chromosomal evolution in primates is presented using chromosomal in situ suppression (CISS) hybridization. Biotin-labeled DNA libraries from flow-sorted human chromosomes are hybridized to chromosome preparations of catarrhines, platyrrhines, and prosimians. By this approach rearrangements of chromosomes that occurred during hominoid evolution are visualized directly at the level of DNA sequences, even in primate species with pronounced chromosomal shuffles.

Animals↗
PubMed1989

Double in situ hybridization in combination with digital image analysis: a new approach to study interphase chromosome topography.

Double in situ hybridization with mercurated and biotinylated chromosome specific DNA probes in combination with digital image analysis provides a new approach to compare the distribution of homologous and nonhomologous chromosome targets within individual interphase nuclei. Here we have used two DNA probes representing tandemly repeated sequences specific for the constitutive heterochromatin of the human chromosomes 1 and 15, respectively, and studied the relative arrangements of these chromosome targets in interphase nuclei of human lymphocytes, amniotic fluid cells, and fibroblasts, cultivated in vitro. We have developed a 2D-image analysis approach which allows the rapid evaluation of large numbers of interphase nuclei. Models to test for a random versus nonrandom distribution of chromosome segments are discussed taking into account the three-dimensional origin of the evaluated 2D-distribution. In all three human diploid cell types the measurements of target-target and target-center distances in the 2D-nuclear image revealed that the labeled segments of the two chromosomes 15 were distributed both significantly closer to each other and closer to the center of the nuclear image than the labeled chromosome 1 segments. This result can be explained by the association of nucleolus organizer regions on the short arm of chromosome 15 with nucleoli located more centrally in these nuclei and does not provide evidence for a homologous association per se. In contrast, evaluation of the interphase positioning of the two chromosome 1 segments fits the random expectation in amniotic fluid and fibroblast cells, while in experiments using lymphocytes a slight excess of larger distances between these homologous targets was occasionally observed. 2D-distances between the labeled chromosome 1 and 15 segments showed a large variability in their relative positioning. In conclusion our data do not support the idea of a strict and permanent association of these homologous and nonhomologous targets in the cell types studied so far.

Cell Nucleolus↗
PubMed1989

Interphase cytogenetics in paraffin embedded sections from human testicular germ cell tumor xenografts and in corresponding cultured cells.

A protocol was developed that allows determination of chromosome aberrations in interphase nuclei from paraffin embedded human tissues. As a model system tissue sections from xenografts derived from three testicular germ cell tumors (two teratocarcinoma and one embryonal carcinoma), as well as sections from normal intestine and testicular tissues, were hybridized with a biotin-labeled DNA probe specific for 1q12. For comparison, isolated nuclei in suspension and metaphase spreads from in vitro cell lines derived from the respective xenografts were assayed. Our results show that interphase tumor cells with aberrant numbers of chromosome 1 can be reliably detected both in vitro and in vivo. Subclones with two and three labeled chromosomes, respectively, could be defined and quantitatively evaluated. Present and future possibilities to pinpoint specific chromosome aberrations directly in cells present in body fluids or in tumor tissues are discussed.

Animals↗
PubMed1989

Supernumerary chromosome 1 in interphase nuclei of atypical germ cells in paraffin-embedded human seminiferous tubules.

The so-called atypical germ cells or cells of carcinoma in situ morphologically resemble neoplastic cells in seminoma. Since seminomas show numerical aberrations of chromosome 1 we have used a DNA probe specific for chromosome region 1q12 to determine whether such aberrations can be detected in atypical germ cell nuclei in paraffin-embedded seminiferous tubules as well. One-third of intratubular nuclei, containing atypical germ cells, consistently showed three hybridization signals in contrast to two signals regularly observed in normal intestine and in spermatogonia. We thus show that cytogenetic studies of precancerous cells can be performed directly on the tissue where these cells originate.

Chromosome Aberrations↗
PubMed1988

Sorting of chromosomes by magnetic separation.

Chromosomes were isolated from Chinese hamster x human hybrid cell lines containing four and nine human chromosomes. Human genomic DNA was biotinylated by nick translation and used to label the human chromosomes by in situ hybridization in suspension. Streptavidin was covalently coupled to the surface of magnetic beads and these were incubated with the hybridized chromosomes. The human chromosomes were bound to the magnetic beads through the strong biotin-streptavidin complex and then rapidly separated from nonlabeled Chinese hamster chromosomes by a simple permanent magnet. The hybridization was visualized by additional binding of avidin-FITC (fluorescein) to the unoccupied biotinylated human DNA bound to the human chromosomes. After magnetic separation, up to 98% of the individual chromosomes attached to magnetic beads were classified as human chromosomes by fluorescence microscopy.

Animals↗
PubMed1988

Delineation of individual human chromosomes in metaphase and interphase cells by in situ suppression hybridization using recombinant DNA libraries.

A method of in situ hybridization for visualizing individual human chromosomes from pter to qter, both in metaphase spreads and interphase nuclei, is reported. DNA inserts from a single chromosomal library are labeled with biotin and partially preannealed with a titrated amount of total human genomic DNA prior to hybridization with cellular or chromosomal preparations. The cross-hybridization of repetitive sequences to nontargeted chromosomes can be markedly suppressed under appropriate preannealing conditions. The remaining single-stranded DNA is hybridized to specimens of interest and detected with fluorescent or enzyme-labeled avidin conjugates following post-hybridization washes. DNA inserts from recombinant libraries for chromosomes 1, 4, 7, 8, 13, 14, 18, 20, 21, 22, and X were assessed for their ability to decorate specifically their cognate chromosome; most libraries proved to be highly specific. Quantitative densitometric analyses indicated that the ratio of specific to nonspecific hybridization signal under optimal preannealing conditions was at least 8:1. Interphase nuclei showed a cohesive territorial organization of chromosomal domains, and laser-scanning confocal fluorescence microscopy was used to aid the 3-D visualization of these domains. This method should be useful for both karyotypic studies and for the analysis of chromosome topography in interphase cells.

Chromosomes, Human↗
PubMed1988

Detection of chromosome aberrations in metaphase and interphase tumor cells by in situ hybridization using chromosome-specific library probes.

Chromosome aberrations in two glioma cell lines were analyzed using biotinylated DNA library probes that specifically decorate chromosomes 1, 4, 7, 18 and 22 from pter to qter. Numerical changes, deletions and rearrangements of these chromosomes were readily visualized in metaphase spreads, as well as in early prophase and interphase nuclei. Complete chromosomes, deleted chromosomes and segments of translocated chromosomes were rapidly delineated in very complex karyotypes. Simultaneous hybridizations with additional subregional probes were used to further define aberrant chromosomes. Digital image analysis was used to quantitate the total complement of specific chromosomal DNAs in individual metaphase and interphase cells of each cell line. In spite of the fact that both glioma lines have been passaged in vitro for many years, an under-representation of chromosome 22 and an over-representation of chromosome 7 (specifically 7p) were observed. These observations agree with previous studies on gliomas. In addition, sequences of chromosome 4 were also found to be under-represented, especially in TC 593. These analyses indicate the power of these methods for pinpointing chromosome segments that are altered in specific types of tumors.

Biotin↗
PubMed1988

Rapid interphase and metaphase assessment of specific chromosomal changes in neuroectodermal tumor cells by in situ hybridization with chemically modified DNA probes.

Repeated DNAs from the constitutive heterochromatin of human chromosomes 1 and 18 were used as probes in nonradioactive in situ hybridization experiments to define specific numerical and structural chromosome aberrations in three human glioma cell lines and one neuroblastoma cell line. The number of spots detected in interphase nuclei of these tumor cell lines and in normal diploid nuclei correlated well with metaphase counts of chromosomes specifically labeled by in situ hybridization. Rapid and reliable assessments of aneuploid chromosome numbers in tumor lines in double hybridization experiments were achieved, and rare cells with bizarre phenotype and chromosome constitution could be evaluated in a given tumor cell population. Even with suboptimal or rare chromosome spreads specific chromosome aberrations were delineated. As more extensive probe sets become available this approach will become increasingly powerful for uncovering various genetic alterations and their progression in tumor cells.

Cell Line↗
PubMed1988

Rapid detection of human chromosome 21 aberrations by in situ hybridization.

Plasmid clones containing up to 94 kilobases of single-copy DNA from band q22.3 of chromosome 21 and a complete pool of insert DNA from a chromosome 21 recombinant library have been used to rapidly detect numerical and structural aberrations of chromosome 21 by in situ hybridization in both metaphase and interphase cells. A trisomic karyotype, diagnostic of Down syndrome, is readily detected in nonmitotic cells because the majority of their nuclei exhibit three discrete foci of hybridization, in contrast to normal diploid cells, which show two foci. Chromosomal translocations involving chromosome 21 sequences were also detected with these probes, and the intranuclear location of 21q22.3 DNA sequences in "normal" human brain neurons was established with the plasmid DNA probe set. These results suggest that chromosome 21-specific probes may have utility in clinical diagnostics, especially by facilitating the direct analysis of interphase cells.

Brain Chemistry↗
PubMed1987

A method for nucleic acid hybridization to isolated chromosomes in suspension.

A procedure was developed to provide differential fluorescent staining of metaphase chromosomes in suspension following nucleic acid hybridization. For this purpose metaphase chromosomes were isolated from a Chinese hamster X human hybrid cell line. After hybridization with biotinylated human genomic DNA, the human chromosomes were visualized by indirect immunofluorescence using antibodies against biotin and fluoresceine-isothiocyanate-(FITC)-labeled second antibodies. This resulted in green fluorescent human chromosomes. In contrast, Chinese hamster chromosomes revealed red fluorescent staining only when counterstained with propidium iodide. Notably, interspecies chromosomal rearrangements could be easily detected. After hybridization and fluorescent staining, chromosomes still showed a well-preserved morphology under the light microscope. We suggest that this procedure may have a useful application in flow cytometry and sorting.

Animals↗
PubMed1986

Detection of chromosome aberrations in the human interphase nucleus by visualization of specific target DNAs with radioactive and non-radioactive in situ hybridization techniques: diagnosis of trisomy 18 with probe L1.84.

The localization of chromosome 18 in human interphase nuclei is demonstrated by use of radioactive and non-radioactive in situ hybridization techniques with a DNA clone designated L1.84. This clone represents a distinct subpopulation of the repetitive human alphoid DNA family, located in the centric region of chromosome 18. Under stringent hybridization conditions hybridization of L1.84 is restricted to chromosome 18 and reflects the number of these chromosomes present in the nuclei, namely, two in normal diploid human cells and three in nuclei from cells with trisomy 18. Under conditions of low stringency, cross-hybridization with other subpopulations of the alphoid DNA family occurs in the centromeric regions of the whole chromosome complement, and numerous hybridization sites are detected over interphase nuclei. Detection of chromosome-specific target DNAs by non-radioactive in situ hybridization with appropriate DNA probes cloned from individual chromosomal subregions presents a rapid means of identifying directly numerical or even structural chromosome aberrations in the interphase nucleus. Present limitations and future applications of interphase cytogenetics are discussed.

Cell Nucleus↗
PubMed1986

Induction of chromosome shattering by ultraviolet light and caffeine: the influence of different distributions of photolesions.

Cells of synchronized and of asynchronously growing cultures of a V79 Chinese hamster line were microirradiated with a low power laser-UV-microbeam of wavelength 257 nm. Ultraviolet light was either focused onto a small part of the nucleus (mode I) or distributed over the whole nucleus (mode II). Following microirradiation, the cells were incubated for 7-20 h with caffeine (1-2 mM) until chromosome preparation was performed. After both modes of microirradiation, shattering of the entire chromosome complement (generalized chromosome shattering, GCS) was observed. It is suggested that the probability by which GCS is induced depends on the total number of DNA lesions rather than on their distribution in the chromatin. The results are consistent with the prediction of a "factor depletion model" which assumes that in a given cell, GCS takes place both in irradiated and non-irradiated chromosomes if the total number of daughter strand-repair sites surpasses a threshold value.

Animals↗
PubMed1986

Two subsets of human alphoid repetitive DNA show distinct preferential localization in the pericentric regions of chromosomes 13, 18, and 21.

We have isolated and characterized two human middle repetitive alphoid DNA fragments, L1.26 and L1.84, which localize to two different sets of chromosomes. In situ hybridization revealed both repeats to have major and minor binding sites on the pericentric regions of several chromosomes. Probe L1.26 maps predominantly to chromosomes 13 and 21. Probe L1.84 locates to chromosome 18. Minor hybridization sites for both probes include chromosomes 2, 8, 9, and 20; in addition, L1.26 revealed minor sites on chromosomes 18 and 22. The binding to these sites strongly depends on hybridization conditions. In Southern blot hybridizations to total human DNA, both L1.26 and L1.84 give the same ladder pattern, with a step size of 170 bp, indicating their presence as tandem repeats, but with different band intensities for each probe. The chromosome-specific nature of particular multimers was confirmed by Southern blot analyses of a human-rodent hybrid cell panel. We conclude that L1.26 and L1.84, with their related sequences, constitute subfamilies of alphoid DNA that are specific for subsets of chromosomes and, in some cases, possibly even for single chromosomes.

Base Sequence↗