Rapid analysis of mouse-hamster hybrid cell lines by in situ hybridization

In situ hybridization techniques for analyzing the murine DNA complement of mouse-hamster hybrid cells are described. Total genomic mouse DNA is labeled with biotin and hybridized without suppression to metaphase spreads from a mouse-hamster hybrid line containing the mouse fusion chromosome X12. Detection via fluorochrome-conjugated avidin reveals mouse chromosomal DNA with high sensitivity and permits the identification of both normal and aberrant murine chromosomes. Conversely, biotinylated total genomic DNA from a hybrid line can be used as a probe on normal mouse metaphase spreads if suppression techniques are employed, facilitating the analysis of mouse chromosomes present in the hybrid line.     

Labeling of the centromeric region on human chromosome 8 by in situ hybridization

Summary Probe DNA that binds preferentially to the centromeric region of human chromosomes 8 was synthesized. Alpha satellite probe DNA molecules were selectively amplified from sorter-purified human chromosomes 8 by in vitro DNA amplification using the polymerase chain reaction (PCR). Probe labeling was performed during PCR by incorporation of biotinylated deoxyuridine. In situ hybridization of unpurified probe DNA comprised of alpha satellite monomer and higher molecular weight DNA fragments with metaphase chromosome spreads showed binding to the centromeric regions of numerous chromosomes. However, blocking with unlabeled total human alphoid DNA dramatically reduced crosshybridization to chromosomes other than 8. Under these conditions, the degenerate probe DNA allowed unambiguous visualization of domains occupied by centromeric DNA of chromosome 8 in metaphase spreads and interphase cell nuclei, thus greatly facilitating the detection of numerical chromosome aberrations in tumor cells. In situ hybridization of size-fractionated alpha satellite DNA identified the monomeric fraction as the major cause of crosshybridization. Alpha satellite dimers and higher molecular weight DNA fragments showed relatively high specificity for human chromosomes 8.     

In situ hybridization at the electron microscope level: hybrid detection by autoradiography and colloidal gold

In situ hybridization has become a standard method for localizing DNA or RNA sequences in cytological preparations. We developed two methods to extend this technique to the transmission electron microscope level using mouse satellite DNA hybridization to whole mount metaphase chromosomes as the test system. The first method devised is a direct extension of standard light microscope level using mouse satellite DNA hybridization to whole mount metaphase chromosomes as the test system. The first method devised is a direct extension of standard light microscope in situ hybridization. Radioactively labeled complementary RNA (cRNA) is hybridized to metaphase chromosomes deposited on electron microscope grids and fixed in 70 percent ethanol vapor; hybridixation site are detected by autoradiography. Specific and intense labeling of chromosomal centromeric regions is observed even after relatively short exposure times. Inerphase nuclei present in some of the metaphase chromosome preparations also show defined paatterms of satellite DNA labeling which suggests that satellite-containing regions are associate with each other during interphase. The sensitivity of this method is estimated to at least as good as that at the light microscope level while the resolution is improved at least threefold. The second method, which circumvents the use of autoradiogrphic detection, uses biotin-labeled polynucleotide probes. After hybridization of these probes, either DNA or RNA, to fixed chromosomes on grids, hybrids are detected via reaction is improved at least threefold. The second method, which circumvents the use of autoradiographic detection, uses biotin-labeled polynucleotide probes. After hybridization of these probes, either DNA or RNA, to fixed chromosomes on grids, hybrids are detected via reaction with an antibody against biotin and secondary antibody adsorbed to the surface of over centromeric heterochromatin and along the associated peripheral fibers. Labeling is on average ten times that of background binding. This method is rapid and possesses the potential to allow precise ultrastructual localization of DNA sequences in chromosomes and chromatin.     

Evidence for integrity of parental genomes in the diploid hybridogenetic water frog Pelophylax esculentus by genomic in situ hybridization

The Western Palearctic water frogs Pelophylax ridibundus and P. lessonae were identified as parental (sexual) species and P. esculentus as their interspecific, hybridogenetically reproducing hybrid with hemiclonal heredity. We used genomic in situ hybridization (GISH) to identify parental chromosomes of P.lessonae and P.ridibundus in diploid P. esculentus karyotypes (2n = 26). GISH probes were made by fluorochrome labeling of total genomic DNA extracted from the sexual progenitors. The labeled probe from one species was hybridized to chromosomes of P. esculentus in the presence of excess of unlabeled genomic DNA from the other species. Thus, the P. lessonae probe was blocked by P. ridibundus unlabeled DNA, and vice versa. We successfully discriminated each of the 13 respective parental chromosomes in metaphase complements of the hybrids according to species-specific hybridization signals. GISH enabled us to confirm additional differences between parental chromosomes in size (smaller chromosomes belong to P. lessonae) and in the presence of DAPI-positive centromeric heterochromatin (detected in chromosomes of P. ridibundus, but not in P. lessonae). The fact that no visible intergenomic exchanges were found in metaphase chromosomes of diploid P. esculentus provides important information on the genomic integrity of hemiclonal transmission and supports hybridogenesis as a reproductive mode at the chromosome level for the specimens examined.     

cDNA cloning and mapping of the human creatine kinase M gene to 19q13

We describe the first isolation of a human creatine kinase M cDNA clone and its mapping of the gene to human chromosome 19. A human creatine kinase M cDNA clone, pJN2CK-M, harboring a 1,160-bp insert, was isolated by colony hybridization with a previously sequenced chicken creatine kinase M cDNA probe. The human cDNA was used as a probe in Southern transfers of TaqI-digested genomic DNA from mouse/human somatic-cell hybrids to localize the human creatine kinase-M gene to chromosome 19. In situ hybridization of the tritiated cDNA probe to metaphase chromosomes of peripheral blood lymphocytes from normal males revealed significant labeling to chromosome 19. These two independent methodologies assign the human creatine kinase-M gene to chromosome 19. Since greater than 69% of the grains of chromosome 19 label band q13, the human creatine kinase-M gene has been mapped to 19q13. On the basis of high-resolution G-banding, the predominant labeling site was 19q13.2-q13.3.     

Structure and chromosomal mapping of a highly polymorphic repetitive DNA sequence from the pseudoautosomal region of the mouse sex chromosomes

The pseudoautosomal region of the Mov15 mouse strain is marked by a Moloney murine leukemia provirus. The sequences flanking the Mov15 provirus were molecularly cloned and shown to consist of a tandemly repeated sequence of 31 nucleotides. Copy number variation of this repeat most likely accounts for the polymorphism in the mouse pseudoautosomal region detected with a probe from the flanking sequences. In situ hybridization to metaphase chromosomes showed heavy labeling of the pairing region of the X and Y chromosomes. The repetitive sequence was also found at the subtelomeric region of three autosomes. A similar level of amplification as the one seen on the sex chromosomes seems to be present on chromosomes 9 and 13. Lower copy number appear to be present on chromosome 4.     

Fluorescentin situ hybridization to soybean metaphase chromosomes

Repetitive DNA sequences were detected directly on somatic metaphase chromosome spreads from soybean root tips using fluorescentin situ hybridization. Methods to spread the forty small metaphase chromosomes substantially free of cellular material were developed using protoplasts. The specific DNA probe was a 1.05 kb internal fragment of a soybean gene encoding the 18S ribosomal RNA subunit. Two methods of incorporating biotin residues into the probe were compared and detection was accomplished with fluorescein-labeled avidin. The rDNA probe exhibits distinct yellow fluorescent signals on only two of the forty metaphase chromosomes that have been counterstained with propidium iodide. This result agrees with our previous analyses of soybean pachytene chromosome [27] showing that only chromosome 13 is closely associated with the nucleolus organizer region. Fluorescentin situ hybridization with the rDNA probe was detected on three of the forty-one metaphase chromosomes in plants that are trisomic for chromosome 13.     

A degenerate alpha satellite probe, detecting a centromeric deletion on chromosome 21 in an apparently normal human male, shows limitations of the use of satellite DNA probes for interphase ploidy analysis.

A degenerate alpha satellite DNA probe specific for a repeated sequence on human chromosomes 13 and 21 was synthesized using the polymerase chain reaction (PCR). Fluorescence in situ hybridization (FISH) with this probe to normal metaphase spreads revealed strong probe binding to the centromeric regions of human chromosomes 13 and 21 with negligible cross-hybridization with other chromosomes. FISH to normal interphase cell nuclei showed four distinct domains of probe binding. However, hybridization with probe to interphase and metaphase preparations from one apparently normal human male resulted in only three major binding domains. Metaphase chromosome analysis revealed a centromeric deletion on one chromosome 21 that caused greatly reduced probe binding. The result suggest caution in the interpretation of interphase ploidy studies performed with chromosome-specific alphoid DNA probes.     

Chromosomal in situ hybridization using yeast artificial chromosomes

Large DNA fragment cloning methods using yeast artificial chromosomes (YACs) have vastly improved the strategies for constructing physical maps of regions of complex genomes, as well as for isolating and cloning genes important for human disease. We present here a simple and rapid method for carrying out in situ hybridization to metaphase chromosomes using isolated YAC clones by labeling DNA directly in agarose gel slices. Nonisotopic labeling and chromosomal in situ hybridization can be used to determine the chromosomal localization of individual YAC clones on human metaphase chromosomes. This method can also be used to characterize YAC clones consisting of single fragments from those that contain concatamerized, and thus artifactual, inserts. This technique also offers a valuable tool to study consistent translocations in neoplastic diseases by identifying YACs that span a specific chromosomal breakpoint.     

Nucleosomes in metaphase chromosomes.

Previous studies of the structure of metaphase chromosomes have relied heavily on electron micrography and have revealed the existence of a 10-nm unit fiber that is thought to generate the native 23-30-nm fiber by higher order folding. The structural relationship of these metaphase fibers to the interphase fiber remains obscure. Recent studies on the digestion of interphase chromatin have revealed the existence of a regularly repeating subunit of DNA and histone, the nucleosome that generates the appearance of 10-nm beads connected by a short fiber of DNA seen on electron micrographs. It was therefore of interest to probe the structure of the metaphase chromosome for the presence of nucleosomal subunits. To this end metaphase chromosomes were prepared from colchicine-arrested cultures of mouse L-cells and were subjected to digestion with stayphylococcal nuclease. Comparison of the early and limit digestion products of metaphase chromosomes with those obtained from interphase nuclei indicates that although significant morphologic changes occur within the chromatin fiber during mitosis, the basic subunit structure of the chromatin fiber is retained by the mitotic chromosome.     

Mapping of the transferrin gene in laboratory rats, mice and man by direct in situ hybridization

Mapping of the gene coding for transferrin was carried out in metaphase chromosomes from bone marrow of laboratory mice and rats as well as from PHA-stimulated human lymphocytes using direct in situ hybridization technique. Plasmid pRTf-17 carrying the insert of rat transferrin cDNA was nick-translated with [125I]dCTP and used as a specific hybridization probe. The total number of silver grains and their distribution along differentially stained chromosomes were determined in 464 metaphase plates (114, 263 and 87 from rat, mouse and man, respectively). The data obtained enable us to assign transferrin gene to chromosome 3 in human and chromosome 9 in mouse. For the first time, the rat transferrin gene was localized on chromosome 7. The most probable sites of transferrin gene localization are 7q31-34, 9F1-3 and 3q21 in rat, mouse and human chromosomes, respectively.     

Bi-color detection of two target DNAs by non-radioactive in situ hybridization

Summary A non-radioactive in situ hybridization technique is described which allows the simultaneous detection of different DNA sequences. To demonstrate the feasibility of the proccdure, metaphases and interphase nuclei of a human-mouse somatic cell hybrid were simultaneously hybridized with mercurated total human DNA and a biotinylated mouse satellite DNA probe. After the hybridization, the probes were detected immunocytochemically using two different and independent affinity systems. By this approach we visualized the two DNA target sequences in metaphase chromosomes and in interphase nuclei with FITC and TRITC fluorescence, or blue (alkaline phosphatase) and brown (peroxidase) precipitated enzyme products. This method not only allows detection of intact chromosomes but also the visualization of rearrangements between parts of human and mouse chromosomes. Furthermore, the technique demonstrates the high topological resolution of nonradioactive in situ hybridizations.This investigation was supported in part by FUNGO, Foundation of Medical Scientific Research in The Netherlands (grant nr 13-54-21)     

PCR amplification and simultaneous digoxigenin incorporation of long DNA probes for fluorescence in situ hybridization.

Nonradioactive in situ hybridization has found widespread applications in cytogenetics. Basic requirements are DNA probes in sufficient amounts and of high specificity as well as a labeling protocol of good reproducibility. The PCR has been of fundamental importance for the amplification of DNA sequences and thus for the production of DNA probes. Meanwhile, PCR protocols for amplification of DNA have reached a high degree of automation. So far, incorporation of labeled nucleotides into these DNA probes has normally been done by nick translation. Here we show that in using the PCR, amplification of a DNA probe larger than one kilobase accompanied by simultaneous incorporation of digoxigenin-11-dUTP can be performed for in situ hybridization experiments. As an example, the DNA probe pUC 1.77 specific for the subcentromeric region q12 of chromosome number 1 was used and hybridized against metaphase chromosomes from human lymphocytes. The labeled chromosome region was detected by anti-digoxigenin-fluorescein, Fab fragments. The experiments were evaluated by digital image analysis of microphotographs.     

Down syndrome with duplication of a region of chromosome 21 containing the CuZn superoxide dismutase gene without detectable karyotypic abnormality

Summary We report the case of an 18-month-old boy with many typical Down syndrome features but a normal cytogenetic analysis. High-resolution banding techniques on lymphocytes and fibroblasts of the propositus and his parents did not show any detectable abnormality including that of trisomy 21 mosaicism. However, CuZn superoxide dismutase (CuZn SOD) in the patient's red cells was increased as in trisomy 21. DNA analysis (Southern blots) using a human CuZn SOD probe showed that the genotype of the propositus contained three CuZn SOD genes. In situ hybridization on metaphase chromosomes with the same probe confirmed the gene location in a segment enclosing the distal part of 21q21 and 21q22.1. There was no significant labeling on other chromosomes of the patient. These results indicate that the Down syndrome phenotype of this patient is due to microduplication of a chromosome 21 fragment containing the CuZn SOD gene.     

Nonradioactive in Situ Hybridization with Digoxigenin Labeled DNA Probes

Nonradioactive in situ hybridization techniques are becoming increasingly important tools for rapid analysis of the topological organization of DNA and RNA sequences within cells. Prerequisite for further advances with these techniques are multiple labeling and detection systems for different probes. Here we summarize our results with a recently developed labeling and detection system. The DNA probe for in situ hybridization is modified with digoxigenin-labeled deoxyuridine-triphosphate. Digoxigenin is linked to dUTP via an 11-atom linear spacer (Dig-[11]-dUTP). Labeled DNA probes were hybridized in situ to chromosome preparations. The hybridization signal was detected using digoxigenin-specific antibodies covalently coupled to enzyme markers (alkaline phosphatase or peroxidase) or to fluorescent dyes. Color reactions catalyzed by the enzymes resulted in precipitates located on the chromosomes at the site of probe hybridization. This was verified by hybridizing DNA probes of known chromosomal origin. The signals were analyzed by bright field, reflection contrast and fluorescence microscopy. The results indicate that the new technique gives strong signals and can also be used in combination with other systems (e.g., biotin) to detect differently labeled DNA probes on the same metaphase plate.     

Generation of Five High-Complexity Painting Probe Libraries from Flow-Sorted Mouse Chromosomes

Mouse metaphase chromosomes were purified by flow sorting from the murine fibroblast cell line Mus spretus clone 5A. We sorted chromosomes that fell into five individual peaks based on the Hoechst 33258/chromomycin A3 DNA histogram: three peaks corresponding to the least amount of DNA and two peaks representing chromosomes with the most DNA content. This is the first example of the successful application of bivariate flow karyotyping to murine chromosome sorting. We then applied primer-directed in vitro DNA amplification using the polymerase chain reaction (PCR) to generate and label larger amounts of chromosome-specific DNA. In situ hybridization showed specific binding of the PCR products to mouse chromosomes Y, 19, 18, 3, and X as well as chromosomes 1 and 2. The combination of chromosome sorting from the M. spretus cell line and PCR proved to be highly valuable for generation of pools of DNA fragments that exhibit specific binding to mouse chromosomes and can be used to identify and delineate mouse metaphase chromosomes.     

Sensitive period for the induction of endoreduplication by rotenone in cultured Chinese hamster cells

Rotenone-induced endoreduplication was investigated in Chinese hamster CHL cells. Cell cycle analyses, using 5-bromo-2-deoxyuridine (BrdU) labeling, revealed that endoreduplicaiton was induced between the G₂-phase and mitotic metaphase. Morphological studies indicated that the chromosomes of cells in metaphase at the time of rotenone exposure immediately aggregated. Within 1 h, however, the aggregated chromosomes began to decondense forming telophase nuclei. Cells with aggregated chromosomes were collected by mitotic selection using the mitotic arrestant TN-16 and then cultured for 30 h following rotenone administration. This population of cells demonstrated an extremely high frequency of endoreduplicated metaphases. Further analysis by BrdU labeling indicated that the aggregated metaphases underwent only one round of DNA replication before endoreduplicated metaphases were formed. The most sensitive period for the induction of endoreduplication by rotenone occurs during mitotic metaphase.by M.F. Trendelenburg     

Oligonucleotide-primed in situ DNA synthesis (PRINS): a method for chromosome mapping, banding, and investigation of sequence organization

Oligonucleotides were annealed to complementary sequences in fixed human metaphase chromosomes and extended with DNA polymerase. The newly synthesized fragments were labeled by incorporating bio-11-dUTP instead of TTP, and the sites of synthesis were detected by immunocytochemistry, using fluorochromes as the reporter molecules. We have obtained clear localization with oligonucleotides from alphoid (centromeric sequences), simple sequence (satellite) DNAs, a variety of Alu-dispersed repeated sequences, and oligonucleotides derived from the Tetrahymena and Trypanosoma telomere-specific sequences. The simple sequence and alphoid oligonucleotides gave results at least comparable to those obtained using the whole molecule as a probe for in situ hybridization, whereas the Alu oligonucleotides produced a diversity of results which depended on the absolute length and location of the oligonucleotide within the Alu sequence. The telomere-specific oligomers also produced a variety of results. The G-rich Trypanosoma oligomer and its complementary C-rich sequence produced strong telomeric signals and some interstitial signals on mouse chromosomes, but only weak telomeric signals on human chromosomes. The G-rich Tetrahymena oligomer produced detectable telomeric signals on human chromosomes. The technique appears to be a valuable extension of present tools for mapping and examining the organization of DNA sequences within chromosomes.     

用C-带和涂染技术检测棕色田鼠Y染色体

采用染色体C 带技术和小鼠整条Y染色体特异探针检测棕色田鼠的Y染色体 ,结果如下 :棕色田鼠雄性个体C 带中期分裂相中 ,X性染色体是亚中部着丝粒染色体 ,在着丝粒处存在着强烈的C阳性带 ,而且在短臂的中间也有一条C阳性带 ,但是没有发现深染的Y染色体。用小鼠整条Y染色体特异探针涂染棕色田鼠的骨髓细胞中期分裂相和间期核 ,以小鼠骨髓细胞中期分裂相和间期核作为对照。涂染结果表明 :棕色田鼠骨髓细胞中期分裂相和间期核涂染信号检出率分别为 0 - 2 %和 3% - 5 % ,两者均呈阴性反应 ,而对照都呈阳性反应。根据实验结果 ,作者认为在棕色田鼠的Y染色体上及整个基因组DNA中不存在小鼠整条Y染色体特异DNA的同源序列 ,其Y染色体上可能没有决定雄性性别的重要基因     

Size heterogeneity of telomeric DNA in mouse meiotic chromosomes

Heterogeneity for the length of telomeric DNA sequences has been found among different mitotic chromosomes in several mammalian species. However, there are no studies reporting such heterogeneity in meiotic chromosomes. To analyse this heterogeneity we have performed fluorescence in situ hybridization with a telomeric (C(3)TA(2))(3) peptide nucleic acid (PNA) probe on spread metaphase chromosomes during both male mouse meiotic divisions. Our results show that independently of the meiotic division, telomeric DNA signals were always surrounded by DAPI-stained chromatin, even at centromeric regions. Moreover, we have found heterogeneity for the size of telomeric DNA signals among different chromosomes, between homologues, and even within a given chromosome. We discuss the functional significance of the location of telomeric DNA in condensed meiotic chromosomes, and then the possible origin for the different polymorphisms found.