Interphase fluorescence in situ hybridization mapping: a physical mapping strategy for plant species with large complex genomes

The chromatin in interphase nuclei is much less condensed than are metaphase chromosomes, making the resolving power of fluorescence in situ hybridization (FISH) two orders of magnitude higher in interphase nuclei than on metaphase chromosomes. In mammalian species it has been demonstrated that within a certain range the interphase distance between two FISH sites can be used to estimate the linear DNA distance between the two probes. The intephase mapping strategy has never been applied in plant species, mainly because of the low sensitivity of the FISH technique on plant chromosomes. Using a CCD (charge-coupled device) camera system, we demonstrate that DNA probes in the 4 to 8 kb range can be detected on both metaphase and interphase chromosomes in maize. DNA probes pA1-Lc and pSh2.5·SstISalI, which contain the maize locia1 andsh2, respectively, and are separated by 140 kb, completely overlapped on metaphase chromosomes. However, when the two probes were mapped in interphase nuclei, the FISH signals were well separated from each other in 86% of the FISH sites analyzed. The average interphase distance between the two probes was 0.50 µm. This result suggests that the resolving power of interphase FISH mapping in plant species can be as little as 100 kb. We also mapped the interphase locations of another pair of probes, ksu3/4 and ksu16, which span theRp1 complex controlling rust resistance of maize. Probes ksu3/4 and ksu16 were mapped genetically approximately 4 cM apart and their FISH signals were also overlapped on metaphase chromosomes. These two probes were separated by an average of 2.32 µm in interphase nuclei. The possibility of estimating the linear DNA distance between ksu3/4 and ksu16 is discussed.     

Low persistence of radiation-induced centromere positive and negative micronuclei in cultured human cells

The micronucleus (MN) assay is widely used both in genetic toxicology and in the biomonitoring of human populations. Lymphocytes, cell lines, and bone marrow and epithelial cells are usually employed as target systems in such studies. However, little effort has been done to assess the persistence of MN in highly proliferative cells. To study the behaviour of MN containing whole chromosomes or acentric fragments, we have performed a time course experiment on the persistence of γ-ray (3 Gy) induced MN in a human lymphoblastoid cell line. The frequency and content of MN were analyzed 1, 3, 7, 14, and 56 days after irradiation by pancentromeric fluorescence in situ hybridization (FISH). We observed a clear induction of both centromere positive and negative MN at completion of the first mitotic division. The frequency of both types of MN drastically declined to basal levels 7 days after irradiation with an identical kinetics. We therefore conclude that centromere positive and negative MN are highly unstable upon cell division, indicating that the MN assay could not be a good biomarker of DNA damage induced by acute treatments in highly proliferative cells. The implication of our findings in biomonitoring and in genotoxicity studies is discussed.     

Evolutionary divergence of human chromosome 9 as revealed by the position of the ABL protooncogene in higher primates

Attempts to solve the fundamental questions regarding the descent of man are dogged by superstitions and unexamined orthodoxies. The origin of humans, established a decade ago based upon cytological analysis of ape chromosomes, continues to be called into question. Although molecular methods have provided a framework for tracing the paths of human evolution, conclusive evidence remains elusive. We have used a single ABL gene probe derived from human chromosome 9 to assess the direction of change in the equivalent ape chromosomes. This approach has resulted in a few surprises which again challenge the prevailing view of early primate evolution based solely on chromosome banding patterns. The ABL protooncogene is present on human chromosome 9 at band q34. Similar DNA sequences presumed to represent an ABL gene, are present on chromosome 11 in chimpanzee (Pan troglodytes) but at a different relative location, indicating that the mechanism of the origin of human chromosome 9 is far more complex than has previously been suggested. Nevertheless, in gorilla (Gorilla gorilla) and orangutan (Pongo pygmaeus), the equivalent to human chromosome band 9 q34 is apparently located on chromosome 13 at a putative telomeric position and no discernible differences could be established. Despite the presence of the ABL protooncogene on human equivalent ape chromosomes, molecular systematics will continue to generate enigmas in the evolutionary context until the entire genome is sequenced.     

Mapping a new frontier; development of integrated cytogenetic maps in plants

Cytogenetic maps, as the name implies, incorporate data from genetic maps with actual cytological features of chromosomes such as centromeres, knobs and, recently, fluorescence in situ hybridization (FISH) signals. Integration of genetic and cytological maps has been accomplished primarily in two ways. The first general strategy is to create a chromosome breakpoint, then determine its cytological position using microscopy, and its position on the genetic map using genetic techniques. A second strategy is by the direct hybridization of genetically mapped sequences onto chromosomes by FISH. The aim of this review is to provide an overview of the state of this field in plants. We review the history and uses of cytogenetic maps, and discuss future directions based on what we have learned. Electronic Publication     

Acrocentric centromere organization within the chromocenter of the human sperm nucleus.

It has recently been reported that in human sperm cells, the centromeres are clustered in a chromocenter in the interior region of the nucleus. The aim of the present study was to determine the intra-chromocenter organization of the five centromeres of the acrocentric chromosomes responsible for the biosynthesis of rRNA. The acrocentric centromeres were labeled by fluorescence in situ hybridization (FISH) after mild decondensation of the sperm nuclei to preserve the tail structure. The tail was used as a topographical marker for the orientation of the nucleus. The following results were obtained: (a) the association among the five centromeres was higher than expected from random distribution; (b) all the centromeres observed were randomly located within the chromocenter, occupying about 87% of the total area of the internal nucleus; (c) a major subpopulation of centromeres was located in a preferred area occupying 8.3% of the total nuclear area, with a peak 0.6 microm on the lateral axis and 1.0 microm on the apical side of the longitudinal axis; and (d) The dispersion of the centromeres was not influenced by the degree of the nuclear decondensation. We conclude that in human sperm nuclei, the acrocentric centromeres are organized within a nonlocalized structural element in the chromocenter. The chromocenter can range from an expanded size of 87% of the whole nucleus to a preferred size of 8.3% independent of the degree of nuclear decondensation. These findings have important implications for nuclear function (rRNA) that is not directly related to sperm cell function or early embryo development.     

Nuclear lamina in plant cells

By using selective extraction and diethylene glycol distearate (DGD) embedment and embedment-free electron microscopy, the nuclear lamina was demonstrated in carrot and Ginkgo male generative cells. Western blotting revealed that the nuclear lamina was composed of A-type and B-type lamins which contained at least 66-ku and 84-ku or 66-ku and 86-ku polypeptides, respectively. These lamin proteins were localized at the nudear periphery as shown by immunogold-labelling. In situ hybridization for light microscope and electron microscope showed that plant cells have the homologous sequences of animal lamin cDNA. The sorting site of lamin mRNA is mainly distributed in the cytoplasm near the nudear envelope. The data have verified that there indeed exists nudear lamina in plant cells.     

染色体畸形变与膀胱癌发生发展的相关性研究

目的:通过荧光原位杂交技术(FISH)结合病理分级,探讨染色体畸形变与膀胱癌发生和发展的关系。方法:采用3、7、9、17号染色体着丝粒探针和9P16区带探针对105例膀胱癌复发患者尿液脱落细胞进行荧光原位杂交,观察膀胱癌复发患者中3、7、9、17号染色体的畸形变情况并分析其与患者临床和病理特征之间的关系。结果:105例膀胱癌复发患者中,3、7、9和17号染色体的非整数倍突变率分别是21.9%、29.5%、12.4%、和36.2%,与患者的性别、年龄无显著相关性(P0.05)。仅7号染色体畸变与膀胱癌的病理分级具有显著相关性(P0.05)。结论:7号染色体畸形变与复发膀胱癌的病理分级显著相关。     

染色体畸形变与膀胱癌发生发展的相关性研究

目的：通过荧光原位杂交技术（FISH）结合病理分级,探讨染色体畸形变与膀胱癌发生和发展的关系。方法：采用3、7、9、17号染色体着丝粒探针和9P16区带探针对105例膀胱癌复发患者尿液脱落细胞进行荧光原位杂交．观察膀胱癌复发患者中3、7、9、17号染色体的畸形变情况并分析其与患者临床和病理特征之间的关系。结果：105例膀胱癌复发患者中,3、7、9和17号染色体的非整数倍突变率分别是21．9％、29．5％、12．4％、和36．2％,与患者的性别、年龄无显著相关性（P〉0．05）。仅7号染色体畸变与膀胱癌的病理分级具有显著相关性[（P〈0．05）。结论：7号染色体畸形变与复发膀胱癌的病理分级显著相关。     

An approach to the validation of novel molecular markers of breast cancer via TMA-based FISH scanning

Tissue microarrays (TMA) are valuable tools for validating results of array-based comparative genomic hybridization (ACGH) and other translational research applications requiring independent verification of genomic gains and losses by fluorescence in situ hybridization (FISH). However, spatial orientation and accurate manual tracking of the TMA cores is challenging and prone to error. Image analysis combined with core tracking software, implemented via an automated FISH scanning workstation, represents a new approach to FISH and TMA-based validation of novel genomic changes discovered by ACGH in breast and other cancers. Automated large-scale tissue microarray validation FISH studies of genomic gains and losses identified by ACGH for breast cancer are feasible using an automated imaging scanner and tracking/classifying software. Furthermore, by leveraging the bifunctional fluorescent and chromogenic properties of the alkaline phosphatase chromogen fast red K and combining the technology with FISH, correlative and simultaneous phenotype/genotype studies may be enabled.     

Characterization of a Wheat- wheatgrass Translocation Line by FISH

The genomic DNA of wheatgrass (Agropyron intermedium (Host) P.B. = Elytrigia intermedia (Host) Nevski = Thinopyrum intermedium (Host) Barkworth and Dewey) was labelled with biotin-16-dUTP as a probe, and genomic DNA of common wheat ( Triticum aestivum L. ) "Chinese Spring" was used for blocking. Wheat-wheatgrass line 33 was examined by fluorescence in situ hybridization (FISH) technique. The terminal regions of a pair of chromosomes showed green fluorescent signals. It has been concluded that chromosome segrnents containing alien genes of wheatgrass are located at the terminal regions of wheat chromosomes in wheat-wheatgrass line 33, and the translocated segments were small. Wheat-wheatgrass line 33 has been proved to be a translocation line with chromosome segments of wheatgrass translocated to the terminal regions of wheat chromoSomes.     

Isolation of carp cDNA clones,representing developmentally-regulated genes,using a subtractive-hybridization strategy

A subtractive-hybridization technique, combined with differential screenings and subsequent whole mount in situ hybridization (ISH) reactions, was used to isolate novel cDNA clones representing developmentally-regulated genes of carp. Small-scale differential screenings of an oocyte and a segmentation-stage cDNA library using oocyte-specific and segmentation stage-specific enriched probes, yielded 75 positive clones. ISH screening showed that 65% (15) of the oocyte-stage clones and 50% (26) of the segmentation-stage clones were indeed stage-specific. Partial sequence analysis suggests that approximately 65% of the 41 stage-specific clones represent novel genes. In addition, an Otxl clone was isolated. Two novel clones and the Otxl clone are of special interest for developmental studies. The clones represent genes that are locally expressed during embryonic development. The expression patterns of Otxl and one of the novel clones suggest functions in specification of the anterior-posterior axis. The three clones provide molecular markers for the study of gastrulation and the patterning of the a-p axis in teleosts.     

GLPp16/CSP17 探针在膀胱尿路上皮癌诊断中的应用

目的:探讨荧光原位杂交技术辅助诊断膀胱尿路上皮癌的可行性。方法:标记为17号染色体着丝粒及9号染色体p16位点9p21区带探针,采用荧光原位杂交技术(Fluorescence in Situ Hybridization FISH)对80例膀胱肿瘤患者尿液间期细胞核进行荧光原位杂交,以20例健康志愿者作为正常对照组,建立阈值。以术后病理结果作为诊断"金标准",对80例膀胱肿瘤患者同时行尿脱落细胞学检查,与FISH进行比较。结果:17号染色体和9p21的畸变率分别为57.5%和63.8%。17号染色体畸变率主要表现为多倍体,与膀胱癌的分级有显著相关性(P<0.01);9号染色体畸变率主要变现为染色体缺失,与膀胱癌分期分级均无相关性(P>0.05)。尿脱落细胞学灵敏度为12.2%,FTSH技术灵敏度为86.5%;两者差异有统计学意义(P<0.01)。结论:荧光原位杂交技术可以作为膀胱尿路上皮癌诊断的一项重要方法,并可能在预后判断中具有重要临床意义。     

植物染色体原位杂交技术的发展与现状

本文主要介绍植物染色体原位杂交技术的发展历史,评述适用于不同研究目的的各种主要原位杂交技术的基本原理和方法,并介绍该技术在植物细胞遗传学领域的应用和发展。 Abstract：In this paper,we briefly introduce the development of in situ hybridization of plant chromosome. The fundamental principle and method of many main kinds of in situ hybridization have been reviewed for different research purposes,and their application and development on plant cytogenetics also been recommended.     

Detection of chromosome 17- and X-bearing human spermatozoa using fluorescence in situ hybridization.

Fluorescence in situ hybridization (FISH) with DNA probes specific to chromosomes 17 and the X has been applied to human ejaculated sperm. After sperm nuclei were decondensed with EDTA and DTT, biotinylated alpha satellite DNA probes TR17 and TRX were separately used on preparations from thirteen healthy donors. After hybridization 96% of sperm were labelled with the TR17 probe and 48% of sperm were labelled with the TRX probe. Frequencies of 0.33% disomic 17 and 0.29% disomic X sperm were found. The frequencies of diploid sperm were assessed as 0.37% using the TR17 probe and 0.20% using the TRX probe which labelled only one half of the sperm; after correcting the result from the X-probe to 0.40% the two frequencies are very similar.