An approach for quantitative assessment of fluorescence in situ hybridization (FISH) signals for applied human molecular cytogenetics.

A number of applied molecular cytogenetic studies require the quantitative assessment of fluorescence in situ hybridization (FISH) signals (for example, interphase FISH analysis of aneuploidy by chromosome enumeration DNA probes; analysis of somatic pairing of homologous chromosomes in interphase nuclei; identification of chromosomal heteromorphism after FISH with satellite DNA probes for differentiation of parental origin of homologous chromosome, etc.). We have performed a pilot study to develop a simple technique for quantitative assessment of FISH signals by means of the digital capturing of microscopic images and the intensity measuring of hybridization signals using Scion Image software, commonly used for quantification of electrophoresis gels. We have tested this approach by quantitative analysis of FISH signals after application of chromosome-specific DNA probes for aneuploidy scoring in interphase nuclei in cells of different human tissues. This approach allowed us to exclude or confirm a low-level mosaic form of aneuploidy by quantification of FISH signals (for example, discrimination of pseudo-monosomy and artifact signals due to over-position of hybridization signals). Quantification of FISH signals was also used for analysis of somatic pairing of homologous chromosomes in nuclei of postmortem brain tissues after FISH with "classical" satellite DNA probes for chromosomes 1, 9, and 16. This approach has shown a relatively high efficiency for the quantitative registration of chromosomal heteromorphism due to variations of centromeric alphoid DNA in homologous parental chromosomes. We propose this approach to be efficient and to be considered as a useful tool in addition to visual FISH signal analysis for applied molecular cytogenetic studies.     

Characterization and application of soybean YACs to molecular cytogenetics

Yeast artificial chromosomes (YACs) are widely used in the physical analysis of complex genomes. In addition to their value in chromosome walking for map-based cloning, YACs represent excellent probes for chromosome mapping using fluorescence in situ hybridization (FISH). We have screened such a library for low-copy-number clones by hybridization to total genomic DNA. Four clones were chosen for chromosome tagging based upon their low or moderate signal. By using degenerate oligonucleotide-primed PCR (DOP-PCR), we were able to use relatively small amounts of soybean YAC DNA, isolated directly by preparative pulsed-field gel electrophoresis, as FISH probes for both metaphase chromosome spreads and interphase nuclei. FISH chromosomal analysis using the three of the clones as probes resulted in relatively simple hybridization patterns consistent with a single homologous locus or two homoeologous loci. The fourth YAC probe resulted in a diffuse hybridization pattern with signal on all metaphase chromosomes. We conclude that YACs represent a valuable source of probes for chromosomal analysis in soybean.     

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.     

Fluorescence intensity profiles of in situ hybridization signals depict genome architecture within human interphase nuclei

An approach towards construction of two-dimensional (2D) and three-dimensional (3D) profiles of interphase chromatin architecture by quantification of fluorescence in situ hybridization (FISH) signal intensity is proposed. The technique was applied for analysis of signal intensity and distribution within interphase nuclei of somatic cells in different human tissues. Whole genomic DNA, fraction of repeated DNA sequences (Cot 1) and cloned satellite DNA were used as probes for FISH. The 2D and 3D fluorescence intensity profiles were able to depict FISH signal associations and somatic chromosome pairing. Furthermore, it allowed the detection of replicating signal patterns, the assessment of hybridization efficiency, and comparative analysis of DNA content variation of specific heterochromatic chromosomal regions. The 3D fluorescence intensity profiles allowed the analysis of intensity gradient within the signal volume. An approach was found applicable for determination of assembly of different types of DNA sequences, including classical satellite and alphoid DNA, gene-rich (G-negative bands) and gene-poor (G-positive bands) chromosomal regions as well as for assessment of chromatin architecture and targeted DNA sequence distribution within interphase nuclei. We conclude the approach to be a powerful additional tool for analysis of interphase genome architecture and chromosome behavior in the nucleus of human somatic cells. The text was submitted by the authors in English.     

Quantitative microscopy after fluorescence in situ hybridization - a comparison between repeat-depleted and non-depleted DNA probes

Complex probes used in fluorescence in situ hybridization (FISH) usually contain repetitive DNA sequences. For chromosome painting, in situ suppression of these repetitive DNA sequences has been well established. Standard painting protocols require large amounts of an unlabeled 'blocking agent', for instance Cot-1 DNA. Recently, it has become possible to remove repetitive DNA sequences from library probes by means of magnetic purification and affinity PCR. Such a 'repeat depleted library probe' was hybridized to the q-arm of chromosome 15 of human metaphase spreads and interphase cell nuclei without any preannealing by Cot-1 DNA. Apart from this, 'standard' FISH conditions were used. After in situ hybridization, microscope images were obtained comparable to those achieved with the #15q library probe prior to depletion. The images were recorded by a true color CCD camera. By digital image analysis using 'line scan' and 'area scan' procedures, the painting efficiency expressed in terms of relative fluorescence signal intensity was quantitatively evaluated. The painting efficiency using the repeat depleted probe of chromosome 15q was compared to the painting efficiency after standard FISH. The results indicate that both types of probes are compatible to a high FISH efficiency. Using equivalent probe concentrations, no significant differences were found for FISH with standard painting probes and repeat depleted painting probes.     

Amplification of the Y chromosome in three murine tumor cell lines transformed in vivo by different human prostate cancers

Summary Conventional and molecular cytogenetic analyses of three murine cancer cell lines that had been induced in male athymic mice by the injection of three different human prostate cancer cell lines revealed selective amplification of the Y chromosome. In particular, analysis of metaphase and interphase nuclei by fluorescence in situ hybridization (FISH) with the mouse Y chromosome-specific DNA painting probe revealed the presence of various numbers of Y chromosomes, ranging from one to eight, with a large majority of nuclei showing two copies (46.5–60.1%). In Interphase nuclei, the Y chromosomes showed distinct morphology, allowing identification irrespective of whether the preparations were treated for 15 min or for 5 h with Colcemid, a chemical known to cause chromosome condensation. However, FISH performed on human lymphocyte cultures with chromosome-specific DNA painting probes other than the Y chromosome did not reveal condensed chromosome morphology in interphase nuclei even after 12 h of Colcemid treatment. Our FISH results indicate that (1) the Y chromosome is selectively amplified in all three cell lines; (2) the mouse Y chromosome number is comparable in both interphase and metaphase cells; (3) the Y chromosome number varies between one and eight, with a large majority of cells showing two or three copies in most interphase nuclei; (4) the condensation of the Y chromosome is not affected by the duration of Colcemid treatment but by its inherent DNA constitution; and (5) the number of copies of the Y chromosome is increased and retained not only in human prostate tumor cell lines but also in murine tumors induced by these prostate tumor cell lines.     

Cytogenetic analysis of human solid tumors by in situ hybridization with a set of 12 chromosome-specific DNA probes

We have applied fluorescent in situ hybridization (FISH) to assess the presence of numerical chromosome aberrations in fresh specimens of human solid tumors of varying histology. For this purpose, a set of 12 biotinylated chromosome-specific, repetitive alpha-satellite DNA probes (for chromosomes 1, 6, 7, 9, 10, 11, 15, 16, 17, 18, X and Y) were hybridized directly to isolated interphase nuclei. Utilizing this approach, we found numerical chromosome changes in all tumors. FISH ploidy profiles were in accordance with flow cytometric DNA histograms of these tumor cells.     

Variations in alphoid DNA sequences escape detection of aneuploidy at interphase by FISH technique.

The advent of a new staining technique, termed fluorescence in situ hybridization (FISH), allows the rapid identification of the genomic constitution of an individual with aneuploidy even in interphase nuclei through the use of a series of chromosome-specific DNA probes, an approach termed "interphase cytogenetics." However, alphoid DNA sequences of every centromere are polymorphic (heteromorphic), and the number of targeted sequences may be below the detection level of a specific DNA probe, thus escaping detection and resulting in the imprecise identification of the chromosomal constitution at interphase. The limitations associated with the FISH technique have dire consequences which are emphasized here with an example in which the presence of an additional chromosome 21 in two siblings born consecutively with trisomy 21 (Down syndrome) was not detected by "interphase cytogenetics." The copy number of alphoid DNA sequences of one of the paternal chromosomes 21 was low and resulted in discordance between domain numbers at interphase and actual chromosome numbers at metaphase in both children. This is an isolated incident that could have led to a misdiagnosis if FISH were the only test employed. Although the advantages of this technology are undeniably enormous, the present finding has made it apparent that precise standards and reliability of the procedure must be established prior to its routine application.     

Detection of aneuploidy involving chromosomes 13, 18, or 21, by fluorescence in situ hybridization (FISH) to interphase and metaphase amniocytes.

Fluorescence in situ hybridization (FISH) with chromosome-specific probes has been applied to detection of numerical aberrations involving chromosomes 13, 18, and 21 in metaphase and interphase amniocytes. High-complexity, composite probes for chromosomes 13, 18, and 21 were used as hybridization probes for this study. These probes were constructed as chromosome-specific libraries in Bluescribe plasmids and are designated pBS-13, pBS-18, and pBS-21. Elements of these probes bind at numerous sites along the target chromosome and, when detected fluorescently, stain essentially the entire long arm of the target chromosome. The target chromosome number (i.e., the number of chromosomes of the type for which the probe was specific) was correctly determined in 20 of 20 samples in which metaphase spreads were analyzed and in 43 of 43 samples in which interphase nuclei were analyzed; all of these studies were conducted in blind fashion. These results suggest the utility of FISH with composite probes for rapid detection of numerical aberrations in metaphase and interphase amniotic cells.     

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.     

植物荧光原位杂交技术的发展及其在植物基因组分析中的应用

荧光原位杂交(FISH)是在染色体、间期核和DNA纤维上定位特定DNA序列的一种有效而精确的分子细胞遗传学方法。20年来,植物荧光原位杂交技术发展迅速:以增加检测的靶位数为目的,发展了双色FISH、多色FISH和多探针FISH鸡尾酒技术;为增加很小染色体目标的检测灵敏度,发展了BAC-FISH和酪胺信号放大FISH(TSA-FISH)等技术;以提高相邻杂交信号的空间分辨力为主要目的,发展了高分辨的粗线期染色体FISH、间期核FISH、DNA纤维FISH和超伸展的流式分拣植物染色体FISH技术。在植物基因组分析中,FISH技术发挥了不可替代的重要作用,它可用于:物理定位DNA序列,并为染色体的识别提供有效的标记;对相同DNA序列进行比较物理定位,探讨植物基因组的进化;构建植物基因组的物理图谱;揭示特定染色体区域的DNA分子组织;分析间期核中染色质的组织和细胞周期中染色体的动态变化;鉴定植物转基因。     

Interphase cytogenetics in pathology: principles, methods, and applications of fluorescence in situ hybridization (FISH)

 Characteristic chromosome aberrations have been identified in various tumors. Fluorescence in situ hybridization (FISH) using specific probes that are generated by vector cloning or in vitro amplification and labeled with fluorescent dyes allow for the detection of these genetic changes in interphase cells. This technique, that is also referred to as ”interphase cytogenetics”, can be performed in cytological preparations as well as in sections of routinely formaldehyde-fixed and paraffin-embedded tissue. In cancer research and diagnostics, interphase cytogenetics by FISH is used to detect numerical chromosome changes and structural aberrations, e.g., translocations, deletions, or amplifications. In this technical overview, we explain the principles of the FISH method and provide protocols for FISH in cytological preparations and paraffin sections. Moreover, possible applications of FISH are discussed. Accepted: 22 July 1997     

Chromosome painting in Arabidopsis thaliana

Chromosome painting, that is visualisation of chromosome segments or whole chromosomes based on fluorescence in situ hybridization (FISH) with chromosome-specific DNA probes is widely used for chromosome studies in mammals, birds, reptiles and insects. Attempts to establish chromosome painting in euploid plants have failed so far. Here, we report on chromosome painting in Arabidopsis thaliana (n = 5, 125 Mb C(-1)). Pools of contiguous 113-139 BAC clones spanning 2.6 and 13.3 Mb of the short and the long arm of chromosome 4 (17.5 Mb) were used to paint this entire chromosome during mitotic and meiotic divisions as well as in interphase nuclei. The possibility of identifying any particular chromosome region on pachytene chromosomes and within interphase nuclei using selected BACs is demonstrated by differential labelling. This approach allows us, for the first time, to paint an entire autosome of an euploid plant to study chromosome rearrangements, homologue association, interphase chromosome territories, as well as to identify homeologous chromosomes of related species.     

Study of fluorescence in situ hybridization for detection of chromosome aberration

The authors applied fluorescence in situ hybridization (FISH) technique for the detection of chromosome aberration in interphase nuclei using the probe specific to alphoid repeats on chromosome 11 and X. Chromosome 11 specific probe showed two major spots in lymphocyte nuclei, while X specific probe showed single spot in male and double spots in female respectively. On the other hand three spots were detected in most of the nuclei from HeLa cells with 11 and X specific probes. We concluded that FISH with the use of chromosome specific probe may become a useful and reliable tool for the detection of chromosome aberration in interphase nuclei.     

Meiotic cytology and chromosome behaviour in wild-type Arabidopsis thaliana

This article reviews the historical development of cytology and cytogenetics in Arabidopsis, and summarizes recent developments in molecular cytogenetics, with special emphasis on meiotic studies. Despite the small genome and small chromosomes of Arabidopsis, considerable progress has been made in developing appropriate cytogenetical techniques for chromosome analysis. Fluorescence in situ hybridization (FISH) applied to extended meiotic pachytene chromosomes has resulted in a standardized karyotype (idiogram) for the species that has also been aligned with the genetical map. A better understanding of floral and meiotic development has been achieved by combining cytological studies, based on both sectioning and spreading techniques, with morphometric data and developmental landmarks. The meiotic interphase, preceding prophase I, has been investigated by marking the nuclei undergoing DNA replication with BrdU. This allowed the subclasses of meiotic interphase to be distinguished and also provided a means to time the duration of meiosis and its constituent phases. The FISH technique has been used to analyse in detail the meiotic organization of telomeres and centromeric regions. The results indicate that centromere regions do not play an active role in chromosome pairing and synapsis; however, telomeres pair homologously in advance of general chromosome synapsis. The FISH technique is currently being applied to analysing the pairing and synapsis of interstitial chromosome regions through interphase and prophase I. FISH probes also allow the five bivalents of Arabidopsis to be identified at metaphase I and this has permitted an analysis of chiasma frequencies in individual bivalents, both in wild-type Arabidopsis and in two meiotic mutants.     

Molecular Cytogenetic Characteristics of Chromosome Imbalance in Spontaneous Human Abortion Cells with Low Proliferative Activity in Vitro

Karyotyping of noncultivated cells of 60 first-trimester spontaneous abortions (blighted ovum and missed abortions) was carried out using fluorescence in situ hybridization (FISH) with centromere-specific DNA probes for all chromosomes of the karyotype. Conventional cytogenetic study of these abortions was impossible because of cell culture failures. The algorithm is proposed for molecular cytogenetic FISH analysis of interphase karyotypes. Chromosome abnormalities were found in 32 fetuses (53.3%). In groups of missed abortions and blighted ovum, the frequency of numerical chromosome abnormalities was 50 and 60%, respectively. Both the numerical chromosome abnormalities typical of spontaneous human abortions (autosomal trisomies, sex chromosome aneuploidy, and polyploidy) and a relatively rare type of genomic imbalance unidentifiable by standard cytogenetic analysis (autosomal monosomies 7, 15, 21, and 22 in mosaic state) were observed. The frequency of these type of chromosome abnormalities comprised 19% of all known karyotype abnormalities determined in spontaneously aborted embryos. Note that the level of confined placental mosaicism in embryos with low cell proliferative activity was 25%, which is substantially higher than the corresponding parameter (1–2%) determined by prenatal diagnosis of chromosome abnormalities in developing embryos. The results of interphase FISH analysis of cells with low proliferative activity in vitro suggest that the pathology of early fetal development and missed abortion in humans are associated with a wider spectrum of chromosome abnormalities.     

Genomic imbalances are confined to non-proliferating cells in paediatric patients with acute myeloid leukaemia and a normal or incomplete karyotype

Leukaemia is often associated with genetic alterations such as translocations, amplifications and deletions, and recurrent chromosome abnormalities are used as markers of diagnostic and prognostic relevance. However, a proportion of acute myeloid leukaemia (AML) cases have an apparently normal karyotype despite comprehensive cytogenetic analysis. Based on conventional cytogenetic analysis of banded chromosomes, we selected a series of 23 paediatric patients with acute myeloid leukaemia and performed whole genome array comparative genome hybridization (aCGH) using DNA samples derived from the same patients. Imbalances involving large chromosomal regions or entire chromosomes were detected by aCGH in seven of the patients studied. Results were validated by fluorescence in situ hybridization (FISH) to both interphase nuclei and metaphase chromosomes using appropriate bacterial artificial chromosome (BAC) probes. The majority of these copy number alterations (CNAs) were confirmed by FISH and found to localize to the interphase rather than metaphase nuclei. Furthermore, the proliferative states of the cells analyzed by FISH were tested by immunofluorescence using an antibody against the proliferation marker pKi67. Interestingly, these experiments showed that, in the vast majority of cases, the changes appeared to be confined to interphase nuclei in a non-proliferative status.     

Molecular cytogenetic characteristics of chromosome imbalance in cells of spontaneous human abortion fetuses with low proliferative activity in vitro

Karyotyping of noncultivated cells of 60 first-trimester spontaneous abortions (blighted ova and missed abortions) was carried out using fluorescence in situ hybridization (FISH) with centromere-specific DNA probes for all chromosomes of the karyotype. Conventional cytogenetic study of these abortions was impossible because of cell culture failures. The algorithm is proposed for molecular cytogenetic FISH analysis of interphase karyotypes. Chromosome abnormalities were found in 32 fetuses (53.3%). In groups of missed abortions and blighted ova, the frequency of numerical chromosome abnormalities was 50 and 60%, respectively. Both the numerical chromosome abnormalities typical of spontaneous human abortions (autosomal trisomies, sex chromosome aneuploidy, and polyploidy) and a relatively rare type of genomic imbalance unidentifiable by standard cytogenetic analysis (autosomal monosomies 7, 15, 21, and 22 in mosaic state) were observed. The frequency of these type of chromosome abnormalities comprised 19% of all known karyotype abnormalities determined in spontaneously perished embryos. Note that the level of confined placental mosaicism in embryos with low cell proliferative activity was 25%, which is substantially higher than the corresponding parameter (1-2%) determined by prenatal diagnosis of chromosome abnormalities in developing embryos. The results of interphase FISH analysis of cells with low proliferative activity in vitro suggest that the pathology of early fetal development and missed abortion in humans are associated with a wider spectrum of chromosome abnormalities.     

Ordering markers in the region of the ataxia-telangiectasia gene (11q22-q23) by fluorescence in situ hybridization (FISH) to interphase nuclei

Fluorescence in situ hybridization (FISH) to interphase nuclei was performed to order probes corresponding to bands 11q22-q23 where the ataxia-telangiectasia (AT) gene(s) have been located. Cosmid probes and one phage probe previously localized to this chromosome 11 region by FISH to metaphase chromosomes, were hybridized to interphase nuclei of the somatic cell hybrid J1a, which contains chromosome 11 as the only human chromosome. Two-color FISH was used with a centromeric reference probe marker. The following order was obtained: cen-D11S385 (CJ52.75)-CJ52.3-D11S384 (CJ52.193) CJ52.114-D11S424 (CJ52.77)-D11S132-NCAM-D11S351 (CJ52.208)-tel. The validity of using the centromeric probe was illustrated by showing that a probe corresponding to 11p13 hybridized more closely to the centromere than a probe corresponding to 11q22-q23, and by using cosmids hybridized three by three.