A Fast Air-dry Dropping Chromosome Preparation Method Suitable for FISH in Plants

Preparation of chromosome spreads is a prerequisite for the successful performance of fluorescence in situ hybridization (FISH). Preparation of high quality plant chromosome spreads is challenging due to the rigid cell wall. One of the approved methods for the preparation of plant chromosomes is a so-called drop preparation, also known as drop-spreading or air-drying technique. Here, we present a protocol for the fast preparation of mitotic chromosome spreads suitable for the FISH detection of single and high copy DNA probes. This method is an improved variant of the air-dry drop method performed under a relative humidity of 50%-55%. This protocol comprises a reduced number of washing steps making its application easy, efficient and reproducible. Obvious benefits of this approach are well-spread, undamaged and numerous metaphase chromosomes serving as a perfect prerequisite for successful FISH analysis. Using this protocol we obtained high-quality chromosome spreads and reproducible FISH results for Hordeum vulgare, H. bulbosum, H. marinum, H. murinum, H. pubiflorum and Secale cereale.     

Multi-locus (ML)-FISH is a reliable tool for nondisjunction studies in human oocytes

In the present study, we developed a fluorescence in situ hybridization (FISH) strategy, which allows a reliable determination of the chromatid number of specific chromosomes in mature human oocytes. 168 unfertilized oocytes were analyzed by dual-color FISH with two direct-labeled locus-specific DNA probes for chromosome 13 and 21. To exclude FISH failures, metaphases with abnormal signal patterns were reanalyzed by multi-locus-FISH (ML-FISH) for chromosome 13 and 21. Following dual-color FISH, abnormal signal patterns were detected in 21 out of 108 metaphases (19.4%). 17 of these metaphases were reanalyzed by ML-FISH. In contrast to the first FISH, seven metaphases showed normal signal patterns after rehybridization, whereas ten metaphases remained abnormal. Out of these real aneuploid metaphases, five showed gain or loss of a single signal (= chromatid), two showed missing double signals (= chromosome) and three showed both. In conclusion, locus-specific FISH probes facilitate differentiation between first meiotic nondisjunction of whole chromosomes and prematurely divided chromatids. Moreover, simultaneous hybridization with a second locus-specific probe on the same chromatid (ML-FISH) helps to differentiate between FISH failures and real meiotic division errors and therefore, allows a more reliable analysis of aneuploidies in human oocytes.     

An improved technique for obtaining well-spread metaphases from plants with numerous large chromosomes

Preparations that contain well-spread metaphase chromosomes are critical for plant cytogenetic analyses including chromosome counts, banding procedures, in situ hybridization, karyotyping and construction of ideograms. Chromosome spreading is difficult for plants with large and numerous chromosomes. We report here a technique for obtaining cytoplasm-free, well-spread metaphases from two Amaryllidaceae species: Sprekelia formosissima (2n = 120) and Hymenocallis howardii (2n = 96). The technique has three main steps: 1) pretreatment to cause chromosome condensation, 2) dripping onto tilted slides coated with a thin layer of pure acetic acid and 3) application of steam and acetic acid to produce cytoplasmic hydrolysis, which spreads the chromosomes.     

A simple reliable procedure for obtaining metaphases from human leukemic bone-marrow aspirates suitable for Giemsa banding

Summary Short incubation of heparinized human leukemic bone-marrow cells in phosphate buffered saline containing colcemid and overnight chilling of fixed cells yields metaphases with elongated and well-spread chromosomes. This technique enables us to do trypsin-Giemsa banding of chromosomes obtained from leukemic marrow cells otherwise difficult to band.     

Preparations of meiotic pachytene chromosomes and extended DNA fibers from cotton suitable for fluorescence in situ hybridization

Fluorescence in situ hybridization (FISH) has become one of the most important techniques applied in plant molecular cytogenetics. However, the application of this technique in cotton has lagged behind because of difficulties in chromosome preparation. The focus of this article was FISH performed not only on cotton pachytene chromosomes, but also on cotton extended DNA fibers. The cotton pollen mother cells (PMCs) instead of buds or anthers were directly digested in enzyme to completely breakdown the cell wall. Before the routine acetic acid treatment, PMCs were incubated in acetic acid and enzyme mixture to remove the cytoplasm and clear the background. The method of ice-cold Carnoy's solution spreading chromosome was adopted instead of nitrogen removed method to avoid chromosomes losing and fully stretch chromosome. With the above-improved steps, the high-quality well-differentiated pachytene chromosomes with clear background were obtained. FISH results demonstrated that a mature protocol of cotton pachytene chromosomes preparation was presented. Intact and no debris cotton nuclei were obtained by chopping from etiolation cotyledons instead of the conventional liquid nitrogen grinding method. After incubating the nuclei with nucleus lysis buffer on slide, the parallel and clear background DNA fibers were acquired along the slide. This method overcomes the twist, accumulation and fracture of DNA fibers compared with other methods. The entire process of DNA fibers preparation requires only 30 min, in contrast, it takes 3 h with routine nitrogen grinding method. The poisonous mercaptoethanol in nucleus lysis buffer is replaced by nonpoisonous dithiothreitol. PVP40 in nucleus isolation buffer is used to prevent oxidation. The probability of success in isolating nuclei for DNA fiber preparation is almost 100% tested with this method in cotton. So a rapid, safe, and efficient method for the preparation of cotton extended DNA fibers suitable for FISH was established.     

短柄草染色体酶解法制片技术

以六倍体短柄草为研究材料,对短柄草的染色体制片方法进行了优化,建立了一种改进的短柄草染色体酶解制片方法。试验结果表明,以45%醋酸固定液固定根尖、酶解时间2h可以获得最佳的根尖染色体制片。此方法不仅可以得到分散良好的有丝分裂中期分裂相,而且还缩短了酶解的时间,提高了制片的效率。     

S基因在甘蓝EDFs上的高分辨率荧光原位杂交定位

植物细胞壁及细胞质的存在和植物染色体所具有的高浓缩特性,限制高效率原位杂交定位在植物细胞内的进行。针对小型染色体芸薹属植物采用常规方法DNA制备纤维的效果不佳的特点,新建制备方法：利用减数分裂前期的染色体为材料,在硅化的玻片上先后通过蛋白酶解和乙醇：乙酸（3：1）的适当处理,采用移动界而法制备EDFs。制备的EDFs比未经伸长处理的染色体在经向和横向方面分别取得较高程度的伸长与膨胀,长度可达到89-257μm,比相应地中期染色体增长30107倍,分辨率可达42．853．0kb。利用SRK和SCR两种探针同时在甘蓝粗线期染色体和EDFs上进行了原位杂交,首次鉴定了S基因座在其单倍体基因组中单拷贝性。在杂交信号检测中尽管未经过信号放大,但仍然可以观察到清晰的绿色信号;经荧光显微镜观察,在单一的EDF上发现两个相距1μm的SCR和SRK的信号点,由此得出局部分辨率为4kb的最高伸长度。     

Chromosomal rearrangements in cattle and pigs revealed by chromosome microdissection and chromosome painting

A pericentric inversion of chromosome 4 in a boar, as well as a case of (2q-;5p+) translocation mosaicism in a bull were analysed by chromosome painting using probes generated by conventional microdissection. For the porcine inversion, probes specific for p arms and q arms were produced and hybridised simultaneously on metaphases of a heterozygote carrier. In the case of the bovine translocation, two whole chromosome probes (chromosome 5, and derived chromosome 5) were elaborated and hybridised independently on chromosomal preparations of the bull who was a carrier of the mosaic translocation. The impossibility of differentiating chromosomes 2 and der(2) from other chromosomes of the metaphases did not allow the production of painting probes for these chromosomes. For all experiments, the quality of painting was comparable to that usually observed with probes obtained from flow-sorted chromosomes. The results obtained allowed confirmation of the interpretations proposed with G-banding karyotype analyses. In the bovine case, however, the reciprocity of the translocation could not be proven. The results presented in this paper show the usefulness of the microdissection technique for characterising chromosomal rearrangements in species for which commercial probes are not available. They also confirmed that the main limiting factor of the technique is the quality of the chromosomal preparations, which does not allow the identification of target chromosomes or chromosome fragments in all cases.     

Chromosome preparations of human blood lymphocytes—Evaluation of techniques

Several parameters of human lymphocyte culturing techniques and metaphase chromosome preparation procedures were studied and quantitatively evaluated in regard to their influence on the results of Q and G banding procedures. The culturing conditions were studied using³H thymidine incorporation as a parameter. A whole blood culturing technique using Ham's F12 medium was found to give optimal and consistent results. Colcemid concentration proved to be of no influence on chromosome contraction or on the number of metaphases obtained over the concentration range investigated. Prolonged exposure to colcemid was found to cause a decrease in the mean chromosome length but the absolute number of metaphases with a low degree of chromosomal contraction hardly decreased. Different spreading techniques were quantitatively analysed and factors important for the spreading of chromosomes were evaluated. Based on the results obtained, an optimal procedure is described which over a period of one year has given consistent results.     

Paint-assisted microdissection-FISH: Rapid and simple mapping of translocation breakpoints in the embryonal rhabdomyosarcoma cell line RD.

BACKGROUND: Spectral karyotyping and multiple fluorophore fluorescence in situ hybridisation (M-FISH) facilitate identification of inter-chromosomal rearrangements, but are of low cytogenetic resolution in mapping translocation breakpoints. Reverse chromosome painting yields increased cytogenetic information but isolation of aberrant chromosomes is technically difficult. We have developed the technique of paint-assisted microdissection FISH (PAM-FISH), which enables microdissection of aberrant chromosomes to be carried out easily and rapidly using relatively simple apparatus. METHODS: A selected chromosome paint is hybridised to abnormal metaphases to label a chromosome of interest, which is then microdissected, amplified, labelled by polymerase chain reaction (PCR), and reverse painted onto extended normal metaphases. RESULTS: PAM-FISH was used to reassess structural chromosomal abnormalities identified by molecular cytogenetics in the rhabdomyosarcoma cell line RD. PAM-FISH improved the analysis of virtually all structural abnormalities, identifying six novel translocations and indicating that seven previously described rearrangements were in fact not present in RD. Accuracy of the breakpoint mapping obtained was confirmed by bacterial artificial chromosome-FISH. CONCLUSIONS: PAM-FISH is ideally suited to analysis of tumour metaphases as it is not affected by poor chromosome morphology. Reagents generated by PAM-FISH are also suitable for other investigations, such as mapping using sequence tagged-site PCR or genomic microarrays. PAM-FISH is technically straightforward and could readily be adopted in a routine cytogenetics laboratory for accurate high-throughput analysis of chromosome breakpoints.     

Chromosome and cytome analysis of the somatic cells of nuclear-chemical production workers with incorporated 239Pu

The analysis of plutonium production factors has been carried out by using two methodical approaches: assessment of chromosomal aberrations level in routine and G-banded metaphases and molecular-cytogenetic investigation of aneugenic/clastogenic damages in cytokinesis-block binuclear lymphocytes by FISH with centromere specific DNA probes. The obtaining data point out for the first time about both aneugenic and clastogenic influences of incorporated 239Pu with activity range from 0.37 to 6.95 kBq. Correlation analysis of chromosome aberrations with cytome abnormalities allowed finding significant connection between number parameters of metaphase and interphase approaches. The results of this study support the suggestion that aberrant chromosomes are involved preferable in aneugenic events. The FISH technique in binucleated cytokinesis-blocked lymphocytes allows extending of detecting spectrum of chromosome damages and glance of aneugenic mechanisms. Correlations between metaphase and interphase-FISH results point out a high sensitivity of FISH cytome assay, which could be used as an independent test for detection both clastogenic and aneugenic environment influences.     

刺鳅X染色体DNA文库的构建

刺鳅（Mastacembelus aculeatus）是具有明显X和Y异形性染色体分化的淡水鱼。本实验室通过显微切割（Microdissection）和兼并引物PCR（DOP-PCR）方法,从雌性刺鳅中期染色体分裂相中分离获得X染色体并扩增其DNA,利用T载体和电转化方法,建立了刺鳅X染色体DNA质粒文库。该文库插入片段的平均长度约为500bp,理论上包含X染色体98％以上的序列。当用荧光原位杂交（FISH）来验证文库的专一性时发现,在无竞争性DNA杂交条件下,整个X和Y染色体上都表现出强烈的杂交信号,并且常染色体上也出现一些随机散布信号;当含有竞争性DNA时,常染色体上的信号消失,仅性染色体上异染色质区域保留有较强信号。就此,本文对刺鳅性染色体上的序列类型进行了探讨。     

Cytological research on the argentophilic nucleolus-organizer regions of the metaphase chromosomes in parthenogenetic mouse embryos]

Silver staining technique visualizing argentophilic nucleolus organizer regions (Ag-NORs) was used for studying parthenogenetic mouse embryos produced by artificial activation of oocytes in Ca(2+)-Mg(2+)-free medium. Ag-NOR-containing chromosomes were detected in metaphases of parthenogenetic embryos during six successive cleavage divisions starting with the two-cell stage. The frequency of metaphases with varying AG-NOR number in diploid parthenogenones was similar to that in the control (fertilized) embryos. Average number of metaphase Ag-NOR chromosomes (calculated per diploid chromosome set) in haploid parthenogenones exceeded that in the control; in some cases all NORs were stained by silver. This is evidence that latent ribosomal cistrons in some chromosomes can be activated.     

Improvement of high‐resolution fluorescence in situ hybridisation mapping on chromosomes of Brassica oleracea var. capitata

The low resolution of chromosome‐based Fluorescence in situ hybridisation (FISH) mapping is primarily due to the structure of the plant cell wall and cytoplasm and the compactness of regular chromosomes, which represent a significant obstacle to FISH. In order to improve spatial resolution and signal detection sensitivity, we provide a reproducible method to generate high‐quality extended chromosomes that are ~13 times as long as their pachytene counterparts. We demonstrate that proteinase K used in this procedure is crucial for stretching pachytene chromosomes of Brassica oleracea in the context of a modified Carnoy's II fixative (6:1:3, ethanol:chloroform:acetic acid). The quality of super‐stretched chromosomes was assessed in several FISH experiments. FISH signals from both repetitive 5S rDNA and single‐copy ARC1 on super‐stretched chromosomes are brighter than those on other different types of chromosome due to enhanced accessibility to targets on stretched pachytene chromosomes. In conclusion, the resulting extended chromosomes are suitable for FISH mapping for repetitive DNA sequences and the localisation of a single‐copy locus, and FISH performed on super‐stretched chromosomes can achieve significantly higher sensitivity and spatial resolution than other chromosome‐based FISH mapping techniques.     

应用荧光原位杂交技术检测人类APPSWE基因在转基因小鼠染色体上的定位及位置效应

应用荧光原位杂交技术研究了人类淀粉样前体蛋白基因瑞典型突变（APPSWE）在转基因小鼠首建、F1及F2代小鼠染色体上的整合及定位,结果在2只首建转基因小鼠中,分别观察80个分裂相,出现杂交信号的核型分别为34及36个,检出率为42.5%和45%;1只F1及1只F2代转基因小鼠中,分别观察100个分裂相,出现杂交信号的核型分别为33及30个,检出率为33%和30%。转基因分别整合在8号、1号、17号和2号染色体上,提示转基因APPSWE已稳定整合到转基因小鼠的染色体上,并通过生殖细胞遗传给予子代,证实转基因在小鼠染色上的整合可能是随机的多点整合,同时,对不同整合位点的转基因小鼠进行了表型研究,结果发现不同整合位点对表型具明显影响。     

Chromosome analysis of a primary carcinoma of the Fallopian tube

Summary It is reported on the first chromosome analysis of a primary carcinoma of the Fallopian tube by direct chromosome preparation. A hypodiploid stem-line and a heteroploidy with a wide range of chromosome number were found. One or several marker chromosomes could be detected in 69 of 83 metaphases investigated. These chromosomal findings are comparable to those of other carcinomas of the genital tract except the carcinoma of the endometrium.     

Total aneuploidy and age-related sex chromosome aneuploidy in cultured lymphocytes of normal men and women

Summary In PHA-cultured lymphocytes, about 8% of metaphases from 32 women were aneuploid compared to 4% of metaphases from 35 men. A significant part of this aneuploidy was characterized by sex chromosome involvement: in women, the loss or gain of X chromosomes; in men, the gain of X chromosomes and the loss or gain of Y chromosomes. The incidence of this aneuploidy was positively age-related for both sexes. Premature division of the X-chromosome centromere was closely associated with X-chromosome aneuploidy in women and men, and appeared to be the mechanism of nondisjunction causing this aneuploidy. Premature centromere division (PCD) indicated a dysfunction of the X-chromosome centromere with aging, and this dysfunction was the basic cause of age-related aneuploidy. A similar mechanism of nondisjunction may operate for the Y chromosome of men, but could not be clearly demonstrated because of the low incidence of Y-chromosome aneuploidy.The balance of the aneuploidy was characterized by chromosome loss and the involvement of all chromosome groups. It was consistent with chromosome loss from metaphase cells damaged during preparation for cytogenetic examination.     

Fusion of 9 beta-satellite and telomere (TTAGGG)n sequences results in a jumping translocation

A newborn was found to have an isochromosome for the short arm of chromosome 9, i(9p) and a jumping translocation of the whole long arm. In 94.4% metaphases, 9q was fused to the telomere of chromosome 19p and, in 5.6% of metaphases, 9q was fused to the telomere of chromosome 8p. The net result was trisomy for the short arm of chromosome 9. With the pan telomere probe, fluorescent in situ hybridization (FISH) investigations found an interstitial telomere on the der(19) and der(8). The 9 beta and classical satellite probes gave a signal only on the long arm of chromosome 9 involved in the jumping translocation. The 9 alpha satellite probe hybridized to i(9p) and not to the other derivative chromosomes. A combination of chromosome 9 (red) and chromosome 19 (green) paint probes used to rapidly screen metaphases for the jumping translocation found 88 metaphases had a der(19)t(9;19) and 4metaphases had a der(8)t(8;9). For the first time, the junction of a jumping translocation has been shown to involve the telomere sequence (TTAGGG)n and beta-satellite sequences by FISH. In this paper, we also review the simultaneous occurrence of an isochromosome for the short arm and translocation of the whole long arm and constitutional jumping translocations.     

<Emphasis Type="Italic">In vitro</Emphasis> root induction in weedy <Emphasis Type="Italic">amaranthus</Emphasis> species to obtain mitotic chromosomes

Summary Mitotic chromosome analysis has proven to be an important tool in monitoring the potential for genetic exchange among related plant species. One major obstacle to using mitotic chromosome analysis in any species is obtaining large numbers of clear, well-spread metaphase chromosomes necessary to perform cytological techniques such as chromosome banding and fluorescent in situ hybridization. The ability to obtain good chromosome spreads is in part determined by the number and morphology of the roots, which contain the metaphase tissue. Many Amaranthus species produce very thin, delicate roots. The technique used in the process described herein provides for much more substantial roots, allowing for higher probability of obtaining well-spread metaphase chromosomes. Seeds were planted in a soilless mixture, and then cuttings and leaves were taken from the plants. The cuttings were sterilized and placed in Murashige and Skoog (MS) media, while leaf tissue was analyzed by flow cytometry, both pre-and post-propagation, to obtain DNA contents. No changes in DNA content were observed. The in vitro procedure produced significantly larger roots than were produced in soilless mix. Furthermore, all of the in vitro roots observed had 32 chromosomes of normal morphology. In vitro root propagation allowed large numbers of roots to be obtained from a single plant, thereby resulting in increased probability of obtaining cells with metaphase chromosomes that reflected the original plants' chromosome numbers and therefore may be used for molecular cytogenetic analysis.     

Fluorescence in situ hybridization of rDNA, telomeric (TTAGGG)n and (GATA)n repeats in the red abalone Haliotis rufescens (Archaeogastropoda: Haliotidae)

The physical location of 18S-5.8S-28S rDNA, telomeric sequences with (TTAGGG)n DNA probe and (GATA)n microsatellites were performed by fluorescence in situ hybridization in chromosomes of red abalone Haliotis rufescens. The karyotype of red abalone showed a diploid number of 36 (8M+9SM+1ST). FISH performed with rDNA probe, showed the location of major ribosomal clusters in the terminal region of the large arms of two submetacentric pairs (chromosome 4 and 5). Localization of heteromorphisms of FISH-rDNA was found between chromosome homologues and sister chromatids in all metaphases analyzed. This indicates that rDNA clusters are variable within the red abalone genome. The variability in the NOR-bearing reported using silver staining in other gastropods and our result are discussed. In addition, the presence of microsatellite (TTAGGG)n and (GATA)n was demonstrated after FISH treatment by DNA probes. The telomeric sequence occurred at the ends of all mitotic chromosomes, while the (GATA)n repetitive was found on chromosomal interstitial zones as well as at the telomeres in abalone chromosomes.