双色荧光原位杂交检测苯系物暴露工人精子9、18号染色体数目畸变

为研究苯系物暴露工人精子常染色体数目畸变.用地高辛标记的9号染色体探针(D9Zl)和生物素标记的18号染色体探针(D18Z1)进行双色荧光原位杂交,测定精子9、18号染色体非整倍体率.车间空气苯时间加权平均浓度(TWA)为86.49mg/m3,高于国家卫生标准1倍,苯暴露工人尿粘糠酸(ttMA)显著高于对照组.共计数14名暴露工人136401条精子,16名对照工人156955条精子.暴露组9、18染色体双体精子率(分别为0.168%±0.063%、0.055%±0.031%)和二倍体精子率(0.073%±0.045%)均高于对照组相应数值(分别为0.050%±0.030%、0.033%±0.025%和0.040%±0.036%);暴露组9、18号染色体缺体率(分别为0.206%±0.047%,0.068%±0.044%)高于对照组值(0.067%±0.037%、0.048%±0.034%).总数目畸变率(0.570%±0.144%)亦高于对照组值(0.218%±0.071%).实验表明接触较高浓度苯可引起长期暴露者精子常染色体非整倍体率增高.     

A new FISH assay to simultaneously detect structural and numerical chromosomal abnormalities in mouse sperm

De novo aberrations in chromosome structure represent important categories of paternally transmitted genetic damage. Unlike numerical abnormalities, the majority of de novo structural aberrations among human offspring are of paternal origin. We report the development of a three-color fluorescence in situ hybridization (FISH) assay (CT8) to detect mouse sperm carrying structural and numerical chromosomal abnormalities. The CT8 assay uses DNA probes for the centromeric and telomeric regions of chromosome 2, and a probe for the subcentromeric region of chromosome 8. The CT8 assay was used to measure the frequencies of sperm carrying certain structural aberrations involving chromosome 2 (del2ter, dup2ter, del2cen, dup2cen), disomy 2, disomy 8, and sperm diploidy. Analysis of approximately 80,000 sperm from eight B6C3F1 mice revealed an average baseline frequency of 2.5 per 10,000 sperm carrying partial duplications and deletions of chromosome 2. Extrapolated to the entire haploid genome, approximately 0.4% of mouse sperm are estimated to carry structural chromosomal aberrations, which is more than fivefold lower than the spontaneous frequencies of sperm with chromosome structural aberrations in man. We validated the CT8 assay by comparing the frequencies of abnormal segregants in sperm of T(2;14) translocation carriers detected by this assay against those detected by chromosome painting cytogenetic analysis of meiosis II spermatocytes. The CT8 sperm FISH assay is a promising method for detecting structural chromosome aberrations in mouse sperm with widespread applications in genetics, physiology, and genetic toxicology.     

Human sperm aneuploidy after exposure to pesticides

This study examined the effect of paternal environmental exposure to pesticides on the frequency of aneuploidy in human sperm. To determine if the chromosome number in germ cells was altered by paternal exposure, multicolor fluorescence in situ hybridization (FISH) analysis was utilized to measure aneuploidy frequencies in the sperm of 40 men (20 exposed, 20 controls). Samples were coded for "blind analysis" to eliminate scorer bias. Aneuploidy and diploidy frequencies were assessed for chromosomes 13, 21, X, and Y. A minimum of 10,000 sperm was scored per donor per chromosome probe with a total of 809,935 sperm scored. Hybridization efficiency was 99%. There were no significant differences in aneuploidy or diploidy frequencies between exposed and control groups, suggesting that the pesticides did not increase the risk of numerical chromosomal abnormalities in these men.     

多色荧光原位杂交检测苯系物接触工人精子1,18号染色体畸变率

为研究苯系物暴露对工人精子染色体的损伤 ,用 4条DNA探针与间期精子核染色体进行多色荧光原位杂交 ,同时检测精子 1号、18号染色体数目畸变和 1号染色体结构畸变 (末端缺失与重复 )。作业车间空气中苯的时间加权浓度 (TWA)为 4 2 2 9mg m3,高于国家卫生标准 (6mg m3)。暴露组工人尿粘糠酸 (ttMA)高于对照组。共计数15例暴露组工人 14 4 2 82条精子 ,14例对照组工人 135 937条精子 ,杂交效率为 99 85 %。非整倍体测定结果 :暴露组精子 1号、18号染色体双体率 (分别为 0 0 88%± 0 0 4 1% ,0 0 87%± 0 0 4 9% )显著高于对照组 1号、18号双体率(0 0 4 5 %± 0 0 2 4 % ,0 0 5 3%± 0 0 2 8% ) ;暴露组 1号、18号染色体缺体率分别为 (0 11%± 0 0 5 9% ,0 0 75 %±0 0 35 % )显著高于对照组相应数值 (0 0 4 8%± 0 0 18% ,0 0 4 5 %± 0 0 2 4 % ) ;而二倍体精子率 ,两组差别无显著性。结构畸变测定结果 :暴露组 1号染色体的末端重复率、末端缺失率 (分别为 0 16 %± 0 0 37% ,0 14 %± 0 0 5 3% )显著高于对照组数值 (分别为 0 0 82 %± 0 0 2 3% ,0 0 6 9%± 0 0 2 8% ) ;暴露组 1号染色体着丝粒重复率及着丝粒缺失率(0 10 %± 0 0 35 % ,0 10 %± 0 0 4 1% )显著性高于对     

Cigarette smoking and aneuploidy in human sperm

Cigarette smoke contains chemicals which are capable of inducing aneuploidy in experimental systems. These chemicals have been shown to reach the male reproductive system, increasing oxidative DNA damage in human sperm and lowering semen quality. We have examined the association between smoking and aneuploid sperm by studying 31 Chinese men with similar demographic characteristics and lifestyle factors except for cigarette smoking. None of the men drank alcohol. These men were divided into three groups: nonsmokers (10 men), light smokers (< 20 cigarettes/day, 11 men), and heavy smokers (> or = 20 cigarettes/day, 10 men). There were no significant differences in semen parameters or in age across groups. Two multi-color fluorescence in situ hybridizations (FISH) were performed: two-color FISH for chromosomes 13 and 21, and three-color FISH for the sex chromosomes using chromosome 1 as an internal autosomal control for diploidy and lack of hybridization. The mean hybridization efficiency was 99.78%. The frequency of disomy 13 was significantly higher in light and heavy smokers than in non-smokers, while no significant differences in the frequency of disomy 21, X or Y were observed across groups. Significant inter-donor heterogeneity in every category of disomic sperm examined was found in both light and heavy smokers, while in nonsmokers only XY disomy showed significant inter-donor differences. Thus, we conclude that cigarette smoking may increase the risk of aneuploidy only for certain chromosomes and that men may have different susceptibilities to aneuploidy in germ cells induced by cigarette smoking. Mol. Reprod. Dev. 59: 417-421, 2001.     

Genomic imbalances associated with methotrexate resistance in human osteosarcoma cell lines detected by comparative genomic hybridization-based techniques

Methotrexate (MTX) is one of the most important drugs for osteosarcoma (OS) treatment. To identify genetic aberrations associated with the development of MTX resistance in OS cells, in addition to the previously reported expression changes of dihydrofolate reductase (DHFR) and reduced folate carrier (RFC) genes, comparative genomic hybridization (CGH)-based techniques were used. The direct comparison between MTX-resistant variants of U-2OS or Saos-2 human OS cell lines with their respective parental cell lines by CGH on chromosomes revealed that development of MTX resistance was associated with gain of the chromosomal regions 5q12-q15 and 11q14-qter in U-2OS variants, and with gain of 8q22-qter in Saos-2 variants. Further analyses by CGH on microarrays demonstrated a progressively increasing gain of mixed lineage leukemia (MLL) gene (11q23) in U-2OS MTX-resistant variants, which was also confirmed by fluorescence in situ hybridization (FISH), in addition to gain of FGR (1p36), amplification/overexpression of DHFR, and slight decrease of RFC expression. In Saos-2 MTX-resistant variants, gain of MYC (8q24.12-q24.13) was detected, together with a remarkable decrease of RFC expression. Further analyses of DHFR, MLL, MYC, and RFC gene status in four additional human OS cell lines revealed that only gain of DHFR and MLL were associated with an inherent lower sensitivity to MTX. These data demonstrate that genetic analyses with complementary techniques are helpful for the identification of new candidate genes, which might be considered for an early identification of MTX unresponsive tumors.     

Fluorescence in situ hybridization using an old world monkey Y chromosome specific probe combined with immunofluorescence staining on rhesus monkey tissues.

To date, there is no commercially available Y chromosome probe that can be used for fluorescence in situ hybridization (FISH) for the male rhesus monkey. We have recently generated a probe for FISH with high specificity to the short arm of the rhesus monkey Y chromosome. In this study, we further describe a method that keeps the integrity of tissue-specific antigenic structures for immunofluorescence staining subsequent to FISH on paraffin-embedded rhesus monkey tissues. We have examined this technique in combination with an epithelial cell-specific marker, cytokeratin 8/18 (CK8/18), on various tissues, including jejunum, liver, kidney, and pancreas. CK8/18 and Y chromosome signals were distinctly seen simultaneously on epithelial cells from the same tissue section from male but not female monkeys. These studies indicate that our FISH immunofluorescence technique can be reliably used to identify and phenotype male cells in paraffin-embedded rhesus monkey tissues.     

Chromosomal domains of chimpanzee are diverged from human as revealed by in situ hybridization using human genomic probe

Utilization of repetitive DNA probes to assess the taxonomic affinity between related species has become the most powerful tool in evolutionary biology today. Consequently, tremendous strides have recently been made towards establishing the phylogenetic relationship of humans with chimpanzee. We employed human genomic proe (P5080 B.5) to identify the degree of divergence of chimpanzee genome from humans. A small protion of structurally distinct genomic areas in chimpanzee could be identified by fluorescencein situ hybridization (FISH) technique when compared to human DNA. The genomic divergence is confined mainly to the chromosomal ends in chimpanzee and may be an important phylogenetic characteristic in human evolution.     

A Review of Fluorescence in Situ Hybridization (FISH): Current Status and Future Prospects

Fluorescence in situ hybridization (FISH) is a powerful technique for detecting DNA or RNA sequences in cells, tissues and tumors. This molecular cytogenetic technique enables the localization of specific DNA sequences within interphase chromatin and metaphase chromosomes and the identification of both structural and numerical chromosome changes. FISH is quickly becoming one of the most extensively used cytochemical staining techniques owing to its sensitivity and versatility, and with the improvement of current technology and cost effectiveness, its use will surely continue to expand. Here we review the wide variety of current applications and future prospects of FISH technology.     

A Review of Fluorescence in Situ Hybridization (FISH): Current Status and Future Prospects

Fluorescence in situ hybridization (FISH) is a powerful technique for detecting DNA or RNA sequences in cells, tissues and tumors. This molecular cytogenetic technique enables the localization of specific DNA sequences within interphase chromatin and metaphase chromosomes and the identification of both structural and numerical chromosome changes. FISH is quickly becoming one of the most extensively used cytochemical staining techniques owing to its sensitivity and versatility, and with the improvement of current technology and cost effectiveness, its use will surely continue to expand. Here we review the wide variety of current applications and future prospects of FISH technology.     

Characters of DNA constitution in the rye B chromosome

We have used chromosome microdissection and microcloning to construct a DNA library of the entire B chromosome （B） of rye. New rye B-specific sequences have been screened from this pool, blasted with other sequences and analyzed to elucidate the characters of DNA constitution and the possible pathway of the origin of the rye B chromosome. We report the discovery of a new sequence that is specific to the rye B centromere.     

Further Syntheses of Lindane Analogs

dl-(1245/36)-4,5,6-Trichloro-l,2,3-trimethoxycyclohexane and dl-(1245/36)-2,3,4,5,6-penta-chloro-1-methylthiocyclohexane were synthesized from (1245/36)-3,4,5,6-tetrachlorocyclo-hexane-1, 2-diol. dl- (1245/36) -1, 4, 5, 6-Tetrachloro-2-methoxy-3-methylthiocyclohexane and dl-(1245/36)-l,4,5,6-tetrachloro-2,3-dimethoxycyclohexane were synthesized from (12345/6)-1, 4, 5, 6-tetrachloro-2, 3-epoxycyclohexane. meso- (1245) -1,2,4,5-Tetrachlorocyclohexane was synthesized from benzene via cyclohexadiene-1,4.     

FISH技术及其在环境微生物监测中的应用

荧光原位杂交（FISH）被广泛应用于微生物群落结构诊断和评价。利用FISH技术在环境样品上直接原位杂交,不仅可测定不可培养微生物的形态特征及丰度,而且可原位分析它们的空间及数量分布。在环境生物监测中有着广阔的应用前景。     

Physical mapping of repetitive sequences and genome analysis in six Elymus species by in situ hybridization

Genome constitution and genetic relationships between six Elymus species were assessed by physical mapping of different repetitive sequences using a technique of sequential fluorescence in situ hybridization and genomic in situ hybridization.The six Elymus species are all naturally growing species in northwest China,namely,E.sibiricus,E.nutans,E.barystachyus,E.xiningensis,E.excelsus,and E.dahuricus.An StStHH genome constitution was revealed for E.sibiricus and StStHHYY for the remainder species.Each chromosome could be clearly characterized by physical mapping with 18S-26S rDNA,5S rDNA,Afa-family,and AAG repeats,and be allocated to a certain genome by genomic in situ hybridization.Two 5S rDNA sites,each in the H and St genomes,and three 18S-26S rDNA sites,two in the St genome and one in the Y genome,were uncovered in most of the species.The strong Afa-family hybridization signals discriminated the H genome from the St and Y genomes.The H and Y genome carried more AAG repeats than St.A common non-Robertsonian reciprocal translocation between the H and Y genomes was revealed in E.barystachyus,E.xiningensis,E.excelsus and E.dahuricus.Comparison of molecular karyotypes strongly suggests that they can be classified into three groups,namely,E.sibiricus,E.nutans,and others.     

FISH技术的临床应用研究

荧光原位杂交（FISH）是染色体显带技术的补充和发展,以人类精子,早期胚胎等间期核及中期染色体为材料,用FISH技术检测染色体的数目异常和微小的结构异常。结果快速准确,显示它较之传统的细胞遗传学技术诊断具有明显的优越性,在临床应用中有广泛的前景。     

The promiscuous and the chaste: frequent allopolyploid speciation and its genomic consequences in American daisies (Melampodium sect. Melampodium; Asteraceae)

Polyploidy, an important factor in eukaryotic evolution, is especially abundant in angiosperms, where it often acts in concert with hybridization to produce allopolyploids. The application of molecular phylogenetic techniques has identified the origins of numerous allopolyploids, but little is known on genomic and chromosomal consequences of allopolyploidization, despite their important role in conferring divergence of allopolyploids from their parental species. Here, using several plastid and nuclear sequence markers, we clarify the origin of tetra- and hexaploids in a group of American daisies, allowing characterization of genome dynamics in polyploids compared to their diploid ancestors. All polyploid species are allopolyploids. Among the four diploid gene pools, the propensity for allopolyploidization is unevenly distributed phylogenetically with a few species apparently more prone to participate, but the underlying causes remain unclear. Polyploid genomes are characterized by differential loss of ribosomal DNA loci (5S and 35S rDNA), known hotspots of chromosomal evolution, but show genome size additivity, suggesting limited changes beyond those affecting rDNA loci or the presence of processes counterbalancing genome reduction. Patterns of rDNA sequence conversion and provenance of the lost loci are highly idiosyncratic and differ even between allopolyploids of identical parentage, indicating that allopolyploids deriving from the same lower-ploid parental species can follow different evolutionary trajectories.     

Optimization of PRINS and C-PRINS for detection of telomeric sequences in Vicia faba

Primed in situ labelling (PRINS) of nucleic acids was developed as an alternative to traditionally used fluorescence in situ hybridization (FISH). Compared to FISH, PRINS is faster and does not require preparation of labelled probes. Nevertheless, the number of applications for physical mapping of DNA sequences on plant chromosomes remains low. This is due to the fact that there are a number of factors which influence the specificity and sensitivity of the reaction. The purpose of this work was to analyse the effect of some of them, including the age of slides, type of Taq DNA polymerase, number and concentration of primers, the presence and concentration of bovine serum albumine and MgCl₂ in the reaction mixture. Furthermore, the effect of various pre-treatments on signal intensity and non-specific fluorescence was studied. A consensus Arabidopsis-type telomeric sequence and Vicia faba mitotic chromosomes were used as a model system. We have found that the age of slides was critical and that under optimal conditions it was possible to achieve relatively high signal to noise ratio. This revised version was published online in July 2006 with corrections to the Cover Date.     

Ferulago trojana (Umbelliferae), a new species from western Turkey

Somatic chromosomes of four species of Ceratozamia , C. hildae , C. kuesteriana , C. mexicana and C. norstogii , and Stangeria eriopus , were observed and compared by the fluorescence in situ hybridization method using 5S ribosomal (rDNA) probes. The four Ceratozamia species and S. eriopus showed the same chromosome number of 2 n  = 16, and had similar karyotypes, comprising 12 metacentric (m), two submetacentric (sm) chromosomes and two telocentric (t) chromosomes. The four Ceratozamia species exhibited a proximal 5S rDNA site in the interstitial region of two m chromosomes. Stangeria eriopus exhibited a distal 5S rDNA site in the interstitial region of two m chromosomes, which probably indicates that the two genera differ in chromosome structure by at least one paracentric inversion. © 2004 The Linnean Society of London, Botanical Journal of the Linnean Society , 2004, 145 , 499–504.     

Molecular and morphologic analyses of expression of ESX1L in different stages of human placental development

The mRNA expression of the ESX1L gene was analyzed by RT-PCR and in situ hybridization in human normal cytogenetically placentas, of different gestational ages. Our RT-PCR analysis showed that ESX1L mRNA is expressed from 5 weeks of gestation until term, suggesting a role not only in trophoblast differentiation but also in the maintenance of the villi and microvasculature. We also observed, by in situ hybridization, that ESX1L mRNA is expressed by cytotrophoblast from chorionic plate, syncytiotrophoblast and stromal cells of all terminal, intermediate and stem villi of term placentas. ESX1L mRNA expression was more pronounced in trophoblast cells of terminal villi than in intermediate and stem villi. In conclusion, ESX1L is expressed during all stages of placental development and is localized to sparse areas of trophoblast in terminal villi in association with cytotrophoblastic cells.     

荧光原位杂交技术及其在植物研究中的应用

荧光原位杂交(FISH)是20世纪生物学领域的一项新技术。FISH应用细胞遗传学和分子生物学的基本原理,作为架设细胞遗传学与分子生物学之间的桥梁,现已被广泛应用于植物学各方面的研究。本文就FISH的基本原理、技术发展及其在植物遗传育种、起源进化、染色体物理图谱构建方面的应用及发展趋势进行了综述。