一种提高体外培养细胞中期分裂相的新方法

为了提高体外培养细胞中期分裂相,用黄牛胎儿成纤维细胞系 （YFF）和西门塔尔小牛成纤维细胞系（CNF）为实验材料,先经4℃低温休克再用秋水仙素处理后制备染色体,比较了不同低温休克处理时间获得的中期分裂相百分率,并运用该方法对20代内YFF和CNF核型变异进行了分析。实验发现,YFF和 CNF经低温休克中期分裂相百分率显著高于对照组（P<0.05）。其中, 20 h低温组中期分裂相（31.7﹪和40.2﹪）高于对照组（4.7﹪和6.4﹪）5倍以上（P<0.01）。实验结果表明,4℃低温休克法是一种提高体外培养细胞中期分裂相的简便方法,适于监测体外培养细胞核型变异。     

小麦根尖细胞同步化诱导和DNA纤维的制备

为了简易快速地获得大量小麦（Triticum aestivumL．）中期染色体和DNA纤维,以普通小麦根尖为材料,采用羟基脲（hydroxyurea,简称HU）,氟乐（trifluralin）结合的双阻断法进行了染色体中期同步化诱导。结果表明,染色体有丝分裂中期指数（metaphaseindex）达70％～80％;以同材料小麦黄化苗提取小麦细胞核,成功制备出小麦DNA纤维。这为研究细胞有丝分裂的调控、染色体形态结构、易位染色体检测和易位染色体片段的精确测量与定位等提供了新的技术支撑。     

从植物细胞核分离大分子量核DNA

研究了从植物中分离百万碱基对级大分子量核DNA的方法。该方法利用差速离心分离植物细胞核,经低熔点琼脂糖块或低熔点琼脂糖微珠包埋,蛋白酶K原位裂解后制备大分子量核DNA。结果表明,选择不同生长时期的材料和不同的包埋细胞核方式对大分子量核DNA的制备有很大的影响,由黄化苗或幼嫩的绿叶为材料分离细胞核,进行胶块包埋是制备大分子量核DNA的最佳条件。利用该法获得的DNA分子量在200kb－5．7Mb之间,主要集中在2．2～5．7Mb之间;每一胶块DNAE量为18～20μg。与包埋原生质体制备大分子量核DNA的方法相比,该方法获得的DNA纯度较高,去除了大部分细胞器DNA的污染;易于被限制性内切酶部分和完全消化,其消化结果具可重复性。该方法操作简单、适用植物种类广泛,用该方法从水稻（OryzasativaL．）、苹果（MaluspumilaMill．）、大豆（Glycinemax（L．）Merr．）、玉米（ZeamaysL．）等多种植物材料中成功地制备了大分子量核DNA。该方法制备的核DNA适用于植物的脉冲交变电泳基因组分析和构建人工细菌染色体文库和人工酵母染色体文库。     

不同预处理对鹰嘴豆根尖细胞染色体制片的影响

采用常规压片法,以鹰嘴豆根尖为材料,研究了秋水仙素、对二氯苯和8-羟基喹啉等预处理剂对积累鹰嘴豆根尖细胞中期分裂相的效果,并比较了不同预处理剂对中期染色体形态的影响。结果表明,3种预处理剂均能使根尖细胞的有丝分裂停留在中期,但0.1%的秋水仙素溶液进行预处理后的中期分裂相,染色体粗短,分散度不理想;0.006 mol/L的8-羟基喹啉溶液进行预处理后的鹰嘴豆中期染色体形态不清晰,边缘缺失;对二氯苯饱和水溶液4 h处理后的根尖细胞染色体背景清晰,缢痕明显,分散度较适宜,适合核型分析。     

分裂中期的CHO细胞能对其染色体DNA进行切除修补吗?

以中国仓鼠细胞CHO-K_1为材料,用放射自显术检查紫外线照射后细胞内DNA的切除修补,没有得到足以证明分裂中期细胞对其染色体DNA进行切除修补的可靠证据。此结果与1977年Ikushima所报告的不同。对于这种修补能力的缺乏,作者认为应考虑到的原因至少有:(1)分裂中期染色体上的DNA处在高度紧密的状态,难于进行切除修补;(2)分裂中期细胞中担任DNA修补合成的多聚酶β的活性可能下降;(3)分裂中期细胞的核膜解体。     

中期染色体外鞘蛋白质的研究

从12例硬皮病患者的抗染色体抗血清中发现4例是抗中期染色体鞘的,用它们和小鼠腹水癌细胞核及全细胞裂解液SDS-PAGE的蛋白印迹相反应,结果显示它们和细胞核裂解液的11条抗原蛋白相结合,而且和全细胞裂解液中除以上的11条外的另8条相结合。     

中期染色体外鞘蛋白质的研究

从１２例硬皮病患者的抗染色体抗血清中发现４例是抗中期染色体鞘的,用它们和小鼠腹水癌细胞核及全细胞裂解液ＳＤＳ－ＰＡＧＥ的蛋白印迹相反应,结果显示它们和细胞核裂解液的１１条抗原蛋白相结合,而且和全细胞裂解液中除以上的１１条外的另８条相结合。     

同步化技术在草鱼染色体显带中的应用及草鱼核型的初步分析

对草鱼细胞进行同步化处理,并在DNA复制的早期和晚期分别掺入BrdU,制备的染色体标本置于CaCl_2溶液中温浴同时用紫外线照射,然后用Giemsa染色即可分别显示出G带和R带。标本中的部分晚前期和早中期分裂相具有高分辨染色体显带特征。用这一技术对草鱼每条染色体进行识别和分析,提出了初步的草鱼核型,发现了四对草鱼染色体具有随体。     

黄瓜花粉母细胞减数分裂行为的研究

研究了华北型、华南型和西南型黄瓜花粉母细胞的减数分裂行为,发现黄瓜细胞核减数分裂的同步性较高,细胞质是同时型分裂。在细胞核分裂的过程中,核仁在前期Ⅰ到中期Ⅰ逐渐消失,在前期Ⅰ再次出现,随后消失;染色体在前期Ⅰ到中期Ⅰ逐渐收缩,变得清晰,至末期Ⅰ解螺旋,变得模糊,在前期I再次清晰。不同生态型黄瓜终变期的染色体构型均以环状二价体为主。在前期Ⅰ和前期Ⅰ,西双版纳黄瓜的核仁都相应地比另外两种生态型黄瓜品种的多,在后期Ⅰ还偶尔出现染色体桥,显示了西双版纳黄瓜变种的特殊性。研究还发现寒冷的气候条件下栽培黄瓜都能够形成高频率的多分体,推测其形成很可能与低温逆境有关。     

“重悬法”制备低等动物染色体

本文介绍利用"离心重悬法"对低等扁虫类生物染色体的制备方法,此法改进了以空气干燥法为主的对低等扁虫类动物染色体的制备方法。结果表明:能得到形态更清晰的中期分裂相,有利于对染色体的进一步统计分析。     

Immunoelectronmicroscopy of metaphase chromosomes

Summary A method for the preparation of ultrathin sections of metaphase chromosomes is described. This method was applied to human metaphase chromosomes, which were immunocytochemically stained with anti-DNA and anti-ribonucleoprotein antibodies, derived from patients with auto-immune disease. Conventionally prepared metaphase spreads as well as cytocentrifuge preparations of chromosome suspensions were studied. The results indicate that the ultrastructure of chromosomes and the immunoreactivity of chromosomal constituents are influenced by the applied preparation methods. In comparison with whole mount preparations, ultrathin sections of immunostained chromosomes allow higher resolution and more precise localization of immunoreactive sites within the chromosomal structure.     

Immunoelectronmicroscopy of metaphase chromosomes

A method for the preparation of ultrathin sections of metaphase chromosomes is described. This method was applied to human metaphase chromosomes, which were immunocytochemically stained with anti-DNA and anti-ribonucleoprotein antibodies, derived from patients with auto-immune disease. Conventionally prepared metaphase spreads as well as cytocentrifuge preparations of chromosome suspensions were studied. The results indicate that the ultrastructure of chromosomes and the immunoreactivity of chromosomal constituents are influenced by the applied preparation methods. In comparison with whole mount preparations, ultrathin sections of immunostained chromosomes allow higher resolution and more precise localization of immunoreactive sites within the chromosomal structure.     

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.     

用于目标序列染色体定位的镜鲤BAC-FISH体系构建

为了构建用于镜鲤(Cyprinus carpio var. specularis)特定基因组序列染色体定位的实验体系, 在细菌人工染色体(Bacterial Artificial Chromosome, BAC)文库筛选池中对已知短序列基因组片段进行PCR扩增, 筛选出包含目标序列的BAC克隆, 提取BAC质粒进行缺刻平移标记制备探针, 开展荧光原位杂交(Fluorescence in situ hybridization, FISH)实验。通过对染色体片前处理、BAC质粒探针制备、C0t-1 DNA封闭基因组重复序列、预杂交、荧光染料选择、信号放大等一系列实验条件和方法的探索优化, 成功实现了目标序列在镜鲤有丝分裂中期染色体上的定位。定位对象既包括在染色体上有单一位点的序列, 如斑马鱼微卫星标记Z6884和Z4268, 也包括在染色体上有多个位点的重复序列, 如黄河鲤性别相关标记CCmf1。来自斑马鱼同一条染色体上的两个微卫星标记被分别定位于镜鲤不同染色体上, 为鲤鱼染色体数目加倍的进化假设提供了一项直接实验证据, 同时将现有遗传连锁图谱与染色体对应起来, 可作为染色体识别和细胞遗传学图谱构建的依据。黄河鲤性别相关重复序列被定位于不少于四条染色体上, 为性别决定相关基因的筛查提供了研究线索。这一BAC-FISH实验体系将成为鲤细胞遗传学图谱构建、基因组进化和比较基因组学研究中的重要研究工具。       

应用荧光原位杂交检测EB病毒BNLF-1基因在转基因小鼠子代染色体上的整合及其定位

应用荧光原位杂交技术研究了EB病毒潜伏膜蛋白基因(BNLF-1)在转基因小鼠子二代染色体上的整合及其定位。结果在两只子二代转基因小鼠中,分别观察80个和60个分裂相,出现杂交信号的核型分别为27和18个,检出率为33.8％和30％。转基因分别整合在14号染色体和10号染色体上。提示转基因BNLF-1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子代;推测转基因原代鼠的转基因整合可能是随机的多位点整合。     

Scanning electron microscopic examination of the secondary constriction of Vero cells

Vero cells (African green monkey kidney in origin) were prepared by the conventional air-drying method and then processed for SEM by a modification of the conductive method based on thiocarbohydrazide-osmium binding [3]. Under SEM, not only metaphase chromosomes but also resting nuclei showed distinct fibres 30 nm in diameter. A few such fibres were found to run across the secondary constriction of the NOR-carrying chromosome.     

Quantification of total genomic DNA and selected repetitive sequences reveals concurrent changes in different DNA families in indica and japonica rice

This paper describes a fluorescence in situ hybridization (FISH) analysis of three different repetitive sequence families, which were mapped to mitotic metaphase chromosomes and extended DNA fibers (EDFs) of the two subspecies of rice (Oryza sativa), indica and japonica (2n=2x=24). The repeat families studied were (1) the tandem repeat sequence A (TrsA), a functionally non-significant repeat; (2) the [TTTAGGG]_n telomere sequence, a non-transcribed, tandemly repeated but functionally significant repeat; and (3) the 5S ribosomal RNA (5S rDNA). FISH of the TrsA repeat to metaphase chromosomes of indica and japonica cultivars revealed clear signals at the distal ends of twelve and four chromosomes, respectively. As shown in a previous report, the 17S ribosomal RNA genes (17S rDNA) are located at the nucleolus organizers (NORs) on chromosomes 9 and 10 of the indica cultivar. However, the japonica rice lacked the rDNA signals on chromosome 10. The size of the 5S rDNA repeat block, which was mapped on the chromosome 11 of both cultivars, was 1.22 times larger in the indica than in the japonica genome. The telomeric repeat arrays at the distal ends of all chromosome arms were on average three times longer in the indica genome than in the japonica genome. Flow cytometric measurements revealed that the nuclear DNA content of indica rice is 9.7% higher than that of japonica rice. Our data suggest that different repetitive sequence families contribute significantly to the variation in genome size between indica and japonica rice, though to different extents. The increase or decrease in the copy number of several repetitive sequences examined here may indicate the existence of a directed change in genome size in rice. Possible reasons for this phenomenon of concurrent evolution of various repeat families are discussed. Received: 9 August 1999 / Accepted: 29 December 1999     

Comparative genomic in situ hybridization (cGISH) analysis of the genomic relationships among Sinapis arvensis, Brassica rapa and Brassica nigra

To further understand the relationships between the SS genome of Sinapis arvensis and the AA, BB genomes in Brassica, genomic DNA of Sinapis arvensis was hybridized to the metaphase chromosomes of Brassica nigra (BB genome), and the metaphase chromosomes and interphase nucleus of Brassica rapa (AA genome) by comparative genomic in situ hybridization (cGISH). As a result, every chromosome of B. nigra had signals along the whole chromosomal length. However, only half of the condensed heterochromatic areas in the interphase nucleus and the chromosomes showed rich signals in Brassica rapa. Interphase nucleus and the metaphase chromosomes of S. arvensis were simultaneously hybridized with digoxigenin-labeled genomic DNA of B. nigra and biotin-labeled genomic DNA of B. rapa. Signals of genomic DNA of B. nigra hybridized throughout the length of all chromosomes and all the condensed heterochromatic areas in the interphase nucleus, except chromosome 4, of which signals were weak in centromeric regions. Signals of the genomic DNA of B. rapa patterned the most areas of ten chromosomes and ten condensed heterochromatic areas, others had less signals. The results showed that the SS genome had homology with AA and BB genomes, but the homology between SS genome and AA genome was clearly lower than that between the SS genome and BB genome.     

Proteome analysis of human metaphase chromosomes

DNA is packaged as chromatin in the interphase nucleus. During mitosis, chromatin fibers are highly condensed to form metaphase chromosomes, which ensure equal segregation of replicated chromosomal DNA into the daughter cells. Despite >1 century of research on metaphase chromosomes, information regarding the higher order structure of metaphase chromosomes is limited, and it is still not clear which proteins are involved in further folding of the chromatin fiber into metaphase chromosomes. To obtain a global view of the chromosomal proteins, we performed proteome analyses on three types of isolated human metaphase chromosomes. We first show the results from comparative proteome analyses of two types of isolated human metaphase chromosomes that have been frequently used in biochemical and morphological analyses. 209 proteins were quantitatively identified and classified into six groups on the basis of their known interphase localization. Furthermore, a list of 107 proteins was obtained from the proteome analyses of highly purified metaphase chromosomes, the majority of which are essential for chromosome structure and function. Based on the information obtained on these proteins and on their localizations during mitosis as assessed by immunostaining, we present a four-layer model of metaphase chromosomes. According to this model, the chromosomal proteins have been newly classified into each of four groups: chromosome coating proteins, chromosome peripheral proteins, chromosome structural proteins, and chromosome fibrous proteins. This analysis represents the first compositional view of human metaphase chromosomes and provides a protein framework for future research on this topic.     

Detection of individual human chromosomes by chromosome in situ suppression hybridization using PCR-amplified bacteriophage library probes

We used the polymerase chain reaction (PCR) to prepare chromosome-specific probes from the bacteriophage λ library LA01NS01, prepared at the Los Alamos National Laboratory from flow sorted human chromosome 1. By using oligonucleotide primers flanking the EcoRI insertion site of the Charon 21A vector, we were able to amplify the human sequences preferentially in the library up to 9.1 kb (maximum insert size). The product of the PCR reaction was nick translated with incorporation of biotinylated residues and used with fluorescence in situ hybridization to observe metaphase chromosomes by fluorescence microscopy. This technique allows for a relatively easy method for preparation of chromosome-specific library probes for “chromosome painting.” The quality of the results obtained by this method compares favorably to those obtained by using bulk-purified library inserts. This method offers potential advantages in terms of cost and east of use.