鸟类羽髓细胞培养方法的建立及其细胞增殖动力学研究

采用常规的外周血培养技术很难获得足够分析的鸟类细胞中期染色体标本。目前为止,对于鸟类的细胞遗传学研究仍采用骨髓制备染色体的方法,但这种方法不能用于对一些稀有珍禽的研究。 为研究鸟类细胞遗传学,我们建立了鸟类羽髓细胞离体短期培养的方法。应用这种方法,经48小时培养就可收获细胞并制备染色体标本。同时,还应用姐妹染色单体差别染色技术(SCD),对羽髓细胞的增殖周期进行了研究并取得成功。 实验表明,我们所建立的鸟类羽髓细胞培养方法具有:(1)取材容易,(2)增殖迅速,(3)操作简便的特点。     

水稻中期染色体和DNA纤维的高效制备技术

水稻中期染色体和DNA纤维制备是水稻分子细胞遗传学研究中的关键技术。目前,这两个技术还有很多不足,该研究建立了高效制备水稻中期染色体和DNA纤维的方法。该方法制备的染色体,分裂相多、杂质少、背景清晰、染色体分散且形态好,水稻根尖分生组织细胞的分裂指数高达25％。植物细胞的细胞壁是制备DNA纤维的最大障碍,所以必须先提取细胞核,然后裂解细胞核释放出DNA纤维。在这个研究中,还建立了一个用刀切法分离细胞核,进而用SDS裂解核膜,用载玻片拖出DNA来制备水稻DNA纤维的方法。该方法制备的DNA纤维多呈平行的细线,背景清晰,伸展的程度均匀,适合于原位杂交。     

水稻减数分裂染色体分析方法

减数分裂粗线期染色体研究技术的发展, 很大程度上克服了水稻(Oryza sativa)细胞遗传研究中较小染色体所带来的研究困难。减数分裂染色体的制备与观察已经成为水稻细胞遗传学研究中的常规方法。该文详细描述了水稻中常用的减数分裂染色体制备、荧光原位杂交和免疫荧光染色的实验方法。     

水稻减数分裂染色体分析方法

减数分裂粗线期染色体研究技术的发展, 很大程度上克服了水稻(Oryza sativa)细胞遗传研究中较小染色体所带来的研究困难。减数分裂染色体的制备与观察已经成为水稻细胞遗传学研究中的常规方法。该文详细描述了水稻中常用的减数分裂染色体制备、荧光原位杂交和免疫荧光染色的实验方法。     

黑斑蛙骨髓细胞染色体标本制备的新方法

染色体标本制备是黑斑蛙分子细胞遗传学研究的基础。为了简化操作程序,缩短实验周期,建立了黑斑蛙染色体制备的一种新方法——骨髓细胞体外短时培养与秋水仙素同步处理法。该方法操作简便,重复性较好,可在较短时间里制备出分裂相较多且形态良好的蛙染色体标本,适用于核型分析、染色体荧光原位杂交、染色体显微分离和单染色体文库构建等多个方面的研究。     

小鼠早期胚胎性别鉴定的研究

用改进的细胞遗传学方法制备染色体标本,鉴定了小鼠晚期桑椹胚(晚桑)、囊胚和扩展囊胚(扩囊)的性别。在实验中,利用小鼠早期胚胎染色体标本制备的理想实验参数,鉴定了242枚小鼠晚桑、囊胚和扩囊的性别,性别鉴定成功率分别为80.4％、99.0％和94.5％。以细胞遗传学方法鉴定小鼠早期胚胎性别的结果为标准,得出用间接免疫荧光法和PCR(聚合酶链反应)扩增SRY(y染色体的性别决定期)部分序列法分别鉴定100枚和26枚小鼠胚胎性别的准确率相应地为74.0％和92.3％。     

染色体微分离和微克隆技术

染色体微分离和微克隆技术是将细胞遗传学和分子遗传学二紧密结合的一项技术,目前已广泛应用于遗传学、医学等研究领域,具有广阔的应用前景。本综述了该技术发展过程中所应用的不同方法,详细介绍了各种方法的步骤及其优缺点,最后探讨了该技术的应用及展望。     

小麦染色体的显微激光分离

探讨了应用氩离子激光进行植物染色体显微激光切割,分离的可行性,应用该技术对普通小麦的体细胞及特定染色体（１Ｂ染色体）实施切割,分离,并且以分离到的单细胞核或单条染色体为模板进行了ＰＣＲ ＤＮＡ扩增。该技术比玻璃针切割分离染色体技术,具有操作方便,容易掌握,且可对整个细胞核进行分离等优点,有利于促进染色体显微操作技术的普及应用。同时,探讨了染色体显微操作技术在细胞遗传学及分子生物学研究领域的应用前景     

蜉蝣目昆虫染色体制备方法探讨

本文对蜉蝣目昆虫染色体制备方法进行了探讨。以蜉蝣稚虫为材料,经秋水仙素水溶液体外培养,采用显微操作技术,结合空气干燥法,可以获得形态良好、分散适中、着色清晰的生殖细胞染色体,为蜉蝣目昆虫细胞遗传学研究提供了一种简便有效的方法。     

额外小染色体的分子细胞遗传学研究

本文应用’H标记的7.3kb的rRNA基因探针进行染色体原位杂交技术,并结合多种细胞遗传学技术对一个额外小染色体的家族进行分析研究。在该家族调查的三代人中有8名成员带有相同的额外小染色体,携带者表型均正常。结果证实该额外小染色体是D组或G组染色体的短臂。并就其额外小染色体的起源,遗传效应及生育等问题进行了扼要的讨论。     

Generation of FISH probes using laser microbeam microdissection and application to clinical molecular cytogenetics

Chromosome microdissection and the reverse FISH technique is one of the most useful methods for the identification of structurally abnormal chromosomes. In particular, the laser microbeam microdissection (LMM) method allows rapid isolation of a target chromosome or a specific region of chromosomes without damage of genetic materials and contamination. Isolated chromosomes were directly amplified by the degenerate oligonucleotide-primed polymerase chain reaction (DOP-PCR), and then the FISH probes labeled with spectrum green- or spectrum red-dUTP were generated by nick-translation. Whole chromosome painting (WCP) probes were successfully generated from only 5 copies of the chromosome. With this method, we produced 24 WCP probes for each human chromosome. We also tried to characterize a marker chromosome, which seemed to be originated from chromosome 11 on conventional banding technique. The marker chromosomes were isolated by the LMM method and analyzed by reverse FISH. We elucidated that the marker chromosome was originated from the short arm of chromosome 5 (5p11-->pter). A fully automated and computer-controlled LMM method is a very simple laboratory procedure, and enables rapid and precise characterization of various chromosome abnormalities.     

New method for generating deletions and gene replacements in Escherichia coli.

We describe a method for generating gene replacements and deletions in Escherichia coli. The technique is simple and rapid and can be applied to most genes, even those that are essential. What makes this method unique and particularly effective is the use of a temperature-sensitive pSC101 replicon to facilitate the gene replacement. The method proceeds by homologous recombination between a gene on the chromosome and homologous sequences carried on a plasmid temperature sensitive for DNA replication. Thus, after transformation of the plasmid into an appropriate host, it is possible to select for integration of the plasmid into the chromosome at 44 degrees C. Subsequent growth of these cointegrates at 30 degrees C leads to a second recombination event, resulting in their resolution. Depending on where the second recombination event takes place, the chromosome will either have undergone a gene replacement or retain the original copy of the gene. The procedure can also be used to effect the transfer of an allele from a plasmid to the chromosome or to rescue a chromosomal allele onto a plasmid. Since the resolved plasmid can be maintained by selection, this technique can be used to generate deletions of essential genes.     

Mapping of 262 DNA markers into 24 intervals on human chromosome 11.

We have extended our mapping effort on human chromosome 11 to encompass a total of 262 DNA markers, which have been mapped into 24 intervals on chromosome 11; 123 of the markers reveal RFLPs. These clones are scattered throughout the chromosome, although some clustering occurs in R-positive bands (p15.1, p11.2, q13, and q23.3). Fifty-two of the markers were found to contain DNA sequences conserved in Chinese hamster, and some of these 52 also cross-hybridized with DNA from other mammals and/or chicken. As the length of chromosome 11 is estimated at nearly 130 cM, the average distance between RFLP markers is roughly 1 cM. The large panel of DNA markers on our map should contribute to investigations of hereditary diseases on this chromosome, and it will also provide reagents for constructing either fine-scale linkage and physical maps or contig maps of cosmids or yeast artificial chromosomes.     

Phylogenetic meta-analysis

Meta-analysis is a powerful statistical technique that combines the results of independent studies to identify general trends. When the species under examination are not independent however, it is also necessary to incorporate phylogenetic information into the analysis. Unfortunately, current meta-analytic approaches cannot account for lack of independence resulting from shared evolutionary history, so a general solution to this problem is lacking. In this article, I derive a model for phylogenetic meta-analysis, so that data across studies may be summarized with evolutionary history explicitly incorporated. The approach takes advantage of common aspects of linear statistical models used by both meta-analysis and the phylogenetic comparative method, thereby allowing them to be analytically combined. In this manner, the correlation structure generated by phylogenetic history can be incorporated directly into the meta-analytic procedure. I illustrate the approach by examining the prevalence of body size clines in mammals. The approach is general, and can also be used to incorporate correlation structure among studies generated by other factors, such as spatial or temporal proximity, or environmental similarity. Therefore, this procedure provides a general statistical template for meta-analytic techniques that can account for attributes that generate nonindependence among studies. Implications of the phylogenetic meta-analysis are discussed.     

A comparison of two methods for evaluating drug-induced chromosome alterations.

Evaluating the technique and procedure for mutagenicity testing in mammals is a prerequisite to the development of a broad spectrum mutagenic assessment program. Two techniques, chromosome examination and micronucleus scoring, show promise but their applicability for mass screening is uncertain. We determined the slide observation time for these two techniques in mice treated orally, subcutaneously, intravenously, and intraperitoneally with cyclophosphamide (CY). In each instance, we detected a dose-response in less observation time by counting micronuclei in polychromatophilic erythrocytes. The simplicity of the scoring method, the ease of micronucleus identification and the rapidity of scoring all suggest the micronucleus test may be favorably integrated into a mutagenicity screening program.     

电子显微镜分离小鼠染色体的研究

本实验对小白鼠的骨髓细胞,制成电镜染色体样品,探索了利用电子显微镜所产生的高度聚焦的电子束,将目标染色体或梁色体处段与其他染色体或染色体片段分离,切割开的过程,检验了电子束分离,切割染色体的可行性和有效性,开发出了一套较为成熟的电子束分离,切割染色体的新技术或方法,使之成为染色体及基因研究的有效手段。     

Banding patterns on lampbrush chromosomes of Triturus marmoratus (Amphibia Urodela) by the Giemsa stain

Lampbrush chromosome preparations from the newt species Triturus marmoratus have been submitted to a banding procedure by using a Giemsa stain technique (C-banding) as well as variants of the method. Centromeres, most of telomeres, the nucleolus organizing region and some segments along the chromosome axes appear to be differently stained. The centromere positions have been indicated on the maps of the lampbrush complement of the species. The possible relationships between banding and chromosome structure and organization are briefly discussed.     

Chromosome-shuffling technique for selected chromosomal segments in Saccharomyces cerevisiae

We describe a novel chromosome engineering technique for shuffling selected regions of chromosomes from two strains in Saccharomyces cerevisiae: The technique starts with the construction of MAT a and MATα strains in which a particular chromosome is split at exactly the same site in both strains such that the split chromosomes generated are marked with different markers. The two strains are then crossed, and the resultant diploid is cultivated in nutrient medium to induce loss of the split chromosome originating from either of the strains. We predicted that some of these clones that are hemizygous for the split chromosome would spontaneously restore a homozygous configuration of the split chromosome during cultivation. We verified this prediction by tetrad analysis and quantitative Southern hybridization analysis, indicating that it is possible to create diploid hybrids in which a selected region of a chromosome from one strain is replaced by the corresponding chromosomal region from another strain. We also found that some chromosomal segments maintain a hemizygous state. This novel technique, which we call ‘chromosome shuffling’, could provide a new tool to analyze phenotypic alterations caused by the replacement or hemizygosity of a selected chromosomal region in not only laboratory but also industrial strains of S. cerevisiae.