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。     

人类中期染色体引物原位延伸标记的研究

染色体上引物原位延伸标记在研究染色体结构和基因定位等方面具有重要意义,分别应用随机引物和ＳＯＸ基因兼并引物人类染色体上进行了原位延伸标记,结果表明,随机引物伸在染色体上呈现明暗相间的带纹样特征。ＳＯＸ基因兼并引物延伸发现了更多的ＳＯＸ基因位座,并进一步证实该家族基因在基因组中是散存在的。     

Mammalian Metaphase Chromosomes with High Molecular Weight DNA isolated at <Emphasis Type="Italic">p</Emphasis>H 10.5

MAMMALIAN metaphase chromosomes may be isolated either at an acid pH^1–4 or at nearly neutral pH^5–7. The only exception is the method of Corry and Cole⁸, performed at pH 9.5. We used alkaline sucrose velocity gradients to determine the size distributions of DNA (molecular weight) in metaphase chromosomes in an attempt to understand their arrangement. Initial experiments yielded 1 × 10⁶ molecular weight DNA (single stranded) pieces regardless of chromosome size. When metaphase cells are lysed directly on the gradient a high molecular weight (1 × 10⁸ for single stranded) is obtained. We therefore examined a large number of chromosome isolation procedures and cytological conditions to determine their effects on the molecular weight of the DNA.     

Specific Heterochromatic Banding of Metaphase Chromosomes Using Nuclear Yellow

The bis-benzimidazole compound nuclear yellow (NY) belongs to the same chemical family as the DNA binding fluorochromes Hoechst 33258 and Hoechst 33342. Spectroscopic studies of NY alone and in the presence of calf thymus DNA show high DNA binding affinity and behavior similar to the Hoechst fluorochromes above. Mitotic metaphase chromosomes from Balb/c mice stained with NY show C-banding and weak G/Q-banding, both of them disappearing after distamycin A (DA) or methyl green (MG) counterstaining. The same staining of human metaphase chromosomes from lymphocyte cultures, however, reveal only faint G/Q-banding (NY) and a characteristic DA-DAPI-like banding (NY-DA, NY-MG). Image analysis of NY stained human chromosomes, confirms that NY is suitable for studying polymorphisms affecting size in the pericentromeric hete-rochromatin of pairs 1, 9 and 16, and shows significant enhancement of NY fluorescence induced by DA in DA-DAPI heterochromatin. Our spectroscopic and cytological results show that NY, either alone or counterstained with DA or MG, can be used for DNA cytochemistry and chromosome banding. Possible mechanisms for the banding patterns induced by NY are discussed.     

Specific Heterochromatic Banding of Metaphase Chromosomes Using Nuclear Yellow

The bis-benzimidazole compound nuclear yellow (NY) belongs to the same chemical family as the DNA binding fluorochromes Hoechst 33258 and Hoechst 33342. Spectroscopic studies of NY alone and in the presence of calf thymus DNA show high DNA binding affinity and behavior similar to the Hoechst fluorochromes above. Mitotic metaphase chromosomes from Balb/c mice stained with NY show C-banding and weak G/Q-banding, both of them disappearing after distamycin A (DA) or methyl green (MG) counterstaining. The same staining of human metaphase chromosomes from lymphocyte cultures, however, reveal only faint G/Q-banding (NY) and a characteristic DA-DAPI-like banding (NY-DA, NY-MG). Image analysis of NY stained human chromosomes, confirms that NY is suitable for studying polymorphisms affecting size in the pericentromeric hete-rochromatin of pairs 1, 9 and 16, and shows significant enhancement of NY fluorescence induced by DA in DA-DAPI heterochromatin. Our spectroscopic and cytological results show that NY, either alone or counterstained with DA or MG, can be used for DNA cytochemistry and chromosome banding. Possible mechanisms for the banding patterns induced by NY are discussed.     

蔗糖梯度离心纯化人类中期染色体

蔗糖梯度离心纯化人类中期染色体①施家琦唐冬生夏家辉（湖南医科大学医学遗传学国家重点实验室,长沙４１００７８）ＰｕｒｉｆｉｃａｔｉｏｎｏｆＨｕｍａｎＭｅｔａｐｈａｓｅＣｈｒｏｍｏｓｏｍｅｓｂｙＳｕｃｒｏｓｅＧｒａｄｉｅｎｔＣｅｎｔｒｉｆｕｇａｔｉｏｎＳ...     

真核生物染色体超级结构研究： Ⅰ.家猪中期染色体超级结构研究

用自行的多级光学放大系统,观察到染色体上１５ｎｍ大小的细微结构,用以研究致密状态的中期染色体超级结构,结合细胞化学技术,用特异性染色显示ＤＮＡ－核蛋白、酸性骨架蛋白在染色体上的构象,本文发表了家猪染色体超级结构的天然形态－螺旋构型照片,描述了染色体螺旋结构的特征及其参数,提出了染色体超级结构的螺旋模型。     

小鼠中期染色体制备方法探讨

目的:寻找小鼠细胞中期染色体制备过程中理想的取材方法和低渗的最佳时间,以及制片时滴片的最佳高度。方法:根据以往小鼠染色体制备方法的基本步骤,在取材方面进行对比,同时分别设置4个加入低渗液的时间和4个滴片高度进行试验对比。结果:各低渗时间段和各滴片高度所制备的结果存在一定的差异性,制备成功率和良好率最高的低渗时间为25 min,滴片高度为20 cm。结论:取材骨髓比较容易和简洁制备中期染色体,最佳低渗时间为25 min,最佳滴片高度为20 cm。     

一种检测人中期染色体原位切口移位的新方法

本研究首次详细描述了在BrdU替代4个细胞周期以上的中期染色体上进行原位染色体切口移位的方法。研究证明,切口移位效率达峰值的最适温度为15—20℃,最佳时间为10—15分钟,DNasc Ⅰ的最佳浓度为2ng/ml。用本方法进行的人外周血淋巴细胞染色体的原位切口移位带型表明,原位切口移位的染色体带型特征与已知的G带、R带有较明显的差异,是另一种新的带型。本方法较用’H-dTTP或Bio-dUTP作标记物进行染色体原位切口移位更简便、快速,不仅可用于活性基因在不同种类基因组内的分布研究,而且可能在细胞遗传学和分子生物学研究中具有更广泛的用途。     

Evolution of Genomic Structures on Mammalian Sex Chromosomes

Throughout mammalian evolution, recombination between the two sex chromosomes was suppressed in a stepwise manner. It is thought that the suppression of recombination led to an accumulation of deleterious mutations and frequent genomic rearrangements on the Y chromosome. In this article, we review three evolutionary aspects related to genomic rearrangements and structures, such as inverted repeats (IRs) and palindromes (PDs), on the mammalian sex chromosomes. First, we describe the stepwise manner in which recombination between the X and Y chromosomes was suppressed in placental mammals and discuss a genomic rearrangement that might have led to the formation of present pseudoautosomal boundaries (PAB). Second, we describe ectopic gene conversion between the X and Y chromosomes, and propose possible molecular causes. Third, we focus on the evolutionary mode and timing of PD formation on the X and Y chromosomes. The sequence of the chimpanzee Y chromosome was recently published by two groups. Both groups suggest that rapid evolution of genomic structure occurred on the Y chromosome. Our re-analysis of the sequences confirmed the species-specific mode of human and chimpanzee Y chromosomal evolution. Finally, we present a general outlook regarding the rapid evolution of mammalian sex chromosomes.     

Chromosomes and DNA of Mus

Using C-banded preparations of Mus dunni it is possible to study the behavior of constitutive heterochromatin in early stages of meiotic prophase. The X and the Y chromosomes, both of which contain a large amount of heterochromatin, lie apart in leptotene but move toward each other during zygotene. They then form the sex vesicle at late zygotene. In autosomes zygotene pairing appears to start from the telomeric ends. The centromere of the Y chromosome associates end-to-end with the terminal end of the long arm of the X chromosome. The autosomal heterochromatic short arms show forked morphology in certain bivalents at pachytene, suggesting probable incomplete synapsis.     

Chromosomes and DNA of Microtus

Newborn Microtus agrestis were given single acute whole-body -irradiation (350, 500, or 750R). C-banded bone marrow preparations showed cells with radiation-induced rearrangements of constitutive heterochromatin of the sex chromosomes, usually the consequence of single events, encompassing a wide spectrum of deletions and translocations. These cells persisted in bone marrow for more than a year after irradiation; however, many cells showing the same redistribution of heterochromatin constituted clones of both deletions and translocations. These results indicate that deletion or rearrangement of constitutive heterochromatin does not impair the capacity of bone marrow cells for further proliferation.     

显微镜光度计扫描测量人中期染色体的面积与DNA相对含量

应用显微镜光度计扫描测量人中期染色体的面积与DNA相对含量,结果显示出人染色体相对面积与相对长度之间密切的相关性。染色体DNA相对含量的高低,与染色体面积大小呈正相关。此外,DNA在染色体二维图形的分布是不均匀的,边缘与着丝粒部位的含量低,在纵向范围内变化较大,在横向范围内的变化较小。     

Comparison Between Two Chromogenic Substrates for Revealing an Immunoperoxidase Reaction of Human Metaphase Chromosomes

To study the binding of an antiadenosine serum to human chromosome DNA, two types of chromogenic reagents were compared. The procedure is as follows: lymphocytes with metaphase chromosomes were spread on slides; some slides were irradiated by UV; all slides were then incubated with antiadenosine rabbit serum and then with antirabbit sheep serum labelled with peroxidase; the latter was revealed in the classical manner by 3,3'-diaminobenzidine (DAB), or, alternatively, by p-phenylenediamine plus pyrocatechol (PPD-PC). The present study shows that the results obtained with PPD-PC were equivalent, if not superior, to those obtained with DAB. In the case of weaker reactions, results with PPD-PC were superior. Furthermore, this reagent has the major advantage of being noncarcinogenic.     

Slide Processing for the Examination of Male Mammalian Meiotic Chromosomes

A rapid method was devised specifically for the cytological identification of translocations in the male mouse at late prophase to metaphase of meiotic division I, but the method should be useful for less specific objectives requiring examination of mammalian testicular material. For the adult mouse, masses of tubules from a single testis, freed of the testicular tunic, are placed in 3 ml of 0.7% sodium citrate for 15-20 min, and subsequently fixed in 50% acetic acid by the addition of 3 ml of glacial acetic acid to the hypotonic citrate. To facilitate handling of individual tubules by preserving their visible structure, the addition of fixative is at a rate which is grossly adjusted so that 2 ml will have been added at the end of 30 sec and the remaining 1 ml by the end of a minute. A single fixed tubule 1-2 cm long is placed lengthwise on a slide and covered with a drop of lactic-acetic orcein made as follows: Add 2 gm of synthetic orcein (G. T. Gurr) to a mixture of 50.0 ml of glacial acetic acid, 42.5 ml of 85% lactic acid, and 7.5 ml of distilled water. After staining for 10 min, a 22 × 50 mm cover slip is placed over the tubule, and it is allowed to stain for an additional 10 min. The majority of germinal cells will not be in late prophase or metaphase of the first meiotic division, therefore many preparations will be useless; however, slides with division figures are radidly selected as follows: Before squashing, examine under a microscope at a magnification of 150, and upon recognition of a single meiotic division, remove the slide and squash the preparation for subsequent detailed examination. As a consequence of the spermatogenic wave that progresses along the length of a tubule, a given slide will usually have many division figures or none at all, hence the limitation of 1 tubule per slide facilitates efficient discarding. Preliminary work with the Chinese hamster suggests that good preparations might be obtained from testes of various mammals when the volume of hypotonic solution is adjusted so as to compensate for varying testicular sizes by maintaining a 15:1 ratio of fluid to estimated volume of tissue.     

Polysome Fractionation and Analysis of Mammalian Translatomes on a Genome-wide Scale

mRNA translation plays a central role in the regulation of gene expression and represents the most energy consuming process in mammalian cells. Accordingly, dysregulation of mRNA translation is considered to play a major role in a variety of pathological states including cancer. Ribosomes also host chaperones, which facilitate folding of nascent polypeptides, thereby modulating function and stability of newly synthesized polypeptides. In addition, emerging data indicate that ribosomes serve as a platform for a repertoire of signaling molecules, which are implicated in a variety of post-translational modifications of newly synthesized polypeptides as they emerge from the ribosome, and/or components of translational machinery. Herein, a well-established method of ribosome fractionation using sucrose density gradient centrifugation is described. In conjunction with the in-house developed “anota” algorithm this method allows direct determination of differential translation of individual mRNAs on a genome-wide scale. Moreover, this versatile protocol can be used for a variety of biochemical studies aiming to dissect the function of ribosome-associated protein complexes, including those that play a central role in folding and degradation of newly synthesized polypeptides.     

Evolution of Mammalian Sex Chromosomes: Cooperation of Genetic and Epigenetic Factors

The X and Y chromosomes of mammals, which significantly differ in structure and genetic composition, are thought to originate from a pair of autosomes. During evolution of sex chromosomes in placental mammals, the degradation of the Y chromosome and inactivation spreading along the X chromosome occurred gradually and in concert. Thus, at the molecular level, the genetic and epigenetic factors interacted toward greater differentiation of the X/Y pair. In this review, in context of a comparison permitting to trace this evolutionary pathway, we consider the structural features of mammalian sex chromosomes focusing on the X-chromosomal genes and the unique epigenetic mechanism of their regulation. Possible causes and consequences of the genes escaping X inactivation and aspects of molecular mechanism of X-chromosome inactivation are discussed. A number of hypotheses are considered on evolutionary relationships of X-chromosome inactivation and other molecular processes in mammals.