Nuclear assembly of demembranated Xenopus sperm in plant cell-free extracts from Nicotiana ovules

The cell-free preparation derived from Nicotiana tabaccum ovules induced chromatin decondensation and pronuclear formation from demembranated Xenopus laevis sperm nuclei. Fluorescent microscope and phase-contrast microscope visualization of assembly intermediates indicated that 95.6% of X. leavis sperm changed their tadpole-like shape to circular shape or elliptical shape after over 1.5 h of incubation. Transmission electron microscope visualization showed that nuclear membrane was assembled around the periphery of the dispersed chromatin. Nuclear envelope of most reassembled nuclei was composed of a double membrane inlaid with a little single membrane. Nucleosome assembly was verified by means of micrococcal nuclease digestion. After 2 to 5 h of incubation, digestion of the product of nuclear assembly with micrococcal nuclease produced at least six nucleosome fragments of about 250 bp each.     

动物克隆过程中核的重新编程

核移植后,供体核会发生核仁结构和组蛋白H1的变化。体细胞型组蛋白H1的消失可作为核重新编程的一种标志,组蛋白H1在中期胞质中比在间期胞质中消失得快。核移植后,供体核的基因表达被重新编程,从而正确进行RNA转录和蛋白质合成。     

Nuclear assembly of purified Crythecodinium cohnii chromosomes in cell—free extracts of Xenopus laevis eggs

Incubation of dinoflagellate Crythecodinium cohnii chromosomes in cytoplasmic extracts of unfertilized Xenopus laevis eggs resulted in chromosomes decondensation and recondensation,nuclear envelope assembly,and nuclear reconstitution.Dinoflagellate Crythecodinium cohnii is a kind of primitive eukaryote which possesses numerous permanently condensed chromosomes and discontinuous double-layered nuclear membrane throughout the cell cycle.The assembled nuclei,being surrounded by a continuous double membrane containing nuclear pores and the uniformly dispersed chromatin fibers are morphologically distinguishable from that of Dinoflagellate Crythecodinium cohnii.However,incubation of dinoflagellate Cyrthecodinium cohnii chromosomes in the extracts from dinoflagellate Crythecodinium cohnii cells does not induce nuclear reconstitution.     

鱼类核移植与重编程

鱼类核移植是动物克隆研究的一个重要领域, 我国学者在上世纪60年代首创了鱼类的核移植研究。以斑马鱼为模式动物, 进行核移植与再程序化研究具有独特的优势。文章总结了鱼类细胞核移植研究的历史、斑马鱼核移植研究概况、以及影响核移植胚胎发育的因素, 特别是核移植胚胎基因组的表观遗传修饰, 如基因组DNA甲基化及组蛋白乙酰化和甲基化等的研究, 将有助于完善克隆技术并提高克隆的成功率, 推动克隆技术的广泛开展和应用。     

Nitrate reductase in cell-free extracts ofAzotobacter

Summary 1. Nitrate reductase activity in cell-free extracts ofAzotobacter vinelandii was obtained. The enzyme is TPNH-linked and shows some stimulation by molybdenum and FMN.2. The cell-free preparations showed a strong DPNH-oxidase activity and also a slight TPNH oxidation following the addition of distilled water. The latter activity could, however, be removed by ultra-centrifugation of the enzyme extracts. However, nitrate reductase seems to be only to a small extent soluble as its main activity was associated with particles. The particles spun down were red in colour suggesting the presence of cytochromes.3. Thick cell suspensions ofA. vinelandii, A. agile, andA. chroococcum showed similar cytochrome spectra. The _max. observed suggest the presence of cytochromes of thec type (_max. at 524 and 552 mµ) anda type (_max. at 590 and 632 mµ).4. No apparent differences were observed between the cytochrome spectra ofA. vinelandii cells grown on molecular and nitrate nitrogen.     

Chromatin assembly in yeast cell-free extracts

A simple method for preparing chromatin assembly extracts has not been available for budding yeast. Here I describe such a method in detail. The assembly extract, a crude 100,000g supernatant, is prepared from cells disrupted in a manual or motorized grinder while they are frozen. The core histones and all soluble protein factors required for chromatin assembly under physiological conditions are present in the extract. Assembly is sensitive to mutation of lysine residues in the amino-terminal tail of histone H4 whose acetylation is associated with nucleosome deposition in vivo. The reaction is ATP dependent, and assembly-driven DNA supercoiling occurs with the same efficiency as in extracts from mammalian somatic cells. This simple system offers a unique opportunity to analyze chromatin metabolism by a combined biochemical and genetic approach that is not feasible for any other model organism.     

细胞抽提物诱导的体细胞重编程

体细胞重编程是指在特定条件下将已分化的体细胞去分化逆转回原始多能状态或转分化为其他类型细胞。目前诱导体细胞重编程的方法主要有：体细胞核移植、细胞融合、特定转录因子转染、细胞抽提物诱导等。近年来抽提物诱导体细胞重编程越来越受到研究者的关注。利用此法, 通过重编程不仅可以获得需要类型的细胞, 而且方便识别与重编程有关的细胞因子, 探求重编程的机制。文章简略概述诱导体细胞重编程的方法, 并重点阐述细胞抽提物诱导体细胞重编程的研究进展。     

应用大肠杆菌染色素DNA在非洲爪蟾卵提取物实现诱导非细胞体系…

近年来我们实验室已成功地利用细胞核体外组装的实验模式,将多种生物的ＤＮＡ在非洲爪蟾卵提取物中实现了非细胞体系核装配。但亲缘关系最远的原核生物的染色体ＤＮＡ是否也能在此真核体系中进行核装配一直没有报道。我们以大肠杆菌染色体ＤＮＡ为材料,研究了它诱导的非细胞体系核装置。在光镜与电镜水平观察了核装配的过程。显微分光光度计扫描显示ＤＮＡ片段在核装配过程中经历了凝集－去凝集的变化。证明大肠杆菌染色体ＤＮＡ也     

Nuclear reprogramming in nuclear transplant rabbit embryos

The first six genetically verified nuclear transplant rabbits have been produced in this study. Individual eight-cell stage embryo blastomeres were transferred and fused with enucleated mature oocytes of which six full-term offspring were produced out of 164 manipulated eggs. The following efficiency rates were determined for the nuclear transplantation procedure: chromosomal removal from oocytes, 92%; fusion rate, 84%; activation rate, 46%; embryo transfer rate, 27%. Additional reasons for the low efficiency rate of nuclear transplant embryos may include limited development due to aging in recipient oocytes and asynchronous transfers of manipulated embryos to recipient females. The successful development to term may have been due to the ability of the mature oocyte to reprogram the eight-cell stage nuclei. The number of cells in blastocysts derived from isolated eight-cell blastomeres (18 +/- .08) was lower than that of nonmanipulated pronuclear (106 +/- 5.1) and nuclear transplant embryos derived from eight-cell stage nuclei (91 +/- 10.2) (p less than 0.001). This evidence along with the significant amount of nuclear swelling in nuclear transplant embryos and a delay in the time of blastocyst formation indicate that nuclear reprogramming had taken place in these embryos. Successful nuclear reprogramming indicates that serial transfers could result in the expanded multiplication of mammalian embryos.     

Nuclear actin in transcriptional reprogramming by oocytes

Comment on: Miyamoto K, et al. Genes Dev 2011; 25:946-58.     

Adenyl cyclase in cell-free extracts ofEscherichia coli

The adenyl cyclase enzyme system was detected in the cells ofEscherichia coli disrupted by sonic treatment. This enzyme activity is located mainly in the cell fraction sedimenting at 2,000×g, i.e. in the cytoplasmic membrane fraction. A prolonged sonication treatment of the cell suspension was followed by the disappearance of activity in the membrane preparation. The pH optimum of the adenyl cyclase inEscherichia coli was on the alkaline side, around pH 9.