RNA Export and Electrokinetic Potential of Nuclei in Germinating Embryos of Cereal Crops

The changes in the contents of major components in the nuclei and nuclear membranes during germination of cereal crop embryos were studied. Treatment with RNase of intact nuclei from both dry and germinating embryos changed the electrokinetic potential (EKP) of the nuclear surface. The interrelations between an increased RNA export from isolated nuclei and increased EKP during germination were shown. The conclusion was drawn that the rate of RNA export from the nuclei affected substantially the EKP value, which opens new possibilities for studying physicochemical properties of the nuclear membrane in relation to the functional state of the genetic apparatus and the physiological state of the plant cell.     

Changes in the composition of phospholipids in nuclear subfractions of wheat seedlings treated with gibberellin

Macromolecule transport between the cytoplasm and nucleus occurs through the nuclear pore complexes with the help of soluble transport factors. The receptors of GA are not yet identified, and the molecular mechanism of plant response to GA is not known so far. We compared the content of phospholipids in the soluble nuclear fraction and nuclear membrane of wheat seedlings on the third and fourth days of germination after treatment with GA. It was shown that GA induced differently directed changes in the composition of phospholipids in nuclear subfractions tested. Changes in the composition of the nuclear membrane are supposed to be involved in the control of gene expression by GA.     

The Effects of N-Terminal Fragments of Immunophilin on Phospholipid Composition of Rat Brain and Human Erythrocyte Membranes

Rat brain slices and human erythrocyte membranes have been incubated in the presence of water-soluble synthetic peptide fragments corresponding to residues 1–9 and 1–15 of the N-terminus of immunophilin and the effects on the phospholipid composition examined. During a 2 h incubation in the presence of 1 nM, 0.1 M, and 10 M concentrations of the peptides there were observed significant and dose-dependent decreases in the amounts of phosphatidylcholine and phosphatidylethanolamine, as well as increases in the amounts of phosphatidylserine and, to a less extent, phosphatidylinositol, cardiolipin and lysophosphatidylcholine. The overall decrease in the neutral phospholipids of rat brain, and no changes in human erythrocyte membranes with the simultaneous increase in the acidic phospholipids, both in brain and erythrocyte membranes, tended to counteract any changes in the phospholipid composition of the material studied. The results are discussed in terms of the possible effects of immunophilin on modulating phospholipid turnover in brain cell erythrocyte membranes.     

The Soluble Nucleotide Content of Germinating Wheat Embryos

Estimates are given of the amounts of adenine, uracil, guanine,and cytosine present in the soluble nucleotides of wheat embryosduring germination in the dark. After 48 hours at 25° Cthe nucleotide content per gramme of dry tissue reaches a maximumlevel when the content of bases is approximately 3.5 µmolesof uracil, 2.8 µmoles of adenine, 0.6 µmoles ofguanine, and 0.5 µmoles of cytosine. The soluble nucleotidecontent of embryos growing at 0 to 5° C is lower than thatof embryos of the same dry weight grown at 25° C. Initiallythe amount of adenine present is greater than the amount ofuracil but after 20 hours of germination at 25° C uracilbecomes the predominant base in the soluble nucleotide fraction.Ion-exchange resin chromatography was used to separate the chiefsoluble nucleotides present in the extract of embryos grownfor four days at 25° C. Uridine diphosphate glucose accountsfor the major part of the uracil and adenosine monophosphatefor most of the adenine. Nicotinamide-adenine dinucleotide wasdetected and identified and also a little uridine 5'-mono-phosphate.The possibility is discussed that most of the adenosine monophosphateis produced by degradation of reduced nicotinamide-adenine dinucleotidephosphate.     

体细胞核移植胚胎核重编程的研究进展

尽管在多种哺乳动物种系中成功制备了体细胞克隆后代,但当前的克隆技术仍有许多亟待解决的问题。体细胞核移植胚胎大多存在许多发育异常,造成了妊娠早期高流产率和出生后高死亡率。有研究认为,克隆胚胎发育障碍的一个重要的原因是供体细胞的遗传重编程不完全。哺乳动物种系中,DNA甲基化是胚胎发育期转录调节的必需步骤,除了单拷贝基因序列外,在基因组很多的区域都可以观测到克隆胚胎的异常甲基化。此外,克隆胚胎的基因印迹也存在异常。     

Nuclear matrix bound terminal deoxynucleotidyl transferase in rat thymus nuclei. I. A possible site for TdT mediated function

Approximately 80% of the terminal deoxynucleotidyl transferase (TdT) in thymus glands from 3–4 week old rats was found to be localized in the nucleus and the remaining 20% in the cytosol. Following endogenous nuclease digestion of the thymus nuclei, 70–85% of the nuclear TdT could be removed by low salt and high salt extractions, whereas 15–30% of the enzyme remained tightly bound to the residual nuclear matrix. Low salt and high salt extracts of the nuclei contained a mixture of 58, 56, 45 and 44 kDa species of TdT whereas only 58 kDa species of the enzyme was found to be associated with the matrix. In addition to TdT, 20–25% of the nuclear DNA polymerase was also tightly bound to the isolated nuclear matrix. These observations lead us to propose that besides being the site of DNA replicationvia-matrix bound replicational complexes [Van der Velden H.M.W. & Wanka F., Molecular Biology Reports 12 (1987): 69], nuclear matrix may also be the site of TdT mediated function and that matrix bound TdT and free TdT could be the functional and nonfunctional forms of the enzyme, respectively, in the thymus gland.Abbreviations dNTP deoxyribonucleoside triphosphate - DTT dithiothreitol - Ig immunoglobulin - PMSF phenylmethylsulfonylfluoride - rNTP ribonucleoside triphosphate - SDS sodium dodecyl sulphate - TCR T cell receptor - TdT terminal deoxynucleotidyl transferase - VDJ variable, diversity and joining segments of Ig or TCR genes     

The Effect of Salinity Stress upon Protein Synthesis of Germinating Wheat Embryos

Two differently salt-sensitive wheat genotypes were imbibedin 0·4 M NaCl for 72 h or, alternatively, for 48 h andthen transferred to water. Seed germination, fresh weight andprotein synthesis in embryos were determined. The followingdifferences were found in the synthesis of in vivo [³⁵S]methionine-labelledproteins during salt imbibition: (a) a general decrease or disappearanceof polypeptides specific to the radicle emergence phase in thesalt-sensitive genotype; (b) a new synthesis of polypeptideswhich are not found during water imbibition and are common toboth genotypes; (c) a differential synthesis of polypeptidesthat are unique to each cultivar. Upon return to water, salt-inducedproteins ceased to be synthesized while proteins associatedwith an advanced germination phase were actively produced. Theseresults suggest that the expression of 'salt stress' proteinsis related to the adaptation process of seeds to salinity aswell as to the genetic constitution of a selected salt-tolerantgenotype.Copyright 1993, 1999 Academic Press Triticum durum, wheat, embryo, salt stress, protein synthesis     

Dietary Modifications of Phospholipid Composition and Biophysical Properties of the Brush Border Membrane Along the Trout Intestine

Brush border membranes (BBM) are isolated from middle and posterior intestine of trout fed either an essential fatty acid-rich diet or a saturated one. The different phospholipid classes are separated, and their fatty acid composition is determined. Fluorescence anisotropy studies are performed using two lipid fluorophores, namely diphenylhexatriene (DPH) and trimethyl-aminodiphenylhexatriene (TMA-DPH). The results indicate that the usual parameters affecting the lipid fluidity such as the phospholipid:protein (PL:PROT), cholesterol:phospholipid (CHOL:PL), and sphingomyelin:phosphatidylcholine (SP:PC) ratios and the unsaturation of the acyl chains are sufficient to explain the fluidity values determined using DPH, but not those obtained with TMA-DPH as a probe. This fluorophore is assessed to be localized only in the external leaflet of the membrane. Hence, it will be affected by the composition of the major phospholipids of this leaflet, sphingomyelin and phosphatidylcholine.     

Microextraction of Nuclear Proteins from Single Maize Embryos

The analysis of DNA binding proteins can be difficult when only small quantities of tissue expressing the desired protein are available. We present a protocol for the preparation of nuclear extracts from as little as 100 mg of tissue. This protocol is well suited for extraction of DNA binding proteins from tissues that are difficult to obtain in large quantities such as maize embryos.     

HeLa细胞核和核骨架含有肌动蛋白

提取HeLa细胞核并制备核骨架标本,以抗肌动蛋白抗体为探针,采用SDS－PAGE、免疫荧光和免疫印迹等方法,对HeLa细胞细胞核和核骨架中的肌动蛋白进行了研究,并用鬼笔环肽荧光染色方法研究了其中的F－肌动蛋白。在荧光显微镜下观察到：代表肌动蛋白的特异性荧光分布在细胞核和核骨架中,说明肌支蛋白是细胞核和核骨架的固有成分;代表F－肌动蛋白的特异性荧光存在于细胞和核骨架中,说明细胞核和核骨架含有F－肌动蛋白。免疫印迹结果进一步肯定了细胞核和核骨架中肌动蛋白的存在。     

Alteration of Nuclear Matrix Protein Composition during Apoptosis in Rat Embryo Cells

Alteration of the nuclear matrix protein composition during active cell death was investigated by high resolution 2-dimensional gel electrophoresis and computer-assisted image analysis. Nuclear matrices were isolated from purified nuclei of a rat embryo cell line showing an immediate apoptotic response to serum reduction. While cell shrinkage and cytoplasmic compaction, characteristic features of apoptosis, were induced, the nuclear matrix protein pattern was not altered 1 h after induction of apoptosis. However, two sets of novel nuclear matrix protein spots appeared with differing kinetics within the following 5 h of apoptosis. They consisted of five and six protein spots, respectively. In addition, the intensity of five nuclear matrix protein spots that had already been present in the uninduced cells increased continuously within an observation period of 12 h. These coincidences point to a potential involvement of the described nuclear matrix proteins in the apoptotic process.     

Nuclear dreams: The malignant alteration of nuclear architecture

Cancer is diagnosed by examining the architectural alterations to cells and tissues. Changes in nuclear structure are among the most universal of these and include increases in nuclear size, deformities in nuclear shape, and changes in the internal organization of the nucleus. These may all reflect changes in the nuclear matrix, a non-chromatin nuclear scaffolding determining nuclear form, higher order chromatin folding, and the spatial organization of nucleic acid metabolism. Malignancy-induced changes in this structure may have profound effects on chromatin folding, on the fidelity of genome replication, and on gene expression. Elucidating the mechanisms and the biological consequences of nuclear changes will require the identification of the major structural molecules of the internal nuclear matrix and an understanding of their assembly into structural elements. If biochemical correlates to malignant alterations in nuclear structure can be identified then nuclear matrix proteins and, perhaps nuclear matrix-associated structural RNAs, may be an attractive set of diagnostic markers and therapeutic targets. J. Cell. Biochem. 70:172–180, 1998. © 1998 Wiley-Liss, Inc.     

Lipins, Lipids and Nuclear Envelope Structure

The lipid composition of biological membranes is crucial for many aspects of organelle function, including growth, signalling, and transport. Lipins represent a novel family of lipid phosphatases that dephosphorylate phosphatidic acid (PA) to produce diacylglycerol (DAG), and perform key functions in phospholipid and triacylglycerol biosynthesis and gene expression. In addition to its role in lipid biosynthesis, the yeast lipin Pah1p and its regulators are required for the maintenance of a spherical nuclear shape. This review summarizes recent advances in our understanding of the yeast lipin Pah1p and highlights the possible roles of phospholipid metabolism in nuclear membrane biogenesis.     

The Glycoprotein Composition of Peripheral Nervous System Myelin Subfractions

Two fractions were isolated by continuous density gradient centrifugation from total particulate matter of rabbit sciatic nerves: a minor fraction, B, consisting of small-sized membrane fragments and a major fraction, C, of characteristic multilayered myelin figures, with maxima at 0.33 and 0.58 M-sucrose, respectively. In comparison with C, fraction B was enriched in CNPase and alkaline phosphatase activities and the P0, 23K and Z proteins, but was virtually devoid of basic protein. The glycoprotein composition of all fractions was examined with four fluorescein isothiocyanate-labelled lectins (WGA, Con A, RCA-60, U.E.). These revealed the presence of six glycoproteins in all fractions with similar lectin binding capacities and molecular weights ranging from 35,500 to 16,000, of which P0 was the predominant component. Material found on the heavy side of fraction C was characterized by the presence of a multitude of glycoproteins which bound variable proportions of the four different lectins, suggesting substantial variations in their carbohydrate moieties. Their absence from the central portion of fraction C points to a location other than that of compact PNS myelin.     

基因枪介导小麦成熟胚遗传转化的影响因素

小麦成熟胚作为转化受体可克服小麦幼胚存在的受季节和幼胚发育阶段限制的缺点。以湖北省小麦品种‘鄂麦12’和模式品种‘Bobwhite’为材料,成熟胚为转化受体,优化基因枪转化法的轰击压力、轰击距离、选择剂等因素,建立以小麦成熟胚为转化受体的高效转化系统。结果表明:小麦成熟胚作为转化受体时,适宜轰击压力和轰击距离组合是900 psi、6 cm;成熟胚对选择剂G418的敏感性强于幼胚,轰击后需要延长恢复时间,选择剂G418的适合浓度为20～40 mg/L。在以上优化条件下小麦成熟胚转化频率达0.3%～0.9%,已初步建立基因枪介导的小麦成熟胚遗传转化系统。     

Effect of NaCl and Polyamines on Plasma Membrane Lipids of Wheat Roots

Caryopses of a salt sensitive wheat cultivar (Triticum aestivum L. cv. Giza 163) were presoaked in 2.5 mM putrescine (Put), 5 mM spermidine (Spd) or 2.5 mM spermine (Spm) for 24 h and then subjected to 150 mM NaCl added to the growth medium for 15 d. Effects of NaCl and polyamines (PAs) on plasma membrane (PM) lipids, phospholipids, fatty acids, and free sterols were determined. NaCl treatment caused a decrease in total phospholipids, increase in saturated fatty acids and altered distribution of sterols and phospholipids. NaCl also induced increase in sterol/phospholipid ratio. PAs treatments (particularly Put and Spd) counterbalanced the NaCl deleterious effects on PM lipids.     

小麦与高冰草体细胞杂种后代的核基因组和叶绿体基因组的遗传特征

以小麦(TriticumaestivumL.)与高冰草(Agropyronelongatum(Host)Nevski)体细胞杂种同一个克隆来源的F2-F6自交系Ⅱ-2、Ⅱ-Ⅰ-8以及由Ⅱ-Ⅰ-8F2分离形成的8-1(F3-F6)为材料,利用小麦叶绿体基因组的微卫星(Microsatellite)特异引物及随机扩增多态性DNA(RAPD)引物进行分析。结果表明,杂种株系的叶绿体基因组组成一致,均以小麦叶绿体基因组为主,仅在rpl14和rpl16基因的间隔序列中检测到双亲的特征带,表明有高冰草的叶绿体DNA在杂种中存在,并稳定遗传至第六代。RAPD分析表明,不同杂种株系中存在不同的高冰草核DNA片段,核基因组在传代中基本稳定。     

哺乳动物核质重组胚后成性重序的机制

研究表明,供体核不完全或者不正确的后成性重序可能是核移植失败的主要原因。本篇文章综述了哺乳动物核质重组胚中存在的几种不同的后成性重序机制,包括DNA甲基化、染色质重构、基因组印记与X染色体失活、端粒维持以及其他后成性遗传机制。理解重组胚后成性重序的机制将有助于我们解决核移植技术中存在的问题,进而促进它的应用。     

Quantitative Analysis of Membrane Protein Transport Across the Nuclear Pore Complex

Nuclear transport of the Saccharomyces cerevisiae membrane proteins Src1/Heh1 and Heh2 across the NPC is facilitated by a long intrinsically disordered linker between the nuclear localization signal (NLS) and the transmembrane domain. The import of reporter proteins derived from Heh2 is dependent on the FG‐Nups in the central channel, and the linker can position the transport factor‐bound NLS in the vicinity of the FG‐Nups in the central channel, while the transmembrane segment resides in the pore membrane. Here, we present a quantitative analysis of karyopherin‐mediated import and passive efflux of reporter proteins derived from Heh2, including data on the mobility of the reporter proteins in different membrane compartments. We show that membrane proteins with extralumenal domains up to 174 kDa, terminal to the linker and NLS, passively leak out of the nucleus via the NPC, albeit at a slow rate. We propose that also during passive efflux, the unfolded linker facilitates the passage of extralumenal domains through the central channel of the NPC .