小麦细胞核仁中rRNA基因转录的超微定位

真核细胞核仁中rRNA基因转录位点是长期以来未能解决的问题。以小麦细胞为研究材料,应用常规电子显微镜技术,观察了小麦细胞核仁纤维中心(Fibrillar centers,FC)内染色质的超微结构;并通过DNA抗体阐明了核仁中DNA位于纤维中心、致密纤维组分(Dense fibrillar component,DFC)以及两者的过渡区域;应用RNA聚合酶I相关转录因子UBF(Upstream binding factor)抗体所做的分析显示,小麦细胞核仁中UBF位于FC与DFC的过渡区域以及DFC中,在FC中没有UBF的存在;进一步借助于RNA/DNA杂合体抗体选择性地直接标记核仁中rRNA基因的转录位点,结果表明了小麦细胞核仁rRNA基因的转录位点是在FC与DFC的过渡区域及DFC中。     

豌豆细胞核仁中rDNA的超微定位和排布构型

利用细胞化学DNA特异染色法——NAMA-Ur特异染色法对豌豆细胞核仁中rDNA的位置及其排布构型进行了原位观察。结果表明,核仁中的rDNA位于纤维中心(FC)以及FC与致密纤维组分的交界处,以环绕FC的形式排布。不同位置的rDNA成分都具有集缩和解集缩两种形态结构,核仁外的核仁伴随染色质经过核仁通道进入核仁,沿FC周边排列,与其中的DNA相连。     

两栖肢场的插值再生和图式调节

French-Bryant-Bryant的增生场图式调节机制被合理地作了修正,从而自洽地测决了同侧嫁接和异侧嫁接致生正常重肢的形成问题:(1)指出细胞插值生长和远向变换约束之间的平衡是一种自发对称性破损机制,会导致全区捷径插值被分区捷径插值局部地(在异侧嫁接)或整体地(在同侧嫁接)取代,分别在全区和分区极大周值差位置致生重肢.(2)关于重肢的手性和细胞来源的理论预测与实验结果吻合.(3)对于异常重肢的结构复杂性也提出了一种可行的解释.     

POLARITIES, CELL DIFFERENTIATION AND PRIMARY INDUCTION IN THE AMPHIBIAN EMBRYO

1. Amphibian eggs are spherical, while the embryos are bilaterally symmetrical. The latter is manifested morphologically when gastrulation begins with the formation of the blastopore at a bilaterally symmetrical (vegetal-dorsal) location on the surface of the embryo. To account for this change in symmetry two polarities (vectors or axes) are required. These need not go through the centre, but if they do, one will go through two poles, called ‘animal’ and ‘vegetal’ in the amphibian embryo, and the other will pass through two points on opposite sides of the egg, one at the ‘dorsal’ and one at the ‘ventral’ side. Together these two polarities define a plane of bilateral symmetry. 2. It may be assumed that one polarity determines that gastrulation begins in the vegetal hemisphere, and the other that it begins at the dorsal side. 3. Judging from the distribution of pigment in the cortex of the egg and that of the yolk-hyaloplasm in the interior, an animal-vegetal polarity is already present in the unfertilized egg. That cytoplasmic components are actually part of the material substrate of this polarity is evident from the fact that the pattern of gastrulation may be upset if the distribution of yolk-hyaloplasm is deranged. 4. At fertilization the pigment border is raised at the side opposite the fertilizing sperm, giving rise to the ‘grey crescent’. The latter confers the first visible bilateral symmetry on the egg, and in fact it determines the presumptive median plane, for blastopore formation begins in the midline of the grey crescent. The dorso-ventral polarity imposed by the sperm is not irreversibly determined. By various experimental means, e.g. restriction of the oxygen supply, it may be inverted. 5. In order to understand the mechanism of the polarities it is necessary to study the processes on which the effects of the polarities are exerted, viz. the process of invagination associated with the formation of the blastopore. It has been known for a long time that at the bottom of the blastoporal groove are located some large flask-shaped cells, called ‘Ruffini's cells’. Various arguments can be mobilized to support the notion that these cells actually are engaged in pulling in the embryonic surface. 6. These cells are the first representatives of a cell type different from the spherical cells which are typical of the early embryo. It may therefore be presumed that Ruffini's cells are the products of the first cell differentiation occurring during amphibian embryogenesis. And it may further be assumed that the polarities somehow control this process. 7. A number of observations suggest that the animal-vegetal polarity is in direct control of the differentiation, ensuring that Ruffini's cells are formed only in the vegetal hemisphere. This point has been corroborated by isolating in cultures small aggregates from various regions of the blastula. When this is done it is found that the only path of differentiation available to animal cells is the formation of small spherical aggregates composed of a mixture of ciliated and non-ciliated cells. In contrast, in cultures of vegetal cells an outgrowth of cells occurs, and these cells share a number of properties with Ruffini's cells, and it is suggested that they are representatives of this cell type. 8. The formation of these cells is suppressed by inhibitors of RNA synthesis and by anaerobiosis induced by KCN. Since oxidative metabolism is apparently required for the differentiation of Ruffini's cells - gastrulation in the intact embryo is suppressed by anaerobiosis - a number of carbohydrate metabolites were scrutinized for their effect on the formation on Ruffini's cells. It was found that at 10 mm lactate completely suppresses their appearance, and indeed all the other cell differentiations that can otherwise be observed in our cell cultures. Since there is a very steep animal-vegetal cytoplasmic gradient in carbohydrate, the content being lowest at the vegetal pole, lactate might potentially be the agent of the animal-vegetal polarity, but there are a number of facts which do not readily support this idea. 9. If animal cells are explanted together with a few vegetal cells, some of the aggregates do not become ciliated, but rather exhibit an outgrowth similar to the one observed with vegetal cells. These animal cells have the same general shape as the vegetal Ruffini's cells, but they are smaller and more pigmented, typical ‘animal’ features. When the cultures are preserved, the cells undergo further differentiation, becoming either ‘mesenchyme’ cells, nerve cells, pigment cells and sometimes even muscle cells may be observed. In the normal embryo these differentiation patterns occur in that part of the animal hemisphere which becomes induced through contact with the vegetal material entering the blastocoel during gastrulation. Thus there is reason to assume that the induction occurring in our cultures is a miniature of the normal induction process. 10. Just as in the sea-urchin embryo, the animal cells in amphibia may become ‘vegetalized’ by addition of Li⁺ to the culture medium. 11. For various reasons it is likely that Ruffini's cells contain heparan sulphate, and in the belief that this substance might be the inductor proper, its effect was tested on animal cells. It turned out that in a concentration of 0·1 ppm it can alter the differentiation pattern of these cells, and we suggest that heparan sulphate, for the time being, is the most likely candidate for the role of primary inductor in the amphibian embryo. 12. The edges of the blastoporal groove, and hence the formation of Ruffini's cells, proceeds gradually around the circumference of the embryo. The effect of the dorso-ventral polarity therefore appears to be concerned with the time at which the cells undergo differentiation, imposing a spatial and a temporal gradient on this phenomenon. The second overt manifestation of the dorso-ventral polarity, next to the formation of the grey crescent, concerns the size of the embryonic cells, the dorsal ones being always smaller than the ventral. This fact suggests the possibility that the polarity may exert its effect by interfering with the process of cell division. 13. The cell divisions in the early embryo are distinguished by being synchronous; all cells are either undergoing mitosis or they are in interphase. The duration of the latter is typically very short. After a certain number of cell divisions, around 10, when the embryos are in the mid-blastula stage, the synchrony is gradually lost, while the interphase becomes considerably prolonged. This peculiar behaviour suggests that the cytoplasm of the early embryonic cells contain some factor which ensures the synchrony. The well-known presence in the early embryo of deoxyriboside-containing material, in an amount corresponding roughly to the total amount of DNA residing in the cell nuclei after 10 cell divisions hinted that deoxyribosides might indeed be the ‘synchrony factor’. 14. This idea was tested first on intact embryos. An excess of deoxyribonucleotides was injected into very early embryos. The result was developmental arrest at a pregastrula stage (no Ruffini's cells formed) in a large percentage of embryos. However, the number of cells was greater than in the controls, and the rate of cell division higher, indicating a delay in the transition to synchrony, thus supporting the proposed mechanism. Furthermore, the deoxynucleotides inhibited cell differentiation and an explanation of this was found in the fact that they also strongly inhibited RNA synthesis. 15. The studies were extended to cell cultures. It was found that deoxyribosides inhibit the differentiation of animal as well as vegetal cells; instead, the cells go on dividing at least for another two rounds. The utilization of added deoxyribosides does not demonstrate that the endogenous substances are similarly utilized. That they are, was indicated by the following experiment: In the presence of cytosine arabinoside, an inhibitor of DNA synthesis de novo, the explanted cells go on dividing an unknown number of times, and then they, animal as well as vegetal cells, undergo differentiation. But in either case these cells are larger (about four times) than the controls. This result suggests that in the experimental cultures the cells go on dividing as long as the cytoplasmic deoxyribosides last and then stop, while the controls synthesize their own DNA for two rounds of division before they undergo differentiation. 16. It is now possible to suggest a mechanism for the dorso-ventral polarity. First it affects the cell size such that the dorsal cells are the smallest. If the cytoplasmic deoxyribosides are evenly distributed at the outset, then small cells must be nearer exhaustion than large ones. A dorso-ventral gradient in cell sue will therefore automatically imply a dorso-ventral gradient in the time at which the cells reach the state in which they can undergo differentiation.     

拟目乌贼卵子发生与卵巢发育

为了丰富拟目乌贼(Sepia lycidas)生物学资料, 为人工育苗与养殖提供理论依据, 采用解剖学和组织学的方法, 对水泥池养殖条件下拟目乌贼卵子发生和卵巢发育进行了研究。结果表明: 经过6个月水泥池养殖, 平均体重为256.34 g, 最大体重达到457.08 g, 个别发育成熟, 绝大部分未达性成熟。卵子发生不同步, 根据细胞形态、细胞大小、滤泡细胞形态和卵黄形成情况可分为卵原细胞阶段(卵原细胞期)、原生质生长阶段(无滤泡期、单层滤泡期和双层滤泡期)、间质生长阶段(滤泡内折早期、滤泡内折中期和滤泡内折晚期)和营养质生长阶段(卵黄发生早期、卵黄发生晚期和成熟期), 共4个阶段10个时期。卵巢发育根据外观形态、性腺指数变化和切面上各期细胞所占的比例, 可分为形成前期、形成期、小生长期、大生长期、成熟前期和成熟期6个时期。拟目乌贼繁殖周期为一年。       

FORMATION AND BEHAVIOR OF PINOSOMES IN THE SEA URCHIN OOCYTE DURING OOGENESIS

Formation and behavior of the pinosomes at the surface of the oocyte during oogenesis in the 4 species of sea urchins, Anthocidaris crassispina, Temnopleurus toreumaticus, Mespilia globulus and Pseudocentrotus depressus, were studied. The plasma membrane of the oocyte is almost smooth at the early stage of oogenesis, although a small number of cytoplasmic processes appear on it, facing the germinal epithelium. At the beginning of vitellogenetic stage many processes appear on the whole surface of the oocyte. Near the base of the fully grown process, the pinosome designated as the α-pinosome is formed. The α-pinosome may play a part in maturation of the yolk granule. The processes shorten as a whole at the time of the breakdown of the germinal vesicle. Formation of the pinosome designated as the β-pinosome begins just before vitellogenetic stage and continues during this stage. The β-pinosome may be directly concerned with the formation of cortical granules.     

SMALL GRANULES IN THE AMPHIBIAN OOCYTE NUCLEUS AND THEIR RELATIONSHIP TO RNA

Small particles (100 to 300 A in diameter) are seen in sections of nucleoli, the loops of the amphibian lampbrush chromosomes, and the Balbiani-ring regions of dipteran salivary-gland chromosomes. All of these structures contain cytochemically demonstrable RNA. Furthermore, the annuli seen on the nuclear envelope are composed of small particles which are similar to or identical with those commonly associated with the endoplasmic reticulum. It seems likely that ribonucleoproteins are organized as small particulates in the nucleus as well as in the cytoplasm.     

TISSUE AND DEVELOPMENTAL SPECIFICITY OF A POLYSIALO-GANGLIOSIDE SPECIES IN THE AMPHIBIAN XENOPUS

Xenopus embryos contain a considerable amount of a polysialo-ganglioside not yet fully characterized; in this paper, we will refer to it as ganglioside X1. Preliminary experiments indicate asialo-GM1 as the core structure of the ganglioside X1 and palmitic and oleic acid as the fatty acids of the ceramide moiety. Further analyses by comparative 2D-TLC with adult fish and chick embryo brains indicate the pentasialilated ganglioside GP1c as the possible structure of X1. In the adult Xenopus, X1 characterizes the ganglioside pattern of the central nervous system while is absent in all the other tested tissues. At least two other more polar (presumably richer in sialic acid) bands are often visible under X1, both in embryos and in brain and spinal cord tissues of adult Xenopus. The persistence of polysialo-gangliosides in the brain and spinal cord of adult amphibians could serve to guarantee a proper functioning of the central nervous system at low body temperature.     

THE REGULATION OF RNA SYNTHESIS AND PROCESSING IN THE NUCLEOLUS DURING INHIBITION OF PROTEIN SYNTHESIS

The effect of protein synthesis inhibition by cycloheximide on nucleolar RNA synthesis and processing has been studied in HeLa cells. Synthesis of 45S RNA precursor falls rapidly after administration of the drug. However, the nucleolar content of 45S RNA remains relatively constant for at least 1 hr because the time required for cleavage of the precursor molecule into its products is lengthened after treatment with cycloheximide. The efficiency of transformation of 45S RNA to 32S RNA remains constant with approximately one molecule of the 32S RNA produced for each cleavage of a molecule of 45S RNA. However, shortly after the cessation of protein synthesis the formation of 18S RNA becomes abortive. The amount of 32S RNA present in the nucleolus remains relatively constant. After long periods of protein synthesis inhibition the 28S RNA continues to be synthesized and exported to the cytoplasm but at a greatly reduced rate. When the protein synthesis inhibitor is removed, a prompt, although partial, recovery in the synthesis rate of 45S RNA occurs. The various aspects of RNA synthesis regulation and processing are discussed.     

A HEAT-SENSITIVE CELLULAR FUNCTION LOCATED IN THE NUCLEOLUS

Striking nucleolar lesions occur in cultured cells after exposure to supranormal temperatures. These lesions appear at 42°C and consist of a loss of the granular ribonucleoprotein (RNP) component and intranucleolar chromatin, and a disappearance of the nucleolar reticulum. The material remaining in the morphologically homogeneous nucleolus is a large amount of closely packed fibrillar RNP. The lesions remain identical as temperature increases to 45°C. These alterations are reversible when the cells are returned to 37°C and are associated with the reappearance of an exaggerated amount of intranucleolar chromatin and granular RNP. High-resolution radioautography indicates that after thermic shock nucleolar RNA synthesis is inhibited whereas extranucleolar sites are preserved: it also suggests that the granular RNP is reconverted to fibrillar RNP probably by simple unraveling. The results prove the existence of heat-sensitive cellular functions in the nucleolus which deal with the DNA-dependent RNA synthesis. The precise site of action is assumed to involve hydrogen bonds, resulting in configurational changes in nucleolar RNP and affecting the stability of the DNA molecule. The subsequent events in nucleolar RNA synthesis are discussed in light of the morphologic and biochemical effects of actinomycin D on the nucleolus.     

THE NUCLEAR PERMEABILITY, INTRACELLULAR DISTRIBUTION, AND DIFFUSION OF INULIN IN THE AMPHIBIAN OOCYTE

[³H]Inulin (mol wt ≈ 5,500) solutions are microinjected into the cytoplasm of mature oocytes of Rana pipiens and the subsequent movement of the solute recorded by quantitative ultralow temperature autoradiography. The autoradiographs show transient cellular diffusion gradients, the influence of the nucleus on these gradients, and the nuclear:cytoplasmic distribution of inulin. Analysis leads to the following conclusions: (a) Inulin diffuses in cytoplasm at about 3 x 10^-6 cm²/s, or one-fifth as rapidly as in water. Most of this decrease is attributable to the increased tortuosity of the diffusional path due to the presence of inclusions and macromolecules. (b) The nuclear envelope is very permeable to inulin; its resistance to inulin's passage is similar to that of cytoplasm. The envelope appears to play a negligible role in regulating the nucleocytoplasmic movement of solutes smaller than macromolecules, (c) Inulin concentrates in the nucleus to four times its cytoplasmic level; this is attributed to solute exclusion from cytoplasmic water. Evidence is presented that among hydrophilic solutes the degree of exclusion increases with molecular size. The potential significance of cytoplasmic exclusion processes to understanding secretion and the intracellular movement of macromolecules is briefly discussed.     

THE FINE STRUCTURE OF OOGENESIS IN MARCHANTIA POLYMORPHA

Archegonium development, beginning with the archegonial initial and culminating in the mature egg, was studied with the electron microscope. The ultrastructural features of the beginning stages in development of the archegonium are relatively similar to one another. Plasmodesmata occur between all adjacent cells at this time. After the secondary central cell is formed these protoplasmic connections are lost, and both axial and parietal cell lineages begin to show signs of ultrastructural differentiation. The mature egg is characterized by cytoplasm rich in ribosomes and larger organelles. Mitochondria and simplified plastids commonly display a juxtaposed association. As far as could be ascertained the numerous plastids and mitochondria in the egg of Marchantia arise through division of preexisting organelles and are not formed anew from evaginations of the nucleus. Blebbing of the nucleus produces polymorphic organelles which appear to be pinched off into the cytoplasm. The mature egg also contains vacuoles and lipid bodies toward its periphery, while dictyosomes and extensive endoplasmic reticulum occur throughout. The space between the wall cells and the mature egg appears to contain an amorphous substance. No extra membrane was observed around the mature egg.     

ANALYSIS OF SODIUM TRANSPORT IN THE AMPHIBIAN OOCYTE BY EXTRACTIVE AND RADIOAUTOGRAPHIC TECHNIQUES

The transport of Na⁺ in mature Eurycea oocytes was studied by quantitative radioautography of ²²Na⁺ using techniques suitable for localization of diffusible solutes, together with conventional extractive techniques. Intracellular Na⁺ consisted of three kinetic fractions: a cytoplasmic fast fraction of about 8.5 µeq/ml H₂O; a cytoplasmic slow fraction of about 58.7 µeq/ml H₂O; and a nuclear fast fraction of about 11.1 µeq/ml H₂O. A nuclear slow fraction, if it exists, does not exceed 5% of the cytoplasmic. The fast fractions represent freely diffusible Na⁺ in the two compartments; the nuclear solvent space is 1.3 times the cytoplasmic. The flux of both fast fractions is determined by the permeability of the cortical membrane, with neither the nuclear membrane nor diffusion in the cytoplasm detectably slowing the flux. The cytoplasmic slow fraction is interpreted to represent Na⁺ bound to nondiffusible constituents which are excluded from the nucleus; these may be yolk platelets, although the widespread observation of Na⁺ binding in other cells, and the high Na⁺/K⁺ selectivity, argues against simple ion-binding to the yolk phosphoprotein.     

GENES IN SPORT AND DOPING

Genes control biological processes such as muscle production of energy, mitochondria biogenesis, bone formation, erythropoiesis, angiogenesis, vasodilation, neurogenesis, etc. DNA profiling for athletes reveals genetic variations that may be associated with endurance ability, muscle performance and power exercise, tendon susceptibility to injuries and psychological aptitude. Already, over 200 genes relating to physical performance have been identified by several research groups. Athletes’ genotyping is developing as a tool for the formulation of personalized training and nutritional programmes to optimize sport training as well as for the prediction of exercise-related injuries. On the other hand, development of molecular technology and gene therapy creates a risk of non-therapeutic use of cells, genes and genetic elements to improve athletic performance. Therefore, the World Anti-Doping Agency decided to include prohibition of gene doping within their World Anti-Doping Code in 2003. In this review article, we will provide a current overview of genes for use in athletes’ genotyping and gene doping possibilities, including their development and detection techniques.