ELECTRON MICROSCOPIC RADIOAUTOGRAPHIC DETECTION OF SITES OF PROTEIN SYNTHESIS AND MIGRATION IN LIVER

The sites of synthesis of proteins and their subsequent migration in rat liver have been studied during a 75 min period after labeling of liver-slice proteins by exposure to leucine-H³ for 2 min. Incorporation of the label into protein began after 1 min and was maximal by 4 min. Electron microscopic radioautography showed that synthesis of proteins in hepatocytes occurs mainly on ribosomes, particularly those in rough endoplasmic reticulum and, to some extent, in nuclei and mitochondria. Most of the newly formed proteins leave the endoplasmic reticulum in the course of 40 min, and concurrently labeled proteins appear in Golgi bodies, smooth membranes, microbodies, and lysosomes. A likely pathway for the secretion of some or all plasma proteins is from typical rough endoplasmic reticulum to a zone of reticulum which is partially coated with ribosomes, to the Golgi apparatus, and thence to the cell periphery. The formation of protein by reticuloendothelial cells was measured and found to be about 5% of the total protein formed by the liver.     

AN ELECTRON MICROSCOPIC STUDY OF NUCLEAR ELIMINATION FROM THE LATE ERYTHROBLAST

The process of expulsion of the nucleus during the transformation of the late erythroblast to reticulocyte is described. Erythroid clones taken from the spleen of lethally irradiated mice transplanted with syngeneic bone marrow were used. 10–12-day old isolated clones were fixed in glutaraldehyde, then in osmium tetroxide. Ultra-thin sections were stained with uranyl acetate and/or lead citrate before examination. Late (orthochromatic) erythroblasts develop pseudopod-like cytoplasmic protrusions into one of which the nucleus gradually penetrates, being deformed by the extrusion through the relatively narrow passage. During the whole process, mitochondria and vesicular and membranous elements are concentrated in the cytoplasm. Once outside the cell, the nucleus reassumes its rounded form. It is surrounded by a narrow rim of cytoplasm and structurally altered plasma membrane and is connected to the rest of the cell by a bridge. Elongated vacuoles appear within this bridge, with a resulting release of the enveloped nucleus which is soon phagocytized by macrophages; this leaves behind the newly formed reticulocyte. During this process, the cytoplasmic protrusions, the agglomeration of mitochondria, and the mode of separation of the nucleus from the rest of the cell are similar to those occurring in mitotic division.     

东方蝾螈早期发育过程中细胞核超微结构的观察

东方蝾螈胚胎发育过程中,从原肠早期到原肠末期无论外胚层或中胚层细胞核内都含有大量的异染色质团块,而到神经板形成后所有细胞核内染色质均呈分散状态。异染色质向常染色质的转变过程发生在原肠末期到神经板形成这段时间里,在原口闭合后4.5小时之前完成,此时期似乎是形态发生的转折点。到发育的后期,细胞核内有一些染色质又会由分散状态转变为凝聚的异染色质团块。预定神经上皮向神经组织分化的决定是个逐渐的过程,这一过程在原肠口闭合时已经开始,到神经板期完成。染色质的分散似乎发生在细胞有了初步决定之后。本文就染色质超微结构变化的意义以及染色质超微结构变化与细胞分化的关系等问题进行了讨论。     

BIOCHEMICAL AND CYTOLOGICAL EXAMINATION ON THE INITIATION OF RIBOSOMAL RNA SYNTHESIS DURING GASTRULATION OF XENOPUS LAEVIS

Embryos of Xenopus laevis at stage 6 were labeled with ¹⁴CO₂ for 4 hr and then allowed to develop under nonradioactive conditions until they reached stage 9, 10, 11 or 12. RNA was extracted and electrophoresed on a polyacrylamide-agarose gel. From the pattern of newly synthesized RNAs, the incorporation into 18S and 28S ribosomal RNAs was measured. At the same time, the specific radioactivity of nucleoside triphosphates in the acid-soluble fraction was determined. On the basis of the results obtained, the absolute amounts of the RNAs synthesized were calculated. The results show that the synthesis of the ribosomal RNAs begins, or is at least markedly activated, around stage 10. Moreover, cytological examination has shown that cells with nucleolated nuclei appeared between stages 9 and 10 and increased thereafter.
Thus, from the results of these studies along two different lines, it can safely be concluded that the initiation of 18S and 28S RNA synthesis takes place around stage 10.     

香鱼成熟卵母细胞过熟进程中卵膜结构变化的扫描电镜观察

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AN AUTORADIOGRAPHIC STUDY OF RNA SYNTHESIS DURING POLLEN EMBRYOGENESIS IN HYOSCYAMUS NIGER (HENBANE)

RNA synthesis during pollen embryogenesis in cultured anther segments of Hyoscyamus niger (henbane) has been followed by autoradiography of ³H-uridine incorporation. Embryogenic divisions were initiated in binucleate pollen grains in which the generative nucleus or both generative and vegetative nuclei synthesized RNA. When the first haploid mitosis in culture resulted in pollen grains with two nearly identical nuclei, those in which both nuclei synthesized RNA became embryogenic. Binucleate pollen grains in which ³H-uridine incorporation was confined exclusively to the vegetative nucleus gradually became starch-filled and nonembryogenic. Based on the degree of involvement of the vegetative nucleus in embryoid formation, some differences were noted between the counts of autoradiographic silver grains over cells cut off by the generative and vegetative nuclei during progressive embryogenesis. The possible significance of RNA synthesis in the nuclei of binucleate pollen grains in determining the pathway of embryogenic divisions is discussed.     

ELECTRON MICROSCOPIC STUDIES ON THE BEHAVIOR OF GLYCOGEN PARTICLES DURING OOGENESIS IN THE SEA URCHIN

The behavior of glycogen particles during oogenesis in the sea urchin was studied by electron microscopy. Before the beginning of oogenesis the nurse cells include many glycogen particles, which are spherical or multiangular in shape and about 600 A in diameter, lying within the vesicle of the large granules and also in the cytoplasm among the granules. There are few glycogen particles in the spaces among the oocytes and the nurse cells. At the early stage of oogenesis the limiting membrane of the large granule breaks locally and the glycogen particles in the vesicle are dispersed into the cytoplasm. The plasma membrane of the nurse cell also breaks in places and glycogen particles are spread throughout the intercellular space. At the beginning of vitellogenesis, β-pinosomes begin to be formed at the periphery of the oocyte; these take in glycogen particles from the outside which are progressively broken into smaller units.     

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.     

AN ELECTRON MICROSCOPIC CYTOCHEMICAL STUDY OF MACROPHAGES DURING UTERINE INVOLUTION

Acid phosphatase was localized at the fine structural level in rat endometrial phagocytes during the period of postpartum involution. These cells showed intense phagocytotic and pinocytotic activities, which were accompanied by the development of abundant lysosomes. Phagosomes acquired their enzymatic complement by fusion with lysosomes; the same appeared to be true in the case of pinocytotic vesicles, but, because of the small size of these vesicles, this point could not be established with certainty. Digestion within some phagolysosomes led to the formation of electron-lucent vacuoles containing solubilized products. Other phagolysosomes showed accumulation of lipid residues in the form of droplets and myelin figures, and the structures acquired the appearance of residual bodies. In many macrophages, overfeeding led to the formation of unusually large numbers of phagolysosomes, which occupied almost the entire cytoplasm with exclusion of other cell organelles. In these cells the presence of abundant lead deposits, apparently free in the cytoplasm suggested an intracytoplasmic release of hydrolases.     

THE RELATIONSHIP BETWEEN RNA SYNTHESIS AND HEMOGLOBIN SYNTHESIS IN AMPHIBIAN ERYTHROPOIESIS : Cytochemical Evidence

A cytochemical study of the relationship between RNA synthesis and hemoglobin synthesis has been performed on splenectomized newts, Triturus viridescens. Employing radioautography, labeled cytidine was incorporated into the RNA of the early developmental stages but was not incorporated in the later stages. Labeled leucine was incorporated into the cellular protein of all stages except mature erythrocytes but was incorporated at a higher level in the later stages. Microphotometric measurements of azure B binding to cytoplasmic RNA revealed a sharp initial increase between the stem cell and proerythroblast followed by a rapid decrease between the basophilic and polychromatophilic stages. The loss of cytoplasmic RNA became more gradual in the late stages and, in the mature erythrocyte, little or no cytoplasmic RNA could be detected. Measurements of cytoplasmic total protein, using fast green staining at pH 2.0, and of heme showed that both curves increased similarly with development, indicating net hemoglobin synthesis. The results are compatible with the hypothesis that, as the stem cell differentiates along erythrocytic lines, a stable "messenger" RNA specifying the production of a given type or types of hemoglobin is formed. This complex probably becomes associated with ribosomal RNA and is retained throughout the process of RBC differentiation.     

花背蟾蜍蝌蚪表皮移植片在角膜诱导中的扫描电镜研究

扫描电镜研究表明,花背蟾蜍蝌蚪在角膜诱导过程中表皮移植片外表面的主要变化是:当移植片细胞中色素消失时,细胞的分泌活动变旺盛,表现在细胞外分泌物增多,这可能与排色素有关。同时移植片细胞表面的微突起和边界嵴日趋细小,移植片透明时它更类似正常角膜。在此期间移植片内表面的主要变化是:细胞外基质不断增加,尤其是胶原纤维的增多和逐渐排列整齐,以及可能由于GAG的合成形成的颗粒状结构的出现和消失,这些都与移植片向角膜上皮分化相关联。由于眼球诱导作用引起的上述变化,都可促进表皮移植片转变为透明的角膜。     

HYBRIDIZATION PROPERTIES OF DNA SEQUENCES DIRECTING THE SYNTHESIS OF MESSENGER RNA AND HETEROGENEOUS NUCLEAR RNA

The relationship of the DNA sequences from which polyribosomal messenger RNA (mRNA) and heterogeneous nuclear RNA (NRNA) of mouse L cells are transcribed was investigated by means of hybridization kinetics and thermal denaturation of the hybrids. Hybridization was performed in formamide solutions at DNA excess. Under these conditions most of the hybridizing mRNA and NRNA react at values of D_ot (DNA concentration multiplied by time) expected for RNA transcribed from the nonrepeated or rarely repeated fraction of the genome. However, a fraction of both mRNA and NRNA hybridize at values of D_ot about 10,000 times lower, and therefore must be transcribed from highly redundant DNA sequences. The fraction of NRNA hybridizing to highly repeated sequences is about 1.7 times greater than the corresponding fraction of mRNA. The hybrids formed by the rapidly reacting fractions of both NRNA and mRNA melt over a narrow temperature range with a midpoint about 11°C below that of native L cell DNA. This indicates that these hybrids consist of partially complementary sequences with approximately 11% mismatching of bases. Hybrids formed by the slowly reacting fraction of NRNA melt within 4°–6°C of native DNA, indicating very little, if any, mismatching of bases. Hybrids of the slowly reacting components of mRNA, formed under conditions of sufficiently low RNA input, have a high thermal stability, similar to that observed for hybrids of the slowly reacting NRNA component. However, when higher inputs of mRNA are used, hybrids are formed which have a strikingly lower thermal stability. This observation can be explained by assuming that there is sufficient similarity among the relatively rare DNA sequences coding for mRNA so that under hybridization conditions, in which these DNA sequences are not truly in excess, reversible hybrids exhibiting a considerable amount of mispairing are formed. The fact that a comparable phenomenon has not been observed for NRNA may mean that there is less similarity among the relatively rare DNA sequences coding for NRNA than there is among the rare sequences coding for mRNA.     

COTTON EMBRYOGENESIS: THE EARLY DEVELOPMENT OF THE FREE NUCLEAR ENDOSPERM

An electron microscope study was made of the central cell and the development of the free nuclear endosperm surrounding the zygote and synergids during the first three days after pollination. The cytoplasm of the central cell, concentrated around the partially-fused polar nuclei, contains many ribosomes, mitochondria and large, dense, starch-containing plastids, some dictyosomes and lipid bodies, and long, single cisternae of rough endoplasmic reticulum (RER) that frequently terminate in whorls. Dense, core-containing microbodies are closely associated with the RER. After fertilization the cytoplasm of the 2-and 4-nucleate endosperm shows an increase in number of dictyosomes, and in amount of RER which becomes stacked in arrays of parallel cisternae. Cup-shaped plastids are associated with many long, helical polysomes. Perinuclear aggregates of dense, granular material also appear after fertilization. Granular aggregates and helical polysomes disappear after the first few divisions of the primary endosperm nucleus. During the second and third days of development there is an increase in dictyosome number and RER proliferation, and endosperm nuclei become deeply lobed. Concurrently, there is a sharp decline in the starch and lipid reserves of the central cell and elaborate transfer walls are formed at the micropylar end of the embryo sac and on the outer surface of the degenerating synergid. The transfer walls contain groups of small, membrane-bound vesicles, and are associated with large numbers of mitochondria and with the smooth endoplasmic reticulum.     

THE SYNTHESIS OF 5S RIBOSOMAL RNA DURING POLLEN DEVELOPMENT

Tradescantia paludosa 5S ribosomal RNA (rRNA) has been characterized with respect to its base composition and relative electrophoretic mobility in comparison with that of E. coli . The period of 5S rRNA synthesis during pollen grain development was determined by pulse labeling the RNA synthesized during a 24 hr period of development with ³²P and then chasing in cold medium until pollen maturity. The period of highest 5S rRNA synthesis was found to occur prior to microspore mitosis. During and following mitosis over a period of 4 days there was a sharp decrease in the amount of 5S RNA synthesized and during the last 48 hr of pollen maturation, no 5S rRNA was synthesized.     

DEVELOPMENT OF FEATHER KERATIN DURING EMBRYOGENESIS OF THE CHICK

The development of keratin in 9 to 17 day embryonic chick feathers has been studied by x-ray diffraction and cytochemical methods. The x-ray diffraction pattern given by the 9-day feathers contains none of the features seen in the adult pattern. In the 10 to 11 day patterns, besides two diffuse rings centered at 4.7 A and 10 A, two sharp, rather weak rings are seen at 35 A and 4.2 A with slight preferred orientations about the equator and the meridian, respectively. At 12 days, in addition to the foregoing, a sharp intense equatorial reflection at ~56 A is observed. On treatment with lipid solvents, the 35 A ring is removed; prolonged extraction removes the 4.2 A ring, while blurring the 56 A reflection and enhancing the central low angle scatter. The 14-day pattern shows, besides all the features seen in the earlier patterns, a 23 A meridional reflection and other meridional and near meridional reflections. All the basic features of the adult pattern are seen at this stage and remain essentially intact on lipid extraction. Beyond 14 days, the pattern remains essentially the same, only improving in clarity and detail. The 4.2 A ring seen in the 10 to 15 day pattern is scarcely detectable in the 16-day pattern. Cytochemical evidence indicates that extensive —S—S bond formation occurs between the 13th and 14th days. It is suggested that lipids serve as a framework for the developing keratin structure which acquires permanent stability through hydrogen bonds and disulfide cross-links. The relation between keratin synthesis and tissue architecture as well as cytodifferentiation is discussed.