NUCLEAR ENVELOPE-ASSOCIATED RESUMPTION OF RNA SYNTHESIS IN LATE MITOSIS OF HELA CELLS

The restitution of RNA synthesis in cultures progressing from metaphase into interphase (G₁) has been investigated in synchronized HeLa S₃ cells by using inhibitors of macro-molecular synthesis and the technique of electron microscope autoradiography. The rate of incorporation of radioactive uridine into RNA approached interphase levels in the absence of renewed protein synthesis. In contrast, maintenance of this rate in G₁ was dependent upon renewed protein synthesis. Restoration of synthesis of heterogeneous nuclear RNA occurred under conditions that inhibited production of ribosomal precursor RNA. In autoradiographs of individual cells exposed to radioactive uridine, silver grains were first detected after nuclear envelope reformation at the periphery of the chromosome mass but before chromosomal decondensation. These data are consistent with the following interpretation. Multiple RNA polymerase activities persist through mitosis and are involved in the initiation of RNA synthesis in early telophase at sites on the nuclear envelope.     

A COMPARISON BETWEEN HETEROGENEOUS NUCLEAR RNA AND POLYSOMAL MESSENGER RNA IN HELA CELLS BY RNA-DNA HYBRIDIZATION

Heterogeneous nuclear RNA (HnRNA) and mRNA from cytoplasmic polyribosomes of HeLa cells have been compared by RNA-DNA hybridization tests. 1 µg of HeLa cell DNA binds 0.05–0.10 µg of either HnRNA or mRNA. In addition, HeLa DNA that is preexposed to unlabeled HnRNA was found to have a reduced capacity to bind either HnRNA or mRNA. The results are compatible with considerable sequence similarity in the two types of RNA but, as is discussed, firm conclusions are precluded by imperfections of the hybridization reaction as presently employed.     

SYNTHESIS OF RNA IN MAMMALIAN CELLS DURING MITOSIS AND INTERPHASE

Chinese hamster cells in the mitotic and G₁ phases of the growth cycle were incubated for 30 or 60 min in suspension tissue culture and pulse-labeled with tritiated uridine. After appropriate chases, washes, and extractions, it was found that all incorporation into the nucleic acid may be accounted for by those cells in interphase. An average of 410 counts was found for incorporation into the cell population (approximately 2.0 x 10⁵ cells) of which over 80% of the cells was initially in mitosis. The increasing number of cells leaving mitosis and entering interphase during the 30 min incubation was theoretically able to account for 470 counts. In addition, short-pulse labeling experiments have shown a consistent linear relationship between the percentage of cells in division and the incorporation of the isotope, which strongly suggests that, if 100% of the cells were in mitosis, the counts would be essentially zero. Thus, the entire label may be attributed to those cells in interphase where portions of the chromosomal material are known to be already extended.     

THE SELECTIVE INTERRUPTION OF NUCLEOLAR RNA SYNTHESIS IN HELA CELLS BY CORDYCEPIN

Cordycepin is an analogue of adenosine lacking the 3'-OH. When incorporated into a growing RNA molecule, cordycepin prevents further elongation, thus producing a prematurely terminated RNA molecule. When HeLa cells are exposed to low concentrations of cordycepin, DNA and protein synthesis are unaffected during short exposure periods. The synthesis of completed ribosomal and ribosomal-precursor (45S) RNA is significantly depressed. Partially completed 45S ribosomal precursor molecules accumulate in the nucleolus. 18S ribosomal RNA can be cleaved from these incomplete precursors, while 32S ribosomal precursor cannot be produced from partially snythesized 45S molecules. The synthesis of transfer RNA is also reduced in the presence of cordycepin. The synthesis of the nuclear heterogeneous RNA species is unaffected by the drug while the cytoplasmic heterogeneous RNA is slightly reduced.     

PROTEIN SYNTHESIS AND RNA SYNTHESIS DURING MITOSIS IN ANIMAL CELLS

Protein synthesis and RNA synthesis during mitosis were studied by autoradiography on mammalian tissue culture cells. Protein synthesis was followed by incubating hamster epithelial and human amnion cells for 10 or 15 minutes with phenylalanine-C¹⁴. To study RNA synthesis the hamster cells were incubated for 10 minutes with uridine-C¹⁴. Comparisons of the synthetic capacity of the interphase and mitotic cells were then made using whole cell grain counts. The rate of RNA synthesis decreased during prophase and reached a low of 13 to 16 per cent of the average interphase rate during metaphase-anaphase. Protein synthesis in the hamster cells showed a 42 per cent increase during prophase with a subsequent return to the average interphase value during metaphase-anaphase. The human amnion cells showed no significant change at prophase but there was a 52 to 56 per cent drop in phenylalanine incorporation at metaphase-anaphase as compared to the average interphase rate. Colcemide was used on the hamster cells to study the effect of a prolonged mitotic condition on protein and RNA synthesis. Under this condition, uridine incorporation was extremely low whereas phenylalanine incorporation was still relatively high. The drastic reduction of RNA synthesis observed under mitotic conditions is believed to be due to the coiled condition of the chromosomes. The lack of a comparable reduction in protein synthesis during mitosis is interpreted as evidence for the presence in these cells of a relatively stable messenger RNA.     

THE PERSISTENCE OF HEMOPOIETIC STEM CELLS IN VITRO

Cells capable of forming colonies in spleens of irradiated mice (CFU) are lost temporarily when bone marrow cells from rats or mice are maintained in culture. Rat marrow CFU go through a minimum at about 3 days after which there is a slow increase in the number of CFU in culture, reaching a maximum at 9 days. Mouse marrow CFU reach a minimum at 3 days and a maximum at 7 days. Some rat marrow CFU persist in culture for as long as 28 days.     

高温诱导HeLa细胞凋亡的相关机理的研究

目的研究不同高温作用后HeLa细胞bcl-2、bax和p53表达的变化.方法人子宫颈癌细胞系(HeLa细胞)分为37℃、40℃、43℃三个温度组,每组经1h相应的温度处理后,以免疫组织化学的方法检测bcl-2、bax和p53的表达并经图象分析仪检测反应产物的光密度并进行统计分析.结果 40℃、43℃组的Bcl-2的平均光密度明显低于37℃组,而43℃组Bax的平均光密度明显高于37℃组和40℃组,P53随着温度的升高平均光密度也随之升高.形态学观察:43℃组均见明显的细胞凋亡.结论温和性高温使HeLa细胞周期受阻,P53表达上调诱导bax基因表达、抑制bcl-2基因的表达,从而导致细胞凋亡.     

MITOCHONDRIAL PROTEIN SYNTHESIS IN HELA CELLS

HeLa cell mitochondrial proteins have been shown to be the products of two separate protein-synthesizing systems; one, the general cellular mechanism, sensitive to inhibition by cycloheximide, the other, a specific mitochondrial system subject to inhibition by low concentrations of chloramphenicol (Galper, J. B., and J. E. Darnell. 1971. J. Mol. Biol 57:363). Preliminary data have suggested that a mitochondrial N-formyl-methionyl-tRNA (f-Met-tRNA) might be the initiator tRNA in the latter (Galper, J. B., and J. E. Darnell. 1969. Biochem. Biophys. Res. Commun. 34:205; 1971. J. Mol. Biol. 57:363). It is demonstrated here that the synthesis of these endogenous mitochondrial proteins is also subject to inhibition by ethidium bromide and decays with a half-life of 1½–2 h in cultures incubated with low concentrations of this dye. The role of formylated f-Met-tRNA as the initiator tRNA in the synthesis of mitochondrial proteins is supported by data from several experiments. The rates of ethidium bromide inhibition of both the charging of f-Met-tRNA and of the synthesis of mitochondrial proteins are strikingly similar. Inhibition by aminopterin of the formylation of f-Met-tRNA greatly depresses the rate of mitochondrial-specific protein synthesis. In the absence of the synthesis of these proteins, respiration, the levels of cytochromes a–a₃ and b, and the number of mitochondrial cristae are decreased. The implications of these findings as they relate to mitochondrial biogenesis are discussed.     

MITOSIS AND THE PROCESSES OF DIFFERENTIATION OF MYOGENIC CELLS IN VITRO

The relation between the mitotic cycle and myoblast fusion has been studied in chick skeletal muscle in vitro. The duration of the cell cycle phases was the same in both early and late cultures. By tracing a cohort of pulse-labeled cells, it was found that myoblast fusion does not occur in S, G₂, or M. Cell surface alterations required for fusion are dependent upon the position of the cell in the division cycle. In early cultures, fusion takes place only after a minimum delay of 5 hr from the time the cell has entered G₁. The mitosis preceding fusion may condition the cell for the abrupt shift in synthetic activity that occurs in the subsequent G₁. In older cultures fusion of labeled cells is diminished. Two factors account for the cessation of fusion in older cultures. First, the number of myogenic stem cells declines, but these cells do not disappear as the cultures mature. Their persistence was demonstrated by labeling dividing mononucleated cells in older cultures and challenging them with nascent myotubes. Some of these labeled cells were incorporated into the forming myotubes. Second, a block to fusion develops during myotube maturation. Well developed myotubes challenged with labeled competent myogenic cells failed to incorporate the labeled nuclei.     

A COMPARISON OF THE HETEROGENEOUS NUCLEAR RNA OF HELA CELLS IN DIFFERENT PERIODS OF THE CELL GROWTH CYCLE

HeLa cells synthesize heterogeneous nuclear RNA (HnRNA) in the G₁, S, and G₂ portions of the cell cycle. HnRNA prepared from these various periods was compared by RNA-DNA hybridization experiments. The results indicated that some of the HnRNA molecules were equivalent at all times in the cell cycle, but limitations in the sensitivity of the hydridization reactions, as well as in the spectrum of hybridizing molecules, restrict the conclusions that can be drawn from these comparisons.     

SOME UNUSUAL FEATURES OF FINE STRUCTURE OBSERVED IN HELA CELLS

HeLa cells from conventional culture media have been studied in thin sections with the electron microscope; in many cases cells were examined in sets of sections cut in series. The fine structure of the cells is described including three unusual features not hitherto reported. It has been found that numerous cells contained rows of parallel smooth surfaced cisternae spaced about 150 mµ apart and communicating with rough surfaced elements of the endoplasmic reticulum. These cisternae resembled "annulate lamellae" but did not contain regular arrays of pores. In many cells an area of juxtanuclear cytoplasm was occupied by a membranous structure composed of closely applied pairs of narrow cisternae either arranged in concentric rings or else extending in several directions in a haphazard manner. Sparse particles were present on the outer membranes of each pair of cisternae. Communications between the double cisternae and other membrane-bounded structures were not observed. A small number of cells contained areas of cytoplasm devoid of organelles and filled with amorphous fuzzy material. The observations recorded are discussed.     

中等纤维在几种人体上皮细胞有丝分裂过程中的行为

本文用间接免疫荧光法和电镜术观察了分别来自人表皮(PcaSE-1)、复层上皮(CNE)和单层上皮(SPC-A-1)的3个上皮细胞系的细胞在有丝分裂过程中中等纤维的行为。结果表明,CNE细胞和SPC-A-1细胞表达两种不同类型的中等纤维系统:角蛋白纤维和波形纤维,而PcaSE-1细胞仅表达角蛋白纤维。当细胞进入有丝分裂时,PcaSE-1细胞的角蛋白纤维维持完整的形态且将有丝分裂纺锤体围绕在细胞中央。相反,在CNE细胞和SPC-A-1细胞中,在细胞有丝分裂时,角蛋白纤维解聚成无定形的胞质小体,然而它们的波形纤维始终保持完整的形态。我们认为(1)在分裂上皮细胞中,角蛋白纤维的解聚与细胞的恶性程度有关,而与间期上皮细胞中是否含有丰富的角蛋白纤维无明显关系。(2)在上皮细胞有丝分裂时,中等纤维可能参于纺锤体的定位和趋中。(3)在分裂CNE细胞中,波形纤维的可能功能是染色体的定位和定向。     

GLOBOID STRUCTURES IN THE CYTOPLASM OF RAPIDLY GROWING HELA CELLS

During the course of electron microscopic study of rapidly growing uninfected HeLa cells, it was found that numerous globoid bodies occurred in the cytoplasm. Reasons are given for suspecting that these structures are mitochondria.     

MITOSIS IN SUSPENSION CULTURES OF HIGHER PLANT CELLS IN A SYNTHETIC MEDIUM

Torrey , J. G., J. Reinert , and N. Merkel . (Harvard U., Cambridge, Mass.) Mitosis in suspension cultures of higher plant cells in a synthetic medium. Amer. Jour. Bot. 49(4): 420–425. Illus. 1962.—A cytological study was made of plant tissue cultures growing in liquid synthetic medium. Mitoses in cell suspension cultures of root callus tissues of Daucus carota L., Convolvulus arvensis L. and Haplopappus gracilis (Nutt.) Gray were found to occur frequently in the first 2 weeks of culture with the highest frequency at about 7 days. No mitoses were observed after 3 weeks, although fresh weight and the number of free-floating cells in the suspension continued to increase for the entire culture period of 4–6 weeks. Mitoses were most frequent in tissue pieces, but occasional mitoses in single isolated cells in suspension were observed in each type of tissue. Normal mitoses were observed in diploid and polyploid cells of all 3 types of tissues cultures. Little evidence of nuclear or chromosomal aberrations was observed in these cultures.     

DNA SYNTHESIS AND MITOSIS IN ERYTHROPOIETIC CELLS

Studies of newt (Triturus or Diemictylus viridescens) erythropoietic cells showed that DNA synthesis and mitosis normally occur throughout most of the developmental process. Mitotic divisions were found in all immature precursor stages from the proerythroblast to the highly hemoglobinized reticulocyte. Mitoses were absent in mature erythrocytes. Radioautographic examination of thymidine-³H incorporation into DNA revealed that all erythroid cells except the mature erythrocyte were labeled. Microphotometric measurements of Feulgen-stained smears showed that all immature stages were undergoing DNA synthesis whereas the mature erythrocyte was inactive. The results obtained from three independent methods clearly demonstrate that (a) no loss of DNA or of chromosomes occurs during erythrocytic development and (b) highly hemoglobinized and, therefore, well-differentiated cells normally do undergo DNA synthesis and mitosis.     

染色体端粒DNA与核骨架的结合关系(简报)

端粒是真核生物染色体的一种特化结构,对于染色体的稳定以及染色体的完全复制有着十分重要的意义。许多种生物的端粒DNA序列已被发现:四膜虫,草履虫为(G_4T_2)_n;人、锥虫、短膜虫为(AG_3T_2)_n;尖毛虫、棘尾虫、游仆虫为(T_4G_4)_n;拟蓝芥菜为(AG_3T_3)_n。     

THE DISTRIBUTION OF SPINDLE MICROTUBULES DURING MITOSIS IN CULTURED HUMAN CELLS

WI-38 and HeLa cells in mitosis have been selected from fixed monolayer cultures and serially sectioned for electron microscopy. Sections perpendicular to the spindle axis permit counting of the number of microtubules at each position on the spindle axis and hence the preparation of tubule distribution profiles. Errors intrinsic to this method are discussed. The changes in the tubule distributions from one mitotic stage to another provide evidence concerning the behavior of the spindle tubules during mitosis. The ratio of the number of tubules passing the chromosomes on the metaphase plate to the maximum number in each half spindle is about 1/2. This ratio changes little in early anaphase, and then decreases in late anaphase at about the same time that a zone of increased tubule number develops at the middle of the interzone. The region where the stem bodies form contains about 3/2 the number of tubules seen elsewhere in the interzone. This ratio is almost constant as the mid-body forms in telophase and then increases to 2/1 in early interphase before the final stages of cytokinesis occur.