NUCLEOLAR AGING IN TETRAHYMENA DURING THE CULTURAL GROWTH CYCLE

Nucelolar morphology was studied by electron microscopy in control and actinomycin D-treated populations of Tetrahymena pyriformis (W) during the cultural growth cycle. Nucleoli exhibit an "aging" cycle concomitant with the cultural growth cycle, but independent of the individual cell cycle. Four different stages in the course of this aging process have been defined. Stage 1 occurs upon inoculation (low number of cells per milliliter) and lasts through lag and accelerating growth phases. In this stage, many small nucleoli are found at the nuclear periphery. In stages 2 and 3, nucleolar fusion begins. Stage 2 dominates the first half of logarithmic growth, and stage 3 dominates the second half. In late decelerating growth phase, the nucleoli enter stage 4. In this stage, only a few large nucleoli are present and these are apparently inactive in ribosome production. In stationary phase, where total RNA remains constant, only stage 4 nucleoli are present. The relative preponderance of granular vs. fibrous components in the nucleoli changes during this cycle, the granular component dominating stage 1 nucleoli and the fibrillar, stage 4 nucleoli. There is a shortening of the intermediate nucleolar stages in the treated cultures; fusion occurs early and is now pronounced. Not enough ribosomes accumulate to carry the treated cultures through the number of generations equivalent to those of the control, which produces a premature stationary phase.     

THE FATE OF MITOCHONDRIA DURING AGING IN TETRAHYMENA PYRIFORMIS

During the growth cycle of Tetrahymena pyriformis the mitochondria undergo changes in position, number, and structure. Ciliates in the logarithmic growth phase possess elongated mitochondria which are aligned along the plasma membrane and are closely associated with the kinetosomes and kinetodesmata. Mitochondria appear to divide across the long axis at this time, resulting in two or more products. Throughout this phase of growth mitochondrial divisions keep pace with cytokinesis so that the population of mitochondria remains at essentially the minimal level. As the ciliates enter the stationary growth phase the mitochondria increase in number, become oval to spherical in shape, and some migrate into the cytoplasm. Intramitochondrial masses of various configurations appear at this time. Some of the mitochondria lying in the cytoplasm become incorporated into vacuoles. Within these vacuoles either a single mitochondrion appears or several mitochondria may be seen along with other cytoplasmic structures. Later in the stationary growth phase the contained mitochondria are dense and the tubules are more compact than normal. Various stages in disorganization of the mitochondria are observed in a single large vacuole. Cytochemical tests reveal the presence of acid phosphatase, suggesting that hydrolysis of the vacuolar contents occurs. Lipid droplets increase in number during the middle and late stationary phase of growth. These events are interpreted as being associated with the normal process of aging in T. pyriformis.     

四膜虫S1营养生长和有性过程中cAMP的含量变化

用放射免疫法测定了四膜虫在群体生长各阶段和在有性过程中cAMP的含量变化。在群体生长的延迟期(Lag phase)之末,即将进入对数期时,cAMP含量出现一个峰值。随着对数期的进展,cAMP含量陆续下降,在对数期之末,cAMP含量降至最低值。群体进入静止期后,细胞内cAMP含量略有回升。 在饥饿诱导接台的过程中,细胞内cAMP含量在开始饥饿的很短时间内即迅速降至一个最低点,并且在整个接合过程中一直保持较低的水平。接合抑制剂伴刀豆球蛋白A(CoaA)和精胺(spermine)等在抑制接合的同时,也阻止饥饿细胞cAMP含量的迅速下降,使之不能达到在正制情况下接合出现时所达到的低水平。 在饥俄细胞的培养液中检出了较高量的cAMP。这可能有助于说明何以饥饿细胞的cAMP含量能够在很短时间内迅速下降。至于排出的cAMP对四膜虫的接合有无促进或其他作用,有特继续研究。     

PHYSIOLOGICAL AND BIOCHEMICAL CHANGES OF CARROT ROOT CALLUS DURING SUCCESSIVE CULTURES

1. The longer the period of stock culture, the more remarkableis the growth inhibition by 8-azaguanine in callus.
2. Chloramphenicol,5-methyltryptophane and mitomycin C exert greaterinhibitionon growth in CCL than in CCS.
3. Bud formation is inhibited bysome concentrations of chloramphenicolwithout accompanyinginhibition of the growth.
4. Cell size and the contents of RNA,DNA, protein and lipid percell of CCL are greater than thoseof CCS, respectively. Thecontents per cell of RNA and lipidin "mitochondrial fraction"are higher in CCL than in CCS.
5. Incorporationof guanine-8-¹⁴C into RNA of CCS occurs rapidlyin the first12 hr and slows down thereafter, but that in CCL-RNAincreasessteadily for 16 hr. This difference in rate of theincorporationafter 12 hr between CCS and CCL is principallydue to the differencein rate of the incorporation into RNAof nuclear, mitochondrialand soluble fractions.

1. The rate of RNA breakdown in CCL wasnot so great as the rateof synthesis.
2. 8-azaguanine (10^–3and 10^–4M) inhibits incorporationof guanine-8.¹⁴C intoRNA of both CCS and CCL during 14 hr,but thereafter (up to25 hr) it inhibits the incorporation intoCCL-RNA alone leavingthat into CCS-RNA unaffected.

1. In CCL 510^–5M 8.azaguaninedoes not affect total radioactivityincorporated into bulk RNA,but inhibits incorporation intoRNA of "mitochondrial fraction".

(Received December 23, 1964; )     

SIMULTANEOUS SYNTHESIS OF HISTONE AND DNA IN SYNCHRONOUSLY DIVIDING TETRAHYMENA PYRIFORMIS

Histone and DNA syntheses have been studied in synchronously dividing Tetrahymena pyriformis GL. During the heat treatment necessary to synchronize cultures of this amicronucleate protozoan, the DNA content of the already polyploid macronucleus increases. When the cells begin synchronous division, their DNA content is reduced in a stepwise process which is closely paralleled by reduction of macronuclear histone content. During cell division, the contents of DNA and histone decrease by slightly more than twofold, and in the subsequent S phase, DNA and histone increase simultaneously to 85% of the values expected if all chromosomes were to double. The first step in the process of reduction of DNA and histone contents is their decrease in excess of twofold, and this is accomplished by removal of extrusion bodies from the nuclei of dividing cells. The second step is a mechanism which allows, in effect, only 70% of the chromatin in the average nucleus to duplicate. Such partial duplication suggests that both histone and DNA syntheses in synchronous Tetrahymena depend upon a regulatory mechanism, the mediating elements of which are localized in only certain chromosomes.     

SECRETION OF ACID HYDROLASES AND ITS INTRACELLULAR SOURCE IN TETRAHYMENA PYRIFORMIS

Axenic Tetrahymena pyriformis, syngen 1, mating type II cells were grown in Cox's defined medium. When washed and transferred into nonnutrient dilute salt solution or resuspended in the defined medium, the intact cells secrete acid hydrolases into the medium. Cells starving in the salt solution release in 5 hr about two-thirds of their β-glucosidase, β-N-acetylglucosaminidase, α-glucosidase, and amylase activities, about one-third of their deoxyribonuclease and phosphatase activities, smaller amounts of ribonuclease, and only a negligible fraction of their proteinase activity and protein content. During this period there is practically no change in the enzyme activities (except for a sudden increase of ribonuclease activity) and protein content of cells and medium together. Cells resuspended in the nutrient medium secrete enzymes as do the starved cells, but replace this loss, so that there is a continuous increase of the activities in the total system. According to isopycnic centrifugation experiments performed in sucrose gradients, the source of the hydrolases is a special population of lysosomes which disappear from the cells during starvation. This population equilibrates in the high density region of the gradients and contains the various acid hydrolases in about the proportion in which these enzymes appear in the medium.     

THE SYNTHESIS OF MICROTUBULE AND OTHER PROTEINS OF THE ORAL APPARATUS IN TETRAHYMENA PYRIFORMIS

Several proteins, including microtubule proteins, have been isolated from the oral apparatus of the ciliate Tetrahymena. The synthesis of these proteins has been studied in relation to formation of this organelle system by the cell. Electron microscopy has shown that the isolated oral apparatus consists primarily of basal bodies, pellicular membranes, and a system of subpellicular microtubules and filaments. Cilia were removed during the isolation; therefore none of the proteins studied was from these structures. Evidence was obtained from the study of total oral apparatus protein which indicates that at least some of the proteins involved in formation of this organelle system may be synthesized and stored in the cytoplasm for use over long periods. This pattern of regulation was found for three individual proteins isolated from the oral apparatus fraction after extraction with a phenol-acetic acid solvent. A different pattern of regulation was found for microtubule proteins isolated from the oral apparatus of Tetrahymena. The data suggest that microtubule proteins, at least in logarithmically growing cells, are not stored in a cytoplasmic pool but are synthesized in the same cell cycle in which they are assembled into oral structures.     

黄瓜果实贮藏过程中某些形态,生化变化及其控制

黄瓜果实采后贮藏过程中,随着其花端部位的种子的不断发育,中部及茎端逐渐萎缩变糠,花端膨大(称为“大头现象”)。花端胎座组织中蛋白质、DNA、RNA 及干重等都随着时间延长呈增加趋势,而中部组织的上述物质的含量则急剧下降。贮藏20天,中部果皮及胎座组织中 DNase 活性分别增加了5和7倍,RNase 活性增加了5和6倍,这两种酶活性在花端组织中则几无变化。实验发现用 BA+GA,处理果实对于延迟花端膨大及中部萎缩有效,并能显著延缓果实叶绿素含量的降低。未授粉果实在贮藏过程中不出现“大头现象”,其蛋白质等物质的含量在果实各部位几乎以相同的速度降低。     

THE NUCLEIC ACIDS OF BASAL BODIES ISOLATED FROM TETRAHYMENA PYRIFORMIS

Pellicular fragments were isolated from ethanol-fixed cells of the holotrichous ciliate Tetrahymena pyriformis by the action of digitonin. The isolated pellicles were further fragmented and the basal bodies of the cilia isolated from them by three methods. The preparations, examined in the electron microscope as embedded sections or negatively stained samples, consisted mainly of somewhat deformed pellicular material, the bulk of which was basal body. DNA was determined by the diphenylamine method and by reaction with DNase, and RNA, by the orcinol method. Nucleic acids were isolated by phenol extraction and analyzed spectrophotometrically and by reaction with RNase. The assays indicated 1.2 to 2.6 per cent RNA, similar to previously published work, but only 0.0 to 1.0 per cent DNA, near enough the sensitivity limits to render the presence of DNA in the preparations uncertain. Although the isolation procedure removed nuclear contents and ribosomes, the nucleic acids could still be a residual contaminant bound to the pellicle during the isolation. Hypotheses of basal body self-duplication, moreover, can be constructed both with and without nucleic acids.     

PARTIAL PURIFICATION AND PHOSPHOTUNGSTATE SOLUBILIZATION OF BASAL BODIES AND KINETODESMAL FIBERS FROM TETRAHYMENA PYRIFORMIS

Previously devised methods for the isolation of basal bodies from ciliate protozoans were found to be inadequate for chemical analysis. We have modified and expanded these procedures and developed a method which gives preparations containing mainly basal bodies and kinetodesmal fibers. This procedure involved fixation of cells in 30% ETOH followed by digitonin or Triton X-100 solubilization and homogenization with a Brinkmann Polytron. This is followed by sucrose gradient centrifugation. Negative staining and thin sectioning revealed these preparations to be substantially more pure than those of previous workers. It was also found that neutralized phosphotungstate (PTA) solubilized many of the components present in fixed Tetrahymena. Neutralized 1.0% PTA solubilized axonemes, cortical, axonemal, and basal body microtubules as well as kinetodesmal fibers. These results have been confirmed by both electron microscope observations and gel electrophoresis of 100,000 g supernatants of the PTA extracts. A solution of 0.1% PTA did not affect the fibers but did solubilize basal bodies. Running 1.0% PTA extracts from our basal body fractions on sodium dodecyl sulfate (SDS) polyacrylamide gels allowed us to tentatively identify the peptides of basal bodies and kinetodesmal fibers. The latter structures appear to consist of a single 21,000 mol wt peptide. These results also suggest that great caution should be taken in interpreting PTA images, especially of microtubules and axonemes.     

THE EFFECT OF 5-BROMODEOXYURIDINE ON DNA REPLICATION AND CELL DIVISION IN TETRAHYMENA PYRIFORMIS

Populations of Tetrahymena pyriformis were grown in a chemically defined medium containing the thymidine analogue 5-bromodeoxyuridine (BUdR). About 65% of the thymidine sites in DNA were substituted by BUdR. During the first generation in the presence of BUdR, all DNA became hybrid. After the following cell division, in about 80% of the cells the second DNA replication round was initiated but no further cell division took place. The cells could be rescued by removing BUdR and adding thymidine. New replication took place before the first cell division. However, although the cells contained double heavy as well as hybrid DNA, only the hybrid DNA was replicated. After a full replication of the hybrid DNA, normal growth was restored. Melting profiles of normal, hybrid, and double heavy DNA indicated a structural change of the double heavy DNA.     

江鲽在卵和卵黄囊期仔鱼发育阶段生化成分的变化

本文研究和测定了江鲽( Platichthys flesus L.)在卵和卵黄囊期仔鱼发育阶段水分、钠、钾、脂肪和蛋白质的含量变化。含水量在卵和仔鱼发育期基本保持在90—92%,在仔鱼进入初次摄食期时可降到89%左右。初孵仔鱼具一大卵黄囊,含水量高达93.3%,起“浮力器官”(buoyancy organ)的作用。钠、钾离子含量在卵受精和仔鱼出膜后波动剧烈,呈“上升—下降—再上升”式型。脂肪和蛋白质含量在卵和仔鱼早期发育和饥饿期均呈明显线性下降。同时,本文还就生化成分变化和海洋浮性鱼卵及其仔鱼的生态习性的关系作了初步探讨。     

MORPHOLOGICAL AND BIOCHEMICAL CHANGES IN RAT SYNAPTOSOME FRACTIONS DURING NEONATAL DEVELOPMENT

A biochemical and quantitative morphologic study of presynaptic endings during postnatal development was carried out in subcellular fractions from cerebral cortex of 1, 4, 8, 12, and 18 day old and adult rats. Crude mitochondrial fractions were subfractionated in Ficoll gradients and all resulting fractions were examined in the electron microscope. Presynaptic terminals and other intact processes were counted. Protein content and enzyme activities were assayed in the fractions and in total brain homogenate. In the first and fourth day of life, most of the presynaptic terminals were found in two "light" fractions, between supernatant and 7.5% Ficoll, where they accounted, respectively, for 6 and 22% of all the processes. Progressively with age, more presynaptic terminals were found in the traditional "synaptosomal" fractions between 7.5 and 13% Ficoll. In that region of the gradient, 40, 54, 75, and 89% of the processes were presynaptic endings at 8, 12, and 18 postnatal days and in the adult animal, respectively. A similar shift from the lighter to the heavier fractions was observed in the distribution of choline acetyltransferase and acetylcholinesterase between days 8 and 12. The rate of increase of the specific activity of these two enzymes paralleled that of the percentage of the presynaptic endings after day 8. This study indicates that subcellular fractions can be used to study formation and maturation of synapses during postnatal development.     

THE MORPHOGENESIS OF BASAL BODIES AND ACCESSORY STRUCTURES OF THE CORTEX OF THE CILIATED PROTOZOAN TETRAHYMENA PYRIFORMIS

Dividing cells of Tetrahymena pyriformis were observed by transmission electron microscopy for signs of morphogenesis of cortical structures. The earliest stage of basal body development observed was of a short cylinder of nine single tubules connected by an internal cartwheel structure. This is set perpendicular to the mature basal body at its anterior proximal surface under the transverse microtubules and next to the basal microtubules. Sequential stages show that the single tubules become triplet tubules and that the "probasal bodies" then elongate and tilt toward the organism's surface while maintaining a constant distance of 75–100 mµ with the "parent." The new basal body after it is fully extended contacts the pellicle, and then assumes a parallel orientation with and moves anterior to the parent basal body. The electron-opaque core in the lumen of the basal body and accessory structures around its outer proximal surface appear after the developing basal body has elongated. These accessory structures associating with their counterparts from other basal bodies and with the longitudinal microtubules may play a role in the final positioning of basal bodies and thus in the maintenance of cortical patterns. Observations on a second sequence of basal body formation suggest that the oral anlage arises by multiple duplication of somatic basal bodies.