THE INTRACELLULAR SITE OF SYNTHESIS OF MITOCHONDRIAL RIBOSOMAL PROTEINS IN NEUROSPORA CRASSA

The intracellular site of synthesis of mitochondrial ribosomal proteins (MRP) in Neurospora crassa has been investigated using three complementary approaches. (a) Mitochondrial protein synthesis in vitro: Tritium-labeled proteins made by isolated mitochondria were compared to ¹⁴C-labeled marker MRP by cofractionation in a two-step procedure involving isoelectric focusing and polyacrylamide gel electrophoresis. Examination of the electrophoretic profiles showed that essentially none of the peaks of in vitro product corresponded exactly to any of the MRP marker peaks. (b) Sensitivity of in vivo MRP synthesis to chloramphenicol: Cells were labeled with leucine-³H in the presence of chloramphenicol, mitochondrial ribosomal subunits were subsequently isolated, and their proteins fractionated by isoelectric focusing followed by gel electrophoresis. The labeling of every single MRP was found to be insensitive to chloramphenicol, a selective inhibitor of mitochondrial protein synthesis. (c) Sensitivity of in vivo MRP synthesis to anisomycin: We have found this antibiotic to be a good selective inhibitor of cytoplasmic protein synthesis in Neurospora. In the presence of anisomycin the labeling of virtually all MRP is inhibited to the same extent as the labeling of cytoplasmic ribosomal proteins. On the basis of these three types of studies we conclude that most if not all 53 structural proteins of mitochondrial ribosomal subunits in Neurospora are synthesized by cytoplasmic ribosomes.     

热休克蛋白代谢过程中Hela细胞热耐受性的变化

HeLa细胞受热应激后,可产生一组热休克蛋白(HSP),其中HSP73/70产量最高,其合成呈现一定的规律性,受热后4h为其合成速率高峰,10h后明显减少,24h恢复正常。随着HSP合成的消失,正常蛋白质合成逐渐恢复。HSP73/70在细胞内分解遵循指数规律,其半衰期为49.9h。HSP合成及分解规律与细胞热耐受性的增加与消退基本吻合,提示二者之间存在着伴随关系,但是否存在量效关系乃至因果关系有待今后进一步探讨。     

RIBONUCLEIC ACID AND PROTEIN SYNTHESIS IN MITOTIC HELA CELLS

HeLa cells arrested in mitosis were obtained in large numbers, with only very slight interphase cell contamination, by employing the agitation method of Terasima and Tolmach, and Robbins and Marcus. Protein synthesis and RNA synthesis were almost completely suppressed in mitotic cells. Active polyribosomes were nearly absent in mitotic cells as compared with interphase cells treated in the same way. Cell-free protein synthesis and RNA polymerase activity were also greatly depressed in extracts of metaphase cells. The deoxyribonucleoprotein (DNP) of condensed chromosomes from mitotic cells was less efficient as a template for Escherichia coli RNA polymerase than was DNP from interphase cells, although isolated DNA from both sources was equally active as a primer. Despite very poor endogenous amino acid incorporation by extracts of metaphase cells, polyuridylate stimulated phenylalanine incorporation by a larger factor in mitotic cell extracts than it did in interphase cell extracts. These results suggest that RNA synthesis is suppressed in mitotic cells because the condensed chromosomes cannot act as a template, and that protein synthesis is depressed at least in part because messenger RNA becomes unavailable to ribosomes. This conclusion was supported by the demonstration that cells arrested in metaphase supported multiplication of normal yields of poliovirus, thereby showing that the mitotic cell is capable of considerable synthesis of RNA and protein.     

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.     

THE EFFECT OF ETHIDIUM BROMIDE ON MITOCHONDRIAL DNA SYNTHESIS AND MITOCHONDRIAL DNA STRUCTURE IN HELA CELLS

The synthesis of mitochondrial DNA (mDNA) in HeLa cells is selectively inhibited by relatively low concentrations of ethidium bromide. After exposure of cells to strongly inhibitory concentrations of the drug, the apparent superhelix density of mDNA is rapidly increased, as judged by its buoyant density in CsCl in the presence of ethidium bromide. Mitochondrial DNA synthesized in the presence of partially inhibitory concentrations of ethidium bromide is also altered in its buoyant density in the presence of the dye, but is more heterogeneous in this respect. However, the change in buoyant density of newly synthesized mDNA may be explained by changes in structure other than a change in superhelix density, as indicated by its increased resistance to digestion by pancreatic DNase.     

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.     

CELL CYCLE EVENTS IN THE HYDROCORTISONE REGULATION OF ALKALINE PHOSPHATASE IN HELA S3 CELLS

The increase in alkaline phosphatase in asynchronous cultures of HeLa S₃ cells grown in medium supplemented with hydrocortisone is characterized by a lag period of 10–12 hr. Present studies utilizing synchronous cell populations indicate: (a) a minimum of 8–10 hr of incubation with hydrocortisone is necessary for maximum induction of alkaline phosphatase; (b) the increase in enzyme activity produced by hydrocortisone is initiated exclusively in the synthetic phase of the cell cycle; (c) alkaline phosphatase activity does not vary appreciably over a normal control cell cycle. Radioactive hydrocortisone is rapidly distributed into HeLa cells irrespective of their position in the cell cycle, indicating that inductive effects are not governed by selective permeability during the cell cycle. Hydrocortisone-1,2-[³H] diffuses back from the cell into the medium when the cells are incubated in fresh medium containing no hydrocortisone, and the alkaline phosphatase induction, under these conditions, is completely reversible.     

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.     

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.     

THE CENTRIOLE CYCLE IN SYNCHRONIZED HELA CELLS

Progression of the HeLa cell through its life cycle is accompanied by centriolar replication and pericentriolar changes that are in synchrony with DNA synthesis and mitosis. The first signs of preparation for replication occur during G₁ at which time the two orthogonal centrioles separate. Replication by budding begins at/or near the initiation of DNA synthesis and is completed by G₂. Pericentriolar changes which probably are causally related to spindle tubule formation occur at this time and include the appearance of vesicles, electron-opaque bodies, and an amorphous pericentriolar halo. These phenomena begin to disappear by late prophase, and the remainder of mitosis manifests decreasing centriolar and pericentriolar activity.     

A COMPARISON OF THE AXONAL TRANSPORT OF TAURINE AND PROTEINS IN THE GOLDFISH VISUAL SYSTEM

Abstract— Radioactive cystathionine, a metabolic precursor of taurine, was injected into the right eye of goldfish. At various times after injection the retina and both optic tecta were extracted with trichloroacetic acid (TCA) and the amount and nature of the radioactivity was determined. Radioactive taurine and inorganic sulfate were present in the TCA-soluble extract of retina and radioactive taurine and a small amount of inorganic sulfate was found in the contralateral optic tectum. That taurine is migrating intraaxonally and is not diffusing in extraaxonal spaces is suggested from experiments in which the migration of taurine was compared with that of [¹⁴C]mannitol, used here as a marker of extracellular diffusion. In the time studied (up to 15 h) mannitol did not migrate to the tectum, whereas taurine was detectable in the tectum as early as 8 h after injection. Since intra-axonal diffusion of amino acids and other small molecules in this system has been ruled out, it is likely that taurine is being transported axonally. The axonal transport of taurine was found to be similar to the fast component of protein transport because: (1) their rates of transport are similar, (2) the transport of both is blocked by the protein synthesis inhibitor cycloheximide, (3) vinblastine, which disrupts neurotubules, appears to have similar effects on both protein and taurine transport, and (4) both rapidly transported proteins and taurine remain mostly intra-axonal once they have been transported to the tectum. Taurine and proteins differ in that rapidly transported proteins are primarily paniculate in nature and localized to a large extent in nerve endings, while taurine is primarily in a soluble fraction and is present in nerve endings only in trace amounts. We suggest that taurine may be loosely linked to a newly synthesized protein in the soma and is then transported along with that protein on a similar conveying mechanism in the axoplasm.     

THE IN VIVO REUTILIZATION OF LYMPHOCYTIC AND SARCOMA DNA BY CELLS GROWING IN THE PERITONEAL CAVITY

An ascites tumor, Sarcoma I, was transplanted to isologous and homologous mice which had been labeled with tritiated thymidine from 1 to 24 hours previously. Radioautographic preparations revealed labeled host lymphocytes emerging to mingle with the transplanted tumor and the subsequent appearance of nuclear radioactivity in the sarcoma. Sarcoma cells cultured subcutaneously or in Millipore diffusion chambers in previously labeled mice did not demonstrate significant radioactivity. Transplantation of washed, H³-thymidine-labeled lymphocytes to non-radioactive, sarcoma-bearing mice was followed by the gradual appearance of nuclear radioactivity in the sarcoma. The label in the sarcoma was entirely removed by deoxyribonuclease but not by ribonuclease treatment prior to radioautography. Intraperitoneal injections of purified, H³-thymidine-labeled sarcoma or lymphoid DNA in normal or tumor-bearing mice were followed by radioactivity appearing in sarcoma or normal peritoneal mononuclear cells. It was concluded that reutilization of DNA and its metabolites may occur in vivo, and the conditions under which reutilization may be detected are discussed.     

玉米精细胞及体细胞原生质体表膜蛋白的比较

以低渗冲击法（改良两步法）及Ｐｅｒｃｏｌｌ密度梯度离心,成功分离纯化生活玉米（Ｚｅａｍ ａｙｓ）精细胞;以混合酶解法制备玉米叶原生质体和愈伤组织原生质体;以ＮＨＳ－生物素标记完整精细胞及原生质体表膜蛋白,进行ＳＤＳ－ＰＡＧＥ和Ｗｅｓｔｅｒｎ ｂｌｏｔ,并以辣根过氧化物酶标亲和素检测被标记的表膜蛋白。结果表明,在精细胞中标记蛋白有４ 种,分子量分别为４８、５９、６７、７９ ｋＤ;叶片原生质体中有５ 种,分子量分别为５４、５８、６６、７１、７８ ｋＤ;愈伤组织原生质体中仅有２ 种,分子量６７ 和８０ ｋＤ。其中４８ ｋＤ蛋白为精细胞所特有,５４ ｋＤ 和７１ ｋＤ蛋白为叶片细胞所特有     

THE RIBOSOMAL RNA OF HAMSTER-MOUSE HYBRID CELLS

The ribosomal RNA (rRNA) of a series of hamster-mouse somatic cell hybrids was studied. Mouse 28S rRNA was separated from its hamster counterpart by a two-step procedure involving sucrose gradient centrifugation of ribosomes and polyacrylamide gel electrophoresis of rRNA. Both hamster and mouse types of rRNA were synthesized in the 11 hybrids tested, including hybrids containing only about one-half the haploid number of either mouse or hamster chromosomes. It appears that, for both hamster and mouse rRNA, when the chromosomes of one species constituted the majority of the chromosomes of a hybrid, a disproportionately higher percentage of rRNA of that species was present in the hybrid. Some hybrid clones, having a majority of mouse chromosomes, had a mouse rRNA cell concentration approximately four to five times higher than the concentration expected from linear extrapolation of the value found for the mouse parental cell line.     

TIME SEQUENCE OF NUCLEAR PORE FORMATION IN PHYTOHEMAGGLUTININ-STIMULATED LYMPHOCYTES AND IN HELA CELLS DURING THE CELL CYCLE

The time sequence of nuclear pore frequency changes was determined for phytohemagglutinin (PHA)-stimulated human lymphocytes and for HeLa S-3 cells during the cell cycle. The number of nuclear pores/nucleus was calculated from the experimentally determined values of nuclear pores/µ² and the nuclear surface. In the lymphocyte system the number of pores/nucleus approximately doubles during the 48 hr after PHA stimulation. The increase in pore frequency is biphasic and the first increase seems to be related to an increase in the rate of protein synthesis. The second increase in pores/nucleus appears to be correlated with the onset of DNA synthesis. In the HeLa cell system, we could also observe a biphasic change in pore formation. Nuclear pores are formed at the highest rate during the first hour after mitosis. A second increase in the rate of pore formation corresponds in time with an increase in the rate of nuclear acidic protein synthesis shortly before S phase. The total number of nuclear pores in HeLa cells doubles from ~2000 in G₁ to ~4000 at the end of the cell cycle. The doubling of the nuclear volume and the number of nuclear pores might be correlated to the doubling of DNA content. Another correspondence with the nuclear pore number in S phase is found in the number of simultaneously replicating replication sites. This number may be fortuitous but leads to the rather speculative possibility that the nuclear pore might be the site of initiation and/or replication of DNA as well as the site of nucleocytoplasmic exchange. That is, the nuclear pore complex may have multiple functions.