THE EFFECT OF HYDROXYUREA AND FLUORODEOXYURIDINE ON CELL CYCLE EVENTS IN THE CHLOROCOCCAL ALGA SCENEDESMUS QUADRICAUDA (CHLOROPHYTA)1

The effect of hydroxyurea and 5-fluorodeoxyuridine (FdUrd) on the course of growth (RNA and protein synthesis) and reproductive (DNA replication and nuclear and cellular division) processes was studied in synchronous cultures of the chlorococcal alga Scenedesmus quadricauda (Turp.) Bréb. The presence of hydroxyurea (5 mg·L^?1)from the beginning of the cell cycle prevented growth and further development of the cells because of complete inhibition of RNA synthesis. In cells treated later in the cell cycle at the time when the cells were committed to division, hydroxyurea present in light affected the cells in the same way as a dark treatment without hydroxyurea; i. e. RNA synthesis was immediately inhibited followed after a short time period by cessation of protein synthesis. Reproductive processes including DNA replication to which the commitment was attained, however, were initiated and completed. DNA synthesis continued until the constant minimal ratio of RNA to DNA was reached. FdUrd (25 mg·L^?1) added before initiation of DNA replication in control cultures prevented DNA synthesis in treated cells. Addition of FdUrd at any time during the cell cycle prevented or immediately stopped DNA replication. However, by adding excess thymidine (100 mg·L^?1), FdUrd inhibition of DNA replication could be prevented. FdUrd did not affect synthesis of RNA, protein, or starch for at least one cell cycle. After removal of FdUrd, DNA synthesis was reinitiated with about a 2-h delay. The later in the cell cycle FdUrd was removed, the longer it took for DNA synthesis to resume. At exposures to FdUrd longer than two or three control cell cycles, cells in the population were gradually damaged and did not recover at all.     

Selective inhibition of the cell cycle of cultured human diploid fibroblasts by aminonucleoside of puromycin

Aminonucleoside (AMS) inhibited the cell cycle of human lung fibroblasts at a point in G1 phase and at another point in G2 phase. Even when this inhibition was fully established, DNA synthesis and mitosis which were in progress proceeded normally. Inhibition of RNA synthesis in the cultures preceded the effects on DNA synthesis and mitosis, but inhibition of protein synthesis could not be detected. These points of potential inhibition do not exist in the cell cycle of HeLa cell, or are not affected by aminonucleoside. Here inhibition of cell proliferation by AMS was less marked, and when inhibition eventually occurred it was not specific for any point of the cell cycle. The rate of entry of the inhibitor was similar in both types of cell.     

Rate of virus-specific RNA synthesis in synchronized chicken embryo fibroblasts infected with avian leukosis virus.

The rate of avian leukosis virus (ALV)-specific RNA synthesis has been examined in bot- uninfected and ALV-infected synchronized chicken embryo fibroblasts. RNA from cells labeled for 2h with [3H]uridine was hybridized with avian myeloblastosis virus poly(dC)-DNA, and the hybridized RNA was analyzed with poly(I)-spephadex chromatography. Approximately 0.5% of the RNA synthesized in ALV-infected cells was detected as virus specific, and no more than a twofold variation in the rate of synthesis was detected at different times in the cell cycle. In synchronized uninfected chicken embryo fibroblasts, approximately 0.03% of the RNA synthesized was detected as virus specific, and no significant variation in the rate of synthesis was observed during the cell cycle. Treatment of ALV-infected chicken embryo fibroblasts with cytosine arabinoside or colchicine was used to block cells at different stages in the cell cycle. The rates of virus-specific RNA synthesis in cells so treated did not differ significantly from the rates in either stationary or unsynchronized virus-infected chicken embryo fibroblasts. These findings support the conclusion that after the initial division of an ALV-infected chicken embryo fibroblast and the initiation of virus RNA synthesis, the rate of virus-specific RNA synthesis is independent of the cell cycle.     

A possible maternal-effect mutant of Xenopus laevis: II. Studies of RNA synthesis in dissociated embryonic cells

Embryos from a female of Xenopus laevis (designated as no. 65) arrest development at gastrulation and are assumed to be ova-deficient mutant. We dissociated these embryos and studied RNA synthesis at different stages. The cells from the ova-deficient embryos reaggregated quite actively as wild-type embryo cells until the late gastrula stage. RNA synthesis was normal at the early blastula stage but greatly inhibited by the late blastula (stage 9.5) stage, when the synthesis of DNA and protein was still not inhibited appreciably. Thus, inhibition in RNA synthesis appears to be the first manifestation of the maternal defect that occurs before the gastrulation arrest.     

A factor in sea urchin eggs inhibits transcription in isolated nuclei by sea urchin RNA polymerase III

Isolated nuclei from sea urchin embryos synthesize RNA at a rate comparable to other animal cell nuclei. All three RNA polymerases are active as judged by alpha-amanitin sensitivity and hybridization to specific cloned DNAs. Extracts were prepared from sea urchin eggs and embryos by extraction with 0.35 M KCl. None of the crude extracts had a large effect on total RNA synthesis. However, extracts from sea urchin eggs inhibited RNA polymerase III activity in nuclei from blastula and gastrula embryos. There was no effect on the synthesis of ribosomal RNA by RNA polymerase I or on the synthesis of two RNA polymerase II products, histone mRNA and the sea urchin analogue of U1 RNA. The inhibitor is present in two different species of sea urchin and has been 50-fold purified by diethylaminoethylcellulose and hydroxylapatite chromatography. The inhibitor is not present in extracts prepared from sea urchin blastula embryos.     

Inhibition of Host Cell Ribosomal Ribonucleic Acid Methylation by Foot-and-Mouth Disease Virus

A study of protein and ribonucleic acid (RNA) synthesis in cells infected by foot-and-mouth disease virus has indicated possible mechanisms of viral control over host cell metabolism. Foot-and-mouth disease virus infection of baby hamster kidney cells resulted in 50% inhibition of host cell protein synthesis at 180 min postinfection. A viral-induced interference with host cell RNA methylation was observed to be more rapidly inhibited than protein synthesis. To determine the nature of methylation inhibition, the kinetics of several host cell methylated RNA species were examined subsequent to virus infection. Data from sucrose zonal centrifugation and methylated albumin kieselguhr chromatography showed that methylation of nuclear RNA was inhibited 50% at 60 min postinfection. Inhibition of nuclear ribosomal RNA precursors and formation of nascent ribosomes correlated with inhibition kinetics of nuclear RNA methylation. It is suggested that the viral interference with the host nuclear RNA methylation is directly responsible for the observed loss of nascent ribosome formation. Moreover, early in the infectious cycle, methylation inhibition of host cell RNA could, in part, account for the cessation of host protein synthesis.     

Control of protein synthesis in human fibroblasts by intracellular potassium

Intracellular potassium ion (K⁺) in cultured human fibroblasts (HF cells) was maintained at reduced steady-state levels by incubating cells in various ouabain concentrations. Small decreases in cell K⁺ had no effect on protein synthesis and cell growth, but when cell K⁺ fell below 60–80% of control levels, the rate of protein synthesis decreased in proportion to further reductions in K⁺. DNA synthesis was also inhibited, presumably because of its dependence on protein synthesis. On the other hand, RNA synthesis remained uninhibited over a wide range of K⁺ concentrations, an effect characteristic of many specific inhibitors of protein synthesis.In ouabain-treated cells neither levels of ATP nor transport of amino acids was limiting for protein synthesis. Loss of activity of messenger or other species of RNA was not responsible for inhibition of protein synthesis, since in the presence of actinomycin D, the rate of protein synthesis could be decreased or increased solely by adjusting cell K⁺. Release from ouabain inhibition restored K⁺ levels, macromolecular synthesis, and cell growth, but there was no resulting synchrony of cell division. In cell populations partially synchronized by serum starvation and refeeding protein synthesis was sensitive to reduction in K⁺ levels throughout the cell cycle.Our quantitative results show that cell K⁺ levels, when sufficiently reduced, can determine the rate of protein synthesis and hence the rate of cell growth.     

Inhibition of Host-Cell Protein and Ribonucleic Acid Synthesis by Newcastle Disease Virus

The mechanisms of Newcastle disease virus-(NDV) induced inhibition of cell protein and ribonucleic acid (RNA) synthesis were investigated. It was observed that the ability of NDV to inhibit cell RNA synthesis is dependent on the virus strain. The inhibitors, azauridine and cycloheximide, were added to cell cultures at different times after infection to study the roles of protein and RNA synthesis in the viral inhibition process. Viral inhibition of cell RNA synthesis and viral inhibition of cell protein synthesis become resistant to cycloheximide at a different time after infection than that in which they become resistant to azauridine. The results indicate that the inhibition of cell RNA synthesis by the Texas strain involves the synthesis of inhibitory proteins which are coded by the viral genome. The Texas and Beaudette strains of NDV appear to employ different mechanisms for the inhibition of host-cell protein synthesis. Viral inhibition of cell protein synthesis does not appear to cause, or be the result of, viral inhibition of cell RNA synthesis.     

灭幼脲对粘虫不育作用的机理

通过一系列组织学观察和生物化学测定,研究了灭幼脲在粘虫Mythimna seporata(Walkcr)雌蛾体内及其所产卵内的作用及影响。 灭幼脲(50ppm)经雌蛾连续取食后,卵巢中DNA、RNA的合成及卵巢对血淋巴中蛋白质的吸取均被促进, 卵巢细胞核中DNA、RNA及蛋白质的含量均比正常雌蛾增加,卵巢发育及卵子形成亦被促进,产卵量正常。粘虫生殖系统未受灭幼脲的抑制。胚胎发育阶段细胞核及线粒体中DNA的含量均因雌蛾取食灭幼脲而降低,但胚胎发育并不中断,幼虫分化可达黑头期。灭幼脲导致粘虫所产卵不能孵化的主要原因在于幼虫分化过程中体壁及气管系统的形成受到严重抑制。体壁皮细胞缩小呈扁平形,无细胞核,连成线状,有断裂;原表皮缺乏或极薄。虫体内无气管分布。幼虫死于卵壳内。灭幼脲的不育作用和直接杀卵作用实质上都是对正在分化幼虫中几丁质合成的抑制。本文对灭幼脲的毒理机制进行了讨论。