Changes in the polyadenylated messenger RNA population during development of Dictyostelium discoideum

The program of gene expression during the life cycle of Dictyostelium discoideum has been assessed by molecular hybridization of cDNA probes with polysomal RNA extracted at the following different stages of development: vegetative growth, interphase (2.5 hr), aggregation (8 hr), postaggregation (12 hr), and preculmination (18 hr). Several different cDNA probes were used. Two probes were prepared from vegetative stage poly(A⁺) RNA, one representing all species present and the other enriched for abundant species. A third cDNA probe was prepared from preculmination stage polysomal RNA and a fourth probe consisted of the preculmination stage cDNA depleted in those species also present at the vegetative stage. Hybridization of the various probes with the different polysomal RNA preparations has revealed developmental changes in the mRNA populations. These changes were not detected in an aggregation less mutant under similar conditions of starvation. Abundant RNA species of vegetative cells were found to drop to low levels, especially during the aggregation period. Fifty percent by mass of the RNA present in polysomes at 18 hr is not present during vegetative growth. Some of the new RNA species appeared during interphase and the remaining during the postaggregation period. A gradual increase in the number of copies per cell of certain RNA species comprising both new species as well as some shared with vegetative cells was observed throughout development. Other results indicated that the composition of polysomal and cytoplasmic RNA is similar during vegetative growth but differs markedly at 18 hr of development. Also, cytoplasmic RNA at 18 hr contained, in addition to polysomal RNA, a large proportion by mass of nonpolysomal RNA similar to vegetative RNA. The number of polysomal RNA species detected by this analysis during vegetative growth and during the preculmination stage were estimated to be 3000 and 3700, respectively. The number of copies of these RNA species ranged between 30 and 2000 per cell during vegetative growth and 3 to 300 per cell in polysomes at 18 hr. Developmentally induced RNAs which were preferentially distributed among abundant and intermediate classes were estimated to number 700–900 species.     

Adenovirus transcription. V. Quantitation of viral RNA sequences in adenovirus 2-infected and transformed cells.

The concentrations, in copies per cell, of viral RNA sequences complementary to different regions of the genome were determined at 8, 18 and 32 hours after infection of human cells with adenovirus type 2: separated strands of fragments of ³²P-labelled adenovirus 2 DNA, generated by cleavage with restriction endonucleases EcoR1, Hpa1 and BamH1, were added to reaction mixtures at sufficient concentrations to drive hybridizations with infected or transformed cell RNA. Under these conditions, the fraction of ³²P-labelled DNA entering hybrid is directly proportional to the absolute amount of complementary RNA in the reaction.At 8 hours after infection in the presence of cytosine arabinoside, “early” viral messenger RNA sequences are present at a frequency of 300 to 1000 copies per cell. The abundance of early mRNA sequences in different lines of adenovirus 2-transformed rat cells is markedly lower than their concentration in lytically infected cells. Moreover, the abundance of early mRNA in a given transformed rat cell line reflects the number of copies of its template DNA sequences per diploid quantity of cell DNA. After the onset of the late phase of the lytic cycle, the abundance of one early mRNA species, that coding for a single-stranded DNA binding protein required for viral DNA replication, is amplified. Viral RNA sequences complementary to regions of the genome coding for other early mRNA sequences remain at the level observed at 8 hours after infection.Exclusively “late” viral mRNA sequences are present over a range of concentrations, 500 to 10,000 copies per cell, depending on the region of the genome. By 18 hours after infection, the nucleus contains approximately three times as much total, viral RNA as the cytoplasm. The abundant nuclear, viral RNA sequences at 18 hours are transcribed from a contiguous region, 65% of the genome in length. In some cases, viral RNA sequences complementary to mRNA sequences are very abundant in the nucleus. When cytoplasmic and nuclear fractions are mixed and incubated under annealing conditions, some mRNA sequences will anneal with more abundant, anti-messenger nuclear RNA sequences to form double-stranded RNA. Such annealing of nuclear, viral RNA to early, cytoplasmic mRNA sequences probably accounts for the inability to detect, by filter hybridization, certain classes of early mRNA sequences during the late stage of infection.     

Nuclear differentiation and nucleic acid synthesis in well-fed exconjugants of Paramecium aurelia

Paramecium aurelia exconjugants contain new macronuclear anlagen and numerous fragments of the old pre-zygotic macronucleus. Macronuclear anlagen develop during the first two cell cycles after conjugation. During this time their volume increases from about 11 m³ to about 3700 m³ and more than 10 doublings of DNA content occur. The rate of DNA synthesis is between two and three times as great as in the vegetative macronucleus. — In macronuclear fragments, however, DNA synthesis is suppressed. The rate of DNA synthesis in macronuclear fragments during the extended first cell cycle after conjugation (11 1/2 hr. vs. 5 1/2 hr. for the vegetative cell cycle) is only about one-third of the rate in vegetative macronuclei and there is only a 65% increase in the mean DNA content of fragments. The rate of fragment DNA synthesis continues to decrease during each of the subsequent two cell cycles. — Unlike the rate of DNA synthesis, the rate of RNA synthesis per unit of DNA is similar in macronuclear anlagen, macronuclear fragments and fully developed macronuclei. Macronuclear fragments continue to synthesize RNA at the normal rate long after the new macronuclei are fully developed. Fragments contribute about 80% of all RNA synthesized during the first two cell cycles after conjugation. RNA synthesis begins very early in the development of macronuclear anlagen and nucleolar material appears during the first half-hour of anlage development. — Chromosome-like structures were never observed during anlage development and there was no evidence of two periods of DNA synthesis separated by a DNA poor stage as has been observed in several hypotrichous Ciliates.     

The program of protein synthesis during sporulation in Bacillus subtilis

The program of protein synthesis was examined during sporulation in Bacillus subtilis as an index of the control of gene expression. At various stages of growth and spore formation, cells of B. subtilis were pulse-labeled with ³⁵S-methionine. Protein was extracted from the radioactively labeled bacteria and then subjected to high resolution one-dimensional and two-dimensional slab gel electrophoresis. We report that sporulating cells restricted or “turned off” the synthesis of certain polypeptides characteristic of the vegetative phase of growth. In certain cases, this “turn off” was prevented in a mutant (SpoOa-5NA) blocked at the first stage of spore formation. Sporulating bacteria also elaborated new polypeptide species that could not be detected in vegetatively growing cells or in cells of the asporogenous mutant SpoOa-5NA in sporulation medium. The synthesis of these sporulation-specific proteins was “turned on” in a temporally defined sequence throughout the period of spore formation. Spore coat protein, for example, was first synthesized at 4 hr after the onset of sporulation, the time at which refractile prespores appeared. Certain sporulation-specific polypeptides including the coat protein were among the most actively produced polypeptides in sporulating cells.     

Changes in the abundance of polyadenylated RNA during slime mould development measured using cloned molecular hybridization probes.

Total Dictyostelium discoideum messenger RNA prepared from cells at the eighth hour of development in suspension culture has been copied into DNA. This DNA was inserted into the plasmid PMB9 and used to transform Escherichia coli. The resulting “clone bank” was screened using an in situ hybridization technique in which replicate copies of a set of clones were hybridized with mRNA isolated from vegetative (non-developing) cells and from cells at the eighth hour of development. The mRNA was labelled in vitro so that the amount of hybridization to a given clone is a measure of the relative abundance of the mRNA complementary to the DNA in that clone. By comparing the amount of hybridization of the mRNA preparations to each clone, it has been possible to identify plasmids containing D. discoideum DNA whose complementary mRNA increases or decreases in abundance during development. These observations are direct proof of a change in mRNA concentration during D. discoideum development for individual high and medium abundance mRNA species. We can estimate from these results the proportion of such mRNA species whose concentration increases significantly during development and we find that only a small fraction show such a change.     

Ribonucleic acid synthesis during fruiting body formation in Myxococcus xanthus.

A method has been devised that allowed us, for the first time, to pulse-label M. xanthus cells with precursors for ribonucleic acid biosynthesis while they were undergoing fruiting body formation. Using this method, we examined patterns of ribonucleic acid (RNA) accumulation throughout the process of fruiting body formation. As development proceeded, the rate of RNA accumulation increased at two periods of the developmental cycle: once just before aggregation and once late in the cycle, when sporulation was essentially completed. In contrast to vegetatively growing cells, in which only stable RNA species are labeled during a 30-min pulse, the majority of radioactivity found in RNA from 30-min pulse-labeled developing cells was found in an unstable heterodisperse fraction that migrated to the 5S to 16S region of sucrose density gradients and sodium dodecyl sulfate-polyacrylamide gels. This pattern of incorporation could not be induced (i) by a shift down of vegetatively growing cells to a nutritionally poor medium, in which the generation time was increased to that of developing cells during the growth phase, or (ii) by plating of vegetative cells onto the same solid-surface environment as that of developing cells, but which surface supported vegetative growth rather than fruiting body formation. Thus, the RNA synthesis pattern observed appeared to be related to development per se rather than to nutritional depletion or growth on a solid surface alone. The radioactivity incorporated into the unstable 5S to 16S RNA fraction accumulated as the pulse length was increased from 10 to 30 min; in contrast, an analogous unstable fraction from vegetative cells decreased as pulse length was increased. This suggested that developmental 5S to 16S RNA was more stable than vegetative cell 5S to 16S RNA (presumptive messenger RNA). However, during a 45-min chase period, radioactivity in 30-min-pulse-labeled developmental 5S to 16S RNA decayed to an extent twice that of developmental RNA located in 16S and 23S regions of sucrose density gradients and was considerably less stable than the 5S, 16S, and 23S RNA species labeled during a 30-min pulse of vegetative cells.     

Stable Messenger Ribonucleic Acid and Germination of Myxococcus xanthus Microcysts

We have examined germination, protein synthesis and ribonucleic acid (RNA) synthesis by microcysts of the fruiting myxobacterium Myxococcus xanthus. The morphological aspects of microcyst formation were completed at about 2 hr after induction had begun. In such microcysts, germination, RNA synthesis, and protein synthesis were inhibited by actinomycin D (Act D). At 6 hr after induction, germination and protein synthesis had become relatively resistant to Act D, whereas RNA synthesis was inhibited by about 95%. Experiments with (3)H-Act D indicated that the deoxyribonucleic acids of both young and old microcysts bind Act D equally. Resistance of germination to Act D was acquired 4 to 5 hr after induction of microcyst formation, and was due to an Act D-sensitive synthesis at that time. Vegetative cells and microcysts were pulsed with uridine-5-(3)H and chased for 60 min; the RNA was extracted and analyzed by means of sucrose density gradient centrifugation and gel electrophoresis. Both microcysts and vegetative cells were found to contain grossly the same types of RNA in the same proportions. RNA pulse-labeled in microcysts was more stable than that in vegetative cells. No particular portions of the microcyst pulse-labeled RNA were selectively stabilized. These data indicate that a stable messenger RNA required for synthesis of germination proteins was synthesized during microcyst formation. This may be the same as the RNA synthesized 4 to 5 hr after initiation of microcyst formation. We suggest that the existence of such stable messenger RNA in microcysts is consistent with the limited biosynthetic activities of such cells.     

Autoradiographic Studies of Uridine-5-H3 Uptake in Entamoeba histolytica in the CLG Medium1

SYNOPSIS. Using uridine-5-H³, “long-term” labeling experiments over a 72 hr growth cycle were done with E. histolytica strain K9 grown in CLG medium with penicillin-inhibited Bacteroides. Autoradiographic analysis revealed that tritium occurs primarily in cytoplasm and rarely the nucleus of amebae. The most extensive cytoplasmic activity was observed during the initial 0–24 hr growth period of amebae as compared to later labeling periods. RNase or RNase followed by DNase extracted a large amount but not all label from amebae. These nucleases were least effective during the initial 24 hr period of growth. Thus it appears that tritium from uridine-5-H³ is not highly specific for RNA in amebae. However, the possibility that such label is associated with RNase-resistant RNA cannot be ruled out. More recent cytochemical studies do indicate the presence of RNase-resistant RNA in the cytoplasm of amebae. The activity found in penicillin-inhibited Bacteroides after uridine-5-H³ labeling and their reaction to the various digestive procedures was similar to amebae at corresponding labeling periods. Therefore at least some of the RNase-resistant material present in the cytoplasm of amebae may be derived from the ingested bacteria; this has been further found by appropriate experiments in which amebae were fed prelabeled bacteria. Nuclear activity when observed (always after 24 hrs growth) was associated either with the periphery of the nucleus and/or the endosome. It was not seen in the nuclear stroma. Some of this activity is RNase-resistant, perhaps representing double or multi-stranded RNA. It therefore appears that RNA is not distributed in the nuclear stroma in “long-term” labeling experiments.     

INFLORESCENCE DEVELOPMENT IN BRASSICA CAMPESTRIS L.

Vegetative seedlings of the Ceres strain Brassica campestris L., a quantitative, long-day plant, were induced to flower by exposure to a 16-hr, long-day cycle. Cytohistological and cytohistochemical changes associated with inflorescence development were examined. Developing shoot apices were classified in vegetative, transitional, and reproductive stages. The vegetative apex possessed a biseriate tunica, central zone, peripheral zone and pith-rib meristem. The transitional stage at 48 hr was marked by an increase in size and by a stratification of the upper cell layers of the shoot apex with a concurrent decrease of apical cytohistochemical zonation. The reproductive stage was initiated at 58 hr by periclinal cell divisions in the 3rd and 4th cell layers of the flank region. Cytohistochemical zonation in the vegetative apical meristem was restored in the floral apex. An “intermediate developmental” phase was not observed between the vegetative and reproductive stage.     

Induction and synthesis of tubulin during the cell cycle and life cycle of Chlamydomonas reinhardi

Two types of tubulin induction are observed in Chlamydomonas reinhardi. One is elicited by flagellar detachment and the other occurs as a normal event of the vegetative cell cycle. In the former case, a strong and extensive induction of tubulin synthesis occurs following deflagellation of cells in all phases of the life cycle [vegetative, gametic, and (early) zygotic]. Synthesis is initiated in all three cell types within 15 min after deflagellation. In gametic and zygotic cells, tubulin synthesis so induced accounts for 15 to 20% of the total protein synthesis during the 1-hr peak period of tubulin production. The ability to support both tubulin synthesis and flagellar regeneration is lost in zygotes at 1.5 hr after the initiation of zygotic development. This alteration represents one of several dramatic shifts in the programming of protein synthesis that occur during the first 4 hr of zygotic differentiation in C. reinhardi. The second (i.e., cell cycle-dependent) type of induction is observed in synchronously growing vegetative cells at ～1.5–2 hr prior to cytokinesis. Tubulin synthesis, in this case, persists at relatively high levels (～5% of the total protein synthesis) for the next 9 hr, i.e., through the entire period of cell division to a time just before the liberation of fully flagellated daughter cells at hr 20 of the cell cycle. Changes in the programming of protein synthesis, and of tubulin synthesis in particular, are discussed in relation to specific physiological and cytological transitions that occur during the growth and differentiation of C. reinhardi.     

Changes in "template-bound" and "free" RNA polymerase activities in isolated nuclei from Xenopus laevis embryos

Nuclei have been isolated from Xenopus laevis embryos and incubated under conditions allowing RNA synthesis to proceed for more than 3 h. The RNA molecules synthesized on the endogenous template are stable, heterogeneous in size and correspond to the activities of the three RNA polymerases.In these in vitro conditions we have determined the extent of activity of the three RNA polymerases during the embryonic development from blastula to swimming tadpole. Our results on isolated nuclei are in good agreement with the changes in RNA synthesis which take place during normal embryonic development.We have measured both the “template-bound” and the “free” activities of each of the three RNA polymerases during development. Amongst the total RNA polymerase activities engaged on the template, the proportion of polymerase I increases as development proceeds: at the blastula stage, there is practically no RNA polymerase I engaged on the template, whereas in swimming tadpoles, RNA polymerase I amounts to about 90% of the RNA polymerases bound to the DNA. Conversely, RNA polymerase I represents the major part of free RNA polymerases in blastula nuclei.Autoradiography of incubated nuclei shows that, at least in swimming tadpoles nuclei, both “free” and “template-bound” RNA polymerase I are localized in the nucleoli.The evolution of “template-bound” RNA polymerase II activity during development is quite different from that of RNA polymerase I: RNA polymerase II activity represents 75% of engaged polymerase activity in blastulae and only 47% at the swimming tadpoles stage.The results suggest that part of the “free” RNA polymerase I activity might progressively become “template-bound” during embryogenesis.     

Cell density,negative proliferation control,and phosphorylation of retinoblastoma protein

Cell density negative control (CDNC) of normal human fibroblast proliferation occurs after stimulation by mitogens with different signal transduction mechanism. Delayed exposure to agents that interfere with CDNC, such as doublestranded RNA and vanadate, reveals the existence of a biochemical event, involved in CDNC, that occurs 5–8 hr after the beginning of mitogenic stimulation. This is earlier than the point of “mitogenic commitment,” defined by the duration of mitogen exposure required for cell cycle entry (8–18 hr). Phosphorylation of the retinoblastoma gene product (pRB) begins 8–10 hr after mitogen stimulation and is nearly complete at 18 hr, just as the first cells enter S-phase. CDNC prevents pRB phosphorylation. Interferon β delays pRB phosphorylation by up to 20 hr but has little effect on the timing of mitogenic commitment. Thus mitogenic commitment is located in time between CDNC and pRB phosphorylation. When agents that cause a release from CDNC are applied to dense, negatively controlled cultures after 18 hr of EGF stimulation, pRB phosporylation occurs 6–8 hr after release. This suggests that the negatively controlled cells process the mitogenic signal but accumulate at a restriction point. The relatively early timing of CDNC-related events in the prereplicative phase raises the possibility that pRB phosphorylation is a consequence rather than a prerequisite for release from cell density negative control. © 1993 Wiley-Liss, Inc.     

Protein and Ribonucleic Acid Synthesis During the Diploid Life Cycle of Allomyces arbuscula

The diploid life cycle of Allomyces arbuscula may be divided into four parts: spore induction, germination, vegetative growth, and mitosporangium formation. Spore induction, germination, and mitosporangium formation are insensitive to inhibition of actinomycin D, probably indicating that stable, pre-existing messenger ribonucleic acid (RNA) is responsible for these developmental events. Protein synthesis is necessary during the entire life cycle except for cyst formation. A system for obtaining synchronous germination of mitospores is described. During germination there is a characteristic increase in the rate of synthesis of RNA and protein although none of the other morphogenetic changes occurring during the life cycle are necessarily accompanied by an appreciable change in the rate of macromolecular synthesis.     

Changes in the Polypeptide Composition during the Ontogenetic Development of the Shoot Apex of Crocus sativus

The shoot apex development during the life cycle of Crocus sativus L. was characterized by light microscopy and two-dimensional gel electrophoresis. Using silver staining of polyacrylamide gels, numerous quantitative and qualitative changes in the populations of polypeptides were observed during transition from vegetative to prefloral and from prefloral to floral stages. Using 80 g protein, we were able to detect 352 polypeptidic spots. In comparison with the vegetative apex, 89 new polypeptides were identified in the prefloral meristem and 29 polypeptides were missing. In the reproductive meristem, 94 new spots were identified and 44 spots were missing. Thus, substantial quantitative and qualitative changes in the populations of polypeptides occurred during the prefloral stage, a point of no return in plant development, i.e., and before floral primordia initiation.     

Expression of c-myc oncogene in rat liver by a dietary manipulation

After rats deprived of protein for several days are fed a meal containing protein, hepatic DNA replication is induced. When nuclear DNA synthesis is stimulated in the normally quiescent rat liver by a dietary manipulation, we examined the changes of the steady-state levels of messenger RNA for c-myc. Levels of c-myc mRNA are gradually elevated approximately 4 to 5-fold above normal in the livers of rats that are fed for several days a diet that lacks protein. After a nutritional shift from a protein-free diet to a diet containing 50% casein, the levels of c-myc mRNA decrease rapidly by 2 h and returned to approximately basal levels after 8 h. Our results suggest that c-myc expression during the prereplicative stage of liver is likely to reflect events associated with entry and progression of hepatocytes into the cell cycle.     

Differentiation in the early chick embryo: effects of bromodeoxyuridine on erythropoiesis

Organ cultures of chick embryos at the definitive streak stage will normally show the first appearance of hemoglobin in erythroid cells after 20 hr of culture. Addition of 5-bromodeoxyuridine (BrdU) will prevent hemoglobin formation when added at the beginning of culture, but the tissue becomes resistant to the inhibitor when addition is delayed for approximately 10 hr. The inhibitory effect of BrdU is canceled or reversed if thymidine replaces BrdU at the beginning of culture or later. Transfer to thymidine containing medium even after 20 hr permits hemoglobin formation to occur at almost the normal time, thus making it unlikely that a complete cell cycle or DNA replication during a complete S period is required for the reversal of inhibition.Even when the appearance of cytologically detectable hemoglobin is inhibited by BrdU, some globin is synthesized and RNA sequences specific for globin are present, but in decreased amount, unless the inhibitor is given very early. BrdU does not affect the synthesis of any particular RNA species or of polyadenylic acid, but it does lower the rate of uptake of adenosine into the ATP pool. While not affecting cell cycle times in cell cultures, BrdU greatly reduces cell numbers.