RNA and protein synthetic patterns during germination of Polysphondylium pallidum microcysts

During microcyst germination in the cellular slime mould Polysphondylium pallidum, an immediate rapid increase in the rate of protein synthesis ([3H]leucine incorporation) is observed within 15 min after the initiation of germination. The data, corrected for amino acid pool changes, reveal that the rate of protein synthesis reaches its peak at 1 1/2 h, after which it decreases. A low level of RNA synthesis ([3H]uridine incorporation) is observed after 1 h and this rate increases markedly after 2 h. Analysis of the RNA species shows a low level of synthesis of all ribosomal RNA's which begins between 1 and 2 h and increases after 2 h. The synthesis of a heterogeneously distributed, poly(A)-containing fraction of RNA (presumptive mRNA) is initiated some time after 2 h and the synthesis of a small molecular weight species in the 4-5S region is observed after 3 h. Thus, it seems that Polysphondylium microcysts show sequentially initiated synthesis of RNA during germination.     

Herpesvirus infection modifies adenovirus RNA metabolism in adenovirus type 5-transformed cells.

The effect of herpes simplex virus (HSV) infection of mRNA metabolism was examined in a system where the fate of specific RNA sequence can be assayed. Adenovirus type 5-transformed rat embryo cell line 107 synthesizes adenovirus-specific RNA (ad-RNA), which functions in the cytoplasm as mRNA. We have utilized ad-RNA as a model for mRNA metabolism, and in a preliminiary study we characterized ad-RNA in the nucleus and cytoplasm by hybridization to filter-bound adenovirus DNA. The results indicated the as-RNA accumulates in the nucleus and that cytoplasmic polyadenylic acid [poly(A)]-containing ad-RNA turns over with a half-life of a few hours. Pulse-chase experiments confirmed these observations and a half-life of about h was determined for the poly(A)-containing cytoplasmic ad-RNA. A second class of ad-RNA remains in the nucleus, where it turns over with a longer hlaf-life (about 24 h). The infection of 107 cells by HSV was restricted at 37 degree C, giving a burst size of 5 PFU per cell and allowing continued host DNA synthesis. Protein synthesis was inhibited greater than 50% by 7 h after infection, and total RNA synthesis was 50% inhibited by 4 h after infection. During the first 8 h after infection, HSV has little effect on the rate of synthesis of ad-RNA as determined by hybridization of nuclear RNA samples, but,during the same period, HSV inhibits the accumulation of poly(A)-containing ad-RNA in the cytoplasm. The degree of this inhibition increases steadily throughout this period and reaches 60% by 6.5 to 8 h after infection. Nosignificant effect was seen on the accumulation of total cellular poly(A)-containing RNA. It was concluded from these experiments that HSV infection alters the metabolism of ad-RNA so as to prevent the normal appearance of the poly(A)-containing mRNA in the cytoplasm. The result for ad-RNA may not represent the behavior of total cellular poly(A)-containing RNA under conditions where infection is restricted.     

Quantitation of vitellogenin messenger RNA in the liver of male xenopus toads during primary and secondary stimulation by estrogen

From livers of estrogen-stimulated female Xenopus toads, large quantities of estrogen-induced, poly(A)-containing RNA could be isolated, showing the same characteristics as vitellogenin mRNA obtained from hormone-treated males.Using cDNA hybridization, vitellogenin mRNA was monitored in the cytoplasmic poly(A)-containing RNA of the liver of male toads during 13 days of primary and the initial phase of secondary stimulation with estrogen.During primary stimulation, low amounts of vitellogenin mRNA, not exceeding 0.18% of the cytoplasmic poly(A)-containing RNA, were first detected after 12 hr of hormone treatment, and vitellogenin mRNA was found to increase on the average to 34% of the cytoplasmic poly(A)-containing RNA on the seventh day of hormone treatment. After 3 days of primary stimulation, accumulation of vitellogenin mRNA leveled off, showing no significant increase in the cytoplasm up to 13 days of hormone treatment. As judged from incorporation of ³²PO₄ into blood plasma proteins of males during primary stimulation, vitellogenin was first detected after 1 day, and its synthesis was found to increase dramatically until the thirteenth day of hormone treatment. This implies that there is a coincidence between appearance and extent of synthesis of vitellogenin and the abundance of vitellogenin mRNA in the cytoplasm, but there is evidence that during later phase of primary stimulation (day 3–13), the increase in synthesis of vitellogenin cannot be attributed anymore to a significant accumulation of vitellogenin mRNA.In male Xenopus, estrogen-induced synthesis of vitellogenin is no more detectable 41 days after hormone injection, and the concentration of vitellogenin mRNA was found to be <0.03% of the cytoplasmic poly(A)-containing RNA. Secondary stimulation by estrogen of these animals results in an at least 30 fold faster accumulation of vitellogenin mRNA in the cytoplasm within the initial 12 hr of hormone treatment. This may explain the faster appearance of vitellogenin in the blood plasma.     

Transport of different RNA species from the nucleus to the cytoplasm in Xenopus laevis neurula cells

Isolated cells from Xenopus laevis neurulae were labeled, and the RNAs extracted from their nuclear and soluble cytoplasmic fractions were analyzed on polyacrylamide gels. In the soluble cytoplasm, 4S RNA emerged very rapidly, and this was immediately followed by the emergence of poly(A)-containing RNA and 18S ribosomal RNA. In contrast, the emergence of 28S ribosomal RNA was delayed by about 2 hr. The size distribution of cytoplasmic poly(A)-containing RNA was much smaller as compared to that of nuclear poly(A)-containing RNA. These results indicate that the newly synthesized RNAs in Xenopus neurula cells are transported from the nucleus to the cytoplasm in a characteristic sequence.     

Axoplasmic transport of RNA containing a polyadenylic acid segment

The nature of the cytoplasmic RNA that appears to migrate along the optic path of the chick has been studied. A considerable proportion of retinally synthesized RNA contains a polyadenylic acid segment. A fraction of this presumptive messenger RNA moves distally to the optic tectum together with nonpolyadenylic acid-containing RNA. The poly(A)-containing and nonpoly(A)-containing RNA classes are transported in roughly the same proportions as their relative retinal cytoplasmic concentrations. The size of the poly(A) segments within the putative messenger RNA (mRNA) did not decrease with time. A proportion of nonmigrating mRNA in retina and optic tectum appeared to have considerable stability, as did transported mRNA.     

Characterization and messenger activity of poly(A)-containing RNA from Chlorella.

Poly(A)-containing RNA was isolated by cellulose column chromatography from total RNA extracted from Chlorella fusca var. vacuolata 211/8p. RNA retained by the column was identified as poly(A)-containing RNA because it contained ribonuclease-resistant tracts, 25 to 55 nucleotides in length, from which not less than 80% of base was found to be adenine after acid hydrolysis. The base composition of poly(A)-containing RNA differed from that of RNA (largely ribosomal) which did not adsorb to cellulose, having a higher adenine content and a lower guanine content. Poly(A)-containing RNA was polydisperse including molecules with mobilities from 10S to 40S with a mean of about 20S. In an in vitro system derived from wheat-germ, protein synthesis was stimulated by adding poly(A)-containing RNA from Chlorella. Optimum conditions were established in this system with respect to the amount of poly(A)-containing RNA added and the concentration of KCl and Mg-2+. It is proposed that, in Chlorella, poly(A)-containing RNA includes cytoplasmic mRNA as has been shown for some other eucaryotic organisms.     

Rapid increase in poly(A) (+) mRNA content following inhibition of protein synthesis

When resting 3T6 cells undergo a serum-induced transition to the growing state, the cytoplasmic content of ribosomal, transfer and messenger RNA increase as the cells prepare for DNA synthesis. The normal linear increase in mRNA content occurs even when the production of ribosomes is blocked. In this paper we determine the effect of inhibiting protein synthesis on the increase in poly(A) (+) mRNA content. Resting cells were serum stimulated in the presence of cycloheximide or puromycin at levels which inhibit protein synthesis by greater than 95%. Cytoplasmic poly(A) (+) mRNA content was determined at various times thereafter. We found that mRNA content increased five to ten times more rapidly in drug treated cells than in control cells stimulated in the absence of inhibitors. mRNA content increased 50–70% by one hour, and 60–90% by two hours following stimulation in the presence of inhibitor, and remained more or less constant thereafter. In contrast, mRNA content increased linearly in control stimulated cultures and did not double until about 15 hours after stimulation. The rapid increase in mRNA content is most likely the result of inhibition of protein synthesis rather than a secondary effect of the drug since the same observations were made in growth stimulated cells if protein synthesis was blocked with either puromycin or cycloheximide. A similar effect was also observed with resting 3T6, exponentially growing 3T6 and growing HeLa cells following exposure to cycloheximide, although the magnitude of the increase was less than that observed with growth stimulated cells. Puromycin had negligible effect on mRNA content in resting or exponentially growing cells. The rapid increase in cytoplasmic poly(A) (+) mRNA content was not due to rapid unbalanced export of nuclear poly(A) (+) RNA into the cytoplasm since there was no decrease in nuclear poly(A) content following serum stimulation in the presence of cycloheximide.     

Essential factors in the kinetic analysis of RNA synthesis in HeLa cells

Further evidence of mRNA in HeLa cells with a half-life two hours or less is given. A kinetic model of RNA synthesis in HeLa cells is described in which equilibration of label occurs first into the acid soluble pool (evidence is given that this pool feeds RNA synthesis) and thence in nuclear and cytoplasmic molecules. The measured accumulation of label in nuclear and cytoplasmic poly(A) is examined with the model and parameters were found which are consistent with the quantitative transfer of nuclear poly(A) to the cytoplasm. The strengths and limitations of the model are discussed.     

Comparison of cytoplasmic and nuclear poly(A)-containing RNA sequences in Xenopus liver cells.

Poly(A)-containing RNAs from cytoplasm and nuclei of adult Xenopus liver cells are compared. After denaturation of the RNA by dimethysulfoxide the average molecule of nuclear poly(A)-containing RNA has a sedimentation value of 28 S whereas the cytoplasmic poly(A)-containing RNA sediments slightly ahead of 18 S. To compare the complexity of cytoplasmic and nuclear poly(A)-containing RNA, complementary DNA (cDNA) transcribed on either cytoplasmic or nuclear RNA is hybridized to the RNA used as a template. The hybridization kinetics suggest a higher complexity of the nuclear RNA compared to the cytoplasmic fraction. Direct evidence of a higher complexity of nuclear poly(A)-containing RNA is shown by the fact that 30% of the nuclear cDNA fails to hybridize with cytoplasmic poly(A)-containing RNA. An attempt to isolate a specific probe for this nucleus-restricted poly(A)-containing RNA reveals that more than 10(4) different nuclear RNA sequences adjacent to the poly(A) do not get into the cytoplasm. We conclude that a poly(A) on a nuclear RNA does not ensure the transport of the adjacent sequence to the cytoplasm.     

Biochemical studies of mammalian oogenesis: kinetics of accumulation of total and poly(A)-containing RNA during growth of the mouse oocyte

Kinetics of accumulation of total and poly(A)-containing RNA have been measured during growth of the mouse oocyte. Total RNA from oocytes isolated at discrete stages of growth was determined by two independent microassays. The full-grown oocyte contained about 0.60 ng of RNA. Kinetics of accumulation of total RNA with respect to oocyte volume were biphasic. Small, growing oocytes (about 30 pl) contained about 0.20 ng of RNA/oocyte. The amount of RNA increased in a quasi-linear fashion until oocyte volume was about 160 pl, at which point there was about 0.57 ng of RNA/oocyte. Thus oocytes about 65% of their final volume had accumulated about 95% of the total amount of RNA present in the fully-grown oocyte. The relative amount of poly (A)-containing RNA in oocytes of various size was determined by in situ hybridization of [3H] poly (U) to ovarian sections from juvenile mice of known age, followed by autoradiography. The kinetics of accumulation of poly (A)-containing RNA were similar to those of total RNA; oocytes about 70% of their final volume had accumulated about 95% of the amount of poly (A)-containing RNA present in the fully-grown oocyte. The poly(A)-containing RNA resided predominantly in the cytoplasm and no obvious cytoplasmic localization was observed. Kinetics of accumulation of total RNA, which is mainly ribosomal, and poly (A)-containing RNA were consistent with levels of RNA polymerases I and II measured by others during oocyte growth (Moore and Lintern-Moore, '78). The number of ribosomes that could be made from the amount of rRNA present at various stages of growth was compared to the actual number of ribosomes calculated from a published morphometric study (Garcia et al., '79). Kinetic differences in accumulation between the theoretical and actual number of ribosomes suggested oocyte ribosomes are recruited into cytoplasmic lattice structures. These structures accumulate during oocyte growth and have been postulated to be a ribosomal storage form. In addition, the results from this study are compared to results derived from lower species.     

Polyribosome formation and poly(A)-containing RNA in embryos of the sand dollar,Dendraster excentricus

Summary The pattern of appearance of ribosomes, newly synthesized mRNA, and poly(A)-containing mRNA in polyribosomes has been examined in sand dollar embryos. From early blastula until shortly before hatching small polyribosomes engaged in histone synthesis predominate. At the time of hatching, when the rate of cell increase is maximal, the proportion of poly(A)-containing RNA in polyribosomes is low. After hatching a new class of large polyribosomes appears and the amount of poly(A)-containing polyribosomal RNA increases. Cordycepin, an inhibitor of RNA adenylylation, prevents the appearance of the large polyribosomes after hatching as well as the increase in poly(A)-containing polyribosomal RNA.     

Polyadenylic acid-containing of rna in unfertilized and fertilized eggs of the rabbit.

Molecular hybridization between ³H-polyuridylic acid and unlabeled RNA prepared from unfertilized rabbit eggs and 10-h postfertilization stage rabbit embryos has been used to measure the amount and subcellular localization of adenylated maternal RNA. The results reported indicate that there is poly (A)-containing RNA (putative messenger RNA) in unfertilized rabbit eggs. The amount of poly (A) in the RNA in rabbit eggs does not increase immediately after fertilization and is located primarily in the ribosomal fraction of the cell. The rate of protein synthesis in fertilized eggs is insensitive to α-amanitin at concentrations which inhibit RNA synthesis. These results suggest that maternal mRNA makes an important contribution to protein synthesis in early stages of cleavage in the rabbit embryo.     

A small nuclear precursor of messenger RNA in the cellular slime mold Dictyostelium discoideum

Previous work (Firtel et al., 1972) showed that messenger RNA from the cellular slime mold Dictyostelium discoideum, like that from mammalian cells, contains a sequence of about 100 adenylic acid residues at the 3′ end. We show here that Dictyostelium nuclei, labeled under a variety of conditions, do not contain material analogous to the large nuclear heterogeneous RNA found in mammalian cells. Rather, the majority of pulse-labeled nuclear RNA that is not a precursor of ribosomal RNA does contain at least one sequence of polyadenylic acid; this RNA, with an average molecular weight of 500,000, appears to be only 20% larger than cytoplasmic messenger RNA.Pulse-labeling experiments show that the nuclear poly(A)-containing RNA is a material precursor of messenger RNA. Whereas previous work showed that over 90% of messenger RNA sequences are transcribed from non-reiterated DNA, we show here that about 25% of nuclear poly (A)-containing RNA is transcribed from reiterated DNA sequences and only 75% from single-copy DNA. We present evidence that a large fraction of the nuclear poly(A)-containing RNA contains, at the 5′ end, a sequence of about 300 nucleotides that is transcribed from repetitive DNA, and which is lost before transport of messenger RNA into the cytoplasm.Based on these and other results, we present a model of arrangement of repetitive and single-copy DNA sequences in the Dictyostelium chromosome.     

Rates of synthesis of major classes of RNA in Drosophila embryos.

We have been successful in labeling to high specific activity (3 × 10⁵ dpm/μg) the RNA synthesized by large numbers of Drosophila embryos. Embryos of various developmental stages were rendered permeable with octane and labeled with [³H]uridine for 1 hr. At each stage the total dpm incorporated into RNA and the specific activity of the UTP pool were measured and used to calculate the absolute rate of RNA synthesis per embryo. This rate increases during embryonic development, from 1 pmole UTP/hr at 2 hr after oviposition to 6 pmoles UTP/hr at 15 hr. The rates of synthesis of nuclear and cytoplasmic poly(A)^? and poly(A)⁺ RNAs were determined by analyzing the fractionated RNAs from each stage by sucrose gradient sedimentation. There is a significant activation of nuclear RNA synthesis at the blastoderm stage (approximately 2 hr after oviposition). After blastoderm, the rates of synthesis of nuclear and cytoplasmic poly(A)^? and poly(A)⁺ RNA per embryo increase continuously; the rate of synthesis of each of these classes per nucleus, however, remains fairly constant. After making corrections for turnover during the labeling period, we find that the rates of synthesis of the major classes of RNA per nucleus at the gastrula stage are: cytoplasmic poly(A)⁺ RNA, 0.06 fg/nucleus-min; hnRNA, 0.86 fg/nucleus-min; and ribosomal RNA, 0.46 fg/nucleus-min. These rates are compared to rates of RNA synthesis in sea urchin embryos.     

Kinetics of synthesis of cytoplasmic messenger-like RNA not associated with ribosomes in HeLa cells

The turn-over of cytoplasmic messenger-like RNA not associated with polyribosomes as well as that of polyribosomal mRNA was investigated by labelling with [3H]uridine in conditions of arrested ribosomal RNA and mitochondrial RNA synthesis. The synthesis of ribosomal RNA was inhibited with toyokamycin and that of mitochondrial RNA with ethidium bromide. In both accumulation kinetics and actinomycin-D-chase experiments, cytoplasmic messenger-like ribonucleoprotein particles and polyribosomes were fractionated by buoyant density centrifugation in CsCl gradients. The half-life of free m1RNA was found to be of 1--2 h whereas the bulk of polyribosomal mRNA was stable over the time period considered (up to 8 h) but with a minor short-lived component. Purification of RNA from polyribosomes labelled under the same conditions and fractionation of it into polyadenylated and non-polyadenylated fractions showed that this short-lived minor component of half-life less than 1 h is non-polyadenylated.     

Cellular DNA replication is independent of the synthesis or accumulation of ribosomal RNA

We have used an antibody against RNA polymerase I to investigate the role of rRNA synthesis and/or accumulation in the control of cell proliferation. The antibody was microinjected directly into the nuclei of quiescent Swiss 3T3 cells that were subsequently stimulated with serum. Under the experimental conditions used, the microinjection of the antibody against RNA polymerase I (RNA pol I) caused a 50–70% decrease in nucleolar RNA synthesis that lasted for at least 17 h, a >90% inhibition in the accumulation of nucleolar RNA, and a 70% inhibition in the accumulation of total cellular RNA. A control IgG, similarly microinjected into Swiss 3T3 cells had no inhibitory effect on either the synthesis or accumulation of nucleolar and cellular RNA. Despite the dramatic effect on the synthesis and accumulation of ribosomal RNA (rRNA) the antibody against RNA pol I was totally ineffective in inhibiting the entry into S phase of serum-stimulated Swiss 3T3 cells. Cells depleted of cellular RNA by metaphase arrest also entered S phase with subnormal amounts of cellular RNA. The results of these experiments clearly indicate that a normal rate of nucleolar RNA synthesis, and a normal rate of accumulation of total cellular RNA are not a prerequisite for the entry of cells into S phase.