Increase In Poly Research Articles

Metabolism of poly(A) sequences during 3T3 cell growth regulation

The metabolism and the intracellular distribution of RNA-associated poly(A) sequences from contact-inhibited and serum-induced proliferating 3T3 cells were examined by hybridization with [ 3H]poly(U). Evidence is presented for the growth-specific changes in the ‘steady-state’ populations of poly(A) sequences as determined by this technique. The results indicate that reinitation of growth in 3T3 cells is accompanied by characteristic changes in total poly(A) content and the size of the ‘steady-state’ poly(A) sequence. The serum-induced state is characterized by: (1) a higher content of RNA-associated poly(A) sequences; (2) the redistribution of poly(A)-containing RNA species from sub-ribosomal to polysomal fraction; and (3) an overall shortening of the RNA-associated poly(A) sequences. The size distribution of newly synthesized poly(A) in both resting and serum-induced cells are similar even though there is a marked reduction in the size of steady-state poly(A) sequences of the RNA from growing cells. The suggestion that there is an enhanced poly(A) processing in serum-induced cells is further substantiated by an increase in poly(A) hydrolyzing activity in growing cells. These findings of the growth-specific cellular pattern of poly(A) sequences in RNA suggest a possible physiological role for the modulation of poly(A) size in the regulation of mRNA expression with respect to cell growth.

Protection by picolinamide, a novel inhibitor of poly (ADP-ribose) synthetase, against both streptozotocin-induced depression of proinsulin synthesis and reduction of NAD content in pancreatic islets

Picolinamide, 2-pyridinecarboxylic acid amide, was found to be a strong inhibitor of poly (ADP-ribose) synthetase of nuclei from rat pancreatic islet cells. Another experiment using isolated pancreatic islets of rats showed that picolinamide protects against streptozotocin-induced depression of proinsulin synthesis as well as against streptozotocin-induced reduction of NAD content. The protection by picolinamide against the NAD depression was considered to be due to the blockage of an increased degradation of NAD mediated by a streptozotocin-induced increase in poly (ADP-ribose) synthetase activity. A possible mechanism of streptozotocin diabetes and its prevention is discussed.

Effect of polyamines on ADP-ribosylation by chick-embryo-liver nuclei

Effects of polyamines on poly(ADP-ribose) formation and DNA synthesis in the chick-embryo-liver nuclei were investigated. When 14-day chick-embryo-liver nuclei were incubated with [ 3H]NAD in the presence of 1 mM spermine, 2.5 mM spermidine, or 3.5 mM putrescine, a 9-fold increase in poly(ADP-ribose) formation was observed. Nuclei treated with nuclease showed high poly(ADP-ribose) synthetase activity as spermine-treated nuclei. However, no further increase in the polymer formation by polyamines was detected in the nuclease-treated nuclei. We found that an increase in the polymer formation by spermine was the result of an increase in both chain length and chain number of the polymer at 2.3- and 6-fold, respectively. The major ADP-ribosylated proteins were determined as two non-histone proteins of M r 130 000 and 70 000. The experiment of DNA synthesis with nuclei ADP-ribosylated in the presence of spermine showed a 7-fold increase in [ 3H]dTMP incorporation into the acid-insoluble fraction. A similar stimulation was also found with nuclei treated with other polyamines, spermidine and putrescine, in the presence of NAD. These results indicate that DNA synthesis in growing tissues containing polyamines at high levels, such as is the case with tumors and the fetus, is stimulated by polyamine-mediated ADP-ribosylation of the nuclear proteins.

The diagnostic value of spectrophotometric analysis of the cerebrospinal fluid in cerebral hematomas

On a clinical basis it is sometimes impossible to distinguish between small intracerebral hematomas and reversible ischemic strokes (RIND). Analysis of the cerebrospinal fluid (CSF) has been used in this context for several years and is still of importance even since the introduction of computerized tomography (CT) of the brain. CSF has been examined in 23 patients with intracerebral hematomas. Typical cell changes with an increase of poly- and mononuclear white cells are seen within the first days after the stroke, whereas clearly pathological spectrophotometric changes are not seen until about one week after the stroke. None of the investigated patients had a significant increase of the absorbance values at the wavelengths 406 and 415 nm (met- and oxyhemoglobin) until 5 days after the stroke.

Poly(ADP-ribose) levels in carcinogen-treated cells

Several reports indicate that intracellular levels of NAD are rapidly decreased by DNA-damaging agents such as strep-tozotocin1–7, neocarzinostatin8,9 and ionising radiation8–11. Similarly, evidence obtained from studies in isolated nuclei and permeabilised cells indicates that DNA damage caused by several alkylating chemicals12–15, X rays16,17 and DNA-hydrolysing enzymes18,19 elicit a considerable increase in the activity of poly(ADP-ribose) polymerase, a chromosomal enzyme which uses NAD as a substrate in the formation of poly(ADP-ribose) in histones and other nuclear proteins20,21. Thus, it has been suggested that DNA-damaging agents lower NAD levels by causing an increase in poly(ADP-ribose) synthesis9,12,13. As DNA damage initiates DNA repair mechanisms, it has also been argued that poly(ADP-ribose) is involved in DNA repair8,12,14,17,22,23. Such possibilities would be strongly supported if an actual increase in the intracellular levels of poly(ADP-ribose) could be shown to occur after induction of DNA damage. We now present data obtained using a new technique which has enabled us to measure the levels of poly(ADP-ribose) in vivo in SV40 virus-transformed 3T3 cells (SVT2). We show that treatment of these cells with the powerful mutagen N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) produces, concomitantly with a decrease in NAD levels, a dramatic increase in the intracellular levels of poly (ADP-ribose).

Turnover and processing of poly(A) in full-grown oocytes and during progesterone-induced oocyte maturation in Xenopus laevis

Changes in the amount and the size of poly(A) sequences during progesterone-induced oocyte maturation were determined by [3H]poly(U) hybridization and by labeling with microinjected [α-32P]ATP and [3H]ATP. Following exposure to progesterone total poly(A) length increases by 10–20 nucleotides, largely due to extension of preexisting poly(A) although there is an increase of 5 to 10% in the number of poly(A) sequences. Immediately following germinal vesicle breakdown (GVBD), poly(A) is degraded with first-order kinetics (t1/2 = 5.7 hr) until about 10 hr after GVBD. This degradation results in the loss of 35–50% of the poly(A) sequences but the size distribution of the remaining poly(A) is unchanged. These changes occur only in oocytes which undergo GVBD, but all the changes in poly(A) content also occur in oocytes enucleated before exposure to progesterone. The poly(A) of ovulated eggs is similar in amount and size to that ofin vitro matured oocytes. The incorporation of [3H]ATP into poly(A) confirms that most of the poly(A) synthesis in both maturing and control oocytes is due to chain extension and turnover rather than tode novo synthesis. The amount of newly synthesized poly(A) is sufficient to allow for the net increase in larger poly(A) sequences by chain extension but not byde novo synthesis. Newly synthesized poly(A) has a size distribution similar to that of steady-state poly(A), and no precursor-like32P-poly(A) molecules are detected. The lengths of poly(A) sequences labeled with [3H]ATP, determined by [3H]AMP:[3H]adenosine ratios, are smaller than those indicated by their electrophoretic mobilities in both control and maturing oocytes. It is argued that the size and stability of oocyte poly(A) sequences are regulated by balanced synthetic and degradative activities of cytoplasmic poly(A) polymerase which result in turnover of the 3′-terminal portion of the poly(A). During progesterone-induced maturation there is a relative increase in poly(A) synthesis until the time of GVBD, when a different degradative activity results in destruction of complete poly(A) sequences. These changes must be cytoplasmically programmed because they occur in the absence of the nucleus.

Stimulation of Polypeptide Polymerization in Escherichia coli Ribosomes by Modification of Ribosomal Sulfhydryl Groups with N‐Bromosuccinimide

Modification of Escherichia coli ribosomes with N‐bromosuccinimide (SucNBr) (molar ratio of reagent to ribosome equal to 10) is accompanied by 100–200% increase in poly(U)‐directed polyphenylalanine synthesis, while no change in peptidyl transferase activity (‘fragment reaction’) was observed. The magnesium dependence of the modified ribosomes in polyphenylalanine synthesis (optimum at 11 mM) is different from that of untreated ones (optimum at 9 mM). Hybridization of SucNBr‐treated and untreated subunits showed that stimulation is produced by modification of the 50‐S subunit. Stimulation is the result of an increase in the rate of polyphenylalanine synthesis without activation of inactive ribosomes present in the preparation. Treatment with SucNBr is also accompanied by the appearance of a peak intermediate between 70‐S ribosomes and 50‐S subunits in sucrose gradient centrifugation. At 7 mM magnesium the intermediate peak appears closer to 50‐S subunits than at 10 mM, disappearing at 20 mM magnesium or after treatment with glutaraldehyde with a concomitant increase in 70‐S ribosomes. These results suggest that the observed intermediate peak is produced by a rapid association/dissociation equilibrium between modified 50‐S and 30‐S subunits with decreased affinity for each other. Modification with 5,5′‐dithio‐bis(2‐nitrobenzoate) (Nbs2) prior to the Suc NBr treatment prevents the stimulation of polyphenylalanine synthesis. When the SucNBr‐stimulated ribosomes are incubated with 2‐mercaptoethanol or dithiothreitol the observed stimulation disappears. These results indicate that stimulation is produced by modification of ribosomal sulfhydryl groups by SucNBr. The stimulatory modification with SucNBr decreases by 5–6 the number of –SH groups per ribosome reactive with Nbs2 while no significant change in the total number of tryptophan residues was observed (tryptophan residues are the next most likely reactive groups in the ribosome).

Polyadenylation of RNA in immature oocytes and early cleavage of Urechis caupo

The poly(A) content of RNA extracted from four stages of immature oocytes, mature oocytes, and cleavage embryos through the eight-cell stage was determined by hybridization with [ 3H]-poly(U). During oogenesis the poly(A) content per cell gradually increases from 0.007 pg of poly(A)/cell in the 10- to 39-μm oocytes to 0.20 pg of poly(A)/cell in the 125-μm mature oocytes. After fertilization there is an additional increase to approximately 1.1 pg of poly(A)/embryo at the two-cell stage which is followed by a slight decline between the two- and eight-cell stages. Most of the increase in poly(A) after fertilization occurs in a 45-min interval coincident with the appearance of the polar bodies. The size distribution of the poly(A) in RNA from the different stages of development was determined based on the length of RNase-resistant poly(U) obtained from poly(U)-poly(A) hybrids. The size distribution of the poly(A) sequences is constant through each stage of development which indicates that the increase in the poly(A) content of the cells is the result of polyadenylation of new RNA sequences.

Synthesis of DNA and poly(adenosine diphosphate ribose) in normal and chronic lymphocytic leukemia lymphocytes.

Peripheral blood lymphocytes were isolated from 9 patients with chronic lymphocytic leukemia (CLL) and 12 normal control donors. The cells were assayed for synthesis of DNA and poly-(adenosine diphosphate ribose) (poly[ADPR]) immediately after isolation and on successive days following their treatment with phytohemagglutinin (PHA). Two different techniques were used to measure DNA synthesis. In the standard technique, DNA synthesis was measured by incubating intact cells with [(3)H]deoxythymidine. In the new technique, the lymphocytes were first rendered permeable to nucleotides, then DNA synthesis was measured by incubating them with [(3)H]deoxythymidine triphosphate in the presence of deoxyATP, deoxyGTP, deoxyCTP, ATP, and Mg(++). Both assays showed the anticipated rise in DNA synthesis after PHA stimulation of normal cells. PHA-stimulated lymphocytes from patients with CLL demonstrated low levels of DNA synthesis in both assay systems. The initial levels of poly(ADPR) synthesis were greater in CLL lymphocytes than in normal cells. Studies with a T-cell-depleted population of normal cells showed the same activity for poly(ADPR) synthesis that was demonstrated by the original population of normal cells. PHA stimulation produced an increase in poly(ADPR) synthesis in both the normal and CLL cells. The increase in poly(ADPR) synthesis in normal cells was coincident with the increase in DNA synthesis. The increase in poly(ADPR) synthesis in the CLL cells was dissociated from the delayed and diminished increase in DNA synthesis. Thus, CLL cells have higher than normal initial levels of poly(ADPR) synthesis. Poly(ADPR) synthesis is dissociated from DNA synthesis in CLL cells whereas it varies directly with DNA synthesis in normal lymphocytes.

Relation of poly(adenosine diphosphoribose) synthesis to DNA synthesis and cell growth

A permeable cell technique has been used to measure the synthesis of DNA and poly(adenosine diphosphoribose) (poly(ADPR)) in mouse L cells subjected to different perturbations of cell growth. Cells leaving log phase growth and entering plateau phase, showed a decrease in DNA synthesis and an associated increase in intrinsic poly(ADPR) synthesis. In contrast to the variations in intrinsic poly(ADPR) synthesis, the total poly(ADPR) synthesis activity, measured in the presence of added DNAase, remained relatively constant during the fluctuations in cell growth status. Cells subjected to acute glucose deficiency also demonstrated a decrease in DNA synthesis and an associated increase in intrinsic poly(ADPR) synthesis. Similarly, cells infected with vaccinia virus demonstrated an abrupt cessation of DNA synthesis associated with an increase in poly(ADPR) synthesis. Treatment of cells with cytosine arabinoside, inhibited cellular DNA synthesis. This was also associated with an increase in the intrinsic activity of poly(ADPR) synthesis. However, in this case, the increase in poly(ADPR) synthesis was associated with an increase in activity of the DNA synthesis complex, despite the overall inhibition of cell DNA synthesis. These studies demonstrate, that in mouse L cells, suppression of DNA synthesis by multiple different physiologic mechanisms is always associated with an increase in intrinsic activity of poly(ADPR) synthesis.

Poly(adenosine dephosphate ribose) metabolism and regulation of myocardial cell growth by oxygen.

Control of the rate of cardiac cell division by oxygen occurs most probably by altering the redox state of a control substance, e.g. NAD(+)right harpoon over left harpoonNADH. NAD(+) (and not NADH) forms poly(ADP-ribose), an inhibitor of DNA synthesis, in a reaction catalysed by poly(ADP-ribose) polymerase. Lower partial pressure of oxygen, which increases the rate of division, would shift NAD(+)-->NADH, decrease poly(ADP-ribose) synthesis, and increase DNA synthesis. Chick-embryo heart cells grown in culture in 20% O(2) (in which they divide more slowly than in 5% O(2)) did exhibit greater poly(ADP-ribose) polymerase activity (+83%, P<0.001) than when grown in 5% O(2). Reaction product was identified as poly(ADP-ribose) by its insensitivity to deoxyribonuclease, ribonuclease, NAD glycohydrolase, Pronase, trypsin and micrococcal nuclease, and by its complete digestion with snake-venom phosphodiesterase to phosphoribosyl-AMP and AMP. Isolation of these digestion products by Dowex 1 (formate form) column chromatography and paper chromatography allowed calculation of average poly(ADP-ribose) chain length, which was 15-26% greater in 20% than in 5% O(2). Thus in 20% O(2) the increase in poly(ADP-ribose) formation results from chain elongation. Formation of new chains also occurs, probably to an even greater degree than chain elongation. Additionally, poly(ADP-ribose) polymerase has very different K(m) and V(max.) values and pH optima in 20% and 5% O(2). These data suggest that poly(ADP-ribose) metabolism participates in the regulation of heart-cell division by O(2), probably by several different mechanisms.

Studies of poly(A+-RNA in mouse hepatoma and cortisone-stimulated rat liver

The content of poly(A)-containing RNA in subcellar fractions has been investigated both in cortisone-treated rat liver and experimental hepatoma cells. The fractions included nuclei, cytoplasm, mitochondria, free and membrane-bound polyribosomes. 1) In both cases of genome activation (cortisone induction and hepatoma cells) an increase in poly(A) content of all subcellular fractions except free polyribosomes was observed. 2) Cortisone was found to induce elongation of poly(A) segments detected in both nuclei and cytoplasm. 3) An increase in the poly(A) block size also was found to be stimulated in nuclei and cytoplasm of hepatoma cells. 4) The observed elongation in poly(A) length occurred against the background of an increase of the population of of poly(A)-RNA's.

Effect of vitamin A nutritional status on the ribonucleic acids of liver, intestinal mucosa and testes of rats.

1. Vitamin A deficiency led to an increase in the oligonucleotide fraction of testes and intestinal mucosa of rats at the expense of high-molecular-weight RNA and 4S RNA, but no such changes were observed in the liver. Retinyl acetate supplementation reversed these effects in both tissues, whereas retinoic acid supplementation was almost equally effective in the mucosa but virtually ineffective in the testes. The ribonuclease activities of all the tissues remained unaffected by the above treatments. 2. The effect of vitamin A deprivation on the acceptor activity of the tRNA of the testes and intestinal mucosa was more pronounced than on the liver tRNA. The testes and mucosal tRNA of the retinoic acid-supplemented rats showed significantly lower charging capacity as compared with the retinyl acetate-supplemented ones. Here also no significant effect was observed on the liver tRNA. 3. Vitamin A deficiency caused a decrease in the percentage of poly(A) in RNA of the mucosa and testes, but not in the liver RNA. The poly(A) contents of both tissues were brought to normal by retinyl acetate supplementation; treatment with retinoic acid led to an appreciable increase in poly(A) in the mucosa, but considerably less increase in poly(A) in the testes. 4. The incorporation of H332PO4 into the rRNA and tRNA of the testes was lowered by vitamin A deficiency, but no such effects was observed in the liver RNA.

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

AbstractWhen 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.

Site of action of iron in the induction of δ-aminolevulinate dehydratase in Neurospora crassa

Summary δ-Aminolevulinate (ALA) dehydratase, the second and rate limiting enzyme of the heme biosynthetic pathway in the mold Neurospora crassa is induced maximally in 30 min by the addition of iron to iron-deficient cultures. The induction of the enzyme is blocked by cycloheximide, protoporphyrin, 8-azaguanine and cordycepin. Iron also brings about an increase in poly(A)-containing RNA synthesis under conditions of induction. The iron dependent increase in poly(A)-containing RNA synthesis is blocked by protoporphyrin. It is suggested that at the time intervals examined, bulk of the messenger RNA synthesized in response to iron addition represents the messenger for ALA dehydratase.

Reovirus-specific enzyme(s) associated with subviral particles responds in vitro to polyribocytidylate to yield double-stranded polyribocytidylate-polyriboguanylate.

In reovirus-infected cells, virus-specific particles accumulate that have associated with them a polyribocytidylate [poly(C)]-dependent polymerase. This enzyme copies in vitro poly(C) to yield the double-stranded poly(C).polyriboguanylate [poly(G)]. The particles with poly(C)-dependent polymerase were heterogeneous in size, with most sedimenting from 300S to 550S. Exponential increase in these particles began at 23 h, and maximal amounts were present by 31 h, the time of onset of exponential growth of virus at 30 degrees C. Maximal amounts of particles with active transcriptase and replicase were present at 15 and 18 h after infection. Thereafter, there was a marked decrease in particles with active transcriptase and replicase until base line levels were reached at 31 h. Thus, the increase in poly(C)-responding particles occurred coincident with the decrease in particles with active transcriptase and replicase. The requirement for poly(C) as template was specific because no RNA was synthesized in vitro in response to any other homopolymer, including 2'-O-methyl-poly(C). Synthesis was optimal in the presence of Mn(2+) as the divalent cation, and no primer was necessary for synthesis. In contrast, the dinucleotide GpG markedly stimulated synthesis in the presence of 8 mM Mg(2+). The size of the poly(C).poly(G) synthesized in vitro was dependent on the size of the poly(C) used as template. This suggested that the whole template was copied into a complementary strand of similar size. The T(m) of the product was between 100 and 130 degrees C. Hydrolysis of the product labeled in [(32)P]GMP with alkali or RNase T2 yielded GMP as the only labeled mononucleotide. This does indicate that the synthesis of the poly(G) strand in vitro did not proceed by end addition to the poly(C) template, but proceeded on a separate strand.

The dynamics of maternal poly(A)-containing mRNA in fertilized sea urchin eggs

Cytoplasmic polyadenylated RNA with the characteristics of sequestered mRNA exists in the unfertilized sea urchin egg. Following egg activation, the amount of poly(A) doubles, but total RNA content stays constant. Chromatography of the RNA on poly(U)-Sepharose shows that the amount of RNA that bears a poly(A) tract increases slightly (approximately 20–30%) during the 2 hr after fertilization. When a cDNA transcript of the poly(A) + mRNA from 2 hr zygotes is reacted against poly(A) + RNA from either eggs or zygotes, the kinetics of reassociation of the two preparations seem identical; hence the RNA sequences bearing poly(A) are the same in eggs and zygotes. Measurement of the length of the poly(A) tract in eggs and zygotes shows an increase in number average length from about 45 bases to 60 bases. Measurement of tract length of poly(A) in two cell zygotes by adenosine/AMP ratios of radioactive RNA shows that the poly(A) tract of the zygote is solely accounted for by radioactive RNA, indicating extensive turnover of the poly(A). It in concluded that the poly(A) tract in these cells is subject to both lengthening and shortening, with the former predominating in this instance. The increase in poly(A) does not involve polyadenylation of different sequences, but is due to an increase in the number of polyadenylated sequences and the length of the poly(A) tracts that they bear.

Complete turnover of poly(A) on maternal mRNA of sea urchin embryos

Measurement of the incorporation of radioactive adenosine into precursor pools and into poly(A) of fertilized sea urchin eggs showed that the amount of adenosine incoporated into poly(A) after a 2 hr incubation approximated the total poly(A) content of the embryos. This was observed whether the incubation was begun at fertilization when the poly(A) content is tripling or at 2.5 hr after fertilization when the poly(A) levels are not changing, and thus indicates that poly(A) turns over continually and completely. The turnover appears to take place on polysomal mRNA, since after either 10 or 120 min of incubation, 75% of the 3H-adenosine incorporated into poly(A) is on polysomes. Poly(A) lengths before and after fertilization are not significantly different, indicating that the increase in poly(A) content reflects the addition of poly(A) sequences onto mRNA molecules which previously contained no poly(A) sequences or only short poly(A) sequences. Both the new as well as the preexisting poly(A) tracts must turn over to produce the incorporation we observe. The radioactive poly(A) tracts measured by alkaline release of adenosine begin as short sequences and gradually extend their lengths until they have reached a size consistent with the idea that the poly(A) sequences have become fully radioactive. This labeling pattern shows that the poly(A) is turning over from the 3′ end terminal probably by a shortening and lengthening mechanism.

Polyadenylation of maternal and newly-synthesized RNA during starfish oocyte maturation

Poly(A)-containing RNA molecules have been characterized during 1-methyladenine (1-MeAd)-induced meiosis in Asterias forbesi oocytes by the formation of ribonuclease-resistant complexes with [ 3H]poly(U). The cytoplasm of primary oocytes arrested in late prophase I contains about 0.33 ng poly(A) per μg RNA whereas the poly(A) content rises to about 1.59 ng/μg RNA after the completion of meiosis. Polyadenylation begins shortly after 1-MeAd addition and continues during and for about 60 min after the completion of meiosis. The characteristics of poly(A) containing RNA molecules and their poly(A) sequences were compared in late prophase I oocytes and ootids. In both developmental stages poly(A) sequences can be detected in the cytoplasm in association with heterodisperse RNA of similar size distribution. Ootids contain a higher proportion of poly(A) in their ribosomal fraction than oocytes. Ootid poly(A) segments are also larger and more heterogeneous than those of oocytes. The accumulation of larger poly(A) segments during meiosis is sensitive to actinomycin D. The inability of ethidium bromide to markedly suppress polyadenylation and the lack of a substantial increase in poly(A)-containing RNA in the mitochondrial fraction suggests that mitochondrial transcripts are not extensively employed as primers. It is concluded that metabolic activation of starfish oocytes is accompanied by the polyadenylation of newly-transcribed RNA rather than the extensive elongation of 3′ terminus of preexisting RNA.

Differential subnuclear distribution of polyadenylate-rich ribonuclei acid during induction of egg-yolk protein synthesis in male Xenopus liver by oestradiol-17 beta.

A 4-8-fold increase in the rate of hepatic nuclear RNA synthesis occurred within 11 h after a single injection of oestradiol-17 beta to male Xenopus to induce egg-yolk protein synthesis. 2. By using a gentle procedure for fractionating nuclei into their major structurally different components [J. R. Tata& B. Baker (1974) Exp. Cell Res. 83. 111-124], it was found that the hormone-induced increase in the total amount of newly made RNA was associated with a 2-10-fold increase in the poly(A) content of nuclear RNA. 3. When the poly (A) content of nuclear RNA was determined by hybridization to poly[3H](U) or specific binding to oligo(dT)-cellulose, most of the increase (10-fold) in poly (A) content of newly synthesized RNA was associated with the euchromatin fractions, whereas the increase was less marked in the other subnuclear fractions. 4. Resolution of nuclear RNA into poly (A)-poor and poly(A)-rich RNA species by chromatography on oligo(dT)-cellulose, followed by polyacrylamide-gel electrophoresis with sodium dodecyl sulphate or in the pressence of 99% formamide, revealed that the hormone caused a preferential enhancement of high-molecular-weight (25S-60S) poly (A)-rich HnRNA (heterogeneous nuclear RNA,) much of which was associated with euchromatin and not with the nuclear sap. 5. Induction of vitellogenin in male frogs was in particular characterized by the appearance of a high-molecular-weight polyadenylated component exhibiting a peak at 35-36S, i.e. a molecular weight of approx. 2.05x10(6)+/-0.15x10(6). Although there is no evidence as yet that such a polyadenylated high-molecular-weight nuclear RNA species contains sequences corresponding to vitellogenin mRNA, it is possible that a high proportion of the most stable form of the putative nuclear precursor to vitellogenin mRNA induced by oestrogen in male Xenopus liver may be only marginally bigger than the cytoplasmic mRNA, and may at any one time be predominantly associated with the euchromatin fraction.