Increase In Poly Research Articles

Modulation of poly(A)polymerase by cAMP in Cicer arietinum

cAMP stimulated the activity of poly(A +)polymerase in chick pea epicotyls with a parallel increase in poly(A +)RNA levels. Experiments with inhibitors of RNA and protein synthesis and fractionation of partially purified enzyme on SDS-PAGE suggested a de novo synthesis of the enzyme.

A mutation impairing alginate production increased accumulation of poly-β-hydroxybutyrate in Azotobacter vinelandii

An algK::Tn 5mutation impairing alginate production in Azotobacter vinelandiiled to a 50% increase in poly--hydroxy-butyrate production to 75% of the dry cell wt.

The expression of poly(ADP-ribose) polymerase during differentiation-linked DNA replication reveals that it is a component of the multiprotein DNA replication complex.

3T3-L1 preadipocytes have been shown to exhibit a transient increase in poly(ADP-ribose) polymerase (PARP) protein and activity, as well as an association of PARP with DNA polymerase alpha, within 12-24 h of exposure to inducers of differentiation, whereas 3T3-L1 cells expressing PARP antisense RNA showed no increase in PARP and are unable to complete the round of DNA replication required for differentiation into adipocytes. The role of PARP in differentiation-linked DNA replication has now been further clarified at both the cellular and enzymological levels. Flow cytometric analysis revealed that control 3T3-L1 cells progressed through one round of DNA replication prior to the onset of terminal differentiation, whereas cells expressing PARP antisense RNA were blocked at the G0/G1 phase of the cell cycle. Confocal microscope image analysis of control S phase cells demonstrated that PARP was localized within distinct intranuclear granular foci associated with DNA replication centers. On the basis of these results, purified replicative complexes from other cell types that had been characterized for their ability to catalyze viral DNA replication in vitro were analyzed for the presence of PARP. PARP exclusively copurified through a series of centrifugation and chromatography steps with core proteins of an 18-21S multiprotein replication complex (MRC) from human HeLa cells, as well as with the corresponding mouse MRC from FM3A cells. The MRC were shown to contain DNA polymerases alpha and delta, DNA primase, DNA helicase, DNA ligase, and topoisomerases I and II, as well as accessory proteins such as PCNA, RF-C, and RP-A. Finally, immunoblot analysis of MRCs from both cell types with monoclonal antibodies to poly (ADP-ribose) revealed the presence of approximately 15 poly(ADP-ribosyl)ated proteins, some of which were further confirmed to be DNA polymerase alpha, DNA topoisomerase I, and PCNA by immunoprecipitation experiments. These results suggest that PARP may play a regulatory role within the replicative apparatus as a molecular nick sensor controlling the progression of the replication fork or modulates component replicative enzymes or factors in the complex by directly associating with them or by catalyzing their poly(ADP-ribosyl)ation.

Nicotinamide Megadosing Increases Hepatic Poly(ADP-Ribose) Levels in Choline-Deficient Rats

Previous work in our laboratory has shown that dietary megadoses of nicotinamide, used in the prevention of diabetes, cause increases in hepatic poly(ADP-ribose). Poly(ADP-ribose) is synthesized from NAD+ by a nuclear enzyme, poly(ADP-ribose)poly-merase, which is activated by DNA strand breaks. The nicotinamide-induced increase in poly(ADP-ribose) could result from an increase in substrate, NAD+, or the induction of strand breaks in DNA. Strand breaks may result from the depletion of single carbon groups, through the excretion of methylated derivatives of nicotinamide. To differentiate between these mechanisms, a 3 ՠ3 factorial experiment was conducted in which rats were fed diets containing various supplements of choline bitartrate (0, 2, 20 g/kg diet) and nicotinamide (0, 1, 2 g/kg diet). At the conclusion of treatments, blood NAD+ and liver lipid, NAD+ and poly(ADP-ribose) levels were determined. Choline deficiency caused the characteristic accumulation of fat in the liver at all levels of nicotinamide. In choline deficient rats, nicotinamide supplements further increased liver lipid concentration. Blood and liver NAD+ concentrations were increased by nicotinamide supplementation, irrespective of choline status. In contrast, liver poly(ADP-ribose) levels were increased by nicotinamide supplementation only in choline deficient rats. These results show that nicotinamide-induced increases in poly(ADP-ribose) levels appear to be dependent on decreased methyl donor status and suggest that adequate choline status is important for preventing some deleterious effects of nicotinamide treatment.

Asparagine-Linked Glycosylation in Schizosaccharomyces pombe: Functional Conservation of the First Step in Oligosaccharide-Lipid Assembly

The gene gpt encoding uridine diphosphate N-acetyl-D-glucosamine:dolichol phosphate N-acetylglucosaminylphosphoryltransferase (L-G1PT) was isolated by screening a Schizosaccharomyces pombe genomic DNA library in λ phage under low-stringency hybridization using the Saccharomyces cerevisiae gene ALG7 as probe. Sequencing 2.4 kb of S. pombe DNA revealed a 1338-bp open reading frame (ORF) encoding a hydrophobic protein of 446 amino acids with a predicted molecular weight of 49,852. The S. pombe protein was 50% identical to the S. cerevisiae protein and 43% identical to the protein from Chinese hamster ovary (CHO) cells. Overexpression of the gpt gene in S. pombe cells increased resistance to tunicamycin 25-fold and increased the specific activity of the enzyme in isolated cell membranes 13-fold. This was accompanied by a 50-fold increase in poly(A)+ RNA hybridizing to the gpt probe. Northern analysis indicated a single 1.8-kb message is transcribed from the gpt gene. The gpt gene is essential for viability of S. pombe. Cells containing a disrupted ORF could be rescued by an expression plasmid containing either the intact S. pombe gpt ORF or the CHO L-G1PT cDNA. The S. pombe gpt gene was mapped to chromosome 2 near top1 and ade1.

Nuclear response of pancreatic islets to interleukin-1β

The mechanisms by which interleukin-1 (IL-1) exerts destructive action on the pancreatic islet β-cells remain elusive. Fragmentation of DNA leading to the activation of poly(ADP-ribose) synthetase was investigated in the present study, by assessing the nuclear response to cytokines in rat pancreatic islets. Nuclear fractions display Mg 2+-dependent poly(ADP-ribose) synthetase activity catalyzing the incorporation of [adenine-U- 14C]NAD, with K a and K m for Mg 2+ and NAD amounting to 0.86 mM and 0.43 mM, respectively. Exposure of the nuclear fraction to rIL-1β (10IU/ml) provoked DNA strand breaks and increased nuclear poly(ADP-ribose) synthetase activity (148.4%, P < 0.01). In intact islets, this nuclear response was observed after 18 h culture in medium containing rIL-1β, with a concomitant decrease in NAD (88.5%). Brief periods of pre-incubation (90 min) with rIL-1β were unable to induce any nuclear activity. Under these conditions, the presence of IFN-α (24 U/ml) and TNF (120 U/ml) was necessary to induce a response to rIL-lβ. Under the latter experimental conditions, a decrease in NAD content was also observed. The nuclear effects of IL-1β were modified by nicotinamide (10 mM), an inhibitor of poly(ADP-ribose) synthetase. It is thus conceivable that an increase in poly(ADP-ribose) synthetase activity together with DNA break is implicated in the beta cytotoxic effect of interleukin-1β.

The 3'-untranslated regions of c-mos and cyclin mRNAs stimulate translation by regulating cytoplasmic polyadenylation.

Early in the development of many animals, before transcription begins, any change in the pattern of protein synthesis is attributable to a change in the translational activity or stability of an mRNA in the egg. As a result, translational control is critical for a variety of developmental decisions, including axis formation in Drosophila and sex determination in Caenorhabditis elegans. Previous work demonstrated that increases in poly(A) length can activate translation, whereas removal of poly(A) can prevent it. In this report we focus on the control of c-mos and cyclin A1, B1, and B2 mRNAs during meiotic maturation and after fertilization of frog eggs. We show that addition and removal of poly(A) from these mRNAs is extensively regulated: The time at which each mRNA receives or loses poly(A), as well as the number of adenosines it gains or loses, differ substantially. Signals in the 3'-untranslated region (UTR) of each mRNA are sufficient to reconstitute both the temporal and quantitative control of poly(A) addition: Chimeric mRNAs in which a luciferase-coding region is joined to the 3' UTRs of cyclin A1, cyclin B1, or c-mos mRNA, receive poly(A) of the same length and at the same time as do the endogenous mRNAs. Moreover, each 3' UTR also regulates translation of the chimeric mRNAs, determining when and how much translation of the luciferase reporter is stimulated during maturation. The magnitude of stimulation in luciferase activity varies from 5- to 100-fold, depending on the 3' UTR. Translational stimulation by each 3' UTR requires poly(A) lengthening, as it is prevented by mutations that prevent that process. These results suggest that the 3' UTRs of cyclin and c-mos mRNAs control not only whether or not an mRNA is turned on during maturation, but when that activation occurs and to what extent. Translational control of c-mos mRNA, which may be achieved through regulation of poly(A) length, may be critical in the activation of maturation, and in the onset of cleavage divisions. Our findings, as well as those of others, suggest that even quite complex patterns of translational activation in the early embryo can be attained through the differential control of a common mechanism.

Stimulation of insulin receptor tyrosine kinase activity by an amino terminal sequence of human growth hormone

The in vitro effect of a hypoglycaemic fragment of human growth hormone containing the sequence H 2N-LeuSerArgLeuPheAsu 11AsnAlaCOOH (Asu 11hGH 613) on tyrosine kinase of rat hepatic insulin receptors was examined. Insulin receptor kinase activity was evaluated using the synthetic polypeptide poly(GluTyr) (4:1) as substrate. The hypoglycaemic Asu 11hGH 613 appeared to enhance the phosphorylation of the exogenous substrate by the stimulation of insulin receptor kinase activity. The levels of poly(GluTyr)(4:1) phosphorylation were significantly higher in the insulin receptor preparations incubated in the presence of the Asu 11hGH 6 613 peptide. A dose dependent stimulation of receptor kinase activity was observed and this stimulatory effect was found to be further enhanced by the addition of increasing concentrations of insulin. In hepatic extracts depleted of insulin receptor, no stimulation of kinase activity by the Asu 11hGH 613 was observed. From these data, it is concluded that the increase of poly (GluTyr)(4:1) phosphorylation is the result of the interaction between the Asu 11hGH 613 and the hepatic insulin receptor.

Participation of Poly(ADP-Ribose) Synthetase in the Process of Norepinephrine-Induced Inhibition of Major Histocompatibility Complex Class II Antigen Expression in Human Astrocytoma-Cells

We previously demonstrated that the expression of a transfected poly(ADP-ribose) synthetase cDNA into macrophage tumor cells inhibited interferon-γ-dependent induction of major histocompatibility complex(MHC) class II antigens. In the present study, we found that addition of norepinephrine to the cultured human astrocytoma STTG1 cells induced mRNA of poly(ADP-ribose) synthetase in 6-14h. Thus, we cultured the cells in the presence of norepinephrine for 24h, and then induced the MHC class II antigen by the addition of interferon-γ. The expression of MHC class II antigen was inhibited, whereas it was not inhibited when norepincphrinc and interferon-γ were simultaneously added into the culture medium. These results suggest that an increase of poly(ADP-ribose) synthetase by norepinephrine cause the inhibition of interferon-γ-mediated MHC class II antigen expression.

Poly(A) tail length of a heat shock protein RNA is increased by severe heat stress, but intron splicing is unaffected

The small heat shock proteins (sHSPs) are induced in all eukaryotes in response to high temperature stress, but are most abundant among members of the plant kingdom where they accumulate in multiple subcellular compartments. We have analyzed the expression of the chloroplast-localized sHSP from Arabidopsis thaliana, HSP21, and characterized the structure of the gene encoding this protein to facilitate future genetic studies on the function of HSP21 in the heat shock response. HSP21 is encoded in Arabidopsis by a single gene whose coding region is interrupted by a single intron. Previous studies have shown that intron processing is disrupted by severe, abrupt heat stress but is protected by pretreatments that induce thermotolerance. The processing of the HSP21 transcript was investigated in response to an abrupt heat stress regime and a gradual heat stress regime, the latter of which is known to confer thermotolerance in plants. Under abrupt stress conditions the HSP21 transcript is somewhat longer than under gradual heat stress conditions. However, the molecular basis for the size difference is not impaired intron splicing, but rather a difference in the length of the poly(A) tail depending on the heat stress regime. The results suggest that an increase in poly(A) tail length may be a generalized response to severe, abrupt heat stress and that poly(A) tail metabolism may be one of numerous cellular processes normally protected in thermotolerant cells from the otherwise damaging effects of high temperature stress.

Antithetical accumulation of myosin heavy chain but not alpha-actin mRNA isoforms during early stages of pressure-overload-induced rat cardiac hypertrophy.

Myocardial response to a hemodynamic overload involves changes in the expression of isogenes encoding myosin heavy chain (MHC) and actin: beta-MHC/alpha-MHC and skeletal/cardiac alpha-actin mRNA isoform ratios are increased. It is not known whether these changes are due to increased accumulations of the two neosynthesized transcripts, beta-MHC and skeletal alpha-actin, or whether the mRNA isoforms normally present, alpha-MHC and cardiac alpha-actin, are concomitantly decreased. To answer these questions, using dot-blot hybridizations, primer extension, and exonuclease VII mapping assays, we have analyzed the content of sarcomeric MHC and actin mRNAs in the poly(A+) RNA in left ventricles of 23-24-day-old rats 18 and 24 hours after a pressure overload induced by stenosis of the thoracic aorta. The results showed a 1.9-fold increase in poly(A+) RNA after the stenosis. Skeletal/cardiac alpha-actin mRNA isoforms were already increased fivefold (from 0.19 to 0.99) at 18 hours, and this was exclusively due to a 5.5-fold increase in skeletal alpha-actin mRNA. At 24 hours, this ratio was increased ninefold (from 0.14 to 1.22), and this was due to a 4.3-fold increase in the level of skeletal alpha-actin mRNAs (p < 0.001) and a 1.9-fold decrease of cardiac alpha-actin mRNA (p < 0.001), restoring the same proportion of sarcomeric actin mRNA in sham-operated and operated rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Purification and characterization of poly(A) polymerase from germinated wheat embryos: enzyme glycosylation

Wheat embryo poly(A) polymerase was purified (1348-fold) to electrophoretic homogeneity by adenosine triphosphate(ATP)-Sepharose and concanavalin A(Con A)-agarose affinity chromatography. The purified polymerase was devoid of cryptic nuclease activity after Con A-agarose affinity chromatography. Thus the Con A-agarose fraction showed a linear increase in poly(A) polymerase activity as a function of time up to 90 min. Fractionation of purified enzyme on native PAGE showed a single protein stained band that coincided with the activity peak of poly(A) polymerase. The molecular weight of poly(A) polymerase was 65 000–70 000 as determined by molecular sieve chromatography. A single subunit of purified poly(A) polymerase (mol. wt., 64 000) was observed on SDS-PAGE. This proved the monomeric nature of the enzyme. The binding of poly(A) polymerase to Con A-agarose and its elution with α-methyl mannopyranoside suggested its glycoprotein nature. The apparent K m of poly(A) polymerase for ATP was 86 μM. The 3H-labelled reaction product of purified enzyme binds to oligo(dT)-cellulose affinity matrix. In addition, the putative poly(A) product was not hydrolysed by RNase A and RNase T i. Thus, it was proved that poly(A) polymerase catalyzes the synthesis of poly(A) sequences.

Poly(ADPR)polymerase expression and activity during proliferation and differentiation of rat astrocyte and neuronal cultures

Poly(ADPR)polymerase (poly(ADPR)P) mRNA and enzymatic activity levels were investigated in primary cultures of rat astrocytes and neurons in the absence or presence of basic fibroblast growth factor (bFGF) and nerve growth factor (NGF), respectively. In cultured rat astrocytes, a biphasic increase in poly(ADPR)P mRNA, associated with enhanced nuclear poly(ADPR)P enzymatic activity, were observed. The first rise in poly(ADPR)P mRNA and enzymatic activity is at the beginning of cell proliferation and the second with the occurrence of cell differentiation. In the presence of bFGF (5 ng/ml) the mRNA peaks and the differentiation-associated poly(ADPR)P enzymatic activity undergoes a 2-fold increase. In neuronal cultures an initial high level of poly(ADPR)P mRNA is followed by a decrease while differentiation is progressively achieved. A limited increase of poly(ADPR)P activity is observed during this phase. In the presence of NGF (50 ng/ml), similar poly(ADPR)P mRNA expression and enzymatic activity patterns are observed. The results suggest that poly(ADPR)P is involved at the onset of nerve-cell proliferation and differentiation.

Increase of poly(ADP-ribose) polymerase mRNA levels during TPA-induced differentiation of human lymphocytes

The non-mitogenic stimulation of human peripheral blood mononuclear cells (PBMC) with low concentrations of the phorbol ester 12- O-tetradecanoylphorbol 13-acatate (TPA) caused a progressive increase in the percent fraction of the cells that were positive for the early activating antigen CD69. At the same time, it caused a progressive increase in the steady-state levels of poly(ADP-ribose) polymerase (pADPRP) transcripts. A further increase in TPA concentration, while inducing the maximal expression of the levels of CD69 activating surface antigen, both in the presence or in the absence of proliferative activity, did not evoke any additional hightening of pADPRP mRNA levels. Time course of PBMC stimulation with a non-mitogenic dose or TPA showed an early increase in the accumulation of pADPRP mRNA, which changed at 8-16 h. and remained high for several days thereafter. On the basis of these data, we suggest flat the increase in pADPRP mRNA may be associated with the commitment of human lymphocytes from a quiescent (G 0) to an activated (G 1) state.

Molecular cloning, expression, and induction of berberine bridge enzyme, an enzyme essential to the formation of benzophenanthridine alkaloids in the response of plants to pathogenic attack.

The berberine bridge enzyme [(S)-reticuline: oxygen oxidoreductase (methylene-bridge-forming), EC 1.5.3.9] is a vesicular plant enzyme that catalyzes the formation of the berberine bridgehead carbon of (S)-scoulerine from the N-methyl carbon of (S)-reticuline in a specific, unparalleled reaction along the biosynthetic pathway that leads to benzophenanthridine alkaloids. Cytotoxic benzophenanthridine alkaloids are accumulated in certain species of Papaveraceae and Fumariaceae in response to pathogenic attack and, therefore, function as phytoalexins. The berberine bridge enzyme has been purified to homogeneity from elicited cell-suspension cultures of Eschscholtzia californica, and partial amino acid sequences have been determined. A cDNA, isolated from a Agt11 cDNA bank of elicited E. californica cell-suspension cultures, coded for an open reading frame of 538 amino acids. The first 22 amino acids constitute the putative signal peptide. The mature protein has a Mr of 57,352, excluding carbohydrate. The berberine bridge enzyme was heterologously expressed in a catalytically active form in Saccharomyces cerevisiae. Southern hybridization with genomic DNA suggests that there is only one gene for the enzyme in the E. californica genome. Hybridized RNA blots from elicited E. californica cell-suspension cultures revealed a rapid and transient increase in poly(A)+ RNA levels that preceded both the increase in enzyme activity and the accumulation of benzophenanthridine alkaloids, emphasizing the integral role of the berberine bridge enzyme in the plant response to pathogens.

Reactive oxygen injury to cultured pulmonary artery endothelial cells: Mediation by poly(ADP-ribose) polymerase activation causing NAD depletion and altered energy balance

The vascular endothelium is a significant site for tissue injury following exposure to reactive oxygen species derived from a number of sources. In order to develop a better understanding of the mechanism(s) of oxidative damage, monolayer cultures of endothelial cells obtained from bovine pulmonary arteries were exposed to reactive oxygen species generated from the oxidation of dihydroxyfumarate (DHF) to diketosuccinate. Exposure to oxidizing DHF caused a loss of cell membrane integrity that was delayed in onset; that is, it did not begin until 2 h after the addition of DHF although reactive oxygen species are produced immediately by DHF in solution. Endothelial cell lysis by DHF was prevented by the simultaneous addition of superoxide dismutase (SOD), catalase (CAT), or deferoximine (DFX). This oxidant-induced lysis was unaffected by N,N,-diphenyl- p-phenylenediamine (DPPD), a potent inhibitor of lipid peroxidation. However, simultaneous addition of 3-aminobenzamide (3AB) and nicotinamide (NA), inhibitors of poly(ADP-ribose) polymerase, prevented cell lysis. Oxidant-induced loss of membrane integrity was preceded by the early appearance of DNA strand breaks, by increased levels of poly(ADP-ribose), the product of polymerase activity, and by depletion of NAD + and ATP, followed by a decline in the energy charge ratio of the cells. None of these intracellular changes occurred when either SOD, CAT, or DFX were added at the same time as DHF, suggesting that O 2, H 2O 2, and HO. mediated these changes. The O 2 appears to be important in the autoxidation reaction of DHF. The latter two reactive oxygen species may be part of cellular-catalyzed Fenton chemistry. The increase in poly(ADP-ribose), depletion of NAD +, and the decline in ATP were also prevented by the addition of 3AB. The oxidant-induced DNA strand breakage was, however, unaffected by either 3AB or NA. Addition of 3AB immediately prior to the onset of cell lysis (2 h after the addition of DHF), prevented cell lysis, i.e., “rescued” the cells when neither SOD, CAT, nor DFX addition were effective. Concurrent with the “rescue” from lysis by 3AB, there was an increase in NAD + content and a return of the energy charge ratio to control levels. The data presented in this study suggests that in endothelial cells, DNA is a very sensitive target for reactive oxygen species and HO. is the likely proximal damaging species. In response to the DNA damage, there is a cascade of intracellular changes consisting of NAD + depletion and alteration in high energy phosphates which will progress to loss of viability and cell membrane integrity unless reversed by inhibitors of poly(ADP-ribose) polymerase.

Activation of human monocyte-derived macrophages by interferon γ is accompanied by increase of poly(ADP-ribose) polymerase activity

We have investigated poly(ADP-ribosyl)ation processes in human monocyte-derived macrophages and the effect of the activating cytokine, interferon γ (IFN-γ) on these processes. IFN-γ was shown to increase the activity of poly(ADP-ribose) polymerase in human macrophages. A 2–3-fold enhancement of poly(ADP-ribose) polymerase activity was observed after 3–4 h of incubation with IFN-γ, whose effects were dose-dependent and maximal at 20–50 U/ml. Staining with anti-poly(ADP-ribose) antibodies and purification of ADP-ribosylated nuclear proteins by affinity chromatography over boronate agarose showed that enhancement of poly(ADP-ribose) polymerase activity by IFN-γ was accompanied by accumulation of poly(ADP-ribose) polymers in nuclear proteins. The effects of IFN-γ on poly(ADP-ribose) polymerase activity were not due to an enhanced accumulation of the message for the enzyme, indicating that the activation of the enzyme activity was due to post-transcriptional modifications. IFN-γ was shown to induce DNA strand breaks in human macrophages. This phenomenon followed the same time-course and was evident with the same doses of IFN-γ that increased poly(ADP-ribose) polymerase activity. Since poly(ADP-ribose) polymerase is known to require DNA nicks for its activity, the capability of IFN-γ to induce DNA strand breaks can explain its effects on poly(ADP-ribosyl)ation processes.

Relationship between poly(ADP-ribose) polymerase activity and DNA synthesis in cultured hepatocytes

Previous studies have demonstrated that an increase in poly(ADP-ribose) polymerase activity could be closely related to DNA replication during liver regeneration and to DNA repair synthesis in different experimental systems. This relationship was further investigated by studying the time course of endogenous and total poly(ADP-ribose) polymerase activity in cultured rat hepatocytes stimulated by epidermal growth factor. This mitogen has been shown to stimulate DNA synthesis in liver cells both in vivo and in vitro. A 6-fold increase in endogenous activity was observed early after epidermal growth factor addition, just before DNA synthesis. A subsequent 4-fold increment in total enzyme activity, concomitant with DNA synthesis, was detected. Orotic acid, which has recently shown mitoinhibitory effect, abolished the epidermal-growth-factor-induced increase in endogenous and total poly(ADP-ribose) polymerase activity, as well as DNA synthesis. On the contrary, 3-aminobenzamide inhibitor of poly(ADP-ribose) polymerase completely suppressed the endogenous activity but only partially modified the increase in total catalytic level and the overall pattern of thymidine incorporation. Taken togheter, these data indicate that, in cultured hepatocytes, the induction of DNA synthesis is supported by an increase poly(ADP-ribose) polymerase activity.

Identification and Characterization of the Poly(A)-Binding Proteins from the Sea Urchin: a Quantitative Analysis

Poly(A)-binding proteins (PABPs) are the best characterized messenger RNA-binding proteins of eucaryotic cells and have been identified in diverse organisms such as mammals and yeasts. The in vitro poly(A)-binding properties of these proteins have been studied intensively; however, little is known about their function in cells. In this report, we show that sea urchin eggs have two molecular weight forms of PABP (molecular weights of 66,000 and 80,000). Each of these has at least five posttranslationally modified forms. Both sea urchin PABPs are found in approximately 1:1 ratios in both cytoplasmic and nuclear fractions of embryonic cells. Quantification in eggs and embryos revealed that sea urchin PABPs are surprisingly abundant, composing about 0.6% of total cellular protein. This is 50 times more than required to bind all the poly(A) in the egg based on the binding stoichiometry of 1 PABP per 27 adenosine residues. We found that density gradient centrifugation strips PABP from poly(A) and therefore underestimates the amount of PABP complexed to poly(A)+ RNA in cell homogenates. However, large-pore gel filtration chromatography could be used to separate intact poly(A)-PABP complexes from free PABP. Using the gel filtration method, we found that the threefold increase in poly(A) content of the egg after fertilization is paralleled by an approximate fivefold increase in the amount of bound PABP. Furthermore, both translated and nontranslated poly(A)+ RNAs appear to be complexed to PABP. As expected from the observation that PABPs are so abundant, greater than 95% of the PABP of the cell is uncomplexed protein.

Identification and characterization of the poly(A)-binding proteins from the sea urchin: a quantitative analysis.

Poly(A)-binding proteins (PABPs) are the best characterized messenger RNA-binding proteins of eucaryotic cells and have been identified in diverse organisms such as mammals and yeasts. The in vitro poly(A)-binding properties of these proteins have been studied intensively; however, little is known about their function in cells. In this report, we show that sea urchin eggs have two molecular weight forms of PABP (molecular weights of 66,000 and 80,000). Each of these has at least five posttranslationally modified forms. Both sea urchin PABPs are found in approximately 1:1 ratios in both cytoplasmic and nuclear fractions of embryonic cells. Quantification in eggs and embryos revealed that sea urchin PABPs are surprisingly abundant, composing about 0.6% of total cellular protein. This is 50 times more than required to bind all the poly(A) in the egg based on the binding stoichiometry of 1 PABP per 27 adenosine residues. We found that density gradient centrifugation strips PABP from poly(A) and therefore underestimates the amount of PABP complexed to poly(A)+ RNA in cell homogenates. However, large-pore gel filtration chromatography could be used to separate intact poly(A)-PABP complexes from free PABP. Using the gel filtration method, we found that the threefold increase in poly(A) content of the egg after fertilization is paralleled by an approximate fivefold increase in the amount of bound PABP. Furthermore, both translated and nontranslated poly(A)+ RNAs appear to be complexed to PABP. As expected from the observation that PABPs are so abundant, greater than 95% of the PABP of the cell is uncomplexed protein.