Stabilization of mRNA following serum-induction of quiescent 3T3 cells

The stability of mRNA has been measured in 3T3 cells in the resting and the growing states, and also during the transition from the resting to the growing state. Pulse labeled poly(A)⁺ mRNA chased with uridine and cytidine supplemented growth medium decayed with a half-life of 6.5 hr in the resting state, 26 hr during the transition from the resting to the growing condition, and 18 hr during serum-stimulated growth. The half-life of poly(A)⁺ mRNA determined by steady state labeling yielded similar results in resting and serum-stimulated 3T3 cells. Thus during the transition from resting to serum-stimulated growth in 3T3 cells poly(A)⁺ mRNA becomes more stable.     

Parallel changes in protein synthesis and messenger RNA content in growing and resting epithelial cells of Xenopus laevis

The regulation of cell growth can be achieved at many levels but ultimately the regulatory factors must alter protein synthesis since growing cells always exhibit an increased rate of protein synthesis compared to resting cells. Some studies using growing and nongrowing mammalian cells have shown that the rate of protein synthesis compared to resting cells. Some studies using growing and nongrowing mammalian cells have shown that the rate of protein synthesis is directly dependent on mRNA content. Other studies have shown that growing and resting cells have similar amounts of mRNA and that protein synthesis is regulated by the proportion of mRNA in polysomes. We have analyzed mRNA content in growing and resting epithelial cells of Xenopus laevis. Quantitation of poly(A)⁺ mRNA by uniform labeling with ³H-uridine and by ³H-poly(U)hybridization demonstrated a direct relationship between mRNA content and the relative rate of protein synthesis in growing and resting cells. Likewise, after serum stimulation of resting cells the increase in mRNA content closely paralleled the increase in protein synthesis. Our results suggest that control of protein synthesis in growing and nongrowing cells is exerted before the translational level.     

Properties of total and poly(A) RNA from exponentially growing and from resting cultures of Tetrahymena thermophila

Total RNA was extracted from exponentially growing and resting cultures of Tetrahymena thermophila. Poly(A)-containing RNA was separated by oligo(dT) affinity chromatography. The following characteristics of both preparations were studied: the changes in sedimentation profiles of newly made RNAs as a function of time, the length of the poly(A) segment, and the capacity of polyadenylated mRNA to code for proteins in vitro. The time-dependent sedimentation profiles of both kinds of RNA changed strikingly with the modes of growth: poly(A)⁺ RNA from heterodisperse in log phase into uniformly and slowly sedimenting in stationary phase, and total RNA from typical ribosomal into heterodisperse with a maximum in the pre-rRNA region. As revealed by the temperature regime developed by Ihle et al. [1] about 80% of all poly(A) RNA molecules carried a poly(A) stretch of less than 50 nucleotides. There was a tendency of the class 0–20 nucleotides to become more frequent in the stationary phase. The polyadenylated mRNAs were translated in the reticulocyte in vitro system. At least one protein of about 26 000 D was translated only in presence of mRNA of growing cells and not with that from resting cells. Another of 3 500 D was found only with mRNA from resting cultures. Three other proteins were translated with different rates according to the culture growth rate. The results demonstrate that the RNA isolated from different phases of culture growth have different dynamic as well as coding properties related to rate of cell multiplication.     

Regulation of repair of naturally occurring DNA strand breaks in lymphocytes

Mouse lymphocytes have been shown to contain DNA strand breaks that were repaired within 2h of onset of culture with mitogen. Inhibitors of ADP ribosylation prevented this repair and blocked cell proliferation. The mitogen concanavalin A caused the internal concentration of NAD⁺, the substrate of the ADP ribose polymerase, to rise to about double that of resting cells within 45 min of stimulation. Addition of 300 μm nicotinamide to the culture in absence of mitogen also resulted in a similar increase in internal [NAD⁺], resulting in increased ADP ribosylation activity (measured in permeabilized cells) and in joining of DNA strand breaks; however, none of the subsequent events of lymphocyte activation such as blast transformation and DNA synthesis occurred. These findings indicate that (1) cellular [NAD⁺] is a rate limiting factor in repair of DNA strand breaks in resting lymphocytes and (2) this repair is necessary but not sufficient for lymphocyte proliferation.     

Potassium ions and the regulation of cell growth in culture

The potassium ion concentration was measured in growing and density-inhibited chick embryo fibroblasts in culture and found to be about 105 mEq/1 in both. Over 90% of the K⁺ exchanged with a single half-time which averaged 19 and 25 min, respectively, for growing and density-inhibited cells. These results show no important differences in K⁺ concentration, compartmentalization or permeability between growing and non-growing cells and indicate that the regulation of growth of these cells does not involve a significant alteration of general K⁺ metabolism.     

Distribution of root mRNA species in other vegetative organs of pea (Pisum sativum L.)

Summary A cDNA library was prepared from, poly(A)⁺ RNA from roots of pea (Pisum sativum L.). Twenty five clones were selected by use of random numbers and used as probes on Northern blots to analyse the distribution of their corresponding mRNA species in other vegetative pea organs: leaf, stem and developing cotyledon. Fifteen cDNA inserts hybridised to single mRNA species, five hybridised to two mRNA species and one hybridised to five homologous mRNAs. Four cDNA clones (16% of those selected) gave no hybridization signals, indicating that the steady state levels of mRNAs were below the detection limit (i.e.less than 2.5 x 10^-5% of poly(A)⁺ RNA). Most of the root mRNAs were represented in all four pea organs as sequences of low and medium abundance. All but two cDNAs encoded mRNA species enhanced in root. However, cDNA clones appeared not to encode mRNA species expressed in a strictly organ-specific manner, as no mRNA unique to root was found. Thus, if organ-unique mRNA species are present, they are only present at a very low level of abundance in the poly(A)⁺RNA population.     

Messenger RNA regulation in human diploid fibroblasts

In resting, non-growing human diploid fibroblasts the amount of rRNA is reduced 1.8-fold, cytoplasmic polysomes are disaggregated, and the level of poly-A RNA (mRNA) is reduced 1.8-fold in relation to growing cells. The distribution of poly-A RNA is altered in resting, non-growing cells so that an average of 64% of the total cytoplasmic poly-A RNA sediments along with particles lighter than 80S (prepolysomal) in sucrose density gradients. By comparison, in growing cells only 30% of the cytoplasmic poly-A RNA sediments in the prepolysomal region. In SDS sucrose gradients, the sedimentation profile of the prepolysomal poly-A RNA from resting cells resembles that of polysomal poly-A RNA from those cells. In contrast, the average size of prepolysomal poly-A RNA from growing cells is much smaller than that of the polysomal poly-A RNA from those cells. These data are compatible with the possibility that resting cell prepolysomal poly-A is untranslated mRNA. Also consistent with this interpretation are experiments which demonstrate that one-quarter to one-third of the prepolysomal poly-A RNA of resting cells is recruited into polysomes in the presence of cycloheximide.     

Localization of host poly(A)+ mRNA in the ribosome profile of mengovirus-infected Ehrlich ascites tumor cells

The distribution of poly(A)⁺ mRNA among polysomes, monosomes, and ribosome-free supernatant fractions after mengovirus infection of Ehrlich ascites tumor (EAT) cells was investigated employing sucrose gradient centrifugation of their corresponding postnuclear supernatants. Poly(A)⁺ mRNA was isolated from sucrose gradient fractions and quantitated in a cell-free protein synthesizing system from uninfected EAT cells. It was also localized by annealing [³H]-poly(U) to the poly(A)-tracts of mRNA present in the sucrose gradient fractions. Both experiments revealed a gradual shift of host poly(A)⁺ mRNA from large to small polysomes and monosomes, respectively, with the time postinfection. The greatest part of host template RNA appears to remain ribosome-bound and only a fraction seems to be detached from the ribosomes in the course of mengovirus infection. At the end of the infectious cycle, 8 h postinfection, approximately 70% of the poly(A)⁺ mRNA detected in uninfected cells is still biologically active, but not translated in vivo, in agreement with data from the [³H] poly(U) hybridization experiment.     

The metabolism of a poly(A) minus mRNA fraction in HeLa cells

About 30% of HeLa cell mRNA lacks poly(A) when labeled in the presence of different rRNA inhibitors. Our method of RNA fractionation precludes contamination of the poly(A)^? mRNA with large amounts of poly(A)⁺ sequences. The poly(A)^? species is associated with polyribosomes, has an average sedimentation value equal to or greater than poly(A)⁺ mRNA, and behaves like the poly(A)⁺ mRNA in its sensitivity to EDTA and puromycin release from polyribosomes. There is very little, if any, hybridization at Rot values characteristic of abundant RNA sequences between the poly(A)^? RNA fractions from total cytoplasm or from polyribosomes and ³H-cDNA made to poly(A)⁺ RNA. This indicates that poly(A)^? mRNA does not arise from poly(A)⁺ mRNA by nonadenylation, deadenylation, or degradation of random abundant mRNA sequences. The rate of accumulation of poly(A)^? mRNA larger than 9S in the cytoplasm parallels the accumulation of poly(A)^? mRNA. The poly(A)^? mRNA is maintained as approximately 30% of the total labeled mRNA in a short (90 min) and in a long (20 hr) time period. These data indicate that poly(A)^? mRNA is not short-lived nuclear or cytoplasmic heterogeneous RNA contamination, and that the half-life of the poly(A)^? mRNA may parallel that of the poly(A)⁺ mRNA. Cordycepin appears to almost completely (95%) inhibit poly(A)⁺ mRNA while only partially (60%) inhibiting the poly(A)^? mRNA. The origin of the cordycepin-insensitive mRNA has not been ascertained.     

Relative amounts of newly synthesized poly(A)+ and poly(A)- messenger RNA during development of Xenopus laevis

The relative amounts of newly synthesized poly(A)⁺ and poly(A)^? mRNA have been determined in developing embryos of the frog Xenopus laevis. Polysomal RNA was isolated and fractionated into poly(A)⁺ and poly(A)^? RNA fractions with oligo(dT)-cellulose. In normal embryos the newly synthesized polysomal poly(A)⁺ RNA has a heterodisperse size distribution as expected of mRNA. The labeled poly(A)^? RNA of polysomes is composed mainly of rRNA and 4S RNA. The amount of poly(A)^? mRNA in this fraction cannot be quantitated because it represents a very small proportion of the labeled poly(A)^? RNA. By using the anucleolate mutants of Xenopus which do not synthesize rRNA, it is possible to estimate the percentage of mRNA which contains poly(A) and lacks poly(A). All labeled polysomal RNA larger than 4S RNA which does not bind to oligo(dT)-cellulose in the anucleolate mutants is considered presumptive poly(A)^? mRNA. The results indicate that about 80% of the mRNA lacks a poly(A) segment long enough to bind to oligo(dT). The poly(A)⁺ and poly(A)^? mRNA populations have a similar size distribution with a modal molecular weight of about 7 × 10⁵. The poly(A) segment of poly(A)⁺ mRNA is about 125 nucleotides long. Analysis of the poly(A)^? mRNA fraction has shown that it lacks poly(A)₁₂₅.     

A comparison of sequence complexity of nuclear and polysomal poly(A)+ RNA from different developmental stages ofXenopus laevis

Summary Nuclear poly(A)⁺ and polysomal poly(A)⁺ RNA were isolated from gastrula and early tadpole stages of the amphibianXenopus laevis. Complementary DNA was synthesized from all RNA preparations. Hybridization reactions revealed that at least all abundant and probably most of the less frequent nuclear and polysomal poly(A)⁺ RNA species present at the gastrula stage are also present at the early tadpole stage. On the other hand, there are nuclear RNA sequences at the latter stage which appear, if at all, only at lower concentrations at the gastrula stage. The polysomal poly(A)⁺ RNA hybridization reactions suggest the existence of polysomal poly(A)⁺ RNA sequences at early tadpole stages which are not present in the corresponding gastrula stage RNA.By cDNA hybridization with poly(A)^– RNA it could be shown that most of the poly(A)⁺ containing RNA sequences transcribed into cDNA were also present within the poly(A)^– RNA. It was estimated, that these sequences are 10 fold more abundant within the poly(A)^– polysomal RNA and 3–6 more abundant within the poly(A)^– nuclear RNA as compared to the poly(A)⁺ RNAs.     

Kinetic study of protein unfolding and refolding using urea gradient electrophoresis

When total cytoplasmic RNA from mouse Friend cells is fractionated using oligo(dT)-cellulose or poly(U)-Sepharose chromatography, approximately 20% of the messenger RNA activity (as measured in the reticulocyte lysate cell-free system) remains in the unbound fraction, even though this contains < 0.5% of the poly(A) (as measured by titration with poly(U)). This RNA, operationally defined as poly(A)^?, is found almost entirely in polysome structures in vivo. Its major translation products, as shown by one-dimensional sodium dodecyl sulphate-containing gels, are the histones and actin. Two-dimensional gels (isoelectric focusing: sodium dodecyl sulphate/gel electrophoresis) show that, with the exception of the mRNAs coding for histones, poly(A)^? mRNA encodes similar proteins to poly(A)⁺ mRNA, though in very different abundances. This is directly confirmed by the arrest of the translation of the abundant poly(A)^? mRNAs after hybridization with a complementary DNA transcribed from poly(A)⁺ RNA.RNA sequences which are rare in the poly(A)⁺ RNA are also found in poly(A)^? RNA, as shown by hybridizing a cDNA transcribed from poly(A)⁺ RNA to total and poly(A)^? polysomal RNA. That this does not simply represent a flow-through of poly(A)⁺ RNA is indicated by (i) the lack of poly(A) by hybridizing to poly(U) in this fraction, (ii) the fact that further passage through poly(U)-Sepharose does not remove the hybridizing sequences, (iii) the very different quantitative distribution of proteins encoded by poly(A)⁺ and poly(A)^? RNAs. We also think that it does not result from removal of poly(A) from polyadenylated RNAs during extraction because RNAs prepared using the minimum of manipulations give similar results. The distribution of both total mRNA and α and β globin mRNAs between poly(A)⁺ and poly(A)^? RNA does not change significantly during the dimethyl sulphoxide-induced differentiation of Friend cells.     

Role of Reactive Oxygen Species in the Radiation Response of Human Hematopoietic Stem/Progenitor Cells

Hematopoietic stem/progenitor cells (HSPCs), which are present in small numbers in hematopoietic tissues, can differentiate into all hematopoietic lineages and self-renew to maintain their undifferentiated phenotype. HSPCs are extremely sensitive to oxidative stressors such as anti-cancer agents, radiation, and the extensive accumulation of reactive oxygen species (ROS). The quiescence and stemness of HSPCs are maintained by the regulation of mitochondrial biogenesis, ROS, and energy homeostasis in a special microenvironment called the stem cell niche. The present study evaluated the relationship between the production of intracellular ROS and mitochondrial function during the proliferation and differentiation of X-irradiated CD34⁺ cells prepared from human placental/umbilical cord blood HSPCs. Highly purified CD34⁺ HSPCs exposed to X-rays were cultured in liquid and semi-solid medium supplemented with hematopoietic cytokines. X-irradiated CD34⁺ HSPCs treated with hematopoietic cytokines, which promote their proliferation and differentiation, exhibited dramatically suppressed cell growth and clonogenic potential. The amount of intracellular ROS in X-irradiated CD34⁺ HSPCs was significantly higher than that in non-irradiated cells during the culture period. However, neither the intracellular mitochondrial content nor the mitochondrial superoxide production was elevated in X-irradiated CD34⁺ HSPCs compared with non-irradiated cells. Radiation-induced gamma-H2AX expression was observed immediately following exposure to 4 Gy of X-rays and gradually decreased during the culture period. This study reveals that X-irradiation can increase persistent intracellular ROS in human CD34⁺ HSPCs, which may not result from mitochondrial ROS due to mitochondrial dysfunction, and indicates that substantial DNA double-strand breakage can critically reduce the stem cell function.     

Proton extrusion and attendant transport phenomena in Candida utilis induced by ethanol

Summary Ion fluxes after ethanol addition to Candida utilis depend crucially on aeration (air versus oxygen). In O₂-aerated non-growing cells ethanol causes an H ⁺ / K ⁺ exchange and an extrusion of acetate and lactate accompanied mostly by K ⁺, and their subsequent reimportation together with H ⁺. Cells from continuous culture display generally stronger acidification and more marked K ⁺ movements than non-growing ones. Offprint requests to: A. Prell     

Sequences coding for proteins expressed in liver and for globin in poly(A)+ and poly(A)− RNA fractions from nuclei and cytoplasm of chicken immature red blood cells

By hybridization with [³H]labeled globin cDNA the contents of globin coding sequences in total nuclear RNA, poly(A)⁺nuclear RNA, poly(A)^--nuclear RNA and polysomal RNA of chicken immature red blood cells was determined to be 0.86%, 20%, 0.42% and 1% respectively. As the poly(A)⁺-fraction comprises only about 2% of total nuclear RNA, globin coding sequences are distributed with 49% in the poly(A)⁺-fraction and with 51% in the poly(A)^--fraction.Part of the mRNA sequences which are found in liver are also transcribed in immature red blood cells. These sequences are enriched in poly(A)⁺-nuclear RNA as the globin coding sequences but their total amount in the poly(A)⁺-fraction is much smaller than in the poly(A)^--fraction.When nuclear RNA from immature red blood cells was translated in an ascites tumor cell-free system, 20% of the newly synthesized proteins were globin chains. The percentage of globin chains in the newly synthesized proteins increased to over 70% when poly(A)⁺-nuclear RNA was translated. Only about 7.5% of globin chains were found in proteins coded by poly(A)^--nuclear RNA.