Messenger RNA (mRNA) and messenger like RNA (mlRNA) were investigated in the cytoplasm of HeLa cells while ribosomal RNA synthesis was arrested. Under these conditions, functional mRNA associated in polyribosomes and cytoplasmic free mlRNA are formed and can be labelled selectively to steady state.All cytoplasmic non-ribosomal RNA sedimenting a t more than 6-7 S exists in the form of ribonucleoprotein complexes which pre-exist in the cell, and are stable upon cell lysis, sedimentation and (after fixation) CsCl density gradient analysis. The functional, true mRNA is contained in a complex of mRNA and protein which bands in association with ribosomes (e = 1.52 to 1.60 g/cm3) in CsCl density gradients or, released by EDTA, a t its own intrinsic density of 1.40-1.88 g/cm3. The cytoplasmic free mlRNA bands as a particle of mlRNA-containing ribonucleoprotein at an identical low density. The molecular weight spectrum of mRNA is identical t o that of mlRNA and the sedimentation pattern of the mRNA-protein complex released from polyribosomes is similar to that of the free mlRNA -protein complex.The physico-chemical separation of mRNA -and mlRNA -protein complexes allowed us to follow their relative kinetics of synthesis and decay. Each type of ribonucleoprotein obeys a different, strictly time-dependent pattern. Label enters the pool of free mlRNA -protein complexes first and may, in a pulse-chase experiment, be partially chased into polyribosomes. At steady state (6 h) 40-60°/, of the labelled RNA remains in the form of free mlRNA-protein particles. These cannot be chased into polyribosomes, the kinetics of mRNA. and mlRNA-protein complexes decay following identical patterns. These findings are in agreement with a model according to which mlRNA from the nucleus first joins the pool of free ribonucleoprotein. Then, the activated mRNA-protein complexes attach to ribosomal subunits and form polyribosomes whereas inactivated mlRNA * protein complexes remain free in the cytoplasm.In order to further strengthen the evidence in favour of the real existence of mRNA-and mlRNA * protein complexes in the cells, the corresponding fractions from sedimentation or CsCl density gradients were observed in the electron microscope. By this method it was possible to see small cytoplasmic particles which have not before been identified. The rounded structures, with diameters ranging from 100 h to 200 8, seem to consist of the coiling of a 35 8 wide pearllike chain which may also be identsed in polyribosomes. The frequency of these particles is highest in the mlRNA. protein band (e = 1.40-1.48 g/cmS). Thus they may correspond to the mRNA -protein complex. However since they share some morphological features with other known biological structures the evidence is not conclusive.t We are desolated to announce to our friends and collegues that Nicole Granbouland died in an accident shortly before the completion of this manuscript. We dedicate this paper to her memory.Unusuul Abbreviations. mRNA, messenger RNA (this term is restricted to function...
Kidney cells, predominantly from Cercopithecus monkeys but also from baboons, were infected in vitro with the SV40 virus. The infectious cycle was studied with the electron microscope by means of thin sections of cells fixed from 3 hours up to 11 days after infection. The frequency of virus formation and various nuclear and cytoplasmic lesions in relation to the infection are described. The virus particles appear in the nucleus in close contact with the chromatin. In a small number of cells they have been observed as early as l0 to 12 hours after infection, but most often they appear 24 to 48 hours afterward. Their mean diameter is 33 m#. They have no membrane and are frequently arranged as crystaMike structures. In addition to the appearance of virus, one observes various lesions in the nucleoplasm and particularly in the nucleolus, which shows an early hypertrophy and produces unusual, dense condensations in contact with the nucleolonema. The importance of these nucleolar lesions and the relationship between the SV40 virus and the polyoma, common wart, and Shope papilloma viruses are discussed.The SV40 virus was discovered by Sweet and Hilleman (38) in Macacus rhesus kidney cells, being identified by the cytopathic effect it produces on the kidney cells of Gercopithecus aethiops sabaeus, a monkey which is not naturally a carrier of this viral agent. The cytopathic effect is characterized by an intense cytoplasmic vacuollzation after several days of infection ("vacuolating agent").Our preliminary electron microscope study on Cercopithecus kidney cells in vitro infected by SV40 virus revealed that the site of development of the virus is in fact entirely nuclear, that the cytoplasmic vacuolization is actually the consequence of its multiplication in the nucleus, and, finally, that its morphological structure is similar to that of the polyoma virus (40). We also observed the importance of nuclear and nucleolar lesions (alterations produced by virus infection) which appear in the course of the infection. These first results are in agreement with the observations made by Gaylord and Hsiung (t5). When it was discovered that the inoculation of this agent into newborn hamsters produced tumors of the sarcoma type (13,17), this virus acquired importance in tumor virus research. A more detailed morphological study of the multiplication of this virus and the cellular lesions it induces appeared necessary to provide a better understanding of the cell-virus interaction following infection with an oncogenic agent.In the course of our study we have paid particular attention to nucleolar alterations during the first stages of infection. In addition, we have been much interested in the comparison of the evolution of this virus with that of the polyoma agent. MATERIAL AND METHODS Cell CulturesPrimary cultures were prepared from kidneys of the African green monkey (Cercopithecus aethiops sa-
Ribosomal, preribosomal (pre-rRNA) and messenger like (mlRNA) RNA molecules from animal cells have been spread in extended form for visualisation in the electron microscope, in order to measure their length. It is shown that the giant nascent pre-rRNA and mlRNA consists of linear molecules of homogeneous shape. Their length ranges up to 3.5 p in the case of pre-rRNA and up to 8.5 p for mlRNA. The correlation of apparent lengths and molecular weight of RNA is discussed. A spacing of 2.45 b, based on the length and the chemically determined molecular weight of E . coli rRNA, allows one to assign molecular weights to rRNA and pre-rRNA that are in fairly good agreement with published data based on sedimentation analysis. However, the 28 S rRNA has twice the length of the 18 S species, which is in disagreement with the mass relation of 2.5-3.0 observed by sedimentation. I n the case of mlRNA the spacing of 3.17 d, derived from the length to molecular weight relation of R 17 phage RNA, is more likely to give correct values than that based on rRNA. This conclusion derives from a discussion of the relation of length and molecular weights in various viral RNA molecules in a range of up to 10' daltons.Two general conclusions may be drawn from the work presented : 1. The high sedimentation rate of pre-rRNA and mlRNA is not based on artificial agglomeration or particular configuration but on the real mass of molecules which contain, in the case of mlRNA, up to 30,000 bases (molecules of up to 50,000 bases are likely to exist also) in covalent linkage.2. The electron microscope may be used to determine molecular weights. The precision of the values obtained may be equal to or better than those determined by sedimentation if correct spacing data are available from reference molecules of the same RNA class.Ever since the detection in animal cells of the precursor to ribosomal RNA (pre-rRNA) and of the giant messenger-like RNA [1--31 the reality of their high molecular weight, estimated on the basis of apparent sedimentation constants (which may reach 80 to 100 S) has been questioned. I n spite of many controls based either on treatments which suppress intermolecular bonds or on specific labelling kinetics, for a long time the occurrence of artificial agglomerates of RNA in sucrose gradients could not be excluded. Since the size of the giant RNA molecules has an implication for theoretical considerations concerning their function in the animal cell we attempted to find a direct proof of their molecular structure and size by visualisation in the electron microscope.Electron The main conclusions of this paper are: a) Linearity exists between the molecular weights calculated from sedimentation constants and the length in electron microscope pictures of the different metabolic forms of rRNA. Estimates of their molecular weights based on the length per nucleotide of E . coli rRNA will be given. It will be shown that the conversion of 45 S to 28 S is based, as has been proposed by Weinberg et al. [15], on a change in length and not i...
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