Uriel Z. Littauer

Ribonucleic acid from Escherichia coli; preparation, characterization and physical properties.

A method has been developed for the isolation of RNA from E. coli “protoplasts”, by extraction with a phenol-water mixture. The RNA preparation contained no detectable DNA and only traces of proteins and polysaccharides. Sedimentation in the ultracentrifuge yielded three boundaries. The two faster moving components were separated from the slower one by (NH4)2SO4 precipitation from a phenol-saturated water solution. From sedimentation, viscosity and light-scattering data the molecular weight was estimated to be of the order of one million. The RNA preparations in their viscosity behavior closely resemble coiling synthetic polyelectrolytes, and behave quite unlike DNA. The viscosity behavior, birefringence of flow, and potentiometric titration data are discussed in terms of a single contractile coil model. The ϵ(P)260 mμ values of the isolated material in 1 M phosphate (pH 7.1) were about 7400. Alkaline hydrolysis or polyribonucleotide phosphorylase action brought about an increase in absorption ranging from 56 to 59% while pancreatic ribonuclease caused a 28–31% increment. At very low concentrations of RNAase the change in optical density remained constant, while viscosity decreased rapidly with time.

Decrease in levels and rates of synthesis of tubulin and actin in developing rat brain.

The cytoplasmic and particulate tubulin content of postnatal rat brains was determined at various stages of development. The amount of tubulin in the soluble fraction was found to increase after birth and levels off at the age of 10-15 days, while the total protein content is still increasing. Indeed, the percentage of tubulin in the soluble fraction is about 33% at birth, stays at this value until day 10, and then decreases to 20% between days 10 and 15. On the other hand, the rate of increase in the level of the particulate tubulin parallels that of the total particulate proteins, and hence there is no change in the percentage of particulate tubulin during brain development. There was close agreement between the tubulin values obtained by the [3H]-colchicine binding assay and those obtained by electrophoretic resolution in sodium dodecylsulfate-polyacrylamide gels. Polyacrylamide gel electrophoresis was also utilized to determine actin levels in developing brains. The percentage of cytoplasmic brain actin also decreased with the age of the rats, from a value of 20% at birth to 10% at day 30, while the percentage of the particulate actin remained constant. The decline in the percentage of cytoplasmic tubulin and actin during brain development can be accounted for by reduction in the proportions of the respective mRNA species. Translation of poly (A)-rich brain mRNA in a wheat-germ cell-free system showed that the percentages of tubulin and actin synthesized decreased gradually with age. Similar results were obtained by analyzing the proteins produced by isolated brain polysomes in a brain cell-free system.

In vitro translation of polyadenylic acid-free rabbit globin messenger RNA☆

Abstract A specific method has been developed for the removal of polyadenylic acid-rich sequences from messenger RNA. The method is based on the processive phosphorolysis of mRNA using molar excess of Escherichia coli polynucleotide phosphorylase at 0 ° C in the presence of 1 m -NaCl. It also enables the determination of the location, length and gross base composition of the poly(A)-rich segment. Under these conditions, it was established that the poly(A)-rich sequence of rabbit globin mRNA is located at the 3 ′ -OH terminus and has an average size of 149 nucleotide residues. After removal of the poly(A)-rich sequence from the mRNA, the remainder of the molecule fails to bind to oligo(dT)-cellulose columns. The poly(A)-rich sequence of rabbit globin mRNA does not interact strongly with other regions of the molecule, as it is phosphorolyzed at the same rate as free poly (A). On the other hand, phosphorolysis of the molecule beyond the poly(A)-rich sequence is rather slow, indicating that this region of the mRNA has a stable secondary structure. The sequence beyond the poly(A)-rich segment was found to be rich in guanosine and uridine residues. The poly(A)-rich sequence of the mRNA present as part of a specific polysomal ribonucleoprotein complex, is protected from the action of polynucleotide phosphorylase, indicating that the attachment of the protecting protein(s) extends to the 3 ′ end of the mRNA. The poly(A)-free mRNA can still be translated in a Krebs ascites tumor cellfree extract. The initial rate of translation of poly(A)-free mRNA, is virtually identical to that found with poly(A)-containing mRNA, in the presence or absence of reticulocyte ribosomal wash fluid. The only difference in the template activity of the mRNA preparations, appears at longer periods of incubation and in the presence of reticulocyte ribosomal wash fluid, when the rate of protein synthesis tends to level off more strongly with poly(A)-free mRNA than with native poly(A)-containing mRNA. On electrophoresis in polyacrylamide gradient gels containing sodium dodecyl sulfate or on cellulose acetate strips the product of the reaction has the same mobility as that of free globin.

The α-subunit of tubulin is preferentially associated with brain presynaptic membrane

Tubulin, the subunit protein of microtubules, is a heterodimer composed of two polypeptides OLand /3of -55 000 1M, each [ 1,2]. The LYand @bunits show microheterogeneity and both have been resolved into several components [3-71. We have recently shown that cytoplasmic tubulin microheterogeneity is most prominent in the brain and increases with rat brain maturation [8]. Tubulin is not only confined to the cytoplasm, as it is found to be associated with various membranes [9-131 including the presynaptic membranes [ 14,151. It was therefore of interest to determine the presence and properties of membrane bound tubulin from brain and compare it to tubulin from the cytoplasmic fraction. By the criteria of molecular weight, isoelectric point, peptide mapping and vinblastine binding we show, in the present study, that synaptosomal membranes isolated from rat cerebral cortex contain significant amounts of tubulin. Furthermore, we find that the (Yand /3-tubulins differ in their association with these membranes.

Induction of differentiation in mouse neuroblastoma cells by hexamethylene bisacetamide.

Abstract Hexamethylene bisacetamide (HMBA), a potent inducer of erythroid differentiation in murine erythroleukemia cells (1), induces differentiation in mouse neuroblastoma cells, as indicated by the extension of neurites and the development of an excitable membrane. HMBA is effective at concentrations 50-fold lower than dimethylsulfoxide (2), another inducer of differentiation in both mouse neuroblastoma and murine erythroleukemia cells.

Bacteriophage Induced Transfer RNA in Escherichia coli

The events taking place after a viral nucleic acid enters a susceptible host cell depend on the specific viral system. In the case of the DNA-containing viruses (1), the nucleic acid serves as a template both for its own replication and for the transcription of viral specific mRNA which is then translated into viral proteins. It has been generally thought that the various components of the host translational system remain unaltered and are utilized for the synthesis of viral proteins; the viral mRNA becomes attached to the preexisting host cell ribosomes, and viral polypeptides are then synthesized by the preexisting host cell tRNA and aminoacyl-tRNA synthetases. This is a rather simplified presentation of the events taking place during synthesis of viral proteins. Recent experiments show that the picture may be more complicated, and, at least in the case of some viruses, a more intricate translational mechanism is involved. Studies from several laboratories show that mammalian and bacterial viruses may induce changes in the translational mechanism of their host cells. These changes were observed in the tRNA, in the enzymes modifying the tRNA (such as tRNA methylases and tRNA thiolases), or in the aminoacyl-tRNA synthetases. The evidence for changes in the translation mechanism which follow virus infection is summarized below. Several modifications of tRNA induced by virus infection have been observed. Thus, the chromatographic profile of leucine tRNA in Escherichia coli is altered after the bacterial cells have been infected with T2 bacterio-

Mitochondrial ribosomal RNA from Aspergillus nidulans: Characterization of a novel molecular species☆☆☆

Abstract Several criteria have been used to compare mitochondrial ribosomal RNA from the fungus, Aspergillus nidulans, with ribosomal RNA from its corresponding cytoplasm and from Escherichia coli cells. Sedimentation velocity values of mitochondrial rRNA (23.5 S; 15.5 s) resembled those of E. coli rRNA and not of the homologous cytoplasmic rRNA (26.5 s; 17.0 s). However, when samples were analyzed by polyacrylamide gel electrophoresis, mitochondrial rRNA appeared to be larger than bacterial rRNA and similar to cytoplasmic rRNA in size. An explanation for the apparent variance in molecular weight estimation by sedimentation and electrophoretic methods was sought on the basis of configurational differences among the various rRNA species. Thermal denaturation studies were carried out at 260 mμ and 280 mμ in order to assess, separately, the contribution of A.U and G.C residues to the ordered structure of the ribosomal RNAs. Mitochondrial rRNA differed from cytoplasmic and bacterial rRNAs in several features. (1) Over the temperature range 10 to 95 °C, mitochondrial rRNA showed a greater percentage change in relative absorbance at 260 mμ than at 280 mμ while cytoplasmic and E. coli rRNA samples exhibited quite the opposite pattern. Based on these data, the G + C content of the ordered regions in the RNA chain were calculated to be 27 and 32% for the heavy and light mitochondrial rRNA components, 55.5 and 51% for the corresponding cytoplasmic ones and 54.5 and 54% for the bacterial rRNA peaks. (2) Thermal denaturation mid-points occurred at considerably lower temperatures for mitochondrial rRNA (Tm260 = 47 °C; Tm280 = 48.5 °C) than for cytoplasmic rRNA (Tm260 = 54.5 °C; Tm280 = 61 °C) or E. coli rRNA (Tm260 = 56 °C; Tm280 = 60 °C). This is interpreted on the basis of differences in the average number of G.C residues per ordered region of the respective molecules. Nucleotide base composition studies were also carried out. They showed mitochondrial rRNA to have a lower G + C content (32%) than the corresponding cytoplasmic rRNA (51%). It is concluded that Aspergillus mitochondrial rRNA represents a unique and novel molecular species differing profoundly from both cytoplasmic and bacterial rRNA types.

Regulation of mRNA levels for microtubule proteins during nerve regeneration

The molecular regulation of tubulin synthesis was investigated in the regenerating goldfish retina. Previous in vivo studies pointed to an increase in tubulin synthesis in the retina during regeneration of the injured goldfish optic nerve. Using labeled cDNA probes, we showed that this increase occurs as a result of enhanced tubulin mRNA levels. Analysis of labeled in vivo products revealed enhanced β2‐tubulin synthesis accompanied by an increase in the level of the low‐M r microtubule‐associated proteins identified as TAU factors. The results are discussed with respect to the possible involvement of these proteins in the process of nerve regeneration.

Sodium-dependent efflux of K+ and Rb+ through the activated sodium channel of neuroblastoma cells

Abstract Passive efflux of 42 K or 86 Rb from differentiated mouse neuroblastoma cells in culture was stimulated up to 8-fold by 10 −4 M veratridine. The increased efflux could be blockedby low concentrations of tetrodotoxin (K i = 4×10 −9 g/ml), and did not occur with other cell types lacking an excitable membrane. The temperature sensitivity of the activated component was much higher than that of the normal passive outflow. It is suggested that the veratridine-dependent, tetrodotoxin-sensitive efflux represents passage of ions through the excitable Na + channel. Replacement of extracellular Na + by Tris + abolished the activation by veratridine. Titration of the Na + requirement resulted in a hyperbolic relationship between external Na + concentration and efflux rate, with an apparent K m of 66.7 mM for Na + . This phenomenon may reflect an interaction between extracellular ions and a regulatory site on the Na + channel.

Cryptic form of mRNA in dormant artemia salina cysts

Abstract Dormant Artemia salina cysts are almost devoid of polysomal structures but contain appreciable quantities of mRNA that sediments mainly as a 40S complex in sucrose gradients. The mRNA can be isolated from this complex and efficiently translated in a wheat germ cell-free system, although the 40S complex itself is inactive. During rehydration of the cysts, mRNA becomes increasingly involved in polysomal complexes which can be actively translated in the cell-free system.