Ribonucleoprotein organization of eukaryotic RNA. XXXII. U2 small nuclear RNA precursors and their accurate 3' processing in vitro as ribonucleoprotein particles

Biosynthetic precursors of U2 small nuclear RNA have been identified in cultured human cells by hybrid-selection of pulse-labeled RNA with cloned U2 DNA. These precursor molecules are one to approximately 16 nucleotides longer than mature U2 RNA and contain 2,2,7-trimethylguanosine "caps". The U2 RNA precursors are associated with proteins that react with a monoclonal antibody for antigens characteristic of small nuclear ribonucleoprotein particles. Like previously described precursors of U1 and U4 small nuclear RNAs, the pre-U2 RNAs are recovered in cytoplasmic fractions, although it is not known if this is their location in vivo. The precursors are processed to mature-size U2 RNA when cytoplasmic extracts are incubated in vitro at 37 degrees C. Mg2+ is required but ATP is not. The ribonucleoprotein structure of the pre-U2 RNA is maintained during the processing reaction in vitro, as are the 2,2,7-trimethylguanosine caps. The ribonucleoprotein organization is of major importance, as exogenous, protein-free U2 RNA precursors are degraded rapidly in the in vitro system. Two lines of evidence indicate that the conversion of U2 precursors to mature-size U2 RNA involves a 3' processing reaction. First, the reaction is unaffected by a large excess of mature U2 small nuclear RNP, whose 5' trimethylguanosine caps would be expected to compete for a 5' processing activity. Second, when pre-U2 RNA precursors are first stoichiometrically decorated with an antibody specific for 2,2,7-trimethylguanosine, the extent of subsequent processing in vitro is unaffected. These results provide the first demonstration of a eukaryotic RNA processing reaction in vitro occurring within a ribonucleoprotein particle.     

Defect in translation of poliovirus RNA at the restrictive temperature.

Translation of the RNA of LSc type 1 poliovirus was examined in vivo at the restrictive temperature (39 °C). During the first two hours of infection at 39 °C the levels of viral polyribosomes were 50% lower than at 35 °C (permissive temperature). During the third hour of infection at 39 °C, only 4 to 10% of the control levels of polyribosomes were observed. Three experiments indicate that the elongation of viral peptides was not occurring properly at 39 °C. First, cultures incubated at 39 °C during the third hour of infection with both [³⁵S]methionine and [³H]uridine exhibit a fourfold increase in the ratio of viral protein/viral RNA in the polyribosome region of sucrose gradients in comparison to controls kept at 35 °C. However, at both temperatures the relative size distribution of polyribosomes was similar. Second, the ratios of released protein/nascent protein after 90-second and 5-minute pulses with [³⁵S]methionine indicate that elongation of peptide chains was inhibited at 39 °C. Third, when initiation of synthesis of viral protein was blocked with 150 mM-NaCl, the polyribosomes disaggregated four to five times more rapidly at 35 °C than at 39 °C. The data indicate that translation of viral RNA is inhibited at the restrictive temperature because of a reduced rate of elongation of viral proteins. The reduced rate of peptide chain elongation at 39 °C was fully reversible when cultures were shifted to 35 °C in the presence of 150 mm-NaCl. The latter finding indicates a conformational change in viral protein at 39 °C.     

Subunits of RNA polymerase in function and structure; Maturation in vitro of core enzyme from Escherichia coli

Reconstitution of Escherichia coli RNA polymerase was found to be markedly enhanced by DNA as well as by the σ subunit. Among discrete steps of subunit assembly, formation of the primary intermediate α₂β complex and subsequent association of the complex with the β′ subunit are not affected by the presence of DNA and the σ subunit; the α₂ββ′ complex thus formed, however, is virtually inactive and is subject to temperature-dependent activation by DNA and the σ subunit. The α₂ββ′ complex is, therefore, a secondary intermediate in the sequence of enzyme formation, or a premature form of core enzyme.In the course of activation of the premature core complex, the subunit σ interacts with both the α₂β complex and the β′ subunit; DNA acts in much the same way. The enzyme, reconstituted in the presence of DNA, is recovered attached to the DNA, added as an enhancer, and initiates RNA synthesis without prior release from the DNA. A limited number of unique DNA sites appear to be concerned with the enzyme maturation.     

In vitro histogenic capacities of limb mesenchyme from various stage mouse embryos

Mesenchyme cells isolated from mouse embryo forelimb buds (stages 15 through 21) and placed in high-density micromass cultures are compared with respect to their in vitro histogenic capacities. Particular emphasis is placed on changes in in vitro chondrogenic capacity. Stage 15 mouse limb cultures form numerous aggregates which uniformly fail to differentiate into cartilage nodules. On the other hand, cartilage nodules are observed in cultures prepared from all subsequent stage limbs, although there is a linear decrease in the size of nodules between stage 16–17 and middle-late stage 21 cultures. This decrease correlates with simultaneous decreases in both the proportion of aggregating cells and the extent of dibutyryl cyclic AMP-stimulated cartilage formation. At the same time, observations indicate that the proportions of nonaggregating and nonchondrogenic mesenchyme, myogenic cells, and, perhaps, fibrogenic mesenchyme are increasing. The only exceptions to these patterns are observed in cultures from middle-late stage 21 limbs, when cartilage differentiation in situ is already extensive. Unlike earlier stage cultures, which form nearly identical numbers of aggregates and nodules, middle-late stage 21 cultures form variable numbers of aggregates, only a few of which differentiate into cartilage nodules. Middle-late stage 21 cultures also contain unexpectedly low numbers of myogenic cells/unit area of culture. Based on changes in the in vitro histogenic capacities, it is concluded that concurrent with a progression of morphogenic events in the limb, there is a progression of changes in the relative proportions of cell subpopulations. Both the existence of the different subpopulations and the changes in their relative proportions can be detected in vitro. Furthermore, it is concluded that cartilage formation in the limbs of both mouse and chick embryos probably occurs according to very similar developmental programs.     

Effect of heat shock on the synthesis of low molecular weight RNAs in drosophila: Accumulation of a novel form of 5S RNA

The synthesis and stability of low molecular weight RNAs following heat shock in Drosophila melanogaster cell cultures have been examined. When cultures are raised from 25°C to 37°C, the synthesis of tRNA and at least two other low molecular weight RNAs continues at the 25°C rate. 5.8S ribosomal RNA and most of the low molecular weight nuclear RNAs are not synthesized. The synthesis of 5S ribosomal RNA is greatly reduced. A large amount of an RNA of about 135 nucleotides in length accumulates at 37°C. Nucleotide sequence analysis reveals that this RNA is a novel form of 5S RNA with approximately 15 additional nucleotides at its 3′ end.     

Trace metal modification of immunocompetence. I. Effect of trace metals in the cultures on in vitro transformation of B lymphocytes.

Lymphocytes obtained from the spleen of Balb/C mice have been subjected to transformation by LPS in the presence of varying concentrations of Pb²⁺, Cd²⁺, or Cr³⁺. Both DNA and protein turnover were followed. It was found that Pb²⁺ and Cr³⁺ are mitogenic over a broad range of concentrations, while Cd²⁺ is slightly mitogenic at very low (10^?6M) concentration and rapidly becomes inhibitory of both [³H]TdR and [³H]ALA uptake. Pb²⁺ appears to stimulate the action of LPS, while Cr³⁺ appears to inhibit. Each of the metals protects the lymphocytes from cell death arising from incubation with LPS. The mechanism of the observed changes is as yet obscure.     

An in vitro model for induction of immunological unresponsiveness to turkey gamma-globulin in primed mouse spleen cells. I. The secondary in vitro response and induction of unresponsiveness.

Spleen cells from mice previously immunized with turkey γ-globulin (TGG) were shown to give a vigorous secondary response in vitro when challenged in Mishell-Dutton cultures with TGG covalently coupled to pig erthrocytes (TGG-PRBC). However, 90–100% of the response could be abrogated by the incorporation of soluble TGG (sTGG) into the culture medium at concentrations greater than 1 mg/ml. Unresponsiveness, as measured by the absence of plaque-forming cells (PFC) in cultures receiving sTGG, was found to be antigen specific in that these cultures were still able to give normal PFC responses to sheep or burro erythrocytes. Spleen cells incubated with sTGG for short periods of time were shown to remain unresponsive after removal of sTGG from the culture and addition of TGG-PRBC. A 1-hr exposure period resulted in greater than 70% Unresponsiveness and a complete unresponsive state required only 8 hr of exposure. In contrast to the continued Unresponsiveness of sTGG-treated cells in vitro, spleen cells incubated with sTGG for 24 hr were fully responsive to an immunogenic challenge with alum-precipitated TGG when they were transferred into irradiated syngeneic mice. These data suggest that the readily induced unresponsive state in cultures of TGG primed cells may involve either a reversible antigen blockade of antigen-sensitive lymphocytes or a peripheral inhibition of reactive cells by suppressor lymphocytes.     

Clustering of the DNA sequences complementary to repetitive nuclear RNA of HeLa cells.

We have studied the hybridization profile of heterogeneous nuclear RNA from HeLa cells across DNA density gradients, and found that components in the high molecular weight fraction of heterogeneous nuclear RNA of HeLa cells hybridize to discrete density fractions on the light and heavy sides of the DNA. The conditions used for hybridization in this work allowed the detection of only those components in the RNA complementary to reiterated sequences in the DNA. These sequences in HnRNA are known to include double-stranded regions, which can be isolated readily. The double-stranded RNA shows a pattern of hybridization across a DNA density gradient which is similar to that of total HnRNA. It is concluded that the repeated sequences in HnRNA are complementary to clusters of repeated sequences in the DNA.     

Levels and modification of methionyl-transfer RNA in preimplantation rabbit embryos.

Transfer RNA with l-methionine acceptor activity was extracted from preimplantation rabbit embryos and purified on reverse-phase-3 columns. The molar quantity of methionine acylated to RNA increases as embryo development proceeds from the 16-cell stage to the 80,000 cell blastocyst stage. However, the quantity of methionyl-tRNA per genome declines 100-fold as the embryo cell number increases. Formylation of methionyl-tRNA illustrated that approximately one-third of tRNA^Met extracted was tRNA_f^Met. Methylation of purified methionyl-tRNA by an adult rabbit liver methylase extract illustrated that two-day preimplantation embryo tRNA is highly hypomethylated relative to tRNA from later stages of development. The hypomethylated methionyl-tRNA was also less effective in ribosome binding studies than more fully methylated methionyl-tRNA present in the later stages of embryo development.     

The regional metabolism of 5-hydroxytryptamine in mouse brain in vitro.

The relative rates of formation of 5-hydroxytryptophol (5-HTOL) and 5-hydroxyindoleacetic acid (5-HIAA) from exogenous 5-hydroxytryptamine, showed regional variations when examined in homogenates of seven separate areas of mouse brain. 5-HTOL production was highest in the cerebellum, and lowest in the corpus striatum, whereas the production of 5-HIAA was greatest in the hypothalamus. Addition of NADPH was shown to increase the formation of the alcohol catabolite in whole brain homogenates. The production of 5-HTOL decreased in the brain homogenates of mice which had previously been injected with phenytoin sodium or oxypertine, with the latter also causing a fall in overall 5-HT metabolism.     

Characterization of Ehrlich ascites tumor cell messenger RNA specifying ribosomal proteins by translation in vitro

We have examined the messenger RNA which codes for the ribosomal proteins in Ehrlich ascites tumor cells. Poly(A)-containing mRNA was isolated from polysomes and fractionated into 11 size classes whose average molecular weights were between 1.8 × 10⁵ and 24 × 10⁵. These mRNAs were used to direct protein synthesis in a fractionated translational system that was derived completely from Ehrlich ascites tumor cells. More than 90% of the ribosomal proteins which we could identify were coded for by mRNAs averaging in size between Mr = 180 × 10³ and 320 × 10³. The small size of these mRNAs indicates that the cytoplasmic mRNAs which specify the ribosomal proteins are monocistronic. We could detect the synthesis of 36 of 48 ribosomal reference proteins as well as 20 additional polypeptides which had characteristics similar to ribosomal protein. The ribosomal proteins were identified on the basis of their positive charge, small size, electrophoretic properties on two-dimensional polyacrylamide gels and chromatographic characteristics on carboxymethyl-cellulose.     

Regulation of energy metabolism in Saccharomyces cerevisiae. Relationships between catabolite repression, trehalose synthesis, and mitochondrial development.

Changes in trehalose accumulation and in cytochromes during diauxic growth in glucose medium were examined in a normal Saccharomyces cerevisiae strain. While no appreciable disaccharide accumulation occurred during most of the logarithmic phase, a rapid synthesis took place during the final stages. The intrinsic capacity of cells to accumulate trehalose was also determined under nonproliferating conditions, in glucose medium lacking a nitrogen source. Cells harvested at an early growth stage had a much lower trehalose accumulation capacity than cells taken after glucose was exhausted from the culture medium. A high trehalose accumulation capacity could also be obtained at any growth stage by using maltose or galactose as carbon source. Since cells grown under various conditions exhibit a correlated change in cytochrome development and in trehalose accumulation capacity, it was concluded that the level of glucose repression determines the concentration and/or state of activation of the trehalose synthetase-trehalase complex. Independent control of trehalose accumulation capacity and mitochondrial biogenesis by the level of glucose repression was shown in two ways: by demonstrating derepression of trehalose accumulation without development of cytochromes a and c in microaerobic cells, and by showing repression-dependent changes in a cytoplasmic respiration-deficient (ρ^?) mutant, which lacked functional mitochondria. Therefore, the capacity of a cell to accumulate trehalose is not regulated solely by the supply of ATP generated by oxidative phosphorylation.     

Secondary structure in transfer RNA genes

The bacterial strand of the heteroduplex of λh80 dglyTsu⁺₃₆tyrTthrT with λh80 carries a cluster of three transfer RNA genes. The bacterial strands of the heteroduplexes of φ80hpsu^+,?_III and φ80hpsu^?_III with φ80h carry two and one genes for tyrosine tRNA, respectively. When these heteroduplexes are spread under weakly denaturing conditions (low formamide), secondary structure features consisting of one or several closely clustered, short duplex regions (folds) are observed. The features map at the positions of the tRNA gene clusters. They are not seen if the DNA is hybridized to Escherichia coli tRNA. It is concluded that the secondary structure features are due to self-complementary sequences in the tRNA genes. In some cases, the duplex folds appear to involve base pairing between sequences on different tRNA genes of a cluster and may also involve the spacer sequences between the tRNA sequences.     

Effect of urethan on polyamine and DNA synthesis in the regenerating rat liver

When a single dose of urethan was injected into the peritoneal cavity of rats immediately after partial hepatectomy, DNA synthesis was delayed by 12 h. The induction of ornithine decarboxylase which was induced biphasically following partial hepatectomy was also reduced and delayed by 14–15 h by the administration of urethan. S-Adenosylmethionine decarboxylase activity in urethan-treated rat liver at 20 h and 29 h after operation was significantly lower than that of untreated animals. This enzyme activity was shown to increase thereafter, reaching a higher level than in untreated rats at 37–42 h. Hepatic spermidine content changed biphasically in a manner similar to DNA synthesis. These results suggest that the activities of ornithine decarboxylase and S-adenosylmethionine decarboxylase may correlate with DNA synthesis and that an increase of spermidine concentration is necessary to DNA synthesis.