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.     

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.     

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.     

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.     

Half-lives of messenger RNA species during growth and differentiation of Dictyostelium discoideum

The half-lives of functional messenger RNAs were determined by a method employing the drugs actinomycin D and daunomycin for the inhibition of mRNA synthesis; the activity of extracted mRNAs was determined by an in vitro translation assay. Several controls indicated that this method yielded reliable values for mRNA half-lives; in particular, the declining rate of protein synthesis in the presence of the drugs is due predominantly to the decay of translatable mRNA. This method was used to determine the half-lives of two specific mRNAs—encoding actin and a protein of MW 51,000—as well as that of total cytoplasmic mRNA activity during growth and at several times in differentiation. The half-lives of at least these two mRNAs were shown to be distinctly different from that of the total mRNA population—about 4 hr. However, no significant change in any of these half-lives was observed between growing and developing cells. Therefore wholesale alterations in the degradation rates of total and at least specific messages do not appear to play a role in the regulation of gene expression during Dictyostelium development.     

Induction of translationally active rat liver glutathione S-transferase B messenger RNA by phenobarbital

Liver poly(A⁺)-RNA was isolated from untreated and phenobarbital-treated rats and translated in cell-free systems derived from wheat germ and rabbit reticulocyte lysates. The primary translation product of glutathione S-transferase B was comprised of two nonidentically sized subunits which comigrated on SDS-polyacrylamide gels with the purified glutathione S-transferase B subunits. The level of translatable glutathione S-transferase B mRNA in rat liver was elevated approximately 3 to 4-fold by phenobarbital administration. Our data suggest that chronic phenobarbital administration to rats increases the amount of cytosolic glutathione S-transferase B via an increase in the functional mRNA level encoding for the enzyme.     

Cell-free Cytoplasmic Polyadenylation of Oogenic RNA

The products of cell-free ATP incorporation mediated by cytoplasmic fractions prepared from unfertilized sea urchin eggs, anucleate egg halves, nucleate egg halves, emetine-treated fertilized eggs, and four-cell embryos have been characterized to determine to what extent the polymers synthesized are poly(A) and to assess the size distribution of the primers adenylated. As judged by alkaline lability, ribonuclease resistance, and retention on poly(U)-impregnated filters, greater than 92% of the label recovered after RNA extraction is present in poly(A). LiCl fractionation indicates that little, if any, free poly(A) is synthesized or cleaved from RNA primers during the reaction, and that 4S RNA is not an effective initiator. In excess of 85% of the poly(A) is associated with RNA having S-values greater than or equal to 18S. Sedimentation profiles of RNA adenylated in the unfertilized egg and anucleate egg half reactions are identical. Suppression of in vivo protein synthesis by emetine alters the profile of RNA subsequently adenylated in vitro. It is proposed that the apparent constraints on the utilization of cytoplasmic RNA or ribonucleoprotein primers of oogenic origin may be effected by RNA-associated proteins capable of regulating the selection and/or extent of their polyadenylation during early embryogenesis.     

The role of hapten-reactive T lymphocytes in the induction of autoimmunity in mice. I. Establishment of the experimental conditions for the termination of immunological tolerance to human gamma globulin.

In order to study the role of hapten-reactive helper T cells in the induction of autoimmunity in mice, an attempt was made to establish an experimental model for the development of hapten-reactive helper T cells and the termination of immunological tolerance against heterologous proteins. Spleen cells taken from mice which were immunized with hapten-isologous protein conjugates (PAB-MGG) demonstrated helper activity for the anti-DNP antibody response of DNP-primed B cells responding to DNP and PAB-conjugated protein, but spleen cells from hapten-heterologous protein conjugate (PAB-HGG)-primed mice could not respond to PAB-determinant. Thus, hapten-reactive helper T cells can develop in mice by the immunization with hapten-isologous protein conjugate, but not with hapten-heterologous protein conjugate. However, spleen cells from mice which had been rendered tolerant by treatment with 2.5 or 0.2 mg of DHGG and then immunized with PAB-HGG could demonstrate helper activity responding to PAB-determinant. This helper activity was PAB-specific, because these spleen cells did not demonstrate helper activity if PAB-determinant was omitted in the primary and the secondary antigen. This helper activity was abrogated by the treatment of spleen cells with anti-θ serum and complement. Thus, hapten-reactive helper T cells were successfully induced by the challenge with hapten-heterologous protein conjugate in carrier-protein tolerant mice. When mice were treated with 2.5 or 0.2 mg of DHGG, no anti-HGG antibody response was induced by the challenge with HGG or PAB-HGG. However, the termination of HGG-tolerance was demonstrated only when the mice were preimmunized with PAB-MGG to raise PAB-rcactive helper T cells, treated with 0.2 mg of DHGG, and then challenged with PAB-HGG. This termination of immunological tolerance was not observed when the mice were preimmunized with PAB-BαA to raise PAB-specific B cells and anti-PAB antibody, or when the mice were treated with 2.5 mg of DHGG. Thus, if HGG-specific B cells remain intact in mice such as treated with low dose of DHGG, these B cells can be activated by some bypass mechanisms in the presence of PAB-reactive helper T cells through the PAB-determinant even in the absence of HGG-reactive helper T cells. These data clearly showed the role of hapten-reactive helper T cells in the termination of immunological tolerance and provide experimental supports to the hypothesis on the termination mechanism proposed by Weigle. The cellular mechanism for the development of hapten-reactive helper T cells in tolerant animals and the cellular mechanism of autoantibody production were discussed on the basis of T-B cell collaboration.     

INvestigation of the basis of reduced metabolic cooperation in mec- cells.

The basis for the apparent loss of metabolic co-operation in a line of mec^? cells derived by selection from a mec⁺ line has been investigated. The results indicate that the loss of pyrimidine deoxyribonucleotide co-operation in these cells is due neither to the dilution of transferred nucleotides by enlarged endogenous pools nor to the failure of distinct internal pools to equilibrate, but rather to a deficiency of intercellular junctions. This deficiency can be reversed by treatment of the cells with db-cAMP and theophylline which results in the restoration of co-operation for TdR nucleotides. The results also indicate that the residual junctional sites in mec^? cells can transfer TdR nucleotides under conditions where the mec⁺ donor pools are expanded.     

The influence of chilling temperature alteration of glyoxysomal succinate levels on isocitratase activity from germinating seedlings

Succinate inhibited isocitratase activity to a greater extent in isolated glyoxysomes of cold-sensitive cotton than in cold-tolerant castor bean seedlings. Exposure of either isolated glyoxysomes or intact seedlings prior to glyoxysome isolation to 5°C appeared to alter the permeability of the glyoxysomal membranes to succinate. Endogenous succinate accumulated in glyoxysomes from 5°C treated cotton seedlings but not in similarly treated castor bean seedlings.     

The role of hapten-reactive T lymphocytes in the induction of autoimmunity in mice. II. Termination of self-tolerance to erythrocytes by immunization with hapten-isologous erythrocytes.

When mice which had been primed with hapten-isologous protein conjugate (PAG-MGG) were challenged with PAB-conjugated isologous mouse erythrocytes (MRBC), they developed Coombs positivity and anemia. However, when mice primed with hapten-heterologous protein conjugate (PAG-HGG) were challenged with PAB-MRBC, neither Coombs positivity nor anemia developed.Since it was demonstrated that PAB-reactive helper T cells were generated by immunization with PAB-MGG but not with PAB-HGG, PAB-reactive helper T cells were considered to play a very crucial role in the induction of autoantibody. These results, as a model for autoantibody production in mice, were discussed on the basis of cellular cooperation mediated by a hapten-mechanism, and are consistent with the hypothesis proposed by Weigle for the mechanism of termination of self-tolerance.     

Antigen modulation of the immune response. VI. Rate of large memory cell appearance in lymph nodes and thoracic duct lymph

We have studied the rate of appearance of memory B-cell subpopulations in the antigen draining lymph nodes and thoracic duct lymph of rats using 1g velocity sedimentation and adoptive transfer. Five days after immunization 100% of the memory response was attributable to large cells. By Days 7, 14, 28, and 77 after priming the large cells contribution to the memory population dropped to 86, 35, 15, and 10% respectively. At the same time the small cell contribution rose from 20% on Day 14 to 46% on Days 28 and 77. The same results were obtained with thoracic duct lymphocytes with the large cells contributing 53% of the response on Day 7 and 20% on Day 150. Appropriate controls were included to show that differential suppression was not responsible for these results. Furthermore, when purified large memory cells were passaged through intermediate hosts for 7 to 11 days, between 76 and 81% of the large cells matured into medium or small lymphocytes. These data show that the earliest memory cells formed after antigen encounter are the blast-like large lymphocytes and that these evolve, through a series of antigen-independent events, first into medium and then small lymphocytes. A model of memory cell development incorporating these results and the results of others is presented.     

The in vivo transformation of phospholipid vesicles to a particle resembling HDL in the rat.

The tumor-specific transplantation antigen (TSTA) in crude three molar potassium chloride (3M KCl) extracts of a chemically-induced, murine fibrosarcoma was purified by ammonium sulfate salt fraction at 20% saturation (S20S) and by polyacrylamide gel electrophoresis (PAGE). Mice, which had been pretreated with the S20S precipitate, displayed retarded outgrowth of a 100-fold supralethal dose of the corresponding, but not of a non-cross-reactive syngeneic tumor. Analysis of PAGE gels by Coomassie Blue staining revealed at least 30 bands in the crude 3M KCl extract, and only two components (Rf 0.34 and 0.43) in the ammonium sulfate fraction. That these two components bore TSTA activity was demonstrated by the observation that the immunoprotective activity of crude 3M KCl extracts was localized to the Rf 0.25–0.50 region. The two components present in the S20S fraction had isoelectric points of 5.05 and 6.9, and estimated molecular weights of 40,000 and 75,000, thus demonstrating the soluble nature of the active principle. These findings offer the prospect of a chemical dissection of the polymorphic TSTA surface markers on MCA-induced murine tumors.     

Brain cyclic AMP levels and the initiation of adult development in the Cecropia silkmoth.

A six-fold increase in the level of brain cyclic AMP is observed in chilled Cecropia pharate adults within 24 hr of transfer from the cold to room temperature. This increase is not observed in pupae chilled for a period insufficient to allow initiation of adult development, nor after injury to diapausing pupae. Other tissues show a variable and minor response during initiation. Injected dibutyryl cAMP will cause initiation in insufficiently chilled pupae, but not in dauer pupae. The possible relationship of this rise in cAMP to the process of initiation is discussed.     

Alterations in the developmental patterns of enzymes in chicken embryos infected with West Nile virus.

Infection of chicken embryos with West Nile (WN) virus, a group B togavirus containing structural lipids, caused a rapidly developing hypertriglyceridemia. Changes in the activity of several hepatic regulatory enzymes in glycolytic and lipogenic pathways occurred during infection. Compared to control values in embryos of the same age (16 days), an 8.8-fold increase in the specific activity of ATP-citrate lyase and a 5.6-fold increase in that of hexokinase were observed on the third day of WN virus infection. Hexose monophosphate shunt dehydrogenase specific activities were elevated twofold in virus-infected livers. Activities of malic enzyme and phosphofructokinase were also elevated in WN virus-infected livers. Malate dehydrogenase and NADP-linked isocitrate dehydrogenase levels showed little or no change during infection. The levels of pyruvate kinase and lactate dehydrogenase were decreased in virus-infected livers. Hepatic acetyl-CoA carboxylase activity was at least twofold higher in virus-infected embryos; however, following removal of low-molecular-weight compounds, the specific activities of this enzyme from infected and control embryos were virtually identical. The results of mixing experiments suggest that the low levels of carboxylase activity in control embryos may be due to the presence of enzyme inhibitor(s) which can be removed by gel filtration.The incorporation of radiolabeled precursors into cellular lipids by liver minces from virus-infected and uninfected embryos was measured. There was a twofold increase in carbohydrate incorporation in virus-infected liver as compared to uninfected liver; [¹⁴C]pyruvic acid was incorporated into lipids to the greatest extent. [1-¹⁴C]acetic acid, [U-¹⁴C]alanine, and [U-¹⁴C]leucine were incorporated very poorly in both infected and control livers. Twice as much [1-¹⁴C]oleic acid or [1-¹⁴C oleic]triolein was incorporated in WN-infected livers as in control. The relative distribution of neutral and polar lipids formed from each precursor was generally similar in infected and uninfected livers as determined by thin-layer chromatography of radiolabeled lipids. Except for a threefold increase in oxidation of [¹⁴C]glucose by virus-infected livers, the oxidations of carbohydrates and fatty acids were similar in infected and uninfected livers. The pentose phosphate pathway appears to be the major pathway utilized in glucose oxidation for both control and virus-infected livers. The results indicate that enhanced flux of metabolites into lipids reflects a virus-induced alteration in embryonic development: The enzyme patterns of infected embryos are more characteristic of older embryos or even newly hatched chicks.     

Histamine H2-receptors in the gastric mucosa: role in acid secretion.

Currently, two major hypotheses dominate thinking about the role of histamine in the regulation of gastric acid secretion. Code has proposed that histamine is the final common mediator of secretagogue action on the parietal cell while Konturek and Grossman have suggested a multi-receptor control of the secretory process. Experimental results derived from the use of recently synthesized histamine H₂-receptor antagonists have been used by both groups to support their hypotheses. Paradoxically, these hypotheses depend on the presumed specificity of the H₂-antagonists in blocking histamine mediated acid secretion while the apparent lack of such secretagogue specificity of the H₂-antagonists is an important basis for the development of the hypotheses. Our review will analyze the experimental evidence which implicates the histamine H₂-receptor in the control of hydrogen ion secretion as well as evidence for and against receptor specificity in the gastric mucosa of histamine H₂-receptor antagonists.