Isolated adrenal cortex cells: ACTH agonists, partial agonists, antagonists; cyclic AMP and corticosterone production

Isolated adrenal cortex cells respond to the addition of ACTH_1–39 or analogs with increased production of cyclic AMP and corticosterone. It is estimated that cyclic AMP production need proceed at less than 20% of maximum to induce maximum corticosterone production. ACTH_1–24, [Lys¹⁷, Lys¹⁸]ACTH_1–8 amide, and ACTH_1–16 amide induce a maximum rate of cyclic AMP and of corticosterone production equal to those of ACTH_1–39. The relative potencies as determined by cyclic AMP and by corticosterone production are in excellent agreement. The analog, ACTH_5–24, induces maximum cyclic AMP production equal to 45% of that of the natural hormone, but as predicted, induces maximum corticosterone production equal to that of ACTH_1–39. The derivative, [Trp(Nps)⁹]ACTH_1–39 induces 77% of maximum corticosterone production and less than 1% of maximum cyclic AMP production. The fragment ACTH_11–24 is a competitive antagonist of ACTH_1–39 for both cyclic AMP and corticosterone production. The observations on agonists, a partial agonist and a competitive antagonist are in harmony with the “second messenger” role assigned to cyclic AMP. A provisional model, based on the fit of the experimental observations to a set of equations, provides expressions of “intrinsic activity,” “receptor reserve”, “sensitivity”, and “amplification” in terms of maximum cyclic AMP production, concentration of ACTH which induces $\text{1}\text{2}$ maximum cyclic AMP production and concentration of cyclic AMP which induces $\text{1}\text{2}$ maximum corticosterone production.     

Angular Alteration of the DNA Helix by <Emphasis Type="Italic">E. coli</Emphasis> RNA Polymerase

IT has been a source of speculation whether the reading of the genetic code of DNA by RNA polymerase involves the disruption of the DNA helix. While circuitous evidence favouring either affirmative or negative answer has been accumulating, direct experiments have been few^1–11. Kosaganov et al. investigated the possibility of a local unwinding of DNA during RNA synthesis by measuring the kinetics of formaldehyde-induced denaturation of DNA during RNA synthesis¹². They concluded that the binding of RNA polymerase did not cause local unwinding but RNA synthesis produced “defects” in the double helix. Unfortunately, the interpretation of formaldehyde-induced denaturation is not clear, nor is the nature of a “defect”.     

<Emphasis Type="Italic">De novo</Emphasis> Synthesis of Transfer and 55 RNA<Superscript>1f</Superscript> in Cleaving Sea Urchin Embryos

SIGNIFICANT changes in RNA metabolism have been described during early sea urchin development. Until recently the only detectable class of RNA synthesized during cleavage stages was that with a low G + C base composition and heterogeneous sedimentation properties (DNA-Jike RNA)¹. The genes for nucleolar ribosomal RNA (26S and 18S) were believed to become active only following gastrulation^2–4 and the products of nuclear transfer RNA (4S) genes were first detected at the mesenchyme blastula stage⁵. Any label in the 4S region of sucrose gradients of RNA from the cleavage stages of embryogenesis was interpreted as reflecting the turnover of the pCpCpA region of pre-existing transfer RNA (tRNA) molecules.     

Methylation of Sindbis virus "26S" messenger RNA.

The main virus-specific messenger RNA species of Sindbis virus-infected hamster cells, the “26S” RNA, has been examined with regard to methylation status. Internal methylated residues and terminal methylated residues were present, in approximately equal amounts. The internal methyl groups were almost all in 5-methylcytosine residues and the terminal methyl groups were mainly in 7-methylguanine residues. Evidence is presented that these latter occur in “capped” 5′-termini with the novel structure m⁷G(5′)pppNp.     

Sex Pheromone in the Aphid <Emphasis Type="Italic">Megoura viciae</Emphasis>

THE adult egg-laying females (oviparae) of most holocyclic aphid species bear on their swollen hind tibiae circular plaques or tubercles which taxonomists variously refer to as sensoria, pseudosensoria, or pseudorhinaria. It has been suggested that they aid the ovipara in fixing her eggs to the host plant¹, that they are sensory receptors², or that they produce a sex pheromone which attracts the males^3–5. Pettersson⁶ has advanced preliminary evidence for the latter function in a species of Schizaphis. Recent studies have shown that these structures (Fig. 1) in Megoura viciae Buckton do indeed secrete a sex pheromone and, moreover, that striking changes in the daily pattern of pheromone release occur as the female ages. In this article these pseudosensoria will be referred to as “scent plaques”, an appropriate term first used by Stroyan⁷.     

Dissociation and Reassociation of Infectious Poliovirus Particles

THE first reconstitution of an infectious virion was achieved when Fraenkel-Conrat and Williams¹ obtained the typical rods of tobacco mosaic virus (TMV) from its components, RNA and protein. Later, the conditions for the “self-assembly” of TMV were improved so that up to 50% of the viral RNA could be coated with the protein. The reconstituted TMV was shown to be infectious and indistinguishable from native virions by several criteria². Recent studies revealed that the reconstitution of TMV is a highly specific multi-step procedure, beginning at the 5′-end of the TMV-RNA^3–5. In the past five years a number of spherical plant viruses have also been reconstituted⁶. In experiments with small RNA-bacteriophages very low efficiencies of reconstitution in the range of 10^–8 to 10^–7 p.f.u. (plaque forming units) per input molecule of RNA were obtained^7,8. Reconstitution was improved by the addition of a minor viral protein, the A-protein, to the mixture of RNA and the structural protein. Even so the efficiency of conversion of RNA into infectious particles was in the range of 2×10^–6 (refs. 9 and 10). We have reported the first successful restoration of poliovirus infectivity lost on dissociation of the virion by urea-mercapto-ethanol treatment¹¹. Here we present evidence for reconstitution of infectious poliovirus particles from a mixture of poliovirus RNA and polypeptides.     

The functional roles of m6A modification in T lymphocyte responses and autoimmune diseases

RNA N6-methyladenosine (m⁶A) modification is abundant in eukaryotes, bacteria and archaea. It is an RNA modification mainly existing in messenger RNA (mRNAs) and has a significant effect on the metabolism and function of mRNAs. m⁶A modification is controlled by three types of proteins, namely methyltransferase as the “writers”, demethylase as the “erasers”, and specific m⁶A recognized protein (YTHDF1–3) as the “readers”. Recent studies have shown that m⁶A modification plays an important role in cancer, viral infection and autoimmune diseases. In this review, we will elaborate on the m⁶A modifications in the homeostasis and differentiation of T cells. Then we will further summarize the effects of m⁶A modification on the T cell responses and T cell-mediated autoimmune diseases. This will advance T cell epigenetics research and provide potential biomarkers and therapeutic targets for autoimmune diseases.     

Reconstitution of a GTPase Activity a 50S Ribosomal Protein from <Emphasis Type="Italic">E. coli</Emphasis>

DURING each step of peptide chain elongation the ribosome shifts up one triplet along the messenger RNA with concomitant movement of the peptidyl-transfer RNA from the donor to the acceptor site. This process, commonly known as translocation, is triggered by a supernatant protein, factor G, which in association with the ribosome cleaves GTP into GDP and inorganic phosphate^1,2 and it has been argued that the energy liberated in this reaction is used “to carry the complex one triplet forward”³.     

Separation of α- and β-Globin Messenger RNAs

THE 10S RNA fraction of reticulocytes from various species contains the haemoglobin messenger RNA^1–4. When this 10S RNA fraction is added to a cell-free system derived from reticulocytes or Krebs II ascites cells, it directs the synthesis of α and β chains of haemoglobin^5–8. The α and β messenger RNA molecules contained in this fraction, however, have not yet been separated and identified. When reticulocyte. RNA of mouse is subjected to electrophoresis on 6% polyacrylamide gels, the 10S fraction contains two major bands and three minor bands⁹, suggesting that the major lOS RNA bands contain the messenger RNAs for the α- and β-globin chains.     

Molecular Basis for Repressor Activity of Qβ Replicase

WITH the purification and characterization of viral replicases, a novel feature of nucleic acid polymerases—stringent template specificity—was recognized^1,2. Qβ replicase, the most extensively studied viral RNA polymerase^2–8, is now known to replicate Qβ RNA², the complementary Qβ minus strand⁹, RNA molecules described as “variants” of Qβ RNA^10,11 and a set of small RNAs of unknown origin which accumulate in Qβ-infected Escherichia coli, collectively designated as “6S RNA”¹². On the other hand, the RNA from phages related distantly, if at all, to Qβ^13,14, such as MS2 or R17 and of other viruses such as TMV² or AMV (Diggelmann and Weissmann, unpublished results) are completely inert as templates, as are ribosomal and tRNA from E. coli². Poly C and C-rich synthetic copolymers at high concentrations elicit synthesis which, however, remains restricted to the formation of a strand complementary to the template^15,16.     

Hybridization maps of early and late messenger RNA sequences on the adenovirus type 2 genome.

Specific fragments of adenovirus type 2 DNA, generated by cleavage with restriction endonucleases endoR.EcoRI, endoR.HpaI and endoR.HindIII were used in hybridization-mapping experiments. The complementary strands of individual cleavage fragments were separated by the method of Tibbetts &; Pettersson (1974). Liquid hybridizations were performed with ³²P-labeled separated strands of cleavage fragments and messenger RNA extracted from cells early and late after adenovirus infection. The fraction of each fragment strand which was represented in “early” and “late” messenger RNA was determined by chromatography on hydroxylapatite. Early messenger RNA was found to be derived from four widely separated regions, two on the 1- and two on the h-strand (h- and l- refer to the strand with heavy and light buoyant density in CsCl when complexed with poly(U, G)). Messenger RNA, present exclusively late after infection, is derived from several locations, predominantly from the l-strand with a major block of continuous sequences extending between positions 0.25 and 0.65 on the unit map of the adenovirus type 2 genome.     

The size and genetic composition of virus-specific RNAs in the cytoplasm of cells producing avian sarcoma-leukosis viruses.

The genome of avian sarcoma virus (ASV) contains four known genes: gag, encoding structural proteins of the viral core; pol, encoding the viral RNA-directed DNA polymerase; env, encoding the glycoprotein(s) of the viral envelope; and src, which is responsible for neoplastic transformation of the host cell. We have located these genes on virus-specific RNAs in cells productively infected with both nondefective and defective strains of ASV by using molecular hybridization with DNAs complementary to specific portions of the ASV genome.The cytoplasm of cells producing nondefective ASV contains three species of polyadenylated virus-specific RNA, each of which has chemical polarity identical to that of the viral genome. The largest species has a molecular weight of 3.3 × 10⁶ daltons and a sedimentation coefficient of 38S, encodes all four viral genes, and is probably identical to the viral genome. A second species has a molecular weight of 1.8 × 10⁶ daltons and a sedimentation coefficient of 28S, and encodes the 3′ half of the viral genome, including env, src and a genetically silent region known as “c.” The smallest species has a molecular weight of 1.2 × 10⁶ daltons and a sedimentation coefficient of 21S, and encodes only src and “c.” All three species of virus-specific RNA contain nucleotide sequences at least partially homologous to a sequence of 101 nucleotides found at the extreme 5′ end of the ASV genome. This sequence may not be present in the portions of the ASV genome which encode the 28S and 21S virus-specific RNAs, and hence may be joined to these RNAs during their maturation from precursor molecules.The size and genetic composition of virus-specific RNAs in cells producing defective deletion mutants reflect the nature of the deletion. Deletions of either src or env eliminate the 28S virus-specific RNA, leaving a 21S RNA (which contains either env and “c” in the case of src deletions or src and “c” in the case of env deletions) and a 35S RNA which is probably identical to the viral genome.Based on these and related results, we propose a model for viral gene expression which conforms to previous suggestions that eucaryotic cells initiate translations only at the 5′ termini of messenger RNAs.     

Adenyl Cyclase Activity in Cultivated Human Skin Fibroblasts

CYCLIC 3′,5′-AMP (cyclic AMP) is an important regulator of cellular processes^{1, 2}. In target cells, hormones stimulate adenyl cyclase, an enzyme which catalyses the conversion of ATP to cyclic AMP, which acts as a “second messenger” on either a membrane or an enzyme system and produces a specific physiological response. Hormonal stimulation of adenyl cyclase in several tissues has been documented².     

Ribonucleic acid synthesis in streptomyces antibioticus: Stable ribonucleic acid species synthesized by young and old cells

In studies of RNA synthesis by intact cells and cell-free extracts of $\text{Streptomyces antibioticus}$, it has been found that 48 hr cells (producing actinomycin) and cell-free extracts are less efficient than 12 hr cells (not producing actinomycin) and extracts in the synthesis of RNA. Analysis of the products of “in vivo” and “in vitro” RNA synthesis by sucrose gradient centrifugation reveals that both 12 and 48 hr cultures and cell-free extracts synthesize ribosomal RNA as well as RNA species of higher and lower molecular weights. However, 50–60% of the ³H-uridine labelled RNA synthesized by intact cells sediments as rRNA as compared with only 5–10% of the cell-free product. The addition of 2 × 10^?5 M actinomycin D to incubation mixtures for cell-free RNA synthesis does not significantly alter the relative amounts of the various RNA species synthesized by 12 or 48 hr extracts.     

Possible Cytoplasmic Precursor of Haemoglobin Messenger RNA

THE “rapidly labelled” RNA of immature erythroblasts includes a rather homogeneous high specific activity RNA in the 9S region of linear sucrose gradients, but when the same RNA is assayed for ability to stimulate protein synthesis in a cell-free system, the peak of activity is found just trailing the 18S ribosomal RNA^1,2. Evidence has been assembled to support the contention that the 9S species of RNA is the haemoglobin messenger RNA³. While investigating the 9S RNA of chicken erythroblasts, we have found conditions in which a well defined rapidly labelled RNA peak could be observed in the 9S and/or the 17S region of the gradient. The concentration of pulse labelled RNA in the 17S region has been reported in diverse systems⁴ and may be a general phenomenon. It is particularly striking in the erythroblast system in which background ribosomal RNA synthesis is at a minimum.     

A novel riboregulator switch system of gene expression for enhanced microbial production of succinic acid

In this paper, a novel riboregulator Switch System of Gene Expression including an OFF-TO-ON switch and an ON-TO-OFF switch was designed to regulate the expression state of target genes between “ON” and “OFF” by switching the identifiability of ribosome recognition site (RBS) based on the thermodynamic stability of different RNA–RNA hybridizations between RBS and small noncoding RNAs. The proposed riboregulator switch system was employed for the fermentative production of succinic acid using an engineered strain of E. coli JW1021, during which the expression of mgtC gene was controlled at “ON” state and that of pepc and ecaA genes were controlled at the “OFF” state in the lag phase and switched to the “OFF” and “ON” state once the strain enters the logarithmic phase. The results showed that using the strain of JW1021, the yield and productivity of succinic acid can reach 0.91 g g^?1 and 3.25 g L^?1 h^?1, respectively, much higher than those using the strains without harboring the riboregulator switch system.