Marshall W. Nirenberg

Monoclonal antibody A2B5 reacts with many gangliosides in neuronal tissue

Monoclonal antibody A2B5, clone 105, obtained from a mouse immunized with 8-day chicken embryo retinas, reacts with most neuronal cells in chicken retina as revealed by immunofluorescence studies [G.S. Eisenbarth, F.S. Walsh, and M. Nirenberg (1979), Proc. Natl. Acad. Sci. USA 75: 4913-4917]. The antibody binds to many antigens, presumably gangliosides, present in ganglioside fractions from chicken brain, chicken retina, and human brain, as detected by autoradiography of thin-layer chromatograms. Most of the antigens, which are found in the mono-, di-, tri-, and polysialoganglioside fractions, do not correspond in chromatographic mobility to any of the major gangliosides of these tissues, as revealed by orcinol reagent. Apart from the fact that only one neuraminidase-labile sialyl residue is required for binding, the carbohydrate sequence recognized by the antibody is not known. There is a qualitative and quantitative change in the ganglioside antigens in chicken brain and retina during development. The less sialylated antigens appear first, followed by the more sialylated ones. The adult tissues contain little ganglioside antigen.

Ornithine and glutamic acid decarboxylase activities in the developing chick retina.

Abstract— γ‐Aminobutyric acid was found in 6‐day‐old chick embryo retina; approx 20% was formed from putrescine and the remainder from glutamic acid. However, at the 18th embryonic day only 1% of the γ‐aminobutyric acid synthesized in retina was derived from putrescine. These results show that γ‐aminobutyric acid is synthesized in chick embryo retina prior to synaptogenesis, and that γ‐aminobutyric acid is synthesized via two pathways. The first pathway involves the conversion of putrescine to γ‐aminobutyric acid; the second path is dependent upon the conversion of glutamic acid to γ‐aminobutyric acid, catalyzed by glutamic acid decarboxylase.

Fine Structure of RNA Codewords Recognized by Bacterial, Amphibian, and Mammalian Transfer RNA

Nucleotide sequences of 50 RNA codons recognized by amphibian and mammalian liver transfer RNA preparations were determined and compared with those recognized by Escherichia coli transfer RNA. Almost identical translations were obtained with transfer RNA from guinea pig liver, Xenopus laevis liver (South African clawed toad), and E. coli. However, guinea pig and Xenopus transfer RNA differ markedly from E. coli transfer RNA in relative response to certain trinucleotides. Transfer RNA from mammalian liver, amphibian liver, and amphibian muscle respond similarly to trinucleotide codons. Thus the genetic code is essentially universal, but transfer RNA from one organism may differ from that of another in relative response to some codons.

Regulatory mechanisms and protein synthesis X. Codon recognition on 30 s ribosomes

Abstract Characteristics of aminoacyl-sRNA binding to 30 s ribosomes were studied. The specificity of RNA codon recognition on 30 s ribosomes resembled the specificity observed with 70 s ribosomes in response to polynucleotide templates; however, at 0 °C, aminoacyl-sRNA was bound to 70 s ribosomes but not to 30 s ribosomes. In addition, the pH optimum of the rate of binding to 30 s ribosomes is pH 6.8 whereas the optimum of the rate of binding to 70 s ribosomes is about pH 9; and Tris—acetate inhibits the binding to 30 s ribosomes at concentrations which do not affect the binding to 70 s ribosomes. Trinucleotide and oligonucleotide templates appear to be active in stimulating aminoacyl-sRNA binding to 30 s ribosomes, but only after incubation at 37 °C of the 30 s particles in 0.02 m -magnesium acetate. Under various conditions studied, no isoleucyl-sRNA was stimulated to bind to 30 s ribosomes in the presence of poly U, whereas this binding occurs readily to 70 s ribosomes. Zone centrifugation studies suggest that the 30 s particles employed in these studies consist of at least two populations of ribosomes, only one of which is active in binding aminoacyl-sRNA in response to polynucleotide templates. The addition of 50 s ribosomes to 30 s subunits increased the binding of aminoacyl-sRNA in response to a polynucleotide template 20 to 30%. Studies on the effects of monovalent cations on the binding of phenylalaninesRNA in response to poly U suggest that 30 s ribosomes may contain at least two sites for specific binding of aminoacyl-sRNAs. The binding of aminoacyl-sRNA to one site is stimulated by ammonium or potassium ions; and the ammonium or potassium ion-stimulated binding is inhibited by lithium ions. Aminoacyl-sRNA binding to the second site is relatively unaffected by ammonium, potassium, lithium or sodium ions.

Sequential Translation of Trinucleotide Codons for the Initiation and Termination of Protein Synthesis

Terminal events in protein synthesis were studied with trinucleotide codons. Initiator and terminator trinucleotides sequentially stimulate N-formyl-methionyl-tRNA binding to ribosomes and the release of free N-formyl-methionine from the ribosomal intermediate. The release factor discovered by Capecchi is also required. The trinucleotides UGA, UAA, and UAG were found to be terminator codons. This pattern of codon degeneracy has not been observed with other trinucleotides and transfer RNA.

Muscarinic acetylcholine receptor regulation by accelerated rate of receptor loss

Sustained activation of muscarinic acetylcholine receptors on neuron-like NG108-15 hybrid cells reduces the number of [3H]quinuclidinyl benzilate binding sites per cell as much as 88%. The response occurs at concentrations of agonists commensurate with those needed to occupy receptors and inhibit adenylate cyclase. Decreases in steady-state receptor levels persist as long as activator remains present. Withdrawal of activator results in a slow increase in receptor levels that is blocked by cycloheximide. Activation shortens receptor half-life by a factor of nearly 4, indicating that regulation occurs at the level of receptor breakdown.

A functional genomic screen for cardiogenic genes using RNA interference in developing Drosophila embryos

Identifying genetic components is an essential step toward understanding complex developmental processes. The primitive heart of the fruit fly, the dorsal vessel, which is a hemolymph-pumping organ, has provided a unique model system to identify cardiogenic genes and to further our understanding of the molecular mechanisms of cardiogenesis. Using RNA interference in developing Drosophila embryos, we performed a genomewide search for cardiogenic genes. Through analyses of the >5,800 genes that cover ≈40% of all predicted Drosophila genes, we identified a variety of genes encoding transcription factors and cell signaling proteins required for different steps during heart development. Analysis of mutant heart phenotypes and identified genes suggests that the Drosophila heart tube is segmentally patterned, like axial patterning, but assembled with regional modules. One of the identified genes, simjang, was further characterized. In the simjang mutant embryo, we found that within each segment a subset of cardial cells is missing. Interestingly, the simjang gene encodes a protein that is a component of the chromatin remodeling complex recruited by methyl-CpG-DNA binding proteins, suggesting that epigenetic information is crucial for specifying cardiac precursors. Together, these studies not only identify key regulators but also reveal mechanisms underlying heart development.

RNA codons and protein synthesis: 15. Dissimilar responses of mammalian and bacterial transfer RNA fractions to messenger RNA codons☆

Abstract Unfractionated aminoacyl-tRNA preparations from guinea pig liver and Escherichia coli respond similarly to most, but not to all, mRNA codons. To clarify the dissimilar responses, liver and E. coli aminoacyl-tRNA preparations were fractionated by column chromatography, and the effects of trinucleoside diphosphate codons upon the binding of purified aminoacyl-tRNA fractions to ribosomes were determined. Sixteen species of mammalian aminoacyl-tRNA were found corresponding to seven amino acids. Codon-anticodon relationships for six amino acids were compared. Only seven universal species of aminoacyl-tRNA were found with both E. coli and liver aminoacyl-tRNA. Seven species of aminoacyl-tRNA were obtained from liver that were not detected with E. coli preparations; conversely, five species of aminoacyl-tRNA were obtained from E. coli that were not found with liver preparations. The results also suggest that some organisms may contain little or no aminoacyl-tRNA for certain codons. For example, liver isoleucine-tRNA responds well to AUA; no response was detected with E. coli isoleucine-tRNA. Liver arginine-tRNA responds well to AGG, but was deficient in response to AGA; E. coli arginine-tRNA was relatively deficient in response to AGA and AGG. One species of E. coli cysteine-tRNA recognizes UGU, UGC and UGA; under identical conditions liver cysteine-tRNA responds to UGU and UGC, but not to UGA. A species of serine-tRNA responding to UAG was obtained from E. coli CR63, but not from E. coli B. These findings are discussed in terms of tRNA suppressors and possible mechanisms for regulating protein synthesis. Mammalian and bacterial aminoacyl-tRNA fractions respond to sets of codons according to wobble base-pairing. Another pattern of degeneracy, in which A, G or U may occupy the third position of synonym triplets, was observed with two peaks of E. coli serine-tRNA.

Use of aminopterin in selecting electrically active neuroblastoma cells

Abstract A marked increase in electrical excitability and process formation occurs in the N-18 clone of mouse neuroblastoma as these cells go from the logarithmic phase of growth to the stationary state in confluent cultures. Even more excitable cells can be selected by growth in culture medium containing 10 −5 M aminopterin which kills about 90% of the cells. Clone 1A-103 does not develop significant processes or exhibit marked electrical excitability under any of the culture conditions studied. Thus, our results show that one or more of the steps required for generation of the action potential is sensitive to regulation in cultured cells. Methods are presented for obtaining populations of either electrically passive cells or electrically excitable cells which can easily be maintained for several weeks. Clones differ markedly in their capacity to extend processes and their ability to generate action potentials.

Opiate receptors as regulators of adenylate cyclase.

This monograph reports on the progress of Nirenbergs neuroblastoma research. The authors find that morphine inhibits enzymatic activity and enzyme production levels. Additionally, hybrid cells show that the number of opiate receptors mediates inhibition of morphine. Results suggest that because drugs may act as enzyme inducers, recovery from the addicted state requires return of adenylate cyclase activity to normal levels.