Skip to main contentU.S. National Library of MedicineU.S. National Library of Medicine


Profiles in Science
   
Pinterest badge Follow Profiles in Science on Pinterest!

The Marshall W. Nirenberg Papers

Title:
Laboratory project: "Cell Recognition and Synapse Formation" pdf (562,114 Bytes) transcript of pdf
Laboratory project: "Cell Recognition and Synapse Formation"
Description:
Item is a photocopy.
Number of Image Pages:
5 (562,114 Bytes)
Date:
1986-09 (September 1986)
Creator:
Nirenberg, Marshall W.
Hilt, Dana
Chin, Hemin
Krueger, Karl
Bray, Patricia
Amaladoss, Benjamin
Yano, Koh
Trisler, G. David
National Heart, Lung, and Blood Institute. Laboratory of Biochemical Genetics
Rights:
This item is in the public domain. It may be used without permission.
Exhibit Category:
From Neuroblastoma to Homeobox Genes, 1976-1992
Relation:
Metadata Record Annual Report of the Laboratory of Biochemical Genetics, October 1, 1985 - September 30, 1986 (September 1986)
Box Number: 6
Folder Number: 30
Unique Identifier:
JJBBSV
Accession Number:
2001-013
Document Type:
Reports
Excerpts
Language:
English
Format:
application/pdf
image/tif
Physical Condition:
Poor
Series: Series III: Laboratory Administration, [1959]-1993
SubSeries: Annual Reports, 1960-1993
Folder: Annual Reports, 1960-1988
Transcript:
Project Number Z01 HL 00009-12 LBG
October 1, 1985 - September 30, 1986
Cell Recognition and Synapse Formation
Principal Investigator:
Marshall Nirenberg, Chief, LBG, NHLBI
Dana Hilt, Staff Fellow, LBG, NHLBI
Hemin Chin, Guest Worker, LBG, NHLBI
Karl Krueger, Staff Fellow, LBG, NHLBI
Patricia Bray, Biologist, LBG, NHLBI
Benjamin Amaladoss, Visiting Fellow, LBG, NHLBI
Koh Yano, Visiting Fellow, LBG, NHLBI
David Trisler, Staff Fellow, LBG, NHLBI
Cooperating Units (if any):
Allen Spiegel, Chief, MDB, NIADDK
Bruce Schrier, LDN, NICHD
Lou Hirsch, Dept. of Biochem., U. of Texas, Dallas, Texas
Lab/Branch: Laboratory of Biochemical Genetics
Section: Section of Molecular Biology
Institute and Location: NHLBI, NIH, Bethesda, Maryland 20205
Total Man Years: 11
Professional: 9
Other: 2
Summary of Work:
[lambda]gtll cDNA libraries derived from human brain poly A+ RNA were screened for recombinants that code for [alpha]-subunits of G signal transduction proteins. Eleven [alpha]s and two [alpha]i clones were characterized. Four species of [alpha]s cDNA were found. A mechanism for generating the four species of [alpha]s mRNA by alternative splicing of precursor RNA was proposed.
Treatment of NG108-15 neuroblastoma-glioma hybrid cells cAMP for several days results in there appearance of voltage-sensitive calcium channels and other ions channels. Twenty cDNA clones were obtained that hybridize to species of poly A+ RNA that increase in abundance 10-90 fold due to treatment of cells with dibutyryl cAMP. Affinity-purified antibodies to the a or y protein subunits of voltage-sensitive calcium channels were used to screen a [lambda]gtll cDNA library. Twenty putative voltage-sensitive calcium channel a subunit cDNA clones and 29 putative y subunit clones were found.
Antigenic molecules termed TOP, which are distributed in a dorsal > ventral concentration gradient in chicken retina, were purified. TOP was shown to be a protein with an Mr of 47,000. The gradient of TOP in the retina is formed early in embryonic development. Thereafter, perpetuation of the gradient does not depend on the continuous presence of an extracellular gradient of diffusible molecules or on maintenance of interactions between cells. Synapses and neuritis in the retina of developing chick embryos were reduced markedly by injection of anti-TOP antibody into the eye.
Project Description:
Objectives:
Our objective is to discover basic mechanisms that regulate the expression of genes.
Major Findings:
Two [lambda]gtll cDNA libraries from human brain were screened with 3 oligodeoxynucleotide probes for recombinants coding for [alpha] subunits of G signal transducing proteins, which couple receptors activated by hormones or light to effectors such as adenylate cyclase or cGMP phosphodiesterase. Fourteen of the 575,000 recombinant clones screened from a human cerebral cortex library were detected with 2 or 3 of the 32P-probes used. DNA inserts from 13 positive clones were sequence partially; 11 clones were identified as [alpha]s cDNA and 2 clones as [alpha]i. The DNA insert from one of the [alpha]s clones was sequenced completely and additional partial sequences were obtained for 10 [alpha]s clones. Four species of [alpha]s cDNA were found that differ in nucleotide sequence in the region that corresponds to [alpha]s amino acid residues 71-88. The clones differ in the codon for [alpha]s amino acid residue 71 (glutamic acid vs. aspartic acid), the presence or absence of codons for the next 15 amino acid residues, and the presence or absence of an adjacent serine residue. A mechanism was proposed for generating 4 species of [alpha]s mRNA by alternative splicing of precursor or RNA transcribed from a single genre.
cDNA from one of the two human [alpha]i clones was sequenced completely (BG-4), and a partial sequence was obtained for the second clone. The first nucleotide residue of BG-4 [alpha]i cDNA corresponds to the 14th residue of the bovine [alpha]i coding sequence and the last residue of BG-4 (1261) is in the 3'-untranslated region. The amino acid sequence derived from the nucleotide sequences reported by others. In addition, the 3'-untranslated regions of BG-4ai cDNA is highly homologous to the 3'-untranslated regions of bovine and rat [alpha]i cDNA. The 3'-untranslated nucleotide sequences of human, bovine, and rat [alpha]s cDNAs also are highly conserved, but differ markedly from [alpha]i 3'-untranslated sequences. These results suggest that the 3'-untranslated regions of [alpha]s and [alpha]i genes and/or mRNA are needed for functions that have not been identified thus far.
In previous studies we have shown that elevation of cAMP levels of NG108-15 neuroblastoma-glioma hybrid cells or neuroblastoma cells for several days results in 10-100 fold increases in the activity of voltage-sensitive calcium channels, 15-45 fold increases in the abundance of synapses with cultured striated muscle cells. In addition, the number of molecules of voltage-sensitive calcium channel protein subunit that binds [3H]-nitrendipine increases 12-fold. We previously obtained about 100 cDNA clones that hybridize to species of mRNA that are more abundant in NG108-15 or NS20-Y cells that had been treated with dibutyryl cAMP for several days then in untreated control cells. Quantitative studies on the extent of increase in abundance of the species of mRNA that respond to dibutyryl cAMP were performed using the cloned cDNA as probes. Twenty cDNA clones were obtained that hybridize to species of poly A+ RNA that increase in abundance 10-90 fold due to treatment of cells with dibutyryl cAMP. Northern blots also were performed and the number of bands of poly A+ RNA that hybridize to each clone cDNA probe and their chain lengths were determined.
Affinity purified antibodies to the a, b, and y protein subunits of voltage-sensitive calcium channels were used to screen a [lambda]gtll cDNA library prepared from poly A+ RNA from rat skeletal muscle. Approximately 20 recombinant clones were found that were identified tentatively as calcium channel a subunit cDNAs. Other cDNA clones were obtained that are putative [Upsilon] subunit clones.
In previous studies a putative cDNA clone for choline acetyltransferase was found. We now have determined the nucleotide sequence of the 1118 bp DNA insert. Partial amino acid sequences of several peptidases were obtained in collaborative studies by Lou Hirsh and his colleagues in Dallas. The [lambda]gtll cDNA library was screened again with 2 new oligodeoxynucleotide probes to different regions of choline acetyltransferase and cDNA clones were obtained that were recognized by both probes. Further studies with these clones are in progress.
Antigenic molecules termed TOP, which are distributed in a dorsal > ventral concentration gradient in chicken retina, are expressed early in development (by 48 hr after fertilization) in the optic cup of chicken embryos and continue to be expressed in retina thereafter. 35S-labeled-TOP-antibody complexes were purified by protein A-Sepharose column chromatography and subjected to NaDodSO4/polyacrylamide gel electrophoresis and autoradiography. TOP also was purified from dorsal retina by anti-TOP IgG-Affigel 10 affinity column chromatography. Both purification methods yielded one major band of protein with an Mr of approximately 47,000. A protein of Mr approximately 47,000 also was purified from chicken embryo brain. Cultured cells dissociated from 8-day chicken embryo retinas accumulated the amount of TOP expected of cells in the intact retina, depending on the position of the cells in the retina. TOP accumulations by cells dissociated from dorsal or ventral retina, mixed in different proportions and cocultured were additive. These results show that TOP is a protein that the gradient of TOP is established early in development, and that perpetuation of the gradient does not depend on the continuous presence of an extracellular gradient of diffusible molecules or on maintenance of interactions between cells. Synapses and neuritis in the retina of developing chick embryos were reduced markedly by injection of anti-TOP antibody into the eye
The addition of bradykinin to NG108-15 cells was shown in previous studies to increase cellular levels of inositol-1,4,5-trisphospahte (IP3) and diacyclglycerol. The newly synthesized IP3 in turn stimulates the release of stored calcium ions into the cytoplasm, thereby activating calcium-dependent K+ channels. The increased efflux of K+ ions results in cell hyperpolarization. This is followed by cell depolarization due to inhibition of M channels, thereby decreasing the rate of K+ efflux from cells via M channels. Additional results now show that inhibition of M channels is due to diacylglycerol and Ca2+ dependent activation of protein kinase C. Several phosphoproteins were detected by two dimensional gel electrophoresis whose synthesis is dependent upon the addition of bradykinin to cells. Whereas, injection of inositol 1,4,5-trisphosphate inside NG108-15 cells results in the release of stored calcium in to the cytoplasm, injection of inositol 1,3,4-triphosphate or inositol 1,3,4,5-tetrakisphosphate has little or no effect on calcium mobilization, but instead results in the activation of nonspecific cation channels. Calcium ions are not required for the activation of the nonspecific cation channels. The nature and significance of these findings warrant further investigation in the light of recent reports that inositol 1,3,4,5-trisphosphate and inositol 1,3,4,5-tetrakisphosphate are present in some tissues and that inositol 1,3,4,5-tetrakisphosphate is synthesized by phosphorylation of inositol 1,2,5-trisphosphate, catalyzed by an appropriate kinase, and that inositol 1,3,4-trisphosphate is formed by dephosphorylation of inositol 1,3,4,5-tetrakisphosphate.
Publications:
1. Higashida, H., Streaty, R.A., Klee, W. and Nirenberg, M.: Bradykinin-activated Transmembrane Singals are Couple via No or Ni to Production of Inositol 1,4,5-trisphosphate, a Second Messenger in NG108-15 Neuroblastoma-glioma Hybrid Cells. Proc. Natl. Acad. Sci., USA 83, 942-946 (1986).
2. Grunwald, G.B., Gierschik, P., Nirenberg, M. and Spiegel, A.: Detection of a-Trasnducin in Retinal Rods but not Cones. Science 231, 856-859 (1986).
3. Moskal, J.R., Trisler, D., Schneider, M.D., and Nirenberg, M.: Purification of a Membrane Protein Distributed in a Topographic Gradient in Chicken Retina. Proc. Natl. Acad. Sci., USA 83, 4730-4733 (1986).
4. Dubois, C., Magnani, J.L., Grunwald, G.B., Spitalnik, S.L., Trisler, G.D., M. Nirenberg, and Ginsburg, V.: Monoclonal Antibody 18B8, Which Detects Synapse-associated Antigens, Binds to Ganglioside Gt3 (II3 (NeuAc)3LacCer). J. Biol. Chem. 261, 3826-3830 (1986).
5. Dubois, C., J.L. Magnani, G.B. Grunwald, S.L. Spitalnik, D. Trisler, M. Nirenberg, and V. Ginsburg. 1985. Monoclonal antibody 18B8, which detects synapse associated antigens, binds to ganglioside GT3 [II3 (NeuAc)3LacCer]. In Glycoconjugates: Proceedings of the VIIIth International Symposium, E.A. Davidson, J.C. Williams, and N.M. DiFerrante, eds. Vol.2, p. 5545, Praeger Publ., New York.
6. Bray, P., Carter, A., Simons, C., Guo, V., P:uckett, C., Kamholtz, J., Spiegel, A., and Nirenberg, M.: Human cDNA Clones for Four Species of Gas Signal Transduction Protein. Proc. Natl. Acad. Sci., USA (In Press).
7. Trisler, D. Synapse formation in retina is influenced by molecules that identify cell position. Current topics in Developmental Biology (In Press).
8. Trisler, D. Synapse formation in avian retina is inhibited by antibody to molecules that identify cell position. Amer. Zool. (In Press).
9. Trisler, D., J. Bekenstein, and M.P. Daniels. Antibody to a molecular marker of cell position inhibits synapse formation in retina. Proc. Natl. Acad. Sci., USA, 83: 4194-4198 (1986)
10. Higashida, H. and Brown, D.A.: Two Polyphosphatidyl Metabolites Control Two K+-Currents In A Neuronal Cell. Nature (In Press).
11. Higashida, H. and Brown, D.A.: Membrane Current Repsonses to Intracellular Injections of Inositol 1,3,4, 5-tetrakisphosphate and Inositol 1,3,4-trisphosphate in NG108-15 Cells. (Submitted to FEBS Letters).
Metadata Last Modified Date:
2010-10-15
Linked Data:
RDF/XML     JSON     JSON-LD     N3/Turtle     N-Triples