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Molecular genetic approach to the study of catecholamines
428
34Molecular
genetics
approach
to the nicotinic
acetylcholine
receptor
(AchR).
GIRAUDAT
J., DEVILLERS-THIERY A., KLARSFELD A. and CHANGEUX J.P. Laboratoire de Neurobiologie Moleculaire Institut Pasteur, 75724 PARIS t:EDEX 15 The nicotinic AchR from T. marmorata electric organ consists of five transmembrane polypeptide chains in the stoiechiometry a2byd . cDNA clones coding for the esubunit, which carries the acetylcholine binding site, wera isolated from a cDNA library constructed from T. marmorata electric organ mRNA (Giraddat.s., 1982, EMS0 J., 1, 713-717). Screening of the clones was done in two successive steps : 1) differentyal colony hybridization enabled us to select clones corresponding to mP.NA species present specifically in T. marmorata electric organ but not in T. marmorata spleen or liver; 2) final identification was done by immunoprecipitating the translation product of singlecDNA clones using monospecific antisera. The complete aminoacid sequence of T. marmorata a-subunit precursor was deduced from nucleotide sequence analysis. We used it to develop a model for the transmembrane organization of the subunit and to propose a possible structure of the ionic channel in the pentameric receptor protein (Devillers-Thiery et al., 1983, Proc. Nat. Acad. Sci. USA, 80, 2067-2071). 3ur results will be discussed w=egard to the data obtained by authors on T. californica AchR. Using Southern blot hybridization, we demonstrated that T. californica eenome contains a single eene coding for the AchR CI-subunit (Klarsfeld et., 1984, &ill0 J., _?, 35-41). Structural and developmental implications will be presented. v
35
MOLECULAR
GENETIC
J. Mallet+,
APPROACH
A. Lamouroux,
u
TO THE STUDY OF CATECHOLAMINES N. Saucon Biguet,
B. Grima and J.F. Julien
(Gif-sur-Yvette,
France)
ABSTRACT
36
NOT RECEIVED
NEUROPEPTIDE DYNAMICS STUDIED IMPLICATIONS. I. Mocchetti, J.P. Schwartz and E. Costa
WITH
A
cDNA
PROBE:
PHARMACOLOGICAL
Brain neuropeptides participate in synaptic function either as primary transmitters or as cotransmitters. In order to define their participation in various pharmacological responses it is important to evaluate the changes in their dynamic state during the action of centrally active drugs. The availability of specific antibodies for neuropeptides has made it possible to measure the changes in neuropeptide content that occur at the level of the nerve terminal. However, since peptides are synthesized as high molecular weight precursors one cannot utilize labeled amino acid incorporation and changes in specific activity to assess biosynthetic rates because of inherent With specific cDNA probes one can measure the mRNA for the technical difficulties. A combination of RIA and hybridization yields an estimation of neuropept ide precursor. neurfpeptide turnover. Daily injections of haloperidol repeated for 3 weeks increase striatal met enkephalin (ME) content and produce an increase of striatal proenkephalin mRNA (PE mRNA). Selective lesion of the nigro-striatal dopamine system with 6-hydroxydopamine also produces an increase of PE mRNA and ME levels, suggesting that dopamine tonically inhibits Fenfluramine elevates the hypothalamic content of ME enkephalinergic turnover in the striatum. without changing PE mRNA levels, suggesting that it acts at the level of peptide utilization. We propose that this approach will allow us to estimate in vivo peptide turnover and thus study the effects of various pharmacological treatment on the dynamic state of peptides.
34Molecular
genetics
approach
to the nicotinic
acetylcholine
receptor
(AchR).
GIRAUDAT
J., DEVILLERS-THIERY A., KLARSFELD A. and CHANGEUX J.P. Laboratoire de Neurobiologie Moleculaire Institut Pasteur, 75724 PARIS t:EDEX 15 The nicotinic AchR from T. marmorata electric organ consists of five transmembrane polypeptide chains in the stoiechiometry a2byd . cDNA clones coding for the esubunit, which carries the acetylcholine binding site, wera isolated from a cDNA library constructed from T. marmorata electric organ mRNA (Giraddat.s., 1982, EMS0 J., 1, 713-717). Screening of the clones was done in two successive steps : 1) differentyal colony hybridization enabled us to select clones corresponding to mP.NA species present specifically in T. marmorata electric organ but not in T. marmorata spleen or liver; 2) final identification was done by immunoprecipitating the translation product of singlecDNA clones using monospecific antisera. The complete aminoacid sequence of T. marmorata a-subunit precursor was deduced from nucleotide sequence analysis. We used it to develop a model for the transmembrane organization of the subunit and to propose a possible structure of the ionic channel in the pentameric receptor protein (Devillers-Thiery et al., 1983, Proc. Nat. Acad. Sci. USA, 80, 2067-2071). 3ur results will be discussed w=egard to the data obtained by authors on T. californica AchR. Using Southern blot hybridization, we demonstrated that T. californica eenome contains a single eene coding for the AchR CI-subunit (Klarsfeld et., 1984, &ill0 J., _?, 35-41). Structural and developmental implications will be presented. v
35
MOLECULAR
GENETIC
J. Mallet+,
APPROACH
A. Lamouroux,
u
TO THE STUDY OF CATECHOLAMINES N. Saucon Biguet,
B. Grima and J.F. Julien
(Gif-sur-Yvette,
France)
ABSTRACT
36
NOT RECEIVED
NEUROPEPTIDE DYNAMICS STUDIED IMPLICATIONS. I. Mocchetti, J.P. Schwartz and E. Costa
WITH
A
cDNA
PROBE:
PHARMACOLOGICAL
Brain neuropeptides participate in synaptic function either as primary transmitters or as cotransmitters. In order to define their participation in various pharmacological responses it is important to evaluate the changes in their dynamic state during the action of centrally active drugs. The availability of specific antibodies for neuropeptides has made it possible to measure the changes in neuropeptide content that occur at the level of the nerve terminal. However, since peptides are synthesized as high molecular weight precursors one cannot utilize labeled amino acid incorporation and changes in specific activity to assess biosynthetic rates because of inherent With specific cDNA probes one can measure the mRNA for the technical difficulties. A combination of RIA and hybridization yields an estimation of neuropept ide precursor. neurfpeptide turnover. Daily injections of haloperidol repeated for 3 weeks increase striatal met enkephalin (ME) content and produce an increase of striatal proenkephalin mRNA (PE mRNA). Selective lesion of the nigro-striatal dopamine system with 6-hydroxydopamine also produces an increase of PE mRNA and ME levels, suggesting that dopamine tonically inhibits Fenfluramine elevates the hypothalamic content of ME enkephalinergic turnover in the striatum. without changing PE mRNA levels, suggesting that it acts at the level of peptide utilization. We propose that this approach will allow us to estimate in vivo peptide turnover and thus study the effects of various pharmacological treatment on the dynamic state of peptides.