(-)1.:;.>,-:r -, 1 1935 - September 30, 1986 c&i:; ? .-r:zgnit%on and Synaptic Formation .I - -3". ..- _- 'L,S! i`?,,$. rr,feri I. . _. z .- : -. fJCJ/Jv/ !bk j'- I ,: &&rs'r.Ell Xirenberg, Chief, I.%, NHLBI . .~7?r::ga:c'. i !,'LuT Nit?. /&r,r,.~,or~, ;;-I;' !/Is:>!<, *,; +;i!rat;,-Jnj nana ;;i-:, Staf-f Fellow, LBG, SHLBI ~eziil; C:tln, Guest Worker, LBG, 3HLBI &T-l :zaladoss, Visiting Fellow, LEG, SHLBI . 7' Icot? L.aPZ, ?;'lsiting Fellow, LBG, NHLBI Davi: Irisler, Staff Fellow, LBG,XHLBI .-A- --_ __.. ---- cGC;Ez.:- `.f j'. -3 (if any) AlIe> Spiegel; Chief, MDB, XIXDDK Bruce Schrier, LDN, NICHD Lou Zrsch, Dept. of Biochem -P.-Y ., U. of Texas, Dallas, Texas LAB;B?".'3- Laborarcry of Biochemical Genetics -.-- - SECTC'. Section of Xolecular Biology -- ItNST+7i-E ,:`.; ~33'TlOrJ -- NHLEiI, !;X, Bethesda, Maryland 20205 TOT&,: *,I;.',. :E; = 5 PRtiFESSIONAL: 11 9 .-...-- ___.- C,.,ECd :===~:Z r-E BOX(ES) czi i'j H~;--,an subjects b (b) Human tissues -- 1%: * rn:;.ors 2 (c) Neither - --- - (E2, interviews .--- SUM ),' f = 'i = = --- __--__ :. Z=`: Vsse standard unreduced tp2 Do nor exceed the space :-3/!`d~d.) -- .--__ i;-7 7 ._ 4-- cDNA libraries derived from human brain poly A+ RNA were screened for _" _..: rse.?.-;' Eie-ler. zs -nants that code for a-subunits of C signal transduction proteins. 2nd two ai clones s!ere characterized. f.OX!P. ' Four species of c+ cDNA were - mechanism for generating the four species of a, mRNA by alternative spiiCir.g Cf precursor RNA was proposed. ';re=,zent of NG108-15 neuroblastoma-glioma hybrid cells CAMP for several days iOriS rss2:lts in thee appearance of voltage-sensitive calcium channels and other c:-z3r;els. poll/ .r- ?.::.r, that Twenty cDNA clones were obtained that hybridize to species of dibz:f;'r:fl CAMP. increase in abundance lo-90 fold due to treatment of cells with vo' Affinity-purified antibodies to the a or Y protein subunits of ^ *'5- "--=-sensitive calcium channels were used to screen a Agtll cDNA library. Tw;ey,t)- ~7.t~ ,,,,tive voltage-sensitive calcium channel a subunit cDNA clones and 29 pL;=a:: -;e Y subunit clones were found. ?.:igenic molecules termed TOP, which are distributed in a dorsal > ventral COr?.c.c-n=- .-*-.. 4. ,-ion gradient in chicken retina, were purified. prc',ei: *~l:h an Mr of 47,000. TOP was shown to be a in srr.Sr;ozic development. The gradient of TOP in the retina is formed early Thereafter, de-a-" perpetuation of the gradient does not -" d..M Sri :he continuous presence of an extracellular gradient of diffusable m0102,Ies or o#n maintenance of interactions between cells. Synapses and ne,;;r- -cc -d-u -. '7 the retina of developing chick embryos were reduced markedly by iy.t:ri' z -* ~.f ~ 4-d-d.. anti-TOP antibody into the eye. f.,:;? 03; ec::VZ is to discover basic mechar.is?s that regulate the expression of :;c+ries , Two ;.gtll cDN.4 libraries from human -brainwere .acreen.ed with 3 oligodeoxynucleotide probes for recombinants coding for 01 subunits of G signal transducing proteins, which couple receptors activated by hormones or light to effecters such as adenylate cyclase or CC'.:? phosphodiesterase. Fourteen of the 575,000 recombinant clones screened,from a human basal ganglia cDNA library and 12 of the 400,000 clones screened from a hunan cerebral cortex library were detected with 2 or 3 of the 32P-probes used. DNA inserts from 13 positive clones were sequenced partially; 11 clones were identified as.ols cDNA and 2 clones 2s .Cfim The DNA insert from one of the us clones was sequenced completely and additional partial sequences were obtained for 10 c+ clones. Four species Gf Us CD% were found that differ in nucleotide sequence in the region that corresponds to c+, amino acid residues 71-88. The clones differ in the codon for "3 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 a b s 8 ;"I :: e o F an adjacent serine residue. A mechanism was proposed for generating h species Gf crs mRI!A by alternative splicing of precursor RNA transcribed from a single 5er.e. CD';.& from one of the two human CLi clones was .sequenced completely (BG-4)) and a partial seqt\ence was obtained for the second clone. The first nucleotide residile of DG-4 ai cDMA corresponds to the 14th residue of the bovine ai coding s e G- ~1 e r, e e and the last residue of BG-11 (1261) is in the 3`-untranslated region. The amino acid sequence derived from the nlucleotide sequence of human BG-4 ai ~3118 is highly !omologous to bovine and rat ai sequences reported by others. In addition, the 3 3' -untranslated region of EG-4 ai cDNA is highly homologous to the -untranslated regions of bovine and rat zi cDNA. The 3'-untranslated nucleotide sequences of human, bovine, ar;ld rat I+ cDNAs also are highly conserved, but differ markedly from ai 3 -untranslated sequences. -These results suggest that the 3 -untranslated regions 0-f as and ai genes and/or mRNA are neede d for functions that have not been identified thus far. In previous studies we have shown that elevation of CAMP levels of NC108-15 neurojlastoaa-gliosa hybrid cells or neuroblastoma cells for several days results in lo-100 fold increases in the activity of voltage-sensitive calcium channels, 15-45 fold increases in spontaneous secretion of acetylcholine at synapses, and 5-15 fold increases in the abundance of synapses with cultured stria:ed m:uscle cells. In addition, the number of molecules of the voltage-sensitive calcium channel protein subunit that binds [3H]-nitrendipine increases l2-fold. We previously obtained about 100 cDNA clones that hybridize to spzies of mRNA that are more abundant in NGlO8-15 or NS20-Y cells that had been tre=t -,ed with dibutyryl CAMP for several days then in untreated control cells. Guantitative studies on the extent Of increase in abundance of the -l- species zr" m-R?3 that respond to d.ibutyry;l 6.XP were performed using the cloned Cl,...? T\,.`A ^_ 23 probes. Twenty cD!iA clones were obtained that hybridize to species of .+ p 3 1 y .a. ;. :; .A. that increase in abundance 1, "-30 fold due to treatment of cells with b i.bu';y;rl CAMP. Northern: blots also were performed and the number of bands of .A -,.* p 0 I y .1 r. . , .-. that hybridize to each cloned c>iiA probe and their chain lengths r:erc determined. Xf,-.L i?`ty purified antib'odss to the a, 0, and Y protein subunits of voltage -sensitive calcium channels were used to screen a Xgtll cDNA library prepared from poly A+ RNA from rat skeletal muscle. Approximate.ly 20. recombinant clones were found that were identi.fied- tentatively as calcium- channel a subunit cDNAs. Other cDNA clones were obtained that are putative Y subunit clones. In previous studies a putative cDNX clone for choline acetyltransferase was found. i.;e now have determined the nucleotide sequence of the 1118 bp DNA insert. Partial amino acid sequences of several peptides derived from choline acetyltransferase by the action of peptidases were obtained in collaborative studies by Lou Hirsh and his colleagues in Dallas. The Xgtll cDNA library was screened again with 2 new oligodeoxynucls otide probes to different regions of choline azetyltransferase and cDNA clones were obtained that were recognized by both probes. Further studies with these clones are in progress. Anti j- -=nic molecules termed TOP, which are distributed in a dorsal > ventral concentra:ion 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 express ed in retina thereafter. 35S-la*aeled-TOP-antibody complexes were purified 3:~ protein A-Sepharose column c?ronatography and subjected to NaDotiSO4/;olyacrylamide gel electrophoresis and autoradiography. TOP also was purified I'r om dorsal retina by anti-TO? IgG-Affigel 10 affinity column chromatography. Both purification methods yielded one major band of protein with an :-II, 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 err-.*bryo retinas accumulated the ar,oun t 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 diffusable molecules or on maintenance of interact ions between cells. Synapses and neurites in the retina of developing chick embryos were reduced markedly by injection of anti-TOP antibody into the eye. The addition of bradykinin to NGlC8-15 cells was shown in previous studies to increase cellular levels of inositol-1,4,5-trisphosphate (IP3) and diacylglycerol. The newly synthesized I?3 in turn stimulates the release of stored Cal? ium ions into the cytoplasm, t-hereby activating calcium-dependent K+ channels. The increased efflux of K+ ions results in cell hyperpolarization. This is followed by cell depolarization d*Je to inhibition of M channels, thereby decreasir.3 the rate of K+ efflux from cells Via M channels. Additional results now shi: that inhibition of 21 channels is due to diacylglycerol and Ca2+ dependen: activation of protein kinase C. Several phosphoproteins were detected -2- Yroject.No. ZOl NL (10009-12 IX `5`14' z-4 5 Ci-ensional gel electrophoresis ~3s~ synthesis is dependent upon the c-i -.ia,n sf *bradykinin to Cells. Whereas, injection of inositol 3, .,.4 i =--:ris?:hosphate inside NC10815 cells results ~fn the release of stored c,2 ! ? i .-. - d - a... in',< the cytoplasm, injection of inositol 1,3,4-trisphosphate or :.nosi:;:. 1,3,4,5'tetrakisphosphate has littie er no effect on calcium .i!73SiliZ?tTOn, but instead results inthe activation of nonspecific catJon &tzr:3:s;- Calcf.um ionsare not r@uired-for tfie-activation of the nonspecific `-catiot: c?,anne.ls. The nature'a k d signific axe of these findings warrant further. I?l'vTS~ +gation in light of recent reports that inositol 1,3,4-trisphosphate and +- inositol--1,3,4.5-tetrakisphosphate-are.present in. some tissyes..and that inositol x.,3-,`r; ,=,- te