Two [lambda]gtll cDNA libraries from human brain were screened with 3 oligodeoxynucleotide probes for recombinants coding
for a subunits of G signal transducing proteins, which couple receptors activated by hormones or light to connectors such
as adenylate cyclase or cGMP 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 human 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 cDNA and 2 clones as [alpha]i.
The DNA insert from one of the as clones was sequenced completely and additional partial sequences were obtained for 10 as
clones. Four species of [alpha]s cDNA were found that differ in nucleotide sequence in the region that corresponds to as
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 as mRNA by alternative splicing of precursor RNA transcribed
from a single gene.
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 sequence of human BG-4 [alpha]i cDNA is highly homologous to bovine and rat [alpha]i sequences reported by others.
In addition, the 3'-untranslated region of BG-4 [alpha]i 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 as 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 spontaneous secretion of acetylcholine at synapses, and 5-15 fold increases in the abundance of synapses with
cultured striated muscle cells. In addition, the number of molecules of the 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 cloned 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
y 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 peptides derived from choline acetyltransferase
by the action of 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
IgC-Affigel 10 affinity column chromatography. Both purification methods yielded one major band of protein with an Mr of
approximately 47,000. A protein of M, 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 diffusable molecules or on maintenance of interactions 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 NG108-15 cells was shown in previous studies to increase cellular levels of inositol-1,4,5-trisphosphate
(IP3) and diacylglycerol. 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 inosito1 1,4,5- trisphosphate inside NC108-15 cells results
in the release of stored calcium into the cytoplasm, injection of inositol 1,3,4-trisphosphate or inositol 1,3,4,5-tetrakisphosphate
has little or no effect on calcium mobilization, but instead results in the activation of nonspecification channels . Calcium
ions are not required for the activation of the nonspecification channels. The nature and significance of these findings
warrant further investigation in light of recent reports that inositol 1,3,4-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 1,4,5-trisphosphate,
catalyzed by an appropriate kinase, and that inositol 1,3,4-trisphosphate is formed by dephosphorylation of inositol 1,3,4,5-tetrakisphosphate.
Immunofluorescence staining on cryostat sections prepared from embryonic brain extract-treated myotubes revealed a precise
colocalization of a 43,000 Mr, cytoplasmic protein (distinct from actin) with newly-formed ACh receptor aggregates. This
result is consistent with a role for the 43,000 Mr protein in receptor immobilization, as suggested indirectly by studies
from other laboratories on fish electric organ and the neuromuscular junction.
We previously showed that partially purified and highly purified fractions from the extracellular matrix of the Torpedo electric
organ induce ACh receptor aggregation in cultured myotubes with a time course similar to that of embryonic pig brain extract.
We now have found that antiserum against a partially purified fraction from Torpedo (700 units/mg protein) can absorb about
60% of the receptor aggregation activity of brain extract. Under the same conditions, 90% of the activity in the Torpedo
fraction was absorbed. This result is consistent with the presence of immunologically related aggregation factors in electric
organ and brain.
We previously showed that neural factor induced formation of ACh receptor aggregates on tetrodotoxin-treated myotubes is associated
with the localized deposition of basal lamina. We now find that embryonic brain extract and ciliary ganglion explants induce
a widespread deposition of basal lamina on non-tetrodotoxin-treated myotubes. Ascorbate oxidase blocks this deposition of
basal lamina, suggesting that ciliary ganglion and embryonic brain extract contain ascorbate-like factors that promote muscle
basal lamina formation. The extensive induction of ACh receptor aggregates by ciliary ganglion explants was only partially
inhibited by ascorbate oxidase, and basal lamina deposition still occurred at the ACh receptor aggregate sites. These results
suggest that the ascorbate-like factor contributes to, but is not primarily responsible for the induction of receptor aggregates.
In addition, they suggest that deposition of basal lamina at receptor aggregates can occur independently of the ascorbate-like
We have been studying hormonal and neurotransmitter-dependent mechanisms that regulate the gene for proenkephalin (pEnk),
the precursor of the opioid peptides methionine- and leucine-enkephalin, in clonal cell lines of neural origin, as well as
in rat brain. NG10S-15 neuroblastoma-glioma hybrid cells and C6 rat glioma cells contain pEnk mRNA, quantitated by blot hybridization.
C6 cells contain a much higher abundance (3-6 pg/ug RNA) but lower enkephalin content than NC-108-15 cells. Treatment of
C6 cells with compounds that activate adenylate cyclase and raise the cAMP concentration (e.g. by a beta-adrenergic receptor
agonist such as (-)- norepinephrine or by forskolin) elevate the pEnk mRNA abundance. Glucocorticoid hormones such as dexamethasone
or cortisol, while having no effect alone on the pEnk mRMA level, potentiate the effect of cAMP elevation, producing maximum
elevations of 8-fold. C6 cells contain proenkephalin but do not process this precursor significantly. Treatment with norepinephrine
and dexamethasone raises the content of proenkephalin 11-fold. Treatment of cells with glucocorticoid and forskolin for 1-6
hr increases pEnk gene transcription at least 2.5 fold. These results suggest that gluccocerticoids and neurotransmitters
that elevate cAMP transcriptionally regulate enkephalin biosynthesis in enkephalinergic cells.
Studies have been initiated on the regulation of expression of the gene for proneuropetide Y (pNPY), the precursor of neuropetide
Y, a putative regulator in the autonomic nervous system. pNPY mRNA is relatively abundant in NG108-15 hybrid cells. Treatment
of these cells with gluccorticoids elevates pNPY mRNA 2-fold.
Two novel neuropeptides having anti-analgesic activity were recently isolated and sequenced by Dr. H. Y. Yang's group.
Their structures are Ala-Gly-Glu-Gly-Leu-Ser-Ser-Tro-Phe-Trp-Ser-Leu-Ala-Ala-Pro-Gln-Arg-Phe-NH2 (A18F-:NH2) and Phe-Leu-Phe-Gln-Pro-Gln-Arg-Phe-NH2.
A rat hypothalamus [lambda]gtll cDNA library was screened with 32P-oligodeoxynucleotides corresponding to portions of these
peptides and putative A18F-NH2 cDNA clones were obtained.
Nearly all prokaryotic genes use the translation initiation codon AUG. However, there are a few examples where GUG or UUG
function as initiation codons in E. coli. The gene for E. coli adenylate cyclase, is one of the genes that uses the unusual
UUG initiation codon. We have investigated the effect of this unusual initiation codon on the expression of the adenylate
cyclase gene by changing the DNA sequence coding for the UUG initiation codon to ATG and GTG, using oligonucleotide-directed
mutagenesis. A comparison of the activities associated with the three codons was made in three different environments: (1)
in the normal environment, with the adenylate cyclase gene expressed from its own promoters, (2) in a transcription fusion
with the adenylate cyclase gene under the transcriptional control of the phage lambda promoter, and (3) in a gene fusion with
the adenylate cyclase gene fused to the E. coli galactokinase gene to generate a fusion protein with galactokinase activity.
In each of the three environments, it was observed that the UUG initiation codon had the lowest efficiency of translation
initiation and the AUG initiation codon had the highest efficiency, while the GUG initiation codon was intermediate. These
results may provide a partial explanation for the finding that the cellular concentration of adenylate cyclase is very low.
In E. coli cAMP plays a crucial role in regulating the expression of inducible genes. The levels of this nucleotide are controlled
primarily by a catabolite-dependent modulation of adenylate cyclase activity. Insight into the mechanism of regulation of
the activity of this enzyme has come primarily from studies of permeable cells. Current information suggests that the phosphoenolpyruvate:
glucose phosphotransferase system (PTS) is intimately involved in the regulation. Additionally, potassium and phosphate ions
play key roles in modulating adenylate cyclase activity. A model for interaction of adenylate cyclase with PTS proteins and
potassium phosphate to form a regulatory complex was proposed previously by us. The purpose of the present study was to test
the proposed model for adenylate cyclase regulation using a reconstitution approach. We found that all of the unique features
of adenylate cyclase characteristic of the regulatory complex observed in permeable cells were reconstituted in cell-free
extracts. The results strongly support the proposal that adenylate cyclase activity is regulated by PTS proteins.