ANNUAL REPORT OF THE
   LABORArIlORY OF BIOCHEMICAL GENETICS
NATIONAL HEART, LUNG, AND BLOOD 'rNSTIT"IJTE
October 1, 1983 through September 30, 1984

   Populations of relatively undifferentiated NG108-15 neuroblastoma-glioma
hybrid cells or NS20-Y neuroblastoma cells can be shifted a more differentiated
state by increasing the levels of cellular CAMP for several days.  In previous
studies -the differentiated cells were shown to possess functional voltage
sensitive channels for Na+, K+, and Ca2+, a Ca2+-dependent K+
channel that is not voltage-sensitive, long neurites, a& small clear vesicles
and large dense-core vesicles; whereas, these components were absent or were
reduced in undifferentiated cells.  Differentiated cells also exhibit higher
specific activities of choline acetyltransferase and acetylcholinesterase,
secrete more acetylcholine when stimulated, and form more synapses with striated
muscle cells than do undifferentiated cells.

  During the past year, poly A+ RNA was obtained from differentiated
NG108-15 and NS20-Y cells, cDNA was synthesized and then cloned using plasmid
pBR322 as the vector.  cDNA corresponding to species of poly A+ RNA that are
more abundant in differentiated cells than in undifferentiated cells were
purified by repetitive hybridization with poly A+ RNA fram undifferentiated
cells; single-stranded nucleic acids then were separated from double-stranded
nucleic acids.  The species of cDNA that increase in abundance as cells
differentiate were cloned.  Some clones will be used as probes to study the
mechanisms of CAMP-dependent regulation of mRNA in neuroblastoma cells.

   Prolonged elevation of cellular CAMP results in an increase in at least one
species of protein that is part of the voltage-sensitive calcium channel
complex.  Concomittantly, cells acquire.functional voltage-sensitive calcium
channels.  Voltage-sensitive calcium channel proteins were purified extensively-
The cDNA libraries that have been constructed will be screened for clones that
correspond to the channel proteins.

   Our previous analysis of glycoproteins synthesized by NGlOS-15 cells grown
.in the presence of PGEl, an activator of adenylate cyclasc, or in the absence
of PGEl, was extended during the past year.  The cells were incubated with
r35sl -methionine solubilized, and the glycoproteins fractionated by wheat
germ agglutinin-, ricin-, or lentil lectin-affinity column chromatography and
then by 2-dimensional polyacrylamide gel electrophoresis.  35S-Glycoproteins
detected by autoradiography were compared with those detected by silver
staining.  Both methods of analysis showed that elevation of intracellular CAMP
levels for several days results in the expression of new glycoproteins, the
disappearance of others, changes in the apparent abundance of some
glycoproteins, and shifts in the p1 of some glycoproteins-  However, silver
staining revealed many glycoproteins that were not detected by autoradiography,
including additional glycoproteins that were expressed only by PGEl-treated
cells.  These results suggest that elevation of cA%P levels of NGlO8-15 cells
for several days affects the expression of genes for some glycoproteins and
alters the post-translational modification. of other glycoproteins.   Current
studies focus on the purification of sufficient amounts of the regulated


glycoproteins to obtain partial amino acid sequences far the proteins and to
obtain antibodies that recognize the proteins.  The6 eDHA libraries that have
been generated will be screened for cDNA that corresponds to some regulated
species of glycoproteins.

  Whether a peripheral neuroblast will give rise to sympathetic or
parasympathetic neurons during differentiation is determined by mechanisms that
regulate the expression of the genes for tyrosine hydrmxylase and choline
acetyltransferase, respectively.  An extracellular protein and calcium ions are
known to be involved in the regulation of these genes, but the mechanisms of     S

regulation are unknown.  Previously, choline acetyltraxsferase from rat brain
was purified to essential'homogeneity and 4 monoclonal antibodies that recognize
the enzyme were obtained.  A large NG108-15 cDNA library was constructed using
the bacteriophage expression vector, Agtll.  Possible &NA clones that direct :.
the synthesis of choline acetyltransferase in E. co12 &ave been detected, but
further work is needed to establish the identity of the clones.

   A monoclonal antibody was obtained previously that recognizes a large
dorsal-ventral concentration gradient of a protein in @asma membranes of
chicken retina cells.  The amount of protein detected is a function of the
position of the cells in retina with-respect to the dorsal-ventral axis of the
retina.  The protein is synthesized by proliferating newroblasts and by
nondividing neurons and the the gradient is formed as tie retina is formed.  The
protein was detected on all cells examined in dorsal am3 middle retina.  Cells
that were dissociated from retina and cultured in vitre+ express the amount of
                                                      --
gradient protein that would be expected of cells in t&z intact retina depending
upon the original position of the cells in the retina..  These results suggest
that the gradient is established by an irreversible, cX.nnally inherited
mechanism and thereafter, the gradient is perpetuated tidependently by each
cell.

   Monoclonal antibody that recognizes the gradient Fotein, or hybridoma     :
cells synthesizing the antibody, were injected into the amniotic cavity of chick
embryos in ovo from the second to the fifth day after Fertilization and into the
         --
vitreal space of chick embryo eyes to determine whether the antibody affects the
development or the spatial organization of the retina.  The retinas of embryos
were continuously exposed to antibody throughout development from the.second to.
the twentieth day after fertilization.  Injection of antibody to the gradient
protein into the eye resulted in a marked reduction of isynapses and neurites in
the inner synaptic layer of the retina; whereas, antibtiies synthesized by
parental P3X63 Ag8 nyeloma cells had no effect.

  RNA was isolated from 14 day chick anbryo retinas and a large cDNA library
was constructed inhgtll that can be used to direct the synthesis of proteins.,..,
specified by the cDNA in E.   coli.  The library currently is being scre'ened for
rkcombinants that direct the synthesis of the gradient protein.  Injection bf
poly A+ RNA from retina into Xenopus laevis oocytes resulted in the synthesis
of the gradient protein.  This assay can be used for the purification of mRNA
for the gradient protein.  The cDNA library also is be%ng screened for
transducin subunits in collaboration with A.  Spiegel.

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  Seventy-six hybridoma cell lines were generated t&at synthesize monoclonal
antibodies that bind to 8 day chick embryo optic tectuzm.  Fifteen hundred
hybridoma lines were generated from spleen cells of m2c.e immunized with the
cervical-thoracic spinal cord and dorsal root ganglia o+:f 8 day chick embryos.
Some of the hybridoma lines synthesize antibodies that recognize antigens that
are restricted to fiber tracts or neuronal cell body r,m&.ons of the spinal
cord-

  Additional information was obtained about other as&igens that are
recognized by monoclonal antibodies.  For example, ant-en 13H9 was shown to be'
a protein with an approximate Mr of 180,000.  The antigen is associated with
cell membranes of all chick retina cells but has not &en detected on neurons or
glia in other parts of the nervous system.  The antigem defines a functional set.
of cells in the nervous system.

   18B8 antigens are first expressed by ganglion neurons and then by other
types of neurons in retina.  The antigens are found on cell soma initially, but-
later in development antigens disappear from cell soma ;and can be seen in a
highly stratified, multi-laminar pattern in the inner qnaptic layer of the
retina and in a circular "organelle" in the outer synapaic layer. The antigens
are expressed by approximately 10% of the cells in ret&na.  In collaboration
with Victor Ginsburg and his colleagues, the antigens Ytere shown to be novel
gangliosides of unknown structure that contain disialyx residues whose abundance
and structure change during development; the location &f the gangliosides in
retina also changes during retinal development.  Most &?f the antigens are
associated with the inner and outer synaptic layers of nretina in late embryo and
adult retina.  In addition, the antigens for many other .monoclonal antibodies
were characterized and in some cases were partially putified.

  A heat-stable, acidic, soluble, bovine brain proten was found that induces
neurite outgrowth from chick embryo cerebral cortical rmeurons at nM
concentrations in defined medium.  The Neurite Extensiorn Factor (NEF) rapidly
stimulates the phosphorylation of a protein with an appnarent M, of 90,000 in
the absence of calcium ions or cyclic nucleotides.  Phelslsphopeptide mapping
results show that the 90,000 M, protein is related to m 87,000 M, protein
that is a major substrate for C kinase in brain.

  Further information has been obtained on the aggre.g&tion of nicotinic
acetylcholine receptors on cultured myotubes induced by neuronal factors. In
experiments using image intensification to directly obsezrve changes in receptor
distribution, and electron microscopy to study changes liin the subsurface
cytoskeleton and extra-cellular matrix, we have demonstrated discrete steps in
the assembly of receptor aggregates from diffuse receprmrs.   The transition from
microaggregates of acetylcholine receptors, which appeazr first, to large dense
aggregates is dependent on temperature and involves an Increase in the stability
of the aggregate.

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   The fine structure was studied of regions of myotubes containing
microaggregates, of nicotinic acetylcholine receptors that form within 90 minutes
of exposure to embryonic brain extract.  A mixture of rhodamine conjugated
$bungarotoxin and peroxidase conjugated toxin-was used so that the formation of
receptor microaggregates could be observed directly and so that the distribution
of acetylcholine receptors on myotube membranes also could be determined.
Small receptor aggregates are converted to larger aggregates that are more
stable than the smaller ones..  Microaggregates form in the presence of embryonic
brain extract and accumulate at temperatures between 18" and 23" but do not form
larger aggregates.  At 36" C aggregates rapidly form from microaggregates.     ;
Microaggregates are destabilized rapidly when brain extract is removed or when
sodium azide is added to inhibit ATP formation.  In contrast, aggregates remain
stable for several hours under these conditions.  Azide reversibly blocks the i
formation of both microaggregates and aggregates at 36" C. Electron micrographs
of microaggregates reveal characteristic mounds in the cell surface, subtended
by loosely organized cytoplasmic filaments.  The receptor aggregates have, in
addition,  an increased association with basal lamina and a characteristic dense.
filamentous structure below the cell membrane.

   The regulation of the gene coding for preproenkephalin, the precursor of
the opioid peptides methionine-and leucinerenkephalin, was investigated.   cDNA
was 'cloned in the Pst I site of pBR322.  A full-length cDNA clone corresponding
to rat striatum preproenkephalin mRNA was found and sequenced.  The primary
structure of the rat preproenkephalin protein deduced from the nucleotide
sequence of the cDXA (269 amino acid residues,Mr 30932) is similar to bovine
and human preproenkephalin (78% and 82% matched amino acid.residues,
respectively) and contains 4 copies of Met-enkephalin, 1 of Leu-enkephalin, 1 of
Met-enkephalin-Arg-Gly-Leu, and 1 of Met-enkephalin-Arg-Phe.  Southern blot .
analysis of rat genomic DNA with a probe prepared from the rat preproenkephalin
clone suggests that the rat contains a single gene for preproenkephalin,  The
relative abundances of rat preproenkephalin mRNA are as follows:  striatum 100,
hypothalamus 11.2, pons + medulla 10.8, spinal cord 10.3, cerebellum 6.1,
midbrain 5.9, frontal cortex 4.6, hippocampus 2.0, thalamus 1.6.
Electroconvulsive shock treatment (1 set per day) of rats for 10 days elicited
increases of 78% and O-14% in the relative abund.ances of preproenkephalin ~RNA
of the hypothalamus and striatum, respectively.

   Preproenkephalin mRNA was detected in NG108-15 mouse neuroblastoma x rat
glioma poly A+ RN4 by Northern blot hybridization.  The-abundance was If650 of
that of the rat striatum and was increased 3.5 fold by treatment of the cells
with the glucocorticoid hormone, dexamethasone, for 4 days. This cell line can
be used for the study of preproenkephalin gene expression.

  Large cDNA libraries were prepared from cat dorsal root ganglion poly A+
RNA and rat spinal cord poly A+ RNA, for the isolation of clones containing*"
cDNA for the precursors of tachykinin neuropeptides.

   Histidyl-proline diketopiperazine (cyclo(His-Pro)), a metabolite of the
thyrotropin releasing hormone, has been reported to inhibit prolactin secretion
from the pituitary, elicit anti-depressant effects, alter cyclic nucleotide


levels, and alter body temperatures.  A search for receptors for cyclo(His-Pro)
revealed specific binding sites for cyclo(His-Pro) in particulate fractions
derived from bovine adrenal cortex or liver, but not in fractions derived from
brain or pituitary.  A single class of binding sites was found with a Kd of
900 ILY .and a maximum number of sites of 92 pmol per mg protein. The binding was
stereospecific and the histidine moiety of the-peptide was a major determinant
of the binding.  The binding sites for cyclo(His-Pro) were inactivated by
incubation of particulate fractions with trypsin or at 100' C.  No metabolism of
cyclo(His-Pro) was detected.

    Some strains of E. coli accumulate toxic levels of methylglyoxal that
inhibit cell growth. -One such strain was isolated and shown to synthesize a
mutant form of the CAMP receptor protein and to lack the gene for adenylate
cyclase.  Growth of the cells on glucose-6-phosphate, but not glucose, resulted.
in premature growth arrest due to the accumulation of methylglyoxal. The
specific activity of phosphofructokinase in the mutant cells was elevated. The
mechanism of growth arrest in the mutant cells was suggested to involve an
increase in the synthesis of triose phosphate via giycolysis with spillover of
metabolites into a pathway leading to the formation of methylglyoxal.