"Annual Report of the Laboratory of Biochemical Genetics" [summary of laboratory projects]
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1984-09 (September 1984)
Nirenberg, Marshall W.
National Heart, Lung, and Blood Institute. Laboratory of Biochemical Genetics
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From Neuroblastoma to Homeobox Genes, 1976-1992
Annual Report of the Laboratory of Biochemical Genetics, October 1, 1983 - September 30, 1984 (September 1984)
Annual Report of the Laboratory of Biochemical Genetics
National Heart, Lung, and Blood Institute
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, and 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 from 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. Concomitantly, 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 NG108-15 cells grown in the presence of PGE1, an activator of adenylate
cyclase, or in the absence of PGE1, was extended during the past year. The cells were incubated with [35S] -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 pI of some glycoproteins. However, silver staining revealed many glycoproteins that were
not detected by autoradiography, including additional glycoproteins that were expressed only by PGE1-treated cells. These
results suggest that elevation of cAMP levels of NG108-15 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. The cDNA 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 hydroxylase 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
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, [lambda]gtll. Possible DNA clones that direct the synthesis of choline acetyltransferase in E. coli have
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
plasma 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 neuroblasts and by nondividing
neurons and the gradient is formed as the retina is formed. The protein was detected on all cells examined in dorsal and
middle retina. Cells that were dissociated from retina and cultured in vitro express the amount of gradient protein that
would be expected of cells in the intact retina depending upon the original position of the cells in the retina. These results
suggest that the gradient is established by an irreversible, clonally inherited mechanism and thereafter, the gradient is
perpetuated independently by each cell.
Monoclonal antibody that recognizes the gradient protein, 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, antibodies synthesized by parental P3X63 Ag8 myeloma cells had
RNA was isolated from 14 day chick embryo retinas and a large cDNA library was constructed in [lambda]gtll that can be used
to direct the synthesis of proteins specified by the cDNA in E. coli. The library currently is being screened for recombinants
that direct the synthesis of the gradient protein. Injection of 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 being screened for transducin subunits in collaboration with A. Spiegel.
Seventy-six hybridoma cell lines were generated that synthesize monoclonal antibodies that bind to 8 day chick embryo optic
tectum. Fifteen hundred hybridoma lines were generated from spleen cells of mice immunized with the cervical-thoracic spinal
cord and dorsal root ganglia of 8 day chick embryos. Some of the hybridoma lines synthesize antibodies that recognize antigens
that are restricted to fiber tracts or neuronal cell body regions of the spinal cord.
Additional information was obtained about other antigens that are recognized by monoclonal antibodies. For example, antigen
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 been detected on neurons or glia in other parts of the nervous system. The antigen 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 synaptic layer of the retina and in a circular "organelle" in the outer synaptic
layer. The antigens are expressed by approximately 10% of the cells in retina. In collaboration with Victor Ginsburg and
his colleagues, the antigens were shown to be novel gangliosides of unknown structure that contain disialyl residues whose
abundance and structure change during development; the location of the gangliosides in retina also changes during retinal
development. Most of the antigens are associated with the inner and outer synaptic layers of retina in late embryo and adult
retina. In addition, the antigens for many other monoclonal antibodies were characterized and in some cases were partially
A heat-stable, acidic, soluble, bovine brain protein was found that induces neurite outgrowth from chick embryo cerebral cortical
neurons at nM concentrations in defined medium. The Neurite Extension Factor (NEF) rapidly stimulates the phosphorylation
of a protein with an apparent M, of 90,000 in the absence of calcium ions or cyclic nucleotides. Phoslsphopeptide mapping
results show that the 90,000 M, protein is related to an 87,000 Mr, protein that is a major substrate for C kinase in brain.
Further information has been obtained on the aggregation of nicotinic acetylcholine receptors on cultured myotubes induced
by neuronal factors. In experiments using image intensification to directly observe changes in receptor distribution, and
electron microscopy to study changes in the subsurface cytoskeleton and extra-cellular matrix, we have demonstrated discrete
steps in the assembly of receptor aggregates from diffuse receptors. The transition from microaggregates of acetylcholine
receptors, which appear first, to large dense aggregates is dependent on temperature and involves an increase in the stability
of the aggregate.
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 [alpha]-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 degrees and 23 degrees but do not form larger aggregates. At 36 degrees
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 information of both microaggregates and aggregates at 36 degrees 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 leucine-enkephalin,
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 cDNA (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 0-14% in the relative abundances of preproenkephalin mRNA of the hypothalamus and striatum,
Preproenkephalin mRNA was detected in NG108-15 mouse neuroblastoma x rat glioma poly A+ RNA by Northern blot hybridization.
The-abundance was 1/650 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 nM 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.