Laboratory project: "Cell Recognition and Synapse Formation"
Item is a photocopy.
Number of Image Pages:
4 (426,327 Bytes)
1988-09 (September 1988)
Nirenberg, Marshall W.
Trisler, G. David
National Heart, Lung, and Blood Institute. Laboratory of Biochemical Genetics
This item is in the public domain. It may be used without permission.
From Neuroblastoma to Homeobox Genes, 1976-1992
Annual Report of the Laboratory of Biochemical Genetics, October 1, 1987 - September 30, 1988 (September 1988)
Project Number: 01 HL 00009-14
Period Covered: October 1, 1987 - September 30, 1988
Title of Project: Cell Recognition and Synapse Formation
Marshall Nirenberg, Chief, LBG, NHLBI
Hemin Chin, Staff Fellow, LBG, NHLBI
Li-Shan Hsieh, Visiting Fellow, LBG, NHLBI
Wu-Hong Tsai, Visiting Fellow, LBG, NHLBI
Maria Giovanni, Staff Fellow, LBG, NHLBI
David Trisler, Guest Worker, LBG, NHLBI
Dana Hilt, Staff Fellow, LBG, NHLBI
Cooperating Units (if any): Bruce Schrier, LDN, NICHD
Lab/Branch: Laboratory of Biochemical Genetics
Section: Section of Molecular Biology
Institute and Location: NHLBI, NIH, Bethesda, Maryland 20892
Total Many Years: 10
Summary Of Work:
1. Treatment of NG108-15 neuroblastoma-glioma hybrid cells results in marked increases in the abundance of certain species
of RNA. Seventeen cDNA clones corresponding to these species of RNA were obtained and the nucleotide sequences of three clones
were determined. DNA clone NG-32 corresponds to mRNA for ATP synthase subunit 6, which is transcribed from the heavy chain
of mitochondrial DNA and codes for a protein that is part of the H+ channel of the ATP-synthase complex. Clone NG-10 DNA
corresponds to another mitochondrial DNA of unknown function, which is transcribed from the light chain of mitochondrial DNA
and may be involved in the initiation of replication of mitochondrial heavy strand DNA.
2. Eleven cDNA clones mere obtained that correspond to mRNA for the [alpha]-subunit of the L-type voltage-sensitive calcium
channel of rat brain. Analysis of the DNA sequence and the deduced amino acid sequence reveals strong homology between brain
ad skeletal muscle calcium channel [alpha]-subunits. Approximately 75% of rat brain [alpha]-subunit amino acid residues that
were defined are either identical to the amino acid residues of rabbit skeletal calcium channel &subunit or are conservative
amino acid replacements.
3. Four novel Drosophila homeobox genes NK-1, -2, -3, and -4 were cloned and partial nucleotide sequences were determined.
One NK-1 cDNA clone was obtained from a cDNA library prepared from poly A+ RNA from 3-12 hr Drosophila embryos, but none was
detected in the 0 to 3 hr embryo library. Six NK-3 cDNA clones were obtained from a library prepared from Drosophila poly
A+ RNA from 0-3 hr embryos and 6 additional clones were obtained from a library prepared for 3-12 hr embryo poly A+ RNA.
The exon-intron structure of the NK-1 gene was determined. One of the two introns found resides within the homeobox.
Previously, many neuroblastoma and related somatic hybrid cell lines were shown to acquire voltage-sensitive ion channels
and other neuronal properties when intracellular cyclic AMP levels were elevated for a number of days. Cells with elevated
cAMP acquire new proteins such as the [alpha]-subunit of voltage-sensitive calcium channels and other proteins of unknown
function. A cDNA library was constructed from poly A+ RNA prepared from NG108-15 neuroblastoma-glioma hybrid cells that
had been treated for 5 days with 1 mM dibutyryl cAMP. The library was screened and 17 cDNA clones were obtained that correspond
to species of RNA that are 3 to 40 times more abundant in cells treated with dibutyryl cAMP than in cells cultured without
this compound. Each cloned cDNA was used as a probe with Northern blots to determine the number of species of poly A+ RNA
responsive to dibutyryl cAMP and the chain length of each species of RNA. The results suggest that the 17 cDNA clones correspond
to species of RNA transcribed from 10 genes. Partial nucleotide sequences of the cDNA inserts from 3 clones were obtained.
Clone NG-32 corresponds to mouse mitochondrial mRNA for ATP synthase subunit 6, a mitochondrial gene. This protein is part
of the H+ channel portion of the mitochondrial ATP synthase complex. Treatment of NGl08-15 cells with dibutyryl cAMP results
in an 8-fold increase in the abundance of mRNA for this protein. The nucleotide sequence of clone NG-10 cDNA was identified
as part of the D-loop region of mouse mitochondrial DNA that contains the origin of replication for the heavy strand of DNA.
The 5'-terminal nucleotide sequence of some molecules of heavy strand mitochondrial DNA is known to consist of a short
segment of RNA that is complimentary to a short light strand miochondrial DNA sequence nearby. Hence, NG-10 cDNA may correspond
to an RNA transcript of the light strand of mitochondrial DNA that serves as a primer for the initiation of heavy strand mitochondrial
DNA synthesis. Treatment of NG108-15 cells with dibutyryl cAMP results in a 40-fold increase in this species of RNA. These
results show that treatment of NG108-15 neuroblastoma-glioma cells with dibutyryl cAMP results in marked increases in the
abundance of RNA transcripts from heavy and light strands of mitochondrial DNA. Further work is needed to determine whether
cAMP regulates mitochondrial biogenesis or the ability to synthesize ATP.
A [lambda]gtll cDNA library was prepared from rat brain poly A+ RNA and screened with oligodeoxynucleotide probes that correspond
to the [alpha]-subunit of L-type voltage-sensitive calcium channels. Eleven positive clones were detected that have cDNA
inserts 1.6-5.5 Kb in length. Nucleotide sequence analysis reveals strong homology as well as differences in the deduced
amino acid sequences of the [alpha]-subunits of rat brain and rabbit skeletal muscle L-type voltage-sensitive calcium channels.
To detect recombinant DNA clones that correspond to novel homeobox genes a Drosophila genomic DNA library was screened with
multiple oligodeoxynucleotide probes, each designed to hybridize to multiple homeobox genes. Five clones that gave positive
signals with 2 or more oligodeoxynucleotide probes exhibited specificities that could not be explained on the basis of known
nucleotide sequences of Drosophila homeobox genes. Nucleotide sequence analysis of the homeobox regions of 4 clones revealed
4 new homeobox genes (NK-1, 2, 3, 4). Two recombinant clones contained identical DNA inserts, each insert contained 2 new
homeobox genes (NK-3 and NK-4). The deduced amino acid sequence of the NK-1 homeobox exhibits the highest homology to the
homeobox regions of deformed, zen-2, and Zen-1 (75, 72, and 71% homology, respectively). The relative homology of the NK-2
homeobox is as follows: NK-4 > NK-3 > NK-1 = IAB-7. The homology of NK-3 is: NK-2 > labial > NK-4 >
NK-1; and NK-4 homology is NK-2 > zen 2 = NK-3 > labial. Genomic DNA fragments from the 4 new homeobox genes were
used to screen cDNA libraries prepared from poly A+ RNA from 0-3 hr Drosophila embryos or from 3-12 hr embryos. One NK-1
cDNA clone was obtained from the 3-12 hr embryo cDNA library, but none was detected in the 0-3 hour embryo libary. Comparison
of the nucleotide sequences of NK-1 cDNA and genomic DNA clones showed that the NK-1 gene has 3 exons. One of the 2 introns
detected resides within the homeobox region.
Significance of the Results
1. The demonstration of dibutyryl cAMP dependent regulation of 2 species of mitochondrial RNA raises questions that can be
addressed in future studies; namely, does cAMP regulate mitochondrial biogenesis or the ability to synthesize ATP?
2. Voltage-sensitive calcium channels are known to play a central role in stimulus-secretion coupling and signal transmission
both within cells and between cells. The DNA clones that were obtained for the [alpha]-subunit of rat brain L-type voltage-sensitive
calcium channels can be used as probes to explore the mechanisms that regulate the [alpha]-subunit gene. The cloned DNA also
can be used to direct the synthesis of the [alpha]-subunit protein of the dihydropyridine-sensitive calcium channel from brain.
Site directed mutagenesis can be used to alter the DNA and explore the relation between calcium channel structure and function.
3. Four novel Drosophila homebox homeobox genes were cloned and partially sequenced. The homeobox family of genes code for
proteins that regulate the expression of genes during development and some determine pathways of differention. These genes
provide an experimental system that can be used to define the mechanisms that regulate the expression of these homeobox genes
as well as regulation of gene expression by homeobox proteins.
1. Bray/P., Carter, Guo, V., Puckett, C., Kamholz, J., Spiegel, A., and Nirenberg, M.: Human cDNA clones for an [alpha]
subunit of Gi Signal-transduction protein. Proc. Natl. Acad. Sci.. USA 84, 5115-5119 (1987).
2. Fitzpatrick, L.A., Chin, H., Nirenberg, M., and Aurbach G.D.: Antibodies to an [alpha] subunit of skeletal muscle calcium
channels regulate parathyroid cell secretion. Proc. Natl. Acad. Sci., USA 85, 2115-2119, (1988).
3. Carter, A., Bardin, C., Collins, R., Simons, C., Bray, P., and Spiegel, A.: Reduced expression of multiple forms of Gs-[alpha]
in pseudohypotarathyroidism type IA. Proc. Natl. Acad. Sci.. USA 84, 7266-7269 (1987).
4. Brown, D.A., and Higashida, H.: Voltage- and calcium-activated potassium currents in mouse neuroblastoma x rat glioma
hybrid cells. J of Physiology 397: 149-165 (1988).
5. Brown, D.A. and Higashida, H.: Membrane current responses of NG108-15 mouse neuroblastoma x rat glioma hybrid cells to
bradykinin. J. of Physiology 397: 167-184 (1988).
6. Brown, D.A. and Higashida, H.: Inositol 1,4,5-trigphosphate and diacylglycerol mimic bradykinin effects on mouse neuroblastoma
x rat glioma hybrid cells. J. of Physiology 397: 185-207 (1988).
7. Higashida, H.: Acetylcholine release by bradykinin, inositol 1,4,5-trisphosphate and phorbol dibutyrate in rodent neuroblastoma
cells. J. of Physiology 397: 209-222 (1988).
8. Trisler, D., and Collins, F.: Corresponding spatial gradients of TOP molecules in the developing retina and optic tectum.
Science 237: 1208-1209 (1987).