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Recombinant DNA Methods in Neuroendocrinology: New Answers to Old Questions

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BOTH neural and endocrine cells respond to local and circulating stimuli, and transduce them into either an increased or decreased synthesis and release of a product which acts via a target membrane receptor to alter the behavior of neighboring or distant cells. An important cellular strateg...

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TIFFACADEMICS
Peptides, Vol. 3, pp. 21%221, 1982.Printed in the U.S.A.




Recombinant DNA Methods
in Neuroendocrinology:
New Answers to Old Questions
L E E E. E I D E N 1

Unit on Neuroendocrinoiogy, Laboratory o f Clinical Science, National Institute o f Mental Health
Bethesda, M D 20205




EIDEN, L. E. Recombinant DNA methods in neuroendocrinology: New answers to old questions. PEPTIDES 3(3)
217-221, 1982.--The development of methods for cloning DNA complementary to specific messenger RNAs encoding
polypeptide hormones has opened new experimental avenues in the neurosciences. Recombinant DNA techniques have
been used to answer questions about the sturcture of polypeptide hormone precursors, the regulation of peptide hormone
production, and the location of peptide-producing cells in nervous and endocrine systems. Here, the application of cDNA
cloning to the quantitation, structural elucidation and tissue localization of mRNAs coding for neuroendocrine peptides is
reviewed.
Recombinant DNA Messenger RNA Pro-opiomelanocortin Enkephalin
Neuropeptide precursor processing Hybridocytochemistry




BOTH neural and endocrine cells respond to local and cir- cases the precursor exists in much smaller amounts in the
culating stimuli, and transduce them into either an increased cell than the final peptide product, and is harder to charac-
or decreased synthesis and release of a product which acts terize and measure. Nevertheless, pulse-chase type experi-
via a target membrane receptor to alter the behavior of ments involving immunological identification of precursor
neighboring or distant cells. An important cellular strategy species as well as direct isolation and sequencing of peptide
for neuroendocrine stimulus-secretion transduction is that hormone precursors have revealed much about precursor-
polypeptide hormones, factors and neurotransmitters are product relationships for several secretory peptides. Exam-
first synthesized as larger protein precursors which are ples include the demonstration of a single high molecular
stored in intracellular vesicles prior to their release [13,33]. weight precursor for adrenocorticotropin, beta-endorphin,
This has become obvious only recently. It was previously beta-lipotropin and alpha-MSH in the pituitary [14,19], the
thought that small peptide hormones/transmitters such as production of vasopressin and oxytocin and their respective
luteinizing hormone-releasing hormone and thyrotropin re- neurophysins from common precursor proteins [6] and the
leasing hormone might be synthesized non-ribosomally on existence of multiple copies of the enkephalin peptides in
protein-enzyme templates, as is the case for the tripeptide adrenal medulla flanked by pairs of basic amino acids within
glutathione [31]. However it now appears, although not larger, presumably precursor molecules [22, 23, 27, 38, 40].
proven, that the mechanism of ribosomal precursor synthe- Regulation of peptide expression under the influence of var-
sis followed by precursor packaging into secretory organ- ious physiological stimuli can also be examined by measur-
elles and subsequent processing to the final hormone will be ing peptide precursor and mature peptide levels directly (for
a valid model for understanding how mature polypeptide example, see [5,26]). These approaches are necessary but
product is made available for release by the neuroendocrine insufficient, however, to reveal regulation of genomic DNA
cell [3,13]. This means that the regulation of neuropeptide and mRNA that ultimately affects peptide secretion.
synthesis, unlike that of the classical biogenic amine neuro- These fundamental neuroendocrine issues are now being
transmitters, can occur directly at four loci at least: tran- addressed by the employment of techniques for the identifi-
scription and maturation of messenger RNA (mRNA), cation and purification of the mRNAs which code for indi-
mRNA translation, precursor protein packaging and metab- vidual neuropeptides, and synthesis of complementary
olism, and the release process itself. DNAs to them. Specifically, recombinant DNA methods
The problems inherent in examining peptide precursor allow (a) measurement of single mRNA species in neuroen-
regulation are multiple: the most obvious is that in most docrine cells and tissues to elucidate how peptide levels are

1Research Associate in the Pharmacology-Toxicology Program of the National Institute of General Medical Sciences, National Institutes of
Health, Bethesda, MD 20205.


217

, 218 EIDEN

modulated by regulation of mRNA transcription, translation plasmid such as pBR322 is opened by digestion with a site-
and stability (b) localization of the cells which produce spe- specific restriction endonuclease, which cleaves only at
cific neuropeptides by in situ visualization of the mRNA specific base sequences [28] and the double-stranded cDNA
coding for them in brain and pituitary and (c) sequencing of is separately modified by addition of homopolymer tails or
cloned DNA complementary to individual mRNA species in double-stranded DNA fragments (linkers) which can later be
order to infer the primary amino acid sequence of peptide site-specifically cleaved, to yield cDNA whose ends are
precursor molecules. Before illustrating with specific exam- capable of joining to the suitably modified exposed ends of
ples, it is first necessary to review briefly the methods cur- the digested plasmid. The modified plasmid and insert are
rently employed in cloning complementary DNA to both annealed; and the circular plasmid now containing the in-
low- and high-abundance mRNAs. serted cDNA is used to transfect a suitable bacterial host
Complementary DNA (cDNA) to a specific mRNA spe- [45]. Bacteria are grown as infected colonies which carry the
cies can be obtained in large quantities as follows: after total plasmid as a self-replicating episome. Plasmid-containing
RNA isolation from the tissue of interest by any of a variety bacteria are selectable because the artificial plasmid carries
of extraction methods [44], total messenger RNA is obtained genes imparting antibiotic resistance to its host. Likewise, if
as the polyadenylated RNA fraction which binds to a second resistance gene is present, its inactivation by inser-
oligodeoxythymidylic acid (OdT) after OdT-cellulose chro- tion of the cDNA at that site allows bacterial colonies con-
matography, since it is thought that most eukaryotic mRNA, taining plasmid to be distinguished from those containing
except that for histone protein, is polyadenylated [11,12]. plasmid with cDNA inserted. Each bacterial colony is a
Commonly, mRNA coding for a neuropeptide even in nerv- clonal representation of a single cDNA insert and therefore
ous tissue relatively rich in that peptide is less than 1% of the representative of a single mRNA is the original population.
total (poly-A)-RNA [8, 16, 20, 44]. Therefore, mRNA can be The so-called "clone bank" or cDNA library must then
enriched by size-fractionation (sucrose gradient or column be screened for the plasmid-cDNA of interest. This can be
chromatography) [44]. The sized fractions can be assayed for accomplished by transferring a portion of each colony to ni-
the ability to direct the synthesis of neuropeptide precursor trocellulose paper, fixing the cellular and plasmid DNA to
in cell-free systems or micro-injected into oocytes [7,43]; the paper, and hybridizing with labeled probe (synthetic
products are detected by antibody precipitation of labeled or oligonucleotide or cDNA to purified mRNA) [18]. Au-
unlabeled protein translated under the direction of the added toradiography reveals the colonies which are positive: these
exogenous mRNA. An alternative approach, necessary if can then be grown on a large scale for isolation of microgram
antibody to the peptide (precursor) is unavailable has re- to milligram quantities of plasmid, and subsequent recovery
cently been described which depends on the availability of of cDNA by digestion of plasmid with the restriction enzyme
limited sequence information about the neruopeptide [35]. A first used for insertion of the cDNA molecule into the plas-
synthetic oligonucleotide can be synthesized based upon the mid.
knowledge that each amino acid in the protein must be coded Cloned cDNA to specific mRNAs coding for single
for by one of the 1-6 possible trinucleotide sequences (the polypeptide gene products are effective analytical tools for
codon). If the codons for each of the amino acids in a 3-5 the quantitative measurement, qualitative localization and
amino acid fragment of the molecule are sufficiently non- rapid sequencing of neuropeptide mRNA. They have been
degenerate a " p o o l " of oligomers can be synthesized which used to answer questions about the localization of
represent all possible combinations for DNA complementary neuropeptide-containing cells and the regulation of peptide
to the RNA coding for this region of the peptide. Thus all of expression in neuroendocrine cells in response to various
the probes will be partially, and one (by definition) fully physiological stimuli. Some of these experiments, which
complementary to a portion of the mRNA. This probe can be point to rather exciting new avenues of neuropeptide re-
used to obtain an estimate of the size of the mRNA coding search, are described below.
for the polypeptide under study by hybridizing end-labeled Complementary DNA to a specific polypeptide mRNA
probe to a Northern blot of the total (poly A)RNA, i.e., RNA can be used to measure both the activity of gene transcrip-
separated on the basis of size on a denaturing gel and trans- tion and the amount of mRNA in the cell, after stimulation
ferred and bound to a membrane paper, providing a physical by a physiological secretagogue. For example, prolactin re-
RNA map of the gel [42]. Synthetic oligonucleotide probes lease from the pituitary is stimulated by thyrotropin releasing
can also be employed to screen fractions during mRNA hormone (TRH) [46]. It is of interest to know how TRH
enrichment procedures. ultimately acts to regulate prolactin expression. Dannies and
Following partial purification (or total purification if the Tashjian were able to demonstrate increased in vitro transla-
RNA itself is to be used as a probe for cDNA screening), tion of prolactin mRNA after exposure of pituitary cells to
enriched RNA is primed for reverse transcription with TRH [10]. Later, cloned cDNA to prolactin mRNA was em-
oligo-dT (complementary to the 3' polyadenylated sequence ployed to assay prolactin mRNA directly by hybridization-
of the mRNAs) to generate full-length cDNA copies. If the kinetic measurements [15]. Prolactin mRNA levels in pitui-
RNA is instead primed with a synthetic oligonucleotide tary tumor cells (GH3) rose after exposure to TRH in parallel
probe, a partial reverse transcript of only the RNA hybridiz- with in vitro translation activity of prolactin mRNA,
ing to the probe results; this can be sequenced immediately demonstrating that TRH increased prolactin levels in these
by the method of Maxam and Gilbert [19] to obtain further cells by a direct increase in mRNA levels (rather than, for
sequence information about the pelatide precursor and to example, activation of translational activity of the
confirm the hybridization specificity of the probe [8, 16, 35]. mRNA)[15]. Finally, direct measurement of nuclear RNA
After denaturation of the RNA-DNA hybrid, a second precursor to prolactin cytoplasmic mRNA using a prolactin
DNA strand complementary to the first is synthesized under mRNA cDNA probe demonstrated that TRH increased
the action of DNA polymerase [43]. The double-stranded prolactin levels by increasing the amount of transcribed
DNA can be inserted into a bacterial plasmid using a wide prolactin mRNA in the nucleus [2]. This showed that TRH
variety of techniques [44,45]. In general, a synthetic circular elevation of cytoplasmic mRNA is a result of increased pro-

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