From the Beckman Research Institute of the City of Hope, Duarte, California 91010
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ABSTRACT |
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The Drosophila cholinergic locus is
composed of two distinct genetic functions: choline acetyltransferase
(ChAT; EC 2.3.1.6), the enzyme catalyzing biosynthesis of
neurotransmitter acetylcholine (ACh), and the vesicular ACh transporter
(VAChT), the synaptic vesicle membrane protein which pumps transmitter
into vesicles. Both genes share a common first exon and the remainder
of the VAChT gene contains a single coding exon residing
entirely within the first intron of ChAT. RNase protection analysis
indicates that all Drosophila VAChT specific transcripts
contain the shared first exon and suggests common transcriptional
control for ChAT and VAChT. Similar types of
genomic organization have been evolutionarily conserved for cholinergic
loci in nematodes and vertebrates, and may operate to ensure coordinate
expression of these functionally related genes in the same cells. The
relative levels of Drosophila ChAT and VAChT mRNA
differ, however, in different tissues or in Cha mutants,
indicating that independent regulation of ChAT and VAChT
transcripts may occur post-transcriptionally. The predicted Drosophila VAChT protein is composed of 578 amino acids and
contains 12 conserved putative transmembrane domains. Full-length VAChT cDNA is 7.2 kilobase long and has unusually long 5- and
3
-untranslated regions (UTR). The 5
-UTR contains a GTG ChAT
translational initiation codon along with three other potential ATG
initiation codons. These features of the VAChT 5
-UTR region suggest
that a ribosome scanning model may not be used for VAChT translation
initiation.
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INTRODUCTION |
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Functional cholinergic neurons make and use acetylcholine (ACh)1 as a neurotransmitter and must coordinately express genes encoding choline acetyltransferase (ChAT, EC 2.3.1.6), for biosynthesis of ACh as well as a vesicular ACh transporter (VAChT), for accumulation of transmitter into synaptic vesicles. The VAChT gene has been cloned from Caenorhabditis elegans (1), Torpedo (2), rat (3-5), and human (3), and was found to encode a protein with 12 putative transmembrane domains which shows broad homology to the functionally characterized vesicular monoamine transporters (6, 7).
In addition to the sequence homology with vesicular monoamine transporters, genetic and biochemical studies have established the functionality of VAChT protein. Non-lethal mutations in the C. elegans VAChT gene (unc17) lead to uncoordinated movements and resistance to normally toxic levels of ACh esterase inhibitors (1, 8, 9). Modest levels of ATP-dependent vesicular transport of ACh have been shown in fibroblasts transfected with rat cDNA clones (3) and in homogenates of PC-12 cells transfected with the human VAChT cDNA (10). Furthermore, microinjection of rat VAChT cDNA into Xenopus embryos has resulted in increased quantal transmitter packaging in motor neurons (11).
In all species examined so far, the VAChT and ChAT genes form a phylogenetically conserved "cholinergic locus" in which the former gene is nested within the first intron of the latter. This unusual genomic arrangement of these two separate but related genetic functions may contribute to the coordinated regulation of both genes in some types of cholinergic neurons and may represent an example of a eukaryotic "operon" (12). In fact, several studies using cell culture systems have indicated coordinate regulatory properties for VAChT and ChAT expression (13-15). In C. elegans, alternative splicing of a common precursor RNA is proposed to be responsible for producing two distinct mRNAs from the cholinergic locus (16). In vertebrates, however, it is likely that ChAT and VAChT specific mRNAs result from a combination of differential promoter usage and/or alternative RNA processing (3, 5, 17, 18).
We have been using Drosophila melanogaster as a model
organism to understand the mechanisms and physiological significance of
cholinergic neuron-specific gene regulation. Our previous studies have
identified the transcriptional regulatory DNA responsible for the
spatial and temporal expression pattern of the Drosophila ChAT gene (19-22). We have also noted that the Drosophila
ChAT gene locus produced an unidentified 7-kb transcript
throughout development in addition to the 4-kb ChAT-specific mRNA
(23, 24). The 7-kb transcript could be detected with a cRNA probe specific to the first exon of ChAT, but not with a probe specific to
the eighth exon (24), indicating that it is transcribed in the same
direction as the ChAT gene, shares at least part of the first exon with authentic ChAT mRNA, and lacks other ChAT specific exons. In this study we have isolated cDNA clones for the 7-kb transcript and demonstrate that it encodes Drosophila VAChT.
In contrast to the vertebrate cholinergic locus, we also demonstrate that all Drosophila VAChT transcripts contain a shared first
exon with ChAT-specific transcripts, indicating that both genetic
functions are under common transcriptional control. The levels of ChAT- and VAChT-specific transcripts also appear to be independently controlled by post-transcriptional mechanisms and the structure of the
VAChT 5-untranslated region (5
-UTR) suggests that
Drosophila VAChT may not use a conventional ribosome
scanning mechanism for initiation of protein translation.
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EXPERIMENTAL PROCEDURES |
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Northern and Southern Blot Analyses-- Northern and Southern blot analyses were carried out according to Sambrook et al. (25). For Northern blots, total RNA was extracted from adult Drosophila heads by the CsCl/guanidinium isothiocyanate method (26). For Southern blot analysis, P1 DNA containing the ChAT and VAChT genes was purified as described in Ref. 27, and digested with either BamHI or SalI. 32P-Labeled probes were prepared from cDNA or genomic DNA clones using the Prime-It random primer labeling kit (Stratagene).
Cloning of VAChT cDNA and Genomic DNA--
A
Drosophila adult head cDNA library constructed in the
ZAP vector (Stratagene) was kindly provided by Dr. C. Hama (National Institute of Neuroscience, Tokyo). Approximately 3.6 × 105 plaques were screened with either
32P-labeled probe d (ChAT and VAChT first exon,
see Fig. 1) or e (ChAT first intron). Positive clones for
probe d were further screened with probe b (ChAT
exon III-VIII) and negative clones were analyzed further as candidates for VAChT cDNA. The Bluescript phagemids containing candidate VAChT
sequences were excised from the
ZAP vector and recircularized in vivo using the ExAssist/SOLR system (Stratagene)
according to the manufacturer's protocol. DNA sequencing was carried
out by the didoxynucleotide chain termination method using the
Sequenase 2.0 kit (U. S. Biochemical Corp.).
Polymerase Chain Reaction (PCR)--
Reverse
transcription-coupled with PCR (RT-PCR) was carried out as described in
Ref. 28 with adult head total RNA. Primer pairs used to amplify a gap
between two partial VAChT cDNA clones (G7 and H3, see below)
and the 3-end of the cDNA were: 5
-TGTCATTGTGGCGGACTT-3
, 5
-GGTACACACATTTATGCT-3
and 5
-CGAATGTCCATTTCGGTAGAA-3
,
5
-GAGAGAGAGAGAGAGAGAGAACTAGTCTCGAGTTTTTTTTTTTTTTTTTT-3
.
Primer Extension--
Primer extension analysis was performed
according to a standard protocol (29), using an oligonucleotide primer
5-CTATTCCTGGCTATACAATGTAGCT-3
. One pmol of the
32P-labeled primer was mixed with 45 µg of adult head
total RNA, incubated at 65 °C for 90 min and 22 °C for 10 min.
The extension reaction was performed at 42 °C for 60 min using
Superscript II (Life Technologies, Inc.), and the reaction product was
analyzed on a 6% polyacrylamide, 7 M urea gel.
RNase Protection Assay--
RNase protection assay was performed
according to Gilman (30). Drosophila ChAT
(XhoI-SacI fragment), VAChT (PstI
fragment), and -tubulin (DraII-EcoRV fragment)
sequences were subcloned into Bluescript (Stratagene). After
restricting the plasmids with PstI, BamHI, or
RsaI, respectively, [32P]UTP-labeled cRNA
probes were prepared using the MAXIscript in vitro
Transcription Kit (Ambion). A probe used to detect both VAChT and ChAT
mRNAs was prepared by using the BamHI-HindIII
fragment from VAChT cDNA cloned into Bluescript II (Stratagene).
The plasmid was digested with BamHI before transcription.
Total RNA was extracted using the RNAzol method (TEL-TEST, Inc.).
Quantitative analysis of transcripts was performed using
32P-labeled ChAT and VAChT antisense probes (2 × 105 cpm/each probe/tube) and a 32P-labeled
-tubulin antisense probe (2 × 104 cpm/tube) mixed
with unlabeled (30 ng/tube)
-tubulin probe. The hybridization
mixture was ethanol precipitated, dissolved in 20 µl of hybridization
buffer (80% formamide in 40 mM Pipes, pH 6.4, 0.4 M NaCl, 1 mM EDTA) and incubated overnight at
50 °C. The mixture was then digested with ribonuclease
T1 (400 units/200 µl; Life Technologies, Inc.) for 30 min
at 37 °C. The ribonuclease digest was electrophoresed in a
denaturing gel, which was dried and exposed to a PhosphorImager screen
overnight. The levels of ChAT, VAChT, and
-tubulin mRNA were
determined by densitometry using a Molecular Dynamics PhosphorImager
and ImageQuant software (Molecular Dynamics, Sunnyvale, CA). Results
are shown as the ratios of the density of the bands protected by either
ChAT or VAChT mRNA to that of bands protected by tubulin
mRNA.
Anti-Drosophila VAChT Polyclonal Antiserum and Western Blot
Analysis--
A cDNA fragment corresponding to a portion of the
predicted hydrophilic VAChT C-terminal domain (Arg-441 to
Asn-546) was amplified by PCR using two oligonucleotide primers
(5-CGGGATCCGAAAGCTGCGCAACATCT-3
, 5
-TGGAATTCATTGGCCACACGGGGCTG-3
), and the amplified
fragment was inserted into the BamHI/EcoRI site
of the pGEX-3X fusion protein expression vector (Phamacia). The fusion
protein was produced and purified according to Smith and Johnson (31).
The VAChT protein fragment was separated from glutathione
S-transferase by incubation with factor Xa (Boehringer
Mannheim) overnight at 30 °C, followed by preparative
SDS-polyacrylamide gel electrophoresis. Polyclonal antiserum was
generated according to a standard procedure described in Harlow and
Lane (32).
Drosophila Stocks and Temperature Shift Experiments-- For temperature shift experiments, wild type Canton-S and two temperature-sensitive mutants of the ChAT gene, Chats1 and Chats2 (33) were reared on a standard diet of cornmeal and agar at 18 °C, 65% humidity, on a 12-h light-dark cycle. Chats1 and Chats2 are point mutations in the ChAT structural gene, which result in thermolabile forms of the enzyme (34). Flies of the same age (3-4 days old) were transferred to a 30 °C incubator for the appropriate period of time. For Western blot analysis, Canton-S and Df(3R)Cha5/TM6 were reared at 25 °C. Df(3R)Cha5 is a deficiency lacking the chromosome region 91B3-91D1 where the ChAT and VAChT genes reside (35).
Prediction of RNA Secondary Structure--
Secondary structure
of Drosophila VAChT mRNA 5-UTR was predicted by energy
minimization using mfold RNA folding prediction program (36).
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RESULTS |
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Northern Blot Analysis of the Cholinergic Locus--
Northern blot
analysis of fly head RNA using a full-length ChAT cDNA probe
reveals 2 transcripts (Fig. 1,
probe a). The 4-kb transcript hybridizes with probes from
ChAT exons III-VIII (Fig. 1, probe b) or a ChAT exon I probe
(probe d). The longer 7-kb transcript can be detected with
either probe a or d but not with probe
b. Probes e and f (prepared from 2 different non-overlapping restriction fragments within the first intron
of ChAT) only detect the 7-kb transcript. A probe prepared from the
5-flanking genomic DNA immediately upstream of the ChAT transcription
start site (probe c) does not detect either transcript.
These results indicate that the 7-kb transcript shares an exon with
ChAT and that the remainder of its sequence is derived form the ChAT
first intron. Based on evolutionary conservation of the cholinergic
locus in other species, the nested 7-kb transcript was expected to
encode Drosophila VAChT.
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Identification of Partial cDNA Clones for the VAChT
Gene--
A Drosophila adult head cDNA library was
screened with probe d which hybridized to both the 7- and
4-kb transcripts (Fig. 1). Forty-two positive clones were isolated and
seven of them were shown to be negative for hybridization with probe
b (a ChAT specific probe). Restriction analysis indicated
that all seven cDNA clones were derived from the same transcript
and partial sequencing of the 5-ends revealed that all clones
contained the 5
-UTR sequence of the ChAT gene. The longest
clone, G7 (3.6 kb), was further analyzed and completely sequenced.
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Detection of the VAChT Protein by Western Blot Analysis-- Western blot analysis of adult head extracts using anti-Drosophila VAChT antiserum (see "Experimental Procedures") detected a rather broad band with an Mr of 65,000 (Fig. 3), which agreed well with the predicted molecular mass of the longest open reading frame from the cDNA sequence. The identity of this band was further confirmed when extracts from the wild type (Canton-S) and flies carrying a VAChT gene-deficient chromosome (Df(3R)Cha5/TM6), were compared. Although overall protein patterns and intensities were indistinguishable between Canton-S and Df(3R)Cha5/TM6 (data not shown), the intensity of the antibody-positive band was reduced approximately to one-half when the extract from Df(3R)Cha5/TM6 was used (Fig. 3).
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Characterization of Full-length VAChT cDNA--
The G7
cDNA clone lacks both the 5- and 3
-regions of the 7-kb
transcript. In a previous study Sugihara et al. (37)
tentatively determined the 5
-end of ChAT exon I by sequencing a primer
extension product obtained using a synthetic primer complimentary to
nucleotides at position 47-70 (relative to the ChAT translation start
site). We have re-examined the 5
-end of this shared exon by carrying out primer extension analysis using primers that corresponds to the
5
-ends of VAChT cDNA clones G1 and G12 (see Fig.
4A). Two major bands and one
minor band were observed following primer extension (Fig.
4B) and the corresponding nucleotides are indicated in Fig.
4A.
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Genomic Organization of the Drosophila Cholinergic
Locus--
Sugihara et al. (24) have previously isolated
and characterized three independent phage clones (gB517, gB7, and gB1)
which contained all 8 exons of Drosophila ChAT and were
missing only a part of the first ChAT intron. We have now isolated this
missing genomic region as part of a P1 clone (DS07917) representing
polytene chromosome region 91C-D (the cytogenetic position of the
ChAT/VAChT locus). This P1 clone hybridized with probe
b, probe c, G7, and H3 (data not shown),
indicating that it contains the entire ChAT gene including
the long first intron containing the complete VAChT cDNA. Southern
blot analysis of DS07917 digested with either BamHI or
SalI showed four or two bands when G7 was used as a probe (Fig. 5A). The 5.5-kb band
(band 3 in Fig. 5A) corresponds to the BamHI
fragment located in the gap between gB517 and gB7. The size of the
first intron of the VAChT and ChAT genes are thus 6.1 and 17.4 kb, respectively. This estimation was further confirmed by
PCR amplification using primers corresponding to the 3-end of genomic
clone gB517 and the 5
-end of the VAChT second exon (data not shown). A
1.8-kb (SalI/BamHI) genomic fragment containing the boundary of the VAChT first intron/exon II was subcloned from DS07917, and the intron/exon boundary was determined. The junction showed a conventional intron/exon sequence boundary, TTAAAG/GTTGTT.
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VAChT mRNA Is Present Only in a Form Which Includes the Shared Exon I-- A class of VAChT mRNAs in rat has recently been identified that does not contain sequences shared with ChAT mRNA (18). To examine the possibility that such mRNAs may also be present in Drosophila an RNase protection assay was carried out with a probe that included the boundary sequence of the shared first exon and the unique VAChT second exon (Fig. 6A). This cRNA probe yielded only two protected fragments which correspond to ChAT and VAChT mRNA (Fig. 6B). The absence of a 238-nucleotide fragment which could correspond to VAChT second exon sequence attached to some other sequence indicates that all detectable VAChT mRNA is present in a form which includes the shared first exon with ChAT.
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Differential Regulation of ChAT and VAChT mRNA Levels-- To examine coordinate regulation of ChAT and VAChT mRNA expression, we determined ChAT and VAChT mRNA levels on the same samples using a quantitative RNase protection assay. cRNA probes were designed to protect a part of the specific coding region for each mRNA (Fig. 7A). Transcript levels were measured in whole flies or in dissected heads or bodies. As shown in Fig. 7B the ratio of VAChT to ChAT transcript is approximately equal in heads while whole flies or bodies show an excess of VAChT transcript relative to ChAT. This result implies that the levels of VAChT and ChAT specific mRNA can be differentially regulated in different parts of the fly.
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DISCUSSION |
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The Drosophila cholinergic locus, like its counterpart
in nematodes and vertebrates, has a unique genomic organization: the VAChT gene is nested within the first intron of the
ChAT gene and both transcripts share a common first exon.
All Drosophila VAChT transcripts appear to share the
complete first exon with ChAT implying that both genetic functions are
under common transcriptional control. Our previous studies identified
the 5 cis-regulatory DNA responsible for the spatial and temporal
expression pattern of the Drosophila ChAT gene using
transgenic animals (19-22). The overall strategy used to regulate ChAT
transcription is to activate or repress several regulatory sites which
are distinct in different types of cholinergic neurons (19-21).
Furthermore, the Drosophila ChAT gene regulatory information
essential for survival has been mapped to a region of the 5
-flanking
DNA 300 bases in length (22). In this sequence the most strongly
footprinted sequence was a 22-base pair region that contains an
octamer-like motif in its center. A Drosophila transcription
factor in the POU family, pdm-1 was shown to bind to the
22-base pair sequence and suggested to regulate ChAT gene
expression (22). The regulatory strategies and mechanisms identified
for Drosophila ChAT expression should be also applicable to
expression of another cholinergic marker, VAChT. In contrast to this
simple situation in Drosophila, some mammalian VAChT
transcripts have been reported to share sequence with ChAT while others
do not (18). These latter VAChT transcripts apparently use alternative
promoters. The nematode cholinergic locus is thought to give rise to
specific ChAT and VAChT transcripts through alternative splicing,
although the situation is complicated by additional trans-splicing
reactions to the first shared exon (16).
The organization of the Drosophila ChAT and VAChT
genes suggests that two distinct transcripts are produced by
alternative RNA processing of a common primary transcript. Usually, the
alternatively processed transcripts code for genetic isoforms or occur
in different cell types or tissues (42). Production of specific VAChT
and ChAT transcripts is unique, however, since two transcripts encode metabolically related but completely distinct genetic functions. Furthermore, both transcripts must be produced in the same cells at the
same times to have proper cholinergic neuron function. RNA processing
to generate specific transcripts for each genetic function may involve
one or both of two possible mechanisms. Alternative 3 splice site
choice between VAChT and ChAT second exons would be responsible for
producing the respective specific transcripts with a shared first exon
from a common pre-mRNA. Alternatively, selection of poly(A)
addition site could be a major determinant for which mRNA is to be
made. It is not yet known which of these potential mechanisms operate
in Drosophila.
Since ChAT and VAChT are under common transcriptional regulatory control in Drosophila, the levels of each transcript are likely to be fixed in different parts of the nervous system. It was somewhat surprising, therefore, to see that the ratio of VAChT to ChAT transcripts varied between heads and bodies as we have observed. Apparently, the levels of each transcript can be regulated independently of each other by post-transcriptional mechanisms in the process of either mRNA production or degradation. As we showed before (40, 41), the ChAT mRNA levels decreased in Chats1 and Chats2 at the non-permissive temperature probably because normal levels of ACh and/or cholinergic neuronal transmission are necessary for keeping the normal ChAT mRNA levels (41). Interestingly, the levels of ChAT and VAChT specific mRNAs can be differentially regulated in the Chats mutant flies. Again a post-transcriptional regulatory phenomenon is likely to account for decreasing levels of ChAT mRNA while the VAChT mRNA levels are held constant. The most appealing rationale for having VAChT and ChAT genetic functions organized as a single genetic locus under common transcriptional control would be to ensure coordinate regulation of the expression of these two genes. The uncoupling of the levels of specific transcripts we have observed in space or in response to a temperature-sensitive structural gene mutant in the ChAT function would perhaps tend to obviate this potential regulatory advantage. Cholinergic neurons could have different physiological demands for biosynthesis and packaging of neurotransmitter in different parts of the nervous system. There may also be differential rates of turnover for the specific transcripts or proteins, thus making it attractive to fine tune the levels of each using post-transcriptional regulatory strategies.
In cultured mammalian cells ChAT and VAChT mRNA levels respond in a coordinated fashion when soluble factors, such as retinoic acid, cAMP, and the leukemia inhibitory factor are added (13-15). In addition, distribution of ChAT and VAChT mRNAs in the adult rat brain is virtually identical (3), suggesting coordinate regulation of these two genes even though specific promoters for ChAT and VAChT may exist (18). ChAT and VAChT mRNA levels, however, can be differentially regulated during rat brain development. VAChT mRNA levels are already present at up to 60% of adult levels during late embryonic stages while ChAT mRNA levels increase primarily during postnatal stages (43). It is not known if the uncoordinated changes in ChAT and VAChT mRNA levels are due to differences in transcription or RNA processing.
The 7.2-kb size of the Drosophila VAChT transcript is
significantly larger than the 2-3-kb transcripts seen in other species (1-5). This difference in mRNA size is attributed primarily to the
unusually long 5- and 3
-UTR in Drosophila VAChT mRNA.
It should be mentioned, however, that a relatively large transcript (~6 kb) has also been detected in a human neuroblastoma cell line (44), using a ChAT cDNA probe. This larger human transcript has not
yet been characterized.
In nematodes and mammals, the exon shared by VAChT and
ChAT does not contain any protein coding sequence. In
Drosophila, however, the 3-half of the shared first exon
corresponds to the ChAT protein coding region and includes an unusual
ChAT GTG translation initiation codon. In addition, the VAChT 5
-UTR
contains three potential translation initiation codons (ATG) which are
all followed by multiple termination codons and thus not likely to mark
the beginning of an open reading frame (Fig. 4A). Most
eukaryotic mRNAs are thought to initiate protein translation using
a ribosome scanning mechanism. Ribosomes bind to a 5
-cap structure and
progressively scan the mRNA until encountering the first AUG codon
which serves as the site of translation initiation (45). The structural
features of the Drosophila VAChT 5
-UTR indicate that
efficient translation initiation cannot be accomplished by the
conventional 5
-cap dependent ribosome scanning mechanism.
One of the alternative mechanisms for Drosophila VAChT
translation initiation is the use of an internal ribosome entry site (IRES). In IRES-mediated translation initiation, ribosomes bypass the
5-CAP by directly binding to an internal sequence and initiate protein
translation at the first AUG downstream of the IRES site. IRES-mediated
translation has been identified in a number of viral as well as
cellular mRNAs (46). Although functional IRES motifs are rather
divergent in sequence (47), Piconoviral IRES elements exhibit highly
conserved secondary structures and contain an polypyrimidine tract
followed by an ATG triplet (48). As shown in Fig.
8 computer analysis of the
Drosophila VAChT 5
-UTR using mfold (36) also predicts
formation of secondary structures. Coupled with the presence of an
extensive polypurine tract between the last upstream ATG and the
initiation codon (Fig. 4A), these features may suggest that
Drosophila VAChT also initiates translation via an
IRES-dependent mechanism. Drosophila ChAT and
VAChT may thus use different translation initiation mechanisms to
achieve differential regulation. In rats, a comparison of the
developmental expression patterns of VAChT mRNA and protein
revealed a large excess of mRNA from late embryonic stages up to
early postnatal ages (43), suggesting possible regulation of VAChT
translation in mammalian nervous system during development.
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ACKNOWLEDGEMENTS |
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We thank Dr. C. Hama for a Drosophila adult head cDNA library and Dr. S. L. Zipursky for P1 Drosophila genomic DNA clones. We also thank Dr. Junko Kasuya for helping carry out primer extension experiments, and Kathy Barbrow and Elvia Gutierrez for excellent technical assistance.
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FOOTNOTES |
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* This work was supported by a grant from National Institutes of Health, National Institute of Neurological Disorders and Stroke (to P. M. S.) and a fellowship from the John Douglas French Alzheimer's Foundation (to T. K.)The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF030197.
To whom the correspondence to be addressed: Div. of Neurosciences,
Beckman Research Institute of the City of Hope, 1450 E. Duarte Rd.,
Duarte, CA 91010. Tel.: 818-301-8364; Fax: 818-301-8908; E-mail:
psalv{at}coh.org.
1
The abbreviations used are: ACh, acetylcholine;
ChAT, choline acetyltransferase; VAChT, vesicular acetylcholine
transporter; UTR, untranslated region; PCR, polymerase chain reaction;
RT-PCR, reverse transcription-coupled polymerase chain reaction; IRES, internal ribosome entry site; Pipes,
piperazine-N,N-bis(2-ethanesulfonic acid); kb,
kilobase pair(s).
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REFERENCES |
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