kephalin (PPenk) mRNA and enkephalin peptides, and may serve as a mammalian model for the invivo study of proenkephalin (Penk) gene expression, peptide ...
DNA AND CELL BIOLOGY Volume 13, Number 1, 1994 Mary Ann Liebert, Inc., Publishers
Pp. 25-35
Cloning and Characterization of Hamster Proenkephalin Gene YUAN-SHAN ZHU,1 ANDREA D. BRANCH,3 HUGH D. ROBERTSON,2 and CHARLES E. INTURRISP
ABSTRACT Our previous studies have shown that the hamster adrenal, like the human, contains high levels of preproenkephalin (PPenk) mRNA and enkephalin peptides, and may serve as a mammalian model for the in vivo study of proenkephalin (Penk) gene expression, peptide biosynthesis, and release. To define further the factors that may regulate hamster Penk gene expression, the hamster Penk gene was isolated from a genomic library prepared from Syrian hamster liver. The hamster Penk gene contains four exons and three introns and encodes 268 amino acids including six copies of Met-enkephalin containing peptides and one copy of Leu-enkephalin. In the 5' upstream region, there are TATA and GC boxes and multiple putative regulatory elements including the cAMP response element, AP-1, AP-2, AP-4, and the glucocorticoid response element (GRE). Possible GREs are also present in the introns. A comparison with the human and the rat Penk genes indicates that both the human and hamster Penk gene contain three introns, while the rat Penk gene has two introns. The intron missing from the rat Penk gene is short and separates the first and second exons of the hamster and human genes. In addition, the hamster and human genes share a region (100 bases) in the 5' upstream sequence that is 98% homologous. It is of interest that Penk gene expression is high in the adrenal medulla of both human and hamster, but is much lower in the rat. These homologous regions and the extra intron may contain regulatory features responsible for a high level of expression in the human and hamster adrenal medulla.
INTRODUCTION of two endogenous opioid in 1975 by Hughes et ai (1975), a number of peptides have been isolated and characterized from mammalian tissues. These opioid peptides are derived from three distinct genes, proenkephalin (Penk), prodynorphin, and proopiomelanocortin (Akil et ai, 1984). The Penk gene encodes the opioid peptides, Met- and Leuenkephalin, which are widely distributed in the central nervous system and several peripheral tissues, and have been postulated to function as neurotransmitters, neuromodulators, and/or neurohormones (Akil et ai, 1984). Because the adrenal gland has been considered the major source of peripheral endogenous opioid peptides, most work has concentrated on this tissue. The adrenal opioid peptides have been demonstrated to be costored and coreleased with catecholamines in the chromaffin cells (Viveros et al.,
Since peptides opioid
the demonstration
1979; Lundberg and Hokfelt, 1983), and may be involved in stress-induced analgesia (Lewis et ai, 1984) and in the modulation of adrenal catecholamine secretion and actions
(Kimura et ai, 1988). Previously, we have shown that the hamster adrenal medulla, like the human, bovine, dog, cat, and guinea pig (Schultzberg et ai, 1978b; Viveros et ai, 1979), contains high levels of preproenkephalin (PPenk) mRNA and enkephalin-containing (EC) peptides, which are 90-fold and 400-fold higher than those in the rat adrenal medulla, respectively (Franklin et ai, 1991a,b). In addition, section of the splanchnic nerve, which innervates the adrenal, produces a profound decrease in the levels of both PPenk mRNA and EC peptides, while the induction of Penk gene expression by reserpine is blocked (Franklin et ai, 1991a,b). These data support the conclusion that transsynaptic activity produces a positive regulation of Penk gene expression in the hamster adrenal, similar to that
Departments of 'Pharmacology and biochemistry, Cornell University Medical tive Diseases, The Rockefeller University, New York, NY 10021. 25
College, 3Center
for Studies of the
Biology of Addic-
ZHU ET AL.
26
shown for most other mammalian species studied to date (Schultzberg et al., 1978a; Franklin et al., 1991a,b). How-
in the rat adrenal appears to be more complex with tonic inhibition being the primary consequence of intact innervation (Kilpatrick et al., 1984; La Gamma et al., 1984; Zhu et al., 1992). Paradoxically, increased impulse activity in rat splanchnic nerve activates the Penk gene (Kanamatsu et al., 1986; DeCristofaro and La Gamma, 1990; Zhu et al., 1993). Thus, both increased and decreased transsynaptic activity increase Penk gene expression in rat adrenal. The basal level of Penk gene expression in the rat adrenal is too low to permit accurate measurement of treatments that diminish the basal level, whereas in the hamster adrenal, both down and up regulation can be measured. Thus, the hamster adrenal may serve as a useful model for the in vivo study of mammalian adrenal medullary Penk gene expression (Franklin et al., 1991a,b). To define further the factors that may regulate hamster Penk gene expression, to obtain some comparative information, and to learn more about the species difference in the basal levels and the regulation of Penk gene expression, we have cloned and characterized the hamster Penk gene and compared its structure with the known Penk gene sequences including human (Comb et al., 1983), bovine (Noda et al., 1982), and rat (Rosen et al., 1984; Yoshikawa et ai, 1984). Our data indicate that the primary structure of the hamster Penk gene is similar to that of the human Penk gene. Furthermore, a high degree of homology among human, hamster, and rat was found in the 5' upstream region of Penk gene. ever,
transsynaptic regulation
of Penk gene
expression
MATERIALS AND METHODS
Preparation of high-molecular-weight genomic DNA from hamster liver The genomic DNA from Syrian hamster liver (8 weeks of age, weighing 80-100 grams) was prepared as described by Davis et al. (1986). The quality and the size of the prepared genomic DNA was evaluated by UV absorbance, gel electrophoresis, and Southern blot analysis.
Preparation and purification of riboprobes A 971-base rat PPenk mRNA riboprobe (sp. act., 6.5 x 108 dpm/jig) was obtained by in vitro transcription and CF11 column purification as described (Zhu et al., 1992) from the pYSEAl plasmid (Yoshikawa et ai, 1984). A 100-base 5' rat PPenk riboprobe (the major portion of exon I of the rat proenkephalin gene; Rosen et al., 1984; Yoshikawa et al., 1984) was prepared by use of a template, which was generated by linearization of pYSECl plasmid (a gift of Dr. Sabol; Yoshikawa et al., 1984) with Dde I digestion, and a 252-base 3' riboprobe was generated from the pYSEAl plasmid (Yoshikawa et at., 1984) that had been linearized with I*vu II. The 5' riboprobe was purified by the combination of 5% polyacrylamide denaturing gel (7 M urea) and CF11 cellulose chromâtography as described before (Branch et al., 1989).
Screening hamster library Syrian hamster genomic library was prepared by Stratagene in the X phage vector FIX II. Recombinant phage were plated out on Escherichia coli PLK17 cells and screened in duplicate by in situ plaque hybridization (Sambrook et al., 1989) with the rat PPenk riboprobe. HybridA
carried out in 1 x TESS buffer (5 mM TV-tris5 mMEDTA, 0.15 M NaCl, 0.25% NaDodS04 pH 7.4) containing 1 x 106 cpm/ml of 32P-labeled riboprobe at 75°C for 4 hr under mineral oil. Filters were washed and exposed to Kodak X-Omat film at -70°C with intensifying screens (Inturrisi et al., 1988). Approximately 1 x 104 recombinant phages were screened and four positive clones (hENK 1-4) were obtained.
ization
was
[hydroxymethyl]methyl-2-amino-ethanesulfonic acid,
DNA sequence
analysis
A 4.8-kb Bam HI fragment from hENK-1 and a 2.1-kb Bam HI fragment from hENK-3 were subcloned into a pGEM 7zf(+) (Promega) vector (named pGEM-4.8 and pGEM-2.1, respectively). A nested set of unidirectional subclones was generated by use of an Erase-a-Base system (ExoIII, Promega). Double-stranded DNA was prepared by use of a Qiagen column. DNA sequences were determined by the dideoxy chain-termination method with the Sequenase 2.0 DNA sequencing kit (USB) with the M13 universal primer, or SP6 promoter primer, or synthetic oligonucleotide primers. The oligonucleotide primers used were TTCGGTTTGGGGCTA (from -49 to -35 in Fig. 2) and CTTTATAATTAGCCC (complementary to bases -26 to -40 in Fig. 2).
Ribonuclease protection mapping The hamster Penk riboprobes were prepared by in vitro as described above and hybridized with total cellular RNA from selected hamster tissues at 75 °C for 4 hr in 30 /tl of 2x TESS hybridization buffer (10 mM Ntris[hydroxymethyl]methyl-2-aminoethanesulfonic acid, 10 mMEDTA, 0.3 M NaCl, 0.5% NaDodS04 pH 7.4) which also contained 150,000-200,000 dpm riboprobes as described elsewhere (Zhu et al., 1992). After hybridization, samples were treated with a 300-/a1 solution containing a high-salt buffer (0.3 M NaCl, 5 mM EDTA, 10 mM TrisHCl pH 7.5), 40 /_g/ml RNase A (Worthington Biochemicals, Freehold, NJ), and 2 itg/ml RNase Tl (Calbiochem, San Diego, CA) at 30°C for 1 hr. Then the samples were phenol-extracted and ethanol-precipitated. Aliquots of the samples were denatured by heating in a formamide-containing dye mixture at 100CC for 2 min and fractionated in 10% polyacrylamide-7 M urea gels (Zhu et al., 1992).
transcription
Primer extension The primer extension was carried out as previously described with a two-step method (Boorstein and Craig, 1989). A synthetic oligonucleotide, AAGGACTCTGAAACG, that is complementary to nucleotides 726-740 (see Fig. 2), was used as a primer. The oligonucleotide was hy-
HAMSTER PROENKEPHALIN GENE
bridized
27
from the hamster adrewith 2 ng of sense transcript of the rat Penk gene (Zhu et ai, 1992) by heating at 80° C for 3 min and incubating at 34°C for 3 hr in 0.3 M NaCl, 10 mMTris-HCl pH 7.5, 2 mMEDTA. Then, the cDNA was radiolabeled by use of [a-32P]dCTP in the presence of 8 fiM of dATP, dGTP, and dTTP, and 15 units of AMV reverse transcriptase (USB) at 37°C for 5 min, and extended in the presence of 1 mM dNTPs, 10 mM DTT, 10 mM Tris-HCl pH 8.4, 6 mM MgCl2, 60 j*g/ml actinomycin D, and 40 units of RNase inhibitor at 42°C for 30 min. The reaction was stopped by addition of 2 /d of 0.5 M EDTA, and the samples were precipitated by ethanol and analyzed by electrophoresis on a 10% polyacrylamide-7 M urea gel to 5 fig of
nal, striatum,
(Zhu
et
or
poly(A)*RNA
liver,
or
ai, 1992).
RESULTS
Isolation and characterization genomic DNA
of hamster
The molecular weight of the isolated hamster genomic DNA was over 50 kb determined by use of a non-denaturing
agarose gel and X DNA (48 kb) as a marker. The ratio of UV OD260/OD28o was 1.75. Southern blot analysis (Sambrook et ai, 1989) of the isolated genomic DNA by use of the rat PPenk riboprobe revealed that a major 3.6-kb Eco RI fragment was radiolabeled (data not shown).
Isolation
of the hamster Penk gene
Approximately 1 x 106 recombinant phage were screened as described in Materials and Methods and four positive clones were identified and named hENK 1 through 4. DNA from these recombinant phage-plaques was purified and analyzed by use of multiple, sequential restriction enzyme digestions and Southern blot hybridizations with the 971-base, 5' or 3' riboprobes prepared from the rat Penk gene. Each of the four positive clones showed a 3.6-kb Eco RI hybridization band by use of either the 971-base-long or 3'-end probes, while only hENK-3 showed a 6-kb Eco RI band hybridized with the 971-base riboprobe and a 2.1-kb Bam HI hybridization band with the 5' riboprobe (data not shown). The results of this characterization are presented in Fig. 1. The results of Southern blot analysis of the four positive colonies and the genomic DNA suggest that the Penk sequence is present in hamster as a single-copy gene. hENK-4
hENK-3
hENK-2 hENK-1 S
EBSB
B
J_
I II I
I
B E
B S
S B
B E B
E
E
-U_U—I_I—L
Exon I II
IV
-\
BamHI 2-2.1 kb
h BamHI 4.8-5kb
Pv P
_M_
FIG. 1.
Il
The map of restriction sites and the sequencing strategy for the hamster proenkephalin (Penk) gene. The upper portion shows the lengths of four clones (hENK-1, hENK-2, hENK-3, and hENK-4), which were identified by in situ plaque hybridization with a rat preproenkephalin riboprobe. Approximately 1 x 10" recombinant phage were screened. One Not I site is located on each end of these clones. These Not I sites are in the FIX II vector arms. The middle portion shows the restriction map and the exon/intron organization of the hamster Penk gene. The map was constructed by analyzing fragments obtained from multiple, sequential enzyme digestions and Southern blot analyses. E, Eco RI; S, Sac I; B, Bam HI; P, Pst I; Pv, Pvu II; N, Nru I. The shaded regions indicate the exons. The lower portion shows the sequencing strategy. The horizontal arrows indicate the direction and extent of sequence determination.
ZHU ET AL.
28
-975 -915 -855 -795 -735 -675 -615 -555 -495 -435 -3 75 -315 -255 -195 -135 -75 -15 46 106 166 226 286 346 4 06 466 526 586 646 7 06 766 826 886 946 1006 1056 1116 1176 1236 12 96 13 56 1416 14 76 1536 15 96 1656 1716 1776 1836 18 96 1956 2 016 2076 2136 2196 2256 2316 2376 2436 2496 2556 2616 2676 2736 2796
gatcctggaa caggtccctc actcctgaca aaacaccctg ggccagattg ttggtttttt gtttaggtta gatttcactg tctgtaacca cagatgggtg cctggagagg tagaaaccca tcaccgcaga
gtgagggagc cccgtggata cactgacatc tgcagcacat atccttcctg tttttatttt
ggggggattg atctaaagga ctaactgaat aaaaaattac
cggctgcgtg ctctccgagc ccctgcttcc cttcagcatc agatgagcaa gccccaagac aagcgttccc tccacaccaa
acacctcccc gacacccccc ttactcatca aacaccccca
acctggaagt cccagaacct ttgtttttga gtattgcttc gaaagttcca tgttagcaag gtggatgccc cgagggtgtc ccctttcaaa cacccccatc cacggtgcca tgcccgcggt gccctcccca gagttcctaa actcgcaccc tccacccccg ggggccgcgt
tagcaacagc tggtcctgga gttttgttat ttttgttttg gaaagccttc gcctactgtg gcctttcgga aggggcaggt
caagtccgga gaacaggatt ttttgtttgt tgtgcctgag aagatccaca cacctgacgc taactgtgca tcgcggaatc
aggtggggga agtccatctg gaaagcagtt gggggggacc tccggcgccc gggagcccct ccgccaqgcg tcggcgcggg ctggcgtagg gcctgcgtca gccqqcqatt
qqggcgcgcg
gcgcgcaggt
ggtttttggt tgttcctttt tgtagcgaca actactgatt aaagtcaggg tcgcaggact caccttctca
ccaaagtgat ctgcgcggtg gctgtgctgg
gctgcagccc cgcctcttcg gtttgqqqct aattataaaq tqqctqtqqc GAGGCAGGCG CTCAGAGCCC CGCAGCCCAA CGACACCGAG
gctgccaacc tccggACAGC GACCGCGACG gtgagttcgc gatctgcccc acacttacct tgtttttctc tctcttgcag CCTGCTCCAT CTGAACGGCG GC/AACgtgag ggtgaccatc acctgttccc taattgcatc catggggacc aggtcctata tcccaagtgc ctgcagaatc tcgccccagg caaccgacgc ggatggatgc caccgggtag ccaggatgct gaagcccccc cggaaagaca gaatggccct aggacaactg gagccactcc tgaccaagtc tgtgggtggg gtctaggaaa gagtatctct aaagcttcct actgggtcac ctttcagagg tcctacagCC CATGGCGCGG TTCCTGAGAC
gcaccttgcc cagctctccg ctgccgcttc
AGTGGCATCT AGGACCCGCT CCTCCAGCAG
tgactttgcc ccccaagact cctaaggcgt gtcccccact ttctcggggc tcctctttgc ccgctcccca gcagcgatct ccaatgcttg cagagaagac agtacáccct aggaagaata ccggggaaga taggtggccc caggaaacag aggaagacag tatgtccccg gggaagcctc atccctgagt aaccccggaa tgtgttgggt gagggtcttg ctctaagacc tcccacctag cggccaggtc cccacgagcg cgtccttctc
TTTGCACCTG GCTGCTAGTG CTTGGGTCCT GCCTCCTGGC TACAGTGCAG GCAG7AATGCA GCCAGGACTG CGCCAAGTGC AGCTACCAC7 TGGTGAGCCC AGGCGACATC AACTTCCTGg tgagtagaat tcttggtgag gggttgtcct tgcgcactca cgaggcagcc ctgcaaacat caagtggtca ctcgcgctca aagtgcttgc
acgagccttt ggtgacctgg gtgacccttc caaaacgttg ccaattttgg aaccagccta tctgggatc- -~3.5 kb-a ctgggctggt gcagcatacc cagcaagctc tccagcaaga ctttttccaa gctcccttgt tccgtgcaag agaaagctgt cttttctctg tgacagtgtg tatactaaag cagataacta gggctctttt agtcatgcat ctccttgttc ttcccagGCG ACAAATGCCT TGAGTTCCCT CTTGCTGGCC GGATGAGCTT GAAGTACGGT CGACCTGCTG AGAGAGCACT CAAAAGAAGC CATGAGAAGG CCTGAAGCGC AGTTCCTGAG GGTGACCCCA TGTTTGTTGT GAAAGCTGTG TGCAGCTATC AAACGTCAAT
TCTCACAAAA TGTGATAGCA AAGAAGTACG TATCCTGTGG GGCTTCATGA AAAGAGCTCC GACAATGATG CCCCAAGTGG GTGGGCCGCC TTTGCGGAGT ATAG7AAAAAA GGCACCCACC CACTTCCTGC ATGTGTGGGG CCGGTATCAT AAATGCTTAC
TCTGGGAGAC TCAACATGTT GAGGGTTCAT AGCCCGAAGA AGAAGGATGC TGGGAACAGG ACAACATGAG AAGACGAAGC CAGAGTGGTG CCCTGCCCTC GATATGGGGG AGCCTGCTGC TTGTACTGTT TCTGTGTTCT GCTGTAAGTT TTGTATATAG
CTGCAAGGAT CAAAGACAGC GAAAAGGTAT AGAAGCGAAT GGATGAGGGC AGACAACCGT CAAGAGGTAT AAAAGAGCTG GATGGACTAC CGATGAGGAA ATTCATGCGA ATCCCCAGTG GCCCTTCTGG CTTGAGTCTC TCTGTTACCT ATATAATAAA
ttggtcttgt gagaagtgac tatcatttgt ctgtgggtgt agagaccgtg ccatcacctg ctatgacaga ataatcactg gctaaaaaac attttattta acaatttgta tgtaaactct aacaaaatta gatgcaatga caatttggtc ctgttttata caatggctaa ttagttgatc taaatttgct caagagttga caccttttcc tcatctccta tccaggattc cagcaagagc tcttgcacac ccttcaccat gaccatagta ggaacctcca tggcatggat aaattaactc acttggcaat cagacaatcc tgtgaattct caaaacactt gatgaatatt tcacaataac cagatatcaa aaatacaagt ttcactttct agttga
atccctatcc
gcttggatcc ctttcgtacc gataagagac tggtccatca
gctctgtctc ctttgaaac_ cattcgcttc tagaaatggc ctggctggaa
TGCACACTGG CTCCTGCAGG AACAAACAGG GGAGGCTTCA GGAGGCGAGA GACACCCTGG GCGAGAGAGG GGGGGCTTCA CAGAAGCGCT CAG7AAGAGAT GCTGAAAGTT TTTTAAAGCC AGCGACTGCC CCTGGATAAC GGGGCTCAGT CATCCCCTJVA CCCATTACCC
AATGTGAAGCTGTCCAAGCC ATGAGAGCCA TGAAGAAGAT TCCTTGCCAA CCAACTCCTC GCCGCCACCA TGAGAGGCCT ATGGGGGCTT ACGGAGGCTT ACTCCAAAGA CTTTCCCAAA TCACCAGTGG TACACTGCCT ACTGGCCTGT CTTTTGGCAG
aacaacagaa
gtgagattaa tcgaactctg agtgggtgaa cgtgttacta attcgttatt
CAACTGCACa tcacggcaaa ggctagactg gcttctttcc cctgacccag taaatgagca aatgcaaaga tgtacagaaa gtgaacgcca
agaaagaact atagctgatg agaagggtac aagctgtttg
29
HAMSTER PROENKEPHALIN GENE
taining part of the 5'-upstream sequence, all of the exon I, II, and III sequences plus introns A and B, and part of the intron C (complementary to nucleotides -86 to 1,034 of the hamster Penk gene shown in Fig. 2) was hybridized to total cellular RNA extracted from hamster adrenal (lane c) and striatum (lane d), tissues containing high levels of
Nucleotide sequence and the primary structure of the hamster Penk gene
Figure 2 shows the nucleotide sequence of the exons and surrounding DNA segments of the hamster Penk gene. A total of 3,800 bases (including 975 bases in the 5' upstream region) of hamster genomic DNA were sequenced to provide information about exons and the surrounding regions. By sequence analysis, mRNA analysis, and comparison with the human and rat Penk genes, the exon/intron junc-
PPenk mRNA. These bands
tions of the hamster Penk gene were determined as shown in Table 1. The sequences flanking the splice donor and acceptor sites obey the GT-AG rule. The hamster Penk gene contains four exons and three introns and encodes 268 amino acids, including a signal peptide, six copies of Metenkephalin-containing peptides, and one copy of Leu-en-
of
Exon/Intron Junctions of the Proenkephalin Gene Hamster, and Rat Sources
Species
Exon 3' boundary
I I
Hum Ham
ACCGCGACG ACCGCGACG
gtgaggcc. gtgagttc.
II II I
Hum Ham Rat
AGCGTCAAC AACGGCAGC
gtgagtga. gtgagtga. gtgagtga.
III III II
Hum Ham Rat
AACTTCCTG AACTTCCTG AACTTCCTG
gtgagtgt. gtgagtag. gtaaggtt.
Exon
not
present when this
site of the hamster Penk gene deduced from sequence analysis (see Fig. 2). The combination of the results obtained by nuclease protection mapping, primer extension, and sequence analysis supports the conclusion that the hamster Penk gene contains four exons and three introns (see Figs. 2, 4A,B, Table 1, and data not shown).
kephalin peptide (see Fig. 3). Figure 4 presents the ribonuclease protection and primer extension analysis of the hamster PPenk mRNA. As shown in Fig. 4A, three predominant bands were present when a 32P-labeled 1,120-base-long hamster riboprobe con-
Table 1. Comparison
were
riboprobe was hybridized to a total cellular RNA extracted from hamster liver (lane b), a tissue that does not express the Penk gene (Franklin et ai, 1991a,b). Primer extension analysis of poly(A)+RNA from the hamster striatum and adrenal, but not the liver, showed a single profound band of 142 bases in length (see Fig. 4B), suggesting a major transcription initiation site in these tissues, and supporting the exon/intron structure and the transcription initiation
from
Human, Exon 5' boundary
Intron (size)
A.
GGCGGCAAC
AsnPheLeu IV IV III
Hum Ham Rat
TTTTAA..AATAAATGC. TTTTAA..AATAAATGC. TTTTGA..AATAAATGC.
PheTerm
B B A .
.
.
(87) (80)
..tccctct-ag ..tctctcttgcag
AGTCGT AGTGGC
(470)...tctctcctcgcag (525)...tctctcct-acag (600)...tctttcca-acag
TCCATG CCCATG CCCATG Met GCTTGC GCGTGC GCATGC
A A
C C B
(3.5kb)....t-ttcccag (3.5kb)....tcttcccag (3.8kb)....actttgcag
AlaCys
.ATAATAAACCTATTACCCCA..3' .ATAATAAACCCATTACCCCA..3' .ATAATAAACCG-TgAaCCCA. .3'
Hum, Human; Ham, hamster. Two possible polyadenylation signals
are
underlined
(B).
The nucleotide sequence of the exons and surrounding DNA segments of the hamster proenkephalin gene. A DNA were sequenced to provide information about exons and the surrounding regions. Exons are shown in uppercase letters; introns, upstream, and downstream sequences are shown in lowercase. Around the TATA box (bold and underlined), there is a 100-base-long segment (underlined) that is 98% homologous to the human proenkephalin gene. Several additional landmarks are identified in the diagram of the hamster gene: the cap site and the poly (A) addition site are in large bold letters; two possible polyadenylation signals are underlined and putative GREs are in bold letters. FIG. 2. total of
3,800 bases of hamster genomic
ZHU ET AL.
30
S GENE
'
Exon
WE
Intron B 525 bp
Exon
IV
Intron C
3500
bp
I
fI
i
AUG
'y J-£mRNA
CapH 5'
Untrans
AATAAA
I c
•il il
«
I
J
Signal
?
UAA 3- Untrans
peptide
-Proenkephalin Segment
L- Poly A
-
FIG. 3. A schematic representation of the hamster Penk gene and mRNA transcript. Also shown are the deduced locations of the Met- and Leu-enkephalin peptides in the translation product. The hamster Penk gene contains four exons and three introns and encodes six copies of Met-enkephalin and one copy of Leu-enkephalin.
Potential cis-acting elements in the 5'-upstream region and intron of the hamster Penk gene Several consensus sequences of promoter elements are present in the hamster Penk gene. A TATA box, TAATTATAAA, was identified in the sequences from -36 to -27 (see Fig. 2). A GC-rich sequence is located in the region from -65 to -52 (Fig. 2). Several putative ris-regulatory elements were present in the 5'-upstream region (see Table 2). Using both an idealized pentadecanucleotide glucocorticoid response element (GRE), 5'-AGAACANNNTGTTCT-3' (Beato, 1989; Freedman, 1992) or a GRE core se-
5'-TGTc/TCT-3' (Hollenberg et ai, 1985), multiple putative GREs were identified in the hamster gene, including two in the 5'-upstream region, from -796 to -782, and from -152 to -238; one in the intron A, from 107 to 121; one in the intron B, from 230 to 244, and one in the intron C, from 1,252 to 1,266 (see Fig. 2).
quence
Comparison of the hamster proenkephalin gene with
human, rat, and bovine proenkephalin genes Figure 5 shows the amino acid alignments of hamster,
70% identical among these four species. The exon/intron structure of the hamster Penk gene appears to be similar to the human. Both the hamster and the human Penk gene contain four exons and three introns with similar sizes, while the rat Penk gene contains three exons and two introns. The short intron that is missing in the rat Penk gene separates exon I and II in both the human and the hamster Penk gene (see Fig. 3). Furthermore, a 100-base region around the TATA box of the hamster Penk gene bases -120 to -21 is identical to the homologous 102-base-long region of the human Penk gene except for two bases that are absent from the hamster gene (Fig. 6). In the same region of rat Penk gene, 5 bases are missing and 6 bases are mismatched compared to this 102-base region of the human Penk gene (Fig. 6). This region contains multiple putative regulatory elements that are conserved among the
three species, including ENKCRE-1, ENKCRE-2, AP-1, and AP-4 sites (Comb et ai, 1988). However, the AP-2 site, CCGCCGGC, presented in both the human and hamster Penk gene (Hyman et ai, 1989), is different in the rat Penk gene (CCGatGGC). In addition, the TATA-box sequences in both the human and the hamster Penk gene are identical (TAATTATAAA), while it is different in the rat Penk gene (TAATTATAgg).
human, bovine, and rat Penk sequences. Each of them contains a signal peptide (amino acids 1-24), six copies of Met-enkephalin, and one copy of Leu-enkephalin. The six Cys residues in the amino terminus and the potential asDISCUSSION paragine-linked glycosylation site, Asn-X-Ser, are conserved in all four species. Several potential phosphorylation sites (Ser and Thr residues) are also conserved in these It has been reported that the basal level and the regulaspecies (see Fig. 5). The amino acid sequences are over tion of Penk gene expression in the rat adrenal are dif-
31
HAMSTER PROENKEPHALIN GENE ferent from other species, including human, guinea pig, dog, bovine, cat, and hamster (Schultzberg et al., 1978a,b; Viveros et al., 1979; Franklin et al., 1991a,b). The primary
of the Penk gene from human and rat sources has been previously elucidated. These data indicate that the exon/intron structure of the human Penk gene (four exons and three introns) is different from that of the rat (three exons and two introns), although a high degree of homology is present in the coding and 5'-upstream regions (Comb et al., 1983; Rosen et al., 1984). In the present study, we have isolated four clones containing sequences homologous to the rat Penk gene from a hamster genomic library by screening with a rat PPenk riboprobe. These structure
clones were characterized and shown to represent a singlecopy hamster Penk gene. The nucleotide sequence of major portions of the hamster Penk was determined, including 975 bases in the 5'-flanking region, all the exons, all of introns A and B, part of intron C, and 577 bases in the 3' downstream region. These results provide essential information about the exon/intron structure of the hamster Penk gene. Our results also indicate that the exon/intron structure of the hamster Penk gene is similar to the human Penk gene (four exons and three introns) but differs from that of the rat Penk gene (three exons and two introns). The analysis of the nucleotide sequence and exon/intron structure of the hamster Penk gene allowed us to deduce
abed
726-1
500-1
249200-
151140-
B 118-
82-
66-
48-
>. 42-
abc
d
A. RNase protection analysis of hamster preproenkephalin mRNA. A. "P-labeled 1,120-base-long hamster riboprobe containing nucleotides complementary to -86 to 1,034 of the hamster Penk gene shown in Fig. 2 was prepared by in vitro transcription. The riboprobe was hybridized with total cellular RNA extracted from hamster liver (lane b), adrenal (lane c), and striatum (lane d) at 75CC for 4 hr. Then, the samples were treated with a solution containing a high salt buffer (0.3 M NaCl, 5 mM EDTA, 10 mM Tris-HCl pH 7.5), 40 jig/ml RNase A, and 2 ni/ml RNase Tl at 30°C for 1 hr. The riboprobe with nuclease digestion omitted is in lane a. Following three sequential phenol extractions and ethanol precipitation, aliquots of the RNA samples were denatured by heating at 100°C for 2 min and fractionated in 10% polyacrylamide7 M urea gels. The size markers are shown on the left side. O, Origin of electrophoresis. B. Primer extension analysis of the hamster proenkephalin gene. A synthetic 15base-long oligonucleotide was hybridized with 5 ni ot poly(A)*RNA prepared from hamster striatum (lane a), adrenal (lane b), and liver (lane c), or a sense transcript prepared from the rat Penk gene (lane d), and the mixture was subjected to reverse transcription. The products were electrophoresed in 10% polyacrylamide-7 M urea gels. DNA size markers are shown on the left side. The expected size of the primer extension product for the sense transcript of rat Penk gene is 147 bases. FIG. 4.
ZHU ET AL.
32
Table 2. Some Putative cw-Regulatory Elements in of the Hamster Penk Gene
the
5'-Flanking Region Percent match
cis-Active Element
Source
Hamster sequence*
cAMP/phorbol CTGCGTCAGCTGCAG TGGCGTA ENKCRE-1 ENKCRE-2 TGCGTCA AP-1 To/TAGTCA AP-2 AP-4
CCGCCGGC CCCCAGGC CAGCTG
""CTGCGTCAGCTGCAG" -""•TGGCGTA"'8 -"TGCGTCA"86 -815TTAcTCA-809 ""TGAtTCA"253 ""TGcGTCA"86 -"CCGCCGGC"70
Human Human Human HeLa cell
Human HeLa cell Human
'"CgCCAGGC""7 "87CAGCTG"82
(# match)
100(15/15) 100 (7/7) 100 (7/7) 86 (6/7) 86 (6/7) 86 (6/7) 100 (8/8) 88 (7/8) 100 (6/6)
"Lowercase letters are sites of mismatch, numbers indicate position from the assigned transcription site in Fig. 2. See References: Comb et al. (1988); Hyman et al. (1989); Franza et al. (1988); Hu et al. (1990); La Gamma et al. (1989); Mitchell and Tjian (1989).
Rat
MAQFLRLCIWLVALGSCLLATVQADCSQDCAKCSYRLVRPGDINFLACTL
Hamster
MARFLRLSAWLLVLGSCLLATVQAECSQDCAKCSYHLVSPGDINFLACTL 50
Human
MARFLTLCTWLLLLGPGLLATVRAECSQDCATCSYRLTOPADINFLACVM
50
Bovine
MARFLGLCTWLLALGPGLLÄTVEAECSQDCATCSYRLARPTDLNPLACTL
50
Rat
ECEGQLPSFKIWETCKDLLQVSKPEFPWDNIDMYKDSSFQDESHLLAKKY
100
Hamster
ECEGQMPSHKIWETCKDLLQVSKPEFPCDSINMFKDSNKQDESHLLAKKY
100
Human
ECEGKLPSLKIWETCKELLQLSKPELPQDGTSTLRENSKPEESHLLAKRY 100
Bovine
ECEGKLPSLKTWETCXELLQLTKLELPPDATSAL-SKQEESHLLAKKY
Rat
GGFMKRYGGFMKKMDELYPVEPEEEANGGEILAKRYGGFMKKDADEGDTL 150
Hamster
GGFMKRYGGFMKKMDELYPVEPEEEANGGE1LAKKYGGFMKKDADEGDTL 150
Human
GGFMKRYGGFMKKMDELYPMEPEEEANGSEILAKRYGGFMKKDAEEDDSL 150
Bovine
GGFMKRYGGFMKKMDELYPLEVEEEANGGEVLGKRYGGFMKKDAEEDDGL 14 7
Rat
ANSSDLLKELLGTGDNRAKDSHOOESTNNDEDSTSKRYGGFMRGLKRSPO 200
Hamster
ANSSDLLKELLGTGDNRAREGRHOESTDND-DNMSKRYGGFMRGLKRSPO 199
Human
ANSSDLLKELLETGDNR--ERSHHODGSDNEEEVSKRYGGFMRGLKRSPO 198
Bovine
GHSSHLLKELLGAGDOR- -EGSLHOEGSDAED-VSKRYGGFMRGLKRSPH 194
50
97
=
Rat
LEDEAKELQKRYGGFMRRVGRPESSMDYQKRYGGFLKRFAESLPSDEEGE 250
Hamster
VEDEAKELOKRYGGFMRRVGRPEWWMDYQKRYGGFLKRFAESLPSDEEAE 24 9
Human
LEDEAKELOKRYGGFMRRVGRPEWWMDYOKRYGGFIiKRFAEALPSDEEGE 24 8
Bovine
LEDETKELOKRYGGFMRRVGRPEWWMDYOKRYGGFLKRFAEPLPSEEEGE 244
Rat
SYSKEVPEMEKRYGGFMRF
26 9
Hamster
SYSKEVPEIEKRYGGFMRF
268
Human
SYSKEVPEMEKRYGGFMRF
26 7
Bovine
SYSKEVPEMEKRYGGFMRF
263
FIG. 5.
the amino acid sequence of hamster Penk, which contains 268 amino acids. There is considerable homology among the Penk amino acid sequences from human, rat, bovine, and hamster sources as shown in Fig. 5. The overall percentage of identical amino acids is about 70% among these four species. Like the human, bovine, and rat Penk, hamster Penk also contains six copies of Met-enkephalin and one copy of Leu-enkephalin. This result further confirmed our previous report that the molar ratio of Met/Leu-enkephalin in hamster Penk from the adrenal medulla was 6:1 as determined by a combination of size exclusion chromatography and radioimmunoassay (Franklin et ai, 1991b). The predicted molecular weight (120 x 268 32,160) of hamster Penk is similar to the previously reported molecular weight (34,000) of Penk from hamster adrenal as determined by Western blot analysis (Franklin
Alignment
of the Penk
protein
sequences from
rat, hamster, human, and bovine sources. The protein sequence of hamster Penk is deduced from its genomic sequence. The Penk rat, human, and bovine sequence was obtained from published cDNA sequences. Met-enkephalin is indicated by boldface and is underlined; Leu-enkephalin is indicated by boldface only. Each amino acid identical to that present in the hamster sequence is indicated by a colon (:).
ai, 1991b). Because potential asparagine-linked glycosylation sites, Asn-X-Ser (positions 152-154 and 180-182 in Fig. 5), are present, glycosylation may contribute to the difference in the molecular weight of hamster Penk estiet
mated from the present calculation and the value deduced by Western blot analysis (Franklin et ai, 1991b). Although the dibasic amino acid in the second Met-enkephalin fragment KK (amino acids 134 and 135 of Fig. 5) in hamster Penk differs from that in rat, human, and bovine Penk, the apparent processing of Penk polypeptide in the adrenal does not differ between rat and hamster (Inturrisi et ai, 1988; Franklin et ai, 1991b). By comparison with the known Penk genomic structures from the human and rat and by use of ribonuclease protection mapping and primer extension, we have demonstrated that the exon/intron structure of the hamster Penk gene is similar to the human Penk gene, which contains four exons and three introns, and differs from the rat Penk gene, which has three exons and two introns. The intron missing from the rat Penk gene is short and separates the first and second exons of the human and hamster Penk
33
HAMSTER PROENKEPHALIN GENE
HUMAN
AGGCGTCGGCGCGGGGCTGGCGTAGGGCCTGCGTCAGCTGCAGCCCGCCG
50
HAMSTER
AGGCGTCGGCGCGGG-CTGGCGTAGGGCCTGCGTCAGCTGCAGCCCGCCG
49
RAT
AG-CGTCGACACGGG-CTGGCGTAGGGCCTGCGTCAGCTGCAGCCCGatG
48
HUMAN
GCGATTGGGGCGCGCGCGCCTCCTTCGGTTTGGGGCTAATTATAAAGTGGCT 102
HAMSTER
GCGATTGGGGCGCGCGCGCCTC-TTCGGTTTGGGGCTAATTATAAAGTGGCT 100
RAT
GCGATTGG--CGCGCGCGCCTC-TTCGGTTTGGGGCTAATTATAsrgGTGGCT
FIG. 6.
Alignment of
a
portion of the nucleotide
97
se-
quences around the TATA box region in the proenkephalin gene from human, hamster, and rat sources. Each nucleotide identical to that present in the human sequence is indicated by a colon (:). The TATA sequences and an AP-2 element identified in the human Penk gene are in boldface and the mismatched bases in the rat Penk gene are in lowercase letters.
gene. This small intron may contain regulatory features responsible for the high-level expression of human and hamster Penk gene in the adrenal as it has previously been shown that the introns can play important roles in control of gene expression (Slater et al., 1985; Mathis et ai, 1989;
Liska et al., 1990; Beenken et al., 1991). However, the significance of this feature in the Penk gene remains to be determined by functional analysis. Analysis of the nucleotide sequence of the hamster Penk gene reveals that the 5'-upstream sequence contains consensus binding sites for multiple transcription factors (Comb et al., 1988). A region from -120 to -21 (100 bases, see Fig. 2) in the 5'-flanking region of the hamster Penk gene is identical to the analogous region (102 bases) of the human Penk gene except for two bases missing in the hamster gene (see Fig. 6). It has been previously demonstrated that this 5'-upstream region in the human Penk gene is important in both the basal and inducible levels of
expression (Comb et al., 1986, 1988; Hyman et al., 1989). Comparison of the nucleotide sequence within this region among human, hamster, and rat Penk genes revealed several differences in the rat Penk gene, although high homology (93.8%, rat vs. human) is present. An AP-2 element (Fig. 6), CCGCCGGC, identified in the human Penk gene (Comb et al., 1988; Hyman et al., 1989), is
also operating in Penk gene remains to be determined. Functional analysis is being carried out in our laboratory to evaluate these cis-regulatory elements and to compare the difference between human, hamster, and rat Penk gene
expression.
The lower expression of the Penk gene in the rat adrenal compared to other species may be due to a yet unidentified tissue-specific inhibitory factor(s). Joshi and Sabol (1991) have reported that in a transfection assay with C6 glioma cells, a short rat Penk/CAT construct (upstream to -190) produced a higher basal level of expression than the longer constructs (upstream to -1,050, -2,700, and -5,800), suggesting that a negative c/s-regulatory element(s) may be located in the distal upstream region of the rat Penk gene. By pharmacological characterization in vivo, we found that the protein synthesis inhibitors, cycloheximide or anisomycin, produced a time- and dose-dependent increase in the levels of PPenk mRNA in the rat adrenal (both cortex and medulla), while the same treatments did not alter
the levels of the PPenk mRNA in the hamster adrenal (Zhu, 1993). These results suggest that some labile protein repressor(s), which inhibit Penk gene expression, may be present in the rat adrenal, but not the hamster adrenal. A similar control mechanism for gene expression has been reported for Penk gene expression in B lymphocytes (Behar et al., 1991) and for other genes, including the IL-2 gene (Zubiaga et ai, 1991), the glycoprotein hormone a-subunit gene (Cox et al., 1990), and immediate-early genes (Morello et al., 1990; Wisdom and Lee, 1991).
ACKNOWLEDGMENT This work was supported in part by National Institute Drug Abuse Grant DA-01457 and by National Institute on Drug Abuse Center Grant DA-05130, which is directed by M.J. Kreek of the Rockefeller University. on
gene
identical in the hamster Penk gene, but is different in the rat Penk gene (CCCatGGC). Hyman et al. (1989) reported that mutation of this site caused a decrease in basal expression by 50% and in cAMP-induced expression by four- to fivefold in a transfection-CAT assay. Furthermore, the sequences of the TATA box among the human, hamster, and rat Penk gene are different. Both the human and the hamster Penk gene have TAATTATAAA, while it is TAATTATAgg in the rat Penk gene (see Fig. 6). The nature of the TATA sequences may play an important role in the control of gene expression as previously reported (Simon et al., 1988; Jameson et al., 1989; Wefald et al., 1990). Wefald et al. (1990) reported that in the human myoglobin gene, the native TATA box, TATAAA, was important in preserving the high-level tissue-specific expression of this gene in muscle in combination with a muscle-specific enhancer. Whether this enhancer-promoter combination is
REFERENCES AKIL, H., WATSON, S.J., YOUNG, E., LEWIS, M.E., KHACHATURIAN, H., and WALKER, J.M. (1984). Endogenous opiods: Biology and function. Annu. Rev. Neurosci. 7, 223-255.
BEATO, M. (1989). Gene regulation by steroid hormones. Cell 56, 335-344. BEENKEN, S.W., KARSENTY, G., RAYCROFT, L., and LOZANO, G. (1991). An intron binding protein is required for transformation ability of p53. Nucleic Acids Res. 19, 47474752.
BEHAR, O.Z., OVADIA, H., POLAKIEWICZ, R.D., ABRAMSKY, O., and ROSEN, H. (1991). Regulation of proenkephalin A messenger ribonucleic acid levels in normal B lymphocytes: Specific inhibition by glucocorticoid hormones and superinduction by cycloheximide. Endocrinology 129, 649-655.
BOORSTEIN, W.R., and CRAIG, E.A. (1989). Primer extension analysis of RNA. Methods Enzymol. 180, 347-369. BRANCH, A.D., BENENFELD, B.J., PAUL, C.P., and ROBERTSON, H.D. (1989). Analysis of ultraviolet-induced RNA-RNA cross-links: A means for probing RNA structurefunction relationships. Methods Enzymol. 180, 418-442.
34
COMB, M., ROSEN, H., SEEBURG, P., ADELMAN, J., and HERBERT, E. (1983). Primary structure of the human proenkephalin gene. DNA 2, 213-229. COMB, M., BIRNBERG, N.C., SEASHOLTZ, A., HERBERT, E., and GOODMAN, H.M. (1986). A cyclic AMP- and phobol ester-inducible element. Nature 323, 353-356. COMB, M., MERMOD, N., HYMAN, S.E., PEARLBERG, J., ROSS, M.E., and GOODMAN, H.M. (1988). Proteins bound at adjacent DNA elements act synergistically to regulate human proenkephalin cAMP inducible transcription. EMBO J. 7, 3793-3805.
COX, G.S., COSGROVE, D.E., SULLIVAN, T.T., and HAAS, M.J. (1990). Induction by cycloheximide of the glycoprotein
hormone alpha-subunit gene in human tumor cell lines and identification of a possible negative regulatory factor. J. Biol. Chem. 265, 13190-13197. DAVIS, L.G., DIBNER, M.D., and BATTEY, J.F. (1986). DNA preparation from cultured cells and tissue. In Basic Methods in Molecular Biology. L.G. Davis, M.D. Dibner, and J.F. Battey, eds. (Elsevier, New York) pp. 47-50. DECRISTOFARO, J.D., and LA GAMMA, E.F. (1990). Bimodal regulation of adrenal opiate peptides by cholinergic mechanisms. Neuroscience 35, 203-210.
FRANKLIN, S.O., ZHU, Y.S., YOBURN, B.C., and INTURRISI, C.E. (1991a). Transsynaptic activity regulates proenkephalin and tyrosine hydroxylase gene expression and the response to reserpine in the hamster adrenal. Mol. Pharmacol. 40, 515-522. FRANKLIN, S.O., YOBURN, B.C., ZHU, Y.-S., BRANCH, A.D., ROBERTSON, H.D., and INTURRISI, C.E. (1991b). preproenkephalin mRNA and enkephalin in normal and denervated adrenals in the Syrian hamster: Comparison with central nervous system tissues. Mol. Brain Res. 10, 241-250. FRANZA, B.R., JR., RAUSCHER, F.J., JOSEPHS, S.F., and CURRAN, T. (1988). The Fos complex and Fos-related antigens recognize sequence elements that contain AP-1 binding sites. Science 239, 1150-1153. FREEDMAN, L.P. (1992). Anatomy of the steroid receptor zinc finger region. Endocrine Rev. 13, 129-145. HOLLENBERG, S.M., WEINBERGER, C, ONG, E.S., CERELLI, G., ORO, A., LEBO, R., THOMPSON, E.B., ROSENFELD, M.G., and EVANS, R.M. (1985). Primary structure and expression of a functional human glucocorticoid receptor cDNA. Nature 318, 635-641. HU, Y.F., LÜSCHER, B., ADMON, A., MERMOD, N., and TJIAN, R. (1990). Transcription factor AP-4 contains multiple dimerization domains that regulate dimer specificity. Genes & Dev. 4, 1741-1752. HUGHES, J., SMITH, T.W., KOSTERLITZ, H.W., FOTHERGILL, L.A., MORGAN, B.A., and MORRIS, H.R. (1975). Identification of two related pentapeptides from the brain with potent opiate agonist activity. Nature 258, 577-579. HYMAN, S.E., COMB, M., PEARLBERG, J., and GOODMAN, H.M. (1989). An AP-2 element acts synergistically with the cyclic AMP- and phorbol ester-inducible enhancer of the human proenkephalin gene. Mol. Cell. Biol. 9, 321-324. INTURRISI, C.E., BRANCH, A.D., ROBERTSON, H.D., HOWELLS, R.D., FRANKLIN, S.O., SHAPIRO, J.R., CALVANO, S.E., and YOBURN, B.C. (1988). Glucocorticoid regulation of enkephalins in cultured rat adrenal medulla. Mol. Endocrinol. 2, 633-640. INTURRISI, C.E., LAGAMMA, E.F., FRANKLIN, S.O., HUANG, T., NIP, T.G., and YOBURN, B.C. (1988). Characterization of enkephalins in rat adrenal medullary expiants. Brain Res. 448, 230-236. JAMESON, J.L., POWERS, A.C., GALLAGHER, G.D., and
ZHU ET AL.
HABENER, J.F. (1989). Enhancer and promoter element interactions dictate cyclic adenosine monophosphate mediated and cell-specific expression of the glycoprotein hormone A-gene. Mol. Endocrinol. 3, 763-772. JOSHI, J., and SABOL, S.L. (1991). Proenkephalin gene expression in C6 rat glioma cells: Potentiation of cyclic adenosine
3',5'-monophosphate-dependent transcription by glucocorticoids. Mol. Endocrinol. 5, 1069-1080.
KANAMATSU, T., UNSWORTH, CD., DILIBERTO, JR., E.J., VIVEROS, O.H., and HONG, J.S. (1986). Reflex splanchnic nerve stimulation increases levels of proenkephalin A mRNA and proenkephalin A-relasted peptides in the rat adrenal medulla. Proc. Nati. Acad. Sei. USA 83, 9245-9249. KILPATRICK, D.L., HOWELLS, R.D., FLEMINGER, G., and UDENFRIEND, U. (1984). Denervation of rat adrenal glands markedly increases preproenkephalin mRNA. Proc. Nati. Acad. Sei. USA 81, 7221-7223. KIMURA, T., KATOH, M., and SATOH, S. (1988). Inhibition by opioid agonists and enhancement by antagonists of the release of catecholamine from the dog adrenal gland in response to splanchnic nerve stimulation: Evidence for the functional role of opioid receptors. J. Pharm. Exp. Ther. 244, 1098-1103. LA GAMMA, E.F., ADLER, J.E., and BLACK, LB. (1984). Impulse activity differentially regulates [Leu]enkephalin and catecholamine characters in the adrenal medulla. Science 224, 1102-1104. LA GAMMA, E.F., GOLDSTEIN, N.K., SNYDER, J.B., JR., and WEISINGER, G. (1989). Preproenkephalin DNA-binding proteins in the rat: 5' Flanking region. Mol. Brain Res. 6, 131140. LEWIS, J.W., CANNON, J.T., and LIEBESKIND, J.C. (1984). Opioid and nonopioid mechanisms of stress analgesia. Science 224, 1102-1104. LISKA, D.J., SLACK, J.L., and BORNSTEIN, P. (1990). A highly conserved intronic sequence is involved in transcriptional regulation of the alpha 1(1) collagen gene. Cell Regulation 1, 487-498. LUNDBERG, J.M., and HOKFELT, T. (1983). Coexistence of peptides and classical neurotransmitters. Trends Neur. Sei. 6, 325-333. MATHIS, J.M., HOUSER, W.H., BRESNICK, E., CIDLOWSKI, J.A., HINES, R.N., PROUGH, R.A., and SIMPSON, E.R. (1989). Glucocorticoid regulation of the rat cytochrome P450c (P450IA1) gene: Receptor binding within intron I. Arch. Biochem. Biophys. 269, 93-105. MITCHELL, P.J., and TJIAN, R. (1989). Transcriptional regulation in mammalian cells by sequence-specific DNA binding proteins. Science 245, 371-378. MORELLO, D., LA VENU, A., and BABINET, C. (1990). Differential regulation and expression of c-jun, c-fos and c-myc proto-oncogenes during mouse liver regeneration and after inhibition of protein synthesis. Oncogene 5, 1511-1519.
NODA, M., FURUTANI, Y., TAKAHASHI, H., TOYOSATO, M., HIROSE, T., INAYAMA, S., NAKANISHI, S., and NUMA, S. (1982). Cloning and sequence analysis of cDNA for bovine adrenal preproenkephalin. Nature 295, 202-206. ROSEN, H., DOUGLASS, J., and HERBERT, E. (1984). Isolation and characterization of the rat proenkephalin gene. J. Biol. Chem. 259, 14309-14313. SAMBROOK, J., FRITSCH, E.F., and MANIATIS, T. (1989). Molecular Cloning: A Laboratory Manual, 2nd ed. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY). SCHULTZBERG, M., LUNDBERG, J.M., HOKFELT, T., TERENIUS, L., BRANDT, J., ELDE, R.P., and GOLDSTEIN, M. (1978a). Enkephalin-like immunoreactivity in gland cells and
nerve
3, 1169-1186.
terminals of the adrenal medulla. Neuroscience
35
HAMSTER PROENKEPHALIN GENE
SCHULTZBERG, M., HOKFELT, T., LUNDBERG, J.M., TERENIUS, L„ ELFVIN, L.-G., and ELDE, R. (1978b). Enkephalin-like immunoreactivity in nerve terminals in sympathetic ganglia and adrenal medulla and in adrenal medullary gland cells. Acta Physiol. Scand. 103, 475-477. SIMON, M.C., FISCH, T.M., BENECKE, B.J., NEVINS, J.R., and HEINTZ, N. (1988). Definition of multiple, functionally distinct TATA elements, one of which is target in the hsp70 promoter for El A regulation. Cell 52, 723-729. SLATER, E.P., RABENAU, O., KARIN, M., BAXTER, J.D., and BEATO, M. (1985). Glucocorticoid receptor binding and activation of a heterologous promoter by dexamethasone in the first intron of the human hormone gene. Mol. Cell. Biol. 5, 2984-2992.
VIVEROS, O.H., DILIBERTO, JR., E.J., HAZUM, E., and CHANG, K.-J. (1979). Opiate-like materials in the adrenal medulla: Evidence for storage and secretion with catecholamines. Mol. Pharmacol. 16, 1101-1108. WEFALD, F.C., DEVLIN, B.H., and WILLIAMS, R.S. (1990). Functional heterogeneity of mammalian TATA-box sequences revealed by interaction with a cell-specific enhancer. Nature 344, 260-262. WISDOM, R., and LEE, W. (1991). The protein-coding region of c-myc mRNA contains a sequence that specifies rapid mRNA turnover and induction by protein synthesis inhibitors. Genes & Dev. 5, 232-243. YOSHIKAWA, K., WILLIAMS, C, and SABOL, S.L. (1984).
Rat brain preproenkephalin mRNA. J. Biol. Chem. 259, 14301-14308. ZHU, Y.S. (1993). "Differential regulation of c-fos, proenkephalin and tyrosine hydroxylase gene expression in the rat adrenal." Ph.D. Thesis, Cornell University.
ZHU, Y.S., BRANCH, A.D., ROBERTSON, H.D., HUANG, T.H., FRANKLIN, S.O., and INTURRISI, CE. (1992). Time of enkephalin mRNA and peptides in cultured rat adrenal medulla. Mol. Brain Res. 12, 173-180.
course
ZHU, Y.S., BRODSKY, M., FRANKLIN, S.O., HUANG, T., and INTURRISI, CE. (1993). Metrazole induces the sequential activation of c-fos, proenkephalin and tyrosine hydroxylase gene expression in the rat adrenal gland: Modulation by glucocorticoid and adrenocorticotropic hormone. Mol. Pharmacol. 44, 328-336. ZUBIAGA, A.M., MUÑOZ, E., and HUBER, B.T. (1991). Superinduction of IL-2 gene transcription in the presence of cycloheximide. J. Immunol. 146, 3857-3863. Address reprint requests to: Dr. Charles E. Inturrisi Cornell University Medical College 1300 York Avenue, LC-524 New York, NY 10021 Received for
publication June 17, 1993; accepted August 2,
1993.
