Molecular Brain Research, 13 (1992) 313-319 © 1992 Elsevier Science Publishers B.V. All rights reserved. 0169-328X/92/$05.00
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BRESM 70411
Organization and complete nucleotide sequence of the gene encoding mouse phenylethanolamine N-methyltransferase Shinji Morita a, Kazuto Kobayashi b, Hiroyoshi Hidaka a and Toshiharu Nagatsu b aDepartment of Pharmacology, Nagoya University School of Medicine, Nagoya (Japan) and blnstitute for Comprehensive Medical Sciences, School of Medicine, Fufita Health University, Toyoake (Japan) (Accepted 26 November 1991)
Key words: Phenylethanolamine N-methyltransferase; Genomic DNA; cDNA; Nucleotide sequence; Mouse
Phenylethanolamine N-methyltransferase (PNMT; EC 2.1.1.28) catalyzes the conversion of norepinephrine to epinephrine, the last step of catecholamine biosynthesis. We have previously reported molecular cloning of cDNA encoding human PNMT and chromosomal localization of its gene (Kaneda et al., J. Biol. Chem., 263 (1988) 7672-7677). In this report, we isolated the chromosomal gene encoding mouse PNMT by cross-hybridization with the human PNMT cDNA. Mouse PNMT gene spanned about 1.8 kb and consisted of 3 exons. Primer extension analysis showed two putative transcription initiation sites. Northern blot analysis and reverse transcription-polymerase chain reaction (RTPCR) revealed the expression of the mouse PNMT mRNA in brain (pons and medulla oblongata) and adrenal gland. Subsequently cDNA encoding mouse PNMT was amplified by RT-PCR and cloned into the plasmid vector. Mouse PMNT gene contained the protein-coding region of 885 bp (295 amino acids) with the predicted molecular weight of 32,627. The deduced amino acid sequence of mouse PNMT revealed the major difference in the N-terminal region, as compared to the human and bovine PNMT sequences. In the 5'-terminal region of the mouse PNMT gene, we found the existence of 23 bp direct repeat sequences, which was not observed in the corresponding regions of the human and bovine PNMT genes. The presence or absence of the direct repeats caused the major difference in the PNMT sequences among species. The typical TATA, GC, and CACCC boxes as well as several sequences homologous to glucocorticoids response elements (GRE) were located in the Y-flanking region of the mouse PNMT gene. INTRODUCTION
We previously r e p o r t e d a full-length c D N A clone encoding h u m a n P N M T 19 and its genomic clone 25. Nucle-
P h e n y l e t h a n o l a m i n e N-methyltransferase ( P N M T ; E C 2.1.1.28) is the terminal enzyme in catecholamine biosynthesis, and catalyzes the t r a n s m e t h y l a t i o n of norepinephrine to epinephrine 1'2°. The enzyme is present at high level in chromaffin cells of adrenal medulla, where epinephrine is synthesized as a h o r m o n e . It is also distributed in epinephrine neurons of m e d u l l a oblongata, hypothalamus, and amygdala of brain 16'17'22, as well as
otide sequence analysis of c D N A clone revealed that h u m a n P N M T consists of 282 amino acid residues with a predicted molecular weight of 30,853. M o r e o v e r , the P N M T gene was assigned to h u m a n c h r o m o s o m e 17 by Southern blot analysis using m o u s e - h u m a n somatic cell hybrids. The analysis of the h u m a n P N M T genomic clone showed that P N M T is e n c o d e d by a single gene that is c o m p o s e d of three exons, and that there are several sequences resembling S p l binding site and glucocorticoids response e l e m e n t ( G R E ) in the Y-flanking region. In the present p a p e r we describe the structures of the gene and c D N A encoding mouse PNMT. The nucleotide and deduced amino acid sequences of mouse P N M T were comp a r e d to the sequence data of P N M T from o t h e r species.
in a small n u m b e r of ganglion and amacrine cells of retina 15, where epinephrine m a y function as a neurotransmitter. The expression levels of P N M T are regulated by several factors in addition to tissue-specific and developmental stage-specific manners. Glucocorticoid h o r m o n e s are known to mainly regulate expression of the P N M T gene 7,sA3. Baetge et al. r e p o r t e d transgenic mice carrying the h u m a n P N M T gene o r a chimeric gene consisting of the h u m a n P N M T gene p r o m o t e r (2 kb) fused to the simian virus 40 large T antigen 2. Their results indicated that the cis-element for a p p r o p r i a t e expression of the h u m a n P N M T gene in adrenal gland and retina is in the 2-kb D N A fragment of the 5 ' - u p s t r e a m region.
MATERIALS AND METHODS
Construction of mouse genomic DNA library Two mouse (DBA/2J) genomic DNA libraries were used in our experiment. A mouse genomic library was purchased from Clontech and consisted of 2 phages which contained 8-22-kb DNA frag-
Correspondence: T. Nagatsu, Institute for Comprehensive Medical Sciences, School of Medicine, Fujita Health University, Toyoake 470-11, Japan.
314 ments digested partially with MboI, in the BamHI site of EMBL3 phage vector. Another library was constructed as follows: Mouse kidney genomic DNA was completely digested with XbaI, and electrophoresed on 0.8% agarose gel. The DNA fragments of about 12-kb were eluted from gel and ligated to EMBL12DNA (Boehringer) digested with XbaI, and packaged into 2 phages using Gigapack plus (Stratagene).
gMPNMT-1 Sal I"
I
Xbal
//
DNA sequence analysis Appropriate DNA fragments were subcloned into the Bluescript M13 + (Stratagene), pUC119 (Takara Shuzo Co.) and pGEM 7Zf + (Promega) vectors. Serial deletion mutants of the proper clones were constructed according to the unidirectional deletion method 2s. Nucleotide sequence was determined by the dideoxy chain-termination method z4 with the Sequenase DNA sequencing kit (Stratagene).
Southern blot analysis of genomic DNA 10/~g of mouse genomic DNA (DBA/2J) was digested with several restriction enzymes and electrophoresed on 0.8% agarose gel. The gel was transferred to a pylon membrane and (Hybond-N, Amersham) in 20 x SSC zr. The membrane was hybridized with 32p-labeled 1.7-kb KpnI-SalI fragment of the mouse PNMT genomic clone.
Northern blot analysis Total RNA was extracted from mouse adrenal glands by guanidine thiocyanate method 1°. Poly(A) ÷ RNA was obtained with oligo(dT)-Latex. 3 #g of poly(A) ÷ RNA was electrophoresed on 2% agarose-formaldehyde gel, and transferred to a nylon membrane in 20 × SSC27. The membrane was hybridized with the 32p-labeled 1.7-kb KpnI-SalI fragment as a probe.
Primer extension analysis A synthetic oligonucleotide, 5'-GTAGTI'GTTGCGGAGATAGG-3', that is complementary to the nucleotide positions from 141 to 160 downstream from the putative initiation codon of mouse PNMT, was used as an extension primer (see Fig. 2). The oligonucleotide was labeled at 5'-end using T4 polynucleotide kinase and [7-32p]ATP at 37°C for 30 min, and the labeled primer was hybridized with 2 pg of mouse adrenal gland poly(A) ÷ RNA, incubated at 37°C for 1 h in the reaction mixture of 80 ktl containing 20 mM Tris-HCl (pH 8.3), 0.25 mM EDTA, 62.5 mM KC1, 10 mM MgC12, 10 mM DTT, 0.25 mM dATP, 0.25 mM dGTP, 0.25 mM dCTP, 0.25 mM dTTP, 75 units of ribonuclease inhibitor, and 400 units of MMLV reverse transcriptase. The product was analysed by electrophoresis on 6% polyacrylamide-7 M urea gel.
Reverse transcription-polymerase chain reaction (RT-PCR) Two oligonucleotides: 5'-CAACAGAAGCATGAACGGTG-3' (forward primer), which corresponds to the nucleotides from -3 to
Sinai I
I -2-kb -
(~15-kb) Kpnl I
Sail" I
g M P N M T / X 12-4 Xbal I
Kpnl
Library screening Mouse genomic DNA libraries were screened by plaque hybridization5 using human PNMT cDNA fragment labeled with [a-32p]dCTP by a multiprime labeling method (Amersham). Hybridization was carried out in 6 × SSC, 5 x Denhardt's, 0.3% SDS, 100 pg/ml denatured salmon sperm DNA, 50% formamide, and labeled probe, at 42°C for 12 h. The filters were washed twice in 2 x SSC containing 0.1% SDS, at 65°C.
i
Kpnl 1
I
Kpnl Sinai Kpnl I I l J t Sac I EcoR V
Kpnl I
Eco47 Ill
Kpnl
I
exon 1
Sphl I
(-12-kb) Xbal
Kpnl I
I
Sac I
I
I
EcoR V
Kpnl I
exon 2 exon 3
Fig. 1. Structure of the mouse PNMT gene. The upper panel shows the restriction map of genomic DNA clones (gMPNMT-I and gMPNMT/X12-4). The lower panel shows the exon/intron organization of the mouse PNMT gene. SalI* indicates the restriction site of SalI in the multicloning sites of EMBL3 vector.
17; and 5'-GGAACTGTCACT TTATTAGGT-3' (reverse primer), which is complementary to the nucleotides from 1,528 to 1,548, were used as RT-PCR primers (see Fig. 2). The first strand of cDNA was yielded by reverse transcription from 1/~g of total RNA using oligo(dT) as a primer. Subsequently, the fragment corresponding to mouse PNMT cDNA was amplified using RT-PCR primers. The resulting PCR products were electrophoresed on 1% agarose gel and visualized with ethidium bromide-staining.
RESULTS
Isolation and characterization of the mouse P N M T gene First, we screened the mouse genomic D N A library purchased from Clontech with human PMNT cDNA as a probe, and three positive clones were isolated from 106 recombinant phages. As they showed the same restriction map, one of them, designated gMPNMT-1, was further analysed. Fig. 1 shows the restriction enzyme map of this clone. Southern blot analysis with several fragments of human PNMT cDNA indicated that gMPNMT-1 lacked the 3'-terminal region of the mouse PNMT gene. Because Southern blot hybridization of mouse genomic D N A detected a single band of 12 kb against the digestion with XbaI (see below), we originally constructed another genomic D N A library containing the XbaI-cut 12-kb fragments. We screened the second mouse genomic library with the 1.7-kb KpnI-SalI fragment prepared from gMPNMT-1 as a probe, and f o u r p o s i t i v e c l o n e s w e r e i s o l a t e d f r o m 2.5 × 105 rec o m b i n a n t p h a g e s . O n e o f t h e m was d e s i g n a t e d g M P NMT/X12-4,
a n d its r e s t r i c t i o n m a p o v e r l a p p e d w i t h
Fig. 2. Nucleotide sequence of the mouse PNMT gene and its flanking regions. The deduced amino acid sequence is shown below the nucleotide sequence. The nucleotides are numbered in the 5' to 3' direction from the major transcription initiation site (marked +1 above the nucleotide). The canonical TATA, CACCC, and GC boxes, as well as putative GRE are shown. The TGA termination codon and polyadenylation signal are underlined. The direct repeat sequences are indicated by horizontal arrows with thick lines. Horizontal arrows with thin lines indicate the location of the oligonucleotide primers used in primer extension analysis and RT-PCR (see Materials and Methods).
315
GRE -~5~: ACCGTGCACTTCTGGGAACAGCCAAGCATTGGAGCTAAATCTGGCAT~ACCAGAGGGTACACTCCATGCTTAGGGGTGG~GTACA~GGCAGAGC -1442 TGTAAACAGGGGGAATGTCTGGTCCCCCTTCCTGTGCCACTGCCCAGCCCTGGCTTCATGTCCAGTTCCTGGCACTGGAGCGTGTCTAGCAGGCC -1347 AGCTGGCTTCAAGGTTTGTTTAAATGATATCTGTGGAGGAGGTTATGGAAGGGGCTGGCACCAGGGCCGTCCTTGGCTGTGCTTGGGGTGTGGAT -1252 GGGGTCAGTGACCCTAAGGCCTGTCAGTTGTAGATCCAGACAGAATCAATCCTTGGCTGGCATCAGGTGTCCCACTACCCCTGGCCTGGGTGGGA -1157 GGACAGGGTTTAAGTTCCTGTCTGTGACCTCTGCAGCTGTTGTGATGATCCCCGACCCAGCCTGGGTGTCTGGCCTTTGGGATAAGGAAGGGACA C A C C C box -1062 CTGGGTAGGACTGGATAGAAGACCAGGACTAT•TTAGCAGAGGCTAGTAACCCTCCCACCCCAGAAAGACATAGGACTTTCTTAGGACTTAAAGG -967 GTCTCTGCTTTAGCAAGACTGGGGATGCTGTTGCCAGGGTAGCTTGCCATTTTGAGAACATGGGAAGGAAGACAGGCAGATTATGTTCCAGATAC -872 CTGGAGCACTAAGCAAGCCTGAAGGCCAGGCCCTGCCATCTTCCTAGTGGGGACACGATGTTGACTGAGGGGTCGGGATCAGGGTAGGGGTGTGG -777 GGATGTCCTGTACCTGCGAAACTGTACCTGCGAAATGCCAAGAGTGCGCATGCGCTGCCCCTCTAGCGGCCGTGGCAGTACCAAGAACATGCTC~ GRE GRE -682 QTACTCTTTGTTCCTACCTGAGTCCAGTGTCCTGGACCTGGTAGGAACATCCTGAAC~AACCATGCTTGCCTGGCCCCCAGATAAGCAGCACATA -587 GCCCTAGAGGCCTGCAGGGGATGCCCGGATGTTCTGCTTGCTAAAAGCATTAGCACGGCTCACCTTCCTTATCTCTGCTGCCATCCGATGCTCAG GRE -492 GGCAGAGACCTGCTCAGGACCCAGGGTCCTCAAGACAGAGGCCAGAACAGAGTGTCCTTTCTGGAGGAGGGAAGGGGTGCTGCAAGATAGAGATG -397 GGTTAGAGGTCTGGAGGTAGGGATGGTATTATAAAGAAGAGGAGTTTGTAAGGGGTGCCCCGAGAGAGGGGAGGAAGTCTGGGAAGGATGCTGGG -302 ACTGGGACTGGGAACACAGGCTAATTTAGACCTCGGGAAAGAGAAGGGGTGAGATGCTGGGCAAAAGACTCTTTGGGAGGTTGGAGAGGAGCTGG -207 GGTA••GCTGGAACTGAGGCTGGGGGTGTAGGGCAG•CCTGGAGCGATCAGGGG•TGGGGGGGGGGGGGTGGAGGGTTTGTTGAGCAAGTGGKAA -112 GCAAGGGTGGGGAGGAGCGATGTTCTAAAGGGCGCCCCCCAGCCTCCGCGCGTCTGTTGCTCAGACACTAACTGAGATGGATGGAGTGACAGAGA CACCC box GC box TATA box -17 TGTGGTGGCCTCAGGCGCCTCATCCCTCAGCAGCCACCCACCCCTGTGACGGAGGGGTCCGGGCGGGGGGACCCAGTGGTAGATAAAGGGATGGG +i 61 G G A G A G G A G G T C T ~ A A ~ A G A A G C ATG AAC GGH G H C T C A HAC C T G AAH CAC GC-T A C A GGG AHT HHC TCA HAC CCG AAG M e t ASh G l y Gly Set Asp Leu Lys His Ala Thr Gly Set Gly Set Asp Pro Lys 133 ~ _ ~ G C A GAG ATG GAC CCT GAC TCC GAC GCT GGC CAG G T A CGT GTC GCC TTG GCT TAC CAG CGC TTC GAG His Ala Ala Glu Met Asp Pro Asp Ser Asp Ala Gly Gln Val Arg Val Ala Leu Ala Tyr Gln Arg Phe Glu 205 CCC CGC G CC TAT CTC C G C AAC AAC TAC GCG CCT CCT CGT GGA GAC CTG AGC AAC CCT GAT GGC GTC GGG CCT Pro Arg Ala Tyr Leu Arg Asn Ash Tyr Ala Pro Pro Arg Gly Asp Leu Set Asn Pro Asp Gly Val Gly Pro 285 TGG AAG C T G CGC TGC ATG G C A CAA GTC TTT GCT ACC G GTGAGCACCGAAACGAGGCATGAGAGAGCAGAACTCATGGGGA Trp Lys Leu Arg Cys Met Ala Gln Val Phe Ala Thr G 380 AAGGTGCCTACCTAGCAGGCACAGACAGGAGCCGGCCTGTAGTCTTAGCGACTAGGACACTGAGGCTTGGAGGACCACTTGAGGCCGGAATTGGA 475 TGACTGACTAGACAGTTTAGAGTCTTTTTCTTTCAAGGGGAGGATGTTTAAGATTGAGAGACGGATGGGGCCAGGTGAGAGAGATAGGGAGAGGG 570 AGGGCGGGAGGGGGAGGTTGGAGAGGCTCATTCCTTGATGTCTCGGGATCGTGATCTCCCCCACTAAATCGTAACTAGAGCTAGTTTCTGAGGGG 665 TGGGACCCGAGTCAGAGGAGCCCTGTCAGCACAGCACTTGCTCCAGATACTACCCTGAAGAAGTGCTGAGGAGAGCACGAGGGGAAGGGGCGGCT 746 GAGCAGGAGCCACTGGTTATCCCTGCCTCTGCTCCACCAG GT GAG GTG TCG GGA CGG GTT CTC ATT GAT ATT GGC TCC GGC ly Glu Val Ser G l y Arg Val Leu Ile Asp Ile Gly Ser Gly 818 CCC ACC ATA TAT C A G C T G CTC AGT GCC TGT GCC CAC TTT GAG GAC ATC ACC ATG A C A GAC TTC TTG G A A GTC Pro Thr Ile Thr Gin Leu Leu Ser Ala Cys Ala His Phe Glu Asp Ile Thr Met Thr Asp Phe Leu Glu Val 890 AAC CGT CAG GAG C T G G G A CTC TGG CTG CGA GAG GAG C C A GGA GCC TTT GAC TGG AGT GTG TAT AGT CAG CAT Ash Arg Gin GIu Leu Gly Leu Trp Leu Arg Glu Glu Pro Gly Ala Phe Asp Trp Set Val Tyr $er Gln His 976 GCC TGC CTC ATT GAG GAC AAG GG GTGAGAGCTGGACTGGCAGCTTCGTGTGCAGCAGTGGGTGGCTGGGGGGGGGGGGCTGCAGAA Ala Cys Leu Ile Glu A s p Lys G1 1058 T GAG TCC TGG CAG GAG AAA G A A CGC CAG CTT GGCTGAGTCTTTGGGGTAGTCCTGAGCCCCGCCTTGTGCCCCCCTGTACAG y Glu Ser Trp Gln Glu Lys Glu Arg Gln Leu 1130 C G A GCG A G G GTG AAG C G A GTC CTG CCT ATC GAT GTG CAC AAG CCC CAG CCC CTG G G A ACT CCC AGT CTG GTC Arg Ala Arg Val Lys Arg Val Leu Pro Ile Asp Val His Lys Pro Gln Pro Leu G,y Thr Pro $er Leu Val 1202 CCT CTG CCT GCC GAC GCC TTG GTC TCT GCC TTC TGC C T G GAG GCT GTG AGC CCA G A T CTT ACT AGC TTC CAG Pro Leu Pro Ala Asp Ala Leu Val Ser Ala Phe Cys Leu Glu Ala Val Set Pro Asp Leu Thr Set Phe Gln 1274 CGG GCT TTG CAT CAC ATC ACC ACA CTG CTG AGG CCC GGG GGT CAT CTC CTC CTC ATT GGG GCC CTG GAG GAG Arg Ala Leu His His Ile Thr Thr Leu Leu Arg Pro Gly Gly His Leu L~u Leu Ile Gly Ala Leu Glu Glu 1346 TCC TGG TAC CTT GCT GGG GAG GCC AGG CTG TCT GTG GTG CCA GTG TCT G A G GAG G A G GTG AGG GAG GCC CTG Ser Trp Tyr Leu Ala Gly Glu Ala Arg Leu Set Val Val Pro Val Set Glu Glu Glu Val Arg Glu Ala Leu 1418 GTC CTT GGT GGT TAC GAG GTC CGA GAG CTT CGC ACC TAC ATC ATG CCT GCC CAC CTC TGC ACG GGG G T A GAT Val Leu GIy Gly Tyr Glu Val Arg Glu Leu Arg Thr Tyr Ile Met Pro Ala His Leu Cys Thr Gly Val Asp 1495 GGGCCCCAAAAATGCCAGGTGTC GAT GTC AAA GGC ATC TTC TTT GCC TGG GCC CAG AAG ATG GAG GTG CAG GTG ~ Asp Val LyS GIy Ile Phe Phe Ala Trp Ala Gln Lys Met Glu Val Gln Val CTGCCTCCAAAGTCCTTATCACCTGAAGTGGAACCTAATAAAGTGACAGTTCCC 9
316 that of gMPNMT-1 (Fig. 1). We d e t e r m i n e d the complete nucleotide sequence of the region covering the mouse P N M T gene (Fig. 2). The exon/intron boundaries were d e t e r m i n e d from the comparison of the mouse D N A sequence with the human P N M T c D N A . The sequences surrounding splice donor and acceptor sites o b e y e d the G T - A G rule 9'21. We performed primer extension analysis with a 20-mer synthetic oligonucleotide as described in Materials and Methods, and two bands (a major and a minor band) were detected as the putative transcription initiation sites (Fig. 3). As shown in Fig. 2, the nucleotide sequence of the mouse P N M T gene was n u m b e r e d beginning with the nucleotide corresponding to the major transcription initiation site. Mouse P N M T was encoded by 295 amirfo acids with the deduced molecular weight of 32,627. The typical polyadenylation signal 6, A A T A A A , was present 60-bp downstream of T G A stop codon. Mouse genomic D N A was analysed by Southern blot hybridization with the 1.7-kb KpnI-SalI fragment containing mouse P N M T gene. The XbaI, EcoRV, and SacI
digestions of genomic D N A gave a single band of 12.0, 8.0, and 7.5 kb, respectively (Fig. 4). These results corr e s p o n d e d to the restriction m a p of the mouse P N M T genomic clones.
Expression of mouse PNMT mRNA and cDNA cloning To confirm the expression of mouse PNMT, N o r t h e r n blot analysis of mouse adrenal p o l y ( A ) ÷ R N A was carried out with the 1.7 kb KpnI-SalI fragment containing the mouse P N M T gene, and a single band of about 1.3 kb was detected (Fig. 5A). Next, total R N A p r e p a r e d from various tissues of mouse was subjected to R T - P C R , as described in Materials and Methods. The P C R products of about 1.0 kb were detected in the brain part (pons and medulla oblongata) and adrenal gland, whereas no products were observed in pancreas and spleen, indicating the tissue-specific expression of mouse P N M T m R N A in brain and adrenal gland (Fig. 5B). The resulting R T - P C R product was subcloned into Bluescript M 1 3 + vector and subjected to nucleotide sequence analysis, to characterize c D N A encoding mouse PNMT. The nucleotide sequence of this c D N A clone coincided with that of exon region of the gene (Fig. 2).
GATC Comparison of nucleotide and deduced amino acid sequences of mouse, bovine, and human PNMT G5' G A G G T C T C* A A C* +1 A G A A G C A T G3'
The predicted amino acid sequence of mouse P N M T was c o m p a r e d to human and bovine P N M T sequences 19 (Fig. 6). The most regions of their amino acid sequences
123 MW(kb) 23.0 -9.4_ 6.6-4.4--
2,3 m 2.0-Fig. 3. Primer extension analysis of the transcription start site of the mouse PNMT gene. Poly(A) + RNA (2 ktg) prepared from mouse adrenal gland was annealed to 20-mer synthetic oligonucleotide labeled at 5'-end, and the mixture was subjected to reverse transcription. The products were electrophoresed on 6% polyacrylamide-7M urea gel. Lanes G, A, T, and C contain the products of the DNA sequence analysis performed with the same oligonucleotide as used in the primer extension reaction. The nucleotide sequence around the putative transcription start sites is shown at the right panel.
Fig. 4. Southern blot analysis of mouse genomic DNA. 10/~g of mouse genomic DNA was digested with either XbaI (lane 1), EcoRV (lane 2), or SacI (lane 3), electrophoresed, and transferred to a nylon membrane. Hybridization was carried out with the 1.7-kb KpnI-SalI fragment of the mouse PNMT genomic clone as a probe.
317
A
B 1
28 S -
2
3
4
MW(I 2.3 2.0
18S1.3 kb
•.~-- 1.0 kb 0.6 Fig. 5. Expression of mouse PNMT mRNA. A: Northern blot analysis of mouse adrenal gland RNA. Poly(A) + RNA (3/~g) was electrophoresed on 2% agarose-formaldehyde gel and transferred to a nylon membrane. Hybridization was carried out with the 1.7-kb KpnI-SalI fragment labeled with 32p. B: RT-PCR of RNA prepared from various mouse tissues. Total RNA samples (1 #g) extracted from adrenal gland (lane 1), pons and medulla oblongata of brain (lane 2), pancreas (lane 3), and spleen (lane 4), were reverse-transcribed to obtain the first strand cDNA. The mouse PNMT cDNA fragments were amplified with RT-PCR primers and analysed on 1% agarose gel.
s h o w e d a h o m o l o g y o f a b o u t 80%, b u t t h e N - t e r m i n a l
P N M T was l o n g e r by e x t r a 11 a m i n o acid r e s i d u e s as
(24 a m i n o acids) and C - t e r m i n a l (5 a m i n o acids) r e g i o n s
c o m p a r e d to the s e q u e n c e s of h u m a n and b o v i n e en-
of the m o u s e P N M T
f r o m the c o r r e s p o n d i n g r e g i o n s of h u m a n a n d b o v i n e
zymes. Fig. 7 shows t h e c o m p a r i s o n of n u c l e o t i d e seq u e n c e s in t h e 5 ' - t e r m i n a l r e g i o n s of t h e m o u s e , h u m a n 2'
P N M T . In particular, t h e N - t e r m i n a l s e q u e n c e of m o u s e
25, and b o v i n e 4 P N M T genes. I n t e r e s t i n g l y , w e f o u n d
Mouse Bovlne Human
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R V:L I D I G P T I Y R: T : L :Z D : ~ G S : : G :'P:T:: :Z Y R T L :I D I G S G P T V Y
L T S F L A 8 F L A:E F V
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Fig. 6. Comparison of the deduced amino acid sequences of mouse, human, and bovine PNMT. The amino acids identical among two or three sequences are enclosed in shadowed boxes. The deduced amino acid sequences of human and bovine PNMT are derived from the data reported by Kaneda et alJ 9 who modified the original sequence of bovine PNMT 3 based on the homology with the human PNMT sequence.
318 1
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MO u se Bovine
AGCAGC
Human
GGCAGC
Mouse
12 27 G1 y S e r G l y S e r A s p P r o L y s H i s A l a A l a G l uMe t A s p P r o A s p S e r GG G A G T G g ¢ T C A G A C e ~ A A G C A C G C S G C A G A G A T G G A C C C T G A C T CC
Bovine
AT GAG CGG GACA GAC CGG AGT CAGG CGG CGG GCGC GGT GCC CGA CTCA M e t S e r G l y T h r A s p A r g S e r G i n A l a A l a G l yAl a V a IF r o A s p S e r 1 16
Human
AT G A G C G G C G C A G A C C G T A G C C C C A A T G C G G G C G C A G C C C C T G A C T C G Me t Se rG ] y A l a A s p A r g S e rP r o A s n A lag i yAl aAl aP r o A s p S e r ] 16
Fig. 7. Comparison of the nucleotide sequences in the 5'-terminal regions of the mouse, human, and bovine PNMT genes. The nucleotide sequence of the mouse PNMT gene is aligned with the human 2'25 and bovine4 sequences. The nucleotides identical to those in mouse PNMT are indicated by asterisks. The 23-bp direct repeat sequences are indicated with horizontal arrows.
two direct repeat sequences of 23 nucleotides, 5 ' - G T G GCTCAGACC~GAAGCACGCT-3', where only the 13th nucleotide is different, in the protein-coding region of the mouse P N M T gene. These characteristic sequences did not exist in the corresponding regions of the human and bovine P N M T genes. Therefore, the major difference in their N-terminal coding regions was based on the presence or absence of the direct r e p e a t sequences. DISCUSSION In this p a p e r , we have described the isolation and characterization of the gene and c D N A encoding mouse PNMT. The complete nucleotide sequence of the mouse P N M T gene was d e t e r m i n e d . It was a single gene which consisted of three exons, spanning approximately 1.8 kb. We could obtain the useful informations involved in the evolution and expression of the P N M T gene by the comparison of the nucleotide and deduced amino acid sequences of mouse, human, and bovine PNMT. In the N-terminal coding region of the mouse P N M T gene, there were the direct repeat sequences c o m p o s e d of 23 nucleotides, which were not observed in the corresponding regions of the human and bovine P N M T
REFERENCES 1 Axelrod, J., Purification and properties of phenylethanolamine N-methyl transferase, J. Biol. Chem., 237 (1962) 1657-1660. 2 Baetge, E.E., Behringer, R.R., Messing, A., Brinster, R.L. and Palmiter, R.D., Transgenic mice express the human phenylethanolamine N-methyltransferase gene in adrenal medulla and retina, Proc. Natl. Acad. Sci. U.S.A., 85 (1988) 3648-3652. 3 Baetge, E.E., Suh, Y.H. and Joh, T.H., Complete nucleotide
genes. The sequence diversity among species gave the major differences in the amino acid sequence of PNMT. F a r a b a u g h et al. previously described that the presence of direct repeats can p r o m o t e deletions in the lac I gene of E. coli 14. Efstratiadis et ah also p r o p o s e d a model for the involvement of short direct r e p e a t sequences in the generation of deletions in the fl-like globin genes during evolution 12. According to their hypotheses, slipped mispairing during D N A replication deletes one of the direct repeats and their surrounding nucleotides. The difference in the presence or absence of the direct repeats in the P N M T gene suggests that these r e p e a t sequences may have been involved in the variation of the gene. One of the direct repeat sequences observed in the mouse P N M T gene may have been r e m o v e d during evolution. We found several transcription regulatory elements in the 5'-flanking region of the mouse P N M T gene. The canonical TATA box 9, T A G A T A A A , was present 32 bp upstream of the putative transcription initiation site. There were two C A C C C boxes 12 at nucleotide - 7 7 to - 7 3 and -1,100 to -1,096, as well as a G C box n at nucleotide -51 to -46. Four nucleotide sequences homologous to glucocorticoids response element ( G R E ) 18 were located at nucleotide -543 to -529, -733 to -719, -777 to -763, and -1,576 to -1,562. Ross et al. d e t e r m i n e d the nucleotide sequence of the rat P N M T gene p r o m o t e r and analysed d e x a m e t h a s o n e - i n d u c e d expression of the P N M T p r o m o t e r using chromaffin cells in primary culture 23. Their data defined the sequence from -528 to -513 in the rat P N M T gene as a functional G R E . The nucleotide sequence, A G A A C A G A G T G T C C T , from -543 to -529 in the mouse P N M T gene completely matches the sequence of the functional G R E identified in the p r o m o t e r of the rat P N M T gene. The functional regulatory elements in the 5'-region of the mouse P N M T gene should be also identified experimentally.
Acknowledgements'. This work was supported by a Grant-in-Aid for Scientific Research, Ministry of Education, Science and Culture, Japan. We would like to thank Drs. M. Katsuki and M. Kimura for helpful discussion and encouragement.
and deduced amino acid sequence of bovine phenylethanolamine N-methyltransferase: partial amino acid homology with rat tyrosine hydroxylase, Proc. Natl. Aead. Sci. U.S.A., 83 (1986) 5454-5458. 4 Batter, D.K., D'Mello, S.R., Turzai, L.M., Hughes III, H.B., Gioio, A.E. and Kaplan, B.B., The complete nucleotide sequence and structure of the gene encoding bovine phenylethanolamine N-methyltransferase, J. Neurosci. Res., 19 (1988) 367-376.
319 5 Benton, W.D. and Davis, R.W., Screening ~.gt recombinant clones by hybridization to single plaques in situ, Science, 196 (1977) 180-182. 6 Birnstiel, M.L., Busslinger, M. and Strub, K., Transcription termination and 3' processing: the end is in site!, Cell, 41 (1985) 349-359. 7 Bohn, M.C., Role of glucocorticoids in expression and development of phenylethanolamine N-methyltransferase (PNMT) in cells derived from the neural crest: a review, Psychoneuroendocrinology, 8 (1983) 381-390. 8 Bohn, M.C., Expression and developement of phenylethanolamine N-methyltransferase (PNMT): role of glucocorticoids, Neurol. Neurobiol., 16 (1986) 245-271. 9 Breathnach, R. and Chambon, P., Organization and expression of eucaryotic split genes coding for proteins, Annu. Rev. Biochem., 50 (1981) 349-383. 10 Chirgwin, J.M., Przybyla, A.E., MacDonald, R.J. and Rutter, W.J., Isolaton of biologically active ribonucleic acid from sources enriched in ribonuclease, Biochemistry, 18 (1979) 5294-5299. 11 Dynan, W.S., Saffer, J.D., Lee, W.S. and Tjian, R., Transcription factor Spl recognizes promoter sequences from the monkey genome that are similar to the simian virus 40 promoter, Proc. Natl. Acad. Sci. USA, 82 (1985) 4915-4919. 12 Efstratiadis, A., Posakony, J.W., Maniatis, T., Lawn, R. M., O'Connell, C., Spritz, R.A., DeRiel, J.K., Forget, B.G., Weissman, S.M., Slightom, J.L., Blechl, A.E., Smithies, O., Baralle, EE., Shoulders, C.C. and Proudfoot, N.J., The structure and evolution of the human fl-globin gene family, Cell, 21 (1980) 653-668. 13 Evinger, M., Joh, T.H. and Reis, D., Transcriptional regulation of phenylethanolanine N-methyltransferase gene expression. In T.H. Joh (Ed.), Catecholamine Genes, Wiley-Liss, New York, 1990, pp. 137-146. 14 Farabaugh, P.J., Schmeissner, U., Hofer, M. and Miller, J.H., Genetic studies of the lac repressor VII. On the molecular nature of spontaneous hotspots in the lac I gene of Escherichia coli, J. Mol. Biol., 126 (1978) 847-863. 15 Hadjiconstantinou, M., Cohen, J. and Neff, N.H., Epinephfine: a potential neurotransmitter in retina, J. Neurochem., 41 (1983) 1440-1444. 16 H6kfelt, T., Fuxe, K., Goldstein, M. and Johansson, O., Evidence for adrenaline neurons in the rat brain, Acta Physiol. Scand., 89 (1973) 286-288. 17 Saavedra, J.M., Palkovits, M., Brownstein, M.J. and Axelrod,
18
19
20 21 22 23
24 25
26 27 28
J., Localization of phenylethanolamine N-methyltransferase in the rat brain nuclei, Nature, 248 (1974) 695-696. Jantzen, H. -M., Str~ihle, U., Gloss, B., Stewart, E, Schmid, W., Boshart, M., Miksicek, R. and Schiitz, G., Cooperativity of glucocorticoid response elements located far upstream of the tyrosine aminotransferase gene, Cell, 49 (1987) 29-38. Kaneda, N., Ichinose, H., Kobayashi, K., Oka, K., Kishi, F., Nakazawa, A., Kurosawa, Y., Fujita, K. and Nagatsu, T., Molecular cloning of cDNA and chromosomal assignment of the gene for human phenylethanolamine N-methyltransfrase, the enzyme for epinephrine biosynthesis, J. Biol. Chem., 263 (1988) 7672-7677. Kirshneer, N. and Goodall, M., The formation of adrenaline from noradrenaline, Biochim. Biophys. Acta, 24 (1957) 658659. Lerner, M.R., Boyle, J.A., Mount, S.M., Wolin, S.L. and Steitz, J.A., Are snRNPs involved in splicing?, Nature, 283 (1980) 220-224. Mezey, E., Phenylethanolamine N-methyltransferase-containing neurons in the limbic system of the young rat, Proc. Natl. Acad. Sci. U.S.A., 86 (1989) 347-351. Ross, M.E., Evinger, M.J., Hyman, S.E., Carroll, J.M., Mucke, L., Comb, M., Reis, D.J., Joh, T.H. and Goodman, H.M., Identification of a functional glucocorticoid response element in the phenylethanolamine N-methyltransferase promoter using fusion genes introduced into chromaffin cells in primary culture, J. Neurosci., 10 (1990) 520-530. Sanger, E, Nicklen, S. and Coulson, A.R., DNA sequencing with chain-terminating inhibitors, Proc. Natl. Acad. Sci. U.S.A., 74 (1977) 5463-5467. Sasaoka, T., Kaneda, N., Kurosawa, Y., Fujita, K. and Nagatsu, T., Structure of human phenylethanolamine N-methyltransferase gene: existence of two types of mRNA with different transcription initiation sites, Neurochem. Int., 15 (1989) 555-565. Southern, E.M., Detection of specific sequences among DNA fragments separated by gel electrophoresis, J. Mol. Biol., 98 (1975) 503-517. Thomas, P.S., Hybridization of denatured RNA and small DNA fragments transferred to nitrocellulose, Proc. Natl. Acad. Sci. USA, 77 (1980) 5201-5205. Yanisch-Perron, C., Vieira, J. and Messing, J., Improved M13 phage cloning vectors and host strains: nucleotide sequence of the Ml3mpl8 and pUC19 vectors, Gene, 33 (1985) 103-119.
313
BRESM 70411
Organization and complete nucleotide sequence of the gene encoding mouse phenylethanolamine N-methyltransferase Shinji Morita a, Kazuto Kobayashi b, Hiroyoshi Hidaka a and Toshiharu Nagatsu b aDepartment of Pharmacology, Nagoya University School of Medicine, Nagoya (Japan) and blnstitute for Comprehensive Medical Sciences, School of Medicine, Fufita Health University, Toyoake (Japan) (Accepted 26 November 1991)
Key words: Phenylethanolamine N-methyltransferase; Genomic DNA; cDNA; Nucleotide sequence; Mouse
Phenylethanolamine N-methyltransferase (PNMT; EC 2.1.1.28) catalyzes the conversion of norepinephrine to epinephrine, the last step of catecholamine biosynthesis. We have previously reported molecular cloning of cDNA encoding human PNMT and chromosomal localization of its gene (Kaneda et al., J. Biol. Chem., 263 (1988) 7672-7677). In this report, we isolated the chromosomal gene encoding mouse PNMT by cross-hybridization with the human PNMT cDNA. Mouse PNMT gene spanned about 1.8 kb and consisted of 3 exons. Primer extension analysis showed two putative transcription initiation sites. Northern blot analysis and reverse transcription-polymerase chain reaction (RTPCR) revealed the expression of the mouse PNMT mRNA in brain (pons and medulla oblongata) and adrenal gland. Subsequently cDNA encoding mouse PNMT was amplified by RT-PCR and cloned into the plasmid vector. Mouse PMNT gene contained the protein-coding region of 885 bp (295 amino acids) with the predicted molecular weight of 32,627. The deduced amino acid sequence of mouse PNMT revealed the major difference in the N-terminal region, as compared to the human and bovine PNMT sequences. In the 5'-terminal region of the mouse PNMT gene, we found the existence of 23 bp direct repeat sequences, which was not observed in the corresponding regions of the human and bovine PNMT genes. The presence or absence of the direct repeats caused the major difference in the PNMT sequences among species. The typical TATA, GC, and CACCC boxes as well as several sequences homologous to glucocorticoids response elements (GRE) were located in the Y-flanking region of the mouse PNMT gene. INTRODUCTION
We previously r e p o r t e d a full-length c D N A clone encoding h u m a n P N M T 19 and its genomic clone 25. Nucle-
P h e n y l e t h a n o l a m i n e N-methyltransferase ( P N M T ; E C 2.1.1.28) is the terminal enzyme in catecholamine biosynthesis, and catalyzes the t r a n s m e t h y l a t i o n of norepinephrine to epinephrine 1'2°. The enzyme is present at high level in chromaffin cells of adrenal medulla, where epinephrine is synthesized as a h o r m o n e . It is also distributed in epinephrine neurons of m e d u l l a oblongata, hypothalamus, and amygdala of brain 16'17'22, as well as
otide sequence analysis of c D N A clone revealed that h u m a n P N M T consists of 282 amino acid residues with a predicted molecular weight of 30,853. M o r e o v e r , the P N M T gene was assigned to h u m a n c h r o m o s o m e 17 by Southern blot analysis using m o u s e - h u m a n somatic cell hybrids. The analysis of the h u m a n P N M T genomic clone showed that P N M T is e n c o d e d by a single gene that is c o m p o s e d of three exons, and that there are several sequences resembling S p l binding site and glucocorticoids response e l e m e n t ( G R E ) in the Y-flanking region. In the present p a p e r we describe the structures of the gene and c D N A encoding mouse PNMT. The nucleotide and deduced amino acid sequences of mouse P N M T were comp a r e d to the sequence data of P N M T from o t h e r species.
in a small n u m b e r of ganglion and amacrine cells of retina 15, where epinephrine m a y function as a neurotransmitter. The expression levels of P N M T are regulated by several factors in addition to tissue-specific and developmental stage-specific manners. Glucocorticoid h o r m o n e s are known to mainly regulate expression of the P N M T gene 7,sA3. Baetge et al. r e p o r t e d transgenic mice carrying the h u m a n P N M T gene o r a chimeric gene consisting of the h u m a n P N M T gene p r o m o t e r (2 kb) fused to the simian virus 40 large T antigen 2. Their results indicated that the cis-element for a p p r o p r i a t e expression of the h u m a n P N M T gene in adrenal gland and retina is in the 2-kb D N A fragment of the 5 ' - u p s t r e a m region.
MATERIALS AND METHODS
Construction of mouse genomic DNA library Two mouse (DBA/2J) genomic DNA libraries were used in our experiment. A mouse genomic library was purchased from Clontech and consisted of 2 phages which contained 8-22-kb DNA frag-
Correspondence: T. Nagatsu, Institute for Comprehensive Medical Sciences, School of Medicine, Fujita Health University, Toyoake 470-11, Japan.
314 ments digested partially with MboI, in the BamHI site of EMBL3 phage vector. Another library was constructed as follows: Mouse kidney genomic DNA was completely digested with XbaI, and electrophoresed on 0.8% agarose gel. The DNA fragments of about 12-kb were eluted from gel and ligated to EMBL12DNA (Boehringer) digested with XbaI, and packaged into 2 phages using Gigapack plus (Stratagene).
gMPNMT-1 Sal I"
I
Xbal
//
DNA sequence analysis Appropriate DNA fragments were subcloned into the Bluescript M13 + (Stratagene), pUC119 (Takara Shuzo Co.) and pGEM 7Zf + (Promega) vectors. Serial deletion mutants of the proper clones were constructed according to the unidirectional deletion method 2s. Nucleotide sequence was determined by the dideoxy chain-termination method z4 with the Sequenase DNA sequencing kit (Stratagene).
Southern blot analysis of genomic DNA 10/~g of mouse genomic DNA (DBA/2J) was digested with several restriction enzymes and electrophoresed on 0.8% agarose gel. The gel was transferred to a pylon membrane and (Hybond-N, Amersham) in 20 x SSC zr. The membrane was hybridized with 32p-labeled 1.7-kb KpnI-SalI fragment of the mouse PNMT genomic clone.
Northern blot analysis Total RNA was extracted from mouse adrenal glands by guanidine thiocyanate method 1°. Poly(A) ÷ RNA was obtained with oligo(dT)-Latex. 3 #g of poly(A) ÷ RNA was electrophoresed on 2% agarose-formaldehyde gel, and transferred to a nylon membrane in 20 × SSC27. The membrane was hybridized with the 32p-labeled 1.7-kb KpnI-SalI fragment as a probe.
Primer extension analysis A synthetic oligonucleotide, 5'-GTAGTI'GTTGCGGAGATAGG-3', that is complementary to the nucleotide positions from 141 to 160 downstream from the putative initiation codon of mouse PNMT, was used as an extension primer (see Fig. 2). The oligonucleotide was labeled at 5'-end using T4 polynucleotide kinase and [7-32p]ATP at 37°C for 30 min, and the labeled primer was hybridized with 2 pg of mouse adrenal gland poly(A) ÷ RNA, incubated at 37°C for 1 h in the reaction mixture of 80 ktl containing 20 mM Tris-HCl (pH 8.3), 0.25 mM EDTA, 62.5 mM KC1, 10 mM MgC12, 10 mM DTT, 0.25 mM dATP, 0.25 mM dGTP, 0.25 mM dCTP, 0.25 mM dTTP, 75 units of ribonuclease inhibitor, and 400 units of MMLV reverse transcriptase. The product was analysed by electrophoresis on 6% polyacrylamide-7 M urea gel.
Reverse transcription-polymerase chain reaction (RT-PCR) Two oligonucleotides: 5'-CAACAGAAGCATGAACGGTG-3' (forward primer), which corresponds to the nucleotides from -3 to
Sinai I
I -2-kb -
(~15-kb) Kpnl I
Sail" I
g M P N M T / X 12-4 Xbal I
Kpnl
Library screening Mouse genomic DNA libraries were screened by plaque hybridization5 using human PNMT cDNA fragment labeled with [a-32p]dCTP by a multiprime labeling method (Amersham). Hybridization was carried out in 6 × SSC, 5 x Denhardt's, 0.3% SDS, 100 pg/ml denatured salmon sperm DNA, 50% formamide, and labeled probe, at 42°C for 12 h. The filters were washed twice in 2 x SSC containing 0.1% SDS, at 65°C.
i
Kpnl 1
I
Kpnl Sinai Kpnl I I l J t Sac I EcoR V
Kpnl I
Eco47 Ill
Kpnl
I
exon 1
Sphl I
(-12-kb) Xbal
Kpnl I
I
Sac I
I
I
EcoR V
Kpnl I
exon 2 exon 3
Fig. 1. Structure of the mouse PNMT gene. The upper panel shows the restriction map of genomic DNA clones (gMPNMT-I and gMPNMT/X12-4). The lower panel shows the exon/intron organization of the mouse PNMT gene. SalI* indicates the restriction site of SalI in the multicloning sites of EMBL3 vector.
17; and 5'-GGAACTGTCACT TTATTAGGT-3' (reverse primer), which is complementary to the nucleotides from 1,528 to 1,548, were used as RT-PCR primers (see Fig. 2). The first strand of cDNA was yielded by reverse transcription from 1/~g of total RNA using oligo(dT) as a primer. Subsequently, the fragment corresponding to mouse PNMT cDNA was amplified using RT-PCR primers. The resulting PCR products were electrophoresed on 1% agarose gel and visualized with ethidium bromide-staining.
RESULTS
Isolation and characterization of the mouse P N M T gene First, we screened the mouse genomic D N A library purchased from Clontech with human PMNT cDNA as a probe, and three positive clones were isolated from 106 recombinant phages. As they showed the same restriction map, one of them, designated gMPNMT-1, was further analysed. Fig. 1 shows the restriction enzyme map of this clone. Southern blot analysis with several fragments of human PNMT cDNA indicated that gMPNMT-1 lacked the 3'-terminal region of the mouse PNMT gene. Because Southern blot hybridization of mouse genomic D N A detected a single band of 12 kb against the digestion with XbaI (see below), we originally constructed another genomic D N A library containing the XbaI-cut 12-kb fragments. We screened the second mouse genomic library with the 1.7-kb KpnI-SalI fragment prepared from gMPNMT-1 as a probe, and f o u r p o s i t i v e c l o n e s w e r e i s o l a t e d f r o m 2.5 × 105 rec o m b i n a n t p h a g e s . O n e o f t h e m was d e s i g n a t e d g M P NMT/X12-4,
a n d its r e s t r i c t i o n m a p o v e r l a p p e d w i t h
Fig. 2. Nucleotide sequence of the mouse PNMT gene and its flanking regions. The deduced amino acid sequence is shown below the nucleotide sequence. The nucleotides are numbered in the 5' to 3' direction from the major transcription initiation site (marked +1 above the nucleotide). The canonical TATA, CACCC, and GC boxes, as well as putative GRE are shown. The TGA termination codon and polyadenylation signal are underlined. The direct repeat sequences are indicated by horizontal arrows with thick lines. Horizontal arrows with thin lines indicate the location of the oligonucleotide primers used in primer extension analysis and RT-PCR (see Materials and Methods).
315
GRE -~5~: ACCGTGCACTTCTGGGAACAGCCAAGCATTGGAGCTAAATCTGGCAT~ACCAGAGGGTACACTCCATGCTTAGGGGTGG~GTACA~GGCAGAGC -1442 TGTAAACAGGGGGAATGTCTGGTCCCCCTTCCTGTGCCACTGCCCAGCCCTGGCTTCATGTCCAGTTCCTGGCACTGGAGCGTGTCTAGCAGGCC -1347 AGCTGGCTTCAAGGTTTGTTTAAATGATATCTGTGGAGGAGGTTATGGAAGGGGCTGGCACCAGGGCCGTCCTTGGCTGTGCTTGGGGTGTGGAT -1252 GGGGTCAGTGACCCTAAGGCCTGTCAGTTGTAGATCCAGACAGAATCAATCCTTGGCTGGCATCAGGTGTCCCACTACCCCTGGCCTGGGTGGGA -1157 GGACAGGGTTTAAGTTCCTGTCTGTGACCTCTGCAGCTGTTGTGATGATCCCCGACCCAGCCTGGGTGTCTGGCCTTTGGGATAAGGAAGGGACA C A C C C box -1062 CTGGGTAGGACTGGATAGAAGACCAGGACTAT•TTAGCAGAGGCTAGTAACCCTCCCACCCCAGAAAGACATAGGACTTTCTTAGGACTTAAAGG -967 GTCTCTGCTTTAGCAAGACTGGGGATGCTGTTGCCAGGGTAGCTTGCCATTTTGAGAACATGGGAAGGAAGACAGGCAGATTATGTTCCAGATAC -872 CTGGAGCACTAAGCAAGCCTGAAGGCCAGGCCCTGCCATCTTCCTAGTGGGGACACGATGTTGACTGAGGGGTCGGGATCAGGGTAGGGGTGTGG -777 GGATGTCCTGTACCTGCGAAACTGTACCTGCGAAATGCCAAGAGTGCGCATGCGCTGCCCCTCTAGCGGCCGTGGCAGTACCAAGAACATGCTC~ GRE GRE -682 QTACTCTTTGTTCCTACCTGAGTCCAGTGTCCTGGACCTGGTAGGAACATCCTGAAC~AACCATGCTTGCCTGGCCCCCAGATAAGCAGCACATA -587 GCCCTAGAGGCCTGCAGGGGATGCCCGGATGTTCTGCTTGCTAAAAGCATTAGCACGGCTCACCTTCCTTATCTCTGCTGCCATCCGATGCTCAG GRE -492 GGCAGAGACCTGCTCAGGACCCAGGGTCCTCAAGACAGAGGCCAGAACAGAGTGTCCTTTCTGGAGGAGGGAAGGGGTGCTGCAAGATAGAGATG -397 GGTTAGAGGTCTGGAGGTAGGGATGGTATTATAAAGAAGAGGAGTTTGTAAGGGGTGCCCCGAGAGAGGGGAGGAAGTCTGGGAAGGATGCTGGG -302 ACTGGGACTGGGAACACAGGCTAATTTAGACCTCGGGAAAGAGAAGGGGTGAGATGCTGGGCAAAAGACTCTTTGGGAGGTTGGAGAGGAGCTGG -207 GGTA••GCTGGAACTGAGGCTGGGGGTGTAGGGCAG•CCTGGAGCGATCAGGGG•TGGGGGGGGGGGGGTGGAGGGTTTGTTGAGCAAGTGGKAA -112 GCAAGGGTGGGGAGGAGCGATGTTCTAAAGGGCGCCCCCCAGCCTCCGCGCGTCTGTTGCTCAGACACTAACTGAGATGGATGGAGTGACAGAGA CACCC box GC box TATA box -17 TGTGGTGGCCTCAGGCGCCTCATCCCTCAGCAGCCACCCACCCCTGTGACGGAGGGGTCCGGGCGGGGGGACCCAGTGGTAGATAAAGGGATGGG +i 61 G G A G A G G A G G T C T ~ A A ~ A G A A G C ATG AAC GGH G H C T C A HAC C T G AAH CAC GC-T A C A GGG AHT HHC TCA HAC CCG AAG M e t ASh G l y Gly Set Asp Leu Lys His Ala Thr Gly Set Gly Set Asp Pro Lys 133 ~ _ ~ G C A GAG ATG GAC CCT GAC TCC GAC GCT GGC CAG G T A CGT GTC GCC TTG GCT TAC CAG CGC TTC GAG His Ala Ala Glu Met Asp Pro Asp Ser Asp Ala Gly Gln Val Arg Val Ala Leu Ala Tyr Gln Arg Phe Glu 205 CCC CGC G CC TAT CTC C G C AAC AAC TAC GCG CCT CCT CGT GGA GAC CTG AGC AAC CCT GAT GGC GTC GGG CCT Pro Arg Ala Tyr Leu Arg Asn Ash Tyr Ala Pro Pro Arg Gly Asp Leu Set Asn Pro Asp Gly Val Gly Pro 285 TGG AAG C T G CGC TGC ATG G C A CAA GTC TTT GCT ACC G GTGAGCACCGAAACGAGGCATGAGAGAGCAGAACTCATGGGGA Trp Lys Leu Arg Cys Met Ala Gln Val Phe Ala Thr G 380 AAGGTGCCTACCTAGCAGGCACAGACAGGAGCCGGCCTGTAGTCTTAGCGACTAGGACACTGAGGCTTGGAGGACCACTTGAGGCCGGAATTGGA 475 TGACTGACTAGACAGTTTAGAGTCTTTTTCTTTCAAGGGGAGGATGTTTAAGATTGAGAGACGGATGGGGCCAGGTGAGAGAGATAGGGAGAGGG 570 AGGGCGGGAGGGGGAGGTTGGAGAGGCTCATTCCTTGATGTCTCGGGATCGTGATCTCCCCCACTAAATCGTAACTAGAGCTAGTTTCTGAGGGG 665 TGGGACCCGAGTCAGAGGAGCCCTGTCAGCACAGCACTTGCTCCAGATACTACCCTGAAGAAGTGCTGAGGAGAGCACGAGGGGAAGGGGCGGCT 746 GAGCAGGAGCCACTGGTTATCCCTGCCTCTGCTCCACCAG GT GAG GTG TCG GGA CGG GTT CTC ATT GAT ATT GGC TCC GGC ly Glu Val Ser G l y Arg Val Leu Ile Asp Ile Gly Ser Gly 818 CCC ACC ATA TAT C A G C T G CTC AGT GCC TGT GCC CAC TTT GAG GAC ATC ACC ATG A C A GAC TTC TTG G A A GTC Pro Thr Ile Thr Gin Leu Leu Ser Ala Cys Ala His Phe Glu Asp Ile Thr Met Thr Asp Phe Leu Glu Val 890 AAC CGT CAG GAG C T G G G A CTC TGG CTG CGA GAG GAG C C A GGA GCC TTT GAC TGG AGT GTG TAT AGT CAG CAT Ash Arg Gin GIu Leu Gly Leu Trp Leu Arg Glu Glu Pro Gly Ala Phe Asp Trp Set Val Tyr $er Gln His 976 GCC TGC CTC ATT GAG GAC AAG GG GTGAGAGCTGGACTGGCAGCTTCGTGTGCAGCAGTGGGTGGCTGGGGGGGGGGGGCTGCAGAA Ala Cys Leu Ile Glu A s p Lys G1 1058 T GAG TCC TGG CAG GAG AAA G A A CGC CAG CTT GGCTGAGTCTTTGGGGTAGTCCTGAGCCCCGCCTTGTGCCCCCCTGTACAG y Glu Ser Trp Gln Glu Lys Glu Arg Gln Leu 1130 C G A GCG A G G GTG AAG C G A GTC CTG CCT ATC GAT GTG CAC AAG CCC CAG CCC CTG G G A ACT CCC AGT CTG GTC Arg Ala Arg Val Lys Arg Val Leu Pro Ile Asp Val His Lys Pro Gln Pro Leu G,y Thr Pro $er Leu Val 1202 CCT CTG CCT GCC GAC GCC TTG GTC TCT GCC TTC TGC C T G GAG GCT GTG AGC CCA G A T CTT ACT AGC TTC CAG Pro Leu Pro Ala Asp Ala Leu Val Ser Ala Phe Cys Leu Glu Ala Val Set Pro Asp Leu Thr Set Phe Gln 1274 CGG GCT TTG CAT CAC ATC ACC ACA CTG CTG AGG CCC GGG GGT CAT CTC CTC CTC ATT GGG GCC CTG GAG GAG Arg Ala Leu His His Ile Thr Thr Leu Leu Arg Pro Gly Gly His Leu L~u Leu Ile Gly Ala Leu Glu Glu 1346 TCC TGG TAC CTT GCT GGG GAG GCC AGG CTG TCT GTG GTG CCA GTG TCT G A G GAG G A G GTG AGG GAG GCC CTG Ser Trp Tyr Leu Ala Gly Glu Ala Arg Leu Set Val Val Pro Val Set Glu Glu Glu Val Arg Glu Ala Leu 1418 GTC CTT GGT GGT TAC GAG GTC CGA GAG CTT CGC ACC TAC ATC ATG CCT GCC CAC CTC TGC ACG GGG G T A GAT Val Leu GIy Gly Tyr Glu Val Arg Glu Leu Arg Thr Tyr Ile Met Pro Ala His Leu Cys Thr Gly Val Asp 1495 GGGCCCCAAAAATGCCAGGTGTC GAT GTC AAA GGC ATC TTC TTT GCC TGG GCC CAG AAG ATG GAG GTG CAG GTG ~ Asp Val LyS GIy Ile Phe Phe Ala Trp Ala Gln Lys Met Glu Val Gln Val CTGCCTCCAAAGTCCTTATCACCTGAAGTGGAACCTAATAAAGTGACAGTTCCC 9
316 that of gMPNMT-1 (Fig. 1). We d e t e r m i n e d the complete nucleotide sequence of the region covering the mouse P N M T gene (Fig. 2). The exon/intron boundaries were d e t e r m i n e d from the comparison of the mouse D N A sequence with the human P N M T c D N A . The sequences surrounding splice donor and acceptor sites o b e y e d the G T - A G rule 9'21. We performed primer extension analysis with a 20-mer synthetic oligonucleotide as described in Materials and Methods, and two bands (a major and a minor band) were detected as the putative transcription initiation sites (Fig. 3). As shown in Fig. 2, the nucleotide sequence of the mouse P N M T gene was n u m b e r e d beginning with the nucleotide corresponding to the major transcription initiation site. Mouse P N M T was encoded by 295 amirfo acids with the deduced molecular weight of 32,627. The typical polyadenylation signal 6, A A T A A A , was present 60-bp downstream of T G A stop codon. Mouse genomic D N A was analysed by Southern blot hybridization with the 1.7-kb KpnI-SalI fragment containing mouse P N M T gene. The XbaI, EcoRV, and SacI
digestions of genomic D N A gave a single band of 12.0, 8.0, and 7.5 kb, respectively (Fig. 4). These results corr e s p o n d e d to the restriction m a p of the mouse P N M T genomic clones.
Expression of mouse PNMT mRNA and cDNA cloning To confirm the expression of mouse PNMT, N o r t h e r n blot analysis of mouse adrenal p o l y ( A ) ÷ R N A was carried out with the 1.7 kb KpnI-SalI fragment containing the mouse P N M T gene, and a single band of about 1.3 kb was detected (Fig. 5A). Next, total R N A p r e p a r e d from various tissues of mouse was subjected to R T - P C R , as described in Materials and Methods. The P C R products of about 1.0 kb were detected in the brain part (pons and medulla oblongata) and adrenal gland, whereas no products were observed in pancreas and spleen, indicating the tissue-specific expression of mouse P N M T m R N A in brain and adrenal gland (Fig. 5B). The resulting R T - P C R product was subcloned into Bluescript M 1 3 + vector and subjected to nucleotide sequence analysis, to characterize c D N A encoding mouse PNMT. The nucleotide sequence of this c D N A clone coincided with that of exon region of the gene (Fig. 2).
GATC Comparison of nucleotide and deduced amino acid sequences of mouse, bovine, and human PNMT G5' G A G G T C T C* A A C* +1 A G A A G C A T G3'
The predicted amino acid sequence of mouse P N M T was c o m p a r e d to human and bovine P N M T sequences 19 (Fig. 6). The most regions of their amino acid sequences
123 MW(kb) 23.0 -9.4_ 6.6-4.4--
2,3 m 2.0-Fig. 3. Primer extension analysis of the transcription start site of the mouse PNMT gene. Poly(A) + RNA (2 ktg) prepared from mouse adrenal gland was annealed to 20-mer synthetic oligonucleotide labeled at 5'-end, and the mixture was subjected to reverse transcription. The products were electrophoresed on 6% polyacrylamide-7M urea gel. Lanes G, A, T, and C contain the products of the DNA sequence analysis performed with the same oligonucleotide as used in the primer extension reaction. The nucleotide sequence around the putative transcription start sites is shown at the right panel.
Fig. 4. Southern blot analysis of mouse genomic DNA. 10/~g of mouse genomic DNA was digested with either XbaI (lane 1), EcoRV (lane 2), or SacI (lane 3), electrophoresed, and transferred to a nylon membrane. Hybridization was carried out with the 1.7-kb KpnI-SalI fragment of the mouse PNMT genomic clone as a probe.
317
A
B 1
28 S -
2
3
4
MW(I 2.3 2.0
18S1.3 kb
•.~-- 1.0 kb 0.6 Fig. 5. Expression of mouse PNMT mRNA. A: Northern blot analysis of mouse adrenal gland RNA. Poly(A) + RNA (3/~g) was electrophoresed on 2% agarose-formaldehyde gel and transferred to a nylon membrane. Hybridization was carried out with the 1.7-kb KpnI-SalI fragment labeled with 32p. B: RT-PCR of RNA prepared from various mouse tissues. Total RNA samples (1 #g) extracted from adrenal gland (lane 1), pons and medulla oblongata of brain (lane 2), pancreas (lane 3), and spleen (lane 4), were reverse-transcribed to obtain the first strand cDNA. The mouse PNMT cDNA fragments were amplified with RT-PCR primers and analysed on 1% agarose gel.
s h o w e d a h o m o l o g y o f a b o u t 80%, b u t t h e N - t e r m i n a l
P N M T was l o n g e r by e x t r a 11 a m i n o acid r e s i d u e s as
(24 a m i n o acids) and C - t e r m i n a l (5 a m i n o acids) r e g i o n s
c o m p a r e d to the s e q u e n c e s of h u m a n and b o v i n e en-
of the m o u s e P N M T
f r o m the c o r r e s p o n d i n g r e g i o n s of h u m a n a n d b o v i n e
zymes. Fig. 7 shows t h e c o m p a r i s o n of n u c l e o t i d e seq u e n c e s in t h e 5 ' - t e r m i n a l r e g i o n s of t h e m o u s e , h u m a n 2'
P N M T . In particular, t h e N - t e r m i n a l s e q u e n c e of m o u s e
25, and b o v i n e 4 P N M T genes. I n t e r e s t i n g l y , w e f o u n d
Mouse Bovlne Human
1 M N
G
s e q u e n c e w e r e largely d i f f e r e n t
G
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40 R R R 80 A T G:E /%.T G E A T G E 120 N.R Q:E N R QIE N R Q::E 160 Q L R A Q L R A (~ L R A 200 /% V S P A V S P A V S P 240 E:A:R L E A R L E /% R L 280 V~D D V
S
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G : S G S D P K H A /% E M M S G :.T:D:.R S~.:Q A ~ G A : V M S:G.A.D R S P N A G.A
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D A G Q V R V A D P:G.L.A A V S A P G 0 /% A V A
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F F F
E P R ~A ~ : L R~N: N: Y ; A P P :R G. D L S :N P D : G V~::G ~P .W:.K~:L R :C M I A Q ~ F E P R ~Y L R N.:N :Y:~:I.P: P.~R:G:D: :IL :-q:CIIP:D:.G ~ G::P ::W ~K: L :R ::C :L: /%: Q T F: E P R A Y :L R N N Y A P P R G ~ D L C N::P::N:G V G P W K : L . R C L:/% Q T F
Mouse Bovine Human
V V V
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Mouse Bovine Human
L G L R L G
L.N L R E E P G AF:D W:S VY .S Q L W L R E E: P :G A :F D N :S: V Y: S Q R W L Q E E P G /% F N W :S M Y S Q
R:~:CL I .E .D K : G R V:::~C:.L: X E :G K G R A C L I E G K G
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R V:L I D I G P T I Y R: T : L :Z D : ~ G S : : G :'P:T:: :Z Y R T L :I D I G S G P T V Y
L T S F L A 8 F L A:E F V
Q Q Q
P: L G P L G P L G
L L S:::A::C :/%::R F: :E D I: T : M : T D F L .:L :S::I: :.G:A~ E :~F E : D . Z T: M ? T :D:F L LiS:/%:~:~C S H F E D I :T~M T DF
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Q R A L H.H~I:T:T.:L:..L:R L ~ E E S::N Y : L /% G Q R A L:::D R Z:.~::..T:.IL L:~R:: P :G:G~:::K~!:IL.ILL : Z:: G : ~ :L:EI:E=S M :Y~L: A :G 0R /% L D . E I : Z ::T::T: :L: L R P G:IG::H :L:L L:~X G /% L::E::E S: W Y L /% G
P V:8:E V:8
E g
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Fig. 6. Comparison of the deduced amino acid sequences of mouse, human, and bovine PNMT. The amino acids identical among two or three sequences are enclosed in shadowed boxes. The deduced amino acid sequences of human and bovine PNMT are derived from the data reported by Kaneda et alJ 9 who modified the original sequence of bovine PNMT 3 based on the homology with the human PNMT sequence.
318 1
Ii
Me tAs n G l y G l y Se r A s p L e u L y s H i s A l a T h r CAA CAGA AGC ATG AAC GGT 05C TCAG ACC TGA AGCA CGC SAC A
MO u se Bovine
AGCAGC
Human
GGCAGC
Mouse
12 27 G1 y S e r G l y S e r A s p P r o L y s H i s A l a A l a G l uMe t A s p P r o A s p S e r GG G A G T G g ¢ T C A G A C e ~ A A G C A C G C S G C A G A G A T G G A C C C T G A C T CC
Bovine
AT GAG CGG GACA GAC CGG AGT CAGG CGG CGG GCGC GGT GCC CGA CTCA M e t S e r G l y T h r A s p A r g S e r G i n A l a A l a G l yAl a V a IF r o A s p S e r 1 16
Human
AT G A G C G G C G C A G A C C G T A G C C C C A A T G C G G G C G C A G C C C C T G A C T C G Me t Se rG ] y A l a A s p A r g S e rP r o A s n A lag i yAl aAl aP r o A s p S e r ] 16
Fig. 7. Comparison of the nucleotide sequences in the 5'-terminal regions of the mouse, human, and bovine PNMT genes. The nucleotide sequence of the mouse PNMT gene is aligned with the human 2'25 and bovine4 sequences. The nucleotides identical to those in mouse PNMT are indicated by asterisks. The 23-bp direct repeat sequences are indicated with horizontal arrows.
two direct repeat sequences of 23 nucleotides, 5 ' - G T G GCTCAGACC~GAAGCACGCT-3', where only the 13th nucleotide is different, in the protein-coding region of the mouse P N M T gene. These characteristic sequences did not exist in the corresponding regions of the human and bovine P N M T genes. Therefore, the major difference in their N-terminal coding regions was based on the presence or absence of the direct r e p e a t sequences. DISCUSSION In this p a p e r , we have described the isolation and characterization of the gene and c D N A encoding mouse PNMT. The complete nucleotide sequence of the mouse P N M T gene was d e t e r m i n e d . It was a single gene which consisted of three exons, spanning approximately 1.8 kb. We could obtain the useful informations involved in the evolution and expression of the P N M T gene by the comparison of the nucleotide and deduced amino acid sequences of mouse, human, and bovine PNMT. In the N-terminal coding region of the mouse P N M T gene, there were the direct repeat sequences c o m p o s e d of 23 nucleotides, which were not observed in the corresponding regions of the human and bovine P N M T
REFERENCES 1 Axelrod, J., Purification and properties of phenylethanolamine N-methyl transferase, J. Biol. Chem., 237 (1962) 1657-1660. 2 Baetge, E.E., Behringer, R.R., Messing, A., Brinster, R.L. and Palmiter, R.D., Transgenic mice express the human phenylethanolamine N-methyltransferase gene in adrenal medulla and retina, Proc. Natl. Acad. Sci. U.S.A., 85 (1988) 3648-3652. 3 Baetge, E.E., Suh, Y.H. and Joh, T.H., Complete nucleotide
genes. The sequence diversity among species gave the major differences in the amino acid sequence of PNMT. F a r a b a u g h et al. previously described that the presence of direct repeats can p r o m o t e deletions in the lac I gene of E. coli 14. Efstratiadis et ah also p r o p o s e d a model for the involvement of short direct r e p e a t sequences in the generation of deletions in the fl-like globin genes during evolution 12. According to their hypotheses, slipped mispairing during D N A replication deletes one of the direct repeats and their surrounding nucleotides. The difference in the presence or absence of the direct repeats in the P N M T gene suggests that these r e p e a t sequences may have been involved in the variation of the gene. One of the direct repeat sequences observed in the mouse P N M T gene may have been r e m o v e d during evolution. We found several transcription regulatory elements in the 5'-flanking region of the mouse P N M T gene. The canonical TATA box 9, T A G A T A A A , was present 32 bp upstream of the putative transcription initiation site. There were two C A C C C boxes 12 at nucleotide - 7 7 to - 7 3 and -1,100 to -1,096, as well as a G C box n at nucleotide -51 to -46. Four nucleotide sequences homologous to glucocorticoids response element ( G R E ) 18 were located at nucleotide -543 to -529, -733 to -719, -777 to -763, and -1,576 to -1,562. Ross et al. d e t e r m i n e d the nucleotide sequence of the rat P N M T gene p r o m o t e r and analysed d e x a m e t h a s o n e - i n d u c e d expression of the P N M T p r o m o t e r using chromaffin cells in primary culture 23. Their data defined the sequence from -528 to -513 in the rat P N M T gene as a functional G R E . The nucleotide sequence, A G A A C A G A G T G T C C T , from -543 to -529 in the mouse P N M T gene completely matches the sequence of the functional G R E identified in the p r o m o t e r of the rat P N M T gene. The functional regulatory elements in the 5'-region of the mouse P N M T gene should be also identified experimentally.
Acknowledgements'. This work was supported by a Grant-in-Aid for Scientific Research, Ministry of Education, Science and Culture, Japan. We would like to thank Drs. M. Katsuki and M. Kimura for helpful discussion and encouragement.
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18
19
20 21 22 23
24 25
26 27 28
J., Localization of phenylethanolamine N-methyltransferase in the rat brain nuclei, Nature, 248 (1974) 695-696. Jantzen, H. -M., Str~ihle, U., Gloss, B., Stewart, E, Schmid, W., Boshart, M., Miksicek, R. and Schiitz, G., Cooperativity of glucocorticoid response elements located far upstream of the tyrosine aminotransferase gene, Cell, 49 (1987) 29-38. Kaneda, N., Ichinose, H., Kobayashi, K., Oka, K., Kishi, F., Nakazawa, A., Kurosawa, Y., Fujita, K. and Nagatsu, T., Molecular cloning of cDNA and chromosomal assignment of the gene for human phenylethanolamine N-methyltransfrase, the enzyme for epinephrine biosynthesis, J. Biol. Chem., 263 (1988) 7672-7677. Kirshneer, N. and Goodall, M., The formation of adrenaline from noradrenaline, Biochim. Biophys. Acta, 24 (1957) 658659. Lerner, M.R., Boyle, J.A., Mount, S.M., Wolin, S.L. and Steitz, J.A., Are snRNPs involved in splicing?, Nature, 283 (1980) 220-224. Mezey, E., Phenylethanolamine N-methyltransferase-containing neurons in the limbic system of the young rat, Proc. Natl. Acad. Sci. U.S.A., 86 (1989) 347-351. Ross, M.E., Evinger, M.J., Hyman, S.E., Carroll, J.M., Mucke, L., Comb, M., Reis, D.J., Joh, T.H. and Goodman, H.M., Identification of a functional glucocorticoid response element in the phenylethanolamine N-methyltransferase promoter using fusion genes introduced into chromaffin cells in primary culture, J. Neurosci., 10 (1990) 520-530. Sanger, E, Nicklen, S. and Coulson, A.R., DNA sequencing with chain-terminating inhibitors, Proc. Natl. Acad. Sci. U.S.A., 74 (1977) 5463-5467. Sasaoka, T., Kaneda, N., Kurosawa, Y., Fujita, K. and Nagatsu, T., Structure of human phenylethanolamine N-methyltransferase gene: existence of two types of mRNA with different transcription initiation sites, Neurochem. Int., 15 (1989) 555-565. Southern, E.M., Detection of specific sequences among DNA fragments separated by gel electrophoresis, J. Mol. Biol., 98 (1975) 503-517. Thomas, P.S., Hybridization of denatured RNA and small DNA fragments transferred to nitrocellulose, Proc. Natl. Acad. Sci. USA, 77 (1980) 5201-5205. Yanisch-Perron, C., Vieira, J. and Messing, J., Improved M13 phage cloning vectors and host strains: nucleotide sequence of the Ml3mpl8 and pUC19 vectors, Gene, 33 (1985) 103-119.
