Characterization DNA Encoding Precursor
Gar Lo, Steve Leqon, Stephen R. Bloom
of Complementary the Rat Neuromedin
Carol Austin,
Simon Wallis,
Zhili Wang,
U
and
Department of Medicine Francis Fraser Lab Royal Postgraduate Medical School Hammersmith Hospital London, W12 ONN, United Kingdom Department of Chemical Pathology (S.L.) Royal Postgraduate Medical School London, W12 ONN, United Kingdom
Neuromedin U (NmU), a peptide originally isolated from porcine spinal cord, is known for its ability to stimulate uterine smooth muscle contraction and to cause selective vasoconstriction. It was subsequently isolated from a number of species. Among the species studied, the five amino acids at the Cterminus of the peptide are totally conserved, suggesting that this region is of major importance. We have cloned and sequenced the cDNA encoding the rat NmU precursor protein using the anchor polymerase chain reaction technique. Sequence analysis revealed that NmU is synthesized as a 174amino acid precursor. Like the precursors of most other small regulatory peptides, it has a hydrophobic signal peptide and a number of paired dibasic amino acids, which may serve as signals for enzymatic cleavage, to release NmU and a series of other peptides. These predicted flanking peptides of NmU show no significant homology with entries in the protein databases searched, and the cDNA likewise shows no homology with entries in the GenBank database. Northern blot analysis using total RNA extracted from different rat tissues shows high levels of NmU mRNA in the ileum, thyroid, and anterior pituitary. Southern blot analysis of rat genomic DNA demonstrates that NmU is a single copy gene. (Molecular Endocrinology 6: 1538-1544, 1992)
NmU-25, the latter having an identical C-terminus as NmU-8 and a 17-amino acid N-terminal extension. Rat NmU however consists of 23 amino acid residues, containing two deletions and nine substitutions compared with porcine NmU-25. High levels of NmU have been detected in the rat gastrointestinal tract, particularly in the ileum, but NmU has also been detected in the pituitary, hypothalamus, spinal cord, thyroid, and many sites in the genitourinary tract (7). NmU contracts isolated rat uterine muscle (l), human bladder (8) and rat stomach muscle (9). It also has selective vasoconstrictor effects on the superior mesenteric arteries in rat and dog (10, 11). A contractile response is seen in the small intestine of man (8) and turtle (12), but NmU has no such effect on the guinea pig ileum (1) or pig jejunum, though NmU was reported to have selective effects on ion transport in the latter (13). As a first step toward understanding the biosynthesis of NmU and its involvement in intestinal absorptive/ secretory processes, we have isolated and sequenced cDNA clones encoding the rat NmU precursor protein.
RESULTS Generation
Primer Selection and Amplification of the NmU Coding Region Two six-amino acid regions located at positions l-6 and 16-21 of rat NmU-23 (2) were chosen for the synthesis of a degenerate oligonucleotide sense primer (NmU-S) and antisense primer (NmU-A), respectively. Restriction sites were included at the 5’-end of these primers to facilitate cloning of the polymerase chain reaction (PCR) product. Based on rat codon preferences (14), an internal oligonucleotide (encoding residues 5-14 of rNmU-23) was synthesized in the
INTRODUCTION Neuromedin U (NmU) was first isolated from porcine spinal cord (1) and subsequently from rat (2, 3), frog (4), dog (5) and rabbit (6) gastrointestinal tissue. Porcine NmU has been isolated as two forms: NmU-8 and OEB8-8809/92/1538-1544$03.00/0 Molecular Endocrinology Copyright 0 1992 by The Endocrine
of Rat NmU cDNA
Socety
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Characterization
of NmU-Encoding cDNA
antisense orientation for monitoring the progress of the cloning procedure. Oligo(dT)-primed rat ileum cDNA was amplified in a PCR using the two mixed primers. A band of the predicted size [82 base pairs (bp)] was visible after electrophoresis of the PCR product on a 1.5% agarose gel containing ethidium bromide (data not shown). After cloning into M13, the recombinant clones containing the NmU sequence were identified by hybridizing with the internal probe. Three clones were sequenced and were found to be identical in the region between the primers (559-586 in Fig. IB). The predicted peptide was in agreement with the published rat NmU sequence
(2). Generation of 3’- and S-Ends of NmU The rest of the NmU sequence was generated in two stages using the anchor PCR protocol (15) outlined in Fig. 1A. Two NmU-specific primers were synthesized using the sequence information obtained above. Before amplification of the 3’-end, rat ileum mRNA was reverse transcribed using the anchor primer BamHl(dT) (Fig. IA). The 3’-end of NmU cDNA was then amplified after two rounds of PCR, first with the mixed sense primer NmUS and the anchor primer, and second with the NmUspecific primer Spl (557-574 in Fig. 1 B) and the anchor primer. A single band (about 160 bp) was visible after gel fractionation of the product and Southern blot analysis showed that this band hybridized to the NmUspecific probe Sp2 (572-589 in Fig. 1B; results not shown). This fragment was then cloned into the plasmid Bluescript. Three clones containing the insert were sequenced. Their sequences were identical except that the poly(A) tail length varied between 14 and 20 bases (557-707 in Fig. 1 B). The approach to cloning of the 5’-end of NmU was similar to that of the 3’-end (Fig. 1A). Rat ileum mRNA reverse transcripts were generated using random primers instead of oligo(dT). Adenosine homopolymer was then added to the 3’-end of the reverse transcripts. Again two rounds of PCR were carried out. In the first round the antisense primer NmU-A was used with the linker(dT) primer. In the second round, the specific primer (Sp2) was used with the linker sequence alone. The use of the linker rather than linker(dT) primer in the second round appeared to reduce nonspecific amplification. After electrophoresis of the PCR product, two bands of approximately 400 and 600 bp were visible. Southern blot analysis with the NmU-specific probe (Spl) shows that both bands hybridize to the probe (data not shown). Both fragments were cloned into M13, and NmU-containing clones were identified by plaque lift hybridization with the NmU-specific probe Spl. Six clones containing the large fragment were sequenced: four had the same 5’-end, and the other two started 20 and 50 bases downstream. The full sequence of 707 bases is shown in Fig. 1 B. Two clones containing the 400-bp fragment were also sequenced and were found to start 190 bp downstream. One of these had a 30-bp deletion (398-427 in Fig. 1B). To investigate the nature of this deletion, a separate PCR
1539
amplification using the original template was carried out with specific primers Sp2 and Sp3 (47-65 in Fig. 1B). Nine clones were sequenced; none had the 30-bp deletion. Efforts have been made to rule out potential artifacts that may have arisen from obtaining the cDNA sequence from amplification of three overlapping regions. A further confirmatory PCR using a 5’-primer located in the 5’-untranslated region (5-20 in Fig. 1 B) and a 3’primer located at the end of the 3’-translated region (620-534 in Fig. 1 B) was carried out. A single band of the expected size was observed after a fraction of the PCR product was analyzed by electrophoresis and the subsequent Southern blotting analysis confirmed that this band hybridized to an NmU-specific probe (557707 in Fig. 1B) (data not shown). In addition, another PCR using primers (353-376 and 620-634 in Fig. 1 B) flanking the NmU coding sequence was performed to rule out the presence of repeated sequences encoding NmU. A single band of 282 bp was observed. Southern blot analysis of the PCR product showed that it hybridized to an NmU-specific probe (353-589 in Fig. 1 B). Northern
Blot Analysis
The size of the NmU message was estimated first from its relative position with ribosomal bands 28s and 18s as shown in Fig. 2A. In order to provide a more accurate size marker, the blot was stripped and rehybridized with a rat galanin cDNA probe (16) which detected an mRNA of slightly higher mobility than NmU mRNA as indicated by the arrows in Fig. 2, A and B. Rat galanin mRNA is 699 bases in length excluding the poly(A) tail (16). The expression of the NmU gene was studied by Northern blot analysis using total RNA extracted from different rat tissues. As shown in Fig. 28, high levels of NmU mRNA were observed in ileum, thyroid, and anterior pituitary. Lower levels of expression were observed in spinal cord, hypothalamus, and stomach. The tissue-specific expression of NmU is consistent with previous reports of NmU peptide distribution (7, 17). Genomic
Southern
Blot Analysis
Rat genomic DNA was digested with different restriction enzymes and then subjected to Southern blot analysis. Two probes were used, one representing bases 355-588 and the other bases 557-707 in Fig. 1B. A single band (6 kb) was detected in the HindIll digest (result not shown). This indicates that NmU is a single copy gene with no closely related sequences in the rat genome. Further experiments using less stringent hybridization conditions failed to detect related sequences (data not shown).
DISCUSSION The primary structure of NmU from several species including rat was known before this work commenced,
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MOL 1540
END0.1992
Vo16No.10
AAAA >
1
f---
NmU-S
NmU-A
-
-1 -
Linket(d;) -Linker -
1
SP2
SP2
AACACCCQCAC~~ACAQQQTACCTQQTTCCCAQTCCAQAQCTQCTTQQAQQQC
001 Qly
TCQ CQA QCA QCT MT 8.r Arg Ah Ala Asn
CAA TTQ QCC QCT QCA ACT 011% La Ala Ala Ala Thr
CTQ CT0 LaU L*U
5’ end PCR
c-
SD3
AQAQATQ Nmt
CQC CGC CCA Arg Arg Pro QCA
Ala
108
QQQ CTC
TCC
QCA
Qly
Ior
Ala
Lmu
TCT CCT CTC CTQ TCA Bmr Pro LOU La Sar
CTQ CTC QCC TQC TOT QCQ QAC QCT 4 TOE AQA LWI Lau Ala Cys Cys Ah Aq Ala Cya Arg
CCA
ATA
TCQ
Or0
110
Sar
CCT CM Pro Qln
AQA
Arg
TTQ TOO MT Lou Trp Am
QAQ ATT CCT 0181 Ile Pro
OTT OAT TCC Val Asp 8.r
Qln
CAQ CCT
Pro
CAQ
Qln
TTA LOU
CCA
CCA
Pro
PI0
QM Qlu
Ala
QCA
Ala
2,, .I 2,, 55
QCT TOT QCQ TCC TTT CT0 Cys Ala 8mr Ph LOU
TCT am
31, 69
(ITT QCA TTQ AQQ AA0 Val Ala LOU I-JAr
CTT L.ll
361 a3
TCT
QM Qlu
QQT ACQ Qly Thr
13 l,, 27
CM ah
Ser
QAA CM Qlu 0111
CT0 La
151
CTA
LOU
TQC COT OTC CTQ AT0 Gym Arg Val La Xat
QAG ATT Qlu Ilm
TTC CAQ AA0 Ph. Gin Lys
ACT QAQ AM Thr Qlu Lys
QCT AA0 Ala Lys
AQQ TTC T T A TTT CAT T A T TCA Arg Pho La Ph Him Tyr Ear
111
TCA MT Bar Asn
407 125
AAQ ACA Lys Thr
PCR
3’ end PCR
BamHl(dT)
SPl
Int*rnal
QAT MT Asp Asn
CAQ AA0 Qln Lyn
QTQ CAT CC0 Val Hia Pro
TTQ QQA MT La Qly Aan
OTT GTQ TCQ TCT OTC Val Val Sor 8er Val
TTQ CTQ CAQ CTC OTT CCT CM La Leu Qln Leu Val PrO Qln
AT0 MO AQA AC MA Ar Nat Lym Arg yr Lym zl 01 QCT CCA AQT QQQ QQA TTC *Pro Ior Gly Qly Ph. . TCA ACA AQT TTC Ier Thr Ia Pho
GTQ MT Val Aan
CCT CAQ QAG CM Pro Qln Qlu Qln
CTG CAT QAQ AQA Las Bin 01~ LAr
QAA TAC CAG Qlu Tyr Qln
TTT TTA TTT AGO CCA LOU PIi* Arg PrQ
Pho
,O, 97 446
529 139
QQT CCT QTA Qly Pro Val
571 153
CQC MT Arg Am
613 197
1
QQQ Qly
110
661 274
~ATQQQATATMAATQTACTCACATTPQTTCTC
707
ATT
TAAAATQQATQCCMATQATCTTTTCA
Fig. 1. Scheme for Amplification of Rat NmU cDNA and the Nucleotide and Predicted Amino Acid Sequences of the Rat NmU Precursor A, Initially the NmU coding sequence was amplified with the mixed sequence primers NmU-S and NmU-A. The 3’-end was amplified using the anchor primer BamHl (dT) with NmU-S and then primer Spl The 5’-end was amplified with the anchor primer, linker(dT) and NmU-A, then linker primer with Sp2. The sequence of the 5’-end was confirmed by sequencing nine clones obtained after amplifying with primers Sp3 and Sp2. All specific primers indicated had 6amHl or EcoRl sites at their 5’-ends. Anchor primers: linker(dT), GGGCGGCCGCAATACGACTCACTATAGGGAGA(T),,; linker, CGCGGATCCAATACGACTCACTATAGGG; BamHl (dT), GACGGGATCC(T),,,. B, Vertical arrow, potential signal peptide cleavage site; horizontal arrows, specific oligonucleotide primers; boxes, putative cleavage sites; brackets, NmU sequence. Nucleotides are numbered to the right of the figure. The numbering of amino acid residues appears below that of the nucleotide. The complete nucleotide sequence shown here has been submitted to the GenBank nucleotide database and assigned the accession number M94555.
but no cDNA sequence was available. Two possible approaches based on the screening of cDNA libraries were considered. The library could be screened with oligonucleotides (14) but preliminary experiments using these as probes to Northern blots were unsuccessful. Alternatively, an expression library could be screened
with an antibody, but it was not clear that available antibodies would recognize epitopes expressed in fusion proteins. Generation of a short portion of NmU cDNA by mixed primer PCR method was considered. This technique has been used for the generation of probes (18, 19). The NmU sequence allowed the selec-
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Characterization
of NmU-Encoding
cDNA
12
1541
3
4
5
6
7
8
11
910
12
13
14
15
A
28-
18-
Fig. 2. Northern
Blot Analysis of Rat RNA A, Thirty micrograms of total rat duodenal RNA with 5 pg ethidium bromide were analyzed RNA was visualized under UV light, and the mobilities of 18s and 28s rRNA were noted. The NmU-specific cDNA probe (297-588 in Fig. 1 B). After autoradiography the blot was stripped probe. The origin is at the top of the figure, and the positions of 28s and 18s rRNA and rat B, Twenty micrograms of total RNA from various rat tissues were analyzed as in A but with The origin is at the top of the figure, and the position of rat galanin mRNA is indicated. Lanes: 5, lung; 6, liver; 7, pancreas; 8, stomach; 9, spleen; 10, uterus; 1 I, brain; 12, hypothalamus; spinal cord. Exposure was for 5 days.
tion of primers fold)-primers
of limited degeneracy (64- and 128with as much as 16,384-fold degeneracy
have been used successfully (20). However, there are only 28 bases between these primers, and this may be close to the lower limit for this technique. The amplified product was only partially resolved from primer dimers, and the sequence determined was only just sufficient for the construction of the specific primers Spl and sp2. The rat NmU cDNA sequence shown in Fig. 16 is 707 bases in length excluding the poly(A) tail. This is likely to represent the full cDNA sequence, as four of the clones have the same 5’-end, and Northern blot analysis indicated that NmU mRNA was very similar in size to rat galanin mRNA [699 bases + poly(A)]. The transcriptional initiation codon was assigned to the most upstream methionine codon, since this is followed by a signal peptide sequence and an open reading frame of 174 codons. The most likely cleavage site of the signal peptide, as predicted by a weight-matrix method (21) is the alanine residue at position 37. If this is indeed the cleavage site in vivo, the signal peptide of NmU has an unusually long 23-residue N-terminal region followed by a typical hydrophobic core region and a more polar C-terminal region. The length of the Nterminal region of a signal peptide can be quite variable
on a formaldehyde/agarose gel. The gel was blotted and probed with an and reprobed with rat galanin cDNA galanin mRNA (arrow) are indicated. no ethidium bromide in the samples. 1, heart; 2, ileum; 3, ileum; 4, kidney; 13, anterior pituitary; 14, thyroid; 15,
(22). One experiment showed that attaching 18 extra residues to the N-terminus of a cloned insulin gene, making the N-terminal region 21 in total, had no effect on its export (23). The predicted mature precursor protein contains four paired dibasic amino acids. The last two flank the NmU peptide and represent sites for proteolytic cleavage releasing the NmU peptide. It is possible that similar cleavage
at the
other
sites
releases
other
active
pep-
tides. These predicted peptides show no significant homology with entries in the NBRF-PIR or SWISS-PRO protein databases, and the cDNA similarly showed no homology with entries in the GenBank database. In the course of amplifying the 5’-end cDNA of NmU, two fragments approximately 400 and 600 bp were obtained. One of the clones, containing the shorter fragment, had a 30-bp deletion. An attempt was made to investigate the nature of this deletion. This region was amplified using specific primers (Sp3 and Sp2), and nine clones were sequenced. However, none contained the deletion, and it seems most likely that this was generated during the course of PCR or cloning procedures. Similarly, the short species seen after amplification of the 5’-end most probably does not represent an mRNA. No such species has been detected on Northern blots, and there are no suitable methionine codons in this region.
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MOL 1542
ENDO.
Vo16No.10
We were aware of two potential problems with the approach used to obtain rat NmU cDNA sequence. First, there might be tandem repeats of NmU as, for instance, is the case with the TRH coding sequence (24). An amplification using a primer pair flanking the NmU coding sequence was carried out. Only a band of the size predicted from the sequence in Fig. 1B was observed demonstrating the absence of tandem repeats. Second, PCR might join separate molecules together, and we have indeed found that the anchor strategy for 5’-end can do this (Howard, A., unpublished observation). Another confirmatory PCR was performed using a 5’-primer in the 5’-untranslated region of NmU cDNA and a 3’-primer downstream from the NmU coding sequence. The result showed that the PCR product was again of the expected size. We therefore conclude that the cDNA sequence presented in Fig. IB represents the mRNA sequence coding for NmU. The Northern blot analysis of different rat tissues (Fig. 2B) generally corresponds with the distribution established by immunological techniques (7, 17, 25). Of particular significance is the failure to detect expression in the uterus. The uterus is the most sensitive tissue to exogenous NmU and yet contains no detectable mRNA and only small amounts of the peptide (7). There are a number of possible explanations for this apparent discrepancy. Although a paracrine role for NmU is generally assumed, an endocrine role cannot be excluded, implying synthesis elsewhere in the body. Alternatively, if NmU in the uterus is released from nerve terminals, the mRNA might again be found elsewhere. Finally, the pharmacological activity seen in the uterus might represent cross-reactivity of NmU with the receptor for some other related peptide. Although this is a possibility, both low stringency genomic Southern blot and Northern blot of uterine mRNA have thus far failed to reveal the presence of any NmU-related sequences. We are interested in the possibility that the smooth muscle contracting activity of NmU might have clinical applications. However, the sequence of human NmU is not known, so it is our intention to clone and sequence the corresponding cDNA. Our recent studies have shown that the rat NmU cDNA probe can detect an mRNA of similar size in human colon and pituitary. We are currently characterizing cDNA clones which could code for the human NmU precursor. In summary, we have used the anchor PCR strategy to characterize cDNA encoding the precursor of NmU. This sequence reveals the presence of NmU-associated peptides at the N-terminal side of NmU. We have demonstrated the expression of NmU in rat anterior pituitary, thyroid, and ileum, and have shown that there is only a single copy of the NmU gene.
MATERIALS
AND
METHODS
RNA Preparation
All tissues were collected from female Wistar rats (Charles Rivers
Breeding
Laboratories,
Margate,
Kent,
UK).
Tissues
were frozen in liquid nitrogen immediately after excision and stored at -70 C. Total RNA was extracted by a single step acid guanidinium thiocyanate-phenol-chloroform extraction method (26). Poly(A)+ RNA was isolated by oligo(dT) cellulose column chromatography (27). Reverse
Transcription
One microgram of rat ileum poly(A)+ RNA in 8 ~1 water was heated to 65 C for 10 min, chilled on ice, and added to 12 ~1 reaction buffer containing 25 mM Tris-HCI, pH 8.3,6 mM MgCIP, 40 mM KCI, 1 mM dithiothreitol, 1 wg oligo(dT)12-,8 or 600 ng random hexamers, and 2 mM of each dNTP. Fifty units of avian myeloblastosis virus reverse transcriptase (Anglian Biotechnology Ltd., Colchester, Essex, UK) and 25 U human placental ribonuclease inhibitor (Amersham, Buckinghamshire, UK) were added, and the reaction was incubated for 1 h at 42 C. .The reaction was then diluted to 0.1 ml with a buffer containina 10 mM Tris-HCI. DH 8.3. 50 mM KCI. 1.5 mM MaCI,. and O.OOi% gelatin. Excess primers in the reierse tran&ip: tion reaction were then removed with a Sephacryl S-200 spun column (Pharmacia, Milton Keynes, UK) following the manufacturer’s instruction. Amplification
of the Coding
Region
This was carried out with one-fifth of the reverse transcripts and 2.5 pg of each mixed primer (NmU-S and NmU-A) in a 50~1 reaction containing 10 mM Tris-HCI, pH 8.3, 50 mM KCI, 1.5 mM MgCI*, 0.001% gelatin, and 0.2 mM of each dNTP. After heating at 95 C for 10 min, 1 ~1 (2.5 U) Taq DNA polymerase (Perkin Elmer-Cetus, Norwalk, CT) was added to the reaction. The mixture was overlaid with mineral oil and subjected to 30 cycles of amplification in a DNA cycler (94 C for 1 min, 45 C for 2 min, and 72 C for 2 min, followed by a final extension at 72 C for 10 min). Amplification
of 3’-End
NmU
cDNA
Five micrograms of rat ileum poly(A)+ RNA were reverse transcribed with 1 wg BamHl(dT) primer as above, alkali hydrolyzed in 0.1 M NaOH at 65 C for 20 min, and then neutralized in 1 M Tris-HCI, pH 7.5. The cDNA was precipitated with 2 vol ethanol using 2 pg tRNA as carrier. It was then resuspended in 20 ~1 water and stored at -20 C. Two rounds of amplification were necessary to give sufficient specificity. The first round was essentially as described for the coding region, except substituting 2 ~1 of the alkali-treated cDNA for the template and 100 ng 6amHl (dT) primer for NmU-A. PCR parameters were 10 cycles of 94 C for 1 min, 40 C for 1 min,
and 72 C for 2.5 min. The PCR reaction was diluted 1OO-fold with water, and one-tenth was then used in the second round PCR reaction, which was as described except 100 ng Spl primer was used instead of primer NmU-S. Amplification
of 5’-End
NmU cDNA
Addition of adenosine homopolymer to the random-primed cDNA was as described by Frohman et al. (15). The tailed cDNA was ethanol precipitated and then resuspended in 50 ~1 water. Two rounds of PCR were necessary to amplify the NmU cDNA specifically. For the first round, 5 ~1 tailed cDNA, 300 ng mixed primer NmU-A, and 100 ng anchor primer linker(dT) were used in a standard 50-~1 PCR reaction. PCR parameters were 10 cycles of 94 C for 40 set, 40 C for 1 min, and 72 C for 2 min. Second round amplification was carried out with one-tenth of the first round PCR product, primers Sp2 and the linker sequence (100 ng). PCR parameters were 30 cycles of 94 C for 40 set, 50 C for 1 min, and 72 C for 2.5 min.
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Characterization
of NmU-Encoding
Oligonucleotide
Hybridization
cDNA
PCR products were separated by electrophoresis, denatured, neutralized, blotted overnight onto Hybond-N membrane (Amersham) (28) and baked at 80 C for 2 h. Oligonucleotide probes were 3’-end labeled with [32P]dCTP by terminal deoxynucleotidyl transferase to a specific activity of 2-5 x 10’ cpm/pg and were used for hybridization at a concentration of 2 no/ml. Hybridizations were carried out in 5x SSC (1 x SSC = O?l5 NaCI, 0.015 Na citrate, pH 7.0)/20 mM Na phosphate, DH 8.8/0.1% sodium dodecvl sulfate (SDSV5x Denhardt’s. and lob fig/ml salmon sperm DNA (28) at 42 C for 2 h. The filters were washed in 5x SSC/O.l% SDS/10 mM Na phosphate, pH 6.8, twice for 30 min at 42 C and hybridized with the internal probe [AGGAAGAAGCCGCCG(GC)(AT)(TG)GGGGCCACAGGGCCCTGGTACTC], then given a final wash in 1 x SSC/O.l% SDS at 42 C for 30 min. Cloning
and Sequencing
of NmU cDNA
After phenol/chloroform extraction and ethanol precipitation the PCR products were digested with restriction endonucleases EcoRl and BamHl and separated by electrophoresis. Regions of the gel containing the specific products were excised, electroeluted, and cloned into an Ml3 or Bluescript plasmid vector. Ml3 recombinants containing NmU cDNA were identified by plaque lift hybridization with a labeled NmUspecific probe. Plasmid clones were characterized by restriction analysis of DNA which was prepared by the boiling method described by Del Sal et al. (29). The recombinant clones were sequenced with Sequenase (U.S. Biochemicals, Cleveland, OH), using the supplier’s recommendation. Multiple isolates were examined to guard against PCR error. Northern
Blot Analysis
After electrophoresis in formaldehyde/l % agarose gel (28) at 80 V for about 2 h the RNA was transferred to Hybond-N membrane (Amersham) and hybridized as described by Reid et al. (30) with a labeled NmU-specific antisense DNA probe. The probe was the 230-bp EcoRl/Hindlll restriction fragment of 5’-NmU cDNA labeled with [3ZP]dCTP as for a randomprimed synthesis (31) but using Sp2 instead of random primers. The specific activity was 6 x lo9 cpm/pg. After hybridization the blot was washed at 60 C for 30 min in a buffer containing 0.25 M Na phosphate, pH 7.2/l mM EDTA/ 2% SDS and finallv washed at 60 C for 30 min in 0.2x SSPE (30 mM NaCI/1.8’m~ NaH2P04/0.2 mM EDTA)/O.l% SDS. After autoradiography, the blot was stripped in.10 mM TrisHCI. DH 7.5/0.1 mM EDTA/0.5% SDS at 80 C for 15 min before reprobing with rat gaianin cDNA Genomic
Southern
Blot Analysis
Rat genomic DNA was prepared from frozen rat brain tissue using a modified method described previously (24). The DNA was digested with restriction endonucleases (Pvull, fstl, and HindIll). After electrophoresis in a 0.7% agarose gel, the DNA was transferred to Hybond-N membranes. The 5’-probe was as for Northern blotting. The other probe was the insert from the clone of the 3’-end, labeled using BamHl(dT) and Spl as primers. Both probes had a specific activity of 2-6 x 10’ cpm/ wg. The hybridization and washing conditions were as described for the Northern blotting. Note
Added
in Proof
Since the submission of this paper, we have discovered that there is an additional 128 bases of untranslated sequence downstream from the cDNA sequence shown in Fig. 1 B. This sequence contains the concensus poly(A) adenylation signal
1543
(AATAAAA) sequence accession
preceding the poly(A) tail by 17 bases. has been deposited in GenBank under number.
The extra the same
Received March 2, 1992. Revision received June 8, 1992. Accepted June 12, 1992. Address requests for reprints to: Prof. S. R. Bloom, Department of Medicine, Second Floor, Francis Fraser Lab, Royal Postgraduate Medical School, Du Cane Road, London W12 ONN, United Kingdom.
REFERENCES 1. Minamino N, Kangawa K, Matsuo H 1985 Neuromedin U8 and U-25: novel uterus stimulating and hypertensive peptides identified in porcine spinal cord. Biochem Biophys Res Commun 130:1078-l 085 2. Conlon JM, Domin J, Thim L, Dimarzo V, Morris HR, Bloom SR 1988 Primary structure of neuromedin U from the rat. J Neurochem 51:988-991 3. Minamino N, Kangawa K, Honzawa M, Matsuo H 1988 Isolation and structural determination of rat neuromedin U. Biochem Biophys Res Commun 156:355-360 4. Domin J, Yiangou Y, Spokes RA, Aitken A, Parmar KB, Chrysanthou BJ, Bloom SR 1989 The distribution and pharmacological action of an amphibian neuromedin U. J Biol Chem 264:20881-20885 5. O’Harte F, Bockman CS, Abel PW, Conlon JM 1991 Isolation, structural characterization and pharmacological activity of dog neuromedin U. Peptides 12: 1 l-l 5 F, Thim L, Conlon JM 1991 Rabbit 6. Kage R, O’Harte neuromedin U-25: lack of conservation of a posttranslational processing site. Regul Peptides 33:191-l 98 7. Domin J, Ghatei MA, Chohan P, Bloom SR 1987 Neuromedin U-a study of its distribution in the rat. Peptides 81779-784 8. Maggi CA, Patacchini R, Giuliani S, Turini D, Barbanti G, Rover0 P, Meli A 1990 Motor response of the human isolated small intestine and urinary bladder to porcine neuromedin U-8. Br J Pharmacol 99186-l 88 9. Benito-Orfila MA. Domin J. Nandha KA. Bloom SR 1991 The motor effect’of neuromedin U on rat stomach in vitro. Eur J Pharmacol 193:329-333 10. Gardiner SM, Compton AM, Bennett T, Domin J, Bloom SR 1990 Reqional hemodvnamic effects of neuromedin U in conscious-rat. Am J Physiol 258:R32-R38 11. Sumi S, lnoue K, Kogire M, Doi R, Takaori K, Suzuki T, Yajima H, Tobe T 1987 Effect of synthetic neuromedin U8 and U-25 novel peptides identified in porcine spinal cord on solanchnic circulation in doas. Life Sci 41 :1585-l 590 CS, Abel PM, Hicks JW, Conlon JM 1989 12. Bockman Evidence that neuromedin U may regulate gut motility in reptiles but not in mammals. Eur J Pharmacol 171:255257 DR, Quito FL 1988 Neuromedin U octapeptide 13. Brown alters ion transport in porcine jejunum. Eur J Pharmacol 155:159-162 14. Lathe R 1985 Synthetic oligonucleotide probes deduced from amino acid sequence data: theoretical and practical considerations. J Mol Biol 183:1-12 15. Frohman MA, Dush MK, Martin GR 1988 Rapid production of full-length cDNAs from rare transcripts: amplification using a single gene-specific oligonucleotide primer. Proc Natl Acad Sci USA 85:8998-9002 16. Kaplan LM, Spindel ER, lsselbacher KJ, Chin WW 1988 Tissue-specific expression of the rat galanin gene. Proc Natl Acad Sci USA 85:1065-l 069 17. Domin J, Steel JH, Adolphus N, Burrin JM, Leonhardt U, Polak JM 1989 The anterior pituitary content of neuromedin U-like immunoreactivity is altered by thyrotrophinreleasing hormone and thyroid hormone status in the rat. J Endocrinol 122:471-476
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MOL 1544
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1992
18. Lee CC, Wu X, Gibbs RA, Cook RG, Muzny DM, Caskey CT 1988 Generation of cDNA probes directed by amino acid sequence: cloning of urate oxidase. Science 239:1288-l 291 19. Girgis SI, Alevizaki M, Denny P, Ferrier GJM, Legon S 1988 Generation of DNA probes of peptides with highly degenerate codons using mixed primer PCR. Nucleic Acids Res 16:10371 20. Kopin AS, Wheeler MB, Leiter AB 1990 Secretin: structure of the precursor and tissue distribution of mRNA. Proc Nat1 Acad Sci USA 87:2299-2303 21. von Heijne G 1986 A new method for predicting signal sequence cleavage sites. Nucleic Acids Res 14:46834690 22. von Heijne G 1985 Signal sequences: the limits of variation. J Mol Biol 184:99-l 05 23. Talmadge K, Brosius J, Gilbert W 1981 An “internal” signal sequence directs secretion and processing of proinsulin in bacteria. Nature 294:176-l 78 24. Lechan RM, Wu P, Jackson IMD, Wolf H, Cooperman S, Mandel G, Goodman RH 1986 Thyrotropin-releasing hormone precursor: characterization in rat brain. Science 231 :159-l 61
Gregory
Pincus
Memorial
25.
26.
27.
28.
29.
30.
31.
Steel JH, Van Noorden S, Ballesta J, Gibson SJ, Ghatei MA, Burrin J, Leonhardt U, Domin J, Bloom SR, Polak JM 1988 Localization of 7B2, neuromedin B, and neuromedin U in specific ceil types of rat, mouse, and human pituitary and extrapituitary tumors. Endocrinology 122:270-282 Chomczynski P, Sacchi N 1987 Single-step method of RNA isolation by acid guanidinium thiocyanate-phenolchloroform extraction. Anal Biochem 162:156-l 59 Aviv J, Leder P 1972 Purification of biologically active globin mRNA by chromatography on oligothymidylic acid cellulose. Proc Natl Acad Sci USA 69:1408-l 412 Sambrook J, Fritsch EF, Maniatis T 1989 Molecular cloning-A laboratory manual, ed 2. Cold Spring Harbor Laboratory, Cold Spring Harbor Del Sal G, Manfioletti G, Schneider C 1988 A one-tube plasmid DNA mini-preparation suitable for sequencing. Nucleic Acids Res 16:9878 Reid RA, John MC, Amasino RA 1988 Deoxyribonuclease I sensitivity of the T-DNA ipt gene is associated with gene expression. Biochemistry 27:5748-5754 Feinberg AP, Vogelstein B 1983 Addendum: a technique for radiolabelling DNA restriction endonuclease fragments to high specific activity. Anal Biochem 137:266-267
Lecture
and Award
The 1992 Gregory Pincus Memorial Lecture will be given jointly by Dr. Shutsung Liao and Dr. Jean D. Wilson on Thursday, November 5, 1992, in the Hoagland-Pincus Center at The Worcester Foundation for Experimental Biology, Shrewsbury, Massachusetts. These scientists, who will receive the Gregory Pincus Medal and Award, will deliver lectures concerning their pioneering work in the androgen field. For further information, please contact: Chairman, mittee, Worcester Foundation for Experimental Massachusetts 01545, Telephone: (508) 842-8921
Gregory Pincus Memorial Lecture ComBiology, 222 Maple Avenue, Shrewsbury, ext. 131, Fax: (508) 842-9632.
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