Biochbnica et Biophysica Acta, 1129(1992) 235-238 © 1992 Elsevier Science Publishers B.V. All rights reserved 0167-4781/92/$05.00
BBAEXP 90301
235
Short Sequence-Paper
Molecular cloning of a cDNA encoding a novel protein related to the neuronal vesicle protein synaptophysin C h i z e n g Z h o n g , D a v i d J. H a y z e r a n d M a r s c h a l l S. R u n g e Dicision of Cardiology', Drawer LL, Emory School of Medicine Atlanta, GA (U.S.A.) (Received 14 October 1991)
Key words: Synaptophysin; Membrane protein: Nucleotide sequenct
The structure of synaptophysin, an integral membrane protein present in synaptic vesicles, is highly conserved. We report the sequence analysis of a clone, HL.5, isolated from a human.erythroleukemia cell cDNA library, that is similar to synaptophysin in DNA and amino acid sequence. The predicted protein product derived from this clone is truncated, and the tissue distribution of HL-$ is different from that of synaptophysin. Thus, HL-5 appears to be a member of a previously undescribed family of synaptophysin-like genes. Synaptophysin is a major integral membrane protein of synaptic vesicles [l]. The distribution of synaptophysin, as determined by Northern blot analysis, is limited to neural tissues, the adrenal medulla, neuroendocrine cells and neoplasms of neural origin. Based on analysis of its deduced amino acid structure, it appears that the protein consists of four intramembrane domains, with amino and carboxy-terminii that are located in the cytoplasm. The regions between intramembrane domains l - l l and I l l - I V have been proposed to lie within the lumen of the synaptic vesicle. The carboxy-terminus is tyrosinc and prolinc-rich [2] (Fig. 2), and has the potential to be extensively phosphorylated [3]. The structure of synaptophysin is highly conserved in three divergent species, rat [2], human [4,5] and cow [6] (97% similarity at the protein level [2,4] (Fig. 2). It has been suggested that synaptophysin plays a role in either membrane movement or maintainance of the integrity of the membrane [6]. During the course of the screening of a eDNA library for G-protein-coupled receptor-encoding clones, we have isolated a clone with a significant level of sequence similarity to synaptophysin. Cultured rat aortic smooth muscle (RASM) cells, grown to passage 4 [7], were the source of mRNA used in the synthesis of first strand eDNA by reverse transcriptase. The polymerase chain reaction (PCR) was employed to amplify the eDNA with degenerate primers derived from consensus sequences of the domains III and Vi of G-coupled receptors [8]. The PCR
Correspondence: M.S. Runge, Empty School of Medicine, Division of Cardiology, P.O. Drawer LL, Atlanta GA 30322, U.S.A.
products were cloned into the vector Bluescript KS using EcoR! and BamHl :~ites present at the 5' exiremi~;es of the primers, and screened by dideoxy sequentmg. One clone, designated rat5, is 0.4 kbp in size, and based on sequence analysis falls into the famil .' of G-protein coupled receptors (data not shown). This t lone was used as a probe to screen a human ery~hropoeitic leukemia (HEL) cDNA library construc~ed in the A vector gti 1. Low stringency washing conditions (42°C, 5 min, 1 × SSC) (1 × SSC = 0.15 M NaCI and (I.(115 M sodium citrate) yielded one clone, designated HL-5, at a frequency of 34 per 1(15 pfu. The 840 nuclcotides of the cloned DNA HL-5 (Fig. 1) includes an open reading frame, of 447 nucleotides encoding a protein of 149 residues (M, = 16 597). There is extensive alignment between the nucleotide (Fig. 1), and the predicted amino acid (Fig. 2), sequences of HL-5 and the corresponding sequences of synaptophysin of rat, human or the cow, with 54% similarity between the HL-5 and synaptophysin nucleotide sequences, and 44% at the amino level. The predicted amino acid sequence of HL-5 has two uninterrupted regions analogous to the putative intramembrane domains I and 11 previously identified in synaptophysin [6]. However, HL-5 appears to terminate prematurely in the third intermembrane domain, due to the loss of a single base. The HL-5 nucleotide and predicted amino acid sequences downstream of the TERM codon, after nucleotide position 450-452, continue the similarities with the synaptophysin sequences and. therefore. it is possible that the single base deletion was a cloning artifact. The interdomain regions are much less conserved between HL-5 and synaptophysin than are the transmembrane domains themselves; most notably, a
236 stretch between domains 1 and I1 displays no evident alignments. HL-5 appears to lack the cytoplasmic tail immediately following the intramembrane domain IV.
10
20
30
40
SO
The nucleotide sequence following the potential TERM codon of HL-5 at nucleotide position 743-745, does not encode tyrosine or proline-rich stretches, in any of
60 T...HLo5
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70
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GTGACCAGTA~TCCCT/tJU51fv, B T . . ,,HL-5
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130
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170
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....
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610
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190
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. . .RA?
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. . . Ht,HHINH
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-CT-C
570
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690
700
710
720
HI,* S
G4AGGAGA¢¢AG¢¢T&G4¢AGT¢¢ATCA~TAeA~TAG¢¢AAGGAGGTAT...
~50
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HUH~i
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300
(AJ~AGGGGGCAC'I'&e.'m.RA~,'L%'I'T¢)
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C--G--A--O
..........
AG'.-TGGGeAO~C----TC-TGCG¢
--A----CAG--,GCCCCG-AA-A...RAT
T........
A G - -'IL'-- -v - ' ~ -
-o~---¢¢G-~-AG-A...HUM~
730
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-G-'JL'~
750
760
770
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.... .....
G-A---&GC-o-To~'Go-GCC--CG-CGo'JL~'GC~-TACoGGCAGG-CCCCG-AG-¢--.,. t~T
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CT&TGACATG'ITGCCAACA~TTATTTG'ITTCTAATA/UUWTAATGGCTT...HLoS TOGG~oCAAGACT-CT&COGGCCt'C-G-G'rI~'To&-¢AAC-CG- * T - C G - G C - G - - A G e . . . RAT -GGGCC,..¢A-GATT-CTACGGGCCT¢-G-GCGGC-&CCCAG*CTGACT-T-GTCAAC-AG... HUMAN
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310
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390
400
410
4~tO llSO
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.....
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CTTTGTTGT...HL-S
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HUMAN
.,.
AGoGG---CGG-GGCT&CGGGCCTC&.~XGACTATGGGCAGCAJ~T.GCTATGGCCA...RAT
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....
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.....
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ACAGGGTGCGCCCACCTCCTt'CTCCAATCAGkTGTAA
..........................
RAT
GCAGGGTGCACCCACCTC¢TTCTCCAATCAGkTGTAG
..........................
HUMAN
oI~----GC---T-...RAT
C°-°GCC--GT'I'°G-C--CA------¢-A--T---T-&-oG--A..-GC¢..G-GG...T....
HUMAN
Fig. I. Alignments of the nucleotide sequences encoding the rat [2] and human [4,5] synaptophysins, with the nucleotide sequence of the HL-5 eDNA. Gaps were introduced to maximize the similarities. No evident alignments were found between nucleotide positions 241-271 inclusive. lntramembrane domains, as defined by Johnston et al. [6] are indicated by the lines above the sequences. Asterisks indicate in-frame termination codons. 3' untranslated regions of the rat and human sequences are not presented.
237 the possible reading frames, analogous to those found in synaptophysin. HL-5 was used as a probe to investigate the gene expression product in the HEL and RASM cell lines by Northern blot analysis (Fig. 3a). A single mRNA species, of 2.5 kb, was found in both cell types. This contrasts with the~ synaptophysin gen¢ which has been reported to be expressed in a variety of neuronal tissues [1-4,6], and undetectable in other tissues of non-neuronal origin, such as the kidney and liver [6], and cultured rat smooth muscle cells [6]. In Southern blot hybridization analysis of human DNA (wash conditions: 0.5 x SSC, 50°C, 1 h), probe HL-5 detected a band of approx. 23 kbp, after digestion with Hindlil, Bglll, EcoRl or Pvull. With a high stringency wash (0.2 × SSC, 60°C, I h), only the 8.0 kbp Eco RI and the 4.5 kbp Pvull fragments were retained. With rat genomic DNA, fewer bands were found. Fig. 3b shows only a 4.7 kb EcoRl fragment, a Pv, ll 3.2 kb band, and a 2.5 kb band. The multiple banding pattern of some of the digests suggests that the HL-5 probe detects members of a multi-gene family.
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E Z Q V N C P F A V T E N K T V T A T F . L R L S - E - A N K T E S A L N I E V E L O L S - D - A N K T E S D L S I E V E L Q L S - D - A N K T K S D L N I E V E - .
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S S . . . . . .
AQ
F YVT F A V E - F - - V - - - A E - F - - V - E - F - - V - -
F
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L Y - - S - - -
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L L - A T - A T - k T
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T,.HL-5 Q,.RAT (]..HUMAN Q..BOVINE
-
V I I I
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O F V V T L V A T F C G
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~ K A L T D Z K AGS VD A - G - S VD A - G - S V D -
FGS
I
VT
-
L A - k L A - A L A - k
I A T G H N M- D P £ M - - D P E H - - D P E -
S HG
E L R D P - - - G E L R D P - - - G E L R O P - - -
-
G
Z -
I -
D K K K
-
Y ............. HL-5 M W r V S T S A W, .HL-5 I
-
F A F A F A -
HW L HWL H W L
-
- S -S - S
- -- -
E -
L P MM H -
C -
K T T T
A G G G
L C ¥..HI,,-5" T - K..RAT T - K..HUHAN T - K..BOVINE
P N V H
K g g g
K Q Q O
V N N N
kbp
A
-23.o
RASM HEL kb 2.5-
-9.4 .......
....
-6,6
-4.4
-2,3 -2.0
5 T ~ N I N E
ZZ O . . N
HBEP
Fig. 3. (A) Northern analysis of poly-adenylated mRNA from RASM and HEL cells. 5 #g of each was denatured with glyoxal [9], analysed on a 1.4% agarose gel and transferred to a nylon filter. The filter was washed with lxSSC, 50°C, 2 x 3 0 min. (B) Southern analysis of human DNA. 10 #g of DNA was digested by Hindlll (H), Bglll (B), EcoR! (E), or Pc, ll (P) and analysed on a 1.0C~- agarose gel, and transferred to a nylon filter. The filter was washed with 0.5 × SSC, 50°C, 1 h. ,~DNA-Hmdlll size markers are indicated.
X Q G R S G F Q I N - L V A - G - F R - F - L V A - G - F R
B
-,.RAT -..HUMAN -, .BOVINE
In summary, the gene encoding the mRNA from which the HL-5 eDNA clone was derived is related to a gene encoding the previously described protein synaptophysin, may be a member of a family of membranebound proteins with a much wider tissue distribution than found with synaptophysin itself. We thank Mark E. Rothenburg for expert technical assistance. This work was supported in part by NIH grants HLA4307 and HL02414. References
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v V
. . . . . . . . . . . . . . .
L V - - V - L V - V L V - - V -
-
L-..RAT L -..ltUHAH L -..BOVINE
F V Y K E T S L H S P S N T S A P H S Q G G Z P P L P E Y H .... - - F - - - G N A A - F N R - - P G A P E K Q - A - G D A Y . . ~ T
- - F - - - G N A A - F L R - - F - - - G H A A - F L R
HL-5*
--PGAPEKQ-A-GDAY..HUI'IAN - - P G A P E K Q - A - G D A Y . . ~ V I N E
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rsHOs..Bovz.g
Fig. 2. A i i g n m e n t s o f t h e t r a n s l a t i o n p r o d u c t s o f t h e s e q u e n c e s i n Fig. l, togetherwiththatofbovinesynaptophysin[6].
1 Wiedenmann, B. and Franke, W.W. (1985) Cell 41. 1017-1028. 2 Leube, R.E., Kaiser, P., Seiter. A., Zimbelmann, R., Franke, W.W., Rehm, H., Knaus, P., Prior, P., kietz. 1t, Reinke. H., Beyreuther, K. and Wiedenmann. B. (19E7) EMBO J. t,. 32613268. 3 Pang, D.T., Wang, J.K.T., Valtorta, F., Benfenati, F. and Greengard, P. (1988) Proc. Natl. Acad. Sci. USA 85. 762-'/6¢~. 4 Sudhof, T.C., Lottspeich, F., Greengard. P.. Mehl. E. and Jahn, R. (1987) Science 238, 1142-1144. 5 Sudhof, T.C., Lottspeich, F., Greengard. P., Mehl. E. and Jalm. R, (1987) Nucleic Acids, Res. 15, 9607. 6 Johnston, P.A., Jahn, R. and Sudhof, T.C. (1989) J. Biol. Chem. 264, 1268-1273.
238 7 Gunther, S., Alexander, R.W., Atkinson, W.J. and Gimbrone, M.A. Jr (1982) J. Cell. Biol. 92, 289-298. 8 Libert, F., Parmentier, M., Lefort, A., Dinsart, C., Van Sande, J., Meamhaut, C., Simons, M-J., Dumont, J,E. and Vassart, G. (1989) Science 244, 569-572.
9 Maniatis, T., Fritsch, E.F. and Sambrook, J. (1982) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor. 10 Bargou, R.C.E.F. and Leube, R.E. (1991) Gene 99, 197-204.
BBAEXP 90301
235
Short Sequence-Paper
Molecular cloning of a cDNA encoding a novel protein related to the neuronal vesicle protein synaptophysin C h i z e n g Z h o n g , D a v i d J. H a y z e r a n d M a r s c h a l l S. R u n g e Dicision of Cardiology', Drawer LL, Emory School of Medicine Atlanta, GA (U.S.A.) (Received 14 October 1991)
Key words: Synaptophysin; Membrane protein: Nucleotide sequenct
The structure of synaptophysin, an integral membrane protein present in synaptic vesicles, is highly conserved. We report the sequence analysis of a clone, HL.5, isolated from a human.erythroleukemia cell cDNA library, that is similar to synaptophysin in DNA and amino acid sequence. The predicted protein product derived from this clone is truncated, and the tissue distribution of HL-$ is different from that of synaptophysin. Thus, HL-5 appears to be a member of a previously undescribed family of synaptophysin-like genes. Synaptophysin is a major integral membrane protein of synaptic vesicles [l]. The distribution of synaptophysin, as determined by Northern blot analysis, is limited to neural tissues, the adrenal medulla, neuroendocrine cells and neoplasms of neural origin. Based on analysis of its deduced amino acid structure, it appears that the protein consists of four intramembrane domains, with amino and carboxy-terminii that are located in the cytoplasm. The regions between intramembrane domains l - l l and I l l - I V have been proposed to lie within the lumen of the synaptic vesicle. The carboxy-terminus is tyrosinc and prolinc-rich [2] (Fig. 2), and has the potential to be extensively phosphorylated [3]. The structure of synaptophysin is highly conserved in three divergent species, rat [2], human [4,5] and cow [6] (97% similarity at the protein level [2,4] (Fig. 2). It has been suggested that synaptophysin plays a role in either membrane movement or maintainance of the integrity of the membrane [6]. During the course of the screening of a eDNA library for G-protein-coupled receptor-encoding clones, we have isolated a clone with a significant level of sequence similarity to synaptophysin. Cultured rat aortic smooth muscle (RASM) cells, grown to passage 4 [7], were the source of mRNA used in the synthesis of first strand eDNA by reverse transcriptase. The polymerase chain reaction (PCR) was employed to amplify the eDNA with degenerate primers derived from consensus sequences of the domains III and Vi of G-coupled receptors [8]. The PCR
Correspondence: M.S. Runge, Empty School of Medicine, Division of Cardiology, P.O. Drawer LL, Atlanta GA 30322, U.S.A.
products were cloned into the vector Bluescript KS using EcoR! and BamHl :~ites present at the 5' exiremi~;es of the primers, and screened by dideoxy sequentmg. One clone, designated rat5, is 0.4 kbp in size, and based on sequence analysis falls into the famil .' of G-protein coupled receptors (data not shown). This t lone was used as a probe to screen a human ery~hropoeitic leukemia (HEL) cDNA library construc~ed in the A vector gti 1. Low stringency washing conditions (42°C, 5 min, 1 × SSC) (1 × SSC = 0.15 M NaCI and (I.(115 M sodium citrate) yielded one clone, designated HL-5, at a frequency of 34 per 1(15 pfu. The 840 nuclcotides of the cloned DNA HL-5 (Fig. 1) includes an open reading frame, of 447 nucleotides encoding a protein of 149 residues (M, = 16 597). There is extensive alignment between the nucleotide (Fig. 1), and the predicted amino acid (Fig. 2), sequences of HL-5 and the corresponding sequences of synaptophysin of rat, human or the cow, with 54% similarity between the HL-5 and synaptophysin nucleotide sequences, and 44% at the amino level. The predicted amino acid sequence of HL-5 has two uninterrupted regions analogous to the putative intramembrane domains I and 11 previously identified in synaptophysin [6]. However, HL-5 appears to terminate prematurely in the third intermembrane domain, due to the loss of a single base. The HL-5 nucleotide and predicted amino acid sequences downstream of the TERM codon, after nucleotide position 450-452, continue the similarities with the synaptophysin sequences and. therefore. it is possible that the single base deletion was a cloning artifact. The interdomain regions are much less conserved between HL-5 and synaptophysin than are the transmembrane domains themselves; most notably, a
236 stretch between domains 1 and I1 displays no evident alignments. HL-5 appears to lack the cytoplasmic tail immediately following the intramembrane domain IV.
10
20
30
40
SO
The nucleotide sequence following the potential TERM codon of HL-5 at nucleotide position 743-745, does not encode tyrosine or proline-rich stretches, in any of
60 T...HLo5
--4~"T-b'Ir~-T~"-oT-~
. . . .
490
S00
510
530
530
540
.*
|
:
g
•
:
-4~.-T...-...---.G-...RAT
AGkT&T T.~_~..~_~TA~"T
~
¢
AAT&TTATTGA
JtJt~t~,, .HL-S
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G. . . .
70
00
00
100
110
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;
II
;
;
G--GC:- -,-,. - G - C ~ , . G - C , . - . . - - C - .,C:. . . . .
G. . . . .
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......
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Sll0
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600
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|
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|
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•
$
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-...C- - ~ A A e A c A - . . r . A - { l a A P ~ ' ~ J t ~ C ~
130
140
IS0
It0
170
100
t
I
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|
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t
--C--,GGAACACA--CA-
....
.l
~
- - ¢ - - C A C . . . . RAT
~
......
--¢--CAC,,..HtJMA#/
ZV GG-GeTTCGGC-GAGC°--G-G---~-¢AJ~r.AJkGA¢OGAGA~-&CAo¢GA--T...RAT
610
U0
630
640
ISO
6qO
•
|
~
$
;
;
~°~-~t~C---.~.-..--G-atA~tAGk¢¢GAGAG'I~'~oG..¢---4~4t~--T-...HUIHUtN
A~TA2TtrG6¢Tt'TeTAAAT&'I'~T
190
~00
~10
|
|
:
~0 |
~30
~40
;
:
TAcs'rt " t ' ~ G ~ a ' l r e r . A ~ ~ S ' r . A i - i - i :
~I~TTTGTG'TA...HL-5
¢-CA---G-G ....
-..¢--G--~--G-~
.....
'IT-o¢--CT'J'A . . . . .
C. . . .
T . . , RAT
¢--¢---G-G
--¢ooG--CC--G-G-
....
T¢--C--CCTG- ....
¢....
T...
....
-G/t&--C-AG-o¢o,,,,C..o----C--,C-CC-&-TG-,AC--..G-TG---oC'roCT-C--C
. . .RA?
G-.GG-CGA. . . .
. . . Ht,HHINH
C-.,.,C-- - - - , , . , C - , , . , C - C ¢ - A - T G - A C - - - G - T G . . . .
-CT-C
570
QO
690
700
710
720
HI,* S
G4AGGAGA¢¢AG¢¢T&G4¢AGT¢¢ATCA~TAeA~TAG¢¢AAGGAGGTAT...
~50
|60
~?0
HUH~i
RAA.. ,HL-5
|00
~SO
300
(AJ~AGGGGGCAC'I'&e.'m.RA~,'L%'I'T¢)
--O~-'lr--G--¢ ....
¢--G--G--'JL'G-...RAT
¢C'~. k. G O G I ~ ¢ A ¢ C , A ~ )
T-AG-'I?-°G--¢ .....
C--G--A--O
..........
AG'.-TGGGeAO~C----TC-TGCG¢
--A----CAG--,GCCCCG-AA-A...RAT
T........
A G - -'IL'-- -v - ' ~ -
-o~---¢¢G-~-AG-A...HUM~
730
740
-G-'JL'~
750
760
770
700
e-- ° . o TCCACCTCTACC~AJiTATAATTAAAG~d4GiUUiTACACTGTATGAAGTATATGTlrGATA... HI,-S
g~
.... .....
G-A---&GC-o-To~'Go-GCC--CG-CGo'JL~'GC~-TACoGGCAGG-CCCCG-AG-¢--.,. t~T
310
3|0
330
340
3S0
360
|
|
;
;
|
;
& ° & o ooGGCo - - ¢ ~ - - ~ - W o ' J ~ C . , G G . - T A C - G O C J k O a o O C C C G - C G - G - - . ,
"T''~'-"4~-T-..G----¢~-...-.-¢.--o..C-.-.G-G~-o--G6ceA¢¢*-...RA? T--°~---GoG-~..o~-~----4:----~.--G-~,-.-T-o~,~.A~--...
HUMAN
190
I100
810
820
030
040
;
*
:
:
,
•
CT&TGACATG'ITGCCAACA~TTATTTG'ITTCTAATA/UUWTAATGGCTT...HLoS TOGG~oCAAGACT-CT&COGGCCt'C-G-G'rI~'To&-¢AAC-CG- * T - C G - G C - G - - A G e . . . RAT -GGGCC,..¢A-GATT-CTACGGGCCT¢-G-GCGGC-&CCCAG*CTGACT-T-GTCAAC-AG... HUMAN
i
310
3QO
390
400
410
4~tO llSO
TGT'I'GG~&G4~Gl%"T~'r~I'GGAT&~J~J~&TGATjkTdI, CA°CTI'~--ACAA---C-~k--Go&¢AJkC--oGGG-
.....
:
CTTTGTTGT...HL-S
1160 :
8"/0
880
."
:
090 _-
900 g
G- . . . .
o¢-G-¢...
RAT
T
TTCA/LTGG/Uk'IT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
CA'¢TT°e-"t"~CJ~'°ACAA-'°¢-GA--G-&-A&C--'GGG--C---¢-G - ....
-C-G-¢...
HUMAN
.,.
AGoGG---CGG-GGCT&CGGGCCTC&.~XGACTATGGGCAGCAJ~T.GCTATGGCCA...RAT
HL-S
CGGCAGI~TGGCA~GGCT&CC,.C__ _-C~"TCAGGGCGACT&TGGGCAGCAJk'.GCTACGGCCC...~
....
IZZ 430
440
;
~
l'Ar.&c'n'GTt'OceJ~/T~
450
460
470
400
910
eQe
•
~ G C A ~ A A A ~ i ~ i r ~ C . . . H L - S
°''"4~'~--G'T'I"'~-~..----.¢-A--T...-'rI~-&
.....
g30 ~ :
~)?~ ~,
***
ACAGGGTGCGCCCACCTCCTt'CTCCAATCAGkTGTAA
..........................
RAT
GCAGGGTGCACCCACCTC¢TTCTCCAATCAGkTGTAG
..........................
HUMAN
oI~----GC---T-...RAT
C°-°GCC--GT'I'°G-C--CA------¢-A--T---T-&-oG--A..-GC¢..G-GG...T....
HUMAN
Fig. I. Alignments of the nucleotide sequences encoding the rat [2] and human [4,5] synaptophysins, with the nucleotide sequence of the HL-5 eDNA. Gaps were introduced to maximize the similarities. No evident alignments were found between nucleotide positions 241-271 inclusive. lntramembrane domains, as defined by Johnston et al. [6] are indicated by the lines above the sequences. Asterisks indicate in-frame termination codons. 3' untranslated regions of the rat and human sequences are not presented.
237 the possible reading frames, analogous to those found in synaptophysin. HL-5 was used as a probe to investigate the gene expression product in the HEL and RASM cell lines by Northern blot analysis (Fig. 3a). A single mRNA species, of 2.5 kb, was found in both cell types. This contrasts with the~ synaptophysin gen¢ which has been reported to be expressed in a variety of neuronal tissues [1-4,6], and undetectable in other tissues of non-neuronal origin, such as the kidney and liver [6], and cultured rat smooth muscle cells [6]. In Southern blot hybridization analysis of human DNA (wash conditions: 0.5 x SSC, 50°C, 1 h), probe HL-5 detected a band of approx. 23 kbp, after digestion with Hindlil, Bglll, EcoRl or Pvull. With a high stringency wash (0.2 × SSC, 60°C, I h), only the 8.0 kbp Eco RI and the 4.5 kbp Pvull fragments were retained. With rat genomic DNA, fewer bands were found. Fig. 3b shows only a 4.7 kb EcoRl fragment, a Pv, ll 3.2 kb band, and a 2.5 kb band. The multiple banding pattern of some of the digests suggests that the HL-5 probe detects members of a multi-gene family.
L N F L K E P L G V V . . . R V V . . . V V . . .
F . . .
A . . .
O -
G E E E
y . . .
T . . .
C G G F K G . . S Y T . . S Y S . . S Y S
p F . . . . . . . . .
R LNE . N Q . H O . N -
T E E E
F . . .
I K V L E W I A S I ~ . H L - 5 . . V - - - Q - V F A - - - . . ~ T . . V .... Q-VFA---..HUMAN . . V---~-VFA---..BOVINE
E Z Q V N C P F A V T E N K T V T A T F . L R L S - E - A N K T E S A L N I E V E L O L S - D - A N K T E S D L S I E V E L Q L S - D - A N K T K S D L N I E V E - .
AS F V Y V Y V ¥ -
OF D k D A E A
P -
P S T T
G VN C C C
I C D VN W K D KGGTTKIF RGGTTKVF QGDFKKIF
. . .
H . . B
L . H O
~ U V
y S . . . . . . . . .
S S . . . . . .
AQ
F YVT F A V E - F - - V - - - A E - F - - V - E - F - - V - -
F
V
F
k A
-
-
L Y - - S - - -
C H S S
I ~ L G MG N G -
L L - A T - A T - k T
Y V
L I - V - V - V
G..14L-5 -. ,RAT -. . H U M A N -. . B O V I N E
¥
G F F F
T,.HL-5 Q,.RAT (]..HUMAN Q..BOVINE
-
V I I I
¥ L L L
ZZZ ~ L Y L D S R K L P M N K N K N K -
O F V V T L V A T F C G
R E H N - G - - M R E H N - G - - H - R E ti H - G - - H -
~ K A L T D Z K AGS VD A - G - S VD A - G - S V D -
FGS
I
VT
-
L A - k L A - A L A - k
I A T G H N M- D P £ M - - D P E H - - D P E -
S HG
E L R D P - - - G E L R D P - - - G E L R O P - - -
-
G
Z -
I -
D K K K
-
Y ............. HL-5 M W r V S T S A W, .HL-5 I
-
F A F A F A -
HW L HWL H W L
-
- S -S - S
- -- -
E -
L P MM H -
C -
K T T T
A G G G
L C ¥..HI,,-5" T - K..RAT T - K..HUHAN T - K..BOVINE
P N V H
K g g g
K Q Q O
V N N N
kbp
A
-23.o
RASM HEL kb 2.5-
-9.4 .......
....
-6,6
-4.4
-2,3 -2.0
5 T ~ N I N E
ZZ O . . N
HBEP
Fig. 3. (A) Northern analysis of poly-adenylated mRNA from RASM and HEL cells. 5 #g of each was denatured with glyoxal [9], analysed on a 1.4% agarose gel and transferred to a nylon filter. The filter was washed with lxSSC, 50°C, 2 x 3 0 min. (B) Southern analysis of human DNA. 10 #g of DNA was digested by Hindlll (H), Bglll (B), EcoR! (E), or Pc, ll (P) and analysed on a 1.0C~- agarose gel, and transferred to a nylon filter. The filter was washed with 0.5 × SSC, 50°C, 1 h. ,~DNA-Hmdlll size markers are indicated.
X Q G R S G F Q I N - L V A - G - F R - F - L V A - G - F R
B
-,.RAT -..HUMAN -, .BOVINE
In summary, the gene encoding the mRNA from which the HL-5 eDNA clone was derived is related to a gene encoding the previously described protein synaptophysin, may be a member of a family of membranebound proteins with a much wider tissue distribution than found with synaptophysin itself. We thank Mark E. Rothenburg for expert technical assistance. This work was supported in part by NIH grants HLA4307 and HL02414. References
gV ~ L N V C V Z F G F L N N I L W G G N AW..HL-5" - - T S - V -T S - - T S -
v V
. . . . . . . . . . . . . . .
L V - - V - L V - V L V - - V -
-
L-..RAT L -..ltUHAH L -..BOVINE
F V Y K E T S L H S P S N T S A P H S Q G G Z P P L P E Y H .... - - F - - - G N A A - F N R - - P G A P E K Q - A - G D A Y . . ~ T
- - F - - - G N A A - F L R - - F - - - G H A A - F L R
HL-5*
--PGAPEKQ-A-GDAY..HUI'IAN - - P G A P E K Q - A - G D A Y . . ~ V I N E
G D A G Y G Q G P G G Y G P O D S Y G P Q G G Y Q P D Y G Q P . . ~ T G D A G Y G Q G P G G Y G P Q D S Y G P Q G G Y Q P D Y G Q P ..HUILIW G O A G Y G Q G P G G Y G F Q D S Y G P Q G G Y Q P ~ Y G Q P . . ~ V I H E
s~GaG~aPOaoyaQOo~oOooAPTsrs.o...~T AasGasayaPOanyGOOayGPoaAPTsrs.os...u.~ as SGGGGYGeOGD YGOOGYGPOGAPTS
rsHOs..Bovz.g
Fig. 2. A i i g n m e n t s o f t h e t r a n s l a t i o n p r o d u c t s o f t h e s e q u e n c e s i n Fig. l, togetherwiththatofbovinesynaptophysin[6].
1 Wiedenmann, B. and Franke, W.W. (1985) Cell 41. 1017-1028. 2 Leube, R.E., Kaiser, P., Seiter. A., Zimbelmann, R., Franke, W.W., Rehm, H., Knaus, P., Prior, P., kietz. 1t, Reinke. H., Beyreuther, K. and Wiedenmann. B. (19E7) EMBO J. t,. 32613268. 3 Pang, D.T., Wang, J.K.T., Valtorta, F., Benfenati, F. and Greengard, P. (1988) Proc. Natl. Acad. Sci. USA 85. 762-'/6¢~. 4 Sudhof, T.C., Lottspeich, F., Greengard. P.. Mehl. E. and Jahn, R. (1987) Science 238, 1142-1144. 5 Sudhof, T.C., Lottspeich, F., Greengard. P., Mehl. E. and Jalm. R, (1987) Nucleic Acids, Res. 15, 9607. 6 Johnston, P.A., Jahn, R. and Sudhof, T.C. (1989) J. Biol. Chem. 264, 1268-1273.
238 7 Gunther, S., Alexander, R.W., Atkinson, W.J. and Gimbrone, M.A. Jr (1982) J. Cell. Biol. 92, 289-298. 8 Libert, F., Parmentier, M., Lefort, A., Dinsart, C., Van Sande, J., Meamhaut, C., Simons, M-J., Dumont, J,E. and Vassart, G. (1989) Science 244, 569-572.
9 Maniatis, T., Fritsch, E.F. and Sambrook, J. (1982) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor. 10 Bargou, R.C.E.F. and Leube, R.E. (1991) Gene 99, 197-204.
