DNA Sequence-the journal of Sequencing and Mapping, Vol. 4, pp. 343-346 Reprints available directly from the publisher Photocopying permitted by license only

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Nucleotide Sequence of Mouse Spermidine Synthase cDNA SANNA MYOHANEN, JARMO WAHLFORS, LEENA ALHONEN and JUHANI JANNE

Department of Biochemistry and Biotechnology, and A.I. Virtanen Institute, University of Kuopio, Kuopio, P.O. Box 1627, FIN-702 7 1 Kuopio, Finland

EMBL L 1 9 3 1 1

The nucleotide sequence of mouse cDNA for spermidine syn- thase appeared to contain 75 nucleotides of 5’ untranslated re- gion, an open reading frame of 909 nucleotides and 297 nucleotides of 3’ untranslated region. The open reading frame encoded a polypeptide of 302 amino acids, displaying 95% sim- ilarity to human and 33% similarity to E. coli spermidine syn- thase. The 3’ flanking region contained an unusual polyadenylation signal AATACA.

KEY WORDS: amino acid sequence, cDNA sequence, mouse, spermidine synthase

Abbreviation: SpdSy, sperrnidine synthase

Spermidine synthase (SpdSy, E.C. 2.5.1.1 6) is an en- zyme of the polyamine biosynthetic pathway, con- vert ing putrescine to spermidine by adding a propylamino group from decarboxylated S-adeno- sylmethionine to it Uanne et al., 1978). The activity of this enzyme is thought to be regulated mainly by the availability of its second substrate, decarboxy- lated S-adenosylmethionine. The latter view is fur- ther supported by recent experiments w i t h transgenic mice carrying the human spermidine syn- thase gene (Kauppinen eta/., 1993). The complete amino acid sequences of both the E. co l i (Tabor and Tabor, 1987) and the human (Wahlfors et a/ . , 1990) spermidine synthase are known, as well as a partial sequence of the bovine enzyme (Eloranta et a/., 1990). The human enzyme displayed only 33% similarity to the E. coli enzyme, but all the known parts of the bovine enzyme were

~

Address for correspondence: Professor J. Janne Tel. +358-71- 163049 FAX +358-71-2811510 E-mail JANNE@MESSI.UKU.FI

highly similar to the human enzyme. We here report the nucleotide sequence of mouse cDNA encoding spermidine synthase. The h g t l l cDNA library, prepared from the mRNA isolated from the mouse kidney, was a gift from Dr. Olli A. Janne, University of Helsinki. The library was screened with radioactively labelled insert of phSDl which contains the whole coding region for the human SpdSy. (Wahlfors et a/., 1990). Two positive clones were isolated, subcloned into pUCl9 and se- quenced complete ly on bo th strands using AutoRead fluorescent sequencing kit and A.L.F auto- mated D N A sequencer (Pharmacia Biotech, Sweden). One of the clones, pmSpd26, appeared to contain the entire coding region for the SpdSy protein, as well as full length 3’ flanking region and 75 nu- cleotides of the 5’ flanking region (Fig. 1 ). Primer ex- tension studies and Northern analyses (results not shown), however, suggested that the ful l length cDNA should contain 200 nucleotides more. The 5’ region present in the clone pmSpd26, was very GC rich (83%) as was the human cDNA. This GC rich region continued far beyond the ATC codon, which indicates the presence of a very strong secondary structure in the mouse SpdSy mRNA. According to the algorithm by Zuker and Stiegler (19811, the first 150 nucleotides of the cDNA are predicted to form a secondary structure with an overall free energy change of -1 02.3 kcalho l . The 3’ flanking region was 297 nucleotides long without the poly(A) tail. This region i s considerably shorter than its human counterpart (658 nu -

343

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344 S . MYOHANEN ETAL.

10 20 30 40 50 60 70 80 c G G G I 7 l ; n ; c ' p G G G G G G G T C C G ~ C G C C C n ; C A G T C C C A

M E

90 100 110 120 130 140 150 160 G C C I I ; G C C C C G A P C c C W X W X A ? C C G % A A G A ~ ~ W

P G P D G P A A P G P A A I R E G W F R E T C S L W 10 20

170 180 190 200 210 220 230 240 E G C U G G C C P ~ a ~ ~ A ~ C 4 T ~ W P G Q A L S L Q V E Q L L H H R R S R Y Q D I L V F R

30 40 50

250 260 270 280 290 300 310 320 AGT~ACGGCAA~lTCn324TG5GTCATCiXG%TAClCAGAl3XAXXXEKCTAEMGAGAT S K T Y G N V L V L D G V I Q C T E R D E F S Y Q E M

60 70 80

330 340 350 3 60 370 380 3 90 400 G ? i m TcA--Ac I A N L P L C S H P N P R K V L I I G G G D G G V L

90 100

410 420 430 440 450 460 470 480 ~ G C A C C C A G n % G A G A T T G A l W + f X A T G K A ~ W G A A G R E V V K H P S V E S V V Q C E I D E D V I E V S K K 110 120 130

490 500 510 520 530 540 550 560 -P clx2xxAm--m F L P G M A V G F S S S K L T L H V G D G F E F M K Q

140 150 160

570 580 590 600 610 620 630 640 G A A C C A P i G A T G C C I l T G A C n . r c A - - m m P N Q D A F D V I I T D S S D P M G P A E S L F K E S

170 180

650 660 670 680 690 700 7 10 720 A T T ~ l G M G A C A ~ l G G C A T ~ ~ ~ ~ ~ ~ C Y Y Q L M K T A L K E D G I L C C Q G E C Q W L H L D 190 200 210

730 740 750 760 770 780 790 800 C I C A T C A A M ; A G A n ; A G G C A ~ ~ C T ~ T ~ ~ A C C I ' A T C L I K E M R H F C K S L F P V V D Y A Y C S I P T Y P

220 230 240

810 820 830 840 850 860 870 880 C A G C C G C C A G A r r G G C T ~ C ~ C C A 4 ~ C ~ ~ ~ ~ ~ ~

S G Q T G F M L C S K N P S T N F R E P V Q Q L T Q 250 260

890 900 910 920 930 940 950 960 C C K C I T l X T - m A Q V E Q M Q L K Y Y N S D M H R A A F V L P E F T R 270 280 290

970 980 990 1000 1010 1020 1030 1040 A A G G C C C I C A A T G A ~ ~ C ~ C C ~ A ~ C ~ ~ ~ C ~ ~ K A L N D I S '

300

1050 1060 1070 1080 1090 1100 1110 1120 r r c A A ~ ~ A C U ~ A C c A G A c r c c

1130 1140 1150 1160 1170 1180 1190 1200 ~ ~ C T G G C C C ~ ~ C ~ C ~ C C ~ ~ ~ A ~ ~ ~ ~ A ~

1210 1220 1230 1240 1250 1260 1270 1280 G I U ; C G ? T C A G C C C C A C G C C T A T A C C A G ~ A C A G C A C ~ A W C C W X A C C C A 4 C W T A C A T G ~ A W

CG

Figure 1 Nucleotide sequence and deduced amino acid sequence of mouse SpdSy cDNA. The numbers above each line represent the nucleotide sequence, while the numbers below the lines refer to the amino acid sequence. The unusual polyadenylation signal AATACA is underlined.

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E. coli Mouse Human

Consensus

E. coli Mouse Human

Consensus

E. coli Mouse Human

Consensus

E. coli Mouse Human

Consensus

E. coli Mouse Human

Consensus

E. coli Mouse Human

Consensus

E. coli Mouse Human

Consensus

MOUSE SPERMIDINE SYNTHASE cDNA

--------- ---- .EKKQ. -H..--.HDQ FGQYFA.DNV .Y.EKTDH.. .--.-..... P......... -........_ ..._..._.. .....-.--. *.-..--... s. . . . . . ... ....-...._ ._........ .-....-... MEPGPDGPAA .GPAAIREGW FRETCSLWPG QALSLQVEQL LHHRRSRYQD

LI1.E". R.MA....V. T......I.H ..MTHV..LA .GHAKH.... - - - . . . - - - - .-........ ...--.._.. ..._.----. ..----.-.. ---...-..- -........- ....--...* ....---..- --.-...--. ILVFRSKTYG NVLVLDGVIQ CTERDEFSYQ EMIANLPLCS HPNPRKVLII

.....AM... .TR.KN...I TMV... AG.V SFCRQY..NH NA-S-DDPRF

....._-... ..._.._... ._...._... E...... ... .V..F..... __......__ ._......_. ........._ Q-........ .I........

GGGDGGVLRE VVKHPSVESV VQCEIDEDVI .VSKKFLPGM A-G-YSSSKL

K.VID..VN. VN-TSQT... ..S.CT..I. .G....TSAF .EGC.RC.NP ,..-..-..- -.-..--._. .-.....-.. ....._._.. . - - - . - . - - . __-- . - . . . - ....-.-._. -..-.....- - - - . _ . .___ . . . - - - . - - - TLHVGDGFEF MKQNQDAFDV IITDSSDPMG PAESLFKESY YQLMKTALKE

G..FVA.NGV CF-QQEEAID SHRKLSHY.S D-GFYQAA.. ..YG.IMT.A ...._..___ _._...._.. .. H..K.... .. D.. ..S.. .......... .. V.... ... ......___. .. Q..Q.... .. A....T.. ....._.... DGILCCQGEC QWLHLDLIKE MR.FC.SLFP W.YAYC.IP TYPSGQIGFM

WATD-DALRH LSTEIIQARF LASGLKCR.. .PAI.T...A ..QYLQD..A . . ._.__.._ R....Q...A .. E. ...... ... M... ... .... T..... ._...._... Q..--P.-.Q .. A....... ... V. ..... .... A..... LCSKNPSTNF .EPVQ.LTQ. QV.QMQLKYY NSD.HRAAnr LPEF-RKAL-

SQP . ..I. . .v. ND.S

345

34 50 50

so

84 100 100

100

134 149 149

150

184 199 199

200

234 249 249

2 50

284 298 298

300

288 302 302

304

Figure 2 Comparison of the amino acid sequences of E. coli (Tabor and Tabor, 1987), mouse and human (Wahlfors eta/, 1990) sperrni- dine synthases. A dot in any of these sequences indicates a similarity between at least two of them, while a dot in the consensus sequence implies a site that i s different in all of the sequences shown. The dashes indicate a missing residue.

cleotides). A common feature for the human and the mouse 3' non-coding sequences was an unusual polyadenylation signal AATACA (Myohanen et a/., 1991). The length of the longest open reading frame in the cDNA was 909 nucleotides coding for a polypep- tide of 302 amino acids. This polypeptide was ap- parently the mouse SpdSy, as i t displayed 95% similarity to the human protein (Fig. 2). This implies differences at 16 locations, 11 of which were non-

conservative. None of these changes, however, nec- essarily affects the overall properties of the protein, as the maps of acidic and basic residues and hy- drophobicity plots for human and mouse polypep- tides appeared to be extremely similar (results not shown). At the nucleotide level, the mouse SpdSy cDNA was 88% similar to the human cDNA. The 5'- and the 3' non-coding regions of human and mouse cDNAs displayed 75% and 65% similarity, respectively. The

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346 S. MYOHANEN ET AL.

most striking similarity at the 5' region was immedi- ately before the ATG codon. This stretch of nu- cleotides was earlier shown (Wahlfors et a/. , 1990) to contain four perfect overlapping Kozak's consen- sus sequences. The overall similarity between E. col i and mam- malian SpdSy polypeptides was only 3370, although there are several regions that are much better con- served (Fig. 2). These include amino acids 60-82, 97-1 19 and 167-1 85 (Fig. 2.), displaying 70%, 74% and 74% similarity, respectively. These regions are apparently essential for the enzymatic activity of the protein. On the other hand, the terminal regions are not conserved to the same degree, as E. coli protein lacks 13 most amino terminal residues, and the most of the differences between E. coli, mouse and human polypeptides can be found near the carboxy terminus. Interestingly, upon comparison our sequence against the whole GenBank database, it appeared that the mouse SpdSy cDNA is very similar to one of the randomly cloned and sequenced cDNAs from the laboratory of Dr . Venter (GenBank entry M78926). It turned out that their sequence was identical to the human SpdSy cDNA (GenBank entry M34338) that was sequenced in our labora-

tory several years ago (Wahlfors eta/., 1990) This work was financially supported by Finnish Foundation for Cancer Research.

(Received 19 November 1993)

REFERENCES

Eloranta, T. O., Wahlfors, J., Alhonen, L., Hyvonen, T. and Janne, J. (1990) in Goldemberg, S. H. and Algranati, I. D. (eds.). Cloning and primary structure of human spermidine synthase. Proceedings of the lnternational Symposium on the Biology and Chemistry of Polyamines (ICSU Press, New York) pp. 91-98.

Janne, I., Poso, H. and Raina, A. (1978). Polyamines in rapid growth and cancer. Biochim. Biophys. Acfa 473, 241-293.

Kauppinen, L., Myohanen, S., Halmekyto, M., Alhonen, L. and Janne, J. (1 993). Transgenic mice over-expressing the human spermidine synthase gene. Biochem. 1.293, 51 3-51 6.

Myohanen, S., Kauppinen, L., Wahlfors, J., Alhonen, L. and Janne, 1. (1 991). Human spermidine synthase gene: structure and chromosomal localization. DNA Cell Biol. 10, 467474.

Tabor, C. W. and Tabor, H. (1987). The speEspeD operon of Escherichia coli. /. Biol. Chem. 262, 16037-1 6040.

Wahlfors, J., Alhonen, L., Kauppinen, L., Hyvonen, T., Janne, J. and Eloranta, T. 0. (1990). Human spermidine synthase: cloning and primary structure. DNA Cell Biol. 9, 103-1 10.

Zuker, M. and Stiegler, P. (1981). Optimal computer folding of large RNA sequences using thermodynamics and auxiliary in- formation. Nucleic Acids Res. 9, 133-1 48.

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