DNA AND CELL BIOLOGY Volume 10, Number 6, 1991 Mary Ann Liebert, Inc., Publishers Pp. 467-474

Human

Spermidine Synthase

Gene: Structure and

Chromosomal Localization

SANNA

MYÖHÄNEN,

LEILA KAUPPINEN, JARMO WAHLFORS, LEENA ALHONEN, and JUHANI JÄNNE

ABSTRACT The human spermidine synthase (EC 2.5.1.16) gene was isolated from a genomic library constructed with DNA obtained from a human immunoglobulin G (IgG) myeloma cell line. Subsequent sequence analyses revealed that the gene comprised of 5,818 nucleotides from the cap site to the last A of the putative polyadenylation signal with 8 exons and 7 intervening sequences. The 5 -flanking region of the gene was extremely GC rich, lacking any TATA box but containing CCAAT consensus sequences. No perfect consensus sequence for the cAMP-responsive element for the AP-1 binding site was found, yet the gene contained seven AP-2 binding site consensus sequences. The putative polyadenylation signal was an unusual AATACA instead of AATAAA. Polymerase chain reaction analysis with DNA obtained from human x hamster somatic cell hybrids indicated that human spermidine synthase genomic sequences segregate with human chromosome 1. Transfection of the genomic clone into Chinese hamster ovary cells displaying a low endogenous spermidine synthase activity revealed that the gene was transiently expressed and hence in all likelihood represents a functional gene.

INTRODUCTION

Themidine,

biosynthesis of the poLYAMiNEs

plished by

putrescine,

and lymphocyte activation (Korpela et al., 1981). In fact, under these conditions, the stimulation of spermidine synthase activity was fully comparable to that of ornithine

sper-

and spermine in mammalian cells is accomthe concerted action of four separate enzymes

ornithine decarboxylase (EC 4.1.1.17), adenosylmethionine decarboxylase (4.1.1.50), spermidine synthase (EC 2.5.1.16), and spermine synthase (EC 2.5.1.22). The two decarboxylases, especially ornithine decarboxylase, have been the subjects of extensive investigation due to their striking inducibility and short half-lives (Jänne et al., 1978). Much less attention has been paid to the two propylamine transferases: spermidine synthase and spermine synthase, apparently because these enzymes are stable and only modestly inducible. The regulation of spermidine and spermine synthases is believed to occur at the level of sub-

adenosylmethionine decarboxylases. Recently, we isolated and sequenced cDNA encoding human spermidine synthase from a human decidual cDNA library (Wahlfors et al., 1990). By using this cDNA as a probe we now have isolated the whole human spermidine synthase gene from a genomic library constructed with DNA from a human myeloma cell line. The gene was subsequently sequenced, its chromosomal location assigned, and its functionality confirmed by transfection experiand



(decarboxylated adenosylmethionine) availability (Jänne et al., 1978). However, there are reports indicating that spermidine synthase, but not spermine synthase, is induced in response to accelerated proliferation such as liver regeneration (Hannonen et al., 1972), hormone-induced growth of tissues (Oka et al, 1977; Käpyaho et al., 1980), strate

ments.

MATERIALS AND METHODS Materials Restriction and other DNA-modifying enzymes were purchased from Boehringer Mannheim, Promega, or New England Biolabs. LambdaGEM Xho I arms were from Promega and M13mpl8 RF DNA from Boehringer Mannheim. Radioactive isotopes [a-32P]dCTP and [7-35S]dATP

Department of Biochemistry & Biotechnology, University of Kuopio, 467

P.O.B.

1627, SF-70211 Kuopio, Finland.

MYOHANEN ET AL.

468

Hybond N nylon membranes were from Amersham. A 2.0 sequencing kit was purchased from U.S. Biochemicals and the AutoRead sequencing kit was from Pharmacia. Sequencing gel reagents were purchased from

and

Sequenase

BioRad or Pharmacia. PCRable DNA system was from BIOS Corporation. DL-[2-'4C]methionine was from Amersham. Chinese hamster ovary cell line CHO-K1 was obtained from the American Type Culture Collection. The cell culture medium F12 was from NordCell and Eagle's minimum essential medium (MEM) from NordVacc; other materials for cell culture were purchased from GIBCO.

Isolation and characterization of a genomic clone Genomic DNA was isolated from human IgG myeloma cell line Sultan 20D with ornithine decarboxylase gene amplification and modest spermidine synthase activity (Hirvonen et al, 1989). A genomic library was constructed into a lambdaGEM12 vector and screened with the nick-translated insert of the cDNA plasmid phSDl (Wahlfors et al., 1990). The insert of a positive clone was subcloned into pUC19 and characterized with Southern blotting and DNA sequencing of double-stranded X template using primers previously constructed for characterization of the cDNA.

DNA

sequencing

The nucleotide sequence of the human spermidine synthase gene was determined using the shotgun strategy (Bankier and Barrel, 1983). An Eco RI fragment of hgSPDSY1 was sheared by sonication or by Alu I or Sau 3A digestions. Fragments were cloned into M13mpl8 and about 100 individual clones were sequenced using the chain-termination method of Sanger et al. (1977). Sequencing reactions and analyses of the reaction products were performed either manually using Sequenase enzyme according to the protocol of U.S. Biochemicals or automatically using Pharmacia's AutoRead fluorescent sequencing kit and A.L.F automated DNA sequencer. dITP was occasionally used instead of dGTP to resolve compressions due to extreme high G/C content of the 5'-flanking region. Double-stranded sequencing was performed occasionally using pUC19 as a vector and following the instructions of the AutoRead sequencing kit. To fill gaps in the sequence or to obtain sequence from the opposite strand, the reverse end of some M13 clones were sequenced using fluorescent reverse M13 primer by the method of Myöhänen and Wahlfors (1991). Sequence data was compared, assembled and analysed with the aid of DNASIS software system (Hitachi America, Ltd. and Pharmacia

Biotechnology). Chromosomal localization Two oligonucleotide primers complementary to fourth and fifth introns of human spermidine synthase gene were synthesized with Applied Biosystems 381A DNA synthesizer. Sequences of the primers were 5-CTGGCTCTGGCCACCTGGTA-3' (4th intron) and 5'-TAAGCATCAGCATCCGGCAG-3' (5th intron), giving a PCR product of

248 bp. PCRable DNA from human x hamster somatic cell hybrids was used as the template in polymerase chain reaction according to BIOS Corporations instructions. Taq DNA polymerase (0.25 U/reaction) and appropriate buffer were from Promega. The amount of both primers was 20 pmole/reaction and the titrated optimal magnesium concentration was 1.5 mM. After denaturation in 96° C for 3 min, 30 cycles in Hybaid thermal cycler were carried out according to the following program: 95°C for 1 min, 62°C for 1 min, and 72°C for 30 sec. Polymerization step in the last cycle was extended to 5 min at 72°C. The analysis of the PCR products was carried out on 1.5% agarose gel stained with ethidium bromide.

Transfection experiments wild-type Chinese hamster ovary cell line CHO-K1 cultured in Nutrient mixture F12/MEM supplemented with 10% fetal calf serum and 50 mg/liter gentamicin. The Eco RI fragment of spermidine synthase genomic clone in pUC19 plasmid, phgSPD65, was used for the transfection experiments. The circular plasmid was transfected into the CHO-K1 cells by the calcium phosphate coprecipitation technique in low pH and low C02. The cells (0.5-1 x 106) were incubated overnight on 10-cm diameter plates in the growth medium with 10 fig of plasmid DNA using buffers and conditions described by Chen and Okayama (1987). After transfection the cells were grown in the F12/MEM medium and spermidine synthase activity was measured by the method of Raina et al. (1983) at 48 and 72 hr after the transfection. Mock transfection was performed with 10 fig of sonicated herring sperm DNA. A

was

RESULTS Isolation and nucleotide sequence spermidine synthase gene

of the human

A human myeloma cell line genomic library constructed into the replacement vector LambdaGEM-12 was screened using the insert of cDNA plasmid phSDl as a probe. Out of 130,000 plaques, one positive X clone was obtained. This clone, designated as hgSPDSYl, had an insert of 10.5 kb (Fig. 1) and was potentially a full-length gene according to Southern blot analyses. This was further confirmed by double-stranded DNA sequencing using oligonucleotides complementary to the cDNA sequence as primers. Sequencing revealed that the clone hySPDSYl contained at least the whole protein coding region of the spermidine synthase gene. The 10-kb Eco RI fragment (Fig. 1) was subcloned into plasmid pUC19 for propagation and designated as phgSPD65. The Eco RI fragment was also used for sequence analysis. The shotgun sequencing approach was used to determine the nucleotide sequence of human spermidine synthase gene and the flanking regions. Ml3 clones from several separate clonings were sequenced to obtain the sequence from both strands. Figure 2 gives the nucleotide sequence of the Pst l-Eco RI fragment containing the whole gene. The human spermidine synthase gene is 5,818 núcleo-

HUMAN SPERMIDINE SYNTHASE GENE

469

I I FIG. 1. Structure of human spermidine synthase gene. a. Restriction enzyme map of the insert in the X clone hgSPDSY1 containing a functional spermidine synthase gene. Restriction sites shown: X, Xho I; E, Eco RI; S, Sac I; H, Hind III; P, Pst I; C, Cla I; B, Bam HI. b. Schematic structure of spermidine synthase gene. Flanking regions and introns are shown as solid line, while open boxes represent untranslated regions and solid boxes represent coding regions. Exons are numbered with Roman numbers. The lengths of coding and untranslated regions in the same exon are marked sepa-

rately.

tides long from the cap site of the mRNA (determined previously by Wahlfors et al, 1990) to the last A of the putative polyadenylation signal. The gene contains 8 exons and 7 introns, with all the junctions conforming to the GT-AG rule. The structure of the gene as well as the lengths of exons and introns are shown in Fig. 1. All the exonic se-

quences match with the cDNA sequence, except nucleotide number 6,456 in the last exon. This nucleotide change is conservative, replacing codon number 297 in the cDNA, GCC, by GCA, both of which encode alanine. The last exon was predicted to be 679 nucleotides long, containing 21 coding nucleotides with the rest being noncoding. The real polyadenylation signal could not be determined by comparison, because the cDNA clone phSDl is not full length at the 3' end (Wahlfors et al, 1990). However, the putative signal AATACA was the best alternative in the near 3'-flanking region. The calculated mRNA size was equal to the one obtained by Northern analysis (about 1.6

kb). Further,

radioactive single-stranded probe complementary to nucleotides 49-336 after the AATACA signal did not hybridize to any mRNA. However, in Southern blot analysis this probe recognized a 7-kb band of spermidine synthase gene. The two longest introns, 3 and 6, contain 4 and 2 Alu repeats, respectively. These particular introns consist almost entirely of /l/«-related sequences and represent more than 30% of th gene. The Alu repeats in spermidine synthase gene show the highest similarity to the "old" subclass (Deininger and Slagel, 1988), as did also the human ornithine decarboxylase gene (Hickok et al, 1990), coding a

another polyamine synthesizing enzyme. A typical CpG-rich island contained about 550 nucleotides in the 5'-flanking region, the first exon and the 5' half of the first intron of spermidine synthase gene. The average G/C percentage of this 1,100-nucleotide region was 78%, but the percentage around the transcriptional start site (from nucleotide 1,200 to nucleotide 1,400) was even

higher,

83%. No TATA box, but two consensus were present, one in the sense strand (nucleotides 1,212-1,216 in Fig. 2) and one in the opposite strand (nucleotides 1,242-1,246 in Fig. 2), 103 and 73 nucleotides away from the transcriptional start site, respectively. Both of these sites are also putative binding sites for transcription factor NF-1 (Mitchell and Tjian, 1989). Eight GC boxes (GGGCGGG) in both strands were present in the promoter region and one was in the first exon of the gene. Among these nine sequences, one appeared to be a part of full consensus sequence of Spl binding site (GCCCCGCCC, at 1,275-1,283 in Fig. 2). According to Mitchell and Tjian (1989), no perfect consensus sequence for CREB or AP-1 (z-juri) binding sites conferring cAMP or TPA inducibility were present in the gene. However, seven AP-2 binding site consensus sequences, also conferring TPA and cAMP inducibility, were found in both orientations (CCCover

CCAAT sequences

CAGGC; at nucleotides 2,391, 3,084, 4,794, 5,241, 5,408, 5,927, and 6,904 in Fig. 2). Whether these putative binding sites are relevant for the transcription of human spermidine synthase gene in vivo is not known. Chromosomal localization PCR was carried out as described in Materials and Methods and reaction products were analyzed by agarose gel electrophoresis. In addition to human genomic DNA control, a single PCR product of the predicted size was obtained with DNA from hybrid cell lines 683, 867, 937, and 1,099 as templates (Table 1). No reaction product was obtained with hamster genomic DNA or with reagent blank. These results clearly map the human spermidine synthase gene to chromosome 1, despite the incorrect positive signal from hybrid cell line 683. According to the manufacturer, this cell line is not supposed to contain chromosome 1. However, our recent studies with genes known to segregate with chromosome 1 have indicated that this particular cell

470

MYOHANEN ET AL.

20

10 i

«

i

*

I

»

*

*

i

l

i

50

40

30 i

i

l

i

i

70

60 »

'

i

'

i

80 i

»

I

ggqgagaggagcccaagccgccggcaggagccagctctcaaggagaaatggaggtaccagagttctccccgctttacaca ttataaactgaggttcccaaaaggggccaggtgtggtggctcacagctgtaatcctagcacttttggaggccgaqqtggg aggatcgaggagttcgaggcaacatagggagactctatctctaccaaaaatttaaaaagtagccaagtatgattqaacac acttgtcccagctactcaggaggctgatgggggaggatcacctgagccccggaggccgagggtgtagtgagccatqatcg atgccactgcactccagcctgggccacaaagtgagaccctgtctcaaaaaaaaataataaaaaaaagggaagqqqttqqc caaggeggettgcctgtgaggcacttggagagtcccacgtggctgtgctggctccaggtcccccagccccttggcccaga ctggtccctcccatcccctccagGATGTCATCCAAGTCTCCAAGAAGTTCCTGCCAGGCATGGCCATTGGCTACTCTAGC

80 160 240 320 400 480 560 610 720 800 880 960 1040 1120 1200 1280 1360 1440 1520 1600 1680 1760 1840 1920 2000 2080 2160 2240 2320 2400 2480 2560 2640 2720 2800 2880 2960 3040 3120 3200 3280 3360 3440 3520 3600 3680 3760 3840 3920 4000 4080 4160 4240 4320 4400 4480 4560 4640

CTCCTCAGACCCCATGGgtaagcagtggatgggccccagggttttctggcagctgcaggtctggaggtcagcctccccca

4800

ctgcaggcgcgcactaccatgcccagctaatttttgtatttttagtacagacggggtttcaccatgttggccaggatcgt

cttgatctcttgacctcgtgatccgcccgcctccgcctccgcctcccacagtgctgggattaccggggtgaaccatcocg

cccaggcacctagcaattctttagcggtcttggfttacctccccittagaaggagcttaaaagcaagcaaggcacattgt

tgcctaggcttgaggcttgctctcacccataaacaacgctgttactctgctctggggacgacacaggaaacgttccccac ctccaggtggaggctgcaaaacgtgtcaaaaccatccctgacataatgtcaagagtagcttatactagtttcatcttcct tccttggcattcgaactgcgtgtggaacaattagcatttaatatttggtaattagcatgcttaatgtgattctgagaagt tctttgacattctcataaaaacagcacattcccacccacccttcaaagagcaagacccagtttgtcaagaaaaattgcgt gccagtcttttctggtgctgaatatgtatgttctgggcctcttcctggacactgctggtaaatttagaaactcgtttaga aaagcacttctctcgtattcaacagcctataggctcatggcgcagaatctaagggaaaatggctaaatccagcttgttaa ttcgcgggctgtgatgattctttccaagtaaataaaaaccctcggttcgccccgacgagccacataatctgttcaaatcc aacaaggaaccagattttggacgcaaagaaggatacgttctactcgccccgtgcaacaacgtaaaccactgtagccgccc gctcccgtgtctccagcccaaaggctgactctccagtccgcacgtcgcagcgctcttgccctccacaccaagcccgagtc ccgcagcccctcgaggccctcggtgcctcccaaccccgagaaggaagcgggggccggtggtgcaccgccccggctgcttg gggcggaggaaggacccggaccccttccgccggcccagcccgccccggaacccgacccggccgcccggccccggccgggc cccacgtggcccctggagcgggccgcactaccctgctgccgccgacggacggcgcgccacagccactctgcgccgctctg cgagccggtggccaatgagcgccaggcgaggccgctttgccattggcgagcgcgggctccgcccccgccggcaggccccg ccccgcgcccgggttaggttgcggcgcgggcggcGGGCGGAGCTGGTCCCGTTGTGCTGCGGCGCCGCGCGGCCTGCAGT CCCGGGCCCGCGCCCCGCGCCGCCCGCCCGCCCGCCATGGAGCCCGGCCCCGACGGCCCCGCCGCCTCCGGCCCCGCCGC CATCCGCGAGGGCTGGTTCCGCGAGACCTGCAGCCTGTGGCCCGGCCAGGCCCTGTCGCTGCAGGTGGAGCAGCTGCTCC

ACCACCGGCGCTCGCGCTACCAGGACATCCTCGTCTTCCGCAGgtaccgccgctgcccgcaggcgcctgccccctaggct cagcccgggccgcctgctgcccgcctcacgggcctctccacgccgggacccaagcgggctggacctcgtcctgccctggc cccctcgccacccctcacaccgcctccctgggctggggctgggactgcgggctggcctcttgggtgggggagtcggagtc tgcgccccgctccacgtgtcagccctcaggacacgtcagagcccgaggagaccccgggtcccaccccggcctccacccgg cggcccgcctgccgttcctcgccacgtgtcaecategetcetcatccctgggacccctaggcgggatggggagaccctec tcacccagggcggcttggggtacgttttccccaccccagagaacccaggtccccgactgtcactccgcccgcagTAAGAC CTATGGCAACGTGCTGGTGTTGGACGGTGTCATCCAGTGCACGGAGAGAGACGAGTTCTCCTACCAGGAGATGATCGCCA

ACCTGCCTCTCTGCAGCCACCCCAACCCGCGAAAGgtaccccagtgtcccctggaacagtgccggacgaggggcggcccc aggtgtgctccgggctcttcccagatgctgcctgcatggttgtcagagaaagtgctagcaaggccaggggcgtcccgcgg aggggtgggggccgacactgacgcggcctcggaatcctagggcagccctggaaggaacttccaggaaaggggacaccggc aegaaagegtttccgagggtagaaaaagatgaggcccgtgggtccgaggggtcagggggtctgcttcaggggcctggggg ctcccagtcctgccagggcccctgccttgactgccccctcctcccagGTGCTGATCATCGGGGGCGGAGATGGAGGTGTC CTGCGGGAGGTGGTGAAGCACCCCTCCGTGGAGTCCGTGGTCCAGTGTGAGATCGACGAGgtgagtgccggcgtagagcc aggtttgagtcctggttctcccagcggccagctgtgccctgaaatggctgcacacccccgagcaaggcaggtagggcctg tttctccatctggaaaacacctggtcggggagggttcagtaggaaaaccagatggcagagggcctggcaggtggtgaggg cacctgcgtggcgagctcttactaaaactgagctgatttttttttttttttttttttgagacagagtttcgctcttgttg cccaggctggagtgtgatggtgcgatctcggctcactgcaacctccacctcctgggctcaagtagttcttctgcctcagc ctccggagtagctgggattacagacatgcgctaccatgcccggctaattttgtatttttagtagagacagggtttctcca tgttggtcaggctggtctcggacctggcgaccacaggtgatccgccagcctcgtcctcccaaagtactgggattacaggc gtgagccaccacgcccagccgactaagctgatttttaatctgagccccaggcaggqccccaagacaqctcaactatttqt acg Liaccccttacactcagtagctgctcactaaaatcatgctacgtgccaggtgttgcccgggtatggggacagtggta gacgacagatcagtccctgccctctaggagctgatgtcgtagttaaaggagacatcagatggccagacgtggtggctcac acctgtaatcccagcactttgggacgccaaggcgggcagatcacctgaggtcaggagttcaagagcagcctggccaacct ggtgaaaccccatttctactaaaaatacaaaaattagccgggcatggtagtgcatgtctgtaaacccagctactagggag gctgaggtggaagaat tacttgagcccgggaggcggaagttgcaatgaaccgagatctcgccactgcactccagcctggg tgacagaggaagaatctgtctcaaaaaaaacaaacaacaaaaatagagacatcaaaqgatqgtctgatgaagqcaaqaca ggggctgggggacaggagaaggcagggttcctgtgaatgcatggggggtggtcagggcaggcctccaggaggtggcgttt gagctgagacctcagtgaaaagcaggtggccgtgtgcagggagggggaggttctcctggccagaggttggaattgcatcc ttctaaaataggaaacaggccaagcgctggtggctcacacctgtaatctcagcactctgggaggctgaggcgggcagatc acaaggtctctactaoaaatacaaQaaaaaaaaaaaatggcccagcttggtggcgtgtgcctgtaatcccaactactcgg gaggctgaggcaggagggtgcagtgagctgagatcgtgccactgcactccagcctgggcagcagagcaagactgtctcga

aaaataaataaataaaataqqaaqcqacaaqaaaqccactcaqatqgqqcqatttqqtctqqaaqqaqqgqataaqqatq

TCGAAGCTGACCCTACATGTGGGTGACGGTTTTGAGTTCATGAAACAGAATCAGGATGCCTTCGACGTGATCATCACTGA 4720

471

HUMAN SPERMIDINE SYNTHASE GENE

'

'

'

I

'

'

4810 I '

4820 '

'

I

'

'

'

'

4830 '

'

I

I

'

4840 I I

'

I

l

4850 I

'

I

4860 '

l ! I

I I

I

I I I

4870 I

I I

'

I

I-

l

4880 l

I

I I

'

'

I

ggccttcagagtaaaggatagagcggcctcccaccccccgaactagagctgtacttttcccttctcatttgttacctgcc ctctgaaacatggctcaggacagtaggcaggagccaggcgactgcccagattcacaagctggtgaccaaggagagtgggg atctggcattgggacactgaggaccctgtgtcctcttcagcctcccctctgctctgaagtggtcagcactggagtggggg caggttctagtcttgaacgaaggcctaggttagaggttcctctgctgtggtgccaatgagactcccccaagaatgggatt caggtgtggatcccccacagacctgggttcagatcctggctctggccacctggtagctgtgtgggtgacagtggccctgg aggtcacagccagactgttcagtgtgtctccctctgtcttccccagGCCCCGCCGAAAGTCTCTTCAAGGAGTCCTATTA CCAGCTCATGAAGACAGCCCTCAAGGAAGATGGTGTCCTCTGCTGCCAGGgtgagccacaggcctggagcactggggcgg ggcggggtggggcagggcaggccctgccggatgctgatgcttaggggcccccagGCGAGTGCCAGTGGCTGCACCTGGAC CTCATCAAGGAGATGCGGCAGTTCTGCCAGTCCCTGTTCCCCGTGGTGGCCTATGCCTACTGCACCATCCCCACCTACCC CAGCGGCCAGATCGGCTTCATGCTGTGCAGCAAGAACCCGgtgagatgggggtgtctgggggtgggggttggggggaagg tgggcataaatagagatccctgcccctgccgggcgcggtggctcacacctgtaacccagcactttgggaggctgaggcgg gcagatcacaaggtcaggagatcgagaccatcttggctaacacggcgaaaccccgtctctactaaaaatacaaaaaaatt agccaggcatggcagcgcgcgcctgtagtcccagctgctggggaggctgaggcaggagaatggcgtgaacccgggaggcg gagcttgcagtgagccgagattgcgccactacattccagcctgggtaacagaggaagattccgactcaaaaaaaaaaaaa aggccctccccaggccaggtgcggtgtctcatgcctgtaattccagcattttgggagaccaaggtgggcggatcacttga ggtcaggagtacaagaccagcctgaccaacatggagaaaccccatcactactcaaaatacaaaaaaaaaaaaaattagcc gggcgtggtggcgcgtacctgaggctgaggcaggagaat.cacttgaacctgggagacagaggt'tgcagtgagctgagatg acqccactgcactccagcgtgqcaacagtqagactccgtctcaaaaaaaaaaaaaaaagtgccccccctgatgtqcccct ggcccggtccccagAGCACGAACTTCCAGGAGCCGGTGCAGCCGCTGACACAGCAGCAGGTGGCGCAGATGCAGCTGAAG TACTACAACTCCGACGTGCACCGCGCCGCCTTTGTGCTGCCCGAGTTTGCCCGCAAGgtgggtggcctgcggggctgggt ggtgggacccagggacccagagcgccctcctgactggcctcatgtccctccagGCACTGAATGATGTGAGCTGAGCCCAG

GCGCCACCACTGATGCCACCCAGGACCTCGGACCTTGGAGCCTGCGGGGTGCCTCGGCCCCTCCAGCCCCGGGCCGGACC TCCTGCTGGCTCTCGCCCACCAACCAAGTGTTACAAGCCCCAGAATGCTGCCCGGCCTGCCCTGCTGGGCGGACTGTCTG TGTGTCTGTCTCTCTGGCGTTCCACCTCCAAGCCTATACCAGCTGTGTACAGCGCCATCTCTCTGCCTTCTGTTGCCCCT

CACTCACCAAACACGTGTATTTATAGCAAAGATTGGAGTCCTGTGTCTCCTGACCTTGGCTGGGCCCAGGCAGGGCCACA TTCACCATTGGGTGCCTCTGGGGTGAGGGTCTGCAGAGGCCTTGCTGGCTGACCCCCAAGTGTCTGCTGCAGGGCTGAGG CTGCAGGCGGGCCATCGTGGATAGCCTGGGGCACAGAGGGTCACCGCAGTCGTCACGTGGGACCCAGAGCTGTCCTGGGA AGCTGACTTAGCTGTCCTTTTACCAAGCCCTTCACAAGGCCACTGGTGACAGCCCCCCAGGGCAGTGGGGTGGGTGAGAT CAGGGTGGGGCTGCCCGGGAGCATTCTCAGAAAAATTGGGGACACTCACAGGTGTAAGTCAGGTCCCATCCAGGTACTCC

AGGGCAAATACAggaaggggtggcggggctggttaccttcggcctttttaagcacatcaggagcttaacactggcccagt gactgtgccctgactccacccggcattcagacttgggttcaaattcccaccatgccccgccccctatgtggacaaattga gaaagcaagtgtgggcaccccaccagggactgcgaggaccagggctgtcccctctccaaggtgctgaactcccgccttcc aggacccaacggtggtgggaggacaggaaaggaaccctctttgcatgggcctgagttgccaacccctttccccaccctgg gcaggggctgggctagcggacgcatcagggagggaggccccactcccagccgaggcagccaccttggagccctaactcac ccgggtatgttttctgggacaccagtgtaagggggattcagtttcgccatcaactctggcttcaggccagtcatagccct 7623 ccagtctccacctgccccccact

4880 4960 5040

5120 5200 5280 5360 5440 5520 5600 5680 5760 5840

5920 6000 6080 6160 6240 6320 6400 6480 6560 6640 6720 6800 6880 6960 7040 7120 7200 7280 7360 7440 7520 7600

FIG. 2. The nucleotide sequence of human spermidine synthase gene. Exons and introns are specified by capital and lowercase letters, respectively. The first nucleotide in the Pst I recognition sequence (see Fig. 1) is designated as nucleotide number 1. The Alu repeats in the 5'-flanking region and in the introns 3 and 6 are underlined. line contains at least

(Kolmer

et

fragments of human chromosome

1

al, 1991).

Expression of human spermidine synthase gene in CHO cells

To verify that the isolated human spermidine synthase clone hgSPDSYl was functional, the plasmid phgSPD65 was transfected into CHO-K1 cells with low endogenous

spermidine synthase activity. Spermidine synthase activity

of the cells transfected with phgSPD65 was three times higher at 48 hr after the transfection than in the cells transfected with herring sperm DNA. When measured at 72 hr after the transfection, the activity was still over two times higher than in the control cells (Table 2).

DISCUSSION The described human spermidine synthase gene probably represents a functional and actively expressed gene. This view is supported by the fact that the gene was transiently

expressed when transferred into Chinese hamster ovary cells (Table 2). Furthermore, the only discrepancy between the cDNA sequence for human spermidine synthase we recently reported (Wahlfors et al, 1990) and the present sequence of the coding region was a conservative replacement of the alanine codon 297 GCC by GCA, the latter triplet still coding for alanine. This difference may have been resulted from the different source of the cDNA (decidual) and genomic (myeloma) libraries used and may just represent DNA polymorphism. The latter possibility has not been proved because no suitable restriction enzyme

that cleaves at this site is available. Based on its promoter structure, the human spermidine synthase gene appears to belong to the so-called "housekeeping" genes characterized by an extreme GC richness and the lack of a TATA box (Dynan, 1986). The transcription factors, such as ETF, are supposed to bind to GC-rich regions of the promoter (Kageyama et al, 1989). If one looks at the promoter region of human spermidine synthase, it is not only GC-rich but contains several 5'-CCCC-3'

MYOHANEN ET AL.

"O

O.

CM

CO

CM CM CM

.

.

m

+

tM O

CM O Cvl

+

.

o

.

CO

+

CM +

.

m

+

CM

CO CM

CM +

+ LO

+ ICI

^3-

+

CM O)

E

o O

CO

E

CM

o

ü

CM CO +

Q

+

+

+

+

8D>++Q

.

++Q + +Q

+

+

+

+

Q

lo io

CM CM

co

CM o CD

C

it*0*0(>)ONOCMSn

Human spermidine synthase gene: structure and chromosomal localization.

The human spermidine synthase (EC 2.5.1.16) gene was isolated from a genomic library constructed with DNA obtained from a human immunoglobulin G (IgG)...
2MB Sizes 0 Downloads 0 Views