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The lipoprotein lipase-encoding human gene: sequence from intron-6 to intron-9 and presence in intron-7 of a 40-million-year-old Alu sequence
Gene, 110 (1992) 257-261 © 1992 Elsevier Science Publishers B.V. All rights reserved. 0378-1119/92/$05.00
257
GENE 06237
The lipoprotein lipase-encoding human gene: sequence from intron-6 to intron-9 and presence in intron-7 of a 40-million-year-old Alu sequence (Recombinant DNA; nucleotide sequencing; triglycerides; fatty acids; illegitimate recombination; phylogeny; exons; pathology; hyperchylomicronemia)
Jean-Claude Chuat a, Alain Raisonnier b, Jacqueline Etienne b and Francis Galiberta * Laborawire d'Hdmatologie Expdrimentale, Centre Hayem, H6pitai St-Louis, Paris (France) Te!.(33-1)42021605: and b Laboratoire de Biochim/e, Facultd de Mddecine St-Antoine, Paris (France) Received by G.Bernardi: 4 April 1991 Revised/Accepted: 8 July/25 September 1991 Received at publishers: 22 October 1991
SUMMARY
The complete nucleotide sequence of the 3877-bp segment spanning the 3' region of intron-6 to the 5' region of intron-9 of the human lipoprotein lipase (LPL)-encoding ten-exert gene, LPL, is reported. An Alu repeat present in intron-7 was found by sequence analysis to belong to the 40-55-million-year-old Alu-Se subclass.
INTRODUCTION
Lipoprotein lipase (LPL) plays a major role in the metabolism of circulating triglyceride-rich lipoproteins. It hydrolyzes chylomicron and VLDL triglycerides, thereby delivering fatty acids to tissues for storage or oxidation. Homology of the LPL gene with other lipase genes such as hepatic lipase (HL) and pancreatic lipase (PL) led to the recognition era new gene family (Kirchgessner et al., 1989). The gene coding for human LPL was assigned to chromosome 8 (p22) (Sparkes et al., 1987). Deficiencies of LPL
Correspondence to: Dr. J. Etienne, Biochimie,Laboratory 507, Facult~ de M~decine St-Antoine, 27 rue Chaligny, 75012 Paris (France) Tel. (33-1)40306249; Fax (33-1)40306490. Abbreviations: aa, amino acid(s); AluJ and Alu-S, [Alu-Sa (Aiu-Sd, AiuSe), Alu-Sc and Alu-Sb] subclasses oftheAiu family(see Fig. 3); bp, base pair(s); HL, hepatic iipase; kb, kilobase(s) or 1000 bp; LPL, lipoprotein lipase; LPL, gene encoding LPL; Myr, millionyears; nt, nucleotide(s); PL, pancreatic lipase; 7SL RNA, small cytoplasmic RNA required for both structural and functional properties of signal recognition protein; VLDL, very low density lipoproteins.
activity are at the origin of pathologic hyperchylomicronerajas. It is only since 1990 that some of these are understood at a molecular level, for instance, mutations such as deletion, insertion or substitution on coding exons have been described. Up to now, only the nt sequence of the exons (Wion et al., 1987), of the intron/exon boundaries and of the 5' upstream region of the gene (Deeb and Peng, 1989; Kirchgessner et al., 1989) have been reported. The human LPL gene is composed of ten exons and nine introns spanning about 30 kb. No intron has been entirely sequenced. However, introns oould also be involved in LPL pathology by mutation ,~t the exon-intron junction (Hata et al., 1990), by partial duplication (Devlin et al., 1990), or by any sort of sequence alteration causing an abnormal folding thereby impeding correct splicing. In this respect, Alu repeats, since they favor illegitimate recombinations, are now viewed as potential troublemakers in pathology (Groffen et al., 1989). The aim of the present study was to determine the nt sequence (3877 bp; Fig. 1) spanning from the last 0.198 kb of intron-6 to the first 0.721 kb of intron-9 with a view to getting more insight in the phylogeny of the lipase gene family.
258 qaatt._caaggt c t g c a t t t t = t a g g t a t g a a c a c t g t ~ f c a t g a t g a a g t c t t t c c a a g c c a c a c c a g t g g t t c o a t g t g t ~ g c a ~ t
ccg~ttga~g ( e x o n 7) a l ot agtgag~tactt ctgtggttotgaatt gcotgaot atttggggtt gtgatatttt eat aaagattgat caacat gtt cgaattt cot ccccaacagTC 320
330
.
100 200
340
PheHisTyrGlnValLys ileHisPheSerGlyThrGluSerGluThrHis ThrAsnGlnAlaPheGluIleSerLeuTyrGlyThrValAlaGluSerG T TCCAT TACCAAGTAAAGATTCAT TT T TCTGGGACTGAGAGTGAAACCCATACCAATCAGGCC TTTGAGATT TCTCTGTATGGCACCGTGGCCGAGAGTG
300
350
luAsnIleProPheThrLe AGAACATCCCATTCACTCTgtgagt agcacaggggc~gcggt =at =atggoaooagt =oct ot cctg=cat aaaocttggt =tgagcagcagaagcagaga gcgatgcot agaaaaoaagt ottt agttaaaaaaat cagaattt caaaattgaggt ottt oct ct attt gat attgagaaaaaaatgott caaatt ggco atttt attttcacttaot agtt at attttttt attt at oat ctt atat ot gttt atttotttt at aaagctgotgtt aaaoaat at aatt aaact at ot c aaaaggttt gacattaaagaaaatgagoaat ggt aacaggaaaccaot ot atagatgtacat ataat atgt acagaaaat at aagt agt aagaagt coat AIu sequence gacaaagtgtt agototttttttttttttttttttttttttttttgagatggagt ot ot ot ot attgccoaggotggagtgcagtgatt cgat ~ c a g o t
400 500 600 700 800
caotgcaa=ct ot acotocoga~ttcaaaoaatt ott ctgtot cagcot cocga~t agotggggotqcaggtg==caccaccat~cccagotaatttttq
~00
t attttt a ~ a g c q a c a g g ~ ot caccatgttggccaagot g ~ oft qaattootgat ot cagqtgat coacccgcot cggcct cccaaagtgotgggat
i000
t acaggt~gagccacoatgcccagcotacccttt act act aatcaaagaaat aaaagt aaggoaacttgat actttt acaatt actagatgaacaaatc
1100
tit aaaaatagocagtgcagacaaggtggtgaagcagaacatgogaaoot acoatgcat cart cacggct agaaccctccaggtgcggaaggt agt attt t aataactttccat agct acaaaat attatt aoat agaagggagtgattttttt ct aat attt at cot aaagaaatagtcaacaaacattttt aaaaaca t oaatt acagt cgt acct at act agcataaattagaaacccagt atccaacattgaggcagtgggt aaatgaatogtggttt at caagtcatt aaaa~ca at ct agccttt aaaaaot at aattgt aggaaacccaggaaaacat agt aaaaaatggaatataaaatc?gaagagaat aaagaat agagaat cgt atgtg tgctatgattgtag~aaataatgtt~aagtatcaa~acaaattgaaaaggaatacatgaaaatgaaaattatatttctgaatgattgacttcaggattt tcttttagaattg~attaaatagt~catgt~attaggataaatg~tggaatgtggatataatttaaaatata~taaatg~at~gac~tt~attttgagt t ctttgttggacatttttgtgoatttttaaaatat cccct aaataat aaagctattt at atttggagaggagaaaaaaaagtggggggcagggagagctg
1200 1300 1400 1500 1600 1700 1800
360
370
(exon 8) uProGluValSerThrAsnLysThrT~rSerPheLeuZleTyrTh=GluValAspIleGl¥ at ctct at aact aaccaaa~tatt gottttttgttt agGCCTGAAGTTTCCACAAATAAGACCTACTCCTTCCTAATTTACACAGAGGTAGATATTGGA 380
390
1900
400
GluLeuLeuMetLeuLysLeuLysTrpLys S e r A s p S e r T y r P h e S e r T r p S e : A s p T r p T r p S e ~ S e r P r o G l y P h e A l a Z l e G l n L y s I l e A r g V a l L GAACTAcTCATGTTGAAG~TcAAATGGAAGAGTGATTcATACTTTAGcTGGT~AGACTGGTGGAG~AGTcCCGG~TTCG~cATT~AGAAGATc~GAGTAA
2000
410
ysAlaGlyGluThrGlnLysL¥ AAGCAGGAGAGACTCAGAAAAAgt aatt aaat gt attttt ctt oott cacttt agaoooccac~tgatgtcaggacct aggggot gt attt oaggggcct t cacaatt cagggagagcttt aggaaaccttgtattt art actgt atgatgtagatttt ottt aggagt=tt ctttt attttottatttttggggggcgg ggggggaag~gacagt atttttgt a~ttcatgtaaggaaaacat aagccotgaatogo~cacagttattoagtgagagctgggatt agaagt caggaat c t cagctt ot oatttggoaot~t~ottgt aagtaoaaaatag~t aggqaaoaaacot cogagatgot ao~tggataatoaaagatt oaaaooaacotott ccagaagggtgagattooaagat a~t c~oaao~gt ot oogoagooco.aoooatgtgtaoooata~aatgaatt aoacagagat cgot at aggatttaaa gctttt at act aaa~gtgotgggatt~tgoaaao~at agtg~gotgt~att~taattt aaaaaaaototaagttaggattgacaaat~attt~t ot~t a gt cattt gcttgtatoaooaaagaagcaaacaaaoaaaoaaaaaa~aaagaaaaagatottggggatggaaatgttat aaagaat ~tttttt acaot ag oaatgt ot agotgaaggoagatgooot aatt o ~ t aatgoagatgot aagagatggoagag~tgatctttt at cat ot ottggtgaaagoocagtaaoat aagaotgot ot agg~tgtotgoatgootgt ota~ot aaatt aaot agottggttgotgaaoaooaggttaggot ~caaatt aooototgatt ~ g a t g t ggootgag~g~gaoagtt aat~a~tgggaat a~oaaaaoaat~acooagoatgat oatgtatt at~t aaaoagtcotgaoagaaotgt aoctttgtgaac 420
2100 2200 2300 2400 2500 2600 2700 2800 2900 3000
430
(exon 9) sVal~lePheCysSerArgGluLysValSerRisLeuGlnLysGlyLys agt gcttttgatt gtt ot aoatggo~t art caoat ccatttt ctt coacagGGTGATCTTCTGT~CTAGGGAGAAAGTGTCTCATTTGCAGAAAGGAAAG
3100
440
AlaProAlaValPheValLysCysHisAspLysSerLeuAsnLysLysSerGl~OP GCACCTGCGGTATTTGTGAAATG~CATGACAAGTCTCTGAATAAGAAGTCAGGCTGgtgagcatt ~tgggot aaagotga~gggoat cot gagcttgca ccotaagggaggcagcttcatgcattcet ctt oacc¢oat ¢aocagoagottgoootgaotoatgtgatoaaagcatt oaat cagt~tttctt agtcctt ctgoatatgtat oaaatgggtotgttg~ttatgcaataottcctotttttttctttotoctottgttt =tcccagcccggacottoaacccaggoacac atttt aggtttt atttt aotoottgaaot aococtgaatott cacti otoottttttotot aotgogtototgotgaotttgcagatgccatotgcagag cat gt aacaoaa~ttt agt agtt googtt ot ggotgt gggt g c a g ~ o t t oocaggatgt art oagggaagt aaaaagat ~caotgcat caoctgcagc oacat agtt oft gait ot ooaagtgooagoat act ocgggacacaoagccaacaggg~t goocoaagcacooatt ot oaaaaccot oaaagotgccaagc aaacagaatgagagtt ataggaaaotgtt ot otcttot atctccaaaoaaot~tgtgoot otttcot aoctgacottt agggot aatcoatgtggcagct gtt agct goat ottt coagagcgt oagt actgagaggaoaot aagoat gt gaoott caot actcct gttct qaatt..o 3877
3200 3300 3400 3500 3600 3700 3800
Fig. 1. Nucleotide sequence (3877 bp) spanning from the 3' end of intron-6 to the 5' end of intron-9 in the human L P L gene (GenBank/EMBL accession No, M76722). The Alu sequence is double underlined. The genomic library used for isolation of clones was a partial S a u 3 A digest of human lymphocyte genomic DNA cloned into the ;tEMBL4 vector (Eladari et al., 1986). It was screened with a I50-mer probe synthetized by the phosphoramidite method. This probe corresponded to the last 14 nt of exon-7 and to the 135 first nt of exert-8 plus 1 nt added to create a 5' B a m H I restriction site for cloning into the MI3 vector. One of the clones thus isolated was fragmented by EcoRI. Two contiguous fragments of 947 bp and 2924 bp were obtained and sequenced by the dideoxy chain-termination method. Three E c o R l sites at the extremities and at the junction between the two contiguous subclones are underlined with squiggly lines.
259 1
EXPEI~.IMENTALAND DISCUSSION
(a) Sequence of part of the LPL gene This 3877-bp sequence is composed of the 3' part of intron-6 (1-198), exon-7 (199-319), intron-'/(320-1839), exon-8 (1840-2022), intron-8 (2023-3051 ), exon-9 (30523156), the stop codon being interrupted by intron-9, and finally the 5' part of intron-9 (3157-3877) (Fig. 1). A glycoprotein consisting of 448 aa, LPL, is synthesized by different cells including adipocytes. After secretion, active LPL is present at the luminal surface of endothelial cells, where it binds to membrane giycosaminoglycans, so no LPL is found in plasma unless it has been previously released by means of heparin injection. Three types of LPL deficiency, named Brunzell's classes (Auwerx et al., 1989), have been described. In class I, LPL is absent in pre- and post-heparin plasma ('null allele' secondary to major defects in the LPL gene); in class II, LPL is released by heparin but has no enzymatic activity; in class Ill, LPL has no enzymatic activity either and furthermore is unable to bind to endothelial cells, so it is spontaneously present in plasma. Knowledge of the sequence of the whole of intron-7 and intron-8 and of part of intron-6 and intron-9 could assist in exploring these types of LPL deficiency at the molecular level.
OGCCGGGCGCGGTGGCTCACGCCTGTAATC GGCTGGGCATGGTGGCTCACACCTGTAATC GGC'~.CATGCCTGTAATC 31
CCAGCACTTTGGGAGGCCGAGGCGGGCGGA CCAGCACTTTGGGAGGCCGAGGCGGGTGGA CCAGCACTTTGGGAGGCCTTGGTGGGTGAA 61 TCACCTGAGGTCAGGAGTTCGAGACCAGCC TCACCTGAGATCAGGAATTCAAGACCAGCT
TCACCTGAGGTCAGGAGTTTGAGACTAGCT
91
TGGCCAACAT~GTGAAACCCCGTCTCTACT TGGCCAACATC~GTGAGACCCTGTCGCTACT TGGCCAACATGGTGAAACCCTGTTTCTACT 121 AAAAATACAAAAA AAAAATACAAAAA A A G (A)s T A C A G A A A Liaker
TTAGCCGGGCGTGGTGG
TTAGCTGGGCATGGTGG TTAGTTGGGCGTGGTGG
151 AlaI CGCGCGCCTGTA ATCCCAGCTACTCGGGAG TGGGCACCTGCAGCCCCAGCTACTCGGGAG
CGGGTGCCTGTAATCCCAGTTACTCAGGAG 181 GCT
GAGGCAGGAGAATCGCTTGAACCCGGG
GCT
GAGGCAGGAGAATCGCTTGAACCGGA
GCTGAGACAGAAGAATTGTTTGAACTCGGG A
211
AGGCGGAGGTTGCAGTGAGCCGAGATCGCG AGGTAGAGGTTGCAGTGAGCTGAGATCGAA A.GCAGAGGTTGCAGTGAGCTAAGATTGCG
241
CCACTGCACTCCAGCCTGGGCGACAGAGCG TCACTGCACTCCAGCCTGGGCAATAGAGAG
CCGCTGCACTCCAGCCTGGGTAACTGAGCG
(b) Identification of an Alu sequence in intron-7 One complete Alu sequence (282 bp), inserted in the opposite direction, has been identified in the middle of intron-7 between 1027 and 746 (Fig. 1). The direct orientation can be aligned with the consensus sequence established by Jurka and Smith (1988) from 125 known Alu sequences (Fig. 2). This Alu sequence is followed by (dA)29. No direct repeats can be found around them. The A + T richness in the noncoding and non-Aiu sequences ofintron7, with a ratio A + T/G + C of 2.3 as compared to 1.6 for the mean human genornic DNA, is noteworthy. The presence of an Alu sequence in intron-7 of the human LPL gene had been mentioned by Deeb and Peng (1989), but no nt sequence of intron-7 was presented.
(c) The Alu sequence of intron-6 Another Alu sequence has been identified in intron-6 of LPL gene by Devlin et al. (1990). This intron was only partially sequenced (0.4 kb out of 2.8 kb) and sequencing of the Alu sequence started from nt position 14 (to 282). This intron-6 dlu sequence is also transposed in the opposite direction relative to the LPL gene and is followed by a (TAAA)t0 tandem repeat°
(d) Alu sequences and genetic rearrangements in pathology The biological role of Alu sequences is unknown. Some of them have been involved in genetic rearrangements. For
271
282
AGACTCCGTCTC AGACTCCATCTC(A) 29 AGACTGTGTCTCAAAAA~AAA)
I0
Fig. 2. Alignmentof A/u sequences. Top, middle, and bottom lines: consensus proposed by Jurka and Smith(1988)and Raisonnier(1991)intron7 (present results), and intron-6 (Devlin et al., 1990)Alu sequences, respectively. The usual hallmarks of Alu sequences (a 120-bp left and a 149-bpnearly homologousright arm, connected by the A-richlinkerwith its TAC triplet) and the Alul restriction site are indicated. instance, in the case of the LPL gene a rearrangement implied a breakpoint in intron-6 with insertion at this level of a 2-kb segment consisting of about the 3' half of exon6 (0.1 kb), followed by the 5' two thirds ofintron-6 (1.9 kb). This duplication resulted in LPL deficiency causing hyperchylomicronemia (Devlin et al., 1990). Actually, the breakpoint in intron-6 is not within the Alu sequence, but in the repeat flanking it. Another example is that of the LDL receptor gene. A recombination between two homologous Aiu sequences of this gene caused a deletion of the exons encoding the transmembrane and cytoplasmic domains of the LDL receptor, so that the resulting nonfunctional receptor could not anchor into the cell membrane (Lehrman et al., 1985). A form of familial hypercholesterolemia can be ascribed to this mechanism. It is known that in human chronic myelocytic leukemia
260 the abnormal chromosome 22 (Philadelphia chromosome) results from translocation between chromosomes 9 and 22. Groffen et al. (1989) by sequencing the region around the breakpoints (gene bcr for chromosome 22, gene abl for chromosome 9) could find Alu-repetitive sequences near, or as part of, the breakpoint in all but one of the investigated patients. These observations suggest that Alu-repetitive sequences may facilitate chromosomal translocations. However, it is not likely that Alu sequences are a prerequisite for this type of chromosomal translocation. At least four different cases of thalassemias have been caused by illegitimate recombination between Alu sequences (Vanin et al., 1983). Because Alu sequences constitute 3-5% of the human genome, these occurrences could be due to chance. However it is quite possible that Alu sequences are hot spots for illegitimate recombination (Groffen et al., 1989).
dated must be placed in one of the Alu subfamilies (Quentin, 1988; Britten et al., 1988; Raisonnier, 1991). According to Jurka and Smith (1988), there are two distinct families of Alu sequences: Alu-J (for Jurka) and Alu-S (for Smith). This classification is based on the different base preferences for a number of diagnostic sequence positions. The two Alu repeats examined here were compared to the Alu sequence consensus proposed by Jurka and Smith (1988) and Raisonnier (1991). By following the procedure proposed by Jurka and Smith (1988) and described in Fig. 3 (top panel), the two Alu repeats clearly belong to the Alu. S family. Aiu-S has in turn been divided into three subclasses: Alu.Sa, Alu-Sb and Alu-Sc. By using another set of diagnostic positions, also shown in Fig. 3 (middle panel), the two Alu sequences were found to belong to the Alu-Sa subclass. Finally, the Alu-Sa subclass is split into Alu-Sd and Alu-Se. As shown in Fig. 3 (bottom panel), Alu sequences of both intron-6 and intron-7 are members of the AIu-Se family.
(e) Classification of the Alu sequences from intron-6 and intron-7 Since Alu sequences have been transposed in the genome at different times in the history of primates, they can be used as paleogenetic markers. For this, the sequence to be
(f) Dating of the Alu sequences from intron-6 and intron-7 An approximate dating of the different Alu subfamilies is given in Fig. 4. The Alu-J family is probably the oldest.
FAMILIES ALU-J AND ALU.S : S7
63
65
70
71
94
lO1 106 163
A C
G A
T C
C G
C T
G C
A G
G A
G A
G A
G A
A G
C T
T A
C T
*
Intron 7
T
A
C
A
T
C
G
G
G
A
A
G
T
A
T
fills
AIu Intron 6
T
A
C
G
T
C
G
A
A
A
A
G
T
A
T
14115
A lu.J Alu.S Alu
194 204 208 220 233 27S
SUBFAMILIES OF ALU.S (-Sa, -Sb and -So) : 65
66
78
88
9S
100
153
163
197
Aiu-Sa Alu.Sb A lu - S e
C
T
T A A
G T T
C T C
T C T
C G T/G
A G G
C G C
T G T
G C G
A lu Alu
C C
T T
T T
G Q
C C
T T
C G
A A
T C
T T
G G
Intron 7 Intron 6
200
219
1011l 10111
SUBFAMILIES OF ALU-Sa (-Sd and -Se) : 244 Alu.Sd A/u.Se
T C
Alu Intron 7 A lu intron 6
C c
Inserllon 204 + 1 A .
272 A G
G G
2/2 212
Fig. 3. Classification of the Alu sequences of intron-7 (present results) and of intron-6 (Devlin et al., 1990) into A/u families and subfamilies. The classification of the two Alu sequences was worked out according to Jurka and Smith (1988) by comparing their sequence to the Alu sequence consensus (see Fig. 2, top line) proposed by Jurka and Smith (1988) and Raisonnier (1991) at the diagnostic positions indicated in the top panel, top line. With more than nine identities (see column marked with asterisk), both intron-6 and intron-7-Alu repeats belong to the Alu-S family. Subclasses of the Alu-S family (Aiu-Sa, -Sb, and -Sc) are based on the diagnostic positions appearing in the middle panel: with more than five identities, both Alu repeats fall in the Alu-Sa family. The Alu-Sa is in turn split into Alu-Sd and Alu-Se from diagnostic positions shown in the bottom panel. (The insertion 264 + 1 means that in Alu-Sd there is an insertion just downstream from position 264): the two repeats examined here can be assigned to Alu-Se.
26! Myr 65-S$
Alu-J Alu-S • Alu-Sa • Alu-Se • Alu.Sb
(i.e., A l u - S d and A l u . S e )
55-40 40-35 35-20
Fig. 4. PaleogenetichistoryoftheAlu sequencefamilies.The time elapsed since Alu repeat individuationwas estimated from the number of random independentmutations,i.e., in the positionsthat are neitherdiagnosticnor CpG (CpG are not informativebecause they are hot spots for substitution, Britten et al., 1988).Alu elementsbelongingto the A lu-Se familywere retrotranscribed before 40 Myr, when New World and Old World monkeys diverged, while those belongingto the Alu-Sc and -Sb familiesare younger (Raisonnier, 1991). For a definitionof Alu families,see legendto Fig. 3. Partial similarity with 7SL RNA, a component of the signal recognition particle, has been shown. Approx. 100 nt at the 5' end and 45 nt at the 3' end of this R N A are homologous with the h u m a n Alu right monomer consensus sequence. This indicates that the Alu sequence may have been retrotranscribed from this R N A after deletion of the central 7SL-specific sequence (Jurka and Smith, 1988). Alu-Sc and Alu-Sb sequences were transposed into the primate genome between 40-Myr-ago and the present time (Britten et al., 1988; Raisonnier, 1991). Thus, according to this evolutionary time scale, the Alu-Se sequences ofintron6 and intron-7 of L P L gone might have been transposed in the primate genome more than 40-Myr-ago. (g) A l u sequences and lipase gone family Comparison of L P L , H L and PL gone organization has
recently enabled Kirchgessner et al. (1989) to propose a model for the evo,lution of this lipase gone family. All these genes are thought to have evolved through multiple rounds of gone duplication plus intron-loss and oxen-shuffling events from an ancestor gone containing 14 introns. As the P L gone has undergone the fewest changes, it can be held to be the closest to the primordial gone. Intron-7 is one of the introns that has been maintained at the same place relative to the exons since the primordial gone. As this intron in L P L includes an Alu repeat, it would be of interest to know if it also does in P L and in the affirmative, to which class i~ belongs. However, it must be borne in mind that L P L and PL ~ave been described in fish (Rasco and Hultin, 198g), birds and mammals, and thus exist ~s separate enzymc,s as early as the fish level on the evolutionary scale. As for HL, it has been described in dog (Greten et al., 1974), rat and man, but has not been hitherto reported in species below the dog level. The successive divergence of this gone family is most likely to be older than the retroposition of the most ancient Alu-J monomeric sequence. Thus, these genes would not contain any orthologous Alu sequence.
ACKNOWLEDGEMENTS This work was supported by grants from the Centre National de la Recherche Scientifique through UPR-41 and from the Institut National de la Sant6 et de la Recherche M6dicale (grant 900 203 to JE). We thank B6atrice Pelletier for typing this manuscript. REFERENCES Auwerx,J.H., Babirak, S.P., Fujimoto,W.Y., lverius, P.H. and Brunzell, J.D.: Detective enzymeprotein in lipoprotein lipase deficiency.Eur. J. Clin. Invest. 19 (1989) 433-437. Britten, RJ., Baron, W.F., Stout, D.B. and Davidson,E.H.: Sources and evolution of human Alu repeated sequences. Prec. Natl. Acad. Sci. USA 85 (1988) 4770-4774. Deeb, S.S. and Pens, R.: Structure of the human lipoproteinlipase gone. Biochemistry28 (1989) 4131-4135. Devlin, R.H., Deeb, S., Brunzell, J. and Hayden, M.R.: Partial gone duplicationinvolvingexon.Aiu interchangeresults in lipoproteinlipase deficiency.Am. J. Hum. Goner. 46 (1990) 112-119. Eladari, M.E., Syed, S.H., Guilhot, S., d'Auriol, L. and Galibert, F.: On the high conservation of the human c-myc first oxen. Biochem. BiGphys. Res. Commun. 140 (1986) 313-319. Greten, H., Sniderman,A.D., Chandler J.G., Steinberg, D. and Brown, W.V.: Exidencefor the hepaticoriginof a canine post-heparinplasma trigl3ceri~e lipase. FEBS Lett. 42 (1974) 157-160. C~roffen,~., Her~lans, A., Grosveld, G. and Heisterkamp, N.: Molecular analysis of chromosome breakpoints. Pros. Nucleic Acid Res. Mol. Biol. 36 (1989) 281-300. Hota, A.., Emi, M., Luc, G., Basdevant, A., Gambert, P., lverius, P.H. and Lalouel,J.M.: Compound heterozygote for lipoproteinlipase de~ciency: Ser---,ThrTM and transition in 3' splice site of intron-2 (AG ~ AA) in the lipoprotein lipase gene. Am. J. Hum. Genet. 47 (1990) 721-726. Jurka, J. and Smith, T.: A fundamentaldivision in the Alu familyof repeated sequences. Prec. NatL.Acad. Sci. USA 85 (1988) 4775-4778. Kirchgessner,T.G., Chuat, J.C., Heinzmann, C., Etienne,J., Guilhot, S., Svenson, K., Ameis,D., Pilon, C., d'Auriol, L., Andalibi,A., Schotz, M.C., Oalibert, F. and Lusis, A.J.: Organization of the human lipoprotein lipase gone and evolution of the lipase gone family. Prec. Natl. Acad. Sci. USA 86 (1989) 9647-9651. Lehrman, M.A., Schneider,WJ., Sudhof, T.C., Brown, M.S., Golstein, J.L. and Russel, D.W.: Mutation in LDL receptor: Alu-Alu recombination deletes exons encoding transmembrane and cytoplasmic domains. Science 227 (1985) 140-146. Quentin, Y.: The Alu familydeveloped through successivewaves of fixation closelyconnected with primate lineagehistory.J. Mol. Evol. 27 (1988) 194-202. Raisonnier,A.: Duplicationof the apolipoproteinC-I goneoccurred about forty millionyears ago. J. Mol. Evol. 32 (1991) 211-219. Rasco, B.A. and Hultin H.O.: A comparison of dogfish and porcine pancreatic lipases. Comp. Biochem. Physiol. 89 (1988) 671-677. Sparkes, R.S., Zollman, S., Klisak, I., Kirchgessner,T.G., KcJmaromy, M.C,. Mohand~st, T., Schotz, M.C. and Lusls, AJ.: Human genes involved in lipolysisof plasma lipoproteins: mapping of loci for lipoprotoin ~ipase to 8p22 and hepatic l[pase to 15q21, Genomics 1 (1987) 138-144. Vanin, E.F., Henth~rn, P.S.,.KiLoussis,D., Gr~sveld,F. and Smithies,O.: Unexpected rdationships between four large deletions in the human p-globin gone c~uster. Cell 35 (1983) 701-709. Wion, K.L., Kirchgessner,T.G., Lusis, A.J., Schotz, M.C. and Lawn, R.M.: Human lipoprotein!/pasocomplementaryDNA sequence. Science 235 (1987) 1638-1641.
257
GENE 06237
The lipoprotein lipase-encoding human gene: sequence from intron-6 to intron-9 and presence in intron-7 of a 40-million-year-old Alu sequence (Recombinant DNA; nucleotide sequencing; triglycerides; fatty acids; illegitimate recombination; phylogeny; exons; pathology; hyperchylomicronemia)
Jean-Claude Chuat a, Alain Raisonnier b, Jacqueline Etienne b and Francis Galiberta * Laborawire d'Hdmatologie Expdrimentale, Centre Hayem, H6pitai St-Louis, Paris (France) Te!.(33-1)42021605: and b Laboratoire de Biochim/e, Facultd de Mddecine St-Antoine, Paris (France) Received by G.Bernardi: 4 April 1991 Revised/Accepted: 8 July/25 September 1991 Received at publishers: 22 October 1991
SUMMARY
The complete nucleotide sequence of the 3877-bp segment spanning the 3' region of intron-6 to the 5' region of intron-9 of the human lipoprotein lipase (LPL)-encoding ten-exert gene, LPL, is reported. An Alu repeat present in intron-7 was found by sequence analysis to belong to the 40-55-million-year-old Alu-Se subclass.
INTRODUCTION
Lipoprotein lipase (LPL) plays a major role in the metabolism of circulating triglyceride-rich lipoproteins. It hydrolyzes chylomicron and VLDL triglycerides, thereby delivering fatty acids to tissues for storage or oxidation. Homology of the LPL gene with other lipase genes such as hepatic lipase (HL) and pancreatic lipase (PL) led to the recognition era new gene family (Kirchgessner et al., 1989). The gene coding for human LPL was assigned to chromosome 8 (p22) (Sparkes et al., 1987). Deficiencies of LPL
Correspondence to: Dr. J. Etienne, Biochimie,Laboratory 507, Facult~ de M~decine St-Antoine, 27 rue Chaligny, 75012 Paris (France) Tel. (33-1)40306249; Fax (33-1)40306490. Abbreviations: aa, amino acid(s); AluJ and Alu-S, [Alu-Sa (Aiu-Sd, AiuSe), Alu-Sc and Alu-Sb] subclasses oftheAiu family(see Fig. 3); bp, base pair(s); HL, hepatic iipase; kb, kilobase(s) or 1000 bp; LPL, lipoprotein lipase; LPL, gene encoding LPL; Myr, millionyears; nt, nucleotide(s); PL, pancreatic lipase; 7SL RNA, small cytoplasmic RNA required for both structural and functional properties of signal recognition protein; VLDL, very low density lipoproteins.
activity are at the origin of pathologic hyperchylomicronerajas. It is only since 1990 that some of these are understood at a molecular level, for instance, mutations such as deletion, insertion or substitution on coding exons have been described. Up to now, only the nt sequence of the exons (Wion et al., 1987), of the intron/exon boundaries and of the 5' upstream region of the gene (Deeb and Peng, 1989; Kirchgessner et al., 1989) have been reported. The human LPL gene is composed of ten exons and nine introns spanning about 30 kb. No intron has been entirely sequenced. However, introns oould also be involved in LPL pathology by mutation ,~t the exon-intron junction (Hata et al., 1990), by partial duplication (Devlin et al., 1990), or by any sort of sequence alteration causing an abnormal folding thereby impeding correct splicing. In this respect, Alu repeats, since they favor illegitimate recombinations, are now viewed as potential troublemakers in pathology (Groffen et al., 1989). The aim of the present study was to determine the nt sequence (3877 bp; Fig. 1) spanning from the last 0.198 kb of intron-6 to the first 0.721 kb of intron-9 with a view to getting more insight in the phylogeny of the lipase gene family.
258 qaatt._caaggt c t g c a t t t t = t a g g t a t g a a c a c t g t ~ f c a t g a t g a a g t c t t t c c a a g c c a c a c c a g t g g t t c o a t g t g t ~ g c a ~ t
ccg~ttga~g ( e x o n 7) a l ot agtgag~tactt ctgtggttotgaatt gcotgaot atttggggtt gtgatatttt eat aaagattgat caacat gtt cgaattt cot ccccaacagTC 320
330
.
100 200
340
PheHisTyrGlnValLys ileHisPheSerGlyThrGluSerGluThrHis ThrAsnGlnAlaPheGluIleSerLeuTyrGlyThrValAlaGluSerG T TCCAT TACCAAGTAAAGATTCAT TT T TCTGGGACTGAGAGTGAAACCCATACCAATCAGGCC TTTGAGATT TCTCTGTATGGCACCGTGGCCGAGAGTG
300
350
luAsnIleProPheThrLe AGAACATCCCATTCACTCTgtgagt agcacaggggc~gcggt =at =atggoaooagt =oct ot cctg=cat aaaocttggt =tgagcagcagaagcagaga gcgatgcot agaaaaoaagt ottt agttaaaaaaat cagaattt caaaattgaggt ottt oct ct attt gat attgagaaaaaaatgott caaatt ggco atttt attttcacttaot agtt at attttttt attt at oat ctt atat ot gttt atttotttt at aaagctgotgtt aaaoaat at aatt aaact at ot c aaaaggttt gacattaaagaaaatgagoaat ggt aacaggaaaccaot ot atagatgtacat ataat atgt acagaaaat at aagt agt aagaagt coat AIu sequence gacaaagtgtt agototttttttttttttttttttttttttttttgagatggagt ot ot ot ot attgccoaggotggagtgcagtgatt cgat ~ c a g o t
400 500 600 700 800
caotgcaa=ct ot acotocoga~ttcaaaoaatt ott ctgtot cagcot cocga~t agotggggotqcaggtg==caccaccat~cccagotaatttttq
~00
t attttt a ~ a g c q a c a g g ~ ot caccatgttggccaagot g ~ oft qaattootgat ot cagqtgat coacccgcot cggcct cccaaagtgotgggat
i000
t acaggt~gagccacoatgcccagcotacccttt act act aatcaaagaaat aaaagt aaggoaacttgat actttt acaatt actagatgaacaaatc
1100
tit aaaaatagocagtgcagacaaggtggtgaagcagaacatgogaaoot acoatgcat cart cacggct agaaccctccaggtgcggaaggt agt attt t aataactttccat agct acaaaat attatt aoat agaagggagtgattttttt ct aat attt at cot aaagaaatagtcaacaaacattttt aaaaaca t oaatt acagt cgt acct at act agcataaattagaaacccagt atccaacattgaggcagtgggt aaatgaatogtggttt at caagtcatt aaaa~ca at ct agccttt aaaaaot at aattgt aggaaacccaggaaaacat agt aaaaaatggaatataaaatc?gaagagaat aaagaat agagaat cgt atgtg tgctatgattgtag~aaataatgtt~aagtatcaa~acaaattgaaaaggaatacatgaaaatgaaaattatatttctgaatgattgacttcaggattt tcttttagaattg~attaaatagt~catgt~attaggataaatg~tggaatgtggatataatttaaaatata~taaatg~at~gac~tt~attttgagt t ctttgttggacatttttgtgoatttttaaaatat cccct aaataat aaagctattt at atttggagaggagaaaaaaaagtggggggcagggagagctg
1200 1300 1400 1500 1600 1700 1800
360
370
(exon 8) uProGluValSerThrAsnLysThrT~rSerPheLeuZleTyrTh=GluValAspIleGl¥ at ctct at aact aaccaaa~tatt gottttttgttt agGCCTGAAGTTTCCACAAATAAGACCTACTCCTTCCTAATTTACACAGAGGTAGATATTGGA 380
390
1900
400
GluLeuLeuMetLeuLysLeuLysTrpLys S e r A s p S e r T y r P h e S e r T r p S e : A s p T r p T r p S e ~ S e r P r o G l y P h e A l a Z l e G l n L y s I l e A r g V a l L GAACTAcTCATGTTGAAG~TcAAATGGAAGAGTGATTcATACTTTAGcTGGT~AGACTGGTGGAG~AGTcCCGG~TTCG~cATT~AGAAGATc~GAGTAA
2000
410
ysAlaGlyGluThrGlnLysL¥ AAGCAGGAGAGACTCAGAAAAAgt aatt aaat gt attttt ctt oott cacttt agaoooccac~tgatgtcaggacct aggggot gt attt oaggggcct t cacaatt cagggagagcttt aggaaaccttgtattt art actgt atgatgtagatttt ottt aggagt=tt ctttt attttottatttttggggggcgg ggggggaag~gacagt atttttgt a~ttcatgtaaggaaaacat aagccotgaatogo~cacagttattoagtgagagctgggatt agaagt caggaat c t cagctt ot oatttggoaot~t~ottgt aagtaoaaaatag~t aggqaaoaaacot cogagatgot ao~tggataatoaaagatt oaaaooaacotott ccagaagggtgagattooaagat a~t c~oaao~gt ot oogoagooco.aoooatgtgtaoooata~aatgaatt aoacagagat cgot at aggatttaaa gctttt at act aaa~gtgotgggatt~tgoaaao~at agtg~gotgt~att~taattt aaaaaaaototaagttaggattgacaaat~attt~t ot~t a gt cattt gcttgtatoaooaaagaagcaaacaaaoaaaoaaaaaa~aaagaaaaagatottggggatggaaatgttat aaagaat ~tttttt acaot ag oaatgt ot agotgaaggoagatgooot aatt o ~ t aatgoagatgot aagagatggoagag~tgatctttt at cat ot ottggtgaaagoocagtaaoat aagaotgot ot agg~tgtotgoatgootgt ota~ot aaatt aaot agottggttgotgaaoaooaggttaggot ~caaatt aooototgatt ~ g a t g t ggootgag~g~gaoagtt aat~a~tgggaat a~oaaaaoaat~acooagoatgat oatgtatt at~t aaaoagtcotgaoagaaotgt aoctttgtgaac 420
2100 2200 2300 2400 2500 2600 2700 2800 2900 3000
430
(exon 9) sVal~lePheCysSerArgGluLysValSerRisLeuGlnLysGlyLys agt gcttttgatt gtt ot aoatggo~t art caoat ccatttt ctt coacagGGTGATCTTCTGT~CTAGGGAGAAAGTGTCTCATTTGCAGAAAGGAAAG
3100
440
AlaProAlaValPheValLysCysHisAspLysSerLeuAsnLysLysSerGl~OP GCACCTGCGGTATTTGTGAAATG~CATGACAAGTCTCTGAATAAGAAGTCAGGCTGgtgagcatt ~tgggot aaagotga~gggoat cot gagcttgca ccotaagggaggcagcttcatgcattcet ctt oacc¢oat ¢aocagoagottgoootgaotoatgtgatoaaagcatt oaat cagt~tttctt agtcctt ctgoatatgtat oaaatgggtotgttg~ttatgcaataottcctotttttttctttotoctottgttt =tcccagcccggacottoaacccaggoacac atttt aggtttt atttt aotoottgaaot aococtgaatott cacti otoottttttotot aotgogtototgotgaotttgcagatgccatotgcagag cat gt aacaoaa~ttt agt agtt googtt ot ggotgt gggt g c a g ~ o t t oocaggatgt art oagggaagt aaaaagat ~caotgcat caoctgcagc oacat agtt oft gait ot ooaagtgooagoat act ocgggacacaoagccaacaggg~t goocoaagcacooatt ot oaaaaccot oaaagotgccaagc aaacagaatgagagtt ataggaaaotgtt ot otcttot atctccaaaoaaot~tgtgoot otttcot aoctgacottt agggot aatcoatgtggcagct gtt agct goat ottt coagagcgt oagt actgagaggaoaot aagoat gt gaoott caot actcct gttct qaatt..o 3877
3200 3300 3400 3500 3600 3700 3800
Fig. 1. Nucleotide sequence (3877 bp) spanning from the 3' end of intron-6 to the 5' end of intron-9 in the human L P L gene (GenBank/EMBL accession No, M76722). The Alu sequence is double underlined. The genomic library used for isolation of clones was a partial S a u 3 A digest of human lymphocyte genomic DNA cloned into the ;tEMBL4 vector (Eladari et al., 1986). It was screened with a I50-mer probe synthetized by the phosphoramidite method. This probe corresponded to the last 14 nt of exon-7 and to the 135 first nt of exert-8 plus 1 nt added to create a 5' B a m H I restriction site for cloning into the MI3 vector. One of the clones thus isolated was fragmented by EcoRI. Two contiguous fragments of 947 bp and 2924 bp were obtained and sequenced by the dideoxy chain-termination method. Three E c o R l sites at the extremities and at the junction between the two contiguous subclones are underlined with squiggly lines.
259 1
EXPEI~.IMENTALAND DISCUSSION
(a) Sequence of part of the LPL gene This 3877-bp sequence is composed of the 3' part of intron-6 (1-198), exon-7 (199-319), intron-'/(320-1839), exon-8 (1840-2022), intron-8 (2023-3051 ), exon-9 (30523156), the stop codon being interrupted by intron-9, and finally the 5' part of intron-9 (3157-3877) (Fig. 1). A glycoprotein consisting of 448 aa, LPL, is synthesized by different cells including adipocytes. After secretion, active LPL is present at the luminal surface of endothelial cells, where it binds to membrane giycosaminoglycans, so no LPL is found in plasma unless it has been previously released by means of heparin injection. Three types of LPL deficiency, named Brunzell's classes (Auwerx et al., 1989), have been described. In class I, LPL is absent in pre- and post-heparin plasma ('null allele' secondary to major defects in the LPL gene); in class II, LPL is released by heparin but has no enzymatic activity; in class Ill, LPL has no enzymatic activity either and furthermore is unable to bind to endothelial cells, so it is spontaneously present in plasma. Knowledge of the sequence of the whole of intron-7 and intron-8 and of part of intron-6 and intron-9 could assist in exploring these types of LPL deficiency at the molecular level.
OGCCGGGCGCGGTGGCTCACGCCTGTAATC GGCTGGGCATGGTGGCTCACACCTGTAATC GGC'~.CATGCCTGTAATC 31
CCAGCACTTTGGGAGGCCGAGGCGGGCGGA CCAGCACTTTGGGAGGCCGAGGCGGGTGGA CCAGCACTTTGGGAGGCCTTGGTGGGTGAA 61 TCACCTGAGGTCAGGAGTTCGAGACCAGCC TCACCTGAGATCAGGAATTCAAGACCAGCT
TCACCTGAGGTCAGGAGTTTGAGACTAGCT
91
TGGCCAACAT~GTGAAACCCCGTCTCTACT TGGCCAACATC~GTGAGACCCTGTCGCTACT TGGCCAACATGGTGAAACCCTGTTTCTACT 121 AAAAATACAAAAA AAAAATACAAAAA A A G (A)s T A C A G A A A Liaker
TTAGCCGGGCGTGGTGG
TTAGCTGGGCATGGTGG TTAGTTGGGCGTGGTGG
151 AlaI CGCGCGCCTGTA ATCCCAGCTACTCGGGAG TGGGCACCTGCAGCCCCAGCTACTCGGGAG
CGGGTGCCTGTAATCCCAGTTACTCAGGAG 181 GCT
GAGGCAGGAGAATCGCTTGAACCCGGG
GCT
GAGGCAGGAGAATCGCTTGAACCGGA
GCTGAGACAGAAGAATTGTTTGAACTCGGG A
211
AGGCGGAGGTTGCAGTGAGCCGAGATCGCG AGGTAGAGGTTGCAGTGAGCTGAGATCGAA A.GCAGAGGTTGCAGTGAGCTAAGATTGCG
241
CCACTGCACTCCAGCCTGGGCGACAGAGCG TCACTGCACTCCAGCCTGGGCAATAGAGAG
CCGCTGCACTCCAGCCTGGGTAACTGAGCG
(b) Identification of an Alu sequence in intron-7 One complete Alu sequence (282 bp), inserted in the opposite direction, has been identified in the middle of intron-7 between 1027 and 746 (Fig. 1). The direct orientation can be aligned with the consensus sequence established by Jurka and Smith (1988) from 125 known Alu sequences (Fig. 2). This Alu sequence is followed by (dA)29. No direct repeats can be found around them. The A + T richness in the noncoding and non-Aiu sequences ofintron7, with a ratio A + T/G + C of 2.3 as compared to 1.6 for the mean human genornic DNA, is noteworthy. The presence of an Alu sequence in intron-7 of the human LPL gene had been mentioned by Deeb and Peng (1989), but no nt sequence of intron-7 was presented.
(c) The Alu sequence of intron-6 Another Alu sequence has been identified in intron-6 of LPL gene by Devlin et al. (1990). This intron was only partially sequenced (0.4 kb out of 2.8 kb) and sequencing of the Alu sequence started from nt position 14 (to 282). This intron-6 dlu sequence is also transposed in the opposite direction relative to the LPL gene and is followed by a (TAAA)t0 tandem repeat°
(d) Alu sequences and genetic rearrangements in pathology The biological role of Alu sequences is unknown. Some of them have been involved in genetic rearrangements. For
271
282
AGACTCCGTCTC AGACTCCATCTC(A) 29 AGACTGTGTCTCAAAAA~AAA)
I0
Fig. 2. Alignmentof A/u sequences. Top, middle, and bottom lines: consensus proposed by Jurka and Smith(1988)and Raisonnier(1991)intron7 (present results), and intron-6 (Devlin et al., 1990)Alu sequences, respectively. The usual hallmarks of Alu sequences (a 120-bp left and a 149-bpnearly homologousright arm, connected by the A-richlinkerwith its TAC triplet) and the Alul restriction site are indicated. instance, in the case of the LPL gene a rearrangement implied a breakpoint in intron-6 with insertion at this level of a 2-kb segment consisting of about the 3' half of exon6 (0.1 kb), followed by the 5' two thirds ofintron-6 (1.9 kb). This duplication resulted in LPL deficiency causing hyperchylomicronemia (Devlin et al., 1990). Actually, the breakpoint in intron-6 is not within the Alu sequence, but in the repeat flanking it. Another example is that of the LDL receptor gene. A recombination between two homologous Aiu sequences of this gene caused a deletion of the exons encoding the transmembrane and cytoplasmic domains of the LDL receptor, so that the resulting nonfunctional receptor could not anchor into the cell membrane (Lehrman et al., 1985). A form of familial hypercholesterolemia can be ascribed to this mechanism. It is known that in human chronic myelocytic leukemia
260 the abnormal chromosome 22 (Philadelphia chromosome) results from translocation between chromosomes 9 and 22. Groffen et al. (1989) by sequencing the region around the breakpoints (gene bcr for chromosome 22, gene abl for chromosome 9) could find Alu-repetitive sequences near, or as part of, the breakpoint in all but one of the investigated patients. These observations suggest that Alu-repetitive sequences may facilitate chromosomal translocations. However, it is not likely that Alu sequences are a prerequisite for this type of chromosomal translocation. At least four different cases of thalassemias have been caused by illegitimate recombination between Alu sequences (Vanin et al., 1983). Because Alu sequences constitute 3-5% of the human genome, these occurrences could be due to chance. However it is quite possible that Alu sequences are hot spots for illegitimate recombination (Groffen et al., 1989).
dated must be placed in one of the Alu subfamilies (Quentin, 1988; Britten et al., 1988; Raisonnier, 1991). According to Jurka and Smith (1988), there are two distinct families of Alu sequences: Alu-J (for Jurka) and Alu-S (for Smith). This classification is based on the different base preferences for a number of diagnostic sequence positions. The two Alu repeats examined here were compared to the Alu sequence consensus proposed by Jurka and Smith (1988) and Raisonnier (1991). By following the procedure proposed by Jurka and Smith (1988) and described in Fig. 3 (top panel), the two Alu repeats clearly belong to the Alu. S family. Aiu-S has in turn been divided into three subclasses: Alu.Sa, Alu-Sb and Alu-Sc. By using another set of diagnostic positions, also shown in Fig. 3 (middle panel), the two Alu sequences were found to belong to the Alu-Sa subclass. Finally, the Alu-Sa subclass is split into Alu-Sd and Alu-Se. As shown in Fig. 3 (bottom panel), Alu sequences of both intron-6 and intron-7 are members of the AIu-Se family.
(e) Classification of the Alu sequences from intron-6 and intron-7 Since Alu sequences have been transposed in the genome at different times in the history of primates, they can be used as paleogenetic markers. For this, the sequence to be
(f) Dating of the Alu sequences from intron-6 and intron-7 An approximate dating of the different Alu subfamilies is given in Fig. 4. The Alu-J family is probably the oldest.
FAMILIES ALU-J AND ALU.S : S7
63
65
70
71
94
lO1 106 163
A C
G A
T C
C G
C T
G C
A G
G A
G A
G A
G A
A G
C T
T A
C T
*
Intron 7
T
A
C
A
T
C
G
G
G
A
A
G
T
A
T
fills
AIu Intron 6
T
A
C
G
T
C
G
A
A
A
A
G
T
A
T
14115
A lu.J Alu.S Alu
194 204 208 220 233 27S
SUBFAMILIES OF ALU.S (-Sa, -Sb and -So) : 65
66
78
88
9S
100
153
163
197
Aiu-Sa Alu.Sb A lu - S e
C
T
T A A
G T T
C T C
T C T
C G T/G
A G G
C G C
T G T
G C G
A lu Alu
C C
T T
T T
G Q
C C
T T
C G
A A
T C
T T
G G
Intron 7 Intron 6
200
219
1011l 10111
SUBFAMILIES OF ALU-Sa (-Sd and -Se) : 244 Alu.Sd A/u.Se
T C
Alu Intron 7 A lu intron 6
C c
Inserllon 204 + 1 A .
272 A G
G G
2/2 212
Fig. 3. Classification of the Alu sequences of intron-7 (present results) and of intron-6 (Devlin et al., 1990) into A/u families and subfamilies. The classification of the two Alu sequences was worked out according to Jurka and Smith (1988) by comparing their sequence to the Alu sequence consensus (see Fig. 2, top line) proposed by Jurka and Smith (1988) and Raisonnier (1991) at the diagnostic positions indicated in the top panel, top line. With more than nine identities (see column marked with asterisk), both intron-6 and intron-7-Alu repeats belong to the Alu-S family. Subclasses of the Alu-S family (Aiu-Sa, -Sb, and -Sc) are based on the diagnostic positions appearing in the middle panel: with more than five identities, both Alu repeats fall in the Alu-Sa family. The Alu-Sa is in turn split into Alu-Sd and Alu-Se from diagnostic positions shown in the bottom panel. (The insertion 264 + 1 means that in Alu-Sd there is an insertion just downstream from position 264): the two repeats examined here can be assigned to Alu-Se.
26! Myr 65-S$
Alu-J Alu-S • Alu-Sa • Alu-Se • Alu.Sb
(i.e., A l u - S d and A l u . S e )
55-40 40-35 35-20
Fig. 4. PaleogenetichistoryoftheAlu sequencefamilies.The time elapsed since Alu repeat individuationwas estimated from the number of random independentmutations,i.e., in the positionsthat are neitherdiagnosticnor CpG (CpG are not informativebecause they are hot spots for substitution, Britten et al., 1988).Alu elementsbelongingto the A lu-Se familywere retrotranscribed before 40 Myr, when New World and Old World monkeys diverged, while those belongingto the Alu-Sc and -Sb familiesare younger (Raisonnier, 1991). For a definitionof Alu families,see legendto Fig. 3. Partial similarity with 7SL RNA, a component of the signal recognition particle, has been shown. Approx. 100 nt at the 5' end and 45 nt at the 3' end of this R N A are homologous with the h u m a n Alu right monomer consensus sequence. This indicates that the Alu sequence may have been retrotranscribed from this R N A after deletion of the central 7SL-specific sequence (Jurka and Smith, 1988). Alu-Sc and Alu-Sb sequences were transposed into the primate genome between 40-Myr-ago and the present time (Britten et al., 1988; Raisonnier, 1991). Thus, according to this evolutionary time scale, the Alu-Se sequences ofintron6 and intron-7 of L P L gone might have been transposed in the primate genome more than 40-Myr-ago. (g) A l u sequences and lipase gone family Comparison of L P L , H L and PL gone organization has
recently enabled Kirchgessner et al. (1989) to propose a model for the evo,lution of this lipase gone family. All these genes are thought to have evolved through multiple rounds of gone duplication plus intron-loss and oxen-shuffling events from an ancestor gone containing 14 introns. As the P L gone has undergone the fewest changes, it can be held to be the closest to the primordial gone. Intron-7 is one of the introns that has been maintained at the same place relative to the exons since the primordial gone. As this intron in L P L includes an Alu repeat, it would be of interest to know if it also does in P L and in the affirmative, to which class i~ belongs. However, it must be borne in mind that L P L and PL ~ave been described in fish (Rasco and Hultin, 198g), birds and mammals, and thus exist ~s separate enzymc,s as early as the fish level on the evolutionary scale. As for HL, it has been described in dog (Greten et al., 1974), rat and man, but has not been hitherto reported in species below the dog level. The successive divergence of this gone family is most likely to be older than the retroposition of the most ancient Alu-J monomeric sequence. Thus, these genes would not contain any orthologous Alu sequence.
ACKNOWLEDGEMENTS This work was supported by grants from the Centre National de la Recherche Scientifique through UPR-41 and from the Institut National de la Sant6 et de la Recherche M6dicale (grant 900 203 to JE). We thank B6atrice Pelletier for typing this manuscript. REFERENCES Auwerx,J.H., Babirak, S.P., Fujimoto,W.Y., lverius, P.H. and Brunzell, J.D.: Detective enzymeprotein in lipoprotein lipase deficiency.Eur. J. Clin. Invest. 19 (1989) 433-437. Britten, RJ., Baron, W.F., Stout, D.B. and Davidson,E.H.: Sources and evolution of human Alu repeated sequences. Prec. Natl. Acad. Sci. USA 85 (1988) 4770-4774. Deeb, S.S. and Pens, R.: Structure of the human lipoproteinlipase gone. Biochemistry28 (1989) 4131-4135. Devlin, R.H., Deeb, S., Brunzell, J. and Hayden, M.R.: Partial gone duplicationinvolvingexon.Aiu interchangeresults in lipoproteinlipase deficiency.Am. J. Hum. Goner. 46 (1990) 112-119. Eladari, M.E., Syed, S.H., Guilhot, S., d'Auriol, L. and Galibert, F.: On the high conservation of the human c-myc first oxen. Biochem. BiGphys. Res. Commun. 140 (1986) 313-319. Greten, H., Sniderman,A.D., Chandler J.G., Steinberg, D. and Brown, W.V.: Exidencefor the hepaticoriginof a canine post-heparinplasma trigl3ceri~e lipase. FEBS Lett. 42 (1974) 157-160. C~roffen,~., Her~lans, A., Grosveld, G. and Heisterkamp, N.: Molecular analysis of chromosome breakpoints. Pros. Nucleic Acid Res. Mol. Biol. 36 (1989) 281-300. Hota, A.., Emi, M., Luc, G., Basdevant, A., Gambert, P., lverius, P.H. and Lalouel,J.M.: Compound heterozygote for lipoproteinlipase de~ciency: Ser---,ThrTM and transition in 3' splice site of intron-2 (AG ~ AA) in the lipoprotein lipase gene. Am. J. Hum. Genet. 47 (1990) 721-726. Jurka, J. and Smith, T.: A fundamentaldivision in the Alu familyof repeated sequences. Prec. NatL.Acad. Sci. USA 85 (1988) 4775-4778. Kirchgessner,T.G., Chuat, J.C., Heinzmann, C., Etienne,J., Guilhot, S., Svenson, K., Ameis,D., Pilon, C., d'Auriol, L., Andalibi,A., Schotz, M.C., Oalibert, F. and Lusis, A.J.: Organization of the human lipoprotein lipase gone and evolution of the lipase gone family. Prec. Natl. Acad. Sci. USA 86 (1989) 9647-9651. Lehrman, M.A., Schneider,WJ., Sudhof, T.C., Brown, M.S., Golstein, J.L. and Russel, D.W.: Mutation in LDL receptor: Alu-Alu recombination deletes exons encoding transmembrane and cytoplasmic domains. Science 227 (1985) 140-146. Quentin, Y.: The Alu familydeveloped through successivewaves of fixation closelyconnected with primate lineagehistory.J. Mol. Evol. 27 (1988) 194-202. Raisonnier,A.: Duplicationof the apolipoproteinC-I goneoccurred about forty millionyears ago. J. Mol. Evol. 32 (1991) 211-219. Rasco, B.A. and Hultin H.O.: A comparison of dogfish and porcine pancreatic lipases. Comp. Biochem. Physiol. 89 (1988) 671-677. Sparkes, R.S., Zollman, S., Klisak, I., Kirchgessner,T.G., KcJmaromy, M.C,. Mohand~st, T., Schotz, M.C. and Lusls, AJ.: Human genes involved in lipolysisof plasma lipoproteins: mapping of loci for lipoprotoin ~ipase to 8p22 and hepatic l[pase to 15q21, Genomics 1 (1987) 138-144. Vanin, E.F., Henth~rn, P.S.,.KiLoussis,D., Gr~sveld,F. and Smithies,O.: Unexpected rdationships between four large deletions in the human p-globin gone c~uster. Cell 35 (1983) 701-709. Wion, K.L., Kirchgessner,T.G., Lusis, A.J., Schotz, M.C. and Lawn, R.M.: Human lipoprotein!/pasocomplementaryDNA sequence. Science 235 (1987) 1638-1641.