Gene, 121 (1992) 237-246 0 1992 Elsevier Science Publishers

GENE

B.V. All rights reserved.

237

0378-l 119/92/$05.00

06764

Sequence of rat lipoprotein (Mouse;

human;

bovine;

lipase-encoding

guinea pig; chicken;

PCR; untranslated

cDNA exon; A+T-rich

sequences;

CpG islands)

Didier Brault a, Lydie No& a, Jacqueline Etienne a, Jocelyne Hamelin b, Alain Raisonnier ‘, Aziz Souli a, Jean-Claude Chuat b, Isabelle Dugail d, Annie Quignard-Boulangi: d, Marcelle Lavau d and Francis Galibert a uLaboratoire de Biochimie et Biologic Molkulaire, Fact& de M&decine St-Antoine, 75012 Paris, France: b C.N.R.S. UPR 41, Centre Hayem, H6pital St-Louis, 75010 Paris. France. Tel. (33-l/42 02 1605; ’ Biochimie. CHU Piti&Salp&i&e, 75013 Paris, France. Tel. (33-l/40 779805; ’ INSERUM France. Tel. (33-l/4633 Received

U I 17. 75006 Paris,

7105

by G. Bernardi:

5 May 1992; Revised/Accepted:

9 July/l2

July 1992; Received

at publishers:

30 July 1992

SUMMARY

A rat lipoprotein lipase (LPL)-encoding cDNA (LPL) has been entirely sequenced and compared to the sequences of all the LPL cDNAs reported in other species. As expected, high homology was found between the coding exons. The putative catalytic triad, Ser’32, Aspire, His24’, according to human numbering, is conserved in rat. As is the case in mouse, an Asn444 present in human LPL is also missing. The major divergences between human, mouse and rat LPLs were observed in the untranslated exon 10, where (i) the rat cDNA exhibits a 157-bp insertion and an 81-bp deletion relative to human; (ii) neither the B 1 repeat nor the homopurine stretch reported in mouse can be recognized, and (iii) the rat cDNA displays several A+T-rich stretches.

INTRODUCTION

Lipoprotein lipase (LPL) is a key enzyme of lipoprotein metabolism that hydrolyzes the triglyceride moiety of chylomicrons and VLDL. It is involved in various pathologies, such as hyperlipemia, atherosclerosis and obesity. For studies on lipoproteins and LPL, rat is one of the most useful laboratory animals. Since it is resistant to this affection, rat is also a model for atherosclerosis research. Moreover, the genetical nature of its obesity makes the

Correspondence to: Dr. J. Etienne, Biochimie, Mtdecine

St-Antoine,

Tel. (33-1)40306249; Abbreviations:

27 rue Chaligny,

aa, amino acid(s); bp, base pair(s); DlT,

dithiothreitol;

or 1000 bp; h (prefix), human;

lipase; LPL, gene encoding

godeoxyribonucleotide;

507, Faculte de

Fax (33-1)40307840.

(prefix), guinea pig; kb, kilobase lipoprotein

Laboratory

75012 Paris, France.

PCR, polymerase

LPL; nt, nucleotide(s); chain reaction;

cytoplasmic RNA required for both structural of signal recognition protein.

oligo, oli-

7 SLRNA,

and functional

gp LPL, small

properties

Zucker rat a valuable model for research in this field. Lastly, cell lines derived from rat tissues lend themselves to studies of LPL gene expression. However, whereas in recent years the sequencing of LPL has been carried out, via cDNA, in five species, human (Wion et al., 1987), bovine (Senda et al., 1987) mouse (Kirchgessner et al., 1987; Semenkovich et al., 1989; Zechner et al., 1991), guinea pig (Enerback et al., 1987) and chicken (Cooper et al., 1989), it has not yet been determined in rat. The aim of this work was to clone and to sequence rat LPL cDNA as a preliminary to metabolic studies on LPL mRNA. Furthermore, it is meant as a contribution to the comparative study of LPL cDNA in different species, with a view to pointing out which aa have been conserved during evolution and are thus likely to belong to domains playing a major role in LPL activity. Four rat cDNA LPL clones were isolated. The longest, a 3.18-kb clone, lacked about 0.20 kb at the 5’ extremity with regard to the mRNA. The missing 5’ fragment was obtained by PCR. The cDNA sequence was compared to

238 of all the LPL cDNAs

the sequences

reported

in other

species. The divergences appearing between rat, mouse and human in the untranslated exon-10 are pointed out.

RESULTS

(b) Comparison of coding exons of rat LPL cDNA with LPL cDNAs from other species

AND DISCUSSION

(a) Nucleotide sequence of the rat cDNA The strategy used for sequencing the clone isolated from a rat cDNA library, as well as the 0.5-kb 5’ fragment obtained by PCR is shown in Fig. 1 and Table I. The clone comprises a total of 3.18 kb. It lacks the first 183 5’ nt coding

region has thus been achieved by sequencing the OS-kb 5’ fragment obtained by PCR. Nucleotide and aa sequences of rat, mouse, human, bovine, guinea pig and chicken have been aligned (Fig. 2).

for mature

LPL. The sequence

1) Clone RP . _. . . . . .) 5

’ 267

of this

(1) High homology

High homology is found between the coding exons of rat and human cDNA LPL, as well as with the other published An exception concerns guinea pig exon 1, which cannot be aligned with exons 1 of other species.

LPL cDNAs.

w19 El3 \“,, __________+ El . _.--. -. ) W5 -------w ..W!.3... w ______* ______+ w21 w9 Wll ______* ______+ WlS ___Y! ________) ____-_____) .--. w3_.._ t 3444 )

Wl ._..._. _)

OAC

3’

3’ 4-&---

2 ) 0.5 kb 5’ fragment

W4

WlO

4_.__.._.__ w2

I

s

RP

W16

w20

, W26

a)

RP ._.._.. __* b)

)

4 Fig. 1. Strategy

(Clontech

for sequencing

Laboratories,

.___._.._ UP

rat LPL cDNA.

The cDNA

Palo Alto, CA). It was screened

library used was prepared

from 4-week-old

with a 150-mer probe synthesized

the last 14 nt of exon-7 and to the 135 first nt of exon-8 of human the Ml3 vector). Four clones were isolated and subcloned

cDNA

LPL (plus

Sprague-Dawley

by the phosphoramidite

rat testicular

method.

1nt added to create a 5’ BumHI restriction

in the EcoRI site of pUC 18. The OS-kb 5’ fragment

was prepared

I

fat cells in lgtl

This probe corresponds

to

site for cloning into

by PCR. Briefly, total RNA

was isolated from rat frozen adipose tissue using the guanidium isothiocyanate/LiCI method as described by Cathala et al. (1983). Total RNA (6 pg) was heated at 65°C for 10 min before priming for cDNA synthesis with 125 ng random hexamer oligos (Pharmacia, Piscataway, NJ) in a 50 ~1 reaction containing

1 x reverse transcriptase

1 mM each dATP,

dGTP,

stopped by incubating This cDNA

dCTP

buffer (50 mM Tris.HCI and dTTP,

pH 8.3/75 mM KCljlO mM DTT/3 mM MgCl,),

and 200 units M-MLV

at 70°C for 10 min. The resulting cDNA

was used as a template

reverse transcriptase

was purified and concentrated

for PCR with (i) an upstream

primer (Table I: PCRl)

20 units RNasin

(BRL). The reaction on a Centricon consisting

(Promega,

was performed

Madison,

WI),

at 37°C for 60 min and

30 column (Amicon

of the first 20 nt transcribed

Corp., Danvers,

MA).

in mouse according

to Zechner et al. (1991). This was attempted in view of the high putative sequence similitude in rat and mouse; (ii) a rat LPL specific downstream primer (Table I: PCR2) complementary to rat nt 382 to 361 (Fig. 2) (plus EcoRI and Hind111 recognition sites). For PCR, samples were denaturated at 94°C (5 min), annealed at 52°C (2 min) and extended at 72°C (2 min)followed by 30cycles of 94”C(l min), 52°C (1 min) and 72°C (2 min) with a final extension of 10 min. The PCR product phosphorylation ing was carried

of approx.

580 bp was excised

and eluted from a 2% Nusieve

LMP

agarose

gel. This fragment

was subcloned

after

into M13mp89 cut by EcoRV and sequenced. The 0.5-kb fragment was inserted in the two possible orientations (a) and (b). Sequencout by the dideoxy method of Sanger et al. (1977), using [C(-35S]dATP, T7 DNA polymerase and the specific probes.

239 TABLE

I

Synthetic

oligos for PCR and for sequencing

the cat LPf. cDNA

(A) Oligos used for PCR PCR 1 = 5 ’ -TGTCAGACTCTCGATTTCTC

PCRZ = 5’-TATAGCCGGCAGACACTGGATA

382-361

(B) Oligos used for sequencing” (1) The 3.18kb

clone UP = TGTAAAACGGCCAGT

RP = AACAGCTT’AGACCATG

= ATGTCCAC~C~AGGGTAC

435-474

W2 = AATCACTGATGGAGGTGGAC

1887-1868

W3 = CGTGTAATTGCAGAGAAGGG

748-767

W4 = AGCCTAATGAAACCAGTCAC

1652-1633

WI

W5 = CGCTCTCAGATGCCCTACAA

997-1016

W6 = TTTCTGTTCCCAGCAACAG

1439-1421

W7 = GGTCAGACTGGTGGAGCAGT

1250-1269

W8 = GCTCCTCACTTTGCACGCAA

1247-1228

El3 = GGACGCTGCAGTGTTTGTGA

1371-1390

WlO = GCCAAGGCAGGATGGTTGAGA

940-920

W9 = GCAGAGAGGAGAAGCATGCC

240-259

W 12 = TGACTTGTACTTCGT-TGTGG

636-617

EI

385-366

= GGGCCT~TA~TCA~AG

349-368

W 14 = ~AAAACCTCAGAGATCTA

W Ii = AAGCAATGGACGACGTGGCT

572-591

W16 = ACT~CAAGATATAGCTGGG

W 13 = TCAGGCTTACCTTGAACTCT

903-922

W 18 = TCCCTGGCACAGAAGATGAC

1343-1324 1100-1081

148-129

W152 = GTACAAGTI-TTAGAGCAGGA

1141-1160

W20 = TACAGAGAAATCTCGAAGGC

W 17 = GCTCGTTGCCGCTCTTTTGT

1279-1298

W22 = TGTATGCCTTGCTGGGGTI’T

868-849

W 19 = CAGAAAGGAAAGAGTCAAGA

1515-1534

W24 = ACATCTACGAAATCCGCATC

624-604

W21=

1773-1792

W26 = TCAll-l-CCCACCAGCTTGGT

401-382

AGCTGTAAATAATGTGTGGG

(2) The 0.3-kb 5’ fragment RP = AACAGCTTAGACCATG

UP = TGTAAAACGGCCAGT

S( = PCRI) = TGTCAGACTCTCGATTTCTC

0 = CTGCTGTGGTTGAAGTGGCA

it The sequence Numbe~ng

of all the probes is given in the 5’ to 3’ direction.

is as in Fig. 2 (coding

sequence)

The left column corresponds

Divergences between the rat sequence and the five other sequences, from the first ATG to the stop codon (1428 nt), are shown in Table II. The ratio of transitions to transver-

TABLE

II

Divergence

between

six LPL cDNA

sequences

(1428 nt from the first

ATG 5’ nt, except for bovine, to the stop codon) Rat vs.:

Mouse

Human

Bovine

Guinea

Chicken

pig Identities Transitions

(I)

Transversions I/V Insertions

(V)

(i)

1358

1242

1151

1163

59

123

136

148

193

114

209 0.923

18 3.277

60 2.050

75 1.813

1023

1.298

0

1

1

1

1

1425

1426

1363

1426

1426

Informative positions” % Divergence’

5.40

12.90

15.55

28.26

18.44

’ For determining the number of positions, each gap was counted as one position, regardless of insertion length, as these gaps are no targets for substitution. Consequently, the insertion of an Asn codon at the end of the protein is counted for one position only rather than three, since obviously the two other positions stitution. b The percentage (1987) equation:

of divergence

to the sense sequence,

and Fig. 3 (exon 10). RP, reverse primer; UP, universal

are not informative was calculated

as regards

to nt sub-

by the Miyamoto

P % = (I + V + i) x lOO/Nb of informative

positions.

et al.

218-199 the right column

to the antisense.

primer.

sions decreases as divergence increases. This could be explained by a double substitution occurring at one position. Paradoxically, the percentage of divergence between rat and guinea pig is greater than between rat and human or human and bovine. However, results tending to place the guinea pig differently on the mammalian phylogenetic tree have been reported (Graur et al., 1991). The longest segments that present homology among the six species studied up to the present time are the end of exon 2 and the beginning of exon 3, This homology has not been previously pointed out. These regions comprisent 42-87, with only three nonconserved aa out of 46; even position 45, which has Thr in chicken, rather than Ser in the five mammals, is functional, since it belongs to the potential ~-glycosylation site, which accepts Ser or Thr (see section bl). Also conserved is the well-known nt 170-217 region (exon 5), with five nonconserved aa out of 48, or else region 234-264 (exon 6) with three nonconserved aa out of 31. But region 287-320, which Semenkovich et al. (1989) indicated as being well conserved at a time when the chicken sequence was not published appears to be less so (seven nonconserved aa out of 34). The ATG in position 1 for the five mammals, or positions 1 and 19 for chicken, which has two alternative start codons (Fig. 2) codes for the N-terminal Met of the hydrophobic signal sequence. The

240 Open Reading Frame

/

rLPt. mLPL hLPL

-174 T&TCT + GTAOCTGTTATGCCCT CcrJ TTT~~~~~TT~T~~ TACTCCTCCTCCGWEAATTCTGCGC CCTGTAACTGTTCT0XCTCXXCTlTAAAGGTTGACTT@XCTACG3ZSC TCCTCCTCCTCAAGGG~AAGCTGCCCACTTCTAGCTGZCCTGCJZATCCCCTTTAAAG~X~%CTT~XTC

rLPL mLPL hLPL

GACXXCTCCGGCTCAACCCTTTBXA TOCOCCTCCTGCTCAACC CXAGCCTCCGOCTG4GCC

rLPL mLPL hLPL bl.PL LPL cge PL

rLPL mLPL hLPL bLPL 2z.L rLPL r?tLPL hLPL bLPL c PL TPL

I

I

TrXCGDkXTGACT

CTATAGTCCTCT TCC4ccMi CTCCAGTETCT ACCGCCAAACCOCGGCTCCAGCCGTCT

’ GCCCGf&-GTTC&CA&CA GAA& CGCCGXTAGTTKZAGCAGAKAGAA~~~G CCGCCCCTTGTAGCTCCTC CAGAGGGA~CGAG

EEEEEW

GAG

ACW,TTTC!CAG4CATC&AGTAAATT 4 W.XTAAG&CCCTGAA d CACAGCTGA ki4 CACTTGT&ATCTG4TTC&TU3ATTA&KTCTGTG A~OATTTCTCAU\CATCGAAAGCAAATTTOCCCTAAOGA AG4GATTTTATCGACATCAAGTAAATTTOCCCTAAC AAAW\TTTTAOAG4CATTG4AAGTAAATTTGCTCTCAGGAG G4AC9CAOIT64GG4CACCTGTUICCTCATT~T~~~~~TCT~G AAAC?ATTATACGG4TATCAGTAAATTT‘XCO34AGG4fXCCCG4 ATGAATTTTGACYYjAATCGI\GAGCAAGTTTTCCTTAAGTTG l l e l 0 l l l l l ArgAspPheSerAsp Ile Glu Sar LysP~ AlaL~Arg Thr ProGl~ AspThr Ata~uA~pThrCysHis Lau lle Peony Lau AlaA~S~r ArgAspPheSerAsp Ile Glu Ser Lys Pha Ala LsuArg Thr Pro Glu AspThr Ala Glu AspThrCys His Lau Ile Pro Gly Leu Ala AspSer ArgAspPhs Ile Asp Ile Glu Ser Lys Pha Ala Lw Arg Thr Pro Glu AspThr Ala Glu AspThrCys His Lsu Ile Pro Gly Val Ala Glu SW LysAspPhsArgAsp lie Glu Ser Lys Phs Ala Lau Arg Thr Pro Glu AspThr Ala Glu AspThrCys His Lau Ile Pro Gly Val Thr Glu Ser L s Asp Tyr Thr Asp Ile Glu Ser Lys Phs Ala Arg Arg Thr Pro Glu Asn Thr Val Glu Asp Thr Cys His Lau lle Pro Gly Val Thr Glu Ser Myet Lys Pi-m Glu Gly Ile Glu Ser Lys Phs SW Lw Arg Thr Pro Ala Glu Pro Asp Glu Asp Val Cys Tyr Lau Val Pro Gly Gln Mat Asp Ser

Ser AsnCts Ser AsnCys

hLPL bLPL

Ala Thr Cys His AlaAsnCys His Ala AsnCys His Ala Gln CysAsn

192 l;: 126 162 102 Val Val Vat ‘2.4 Vat Leu

Phe Asn Phe Asn PheAsn PheAsn

l * l e*e His Ser SW Lys Thr Pha Val Val His Ser SW Lys Thr Pha Val Val His His His His

SW Ser Ser Thr

SW SW SW Ser

Lys Lys Lys Lys

Thr Thr Thr Thr

Phe Pha Pha Pha

Met Val Met Val

Val Val Val Val

c:: 291 225 261 291

*ee*****ee***eeeee Ile Ile

His Gly Trp Thr Val Thr Gly Mel Tyr Glu Ser Trp Val Pro Lys Lau Val Ala His Gly Trp Thr Val Thr Gly Met Tyr Glu Ser Trp Val Pro Lys Leu Val Ala

Ile 11s Ile Ile

His His His His

Gly Gly Qly Gly

Trp Trp Trp Trp

Thr Thr Thr Thr

Val Vat Vat Val

Thr Thr Thr Thr

Gly Gly Gly Gly

Met Met Met Mat

Ttr Tyr Tyr Tyr

Glu Glu Glu 610

Ser Ser Ser Ser

Trp Trp Trp Trp

Val Val Val Val

Pro Pro Pro Pro

Lys Lys Lys Lys

Leu Leu Leu Leu

rLPL InLPL hLPL bLF3. LPL 9e c PL

Ala Ala Ala Ala

rLPL mLPL hLPL bLPL

rLPL IllLPL

GTGOGAAAT b TGT-LTCATCAA k TGfXTGWIGkAAGAATTT J CTACWCCT 1 GACAATGT &ACCTCTTAG~GTA~GTCT~G~GCCC.AT GTGGG9AATGATCiT-GATTCATCAACT~T~~~~TT~~A~TA~~~~~TCTTA~A~~TT~~~T TTATCAACTG04TGGAc33WWGTTTAACTACCCTCT~~AT~CC4TCTCTTGGGATACA~TT~~T GTGf33CAGGATOTGGCCWGT GGCGGPTGAATTTAACTATCCCCTGGGCAATGTGCATCTCTT~TACAGCCTT~T GTGWACAGGATGT GGCCAAGTTTATOAACTGGAT GTTG6909AGATGTAGCCA~TTATCAACTOOATGG4aC GIGOG4AAEOATFTTGCCATGITCATTGATIGG9TGG(\GT l eeeeeeeeeee V& $y AsnA?pV% A?a Arg P% lie Asn Tfp Leu Glu Glu Glu Pt Asn TTr Pro Leu AspAsn Val His Lau Leu Gly Tyr Val Gly A:n Asp Val Ala Arg Phe lie Asn Trp Met Glu Glu Glu Pha Lys Tyr Pro Leu AspAsn Val His Lau Lau Gly

hLPL bLPL LPL c9: PL

Val Gly Vs.1 Gly Val Gly Val Gly

(continued

on

Lau Lsu Lau Leu

p.

Tyr Tyr Tyr Tyr

&In Gln Glu Lys

Lys Arg Glu Pro Lys Arg Glu Prc Lys Arg Glu Pro Lys Arg Glu Pro

Asp Asp Asp Asp

Val Val Val Val

Ala Arg Ala Lys Ala Arg AlaMet

Asp Asp Asp Asp

Pha Phs Phs Pb

Ser Ser Ser Ser

Ile Met Ile Ile

Asn Asn Asn Asn

Vat Vat Val Val

Asn Trp Asn Trp Asn Trp AspTrp

lie Val Ile Val Ile Val Ile Val

Met Met Met Met

Glu Ala Glu Glu

Val Val Val Val

Glu Asp Asp Glu

Asp Asp Asp Asp

Glu Glu Glu Lys

Trp Tip Trp Trp

V’,l Val Val Val

l l l l l l l l e l l l Leu TyrArgAla Gln Gin His Tyr Pro Val Ser Ala Gly Tyr Thr Lys Leu Lau Tyr Arg Ala Gln Gln His Tyr Pro Val Ser Ala Gly Tyr Thr Lys Leu

SW

Lw Arg Ala Lau SW Arg Ala La! Ar Arg Ala Lau Va I Arg Ala

PhaAsn PhaAsn Phe Lys PheAsn

Tyr Tyr Tyr Tyr

Pro Pro Ser Pro

Gin Gin Gln Gln

Glu Gln His Gln

Lsu AspAsn Lau Gly Asn ValAspAsn LsuAsnAsn

His His His His

Val Val Val Val

Tyr Tyr Tyr Tyr

His His His His

Pro Pro Pro Pro

Lsu Lsu Lw Lau

Val Val Glu Val

Leu Leu Lsu Lsu

Ser Set Ser Ssr

Gly Gly Gly Gly

Ala Ala Ala Ala

Gly Gly Asp Ala

Tyr Tyr Tyr Tyr

Thr Thr Thr Thr

Lzs Lys Lys Lys

:II

Ala Ala Ala Asp

+ GTAT ~-CAT!ATC~~~T~TA!ACCG GCAACATTATC09GTGTCAOCTGGCTACACCAACCTG TACACCAAACTG GZAGCATTATCCAGTGTCTGCAGWTACACZAAGCTG GQ3XCAWXCATTACCCAGGTCTGCGGACTACACCAA~TG

COXTATA~AAAGWi4ACkTGACTCCAA 4 GTCATTGTA&TAGACTGGT GCCCTGiftCAAWIGAWUlCCT~ATGTUITTGTAG OCCCTGTACAA~AW\WU\CCAGI\CTCCAATOTCI\TTGT GCCTTGTACAAG9GGG4ACCGGACTCCAACOTCATCOTOO GCTCTGTACAAOAGGOAACCAGACTCCAATGTUITTGTOO GCTCTGTACAAOAGOGAACCTGATTCAAAT~~TTGTTTG l ee*eeeeeee*eeeee Ala Lsu Tyr LysArg Glu ProAspSerAsnVal Ile Val ValAspTrp Ala Lsu Tyr Lys Arg Glu ProAsp Ser Asn Val Ile Val ValAspT;p

OPL TPL

Fig. 2

l l His PhaAsn His PheAsn

rLPL mLPL

2% LPI. cBe PL

1; -1

Met Asn tie AspArg Lys Ire Lau Asn Lys Ala Leu Ala Lys Glu Lys Val Met Glu Arg Gly Arg Gly Met Gly Lys Tyr Ala Lw Lsu Ala Val Lsu Cys LauCys Lsu Arg Gly Ala Ala Gly

3EL

rLPL InLPL

-72

_Sienal eplide Met Glu g er Lys Ala Lsu Leu Lau Val Ala Leu Gly Val Trp Leu Gln Ser Leu Thr Ala Phs Arg Gly Gly Val Ala Ala Met Glu Ser Lys Ala Lsu Lau Lsu Val Val Leo Qly Val Trp Lau Gln SW Lau Thr Ala PI-e Arg Gly Gly Val Ala Ala Met Glu SW Lys Ala Leu Leu Val Lsu Thr Lw Ala Val Trp Lau Gln Ser Leu Thr Ala Ser Arg Gly Gly Val Ala Ala

rLPL nlLPL hLPL bLPL

%F

:p:

Leu Leu Leu Lw

390 390 390 324 360 300 103 103 103 105 103 105

469 469 489 423 459 460 136 136 136 136 136 136

241)

first aa of the mature protein is conserved in four of the six species. It is Ala in rat, human, mouse and guinea pig. In rat, human and mouse, this Ala is the third of a stretch of three Ala, while there is no such stretch in guinea pig. There are two more N-terminal aa in bovine, -Asp-Arg, followed by Ile instead of Ala and in chicken, Ser-Asp followed by Pro instead of Ala. In rat, we found that aa 444 of the

human sequence (Asn) is missing, as was the case in mouse (Zeckner et al., 1991). In chicken, the C-terminal sequence is 15 aa longer (see below for an explanation). All these particular differences result in hLPL and gpLPL being 448 aa long, while rat and mouse are 447 (448 -1) bovine 450 (448 + 2), and chicken 465 (448 + 2 + 15).

241

rLPL mLPL hLPL bLPL LPL Be c PL

GCT~T~TTGCA~OCT~~TM~~~~~TTACT~T~T~~T~~T~CTTT~~AT~~~GT GCT~T~ATTGCAGOI\AGTCT~~TM~~~~~TM~TTA~T~~T~~TMCTT~~AT~~~T~GT GCTOCTGGCGTTGCTGGGAGTCOGACCAATACGAAGGTCAGT GAAAAAGGTOI\ACAGPIATTACTOOTCT~T~T~T~~~T~T~.~~T~T~T~TATC ~T~T~TT”.‘~~TTAAeW l eeeee*e*e l l Pro Ala Gly Pro As” Phs Ala Ala Gly Val Ala Gly Se, Leu Asn Lys Lys Val As” Arg Ile Thr Gly Leu rLPL P;o Ala Gly Pro As” Pi-m Ala Ala Gly Val Ala GJy Se, Leu Thr As” Lys Lys Val As” Arg Ile Thr Gly Lsu mLPL * P;o Ala Gly Pro As” Phe hLPL Ala Ala Gly Its Ala &y Se, Lsu Thr As” Lys Lys Val As” Arg Ile Thr kly Lsu Pro Ala Gly Pro As” Phs bLPL Ala Ala Gly Ile Ala Gly Se, Lsu Thr As” L s Lys Val As” Arg Ile Thr Gly Lsu Pro Ala Gly Pro Am Phs LPL Ala Ala Gly Val Ala Gly Se, Arg Thr As” T 6 r Lys Val Se, Arg Ile Thr Gly Lsu Pro AlaGly ProThrPhsGlu cge PL Ala AlaGly Ile AlaGly Se, LsuThrLys LysLysValAsnArg Ile Thr Gly LB”

Th,

. Gl” Tyr Ala Glu Ala Glu Tyr Ala Gl” Ala

Pro Se, Pro SW

169 169

Gl” Tyr Ala Glu Ala Pro Se, Glu Tyr Ala Glu Ala Pro Se, Glu Tyr Ala Glu Ala Thr Se, Tyr AlaAspAla Pro Ile

169 171 169 171

66.7 667 607 621 657 667

rLPL

KY-

bLPL LPL ge c PL rLPL mLPL

hLPL bLPL LPL cge PL

_-__.-...__.. Pro

l eeeeee l eeeeee Arg Leu SW AspAsp Ala AspPhs Val AspVal Arg Lsu Ser ProAspAspAlaAspPhs ValAspVal * Arg Lsu Ser Pro AspAsp Ala AspPhs ValAsp Val Arg Lsu Ser Pro AspAsp Ala AspPhs Val AspVal Arg Lsu SW ProAspAsp Ala Gln PheVal AspVal Arg Lsu SW Pro AspAsp AlaAsp Phs Val AspVal

l l l l l l eeeeeeeeee Lsu His Thr Phe ThrArg Gly SW Pro Gly Arg Ser Ile Gly II8 Lsu His Thr PhsThr Arg G:y Ser Pro Gly Arg Ser Ile Gly Ile * Lsu His Thr Phs Thr Arg Gly SW Pro Gly Arg SW Ile Gly Ile Lsu His Thr Phs Thr Arg Gly SW Pro Gly Arg Ser Ile Gly Ile Lsu His Thr PheThr Arg Gly Ser Pro Gly Arg SW Ile Gly Ile Lsu His Thr Tyr Thr Arg Gly Ser ProAspArg Ser Ile Gly Ile

Gln Lys Pro Val Gly His Gin Lys Pro Val Gly HIS

202 202

Gln Gl” Gln Gln

202 204 202 204

Lys Lys Lys Lys

Pro Pro Pro Pro

Val Val Val Val

Gly Gly Gly Gly

His His His His

766 766 766 720 756 706

,LPL mLPL hLPL bLPL LPL cBe PL rLPL mLPL

235 237 235 237

hLPL bLPL LPL cgP PL

rLPL mLPL hLPL bLPL LPL cge PL ,LPL mLPL

CTAGTGAAGTGCTCCCACGAGCGCTCCATTCATCTCTTCAA CTGGTG9AGTGCTCCCAC~T~T~TCTCTCTTCA CTAGT~GTGCTCCCATGAGCOCTCCATTCATCTCTTCG CTGGTOPIAOT~TCTCATOAACGPITCCATCTCTT~TT~CT~CT~TCTAT~~e~~~T~TA~T~A~~A~G l eeeeee*eeeooeeeee Glu Arg Ser Ile His Lsu Phs Ile Asp Ser Lsu Leu Asn Glu Glu Asn PTo St, Lys A7.s TT,A?gCTsAsn Se, L:s &J Glu Arg Ser Ile His Leu Phs Ile Asp SW Lsu Leu Asn Glu Glu Asn Pro Ser Lys Ala Tyr Arg Cys Asn SW Lys Glu Glu Glu Glu Glu

hLPL bLPL ZK’

rLPL mLPL hLPL bLPL LPL cge PL rLPL mLPL hLPL bLPL LPL cge PL

rLPL mLPL hLPL bLPL LPL cge PL rLPL mLPL hLPL bLPL LPL cge PL

Fig. 2. (continued

A?g Arg Arg Arg

S*r Ser SW Ser

Ile Ile Ile Ile

His His His His

Leu Lsu Lsu Lsu

Phs Phe Phs Phs

II8 Ile Ile Ile

A:p Asp Asp Asp

Ser Ser Ser Ser

Leu Lsu Lsu Lsu

Leu Leu Leu Lsu

Asn Asn Asn Tyr

Glu Glu Glu Glu

Glu Glu Glu Glu

Asn Asn Asn Lys

Pro Pro Pro Pro

~TTTGAGAAAOGGCTCTGCTTGAGTTGTAGAAAGPJ\TG ~TTT~-OGTCTCTG~~T~~~~T~~~T-TA~~T~~~~~~~~~-~TG ~TTTGAAAAAOGGCTCT~T~~T~~M~~T~~~~~TA~~T~A~~~~~~~~-~TG G9ACCGTTGCAACAACTTGTATAAAGTCAACAGAGTGTG GXTTTG4WGajCCTCTGCCTAA~Tl l eeeeeeeeeeeee*e* l l Ala Phe Glu Lys Gly Lsu Cys Lsu Se, Cys Arg Lys As” Arg Cys As” As” Val Gly Tyr Glu Ile Asn Ala Phs Glu Lys Gly Lsu Cys Leu Se, Cys Arg Lys Asn Arg Cys AsnAs” Leu Gly Tyr Gl” Ile Asn Ala Phs Glu Lys Gly Lsu Cys Leu Se, Cys Arg Lys Asn Arg CysAsnAs” Lsu Gly Tyr Glu II8 As” Ala Phs Glu Lys Gly Lsu Cys Lsu SerCys Arg LysAs”ArgCysAsnAsnMeI Gly Tyr Glu Ile As” Ala Phs Glu Lys Gly Lsu Cys Leu Se, Cys Arg LysAs”ArgCysAs”As” Val Gly Tyr Gl” Ile As” Ala Phs Glu Lys Gly Leu Cys Leu Se, Cys Arg LysAs”ArgCysAsnAs” Lsu Gly Tyr Lys Val As”

Gt CAAGATTCA A TTTTCTGGP, TACCTGAA d CTCGCTCT d GATGXCTA &AA AGTATTCkATTACCAA TACCTGPJIGACTCGCTCTC~T~TACAAAGTGTTCC TACCTGPIP~CTCGTTCTCAGATGCCCTACAAAGTCTT~TTA-~~~TT~TTTTTCT-CT~~~~~TA-T~~C TACCTGAA~CTCGTTCTCAG4TGCCTTACAAAGTCTTCCC TACCT09AGACTCCCTCACA~T~TACAAAGTGTTWC TACTTG4AGACCCGT~TC~T~TACAAAGTCTTC l eeeoeeeeeeem T:r L’, L;s T!, A?g SW Gln Met Pro Tyr Lys Val Phs His Tyr Gln Val Lys Ile His P’;, Ser G?y Tyr Leu Lys Thr Arg Ser Gln Mel Pro Tyr Lys Val Phs His Tyr Gln Val Lys Ile His Phs SW Gly Tyr Lsu Lys Thr Arg Ser Gln Met Pro Tyr Lys Val Phe His Tyr Gln Val Lys Ile His Phs SW Gly Tyr Lsu Lys Thr Arg Ser Gln Met Pro Tyr Lys Val Phs His Tyr Gln Val Lys 11s His Phs Ser Gly Tyr Lsu Lys Thr Arg Ser Gln Met Pro Tyr Lys Val Phs His Tyr Gln Val Lys Ile Tyr Phs Ser Gly Tyr Lsu Lys Thr Arg Ala Gln Met Pro Tyr Lys Val Phs His Tyr Gln Val Lys Ile HIS Phs Phs Gly

Ser SW Ser Ser

Lys Lys Lys Mel

Ala Ala Ala Ala

Tt, Tyr Tyr Tyr

Arg Arg Arg Arg

Cys Cys Cys Cys

Ser Asn Asn Asn

Ser Ser Ser Thr

Lys Lys Lys Lys

Glu Glu Glu Glu

665 665 085 619 655 605 266 266 266 270 266 270

964 964 964 916 954 964 Lys Lys Lys Lys Lys Arg

l Val Val Val Val Val Val

l Arg Arg Arg Arg Arg Arg

Ala Ala Ala Ala Ala Thr

ACTGAGAAT d

Thr Thr Thr Thr Thr Lys

Glu Glu Glu Glu Glu Thr

Asn Asn Ser Ser Thr Asn

. Lys Lys Lys Lys Lys Lys

l Arg Arg Arg Arg Arg Arg

Se, Se, Se, Se, Se, As”

CAAGCAAAA

Asp Gly Glu Asn Thr Val

Lys L s T yhr Thr Thr Thr

Gln Gln His Tyr Tyr Lys

Se, Se, Se, SW Se, Thr

l Lys Lys Lys Lys Lys Lys

l Met Met Met Mel Met Met

kwc.acc

AsnAsn His Asn Thr Am Thr Asn Thr Asn Val Asp

C% Gln Gln Gln Gln Gln

301 301 301 z 303

1063 1063 1063 1017 1053 1063 Ala Ala Ala Ala Ala Pro

334 E 336 334 336

on p. 242)

The putative catalytic triads Ser13*, AsP’~~, His241 coded by exons 4, 5 and 6 in human is conserved in rat, with the same aa in the same positions, the codon for Ser13*, however, being AGC in human and AGT in rat. Ser13* occurs in the consensus sequence Gly-Xaa-Ser-Xaa-Gly present in serine proteinases and in human pancreatic lipase. It has

been shown that hydrolysis of tri- and monoacylglycerol by LPL stems from a common active site. The catalytic site and the heparin-binding domain reside on two separate folded domains. Although the heparin-binding domain of LPL has not yet been definitely assigned, this site was shown by peptide cleavage experiments (Olivecrona et al.,

242

1182 1162

1LPL mLPL hLPL bLPL LPL c PL ge rLPL mLPL hLPL bLPL LPL cge PL

rLPL mLPL hLPL bLPL LPL cge PL rLPL mLPL hLPL bLPL ZK’

rLPL I$?blPL LPL cBe PL rLPL mLPL hLPL bLPL splPL CLPL

1::: 1t52 1182 & Phe Phe Phe Phe Phe

Glu Glu Glu Glu Glu Leu

It, Ile Ile I le Ile lie

Sir Ser Ser Ser Ser Ser

t Leu Leu Leu Leu Leu

T-r Tyr Tyr Tyr Tyr Tyr

T:r Thr Thr Thr Thr Thr

Val Val Val Val Val Leu

l Glu Glu Glu Glu Glu Glu

Ala Ala Ala Ala Ala Asp

e**ea**ee**e Ser Giu As” Ser Glu As” Ser Glu As” Ser Glu As” Ser Glu As” Ser Glu Asn

ile Pro Phe Thr Leu Pro Glu Ile Pro Phe Thr Leu Pro Glu Ile Pro Phe Thr Lw Pro GIu Ile Pro Phe Thr Leu Pro Glu Ile Pro Phe Thr Leu Pro Glu Ile Pro Plm Thr Leu Pro Glu

Val Val Val Val Val Val

Ser Ser Ser Ser Ser Ser

Thr Thr Thr Thr Ala Ser

A:” Lys As” Lys As” Lys As” Lys AsnAsn As” Lys

l Thr Thr Thr Thr Thr Thr

Tyr Tyr Tyr Tyr Tyr Phe

l Ser Ser Ser Ser Ser Ser

l Phe Ptw Phe Phe Phe Phe

l Lw Leu Leu Leu Leu Leu

lie Ile I le Leu Ile lie

T:r Tyr Tyr Tyr Tyr Tyr

367 367 367 369 367 369

1281 1261 1281

l eeeee l ee Thr Glu Val Asp I le Gly Glu Leu Leu Met Met L;s t Lys T:p Thr Glu Val Asp 118 Gly Glu Leu Lw Met Met Lys Leu Lys Trp * Thr Glu Val Asp Ile Gly Glu Leu Lea Me( Leu Lys Leu Lys Trp Thr Glu Val Asp Ile Cly Glu Leu Leu Mel Leu Lys Leu Lys Trp Thr Glu Val Asp Ile Gly Glu Leu Leu Met Leu Lys Leu Lys Trp Thr Glu Val Asp lie Gly AspLeu Leu Met Leu Lys Leu Gl” Trp

I

I

I

ATUMCNAW\TCCG4GTG4AACD.%XA~GACTCAGAA ATCGAG9GGATCCGAOTOAAAGCCOGI\OAGACTCAGAAA ATTCAGAAGATCA~OTAAAAGCAGGAGPIGACTCAGAA ATTEYrJ\AGAT~WIG~~~~CT~~~~~TCTTCT~T~ ATCGAAAAG9TCAG9GTAAAAOCAOU\GAGR04CAG4AA ATTCAGAG4GT~GAGTG9AGTCRGOCUIAACTCAG4AA l eeeee . eee Ile Glu Lys Ile Arg Val Lys Ala Gly Glu Thr Gln I le Glu Arg Ile Arg Val Lys Ala Gly Glu Thr Gln Ile Gln Lvs Ile Am Val Lvs Ala Glv Glu Thr Gin Ile Gly c(‘s lie Ari Val L,, Ala Glj, Glu Thr Gln lfe Glu Lys II8 Arg Val Lys Ala Gly Glu Thr Gin I le Gln Arg Val Arg Val Lys Ser Gly Glu Thr Gln

I rLPL “lLPL hLPL bLPL

‘.i?y Gly Gly Gly Gly Gly

I

1::: 1281 Lys Ser Asp Ser T yr Pk S:r T:p Ser Asp Ttp T:p Ser SW Pro Ser P: Val Mei Ser Asp Ser Tyr Phe Ser Trp ProAsp Trp Trp Ser SW Pro Ser Phe Val Lys Ile Ile Glu

SerAsp Ser Ser Asp Ser Thr Glu Ser Lys AspThr

Tyr Tyr Tyr Phe

Phe Phe Phe phe

SW SW Ser Ser

Trp Trp Trp Trp

SerAsp Ser As” Ser Ser Ser Asn

Trp Trp Trp Trp

Trp Trp Trp Trp

Ser Ser Gly Thr

SW Ser Arg Pro

Pro Pro Pro Phe

Gly Gly Thr Ala

Plw Ala PheAsp Phe Thr Phe Thr

1380 1380 1380 1314 1350 1360

Ad, GGTCATCT!CTGTGCCAC~~~T~CT~~CT~~A~~~~T~A ~TGTCTTATCTOCAOA~A~~~~~~

l

Lys Lys Lvs L+ Lys Lys

Lys Lys Lvs L,, Lys Lys

Val Val Val Val lie Vat

Ile Ile Ile lie Vat Val

*

PheCys Ala PheCys PheCvs PheC;s PheCys Phe Cys

Ala Ser Ser Ser Ser

l Arg Arg Ara Ar; Arg Arg

Glu Glu Glu Glu Glu Asp

Lys Lys Lvs Ljrs Lys Gly

I

I

Val Val Val Met Vat Ser

l Ser Ser Ser Ser Ser Ser

His His His Tyr Lys Arg

e Lw Leu Leu L&w Leo Leu

I

Gln Gln Gln Gl” Gin Gly

400 402 400 402

l Lys Lys Lvs L$s Lys Lys

l Gly Gly Glv Glj, Gly Gly

LysAspArg Lys Asp Ser Lvs Ala Pro Lis Sar Pro Lys Glu Ala Glu Glu Ala

I

Ala Ala Ala Vat Pro Ala

433 433 433 435 433 435

I

GTGTTTGTWAATGCCATGKAAGTCTCTG GTGTTTGTGAAATWCAT69CAAGTCTCTG

ZKL rLPL mLPL

l l l l Val Phe Val Lys Cys His Asp Lys Ser Leu Val Phe Val Lys Cys His Asp Lys Ser Lw

hLPL bLPL gpLPL CLPL

Val Ile Val Ile

Fig. 2. Nucleotidc

Val Val Val Val

sequences

guinea pig and chicken aa (although

Pha Pha Phe Ptw

Lys Cys LysCys Lys Cys Lys Cys

Lys Lys Lys Gln

mouse (mLPL), bovine (bLPL), guinea

pig

of chicken

are translated

again in Fig. 3 in order to align all the exon-10 in the six species are indicated

and deduced

The beginning

aa of chicken

*

Ser Lw Asn Lys Lys Ser LeuAsn Arg Lys Ser Lou Asn Lys Lys Pro Val Ser Arg Lys

of rat ML cDNA

sequences.

the C-terminal

His Asp His Asp His Asp LWJ Glu

. l Lys Lys SW GtySlop Lys Lys Set GlyStop

sequences.

(gpLPL)

SW Ser SW Arg

447 447

Slop

Gly GlyStop GlySlop Gly Gly Ala

aa sequence.

448 450 448 Lys Lys Ala Ser Lys Glu Asn SW Ala

The nt sequence of rat LPL cDNA.

exon-10 sequence

from the beginning

1987) to be located in the middle part of the C-terminal region. A candidate region is Ly~~~~-Lys”“” (numbered as in human LPL), that harbours a cluster of positively charged aa. Thus, 5 aa out of 9 (italicised) are positively charged (KVRARRSSK). This sequence, located in exon 6 (human gene), is entirely conserved in terms of aa with, however, four codons differing at the third base in art, mouse, human, bovine and guinea pig (Enerbgck et al., 1987). In chicken, there is only functional homology and no identity, RVRTKRNTK. Cooper et al. (1989) proposed three other candidate regions by analogy with the heparinbinding regions of apolipoproteins E and B. Such a region is found in 279-282 (human numbering), where RKNR is conserved in all six species (in terms of aa not codons). The ten conserved Cys of the previously described species are also conserved in rat (Cys 27, 40, 216, 239, 264,

465

with human, mouse, bovine,

as predicted

mutations

all the C-terminal

and not from the end of exon-9 as for the other 5 cDNAs)

and chicken (cLPL). The aaof the catalytic

by black spots; those involved in missense

Comparison

is written twice, once in this figure, in order to gather

of exon-10

Aligned aa sequences

His Glu SW AlaStop

from LPL cDNA

clones for rat (rLPL),

human

and

(hLPL),

triad (Ser’j2, AsP’~~, His2” ) are boxed. Those aa conserved

in human

are indicated

by asterisks.

275, 278, 283,418,438). The sequences around these Cys are highly conserved. It has been shown for bovine LPL that all Cys were disulfide-bonded (Yang et al., 1989). Two N-glycosylation sites, Asn43 and Asn359, that correspond to the consensus sequence Asn-Xaa-Ser/Thr where Pro is excluded as Xaa and that have been previously shown to be conserved in all five species, are also conserved in rat. At the present time, the LPI, site of interaction with apolipoprotein CII has not yet been identified. Similarly, the site involved in the formation of the LPL homodimer, the active predominant form of LPL bound to endothelial heparan sulfate, remains to be established. Interestingly, the aa involved in the missense mutations already known to be responsible for human LPL deficiency are all conserved in all six species, with two apparent ex-

243 ceptions.

These

following.

In exon 3: Tyr61 (+ Stop), Va169 (+Leu),

aa, that are indicated

in Fig. 2, are the Trp*’

(+Arg),

Lys102 (+insertion + frameshift), Glnlo6 (- Stop); in exon 4: His136 (-+ Arg), Gly14* (4 Glu), G~Y’~~ (4 Ser); in exon 5: Asp156 (+Gly or +Asn), Pro15’ (+Arg), Ala’76 (+Thr), Gly”’ (-+Glu), Ile’94 (+Thr), Asp204 (+Glu), Ile205 (+ Ser), Pro*” (+Leu), Cys216 (+ Ser), Ala221 (+frameshift); in exon 6: Arg243 (-+His), Ser244 (+Thr), Tyr262 (+ Stop); in exon 8: Trp 382 Asp250 (+Asn), (+ Stop); in exon 9: Ser447 (+ Stop). One exception occurs in Ser447, but this exception is more apparent than real, because this mutation has been likened to a polymorphism rather than to a source of metabolic pathology. In fact, the two last aa of LPL are not necessary for LPL activity. Another exception is Glnlo6, but in this case we are dealing with premature termination of the peptide chain rather than with a missense mutation. For references concerning these mutations, see a review by Etienne et al. (1992). (2) CG content CG sequences are known to be rather unevenly distributed in the genome. CGs have been lost in the course of evolution, leaving vertebrates with a remarkable deficiency of this dinucleotide in the different genes (where CG/GC: is approx. 0.1) except in the regions surrounding the promoters of housekeeping genes, the CpG island, where CC/CC is approx. 1. LPL exons 2 to 9 are relatively rich in CG. The ratios CG/GC are 0.41,0.42,0.43,0.47,0.53,0.26 for rat, mouse, human, bovine, guinea pig and chicken, respectively. The role of CG enrichment in these exons cannot be presently explained. (c) Comparison of the sequences of the untranslated exon 10 The 3.18-kb clone alone covers the whole of rat exon 10, while several overlapping clones in mouse and in human were necessary to achieve this. The untranslated exon 10 of rat LPL cDNA is 2019 nt long, i.e., it is longer than the total of the nine other coding exons (1160 nt). This exon is 1948 nt long in human (Wion et al., 1987) and 2353 nt in mouse (Zechner et al., 1991). Gene LPL organization is known for human (Deeb and Peng, 1989; Kirchgessner et al., 1989), mouse (Zechner et al., 1991), and chicken (Cooper et al., 1992). Exon 10 starts with A, the last nt of the stop codon TGA that was interrupted by intron 9. This is the only known example so far of stop codon interruption by an intron (3.1-kb long in human and mouse) or with the last noncoding exon containing only the third nt of the stop codon and overriding in length the total of the coding exons located upstream. Remarkably, in chicken (Cooper et al., 1992) the last codon in exon 9 interrupted by intron 9 is GG/T (Gly)

rather than TG/A (stop codon), as is the case in human and the four other species.

Chicken

exon 10 thus presents

a

short ORF translated into Gly + 14 aa up to the next stop codon. This explains why the chicken aa sequence is longer, not because exon 9 is longer, but because there is no stop codon between exon 9 and exon 10 (Figs. 2 and 3). Zechner et al. (1991) have reported in mouse an insert (relative to man) consisting of (i) a Bl repetitive element (Kalb et al., 1983) (belonging to the Ah family) of 152 nt followed by (ii) a homopurine stretch of 169 nt consisting solely of A and G residues. This insert is flanked just upstream (GAAAATGAGCTTATAA) and downstream (GAAAATGAGCTTGTAA) by a 16-nt direct repeat. (differing only where italicised; see Fig. 3.) Six different mouse strains were tested by Zechner et al. (1991) and the B 1 element was found in exon 10 of all these. The mouse insert was not described by Kirchgessner et al. (1987) because their clone stopped about 250 nt ahead of it. In rat, both the Bl repetitive element and the homopurine stretch are missing. Consequently, only one of the two repeats is present. One copy of this direct repeat is found in human exon 10 and bovine exon 10, which likewise lack the mouse-specific Bl element (Fig. 3). Zechner et al. (1991) have reported in mouse a 195-nt region, located just after the 3’ direct repeat following the insert, that has no homology to human 3’ cDNA. We have also found this region in rat, where it includes a stretch of 157 nt that is highly homologous to the 3’ part of this mouse sequence. However, it is deleted in man. This clearly appears when the sequences of mouse, human and rat are aligned. The 157-nt stretch was compared to known rodent repetitive elements, such as mouse Bl (or Ah type I), also present in rat (Kalb et al., 1983); rat B2 (Bains et al., 1989) (or Ah type 2) (Kalb et al., 1989) and rat newtype (Kalb et al., 1989) both also present in mouse. There is no homology with any of these. Of course, there is no homology with 7 SLRNA either, since this element, which is assumed to be the progenitor of the Bl family, is itself homologous to the Bl element (Quentin, 1989). However, a sequence of this mouse/rat insert can be recognized in other genes: 930CCTGCCTTGGCTTCCTGAGTGCTGGGA956 (Fig. 3). Its percentage of homology (interrogation of the data bank GenBank) is 74% with the murine eosinophil differentiation factor (interleukin 5)-encoding gene, 88 % with the hamster replication-initiation locus, 88 % with mouse pim-1 proto-oncogene, 85% with mouse p-2 microglobulin-encoding gene, 8 1 y0 with human cosmid clone HDAB, 85% with human PRT, 77% with rat heme oxygenase-encoding gene, 74% with R. norvegicus toninencoding gene, 66% with human apolipoprotein E-encoding gene, 88 y0 with HSAG-1 middle repetitive element, 81 y0 with the mouse surfeit locus, 74% with the mouse

244 Exons 10

rLPL mLPL hLPL bLPL LPL cge PL

’

c&4 cw4AkGCATCTGA G-+TCTTTGudCCGAAGAAA A TGAAGTAAA!TTTATTT AAkAA AATAkC TTGTTT CAAGA GAAGAAA GCATCCOAGTTCTTTGUGCAGUGUAACAMGTAAATTTAATTT AAAAMATAATACCC TTGTTT CAGAACAMG9ACGGCATOT~TTCT~~~T~~~~~MCTTTTAC AAAA CATACCCAGTGTTT CGGAAGAAAGUCAGCATATWTTCTATWAGAATG AAGTAACTTTTAC MM GATGCCCAGTGCTTT CAGAAAW~TTAGTACTWGCTCTGTGAAOAACA AAATWTTTTAC AAAA GATGTTCAGTGCTTT AAAGAAAATTCTGCACACGAGTCTGCT~~AAG ACACT-GG4GGATGGTCTGTTCA

rLPL mLPL hLPL bLPL LPL cge PL

GGG TGTTTkAA GTGGA + TTTCCTGA G+ ATTAATCCCkCTATA TC + TGTTAGTTdAT GGG TGTTTCiUA GTGGGTTTTCCTOAGTATTMTcCCAf3ZTCTA TCTTGTTAGTTAAAC CGGGTGTTTCAAAAGTGGATTTTCCTGAATATTAATCCCATTCA ACi4 TGOT Ci4AATGTGWTTTTCCGGAGTATTAACCCCA~TCTAGCCTTATTAGTTATTT Go0 TGCTTAAAA GTWTTTTCCTWGTATTAATCCCAQZTATCTCCT~TTAGTTC.UC TCCCTGT G9ACTOGATGTTCA09ACCAAATATATATA~TATC

AGAAG4 L GTGTCAAA + ATTAAAA CK&TMCAC~ AOlUIOACAGTCTWAATATTAAACOOTOGCTAACCCCA

rLPL rnLPL hLPL bLPL LPL cge PL

CCTAAT G&G ATAGCA+GTCCTCCA~TCAG4AG9’CAGCAGAGA~GAAGCATCTCTTAT~T~TT~~-TCAT+ ACGTGPI G& GGGTGAGGM TCTAATOOCCC ATAWAaTTCTTCCAGCATCAGU~ CATCAGGCAGGWAAACATOGTCTTGTATCCCTTAAGAAG~XATCATT ATTTATGOGG TATAGTGGCCAAATAGCACATCCTccAAcGrT~~ CAGTGGATCAT WAAAGTOCTGTTTTG TCCTTTGAGAAAWAATAAT ATTTGAGGTG TATAGTGXCAAA TAOCACATCTTCCMCATTAAAAAAA TAACAGAT AT WAAAGCACTGCATTCTGTCTTTT WAAAAATATGAGT AT TAAGGCC TCTAGTGGGTA ACTCCA ATCCTC AGCATTA AA09 TGGTA ATCG CS,AGC CCGTGTT TGTCT TTOAGOGACAAA A TTGTAAGGAAGTCTCAGACAAAAGTTACTAACCATAATTA CATTATCTGATAGTTAAGAACAAAW,AACCcCT(3ZAACAACTTccGAAAG

rLPL mLPL hLPL bLPL LPL cge PL

TGTTCCCAA JXATA CAAGACTCC!TCATGTGA &A TTTGGTdTGGTCT d TTAGTAAGGkCTCTTATT~TCATTAGAT A TCTGAGG TGTTCCCAACAATA TAAGACTCC4TCATGTGACTTTGGTcAT~CTAAAATTAGTAAGAAC TCTC%GG TGTTT04GCOCAOAGTAAMTAAGGCTCCTTCATGTGGCGTCTT TATTTAAAATGATAAAATAATCAGATCTCTTCATGTAGTAT TTTTAATTGGGATTCTQXT TGTKCCA TAGTAATATCC GCTTGAGTGTTAGGAAOTAATAAGTAATTTT~TTAATC~~~TT~~~~TTCTT~T~~T~TCTTTA~~TTT~A~ATTTATT

rLPL rnLPL hLPL bLPL CLPL

GACCTTCTkAAGTTCTC + TGAAGTCT TT AAATTd MTATAkAACAACA T + TTTTTGTGC!GTWTCAdTCCATTTCT + TAGCAGT TT ATATTGA G4CCTTTTCAMGTTTTCTCGTCT AATATAGACAATA TTTTTTGTGGCATGAGTCAGGTCCATTTCTTTAOCGGT CTTTTCCGCGGCA CCAATCAGACTCATCTACACQZAGTATG TC GOACTGPI GGCCTTCTCAAACTTTACTCTAAGTCTCCAAGAATACAGAAATG TCTAG4CTG4TAACCTTCTCAGAGTTTTCTCCG4GTCT AAATATAOOAAGTAAGTTTTTTT@XXGTCi4GTAGGKccGTTTAccTATcAATCAA GAAAAGATGCAAAGCTCTOTACCTMjTCCTCTTTTTAGTGTTTTT

rLPL mLPL hLPL bLPL CLPL

190 192 193 177 161 195

AGGAGACAGTCTCAMTACTAAAA CTAATTCA G~GS~GVITCTCAAATACTAAAAAGTGACTMTTCA TTC

’

’

T

T+ TT TT

267 269 260 274 264 294

360 363 366 372 263 394

T& TG

470 452 464 466 494

C!TTTACCAT d GG4TATA&CCCTACCA~TAAAATA AAACAGCTGkCCA TTGTAkTAGTT J TAAATAAAG&CAGUjA~T~GACTT AAACACCTGGCCT TTWAACTAGTTTTTTTTTACCATT~TATATTCCCCCCA CCAAAAAAAAAAAAAAAAAAAAGTAACCAGGAACGTGTGACTTG TGATGTTTTAGAATGA TTCCCTCTTGCTATTGCAATGTCXTCCA~cGTC4 ACCAGGAACATGTAACTTG 2 TCCCTACTTTCTTGGAATTACTCTCCTCTTGGAA ACCAGGAACTAGTGACTTG TGTTGTTCTCCTGGCCTTGACTGAATTTTCAGGCAC;TTTCTCAT

+

567 551 556 545 594

rLPL mLPL hLPL bLPL CLPL

GfJ TTCCTGA &TT @XAAGCA~TGWGA~GGCTCGT Ac&WCAdC CWTACkTCAGTA d GGTACAAAA k TAG4 GCAAAAGCAGTTGAAG4CATGOCTCATGAAGTCCTGACCCTT GGTCCCACCACAAC AAAGTACAAGTC’MCAGAGTACAAMCCTAGA GAGAGGGACGAAGAAAG CTGATAAA CACAGAGGTTTTAAACAGTCCC TACCATTGGCCTGCATCATGC+IAAGTTACAAATTcAAGSAGATA GAG4TAGAAATG4AGAATAGAGTTGATAAAGCACTGAAccTTTAAAC CCCCTCTACGGTTGGT TGCATCATAACTAAGTTACCAATTAAAGGAGATA GGAAAAAAATACCCTTGTGTGTA04GCTATAACAGAGATGTTTAC

’

rLPL mLPL hLPL bLPL CLPL

CT &I G T A ATTCTTAGTTIGACTTCAA GTTTTATOOC + TAATTCCTC!GTCTTTT AAAAACGT $A: ?:::;:;;;:A CTGAG TAATTCTTAATAGACTTGAA TTTTTATGXTTMTCCTTCTATCTTTTAAATATTT TA AMTCT AGATCAATTAATTCTTAATA~TTTATCGTTT ATTGCTTMTCCCTCTCTCCCCCTTCTTTTTT GTCTCAAMTTATATTATAATA TATAMGTTGWATCAATTAMTCT TTAACAGTTT ATGGTTTAGTATTTCCCCTTCCTTTTCCT~TTTGTCTCAAGATTATATTTTAATA TmGTAGAT-ATOCTTCTTA TATAATCTGTTMGATATGTAATCCTATGCACCTTAT ~AGCAATAGC~~G~~~SU~

rLPL mLPL hLPL bLPL

TTATTCTC + AGACAGAT G-+TGAAA T&TTGTGA TTGTTCTCTOGATAGATGT ATGTTCTCTWGTAGGTGT GTTTTTCCCTAGATAGGCTTTTWACTGTTA

I

I

I

I

I

I

656 640 651 642 694

733 717 746 741 707

I

TGGZAATGGTGGCOCTCACCTTTMTcccA@ZACTTGGZAG3XGAG%AGWGGATTTC

766 616 762 778

rLPL mLPL hLPL bLPL

916

rLPL mLPL hLPL bLPL

1016

rLPL mLPL hLPL bLPL

rLPL rnLPL hLPL bLPL

I

I

I

I

AAGAAA~MGAMGAAAGAAAGAAAGAAA~M~A~AA~~-~AA~A

I

T +

Insertion G&c TGGCTGA!TTTATTTCTATGTTTGCT GAGCTCiKi&AATAAT + TCTTGAGAAAAGGAATACT TTGCTG4AAGACAAAMTOTAGGTTGATTTTTACTTCTCTTTTTT~TTTCTT~A~~~TT~~T~T~~~A~CT~TA~ GTGCAGUAAAAAAAA ACAGAGGWAAAAMT

d

’

T GGTGACATA TGAAT! TGAGGTGACACA TAAATT CCTCAGCTGACACATAATTTGAATG TCATCAGCTGATACAGAATTTTAACT

+ GTCCCACT &ATcTGA&TwzcAAJ

rLPL mLPL hLPL bLPL

ACTAAACTA + GTACTTCAGbCTTACCTTdACTCTCAA ACTAAACTATGTATTTCAGGCTGGCCTTGAACTCTCAACCTTT

rLPL mLPL hLPL bLPL

TAGAACA&TTCAAT TTCTTAGT d TTTTCACCAd GCC~ATCGTTAG&TTT~TT~GKTCATC!TG~CCO l&T TTCTTACTTGTTTTCATCAATTTGAAAT GCCCAATATCCAATACTTTGTATTTCATTTG~GACTCATCT~CGCCATGC~TCTGTCACACTT~~~~~~A T CCAMTGATTTTCATCAATTTAAAATCATTCAATATCTU\G T CACAATTACATTCA TTGAAGTCCTTTAATGT GATAGTTACTGTTCA TTTTAWCTTATTTCAQXATGCTTcAGTcAG4CTTTOAGATG

Fig. 3 (continued

c&A TCCTGCCT!GGCTTCCTdGT~TG&CTT~TMc&ATAATTTTA OATA G4TA

on

p.

707 1109 606 604

+ TATCAGATT

867 1209 824 620

+

967 1309 650 646

1

1066 1406 949 937

CCGTAATTTTATTATTAGATTC CTGTAATTTTATTATCAGACTT

245)

kidney androgen-regulated protein-encoding gene and 88 % with human cytochrome P450 IIEl-encoding gene. Another interesting feature of the rat sequence that was not pointed out by Zechner et al. (1991) in mouse, but again

clearly appears upon alignment of the three sequences, is a deletion of 8 1 nt (rat)/7 1 nt (mouse) relative to man. This rat deletion corresponds to a fragment spanning 15911671 in human (Fig. 3).

245

’

rLPL mLPL hLPL bLPL

ad TTTTTTTTTC ICAAGTTTTA!WCAGG~ T>TCWT~cTGCAcT G&MAGT TCACATTAA + TTCTAGTTT 1 GMGTOA AWGTTTTA~XAGGACCATTTTTTTTT TCAAGTTCUATTCTGCACTGT-GT TCACATTAATTTCTAGTTTAaTGTGA TCTCTTTTOTTCCTCrCTTTGAU\TOAAAAGATAGGTTTGTTTT TCTCTTTC TCCCTAACTTTGATA GAAA CACGTATTCAC09GTA CTTTGCATCAT-GTCCTACAAATTTTAGWAAA~TTGTTTTT

1173 1493 1044 1027

rLPL mLPL hLPL bLPL

ACCACGTAG TCG+d TTACTAG&AATGTGTA+AT C&A TGCTTGTk.4CTGTT&-GTGkiWCCTTC+ATTGTGATA~ GM TGCTTGTAAACTOCT GT-GMAAGWXCTCAACTGTAAT~~ ATCACATWG TTGAAA TTACTAGAAAATGTOCATAT GCAGTGCTTGTAAACCATCGCGTGCAATO ACTAAGTUAA~OOAGAGOTTCCTOGGG T(XjPTTCCTAA ACTAAGTAAAGWACA~AAAGTTACAGTCA GTCATATAGTCACTOCCAGTGCTTA AAACCGTTGTG64CXATGGGATcAATT~TATATCCA

1262 1560 1117 1124

rLPL mLPL hLPi_ bLPL

CCATAOACA &r ACCAGGCT CTATAGUAGTACCAGGATTGTTGCcGCTGTTTTGTTTTAcCTT~AA.4AcAAGWAAAAATCMTAATGAACiUT ~~TGGI\GTACCATGA~T~TATTT~T~~~~~ AACAAC ATTGTCATWGTTG

rLPL k? bLPL

CJA TATAAAAT&TAAAAAAdAAAATAAAA CAAGPITCT d TATGTT CA&TTGCTTTTkTATTCAT CGAGPlTCTCACATTTT CAGATTGCTTTTATTATTCATTAATGTAAAAAAATAAAO CTAOATCTCCTATTTTTGWAATGCTCTT CTACGTATAAATATWTGTUG CTAWTCTCATATTTT CAGUTGCTTTT CTATATATTATTATCAAATGTAGAG

rLPL mLPL hLPL bLPL

+ GAACAC ~TACACA ~-~cAAGAGc+TT~~TTC~ATGTCAAA AACAGAAAC 1,GTGUA TGk-TGWTATC CAACCCA CAT~CCCCACAAGTGTAGTCGTCATTCAATGTAA AGCAGAAACTFTGAAA TTTGTGGGTATCTGUCAC AACCCGA CTGTGAAAGTATGTG ATATCTWC4CATACTAGCZTCTGCATGTGTGTTG CA CGTATGAAECCMCATACACG9TTATTGCTCAGCATGGAAA AATCCAAACTGT04ATGTGTGTGGGTGTCTGpJ\CACATATT

rLPL mLPL hLPL bLPL

CSGTCAAGA L TATAC’XT kI TATGTCAG’ GKEAAGCGPTATACTGTATGTTAG TCAAWGATGTATTGGAACATGT~GTA~ TGATTAAGAOGTACATTAGAACACACTGA

rLPL mLPL hLPL bLPL

ATAGGA c+d AGTGKT ATAGG4GUAGOT~CCWTTTCATCA GTAKWXAATGTTGTGATTAA~T~T~~TTGGAAT GTAGAATGAATGCTGTGATTGKATGVXCA~TTGMATC+IATTCTCTCT

rLPL mLPL hLPL

ATTCA T G&I GTATATA C& ATTCA TTACAGTATATACACATCCACATGCA ATTAAATTTCTOGATTTOOGTTGTGACCCAGGGTGCATTAT

rLPL mLPL hLPL

TACATATC + CAATGATGC + TTGACTTTA & TTTTATTT TTAGCTGT TACATATGTWT~TGCTTTAOCTTTTCAATTT ATTTATTAGCTGTAAATA TTATATATATCAAGG9TGTTCTGGCTTTTA~TTTTTACATT

rLPL mLPL hLPL

G+ GTCCACCT ~ATCAGTGAT+GTCT GTGGCTATCkGGG TGTA&TTTGTGGTbCTAACTCT GTGGTTATCTGCAG TATAGATTTGTGGTCCTAACTTTGTGTCCGTCTCCATCCAGTA GTGETATCTGCATTTATAAAAATGTFTajTOCTAACTGTATGr GTCTTTATCAGTGATGGTCT

rLPL mLPL hLPL

~GGAAG4AAATAAAC+C~T~~~-T~TCT~TTT CAATOGAAT GCTTTT MAA~AGUAACTCACCTGTGTGAAGUATWTATCTGCTTT CAATCSAATAGGCTTTT ATCSAATGGGCTTTAACAAAACAAG#,AWAAcGTACTTAACTGTGTC&WAAATCXUTcA~TTT

rLPL mLPL

CGCG TAAC

bT~&TTmTTTAcTATd

window

TATTTATOA TATTTATGAA TAATCAA~GTG4GTMACAACTATTTATAAA TAGTCAAG4GTG4GTGUCATTTATTTATAAA

1194 1166

1431 1752 1279 1272

TATCTCAGA Gd TGTTGCTGd &AA GTTC AAGATC TATCTCAGA%CTATAGCTGGG ATGTCAAATATCTCAG4GATAGCTGGG AAGTCAAATATTTCAG4GATTACTGAA

’

’

’

’

’

Deletion

’

1525 1646 1374 1367

1621 1942 1470 1463

GATGTA c&A TATGTCA TATGTATMTTTGTTAC G4TGTAT~TTTGTTGTTGTCUXTTTATTAATTC GTTGTGTGKTTGTaaaaaaaaa

1712 2034 1570 1539

’

’

KGAAAAACTTTG

1731 2063 1670

,&TA ATaOC-mdmA mTTdt,t,AwCT&mTGd 1620 ATGTGTGOGTATGTMWT GCTTGTAAACACT-GTCTGTT 2159 1770

&TGAGCCAA&TC

ACTCTdTCi4A

CACAGAGCCAACTC

ACTCTT

CAGC

A 2256 1921 1652

TC&.WACGTTT~ATCAT CTGKSACATTTTATCATGATAC TTGKAACATTTTATTACCaa

L

2015 2349 1946 2019 2353

of exon-10 in six species. The polyadenylation

signals are boxed. GenBank/EMBL

and that of the five other species was made using NUCALN manually

A 1334 1669

I

TGAACiUT’

'

size = 20; gap penalty = 7. Aligned

was optimized

L

TkTTCC4GAT~TOCTGTkCTACXXcTTkCTAGG4G&TTGGTTGTk!CTATGTAA+ TAGTTCCAGPITATCCTKiMTGTTAGCCCTTCCTAGOI\ATGTAAT TTGTTCCTG4TGTGCXXGCTTCG4cCCTTTCTCTG4Ci4GKNTGkTCGECCTATAAATA ACTCCTTCATCTGAGAGATACGGTTGRXCTGTACAAA GTACiUAATGGTTCCAGT@ZTjTGCCGGI\AC

A

A

T

ACi4AG AWAG

’

Fig. 3. The nt sequence the rat sequence

’

for a minimum

sequences

were transferred

rate of divergence,

respecting

(Wilbur and Lipman,

into a WingZ

spreadsheet

the choice order: transitions>

We have also sequenced another independent 2.5kb clone, that was shorter than the 3.18-kb clone at the 5’ end but identical to it at its 3’ end. It provided confirmation of the 157-bp insertion and of the 8 1-bp deletion and was also devoid of the mouse Bl element and of the homopurine stretch insert. The consequences of the presence of these inserts and deletions in the untranslated region of mRNA remain to be elucidated. (d) Particular features of rat cDNA LPL exon 10 (I) Polyadenylation signals Mouse and human LPL cDNA have two polyadenylation signals. It is likely that only one of these is functional in rat, since a single LPL mRNA species (3.6 kb) was isolated from different rat tissues (Kirchgessner et al., 1987; Semenkovich et al., 1989). However, eight AATAAA se-

accession

No. L03294.

Alignment

1983) with the following parameters: (Informix,

USA) and alignment

between

K-tuple size = 3;

of the six sequences

transversions>gaps.

quences (four of which overlap) can be found (Fig. 3). These A+T-rich sequences might be involved in mRNA stability. (2) Richness in A+T exclusive sequences Our attention focused on elements rich in A+T, such as: 22-nt: TAAATTTTATTTAAAAAAAATA (64-85); 1 lnt: AAATATTAAAA (167- 177); 20-nt: AAATAAAATAAATAAATAAA (526-545); 9-nt: ATTTTATTT (816824); 13-nt: ATAATTTTATTAT (968-980); 12-nt: ATTTTTTTTTTA (1163-l 174); 14-nt: TATAAAAAAATAAT (1304-1317); lo-nt: AATTATTTAT (1322-1331); 30-nt: AATATAAAAT(G)TTAAAAAAAAAAAATAAAA (1373-1402); 18-nt: TTTAAATTTTATTTATTA (1756-1773). The role of these A+T-rich motifs is presently unknown. It was suggested that A+T-rich motifs such as ATTTA could play some part in the instability of mRNAs (Caput et al., 1986; Shaw et al., 1986; Wilson and Treisman, 1988;

246 Brawerman 1989) or else could be repressive elements decreasing translation efficiency (Kruys et al., 1987; 1989; Han et al., 1990). They presumably play a role in the secondary or tertiary structure of RNA and might be involved, whether directly or indirectly, in the recognition of mRNA by specific degradation endonucleases. It must be pointed out that the consensus sequence AATAAATAAATAAA,

which has been described

(Reeves

and Magnuson, 1990) in the 3’-untranslated region of a variety of genes involved in cellular stimulation (lymphokines, cytokines and proto-oncogenes), is also found in rat (nt 1692-1705). The half-life of LPL mRNA in different species is poorly documented. In view of the present results, it would be interesting to study the stability of all these mRNA and to investigate

the role of their A+T-rich

Han, J., Brown, T. and Beutler, B.: Endotoxin-responsive trol cachectin/tumor necrosis factor biosynthesis level.: J. Exp. Med. 171 (1990) 465-475. Kalb, V.F., Glasser, tive elements

S., King, D. and Lingrel, J.B.: A cluster of repeti-

within a 700 base pair region in the mouse gcnome.

Nucleic Acids Res. 11 (1983) 2177-2184. Kirchgessner,

T.G.,

sequence

Svenson,

of cDNA

K.L., Lusis, A.J. and Schotz,

encoding

lipoprotein

M.C.: The

lipase. J. Biol. Chem.

262

C., Etienne, J., Guilhot,

S.,

(1987) 8463-8466. Kirchgessncr,

T.G., Chuat, J.C., Heinzmann,

Svenson,

K., Ameis, D., Pilon, C., D’Auriol,

M.C., Galibert,

L., Andalibi,

F. and Lusis, A.: Organization

A., Schotz,

of the human

lipo-

protein lipase gene and evolution of the lipase gene family. Proc. Natl. Acad.

Sci. USA 86 (1989) 9647-9651.

Kruys, V., Wathelet,

M., Poupart,

P., Contreras,

J. and Huez, G.: The 3’ untranslated beta mRNA has an inhibitory

R., Fiers, W., Content,

region of the human interferon-

effect on translation.

Proc. Natl. Acad.

Sci. USA 84 (1987) 6030. Kruys, V., Marinx,

sequences.

sequence con-

at the translational

lational

O., Shaw, G., Deschamps,

blockade

imposed

J. and Huez, G.: Trans-

by cytokines-derived

UA-rich

sequcnccs.

Science 245 (1989) 852-855. Miyamoto,

ACKNOWLEDGEMENTS

M.M.,

Slightom,

tions of humans

We thank Dr. Michel Bouillot (Genset) for his stimulating participation in the construction of the synthetic probe. This work was supported by grants from Institut National de la Sante et de la Recherche Mtdicale (grant 900 203 to J.E.) and from the Centre National de la Recherche Scientifique through UPR41. We thank Beatrice Pelletier for typing the manuscript.

globin region. Olivecrona,

J.L. and Goodman,

and African

rela-

in the $n-

Science 238 (1987) 369-373.

T. and Bcngtsson-Olivecrona,

milk-the model enzyme in lipoprotein J. (Ed.),

M.: Phylogenetic

apes from DNA sequences

Lipoprotein

Lipase.

G.: Lipoprotein lipasc research.

Evener

lipase from

In: Borensztajn,

Publishers,

Chicago,

1987,

pp. 15-28. Quentin,

Y.: Succcssivc

history. Reeves,

waves of fixation of B 1 variants

R. and Magnuson,

pression

in rodent lineage

J. Mol. Erol. 28 (1989) 299-305. N.S.:

of mammalian

Mechanisms

cytokine

regulating

transient

genes and cellular oncogenes.

exProg.

Nucleic Acid. Res. Mol. Biol. 38 (1990) 241-282. Sanger, F., Nicklen,

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