The primary structure of mouse saposin

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Vo1.184, No. 3,1992

Pages 1266-1272

May 15,1992

THE PRIMARY STRUCTURE OF MOUSE SAPOSIN Masahiko Tsuda I'* , Takeshi Sakiyama I, Hideya Endo 2 1 and Teruo Kitagawa IDepartment of Pediatrics, Nihon University, 8, l-Chome, Kandasurugadai,

School of Medicine,

Chiyoda-ku, Tokyo I01, Japan

2Medical Research Institute, Nihon University, 30-1, Kamimachi, Ohyaguchi,

Itabashi-ku,

School of Medicine,

Tokyo 173, Japan

Received March I, 1992

SUMMARY: The primary structure of mouse sphingolipid activator protein (saposin) was determined by cDNA sequencing. The amino acid sequence predicted by the cDNA sequence revealed that mouse saposin was highly homologous to human saposin and also to rat sertoli cell glycoprotein. Mouse saposin also has four functional domains, which are structurally similar to each other, and each domain has cysteines, prolines, and a potential glycosylation site at an almost identical position. An amino acid comparison between human and mouse saposins revealed that the similarity was approximately 70%, and human saposin lacks thirty-one amino acids between domains C and D. Heterogeneities of mRNA were found in both the coding and noncoding regions. © 1992 Academic Press, Inc.

Saposin proteins

is

that

a is

family known

of to

small,

heat

activate

stable,

a number

and

highly

of lysosomal

glycosylated

hydrolases

(i).

Saposin B, originally designated as SAP-I, is also a sulfatase activator, and the genetic deficiency of this protein results in clinical features similar to those

observed

originally

in

called

metachromatic SAP-2,

and

the

leukodystrophy deficiency

clinical features of Gaucher's disease (4, 5). and it was revealed

that four different

and

the

possibility

3).

this

protein

of

processing therapies

(6, 7).

for

it is essential to use an excellent animal model.

technology

has

it

*To whom correspondence

possible

to create

should be addressed.

0006-291X/92 $4.00 Copyright © 1992 by A~'aclemic Press, Inc. All rights of reproduction in any form reserved.

1266

leads

C

was

to the

Human saposin has been cloned,

disorders,

made

Saposin

saposins were derived from the same

large precursor and generated by proteolytic pathophysiology

of

(2,

mouse

models

To study the

lysosomal

storage

Recent molecular by

the

transgenic

Vol. 184, No. 3, 1992

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

method and gene targeting. mouse model,

As the first step in creating a saposin-deficient

we initiated

the isolation of mouse saposin cDNA, and here we

report the primary structure of mouse saposin precursor, presaposin.

MATERIALS AND METHODS Isolation of Mouse Saposin cDNA A mouse liver eDNA library (purchased from Stratagene, La Jolla, CA) was screened by plaque hybridization. Human saposin cDNA obtained from ATCC was used as a probe. Human saposin cDNA was labeled with [32p]-dCTP (NEN, Boston, MA) by means of the random priming method using a commercially available kit (Takara, Tokyo, Japan). Positive clones were purified, and in vivo excised to the plasmid bluescript (Stratagene, La Jolla, CA). Inserts were digested with EcoRl (Takara, Tokyo, Japan) and sized on a 1% agarose gel. A mouse brain cDNA library (Clontech, Palo Alto, CA) was also screened to identify the 5'-end nucleotide sequence. Mouse saposin cDNA isolated from the liver library described above was used as a probe. Nucleotide Sequence Determination Several clones were characterized by their nucleotide sequence and restriction enzyme map. Restriction enzymes were purchased from Takara, Tokyo, Japan. Insert DNA was digested with several restriction enzymes and each fragment was further subcloned into the sequence plasmid pG~M-4Z (Promega, Madison, WI) and directly subjected to nueleotide sequencing by the dideoxynucleotide chain termination method (8), using an alkaline-denaturated plasmid as a template (9). Nucleotide sequencing was performed with commercially available kits (USB, Cleveland OH, or Pharmacia, UPSAIA, Sweeden). Computer Analysis Computer analyses for nucleotide and amino acid comparison were performed with the software program GENETYX (SDC, Tokyo). RESULTS AND DISCUSSION The mouse liver cDNA library was screened by hybridization with a probe specific

to the

plaques result

were

initially

indicated

saposin mRNA.

C-terminus

that

of human

obtained

saposin.

from

approximately

the 0.005%

Approximately

1 X of

i0

6

phages

the mRNA

All positive clones were plaque-purified,

in

fifty positive screened. the

This

liver

was

in vivo excised to

the plasmid bluescript, and then the inserts were sized on a 1% agarose gel. The longest clone designated as MSAP-24 was sequenced.

The sequence analysis

of MSAP-24 revealed a close similarity to human saposin cDNA as well as rat sertoli cell glycopr0tein.

However, the 5'-end of this clone was rearranged.

MSAP-24 was then used as a probe to screen a mouse brain cDNA library, and a longer

clone

analysis.

designated

as

MSAP-4

was

obtained

and

Several other clones were also sequenced,

mRNA were observed.

subjected

to

sequence

and heterogeneities

of

The nucleotide sequence and deduced amino acid sequence

1267

Vol. 184, No. 3, 1992

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

18 20 30 40 50 00 70 BO 90 GCGCCATGTACG~CTCGCCC.TCTTCGCCAGCCTTCTG~C~ACCGCTCTGACCA~CCCTGTCCAA~ACCCGAA~ACATGC~CTGGG~G~T MetTy~A~aL~A~aLeuPheA~a~erLe~LeuA~aThrA~aLeuThrSerP~Va1~nAspPr~LysThrCys~e~G~yG~ySer ]OO 110 120 130 140 150 , 180 178 180 CAG•AGTGCTGTGCAGA•ATGTGAA•ACGG•GGTGGACTGT••GG•CGT•AAGCACTG•CAGCAGA•GG•CTG•AGCAAGCCCA•AGCGA

A•aVa•LeuCysArgAspVa•LysThrA•aVa•AspCy•G••A•aVa•Lys•isCy•G•n••n•etV••Trp•e•LysP••ThrA•aL•s 180 200 210 228 230 240 250 288 270 AATCCCT•CCTT•CGACATAT•CAAAAC•GT•GTCACCGAAGCT•G•AACTTGCTGAAAGA•AAT•CTACGCAGGAGGA•ATCC•TCA•T

~erLeuPr~CysAsp~eCy~L~sThrVa~Va~ThrG~uA~a~yA~nLeuLeuLy~AspA~nA~aThrG~nG~uG~u~eLeu~isT~r 280 290 300 310 320 330 340 560 380 ACCT••AGAAGACCTGTGAGTGGATTCATGACTCCA•CCTG•C•GCCTCGTGCAAGGAGGTGGTTGACTCT•ACCTGCCTGTCATCCTGG

LeuG•uLysTh••y•G•uTrp••e•isAsp•erSerLeuSerA•a•erCysL••G•uVa•Va•Asp•erTy•LeuPr•Va•••eLeuAsp 370

380

380

400

410

420

439

440

450

ACATGATTAAGGGCGA•ATGAGCAACCCTGGGGAA•T•T•CTCT•CGCTCAACCTCTGCCAGTCCCTTCAGGAGTACTTG•CC•A•CAAA ~t1~eLy~G~yG~MetS~rAsnPr~G~yG~Va~ys~rA~aLeuAsnLeuCysG~nSerLeuG~nG~uTyrLeuA~aG~uG~nAsn 460 470 460 490 500 510 820 530 540 AC•AGAAAC•GCTTGAGTCCAACAAGATCCC•GA•GT•GACA•G•CCCGTGTGGTT•CCCCCTTCA••TCCAACA•CCC•CTCCT•CTGT

G•nLysG•nLe•G•uSerAsnL•s1•ePr•G•uVa•A•p••tA•aArgVa•Va•A•aPr•PheMet•erA•n••ePr•LeuLeuLeuTyr 550

880

570

580

590

800

810

820

830

ACCCTCAGGATCACCCCCGCAGCCAGCCCCAACCTAAGGCTAACGAGGACGT•TGC•AGGACTGTA••AA•C•GGTGTCTGATGTCCAGA P••G•nAsp•isP••Ar••erG•nPr•G•nP••LysA•aAsnG•uAspVa•CysG•nAspCysMetLysLeuVa•SerAspVa•G•n•hr 840 888 890 670 885 890 700 710 720 CTGCTGTGAAGACCAACTC••GCTTTAT•CA•GGCTTCGT•GACCACGTGAAG•AGGATTGTGACCGCTTGGGGCCAGGC•T•TCTGACA

A•aVa•L•sThrAsnSerSerPhe1•e••nG•yPheVa•AspH•s•a•L•s••uAspCysAspArgLeuG•yP••G••Va••erAsp•1e 730 740 750 780 770 780 790 800 810 TAT•CAAGAACTACGT•GACCA•TATTCCGAGGTCT•TGT•CA•AT•TTGAT•CACAT•CAG•ATCA•CAACCCAAG•AAATCTG•GTGC CysLysAsnTyrVa•AspG•nTyrSe•G•uVa•Cy•Va•••nMetLeuMet•i•MetG•nAspG•nG•nPr•Lys••u1•eCysVa•Leu 820 530 840 850 860 870 880 890 800 TG••TGG•TTCTGTAATGAGGTCAAGAGAGTGCCAATGAAGACTCTGGTCCCT•CCA•CGA•ACCATTAAGAACATCCTCCCTGCCCT•G

A~aG~yPheCysAsnG~uVa~LysAr~a~Pr~MetLysThrLeuVa~Pr~A~aThrG1uThr1~eLysAsn~eLeuPr~A~aLeu~u 910

820

930

940

850

980

970

980

890

AGATGATGGAC•CCTATGA•CA•AATCTGGTCCAG•CCCACAATGTGATTTTATGC•A•ACCTGTCA•TTT•T•AT•AATAAGTTTTCTG Met•etAspP••TyrG•aG•nAsnLeuV••••nA•a••sAsnVa•••eLeuCysG•nTh•CysG•nPheVa••etAsnLysPheSerG•u 1000 1010 1020 1050 1040 1050 1080 1070 1080 AGCTGA•TGTCAATAATGC•ACT•AGGAGCTCCTAGTTAAA•GTTTGAGCAACGCATGCGGA•TGCTCCCC•ATCCTGCCAGAACCAAGT

Leu••eVa•AsnAsnA•aTh•••u••uLeuLeuVa•Ly•••yLeuSerAsnA•aCysG•yVa•LeuP••A•pPr•A•a•rgThrLysCys 1090 II00 III0 1120 1130 1140 1150 I158 1170 GCCAGGAGGTG•TG••AACATTTGG•CCCTCCCT•TT••ACATCTTTATCCATGAGGTAAACCCCA•CTCTCT•T•CGGTGTGATCGGCC

G~nG~u~a~Va~G~yThrPheG~yPr~erLeu~euAsp~1ePhe~1e~sG~uVa~AsnPr~SerSerLeuCysG~yVa~1~eG~Leu 1180 I190 1200 1210 1220 1230 1240 1250 1260 TCT••GCTGCCCGCCCGGA•TTG•TGGAGGCACTTGAGCAGC•TGCGCCAGCCATTGTATCT•CACTGCTCAAA•AGCCCACACCGCCAA CysA~aA~aA~Pr~uLeuVa~G~uA~aLeu~uG1nP~A~aPr~A~a~eVa~Ser~aL~uLeu~ysG~uPr~ThrPr~Pr~Lys 1270 1280 1290 1300 1510 1320 1330 1340 1550 A•CAGCCCG•ACAG•CCAA•CA•TCGGCATTGCCCGCCCATGTGCCTCCTCAGA••AATGGT•GGTTCTGTGAGGT•TGCAAGAAACTGG

G••Pr•A•a••nPr•LysG•nSerA•aLeuPr•A•aHis•a•Pr•Pr•••nLy•Asn••yG•yPheCysG•uVa•CysL•sL•sLeuVa• 1360

1370

1380

1390

1400

1410

1420

1430

1440

TCCTCTATTTG•AACATA•C•TG•AGAAAAA•A•CACCAAG•AG•AAATCCTGGCCGCACTTGAGAAG••CTGCAGCTTCCTGCCAGACC LeuTyrLeu~uHisAsnLeuG~uLysAsn~erTh~LysG~uG~u~eLeuA~aA~aLeuG~aLysG~yCy~SerPheLeuPr~AspPr~ 1450

1480

1470

1480

1490

1500

1510

1520

1530

CTTACCA•AAGCAGTGCGAT•ACTTT•T••CT•A•TATGAGCC•TTGCTATT••AGATCCTCGTG•AA•TGATGGATC•TGGATTTGTGT TyrG~nLysG~nCy~AspAspPheVa~A~aG~uTyPG~uP~LeuLeuLeuG~u~eLeuVa~G~uVa~MetAspPr~yPheVa~Cys 1840

1550

1580

1570

1590

1590

1800

1510

1620

GCT•GAAAATTGGA•T•TGCCCTTCTGCCTATAAG•T••T•CTG•GAAC•GAGAAGTGTGT•TGGG•C•CTA•CTACTGGTGTCAG•ACA SerLys~eG~yVa~Cy~Pr~SerA~aTy~L~sL~uLeuL~uG~yT~rG~uLysCys~TrpG~yPr~erTyrTrpCysG1nAsnMet 1830 1640 1850 1880 1670 1880 1890 1700 1710 T••AGACTGCCGC••GAT•CAATGCT•TCGATCATTGCAAA•GCCATGTGTG•AACTAGTTTCCCAGCT•CAGAA•TCACCTACTTGTGG GluThrAlaAlaArgCysAsnAlaValAspHlsCysLysArgHisValTrpAsn 1720

1730

1740

1750

1780

1770

1780

1780

1800

GTCTA~G~TAATGAACACATAGATCTATTTGACTTAATAAGTAGGAACCCCCTTTGCCCTTCCCCCATCTC~TCTCCCTTACTGTAGCAT 1810

1820

1830

1850

1840

1860

1870

1890

1890

TTCTGTCATGTAAGAGGTGCTGACA•CCACTTCCGTGTCCCCTTTCTGCTCGAAGGATGA•GATACCTT•GGACATCGCTC•CCGGCTGC 1800 1910 1820 1930 1940 1950 1960 1970 1980 CCTTTTCACCCACCTGCTG•A•G••••••GT•A•CCAGAGGGCA•GAGC•TTTTCTGAGCCCTTTCTTGGTGTGTGGGGGATCTAT••CC 1990

2000

2010

2020

2030

2040

2050

2080

2070

ATCTCCTAC•ATGAGGG•GCTACCCAGCTTCCT•T••TACCAAG•A•TTAT••T•GAT•ATTAGAAGCACAGAAT•ATCAGGCC•T•AGA 2080

2090

2100

2110

2120

2130

2140

2150

2150

~CGAT~GAATG~C~ATTGTCATAGCACAGAGATTTCAGAAGCA~CTGCAG~TGGCTTGCTTGGGATGTTGCTGTCCCTGGGTCA~CCTTC 2170 2190 2190 2200 2210 2220 2230 2240 2250 CATTCTGCTTTCCTGTCTTCCCGTC•GCCTTGTTGGGGTTCTGTGGGGTAGGGTGGGGAG•GGAAACTTGTGAATGTAACTTGCCT•TGC 2280 2270 2280 2290 2300 2310 2320 2530 2340 ~GT~ACGTTCACGTGGGCCTGGTCTTTTGTGTGTGAGG~CCTTGACCGT~TG~CCTCTGCCTGGACTGT~T~GGGTCCT~CACGGCTT 2550 2560 2370 2380 2390 2400 2410 2420 2430 ••CCACCAC•TGTA•CTCTTGTT•ACCTGCCTGTTCACCTCATGAGTGAAGC•T•TGCC•G•CAG•G•GCCATGAACTGA•G•GTCTCTG 2440

2480

2480

2470

2480

2490

2500

2510

2520

T•TA•AGTAGAAGCTTCCTGTGC•TCC•GTTG•CAGGAGACAGC•TGTGCAGTTAAATGGAC•TAGATTTT•TTTTGCACTAAAGT•TCT 2530

2540

2550

2580

GTGACTTAATAAAGTTCTGTTAACCAACAGAA~AAAAAAAAAAAAA

Fig. i. Nucleotide sequence of the longest mouse saposin cDNA and deduced amino acid sequence. The putative polyadenylation signal is underlined. The amino acids are designated by three-letter codes.

1268

VOI. 184, No. 3, 1992

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

bIYALFLLASL LGAALAGPVL GLKECTRGSA VWCQNVLTAS DCGAVKHCLQ

HUNAN RAT NOUSE

****A-F*** ,AT**TS**Q DL~T*SG*** *L*RD*K**V **G*****Q* TVWNKPTVF~ LPCDICKDVV TAAGDMLKDN ATEEEILVYL EKTCDWLPLP M**S***A*]* *******T** *E**NL**** *******H** ****A*IHDS N**S***A*[* *******T** *E**NL**** **Q****H** ****E*IHDS NNSASCKEIV DSYLPVILDI IKGEMSRPGE VCSALNLCES LQK~LAELNH

QKQLESNKIP ELDMTEVVAP FMANIPLLLY PQDGPRSKPQ PKDN~GDVCQD *R***~*-** *V*LAR**** **S******* ***R***Q** **A*[E***** *K**-***** *V-MAR**** **S******* ***H***Q** **A*[E***** CIQMVTDIQT AVRTNSTFVQ ALVEHVKEEC DRLGPGMADI CKNYISQYSE *MKL*T**** ******S*** G*~D****D* ***~**VS** ****VD**** *NKL*S*V** **K***S*I* GF~D****D* ******VB** ****VD**** IAIQNNMHMQ PKEICALVGF CDEV~EMPMQ TLVPAKVASK NVIPALELVE V.V******* *****VM*** ****[*RV**R *****TE*I~ *IL*****TD VCV**L**** *****VLA** ~N**]*RV**K *****TETI~ *IL***~NMD PIKKHEVPAK SDVYCEVCEL LVKEVTKLID NNKTEKEILD AFDKMCSKLP ~YEQDVIQ*Q NVIF*Q**Q* VMRKLSE**I **A**ELLIK GLS*A**L** *YEQNLVQ*H NVIL*QT*QF VMNKFSE*~I **A**ELLVK GLSNA*GV** KSLSEECQEV VDTYGSSILS VLLEEVSPEL VCSMLHpCSG] TR ........ APA*TK**** LV*F*P*L*D **MH**N*NF L*GVISL**~ NPNLVGTLEQ DPARTK**** ~G*F*P*L*D IFIH**N*SS L*GVIGL*AA] RPELVEALEQ ....................... LPALTVH VTQPKDC~FC EVCKKLVGYL PAAAIVSALP KEPAPPKQPE EPKQS**RA* *PPQ*N*I**~ *******I** PAPAIVSALL KEPTPPKQPA QPKQS**PA* *PPQ*N~I~** *~**~**L~*

DNNLEKNSTK QEILAALEKG CSFLPDPYQK QCDQFVAEYE PVLIEILVEV

NDPSFVCLKI GACPS~HKPL LGTEKCIYGP SYWCQNTETA ARCNAVEHCK *~***~S** ~V*~J*Y*L* ******VW** G****~S*** *Q**~*D*** ~**G***S** *V***I*Y*L* ******VW** G*****M*** *Q****D*** RHVWN

Fig. 2. Comparison of the amino acid sequence of mouse saposin with those of human saposin and rat sertoli cell glycoprotein. Identical residues are identified by an asterisk. Each missing amino acid is indicated by a dash.

of m o u s e human

saposin

saposin,

is s h o w n

mouse

Figure

2.

acids.

Although

3'-end,

a mouse has

acid

sequence

70%. and

There

The rat

the

these

the

are

a

single

a human

and

i, rat

large

eDNA has

and

the

sertoli open

two

cell

and

glycoprotein

sequence

further

identical.

acid

study

support

of

frame

an approximate

90%

saposin

the e v i d e n c e

It is i n t e r e s t i n g

1269

that

eDNA

has

sertoli

that m i c e

3'

and

in

amino at

the

and a m i n o

both approximately

between

the

shown 557

The n u c l e o t i d e were

between

(AATAAA)

comparison

to

is

encoding

signals

sequence

extended

mouse

comparison

glycoprotein

signal.

sequence

revealed

similarity

cell

acid

polyadenylation

to the k n o w n h u m a n amino

amino

reading

o n l y one p o l y a d e n y l a t i o n

expression

results

saposin

is

nucleotide

nucleotide

Although

saposin,

similarities

sertoli

in F i g u r e

mouse

similarity,

noncoding not

cell

saposin

been

region.

performed,

glycoprotein

rats h a v e

and

and

a thirty-one

Vol. 184, No. 3, 1 9 9 2

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Saposin A B C D

- S L P C D I C K T W T - - E A G N L L K D N A T - Q E E I LHY-- L E K T C E W I H D S S L S EDV-CQDCMKLVSDVQTA--VKTNSSFIQGFVD--HVKEDCDRL-GPGVS VI L - C Q T C Q F V M N K F - - S E L I V N N A T - - E E L L - V K G L S N A C G V L P D P A - S - G F - C E V C K K L V L Y L E H - N L E K - N S T - K E E I L-- A A L E K G C S F L P D P ---

Consensus

- - F - C Q - C K K V V - - - E - - N L - K - N A T - - E E I L .... L E K - C - - L P D P - L S

Saposin A B C D

AS .... CKEVVDSYLPVILD-MIKGE-MSNPGEVCSALNLCQS .... DICKNYVDQYSEVCVQMLM---HM-QPKEICVLAGFCNE T .... KCQEVVGTFGPSLLDIFI--HEV-NPSSLCGVIGLCAA

consensus

......

--YQKQCDDFVAEYEPLLLEILV--EVM-DPGFVCSKIGVCPS CKEVVD-Y-PVLLDILI--E-M-NP-EVCS-IGLC-S

Fig. 3. Multiple alignment of the four functional domains. A consensus sequence is given where two or more identical amino acids align. Closed triangles represent residues which align in all four domains, while the dash indicated alignment of the four potential N-glycosylation sites in mouse saposin.

amino acid insertion between domains C and D. homologous among those species.

Domains A and D were highly

On the other hand, domains B and C were less

homologous. Molecular analyses of the sulfatide activator deficiency and the variants of Gaucher's identified.

disease

have

of

saposin

substitution. activator

and

several mutations

have

been

For sulfatide activator protein deficiency, three mutations have

been identified so far. codon

been performed,

B,

Kretz et al. found a C to T transition in the 23rd

resulting

in

a

threonine

to

isoleucine

amino

acid

This base change eliminates the glycosylation signal in this

protein

(i0).

Zhang

et

al.

found

a

33-base

insertion

between

nucleotides 777 and 778 (numbered from the A of the ATG initiation codon)(ll). Most recently, Holtschmidt et al. found a G to C transversion that eliminated a cysteine residue in the 47th codon of saposin B (12). Gaucher's disease,

a G to T transversion resulting

in the

In a variant

of

substitution of

phenylalanine for cysteine in the 72nd codon of saposin C was identified (13). All of these residues found mutated in these disorders were conserved among humans, mice, and rats, which indicates the significance of these residues. Figure 3 shows the multiple alignment of four junctional domains. alignment

position of six cysteine

potential

glycosylation

site

in

residues,

each

domain

one was

proline

A complete

residue,

observed.

A

and

one

consensus

sequence for two or more identical amino acids occurring in the same position of the alignment suggested that the saposin gene arose by gene duplication.

1270

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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

mouse + 9

256 M H M Q D Q q P K E ATG CAC ATG CAG GAT CAG CAA CCC AAG GAA

mouse + 6

ATG CAC ATG --- GAT CAG CAA CCC AAG GAA

mouse

ATG CAC ATG ......... CAA CCC AAG GAA

s

5

AATAAAGTTCTTGTTAACCAACAGAAAAAAAA

mouse + 3

AATAAAGTTCTTGTTAACCAAC--AAAAAAAA

mouse

AATAAAGTTCTTGTTAACC .....AAAAAAAA

mouse

+

Fig. 4. The heterogeneities found in mouse saposin mRNA. A: In the coding region, a 9-base insertion (CAGGATCAG) or a 6-base insertion (GATCAG) was found. The number above Met indicates the amino acid number counting from initiation methionine. B: In the noncoding region, a 5-base insertion (AACAG) or a 3-base insertion (AAC) was found just before the poly(A)tail. Poly(A) attaching signals are underlined.

Sequence

analysis

heterogeneitles Besides

the

contained region. it was there

several

mRNA,

additional

there

stretch

These heterogeneities at exactly

were

either

disclose

the

were of

the same position three

or

five

both the coding and noneoding mouse

revealed

eDNA

should

mechanism

of

model of saposin-deficiency

at

either

presence

of

regions of the mRNA (Fig. 4).

least nine

the

two or

additional six

bases

mRNAs

which

the

coding

in

were also found in human saposln eDNA (12), and

poly (A) track in some clones.

The

clones

both in the coding and noneoding

shortest

an

of

and

sequence.

additional

base

The significanees

In the noncoding insertions

just

region,

before

of these heterogenelties

the in

regions have yet to be determined. facilitate

gene

studies

duplication

as

on well

developed by the molecular

genomic as

organization

providing

an

to

animal

approach.

ACKNOWLEDGMENTS This work was supported in part by Grants in Aid for the Scientific Research on Priority Areas, Molecular Basis of Single-gene Disorders from the Ministry of Education, Science and Culture, Japan.

REFERENCES i. O'Brien, J.S. and Kishimoto, Y. (1991) FASEB J. 5, 301-308 2. Stevens, R.L., Fluharty, A.L., Kihara, H., Kaback, M.M., Shapiro, L.J., Marsh, B., Snadhoff, K. and Fischer, G. (1981) Am. J. Hum. Genet. 33, 900-906 3. Inui, K., Emmett, M. and Wenger D.A. (1983) Proc. Natl. Acad. Sci. USA 80, 3074-3077 1271

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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

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