Nucleotide sequence of mouse 5-aminolevulinic acid synthase cDNA and expression of its gene in hepatic and erythroid tissues

55

Gene, 48 (1986) 55-63 Elsevier GEN 01788

of mouse Saminolevulinic hepatic and erythroid tissues

Nucleotide

acid synthase cDNA and expression of its gene in

sequence

(Recombinant homology)

DNA; heme synthesis; Eschetichia cofi complementation;

liver; spleen; chicken and mouse

David S. Schoenhaut” and Peter J. Curtisb* a UniversityofPennsylvania. Graduate Group in Biology, Philadelphia, PA 19104 Tel. (215)898-3860, and b The Wistar Institute of Anatomy and Biology, Philadelphia, PA 19104 (U.S.A.) Tel. (215)898-3859 (Received

May 30th, 1986)

(Revision

received

(Accepted

August

August

15th, 1986)

30th, 1986)

SUMMARY

The cDNA coding for 5aminolevulinic acid (ALA) synthase (EC 2.3.1.37) in both liver and anemic spleen of the mouse has been cloned. The liver clone was selected by complementation of an Escherichiu cofi hemA mutant. Erythroid clones were obtained by screening a cDNA library made from mouse anemic spleen RNA, using the liver cDNA as a probe. The sequences of the spleen-derived and liver-derived cDNAs are identical. The nucleotide sequence and predicted amino acid (aa) sequence of a 1.6kb spleen-derived cDNA is presented. The mouse ALA synthase aa sequence displays extensive homology to ALA synthase of chick embryonic liver. The ALA synthase mRNA, detected by Northern blot analysis, was the same size, approx. 2.3 kb, in mouse liver, anemic spleen, and mouse erythroleukemia cells. It is therefore unlikely that different isozymic forms of ALA synthase are present in mouse erythroid and hepatic tissue and this is not the basis for the different effects of heme and porphyrinogenic compounds on the expression of liver and erythroid ALA synthase.

INTRODUCTION

The enzyme, ALA synthase (EC 2.3.1.37) catalyzes the first reaction of the heme biosynthetic path* To

whom

correspondence

and reprint

requests

should

be

addressed. Abbreviations:

aa, amino acid(s); AIA, allylisopropylacetamide;

ALA, 5-aminolevulinic

acid; Ap, ampicillin;

sulfoxide;

kb, kilobases

or kilobase pair(s); MEL, mouse erythro-

leukemia;

nt, nucleotide(s);

resistance;

OTC, ornithine

DMSO,

dimethyl-

transcarbamylase;

SSC, 0.15 M NaCl, 0.015 M Na, .citrate,

R,

pH 7.0.

way in animals. ALA synthase is the rate-limiting step in porphyrin biosynthesis and the major control point of heme production in liver (Kappas et al., 1983). The enzyme is located in the mitochondrial matrix and is derived from a higher M, cytoplasmic precursor (Yamauchi et al., 1980; Ades and Harpe, 1981). Processing of the precursor has been shown in chicken liver to involve cleavage of an N-terminal presequence that is probably required for targeting to the mitrochondrial matrix (Borthwick et al., 1985). A cytoplasmic precursor of rat ALA synthase has also been identified (Yamauchi et al., 1980).

0378-l 119/86/$03.50 0 1986 Elsevier Science Publishers B.V. (Biomedical Division)

56

Since heme is required for the cytochromes of the mitochondrial electron transport chain, ALA synthase is presumably present in all tissues. However, ALA synthase activity can be induced to significantly higher levels in liver where large amounts of heme are required for microsomal detoxification systems, and its activity increases during the differentiation of erythroid cells where heme is needed for hemoglobin synthesis. ALA synthase appears to be regulated differently in these tissues. In the liver, enzyme levels are repressed by heme and increased by porphyrinogenic drugs such as AIA and 3,5-diethoxycarbonyl- 1,Cdihydrocollidine. In contrast, heme either increases or does not affect ALA synthase activity in erythroid cells and little or no effect of porphyrinogenic compounds is observed (Kappas et al., 1983). ALA synthase activity is induced in the spleens of polycythemic mice injected with erythropoietin (Nakao et al., 1968; Sassa et al., 1975) and in MEL cells treated with DMSO (Ebert and Ikawa, 1974; Sassa, 1976). Liver-specific and erythroid-specific forms of ALA synthase which differ in M, and reactivity to ALA synthase-specific antibody have been found in chicken (Watanabe et al., 1983). ALA synthase cDNAs have been cloned from chicken liver (Borthwick et al., 1984) and erythroid cells (Yamamoto et al., 1985). Different ALA synthase mRNAs are expressed in chicken liver and erythroid tissue (Yamamoto et al., 1985), however, only one ALA synthase gene has been detected in the chicken genome (Maguire et al., 1986). To investigate the molecular basis for differential regulation of hepatic and erythroid ALA synthase, we have cloned cDNA from mouse liver and erythroid cells. The liver ALA synthase cDNA was selected by its ability to complement an E. coli hem4 mutant. We present the nt sequence and predicted aa sequence of the mouse erythroid ALA synthase clone and data which indicate that a single mRNA species is expressed in mouse liver, anemic spleen and MEL cells.

MATERIALS

AND

METHODS

(a) Ceils

The E. coli hem4 mutant (supplied by Dr. A. Sasarman, University of Montreal4 Montreal,

Canada) required 50 pg/ml ALA (Sigma, St. Louis, MO) for growth. MEL cells (clone GM86, a 745 derivative supplied by the Institute for Medical Research, Camden, NJ) were cultured in Dulbecco’s modified Eagle’s medium (Flow Laboratories, McLean, VA) supplemented with 876 mg/l of rghmunine, 0.17% sodium bicarbonate, 100 mg/l of streptomycin, 200 units/ml of penicillin, and 10% calf serum. For induction, cells were seeded at lo5 cells/ml and incubated for 24 h. DMSO was then added to 1.8 y0 and cells were incubated for another 24 h. An equivalent volume of medium containing 1.8% DMSO was then added and these cultures were incubated for 2 days before harvesting. (b) The mouse liver cDNA expression library

Six- to eight-week-old mice (BALB/c, Harlan Sprague-Dawley, Indianapolis, IN) were injected intraperitoneally with AIA (supplied by Hoffman-La Roche, Nutley, NJ), 4 mg/lOO g in 95% ethanol 4 times at 12-h intervals. ALA synthase activity was assayed in mitochondria prepared from the livers of control and treated mice by the radiometric method of Scotto et al. (1983). Poly(A) +RNA was prepared from. the livers of AIA-treated mice and used to synthesize cDNA, which was cloned into the PstI site of pUC8 by oligo(dG) : oligo(dC) tailing (Curtis, 1983). Plasmid pUC8 was kindly supplied by Dr. J. Messing, University of Minnesota (St. Paul, MN). The hybrid DNA was transformed into E. coli DHl (Ham&an, 1983) to give approx. 250 000 colonies from which plasmid was prepared to constitute the library. The plasmid library was subsequently transformed into the E. coli hemA mutant and plated on LB agar containing 15 pg Ap/ml. To estimate the total number of transformants, an aliquot was plated on LB agar with 15 pg Ap/ml plus 50 pg ALA/ml. ALA synthase activity in transformed E. coli cells was assayed as previously described (Burnham, 1970). A mouse anemic spleen cDNA library in E. coli DH 1 was screened by hybridization with radioactive probes, that were synthesized from the cloned cDNA insert using calf thymus primers (Summers, 1975). The probes were hybridized to filters in 6 x SSC at 65°C (Curtis, 1983). The filters were washed at 65°C in 2 x SSC, 0.5% sodium dodecyl sulfate.

57

(c) Sequence analysis

TABLE

I

Effect of AIA on ALA synthase

Dideoxy sequencing (Sanger et al., 1977) was performed by cloning the fragments of interest into either M13mp18 or M13mp19 (Norrander et al., 1983). Bacteriophage and JM103 host bacteria were kindly supplied by Dr. J. Messing (University of Minnesota, St. Paul, MN). Sequencing of the inserts was accomplished by exonuclease III (New England Biolabs) digestion of M 13mp 19 clones after cleavage of replicative form of the phage with Sac1 and BumHI essentially as described by Henikoff (1984) except that the exonuclease III digestion was carried out at 25°C and aliquots were removed at 2-min intervals. Comparison of chicken and mouse aa sequences was performed by the PRTLN program of Wilbur and Lipman (1983). (d) Analysis

of RNA

RNA was isolated from mouse anemic spleen, livers from mice treated with AIA as above, and MEL cells as previously described (Curtis, 1983). Poly(A)’ RNA was prepared by oligo(dT) cellulose chromatography. For Northern blot analysis, RNA was electrophoresed in a 1.2% agarose gel containing 3% formaldehyde and transferred to nitrocellulose (Thomas, 1980; Maniatis et al., 1982). The filter was hybridized in 5 x SSC, 4 x Denhardt’s solution (Denhardt, 1966) and 16 pg/ml denatured and sonicated salmon sperm DNA. Radioactive probes were prepared from cloned cDNA insert using calf thymus primers (Summers, 1975).

AIA (mg/lOO g mouse)

in mouse

ALA synthase

(nmol/mg

0

liver * protein/h)’

0.26

10

1.88

20

2.25

50

5.26

4 (4 times)b

21.10

10 (4 times)b

18.50

a Starved

mice were injected

with AIA dissolved

in 0.1 ml 95 %

ethanol. b Multiple c Livers

injections

were given at 12-h intervals.

were removed

mitochondria of Scott0

16 h after the final injection;

were assayed

for ALA synthase

isolated

by the procedure

et al. (1983).

AIA-injected mouse and used as template to synthesize cDNA. A bacterial expression library was constructed by inserting tailed, double-stranded cDNA into the PstI site of plasmid pUC8. The mouse liver cDNA library was used to transform the E. coli herd mutant which is deficient in ALA synthase. Transformants were plated on Ap plates both in the absence and presence of ALA. Of approx. 100 000 transformants for ApR, 50 colonies were identified that did not require ALA for growth and were divided randomly into five pools. Plasmid from the five pools was used to transform the hemA mutant and plated as before. As shown in Table II, 20 to 70% of the ApR colonies were able to grow in the absence of ALA. Two colonies from each pool of transformants were selected and rescreened as TABLE

II

Transformation RESULTS

mids

(a) Selection of mouse liver ALA synthase cDNA by complementation in Escherichia cofi

Plasmid

Injection of chick embryos and rats with AIA induces a marked increase of ALA synthase activity in liver, which is accompanied by an increase in ALA synthase mRNA (Yamauchi et al., 1980; Brooker et al., 1980). Injection of mice with AIA also results in an increase in liver ALA synthase activity 1976; Table I). Therefore, (Igarashi et al., poly(A) + RNA was prepared from the liver of an

activity

pool

of Escherichia coli hemA mutant

Number

of colonies”

Medium

ALA -

by pooled plas-

% ALA+ Medium

ALA +

1

197

279

70

2

2096

8742

24

3

345

464

74

4

1574

5245

30

5

1212

4230

29

a Plasmid

pools

(Hanahan,

1983), which was plated onto LB agar in the absence

(ALA-)

were

and presence

transfected

into E. coli hemA

(ALA ‘) of 50 pg ALA/ml.

mutant

58 TABLE III ALA symthase activity in E. coli transformants a

0.9

P

1.2

P 0.15

0.55

P

5-1

0.65

I

6 ALA synthase (nmol/h/mg protein) 12 Wild type hemA

PML~-~~ PML~-~~

0.025 <0.016 3.700 13.200

-

0.65 -

i H

1.2 H P’;

0.62

20 ” Assay was performed essentially as described by Burnham (1970). Wild type is E. coli strain HBlOl. b Refers to hem4 transformants.

above. Plasmid DNA prepared from these colonies transferred the ALA-independent growth phenotype to 80 to 100% of this third set of ApR transformants. Digestion of plasmid from the 10 colonies with PslI gave two fragments which had identical sizes of 0.9 and 0.65 kb in each sample. ALA synthase activity was assayed in two of the transformants derived from the mouse liver cDNA library (designated pML4-2 and pML2-2), in the hemA mutant, and in wild-type E. coli. The results, presented in Table III, indicate an approximate 150- to 500-fold increase in ALA synthase activity in transformants when compared with wild-type E. coli while the activity in the hem4 mutant was not detectable. (b) Selection of erythroid ALA synthase clones

A mouse anemic spleen cDNA library was screened under nonstringent conditions with a probe prepared from the 0.9- and 0.65kb PstI fragments of pML5-1. 20 colonies which gave the strongest response were selected for analysis. Plasmid DNA was prepared from these colonies, digested with PstI, and electrophoresed. Two of the plasmids selected from the mouse anemic spleen cDNA library, pMS6 and pMS20, contained the largest inserts (1.35 and 1.82 kb, respectively) while a third clone, pMS 12, was representative of a group of clones containing a 1.2-kb insert. All of the erythroid inserts analyzed contained an internal &I site. The pMS (mouse spleen) clones were analyzed by Southern (1975) blotting using the 0.9- and 0.65-kb PstI fragments of pML5-1 as probes in separate hybridizations. The two PstI fragments derived from each erythroid clone hybridized exclusively to one or the other fragment of pML5-1, indicating that the PstI site was conserved in both the

Fig. 1. Mouse ALA synthase cDNA clones. Clones from mouse liver (pML5-1) and anemic spleen (pMS6, 12, 20) are aligned with respect to the common internal PSI site (P). Numbers above the lines refer to the approximate length of each PstI fragment in kb. Arrows indicate the extent and direction of sequencing of each clone. H, HindIII; X, XbaI.

liver and anemic spleen ALA synthase cDNA sequences. Digestion of pMLS-1 with Hind111 revealed that the 0.9-kb PstI fragment is adjacent to the 1acZ promoter in pUC8 and thus represents the 5’ portion of the gene. Therefore it was possible to orientate and compare the different ALA synthase cDNA clones (Fig. 1). (c) Sequence analysis of ALA synthase cDNA

The PstI fragments from pML5-1, pMS6, pMS 12 and pMS20 were purified by preparative agarose gel electrophoresis and individually ligated into the PstI site of M13mp18 for DNA sequencing. Approx. 250 nt were sequenced from both orientations of the insert in each Ml3 clone (Fig. 1) resulting in the comparison of about 1 kb of DNA from three noncontiguous regions ofthe plasmid clones (the internal PstI region, and the 5’ and 3’ ends). The identical sequence was observed in all of these clones. To sequence the entire erythroid cDNA we chose the 1.2-kb fragment from pMS20 and the 0.65-kb fragment from pMS 12, which together represent the longest erythroid sequence obtained (1.85 kb). Each fragment was cloned into the PstI site of M13mp19 in both orientations. The method of Henikoff (1984)

G::

TGG TTC GTC UC AGT GCA GGG CAA CAG 111 GGG UC Trp Phe “I, Leu Ser Ala Gly Gin Gin Asp Phe Gly Le”

60 AGG ATG GTGGCR GCR GCT ATG TTG CTA CGG KC TGT CCA GTG CTC TC, ,:i Arg MET “al Ala Ala Ala M;‘, Le” Le” Arg Ser Cyr Pro “a, ku Ser Gin

59

L50 180 120 GGC CCC ACA GGC UC CTG GGC AAA GTG GCT AA.4 ACC ,AC CAG TTC CTA TTT AGT ATT GGA CGC TGC CCC ATC CTG GCC ACT CAA GGA CCA Gly Pro Thr Gly ie" Le" Gly Lye "a1 Ala Lys Thr Tyr Gin Phe Leu Phe Ser Ile Gly Arg Cyo Pro Ile Leu A,a Thr Gin Gly Pro 6" 40 240 21" ml ACC TG, TC, CAA ATC CA, CT, RAG GCA ACC A.&GGC, CGA C&A WA CTC CAR GAC AGG RAG RGC RAG A,, GTG CAG AGC GCA GC, CCA GAA Thr Cyr SW Gin Ile His Le" Lys Ala Thr Lyr Ala Gly Gly Glu Leu Gl" Rrp Arg Lys Ser Lyr Ile "al G,n APT Ala !+,a Pro Glu -80 k 330 36” 300 + GTT CAR GAG GA1 GTC RAG ACT TTC MG RCA GAC CTG CTG AK ACC ATG GA1 TCA ACC AK CGA AGC CA, TCA TTT CC, AGT TTC CAG GAG "a, Gin Glu Asp "a, Lys Thr Phe Lys Thr Asp Le" Le" Ser Thr ME, ASP Ser Thr Ti,rArg Ser HIS Ser Pne Pro Ser PIW Gin Glu 100 120

CC.4 GAG CAG ACT GAA GGG GCA Pro Gl” Gin Thr Glu Gly Ala

4Z” 390 45” GTT CCC CAC CTG ATT CRG MC RAT ATG ACT GGA AGC CAG GCT TTC GGT TAT GAC CAR 111 TTC AGR GAC “a, Pro Hi5 Le” Lie G,” As" Asn MET Thr Giy Ser ;;; A,a Phe Gly Tyr Rrp 6," Phe Pne Arg Asp

510 480 540 AAG ATC ATG GAG AAG AAA CAG GAC CAC KC TAC CGT GTG TTC RAG AC, GTG RAT CGT TGG GC, AA, GCC TAC CCC T,, G&C CIA CAC TTC Lys Ile "ET Glu Lys Lys Gin Asp His 16"; Tyr APT "al Phe iyr Thr "al AS" Arg Trp Ala Am Ala Tyr Pro Phe Ala Gin HII Phe 180

570 600 630 KC GAG GCA TCT ATG GCA TCA LAG GA1 GTT TCT GTT TGG TGT AG, AA, GAC TAT TTG GGC ATA AGC AGA CRC CC, CG, GTC TTG CAG GCC Ser Gl" Ala SW MET Ala Ser Lys Asp "11 Ser "a, TV Cys Ser Am ASP Tyr Le" Gly Ile Ser Arg "IS PPO kg "al Leu Gin AIa 200

690 660 720 ATA GAG GAG AtC CTG AAG MT CAT GGA GCT CGA GCT GGG GGC ACT CGC AA, ATC TCA C&T ACC AGC AAG 111 CAT GTG GAG CT, &.,A CAG Ile GI" Glu Thr Leu iys AS" "IS Gly Ala Gly Ala Gly Gly Thr Arg AS" ,,e Ser Gly Thr Ser Lys Phe ",I "a, Glu Leu Giu Gin 220 210 IBO 810 TG, TTT GTG GCC AA, GA, TC, ACT CTC TTT RCA CTG GCC MG Cys Phe "a, ;A; Am ASP SW Thr Leu Pne Thr Leu Ala Lys

150 GAG CTG GC, GAA CTA CRC CAG MA GAC TCA GCT CTG CTC TTC TLC KC Glu Le" Ala Glu Leu "IS Gin Lyr Asp SW Ala Leu ie" Phe Ser SW

CT, CTG CCA GGG TGT GAG AK Le” Leu Pro Giy Cys Gl” Ile

810 840 TAC TCA GA.1 GCR GGC AAT CAT GCC KC ATG ATC CAR GGC ATT Tyr Ser Asp Ala Gly As" HIS Ala Ser MET Ile Gin Gly Ile 280

1020 TCC ATG GA1 GGT GCC ATC TGT CC, CTG GAG GAA TTG TGT GA, Ser MET Asp Gly Ala Ile Cys Pro Le” Glu Glu Le” Cys Asp 340 ,110 GTA GGA CTG TAT GGA GCt CGG GGT GCA GGT AK “11 Giy Le” Tyr Gly Ala Arg Gly Ala Gly Ile

90” CGC MC AGT GGT GCA GC‘ AAG 111 GTC Arg Am Se? Gly Ala Ala Lys PM “a, 3”O

,050 lo*0 GTG GCC CRC CAG TAT GGA GCC CTG AK TTC GTR GA, GAA GTC CAT GCT “11 A,. "11 Gin Tyr Gly 416 Leu Thr Phe "al ASP Glu "al "IS Ala 36"

114” LllO GGG GAG CGT GA, GGA ATT ATG CRC RAG CT, GAC ATC ATC TCT GGA AC, CT, GGC AAG Gly Glu Arg ASP Gly Ile "ET "IS 2; ieu ASP Ile Lie SW Gly Thr Leu Giy Lyr

120" GCC 11, GGT TGC GTC GGT GGC TAT ATA GCC RGC ACT CGG GAC TTG GTG GAC ATG KC TAC GCT GCA GGC TTC ATC TTT ACC'::? AI3 Phe Gly Cyr "al GIy Gly Tyr ile Ala Ser Tiv Arg ASP Leu "al Asp MET "al Arg Ser Tyr Ala 416 Gly Phe Ile Phe Ti,r Tnr 400 42"

GTG’:::

,290 1320 TCA CTG CC, CCC ATG AK CTC TCT GGG GCT CTA GAA TCT GTG CGC CTA CTC RAG GGA GAG GAG GGT CAA GCC CTG AGG CGG GCA CAC?:: Ser Le" PTO Pm NE, MET Le" Se? Gly Fld Leu Glu Ser "al Arg Le" Le" Lys Gly Glu Gl" Gly Gin A11 Le" Arg Arc,Ala HIS Gin 440 ,380 CGC MT GTC AAA CAC ATG CGC CAG CTG CTA ATG GAC AGG GGC TTT CC, GTT AK CCC% CCC AGC CAC ATC RTC CCC ATC AGG GTGl:L: Arg Asn "a1 Lyr HIS ME, Arg Gl" Leu Leu HE, ASP Arg Giy Phe Pro "al Ile Pro CYP PPO Ser "IS Ile Ile Pro Ile Arg "a, Gly 460 Ok!"

AAT GCA GCA CTC AK AGC RAG AK As" Ala Ala Le” AS” Ser Lys Ile

1470 ,500 TGT GA1 CT1 CTG CTC KC AAG CRC AGC ATC TAT GTG CAG GCC AK Cys Asp Le” Leu Le” SW Lys “1s Ser Lie Tyr “a, Gin Ala Ile 50"

AN TAC CCA At, Asn Tyr Pro Thr

GTG

CC,‘;::

“a,

Pro

Arg

156” GGT GAG GAG CTA CTG CGC TTG GCC CCC TCC CCC CAC CAC RGC CCT CAG ATG ATG GAA% TTT GTG GAG AAG CR, CTG CT,? GCC TGG?:; Gly G1u Glu ieu le" Arg Le" Aia Pro Ser Pro "IS HIS Ser Pm Gin ME, MET Glu Ann Phe "al GI" Lys Leu ku leu RIa Trp Thr 520 54" ,610 GAG G,G GGG CTG CCC CTC CAA GA1 GTCl TCT GTG GCT GCA TGC MC TTC TGT CRT ,Gl'% GTG CRC 111 GRA CT, ATb AGC GAG TGG';:: Glu "a1 Gly Leu Pro Le" Gin ASP "al Ser "al 416 Ala Cys As" Phe Cys "II Arg Pro "al "1s he Glu Leu "ET Ser Gl" ,PP Gl" 56" L14" CGA KC TAC T,, GGG AAC ATG GGA CCC CAR TAT GTT ACC ACC TAT GC, z Aq Ser Tyr Phe Gly Am HE, Gly PPO Gin Tyr "al Thr Thr Tyr AIa 580

177" GGRGCCAGC,GCC,,GGATG~~AG~,~~A~~,G~A~~~~~~~,GGGG~,GGG

,830 1%" C,TCCTCC,GCTC,CTGCTTTCC,GTGTGCT,CTGGCTGACTTGR,,C,GAA~~~AG~AA~,,GAAA~A~~~~,AAAAA

Fig. 2. Nucleotide

sequence

and predicted

to nt; below to aa. The polyadenylation the start of the shorter by a small upward

cDNA

sequence

amino acid sequence

of mouse ALA synthase

signal at nt 1866 is indicated obtained

by a shaded

from the liver library. The hypothetical

arrow at Gln 77. The basic residues

which align with arginine

cDNA.

Numbers

box. The bold downward

residues

cleavage

above the sequence arrow

site of the pre-sequence

in the human

refer

at nt 309 identifies

OTC precursor

is designated are underlined.

60

Mouse

Chicken Fig. 3. Homology chicken

Nbetween

and the hypothetical

mouse

and chicken

cleavage

liver ALA synthase.

Arrows

show the position

site in the mouse. The N and C termini are indicated.

of the leader

Intensity

peptide

of shading

cleavage

represents

site in

the degree

of homology.

was used to generate a series of overlapping

deletions

of the inserts in each orientation for complete sequencing ofboth strands. In addition, the sequence was confirmed across the PstI site after subcloning a HindIII-XbaI fragment containing this region into M13mp19. Translation of the nt sequence (1896 nt) revealed one open reading frame beginning at the first nt. The first stop codon appears at nt 1759 and is followed by approx. 130 nt of untranslatable sequence that contains an AATAAA polyadenylation signal at nt 1866 (Fig. 2). This cDNA can code for a polypeptide which has a maximum size of 586 aa and 64.6 kDa.

(d) Homology to chick ALA synthase A comparison of the predicted aa sequence of mouse erythroid ALA synthase with that of chick liver ALA synthase (Borthwick et al., 1985) revealed extensive homology between the mouse and the chicken enzymes. The homology is approx. 80% over 300 residues between aa 265 and 570 in the chick and 216 to 516 in the mouse. This homology decreases when proceeding outward from this region towards the N- and C-termini, and is least at the extreme N-terminal region (Fig. 3).

pre-sequence

of human

OTC

is devoid

Lys 68, Lys 71, and Gln 77 can be aligned precisely with Arg -27, Arg -10, Arg -7, and Gln -1 of OTC (Fig. 2). It should also be noted that the mouse liver cDNA codes for a functional enzyme in E. coli, but is 103 aa shorter at the N-terminus than the erythroid sequence (Fig. 2). Clearly, the first 100 aa are not essential for an active enzyme as has been indicated by the results of Srivastava et al. (1982) and Maguire et al. (1986). On the basis of these assumptions, the mature form of mouse ALA synthase may be as small as 56 kDa.

(f) The level of ALA synthase mRNA The steady-state levels of ALA synthase mRNA present in mouse erythroid and hepatic tissues were estimated by Northern blot analysis using 32P-

B

A 1 28s

2

1

3 1 t

-

2

3

28s I

(e) Presence of a leader sequence From a comparison of leader sequences of proteins translocated into the mitochondrial matrix it has been possible to state only that the presequence is predominantly basic. A functional role for specific basic residues in the leader sequence of human OTC has been demonstrated for targeting this enzyme to the mitochondrial matrix (Horwich et al., 1985a,b). Examination of the N-terminal region of the mouse ALA synthase sequence revealed only two acidic but seven basic residues in the first 77 aa while the

of acidic

residues but contains four arginine residues (Horwith et al., 1984). Remarkably, if the cleavage site of the mouse ALA synthase precursor is taken as Gln 77, then Arg 51,

18s

-

Fig. 4. Northern

blot analysis of ALA synthase

mRNA. (A) 5 pg

poly(A) + RNA from: mouse liver (lane I), liver of AIA-treated mouse (lane 2), and mouse anemic spleen (lane 3). (B) 20 ng total RNA from mouse anemic spleen (lane I), uninduced (lane 2), MEL (lane 3). Markers

cells

induced

used were mouse

ments from the ALA synthase probe. Exposure

for 3 days cDNA

rRNA.

MEL cells

1.8%

Labeled

in pMS20

times were: 36 h for lanes

(A); 16 h for lanes 1, 2 and 3 (B).

with

DMSO

PsrI frag-

were used as

1and 2,3 h for lane 3

61

labeled probes derived from the ALA synthase erythroid cDNA (Fig. 4). In mouse liver, anemic spleen and MEL cells, a hybridizable band of RNA was observed with an apparent size of approx. 2.3 kb. The level of ALA synthase mRNA was considerably lower in the liver than in erythroid cells, so that poly(A)’ RNA was prepared for the comparison of normal and AIA-treated livers. A considerable increase in the steady-state level of ALA synthase mRNA was observed in the treated livers, similar to the increases observed in chick embryo liver (Borthwick et al., 1984). A large increase in the steady-state level of ALA synthase mRNA was also observed upon induction of MEL cells, though this increase appears to be greater than the increase in enzyme activity previously observed during induction (Sassa et al., 1975; Ebert and Ikawa, 1974; Sassa, 1976).

DISCUSSION

A mouse liver ALA synthase cDNA has been cloned by its ability to complement an E. coli mutant deficient in this enzyme. The ability of mammalian sequences to complement E. coli mutants provides a powerful approach to the cloning of enzymes of metabolic pathways common to E. coli and animal cells. Of particular interest would be those enzymes whose deficiencies are responsible for inborn errors of metabolism. The identity between nt sequences of the mouse liver and erythroid ALA synthase cDNA clones, and the detection of a single RNA species in these tissues, raises the possibility that a single ALA synthase gene is expressed in mouse liver and erythroid cells. These results contrast with data from chicken where liver and erythroid-specific ALA synthase isozymes exist (Watanabe et al., 1983). In the chicken liver, the ALA synthase precursor is 73 kDa and is processed to a 65kDa mitochondrial form (Ades and Harpe, 1981), while in chicken erythroid cells, the enzyme appears to be synthesized as a 55-kDa precursor that is found as a 53-kDa protein in mitochondria (Watanabe et al., 1983). In addition, a cloned chicken erythroid cDNA identified a 2.8-kb mRNA species in liver and a 2.0-kb mRNA in erythroid cells which were distinguishable by hybrid-

ization efficiency (Yamamoto et al., 1985). Similar to the chicken, the rat liver enzyme is synthesized as a 75-kDa precursor which is processed to a 66- or 70-kDa protein (Yamamoto et al., 1982; Srivastava et al., 1982). Earlier studies have shown that the tissue-specific regulation of mouse ALA synthase is similar to that observed in the chick, where enzyme activity in the liver is repressed by heme and stimulated by porphyrinogenic agents, while erythroid ALA synthase activity is relatively insensitive to these compounds (Kappas et al., 1983; Wada et al., 1967). Although the simplest interpretation of our data is that one ALA synthase gene codes for the identical enzyme expressed in mouse liver and erythroid cells, alternative explanations may be put forth that would better reconcile our results with the presence of distinct, differently regulated ALA synthases in the chick. Tissue-specific regulation of transcription of a single gene may be accomplished by the use of a separate promoter in each cell type (Shaw et al., 1985). Processing of the transcript could in this case result in the expression of identical mRNAs in the two tissues. Alternatively the approx. 2.3-kb RNAs identified in the liver and erythroid cells may arise from two different but highly homologous genes which may differ only in sequences present at the 5’ end of the RNA or in untranscribed or untranslated regions. The mouse anemic spleen ALA synthase mRNA appears to be about 350 nt longer at the 5’ end than the pMS20 cDNA clone, as judged by primer extension analysis using mouse anemic spleen poly(A)+ RNA as a template (unpublished observation). However, we have presented evidence in this paper that the cDNA may contain the essential sequence for the mature, mitochrondrial enzyme and at least part of an N-terminal presequence. This is based on the similarity of the N-terminal region to the leader peptides of other mitochondrial proteins in overall basic character. Additional evidence that the N-terminal sequences may not be part of the mature enzyme is that this region does not appear to play a critical role in catalysis, as suggested by the observations that the liver cDNA clone (pMLS-1) lacking this region is functional in E. coli and these sequences are not significantly conserved between the chicken liver and mouse enzymes. In the light of our results it will be particularly

62

interesting to determine how the expression of the mouse ALA synthase gene is regulated to produce in all cells a low constitutive level of the enzyme, while an increased level of the enzyme occurs in the liver in response to heme depletion and in erythroid cells during erythropoiesis.

Henikoff,

S.: Unidirectional

creates

targeted

digestion

breakpoints

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