High levels of aldehyde dehydrogenase transcripts in the undifferentiated chickretina

Exp. Eye Res. (1992) 54, 297-305

High Levels of Aldehyde Dehydrogenase Transcripts in the Undifferentiated Chick Retina ROSELINE GODBOUT

Molecular Genetics and Carcinogenesis Laboratory, Cross Cancer Institute and Department of Biochemistry, University of Alberta, Edmonton, Alberta T6G 1Z2, Canada (Received Houston 22 January 1991 and accepted in revised form 6 May 1991) A cDNA clone corresponding to chicken aldehyde dehydrogenase (ALDH) mRNA was isolated from a library representing the polyadenylated RNAs expressed in the retina of day 3"5 chick embryos. The profile of ALDH RNA expression was examined in different tissues as well as at different stages of development in the chick embryo. A notable feature of this analysis was the high level of ALDH transcripts found in the undifferentiated cells of the retina. A 20-fold decrease in ALDH RNA levels was observed upon retinal differentiation, in contrast to the kidney, liver and gut where tissue maturation was accompanied by an increase in ALDH mRNA levels. The observations reported here suggest an important role for the ALDH enzyme in retinal development. One possibility is that retinal, the aldehyde form of vitamin A, serves as a substrate for ALDH in the developing retina, resulting in the formation of retinoic acid which has been implicated in various differentiation processes. Key words: aldehyde dehydrogenase; retinal development; gene expression ; cDNA sequence ; carbonic anhydrase II: retinoic acid. 1. Introduction Aldehyde dehydrogenases form a complex group of enzymes which can oxidize a wide range of biological aldehydes to the corresponding acids (Pietruszko, 1983). ALDH activity has been reported in most m a m m a l i a n tissues, including ocular tissues, such as lens, cornea and retina (Jedziniak and Rokita, 1983; Tipton, 1985; Holmes and VandeBerg, 1986). Although the precise function of ALDHs is not known, they have been postulated to be involved in the oxidation of acetaldehyde derived from ethanol and in the conversion of biogenic aldehydes, such as those derived from dopamine, to yield the corresponding acids (Turner, Illingworth and Tipton, 1974; Weiner, 1979). Proposed roles for ALDHs in ocular tissues include involvement in the lipid peroxidation process, ethanol metabolism, carbohydrate metabolism and the visual cycle of the retina (Holmes and VandeBerg, 1986). Based on primary amino acid sequence, three classes of ALDHs have been defined in m a m m a l s : cytosolic, mitochondrial and a third class which includes tumour ALDHs (Hempel and Lindahl, 1989). The degree of homology within each ALDH class in m a m m a l s is very high. For example, h u m a n cystolic ALDH shares over 90% homology with horse cytosolic ALDH (Hempel, yon Bahr-Lindstr6m and J6rnvall, 1984; von BahrLindstr6m, Hempel and ]6rnvall, 1984) while only 70% homology is observed between the h u m a n cytosolic and mitochondrial enzymes (Hempel, Kaiser and J6rnvall, 1985). Rat class 3 ALDH primary amino acid structure differs substantially from that of class 1 and class 2 ALDHs (29'8 and 28% identity with class 1 and class 2 ALDHs, respectively, with the 0014-4835/92/020297+09 $03.00/0

introduction of gaps) (Hempel, Harper and Lindahl, 1989). Non-mammalian ALDH cDNAs have recently been cloned from Aspergillus nidulans and Pseudomonas oleovorans (Pickett et al., 1987; Kok et al., 1989). When amino acid alignment is improved by introducing several gaps, sequence homology to m a m malian ALDHs as high as 4 9 % (A. nidulans) and 37% (P. oleovorans) is observed. Dunn et al. (1989) have analysed the tissue distribution of the 2.3 kilobase rat ALDH-phenobarbitol (PB)mRNA. ALDH-PB is believed to be the rat homologue of the h u m a n cytosolic ALDH-1 because of the remarkable similarity between the two sequences. High levels of ALDH-PB RNA were found in the kidney and lung, intermediate levels in the liver and low levels in the colon, brain, heart and small intestine. In the course of defining genes involved in retinal differentiation, a chicken ALDH cDNA was isolated. The predicted amino acid sequence deduced from the cDNA sequence demonstrated extensive homology to the h u m a n cytosolic ALDHs. While high levels of ALDH mRNA were present in the neuro-ectodermal cells of the undifferentiated neural retina, a considerable decrease in RNA levels was observed by day 19 of incubation when the retina consists of differentiated neuronal, glial and photoreceptor cells.

2. Materials and Methods

Preparation of eDNA Library Neural retinas at day 3 to 3-5 of incubation, representing stages 2 0 - 2 1 as defined by Hamilton (1952), were dissected using a Zeiss StemiSV8 microscope and immediately frozen in liquid nitrogen. © 1992 Academic Press Limited

298

RNA was isolated by phenol extraction and lithium chloride precipitation (Maniatis, Fritsch and Sambrook, 1989) and enriched for polyadenylated [poly(A) +] sequences by oligodeoxythymidylate [oligo(dT)]-cellulose chromatography (Aviv and Leder, 1972). Complementary DNA (cDNA) produced from an estimated 1 #g of poly(A) + RNA obtained from the retinas of approximately 100 embryos was prepared using AMV reverse transcriptase (Life Sciences, St Petersburg, FL, U.S.A.) for synthesis of the first strand and RNAse H and Klenow polymerase I for the second strand. The double-stranded cDNA was ligated to a 50-fold molar excess of linkers. After digestion with EcoR I, the cDNA was size-selected (> 400 base pairs) on a polyacrylamide gel and figated to EcoR I-digested 2tZAPII (Stratagene, La Jolla, CA, U.S.A.) to generate a cDNA library of 5 x 105 independent phages.

Screening of eDNA Library Sequences preferentially expressed in the retina were selected using a differential (plus/minus) screening strategy designed to identify transcripts expressed at moderate to high levels. Replica filters were hybridized with a2P-labelled single-stranded eDNA derived from either reverse transcribed day 3'5 retina poly(A) + RNA (probe A) or day 3-5 heart poly(A) + RNA (probe B). Plaques hybridizing to probe A but not to probe B underwent secondary and tertiary rounds of screening. The filters (HATF nitrocellulose from Millipore, Mississauga, Ontario, Canada) were hybridized in 50 % formamide containing dextran sulphate (Wahl, Stern and Stark, 1979) for 24-36 hr. Phagemids containing cDNAs of interest were excised from AZAPII following the suppliers' directions (Stratagene).

RNA Analysis Poly (A) + RNA was extracted from the indicated tissues and stages of development (days of incubation in parenthesis): retina (3'5, 5, 10, 19), brain (3"5, 5, 10, 19), liver (5, 6/7, 16), gut (10, 16), stomach (16), kidney (7, 16), lung (16), lens (3.5*, 16), retinal pigmented epithelium (3.5*, 16), human fetal retina (week 10/11 of gestation). [The asterisk indicates that the tissue isolated at this stage of development was contaminated with surrounding tissue (see Results and Fig. 2 legend).] Poly (A)+RNAs were electrophoresed in a 6% formaldehyde-l.5% agarose gel in MOPS buffer (20 mM morpholinepropanesulphonic acid, 5 mM sodium acetate, 1 mM EDTA, pH 7"0) after denaturation of the RNA at 65°C for 15 min in 70 TOformamide-6 % formaldehyde-MOPS buffer. The RNA was transferred to nitrocellulose filters (Amersham, Arlington Heights, IL, U.S.A.) in 3 M sodium chloride-0'3 M sodium citrate (Thomas, 1980) and the filters hybridized as described (Wahl et al., 1979). The DNA probes, a2p_ labelled by nick translation, included the following

R. G O D B O U T

gel-purified fragments: the 1.7 kb EcoR I insert from clone 293, the 1.4kb carbonic anhydrase-IIcDNA insert from clone 374 (Godbout et al., 1990), the mouse 0c-actin eDNA (Minty et al., 1981), and the human ALDH-1 cDNA (Hsu et al., 1985). To compare the intensity of the bands obtained after autoradiography in day 3'5 versus day 19 retina, the bands were quantitated by densitometric scanning of autoradiograms at different exposures to ensure that the intensity of the signal obtained was directly proportional to the number of 82p-labelled molecules hybridized to each RNA band.

Southern Blot Analysis Genomic DNA was isolated (Maniatis et al., 1989) from day 3.5 chick embryo bodies. Ten micrograms of DNA were digested with EcoR I and Hind III, respectively, and electrophoresed in a 1 To agarose gel in 40 mM Tris-acetate and 1 mM EDTA (pH 7"2). The marker utilized was Hind lII digested AC1857 DNA. After denaturation (0.5 M NaOH, 1.5 M NaC1) and neutralization (0.5 M Tris-HC1, pH 7"0 and 3 M NaC1), the DNA was transferred to nitrocellulose as described above. The conditions for prehybridization and hybridization were as described previously (Wahl et al., 1979).

DNA Sequencing Sequencing was by the dideoxynucleotide chain termination method (Sanger, Nicklen and Coulson, 1977) using Klenow polymerase I (Boehringer Mannhelm, Dorval, Quebec, Canada) as modified for doublestranded DNA templates (Mierendorf and Pfeffer, 1987). A sequential deletion strategy (Henikoff, 1987) was utilized in order to obtain overlapping sequences. Briefly, clone 293 was digested with Kpn I and Sal I in order to generate a 3' overhang and a 5' overhang, respectively. Aliquots of DNA were then digested with exonuclease III for increasingly longer periods of time at 30°C, treated with mung bean nuclease and bluntend ligated using T4 DNA ligase. For nucleotide sequencing of the opposite strand, the eDNA insert was excised with EcoR I and ligated in EcoR I-digested pBluescript. Inserts ligated in the opposite direction were identified using diagnostic restriction enzyme sites and generation of overlapping sequences was as described above. The Fasta DNA database (Pearson and Lipman, 1988) was used to search for sequence homology and the IBI/Pustell Sequence Analysis Programs to determine the correct reading frame. 3. Results

Isolation and Characterization of Clone 293 A eDNA library in AZAPII was constructed representing the poly(A) + RNAs expressed in the chick retina at day 3"5 of incubation. At this stage of

ALDEHYDE

DEHYDROGENASE Lanes:

I

2

299

m R N A IN THE D E V E L O P I N G R E T I N A 3

4

5

6

7

8

9

I0

II

12

13

FIG. 1. Northern blot analysis of clone 293 mRNA expression in tissues of the chick embryo. Two micrograms of poly(A)+ RNA derived from the indicated tissues were added to lanes 1-13 as follows: (1) day 5 retina: (2) day 19 retina; (3) day 5 liver; (4) day 6/7 liver; (5) day 16 liver; (6) day 10 gut; (7) day 16 gut; (8) day 16 stomach (including gizzard); (9) day 7 kidney; (1 O) day 16 kidney; (11) day 16 lung; (12) day 16 lens; and (13) day 16 pigmented epithelium. The filter was hybridized with clone 293 (A), and mouse a-actin (B). The extra bands obtained in (B) represent tissue-specific actin mRNAs (Bergsma et al., 1985). Densitometric scanning of lanes 1 and 2 was using different autoradiograms at different exposures to ensure linearity of signal intensity. The asterisks in (A) indicate the position of the 28s and 18s rRNAs. development, the retina consists of undifferentiated neuro-ectodermal cells (Moscona, 1983). Replicate plaque lifts were hybridized with 32p-labelled singlestranded cDNA probes derived from: (1) d a y 3 . 5 retina; and (2) d a y 3 ' 5 heart. From an initial 5 0 0 0 0 p h a g e s , 80cDNA clones were selected for further analysis after undergoing low-density secondary and tertiary rounds of screening. The tissueand stage-specific RNA expression profile of one of these cDNAs (clone 293) was studied in detail. Very high transcript levels were observed upon hybridizing the 1.7 kb cDNA insert of clone 293 to mRNA derived from day 5 retina. At day 19, however, RNA levels were 20-fold lower. In contrast, the concentration of clone 293 mRNA in the liver was very low at early stages of development. This was followed by a substantial increase in RNA levels by day 16 of incubation. Two additional tissues, the kidney and gut, also demonstrated elevated levels of RNA expression at later developmental stages. The stomach, lung, lens and pigmented epithelium at day 16 of incubation had very low levels of clone 293 mRNA. Interestingly, the apparent size of the transcript in the retina was different from that of other non-ocular tissues expressing clone 293. This difference in electrophoretic mobility was observed in all experiments and is clearly demonstrated in Fig. 1 (A) (compare lanes 2

and 3). The size of the retinal transcript was estimated to be approximately 2.5 kb by comparison to RNA molecular weight markers (Boehringer Mannheim) and actin mRNA (data not shown). Probing with actin DNA ensured that a similar amount of RNA was loaded in each lane. It should be noted that day 16 liver mRNA consistently generated a signal of lower intensity (Bergsma, Chang and Schwartz, 1985) when hybridized to actin DNA. A more detailed analysis of the developing retina indicated that clone 293 RNA levels are virtually identical in the day 3-5 and day 5 retina (Fig. 2, lanes 3 and 4), but reduced in the day 10 retina (lane 5), with a further decrease at day 19, 2 days prior to hatching (lane 6). At day 3.5, pigmentation of the eye is faint but distinct, allowing the dissection of the retina from the pigmented epithelium immediately surrounding it. Although considerable care was taken to include only retinal tissue in the dissections, the possibility remained that very high expression in a small amount of contaminating pigmented epithelium could account for an 'apparent' high level of transcripts in the retina. To exclude this possibility, poly(A) + RNA was extracted from the tissues external to the retina including the pigmented epithelium, choroid and sclera. As seen in Fig. 2(A) (lane 2), the level of clone 293 mRNA expression is much lower in

300 .Qrle8 ;

R. G O D B O U T t

2

.3

4

5

6

7

8

9

I0

is expressed at very high levels in the n e u r o ectodermal cells of the chick retina and becomes restricted to Mfiller glial cells upon differentiation of the precursor stem cells (Moscona, 1983; Moscona and Linser, 1983). Upon reprobing the filter with CA-II cDNA, a similar pattern of RNA expression was observed in the day 3"5 to day 10 retina for both CA-II and c l o n e 2 9 3 [Fig. 2(B)]. In contrast to CA-II RNA, however, clone 293 transcript levels decreased substantially from day 10 to day 19. Although expressed at low levels, CA-II RNA could be detected in the brain from day 3.5 to day 19.

Clone 293 Represents Chicken Aldehyde Dehydrogenase mRNA

FIG. 2. Northern blot analysis of clone 293 and carbonic anhydrase-II expression in the developing chick retina and brain. Poly (A)+ RNAs extracted from the following tissues are included in lanes 1-10 as follows: (1) day 3-5 lens and cornea, including the anterior portion of the retina; (2) day 3"5 pigmented epithelium, choroid and sclera, including some contaminating retinal material (which cannot easily be removed from the pigmented epithelium)', (3-6) retina (day 3'5, 5, 10 and 19 of incubation, respectively); and (7-10) brain (day 3"5, 5, 10 and 19, respectively). Two micrograms of poly(A) + RNA were loaded per lane for all tissues except those obtained at day 3"5 when only 1 #g of poly(A) + RNA was loaded per lane. The filter was hybridized with clone 293 (A), carbonic anhydrase-II cDNA (B) and mouse ct-actin (C). this tissue preparation and probably results from a small amount of retinal contamination. Similarly, dissection of day 3"5 lens and cornea, including the anterior portion of the retina, indicated that clone 2 9 3 m R N A levels in the lens and cornea are substantially lower than that found in the retina (lane 1). These results suggest that the major site of clone 293 transcription in the immature eye is the retina. The retina is derived from the same primitive neuro-ectoderm that forms the brain. One might, therefore, expect that some of the genes expressed at the early stages of retinal development would also be expressed in the immature brain. However, clone 293 mRNA was not detected in the brain from day 3.5 to day 19 [Fig. 2(A), lanes 7-10]. A second clone isolated from the day 3.5 cDNA library contained a l'4-kb EcoR I insert corresponding to CA-II mRNA (Godbout et al., 1990). The CA-II gene

Sequencing of the 1'7 kb cDNA clone and a search for nucleotide homology using the GenBank DNA database revealed extensive homology with a number of m a m m a l i a n ALDHs as well as a more limited homology to A. nidulans and P. oleovorans ALDHs (Pickett et al., 1987; Kok et al., 1989) [Fig. 3(A)]. In an attempt to obtain a more complete cDNA clone and to verify the nucleotide sequence at the 5' and 3' ends of clone 293, as well as to eliminate the possibility of cloning artefacts, a second clone was obtained from a day 7 chick retina cDNA library using clone 293 as a probe. This cDNA (clone 640; 1876 bp) contained an additional 150 bp and 20 bp at the 5' and 3' ends of the cDNA, respectively [Fig. 3(B)]. An in-frame methionine codon was located 286 bp downstream from the 5' end of the cDNA. The chicken ALDH cDNA was found to encode a predicted protein of 509 amino acids, in contrast to the m a m m a l i a n cytosolic ALDH genes which encode a 501 amino acid protein (including the start codon, methionine) (Hempel, von Bahr-Lindstr6m and J6rnvall, 1984; Dunn et al., 1989) and the h u m a n mitochondrial ALDH gene which encodes a 517 amino acid protein (including a 17 amino acid leader peptide) (Hsu, Bendel and Yoshida, 1988). The highest level of homology was with the h u m a n cytosolic ALDH-1 (83%) and rat ALDH-PB (78 %). A somewhat lower level of homology was observed with the h u m a n mitochondrial protein (68 %, not including the leader peptide). There was no homology between the 17 amino acid leader peptide of the mitochondrial protein and the first nine amino acids encoded by the chicken ALDH mRNA.

Genomic Structure of the Chicken ALDH Gene The difference in molecular weight observed between liver and retina ALDH RNA could be due to alternative splicing, alternative transcription initiation or termination sites, or the presence of different genes encoding distinct mRNA species. To test the latter possibility, a Southern blot was prepared using chicken genomic DNA digested with EcoR I and Hind III, respectively. Figure 4 shows the fragments obtained

ALDEHYDE

DEHYDROGENASE

mRNA

IN

THE

DEVELOPING

RETINA

301

(~) TI'C(M"~TGATAAACACAGAACTGAAGTAAGAATCTAAGTA~'CATI']'G-Fi'C,CTGTTACTGAATCGTGCATATATAGAGGTCATGA C I ' ~ AAA~CT~ACr

AA~A~CAA~T~AAAATCTCr~

AC~'GCCTCCTG

AC AC AA AC3"~AC

A -

P

L

P

$

A

A

T

Q

M

s

s

-

A

TO

M

S

S

S

G

~'M L

K

x

R

.

A

+

L .

.

.

.

.

.

L

R

.

.

.

.

.

.

.

G

R

.

.

.

.

.

.

W

.

. .

. .

.

AAA

CTC

~

TCI

ACT

K

L

F

$

P

Y

V

t

-

T

H

¥

.

.

.

M

.

. .

.

.

.

.

S

.

.

. .

. .

CTA

ATG G A T

T

A

Y

L

M

I

S

D

y

EGAG

cCT

VCT"r

"

Q II

L

Q

.

. R

H

.

.

.

.

.

.

C

.

.

.

L

.

.

.

L

.

.

.

.

.

.

N

K

. Y

. .

.

c

.

.

.

.

. .

. .

. .

T

T

Y

v

L k

.

A . .

GTT

CCA

ATG GAT

GGA

AAC pTrr

V I

P

lM

G

N D

^

~

K

_

p

:

:

:

:

:

c,

Q

.

s .

CGT

GGGA

~AA

.

.

~TT

.

M

G

S

v

X

N

v

A A

. .

.

.

A

.

.

T .

.

.

. H

.

:

:

.

L

Y

S

E

S 5

_

.

.

P .

.

.

.

N .

[

. .

. .

. .

S

.

.

. .

.

I

.

D S

.

F

A

M

s

v

-

-

c

v

L

.

. .

GAT ° .

GAG T I T [~ F

. .

.

S

.

.

T

~TG

V~

L

I

.

g

.

. .

.

. .

-

-

GCA

GG&

E

.

.

A

.

. .

.

.

.

CCA

. .

.

L

I*

.

.

.

F

.

.

TTT

G

.

.

TA T GM

IL

.

(3 .

.

A

. .

D

L

.

.

.

.

.

.

.

CCC R

.

CGG R

.

.

T

.

. .

.

D

.

E

.

.

AIT I

G A A CGA E R

E

V V

A .

.

V

.

.

. .

E

-

V

.

.

.

.

V

.

.

.

.

~TC

Q

A

.L .

. .

CCT

GG&

AAG

G'TG

. . .

V .

~IT

cTCT

TCGT

-

A

T

^-

-

-

ss

.. . . .

ATT

GTG

CCA

GCC

TT[

CGA

V

P

.

.

I .

.

.

.

F

.

.

K O .

E V

G

L

F

P

A

qH

-

.

.

A

l .

.

.

.

H

I

.

A -

G

. . . . . . .

D

M

G

-

E E

N

Y

T

A. . . . . . . . . . . .

R T

A

.

K

.

.

E

.

.

Q

ATA

E

.

.

.

F~ .

GGA

G

. . .

. . . . . . . . . . .

.

f

H

-

F

N

v

F

.

.

.

~CT

V~

Q

O -

M . .

.

T~Fr F

.

.

.

.

.

.

.

.

.

.

ACC AAA GAC T K O

ATI" C A T I D

A A A CCA K A

CTG L

ACA ~T T F

.

.

.

.

R

N $

Y

.

.

L L .

.

E .

.

G . .

.

.

N s .

.

.

C . . .

R .

.

. .

.

.

.

I

G . M .

.

.

E .

Y .

L V [

C

GCA A

TCT S

GCT A

s S s

Q

.

Q

E A . .

. Q

.

.

.

.

C

.

G A T CCT D P

CTG L

"IrA L

CCT P

CG'T G T A G V

CAG C A A GGC Q Q G

-

P -

D O

S S

K

-

N N

E S

. .

l

-

-

N

-

I"

.

FT~

IA. . .Q . . . P. . . T

. .

.

.

.

I R .

. .

.

.

.

.

A . .

K

~

.

.

.

X

.

.

I

S

.

S

L

A O . . . . . .

D

.

.

E

K

G

R

.

V~

. D

.

.

.

.

.

-

CCT P

.

-" .

I

K

.

.

.

X

G

. .

A

.

.

. .

. .

.

N

. .

.

.

.

.

N

.

.

.

.

.

S

.

.

T . P

.

.

.

.

.

GAC A A G O K

G

-

.

C

-

Q

.

. .

S. . . . . N

. .

Y . . -

O .

.

K A

Y

O

T. . . . D. . . . Q

.

.

. .

A

. .

A

.

A

V

C

-

.

-

~

GGA GGA C G

FTTT

c

.

V

Y

F

II

. .

. .

. . .

. .

Y . .

.

C T T CAG L Q .

.

T . . .

.

CCT A

. . .

E .

GGG ACA GTG TGG G "[ V W

.

.

. .

. .

.

.

.

.

. .

K .

. L

.

.

.

.

.

.

.

.

T -

GTr V

.

TAT Y

. .

.

T -

AAT TGT N C

.

. .

.

-

.

.

V M V

A

.

K -

v .

P .

A G T , GCA S A

T~T F

TCA 5

GCT A

O L c

.

c -

-

v T

Q .

$

5

L v

v

K .

. . .

. .

N . . .

S

V

C A G TGT Q C

CCT P

s .

.

. .

. .

F~TT . .

.

AAG ATG TCA K M 5

S

.

.

.

.

--

. . .

. . I~l

Bg

200

14

4-00

B

600

800

B Hp B

1000

1200

E

1400

1600

1800

295

640

FIG. 3. A , Nucleotide sequence and derived amino acid sequence of chicken aldehyde dehydrogenase cDNA and comparison of the deduced amino acid sequence with that of human and rat ALDHs. The initiating codon is underlined. The order of comparison from top to bottom is chicken ALl)H, human mitochondrial ALDH-2 (Hsu et al., 1988), rat ALDH-PB (Dunn et al., 1989) and human cytosolic ALDH-1 (Hempel et al., 1984). The 17 amino acid leader peptide of ALDH-2 is included. Amino acids that are identical with chicken ALDH are indicated by a dash. This sequence has been submitted to the EMBL/GenBank DNA databases with the accession number X58869. B, Restriction enzyme map of chicken ALDH cDNA. The nucleotide sequence presented in (A) was derived from clones 293 and 640 as indicated by the solid lines. Clone 293 was sequenced in both orientations. The proposed translation initiation site is indicated by the arrow. Restriction enzymes are B (BamH I),

(Bgl II), E (EcoRI), H (HindIII),

Hp

1440

Y

(a)

Bg

IU~O

.

L

. .

vGTT

II

•. . . . . . . . . . . . . . .

C

.

CAG FTTT C A A Q Q

T .

FTTT

.

.

.

.

.

.

GAG E

E

V~

F

CTT L . . . . . . .

T ~ A T C C T ] ' G A T C T C C A G A A I T r A C C T T C T [ C A A A T C A A T C C G A A A / ~ A A G C T AT T I C A A T T ~

E

7~0

.

T

.

.

s

A

L

.

.

T

GCA

A .

E

.

C .

GCA

G

R

. .

~V M

GGA

.

E . . .

.

.

.

.

. .

.

.

V .

.

V

v

.

CAA ATT Q l

. . . . . .

GV T ]

.

OCT

A

.

. . . . . . . . . . . . . . . . . . . . . . . . . . v

.

Y F

E

.

.

T

.

N

.

.

T .

^ A

GGG G

.

.

.

.

.

.

G

.

V -

T

T

.

.

CTT L

T !

P S

.

.

A v

V~

.

ACC

.

.

.

.

.

.

V V

. .

.

. .

!

ACA V

..

.

.

.

.

.

.

. .

CEC

R

R -

M L

.

C

. . .

v .

VGT~ ACA LCT. . . .

K .

TAC ACT Y T

I

.

Q

. . .

.

.

......

S -

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

.

.

N . . .

G

.

. y Y

T S S $

FTTC 48O

-

G

.

AAG K

L . .

. .

K S

EQ .

F

.

~ G -

.

V

.

. .

A -

G .

V .

.

G

-

o .

. . . . . .N G

Gp C C T A

cG

.

O

.

G

N

.

s ~

~TC

&AT

AGCA

, I

L

.

~

V

.

. .

.

~

. . . .

G

. .

.

~

P

. .

.

L V

Q

. .

K R

. .

A

GCA AAG A K

.

C

A

N

.

V

v

s

.

E . . .

. .

w

.

AGC S

$ .

.

.

.

. .

M

.

.

.

A

.

GTIV

.

.

.

D

.

.

P

A

c

C C T AT]" T A T e 1 Y

V

V

~ .

N

ACT

L

A

-

L M M

S S

T

v

K

T

. .

CCA

P

[3

Q

G N A

R K

T

l

D

G

GAG

D

-

A

GGT

AAA

M

. .

E

H

ATT

II

. .

"

-

CAT

T~A

Ill

. .

"

[A. . . . . . . . .

| Y

TCC

S

M L

Q

K

G .

K. . . ., . . L. . . D. . . L . . .] . . .E . . S. . . G. . . K. . . K . . .E . . .G . . .A . . K. . . L . . . E. . . C. . .C . . .G . . G. . . P. . . w . . .G .

. .

. .

T

AGCT . . . . . . . .

D

GGA

S

GAG g

.

.

M I

L

A

Q

A .

ATG

-

R

.

. .

W . .

TAC

.

.

D

.

.

A .

LCTT

.

R

LIrA

.

.

C y

GCC

.

.

ACT

.

Y

ACT

.

.

.

A .

R -

4~G

.

.

.

s

N

I L

C

.

.

.

.

o

T C

A

S

.

.

.

.

H .

K

E

T T

.

.

.

.

[ L

~

.

.

E

.

.

.

.

E .

.

C V

ACA

.

.

.

V

v

~IG

.

.

N .

A M

CAA .

.

V

.

.

V

.

.

.

. .

L

GAA

.

L

.

.

. .

A A A ATC

GCG

Q

.

.

. .

GAT

~;

.

.

-

.

. .

GCT

FTT. . . . P

.

.

E

.

. .

TC,G

i

.

.

.

.

N . .

GCA C C C

i

E

.

.

. .

D

.

K Q .

TGT

:

.

.

.

TAC

IA . . . . . . . . p w N . . . .

.

.

.

T N

A T A CGC

:

.

.

.

C I!

Y

ACA

:

A

. .

F Q Q

K

AAA

:

.

. .

.

V

AIT

~

.

.

E

.

I

E

I"GT

L

.

.

.

K

C,CT

G D

.

.

P

GGT

:

.

O

E

.

:

.

.

.

. .

i

.

T

.

. . .

:

.

~

A

~

.

Q

A

L

]~ .

Q

.l

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

IA

K .

.

K

.

.

.

.

.

TT

.

.

-

v .

.

R

.

.

.

L

L

R

P

.

V

A G A C C r AGGA AATCAAGCCC]'GA T T ~

A AC

N -

.

CCA

K

A

.

.

y

AAA

P

A

V

-

VC]"G

E

E

V

GGT

P

6

T

s

v v

T

E

.

t.

T C ~ C A ~

"ira

N

-

.

^ -

ACrCTG ACAGTAGTCCrCATCC.CACA C C r f A ~ C A C T ( ~ C C A ? ~ I T T G A A A T A I " F A C

A ~C

T

. .

TAC

~AT

.

A

.

. .

.

ACC~A~C

D

T

.

.

D

.

. .

.

GCC

N RACA

.

.

T

. .

S

I

R

. .

FTTT TACA

V

.

.

.

. .

L

T

.

R

P

R

V

.

~

G

P

P

. .

s

L

F

I I

ACA

p

P

.

.

S

.

s

F

T

. .

.

C

.

c

A

R

N

.

£

A

A~

(HpaII), S (Sau I).

upon hybridizing the filter with the 1" 7 kb insert from clone 2 9 3 . A total of five and four bands were present in lanes 1 and 2, respectively, accounting for 1 6 - 1 8 kb of DNA sequence. W h e n a 3 0 0 bp BamHI fragment mapping from + 6 4 0 to + 9 4 0 w a s utilized

as a probe, t w o bands were obtained with EcoR Idigested DNA and one band with Hind III-digested DNA (indicated by dots in Fig. 4). The limited n u m b e r of bands hybridizing to the 3 0 0 bp fragment suggests that a single gene codes for the m R N A species

302 M

R. G O D B O U T E

N

2~

9.4

6"6

4-4

the retina at early stages of development. The high level of homology to h u m a n cytosolic ALDH would suggest that the most likely form of ALDH to be expressed at high levels in the immature h u m a n retina is ALDH-1. Total RNA was extracted from the retinas of two human fetuses at approximately 10 weeks gestation. Chick and h u m a n fetal retina RNAs were probed with both the chicken ALDH and h u m a n ALDH-1 cDNAs. Under stringent hybridization and wash conditions, no signal was detected in the lane containing h u m a n retina RNA upon hybridization with chicken ALDH cDNA [Fig. 5(A), lane 2]. However, a very weak signal could be detected when the stringency was reduced [Fig. 5(B)]. When the h u m a n cytosolic ALDH-1 cDNA was utilized as a probe, a signal of higher intensity was obtained in the lane containing the chick retina RNA than that containing human fetal retina RNA [Fig. 5(C)]. Probing for actin sequences demonstrated that similar levels of poly(A) + RNA were present in both lanes [Fig. 5(D)]. These results indicate that ALDH transcription is considerably higher in the undifferentiated chick retina than in the h u m a n fetal retina at 10 weeks gestation.

2-5

4. Discussion 2-0

FIG. 4. Southern blot analysis of chicken ALDH gene. Ten micrograms of DNA were loaded in lane 1 (EcoR I-digested) and tane 2 (Hind III-digested). The filter was hybridized with the 1.7 kb insert from clone 293. An identical filter was hybridized with a 300 bp BamH 1 fragment (+640 to + 940 bp) obtained from clone 293. The bands hybridizing to the 300 bp probe are indicated by a dot. Size markers (in kilobase pairs) are indicated in lane M. hybridizing to clone 293, especially in light of the fact that the human mitochondrial ALDH (h-ALDH-2) gene contains at least 13 exons spanning approximately 44 kb (Hsu et al., 1988). It should be noted, however, that the comparatively low intensity of the 5 kb and 3 kb bands in lanes 1 and 2, respectively, could indicate the presence of a second gene with a lower affinity for clone 293. Alternatively, these may simply represent DNA fragments which hybridize to a very small portion of the probe.

E~:pression of ALDH mRNA in the Human Fetal Retina Although ALDH enzymatic levels have been measured in adult primate retina (Holmes and VandeBerg, 1986), these studies have not been extended to include

Although mammalian ALDHs have been extensively studied over the last twenty years, their function in the various tissues in which they are expressed remains unclear. In general, adult tissues have been examined for ALDH activity and most of the roles postulated for these enzymes are associated with tissue maturation. In agreement with the hypothesis that ALDH activity increases during development (Pikkarainen, 1971; Lindahl, 1977), many of the tissues analysed in the developing chick embryo demonstrated substantially higher RNA levels for this gene at later stages of differentiation, for example, the liver and gut. An exception is the neural retina which showed very high levels of ALDH RNA at day 3'5 of incubation, a stage when the retina consists mainly of undifferentiated precursor cells. At this time, one can only speculate as to why ALDH RNA is expressed at such high levels at the early stages of chick retina development, undergoing a 20-fold reduction in steady-state levels by day 19 of incubation. The ALDHs have been found to have a wide substrate specificity. Agents whose metabolic fate is potentially affected by ALDH include ethanol, formaldehyde, dopamine, serotonin, histamine, retinol (Manthey and Sladek, 1989), and medium chain-length aldehydes, such as 4-hydroxynonenal, generated by the lipid peroxidation process (Esterbauer, Zollner and Lang, 1985). Lipid peroxidation by-products have been reported in ocular tissues (Bhuyan, Bhuyan and Podos, 1981; Crabbe, Ting and Halder, 1982) and illumination is known to increase lipid peroxidation of retinal membranes (Kagan et al., 1973). If lipid peroxidation by-products are important substrates for retinal ALDH, one would

ALDEHYDE

DEHYDROGENASE

m R N A IN T H E D E V E L O P I N G

A Lones:

I

RETINA

B 2

I

303 C

2

!

D 2

1

2

Fro. 5. ALDH expression in chick retina and fetal human retina. One microgram of day 3-5 chick retina poly(A)÷ RNA and 30/~g of pooled 10 week human retina total RNA [estimated to be equivalent to approximately 1/zg of poly(A)+ RNA] were loaded in lanes 1 and 2, respectively. The filter was sequentially hybridized with: (A) clone 293 (chicken ALDH) cDNA under stringent conditions (hybridization in 50% formamide, washes at 55°C in 0.1 × SSC, 0'1%'SDS); (B) clone 293 under reduced stringency (hybridization in 30% formamide at 42°C, washes at 37°C in 0"5 x SSC, 0.1% SDS); (C) human ALDH-1 under reduced stringency; and (D) mouse c~-actin. Note that there is some cross-hybridization to the human 28s and 18s rRNAs when the chicken ALDH probe is analysed under low stringency. The asterisk indicates the position of the very weak human ALDH mRNA signal. expect a requirement for higher levels of ALDH in the differentiated retina rather than the undifferentiated tissue. In fact, most of the putative substrates for ALDH in the retina appear to be associated with tissue maturation (Holmes and VandeBerg, 1986). A possible substrate for ALDH activity in the immature retina may be retinal (Manthey and Sladek, 1989), the aldehyde form of vitamin A. One of the richest sources of retinoids in the body is the retinal pigmented epithelium which controls the supply of nutrients and metabolites to the retina (Bridges et al., 1983). In the differentiated retina, opsin traps retinal as fast as it appears to form visual pigments. If the chicken retina ALDH can efficiently utilize retinal as a substrate, this would lead to the formation of retinoic acid which has been implicated in epithelial cell differentiation (Brockes, 1989). Recent evidence suggests that retinoic acid may also have an important influence on certain components of the nervous system (Momoi et al., 1990; Maden, Ong and Chytil, 1990). Whether a reduction in retinal levels or an increase in retinoic acid levels has a biological effect in the differentiating chick retina remains to be determined. In this study, ALDH RNA levels appeared to be substantially lower in the h u m a n fetal retina compared to the chick retina. Since it was only possible to examine the human retina at approximately 10 weeks gestation, representing a comparatively later stage of development than day 3.5 in the chick embryo (McDonnell, 1989), the possibility remains that higher levels of ALDH RNA are present prior to 10 weeks in the developing h u m a n retina. Alternatively, high levels of ALDtt RNA may be found in tissues proximal to the retina, such as the pigmented epithelium, which

could then provide the end-product of the enzymatic reaction to the adjacent tissue. In this regard, it should be noted that cellular retinoic acid binding protein (CRABP) is virtually absent in bovine pigmented epithelium but abundant in the retina (Saari et al., 1977). In contrast, cellular retinol binding protein (CRBP)--proposed to enhance the uptake of retinol into cells and to facilitate its intracellular utilization (Levin et al., 1988)--is more abundant in the pigmented epithelium than in the retina (Saari et al., 1977). To date, few genes have been identified as being specific to or preferentially expressed in the immature retina. One exception is CA-II which is expressed at very high levels in the undifferentiated chick retina. Upon retinal maturation, CA-II expression becomes restricted to Mfiller glial cells (Moscona, 1983; Moscona and Linser, 1983), It has been postulated that carbonic anhydrase could have a role in the inhibition of ALDH since it catalyses the very rapid equilibrium between acetaldehyde, as well as other aldehydes, and their hydrates (Pocket and Dickerson, 1968; Sheridan, Deakyne and Allen, 1980). Aldehyde hydrates strongly inhibit ALDH activity. It is interesting that not only are both CA-II and ALDH mRNAs present at high levels in the immature retina but that they both undergo a decrease in RNA levels upon retinal differentiation. If CA-II does have an ALDH inhibitory function, a tight relationship may exist between the two enzymes during retinal differentiation with CA-II controlling the activity of ALDH. The high degree of homology between chicken and mammalian ALDHs suggests similar biological functions for the enzyme in these species. All mammalian

304

class I cytosotic ALDHs studied to date consist of 500 amino acids (not including the initiating codon). The mitochondrial ALDH has an additional leader peptide at its 5' end (Hsu et al., 1988). Although the chicken ALDH RNA encodes a predicted 509 amino acid protein, it is thought to represent a cytosolic form of the en2yme based on sequence analysis. The amino acid homology between the h u m a n and chicken ALDHs extends throughout the sequence with the exception of the first few residues at the 5' end. Segments found to be highly conserved in the mammalian ALDHs and which are thought to represent the coenzyme binding site (e.g. amino acids at positions 194-248, and glycine distribution at positions 2 1 3 - 2 2 9 and 2 4 5 - 2 5 0 ) (Hempel and Lindahl, 1989) are also highly conserved in the chicken ALDH. However, the areas of homology extend beyond this area; for example, 6 7 / 7 1 residues are conserved from positions 3 4 1 - 4 1 1 . In summary, the discovery that ALDH RNA is expressed at very high levels in the immature chick retina is intriguing, giving rise to speculations as to its possible function in this tissue. A reduction in the levels of a specific aldehyde, or an increase in the levels of an acid produced as the result of the enzymatic reaction may be critical to the development of the mature retina. Whether a requirement for high levels of ALDH is restricted to the chicken needs to be verified by analysing ocular tissues at different developmental stages in other species. The high level of homology between h u m a n ALDH-1 and the chicken ALDH would suggest a similar role for this enzyme in these two distantly related species.

Acknowledgements I am grateful to Dr Yoshida for providing the human aldehyde dehydrogenase-1 probe and to Shelby Hunt and Diane Bradshaw for expert assistance in the preparation of the manuscript. Many thanks to Randy Andison for excellent technical assistance and to Rufus Day and Chris Upton for carrying out the nucleotide homology search. This work was supported by the Medical Research Council of Canada and the Alberta Heritage Foundation for Medical Research. References Aviv, H. and Leder, P. (1972). Purification of biologically active globin mRNA by chromatography on oligo thymidylic acid-cellulose. Proc. Natl. Acad. Sci. U.S.A. 69, 1408-12. Bergsma, D.J., Chang, K.S. and Schwartz, R.J. (1985). Novel chicken actin gene: third cytoplasmic isoform. Mol. Cell. Biol. 5, 1151-62. Bhuyan, K.C., Bhuyan, D.K. and Podos, S.M. (1981). Evidence of increased lipid peroxidation in cataracts. IRCS Med. Sci. 9, 126-7. Bridges, C. D. B., Fong, S.-L., Liou, G. I., Alvarez, R. A. and Landers, R. A. (1983). Transport, utilization and metabolism of visual cycle retinoids in the retina and pigment epithelium. Prog. Retinal Res. 2, 137-62. Brockes, J. P. (1989): Retinoids, homeobox genes, and limb morphogenesis. Neuron 2, 1285-94.

R. G O D B O U T

Crabbe, M. J. C., Ting, H.-H. and Halder, A. B. (1982). Lens aldehyde reductase and dehydrogenase, and their possible involvement in diabetes and cataract formation. In Enzymology of Carbonyl Metabolism : Aldehyde Dehydrogenase and Aldo/Keto Reductase (Eds Weiner, H. and Wermuth, B.). Pp. 329-46. Alan R. Liss: New York. Dunn, T.J., Koleske, A.J., Lindahl, R. and Pitot, H.C. (1989). Phenobarbital-inducible aldehyde dehydrogenase in the rat. ]. Biol. Chem. 264, 13057-65. Esterbauer, H., Zoliner, H. and Lang, J. (1985). Metabolism of the lipid peroxidation product 4-hydroxynonenal by isolated hepatocytes and by liver cytosolic fractions. Biochem. J. 228, 363-73. Godbout, R., Andison, R., Upton, C. and Day, R. (1990). Utilization of the second polyadenylation signal at the 3' end of the chicken carbonic anhydrase II gene. Nucleic Acids Res. 18, 1049. Hamilton, H. L. (1952). An introduction to embryology. In Lillies's Development of the Chick. Holt, Rinehart and Winston: New York. Hempel, J., Harper, K. and Lindahl, R. (1989). Inducible (Class 3) aldehyde dehydrogenase from rat hepatocellular carcinoma and 2,3,7,8-tetrachlorodibenzo-pdioxin-treated liver: distant relationship to the class 1 and 2 enzymes from mammalian liver cytosol/ mitochondria. Biochemistry 28, 1160-7. Hempel, J., Kaiser, R. and J6rnvall, H. (1985). Mitochondrial aldehyde dehydrogenase from human liver: primary structure, differences in relation to the cytosolic enzyme and functional correlations. Eur. ]. Biochem. 153, 13-28. Hempel, J. and Lindahl, R. (1989). Class III aldehyde dehydrogenase from rat liver: superfamily relationship to classes I and II and functional interpretations. In Enzymology and Molecular Biology of Carbonyl Metabolism, Vol. 2. (Eds Weiner, H. and Flynn, T.G.). Pp. 3-17. Alan R. Liss: New York. Hempel, J., yon Bahr-Lindstr6m, H. and J6rnvall, H. (1984). Aldehyde dehydrogenase from human liver. Primary structure of the cytoplasmic isoenzyme. Eur. ]. Biochem. 141, 21-35. Henikoff, S. (1987). Unidirectional digestion with exonucleaseIII in DNA sequence analysis. Methods EnzymoI. 155, 156-65. Holmes, R.S. and VandeBerg, J.L. (1986). Ocular NADdependent alcohol dehydrogenase and aldehyde dehydrogenase in the baboon. Exp. Eye Res. 43,383-96. Hsu, L. C., Bendel, R. E. and Yoshida, A. (1988). Genomic structure of the human mitochondrial aldehyde dehydrogenase gene. Genomics 2, 57-65. Hsu, L. C., Tani, K., Fujiyoshi, T., Kurachi, K. and Yoshida, A. (1985). Cloning of cDNAs for human aldehyde dehydrogenase 1 and 2. Proc. Natl. Acad. Sci. U.S.A. 82, 3771-5. Jedziniak, J. and Rokita, J. (1983). Aldehyde metabolism in the human lens. Exp. Eye Res. 37, 119-27. Kagan, V., Schvedova, A., Novikov, K. and Kozlov, Y. (1973). Light induced free radical oxidation of membrane lipid photoreceptors of frog retina. Biochim. Biophys. Acta 330, 76-9. Kok, M., Oldenhuis, R., van der Linden, M.P.G., Meulenberg, C.H.C., Kingma, J. and Witholt, B. (1989). The Pseudomonas oleovorans alkane hydroxylase gene. I. Biol. Chem. 264, 5442-51. Levin, M. S., Locke, B., Yang, N. C., Li, E. and Gordon, J. I. (1988). Comparison of the ligand binding properties of two homologous rat apocellular retinol-binding proteins expressed in Escherichia coll. J. Biol. Chem. 263, 17715-23. Lindahl, R. (1977). Aldehyde dehydrogenase in 2-acetamidi-

A L D E H Y D E D E H Y D R O G E N A S E m R N A IN THE D E V E L O P I N G RETINA

dofluorene-induced rat hepatomas. Eur. J. Biochem. 164, 119-23. McDonnell, J. M. (1989). Ocular embryology and anatomy. In Retina (Ed. Ryan, S.J.). Pp. 1-16. C.V. Mosby: St Louis, MO. Maden, M., Ong, D.E. and Chytil, F. (1990). Retinoidbinding protein distribution in the developing nervous system. Development 109, 75-80. Maniatis, T., Fritsch, E,F. and Sambrook, J. (1989). Molecular Cloning: A Laboratory Manual, 2nd edn. Cold Spring Harbor Lab: Cold Spring Harbor, New York. Manthey, C. L. and Sladek, N. E. (1989). Aldehyde dehydrogenase-catalyzed bioinactivation of cyclophosphamide. In Enzymology and Molecular Biology of Carbonyl Metabolism, Vol. 2. (Eds Weiner, H. and Flynn, T.G.). Pp. 49-63. Alan R. Liss: New York. Mierendorf, R. C. and Pfeffer, D. (1987). Direct sequencing of denatured plasmid DNA. In Methods Enzymol. 152, 556-62. Minty, A. J., Caravatti, M., Robert, B., Cohen, A., Daubas, P., Weydert, A., Gros, F. and Buckingham, M. E. (1981). Mouse actin messenger RNAs. ]. Biol. Chem. 256, iO08-14. Momoi, M., Yamagata, T., Ichihashi, K., Yanagisawa, M., Yamakado, M. and Momoi, T. (1990). Expression of cellular retinoic acid binding protein in the developing nervous system of the mouse embryo. Dev, Brain Res. 54, 161-7. Moscona, A. A. (1983). On glutamine synthetase, carbonic anhydrase and M/iller glia in the retina. Prog. Retinal Res. 2, 111-35. Moscona, A. A. and Linser, P. (1983). Developmental and experimental changes in retinal glia cells: cell interactions and control of phenotype expression and stability. Curr. Topics Dev. Biol. 18, 155-88. Pearson, W. R. and Lipman, D. J. (1988). Improved tools for biological sequence comparison, Prot. Natl. Acad. ScL U.S.A. 85, 2444-8. Pickett, M., Gwynne, D. I., Buxton, F. P., Elliott, R., Davies, R.W., Lockington, R.A., Scazzocchio, C. and SealyLewis, H.M. (1987). Cloning and characterization of the aldA gene of Aspergillus nidulans. Gene 51, 217-26. Pietruszko, R. (1983). Aldehyde dehydrogenase isozymes. In Isozymes: Current Topics in Biological and Medical

20

305

Research. (Eds Rattazzi, M.C., Scandalios, ]. G. and Whitt, G. S.). Pp. 195-217. Alan R. Liss: New York. Pikkarainen, P.H. (1971). Aldehyde-oxidizing capacity during development in human and rat liver. Ann. Med. Exp. Biol. Fenn. 49, 151-6. Pocker, Y. and Dickerson, D.C. (1968). The catalytic versatility of erythrocyte carbonic anhydrase. V. Kinetics studies of the enzyme catalyzed hydration of aliphatic aldehydes. Biochemistry 7, 1995-2004. Saari, J. C., Bunt, A.H., Futterman, S. and Berman, E. R. (1977). Localization of cellular retinol-binding protein in bovine retina and retinal pigment epithelium, with a consideration of the pigment epithelium isolation technique. Invest. Ophthalmol. Vis. Sci. 16, 797-806. Sanger, F., Nicklen, S. and Coulson, A.R. (1977). DNA sequencing with chain-terminating inhibitors. Proc. Natl. Acad. Sci. U.S.A. 74, 5463-6. Sheridan, R.P., Deakyne, C.A. and Allen, L.C. (1980). Acetaldehyde hydrate and carbonic anhydrase: possible roles in the inhibition of brain aldehyde dehydrogenase. Adv. Exp. Med. Biol. 132, 705-13. Thomas, P. S. (1980). Hybridization of denatured RNA and small DNA fragments transferred to nitrocellulose. Proc. Natl. Acad. ScL U.S.A. 77, 5201-5. Tipton, K.F. (1985). Aldehyde dehydrogenases. In Enzymology of Carbonyl Metabolism, Vol. 2, (Eds Weiner, H. and Wermuth, B.). Pp. 3-13. Alan R. Liss: New York. Turner, A.J., Illingworth, J.A. and Tipton, K.F. (1974). Simulation of biogenic amine metabolism in the brain. Biochem. ]. 144, 353-60. yon Bahr-Lindstr6m, H., Hempel, J. and J6rnvall, H. (1984). The cytoplasmic isoenzyme of horse liver aldehyde dehydrogenase. Relationship to the corresponding human isoenzyme. Eur. ]. Biochem. 141, 37-42. Wahl, G.M., Stern, M. and Stark, G.R. (1979). Efficient transfer of large DNA fragments from agarose gels to diazobenzyloxymethyl-paper and rapid hybridization by using dextran sulfate. Proc. Natl. Acad. Sci. U.S.A. 76, 3683-7. Weiner, H. (1979). Aldehyde dehydrogenase: mechanism of action and possible physiological role. In Biochemistry and Pharmacology of Ethanol. (Eds Majchrowicz, E. and Noble, E. P.). Pp. 107-124. Alan R. Liss: New York.

EER 54