Gene, 99 (1991) 181-189

181

Elsevier

GENE

03951

Molecular characterization (Citrullinemia;

of the murine argininosuccinate

urea cycle; cDNA;

recombinant

DNA;

Linda C. Surha,b,*, Arthur L. Beaudet”,’ and William

promoter;

synthetase locus

genome

organization;

exon; intron)

E. O’Brien”*b*c

“Institute for Molecular Genetics and bDepartment of Biochemistry, Baylor College of Medicine, Houston, TX 77030 (U.S.A.); Hughes Medical Institute, Houston, TX 77030 (U.S.A.) Received by J. Piatigorsky: 28 August Revised: 18 September 1990 Accepted: 1 November 1990

and ‘Howard

1990

SUMMARY

The cDNA and gene encoding murine argininosuccinate synthetase were cloned and characterized. The cDNA sequence predicts a peptide of 412 amino acids (aa) including the initiator methionine. There is 98% identity with the aa sequence of the human enzyme. The 3’-untranslated region of the cDNA includes two regions of sequence which are conserved between mouse, rat, human and cow. The murine gene contains 16 exons with the start codon occurring in exon 3. Although alternative splicing occurs in primates to include or exclude exon 2, exon 2 sequences were included in the murine mRNA in all tissues and developmental stages examined. The inclusion of exon 2 in murine mRNA, compared to the usual exclusion in human mRNA, may be explained by differences in the donor splice sequences for exon 2.

INTRODUCTION

Argininosuccinate synthetase (ASS; EC 6.3.4.5) functions in the urea cycle ofureotelic animals and catalyzes the conversion of citrulline, aspartic acid and ATP to argininosuccinic acid, AMP and PP,. The highest enzyme activity is found in liver and kidney, but low levels of activity are found in most other tissues. The enzyme has been purified from rat (Saheki et al., 1975), human (O’Brien, 1979), and bovine (Rochovansky et al., 1977) liver. In all cases the enzyme is a homotetramer with a subunit A4,. of approx. 46000 and with no known evidence of posttransCorrespondence to: Dr. W.E. O’Brien, Baylor

College

(713)798-4795; *Present

of

Medicine,

Institute

Houston,

77030

Genetics,

(U.S.A.)

Tel.

Fax (713)797-6718.

address:

Department

of Genetics,

Eastern Ontario, 401 Smyth Rd., Ottawa, Tel. (613)737-2720. Abbreviations:

for Molecular

TX

aa, amino

acid(s);

ASS,

Children’s

Ontario,

Hospital

KlH

8Ll (Canada)

argininosuccinate

synthetase;

of

ASS, gene encoding ASS; bp, base pair(s); kb, kilobase or 1000 bp; nt, nucleotide(s); oligo, oligodeoxyribonucleotide; PP,, inorganic pyrophosphate;

TF, transcription

0378-I 119/91/$03.50

0

factor;

1991 Elsevier

fsp, transcription

Science Publishers

start point(s).

B.V.

lational modification. The cDNA sequence for the human (Bock et al., 1983), bovine (Dennis et al., 1989) and rat (Surh et al., 1988) enzymes are known. Genetic deficiency of ASS causes citrullinemia in humans (Beaudet et al., 1986) and in cattle (Harper et al., 1986; Dennis et al., 1989). Regulation of expression of the enzyme has been studied extensively in vivo and in vitro as has been reviewed (Jackson et al., 1986a; Beaudet et al., 1986). Expression of the gene is not observed during fetal development of the rodent until just before birth, at which time there is a marked increase in expression that continues for several days until adult levels are reached (Raiha, 1976; Adcock and O’Brien, 1984; Morris et al, 1989). This developmental profile of expression of ASS is coordinated with the other four genes of the urea cycle. Activities of this enzyme and of other urea-cycle enzymes are increased in response to higher dietary protein intake in the rat (Schimke, 1962) and in macaques (Nuzum and Snodgrass, 1971). In cultured human cells, expression of the enzyme is repressed by high concentrations of Arg in the culture medium (Jacoby, 1974; Su et al., 1981; Irr and Jacoby, 1978; Schimke,

182 1: AGCCCTCTGC CGCCGTCTGC CACTGCGCCT GGGCTCACTG AGTGGTTCAT CTGGCCAGGA AAGCAGACTA CACGGACTCC AGGGACCTGT ACCTATAATC CAAGACAAG 110: ATG TCC AGC AAG GGC TCT GTG GTT CTG GCC TAC AGT GGT GGC CTG GAC ACC TCC TGC ATC CTC GTG TGG CTG AAG GAA CAA GGC TAT GAT 1: Met Ser Ser Lys Gly Ser Val Val Leu Ala Tyr Ser Gly Gly Leu Asp Thr Ser Cys Ile Leu Val Trp Leu Lys Glu Gln Gly Tyr Asp ZOO: GTC ATC GCC TAC CTG GCC AAC ATT GGC CAG AAG GAA GAC TTT GAG GAA GCC AGG AAG AAG GCG CTG AAG CTT GGG GCC AAA AAG GTG TTC 31: Val Ile Ala Tyr Leu Ala Asn Ile Gly Gln Lys Glu Asp Phe Glu Glu Ala Arg Lys Lys Ala Leu Lys Leu Gly Ala Lys Lys Val Phe 290: ATT GAG GAT GTG AGC AAG GAA TTT GTG GAA GAG TTC ATC TGG CCT GCT GTC CAG TCC AGT GCA CTC TAC GAG GAC CGC TAT CTC CTG GGC 61: Ile Glu Asp Val Ser Lys Glu Phe Val Glu Glu Phe Ile Trp Pro Ala Val Gin Ser Ser Ala Leu Tyr Glu Asp Arg Tyr Leu Leu Gly 380: ACC TCT CTC GCC AGG CCT TGC ATA GCT CGC AGA CAG GTG GAG ATT GCC CAG CGT GAA GGG GCC AAG TAT GTG TCT CAC GGC GCC ACG GGA 91: Thr Ser Leu Ala Arg Pro Cys Ile Ala Arg Arg Gln Val Glu Ile Ala Gln Arg Glu Gly Ala Lys Tyr Val Ser His Gly Ala Thr Gly 470: AAG GGG AAT GAC CAG GTC CGC TTT GAG CTC ACC TGC TAT TCA CTG GCA CCC CAG ATT AAG GTC ATC GCT CCC TGG AGG ATG CCT GAG TTT 121: Lys Gly Asn Asp Gln Val Arg Phe Glu Leu Thr Cys Tyr Ser Leu Ala Pro Gin Ile Lys Val Ile Ala Pro Trp Arg Met Pro Glu Phe 560: TAC AAC CGG TTC AAG GGC CGA AAT GAT CTG ATG GAG TAT GCA AAG CAA CAC GGA ATC CCC ATC CCT GTC ACC CCC AAG AGC CCC TGG AGT 151: Tyr Asn Arg Phe Lys Gly Arg Asn Asp Leu Met Glu Tyr Ala Lys Gln His Gly Ile PLO Ile Pro Val Thr Pro Lys Ser Pro Trp Ser 650: ATG GAT GAA AAC CTC ATG CAC ATC AGC TAT GAG GCT GGG ATC CTG GAA AAC CCC AAG AAT CAA GCA CCT CCG GGT CTC TAC ACA AAA ACT 181: Met Asp Glu Asn Leu Met His Ile Ser Tyr Glu Ala Gly Ile Leu Glu Asn Pro Lys Asn Gin Ala Pro Pro Gly Leu Tyr Thr Lys Thr 740: CAG GAC CCT GCC AAA GCA CCC AAC AGC CCA GAT GTC CTT GAG ATA GAA TTC AAA AAA GGG GTC CCT GTG AAG GTG ACC AAC ATC AAA GAT - 211: Gln Asp Pro Ala Lys Ala Pro Asn Ser Pro Asp Val Leu Glu Ile Glu Phe Lys Lys Gly Val Pro Val Lys Val Thr Asn Ile Lys Asp 830: GGC ACA ACC CGC ACC ACA TCC CTG GAA CTC TTC ATG TAC CTG AAC GAA GTT GCG GGC AAG CAC GGA GTG GGT CGC ATT GAC ATC GTG GAG 241: Gly Thr Thr Arg Thr Thr Ser Leu Glu Leu Phe Met Tyr Leu Am

Glu Val Ala Gly Lys His Gly Val Gly Arg Ile Asp Ile Val Glu

920: AAC CGC TTC ATT GGA ATG AAG TCC CGA GGT ATC TAC GAG ACC CCA GCA GGG ACC ATC CTT TAC CAC GCT CAT TTA GAC ATA GAG GCC TTC 271: Asn Arg Phe Ile Gly Met Lys Ser Arg Gly Ile Tyr Glu Thr Pro Ala Gly Thr Ile Leu Tyr His Ala Hx 1010: ACG ATG

Leu Asp Ile Glu Ala Phe

GAT CGG GAA GTA CGC AAA ATC AAG CAG GGC CTG GGC CTC AAA TTC GCA GAG CTC GTA TAC ACA GGT TTC TGG CAC AGC CCT GAA

301: Thr Met Asp Arg Glu Val Arg Lys Ile Lys Gln Gly Leu Gly Leu Lys Phe Ala Glu Leu Val Tyr Thr Gly Phe Trp His Ser Pro Glu 1100: TGT GAA TTT GTT CGC CAC TGT ATC CAG AAG TCC CAG GAG CGG GTA GM

GGG AAG GTG CAG GTG TCT GTC TTC AAG GGC CAP. GTG TAC ATC

331: Cys Glu Phe Val Arg His Cys Ile Gln Lys Ser Gln Glu Arg Val Glu Gly Lys Val Gln Val Ser Val Phe Lys Gly Gin Val Tyr Ile 1190: CTC GGT CGG GAG TCT CCA CTT TCA CTC TAC AAT GAA GAG CTG GTG AGC ATG AAC GTG CAG GGC GAC TAT GAG CCC ATC GAC GCC ACT GGC 361: Leu Gly Arg Glu Ser Pro Leu Ser Leu Tyr Asn Glu Glu Leu Val Ser Met Asn Val Gln Gly Asp Tyr Glu Pro Ile Asp Ala Thr Gly 1280: TTC ATC AAT ATC AAC TCG CTC AGG CTG AAG GAG TAC CAT CGC CTT CAG AGC AAG GTC ACT GCC AAA TAG ACCCTGACAA AGACGGAGCG GGCC'I 391: Phe Ile Am

Ile Am

Ser Leu Arg Leu Lys Glu Tyr His Arg Leu Gin Ser Lys Val Thr Ala Lys ***

1374: CCCCA CTCTGCAGCT CTCCCAGGCT TCAGCATTAA TTGTTGTGAT AAATTTGTAA TTGTAGCTTG TTCTCCACCA CCTGACTGGG GCTGCTGTGT CCCCCCCCGCC CC 1483: CCCACAGC CTTTGTTCCC TGGTCCCCTA TAGCCTACAA AAGTGGTCAT CCAAGGGAAG GGAGGGTGGC

Fig. 1. The nt and deduced

aa sequence

nt were derived from genomic Laboratories.

All libraries

DNA labeling method in the laboratory NIH3T3

were probed

(Feinberg

package

cells and was obtained

written

from murine ASS cDNA. The A cDNA library prepared

rspis designated

and Vogelstein, from Stratagene.

(Surh et al., 1988), radiolabeled

1984). Three mouse genomic Institute

of Technology)

The libraries

+ 1. The most 5’ (nt + 1 to +28) and 3’ (+ 1338 to terminus)

from DBA/2J mouse liver RNA (Edenberg

using a rat ASS cDNA clone, pASR2

of either Dr. L. Hood (California

into Ml3mpl8 or Ml3mpl9, previously described (Sanger, denote

sequencing.

GGGCAGCTGC AG

were screened

libraries

et al., 1985) was obtained with [a-32P]dCTP

were utilized. Two libraries

or Dr. J. Weis (Harvard as described

(Sambrook

University).

from Clontech

by the random

were prepared The third library

et al., 1989). DNA fragments

primer

from BALB/c

mice

was prepared

from

were cloned either

or into pTZl8U or pTZl9U. Dideoxynucleotide chain-termination sequencing with [a-35S]dATP was performed as 1981; Biggin et al., 1983; Tabor and Richardson, 1987). Computer analyses were performed using the Eugene/Sam software

by the Molecular

Biology Information

Resource

in the Department

of Cell Biology (Baylor

College of Medicine).

The three asterisks

stop codon.

1964). The regulatory sequences necessary for metabolite repression in cultured cells are present in the 5’ region of the gene (Jackson et al., 1986b; Boyce et al., 1986), and there is evidence that this regulation is primarily due to changes in transcription (Jackson et al., 1988). ASS is overexpressed in canavanine-resistant, cultured cells (Jacoby,

1978; Su et al., 1981). The over-expression is associated with increased transcription, but there is no gene amplitication (Jackson et al., 1988). The mechanism of over-expression of the gene is unknown (Jackson et al., 1988; Boyce and Freytag, 1989; Boyce et al., 1989). Alternative splicing occurs for human ASS (Freytag

183 et al., 1984a), resulting in the presence or absence of exon 2,

identity between any two species. Comparison

but this does not alter the coding sequence

sequence

since the ATG

with that of the sequences

of the murine

from either of two

Methanosarcina

barkerii

start codon occurs in exon 3. The biological significance, if any, ofthis alternative splicing has not been determined. We

Methanococcus

now report the determination of the mouse cDNA sequence and characterization of the mouse ASS. Splicing of the 5’

conservative 1988).

exons of the mouse and human genes was compared demonstrating significant differences between the species but no major developmental or tissue-specific differences within a

The nt sequence of the murine cDNA includes 109 bp of 5’-untranslated sequence and at least 220 bp of 3’-untranslated sequence. The sequence surrounding the start codon

Archaebacteria

deviates

species.

species,

or

vannielii, reveals a 38% aa identity

changes

are considered)

from the consensus

of Kozak

(Morris

(54% if and Reeve,

(1984), and differs

between mouse and human but is identical between mouse and rat. The murine and human cDNA sequences have RESULTSAND

DISCUSSION

(a) Murine ASS cDNA The cDNA clone for rat ASS was used to isolate two overlapping clones from a cDNA library prepared from the liver of a DBA/2J mouse. The cDNA sequence depicted in Fig. 1 includes some 5’- and 3’-untranslated sequences derived from genomic clones to be described below. The cDNA sequence predicts a primary translation product of 412 aa, including the initiation Met, and this is identical in size to the human, rat and bovine enzyme. The aa sequence of murine ASS is 98.1 y0 identical with the human enzyme (98.3% including conservative aa substitutions). There is remarkable conservation of aa sequences between murine, rat (Surh et al., 1988), bovine (Dennis et al., 1989) and human (Bock et al., 1983) ASS with a minimum of 95%

89% nt identity in the coding region with 136 nt differences, 93% of which occur in the third position of the codon. There is low sequence similarity in the 5’-untranslated region (30%) but higher conservation lated region (70%).

in the 3’-untrans-

Comparison of the 3’-untranslated region for ASS from four mammalian sequences reveals two significant regions of homology (Fig. 2). The first region is approx. 42 nt in length and consists of 74% A+T residues. The second region of homology is approx. 50 nt 3’ of the first and contains 57 nt with 50 y0 A + T. A + T-rich regions, and the AUUUA motif in particular, have been shown to be associated with rapid mRNA turnover in short-lived mRNAs including some inflammatory response proteins (Shaw and Kamen, 1986; Caput et al., 1986; Jones and Cole, 1987; Brawerman, 1989). The AUUUA sequence is not present

*** mouse rat human cow

GCCAAATAGaccc.. ---------_--

tgacaaagacggagcgggcctcc.ccactctgcag --------- . -- . c---------

mouse rat human cow

ctctcccaggcttcagcat

-------------gcg~----~--g-agct---g-c~---~-~~-~~--a-~----------------agc------t-ca-agct---g-cg-c~---g-cg-c~----~-a-cc ------------:,,----~

aattgttgtgataaatttgtaattgtagct

a--c----a-tacag--gc---------------~-----------ga-a--_---_a_tg-agctgc-----______----~-----g-----ga-accacctgactggggctgctgtgtccccccccg...ccccc

mouse rat human cow

-------

mouse rat human cow

ccacagc.... ----ccacagg ---g....... ---atagtc-

--gg---g-a-c-_

.tag--g-g-tg--agg.......--

ctttgttccctggtcccc.tatagcctacaaaagtggtc __ -___________________ ------------------c-ga-----g----c--t---

mouse rat human rat human Fig. 2. Interspecies compared

comparison

as indicated.

Terminal

caattaa caattaaaagagaaaaaaaaaaa

of the 3’-untranslated

region of the ASS cDNA. The nt sequences

nt ofthe coding sequence

from the cDNA

are shown in upper case with the translational

indicate identity between mouse and other nt. Optimal alignment is generated are boxed. The polyadenylation site is underlined in rat and human.

by conservatively

of mouse, rat, human

stop codon, TAG, marked

and cow are

by asterisks.

Dashes

placed gaps (dots). Two large regions of near 100% identity

184

map for murine ASS. A partial EcoRI (E) restriction

Fig. 3. Restriction vertical

lines with introns

and flanking

sequence

clones. Clones 134s and 142s were derived recombinant

libraries.

and 1102-30;

a predicted

site marked

map is shown spanning

by the horizontal

from an NIH3T3

The EcoRI polymorphic

the gap includes

represented

genomic

recombinant

by an asterisk

is present

EcoRI site in exon 10 as determined

I*

B

1

%217201-

2

3

,-

160147-

123-

while the others

of the fsp by primer extension. (upper-case

(Panel A) The nt sequence

letters) is shown with the upstream

genomic sequence (lower-case letters). The position ofthe antisense oligo used in primer extension is boxed. The multiple termination sites for transcription

were from two different

Balb/c genomic

in Balb/c DNA but not in 3T3 DNA. A gap exists between

142s

from the cDNA.

5’-end

are indicated

(horizontal

bracket)

within

a loose

consensus, YYCAYYYYY, with the adenine predicted as the preferred fsp (A, indicated by an asterisk). The position of the TATAA box is underlined. (Panel B) Primer-extension products produced by reverse transcriptase

library,

by numbered, phage genomic

mRNA

of ASS. There is also evi-

(b) The murine ASS A total of 37 phage 1 clones were isolated by screening three different murine genomic DNA libraries using rat cDNA as a probe. There are numerous processed dispersed pseudogenes for human ASS (Freytag et al., 1984b), and Southern blotting analysis and preliminary chromosome mapping data suggested that numerous pseudogenes also exist in the mouse (Nakamura et al., 1985). Genomic clones hybridizing with both the 5’ and 3’ portions of the cDNA probe were presumed to contain pseudogenes and generally were not studied further. A repeat-free fragment from clone 13C was used to rescreen the library to obtain clones A34S and A42S. Seven unique genomic clones were studied in detail using restriction mapping and sequencing. These clones demonstrated that the murine ASS locus spans at least 50 kb (Fig. 3). One additional EcoRI site was found in a genomic clone from Balb/c DNA but was not present

;::--

for murine ASS cDNA

are indicated

dence that the 3’-untranslated region can influence translational efficiency. A critical role has been shown for the 3’-noncoding region in translational activation of dormant mRNAs as part of murine oocyte maturation (Strickland et al., 1988). The 3’-untranslated region of interferon-p mRNA has a pronounced inhibitory effect on translation in some in vitro systems (Kruys et al., 1987). Although these conserved sequences suggest some functional importance for this 3’-untranslated region, a computer search of nt sequence databases did not identify any closely related regions in other genes.

tataaccctggatgcgcgcctctctcAGCCC TCTGCCGCCGTCTGCCACTGCGCCTGGGCTC ACTGAGTGGTTCATCTGGCCAGGAAAGCAGA CTACACGGACTCCAGGGACCTGTACCTATAA TCCAAG&AAGm

reverse

50 kb. Exon positions

in the 3’untranslated

A

Fig. 4. Mapping

approx.

line. The lower part of the figure shows the overlapping

were analyzed

on a 6”/, polyacrylamide

- 8 M urea gel. The

labeled

primer

in panel A was hybridized

to 20 pg (lane 1) and

5 pg (lane 2) of poly(A) + RNA isolated from adult mouse liver or to 20 pg of yeast tRNA (lane 3). Total cellular guanidinium-isothiocyanate

and

RNA was isolated

pelleting

through

either by using

a CsCl

cushion

(Chirgwin

et al., 1979), or by sequential ethanol precipitations from guanidine. HCl (MacDonald et al., 1987). Poly(A) + RNA was isolated by affnity chromatography with oligo(dT),,.,,-cellulose (Aviv and Leder, 1972) using the manufacturer’s specifications (Collaborative Research). A4, markers, in bp (numbers HpaII digests of pBR322.

on left side), were derived from “P-labeled,

185 in clones from NIH3T3 DNA (Fig. 3). This represents an authentic strain polymorphism which can be demonstrated

In an attempt to identify conserved sequences which might be functionally important in the promoter region, the

by Southern blot and has been used for linkage mapping of the gene to the proximal region of mouse chromosome 2 (Jackson et al., 1990). There is a gap of unknown size in the genomic sequence between clones 142s and ,?102-30; this

5’-flanking sequences from the mouse and human (Jinno et al., 1985) were compared (Fig. 5). The 267 bp upstream region from the tsp is G + C-rich in mouse (73 %) as is the

gap includes exon 10. Some of the genomic clones initially appeared contain portions of the expressed gene based

same region in human (81%). There is a TATAA sequence (Breathnach and Chambon, 1981) in mouse and human,

likely to on their

but there is no CCAAT consensus sequence (Grosveld et al., 1982) in either gene. The TATAA sequence is embedded in a larger, highly conserved, region. There are

hybridization pattern but subsequently were proven to represent fragments of pseudogenes. On sequencing, these clones showed regions of 70-80% nt identity with the cDNA, evidence of a few hundred

other conserved sequences upstream and downstream from the tsp. The conservation of sequence in the first portion of exon 1 may be consistent with a conserved recognition site

of processed structure, and interruptions bp of extraneous DNA with no recogniz-

for a TATAA binding protein, since it is known that TFIID can protect a region from -40 to + 20 or + 30 in footprinting

able splice junctions. These sequences are presumed to represent ancient pseudogenes which have diverged signilicantly and have been interrupted by additional rearrangements or insertions (data not shown). Primer extension analysis was performed using an oligo primer in the region believed to represent the third exon. Termination occurred over a region of 5 to 6 adjacent nt to yield a fragment of about 123 nt (Fig. 4). In this region, there is a loose consensus sequence for transcription initiation, Y-Y-C-A-Y-Y-Y-Y-Y (Corden et al., 1980), except for the presence of a purine, guanine, following adenine. The tsp for the human gene was mapped a few nt downstream from this site (Freytag et al., 1984a).

M

analyses (Parker and Topol, 1984; Nakajima et al., 1988; Horikoshi et al., 1989). Human ASS has multiple GC boxes or hexanucleotide core sequences for Spl binding in both orientations (Dynan and Tjian, 1985; Kadonaga et al., 1986). There is only one GC box in the mouse at position -92 and this is conserved relative to the human. A 15-bp sequence in the human gene shows conservation with two other urea-cycle enzymes, ornithine transcarbamylase and arginase, but was not present within the available murine sequence (Ohtake et al., 1988). The exon-intron boundaries were sequenced for almost

ccagggtctcatgaccaaggaatccccagggagcgagcgagcagctgcagggggtqggct~ag......~q~ag

Ii --ga-ag-a-cc-cttccc-gg.--------ag-a-gcgtaga~g~-~~~g~q~~~g~gg~----M cccccgagtgaccgcgcagt~agtggaggctga~g~ccggggcgctgtgacgcaca~gttcc~ w ~~~ccc-gatcgccactcgccgg--act-g-a-c-aggc

Fig. 5. The nt sequence

of the 5’-flanking

sequences

letters. The

enclosed between

in lower-case

in (boxes with orientation

indicated

mouse and human is designated

complementary was annealed

region of murine (M) and human

tspof mouse is designated by arrowhead).

by dashes.

to nt + 106 to + 127 (relative to the to mouse

Optimal

(H) ASS. Exon 1 sequence

+ 1. The TATAA AP2 consensus alignment

consensus

sequences

is generated

sequence

are designated

by conservatively

tsp)of the mouse cDNA and was end-labeled

liver poly(A)+ RNA for 1 h at 50°C in 10 ~1 of hybridization

is in upper-case is underlined by arrows

letters,

and putative

5’-flanking

and intron

Spl-binding

over the region.

Sequence

sites are identity

placed gaps (dots). A 22-nt oligo was synthesized

using T4 polynucleotide

buffer containing:

kinase. The end-labeled

6 units avian myeloblastosis

primer

virus reverse

transcriptase (Life Sciences)/10 mM dithiothreitol/0.4 pg actinomycin D/0.3 units RNasin (Promega, Madison, WI)/05 mM each of dATP, dTTP, dGTP and dCTP. Reaction products were ethanol-precipitated and separated by electrophoresis on denaturing 6% polyacrylamide - 8 M urea gels.

186 EXON 3' Splice (C/t)ll~ca~ ~AG......................G wccctggatgcgcgcctctctc aatatcacatttactttcacag atatctttcttgctccctccag actggctgtttctgctccacag ctcatagctgctctccctttag atacccttatcttccattccag gacccctttgtcctgtctgcag tccgttttgccttcacctgtag caactcatctctctccctgcag

AGC. AGT. ACA. GCC. GTG. GGG. GTC. CAA. CTA. AAT. ctctcctccctctctgccccag GGG. GGG. tggatctag GTA. cattcattgtttcttcctgcag GTT. ctgcag CAT. GCT.

Fig. 6. Exon-intron

boundaries.

The consensus

sequences

the murine ASS gene shown below. Exon sequences relative to the asp at + 1, with the exon order indicated numbers:

MUSASSAOI-12

proposed

(EXl-16).

(I-EXl-40).....CTG (41-EX2-104)....AAG :;105-EX3-214)....CTG .(215-EX4_283)....AAG .(284-EX5-472)....AAG .(473-EX6-529)....AAG .(530-EX7-604)....AAG .(605-EX8-675)....CAG .(676-Ex9-706)....AAG .(707-EXlO-797)...AAG .(798-EXll-882)...TGC .(883-8X12-947)...GAG (948-EX13-1079)..CAG i1080-EX14-1236)..GAG (1237-EX15-1301)..CAG (1302-EX16)

for splice junctions

are in upper-case

5' Splice gtgagt

gtattttccaccttt

gtgaggaagtgcctt gtaagacgatgagaa gtaaggctatggatg gtaagatgttactcc gtacatctctgcctc gtaagcccaacccct

gtaagccactttgcc gtgagtgtccacagt gtatgtccgagtccc gtaggctccctgtgc

gtaagagagaaactc

are shown at the top, with the actual boundaries

letters, introns in lower-case

More extensive

gtaagaagttccggg

information

letters. Corresponding

on intron sequences

cDNA positions

is available

and MUSASSB.

A

ECORI

Pst I

Hhdlll

I 307 Bases

Probe:

Predicted

Fragments:

(+)

exon 2

(-)

exon 2

242 Bases 170 Bases

FETUS

ADULT

I

CELLS

In

i .

w*-8

-404

“’

-309

_.:l -160

Fig. I.

determined

for

are numbered

from GenBank.

Accession

187 that human ASS mRNA

consisted

predominantly

cules which lacked exon 2, whereas

baboon

of mole-

liver mRNA

included exon-2 sequences (Freytag et al., 1984a). Recently, numerous human cDNA clones were isolated, and all lacked mouse

exon-2 cDNA

sequences.

sequences (Kobayashi et al., 1990). The clones described above include exon-2

To assess the potential

significance

of alterna-

tive splicing of exon 2 in the mouse, a study of different tissues and developmental stages was undertaken. Total RNA was isolated from adult tissues of DBA/2J mice which was the strain used in the construction of the cDNA library. RNA was isolated from the total fetus, placenta and

217-mm

fetal liver taken at day 18 of gestation. Total RNA was isolated from NIH3T3 and clone-1D cell lines. A &I-

190-aim 180-

Fig. 8. Sl nuclease human-specific, derived

analysis

antisense

from human

individual

fetus (A), human

RNA sources

each lane. Negative

of alternative

and quantity

control

bp on the left margin)

splicing

in the human.

RNA probe was hybridized

adult (B) and baboon

(C). The

in pg of RNA are indicated are identical

A

with total RNA

is yeast tRNA (tRNA). M, markers

and methods

Hind111 fragment from pASM12 which contains exon 2 was subcloned in pTZ19R and used to make uniformly 32P-labeled, antisense RNA probe from the T7 RNA promoter (Fig. 7A). The predicted size of the RNA probe was 307 nt, and the predicted sizes of the protected RNA fragments were 242 nt including exon 2 and 178 nt excluding exon 2. A protected fragment of 242 nt was observed in all instances, whether from adult tissue, fetal tissue or cultured cells (Fig. 7B). The multiple bands in this region were secondary to Sl-nuclease nicking. On longer exposures (data not shown), no fragments of approx. 178 nt were observed. This indicated that exon 2 was present in the mature mRNA molecules for murine ASS in all tissues analyzed. A similar study was performed using a human probe, which was hybridized to adult and fetal human tissues (Fig. 8). Undigested probe was 388 nt. Human tissues, both adult and fetal, predominantly lacked exon 2 and protected fragments with a mobility of 170 nt. There was a small amount of a larger protected fragment, at 227 nt, indicating that some mRNA molecules included

above (sizes in

to those in Fig. 7B.

all of the murine gene (Fig. 6). Most of the human exonintron boundaries have also been sequenced (unpublished data), and the data indicate 16 exons in the murine and the human gene. In particular, exons 10, 12 and 16, which have not been completely sequenced in mouse, have been sequenced in human ASS. All available exon-intron boundaries are identical between the two species, and the AG-GT splice consensus dinucleotides (Mount, 1982) are present in all cases.

exon 2. It is curious that ornithine aminotransferase, which undergoes similar metabolic regulation as the urea-cycle enzymes, displays a mixed RNA population in retinoblastoma where there are transcripts which both include and exclude a 5’-untranslated second exon (Fagan et al., 1989). However, exon 2 is uniformly absent in the mRNA

(c) Alternative splicing of exon 2 The initial cDNA clone for the human enzyme contained exon-2 sequences, but nuclease protection studies indicated Fig. 7. Sl nuclease S 1 nuclease promoter. without

of alternative

The construct

splicing in the mouse. (A) Schematic

was linearized

As shown below the construct, from various

source and quantity

the predicted

tissues. Total RNA isolated

(in pg) of RNA used in each analysis

digests of pBR322

length of undigested showing

from fetal or adult tissues,

to the manufacturer’s

protocol.

Hybridization

uniformly

obtained

or cultured

is shown above each lane. Undigested

and the sizes are shown in bp. The plasmid, were used to prepare

probe is 307 nt, protected

the products

pTZASM-ph,

from the last few nt of exon 1 to a Hind111 site in exon 4) cloned into pTZl9R human cDNA sequences

representation of the RNA probe and the predicted products following “P-labeled, antisense probe was synthesized from the T7 RNA

at the unique EcoRI site; uniformly

exon 2 is 178 nt (Bases = nt). (Panel B) Autoradiogram

RNA derived HpaII

analysis

digestion.

labeled, antisense

was performed

overnight

containing

and the plasmid,

product

containing

after Sl nuclease

digestion

cells are demarcated

exon 2 is 242 nt and product of RNA:DNA

by horizontal

hybrids

from

square brackets.

The

probe is shown at far left. M, markers mouse cDNA pGEMGH-he

sequences

(Herman

RNA probes using T7 RNA polymerase

at 45°C in a final volume of 30 ~1 containing

were “P-labeled,

+29 to +271 (which extends

et al., 1989) containing (Promega,

Madison,

analogous

WI) according

0.5 to 50 ng of total RNA/lo5

cpm

of RNA probe/80% formamide/ mM Pipes pH 6.7/0.4 M NaCI/l mM EDTA. S 1 nuclease digestion was then performed following addition of 300 nl ice-cold S 1 nuclease buffer pH 4.6, containing 33 mM Na acetate/50 mM NaCl/0.03 mM ZnS0,/70 to 100 units of S 1 nuclease with incubation for 1 h at 37°C. The protected

fragments

were analyzed

by electrophoresis

on a 6% polyacrylamide

- 8 M urea gel.

188 of ornithine

aminotransferase

from normal

human

tissues

including liver, kidney and retina (Mitchell et al., 1988). At the present time, biological significance has not been demonstrated

for the alternative

thine aminotransferase

splicing in either ASS or orni-

RNA.

and Chambon,

P.: Promoter

genes. Science

209 (1980) 1406-1414.

Dennis,

J.A., Healy,

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fellowship from the Medical Research to L.C.S. The nt sequences reported in

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