Download Nucleotide Sequence and Organization of the Rat Heme Oxygenase

THEJOURNAL
OF BIOLOGICAL
Vol. 262, No. 14,Issue of May 15, pp. 6795-6802 1987
Printed in CIT.S.A.
CHEMISTRY
0 1987 by The American Society of Biological Chemists, Inc
Nucleotide Sequence and Organization
of the Rat Heme
Oxygenase Gene*
(Received for publication, September 8,1986, andin revised form, January 8,1987)
Rita M.Muller, Hayao TaguchiS, and
Shigeki Shibaharas
From the Friedrich Miescher-Institut, CH-4002 Basel, Switzerland
The microsomal heme oxygenase cleavesthe heme ring at
the a-methene bridge to form biliverdin, a tetrapyrrole green
pigment (1).Biliverdin is subsequently converted to bilirubin,
a yellow bile pigment, by biliverdin reductase (2). Under
physiolo~calconditions, the activity of heme oxygenase is
highest in the spleen, where senescent erythrocytes are sequestrated and destroyed (3). Heme oxygenase activity is
highly inducible by its substrate heme (3-7) and by various
non-heme substances such as heavy metals (8,9), bromobenzene (lo), and endotoxin (11){reviewedin Ref. 12).
We are particularly interested in the induction of heme
oxygenase by its substrateheme because 1)heme is an essential component of hemoglobin and other hemoproteins, and
is required in all animals cells, 2) hemin appears to induce
heme oxygenase at the transcriptional level (13-15), and 3)
hemin also regulates the activity of S-aminolevulinate synthase, a key enzyme for heme biosynthesis (reviewed in Ref.
16). Thus, heme seems to regulate both its biosynthesis and
degradation. Heme also regulates the transcription of yeast
-
* The costs of publication of this article were defrayed in part by
the payment of page charges. This article must therefore be hereby
marked “advertisement” in accordance with 18 U.S.C. Section 1734
solely to indicate this fact.
The nucleotide sequence(s)reported in thispaper has been submitted
to the GenBankTM/EMBL Data Bank withaccessionnumberfs)
502722.
$ Present address: Dept. of Agricultural Chemistry, The University
of Tokyo, Tokyo 113, Japan.
$ To whom correspondence should be sent: Friedrich MiescherInstitut, P. 0. Box 2543, CH-4002 Basel, Switzerland.
by in situ plaque hybridization (20). The hybridization probes used
were an equimolar mixture of the EcoRI-EcoRI fragment (nucleotide
residues -101-88) and theEcoRI-HindIII fragment (nucleotide residues 88-971) excised from the rat heme oxygenase cDNA, XRHO6
(E), andlabeled with [(r-32P]dCTPby random priming method (21).
The numbers in parentheses, shown together with restriction enzymes, indicate the 5”terminal nucleotide generated by cleavage.
Hybridization-positive phage clones were isolated by repeated plaque
purification, and the isolated DNA inserts were subcloned in pUC8
plasmid (22). The subcloned DNA fragments were used for further
analysis, and nucleotide sequences were determined (23).
SI ~ ~ ~ a s e - ~Analysis-Total
p i n g
RNA was isolated from rat
spleen (24) for S1-mapping analysis, as spleen is abundant in heme
oxygenase mRNA (15, 25). The EcoRI-BamHI DNA fragment (nucleotide residues -749-372) was partially digested with XhoI. The
obtained EcoRI-XhoI fragment (nucleotide residues -749-69) was
labeled at its 5‘ ends with [y-3ZP]ATPusing T4 polynucleotide kinase
and then digested with NdeI. The resulting NdeI-XhoI fragment
(nucleotide residues -139-69) (20 fmol) was hybridized with spleen
RNA (20 pg) a t 45 ‘C for 3 h in 10 pl of 80% formamide containing
0.4 M NaCl, 1m M EDTA, and 40 mM Pipes,’ pH 6.4 (26), and digested
with S1 nuclease (180 units/ml) a t 37 “C for 30 min. The products
were fractionated by electrophoresis on a sequencing gel.
Analysis of Promoter Function of the 5 ’ - F ~ n kRegion-The
~~
EcoRI-BamHI fragment (nucleotide residues -748-373)was transcribed in the presence of [C~-~’P]GTP
using HeLa cell lysates according to the instru~ions
of the supplier. The concentrations of template
DNA and a-amanitin was 12 pg/ml and 1 pg/ml, respectively. The
products of in vitro transcription were analyzed as described (27). We
also used the subcloned plasmid harboring the EcoRI-EcoRI fragment
(nucleotide residues -748-2116) as a template and determined the 5‘
end of the transcriptsby S1 nuclease-mapping analysis. The S1probe
was the HindIII-BamHI fragment (nucleotide residues -549-373)
end-labeled at theBamHI site.
The abbreviations used are: Pipes, 1,4-piper~inediethanesulfonic
acid; kb, kilobase pairs; bp, base pairs.
6795
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Heme oxygenase, an essential enzyme of heme catab-iso-l-cytochrome c gene (17).
In order to understand the molecular mechanisms of inducolism, is inducibleby its substrate heme, by heavy
metals, and by various other substances.To study the tion of heme oxygenase, we have isolated and characterized
molecular mechanisms of the induction of heme oxy- the genomic clones for rat heme oxygenase. We show the
genase, we isolated the heme oxygenase gene from a complete nucleotide sequence of the heme oxygenase gene
rat genomic DNA library using cloned cDNA as hy- and its 5“flanking region.
bridization probes and determined
its complete nucleoEXPERIMENTAL PROCEDURES
tide sequence. The gene is composed of 6830 nucleotides, and is organized in four introns andfive exons.
Materials-Sprague-Dawley rats were used as sources of DNA and
The transcription initiation site was identified by S1 RNA. Restriction endonucleases were purchased from Bethesda Renuclease mappinganalysis. Using HeLa cell lysate, we search Laboratories, New England BioLab, and Pharmacia; bactericonfirmed that the transcription of cloned hemeoxy- ophage lambda packaging kits and DNA of EMBL4 from Amersham
genase gene is initiated accurately at the assignedini- Corp.; DNA polymerase I (Klenow fragment), T4 pol~ucleotide
tiation site. In the 5”flanking region of the heme ox- kinase, DNA ligase and nuclease S1 from Phannacia; HeLa cell lysate
ygenase gene, we foundseveral potential bindingsites from Bethesda Research Laboratories; [(u-~*P]~CTP and [y3*P]ATP
Amersham Corp.
for different transcription factors: a transcription
fac- from
Ctoning and Sequencing of Genomic DNA Encoding Rat Heme
tor Spl, a positive regulator for the controlof amino Oxygenase-A genomic DNAlibrary, constructed using EcoRI partial
acid synthesis (GCN4), a heat shock transcription
fac- digests of Sprague-Dawley rat liver DNA, was kindly provided by Dr.
tor, and a metal-dependent transcription factor. Fur- J. Bonner, Phytogen, Pasadena, CA (18). We also constructed our
l containsthesequencethat
thermore,theintron
own rat genomic DNA library in EMBL4, a lambda replacement
shows about 65% homology to that of the neuronal vector (IS), using Sau3AI partial digests of rat liver DNA. These
libraries were screened for DNA segments encoding heme oxygenase
identifier sequence, a possible enhancer element.
6796
Structure of the Heme Oxygenase Gene
RESULTS AND DISCUSSION
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FIG. 1. Restriction map and sequencing strategyof cloned rat genomicDNA encoding heme oxygenase.The direction of transcription is from left to right. A, restriction map of cloned rat heme oxygenase gene.All
existing sites for restriction enzymes indicated are shown, except for the region contained inthe 3'-EcoRI fragment
(about 8 kb) of XRHO1. The locations of exons are indicated by open boxes (untranslated regions) and by closed
boxes (protein coding region). B, sequencing strategy. The arrows indicate the direction and extent of sequence
determinations. The short uertical lines at the end of arrows indicate the sites of 5' end labeling. The restriction
sites used for 5' end labeling were abbreviated as follows: A , AuaII; B , BamHI; C, AccI; D,DdeI; E, EcoRI; F,
FnulHI; G, BglII; H , HindIII; I,H i n f f J , BanII; L, ClaI; M , SmaI; N , NdeI; 0,NcoI; P, PuuII; Q, TaqI; R, RsaI; S ,
PstI; T , BstNI; U, AluI; W, SstI; X , XhoI; Y, XbaI; 2,HpaII. A slashed box indicates the NdeI-XhoI fragment
labeled at theXhoI site, which was used for S 1 nuclease-mapping analysis (see Fig. 3 ) .
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Isolation of Rat Genomic DNAEncoding Heme OxygenaseThe rat genomic library used initially was a collection of
recombinant phage that contain rat liver DNA fragments
generated by partial digestion with EcoRI and joined to theX
Charon 4A arms (18). From about 1.5 x lo6 plaques, we
isolated and characterized one hybridization-positive clone
(XRHOl). XRHOl harbors a DNA insert composed of two
EcoRI fragments of about 7 kb and 8 kb in the 5' to 3'
direction (Fig. lA). Restriction mapping and blot hybridization analysis indicated that the DNA fragment carried by
XRHOl does not contain the 5' upstream region from the
internal EcoRI site of rat cDNA (15). Therefore, using an
EcoRI-EcoRI fragment (nucleotide residues -101-88) excised
from a cDNA clone, XRHO6 (15), we screened the same library
(about 2 X lo6 plaques), but we could not isolate the phage
clone carrying 5' upstream region. Therefore, we decided to
construct our own library using Sau3AI partial digests of rat
liver DNA. From 1 X lo6 plaques, we isolated four positive
clones using a same EcoRI-EcoRI probe. One of them,
XRH05, carries a DNA fragment encoding the 5' upstream
region. The clone, XRH05, harbors a DNA insert composed
of 5 EcoRI fragments of about 400,900, and 500 bp, and 2.8,
7, and 3.9 kb in the 5' to 3' direction (Fig. lA). The DNA
fragment carried by both overlapping genomic clones, XRHOl
and XRH05, encoded the entire region of heme oxygenase
gene (Fig. lA). Blot hybridization analysis of rat liver DNA
indicated that the cloned heme oxygenase gene retains the
same sequence organization asinthe
genomicDNA and
suggested that there is a single gene for heme oxygenase in
rat genome (data not shown).
deterStructure of theRat HemeOxygenaseGene-We
mined the nucleotide sequence of the entire rat heme oxygen-
ase gene and about 1.4 kb of the 5'-flanking region according
to thestrategy shown (Fig. 1B). We determined the sequence
in both directions. The ratheme oxygenase geneis composed
of 6830 nucleotides and is divided by four introns into five
exons (Fig. 2), which are identified by comparison with the
cDNA sequence.
To identify the transcription initiation site, we used S1
nuclease-mapping analysis. The S1 probe was the NdeI-XhI
fragment (nucleotide residues -139-69) labeled at the X h I
site and is indicated by a sloshed box in Fig. 1B. The results
show the presence of several protected fragments aroundone
major fragment (Fig. 3), suggesting that the transcription of
the heme oxygenase genestarts atone main site. By comparing the position of the major protected fragment with sequence
ladder of the S1 probe, we assign the A residue of nucleotide
1 as the tentative transcription initiation site (indicated by
an arrow in Fig. 3). In this analysis, we assume that the
mobility of the protected fragment is about 1 residue behind
that of the products in the sequence ladder (28). The A residue
of nucleotide 1 is also the first nucleotide of the full length
cDNA, pRHOl (15),andthereare
no plausible acceptor
sequences of intron near this A residue. Therefore, we conclude that thetranscription starts mainly at theA residue of
nucleotide 1. The lengths of five exons are 151,121,492,100,
and 693 bp in the 5' to 3' direction.
Promoter Activity of 5'-Flanking Region of the Rat Heme
Oxygenase Gene-There are no typical TATA-boxes (29) and
CAAT-boxes (30) in the 5"flanking region of the heme oxygenase gene. However,one TATA-like sequence, TACTTAA,
is located 28 bp upstream from the transcription initiation
site (Fig. 2). The promoter function of this TATA-like sequence was analyzed using HeLa cell lysate (Fig. 4). The
transcription of the truncated DNA fragment containing a
TATA-like sequence gave rise to the product of about 390
6797
~ t r ~ ~ofuthe
r eHeme Oxygenase Gene
-1321
A~GCTTG~TTCTA~TfTCCAAAAT6lT~6A6llGGlTlllCT66AGA~lClCAGG~lTAACAAAACA
-1201
~~GACACAAAAAGTGRA6TC~llGlT6A6AlCbAAAGlCA~lllAGA6lAG6AGAGAl6G~C66Gl66TlCACA6l~Cll~ClGCCClTACA6A66ACCT~AATTCAGllCCClACAT66
120
ACA6TC6CCAGTC6CCTECA6AGlllCC6CClCCAbCCA6C6R6l66A6CCl6CC66~GCA6A6CCAlCTC6~GC6GA6CCC66ffiCCl6AA6CG~6C66~6CCA6CCT6AACl~6CCCA
-Exon
1
210
6TCCC6C6~6A6C6CCCACA6CTC6RCA66CAA6C6~ClRCll6G66AC666~~Cl6l6Al6TCCC6C6lCCCTl6lCCA6CClA6AA~l6CA66Cl6C666ACCAA66bAC6A6ll6
I
Intron 1
3b0
CC6CCCA6TCTRCCTGTAGCCRl6G6CA6CT66R6G6A6A~bRl6lGAlTC~CCCClCl6ACAC6Cl66A66l6ACC66Al6G6l6l6lCll6lAGlAlCCR66Tll6R6GGAl6bACA6
Downloaded from www.jbc.org by on December 1, 2006
-241
~GdCTTG6GGCCC6G~TGGG~CbdlCCCTGG6TlTGCCC~~CA6CAlCTCACbGClAGAR~lGTClCCTAGTlCT66AAC~TlCCA6AlTCCl6A6A66C~66ClAClCCT6CClTCCTA
7-20
6TA6~TCAA6TT66CCTTGAACTCC86TTCGCTTCCCTll6CTTCCl6A6lACl6A6AlT6AA66l6CCCC6CCbCCACbCll66CllCl~llC&lCClCClCCTCClCClCClCCllCl
840
-
CCTCTTCCTCCT~~TCCTCTTCCTCT~CllCCTCCllClCClCllCCTCClTClCClClTl6AlllTTlll6bGAC66A6CGCClClTl6lA6CCCl6GAl~lCTlCAClCl6lAGAlCA
960
66CT66CCTA6~ACTC66~6ATCT6CCTGCCTCl6CClCCClAGl6Cl6AGAllACA66CCT6CACCAClCl6lCCAGClClTlTllClllll~TCCllTlRlAAAGCC~~lTll~T~~l
.
1080
TAT~TTTTT~TTf~~A6C6TCTCG~6l~AC~llTA~lllbTCGl6CAT6lAGA6TG6CCACAlAlAl6ACACAClAA~lAl6l66AGGTC~6AGllCGAClTG~AGAAA6TCACllCTCA
1200
6TCT~CC~f6TG6CTTCCA~G~AA6AT~66Cl?ATCA6GT66CAA6lGC6TllCCl66Cl6A6CTlACACT6lCTAGlCClCACCTC~~~C~Tll~6CA6~~~G6~l~~~~C?T~~~~~T
FIG.2. Nucleotide sequence of the rat heme oxygenase gene. The nucleotide sequence of the message
strand is shown. Nucleotide residues are numbered in the 5' to 3' direction, beginning with the transcription
initiation site,and thenucleotides on the 5' side of residue 1are indicated by negative numbers. Exons are indicated
by thick solid lines. The transcription initiation site is marked with an asterisk. The ATG codon for initiating
methionine and the TAA termination codon are indicated by an owrline. A TATA-like sequence is indicated by
ouerline and underline. The potential binding sites for different transcription factors areindicated as follows: Spl,
solid lines;GCN4, dotted lines; beat shock t r a n ~ ~ p t i factor,
o n double u ~ r l i n e sand
; me^-dependent transcription
factor, double dotted lines. Four copies of Alu-type sequences are indicated by thin underlines. The identifier-like
sequence was indicated by an ouerline. The direct repeatscomposed of 42 pyrimidines are indicated by underlines
with an arrowhead.
6798
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.
3360
.
3840
.
3960
66T66CCC~C6C~T~T~CCC6Cl~CCT666l6ACClClC~66666lCA66lCCl6~~6A~6~ll6C6C~6A~66CC~l66CCll6CC~~6ClCl6666~~66CCl66CllllTlC~CCll
CCT6T666~66~6TG~T~CA6666~A6llCCCl6Al6lC~ll6ClClCll6l6l66l666Cl6l6AlCClCl6RC~~l66l6lA6Cl66ACCl6666l6CCl6~6l~Cl66l6C~l66CC
A66CCA6CI\CA6TCCA66CllC66~T6Cl6AC~l6lCCC~66l66CRTlT6CllCClTTTClTT6~TTT6CRTT6RT6T6TT6CCCRC666T~T~TCT6TlT66T6TC~6ATTC6ll666
.
4080
66T66~6CTACA6AC~6CT666~6CT6CC~CAG666~~CC666~~Tl6~ACCC6CAlCClCl66~6~6C~AClA~C6ClCllA~lClll6~~ClAlCTll~6ClC66l6llll6~6C~~6
,
4200
6CCCCG~AA66C~6~6CT66666~6l~6~Cl6lCCl~lll~l~Cl~l666l6Cl~l66l66TC~666llCC~~lll666ACClCCl6lClClCCCCAClllC~6CCl6A6~6CCllT~~~
FIG. 2"Continued
6799
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FIG. 2-Continued
6800
Structure of Oxygenase
the
Gene
Heme
A
C
T
significance. Three copies of GGGCGG, a basic motif of the
binding site for Spl (reviewed in Ref. 31), are located at
G A C C T 1 2
positions (-520) to (-526), (-99) to (-91) and (-58) to (-52).
The first of these three is in a 3’ to 5’ orientation. The second
sequence,GGGGCGGGG,is completely identical with the
consensus sequences of Spl binding sites (33). Two copiesof
TGACTC are located at positions (-138) to (-143) and (-43)
to (-38). The first copyis in a 3‘ to 5’ orientation. This
hexanucleotide is the binding site for the transcriptionfactor
GCN4, which is responsible for general amino acid control
regulation in yeast (reviewed in Ref.32). In yeast, many
different amino acid biosynthetic pathways are under a comG
mon control and activated in response to starvation for any
C
single amino acid. Furthermore, a sequence with an almost
G
perfect dyad symmetry, TCTGGAACCTTCCAGA, islocated
A
273 bpupstream from the transcription initiationsite (double
G
underlines in Fig. 2) and is completely identical to the con-T
sensus sequence of heat shock element, C--GAA--TTC--G,
G
which can bind the heat shock transcription factor, a posiT
tive regulator of the transcription of the heat shock proC
tein genes (reviewed in Ref. 33). The regulatory region also
A
contains a sequence with an imperfect dyad symmetry,
GGGTGCTGCACTC (nucleotide residues -581 to -569),
which contains two copies of core sequences of metal regulatory elements found in metallothionein genes (34). However,
the heme oxygenase gene contains no heme-responsive element of the yeast iso-1-cytochrome c gene (17).
We are particularly interested in the presence of a heat
shock element because of the similarities in the natureof the
induction of heat shock proteins and of heme oxygenase.Like
heme oxygenase, heat shock proteins are induced by various
reagents or conditions (reviewed in Ref. 35), which seem to
be stressful for the cells or tissues. Recently, we found that
both activity and mRNA of heme oxygenaseincreased following heat-treatment of rat glioma cells?
Features of Intron Sequences-The lengths of four introns
are 1900, 826, 1508,and 1039 bp in the 5‘ to 3’ direction. All
FIG. 3. S1 nuclease-mappinganalysis of the 5’ end of heme the exon/intron boundaries follow the GT/AG rule (36) and
oxygenase mRNA. The S1 probe was hybridizedto spleen RNA or are consistent with the consensus sequences for donor and
yeast tRNA, digestedwith S1 nuclease, and fractionatedon a sequenc- acceptor sites of RNA splicing (37), with the exception of the
ing gel together with the sequencing ladder of the S1 probe (NdeI- donor site of the intron 1 (Fig. 2). The intron 1begins with a
XhoI fragment labeledat the XhoI site, see Fig. 1B).Complementary GC dinucleotide instead of a GT dinucleotide. To exclude the
nucleotides aroundthe initiation sites are shownalong the sequencing possibility of sequencing error, we confirmed this donor seladder. The predominant transcriptionsite is indicated by an arrow.
Lane I, the protected fragmentswith spleen RNA, andlane 2, control quence several times using different subclones. Interestingly,
the same deviation of donor sequences was reported in four
sample with yeast tRNA.
other examples: chick (38) and duck aD-globin genes (39),
murine a*-crystallin gene
(40) and human y subunit prenucleotides (indicated by an arrow in Fig. 4 4 , Lane a), which cursor gene of muscle acetylcholine receptor (41). In all these
was not detectable in the presence of a-amanitin (lane b). cases, the deviated donor sequences have a common sequence,
The size of this product by RNA polymerase I1 is consistent AG/GCAAGwhich is similar to the consensus sequences
with the expected size of the transcripts (376 nucleotides). except for the C residue. In the case of duck aD-globingene,
Then, we used subcloned plasmid as a template and deter- the deviated donor sequence of the intron was functional in a
mined the 5’ end of the RNAs synthesized in vitro by S1 transient expression system (39). Therefore, the AG/GCAAG
is a functional donor sequence of some introns. Like the
nuclease-mapping analysis (Fig. 4B). The S1 probe was the
murine aA2-crystallin gene (40), a possibility of an alternative
HindIII-BarnHI fragment (nucleotide residues -549-373) lasplicing for heme oxygenase mRNA not
is excluded, although
beled at the BarnHI site, which is located in the intron 1. The
the resulting truncated polypeptide is only 17-amino acid
major protected band of about 390 nucleotides was sensitive residues due to thepresence of termination codon TGAin the
to a-amanitin indicated by an arrow (Fig. 4B,lane a).Another intron 1 (nucleotide residues 180-182). Within the intron 1,
strong signal of about 900 nucleotides was probably due to there areno open reading frames which start with a potential
the presence of protected S1 probe, which was not digested initiating methionine and encode a long peptide.
with S1 nuclease. Therefore, the transcription of the cloned
In the rat
heme oxygenase gene region,
we found four copies
heme oxygenase geneis initiated accurately and efficiently at of repeated sequences whichare similar (about 70%) to those
or near the assigned transcription initiationsite.
of rat Alu-like sequence (42). As underlined in Fig.2, one
Features of 5’-Flanking Region- In the regulatory region copy is in the 5’-flanking region, nucleotide residues (-1067)
of the rat heme oxygenase gene, we found several potential
binding sites for known transcription factors of regulatory
* S. Shibahara andR. M. Muller, manuscriptin preparation.
+ +
+
“\
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v
6801
Structure of the Heme Oxygenase Gene
A
FIG. 4. Promoter activity of 5’flanking regionof the rat hemeoxygenase gene.A, in uitro transcripts of
truncated DNA template containing a
putative
TATA-like
sequence.
The
EcoRI-BamHI
fragment
(nucleotide
residues -748-373) was transcribed in the
absence (lane a) or in the presence of aamanitin (lane b). The size markers were
end-labeled bX174RFDNA fragments
generated by the digestion with Hue111
and were given in base pairs (lane c). B,
identification of the 5‘ end ofRNAs
synthesized in uitro. The subcloned plasmid (40 pg/ml) was transcribed in the
absence (lane a) or in the presence of aamanitin (lane b), and the products were
analyzed by S1 nuclease mapping. The
size markers (lane c) were same as in A.
B
a b c
a b c
18%
872
603
.
)
310
271/ 281
234
310
271/281
194
194
118
118
234
1184
GGArrGAACTTAGGACTTCAAACATTCTAGGCAAGCGCTTTACCCATAAGGTA~GTAT~G~CC1249
1181
GGACCGAACCCAGGGCCUUGCGCUUCCUAGGCAAGCGCUCGCUAAAUCCCCAGCCCC 1116
**
*I
**+*
**
* +**+ +
FIG. 5. Homology between the intron1of the heme oxygenase geneand the rat identifier sequence.
The top line shows the sequence of the intron 1 (nucleotide residues 1184-1249) and the bottom line shows the
complementary strand of identifier sequence (nucleotide residues 1181-1116)
of clone plB308 (44). Asterisks
indicate nucleotide differences.
1)
REFERENCES
S., and Schmid, R. (1969) J. Bwl.
Chem. 244,6388-6394
2. Tenhunen, R., Ross, M. E., Marver, H. S., and Schmid, R. (1970)
the heme oxygenase gene contains the sequence (nucleotide
Biochemistry 9,298-303
residues 1184-1249) that shows about 65% homology to the
3. Tenhunen, R., Marver, H. S., and Schmid, R. (1970)J. Lab. Clin.
complementary strand of the neuronal identifier sequences
Med. 75,410-421
(43, 44) (Fig. 5). The identifier sequence, a middle-repetitive
4. Pimstone, N. R., Engel, P., Tenhunen, R., Seitz, P. T., Marver,
element, is located preferentiallyin introns of neuronal-speH. S., and Schmid, R. (1971) J. Clin. Inuest. 50. 2042-2050
as a positive regulator 5. Pimstone, N. R., Tenhunen, R., Seitz, P. T., Maker, H. S., and
cific genes (44)and recently is proposed
Schmid, R. (1971) J. Exp. Med. 133, 1264-1281
of neuronal-specific gene expression (45). The intron 1 also
6. Gemsa, D., Woo, C. H., Fudenberg, H. H., and Schmid, R. (1973)
contains incomplete direct repeats of 42 bp, which are comJ. Clin. Inuest. 52, 812-822
posed only of pyrimidines (indicatedby the underline with a n
7. Shibahara, S., Yoshida, T.,and Kikuchi, G. (1978) Arch. Biochem.
arrowhead in Fig. 2). Incidentally, these direct repeats show
Biophys. 188,243-250
of protein
about 66%homology to the complementary strand
8. Maines, M. D., and Kappas, A. (1974) Proc. Natl. Acad. Sci. U.
S. A. 71,4293-4297
coding region of yeast heat shock protein-90 gene (nucleotide
9. Maines, M. D., and Kappas, A. (1976) Biochem.J. 154,125-131
residues 707-632) (46).
10. Guzelian, P. S., and Elshourbagy, N. A. (1979) Arch. Biochem.
The heme oxygenase gene and its 5”flanking region conBiophys. 196,178-185
tains a number of sequences of potential regulatory signifi11. Bissell, D.M.,
and Hammaker, L. E.(1976)Arch.Biochem.
cance. Particularly, the presenceof a heat shock element led
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us to find that hemeoxygenase is a heat shock protein.2 The
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780-908 and 1645-1795; and one copy is in the intron 3,
nucleotide residues 4312-4437. Furthermore, the intron 1 of
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