Vol. 185, No. 2, 1992 June 15, 1992
AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 489-495
OF ITS PROMOTER
Arvind Chopra’, Dalton L. Ferreira-Alvest Pierre Sirois’ and Jean-PaulThirion”
Departmentsof ’ Microbiology and ‘Pharmacology, Faculty of Medicine, University of Sherbrooke, Sherbrooke(P.Q.) Canada JlH 5N4
The guinea pig 5lipoxygenase (5LO) gene and its promoter were cloned from a guinea pig genomic DNA library. Sequencinganalysisof the guinea pig promoter revealed that expressionof the 5-LO genein this rodent is probably governedby cis acting nucleotidesequences quite similar to the humangene. Nucleotide sequences that bind factors like Sp-1, AP-2, NF-kB and c-Ha-rus - were identified in the guineapig S-LO promoter region. Q1992Academic Press,Inc. SUMMARY:
Leukotrienes(LT) are metabolitesof arachidonicacid that arereleasedby leukocytesduring hypersensitivity and inflammatory reactions(1). Thesesubstances arehighly active pharmacological mediatorsandcontrol many cell functionsresponsiblefor bronchoconstrictionassociated with asthma. They are synthesizedfrom arachidonicacid in a cascadeof reactionsof which the initial two are catalyzed by the enzyme 5lipoxygenase (S-LO; E.C.22.214.171.124). Due to its key position in the synthesisof LT, various laboratoriesare investigatingthe regulationof the 5LO gene. Recently the nucleotide sequencesof the human5-LO cDNA and its promoter have beenreported (2,3). Guineapigs (gp) have beenusedextensively asan animalmodelto study asthmabecausethe pulmonary system of this animal respondsquite similarly to the human during hypersensitivity reactions(4,5). Although the molecularbiology of the human5-LO hasbeeninvestigatedto some extent (2,3), no information is availableat the nucleic acid level about the gp enzyme. In this paper, as a first step to study the similaritiesand differencesbetweenthe humanand gp 5-LO genes,we report the cloning, partial sequenceand featuresof the gp 5-LO geneand its promoterin relation to the human5-LO sequence. *To whomcorrespondence should be addressed. +On sabbaticalleave from the Departmentof Pharmacology,Biological SciencesInstitute, University of Minas Genus, Belo Horizonte, Brazil 30 000, P.O. Box 2486. 0006-291X/92
Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
Bacterial and phase strains: DNA extraction and probes The human 5-LO cDNA, cloned in pUC13, was a gift from Dr. J. Evans (Merck Frosst Lab., Montreal). An appropriate bacterial strain was transformed with this construct to amplify the human cDNA and the nick translated (6) 1735 bp EcoRJ-Bam HI fragment (Fig. 1) was used as a probe to detect the gp 5LO on genomic DNA Southern blots. Other fragments (designated a to f; Fig. 1) from the human cDNA were purified either on acrylamide or agarose gels to yield the l-142 bp, l290 bp, 291-1040 bp, 1041-1416 bp, 1417-1706 bp and 1707-2505 bp oligonucleotides which were used as probes during this study. The EcoRI-Barn HI cDNA fragment was also used for initial screening (7) of the gp genomic DNA library (see below) and the other probes (a to f) were employed to distinguish bewteen the various phages isolated during screening.
Genomic DNA Southern blots The gp liver and human placental genomic DNA were isolated (9) and digested with different restriction enzymes. The fragments were separated on a 1 % agarose gel and transfered onto nylon membranes by standard procedures (10). The blots were hybridized with the 1.735 Kb Eco RIBamHI probe as before (11). Briefly, prehybridization and hybridization were at 42“C in a 50 % formamide buffer containing 5 x SSC, 50 mM sodium phosphate (pH 7.4) and 0.25 mg/ml salmon sperm DNA. The blots were washed thrice (20 min each) at room temperature in 2 x SSC and 0.1 % SDS and the stringent washings were done twice (30 min each) at 50°C in 0.1 x SSC and 0.1 % SDS. The blots were visualized by autoradiography at -70°C for 2-5 days using intensifying screens.
Isolation of Dhase recombinants carrviw different parts of the S-LO PID gene A gp genomic DNA library in phage lambda Fix II was obtained from Stratagene (La Jolla, CA) and screened with the nick-translated EcoRI-BamHI fragment or the other probes (a to f) as described above.
Seauencine analvsis DNA sequencing was carried out in Ml3 mp18 (12) using dideoxynucleotides as chain terminators (13) and the sequencing kit from Pharmacia. Nucleic acid sequences were analysed with the Pustell sequence analysis program (International Biotechnologies Inc., CT.).
RESULTS AND DISCUSSION Preliminary experiments were carried out to determine conditions under which the human 5 LO probe would hybridize with the gp DNA on Southern blots. About 10 pg of either human or gp DNAs were digested with about 100 units of different restriction enzymes, electrophoresed, blotted and hybridized with the probe as described in Materials and Methods. The Southern blot (Fig. 2), revealed that in both human and gp DNA, Eco RI and Hind III generated fragments of 2 to 12 Kb which hybridized with the human probe. With Pst I mostly fragments smaller than 4 Kb were generated.
This indicated that genes from both species had high homology regions that were
conserved during evolution.
We then screened the gp genomic DNA library using the same 1.735
Kb probe and isolated 32 phages. Of these, fifteen were retained and analysed further by the plaque hybridization technique with the various probes (a to f) detailed under Materials and Methods. The 490
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2507 I 3 E
Fig. 1: Restriction map of human 5-LO cDNA indicating the different fragmentsused as probes.
Fragmentsa to fare respectively1-142,l-290,291-1040,1041-1416,1417-1706and1707-2507. The EcoRI-BamHI, 1.735kb fragmentwasusedto initially detectandscreenfor the 5LO in gp genomicDNA andits library.
various isolateshybridized with the different probes(Table 1) indicating that eachphageprobably containeddistinct segmentsof the gp 5-LO gene. Other experiments(data not presented)with the different phageDNAs showedthat the gp genewas at least20 kb in size. Phages22,25,26 and 86 hybridized only with the short 142 bp 5’ end fragment of the human 5-LO cDNA (probe a). Among thesephages,we found phage 26
Fig. 2: Southernblotsof guineapig liver andhumanplacentagenomicDNA, probedwith the human5-
lipoxygenasecDNA. LanesA, B andC areguineapig DNA, lanesD, E andF arehumanDNA. LanesA andD, Eco RI digest;B andE, Hind III digest;C andF, PstI digest. Size markersare represented by numbersandbarson the left handside.
Vol. 185, No. 2, 1992
TABLE 1 Characteristics of different phagesisolatedfrom guineapig genomicDNA library PHAGES (CLONES)
(+) Indicateshybridization with the probe.
contained the largest gp genomic DNA insert (about 10 Kb in size) and its restriction map was prepared(Fig. 3). For identification of DNA fragmentscontainingthe 5’ end of gp 5-LO genein the phage26 insert, either the 142 or the 290 bp probe (a or b) wasused. After Pst I digestiontwo gp DNA were identified to contain portionsof the 5’ end. Both fragments(143 and640 bp, respectively designated5-LO A and 5-LO B) were subclonedin Ml3 mp18 and sequenced(Fig. 4) asdescribed in Materials and Methods. Clone 5-LO A containedpartsof the 5-LO N-terminal exon and anintron. The larger clone, 5-LO B, containedrest of the above exon along with the promoter region of the gene. Sequencefrom the two clonescould easily be aligned becausethe gp 5-LO amino acid sequenceencoded by one of the three reading frames of the clones, exhibited more than 95 % homology with the N terminal amino acid sequenceof the human 5-LO. Only two amino acids, isoleucineand phenylalanineat positions27 and 42 in the gp (Fig. 4) are respectively replacedby valine and proline in the human5-LO. Since 5-LO from both sourceshave a similar function, we believe thesesubstitutionsare not related to enzyme function. Theseresultsalsosuggestthat the 5’
, 100bp P *
Fig. 3: Restrictionmap of the pUC18 insert(derived from LambdaFix II, clone 26). Arrows show directionof sequencing (Universalprimerl +; Syntheticprimer4). Boxed arearepresents Nterminalexon of guineapig 5-lipoxygenasegene. Different restrictionenzymeswereA, Ava II; B, Bam HI; E, Eco RI; H, Hind III; K, Kpn I, P, PstI; Pv, PvuII and X, XbaI. 492
185, No. 2, 1992
Pst CTG GAG AGC TCC CGA GTC CCT CCC ACC CTC TCC TAA GCC CCG GGG ACT CGC CCT GCT AP-2
CAA AGG CCC CGG CGA GGC AGC GAT CCC GGC TGG TAC GCC ATG GGC CCC AGC GGA ACT
GGC CGG AGC GGC TGA GGC TGA GGC AAG GCA GAT GCA GGC CGT AGA AGG GAA GAA GAG
CGA ACT GCG GGA AGC CAG GGC GGA TCC TCA GGG CTG AGA CCT GGG GGGGT sp-1 NF-kB
GGA GAG GGG GGG CGT GGC CAA GAG CCC CCG GGT GGG CGG AGT CTG GGT TTG TGG GCT sp-1
TGG TTT TGT GTG GGC GGG GCT TGA GCC GGG AGT TTG TGC GGG ACC TGG GTC TAG ACG sp-1
AGG TGG TGC TAO'TAC
CGG GGC AGG GGC AGG GGA GGG GCT CTG GAG GCA GGG CAT CTG TCA GTG GCC AGC TGG
ACT GAC AGG CGG CCC GGC CCC AGC ACC GCG GGA TCT GAG GAG GCA CTC GGA CCT GGG
Promoter +j CGC TTG AGC TCC CCC AGC CGG CGC CCC ACC ATG CCG TCC TAC ACG GTG ACC GTG GCC Met Pro Ser Tyr Thr Val Thr Val Ala
CAG GGG GCG AGG TGG GGG CGG GGC CTA GAG AGG CCG GGT GGG c-Ha-ras sp-1
ACC GGC AGC CAG TGG TTC GCC GGC ACC GAC GAC TAC ATC TAG CTG AGC CTC ATC GGC Thr Gly Ser Gin Trp Phe Ala Gly Thr Asp Asp Tyr Ile Tyr Leu Ser Leu Ile Gly
Pst I TCT GCG GGC TGC AGC GAG AAG CAC CTG CTG GAC AAG GCC TTC TAC AAT GAG TTC GAG Ser Ala Gly Cys Ser Glu Lys His Leu Leu Asp Lys Ala Phe Tyc Am Asp Phe Glu
p Intron CGC GGC GCG GTG AGC CTG GCG CGG GCC TGC GCG GGC GGG CAG GCG GGG ACC TGC AU+ Arg Gly Ala Val sp-1
GGG GAC CTT GCA GAA GGG GGA CTT GCA GAA GAG AA~ CTG CAG Pst I
Fig. 4: Nucleotidesequence of the 5’ andpromoterregionof guineapig Slipoxygenasegene. Various regulatoryelementsin the sequence areunderlinedandlabelled. Underlinedammoacidsshow differencesbetweenthe guineapig and human5LO aminoacid sequence(seetext for details).
N-terminal may not be a part of the enzymecatalytic sitebecauseproline in contrastto phenylalanine would drastically changethe tertiary structureof the protein. Beyond aminoacid 51, no aminoacid homology betweenthe two sequences wasdetectedsuggestingthat this wasthe end of the first exon in the gp DNA. This was confirmed by the presenceof the CTC/AGCCTG sequenceat the exonintron junction which is in confirmation of the GT-AG rule (14,lS). From the readingframe of the identified exon we concludedthat the region betweennucleotides-543 and -1 was therefore the gp 5-LO promoter. Further analysis(Table 2) of the gp promoter sequencerevealed someinteresting features which were attributed to cis-regulatory elementsin
systems. The gp promoter lacked the
CCAAT and TATA sequenceslike its humanhomologue(3) and was C + G rich. Among the various cis-acting gene regulatory elementsthe AP-2 binding sequence(at -496) showed66 % homology with the concensussequencesuggestingthat expressionof this genein gp andhumansmay 493
Vol. 185, No. 2, 1992
TABLE 2 Transcription and other factor binding sequences found in guinea pig 5-lipoxygenase promoter FACTOR
ACTCGCCCECT (66 %)* consensus sequence AGTCCCCAGGCT
GGGAGTCGGC (80 %) consensus sequence GGGANNYYCC
GGGGGCGAGGTGGGGGCG GAGGTGGGGGGCGGGGCC consensus sequence GGGGCGGGGGCGGGGGCG
-353; -280 -244; -169 -14.5; +I74
GGGCGG (100 %)
(77 %) (77 %)
consensus sequence GGGCGG *Numbers in parentheses represent percent homology with consensus sequence.
(3). There was one binding sequenceat -327 for the NFkB factor which exhibited 80 %
with the consensus sequence (3,16).
There were two overlapping
sequences (at -182 and -
176) for c-Ha-= (17) that exhibit 77 % homology with the concensussequences.Six Sp-1 binding sites (at -353, -280, -244, -169, -145 and +174) exhibiting 100 % homology with the concensus sequencewere identified in the gp sequence. As with the humangene, it appearsthat the Sp-1 regulatory elementsplay an important role in expressionof the gp S-LO gene. The significanceof the Sp-1 binding sequencein the intron (at +174) is unknown.
-142 to -184 several
overlapping sequences for different factors like c-Ha-= and Sp-1 were evident. We suggestthese overlapping sequencesin the gp 5-LO promoter may have similar functions as the Sp-1 assembly reported in the human 5-LO promoter (3). Since the gp and human 5-LO geneshave similar regulatory elements,it strongly indicatesthat the enzyme from both speciesprobably have similar expressionmechanismand ancestory. Our observationsand further studiesin this direction would be useful to explain why guineapigs and humanshave similar responsesduring an asthmacrisis.
We thank Mr. Denis Tang for his help with the computerprogramand
Ms. CarmenLabrecquefor secretarialassistance.This investigation wassupportedby the Medical ResearchCouncil
PS is in receipt of an MRC Scientist Award. 494
Vol. 185, No. 2, 1992
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