Characterization of the rabbit CYP1A1 and CYP1A2 genes: Developmental and dioxin-inducible expression of rabbit liver P4501A1 and P4501A2

ARCHIVES

OF BIOCHEMISTRY

AND

BIOPHYSICS

Vol. 294, No. 2, May 1, pp. 707-716, 1992

Characterization of the Rabbit CYPIAI and CYPIAZ Genes: Developmental and Dioxin-Inducible Expression of Rabbit Liver P4501Al and P4501A2 David

K. Strom,l

Hans Postlind,’

and Robert

H. Tukey3

Departments of Medicine and Pharmacology, UCSD Cancer Center, 0812, Cancer Genetics Program, University of California at San Diego, IA Jolla, California 92093-0812

Received October 24, 1991, and in revised form January

17, 1992

In adult rabbits, the CYPlAl and CYPlA2 genes are expressed constitutively. Exposure to 2,3,7&tetrachlorodibenzo-p-dioxin (TCDD) leads to elevations in both CYPlAl and CYPlA2 gene products (S. T. Okino et al., 1985, Proc. Natl. Acad. Sci. USA 82, 5310-5314). In this report, we have characterized the rabbit CYPlAl and CYPlA2 genes, and analyzed the pattern of expression of these genes in neonatal animals following exposure to TCDD. Genomic clones encoding the entire rabbit CYPlAl and CYPlA2 genes were characterized. Restriction enzyme analysis and partial DNA sequence analysis identified the seven exons for the CYPlAl and CYPlA2 genes. Primer extension analysis using mRNA from TCDD-treated neonatal rabbits helped confirm the start of transcription for the CYPlA genes. The length of the noncoding first exon of the CYPlAl gene was 74 bases, compared to 90 and 88 bases for the human and rodent CYPlAl genes. The length of the noncoding CYPlA2 gene first exon was 53 bases, similar to its counterpart in human and rodents. DNA sequence analysis of the 5’ regulatory regions and comparison to the rodent and human CYPl genes demonstrated that the rabbit CYPlAl and CYPlA2 genes were most similar to their human orthologs. The 5’ region of the CYPlAl gene contained several consensus dioxin (Ah)-receptor responsive elements (XREs), while no functional XRE sequences were identified in the CYPlA2 gene. When expression of the two genes were monitored, a small amount of constitutive P4501Al mRNA was detected in neonatal rabbits from the ages of 1 to 17 days, while P4501A2 mRNA levels could not be observed until S’ Present address: Department of Tumor Cell Biology, St. Jude Children’s Hospital, 332 North Lauderdale, P.O. Box 318, Memphis, TN 38101-0318. ’ Present address: Department of Pharmaceutical Biochemistry, Biomedical Center, Box 578, S-751 23 Uppsala, Sweden. ’ To whom correspondence should be addressed. 0003-9661/92 $3.00 Copyright 0 1992 by Academic Press, All rights of reproduction in any form

12 days postpartum. In response to TCDD treatment, P4501Al mRNA levels were inducible at all neonatal time points, while P4501A2 mRNA levels could not be induced until the animals were 3-5 days postpartum. While the dioxin Ah-receptor most likely plays a major role in the induction of these genes by TCDD, early expression of the CYPlAl and CYPlAQ genes is differentially regulated in a developmental fashion. o 1992 Academic

Press,

Inc.

Cytochrome P45Os collectively constitute a “super” gene family, with the evolution of a large number of P45Os that participate in many different monoxygenase reactions (l-3). The functional characteristics of many different P45Os are often accentuated in animals by the administration of agents that increase the physical abundance of the proteins, a phenomenon called induction. Dependent upon the type of chemical used to enhance P450 inducibility, the mechanisms contributing to the rate limiting events have been shown to be controlled at different cellular and molecular levels. Events that are controlled at the level of gene transcription (4-7) and post-transcriptional regulation such as mRNA and protein stabilization (%-lo), have all been implicated in P450 inducibility. In adult animals, treatment with polycyclic aromatic hydrocarbons such as 3-methylcholanthrene (3MC)4 or halogenated aromatic hydrocarbons such as 2,3,7&tetrachlorodibenzo-p-dioxin (TCDD) results in the induction

4 Abbreviations used: TCDD, 2,3,7,6tetrachlorodibenzo@ioxin; 3MC, 3-methylcholanthrene; PAH, polycyclic aromatic hydrocarbon; BTE, basal transcriptional element; XRE, xenobiotic responsive element; DMSO, dimethyl sulfoxide; DTT, dithiothreitol. 707

Inc. reserved.

708

STROM,

POSTLIND.

of P4501A15 and P4501A2 (11-17). While several reports have indicated that the induction of P4501A2 by PAHs occurs by post-transcriptional events (18-20), a recent report demonstrates that the induction of P4501Al and P4501A2 mRNAs by TCDD results solely from transcriptional activation of the Cypl a-l and Cypl a-2 genes (21). Analysis of P4501Al benzo[a]pyrene hydroxylase activity and quantitation of RNA levels indicate that the induction of P4501Al can occur in many different tissues (22-27), suggesting that there does not exist tight tissuespecific control of the CYPlAl gene. The molecular events associated with transcriptional activation of the CYPlAl gene involve binding of TCDD or 3MC with high affinity to the cytosolic dioxin Ah-receptor (28, 29), followed by nuclear translocation (30-32) and binding to specific enhancer sequences flanking the CYPlAl gene promoter (33,34). Interestingly, P4501A2 mRNA has been observed only in the liver and nasal mucosa (35, 36), indicating that the regulation of CYPlA2 gene is controlled by allelic factors that influence the tissue-specific expression of this gene. Transfection and expression of the human CYPlA2 gene in human hepatic-derived HepG2 and not in the breast carcinoma MCF7 cells confirm that the expression of P4501A2 is regulated by tissue-specific factors (37). Differences have also been observed in the developmental expression of P4501Al and P4501A2 in response to induction. The expression of the CYPlAl gene has been detected constitutively in fetal rodent tissues (38) and is inducible in the fetus when the pregnant animals are administered polycyclic aromatic hydrocarbons (3840). Exposure of pregnant mice to 3MC results in a differential pattern of expression of the CyplA genes in the newborns, with P4501A2 mRNA expression detectable l-2 days following birth (41). In rabbit liver, developmental differences in the TCDD-inducible enzyme activities catalyzed by P4501Al and P4501A2 have also been reported (22,42,43), with increases in P4501Al activities occurring in newborns and elevations in P4501A2 activities not detectable until 1 to 2 weeks later in the older neonatal rabbits. P4501A2 induction was observed only in adult rabbits, while transplacental treatment by TCDD demonstrated induction in newborn rabbits of only P4501Al (42). These results indicated that rabbits offered a unique opportunity to study developmental expression of the CYPl genes, since the differential response between CYPlAl and CYPlA2 gene expression was separated by 7 to 14 days, compared to 1 to 2 days as observed in rodents. The rabbit P4501Al and P4501A2 cDNAs have previously been identified in our laboratory (12), as well as in several other laboratories (44,45). In efforts to understand the molecular events associated with the differences ’ The genes encoding cytochromes P4501Al and P4501A2 are designated CYPlAl and CYPlA2 in accordance with the proposed nomenclature for P450 genes and proteins (65).

AND

TUKEY

in the expression of the CYPlAl and CYPlA2 genes in rabbits, experiments were initiated using the rabbit cDNAs (12) to characterize these genes. Experiments have also been conducted using primer extension and Northern blot analysis to monitor the levels of the CYPlAl and CYPlA2 gene products in neonatal rabbits following exposure to TCDD. EXPERIMENTAL

PROCEDURES

Material;. Restriction endonucleases were purchased from either New England Biolabs or Pharmacia and were used as recommended by the suppliers. Ml3 Sequencing kits, Hybond-N+ nylon nucleic acid transfer membranes, d-5’-[a-s*P]CTP (3000 Ci/mmol), d-5’-[a-3SS]ATP (600 Ci/mmol), and 5’-[y-“P]ATP (3000 Ci/mmol) were purchased from Amersham. DNA polymerase, T4 DNA ligase, and oligo(dT)-cellulose were purchased from Boehringer-Mannheim Biochemical. Reverse transcriptase was purchased from Life Sciences. The plasmids pBluescript II KS/SK and phage packaging extracts were purchased from Stratagene. Nitrocellulose BA85 disk membranes used for phage DNA transfer were purchased from Schleicher & Schuell. Agarose was purchased from the FMC Corp. Treatment of animal; and isolation of RNA. Newborn New Zealand white rabbits from Simonson Laboratories (San Diego) were purchased at different ages. 2,3,7,8-Tetrachlorodibenzo-p-dioxin was dissolved in DMSO at a concentration of 150 nM and suspended in corn oil prior to injection. Rabbits received a single intraperitoneal injection of 10 pg/ kg TCDD and were sacrificed by cervical dislocation 12-18 h after treatment. The livers were purfused with an ice-cold solution of 1.15% KCl. From 2-5 g of liver, total RNA was isolated by the guanidine-HCL procedure (46). Poly(A)+ RNA was prepared by oligo(dT)-cellulose chromatography (47). Selection of probes. The rabbit liver P4501Al and P4501A2 cDNAs were isolated from a pBR322 library (48), and regions of each cDNA subcloned into pBluescript KS vectors (Stratagene, La Jolla, CA). Recombinant cDNA clones encoding the full-length P4501Al and P4501A2 mRNAs have been characterized (44, 45), and based upon homology comparisons between the two cDNAs, specific 5’ oligonucleotides were synthesized. The 5’-specific P4501Al oligonucleotide 5’-ACCCGAGGTTTTGAGGCTCC-3’ and the B-specific P4501A2 oligonucleotide 5’-ACCTTAGGCCTCGAGGCCCT-3’ were used in primer extension analysis and as probes to detect RNA by Northern blot analysis. Northern blot analysis. Analysis of P4501Al and P4501A2 transcripts were determined following electrophoresis of poly(A)+ on 1% denaturing agarose gels containing formaldehyde (49). Following transfer of the RNA to Hybond-N+ as described by Thomas (501, the filters were hybridized with nick-translated 3’-specific cDNAs (4) or with end-labeled 5’-specific P4501Al and P4501A2 oligonucleotides as described (51). Isolation of genomic clones. DNA was isolated from rabbit spleen (52) and a genomic library constructed in the X replacement vector XEMBL 3, as previously described (51, 53). Escherichti coli K802 was transfected with the packaged DNA, and the amplified libraries were screened with nick-translated cDNA inserts from the rabbit liver P4501Al and P4501A2 cDNAs. From approximately 5 X 10’ recombinant phage clones, an initial screen was conducted for 3’-specific P4501Al and P4501A2 cDNA clones, and over 20 recombinant clones were purified. Restriction enzyme and Southern blot analysis with 3’ and 5’ portions of the cDNAs indicated that the entire CYPlAl gene had been cloned in a single fragment. To identify clones that encoded the CYPlA2 gene, the P4501A2 5’-specific oligonucleotide and 5’ portions of P4501A2 cDNA were used to purify recombinants that encoded the 5’ regions of the gene. Restriction enzyme analysis and partial DNA sequence confirmed that clones XlA2A and XlA2B overlapped and encoded that entire CYPlA2 gene. Primer extension analysis. The 5’ P4501Al and P4501A2 complementary oligonucleotides were end-labeled with T4 DNA polynucleotide

RABBIT

CYPlAl

A). CYPl Al Gene a cl w

AND

CYPlA2

AND

P 5 a!

s a

z 8 w

I

XYPl Al I ’

DEVELOPMENTAL

?Pa E 58 mmw

709

ra E% h!iLz w8

I

I

EXPRESSION

i H5OObp

1’

I

I “b

I *500bp -“c,

5. -

B) CYPlA2 Gene hCYP1 A2-A I

EYPl A2-B I

FIG. 1. Restriction endonuclease analysis of X-EMBL 3 clones encoding the CYPlAl and CYPlAZ genes. Using 5’ and 3’portions of the rabbit P4501Al and P4501A2 cDNA clones, three genomic clones were isolated and characterized by restriction enzyme analysis and partial DNA sequence analysis. DNA fragments from each gene were subcloned into pBluescript vectors for DNA sequence analysis. Location of exon sequences for both genes were identified by Southern blot analysis and DNA sequence. Boxes represent the position of oligonucleotide primers that were generated from cDNA sequence data (12,44) and which were homologous to the CYPlAl or CYPlA2 exon gene sequences, while slashes represent the start of the DNA sequence from individual subclones.

kinase in the presence of [y-32P]ATP. Approximately 1 X 10’ cpm were added to 3 pg mRNA in 30 ~1 10 mM Tris-HCl, pH 7.5, containing 1 mM EDTA and 0.25 M KCl. The samples were heated to 90°C for 2 min and incubated at 37°C overnight. After the annealing step, 170 ~1 of 0.3 M sodium acetate was added and the RNA precipitated with 500 al of EtOH. The pellet was rinsed once with a solution of EtOH/O.l M sodium acetate (3:l) mixture and then air dried. The pellet was resuspended in 25 pl of a reaction mixture containing 75 mM Tris-HCl, pH 7.5,12 mM MgClz, 15 mM DTT, 3 pg/ml actinomycin D, 0.75 mM each of dATP, dGTP, dCTP, and d’l.TP, 10 units/ml Rnasin, and 5 units/ml reverse transcriptase, and incubated at 37°C for 30 min. The reactions were terminated by adding 1 ~10.5 M EDTA and 181 of 1 mg/ml pancreatic ribonuclease A, and incubated at 37°C for 30 min. A total of 100 ~1 of 0.3 M ammonium acetate was added and extracted with 125 ~1 of phenol/ chloroform/isoamyl alcohol (5/4/l), and precipitated with 300 pl of EtOH. The pellets were resuspended in 6 ~1 of 60% formamide containing 5 mM NaOH, boiled, and subjected to electrophoresis in 7.5% acryiamideDNA sequencing gels. To determine the length of the extension products, a single-stranded M13mp18 vector was sequenced using the universal primer. DNA sequence analysis. Restriction fragments derived from the genomic clones were isolated and cloned into pBluescript KS or SK. The DNA sequence was generated directly from the double-stranded plasmids by the chain termination method (54,55).

RESULTS

Characterization of rabbit CYPl Al and CYPlA2. A restriction endonuclease map of the X-EMBL3 genomic clones encoding the CYPlAl and CYPlA2 genes and the

sequencing strategy of the different subclones is shown in Fig. 1. While DNA sequencing proved to be useful in positioning the location of several of the introns and exons, it was performed to conclusively identify the CYPlAl and CYPlA2 genomic clones. Southern blot analysis using the cDNAs demonstrated that the CYPlAl and CYPlA2 genes were single-copy genes in the rabbit genome (data not shown), but due to the high sequence homology between the cDNAs (>80% within the coding region), we felt it important to conduct sequencing at different exon positions to confirm the identity of the genes. DNA sequence analysis across the different exon-intron junctions revealed the presence of the consensus GT-AG RNA splice sites. The positions of the exon-intron junctions that were not sequenced were determined by restriction enzyme analysis and Southern blot analysis using different portions of the rabbit P4501Al and P4501A2 cDNAs. The partial DNA sequence analysis of the CYPlAl and CYPlA2 gene subclones matched that of previously published P4501Al and P4501A2 cDNAs (12,44,45). As determined for other species, both rabbit genes contain six coding exons, numbers 2-7, and a single 5’ noncoding exon. Identification of the transcriptional start site. To determine the length of the rabbit liver P4501Al and P4501A2 mRNAs and locate the start of transcription on

710

STROM,

POSTLIND,

the CYPlAl and CYPlA2 genes, oligonucleotides were generated to the published sequence (44) and employed in primer extension experiments. When the two mRNAs were aligned for homology comparison, a divergent region 90 bases 3’ from the initiation methionine codon ATG was used to generate antisense oligonucleotides to the mRNAs. The P4501Al oligonucleotide 5’-ACCCGAGGTTTTGAGGCTCC-3’ recognized bases 205-186 and the P4501A2 oligonucleotide 5’-ACCTTAGGCCTCGAGGCCCT-3’ recognized bases 151-132 (44). Alignment of the two mRNAs showed a mismatch of 7 bases between the two oligonucleotides, which would allow for specific annealing of the respective oligonucleotides to the mRNAs. Since the expression of CYPlAl and CYPlA2 responds differently in neonatal rabbits to the actions of TCDD, poly(A)+ selected RNA from 3- and 21-day-old TCDDtreated and untreated rabbits were used as templates in primer extension experiments (Fig. 2). With the P4501Al primer, two major extension products of 210 and 212 bases and a minor product of 221 to 222 bases from animals treated with TCDD were observed. No extension products were observed using mRNA from untreated rabbits. When the P4501A2 oligonucleotide was employed, two extension products of 157 and 154 bases was observed when mRNA from the 21-day-old TCDD-treated rabbit was used as a template. There were no detectable extension products when mRNA was employed from the 3-day-old TCDDtreated rabbits. Since the same preparations of mRNA were employed for primer extension with the P4501Al and P4501A2 oligonucleotides, the differences in detection of P4501Al and P4501A2 gene products between 3-dayold rabbits treated with TCDD confirms that developmental factors contribute to the inducible expression of the rabbit CYPlA2 gene. To localize the start of transcription for the CYPlAl and CYPlA2 genes, the length of the extension products as determined from the M13mp18 sequence was compared to the DNA sequence generated for the first exon and some 5’ flanking DNA of both genes. For the CYPlAl gene, the major 210- and 212-base-pair extension products aligned with cytosine residues, while a faint 221- to 222base-pair extension product corresponded to either a thymidine or a cytosine residue (Figs. 2 and 3). The two CYPlA2 gene start sites both corresponded to cytosine bases (Figs. 2 and 4). When the positions of the transcriptional start sites were compared to the published sequence of the P4501Al and P4501A2 cDNAs (44,45), 57 bases of the P4501Al and 3-6 bases of the P4501A2 mRNAs were not represented in the previously published sequences. Both the CYPlAl and CYPlA2 genes from all species contain a 5’ noncoding first exon. In the human (56) and rodent (57,58) CYPlAl gene, this exon is 90 and 87 base pairs, respectively, while in the rabbit, the noncoding exon is only 74 base pairs (Fig. 3). Allowing for gaps, exon 1 of the rabbit CYPlAl gene is 70% similar in sequence to

AND

TUKEY P4501Al

M-13

P4MlAZ

1234ACGT5678 CYPl.41 CAPSrrE

SEglJENce CYPlA2 CAP-SITE sEglmNce

: E 221, T \ : :

221L 112 210-

C : A

212-

E

210-

P : t / G

FIG. 2. Primer extension analysis using P4501Al and P4501A2 mRNA. Approximately 3 pg of mRNA from 3-day control (lanes 1 and 5), 3-day induced (lanes 2 and 6), 21-day control (lanes 3 and 7), and 21-day induced (lanes 4 and 8) was annealed to the P4501Al-specific oligonucleotide 5’-ACCCGAGGTTTTGAGGCTCC-3’ (lanes l-4) or the P4501A2-specific oligonucleotide 5’-ACCTTAGGCCTCGAGGCCCT-3’ (lanes 5-8). A sequencing ladder was generated using M13mp18. The corresponding CYPlAl and CYPlA2 gene sequences encoding parts of exon 1 were generated from the X-EMBL 3 genomic clones. The identities of the transcriptional start sites were calculated based upon the length of the extended RNA products, the identity of the exon-intron boundary flanking exon 1, and the homology of the exon 1 DNA sequence with that of cDNA sequence published for the P4501Al and P4501A2 cDNAs (44, 45).

the its counterpart in rodent and human. The rabbit CYPlA2 noncoding first exon is 53 bases (Fig. 4), in comparison to 53 for the human (37) and 51 and 52 base pairs for the mouse (57) and rat (59), respectively. The first exon of the rabbit CYPlA2 gene is 70% identical to that in the rodent and human CYPlA2 genes. Comparison of 5’ flanking DNA sequences. Approximately 1 kb of DNA was sequenced in the 5’ direction of exon 1 for both the CYPlAl and CYPlA2 genes, and DNA alignments were performed against all the sequences in the Genbank DNA sequence data bank. The rabbit CYPlAl gene sequences were 60 and 52%, similar to the flanking sequences of the human and rodent CYPlAl genes, respectively. An alignment of the rabbit and human 5’ flanking sequences of the CYPlAl gene is shown in Fig. 5A. Several regions of high sequence similarity are observed with the CYPlAl genes (56,60). The first region (83% homology) is from -1 to -66 and contains the prox-

RABBIT

CYPlAl

AND

CYPlA2

AGTiGGCTGTCGCiGGCCTCcAAicccTTc&&cTccT~GGcTGc~GG

AND

-901

TCTCCCCGGCCAGGTGGCGCE;GGGAGGGGCSGCCACCAtCCCTGCA

-821

GGTGCTCCGACGCGGGCGGGE;GAGCtTCG~ATTCCCCCC~CGCACCCTG

-781

GCCGGTCCCGGGGGGCCCCCiCTAGCCCTTGCGCTCTGTA~CCCCCGC~GTACTAGCG

-721

ACCCCCTGGCCTCCTTCCTGGACCCAGGGGCAACCTCGAG~GCCAGGGTC~GGCGGGAGG -661 TGGCCCTGGGiTGCCACGCGCCTGACCCCGGAGGCGGCGC~CGGAGAGCC~GCGCGACCC -601 TCGGTCCGTGAGCGCGACGTiCGCCGGGTCGTCGCGCCCT~GGGGACGGG~CCAGCTCAG -541 CGGCGCCTCTGGCCTTCCGGCGGCCGTGACCTCGGGCACA~GGTCGCTGA~CTTC~

-481

~GCAGGcncTCAGTAnCCCCGGGGGAGCGGTCGCCACG~GCAGCCC~GCCTGG

-421

CCGGTTGGCTbGGCAGCGnniGGGGGAAGGGGGACTGGCA~CGCCGTGTC~CGGTCGGTG

-361

CAAAAATGATGTCGGGAGATcCGGATCTCCCGACGGAGCC~CTCAGGCCC~CTTTTCCGG

-301

GTGAGCTCCAbCTTCTGCCTSAAACCCCCGCGATGCATAACTGGG

-241

GGTCCCAGGTATCCGCAGCCiCCACCCCTTATGTGCAGGC~CCTAGATGA~GCCTCCCTG

-181

TGGAGGTCTTliTCTGTCCCCiCCTCCCCCTiCTCGTCCCA~CCTGGAGGG~CCAGCCCC~

-121

SPl

DEVELOPMENTAL

711

EXPRESSION

close to the TATA box region between -175 and -1. Shown in Fig. 5B is the alignment with the human CYPlA2 gene. Two direct repeats are present at -70 and -90 bases from the start of transcription. The distal repetitive element is completely conserved between human and rabbit, while the proximal repetitive element shares 8 of 10 bases in common with the human gene. Although the approximate position of these elements is conserved in rat, mouse, human, and rabbit, these sequences are less conserved in the rat and mouse CYPlA2 genes (data not shown). There is no known function for these repetitive sequences. In addition, a consensus XRE sequence (CACGC-A) is present on the rabbit CYPlA2 gene at bases -493 to -499. This is notable since the rat, mouse, and human CYPlA2 genes do not contain consensus XRE elements. To determine if the 5’ flanking region of the rabbit CYPlA2 gene contained elements that could be recognized by the

~GCAGCcCTCCCTCTCcCCCCCACCC~CCGGGGCAG~CCCTCCCTC~CCCCTCCAG

-61

TAnAGGAAGGCGTGGCCACAcGCAGCCTCCiATAAAGTGCCC~~GCCCTCA~C

-1

GTGAGTGCAbGTGAGTGTTCCTTTCACTCCCTGTGAAAGGCATTTTG~TGCCTACAGG

t134

-821

GTTTCCTAGGGAGCAGCAGAGGCCGGGATiGTCCTCAGTGGCCAGTCAG~GCAGCCCCCA

+I94

-781

GTAGGGCACiGAGGCCGGGbGAAGGACAGGACTTAGCCCAGTTCCTACG~CGCGTCCCAG

t254

-721

ACCTGTGCGiACAGACACCAGCCCTGGTCGGGACTCCAC~GGCAGCTCC~GCTCCATGCC

t314

-661

CACACACCCiGGGCAAGCTT

t334

-601

-901

FIG. 3. DNA sequence of the 5’ region of the CYPlAl gene. Part of intron 1, exon 1, and 950 bases 5’to the start of transcription are shown. The location of exon 1 is shown in bold and the arrows indicate the bases represented as the probable start sites for transcription, as demonstrated in Fig. 2. Two dioxin Ah-receptor enhance sequences, termed XREs, and consensus SPl sites are shown. The presumed TATAA box is shown in bold and underlined. The asterisk (*) indicates the position of the furthest 5’ base identified in the rabbit P4501Al cDNA (44).

-541 -481 -421 -361 -301 -241 -181 -121

imal promoter elements. The second region (84% homology) is from -428 to -560 and contains a proximal dioxin Ah-receptor enhancer sequence and one SPl binding site. The final region (80% homology) is from -874 to -953. This region contains the distal XRE of the rabbit CYPlAl gene. The TATA box regions are very similar, and the BTE (61), or basal transcriptional elements, are also conserved. Along with the XRE sequences, the BTE were found to be crucial for high inducible expression of the CYPlAl gene in the presence of polycyclic aromatic hydrocarbons (61). Interestingly, a region between -645 and -800 of the human CYPlAl gene has been shown to contain two negative regulatory elements that are similar among the rat, mouse, and human CYPlAl genes (62), but are not conserved in the rabbit CYPlAl gene. The highest degree of similarity with the rabbit CYPlA2 gene occurred in primarily one region when compared to the human and rodent CYPlA2 genes (37). While there was little sequence similarity between bases -905 to -175, there existed a high degree of similarity

-61 -1 1

1

l

CCGCCGTGGACAGCCCCCACAGCCCCGCCGATCTWU\GCACAG

t53

t113 t173 CTCCATTTTCCAAGGAGCGbTTTAACCTAGGACATCTCCAGGGTCCCT~CTCGGAGTGG

t233

GGGAGAGGAbAGGAGGGTCtTTGCTAGCCACGAGGGTGGCG~CTGCTGCCCA

t293

ACCCCAGCCiCCCAGGCTGiGATGGACAG~GACTCACTC~TCATTGGGG~CGGAGGGAGG

t353

CACCCTTCCiGGTATGGACcAGAAGCTGTiTGTTGCAGC~CTGGGACCT~GCCTCCTCCG

t413

TGTCACTGGGTGGGG

t428

FIG. 4. DNA sequence of the 5’ region of the CYPlA2 gene. As in the previous figure, the DNA sequence of part of intron 1, all of exon 1, and some the 5’ flanking DNA is shown. The arrows indicate the probable position of transcriptional initiation of the CYPlA2 gene. An SPl and a potential dioxin Ah-receptor enhance XRE sequence are underlined. In addition, two identical sequences that are repeated, REl and RE2, are also shown. The asterisk (:) indicates the position of the furthest 5’ base identified in the rabbit P4501A2 cDNA (44).

712

STROM,

POSTLIND,

AND

TUKEY RRE

-547

GCTGTCCTTGGCAGCCTGTGAGAGATTCTGTGTGCTGGCTCT

-563

TATGTC

-487

GGC~TCTCCGGAGCAGCCCTTGTCTATTCTGGCCCAGCGATTCTGGCCG~GCC

A

IIII

I

lllll

I II

TCTTAGTCTTTTTG

IIII

I

CATAAGGGGGGC

III

IIllIllll

CTCTCTTTAGGATGCAAAATCT

SPl

I Illll -509

TTATCATC

-915

I I ATCCGCCCATCTCGGCCTCTCAAAGTGCTGGGATTACA

-845

ATGACAGATAAAGA

II

Ill1

II

III

TGGTGGATAGA

III

IllllllI

TAGGTAGACAGAGGATGGACAGATAATCATCT

I III1

I

II

llllll

CCTAGAGGTGTACATTTTTTMCAGMCCATTCMMGCAGGTTGTGGGGATCATGA

-789

CTTATTGAGAGATGATTGAT

I

II II

CA

AAATGATAATAGATGGATGACAGGGAGCGATGATAGAGC

lllll

IIllllII

II

I

-367

AGGAACGTGAGGGMCTTTCTAGAAGGCT

TTCCATGCTACAGCATTAATCTCCTAAGAATA

-725

ATTTGMAGCAGGCTGCCGGTGTCACGGCACTCCACCCCTAACCAGCACCCCCCTGTCC

Ill

II

III

AGGATACACTCCCACATACCAT

II

II I

GACA

lllll

III CCATTTA

IIIII

IIIII

lllll

I II

II II I

CTGTTTGGTTTTGCAGGTTGTTGGAGGGTACTACA

I

CAGGCCMGA

IIII

GMGA

TGCGGAGCCGGCTGGAGT

llllllIllI

III

III

-400

ACATCCCTCTGGAGAGGGGCCGTGACCCTGGTTGGCCTAGACTGAGTGCCCTGGCA~GC

-310

CATAGGTTGTTGGGGAGGTGGCACTAGAGGGGCAGCGTGGGCCCCTG~C~GTGCC

I

I I II

lllll

IIII

-340

TCTTCCTCATGTGTGCAGTGGGA

-251

GCAGGGACCCT CC

I

CTCT

lllllIll

III

III

C

lllll

III

MGMGCCCAGATCAGTCCAAAGGCCTAACCCCCAC GGGGACA GCGCCCACT CCCAGCCCCCT

IIII

IIII

llIllllII

-281

TCCCAGACCCTACCCTACTCTTCAGAGMATAGGCTCCCIAG

-201

C

CCTGGAGGGMGCGGATCC

I

IIII

I IIII

-797

III AGTGGC

TGGMCACGC

III II I I

-056

lllll

-457

I

GGTGCGAGCCATCGCGCCTGG

II

CTTGGGCTGAMATCAGG

CTCATGTCGCACGAGCMCCTCATTTGCTCTTTAGTTTCTCACTGTGTCTCCCAG~GGC

I CGATCCATAGCMGACAGATTGATAGGTAGACAGAGCATCCCAG

I III

CACTTGGGGA

-427

I

-905

I Illll

TCTTCTTTTC

GGCCTG

IIII

G ATCCTCTCTGGCCCAGCCCCAGGGAGCTGGCCT

llIllIIll

IIIIIIIIIIIII

III

CTGTACCTTCATCCCCAGGGACCCAGCACCCCTTCT~CCTATCCCC~~GTCACCCT

-147

GGG CCTGGGGAGTGGGCGGACGTGAGGGCTCATGGCCCAAAGCCGAGGGTTGA

E

IIIIIIIIIIIIIII

II

IIIIIIIIIIIIIIIIIIIIll1ll

-739

CTCTGTTCACACCTMAAAMTTTACATTTATTCCAG~TATCATCTMTCTCCAGTCCG

-161

GGGTCTTAGGTAGTAGGTGGAGCTGAGGGATAATGGCCC

-666

ACAAACCAGGGTACCCCTCACACCCGTGCTGCTGTGTGCAGA

-92

UACTTTGTTCAGTGATCTTGATGTCAGGTG

ACCAGGWTGA

IIIIIIIIIIlIIIIIIIII

llllllllll

RE 1

III

II I

III

II

III

II I

-679

TGCTTACATGTCCCCMTTGTCCCCMMCATCTTTTATCTTTTATAGATTTTTTTMMTTTTGTT

-607

TCAGCCCAGGATGCCGTCTCCCGGCCACGGTCACGGTCACATGTCCCCAGTTCTCCCGGA

II -619

I

III

II

III

TAAATGCCATATCCMTCGATATGGCA

III

III

ATCAAATGCAAA

III

III

MGGCMGAGTTGATCCT~

REZ

III1 II lllllll

-102

CAACTTTGTTCAGTGATCCAGClTTUTATCAGGTGATCAGGACAACCAGG~CTGA

-41

TAGGTGGTAGGGAA~TGCCCC

II

TCCATATTGCATTTGGT

III

-221

I Illllll

AGGMGMGACCAGCCC

III

IIII II I I llllll -42

CTCGGCCGGAGCCTG

II III

I lllll

TAGGGGGCGGTGTT~GGCCACTCACCTAGAGCCAG

IIII

R&bitCWlAZ

I ~cYPlA2

FIG. 5. Alignment of 5’ sequences of the rabbit and human CYPl genes. (A) Using the NALIGN program in PCGENE (IntelliGenetics, Inc.), the 5’ flanking DNA was aligned to that of the human CYPlAl gene. The conserved XRE sequences are outlined, as well as an SPl site. Two negative regulatory sequences, NREl and NREZ, are underlined in the human sequence. (B) An alignment of the rabbit and human CYPlA2 genes. Potential regulatory sequences identified in the rabbit sequence are underlined. REl and RE2 represent the location of two repetitive sequences in the rabbit CYPlA2 gene.

dioxin Ah-receptor, a portion of DNA containing the rahbit CYPlA2 gene promoter and 2000 bases of flanking DNA was placed 5’ to a chloramphenicol acetyl transferase gene and transfected into human HepG2 cells. When the cells were treated with TCDD or 3MC, no induction of chloramphenicol acetyl transferase activity could be detected (data not shown). This would indicate that the XRE sequence identified on the CYPlA2 gene does not serve as an enhancer sequence to modify the transcriptional activity of the gene during exposure to polycyclic aromatic hydrocarbons. Developmental expression of P4501Al and P4501A2. To examine in greater detail the developmental expression of the CYPlA2 gene as observed in the primer extension studies, neonatal rabbits of different ages were examined

for their ability to regulate P4501Al and P4501A2. Northern blot analysis of mRNA selected from rabbit liver RNA was analyzed to determine the levels of P4501Al or P4501A2 mRNA, as shown in Fig. 6. In rabbits that were untreated, a minor amount of constitutive P4501Al mRNA could be detected at all ages, with a substantially higher abundance observed in rabbits that were 21 days and older. In contrast, P4501A2 mRNA was not detected in newborns, but could be observed in rabbits that were about 12 days old. To examine the ability of TCDD to induce the CYPlAl and CYPlA2 genes, P4501Al and P4501A2 mRNA levels were analyzed from neonatal rabbits of different ages following treatment with TCDD (Fig. 7). At all ages, induced P4501Al mRNA could be detected. However, using the

RABBIT

CYPlAl

AND

CYPlA2

AND

DEVELOPMENTAL

713

EXPRESSION XRE

-522

GGCGGCCGTGACCTCGGGCACAGGGTCGCTGAGCTTCTUC

-538

CCC CCCGTGACCTCAGGGCTGGGGTCGCAGCGCTTCTCkCGCbCCGGGACTCAGTAA

-462

CCCCGGGGGAGCGGTCGCCAC GGGCAGCCCWCCTGGCCGGTTGGCTGGGCAGC

-479

CCCCGGGMGGAGGTCACCACGGGGCAGCCC~CGCCTGCCGAGT

III

B

IllllllllI

lllllll

II

I IIIIIIIIIIIIIII

IIIIIIIIIIII

SPl

Illllll

I IIII III1 IIIIIIIIIIIIIII

I IIII

I IIII

II

CCTGGTAGGC

XRE -953

AGTTGGCTGTCGCGGGCCTCCAAGCCCllCT

I llllll

IIIIIIIIIII

CACGCAACTCCTGGGCTGCGGGlClCCCC

I IIIIIIIIIIIIII

Illllll

-1014

GGGTGGCTG CGCGGGCCTCC GGTCCTTCTCNXCMCGCCTGGGCACCGCGCCT

-893

GGCCAGGTGGCGCGGGGAGGGGCCGGGCCACCTCTGGCCTCTGCAAGCCT

lllllIllll

lllllll

llllll

IIIIIIIllll

II

II

I

I I IIII

CGCCGTGTCCCGGTCGGTGCAAAAATGATGTCG

IIIIIIIIIIIIIIIIIlIIIIIIIII

I

-420

TGTAGCGCTGGGGAGGCATCTGCACGCCCAGCGTTCCACTATGACGAAGA

GCAGGTG

-346

GGAGATCCGGATCTCC CGACGGAGCCCCTCAGGCCCCCTTTTCCGGGTGAGCTCCAGCT

-360

GGAGTCCCCGCGCCCCAGGATGGAGCTTCCCGTACCCTCTCTTCGGGCTGTCCTGGGACT

-287

TCTGCCTCAAACCC

CCGCG

ATGCA T

III llllll

II II

IIII I

III1 III

IIIII

GGCCAGGTGGGGCGGGGACGGGCCGCCTGACCTCTGCCCCCTAGAGGGATGTCGCCGGCG

-836

CTCCGACGC

G GGCGGGGG AGCCTCCGCGCCCCTTCGCA

II

IIllllIIII

GCAC

TTCCCCCCTC

II I Illllll

I Illll

GAATGGGGGMGGGGGACTGGCAC

CCC

-957

III

-403

II I II II

I I

III

Illllll

Ill

II

IIIIIIIIIIII

II

II

AAGGGGGTGCCTTGTAACTG

III

llllll

II II

-897

CACGCAAGCTAGAGCCGGGGGTAGGGTGGGGGCTCCGCGCCAGGTGCCCCCTCCGTGGTC

-300

TCTCCCTCAAGCCCCCTCCTCGGCTGGGTTCTGCACTGCCCTTGGGACGCCTTGGAATTG

-784

CCTGG

CCG GTC CCGGGGGGCCCCCTCTAGCCCTTGCGCTCTG

-242

GGGGTCCCAGGTATCCGCAGCCTCCACCCCT

TATGTGCAGGCACCTAGATGAGGCCTCC

Illll

III III

II I IIIIII

lllll

I lllllll

III

TAGCCCCCGCAAA

lllll

II

IIII

-837

CCTGGGCCCGAGTCTTTCCGTGGCCCCCCGC CGCCGGATTTCTGTGCTCTGCCAATCAAA

-729

GTACTAGCGACCCCCTG

I I lllll

lllllll

llllllll

Illll

I

III TCC

-240

GGACTTCCAGGTGTTCCCAGCCCTCACCCCTCTATGTCAGGCACCGAGATGTG

-183

CTGTGGAGGTCTTGTCTGTCCCCTCCTCCCCCTTCTCGTCCCACCCTGGAGGGCCCAGCC

-183

CATAGTCGG

-123

CCCGCCCCGCAGCCCTCCCTCTCCCCCCCACCCACCGGGGCAGCCCCTCCCTCCCCCCTC

-126

CTCCAATCCCAGAGAGACCAGCCCGGTTCAGGCTGCTTCTCCCTCCATCTCAGCTCGCTC

-63

CAGTAAAGGAAGGCGTGGCCACACG

C AC CCTCC-GGTGGCAGTGCCCTGGC

III

I II II lIIIIIIIIIIIIIIIIIII

RRE 1

I Illlll

lllll

GCC

TCCTTCCTGGACCCAGGGG

II II II II

I III

III

CAACCTCGAGG

I

I

II III1

-777

GCACTAGCCACCCCCTCAAGGGCCGGTGGGTCCTGGCTGGAGG

-679

GCCAGGG

TCCGGCGGGAGGTGGCCCT G GGTTGCCACGCGCC

TGA

I I I I

II I

III

I II IIIllllII

lllll

II

II

IIIIII

I

TTCT TGCCCACCCGACCCCCCACCCCCGCCGCCCTCCGCCACCTTTCT

RRE 2

-717 -635

IIII

IIIIIIIIIIIIII

GACCGCGCGTTGCAATCAGCACTAAGGCGATCCTAGAGGCTGCGAGGAGCCGCTAGTGAG CCCCG GAGGCGGCGCC CGGAGAGCCCGCGCGA CCCTCGGTCCGTGAGCGCGACGT

IIIlllIllll

I III

III II II II

IIIIII

-657

CGCTCAGCGAGCCTGCCCCTTCGCCATCCATCCTCC

-580

TCGCCGGGTCG TCGCGCCCTGGGGGACGGGGCCAGCTCAGCGGCG CCTCTGGCCTTCC

II I IIII -597

II II llIllIllI

II lllll

TCAGCTAGTCGCCCGGGCTCTGGGGGACAGGTCCAGC

I llllll

I I

II

Ill1 IIIIIIIIIIIIIII

II

II I I

I

lllll

-66

CAGGGAAGG AGGCGTGGCCACACGTACAAGCCCGCC~GGTGGCAGTGCCTTCAC

-7

CCTCACC

Rabbit

IIIII

I I

CYPlAl

IIIIIII

IIllIIIIIIIII

CCGCGGCGCCCTCTGGCCTTCC

I III

-7

CCTCACC

W

CYPlAl

FIG. B-Continued

same preparations of mRNA, mild induction of P4501A2 mRNA was observed at 3 days with a significant increase in mRNA observed at 5 days. While both P4501Al and P4501A2 mRNAs are enhanced in neonatal rabbits following exposure to TCDD, these results indicate that developmental factors onset after birth appear to play an important role in controlling the inducible expression of the CYPlA2 gene. DISCUSSION

The rabbit CYPlAl and CYPlA2 genes are structurally similar to their homologs in rodents and human. Each gene contains six introns and seven exons, with the first exons consisting of 5’ noncoding DNA. Primer extension analysis using an mRNA and DNA sequence of exon 1 and the 5’ flanking DNA demonstrated that the length of the exon 1 for both genes was comparable to their counterparts in other species. DNA sequence

analysis of 5’ flanking regions of both genes identified several putative c&acting elements involved in transcriptional regulation. The CYPlAl gene contains several XRE elements (33,34) and the BTE sequence (61), both necessary elements that appear to be important for transcriptional activation by the dioxin Ah-receptor. While the flanking region of the rabbit CYPlAl gene is similar to that of the mouse and rat CYPlAl gene, it exhibits 60% sequence similarity to the human yet only 52% similarity to the rodent CYPlAl genes. In contrast, just the promoter region of the rabbit CYPlA2 (bases -1 to -187) gene conveyed a high degree of sequence identity to other species, indicating that this region may be important for overall expression of the gene. While regulatory sequences involved in the tissue-specific expression of the human CYPlA2 gene have been identified (37), transfection and the lack of expression of the rabbit CYPlA2 gene promoter region and 2000 bases of flanking DNA linked to a reporter gene indicate that

714

STROM,

AGE Jdays)

2

4

8 12 15 1721

2328

POSTLIND,

A

P4501Al

P4501A2

actln

P4501A2

AND

TUKEY

the rabbits were 3-5 days old. In addition, when pregnant rabbits were treated with TCDD, the newborn animals displayed large increases in only P4501Al mRNA (data not shown), consistent with some previous reports that only P4501A2 is induces in hero (38, 40). The delay in the ability of TCDD to induce rabbit liver P4501A2 mRNA is consistent with previous observations on the regulation of this gene product in rodents and rabbits (13, 63). It has recently been proposed that the constitutive expression of the CYPlAl and CYPlA2 genes in the late neonatal stages of development is triggered by dietary changes following weaning (64). For most of the experiments that we conducted, the induction of the CYPlA2 gene by TCDD could not be elicited until the rabbits were about 5-8 days old, approximately the same developmental time period which proceeds constitutive expression of the CYPlA2 gene. The parallelism between constitutive expression and the ability to induce P4501A2 mRNA with TCDD indicates that the developmental onset of certain transcriptional factors necessary for expression may be a prerequisite for induction by TCDD. Since the CYPlA2 gene is transcriptionally regulated by TCDD and PAHs in a dioxin Ah-receptor dependent fashion (6, 21), the experiments that we have conducted in rabbits suggest AGE (days) TCDD

1

1 +

3

3 +

5

5 +

21 21 +

FIG. 6. Northern blot analysis of P4501Al and P4501A2 mRNA in untreated neonatal rabbits. (A) Poly(A)+ RNA was isolated from rabbits at different ages as described under Experimental Procedures. mRNA (1 pg) from each sample was electrophoresed under denaturing conditions in a 0.8% agarose gel, transferred to nitrocellulose, and hybridized with either a 32P-labeled 3’ P4501Al cDNA clone (12) encoding bases 17412022 [equivalent to bases 2047-2327 (44)] or a 3’ P4501A2 clone (12) encoding bases 1282-1746 [equivalent to bases 1603-2084 (44)]. After stripping the Northern blot, it was hybridized with a human actin cDNA probe (66). (B) Overexposure of lA2 Northern blot in A.

similar responsive elements are not present on this region of the rabbit CYPlA2 gene. As a function of age, the constitutive expression of the CYPlAl and CYPlA2 genes as measured by RNA abundance show slightly different patterns. In all of our experiments, a small amount of P4501Al mRNA was detected in l-day-old newborns, followed by a significant increase in mRNA accumulation in 17-day-old animals. We were unable to detect any constitutive expression of the CYPlA2 gene product until rabbits were 8 to 12 days old, followed by steady increases to adult levels by 21 days. The most striking differences in developmental expression of the two genes were observed following treatment of neonatal rabbits with TCDD. P4501Al mRNA was enhanced by TCDD treatment at all ages, while induction of P4501A2 mRNA did not occur until

actin

I

FIG. 7. Northern blot analysis of P4501Al and P4501Al mRNA from neonatal rabbits treated with TCDD. Rabbits at different ages were treated with 10 rg/kg TCDD for 12 h, and mRNA was isolated from the liver. Untreated rabbits at the same age served as controls. Using 1 pg of mRNA, two Northern blots were prepared as described under Experimental Procedures. One was probed with a P45OlAl-specific oligonucleotide, 5’-ACCCGAGGTTTTGAGGCTCC-3’, and the other with a P4501A2-specific oligonucleotide, 5’-ACC’ITAGGCCTCGAGGCCCT3’. After autoradiography for 2 days, the filter probed with the P4501A2 oligonucleotides was stripped and hybridized with the human actin cDNA.

RABBIT

CYPlAl

AND

CYPlA2

AND

that the CYPlA2 locus is most likely in an inactive state until the stages of middle to late neonatal development. Developmental maturation of the CYPlA2 gene to a transcriptionally active state may facilitate the ability of the dioxin Ah-receptor to promote transcriptional activation of this gene. However, the developmental restrictions on the CYPlA2 gene can be overridden by larger doses or repeated treatment with PAHs to pregnant animals (39), suggesting that the actions of the dioxin Ahreceptor on regulating the CYPlA2 gene can mimic those of the CYPlAl gene. While active dioxin Ah-receptor enhancer XRE elements have not been identified on the CYPlA2 gene from any species, transcriptional activation by TCDD in neonatal and adult liver (21) most likely proceeds by an association of the nuclear dioxin Ah-receptor with &-acting enhancer sequences in a fashion similar to the transcriptional activation of the CYPlAl gene. Only in extrahepatic tissues, where the induction of the CYPlA2 gene by inducers does not occur (21), tissue-specific regulatory factors play a dominant role in suppressing activation of this gene. Although inducers such as TCDD and PAHs interact with the dioxin Ah-receptor to promote transcriptional activation of the CYPlA2 gene and induction of P4501A2, other allelic influences such as developmental and tissue-specific factors underlie the expression of this gene. ACKNOWLEDGMENTS This work was supported in part by USPHS Grant CA37139. R.H.T. is a recipient of a Senior Faculty Research Award, FRA-12, from the American Cancer Society. Part of this work was conducted in partial fulfillment for the requirements of a Ph.D. thesis (D.K.S.).

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EXPRESSION

11. Tukey, R. H., Nebert, D. W., and Negishi, M. (1981) J. Biol. Chem.

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38. Omiecinski, C. J., Redlich, C. A., and Costa, P. (1990) Cancer Res. 50,4315-4321.

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39. Marie, S., Anderson, 2059-2063.

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40. Dey, A., Westphal, H., and Nebert, D. W. (1989) Proc. Natl. Acad. Sci. USA 86, 7446-7450.

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54. Sanger, F., Coulson, A. R., Barrell, B. G., Smith, A. J. H., and Roe, B. A. (1980) J. Mol. Biol. 143,161-178. 55. Carlson, J., and Messing, J. (1984) J. Biotechnol. 1, 253-264. 56. Kawajiri, K., Watanabe, J., Gotoh, O., Tagashira, Y., Sogawa, K., and Fujii-Kuriyama, Y. (1986) Eur. J. Biochem. 159,219-225. 57. Gonzalez, F. J., Kimura, S., and Nebert, D. W. (1985) J. Biol. Chem. 260,5040-5049. Y. (1984) 58. Sogawa, K., Gotoh, O., Kawajiri, K., and Fujii-Kuriyama, Proc. Natl. Acad. Sci. USA 81, 5066-5070. 59. Sogawa, K., Gotoh, O., Kawajiri, K., Harada, T., and Fujii-Kuriyama, Y. (1985) J. Biol. Chem. 260, 5026-5032.

60. Jaiswal, A. K., Gonzalez, F. J., and Nebert, D. W. (1985) Nucleic AcidsRes. 13,4503-4520. 61. Yanagida, A., Sogawa, K., Yasumoto, K.-I., and Fujii-Kuriyama, Y. (1990) Mol. Cell. Biol. 10, 1470-1475. 62. Hines, R. N., Mathis, J. M., and Jacob, C. S. (1988) Curcinogenesis 9, 1599-1605.

63. Guenthner, T. M., and Nebert, D. W. (1978) Eur. J. Biochem. 91, 449-456. 64. Pineau, T., Daujat, M., Pichard, L., Girard, F., Angevain, J., Bonfils, C., and Maurel, P. (1991) Eur. J. Biochem. 197, 145-153. 65. Nebert, D. W., Nelson, D. R., Coon, M. J., Estabrook, R. W., Feyereisen, R., Fujii-Kuriyama, Y., Gonzalez, F. J., Guengerich, F. P., Gunsalus, I. C., Johnson, E. F., Loper, J. C., Sato, R., Waterman, M. R., and Waxman, D. J. (1991) DNA Cell Biol. 10, 1-14. 66. Gunning, P., Ponte, P., Okayama, H., Engel, J., Blau, H., and Kedes, L. (1983) Mol. Cell. Biol. 3, 787-795.