©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Alternative mRNA Splicing and Differential Promoter Utilization Determine Tissue-specific Expression of the Apolipoprotein B mRNA-editing Protein (Apobec1) Gene in Mice
STRUCTURE AND EVOLUTION OF Apobec1 AND RELATED NUCLEOSIDE/NUCLEOTIDE DEAMINASES (*)

Makoto Nakamuta (1), Kazuhiro Oka (1), Julia Krushkal (2), Kunihisa Kobayashi (1) (3)(§), Mikio Yamamoto (4), Wen-Hsiung Li (2), Lawrence Chan (1)

From the (1) Departments of Cell Biology and Medicine, Baylor College of Medicine, Houston, Texas 77030, the (2) Center for Demographic and Population Genetics, University of Texas, Houston Texas 77225, the (3) Children's Nutrition Research Center, Houston, Texas 77030, and the (4) Department of Biochemistry II, National Defense Medical College, 3-2 Namiki, Tokorozawa, Saitama 359, Japan

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Apolipoprotein (apo) B mRNA editing consists of a CU conversion involving the first base of the codon CAA, encoding Gln 2153, to UAA, a stop codon. Editing occurs in the intestine only in most mammals, and in both the liver and intestine in a few mammalian species including mouse. We have cloned the cDNA for the mouse apoB mRNA editing protein, apobec1. Expression of mouse apobec1 cDNA in HepG2 cells results in the editing of the intracellular apoB mRNA. The cDNA predicts a 229-amino acid protein showing 92, 66, and 70% identity to the rat, rabbit, and human proteins, respectively. Based on the estimated values of divergence of apobec1 sequences in terms of the numbers of synonymous and non-synonymous substitutions per site, we found that apobec1 is a fairly rapidly evolving protein. Sequence comparison among mammalian apobec1 sequences has permitted the identification of seven conserved regions that may be functionally important for editing activity. We present a phylogenetic tree relating apobec1 sequences to double-stranded RNA adenosine deaminase and other nucleotide/nucleoside deaminases. Northern blot analysis indicates that apobec1 mRNA exists in two different sizes, a 2.2-kilobase (kb) form in small intestine and a 2.4-kb form in liver, spleen, kidney, lung, muscle, and heart. To study the molecular basis for the different sized apobec1 mRNAs, we cloned the apobec1 gene and characterized its exon-intron organization together with the sequences expressed in the hepatic and intestinal mRNA. The mouse apobec1 gene contains 8 exons and spans 25 kb, and is located in chromosome 6. The major hepatic mRNA contains all 8 exons, whereas the major small intestinal mRNA misses the first 3 exons and its transcription is initiated in exon 4. The intestinal mRNA also contains at its 5` end a unique 102-nucleotide piece that is absent in the liver mRNA. We also identified two alternatively spliced hepatic apobec1 mRNAs with different acceptor sites in exon 4. Transient expression studies using promoter-reporter gene constructs in HeLa, Hepa, and Caco-2 cells indicate that the 5`-flanking sequences of the liver mRNA (i.e. upstream of exon 1) have predominantly hepatic promoter activity and the 5`-flanking sequences of the major small intestine mRNA (i.e. upstream of exon 4) have preferential intestinal promoter activity. We conclude that alternative mRNA splicing and differential promoter utilization determine the tissue-specific expression of the apobec1 gene in mice. Our observations provide information on unique aspects of the molecular basis of the tissue-specific expression of apobec1 mRNA and the evolutionary basis of RNA editing.


INTRODUCTION

RNA editing appears to be a common biological phenomenon among plants and protozoa (1, 2, 3) . In mammals, there are only a few well documented instances of mRNA editing (4, 5) ; mRNAs that have been found to be subject to editing include those for apolipoprotein (apo)() B (6, 7) , glutamate-activated receptor channel (8, 9) , and possibly Wilms tumor WT1 gene (10) and others (e.g. Ref. 11). ApoB mRNA editing involves a single base CU conversion of the first base of a codon CAA, encoding Gln-2153, in apoB-100 mRNA, to UAA, a translational termination codon, in apoB-48 mRNA. The unedited mRNA encodes apoB-100, which contains 4536 amino acid residues, whereas the edited mRNA encodes apoB-48, which contains 2152 residues. In humans, apoB-100 is synthesized exclusively in the liver, and is an essential component of hepatic very low density lipoprotein (VLDL) and its metabolic products, intermediate density lipoprotein and low density lipoprotein (LDL) (12). ApoB-48 is synthesized in the small intestine in all mammals, and is an obligatory structural component of chylomicrons (12) . In rats and mice, apoB-48 is also synthesized in the liver and apoB-48 can be detected in hepatic VLDL particles in these animals. However, apoB-48-containing VLDL particles do not contribute to LDL production (13). ApoB-48 is colinear with the N-terminal 48% of apoB-100. ApoB-100 serves as a ligand for the LDL receptor and is an essential component of lipoprotein (a), an LDL-like lipoprotein containing apo(a) covalently linked to apoB-100 (14) . The putative LDL receptor-binding domain (15) and apo(a) attachment site (16, 17) in apoB-100 both reside in the C-terminal half of the molecule which is absent in apoB-48. As a result of this structural difference, apoB-48 is incapable of binding to the LDL receptor or contributing to lipoprotein (a) production.

ApoB mRNA editing activity can be demonstrated in extracts of rat and mouse liver and small intestine as well as other tissues (18, 19, 20, 21) . The proposal that editing is mediated by a multicomponent enzyme complex, designated an editosome (22, 23) , was supported by the recent finding of Teng et al.(24) that a cloned rat apoB mRNA editing protein, designated apobec1 (for apolipoprotein B mRNA editing component-1), by itself is incapable of editing synthetic mini-apoB mRNA substrates in vitro. Editing by rat apobec1 requires the participation of tissue complementation factor(s) that appear to be widely distributed (24, 25) .

The deduced amino acid sequence of apobec1 from rat (24) , rabbit (26) , and humans (27, 28) shows limited similarity to dCMP/CMP and cytidine/deoxycytidine deaminases (29) . Apobec1 appears to be a zinc-requiring cytidine deaminase (29, 30) . Apobec1 mRNA distribution differs among different mammalian species. In human and rabbit, the major site of expression is the small intestine (26, 27, 28) (low level expression is also detected in colon in the rabbit (26) ), whereas in the rat, it is also expressed in varying amounts in liver, spleen, kidney, lung, and colon (24) . Interestingly, the rat hepatic and small intestinal apobec1 mRNAs differ significantly in size, being predominantly 1.24 kb in the liver and 1.0 kb in the small intestine (24). The genomic structure of rat apobec1 and the molecular basis for the size difference between mRNAs from the two tissues are currently unknown.

In this article, we have cloned the cDNA and genomic sequences of mouse apobec1. The cDNA predicts a 229-residue protein. Computational analysis of apobec1 sequence from different mammalian species indicates that it is a fairly rapidly evolving protein. Seven regions of relative sequence conservation can be identified. Mouse apobec1 mRNA is expressed in small intestine, liver, spleen, kidney, lung, muscle, and heart. It shows tissue-specific size difference in that the predominant hepatic mRNA is 2.4 kb (which is similar to that in other tissues except small intestine), and the major small intestinal mRNA is 2.2 kb. To investigate the molecular basis for this observation, we determined the structure of the apobec1 chromosomal gene and the structure of the mRNAs expressed in the two tissues. Our analysis indicates that the apobec1 gene is located in mouse chromosome 6. It contains 8 exons and spans 25 kb. We found that the major hepatic mRNA contains all 8 exons. In contrast, the major small intestinal mRNA misses the first 3 exons and its transcription is initiated in exon 4 with the mRNA containing exon 48. Exon 4 contained a 102-nt piece unique to the 5` end of intestinal mRNA. Two alternatively spliced mRNAs with different acceptor sites in exon 4 were also identified in the hepatic species. Transient expression studies using promoter-reporter constructs in transfected cells indicate that the 5`-flanking sequences of the liver mRNA (i.e. upstream of exon 1) have predominantly hepatic promoter activity and the 5`-flanking sequences of the small intestinal mRNA (i.e. upstream of exon 4) have preferential intestinal promoter activity. Our studies provide information on unique aspects of apobec1 gene expression that will be useful for future investigations on the tissue-specific regulation and evolutionary basis of RNA editing in mammals.


MATERIALS AND METHODS

Mouse Small Intestine and Liver cDNA Cloning

A ZAP II mouse small intestine cDNA library and a ZAP liver cDNA library (Stratagene) were screened with a 0.7-kb fragment of the rat apobec1 cDNA amplified from rat liver poly(A) RNA by reverse transcription-PCR described previously (28) . 2 10 plaque-forming units were screened from the small intestine cDNA library; seven clones were identified. Three positive clones (EPS 4, EPS 5, and EPS 17) were chosen for further analysis. We screened a total of 1.4 10 plaque-forming units from the liver cDNA library and identified five independent cDNA clones. All five were characterized by restriction mapping and the two longest clones (EPL 28, and EPL 31) were chosen for complete sequence analysis. Sequencing of cDNAs subcloned into pBluescript (Stratagene) was performed with the dideoxy chain termination technique (31) using Sequenase version 2.0 (U. S. Biochemical Corp.). The sequences presented have been established from data obtained from multiple sequence determinations and, for the most part, from both DNA strands.

Construction of Mouse Apobec1 Expression Vector

The protein coding region of mouse apobec1 cDNA was amplified by polymerase chain reaction (PCR) using synthetic oligonucleotides. The sequences of oligonucleotides were 5`-GAAGGGATCCGCCGCCACCATGAGTTCCGAGACAGGCCC-3` for the 5` upstream primer, and 5`-CGCGGGAATTCTCCCAGAAGTCATTTCAACCCT-3` for the 3` downstream primer. The 5` forward primer contained Kozak consensus sequence at the translation initiation site (32) . Artificial BamHI and EcoRI restriction enzyme sites (underlined) were incorporated in these sequences for cloning purposes. The PCR product was subcloned into pBluescript II KS and sequenced. The insert was removed by digestion with ClaI and BamHI and subcloned into the BglII and ClaI sites of the expression vector pCMV4 (33) .

Expression of Mouse Apobec1 in HepG2 Cells and Primer Extension Assay for ApoB mRNA Editing

HepG2 cells were purchased from ATCC and maintained in minimum essential medium supplemented with 10% fetal bovine serum, penicillin (50 units/ml), and streptomycin (50 µg/ml). 3 10 cells were seeded in 6-cm culture dishes the day before transfection. Transfection was carried out with 3.8 µg of pCMV4 containing mouse apobec1 cDNA and 0.2 µg of pMC1neo poly(A) (Stratagene) by using Lipofectamine reagent (Life Technologies Inc.) according to the manufacturer's instructions. Stable transfectants were selected in media supplemented with 1 mg/ml G418 (Life Technologies). After 3 weeks, G418-resistant colonies were transferred to a 24-well plate. Finally they were grown in 6-well plates. We prepared total cellular RNA using Ultraspec II and screened for the expression of apobec1 mRNA by Northern blot analysis. The RNA preparation was treated with RNase-free DNase I (Stratagene), then proteinase K, extracted with phenol-chloroform, precipitated with ethanol, and suspended in water.

Total apoB mRNA was amplified by reverse transcriptase-PCR. Reverse transcription was performed at 42 °C for 30 min in a total volume of 10 µl of reverse transcriptase buffer containing 1 PCR buffer II (Perkin-Elmer), 2.5 mM MgCl, 250 µM each dNTP, 25 pmol of 3` downstream oligonucleotide (5`-CACGGATATGATAGTGCTCATCAAGAC-3`), 40 mM dithiothreitol, 20 units of RNasin (Promega), and 20 units of SuperScript II reverse transcriptase (Life Technologies). At the end of the reaction, each reaction mixture was added to a tube containing 40 µl of a solution containing (1 PCR buffer II, 1.25 mM MgCl, 187.5 µM each dNTP, 25 pmol of 5` upstream primer (5`-CTGACTGCTCTLACAAAAAAGTATAGA-3`), and 2 units of Taq DNA polymerase (Promega)). PCR was performed for 35 cycles of 20 s at 95 °C, 20 s at 55 °C, and 1 min at 72 °C. For each RNA sample, a negative control was run in the absence of reverse transcriptase.

The degree of editing was determined by primer extension assay (18, 34) . The reverse transcriptase-PCR products were purified by Qiaquick PCR purification kit (Qiagen). One to two µl (50 ng) of purified PCR products was denatured for 5 min in boiling water under 10 µl of mineral oil and annealed for 30 min at 37 °C in a 7-µl solution containing 2 µl of 5 first strand synthesis buffer (Life Technologies), 5 10 cpm of human apoB complementary oligonucleotide (5`-ATCATAACTATCTTTAATATACTGA-3`) 5`-end labeled by [-P]ATP. After annealing, 1 µl of 1 M dithiothreitol, 0.5 µl of 10 mM dATP, dCTP, and dTTP, 1 µl of 5 mM ddGTP, and 20 units of SuperScript II reverse transcriptase were added in a total volume of 3 µl and primer extension was performed for 30 min at 37 °C. The reaction was stopped by the addition of 4 µl of sequence loading buffer. The extension product was heated for 10 min at 70 °C and loaded on a 6% denaturing acrylamide gel. The dried gel was exposed to XAR-5 film and the autoradiograph was scanned by densitometer.

Northern Blot Analysis

We isolated total mouse RNA from the small intestine and the liver by Ultraspec RNA Isolation System (Biotecx). Poly(A) RNA was prepared by using an oligo(dT)-cellulose column (Life Technologies, Inc.). 2 µg of poly(A) RNA was size-fractionated on a 1.5% agarose gel in MOPS buffer (20 mM), pH 7.0, transferred to a Hybond-N nylon membrane (Amersham). This membrane and the multiple tissue Northern blot membrane (Mouse MTN Blot 7762-1, Clontech) were probed by a random-primed P-labeled mouse apobec1 cDNA probe (complete coding region) according to the manufacturer's instructions. The filters were hybridized at 42 °C for 16 h and then washed sequentially with 2 SSC, 0.05% SDS, and 2 SSC, 0.1% SDS, and finally with 0.1 SSC, 0.1% SDS at 65 °C for 30 min. (1 SSC = 15 mM sodium citrate, 150 mM sodium chloride, pH 9.0.) The filters were exposed to x-ray films with two intensifying screens at -80 °C for 24-72 h.

Analysis of Apobec1 from Different Species

The rat, rabbit, and human apoB mRNA editing protein sequences were retrieved from the GenBank data base by using the NCBI Retrieve e-mail server (35) . The numbers of the non-synonymous and synonymous substitutions per site between sequences were estimated by the Li (36) method.

To identify conserved regions within the sequences of the apoB mRNA editing protein, the sliding window approach was applied to the alignment of these sequences. For every window, the average proportion of differences per sequence pair per window position was computed and assigned to the position in the middle of the window.

Phylogenetic Analysis of Apobec1 and Homologous Sequences

To study the relationships among the apoB mRNA editing proteins and different nucleotide and nucleoside deaminases, a phylogenetic tree was reconstructed on the basis of a region conserved among the different groups of sequences.

Search for homologous sequences was performed throughout the GenBank (35), EMBL (37) , PIR International (38) , and SWISS-PROT (39) data bases by using the NCBI Blast e-mail server (40) to identify the sequences of nucleotide and nucleoside deaminases and related sequences. Sequences were extracted by using the NCBI Retrieve e-mail server. The total number of sequences included in the analysis was 20, including apobec1 sequences from four mammalian species. Two human apobec1 sequences were included because there were minor differences between them. The sequences examined and their identification numbers are listed in .

The region of homology among different sequences was identified after the sequences were aligned by the manual alignment program from the VOSTORG package for phylogenetic analysis (41) . Positions including large gaps were not included in the analysis. In total, 34 positions with reliable alignment were used for the tree inference.

The tree was inferred on the basis of amino acid distances. The observed proportions of differences between sequences were corrected for multiple substitutions by the empirical formula (42) . The phylogenetic tree was inferred by the neighbor-joining method (43) . The root position was determined by the UPGMA method (44) .

Mouse Genomic DNA Cloning

We screened a total of 1.5 10 plaque-forming units of a DASH II mouse genomic library (Stratagene) with the P-labeled mouse apobec1 cDNA probe and identified three mouse apobec1 subgenomic clones ( mgE5, mgE7, and mgE9). Subgenomic DNA fragments carrying exons and the 5`-flanking region were characterized by restriction mapping and Southern blotting. After subcloning into pBluescript II KS (Stratagene), the sequences of exons, exon/intron boundaries, and the 5`-flanking region were determined.

Chromosomal Mapping of the Apobec1 Gene

We purchased the BSS panel 2 Southern blot filter sets from the Jackson Laboratory, ME. These contain DNA from panels of 94 backcross animals from the cross ((C57BL6/Ei SPRET/Ei) SPRET/Ei). Mouse apobec1 cDNA was P-labeled by random priming and used as hybridization probe. The conditions for prehybridization, hybridization, and subsequent washes were those recommended by the Jackson Laboratory. The filters were exposed to x-ray films at -80 °C for 72 h.

RNase Protection Assay

A HindIII-AvaI fragment of subgenomic DNA (276 bp) including exon 4 and its 5`-flanking region was subcloned into SmaI site of pBluescript II KS (Stratagene), and cut with EcoRI to generate linearized DNA template. An antisense RNA probe was synthesized using the DNA template, T7 RNA polymerase [-P]UTP (3,000 Ci/mmol, Amersham), and T3/T7 in vitro Transcription System (Clontech). We performed RNase protection assays by using RPA II kit (Ambion) according to the manufacturer's instructions. Briefly, 6 µg of poly(A) RNA were hybridized with 3 10 cpm of the P-labeled RNA probe at 55 °C for 16 h after denaturation at 90 °C for 5 min. The annealed mixture was incubated at 37 °C for 30 min with RNase (A/T1). The protected fragments were precipitated and analyzed on a 6% urea-polyacrylamide gel, followed by autoradiography.

Construction of Promoter-reporter DNA Constructs for in Vitro Transfection

An EcoRI-HindIII subgenomic DNA fragment including exon 1 and its 5`-flanking region (1598-bp 5`-flanking DNA + 72-bp exon 1) was subcloned into the HindIII site of pCAT-Basic Vector (Promega) and pCAT-Enhancer Vector (Promega) to be tested for promoter activity. These plasmids were designated pCAT-EX1 and pCAT-EX1E, respectively. A PstI-PstI DNA fragment including exon 4 and its 5`-flanking region (1575-bp 5`-flanking DNA + 168-bp intestinal exon 4) was also subcloned into the PstI site of pCAT-Basic and pCAT-Enhancer, designated pCAT-EX4 and pCAT-EX4E, respectively. As a positive control, we used pCAT-Promoter Vector (Promega), which contains an SV40 promoter upstream from the CAT gene of pCAT-Basic.

Transient Transfection and Chloramphenicol Acetyltransferase (CAT) Assays

HeLa cells were cultured in minimal essential medium supplemented with Earle's salts, 0.1 mM non-essential amino acid solution (Life Technologies, Inc.), and 10% fetal bovine serum. Mouse hepatoma cells (Hepa) were cultured in Dulbecco's modified Eagle's medium with 10% fetal bovine serum. Caco-2 (a human intestinal carcinoma cell line) cells were cultured in minimal essential medium with 1 mM sodium pyruvate (Life Technologies, Inc.), 0.1 mM nonessential amino acid, 20% fetal bovine serum, and were used at confluency for 1 week (45) . All cultures were maintained at 37 °C with 5% CO. Media for all cultures routinely included 100 units/ml penicillin and 100 µg/ml streptomycin (Life Technologies, Inc.). Approximately 24 h before transfection, tissue culture flasks were trypsinized, and 4 10 cells were seeded in a six-well plate. Cells were co-transfected using 6 µl of Lipofectamine reagent (Life Technologies, Inc.) with 1.5 µg of CAT gene recombinant plasmid and 0.1 µg of the internal reference plasmid pRSV--Gal, in which the -galactosidase gene is driven by the Rous sarcoma virus promoter (46) , according to the manufacturer's instructions. Cells were exposed to lipid-DNA complexes for 24 h under standard culture conditions without antibiotics and then were fed their complete growth medium. CAT assays were performed 48 h after transfection.

We determined CAT activity using Quan-T-CAT assay system (Amersham) and CAT Enzyme Assay System (Promega) according to the manufacturer's instructions. Each CAT gene recombinant plasmid was tested in four independent transfection experiments. The -galactosidase assay was performed by using -Galactosidase Enzyme Assay System (Promega). CAT activity was corrected for differences in transfection efficiency between flasks based on the results of -galactosidase assay internal control.


RESULTS

Cloning and Sequencing of Mouse Liver and Intestinal Apobec1 cDNAs

By screening ZAP cDNA libraries constructed from mouse small intestine and liver mRNAs, we identified seven intestinal and five hepatic cDNA clones that hybridized strongly to a P-labeled rat apobec1 cDNA probe. We selected the three longest intestine clones and two longest liver clones for complete sequence analysis. All cDNAs contain an open reading frame that encodes a 229-amino acid residue sequence (Fig. 1) that can be readily aligned with the rat, rabbit, and human apobec1 sequences.


Figure 1: Complementary DNA and deduced amino acid sequence of mouse apobec1 and alignment with other mammalian apobec1 sequences. Only the coding region of the cDNA is shown. For the amino acid sequence from other mammals, only residues that are different from the mouse sequence are shown. The GenBank accession numbers are shown on the left. There are two human apobec1 sequences that are slightly different. Both are included in this analysis.



Expression of Mouse Apobec1 cDNA in Human Hepatoma Cells

The mouse apobec1 cDNA clone was identified by cross-hybridization to the cloned rat cDNA and its sequence shows substantial homology to the other mammalian apobec1 cDNAs. We wished to confirm that it can be expressed functionally as an apoB mRNA-editing protein. The complete coding region of the mouse apobec1 cDNA was subcloned into the expression vector pCMV4 (33) . It was transfected into a human hepatoma cell line, HepG2, which has no editing activity and contains 100% unedited apoB mRNA. Total RNA was isolated from untransfected HepG2 cells, and cells stably transfected with an apobec1 cDNA expression vector or vector alone, without apobec1 insert, and the degree of apoB mRNA editing was measured by primer extension (18, 34) . In the cells that were stably transfected with the mouse apobec1 vector, the endogenous apoB mRNA contained 13.0 ± 5.8% edited (apoB-48) mRNA compared to 0% in nontransfected controls and those transfected with the vector only (data not shown). Therefore, the cloned mouse apobec1 cDNA encodes a functional protein whose expression in HepG2 cells is sufficient to produce editing of the endogenous human apoB mRNA.

Structure of Mouse Apobec1 and Evolution of the Apobec1 Gene

The predicted mouse apobec1 amino acid sequence (Fig. 1) showed a 92, 66, and 70% identity to the rat, rabbit, and human proteins, respectively (excluding the 7-residue C-terminal extension in the human and rabbit sequences). A number of putative functional sequence motifs are found in human (27, 28) , rat (47) , and rabbit (26) apobec1. The mouse enzyme contains consensus phosphorylation sites for protein kinase C (residues 13-15), cAMP and cGMP-dependent protein kinase (residues 33-36) and casein kinase II (residues 45-48). It also contains an N-linked glycosylation consensus sequence (residues 57-59) which is also present in the rabbit and human sequences but not in the rat sequence. Studies in vitro suggest that this site is not utilized in human apobec1 (28) . In addition, we identified two consensus N-myristoylation sites (residues 45-50 and 188-196) in the predicted mouse apobec1 sequence. An interesting feature of the other mammalian apobec1 sequences is the presence of two overlapping heptad leucine-rich repeats. Similar motifs can be identified in the mouse sequence spanning residues 182-203 and 189-210. The functional significance of these consensus sequence motifs identified above is unknown.

Divergence among Apobec1 Sequences from Different Species

The availability of cDNA and deduced amino acid sequence of apobec1 from four different mammalian species has enabled us to calculate the rates of nucleotide substitution during evolution. illustrates the estimated values of divergence of apobec1 sequences in terms of the numbers of synonymous and non-synonymous substitutions per site. The divergence values for both synonymous and non-synonymous substitutions show that the rat and mouse sequences are much more closely related to each other than to the rabbit or human sequences. Also, both mouse and rat proteins are 7 amino acid residues shorter in length than the rabbit and human sequences. This sequence divergence reflects the species divergence, as mouse and rat are phylogenetically close to each other.

The estimated proportion of synonymous substitutions between the mouse and rat apobec1 sequences is 15.8%. This value is slightly higher than the corresponding value of 14.4% computed by Li (36) for a large set of coding sequences from mouse and rat genes. The estimated proportion of the non-synonymous substitutions (4.0%) is considerably higher than the corresponding value (1.5%) computed by Li (36) for various sequence data. Thus, apobec1 is not a conservative protein. The divergence between the mouse and rat apobec1 sequences, however, is lower than that for apoB. The latter protein is a product of apoB mRNA, which is a substrate for apobec1. The value of divergence between the mouse and rat apoB-100 is approximately 7.6% for non-synonymous substitutions (36) .

Conserved Regions within Apobec1 Sequences

Fig. 2 shows the average divergence at the amino acid positions within each window of the aligned apobec1 sequences. The divergence values were computed using a sliding window size of 5 residues. Several conserved regions are identified by this analysis. These regions include positions 15-20, 31-35, 37-42, 61-66, 84-93, 127-131, 155-159, and 189-193. Many positions from these regions may be functionally important for nucleotide and nucleoside deaminase regions, as was previously suggested by other authors (26, 28, 48, 49, 50) . In particular, positions 15-20 and 31-34 fall into the potential nuclear localization motif. The regions 61-66 and 84-93 are a part of the putative apobec1 catalytic domain, where His and Cys are potential zinc-chelating sites, and Glu may be essential for deaminase activity. The putative zinc-coordinating Cys is also conserved among all sequences studied. The positions 189-193 fall into two completely conserved Pro residues flanked on either side by leucine-rich domains which may be involved in protein-protein interactions. Therefore, the functionally important regions of apobec1 in humans, rabbits, rats, and mice seem to be a subject for a negative selection which leads to conservation of these regions.


Figure 2: Average proportions of substitutions per amino acid position per sequence pair computed by using a sliding window of the size 5. The smaller divergence values correspond to the more conserved positions.



Relationship of Apobec1 to Other Nucleotide and Nucleoside Deaminases

Fig. 3 displays the tree inferred by the neighbor-joining method for the region that is shared by the apobec1s and other deaminases whose sequences are available from various DNA data bases. This region includes the known catalytic center of nucleoside and nucleotide deaminases (50) . The tree inferred reveals a distinct separation between apobec1 and the other deaminases.


Figure 3: Phylogenetic tree inferred by the neighbor-joining method for the region conserved within apobec1 sequences and different related deaminases. The horizontal scale in the bottom of the figure indicates the numbers of amino acid substitutions per position along the tree branches. The length of the tree branches was computed by the neighbor-joining method.



According to the UPGMA, the tree root is located between the apobec1 sequences and the other deaminases. According to this position of the root, the nucleotide deaminases, in particular dCMP/CMP deaminases, belong to the more ancient group than the nucleoside deaminases (cytidine/deoxycytidine deaminases). We included in our analysis the sequence of the human double-stranded RNA adenosine deaminase (DRADA) and the homologous region from the nematode mitochondrial gene T20H4.4. According to the tree shown in Fig. 3, both sequences are related to each other and they diverged from the group of cytosine nucleotide deaminases.

Among the sequences that belong to each particular enzyme type, the branch length was different for different species lineages; e.g. the mouse apobec1 sequence has accumulated more mutations than did the corresponding human apobec1. Also, the dCMP/CMP deaminase in the nematode Caenorhabditis elegans has accumulated more substitutions than the human sequence. Similarly, the branch leading to the Mycoplasma pirim cytidine/deoxycytidine deaminase is much longer than the branch for the analogous Bacillus subtilis sequence. The latter branch is in turn longer than that of the human sequence. The difference in the numbers of substitutions can be related to the generation-time effects, as was pointed out earlier by Wu and Li (51) by using the example of rodent versus human sequences. On the other hand, the catalytic region is important for deaminase function, and therefore it has been conserved in distant sequences. Thus, one may assume that selection forces may also have played a role in the differences in rates among lineages.

The fundamental division of the different deaminase types and the presence of these sequences in distant species may suggest that the divergence of the main groups of nucleotide and nucleoside deaminases and related sequences including apobec1 may have occurred at the very early stages of evolution, as was noted previously by Bhattacharya et al.(49) . These very distantly related sequences, however, still share a conserved domain of homology, which means that the functional constraints have been important in the evolution of these sequences.

Northern Blot Analysis of Apobec1 mRNA in Mouse Tissues

We used the mouse apobec1 cDNA as a probe to examine the distribution of apobec1 mRNA in various mouse tissues by Northern blot analysis (Fig. 4). We found that apobec1 mRNA is widely distributed. In most tissues, the major mRNA band measures 2.3-2.5 kb; in the small intestine it measures 2.1-2.3 kb. The level of expression appears to be highest in the small intestine, liver, and spleen, followed by the kidney, lung, muscle, and heart. It is barely detectable in testis and brain. There is also a band of 4.6 kb of lower intensity in most of the tissues that express the 2.1-2.5-kb mRNA species.


Figure 4: Northern blot analysis of apobec1 mRNA expression in various mouse tissues. 2 µg of poly(A) RNA was loaded for each sample. The left two lanes represent mouse intestinal and hepatic poly(A) RNA isolated in our laboratory. The 8 samples on the right are from Mouse MTN Blot #7762 from Clontech.



Cloning and Structural Characterization of the Mouse Apobec1 Gene

We noted sequence differences in the 5` region of the mouse small intestine and liver cDNAs (see below). In order to determine the molecular basis of this observation, we cloned and characterized the chromosomal gene of mouse apobec1. We isolated three -phage clones, designated mgE5, mgE7, and mgE9, from a DASH-II mouse genomic library by hybridization to a mouse apobec1 cDNA probe. We characterized these three overlapping clones by restriction mapping and partial sequence analysis; all the exons, the exon-intron junctions, and the 3`-flanking region of the last exon (no. 8), as well as the 5`-flanking regions of exon 1 and exon 4 were completely sequenced. The complete genomic structure of mouse apobec1 is presented in Fig. 5. The restriction map of the apobec1 gene in mouse genomic DNA completely matched that predicted from the structure of these genomic clones. Furthermore, there is no difference between the cDNA sequence and the corresponding exon sequence in these clones.


Figure 5: Structure of mouse apobec1 gene and mRNAs. The restriction map, genomic clones, and genomic structure are as shown in the top half of the figure. The liver-type and intestine-type mRNAs as deduced from the genomic structure and structure of the corresponding cDNAs, primer-extension assay and RNase protection assay are as shown in the bottom half of the figure. The numbers above each mRNA structure indicate the various exons from which specific portions of the mRNAs are transcribed. The stippled box represents a sequence present in the intestine-type mRNA only. It is part of intron 3 that is spliced out from the liver-type mRNA. There are two alternatively spliced species of liver-type mRNA. The region present in the longer species only is indicated by a cross.



The mouse apobec1 gene spans 25 kb. It contains eight exons and seven introns. The exons vary in size from 28 bp (exon 5) to 1144 bp (exon 8). The sizes of the individual exons and introns and the sequences immediately flanking the exon-intron junctions are presented in I. All intron sequences at these junctions follow the gt-ag rule. The first 3 introns occur in the 5`-untranslated region of the liver-type mRNA (see below). Introns 47 interrupt the coding region of the gene at codon 6, 15, 148, and 187, respectively.

Alternative Transcription Initiation and Differential Exon Utilization Determine Mouse Apobec1 mRNA Expression in Liver and Small Intestine

The sequence of the coding region was identical among the mouse apobec1 cDNA clones isolated from the liver and small intestine. However, in the 5` end of the mRNA, the sequences were different. A comparison of the genomic structure and cDNA sequences permitted us to deduce the structure organization of the liver and small intestine apobec1 mRNA with respect to the mouse apobec1 gene (Fig. 5).

The liver-type apobec1 mRNAs are transcribed from exons 18. We detected 2 species of liver-type mRNA that contained 2270 and 2214 nt, respectively, plus the poly(A) tail. The difference between the 2 species consists of an alternative 3` splice acceptor site in exon 4 so that a 56-nt piece of the mRNA in the larger species (marked with a cross in Fig. 5) is missing in the smaller mRNA. This alternative splicing pattern was revealed by the structure of the two liver cDNA clones, EPL 28 and EPL 31 (Fig. 5) and was confirmed by PCR analysis of mouse liver poly(A) RNA. The smaller species of mRNA accounts for >95% and the larger species, <5% of the total liver apobec1 mRNA. Since the insertion/deletion in the mRNA occurs in the 5`-untranslated region of the mRNAs, it does not affect the primary structure of apobec1, the protein product encoded by both species of mRNA. The sequence of the 5`-untranslated region of the liver-type apobec1 mRNA is shown in Fig. 6 , A and B. It contains 565 and 509 nt, respectively, for the two alternatively spliced mRNAs.


Figure 6: Sequence of the mouse apobec1 gene. A, 5`-flanking DNA, 5`-untranslated region (which includes exons 1, 2, and 3). B, part of intron 3 immediately 5` to exon 4, a region of intron 3 that is expressed in the intestine-type mRNA (underlined), and exon 4 (which includes the rest of the 5`-untranslated region the first five and one-third codons of apobec1). C, 3`-untranslated region and 3`-flanking DNA of the mouse apobec1 gene. The 5`- and 3`-flanking DNA and intron sequences are in small letters. The exons are in capital letters. Location of potential transcription factor binding sites for AP-1 (60), AP-2 (61), AP-3 (62), octamer-binding transcription factor-1 (OCT) (63), half-sites for glucocorticoid/androgen receptor (BR/AR) (64, 65), and estrogen receptor (ER) (66) are indicated by single underlines. Potential metal-responsive elements of the metallothionein 1 gene promoter (MT-1) (67), (MBF-1) (68), retinoic acid response element (RARE) (69), sterol regulatory element (SRE) (70) are also shown. Nucleotides that differ from the consensus sequence are indicated by double underlines.A, the two major transcription initiation sites for the liver-type mRNA are marked with a dot, the more 3` situated site appears to be the dominant one. B, the two major transcription initiation sites for the intestine-type mRNA are marked with a dot. The two alternative 3` splice acceptor sites for the two species of liver-type mRNA are marked by arrows. C, a putative variant polyadenylation signal is underlined.



The major mouse small intestine apobec1 mRNA contains 1965 nt, plus the poly(A) tail. Its transcription is initiated from exon 4 and the mRNA misses the first 3 exons of the apobec1 gene which are unique to the liver-type mRNAs. The small intestine mRNA contains a 102-nt sequence in the 5` end (stippled box in Fig. 5and Fig. 7) that is unique to the mRNA expressed in this tissue. It is missing in the liver-type mRNA because the exon 4 3`-acceptor site of the latter occurs downstream to it and this piece of RNA is spliced out from the mature liver-type apobec1 mRNAs. The 5`-untranslated region of the small intestine apobec1 mRNA contains 260 nt and its sequence is displayed in Fig. 6B. There is no difference in the other parts (coding region (687 nt) and 3`-untranslated region (1018 nt), Fig. 6C)) of the apobec1 mRNA from liver or small intestine. A variant polyadenylation signal consensus sequence, AGTAAA (52, 53) , is identified 20 nt upstream of the poly(A) site.


Figure 7: Mapping of intron 3/exon 4 boundaries and 5`-end of apobec1 mRNA by ribonuclease protection. The experimental protocol is described under ``Experimental Procedures.'' The stippled box represents the 5` end of intestine-type mRNA that is unique to this tissue; it corresponds to the stippled box in Fig. 5. Two alternatively spliced liver-type apobec1 mRNAs differ by the presence or absence of a 56-nt piece of RNA (marked by a cross, and referring to similar box in Fig. 5). The occurrence of two alternatively spliced liver-type apobec1 mRNAs is confirmed by sequence analysis of cDNAs that show the insertion/deletion (see Fig. 5). Arrows: unmarked, undigested RNA probe; *, intestine RNA-protected product;**, longer liver RNA-protected product;***, shorter liver RNA-protected product.



The mRNA structures presented in Fig. 5 represent the major species of apobec1 mRNA in the two tissues. In the liver, by Northern blot analysis (Fig. 4), essentially all the mRNA was of the high molecular weight (``liver-type'') form. Very small amounts, representing <5%, of the ``intestinal-type'' mRNA was detected by RNase protection (Fig. 7). In the small intestine, in addition to the low molecular weight (intestinal-type) species, small amounts (20%) of the larger liver-type mRNA could be detected both by Northern blot analysis (Fig. 5) and by RNase protection (Fig. 7). Therefore, the tissue specificity of expression of these two forms of apobec1 mRNA is not absolute in either organ, although the major species of mRNA is present at a concentration substantially higher than that of the minor species in each tissue.

Structure of DNA Flanking the 5` End of Exon 1 and Exon 4 and Assay for Promoter-reporter Gene Activity by Transient Transfection

We determined the nucleotide sequence of 1.5 kb of the DNA flanking the 5` end of exon 1 of mouse apobec1 (Fig. 6A). A variant TATA box, TAAA, can be identified at position -23 to -19, and a putative CAT box with the sequence CACT is present at position -58 to -55. A number of consensus sequences for potential interaction with trans-acting factors can be identified (Fig. 6A), including sites for AP-3 (-247 to -240), GR/AR (-290 to -284), OCT (-300 to -293), RARE (-355 to -348), AP-1 (-753 to -747), GR (-996 to -991), MT-1 (-1256 to -1251), and AP-2 (-1280 to -1273). The functional significance of these sequence motifs in apobec1 gene expression is unknown.

Intron 3 of the apobec1 gene spans 6.4 kb. It flanks the 5` end of exon 4 which coincides with the transcription initiation site for the intestinal type of mouse apobec1 mRNA. We determined the nucleotide sequence of 1.5 kb of intron 3 immediately 5` upstream to the first exon of the intestinal mRNA (which continues into exon 4 of the apobec1 gene shown in Fig. 6B). A TATA box is identified at position -28 to -25 (for convenience, we designated the first nucleotide 5` upstream of the intestinal mRNA as number -1). A putative CAT-box sequence, CCACT, is present at position -70 to -66. Again, a number of potential sites for binding of trans-acting factors are present in this region of intron 3 (Fig. 6B), including sites for OCT (-233 to -226), AP-1 (-256 to -250, -1133 to -1126 and -1213 to -1206), MBF-1 (-271 to -265 and -1236 to -1220), SRE (-522 to -515), AP-2 (-731 to -724), AP-3 (-943 to -938), MT-1 (-993 to -988), GR/AR (-1145 to -1140), and ER (-1315 to -1311). Again, the functional significance of these sites is unknown.

The expression of the liver and intestine-type of apobec1 mRNAs is regulated by tissue-specific factors. We examined whether the sequences immediately flanking the 5` end of the liver-type mRNA have promoter activity different from those flanking the intestine-type mRNA in the context of their ability to direct the expression of a reporter gene in three types of cells in vitro: (i) HeLa, an epithelial non-liver, non-intestine cell line; (ii) Hepa, a mouse hepatoma cell line; and (iii) Caco-2, a human colon carcinoma cell line. The two specific DNA-reporter constructs used contain CAT as a reporter gene linked to the DNA to be tested: (i) pCAT-EX1 that contains 1.7 kb of the DNA flanking the 5` end of exon 1, and (ii) pCAT-EX4 that contains 1.7 kb of the DNA flanking the 5` end of exon 4. Two control plasmids were used: pCAT-Basic, which contains the CAT gene alone, and pCAT-Promoter, which contains an SV40-Promoter upstream from the CAT gene.

The four different promoter-CAT constructs were individually transfected into HeLa, Hepa, or Caco-2 cells. The relative level of CAT expression in these cells is shown in Fig. 8A. It is evident that pCAT-Promoter gave consistently good expression, and pCAT-Basic, little expression in all three types of cells. In both the HeLa cells and Hepa cells, pCAT-EX1 expression was strong and roughly matched that of pCAT-Promoter. pCAT-EX4 had very low activity in these cells. In Caco-2 cells, the relative levels of expression of the two apobec1 promoter-CAT constructs were quite different from those in the other two cell types. pCAT-EX4 was expressed at the highest level in these cells. pCAT-Promoter and pCAT-EX1 were expressed at somewhat lower levels, 60-75% that of pCAT-EX4 but still much higher than pCAT-Basic, which was expressed at a barely detectable level. Similar results were obtained when we analyzed CAT activity by a quantitative scintillation assay (Fig. 8B) instead of the chromatography assay (Fig. 8A). The results are consistent with the presence of liver-specific elements in the 5`-flanking DNA upstream of exon 1, and of intestine-specific elements in the DNA sequences flanking exon 4, and, to a lesser extent, those flanking exon 1 as well.


Figure 8: CAT activity of promoter-reporter gene constructs transfected in HeLa, Hepa, and Caco-2 cells. A, enzyme assay system (Promega) was used to display the acetylated chloramphenicol products. B, Quan-T-CAT Assay System (Amersham) was used to compare the activity of the four constructs in each cell line. The bars represent standard deviations.



Localization of the Mouse Apobec1 Gene to Chromosome 6

The mouse chromosomal location of apobec1 was determined by interspecific backcross analysis using progeny derived from matings of (C57BL6/Ei SPRET/Ei) SPRET/Ei. Southern blots using apobec1 cDNA as a hybridization probe indicate that the apobec1 locus was polymorphic for PuvII, producing a 9.5-kb band for C57BL/6 and a 3.6-kb band for Mus spretus (Fig. 9A). We used BSS panel 2 blots from Jackson Laboratory for PuvII to determine the segregation pattern of the apobec1 locus. Mouse apobec1 was mapped to chromosome 6 by this technique. The segregation patterns of this locus and flanking genes in the 94 backcross animals that were typed for all loci are shown in Fig. 9B. A partial chromosome 6 linkage map showing the location of apobec1 is presented in Fig. 9C.


Figure 9: Chromosomal localization of mouse Apobec1 locus. A, Southern blot analysis of PvuII-digested mouse DNA from C57BL/6, M. spretus, and backcross mouse heterozygous for the two alleles. B, segregation pattern of Apobec1 in backcross animals that were typed for all loci. The filled boxes represent the presence of a C57BL/6 allele, and open boxes represent the presence of an M. spretus allele. Stippled boxes indicate that the locus was not typed. The number of offspring inheriting each type of chromosome is listed at the bottom of each column. R, recombination frequency for each interval. SE, standard error. C, partial chromosome 6 linkage map showing the location of apobec1 in relation to linked genes. Relative recombination distance in 10 centimorgans (10 cM) is as indicated.




DISCUSSION

ApoB mRNA editing is a mechanism by which the organism determines how much apoB-48 is synthesized in place of apoB-100 in a specific tissue. Because of the very different functions of these two species of apoB, apoB mRNA editing has major physiological and clinical implications (4, 5, 12) . All apoB-100-containing lipoproteins, including VLDL, intermediate density lipoprotein, LDL, and lipoprotein (a), are highly atherogenic, especially when they are present in high concentrations. ApoB mRNA editing down-regulates the amount of apoB-100 production. Teng et al.(47) recently showed that somatic gene transfer of rat apobec1 into the liver of mice essentially eliminates apoB-100 and plasma LDL without affecting plasma high density lipoproteins, an anti-atherogenic species of lipoprotein. If a similar strategy can be safely applied to humans, apobec1 may have substantial potential as a therapeutic gene.

The mouse normally edits apoB mRNA both in the liver and small intestine. Approximately 60-70% of the hepatic apoB mRNA and some 95% of the small intestinal mRNA is in the edited (apoB-48) form. The presence of substantial degrees of apoB mRNA editing in the liver is also shared by rat and, to a lesser extent, by horse and dog (54) . In all other mammalian species examined to date, apoB mRNA editing is largely confined to the intestine (54) . Teng et al.(24) first reported the cloning of rat apobec1 cDNA. Northern blot analysis showed that rat apobec1 mRNA was detected in multiple tissues. Interestingly, in the rat, the size of apobec1 mRNA in the small intestine is 1.0 kb, whereas that in all other tissues including the liver is 1.24 kb. The structural basis of the different sized mRNAs in different tissues of the rat is unknown. In this study, we found that in the mouse, small intestinal apobec1 mRNA is also smaller than the mRNA in other tissues, being 2.2 kb in the intestine compared to 2.4 kb in the liver and other tissues (Fig. 4).

In order to determine the molecular basis of the different sized apobec1 mRNA in mouse liver and intestine, we cloned the apobec1 gene and determined its chromosomal location and exon-intron organization. We mapped the mouse apobec1 locus to a region of chromosome 6 which is syntenic to a region in the short arm of human chromosome 12. By our analysis, apobec1 is immediately proximal to Hcph (Fig. 9, B and C). The human homolog of Hcph, PTPN6, has been localized to chromosome 12p12-p13 (55, 56) . The human APOBEC1 locus has also been localized to chromosome band 12p13.1-p13.2 by fluorescence in situ hybridization (28) . Based on the synteny between the two loci, we conclude that the apobec1 gene cloned in this study is a true homolog of the human APOBEC1 gene.

Analysis of apobec1 mRNA and genomic structure indicates that alternative mRNA splicing and differential promoter utilization determine the tissue-specific expression pattern of the apobec1 gene in mice; they also account for the size difference of apobec1 mRNA in liver and small intestine. The differences between hepatic and intestinal apobec1 mRNA involve the 5`-untranslated region; the coding region, and hence, the deduced apobec1 amino acid sequence, is identical in both tissues. This finding is significant because it excludes differences in apobec1 structure as the basis for different apoB mRNA editing activity in the two tissues. Although we do not have any direct evidence for it, it is likely that this conclusion also holds true for other mammalian species, e.g. rat, horse, and dog, that edit apoB mRNA in both liver and intestine.

ApoB mRNA editing has been observed only in mammals and does not occur in chickens. Therefore, the process seems to have evolved after the divergence of the avian species from mammals. Taking advantage of the availability of apobec1 sequence from four different mammalian species (human, rabbit, rat, and mouse), we have examined the rate of nucleotide substitution in apobec1, a quantitative measure of how tightly conserved is this mammalian enzyme during evolution. As summarized under ``Results,'' we found that apobec1 is not a conservative protein, having a non-synonymous substitution rate 2-3-fold higher than the value computed from a large set of coding sequences from mouse and rat genes (36) . This observation is interesting. We speculate that the relatively high rate of nucleotide substitution of apobec1 may be related partly to the non-conservative nature of its substrate, apoB mRNA, which seems to be an even more rapidly evolving gene. Additional information on the specific interactions between apobec1 and apoB mRNA will be needed to see if this hypothesis is correct.

To identify conserved and therefore potentially functionally important regions in apobec1, we examined the pattern of amino acid conservation over short sequence segments. Using this approach, we have identified seven regions in apobec1 that are better conserved than the rest of the molecule (see ``Results'' and Fig. 2). Interestingly, several of these regions coincide with some previously identified possible functional regions, e.g. a potential nuclear localization motif (26) , putative catalytic domain, and zinc-chelating and zinc-coordinating regions (48, 49) . Also of interest is the complete conservation of two Pro residues that are juxtaposed to leucine-rich sequences on either side. There is evidence that apobec1 exists as a homodimer (28) . It also requires tissue complementation factor(s) to express editing activity (24, 25, 26) . These leucine-rich sequence motifs have been postulated to participate in dimerization (5, 28) and/or interaction with complementation factors (24, 25, 26, 27, 28) . The strong conservation of the double Pro residues and their flanking leucine-rich motifs suggests that this region is important for the functional integrity of apobec1. Indeed, deletion of the entire leucine-rich domain in rat apobec1 eliminates editing activity (24) .

Sequence similarity between rat apobec1 and the other nucleotide/nucleoside deaminases was first noted by Navaratnam et al.(29) . We have constructed a phylogenetic tree for the region conserved among these enzymes (Fig. 3). We compared 20 different sequences to infer the phylogenetic relationship using the UPGMA method. The tree root by our computation is located between the apobec1 sequences and the other deaminases, and is different from that proposed by Bhattacharya et al.(49) , who analyzed eight sequences using the program KITSCH. It is not clear which phylogenetic tree is more reliable. To determine the root position reliably, longer regions of sequence homology would be needed, or reliable outgroup sequences should be used. Neither alternative is available at the present moment, since the deaminase sequences are highly divergent and since the homology between distant sequences does not extend far beyond the catalytic center.

Apart from apobec1, the only other enzyme that has been implicated in mRNA editing in mammals to date is DRADA. DRADA converts adenosines to inosines by a hydrolytic deaminase reaction (57) . The exact biological function of DRADA in vivo is still unclear. However, there is suggestive evidence that it may be involved in the editing of glutamate-gated ion channel subunit mRNAs in mammalian brain (5, 8, 9). Interestingly, DRADA, an adenosine deaminase, shows sequence similarity to apobec1 and other cytidine/cytosine deaminases (Ref. 50, and see Fig. 3) but no sequence homology to other known adenosine deaminases (50, 58) . As suggested by Kim et al.(50, 59) , the catalytic mechanism of DRADA might be more similar to that of apobec1 and the cytidine/cytosine deaminases than to the other adenosine deaminases.

Apobec1 cDNA has been cloned from human, rabbit, rat, and mouse. Northern blot analysis indicates that apobec1 mRNA is expressed in the intestine only in human and rabbit, but in both intestine and liver (as well as other tissues) in rat and mouse. The evolutionary basis for the expression of apobec1 in both liver and intestine in the two latter species is unknown. The differential promoter utilization in the mouse gene suggests that this rodent species has acquired the capacity to express apobec1 mRNA in the liver because of the evolutionary acquisition of a liver-specific promoter 12 kb upstream to the intestinal mRNA transcription site, and the recruitment of three liver-specific exons into the 5`-untranslated region of an mRNA which shares common sequence in the rest of the molecule with the intestinal apobec1 gene transcript. Experiments using transient expression of promoter-reporter gene constructs in vitro (Fig. 8, A and B) support this hypothesis. This model would predict that animals that express apobec1 mRNA in the intestine only but not in the liver should have an apobec1 gene containing only the last five exons (numbers 48, Fig. 5). The genomic structure of apobec1 is currently unknown in animals that express apobec1 mRNA in the intestine only. However, the relatively small size (of 1.1 kb) of apobec1 mRNA in both humans (27, 28) and rabbits (26) is consistent with this interpretation. Detailed structural analysis of the gene and mRNA for these two mammalian species will be needed to verify the validity of this hypothesis.

  
Table: List of sequences used for phylogenetic analysis


  
Table: Rates of nucleotide substitution in apobec1 as defined by number of substitutions per synonymous site (below diagonal) and per nonsynonymous site (above diagonal)


  
Table: Exon-intron organization of the mouse apobec1 gene

Upper case letters indicate exon and lower case letters, intron sequences. The splice junctions are marked by arrows for the first intron only.



FOOTNOTES

*
This work was supported by Grant HL27341 from the National Institutes of Health for a Specialized Center of Research in Arteriosclerosis at Baylor College of Medicine (to L. C.) and a grant from the National Institutes of Health (to W.-H. L.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

GenBank accession numbers for the apobec1 sequences are: for exon no. 1, U21944; 2, U21945; 3, U21946; 4, U21947; 5, U21948; 6, U21949; 7, U21950; 8, U21951. For liver cDNAs, minor (longer) transcript, U22262; major (shorter) transcript, U22263; intestine cDNA, U22264.

§
Supported by a postdoctoral fellowship from the Children's Nutririon Research Center in Houston.

The abbreviations used are: apo, apolipoprotein; VLDL, very low density lipoproteins; LDL, low density lipoproteins; PCR, polymerase chain reaction; CAT, chloramphenicol acetyltransferase; apobec1, apolipoprotein B mRNA editing component 1; DRADA, double-stranded RNA adenosine deaminase; bp, base pair(s); nt, nucleotide(s); kb, kilobase pair(s); MOPS, 4-morpholinepropanesulfonic acid; UPGMA, unweighted pair group method with arithmetic mean.


ACKNOWLEDGEMENTS

We thank Sally Tobola for expert secretarial assistance, Lucy Rowe with preparation of the mouse chromosomal mapping data, and Kazumi Ishimura-Oka and Julie Martinez for assistance with the tissue-culture experiments.


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