From the
Apolipoprotein (apo) B mRNA editing consists of a C
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)
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
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
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 (
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.
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) .
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
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 (
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.
The liver-type apobec1 mRNAs are transcribed from
exons 1
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
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
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,
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
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
Upper case letters indicate exon and lower case
letters, intron sequences. The splice junctions are marked by arrows
for the first intron only.
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.
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.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
U
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.
(
)
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 C
U 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.
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
4
8. 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.
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.
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.
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.
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.
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.
-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.
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.
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.
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 4
7 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).
8. 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.
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.
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.
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.
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).
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
4
8, 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
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.