1. health and fitness
2. disease

Hereditary Overexpression of Adenosine Deaminase in Erythrocytes:
Studies in Erythroid Cell Lines and Transgenic Mice
By Everett H. Chen and Beverly S. Mitchell
Overexpression of adenosine deaminase (ADA) in red blood
cells is characterized by a marked, tissue-specific increase
in levels of structurally normal ADA mRNA and enzymatic
activity in the erythrocytes of affected individuals, leading
to adenosine triphosphate (ATP) depletion and hemolytic
anemia. This autosomal dominant trait is linked to theADA
gene. To investigate the molecular mechanism responsible
for this disorder, we examined relative reporter gene activity
using constructs containing 10.6 kb of 5’ flanking sequence
and 12.3 kb of the first intron of the ADA gene from the
normal and mutant alleles. No differences in chloramphenicol acetyltransferase (CAT) activity were found in transient
transfection experiments using erythroleukemia cell lines.
Transgenic mice containing the ADA constructs expressed
CAT in the appropriate tissue-specific fashion, with 10’- to
104-foldhigher activity in thethymus. However, CAT activities in erythrocytes and bone marrow of mice containing
high transgene copy numbers did not differ between the
normal and mutant alleles. These results indicate that the
mutation responsible for ADA overexpression is unlikely to
reside in the 5’ and promoter regions or in the regulatory
regions of the first intron. It is possible that the erythroidspecific overexpression of ADA results from a mutation at
some distance from the gene or requires an interaction of a
proximal mutation with more distal DNA elements.
0 1994 by The American Society of Hematology.
A
the amount of catalyticallynormal
ADA protein and a
markedincrease in thelevel of structurallynormalADA
I ~ R N Ait, ~seems likely that the defect is due to increased
production in erythroidprecursors. From linkage analysis,
we have determined that the mutation causing RBC-specific
ADA overproduction lies within or nearthe ADA IOCUS.~
This mutation most likely exists in a region of the gene that
affects either transcription or pre-mRNA processing, since
the mRNA is normal in sequence. In order to search for the
mutation in potential regulatory regions 5‘ to and within the
first intron of the 32-kb ADA gene: we generated reporter
geneconstructsfrom
thenormal and putativelyaberrant
ADA alleles and looked for differencesinexpression
in
erythroid cells using transient transfection assays and
in a
transgenic mouse model.
DENOSINE DEAMINASE (ADA) is a purine catabolic
enzyme that catalyzes the deamination of adenosine
to inosine and 2’-deoxyadenosine to 2‘-deoxyinosine. The
ADA gene is expressedin all tissues, and thus may be categorized as a “housekeeping” gene. However,the level of
expression varies by more than 1,000-fold in different tissues
and developmental states, with the highest level of
expressionin corticalthymocytesand in T lymphoblasts.’This
high level of expression in immature T cells may be critical
for T-cell development, becausemutations in the ADA gene
that abolish its function cause T-cell depletion and severe
combined immunodeficiency disease.’
In contrast to immature T cells, red blood cells (RBCs)
normally have low amounts of ADA activity. Mutations in
the ADAgene thatresult in decreasedor absentactivity
have noeffecton
the function or longevityof theRBC;
however,the
tissue-specificoverproduction
of ADA in
RBCs causes hemolytic anemia. Individuals
with this autosomal dominant disorder haveshortened “Cr-labeled RBC survival, elevated reticulocyte count, splenomegaly, and mild
hyperbilirubinemia. RBCsfrom affected individuals have
40- to 70-fold increased levels of ADA activity, leading to
the increased catabolism of adenosine and adenosinetriphosphate (ATP) depletion, while ADA activities in leukocytes
and fibroblasts are normal.’ The specific molecular defect
underlying the tissue-specific enzyme overexpression has not
been elucidated.
Since the disorderis associated with a 70-fold increase in
From the Departments of Pharmacology and Medicine, Universir.y
of North Carolina, Chapel Hill; and the Graduate Program in Cellular Molecular Biology, UniversiQ of Michigan, Ann Arbor.
Submitfed January 25, 1994; accepted June H, 1994.
Supported by Grant No. I-RO1-A124012from the National Institutes of Health (NIH).
Address reprint requests to Beverly Mitchell, MD, 1106 FLOB
CB#7365, Universiv of North Carolina, Chapel Hill, NC 275997365.
The publication cvstsof this article were defrayed in pctrt by page
charge payment. This article must therefore be herebv marked
“advertisement” in accordance with 18 U.S.C. section 1734 solely to
indicate this fact.
0 1994 by The American Society of Hematology
0006-4971/94/8407-0034$3.00/0
2346
MATERIALS AND METHODS
Cell culture. K562(obtainedfrom
Dr FrancisCollins,NIH)
and K562-BM’ (obtained from Dr George Atweh, SUNY-Brooklyn)
erythroleukemia cells were maintainedin RPMI-1640 with 10%fetal
calf serum. Mouse erythroleukemia (MEL) cells obtained from
Dr
Mike Clarke (University of Michigan) were grown in Dulbecco’s
modified essentialmedium(DMEM)
with 10% fetal calf serum.
Hemoglobin synthesis in K562 cells was induced by treatment with
20 pmol/L hemin for 4 days.
DNase I hypersensitive sites. Hypersensitivesitesweredeterdescribed
10’ cells
mined by an
adaptation of a previously
werewashedwithcoldphosphate-bufferedsaline,resuspension
buffer (RSB) (10 mmol/L Tris, pH 7.4, 10 mmolL NaCI, 5 mmol/
L MgCL), and RSB + 0.5% NP-40. Nuclei were resuspended in 4.5
mL RSB + I mmol/LCaCI2,dividedinto0.5mLaliquots,
and
digested with DNase l (Worthington, Freehold, NJ) atfinal
a concentration of 0 to 2 b,g/mL at 37°C for 15 minutes. The reactions were
stopped with 600 rnrnol/L NaCI, 20 mmol/L Tris, pH 7.4, 5 mmol/
L EDTA, 1 % sodium dodecyl sulfate (SDS). DNA was purified by
proteinase K treatment, phenoVchoroform extraction,andethanol
precipitation. DNase-digested DNA was cut with Hind111 or BamHI,
electrophoresed on a 1% agarose gel, and blotted to nitrocellulose.
Probes were generated by polymerase chain reaction (PCR) amplification of ADA gene hequences and labeled as described.’
Library eonstruction und cloning. GenomicDNA from anaffected individual (E.L.) was prepared from Epstein-Barr virus-transformedBlymphoblastsanddigestedtocompletion
with BamH1.
DNA migrating between 9 and 23 kb on an agarose gelwas excised.
purified with Geneclean (Bio 1 0 1 , La Jolla. CA), and ligated into
the Lambda Fix I1 vector (Stratagene. La Jolla. CA) at the Xho 1
Blocld, Vol 84, No 7 (October l ) , 1994:pp 2346-2353
2347
ADA OVEREXPRESSION IN ERYTHROCYTES
cell stage were transferred to day 0.5 postcoitum pseudopregnant
CD-l females. C57BL/6J X SJL/J mice were obtained from The
Jackson Laboratory (Bar Harbor, ME), and CD-l mice were obtained
from Charles River (Wilmington, MA). Founder transgenic mice
were mated to C57BL/6J mice. All procedures using mice were
approved by the University of Michigan Committee on Use and
Care of Animals and the University of North Carolina Institutional
Animal Care and Use Committee. All work was conducted in accord
with the principles and procedures outlined in the NIH Guidelines
for the Care and Use of Experimental Animals.
To assay for the presence of the transgene, mouse tail DNA was
preparedI6 and amplified by PCR using primers specific for the human ADA gene and primers specific for the mouse p-globin gene
as an internal control. Southern blot analysis was used to determine
the different lineages in the F, generation. In addition to the bands
expected from the usual head-to-tail integration pattern, other bands
were observed that most likely represented partial digestion products,
rearrangements, or deletions. To get an approximate estimate of
copy number, we performed slot blot assays using serial dilutions
of pADA CAT 1 1/12 in 10 pg of mouse genomic DNA as standards.
Quantitation was performed with a phosphorimager (Molecular Dynamics, Sunnyvale, CA).
Preparation of adult mouse tissue extracts and CAT assays were
performed as described by Aronow et al.' Blood was collected by
retroorbital bleeding or cardiac puncture, and RBCs were purified
on Histopaque 1083 (Sigma, St Louis, MO). Solid tissues were
homogenized in 300 pL 0.25 m o m Tris, pH 8, with a Polytron
homogenizer. All tissues were subjected to three freeze-thaw cycles
and centrifugation. Protein concentrations of the supernatants were
determined by the method of Bradford" using bovine serum albumin
as a standard. CAT assays were performed onall tissues from a
single mouse on the same day. Twenty microliters of extract was
heated to 63°C for 10 minutes before addition of acetyl CoA (25
pg) and ['4C]chloramphenicol (0.05 pCi) for a 2-hour incubation.
Extracts were diluted in 10 mg/mL bovine serum albumin if necessary to ensure linear assay conditions. Acetylated products were
separated by thin layer chromatography and visualized by autoradiography. Quantitation was performed by counting the monoacetylated product spots in a liquid scintillation counter.
site by partial fill-in using a 3:l vector:insert ratio. DNA was packaged using Gigapack I1 Gold extract (Stratagene), and Tap 90 cells
were infected. Plaques, 5.4 X lo5, were screened for the 12.6-kb
promoter and 12.3-kb intron fragments. Eight promoter clones and
9 intron clones were plaque-purified. Phage DNA was prepared by
the plate lysis method" with the addition of a second polyethylene
glycol precipitation.
Promoter chloramphenicol acetyltransferase (CAT) constructs
were made by ligating the 2.2-kb NcoI genomic fragments containing
2.1 kb of promoter and 98 bp of 5' untranslated sequence into the
pCAT-Basic vector (Promega, Madison, WI) at the XbaI site to form
pADA CAT 2.2. The 8 clones were sequenced using a primer to an
AluVpA" element within the insert to distinguish the two alleles?
To obtain additional 5' sequences, the 10.5-kb EagI-Sal1 fragments
from the phage genomic clones were ligated into pADA CAT 2.2
digested with EagI and San to generate pADA CAT 11.
The 12.3-kb BarnHI intron fragments were subcloned into pADA
CAT 1 1 at the BamHI site 3'to the CAT gene. pADA CAT 11
(mutant and wild-type) vectors were digested with BarnHI and partially filled in with dGTP and dATP, whereas the intron inserts were
digested with Sal1 and partially filled inwith dCTP and d'TTP.
Ligation yielded pADA CAT 11/12.
Transient transfection assays. K562, K562-BM, and MEL cells
were grown to log phase, washedwith serum-free medium, and
resuspended in serum-free mediumat IO' cells/400 pL. Plasmid
DNA was prepared with Qiagen columns (Chatsworth, CA), followed by centrifugation to remove resin and phenolkhloroform extractions of the supernatant. Forty micrograms of DNA in 100 pL
of phosphate-buffered saline was added to the cells in constant molar
amounts. Electroporation was performed using a Bio-rad Gene
Pulser (Richmond, CA) and 0.4 cm cuvettes; settings were 240 V,
960 pF for K562 lines, and 280 V, 960 pF for MEL cells. After 48
hours, cells were harvested and extracts prepared by three freezethaw cycles, heating to 60°C for 10 minutes,"and collecting the
supernatant. CAT assays were performed by incubating 50 pL of
extract with 0.1 pCi of ['4C]chloramphenicoland 25 pg of n-butyryl
CoA in 125 pL for 2 hours. Reactions were stopped with xylenes,
and the reaction products were extracted and counted.13
Transgenic mouse assays. The -25-kb ADA CAT 11/12 inserts
were liberated from the plasmid vector by digestion with San and
PvuI, separated by agarose gel electrophoresis using Seaplaque-GTG
(FMC, Rockland, ME), extracted with phenol, and precipitated with
ethanol. The DNA was further purified using a Qiagen Tip-20 column and dissolved in 10 mmoUL Tris, pH 7.4,0.25 mmoUL EDTA.l4
Transgenic mice were prepared by the Transgenic Animal Model
Core of the University of Michigan's Biomedical Research Core
facilities. The purified DNA was microinjected into F2 hybrid zygotes from C57BU6J X SJL/J parents at a concentration of 2 to 3
nglpL." After overnight incubation, the eggs that survived to the 2-
RESULTS
To determine potential erythroid-specific regulatory sites
for ADA expression, the ADA gene in K562 erythroleukemia cells was examined for DNase I hypersensitive sites.
Although this assay was, of necessity, performed in a leuke-
mic cell line and not in cells from an affected individual,
we hypothesized that the resulting hypersensitive sites might
still be informative. Figure 1 shows the 5' and first intron
2,
1-
BamH 1
Fig 1. Schemaof the 5' and
first intron regions of the ADA
gene examined for DNase I hy& d l and
persensitive
sites.
Hindlll sites and major DNase I
hypersensitive sites (HSJare indicated.Probes 1 and 2 were
used to scan the 5' flanking region, first exon, and part of the
first intron. Probes 3 and 4 were
used to scanmuchof the first
intron.
2.6 kb
-3
BamH 1
HS
12.3 kb
BamH 1
HS V
l
l
I
""l
Exon 2
Exon 1
-
1
4
7.6 kb
Hind 111
Hind 111
1
9.5 kb 4
Hind 111
1 kb
CHEN AND MITCHELL
2348
Untreated
A.
DNWIUWW 0
1mQ
a
0
37
Hemin-induced
2
c
DNarel(!qmO
1.w
0
0
0
37
2
5
n
76L
7.6 Rh
c
m
12.3Rh
lZ3kb
c
c
regions scanned with this technique. The HindIII digest was
first hybridized with probe 1 to examine the 7.6-kb region
surrounding the first exon (Fig 2A). A strong hypersensitive
site, indicated by the arrow, was found at the exon l-intron
1 junction (see also HS in Fig l ) , and several minor sites
were observed further 5'. Hemin, a weak inducer of hemoglobin synthesis and hence of erythroid differentiation, had
no detectable effect on DNase hypersensitivity, but induced
benzidine positivity in only 30% of treated cells. The hypersensitive sites were confirmed by scanning the 12.6-kb
BarnHI fragment containing 10.5 kb of upstream sequence,
exon 1 and 2 kb of intronwith a second probe from the
opposite side (data not shown).
BarnHI digestion also released the adjacent 12.3-kb fragment encompassing most of the first intron (Fig l ) . There
was a strong hypersensitive site8 to 9 kb into thefirst
intron (Fig 2B) that closely mapped to a previously reported
hypersensitive site' (see also HS V in Fig 1). Interestingly,
hemin treatment decreased the intensity of this site, suggesting that erythroid differentiation might decrease protein
binding to this site. This hypersensitive site was confirmed
by digestion of the DNase-treated DNAwith HindIII and
labeling with a different probe (data not shown).
Genomic DNA from immortalized B lymphoblasts from
an affected individual was digested to completion with
BarnHI and used to prepare a library containing 9- to 23kb fragments. Eight promoter clones were isolated, and the
mutant and wild-type alleles were identified by sequencing
through an AluVpA" region. Nine intron clones were isolated and sequenced in the region mapping to hypersensitive
site V. Six of 9 clones hadan A to C transversion when
compared with the published sequence: creating a new
Fig 2. (A) DNase I hypersensitive sites of Hindlll-digested DNA
hybridized with probe 1. (B)
DNase I hypersensitivesites of
BarnHI-digested DNA hybridized
with probe 3. Major hypersensitive sites are indicatedby
the
arrows.
BstNI site. To determine whether this basechange was linked
to the mutant allele, we traced the inheritance of the BstNI
restriction fragment length polymorphism (RFLP). PCRof
hypersensitive site V was done on DNA samples from 7
members of the immediate family, and the BstNI RFLP was
found to be associated with the unaffected allele. Thus, the
A to C transversion at hypersensitive site V of the first intron
was a neutral polymorphism in the wild-type allele.
The separation andidentification of thetwo alleles allowed us to compare their expression using transient transfection assays. Three pairs of plasmids were prepared: pADA
CAT 2.2, containing 2.1 kb of promoter and 98 bpof 5'
untranslated sequence; pADA CAT I 1, containing 10.6 kb
of upstream sequence; andpADA CAT 11/12, containing
both 10.6 kb of upstream sequence and 12.3 kb of intron I
(Fig 3). Three cell lines were tested: K562 erythroleukemia
cells, which express small amounts of embryonic and fetal
hemoglobins; K562-BM cells, which express a small amount
of adult &globin as well; and MEL cells, which express
some adult mouse globin. As seen in Fig 3, no significant
differences in CAT activities were observed between the two
alleles, regardless of construct or cell type.
Since the erythroleukemia cell lines do not recapitulate
normal erythroid differentiation, itremained possible that
the mutant allele might be overexpressed as a consequence
of its failure to undergo normal downregulation during the
maturation of normal erythroid progenitors. In addition, it
seemed possible that integration of the construct into the
genome might be required for its regulation. We therefore
made transgenic mice containing either mutant or wild-type
ADA CAT 11/12. All founder mice were bred, and only FI
mice were analyzed to avoid the problemofmosaicism.
2349
ADA OVEREXPRESSION IN ERYTHROCYTES
Exon 2
Exon l
I
B/S
I
N
..._I
I
/l
E N
I
B/s
B/S
-
Relalwe CAT Actlvlty
1 kb
Fig 3. Results
of
transient
transfection assaysusing
mutant and wild-type pADA CAT
constructs. Shown at the top is
a map of the restriction sites
used to make the CAT
constructs. (BAS) B a d 1 or Sal,
B s d l in the endogenous
ADA
gene, and Sal from the polylinker
of
the phage genomic
clones; (NI Ncd; (E) Eagl. Mutant
and wild-type constructs were
transfected into MEL, K562-BM,
and K562 cells. Relative CAT activities are shown in the adjacent table.
Mut
MEL
K562-BM
K562
1
1
1
23
14.4
41
4.2
21.3
S.?
46
12.3
26
64
16.8
2.3
4.7
31.2
2.3
4.8
32.0
2.2
pADA CAT 2.2
W1
m
-
Mu1
pADA CAT 11
4
Wt
pADACAT 11/12
wt
Twelve lines of mice with the mutant allelic construct and
7 lines with thewild-type allelic construct wereobtained. All
mice with either the mutantor wild-type transgene expressed
CAT activity. Those mice with low copy
numbers of the
transgene (generally < 10 copies) expressed CAT
in the thymus, spleen, and marrow
in a consistent fashion,with expression in the thymus being greater by at least two orders of
magnitude (Fig 4A). Expression in the brain, liver, and kidney was not always detectable, and no expression was detected in RBCs with either allele. Treatment of mice with
phenylhydrazine to induce hemolysis andreticulocytosis did
not induce CAT activity in RBCs (data not shown).
In order to obtain detectable RBC CAT expression, we
examined the mice with transgenes in high copy numbers
(Fig 4B). Sevenlineshadthemutant
allelictransgenein
high copy numbers, and 3 lines had the wild-type allele in
high copy numbers. Again, thymus had the highest level of
expression; marrow and spleenhad 100-fold less. Expression
in the brain, liver, and kidney was lower than in the thymus
by 1,000- to 10,000-fold. CAT activities in RBCs were low,
but easily detectable in these mice. There was no consistent
difference in the levelof expression between mutant and
wild-typeallelic constructs in RBCs. Table 1 shows RBC
CAT activity normalized to CAT activity in the thymus or
kidney. In 6 lines with the mutant allelic construct, normalized RBC CAT activities were not significantly greater than
in the 3 lines with the wild-type allelic construct. Only line
978413 had significantly elevated RBC CAT activity, a finding which may be attributed to itsintegration site in the
genome.
DISCUSSION
Elevated adenosine deaminase levels in RBCs have been
reported in numerous hematological disorders, such as Diamond-Blackfan syndrome," AIDS," certain instances of anisopoikilocytosis," myelodysplastic and myeloproliferative
syndromes," and primaryacquiredsideroblastic
anemia."
ADA activity in these disorders is usually elevated by 2- to
10-fold andis most likely secondary to defectiveerythropoiesis. When ADA activity is 40- to 110-fold above normal,
it istheprimary
defectandcauses hemolytic anemia by
depleting the RBC of ATP. Four casesof ADA overproduction associated
with
hemolytic
anemia
have
been rep ~ r t e d , ~but
. ~only
~ - ~one
~ kindred has been demonstrated to
have an autosomal dominant mode of inheritance with the
genetic defect linked to the ADA 10cus.~ This
association
made it feasible to attempt toidentify the precise molecular
defect in this disorder.
In earlier studies, we had determined that increased RBC
ADA activity was due to an increased
level of ADA mRNA.4
A cDNA clone from areticulocytelibrary
of an affected
individual was completely normal in sequence. This result
was confirmed in other family members aswell by chemical
mismatchcleavageanalysis
(E. Chen, unpublished data).
Furthermore, in six other clones from the
reticulocyte library,
the 5' and 3' untranslated regions were examined for base
alterations. None were found, suggesting that cytoplasmic
ADAmRNAwas
unlikely tobemore
stable. Whenthe
1. l-kb XhoI fragment encompassing the proximal promoter
region of the ADA gene was cloned from
an affected individual, no sequencealterations were found inthe 8 clones examined (E. Chottiner, unpublished data). From these findings,
it was clear that the mutation was very unlikely to reside in
the coding region or in the promoter of the gene. We therefore proceeded to look for sitesof ADA gene regulation
both in more distal 5' regions andwithin the first intron. This
intron contains a classical enhancer that boosts expressionin
T-lymphoblast cell lines, as well as a locus control region
(LCR) that confers copy number-dependent, position-independent expression of atransgeneinthethymus.26
Other
regions of the ADA gene, including the other introns and
the 3' flanking sequences, may also contain regulatory regions, but none have been described thus far.
To locate possible erythroid-specific regulatory regions,
CHEN AND MITCHELL
4 nnnn
I
uuuu
l
A
1000
100
10
1
.l
.01
.001
Thymus
rnut wt
10000
Spleen
rnut wt
RBC
rnut wt
Marrow
rnut wt
Brain
mut wt
Liver
rnut wt
Kidney
rnut wt
4
il4
B
rll
Thymus
rnut wt
Spleen
rnut wt
RBC
rnut wt
Marrow
rnut wt
Brain
mut wt
Liver
rnut wt
Kidney
rnut wt
Fig 4. CAT expression in transgenic mouse tissues. (A) ADA CAT 11/12 expressionin mice with the transgene in low copy numbers. Shaded
bars represent the five independent lines containing the mutant allelic construct; open bars represent the four lines containing the wild-type
allelic construct. (B) ADA CAT 11/12 expression in mice with the transgene in high copy numbers. Shaded bars represent the seven lines
containing the mutant allelic construct; open bars represent the three lines containing the wild-type allelic construct.
the ADA gene was scanned lor DNase 1 hypersensitive sites
to 14.3
from 10.6 kbupstream of the translationstartsite
kb i n t o the first intron. Two rnajor hypersensitive sites were
found in K562 erythroleukemia cells. Thc one at the first
exonwashemin-insensitiveand
corresponds to the region
reported to he involved in transcriptional attenuation272X (discussed below). The othermajorsite
was in the middle of
the first intron, and corresponds to hypersensitive site V , as
2351
ADA OVEREXPRESSION IN ERYTHROCYTES
Table 1. Ratio of Transgenic Mouse RBC CAT Activity to Thymus
or Kidney CATActivity
1.9
Mutant lines
9974 a
9774 b
9774 a'
9774 d
9784 b
9782 a
9773
Wild-type lines
8951 a
8951 b
8951 c
RBC/Thymus ( ~ 1 0 ' )
RBC/Kidney ( ~ 1 0 ' )
3.3
2.5
10.5
6.2
500
4.2
6.6
0.83
1.o
0.69
2.5
19
0.84
0.28
4.7
1.4
1.1
1.1
0.70
described by Aronow et al.' This site is found in many tissues
and cell lines, including human thymus, transgenic thymus,
transgenic spleen, Molt4 T lymphoblasts, and CEM T
lymphoblasts, as a minor hypersensitive site. Hypersensitive
site V is very strong in fibroblasts, but is not seen in the Bcell line GM 3638, and is distinct from the site responsible
for the very high levels of thymic expression. Whether or not
these hypersensitive sites exist in the erythroid progenitors of
affected individuals or constitute important sites for ADA
down-regulation, which, when disrupted, would increase
ADA expression, could not be determined from these studies.
To determine the physiologic significance of these regions, we examined the relative CAT expression of constructs containing the mutant and wild-type alleles. In our
transient transfection assays, using three cell lines that represent to some extent different developmental states of erythroid cells, we saw no difference in expression between the
two alleles. Transient transfection into erythroid cell lines
has some drawbacks, which have been similarly encountered
by those studying the non-deletion form of hereditary persistence of fetal hemoglobin (HPFH), where point mutations
in the y-globin gene promoters are responsible for the phenotype.29In transient transfection of K562 cells with the - 175
T -+ C y-gene, there is only a 3- to 4-fold increase in expression compared with the wild-type promoter. In vivo, however, this HPFW gene mutation increases expression by over
l O O - f ~ l dOther
. ~ ~ HPFH mutations (-202 C + G, - 196 C +
T, and - 117 G + A) have no effect in transient transfection
assays." Several possibilities could explain both our results
and those in the HPFH experiments. First, the transfected
gene may not include all the necessary sequence information.
Second, it is possible that overexpression would be manifest
only after stable integration into the genome. The chicken
lysozyme gene 'A elements'/matrix attachment regions3' or
the human interferon p gene scaffold attached regions33enhance expression in stable transfection assays butnot in
transient assays, as does the &globin LCR (except for hypersensitive site-2), which exerts its effect by creating open
chromatin regions or by acting as a boundary element.14
Third, if ADA overexpression were the result oflackof
downregulation during erythroid development, experiments
in cell lines wouldonly provide a static view at a single
stage of erythropoiesis.
Because of these potential limitations, we also compared
the expression of mutant versus wild-type alleles in a
transgenic mouse model. Our transgene resembled ADA
CAT 4/12, as described by Aronow et al.' They used CAT
as a reporter gene, and their largest transgene contained 4
kb of 5' flanking sequence (compared with our 10.6) and a
12.8-kb BssHII first intron fragment (comparable with our
12.3-kb BumHI fragment). The pattern of expression in our
mice as compared with their mice was the same: thymus
had by far the highest activity, followed by spleen and marrow, and then brain and liver. Transgene activities paralleled
endogenous ADA activities in human tissues except in the
marrow, where it paralleled the mouse expression level. The
absolute levels of CAT activity in our mice were 5- to 50fold lower than those of Aronow et all in all tissues, and we
speculate that the additional 6.6 kb of 5' flankingregion
contains repressor activity.
No difference in RBC expression was apparent between
the two alleles. There are several possible explanations for
this finding. First, andwe believe most likely, the causal
mutation is notin our transgene. It could be further 5' to
the 10.6-kb fragment, in other introns, or at the 3' end.
Second, the transgene could contain the causal mutation, but
the mutation maynotbe
expressed outside of its native
genomic context. For example, the causal mutation may have
to interact with normal LCR elements that are not present
in our constructs, but are present in more distant regions of
chromosome 20. Other possibilities exist, but are far less
likely. For example, failure to detect overexpression could
be due to the absence in the construct of the last 31 bp of
exon l and the adjoining 2 kb of first intron. However, we
have not found any sequence differences between the two
alleles within the initial 594 bases of the first intron, the
region that has been implicated in transcriptional attenuation
of ADA gene e x p r e s ~ i o n . ~The
~ , ~demonstration
'
of correct
tissue-specific expression of our transgene in the absence of
this region makes its functional role uncertain. Indeed, there
is increasing evidence that transcriptional attenuation of
mammalian genes may be an artifact of the xenopus oocyte
system used to study this p h e n ~ m e n o n . ~It~is, 'also
~ conceivable that the appropriate trans-acting factor(s) involved in
RBCADA overexpression are notpresentin murine erythroid precursors. However, the mouse model has served
very well for studies of globin gene regulation and we are
not aware of any examples of erythroid-specific transcription
factor discrepancies between mice and humans.
Further extension of this work would require the cloning
of the entire mutant and wild-type ADA genes, including
large 5' and 3' flanking regions, in yeast artificial chromos o m e ~or~bacterial
~
artificial chromo~omes.~'Until better
systems develop for studying human erythropoiesis, introduction of this large transgene into the mouse remains the
most direct approach. The feasibility of introducing large
transgenes has been demonstrated r e ~ e n t l y . ~Elucidating
~.~'
the mechanism of ADA overexpression in RBCs could provide new insights into the regulation of gene expression
during erythroid development.
2352
CHEN AND MITCHELL
18. GladerBE,Backer
K, DiamondLK:Elevatederythrocyte
adenosine deaminase activity in congenital hypoplastic anemia. N
We thank David Ginsburg for his continuous encouragement and
Engl J Med 309:1486. 1983
advice. Wealso thank Craig Thompson for the DNase hypersensitive
19. CowanMJ, Brady RO. Widder KJ: Elevatederythrocyte
siteassay,DeborahGumuciofortheK562transienttransfection
adenosine deaminase activity in patients with acquired immunodetiassay, Sally Camper and Thom Saunders for generation of transgenic
ciency syndrome. Proc Natl Acad Sci USA 83: 1089, 1986
mice, Bruce Aronow and Dan Wiginton for sharing their experience
20. Novelli G, Stocchi V, Giannotti A, Magnani M, Dallapiccola
and technical knowledge, and Elaine Chottiner and Tom Gribbin for
B:
Increasederythrocyteadenosinedeaminaseactivitywithout
their initial efforts on this project.
haemolytic anaemia. Hum Hered 3 6 3 7 . 1986
21. Van Der WeydenMB, Harrison C.Hallam L, McVeigh D.
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