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Brief report
Human erythroid porphobilinogen deaminase exists in 2 splice variants
Alexander N. Gubin and Jeffery L. Miller
Human porphobilinogen deaminase
(PBGD) is, reportedly, encoded by 2 distinct messenger RNAs (mRNAs) transcribing from a single gene. The ubiquitous
form of the PBGD gene product is often
used as an endogenous reference in gene
expression studies because it is pseudogene free and has minimal transcriptional
variability among tissues. A distinct erythroid-specific gene product has also
been described because of the alternate
splicing of the gene. Here is reported the
existence of an additional erythroid-specific isoform of PBGD mRNA in primary
cells. (Blood. 2001;97:815-817)
© 2001 by The American Society of Hematology
Introduction
The third enzyme of the heme synthesis pathway, porphobilinogen
deaminase (PBGD), is expressed as 2 isoforms: the so-called
“housekeeping” form present in all cell types, and a second isoform
expressed only in erythroid cells.1-4 The autosomal dominant
disorder acute intermittent porphyria (AIP) is caused by mutations
in the PBGD gene.5 Clinically, AIP gene variants may or may not
affect enzymatic levels within erythroid cells.3,6,7 One study
reported low erythrocyte PBGD activity in blood donors, whereas
lymphocyte PBGD activity was within the normal range.8 A
possible mutation within the erythroid-specific part of the PBGD
gene was proposed to explain this finding, but was not further
explored. On the basis of the extensive genetic studies of AIP,
genomic analyses have also shown that PBGD is a pseudogenefree, single-copy gene, thus making it a useful control for RNA
analyses.9-14 We sought to use PBGD expression as both an
erythroid-specific and housekeeping control for our studies of
erythroid-cell gene expression patterns. Unexpectedly, our efforts
revealed the existence of 2 distinct erythroid-specific PBGD
messenger RNA (mRNA) transcripts.
Study design
Reverse transcriptase–polymerase chain reaction
Total RNA was purified from CD34⫹ cells collected on specified days after
growing in erythropoietin-supplemented media using TRIzol reagent (Life
Technologies, Rockville, MD) according to the manufacturer’s directions.
Reverse transcription (RT) was performed using oligo- (dT) primer and
Superscript Reverse Transcriptase (Life Technologies) as suggested by the
manufacturer at 42°C for 50 minutes. One microliter of RT reaction was
used in 50 ␮L polymerase-chain reaction (PCR) amplification with
appropriate primers as follows: initial denaturation at 95°C for 1 minute,
denaturing at 95°C for 15 seconds, annealing at 68°C for 15 seconds, and
extension at 72°C for 20 seconds for 28 cycles. PCR products were
separated in 1.2% agarose gel and stained with ethidium bromide. For
PBGD amplification, the following primers were used: 5⬘-ACACAGCCTACTTTCCAAGCGGAGCCAT-3⬘ (primer 1), 5⬘-TGGATCCCTGAGGAGGGCAGAAG-3⬘ (primer 2), 5⬘-TCTTGTCCCCTGTGGTGGACATAGCAAT-3⬘ (primer 3). Primers 1 and 3 specifically amplify the
From the Laboratory of Chemical Biology, National Institute of Diabetes and
Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD.
Submitted August 15, 2000; accepted September 29, 2000.
Reprints: J. L. Miller, Laboratory of Chemical Biology, Bldg 10, Rm 9B17,
National Institute of Diabetes and Digestive and Kidney Diseases, National
BLOOD, 1 FEBRUARY 2001 䡠 VOLUME 97, NUMBER 3
housekeeping PBGD. Primers 2 and 3 specifically amplify an erythroid
PBGD. The PCR products were completely sequenced using a dRhodamine
DNA sequencing kit (PE Biosystems, Foster City, CA).
Northern blotting
Human Immune System Multiple Tissue Northern (MTN) Blot II was
processed as recommended by the manufacturer (Clontech, Palo Alto, CA).
Briefly, the membrane was prehybridized in ULTRAhyb hybridization
buffer (Ambion, Austin, TX) at 42°C for 30 minutes. The buffer was
replaced with the fresh ULTRAhyb containing 32P-labeled probe and
hybridized at 42°C for 16 hours. The membrane was washed in low- and
high-stringency buffers (Ambion) according to the manufacturer’s protocol
and subjected to autoradiography. Probes specific for the housekeeping
(probe H) and an erythroid alternative (probe EA) part of PBGD used in
Northern blotting were generated by PCR, purified by QIAquick PCR
Purification Kit (Qiagen, Valencia, CA) and labeled using the DNAlabeling beads (⫺dCTP) (Amersham Pharmacia Biotech, Piscataway, NJ).
Primers 5⬘-GGTACTGAGGAAGGTTAAAGGGA -3⬘ and 5⬘-AGGATTACCCCTCGAACACCTGCAGAAAAAGTCCCCAGGT-3⬘ were used to amplify the 4138-4315 portion (corresponding to intron between exons 2 and
3) of the PBGD gene (GenBank accession number M95623). Primers
5⬘-GGCTCTGCGGAGACCAGGAGTCAG-3⬘ and 5⬘-TTACCAGACATGGCTCCGCTTGGAA-3⬘ were used to amplify the 959-1101 region (in
exon 1) of the gene. Genomic DNA from K562 cells was used as a template
for PCR amplification.
Results and discussion
The PBGD gene comprises 15 exons encoded within a 10-kilobase
(kb) region of chromosome 11q23.3.4 Figure 1 depicts the structure
of the human PBGD mRNA. Different isoforms are produced
through alternative splicing of exon 1 and exon 2. Splicing exon 1
to exon 3 results in expression of the housekeeping form of PBGD
(PBGD-H). The combination of exon 2 and exon 3 produces an
erythroid form of PBGD (PBGD-E). Figure 2A shows the results of
PT-PCR amplification of PBGD gene products in CD34⫹ cells
grown in the presence of erythropoietin. Cells were collected over
Institutes of Health, Bethesda, MD 20892; email: [email protected].
The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked ‘‘advertisement’’ in accordance with 18 U.S.C. section 1734.
© 2001 by The American Society of Hematology
815
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816
BLOOD, 1 FEBRUARY 2001 䡠 VOLUME 97, NUMBER 3
GUBIN and MILLER
Figure 1. Structure of human PBGD transcripts. Probe H: Probe specific for
housekeeping PBGD. Probe EA: Probe specific for erythroid alternative PBGD.
Relevant exons are shown. AUG and UAA depict start and stop codons, respectively.
12 days, total RNA was purified, reverse transcribed with oligo(dT) primer, and amplified using housekeeping or erythroidspecific primers for PBGD. Primers 1 and 3 were used to amplify
housekeeping PBGD (expected product size is 213 base pairs [bp]),
and primers 2 and 3 were used to amplify erythroid PBGD
(expected product size is 177 bp). Although the PBGD-H product
was present in all samples, the PBGD-E9 PCR product (sequence
confirmed) was amplified only after several days in culture
consistent with erythroid cell commitment and differentiation.
Additionally, a 353-bp product was amplified with primers 2 and 3
(PBGD-EA). Complete sequencing of PBGD-EA revealed an
alternatively spliced form of erythroid PBGD containing the intron
between exons 2 and 3, thus extending the 5⬘ untranslated region of
the erythroid transcript by 176 bp. Two downstream introns
between exons 3, 4, and 5 were correctly spliced in both 177-bp and
353-bp PR-PCR products.
Because PBGD-EA has not been reported elsewhere, we
attempted to confirm the existence of this transcript in primary
tissues (Figure 2B). Human Immune System MTN Blot II (Clontech) was hybridized with probes specific either for the housekeeping (probe H) or the alternate erythroid (probe EA) forms of PBGD.
The probe EA (Figure 1) corresponds to intron between exons 2
and 3. Hybridization with the probe H produced a band of expected
size (about 1.5 kb) in all 6 tested tissues. In contrast, a distinct
1.5-kb band was observed after hybridization with the probe EA
only in bone marrow and fetal spleen. This confirms the erythroidspecific expression of PBGD-EA and provides evidence for its
presence in primary tissues. A computer-assisted BLAST query of
the expressed sequence tag (EST) database with the 176-bp
intronic sequence identified 3 additional human ESTs (GenBank
accession nos. T84131, R06321, and T57423) that retain this
intronic fragment in processed transcripts. All 3 ESTs were derived
from fetal liver or spleen libraries supporting our hypothesis that
PBGD-EA represents an erythroid-specific isoform. BLAST queries failed to identify any EST currently deposited in GenBank that
match the human PBGD-E isoform.
Erythroid-specific transcription of the PBGD gene has been
examined previously in the context of promoter transactivation.15,16
We report here that human PBGD is expressed as 3 different
transcripts, 2 being erythroid specific. The additional 176-bp
fragment of PBGD-EA isoform contains at least one known
polymorphism in humans.5 The existence of this second erythroid
PBGD isoform has not been reported elsewhere, despite extensive
Figure 2. Confirmation of existence of the PBGD erythroid alternative transcript. (A) RT-PCR assay of PBGD transcipts. Total RNA from CD34⫹ cells collected
on indicated (0-12) days after addition of erythropoietin was used for RT-PCR as
described in study design. M: 100-bp DNA ladder (Life Technologies). N: No DNA
added control. (B) Northern blot analysis of PBGD-H (left panel) and PBGD-EA (right
panel) expression in human hematologic tissues. Lanes 1 to 6 contain, in order, 2 ␮g
polyA⫹ RNA from human spleen, lymph node, thymus, peripheral blood leukocytes,
bone marrow, and fetal liver. 32P-labeled probe H (panel A) or probe EA (panel B) was
used for hybridization. RNA size marker bands are indicated in between the blots.
analyses of this gene over the last 15 years. Interestingly, a previous
study of erythroid-specific PBGD expression failed to reveal an
alternate erythroid transcript in HEL cells.9 Despite our clear
demonstration of PBGD-EA in primary erythroid cell cultures,
bone marrow, and fetal liver, we were unable to show expression of
the alternate transcript in HEL cells (not shown). Our discovery
highlights the prospects of identifying unexpected, primary tissuespecific gene expression patterns even among extensively studied
genes. Erythroid-specific isoforms involving the first 3 exons of the
second (5-aminolevulinate dehydratase)17 and the fourth (uroporphyringen III synthase)18 heme biosynthesis enzymes have also
been described. Transcripts from an alternate promoter within the
first intron of the NF-E2 are also associated with erythroid
specificity.19,20 Similarly, alternate transcriptional start sites and
splicing result in the erythroid band 4.1 gene products.21 In all
cases, the erythroid-specific mRNAs contain an alternate 5⬘ region.
These data suggest erythroid-specific cell development is accomplished at the genomic level via multiple mechanisms, including
shared patterns of gene organization, transactivation, and RNA
maturation.
Acknowledgment
We thank the National Institutes of Health Department of Transfusion Medicine for providing the primary cells used in this study.
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2001 97: 815-817
doi:10.1182/blood.V97.3.815
Human erythroid porphobilinogen deaminase exists in 2 splice variants
Alexander N. Gubin and Jeffery L. Miller
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