(Received for publication, July 25, 1995)
From the
Band 7.2b is an integral membrane phosphoprotein absent from the
erythrocyte membranes of patients with hereditary hydrocytosis, a
hemolytic anemia inherited in an autosomal dominant fashion and
characterized by stomatocytic red blood cells with abnormal
permeability to Na and K
. The precise
role of band 7.2b is unknown, but it may interact with other proteins
of the junctional complex of the membrane skeleton. To gain additional
insight into the structure and function of this protein and to provide
the necessary tools for further genetic studies of hydrocytosis
patients, we determined the sequence of the full-length human band 7.2b
cDNA, characterized the genomic structure of the band 7.2b gene,
studied its pattern of expression in different tissues, and
characterized the promoter of the gene. The composite band 7.2b gene
cDNA was 3047 base pairs in length. Northern blot analysis revealed a
wide tissue distribution of expression of the band 7.2b gene, with
utilization of alternative polyadenylation signals generating
transcripts of 2.2 and 3.1 kilobases. Cloning of the band 7.2b
chromosomal gene revealed that it is composed of seven exons
distributed over 40 kilobases of DNA. The band 7.2b gene promoter was
identified as a TATA-less, (G + C)-rich promoter with a typical
InR recognition sequence and a single transcription initiation site. It
directed high level expression of a reporter gene in both erythroid and
nonerythroid cells. An imperfect simple sequence repeat polymorphism
was identified in the 5`-flanking DNA, and an assay was developed for
its analysis by PCR.
Hereditary stomatocytosis consists of a heterogeneous group of disorders characterized by the presence of mouth-shaped (stomatocytic) erythrocytes on peripheral blood smears. The clinical severity of hereditary stomatocytosis is variable; some patients experience hemolysis and anemia, while others are asymptomatic(1) . The red cell membranes of these patients usually exhibit abnormal permeability to the univalent cations sodium and potassium, with resultant modification of intracellular water content(2, 3, 4, 5) .
Hereditary
hydrocytosis is one subset of the stomatocytosis syndromes. Hydrocytic
erythrocytes are characterized by an increased intracellular sodium and
decreased intracellular potassium content(6, 7) . The
transport rate of potassium via both the
Na/K
pump and the
Na
, K
/2Cl
cotransport system is increased. It has been hypothesized that
there is an ion leak in these erythrocytes, with an inadequate
compensatory increase in transport by the
Na
/K
pump.
The red cell membranes
of many hydrocytosis patients lack a 31-kDa protein, band
7.2b(5, 8, 9, 10, 11, 12, 13) .
Band 7.2b is an integral membrane phosphoprotein whose function is not
completely understood. It has been hypothesized that band 7.2b may
support, activate, or regulate an as yet unidentified associated ion
channel(7) . Recent evidence showing a potential interaction
between band 7.2b and the membrane skeleton protein adducin
suggests that band 7.2b may be a part of the junctional complex of the
membrane skeleton(14) . In this capacity, band 7.2b may
participate in a variety of specialized cellular
functions(15) . Protein immunoblotting has shown the presence
of band 7.2b reactivity in a wide variety of tissues and in a wide
cross-species distribution(12, 16) . The human band
7.2b protein has been purified, and partial cDNAs corresponding to its
coding region have been
cloned(12, 13, 16, 17) . Band 7.2b
shares no sequence homology to other known proteins.
To gain
additional insight into the structure and function of this protein and
provide the necessary tools for further genetic studies of hydrocytosis
patients, we constructed a composite full-length human band 7.2b cDNA
containing the 5`- and 3`-untranslated sequences compared with
previously cloned cDNAs. We also cloned the chromosomal gene encoding
band 7.2b, characterized the genomic structure of the band 7.2b gene,
studied its pattern of expression in different tissues, and identified
its promoter. An imperfect simple sequence repeat (SSR) ()polymorphism in the band 7.2b gene was identified, and a
PCR-based assay for its analysis was developed.
Figure 1: Structure of the human band 7.2b cDNA. A diagram of the human band 7.2b cDNA is shown. The open reading frame is denoted by the box. The locations of the initiation and termination codons, polyadenylation signals, and an Alu repeat are shown. Sequence obtained by 5` RACE is denoted by a hatched box. Probes used in genomic library screening and Northern blotting (see ``Materials and Methods'') are shown.
Figure 4: Genomic organization of the human band 7.2b gene. Five overlapping clones containing the band 7.2b gene were isolated from a human genomic DNA library. These clones spanned a distance of over 40 kb. A restriction map for EcoRI (E) is shown. Individual exons (not to scale) are denoted by closed boxes. The location of an imperfect simple sequence polymorphism in the 5`-flanking DNA of the band 7.2b gene is indicated by an arrow and SSR.
Figure 2: Mapping the 5` end of the human band 7.2b cDNA. Primer extension analysis of the 5` end of human band 7.2b mRNA. Primer extension was carried out using 10 µg of K562, HEL, and HL60 total RNA or tRNA as template. The size of the extension products, 141 nucleotides, indicates that the 5` end of the mRNA is located at position -61 relative to the adenosine of the initiator methionine codon. The cDNA sequence of this additional 5`-untranslated cDNA was determined by 5` RACE and is shown in Fig. 3.
Figure 3: Nucleotide sequence with encoded amino acid sequence of human band 7.2b cDNA. The composite nucleotide sequence shown was determined from 5` RACE products and limited sequencing of genomic DNA clones. The composite band 7.2b cDNA is 3047 bp in length, predicting a 288-amino acid polypeptide of 31 kDa, consistent with the mobility previously observed on SDS-polyacrylamide gel electrophoresis gels. The initiator methionine, ATG, the termination codon, TAG, and both polyadenylation signals, AATAAA, are underlined. The locations of exons are denoted by triangles.
Secondary structure predictions of the band 7.2b protein predict the
presence of three domains, a highly charged NH-terminal
domain, a hydrophobic stretch that encodes a potential
membrane-spanning domain, and a COOH-terminal domain composed of
sheet and
helix. There is no concordance between the exon
organization of the gene and the location of these three domains in the
protein.
Figure 5:
An Imperfect SSR polymorphism in the human
band 7.2b gene. A, identification of polymorphic alleles using
a PCR-based assay. A SSR was identified in the 5`-flanking DNA of the
band 7.2b gene. Oligonucleotide primers corresponding to sequences
flanking the SSR, one of which was end-labeled with
[P]ATP, were used to amplify genomic DNA in a
PCR reaction. Amplification products were denatured and electrophoresed
in an acrylamide gel, the gels were autoradiographed, and the results
were analyzed. Five alleles of varying size were identified in
individuals from different racial backgrounds using this PCR-based
assay of genomic DNA, with the most common allele arbitrarily called N. B, sequence of the imperfect simple sequence repeat.
Differences in the sequences of the five identified alleles are shown.
The most common allele is denoted as N.
Hereditary xerocytosis, also known as dehydrated stomatocytosis, is a clinically less severe, co-dominantly inherited stomatocytic syndrome without band 7.2b deficiency(6, 7) . The role of band 7.2b in hereditary xerocytosis, if any, is unknown. We used this polymorphism to study the two affected homozygous probands of a xerocytosis kindred. The probands are the products of a consanguineous mating (VI-30 and VI-31 in (34) ) and are presumably homozygous for a single mutation. One proband was homozygous for the band 7.2b gene common allele(N, N); the other was heterozygous for alleles N and N + 2, suggesting that a mutation of the band 7.2b gene is not responsible for the stomatocytosis observed in this kindred.
Figure 6:
Northern blot Analysis. Top,
samples of 2 µg of poly(A) RNA from various human
tissues were hybridized to a [
P]dCTP-labeled
cDNA fragment (probe 1). Abundant message was detected in all tissues
examined except brain, colon, and ovary. Middle, the same
blots were stripped and rehybridized to probe 2. Bottom, the
same blots were stripped and hybridized with a 2.0-kb human
-actin
cDNA probe as a control for loading.
Figure 7: 5`-Flanking genomic DNA sequence. The nucleotide sequence of the 5`-flanking genomic DNA of the human band 7.2b gene is shown. Consensus sequences for potential DNA-protein binding sites are underlined. The locations of a recognition sequence for a transcription initiator (InR) site and the initiator methionine codon are double underlined. The junction between exon 1 and intron 1 is shown by the inverted triangle.
To investigate if the region from -531 to +37 was capable of directing expression of a reporter gene, test plasmids pHB7-forward or pHB7-reverse (Fig. 8) were transiently transfected into erythroid (K562) or nonerythroid (NIH3T3) cells. The relative luciferase activity was determined 24 h after transfection and compared with the activity obtained with the parental promoterless vector. As shown in Fig. 8, the putative band 7.2b promoter plasmid, pHB7-forward, directed high level expression of the luciferase reporter gene in both erythroid and nonerythroid cells. In addition, the plasmid with the promoter in reverse orientation, pHB7-reverse, also directed expression in both cell lines but at a lower level, suggesting a bidirectional capability of the band 7.2b promoter.
Figure 8: Activity of the band 7.2b gene promoter in erythroid and nonerythroid cell lines in transient transfection assays. Plasmids containing 5`-flanking DNA of the band 7.2b gene inserted upstream of the firefly luciferase gene were transfected into K562 or NIH3T3 cells as described. Relative luciferase activity was expressed as that obtained from the test plasmids versus the activity obtained from the promoterless plasmid pGL2B plasmid, taking into account the transfection efficiency. The data are means ± S.D. of at least six independent transfection experiments. The band 7.2b gene fragment displays bidirectional activity in erythroid and nonerythroid cells.
The function of band 7.2b protein is unknown. Its importance, however, is underscored by its wide tissue and species distribution. In humans, Northern blot analysis detected message in essentially every tissue analyzed except brain. Protein immunoblotting using a polyclonal anti-human band 7.2b antibody showed reactivity in human liver and kidney tissue but none in brain, cardiac tissue, or ileum(12) . Reactivity to a monoclonal antibody directed against human erythrocyte band 7.2b has been observed in the erythrocyte membranes of a wide variety of species including frog, rat, chicken, rabbit, pig, cow, and sheep(16) .
Genetic analyses of patients with hereditary stomatocytosis (hydrocytosis) have been previously hampered by the lack of knowledge of the genomic sequences of the band 7.2b gene, including promoter sequences, as well as by the lack of suitable polymorphic markers in the gene. Although hereditary hydrocytosis appears to be a dominantly inherited condition and affected individuals are presumed to be heterozygous for the disorder, there is complete deficiency of band 7.2b protein(5, 8, 9, 10, 11, 12, 13) . This observation suggests a dominant-negative effect, where defective band 7.2b protein may interact with wild-type band 7.2b protein, forming an unstable oligomeric protein complex. Band 7.2b protein does appear to form oligomers. Alternatively, the defect may be present in a protein that interacts with band 7.2b, with the abnormal interaction leading to secondary degradation of band 7.2b protein. Band 7.2b-deficient hydrocytosis patients apparently do not suffer any nonhematologic symptomatology(7) .
A variety of mutations causing human disease have been described that affect RNA processing and translation (reviewed in (36) ). These mutations may be associated with dramatic decreases in steady state mRNA levels. This fact has important implications for mutation detection methods. Reverse transcriptase-PCR-based techniques are unlikely to detect mutations with markedly decreased mutant mRNA levels, necessitating study of these mutations at the level of genomic DNA(36) . A recently described nonsense mutation in the band 3 gene associated with decreased mRNA levels, band 3 Noirterre, was not detected using cDNA mutation detection techniques but was only detected in genomic DNA(37) . Characterization of the genomic structure of the band 7.2b gene will allow structural studies of the band 7.2b gene in patients with hereditary hydrocytosis.
Cleavage of primary mRNA transcripts and the addition of poly(A) to the newly formed 3` end of the transcript downstream of the highly conserved polyadenylation signal AAUAAA are features of almost all eukaryotic mRNAs(38) . 3`-untranslated regions can have an important influence on mRNA function including translation, localization, stability, and in some cases even gene transcription (39) . Alternative polyadenylation of 3`-untranslated regions may play a role in determining developmental or tissue-specific preferences for various mRNA transcripts(39, 40) . The role of the two band 7.2b gene transcripts that vary in their 3`-untranslated regions is unknown.
The bidirectional activity of the band 7.2b gene promoter is similar to that observed in a wide variety of gene promoters, particularly housekeeping gene promoters such as those of the genes encoding dihydrofolate reductase, HMG CoA reductase, and hypoxanthine phosphoribosyltransferase(41, 42, 43) . Studies are currently ongoing to further characterize the band 7.2b gene promoter and to determine if it truly has bidirectional activity.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U33925[GenBank], U33926[GenBank], U33927[GenBank], U33928[GenBank], U33929[GenBank], U33930[GenBank], and U33931[GenBank].