©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Four Variant Chicken Erythroid AE1 Anion Exchangers
ROLE OF THE ALTERNATIVE N-TERMINAL SEQUENCES IN INTRACELLULAR TARGETING IN TRANSFECTED HUMAN ERYTHROLEUKEMIA CELLS (*)

(Received for publication, December 13, 1994; and in revised form, May 5, 1995)

Kathleen H. Cox Tracy L. Adair-Kirk John V. Cox (§)

From the Department of Microbiology and Immunology, University of Tennessee Health Science Center, Memphis, Tennessee 38163

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

Four variant AE1 anion exchangers with predicted molecular masses of 99, 102, 104, and 108 kDa are expressed in chicken erythroid cells. These variant polypeptides differ in sequence only at the N terminus of their cytoplasmic domains. Molecular analyses have shown that transcripts derived from both of the erythroid-specific promoters, P1 and P2, encode all four of these AE1 anion exchanger variants. However, quantitative RNase protection analyses have shown that the transcripts derived from the P1 promoter are much more prevalent than those derived from the P2 promoter. Reverse transcriptase polymerase chain reaction studies have indicated that the extensive diversity in the transcripts derived from the AE1 gene occurs both in primitive and definitive lineage erythroid cells. Transient transfection analyses using human erythroleukemia cells have investigated the functional significance of the alternative sequences at the N terminus of these variant exchangers. These studies have shown that the erythroid AE1 variants are sorted to different membrane compartments in these cells. The 99- and 102-kDa variants are primarily sorted to the plasma membrane, whereas the 108-kDa variant is retained in a perinuclear compartment. These results suggest that the alternative N-terminal cytoplasmic sequences of these polypeptides may serve as signals to direct these variant transporters to different membrane compartments within cells.


INTRODUCTION

The erythroid AE1 anion exchanger (band 3) possesses dual functions that are segregated into distinct domains. The C-terminal membrane-spanning region of the polypeptide primarily mediates the electroneutral exchange of bicarbonate for chloride(1) . This electroneutral exchange reaction allows the CO(2) generated in respiring tissues to be carried to the lungs in the form of bicarbonate where it is expelled. The N-terminal cytoplasmic domain of the AE1 anion exchanger provides an attachment site for the erythroid membrane cytoskeleton through its interaction with ankyrin(2) , protein 4.1(3) , and protein 4.2(4) .

Recent analyses have indicated that the gene that encodes the erythroid AE1 anion exchanger also encodes the electroneutral anion exchanger that has been characterized in the basolateral membrane of the A-intercalated cell of the kidney collecting duct(5) . These studies have shown that the AE1 anion exchanger transcripts that accumulate in human(6) , mouse(7) , rat(8) , and chicken (9) kidney differ from the erythroid AE1 transcripts in the sequences present at their 5` ends, resulting in polypeptides that only differ from their erythroid counterparts in the sequences present at the N terminus of their cytoplasmic domains. This type of N-terminal cytoplasmic diversity has also been shown to exist among the AE1 anion exchangers expressed in chicken erythroid cells. Two variant erythroid AE1 anion exchanger transcripts, AE1-1 and AE1-2, which are derived by alternative transcriptional initiation and differential RNA splicing(10, 11) , encode polypeptides with alternative N-terminal cytoplasmic domains.

The studies described here, in conjunction with previous analyses, have indicated that a total of 14 AE1 anion exchanger transcripts accumulate in chicken erythroid cells. Twelve of these transcripts are derived from the two erythroid-specific promoters of the AE1 gene, P1 and P2 (11) . The other two transcripts, AE1-3 and AE1-5, whose expression is regulated by the P3 promoter of the AE1 gene, (^1)accumulate both in erythroid and in kidney cells(9) . Although 12 AE1 transcripts are derived from the two erythroid-specific promoters, they encode only four different structural variants with predicted molecular masses of 99, 102, 104, and 108 kDa. This observed diversity is consistent with immunoblotting studies that have shown that four anion exchanger species of 95, 99, 102, and 108 kDa accumulate in chicken erythroid membranes(9) . Like the variant AE1 anion exchangers that have been previously characterized, the polypeptides encoded by these variant AE1 transcripts differ only in the sequences present in their N-terminal cytoplasmic domains. Transfection analyses have indicated that the 108-kDa AE1 variant is primarily retained in a perinuclear compartment when transiently expressed in human erythroleukemia (HEL) (^2)cells. In contrast, the 99- and 102-kDa AE1 variants primarily accumulate in the plasma membrane of transfected HEL cells. These results suggest that the alternative N-terminal cytoplasmic sequences of these variant transporters may be involved in targeting these polypeptides to different membrane compartments within cells.


MATERIALS AND METHODS

Isolation of cDNAs Corresponding to the 5` Ends of the Variant Chicken Erythroid AE1 Transcripts

cDNAs corresponding to the 5` ends of the variant chicken erythroid AE1 transcripts were cloned using a reverse transcriptase polymerase chain reaction (RT-PCR) rapid amplification of cDNA ends (RACE) protocol(9) . Briefly, poly(A) RNA isolated from 10-day embryonic erythroid cells was reverse transcribed using an oligonucleotide corresponding to nucleotides 1667-1687 of the erythroid AE1-1 transcript(10, 11) . This first strand cDNA was tailed with dATP using terminal deoxynucleotidyl transferase and used as a template for PCR using an antisense primer corresponding to nucleotides 362-382 of the AE1-1 transcript and a R(I)dT sense primer (the R(I) sequence at the 5` end of this primer contained the sequence for a restriction endonuclease site). The products of the first round of PCR were then used as the template for a second round of PCR using an antisense primer corresponding to nucleotides 298-318 of the AE1-1-transcript (this antisense primer contained the restriction endonuclease site found in the R(I) sequence at its 5` end) and a sense primer corresponding to R(I). The amplification products resulting from this PCR reaction were digested with the restriction endonuclease, subcloned into a pGEM-3 vector (Promega), and sequenced using the dideoxy chain termination method.

Construction and Screening of a Chicken Genomic DNA Library

Chicken genomic DNA was isolated from chicken liver(12) . Restriction fragments resulting from the BamHI digestion of this genomic DNA were ligated into a BamHI-cut ZAP Express vector (Stratagene). This phage library was screened by plaque filter hybridization using a P-labeled cDNA probe of 228 nucleotides that initiated with nucleotide 14 of exon 4 and included all of exons 5 and 6. This resulted in the isolation of two contiguous genomic clones of 1.6 and 1.2 kilobases pair. pBK-CMV phagemid DNA containing each of these genomic inserts was prepared and sequenced by the dideoxy chain termination method using specific oligonucleotides as primers.

Generation of cDNAs Corresponding to the 3` End of Exon 6 Containing AE1 Transcripts

cDNAs corresponding to the 3` end of exon 6 containing AE1 transcripts were generated by RT-PCR. Poly(A) RNA isolated from 10-day embryonic erythroid cells was reverse transcribed using an oligonucleotide corresponding to nucleotides 3017-3037 of the erythroid AE1-1 anion exchanger transcript(10, 11) . This primer is complementary to a sequence in the 3`-untranslated region of all the chicken AE1 transcripts(9, 10) . This first strand cDNA was used as the template for PCR using an antisense primer corresponding to nucleotides 2978-2998 in the 3` untranslated region of the AE1-1 transcript (10, 11) and a sense primer corresponding to nucleotides 1-21 of exon 6. The PCR amplification primers contained the sequence for a restriction endonuclease site at their 5` end. The amplification products resulting from this PCR reaction were digested with the restriction endonuclease and subcloned into a pGEM-3 vector, and the entire sequence of each clone was determined by the dideoxy chain termination method of sequencing using specific oligonucleotides as primers. Multiple cDNAs were sequenced to insure that any sequence differences observed between these exon 6 containing transcripts and the previously characterized AE1 transcripts (9, 10) were not due to the insertion of incorrect nucleotides by Pfu DNA polymerase (Stratagene) during PCR amplification.

Analysis of the Expression of the Variant Chicken Erythroid AE1 Transcripts in Primitive and Definitive Erythroid Cells by RT-PCR

RNA was isolated (12) from 10-day embryonic erythroid cells and from 3-day-old chicken embryos. Following purification of poly(A) RNA, 2 µg of poly(A) RNA from each source was reverse transcribed using an oligonucleotide complementary to nucleotides 3017-3037 of the erythroid AE1-1 transcript(10, 11) . This first strand cDNA was used as the template for PCR amplifications using an antisense primer complementary to nucleotides 74-95 of exon 6 (6AS) of the AE1 gene and one of three different sense primers. 1S corresponded to nucleotides 11-31 in the 5`-untranslated first exon of the erythroid AE1-1 transcript(10, 11) , 1`S corresponded to the 21 nucleotides 5` of the putative transcription initiation site of the AE1-1 transcript in the AE1 gene(11) , and 2S corresponded to nucleotides 174-194 in the 5` untranslated first exon of the AE1-2 transcript(10, 11) . PCR amplifications were also carried out using each of these sense primers in combination with an antisense primer complementary to nucleotides 54-74 of exon 10 (10AS) of the AE1 gene. Each primer contained 9 additional nucleotides at its 5` end that included the sequence for a restriction endonuclease site. The resulting PCR amplification products were resolved on a 1.5% agarose gel and stained with ethidium bromide. The amplification products were then transferred to nitrocellulose, and the filters were incubated with a P-labeled oligonucleotide corresponding either to nucleotides 11-31 in exon 3, nucleotides 50-70 in exon 3, nucleotides 14-34 in exon 4, or nucleotides 1-21 in exon 6 of the AE1 gene as described previously(9) . Following washing, hybridizing species were detected by autoradiography using an intensifying screen. Specific hybridizing species were isolated on a low melting point agarose gel, digested with the appropriate restriction endonuclease, subcloned into a pGEM-3 vector, and sequenced by the dideoxy chain termination method.

RNA Blotting Analysis

200 ng of poly(A) RNA was isolated from 10-day embryonic erythroid cells(12) , electrophoresed on a formaldehyde agarose gel, and transferred to nitrocellulose. The nitrocellulose filter was probed with a P-labeled antisense in vitro transcript corresponding to nucleotides 2790-3068 of the erythroid AE1-1 transcript(10, 11) . This sequence is present at the 3` end of all the previously characterized AE1 transcripts(9, 10) . Alternatively, an identical blot was probed with a P-labeled antisense in vitro transcript corresponding to nucleotides 1-95 of exon 6. 2 10^6 cpm/ml of each of these P-labeled probes was incubated with the nitrocellulose filters in 50% formamide, 0.1 M PIPES, pH 6.8, 0.5 M NaCl, 10 Denhardt's solution, and 0.2% SDS for 16 h at 68 °C. The filters were then incubated for 2 h at 75 °C, followed by washing in 0.2 SSC and 0.01% SDS at 68 °C. Hybridizing species were detected by autoradiography using an intensifying screen.

RNase Protection Analysis

P-labeled antisense transcripts were synthesized from the cDNA templates illustrated in Fig. 5A and 6C using SP6 RNA polymerase. 300,000 cpm of each P-labeled probe was hybridized with 1 µg of poly(A) RNA isolated from 10-day embryonic erythroid cells in 80% formamide, 40 mM PIPES, 0.4 M NaCl, and 1 mM EDTA for 16 h at 50 °C. Following hybridization, the samples were digested with 0.3 µg of RNase A and 6.6 units of RNase T1 for 1 h at 30 °C, deproteinized by treatment with proteinase K, phenol-extracted, and precipitated(12) . The samples were then resuspended, electrophoresed on a 7 M urea, 6% polyacrylamide gel, and the protected fragments were visualized by autoradiography using an intensifying screen.


Figure 5: Quantitation of the abundance of the four structural AE1 anion exchanger variants by RNase protection analyses. The relative abundance of the four AE1 anion exchanger structural variants was determined by RNase protection analyses. The structure of each construct used to generate antisense RNA probes is illustrated in A. The black boxes in each construct represent vector sequences. P-labeled antisense transcripts corresponding either to exon 4 (B, lanes 4-6), exons 3 and 4 (B, lanes 1-3), or exons 3, 4, 5, and 6 (B, lanes 7-9) were synthesized in vitro and hybridized to 1 µg of poly(A) RNA isolated from 10-day embryonic erythroid cells (lanes 2, 5, and 8). Following hybridization, samples were digested with RNase, and the products were resolved on a 6% polyacrylamide, 7 M urea gel. In B, lanes 1, 4, and 7 correspond to the probe alone, and lanes 3, 6, and 9 correspond to control hybridizations and digestions carried out in the presence of the probe and tRNA alone. The protected fragments corresponding to exon 4; exons 3 and 4; exons 4, 5, and 6; and exons 3, 4, 5, and 6 are indicated on the right hand side of the figure. Markers in B correspond to in vitro transcripts of known size.



Quantitation of the Relative Abundance of the Variant Erythroid AE1 Transcripts

The autoradiograms resulting from the RNase protection assays were scanned, and the percentage of label in each fragment determined using the ScanAnalysis software from Biosoft. The autoradiographic exposures used for this analysis were those in which individual bands were not yet saturated. The values for the relative abundance of the individual AE1 variants represents the average value obtained from two independent experiments, in which virtually identical results were obtained.

Transient Expression of the Variant Erythroid AE1 Anion Exchangers in HEL Cells

Full-length cDNAs corresponding to the 99-, 102-, and 108-kDa erythroid AE1 anion exchangers were subcloned into the pcDNA3 eukaryotic expression vector (Invitrogen) downstream of the constitutive immediate early gene promoter of cytomegalovirus. In each construct, the sequence immediately upstream of the translation initiation codon has been removed and replaced with a Kozak sequence (ACCACC) (14) to enhance translation initiation efficiency. Each construct was transfected into HEL cells by a modification of the calcium phosphate technique(15) , and after 48 h, the cells were harvested and fixed on coverslips by incubating in 0.5% paraformaldehyde in PBS for 5 min at room temperature. Following permeabilization in 0.5% Triton X-100 in PBS, the cells were incubated with a 1:3,000 dilution of an AE1 anion exchanger-specific peptide antibody, pAE1A, in PBS containing 0.5% Triton X-100. This antibody has been generated against a peptide corresponding to amino acids 185-203 of the erythroid AE1-1 anion exchanger(9) . The cells were then washed and incubated with donkey anti-rabbit IgG conjugated to lissamine and following washing immunoreactive polypeptides were visualized using a Bio-Rad confocal laser scanning microscope. The transfection efficiency in these experiments ranged from 0.3-2% of the total cell population. As a control for the specificity of staining, the pAE1A antibodies were incubated with the transfected HEL cells in the presence of 10 µg/ml of the peptide they were generated against or in the presence of 10 µg/ml of a nonspecific peptide.


RESULTS

Identification and Characterization of Variant Chicken Erythroid AE1 Anion Exchanger Transcripts

Previous analyses have indicated that two erythroid transcripts, AE1-1 and AE1-2, are derived from the chicken AE1 anion exchanger gene by alternative transcriptional initiation and differential RNA splicing(11) . These transcripts, which differ only at their 5` ends, encode anion exchangers that possess alternative N-terminal cytoplasmic domains. Recent studies have further characterized three additional transcripts derived from the chicken AE1 gene, AE1-3, AE1-4, and AE1-5, which accumulate in chicken kidney(9) . Like the erythroid transcripts, the variant kidney AE1 transcripts differ only at their 5` ends and encode predicted polypeptides with alternative N-terminal cytoplasmic domains. RT-PCR analyses have shown that the AE1-3 and AE1-5 transcripts also accumulate in erythroid cells (9) . The structure at the 5` end of each of these variant transcripts is illustrated in Fig. 1A.


Figure 1: Structure of the variant chicken AE1 anion exchangers. The exon organization of the variant chicken AE1 anion exchangers is illustrated diagrammatically in A. The predicted site of translation initiation of each variant, as well as the location of the antisense primer (arrow in AE1-1) used for the RT-PCR RACE analyses are indicated. Boxes containing different patterns at the 5` ends of the AE1-3, AE1-4, and AE1-5 transcripts correspond to the variant specific exons of these transcripts. The boxes that are shaded are held in common among all of the variant transcripts. The location of the peptide, pAE1A, that was used to generate AE1 anion exchanger-specific antibodies is indicated. The nucleotide and predicted amino acid sequence of exons 5 and 6 is illustrated in B. The arrows in B mark the location of splice junctions.



To determine whether additional diversity exists at the 5` ends of the chicken erythroid AE1 transcripts, RT-PCR RACE using an antisense primer corresponding to nucleotides 298-318 of the AE1-1 transcript has been used to amplify the 5` ends of the erythroid AE1 transcripts. The location of the antisense primer used for these analyses is indicated by the arrow in Fig. 1A. The amplification products resulting from this protocol were subcloned into a pGEM-3 vector, and 20 clones were isolated and sequenced. Eight clones were identical to the sequence of the AE1-1 and AE1-2 transcripts, and their 5` ends corresponded to various sites within exon 4 of the AE1 gene (Fig. 1A). These clones presumably resulted from premature termination during the reverse transcriptase reaction. Six clones were identical to the AE1-1 transcript, and their 5` ends corresponded to the nucleotide immediately 3` of the previously defined transcription start site of the erythroid AE1-1 transcript (11) . Four clones extended further 5` in the AE1 gene than the putative transcription initiation site of the AE1-1 transcript(11) , with the largest clone extending 21 nucleotides upstream of this site. Whether the erythroid AE1-1 transcripts initiate at a site upstream of that previously determined (11) will be addressed in the RNase protection analyses described below.

Two additional clones were isolated that contained a novel 153-nucleotide insert between exon 4 and exon 10. Exon 10 is the first exon that is held in common among all of the variant erythroid and kidney AE1 transcripts (Fig. 1). Data base searches have shown that the amino acid sequence encoded by this 153-nucleotide insert (Fig. 1B) is not homologous to the sequence of any of the previously characterized anion exchanger gene members. Sequence analysis of the region of the AE1 gene that contains this 153-nucleotide insert has shown that this sequence is derived from two exons, 5 and 6, that are separated by an intron of 102 nucleotides (Fig. 2A). Exons 5 and 6 lie immediately upstream of the exons at the 5` ends of the variant kidney AE1 transcripts, AE1-3, AE1-4, and AE1-5.^1


Figure 2: Analysis of the structure of AE1 transcripts containing exons 5 and 6 by RT-PCR. Poly(A) RNA isolated from 10-day embryonic erythroid cells was reverse transcribed using a primer complementary to nucleotides 3017-3037 of the erythroid AE1-1 transcript(10, 11) . This first strand cDNA was PCR amplified using an antisense primer complementary to nucleotides 74-95 of exon 6 (B, lanes 1-6) or an antisense primer complementary to nucleotides 54-74 of exon 10 (B, lanes 7-12). Each of these antisense primers was used in combination with three different sense primers; 1S (B, lanes 1, 2, 7, and 8), 1`S (B, lanes 3, 4, 9, and 10), or 2S (B, lanes 5, 6, 11, and 12). The location of the sequences in the AE1 gene corresponding to each primer is illustrated in A. The even-numbered lanes in B correspond to PCR amplifications carried out in the absence of first strand cDNA template. The amplification products were electrophoresed on a 1.5% agarose gel and stained with ethidium bromide (B). Alternatively, the amplification products were transferred to nitrocellulose and probed with a P-end-labeled oligonucleotide corresponding either to nucleotides 11-31 in exon 3 (C), nucleotides 14-34 in exon 4 (D), or nucleotides 1-21 in exon 6 (E). The organization and size of the exons at the 5` end of the AE1 gene are depicted in A. The filled box at the 5` end of exon 2 corresponds to the 21 nucleotides 5` of the previously defined transcription initiation site of the erythroid AE1-1 transcript(11) . Open boxes correspond to introns in the variant erythroid AE1 transcripts and are not drawn to scale. Exons 7, 8, and 9, which are not illustrated in this diagram, are not part of the transcripts derived from the P1 and P2 promoters. The markers in B correspond to pGEM-3 DNA digested with HinfI restriction endonuclease.



The 5` termini of both of the cDNAs that contained exons 5 and 6 resided within exon 4 of the AE1 gene (Fig. 1A). Because the AE1-1 and AE1-2 transcripts, which are derived from the P1 and P2 promoters, respectively, each contain exon 4 of the AE1 gene, it was possible that transcripts containing exons 5 and 6 initiated transcription from one or both of these erythroid-specific AE1 promoters. To further investigate the structure of transcripts containing exons 5 and 6, RT-PCR analyses have amplified the sequence at the 5` end of AE1 transcripts present in RNA isolated from 10-day embryonic erythroid cells. Poly(A) erythroid RNA was reverse transcribed using a primer complementary to nucleotides 3017-3037 of the erythroid AE1-1 transcript. This first strand cDNA was PCR amplified using an antisense primer complementary to a sequence in exon 6 (6AS in Fig. 2A) in combination with one of three different sense primers. The first sense primer, 1S, corresponded to nucleotides 11-31 of the erythroid AE1-1 transcript (10, 11) . The second sense primer, 1`S, corresponded to the 21 nucleotides 5` of the putative transcription initiation site of the erythroid AE1-1 transcript in the AE1 gene(11) . The 1`S sequence was contained in one of the cDNA clones originally isolated by RT-PCR RACE. The third sense primer, 2S, corresponded to a sequence in the 5` untranslated first exon of the erythroid AE1-2 transcript(11) . The location of these sense primers is illustrated in Fig. 2A. Interestingly, amplifications using either the 1S (Fig. 2B, lane 1), 1`S (Fig. 2B, lane 3), or 2S (Fig. 2B, lane 5) sense primer resulted in two amplification products.

To determine the origin of these species, the amplification products were transferred to nitrocellulose, and the filters were probed with P-end-labeled oligonucleotides corresponding either to nucleotides 11-31 in exon 3 (Fig. 2C) or to nucleotides 14-34 in exon 4 (Fig. 2D) of the AE1 gene. Both of the amplification products in each lane hybridized with the exon 4-specific oligonucleotide (Fig. 2D, lanes 1, 3, and 5), whereas only the larger product hybridized with the exon 3-specific oligonucleotide (Fig. 2C, lanes 1, 3, and 5). The observation that the exon 3-specific oligonucleotide weakly hybridized with the amplification products generated with the 1S and 1`S sense primers (Fig. 2C, lanes 1 and 3) was not observed when an oligonucleotide corresponding to nucleotides 50-70 in exon 3 was used as a probe. The hybridization pattern obtained with the oligonucleotide corresponding to nucleotides 50-70 in exon 3 mirrored the ethidium bromide staining pattern observed for the larger amplification product in each lane (data not shown). Sequence analysis of the PCR products in Fig. 2B (lanes 1-6) that hybridized with the exon 3- and exon 4-specific oligonucleotides verified that these products were derived from AE1 transcripts that contain exons 4, 5, and 6, and either lack or contain exon 3.

These results indicate that some of the transcripts initiating with the 5`-untranslated first exon of both the AE1-1 and the AE1-2 transcripts contain exons 5 and 6. In addition, they indicate that AE1 transcripts derived from both the P1 and P2 promoters in 10-day embryonic erythroid cells either lack (Fig. 2, lower band in each amplification) or contain (Fig. 2, upper band in each amplification) exon 3 of the AE1 gene. Finally, the data indicate that at least some of the transcripts derived from the P1 promoter initiate transcription upstream of the previously defined transcription initiation site(11) , because an identical profile of PCR amplification products was obtained when using the 1S or 1`S sense primers (compare lanes 1 and 3 of Fig. 2B).

To further investigate the structure of AE1 transcripts containing exon 6, 200 ng of poly(A) erythroid RNA was electrophoresed on a formaldehyde-agarose gel, transferred to nitrocellulose, and probed either with a P-labeled antisense transcript complementary to nucleotides 2790-3068 of the erythroid AE1-1 transcript (Fig. 3, lane 1; this sequence is present in all the previously characterized chicken AE1 transcripts) or with a P-labeled antisense transcript complementary to nucleotides 1-95 of exon 6 (Fig. 3, lane 2). This analysis revealed that the exon 6-specific probe hybridizes with a major transcript of 4.7 kilobases, as well as minor species that range in size from 4.8 to 5.1 kilobases (Fig. 3, lane 2). In contrast, a diffuse RNA species that was centered at 4.6 kilobases was detected with the probe that recognizes all of the AE1 variants (Fig. 3, lane 1). The major exon 6-containing transcript comigrates with the slower migrating transcripts recognized by the probe that detects all of the variants, consistent with the additional 153 nucleotides contained within exons 5 and 6. This result suggested that like the previously characterized AE1 transcripts, the transcripts containing exons 5 and 6 may only differ from the other variants in the sequences present at their 5` ends. To insure this was the case, cDNAs have been cloned by RT-PCR using an exon 6-specific sense primer and an antisense primer complementary to a sequence in the 3` untranslated region of all the chicken erythroid and kidney AE1 cDNAs that have been characterized. Sequence analysis of these clones has indicated that the sequence 3` of exon 6 is identical to the sequence of the other AE1 variants.


Figure 3: RNA blotting analysis of the exon 6-containing transcripts in chicken erythroid cells. 200 ng of poly(A) RNA isolated from 10-day embryonic erythroid cells was electrophoresed on a formaldehyde-agarose gel and transferred to nitrocellulose. These nitrocellulose filters were probed either with a P-labeled antisense transcript complementary to nucleotides 2790-3068 of the erythroid AE1-1 transcript (lane 1) or with a P-labeled antisense transcript complementary to nucleotides 1-95 of exon 6 (lane 2). Following washing, the hybridizing species were detected by autoradiography using an intensifying screen. Markers correspond to transcripts of known size.



The analyses described above have indicated that transcripts containing exons 5 and 6 are derived from both the P1 and P2 promoters of the AE1 gene, and these transcripts either lack or contain exon 3. Additional RT-PCR studies have examined whether similar complexity exists among the AE1 transcripts that lack exons 5 and 6. In this experiment, the first strand cDNA was amplified with the same three sense primers described above, 1S, 1`S, and 2S (Fig. 2A), and with an antisense primer complementary to nucleotides 54-74 in exon 10 (10AS in Fig. 2A), which is the first exon held in common among all of the variant AE1 transcripts(9, 10) . Analysis of the resulting amplification products revealed two major ethidium bromide-stained species (Fig. 2B, lanes 7, 9, and 11). To determine the origin of these species, the amplification products were transferred to nitrocellulose and probed with P-labeled oligonucleotides corresponding to a sequence in exon 3 (Fig. 2C), a sequence in exon 4 (Fig. 2D), or a sequence in exon 6 (Fig. 2E) of the AE1 gene. The two major species (Fig. 2B, lanes 7, 9, and 11) in each amplification correspond to transcripts that lack exons 5 and 6 and either lack (the faster migrating species) or contain (the slower migrating species) exon 3. Additional species are also visible in the autoradiograms that correspond to transcripts containing exons 5 and 6. Longer exposure of the autoradiogram in Fig. 2C has indicated that the slower migrating species that hybridizes with the exon 6-specific probe (Fig. 2E, lanes 7, 9, and 11) also hybridizes with the exon 3-specific probe (data not shown). The faster migrating species that hybridizes with the exon 6-specific probe (Fig. 2E, lanes 7, 9, and 11) presumably corresponds to transcripts containing exons 5 and 6 that lack exon 3. As mentioned above, the difference in the extent of hybridization of the PCR products derived from transcripts from the P1 (Fig. 2C, lanes 7 and 9) and P2 (Fig. 2C, lane 11) promoters with the exon 3-specific probe is not observed when another exon 3-specific oligonucleotide is used as a probe (data not shown). These results indicate that PCR reactions using each of these sense primers result in four amplification products, indicating that at least four erythroid AE1 transcripts initiate transcription from both the P1 and P2 promoters.

The exon organization at the 5` end of each of the erythroid AE1 transcripts is illustrated in Fig. 4. Because the four variants derived from each of the promoters have identical coding sequences, we have named the variants AE1-1a, AE1-1b, AE1-1c, AE1-1d, AE1-2a, AE1-2b, AE1-2c, and AE1-2d. The 1 and the 2 in the nomenclature refer to the promoter from which the transcript was derived, and a, b, c, and d refer to the four different structural variants. Finally, four additional AE1 transcripts, AE1-1`a, AE1-1`b, AE1-1`c, and AE1-1`d, are expressed in erythroid cells. These transcripts initiate transcription at least 21 nucleotides upstream (filled box at the 5` end of exon 2 in Fig. 2A) of the previously defined transcription initiation site of the AE1-1 transcript. Whether all of the AE1-1 variants initiate transcription from this site will be examined in the RNase protection analyses described below.


Figure 4: Exon organization at the 5` end of the variant transcripts derived from the erythroid-specific promoters of the chicken AE1 gene. The exon organization at the 5` end of the variant transcripts derived from the P1 and P2 promoters of the AE1 gene is illustrated. The 1 and 2 in the nomenclature refer to the promoter from which the transcript was derived, and a, b, c, and d refer to the four different structural variants. The filled box at the 5` end of the variant AE1-1 transcripts represents the sequence present in transcripts that initiate from the minor transcription initiation site of the P1 promoter. The regions that are shaded are held in common among all of the variant transcripts.



Determination of the Relative Abundance of the Variant Erythroid AE1 Transcripts by RNase Protection Analyses

The PCR amplification products derived from transcripts containing exons 5 and 6 (Fig. 2E, lanes 7, 9, and 11) are much less prevalent than the amplification products derived from AE1 transcripts that lack exons 5 and 6 (Fig. 2D, lanes 7, 9, and 11). To determine if this result accurately reflects the relative abundance of the individual AE1 variants, RNase protection analyses have been carried out. The regions at the 5` end of the variant AE1 cDNAs that were used as templates for the synthesis of P-labeled antisense transcripts are illustrated in Fig. 5A. Following synthesis of these antisense transcripts, each was hybridized to 1 µg of poly(A) RNA isolated from 10-day embryonic erythroid cells, and the resulting hybrids were digested with RNase and electrophoresed on a denaturing urea/acrylamide gel. Hybridization of the probe corresponding to exon 4 alone to erythroid RNA resulted in a protected fragment of 88 nucleotides as predicted (Fig. 5B, lane 5), whereas hybridization of the probe corresponding to exons 3 and 4 to erythroid RNA resulted in two protected fragments of 165 and 92 nucleotides (Fig. 5B, lane 2). The larger of these fragments corresponds in size to exons 3 and 4 together, whereas the smaller fragment corresponds in size to exon 4 alone. This result indicates that exon 3 is only present in transcripts that also contain exon 4, consistent with the RT-PCR analysis. Multiple protected fragments of 317, 244, 165, and 92 nucleotides resulted from the hybridization of the probe corresponding to exons 3, 4, 5, and 6 to erythroid RNA (Fig. 5B, lane 8). The largest fragment corresponded in size to exons 3, 4, 5, and 6; the fragment of 244 nucleotides corresponded in size to exons 4, 5, and 6; the fragment of 165 nucleotides corresponded in size to exons 3 and 4; and the fragment of 92 nucleotides corresponded in size to exon 4. Some of the fragments in these protection analyses are slightly larger than would be predicted from the sizes of the exons that make them up. This discrepancy in size is due to the conserved nature of the splice donor sequences at the 3` end of each of the exons. This is most clearly illustrated by comparing the fragment corresponding to exon 4 in each of the protections. When exon 4 alone was used as a probe, the predicted fragment of 88 nucleotides was observed (Fig. 5B, lane 5). However, the conserved splice donor sequences at the 3` end of exons 1, 2, and 3 results in a larger protected fragment for exon 4 when constructs corresponding to exons 3 and 4 or exons 3, 4, 5, and 6 are used as probes (Fig. 5B, lanes 2 and 8).

Densitometric scanning of these protection analyses has revealed that transcripts containing exons 5 and 6 comprise 12% of the erythroid AE1 transcripts. The value obtained for the percentage of AE1 transcripts containing exons 5 and 6 from this quantitative protection analysis is much higher than the PCR analyses had suggested. In addition, these experiments have shown that the transcripts lacking exon 3 comprise 37% of the total AE1 transcripts.

Additional RNase protection analyses have determined the percentage of exon 3- and exon 4-containing transcripts that are derived from the P1 and P2 promoters of the AE1 gene. For these studies, constructs containing exons 3 and 4 were generated that initiated either with the 5`-untranslated first exon of transcripts derived from the P2 promoter or with the 5`-untranslated first exon of transcripts derived from the P1 promoter (Fig. 6C). The RNA probe corresponding to transcripts derived from the P1 promoter possesses 21 additional nucleotides at its 5` end that are not present in the previously characterized AE1-1 transcript(11) . These additional nucleotides correspond to the 21 nucleotides in the AE1 gene that are immediately upstream of the putative transcription initiation site of the AE1-1 transcript(11) . P-labeled in vitro transcripts derived from these constructs were hybridized to 1 µg of poly(A) RNA isolated from 10-day embryonic erythroid cells. Following RNase digestion, protected fragments of 92 and 168 nucleotides were observed with each probe (Fig. 6, A and B, lanes 2). These fragments correspond to exon 4 alone and exons 3 and 4 together. Again these protection fragments are slightly larger than predicted due to the conserved sequences at the 3` end of each of the exons. In addition, a full-length protected product was observed in each of the protections. However, the full-length protected fragments corresponding to transcripts derived from the P1 promoter (Fig. 6B, 2-3-4 and 2`-3-4) are much more prominent than the full-length protected fragment corresponding to the transcripts derived from the P2 promoter (Fig. 6A, 1-3-4). The additional bands in the protection analyses in Fig. 5and Fig. 6that were absent in control lanes were reproducibly observed, and at this time their origin is unknown. Densitometric scanning of the protections in Fig. 6has indicated that 99% of the exon 3- and exon 4-containing transcripts initiate from the P1 promoter, whereas only 1% initiate from the P2 promoter. Furthermore, this analysis has revealed that 5% of the transcripts derived from the P1 promoter initiate transcription at a site upstream of the previously defined start site for transcription (protected fragment marked 2`-3-4 in Fig. 6B), whereas 95% of the transcripts derived from this promoter initiate transcription from the previously defined start site (11) (protected fragment marked 2-3-4 in Fig. 6B).


Figure 6: Quantitation of the relative abundance of the transcripts derived from the two erythroid-specific AE1 promoters by RNase protection analyses. Two constructs were generated in the pGEM-3 vector that contained exons 3 and 4 (C). The first construct initiated with the 5`-untranslated first exon of the variant AE1-2 transcripts (A, lanes 1-3), and the second construct initiated with the 5`-untranslated first exon of the variant AE1-1 transcripts (B, lanes 1-3). The cross-hatched box corresponds to the 21 nucleotides 5` of the previously defined transcription initiation site of the erythroid AE1-1 transcript in the AE1 gene(11) , and the black boxes in each construct represent vector sequences (C). P-labeled antisense transcripts were generated from each construct and hybridized to 1 µg of poly(A) RNA isolated from 10-day embryonic erythroid cells (A and B, lanes 2). Following hybridization, samples were digested with RNase, and the products were resolved on a 6% polyacrylamide/7 M urea gel. Lanes 1 in A and B correspond to the probe alone. Lanes 3 in A and B correspond to control hybridizations and digestions carried out in the presence of the probe and tRNA alone. The protected fragments corresponding to exon 4; exons 3 and 4; exons 1, 3, and 4; exons 2, 3, and 4; and exons 2`, 3, and 4 are indicated on the right hand side of the figures. Markers in A and B correspond to in vitro transcripts of known size.



Both of the Erythroid-specific AE1 Promoters Are Active in Primitive and Definitive Lineage Erythroid Cells

The results described above have shown that four identical structural variants are derived from both of the erythroid-specific AE1 promoters in 10-day embryonic erythroid cells. The erythroid cells present in 10-day chicken embryos are almost entirely of definitive lineage in origin (16) . Erythroid cells of the definitive lineage, which persist into adult life, begin to enter the circulation on day 5 of development. Prior to day 5, erythroid cells in chicken embryos are exclusively derived from cells of the primitive lineage(16) . To determine if the same complexity of transcripts is present in primitive lineage erythroid cells, RT-PCR analyses identical to those described above have been carried out using RNA isolated from 3-day chicken embryos. Although it is possible that transcripts detected in analyses with embryonic RNA may be derived from cell types other than erythroid cells, in situ hybridization studies have indicated that erythroid cells are the only cell type that accumulates detectable levels of AE1 transcripts in 3-day-old chicken embryos (data not shown). These RT-PCR studies have shown that all of the variant species detected in RNA isolated from 10-day embryonic erythroid cells are also present in primitive lineage erythroid cells (Fig. 7). However, the transcripts containing exons 5 and 6 that are derived from the P2 promoter are more prevalent in 10-day embryonic erythroid cells (Fig. 2E, lane 5) than in 3-day embryonic RNA (Fig. 7D, lane 5). Although this result suggests there may be lineage-specific differences in the relative abundance of individual variants, the same diversity observed in the AE1 transcripts present in definitive lineage erythroid cells is also detected in primitive lineage erythroid cells.


Figure 7: Expression of the variant AE1 transcripts in chicken erythroid cells of the primitive lineage. Poly(A) RNA isolated from 3-day-old chicken embryos was reverse transcribed using a primer complementary to nucleotides 3017-3037 of the erythroid AE1-1 transcript(10, 11) . This first strand cDNA was PCR amplified using an antisense primer complementary to nucleotides 74-95 of exon 6 (lanes 1-6) or an antisense primer complementary to nucleotides 54-74 of exon 10 (lanes 7-12). Each of these antisense primers was used in combination with three different sense primers: 1S (lanes 1, 2, 7, and 8), 1`S (lanes 3, 4, 9, and 10), or 2S (lanes 5, 6, 11, and 12). The location of the sequence in the AE1 gene corresponding to each primer is illustrated in Fig. 2A. The even-numbered lanes correspond to PCR amplifications carried out in the absence of first strand cDNA template. The amplification products were electrophoresed on a 1.5% agarose gel and stained with ethidium bromide (A). Alternatively, the amplification products were transferred to nitrocellulose and probed with a P-end-labeled oligonucleotide corresponding either to nucleotides 11-31 in exon 3 (B), nucleotides 14-34 in exon 4 (C), or nucleotides 1-21 in exon 6 (D). The markers correspond to pGEM-3 DNA digested with HinfI restriction endonuclease.



Variant Chicken Erythroid AE1 Anion Exchangers Are Targeted to Different Membrane Compartments in Transfected HEL Cells

The variant erythroid AE1 transcripts encode predicted polypeptides with alternative N-terminal cytoplasmic domains. AE1-1a, AE1-1`a, and AE1-2a encode identical polypeptides with a predicted molecular mass of 98,801 daltons; AE1-1b, AE1-1`b, and AE1-2b encode identical polypeptides with a predicted molecular mass of 102,352 daltons; AE1-1c, AE1-1`c, and AE1-2c encode identical polypeptides with a predicted molecular mass of 104,383 daltons; and AE1-1d, AE1-1`d, and AE1-2d encode identical polypeptides with a predicted molecular mass of 107,935 daltons. To begin to investigate the functional significance of these alternative N-terminal sequences, the intracellular localization of variant AE1 transporters that have been transiently expressed in HEL cells has been determined. Initial studies examined the intracellular localization of the 99-kDa variant that contains exon 4 alone (Fig. 8A), the 102-kDa variant that contains exons 3 and 4 (Fig. 8B), and the 108-kDa variant that contains exons 3, 4, 5, and 6 (Fig. 8C). Each of these variants was subcloned into the pcDNA3 vector downstream of the constitutive immediate early gene promoter of cytomegalovirus. Each construct was transfected into HEL cells, and 48 h following transfection the cells were harvested and fixed on coverslips by incubating in 0.5% paraformaldehyde in PBS. The cells were then permeabilized in 0.5% Triton X-100 in PBS, and the intracellular localization of the variant AE1 anion exchangers was determined by incubating with an antibody, pAE1A(9) , that has been generated against a peptide corresponding to amino acids 185-203 of AE1-1 (Fig. 1A). Immunoreactive polypeptides were detected by incubating the cells with donkey anti-rabbit IgG conjugated to lissamine and visualized by confocal laser scanning microscopy.


Figure 8: Immunolocalization of the variant erythroid AE1 anion exchangers in transfected HEL cells. cDNAs encoding the 99- (A), 102- (B), and 108-kDa (C) erythroid AE1 variants were subcloned into the pcDNA3 vector and transfected into HEL cells. 48 h following transfection, the cells were fixed, permeabilized in 0.5% Triton X-100 in PBS, and incubated with the pAE1A antibody. Following washing in PBS, the cells were incubated with donkey anti-rabbit IgG conjugated to lissamine. The images in A-C correspond to 0.5-µm optical sections collected on a Bio-Rad confocal laser scanning microscope. Each image is approximately 1.5 µm from the top of the cell. The arrows in C mark the location of the plasma membrane in this transfected cell. The fluorescence surrounding the plasma membrane of the cell in B is due to the background staining of immediately adjacent, nontransfected cells. The bar is 3 µm.



These analyses revealed that the variant AE1 anion exchangers were targeted to different membrane compartments in transfected HEL cells (Fig. 8). The 99-kDa anion exchanger, which contains exon 4, accumulated exclusively in the plasma membrane of transfected HEL cells (Fig. 8A). In >80% of the cells expressing the 102-kDa anion exchanger, which contains exons 3 and 4, this variant transporter also accumulated exclusively in the plasma membrane (Fig. 8B). Although the vast majority of staining for the remainder of the cells expressing the 102-kDa variant was also restricted to the plasma membrane, some staining was observed in a perinuclear compartment of these cells (data not shown). In contrast to the patterns of localization observed for the 99- and 102-kDa variants in transfected HEL cells, the 108-kDa anion exchanger, which contains exons 3, 4, 5, and 6, accumulated in a perinuclear compartment of transfected cells (Fig. 8C). The vesicular perinuclear staining observed for the 108-kDa variant was often in the form of a cap that was restricted to one side of the nucleus. However, the staining was sometimes seen to entirely surround the nucleus (Fig. 8C). Identical results were obtained in four separate experiments in which at least 100 cells were scored for each of the variant constructs. In addition, immunolocalization analyses using secondary antibody alone or carried out in the presence of the peptide against which the pAE1A antiserum was generated exhibited background levels of staining, whereas a nonspecific peptide had no effect on the staining patterns described above (data not shown). At this time, the identity of the intracellular compartment where the 108-kDa AE1 variant accumulates is not known. Nonetheless, these data suggest that the alternative N-terminal sequences of the erythroid AE1 anion exchangers serve as signals to direct these variant transporters to different membrane compartments within cells.


DISCUSSION

The results described here have indicated that transcripts encoding polypeptides of 99, 102, 104, and 108 kDa are derived from both of the erythroid-specific promoters, P1 and P2, of the chicken AE1 gene. The predicted sizes of these polypeptides agree reasonably well with previous immunoblotting analyses that have demonstrated the presence of four AE1 anion exchanger species of 95, 99, 102, and 108 kDa in membranes isolated from chicken erythroid cells(9) . This cDNA cloning analysis has indicated that the diversity observed among the four variant chicken erythroid AE1 anion exchanger polypeptides is restricted to the N terminus of their cytoplasmic domains. Similar N-terminal cytoplasmic diversity has also been observed among the three variant anion exchangers, AE1-3, AE1-4, and AE1-5, that are encoded by the AE1 gene in chicken kidney(9) . Immunolocalization analyses have suggested that the alternative N-terminal cytoplasmic sequences of the kidney AE1 anion exchangers may be involved in targeting these variant transporters to different membrane compartments in kidney epithelial cells. (^3)Similarly, the different patterns of intracellular localization exhibited by the variant erythroid AE1 anion exchangers that have been transiently expressed in HEL cells suggest that the alternative N-terminal sequences of these polypeptides may serve as signals to target these transporters to different membrane compartments within these cultured cells.

Previous studies have shown that murine erythroid AE1 anion exchangers exclusively accumulate in a pre-Golgi compartment rather than in the plasma membrane of transfected human embryonic kidney cells(18) . Anion transport assays (18) and the ability of the transfected murine AE1 anion exchanger to be coimmunoprecipitated with the anion exchanger binding domain of ankyrin (19) have suggested that the retention of the murine AE1 anion exchanger in this perinuclear compartment is not due to misfolding. The data presented here have shown that the 99- and 102-kDa chicken AE1 variants are transported to the plasma membrane of transfected HEL cells, which upon stimulation can differentiate into erythroid cells, whereas the 108-kDa AE1 variant is retained in a perinuclear compartment. The observation that some of the AE1 variants undergo transport to the plasma membrane in transfected HEL cells indicates that HEL cells, unlike the human embryonic kidney cells, contain all of the components necessary for the plasma membrane targeting of these anion exchangers. Furthermore, the demonstration that the cellular sorting machinery of HEL cells can target these electroneutral transporters to the plasma membrane suggests that the 108-kDa AE1 variant, which accumulates in a perinuclear compartment of transfected HEL cells, may be specifically retained in the endoplasmic reticulum or Golgi.

Immunolocalization analyses of other investigators have shown that antibodies generated against a peptide corresponding to the C terminus of the murine erythroid AE1 anion exchanger recognize a polypeptide of 115 kDa that is localized to the Golgi complex of a variety of cell types(20) , suggesting that AE1 anion exchanger-like polypeptides may participate in the maintenance of pH equilibrium in this organelle. The observation that the 108-kDa erythroid AE1 variant is preferentially retained in a perinuclear compartment of transfected HEL cells, whereas variants lacking exons 5 and 6 are primarily transported to the plasma membrane, suggests that the amino acid sequence of exons 5 and 6 may serve as a signal for retention in the endoplasmic reticulum or Golgi. Short cytoplasmic sequences have previously been shown to serve as retrieval signals for some type I membrane proteins to the endoplasmic reticulum (21, 22) and the trans Golgi network(23, 24) . However, these sequences share no obvious similarity with the sequence of exons 5 and 6. The recent demonstration that isoforms of ankyrin (25) and spectrin (26) are localized to the Golgi complex suggests the possibility that variants containing exons 5 and 6 associate with the Golgi-specific isoforms of these membrane cytoskeletal polypeptides, thereby retaining these variants in this intracellular compartment. Alternatively, the sequence of exons 5 and 6 could act by inhibiting the ability of variants containing this sequence to interact with elements of the HEL transport machinery involved in transport to the plasma membrane, or these cells may simply lack the elements required for the plasma membrane transport of this variant. Although we cannot rule out that the retention of variants containing exons 5 and 6 in this as yet undefined perinuclear compartment of HEL cells is due to misfolding of the polypeptides, the fact that the variants lacking exons 5 and 6 are sorted to the plasma membrane suggests that this possibility is unlikely.

Previous studies have shown that the extreme N terminus of the human erythroid AE1 anion exchanger is one of several regions of this polypeptide that are involved in mediating its association with erythroid ankyrin(27, 28) . Furthermore, recent studies have shown that the alternative N-terminal sequences of the variant chicken kidney AE1 anion exchangers affect their capacity to associate with the detergent insoluble membrane cytoskeleton.^3 At this time there is no evidence that the diversity at the N terminus of the variant chicken erythroid AE1 anion exchangers affects their ability to associate with the membrane cytoskeleton. However, it is possible that the alternative N-terminal sequences of these variant transporters may regulate their interaction with the membrane cytoskeleton. Regulating this interaction may in turn provide a mechanism for targeting these variant polypeptides to different membrane compartments within cells.

The studies described above have shown that transcripts encoding each of the variant AE1 anion exchanger polypeptides are derived from both the P1 and P2 promoters of the AE1 gene. RNase protection analyses have shown that the transcripts derived from the P1 promoter are much more prevalent than the transcripts derived from the P2 promoter. In addition, these studies have shown that transcripts derived from the P1 promoter initiate transcription from two sites. The primary site of transcription initiation from the P1 promoter corresponds to that previously defined from primer extension analyses(11) . Although the secondary transcription initiation site from the P1 promoter has not been precisely mapped, cDNA cloning and RNase protection analyses have shown that it lies at least 21 nucleotides upstream of the primary transcription start site. Initiation of transcription from multiple sites has also been shown to occur at the erythroid-specific promoter of the AE1 anion exchanger gene in both mouse (29) and rat(30) . Interestingly, the transcripts that initiate from the secondary initiation site of the P1 promoter also encode each of the four AE1 structural variants. The diversity observed among the transcripts derived from the P1 and P2 promoters is made even more remarkable by the fact that the 12 variant AE1 transcripts encode only four different structural polypeptides.

Although transcription from alternative promoters that results in the production of polypeptides that differ at their N terminus has been reported for several genes(13, 17, 31) , the chicken erythroid AE1 anion exchanger transcripts are unique in that variant transcripts encoding identical structural polypeptides are simultaneously derived from two separate promoters. Previous studies have also shown that the use of alternative promoters can be developmentally regulated(17) . To investigate whether the two erythroid-specific AE1 promoters, P1 and P2, are differentially regulated during development, RT-PCR analyses have determined the array of AE1 transcripts expressed in erythroid cells in 3-day-old and 10-day-old chicken embryos. The erythroid cells isolated from 10-day-old chicken embryos are almost entirely of definitive lineage in origin, whereas the erythroid cells in 3-day-old embryos are exclusively derived from cells of the primitive lineage (16) . These studies have shown that each of the 12 variant AE1 transcripts that accumulate in erythroid cells isolated from 10-day-old embryos also accumulate in erythroid cells isolated from 3-day-old embryos. Because the erythroid AE1 anion exchanger is involved in the maintenance of pH equilibrium in the plasma, the redundancy observed in the gene products derived from the two erythroid-specific AE1 promoters may provide a mechanism to insure the high level expression of these electroneutral transporters during all stages of chicken embryonic development. Alternatively, these two promoters may differentially respond to different environmental stimuli, thereby increasing the capacity of erythroid cells to regulate the expression of the anion exchanger under different environmental conditions.


FOOTNOTES

*
This research was supported by Grant 91-009920 from the National Chapter of the American Heart Association, Grant IN-176-B from the American Cancer Society (to J. V. C.), and a grant from the National Kidney Foundation of West Tennessee, Inc. (to K. H. C.). Oligonucleotides and peptides were provided by the Molecular Resource Center Synthesis Facility, University of Tennessee, Memphis. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dept. of Microbiology and Immunology, University of Tennessee, Memphis, 858 Madison Ave., Memphis, TN 38163. Tel.: 901-448-7080; Fax: 901-448-8462.

(^1)
K. H. Cox and J. V. Cox, unpublished observations.

(^2)
The abbreviations used are: HEL, human erythroleukemia; RT, reverse transcriptase; PCR, polymerase chain reaction; RACE, rapid amplication of cDNA ends; PIPES, 1,4-piperazinediethanesulfonic acid; PBS, phosphate-buffered saline.

(^3)
J. Cox, unpublished observations.


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