Identification of the full-length AE2 (AE2a) isoform as the Golgi-associated anion exchanger in fibroblasts.

Na(+)-independent Cl(-)/HCO(3)(-) exchangers (AE1, AE2, AE3) are generally known as ubiquitous, multispanning plasma membrane proteins that regulate intracellular pH and transepithelial acid-base balance in animal tissues. However, previous immunological evidence has suggested that anion exchanger (AE) proteins may also be present in intracellular membranes, including membranes of the Golgi complex and mitochondria. Here we provide several lines of evidence to show that an AE protein is indeed a resident of the Golgi membranes and that this protein corresponds to the full-length AE2a isoform in fibroblasts. First, both the N- and C-terminal antibodies to AE2 (but not to AE1) detected an AE protein in the Golgi membranes. Golgi localization of this AE2 antigen was evident also in cycloheximide-treated cells, indicating that it is a true Golgi-resident protein. Second, our Northern blotting and RT-PCR analyses demonstrated the presence of only the full-length AE2a mRNA in cells that show prominent Golgi staining with antibodies to AE2. Third, antisense oligonucleotides directed against the translational initiation site of the AE2a mRNA markedly inhibited the expression of the endogenous AE2 protein in the Golgi. Finally, transient expression of the GFP-tagged full-length AE2a protein resulted in predominant accumulation of the fusion protein in the Golgi membranes in COS-7 and CHO-K1 cells. Golgi localization of the AE2a probably involves its oligomerization and/or association with the recently identified Golgi membrane skeleton, because a substantial portion of both the endogenous AE2a and the GFP-tagged fusion protein resisted detergent extraction in cold. (J Histochem Cytochem 49:259-269, 2001)     

Nucleotide sequence of rat alpha 1-acid glycoprotein messenger RNA

The complete nucleotide sequence of rat alpha 1-acid glycoprotein (alpha 1-AGP) mRNA has been determined from cloned double-stranded cDNA. The coding portion of the mRNA was bounded at the ends by a 5'-untranslated region of 35 nucleotides in length and a 3'-untranslated region of 119 nucleotides in length. The 3'-untranslated region contains the characteristic AAUAAA sequence ending 18 nucleotides from the 3'-terminal poly(A) segment. The 5'-region of the mRNA contains two in-phase AUG codons separated by 12 nucleotides. Comparison with the known NH2-terminal amino acid sequence of serum rat alpha 1-AGP suggests that the primary translation product of the mRNA contains an additional 14 or 18 amino acids that are not present in the mature form of the protein, which contains 187 amino acids. The inferred amino acid sequence of rat alpha 1-AGP and the known amino acid sequence of human alpha 1-AGP have several regions of identity clustered in the NH2-terminal portion of the proteins. The carboxyl-terminal regions show significantly less homology. Six potential asparagine glycosylation sites are found in the rat sequence, and four of these sites are in positions similar to known glycosylation sites in the human protein. Furthermore, three of these potential glycosylation sites are in a region that exhibits extensive amino acid sequence conservation, suggesting that this region may be important for the biological function of alpha 1-AGP.     

Partial Characterization of a Human ZincDeficiency Syndrome by Differential Display

The effect of the acrodermatitis enteropathica mutation (AE) on gene expression was investigated using differential display. Two differentially expressed cDNAs were partially characterized. The NA8 cDNA (HT₁₁A anchor and HAP 8 random primer pair) was expressed in greater quantity in normal fibroblasts, was 249 bp, and hybridized to three mRNA species (2 kb, 1 kb, 0.8 kb). Northern blot analysis indicated that the relative amounts of the AE mRNA species were reduced by 73%, 75%, and 52%, respectively. The cDNA sequence exhibited 92–93% homology to the human cytochrome oxidase subunit II, as analyzed through the GenBank database. The AEG4 cDNA species (HT₁₁G anchor and HAP 4 random, primer pair) was expressed in greater quantity in AE fibroblasts, was 197 bp, and hybridized to two mRNA species (9 kb, 4 kb). Northern blot analysis indicated that the 9-kb mRNA species was present equally in AE and normal cells, but the 4-kb mRNA species was only present in the AE fibroblasts. The cDNA sequence exhibited 92% homology to LINE1 human retrotransposons, as analyzed through the GenBank database. The functional relationship between the mutation and the reduced expression of cytochrome oxidase subunit II is unknown at this time and needs to be addressed. The increased expression of the LINE1 element in AE fibroblasts may be indicative of an insertion mutation affecting the mRNA of a protein involved in zinc transport, a prospect which requires further investigation.     

The primary structure of rat ribosomal protein S25.

The amino acid sequence of the rat 40S ribosomal subunit protein S25 was deduced from the sequence of nucleotides in a recombinant cDNA. Ribosomal protein S25 has 125 amino acids and has a molecular weight of 13,733. Hybridization of the cDNA to digests of nuclear DNA suggests that there are 19 to 22 copies of the S25 gene. The mRNA for the protein is about 550 nucleotides in length. Rat S25 is homologous to ribosomal proteins from other eukaryotes (human and yeast).     

Complete nucleotide sequence of ovine alpha-lactalbumin mRNA

The nucleotide sequence of ovine alpha-lactalbumin mRNA has been determined by chemical sequencing of two cDNA recombinant plasmids and a primer extension product. Ovine alpha-lactalbumin mRNA contains 723 nucleotides (excluding the poly(A) tail), with a 5' non-coding region of 26 nucleotides, followed by the 426 nucleotides of the coding region which determines a sequence signal of 19 amino acid residues and the 123 amino acid residues of mature alpha-lactalbumin. The coding region is followed by a 3' untranslated sequence of 271 nucleotides. The derived amino acid sequence of ovine pre-alpha-lactalbumin differs from that of its bovine counterpart by 8 amino acid substitutions, all but one originating from single mutations. Comparison of sequences of guinea pig, rat and human alpha-lactalbumin mRNAs with their ovine and bovine counterparts has revealed that these molecules have rapidly evolved. The highest degree of conservation was observed in the region coding for the mature protein and corresponds essentially to sequences which interact with UDP-galactosyltransferase and Ca2+ ions.     

The primary structure of rat ribosomal protein L38.

The amino acid sequence of the rat 60S ribosomal protein L38 was deduced from the sequence of nucleotides in three recombinant cDNAs. Ribosomal protein L38 has 69 amino acids (the NH2-terminal methionine is removed after translation of the mRNA) and has a molecular weight of 8,081. Hybridization of the cDNA to digests of nuclear DNA suggests that there are 11-13 copies of the L38 gene. The mRNA for the protein is about 450 nucleotides in length.     

The primary structure of rat ribosomal protein L21

The covalent structure of rat ribosomal protein L21 was deduced from the sequence of nucleotides in a recombinant cDNA and confirmed from the NH2-terminal amino acid sequence of the protein. Ribosomal protein L21 contains 159 amino acids (the NH2-terminal methionine is removed after translation of the mRNA) and has a molecular weight of 18,322. Hybridization of the cDNA to digests of nuclear DNA suggests that there are 16-23 copies of the L21 gene. The mRNA for the protein is about 680 nucleotides in length.     

Sequence of human adenosine deaminase cDNA including the coding region and a small intron

The nucleotide sequence for an unusual, cloned human adenosine deaminase cDNA has been determined. Contained within a sequence of 1535 nucleotides is a coding sequence of 1089 nucleotides that encodes a protein of 40,762 daltons. The coding sequence is interrupted by a non-coding region containing 76 nucleotides. Both the 3' and 5' ends of this region have consensus sequences generally associated with splice sites. The 3' untranslated sequence contained 308 nucleotides, including a polyadenylation signal sequence 20 nucleotides from the end. The cloned cDNA appears to correspond to a nuclear mRNA precursor which contains a small intron.     

Sequence of the pS2 mRNA induced by estrogen in the human breast cancer cell line MCF-7.

We present the complete sequence of an mRNA which is induced by estrogen in the human breast cancer cell line MCF-7 [pS2 mRNA, Masiakowski et al., Nucleic Acids Res. 10, 7895-7903 (1982)]. Primer extension and cloning of double-stranded cDNA (ds-cDNA) into a vector designed to make full-length cDNA were used to determine the sequence of the fifteen 5'-terminal nucleotides which were not present in the original pS2 ds-cDNA clone. The mRNA sequence has a major open reading frame encoding 84 amino-acids, flanked by a 40 nucleotide 5'-untranslated region and a 198 nucleotide 3'-untranslated region preceding the polyA tail. The 3'-untranslated region contains a polyadenylation signal, AUUAAA, 14 nucleotides upstream from the polyA tail. The derived protein sequence contains a putative signal peptide region suggesting that the protein may be secreted. The nucleotide and derived amino-acid sequences were compared to previously determined sequences, particularly to those of hormone-regulated proteins and growth factors, and no obvious similarities were observed.     

Characterization of the 5'-flanking region of the human multidrug resistance protein 2 (MRP2) gene and its regulation in comparison withthe multidrug resistance protein 3 (MRP3) gene.

The multidrug resistance proteins MRP2 (symbol ABCC2) and MRP3 (symbol ABCC3) are conjugate export pumps expressed in hepatocytes. MRP2 is localized exclusively to the apical membrane and MRP3 to the basolateral membrane. MRP2 mRNA is expressed at a high level under normal conditions, whereas MRP3 mRNA expression is low and increases only when secretion across the apical membrane by MRP2 is impaired. We studied some of the regulatory properties of the two human genes using transient transfection assays with promoter-luciferase constructs in HepG2 cells and cloned fragments of 1229 nucleotides and 1287 nucleotides of the MRP2 and MRP3 5'-flanking regions, respectively. The sequence between nucleotides -517 and -197 was decisive for basal MRP2 expression. Basal promoter activity of MRP3 was only 4% of that measured for MRP2. At submicromolar concentrations, the histone deacetylase inhibitor trichostatin A reduced the MRP2 reporter gene activity and expression of the protein. Disruption of microtubules with nocodazole decreased gene and protein expression of MRP2 and increased MRP3 reporter gene activity. The genotoxic 2-acetylaminofluorene decreased the activity of the human MRP2 reporter gene construct, but increased MRP3 gene activity and enhanced the amounts of mRNA and protein of MRP2 and MRP3. Thus, regulation of the expression of these ATP-dependent conjugate export pumps is not co-ordinate, but in part inverse. The inverse regulation of the two MRP isoforms is consistent with their distinct localization, their different mRNA expression under normal and pathophysiological conditions, and their different directions of substrate transport in polarized cells.     

Molecular cloning and nucleotide sequence of a bovine alpha-lactalbumin cDNA

A cDNA clone for the bovine milk protein, alpha-lactalbumin (alpha LA), has been identified using a rat cDNA probe. The bovine cDNA clone is 703 nucleotides (nt) long, contains 8 nt of 5'-untranslated sequence and 269 nt of 3'-untranslated sequence. When compared with previously reported sequences, the bovine alpha LA mRNA sequence has 74% similarity with rat alpha LA mRNA, 79% similarity with human mRNA and 74% similarity with guinea pig mRNA.     

Molecular cloning and primary structure of rat thyroxine-binding globulin

Rat thyroxine-binding globulin (TBG) cDNAs were isolated from a rat liver cDNA library by using a human TBG cDNA as a probe. From two overlapping cDNA inserts, an aligned cDNA sequence of 1714 nucleotides was obtained. There was 70% homology with human TBG cDNA over the span of 1526 nucleotides. In order to confirm that the cloned cDNA encodes rat TBG and to localize the NH2-terminal amino acid of the mature molecule, the protein was purified by affinity chromatography and subjected to direct protein microsequencing. The NH2-terminal amino acid sequence was identical with that deduced from the nucleotide sequence. The rat TBG cDNA sequenced consisted of a truncated leader sequence (35 nucleotides), the complete sequence encoding the mature protein (1194 nucleotides) and the 3'-untranslated region (485 nucleotides), containing two polyadenylation signals. It was deduced that rat TBG consists of 398 amino acids (Mr = 44,607), three NH2-terminal residues more than human TBG, with which it shares 76% homology in primary structure. Of the six potential N-glycosylation sites, four are located in conserved positions compared to human TBG. Northern blot analysis of rat liver revealed an approximately 1.8-kilobase TBG mRNA. Its amount increased markedly following thyroidectomy and decreased with thyroxine treatment in a dose-dependent manner.     

The primary structure of rat ribosomal protein S19

The covalent structure of rat ribosomal protein S19 was deduced from the sequence of nucleotides in a recombinant cDNA and confirmed from the NH2-terminal amino acid sequence of the protein. Ribosomal protein S19 contains 144 amino acids (the NH2-terminal methionine is removed after translation of the mRNA) and has a molecular weight of 15,944. Hybridization of the cDNA to digests of nuclear DNA suggests that there are 15-18 copies of the S19 gene. The mRNA for the protein is about 640 nucleotides in length. Rat S19 is related to Saccharomyces cerevisiae S16A and to Halobacterium marismortui S12.     

The primary structure of rat ribosomal protein L17

The amino acid sequence of the rat 60S ribosomal subunit protein L17 was deduced from the sequence of nucleotides in two recombinant cDNAs. Ribosomal protein L17 has 184 amino acids and has a molecular weight of 21,383. Hybridization of the cDNA to digests of nuclear DNA suggests that there are 17-19 copies of the L17 gene. The mRNA for the protein is about 720 nucleotides in length. Rat L17 is homologous to human L17 and related to Saccharomyces cerevisiae YL17, Halobacterium marismortui L23, Halobacterium halobium L22e, Escherichia coli L22 and other members of the prokaryotic L22 family.     

Regulation of DRA and AE1 in rat colon by dietary Na depletion

Two distinct Cl/anion exchange activities (Cl/HCO(3) and Cl/OH) identified in apical membranes of rat distal colon are distributed in cell type-specific patterns. Cl/HCO(3) exchange is expressed only in surface cells, whereas Cl/OH exchange is localized in surface and crypt cells. Dietary Na depletion substantially inhibits Cl/HCO(3) but not Cl/OH exchange. We determined whether anion exchange isoforms (AE) and/or downregulated in adenoma (DRA) are expressed in and related to apical membrane anion exchanges by examining localization of AE isoform-specific and DRA mRNA expression in normal and Na-depleted rats. Amplification of AE cDNA fragments by RT-PCR with colonic mRNA as template indicates that AE1 and AE2 but not AE3 mRNAs are expressed. In situ hybridization study revealed that AE1 mRNA is expressed predominantly in surface but not crypt cells. In contrast, AE2 polypeptide is expressed in basolateral membranes and DRA protein is expressed in apical membranes of both surface and crypt cells. AE1 mRNA is only minimally present in proximal colon, and DRA mRNA abundance is similar in distal and proximal colon. Dietary Na depletion reduces AE1 mRNA abundance but did not alter DRA mRNA abundance. This indicates that AE1 encodes surface cell-specific aldosterone-regulated Cl/HCO(3) exchange, whereas DRA encodes aldosterone-insensitive Cl/OH exchange.