Use Of Alternative Polyadenylation Sites Research Articles

Regulation of oxytocin receptor gene expression in sheep: tissue specificity, multiple transcripts and mRNA editing

The increase in uterine oxytocin receptor concentrations over the late luteal phase of the oestrous cycle in sheep is thought to play an important role in the regulation of the duration of the cycle by facilitating the effect of oxytocin on uterine prostaglandin release. Experiments indicated that oxytocin receptor mRNA expression in the endometrium was high at oestrus compared with at days 2, 7 and 12 of the oestrous cycle. The amount of oxytocin receptor mRNA expression in the pituitary gland did not show any significant differences during the oestrous cycle. Oxytocin receptor cDNA was obtained and characterized from ovine uterine endometrium on day 15 of the oestrous cycle, using RT-PCR techniques, to study the mechanisms underlying the resolution of oxytocin receptor expression. The cDNA sequence for the oxytocin receptor gene in sheep was found to be similar to that described previously, except for a difference of seven nucleotides. These nucleotide differences resulted in changes in four of the deduced amino acids in the oxytocin receptor sequence. The heterogeneity of the different sized oxytocin receptor transcripts in sheep is due, at least in part, to the alternative use of polyadenylation sites. Northern hybridization confirmed that the oxytocin receptor gene is expressed in ovine corpus luteum. The investigations on oxytocin receptor gene expression indicate that the patten of oxytocin receptor gene expression in sheep is not only tissue-specific, but also highly function-related. Evidence was obtained of mRNA editing in both the coding and the 3'-untranslated (3'UTR) regions of oxytocin receptor gene transcripts in ovine endometrium; this was the first demonstration of this phenomenon for oxytocin receptor mRNA. The present results indicate that the observed differences in oxytocin receptor mRNA sequences for the different oxytocin receptor populations in endometrium are due to mRNA editing. mRNA editing of oxytocin receptor transcripts may be reflected in changes in the amino acid composition of the carboxyl terminus of the receptor, which would explain the differences in the observed responses to an oxytocin challenge.

Regulation of oxytocin receptor gene expression in sheep: tissue specificity, multiple transcripts and mRNA editing

The increase in uterine oxytocin receptor concentrations over the late luteal phase of the oestrous cycle in sheep is thought to play an important role in the regulation of the duration of the cycle by facilitating the effect of oxytocin on uterine prostaglandin release. Experiments indicated that oxytocin receptor mRNA expression in the endometrium was high at oestrus compared with at days 2, 7 and 12 of the oestrous cycle. The amount of oxytocin receptor mRNA expression in the pituitary gland did not show any significant differences during the oestrous cycle. Oxytocin receptor cDNA was obtained and characterized from ovine uterine endometrium on day 15 of the oestrous cycle, using RT-PCR techniques, to study the mechanisms underlying the resolution of oxytocin receptor expression. The cDNA sequence for the oxytocin receptor gene in sheep was found to be similar to that described previously, except for a difference of seven nucleotides. These nucleotide differences resulted in changes in four of the deduced amino acids in the oxytocin receptor sequence. The heterogeneity of the different sized oxytocin receptor transcripts in sheep is due, at least in part, to the alternative use of polyadenylation sites. Northern hybridization confirmed that the oxytocin receptor gene is expressed in ovine corpus luteum. The investigations on oxytocin receptor gene expression indicate that the patten of oxytocin receptor gene expression in sheep is not only tissue-specific, but also highly function-related. Evidence was obtained of mRNA editing in both the coding and the 3'-untranslated (3'UTR) regions of oxytocin receptor gene transcripts in ovine endometrium; this was the first demonstration of this phenomenon for oxytocin receptor mRNA. The present results indicate that the observed differences in oxytocin receptor mRNA sequences for the different oxytocin receptor populations in endometrium are due to mRNA editing. mRNA editing of oxytocin receptor transcripts may be reflected in changes in the amino acid composition of the carboxyl terminus of the receptor, which would explain the differences in the observed responses to an oxytocin challenge.

The expression profile for the tumour suppressor gene PTEN and associated polymorphic markers.

PTEN, a putative tumour suppressor gene associated with prostate and other cancers, is known to be located within the chromosomal region 10q23.3. Transcription of the PTEN gives rise to multiple mRNA species. Analyses by Northern blots, using cell lines which express PTEN together with cell lines which have lost the PTEN or carry a truncated version of the gene, has allowed us to demonstrate that the pseudogene is not transcribed. In addition, 3′ RACE studies confirmed that the multiple mRNA species arising from the gene probably result from the use of alternative polyadenylation sites. No evidence for tissue- or cell-specific patterns of transcription was found. Analysis by 5′ RACE placed the putative site for the start of transcription around 830 bp upstream of the start codon. A map of the location of the PTEN gene with a series of overlapping YAC, BAC and PACs has been constructed and the relative position of eight microsatellite markers sited. Two known and one novel marker have been positioned within the gene, the others are in flanking regions. The more accurate location of these markers should help in future studies of the extent of gene loss. Several polymorphisms were also identified, all were within introns. Four of the common polymorphisms appear to be linked. In blood, DNA from 200 individuals, including normal, BPH and prostate cancer patients, confirmed this link. Only two samples of 200 did not carry the linked haplotype, both were patients with advanced prostate cancer. It is possible that such rearrangements within PTEN could be evidence of predisposition to prostate cancer in this small number of cases. © 2000 Cancer Research Campaign

Characterization and Screening for Mutations of the Growth Arrest-Specific 11 (GAS11) andC16orf3Genes at 16q24.3 in Breast Cancer

Loss of heterozygosity involving the long arm of chromosome 16 is a frequent event seen in a number of human carcinomas, including breast, prostate, hepatocellular, and ovarian cancers. A region found to be commonly deleted in breast and prostate carcinomas is located at 16q24.3, which suggests the presence of a tumor suppressor gene that may be altered in these two malignancies. A detailed physical and transcription map of this region that includes the loci defining the smallest region of deletion has been constructed. This report describes the characterization of a transcript located in this region, the growth arrest-specific 11 (GAS11) gene, which was viewed as a potential tumor suppressor gene due to the expression of its mouse homolog specifically during growth arrest. The gene consists of 11 exons spanning approximately 25 kb. Northern blot analysis identified two ubiquitously expressed mRNAs of 3.4 and 1.8 kb produced by the use of alternative polyadenylation sites. Another gene,C16orf3(chromosome 16 open reading frame 3), was found to lie within intron 2 ofGAS11.This gene appears intronless, is transcribed in the orientation opposite to that ofGAS11,and is expressed at low levels. These genes were examined for mutations in breast tumor DNA, and both were excluded as tumor suppressor genes involved in breast cancer.

A variant transcript encoding an isoform of the human protein tyrosine kinase EPHB2 is generated by alternative splicing and alternative use of polyadenylation signals.

We previously isolated and characterized cDNA clones of DRT (EPHB2), encoding a receptor protein-tyrosine kinase of the EPH family. Northern blot analysis showed that EPHB2 transcripts are expressed in three sizes of approximately 4, 5, and 11 kb, suggesting that these transcripts are generated by alternative splicing and/or alternative use of polyadenylation sites. To explore this possibility, we isolated additional EPHB2 cDNA clones, including clone 5K-1, by re-screening the human fetal brain cDNA library. Nucleotide sequence analysis of clone 5K-1 revealed that it represents a variant transcript of EPHB2 (EPHB2v). Relative to the EPHB2 cDNA sequence previously reported, clone 5K-1 has two coding region deletions of 3 and 93 nucleotides. Nucleotide sequence analyses of EPHB2 genomic DNA fragments corresponding to these deletions suggest that the EPHB2v transcript is generated by alternative splicing. The 3' end of clone 5K-1 contains a polyadenosine stretch preceded by a potential polyadenylation signal, which is not used to generate the EPHB2 transcript. Taken together, these data indicate that EPHB2v is generated by both alternative splicing and alternative use of polyadenylation sites. The EPHB2v protein lacks one arginine residue that resides immediately following the EPHB2 transmembrane domain. In contrast, as a result of the frame shift caused by the 93 nucleotide deletion, the C-terminus of the EPHB2v protein is longer by 70 amino acids than that of EPHB2. We also show that the human neuroblastoma cell line SY5Y and NTera-2N neurons express high levels of EPHB2 and lower levels of EPHB2v. These structural variations found between the EPHB2 and EPHB2v proteins may reflect functional heterogeneity of EPHB2.

Characterization of a Novel Gene, C21orf2, on Human Chromosome 21q22.3 and Its Exclusion as the APECED Gene by Mutation Analysis

Exon trapping was performed from a partial cosmid, PAC, and P1 clone contig from human chromosome 21 betweenMX1and 21qter to identify genes that may be involved in the pathogenesis of Down syndrome or several of the genetic diseases that map to chromosome 21q22.3. One 19-bp exon showed identity to three ESTs. The complete sequence of the EST clones, RT-PCR, and cDNA library screening were used to determine the full-length cDNA sequence of 2.2 kb with an open reading frame of 256-amino-acids. The putative 256-amino-acid peptide has homology with a hypotheticalCaehorhabditis elegansprotein of unknown function. Northern blot analysis of this gene, termed C21orf2 (chromosome 21 open reading frame 2), revealed two ubiquitously expressed mRNAs of 2.2 and 1.2 kb produced by use of alternative polyadenylation sites. Hybridization of the EST clones to a cosmid contig in chromosome 21q22.3 mapped C21orf2 just distal toPFKL,a critical mapping region for several genetic diseases. Comparison to publicly available genomic sequence, and additional data, revealed that the gene is split into seven exons over 10.5 kb, further refining the mapping position to only 1.2 kb distal toPFKLwith the direction of transcription toward the centromere. The 5′UTR is contiguous withD21S400,and intron 2 contains a 52-bp VNTR polymorphism. Given its mapping position, C21orf2 is a candidate for involvement in disorders including autoimmune polyglandular disease type I (also called autoimmune polyendocrinopathy candidiasis ectodermal dystrophy or APECED) and the autosomal nonsyndromic deafness loci, DFNB8 and DFNB10. Mutation analysis using sequencing of RT-PCR and genomic DNA-derived PCR products, SSCP, and Southern and Northern blot analyses in APECED patients excluded C21orf2 as the gene for APECED.

Expression of Members of a Novel Membrane Linked Metalloproteinase Family (ADAM) in Human Articular Chondrocytes

Using resting chondrocytes derived from human articular femoral head and a conditionally immortalised human articular chondrocyte cell line we have studied the expression of members of the novel metalloproteinase/disintegrin family termed ADAM. Using RT-PCR we can detect the expression of ADAM-12 a novel family member isolated from myeloma cells [1]. We also find expression of ADAM 10 a functional metalloproteinase/disintegrin first isolated from bovine brain and ADAM-15 a metallodisintegrin isolated from mammary derived epithelial cells. Northern blotting was used to confirm expression. One main transcript is visible for ADAM-12 whereas both ADAM-10 and ADAM-15 have multiple transcripts indicating possible RNA variants potentially derived from alternative splicing or alternative use of polyadenylation sites. Since chondrocytes are proposed as an important source of metalloproteinase enzymes involved in joint pathology the potential relevance of the expression of these molecules to connective tissue disorders is discussed.

Expression of the release factor eRF1 (Sup45p) gene of higher eukaryotes in yeast and mammalian tissues

Polypeptide chain termination in eukaryotic cells is mediated in part by the release factor eRF1 (Sup45p). We have isolated and characterised cDNAs encoding this translation factor from Syrian hamster ( Mesocricetus auratus) and human ( Homo sapiens) Daudi cells. Comparison of the deduced amino acid sequence of these new eRF1 (Sup45p) sequences with those published for Saccharomyces cerevisiae Arabidopsis thaliana, Xenopus laevis and human indicates a high degree of amino acid identity across a broad evolutionary range of species. Both the 5′ and 3′ UTRs of the mammalian eRF1 (Sup45p)-encoding cDNAs show an unusually high degree of conservation for non-coding regions. In addition, the presence of two different lengths of 3′ UTR sequences in the mammalian eRF1 (Sup45p) cDNAs indicated that alternative polyadenylation sites might be used in vivo. Northern blot analysis demonstrated that eRF1 (Sup45p) transcripts of differing length, consistent with the use of alternative polyadenylation sites, were detectable in a wide range of mammalian tissues. The Xenopus, human and Syrian hamster eRF1 (Sup45p) cDNAs were shown to support the viability of a strain of S cerevisiae carrying an otherwise lethal sup45::HIS3 gene disruption indicating evolutionary conservation of function. However, the yeast strains expressing the heterologous eRF1 (Sup45p) showed a defect in translation termination as defined by an enhancement of nonsense suppressor tRNA activity in vivo. Western blot analysis confirmed that Xenopus eRF1 (Sup45p) was primarily ribosome-associated when expressed in yeast indicating that the ribosome-binding domain of eRF1 (Sup45p) is also conserved.

Multiple pathways for Steel regulation suggested by genomic and sequence analysis of the murine Steel gene.

The Steel (Sl) locus encodes mast cell growth factor (Mgf) that is required for the development of germ cells, hematopoietic cells and melanocytes. Although the expression patterns of the Mgf gene are well characterized, little is known of the factors which regulate its expression. Here, we describe the cloning and sequence of the full-length transcription unit and the 5' flanking region of the murine Mgf gene. The full-length Mgf mRNA consists of a short 5' untranslated region (UTR), a 0.8-kb ORF and a long 3' UTR. A single transcription initiation site is used in a number of mouse tissues and is located just downstream of binding sites for several known transcription factors. In the 5' UTR, two ATGs were found upstream of the initiator methionine and are conserved among different species, suggesting that Mgf may be translationally regulated. At least two Mgf mRNAs are produced by alternative use of polyadenylation sites, but numerous other potential polyadenylation sites were found in the 3' UTR. In addition, the 3' UTR contains numerous sequence motifs that may regulate Mgf mRNA stability. These studies suggest multiple ways in which expression of Mgf may be regulated.

Use of alternative polyadenylation sites in the synthesis of mRNAs encoding the interferon-induced tryptophanyl tRNA synthetase

The interferon-mediated induction of the gene encoding the human tryptophanyl tRNA synthetase (WRS) results in the production of two mRNA species differing in size by approximately 800 base pairs (bp). Two distinctly sized cDNAs differing by approximately 800 bp were isolated from a cDNA library generated from mRNA prepared from IFN-γ-treated cells. Northern blot analysis using cDNA probes recognizing different regions of the WRS mRNA reveals distinctly sized mRNAs differing in the length of their 3′ untranslated regions. Differential display analysis using oligo dT primers demonstrates that the different sized WRS mRNAs result from alternative polyadenylation of this transcript.

RNA Expression and Chromosomal Location of the Mouse Long-Chain Acyl-CoA Dehydrogenase Gene

The cDNA for mouse long-chain acyl-CoA dehydrogenase (Acadl, gene symbol; LCAD, enzyme) was cloned and characterized. The cDNA was obtained by library screening and reverse transcription-polymerase chain reaction (RT-PCR). The deduced amino acid sequence showed a high degree of homology to both the rat and the human LCAD sequence. Northern analysis of multiple tissues using the mouse Acadl cDNA as a probe showed two bands in all tissues examined. We found a total of three distinct mRNAs for Acadl. These three mRNAs were encoded by a single gene that we mapped to mouse chromosome 1. The three transcripts differed in the 3′ untranslated region due to use of alternative polyadenylation sites. Quantitative evaluation of a multitissue Northern blot showed a varied ratio of the larger transcript as compared with the smaller transcripts.

Use of Alternative Polyadenylation Sites by the Chicken S6 Ribosomal Protein Gene

Two chicken ribosomal protein S6 cDNAs were isolated from a liver cDNA library and their DNA sequence determined. Each of the cDNAs was found to use a different polyadenylation signal and to be polyadenylated at a different site. This is the first observation of alternative Poly(A) site usage by a ribosmal protein gene. Northern blot analysis revealed that the chicken rpS6 transcripts can be alternatively polyadenylated in brain tissue. Southern blot analysis indicates that there is only one rpS6 gene in the chicken genome. The long open reading frame of the chicken rpS6 gene specifies 249 amino acids which show high homology with rpS6 homologues from other species. The four serine residues in rpS6 which are major sites of phosphorylation in mammals are conserved in chicken.

Drifter, a Drosophila POU-domain transcription factor, is required for correct differentiation and migration of tracheal cells and midline glia.

The Drosophila drifter (dfr) gene, previously referred to as Cf1a, encodes a POU-domain DNA-binding protein implicated as a neuron-specific regulator in the developing central nervous system (CNS). We have isolated full-length dfr cDNA clones that encode a 46-kD protein containing the conserved POU-domain DNA-binding domain. The use of alternate polyadenylation sites produces two dfr mRNA transcripts that are first expressed in stage 10 embryos at 5- to 6-hr of development. A specific anti-dfr polyclonal antiserum generated against a dfr-glutathione S-transferase fusion protein recognizes a 46-kD protein on Western blots and has been used to analyze the cell-specific distribution of dfr protein during embryonic development. dfr protein is distributed in a complex expression pattern including the tracheal system, the middle pair of midline glia, and selected CNS neurons. We have carried out a genetic characterization of the dfr locus, previously localized to region 65D of the third chromosome, by generating a series of overlapping deficiencies between 65A and 65E1 that were used to isolate dfrE82, an EMS-induced lethal allele. Analysis of dfrE82 mutant embryos shows a disruption of the developing tracheal tree as well as commissural defects in the developing CNS. Based on an examination of a cell-specific marker for tracheal cells and midline glia, these defects appear to be caused by a failure of these cells to follow their characteristic routes of migration. The dfrE82 tracheal phenotype is rescued by a dfr minigene present as a P-element transposon expressing wild-type dfr protein in tracheal cells. These results suggest that the dfr protein plays a fundamental role in the differentiation of tracheal cells and midline glia possibly by regulating the expression of essential cell-surface proteins required for cell-cell interactions involved in directed cell migrations.

Structure and expression of the gene encoding phosphofructokinase (PFK) in Drosophila melanogaster.

The gene from Drosophila melanogaster that encodes phosphofructokinase has been isolated and its structure and expression characterized. Southern blots of genomic DNA, comparison of multiple clones, and in situ hybridization to salivary gland chromosomes indicate that the Drosophila genome contains a single copy of the gene which encodes phosphofructokinase (PFK) that is located at cytogenetic position 46D/E on the polytene map. Analysis of the gene structure reveals a single coding region that spans 6.5 kilobase pairs and is composed of eight exons and seven introns. Three developmentally regulated transcripts are generated through the use of alternative polyadenylation sites. Transcription initiation occurs at a single site. The pattern of transcript accumulation during development is similar to other genes of Drosophila which encode enzymes of the glycolytic pathway. Deficiency analysis of the 46D/E region has identified a lethal complementation group associated with reduced PFK activity, thereby establishing the vital role of PFK function in Drosophila.

Cell Density Regulates Differential Production of bFGF Transcripts

In vitro cultures of human retinal pigment epithelial (RPE) cells were used to study the regulation of basic fibroblast growth factor (bFGF) gene expression. Four transcripts of 7.0, 3.7, 2.2, and 1.2 kB are produced from the bFGF gene. Increasing cell density has a profound effect on the expression of the 7.0 kB transcript relative to the 3.7 kB transcript. Here, evidence is presented suggesting that posttranscriptional processing events are responsible for differential expression of the 7.0 and 3.7 kB bFGF transcripts as a function of cell density. Primer extension analysis demonstrates that these two transcripts originate from a single transcription initiation site. Determination of the half-lives of the 7.0 and 3.7 kB transcripts at confluent cell density did not explain the relative expression of these mRNAs. These differences may arise from the use of alternative polyadenylation sites in the 3' untranslated region (UTR) as a function of cell density. Polysomal analysis indicates no selective translation of any of the four bFGF transcripts in RPE cells.

Can, a putative oncogene associated with myeloid leukemogenesis, may be activated by fusion of its 3' half to different genes: characterization of the set gene.

The translocation (6;9)(p23;q34) in acute nonlymphocytic leukemia results in the formation of a highly consistent dek-can fusion gene. Translocation breakpoints invariably occur in single introns of dek and can, which were named icb-6 and icb-9, respectively. In a case of acute undifferentiated leukemia, a breakpoint was detected in icb-9 of can, whereas no breakpoint could be detected in dek. Genomic and cDNA cloning showed that instead of dek, a different gene was fused to can, which was named set. set encodes transcripts of 2.0 and 2.7 kb that result from the use of alternative polyadenylation sites. Both transcripts contain the open reading frame for a putative SET protein with a predicted molecular mass of 32 kDa. The set-can fusion gene is transcribed into a 5-kb transcript that contains a single open reading frame predicting a 155-kDa chimeric SET-CAN protein. The SET sequence shows homology with the yeast nucleosome assembly protein NAP-I. The only common sequence motif of SET and DEK proteins is an acidic region. SET has a long acidic tail, of which a large part is present in the predicted SET-CAN fusion protein. The set gene is located on chromosome 9q34, centromeric of c-abl. Since a dek-can fusion gene is present in t(6;9) acute myeloid leukemia and a set-can fusion gene was found in a case of acute undifferentiated leukemia, we assume that can may function as an oncogene activated by fusion of its 3' part to dek, set, or perhaps other genes.

Can, a Putative Oncogene Associated with Myeloid Leukemogenesis, May Be Activated by Fusion of Its 3‣ Half to Different Genes: Characterization of the set Gene

The translocation (6;9)(p23;q34) in acute nonlymphocytic leukemia results in the formation of a highly consistent dek-can fusion gene. Translocation breakpoints invariably occur in single introns of dek and can, which were named icb-6 and icb-9, respectively. In a case of acute undifferentiated leukemia, a breakpoint was detected in icb-9 of can, whereas no breakpoint could be detected in dek. Genomic and cDNA cloning showed that instead of dek, a different gene was fused to can, which was named set. set encodes transcripts of 2.0 and 2.7 kb that result from the use of alternative polyadenylation sites. Both transcripts contain the open reading frame for a putative SET protein with a predicted molecular mass of 32 kDa. The set-can fusion gene is transcribed into a 5-kb transcript that contains a single open reading frame predicting a 155-kDa chimeric SET-CAN protein. The SET sequence shows homology with the yeast nucleosome assembly protein NAP-I. The only common sequence motif of SET and DEK proteins is an acidic region. SET has a long acidic tail, of which a large part is present in the predicted SET-CAN fusion protein. The set gene is located on chromosome 9q34, centromeric of c-abl. Since a dek-can fusion gene is present in t(6;9) acute myeloid leukemia and a set-can fusion gene was found in a case of acute undifferentiated leukemia, we assume that can may function as an oncogene activated by fusion of its 3' part to dek, set, or perhaps other genes.

Molecular cloning of the major cell surface heparan sulfate proteoglycan from rat liver.

We have used an antiserum raised against a purified heparan sulfate proteoglycan (HSPG) preparation isolated from rat liver to screen a lambda gt11 expression library and have obtained overlapping cDNA clones that contain the full-length coding sequence of an HSPG core protein capable of spanning the plasma membrane. The open reading frame of the rat cDNA encodes a protein of 211 amino acids. The predicted protein sequence (23 kDa) has a high degree of homology with the published partial sequence of the human lung fibroblast HSPG, fibroglycan. The deduced protein sequence contains a 24-amino acid transmembrane domain and a 33-amino acid cytoplasmic domain, both of which are identical with the corresponding regions of human fibroglycan and are highly homologous to the human, hamster, and mouse epithelial HSPG, syndecan. The putative ectodomain, which has 85% homology to fibroglycan, contains three possible glycosaminoglycan attachment sites that may be occupied by heparan sulfate chains. The major 49-kDa core protein in the liver HSPG preparation was found to be reactive to an antibody that specifically recognizes the cytoplasmic domain of fibroglycan. We have used the full-length cDNA clone to analyze the expression of this transmembrane core protein gene in whole tissues and several epithelial and fibroblastoid cell lines. It hybridizes to three mRNA species in all cell and tissue types examined, but in liver, isolated hepatocytes, and kidney, an additional 0.8-kilobase mRNA was detected. The three common messages arise from differential use of alternative polyadenylation sites, whereas the fourth tissue-restricted RNA species represents a related gene transcript. The rat equivalent of human fibroglycan therefore appears to be the major transmembrane proteoglycan in liver, and its widespread expression in many diverse tissues and cells suggests that it plays an important role in cellular interactions.

Use of alternative polyadenylation sites for tissue-specific transcription of two angiotensin-converting enzyme mRNAs.

The pulmonary isozyme of rabbit angiotensin-converting enzyme (ACE) is encoded by an mRNA of about 5 kb. cDNA clones corresponding to different parts of this mRNA have been isolated and the complete nucleotide sequences of both the coding and non-coding regions of the mRNA have been determined. The encoded protein has 1309 residues with a 33 amino acids-long signal peptide at the amino terminus and a potential membrane-anchoring domain near the carboxyl terminus. There is a strong sequence homology between two regions of the rabbit cDNA and between the rabbit, human, and mouse cDNAs. Comparison of the nucleotide sequences of the 3' untranslated regions of rabbit pulmonary and testicular ACE cDNAs revealed that the testicular cDNA is nested within the pulmonary cDNA at the 3' end. A rabbit genomic clone encompassing this region was isolated and partially sequenced. It was shown that the gene contains two potential polyadenylation sites 628 bp apart within one exon. Northern analyses with an appropriate oligonucleotide probe confirmed that the proximal polyadenylation site is used exclusively for terminating the testicular mRNA whereas the distal one is used exclusively for the pulmonary mRNA. These results demonstrated that the transcription of the two mRNAs encoding the two ACE isozymes not only initiates at two alternative tissue-specific sites which are 5.7 kb apart but the mRNAs also get polyadenylated at two alternative sites which are 628 bp apart.

Characterization of Rat ARPP‐21 mRNA: Sequence Analysis, Tissue Distribution, and Regulation

ARPP-21 (cyclic AMP-regulated phosphoprotein; Mr = 21,000) is a cytosolic neuronal phosphoprotein that is highly enriched in the striatum and in other dopaminoceptive regions of the brain. The state of phosphorylation of ARPP-21 is also regulated by vasoactive intestinal peptide in intact cells. We previously reported the sequence analysis of bovine ARPP-21 cDNA and have now characterized rat ARPP-21 cDNA to study further the molecular biology of this protein. The sequence of the coding region is 82 and 85% identical at the nucleotide and amino acid levels, respectively, between the two species. There are two major classes of clones, differing only in the lengths of their 3' untranslated ends, suggesting that the different ARPP-21 mRNAs are derived from the use of alternate polyadenylation sites. Both major mRNA species, 2.6 and 0.7 kb, are present at the highest concentration in the striatum, followed by the cortex, consistent with previous immunocytochemical results. Southern blot analysis reveals a simple hybridization pattern, consistent with the presence of a single rat gene encoding ARPP-21. The steady-state levels of the ARPP-21 mRNAs are developmentally regulated but, in the neonatal and mature animal, are not altered following 6-hydroxydopamine lesions of the substantia nigra or by pharmacologic treatments that up-regulate the D1- or D2-dopamine receptors.