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Results for “Alternative Splicing”

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PubMed1996

A new alternative splice variant of the mouse Fas antigen with a deletion in the N-terminal portion of the extracellular domain.

The Fas antigen (Fas/APO-1/CD95) has been shown to induce apoptosis when bound to a monoclonal anti-Fas antibody or Fas ligands. Recently, a new soluble human Fas isoform which lacks the transmembrane domain due to alternative splicing has been isolated; however, no mouse Fas isoforms have been reported so far. Analysis of Fas transcripts by RT-PCR detected no Fas transcripts corresponding to the human soluble Fas isoform in mouse thymus, spleen and liver. However, we detected a new isoform with a 117-bp deletion in the second exon in various mouse tissues and cell lines. This isoform, termed truncated Fas (T-Fas), can be generated by alternative splicing and lacks the N-terminal portion of the extracellular domain just after the signal sequence. Since the deletion involves the first cysteine-rich motif believed to be necessary for binding to the Fas ligand, the T-Fas protein may lack the ability to induce apoptosis. The expression of T-Fas relative to that of the normal Fas varies considerably among mouse tissues and cell lines, suggesting preferential transcription of the T-Fas isoform in certain cell types.

Alternative Splicing↗

PubMed2002

Alternative splicing of the IIICS domain in fibronectin governs the role of the heparin II domain in fibrillogenesis and cell spreading.

The Heparin (Hep) II-binding domain of fibronectin regulates the formation of focal adhesions and actin stress fibers and hence plays an important role in cell spreading, migration, and fibronectin fibrillogenesis. Using human skin fibroblast cultures, we demonstrate that alternative splicing of the neighboring IIICS domain may regulate the activities of the Hep II domain in cell spreading and fibronectin fibrillogenesis. Recombinant Hep II domains, adjacent to either the IIICS domain or the H89 splice variant that contains the amino-terminal sequence of the IIICS domain, blocked fibronectin fibrillogenesis and required sulfated proteoglycans to mediate cell spreading. If the Hep II domain was adjacent to either the H0 or H95 splice variants, which both lack the amino terminus of the IIICS domain, fibrillogenesis was not inhibited and cell spreading was independent of a sulfated proteoglycan-mediated mechanism. The effect of the splice variants on the Hep II domain could be mimicked using a Hep II domain that contained only 6 amino acids from the III(15) repeat or 10 amino acids from the IIICS domain suggesting that sequences proximal to the III(14) repeat determined the role of the Hep II domain in these processes. We propose that alternative splicing of the IIICS domain modulates interactions between heparan sulfate proteoglycans and the Hep II domain and that this serves as a mechanism to control the biological activities of fibronectin.

Alternative Splicing↗

PubMed2006

Conservation and expression of a novel alternatively spliced Evi1 exon.

The Evi1 transcriptional repressor protein is expressed in a developmentally regulated manner, is essential for normal development, participates in regulating cell proliferation and differentiation of cells of haemopoietic and neuronal origin and contributes to the progression of leukaemia. In this report we describe a new murine Evi1 gene transcript (Delta105) that contains two alternatively spliced regions encoding a 9 amino acid insertion (Rp+9) within the repressor domain (Rp) and a 105 amino acid C-terminal truncation. Abundant levels of the 105 amino acid truncated protein are observed in murine leukaemia cells. The combined primary sequence alterations do not affect the DNA binding, transcriptional repressor or CtBP2 protein binding properties of Evi1 but they do reduce its transforming and cell proliferation stimulating activities. Reduced transforming activity is most likely due to the C-terminal truncation as the activity of Evi1 containing either Rp or Rp+9 is indistinguishable. Both isoforms exist in all murine tissues and cell lines examined. However, only the Rp+9 alternative splice variant is also found in humans and other vertebrates. Murine and human forms of Evi1 with Rp or Rp+9 exist. The additional 9 amino acids are encoded by a conserved 27 nucleotide exon, the overall structural organisation of the gene being preserved in the two species. The function of the Rp+9 and Delta105 splice variants is unknown although the conservation of Rp+9 throughout evolution in vertebrate species suggests it is essential to the broad spectrum of biological activities attributed to this developmentally essential protein.

Alternative Splicing↗

PubMed2004

Alpha CP-4, encoded by a putative tumor suppressor gene at 3p21, but not its alternative splice variant alpha CP-4a, is underexpressed in lung cancer.

alpha CP-4 is an RNA-binding protein coded by PCBP4, a gene mapped to 3p21, a common deleted region in lung cancer. In this study we characterized the expression of alpha CP-4 and alpha CP-4a, an alternatively spliced variant of alpha CP-4, in lung cancer cell lines and non-small cell lung cancer (NSCLC) samples from early stage lung cancer patients. In NSCLC biopsies, an immunocytochemical analysis showed cytoplasmic expression of alpha CP-4 and alpha CP-4a in normal lung bronchiolar epithelium. In contrast, alpha CP-4 immunoreactivity was not found in 47% adenocarcinomas and 83% squamous cell carcinomas, whereas all of the tumors expressed alpha CP-4a. Besides, lack of alpha CP-4 expression was associated with high proliferation of the tumor (determined by Ki67 expression). By fluorescence in situ hybridization, >30% of NSCLC cell lines and tumors showed allelic losses at PCBP4, correlating with the absence of the protein. On the other hand, no mutations in the coding region of the gene were found in any of the 24 cell lines analyzed. By Northern blotting and real-time reverse transcription-PCR, we detected the expression of alpha CP-4 and alpha CP-4a messages in NSCLC and small cell lung cancer cell lines. Our data demonstrate an abnormal expression of alpha CP-4 in lung cancer, possibly associated with an altered processing of the alpha CP-4 mRNA leading to a predominant expression of alpha CP-4a. This may be considered as an example of alternative splicing involved in tumor suppressor gene inactivation. Finally, induction of alpha CP-4 expression reduced cell growth, in agreement with its proposed role as a tumor suppressor, and suggesting an association of this RNA-binding protein with lung carcinogenesis.

Aged↗

PubMed2007

A tale of two templates: automatically resolving double traces has many applications, including efficient PCR-based elucidation of alternative splices.

Trace Recalling is a novel method for deconvoluting double traces that result from simultaneously sequencing two DNA templates. Trace Recalling identifies up to two bases at each position of such a trace. The resulting ambiguity sequence is aligned to the genome, identifying one template sequence. A second template sequence is then inferred from this alignment. This technique makes possible many exciting biological applications. Here we present two such applications, alternate splice finding and elucidation of multiple insertion sites in a random insertional mutagenesis library. Our results demonstrate that RT-PCR followed by Trace Recalling is a more efficient and cost effective way to find alternate splices than traditional methods. We also present a method for mapping double-insertion events in a random insertional-mutagenesis library.

Algorithms↗

PubMed2006

Differential expression of alternatively spliced mRNA forms of the insulin-like growth factor 1 receptor in human neuroendocrine tumors.

The activation of the insulin-like growth factor 1/IGF1 receptor system (IGF1/IGF1R) is a critical event in the transformation and tumorigenicity processes in a wide variety of human tumors. The IGF1/IGF1R system has been recently studied in carcinoid tumors that often arise in the gastrointestinal tract; these tumors are characterized by hypersecretion of bioamines and neuropeptides, leading to functional tumor disease. Two alternatively spliced IGF1R mRNA transcripts have been described to differ by only three nucleotides (CAG) in the coding sequence, resulting in an amino-acid change from the originally described Thr-Gly to an Arg in the extracellular portion of the receptor beta subunit. In transfected Chinese hamster ovary cells, the form without CAG (CAG-) exhibited an approximate 2-fold increase in IGF1 stimulation of activities required for its mitogenic properties. In this study, we examine the relative expression of the two IGF1R mRNA isoforms by a semiquantitative RT-PCR approach using highly standardized conditions, beta-2 microglobulin (B2M) as a reference gene and gel imaging analysis. We analyzed a large series of human neuroendocrine tumors (32 samples) and 9 normal tissues. A significant higher expression of both isoforms in the tumor samples (approximately 2-fold increase) was found, while a constant CAG+/CAG- IGF1R mRNA isoforms of an approximate 3:1 ratio was observed in all tumoral and normal cell types studied. The phylogenetic study of the IGF1R locus in several species suggests that human IGF1R CAG- mRNA isoform is evolutionarily more recent compared to the IGF1R CAG+ mRNA isoform and it could be used by the splicing apparatus at this intron/exon junction with a lower efficiency. This study highlights the relevance of IGF1R mRNA expression in neuroendocrine tumor cells, and the constant presence of 'subtle' alternative splicing for the IGF1R locus.

Adult↗

PubMed1997

Selective increase in specific alternative splice variants of tyrosinase in murine melanomas: a projected basis for immunotherapy.

Melanomas tend to become less pigmented in the course of malignant progression. Thus, as proliferation increases, the tumors are decreasingly characterized by the tissue-specific phenotype of normally differentiated melanocytes. To learn whether the decline in melanization is associated with a shift from constitutive to alternative splicing of some pigment gene pre-mRNAs, melanomas were collected from Tyr-SV40E transgenic mice of the standard C57BL/6 strain. The mRNAs of the tyrosinase gene, which has a key role in melanogenesis, were analyzed by quantitative reverse transcriptase-PCR in 34 samples from 16 cutaneous tumors and 9 metastases. The cutaneous tumors included some cases with distinct melanotic and amelanotic zones, which were separately analyzed. All tyrosinase transcripts found in the melanomas were also found in normal skin melanocytes. However, the Delta1b and Delta1d alternatively spliced transcripts, due to deletions within the first exon, were specifically augmented in most of the tumors over their very low levels in skin; the exceptions were some all-amelanotic tumors in which no tyrosinase transcripts were detected. The level of Delta1b rose as high as 11.3% of total tyrosinase mRNAs as compared with 0.6% in skin; Delta1d reached 4.0% as compared with 0. 8% in skin. Expression of these splice variants was highest in the melanotic components of zonal primary tumors, relatively lower in their amelanotic components, and still lower in all-amelanotic primary tumors and amelanotic metastases. The increase in Delta1b and Delta1d transcripts may be predicted to increase the levels of unusual peptides, which could have antigenic potential in the tumors, especially in the relatively early phases of malignancy. Analyses of the alternative transcripts of other pigment genes may identify additional candidate antigens, ultimately enabling melanoma cells in all phases of the disease to be represented as a basis for immune intervention.

Alternative Splicing↗

PubMed1999

Extensive alternative splicing of interleukin-7 in malignant hematopoietic cells: implication of distinct isoforms in modulating IL-7 activity.

Interleukin-7 (IL-7) plays a pivotal role in early stages of normal B and T cell development. In addition, IL-7 stimulates the proliferation of both antitumor reactive cells and a number of T and B cell malignancies, underlining its significance for leukemogenesis. However, its exact role in the process of pathologic maturation of lymphocytes and regulation of the immune response is not completely understood. As alternative splicing of pre-mRNA has been shown to be involved in the control of gene expression, and splicing-derived protein isoforms with antagonistic activity have been found, we assessed the mRNA-expression of IL-7 and its previously described alternative splice variant lacking exon 4, IL-7delta4, in leukemic cells from children with acute lymphoblastic leukemia (ALL). PCR of full-length IL-7 cDNA enabling the competitive amplification of both variants led to the amplification of diverse unexpected PCR products. The sequence data demonstrated the existence of three additional in-frame splice variants resulting from exon skipping of exon 3 or exon 5 or both in combination with exon 4. We named these IL-7delta3/4, IL-7delta4/5, and IL-7delta3/4/5. Furthermore, three out-of-frame splice variants were identified, IL-7(-56bpExon2), IL-7delta4(-56bpExon2), and IL-7delta3/4/5(-56bpExon2), in which, in addition to the aforementioned exon skipping, 56 bp of the 3' end of exon 2 are omitted. Our results led us to assume that splicing-derived IL-7 isoforms play a potential role in modulating IL-7-mediated biologic effects. Further studies are required to clarify the significance of the diverse IL-7 protein isoforms for the regulation of IL-7 function and the pathogenesis of leukemia.

Alternative Splicing↗

PubMed1993

The 67-kD elastin/laminin-binding protein is related to an enzymatically inactive, alternatively spliced form of beta-galactosidase.

We and others have previously shown that a 67-kD cell surface elastin/laminin-binding protein (EBP) is responsible for cell adhesion to elastin and laminin and for mediating the process of elastin fiber assembly, but the nature of this protein was unknown. In this report we provide evidence that a 67-kD catalytically inactive form of beta-galactosidase produced by alternative splicing demonstrates immunological and functional similarity and sequence homology to the 67-kD EBP, suggesting that the two might be the same. Antibody prepared to a synthetic peptide, N-Ac-GSPSAQDEASPL, corresponding to a frame-shift-generated sequence unique to the alternatively spliced form of human beta-galactosidase, also recognized sheep EBP both on Western blotting and in aortic tissue. Furthermore, this synthetic peptide (S-GAL) binds to elastin and laminin, but not to fibronectin, collagen I, or collagen III. Moreover, both tropoelastin and laminin which bind to S-GAL peptide affinity columns can be specifically eluted from them with an excess of free S-GAL peptides. In addition, sequence homology among this splice variant of human beta-galactosidase, sheep EBP, and NH2-terminal sequences of some elastases suggests that these proteins share a common ligand-binding motif that has not been previously recognized.

Alternative Splicing↗

PubMed2004

Tissue-specific expression and regulation of the alternatively-spliced forms of lysyl hydroxylase 2 (LH2) in human kidney cells and skin fibroblasts.

Lysyl hydroxylases 1, 2, and 3 catalyse the hydroxylation of specific lysines in collagen. A small percentage of these hydroxylysine residues are precursors for the cross-link formation essential for the tensile strength of collagen. Lysyl hydroxylase 2 (LH2) exists as two alternatively-spliced forms; the long transcript (the major ubiquitously-expressed form) includes a 63 bp exon (13A) that is spliced out in the short form (expressed, together with the long form, in human kidney, spleen, liver, and placenta). This study shows that this alternative splicing event can be regulated by both cell density and cycloheximide (CHX). Although only the long form of LH2 is detected in untreated confluent human skin fibroblasts, after 24 h treatment with CHX the short LH2 transcript is also expressed. In kidney cells, in which both LH2 transcripts are equally expressed, the long LH2 transcript is significantly decreased after 24 h CHX treatment, whereas expression of the short transcript is slightly increased. This suggests that, in kidney cells, the splicing mechanism for the inclusion of exon 13A in LH2 requires a newly-synthesized protein factor that is suppressed by CHX, whereas, in skin fibroblasts in which levels of LH2 (long) are unaffected, CHX appears to suppress a factor that inhibits exclusion of exon 13A, thereby promoting expression of LH2 (short). As these alternate transcripts of LH2 may have specificity for hydroxylation of lysines in either telopeptide or helical collagen domains, their relative expression determines the type of cross-links formed, thereby affecting collagen strength. Therefore, any perturbation of the regulation of LH2 splicing could influence the stability of the extracellular matrix and contribute to specific connective tissue disorders.

Alternative Splicing↗

PubMed1996

Alternative splicing of the human IgA Fc receptor CD89 in neutrophils and eosinophils.

Receptors for the Fc portion of IgA (Fc alpha R) trigger important immunological elimination processes against IgA-coated targets. Investigation of human Fc alpha R (CD89) transcripts in neutrophils, eosinophils and a monocyte-like cell line, THP-1, with the use of reverse transcriptase PCR, Northern blotting and RNase protection analysis, has provided evidence in these cell types for at least two distinct transcripts generated by alternative splicing. The cDNAs derived from the two major transcripts of both neutrophils and eosinophils have been cloned and sequenced. For both cell types, the larger clone represents the previously described full-length receptor, whereas the second, shorter, splice variant lacks the entire second, membrane-proximal, Ig-like domain. Stable CHO-K1 transfectants have been obtained for both full-length and truncated variant neutrophil receptors. Whereas the full-length receptor is recognized by a panel of five anti-Fc alpha R monoclonal antibodies (mAbs), the shorter variant is bound weakly by only two of the antibodies, suggesting that the epitopes recognized by the majority of the mAbs lie at least in part in the second Ig-like domain of Fc alpha R. Both full-length and splice variant forms of the receptor bind secretory IgA, but the weak binding to serum IgA seen with the full-length receptor is not evident with the shorter variant. Alternative splicing might therefore serve as a means of diversifying Fc alpha R structure and function.

Alternative Splicing↗

PubMed1995

Expression of human chromosome 19p alpha(1,3)-fucosyltransferase genes in normal tissues. Alternative splicing, polyadenylation, and isoforms.

The human alpha(1,3)-fucosyltransferase genes FUT3, FUT5, and FUT6 form a cluster on chromosome 19p13.3. Expression was studied using reverse transcriptase-polymerase chain reaction, rapid amplification of cDNA ends, and Northern analyses. FUT3 and FUT6 were expressed at high levels, while FUT5 expression was lower and restricted to fewer cell types. Alternatively spliced transcripts were identified for FUT3 and FUT6 in kidney, liver, and colon. A 2.37-kilobase pair (kb) FUT3 transcript, detected at high levels in kidney and colon, was absent in liver. FUT6 expression was characterized by a 3.5-kb transcript present in kidney and liver, and a 2.5-kb transcript in colon and liver. Two polyadenylation sites were shown for FUT5, but absence of consensus sequences suggests reduced efficiency for cleavage and polyadenylation. Two polyadenylation sites were also shown for FUT6, with the alternatively spliced downstream signal in tissues expressing high levels of FUT6. In these tissues, additional splicing results in isoforms with catalytic domain deletions. No detectable alpha(1,3)- or alpha(1,4)-fucosyltransferase activity was found in assays of cells transfected with FUT6 isoform cDNAs. Thus, tissue-specific post-transcriptional modifications are associated with expression patterns of FUT3, FUT5, and FUT6.

Alternative Splicing↗

PubMed1999

Transfer of rps14 from the mitochondrion to the nucleus in maize implied integration within a gene encoding the iron-sulphur subunit of succinate dehydrogenase and expression by alternative splicing.

The maize mitochondrial genome does not contain a gene coding for ribosomal protein S14. In this paper we show that the functional rps14 gene was translocated to the nucleus and acquired the signals conferring expression and product targeting to the mitochondrion in a way not previously described. Transferred rps14 was found integrated between both exons of a gene encoding the iron-sulphur subunit of the respiratory complex II (sdh2). Sdh2 exon 1 and rps14 were separated by a typical plant nuclear intron that was spliced to give a mature poly(A)+ mRNA of 1.4 kb. This processed mRNA encoded a chimeric SDH2 (truncated)-RPS14 polypeptide, and we show that this chimeric polypeptide is targeted into isolated plant mitochondria, where it is proteolytically processed in a complex way. An alternative splicing event utilizing the same 5' splice site and a different downstream 3' splice site generated a second mature poly(A)+ mRNA of 1.3 kb that contained both sdh2 exons. This sdh2 transcript encoded an SDH2 polypeptide highly conserved compared with its homologues in other organisms, and it contained the three cysteine-rich clusters that made up the three non-heme iron-sulphur centres responsible for electron transport. To our knowledge, these results constitute the first evidence of alternative splicing playing a role in the expression and targeting of two mitochondrial proteins with different functions from the same gene.

Alternative Splicing↗

PubMed2005

ECgene: genome-based EST clustering and gene modeling for alternative splicing.

With the availability of the human genome map and fast algorithms for sequence alignment, genome-based EST clustering became a viable method for gene modeling. We developed a novel gene-modeling method, ECgene (Gene modeling by EST Clustering), which combines genome-based EST clustering and the transcript assembly procedure in a coherent and consistent fashion. Specifically, ECgene takes alternative splicing events into consideration. The position of splice sites (i.e., exon-intron boundaries) in the genome map is utilized as the critical information in the whole procedure. Sequences that share any splice sites are grouped together to define an EST cluster in a manner similar to that of the genome-based version of the UniGene algorithm. Transcript assembly is achieved using graph theory that represents the exon connectivity in each cluster as a directed acyclic graph (DAG). Distinct paths along exons correspond to possible gene models encompassing all alternative splicing events. EST sequences in each cluster are subclustered further according to the compatibility with gene structure of each splice variant, and they can be regarded as clone evidence for the corresponding isoform. The reliability of each isoform is assessed from the nature of cluster members and from the minimum number of clones required to reconstruct all exons in the transcript.

Algorithms↗

PubMed2005

Structure and transcription promoter activity of mouse flap endonuclease 1 gene: alternative splicing and bidirectional promoter.

Flap endonuclease 1 (FEN1) is a structure-specific nuclease involved in DNA replication and repair. The mouse Fen1 gene, which has two exons, is located immediately adjacent to the gene corresponding to full-length cDNA of Riken 1810006K21 (1810006K21Rik) in a head-to-head orientation. Transcription initiation sites of each gene are 274 bp apart in the mouse genome. The spacer sequence between the bidirectional genes contains a CpG island, but lacks the typical TATA box. In the present study, transcription of the mFen1 gene was started from two initiation sites, and the first noncoding exon was spliced to the second exon using two different splicing donor sites, producing 3 kinds of mFen1 transcripts. A 594-bp fragment between the mFen1 and 1810006K21Rik genes, which contains two conserved sequence blocks (CSB) between mouse and human sequence, functions as a bidirectional promoter. The multiple cis-elements, including an Ets-binding site and E-box in the CSB, are involved in activation or repression of transcription in both directions. Interestingly, the E-box activates mFen1 transcription and simultaneously represses promoter activity in the opposite direction. Mutation of either splicing donor site of the mFen1 gene produced limiting alternative splicing products, but did not affect luciferase activity. In contrast, the splicing-defective mutation produced by disruption of the acceptor site completely lacked luciferase activity, indicating that the splicing has a significant effect on production of luciferase protein by shortening the 5'-untranslated region of the mRNA.

5' Untranslated Regions↗

PubMed2002

Alternative splicing of the 5'-sequences of the mouse EAAT2 glutamate transporter and expression in a transgenic model for amyotrophic lateral sclerosis.

Glutamate-mediated neurotoxicity and a reduced expression of the excitatory amino acid transporter 2 (EAAT2) have been described in the pathogenesis of several acute and chronic neurological conditions. EAAT2 is the major carrier of glutamate in the mammalian brain. However, the principles of EAAT2 expression regulation are not fully understood. For the human brain, extensive alternative splicing of the EAAT2 RNA has been shown. To delineate the complex RNA regulation of EAAT2 we investigated whether the murine species is a suitable model for the study of EAAT2 splicing events. We identified five splice variants (mEAAT2/5UT1-5) encoding different 5'-untranslated sequences and two distinct N-termini of the putative EAAT2 polypeptide. In the murine CNS we found a region-specific expression pattern of the novel 5'-variants of EAAT2 as shown by in situ hybridization, dot blotting and competitive reverse transcription polymerase chain reaction. Furthermore, we performed an expression analysis of the EAAT2 splice variants in the spinal cord of a transgenic model (SOD1G93A) of amyotrophic lateral sclerosis, a motor neurone disease for which altered splicing of EAAT2 has been discussed. We found an increased expression of mEAAT2/5UT4 and a reduction of mEAAT2/5UT5 in the early course of the disease. We conclude that alternative splicing of 5'-sequences may contribute to the regional expression of the EAAT2 RNA and was altered in the pre-symptomatic stage of the SOD1G93A-mouse model for amyotrophic lateral sclerosis.

5' Untranslated Regions↗

PubMed1998

Alternative splicing and genomic organization of the L5-67 gene of Aplysia californica.

The L5-67 gene was first identified on the basis of its high expression level in the LUQ neurons, a group of four giant cells located in the left upper quadrant of the abdominal ganglion of Aplysia californica. Its mRNA and peptides were later shown to be present in these cells, as well as in about 100 other smaller neurons in the CNS. L5-67 propeptide and/or mature peptides are also present in peripheral organs, particularly in the kidney, which is the target of most LUQ processes. Using RT-PCR, we show the presence of an alternatively spliced L5-67 transcript arising from the exclusion of the fourth exon from the mature mRNA. This alternative splicing event occurs specifically in the kidney, although we could not identify the cells in which it takes place. Translation of this transcript generates a 52 amino acid (aa) propeptide in which the first N-terminal 45 aa are identical to the original L5-67 propeptide. The last seven C-terminal aa are unrelated to the previously characterized L5-67 peptides due to a change in the open reading frame.

Alternative Splicing↗

PubMed1993

Use of the polymerase chain reaction for the detection of alternatively spliced mRNAs of plasma membrane calcium pump.

Polymerase chain reaction [PCR, reverse transcriptase-PCR (RT-PCR)] has been used to amplify the mRNA subspecies of the plasma membrane calcium pump isoform 1 (PMCA1) in total RNA extracted from hamster tissues. Two primers were synthesized that encompass the site at which a 154-bp exon is included totally (PMCA1a), partially (PMCA1c and d), or completely excluded (PMCA1b) in the carboxy-terminal regulatory region. PCR amplification revealed two bands (PMCA1b and 1c) that are more abundant in various tissues, while Southern hybridization of the samples after PCR amplification has detected two additional mRNA variants corresponding to PMCA1a and 1d. The distribution of these mRNA variants are tissue specific and correlate well with the pump protein distribution patterns on immunoblot. Since these multiple bands on the immunoblot are not derived from proteolysis, it is suggested that they represent the PMCA1 isozymes encoded by these alternatively spliced mRNAs. To our knowledge, this is the first report to show all four alternatively spliced mRNAs that are simultaneously detected in one single RNA sample using PCR technique. Since these isozymes are different in their regulatory domain, their tissue-specific expression may be physiologically important.

Alternative Splicing↗