RESUMO
Mouse cDNA sequences encoding alpha, beta, and gamma adducins were cloned from a mouse reticulocyte cDNA library. The purified clones contain alternatively spliced exons from all three adducin genes. In the case of alpha and beta, the inclusion of the alternatively spliced exons results in truncated polypeptide isoforms (called alpha-2 and beta-2). The mouse predicted amino acid sequences are compared with published rat and human sequences. For completion of this comparison, cDNA encoding the rat beta-1 carboxy terminus was cloned by PCR. The carboxy terminal region containing MARCKS homology, calmodulin-binding region-2, and spectrin-actin-binding site, is conserved among alpha-1, beta-1, and gamma-1 isoforms in mouse, rat, and humans. We also report here the localization of the gene encoding gamma adducin (Add3) to murine Chr 19, in a region that shows conserved synteny with human Chr 10.
Assuntos
Processamento Alternativo/genética , Proteínas de Ligação a Calmodulina/genética , Mapeamento Cromossômico , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , Proteínas do Citoesqueleto/genética , Primers do DNA/química , DNA Complementar/química , Éxons/genética , Biblioteca Gênica , Humanos , Camundongos , Dados de Sequência Molecular , Ratos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Alinhamento de Sequência , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Homologia de Sequência do Ácido Nucleico , Células Tumorais CultivadasRESUMO
Adducins are a family of cytoskeleton proteins encoded by three genes (alpha, beta, gamma). In a comprehensive assay of gene expression, we show the ubiquitous expression of alpha- and gamma-adducins in contrast to the restricted expression of beta-adducin. beta-adducin is expressed at high levels in brain and hematopoietic tissues (bone marrow in humans, spleen in mice). To elucidate adducin's role in vivo, we created beta-adducin null mice by gene targeting, deleting exons 9-13. A 55-kDa chimeric polypeptide is produced from the first eight exons of beta-adducin and part of the neo cassette in spleen but is not detected in peripheral RBCs or brain. beta-adducin null RBCs are osmotically fragile, spherocytic, and dehydrated compared with the wild type, resembling RBCs from patients with hereditary spherocytosis. The lack of beta-adducin in RBCs leads to decreased membrane incorporation of alpha-adducin (30% of normal) and unexpectedly promotes a 5-fold increase in gamma-adducin incorporation into the RBC membrane skeleton. This study demonstrates adducin's importance to RBC membrane stability in vivo.
Assuntos
Proteínas de Ligação a Calmodulina/genética , Proteínas de Ligação a Calmodulina/fisiologia , Eritrócitos/citologia , Anemia/genética , Animais , Northern Blotting , Células Precursoras Eritroides/metabolismo , Éxons , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Modelos Genéticos , Mutagênese , Concentração Osmolar , Fragilidade Osmótica/genética , Poli A/análise , RNA/análise , Esferocitose Hereditária/genética , Baço/metabolismo , Distribuição TecidualRESUMO
The intron-exon organization of the human beta-adducin gene (ADD2) has been determined from overlapping genomic clones. The gene spans over 100 kb on chromosome 2p13 and comprises 17 exons. Seven of the exons are identical in size to the corresponding exons of the alpha-adducin gene (4p16.3), suggesting gene duplication. A 275-bp fragment 5' to exon 1 demonstrates strong promoter activity in a transient transfection assay. Within 333 bp 5' of the first exon can be found several putative transcription factor-binding sites: three SP1 sites, one GATA site, three MZF1 sites, one p300 site, and one c-Ets site. Alternatively spliced exons in the 3' region are described and contain distinct coding regions, stop codons, and 3'UTR, corresponding to previously published beta-adducin cDNA sequences beta-1 and beta-2. The alternative splice sites for the smallest adducin isoform, beta-3, are alternative donor and acceptor sites within exons 7 and 12. The most recently described isoform, beta-4, includes an alternative exon (exon 15) that results in a frame shift and early termination. Intron-exon splice sites are presented for all 17 exons and conform to the consensus sequences for mammalian splice sites. These results will be useful in further analysis of tissue-specific expression of adducin isoforms and in analysis of DNA from patients with diseases mapping to this region of chromosome 2.
