RESUMEN
Profilin is a small (12-15 kDa) actin- and phospholipid-binding protein previously known only from studies on animals and lower eukaryotes but recently identified as a birch pollen allergen. Here we have identified and characterized three members of the profilin multigene family from the plant Zea mays. Two cDNAs isolated from a maize pollen library (ZmPRO 1 and ZmPRO 3) each have a single, large open reading frame encoding a putative polypeptide 131 amino acids long with a predicted molecular weight of approximately 14 kDa. A third maize pollen cDNA (ZmPRO 2) has two in-frame translation initiation codons. Use of the first ATG would result in a polypeptide 137 amino acids long with a molecular weight of 14.8 kDa. The three maize profilins are highly homologous to each other (> 90% nucleotide and amino acid sequence identity) as well as other plant profilins but show far less similarity (30-40% amino acid sequence identity) to animal and lower eukaryote profilins. Multiple sequence alignments indicate that only nine residues are shared by all eukaryotic profilins examined. However, limited comparisons reveal domains in the NH2 and COOH termini that have a high degree of similarity suggesting functional conservation. The maize gene family size is estimated to contain three to six members based on Southern blot experiments with gene-specific and coding region probes. Northern blot analysis demonstrates that the three maize profilin cDNAs characterized here are utilized in a tissue-specific manner and are anther or pollen specific.
Asunto(s)
Proteínas Contráctiles , Genes de Plantas , Proteínas de Microfilamentos/genética , Familia de Multigenes , Zea mays/genética , Actinas/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Northern Blotting , Southern Blotting , Western Blotting , ADN , Proteínas de Microfilamentos/metabolismo , Datos de Secuencia Molecular , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Profilinas , Homología de Secuencia de Aminoácido , Homología de Secuencia de Ácido Nucleico , Transcripción GenéticaRESUMEN
Immunological studies have shown that plants contain intermediate-filament antigens, but it is not known whether these proteins are capable in themselves of forming filaments. To address this problem, a detergent-resistant and high-salt-insoluble fraction from carrot (Daucus carota L.) suspension cells was solubilized with 9 M-urea and then subjected to a two-step dialysis procedure, devised for the reconstitution of animal intermediate filaments. This induced the self-assembly of 10 nm filaments and large bundles of filaments. The predominant components of reconstituted material were polypeptides with apparent molecular masses between 58 and 62 kDa. These polypeptides immunoblotted with two monoclonal antibodies known to show broad cross-reactivity with intermediate filaments across the phylogenetic spectrum. This establishes that the antigens are able to self-assemble into intermediate-sized filaments.