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Abstract Five different type I keratins from a teleost fish, the rainbow trout Oncorhynchus mykiss , have been sequenced by cDNA cloning and identified at the protein level by peptide mass mapping using MALDI-MS. This showed that the entire range of type I keratins detected biochemically in this fish has now been sequenced. Three of the keratins are expressed in the epidermis (subtype Ie), whereas the other two occur in simple epithelia and mesenchymal cells (subtype Is). Among the Is keratins is an ortholog of human K18; the second Is polypeptide is clearly distinct from K18. We raised a new monoclonal antibody (F1F2, subclass IgG1) that specifically recognizes trout Is keratins, with negative reactions on zebrafish. A phylogenetic tree has been constructed from a multiple alignment of the rod domains of the new sequences together with type I sequences from other vertebrates such as shark, zebrafish, and human; a recently sequenced lamprey Is keratin was applied as outgroup. This tree shows one branch defining the K18 orthologs and a second branch containing all other type I keratins (mostly subtype Ie). Within this second branch, the teleost keratins form a separate, highly bootstrap-supported twig. This tree leaves little doubt that the teleost Ie keratins diversified independently from the mammalian Ie keratins. [ABSTRACT FROM AUTHOR]

Abstract From a teleost fish, the rainbow trout Oncorhynchus mykiss , we have cloned and sequenced cDNAs encoding five different type II keratins. The corresponding protein spots, as separated by 2D-PAGE of trout cytoskeletal preparations, have been identified by peptide mass mapping using MALDI mass spectrometry. Three of the sequenced keratins are expressed in the epidermis (subtype IIe), and two in simple epithelia and mesenchymal cells (subtype IIs). The IIs keratins are both orthologs of human K8. This leaves unsequenced only the trace component S3 of the biochemically established trout keratin catalog. A phylogenetic tree has been constructed from a multiple alignment of the rod domains of the new keratin sequences together with type II sequences from other vertebrates such as shark, zebrafish, and human; lamprey K8 (recently sequenced in our laboratory) has been used as outgroup. This tree suggests, in a highly bootstrap-supported manner, that the teleost IIe keratins diversified independently from the mammalian IIe keratins. In contrast, all the species investigated express K8-like keratins, suggesting that the different IIe branches evolved from K8-like progenitors. The tree also indicates that the published zebrafish sequences represent IIe keratins and that the biochemically identified K8 ortholog in zebrafish has not yet been sequenced. [ABSTRACT FROM AUTHOR]