We have cloned cytoplasmic intermediate filament (IF) proteins from a large number of invertebrate phyla using cDNA probes, the monoclonal antibody IFA, peptide sequence information, and various RT-PCR procedures. Novel IF protein sequences reported here include the urochordata and nine protostomic phyla, i.e., Annelida, Brachiopoda, Chaetognatha, Echiura, Nematomorpha, Nemertea, Platyhelminthes, Phoronida, and Sipuncula. Taken together with the wealth of data on IF proteins of vertebrates and the results on IF proteins of Cephalochordata, Mollusca, Annelida, and Nematoda, two IF prototypes emerge. The L-type, which includes 35 sequences from 11 protostomic phyla, shares with the nuclear lamins the long version of the coil 1b subdomain and, in most cases, a homology segment of some 120 residues in the carboxyterminal tail domain. The S-type, which includes all four subfamilies (types I to IV) of vertebrate IF proteins, lacks 42 residues in the coil 1b subdomain and the carboxyterminal lamin homology segment. Since IF proteins from all three phyla of the chordates have the 42-residue deletion, this deletion arose in a progenitor prior to the divergence of the chordates into the urochordate, cephalochordate, and vertebrate lineages, possibly already at the origin of the deuterostomic branch. Four phyla recently placed into the protostomia on grounds of their 18S rDNA sequences (Brachiopoda, Nemertea, Phoronida, and Platyhelminthes) show IF proteins of the L-type and fit by sequence identity criteria into the lophotrochozoic branch of the protostomia.

We report sequences for nuclear lamins from the teleost fish Danio and six invertebrates. These include two cnidarians (Hydra and Tealia), one priapulid, two echinoderms, and the cephalochordate Branchiostoma. Combining these results with earlier data on Drosophila, Caenorhabditis elegans, and various vertebrates, the following conclusions on lamin evolution can be drawn. First, all invertebrate lamins resemble in size the vertebrate B-type lamin. Second, all lamins described previously for amphibia, birds and mammals as well as the first lamin of a fish, characterized here, show a cluster of 7 to 12 acidic residues in the tail domain. Since this acidic cluster is absent from all invertebrate lamins including that of the cephalochordate Branchiostoma, it was acquired with the vertebrate lineage. The larger A-type lamin of differentiated cells must have arisen subsequently by gene duplication and insertion of an extra exon. This extra exon of the vertebrate A-lamins is the only major change in domain organization in metazoan lamin evolution. Third, the three introns of the Hydra and Priapulus genes correspond in position to the last three introns of vertebrate B-type lamin genes. Thus the entirely different gene organization of the C. elegans and Drosophila Dmo genes seems to reflect evolutionary drift, which probably also accounts for the fact that C. elegans has the most diverse lamin sequence. Finally we discuss the possibility that two lamin types, a constitutively expressed one and a developmentally regulated one, arose independently on the arthropod and vertebrate lineages.

The evolutionary relationship of muscle and nonmuscle actin isoforms in deuterostomia was studied by the isolation and characterization of two actin genes from the cephalochordate Branchiostoma lanceolatum and two from the hemichordate Saccoglossus kowalevskii The Branchiostoma genes specify a muscle and a nonmuscle actin type, respectively. Together with earlier results on muscle actins from vertebrates and urochordates, a N-terminal sequence signature is defined for chordate muscle actins. These diagnostic amino acid residues separate the chordates from the echinoderms and other metazoa. Although the two Saccoglossus actins characterized so far lack the diagnostic residues, in line with the presumptive phylogenetic position of hemichordates outside the chordates, a definitive conclusion can only be expected once the full complement of actin genes of Saccoglossus is established. Comparison of the intron patterns of the various deuterostomic actin genes shows that intron 330-3, which is present in all vertebrate genes, is conspicuously absent from nonvertebrate genes. The possible origin of this intron is discussed.

Materials and methods