LAUSR

ABSTRACT Although the Tumor necrosis factor gene superfamily seems to be very conserved in vertebrates, phylogeny, tissue expression, genomic and gene organization, protein domains and polymorphism analyses showed that a strong change has happened mostly in invertebrates in which protochordates were a constraint during the immune‐molecules history and evolution. RT PCR was used to investigate differential gene expression in different tissues. The expression shown was greater in the pharynx. Single‐nucleotide polymorphism has been investigated in Ciona intestinalis Tumor necrosis factor alpha (CiTNF&agr;) mRNA isolated from the pharynx of 30 ascidians collected from Licata, Sicily (Italy), by denaturing gradient gel electrophoresis (DGGE). For this analysis, CiTNF&agr; nucleotide sequence was separated into two fragments, TNF‐1 and ‐2, respectively, of 630 and 540 bp. We defined 23 individual DGGE patterns (named 1 to 10 for TNF‐1 and 1 to 13 for TNF‐2). Five patterns for TNF‐1 accounted for <10% of the individuals, whereas the pattern 13 of TNF‐2 accounted for >20% of the individuals. All the patterns were verified by direct sequencing. Single base‐pair mutations were observed mainly within COOH‐terminus, leading to 30 nucleotide sequence variants and 30 different coding sequences segregating in two main different clusters. Although most of the base mutations were silent, four propeptide variants were detected and six amino acid replacements occurred within COOH‐terminus. Statistical tests for neutrality indicated negative selection pressure on signal and mature peptide domains, but possible positive selection pressure on COOH‐terminus domain. Lastly we displayed the in silico 3D structure analysis including the CiTNF&agr; variable region. HighlightsReal time PCR analysis revealed that CiTNF&agr; mRNA was expressed mainly in the pharynx.Gene organization and phylogeny of CiTNF&agr; clarify the evolution of TNF family.Single‐nucleotide polymorphism analysis show an unexpected degree of variability.Tests for neutrality indicated a positive selection pressure for COOH‐terminus domain.