Immunophylogenetic aspects of a gorgonian coral

One goal of comparative immunology is to derive inferences about evolutionary pathways in the development of immune-defense systems. Almost 700 million years ago, a major divergence occurred in the phylogeny of animals, spitting all descendants into either the protostome or deuterostome (includes vertebrates) lineages. Genes have evolved independently along these lineages for that amount of time. Cnidarians originated before that divergence event, and can hold clues as to which immune response genes are homologous to both lineages. This work uses the gorgonian coral, Swiftia exserta, for two major reasons: (1) because of their phylogenetic position, corals are an important animal model in studies concerning the phylogeny of immune-response genes, and (2) nothing is known about the genes controlling immunocompetence in corals. The work described here has important implications in both innate and adaptive immunity. ^ The vertebrate complement system is a major component of innate immunity. C3 is a critical component of the three pathways of complement. Because of its opsonic properties, a C3-like protein is expected to have evolved early. However, currently available data suggests that complement-like components are unique to the deuterostome lineage. This work describes the cloning and characterization of a C3-like gene from S. exserta. The deduced polypeptide sequence reveals conservation of multiple, functionally critical, sites while sharing physiochemical and structural properties with the complement components C3/C4/C5. ^ Antigen processing, via intracellular enzymatic proteasomes, is a major requirement of vertebrate adaptive immunity. These organelles have a catalytic core, through which pass intracellular proteins for degradation into peptides presentable to the immune system. LMP 7 is one component of the paralogous “immuno-proteasome”. LMP 7 is a paralog of the ubiquitous LMP X, but is restricted to vertebrates. While LMP 7 is absent in the coral, this work describes a coral LMP X gene. Phylogenetic analyses, along with hydropathy profiling of a critical portion of the invertebrate and vertebrate paralogous genes, suggests that some invertebrates have two diverging LMP X genes. In some cases, one LMP X protein shares characteristics with vertebrate LMP 7. This work presents new evidence for how the LMP X and 7 genes evolved. ^

Insulin-like growth factor-I gene expression as a growth indicator in Nile tilapia Oreochromis niloticus L.

Somatic growth in fishes is regulated by a variety of hormones. A central step in this hormonal network is the growth hormone-insulin-like growth factor-I (IGF-I) axis. Studies conducted evaluated the relationship of hepatic IGF-I (hIGF-1) mRNA with growth as affected by feeding regimes (satiation or restricted level; daily or alternate-day feeding), temperatures (high, ambient, low) and by social stress. To develop a cellular means for the quantification of hIGF-I mRNA levels in O. niloticus, hIGF-I cDNA was isolated and cloned. The partial sequence of IGF-I cDNA encodes for signal peptide, mature protein and a portion of the E-domain. A sensitive TaqMan quantitative reverse transcription-polymerase chain reaction (qRT-PCR) assay was developed based on the mature IGF-I. Using the developed qRT-PCR assay a significant positive correlation was observed between hIGF-I mRNA levels and growth rate of fish reared under different feeding regimes (r = 0.64) and temperature conditions (r = 0.64). On the dynamics of hIGF-I gene expression in response to elevated temperature, hIGF-I mRNA levels were significantly elevated after at least 2 days of exposure to warm temperature. This validates the concept that hIGF-I gene expressions are sufficiently sensitive to be used as a rapid growth rate indicator for O. niloticus. The hIGF-I levels have a significant positive correlation with specific growth rate (length; r = 0.92), and with condition factor (r = 0.55). On the effect of social stress, differential alterations in growth rates between the dominant and subordinates were observed which was attributed more to behavioral changes as transduced by physiological regulators. The fish's relative position in the social hierarchy was consistently reflected in the levels of hIGF-I mRNA and the eye color pattern. Subordination depressed hIGF-I levels while dominance stimulated it. These findings have shown that hGF-I level remained positively correlated to growth rate as affected by feeding regime, temperature and social stress. This suggests that hIGF-I plays a key role in controlling growth in O. niloticus and indicates that IGF-I mRNA quantification could prove useful for the rapid assessment of growth rate in this species of fish.

Construction and screening of A cDNA library for the C3 gene(s) of the Nurse Shark (Ginglymostoma Cirratum)

Mammalian C3 is a complement protein which consists of an α chain (125kDa) and β chain (75kDa) held together by a disulfide bond. The a chain contains a conserved thiolester site which provides the molecule with opsonic properties. The protein is synthesized as a single pro-C3 molecule which is post-translationally modified. C3 genes have been identified in organisms from different phyla, however, the shark C3 gene remains to be cloned. Sequence data from the shark will contribute to understanding further the evolution of this key protein. To obtain additional sequence data for shark C3 genes a cDNA library was constructed and screened with a DIG-labeled C3 probe. Fifty clones were isolated and sequenced. Analysis identified four sequences that yielded positive alignments with C3 of a variety of organisms including human C3. Deduced amino acid sequence analysis confirmed a β/α cut site (RRRR), the CR3 and properdin binding sites, the catalytic histidine, and the reactive thiolester sequence. In the shark there are at least two C3-like genes as the gene sequence obtained is distinct from that previously described.