Genetic characterization of hybridization and introgression between anadromous rainbow trout (Oncorhynchus mykiss irideus) and coastal cutthroat trout (O. clarki clarki).

Interspecific hybridization represents a dynamic evolutionary phenomenon and major conservation problem in salmonid fishes. In this study we used amplified fragment length polymorphisms (AFLP) and mitochondrial DNA (mtDNA) markers to describe the extent and characterize the pattern of hybridization and introgression between coastal rainbow trout (Oncorhynchus mykiss irideus) and coastal cutthroat trout (O. clarki clarki). Hybrid individuals were initially identified using principle coordinate analysis of 133 polymorphic AFLP markers. Subsequent analysis using 23 diagnostic AFLP markers revealed the presence of F1, rainbow trout backcross, cutthroat trout backcross and later-generation hybrids. mtDNA analysis demonstrated equal numbers of F1 hybrids with rainbow and cutthroat trout mtDNA indicating reciprocal mating of the parental types. In contrast, rainbow and cutthroat trout backcross hybrids always exhibited the mtDNA from the recurrent parent, indicating a male hybrid mating with a pure female. This study illustrates the usefulness of the AFLP technique for generating large numbers of species diagnostic markers. The pattern of hybridization raises many questions concerning the existence and action of reproductive isolating mechanisms between these two species. Our findings are consistent with the hypothesis that introgression between anadromous populations of coastal rainbow and coastal cutthroat trout is limited by an environment-dependent reduction in hybrid fitness.

A minichromosome carrying a pigmentation gene and brook trout DNA sequences in transgenic rainbow trout.

We describe the transmission of an introduced minichromosome of brook trout (Salvelinus fontinalis) origin, carrying a pigmentation gene, through three generations in rainbow trout (Oncorhynchus mykiss). The minichromosome was originally introduced into gynogenetic albino rainbow trout using gamma-irradiated brook trout sperm. In the third generation, the presence of the minichromosome was correlated with pigmentation. A brook trout specific interspersed repeat DNA sequence, Fok I, was also correlated with pigmentation in these individuals. This system, the first clearly documented example of induced chromosome mediated gene transfer at the organismal level, could have applications in studies of gene mapping, development, gene regulation, and chromosome function.

Fibronectin attachment is permissive for IL-1 mediated gene regulation.

This study examines the effect of cell-matrix interaction on IL-1 induced gene regulation. In fibroblasts and smooth muscle cells attached to fibronectin, IL-1 caused a pronounced reduction in proteoglycan synthesis, while no reduction occurred in cells plated on bare plastic. Further, fibronectin attachment was permissive for IL-1 mediated suppression of both versican and collagen mRNA levels, initially noted after 4-6 h of IL-1 incubation. Attachment to vitronectin was less potent in influencing regulation, and collagen had no effect, suggesting specificity of the matrix modulation of the IL-1 induced response. Similar fibronectin induced dependence was demonstrated for IL-1 regulation of IL-6 gene expression, supporting the notion of a general effect of fibronectin receptor engagement on IL-1 induced signal transduction.

Fibronectin attachment activates the NF-kappa B p50/p65 heterodimer in fibroblasts and smooth muscle cells.

The nuclear factor kappa B, NF-kappa B, is activated by numerous inflammatory mediators, regulating biological responses that in part are dependent on the extracellular matrix. Analyses of the influence of cell-matrix interaction on NF-kappa B activation revealed that attachment to fibronectin caused an increase in NF-kappa B activity corresponding to 3-fold the level in cells plated on bare plastic. This effect was dependent on the degree of attachment and appeared to involve both the RGD motif of the cell-binding domain, and the heparin-binding domain of fibronectin. Fibronectin attachment specifically caused activation of the p50/p65 heterodimeric form of NF-kappa B, resulting in an increase of this complex to a level similar to that induced by cytokine stimulation. These data show that cell attachment has a pronounced influence on the level of NF-kappa B activity, affecting specific dimeric complexes, and suggest this to be a pathway through which effects depending on cell-matrix interaction can regulate cell behavior and cytokine responses.

Interleukin-1 beta regulation of fibroblast proteoglycan synthesis involves a decrease in versican steady-state mRNA levels.

This study investigates the effects of interleukin (IL)-1 beta on proteoglycan metabolism by fibroblasts surrounded by endogenous extracellular matrix. In both three-dimensional matrix cultures and long-term monolayer cultures IL-1 beta caused a significant decrease in synthesis and deposition of sulphated proteoglycans, but had no effect on release of deposited material. The decrease in synthesis became successively more pronounced, and corresponded to 40-60% of the control after 72 h incubation. The reduction was almost totally accounted for by an effect on the chondroitin ABC-lyase-sensitive proteoglycans. Gel electrophoresis showed a significant decrease in a high-molecular-mass chondroitin ABC-lyase-sensitive proteoglycan after incubation with IL-1 beta. Northern-blot analyses of total RNA revealed a pronounced decrease in the steady-state mRNA levels of versican, the large chondroitin sulphate, with levels corresponding to 10-30% of controls. In comparison, the steady-state mRNA level for decorin, the major sulphated proteoglycan synthesized by the cells, was only slightly affected. The prominent decrease in synthesis of sulphated proteoglycans induced in long-term fibroblast cultures, including the pronounced decrease in versican steady-state mRNA levels, is likely to have a significant effect on the structure of the extracellular matrix. Induction of this type of change may constitute a significant mechanism whereby IL-1 beta can affect the properties of connective tissue during inflammation and wound healing.