Atlantic salmon HNF-3/forkhead: cDNA sequence, evolution, expression, and functional analysis

We report the isolation and characterization of a cDNA encoding an HNF-3 family member (as HNF-3) from Atlantic salmon (Salmo salar L). The important functional domains of HNF-3 proteins that have been characterized previously are revealed by segments of high identity along the alignment of the asHNF-3 with winged helix/forkhead amino acid sequences isolated from other species. A comparison of asHNF-3 cDNA and genomic DNA indicated that there were no introns present in the asHNF-3 gene. Expression of asHNF-3 protein in adult salmon tissues was not exclusive to liver but was also present in the pancreas and intestine. An RT-PCR analysis performed on salmon development showed that asHNF3 expression is detectable before gastrulation at the mid blastula transition stage. Functional analysis of the asHNF-3 protein using a characterized HNF-3 consensus binding site demonstrated that the protein can recognize and bind to specific HNF-3 consensus sequences. We also report the identification of a novel HNF3 binding site in the promoter of the Atlantic salmon transferrin gene.     

Antifreeze protein gene transfer in Atlantic salmon.

Salmonids freeze to death if they come into contact with ice. Many marine fish species that inhabit icy sea waters synthesize antifreeze proteins (AFP) to protect them from freezing. Production of stable lines of freeze-resistant salmon and other species would greatly facilitate development of sea-pen aquaculture in many regions. We successfully introduced winter flounder AFP genes into Atlantic salmon. Research to date indicates stable genomic integration and low levels of expression of winter flounder AFP genes in a small number (approximately 3%) of salmon developed from microinjected eggs. Inheritance of the AFP gene by offspring (F1) from crosses between transgenic and wild-type salmon revealed that the transgenic flounders (F0) were germ-line mosaics. Low levels of AFP precursors could be detected in the blood of all these transgenic offspring (F1). Approximately 50% of the progeny produced by crosses between transgenic F1 and wild-types contained the AFP genes. These results demonstrate that stable germ-line transformed Atlantic salmon can be produced.     

HNF-4 increases activity of the rat Apo A1 gene.

Apolipoprotein A1 (Apo A1) is the major protein component of high density lipoprotein (HDL) particles. HDL particles mediate the removal of cholesterol from extra-hepatic tissues via a process known as reverse cholesterol transport. Augmented production of Apo A1 will likely be beneficial to those who suffer from the consequences of hypercholesterolemia. One approach to increase expression of the protein is to identify nuclear factor(s) that enhance Apo A1 promoter activity. Therefore, we have used transient transfection to study a limited portion (-474 to -7) of the gene and showed that a cis-regulatory element, site C had a permissive effect on the ability of an adjacent site B to increase promoter activity by 30-fold. The importance of element C prompted us to identify the factor(s) that interact with this site. Results showed that HNF-4, a new member of the thyroid/steroid hormone receptor superfamily interacts with site C to enhance activity of the promoter. Based on this observation and that of the known inhibitory effects of ARP-1 on site C, we postulate a model which may account for the tissue-specific expression of the rat Apo A1 gene.     

A gene coding for a novel protein specific to the olfactory rosettes of Atlantic salmon

We have isolated a 1.6 kb clone from a cDNA library made from the olfactory rosettes of the Atlantic salmon (Salmo salar). The clone contains a 1200 bp, open reading frame (named OSC) which codes for a protein with 400 amino-acid residues (Oscp). The mRNA corresponding to OSC is strongly expressed in the olfactory rosettes and weakly expressed in gills but is expressed in only these two tissues. This suggests that Oscp may have a specific and important role in olfaction. The sequence of Oscp suggests that it is not globular. Predictions show only a small fraction of alpha-helix. Oscp is hydrophilic but with the number of positively charged residues equal to the number of negatively charged residues. No closely similar protein can be found on the basis of homology searches or hydrophobicity comparisons. However, a 44 residue segment (G300 through K343) is significantly homologous to a segment of alpha-lactalbumin (G51 through K94). The similarities include the 19 residues of the "alpha- lactalbumin-lysozyme C signature," the ten residues of the Ca(2+) binding elbow and the four cysteine residues which provide two key disulfide links in alpha-lactalbumin and lysozyme C. Two more Cys residues are also very similarly placed. We conclude that the gene OSC codes for a unique protein which most likely contains a specific site for binding Ca(2+) and plays a unique role in the signal pathway of olfaction in salmon.     

Spatio-temporal effects of river regulation on habitat quality for Atlantic salmon fry

Many upland rivers in the Northern Hemisphere contain important habitat for Atlantic salmon (Salmo salar L.). Owing to their sensitivity to environmental change, salmon are often used as bio-indicators. In Scotland, rivers containing potentially suitable habitat for salmon fry are often also regulated for hydropower. Regulated flow regimes can differ substantially spatially and temporally. Thus, where river management may be needed to maintain, restore, and protect their ecological functioning, this needs to be based on evidence of such spatio-temporal effects. This study investigated the effects of different types of river regulation on the hydraulic characteristics of downstream river reaches and the inferred consequences for salmon fry using hydraulic habitat quality models. The study focussed on the River Lyon (390 km²), a tributary of the Tay (4587 km²), Scotland, UK. Hydraulic habitat variability was assessed for three reach-scale sites with contrasting flow regimes characterised by (a) releases from hydropower generation, (b) compensation flow and (c) partly re-naturalised flow conditions. For each site, high resolution Digital Terrain Models (DTMs) were developed from bathymetric surveys and 2D hydraulic models were used to assess hydraulic characteristics. Discharge time series were used to simulate hydraulic conditions for regulated and simulated natural flows. Depth and velocity data were extracted from the hydraulic models and used to infer habitat quality using a habitat model developed for Atlantic salmon fry in similar-sized Scottish rivers. Results showed the effects of regulation can vary substantially within reaches and between seasons. Comparison to natural flow regimes suggested that flow alteration has a variable influence on habitat quality depending on the type of regulation and time of year. This work has improved understanding of the effects of regulation on biophysical processes and may also be useful for managing trade-offs between management, restoration, and societal benefits.     

Mutant HNF-1alpha and mutant HNF-1beta identified in MODY3 and MODY5 downregulate DPP-IV gene expression in Caco-2 cells

Dipeptidylpeptidase IV (DPP-IV) is a well-documented drug target for the treatment of type 2 diabetes. Hepatocyte nuclear factors (HNF)-1alpha and HNF-1beta, known as the causal genes of MODY3 and MODY5, respectively, have been reported to be involved in regulation of DPP-IV gene expression. But, it is not completely clear (i) that they play roles in regulation of DPP-IV gene expression, and (ii) whether DPP-IV gene activity is changed by mutant HNF-1alpha and mutant HNF-1beta in MODY3 and MODY5. To explore these questions, we investigated transactivation effects of wild HNF-1alpha and 13 mutant HNF-1alpha, as well as wild HNF-1beta and 2 mutant HNF-1beta, on DPP-IV promoter luciferase gene in Caco-2 cells by means of a transient experiment. Both wild HNF-1alpha and wild HNF-1beta significantly transactivated DPP-IV promoter, but mutant HNF-1alpha and mutant HNF-1beta exhibited low transactivation activity. Moreover, to study whether mutant HNF-1alpha and mutant HNF-1beta change endogenous DPP-IV enzyme activity, we produced four stable cell lines from Caco-2 cells, in which wild HNF-1alpha or wild HNF-1beta, or else respective dominant-negative mutant HNF-1alphaT539fsdelC or dominant-negative mutant HNF-1betaR177X, was stably expressed. We found that DPP-IV gene expression and enzyme activity were significantly increased in wild HNF-1alpha cells and wild HNF-1beta cells, whereas they decreased in HNF-1alphaT539fsdelC cells and HNF-1betaR177X cells, compared with DPP-IV gene expression and enzyme activity in Caco-2 cells. These results suggest that both wild HNF-1alpha and wild HNF-1beta have a stimulatory effect on DPP-IV gene expression, but that mutant HNF-1alpha and mutant HNF-1beta attenuate the stimulatory effect.     

The regulation of forkhead/HNF-3beta expression in the Ciona embryo

The Ciona forkhead/HNF-3beta gene (Ci-fkh) is expressed in the primary axial tissues of the developing tadpole, including the notochord, endoderm, and rudimentary floor plate of the CNS. In an effort to determine the basis for this complex pattern of expression we have conducted a detailed analysis of the Ci-fkh 5'-regulatory region. Different 5' sequences were attached to a lacZ reporter gene and analyzed in electroporated Ciona embryos. A short regulatory sequence (AS) located approximately 1.7 kb upstream of the transcribed region is shown to be essential for expression in all three axial tissues. The proximal 20 bp of the AS contains overlapping Snail repressor elements and a T-box motif. Deleting these sequences causes the loss of reporter gene expression in the endoderm, as well as expanded expression in the neural tube. These results suggest that a T-box gene such as Ci-VegTR activates Ci-fkh expression in the endoderm, while the Ci-Sna repressor excludes expression from the lateral ependymal cells and restricts the Ci-fkh pattern to the rudimentary floor plate in ventral regions of the neural tube. We also present evidence for Ci-fkh positive autofeedback, whereby the Ci-Fkh protein binds to critical activator sites within the Ci-fkh 5'-regulatory region and helps maintain high levels of expression. We discuss these results with respect to forkhead/HNF-3beta regulation in vertebrates.     

Quantification of Atlantic salmon type-I interferon using an Mx1 promoter reporter gene assay

We here describe an assay for the detection of interferon-like activity in Atlantic salmon based on the transient transfection of chinook salmon embryo cells (CHSE-214 cells) with a rainbow trout Mx1 promoter linked to a luciferase reporter. A beta-galactosidase gene under the control of a constitutively expressed beta-actin promoter was used as a transfection standard, and luciferase and beta gal expression were measured by a commercially available kit. Interferon containing supernatants from poly I:C- or CpG-stimulated leucocytes added to transfected CHSE-cells induced high luciferase expression (>60-fold induction compared to supernatants from non-stimulated cells). There was no response to supernatants from LPS- and ConA/PMA-stimulated leucocytes, demonstrating the specificity for type I interferon-like activity. Duplicate samples analysed using a cell protection assay for detection of antiviral activity correlated well with levels obtained by the Mx1 promoter reporter gene assay (R2=0.97), confirming the reporter assay as a reliable substitute for the standard antiviral assay. The Mx reporter gene assay also has advantages in terms of sensitivity, high dynamic range and reliability over the conventional cell protection assay.     

A role for Rac3 GTPase in the regulation of autophagy

The process of autophagy is situated at the intersection of multiple cell signaling pathways, including cell metabolism, growth, and death, and hence is subject to multiple forms of regulation. We previously reported that inhibition of isoprenylcysteine carboxylmethyltransferase (Icmt), which catalyzes the final step in the post-translational prenylation of so-called CAAX proteins, results in the induction of autophagy which enhances cell death in some cancer cells. In this study, using siRNA-mediated knockdown of a group of small GTPases that are predicted Icmt substrates, we identify Rac3 GTPase as a negative regulator of the process of autophagy. Knockdown of Rac3, but not the closely related isoforms Rac1 and Rac2, results in induction of autophagy. Ectopic expression of Rac3, significantly rescues cells from autophagy and cell death induced by Icmt inhibition, strengthening the notion of an isoform-specific autophagy regulatory function of Rac3. This role of Rac3 was observed in multiple cell lines with varying Rac subtype expression profiles, suggesting its broad involvement in the process. The identification of this less-studied Rac member as a novel regulator provides new insight into autophagy and opens opportunities in identifying additional regulatory inputs of the process.