Genotype-Temperature Interaction in the Regulation of Development,Growth, and Morphometrics in Wild-Type,and Growth-Hormone Transgenic Coho Salmon

Background

The neuroendocrine system is an important modulator of phenotype, directing cellular genetic responses to external cues such as temperature. Behavioural and physiological processes in poikilothermic organisms (e.g. most fishes), are particularly influenced by surrounding temperatures.

Methodology/Principal Findings

By comparing the development and growth of two genotypes of coho salmon (wild-type and transgenic with greatly enhanced growth hormone production) at six different temperatures, ranging between 8° and 18°C, we observed a genotype-temperature interaction and possible trend in directed neuroendocrine selection. Differences in growth patterns of the two genotypes were compared by using mathematical models, and morphometric analyses of juvenile salmon were performed to detect differences in body shape. The maximum hatching and alevin survival rates of both genotypes occurred at 12°C. At lower temperatures, eggs containing embryos with enhanced GH production hatched after a shorter incubation period than wild-type eggs, but this difference was not apparent at and above 16°C. GH transgenesis led to lower body weights at the time when the yolk sack was completely absorbed compared to the wild genotype. The growth of juvenile GH-enhanced salmon was to a greater extent stimulated by higher temperatures than the growth of the wild-type. Increased GH production significantly influenced the shape of the salmon growth curves.

Conclusions

Growth hormone overexpression by transgenesis is able to stimulate the growth of coho salmon over a wide range of temperatures. Temperature was found to affect growth rate, survival, and body morphology between GH transgenic and wild genotype coho salmon, and differential responses to temperature observed between the genotypes suggests they would experience different selective forces should they ever enter natural ecosystems. Thus, GH transgenic fish would be expected to differentially respond and adapt to shifts in environmental conditions compared with wild type, influencing their ability to survive and interact in ecosystems. Understanding these relationships would assist environmental risk assessments evaluating potential ecological effects.     

Increased intrinsic growth rate is advantageous even under ecologically stressful conditions in coho salmon (<Emphasis Type="Italic">Oncorhynchus kisutch</Emphasis>)

Growth rate is an ecologically important trait, affecting the energy acquisition from, and provisioning to, the surrounding community. One of many costs suggested to counteract the evolution of increased intrinsic growth rate is an associated reduction in tolerance to conditions of nutrient stress. Here we test this concept with individuals possessing experimentally increased intrinsic growth rates (growth hormone transgenic coho salmon, Oncorhynchus kisutch) relative to wild genotypes. Using a series of three experiments, survival and growth of both genotypes were assessed on a physiological and behavioral level while varying food abundance, social interactions, and predation risk. Only in complete absence of exogenous food in newly emerged fry did the high intrinsic growth rate appear costly with a shorter average survival time compared to wild-type (Exp. 1). In experiment 2, genotypes with elevated intrinsic growth showed equal or higher survival and growth than wild-type genotypes In a third experiment, adding very limited amounts of food and allowing for social interactions in a simulated natural environment benefited transgenic individuals relative to wild-types, but at similar magnitudes in both the absence and presence of predators. Populations with transgenic individuals present did not crash under these competitive conditions as previously reported when studied in simple environments where hiding and attack escape were not possible. Our data suggest that transgenic fish have a greater scope for growth under most conditions, but are not obligated to use this capability. Physiological (e.g. appetite and conversion efficiency) and behavioral traits (e.g. competitive ability and risk-taking) found previously to correlate positively with intrinsic growth rate in the transgenic strain likely aided in their survival and growth, even under food limited conditions. Hence, at least in coho salmon, intrinsic growth rate does not appear to strongly affect survival under nutrient stress.     

A selection strategy in plant transformation based on antisense oligodeoxynucleotide inhibition

Antisense oligodeoxynucleotide (asODN) inhibition was developed in the 1970s, and since then has been widely used in animal research. However, in plant biology, the method has had limited application because plant cell walls significantly block efficient uptake of asODN to plant cells. Recently, we have found that asODN uptake is enhanced in a sugar solution. The method has promise for many applications, such as a rapid alternative to time‐consuming transgenic studies, and high potential for studying gene functionality in intact plants and multiple plant species, with particular advantages in evaluating the roles of multiple gene family members. Generation of transgenic plants relies on the ability to select transformed cells. This screening process is based on co‐introduction of marker genes into the plant cell together with a gene of interest. Currently, the most common marker genes are those that confer antibiotic or herbicide resistance. The possibility that traits introduced by selectable marker genes in transgenic field crops may be transferred horizontally is of major public concern. Marker genes that increase use of antibiotics and herbicides may increase development of antibiotic‐resistant bacterial strains or contribute to weed resistance. Here, we describe a method for selection of transformed plant cells based on asODN inhibition. The method enables selective and high‐throughput screening for transformed cells without conferring new traits or functions to the transgenic plants. Due to their high binding specificity, asODNs may also find applications as plant‐specific DNA herbicides.     

Modular and configurable optimal sequence alignment software: <Emphasis Type="Italic">Cola</Emphasis>

Background

The fundamental challenge in optimally aligning homologous sequences is to define a scoring scheme that best reflects the underlying biological processes. Maximising the overall number of matches in the alignment does not always reflect the patterns by which nucleotides mutate. Efficiently implemented algorithms that can be parameterised to accommodate more complex non-linear scoring schemes are thus desirable.

Results

We present Cola, alignment software that implements different optimal alignment algorithms, also allowing for scoring contiguous matches of nucleotides in a nonlinear manner. The latter places more emphasis on short, highly conserved motifs, and less on the surrounding nucleotides, which can be more diverged. To illustrate the differences, we report results from aligning 14,100 sequences from 3' untranslated regions of human genes to 25 of their mammalian counterparts, where we found that a nonlinear scoring scheme is more consistent than a linear scheme in detecting short, conserved motifs.

Conclusions

Cola is freely available under LPGL from https://github.com/nedaz/cola.

     

Differences in proliferation rate between CADASIL and control vascular smooth muscle cells are related to increased TGFβ expression

Cerebral autosomal‐dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is a familial fatal progressive degenerative disorder. One of the pathological hallmarks of CADASIL is a dramatic reduction of vascular smooth muscle cells (VSMCs) in cerebral arteries. Using VSMCs from the vasculature of the human umbilical cord, placenta and cerebrum of CADASIL patients, we found that CADASIL VSMCs had a lower proliferation rate compared to control VSMCs. Exposure of control VSMCs and endothelial cells (ECs) to media derived from CADASIL VSMCs lowered the proliferation rate of all cells examined. By quantitative RT‐PCR analysis, we observed increased Transforming growth factor‐β (TGFβ) gene expression in CADASIL VSMCs. Adding TGFβ‐neutralizing antibody restored the proliferation rate of CADASIL VSMCs. We assessed proliferation differences in the presence or absence of TGFβ‐neutralizing antibody in ECs co‐cultured with VSMCs. ECs co‐cultured with CADASIL VSMCs exhibited a lower proliferation rate than those co‐cultured with control VSMCs, and neutralization of TGFβ normalized the proliferation rate of ECs co‐cultured with CADASIL VSMCs. We suggest that increased TGFβ expression in CADASIL VSMCs is involved in the reduced VSMC proliferation in CADASIL and may play a role in situ in altered proliferation of neighbouring cells in the vasculature.     

The crystal structure of staphylococcal enterotoxin type D reveals Zn2+-mediated homodimerization.

Bacterial superantigens, including the staphylococcal enterotoxins, are the most potent activators of T cells known and have been suggested as a causative factor in Gram-positive shock in humans. Staphylococcal enterotoxin D (SED) is dependent upon Zn2+ for high affinity interactions with MHC class II molecules and thus SED was co-crystallized with Zn2+. The crystal structure of SED has been determined in two different space groups, at 2.3 and 3.0 A resolution respectively. The three-dimensional structure of SED is similar to structures of other bacterial superantigens, although this study has revealed that SED has the unique capability of forming dimers in the presence of Zn2+. The high affinity Zn2+ site used in dimer formation is located on the surface of the beta-sheet in the C-terminal domain. Two bound metal ions are coordinated by residues from both molecules in the dimer interface and thus contribute directly to formation of the dimer. A second Zn2+ site is located on the surface close to the domain interface of the molecule. The unique feature of SED in forming a Zn2+-dependent homodimer seems to facilitate novel and biologically relevant multimeric interactions with MHC class II molecules, as shown by the induction of cytokine mRNA in human monocytes when exposed to SED and SED mutants.     

Effects of β-Amyloid-(25-35) Peptides on Radioligand Binding to Excitatory Amino Acid Receptors and Voltage-Dependent Calcium Channels: Evidence for a Selective Affinity for the Glutamate and Glycine Recognition Sites of the NMDA Receptor

The neurotoxic fragment corresponding to residues 25-35 of the -amyloid (A) peptide [A-(25-35)] has been shown to exert effects on (+)-[³H]5-methyl-10,11-dihydro-5H-dibenzo[a,d]-cyclohepten-5,10-imine maleate ([³H]MK-801) binding to the cation channel of the N-methyl-D-aspartate (NMDA) receptor. In the present study, we investigated whether the amidated and carboxylic acid C-terminated forms of A-(25-35) [A-(25-35-NH₂) and A-(25-35-COOH), respectively] exert effects on other excitatory amino acid receptor and cation channel types in rat cortical membranes. Both A-(25-35-NH₂) and A-(25-35-COOH) gave statistically significant dose-dependent inhibitions of [³H]glutamate and [³H]glycine binding to the agonist recognition sites of the NMDA receptor. Ten M A-(25-35-NH₂) and A-(25-35-COOH) gave 25% and 20% inhibitions of [³H]glutamate binding and 75% and 70% inhibitions of [³H]glycine binding, respectively. A-(25-35-NH₂), but not A-(25-35-COOH), gave a small (ca. 17% at 10 M) statistically significant increase of [³H]amino-3-hydroxy-5-methylisoxazole-4-propionate ([³H]AMPA) binding. [³H]kainate binding was not significantly affected by either peptide. Similarly, neither peptide affected either the maximal level or EC₅₀ value for calcium stimulation of [³H]nitrendipine binding. It is concluded that A-(25-35) shows slight affinity for the agonist recognition sites of the NMDA receptor, but not for other excitatory amino acid receptor types or for L-type voltage-dependent calcium channels.