Estrogen hormones interact with cellular ERs to exert their biological effects in vertebrate animals. Similar to other animals, fishes have two distinct ER subtypes, ERalpha (NR3A1) and ERbeta (NR3A2). The ERbeta subtype is found as two different isoforms in several fish species because of a gene duplication event. Although predicted, two different isoforms of ERalpha have not been demonstrated in any fish species. In the rainbow trout (Oncorhynchus mykiss), the only ER described is an isoform of the ERalpha subtype (i.e. ERalpha1, NR3A1a). The purpose of this study was to determine whether the gene for the other ERalpha isoform, ERalpha2 (i.e., NR3A1b), exists in the rainbow trout. A RT-PCR and cloning strategy, followed by screening a rainbow trout BAC library yielded a unique DNA sequence coding for 558 amino acids. The deduced amino acid sequence had a 75.4% overall similarity to ERalpha1. Both the rainbow trout ERbeta subtypes, ERbeta1 [NR3A2a] and ERbeta2, [NR3A2b] which were previously unknown in this species, were also sequenced as part of this study, and the amino acid sequences were found to be very different from the ERalphas (approximately 40% similarity). ERbeta1 and ERbeta2 had 594 and 604 amino acids, respectively, and had 57.6% sequence similarity when compared to one another. This information provides what we expect to be the first complete nuclear ER gene family in a fish. A comprehensive phylogenetic analysis with all other known fish ER gene sequences was undertaken to understand the evolution of fish ERs. The results show a single ERalpha subtype clade, with the closest relative to rainbow trout ERalpha2 being rainbow trout ERalpha1, suggesting a recent, unique duplication event to create these two isoforms. For the ERbeta subtype there are two distinct subclades, one represented by the ERbeta1 isoform and the other by the ERbeta2 isoform. The rainbow trout ERbeta1 and ERbeta2 are not closely associated with each other, but instead fall into their respective ERbeta subclades with other known fish species. Real-time RT-PCR was used to measure the mRNA levels of all four ER isoforms (ERalpha1, ERalpha2, ERbeta1, and ERbeta2) in stomach, spleen, heart, brain, pituitary, muscle, anterior kidney, posterior kidney, liver, gill, testis and ovary samples from rainbow trout. The mRNAs for each of the four ERs were detected in every tissue examined. The liver tended to have the highest ER mRNA levels along with the testes, while the lowest levels were generally found in the stomach or heart. The nuclear ERs have a significant and ubiquitous distribution in the rainbow trout providing the potential for complex interactions that involve the functioning of many organ systems.

We isolated the barley stem rust resistance genes Rpg5 and rpg4 by map-based cloning. These genes are colocalized on a 70-kb genomic region that was delimited by recombination. The Rpg5 gene consists of an unusual structure encoding three typical plant disease resistance protein domains: nucleotide-binding site, leucine-rich repeat, and serine threonine protein kinase. The predicted RPG5 protein has two putative transmembrane sites possibly involved in membrane binding. The gene is expressed at low but detectable levels. Posttranscriptional gene silencing using VIGS resulted in a compatible reaction with a normally incompatible stem rust pathogen. Allele sequencing also validated the candidate Rpg5 gene. Allele and recombinant sequencing suggested that the probable rpg4 gene encoded an actin depolymerizing factor-like protein. Involvement of actin depolymerizing factor genes in nonhost resistance has been documented, but discovery of their role in gene-for-gene interaction would be novel and needs to be further substantiated.actin depolymerizing factor ͉ barley ͉ disease resistance domains ͉ map-based cloning

The molecular pathways in embryonic vertebrates leading to gonad formation in each sex are incompletely understood. The purpose of this study was to identify novel genes that could be associated with sex-specific gonadal differentiation in a fish, the rainbow trout (Oncorhynchus mykiss). This study was facilitated by a custom microarray based on 7,671 genes derived from embryonic rainbow trout gonad cDNA libraries and public databases. Gonad samples for total RNA isolation were obtained from pvasa-green fluorescent protein (pvasa-GFP) transgenic rainbow between 300 and 700 degree-days of development post-fertilization. The transgenic fish permitted the collection of gonads from embryonic rainbow trout during the period of molecular sex differentiation in advance of any morphologically distinguishable characteristics of sex. A bioinformatic method was used with the microarray data that looked for strong associations in gene expression patterns between known sex differentiation genes (the target genes) and novel genes (the target-associated genes) previously not allied with sex differentiation in fishes. The expression patterns of representative target genes from both sexes and their target-associated genes were independently confirmed by real-time reverse transcription polymerase chain reaction to support the validity of the bioinformatic method employed. Numerous novel genes were identified in the gonads of embryonic female and male rainbow trout that could be involved in sex-specific differentiation pathways in this fish.

The rainbow trout endocrine system is sensitive to changes in annual day length, which is likely the principal environmental cue controlling its reproductive cycle. This study focuses on the endocrine regulation of vitellogenin (Vg) protein synthesis, which is the major egg yolk precursor in this fish species. We present a model of Vg production in female rainbow trout which incorporates a biological pathway beginning with sex steroid estradiol-17β levels in the plasma and concluding with Vg secretion by the liver and sequestration in the oocytes. Numerical simulation results based on this model are compared with experimental data for estrogen receptor mRNA, Vg mRNA, and Vg in the plasma from female rainbow trout over a normal annual reproductive cycle. We also analyze the response of the model to parameter changes. The model is subsequently tested against experimental data from female trout under a compressed photoperiod regime. Comparison of numerical and experimental results suggests the possibility of a time-dependent change in oocyte Vg uptake rate. This model is part of a larger effort that is developing a mathematical description of the endocrine control of reproduction in female rainbow trout. We anticipate that these mathematical and computational models will play an important role in future regulatory toxicity assessments and in the prediction of ecological risk.

This study used a real-time quantitative polymerase chain reaction (qPCR) method based on the growth hormone pseudogene (GHp) in chinook salmon (Oncorhynchus tshawytscha) to determine genetic sex. The GHp is present as a single copy in the genome of the male chinook salmon but is absent in the female, providing a means of using this real-time qPCR method to discriminate genetic sex. Comparisons between genomic DNA samples from 2 geographically distinct populations of chinook salmon (Columbia River, Washington, and Yukon River, Alaska) showed, within each population examined, that the males were clearly differentiated from the females. There were no interpopulation differences between males or females. The advantages of this real-time qPCR method are that it is rapid, is amenable to high sample throughput, and provides an accurate numerical value that allows comparisons between samples by statistical methods.