Sandra S. Ristow

Status and opportunities for genomics research with rainbow trout

The rainbow trout (Oncorhynchus mykiss) is one of the most widely studied of model fish species. Extensive basic biological information has been collected for this species, which because of their large size relative to other model fish species are particularly suitable for studies requiring ample quantities of specific cells and tissue types. Rainbow trout have been widely utilized for research in carcinogenesis, toxicology, comparative immunology, disease ecology, physiology and nutrition. They are distinctive in having evolved from a relatively recent tetraploid event, resulting in a high incidence of duplicated genes. Natural populations are available and have been well characterized for chromosomal, protein, molecular and quantitative genetic variation. Their ease of culture, and experimental and aquacultural significance has led to the development of clonal lines and the widespread application of transgenic technology to this species. Numerous microsatellites have been isolated and two relatively detailed genetic maps have been developed. Extensive sequencing of expressed sequence tags has begun and four BAC libraries have been developed. The development and analysis of additional genomic sequence data will provide distinctive opportunities to address problems in areas such as evolution of the immune system and duplicate genes.

Physical and genetic mapping of the rainbow trout major histocompatibility regions: evidence for duplication of the class I region

One of the most unexpected discoveries in MHC genetics came from studies dealing with the teleost MHC. Initially discovered in zebrafish, the MHC class I and II regions of all bony fish are not linked. Previous segregation analysis in trout suggested that the class I and II regions reside on completely different chromosomes. To learn more about MHC genomics in trout, we have isolated BAC clones harboring class Ia and Ib loci, a single BAC clone containing an MH class II gene (DAB), as well as BAC clones containing the ABCB2 gene. Upon PCR and sequence confirmation, BAC clones were labeled and used as probes for in situ hybridization on rainbow trout metaphase chromosomes for determination of the physical locations of the trout MH regions. Finally, SNPs, RFLPs, and microsatellites found within the BAC clones allowed for these regions to be assigned to specific linkage groups on the OSU × Hotcreek (HC) and OSU × Arlee (ARL) genetic linkage maps. Our data demonstrate that the trout MH regions are located on at least four different chromosomes and the corresponding linkage groups, while also providing direct evidence for the partial duplication of the MH class I region in trout.

Nonspecific cytotoxic cells of rainbow trout (Oncorhynchus mykiss) kill YAC-1 targets by both necrotic and apoptic mechanisms

Nonspecific cytotoxic cells (NCC) have been identified in a number of fish species and are thought to be evolutionary progenitors of mammalian natural killer cells. We show here that trout NCC are functionally similar to cytotoxic cells of higher vertebrates in that they mediate cytotoxicity through both mechanisms of apoptosis and necrosis. To demonstrate that trout NCC inflict apoptic and necrotic lesions in tumor target cells, DNA fragmentation and 51chromium release assays were conducted using leukocytes isolated from peripheral blood, spleen, and anterior kidney. At effector-target ratios of 25:1, 50:1, 100:1, and 200:1, the release of thymidine-labeled DNA fragments and the release of 51chromium from YAC-1 target cells paralleled one another. Percent chromium release and DNA fragmentation increased when effector:target incubation times were extended from 4 to 18 h. As evidenced in agarose gels, the pattern of fragmentation induced by trout effector cells was identical to that produced by BALB/c NK cells. Similar to human and murine NK cells, trout NCC were maximally inhibited by 50 mM mannose-6-phosphate. Morphologic characteristics of rainbow trout NCC were examined using light and electron microscopy. Photomicrographs of effector:target cell mixtures after a 1 h incubation show NCC binding to target YAC-1 cells. Transmission electron micrographs of the conjugates revealed that the cells responsible for killing are small (4.2-4.5 microns), agranular mononuclear leukocytes.

A single major chromosomal region controls natural killer cell-like activity in rainbow trout

We report the identification of a single major chromosomal region controlling natural killer (NK) cell-like activity in rainbow trout (Oncorhynchus mykiss). A genetic map based on 484 AFLP and 39 microsatellite genotypes from 106 doubled haploid fish was constructed. These fish were produced by androgenesis from a hybrid of two clonal lines divergent in NK-like activity. NK-like activities for 75 of the doubled haploids were quantified by an in vitro chromium release assay utilizing 51Cr-labeled YAC-1 target cells. Composite interval mapping revealed a single major quantitative trait locus (QTL) associated with NK-like activity in this rainbow trout model. Genetic mapping revealed this QTL to also be unlinked to: fragmented MHC class I and MHC class II regions, the leukocyte receptor cluster, the natural killer cell enhancement factor (NKEF) gene, the RAG-1 gene, and two QTL associated with resistance to infectious pancreatic necrosis virus in rainbow trout. Collectively, these results extend the utility of rainbow trout as an immunological model and are consistent with the idea that a single chromosomal region homologous to the natural killer cell complex (NKC) located on syntenic portions of mouse chromosome (Chr) 6, human Chr 12, and rat Chr 4 may exist in a lower vertebrate model.

Cloning, characterization and genomic structure of the natural killer cell enhancement factor (NKEF)-like gene from homozygous clones of rainbow trout (Oncorhynchus mykiss)

Natural killer cell enhancement factor (NKEF) belongs to the antioxidant protein family. In the human, NKEF has the ability to enhance natural killer cell cytotoxic activity in vitro. In the present work, the cDNAs of NKEF from three strains of homozygous clones of rainbow trout were cloned from the splenic cDNA library of one of the strains, OSU142, and then by RT-PCR for the Hot Creek (HC) and Arlee (AR) strains. The HC sequence has 99% sequence identity with both OSU142 and AR. OSU142 and AR have only one nucleotide difference in the cDNA sequence. All three sequences have the same deduced NKEF peptide, which contains 199 amino acids. The 6. 5 kb genomic DNA of OSU142 containing NKEF was sequenced and contains six exons and five introns. Tissue specific expression of NKEF was studied by RT-PCR in eight different tissues of OSU142 and revealed that all tissues expressed NKEF. A southern blot revealed that the gene for NKEF is present in a single copy. The cDNA and amino acid sequences of trout NKEF have high similarity with human, rat, mouse and carp sequences, therefore, indicating that NKEF is a very conserved gene.

Infectious Hematopoietic Necrosis (IHN) and Viral Hemorrhagic Septicemia (VHS): Detection of Trout Antibodies to the Causative Viruses by Means of Plaque Neutralization, Immunofluorescence, and Enzyme-Linked Immunosorbent Assay

Abstract Sera collected from cultured rainbow trout Oncorhynchus mykiss surviving outbreaks of infectious hematopoietic necrosis (IHN) or viral hemorrhagic septicemia (VHS) were examined for the presence of antibodies to both of the causative viruses, infectious hematopoietic necrosis virus (IHNV) and Egtved virus (viral hemorrhagic septicemia virus: VHSV). Sera were screened with three serological tests: 50% plaque neutralization test (PNT), immunofluorescence (IF), and enzyme-linked immunosorbent assay (ELISA). In sera from 20 rainbow trout surviving IHN, antibodies to IHNV were detected in 9 fish by PNT, in 12 fish by IF, and in 9 fish by ELISA. In these sera, antibodies cross-reacting with VHSV were rare (detected in 0 fish by PNT, in 1 by IF, and in 1 by ELISA). In sera from 20 rainbow trout surviving VHS, antibodies to VHSV were detected in 9 fish by PNT, in 16 fish by IF, and in 18 fish by ELISA. A considerable percentage of the VHS-survivor sera contained antibodies that cross-reacted with IHNV, a...

Inhibition of natural killer cell activity in vitro by alcohols.

Abstract The effect of the addition of small quantities of alcohols to cocultures of natural killer effector cells from C57BL 6 mice and BDIX rats with YAC-1 tumor cell targets has been studied. The order of inhibition of NK cell-mediated killing is 1-butanol > 1-propanol > 2-propanol > ethanol > methanol. The inhibition of killing due to the addition of alcohol correlates with decreases of effector-target cell binding. Therefore caution should be exercised in interpreting results of cellular experiments in which these alcohols have been used to solubilize inhibitors.

Development of Monoclonal Antibodies that Recognize a Type-2 Specific and a Common Epitope on the Nucleoprotein of Infectious Hematopoietic Necrosis Virus

Abstract Two monoclonal antibodies were produced against the nucleoproteins of two strains of infectious hematopoietic necrosis virus (IHNV). One antibody, 1NDW14D, obtained by immunizing BALB/c mice with the nucleoprotein from Dworshak IHNV strain DW2, universally recognized IHNV in tests of direct and indirect fluorescence. The second antibody, 2NH105B, obtained by immunization with the nucleoprotein from an IHNV strain isolated from rainbow trout Oncorhynchus mykiss (formerly Salmo gairdneri) in the Hagerman Valley, Idaho, recognized biochemical type-2 IHNV. Both antibodies, 1NDW14D and 2NH105B, when conjugated with fluorescein, can be used in a direct fluorescence test that is more rapid than the previous methods of detecting the virus.

Coding sequences of the MHC ii β chain of homozygous rainbow trout (Oncorhynchus mykiss)1

Abstract Six lines of homozygous rainbow trout ( Oncorhynchus mykiss ) from different genetic and geographical backgrounds have been produced as aquatic models for biomedical research by the chromosome set manipulation techniques of androgenesis and gynogenesis Messenger RNA from spleens was extracted and the MHC II B cDNA sequences amplified by RT–PCR were cloned into plasmids Sequences of the MHC II β 2 domains were highly conserved between the different plasmids from the same and different lines of trout Most of the variability among sequences was found in the amino terminal half of the β 1 domain which corresponds with the peptide binding region of the MHC II molecule This diversity suggests that the different lines of trout may exhibit differences in immune response Rainbow trout MHC II B sequences were similar to the MHC II B sequences of the Pacific salmon ( O gorbuscha O tshawytscha O nerka O masou O kisutch ) Southern blot analysis performed on the restricted DNA of the OSU and Hot Creek trout and the doubled haploid progeny produced by androgenesis from OSU×Hot Creek hybrids indicates that two distinct genes encode the MHC II B sequences and that these genes are unlinked

Arlee line of rainbow trout (Oncorhynchus mykiss) exhibits a low level of nonspecific cytotoxic cell activity

Nonspecific cytotoxic cell (NCC) activity was assessed in the peripheral blood of four isogenic lines of rainbow trout (Oncorhynchus mykiss) which were derived by the chromosome set manipulation technique of androgenesis. In these fish, whose isogenicity was previously confirmed by multilocus DNA fingerprint analysis, NCC activity was studied by the release of 51Cr from YAC-1 targets. Two groups of trout (the homozygous Arlee 12 line and the heterozygous hybrid of the Arlee 63 and Arlee 12 lines) had significantly lower levels of NCC activity in peripheral blood than either outbred rainbow trout or other lines with Hot Creek or hybrid Arlee x Hot Creek ancestry. The low NCC activity in the Arlee line appears to be inherited as a recessive trait. Peripheral blood cells of the trout mediated lectin dependent cellular cytotoxicity (LDCC) with the addition of phytohemagglutinin to co-cultures of effector cells and YAC-1 cells. The low NCC activity in the peripheral blood of these fish is not due to a condition analogous to the NCC-deficient Chediak-Higashi syndrome of man or the beige mutation of mice.