The sex determining loci and sex chromosomes in the family salmonidae.

Salmonids are descended from a common ancestor that underwent an autotetraploidization event. After a whole genome duplication species could deal with sex determination by deleting one copy of SEX, the sex determining locus, or by recruiting a duplicated transcription factor to become a novel sex determining gene. It is not known which if any of these strategies salmonids adopted, but it appears that they all have primarily a genetic mechanism of sex determination with male heterogamety. The sharing of sex-linked markers on the X and Y chromosomes and the difficulty in identifying Y-specific markers indicate that X and Y chromosomes in salmonids have a large pseudoautosomal region and a small sex determining region. Linkage analyses suggest that either SEX differs in different lineages or else has remained the same and moved by transposition to different chromosomes. The identification of the sex chromosomes in salmonid species has not resolved this issue. It is clear that salmonids are at an early stage in sex chromosome differentiation and therefore provide a wonderful opportunity to study the evolution of sex determination. The availability of a reference salmonid genome sequence would provide an important resource for research in this area.

Rainbow smelt (Osmerus mordax) genomic library and EST resources.

Genomic resources in rainbow smelt (Osmerus mordax) enable us to examine the genome duplication process in salmonids and test hypotheses relating to the fate of duplicated genes. They further enable us to pursue physiological and ecological studies in smelt. A bacterial artificial chromosome library containing 52,410 clones with an average insert size of 146 kb was constructed. This library represents an 11-fold average coverage of the rainbow smelt (O. mordax) genome. In addition, several complementary deoxyribonucleic acid libraries were constructed, and 36,758 sequences were obtained and combined into 12,159 transcripts. Over half of these transcripts have been identified, several of which have been associated with cold adaptation. These basic resources show high levels of similarity (86%) to salmonid genes and provide initial support for genome duplication in the salmonid ancestor. They also facilitate identification of genes important to fish and direct us toward new technologies for other studies in fish biology.

Glutamine synthetase in tilapia gastrointestinal tract: zonation, cDNA and induction by cortisol.

Glutamine synthetase, an enzyme generally associated with ammonia detoxication in the vertebrate brain and with hepatic nitrogen turnover in mammals, shows substantial activities in the gastrointestinal tract of teleostean fishes. Enzyme activity is highest in the central area of the stomach and reveals a distinct distribution pattern in stomach and along the intestine of tilapia (Oreochromis niloticus), rainbow trout (Oncorhynchus mykiss) and copper rockfish (Sebastes caurinus). In all three species, intestinal activity peaks in the distal region of the intestine. The brain contains the highest titre of the enzyme (46 U g(-1) in tilapia brain versus 15 U g(-1) in tilapia stomach), but because of the relative mass of the stomach, the largest glutamine synthetase pool in tilapia body appears to be localized in the stomach. Activities in white and red muscle are very modest at 0.1% of the brain. Independent of distribution, peak activities of glutamine synthetase in selected areas of tilapia stomach and intestine are significantly (two- to fourfold) increased after a 5-day treatment with an intraperitoneal cortisol deposit. Cortisol also increases glutamine synthetase activity in tilapia liver, white and red muscle, while activities in brain remain unaffected. We cloned and sequenced the predominant transcript of tilapia stomach glutamine synthetase (about 1.9 kb), encoding a 371-amino acid peptide. The open reading frame shows considerable identity with glutamine synthetase in toadfish (92% at peptide level, 87% at nucleotide level), but possesses a longer 3'-untranslated region than the toadfish. The tilapia glutamine synthetase mRNA contains a remnant of a putative mitochondrial leader sequence, but without a conserved second site for initiation of translation. We also find evidence for additional transcripts of glutamine synthetase in tilapia, suggesting multiple genes. Finally, we present evidence for similar abundance of glutamine synthetase transcripts in all regions of rockfish intestine. The physiological significance of the presence of glutamine synthetase in teleostean intestine is discussed.

Regulation and expression of gonadotropin-releasing hormone gene differs in brain and gonads in rainbow trout.

The GnRH gene is transcribed in both the brain and gonads. GnRH in the brain is critical for reproduction, but the function and importance of GnRH in the ovary and testis is not clear. In this study we examine whether regulation of the GnRH gene is distinct in the brain and gonads, whether the regulation of the GnRH gene in the gonads is altered after genome duplication, and whether the regulatory region of the GnRH gene is tightly conserved in vertebrates. From ovary and testis, we isolated and sequenced for the first time two different genes and their complementary DNAs that encode the identical peptide known as salmon GnRH. Rainbow trout were selected because they are tetraploid due to genome duplication. A downstream promoter is used in the brain and gonads by salmon GnRH messenger RNA1 (mRNA1) and mRNA2, but mRNA2 also uses an upstream promoter only in the gonads. Two types of long mRNA2 transcripts in ovary and testis both use an alternative start site at position 323; one of these types also retains intron 1. This long 5'-untranslated region is a likely site for distinct regulation of mRNA in the gonad. Additional evidence for separate regulation is that a different expression pattern exists in brain and gonads for GnRH mRNAs during development and maturation. Gene duplication did not alter the encoded peptide, but changed the expression pattern and resulted in complete divergence of the promoter sequence from position -215. A comparison of the mammalian and trout GnRH genes reveals that the promoters are without sequence identity except for a few consensus sites in both regulatory regions. The duplicated trout genes provide a model to study a critical gene whose product controls reproduction in all vertebrates.

Evidence for gonadotropin-releasing hormone peptides in the ovary and testis of rainbow trout.

GnRH is usually classified as a neuropeptide that is synthesized in the brain. Recent evidence indicates that GnRH mRNA is present also in the ovary and testis. However, isolation of the peptide from testis has not been reported. We used HPLC and specific RIAs to determine whether the GnRH peptide can be detected in gonads, the developmental stage at which the peptide is expressed, and the number of molecular forms of GnRH that are present in the ovary and testis. Extracts of immature and mature ovarian and testicular tissue were examined from 17- to 21-mo-old rainbow trout (Oncorhynchus mykiss). For the first time, GnRH peptides were isolated from testis and identified by HPLC-RIA with specific antisera and by elution position compared with synthetic standards. GnRH peptides were also present in the ovary. In addition, multiple forms of GnRH, including a form not normally detected in the brain of trout, were shown to be present in the gonads. During development, GnRH peptides were expressed only at specific stages in the gonads, which may explain the inability to detect and isolate the GnRH peptides from gonads in earlier studies.

Regulation and expression of gonadotropin-releasing hormone in salmon embryo and gonad.

This study examines whether gonadotropin-releasing hormone (GnRH) expression in gonads differs from that in brain as to use of alternative promoters and time of expression in early development and reproductive maturation. Within ovary, testis, and embryos, the transcripts from sGnRH gene 1 or gene 2 were expressed using either a conventional or upstream start site and conventional and alternative splicing. In immature sockeye salmon gonads, sGnRH mRNA was expressed briefly during the first 2 years, but in precociously mature fish was expressed for 2 months in the second year. Also, a cDNA encoding another form of GnRH (chicken GnRH-II) was isolated from the gonads. We conclude that differential regulation of GnRH occurs in embryos, ovary and testis compared to brain in salmonids and that more than one form of GnRH is expressed in the salmonid gonad.

Characterization of the Pacific salmon gonadotropin-releasing hormone gene, copy number and transcription start site.

Multiple forms of gonadotropin-releasing hormone (GnRH) have been shown to exist in all vertebrates examined except recently-evolved placental mammals. To study the origin and regulation of the GnRH genes in a Pacific salmon (Oncorhynchus nerka), we isolated and sequenced the salmon form of GnRH. The Southern blot shows a single band that strongly hybridizes to a probe for the gene reported here and weaker bands that may represent genes for related forms of GnRH. There is strong conservation of sequence in the hormone coding region and of the gene organization between fish and mammals. However, the GnRH-associated peptide (GAP) shows very little sequence identity with the mammalian GAPs, questioning its physiological role. We also show for the first time the transcriptional start site for a GnRH gene in a non-mammalian species. Interestingly, a large segment of 1152 nucleotides in the promoter region of the Pacific salmon GnRH gene is missing compared with the Atlantic salmon (Salmo salar) gene. These gene rearrangements suggest that these two salmonid species, which have been geographically separated for 8-15 million years, have evolved promoters with different mechanisms for control and transcription of GnRH.