Several subfamilies of the salmonid Hpa I short interspersed element (SINE) family were isolated from salmonid genomes and were sequenced. For each genomic locus that represented the subfamily, amplification by PCR of the orthologous loci in the 12 fish aflowed us to determine the order of branching of the Pacific salmonid species. The deduced phylogeny suggests three evolutionary lines, namely, a line of chum salmon, pink salmon, and kokanee; a line of coho salmon and chinook salmon; and a line ofsteelhead trout. Our data also support a change in the phylogenetic ament of steelhead trout from Salmo to Oncorhynchus. We present here an extensive phylogenetic tree constructed from an analysis of differential insertion of SINEs, and we propose that SINE insertion analysis is one of the best available methods for clarifying the order of divergence of closely related species.A retroposon is defined as a nucleotide sequence, present initially as a cellular RNA transcript, that has been reincorporated into the genome, presumably via a cDNA intermediate. Retroposons constitute roughly 10% of the human genome and are similarly abundant in other mammalian genomes (1, 2). As a result, the remarkable fluidity of eukaryotic genomes reflects the contributions of retroposition (2) as well as mechanisms operating at the DNA level such as mutation and recombination (1-4). Retroposons can be unique to one species, a few species, a genus, or in some cases a family. Retroposition is therefore a specialized form of gene duplication, which is believed to be of major importance in the creation of genetic diversity during evolution (5).Nonviral retroposons are classified into three main groups: processed retropseudogenes, LINEs (long interspersed elements), and SINEs (short interspersed elements) (6). Except for the rodent type 1 and human Alu families (7,8), all of the SINE families examined to date have been shown to be derived from tRNAs (9)(10)(11)(12)(13)(14). In contrast to DNA transposable elements, which are often capable of being excised precisely, SINEs appear to be inserted irreversibly and should therefore provide an ideal evolutionary and phylogenetic marker (4).The Pacific salmon and trout (Oncorhynchus) not in other species (16). These results suggest that these SINEs were amplified specifically within certain salmonid lineages during evolution.Our data prompted us to attempt to construct a phylogenetic tree for the salmonid species by using SINE insertions as irreversible events that would serve as informative markers of evolution. In this report, we present a characterization of the four subfamilies of the Hpa I family.t These subfamilies were amplified in the four different ancestral species within the genus Oncorhynchus. Such characterization provides a highly reliable order of branching of the various species of Oncorhynchus. MATERIALS AND METHODSExperiments were performed by using standard techniques (18)(19)(20)(21).The fish species examined in this study and their geographic sources are listed in Table ...
Ninety-nine members of the salmonid HpaI and AvaIII families of short interspersed repetitive elements (SINEs) were aligned and a general consensus sequence was deduced. The presence of 26 correlated changes in nucleotides (diagnostic nucleotides) from those in the consensus sequence allowed us to divide the members of the HpaI family into 12 subfamilies and those of the AvaIII family into two subfamilies. On the basis of the average sequence divergences and the phylogenetic distributions of the subfamilies, the relative antiquity of the subfamilies and the process of sequential changes in the respective source sequences were inferred. Despite the higher mutation rates of CG dinucleotides in individual dispersed members, no hypermutability of CG positions was observed in changes in the source sequences. This result suggests that sequences of SINEs located in a nonmethylated or hypomethylated genomic region could have been selected as source sequences for retroposition and/or that some CG sites are the parts of recognition sequences of retropositional machineries.
Fourteen members ofthe Hpa I subfilies of tRNA-derived SINEs in particular salmonid species were isolated from genomic libraries of chum salmon, koknee, coho salmon, masu salmon, and s e. Ali t of the sequences of these 14 members, together with those of4 members already published, 3 of which were previously d sted to have been amplied speificaly in certain lineages, revealed the presence of five sbfali with particular disc nucleotides. The amplification of members of the same subfamily in different salmonid lineages and the amplification of members of different subfamilies in the same saAmonid lineage suggest that multiple dispersed loci were responsible for amplification or, alternatively, that SINEs were transmitted horizontally between species. These two possibilities are not mutually exclusive. Our results also indicate that the Hpa I SINEs in salmonids behave like parasites. The amplifiation of these
Salmonid species contain numerous short interspersed repetitive elements (SINEs), known collectively as the HpaI family, in their genomes. Amplification and successive integration of individual SINEs into the genomes have occurred during the evolution of salmonids. We reported previously a strategy for determining the phylogenetic relationships among the Pacific salmonids in which these SINEs were used as temporal landmarks of evolution. Here, we provide evidence for extensive genomic rearrangements that involved retropositions and deletions in a common ancestor of all the Pacific salmon and trout. Our results provide genetic support for the recent phylogenetic reassignment of steelhead and related species from the genus Salmo to the genus Oncorhynchus. Several other informative loci identified by insertions of HpaI SINEs have been isolated, and previously proposed branching orders of the Oncorhynchus species have been confirmed. The authenticity of our phylogenetic tree is supported both by the isolation of more than two informative loci per branching point and by the congruence of all our data, which suggest that the period between succesive speciations was sufficiently long for each SINE that had been amplified in the original species to become fixed in all individuals of that species.
The gag gene of a Japanese feline immunodeficiency virus (FIV) isolate, designated as FIV TM 2, was expressed in Escherichia coli as a fusion protein with TrpE. Using this expressed protein, an enzyme-linked immunosorbent assay was developed for detection of antibodies to FIV gag protein in feline sera. With serum samples from a cat experimentally infected with FIV, it was demonstrated that the period of seroconversion detected by this method corresponded to that by Western blotting.
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