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 ...

We have identified cis-regulatory sequences acting on Otx2expression in epiblast (EP) and anterior neuroectoderm (AN) at about 90 kb 5′ upstream. The activity of the EP enhancer is found in the inner cell mass at E3.5 and the entire epiblast at E5.5. The AN enhancer activity is detected initially at E7.0 and ceases by E8.5; it is found later in the dorsomedial aspect of the telencephalon at E10.5. The EP enhancer includes multiple required domains over 2.3 kb, and the AN enhancer is an essential component of the EP enhancer. Mutants lacking the AN enhancer have demonstrated that these cis-sequences indeed regulate Otx2 expression in EP and AN. At the same time, our analysis indicates that another EP and AN enhancer must exist outside of the –170 kb to +120 kb range. In Otx2ΔAN/– mutants, in which one Otx2allele lacks the AN enhancer and the other allele is null, anteroposterior axis forms normally and anterior neuroectoderm is normally induced. Subsequently, however, forebrain and midbrain are lost, indicating that Otx2 expression under the AN enhancer functions to maintain anterior neuroectoderm once induced. Furthermore, Otx2 under the AN enhancer cooperates with Emx2 in diencephalon development. The AN enhancer region is conserved among mouse, human and Xenopus; moreover, the counterpart region in Xenopus exhibited an enhancer activity in mouse anterior neuroectoderm.

XX gonads differentiate into ovaries, a morphologic event evident by embryonic day 13.5 (E13.5) in mice. To identify early markers of oogenesis, sex-specific urogenital ridge cDNA libraries were constructed from E12-13 embryos. After mass excision and isolation of plasmid DNA, approximately 4800 expressed sequence tags were determined and compared to existing databases. Few cDNAs were specifically expressed in the urogenital ridge, but one, designated GPBOX, encodes a 227-amino-acid homeobox protein that is first expressed at E10.5 in the embryo as well as in the extraembryonic tissues. The Gpbox gene is single copy in the mouse genome and is located on the X chromosome in close proximity to two other homeobox genes, Pem and Psx1. Within the embryo, its expression is limited to the gonad, and transcripts are not detected in adult tissues. Although comparable levels are initially present in both sexes, GPBOX transcripts accumulate faster in female germ cells and peak at E12.5 when they are present in fivefold greater abundance than in males. The persistence of GPBOX transcripts in female germ cells until E15.5 and their virtual disappearance in males by E13.5 suggest that Gpbox may play a role in mammalian oogenesis.

For normal fertilization in mammals, it is important that functionally mature sperm are motile and have a fully formed acrosome. The glycosyltransferase-like gene, human polypeptide N-acetylgalactosaminyltransferase-like protein 5 (GALNTL5), belongs to the polypeptide N-acetylgalactosamine-transferase (pp-GalNAc-T) gene family because of its conserved glycosyltransferase domains, but it uniquely truncates the C-terminal domain and is expressed exclusively in human testis. However, glycosyltransferase activity of the human GALNTL5 protein has not been identified by in vitro assay thus far. Using mouse Galntl5 ortholog, we have examined whether GALNTL5 is a functional molecule in spermatogenesis. It was observed that mouse GALNTL5 localizes in the cytoplasm of round spermatids in the region around the acrosome of elongating spermatids, and finally in the neck region of spermatozoa. We attempted to establish Galntl5-deficient mutant mice to investigate the role of Galntl5 in spermiogenesis and found that the heterozygous mutation affected male fertility due to immotile sperm, which is diagnosed as asthenozoospermia, an infertility syndrome in humans. Furthermore, the heterozygous mutation of Galntl5 attenuated glycolytic enzymes required for motility, disrupted protein loading into acrosomes, and caused aberrant localization of the ubiquitin-proteasome system. By comparing the protein compositions of sperm from infertile males, we found a deletion mutation of the exon of human GALNTL5 gene in a patient with asthenozoospermia. This strongly suggests that the genetic mutation of human GALNTL5 results in male infertility with the reduction of sperm motility and that GALNTL5 is a functional molecule essential for mammalian sperm formation.