The Okinawa spiny rat, Tokudaia muenninki, is the only species with a Y chromosome in the genus Tokudaia. Its phylogenic relationship with two XO/XO species, Tokudaia osimensis and Tokudaia tokunoshimensis, lacking a Y chromosome and the mammalian sex-determining gene SRY, is unknown. Furthermore, there has been little cytogenetic analysis of the sex chromosomes in T. muenninki. Therefore, we constructed molecular phylogenetic trees with nucleotide sequences of cyt b, RAG1, and IRBP. All trees strongly supported that T. muenninki was the first to diverge from the Tokudaia ancestor, indicating that loss of the Y chromosome and SRY occurred in the common ancestor of the two XO/XO species after T. muenninki diverged. We found that the X and Y chromosomes of T. muenninki consisted of large euchromatic and heterochromatic regions by conducting G- and C-banding analyses. PCR, Southern blotting, and FISH revealed that T. muenninki males had multiple SRY copies on the long arm of the Y chromosome. At least three of 24 SRY sequences contained a complete open reading frame (ORF). A species-specific substitution from alanine to serine was found in all copies at the DNA-binding surface within the HMG-box, suggesting that it occurred in an original SRY.

The Ryukyu spiny rat, Tokudaia osimensis, has an XO/XO sex chromosome constitution, lacking a Y chromosome and the mammalian sex-determining gene SRY. To investigate the Y-loss event, we traced three proto-Y-linked genes, RBMY1A1, EIF2S3Y, and KDM5D, in the genome. The original Y-linked RBMY1A1 was lost as well as SRY, and the remaining RBMY1A1 was a processed pseudogene on autosome. In contrast, EIF2S3Y and KDM5D were conserved in genomes of both sexes as a result of their translocation from the Y chromosome to the X chromosome and/or autosomes. Furthermore, these genes were expressed in gonads and brains of both sexes. Our study indicated a loss of Y-linked genes with important male functions to be necessary for the Y chromosome to disappear. These functions might have been retained through the acquisition of new genes, and therefore, the Y-loss has had no harmful effect on the maintenance of this species.

The use of mutant mice plays a pivotal role in determining the function of genes, and the recently reported germ line transposition of the Sleeping Beauty (SB) transposon would provide a novel system to facilitate this approach. In this study, we characterized SB transposition in the mouse germ line and assessed its potential for generating mutant mice. Transposition sites not only were clustered within 3 Mb near the donor site but also were widely distributed outside this cluster, indicating that the SB transposon can be utilized for both region-specific and genome-wide mutagenesis. The complexity of transposition sites in the germ line was high enough for large-scale generation of mutant mice. Based on these initial results, we conducted germ line mutagenesis by using a gene trap scheme, and the use of a green fluorescent protein reporter made it possible to select for mutant mice rapidly and noninvasively. Interestingly, mice with mutations in the same gene, each with a different insertion site, were obtained by local transposition events, demonstrating the feasibility of the SB transposon system for region-specific mutagenesis. Our results indicate that the SB transposon system has unique features that complement other mutagenesis approaches.

Silencing of transposable elements occurs during fetal gametogenesis in males via de novo DNA methylation of their regulatory regions. The loss of MILI (miwi-like) and MIWI2 (mouse piwi 2), two mouse homologs of Drosophila Piwi, activates retrotransposon gene expression by impairing DNA methylation in the regulatory regions of the retrotransposons. However, as it is unclear whether the defective DNA methylation in the mutants is due to the impairment of de novo DNA methylation, we analyze DNA methylation and Piwi-interacting small RNA (piRNA) expression in wild-type, MILI-null, and MIWI2-null male fetal germ cells. We reveal that defective DNA methylation of the regulatory regions of the Line-1 (long interspersed nuclear elements) and IAP (intracisternal A particle) retrotransposons in the MILI-null and MIWI2-null male germ cells takes place at the level of de novo methylation. Comprehensive analysis shows that the piRNAs of fetal germ cells are distinct from those previously identified in neonatal and adult germ cells. The expression of piRNAs is reduced under MILI- and MIWI2-null conditions in fetal germ cells, although the extent of the reduction differs significantly between the two mutants. Our data strongly suggest that MILI and MIWI2 play essential roles in establishing de novo DNA methylation of retrotransposons in fetal male germ cells.

BACKGROUND: Defensins comprise a large family of cationic antimicrobial peptides that are characterized by the presence of a conserved cysteine-rich defensin motif. Based on the spacing pattern of cysteines, these defensins are broadly divided into five groups, namely plant, invertebrate, alpha-, beta-, and theta-defensins, with the last three groups being mostly found in mammalian species. However, the evolutionary relationships among these five groups of defensins remain controversial. RESULTS: Following a comprehensive screen, here we report that the chicken genome encodes a total of 13 different beta-defensins but with no other groups of defensins being discovered. These chicken beta-defensin genes, designated as Gallinacin 1-13, are clustered densely within a 86-Kb distance on the chromosome 3q3.5-q3.7. The deduced peptides vary from 63 to 104 amino acid residues in length sharing the characteristic defensin motif. Based on the tissue expression pattern, 13 beta-defensin genes can be divided into two subgroups with Gallinacin 1-7 being predominantly expressed in bone marrow and the respiratory tract and the remaining genes being restricted to liver and the urogenital tract. Comparative analysis of the defensin clusters among chicken, mouse, and human suggested that vertebrate defensins have evolved from a single beta-defensin-like gene, which has undergone rapid duplication, diversification, and translocation in various vertebrate lineages during evolution. CONCLUSIONS: We conclude that the chicken genome encodes only beta-defensin sequences and that all mammalian defensins are evolved from a common beta-defensin-like ancestor. The alpha-defensins arose from beta-defensins by gene duplication, which may have occurred after the divergence of mammals from other vertebrates, and theta-defensins have arisen from alpha-defensins specific to the primate lineage. Further analysis of these defensins in different vertebrate lineages will shed light on the mechanisms of host defense and evolution of innate immunity.

Meltrin alpha (ADAM12) is a metalloprotease-disintegrin whose specific expression patterns during development suggest that it is involved in myogenesis and the development of other organs. To determine the roles Meltrin alpha plays in vivo, we generated Meltrin alpha-deficient mice by gene targeting. Although the number of homozygous embryos are close to the expected Mendelian ratio at embryonic days 17 to 18, ca. 30% of the null pups born die before weaning, mostly within 1 week of birth. The viable homozygous mutants appear normal and are fertile. Most of the muscles in the homozygous mutants appear normal, and regeneration in experimentally damaged skeletal muscle is unimpeded. In some Meltrin alpha-deficient pups, the interscapular brown adipose tissue is reduced, although the penetrance of this phenotype is low. Impaired formation of the neck and interscapular muscles is also seen in some homozygotes. These observations suggest Meltrin alpha may be involved in regulating adipogenesis and myogenesis through a linked developmental pathway. Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is a candidate substrate of Meltrin alpha, and we found that TPA (12-O-tetradecanoylphorbol-13-acetate)-induced ectodomain shedding of HB-EGF is markedly reduced in embryonic fibroblasts prepared from Meltrin alpha-deficient mice. We also report here the chromosomal locations of Meltrin alpha in the mouse and rat.

All snake species exhibit genetic sex determination with the ZZ/ZW type of sex chromosomes. To investigate the origin and evolution of snake sex chromosomes, we constructed, by FISH, a cytogenetic map of the Japanese four-striped rat snake (Elaphe quadrivirgata) with 109 cDNA clones. Eleven of the 109 clones were localized to the Z chromosome. All human and chicken homologues of the snake Z-linked genes were located on autosomes, suggesting that the sex chromosomes of snakes, mammals, and birds were all derived from different autosomal pairs of the common ancestor. We mapped the 11 Z-linked genes of E. quadrivirgata to chromosomes of two other species, the Burmese python (Python molurus bivittatus) and the habu (Trimeresurus flavoviridis), to investigate the process of W chromosome differentiation. All and 3 of the 11 clones were localized to both the Z and W chromosomes in P. molurus and E. quadrivirgata, respectively, whereas no cDNA clones were mapped to the W chromosome in T. flavoviridis. Comparative mapping revealed that the sex chromosomes are only slightly differentiated in P. molurus, whereas they are fully differentiated in T. flavoviridis, and E. quadrivirgata is at a transitional stage of sex-chromosome differentiation. The differentiation of sex chromosomes was probably initiated from the distal region on the short arm of the protosex chromosome of the common ancestor, and then deletion and heterochromatization progressed on the sex-specific chromosome from the phylogenetically primitive boids to the more advanced viperids.

Chicken genome mapping is important for a range of scientific disciplines. The ability to distinguish chromosomes of the chicken and other birds is thus a priority. Here we describe the molecular cytogenetic characterization of each chicken chromosome using chromosome painting and mapping of individual clones by FISH. Where possible, we have assigned the chromosomes to known linkage groups. We propose, on the basis of size, that the NOR chromosome is approximately the size of chromosome 22; however, we suggest that its original assignment of 16 should be retained. We also suggest a definitive chromosome classification system and propose that the probes developed here will find wide utility in the fields of developmental biology, DT40 studies, agriculture, vertebrate genome organization, and comparative mapping of avian species.

We have reported on Spred-1 and Spred-2, which inhibit MAP kinase activation by interacting with c-kit and ras/raf. Here, we report the cloning of a third member in this family, Spred-3. Spred-3 is expressed exclusively in the brain and its gene locates in chromosome 19q13.13 in human. Like Spred-1 and -2, Spred-3 contains an EVH1 domain in the N-terminus and a Sprouty-related cysteine-rich region (SPR domain) in the C-terminus that is necessary for membrane localization. However, Spred-3 does not possess a functional c-kit binding domain (KBD), since the critical amino acid Arg residue in this region was replaced with Gly in Spred-3. Although Spred-3 suppressed growth factor-induced MAP kinase (Erk) activation, inhibitory activity of Spred-3 was lower than that of Spred-1 or Spred-2. By the analysis of chimeric molecules between Spred-3 and Spred-1, we found that the SPR domain, rather than KBD, is responsible for efficient Erk suppression. The finding of Spred-3 revealed the presence of a novel family of regulators for the Ras/MAP kinase pathway, each member of which may have different specificities for extracellular signals.

Impacts of genetic polymorphisms of the ATP-binding cassette (ABC) transporter BCRP/MXR1/ABCP (ABCG2) on drug response have been implicated; however, the hitherto reported data involve some inconsistencies. To re-evaluate the effect of single nucleotide polymorphisms (SNP) of ABCG2 in vitro, we created a total of seven variant cDNAs (V12M, Q141K, F208S, S248P, F431L, S441N and F489L) by site-directed mutagenesis and stably expressed each of them in Flp-In-293 cells using the Flp recombinase system. Multicolor fluorescence in situ hybridization mapping analysis revealed that one single copy of ABCG2 cDNA was incorporated into the telomeric region of chromosome 12p. It was proven that mRNAs of those integrated ABCG2 variants were expressed evenly in Flp-In-293 cells. However, the protein expression levels varied among those variants. In particular, expression of the F208S and S441N variants was markedly low, suggesting the instability of these variant proteins. Drug resistance profiles of Flp-In-293 cells expressing two major SNP variants (V12M and Q141K) toward the drug SN-38 demonstrated that the IC50 value (drug concentrations producing a 50% reduction of cell growth) for Q141K was approximately 50% of that for wild type. The contributions of the minor SNP variants (F208S, S248P, F431L, S441N and F489L) to drug resistance toward SN-38, mitoxantrone, doxorubicin, daunorubicin or etoposide were significantly lower than wild type. Based on our functional validation, the above-mentioned non-synonymous polymorphisms as well as acquired mutants (R482G and R482T) of ABCG2 were classified into four groups. Furthermore, new camptothecin analogs synthesized by our research group had potent effects in circumventing ABCG2-mediated drug resistance without any influence from major non-synonymous polymorphisms.

Heparan sulphate 6- O -sulphotransferase (HS6ST) catalyses the transfer of sulphate from adenosine 3'-phosphate, 5'-phosphosulphate to the 6th position of the N -sulphoglucosamine residue in HS. We previously described the occurrence of three isoforms of mouse HS6ST, mHS6ST-1,-2, and -3 [Habuchi, Tanaka, Habuchi, Yoshida, Suzuki, Ban and Kimata (2000) J. Biol. Chem. 275, 2859-2868]. In the present study, we have characterized HS6ST-2 and HS6ST-1 human isologues, including their chromosomal localizations. In the process of their cDNA cloning, we found two forms of HS6ST-2: the original (hHS6ST-2) and a short form (hHS6ST-2S) with 40 amino acids deleted. Both hHS6ST-2 and hHS6ST-2S catalysed the same sulphation reaction, but their preferences for sulphation sites in HS substrates were different. Dot-blot analysis of the two forms showed that the original form was exclusively expressed in adult and foetal brain tissues, whereas the short form was expressed preferentially in ovary, placenta and foetal kidney, suggesting that the expression of two forms of hHS6ST-2 is strictly regulated to yield tissue-dependent differences in the fine structure of HS. A refined analysis of their reaction products has led us to another finding, that HS6STs could also transfer sulphate to N -sulphoglucosamine residues located at the non-reducing terminal of HS with high affinity.

In the XX/XY sex-determining system, the Y-linked SRY genes of most mammals and the DMY/Dmrt1bY genes of the teleost fish medaka have been characterized as sex-determining genes that trigger formation of the testis. However, the molecular mechanism of the ZZ/ZW-type system in vertebrates, including the clawed frog Xenopus laevis, is unknown. Here, we isolated an X. laevis female genome-specific DM-domain gene, DM-W, and obtained molecular evidence of a W-chromosome in this species. The DNA-binding domain of DM-W showed a strikingly high identity (89%) with that of DMRT1, but it had no significant sequence similarity with the transactivation domain of DMRT1. In nonmammalian vertebrates, DMRT1 expression is connected to testis formation. We found DMRT1 or DM-W to be expressed exclusively in the primordial gonads of both ZZ and ZW or ZW tadpoles, respectively. Although DMRT1 showed continued expression after sex determination, DM-W was expressed transiently during sex determination. Interestingly, DM-W mRNA was more abundant than DMRT1 mRNA in the primordial gonads of ZW tadpoles early in sex determination. To assess the role of DM-W, we produced transgenic tadpoles carrying a DM-W expression vector driven by approximately 3 kb of the 5'-flanking sequence of DM-W or by the cytomegalovirus promoter. Importantly, some developing gonads of ZZ transgenic tadpoles showed ovarian cavities and primary oocytes with both drivers, suggesting that DM-W is crucial for primary ovary formation. Taken together, these results suggest that DM-W is a likely sex (ovary)-determining gene in X. laevis.