Interferon-induced transmembrane protein-1 competitively blocks Ephrin receptor A2-mediated Epstein-Barr virus entry into epithelial cells.

Epstein-Barr virus (EBV) can infect both B cells and epithelial cells (ECs), causing diseases such as mononucleosis and cancer. It enters ECs via Ephrin receptor A2 (EphA2). The function of interferon-induced transmembrane protein-1 (IFITM1) in EBV infection of ECs remains elusive. Here we report that IFITM1 inhibits EphA2-mediated EBV entry into ECs. RNA-sequencing and clinical sample analysis show reduced IFITM1 in EBV-positive ECs and a negative correlation between IFITM1 level and EBV copy number. IFITM1 depletion increases EBV infection and vice versa. Exogenous soluble IFITM1 effectively prevents EBV infection in vitro and in vivo. Furthermore, three-dimensional structure prediction and site-directed mutagenesis demonstrate that IFITM1 interacts with EphA2 via its two specific residues, competitively blocking EphA2 binding to EBV glycoproteins. Finally, YTHDF3, an m6A reader, suppresses IFITM1 via degradation-related DEAD-box protein 5 (DDX5). Thus, this study underscores IFITM1's crucial role in blocking EphA2-mediated EBV entry into ECs, indicating its potential in preventing EBV infection.

Methylosome protein 50 is necessary for oogenesis in medaka.

Methylosome protein 50 (Mep50) functions as a partner to protein arginine methyltransferase 5. MEP50 serves as a coactivator for both the androgen receptor and estrogen receptor in humans. Mep50 plays a crucial role in the development of germ cells in Drosophila. The precise role of Mep50 in oogenesis remains unclear in vertebrates. The objective of this study was to investigate the role of Mep50 in oogenesis in medaka fish. Disruption of Mep50 resulted in impaired oogenesis and the formation of multiple oocyte follicles in medaka. RNA-seq analysis revealed significant differential gene expression in the mutant ovary, with 4542 genes up-regulated and 1264 genes down-regulated. The regulated genes were found to be enriched in cellular matrices and ECM-receptor interaction, the Notch signaling pathway, the PI3K-Akt signaling pathway, the MAPK signaling pathway, the Hippo signaling pathway, and the Jak-Stat pathway, among others. In addition, the genes related to the hypothalamus-pituitary-gonad axis, steroid metabolism, and IGF system were impacted. Furthermore, the mutation of mep50 caused significant alterations in alternative splicing of pre-mRNA in ovarian cells. Quantitative RT-PCR results validated the findings from RNA-seq analysis in the specific genes, including akt2, map3k5, yap1, fshr, cyp17a, igf1, ythdc2, cdk6, and col1, among others. The findings of this study demonstrate that Mep50 plays a crucial role in oogenesis, participating in a diverse range of biological processes such as steroid metabolism, cell matrix regulation, and signal pathways. This may be achieved through the regulation of gene expression via mRNA splicing in medaka ovarian cells.

Alterations of plasma exosomal proteins and motabolies are associated with the progression of castration-resistant prostate cancer.

BACKGROUND: Current diagnosis tools for prostate cancer (PCa) such as serum PSA detection and prostate biopsy cannot distinguish dormant tumors from invasive malignancies, either be used as prognosis marker for castration resistant prostate cancer (CRPC), the lethal stage of PCa patients. Exosomes have been widely investigated as promising biomarkers for various diseases. We aim to characterize the proteomic and metabolomic profile of exosomes and to evaluate their potential value for the diagnosis of PCa, especially CRPC. We also investigate the functions of some specific exosome biomarkers in the progression of CRPC. METHODS: Integrated proteomics and metabolomics analysis were performed for plasma-derived exosomes collected from tumor-free controls (TFC), PCa and CRPC patients. Expression of specific exosomal proteins were further validated by targeted 4D-parallel reaction monitoring (PRM) mass spectrometry among the three cohorts. Tissue distribution and functional role of exosomal protein LRG1 was studied in clinical PCa tissue samples and cell line models. RESULTS: Three potential exosomal protein markers were identified. The apolipoprotein E level in PCa samples was 1.7-fold higher than that in TFC (receiver operating characteristic value, 0.74). Similarly, the levels of exosome-derived leucine-rich alpha2-glycoprotein 1 (LRG1) and inter-alpha-trypsin inhibitor heavy chain H3 (ITIH3) in the CRPC group were 1.7 and 2.04 times, respectively, higher than those in the PCa group (ROC values, 0.84 and 0.85, respectively), indicating that LRG1 and ITIH3 could serve as predictive markers for CRPC. For metabolomic evaluation of exosomes, a series of differentially expressed metabolites were identified, and a combined metabolite panel showed ROC value of 0.94 for distinguishing PCa from TFC and 0.97 for distinguishing CRPC from PCa. Immunohistochemistry of tissue microarray showed that LRG1 protein was significantly upregulated in advanced prostate cancer and functional assay revealed that ectopic expression of LRG1 can significantly enhance the malignant phenotype of prostate cancer cells. More importantly, PCa cell derived LRG1-overexpressed exosomes remarkably promoted angiogenesis. CONCLUSION: Integration of proteomics and metabolomics data generated proteomic and metabolic signatures of plasma exosomes that may facilitate discrimination of CRPC from PCa and TFC patients, suggesting the potential of exosomal proteins and metabolites as CRPC markers. The study also confirmed the important role of exosomal protein LRG1 in PCa malignant progression.

A novel ferroptosis-related gene prognostic index for prognosis and response to immunotherapy in patients with prostate cancer.

Background: Prostate cancer (PCa) is among the leading causes of cancer death worldwide. Ferroptosis refers to an iron-dependent form of regulated cell death and is involved in prostate tumorigenesis. A few ferroptosis-related gene signatures have been developed to predict the prognosis for PCa patients. However, previous signatures were typically established based on biochemical recurrence-free survival, which has proven not to be a good surrogate for overall survival (OS). This study aimed to construct a novel ferroptosis-related gene prognostic index (FRGPI) to predict disease-free survival (DFS) and response to immunotherapy for PCa patients after radical prostatectomy. Methods: Gene expression and clinicopathological data on PCa patients were obtained from the TCGA database. Ferroptosis-related hub genes associated with DFS of PCa patients were identified by an in-depth bioinformatics analysis using a novel and comprehensive algorithm based on functional enrichment, consensus clustering, weighted gene co-expression network analysis (WGCNA), and protein-protein interaction (PPI) network construction. The FRGPI was established on the basis of the genes selected using multivariate cox regression analysis and further validated in two additional PCa cohorts. Next, the clinicopathological, molecular, and immune profiles were characterized and compared between FRGPI-high and FRGPI-low subgroups. Finally, the predictive role of the FRGPI in response to immunotherapy was estimated using a metastatic urothelial cancer cohort treated with an anti-PD-L1 agent. Results: The FRGPI was constructed based on four genes (E2F1, CDC20, TYMS, and NUP85), and FRGPI-high patients had worse DFS than FRGPI-low patients. Multivariate cox regression analysis revealed that FRGPI could act as an independent prognostic factor for PCa patients after radical prostatectomy. A prognostic nomogram comprising the FRGPI and other clinicopathological parameters was established to predict the DFS for PCa patients quantitatively. In addition, comprehensive results demonstrated that high FRGPI scores showed a significantly positive correlation with worse clinicopathological features, higher mutation counts, increased frequency of copy number variations (CNVs), higher homologous recombination deficiency (HRD) and immune scores, higher mRNAsi, and more importantly, enhanced sensitivity to immunotherapy. Conclusions: FRGPI is not only a promising and robust prognostic biomarker, but also a potential indicator of immunotherapeutic outcomes for PCa patients after radical prostatectomy.

Alternative splicing of medaka bcl6aa and its repression by Prdm1a and Prdm1b.

Bcl6 and Prdm1 (Blimp1) are a pair of transcriptional factors that repressing each other in mammals. Prdm1 represses the expression of bcl6 by binding a cis-element of the bcl6 gene in mammals. The homologs of Bcl6 and Prdm1 have been identified in teleost fish. However, whether these two factors regulate each other in the same way in fish like that in mammals is not clear. In this study, the regulation of bcl6aa by Prdm1 was investigated in medaka. The mRNA of bcl6aa has three variants (bcl6aaX1-X3) at the 5'-end by alternative splicing detected by RT-PCR. The three variants can be detected in adult tissues and developing embryos of medaka. Prdm1a and prdm1b are expressed in the tissues and embryos where and when bcl6aa is expressed. The expression of prdm1a was high while the expression of bcl6aa was low, and vice versa, detected in the spleen after stimulation with LPS or polyI:C. In vitro reporter assay indicated that bcl6aa could be directly repressed by both Prdm1a and Prdm1b in a dosage-dependent manner. After mutation of the key base, G, of all predicted binding sites in the core promoter region of bcl6aa, the repression by Prdm1a and/or Prdm1b disappeared. The binding site of Prdm1 in the bcl6aa gene is GAAAA(T/G). These results indicate that both Prdm1a and Prdm1b directly repress the expression of bcl6aa by binding their binding sites where the 5'-G is critical in medaka fish.

Kinesin family member 3A stimulates cell proliferation, migration, and invasion of bladder cancer cells in vitro and in vivo.

Bladder cancer is one of the most common malignant tumors of the urinary system, with high morbidity and mortality. At present, the survival rates and prognosis of patients with bladder cancer are still relatively low; thus, there remains a need to improve prognosis by identifying novel targets. Kinesins (kinesin superfamily proteins) are a series of microtubule-based motor proteins that mediate various types of cellular processes. Kinesin family member 3A (KIF3A) is critical for cytoplasm separation in mitosis, and it has been reported to be misexpressed in multiple types of cancer. However, its effects on the progression and development of bladder cancer remain unclear. Herein, we report that KIF3A is highly expressed in human bladder cancer. We identified a significant correlation between KIF3A and clinical features, including clinical stage (P = 0.047), pathological tumor status (P = 0.045), lymph node status (P = 0.041) and metastasis (P = 0.035). KIF3A expression was also correlated with poor prognosis of patients with bladder cancer. Our results further indicated that KIF3A ablation resulted in cell cycle arrest; blocked the proliferation, migration and invasion of bladder cancer cells in vitro; and restrained tumor growth in mice in a microtubule-dependent manner. In summary, our findings suggest that KIF3A is a potential therapeutic target for bladder cancer.

Performance of Dual-Source CT in Calculi Component Analysis: A Systematic Review and Meta-Analysis of 2151 Calculi.

OBJECTIVE: To evaluate the performance of dual-source computed tomography (DSCT) in the component analysis of all types of calculi by doing a systematic review and meta-analysis. METHODS: We searched MEDLINE, Embase, Scopus, and CNKI up to February 28, 2020, for in vivo studies investigating the performance of DSCT in the component analysis of calculi. We pooled the sensitivity, specificity, and areas under the summary receiver operating characteristic (AUROC) curves using a random-effect model in the meta-analysis. Publication bias was evaluated using Deek's funnel plot asymmetry test. RESULTS: This analysis included a total of 37 studies in 1840 patients with 2151 calculi (462 uric acid [UA], 1383 calcium oxalate [CaOx], 55 cystine [Cys], 197 hydroxyapatite [HA], and 54 struvite [SV]). Using DSCT, the pooled accuracy for diagnosing UA (sensitivity, 0.95; specificity, 0.99), CaOx (0.98; 0.93), Cys (0.99; 0.99), HA (0.91; 0.99), and SV (0.42; 0.98) was calculated, respectively. The AUROC value was 0.99, 0.99, 1.00, 0.99, and 0.93, respectively. The P values for publication bias test were .49, .70, .07, .04, and .19, respectively. CONCLUSION: Dual-source computed tomography has high sensitivity and specificity for the component analysis of UA, CaOx, Cys, and HA calculi in vivo. This tool may have the potential to replace the current analysis tool in vitro in diagnosing calculi.

The Proteins Interacting with Prmt5 in Medaka (Oryzias latipes) Identified by Yeast Two-Hybridization.

BACKGROUND: Prmt5 plays major role in regulation of gene expression, RNA processing, cell growth and differentiation, signal transduction, germ cell development, etc., in mammals. Prmt5 is also related to cancer. Knowing the proteins interacting with Prmt5 is important to understand Prmt5's function in cells. Although there have been reports on proteins binding with Prmt5 in mammals, the partner proteins of Prmt5 in fish are still unclear. OBJECTIVES: The objective was to obtain proteins that bind with Prmt5 in medaka, a model fish. METHODS: Yeast two hybridization was adopted to achieve the objective. Medaka Prmt5 was used as a bait to fish the prey, binding proteins in a cDNA library of medaka. Co-immunoprecipitation and in silicon analysis were performed to study the interaction of medaka Mep50 and Prmt5. RESULTS: Eight proteins were identified to bind with Prmt5 from 69 preliminary positive colonies. The binding proteins are methylosome protein 50 (Mep50), apolipoprotein A-I-like (Apo-AI), PR domain containing protein 1a with zinc fingers (Prdm1a), Prdm1b, T-cell immunoglobulin mucin family member 3 (Tim-3), phosphoribosylaminoimidazole carboxylase and phosphoribosylaminoimidazolesuccinocarboxamide synthase (Paics), NADH dehydrogenase subunit 4 (ND4) and sciellin (Scl). Co-immunoprecipitation confirmed the interaction of medaka Prmt5 and Mep50. Predicted structures of medaka Prtm5 and Mep50 are similar to that of human PRMT5 and MEP50. CONCLUSION: Medaka Mep50, Prdm1a, Prdm1b, Apo-AI, Tim-3, Paics, ND4, and Scl bind with Prmt5.

Bcl6aa and bcl6ab are ubiquitously expressed and are inducible by lipopolysaccharide and polyI:C in adult tissues of medaka Oryzias latipes.

B-cell lymphoma-6 (Bcl6) is a transcriptional repressor that plays important roles in various physiological activities such as innate and adaptive immune response, lymphocyte differentiation, and cell cycle regulation in mammals. Two homologs of Bcl6a, namely Bcl6aa and Bcl6ab, are identified in teleost fish including medaka Oryzias latipes. The expression profiles of bcl6aa and bcl6ab in medaka were studied using reverse-transcription polymerase chain reaction and in situ hybridization. The transcripts of bcl6aa and bcl6ab were detected from very early embryos such as the four-cell stage until hatching. Bcl6aa and bcl6ab were clearly detected in the embryonic body from 5 days postfertilization onward by in situ hybridization. Bcl6aa was specifically expressed in the retina, whereas bcl6ab was expressed in entire embryonic body. The results referred to that both bcl6aa and bcl6ab originate maternally in the zygotes and may play major roles in embryogenesis of medaka. The transcripts of bcl6aa and bcl6ab were detected in all examined adult tissues, including immune organs such as the gill, spleen, kidney, liver, and intestine. The expression of bcl6aa and bcl6ab in the liver, spleen, head-kidney, and intestine could be upregulated or downregulated by lipopolysaccharide and polyriboinosinic-polyribocytidylic acid. These results indicate that both bcl6aa and bcl6ab may be involved in immune response in medaka.

Expression of the alternative splicing variants of bcl6b in medaka Oryzias latipes.

Bcl6B, also known as BAZF, plays important roles in the immune response, repression of cancers, and maintenance of spermatogonial stem cells in mammals. In this study, the homologous gene bcl6b and its 5 alternative splicing variants, namely bcl6bX1 to bcl6bX5, were isolated from medaka fish, Oryzias latipes. Medaka bcl6b possesses conserved domains such as BTB domain, RD2 domain and four zinc fingers. Medaka bcl6bX1 to bcl6bX3 possess all three previously mentioned domains with minor differences in sequences. Medaka bcl6bX4 possesses only the BTB domain due to premature stopping, and bcl6bX5 possesses both the BTB domain and zinc fingers without the RD2 domain. Medaka bcl6b was expressed in the tissues including the brain, heart, gill, muscle, spleen, kidney, intestine, ovary and testes of adult fish. Medaka bcl6b was expressed in the embryos from very early stage, and could be detected clearly in the developing eyes by RT-PCR and in situ hybridization. Medaka bcl6b could respond to the stimuli of polyI:C and LPS in the kidney and spleen. Medaka bcl6bX1 to bcl6bX3 were the majority of the variants expressed in the adult tissues and the embryos, and were the major response to the stimulation of polyI:C and LPS in the spleen. These results suggested that bcl6b, including its isoforms, could function in various tissues and embryogenesis. Moreover, bcl6b might be a factor for immune response in medaka.

Expression pattern and functional analysis of fundc1 in rare minnow (Gobiocypris rarus).

Fundc1 is a mitochondrial outer membrane protein and plays important roles in mitochondria fission and hypoxia-induced mitophagy in mammalian cells. However, there is no relevant report of fundc1 in fish. In the present study, we cloned a 942bp fundc1 cDNA from rare minnow. The cDNA, designated as Grfundc1 cDNA, contains an open reading frame (ORF) of 459bp which encodes a polypeptide of 152 amino acid residues. Comparisons of deduced amino acid sequences demonstrated that Grfundc1 was highly homologous with those of other vertebrates. RT-PCR and real time PCR detection revealed that the transcripts of Grfundc1 were not detectable in the unfertilized eggs and had high levels at blastula and gastrula stages. Whole mount in situ hybridization analysis observed that Grfundc1 was ubiquitously expressed at early stage and later riched in specific regions, such as brain, branchial arch, eye and somite during embryogenesis. Grfundc1 was expressed in all the tissues of rare minnow adult, including brain, liver, gill, eyes, heart, kidney, intestine, muscle, testis and ovary. The expression of Grfundc1 in the brain, gill, heart and eye of rare minnow adult was significantly down-regulated by hypoxia. Similar hypoxic response was observed in the rare minnow embryos at 48hpf following hypoxia exposure. Functional analysis showed that knockdown of Grfundc1 significantly caused defects in the body axis and dorsal neural tissues of rare minnow embryos. These results indicate that Grfundc1 may play important roles in embryogenesis in fish.

Impact of reduced-radiation dual-energy protocols using 320-detector row computed tomography for analyzing urinary calculus components: initial in vitro evaluation.

OBJECTIVE: To evaluate the impact of reduced-radiation dual-energy (DE) protocols using 320-detector row computed tomography on the differentiation of urinary calculus components. MATERIALS AND METHODS: A total of 58 urinary calculi were placed into the same phantom and underwent DE scanning with 320-detector row computed tomography. Each calculus was scanned 4 times with the DE protocols using 135 kV and 80 kV tube voltage and different tube current combinations, including 100 mA and 570 mA (group A), 50 mA and 290 mA (group B), 30 mA and 170 mA (group C), and 10 mA and 60 mA (group D). The acquisition data of all 4 groups were then analyzed by stone DE analysis software, and the results were compared with x-ray diffraction analysis. Noise, contrast-to-noise ratio, and radiation dose were compared. RESULTS: Calculi were correctly identified in 56 of 58 stones (96.6%) using group A and B protocols. However, only 35 stones (60.3%) and 16 stones (27.6%) were correctly diagnosed using group C and D protocols, respectively. Mean noise increased significantly and mean contrast-to-noise ratio decreased significantly from groups A to D (P <.05). In addition, the effective dose decreased markedly from groups A to D at 3.78, 1.81, 1.07, and 0.37 mSv, respectively. CONCLUSION: Decreasing the DE tube currents from 100 mA and 570 mA to 50 mA and 290 mA resulted in 96.6% accuracy for urinary calculus component analysis while reducing patient radiation exposure to 1.81 mSv. Further reduction of tube currents may compromise diagnostic accuracy.

Gene amplification and functional diversification of melanocortin 4 receptor at an extremely polymorphic locus controlling sexual maturation in the platyfish.

In two swordtail species of the genus Xiphophorus, the onset of puberty has been shown to be modulated at the P locus by sequence polymorphism and gene copy-number variation affecting the type 4 melanocortin hormone receptor Mc4r. The system works through the interaction of two allelic types, one encoding wild type and the other dominant-negative receptors. We have analyzed the structure and evolution of the P locus in the platyfish Xiphophorus maculatus, where as many as nine alleles of P determining the onset of sexual maturity in males and females, fecundity in females, and adult size in males are located on both the X and Y chromosomes in a region linked to the master sex-determining locus. In this species, mc4r has been amplified to up to 10 copies on both the X and Y chromosomes through recent large serial duplications. Subsequently, mc4r paralogues have diverged considerably into many different subtypes. Certain copies have acquired new untranslated regions through genomic rearrangements, and transposable element insertions and other mutations have accumulated in promoter regions, possibly explaining observed deviations from the classical mc4r transcriptional pattern. In the mc4r-coding sequence, in-frame insertions and deletions as well as nonsense and missense mutations have generated a high diversity of Mc4r-predicted proteins. Most of these variants are expressed in embryos, adults, and/or tumors. Functional receptor characterization demonstrated major divergence in pharmacological behavior for Mc4r receptors encoded by different copies of platyfish mc4r, with differences in constitutive activity as well as binding and stimulation by hormones. The high degree of allelic and copy-number variation observed between individuals can explain the high level of polymorphism for sexual maturation, fecundity, and body size in the platyfish: multiple combinations of Mc4r variants with different biochemical properties might interact to modulate the melanocortin signaling that regulates the hypothalamus-pituitary-gonadal axis.

Anti-tumor effects of Astragalus on hepatocellular carcinoma in vivo.

OBJECTIVE: The objective of the present study is to investigate the anti-proliferation activity of Astragalus on human hepatocellular carcinoma (HCC) cells and its mechanism. MATERIALS AND METHODS: Hepatic cancer H22 bearing mice were used to study the anti-hepatocarcinoma activity of Astragalus in vivo. The growth curve and inhibitory rate of tumor growth were measured. Cell apoptosis of each group was measured by flow cytometry (FCM). Protein expression of Bax and Bcl-2 were analyzed by immunohistochemistry (IHC). The Statistical Package for Social Sciences version 13.0 (SPSS Inc, Chicago, IL) was used for standard statistical analysis including one-way ANOVA and Student's t-test. A value of P<0.05 was considered to be statistically significant. RESULTS: Astragalus significantly inhibited the growth of H22 carcinoma, with an inhibitory rate of 17.28-52.36%. FCM and immunohistochemical assay show that the cell apoptosis rate and protein expression of Bax and Bax/Bcl-2 ratio of H22 transplanted tumor in Astragalus treated group were significantly higher than the control group (P<0.05). The protein expression of Bcl-2 was significantly lower than control (P<0.05). CONCLUSION: The results of the present study suggest that Astragalus has significant anti-tumor effect in vivo in inducing apoptosis of H22 tumor cells by promoting protein expression of Bax, decreasing protein expression of Bcl-2 gene, and markedly increasing the Bax/Bcl-2 ratio.

Zisupton--a novel superfamily of DNA transposable elements recently active in fish.

Transposable elements are widespread mobile DNA sequences able to integrate into new locations within genomes. Through transposition and recombination, they significantly contribute to genome plasticity and evolution. They can also regulate gene expression and provide regulatory and coding sequences (CDSs) for the evolution of novel gene functions. We have identified a new superfamily of DNA transposon on the Y chromosome of the platyfish Xiphophorus maculatus. This element is 11 kb in length and carries a single CDS of 24 exons. The N-terminal part of the putative protein, which is expressed in all adult tissues tested, contains several nucleic acid- and protein-binding domains and might correspond to a novel type of transposase/integrase not described so far in any transposon. In addition, a testis-specific splice isoform encodes a C-terminal Ulp1 SUMO protease domain, suggesting a function in posttranslational protein modification mediated by SUMO and/or ubiquitin small peptides. Accordingly, this element was called Zisupton, for Zinc finger SUMO protease transposon. Beside the Y-chromosomal sequence, five other very similar copies were identified in the platyfish genome. All copies are delimited by 99-bp conserved subterminal inverted repeats and flanked by copy-specific 8-nt target site duplications reflecting their integration at different positions in the genome. Zisupton elements are inserted at different genomic locations in different poeciliid species but also in different populations of X. maculatus. Such insertion polymorphisms between related species and populations indicate relatively recent transposition activity, with a high degree of nucleotide identity between species suggesting possible implication of horizontal gene transfer. Zisupton sequences were detected in other fish species, in urochordates, cephalochordates, and hemichordates as well as in more distant organisms, such as basidiomycete fungi, filamentous brown algae, and green algae. Possible examples of nuclear genes derived from Zisupton have been identified. To conclude, our analysis has uncovered a new superfamily of DNA transposons with potential roles in genome diversity and evolutionary innovation in fish and other organisms.

Helitron Transposons on the Sex Chromosomes of the Platyfish Xiphophorus maculatus and Their Evolution in Animal Genomes.

The sex-determining region of the sex chromosomes of the platyfish Xiphophorus maculatus contains several copies of a recently described class of DNA transposons called Helitrons, which probably transpose through a mechanism involving rolling circle replication. The unique open reading frame of platyfish elements encodes a 2816 amino-acid protein with helicase and replication initiator (Rep) domains, which are hallmarks of Helitrons. Like previously described elements from zebrafish but unlike sequences from plants, insects and nematodes, platyfish Helitrons also encode a C-terminal apurinic-apyrimidinic-like endonuclease probably captured from a non-long-terminal- repeat retrotransposon. A cysteine protease domain related to the Drosophila ovarian tumor (OTU) protein was identified in the N-terminal part of the platyfish sequence. Putative endonuclease and protease have been acquired sequentially at least 600 million years ago and maintained functional in elements from sea urchin, lancelet and teleost fish, implying an important role for these domains in the transposition mechanism. Apparently intact Helitron elements are transcribed in Xiphophorus, and insertion polymorphism was observed between related fishes in different poeciliids. These observations suggest that Helitron transposons are still active in the genome of platyfish and related species, where they might play a role in the evolution of sex chromosomes and other genomic regions.

Molecular analysis of the sex-determining region of the platyfish Xiphophorus maculatus.

Due to the presence of genetically well-defined sex chromosomes, with a relatively restricted sex-determination region containing markers identified at the molecular level, the platyfish Xiphophorus maculatus is one of the best models for the positional cloning of a master sex-determining gene in fish. Both male and female heterogametes and three different types of sex chromosomes have been described in the platyfish, with several loci involved in pigmentation, melanoma formation, and sexual maturity closely linked to the master sex-determining locus. Using the melanoma-inducing oncogene Xmrk, its protooncogenic counterpart egfrb, as well as other X- and Y-linked molecular markers, bacterial artificial chromosome (BAC) contigs have been assembled for the sex-determining region of X. maculatus, which was mapped by fluorescent in situ hybridization to the subtelomeric region of the sex chromosomes. Initial sequence analysis of these contigs revealed several gene candidates and uncovered syntenies with different mammalian and chicken autosomes, supporting an independent origin of sex chromosomes in platyfish and tetrapods. Strikingly, the sex determination region of the platyfish is very instable and frequently undergoes duplications, deletions, and transpositions. This instability might be linked to the high genetic variability affecting sex determination and other sex-linked traits in Xiphophorus.

The Xmrk oncogene can escape nonfunctionalization in a highly unstable subtelomeric region of the genome of the fish Xiphophorus.

The Xmrk oncogene involved in melanoma formation in the fish Xiphophorus was formed relatively recently by duplication of the epidermal growth factor co-orthologue egfrb. In the platyfish X. maculatus, Xmrk is located close to the major sex-determining locus in a subtelomeric region of the X and Y sex chromosomes that frequently undergoes duplications and other rearrangements. This region accumulates repetitive sequences: more than 80% of the 33-kb region 3' of Xmrk is constituted by retrotransposable elements. The high degree of nucleotide identity between X- and Y-linked sequences and the rarity of gonosome-specific rearrangements indicated that the instability observed was not a manifestation of gonosome-specific degeneration. Seven other duplicated genes were found, all corresponding, in contrast to Xmrk, to pseudogenes (nonfunctionalization). Functional persistence of Xmrk in a highly unstable region in divergent Xiphophorus species suggests a beneficial function under certain conditions for this dispensable and potentially injurious gene.