The Genomic and Genetic Toolbox of the Teleost Medaka (Oryzias latipes)

The Japanese medaka, Oryzias latipes, is a vertebrate teleost model with a long history of genetic research. A number of unique features and established resources distinguish medaka from other vertebrate model systems. A large number of laboratory strains from different locations are available. Due to a high tolerance to inbreeding, many highly inbred strains have been established, thus providing a rich resource for genetic studies. Furthermore, closely related species native to different habitats in Southeast Asia permit comparative evolutionary studies. The transparency of embryos, larvae, and juveniles allows a detailed in vivo analysis of development. New tools to study diverse aspects of medaka biology are constantly being generated. Thus, medaka has become an important vertebrate model organism to study development, behavior, and physiology. In this review, we provide a comprehensive overview of established genetic and molecular-genetic tools that render medaka fish a full-fledged vertebrate system.     

Medaka unextended-fin mutants suggest a role for Hoxb8a in cell migration and osteoblast differentiation during appendage formation

Hoxb8 has been suggestively implicated in the formation of the zone of polarizing activity (ZPA) in the limb bud. However, as hoxb8-/- mice did not show any defects in their limb development, the role of Hoxb8 during limb development has not been fully elucidated. Here, we report the identification of the medaka hoxb8a mutant, unextended-fin (ufi), in which all the fin tissues were malformed. Since the abnormal phenotype was observed in the caudal fin, the ufi phenotype suggests that the medaka Hoxb8a has a fundamental role in the formation of appendages protruding from the trunk. Our analyses revealed that the expression of wnt5a, a regulator of cell migration that signals through the non-canonical Wnt/Ca2+ pathway, was down-regulated in the ufi fin-folds. In fact, we found that the proximal-distal cell migration was impaired in ufi mutants and that the defect could be reversed by the injection of a Wnt5a protein. Moreover, we show herein that the numbers of proliferating cells and osteoblastic cells were increased in the ufi mutants. According to these results, we propose that the medaka Hoxb8a protein functions in the outgrowth of appendages through the regulation of cell migration and osteoblast differentiation.     

端粒酶催化亚基基因在小鼠 胚胎发育早期的转录活性

用 RT-PCR 引物分别扩增成年昆明 (KM) 小鼠睾丸、脾脏、肾脏、肝脏和胸腺组织的总 RNA 发现,端粒酶催化亚基基因 tert 在这些组织中都有转录,目标产物正确组装到 PMD 18-T 载体后测序,结果与已知 cDNA 序列一致 . PMSG/hCG 超数排卵方法获得 KM 小鼠成熟卵母细胞和 CZB 溶液体外培养的胚胎 (KM ♀× KM ♂ ) ,用酸性 Tyrode's 溶液消化透明带后,采用巢式 RT-PCR ,同时分析 tert 基因和持家基因 hprt 的转录发现,对于单个样品来说 , 全部卵母细胞 (15 h-post hCG , 10/10) 都存在 hprt 转录本,其中,只有 40% (4/10) 还同时存在 tert 转录本 . 原核形成初期 (20 h-post hCG , 6/6) 和原核晚期 (30 h post-hCG , 8/8) 的受精卵,以及发育至 2-C 早期的胚胎 (35 h-post hCG , 7/7) 都不转录 tert 基因,只有 hprt mRNA 存在; 2-C 晚期 (50 h-post hCG) 时,两个基因同时转录 (4/8) 和一个基因单独转录 (4/8) 的胚胎各占 50% ;从 4-C 阶段 (65 h-post hCG , 4/4) 开始,包括 8-C 阶段 (75 h-post hCG , 4/4) ,桑椹胚阶段 (93 h-post hCG , 4/4) ,直至囊胚阶段 (118 h-post hCG , 4/4) ,所有的胚胎都同时转录 tert 和 hprt 基因,而且转录水平明显升高 . 以 20 枚胚胎量为模板进行 RT-PCR 发现,原核早期,原核晚期的胚胎中仍然没有 tert 基因转录,只有 hprt mRNA ,但是,在 2-C 早期胚胎中同时检测到了 hprt 和 tert 两种 mRNA. 结果表明,持家基因 hprt 在成熟卵母细胞受精前后,以及胚胎早期发育过程中均存在转录本 . 40% 卵母细胞中存在的 tert mRNA 在受精后很快降解,检测不到;胚胎基因组在 2-C 早期开始转录 tert mRNA ,转录水平逐渐上升 . 结果暗示,小鼠胚胎的基因组 DNA 在 2-C 早期开始启动,功能基因 tert 也在此时开始转录,可能与胚胎发育初期的染色体保护有关 .     

Characterization of 10 MADS-box genes from Pyrus pyrifolia and their differential expression during fruit development and ripening

We cloned 10 Japanese pear (Pyrus pyrifolia) MIKC-type II MADS-box genes, and analyzed their expression during fruit development and ripening. PpMADS2-1 was APETALA (AP)1-like; PpMADS3-1 was FRUITFULL (FUL)/SQUAMOSA (SQUA)-like; PpMADS4-1 was AGAMOUS-like (AGL)6; PpMADS5-1 and PpMADS8-1 were SUPPRESSOR OF OVEREXPRESSION OF CONSTANS (SOC)-like; PpMADS9-1, PpMADS12-1, PpMADS14-1 and PpMADS16-1 were SEPALLATA (SEP)-like; while PpMADS15-1 was AGL/SHATTERPROOF (SHP)-like. Phylogenetic analysis showed their grouping into five major clades (and 10 sub-clades) that was consistent with their diverse functional types. Expression analysis in flower tissue revealed their distinct putative homeotic functional classes: A-class (PpMADS2-1, PpMADS3-1, PpMADS4-1, and PpMADS14-1), C-class (PpMADS15-1), E-class (PpMADS9-1, PpMADS12-1, and PpMADS16-1) and E (F)-class (PpMADS5-1 and PpMADS8-1). Differential gene expression was observed in different fruit tissues (skin, cortex and core) as well as in the cortex during the course of fruit development and ripening. Collectively, our results suggest their involvement in the diverse aspects of plant development including flower development and the course of fruit development and ripening.     

Thermodynamic characterization of the Saccharomyces cerevisiae telomerase RNA pseudoknot domain in vitro

Recent structural and functional characterization of the pseudoknot in the Saccharomyces cerevisiae telomerase RNA (TLC1) has demonstrated that tertiary structure is present, similar to that previously described for the human and Kluyveromyces lactis telomerase RNAs. In order to biophysically characterize the identified pseudoknot secondary and tertiary structures, UV-monitored thermal denaturation experiments, nuclear magnetic resonance spectroscopy, and native gel electrophoresis were used to investigate various potential conformations in the pseudoknot domain in vitro, in the absence of the telomerase protein. Here, we demonstrate that alternative secondary structures are not mutually exclusive in the S. cerevisiae telomerase RNA, tertiary structure contributes 1.5 kcal mol(-1) to the stability of the pseudoknot (≈ half the stability observed for the human telomerase pseudoknot), and identify additional base pairs in the 3' pseudoknot stem near the helical junction. In addition, sequence conservation in an adjacent overlapping hairpin appears to prevent dimerization and alternative conformations in the context of the entire pseudoknot-containing region. Thus, this work provides a detailed in vitro characterization of the thermodynamic features of the S. cerevisiae TLC1 pseudoknot region for comparison with other telomerase RNA pseudoknots.     

Effects of Doxycycline on gene expression in Wolbachia and Brugia malayi adult female worms in vivo

Background

Most filarial nematodes contain Wolbachia symbionts. The purpose of this study was to examine the effects of doxycycline on gene expression in Wolbachia and adult female Brugia malayi.

Methods

Brugia malayi infected gerbils were treated with doxycycline for 6-weeks. This treatment largely cleared Wolbachia and arrested worm reproduction. RNA recovered from treated and control female worms was labeled by random priming and hybridized to the Version 2- filarial microarray to obtain expression profiles.

Results and discussion

Results showed significant changes in expression for 200 Wolbachia (29% of Wolbachia genes with expression signals in untreated worms) and 546 B. malayi array elements after treatment. These elements correspond to known genes and also to novel genes with unknown biological functions. Most differentially expressed Wolbachia genes were down-regulated after treatment (98.5%). In contrast, doxycycline had a mixed effect on B. malayi gene expression with many more genes being significantly up-regulated after treatment (85% of differentially expressed genes). Genes and processes involved in reproduction (gender-regulated genes, collagen, amino acid metabolism, ribosomal processes, and cytoskeleton) were down-regulated after doxycycline while up-regulated genes and pathways suggest adaptations for survival in response to stress (energy metabolism, electron transport, anti-oxidants, nutrient transport, bacterial signaling pathways, and immune evasion).

Conclusions

Doxycycline reduced Wolbachia and significantly decreased bacterial gene expression. Wolbachia ribosomes are believed to be the primary biological target for doxycycline in filarial worms. B. malayi genes essential for reproduction, growth and development were also down-regulated; these changes are consistent with doxycycline effects on embryo development and reproduction. On the other hand, many B. malayi genes involved in energy production, electron-transport, metabolism, anti-oxidants, and others with unknown functions had increased expression signals after doxycycline treatment. These results suggest that female worms are able to compensate in part for the loss of Wolbachia so that they can survive, albeit without reproductive capacity. This study of doxycycline induced changes in gene expression has provided new clues regarding the symbiotic relationship between Wolbachia and B. malayi.     

Interleukin-13 interferes with CFTR and AQP5 expression and localization during human airway epithelial cell differentiation

Interleukin-13 (IL-13) is a central regulator of Th2-dominated respiratory disorders such as asthma. Lesions of the airway epithelial barrier frequently observed in chronic respiratory inflammatory diseases are repaired through proliferation, migration and differentiation of epithelial cells. Our work is focused on the effects of IL-13 in human cellular models of airway epithelial cell regeneration. We have previously shown that IL-13 altered epithelial cell polarity during mucociliary differentiation of human nasal epithelial cells. In particular, the cytokine inhibited ezrin expression and interfered with its apical localization during epithelial cell differentiation in vitro. Here we show that CFTR expression is enhanced in the presence of the cytokine, that two additional CFTR protein isoforms are expressed in IL-13-treated cells and that part of the protein is retained within the endoplasmic reticulum. We further show that aquaporin 5 expression, a water channel localized within the apical membrane of epithelial cells, is completely abolished in the presence of the cytokine. These results show that IL-13 interferes with ion and water channel expression and localization during epithelial regeneration and may thereby influence mucus composition and hydration.     

Exclusive paternal expression and novel alternatively spliced variants of epsilon-sarcoglycan mRNA in mouse brain

Mutations of SGCE encoding epsilon-sarcoglycan cause myoclonus-dystonia. SGCE is paternally expressed; however, 5-10% of patients show maternal inheritance of the disease. We found Sgce was exclusively paternally expressed in mice by using a novel polymorphism marker. The result was confirmed in Sgce heterozygous knockout mice. This finding suggests that maternally inherited myoclonus-dystonia may not result from maternal expression of SGCE. Furthermore, we report a new family of alternatively spliced Sgce mRNA expressed in the brain coding for different C-terminal sequences possessing a PDZ-binding motif. Our results provide a better basis for diagnosis and understanding of the pathogenesis of myoclonus-dystonia.     

Cloning and expression of multiple integral membrane proteins from Mycobacterium tuberculosis in Escherichia coli

Seventy integral membrane proteins from the Mycobacterium tuberculosis genome have been cloned and expressed in Escherichia coli. A combination of T7 promoter-based vectors with hexa-His affinity tags and BL21 E. coli strains with additional tRNA genes to supplement sparsely used E. coli codons have been most successful. The expressed proteins have a wide range of molecular weights and number of transmembrane helices. Expression of these proteins has been observed in the membrane and insoluble fraction of E. coli cell lysates and, in some cases, in the soluble fraction. The highest expression levels in the membrane fraction were restricted to a narrow range of molecular weights and relatively few transmembrane helices. In contrast, overexpression in insoluble aggregates was distributed over a broad range of molecular weights and number of transmembrane helices.     

BRCC2 inhibits breast cancer cell growth and metastasis in vitro and in vivo via downregulating AKT pathway

In our previous study, we demonstrated that the BRCC2 (breast cancer cell 2) gene is a proapoptotic molecule that interacts with Bcl-X_L. BRCC2 downregulation is associated with poor disease-free and overall survival in breast cancer. In this study, we aimed to investigate the role of BRCC2 in tumor suppression in breast cancer. In clinical breast cancer samples, we found that BRCC2 expression was significantly downregulated in cancer lesions compared with paired normal breast tissues. By silencing or overexpressing BRCC2 in breast cancer cells, we found that BRCC2 could inhibit cell growth and metastasis in vitro. An in vivo assay showed that BRCC2 not only dramatically inhibited breast cancer cell xenograft formation and growth but also inhibited breast cancer cell metastasis in a lung metastasis model. Moreover, we demonstrated that BRCC2 inhibited breast cancer metastasis via regulation of the Akt pathway. Thus, our study provided evidence that BRCC2 functions as a novel tumor suppressor in breast cancer and may be a potential therapeutic target for breast cancer management.