The Dr-nanos gene is essential for germ cell specification in the planarian Dugesia ryukyuensis

Homologs of nanos are required for the formation and maintenance of germline stem cell (GSC) systems and for gametogenesis in many metazoans. Planarians can change their reproductive mode seasonally, alternating between asexual and sexual reproduction; they develop and maintain their somatic stem cells (SSCs) and GCSs from pluripotent stem cells known as neoblasts. We isolated a nanos homolog, Dr-nanos, from the expressed sequence tags (ESTs) of the sexualized form of Dugesia ryukyuensis. We examined the expression of Dr-nanos in asexual and sexualized planarians by in situ hybridization and analyzed its function using RNA interference (RNAi) together with a planarian sexualization assay. A nanos homolog, Dr-nanos, was identified in the planarian D. ryukyuensis. Dr-nanos expression was observed in the ovarian primordial cells of the asexual worms. This expression increased in proportion to sexualization and was localized in the early germline cells of the ovaries and testes. In X-ray-irradiated worms, the expression of Dr-nanos decreased to a large extent, indicating that Dr-nanos is expressed in some subpopulations of stem cells, especially in GSCs. During the sexualization process, worms in which Dr-nanos was knocked down by RNAi exhibited decreased numbers of oogonia in the ovaries and failed to develop testes, whereas the somatic sexual organs were not affected. We conclude that Dr-nanos is essential for the development of germ cells in the ovaries and testes and may have a function in the early stages of germ cell specification, but not in the development of somatic sexual organs.     

Signification of the sexualizing substance produced by the sexualized planarians

Asexual worms of an exclusively fissiparous strain (the OH strain) of the planarian Dugesia ryukyuensis keep developing hermaphroditic reproductive organs and eventually undergo sexual reproduction instead of asexual reproduction, namely fission, if they are fed with sexually mature worms of an exclusively oviparous planarian, Bdellocephala brunnea, suggesting that the sexually mature worms has a sexualizing substance(s). The fully sexualized worms no longer need the feeding on sexual worms to maintain the sexuality. Here, we demonstrate that the sexualized worms produce enough of their own sexualizing substance similar to that contained in B. brunnea. In case of surgical ablation of the sexualized worms, the fragments with sexual organs regenerate to become sexual, while those without sexual organs, namely head fragments, regenerate to return to the asexual state. The asexual regenerants from the sexualized worms are also fully sexualized by being fed with B. brunnea. Additionally, it was reported that head region in sexually mature worms lacks the putative sexualizing substance necessary for complete sexualization (Sakurai, 1981). These results suggest that the fragments without sexual organ lack enough of an amount of the putative sexualizing substance and the sexuality is maintained by the sexualizing substance contained in the sexualized worms.     

Characterization of novel genes expressed specifically in the sexual organs of the planarian Dugesia ryukyuensis

The planarian Dugesia ryukyuensis reproduces both asexually (fissiparous) and sexually (oviparous) and can switch from the asexual mode to the sexual mode. By feeding with mature Bdellocephala brunnea oviparous worms, the fissiparous worms, which do not possess sexual organs, can be converted to fully sexualized worms in a process termed sexualization. As sexualization proceeds, the sexual organs are formed uniformly and five stages (stages 15) of the process have been identified histologically. In order to clarify the sexualization process, we attempted to isolate the genes expressed specifically at stage 5 by the differential display method. We isolated five genes expressed in the testis and two genes expressed in the yolk gland, which is an organ specific to sexualized worms. By BLAST search, one of the testis-specific genes was coded as testis-specific alpha-tubulin and two yolk gland-specific genes are similar to ribose-phosphate pyrophosphokinase I and F-box/SPRY-domain protein 1. Drs1, Drs2 and Drs3 were expressed in spermatocytes and spermatids from the early stage of spermatogenesis and Drs4 and Drs5 were expressed in spermatogonia, spermatocytes and spermatids. These genes are useful markers for elucidating the sexualization process.     

Seasonal changes in the sexualization of the planarian Dugesia ryukyuensis

Asexual individuals in a fissiparous clone of the planarian Dugesia ryukyuensis develop hermaphroditic sexual organs and eventually undergo sexual reproduction instead of asexual reproduction if they are fed with the adults of Bdellocephala brunnea, an oviparous planaria. The experimental sexualization means that the adults of B. brunnea contain a putative sexualizing substance(s), which is the first candidate for the chemical(s) responsible for switching from asexual to sexual reproduction in metazoans. In the present study, the feeding experiment over two consecutive years revealed that the experimental sexualization has seasonal changes. In summer, the asexual individuals were not fully sexualized, though they developed a pair of ovaries. The developing ovaries degenerate if the feeding is stopped. On the contrary, in winter, they developed all the sexual organs. The sexual organs keep developing even if the feeding is stopped after a certain critical point named the point-of-no-return. It was demonstrated that the extreme difference of the sexualization was attributed to the seasonal change of the quality and/or quantity of the sexualizing substance contained in B. brunnea, as well as the minor change of the susceptibility to the sexualizing substance in the asexual individuals. On the other hand, the histological research of B. brunnea revealed that the degree of the maturation of the sexual organs varied extremely through a year. Taking these results into account, we suggest that the production of the sexualizing substance has no direct relation to any particular mature sexual organs.     

Identification and development of polymorphic EST-SSR markers by sequence alignment in pepper, Capsicum annuum (Solanaceae)

? Premise of the study: The redundancies in expressed sequence tags (ESTs) in the National Center for Biotechnology Information sequence database were used to identify and develop polymorphic simple sequence repeat (SSR) markers for pepper (Capsicum annuum). ? Methods and Results: Sixty-eight polymorphic SSR loci were identified in the contigs (containing redundant ESTs) generated by assembling 118060 pepper ESTs from the public sequence database. Thirty-three SSR markers exhibited polymorphism among 31 pepper varieties, with alleles per SSR marker ranging from two to six. The mean observed and expected heterozygosity were 0.28 and 0.39, respectively. There were 18 SSR markers with a motif repeat number of less than five, accounting for 55% of the total. ? Conclusions: We demonstrated the value of mining the redundant sequences in public sequence databases for the development of polymorphic SSR markers, which can be used for marker-assisted breeding in pepper.     

银杏EST序列中微卫星的分布特征

本文利用从NCBI下载的21 590条银杏EST序列,分析了银杏(表达序列标签微卫星)EST-SSR在银杏EST序列的分布和比较了在不同长度EST序列中的SSR特性.在剔除冗余和低质量序列后,得到总长为5 708.385 kb的无冗余EST序列7 961条,发现了405个EST序列(5.09%)含有475个SSR,长度400-1000 bp的EST序列含SSR位点数为445个,占SSR总数的93.68%.二核苷酸和三核苷酸基元类型是银杏EST-SSR的主要类型,分别占SSR总数的73.89%和24.00%,最常见的SSR基元是:(AT)_n、(AG)_n、(AC)_n、(AAG)_n和(AAT)_n.通过对银杏EST序列中SSR位点信息的发掘分析,为有针对性地设计EST-SSR引物,开发银杏EST-SSR分子标记奠定基础.     

Switching from asexual to sexual reproduction in the planarian Dugesia ryukyuensis: change of the fissiparous capacity along with the sexualizing process

Asexual worms of fissiparous strain of the planarian Dugesia ryukyuensis switch from asexual to sexual reproduction, if they are fed with sexually mature worms of Bdellocephala brunnea. This suggests that the sexually mature worms have a sexualizing substance(s) that induces the sexuality in the asexual worms. Here, we found by analysis of the sexualization that the cessation of the fission, namely their asexual reproduction, occurs immediately after the acquisition of sexuality. This result suggests that the downstream mechanisms induced by the putative sexualizing substance in B. brunnea become responsible for the cessation of fission. We also found that the decapitation triggers fission in the worms even after the acquisition of sexuality if they are not sexually mature, while the fully sexualized worms never fission even though they are decapitated. This result suggests that the cessation of fission takes place via at least two steps: (1) the mechanisms associated with the cephalic system; (2) other mechanisms independent of cephalic control.     

Transcriptional pattern of a novel gene,expressed specifically after the point-of-no-return during sexualization,in planaria

We have investigated sexualization of asexual worms in the planarian Dugesia ryukyuensis. During sexualization there is a point from which an animal cannot return to the asexual state (point-of-no-return). To isolate the genes related to the point-of-no-return, we performed differential screening and isolated one novel gene that was expressed specifically in yolk glands of the worms after the point-of-no-return and named it Dryg. It encoded 655 amino acids with a predicted molecular mass of 79 kDa. We performed a series of experiments using Dryg as a molecular marker in the yolk gland. At first, we monitored how the yolk gland was formed during sexualization. The expression in sexualizing worms at stage 3 is limited to a single type of cell that has characteristics of neoblasts, the totipotent somatic cells; however, the expression is observed in the yolk gland in sexualized worms. Furthermore, we monitored yolk glands for expression during regeneration. The original yolk glands seem to disappear after ablation, then new yolk glands appeared along the ventral nerve cords. Because this expression pattern looks like that of sexualizing worms at stage 3, we speculate that yolk gland cells may differentiate from neoblasts during regeneration as observed during sexualization.     

Identification and characterization of a new family of C-type lectin-like genes from planaria Girardia tigrina

A novel family of C-type lectin-like genes, denoted multidomain free lectin (MDFL), was identified in the freshwater planaria Girardia (Dugesia) tigrina. We cloned several genes that encode proteins comprising a signal peptide and a number of consecutive C-type lectin-like domains (CTLDs) interconnected by short linker stretches. Analyses of genomic organization, CTLD amino acid sequences, and the overall architecture of these proteins indicate that planarian proteins are a separate family of C-type lectin-like proteins. These genes are expressed in specifically differentiated gland cells of planaria and the corresponding proteins are excreted as components of the planarian body surface mucus.     

Generation of 919 expressed sequence tags from immature flower buds and gene expression analysis using expressed sequence tags in the model plant Lotus japonicus

Lotus japonicus has received increased attention as a potential model legume plant. In order to study gene expression in reproductive organs and to identify genes that play a crucial function in sexual reproduction, we constructed a cDNA library from immature flower buds containing anthers at the stage of developing tapetum cells in L. japonicus, and characterized 919 expressed sequence tags (ESTs) randomly selected from a cDNA library of the immature flower buds. The 919 ESTs analyzed were clustered into 821 non-redundant EST groups. As a result of a database search, 436 groups (53%) out of the 821 groups showed sequence similarity to genes registered in the public database. Out of these 436 groups, 109 groups showed similarity to genes encoding hypothetical proteins whose function had not yet been estimated. Three hundred eighty five groups (47%) showed no significant homology to known sequences and were classified as novel sequences. A comparison of 821 non-redundant EST sequences and EST sequences derived from the whole plant L. japonicus revealed that 474 EST sequences derived from immature flower buds were not found in the EST sequences of the whole plant. In order to confirm the expression pattern of potential reproductive-organ specific EST clones, nine clones, which were not matched to ESTs derived from the whole plant, were selected, and RT-PCR analysis was performed on these clones. As a result of RT-PCR, we found two novel anther specific clones. One clone was homologous to a gene encoding human cleft lip and palate associated transmembrane protein (CLPTM1) like protein, and the other clone did not show a significant similarity to any genes deposited in the public database. These results indicate that ESTs analyzed here represent a valuable resource for finding reproductive-organ specific genes in Lotus japonicus.     

A large scale analysis of cDNA in Arabidopsis thaliana: generation of 12,028 non-redundant expressed sequence tags from normalized and size-selected cDNA libraries.

For comprehensive analysis of genes expressed in the model dicotyledonous plant, Arabidopsis thaliana, expressed sequence tags (ESTs) were accumulated. Normalized and size-selected cDNA libraries were constructed from aboveground organs, flower buds, roots, green siliques and liquid-cultured seedlings, respectively, and a total of 14,026 5'-end ESTs and 39,207 3'-end ESTs were obtained. The 3'-end ESTs could be clustered into 12,028 non-redundant groups. Similarity search of the non-redundant ESTs against the public non-redundant protein database indicated that 4816 groups show similarity to genes of known function, 1864 to hypothetical genes, and the remaining 5348 are novel sequences. Gene coverage by the non-redundant ESTs was analyzed using the annotated genomic sequences of approximately 10 Mb on chromosomes 3 and 5. A total of 923 regions were hit by at least one EST, among which only 499 regions were hit by the ESTs deposited in the public database. The result indicates that the EST source generated in this project complements the EST data in the public database and facilitates new gene discovery.     

CHARACTERIZATION OF EXPRESSED SEQUENCE TAGS GENERATED FROM SKIN CDNA CLONES OF Equus caballus BY SINGLE PASS SEQUENCING

A cDNA library was built using RNA extracted from the skin tissue of an adult horse. The library was primed with oligo (dT) and sequences were directionally inserted in order to produce an expression library. The library has 5.8X 10⁵ plaque forming units with 99.6% recombinant phage. The average insert size is 1.3 Kbp. Three hundred and thirteen expressed sequence tags (ESTs) were generated from sequencing of the 5 prime end of randomly selected skin cDNA clones. The ESTs were sequenced on an ABI 377 using Big-Dye chemistry. A similarity search was performed on each EST using the NCBI non-redundant protein database and 206 ESTs were putatively identified. Twenty six percent of the identified ESTs were redundant. The ESTs were categorized by function. The most frequently identified functional class was translational proteins.     

Characterization of expressed sequence tags generated from skin cDNA clones of Equus caballus by single pass sequencing

A cDNA library was built using RNA extracted from the skin tissue of an adult horse. The library was primed with oligo (dT) and sequences were directionally inserted in order to produce an expression library. The library has 5.8X 10(5) plaque forming units with 99.6% recombinant phage. The average insert size is 1.3 Kbp. Three hundred and thirteen expressed sequence tags (ESTs) were generated from sequencing of the 5 prime end of randomly selected skin cDNA clones. The ESTs were sequenced on an ABI 377 using Big-Dye chemistry. A similarity search was performed on each EST using the NCBI non-redundant protein database and 206 ESTs were putatively identified. Twenty six percent of the identified ESTs were redundant. The ESTs were categorized by function. The most frequently identified functional class was translational proteins.     

甜瓜EST序列中微卫星的分布特征

GenBank中35547条甜瓜EST经去冗余处理后,得到总长度为250.3Mb的无冗余EST34438条。这些序列中有2813个微卫星简单重复序列（Simple sequence repeat,SSR）,分布于2107条EST中,出现频率为8.16%,平均分布距离为8.90kb。三核苷酸重复是主导重复类型,占SSR总数的47.14%;其次是二核苷酸和单核苷酸重复,分别占SSR总数的20.72%和16.99%。AAG/TTC是优势重复基元,占微卫星总数的29.26%,AG/CT和A/T分别占14.61%和16.25%。在所有的SSR中,重复次数为4～10次的占70.32%,长度为12～20bp的占51.12%。并对这些SSR的多态性潜能进行了评价。     

陆地棉EST长度多态性与其SSR分布特征相关性分析

目的:分析陆地棉EST长度多态性与其SSR分布特征的相关性。方法:从NCBI公共数据库下载陆地棉EST序列,应用SSRIT搜索SSR,分析20 000条无冗余的EST序列。结果:在剔除低质量和冗余的序列后,得到全长为7 363.878kb的无冗余EST序列7 322条,其中含有SSR位点的EST序列数520条,占被分析EST比例的2.60%。长度在400bp以下的EST序列含SSR的比例为1.46%;长度在400bp以上的EST序列含SSR的比例为8.94%。在1～6bp的重复基元中,二核苷酸重复基元的SSR重复频率最高,占总数的63.46%,其次是三核苷酸,占总数的34.04%。二核苷酸类型(AG)n、(AT)n和三核苷酸类型(AAG)n、(ACC)n、(ACT)n、(AAT)n是SSR的主要重复基元。结论:棉花EST-SSR可用于棉花分子标记,为有针对性设计陆地棉EST-SSR引物奠定基础。