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Sample records for hybridization ssh cdna

  1. cDNA amplification by SMART-PCR and suppression subtractive hybridization (SSH)-PCR.

    Science.gov (United States)

    Hillmann, Andrew; Dunne, Eimear; Kenny, Dermot

    2009-01-01

    The comparison of two RNA populations that differ from the effects of a single-independent variable, such as a drug treatment or a specific genetic defect, can identify differences in the abundance of specific transcripts that vary in a population-dependent manner. There are a variety of methods for identifying differentially expressed genes, including microarray, SAGE, qRT-PCR, and DDGE. This protocol describes a potentially less sensitive yet relatively easy and cost-effective alternative that does not require prior knowledge of the transcriptomes under investigation and is particularly applicable when minimal levels of starting material, RNA, are available. RNA input can often be a limiting factor when analyzing RNA from, for example, rigorously purified blood cells. This protocol describes the use of SMART-PCR to amplify cDNA from sub-microgram levels of RNA. The amplified cDNA populations under comparison are then subjected to suppression subtractive hybridization (SSH-PCR), a technique that couples subtractive hybridization with suppression PCR to selectively amplify fragments of differentially expressed genes. The final products are cDNA populations enriched for significantly over-represented transcripts in either of the two input RNA preparations. These cDNA populations may then be cloned to make subtracted cDNA libraries and/or used as probes to screen subtracted cDNA, global cDNA, or genomic DNA libraries.

  2. Gene expression profiling of osteoclast differentiation by combined suppression subtractive hybridization (SSH) and cDNA microarray analysis.

    Science.gov (United States)

    Rho, Jaerang; Altmann, Curtis R; Socci, Nicholas D; Merkov, Lubomir; Kim, Nacksung; So, Hongseob; Lee, Okbok; Takami, Masamichi; Brivanlou, Ali H; Choi, Yongwon

    2002-08-01

    Bone homeostasis is maintained by the balanced action of bone-forming osteoblasts and bone-resorbing osteoclasts. Multinucleated, mature osteoclasts develop from hematopoietic stem cells via the monocyte-macrophage lineage, which also give rise to macrophages and dendritic cells. Despite their distinct physiologic roles in bone and the immune system, these cell types share many molecular and biochemical features. To provide insights into how osteoclasts differentiate and function to control bone metabolism, we employed a systematic approach to profile patterns of osteoclast-specific gene expression by combining suppression subtractive hybridization (SSH) and cDNA microarray analysis. Here we examined how gene expression profiles of mature osteoclast differ from macrophage or dendritic cells, how gene expression profiles change during osteoclast differentiation, and how Mitf, a transcription factor critical for osteoclast maturation, affects the gene expression profile. This approach revealed a set of genes coordinately regulated for osteoclast function, some of which have previously been implicated in several bone diseases in humans.

  3. [Construction of suppression subtractive hybridization (SSH) library of copepod Pseudodiaptomous annandalei and its ferritin cDNA cloning and differential expression under nickel stress].

    Science.gov (United States)

    Jiang, Jie-Lan; Wang, Gui-Zhong; Wu, Li-Sheng; Li, Shao-Jing

    2012-07-01

    To study the molecular response mechanisms of copepod to nickel stress, a suppression subtractive hybridization (SSH) cDNA library of Pseudodiaptomous annandalei under nickel stress was constructed by using SSH technique, and a total of 140 clones were randomly picked from the growing colonies and identified by PCR. The recombinant rate of the library was 98.6%, and the volume of the library was 1.12 x 10(6) cfu. After the recombinant plasmids were sequenced, a partial cDNA fragment of ferritin was recognized based on BLAST searches in NCBI, with a size of 859 bp and continuously encoding 170 amino acid residues. The semi-quantitative PCR results showed that the ferritin cDNA under 24 h nickel stress was distinctly up-regulated. The successful construction of the SSH library and the obtaining of ferritin cDNA fragment would supply basis for the further study of the molecular response mechanisms of copepod to nickel stress.

  4. Construction and analysis of SSH cDNA library of human vascular endothelial cells related to gastrocarcinoma

    OpenAIRE

    2003-01-01

    AIM: To construct subtracted cDNA libraries of human vascular endothelial cells (VECs) related to gastrocarcinoma using suppression substractive hybridization (SSH) and to analyze cDNA libraries of gastrocarcinoma and VECs in Cancer Gene Anatomy Project (CGAP) database.

  5. Combining SSH and cDNA microarrays for rapid identification of differentially expressed genes.

    Science.gov (United States)

    Yang, G P; Ross, D T; Kuang, W W; Brown, P O; Weigel, R J

    1999-03-15

    Comparing patterns of gene expression in cell lines and tissues has important applications in a variety of biological systems. In this study we have examined whether the emerging technology of cDNA microarrays will allow a high throughput analysis of expression of cDNA clones generated by suppression subtractive hybridization (SSH). A set of cDNA clones including 332 SSH inserts amplified by PCR was arrayed using robotic printing. The cDNA arrays were hybridized with fluorescent labeled probes prepared from RNA from ER-positive (MCF7 and T47D) and ER-negative (MDA-MB-231 and HBL-100) breast cancer cell lines. Ten clones were identified that were over-expressed by at least a factor of five in the ER-positive cell lines. Northern blot analysis confirmed over-expression of these 10 cDNAs. Sequence analysis identified four of these clones as cytokeratin 19, GATA-3, CD24 and glutathione-S-transferase mu-3. Of the remaining six cDNA clones, four clones matched EST sequences from two different genes and two clones were novel sequences. Flow cytometry and immunofluorescence confirmed that CD24 protein was over-expressed in the ER-positive cell lines. We conclude that SSH and microarray technology can be successfully applied to identify differentially expressed genes. This approach allowed the identification of differentially expressed genes without the need to obtain previously cloned cDNAs.

  6. Construction and analysis of SSH cDNA library of human vascular endothelial cells related to gastrocarcinoma

    Institute of Scientific and Technical Information of China (English)

    Yong-Bo Liu; Zhao-Xia Wei; Li Li; Hang-Sheng Li; Hui Chen; Xiao-Wen Li

    2003-01-01

    AIM: To construct subtracted cDNA libraries of human vascular endothelial cells (VECs) related to gastrocarcinoma using suppression substractive hybridization (SSH) and to analyze cDNA libraries of gastrocarcinoma and VECs in Cancer Gene Anatomy Project (CGAP) database.METHODS: Human VECs related to gastric adenocarcinoma and corresponding normal tissue were separated by magnetic beads coupled with antibody CD31 (Dynabeads CD31). A few amount of total RNA were synthesized and amplified by SMARTTM PCR cDNA Synthesis Kit. Then, using SSH and T/A cloning techniques, cDNA fragments of differentially expressed genes in human VECs of gastric adenocarcinoma were inserted into JM109 bacteria. One hundred positive bacteria clones were randomly picked and identified by colony PCR method. To analyze cDNA libraries of gastrocarcinoma and VECs in CGAP database, the tools of Library Finder,cDNA xProfiler, Digital GENE Expression Displayer (DGED),and Digital Differential Display (DDD) were used.RESULTS: Forward and reverse subtraction cDNA libraries of human VECs related to gastrocarcinoma were constructed successfully with SSH and T/A cloning techniques. Analysis of CGAP database indicated that no appropriate library of VECs related to carcinoma was constructed.CONCLUSION: Construction of subtraction cDNA libraries of human VECs related to gastrocarcinoma was successful and necessary, which laid a foundation for screening and cloning new and specific genes of VECs related to gastrocardnoma.

  7. Suppression subtractive hybridization (SSH) for isolation and characterization of genes related to testicular development in the giant tiger shrimp Penaeus monodon.

    Science.gov (United States)

    Leelatanawit, Rungnapa; Klinbunga, Sirawut; Aoki, Takashi; Hirono, Ikuo; Valyasevi, Rudd; Menasveta, Piamsak

    2008-11-30

    Suppression subtractive hybridization (SSH) cDNA libraries of the giant tiger shrimp, Penaeus monodon, were constructed. In total, 178 and 187 clones from the forward and reverse SSH libraries, respectively, of P. monodon were unidirectionally sequenced. From these, 37.1% and 53.5% Expressed Sequence Tags (ESTs) significantly matched known genes (E-value 0.05).

  8. Suppression Subtractive Hybridization (SSH) and its modifications in microbiological research.

    Science.gov (United States)

    Huang, Xiaowei; Li, Yunxia; Niu, Qiuhong; Zhang, Keqin

    2007-09-01

    Suppression subtractive hybridization (SSH) is an effective approach to identify the genes that vary in expression levels during different biological processes. It is often used in higher eukaryotes to study the molecular regulation in complex pathogenic progress, such as tumorigenesis and other chronic multigene-associated diseases. Because microbes have relatively smaller genomes compared with eukaryotes, aside from the analysis at the mRNA level, SSH as well as its modifications have been further employed to isolate specific chromosomal locus, study genomic diversity related with exceptional bacterial secondary metabolisms or genes with special microbial function. This review introduces the SSH and its associated methods and focus on their applications to detect specific functional genes or DNA markers in microorganisms.

  9. [Development of new SSR markers from EST of SSH cDNA libraries on rose fragrance].

    Science.gov (United States)

    Yan, Hui-Jun; Zhang, Hao; Xie, Ji-Rong; Li, Shu-Fa; Jian, Hong-Ying; Qiu, Xian-Qin; Wang, Qi-Gang; Wang, Ji-Hua; Tang, Kai-Xue

    2009-09-01

    The new SSR markers of rose related fragrance were developed based on the SSH cDNA libraries of rose floral scent mutant. In this study, 10 EST-SSRs (2.6%) from 391 ESTs in the libraries were identified. Six EST-SSRs primers were designed to sequence flanking SSRs. The primer pairs designed were screened on the wild-type Jinyindao, which has flowers full of pleasant scent, and the mutant-type Wangriqinghuai without perceivable floral scent. Five primer pairs were amplified effectively in Jinyindao and Wangriqinghuai, and 3 were polymorphic between Jinyindao and Wangriqinghuai. Eighteen rose cultivars including fragrant roses and nonfragrant roses were identified by the five prime pairs. These results proved that EST-SSR markers are effective markers to identify the polymorphism of the rose.

  10. Differential gene expression profiling in aggressive bladder transitional cell carcinoma compared to the adjacent microscopically normal urothelium by microdissection-SMART cDNA PCR-SSH.

    Science.gov (United States)

    Wang, H T; Ma, F L; Ma, X B; Han, R F; Zhang, Y B; Chang, J W

    2006-01-01

    Identifying novel and known genes that are differentially expressed in aggressive bladder transitional cell carcinoma (BTCC) has important implications in understanding the biology of bladder tumorigenesis and developing new diagnostic and therapeutic agents. In this study we identified the differential gene expression profiles comparing tumor to the adjacent microscopically normal mucosa by manual microdissection on frozen sections. The RNAs extracted from microdissected tissues were amplified by SMART cDNA PCR technology to generate forward subtractive cDNA library by suppressive subtractive hybridization (SSH). We obtained 376 positive clones, one hundred clones of aggressive BTCC subtracted cDNA library were selected at random and inserts were reamplified by PCR. After differential screening by reverse dot blotting, 73 positive clones, that contend inserts putatively upregulated in aggressive BTCC, were further analysed by DNA sequencing, GenBank and EST database searching. Sequencing results showed that 66 clones stand for 23 known genes and 7 clones for three new EST (Genbank number: DN236875, DN236874 and DN236873). In conclusion, microdissection-SMART cDNA PCR-SSH allowed for an efficient way to identify aggressive BTCC-specific differential expressed genes that may potentially be involved in the carcinogenesis and/or progression of aggressive BTCC. These differentially expressed genes may be of potential utility as therapeutic and diagnostic targets for aggressive BTCC.

  11. Construction of cDNA subtractive library from pearl oyster ( Pinctada fucata Gould) with red color shell by SSH

    Science.gov (United States)

    Guan, Yunyan; Huang, Liangmin; He, Maoxian

    2011-05-01

    The molecular basis of color polymorphism in the shells of the pearl oyster Pinctada fucata is largely unknown. We developed a red-shelled family line and used suppression subtractive hybridization (SSH) to screen for differentially expressed genes in red- and non-red-shelled pearl oysters. We constructed forward and reverse cDNA subtractive libraries consisting of 2 506 and 797 clones, respectively. Among 343 randomly selected clones in the forward library, 304 sequences were identified in GenBank using BLASTx and BLASTn. Of the 304 sequences, 13 showed no similarity to known sequences and 291 were matched with known genes of the pearl oyster, including shematrin-1, shematrin-2, shematrin-6, shematrin-7, nacrein, nacrein-like protein, aspein for shell matrix protein, glycine-rich protein, mantle gene 5, 28S, EST00031, EST00036, 16S, and COI. In the reverse library, 7 clones were sequenced and analyzed by BLAST. Two sequences shared similarity with EST00036 from the P. fucata subtraction cDNA library, four with the P. fucata mitochondrial gene for 16S rRNA and 1 with P. fucata shematrin-2. We evaluated the expression of 12 genes from the forward library using RT PCR. Two sequences matched with 16S and COI so were considered to be false positives. The remaining 10 sequences were differentially expression in the red-shelled pearl oysters. Our results suggest that differential expression of these genes may be related to color variation in the red-shelled family line of the pearl oyster.

  12. Construction of cDNA subtractive library from pearl oyster (Pinctada fucata Gould) with red color shell by SSH

    Institute of Scientific and Technical Information of China (English)

    GUAN Yunyan; HUANG Liangmin; HE Maoxian

    2011-01-01

    The molecular basis of color polymorphism in the shells of the pearl oyster Pinctada fucata is largely unknown. We developed a red-shelled family line and used suppression subtractive hybridization (SSH) to screen for differentially expressed genes in red- and non-red-shelled pearl oysters. We constructed forward and reverse cDNA subtractive libraries consisting of 2 506 and 797 clones, respectively.Among 343 randomly selected clones in the forward library, 304 sequences were identified in GenBank using BLASTx and BLASTn. Of the 304 sequences, 13 showed no similarity to known sequences and 291 were matched with known genes of the pearl oyster, including shematrin-1, shematrin-2, shematrin-6,shematrin-7, nacrein, nacrein-like protein, aspein for shell matrix protein, glycine-rich protein, mantle gene 5, 28S, EST00031, EST00036, 16S, and COI. In the reverse library, 7 clones were sequenced and analyzed by BLAST. Two sequences shared similarity with EST00036 from the P. fucata subtraction cDNA library, four with the P. fucata mitochondrial gene for 16S rRNA and 1 with P. fucata shematrin-2. We evaluated the expression of 12 genes from the forward library using RT PCR. Two sequences matched with 16S and COI so were considered to be false positives. The remaining 10 sequences were differentially expression in the red-shelled pearl oysters. Our results suggest that differential expression of these genes may be related to color variation in the red-shelled family line of the pearl oyster.

  13. Construction of a metastasis-associated gene subtracted cDNA library of human colorectal carcinoma by suppression subtraction hybridization

    Institute of Scientific and Technical Information of China (English)

    Li Liang; Yan-Qing Ding; Xin Li; Guang-Zhi Yang; Jun Xiao; Li-Chun Lu; Jin-Hua Zhang

    2004-01-01

    AIM: To construct a differentially-expressed gene subtracted cDNA library from two colorectal carcinoma (CRC) cell lines with different metastatic phenotypes by suppression subtractive hybridization.METHODS: Two cell lines of human CRC from the same patient were used. SW620 cell line showing highly metastatic potential was regarded as tester in the forward subtractive hybridization, while SW480 cell line with lowly metastatic potential was treated as tester in the reverse hybridization. Suppression subtractive hybridization (SSH)was employed to obtain cDNA fragments of differentially expressed genes for the metastasis of CRC. These fragments were ligated with T vectors, screened through the bluewhite screening system to establish cDNA library.RESULTS: After the blue-white screening, 235 white clones were picked out from the positive-going hybridization and 232 from the reverse. PCR results showed that 200-700 bp inserts were seen in 98% and 91% clones from the forward and reverse hybridizations, respectively.CONCLUSIONS: A subtractive cDNA library of differentially expressed genes specific for metastasis of CRC can be constructed with SSH and T/A cloning techniques.

  14. Suppression subtractive hybridization: a method for generating differentially regulated or tissue-specific cDNA probes and libraries.

    OpenAIRE

    1996-01-01

    A new and highly effective method, termed suppression subtractive hybridization (SSH), has been developed for the generation of subtracted cDNA libraries. It is based primarily on a recently described technique called suppression PCR and combines normalization and subtraction in a single procedure. The normalization step equalizes the abundance of cDNAs within the target population and the subtraction step excludes the common sequences between the target and driver populations. In a model sys...

  15. Identification of genes involved in the response of haemocytes of Penaeus japonicus by suppression subtractive hybridization (SSH) following microbial challenge.

    Science.gov (United States)

    He, Nanhai; Liu, Haipeng; Xu, Xun

    2004-08-01

    Penaeus japonicus were injected with a heat-killed microorganism suspension and 291 randomly selected cDNA fragments generated by suppression subtractive hybridization (SSH) were sequenced. A total of 71 cDNA clones corresponding to 25 genes were found to have enhanced expression, of which eight are found for the first time in shrimp. The most abundant gene in the subtractive library was Kunitz-type protease inhibitor, clearly indicating this protease inhibitor in the response. A number of genes encoding signaling molecules, such as Ras-related nuclear protein (Ran), growth factor receptor bound protein (Grb), TGF-beta receptor interacting protein, integrin binding protein and interferon receptor bound protein were found for the first time in the shrimp, and they may be involved in the regulation of the host defense against the injected microbes. Furthermore, cDNAs of chaperonin, proteasome, antioxidant as well as genes associated with actin reorganization, which may be necessary for phagocytosis and encapsulation, were also expressed at a higher level after the challenge. These results may facilitate the understanding of shrimp immune responses.

  16. Identification of some unknown transcripts from SSH cDNA library of buffalo follicular oocytes.

    Science.gov (United States)

    Rajput, S K; Kumar, P; Roy, B; Verma, A; Pandey, H P; Singh, D; De, S; Datta, T K

    2013-03-01

    A buffalo oocyte-specific subtracted cDNA library was constructed to identify exclusively or preferentially oocyte-expressed genes. The library represented an enriched population of transcripts obtained from oocytes of diverse ovarian follicular origin and at different stages of in vitro maturation. A total of 1173 high-quality sequences of oocyte-specific genes were clustered into 645 unique sequences, out of which 65.76% were represented as singlets and 34.26% as contig expressed sequence tags (ESTs; clusters). Analysis of sequences revealed that 498 of these sequences were identified as a known sequence in mammalian species including buffalo, 103 as uncharacterized ESTs and 44 unknown sequences including 1 novel EST, so far not reported in any species. Gene ontology annotation classified these sequences into functional categories of cellular events and biological processes associated with oocyte competence. Expression status of the isolated unknown ESTs confirmed that many of these are expressed in oocytes exclusively and in others preferentially, some in excess of 80-fold greater in comparison with a variety of somatic tissues. The isolated novel EST was detected to be expressed exclusively in oocytes and testicular cells only. To our knowledge, this is the first report giving a detailed transcriptome account of oocyte-expressed genes in buffalo. This study will provide important information on the physiological control of oocyte development, as well as many questions yet to be addressed on the reproductive process of buffalo.

  17. The mining of pearl formation genes in pearl oyster Pinctada fucata by cDNA suppression subtractive hybridization.

    Science.gov (United States)

    Wang, Ning; Kinoshita, Shigeharu; Nomura, Naoko; Riho, Chihiro; Maeyama, Kaoru; Nagai, Kiyohito; Watabe, Shugo

    2012-04-01

    Recent researches revealed the regional preference of biomineralization gene transcription in the pearl oyster Pinctada fucata: it transcribed mainly the genes responsible for nacre secretion in mantle pallial, whereas the ones regulating calcite shells expressed in mantle edge. This study took use of this character and constructed the forward and reverse suppression subtractive hybridization (SSH) cDNA libraries. A total of 669 cDNA clones were sequenced and 360 expressed sequence tags (ESTs) greater than 100 bp were generated. Functional annotation associated 95 ESTs with specific functions, and 79 among them were identified from P. fucata at the first time. In the forward SSH cDNA library, it recognized mass amount of nacre protein genes, biomineralization genes dominantly expressed in the mantle pallial, calcium-ion-binding genes, and other biomineralization-related genes important for pearl formation. Real-time PCR showed that all the examined genes were distributed in oyster mantle tissues with a consistence to the SSH design. The detection of their RNA transcripts in pearl sac confirmed that the identified genes were certainly involved in pearl formation. Therefore, the data from this work will initiate a new round of pearl formation gene study and shed new insights into molluscan biomineralization.

  18. Suppression subtraction hybridization (SSH) and macroarray techniques reveal differential gene expression profiles in brain of sea bream infected with nodavirus.

    Science.gov (United States)

    Dios, S; Poisa-Beiro, L; Figueras, A; Novoa, B

    2007-03-01

    Despite of the impact that viruses have on aquatic organisms, relatively little is known on how fish fight against these infections. In this work, the brain gene expression pattern of sea bream (Sparus aurata) in response to nodavirus infection was investigated. We used the suppression subtractive hybridization (SSH) method to generate a subtracted cDNA library enriched with gene transcripts differentially expressed after 1 day post-infection. Some of the ESTs from the infected tissues fell in gene categories related to stress and immune responses. For the reverse library (ESTs expressed in controls compared with infected tissues) the most abundant transcripts were of ribosomal and mitochondrial nature. Several ESTs potentially induced by virus exposure were selected for in vivo expression studies. We observed a clear difference in expression between infected and control samples for two candidate genes, ubiquitin conjugating enzyme 7 interacting protein, which seems to play an important role in apoptosis and the interferon induced protein with helicase C domain 1 (mda-5) that contributes to apoptosis and regulates the type I IFN production, a key molecule of the antiviral innate response in most organisms.

  19. [Construction of subtractive cDNA libraries of the sporogony stage of Eimeria tenella by suppression subtractive hybridization].

    Science.gov (United States)

    Han, Hong-Yu; Lin, Jiao-Jiao; Zhao, Qi-Ping; Dong, Hui; Jiang, Lian-Lian; Wang, Xin; Han, Jing-Fang; Huang, Bing

    2007-11-01

    In order to clone and identify differentially expressed genes in the sporogony stage of Eimeria tenella, the cDNAs from unsporulated oocysts and sporulated oocysts of E. tenella were used as driver, respectively, the cDNAs from sporozoites of E. tenella was used tester, Two subtractive cDNA libraries of sporozoites were constructed by using the technique of suppression subtractive hybridization (SSH). the cDNAs from unsporulated oocysts was used driver, the cDNAs from sporulated ooceysts was used tester, one subtractive cDNA library of sporulated oocysts was constructed. PCR amplification revealed that the two subtractive cDNA libraries of sporozoites and one subtractive cDNA library of sporulated oocysts contained approximated 96%, 96% and 98% recombinant clones, respectively. Fifty positive clones were sequenced and analyzed in GenBank with Blast search from three subtractive cDNA libraries, respectively, thirteen unique sequences were found from the subtractive cDNA library of sporulated oocysts, eight ESTs shared significant identity with previously described. A total of forty unique sequences were obtained from the two subtractive cDNA libraries, nine ESTs shared significant identity with previously described, the other sequences represent novel genes of E. tenella with no significant homology to the proteins in Genbank. These results have provided the foundation for cloning new genes of E. tenella and further studying new approaches to control coccidiosis.

  20. Construction of Hypericin Gland-Specific cDNA Library via Suppression Subtractive Hybridization.

    Science.gov (United States)

    Singh, Rupesh Kumar; Hou, Weina; Franklin, Gregory

    2016-01-01

    Hypericin, an important determinant of the pharmacological properties of the genus Hypericum, is considered as a major molecule for drug development. However, biosynthesis and accumulation of hypericin is not well understood. Identification of genes differentially expressed in tissues with and without hypericin accumulation is a useful strategy to elucidate the mechanisms underlying the development of the dark glands and hypericin biosynthesis. Suppression Subtractive Hybridization (SSH) is a unique method for PCR-based amplification of specific cDNA fragments that differ between a control (driver) and experimental (tester) transcriptome. This technique relies on the removal of dsDNA formed by hybridization between a control and test sample, thus eliminating cDNAs of similar abundance, and retaining differentially expressed or variable in sequence cDNAs. In our laboratory we applied this method to identify the genes involved in the development of dark glands and accumulation of hypericin in Hypericum perforatum. Here we describe the complete procedure for the construction of hypericin gland-specific subtracted cDNA library.

  1. [Gene expression profiling by suppression subtractive hybridization (SSH): a example for its application to the study of lymphomas].

    Science.gov (United States)

    Villalva, C; Trempat, P; Zenou, R C; Delsol, G; Brousset, P

    2001-03-01

    Suppression subtractive hybridization (SSH) was used to isolate genes that were differentially expressed in anaplastic lymphoma kinase (ALK)-positive and ALK-negative anaplastic large cell lymphoma. In addition, this approach was applied to Hodgkin's disease cases with different clinical outcomes. SSH combines a normalization step that equalizes the abundance of cDNAs within the sequences to be tested and a subtraction step that excludes the common sequences between the target and the control. In a model system, the SSH technique enriches for rare sequences up to 5,000-fold in one round. We have isolated several genes whose expression varied significantly with regard to the tumour subtypes. There were different genes with known or unknown functions. We aim to compare the results of the SSH approach with those obtained with high density filters. In a near future, we would like to design DNA chips specific of each pathology that could be used for clinical purposes (evaluation of prognosis and therapeutic response).

  2. Efficacy of SSH PCR in isolating differentially expressed genes

    Directory of Open Access Journals (Sweden)

    Cai Li

    2002-05-01

    Full Text Available Abstract Background Suppression Subtractive Hybridization PCR (SSH PCR is a sophisticated cDNA subtraction method to enrich and isolate differentially expressed genes. Despite its popularity, the method has not been thoroughly studied for its practical efficacy and potential limitations. Results To determine the factors that influence the efficacy of SSH PCR, a theoretical model, under the assumption that cDNA hybridization follows the ideal second kinetic order, is proposed. The theoretical model suggests that the critical factor influencing the efficacy of SSH PCR is the concentration ratio (R of a target gene between two cDNA preparations. It preferentially enriches "all or nothing" differentially expressed genes, of which R is infinite, and strongly favors the genes with large R. The theoretical predictions were validated by our experiments. In addition, the experiments revealed some practical limitations that are not obvious from the theoretical model. For effective enrichment of differentially expressed genes, it requires fractional concentration of a target gene to be more than 0.01% and concentration ratio to be more than 5 folds between two cDNA preparations. Conclusion Our research demonstrated theoretical and practical limitations of SSH PCR, which could be useful for its experimental design and interpretation.

  3. Isolation and characterization of genes functionally involved in ovarian development of the giant tiger shrimp Penaeus monodon by suppression subtractive hybridization (SSH).

    Science.gov (United States)

    Preechaphol, Rachanimuk; Klinbunga, Sirawut; Khamnamtong, Bavornlak; Menasveta, Piamsak

    2010-10-01

    Suppression subtractive hybridization (SSH) libraries between cDNA in stages I (previtellogenic) and III (cortical rod) ovaries of the giant tiger shrimp (Penaeus monodon) were established. In all, 452 ESTs were unidirectionally sequenced. Sequence assembly generated 28 contigs and 201 singletons, 109 of which (48.0%) corresponding to known sequences previously deposited in GenBank. Several reproduction-related transcripts were identified. The full-length cDNA of anaphase promoting complex subunit 11 (PmAPC11; 600 bp with an ORF of 255 bp corresponding to a polypeptide of 84 amino acids) and selenoprotein Mprecursor (PmSePM; 904 bp with an ORF of 396 bp corresponding to a polypeptide of 131 amino acids) were characterized and reported for the first time in penaeid shrimp. Semiquantitative RT-PCR revealed that the expression levels of PmSePM and keratinocyte-associated protein 2 significantly diminished throughout ovarian development, whereas Ser/Thrcheckpoint kinase 1 (Chk1), DNA replication licensing factor mcm2 and egalitarian were down-regulated in mature ovaries of wild P. monodon (p < 0.05). Accordingly, the expression profiles of PmSePM and keratinocyte-associated protein 2 could be used as biomarkers for evaluating the degree of reproductive maturation in domesticated P. monodon.

  4. Isolation and characterization of genes functionally involved in ovarian development of the giant tiger shrimp Penaeus monodon by suppression subtractive hybridization (SSH

    Directory of Open Access Journals (Sweden)

    Rachanimuk Preechaphol

    2010-01-01

    Full Text Available Suppression subtractive hybridization (SSH libraries between cDNA in stages I (previtellogenic and III (cortical rod ovaries of the giant tiger shrimp (Penaeus monodon were established. In all, 452 ESTs were unidirectionally sequenced. Sequence assembly generated 28 contigs and 201 singletons, 109 of which (48.0% corresponding to known sequences previously deposited in GenBank. Several reproduction-related transcripts were identified. The full-length cDNA of anaphase promoting complex subunit 11 (PmAPC11; 600 bp with an ORF of 255 bp corresponding to a polypeptide of 84 amino acids and selenoprotein M precursor (PmSePM; 904 bp with an ORF of 396 bp corresponding to a polypeptide of 131 amino acids were characterized and reported for the first time in penaeid shrimp. Semiquantitative RT-PCR revealed that the expression levels of PmSePM and keratinocyte-associated protein 2 significantly diminished throughout ovarian development, whereas Ser/Thr checkpoint kinase 1 (Chk1, DNA replication licensing factor mcm2 and egalitarian were down-regulated in mature ovaries of wild P. monodon (p < 0.05. Accordingly, the expression profiles of PmSePM and keratinocyte-associated protein 2 could be used as biomarkers for evaluating the degree of reproductive maturation in domesticated P. monodon.

  5. A Mathematical Model for Suppression Subtractive Hybridization

    OpenAIRE

    2002-01-01

    Suppression subtractive hybridization (SSH) is frequently used to unearth differentially expressed genes on a whole-genome scale. Its versatility is based on combining cDNA library subtraction and normalization, which allows the isolation of sequences of varying degrees of abundance and differential expression. SSH is a complex process with many adjustable parameters that affect the outcome of gene isolation.We present a mathematical model of SSH based on DNA hybridization kinetics for assess...

  6. Construction and analysis of an SSH cDNA library of early heat-induced genes of Vigna aconitifolia variety RMO-40.

    Science.gov (United States)

    Rampuria, Sakshi; Joshi, Uma; Palit, Paramita; Deokar, Amit A; Meghwal, Raju R; Mohapatra, T; Srinivasan, R; Bhatt, K V; Sharma, Ramavtar

    2012-11-01

    Moth bean ( Vigna aconitifolia (Jacq.) Marechal) is an important grain legume crop grown in rain fed areas of hot desert regions of Thar, India, under scorching sun rays with very little supplementation of water. An SSH cDNA library was generated from leaf tissues of V. aconitifolia var. RMO-40 exposed to an elevated temperature of 42 °C for 5 min to identify early-induced genes. A total of 488 unigenes (114 contigs and 374 singletons) were derived by cluster assembly and sequence alignment of 738 ESTs; out of 206 ESTs (28%) of unknown proteins, 160 ESTs (14%) were found to be novel to moth bean. Only 578 ESTs (78%) showed significant BLASTX similarity (<1 × 10(-6)) in the NCBI non-redundant database. Gene ontology functional classification terms were retrieved for 479 (65%) sequences, and 339 sequences were annotated with 165 EC codes and mapped to 68 different KEGG pathways. Four hundred and fifty-two ESTs were further annotated with InterProScan (IPS), and no IPS was assigned to 153 ESTs. In addition, the expression level of 27 ESTs in response to heat stress was evaluated through semiquantitative RT-PCR assay. Approximately 20 different signaling genes and 16 different transcription factors have been shown to be associated with heat stress in moth bean for the first time.

  7. Instrumented SSH

    Energy Technology Data Exchange (ETDEWEB)

    Campbell, Scott; Campbell, Scott

    2009-05-27

    NERSC recently undertook a project to access and analyze Secure Shell (SSH) related data. This includes authentication data such as user names and key fingerprints, interactive session data such as keystrokes and responses, and information about noninteractive sessions such as commands executed and files transferred. Historically, this data has been inaccessible with traditional network monitoring techniques, but with a modification to the SSH daemon, this data can be passed directly to intrusion detection systems for analysis. The instrumented version of SSH is now running on all NERSC production systems. This paper describes the project, details about how SSH was instrumented, and the initial results of putting this in production.

  8. Analysis of differentially expressed genes in the precocious line of Eimeria maxima and its parent strain using suppression subtractive hybridization and cDNA microarrays.

    Science.gov (United States)

    Dong, Hui; Lin, Jiaojiao; Han, Hongyu; Jiang, Lianlian; Zhao, Qiping; Zhu, Shunhai; Huang, Bing

    2011-04-01

    The precocious line of Eimeria spp., obtained by repeated passages of oocysts initially collected from feces of previously infected chickens, has unique phenotypes and plays an important role in immunizing chickens against coccidiosis. However, the genetic basis of precocious phenotype in Eimeria is still poorly understood. To investigate gene expression changes in sporulated oocysts between the precocious line of E. maxima and its parent strain, subtractive cDNA libraries were constructed by suppression subtractive hybridization (SSH). A total of 3,164 cDNA fragments were selected from the SSH cDNA libraries to fabricate cDNA microarrays and further identify the differentially expressed genes. The credibility of the microarray data was verified by real-time PCR. A total of 360 valid expressed sequence tags (ESTs) were obtained, which represented 32 unique sequences. Twenty-one genes were validated as downregulated and 11 genes as upregulated in the precocious line. Homology searching of the public sequence database showed that six genes encoded proteins homologous with previously reported proteins, including rhomboid-like protein and transhydrogenase of E. tenella, serpin, and cation-transporting ATPase of E. acervulina, a heat-shock protein of E. maxima, and a conserved hypothetical protein of Toxoplasma gondii. Thus, the remaining 26 ESTs have not been previously reported. Further characterization of these differentially expressed genes will be useful in understanding the genetic basis for the precocious phenotype in Eimeria spp.

  9. Suppression subtractive hybridization and its application to fish gene cloning%抑制消减杂交(SSH)及其在鱼类基因克隆中的应用

    Institute of Scientific and Technical Information of China (English)

    程起群

    2004-01-01

    There are 10 percent to 15 percent genes expression in certain cells during the life time of fishes like other vertebrates. The genes were different at different development stage, under different physiological conditions, and in different kinds of cells. So comparing the differences of gene expression in different cells can help us understand the genetic nature of phenotypic differences, and understand the basic information of life period, and find the genes in relation to development and diseases, and finally benefit mankind. Several methods were developed to clone differential expression gene in recent years. They are subtractive hybridization (SH), differential display (DD),representional difference analysis (RDA), and so on. These methods all have postive influences on cloning special genes, but they all have some defects, such as higher false-positive, lower replication, lower sensitive and difficulty to manipulate. Suppression subtractive hybridization (SSH) was developed by Diatchenko et al in 1996. SSH was based on suppression PCR and combines normalization and subtraction in a single procedure. It is a more effective and convenient method than all others mentioned above. The principle and the rules of manipulation of SSH in detail was illuminated and the novel genes cloned by SSH was listed. They are immune related genes and reproduction and development related genes. The reproduction and development related genes are as follows: ZP3, Cyclin A2,CBI02, YA2, FSTRAP. The immune related genes are as follows: NKEF(natural killer enhancing factor), CC chemokine, CXCR1, CXCR2, CXCR4, AIF-1(allograft inflammatory factor-1), IL-1β(inteleukin-1), FcεRIγ(γ submit of high affinity Fc receptor for IgE), SSA(serum amyloid A), LECT2 (leucocyte cell-derived hemotaxin 2), GMFβ(glia maturation factorβ), CD45, Lysozyme C, PBEF (Pre-B cell enhancing factor), C-type lectin,PTX(Pentraxin), IL-1RⅡ, IL-8-1ike CXC chemokine, TF(tissue factor), trout chemokine 2, TNF decoy

  10. [Cold induced cDNA library construction of highland barley (Hordeum vulgare L. var. nudum Hk. f.) using suppression subtractive hybridization technology].

    Science.gov (United States)

    He, Tao; Jia, Jing Fen

    2008-12-01

    Cold-induced genes of highland barley (Hordeum vulgare L. var. nudum Hk. f.) were studied using suppression subtractive hybridization (SSH) technique. The cDNA from the materials treated with 4 degrees C was used as "tester", and that from the materials growing in green house (20+/-2 degrees C) as "driver". A subtractive library of highland barley including 640 cDNA clones was constructed in this study. Enzyme digestion of 32 clones chosen randomly from the library indicated that 87.5% of them contained inserts. The cDNA inserts of 16 clones were sequenced. Blast search analyses showed that these cDNAs were homologies to genes encoding the following proteins: metallothionein, protein kinase, ethylene signal transcription factor, bZIP transcription factor, zing finger transcription factor, ribulose-1,5-bisphosphate carboxylase, ribosomal protein, sodium: hydrogen antiporter, catalase, NADPH-cytochrome reductase, ascorbate peroxidase, DNA binding protein, and sugar transporter-like protein. These results indicated that the cDNA clones in the library were related to cold-induced genes, and suggested that the cold-tolerant mechanism of highland barley might be a complicated, interactive system involving multiple approaches and genes. Construction of subtractive cDNA library provided an advantage for further studies to isolate and clone cold-induced genes in highland barley.

  11. Identification of Differentially Expressed Genes During Anther Abortion of Taigu Genic Male Sterile Wheat by Combining Suppression Subtractive Hybridization and cDNA Array

    Institute of Scientific and Technical Information of China (English)

    Qing-Shan Chang; Rong-Hua Zhou; Xiu-Ying Kong; Zeng-Liang Yu; Ji-Zeng Jia

    2006-01-01

    Taigu Genic Male Sterile Wheat (TGMSW; Triticum aestivum L.), a dominant genic male sterile germplasm, is of considerable value in the genetic improvement of wheat because of its stable inherence, complete male abortion, and high cross-fertilization rate. To identify specially transcribed genes in sterile anther, a suppression subtractive hybridization (SSH) library was constructed with sterile anther as the tester and fertile anther as the driver. A total of 2 304 SSH inserts amplified by polymerase chain reaction were arrayed using robotic printing. The cDNA arrays were hybridized with 32P-labeled probes prepared from the RNA of forward- and reverse-subtracted anthers. Ninety-six clones were scored as upregulated in sterile anthers compared with the corresponding fertile anthers and some clones were selected for sequencing and analysis in GenBank. Based on their putative functions, 87 non-redundant clones were classified into the following groups: (i) eight genes involved in metabolic processes; (ii) four material transportation genes;(iii) three signal transduction-associated genes; (iv) four stress response and senescence-associated protein genes; (v) seven other functional protein genes; (vi) five genes with no known function; and (vii)another 56 genes with no match to the databases. To test the hybridization efficiency, eight genes were selected and analyzed by Northern blot. The results of the present study provide a comprehensive overview of the genes and gene products involved in anther abortion in TGMSW.

  12. Identification of Botrytis cinerea genes up-regulated during infection and controlled by the Galpha subunit BCG1 using suppression subtractive hybridization (SSH).

    Science.gov (United States)

    Schulze Gronover, Christian; Schorn, Corinna; Tudzynski, Bettina

    2004-05-01

    The Galpha subunit BCG1 plays an important role during the infection of host plants by Botrytis cinerea. Delta bcg1 mutants are able to conidiate, penetrate host leaves, and produce small primary lesions. However, in contrast to the wild type, the mutants completely stop invasion of plant tissue at this stage; secondary lesions have never been observed. Suppression subtractive hybridization (SSH) was used to identify fungal genes whose expression on the host plant is specifically affected in bcg1 mutants. Among the 22 differentially expressed genes, we found those which were predicted to encode proteases, enzymes involved in secondary metabolism, and others encoding cell wall-degrading enzymes. All these genes are highly expressed during infection in the wild type but not in the mutant. However, the genes are expressed in both the wild type and the mutant under certain conditions in vitro. Most of the BCG1-controlled genes are still expressed in adenylate cyclase (bac) mutants in planta, suggesting that BCG1 is involved in at least one additional signaling cascade in addition to the cAMP-depending pathway. In a second SSH approach, 1,500 clones were screened for those that are specifically induced by the wild type during the infection of bean leaves. Of the 22 BCG1-controlled genes, 11 also were found in the in planta SSH library. Therefore, SSH technology can be successfully applied to identify target genes of signaling pathways and differentially expressed genes in planta.

  13. Preparation of fluorescent-dye-labeled cDNA from RNA for microarray hybridization.

    Science.gov (United States)

    Ares, Manuel

    2014-01-01

    This protocol describes how to prepare fluorescently labeled cDNA for hybridization to microarrays. It consists of two steps: first, a mixture of anchored oligo(dT) and random hexamers is used to prime amine-modified cDNA synthesis by reverse transcriptase using a modified deoxynucleotide with a reactive amine group (aminoallyl-dUTP) and an RNA sample as a template. Second, the cDNA is purified and exchanged into bicarbonate buffer so that the amine groups in the cDNA react with the dye N-hydroxysuccinimide (NHS) esters, covalently joining the dye to the cDNA. The dye-coupled cDNA is purified again, and the amount of dye incorporated per microgram of cDNA is determined.

  14. Transcription profiles of boron-deficiency-responsive genes in citrus rootstock root by suppression subtractive hybridization and cDNA microarray.

    Science.gov (United States)

    Zhou, Gao-Feng; Liu, Yong-Zhong; Sheng, Ou; Wei, Qing-Jiang; Yang, Cheng-Quan; Peng, Shu-Ang

    2014-01-01

    Boron (B) deficiency has seriously negative effect on citrus production. Carrizo citrange (CC) has been reported as a B-deficiency tolerant rootstock. However, the molecular mechanism of its B-deficiency tolerance remained not well-explored. To understand the molecular basis of citrus rootstock to B-deficiency, suppression subtractive hybridization (SSH) and microarray approaches were combined to identify the potential important or novel genes responsive to B-deficiency. Firstly four SSH libraries were constructed for the root tissue of two citrus rootstocks CC and Trifoliate orange (TO) to compare B-deficiency treated and non-treated plants. Then 7680 clones from these SSH libraries were used to construct a cDNA array and microarray analysis was carried out to verify the expression changes of these clones upon B-deficiency treatment at various time points compared to the corresponding controls. A total of 139 unigenes that were differentially expressed upon B-deficiency stress either in CC or TO were identified from microarray analysis, some of these genes have not previously been reported to be associated with B-deficiency stress. In this work, several genes involved in cell wall metabolism and transmembrane transport were identified to be highly regulated under B-deficiency stress, and a total of 23 metabolic pathways were affected by B-deficiency, especially the lignin biosynthesis pathway, nitrogen metabolism, and glycolytic pathway. All these results indicated that CC was more tolerant than TO to B-deficiency stress. The B-deficiency responsive genes identified in this study could provide further information for understanding the mechanisms of B-deficiency tolerance in citrus.

  15. Comparison of RNA expression profiles on generations of Porphyra yezoensis (Rhodophyta, based on suppression subtractive hybridization (SSH

    Directory of Open Access Journals (Sweden)

    Shen Songdong

    2011-10-01

    Full Text Available Abstract Background Porphyra yezoensis Ueda is one of the most important edible seaweed, with a dimorphic life cycle which consists of gametophyte as macroscopical blade and sporophyte as microscopic filamentous. Conspicuous differences exist in the two generations, such as morphology, cell structure, biochemistry, physiology, and so on. The developmental process of Porphyra yezoensis has been studied thoroughly, but the mechanism is still ambiguous and few studies on genetic expression have been carried out. In this study, the suppression subtractive hybridization (SSH method conducted to generate large-scale expressed sequence tags (EST is designed to identify gene candidates related to the morphological and physiological differences between the gametophytic and sporophytic generations of Porphyra yezoensis Ueda. Findings Each 300 clones of sporophyte and gametophyte cells were dipped onto the membrane for hybridization. The result of dot-blot suggested there were 222 positive clones in gametophyte library and 236 positive clones in sporophyte library. 383 positive clones of strongest signals had been sequenced, and 191 EST sequences of gametophyte and 192 of sporophyte were obtained. A total of 196 genes were obtained, within which 104 genes were identified from the gametophyte and 92 from the sporophyte. Thirty-nine genes of the gametophyte and 62 genes of the sporophyte showed sequence similarity to those genes with known or putative functions which were classified according to their putative biological roles and molecular functions. The GO annotation showed about 58% of the cellular component of sporophyte and gametophyte cells were mainly located in cytoplasm and nucleus. The special genes were located in Golgi apparatus, and high expression in plastid, ribosome and endoplasmic reticulum. The main biological functions of gametophyte cells contributed to DNA repair/replication, carbohydrate metabolism, transport and transcription

  16. Construction of a hepatic stellate cells subtracted cDNA library of differentially expressed genes in normal mice and mice with Schistosomiasis japonica*

    OpenAIRE

    2005-01-01

    To construct a hepatic stellate cells (HSCs) subtracted cDNA library to find differentially expressed genes in normal mice and mice infected with Schistosoma japonicum (S. japonicum). Suppression subtractive hybridization (SSH) was used. The cDNA fragments of normal mouse were compared to those of schistosoma-infected mice to find differentially expressed genes. Then differentially expressed cDNA fragments were directly inserted into T/A cloning vector to set up the subtractive library. Ampli...

  17. Pro OpenSSH

    CERN Document Server

    Stahnke, Michael

    2006-01-01

    SSH, acronym for Secure Socket Shell, is for users and administrators wishing to establish secure communication between disparate networks. 'Pro OpenSSH', authored by two Fortune 100 system administrators, provides readers with a highly practical reference for configuring and deploying OpenSSH in their own environment.

  18. Suppression subtractive hybridization (SSH) combined with bioinformatics method: an integrated functional annotation approach for analysis of differentially expressed immune-genes in insects.

    Science.gov (United States)

    Badapanda, Chandan

    2013-01-01

    The suppression subtractive hybridization (SSH) approach, a PCR based approach which amplifies differentially expressed cDNAs (complementary DNAs), while simultaneously suppressing amplification of common cDNAs, was employed to identify immuneinducible genes in insects. This technique has been used as a suitable tool for experimental identification of novel genes in eukaryotes as well as prokaryotes; whose genomes have been sequenced, or the species whose genomes have yet to be sequenced. In this article, I have proposed a method for in silico functional characterization of immune-inducible genes from insects. Apart from immune-inducible genes from insects, this method can be applied for the analysis of genes from other species, starting from bacteria to plants and animals. This article is provided with a background of SSH-based method taking specific examples from innate immune-inducible genes in insects, and subsequently a bioinformatics pipeline is proposed for functional characterization of newly sequenced genes. The proposed workflow presented here, can also be applied for any newly sequenced species generated from Next Generation Sequencing (NGS) platforms.

  19. Identification of differentially expressed genes in normal mucosa, adenoma and adenocarcinoma of colon by SSH

    Institute of Scientific and Technical Information of China (English)

    Min-Jie Luo; Mao-De Lai

    2001-01-01

    AIM To construct subtracted cDNA libraries and further identify differentially expressed genes that are related to the development of colorectal carcinoma (CRC).``METHODS Suppression subtractive hybridization(SSH)was done on cDNAs of normal mucosa, adenoma and adenocarcinoma tissues from the same patient. Three subtracted cDNA libraries were constructed and then hybridized with forward and backward subtracted probes for differential screening. Positive clones from each subtracted cDNA library were selected for sequencing and BLAST analysis. Finally, virtual Northern Blot confirmed such differential expression.``RESULTS By this way, there were about 3 -4× l02clones identified in each subtracted cDNA library, in which about 85% positive clones were differentially screened. Sequencing and BLAST homology search revealed some clones containing sequences of known gene fragments and several possibly novel genes showing few or no sequence homologies with any known sequences in the database.``CONCLUSION All results confirmed the effectiveness and sensitivity of SSH. The differentially expressed genes during the development of CRC can be used to shed light on the pathogenesis of CRC and be useful genetic markers for early diagnosis and therapy.``

  20. Identification of differentially-expressed genes potentially implicated in drought response in pitaya (Hylocereus undatus) by suppression subtractive hybridization and cDNA microarray analysis.

    Science.gov (United States)

    Fan, Qing-Jie; Yan, Feng-Xia; Qiao, Guang; Zhang, Bing-Xue; Wen, Xiao-Peng

    2014-01-01

    Drought is one of the most severe threats to the growth, development and yield of plant. In order to unravel the molecular basis underlying the high tolerance of pitaya (Hylocereus undatus) to drought stress, suppression subtractive hybridization (SSH) and cDNA microarray approaches were firstly combined to identify the potential important or novel genes involved in the plant responses to drought stress. The forward (drought over drought-free) and reverse (drought-free over drought) suppression subtractive cDNA libraries were constructed using in vitro shoots of cultivar 'Zihonglong' exposed to drought stress and drought-free (control). A total of 2112 clones, among which half were from either forward or reverse SSH library, were randomly picked up to construct a pitaya cDNA microarray. Microarray analysis was carried out to verify the expression fluctuations of this set of clones upon drought treatment compared with the controls. A total of 309 expressed sequence tags (ESTs), 153 from forward library and 156 from reverse library, were obtained, and 138 unique ESTs were identified after sequencing by clustering and blast analyses, which included genes that had been previously reported as responsive to water stress as well as some functionally unknown genes. Thirty six genes were mapped to 47 KEGG pathways, including carbohydrate metabolism, lipid metabolism, energy metabolism, nucleotide metabolism, and amino acid metabolism of pitaya. Expression analysis of the selected ESTs by reverse transcriptase polymerase chain reaction (RT-PCR) corroborated the results of differential screening. Moreover, time-course expression patterns of these selected ESTs further confirmed that they were closely responsive to drought treatment. Among the differentially expressed genes (DEGs), many are related to stress tolerances including drought tolerance. Thereby, the mechanism of drought tolerance of this pitaya genotype is a very complex physiological and biochemical process, in

  1. Extraction of high quality of RNA and construction of a suppression subtractive hybridization (SSH) library from chestnut rose (Rosa roxburghii Tratt).

    Science.gov (United States)

    Xu, Qiang; Wen, Xiaopeng; Tao, Nengguo; Hu, Zhiyong; Yue, Hailin; Deng, Xiuxin

    2006-04-01

    Chestnut rose (Rosa roxburghii Tratt) is a rare fruit crop of promising economical importance in fruit and ornamental exploitation in China. Isolation of high quality RNA from chestnut rose is difficult due to its high levels of polyphenols, polysaccharides and other compounds, but a modified CTAB extraction procedure without phenol gave satisfactory results. High concentrations of PVP (2%, w/v), CTAB (2%, w/v) and beta-mercaptoethanol (4%, v/v) were used in the extraction buffer to improve RNA quality. The average yield was about 200 microg RNA g(-1) fresh leaves. The isolated RNA was of sufficient quality for construction of suppression subtraction hybridization (SSH) library, which allowed the isolation of several pathogen-induced defense genes.

  2. Human cDNA mapping using fluorescence in situ hybridization

    Energy Technology Data Exchange (ETDEWEB)

    Korenberg, J.R.

    1993-03-04

    Genetic mapping is approached using the techniques of high resolution fluorescence in situ hybridization (FISH). This technology and the results of its application are designed to rapidly generate whole genome as tool box of expressed sequence to speed the identification of human disease genes. The results of this study are intended to dovetail with and to link the results of existing technologies for creating backbone YAC and genetic maps. In the first eight months, this approach generated 60--80% of the expressed sequence map, the remainder expected to be derived through more long-term, labor-intensive, regional chromosomal gene searches or sequencing. The laboratory has made significant progress in the set-up phase, in mapping fetal and adult brain and other cDNAs, in testing a model system for directly linking genetic and physical maps using FISH with small fragments, in setting up a database, and in establishing the validity and throughput of the system.

  3. Identification and characterization of genes differentially expressed in X and Y sperm using suppression subtractive hybridization and cDNA microarray.

    Science.gov (United States)

    Chen, Xiaoli; Yue, Yang; He, Yanan; Zhu, Huabin; Hao, Haisheng; Zhao, Xueming; Qin, Tong; Wang, Dong

    2014-10-01

    Differential expression of genes leads to variations in the phenotypes of X and Y sperm, although some differentially expressed gene products are shared through intercellular bridges. Genes differentially expressed in bovine X and Y sperm were identified by a combination of suppression subtractive hybridization (SSH), cDNA microarray, and sequence-homology analysis. Microarray data and Significance Analysis of Microarrays software were used to identify 31 differentially expressed genes, only four of which were previously identified. These genes are involved in fundamental life processes of mature sperm, and may be associated with the differences between X and Y sperm since 27 versus 4 were upregulated in X versus Y sperm, respectively. The levels of expression of seven genes-including the known genes UTY, DPH3, CYTB, and ISCU, and the unknown genes X + Y contig 41, X + Y contig 18, and Y + X contig 16-were validated by quantitative real-time PCR, and some genes were clearly differentially expressed by X and Y sperm, despite the presence of intercellular bridges among spermatids. These results provide a theoretical basis for research on gene expression during sperm development, as well as on sex control at the level of sperm.

  4. Comparative transcript profiling of gene expression between seedless Ponkan mandarin and its seedy wild type during floral organ development by suppression subtractive hybridization and cDNA microarray.

    Science.gov (United States)

    Qiu, Wen-Ming; Zhu, An-Dan; Wang, Yao; Chai, Li-Jun; Ge, Xiao-Xia; Deng, Xiu-Xin; Guo, Wen-Wu

    2012-08-16

    Seedlessness is an important agronomic trait for citrus, and male sterility (MS) is one main cause of seedless citrus fruit. However, the molecular mechanism of citrus seedlessness remained not well explored. An integrative strategy combining suppression subtractive hybridization (SSH) library with cDNA microarray was employed to study the underlying mechanism of seedlessness of a Ponkan mandarin seedless mutant (Citrus reticulata Blanco). Screening with custom microarray, a total of 279 differentially expressed clones were identified, and 133 unigenes (43 contigs and 90 singletons) were obtained after sequencing. Gene Ontology (GO) distribution based on biological process suggested that the majority of differential genes are involved in metabolic process and respond to stimulus and regulation of biology process; based on molecular function they function as DNA/RNA binding or have catalytic activity and oxidoreductase activity. A gene encoding male sterility-like protein was highly up-regulated in the seedless mutant compared with the wild type, while several transcription factors (TFs) such as AP2/EREBP, MYB, WRKY, NAC and C2C2-GATA zinc-finger domain TFs were down-regulated. Our research highlighted some candidate pathways that participated in the citrus male gametophyte development and could be beneficial for seedless citrus breeding in the future.

  5. Chromosomal Localization of DNA Amplifications in Neuroblastoma Tumors Using cDNA Microarray Comparative Genomic Hybridization

    Directory of Open Access Journals (Sweden)

    Ben Beheshti

    2003-01-01

    Full Text Available Conventional comparative genomic hybridization (CGH profiling of neuroblastomas has identified many genomic aberrations, although the limited resolution has precluded a precise localization of sequences of interest within amplicons. To map high copy number genomic gains in clinically matched stage IV neuroblastomas, CGH analysis using a 19,200-feature cDNA microarray was used. A dedicated (freely available algorithm was developed for rapid in silico determination of chromosomal localizations of microarray cDNA targets, and for generation of an ideogram-type profile of copy number changes. Using these methodologies, novel gene amplifications undetectable by chromosome CGH were identified, and larger MYCN amplicon sizes (in one tumor up to 6 Mb than those previously reported in neuroblastoma were identified. The genes HPCAL1, LPIN1/KIAA0188, NAG, and NSE1/LOC151354 were found to be coamplified with MYCN. To determine whether stage IV primary tumors could be further subclassified based on their genomic copy number profiles, hierarchical clustering was performed. Cluster analysis of microarray CGH data identified three groups: 1 no amplifications evident, 2 a small MYCN amplicon as the only detectable imbalance, and 3 a large MYCN amplicon with additional gene amplifications. Application of CGH to cDNA microarray targets will help to determine both the variation of amplicon size and help better define amplification-dependent and independent pathways of progression in neuroblastoma.

  6. Suppression subtractive hybridization.

    Science.gov (United States)

    Ghorbel, Mohamed T; Murphy, David

    2011-01-01

    Comparing two RNA populations that differ from the effects of a single independent variable, such as a drug treatment or a specific genetic defect, can establish differences in the abundance of specific transcripts that vary in a population dependent manner. There are different methods for identifying differentially expressed genes. These methods include microarray, Serial Analysis of Gene Expression (SAGE), and quantitative Reverse-Transcriptase Polymerase Chain Reaction (qRT-PCR). Herein, the protocol describes an easy and cost-effective alternative that does not require prior knowledge of the transcriptomes under examination. It is specifically relevant when low levels of RNA starting material are available. This protocol describes the use of Switching Mechanism At RNA Termini Polymerase Chain Reaction (SMART-PCR) to amplify cDNA from small amounts of RNA. The amplified cDNA populations under comparison are then subjected to Suppression Subtractive Hybridization (SSH-PCR). SSH-PCR is a technique that couples subtractive hybridization with suppression PCR to selectively amplify fragments of differentially expressed genes. The resulting products are cDNA populations enriched for significantly overrepresented transcripts in either of the two input RNAs. These cDNA populations can then be cloned to generate subtracted cDNA library. Microarrays made with clones from the subtracted forward and reverse cDNA libraries are then screened for differentially expressed genes using targets generated from tester and driver total RNAs.

  7. Construction of subtracted cDNA library of diferentially expressed genes of multidrug resistant tubercle bacillus by suppression subtracted hybridization%应用抑制消减杂交技术构建耐多药结核杆菌差异表达基因消减cDNA文库

    Institute of Scientific and Technical Information of China (English)

    张运玲; 郑改焕; 刘芮汐; 彭哲; 李奇志; 幸琳琳; 朱朝敏

    2012-01-01

    Objective: To build the subtracted cDNA library of differentially expressed genes of multidrug-resistant tubercle bacillus (MDR-TB)and to further discuss the molecular mechanism of MDR-TB. Methods:Tester was MDR-TB and Driver was sensitive tuberculosis. Suppression subtractive hybridization (SSH) and T/A cloning technology were done to build the subtracted cDNA library of differentially expressed genes of MDR-TB. Results:The subtracted cDNA library of differentially expressed genes of MDR-TB was successfully built and 113 differentially expressed cDNA fragements of MDR-TB were obtained. Sequencing and homology analysis showed that 5 of them were novel cDNA sequences and 5 sequenced genes were reported to be related with MDR in TB. Conclusions: SSH is an effective method for screening new function genes. Many genes both known and unknown are in correlation with MDR in TB. Discovery of these genes provides a solid foundation for the explanation of MDR mechanism in TB.%目的:构建结核杆菌耐多药株与敏感株的差异表达消减cDNA文库,进一步探索结核杆菌耐多药的分子机制.方法:以耐多药菌株cDNA为实验组(Tester),敏感株cDNA为驱动组(Driver)应用抑制消减杂交(Suppression subtractive hybridization,SSH)技术结合T/A克隆技术构建结核杆菌耐多药株与敏感株的差异表达消减cDNA文库.结果:成功构建了耐多药结核菌株差异表达消减cDNA文库,获得113个差异表达cDNA片段.结论:研究表明SSH技术是筛选新功能基因的有效方法;多种已知或未知基因均参与了结核杆菌耐多药的调节,大规模筛选与克隆这些基因为进一步研究结核杆菌耐多药机制的产生奠定了必要的理论基础.

  8. High-throughput screening of suppression subtractive hybridization cDNA libraries using DNA microarray analysis

    CSIR Research Space (South Africa)

    Van den Berg, N

    2004-11-01

    Full Text Available DNAs to the SSH libraries arrayed on glass slides, two values were calculated for each clone, an enrichment ratio 1 (ER1) and an enrichment ratio 2 (ER2). Graphical representation of ER1 and ER2 enabled the identification of clones that were likely to represent up...

  9. Construction of subtracted cDNA libraries of gastrocarcinoma and normal tissue with suppression subtractive hybridization and their quality analysis%人胃癌抑制消减cDNA文库的构建及文库质量分析

    Institute of Scientific and Technical Information of China (English)

    吴岚军; 毛秉智; 王升启

    2001-01-01

    Objective: To construct subtracted cDNA libraries of stomach tumors and normal stomach tissue using suppression subtractive hybridization(SSH).Methods: cDNA Library subtraction was performed using the protocol described in the Clontech PCR-Select cDNA Subtraction Kit. cDNA was synthesized from 2 μg of poly A+RNA from the tumor and normal tissues using AMV reverse transcriptase. The tester and driver cDNAs were digested with RsaⅠ, a four-base-cutting restriction enzyme that yields blunt ends. The tester cDNA was then subdivided into two portions, and each was ligated with different cDNA adaptor. Two hybridizations were performed. In the first, an excess of driver was added to each sample of tester. Hybridization kinetics led to equalization and enrichment of differentially expressed sequences. During the second hybridization, the two primary hybridization samples were mixed together without denaturing and thus the templates were generated from differentially expressed sequences for PCR amplification. Using suppression PCR, only differentially expressed sequences were amplified exponentially and after second PCR amplification the background was reduced and differentially expressed sequences were further enriched. The cDNAs were then directly inserted into a T/A cloning vector to generate a stomach tumor-specific subtracted cDNA library. Results: The amplified library contained 800 positive clones. Plasmid inserts were PCR amplified and showed 250-700 bp inserts. Conclusions: The successfully constructed subtracted cDNA library of gastrocarcinoma and normal tissue enables us to compare two populations of mRNA and obtain clones of genes that expressed in one population but not in the other.The characterization of these genes will allow them to be exploited for their diagnostic and therapeutic potentials.%目的:构建人胃癌抑制消减cDNA文库,为进一步大批量筛选、克隆胃癌特异性表达的基因奠定基础。方法:从胃癌和

  10. [Construction and analysis of a forward and reverse subtractive cDNA library from leaves and stem of Polygonum sibiricum Laxm. under salt stress].

    Science.gov (United States)

    Liu, Guan-Jun; Liu, Ming-Kun; Xu, Zhi-Ru; Yan, Xiu-Feng; Wei, Zhi-Gang; Yang, Chuan-Ping

    2009-04-01

    Using cDNAs prepared from the leaves and stems of Polygonum sibiricum Laxm. treated with NaHCO3 stress for 48 h as testers and cDNAs from unstressed P. sibiricum leaves and stems as drivers library, suppression subtractive hybridization (SSH) was employed to construct a cDNA subtracted library, which contained 2 282 valid sequences including 598 ESTs in the stems forward SSH library and 490 ESTs in the stem reverse SSH library, 627 ESTs in the leaf forward SSH library and 567 in the leaf reverse SSH library. According to the functional catalogue of MIPs and the comparison of the reverse and forward SSH libraries of the stem and leaf, the responses to NaHCO3 stress were different between leaf and stem, except for the same trend in cell rescue defense and transport facilitation. The trend in the metabolism, energy, photosynthesis, protein synthesis, transcription, and signal transduction was opposite. RT-PCR analysis demonstrated that the expression of 12 putative stress related genes in the NaHCO3-treated leaves and stems was different from that in the untreated leaves and stems. This indicated that different mechanisms might be responsible for reactions of leaf and stem in P. sibiricum. The results from this study are useful in understanding the molecular mechanism of saline-alkali tolerance in P. sibiricum.

  11. Social Impact of SSH Research

    DEFF Research Database (Denmark)

    Johansson, Lasse Gøhler; Grønvad, Jonas Følsgaard; Pedersen, David Budtz

    2016-01-01

    of making social impact. However, the social sciences and humanities (SSH) have only played a marginal role in the societal challenges of Horizon2020. According to Science Europe, the pan-European association for research councils and foundations, only 26.7 percent of the topics under the Horizon2020’s...... societal challenges explicitly invite contributions from SSH research. If we look at the humanities in isolation, it is only around 10 percent of the topics. The marginal role of SSH in Horizon2020 is, among other things, the result of an inadequate understanding of the social impact of SSH research...... and of inadequate instruments for measuring the impact of SSH research. In this paper we address the following questions: 1) how can we understand the social impact of SSH research? And 2) how can we meaningfully measure or assess it? These questions are addressed through a survey of the current scientific...

  12. Social Impact of SSH Research

    DEFF Research Database (Denmark)

    Johansson, Lasse Gøhler; Grønvad, Jonas Følsgaard; Pedersen, David Budtz

    2016-01-01

    societal challenges explicitly invite contributions from SSH research. If we look at the humanities in isolation, it is only around 10 percent of the topics. The marginal role of SSH in Horizon2020 is, among other things, the result of an inadequate understanding of the social impact of SSH research...... of making social impact. However, the social sciences and humanities (SSH) have only played a marginal role in the societal challenges of Horizon2020. According to Science Europe, the pan-European association for research councils and foundations, only 26.7 percent of the topics under the Horizon2020’s...... and of inadequate instruments for measuring the impact of SSH research. In this paper we address the following questions: 1) how can we understand the social impact of SSH research? And 2) how can we meaningfully measure or assess it? These questions are addressed through a survey of the current scientific...

  13. Evolutionary conservation and DNA binding properties of the Ssh7 proteins from Sulfolobus shibatae

    Institute of Scientific and Technical Information of China (English)

    CHEN; Xulin(陈绪林); GUO; Rong(郭荣); HUANG; Li(黄力); Ray; Hong

    2002-01-01

    The thermoacidophilic archaeon Sulfolobus shibatae synthesizes a large amount of the 7-ku DNA binding proteins known as Ssh7. Our hybridization experiments showed that two Ssh7-encoding genes existed in the genome of S. Shibatae. These two genes, designated ssh7a and ssh7b, have been cloned, sequenced and expressed in Escherichia coli. The two Ssh7 proteins differ only at three amino acid positions. In addition, the cis-regulatory sequences of the ssh7a and ssh7b genes are highly conserved. These results suggest the presence of a selective pressure to maintain not only the sequence but also the expression of the two genes. We have also found that there are two genes encoding the 7-ku protein in Sulfolobus solfataricus. Based on this and other studies, we suggest that the gene encoding the 7-ku protein underwent duplication before the separation of Sulfolobus species. Binding of native Ssh7 and recombinant (r)Ssh7 to short duplex DNA fragments was analyzed by electrophoretic mobility shift assays. Both native and recombinant forms of the protein behaved in a similar fashion in the assays, suggesting that the interaction of Ssh7 with DNA is not affected either by specific lysine methylation found in the native Ssh7 proteins or by the difference between the two Ssh7 isomers in amino acid sequence. Our data show that Ssh7 binds duplex DNA fragments with a binding size of ~ 6.6 base pairs and an apparent dissociation constant of (0.7-1.0)×10-7 mol/L under the assay conditions employed in the present study. In addition, Ssh7 binds more tightly to negatively supercoiled DNA than to linear or relaxed DNA.

  14. Isolation of cDNAs of scrapie-modulated RNAs by subtractive hybridization of a cDNA library.

    OpenAIRE

    1988-01-01

    We have developed a subtractive cloning procedure based on the hybridization of single-stranded cDNA libraries constructed in pi H3M, a vector containing the phage M13 origin of replication. We have used this strategy to isolate three transcripts whose abundance is increased in scrapie-infected brain. DNA sequence analysis showed that they represent glial fibrillary acidic protein, metallothionein II, and the B chain of alpha-crystallin; the latter two may represent a response to stress.

  15. [Identification and analysis of differentially expressed genes during wood formation in Chinese fir by SSH].

    Science.gov (United States)

    Wang, Gui-Feng; Gao, Yan; Yang, Li-Wei; Shi, Ji-Sen

    2007-04-01

    Wood is an important raw material for the global industry with rapidly increasing demand. To isolate the differentially expressed genes in xylogenesis of Chinese fir [Cunninghamia lanceolata (Lamb.) Hook], a forward subtractive cDNA library was constructed using suppression subtractive hybridization (SSH) method, which was performed using the cDNA from the mutant Dugansha clone as the tester and the cDNA from the normal Jurong 0 clone as the driver. Six hundred and eighteen clones were obtained. Recombinants were identified using PCR with universal T7 and SP6 primers and using EcoR digestion. To further eliminate false positive, dot hybridization was used with four DIG-labeled probes (FSP, RSP, UTP, and UDP). Real-time PCR was performed to confirm the results. A total of 260 unique ESTs were obtained, 60% of the ESTs exhibiting homologies with proteins of known function fell into 4 major classes: metabolism, cell wall biogenesis and remodeling, signal transduction and stress. The systematic analysis of genes involved in wood formation in Chinese fir provides valuable insights into the molecular mechanisms involved in xylem differentiation, is important resources for forest research directed toward understanding the genetic control of wood formation and future endeavors to modify wood and fiber properties for industrial use.

  16. Human secreted carbonic anhydrase: cDNA cloning, nucleotide sequence, and hybridization histochemistry

    Energy Technology Data Exchange (ETDEWEB)

    Aldred, P.; Fu, Ping; Barrett, G.; Penschow, J.D.; Wright, R.D.; Coghlan, J.P.; Fernley, R.T. (The Howard Florey Institute of Experimental Physiology and Medicine, Parkville, Victoria (Australia))

    1991-01-01

    Complementary DNA clones coding for the human secreted carbonic anhydrase isozyme (CAVI) have been isolated and their nucleotide sequences determined. These clones identify a 1.45-kb mRNA that is present in high levels in parotid submandibular salivary glands but absent in other tissues such as the sublingual gland, kidney, liver, and prostate gland. Hybridization histochemistry of human salivary glands shows mRNA for CA VI located in the acinar cells of these glands. The cDNA clones encode a protein of 308 amino acids that includes a 17 amino acid leader sequence typical of secreted proteins. The mature protein has 291 amino acids compared to 259 or 260 for the cytoplasmic isozymes, with most of the extra amino acids present as a carboxyl terminal extension. In comparison, sheep CA VI has a 45 amino acid extension. Overall the human CA VI protein has a sequence identity of 35 {percent} with human CA II, while residues involved in the active site of the enzymes have been conserved. The human and sheep secreted carbonic anhydrases have a sequence identity of 72 {percent}. This includes the two cysteine residues that are known to be involved in an intramolecular disulfide bond in the sheep CA VI. The enzyme is known to be glycosylated and three potential N-glycosylation sites (Asn-X-Thr/Ser) have been identified. Two of these are known to be glycosylated in sheep CA VI. Southern analysis of human DNA indicates that there is only one gene coding for CA VI.

  17. Dicty_cDB: SSH308 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available SS (Link to library) SSH308 (Link to dictyBase) - - - Contig-U13027-1 SSH308P (Link to Original site) SSH...308F 417 SSH308Z 615 SSH308P 1032 - - Show SSH308 Library SS (Link to library) Clone ID SSH...e URL http://dictycdb.biol.tsukuba.ac.jp/CSM/SS/SSH3-A/SSH308Q.Seq.d/ Representative seq. ID SSH...308P (Link to Original site) Representative DNA sequence >SSH308 (SSH308Q) /CSM/SS/SSH3-A/SSH...sill fllqeidvl*ka*nvlyqksilnilivildlklieklldvysllkqefiihql*sshklh kcitinnnhnnnimh

  18. Dicty_cDB: SSH490 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available SS (Link to library) SSH490 (Link to dictyBase) - - - Contig-U15458-1 SSH490P (Link to Original site) SSH...490F 364 SSH490Z 515 SSH490P 879 - - Show SSH490 Library SS (Link to library) Clone ID SSH... URL http://dictycdb.biol.tsukuba.ac.jp/CSM/SS/SSH4-D/SSH490Q.Seq.d/ Representative seq. ID SSH...490P (Link to Original site) Representative DNA sequence >SSH490 (SSH490Q) /CSM/SS/SSH4-D/SSH4...s l**rysfycsmv*tc*nl*c*s*tt*yfiinw**rssng*yynqsfiktks*stsshl*n i--- ---PSIVNEILKSIVAQFNASQLITQREQVSRLIFKRLVD

  19. Dicty_cDB: SSH379 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available SS (Link to library) SSH379 (Link to dictyBase) - - - Contig-U15118-1 SSH379P (Link to Original site) SSH...379F 371 SSH379Z 582 SSH379P 953 - - Show SSH379 Library SS (Link to library) Clone ID SSH... URL http://dictycdb.biol.tsukuba.ac.jp/CSM/SS/SSH3-D/SSH379Q.Seq.d/ Representative seq. ID SSH...379P (Link to Original site) Representative DNA sequence >SSH379 (SSH379Q) /CSM/SS/SSH3-D/SSH3...: ti*lcltkrkttylhllkil*svvlpvvsqklllhqlnvlnyyykfnllqlklllinntk vssivllefqknkvsshy

  20. Dicty_cDB: SSH306 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available SS (Link to library) SSH306 (Link to dictyBase) - - - Contig-U16505-1 SSH306P (Link to Original site) SSH...306F 388 SSH306Z 544 SSH306P 932 - - Show SSH306 Library SS (Link to library) Clone ID SSH... URL http://dictycdb.biol.tsukuba.ac.jp/CSM/SS/SSH3-A/SSH306Q.Seq.d/ Representative seq. ID SSH...306P (Link to Original site) Representative DNA sequence >SSH306 (SSH306Q) /CSM/SS/SSH3-A/SSH3...emcrf**grfgycq*irnpsikstslftkcsirvctfi nygie*kdp--- ---kqslisvsst*gl*tkfgnnvmsshytlh*ilklniw*tlnpskr*emvl*ls

  1. Dicty_cDB: SSH325 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available SS (Link to library) SSH325 (Link to dictyBase) - - - Contig-U12240-1 SSH325P (Link to Original site) SSH...325F 211 SSH325Z 719 SSH325P 930 - - Show SSH325 Library SS (Link to library) Clone ID SSH... URL http://dictycdb.biol.tsukuba.ac.jp/CSM/SS/SSH3-B/SSH325Q.Seq.d/ Representative seq. ID SSH...325P (Link to Original site) Representative DNA sequence >SSH325 (SSH325Q) /CSM/SS/SSH3-B/SSH3...knfqkqffp*lrixkixxnlikkkkkkxlxvxp*x*f**nkkkkkrkknph*kkth ththhnthtlyiilnlhsshynip

  2. Dicty_cDB: SSH277 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available SS (Link to library) SSH277 (Link to dictyBase) - - - Contig-U14946-1 SSH277P (Link to Original site) SSH...277F 336 SSH277Z 415 SSH277P 751 - - Show SSH277 Library SS (Link to library) Clone ID SSH... URL http://dictycdb.biol.tsukuba.ac.jp/CSM/SS/SSH2-D/SSH277Q.Seq.d/ Representative seq. ID SSH...277P (Link to Original site) Representative DNA sequence >SSH277 (SSH277Q) /CSM/SS/SSH2-D/SSH2...lmnylrksls ivllvpfhmqa*sxpsflvih*avsklllfkkkkkkxeiyf*iiikwkkkkktfkktlkn pynkkl**lknqkkkkkkkkk Frame C: kkkkyf*kkkifeyfffflssh

  3. Dicty_cDB: SSH109 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available SS (Link to library) SSH109 (Link to dictyBase) - G02274 DDB0168040 Contig-U05306-1 SSH...109E (Link to Original site) - - - - - - SSH109E 493 Show SSH109 Library SS (Link to library) Clone ID SSH...riginal site URL http://dictycdb.biol.tsukuba.ac.jp/CSM/SS/SSH1-A/SSH109Q.Seq.d/ ...Representative seq. ID SSH109E (Link to Original site) Representative DNA sequence >SSH109 (SSH109Q) /CSM/SS/SSH1-A/SSH...lkkviikmiinifffkkkkkkkk Homology vs CSM-cDNA Score E Sequences producing significant alignments: (bits) Value SSH109 (SSH

  4. Construction and Sequence Analysis of SSH cDNA Library form Aphid-resistant Sorghum%高粱广谱抗蚜基因SSH文库的构建及其序列分析

    Institute of Scientific and Technical Information of China (English)

    齐金凤; 孙权; 常金华; KhalidHussain; 林凤

    2012-01-01

    The aim was to study broad-spectrum anti-aphid gene in sorghum and sorghum aphid interactions in the molecular genetic basis and mechanism, and to explore and use the information of aphid resistance genes. In this study, 'Henongl6' with aphid-resistance genes and 'Qian3' susceptible to sorghum aphid were used as the tester and the driver and vice versa to construct SSH library, so that the aphid resistance genes were enriched effectively, then the differential gene sequence was analyzed. 200 positive clones were randomly selected and sequenced. 18 open reading frame (ORF) sequences were found through the NCBI ORF Finder online prediction. Then the 18 ORF sequences were blasted and analyzed using online Blastx. The results showed that the 18 ORF sequences were highly related with putative oxygen-evolving enhancer protein in chloroplast, adenylate cyclase-associated protein, ribosomal protein, putative senescence-associated protein. The contigs without ORF were blasted and analyzed using online Blastx and Blastn, and 66 ESTs had highly homologous to genes sequences with known disease resistance in many plants, such as ribosomal protein,transport membrane protein, NBS-LRR disease resistance protein family-1, zinc finger protein, ATP synthase, NADH dehydrogenase, retrotransposon, Adenylyl cyclase-associated protein, and bZIP transcription factor etc.. These homologous genes involved in secondary metabolism, energy metabolism, membrane transport, signal transduction and transcription regulation and so on. The SSH library of sorghum aphid resistance gene was constructed successfully, which indicated that the SSH technique in the mechanism of aphid resistance of sorghum was a feasibility study. The results laid the foundation for further studying on sorghum aphid-resistance gene function.%为了从分子水平上解析高粱广谱抗蚜基因在高粱与蚜虫互作中的分子遗传基础和作用机理,为发掘并利用抗蚜基因提供可靠的信息支持.

  5. Dicty_cDB: SSH236 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available SS (Link to library) SSH236 (Link to dictyBase) - - - Contig-U12328-1 SSH236F (Link to Original site) SSH...236F 189 - - - - - - Show SSH236 Library SS (Link to library) Clone ID SSH236 (Link to...ycdb.biol.tsukuba.ac.jp/CSM/SS/SSH2-B/SSH236Q.Seq.d/ Representative seq. ID SSH23...6F (Link to Original site) Representative DNA sequence >SSH236 (SSH236Q) /CSM/SS/SSH2-B/SSH236Q.Seq.d/ CTTTT.../VF/VFI8-C/VFI851Q.Seq.d/ 375 e-103 SSH237 (SSH237Q) /CSM/SS/SSH2-B/SSH237Q.Seq.d/ 375 e-103 SSH236 (SSH236Q) /CSM/SS/SSH2-B/SSH

  6. Dicty_cDB: SSH550 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available SS (Link to library) SSH550 (Link to dictyBase) - - - Contig-U15330-1 SSH550E (Link... to Original site) - - - - - - SSH550E 258 Show SSH550 Library SS (Link to library) Clone ID SSH550 (Link to...ycdb.biol.tsukuba.ac.jp/CSM/SS/SSH5-C/SSH550Q.Seq.d/ Representative seq. ID SSH55...0E (Link to Original site) Representative DNA sequence >SSH550 (SSH550Q) /CSM/SS/SSH5-C/SSH550Q.Seq.d/ CGAAT...55Q) /CSM/SS/SSL4-C/SSL455Q.Seq.d/ 456 e-128 SSH550 (SSH550Q) /CSM/SS/SSH5-C/SSH550Q.Seq.d/ 456 e-128 SSH134 (SSH134Q) /CSM/SS/SSH

  7. Identification of differentially expressed genes in esophageal cancer through SSH in com- bination with high throughput reverse Northern screening

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    To understand the molecular mechanisms of carcinogenesis of esophagus and to isolate genes with different expression levels in esophageal cancer, suppression subtractive hybridization (SSH) was combined with PCR-based cDNA synthesis and reverse Northern on the cancer tissues and matched almost normal mucosa using 5 microgram of total RNA as starting marterial. Eight genes were found expressed differentially in esophageal cancer, in which 5 were known genes and 3 were novel ones; and 6 were down-regulated in cancer tissues, while 2 were up-regulated; 6 were of mid-high abundance and 2 were of low abundance in esophagus. The results revealed that alteration in expression level of multiple genes underlied the initiation and development of esophageal cancer. The differentially expressed genes identified in this study such as liporcotinⅠ, cystatin A, cystatin B, cytokeratin 13 may play roles in dedifferentiation, transformation and malignant proliferation of esophageal cancer. The combination of SSH with PCR-based double- strand cDNA synthesis and high throughput reverse Northern screening is an efficient way to isolate differentially expressed genes from microgram of total RNA.

  8. Dicty_cDB: SSH596 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available SS (Link to library) SSH596 (Link to dictyBase) - - - Contig-U10001-1 SSH596P (Link to Original site) SSH...596F 354 SSH596Z 451 SSH596P 805 - - Show SSH596 Library SS (Link to library) Clone ID SSH... URL http://dictycdb.biol.tsukuba.ac.jp/CSM/SS/SSH5-D/SSH596Q.Seq.d/ Representative seq. ID SSH...596P (Link to Original site) Representative DNA sequence >SSH596 (SSH596Q) /CSM/SS/SSH5-D/SSH5...re E Sequences producing significant alignments: (bits) Value SSH596 (SSH596Q) /CSM/SS/SSH5-D/SSH

  9. Dicty_cDB: SSH571 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available SS (Link to library) SSH571 (Link to dictyBase) - - - Contig-U13239-1 SSH571P (Link to Original site) SSH...571F 228 SSH571Z 189 SSH571P 417 - - Show SSH571 Library SS (Link to library) Clone ID SSH... URL http://dictycdb.biol.tsukuba.ac.jp/CSM/SS/SSH5-C/SSH571Q.Seq.d/ Representative seq. ID SSH...571P (Link to Original site) Representative DNA sequence >SSH571 (SSH571Q) /CSM/SS/SSH5-C/SSH5...core E Sequences producing significant alignments: (bits) Value SSH571 (SSH571Q) /CSM/SS/SSH5-C/SSH571Q.Seq.

  10. Dicty_cDB: SSH151 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available SS (Link to library) SSH151 (Link to dictyBase) - - - Contig-U01152-1 SSH151P (Link to Original site) SSH...151F 286 SSH151Z 654 SSH151P 940 - - Show SSH151 Library SS (Link to library) Clone ID SSH... URL http://dictycdb.biol.tsukuba.ac.jp/CSM/SS/SSH1-C/SSH151Q.Seq.d/ Representative seq. ID SSH...151P (Link to Original site) Representative DNA sequence >SSH151 (SSH151Q) /CSM/SS/SSH1-C/SSH1...omology vs CSM-cDNA Score E Sequences producing significant alignments: (bits) Value SSH151 (SSH151Q) /CSM/SS/SSH1-C/SSH

  11. Dicty_cDB: SSH269 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available SS (Link to library) SSH269 (Link to dictyBase) - - - Contig-U01518-1 SSH269P (Link to Original site) SSH...269F 381 SSH269Z 406 SSH269P 787 - - Show SSH269 Library SS (Link to library) Clone ID SSH... URL http://dictycdb.biol.tsukuba.ac.jp/CSM/SS/SSH2-C/SSH269Q.Seq.d/ Representative seq. ID SSH...269P (Link to Original site) Representative DNA sequence >SSH269 (SSH269Q) /CSM/SS/SSH2-C/SSH2...lignments: (bits) Value SSH269 (SSH269Q) /CSM/SS/SSH2-C/SSH269Q.Seq.d/ 1078 0.0 S

  12. Dicty_cDB: SSH644 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available SS (Link to library) SSH644 (Link to dictyBase) - - - Contig-U15077-1 SSH644P (Link to Original site) SSH...644F 233 SSH644Z 229 SSH644P 462 - - Show SSH644 Library SS (Link to library) Clone ID SSH... URL http://dictycdb.biol.tsukuba.ac.jp/CSM/SS/SSH6-B/SSH644Q.Seq.d/ Representative seq. ID SSH...644P (Link to Original site) Representative DNA sequence >SSH644 (SSH644Q) /CSM/SS/SSH6-B/SSH6...268 7e-71 SSK652 (SSK652Q) /CSM/SS/SSK6-C/SSK652Q.Seq.d/ 268 7e-71 SSH644 (SSH644Q) /CSM/SS/SSH6-B/SSH

  13. Dicty_cDB: SSH168 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available SS (Link to library) SSH168 (Link to dictyBase) - - - Contig-U15567-1 SSH168P (Link to Original site) SSH...168F 329 SSH168Z 641 SSH168P 970 - - Show SSH168 Library SS (Link to library) Clone ID SSH... URL http://dictycdb.biol.tsukuba.ac.jp/CSM/SS/SSH1-C/SSH168Q.Seq.d/ Representative seq. ID SSH...168P (Link to Original site) Representative DNA sequence >SSH168 (SSH168Q) /CSM/SS/SSH1-C/SSH1... producing significant alignments: (bits) Value SSH168 (SSH168Q) /CSM/SS/SSH1-C/SSH

  14. Dicty_cDB: SSH570 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available SS (Link to library) SSH570 (Link to dictyBase) - - - Contig-U15676-1 SSH570P (Link to Original site) SSH...570F 229 SSH570Z 192 SSH570P 421 - - Show SSH570 Library SS (Link to library) Clone ID SSH... URL http://dictycdb.biol.tsukuba.ac.jp/CSM/SS/SSH5-C/SSH570Q.Seq.d/ Representative seq. ID SSH...570P (Link to Original site) Representative DNA sequence >SSH570 (SSH570Q) /CSM/SS/SSH5-C/SSH5...M-cDNA Score E Sequences producing significant alignments: (bits) Value SSH570 (SSH570Q) /CSM/SS/SSH5-C/SSH5

  15. Dicty_cDB: SSH503 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available ycdb.biol.tsukuba.ac.jp/CSM/SS/SSH5-A/SSH503Q.Seq.d/ Representative seq. ID SSH50...3F (Link to Original site) Representative DNA sequence >SSH503 (SSH503Q) /CSM/SS/SSH5-A/SSH503Q.Seq.d/ AGAAA

  16. Avoiding cross hybridization by choosing nonredundant targets on cDNA arrays

    DEFF Research Database (Denmark)

    Nielsen, Henrik Bjørn; Knudsen, Steen

    2002-01-01

    PROBEWIZ designs PCR primers for amplifying probes for cDNA arrays. The probes are designed to have minimal homology to other expressed sequences from a given organism. The primer selection is based on user-defined penalties for homology, primer quality, and proximity to the 3' end.......PROBEWIZ designs PCR primers for amplifying probes for cDNA arrays. The probes are designed to have minimal homology to other expressed sequences from a given organism. The primer selection is based on user-defined penalties for homology, primer quality, and proximity to the 3' end....

  17. Construction of the subtractive cDNA library of injured adult and fetal rabbit skins

    Institute of Scientific and Technical Information of China (English)

    张波; 刘大维; 王正国; 朱佩芳; 周继红; 蒋建新

    2004-01-01

    Objective: Early gestational mammalian fetuses possess the amazing ability to heal cutaneous wounds in a scarless fashion. Over the past years, scientists have been working to decipher the mechanisms underlying this regenerative repair. The remarkable phenotypic differences between fetal and adult healings behoves us to learn their characteristics in genetics, which represents potentially important mechanisms involved in wound repair observed in fetal versus adult tissues. In this sense, it is reasonable to construct subtractive cDNA library for future research.Methods: Middle laparotomy and hysterotomy were performed on pregnant rabbits at 20-day gestation to expose the fetal back, and a longitudinal incision through the skin was made on the back of the fetus. The traumatized fetal skin was harvested 12 hours post-operation, the fetus control and traumatized adult skin specimens were taken at the same time. dscDNA was synthesized from total RNA of skin samples with SMART technology. Taking one of the three samples as Tester respectively and the other two as Drivers, we obtained 1 forward and 2 reverse hybridization products. After being amplified with selective polymerase chain reaction, the products were inserted into a vector, and then transferred into E.coli HB101. The colonies were screened afterwards. Results: The wounded fetuses were alive for a long time even after birth. Every determinant step, such as RNA isolation, cDNA synthesis, Rsa I digestion, adaptor ligation and hybridization, was well-operated. Subtractive efficiency identification demonstrated that the suppression subtractive hybridization (SSH) was successful. Insertion into vector and transferring to E.coli were satisfactory. Conclusions: Instead of classic SSH, an improved SSH with 2 Drivers was applied for the experiment. Results confirmed that the improved program was reasonable and correct in both theory and practice. The subtractive cDNA library we have obtained is going to be used for

  18. Avoiding cross hybridization by choosing nonredundant targets on cDNA arrays

    DEFF Research Database (Denmark)

    Nielsen, Henrik Bjørn; Knudsen, Steen

    2002-01-01

    PROBEWIZ designs PCR primers for amplifying probes for cDNA arrays. The probes are designed to have minimal homology to other expressed sequences from a given organism. The primer selection is based on user-defined penalties for homology, primer quality, and proximity to the 3' end....

  19. Dicty_cDB: SSH535 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available SS (Link to library) SSH535 (Link to dictyBase) - - - Contig-U15335-1 SSH535E (Link... to Original site) - - - - - - SSH535E 247 Show SSH535 Library SS (Link to library) Clone ID SSH535 (Link to...ycdb.biol.tsukuba.ac.jp/CSM/SS/SSH5-B/SSH535Q.Seq.d/ Representative seq. ID SSH53...5E (Link to Original site) Representative DNA sequence >SSH535 (SSH535Q) /CSM/SS/SSH5-B/SSH535Q.Seq.d/ TGGTA... Acid sequence (All Frames) Frame A: w*qmrfgi*ssshnw*rsrfskilr*sip*nlcqdscqr*gsfl

  20. Dicty_cDB: SSH832 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available SS (Link to library) SSH832 (Link to dictyBase) - G02305 DDB0231538 Contig-U14509-1 SSH...832E (Link to Original site) - - - - - - SSH832E 995 Show SSH832 Library SS (Link to library) Clone ID SSH...riginal site URL http://dictycdb.biol.tsukuba.ac.jp/CSM/SS/SSH8-B/SSH832Q.Seq.d/ ...Representative seq. ID SSH832E (Link to Original site) Representative DNA sequence >SSH832 (SSH832Q) /CSM/SS/SSH8-B/SSH...i*iyiykink*ng**cks*rifrcr**kikrw*ll*nvwwwffkirrc sfrlykgskfi*nvkemgssrcsiskssrmlfkgik*t*csiklcfssrll*krkcyrcn hmfessh

  1. Dicty_cDB: SSH711 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available SS (Link to library) SSH711 (Link to dictyBase) - - - - SSH711Z (Link to Original site) - - SSH...711Z 382 - - - - Show SSH711 Library SS (Link to library) Clone ID SSH711 (Link to dictyBase) At...las ID - NBRP ID - dictyBase ID - Link to Contig - Original site URL http://dictycdb.biol.tsukuba.ac.jp/CSM/SS/SSH7-A/SSH...711Q.Seq.d/ Representative seq. ID SSH711Z (Link to Original site) R...epresentative DNA sequence >SSH711 (SSH711Q) /CSM/SS/SSH7-A/SSH711Q.Seq.d/ XXXXXXXXXXTCACGTTCAGCTCGTGCCGGTAT

  2. Dicty_cDB: SSH880 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available SS (Link to library) SSH880 (Link to dictyBase) - G02308 DDB0191109 Contig-U13429-1 SSH...880Z (Link to Original site) - - SSH880Z 601 - - - - Show SSH880 Library SS (Link to library) Clone ID SSH...riginal site URL http://dictycdb.biol.tsukuba.ac.jp/CSM/SS/SSH8-D/SSH880Q.Seq.d/ ...Representative seq. ID SSH880Z (Link to Original site) Representative DNA sequence >SSH880 (SSH880Q) /CSM/SS/SSH8-D/SSH...hilf*if*lf*lvvl*qhvvfiy slmvysivlldlylvfiifwlvfvlysskssshkn**iflvfihiglvkvllfl*lvy*s leivdsf*qlvll*lplvlfv*f

  3. Dicty_cDB: SSH508 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available SS (Link to library) SSH508 (Link to dictyBase) - - - - SSH508E (Link to Original site) - - - - - - SSH...508E 470 Show SSH508 Library SS (Link to library) Clone ID SSH508 (Link to dictyBase) At...las ID - NBRP ID - dictyBase ID - Link to Contig - Original site URL http://dictycdb.biol.tsukuba.ac.jp/CSM/SS/SSH5-A/SSH...508Q.Seq.d/ Representative seq. ID SSH508E (Link to Original site) R...epresentative DNA sequence >SSH508 (SSH508Q) /CSM/SS/SSH5-A/SSH508Q.Seq.d/ GTTTTTAAAAAATTTATAAAAATAAAAATTATT

  4. Dicty_cDB: SSH324 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available SS (Link to library) SSH324 (Link to dictyBase) - - - Contig-U03732-1 SSH324Z (Link... to Original site) - - SSH324Z 644 - - - - Show SSH324 Library SS (Link to library) Clone ID SSH324 (Link to...ycdb.biol.tsukuba.ac.jp/CSM/SS/SSH3-A/SSH324Q.Seq.d/ Representative seq. ID SSH32...4Z (Link to Original site) Representative DNA sequence >SSH324 (SSH324Q) /CSM/SS/SSH3-A/SSH324Q.Seq.d/ XXXXX...SQMNPMAYPDLQLPPVAE*ik****yntx x**npkkqqntilqpllhkxxkkkkikk*q*kkhqhkrihinvyspsshqq

  5. Dicty_cDB: SSH667 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available SS (Link to library) SSH667 (Link to dictyBase) - - - Contig-U12865-1 SSH667Z (Link... to Original site) - - SSH667Z 358 - - - - Show SSH667 Library SS (Link to library) Clone ID SSH667 (Link to...ycdb.biol.tsukuba.ac.jp/CSM/SS/SSH6-C/SSH667Q.Seq.d/ Representative seq. ID SSH66...7Z (Link to Original site) Representative DNA sequence >SSH667 (SSH667Q) /CSM/SS/SSH6-C/SSH667Q.Seq.d/ XXXXX...lkmnplivliqkqklvtklnqrlmplplviphqpllsmfcf*lhlll*lls f Frame C: ---tkfsshqrfkyr*q*

  6. Dicty_cDB: SSH893 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available SS (Link to library) SSH893 (Link to dictyBase) - - - Contig-U16327-1 SSH893F (Link to Original site) SSH...893F 385 - - - - - - Show SSH893 Library SS (Link to library) Clone ID SSH893 (Link to...ycdb.biol.tsukuba.ac.jp/CSM/SS/SSH8-D/SSH893Q.Seq.d/ Representative seq. ID SSH89...3F (Link to Original site) Representative DNA sequence >SSH893 (SSH893Q) /CSM/SS/SSH8-D/SSH893Q.Seq.d/ AAAAA...SLLTYANAYDYFTTTLANQNPVCASVDVI QNVCTEVCGRFVRYIPDATNTNQFTFAEYTTNQCTVQVTPAVTNTFTCADQTSSHALGSD WSGVCKI--- Transl

  7. Dicty_cDB: SSH429 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available SS (Link to library) SSH429 (Link to dictyBase) - - - Contig-U14705-1 SSH429Z (Link... to Original site) - - SSH429Z 564 - - - - Show SSH429 Library SS (Link to library) Clone ID SSH429 (Link to...ycdb.biol.tsukuba.ac.jp/CSM/SS/SSH4-B/SSH429Q.Seq.d/ Representative seq. ID SSH42...9Z (Link to Original site) Representative DNA sequence >SSH429 (SSH429Q) /CSM/SS/SSH4-B/SSH429Q.Seq.d/ XXXXX...qlsktmlw*hvlfnftk*pilfn*flwkhkclwsssim*wcslfhs**stfwl w*ifkslqkw*mc*spnl*cwsshvg*trcwyddy*citnylsrshwwiimwle

  8. Dicty_cDB: SSH447 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available SS (Link to library) SSH447 (Link to dictyBase) - - - Contig-U13429-1 SSH447Z (Link... to Original site) - - SSH447Z 514 - - - - Show SSH447 Library SS (Link to library) Clone ID SSH447 (Link to...ycdb.biol.tsukuba.ac.jp/CSM/SS/SSH4-B/SSH447Q.Seq.d/ Representative seq. ID SSH44...7Z (Link to Original site) Representative DNA sequence >SSH447 (SSH447Q) /CSM/SS/SSH4-B/SSH447Q.Seq.d/ XXXXX...-anqlhilf*if*lf*lvvl*qhvvfiyslmvysivlldlylvfiifwlvfvlyssks sshkn**iflvfihiglvkvllfl*lvy*sleivdsf*qlvll*lplvl

  9. Dicty_cDB: SSH326 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available SS (Link to library) SSH326 (Link to dictyBase) - - - - SSH326E (Link to Original site) - - - - - - SSH...326E 545 Show SSH326 Library SS (Link to library) Clone ID SSH326 (Link to dictyBase) At...las ID - NBRP ID - dictyBase ID - Link to Contig - Original site URL http://dictycdb.biol.tsukuba.ac.jp/CSM/SS/SSH3-B/SSH...326Q.Seq.d/ Representative seq. ID SSH326E (Link to Original site) R...epresentative DNA sequence >SSH326 (SSH326Q) /CSM/SS/SSH3-B/SSH326Q.Seq.d/ AATTCAATTATAAACAACAAATTAAAAACTCAA

  10. Construction and selection of subtracted cDNA library of mouse hepatocarcinoma cell lines with different lymphatic metastasis potential

    Institute of Scientific and Technical Information of China (English)

    Li Hou; Jan-Wu Tang; Xiao-Nan Cui; Bo Wang; Bo Song; Lei Sun

    2004-01-01

    AIM: In order to elucidate the molecular mechanism of lymphatic metastasis of hepatocarcinoma, we detected the difference of gene expression between mouse hepatocarcinoma cell lines Hca-F and Hca-P with different lymphatic metastasis potential.METHODS: cDNA of Hca-F cells was used as a tester and cDNA of Hca-P cells was used as a driver. cDNAs highly expressed in Hca-F cells were isolated by the suppression subtractive hybridization (SSH) method. The isolated cDNA was cloned into T/A cloning vector. The ligation products were transformed into DH5 α competent cells. Individual clones were randomly selected and used for PCR amplification.Vector DNA from positive clones was isolated for sequencing.RESULTS: There were 800 positive clones in amplified subtracted cDNA library. Random analysis of 160 clones with PCR showed that 95% of the clones contained 100-700 bp inserts. Analysis of 20 sequenced cDNA clones randomly picked from the SSH library revealed 4 known genes (mouse heat shock protein 84 ku, DNA helicase, ribosomal protein S13 ,ethanol induced 6 gene) and 3 expressed sequence tags (ESTs). Four cDNAs showed no homology and presumably represent novel genes.CONCLUSION: A subtracted cDNA library of differentially expressed genes in mouse heptocarcinoma cell lines with different lymphatic metastasis potential was successfully constructed with SSH and T/A cloning techniques. The library is efficient and lays a solid foundation for searching new lymphatic metastasis related genes. The expression of mouse heat shock protein gene, DNA helicase and other 4 novel gene may be different between mouse heptocarcinoma cell lines with different lymphatic metastasis potential.

  11. Dicty_cDB: SSH791 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available SS (Link to library) SSH791 (Link to dictyBase) - - - Contig-U03296-1 SSH791E (Link to Original site) SSH...791F 370 SSH791Z 306 SSH791P 676 SSH791E 504 Show SSH791 Library SS (Link to library) Clone ID SSH...nal site URL http://dictycdb.biol.tsukuba.ac.jp/CSM/SS/SSH7-D/SSH791Q.Seq.d/ Representative seq. ID SSH...791E (Link to Original site) Representative DNA sequence >SSH791 (SSH791Q) /CSM/SS/SSH7-D/SSH...lignments: (bits) Value SSL655 (SSL655Q) /CSM/SS/SSL6-C/SSL655Q.Seq.d/ 999 0.0 SSH791 (SSH

  12. Dicty_cDB: SSH770 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available SS (Link to library) SSH770 (Link to dictyBase) - G21033 DDB0217014 Contig-U09425-1 SSH...770P (Link to Original site) SSH770F 255 SSH770Z 378 SSH770P 633 - - Show SSH770 Library SS (Link to library) Clone ID SSH...ontig-U09425-1 Original site URL http://dictycdb.biol.tsukuba.ac.jp/CSM/SS/SSH7-C/SSH...770Q.Seq.d/ Representative seq. ID SSH770P (Link to Original site) Representative DNA sequence >SSH770 (SSH770Q) /CSM/SS/SSH...vk Homology vs CSM-cDNA Score E Sequences producing significant alignments: (bits) Value SSH770 (SSH770Q) /CSM/SS/SSH7-C/SSH

  13. Dicty_cDB: SSH681 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available SS (Link to library) SSH681 (Link to dictyBase) - G00800 DDB0189403 Contig-U09422-1 SSH...681P (Link to Original site) SSH681F 304 SSH681Z 355 SSH681P 659 - - Show SSH681 Library SS (Link to library) Clone ID SSH...ontig-U09422-1 Original site URL http://dictycdb.biol.tsukuba.ac.jp/CSM/SS/SSH6-D/SSH...681Q.Seq.d/ Representative seq. ID SSH681P (Link to Original site) Representative DNA sequence >SSH681 (SSH681Q) /CSM/SS/SSH...cing significant alignments: (bits) Value SSH681 (SSH681Q) /CSM/SS/SSH6-D/SSH681Q

  14. Dicty_cDB: SSH647 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available SS (Link to library) SSH647 (Link to dictyBase) - G00798 DDB0216578 Contig-U01803-1 SSH...647P (Link to Original site) SSH647F 323 SSH647Z 337 SSH647P 660 - - Show SSH647 Library SS (Link to library) Clone ID SSH...ontig-U01803-1 Original site URL http://dictycdb.biol.tsukuba.ac.jp/CSM/SS/SSH6-B/SSH...647Q.Seq.d/ Representative seq. ID SSH647P (Link to Original site) Representative DNA sequence >SSH647 (SSH647Q) /CSM/SS/SSH...cing significant alignments: (bits) Value SSH647 (SSH647Q) /CSM/SS/SSH6-B/SSH647Q

  15. Dicty_cDB: SSH344 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available SS (Link to library) SSH344 (Link to dictyBase) - G02281 DDB0190364 Contig-U04680-1 SSH...344P (Link to Original site) SSH344F 378 SSH344Z 530 SSH344P 908 - - Show SSH344 Library SS (Link to library) Clone ID SSH...ontig-U04680-1 Original site URL http://dictycdb.biol.tsukuba.ac.jp/CSM/SS/SSH3-B/SSH...344Q.Seq.d/ Representative seq. ID SSH344P (Link to Original site) Representative DNA sequence >SSH344 (SSH344Q) /CSM/SS/SSH... significant alignments: (bits) Value SSH344 (SSH344Q) /CSM/SS/SSH3-B/SSH344Q.Seq.d/ 844 0.0 SSL102 (SSL102Q

  16. Dicty_cDB: SSH262 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available SS (Link to library) SSH262 (Link to dictyBase) - G02279 DDB0232044 Contig-U10243-1 SSH...262P (Link to Original site) SSH262F 411 SSH262Z 346 SSH262P 757 - - Show SSH262 Library SS (Link to library) Clone ID SSH...ontig-U10243-1 Original site URL http://dictycdb.biol.tsukuba.ac.jp/CSM/SS/SSH2-C/SSH...262Q.Seq.d/ Representative seq. ID SSH262P (Link to Original site) Representative DNA sequence >SSH262 (SSH262Q) /CSM/SS/SSH...omology vs CSM-cDNA Score E Sequences producing significant alignments: (bits) Value SSH262 (SSH262Q) /CSM/SS/SSH2-C/SSH

  17. Dicty_cDB: SSH842 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available SS (Link to library) SSH842 (Link to dictyBase) - - - Contig-U16382-1 SSH842E (Link... to Original site) - - - - - - SSH842E 344 Show SSH842 Library SS (Link to library) Clone ID SSH842 (Link to...ycdb.biol.tsukuba.ac.jp/CSM/SS/SSH8-B/SSH842Q.Seq.d/ Representative seq. ID SSH84...2E (Link to Original site) Representative DNA sequence >SSH842 (SSH842Q) /CSM/SS/SSH8-B/SSH842Q.Seq.d/ CACAT...alignments: (bits) Value SSH842 (SSH842Q) /CSM/SS/SSH8-B/SSH842Q.Seq.d/ 541 e-153 VSD831 (VSD831Q) /CSM/VS/V

  18. Dicty_cDB: SSH477 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available SS (Link to library) SSH477 (Link to dictyBase) - G02285 DDB0167090 Contig-U05359-1 SSH...477F (Link to Original site) SSH477F 280 - - - - - - Show SSH477 Library SS (Link to library) Clone ID SSH...riginal site URL http://dictycdb.biol.tsukuba.ac.jp/CSM/SS/SSH4-D/SSH477Q.Seq.d/ ...Representative seq. ID SSH477F (Link to Original site) Representative DNA sequence >SSH477 (SSH477Q) /CSM/SS/SSH4-D/SSH...ficant alignments: (bits) Value SSH477 (SSH477Q) /CSM/SS/SSH4-D/SSH477Q.Seq.d/ 133 2e-30 SSL641 (SSL641Q) /C

  19. Dicty_cDB: SSH783 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available SS (Link to library) SSH783 (Link to dictyBase) - - - Contig-U16441-1 SSH783Z (Link... to Original site) - - SSH783Z 361 - - - - Show SSH783 Library SS (Link to library) Clone ID SSH783 (Link to...ycdb.biol.tsukuba.ac.jp/CSM/SS/SSH7-D/SSH783Q.Seq.d/ Representative seq. ID SSH78...3Z (Link to Original site) Representative DNA sequence >SSH783 (SSH783Q) /CSM/SS/SSH7-D/SSH783Q.Seq.d/ XXXXX.../CSM/VS/VSA8-A/VSA811Q.Seq.d/ 664 0.0 SSH783 (SSH783Q) /CSM/SS/SSH7-D/SSH783Q.Seq

  20. Dicty_cDB: SSH351 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available SS (Link to library) SSH351 (Link to dictyBase) - - - Contig-U01797-1 SSH351F (Link to Original site) SSH...351F 189 - - - - - - Show SSH351 Library SS (Link to library) Clone ID SSH351 (Link to...ycdb.biol.tsukuba.ac.jp/CSM/SS/SSH3-C/SSH351Q.Seq.d/ Representative seq. ID SSH35...1F (Link to Original site) Representative DNA sequence >SSH351 (SSH351Q) /CSM/SS/SSH3-C/SSH351Q.Seq.d/ AAAAA... Homology vs CSM-cDNA Score E Sequences producing significant alignments: (bits) Value SSH580 (SSH580Q) /CSM/SS/SSH5-D/SSH

  1. Dicty_cDB: SSH202 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available SS (Link to library) SSH202 (Link to dictyBase) - G21004 DDB0169430 Contig-U01777-1 SSH...202P (Link to Original site) SSH202F 307 SSH202Z 410 SSH202P 717 - - Show SSH202 Library SS (Link to library) Clone ID SSH...ontig-U01777-1 Original site URL http://dictycdb.biol.tsukuba.ac.jp/CSM/SS/SSH2-A/SSH...202Q.Seq.d/ Representative seq. ID SSH202P (Link to Original site) Representative DNA sequence >SSH202 (SSH202Q) /CSM/SS/SSH...kkkkkkkkkkke Homology vs CSM-cDNA Score E Sequences producing significant alignments: (bits) Value SSH202 (SSH202Q) /CSM/SS/SSH

  2. Dicty_cDB: SSH543 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available SS (Link to library) SSH543 (Link to dictyBase) - - - Contig-U05038-1 SSH543E (Link... to Original site) - - - - - - SSH543E 353 Show SSH543 Library SS (Link to library) Clone ID SSH543 (Link to...ycdb.biol.tsukuba.ac.jp/CSM/SS/SSH5-B/SSH543Q.Seq.d/ Representative seq. ID SSH54...3E (Link to Original site) Representative DNA sequence >SSH543 (SSH543Q) /CSM/SS/SSH5-B/SSH543Q.Seq.d/ ATTTT...roducing significant alignments: (bits) Value SSH543 (SSH543Q) /CSM/SS/SSH5-B/SSH543Q.Seq.d/ 349 2e-95 SSE66

  3. Dicty_cDB: SSH342 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available SS (Link to library) SSH342 (Link to dictyBase) - - - Contig-U10784-1 SSH342F (Link to Original site) SSH...342F 339 - - - - - - Show SSH342 Library SS (Link to library) Clone ID SSH342 (Link to...ycdb.biol.tsukuba.ac.jp/CSM/SS/SSH3-B/SSH342Q.Seq.d/ Representative seq. ID SSH34...2F (Link to Original site) Representative DNA sequence >SSH342 (SSH342Q) /CSM/SS/SSH3-B/SSH342Q.Seq.d/ CAAAA...nificant alignments: (bits) Value SSH342 (SSH342Q) /CSM/SS/SSH3-B/SSH342Q.Seq.d/ 672 0.0 SLE452 (SLE452Q) /C

  4. Dicty_cDB: SSH859 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available SS (Link to library) SSH859 (Link to dictyBase) - - - Contig-U16289-1 SSH859Z (Link... to Original site) - - SSH859Z 469 - - - - Show SSH859 Library SS (Link to library) Clone ID SSH859 (Link to...ycdb.biol.tsukuba.ac.jp/CSM/SS/SSH8-C/SSH859Q.Seq.d/ Representative seq. ID SSH85...9Z (Link to Original site) Representative DNA sequence >SSH859 (SSH859Q) /CSM/SS/SSH8-C/SSH859Q.Seq.d/ XXXXX...-A/SSI621Q.Seq.d/ 609 e-173 SSI264 (SSI264Q) /CSM/SS/SSI2-C/SSI264Q.Seq.d/ 609 e-173 SSH859 (SSH859Q) /CSM/SS/SSH8-C/SSH

  5. Dicty_cDB: SSH123 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available SS (Link to library) SSH123 (Link to dictyBase) ssh123 G01233 DDB0217220 Contig-U01...Library SS (Link to library) Clone ID SSH123 (Link to dictyBase) Atlas ID ssh123 NBRP ID G01233 dictyBase ID...765-1 | Contig-U16448-1 SSH123P (Link to Original site) SSH123F 377 SSH123Z 186 SSH123P 563 - - Show SSH123 ...tp://dictycdb.biol.tsukuba.ac.jp/CSM/SS/SSH1-A/SSH123Q.Seq.d/ Representative seq. ID SSH123P (Link to Origin...al site) Representative DNA sequence >SSH123 (SSH123Q) /CSM/SS/SSH1-A/SSH123Q.Seq.d/ TTTTTTTTTTAAAATTTTTCTTT

  6. Dicty_cDB: SSH895 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available SS (Link to library) SSH895 (Link to dictyBase) - - - Contig-U07719-1 SSH895Z (Link... to Original site) - - SSH895Z 590 - - - - Show SSH895 Library SS (Link to library) Clone ID SSH895 (Link to...ycdb.biol.tsukuba.ac.jp/CSM/SS/SSH8-D/SSH895Q.Seq.d/ Representative seq. ID SSH89...5Z (Link to Original site) Representative DNA sequence >SSH895 (SSH895Q) /CSM/SS/SSH8-D/SSH895Q.Seq.d/ XXXXX...) /CSM/SS/SSI4-C/SSI455Q.Seq.d/ 1059 0.0 SSH895 (SSH895Q) /CSM/SS/SSH8-D/SSH895Q.Seq.d/ 1059 0.0 SSE774 (SSE

  7. Dicty_cDB: SSH279 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available SS (Link to library) SSH279 (Link to dictyBase) - - - Contig-U10253-1 SSH279E (Link... to Original site) - - - - - - SSH279E 427 Show SSH279 Library SS (Link to library) Clone ID SSH279 (Link to...ycdb.biol.tsukuba.ac.jp/CSM/SS/SSH2-D/SSH279Q.Seq.d/ Representative seq. ID SSH27...9E (Link to Original site) Representative DNA sequence >SSH279 (SSH279Q) /CSM/SS/SSH2-D/SSH279Q.Seq.d/ CATAA...SS/SSI1-C/SSI155Q.Seq.d/ 349 2e-95 SSH279 (SSH279Q) /CSM/SS/SSH2-D/SSH279Q.Seq.d/ 349 2e-95 own update 2002.

  8. Dicty_cDB: SSH814 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available SS (Link to library) SSH814 (Link to dictyBase) - - - Contig-U13888-1 SSH814Z (Link... to Original site) - - SSH814Z 529 - - - - Show SSH814 Library SS (Link to library) Clone ID SSH814 (Link to...ycdb.biol.tsukuba.ac.jp/CSM/SS/SSH8-A/SSH814Q.Seq.d/ Representative seq. ID SSH81...4Z (Link to Original site) Representative DNA sequence >SSH814 (SSH814Q) /CSM/SS/SSH8-A/SSH814Q.Seq.d/ XXXXX...M681 (SSM681Q) /CSM/SS/SSM6-D/SSM681Q.Seq.d/ 993 0.0 SSH814 (SSH814Q) /CSM/SS/SSH8-A/SSH814Q.Seq.d/ 993 0.0

  9. Dicty_cDB: SSH196 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available SS (Link to library) SSH196 (Link to dictyBase) - - - Contig-U03803-1 SSH196Z (Link... to Original site) - - SSH196Z 554 - - - - Show SSH196 Library SS (Link to library) Clone ID SSH196 (Link to...ycdb.biol.tsukuba.ac.jp/CSM/SS/SSH1-D/SSH196Q.Seq.d/ Representative seq. ID SSH19...6Z (Link to Original site) Representative DNA sequence >SSH196 (SSH196Q) /CSM/SS/SSH1-D/SSH196Q.Seq.d/ XXXXX...re E Sequences producing significant alignments: (bits) Value SSH196 (SSH196Q) /CSM/SS/SSH1-D/SSH196Q.Seq.d/

  10. Dicty_cDB: SSH732 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available SS (Link to library) SSH732 (Link to dictyBase) - - - Contig-U12272-1 SSH732F (Link to Original site) SSH...732F 336 - - - - - - Show SSH732 Library SS (Link to library) Clone ID SSH732 (Link to...ycdb.biol.tsukuba.ac.jp/CSM/SS/SSH7-B/SSH732Q.Seq.d/ Representative seq. ID SSH73...2F (Link to Original site) Representative DNA sequence >SSH732 (SSH732Q) /CSM/SS/SSH7-B/SSH732Q.Seq.d/ AAAAA...t alignments: (bits) Value SSL833 (SSL833Q) /CSM/SS/SSL8-B/SSL833Q.Seq.d/ 402 e-111 SSH732 (SSH732Q) /CSM/SS/SSH7-B/SSH

  11. Dicty_cDB: SSH726 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available SS (Link to library) SSH726 (Link to dictyBase) - - - Contig-U15185-1 SSH726E (Link to Original site) SSH...726F 290 SSH726Z 187 SSH726P 477 SSH726E 365 Show SSH726 Library SS (Link to library) Clone ID SSH...nal site URL http://dictycdb.biol.tsukuba.ac.jp/CSM/SS/SSH7-B/SSH726Q.Seq.d/ Representative seq. ID SSH...726E (Link to Original site) Representative DNA sequence >SSH726 (SSH726Q) /CSM/SS/SSH7-B/SSH...%: mitochondrial 28.0 %: cytoplasmic 20.0 %: nuclear 4.0 %: plasma membrane 4.0 %: peroxisomal >> prediction for SSH

  12. Dicty_cDB: SSH403 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available SS (Link to library) SSH403 (Link to dictyBase) - - - Contig-U04637-1 SSH403Z (Link... to Original site) - - SSH403Z 563 - - - - Show SSH403 Library SS (Link to library) Clone ID SSH403 (Link to...ycdb.biol.tsukuba.ac.jp/CSM/SS/SSH4-A/SSH403Q.Seq.d/ Representative seq. ID SSH40...3Z (Link to Original site) Representative DNA sequence >SSH403 (SSH403Q) /CSM/SS/SSH4-A/SSH403Q.Seq.d/ XXXXX...SSJ8-C/SSJ869Q.Seq.d/ 912 0.0 SSH403 (SSH403Q) /CSM/SS/SSH4-A/SSH403Q.Seq.d/ 912 0.0 SSG441 (SSG441Q) /CSM/S

  13. Dicty_cDB: SSH260 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available SS (Link to library) SSH260 (Link to dictyBase) - - - Contig-U15823-1 SSH260Z (Link... to Original site) - - SSH260Z 561 - - - - Show SSH260 Library SS (Link to library) Clone ID SSH260 (Link to...ycdb.biol.tsukuba.ac.jp/CSM/SS/SSH2-C/SSH260Q.Seq.d/ Representative seq. ID SSH26...0Z (Link to Original site) Representative DNA sequence >SSH260 (SSH260Q) /CSM/SS/SSH2-C/SSH260Q.Seq.d/ XXXXX.../VFF3-C/VFF363Q.Seq.d/ 361 8e-99 SSL121 (SSL121Q) /CSM/SS/SSL1-A/SSL121Q.Seq.d/ 361 8e-99 SSH260 (SSH260Q) /CSM/SS/SSH2-C/SSH

  14. Dicty_cDB: SSH128 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available SS (Link to library) SSH128 (Link to dictyBase) - G21001 DDB0201637 Contig-U04054-1 SSH...128Z (Link to Original site) - - SSH128Z 620 - - - - Show SSH128 Library SS (Link to library) Clone ID SSH...riginal site URL http://dictycdb.biol.tsukuba.ac.jp/CSM/SS/SSH1-B/SSH128Q.Seq.d/ ...Representative seq. ID SSH128Z (Link to Original site) Representative DNA sequence >SSH128 (SSH128Q) /CSM/SS/SSH1-B/SSH...ant alignments: (bits) Value SSH128 (SSH128Q) /CSM/SS/SSH1-B/SSH128Q.Seq.d/ 1207 0.0 SLE645 (SLE645Q) /CSM/S

  15. Dicty_cDB: SSH433 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available SS (Link to library) SSH433 (Link to dictyBase) - - - Contig-U15600-1 SSH433F (Link to Original site) SSH...433F 336 - - - - - - Show SSH433 Library SS (Link to library) Clone ID SSH433 (Link to...ycdb.biol.tsukuba.ac.jp/CSM/SS/SSH4-B/SSH433Q.Seq.d/ Representative seq. ID SSH43...3F (Link to Original site) Representative DNA sequence >SSH433 (SSH433Q) /CSM/SS/SSH4-B/SSH433Q.Seq.d/ AAATT...t alignments: (bits) Value SSH433 (SSH433Q) /CSM/SS/SSH4-B/SSH433Q.Seq.d/ 297 6e-80 SSF237 (SSF237Q) /CSM/SS

  16. Dicty_cDB: SSH518 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available SS (Link to library) SSH518 (Link to dictyBase) - - - Contig-U16252-1 SSH518E (Link... to Original site) - - - - - - SSH518E 456 Show SSH518 Library SS (Link to library) Clone ID SSH518 (Link to...ycdb.biol.tsukuba.ac.jp/CSM/SS/SSH5-A/SSH518Q.Seq.d/ Representative seq. ID SSH51...8E (Link to Original site) Representative DNA sequence >SSH518 (SSH518Q) /CSM/SS/SSH5-A/SSH518Q.Seq.d/ AAAGA...-B/SSI448Q.Seq.d/ 672 0.0 SSH518 (SSH518Q) /CSM/SS/SSH5-A/SSH518Q.Seq.d/ 672 0.0 SLF154 (SLF154Q) /CSM/SL/SL

  17. Dicty_cDB: SSH619 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available SS (Link to library) SSH619 (Link to dictyBase) - - - Contig-U04437-1 SSH619Z (Link... to Original site) - - SSH619Z 611 - - - - Show SSH619 Library SS (Link to library) Clone ID SSH619 (Link to...ycdb.biol.tsukuba.ac.jp/CSM/SS/SSH6-A/SSH619Q.Seq.d/ Representative seq. ID SSH61...9Z (Link to Original site) Representative DNA sequence >SSH619 (SSH619Q) /CSM/SS/SSH6-A/SSH619Q.Seq.d/ XXXXX... Homology vs CSM-cDNA Score E Sequences producing significant alignments: (bits) Value SSH619 (SSH619Q) /CSM/SS/SSH6-A/SSH

  18. Dicty_cDB: SSH436 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available SS (Link to library) SSH436 (Link to dictyBase) - - - Contig-U12201-1 SSH436Z (Link... to Original site) - - SSH436Z 551 - - - - Show SSH436 Library SS (Link to library) Clone ID SSH436 (Link to...ycdb.biol.tsukuba.ac.jp/CSM/SS/SSH4-B/SSH436Q.Seq.d/ Representative seq. ID SSH43...6Z (Link to Original site) Representative DNA sequence >SSH436 (SSH436Q) /CSM/SS/SSH4-B/SSH436Q.Seq.d/ XXXXX...0.0 SSL223 (SSL223Q) /CSM/SS/SSL2-A/SSL223Q.Seq.d/ 694 0.0 SSH436 (SSH436Q) /CSM/SS/SSH4-B/SSH436Q.Seq.d/ 69

  19. Dicty_cDB: SSH176 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available SS (Link to library) SSH176 (Link to dictyBase) - - - Contig-U09430-1 SSH176E (Link... to Original site) - - - - - - SSH176E 415 Show SSH176 Library SS (Link to library) Clone ID SSH176 (Link to...ycdb.biol.tsukuba.ac.jp/CSM/SS/SSH1-D/SSH176Q.Seq.d/ Representative seq. ID SSH17...6E (Link to Original site) Representative DNA sequence >SSH176 (SSH176Q) /CSM/SS/SSH1-D/SSH176Q.Seq.d/ AAACT...) /CSM/SS/SSI3-C/SSI361Q.Seq.d/ 692 0.0 SSH176 (SSH176Q) /CSM/SS/SSH1-D/SSH176Q.Seq.d/ 692 0.0 SSF336 (SSF33

  20. Dicty_cDB: SSH602 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available SS (Link to library) SSH602 (Link to dictyBase) - - - Contig-U13413-1 SSH602F (Link to Original site) SSH...602F 311 - - - - - - Show SSH602 Library SS (Link to library) Clone ID SSH602 (Link to...ycdb.biol.tsukuba.ac.jp/CSM/SS/SSH6-A/SSH602Q.Seq.d/ Representative seq. ID SSH60...2F (Link to Original site) Representative DNA sequence >SSH602 (SSH602Q) /CSM/SS/SSH6-A/SSH602Q.Seq.d/ CTTGT...SI2-C/SSI258Q.Seq.d/ 527 e-149 SSH602 (SSH602Q) /CSM/SS/SSH6-A/SSH602Q.Seq.d/ 527

  1. Dicty_cDB: SSH479 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available SS (Link to library) SSH479 (Link to dictyBase) - - - Contig-U11350-1 SSH479Z (Link... to Original site) - - SSH479Z 544 - - - - Show SSH479 Library SS (Link to library) Clone ID SSH479 (Link to...ycdb.biol.tsukuba.ac.jp/CSM/SS/SSH4-D/SSH479Q.Seq.d/ Representative seq. ID SSH47...9Z (Link to Original site) Representative DNA sequence >SSH479 (SSH479Q) /CSM/SS/SSH4-D/SSH479Q.Seq.d/ XXXXX...g significant alignments: (bits) Value SSH479 (SSH479Q) /CSM/SS/SSH4-D/SSH479Q.Seq.d/ 1078 0.0 SSE215 (SSE21

  2. Dicty_cDB: SSH861 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available SS (Link to library) SSH861 (Link to dictyBase) - - - Contig-U10970-1 SSH861Z (Link... to Original site) - - SSH861Z 570 - - - - Show SSH861 Library SS (Link to library) Clone ID SSH861 (Link to...ycdb.biol.tsukuba.ac.jp/CSM/SS/SSH8-C/SSH861Q.Seq.d/ Representative seq. ID SSH86...1Z (Link to Original site) Representative DNA sequence >SSH861 (SSH861Q) /CSM/SS/SSH8-C/SSH861Q.Seq.d/ XXXXX.../VFD762Q.Seq.d/ 876 0.0 VFB810 (VFB810Q) /CSM/VF/VFB8-A/VFB810Q.Seq.d/ 876 0.0 SSH861 (SSH861Q) /CSM/SS/SSH8-C/SSH

  3. Dicty_cDB: SSH218 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available SS (Link to library) SSH218 (Link to dictyBase) - G21005 DDB0185803 Contig-U05324-1 SSH...218E (Link to Original site) - - - - - - SSH218E 360 Show SSH218 Library SS (Link to library) Clone ID SSH...riginal site URL http://dictycdb.biol.tsukuba.ac.jp/CSM/SS/SSH2-A/SSH218Q.Seq.d/ ...Representative seq. ID SSH218E (Link to Original site) Representative DNA sequence >SSH218 (SSH218Q) /CSM/SS/SSH2-A/SSH...fvf Homology vs CSM-cDNA Score E Sequences producing significant alignments: (bits) Value SSH218 (SSH218Q) /CSM/SS/SSH2-A/SSH

  4. Dicty_cDB: SSH704 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available SS (Link to library) SSH704 (Link to dictyBase) ssh704 - - - SSH704P (Link to Original site) SSH704F 228 SSH...704Z 260 SSH704P 488 - - Show SSH704 Library SS (Link to library) Clone ID SSH704 (...Link to dictyBase) Atlas ID ssh704 NBRP ID - dictyBase ID - Link to Contig - Original site URL http://dictyc...db.biol.tsukuba.ac.jp/CSM/SS/SSH7-A/SSH704Q.Seq.d/ Representative seq. ID SSH704P... (Link to Original site) Representative DNA sequence >SSH704 (SSH704Q) /CSM/SS/SSH7-A/SSH704Q.Seq.d/ GGTGGTT

  5. Dicty_cDB: SSH153 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available SS (Link to library) SSH153 (Link to dictyBase) - - - Contig-U16354-1 SSH153E (Link... to Original site) - - - - - - SSH153E 615 Show SSH153 Library SS (Link to library) Clone ID SSH153 (Link to...ycdb.biol.tsukuba.ac.jp/CSM/SS/SSH1-C/SSH153Q.Seq.d/ Representative seq. ID SSH15...3E (Link to Original site) Representative DNA sequence >SSH153 (SSH153Q) /CSM/SS/SSH1-C/SSH153Q.Seq.d/ ATTAA...7-B/SSK729Q.Seq.d/ 1106 0.0 SSH153 (SSH153Q) /CSM/SS/SSH1-C/SSH153Q.Seq.d/ 1106 0

  6. Dicty_cDB: SSH831 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available SS (Link to library) SSH831 (Link to dictyBase) - G02304 DDB0231410 Contig-U13894-1 SSH...831Z (Link to Original site) - - SSH831Z 574 - - - - Show SSH831 Library SS (Link to library) Clone ID SSH...riginal site URL http://dictycdb.biol.tsukuba.ac.jp/CSM/SS/SSH8-B/SSH831Q.Seq.d/ ...Representative seq. ID SSH831Z (Link to Original site) Representative DNA sequence >SSH831 (SSH831Q) /CSM/SS/SSH8-B/SSH...nt alignments: (bits) Value SSH831 (SSH831Q) /CSM/SS/SSH8-B/SSH831Q.Seq.d/ 954 0.0 AFO821 (AFO821Q) /CSM/AF/

  7. Dicty_cDB: SSH618 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available SS (Link to library) SSH618 (Link to dictyBase) - - - Contig-U15932-1 SSH618Z (Link... to Original site) - - SSH618Z 371 - - - - Show SSH618 Library SS (Link to library) Clone ID SSH618 (Link to...ycdb.biol.tsukuba.ac.jp/CSM/SS/SSH6-A/SSH618Q.Seq.d/ Representative seq. ID SSH61...8Z (Link to Original site) Representative DNA sequence >SSH618 (SSH618Q) /CSM/SS/SSH6-A/SSH618Q.Seq.d/ XXXXX...CSM/SS/SSK1-A/SSK110Q.Seq.d/ 638 0.0 SSI103 (SSI103Q) /CSM/SS/SSI1-A/SSI103Q.Seq.d/ 638 0.0 SSH618 (SSH618Q) /CSM/SS/SSH6-A/SSH

  8. Dicty_cDB: SSH220 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available SS (Link to library) SSH220 (Link to dictyBase) - - - Contig-U16394-1 SSH220E (Link... to Original site) - - - - - - SSH220E 277 Show SSH220 Library SS (Link to library) Clone ID SSH220 (Link to...ycdb.biol.tsukuba.ac.jp/CSM/SS/SSH2-A/SSH220Q.Seq.d/ Representative seq. ID SSH22...0E (Link to Original site) Representative DNA sequence >SSH220 (SSH220Q) /CSM/SS/SSH2-A/SSH220Q.Seq.d/ ATTTA...Sequences producing significant alignments: (bits) Value SSH220 (SSH220Q) /CSM/SS/SSH2-A/SSH

  9. Dicty_cDB: SSH309 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available SS (Link to library) SSH309 (Link to dictyBase) - - - Contig-U16603-1 SSH309Z (Link... to Original site) - - SSH309Z 633 - - - - Show SSH309 Library SS (Link to library) Clone ID SSH309 (Link to...ycdb.biol.tsukuba.ac.jp/CSM/SS/SSH3-A/SSH309Q.Seq.d/ Representative seq. ID SSH30...9Z (Link to Original site) Representative DNA sequence >SSH309 (SSH309Q) /CSM/SS/SSH3-A/SSH309Q.Seq.d/ XXXXX.../SS/SSK7-C/SSK766Q.Seq.d/ 1122 0.0 SSH309 (SSH309Q) /CSM/SS/SSH3-A/SSH309Q.Seq.d/

  10. Dicty_cDB: SSH751 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available SS (Link to library) SSH751 (Link to dictyBase) - - - Contig-U01248-1 SSH751Z (Link... to Original site) - - SSH751Z 323 - - - - Show SSH751 Library SS (Link to library) Clone ID SSH751 (Link to...ycdb.biol.tsukuba.ac.jp/CSM/SS/SSH7-C/SSH751Q.Seq.d/ Representative seq. ID SSH75...1Z (Link to Original site) Representative DNA sequence >SSH751 (SSH751Q) /CSM/SS/SSH7-C/SSH751Q.Seq.d/ XXXXX...188 (SSM188Q) /CSM/SS/SSM1-D/SSM188Q.Seq.d/ 472 e-132 SSH751 (SSH751Q) /CSM/SS/SSH7-C/SSH751Q.Seq.d/ 472 e-1

  11. Dot-Blot Hybridization for Detection of Five Cucurbit Viruses by Digoxigenin-Labelled cDNA Probes

    Institute of Scientific and Technical Information of China (English)

    MENG Juan; GU Qin-sheng; LIN Shi-ming; PENG Bin; LIU Li-feng; TIAN Yan-ping; LI Li

    2007-01-01

    Dot-blot hybridization was applied in this paper to detect five viruses infecting cucurbitaceous crops,Zuccini yellow mosaic virus(ZYMV),Watermelon mosaic virus(WMV),Cucumber mosaic virus(CMV),Papaya ringspot virus watermelon strain(PRSV-W)and Squash mosaic virus(SqMV),as a good alternative assay in seed health test and epidemiological and transgenic research.Digoxigenin-labelled cDNA probes of the five viruses were synthesized by PCR with the specific primers and applied in dot-blot hybridization to detect five viruses in crude extraction of the infected leaves.And three SqMV probes of different lengths(0.55,1.6,and 2.7 kb,respectively)were designed to investigate the effect of hybridization.The results showed that the sensitivity for detecting the crude extraction of infected leaves by ZYMV,WMV,CMV,PRSV-W,and SqMV was down to 1:160,1:160,1:320,1:160,and 1:320,respectively.Three SqMV probes of different length showed no differences on the sensitivity and specificity.The digoxigenin-labelled probes prepared by PCR could be used for accurate and rapid identification of 5 viruses infecting cucurbitaceous crops with good stabilities,sensitivities,specificity,and reproducibilities.

  12. Screening and cloning of differentially expressed genes in placentas from patients of pregnancy-induced hypertension by suppression subtractive hybridization

    Institute of Scientific and Technical Information of China (English)

    尹国武; 姜锋; 李东红; 姚元庆

    2003-01-01

    Suppresssion subtractive hybridization (SSH) was preformed to compare gene expression profiles of PIH patients and normal pregnancy placentas. The subtractive cDNA library of PIH placenta was set up and screedned. Differential cDNAs were cloned, and sequenced by T 7 primer methodology. One hundred and three differential cDNAs were isolated by SSH. Sequencing and BLAST analysis showed 90 inserts shared more than 95% homolog with sequences in the GenBank/EMBL database. We identified 36 putative genes including pregnancy-specific glycoprotein gene (BC005924), serine protease inhibitor gene(BC012868), VEGFR-1 gene(AF063657, etc.

  13. Hybridization-based antibody cDNA recovery for the production of recombinant antibodies identified by repertoire sequencing.

    Science.gov (United States)

    Valdés-Alemán, Javier; Téllez-Sosa, Juan; Ovilla-Muñoz, Marbella; Godoy-Lozano, Elizabeth; Velázquez-Ramírez, Daniel; Valdovinos-Torres, Humberto; Gómez-Barreto, Rosa E; Martinez-Barnetche, Jesús

    2014-01-01

    High-throughput sequencing of the antibody repertoire is enabling a thorough analysis of B cell diversity and clonal selection, which may improve the novel antibody discovery process. Theoretically, an adequate bioinformatic analysis could allow identification of candidate antigen-specific antibodies, requiring their recombinant production for experimental validation of their specificity. Gene synthesis is commonly used for the generation of recombinant antibodies identified in silico. Novel strategies that bypass gene synthesis could offer more accessible antibody identification and validation alternatives. We developed a hybridization-based recovery strategy that targets the complementarity-determining region 3 (CDRH3) for the enrichment of cDNA of candidate antigen-specific antibody sequences. Ten clonal groups of interest were identified through bioinformatic analysis of the heavy chain antibody repertoire of mice immunized with hen egg white lysozyme (HEL). cDNA from eight of the targeted clonal groups was recovered efficiently, leading to the generation of recombinant antibodies. One representative heavy chain sequence from each clonal group recovered was paired with previously reported anti-HEL light chains to generate full antibodies, later tested for HEL-binding capacity. The recovery process proposed represents a simple and scalable molecular strategy that could enhance antibody identification and specificity assessment, enabling a more cost-efficient generation of recombinant antibodies.

  14. Isolation and identification of genes expressed differentially in rice inflorescence meristem with suppression subtractive hybridization

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    A subtracted cDNA library of rice (Oryza sativa L.) inflorescence meristem (IM) was constructed using the sup-pression subtractive hybridization (SSH) method. The cDNAs of the rice shoot apical meristem (SAM) were used as "driver" and inflorescence meristem (IM) as "tester" in the experiment, respectively. Forty of 250 randomly chosen cDNA clones were identified by differential screening, which were IM-specific or IM-highly expressed. Most of the rice IM cDNAs cloned by SSH appear to represent rare transcripts, 40% of which were derived from truly differentially ex-pressed genes. Of all the forty sequenced cDNA inserts, eleven contain the regions with 60%-90% identity to their homolog in GenBank, eighteen are expected to be new genes, only two correspond to published rice genes.

  15. Isolation of genes involved in the preventive effect of electroacupuncture at Fenglong acupoint (ST40) on hypercholesterolemia mice by suppression subtractive hybridization (SSH) combined with negative subtraction chain (NSC) technology.

    Science.gov (United States)

    Li, Xingjie; Zhang, Yizheng; Yan, Wenqi; Kang, Jinmei; Kang, Yaoxia; Lie, Min

    2006-01-01

    We have shown that electroacupuncture (EA) at Fenglong acupoint (ST40) has the cholesterol-lowering effect in hypercholesterolemia mice. The present study was designed to study preventive effect of EA at ST40 on hypercholesterolemia. C57BL/6j mice were randomly divided into normal group (NG), hypercholesterolemia group (HG) and EA prevention group (EPG). NG were fed chow, HG a hypercholesterolemic diet (HD), and EPG the same HD and received EA treatment simultaneously. Lipid profile of both the plasma and liver indicated that EA at ST40 had preventive effect on hypercholesterolemia. Compared with corresponding values in the HG mice, the levels of the hepatic total cholesterol and total triglyceride in the EPG mice lowered 45% and 23% respectively, and the levels of plasma total-, LDL-, and HDL-cholesterol in the EPG mice lowered 39%, 37% and 39% respectively. Eleven genes whose expressions were up-regulated in EPG mice compared with HG were isolated using suppression subtractive hybridization (SSH) combined with negative subtraction chain (NSC) technology, and then confirmed by dot-blot assay. Except two genes whose functions were still unknown, the others were mainly involved in cholesterol metabolism, lipid metabolism, glucose metabolism and immune response. The potential molecular mechanism of preventive effect was discussed.

  16. Dicty_cDB: SSH876 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available SS (Link to library) SSH876 (Link to dictyBase) - - - Contig-U14096-1 SSH876Z (Link... to Original site) - - SSH876Z 603 - - - - Show SSH876 Library SS (Link to library) Clone ID SSH876 (Link to...ycdb.biol.tsukuba.ac.jp/CSM/SS/SSH8-D/SSH876Q.Seq.d/ Representative seq. ID SSH87...6Z (Link to Original site) Representative DNA sequence >SSH876 (SSH876Q) /CSM/SS/SSH8-D/SSH876Q.Seq.d/ XXXXX...1Q.Seq.d/ 658 0.0 SSK159 (SSK159Q) /CSM/SS/SSK1-C/SSK159Q.Seq.d/ 658 0.0 SSH876 (SSH876Q) /CSM/SS/SSH

  17. Dicty_cDB: SSH724 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available SS (Link to library) SSH724 (Link to dictyBase) - - - Contig-U04708-1 SSH724Z (Link... to Original site) - - SSH724Z 323 - - - - Show SSH724 Library SS (Link to library) Clone ID SSH724 (Link to...ycdb.biol.tsukuba.ac.jp/CSM/SS/SSH7-A/SSH724Q.Seq.d/ Representative seq. ID SSH72...4Z (Link to Original site) Representative DNA sequence >SSH724 (SSH724Q) /CSM/SS/SSH7-A/SSH724Q.Seq.d/ XXXXX...) /CSM/SS/SSK5-C/SSK552Q.Seq.d/ 182 2e-45 SSJ422 (SSJ422Q) /CSM/SS/SSJ4-A/SSJ422Q.Seq.d/ 182 2e-45 SSH724 (SSH724Q) /CSM/SS/SSH

  18. Dicty_cDB: SSH847 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available SS (Link to library) SSH847 (Link to dictyBase) - - - Contig-U01248-1 SSH847E (Link to Original site) SSH...847F 283 SSH847Z 610 SSH847P 893 SSH847E 791 Show SSH847 Library SS (Link to library) Clone ID SSH...nal site URL http://dictycdb.biol.tsukuba.ac.jp/CSM/SS/SSH8-B/SSH847Q.Seq.d/ Representative seq. ID SSH...847E (Link to Original site) Representative DNA sequence >SSH847 (SSH847Q) /CSM/SS/SSH8-B/SSH...ames) Frame A: kkkkklknflkifpkpcrfffhikilflhs*f*syqnikngssss*mlqilqkqtihqi* ilsctrc*nqnfrfrs*esin**ipimcsshl

  19. Dicty_cDB: SSH381 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available SS (Link to library) SSH381 (Link to dictyBase) - - - Contig-U12167-1 SSH381E (Link to Original site) SSH...381F 299 SSH381Z 440 SSH381P 739 SSH381E 693 Show SSH381 Library SS (Link to library) Clone ID SSH...nal site URL http://dictycdb.biol.tsukuba.ac.jp/CSM/SS/SSH3-D/SSH381Q.Seq.d/ Representative seq. ID SSH...381E (Link to Original site) Representative DNA sequence >SSH381 (SSH381Q) /CSM/SS/SSH3-D/SSH...vttqnvklfstq*thyvqlpgfqnglnkkplisshqt efkyii*iinssinnlfkkkekkknimi*ikkmenfkkikkkkkkkkkkk Homology vs CSM-cDN

  20. Suppression subtractive hybridization (SSH)-based method for estimating Cd-induced differences in gene expression at cultivar level and identification of genes induced by Cd in two water spinach cultivars.

    Science.gov (United States)

    Huang, Baifei; Xin, Junliang; Yang, Zhongyi; Zhou, Yihui; Yuan, Jiangang; Gong, Yulian

    2009-10-14

    The abilities to accumulate cadmium (Cd) are different among cultivars (cv.) in many species. The characteristic of Cd concentration among cultivars is heritable and is probably controlled by genes, but rather limited information about the relevant genes in vegetable crops has been published. In the present study, a suppression subtractive hybridization (SSH) approach was used to identify genes induced by Cd in two water spinach (an important vegetable in southern China) cultivars that differ in Cd accumulation in their edible parts. The two cultivars were cv. Qiangkunqinggu (QK), a low Cd accumulative cultivar and cv. Taiwan 308 (TW), a high Cd accumulative cultivar. In the construction of QK and TW libraries, the plants without Cd treatment were taken as drivers and the plants exposed to 6 mg L(-1) Cd for 24 h as testers. Four hundred clones were sequenced, and 164 nonrepeated sequences (112 from the QK library and 52 from the TW library) were assigned to being functional genes or proteins. A tremendous difference in Cd-induced gene expressions between the two libraries was observed. In the QK library, genes implicated in disease/defense comprised one of the largest sets (20.6%), whereas the proportion was only 8.8% in the TW library. An MT3 gene (Q5), a wound inductive gene (Q22), an antioxidation relevant gene (Q34), a lectin gene (Q45), an f-box family protein gene (Q319), a 20S proteasome subunit gene (T17), a multidrug resistance associated protein gene (T156), and a cationic amino acid transporter gene (T218) were selected to compare semiquantitatively their expression between cv. QK and cv. TW using the RT-PCR method, and obvious differences were detected. The relationships between the identified differences in the expressions of the genes and the Cd accumulation of the two cultivars were discussed, and it was concluded that the SSH approach is useful for finding the difference in expression of Cd-induced gene even at the cultivar level and is applicable

  1. Identification of flowering-related genes between early flowering trifoliate orange mutant and wild-type trifoliate orange (Poncirus trifoliata L. Raf.) by suppression subtraction hybridization (SSH) and macroarray.

    Science.gov (United States)

    Zhang, Jin-Zhi; Li, Zhi-Min; Yao, Jia-Ling; Hu, Chun-Gen

    2009-02-01

    To gain a better understanding of gene expression in early flowering trifoliate orange mutant (precocious trifoliate orange, Poncirus trifoliata L. Raf.), we performed suppression subtractive hybridization, which allowed identification of flowering-related genes in the mutant and the wild type in the juvenile phase. Using macroarray analysis, we identified 125 and 149 non-redundant expressed sequence tags (ESTs) in the forward-subtracted and the reverse-subtracted library. These cDNAs covered a broad repertoire of flowering development related genes, provided helpful information for understanding genetic mechanism underlying the signaling and regulation in transition from the vegetative to reproductive phase. We have investigated the temporal and spatial expression pattern of some SSH-enriched flowering-related genes in the mutant and the wild type. Of these genes, three genes (BARELY ANY MERITED, FLOWERING LOCUS T and TERMINAL FLOWER1) encoding proteins previously reported to be associated with, or involved in, developmental processes in other species were identified and further investigated by in situ hybridization. Specific spatial and/or temporal patterns were detected, and differences were observed between the mutant and the wild type during flower development. Meanwhile, the temporal expression of these genes was further examined by real-time PCR, the results showed that FT and BAM transcripts accumulated to higher levels and TFL1 transcripts accumulated to lower levels in mutant juvenile tissues relative to wild-type juvenile tissues. In the adult stage, FT, BAM and TFL1 expression patterns were closely correlated with flowering development, suggesting that these three genes may play a critical role in the early flowering process of precocious trifoliate orange.

  2. Growth hormone regulation of rat liver gene expression assessed by SSH and microarray.

    Science.gov (United States)

    Gardmo, Cissi; Swerdlow, Harold; Mode, Agneta

    2002-04-25

    The sexually dimorphic secretion of growth hormone (GH) that prevails in the rat leads to a sex-differentiated expression of GH target genes, particularly in the liver. We have used subtractive suppressive hybridization (SSH) to search for new target genes induced by the female-characteristic, near continuous, pattern of GH secretion. Microarrays and dot-blot hybridizations were used in an attempt to confirm differential ratios of expression of obtained SSH clones. Out of 173 unique SSH clones, 41 could be verified as differentially expressed. Among these, we identified 17 known genes not previously recognized as differentially regulated by the sex-specific GH pattern. Additional SSH clones may also represent genes subjected to sex-specific GH regulation since only transcripts abundantly expressed could be verified. Optimized analyses, specific for each gene, are required to fully characterize the degree of differential expression.

  3. [Construction of SSH library from haemocyte of variously colored abalone challenged with bacteria and differential expression analysis of macrophage expressed protein].

    Science.gov (United States)

    Ren, Hong-Lin; Xu, Dan-Dan; Qiao, Kun; Cai, Ling; Huang, Wei-Bin; Zhang, Nai; Wang, Ke-Jian

    2008-08-01

    Abalones are considered to be the most precious delicacy from the sea, and become very important commercial seafood in aquaculture worldwide. Variously colored abalone (Haliotis diversicolor Reeve, 1846) has been widely cultured on the southeast coast for more than twenty years. However, abalone culture frequently suffers from bacterial infection and mass mortality of reared abalones causes serious economic losses. Unfortunately, knowledge of the defense mechanism in this animal is still lacking. In this study, using suppression subtractive hybridization (SSH) technology, a forward SSH library was constructed from haemocytes of H. diversicolor, with the content of 1.37x10(6) pfu and the recombinant rate of 98.18%. After the recombinant plasmids were sequenced, partial cDNA of macrophage expressed protein (MEP) was recognized based on BLAST searches in NCBI, with the size of 1,551 bp, and continuously encoding 517 amino acids. Semi-quantitative PCR and quantitative real-time PCR results showed that MEP cDNA was distinctly up-regulated in haemocytes of the bacterial-challenged group compared to the unchallenged group. The gene information obtained from this library will provide new insights into the immune mechanism of H. diversicolor and facilitate future study of target genes involved in the response to invading microorganisms.

  4. Dicty_cDB: SSH606 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available SS (Link to library) SSH606 (Link to dictyBase) ssh606 - - Contig-U14087-1 SSH606Z ...(Link to Original site) - - SSH606Z 590 - - - - Show SSH606 Library SS (Link to library) Clone ID SSH606 (Li...nk to dictyBase) Atlas ID ssh606 NBRP ID - dictyBase ID - Link to Contig Contig-U14087-1 Original site URL h...ttp://dictycdb.biol.tsukuba.ac.jp/CSM/SS/SSH6-A/SSH606Q.Seq.d/ Representative seq. ID SSH...606Z (Link to Original site) Representative DNA sequence >SSH606 (SSH606Q) /CSM/SS/SSH6-A/SSH606Q.Se

  5. Dicty_cDB: SSH359 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available SS (Link to library) SSH359 (Link to dictyBase) - G21014 DDB0231845 Contig-U12241-1 SSH...359P (Link to Original site) SSH359F 432 SSH359Z 645 SSH359P 1077 - - Show SSH359 Library SS (Link to library) Clone ID SSH...Contig-U12241-1 Original site URL http://dictycdb.biol.tsukuba.ac.jp/CSM/SS/SSH3-C/SSH...359Q.Seq.d/ Representative seq. ID SSH359P (Link to Original site) Representative DNA sequence >SSH359 (SSH359Q) /CSM/SS/SSH...3-C/SSH359Q.Seq.d/ ATTCAATAAATAATCATGATATTGAAATTAAAAATCCAGAAAATAATAAAAATTATAATA ATATTAA

  6. Dicty_cDB: SSH823 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available SS (Link to library) SSH823 (Link to dictyBase) ssh823 - - Contig-U04575-1 SSH823F ...(Link to Original site) SSH823F 185 - - - - - - Show SSH823 Library SS (Link to library) Clone ID SSH823 (Li...nk to dictyBase) Atlas ID ssh823 NBRP ID - dictyBase ID - Link to Contig Contig-U04575-1 Original site URL h...ttp://dictycdb.biol.tsukuba.ac.jp/CSM/SS/SSH8-A/SSH823Q.Seq.d/ Representative seq. ID SSH...823F (Link to Original site) Representative DNA sequence >SSH823 (SSH823Q) /CSM/SS/SSH8-A/SSH823Q.Se

  7. Dicty_cDB: SSH536 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available SS (Link to library) SSH536 (Link to dictyBase) - - - Contig-U01795-1 | Contig-U16069-1 SSH...536P (Link to Original site) SSH536F 335 SSH536Z 129 SSH536P 464 - - Show SSH536 Library SS (Link to library) Clone ID SSH...95-1 | Contig-U16069-1 Original site URL http://dictycdb.biol.tsukuba.ac.jp/CSM/SS/SSH5-B/SSH...536Q.Seq.d/ Representative seq. ID SSH536P (Link to Original site) Representative DNA sequence >SSH536 (SSH...536Q) /CSM/SS/SSH5-B/SSH536Q.Seq.d/ ACCAATTCATAACTCTTTACAAAATATTTTTAAAAATGCAAATAGGTCAAATTTAAATAA

  8. Dicty_cDB: SSH288 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available SS (Link to library) SSH288 (Link to dictyBase) - - - Contig-U13975-1 SSH288Z (Link... to Original site) - - SSH288Z 574 - - - - Show SSH288 Library SS (Link to library) Clone ID SSH288 (Link to...ycdb.biol.tsukuba.ac.jp/CSM/SS/SSH2-D/SSH288Q.Seq.d/ Representative seq. ID SSH28...8Z (Link to Original site) Representative DNA sequence >SSH288 (SSH288Q) /CSM/SS/SSH2-D/SSH288Q.Seq.d/ XXXXX...0 SSI764 (SSI764Q) /CSM/SS/SSI7-C/SSI764Q.Seq.d/ 767 0.0 SSI633 (SSI633Q) /CSM/SS/SSI6-B/SSI633Q.Seq.d/ 767 0.0 SSH448 (SSH

  9. Dicty_cDB: SSH663 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available SS (Link to library) SSH663 (Link to dictyBase) - - - Contig-U01805-1 | Contig-U11005-1 SSH...663P (Link to Original site) SSH663F 221 SSH663Z 245 SSH663P 466 - - Show SSH663 Library SS (Link to library) Clone ID SSH...05-1 | Contig-U11005-1 Original site URL http://dictycdb.biol.tsukuba.ac.jp/CSM/SS/SSH6-C/SSH...663Q.Seq.d/ Representative seq. ID SSH663P (Link to Original site) Representative DNA sequence >SSH663 (SSH...663Q) /CSM/SS/SSH6-C/SSH663Q.Seq.d/ ACATACTTATATAAAAGTACATATAAGTAAATTTATTATATGTTAAAACATATTATTTAT

  10. Dicty_cDB: SSH475 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available SS (Link to library) SSH475 (Link to dictyBase) ssh475 - - Contig-U12274-1 SSH475F ...(Link to Original site) SSH475F 187 - - - - - - Show SSH475 Library SS (Link to library) Clone ID SSH475 (Li...nk to dictyBase) Atlas ID ssh475 NBRP ID - dictyBase ID - Link to Contig Contig-U12274-1 Original site URL h...ttp://dictycdb.biol.tsukuba.ac.jp/CSM/SS/SSH4-D/SSH475Q.Seq.d/ Representative seq. ID SSH...475F (Link to Original site) Representative DNA sequence >SSH475 (SSH475Q) /CSM/SS/SSH4-D/SSH475Q.Se

  11. Dicty_cDB: SSH623 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available SS (Link to library) SSH623 (Link to dictyBase) - - - Contig-U11902-1 | Contig-U13418-1 SSH...623P (Link to Original site) SSH623F 177 SSH623Z 611 SSH623P 788 - - Show SSH623 Library SS (Link to library) Clone ID SSH...02-1 | Contig-U13418-1 Original site URL http://dictycdb.biol.tsukuba.ac.jp/CSM/SS/SSH6-A/SSH...623Q.Seq.d/ Representative seq. ID SSH623P (Link to Original site) Representative DNA sequence >SSH623 (SSH...623Q) /CSM/SS/SSH6-A/SSH623Q.Seq.d/ TAAAAATCAAAAAAATAAAATTTTACCAAATACATTTTCACCAAATCTACAACCTCAACA

  12. Dicty_cDB: SSH630 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available SS (Link to library) SSH630 (Link to dictyBase) ssh630 G02291 DDB0229933 Contig-U12011-1 SSH...630P (Link to Original site) SSH630F 364 SSH630Z 282 SSH630P 646 - - Show SSH630 Library SS (Link to library) Clone ID SSH...630 (Link to dictyBase) Atlas ID ssh630 NBRP ID G02291 dictyBase ID DDB0229933 Link t...o Contig Contig-U12011-1 Original site URL http://dictycdb.biol.tsukuba.ac.jp/CSM/SS/SSH6-B/SSH...630Q.Seq.d/ Representative seq. ID SSH630P (Link to Original site) Representative DNA sequence >SSH630 (SSH

  13. Dicty_cDB: SSH476 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available SS (Link to library) SSH476 (Link to dictyBase) ssh476 G00791 DDB0229928 Contig-U09415-1 SSH...476P (Link to Original site) SSH476F 340 SSH476Z 491 SSH476P 831 - - Show SSH476 Library SS (Link to library) Clone ID SSH...476 (Link to dictyBase) Atlas ID ssh476 NBRP ID G00791 dictyBase ID DDB0229928 Link t...o Contig Contig-U09415-1 Original site URL http://dictycdb.biol.tsukuba.ac.jp/CSM/SS/SSH4-D/SSH...476Q.Seq.d/ Representative seq. ID SSH476P (Link to Original site) Representative DNA sequence >SSH476 (SSH

  14. Dicty_cDB: SSH286 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available SS (Link to library) SSH286 (Link to dictyBase) - - - Contig-U12201-1 SSH286Z (Link... to Original site) - - SSH286Z 573 - - - - Show SSH286 Library SS (Link to library) Clone ID SSH286 (Link to...ycdb.biol.tsukuba.ac.jp/CSM/SS/SSH2-D/SSH286Q.Seq.d/ Representative seq. ID SSH28...6Z (Link to Original site) Representative DNA sequence >SSH286 (SSH286Q) /CSM/SS/SSH2-D/SSH286Q.Seq.d/ XXXXX...M319 (SSM319Q) /CSM/SS/SSM3-A/SSM319Q.Seq.d/ 712 0.0 SSI506 (SSI506Q) /CSM/SS/SSI5-A/SSI506Q.Seq.d/ 712 0.0 SSH286 (SSH

  15. Dicty_cDB: SSH138 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available SS (Link to library) SSH138 (Link to dictyBase) - G00782 DDB0231824 Contig-U01120-1 SSH...138P (Link to Original site) SSH138F 379 SSH138Z 625 SSH138P 1004 - - Show SSH138 Library SS (Link to library) Clone ID SSH...Contig-U01120-1 Original site URL http://dictycdb.biol.tsukuba.ac.jp/CSM/SS/SSH1-B/SSH...138Q.Seq.d/ Representative seq. ID SSH138P (Link to Original site) Representative DNA sequence >SSH138 (SSH138Q) /CSM/SS/SSH...1-B/SSH138Q.Seq.d/ TTTTTTTTTTCCTCACACAAAAATAAATAAAAAAATAAAAATAAAAATAAATAATTATAA TTGAAGA

  16. Dicty_cDB: SSH370 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available SS (Link to library) SSH370 (Link to dictyBase) - - - Contig-U16289-1 SSH370F (Link to Original site) SSH...370F 359 - - - - - - Show SSH370 Library SS (Link to library) Clone ID SSH370 (Link to...ycdb.biol.tsukuba.ac.jp/CSM/SS/SSH3-C/SSH370Q.Seq.d/ Representative seq. ID SSH37...0F (Link to Original site) Representative DNA sequence >SSH370 (SSH370Q) /CSM/SS/SSH3-C/SSH370Q.Seq.d/ AATAA...VS/VSJ2-D/VSJ277Q.Seq.d/ 359 2e-98 SSK265 (SSK265Q) /CSM/SS/SSK2-C/SSK265Q.Seq.d/ 359 2e-98 SSH370 (SSH

  17. Dicty_cDB: SSH463 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available SS (Link to library) SSH463 (Link to dictyBase) - - - Contig-U13869-1 - (Link to Or...iginal site) - - - - - - SSH463E 454 Show SSH463 Library SS (Link to library) Clone ID SSH463 (Link to dicty...iol.tsukuba.ac.jp/CSM/SS/SSH4-C/SSH463Q.Seq.d/ Representative seq. ID - (Link to ...Original site) Representative DNA sequence >SSH463 (SSH463Q) /CSM/SS/SSH4-C/SSH463Q.Seq.d/ ATATAATCATAAAATGG...logy vs CSM-cDNA Score E Sequences producing significant alignments: (bits) Value SSH645 (SSH645Q) /CSM/SS/SSH6-B/SSH

  18. Dicty_cDB: SSH589 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available SS (Link to library) SSH589 (Link to dictyBase) - G21023 DDB0233426 Contig-U13415-1 | Contig-U15575-1 SSH...589P (Link to Original site) SSH589F 380 SSH589Z 453 SSH589P 833 - - Show SSH589 Libra...ry SS (Link to library) Clone ID SSH589 (Link to dictyBase) Atlas ID - NBRP ID G21023 dictyBase ID DDB023342...cdb.biol.tsukuba.ac.jp/CSM/SS/SSH5-D/SSH589Q.Seq.d/ Representative seq. ID SSH589P (Link to Original site) R...epresentative DNA sequence >SSH589 (SSH589Q) /CSM/SS/SSH5-D/SSH589Q.Seq.d/ ATAAAGATTTATCCCCAAATCCAAAAAAAAAAG

  19. Dicty_cDB: SSH604 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available SS (Link to library) SSH604 (Link to dictyBase) ssh604 - - Contig-U13416-1 | Contig-U16394-1 SSH...nk to library) Clone ID SSH604 (Link to dictyBase) Atlas ID ssh604 NBRP ID - dictyBase ID - Link to Contig C...604P (Link to Original site) SSH604F 347 SSH604Z 268 SSH604P 615 - - Show SSH604 Library SS (Li...ontig-U13416-1 | Contig-U16394-1 Original site URL http://dictycdb.biol.tsukuba.ac.jp/CSM/SS/SSH6-A/SSH...604Q.Seq.d/ Representative seq. ID SSH604P (Link to Original site) Representative DNA sequence >SSH604 (SSH

  20. Dicty_cDB: SSH183 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available SS (Link to library) SSH183 (Link to dictyBase) - - - Contig-U15632-1 SSH183E (Link... to Original site) - - - - - - SSH183E 254 Show SSH183 Library SS (Link to library) Clone ID SSH183 (Link to...ycdb.biol.tsukuba.ac.jp/CSM/SS/SSH1-D/SSH183Q.Seq.d/ Representative seq. ID SSH18...3E (Link to Original site) Representative DNA sequence >SSH183 (SSH183Q) /CSM/SS/SSH1-D/SSH183Q.Seq.d/ ATTAA...vfgyqlilhfy*vi**ldplvlis Homology vs CSM-cDNA Score E Sequences producing significant alignments: (bits) Value SSH183 (SSH

  1. Dicty_cDB: SSH416 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available SS (Link to library) SSH416 (Link to dictyBase) - G23718 DDB0205744 Contig-U00298-1 SSH...416E (Link to Original site) SSH416F 257 SSH416Z 243 SSH416P 500 SSH416E 388 Show SSH416 Library SS (Lin...Contig Contig-U00298-1 Original site URL http://dictycdb.biol.tsukuba.ac.jp/CSM/SS/SSH4-A/SSH...416Q.Seq.d/ Representative seq. ID SSH416E (Link to Original site) Representative DNA sequence >SSH416 (SSH...416Q) /CSM/SS/SSH4-A/SSH416Q.Seq.d/ GTTGAATCTTTAAATAGATTATATGCTTTGGAATGTTTAGATATTAGTAAGAATAACATT

  2. Dicty_cDB: SSH526 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available SS (Link to library) SSH526 (Link to dictyBase) - - - Contig-U01794-1 | Contig-U16394-1 SSH...526P (Link to Original site) SSH526F 327 SSH526Z 170 SSH526P 497 - - Show SSH526 Library SS (Link to library) Clone ID SSH...94-1 | Contig-U16394-1 Original site URL http://dictycdb.biol.tsukuba.ac.jp/CSM/SS/SSH5-B/SSH...526Q.Seq.d/ Representative seq. ID SSH526P (Link to Original site) Representative DNA sequence >SSH526 (SSH...526Q) /CSM/SS/SSH5-B/SSH526Q.Seq.d/ ATTGAATCCAGCGGTAACAGTTGGTTGTGTTACAACTGGTAGAATGGGAATTCTCAATGG

  3. Dicty_cDB: SSH534 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available SS (Link to library) SSH534 (Link to dictyBase) - - - Contig-U05020-1 SSH534E (Link... to Original site) - - - - - - SSH534E 365 Show SSH534 Library SS (Link to library) Clone ID SSH534 (Link to...ycdb.biol.tsukuba.ac.jp/CSM/SS/SSH5-B/SSH534Q.Seq.d/ Representative seq. ID SSH53...4E (Link to Original site) Representative DNA sequence >SSH534 (SSH534Q) /CSM/SS/SSH5-B/SSH534Q.Seq.d/ CTATT...-C/VSI267Q.Seq.d/ 476 e-133 VSG392 (VSG392Q) /CSM/VS/VSG3-D/VSG392Q.Seq.d/ 476 e-133 SSH534 (SSH

  4. Suppression subtractive hybridization coupled with microarray analysis to examine differential expression of genes in Japanese flounder Paralichthys olivaceus leucocytes during Edwardsiella tarda and viral hemorrhagic septicemia virus infection.

    Science.gov (United States)

    Matsuyama, Tomomasa; Fujiwara, Atushi; Takano, Tomokazu; Nakayasu, Chihaya

    2011-10-01

    Transcriptional changes in the peripheral blood leucocytes (PBL) of Japanese flounder Paralichthys olivaceus challenged by Edwardsiella tarda and viral hemorrhagic septicemia virus (VHSV) were investigated using suppression subtractive hybridization (SSH) coupled with cDNA microarray analysis. First, we constructed an SSH cDNA library using mRNA samples isolated from PBL of P. olivaceus that had been experimentally infected with E. tarda. We then examined the transcriptional changes occurring in the PBL due to E. tarda and VHSV infection using a cDNA microarray produced using clones produced from the SSH library. A total of 565 and 180 cDNA sequences corresponding to mRNA species that are either up- or down-regulated by E. tarda infection were isolated by SSH. While host gene expression responses in response to E. tarda and VHSV infection share several response elements, distinct patterns of gene expression were also observed. Specifically, E. tarda infection enhanced the expression of cell adhesion molecules while VHSV enhanced the expression of interferon and proteasome-related genes. In challenge trials of the two infectious agents, expression profiles of chemokines were also observed to differ. The results indicated that distinguishing between viral and bacterial infection is possible based on the RNA expression profiles of PBL from infected fish.

  5. Isolation of novel non-HLA gene fragments from the hemochromatosis region (6p21. 3) by cDNA hybridization selection

    Energy Technology Data Exchange (ETDEWEB)

    Goei, V.L.; Capossela, A.; Gruen, J.R.; Parimoo, S.; Chu, T.W. (Yale Univ. School of Medicine, New Haven, CT (United States))

    1994-02-01

    It has previously been shown that cDNA hybridization selection can identify and recover novel genes from large cloned genomic DNA such as cosmids or YACs. In an effort to identify candidate genes for hemochromatosis, this technique was applied to a 320-kb YAC containing the HLA-A gene. A short fragment cDNA library derived from human duodenum was selected with the YAC DNA. Ten novel gene fragments were isolated, characterized, and localized on the physical map of the YAC. 39 refs., 4 figs., 3 tabs.

  6. Immobilization and Hybridization of cDNA Arrays on the Glass Surface

    Institute of Scientific and Technical Information of China (English)

    谢文章; 孙宝全; 周玉祥; 程京

    2002-01-01

    DNA microarray is a powerful tool allowing simultaneous detection of many different target molecules in one sample. The efficiency of the array mainly depends on the sequence of the capture probes and the way in which they are attached to the support. Coupling process needs to be quick, reproducible and compatible with automatic spotting devices dispensing tiny drops of liquid on the glass surface. Coupling strategies were evaluated in light of grafting DNA onto the glass surface. The results indicate that attaching unmodified DNA to a poly-L-lysine coated glass surface is a preferred method in fabricating DNA microarrays. In this paper, both the coupling procedure and the hybridization efficiency have been optimized. The unmodified double-stranded DNA in high salt solution being spotted onto the poly-L-lysine coated glass surface enhances the adsorption of DNA onto the poly-L-lysine layer.

  7. A gene encoding a protein with a proline-rich domain (MtPPRD1), revealed by suppressive subtractive hybridization (SSH), is specifically expressed in the Medicago truncatula embryo axis during germination.

    Science.gov (United States)

    Bouton, Sophie; Viau, Laure; Lelièvre, Eric; Limami, Anis M

    2005-03-01

    A gene MtPPRD1, encoding a protein of 132 amino acids containing a proline-rich domain (PRD), has been revealed by suppressive subtractive hybridization (SSH) with two mRNA populations of embryo axes harvested immediately before and after radicle emergence. Although at the protein level MtPPRD1 showed low homology with plant lipid transfer proteins (LTPs), it did exhibit the eight cysteine residues conserved in all plant LTPs, a characteristic signature that allows the formation of a hydrophobic cavity adapted for loading hydrophobic molecules. Expression studies of MtPPRD1 have been carried out by quantitative real time RT-PCR throughout germination and post-germination processes in control seeds and seeds in which germination was delayed by abscisic acid (ABA) or the glutamine synthetase inhibitor methionine sulphoximine (MSX) treatments. The results showed that MtPPRD1 expression is developmentally regulated, induced in the embryo axis immediately before radicle emergence, reaches its maximum expression and declines during the early post-germination phase. Organ specificity studies showed that, except for a low and probably constitutive expression in roots, MtPPRD1 is specifically expressed in the embryo axis. Based on both experimental and in silico studies several putative roles are proposed for MtPPRD1 in Medicago truncatula, this protein can intervene (i) as an LTP in membrane biogenesis and regulation of the intracellular fatty acid pool by binding and transferring fatty acids and phospholipids between membranes, (ii) in the control of a developmental process specific to late germination and to early phases of post-germination, and (iii) and/or pathogen defence.

  8. Dicty_cDB: SSH126 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available SS (Link to library) SSH126 (Link to dictyBase) - - - Contig-U10885-1 SSH126Z (Link... to Original site) - - SSH126Z 627 - - - - Show SSH126 Library SS (Link to library) Clone ID SSH126 (Link to...ycdb.biol.tsukuba.ac.jp/CSM/SS/SSH1-B/SSH126Q.Seq.d/ Representative seq. ID SSH12...6Z (Link to Original site) Representative DNA sequence >SSH126 (SSH126Q) /CSM/SS/SSH1-B/SSH126Q.Seq.d/ XXXXX...ifyyql llfslfqlqfyhyffkiiiiiiiirkikkkk Homology vs CSM-cDNA Score E Sequences producing significant alignments: (bits) Value SSH

  9. Dicty_cDB: SSH574 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available SS (Link to library) SSH574 (Link to dictyBase) - G00795 DDB0191628 Contig-U03732-1 SSH...574P (Link to Original site) SSH574F 319 SSH574Z 351 SSH574P 670 - - Show SSH574 Library SS (Link to library) Clone ID SSH...ontig-U03732-1 Original site URL http://dictycdb.biol.tsukuba.ac.jp/CSM/SS/SSH5-D/SSH...574Q.Seq.d/ Representative seq. ID SSH574P (Link to Original site) Representative DNA sequence >SSH574 (SSH574Q) /CSM/SS/SSH...fis**se*KNILFIKKKKTHTIY IKNSYKMSKVAALGWGTLVYLGVGLLLAIYPPFVTDKPLGRVCFI--- ---iii*ytriikskkttkyyfttpvt*ttkkkkikk*q*kkhqhkrihinvyspssh

  10. Dicty_cDB: SSH793 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available SS (Link to library) SSH793 (Link to dictyBase) - - - Contig-U15497-1 SSH793Z (Link... to Original site) - - SSH793Z 375 - - - - Show SSH793 Library SS (Link to library) Clone ID SSH793 (Link to...ycdb.biol.tsukuba.ac.jp/CSM/SS/SSH7-D/SSH793Q.Seq.d/ Representative seq. ID SSH79...3Z (Link to Original site) Representative DNA sequence >SSH793 (SSH793Q) /CSM/SS/SSH7-D/SSH793Q.Seq.d/ XXXXX...al 4.0 %: mitochondrial 4.0 %: peroxisomal >> prediction for SSH793 is cyt 5' end seq. ID - 5' end seq. - Le

  11. Dicty_cDB: SSH234 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available SS (Link to library) SSH234 (Link to dictyBase) - - - Contig-U16581-1 SSH234E (Link... to Original site) - - - - - - SSH234E 564 Show SSH234 Library SS (Link to library) Clone ID SSH234 (Link to...ycdb.biol.tsukuba.ac.jp/CSM/SS/SSH2-B/SSH234Q.Seq.d/ Representative seq. ID SSH23...4E (Link to Original site) Representative DNA sequence >SSH234 (SSH234Q) /CSM/SS/SSH2-B/SSH234Q.Seq.d/ CAATT...%: vacuolar 4.0 %: cytoskeletal >> prediction for SSH234 is cyt 5' end seq. ID - 5' end seq. - Length of 5'

  12. Dicty_cDB: SSH461 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available SS (Link to library) SSH461 (Link to dictyBase) - - - Contig-U16709-1 SSH461E (Link... to Original site) - - - - - - SSH461E 509 Show SSH461 Library SS (Link to library) Clone ID SSH461 (Link to...ycdb.biol.tsukuba.ac.jp/CSM/SS/SSH4-C/SSH461Q.Seq.d/ Representative seq. ID SSH46...1E (Link to Original site) Representative DNA sequence >SSH461 (SSH461Q) /CSM/SS/SSH4-C/SSH461Q.Seq.d/ TTTAG...gi 4.0 %: vesicles of secretory system 4.0 %: cytoplasmic >> prediction for SSH46

  13. Dicty_cDB: SSH311 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available SS (Link to library) SSH311 (Link to dictyBase) - - - Contig-U12223-1 SSH311Z (Link... to Original site) - - SSH311Z 508 - - - - Show SSH311 Library SS (Link to library) Clone ID SSH311 (Link to...ycdb.biol.tsukuba.ac.jp/CSM/SS/SSH3-A/SSH311Q.Seq.d/ Representative seq. ID SSH31...1Z (Link to Original site) Representative DNA sequence >SSH311 (SSH311Q) /CSM/SS/SSH3-A/SSH311Q.Seq.d/ XXXXX.../ 301 6e-81 SSK705 (SSK705Q) /CSM/SS/SSK7-A/SSK705Q.Seq.d/ 301 6e-81 SSI190 (SSI190Q) /CSM/SS/SSI1-D/SSI190Q.Seq.d/ 301 6e-81 SSH

  14. Dicty_cDB: SSH653 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available SS (Link to library) SSH653 (Link to dictyBase) - - - Contig-U15497-1 SSH653Z (Link... to Original site) - - SSH653Z 342 - - - - Show SSH653 Library SS (Link to library) Clone ID SSH653 (Link to...ycdb.biol.tsukuba.ac.jp/CSM/SS/SSH6-C/SSH653Q.Seq.d/ Representative seq. ID SSH65...3Z (Link to Original site) Representative DNA sequence >SSH653 (SSH653Q) /CSM/SS/SSH6-C/SSH653Q.Seq.d/ XXXXX... 1.00 40.0 %: cytoplasmic 36.0 %: nuclear 20.0 %: cytoskeletal 4.0 %: peroxisomal >> prediction for SSH653 i

  15. SSH reveals a linkage between a senescence-associated protease and Verticillium wilt symptom development in lettuce (Lactuca sativa)

    Science.gov (United States)

    Suppression subtractive hybridization (SSH) was employed to identify lettuce (Lactuca sativa) genes that are differentially expressed in symptomatic leaves infected with Verticillium dahliae. Genes expressed only in symptomatic leaves included those with homology to pathogenesis-related (PR) protei...

  16. Human cDNA mapping using fluorescence in situ hybridization. Progress report, April 1--December 31, 1992

    Energy Technology Data Exchange (ETDEWEB)

    Korenberg, J.R.

    1993-12-31

    The ultimate goal of this proposal is to create a cDNA map of the human genome. Mapping is approached using the techniques of high resolution fluorescence in situ hybridization (FISH). This technology and the results of its application are designed to rapidly generate whole genome as tool box of expressed sequence to speed the identification of human disease genes. The results of this study are intended to dovetail with and to link the results of existing technologies for creating backbone YAC and genetic maps. In the first eight months, this approach will generate 60--80% of the expressed sequence map, the remainder expected to be derived through more long-term, labor-intensive, regional chromosomal gene searches or sequencing. The laboratory has made significant progress in the set-up phase, in mapping fetal and adult brain and other cDNAs, in testing a model system for directly linking genetic and physical maps using FISH with small fragments, in setting up a database, and in establishing the validity and throughput of the system.

  17. Molecular cloning and expression of a cDNA encoding a hybrid histidine kinase receptor in tropical periwinkle Catharanthus roseus.

    Science.gov (United States)

    Papon, N; Bremer, J; Vansiri, A; Glévarec, G; Rideau, M; Creche, J

    2006-09-01

    Signalling pathways involving histidine kinase receptors (HKRs) are widely used by prokaryotes and fungi to regulate a large palette of biological processes. In plants, HKRs are known to be implicated in cytokinin, ethylene, and osmosensing transduction pathways. In this work, a full length cDNA named CRCIK was isolated from the tropical species CATHARANTHUS ROSEUS (L.) G. Don. It encodes a 1205 amino acid protein that belongs to the hybrid HKR family. The deduced amino acid sequence shows the highest homology with AtHK1, an osmosensing HKR in ARABIDOPSIS THALIANA. In return, CrCIK protein shares very low identity with the other 10 ARABIDOPSIS HKRs. Southern blot analysis indicates that the CRCIK corresponding gene is either present in multiple copies or has very close homologues in the genome of the tropical periwinkle. The gene is widely expressed in the plant. In C. ROSEUS C20D cell suspension, it is slightly induced after exposure to low temperature, pointing to a putative role in cold-shock signal transduction.

  18. Identification and analysis of differentially expressed genes involved in dark-induced photoperiod response and senescence of soybean leaves by suppression subtractive hybridization

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    A cDNA subtractive library enriched for dark-induced up-regulated ESTs was constructed by suppression subtractive hybridization(SSH) from leaf tissues of soybean cultivar DongNong L13 treated with short-day(8-h light/16-h dark) and long-day(16-h light/8-h dark) conditions.A total of 148 clones were sequenced,representing 76 unique ESTs which corresponded to about 20% of 738 clones from the cDNA library and showed a significant up-regulation of at least three fold verified by dot blot hybridization.The putat...

  19. Construction of Yeast Two-hybrid cDNA Library of Verticillium dahliae%大丽轮枝菌酵母双杂交 cDNA 文库的构建

    Institute of Scientific and Technical Information of China (English)

    康静敏; 刘坤; 刘严; 张怡; 李成伟; 谭光轩

    2015-01-01

    为了分离克隆与植物互作的大丽轮枝菌致病相关蛋白基因,利用 SMART 技术构建了大丽轮枝菌酵母双杂交 cDNA 文库。结果表明,构建的文库滴度为5.2×107 cfu/mL,库容为3.9×107 cfu;文库 cDNA 插入片段长度主要分布在0.5~2.0 kb,平均为1 kb;文库重组率为96%。表明文库质量较好,为筛选与植物互作的大丽轮枝菌致病蛋白基因奠定了基础。%To seek out the V. dahliae proteins involved in the interaction of plant with V. dahliae,a yeast two-hybrid library of V. dahliae was constructed using SMART technique. Results showed that the titer of cDNA library was 5. 2 × 107 cfu/mL,the library contained 3. 9 × 107 cfu independent clones,the size distribution of insert frag-ments ranged from 0. 5 -2. 0 kb,and the recombination rate was about 96%. These data demonstrated that the li-brary could meet the requirements of standard cDNA library,which laid a foundation for screening the interaction proteins from V. dahliae.

  20. Dicty_cDB: SSH190 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available SS (Link to library) SSH190 (Link to dictyBase) - - - Contig-U16136-1 SSH190F (Link to Original site) SSH...190F 324 - - - - - - Show SSH190 Library SS (Link to library) Clone ID SSH190 (Link to...ycdb.biol.tsukuba.ac.jp/CSM/SS/SSH1-D/SSH190Q.Seq.d/ Representative seq. ID SSH19...0F (Link to Original site) Representative DNA sequence >SSH190 (SSH190Q) /CSM/SS/SSH1-D/SSH190Q.Seq.d/ AGATA...yis***************iykqntnknikne*ftc*r*frl*ryyg*rvl ng*ye*tw*nfssh*il**f*r*f*r*i*r

  1. Dicty_cDB: SSH552 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available SS (Link to library) SSH552 (Link to dictyBase) - - - Contig-U13930-1 SSH552F (Link to Original site) SSH...552F 298 - - - - - - Show SSH552 Library SS (Link to library) Clone ID SSH552 (Link to...ycdb.biol.tsukuba.ac.jp/CSM/SS/SSH5-C/SSH552Q.Seq.d/ Representative seq. ID SSH55...2F (Link to Original site) Representative DNA sequence >SSH552 (SSH552Q) /CSM/SS/SSH5-C/SSH552Q.Seq.d/ AAACA...mkkltlvlaisskrevlnllmert*pifk--- Frame B: nkykykkkky**in**ifnk*inkin*ndcqsfqinypriihfisqrwfnfslwc*nyfp kc*kr*r*sr*kssh

  2. Cost-effective sequencing of full-length cDNA clones powered by a de novo-reference hybrid assembly.

    Science.gov (United States)

    Kuroshu, Reginaldo M; Watanabe, Junichi; Sugano, Sumio; Morishita, Shinichi; Suzuki, Yutaka; Kasahara, Masahiro

    2010-05-07

    Sequencing full-length cDNA clones is important to determine gene structures including alternative splice forms, and provides valuable resources for experimental analyses to reveal the biological functions of coded proteins. However, previous approaches for sequencing cDNA clones were expensive or time-consuming, and therefore, a fast and efficient sequencing approach was demanded. We developed a program, MuSICA 2, that assembles millions of short (36-nucleotide) reads collected from a single flow cell lane of Illumina Genome Analyzer to shotgun-sequence approximately 800 human full-length cDNA clones. MuSICA 2 performs a hybrid assembly in which an external de novo assembler is run first and the result is then improved by reference alignment of shotgun reads. We compared the MuSICA 2 assembly with 200 pooled full-length cDNA clones finished independently by the conventional primer-walking using Sanger sequencers. The exon-intron structure of the coding sequence was correct for more than 95% of the clones with coding sequence annotation when we excluded cDNA clones insufficiently represented in the shotgun library due to PCR failure (42 out of 200 clones excluded), and the nucleotide-level accuracy of coding sequences of those correct clones was over 99.99%. We also applied MuSICA 2 to full-length cDNA clones from Toxoplasma gondii, to confirm that its ability was competent even for non-human species. The entire sequencing and shotgun assembly takes less than 1 week and the consumables cost only approximately US$3 per clone, demonstrating a significant advantage over previous approaches.

  3. Microarray and suppression subtractive hybridization analyses of gene expression in hybrid poplar (Populus alba × Populus tremula var. glandulosa) cell suspension cultures after exposure to NaCl.

    Science.gov (United States)

    Bae, Eun-Kyung; Lee, Hyoshin; Lee, Jae-Soon; Noh, Eun-Woon; Choi, Young-Im; Lee, Byung-Hyun; Choi, Dong-Woog

    2012-09-01

    The gene expression profiles of hybrid poplar (Populus alba × Populus tremula var. glandulosa) cells in suspension culture after exposure to salinity (NaCl) induced stress were examined by constructing two suppression subtractive hybridization (SSH) libraries. cDNA from non-treated cells was used as a driver and cDNA samples from cell suspension cultures exposed to 150 mM NaCl for 2 or 10 h were used as testers. Randomly selected clones from each SSH library were sequenced and 727 high-quality expressed sequence tags (ESTs) were obtained and analyzed. Four novel ESTs were identified. Between the two libraries, 542 unique SSH clones were selected for placement on a cDNA microarray. In total, 18 differentially expressed genes were identified with 4 and 12 genes being significantly differentially expressed 2 and 10 h after the treatment, respectively. Genes related to metabolism and protein synthesis and several genes whose protein products are implicated in salt or other abiotic stress-related responses were expressed in the salt-stressed cells.

  4. [Study on gene expression of Tamarix under NaHCO3 stress using SSH technology].

    Science.gov (United States)

    Yang, Chuan-Ping; Wang, Yu-Cheng; Liu, Gui-Feng; Jiang, Jing

    2004-09-01

    The gene expression of Tamarix androssowii under NaHCO3 stresses is studied by using SSH technique, in which the cDNA from the materials treated with NaHCO3 solution is as tester and the cDNA from the materials in normal growth is as driver. Total 36 genes related to NaHCO3 stress were obtained through Northern hybridization. Blastx analysis showed that the proteins encoded by these genes were homologous to the following proteins: the antioxidant enzymes catalase and peroxiredoxin; trehalose phosphatase, which was related to trehalose synthesis; a few regulation proteins such as bZIP transcription factor, MADS-box protein, glycine-rich RNA-binding proteins, CCCH-type zinc finger protein and F-box protein etc; early light-induced protein, which could protect and/or repair the photosynthetic apparatus damage induced by stress; cysteine proteinase and vacuolar processing enzyme that can make function in plant cell death, and lipid transfer protein precursor, polyubiquitin, chalcone synthase, NADP-dependent isocitrate dehydrogenase, salt-induced S12 protein, and oxygen-evolving enhancer protein 1 etc. Among 36 genes obtained, the proteins encoded three genes were homologous to 3 putative proteins: HAK2, calcium-binding protein and RNA-binding protein, respectively. In addition, 6 new salt stress response squences were found. The result indicated that the salt-tolerant mechanism of Tamarix androssowii may be a complicated, interactive system involving multiple approaches and multiple genes, but not only a single salt gland-depended approach.

  5. Identification of differentially expressed genes in lung tissues of nickel-exposed rats using suppression subtractive hybridization.

    Science.gov (United States)

    Zhang, Jing; Zhang, Jun; Fan, Yingying; Liu, Lihong; Li, Mengjie; Zhou, Yang; Shao, Zhihua; Shi, Hongjun; Wang, Ying

    2011-11-01

    Occupational exposure to nickel compound, such as nickel refining, electroplating, and in conjunction with other metals, is harmful to the health, causing respiratory distress, and lung and nasal cancer. In this work, the different gene expression patterns of lung tissues from nickel-exposed rats and controls were investigated. The suppression subtractive hybridization (SSH) method was used to generate two subtracted cDNA libraries with gene transcripts differentially expressed after nickel inducing. Dot-blot hybridizations were used to confirm differential ratios of expression of obtained SSH clones. Out of 768 unique SSH clones, which were chosen randomly from the two subtraction libraries (384 of each), 319 could be verified as differentially expressed. According to blast screening and functional annotation, 28% genes in nickel-induced cDNA library were related to cell differentiation, whereas 21% in driver library were related to oxygen transport. Two novel expressed sequence tags (ESTs; NCBI Accession No. FC809414 and No. FC809411) in nickel-induced cDNA library were obtained. The genes detected in the present study are probably important genes associated with nickel-induced lung cancer.

  6. Selection of genes associated with variations in the Circle of Willis in gerbils using suppression subtractive hybridization.

    Science.gov (United States)

    Li, Zhenkun; Huo, Xueyun; Zhang, Shuangyue; Lu, Jing; Li, Changlong; Guo, Meng; Fu, Rui; He, Zhengming; Du, Xiaoyan; Chen, Zhenwen

    2015-01-01

    Deformities in the Circle of Willis (CoW) can significantly increase the risk of cerebrovascular disease in humans. However, the molecular mechanisms underlying these deformities have not been understood. Based on our previous studies, variations in the CoW of gerbils are hereditary. A normal CoW is observed in approximately 60% of gerbils, a percentage that also applies to humans. Thus, gerbil is an ideal experimental model for studying variations in the CoW. To study the mechanisms underlying these variations, we selected genes associated with different types of the CoW using suppression subtractive hybridization (SSH). After evaluating the efficiency of SSH using quantitative real-time polymerase chain reaction (qPCR) on subtracted and unsubtracted cDNA and Southern blotting on SSH PCR products, 12 SSH libraries were established. We identified 4 genes (CST3, GNAS, GPx4 and PFN2) associated with variations in the CoW. These genes were identified with qPCR and Western blotting using 70 expressed sequence tags from the SSH libraries. Cloning and sequencing allowed us to demonstrate that the 4 genes were closely related to mouse genes. We may assume that these 4 genes play an important role in the development of variations in the CoW. This study provides a foundation for further research of genes related to development of variations in the CoW and the mechanisms of dysmorphosis of cerebral vessels.

  7. Selection of genes associated with variations in the Circle of Willis in gerbils using suppression subtractive hybridization.

    Directory of Open Access Journals (Sweden)

    Zhenkun Li

    Full Text Available Deformities in the Circle of Willis (CoW can significantly increase the risk of cerebrovascular disease in humans. However, the molecular mechanisms underlying these deformities have not been understood. Based on our previous studies, variations in the CoW of gerbils are hereditary. A normal CoW is observed in approximately 60% of gerbils, a percentage that also applies to humans. Thus, gerbil is an ideal experimental model for studying variations in the CoW. To study the mechanisms underlying these variations, we selected genes associated with different types of the CoW using suppression subtractive hybridization (SSH. After evaluating the efficiency of SSH using quantitative real-time polymerase chain reaction (qPCR on subtracted and unsubtracted cDNA and Southern blotting on SSH PCR products, 12 SSH libraries were established. We identified 4 genes (CST3, GNAS, GPx4 and PFN2 associated with variations in the CoW. These genes were identified with qPCR and Western blotting using 70 expressed sequence tags from the SSH libraries. Cloning and sequencing allowed us to demonstrate that the 4 genes were closely related to mouse genes. We may assume that these 4 genes play an important role in the development of variations in the CoW. This study provides a foundation for further research of genes related to development of variations in the CoW and the mechanisms of dysmorphosis of cerebral vessels.

  8. Analysis of immune system gene expression in small rheumatoid arthritis biopsies using a combination of subtractive hybridization and high-density cDNA arrays.

    Science.gov (United States)

    Zanders, E D; Goulden, M G; Kennedy, T C; Kempsell, K E

    2000-01-13

    Subtractive hybridization of cDNAs generated from synovial RNA which had been isolated from patients with rheumatoid arthritis (RA) or normal controls was used in conjunction with high-density array hybridization to identify genes of immunological interest. The method was designed to detect gene expression in small needle biopsy specimens by means of a prior amplification of nanogram amounts of total RNA to full-length cDNA using PCR. The latter was cut with Rsa I, ligated with adapters, hybridized with unmodified driver cDNA, and subjected to suppression subtraction PCR. Differentially expressed products were cloned into E. coli and picked into 384 well plates. Inserts were obtained by PCR across the multiple cloning site, and the products arrayed at high density on nylon filters. The subtracted cDNAs were also labelled by random priming for use as probes for library screening. The libraries chosen were the subtracted one described above and a set of 45,000 ESTs from the I.M. A.G.E consortium. Clones showing positive hybridization were identified by sequence analysis and homology searching. The results showed that the subtracted hybridization approach could identify many gene fragments expressed at different levels, the most abundant being immunoglobulins and HLA-DR. The expression profile was characteristic of macrophage, B cell and plasma cell infiltration with evidence of interferon induction. In addition, a significant number of sequences without matches in the nucleotide databases were obtained, this demonstrates the utility of the method in finding novel gene fragments for further characterisation as potential members of the immune system. Although RA was studied here, the technology is applicable to any disease process even in cases where amounts of tissue may be limited.

  9. Specific detection of neuronal cell bodies: in situ hybridization with a biotin-labelled neurofilament cDNA probe.

    NARCIS (Netherlands)

    P. Liesi; J-P. Julien (Jean-Pierre); P. Vilja; F.G. Grosveld (Frank); L. Rechardt

    1986-01-01

    textabstractWe have used a biotinylated, 300-nucleotide cDNA probe which encodes the 68,000 MW neurofilament protein to detect neurofilament-specific mRNA in situ. The neurofilament message specifically demonstrates the neuronal cell bodies, in contrast to the usual antibody staining which detects t

  10. B-G cDNA clones have multiple small repeats and hybridize to both chicken MHC regions

    DEFF Research Database (Denmark)

    Kaufman, J; Salomonsen, J; Skjødt, K

    1989-01-01

    We used rabbit antisera to the chicken MHC erythrocyte molecule B-G and to the class I alpha chain (B-F) to screen lambda gt11 cDNA expression libraries made with RNA selected by oligo-dT from bone marrow cells of anemic B19 homozygous chickens. Eight clones were found to encode B-G molecules which...

  11. Cutis laxa: reduced elastin gene expression in skin fibroblast cultures as determined by hybridizations with a homologous cDNA and an exon 1-specific oligonucleotide

    Energy Technology Data Exchange (ETDEWEB)

    Olsen, D.R.; Fazio, M.J.; Shamban, A.T.; Rosenbloom, J.; Uitto, J.

    1988-05-15

    Fibroblast cultures were established from six patients with cutis laxa, and elastin gene expression was analyzed by RNA hybridizations with a 2.5-kilobase human elastin cDNA or an exon 1-specific 35-base oligomer. Northern analyses using either probe detected mRNA transcripts of approx. 3.5 kilobases, and no qualitative difference between the control and cutis laxa mRNAs was detected. However, quantitation of the elastin mRNA abundance by slot blot hybridizations revealed markedly reduced levels in all cutis laxa cell strains. Assuming equal translational activity of the control and cutix laxa mRNAs, the reduced mRNA levels could result in diminished elastin production, providing an explanation for the paucity of elastic fibers in the skin and other tissues in cutis laxa.

  12. Chemiluminescent detection of sequential DNA hybridizations to high-density, filter-arrayed cDNA libraries: a subtraction method for novel gene discovery.

    Science.gov (United States)

    Guiliano, D; Ganatra, M; Ware, J; Parrot, J; Daub, J; Moran, L; Brennecke, H; Foster, J M; Supali, T; Blaxter, M; Scott, A L; Williams, S A; Slatko, B E

    1999-07-01

    A chemiluminescent approach for sequential DNA hybridizations to high-density filter arrays of cDNAs, using a biotin-based random priming method followed by a streptavidin/alkaline phosphatase/CDP-Star detection protocol, is presented. The method has been applied to the Brugia malayi genome project, wherein cDNA libraries, cosmid and bacterial artificial chromosome (BAC) libraries have been gridded at high density onto nylon filters for subsequent analysis by hybridization. Individual probes and pools of rRNA probes, ribosomal protein probes and expressed sequence tag probes show correct specificity and high signal-to-noise ratios even after ten rounds of hybridization, detection, stripping of the probes from the membranes and rehybridization with additional probe sets. This approach provides a subtraction method that leads to a reduction in redundant DNA sequencing, thus increasing the rate of novel gene discovery. The method is also applicable for detecting target sequences, which are present in one or only a few copies per cell; it has proven useful for physical mapping of BAC and cosmid high-density filter arrays, wherein multiple probes have been hybridized at one time (multiplexed) and subsequently "deplexed" into individual components for specific probe localizations.

  13. [Construction and analysis of subtractive cDNA library of Phellodendron amurense under drought stress].

    Science.gov (United States)

    Wang, Huimei; Wang, Yanbing; Zu, Yuangang; Sun, Lianhui

    2008-02-01

    With cDNA from Phellodendron amurense seedlings treated with drought stress as tester and cDNA from this plant in normal growth as driver, we construct cDNA subtracted library using suppression subtractive hybridization (SSH). In the library, the rate of recombination was 95%, the size of inserts was 300-800 bp. Two hundred and sixty-five new genes were obtained by DNA sequencing 816 positive clones picked randomly, and partitioned to 16 classes after nucleotide Blast and BlastX homological analysis against NT, NR, SWISSPROT, KEGG database. Forty-four drought stress associated genes, such as heat shock protein cognate 70, dehydration responsive protein 22, universal stress protein, metallothionein II, late embryogenesis abundant protein, were obtained, which made 16.6% of the overall genes. These genes included osmotic regulator, signal component regulatory protein and antioxidant enzyme. The research had established a basis for cloning stress resistance genes and further studying genes expression in P. amurense seedlings under drought stress.

  14. Analysis of Differential Gene Expression Pattern in Brassica napus Hybrid Huayouza 6 and Its Parents Using Arabidopsis cDNA Microarray

    Institute of Scientific and Technical Information of China (English)

    SHEN Jun-ru; WU Jian-yong; ZHANG Jian; LIU Ping-wu; YANG Guang-sheng

    2006-01-01

    Huayouza 6, a new semi-winter Brassica napus variety with high-yield, good quality, prematurity and extensive adaptability, was derived from the cross between the female parent 8086A and male parent 7-5. Two cDNA-based Arabidopisis microarray were used to analyze gene differential expression in bud of an elite B. napus hybrid Huayouza6 and its parents,in which there were 83 over-expression transcripts and 331 under-expression transcripts between Huayouza 6 and its female parent 8086A and 94 over-expression transcripts, and 423 under-expression transcripts were demonstrated betweenHuayouza 6 and its male parent 7-5. Further analysis showed that there were significant number of genes responsible for photosynthesis, and its implication for heterosis was discussed. Northern analysis of phosphoribulokinase coincided with its expression pattern derived from hybridization of Arabidopsis cDNA microarray and B. napus mRNA, this system of heterologous hybridization analysis should be applicable to other close relatives of Arabidopsis thaliana.

  15. Generation of high-affinity fully human anti-interleukin-8 antibodies from its cDNA by two-hybrid screening and affinity maturation in yeast.

    Science.gov (United States)

    Ding, Ling; Azam, Mark; Lin, Yu-Huei; Sheridan, James; Wei, Shuanghong; Gupta, Gigi; Singh, Rakesh K; Pauling, Michelle H; Chu, Waihei; Tran, Antares; Yu, Nai-Xuan; Hu, Jiefeng; Wang, Wei; Long, Hao; Xiang, Dong; Zhu, Li; Hua, Shao-Bing

    2010-10-01

    We have developed a technology for rapidly generating novel and fully human antibodies by simply using the antigen DNA. A human single-chain variable fragment (scFv) antibody library was constructed in a yeast two-hybrid vector with high complexity. After cloning cDNA encoding the mature sequence of human interleukin-8 (hIL8) into the yeast two-hybrid system vector, we have screened the human scFv antibody library and obtained three distinct scFv clones that could specifically bind to hIL8. One clone was chosen for further improvement by a novel affinity maturation process using the error-prone PCR of the scFv sequence followed by additional rounds of yeast two-hybrid screening. The scFv antibodies of both primary and affinity-matured scFv clones were expressed in E. coli. All purified scFvs showed specific binding to hIL8 in reciprocal coimmunoprecipitation and ELISA assays. All scFvs, as well as a fully human IgG antibody converted from one of the scFv clones and expressed in the mammalian cells, were able to effectively inhibit hIL8 in neutrophil chemotaxis assays. The technology described can generate fully human antibodies with high efficiency and low cost.

  16. Isolation of mycoparasitic-related transcripts by SSH during interaction of the mycoparasite Stachybotrys elegans with its host Rhizoctonia solani.

    Science.gov (United States)

    Morissette, Danielle C; Dauch, Amélie; Beech, Robin; Masson, Luke; Brousseau, Roland; Jabaji-Hare, Suha

    2008-02-01

    Mycoparasitism by antagonistic fungi involves changes in the biochemistry and physiology of both partners. Analysis of genes that are expressed during mycoparasite-host interaction represents a powerful strategy to obtain insight into the molecular events underlying these changes. The aim of this study is to identify genes whose expression is upregulated when the mycoparasite Stachybotrys elegans is in direct confrontation with its host Rhizoctonia solani. Suppression subtractive hybridization (SSH) was used to create a subtracted cDNA library, and differential screening was applied to identify the over-expressed transcripts. We report the analysis of 2,166 clones, among which 47% were upregulated during mycoparasitism. Two hundred and sixty-one clones were sequenced that corresponded to 94 unique genes. Forty-four of these were identified as novel genes, while the remainder showed similarity to a broad diversity of genes with putative functions related to toxin production, pathogenicity, and metabolism. As a result of mycoparasitism, 15 genes belonged to R. solani among which 9 genes were assigned putative functions. Quantitative RT-PCR was used to examine the upregulation of 12 genes during the course of mycoparasitism. Seven genes showed significant upregulation at least at one-time point during interaction of the mycoparasite with its host. This study describes a first step toward knowledge of S. elegans genome. The results present the useful application of EST analysis on S. elegans and provide preliminary indication of gene expression putatively involved in mycoparasitism.

  17. Analysis of SSH library of rice variety Aganni reveals candidate gall midge resistance genes.

    Science.gov (United States)

    Divya, Dhanasekar; Singh, Y Tunginba; Nair, Suresh; Bentur, J S

    2016-03-01

    The Asian rice gall midge, Orseolia oryzae, is a serious insect pest causing extensive yield loss. Interaction between the gall midge and rice genotypes is known to be on a gene-for-gene basis. Here, we report molecular basis of HR- (hypersensitive reaction-negative) type of resistance in Aganni (an indica rice variety possessing gall midge resistance gene Gm8) through the construction and analysis of a suppressive subtraction hybridization (SSH) cDNA library. In all, 2,800 positive clones were sequenced and analyzed. The high-quality ESTs were assembled into 448 non-redundant gene sequences. Homology search with the NCBI databases, using BlastX and BlastN, revealed that 73% of the clones showed homology to genes with known function and majority of ESTs belonged to the gene ontology category 'biological process'. Validation of 27 putative candidate gall midge resistance genes through real-time PCR, following gall midge infestation, in contrasting parents and their derived pre-NILs (near isogenic lines) revealed induction of specific genes related to defense and metabolism. Interestingly, four genes, belonging to families of leucine-rich repeat (LRR), heat shock protein (HSP), pathogenesis related protein (PR), and NAC domain-containing protein, implicated in conferring HR+ type of resistance, were found to be up-regulated in Aganni. Two of the reactive oxygen intermediates (ROI)-scavenging-enzyme-coding genes Cytosolic Ascorbate Peroxidase1, 2 (OsAPx1 and OsAPx2) were found up-regulated in Aganni in incompatible interaction possibly suppressing HR. We suggest that Aganni has a deviant form of inducible, salicylic acid (SA)-mediated resistance but without HR.

  18. Genomic comparison of Yersinia pestis and Yersinia pseudotuberculosis by combination of suppression subtractive hybridization and DNA microarray

    DEFF Research Database (Denmark)

    Wang, Xiaoyi; Zhou, Dongsheng; Qin, Long

    2006-01-01

    a combination of suppression subtractive hybridization (SSH) and comparative genomic hybridization with DNAs from a diverse panel of Y. pestis and Y. pseudotuberculosis strains. SSH followed by BLAST analysis revealed 112 SSH fragments specific to strain ATCC29833, compared to the genomic sequence data of Y...

  19. [Construction and analysis of the SSH library with the resistant wheat near-isogenic line and its susceptible parent infected by Puccinia striiformis Westend. f. sp. tritici].

    Science.gov (United States)

    Shu, Wei; Chen, Xiao-Hong; Niu, Yong-Chun

    2011-09-01

    To analyze the differentially expressed genes between resistant and susceptible wheat near-isogenic lines infected by Puccinia striiformis Westend. f. sp. tritici, a subtractive library containing about 1300 clones was constructed using suppression subtractive hybridization (SSH) in which the cDNA from resistant Yr4/6 × Taichung 29 seedlings inoculated with race CY26 was used as the tester, and the corresponding cDNA from susceptible Taichung 29 as the driver. Six hundred clones from the library were analyzed with reverse Northern blot. The positive clones were further tested by Northern blotting analysis. Twelve clones were verified and showed significant difference. By means of sequencing and BlastX analysis, six function-known differentially expressed sequences were detected, and their putative products were leucine-rich repeat protein, catalase, thioredoxin H-type, RNA binding protein, ascorbate peroxidase, and heat shock protein, respectively. Among them, leucine-rich repeat protein belongs to signal transduction protein, and others belong to defense response protein.

  20. Construction of a hepatic stellate cells subtracted cDNA library of differentially expressed genes in normal mice and mice with Schistosomiasis japonica

    Institute of Scientific and Technical Information of China (English)

    Zheng Min; Wu Yi-jun; Cai Wei-min; Weng Hong-lei; Liu Rong-hua

    2005-01-01

    To construct a hepatic stellate cells (HSCs) subtracted cDNA library to find differentially expressed genes in normal mice and mice infected with Schistosomajaponicum (S. japonicum). Suppression subtractive hybridization (SSH) was used. The cDNA fragments of normal mouse were compared to those of schistosoma-infected mice to find differentially expressed genes.Then differentially expressed cDNA fragments were directly inserted into T/A cloning vector to set up the subtractive library.Amplification of the library was carried out with transformation of DH5α. The amplified library contained more than 400 positive bacterial clones, which were then hybridized with forward and backward subtracted probes for differential screening. One hundred positive bacterial clones were randomly selected for sequencing and BLAST analysis. Finally, virtual Northern Blot confirmed such differential expression. The subtracted cDNA library of differentially expressed genes of HSCs was constructed successfully,the library is efficient and lays foundation for screening and cloning new and specific genes of schistosomiasis.

  1. Suppression subtractive hybridization reveals differential gene expression in sunflower grown in high P.

    Science.gov (United States)

    Padmanabhan, Priya; Sahi, Shivendra V

    2011-06-01

    Sunflower (Helianthus annuus L.) is a commercially important oilseed crop. Previous studies proved that this crop is a promising plant species for phytoextraction of excess soil phosphorus (P) because of its superior P accumulating characteristics. Suppression subtractive hybridization (SSH) strategy was employed to isolate and characterize genes that are induced in response to high P in this crop. SSH library was prepared using cDNA generated from plants treated with high P as the 'tester'. Based on the results of dot blot analysis, 360 positive cDNA clones were selected from the SSH library for sequencing. A total of 89 non-redundant expressed sequence tags (ESTs) were identified as high P-responsive genes and they were classified into 6 functional groups. Several genes involved in metabolism showed markedly preferential expression in the library. For further confirmation, thirteen of the representative ESTs were selected from all categories for RT-PCR analysis and the results showed up-regulation of these genes in response to high P-treatment. The gene expression data derived from this study suggested that several of the up-regulated genes identified under high P-treatment might be involved in P-accumulation and tolerance in this plant.

  2. The SSH + Ext human resources management system%SSH+Ext人力资源管理系统

    Institute of Scientific and Technical Information of China (English)

    只春升

    2013-01-01

    本文在研究当前形势的情况下,提出了一种基于SSH+EXT技术的人力资源管理系统,对系统的需求和总体框架的设计进行了分析,并且描述了基于SSH+EXT技术的人力资源管理系统的设计过程.

  3. Comparative analysis of gene expression at early seedling stage between a rice hybrid and its parents using a cDNA microarray of 9198 uni-sequences

    Institute of Scientific and Technical Information of China (English)

    HUANG; Yi; LI; Lihua; CHEN; Ying; LI; Xianghua; XU; Caiguo; WANG; Shiping; ZHANG; Qifa

    2006-01-01

    Using a cDNA microarray consisting of 9198 expressed sequence tags, we surveyed the gene expression profiles in shoots and roots of a rice hybrid, Liangyoupei 9 and its parents Peiai 64s and 93-11 at 72 h after germination. A total of 8587 sequences had detectable signals in both shoots and roots of the three genotypes. A total of 1571 sequences exhibited significant (P<0.01) expression differences in shoots or roots among the three genotypes, of which 121 showed expression polymorphisms in both shoots and roots, and 870 revealed significant expression differences between the hybrid and one of the parents. The expression polymorphism of the sequences was associated with the functional categories of the sequences. They occurred more frequently in categories of carbohydrate, energy and lipid metabolisms and stress response than expected, while less frequently in categories of amino acid metabolism, transcription and translation regulation, and signal transduction. A total of 214 sequences exhibited significant (P<0.05) mid-parent heterosis in expression, of which 117 had homology to genes with known functions, assigned in the categories of basic metabolism, genetic information processing, cell growth and death, signal transduction, transportation and stress response. The results may provide useful information for exploring the relationship between gene expression polymorphism and phenotypic variation, and for characterizing the molecular mechanism of seedling development and heterosis in rice.

  4. SSH adequacy to preimplantation mammalian development: Scarce specific transcripts cloning despite irregular normalisation

    Directory of Open Access Journals (Sweden)

    Renard JP

    2005-11-01

    Full Text Available Abstract Background SSH has emerged as a widely used technology to identify genes that are differentially regulated between two biological situations. Because it includes a normalisation step, it is used for preference to clone low abundance differentially expressed transcripts. It does not require previous sequence knowledge and may start from PCR amplified cDNAs. It is thus particularly well suited to biological situations where specific genes are expressed and tiny amounts of RNA are available. This is the case during early mammalian embryo development. In this field, few differentially expressed genes have been characterized from SSH libraries, but an overall assessment of the quality of SSH libraries is still required. Because we are interested in the more systematic establishment of SSH libraries from early embryos, we have developed a simple and reliable strategy based on reporter transcript follow-up to check SSH library quality and repeatability when starting with small amounts of RNA. Results Four independent subtracted libraries were constructed. They aimed to analyze key events in the preimplantation development of rabbit and bovine embryos. The performance of the SSH procedure was assessed through the large-scale screening of thousands of clones from each library for exogenous reporter transcripts mimicking either tester specific or tester/driver common transcripts. Our results show that abundant transcripts escape normalisation which is only efficient for rare and moderately abundant transcripts. Sequencing 1600 clones from one of the libraries confirmed and extended our results to endogenous transcripts and demonstrated that some very abundant transcripts common to tester and driver escaped subtraction. Nonetheless, the four libraries were greatly enriched in clones encoding for very rare (0.0005% of mRNAs tester-specific transcripts. Conclusion The close agreement between our hybridization and sequencing results shows that the

  5. B-G cDNA clones have multiple small repeats and hybridize to both chicken MHC regions

    DEFF Research Database (Denmark)

    Kaufman, J; Salomonsen, J; Skjødt, K

    1989-01-01

    in turn react with authentic B-G proteins. None of the clones represent a complete message, some--if not all--bear introns, and none of them match with any sequences presently stored in the data banks. The following new information did, however, emerge. At least two homologous transcripts are present......We used rabbit antisera to the chicken MHC erythrocyte molecule B-G and to the class I alpha chain (B-F) to screen lambda gt11 cDNA expression libraries made with RNA selected by oligo-dT from bone marrow cells of anemic B19 homozygous chickens. Eight clones were found to encode B-G molecules which...... could explain the bewildering variation in size of B-G proteins within and between haplotypes. Southern blots of genomic chicken DNA gave complex patterns for most probes, with many bands in common using different probes, but few bands in common between haplotypes. The sequences detected are all present...

  6. Identification of immune response-related genes in the Chinese oak silkworm, Antheraea pernyi by suppression subtractive hybridization.

    Science.gov (United States)

    Liu, Qiu-Ning; Zhu, Bao-Jian; Wang, Lei; Wei, Guo-Qing; Dai, Li-Shang; Lin, Kun-Zhang; Sun, Yu; Qiu, Jian-Feng; Fu, Wei-Wei; Liu, Chao-Liang

    2013-11-01

    Insects possess an innate immune system that responds to invading microorganisms. In this study, a subtractive cDNA library was constructed to screen for immune response-related genes in the fat bodies of Antheraea pernyi (Lepidoptera: Saturniidae) pupa challenged with Escherichia coli. Four hundred putative EST clones were identified by suppression subtractive hybridization (SSH), including 50 immune response-related genes, three cytoskeleton genes, eight cell cycle and apoptosis genes, five respiration and energy metabolism genes, five transport genes, 40 metabolism genes, ten stress response genes, four transcription and translation regulation genes and 77 unknown genes. To verify the reliability of the SSH data, the transcription of a set of randomly selected immune response-related genes were confirmed by semi-quantitative reverse transcription-PCR (RT-PCR) and real-time quantitative reverse transcription-PCR (qRT-PCR). These identified immune response-related genes provide insight into understanding the innate immunity in A. pernyi.

  7. Linkage of cDNA expression profiles of mesencephalic dopaminergic neurons to a genome-wide in situ hybridization database

    Directory of Open Access Journals (Sweden)

    Simon Horst H

    2009-01-01

    Full Text Available Abstract Midbrain dopaminergic neurons are involved in control of emotion, motivation and motor behavior. The loss of one of the subpopulations, substantia nigra pars compacta, is the pathological hallmark of one of the most prominent neurological disorders, Parkinson's disease. Several groups have looked at the molecular identity of midbrain dopaminergic neurons and have suggested the gene expression profile of these neurons. Here, after determining the efficiency of each screen, we provide a linked database of the genes, expressed in this neuronal population, by combining and comparing the results of six previous studies and verification of expression of each gene in dopaminergic neurons, using the collection of in situ hybridization in the Allen Brain Atlas.

  8. Human cDNA mapping using fluorescence in situ hybridization. Progress report, April 1, 1992--December 31, 1992

    Energy Technology Data Exchange (ETDEWEB)

    Korenberg, J.R.

    1993-03-04

    Genetic mapping is approached using the techniques of high resolution fluorescence in situ hybridization (FISH). This technology and the results of its application are designed to rapidly generate whole genome as tool box of expressed sequence to speed the identification of human disease genes. The results of this study are intended to dovetail with and to link the results of existing technologies for creating backbone YAC and genetic maps. In the first eight months, this approach generated 60--80% of the expressed sequence map, the remainder expected to be derived through more long-term, labor-intensive, regional chromosomal gene searches or sequencing. The laboratory has made significant progress in the set-up phase, in mapping fetal and adult brain and other cDNAs, in testing a model system for directly linking genetic and physical maps using FISH with small fragments, in setting up a database, and in establishing the validity and throughput of the system.

  9. [Differential gene expression in incompatible interaction between Lilium regale Wilson and Fusarium oxysporum f. sp. lilii revealed by combined SSH and microarray analysis].

    Science.gov (United States)

    Rao, J; Liu, D; Zhang, N; He, H; Ge, F; Chen, C

    2014-01-01

    Fusarium wilt, caused by a soilborne pathogen Fusarium oxysporum f. sp. lilii, is the major disease of lily (Lilium L.). In order to isolate the genes differentially expressed in a resistant reaction to F. oxysporum in L. regale Wilson, a cDNA library was constructed with L. regale root during F. oxysporum infection using the suppression subtractive hybridization (SSH), and a total of 585 unique expressed sequence tags (ESTs) were obtained. Furthermore, the gene expression profiles in the incompatible interaction between L. regale and F. oxysporum were revealed by oligonucleotide microarray analysis of 585 unique ESTs comparison to the compatible interaction between a susceptible Lilium Oriental Hybrid 'Siberia' and F. oxysporum. The result of expression profile analysis indicated that the genes encoding pathogenesis-related proteins (PRs), antioxidative stress enzymes, secondary metabolism enzymes, transcription factors, signal transduction proteins as well as a large number of unknown genes were involved in early defense response of L. regale to F. oxysporum infection. Moreover, the following quantitative reverse transcription PCR (QRT-PCR) analysis confirmed reliability of the oligonucleotide microarray data. In the present study, isolation of differentially expressed genes in L. regale during response to F. oxysporum helped to uncover the molecular mechanism associated with the resistance of L. regale against F. oxysporum.

  10. Screening for Novel Binding Proteins Interacting with Human Papillomavirus Type 18 E6 Oncogene in the Hela cDNA Library by Yeast Two-Hybrid System

    Institute of Scientific and Technical Information of China (English)

    Shuang LI; Ping LIU; Ling XI; Xuefeng JIANG; Jianfeng ZHOU; Shixuan WANG; Li MENG; Yunping LU; Ding Ma

    2008-01-01

    To screen for novel binding proteins interacting with high-risk HPV 18 E6 oncogene, the strain AH109 was transformed with pGBKT7-HPV18 E6 plasmid, and subsequent transference was utilized to screen for interacting proteins with HPV 18 E6 in human Hela cDNA library. HPVl8 E6 mRNA was expressed in yeast and there was no self-activation and toxicity in AH109. Seven proteins that interacted with HPV18 E6, including transmembrane protein 87B, phosphonoformate im- muno-associated protein 5, vimentin, KM-HN-1 protein, dedicator of cytokinesis 7, vaccinia related kinase 2 and a hypothetical protein, were identified. It was suggested that yeast two-hybrid system is an efficient for screening interacting proteins. The high-risk HPV 18 E6 oncogene may interact with the proteins, which may be associated with signal transduction and transeriptional control, epithelial cell invasion and migration, as well as humoral and cellular immune etc. This investigation provides functional clues for further exploration of potential oncogenesis targets for cancer biotherapy.

  11. Screening of genes related to sulfide metabolism in Urechis unicinctus (Echiura, Urechidae) using suppression subtractive hybridization and cDNA microarray analysis.

    Science.gov (United States)

    Shi, Xiaoli; Shao, Mingyu; Zhang, Litao; Ma, Yubin; Zhang, Zhifeng

    2012-09-01

    Exogenous sulfide can generally induce metabolic injuries in most organisms and even cause death. However, organisms inhabiting intertidal zones, hydrothermal vents, and cold seeps, can tolerate, metabolize, and utilize sulfide. In this study, both suppression subtractive hybridization and cDNA microarray analysis were employed to screen sulfide metabolism-related genes from the body wall in echiuran worm Urechis unicinctus, a marine sediment species. A total of 3456 monoclones were isolated and 82 were identified as differentially expressed genes in worms exposed to 50 μM sulfide for 24 h, compared to controls. The identified genes encoded proteins with multiple processes, including metabolism, cellular process, biological regulation, response to stimulus, multicellular organismal process, localization, development, and cellular component organization. Eight genes, serase, vacuolar protein, src tyrosine kinase, sulfide oxidase-like oxidoreductase, aprataxin, SN-RNP, aminopeptidase, and predicted protein, were selected to verify expression in the worm using qRT-PCR. The agreement of gene expression evaluation was 62.5% between the results of microarray analysis and qRT-PCR. These new data will provide clues for further probing of the molecular mechanism of sulfide metabolism.

  12. SNP, ARMS and SSH authentication of medicinal Dendrobium officinale KIMURA et MIGO and application for identification of Fengdou drugs.

    Science.gov (United States)

    Ding, Ge; Zhang, Daizhen; Feng, Zhenyu; Fan, Wenjing; Ding, Xiaoyu; Li, Xuexia

    2008-04-01

    Dried stems of Dendrobium officinale have been used as crude drugs in traditional Chinese medicine (TCM) with good tonic efficacy. Sequences of chloroplast, nuclear and mitochondria genes and the method of genomic DNA (gDNA) suppression subtraction hybridization (SSH) were used to authenticate different populations during the process of good agriculture practice (GAP) and crude drug quality control. Six populations could be authenticated successfully by nine single sucleotide polymorphism (SNP) sites and six pairs of diagnostic primers for amplification refractory mutation system (ARMS) were also designed to identify six populations on the basis of single nucleotide polymorphism (SNPs). The remainder two populations (JSR, GGL) with the same sequences could be authenticated by SSH. One population-specific fragment was obtained by SSH and a pair of specific primers (SSH-JB01, SSH-JB02) on the specific sequence was designed to authenticate GGL population from the other populations tested. As the resultants were population-specific, the botanic origins of fifty "Fengdou" drug samples from markets could be classified. It is evident that the combined methods provide a high throughput and reliable approach for identification of D. officinale plants and "Fengdou" drugs.

  13. Development of a cDNA microarray of zebra mussel (Dreissena polymorpha) foot and its use in understanding the early stage of underwater adhesion.

    Science.gov (United States)

    Xu, Wei; Faisal, Mohamed

    2009-05-01

    The underwater adhesion of the zebra mussel (Dreissena polymorpha) to substrates is a complex process that is controlled by a delicate apparatus, the byssus. As a critical activity of the byssus glands embedded in the zebra mussel feet, byssogenesis is highly active to produce numerous byssal threads from the settled juvenile stage through the adult stage in its life cycle. This lifelong activity helps the zebra mussel to firmly attach to substrata underwater, thereby causing severe economic and ecologic impacts. In an attempt to better understand the zebra mussel's byssus activity, a cDNA microarray (ZMB) including 716 genes, generated from a Suppression Subtractive Hybridization (SSH) cDNA library, was printed and used for the comparison of gene expression during zebra mussel adhesion and non-adhesion. To better understand the byssogenesis mechanism, RNA samples from the zebra mussel feet with byssogenesis and without byssogenesis were used in a two-color hybridization to reveal the gene differential expression in the two states. Based on the P values (Pbyssus cDNA microarray is an efficient tool for the studies of differential gene expression in different byssogenesis states, thereby revealing important details of the underwater adhesion.

  14. “A STUDY ON SECURE SHELL (SSH PROTOCOL”

    Directory of Open Access Journals (Sweden)

    NIDHI KANDHIL

    2011-08-01

    Full Text Available Secure Shell (SSH provides an open protocol for securing network communications that is less complex and expensive than hardware-based VPN solutions. Secure Shell client/server solutions provide command shell, file transfer, and data tunneling services for TCP/IP applications. SSH connections provide highly secure authentication, encryption, and data integrity to combat password theft and other security threats. VanDyke Software® clients and servers are mature native Windows implementations that offer a range of SSH apabilities and are interoperable with SSH software on other platforms.

  15. Construction of the Subtracted cDNA Library of Striatal Neurons Treated with Long-term Morphine

    Institute of Scientific and Technical Information of China (English)

    Bo Bai; Hai-qing Liu; Jing Chen; Ya-lin Li; Hui Du; Hai Lu; Peng-li Yu

    2011-01-01

    Objective To construct a morphine tolerance model in primarily cultured striatal neurons, and screen the differentially expressed genes in this model using suppression subtractive hybridization (SSH).Methods Sbtracted cDNA libraries were constructed using SSH from normal primarily cultured striatal neurons and long-term morphine treated striatal neurons (10-5 mol/L for 72 hours). To check reliability of the cell culture model, RT-PCR was performed to detect the cAMP-responsive element-binding protein (CREB) mRNA expression. The subtracted clones were prescreened by PCR. The clones containing inserted fragments from forward libraries were sequenced and submitted to GenBank for homology analysis. And the expression levels of genes of interest were confirmed by RT-PCR. Results CREB mRNA expression showed a significant increase in morphine treated striatal neurons (62.85±1.98) compared with normal striatal neurons (28.43±1.46, P<0.01). Thirty-six clones containing inserted fragments were randomly chosen for sequence analysis. And the 36 clones showed homology with 19 known genes and 2 novel genes. The expression of 2 novel genes, mitochondrial carrier homolog 1 (Mtch1; 96.81±2.04 vs. 44.20±1.31, P<0.01) and thymoma viral proto-oncogene 1 (Akt1; 122.10±2.17 vs. 50.11 ±2.01, P<0.01), showed a significant increase in morphine-treated striatal neurons compared with normal striatal neurons. Conclusions A reliable differential cDNA library of striatal neurons treated with long-term morphine is constructed. Mtchl and Akt1 might be the candidate genes for the development of morphine tolerance.

  16. SSH 框架技术分析%Analysis of the SSH Framework Technology

    Institute of Scientific and Technical Information of China (English)

    杜素芳

    2014-01-01

    This paper analyzes the technical advantages of SSH framework and its constituent parts, and thor-oughly introduces the functions of each part.%本文分析了SSH框架技术的优势及各组成部分,并对各部分的功能特点进行了详细阐述。

  17. SSH compromise detection using NetFlow/IPFIX

    NARCIS (Netherlands)

    Hofstede, Rick; Hendriks, Luuk

    2015-01-01

    Dictionary attacks against SSH daemons are a common type of brute-force attack, in which attackers perform authentication attempts on a remote machine. By now, we are used to observing a steady number of SSH dictionary attacks in our networks every day; however, these attacks should not be underesti

  18. SSHCure: SSH Intrusion Detection using NetFlow and IPFIX

    NARCIS (Netherlands)

    Hendriks, Luuk; Hofstede, Rick; Sperotto, Anna; Pras, Aiko

    2014-01-01

    With this poster, we present our SSH Intrusion Detection System named SSHCure: it is the first IDS capable of distinguishing successful attacks from unsuccessful attacks, thus detecting actual compromises. As powerful as SSH is to administrators, as attractive it is to anyone with malicious intents.

  19. Use of SSH on a compartmented mode workstation

    Energy Technology Data Exchange (ETDEWEB)

    Tolliver, J.S. [Oak Ridge National Lab., TN (United States); Dillow, D. [Lockheed Martin Energy Systems, Oak Ridge, TN (United States)

    1997-08-01

    SSH stands for {open_quotes}Secure Shell.{close_quotes} It is now a user shell like csh or ksh. Instead it is a widely-used means to accomplish secure, encrypted communication among cooperating nodes. It is a secure replacement for the {open_quotes}r-commands{close_quotes}, rlogin, and rcp. SSH is free for noncommercial use and builds and runs on most any Unix platform. A Compartmented Mode Workstation (CMW) is an example of a secure or {open_quotes}trusted{close_quotes} operating system. The use of SSH on a CMW introduces security problems unless the SSH source code is modified to take advantage of the security features of the CMW. This paper describes the port and use of SSH on one particular brand of CMW.

  20. Proposal and Implementation of SSH Client System Using Ajax

    Science.gov (United States)

    Kosuda, Yusuke; Sasaki, Ryoichi

    Technology called Ajax gives web applications the functionality and operability of desktop applications. In this study, we propose and implement a Secure Shell (SSH) client system using Ajax, independent of the OS or Java execution environment. In this system, SSH packets are generated on a web browser by using JavaScript and a web server works as a proxy in communication with an SSH server to realize end-to-end SSH communication. We implemented a prototype program and confirmed by experiment that it runs on several web browsers and mobile phones. This system has enabled secure SSH communication from a PC at an Internet cafe or any mobile phone. By measuring the processing performance, we verified satisfactory performance for emergency use, although the speed was unsatisfactory in some cases with mobile phone. The system proposed in this study will be effective in various fields of E-Business.

  1. Identification of cold responsive genes in Pacific white shrimp (Litopenaeus vannamei) by suppression subtractive hybridization.

    Science.gov (United States)

    Peng, Jinxia; Wei, Pinyuan; Chen, Xiuli; Zeng, Digang; Chen, Xiaohan

    2016-01-10

    The Pacific white shrimp (Litopenaeus vannamei) is one of the most widely cultured shrimp species in the world. Despite L. vannamei having tropical origins, it is being reared subtropically, with low temperature stress being one of the most severe threats to its growth, survival and distribution. To unravel the molecular basis of cold tolerance in L. vannamei, the suppression subtractive hybridization (SSH) platform was employed to identify cold responsive genes in the hepatopancreas of L. vannamei. Both forward and reverse cDNA libraries were constructed, followed by dot blot hybridization, cloning, sequence analysis and quantitative real-time PCR. These approaches identified 92 cold induced and 48 cold inhibited ESTs to give a total of 37 cold induced and 17 cold inhibited contigs. Some of the identified genes related to stress response or cell defense, such as tetraspanins (TSPANs), DEAD-box helicase, heat shock proteins (HSPs) and metallothionein (MT), which were more abundant in the forward SSH library than in the reverse SSH library. The most abundant Est was a tetraspanin-8 (TSPAN8) homolog dubbed LvTSPAN8. A multiple sequence alignment and transmembrane domain prediction was also performed for LvTSPAN8. LvTSPAN8 expression was also examined in the gills, muscle, heart and hepatopancreas following cold exposure and showed the highest expression levels in the hepatopancreas. Overall, this study was able to identify several known genes and novel genes via SSH that appear to be associated with cold stress and will help to provide further insights into the molecular mechanisms regulating cold tolerance in L. vannamei.

  2. SSH Analysis of Endosperm Transcripts and Characterization of Heat Stress Regulated Expressed Sequence Tags in Bread Wheat.

    Science.gov (United States)

    Goswami, Suneha; Kumar, Ranjeet R; Dubey, Kavita; Singh, Jyoti P; Tiwari, Sachidanand; Kumar, Ashok; Smita, Shuchi; Mishra, Dwijesh C; Kumar, Sanjeev; Grover, Monendra; Padaria, Jasdeep C; Kala, Yugal K; Singh, Gyanendra P; Pathak, Himanshu; Chinnusamy, Viswanathan; Rai, Anil; Praveen, Shelly; Rai, Raj D

    2016-01-01

    Heat stress is one of the major problems in agriculturally important cereal crops, especially wheat. Here, we have constructed a subtracted cDNA library from the endosperm of HS-treated (42°C for 2 h) wheat cv. HD2985 by suppression subtractive hybridization (SSH). We identified ~550 recombinant clones ranging from 200 to 500 bp with an average size of 300 bp. Sanger's sequencing was performed with 205 positive clones to generate the differentially expressed sequence tags (ESTs). Most of the ESTs were observed to be localized on the long arm of chromosome 2A and associated with heat stress tolerance and metabolic pathways. Identified ESTs were BLAST search using Ensemble, TriFLD, and TIGR databases and the predicted CDS were translated and aligned with the protein sequences available in pfam and InterProScan 5 databases to predict the differentially expressed proteins (DEPs). We observed eight different types of post-translational modifications (PTMs) in the DEPs corresponds to the cloned ESTs-147 sites with phosphorylation, 21 sites with sumoylation, 237 with palmitoylation, 96 sites with S-nitrosylation, 3066 calpain cleavage sites, and 103 tyrosine nitration sites, predicted to sense the heat stress and regulate the expression of stress genes. Twelve DEPs were observed to have transmembrane helixes (TMH) in their structure, predicted to play the role of sensors of HS. Quantitative Real-Time PCR of randomly selected ESTs showed very high relative expression of HSP17 under HS; up-regulation was observed more in wheat cv. HD2985 (thermotolerant), as compared to HD2329 (thermosusceptible) during grain-filling. The abundance of transcripts was further validated through northern blot analysis. The ESTs and their corresponding DEPs can be used as molecular marker for screening or targeted precision breeding program. PTMs identified in the DEPs can be used to elucidate the thermotolerance mechanism of wheat-a novel step toward the development of "climate-smart" wheat.

  3. SSH analysis of endosperm transcripts and characterization of heat stress regulated expressed sequence tags in bread wheat

    Directory of Open Access Journals (Sweden)

    Suneha Goswami

    2016-08-01

    Full Text Available Heat stress is one of the major problems in agriculturally important cereal crops, especially wheat. Here, we have constructed a subtracted cDNA library from the endosperm of HS-treated (42°C for 2 h wheat cv. HD2985 by suppression subtractive hybridization (SSH. We identified ~550 recombinant clones ranging from 200 to 500 bp with an average size of 300 bp. Sanger’s sequencing was performed with 205 positive clones to generate the differentially expressed sequence tags (ESTs. Most of the ESTs were observed to be localized on the long arm of chromosome 2A and associated with heat stress tolerance and metabolic pathways. Identified ESTs were BLAST search using Ensemble, TriFLD and TIGR databases and the predicted CDS were translated and aligned with the protein sequences available in pfam and InterProScan 5 databases to predict the differentially expressed proteins (DEPs. We observed eight different types of post-translational modifications (PTMs in the DEPs corresponds to the cloned ESTs—147 sites with phosphorylation, 21 sites with sumoylation, 237 with palmitoylation, 96 sites with S-nitrosylation, 3066 calpain cleavage sites, and 103 tyrosine nitration sites, predicted to sense the heat stress and regulate the expression of stress genes. Twelve DEPs were observed to have transmembrane helixes (TMH in their structure, predicted to play the role of sensors of HS. Quantitative Real-Time PCR of randomly selected ESTs showed very high relative expression of HSP17 under HS; up-regulation was observed more in wheat cv. HD2985 (thermotolerant, as compared to HD2329 (thermosusceptible during grain-filling. The abundance of transcripts was further validated through northern blot analysis. The ESTs and their corresponding DEPs can be used as molecular marker for screening or targeted precision breeding program. PTMs identified in the DEPs can be used to elucidate the thermotolerance mechanism of wheat – a novel step towards the development of

  4. SSH Analysis of Endosperm Transcripts and Characterization of Heat Stress Regulated Expressed Sequence Tags in Bread Wheat

    Science.gov (United States)

    Goswami, Suneha; Kumar, Ranjeet R.; Dubey, Kavita; Singh, Jyoti P.; Tiwari, Sachidanand; Kumar, Ashok; Smita, Shuchi; Mishra, Dwijesh C.; Kumar, Sanjeev; Grover, Monendra; Padaria, Jasdeep C.; Kala, Yugal K.; Singh, Gyanendra P.; Pathak, Himanshu; Chinnusamy, Viswanathan; Rai, Anil; Praveen, Shelly; Rai, Raj D.

    2016-01-01

    Heat stress is one of the major problems in agriculturally important cereal crops, especially wheat. Here, we have constructed a subtracted cDNA library from the endosperm of HS-treated (42°C for 2 h) wheat cv. HD2985 by suppression subtractive hybridization (SSH). We identified ~550 recombinant clones ranging from 200 to 500 bp with an average size of 300 bp. Sanger's sequencing was performed with 205 positive clones to generate the differentially expressed sequence tags (ESTs). Most of the ESTs were observed to be localized on the long arm of chromosome 2A and associated with heat stress tolerance and metabolic pathways. Identified ESTs were BLAST search using Ensemble, TriFLD, and TIGR databases and the predicted CDS were translated and aligned with the protein sequences available in pfam and InterProScan 5 databases to predict the differentially expressed proteins (DEPs). We observed eight different types of post-translational modifications (PTMs) in the DEPs corresponds to the cloned ESTs-147 sites with phosphorylation, 21 sites with sumoylation, 237 with palmitoylation, 96 sites with S-nitrosylation, 3066 calpain cleavage sites, and 103 tyrosine nitration sites, predicted to sense the heat stress and regulate the expression of stress genes. Twelve DEPs were observed to have transmembrane helixes (TMH) in their structure, predicted to play the role of sensors of HS. Quantitative Real-Time PCR of randomly selected ESTs showed very high relative expression of HSP17 under HS; up-regulation was observed more in wheat cv. HD2985 (thermotolerant), as compared to HD2329 (thermosusceptible) during grain-filling. The abundance of transcripts was further validated through northern blot analysis. The ESTs and their corresponding DEPs can be used as molecular marker for screening or targeted precision breeding program. PTMs identified in the DEPs can be used to elucidate the thermotolerance mechanism of wheat—a novel step toward the development of

  5. SSH Honeypot: Building, Deploying and Analysis

    Directory of Open Access Journals (Sweden)

    Harry Doubleday

    2016-05-01

    Full Text Available This article is set to discuss the various techniques that can be used while developing a honeypot, of any form, while considering the advantages and disadvantages of these very different methods. The foremost aims are to cover the principles of the Secure Shell (SSH, how it can be useful and more importantly, how attackers can gain access to a system by using it. The article involved the development of multiple low interaction honeypots. The low interaction honeypots that have been developed make use of the highly documented libssh and even editing the source code of an already available SSH daemon. Finally the aim is to combine the results with the vastly distributed Kippo honeypot, in order to be able to compare and contrast the results along with usability and necessity of particular features. Providing a clean and simple description for less knowledgeable users to be able to create and deploy a honeypot of production quality, adding security advantages to their network instantaneously.

  6. Using SSH Technology to Identify Relevant Genes Resistant to Cucumber Downy Mildew%利用SSH技术鉴定黄瓜抗霜霉病相关基因

    Institute of Scientific and Technical Information of China (English)

    刘大军; 秦智伟; 周秀艳; 辛明; 武涛

    2014-01-01

    Adopting SSH technology,this paper studied on the difference expressive genes of downy mildew resistant cucumber inbred line ‘M801-3-1’ before and after the infection of downy mildew.Disease-resistant cucumber material vaccinated downy mildew and unvaccinated cDNA library was constructed using SSH technology.Forty-eight positive clones were obtained by reverse Northern blot hybridization detection, and 14 UniESTs were obtained including 8 singletons and 6 contigs via molecular biology software.The SSH-EST genes function analysis indicated that oxidative stress resistance and chloroplast reconstruction and protection mechanisms had very important role on disease resistance.At the same time,the paper put forward that Clpb,HSP70,HSP22 and peptidyl-prolyl cis-trans isomerase might be involved in the R gene-mediated defense reaction,and smHSP might be the ‘defending target’ of R protein,responsible for monitoring intracellular exception.This study has provided a key evidence for revealing the relations between active oxygen mechanism and R gene mediate protection mechanism.%采用SSH技术对黄瓜抗霜霉病自交系M801-3-1侵染霜霉病菌前后的差异表达基因进行了研究。利用 SSH 技术构建抗病黄瓜材料接种霜霉病菌和未接种差异表达的差减cDNA文库。经反向Northern blot杂交检测,共得到48个阳性克隆。利用分子生物学软件对测序后的序列进行质量检测和聚类、拼接,共得到14个UniESTs,其中包括8个singletons和6个contigs。通过SSH-EST代表基因功能的分析,说明抗氧化胁迫能力和叶绿体的重建和保护机制对抗病品种抗病有重要作用。同时,提出SSH-EST代表的clpb、HSP70、HSP22和肽脯氨酰顺反异构酶还可能参与了R基因介导的防御反应,smHSP有可能就是R蛋白的“保卫靶”,负责监测细胞内的异常,这一发现为揭示活性氧机制和R基因介导的防卫机制之间的关系提供了关键证据。

  7. Use of suppression subtractive hybridization to identify downy mildew genes expressed during infection of Arabidopsis thaliana.

    Science.gov (United States)

    Bittner-Eddy, Peter D; Allen, Rebecca L; Rehmany, Anne P; Birch, Paul; Beynon, Jim L

    2003-11-01

    SUMMARY Peronospora parasitica is an obligate biotrophic oomycete that causes downy mildew in Arabidopsis thaliana and Brassica species. Our goal is to identify P. parasitica (At) genes that are involved in pathogenicity. We used suppression subtractive hybridization (SSH) to generate cDNA libraries enriched for in planta-expressed parasite genes and up-regulated host genes. A total of 1345 clones were sequenced representing cDNA fragments from 25 putative P. parasitica (At) genes (Ppat 1-25) and 618 Arabidopsis genes. Analyses of expression patterns showed that 15 Ppats were expressed only in planta. Eleven Ppats encoded peptides with homology (BlastP values planta-expressed genes from P. parasitica (At) that complements other gene discovery approaches such as EST sequencing.

  8. Identification of differentially expressed radiation-induced genes in cervix carcinoma cells using suppression subtractive hybridization

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Jun Sang; Lee, Young Sook; Lee, Jeung Hoon; Lee, Woong Hee; Seo, Eun Young; Cho, Moon June [Chungnam National University, Daejeon (Korea, Republic of)

    2005-03-15

    A number of genes and their products are induced early or late following exposure of cells to ionizing radiation. These radiation-induced genes have various effects of irradiated cells and tissues. Suppression subtractive hybridization (SSH) based on PCR was used to identify the differentially expressed genes by radiation in cervix carcinoma cells. Total RNA and poly (A){sup +} mRNA were isolated from irradiated and non-irradiated HeLa cells. Forward-and reverse-subtracted cDNA libraries were constructed using SSH. Eighty-eight clones of each were used to randomly select differentially expressed genes using reverse Northern blotting (dot blot analysis). Northern blotting was used to verify the screened genes. Of the 176 clones, 10 genes in the forward-subtracted library and 9 genes in the reverse-subtracted library were identified as differentially expressed radiation-induced genes by PCR-select differential screening. Three clones from the forward-subtracted library were confirmed by Northern blotting, and showed increased expression in a dose-dependent manner, including a telomerase catalytic subunit and sodium channel-like protein gene, and an ESTs (expressed sequence tags) gene. We identified differentially expressed radiation-induced genes with low-abundance genes with SSH, but further characterization of theses genes are necessary to clarify the biological functions of them.

  9. Identification of differentially expressed genes in parasitic phase Miamiensis avidus (Ciliophora: Scuticociliatia) using suppression subtractive hybridization.

    Science.gov (United States)

    Lee, Eun Hye; Kim, Ki Hong

    2011-04-06

    Miamiensis avidus, a causative agent of scuticociliatosis in cultured marine fish, can live not only in seawater as a free-living organism but also in fish as a parasite. In this study, a cDNA library of representative mRNAs more specific to parasitic phase M. avidus was generated using suppression subtractive hybridization (SSH), and 520 clones selected from the SSH library were single-run sequenced. The differential gene expression patterns were confirmed by semi-quantitative reverse-transcription PCR. Of the 510 SSH clones, 21 clones of 6 putative genes did not match sequences in the public database. The expectation values (E-values) of 117 clones encoding 9 putative genes were greater than 1 x 10(-5). The other 372 clones that met the criterion of E value <1 x 10-5 were matched to 26 known sequences in the database. Genes associated with signal transduction, cell proliferation, membrane transportation, protein translocation, and transcription regulation were preferentially expressed in parasitic phase M. avidus. The differential gene expression may be needed for the ciliates to survive in the host fish, and the corresponding proteins might be used as antigen candidates for development of scuticociliatosis vaccines.

  10. Over-expressed Genes Detected by Suppression Subtractive Hybridization in Carcinoma Derived From Transformed 16HBE Cells Induced by BPDE

    Institute of Scientific and Technical Information of China (English)

    SHE-JUAN AN; JIA-KUN CHEN; LI-LI LIU; YAN-FENG ZHAO; XUE-MIN CHEN

    2005-01-01

    Objective To screen the over differentially expressed genes in carcinoma induced by BPDE-transformed 16HBE cells (16HBE-C cells). Methods The suppression subtractive hybridization (SSH) method was performed to profile differentially expressed genes between 16HBE-C cells and 16HBE cells. The cDNA fragments of differentially expressed genes were inserted into TA cloning vector and transformed competent E. coli strain. Positive clones were randomly picked up and identified by the colony PCR method. Dot blot was used to test the same source with the tester. The differentially expressed cDNA fragments were sequenced and compared with known genes and EST database in Genbank. Results Eight known genes were over-expressed in 16HBE-C cells including eukaryotic translation elongation factor 1 alpha 1, HIF-1 responsive RTP801, ribosomal protein L10 (RPL10), ribosomal protein S29 (RPS29), mitochondrion related genes, and laminin receptor 1. Three differentially expressed cDNA fragments could not be matched to the known genes but to the EST database. Conclusion The SSH method can detect differentially expressed genes between 16HBE-C and 16HBE cells. BPDE-induced carcinogenesis may be related to alteration of at least eight known genes and three unknown genes. These expression data provide a clue to further cloning novel genes and studying functions in BPDE-induced carcinoma.

  11. Isolation, identification and expression analysis of salt-induced genes in Suaeda maritima, a natural halophyte, using PCR-based suppression subtractive hybridization

    Directory of Open Access Journals (Sweden)

    Sahu Binod B

    2009-06-01

    Full Text Available Abstract Background Despite wealth of information generated on salt tolerance mechanism, its basics still remain elusive. Thus, there is a need of continued effort to understand the salt tolerance mechanism using suitable biotechnological techniques and test plants (species to enable development of salt tolerant cultivars of interest. Therefore, the present study was undertaken to generate information on salt stress responsive genes in a natural halophyte, Suaeda maritima, using PCR-based suppression subtractive hybridization (PCR-SSH technique. Results Forward and reverse SSH cDNA libraries were constructed after exposing the young plants to 425 mM NaCl for 24 h. From the forward SSH cDNA library, 429 high quality ESTs were obtained. BLASTX search and TIGR assembler programme revealed overexpression of 167 unigenes comprising 89 singletons and 78 contigs with ESTs redundancy of 81.8%. Among the unigenes, 32.5% were found to be of special interest, indicating novel function of these genes with regard to salt tolerance. Literature search for the known unigenes revealed that only 17 of them were salt-inducible. A comparative analysis of the existing SSH cDNA libraries for NaCl stress in plants showed that only a few overexpressing unigenes were common in them. Moreover, the present study also showed increased expression of phosphoethanolamine N-methyltransferase gene, indicating the possible accumulation of a much studied osmoticum, glycinebetaine, in halophyte under salt stress. Functional categorization of the proteins as per the Munich database in general revealed that salt tolerance could be largely determined by the proteins involved in transcription, signal transduction, protein activity regulation and cell differentiation and organogenesis. Conclusion The study provided a clear indication of possible vital role of glycinebetaine in the salt tolerance process in S. maritima. However, the salt-induced expression of a large number of genes

  12. [Construction and analysis of SSH library of Gossypium barbadense upon infection with Verticillium dahliae].

    Science.gov (United States)

    Zhu, Long-Fu; Tu, Li-Li; Zhang, Xian-Long; Nie, Yi-Chun; Guo, Xiao-Ping; Xia, Qi-Zhong

    2005-05-01

    Roots were collected from the seedlings inoculated with pathogen Verticillium dahliae after 2, 4, 8, 12, 24, 48, 72 and 96 hours for total RNA extraction. The cDNAs from the inoculated seedlings were used as the tester and those from the control seedlings as the driver. SSH method was employed to find the differently expressed cDNAs responding to the pathogen. T/A clone library was constructed containing 534 clones. The cDNA inserts were amplified from the bacterial clones directly with M13 primers by PCR. The size of the products ranged 0.2 - 1.2 kb with an average size of 0.5 kb. The SSH products were dotted on nylon filters, and the positive clones were screened by virtual Northern blotting with probes of the two kinds of initiative cDNAs. Totally 78 clones which were up-regulated and putatively involved in the defense response of G. barbadense were identified and sequenced. Sequence similarity searches were performed with the Blastn and Blastx. Most of them showed high or partial homology to genes or ESTs induced by different stresses in Arabidopsis thaliana and other species,such as the pathogenesis-related 10 family of G. hirsumtum and disease resistance-responsive family protein in Arabidopsis thaliana. The results would be helpful to understand the molecular mechanisms of disease response in cotton.

  13. ARC Code TI: Middleware Using Existing SSH Hosts (Mesh)

    Data.gov (United States)

    National Aeronautics and Space Administration — Mesh is a secure, lightweight grid middleware that is based on the addition of a single sign-on capability to the built-in public key authentication mechanism of SSH...

  14. 玉米弯孢叶斑病菌酵母双杂交cDNA文库的构建及评价%Construction and characterization of yeast two hybrid cDNA library of Curvularia lunata

    Institute of Scientific and Technical Information of China (English)

    荆晶; 刘铜; 陈捷

    2012-01-01

    由新月弯孢[Curvularia lunata (Wakker)Boed]引起的玉米弯孢叶斑病是我国玉米生产区的一种重要性叶斑病,曾在20世纪90年代给我国玉米生产造成较大损失.目前对该病原菌致病类型鉴定与致病相关基因、病害发生规律、综合防治等方面均有较好的研究基础[1],并且在前期的研究中发现该病菌调控毒素形成的相关基因与色素合成相关基因在表达上存在某种关联性[2],由于这2个基因均为致病相关基因,因此研究两基因表达的蛋白的关联性对于全面揭示病菌致病机理和调控网络具有重要意义.%BD SMART technique and LD-PCR were applied to synthesize double strand cDNA(ds cDNA). The ds cDNA and pGADT7-Rec were transfered into competent yeast cell to construct yeast two-hybrid cDNA library of Curvularia lunata by homologous recombination method. The results showed that library capacitance was 2.46×105 and transformation rate was 2.13×105/μg pGADT7-Rec. The plaque titer of the library was 2.5×108 pfu/mL and the recombination frequency was 88. 24%. The length of inserted cDNA fragment was ranged from 0.4 kb to 3 kb in average. This is the first time to use BD SMART technique to construct yeast two-hybrid cDNA library of C. lunata by homologous recombination.

  15. Dicty_cDB: SSH130 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available SS (Link to library) SSH130 (Link to dictyBase) - G21002 DDB0190562 Contig-U05309-1...k to library) Clone ID SSH130 (Link to dictyBase) Atlas ID - NBRP ID G21002 dictyBase ID DDB0190562 Link to ...Contig Contig-U05309-1 Original site URL http://dictycdb.biol.tsukuba.ac.jp/CSM/S...NYILYPTSPPLLSECF SVSYPPPPFLNCNNNSNNSSPINSPKSFNNNNHSSNNFNITNVNNFYFNNNLNNSGSGST SSGGNSGTPPSPIQIPIICT...KKKKKINKMYQPXINNNYILYPTSPPLLSECF SVSYPPPPFLNCNNNSNNSSPINSPKSFNNNNHSSNNFNITNVNNFYFNNNLNNSGSGST SSGGNSGTPPSPIQIPIICT

  16. Isolating Soil Drought-Induced Genes from Maize Seedling Leaves Through Suppression Subtractive Hybridization

    Institute of Scientific and Technical Information of China (English)

    LI Hui-yong; HUANG Su-hua; SHI Yun-su2; SONG Yan-chun; ZHAO Jiu-ran; WANG Feng-ge; WANG Tian-yu; LI Yu

    2007-01-01

    In this study, a forward cDNA library was constructed by suppression subtractive hybridization using seedling leaves of CN165, a drought-tolerant maize inbred line. In the suppression subtractive hybridization (SSH) library, 672 positive clones were picked up randomly. After polymerase chain reaction (PCR) of each clone, all the single clones were sequenced. Totally 598 available sequences were obtained. After cluster analysis of the EST sequences, 80 uniESTs were obtained, among which 57 uniESTs were contigs and 23 uniESTs were singlets. The results of BLASTN showed that all the uniESTs had homologous sequences in the nr database. The BLASTX results indicated that 68 uniESTs had significant protein homology, 8 uniESTs with homology of unknown proteins and putative proteins, and 4 uniESTs without protein homology. Those drought stress-induced genes were involved in many metabolism pathways to regulate plant growth and development under drought stress.

  17. Establishing cDNA substractive library of kidney Ying deficiency of lupus nephyritis by Suppression substractive hybridization%狼疮性肾炎肾阴虚证cDNA消减文库的构建

    Institute of Scientific and Technical Information of China (English)

    李玉萍; 罗仁; 赵晓山; 聂晓莉; 李俊

    2006-01-01

    目的:采用抑制性消减杂交(suppression subtractive hybridization,SSH)技术构建汉族人狼疮性肾炎(Lupus Nephritis LN)肾阴虚证cDNA消减文库.方法:选择汉族人LN且中医辨证为肾阴虚证的患者以及正常人作为其对照组,进行正向和消减杂交.采用Trizol一步法提取总RNA,用SMART(Switch Mechanism At 5 end of RNA Template)技术逆转录并扩增总cDNA,用RsaI酶切基因组cDNA成大小不等的片段,分别与两种不同的接头连接,进行两次消减杂交及两次抑制性PCR,将PCR产物与U载体连接,经蓝白斑筛选后,再用PCR方法插入片段筛选出阳性重组质粒,构建LN肾阴虚证消减文库.结果:用SSH方法筛选出了LN肾阴虚证的差异cDNA片段,得到了450个白色克隆,再经PCR方法快速筛选出阳性重组质粒,从而成功地构建了LN肾阴虚证的cDNA消减文库.结论:SSH技术能够快速有效地分离差异cDNA片段以构建LN肾阴虚证cDNA消减文库,为进一步筛选和克隆LN肾阴虚证相关基因奠定了基础.

  18. Differentially expressed genes of human microvascular endothelial cells in response to anti-dengue virus NS1 antibodies by suppression subtractive hybridization.

    Science.gov (United States)

    Yin, Yue; Jiang, Lan; Fang, Danyun; Jiang, Lifang; Zhou, Junmei

    2013-06-01

    It has been previously shown that anti-dengue virus (DENV) nonstructural protein NS1 antibodies could act as autoantibodies that direct against one or more of the host's own proteins, which has potential implications for dengue hemorrhagic fever pathogenesis. In the present study, we have employed suppression subtractive hybridization (SSH) to identify the differentially expressed genes from human microvascular endothelial cells (HMEC-1) in response to anti-dengue virus type 2 NS1 antibodies (anti-DENV2 NS1 Abs). A total of 35 clones from the SSH cDNA library were randomly selected for further analysis using bioinformatics tools after vector screening. After searching for sequence homology in NCBI GenBank database with BLASTN and BLASTX programs, 23 obtained sequences with significant matches (E-values <1×10(-4)) in the SSH library. The predicted genes in the subtracted library include immune response molecules (CD59 antigen preproprotein preproprotein, MURR1), signal transduction molecules (Nuclear casein kinase and cyclin-dependent kinase substrate 1), calcium-binding proteins (S100A6, Annexin A2 isoform 1/2), and cell-membrane component (Yip1 domain family). From these clones, 5 upregulated genes were selected for differential expression profiling by real-time RT-PCR to confirm their upregulated status. The results confirmed their differential upregulation, and thus verified the success of SSHs and the likely involvement of these genes in dengue pathogenesis.

  19. Isolation and analysis of water stress induced genes in maize seedlings by subtractive PCR and cDNA macroarray.

    Science.gov (United States)

    Zheng, Jun; Zhao, Jinfeng; Tao, Yazhong; Wang, Jianhua; Liu, Yunjun; Fu, Junjie; Jin, Ying; Gao, Peng; Zhang, Jinpeng; Bai, Yunfeng; Wang, Guoying

    2004-08-01

    In order to identify genes induced during the water stress response in maize (Zea mays) seedlings, suppression subtractive hybridization (SSH) was performed using mixed cDNAs prepared from maize seedlings treated with 20% PEG as testers and cDNAs from unstressed maize seedlings as drivers. A forward subtractive cDNA library was constructed, from which 960 recombinant colonies were picked and amplified. Through differential screening of the subtractive cDNA library, 533 clones were identified as water stress induced. After sequencing, 190 unique expressed sequence tags (ESTs) were obtained by clustering and blast analysis, which included transcripts that had previously been reported as responsive to stress as well as some functionally unknown transcripts. The ESTs with significant protein homology were sorted into 13 functional categories. A cDNA marcoarray containing the 190 unique ESTs was used to analyze their expression profiles in maize seedling during both PEG treatment and natural drought. The results indicated that 67 ESTs in leaves and 113 ESTs in roots were significantly up-regulated by PEG-stress. 123 ESTs were found to be up-regulated for at least one time-course point in either maize leaves or roots. Correspondingly, 163 ESTs were significantly up-regulated by drought stress. Results from the hierarchical cluster analysis suggest that the leaves and roots of maize seedlings had different expression profiles after PEG treatment and that there was a lot of overlap between PEG- and drought-stress induced up-regulated transcripts. A set of transcripts has been identified, which have significantly increased expression and probably involved in water stress signaling pathway based on data analysis.

  20. Construction and evaluation of yeast two-hybrid cDNA library of Diaphorina citri (Hemiptera: Psyllidae)%柑橘木虱酵母双杂交cDNA文库的构建及评价

    Institute of Scientific and Technical Information of China (English)

    马晓芳; 陈国庆; 张学潮; 徐海君

    2013-01-01

    In order to study the interacting proteins between the Asian citrus psyllid ( Diaphorina citri) and Candidatus Liberibacter asiaticus ( CLas) which is the pathogenic bacterium causing Huanglongbing, yeast two-hybrid cDNA library of D. citri was constructed using the Switching Mechanism at 5' End of the RNA Transcript (SMART) technique. The total RNA was isolated from the citrus psyllid adults bred in the laboratory and subjected to reverse transcription, and the double-strand cDNAs ( ds cDNAs) were synthesized. The ds cDNAs were ligated with homologous adapter and purified by the chromatography column. By using homologous recombination reaction, the ds cDNAs were transformed into the Y187 competent cell with the library plasmid pGADT7-Rec to construct yeast two-hybrid cDNA library. Detection of the library indicated that it contained more than 106 independent clones, the titer of the amplified library was 2. 23 × 10 cfu/mL, and the average size of inserts was above 750 bp in the cDNA library. These results demonstrate that the library meets the requirements of the standard cDNA library. Moreover, two membrane proteins, ORF420 and ORF3420, from ( CLas) were used as bait proteins to screen the interacting proteins in the library, but no positive clone was screened in the tests. The yeast two-hybrid cDNA library of D. citri will be useful for the research on the interaction between insect vectors and C. Liberibacter asiaticus in the future.%为了探索研究柑橘木虱Diaphorina citri与柑橘黄龙病(Huanglongbing,HLB)病原菌的相互作用蛋白,本研究运用RNA转录中5 '末端转换机制(Switching Mechanism at 5'End of the RNA Transcript,SMART)技术构建了柑橘木虱的酵母双杂交cDNA文库.以实验室饲养的柑橘木虱为材料,提取总RNA,经反转录后合成ds cDNA,两端添加同源重组序列,并用层析柱纯化;ds cDNA与文库质粒pGADT7-Rec在酵母Y187感受态细胞内发生同源重组,柑橘木虱cDNA重组到文库质粒

  1. 罗汉果果实不同发育时期SSH文库的构建%Construction of SSH-cDNA library from different developmental stages of Siraitia grosvenorii

    Institute of Scientific and Technical Information of China (English)

    唐其; 邱德有; 马小军; 莫长明; 付伟

    2011-01-01

    In order to reveal the differentially-expressed genes involved in triterpenoid saponin biosynthesis of Siraitia grosvenorii, 50 DAF(days after flowering)and 70 DAF fruits from different developmental stages were employed to construct subtractive cDNA library by suppression subtractive hybridization(SSH)in this study. Total 641 positive clones were selected randomly and sequenced from the forward-subtracted cDNA library(70 DAF as the tester and 50 DAF as the driver), and 622 high quality sequences were obtained. The rate of the recombination fraction was above 96%. The distribution of inserted fragments ranged from 101 bp and 934 bp,and the average fragment size was 500 bp. Followed by BLASTN and BLASTX for sequences dates,201 ESTs showed no significant matches to any other sequences in NCBI nucleotide sequence database,they were probably novel genes. The other 421 ESTs carried with remarkable identity to proteins with known function in the database, which fell into several functional categories including energy and secondary metabolism, transcription factors,ripening, senescence and pathogen-resistance. Further results indicated that the cDNA library quality were conformed to SSH library standard and could be used in further studies, also would provide reference for studying the differentially expressed genes, exploring molecular mechanism of triterpenoid saponin biosynthesis,and increasing productivity and quality of mogrosides in S. grosvenorii.%以罗汉果授粉后50 d和70 d果实为材料,利用抑制消减杂交技术构建了罗汉果果实在不同生育时期皂苷生物合成相关基因的差减cDNA文库.在正向差减文库(70 d为tester,50 d果实为driver)中随机挑选了641个cDNA阳性克隆测序,得到622条有效序列,重组率在96%以上.外源片段的长度分布在101~934 bp之间,平均长度约500 bp.BLASTN结果显示:201个ESTs序列没有同源序列,可能是新基因;另外421个ESTs序列在核酸数据库中存在同源

  2. Identification of Differentially Expressed Genes in Sweetpotato Storage Roots Between Kokei No. 14 and Its Mutant Nongdafu 14 Using PCR-Based cDNA Subtraction

    Institute of Scientific and Technical Information of China (English)

    CHEN Wei; ZHAI Hong; YANG Yuan-jun; HE Shao-zhen; LIU De-gao; LIU Qing-chang

    2013-01-01

    The contents of carotenoids in the storage root of sweetpotato, Ipomoea batatas (L.) Lam. vary dramatically among different cultivars. However, so far little is known about the regulation of carotenoids synthesis in sweetpotato. In our laboratory, we identified a novel sweetpotato mutant, Nongdafu 14, which is a homogenous mutant derived from the wild type Kokei No. 14. The contents of carotenoids in the storage root of Nongdafu 14 were analyzed using high performance liquid chromatography (HPLC), and it was found that the amount of carotenoids, b-carotene, lutein and zeaxantion, three major types of carotenoids in sweetpotato storage roots, increased 2-26 folds in Nongdafu 14 compared to Kokei No. 14. Suppression subtractive hybridization (SSH) was used to identify genes that were differentially expressed in Nongdafu 14, and a differentially expressed cDNA library was constructed using the cDNA of Nongdafu 14 storage roots as tester and that of Kokei No. 14 storage roots as driver. Out of the 1 530 clones sequenced, we identified 292 nonredundant ESTs. GO and KEGG analyses of these differentially expressed ESTs indicated that diverse metabolism pathways were affected and candidate genes involved in regulation of carotenoids synthesis are suggested.

  3. Dicty_cDB: SSH722 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available 2.y1 Plasmodium falciparum 3D7 asexual cDNA Plasmodium falciparum cDNA 5' similar...2 |BU498482.1 PfESToab97g07.y1 Plasmodium falciparum 3D7 asexual cDNA Plasmodium falciparum cDNA 5' similar ...equence. 46 0.23 1 BU498405 |BU498405.1 PfESToab96g09.y1 Plasmodium falciparum 3D7 asexual cDNA Plasmodium f

  4. Screening of genes for proteins interacting with the PS1TP5 protein of hepatitis B virus: probing a human leukocyte cDNA library using the yeast two-hybrid system

    Institute of Scientific and Technical Information of China (English)

    ZHANG Jian-kang; ZHAO Long-feng; CHENG Jun; GUO Jiang; LUN Yong-zhi; HONG Yuan

    2006-01-01

    Background The hepatitis B virus (HBV) genome includes S, C, P and X regions. The S region is divided into four subregions of pre-pre-S, pre-S1, pre-S2 and S. PS1TP5 (human gene 5 transactivated by pre-S1 protein of HBV) is a novel target gene transactivated by the pre-S1 protein that has been screened with a suppression subtractive hybridization technique in our laboratory (GenBank accession: AY427953). In order to investigate the biological function of the PS1TP5 protein, we performed a yeast two-hybrid system 3 to screen proteins from a human leukocyte cDNA library interacting with the PS 1TP5 protein.Methods The reverse transcription polymerase chain reaction (RT-PCR) was performed to amplify the gene of PS1TP5 from the mRNA of HepG2 cells and the gene was then cloned into the pGEM-T vector. After being sequenced and analyzed with Vector NTI 9.1 and NCBI BLAST software, the target gene of PS1TP5 was cut from the pGEM-T vector and cloned into a yeast expression plasmid pGBKT7, then "bait" plasmid pGBKT7-PS 1TP5 was transformed into the yeast strain AH109. The yeast protein was isolated and analyzed with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting hybridization.After expression of the pGBKT7-PS1TP5 fusion protein in the AH109 yeast strain was accomplished, a yeast two-hybrid screening was performed by mating AH 109 with Y 187 containing a leukocyte cDNA library plasmid.The mated yeast was plated on quadruple dropout medium and assayed for α-gal activity. The interaction between the PS1TP5 protein and the proteins obtained from positive colonies was further confirmed by repeating the yeast two-hybrid screen. After extracting and sequencing of plasmids from blue colonies we carried out a bioinformatic analysis.Results Forty true positive colonies were selected and sequenced, full length sequences were obtained and we searched for homologous DNA sequences from GenBank. Among the 40 positive colonies, 23 coding genes

  5. 红笛鲷头肾消减cDNA文库的构建与分析%Construction and analysis of subtractive cDNA library of head kidney in humphead snapper,Lutjanus sanguineus

    Institute of Scientific and Technical Information of China (English)

    张新中; 吴灶和; 简纪常; 鲁义善

    2011-01-01

    应用抑制性消减杂交技术(SSH)构建红笛鲷(Lutjanus sanguineus)头肾消减cDNA文库,筛选红笛鲷免疫相关基因的EST.以哈氏弧菌(Vibrio harveyi)灭活疫苗体内诱导红笛鲷为实验组,以注射无菌生理盐水的红笛鲷为驱动组,通过SSH技术构建红笛鲷头肾消减.DNA文库.利用PCR技术和斑点杂交对文库进行筛选,从2 424个含插人片段的阳性克隆中筛选了680个克隆在上海生工进行了序列测定.使用BLASTx和BLASTn工具对获得的ESTs与GenBank数据库进行同源性比较并根据相似性序列的名称通过GO法对ESTs进行注释.结果获得了30个与红笛鲷免免疫防御相关基因的EST,如组织相容性抗原复合物基因(MHC I和MHCII),免疫球蛋白基因(IgH和IgL)、热休克蛋白基因(HSP10,HSP70和HSP90)等.本研究构建了哈氏弧菌灭活疫苗免疫后与正常组织差异表达的消减cDNA文库,并获得一批与红笛鲷免疫防御相关的ESTs,旨在为探讨红笛鲷分子免疫防御机制、筛选参与免疫防御调控相关的功能基因,揭示红笛鲷免疫抗病机制、提高机体抗病力、实现遗传改良奠定基础.%A subtracted cDNA library of humphead snapper (Lutjanus sanguineus) was constructed by suppression subtractive hybridization technology (SSH) to screen immune-related EST. The cDNA library has been constructed by the mRNA of the test group and driven by SSH. Differential ESTs from the subtracted cDNA library have been identified by both PCR technology and dot blot hybridization. Six hundred and eighty positive clones were sequenced by Sangon Biological Engineering Technology & Services Co., Ltd. The homology of the sequences was analyzed by BLASTx tool and BLASTn tool in GenBank database. Functional distribution was performed based on the features of ESTs by gene ontology annotation (GO) and 30 immune-related ESTs of L. sanguineus, such as major histocompatibility complex gene (MHC Ⅰ and MHC Ⅱ ), immunoglobulin gene

  6. [Whole cDNA sequence cloning and expression of chicken L-FABP gene and its relationship with lipid deposition of hybrid chickens].

    Science.gov (United States)

    Yu, Ying; Wang, Dong; Sun, Dong-Xiao; Xu, Gui-Yun; Li, Jun-Ying; Zhang, Yuan

    2011-07-01

    Liver fatty acid-binding protein (L-FABP) is closely related to intracellular transportation and deposition of lipids. A positive differential displayed fragment was found in the liver tissue among Silkie (CC), CAU-brown chicken (CD), and their reciprocal hybrids (CD and DC) at 8 weeks-old using differential display RT-PCR techniques (DDRT-PCR). Through recycling, sequencing, and alignment analysis, the fragment was identified as chicken liver fatty acid-binding protein gene (L-FABP, GenBank accession number AY321365). Reverse Northern dot blot and semi-quantitative RT-PCR revealed that the avian L-FABP gene was over-expressed in the liver tissue of the reciprocal hybrids (CD and DC) compared to their parental lines (CC and DD), which was consistent with the fact that higher abdomen fat weight and wider inter-muscular fat width observed in the reciprocal hybrids. Considering the higher expression of L-FABP may contribute to the increased lipid deposition in the hybrid chickens, the functional study of avian L-FABP is warranted in future.

  7. SSHCure: a flow-based SSH intrusion detection system

    NARCIS (Netherlands)

    Hellemons, Laurens; Hendriks, Luuk; Hofstede, Rick; Sperotto, Anna; Sadre, Ramin; Pras, Aiko

    2012-01-01

    SSH attacks are a main area of concern for network managers, due to the danger associated with a successful compromise. Detecting these attacks, and possibly compromised victims, is therefore a crucial activity. Most existing network intrusion detection systems designed for this purpose rely on the

  8. Transcripts of pectin-degrading enzymes and isolation of complete cDNA sequence of a pectate lyase gene induced by coffee white stem borer (Xylotrechus quadripes) in the bark tissue of Coffea canephora (robusta coffee).

    Science.gov (United States)

    Bharathi, Kosaraju; Santosh, P; Sreenath, H L

    2017-05-01

    Of the two commercially cultivated coffee (Coffea) species, C. arabica (arabica) is highly susceptible and C. canephora (robusta) is highly resistant to the insect pest Xylotrechus quadripes (Coleoptera: Cerambycidae), commonly known as coffee white stem borer (CWSB). We constructed a forward-subtracted cDNA library by Suppression Subtractive Hybridization (SSH) from robusta bark tissue for profiling genes induced by CWSB infestation. Among the 265 unigenes of the SSH EST library, 7 unigenes (5 contigs and 2 singletons) matching different pectin-degrading enzymes were discovered. These ESTs matched one pectate lyase, three polygalacturonases, and one pectin acetylesterase gene. Quantitative real-time PCR (qRT-PCR) revealed that CWSB infestation strongly induces the pectate lyase gene at 72 h. Complete cDNA sequence of the pectate lyase gene was obtained through 3' and 5' RACE reactions. It was a 1595 bp long sequence that included full CDS and both UTRs. Against C. canephora genome sequences in Coffee Genome Hub database ( http://coffee-genome.org/ ), it had 22 matches to different pectate lyase genes mapped on 9 of the 11 pseudochromosomes, the top match being Cc07_g00190 Pectate lyase. In NCBI database, it matched pectate lyase sequences of several plants. Apart from C. canephora, the closest pectate lyase matches were from Sesamum indicum and Nicotiana tabacum. The pectinolytic enzymes discovered here are thought to play a role in the production of oligogalacturonides (OGs) which act as Damage-Associated Molecular Pattern (DAMP) signals eliciting innate immunity in plants. The pectate lyase gene, induced by CWSB infestation, along with other endogenous pectinolytic enzymes and CWSB-specific elicitors, may be involved in triggering basal defense responses to protect the CWSB-damaged tissue against pathogens, as well as to contain CWSB in robusta.

  9. Screening of genes of proteins interacting with p7 protein of hepatitis C virus from human liver cDNA library by yeast two-hybrid system

    Institute of Scientific and Technical Information of China (English)

    Yan-Ping Huang; Xue-Song Gao; Dong Ji; Shu-Mei Lin; Yan-Wei Zhong; Qing Shao; Shu-Lin Zhang; Jun Cheng; Lin Wang; Jiang Guo; Yan Liu; Yuan Yang; Li-Ying Zhang; Gui-Qin Bai

    2005-01-01

    AIM: To investigate the biological function of p7 protein and to look for proteins interacting with p7 protein in hepatocytes.METHODS: We constructed p7 protein bait plasmid by doning the gene of p7 protein into pGBKT7, then transformed it into yeast AH109 (a type). The transformed yeast was mated with yeast Y187 (α type) containing liver cDNA library plasmid, pACT2 in 2xYPDA medium. Diploid yeast was plated on synthetic dropout nutrient medium (SD/-Trp-Leu-His-Ade) containing x-α-gal for selection and screening. After extracting and sequencing of plasmids from blue colonies, we performed sequence analysis by bioinformatics.RESULTS: Fifty colonies were selected and sequenced.Among them, one colony was Homo sapiens signal sequence receptor, seven colonies were Homo sapiens H19, seven colonies were immunoglobulin superfamily containing leucine-rich repeat, three colonies were spermatid peri-nuclear RNA binding proteins, two colonies were membrane-spanning 4-domains, 24 colonies were cancer-associated antigens, four colonies were nucleoporin 214 ku and two colonies were CLL-associated antigens.CONCLUSION: The successful cloning of gene of protein interacting with p7 protein paves a way for the study of the physiological function of p7 protein and its associated protein.

  10. Differential cDNA cloning by enzymatic degrading subtraction (EDS).

    OpenAIRE

    1994-01-01

    We describe a new method, called enzymatic degrading subtraction (EDS), for the construction of subtractive libraries from PCR amplified cDNA. The novel features of this method are that i) the tester DNA is blocked by thionucleotide incorporation; ii) the rate of hybridization is accelerated by phenol-emulsion reassociation; and iii) the driver cDNA and hybrid molecules are enzymatically removed by digestion with exonucleases III and VII rather than by physical partitioning. We demonstrate th...

  11. Identiifcation of genes upregulated by recombinant inter feron-alpha in HepG2 cells by suppressive subtractive hybridization analysis

    Institute of Scientific and Technical Information of China (English)

    Jian-Hui Qu; Jun Cheng; Ling-Xia Zhang; Li-Ying Zhang; Yan-Wei Zhong; Yan Liu; Lin Wang; Jiu-Zeng Dai; Dong-Ping Xu

    2007-01-01

    BACKGROUND:Interferon-alpha (IFN-α) is an important cytokine with multiple functions, but the target genes transactivated by IFN-αremain largely unknown. A study of such genes will help to understand the mechanism of function of IFN-α. To isolate the gene transcripts speciifcally upregulated by IFN-α in HepG2 cells, we conducted suppressive subtractive hybridization (SSH) analysis. METHODS: SSH was used to analyze the target genes transactivated by recombinant IFN-α protein, and a subtractive cDNA library was constructed from HepG2 cells treated with recombinant IFN-α (rIFN-α, 2000 IU/ml) for 16 hours as tester, and cells not treated with rIFN-αas driver. The SSH PCR products from the library were cloned into pGEM-T easy vector and with BLASTX, the positive clones were randomly selected, sequenced and compared to the database in GenBank of the 35 differentially expressed gene fragments from the library, 6 clones showed signiifcant homology to other known proteins. RESULTS: The subtractive cDNA library of genes upregulated by IFN-α was constructed successfully. rIFN-α upregulated the expression of the RAN binding protein 5 (RANBP5), NADH dehydrogenase, exosome component 3 (EXOSC3), zinc ifnger RNA binding protein, Dickkopf homolog 1 (DKK1) and acetyl-coenzyme A acetyltransferase 2 (ACAT2). CONCLUSIONS: These results suggest that rIFN-α can upregulate the expression of important genes to exert its functions, and provide new clues for discovering the molecular mechanisms of action of IFN-α.

  12. Dicty_cDB: SSH565 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available BQ596432 |BQ596432.1 PfESToab33g01.y1 Plasmodium falciparum 3D7 asexual cDNA Plasmodium falciparum cDNA 5' ...NA sequence. 52 6e-11 3 BI816352 |BI816352.1 PfESToaa39d09.y1 Plasmodium falciparum 3D7 asexual cDNA Plasmod

  13. Dicty_cDB: SSH675 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available oab33g01.y1 Plasmodium falciparum 3D7 asexual cDNA Plasmodium falciparum 3D7 cDNA 5' similar to TR:Q94652 Q9... BI816352 |BI816352.1 PfESToaa39d09.y1 Plasmodium falciparum 3D7 asexual cDNA Plasmodium falciparum cDNA 5' ...6952 |BQ596952.1 PfESToab24g06.y1 Plasmodium falciparum 3D7 asexual cDNA Plasmodi

  14. Genetic differences between two Leishmania major-like strains revealed by suppression subtractive hybridization.

    Science.gov (United States)

    Wu, Ângela C A; Freitas, Michelle A R; Silva, Soraia de O; Nogueira, Paula M; Soares, Rodrigo P; Pesquero, João Bosco; Gomes, Maria A; Pesquero, Jorge L; Melo, Maria N

    2015-01-01

    Leishmania major, the causative agent of zoonotic leishmaniasis, is restricted to Old World countries. Molecular and biochemical techniques have been used to identify some L. major-like isolated in South America including Brazil. Here, two L. major-like strains, one virulent (BH49) and one non-virulent (BH121), were subjected to suppression subtractive hybridization (SSH) technique in order to identify differentially expressed genes. SSH technique identified nine cDNA fragments exhibiting high homology to previously sequenced L. major genes. Five cDNAs (four specific for BH49 and one for BH121) were confirmed by RT-PCR. Among those differentially expressed subtracted genes, some were involved in physiological processes including metabolism, translation and destination of proteins, production of energy, virulence factors and unknown functions. Western-blot analysis confirmed a higher expression level of β-1,3-galactosyl residues in L. major-like lipophosphoglycan (LPG). This molecular analysis opens the possibility for identification of potential virulence factors not only in different strains, but also in others species of Leishmania.

  15. Analysis of gene expression in granulosa cells of ovine antral growing follicles using suppressive subtractive hybridization.

    Science.gov (United States)

    Chen, A Qin; Wang, Zheng Guang; Xu, Zi Rong; Yu, Song Dong; Yang, Zhi Gang

    2009-10-01

    Follicular growth, development and ovulation are highly ordered processes that involve the expression of many genes under precise temporal and spatial regulation. However, information on stage-specific gene expression during the antral follicle phase in sheep is not well understood. In the present study, suppressive subtractive hybridization (SSH) was performed to screen genes that were differentially expressed in the granulosa cells between large follicles (LF, >5mm) and small follicles (SF, 3-5mm), and subtractive cDNA library was constructed. Furthermore, with dot-blot analysis, a total of 90 clones randomly selected from the library were proven to be differentially expressed in the granulosa cells. Among these, 38 exhibited high homology to known genes, 14 sequences were corresponding to novel expressed sequence tags (ESTs). Four ESTs, LAPTM4A, SERPINE2, GSTA1, and INHBA, were further examined the reproducibility of the SSH data by the real-time quantitative PCR. Results confirmed an increase expression of respective mRNA in granulosa cells of large follicles compared with that of small follicles. It is concluded that we have identified several genes (known or unknown) that may effect follicular growth, dominance or ovulation in ewes.

  16. 多快好省地实施OpenSSH

    Institute of Scientific and Technical Information of China (English)

    赵长林

    2008-01-01

    虽然使用代替口令的公钥认证是增强SSH传输安全性的一个好方法,但传输SSH的统一性密钥却也是一件令人不快的事。而ssh-copy-id这个包括在OpenSSH之内的小程序使得这个过程变得格外简单。

  17. Critical Success Factors (CSFs for achieving sustainable social housing (SSH

    Directory of Open Access Journals (Sweden)

    Akanbi Olusayo Oyebanji

    2017-06-01

    Full Text Available The overarching objective of social housing is to meet housing needs, particularly those of the vulnerable households – low and middle income earners. However, there is evidence to show that social housing is not adequately supported to achieve sustainable goals despite its significance for addressing the housing crisis. The aim of this study is to determine the Critical Success Factors (CSFs for achieving Sustainable Social Housing (SSH from economic, environmental and social perspectives for meeting housing needs. The document content analysis approach involving relevant literature resources was used for generating the success factors (SFs for achieving SSH. Findings from this approach were refined before using them in preparing a questionnaire used to gather data from housing authorities (public and private non-profit social housing organisations in England and they were asked to rank the criticality level of the identified success factors. The data gathered through the relevant documents and respondents were analysed respectively with NVivo and Statistical Package for Social Science (SPSS. Findings revealed some of the CSFs for achieving SSH for meeting housing needs as: adequate funding and provision, affordability, efficient economic planning, appropriate construction technology, environmental protection, use of environmental friendly materials, effective land use planning, appropriate design, security of lives and property, provision of social services and ensuring social cohesion. The paper recommends the use of efficient sustainable development (SD strategies and legal and institutional frameworks for monitoring and evaluating the delivery of SSH. The Government must embark on effective housing programmes for ensuring adequate provision of social housing that is sustainable for meeting housing needs in the short and long-run. There is need for the Government to regularly provide financial supports to social housing providers and users

  18. A method for generating subtractive cDNA libraries retaining clones containing repetitive elements.

    OpenAIRE

    1997-01-01

    Here we describe a two-stepped photobiotin-based procedure to enrich a target (canine retinal) cDNA library for tissue specific clones without removing those containing repetitive ( SINE ) elements, despite the presence of these elements in the driver population. In a first hybridization excess SINE elements were hybridized to a driver (canine cerebellar) cDNA. In a second hybridization target cDNA was added to this reaction. The resulting cDNA library was enriched for retinal specific clones...

  19. Dicty_cDB: SSH387 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available BI814872 |BI814872.1 PfESToaa07d11.y1 Plasmodium falciparum 3D7 asexual cDNA Pla...-05 2 BI814952 |BI814952.1 PfESToaa08d03.y1 Plasmodium falciparum 3D7 asexual cDNA Plasmodium falciparum cDN...52 9e-05 2 BU495228 |BU495228.1 PfESToab70a10.y1 Plasmodium falciparum 3D7 asexual cDNA Plasmodium falciparu...Q576846.1 PfESToab10e05.y1 Plasmodium falciparum 3D7 asexual cDNA Plasmodium falc...86 |BU498486.1 PfESToab97g12.y1 Plasmodium falciparum 3D7 asexual cDNA Plasmodium falciparum cDNA 5' similar

  20. Dicty_cDB: Contig-U05900-1 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available 39018 ) 1278280 NCCCWA 03RT Oncorhynchus mykiss cDNA clon... 50 0.15 1 ( CN809704 ) 2O5 Suppression subtractive hybridization... (SSH) l... 50 0.15 1 ( CN809695 ) 2E1 Suppression subtractive hybridization... (SSH) l... 50 0.15 1 ( CN809694 ) 2I24 Suppression subtractive hybridization (SSH) ... 50 0.15 ...1 ( CN809693 ) 1B14 Suppression subtractive hybridization (SSH) ... 50 0.15 1 ( CN809692 ) 2D5 Suppression subtractive hybridization... (SSH) l... 50 0.15 1 ( CN809691 ) 2D8 Suppression subtractive hybridization (SSH) l

  1. Application of SSH and quantitative real time PCR to construction of gene expression profiles from scallop Chlamys farreri in response to exposure to tetrabromobisphenol A.

    Science.gov (United States)

    Gong, Xiaoli; Pan, Luqing; Miao, Jingjing; Liu, Na

    2012-11-01

    TBBPA-induced genes were identified using suppression subtractive hybridization (SSH) from Chlamys farreri. A total of 203 and 44 clones from SSH forward and reverse library were respectively obtained including cellular process, immune system process, response to stimulus, metabolic process and signaling etc. Differential gene expressions were compared between scallops from control and TBBPA treatment groups (400 μg/L, 15 days) using quantitative real time RT-PCR. For further research, eight significant genes expression from scallops exposed to TBBPA (0; 100; 200; 400 μg/L) sampling at 0, 1, 3, 6 and 15 days, were utilized for Q-RT-PCR. The results revealed that the expression level of most selected cDNAs was dominantly up-regulated or down-regulated in the TBBPA-induced scallops. These findings provide basic genomic information of the bivalve and the selected genes may be the potential molecular biomarkers for TBBPA pollution in aquatic environment.

  2. Dicty_cDB: SSH762 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available 4 |CD392624.1 Gm_ck11824 Soybean induced by Salicylic Acid Glycine max cDNA 3', m...t lab Zea mays cDNA, mRNA sequence. 42 4.4 1 CD417162 |CD417162.1 Gm_ck7685 Soybean induced by Salicylic Aci...d Glycine max cDNA 3', mRNA sequence. 42 4.4 1 CD408286 |CD408286.1 Gm_ck34391 Soybean induced by Salicyli...1 Gm_ck25879 Soybean induced by Salicylic Acid Glycine max cDNA 3', mRNA sequence

  3. Dicty_cDB: SSH558 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available osoma mansoni, Phil LoVerde/Joe Merrick Schistosoma mansoni cDNA clone SMNAS44 5'...histosoma mansoni, Phil LoVerde/Joe Merrick Schistosoma mansoni cDNA clone SMNCG92 5' end similar to ATPase ...2 Schistosoma mansoni, Phil LoVerde/Joe Merrick Schistosoma mansoni cDNA clone SMNCH20 5' end similar to ATP...cum cDNA, mRNA sequence. 54 0.002 1 AI067329 |AI067329.1 EST209007 Schistosoma mansoni, Phil LoVerde/Joe

  4. Biomphalaria glabrata transcriptome: cDNA microarray profiling identifies resistant- and susceptible-specific gene expression in haemocytes from snail strains exposed to Schistosoma mansoni

    Directory of Open Access Journals (Sweden)

    Rollinson David

    2008-12-01

    Full Text Available Abstract Background Biomphalaria glabrata is an intermediate snail host for Schistosoma mansoni, one of the important schistosomes infecting man. B. glabrata/S. mansoni provides a useful model system for investigating the intimate interactions between host and parasite. Examining differential gene expression between S. mansoni-exposed schistosome-resistant and susceptible snail lines will identify genes and pathways that may be involved in snail defences. Results We have developed a 2053 element cDNA microarray for B. glabrata containing clones from ORESTES (Open Reading frame ESTs libraries, suppression subtractive hybridization (SSH libraries and clones identified in previous expression studies. Snail haemocyte RNA, extracted from parasite-challenged resistant and susceptible snails, 2 to 24 h post-exposure to S. mansoni, was hybridized to the custom made cDNA microarray and 98 differentially expressed genes or gene clusters were identified, 94 resistant-associated and 4 susceptible-associated. Quantitative PCR analysis verified the cDNA microarray results for representative transcripts. Differentially expressed genes were annotated and clustered using gene ontology (GO terminology and Kyoto Encyclopaedia of Genes and Genomes (KEGG pathway analysis. 61% of the identified differentially expressed genes have no known function including the 4 susceptible strain-specific transcripts. Resistant strain-specific expression of genes implicated in innate immunity of invertebrates was identified, including hydrolytic enzymes such as cathepsin L, a cysteine proteinase involved in lysis of phagocytosed particles; metabolic enzymes such as ornithine decarboxylase, the rate-limiting enzyme in the production of polyamines, important in inflammation and infection processes, as well as scavenging damaging free radicals produced during production of reactive oxygen species; stress response genes such as HSP70; proteins involved in signalling, such as importin 7

  5. Suppression subtractive hybridization coupled with microarray analysis to examine differential expression of genes in virus infected cells

    Directory of Open Access Journals (Sweden)

    Munir Shirin

    2004-01-01

    Full Text Available High throughput detection of differential expression of genes is an efficient means of identifying genes and pathways that may play a role in biological systems under certain experimental conditions. There exist a variety of approaches that could be used to identify groups of genes that change in expression in response to a particular stimulus or environment. We here describe the application of suppression subtractive hybridization (SSH coupled with cDNA microarray analysis for isolation and identification of chicken transcripts that change in expression on infection of host cells with a paramyxovirus. SSH was used for initial isolation of differentially expressed transcripts, a large-scale validation of which was accomplished by microarray analysis. The data reveals a large group of regulated genes constituting many biochemical pathways that could serve as targets for future investigations to explore their role in paramyxovirus pathogenesis. The detailed methods described herein could be useful and adaptable to any biological system for studying changes in gene expression.

  6. Molecular cloning of lupin leghemoglobin cDNA

    DEFF Research Database (Denmark)

    Konieczny, A; Jensen, E O; Marcker, K A

    1987-01-01

    Poly(A)+ RNA isolated from root nodules of yellow lupin (Lupinus luteus, var. Ventus) has been used as a template for the construction of a cDNA library. The ds cDNA was synthesized and inserted into the Hind III site of plasmid pBR 322 using synthetic Hind III linkers. Clones containing sequences...... its nucleotide sequence was consistent with known amino acid sequence of lupin Lb II. The cloned lupin Lb cDNA hybridized to poly(A)+ RNA from nodules only, which is in accordance with the general concept, that leghemoglobin is expressed exclusively in nodules. Udgivelsesdato: 1987-null...

  7. Molecular cloning of lupin leghemoglobin cDNA

    DEFF Research Database (Denmark)

    Konieczny, A; Jensen, E O; Marcker, K A

    1987-01-01

    Poly(A)+ RNA isolated from root nodules of yellow lupin (Lupinus luteus, var. Ventus) has been used as a template for the construction of a cDNA library. The ds cDNA was synthesized and inserted into the Hind III site of plasmid pBR 322 using synthetic Hind III linkers. Clones containing sequences...... its nucleotide sequence was consistent with known amino acid sequence of lupin Lb II. The cloned lupin Lb cDNA hybridized to poly(A)+ RNA from nodules only, which is in accordance with the general concept, that leghemoglobin is expressed exclusively in nodules. Udgivelsesdato: 1987-null...

  8. Construction and analysis of the cDNA subtraction library of yeast and mycelial phases of Sporothrix globosa isolated in China: identification of differentially expressed genes.

    Science.gov (United States)

    Hu, Qing-bi; He, Yu; Zhou, Xun

    2015-12-01

    Species included in the Sporothrix schenckii complex are temperature-dependent with dimorphic growth and cause sporotrichosis that is characterized by chronic and fatal lymphocutaneous lesions. The putative species included in the Sporothrix complex are S. brasiliensis, S. globosa, S. mexicana, S. pallida, S. schenckii, and S. lurei. S. globosa is the causal agent of sporotrichosis in China, and its pathogenicity appears to be closely related to the dimorphic transition, i.e. from the mycelial to the yeast phase, it adapts to changing environmental conditions. To determine the molecular mechanisms of the switching process that mediates the dimorphic transition of S. globosa, suppression subtractive hybridization (SSH) was used to prepare a complementary DNA (cDNA) subtraction library from the yeast and mycelial phases. Bioinformatics analysis was performed to profile the relationship between differently expressed genes and the dimorphic transition. Two genes that were expressed at higher levels by the yeast form were selected, and their differential expression levels were verified using a quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR). It is believed that these differently expressed genes are involved in the pathogenesis of S. globosa infection in China.

  9. Construction and analysis of the cDNA subtraction library of yeast and mycelial phases of Sporothrix globosa isolated in China: identification of differentially expressed genes*

    Science.gov (United States)

    Hu, Qing-bi; He, Yu; Zhou, Xun

    2015-01-01

    Species included in the Sporothrix schenckii complex are temperature-dependent with dimorphic growth and cause sporotrichosis that is characterized by chronic and fatal lymphocutaneous lesions. The putative species included in the Sporothrix complex are S. brasiliensis, S. globosa, S. mexicana, S. pallida, S. schenckii, and S. lurei. S. globosa is the causal agent of sporotrichosis in China, and its pathogenicity appears to be closely related to the dimorphic transition, i.e. from the mycelial to the yeast phase, it adapts to changing environmental conditions. To determine the molecular mechanisms of the switching process that mediates the dimorphic transition of S. globosa, suppression subtractive hybridization (SSH) was used to prepare a complementary DNA (cDNA) subtraction library from the yeast and mycelial phases. Bioinformatics analysis was performed to profile the relationship between differently expressed genes and the dimorphic transition. Two genes that were expressed at higher levels by the yeast form were selected, and their differential expression levels were verified using a quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR). It is believed that these differently expressed genes are involved in the pathogenesis of S. globosa infection in China. PMID:26642182

  10. Rabbit muscle creatine phosphokinase. CDNA cloning, primary structure and detection of human homologues.

    Science.gov (United States)

    Putney, S; Herlihy, W; Royal, N; Pang, H; Aposhian, H V; Pickering, L; Belagaje, R; Biemann, K; Page, D; Kuby, S

    1984-12-10

    A cDNA library was constructed from rabbit muscle poly(A) RNA. Limited amino acid sequence information was obtained on rabbit muscle creatine phosphokinase and this was the basis for design and synthesis of two oligonucleotide probes complementary to a creatine kinase cDNA sequence which encodes a pentapeptide. Colony hybridizations with the probes and subsequent steps led to isolation of two clones, whose cDNA segments partially overlap and which together encode the entire protein. The primary structure was established from the sequence of two cDNA clones and from independently determined sequences of scattered portions of the polypeptide. The reactive cysteine has been located to position 282 within the 380 amino acid polypeptide. The rabbit cDNA hybridizes to digests of human chromosomal DNA. This reveals a restriction fragment length polymorphism associated with the human homologue(s) which hybridizes to the rabbit cDNA.

  11. Identification of the immune expressed sequence tags of pearl oyster (Pinctada martensii, Dunker 1850) responding to Vibrio alginolyticus challenge by suppression subtractive hybridization.

    Science.gov (United States)

    Wang, Yanhong; Fu, Dingkun; Luo, Peng; He, Xiaocui

    2012-09-01

    One hemolymph subtracted cDNA library of pearl oyster (Pinctada martensii, Dunker 1837) was constructed using the suppression subtractive hybridization (SSH) in response to Vibrio alginolyticus. A total of 1089 clones were sequenced. All the consensuses were recognized based on the BLAST searches in NCBI, and revealed that 376 (58%) of them had no significant matches to reported sequences in the database. 267 ESTs were in significant matches after homologous sequence searches. Hypothesized genes inferred from EST sequences were categorized into six groups according to their putative biological functions: replication, transcription and translation; cellular processes; responded to stimuli; metabolism and biosynthesis; signal transduction genes; "other" category. The five genes, pearlin gene promoter PGPPm, serine/threonine kinase STKPm, limbic system-associated membrane protein LSAMPPm, nacrein gene intron 6 NGIPm6 and ferritin-like protein FLPPm, were analyzed using real-time PCR. All these genes were significantly expressed after V. alginolyticus challenge.

  12. Transactivating effect of complete S protein of hepatitis B virus and cloning of genes transactivated by complete S protein using suppression subtractive hybridization technique

    Institute of Scientific and Technical Information of China (English)

    Gui-Qin Bai; Yan Liu; Jun Cheng; Shu-Lin Zhang; Ya-Fei Yue; Yan-Ping Huang; Li-Ying Zhang

    2005-01-01

    AIM: To investigate the transactivating effect of complete S protein of hepatitis B virus (HBV) and to construct a subtractive cDNA library of genes transactivated by complete S protein of HBV by suppression subtractive hybridization (SSH) technique and to clone genes associated with its transactivation activity, and to pave the way for elucidating the pathogenesis of hepatitis B virus infection.METHODS: pcDNA3.1(-)-complete S containing full-length HBV S gene was constructed by insertion of HBV complete S gene into BarmH-I/Kpn I sites. HepG2 cells were cotransfected with pcDNA3.1(-)-complete S and pSV-lacZ.After 48 h, cells were collected and detected for the expression of β-galactosidase (β-gal). Suppression subtractive hybridization and bioinformatics techniques were used.The mRNA of HepG2 cells transfected with pcDNA3.1(-)-complete S and pcDNA3.1(-) empty vector was isolated,and detected for the expression of complete S protein by reverse transcription polymerase chain reaction (RT-PCR)method, and cDNA was synthesized. After digestion with restriction enzyme RcaI, cDNA fragments were obtained.Tester cDNA was then divided into two groups and ligated to the specific adaptors 1 and 2, respectively. After tester cDNA had been hybridized with driver cDNA twice and underwent nested PCR twice, amplified cDNA fragments were subcloned into pGEM-Teasy vectors to set up the subtractive library. Amplification of the library was carried out within E. coli strain DH5α. The cDNA was sequenced and analyzed in GenBank with BLAST search after polymerase chain reaction (PCR) amplification.RESULTS: The complete S mRNA could be detected by RT-PCR in HepG2 cells transfected with the pcDNA3.1(-)-complete S. The activity of β-gal in HepG2 cells transfected with the pcDNA3.1(-)-complete S was 6.9 times higher than that of control plasmid. The subtractive library of genes transactivated by HBV complete S protein was constructed successfully. The amplified library contains 86

  13. Construction and analysis of liver suppression subtractive hybridization library of silver carp (Hypophthalmichthys molitrix) intraperitoneally injected with microcystin-LR.

    Science.gov (United States)

    Qu, Xiancheng; Zhang, Kaiyue; Cui, Zhihui; Zhang, Yong; Jiang, Jiaoyun; Feng, Long; Liu, Qigen

    2011-09-01

    Microcystin-LR (MC-LR) is the most frequently studied cyclic heptapeptide hepatotoxin produced by cyanobacteria. The toxin accumulates rapidly in the liver where it exerts most of its damage, but the molecular mechanisms behind its toxicity remain unclear. Here, suppression subtractive hybridization (SSH) was used to identify alterations in gene transcription of the silver carp (Hypophthalmichthys molitrix) after exposure to MC-LR. After hybridization and cloning, the forward and reverse subtractive cDNA libraries were obtained. At random, 150 positive clones (70 forward and 80 reverse) were selected and sequenced from the subtractive libraries, which gave a total of 88 gene fragment sequences (48 forward and 40 reverse). Sequencing analysis and homology searches showed that these ESTs represented 75 unique genes and 13 duplicates. Of the 75 unique genes, 38 shared high homology with fish genes of known functions, including immune-related genes, transporters and some involved in cell metabolism. Four sequenced genes (Fs59, Fs70, Rs2 and Rs15) were analyzed further using semi-quantitative RT-PCR. The genes from the forward library (Fs59 and Fs70) were found to be transcriptionally upregulated, while the genes from the reverse library (Rs2 and Rs15) were found to be transcriptionally downregulated. These results confirmed the successful construction of the subtractive cDNA library that was enriched for genes that were differentially transcribed in the silver carp liver challenged with MC-LR.

  14. Characterization of resistance to pine wood nematode infection in Pinus thunbergii using suppression subtractive hybridization

    Directory of Open Access Journals (Sweden)

    Hirao Tomonori

    2012-01-01

    Full Text Available Abstract Background Pine wilt disease is caused by the pine wood nematode, Bursaphelenchus xylophilus, which threatens pine forests and forest ecosystems worldwide and causes serious economic losses. In the 40 years since the pathogen was identified, the physiological changes occurring as the disease progresses have been characterized using anatomical and biochemical methods, and resistant trees have been selected via breeding programs. However, no studies have assessed the molecular genetics, e.g. transcriptional changes, associated with infection-induced physiological changes in resistant or susceptible trees. Results We constructed seven subtractive suppression hybridization (SSH cDNA libraries using time-course sampling of trees inoculated with pine wood nematode at 1, 3, or 7 days post-inoculation (dpi in susceptible trees and at 1, 3, 7, or 14 dpi in resistant trees. A total of 3,299 sequences was obtained from these cDNA libraries, including from 138 to 315 non-redundant sequences in susceptible SSH libraries and from 351 to 435 in resistant SSH libraries. Using Gene Ontology hierarchy, those non-redundant sequences were classified into 15 subcategories of the biological process Gene Ontology category and 17 subcategories of the molecular function category. The transcriptional components revealed by the Gene Ontology classification clearly differed between resistant and susceptible libraries. Some transcripts were discriminative: expression of antimicrobial peptide and putative pathogenesis-related genes (e.g., PR-1b, 2, 3, 4, 5, 6 was much higher in susceptible trees than in resistant trees at every time point, whereas expression of PR-9, PR-10, and cell wall-related genes (e.g., for hydroxyproline-rich glycoprotein precursor and extensin was higher in resistant trees than in susceptible trees at 7 and 14 dpi. Conclusions Following inoculation with pine wood nematode, there were marked differences between resistant and susceptible trees

  15. Identification of C4 photosynthesis metabolism and regulatory-associated genes in Eleocharis vivipara by SSH.

    Science.gov (United States)

    Chen, Taiyu; Ye, Rongjian; Fan, Xiaolei; Li, Xianghua; Lin, Yongjun

    2011-09-01

    This is the first effort to investigate the candidate genes involved in kranz developmental regulation and C(4) metabolic fluxes in Eleocharis vivipara, which is a leafless freshwater amphibious plant and possesses a distinct culms anatomy structure and photosynthetic pattern in contrasting environments. A terrestrial specific SSH library was constructed to investigate the genes involved in kranz anatomy developmental regulation and C(4) metabolic fluxes. A total of 73 ESTs and 56 unigenes in 384 clones were identified by array hybridization and sequencing. In total, 50 unigenes had homologous genes in the databases of rice and Arabidopsis. The real-time quantitative PCR results showed that most of the genes were accumulated in terrestrial culms and ABA-induced culms. The C(4) marker genes were stably accumulated during the culms development process in terrestrial culms. With respect to C(3) culms, C(4) photosynthesis metabolism consumed much more transporters and translocators related to ion metabolism, organic acids and carbohydrate metabolism, phosphate metabolism, amino acids metabolism, and lipids metabolism. Additionally, ten regulatory genes including five transcription factors, four receptor-like proteins, and one BURP protein were identified. These regulatory genes, which co-accumulated with the culms developmental stages, may play important roles in culms structure developmental regulation, bundle sheath chloroplast maturation, and environmental response. These results shed new light on the C(4) metabolic fluxes, environmental response, and anatomy structure developmental regulation in E. vivipara.

  16. An SSH library responsive to azadirachtin A constructed in Spodoptera litura Fabricius cell lines.

    Science.gov (United States)

    Yan, Chao; Zhang, Zhi-Xiang; Xu, Han-Hong

    2012-05-31

    The present study revealed differentially expressed genes responsive to azadirachtin A (Aza) in Spodoptera litura cell line through suppression subtractive hybridization. In the Aza-responsive SSH library, approximately 270 sequences represent 53 different identified genes encoding proteins with various predicted functions, and the percentages of the gene clusters were 26.09% (genetic information processing), 11.41% (cell growth and death), 7.07% (metabolism), 6.52% (signal transduction/transport) and 2.72% (immunity), respectively. Eleven clones homologous to identified genes were selected to be confirmed through quantitative real time polymerase chain reaction. Among the eleven clones validated, all but one transcript of lipase showed an increase in SL cell line collected from ETA, whereas the transcripts of other genes were lower in the SL cell line collected from ETA compared with that of UETA. These genes were considered to be related to the response of SL cell line to Aza. These will provide a new clue to uncover the molecular mechanisms of Aza acting on SL cell line.

  17. [Bioinformatic analysis of adenoma-normal mucosa SSH library of colon].

    Science.gov (United States)

    Lü, Bing-Jian; Cui, Jing; Xu, Jing; Zhang, Hao; Luo, Min-Jie; Zhu, Yi-Min; Lai, Mao-De

    2006-04-01

    We established a colonic adenoma-normal mucosa suppressive subtraction hybridization (SSH) library in 1999. In this study, we wanted to explore the expression profile of all candidate genes in this library. We developed an EST pipeline which contained two in-house software packages, nucleic acid analytical software and GetUni. The nucleic acid analytical software, an integrator of the universal bioinformatics tools including phred, phd2fasta, cross_match, repeatmasker and blast2.0, can blast sequences of differential clones with the downloaded non-redundant nucleotide (NR) database. GetUni can cluster these NR sequences into Unigene via matching with the downloaded Homo Sapiens UniGene database. Sixty-two candidate genes in A-N library were obtained via the high throughput automatic gene expression bioinformatics pipeline. Gene Ontology online analysis revealed that ribosome genes and immunity-regulating genes were the two most common categories in the KEGG or Biocarta Pathway. We also detected the expression of 2 genes with highest hits, Reg4 and FAM46A, by semi-quantitative RT-PCR. Both genes were up-regulated in 10 or 9 out of 10 adenomas in comparison with the paired normal mucosa, respectively. The candidate genes in A-N library would be of great significance in disclosing the molecular mechanism underlying in colonic adenoma initiation and progression.

  18. Application of SSH and a macroarray to investigate altered gene expression in Mytilus edulis in response to exposure to benzo[a]pyrene.

    Science.gov (United States)

    Brown, M; Davies, I M; Moffat, C F; Craft, J A

    2006-07-01

    The lack of genomic resources for aquatic invertebrates restricts their use as sentinel species in coastal environments. It is known that where genomic data are not available, suppression subtractive hybridisation (SSH) can generate cDNA libraries representative of pollutant-responsive gene transcription in aquatic vertebrates. To assess whether the approach was equally suited to aquatic invertebrates, altered gene expression in digestive gland of the mussel, Mytilus edulis, in response to exposure to benzo[a]pyrene (BaP) (1 mg/l) was investigated with SSH and a nylon macroarray. Screening of the subtracted libraries showed 112/250 up-regulated and 25/55 down-regulated clones were positive for differential expression and characterisation of these identified 87 with unique sequence suitable for array on a nylon membrane. The transcripts isolated were from a diverse range of genes involved in general stress, oxidative stress, cell adhesion, transcriptional and translational regulation, transport mechanisms, energy metabolism, cell metabolism, lipid metabolism, protein turnover and activation, lysosomal activity and 22 cryptic clones. Subsequent use of the clones in macroarray format to analyse expression of BaP-responsive genes (0 vs 4 day exposed) showed 0-100-fold increased levels of the forward-subtracted probes and between 0 and 0.1-fold down-regulation of the reverse-subtracted probes. Only 15% of the clones showed less than 2-fold change in expression. The gene ontology of the transcripts isolated demonstrates that BaP elicits a multitude of responses with a major feature being disruption of cellular redox status. The results indicate that the use of SSH and a macroarray is a robust method to discover novel pollutant-responsive genes in aquatic invertebrates.

  19. Isolation and identification of novel genes involved in artemisinin production from flowers of Artemisia annua using suppression subtractive hybridization and metabolite analysis.

    Science.gov (United States)

    Liu, Shuoqian; Tian, Na; Li, Juan; Huang, Jianan; Liu, Zhonghua

    2009-11-01

    Malaria is a global health problem that threatens 300-500 million people and kills more than one million people annually. Artemisinin is highly effective against multidrug-resistant Plasmodium falciparum and it has been widely used as part of the artemisinin-based combination therapies against malaria. To elucidate the biosynthetic pathway of artemisinin and to clone related genes in Artemisia annua, differentially expressed genes between blooming flowers and flower buds were isolated and characterized by a combined approach of suppression subtractive hybridization (SSH) and metabolite analysis. A total of 350 cDNA clones from a subtractive cDNA library were randomly picked, sequenced and analyzed and 253 high-quality sequences were obtained. BLASTX comparisons indicated that about 9.9 % of the clones encoded enzymes involved in isoprenoid (including artemisinin) biosynthesis. The expression of 4 gene transcripts involved in artemisinin biosynthesis was examined by RT-PCR and the results confirmed the higher expression of these transcripts in blooming flowers than in flower buds. In addition, 2 putative transcript factors transparenta testa glabra 1 (TTG1) and ENHANCER OF GLABRA3 (GL3), which promote trichome initiation, were presented in the library. Finally, this study demonstrated that the increase of expression level of the putative TTG1 gene correlated with the improvement of glandular trichome density and artemisinin production in A. annua leaves. The subtractive cDNA library described in the present study provides important candidate genes for future research in order to increase the artemisinin content in A. annua.

  20. Identification of H. Pylori strain specific DNA sequences between two clinical isolates from NUD and gastric ulcer by SSH

    Institute of Scientific and Technical Information of China (English)

    Feng-Chan Han; Min Gong; Han-Chong Ng; Bow Ho

    2003-01-01

    AIM: The genomes of Helicobacter pylori(H. pylori) from different individuals are different. This project was to identify the strain specific DNA sequences between two clinical H. pylori isolates by suppression subtractive hybridization (SSH).METHODS: Two clinical H. pylori isolates, one from gastric ulcer (GU, tester) and the other from non-ulcer dyspepsia (NUD, driver), were cultured and the genomic DNA was prepared and submitted to AluⅠdigestion. Then two different adaptors were ligated respectively to the 5′-end of two aliquots of the tester DNA fragments and SSH was made between the tester and driver DNA. The un-hybridized tester DNA sequences were amplified by two sequential PCR and cloned into pGEM-T-Easy Vector. The tester strain specific inserts were screened and disease related DNA sequences were identified by dot blotting.RESULTS: Among the 240 colonies randomly chosen, 50contained the tester strain specific DNA sequences. Twenty three inserts were sequenced and the sizes ranged from 261 bp to 1 036 bp. Fifteen inserts belonged to the H.pylori plasmid pHPO100 that is about 3.5 kb and codes a replication protein A. Other inserts had patches of homologous to the genes of H. pylori in GenBank. Various patterns of dot blots were given and no GU strain unique DNA sequences were found when 4 inserts were used as probes to screen the genomic DNA from 27 clinical isolates, 8 from GU, 12 from duodenum ulcer (DU), 4 from GU-DU, 2 from NUD and 1from gastric cancer (GC). But a 670 bp DNA fragment (GU198)that was a bit homologous to the 3′-end of the gene of thymidylate kinase was positive in 7 GU strains (7/8), 3 GUDU strains (3/4) and 3 DU strains (3/12). A 384 bp fragment (GU79) of the replication gene A (repA) was positive only in 4 H, pylori isolates, 2 from GU and 2 from GU-DU.CONCLUSION: Differences exist in the genes of different H.pylori isolates. SSH is very effective to screen H. pylori strain specific DNA sequences between two clinical isolates

  1. [Construction and analysis of subtractive cDNA library associated with multidrug resistance of acute leukemia].

    Science.gov (United States)

    Ji, Lei; Zhang, Wang-Gang; Liu, Jie; Liu, Xin-Ping; Yao, Li-Bo

    2004-08-01

    The study was aimed to construct subtractive cDNA library associated with multidrug resistance (MDR) of acute leukemia for screening genes related to MDR in leukemia. The improved PCR-based subtractive hybridization was performed to clone differential genes between HL-60/VCR and HL-60 cell line. The mRNA of HL-60/VCR and HL-60 cell line were isolated. Then the mRNA of HL-60/VCR group was reversely transcribed into cDNA by Cap-Finder method, and the mRNA of HL-60 was reversely transcribed into cDNA by ordinary method to be marked by biotin for the hybridization next with HL-60/VCR cDNA. After hybridizing, filtrating through the sephacryl S-400 column, absorbing by the magnetic beads, and amplifying by PCR method, the fragments were cloned by T-A method and the cDNA library was constructed. Then the quality of cDNA library was identified by dot-blotting hybridization method. The results showed that after constriction, the library demonstrated its good quality. There was a high proportion of large fragments in this library. From small amount of samples a large amount of candidate fragments could be screened rapidly at once by dot-blotting hybridization. It is concluded that a differentially-expressed subtractive cDNA library in MDR of leukemia with high quality and larger fragments can be efficiently constructed by improving subtractive hybridization and selective PCR method.

  2. Procedure for normalization of cDNA libraries

    Science.gov (United States)

    Bonaldo, Maria DeFatima; Soares, Marcelo Bento

    1997-01-01

    This invention provides a method to normalize a cDNA library constructed in a vector capable of being converted to single-stranded circles and capable of producing complementary nucleic acid molecules to the single-stranded circles comprising: (a) converting the cDNA library in single-stranded circles; (b) generating complementary nucleic acid molecules to the single-stranded circles; (c) hybridizing the single-stranded circles converted in step (a) with complementary nucleic acid molecules of step (b) to produce partial duplexes to an appropriate Cot; (e) separating the unhybridized single-stranded circles from the hybridized single-stranded circles, thereby generating a normalized cDNA library.

  3. 基于SSH+Ajax框架技术的MMS的研究与实现

    Institute of Scientific and Technical Information of China (English)

    张少龙

    2012-01-01

    针对B/S的MMS的建设与应用,提出基于SSH+Ajax技术架构的解决方案并具体实现,结果证明,SSH(Struts、Spring、Hibernate)技术架构能提高系统的可维护性、可扩展性并提高系统开发效率。

  4. Effects of a supersulfated low molecular weight heparin (IK-SSH) on different hemostatic parameters.

    Science.gov (United States)

    Glusa, E; Barthel, W; Schenk, J; Radziwon, P; Butti, A; Markwardt, F; Breddin, K H

    1998-01-01

    In a phase I trial effects of a new supersulfated low molecular weight heparin (IK-SSH) on different hemostatic parameters were investigated in healthy volunteers. Parameters studied were activated partial thromboplastin time (aPTT), thrombin time, Heptest, anti-activated factor II (anti-FIIa) and anti-activated factor X (anti-FXa) activity, platelet adhesion, platelet count, platelet-induced thrombin generation time (PITT), bleeding time, antithrombin III, fibrinogen and several safety parameters. After single intravenous (i.v.) injections of IK-SSH (0.14, 0.33 and 0.66 mg/kg) aPTT, Heptest and PITT were strongly and dose-dependently prolonged. After ascending subcutaneous (s.c.) doses of IK-SSH (0.33, 0.66 and 1 mg/kg) aPTT, Heptest and PITT were prolonged in a dose-dependent manner. Repeat s.c. injections of 1 mg/kg IK-SSH for 5 days markedly prolonged aPTT, Heptest and PITT. No cumulative effects were observed. Anti-FIIa and anti-FXa activity were not or only slightly increased. Bleeding time, thrombin time and platelet adhesion were not significantly changed after i.v. and s.c. injections of IK-SSH. However, tissue factor pathway inhibitor (TFPI) concentration was markedly increased after each injection of IK-SSH and returned to the preinjection value 24 h later. IK-SSH prolongs aPTT, Heptest and PITT in a similar manner as other low molecular weight heparins but without significantly affecting thrombin time, FIIa and FXa activity. The release of TFPI may well be responsible for the prolongation of aPTT, Heptest and PITT. IK-SSH may be further developed as an antithrombotic agent.

  5. Construction of subtractive cDNA Library of apoptosis-related genes in NB4 cells treated by arsenic trioxide%用抑制性差减杂交构建As_2O_3诱导的NB4细胞凋亡相关基因文库

    Institute of Scientific and Technical Information of China (English)

    狄春红; 顾少华; 谭晓华; 仙玲玲; 吴奇涵; 杨磊

    2009-01-01

    Objective: Construct the gene library of apoptosis related genes in acute promyelocytic leukemia (APL) cell line NB4 cells treated by arsenic trioxide to clarify the apoptotic mechanism of NB4 cells. Method: APL cell line NB4 cells treated with or without arsenic trioxide for 24 hours. Total RNA was extracted and suppress subtractive hybridization (SSH) was conducted according to the manual. With the cDNA of the apoptosis cells as the tester and that of control cells as the driver, forward and reverse hybridization was performed- Differentially expressed genes were linked with pGEM-Teasy cloning vector and transformed into E.coli DH5α. The positive clones were screened by blue and white spot. PCR were used to amplify these genes. Result: The subtractive cDNA libraries related with apoptosis of NB4 cells were successfully constructed. Conclusion: The constructed subtractive libraries are suitable for further study on the functional genes associated with apoptosis of NB4 cells induced by arsenic trioxide.%目的: 构建三氧化二砷(As_2O_3)诱导的急性早幼粒细胞白血病细胞株NB4细胞凋亡相关基因文库.方法:用含4 μmol·L~(-1)As_2O_3和正常培养基培养NB4细胞24 h,抽提总RNA,经逆转录酶合成双链cDNA,分别以砷诱导凋亡组和对照组作为tester和driver,进行双向抑制性差减杂交(supptess-ion sublxactive hybridization,SSH),筛选As_2O_3诱导的NB4细胞凋亡相关基因,将差异表达基因进行PCR扩增并与pGEM-Teasy克隆载体连接,转化DH5 α大肠杆菌,经蓝白斑筛选获得白色阳性克隆,PCR扩增出未知基因片段.结果:成功构建了分别代表在NB4细胞中表达上调和下调的基因文库.结论: 经双向抑制性差减杂交获得了NB4细胞差异表达基因文库,为克隆NB4细胞凋亡相关基因奠定了基础.

  6. Construction and characterization of a normalized whole-life-cycle cDNA library of rice

    Institute of Scientific and Technical Information of China (English)

    2003-01-01

    A cDNA library with genomic complete coverage is a powerful tool for functional genomic studies. For studying the functions of rice genes on a large scale, a normalized whole-life-cycle cDNA library is constructed based on the strategy of saturation hybridization with genomic DNA using rice cultivar Minghui 63, an elite restorer line for a number of rice hybrids that are widely cultivated in China. This library consists of cDNA from 15 directionally cloned cDNA libraries constructed with different tissues from 9 developmental stages. For normalization, the denatured plasmids purified from the 15 directionally cloned libraries are mixed and hybridized with saturated genomic DNA labeled with magnetic beads in two complementary systems. Well-matched plasmids are captured from the hybridized genomic DNA and electroporated into competent DH10B E. coli for construction of the normalized whole-life-cycle cDNA library. This library consists of 62000 clones with an average insert length about 1.4 kb. Inverse Northern blotting shows that this cDNA library included many rarely expressed genes and tissue-specific genes. Sequencing of 10750 cDNA clones of this library reveals 6399 unique ESTs (expressed sequence tags), indicating that the non-redundancy of the library is about 59.5%. This library has been used to make cDNA microarrays for functional genomic studies.

  7. Screening of UV-B-induced genes from apple peels by SSH: possible involvement of MdCOP1-mediated signaling cascade genes in anthocyanin accumulation.

    Science.gov (United States)

    Peng, Ting; Saito, Takanori; Honda, Chikako; Ban, Yusuke; Kondo, Satoru; Liu, Ji-Hong; Hatsuyama, Yoshimichi; Moriguchi, Takaya

    2013-07-01

    Suppression subtractive hybridization (SSH) was employed to identify candidate genes involved in red coloration in apple peel with the ultraviolet (UV)-B-treated 'Mutsu'. After reverse Northern blotting verification, nearly 80 clones were successfully sequenced. Large portions of the expressed sequence tags (ESTs) are well characterized anthocyanin biosynthesis-related genes, such as chalcone synthase (11A5), flavonol synthase (12F3), anthocyanidin synthase (11H5) and UDP-glycosyl transferase (14A12) whose presence proved the success of SSH. Eight ESTs were selected for quantitative real-time polymerase chain reaction analysis and their expressions were all elevated in 'Induction', further confirming the reliability of the SSH library. One EST, 11F4 (CONSTITUTIVE PHOTOMORPHOGENIC 1: COP1) with putative function in light signal relay was further analyzed in 'Mutsu' and 'Tsugaru', along with MdHY5 (ELONGATED HYPOCOTYL 5: the downstream target of COP1), MdMYB22 (a possible flavonol-specific activator under the regulation of HY5, belonging to the SG7/PRODUCTION OF FLAVONOL GLYCOSIDES family) and MdMYBA. Results showed that MdCOP1, MdHY5, MdMYB22 and MdMYBA were all UV-B inducible genes and anthocyanin accumulation occurred after their increased expressions. Moreover, their expressions and anthocyanin content were enhanced under UV-B plus 17°C treatment. The presence of G box, a known consensus binding site of HY5, in the MdMYBA promoter region implicated that it could be regulated by MdHY5, which was verified by the result of the yeast one-hybrid analysis. Our data suggested that UV-B irradiation would induce the utmost upstream light signaling factor, MdCOP1, which activates MdHY5 signaling by binding to the promoter regions of MdMYBs, and finally leads to the red coloration of apple peels.

  8. Dicty_cDB: SSH103 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available |AI067610.1 EST209292 Schistosoma mansoni, Phil LoVerde/Joe Merrick Schistosoma m...ST269616 Schistosoma mansoni female, Phil LoVerde/Joe Merrick Schistosoma mansoni cDNA clone SMFAA90 5' end

  9. Dicty_cDB: SSH725 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available 1 CAB20007_IIa_Fa_H10 Cabernet Sauvignon Flower bloom - CAB2 Vitis vinifera cDNA clone CAB20007_IIa_Fa_H10 5...', mRNA sequence. 54 3e-12 2 CF211161 |CF211161.1 CAB20007_IIa_Ra_H10 Cabernet Sauvignon Flower bloom - CAB2

  10. Normalized cDNA libraries

    Science.gov (United States)

    Soares, Marcelo B.; Efstratiadis, Argiris

    1997-01-01

    This invention provides a method to normalize a directional cDNA library constructed in a vector that allows propagation in single-stranded circle form comprising: (a) propagating the directional cDNA library in single-stranded circles; (b) generating fragments complementary to the 3' noncoding sequence of the single-stranded circles in the library to produce partial duplexes; (c) purifying the partial duplexes; (d) melting and reassociating the purified partial duplexes to moderate Cot; and (e) purifying the unassociated single-stranded circles, thereby generating a normalized cDNA library.

  11. Construction and characterization of a yeast two-hybrid cDNA library from a FAT10-overexpressing human hepatic carcinoma cell line Hep3B%类泛素FAT10高表达肝癌细胞株Hep3B酵母双杂交cDNA文库的构建

    Institute of Scientific and Technical Information of China (English)

    余新; 刘天; 德洪波; 李国惠; 邵江华

    2011-01-01

    AIM: To construct a yeast two-hybrid cDNA library from a FAT10-overexpressing human hepatic carcinoma cell line Hep3B.METHODS: Total RNA was prepared from Hep3B cells and used to purify poly (A) mRNA.Double-stranded cDNA was synthesized from the purified mRNA, ligated to EcoR Ⅰ adaptor,digested with EcoR Ⅰ/Xho Ⅰ enzymes, and then cloned into the pGADT7 vector.The recombinant vector was transformed into E.coli DH10B to obtain a primary cDNA library.The primary library was amplified and used to determine the size of cDNA inserts through enzyme digestion.RESULTS: The primary cDNA library contained 1.03 × 106independent clones.The titer of the cDNA library was estimated to be 2.50 × 106 cfu/mL, and that of the amplified library was 3.60 × 109 cfu/mL.The size of the inserts varied from 0.5 to 3.5 kb, with an average value of about 2.0 kb.CONCLUSION: A yeast two-hybrid cDNA library has been successfully generated from FAT10-over-expressing Hep3B ceils and can be used for future screening of proteins interacting with FAT10.%目的:构建类泛素FAT10高表达肝癌细胞株Hep3B的酵母双杂交用cDNA文库.方法:从肝癌细胞Hep3B中提取总RNA,分离mRNA.利用反转录酶M-MLV与Oligo(dT)AnchorPrimer合成1 Strand cDNA,用E.coli DNAPolymerase与E.coli DNA Ligase将RNA链置换成DNA链,合成2 Strand cDNA.将双链cDNA与EcoR Ⅰ Adaptor连接,然后用EcoR Ⅰ /Xho Ⅰ进行酶切.使用Spin Column除去短链cDNA与pGADT7载体连接,转化入E.coli DH10B,建成原始文库.然后对其进行扩增并随机挑取单菌落,酶切鉴定重组子插入片段大小.结果:提取的总RNA降解少且分子完整;RNA纯度高,相对分子质量为400-5000 bp;成功合成双链cDNA,均符合建库要求:库容量达到1.03×10克隆,原始文库滴度为2.50×10 cfu/L,扩增后的文库滴度为3.60×10 cfu/L.插入片段大小分布为0.5-3.5 kb,平均长度约为2.0 kb.结论:所构建文库的各项指标均达到要求,为筛选FAT10作用蛋白奠定了重要基础.

  12. 酵母双杂交筛选胎肾上腺cDNA文库中HNP-1结合蛋白%SCREENING THE GENE SEQUENCES OF THE INTERACTION PROTEINS OF HNP-1 FROM HUMAN FETAL ADRENAL CDNA LIBRARY BY YEAST TWO HYBRID SYSTEM

    Institute of Scientific and Technical Information of China (English)

    杜润滋; 邓璐霞; 黄宁; 罗朝志

    2011-01-01

    [Objective] To screen proteins binding with α-defensin (HNP-1) mature peptide from placenta cDNA libraty by yeast two hybrid technique. [Methods] The cDNA fragment encoding HNP-1 mature peptide was amplified by polymera-sechain reaction (PCR) and constructed into pGBK-T7 vector as the bait plasmid in yeast two hybrid system 3. Subsequently , the RNA from fetal adrenal gland was obtained and then transformed into cDNA library using SMART technology. The fetal adrenal cDNA library was screened with pGBKT7-HNP-1 as bait plasmid by yeast-two hybrid system Matchmaker Lexa. Finally, the positive clone was obtained by PCR and then identified by sequence. Then the interaction between them was determined by GST pull down in vitro and coimmunoprecipitates experiments in vivo. [Results] Bait and cDNA library have been constructed successfully and transformed into yeast. Then the interaction protein was found-melanocortin 2 receptor (ACTHR), CCAAT-enhancer-binding proteins (C/EBP), Tramembrane trafficking protein (TMP21), low density lipoprotein receptor-related protein 6 (LRP6). Therefore, melanocortin 2 receptor (ACTH-R) was determined into the major subjects. And bands which can demonstrate the relationship between HNP-1 and ACTH-R was obtained in GST pull down and coimmunoprecipitates experiments. [Conclusion] ACTH-R can bind to HNP-1 we obtained from fetal adrenal cDNA library and it may play important roles in the function of HNP-1 mature peptide.%[目的]筛选胎肾上腺cDNA文库中与α防御素HNP-1成熟肽具有相互作用的蛋白分子.[方法]通过聚合酶链反应(PCR)成功获得HNP-1成熟肽基因插入酵母表达载体pGBK-T7中构建诱饵质粒,同时提取胎肾上腺RNA,SMART技术制备人胎肾上腺cDNA文库,并采用Matchmaker LexA酵母双杂交系统从胎肾上腺cDNA文库中筛选与HNP-1成熟肽相互作用的蛋白.最后通过PCR筛选获得阳性克隆并测序,而后经回转实验,GST pull down以及免疫共沉淀再次验证

  13. [Valuation for usefulness of selected chromosomal markers for Bacillus anthracis identification. II. Valuation for markers SSH and rpoB].

    Science.gov (United States)

    Zasada, Aleksandra Anna; Jagielski, Marek

    2006-01-01

    The article presents results of valuation for B. anthracis-specificity and usefulness for its identification obtained for different chromosomal markers. In the second part of the study markers SSH241, SSH196, SSH163, SSH133 as well as a fragment of the house-keeping gene rpoB were analyzed. For the investigation MSSCP and multiplex-PCR assays were used. There were also tested different techniques of electrophoresis. The results gave an information about specificity of tested markers and their usefulness for B. anthracis identification.

  14. Dicty_cDB: SSH878 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available M library Mus musculus genomic clone UUGC1M0399K06 R, DNA sequence. 48 0.076 1 BU572652 |BU572652.1 PA__Ea0001H21f Almond developing...nce. 48 0.076 1 BU573578 |BU573578.1 PA__Ea0004H03f Almond developing seed Prunus dulcis cDNA clone PA__Ea00

  15. Construction and analysis of gonad suppression subtractive hybridization libraries for the rice field eel, Monopterus albus.

    Science.gov (United States)

    Qu, Xiancheng; Jiang, Jiaoyun; Shang, Xiaoli; Cheng, Cui; Feng, Long; Liu, Qigen

    2014-04-25

    The objective of this study was to investigate gene transcription profiles of the stage IV ovary and the ovotestis of the rice field eel (Monopterus albus) in an attempt to uncover genes involved in sex reversal and gonad development. Suppression subtractive hybridization (SSH) libraries were constructed using mRNA from the stage IV ovary and the ovotestis. In total 100 positive clones from the libraries were selected at random and sequenced, and then expressed sequence tags (ESTs) were used to search against sequences in the GenBank database using the BLASTn and BLASTx search algorithms. High quality SSH cDNA libraries and 90 ESTs were obtained. Of these ESTs, 43 showed high homology with genes of known function and these are associated with energy metabolism, signal transduction, transcription regulation and so on. The remaining 47 ESTs shared no homology with any genes in GenBank and are thus considered to be hypothetical genes. Furthermore, the four genes F11, F63, R11, and R47 from the forward and reverse libraries were analyzed in gonad, brain, heart, spleen, liver, kidney and muscle tissues. The results showed that the transcription of the F11 and F63 genes was significantly increased while the expression of the R11 and R47 genes was significantly decreased from IV or V ovary. In addition, the results also indicated that the four genes' expression was not gonad-tissue specific. This results strongly suggested that they may be involved in the rice field eel gonad development and/or sex reversal.

  16. 巴西橡胶树胶乳均一化酵母双杂交cDNA文库构建%Construction of a normalized yeast two-hybrid cDNA library of the latex of rubber tree (Hevea brasiliensis Müll. Arg.)

    Institute of Scientific and Technical Information of China (English)

    余海洋; 张宇; 王萌; 覃碧

    2016-01-01

    In this study, the latex of rubber tree (Hevea brasiliensis) clone ‘Reyan 7-33-97’ was used as plant material. SMART® cDNA synthesis technology was used to generate the ifrst strand cDNA, and then long distance PCR (LD-PCR) was used to amplify double-strand cDNA (ds-cDNA). The ds-cDNA was normalized by duplex-speciifc nuclease (DSN). The normalized cDNA was puriifed by running CHROMA SPIN+TE-400 column to remove short cDNA fragments. The puriifed cDNA and linear vector pGADT7-Rec were co-trans-formed into competent Y187 yeast cell to generate a normalized yeast two-hybrid cDNA library. The results show that the cDNA of the latex of rubber tree was wide range of fragment sizes and with uneven abundance before normalization. After normalized by DSN and puriifed using CHROMA SPIN+TE-400 column, cDNA below 500 bp had been removed efficiently, and high abundance of cDNA had been reduced significantly. Moreover, RT-PCR revealed that the transcripts of two housekeeping genes18S rRNA andβ-actinwere decreased signiifcantly after normalization. The harvested library had 1.26×106 independent clones. The titer of the library was up to 3.23×107 cfu·mL-1, the recombination rate was 87%, and the average insert size was more than 1.0 kb. In this study, a normalized yeast two-hybrid cDNA library from the latex of rubber tree has been successfully established, which provides a reference for studying natural rubber biosynthetic pathway and its molecular regulation mechanism in rubber tree.%以巴西橡胶树无性系‘热研7-33-97’胶乳为材料,采用SMART® cDNA合成技术反转录合成cDNA第一链,并通过LD-PCR合成双链cDNA (ds-cDNA),采用双链特异性核酸酶(DSN)对ds-cDNA进行均一化处理,并经过CHROMA SPIN+TE-400柱子去除短片段的cDNA,纯化后的cDNA和线性化载体pGADT7-Rec共转化酵母Y187感受态细胞构建均一化酵母双杂交cDNA文库。结果显示:均一化之前橡胶树胶乳cDNA片段分布较

  17. Cloning and expression analysis of MBLL cDNA

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    The mbl (muscleblind) gene of Drosophila encodes a nuclear protein which contains two Cys3His motifs. The mutation of mbl gene will disturb the differentiation of all the Drosophila's photoreceptors. Primers have been designed according to human EST086139, which is highly homologous to mbl gene. Human fetal brain cDNA library has been screened and a novel cDNA clone has been obtained. The 2595 bp cDNA, designated MBLL (muscleblind-like), contains an open reading frame which encodes 255 amino acids and has 4 Cys3His motifs (GenBank Acc. AF061261). The amino acids sequence shares high homology to Drosophila's mbl. The Northern blot and RNA dot blot hybridization of 43 human adult tissues and 7 fetal tissues show that MBLL is a widely expressed gene, but the expression amounts differ in these tissues.

  18. Using ssh as portal - The CMS CRAB over glideinWMS experience

    CERN Document Server

    Belforte, Stefano; Letts, James; Fanzago, Federica; Saiz Santos, Maria Dolores; Martin, Terrence

    2013-01-01

    The User Analysis of the CMS experiment is performed in distributed way usingboth Grid and dedicated resources. In order to insulate the users from the details of computing fabric, CMS relies on the CRAB (CMS Remote Analysis Builder) package as an abstraction layer. CMS has recently switched from a client-server version of CRAB to a purely client-based solution, with ssh being used to interface with HTCondor-based glideinWMS batch system. This switch has resulted in significant improvement of user satisfaction, as well as in significant simplification of the CRAB code base and of the operation support. This paper presents the architecture of the ssh-based CRAB package, the rationale behind it, as well as the operational experience running both the client-server and the ssh-based versions in parallel forseveral months.

  19. Study of transactivating effect of pre-S2 protein of hepatitis B virus and cloning of genes transactivated by pre-S2 protein with suppression subtractive hybridization

    Institute of Scientific and Technical Information of China (English)

    Dong Ji; Jun Cheng; Guo-Feng Chen; Yan Liu; Lin Wang; Jiang Guo

    2005-01-01

    AIM: To investigate the transactivating effect of pre-S2 protein of hepatitis B virus (HBV) and construct a subtractive cDNA library of genes transactivated by pre-S2 protein with suppression subtractive hybridization (SSH)technique, and to pave the way for elucidating the pathogenesis of HBV infection.METHODS: pcDNA3.1(-)-pre-S2 containing pre-S2 region of HBV genome was constructed by routine molecular methods. HepG2 cells were cotransfected with pcDNA3.1 (-)-pre-S2/pSV-lacZ and empty pcDNA3.1(-)/pSV-lacZ.After 48 h, cells were collected and detected for the expression of β-galactosidase (β-gal). SSH and bioinformatics techniques were used, the mRNA of HepG2 cells transfected with pcDNA3.1(-)-pre-S2 and pcDNA3.1(-) empty vector was isolated, respectively, cDNA was synthesized. After digestion with restriction enzyme RsaI, cDNA fragments were obtained. Tester cDNA was then divided into two groups and ligated to the specific adaptor 1 and adaptor 2, respectively. After tester cDNA was hybridized with driver cDNA twice and underwent two times of nested PCR, amplified cDNA fragments were subcloned into pGEM-Teasy vectors to set up the subtractive library.Amplification of the library was carried out with E. coli strain DH5α. The cDNA was sequenced and analyzed in GenBank with Blast search after PCR.RESULTS: The pre-S2 mRNA could be detected in HepG2 cells transfected with pcDNA3.1(-)-pre-S2 plasmid. The activity of β-gal in HepG2 cells transfected with pcDNA3.1 (-)-pre-S2/pSV-lacZ was 7.0 times higher than that of control plasmid (P<0.01). The subtractive library of genes transactivated by HBV pre-S2 protein was constructed successfully. The amplified library contains 96 positive clones. Colony PCR showed that 86 clones contained 200-1 000 bp inserts. Sequence analysis was performed in 50 clones randomly, and the full length sequences were obtained with bioinformatics method and searched for homologous DNA sequence from GenBank, altogether 25 coding sequences

  20. Screening differentially expressed genes of urine exfoliated urothelial cells in transitional cell carcinoma by SSH%膀胱癌患者尿脱落细胞抑制消减文库的构建及差异基因的初步筛选

    Institute of Scientific and Technical Information of China (English)

    张冲; 郭柏鸿; 张志华; 吏葆光; 车团结; 陈一戎

    2009-01-01

    目的 应用抑制性消减杂交方法 筛选膀胱移行细胞癌患者与正常人尿脱落细胞差异表达基因.方法 分离膀胱移行细胞癌患者与正常人尿液中总mRNA,用SMART技术反转录成cDNA,经过酶切、接头连接、两轮消减杂交及两轮抑制性PCR,使得差异表达的DNA片段得以富集.PCR产物与T/A载体连接并转化大肠杆菌XL-blue构建差异表达基因的cDNA消减文库.文库扩增后随机挑取克隆进行酶切、测序及同源性分析.结果 PCR鉴定有317个克隆载有主要在200~900bp之间呈随机分布的插入片段,片段插入率达93.2%,证实建库成功.对20个质粒测序结果 经同源性比对分析,其中20个片段源于17个已知基因,1个克隆在GenBank中未检索到与其有相似性的基因序列,表明它们可能为BTCC差异表达的新基因.结论 该消减杂交文库质量町靠,它的成功构建为进一步筛选、克隆膀胱肿瘤差异表达基因提供了依据.也为膀胱肿瘤诊断基因芯片的研究与开发奠定了基础.%Objectives To Screening transitional" differentially suppression subtractive eDNA library in transitional cell carcinoma of bladder (BTCC) and normal urine exfoliated urothelial cells. Methods Total mRNA was isolated from BTCC and normal exfoliatod urothelial calls in urine, respectively. Then double-strand eDNA was synthesized and restricted by Hae Ⅲ. eDNA of BTCC was divided into two groups and ligated with either adaptor Ⅰ or adaptor 2. After hybridized twice normal exfoliated urothelial cells eDNA underwent nested PCR, the PCR products were cloned into PGM-T vector and transformed to E. Eoli JM109. Some pesitive clones were randomly picked up, digested, sequenced and homologous analyzed. Results The SSH library contained about 400 positive clones. Random analysis of 384 clones with enzyme restriction showed that 317clones contained cDNA fragments which were mainly between 200~900bp. The inserted rate reached to 82

  1. Application of an improved cDNA competition technique to identify prostate cancer-associated gene.

    Science.gov (United States)

    Rinaldy, A R; Steiner, M S

    1999-11-01

    A technique to improve cDNA library screening was developed by using mixed probes derived from two closely related cDNA populations of high-metastatic MAT-LyLu and low-metastatic AT-1 Dunning R3227 rat prostate cancer sublines. The technique required the generation of a cDNA library from each subline followed by polymerase chain reaction (PCR) amplification of the cDNA insert population. The PCR products derived from the first library were radiolabeled and mixed with an excess amount of PCR products from the second library. The mixture and an excess amount of both the lambda and pBluescript DNA were used as a probe to screen the first cDNA library. This mixed probe (designated the competition probe) differentially cross-hybridized with the plaque lift of the screened first cDNA library. Weak radioactive signals indicated the cross-hybridization of cDNA sequences common to the competition probe mixture and the first cDNA library, whereas strong signals implied unhybridized unique or abundant cDNA sequences in the first cDNA library. The reproducibility of this technique was confirmed by showing that the full-length cDNA clones were associated with the phenotype of the screened first cell line. The isolated clones were characterized as rat nucleolar protein, rat mitochondrial genes coding for 16S and 12S rRNAs, and rat tRNAs specific for valine and phenyl-alanine. This result is consistent with the fact that the first cell line, MAT-LyLu, is metabolically more active than are AT-1 cells because of higher gene dosage or amplification of nucleolar and mitochondrial RNA and its associated genes. Another clone which had a strong signal represented a novel gene associated with the MAT-LyLu cancer phenotype.

  2. Suppression Subtractive Hybridization Identified Genes Differentially Expressed in a Multidrug Resistance Cell Line of Human Lung Adenocarcinoma%肺腺癌多药耐药细胞特异表达基因的克隆与鉴定

    Institute of Scientific and Technical Information of China (English)

    陈杰; 钱桂生; 黄桂君; 熊玮; 李靖

    2001-01-01

    Objective: The aim of this study was to clone and screen multidrug resistance related gene of human adenocarcinoma cell. Methods: The suppression subtractive hybridization (SSH) was performed on human adenocarcinoma multidrug resistance cell line (SPC-A-1/CDDP, as tester) and human adenocarcinoma cell line (SPC-A-1, as driver). After the subtracted cDNA library being constructed, the dot blots was used to screen the subtracted cDNA library with forward and reverse-subtracted cDNA probes. The differentially expressed cDNA fragments in SPC-A-1/CDDP was sequenced and analyzed through Genbank with Blast search. The novel cDNA sequences were analyzed by Northern blots. Results: A high quality subtracted cDNA library was constructed. Twenty-three differentially expressed cDNA fragments in SPC-A-1/CDDP were identified. Two of them were novel cDNA sequences and the others had 93%-100% homology with the known genes respectively. Northern blots indicated the novel cDNA sequences only expressed in SPC-A-1/CDDP cell. Conclusion: The novel cDNA sequences might be multidrug resistance related genes in human lung adenocarcinoma. SSH is a powerful technique to identify differentially expressed genes.%目的:克隆和筛选肺腺癌多药耐药细胞特异表达基因。方法:将肺腺癌多药耐药细胞(SPC-A-1/CDDP)作为实验方,肺腺癌细胞(SPC-A-1)作为对照方,应用抑制消减杂交技术,构建实验方特异表达cDNA消减文库;用斑点杂交法初步筛选cDNA消减文库后,将获得的阳性克隆进行测序和同源性分析(Genbank),对新的cDNA序列进行Northern blot杂交验证。结果:建立了一个肺腺癌多药耐药细胞(SPC-A-1/CDDP)特异表达cDNA消减文库,斑点杂交法初步筛选显示23个克隆中有SPC-A-1/CDDP特异表达cDNA片断,测序和同源性分析表明2个cDNA片断为新序列,其余cDNA片断与已知基因有93%~100%的同源性,Northern blot杂交结果表明2

  3. Transcriptome responses in the rectal gland of fed and fasted spiny dogfish shark (Squalus acanthias) determined by suppression subtractive hybridization.

    Science.gov (United States)

    Deck, Courtney A; McKay, Sheldon J; Fiedler, Tristan J; LeMoine, Christophe M R; Kajimura, Makiko; Nawata, C Michele; Wood, Chris M; Walsh, Patrick J

    2013-12-01

    Prior studies of the elasmobranch rectal gland have demonstrated that feeding induces profound and rapid up regulation of the gland's ability to secrete concentrated NaCl solutions and the metabolic capacity to support this highly ATP consuming process. We undertook the current study to attempt to determine the degree to which up regulation of mRNA transcription was involved in the gland's activation. cDNA libraries were created from mRNA isolated from rectal glands of fasted (7days post-feeding) and fed (6h and 22h post-feeding) spiny dogfish sharks (Squalus acanthias), and the libraries were subjected to suppression subtractive hybridization (SSH) analysis. Quantitative real time PCR (qPCR) was also used to ascertain the mRNA expression of several genes revealed by the SSH analysis. In total the treatments changed the abundance of 170 transcripts, with 103 up regulated by feeding, and 67 up regulated by fasting. While many of the changes took place in 'expected' Gene Ontology (GO) categories (e.g., metabolism, transport, structural proteins, DNA and RNA turnover, etc.), KEGG analysis revealed a number of categories which identify oxidative stress as a topic of interest for the gland. GO analysis also revealed that branched chain essential amino acids (e.g., valine, leucine, isoleucine) are potential metabolic fuels for the rectal gland. In addition, up regulation of transcripts for many genes in the anticipated GO categories did not agree (i.e., fasting down regulated in feeding treatments) with previously observed increases in their respective proteins/enzyme activities. These results suggest an 'anticipatory' storage of selected mRNAs which presumably supports the rapid translation of proteins upon feeding activation of the gland.

  4. The effect of column purification on cDNA indirect labelling for microarrays

    Directory of Open Access Journals (Sweden)

    Kiss John Z

    2007-06-01

    Full Text Available Abstract Background The success of the microarray reproducibility is dependent upon the performance of standardized procedures. Since the introduction of microarray technology for the analysis of global gene expression, reproducibility of results among different laboratories has been a major problem. Two of the main contributors to this variability are the use of different microarray platforms and different laboratory practices. In this paper, we address the latter question in terms of how variation in one of the steps of a labelling procedure affects the cDNA product prior to microarray hybridization. Results We used a standard procedure to label cDNA for microarray hybridization and employed different types of column chromatography for cDNA purification. After purifying labelled cDNA, we used the Agilent 2100 Bioanalyzer and agarose gel electrophoresis to assess the quality of the labelled cDNA before its hybridization onto a microarray platform. There were major differences in the cDNA profile (i.e. cDNA fragment lengths and abundance as a result of using four different columns for purification. In addition, different columns have different efficiencies to remove rRNA contamination. This study indicates that the appropriate column to use in this type of protocol has to be experimentally determined. Finally, we present new evidence establishing the importance of testing the method of purification used during an indirect labelling procedure. Our results confirm the importance of assessing the quality of the sample in the labelling procedure prior to hybridization onto a microarray platform. Conclusion Standardization of column purification systems to be used in labelling procedures will improve the reproducibility of microarray results among different laboratories. In addition, implementation of a quality control check point of the labelled samples prior to microarray hybridization will prevent hybridizing a poor quality sample to expensive

  5. Preparation of cDNA libraries from vascular cells.

    Science.gov (United States)

    Lieb, M E; Taubman, M B

    1999-01-01

    The vast majority of past and present efforts in the molecular cloning of expressed sequences involve isolation of clones from cDNA libraries constructed in bacteriophage lambda (1,2). As discussed in Chapter 6 , screening these cDNA libraries using labeled probes remains the most straightforward method to isolate full length cDNAs for which some partial sequence information is known. Although the availability of high quality reagents and kits over the past decade has made the process of library construction increasingly straightforward, generation of high-quality libraries is a task that still requires a fair amount of dedicated effort. Because alternative PCR-based cloning strategies have become increasingly popular alternatives to cDNA library screening, it is useful to consider the advantages and disadvantages of each strategy before embarking on a project to construct a cDNA library (Table 1). In our opinion, it is worthwhile to construct a cDNA library when the transcript of interest is not exceedingly rare (i.e., can readily be detected by Northern blot analysis of total RNA), when multiple cDNAs will need to be cloned over a period of time, and in situations where occasional mutations can not be tolerated (for example, if the cDNA is to be expressed in mammalian cells to examine function). In situations where the transcript of interest is expressed at exceedingly low levels, or when only a single cDNA needs to be cloned, a PCR-based strategy should be considered. When the tissue source is precious (such as a unique clinical specimen), successful construction of a phage library provides a resource that can be amplified and used for multiple cloning projects over many years, but runs the risk of consuming the available RNA if the library construction fails. Table 1 Comparison of Relative Advantages of cDNA Cloning from Lambda Phage Libraries by Plaque Hybridization Compared to Newer PCR- Based Strategies Lambda phage cDNA library PCR-based strategy Freedom

  6. Reconstruction of ocean velocities from the synergy between SSH and SST measurements

    Science.gov (United States)

    Isern-Fontanet, Jordi; Turiel, Antonio

    2013-04-01

    Recent advances in our understanding of the dynamics in the upper layers of the ocean have allowed us to develop methodologies to recover high resolution velocities from surface measurements such as Sea Surface Heights (SSH) and Sea Surface Temperatures (SST). These methods are based on the combined use of advanced signal processing techniques, such as wavelet analysis and singularity analysis, with dynamical approaches such as the Surface Quasi-Geostrophic (SQG) equations. Within the SQG framework, SSH and SST are closely related, which can be exploited to develop a synergetic approach that combines existing satellite measurements of these fields that can be used to recover subsurface buoyancy anomaly, surface and subsurface horizontal velocities and vertical velocities in the upper 300-500 m. Sentinel-3 satellite will follow its predecessors, ERS-1/2 and Envisat, and will provide simultaneous measurements of SST (SLSTR instrument) and SSH (SRAL and auxiliary instruments) that can be combined to produce high resolution surface currents. To test the feasibility of this approach for Sentinel-3 satellites we have reconstructed surface currents from AATSR and RA data provided by Envisat and compared results against independent SSH measurements provided Jason-1/2 platforms.

  7. Ssh4, Rcr2 and Rcr1 affect plasma membrane transporter activity in Saccharomyces cerevisiae.

    Science.gov (United States)

    Kota, Jhansi; Melin-Larsson, Monika; Ljungdahl, Per O; Forsberg, Hanna

    2007-04-01

    Nutrient uptake in the yeast Saccharomyces cerevisiae is a highly regulated process. Cells adjust levels of nutrient transporters within the plasma membrane at multiple stages of the secretory and endosomal pathways. In the absence of the ER-membrane-localized chaperone Shr3, amino acid permeases (AAP) inefficiently fold and are largely retained in the ER. Consequently, shr3 null mutants exhibit greatly reduced rates of amino acid uptake due to lower levels of AAPs in their plasma membranes. To further our understanding of mechanisms affecting AAP localization, we identified SSH4 and RCR2 as high-copy suppressors of shr3 null mutations. The overexpression of SSH4, RCR2, or the RCR2 homolog RCR1 increases steady-state AAP levels, whereas the genetic inactivation of these genes reduces steady-state AAP levels. Additionally, the overexpression of any of these suppressor genes exerts a positive effect on phosphate and uracil uptake systems. Ssh4 and Rcr2 primarily localize to structures associated with the vacuole; however, Rcr2 also localizes to endosome-like vesicles. Our findings are consistent with a model in which Ssh4, Rcr2, and presumably Rcr1, function within the endosome-vacuole trafficking pathway, where they affect events that determine whether plasma membrane proteins are degraded or routed to the plasma membrane.

  8. Molecular cloning and mammalian expression of human beta 2-glycoprotein I cDNA

    DEFF Research Database (Denmark)

    Kristensen, Torsten; Schousboe, Inger; Boel, Espen

    1991-01-01

    Human β2-glycoprotein (β2gpI) cDNA was isolated from a liver cDNA library and sequenced. The cDNA encoded a 19-residue hydrophobic signal peptide followed by the mature β2gpI of 326 amino acid residues. In liver and in the hepatoma cell line HepG2 there are two mRNA species of about 1.4 and 4.3 k......, respectively, hybridizing specifically with the β2gpI cDNA. Upon isoelectric focusing, recombinant β2gpI obtained from expression of β2gpI cDNA in baby hamster kidney cells showed the same pattern of bands as β2gpI isolated from plasma, and at least 5 polypeptides were visible...

  9. Suppression subtractive hybridization to obtain differential genes associated kidney yang defficiency of IgA nephropathy%运用消减杂交技术构建IgA肾病肾阳虚证的相关基因文库

    Institute of Scientific and Technical Information of China (English)

    李玉萍; 罗仁; 孙晓敏; 聂晓莉; 赵晓山; 杨海文

    2011-01-01

    Objective To construct cDNA substractive library of kidney yang defficiency of IgA nephropathy ( IgAN)by suppression subtractive hybridization ( SSH).Methods The research related genes of kidney yang defficiency syndrome of IgAN.Microamount RNA was amplified.SSH and gene cloning were used to obtain differential genes associated kidney yang defficiency of IgAN.Result A Total of 537 clones were obtained.276 from the forward subtractive library and 261 from the reverse subtractive library.Conclusion The cDNA subtractive library is successfully constructed and it may provide a solid foundation for screening and cloning the genes of kidney yang defficiency.%目的 采用抑制性消减杂交技术构建汉族人IgA肾病肾阳虚证cDNA消减文库.方法 以辨病与辨证相结合,以IgA肾病肾阳虚证入手,应用RNA微量扩增、抑制性消减杂交、基因克隆等技术和方法,构建IgA肾病肾阳虚证消减文库.结果 该研究成功地构建了IgA肾病肾阳虚证的cDNA消减文库,其中正向消减文库获得276个阳性克隆,反向消减文库获得261个阳性克隆.结论 该研究初步构建了IgA肾病肾阳虚证的cDNA消减文库,为进一步筛选和克隆肾阳虚证的相关基因奠定了基础.

  10. Isolating Cd2+-induced genes from Fuyang soybean seedlings roots through suppression subtractive hybridization%应用抑制性差减杂交法分离法分离阜豆苗期根系镉胁迫诱导表达的基因

    Institute of Scientific and Technical Information of China (English)

    赵胡

    2012-01-01

    以1/2Hoagland溶液培养的阜豆幼苗根系为对照群体,含Cd2+浓度为100 μM营养液处理的为目标群体,进行抑制差减杂交.用经过对照组cDNA差减目标组cDNA构建了一个含有大约600个独立克隆的差减文库.随机挑取部分克隆进行菌落PCR鉴定,表明插入的片段大小均在250 ~ 800 bp.对已确认的6个阳性克隆测序,序列分析和同源性比较,表明它们与镉胁迫有关.%A subtracted cDNA library of Fuyang soybean seedlings roots specific to Cd2+ stress was constructed by suppression subtractive hybridization ( SSH). It was composed of approximately 600 individual recombinant clones. SSH was performed between two groups of Fuyang soybean seedlings, the group of Fuyang soybean seedlings cultivated in 1/2 Hoagland'Ls (H) solution as driver and the other treated by Cd2+ stress of 1/2 Hoagland'Ls solution as tester. The recombinant clones from the subtracted cDNA library were chosen randomly and PCR identification of colonies was performed. Result showed that insertions were ranged from 250 to 800bp. Six confirmed positive clones were sequenced. It indicated that they were closely related to cadmium stress.

  11. Multidrug resistance associated genes of leukemia separated by suppression subtractive hybridization%抑制性差减杂交技术分离白血病多药耐药基因

    Institute of Scientific and Technical Information of China (English)

    王楠; 潘喆; 袁宏

    2016-01-01

    Objective To isolate and identify differential expression genes associated with multidrug resistance of leukemia . Methods Differential expression genes between leukemia cell line K 562 and resistant cell lines K562/DOX were isolated by using suppression subtractive hybridization (SSH) technique .Total RNA were extracted .cDNA were synthesized and digested by restric-tion enzyme Rsa Ⅰ ,then connected with adopter1 and adopter2R ,and linked with pMD19-T vector .Constructed vectors were trans-ferred into E .coli .Subtracted cDNA library was constructed ,and the positive clones were screened according to base sequences and homologous sequences .The differential expression genes were indentified by comparison analysis of Gene Bank database .Results A total of 220 differential expression genes were sequenced ,including hemoglobin ,ribosomes and mitochondria related genes ,and heat shock factor binding protein 1 (HSPB1) gene and other genes .Conclusion SSH method and molecular cloning technique could be used to construct subtracted cDNA library of differential expression genes between drug resistant and not -resistant leukemia cells , which might be useful for further screening and cloning of differential expression genes of multidrug resistant tumor cells .%目的:分离及鉴定与白血病多药耐药性相关的差异表达基因。方法采用抑制性差减杂交(SSH)技术分离非耐药细胞株 K562与耐药细胞株 K562/DOX 差异表达基因。提取总 RNA ,逆转录合成 cDNA ,经限制性内切酶 Rsa Ⅰ酶切后,分别与不同的接头(adopter1和 adopter2R)连接;连接产物插入 pMD19-T 载体后转入大肠埃希菌中,构建 cDNA 差减文库;挑取阳性克隆提取质粒进行测序及同源序列分析,确定差异表达基因。结果筛选获得220个差异表达基因,包括血红蛋白、核糖体和线粒体等相关基因,以及热休克因子结合蛋白(HSPB1)基因等其他基因。结论采用 SSH

  12. SSH gene expression profile of Eisenia andrei exposed in situ to a naturally contaminated soil from an abandoned uranium mine.

    Science.gov (United States)

    Lourenço, Joana; Pereira, Ruth; Gonçalves, Fernando; Mendo, Sónia

    2013-02-01

    The effects of the exposure of earthworms (Eisenia andrei) to contaminated soil from an abandoned uranium mine, were assessed through gene expression profile evaluation by Suppression Subtractive Hybridization (SSH). Organisms were exposed in situ for 56 days, in containers placed both in a contaminated and in a non-contaminated site (reference). Organisms were sampled after 14 and 56 days of exposure. Results showed that the main physiological functions affected by the exposure to metals and radionuclides were: metabolism, oxireductase activity, redox homeostasis and response to chemical stimulus and stress. The relative expression of NADH dehydrogenase subunit 1 and elongation factor 1 alpha was also affected, since the genes encoding these enzymes were significantly up and down-regulated, after 14 and 56 days of exposure, respectively. Also, an EST with homology for SET oncogene was found to be up-regulated. To the best of our knowledge, this is the first time that this gene was identified in earthworms and thus, further studies are required, to clarify its involvement in the toxicity of metals and radionuclides. Considering the results herein presented, gene expression profiling proved to be a very useful tool to detect earthworms underlying responses to metals and radionuclides exposure, pointing out for the detection and development of potential new biomarkers.

  13. Diversity Suppression-Subtractive Hybridization Array for Profiling Genomic DNA Polymorphisms

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    Genomic DNA polymorphisms are very useful for tracing genetic traits and studying biological diversity among species. Here, we present a method we call the "diversity suppression-subtractive hybridization array" for effectively profiling genomic DNA polymorphisms. The method first obtains the subtracted gDNA fragments between any two species by suppression subtraction hybridization (SSH) to establish a subtracted gDNA library,from which diversity SSH arrays are created with the selected subtracted clones. The diversity SSH array hybridizes with the DIG-labeled genomic DNA of the organism to be assayed. Six closely related Dendrobium species were studied as model samples. Four Dendrobium species as testers were used to perform SSH. A total of 617 subtracted positive clones were obtained from four Dendrobium species, and the average ratio of positive clones was 80.3%. We demonstrated that the average percentage of polymorphic fragments of pairwise comparisons of four Dendrobium species was up to 42.4%. A dendrogram of the relatedness of six Dendrobium species was produced according to their polymorphic profiles. The results revealed that the diversity SSH array is a highly effective platform for profiling genomic DNA polymorphisms and dendrograms.

  14. Suppression subtractive hybridization and comparative expression analysis to identify developmentally regulated genes in filamentous fungi.

    Science.gov (United States)

    Gesing, Stefan; Schindler, Daniel; Nowrousian, Minou

    2013-09-01

    Ascomycetes differentiate four major morphological types of fruiting bodies (apothecia, perithecia, pseudothecia and cleistothecia) that are derived from an ancestral fruiting body. Thus, fruiting body differentiation is most likely controlled by a set of common core genes. One way to identify such genes is to search for genes with evolutionary conserved expression patterns. Using suppression subtractive hybridization (SSH), we selected differentially expressed transcripts in Pyronema confluens (Pezizales) by comparing two cDNA libraries specific for sexual and for vegetative development, respectively. The expression patterns of selected genes from both libraries were verified by quantitative real time PCR. Expression of several corresponding homologous genes was found to be conserved in two members of the Sordariales (Sordaria macrospora and Neurospora crassa), a derived group of ascomycetes that is only distantly related to the Pezizales. Knockout studies with N. crassa orthologues of differentially regulated genes revealed a functional role during fruiting body development for the gene NCU05079, encoding a putative MFS peptide transporter. These data indicate conserved gene expression patterns and a functional role of the corresponding genes during fruiting body development; such genes are candidates of choice for further functional analysis.

  15. Differentially expressed genes of virulent and nonvirulent Entamoeba histolytica strains identified by suppression subtractive hybridization.

    Science.gov (United States)

    Freitas, Michelle A R; Alvarenga, Ângela C; Fernandes, Helen C; Gil, Frederico F; Melo, Maria N; Pesquero, Jorge L; Gomes, Maria A

    2014-01-01

    Entamoeba histolytica is a parasite which presents capacity to degrade tissues and therefore has a pathogenic behavior. As this behavior is not shown by all strains, there have been several studies investigating molecular basis of the cytotoxicity process. Using the suppression subtractive hybridization (SSH) technique, differential gene expressions of two E. histolytica strains, one virulent (EGG) and one nonvirulent (452), have been analyzed with the purpose of isolating genes which may be involved with amoebic virulence. Nine cDNA fragments presenting high homology with E. histolytica previously sequenced genes were subtracted. Of these, four genes were confirmed by RT-PCR. Two coding for hypothetical proteins, one for a cysteine-rich protein, expressed only in the virulent strain, EGG and another one, coding for grainin 2 protein, exclusive from 452 strain. This study provided new insight into the proteins differences in the virulent and nonvirulent E. histolytica strains. We believe that further studies with these proteins may prove association of them with tissue damage, providing new perceptions to improve treatment or diagnosis of the invasive disease.

  16. Differentially Expressed Genes of Virulent and Nonvirulent Entamoeba histolytica Strains Identified by Suppression Subtractive Hybridization

    Directory of Open Access Journals (Sweden)

    Michelle A. R. Freitas

    2014-01-01

    Full Text Available Entamoeba histolytica is a parasite which presents capacity to degrade tissues and therefore has a pathogenic behavior. As this behavior is not shown by all strains, there have been several studies investigating molecular basis of the cytotoxicity process. Using the suppression subtractive hybridization (SSH technique, differential gene expressions of two E. histolytica strains, one virulent (EGG and one nonvirulent (452, have been analyzed with the purpose of isolating genes which may be involved with amoebic virulence. Nine cDNA fragments presenting high homology with E. histolytica previously sequenced genes were subtracted. Of these, four genes were confirmed by RT-PCR. Two coding for hypothetical proteins, one for a cysteine-rich protein, expressed only in the virulent strain, EGG and another one, coding for grainin 2 protein, exclusive from 452 strain. This study provided new insight into the proteins differences in the virulent and nonvirulent E. histolytica strains. We believe that further studies with these proteins may prove association of them with tissue damage, providing new perceptions to improve treatment or diagnosis of the invasive disease.

  17. Screening of aplastic anaemia-related genes in bone marrow CD4+ T cells by suppressive subtractive hybridization

    Institute of Scientific and Technical Information of China (English)

    ZHENG Miao; LIU Wen-li; FU Jin-rong; SUN Han-ying; ZHOU Jian-feng; XU Hui-zhen

    2007-01-01

    Background CD4+ T cells play a crucial role in the pathogenesis of aplastic anaemia. However, the mechanisms of over-proliferation, activation, infiltration of bone marrow and damage to haematopoietic cells of CD4+ T cells in aplastic anaemia are unclear. Therefore, we screened differentially expressed genes of bone marrow CD4+ T cells of aplastic anaemia patients and normal donors by suppressive subtractive hybridization to investigate the pathogenesis of aplastic anaemia.Methods The bone marrow mononuclear cells of a first visit aplastic anaemia patient and a healthy donor of the same age and sex were isolated using lymphocyte separating medium by density gradient centrifugation. With the patients as "tester" and donor as "driver", their CD4+T cells were separated with magnetic bead sorting and a cDNA library established by suppressive subtractive hybridization. Then 15 of the resulting subtracted cDNA clones were randomly selected for DNA sequencing and homological analysis. With semiquantitative RT-PCR, bone marrow samples from 20 patients with aplastic anaemia and 20 healthy donors assessed the expression levels of differentially expressed genes from SSH library.Results PCR detected 89 clones in the library containing an inserted fragment of 100 bp to 700 bp. Among 15 sequenced clones, 12 were known genes including 3 repeated genes. Compared with normal donors, there were 9/12 genes over-expressed in bone marrow CD4+T cells of patients with aplastic anaemia. The effects of these genes included protein synthesis, biology oxidation, signal transduction, proliferative regulation and cell migration. Not all these genes had been reported in the mechanisms of haematopoietic damage mediated by CD4+ T cells in aplastic anaemia.Conclusions Screening and cloning genes, which regulate functions of CD4+ T cells, are helpful in elucidating the mechanisms of over proliferation, activation, infiltrating bone marrow and damaging haematopoietic cells of CD4+ T cells in

  18. Construction of Subtracted cDNA Library of the Early Growth Plate in Broiler Chickens with Thiram-induced Tibial Dyschondroplasia%福美双诱发肉鸡胫骨软骨发育不良早期生长板消减cDNA文库的构建

    Institute of Scientific and Technical Information of China (English)

    宁官保; 郭定宗; 田文霞; 王瑞; 覃平; 乔建钢; 李宏全; 李家奎; 毕丁仁; 潘思轶

    2011-01-01

    [Objective] An experiment was conducted to construct subtracted cDNA library for selecting time series genes differentially expressed in the TD growth plate of broiler chickens at the early stage with cDNA microarray. [Method] AVIAN (AV) broiler chicks at 7 days of age were randomly divided into two groups. After fasting overnight, they were fed with regular diet (control) or the same diet containing 100 mg/kg thiram for 48h to induce TD (thiram diet-fed). Forward and reverse-subtracted eDNA libraries were generated by suppression subtractive hybridization technology (SSH). Identification of the inserted cDNA fragments in subtractive library was done using PCR. One hundred clones were randomly selected for further DNA sequencing, blast homology analysis and function prediction. [Result] A total of 2 227 positive clones were obtained and the size of inserts was between 200 bp and 1 000 bp. There were 97 homologous gene sequences shared more than 99% identity with genes known in chicken (Gallus gallus). Non-redundancy of sequenced genes was 68.7%. Meanwhile, 3 clones were found to be novel EST as no functional clues were associated with them by bioinformatic analysis. Most of these genes are involved in matrix formation,endochondral ossification and remodelling, developmental regulation, signal transduction, electron transport in mitochondrial respiratory chain and vascularization. [ Conclusion ] Successfully produced cDNA library would make a good foundation for further printing cDNA microarray, screening differential expression genes of TD growth plates at different stages, and also may provide new insights into understanding the pathogenesis of TD.%[目的]为应用cDNA芯片技术筛选肉鸡胫骨软骨发育不良(TD)时序性差异表达基因,本研究构建了早期生长板消减cDNA文库.[方法]将7日龄AV肉鸡随机分为两组,对照组(control,C)饲以基础日粮,试验组(thiram diet-fed,T)饲以基础日粮添加福美双100 mg·kg(-1),2 d

  19. Establishment of Self-incompatibility Gene cDNA Microarray to Identify S-genotypes of Pyrus pyrifolia

    Directory of Open Access Journals (Sweden)

    Jiang Nan

    2015-11-01

    Full Text Available Based on the cDNA sequences from hyper variable (HV regions of identified 52 S-alleles in Oriental pear cultivars, S-RNase cDNA probes were designed, and a cDNA microarray for S-RNase detections was established. Each microarray contained 240 sites from 55 cDNA probes, including all specific cDNA sequences from the HV regions of the S-alleles. Using the cDNA of pistils of tested pear cultivars as template and Cy3 fluorescently labeling primers by PCR amplification, microarray hybridization detected the S-genotype of each pear cultivar. The genotypes inferred from the cDNA microarray hybridization signals of pear cultivars such as ‘Lijiang Huangsuanli’, ‘Xiuyu’, ‘Midu Yuli’, ‘Baimianli’, and ‘Deshengxiang’ were similar to the known genotypes of all tested cultivars. The S-RNase cDNA microarrays and the oligonucleotide gene chips were then used to conduct parallel testing of 24 P. pyrifolia cultivars with unknown S-genotypes. In conclusion, the construction of cDNA microarrays has further improved the pear S-RNase detection platform.

  20. Heavy-tailed distribution of the SSH Brute-force attack duration in a multi-user environment

    Science.gov (United States)

    Lee, Jae-Kook; Kim, Sung-Jun; Park, Chan Yeol; Hong, Taeyoung; Chae, Huiseung

    2016-07-01

    Quite a number of cyber-attacks to be place against supercomputers that provide highperformance computing (HPC) services to public researcher. Particularly, although the secure shell protocol (SSH) brute-force attack is one of the traditional attack methods, it is still being used. Because stealth attacks that feign regular access may occur, they are even harder to detect. In this paper, we introduce methods to detect SSH brute-force attacks by analyzing the server's unsuccessful access logs and the firewall's drop events in a multi-user environment. Then, we analyze the durations of the SSH brute-force attacks that are detected by applying these methods. The results of an analysis of about 10 thousands attack source IP addresses show that the behaviors of abnormal users using SSH brute-force attacks are based on human dynamic characteristics of a typical heavy-tailed distribution.

  1. Solving Energy Levels for SSH Hamiltonian Describing Peierls Phase Transition by Virtue of Invariant Eigen-operator Method

    Institute of Scientific and Technical Information of China (English)

    P.S. Vyas; FAN Hong-Yi; P.N. Gajjar; WU Hao; B.Y. Thakore; A.R. Jani

    2008-01-01

    We show that the recently proposed invariant eigen-operator (IEO) method can be successfully applied to solving energy levels for SSH Hamiltonian describing Peierls phase transition. The electronic energy band of compound lattice is also studied by IEO method.

  2. Applications of Suppression Subtractive Hybridization (SSH) in Identifying differentially expressed transcripts in Ascochyta rabiei

    Science.gov (United States)

    Introduction – Ascochyta rabiei, casual agents of chickpea ascochyta blight, is divided into two pathotypes based on virulence levels. Genetic mechanisms of this phenotypic differentiation are poorly understood. This research is directed toward understanding molecular differences between the two pa...

  3. Phenol emulsion-enhanced DNA-driven subtractive cDNA cloning: isolation of low-abundance monkey cortex-specific mRNAs.

    OpenAIRE

    1988-01-01

    To isolate cDNA clones of low-abundance mRNAs expressed in monkey cerebral cortex but absent from cerebellum, we developed an improved subtractive cDNA cloning procedure that requires only modest quantities of mRNA. Plasmid DNA from a monkey cerebellum cDNA library was hybridized in large excess to radiolabeled monkey cortex cDNA in a phenol emulsion-enhanced reaction. The unhybridized cortex cDNA was isolated by chromatography on hydroxyapatite and used to probe colonies from a monkey cortex...

  4. Cloning of cDNA encoding steroid 11. beta. -hydroxylase (P450c11)

    Energy Technology Data Exchange (ETDEWEB)

    Chua, S.C.; Szabo, P.; Vitek, A.; Grzeschik, K.H.; John, M.; White, P.C.

    1987-10-01

    The authors have isolated bovine and human adrenal cDNA clones encoding the adrenal cytochrome P-450 specific for 11..beta..-hydroxylation (P450c11). A bovine adrenal cDNA library constructed in the bacteriophage lambda vector gt10 was probed with a previously isolated cDNA clone corresponding to part of the 3' untranslated region of the 4.2-kilobase (kb) mRNA encoding P450c11. Several clones with 3.2-kb cDNA inserts were isolated. Sequence analysis showed that they overlapped the original probe by 300 base pairs (bp). Combined cDNA and RNA sequence data demonstrated a continuous open reading frame of 1509 bases. P450c11 is predicted to contain 479 amino acid residues in the mature protein in addition to a 24-residue amino-terminal mitochondrial signal sequence. A bovine clone was used to isolate a homologous clone with a 3.5-kb insert from a human adrenal cDNA library. A region of 1100 bp was 81% homologous to 769 bp of the coding sequence of the bovine cDNA except for a 400-bp segment presumed to be an unprocessed intron. Hybridization of the human cDNA to DNA from a panel of human-rodent somatic cell hybrid lines and in situ hybridization to metaphase spreads of human chromosomes localized the gene to the middle of the long arm of chromosome 8. These data should be useful in developing reagents for heterozygote detection and prenatal diagnosis of 11..beta..-hydroxylase deficiency, the second most frequent cause of congenital adrenal hyperplasia.

  5. Identification of differentially expressed genes after partial rat liver ischemia/reperfusion by suppression subtractive hybridization

    Institute of Scientific and Technical Information of China (English)

    Christine Fallsehr; Christina Zapletal; Michael Kremer; Resit Demir; Magnus von Knebel Doeberitz; Ernst Klar

    2005-01-01

    AIM: To identify potential diagnostic target genes in early reperfusion periods following warm liver ischemia before irreversible liver damage occurs.METHODS: We used two strategies (SSH suppression subtractive hybridization and hybridization of cDNA arrays)to determine early changes in gene expression profiles in a rat model of partial WI/R, comparing postischemic and adjacent nonischemic liver lobes. Differential gene expression was verified (WT/R; 1 h/2 h) and analyzed in more detail after warm ischemia (1 h) in a reperfusion time kinetics (0, 1, 2 and 6 h) and compared to untreated livers by Northern blot hybridizations. Protein expression was examined on Western blots and by immunohistochemistry for four differentially expressed target genes (Hsp70,Hsp27, Gadd45a and IL-1rl).RESULTS: Thirty-two individual WI/R target genes showing altered RNA levels after confirmation by Northern blot analyzes were identified. Among them, six functionally uncharacteristic expressed sequences and 26 known genes (12 induced in postischemic liver lobes, 14 with higher transcriptional expression in adjacent nonischemic liver lobes). Functional categories of the verified marker genes indicate on the one hand cellular stress and tissue damage but otherwise activation of protective cellular reactions (AP-1 transcription factors, apoptosis related genes, heat shock genes). In order to assign the transcriptional status to the biological relevant protein level we demonstrated that Hsp70, Hsp27, Gadd45a and IL-1rI were clearly up-regulated comparing postischemic and untreated rat livers, suggesting their involvement in the WI/R context.CONCLUSION: This study unveils a WI/R response gene set that will help to explore molecular pathways involved in the tissue damage after WI/R. In addition, these genes especially Hsp70and Gadd45a might represent promising new candidates indicating WI/R liver damage.

  6. Profiling of differentially expressed genes using suppression subtractive hybridization in an equine model of chronic asthma.

    Directory of Open Access Journals (Sweden)

    Jean-Pierre Lavoie

    Full Text Available BACKGROUND: Gene expression analyses are used to investigate signaling pathways involved in diseases. In asthma, they have been primarily derived from the analysis of bronchial biopsies harvested from mild to moderate asthmatic subjects and controls. Due to ethical considerations, there is currently limited information on the transcriptome profile of the peripheral lung tissues in asthma. OBJECTIVE: To identify genes contributing to chronic inflammation and remodeling in the peripheral lung tissue of horses with heaves, a naturally occurring asthma-like condition. METHODS: Eleven adult horses (6 heaves-affected and 5 controls were studied while horses with heaves were in clinical remission (Pasture, and during disease exacerbation induced by a 30-day natural antigen challenge during stabling (Challenge. Large peripheral lung biopsies were obtained by thoracoscopy at both time points. Using suppression subtractive hybridization (SSH, lung cDNAs of controls (Pasture and Challenge and asymptomatic heaves-affected horses (Pasture were subtracted from cDNAs of horses with heaves in clinical exacerbation (Challenge. The differential expression of selected genes of interest was confirmed using quantitative PCR assay. RESULTS: Horses with heaves, but not controls, developed airway obstruction when challenged. Nine hundred and fifty cDNA clones isolated from the subtracted library were screened by dot blot array and 224 of those showing the most marked expression differences were sequenced. The gene expression pattern was confirmed by quantitative PCR in 15 of 22 selected genes. Novel genes and genes with an already defined function in asthma were identified in the subtracted cDNA library. Genes of particular interest associated with asthmatic airway inflammation and remodeling included those related to PPP3CB/NFAT, RhoA, and LTB4/GPR44 signaling pathways. CONCLUSIONS: Pathways representing new possible targets for anti-inflammatory and anti

  7. Investigation of bipolaron formation in the Su-Schrieffer-Heeger (SSH) model and various extensions

    Science.gov (United States)

    Sous, John; Berciu, Mona; Krems, Roman

    We develop a variational scheme for studying the stability of bipolarons in one-dimensional systems. In particular, we consider the SSH, Holstein, and, Breathing-Mode models along with combinations of these couplings and with other extended variations. We derive equations of motions under the variational approximation and solve numerically for the two-particle Green's function. We study the stability of bipolarons under different conditions and for fermonic and bosonic particles.

  8. The design of student achievement management system based on SSH2%基于SSH2学生成绩管理系统的设计

    Institute of Scientific and Technical Information of China (English)

    李留青; 张莹莹

    2013-01-01

    Along with the increase in student Numbers,how can the effective management of student achievement information,establish a set of suitable for the university student achievement management system,is the construction of digital information required to solve important problems in colleges and universities.This paper designs an SSH2 based on student achievement management system,can realize the student achievement of entry,query,modify,add and manage of students,and other functions.%  随着学生数量的增多,如何能有效的管理学生成绩信息,建立一套适合本校学生成绩管理系统,是各高校数字信息建设所必需解决的重要问题。本文设计了基于SSH2学生成绩管理系统,可以实现对学生成绩的录入、查询、修改、学生的添加和管理等功能。

  9. An improved, SSH-based method to automatically identify mesoscale eddies in the ocean

    Institute of Scientific and Technical Information of China (English)

    WANG Xin; DU Yun-yan; ZHOU Cheng-hu; FAN Xing; YI Jia-wei

    2013-01-01

      Mesoscale eddies are an important component of oceanic features. How to automatically identify these mesoscale eddies from available data has become an important research topic. Through careful examination of existing methods, we propose an improved, SSH-based automatic identification method. Using the inclusion relation of enclosed SSH contours, the mesoscale eddy boundary and core(s) can be automatically identified. The time evolution of eddies can be examined by a threshold search algorithm and a tracking algorithm based on similarity. Sea-surface height (SSH) data from Naval Research Laboratory Layered Ocean Model (NLOM) and sea-level anomaly (SLA) data from altimeter are used in the many experiments, in which different automatic identification methods are compared. Our results indicate that the improved method is able to extract the mesoscale eddy boundary more precisely, retaining the multiple-core structure. In combination with the tracking algorithm, this method can capture complete mesoscale eddy processes. It can thus provide reliable information for further study of reconstructing eddy dynamics, merging, splitting, and evolution of a multi-core structure.

  10. Efficient and simpler method to construct normalized cDNA libraries with improved representations of full-length cDNAs

    Energy Technology Data Exchange (ETDEWEB)

    Soares, Marcelo Bento (New York, NY); Bonaldo, Maria de Fatima (New York, NY)

    1998-01-01

    This invention provides a method to normalize a cDNA library comprising: (a) constructing a directionally cloned library containing cDNA inserts wherein the insert is capable of being amplified by polymerase chain reaction; (b) converting a double-stranded cDNA library into single-stranded DNA circles; (c) generating single-stranded nucleic acid molecules complementary to the single-stranded DNA circles converted in step (b) by polymerase chain reaction with appropriate primers; (d) hybridizing the single-stranded DNA circles converted in step (b) with the complementary single-stranded nucleic acid molecules generated in step (c) to produce partial duplexes to an appropriate Cot; and (e) separating the unhybridized single-stranded DNA circles from the hybridized DNA circles, thereby generating a normalized cDNA library. This invention also provides a method to normalize a cDNA library wherein the generating of single-stranded nucleic acid molecules complementary to the single-stranded DNA circles converted in step (b) is by excising cDNA inserts from the double-stranded cDNA library; purifying the cDNA inserts from cloning vectors; and digesting the cDNA inserts with an exonuclease. This invention further provides a method to construct a subtractive cDNA library following the steps described above. This invention further provides normalized and/or subtractive cDNA libraries generated by the above methods.

  11. Efficient and simpler method to construct normalized cDNA libraries with improved representations of full-length cDNAs

    Energy Technology Data Exchange (ETDEWEB)

    Soares, M.B.; Fatima Bonaldo, M. de

    1998-12-08

    This invention provides a method to normalize a cDNA library comprising: (a) constructing a directionally cloned library containing cDNA inserts wherein the insert is capable of being amplified by polymerase chain reaction; (b) converting a double-stranded cDNA library into single-stranded DNA circles; (c) generating single-stranded nucleic acid molecules complementary to the single-stranded DNA circles converted in step (b) by polymerase chain reaction with appropriate primers; (d) hybridizing the single-stranded DNA circles converted in step (b) with the complementary single-stranded nucleic acid molecules generated in step (c) to produce partial duplexes to an appropriate Cot; and (e) separating the unhybridized single-stranded DNA circles from the hybridized DNA circles, thereby generating a normalized cDNA library. This invention also provides a method to normalize a cDNA library wherein the generating of single-stranded nucleic acid molecules complementary to the single-stranded DNA circles converted in step (b) is by excising cDNA inserts from the double-stranded cDNA library; purifying the cDNA inserts from cloning vectors; and digesting the cDNA inserts with an exonuclease. This invention further provides a method to construct a subtractive cDNA library following the steps described above. This invention further provides normalized and/or subtractive cDNA libraries generated by the above methods. 25 figs.

  12. Efficient and simpler method to construct normalized cDNA libraries with improved representations of full-length cDNAs

    Science.gov (United States)

    Soares, Marcelo Bento; Bonaldo, Maria de Fatima

    1998-01-01

    This invention provides a method to normalize a cDNA library comprising: (a) constructing a directionally cloned library containing cDNA inserts wherein the insert is capable of being amplified by polymerase chain reaction; (b) converting a double-stranded cDNA library into single-stranded DNA circles; (c) generating single-stranded nucleic acid molecules complementary to the single-stranded DNA circles converted in step (b) by polymerase chain reaction with appropriate primers; (d) hybridizing the single-stranded DNA circles converted in step (b) with the complementary single-stranded nucleic acid molecules generated in step (c) to produce partial duplexes to an appropriate Cot; and (e) separating the unhybridized single-stranded DNA circles from the hybridized DNA circles, thereby generating a normalized cDNA library. This invention also provides a method to normalize a cDNA library wherein the generating of single-stranded nucleic acid molecules complementary to the single-stranded DNA circles converted in step (b) is by excising cDNA inserts from the double-stranded cDNA library; purifying the cDNA inserts from cloning vectors; and digesting the cDNA inserts with an exonuclease. This invention further provides a method to construct a subtractive cDNA library following the steps described above. This invention further provides normalized and/or subtractive cDNA libraries generated by the above methods.

  13. Construction and analysis of subtractive cDNA library of recovery body wall in sea cucumber Apostichopus japonicus%仿刺参体壁创伤修复消减文库的构建与分析

    Institute of Scientific and Technical Information of China (English)

    秦艳杰; 李霞; 张慧敏; 王雪

    2013-01-01

    应用抑制性消减杂交技术(SSH),构建了仿刺参Apostichopus japonicus(体质量为65 ~90 g)正常体壁及创伤修复(24、48、72、96、120 h后)体壁的消减cDNA文库,利用PCR和斑点杂交技术对文库进行筛选,随机挑取的768个克隆中发现292个阳性克隆,对其中信号强度较强的224个阳性克隆进行测序,得到208个有效EST序列.经BlastX工具对获得的EST与GenBank数据库进行比对分析,结果有171个EST序列与数据库中的基因同源(e≤0.001,相似性>40%),其中153个为未知基因,18个为已知功能或已命名基因,包括在创伤及修复的体壁中上调表达的β微管蛋白、微管蛋白α-1链、肌动蛋白、肌动蛋白ike 7B类似物、细胞色素c氧化酶亚基Ⅰ、tRNA假尿苷合成酶A、GTP酶、细胞分裂周期2类似蛋白、有丝分裂原活化蛋白激酶、homeobox蛋白、延伸因子1A、核糖体蛋白L30、核糖体蛋白L17、60S酸性核糖体蛋白PO、26S蛋白酶调节亚基、泛素特异性肽酶24、大肠癌血清抗原3、清道夫受体蛋白12等.本研究结果可为探讨刺参体壁再生过程和分子机理,以及筛选刺参体壁创伤修复过程中相关功能基因的研究提供基础依据.%A subtracted cDNA library of sea cucumber Apostichopus japonicus(body weight 65-90 g) was constructed by suppression subtractive hybridization technology (SSH) to screen EST associated with recovery body wall.The cDNA library of the test group has been constructed by the mRNA of the body walls 24,48,72,96 and 120h after the operation,and those with no operation as the control group.Differential EST from the subtracted cDNA library have been identified by both PCR technology and dot blot hybridization.Two hundred and ninety-two positive clones were observed from total 768 clones,and 224 positive ones were sequenced.Two hundred and eight EST were found and analyzed by BlastX tool in GenBank database,in which 171 EST were homologous with sequences

  14. cDNA cloning, sequence analysis, and chromosomal localization of the gene for human carnitine palmitoyltransferase

    Energy Technology Data Exchange (ETDEWEB)

    Finocchiaro, G.; Taroni, F.; Martin, A.L.; Colombo, I.; Tarelli, G.T.; DiDonato, S. (Istituto Nazionale Neurologico C. Besta, Milan (Italy)); Rocchi, M. (Istituto G. Gaslini, Genoa (Italy))

    1991-01-15

    The authors have cloned and sequenced a cDNA encoding human liver carnitine palmitoyltransferase an inner mitochondrial membrane enzyme that plays a major role in the fatty acid oxidation pathway. Mixed oligonucleotide primers whose sequences were deduced from one tryptic peptide obtained from purified CPTase were used in a polymerase chain reaction, allowing the amplification of a 0.12-kilobase fragment of human genomic DNA encoding such a peptide. A 60-base-pair (bp) oligonucleotide synthesized on the basis of the sequence from this fragment was used for the screening of a cDNA library from human liver and hybridized to a cDNA insert of 2255 bp. This cDNA contains an open reading frame of 1974 bp that encodes a protein of 658 amino acid residues including 25 residues of an NH{sub 2}-terminal leader peptide. The assignment of this open reading frame to human liver CPTase is confirmed by matches to seven different amino acid sequences of tryptic peptides derived from pure human CPTase and by the 82.2% homology with the amino acid sequence of rat CPTase. The NH{sub 2}-terminal region of CPTase contains a leucine-proline motif that is shared by carnitine acetyl- and octanoyltransferases and by choline acetyltransferase. The gene encoding CPTase was assigned to human chromosome 1, region 1q12-1pter, by hybridization of CPTase cDNA with a DNA panel of 19 human-hanster somatic cell hybrids.

  15. Transcriptional profiling of genes involved in embryogenic, non-embryogenic calluses and somatic embryogenesis of Valencia sweet orange by SSH-based microarray.

    Science.gov (United States)

    Ge, Xiao-Xia; Chai, Li-Jun; Liu, Zheng; Wu, Xiao-Meng; Deng, Xiu-Xin; Guo, Wen-Wu

    2012-10-01

    Somatic embryogenesis (SE) is a most promising technology that is used for in vitro germplasm conservation and genetic improvement via biotechnological approaches in citrus. Herein, three suppression subtractive hybridization (SSH) libraries were constructed using calluses of Citrus sinensis cv. 'Valencia' to explore the molecular mechanisms that underlie the SE in citrus. A total of 880 unisequences were identified by microarray screening based on these three SSH libraries. Gene ontology analysis of the differentially expressed genes indicated that nucleolus associated regulation and biogenesis processes, hormone signal transduction, and stress factors might be involved in SE. Transcription factors might also play an important role. LEC1/B3 domain regulatory network genes (LEC1, L1L, FUS3, ABI3, and ABI5) were isolated in citrus SE. Some new transcription factors associated with citrus SE, like a B3 domain containing gene and HB4, were identified. To understand the influence of these isolated genes on SE competence, their expression profiles were compared among callus lines of seven citrus cultivars with different SE competence. The expression dynamics suggested that these genes could be necessary for the SE initiation and might play a role in embryogenic competence maintenance in different cultivars. On the basis of gene expression profiles, an overview of major physiological and biosynthesis processes at different developmental stages during citrus SE is presented. For the first time, these data provide a global resource for transcriptional events important for SE in citrus, and the specific genes offer new information for further investigation on citrus SE maintenance and development.

  16. Gene expression profiling related to powdery mildew resistance in wheat with the method of suppression subtractive hybridization

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    "Bainong 3217×Mardler" BC5F4 wheat line at the initial stage of inoculation with powdery mildew pathogen (Erysiphe graminis DC) was used to construct a suppression subtractive hybridization (SSH) cDNA library. Totally 760 ESTs were obtained through sequencing. Similarity analysis of ESTs based on BLASTn and BLASTx with the sequences in GenBank, in combination with macroarray differential screening, revealed that 199 ESTs of 65 kinds were known to be functionally disease resistance related. Based on the gene expression profiling in the present study, it is postulated that salicylic acid (SA) and MAP-related signal transduction pathways were involved in powdery mildew resistance in wheat. System acquired resistance genes were predominant in terms of kinds and quantity. With the initiation of cell defense reaction, the genes conferring anti-oxida- tion substances were largely expressed and thus cell protection mechanism was activated. Much evidence revealed that phenylpropanes metabolic pathway was involved in phytoalexin synthesis in wheat powdery mildew resistance. Genes conferring some enzymes of structural modification of cell walls and proteinase inhibitors inhibiting pathogen growth were also detected. The genes controlling a few proteinases (mainly cysteine proteinase) had a considerable redundancy of expression.

  17. Isolation of 24 novel cDNA fragments from microdis—sected human chromosome band

    Institute of Scientific and Technical Information of China (English)

    ZHANGMIN; LONGYU; 等

    1998-01-01

    The strategy of isolating the band0specific expression fragments from a probe pool generated by human chromosome microdissection was reported.A chromosome 14q 24.3 band-specific single copy DNA pool was constructed based on this probe pool.Using total DNA of the pool as probe to hybridize the human marrow cDNA library,68 primary positive clones were selected from 5×105 cDNA clones.Among these primary clones,32 secondary clones were obtained after second-round screening and designed as cFD14-1-32.Finally,24 band-specific expression fragments were identified from these 32 positive clones by DNA hybridization.Those band-specific clones can hybridize to both 14q24.3 DNA and human genomic DNA but cann't hybridize to 17q11-12 DNA,Partial sequences of 13 fragments of them were sequenced and idenfified as novel cDNA sequences,and these sequences were proved to have some homology with known genes in NCBI database.Analysis of expression spectrum of cFD 14-1 suggested that the cDNA fragments thus obtained should be used to isolate the genes can not been cloned in 14q24.3 region.

  18. 与人巨细胞病毒UL128两种不同结构蛋白相互作用蛋白的筛选与分析%Screening of protein interacting with the transcript of UL128 gene showed two protein patterns by yeast two-hybrid from human fetus brain cDNA library

    Institute of Scientific and Technical Information of China (English)

    任高伟; 崔鑫; 马艳萍; 齐莹; 阮强; 孙峥嵘

    2010-01-01

    Objective Using yeast two-hybrid system to screen the proteins which can interact with the human cytomegalovirus (HCMV) UL128 which have two difference transcription structure from human fetus brain cDNA library, and compare the difference with structure and function of interacting proteins. Methods Two fragments of UL128 were amplified by 3'RACE and 5'RACE technology, the length are 519 bp and 642 bp, respectively. The "bait plasmid" (named as pGBKT7-UL128-519 bp and pGBKT7-UL128-642 bp) was constructed successfully. Using pGBKT7-UL128-519 bp and pGBKT7-UL128-642 bp as a bait, a human fetus brain cDNA was screened and the proteins interacting with UL128-519 bp and UL128-642 bp encoded protein were searched, and the positive clones were sequenced and analyzed by bioinformatic methods. Results EFEMP2 interacting with HCMV UL128-519 bp were identified, THY-1 interacting with HCMV UL128-642 bp were identified. Conclusion EFEMP2 and THY-1 proteins interacting with HCMV UL128-519 bp and UL128-642 bp in human fetus brain cDNA library were successfully screened, but same proteins weren't found from the proteins interacting with UL128-519 bp and UL128-642 bp protein, UL128-519 bp and UL128-642 bp protein may be play an different effect in the process of infect by HCMV.%目的 利用酵母双杂交系统从人胎脑cDNA文库中筛选与两种不同转录结构的人巨细胞病毒(HCMV)UL128编码蛋白相互作用的蛋白,比较两者相互作用蛋白之间的异同点.方法 通过3'RACE和5'RACE技术扩增出两种HCMV UL128片段,其大小分别为519 bp和642 bp,并将其成功构建到酵母诱饵表达载体pGBKT7中.将以上两种酵母表达载体分别转化到酵母菌AH109中,再将文库DNA转化到已含有酵母表达载体的AH109中,筛选与两种片段大小不同的UL128编码蛋白相互作用的人胎脑蛋白,并对筛选得到的阳性克隆进行测序和生物信息学分析.结果 筛出EFEMP2与UL128-519 bp编码蛋白相互作用,THY-1

  19. SSH-BM-I, a tryptamine derivative, stimulates mineralization in terminal osteoblast differentiation but inhibits osteogenesis of pre-committed progenitor cells.

    Science.gov (United States)

    Mikami, Yoshikazu; Somei, Masanori; Tsuda, Hiromasa

    2011-01-01

    SSH-BM-I was synthesized from tryptamine by using a newly developed synthetic method, and it has structural similarity to bromomelatonin. Recently, it had been reported that SSH-BM-I increases osteoblasts in scales of gold fish. However, the effect of SSH-BM-I on osteoblast differentiation in mammalian cells has not yet been examined. Therefore, this study examined the effect of SSH-BM-I on osteoblast differentiation in mesenchymal progenitor-like cells and mature osteoblast-like cells. SSH-BM-I enhanced terminal osteoblast differentiation, as indicated by mineralization, which was accompanied by upregulation of the osteogenic marker genes bone sialoprotein (BSP) and osteocalcin (OC). However, in mesenchymal progenitor ROB-C26 cultures, no mineralized nodules were observed regardless of SSH-BM-I treatment, although BMP-2 was able to induce nodule formation in these cells. Furthermore, BMP-2-induced nodule formation was suppressed by SSH-BM-I treatment in ROB-C26 cultures. We further investigated the impact of the timing and duration of SSH-BM-I treatment on osteoblast differentiation. The effect of SSH-BM-I treatment on osteoblast differentiation of ROB-C26 in the presence of BMP-2 switches from negative to positive sometime between day 6 and 9, because SSH-BM-I treatment enhanced the formation of mineralized nodules when it was started on day 9, but suppressed nodule formation when it was started at day 6 or earlier. These results suggest that the stimulatory effects of SSH-BM-I on the formation of mineralized nodules depend on the degree of cell differentiation.

  20. Identification of differentially expressed genes in Mongolian sheep ovaries by suppression subtractive hybridization.

    Science.gov (United States)

    He, Xiaolong; Li, Bei; Wang, Feng; Tian, Chunying; Rong, Weiheng; Liu, Yongbin

    2012-07-01

    Fecundity is an important trait in sheep. Because it is directly related to production costs and efficiency, it has great economic impact in sheep husbandry. Because Mongolian sheep are a longstanding, indigenous breed, they are genetically related to most other breeds of sheep in China. The study of genes related to reproductive traits is essential to improving the fecundity of Mongolian sheep. In the present study, suppression subtractive hybridization (SSH) was performed using forward and reverse nested primers on cDNA libraries from ovarian tissue of single-bearing (S) and biparous (B) Mongolian sheep (MS). This yielded 768 clones. The length of the inserted fragments ranged from 150 to 1000 bp. From these, dot blot hybridization followed by sequencing and homology blast search in GenBank resolved 373 differentially expressed clones, representing 185 gene sequences (homology >85% and length >200 bp), 10 expressed sequence tags (ESTs; homology >95% and length >100 bp), and 4 unknown ESTs. The analysis of the differentially expressed gene functions allowed these genes to be categorized into seven groups: cell/body or immune defense, metabolism, transportation, nucleic acid modification, cell development, signal transduction, and cell structure. Four differentially expressed genes, a disintegrin and metalloproteinase with thrombospondin motifs 1 (ADAMTS1), inhibitor of DNA binding 3 (ID3), bone morphogenetic protein 6 (BMP6), and integrin beta 1 (ITGB1), were randomly selected and verified using relative quantitative real-time polymerase chain reaction (RQ-PCR). The expression of these genes in BMS ovaries was 30.06, 11.55, 0.82, and 1.12-fold that of SMS ovaries, respectively.

  1. Suppression subtractive hybridization identified genes differentially expressed in a human lung adenocarcinoma multidrug resistance cell line%应用抑制消减杂交(SSH)克隆肺腺癌多药耐药细胞特异表达基因

    Institute of Scientific and Technical Information of China (English)

    陈杰; 钱桂生; 等

    2001-01-01

    目的 克隆和筛选肺腺癌多药耐药细胞特异表达基因。方法 将肺腺癌多药耐药细胞(SPC-A-1/CDDP)作为实验组,肺腺癌细胞(SPC-A-1)作为对照组,应用抑制消减杂交技术,构建实验组特异表达cDNA消减文库;用斑点杂交初步筛选cDNA消减文库后,将获得的阳性克隆进行测序和同源性分析(Genebank)。结果 建立了一个肺腺癌多药耐药细胞(SPC-A-1/CDDP)特异表达cDNA消减文库,斑点杂交初步筛选显示23个克隆中有SPC-A-1/CDDP特异表达cDNA片断,测序和同源性分析表明2个cDNA片断为新序列,其余cDNA片断与已知基因有96%~100%的同源性。结论 2个新的cDNA序列可能为未知肺腺癌多药耐药相关基因序列;抑制消减杂交是克隆特异表达基因的有效方法。%Objective To clone and screen multidrug resistance related gene of human adenocarcinoma cell. Methods Suppression subtractive hybridization (SSH) was performed on human adenocarcinoma multidrug resistance cell line (SPC-A-1/CDDP, as tester, which was established from cell line SPC-A-1 under the inducement of cisplatin) and human adenocarcinoma cell line (SPC-A-1, as driver). After the construction of subtracted cDNA library, dot blot was used to screen the subtracted cDNA library with forward- and reverse-subtracted cDNA probes. The differentially expressed cDNA fragments in SPC-A-1/CDDP was sequenced and analyzed in Genebank with Blast search. Results A subtracted cDNA library of high quality was constructed. Twenty-three differentially expressed cDNA fragments in SPC-A-1/CDDP were identified. Two of them were novel cDNA sequences and the others shared 96%~100% homology with the known sequence. Conclusion The novel cDNA sequences might be the human lung adenocarcinoma multidrug resistance related genes. SSH is an effective approach to identify differentially expressed genes.

  2. Construction and analysis of suppression subtractive cDNA libraries of continuous monoculture Rehmannia glutinosa%连作地黄cDNA消减文库的构建及分析

    Institute of Scientific and Technical Information of China (English)

    张重义; 范华敏; 杨艳会; 李明杰; 李娟; 许海霞; 陈军营; 陈新建

    2011-01-01

    目的:通过构建连作地黄cDNA消减文库,探讨地黄连作障碍的分子机制.方法:利用抑制性消减杂交(SSH)技术构建连作地黄的正反消减文库,通过蓝白斑筛选、PCR的方法鉴定出阳性克隆,并对其进行测序和生物信息学分析.结果:连作地黄cDNA消减文库构建成功,正向和反向消减文库均筛选了300个阳性克隆.测序结果表明:正库、反库分别获得232条、214条特异的EST序列;经NCBI数据库分析,正库、反库中分别有200,195条EST序列的基因具有蛋白功能注释;COG基因功能预测结果表明,正库、反库中分别有60,61条EST序列具有相应的的基因功能分类,涉及21个代谢途径.结论:差异表达基因的功能注释表明,连作对地黄体内的基因表达具有深刻的影响.本研究筛选地黄响应连作的关键基因,为揭示地黄连作障碍的分子机制奠定了基础.%Objective: To explore the molecular mechanism of continuous monoculture problem by constructing the eDNA libraries of continuous monoculture Rehmannia glutinosa. Method: To use the suppression subtractive hybridization (SSH) technique to construct the forward and reverse subtractive cDNA libraries of continuous monoculture R. glutinosa to adopt blue-white colony sereening and PCR to detect the positive clones which would be sequenced and analyzed by bioinformatics. Result: The subtracted cDNA libraries of continuous monoculture R. glutinosa. were successfully constructed, and the result showed that the forward and reverse subtracted libraries obtained 300 positive clones, respectively. The forward and reverse libraries got different ESTs, and produced 232 ( forward library) and 214 ( reverse library) unique ESTs by sequencing. Based on homology search of BLASTX and BLASTN in NCBI, 200 and 195 of unique ESTs were homologous to known genes in the forward and reverse libraries, respectively. Categories of orthologous group (COG) showed that the forward and reverse

  3. 干旱胁迫的水稻根高效酵母双杂交体系建立%Construction and High-efficient Screenings of a Yeast Two-Hybrid cDNA Library from the Drought-stressed Roots of Rice

    Institute of Scientific and Technical Information of China (English)

    付坚; Linkun Gu; 郭怡卿; Liyuan ZHANG; 王玲仙; 李定琴; 王波; Jeff Qingxi SHEN; 程在全

    2013-01-01

    采用SMART技术在酵母菌株AH109中构建了干旱胁迫下水稻根部全长cDNA文库,采用改进的滤膜杂交方法建立了高效酵母杂交体系,以抗旱相关水稻转录调控因子OsWRKy71基因作为诱饵对该方法进行检验.实验获得的酵母杂交文库容量为4.9×10 6,平均插入片段为800 bp,达到了基因分离和克隆等后续研究的标准.同时采用滤膜杂交法将文库的杂交效率提高到了6.9%,是液体杂交法的6倍以上.该方法不需要特殊的设备,且具有低消耗和高效率的特点,可用于高通量酵母双杂交分析.%By using the SMART technique, a yeast two-hybrid cDNA library of rice roots was constructed and utilized to study protein-protein interactions induced by drought. An optimized high efficiency membrane mating method, substituting for liquid mating method, was developed for library screenings. As results, there are about 4. 9 × 106 clones in the library with the averaged size of the insert fragments to be about 800 base pairs. To verify the efficiency of the membrane mating method, OsWRKY71 , a well-studied gene encoding a transcriptional repressor of gibberellin signaling, was used as a bait to screen this library. The mating efficiency was shown to be as high as 6. 9% , which was six times higher than that of the liquid mating method. This high efficient mating method is simple, cost-effective and suitable for large scale yeast hybrid analyses.

  4. SEA SURFACE HEIGHT (SSH) CHANGE AND ITS RELATIONSHIP WITH WIND STRESS IN THE NORTH PACIFIC OCEAN%北太平洋海表面高度(SSH)与风应力变化的关系

    Institute of Scientific and Technical Information of China (English)

    黄琳; 孙佳; 杨逸秋; 袁逸凡

    2013-01-01

    利用1958-2008年的北太平洋海表面高度和风应力资料,并与ENSO和PDO指数进行相关分析.结果发现,风应力及其经向分量主要通过季节振动影响海表面高度(SSH)的年周期变化,纬向风应力主要通过多年振动影响SSH的ENSO和PDO周期.纬向风应力和SSH均以黑潮延伸体主轴为界,两侧呈现出相反的升降趋势,SSH为北降南升,纬向风应力南降北升.风应力和SSH升降趋势相同,均表现为“上升—下降—上升”的变化特征.在地形变化剧烈、等深线南北分布的海区,西风增强会导致SSH升高,且西侧升高较为明显.北风增强将导致北太平洋西岸SSH升高,东岸SSH降低.%Correlation analysis between sea surface height, wind stress and index of ENSO and PDO for the period of 1958-2008 shows in the North Pacific Ocean, wind stress and its meridional component affect the annual cycle of SSH mainly through the seasonal vibration, and the zonal component affect the ENSO and PDO cycles of the SSH through years of vibration. Separated by the Kuroshio extension spindle, the zonal wind stress and SSH show different trends at the same side: SSH decreases at the north while increasing at the south, and the zonal wind stress decreases at the south while increasing at the north. Wind stress and SSH has the same trend: "increase-decrease-increase". The westerly enhanced the SSH rise in sea area where the topography is steep and isobaths distribute along the north-south direction, and the west side increased more significantly. The north wind enhancement led to SSH rise at the west coast of the North Pacific and drop at the east coast.

  5. Study on Wusan Granule Anti-tumor Related Target Gene Screened by Cdna Microarray

    Institute of Scientific and Technical Information of China (English)

    YOU Zi-li; SHI Jin-ping; CHEN Hai-hong

    2006-01-01

    To screen Wusan Granule anti-tumor related target gene using cDNA microarray technique, both mRNA from Lewis lung carcinoma tissues treated by Wusan Granule and untreated control are reversibly transcribed to prepare cDNA probes which are labeled by Cy5 and Cy3. Then, the probes are hybridized to the mice cDNA microarray type MGEC-20S. After hybridization, the cDNA microarray is scanned by ScanArray 3 000 scanner and the data is analyzed by ImaGene 3 software to screen the differentially expressed genes. There are 45 differentially expressed genes including 18 known genes and 27 unknown genes between the two groups, and among them, 20 elevated genes and 25 reduced genes are identified. Additionally, the genes related to invasion and metastasis of malignant carcinomas are down-regulated and the genes related to apoptosis are up-regulated. The cDNA microarray technique is a high-throughput approach to screen the Wusan Granule anti-tumor related target genes, which allow us to explore the molecular biological mechanism on a genomic scale.

  6. Cloning and expression of a cDNA encoding ribosomal protein S4 from Rice (Oryza sativa)

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    A cDNA clone, pS4, has been isolated from a cDNA library prepared from rice anthers of about 1.0 mm in length. DNA sequence analysis and database search show that the cDNA encodes a protein which is highly homologous to eukaryotic 80S ribosomal protein subunit 4 (S4). Northern hybridization indicates that this gene expresses in all tissues analyzed although the expression level varies and it cannot be induced by mechanical wounding in leaves. Southern blot analysis demonstrates that this rice S4 gene is from a multigene family.

  7. ASSOCIATION OF DIFFERENTIALLY EXPRESSED cDNA FRAGMENT OF FGG WITH HEPATOCELLULAR CARCINOMA

    Institute of Scientific and Technical Information of China (English)

    范秉琳; 朱武凌; 邹国林; 段芳龄

    2002-01-01

    Objective: To identify a cDNA clone from the subtracted library of human hepatocellular carcinoma (HCC). Methods: Suppression subtractive hybridization was used to isolated a panel of genes that are differentially expressed in hepatocellular carcinoma as compared with cirrhotic liver. T/A cloning method was used to construct a subtracted cDNA library. DNA sequencing analysis and Northern blot analysis were also utilized. Results: The cloned cDNA is 787 nucleotides in length and contains an open reading frame of 230 amino acids, which is a cDNA fragment of reported human fibrinogen, gamma polypeptide (FGG). Northern analysis revealed that this gene was overexpressed in two hepatocellular carcinoma cell lines, SMMC-7721 and HepG2. Conclusion: Sequence identity proved the cDNA clone fragment of as FGG gene. Differential expression of the cDNA fragment in HCC suggested that FGG is related to HCC, indicating a new clue for developing a novel diagnostic and prognostic marker.

  8. Analysis of gene expression profile of aspermia using cDNA microarray

    Institute of Scientific and Technical Information of China (English)

    杨波; 高晓康; 王禾; 刘贺亮; 陈宝琦; 秦荣良; 康福霞; 邵国兴; 邵晨

    2003-01-01

    Objective: To identify the differential gene expression profiles between the normal and aspermia human testes utilizing cDNA microarray. Methods: cDNA probes were prepared by labeling mRNA of aspermia testes tissues with Cy5-dUTP and mRNA of normal testes tissues with Cy3-dUTP respectively through reverse transcription. The mixed cDNA probes were then hybridized with 4096 cDNA arrays (4096 unique human cDNA sequences), and the fluorescent signals were scanned by ScanArray 3000 scanner (General Scanning, Inc.). The values of Cy5-dUTP and Cy3-dUTP on each spot were analyzed and calculated by ImaGene 3.0 software (BioDiscovery, Inc.). Differentially expressed genes were screened according to the criterion that the absolute value of natural logarithm of the ratio of Cy5-dUTP to Cy3-dUTP was greater-than 2.0 or less-than 0.5. A randomly chosen gene RAP1A was studied by in situ hybridization to evaluate the accuracy of the results. Results: 623 differential expressed genes related to aspermia were found. There were 303 up-expressed genes and 320 down-expressed genes. A distinct up-expressed gene RAP1A was confirmed by in situ hybridization. Conclusions: Screening the differential gene expression profiles between the normal and aspermia human testis by cDNA microarray can be used in the study of aspermia-related genes and the further research due to its properties, RAP1A may play some roles in the development and progression of aspermia.

  9. Identification of proteins involved in the functioning of Riftia pachyptila symbiosis by Subtractive Suppression Hybridization

    Directory of Open Access Journals (Sweden)

    Lallier François H

    2007-09-01

    Full Text Available Abstract Background Since its discovery around deep sea hydrothermal vents of the Galapagos Rift about 30 years ago, the chemoautotrophic symbiosis between the vestimentiferan tubeworm Riftia pachyptila and its symbiotic sulfide-oxidizing γ-proteobacteria has been extensively studied. However, studies on the tubeworm host were essentially targeted, biochemical approaches. We decided to use a global molecular approach to identify new proteins involved in metabolite exchanges and assimilation by the host. We used a Subtractive Suppression Hybridization approach (SSH in an unusual way, by comparing pairs of tissues from a single individual. We chose to identify the sequences preferentially expressed in the branchial plume tissue (the only organ in contact with the sea water and in the trophosome (the organ housing the symbiotic bacteria using the body wall as a reference tissue because it is supposedly not involved in metabolite exchanges in this species. Results We produced four cDNA libraries: i body wall-subtracted branchial plume library (BR-BW, ii and its reverse library, branchial plume-subtracted body wall library (BW-BR, iii body wall-subtracted trophosome library (TR-BW, iv and its reverse library, trophosome-subtracted body wall library (BW-TR. For each library, we sequenced about 200 clones resulting in 45 different sequences on average in each library (58 and 59 cDNAs for BR-BW and TR-BW libraries respectively. Overall, half of the contigs matched records found in the databases with good E-values. After quantitative PCR analysis, it resulted that 16S, Major Vault Protein, carbonic anhydrase (RpCAbr, cathepsin and chitinase precursor transcripts were highly represented in the branchial plume tissue compared to the trophosome and the body wall tissues, whereas carbonic anhydrase (RpCAtr, myohemerythrin, a putative T-Cell receptor and one non identified transcript were highly specific of the trophosome tissue. Conclusion Quantitative PCR

  10. Isolation of biosynthesis related transcripts of 2,3,5,4'-tetrahydroxy stilbene-2-O-β-D-glucoside from Fallopia multiflora by suppression subtractive hybridization

    Directory of Open Access Journals (Sweden)

    Wei Zhao

    2014-07-01

    Full Text Available 2,3,5,4'-tetrahydroxy stilbene-2-O-ß-D-glucoside (THSG exerts multiple pharmacodynamic actions, found in Fallopia multiflora, but the biosynthesis pathway of THSG is still unclear. To clear this ambiguity, we constructed suppression subtractive hybridization (SSH libraries to screen the genes involved in THSG biosynthesis from two F. multiflora varieties, which vary significantly in THSG content. Twelve non-redundant differentially expressed sequence tags were obtained and the full lengths of 4 unreported fragments were amplified by rapid amplification of cDNA ends. We totally got 7 full-length transcripts, and all of them were aligned to the transcriptome and digital gene expression tag profiling database of four F. multiflora tissues (root, stem and leaf from Deqing F. multiflora and another root from Chongqing F. multiflora; data unpublished using local BLAST. The results showed that there was a significant, organ specific difference in the expression of fragments and full-length sequences. All the sequences were annotated by aligning to nucleotide and protein databases. Kyoto Encyclopedia of Genes and Genomes pathway analysis indicated that THSG biosynthesis was correlated with multiple life activities.

  11. Differentially expressed genes in a flock of Chinese local-breed chickens infected with a subgroup J avian leukosis virus using suppression subtractive hybridization

    Directory of Open Access Journals (Sweden)

    Guiping Zhao

    2010-01-01

    Full Text Available Avian leukosis virus subgroup J (ALV-J is a new type of virus that mainly induces myeloid leukosis (ML in chickens. To further elucidate the pathogenesis of ALV-J infection and tumor development, expression profiles from the bone marrow tissue of 15 infected and 18 non-infected birds from a local-breed poultry-farm under naturally infected conditions, were analyzed by suppression-subtractive hybridization. The birds were diagnosed as ML+ (or ML- by specific ALV-J detection methods, involving serological tests for antigens and antibodies, and RT-PCR to detect viral RNA. A total of 59 partial gene sequences were revealed by differential screening of 496 forward and 384 reverse subtracted cDNA clones. Of these, 22 identified genes, including 8 up-regulated and 14 down-regulated, were related to immune functions, these genes being, MHC B-G antigen, translationally-controlled tumor protein (TPT1/TPTC, transferrin and ferritin, hemoglobin and Carbonic anhydrase. Four of the down-regulated genes were selected for further analysis, in view of their predicted roles in infection and immunity by real-time qRT-PCR, using RNA collected from the same birds as those used for SSH. The four genes were expressed at significantly lower levels (p < 0.001 in ALV-J infected birds than in non-infected ones.

  12. Identification of genes involved in indole-3-butyric acid-induced adventitious root formation in nodal cuttings of Camellia sinensis (L.) by suppression subtractive hybridization.

    Science.gov (United States)

    Wei, Kang; Wang, Liyuan; Cheng, Hao; Zhang, Chengcai; Ma, Chunlei; Zhang, Liqun; Gong, Wuyun; Wu, Liyun

    2013-02-10

    The plant hormone auxin plays a key role in adventitious rooting. To increase our understanding of genes involved in adventitious root formation, we identified transcripts differentially expressed in single nodal cuttings of Camellia sinensis treated with or without indole-3-butyric acid (IBA) by suppressive subtractive hybridization (SSH). A total of 77 differentially expressed transcripts, including 70 up-regulated and 7 down-regulated sequences, were identified in tea cuttings under IBA treatment. Seven candidate transcripts were selected and analyzed for their response to IBA, and IAA by real time RT-PCR. All these transcripts were up regulated by at least two folds one day after IBA treatment. Meanwhile, IAA showed less positive effects on the expression of candidate transcripts. The full-length cDNA of a F-box/kelch gene was also isolated and found to be similar to a group of At1g23390 like genes. These unigenes provided a new source for mining genes related to adventitious root formation, which facilitate our understanding of relative fundamental metabolism.

  13. Toxic effect on tissues and differentially expressed genes in hepatopancreas identified by suppression subtractive hybridization of freshwater pearl mussel (Hyriopsis cumingii) following microcystin-LR challenge.

    Science.gov (United States)

    Yang, Ziyan; Wu, Hongjuan; Li, Yuan

    2012-07-01

    Microcystins are a family of potent hepatotoxins produced by freshwater cyanobacteria and can cause animal intoxications and human diseases. In this study, the effect of microcystin-LR (MC-LR) on the tissues of freshwater pearl mussel (Hyriopsis cumingii) was evaluated and differentially expressed genes in the hepatopancreas of the mussel exposed to MC-LR were identified. HPLC analysis of cell extracts from various tissues of the mussel indicated that the hepatopancreas had the highest MC-LR levels (55.78 ± 6.73 μg g⁻¹ DW) after 15-day exposure. The MC-LR concentration in gill or muscle was an order of magnitude less than in hepatopancreas or gonad. Subtractive cDNA library was constructed by suppression subtractive hybridization (SSH), and ∼400 positive clones were sequenced, from which 98 high quality sequences were obtained by BLAST analysis. The screening identified numerous genes involved in apoptosis, signal transduction, cytoskeletal remodel, innate immunity, material and energy metabolism, translation and transcription which were extensively discussed. The results of this study add large amount of information to the mussel genome data, and for the first time present the basic data on toxicity effect of MC-LR on mussel.

  14. Differentially expressed genes in a flock of Chinese local-breed chickens infected with a subgroup J avian leukosis virus using suppression subtractive hybridization.

    Science.gov (United States)

    Zhao, Guiping; Zheng, Maiqing; Chen, Jilan; Wen, Jie; Wu, Chunmei; Li, Wenjuan; Liu, Libo; Zhang, Yuan

    2010-01-01

    Avian leukosis virus subgroup J (ALV-J) is a new type of virus that mainly induces myeloid leukosis (ML) in chickens. To further elucidate the pathogenesis of ALV-J infection and tumor development, expression profiles from the bone marrow tissue of 15 infected and 18 non-infected birds from a local-breed poultry-farm under naturally infected conditions, were analyzed by suppression-subtractive hybridization. The birds were diagnosed as ML+ (or ML-) by specific ALV-J detection methods, involving serological tests for antigens and antibodies, and RT-PCR to detect viral RNA. A total of 59 partial gene sequences were revealed by differential screening of 496 forward and 384 reverse subtracted cDNA clones. Of these, 22 identified genes, including 8 up-regulated and 14 down-regulated, were related to immune functions, these genes being, MHC B-G antigen, translationally-controlled tumor protein (TPT1/TPTC), transferrin and ferritin, hemoglobin and Carbonic anhydrase. Four of the down-regulated genes were selected for further analysis, in view of their predicted roles in infection and immunity by real-time qRT-PCR, using RNA collected from the same birds as those used for SSH. The four genes were expressed at significantly lower levels (p < 0.001) in ALV-J infected birds than in non-infected ones.

  15. Cloning chromosome specific genes by reciprocal probing of arrayed cDNA and cosmid libraries

    Energy Technology Data Exchange (ETDEWEB)

    Yazdani, A.; Lee, C.C.; Wehnert, M. [Baylor College of Medicine, Houston, TX (United States)] [and others

    1994-09-01

    A human gene map will greatly facilitate the association of genes to single locus diseases and provide candidates for genes involved in complex genetic traits. Given the estimated 100,000 human genes an integrated strategy with a high throughput approach for isolation and mapping of expressed sequences is needed to create such a gene map. We have developed an approach that allows high throughput gene isolation and mapping using arrayed genomic and cDNA lambda libraries. Reciprocal probing of the arrayed genomic and cDNA cosmic libraries can rapidly establish cDNA-cosmid associations. Fluorescence in situ hybridization (FISH) chromosomal mapping and expressed sequence tag/sequence tag site (EST/STS) primers generated from DNA sequence of PCR-based mapping using somatic hybrid cell line mapping panels were utilized to characterize further the hybridization-based cDNA cosmid association. We have applied this approach to chromosome 17 using a placental cDNA library and have identified a total of 30 genes out of which 11 are novel. Furthermore seven cDNAs were mapped to 17q21 in this study, providing novel candidate genes for BRCA-1 gene for early onset breast cancer. The results of our study clearly show that an integration of an expression map into physical and genetic maps can provide candidate genes for human diseases that have been mapped to specific regions. This approach combined with the current physical mapping efforts could efficiently provide a detailed human gene map.

  16. Cloning Full-Length cDNAs from Vascular Tissues and Cells by Rapid Amplification of cDNA Ends (RACE) and RT-PCR.

    Science.gov (United States)

    Shen

    1999-01-01

    The isolation of full-length cDNAs remains a frequent task undertaken in many laboratories. A full-length cDNA is often desirable for one of the following purposes: 1) to complete the sequence of a partial cDNA cloned by library screenings or the yeast one- or two-hybrid system; 2) to derive the cDNA sequence encoding a protein, based on peptide sequences; 3) to obtain the sequence of a reported cDNA for functional analysis or expression studies; and 4) to define exon/intron boundaries of a cloned gene or determine transcription start site(s) of a promoter.

  17. Mobile phone ordering system based on Android and SSH2%基于Android和SSH2的手机订餐系统

    Institute of Scientific and Technical Information of China (English)

    张国平; 黄淼; 褚龙现

    2016-01-01

    For the convenience that the users is not restricted to time and space to get the ordering information, this paper uses the technology based on Android and SSH2, designs and develops a Online ordering system. The system is consists of mobile phone client and server,The mobile client of the system can realize the functions of login for registered users, query for Food infromation,build for order and query for order information. The backstage server can realize the functions of user management, user orders and search , shopping cart view and search,restaurant management, food management restaurant orders and serach. The system can realize the more business opportunities for the restaurant seller, and at the same time to provide more convenience for customers. Experimental results show that the system has the advantages of simple operation, strong portability, fast loading speed and less resource-intensive, and thus reaching the design requirements.%为方便用户不受时间和空间的限制,便能快速订餐,本文采用Android和SSH2技术设计并开发一个网上订餐系统,该系统由手机客户端和后台服务器端两部分组成,手机客户端实现用户的注册登录、菜品信息查询、生成订单和订单信息查询等功能;后台服务器端实现了用户管理,用户订单查看与检索,购物车查看与检索,餐厅管理,菜品管理,餐厅订单查看与检索等功能。开发的系统能给餐厅卖家提供更多商机,同时也给顾客提供更多的便利。实验表明,该系统具有操作简便、扩展性强、加载速度快和占用资源少等优点,达到了设计要求。

  18. cDNA sequence quality data - Budding yeast cDNA sequencing project | LSDB Archive [Life Science Database Archive metadata

    Lifescience Database Archive (English)

    Full Text Available Budding yeast cDNA sequencing project cDNA sequence quality data Data detail Data name cDNA sequence quality... data Description of data contents Phred's quality score. PHD format, one file to a single cDNA data, and co...ription Download License Update History of This Database Site Policy | Contact Us cDNA sequence quality data - Budding yeast cDNA sequencing project | LSDB Archive ...

  19. cDNA cloning of human myeloperoxidase: decrease in myeloperoxidase mRNA upon induction of HL-60 cells

    Energy Technology Data Exchange (ETDEWEB)

    Weil, S.C.; Rosner, G.L.; Reid, M.S.; Chisholm, R.L.; Farber, N.M.; Spitznagel, J.K.; Swanson, M.S.

    1987-04-01

    Myeloperoxidase (MPO), the most abundant neutrophil protein, is a bacteriocidal component of the primary granules and a critical marker in distinguishing acute myelogenous leukemia from acute lymphoid leukemia. A cDNA clone for human MPO was isolated by immunologic screening of human hematopoietic lambdagt11 expression vector libraries with specific anti-MPO antibody. The identity of the cDNA clone was confirmed by finding that (i) epitope-selected antibody against this clone recognizes purified MPO and MPO in human promyelocytic (HL-60) cell lysates by immunoblot analysis, and that (ii) hybrid section of HL-60 mRNA with this cDNA clone and translation in vitro results in the synthesis of an 80-kDa protein recognized by the anti-MPO antiserum. RNA blot analysis with this MPO cDNA clone detects hybridization to two polyadenylylated transcripts of approx. = 3.6 and approx. = 2.9 kilobases in HL-60 cells. No hybridization is detected to human placenta mRNA. Upon induction of HL-60 cells to differentiate by incubation for 4 days with dimethyl sulfoxide, a drastic decrease in the hybridization intensity of these two bands is seen. This is consistent with previous data suggesting a decrease in MPO synthesis upon such induction of these cells. The MPO cDNA should be useful for further molecular and genetic characterization of MPO and its expression and biosynthesis in normal and leukemic granulocytic differentiation.

  20. Design and Implementation of Paperless Examination System Based on SSH2%基于SSH2的无纸化考试系统的设计与实现

    Institute of Scientific and Technical Information of China (English)

    周晨; 陆正; 高莺; 查艳芳; 程龙

    2013-01-01

    本文针对无纸化考试系统,通过SSH2技术架构,描述了系统用户管理,题库管理,试卷生成和考试与分析系统等功能的实现。应用表明本系统实现了基本功能。同时,由于SSH2的使用,系统易于快速实现,具有良好可扩展性和可维护性。%In light of the paperless examination system which includes system user management, question bank management, test paper auto-generation, test and result analysis management, this paper describes that the realization method based on the technology architecture for SSH2. It is shown by the application that the system supplies the based function. At the same time, because of the application of SSH2 framework,the system is easy to realize, and has a good extendibility and maintainability.

  1. Construction of differentially expressed cDNA libraries of aldehyde dehydrogenase with high and low activity from tongue squamous carcinoma Tca8113 cell line%基于舌鳞癌Tca8113细胞醛脱氢酶活性不同构建差异表达基因cDNA文库

    Institute of Scientific and Technical Information of China (English)

    孙守娟; 季平; 邓诚; 李颖; 邹波; 漆小娟

    2012-01-01

    Objective To construct the differentially expressed cDNA libraries of aldehyde dehydro genase with high and low activity (ALDHhigh/ALDHlow) from tongue squamous carcinoma Tca8113 cell line. Methods Expression of stem cell marker ALDH was detected, and ALDHhighand ALDHlow cells were collected by Aldefluor assay combined with flow cytometry. Differentially expressed genes of total RNA that was extracted from the two cell subpopulations by Trizol were screened and amplified by suppressing subtractive hybridization ( SSH) , and the PCR products were connected with pMD18-T vector and then transfected into E. Coli DH5a for amplification. Enzyme digestion, gene sequencing and homology analysis were performed in 24 positive clones that were randomly picked from each library. Results Two subproportions of ALDHhigh and ALDHlowwere " screened out, and ALDHhigh cells in Tca8113 cells accounted for 2. 5%. RNA D(260)/D(280) of ALDHhigh and ALDHlow were 1. 93 and 1. 92, respectively. Two-directional subtractive cDNA libraries of ALDHhigh and ALDHlow were constructed, and each library comprised 500 clones. PCR analysis of 24 clones randomly picked from each library showed that insert-fragments distributed in 200 - 700 bp, and no false positive clones were detected. Gene sequencing result that was analyzed and indexed by PubMed showed that cancer related genes included SLC25A13, KLHL2, NPC1, WAPL, BARD1, Notch2 and EEF2K. Conclusion Two-directional subtractive cDNA libraries of ALDHhigh and ALDHlow cells were successfully constructed.%目的 构建舌鳞癌Tca8113细胞系中高、低醛脱氢酶活性(high/low aldehyde dehydrogenase activity,ALDHhigh/ALDHlow)细胞差异表达基因cDNA文库.方法 用流式细胞仪检测ALDEFLUOR(R)染色的Tca8113细胞中干细胞标志物ALDH的表达,并收集ALDHhigh和ALDHlow细胞;用Trizol分别提取两亚群细胞的总RNA;用抑制性消减杂交(SSH)对2组RNA进行差异基因筛选和扩增,扩增产物与pMD18-T载体

  2. Are ‘STEM from Mars and SSH from Venus’?: Challenging disciplinary stereotypes of research’s social value

    NARCIS (Netherlands)

    Olmos-Penuela, Julia; Benneworth, Paul; Castro-Martinez, Elena

    2014-01-01

    There is a reasonably settled consensus within the innovation community that science, technology, engineering and mathematics (STEM) research is more ‘useful’ to societies than other types of research, notably social sciences and humanities (SSH) research. Our paper questions this assumption, and se

  3. Safety surrogate histograms (SSH): A novel real-time safety assessment of dilemma zone related conflicts at signalized intersections.

    Science.gov (United States)

    Ghanipoor Machiani, Sahar; Abbas, Montasir

    2016-11-01

    Drivers' indecisiveness in dilemma zones (DZ) could result in crash-prone situations at signalized intersections. DZ is to the area ahead of an intersection in which drivers encounter a dilemma regarding whether to stop or proceed through the intersection when the signal turns yellow. An improper decision to stop by the leading driver, combined with the following driver deciding to go, can result in a rear-end collision, unless the following driver recognizes a collision is imminent and adjusts his or her behavior at or shortly after the onset of yellow. Considering the significance of DZ-related crashes, a comprehensive safety measure is needed to characterize the level of safety at signalized intersections. In this study, a novel safety surrogate measure was developed utilizing real-time radar field data. This new measure, called safety surrogate histogram (SSH), captures the degree and frequency of DZ-related conflicts at each intersection approach. SSH includes detailed information regarding the possibility of crashes, because it is calculated based on the vehicles conflicts. An example illustrating the application of the new methodology at two study sites in Virginia is presented and discussed, and a comparison is provided between SSH and other DZ-related safety surrogate measures mentioned in the literature. The results of the study reveal the efficacy of the SSH as complementary to existing surrogate measures.

  4. Identification of low Ca(2+) stress-induced embryo apoptosis response genes in Arachis hypogaea by SSH-associated library lift (SSHaLL).

    Science.gov (United States)

    Chen, Hua; Zhang, Chong; Cai, Tie Cheng; Deng, Ye; Zhou, Shuangbiao; Zheng, Yixiong; Ma, Shiwei; Tang, Ronghua; Varshney, Rajeev K; Zhuang, Weijian

    2016-02-01

    Calcium is a universal signal in the regulation of wide aspects in biology, but few are known about the function of calcium in the control of early embryo development. Ca(2+) deficiency in soil induces early embryo abortion in peanut, producing empty pods, which is a general problem; however, the underlying mechanism remains unclear. In this study, embryo abortion was characterized to be caused by apoptosis marked with cell wall degradation. Using a method of SSH cDNA libraries associated with library lift (SSHaLL), 62 differentially expressed genes were isolated from young peanut embryos. These genes were classified to be stress responses, catabolic process, carbohydrate and lipid metabolism, embryo morphogenesis, regulation, etc. The cell retardation with cell wall degradation was caused by up-regulated cell wall hydrolases and down-regulated cellular synthases genes. HsfA4a, which was characterized to be important to embryo development, was significantly down-regulated under Ca(2+) -deficient conditions from 15 days after pegging (DAP) to 30 DAP. Two AhCYP707A4 genes, encoding abscisic acid (ABA) 8'-hydroxylases, key enzymes for ABA catabolism, were up-regulated by 21-fold under Ca(2+) -deficient conditions upstream of HsfA4a, reducing the ABA level in early embryos. Over-expression of AhCYP707A4 in Nicotiana benthamiana showed a phenotype of low ABA content with high numbers of aborted embryos, small pods and less seeds, which confirms that AhCYP707A4 is a key player in regulation of Ca(2+) deficiency-induced embryo abortion via ABA-mediated apoptosis. The results elucidated the mechanism of low Ca(2+) -induced embryo abortion and described the method for other fields of study.

  5. LD-RTPCR:\tA NEW METHOD FOR LABELLING TRACE cDNA MICROARRAY PROBE

    Institute of Scientific and Technical Information of China (English)

    范保星; 孙敬芬; 梁好; 王升启; 周平坤; 吴德昌

    2002-01-01

    Objective: To explore the usefulness of long distance reverse transcript combining linear amplification (LD-RTPCR) in labeling slight trace probe used for cDNA microarray. Methods: Total RNA from BEP2D cells was extracted and labeled by two different methods, LD-RTPCR with Cy3-dCTP as fluorescent dye and traditionally used RNA reverse transcript (RT) with Cy5-dCTP as fluorescent dye. Then, the probes labeled by two methods were mixed equally and hybridized with the cDNA microarray. Results: Scan and analysis of the microarray showed that the two methods labeled probes had consistent results. Conclusion: LD-RTPCR was proved useful for labeling cDNA microarray probe, especially for limited RNA material.

  6. Profiling gene expression patterns of nasopharyngeal carcinoma and normal nasopharynx tissues with cDNA microarray

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    5 μg of total RNAs from normal nasopharynx and nasopharyngeal carcinoma tissue have been labeled with α-32P-dCTP during reverse transcription. The synthesized cDNA probes have been hybridized to high-density cDNA microarray containing 5184 genes or expression sequence tags (ESTs). Then image analysis software has been applied to comparing their expression profiles. Results show that 187 ESTs were of density value above 200 in nasopharyngeal carcinoma tissue while there were 307 such ESTs in normal nasopharynx tissue; 38 ESTs were strongly expressed in nasopharynx, but weakly expressed in nasopharyngeal carcinoma; 48 ESTs were strongly expressed in nasopharyngeal carcinoma, but weakly expressed in normal nasopharynx. These results suggest that there may exist some new differentially expressed genes involved in nasopharyngeal carcinoma development. Furthermore, the results strongly indicate that high-density cDNA microarray is a powerful and efficient tool for large-scale screening differentially expressed genes.

  7. SENSE shimming (SSH): A fast approach for determining B(0) field inhomogeneities using sensitivity coding.

    Science.gov (United States)

    Splitthoff, D N; Zaitsev, M

    2009-11-01

    The pursuit of ever higher field strengths and faster data acquisitions has led to the construction of coil arrays with high numbers of elements. With the sensitivity encoding (SENSE) technique, it has been shown that the sensitivity of those elements can be used for spatial image encoding. Here, a proof-of-principle is presented of a method that can be considered an extreme case of the SENSE approach, completely abstaining from using encoding gradients. The resulting sensitivity encoded free-induction decay (FID) data are then not used for imaging, but for determining B(0) field inhomogeneity distribution. The method has therefore been termed "SENSE shimming" (SSH). In phantom experiments the method's ability to detect inhomogeneities of up to the second order is demonstrated.

  8. Using ssh and sshfs to virtualize Grid job submission with RCondor

    Science.gov (United States)

    Sfiligoi, I.; Dost, J. M.

    2014-06-01

    The HTCondor based glideinWMS has become the product of choice for exploiting Grid resources for many communities. Unfortunately, its default operational model expects users to log into a machine running a HTCondor schedd before being able to submit their jobs. Many users would instead prefer to use their local workstation for everything. A product that addresses this problem is RCondor, a module delivered with the HTCondor package. RCondor provides command line tools that simulate the behavior of a local HTCondor installation, while using ssh under the hood to execute commands on the remote node instead. RCondor also interfaces with sshfs, virtualizing access to remote files, thus giving the user the impression of a truly local HTCondor installation. This paper presents a detailed description of RCondor, as well as comparing it to the other methods currently available for accessing remote HTCondor schedds.

  9. Discovery and analysis of pancreatic adenocarcinoma genes using cDNA microarrays

    Institute of Scientific and Technical Information of China (English)

    Gang Jin; Xian-Gui Hu; Kang Ying; Yan Tang; Rui Liu; Yi-Jie Zhang; Zai-Ping Jing; Yi Xie; Yu-Min Mao

    2005-01-01

    AIM: To study the pathogenetic processes and the role of gene expression by microarray analyses in expediting our understanding of the molecular pathophysiology of pancreatic adenocarcinoma, and to identify the novel cancer-associated genes.METHODS: Nine histologically defined pancreatic head adenocarcinoma specimens associated with clinical data were studied. Total RNA and mRNA were isolated and labeled by reverse transcription reaction with Cy5 and Cy3 for cDNA probe. The cDNA microarrays that represent a set of 4 096 human genes were hybridized with labeled cDNA probe and screened for molecular profiling analyses.RESULTS: Using this methodology, 184 genes were screened out for differences in gene expression level after nine couples of hybridizations. Of the 184 genes,87 were upregulated and 97 downregulated, including 11 novel human genes. In pancreatic adenocarcinoma tissue, several invasion and metastasis related genes showed their high expression levels, suggesting that poor prognosis of pancreatic adenocarcinoma might have a solid molecular biological basis.CONCLUSION: The application of cDNA microarray technique for analysis of gene expression patterns is a powerful strategy to identify novel cancer-associated genes, and to rapidly explore their role in clinical pancreatic adenocarcinoma. Microarray profiles provide us new insights into the carcinogenesis and invasive process of pancreatic adenocarcinoma. Our results suggest that a highly organized and structured process of tumor invasion exists in the pancreas.

  10. Cloning and expression of a cDNA for mouse prostaglandin E receptor EP2 subtype.

    Science.gov (United States)

    Honda, A; Sugimoto, Y; Namba, T; Watabe, A; Irie, A; Negishi, M; Narumiya, S; Ichikawa, A

    1993-04-15

    A functional cDNA clone encoding mouse EP2 subtype of prostaglandin (PG) E receptor was isolated from a mouse cDNA library by cross-hybridization with the mouse EP3 subtype PGE receptor cDNA. The mouse EP2 receptor consists of 513 amino acid residues with putative seven-transmembrane domains. In contrast to EP3 receptor, this receptor possesses long third intracellular loop and carboxyl-terminal tail. [3H] PGE2 specifically bound to the membrane of mammalian COS cells transfected with the cDNA. The binding to the membrane was displaced with unlabeled PG in the order of PGE2 = PGE1 > iloprost > or = PGF2 alpha > or = PGD2. The binding was also inhibited by misoprostol, an EP2 and EP3 agonist, but not by sulprostone, an EP1 and EP3 agonist, and SC-19220, an EP1 antagonist. PGE2 markedly increased cAMP level in COS cells transfected with the cDNA. These results suggest that this receptor is EP2 subtype. Northern blot analysis demonstrated that the EP2 mRNA is widely expressed in various tissues, the abundant expression being observed in ileum, thymus, and mastocytoma P-815 cells.

  11. Identification of Differentially Expressed Genes in Metastatic and Non-Metastatic Nasopharyngeal Carcinoma Cells by Suppression Subtractive Hybridization

    Directory of Open Access Journals (Sweden)

    Xu-Yu Yang

    2005-01-01

    Full Text Available Background & Objective: Nasopharyngeal carcinoma (NPC is an epithelial neoplasm with high occurrence rates in southern China. The disease often metastasizes to regional lymphnodes at a very early stage. Local recurrences and metastasis occur frequently in patients with NPC and are a leading cause of death, despite improvements on treatment modalities. The molecular mechanism underlying the metastasis of nasopharyngeal carcinoma remains poorly understood, however, and requires additional elucidation. The aim of this study was to explore possible NPC gene candidates that may play key roles in NPC metastasis. Methods: Subtractive suppression hybridization (SSH was performed to isolate differentially expressed clones between the metastatic 5-8F and non-metastatic 6-10B nasopharyngeal carcinoma cell lines. Differentially expressed clones were screened and confirmed by reverse Northern blotting. The sequences of cDNA fragments were subsequently analyzed and compared to known sequences in Genbank. Results & Discussion: The SSH library contained thousands of positive clones. Random analysis of 300 clones by PCR demonstrated that 269 clones contained inserted fragments. Reverse Northern blot confirmed that 20 out of 192 clones examined were significantly up-regulated in the 5-8F cell line. Among these 20 clones, 16 were previously identified genes (flotilin-2, ezrin, pim-3, fli-1, mel, neugrin, znf216, ASB1, raly, UBE2A, keratin6A, TMED7, EIF3S9, FTL, two ribosomal proteins RPL21 and RPL16, two were predicted genes (c9orf74 and MDS006, and two sequences shared no homology with known genes listed in GenBank and may represent novel genes. The proposed functions of the genes identified in this study include cell signal transduction, cell survival, transcription regulation, cell mobility, protein synthesis, and DNA damage repair. Flotillin-2, fli-1, pim-3 and ezrin have previously been reported to be associated with tumor metastasis and progression. The

  12. Identification and cloning of the cDNA of a Rb-associated protein RAP140a

    Institute of Scientific and Technical Information of China (English)

    李权; 闻宏; 敖世洲

    2000-01-01

    Rb exerts important physiological functions in cell-cycle control, gene expression, cell differentiation, apoptosis, development and tumorigenesis by interacting with many cellular proteins. Using human partial Rb as bait, we screened a human fetal brain cDNA library through yeast two-hybrid system and obtained six novel cDNA fragments. Among them, one cDNA fragment corresponds to two different transcripts, 7 kb and 9 kb in Northern blot analysis. These two transcripts showed uniform distribution in various human tissues. We cloned the full-length cDNA of a 7.2 kb transcript through three times PCR amplifications. It was named RAP140a and predicted to encode a 1 233 amino acids hydrophilic protein. RAP140a was mapped to chromosome 3p13-p14.1. RAP140a may be functionally related to the intracellular translocation of Rb or other proteins.

  13. Identification and cloning of the cDNA of a Rb-associated protein RAP140a

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Rb exerts important physiological functions in cell-cycle control, gene expression, cell differentiation, apoptosis, development and tumorigenesis by interacting with many cellular proteins. Using human partial Rb as bait, we screened a human fetal brain cDNA library through yeast two-hybrid system and obtained six novel cDNA fragments. Among them, one cDNA fragment corre-sponds to two different transcripts, 7 kb and 9 kb in Northern blot analysis. These two transcripts showed uniform distribution in various human tissues. We cloned the full-length cDNA of a 7.2 kb transcript through three times PCR amplifications. It was named RAP140a and predicted to encode a 1 233 amino acids hydrophilic protein. RAP140a was mapped to chromosome 3p13-p14.1. RAP140a may be functionally related to the intracellular translocation of Rb or other proteins.

  14. Identification of two novel nodule-specific genes from Astragalus sinicus L. by suppressive subtractive hybridization

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    To identify the genes involved in nodule formation and to increase usable molecular probes, a cDNA library of Astragalus sinicus genes specifically expressed in infected roots by Mesorhizobium huakuii 7653R is generated using a PCR-based suppressive subtractive hybridization (SSH) technique with two mRNA populations of infected and uninfected control roots. Two nodule-specific genes, AsIIC259 and AsG2511, are identified from infected roots of A. sinicus. The amino acid sequences deduced from the open reading frames (ORFs) reveal that AsIIC259 and AsG2511 encodes a polypeptide with 134 and 58 amino acids, respectively. A signal peptide sequence is predicted with high probability at the N-termini of the AsIIC259 and AsG2511. The motif searches show that the deduced polypeptide of AsIIC259 contains two N-glycosylation sites, a cAMP- and cGMP-dependent protein kinase phosphorylation site and a casein kinase II phosphorylation site. BLASTP searches reveal that AsIIC259 putative protein displays a low degree of similarity to a unique nodulin from Lupinus luteus nodules. No significant identity is displayed over the predicted polypeptides of AsG2511 with any published sequences. Virtual Northern blot and semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) analyses indicate that the two genes are expressed exclusively in inoculated roots and that their expression is 2-4 d later than that of the leghaemoglobin (Lb) gene during nodule development.

  15. Cloning of human brevican cDNA and expression of its mRNA in human glioma

    Institute of Scientific and Technical Information of China (English)

    韩唏; 董艳; 由振东; 何成; 卢亦成

    2003-01-01

    Objective:To clone the cDNA of human brevican secreting isoform and to investigate its mRNA expression in human glioma.Methods:The full-length cDNA of human brevican secreted isoform was cloned from a human ahaplastic astrocytoma by RT-PCR,and the expression of human brevican mRNA in 22 cases of human glioma and 13 cases of non-glial brain tumors were investigated by in situ hybridization.Results:The cDNA which including the whole open reading frame of human brevican secreted isoform was obtained.In situ hybridization showed that brevican positive cells were present in all of the 22 cases of gliomas(100%),whereas none were found in the 13 cases of non-glial and metastasis brain tumors examined.Conclusion:The results suggest that brevican mRNA is highly and specifically expressed in human glioma.

  16. Suppression subtractive hybridization profiles of radial growth phase and metastatic melanoma cell lines reveal novel potential targets

    Directory of Open Access Journals (Sweden)

    Espreafico Enilza M

    2008-01-01

    Full Text Available Abstract Background Melanoma progression occurs through three major stages: radial growth phase (RGP, confined to the epidermis; vertical growth phase (VGP, when the tumor has invaded into the dermis; and metastasis. In this work, we used suppression subtractive hybridization (SSH to investigate the molecular signature of melanoma progression, by comparing a group of metastatic cell lines with an RGP-like cell line showing characteristics of early neoplastic lesions including expression of the metastasis suppressor KISS1, lack of αvβ3-integrin and low levels of RHOC. Methods Two subtracted cDNA collections were obtained, one (RGP library by subtracting the RGP cell line (WM1552C cDNA from a cDNA pool from four metastatic cell lines (WM9, WM852, 1205Lu and WM1617, and the other (Met library by the reverse subtraction. Clones were sequenced and annotated, and expression validation was done by Northern blot and RT-PCR. Gene Ontology annotation and searches in large-scale melanoma expression studies were done for the genes identified. Results We identified 367 clones from the RGP library and 386 from the Met library, of which 351 and 368, respectively, match human mRNA sequences, representing 288 and 217 annotated genes. We confirmed the differential expression of all genes selected for validation. In the Met library, we found an enrichment of genes in the growth factors/receptor, adhesion and motility categories whereas in the RGP library, enriched categories were nucleotide biosynthesis, DNA packing/repair, and macromolecular/vesicular trafficking. Interestingly, 19% of the genes from the RGP library map to chromosome 1 against 4% of the ones from Met library. Conclusion This study identifies two populations of genes differentially expressed between melanoma cell lines from two tumor stages and suggests that these sets of genes represent profiles of less aggressive versus metastatic melanomas. A search for expression profiles of melanoma in

  17. [The effect of SSH&H on the lifespan and spontaneous cancer development in transgenic mice with HER-2/neu mutation].

    Science.gov (United States)

    Tyndyk, M L; Popovich, I G; Anikin, I V; Egormin, P A; Iurova, M N; Zabezhinskiĭ, M A; Anisimov, V N

    2012-01-01

    10 months old mice receiving SSH&H with daily food increased the lifespan in comparison to the control group. The maximal lifespan was increased by 1,6 months. For the long-living 10% group the mean lifespan increased by 8,7% compared to the control group (pSSH&H on the neoplastic rate in transgenic mice with HER-2/neu mutation.

  18. Suppression substractive hybridisation (SSH) and real time PCR reveal differential gene expression in the Pacific cupped oyster, Crassostrea gigas, challenged with Ostreid herpesvirus 1

    OpenAIRE

    2011-01-01

    Virus-induced genes were identified using suppression subtractive hybridisation (SSH) from Pacific cupped oyster, Crassostrea gigas, haemocytes challenged by OsHV-1. A total of 304 clones from SSH forward library were sequenced. Among these sequences, some homologues corresponded to (i) immune related genes (macrophage express protein, IK cytokine, interferon-induced protein 44 or multicopper oxidase), (ii) apoptosis related genes (Bcl-2) and (iii) cell signalling and virus receptor genes (gl...

  19. Správa veřejných klíčů SSH v programech FreeIPA a SSSD

    OpenAIRE

    Cholasta, Jan

    2012-01-01

    SSH je jeden z nejpoužívanějších protokolů pro vzdálený přístup v Internetu. SSH je flexibilní a rozšiřitelný protokol, který se skládá ze tří hlavních součástí: SSH transportního protokolu, který obstarává důvěrnost, integritu a autentizaci serveru, SSH autentizačního protokolu, který obstarává autentizaci uživatelů a SSH spojovacího protokolu, který obstarává multiplexování více kanálů různých typů (interaktivní sezení, přesměrování TCP/IP spojení, atd.) do jednoho spojení. OpenSSH je jedna...

  20. SSH & the City. A Network Approach for Tracing the Societal Contribution of the Social Sciences and Humanities for Local Development

    Energy Technology Data Exchange (ETDEWEB)

    Robinson-Garcia, N.; Van Leeuwen, T.; Rafols, I.

    2016-07-01

    Current evaluation frameworks in research policy were designed to address: 1) life and natural sciences, 2) global research communities, and; 3) scientific impact. This is problematic, as they do not adapt well to SSH scholarship, to local interests, or to consider broader societal impacts. This paper discusses three different evaluation frameworks and proposes a methodology to operationalize them and capture societal interactions between social sciences and humanities (SSH) researchers and their local context. To capture such interactions, we propose the use of social media and web-link analysis to identify interactions between academics and local stakeholders. We consider that the power of these tools is not so much on understanding their meaning as ‘acts’ to develop impact or visibility metrics whenever a mention to a research article is made, but as proxies for personal interactions. We offer some examples of the expected social networks we aim at developing for two Spanish cities: Granada and Valencia. (Author)

  1. Cloning, expression and mapping of the full-length cDNA of human CCTβ subunit

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Chaperonins assist the proper folding of target proteins without being a part of the substrates. The eukaryotic cytosolic chaperonin, CCT-Chaperonin Containing TCP-1 (tailless complex polypeptide-1), is mainly involved in the formation of cytoskeletal proteins and is essential for cell viability. Mammalian CCT is commonly a protein complex composed of 7-9 subunit species. We have isolated a novel full-length cDNA from human testis cDNA library. This cDNA of 1935 bp contains a 1605 bp open reading frame (ORF) encoding 535 amino acids (aa). The deduced protein of the cDNA is highly homologous to the CCTβ subunit of saccharomyces cerevisiae, schizosaccharomyces pombe, caenorhabditis elegans and mouse, etc. Especially high homology (97%) is found between the deduced protein and mouse CCTb. On the basis of such high homology, the protein encoded by the new gene was proposed to be a human CCTβ subunit. Northern hybridization showed that human CCTβ gene is expressed as a transcript of about 2.0 kb in various tissues. Overexpression was seen in testis with the expression level 3-24 times of those in other tissues. The CCTβ gene was mapped to human chromosome 12q14 by Radiation Hybrid Mapping. Through homologous search, the 5′-end of the cDNA sequence was found to share intermittent regional homology with the 3′-end of human genomic sequence (U91327). The genomic structure of the 5′-end of CCTβ was also described in detail through comparative analysis.

  2. Yeast two-hybrid screen.

    Science.gov (United States)

    Makuch, Lauren

    2014-01-01

    Yeast two-hybrid is a method for screening large numbers of gene products (encoded by cDNA libraries) for their ability to interact with a protein of interest. This system can also be used for characterizing and manipulating candidate protein: protein interactions. Interactions between proteins are monitored by the growth of yeast plated on selective media.

  3. Characterization of expressed sequence tags obtained by SSH during somatic embryogenesis in Cichorium intybus L

    Directory of Open Access Journals (Sweden)

    Quillet Marie-Christine

    2007-06-01

    Full Text Available Abstract Background Somatic embryogenesis (SE is an asexual propagation pathway requiring a somatic-to-embryonic transition of differentiated somatic cells toward embryogenic cells capable of producing embryos in a process resembling zygotic embryogenesis. In chicory, genetic variability with respect to the formation of somatic embryos was detected between plants from a population of Cichorium intybus L. landrace Koospol. Though all plants from this population were self incompatible, we managed by repeated selfing to obtain a few seeds from one highly embryogenic (E plant, K59. Among the plants grown from these seeds, one plant, C15, was found to be non-embryogenic (NE under our SE-inducing conditions. Being closely related, we decided to exploit the difference in SE capacity between K59 and its descendant C15 to study gene expression during the early stages of SE in chicory. Results Cytological analysis indicated that in K59 leaf explants the first cell divisions leading to SE were observed at day 4 of culture. In contrast, in C15 explants no cell divisions were observed and SE development seemed arrested before cell reactivation. Using mRNAs isolated from leaf explants from both genotypes after 4 days of culture under SE-inducing conditions, an E and a NE cDNA-library were generated by SSH. A total of 3,348 ESTs from both libraries turned out to represent a maximum of 2,077 genes. In silico subtraction analysis sorted only 33 genes as differentially expressed in the E or NE genotype, indicating that SSH had resulted in an effective normalisation. Real-time RT-PCR was used to verify the expression levels of 48 genes represented by ESTs from either library. The results showed preferential expression of genes related to protein synthesis and cell division in the E genotype, and related to defence in the NE genotype. Conclusion In accordance with the cytological observations, mRNA levels in explants from K59 and C15 collected at day 4 of SE

  4. SSH-2 measurements of cirrus at 18-28 micrometers from the King Air during FIRE 2

    Science.gov (United States)

    Griffin, Michael K.

    1993-01-01

    In November of 1991, the First ISCCP (International Satellite Cloud Climatology Project) Regional Experiment (FIRE) Phase II cirrus study took place at Coffeyville, Kansas. The field experiment incorporated instrumentation from surface, aircraft, and satellite to attempt to define the optical, radiative, and microphysical characteristics of these high altitude, predominantly ice clouds. The NCAR King Air research aircraft was outfitted with a variety of radiative and microphysical instrumentation for the FIRE II project. Included for this project was the SSH-2, a 16-channel passive radiometer. The SSH-2 was originally designed as a space-qualified infrared (IR) temperature and water vapor sounder for deployment onboard the Defense Meteorological Satellite Program (DMSP) series of environmental satellites. For this experiment, only those channels associated with the water vapor profiling function have been examined although downwelling radiance measurements were taken at all channels during the project. With supporting information from the aircraft telemetry observations it may be possible to relate these SSH-2 measurements to cloud radiative and microphysical properties. The following sections will describe the spectral characteristics of the instrument, the calibration scheme used to convert the raw measured counts into calibrated radiances, and the case studies that will be covered in this paper. This will be followed by a discussion of the results of this preliminary investigation and a description of future work to be done.

  5. 应用抑制性差减杂交技术构建铁皮石斛差减cDNA文库的探讨%Construction of Subtractive cDNA Library from the Leaves of Dendrobium candidum Wall. ex Lindl. by Suppression Subtractive Hybridization Technology

    Institute of Scientific and Technical Information of China (English)

    魏小勇

    2005-01-01

    [目的]探讨构建铁皮石斛抑制差减文库的方法,为克隆石斛碱生物合成相关功能基因奠定基础.[方法]采用抑制性差减杂交(SSH)技术,分别以4年生和1年生铁皮石斛叶片互补脱氧核糖核酸(cDNA)作为检测子(tester)与驱赶子(driver),将所获得差减cDNA片段克隆入质粒表达载体(PointTMXa-1 T-Vector),转化大肠杆菌JM109,聚合酶链反应(PCR)扩增鉴定插入片段.[结果]成功地构建了与石斛碱生物合成相关的差减cDNA文库,获得的正、反向差减文库分别含560、220个重组子;插入片段的平均大小为550bp.[结论]所构建的差减cDNA文库适合进一步克隆分析石斛碱相关功能基因研究.

  6. Tailed pooled suppression subtractive hybridization (PSSH) adaptors do not alter efficiency.

    Science.gov (United States)

    Gerrish, Robert S; Gill, Steven R

    2010-11-01

    Suppression Subtractive Hybridization (SSH) and its derivative, Pooled Suppression Subtractive hybridization (PSSH), are powerful tools used to study variances larger than ~100 bp in prokaryotic genome structure. The initial steps involve ligating an oligonucleotide of known sequence (the "adaptor") to a fragmented genome to facilitate amplification, subtraction and downstream sequencing. SSH results in the creation of a library of unique DNA fragments which have been traditionally analyzed via Sanger sequencing. Numerous next generation sequencing technologies have entered the market yet SSH is incompatible with these platforms. This is due to the high level of sequence conservation of the oligonucleotide used for SSH. This rigid adherence is partly because it has yet to be determined if alteration of this oligonucleotide will have a deleterious impact on subtraction efficiency. The subtraction occurs when non-unique fragments are inhibited by a secondary self-pairing structure which requires exact nucleotide sequence. We determine if appending custom sequence to the 5' terminal ends of these oligonucleotides during the nested PCR stages of PSSH will reduce subtraction efficiency. We compare a pool of ten S. aureus clinical isolates with a standard PSSH and custom tailed-PSSH. We detected no statistically significant difference between their subtraction efficiencies. Our observations suggest that the adaptor's terminal ends may be labeled during the nested PCR step. This produces libraries labeled with custom sequence. This does not lead to loss of subtraction efficiency and would be invaluable for groups wishing to combine SSH or PSSH with their own downstream applications, such as a high throughput sequencing platform.

  7. Lectin cDNA and transgenic plants derived therefrom

    Science.gov (United States)

    Raikhel, Natasha V.

    2000-10-03

    Transgenic plants containing cDNA encoding Gramineae lectin are described. The plants preferably contain cDNA coding for barley lectin and store the lectin in the leaves. The transgenic plants, particularly the leaves exhibit insecticidal and fungicidal properties.

  8. Lysosomal {beta}-mannosidase: cDNA cloning and characterization

    Energy Technology Data Exchange (ETDEWEB)

    Chen, H.; Leipprandt, J.R.; Traviss, C.E. [Michigan State Univ., East Lansing, MI (United States)] [and others

    1994-09-01

    Lysosomal {beta}-mannosidase is an exoglycosidase that cleaves the single {beta}-linked mannose residue from the non-reducing end of all N-linked glycoprotein oligosaccharides. Deficiency of this enzyme results in {beta}-mannosidosis, a severe neurodegenerative disease in goats and cattle. The human cases described have a milder, highly variable presentation. Study of the molecular pathology of this disease in ruminants and humans and development of the animal model for gene therapy studies required cloning of the gene for {beta}-mannosidase has been cloned. {beta}-Mannosidase cDNA were obtained from a bovine thyroid cDNA library by screening with mixed oligonucleotides derived from peptide sequences resulting from microsequencing of bovine {beta}-mannosidase peptides. A total of six independent positive clones were identified from 5 x 10{sup 5} plaques, covering about 80% of the C-terminal region. The missing 5{prime} region was obtained using 5{prime} RACE. The full-length construct contains 3852-bp nucleotides, encoding 879 amino acids. The initiation codon is followed by 17 amino acids containing the characteristics of a typical signal peptide sequence. The deduced amino acid sequence is colinear with all peptide sequences determined by protein microsequencing. Northern blot analysis demonstrated a 4.2 kb single transcript in various tissues from both normal and affected goats and calves. The mRNA level was decreased in affected {beta}-mannosidosis animals. The gene encoding {beta}-mannosidase was localized on human chromosome 4 by Southern analysis of rodent/human somatic cell hybrids. The mutation in bovine {beta}-mannosidosis has been identified. This is the first report of cloning of the {beta}-mannosidase gene.

  9. Screening of proteins binding to mouse cytomegalovirus M122 protein from mouse brain cDNA library by yeast two-hybrid system%酵母双杂交技术筛选小鼠脑cDNA文库中鼠巨细胞病毒即刻早期蛋白M122相互作用蛋白的研究

    Institute of Scientific and Technical Information of China (English)

    王慧; 周玉峰; 舒赛男; 罗丹; 田佳; 张慧娟; 杜小弋; 方峰

    2010-01-01

    histone acetyltransferase(monocytic leukemia)4,Myst4]、透明质酸和蛋白多糖连接蛋白1(hyaluronan and proteoglycan link protein 1,Hapln1)、自噬相关蛋白3(autophagy-related 3,Atg3)、精氨酸/色氨酸富集的剪切因子5(splicing factor,arginine/serine-rich 5,Sfrs5)、C3HC型锌指蛋白(zinc finger,C3HC-type containing 1,Zc3hc1)、硫氧还蛋白相关的跨膜蛋白1(thioredoxin-related transmembrane protein 1,Txndc1)、接头蛋白复合物AP-1亚单位(adaptor protein complex AP-1,gamma 1 subunit,Aplg1)和Cul1蛋白(cullin 1,Cul1).回返验证实验进一步证实这些蛋白与M122蛋白能够在酵母细胞AH109发生相互作用,但Aplg1和Cul1被证实具有自激活作用.结论 筛选到的其中19种已知基因编码的蛋白可能与巨细胞病毒的致病机制相关,但仍需进一步的验证.%Objective To investigate the biological function of M122 in pathogenesis of MCMV in developmental brain disorders and brain damage, screening for mouse brain cDNA library interacting with M122 was performed by a yeast two-hybrid system. Methods The reconstructed bait plasmid pGBKT7-M122 was transformed into yeast cells AH109 and screened on the nutrient deficiency medium SD/-Trp. After express of the bait protein in AH109 yeast strains was detected by Western blot analysis, yeast-two hybrid screening was performed by mating AH109 with Y187 containing mouse brain cDNA library plasmid. The diploid yeast cells were plated on the nutrient deficiency medium SD/-Trp/-Leu/-His/-Ade. The second screening was performed with SD/-Trp/-Leu/-His/-Ade containing X-α-gal. The plasmids in positive colonies were extracted and transformed into E. coli JM109 cells. After plasmid DNA in JM109 cells were extracted form positive colonies and sequenced, the results were analyzed by bioinformatic methods. The interactions between M122 protein and the protein obtained from positive colonies were further confirmed by repeating yeast-two hybrid. Then, autoactivations of

  10. Event Detection and Visualization of Ocean Eddies based on SSH and Velocity Field

    Science.gov (United States)

    Matsuoka, Daisuke; Araki, Fumiaki; Inoue, Yumi; Sasaki, Hideharu

    2016-04-01

    Numerical studies of ocean eddies have been progressed using high-resolution ocean general circulation models. In order to understand ocean eddies from simulation results with large amount of information volume, it is necessary to visualize not only distribution of eddies of each time step, but also events or phenomena of eddies. However, previous methods cannot precisely detect eddies, especially, during the events such as eddies' amalgamation, bifurcation. In the present study, we propose a new approach of eddy's detection, tracking and event visualization based on sea surface height (SSH) and velocity field. The proposed method detects eddies region as well as streams and currents region, and classifies detected eddies into several types. By tracking the time-varying change of classified eddies, it is possible to detect not only eddies event such as amalgamation and bifurcation but also the interaction between eddy and ocean current. As a result of visualizing detected eddies and events, we succeeded in creating the movie which enables us to intuitively understand the region of interest.

  11. Cloning of a cDNA for steroid sulfatase: frequent occurrence of gene deletions in patients with recessive X chromosome-linked ichthyosis

    Energy Technology Data Exchange (ETDEWEB)

    Bonifas, J.M.; Morley, B.J.; Oakey, R.E.; Kan, Y.W.; Epstein, E.J. Jr.

    1987-12-01

    A human steroid sulfatase cDNA 2.4 kilobases long was isolated from a human placental lambda gt11 cDNA expression library. The library was screened with monospecific rabbit antibodies elicited by injection of steroid sulfatase protein purified from human placentas. Hybridization of the cDNA with EcoRI-digested genomic DNA indicated that patients from 14 of 15 apparently unrelated families have gross deletions of the gene for steroid sulfatase. One patient had genomic DNA fragments that were identical to those from normal individuals, indicating the absence of any major deletions as the cause of his lack of steroid sulfatase enzyme activity.

  12. Determination of Mean Dynamic Topography (MDT) to Bridge Geoid and Mean Sea Surface Height (SSH) with a New Elliptic Equation

    Science.gov (United States)

    Chu, P. C.

    2016-12-01

    Mean dynamic topography (MDT, η) bridges the geoid and the mean sea surface (from satellite altimetry) and constrains large scale surface geostrophic circulations. It can be estimated from either satellite or underwater ocean temperature (T) and salinity (S) data. Satellite altimeter measures sea surface height (SSH) with high precision and unique resolution above a reference ellipsoid (not geoid). Two Gravity Recovery and Climate Experiment (GRACE) satellites launched in 2002, provide data to compute the marine geoid [called the GRACE Gravity Model (GGM)] (see website: http://www.csr.utexas.edu/grace/). The MDT is the difference of altimetry-derived mean SSH and the mean marine geoid (using GGM or pre-GRACE gravity model such as EGM96). A major difficulty arises that the spatial variations in mean SSH and marine geoid are approximately two orders of magnitude larger than the spatial variations in η.The second approach (using T, Sdata) is based on geostrophic balance, which is at the minimum energy state in the linear Boussinesq primitive equations with conservation of potential vorticity. In this paper, a new elliptic equation, -[∂x(gh/f2)∂xη+∂y(gh/f2)∂yη]+η = (g/f2)(∂C/∂x-∂B/∂y)is derived to determine MDT with H the water depth, g the gravitational acceleration, and coefficients (B, C) depend on 3D mean temperature (T) and salinity (S) data. Numerical approach transforms the elliptic equation into a set of well-posed linear algebraic equations of η at grid points. The solution for the North Atlantic Ocean (100oW-6oW, 7oN-72oN) on 1oX1ogrids with the coefficients (B, C) calculated from the three-dimensional (T, S) data of the NOAA National Centers for Environmental Information (NCEI) World Ocean Atlas 2013 version 2 (http://www.nodc.noaa.gov/OC5/woa13/woa13data.html) and H from the NOAA ETOPO5 (https://www.ngdc.noaa.gov/mgg/fliers/93mgg01.html), compares well with the difference (also considered as the MDT) between the time-averaged SSH and

  13. Human sulfotransferase SULT1C1: cDNA cloning, tissue-specific expression, and chromosomal localization

    Energy Technology Data Exchange (ETDEWEB)

    Her, Chengtao; Weinshilboum, R.M. [Mayo Foundation, Rochester, MN (United States); Kaur, G.P. [Temple Univ. Medical School, Philadelphia, PA (United States)] [and others

    1997-05-01

    We have isolated and sequenced a cDNA that encodes an apparent human orthologue of a rat sulfotransferase (ST) cDNA that has been referred to as {open_quotes}ST1C1{close_quotes} - although it was recently recommended that sulfotransferase proteins and cDNAs be abbreviated {open_quotes}SULT.{close_quotes} The new human cDNA was cloned from a fetal liver-spleen cDNA library and had an 888-bp open reading frame. The amino acid sequence of the protein encoded by the cDNA was 62% identical with that encoded by the rat ST1C1 cDNA and included signature sequences that are conserved in all cytosolic SULT enzymes. Dot blot analysis of mRNA from 50 human tissues indicated that the cDNA was expressed in adult human stomach, kidney, and thyroid, as well as fetal kidney and liver. Northern blot analyses demonstrated that the major SULT1C1 mRNA in those same tissues was 1.4 kb in length. We next determined the partial human SULT1C1 gene sequence for a portion of the 5{prime}-terminus of one intron. That sequence was used to design SULT1C1 gene-specific primers that were used to perform the PCR with DNA from human/rodent somatic cell hybrids to demonstrate that the gene was located on chromosome 2. PCR amplifications performed with human chromosome 2/rodent hybrid cell DNA as template sublocalized SULT1C1 to a region between bands 2q11.1 and 2q11.2. 14 refs., 2 figs.

  14. Identification of differentially expressed genes in uveal melanoma using suppressive subtractive hybridization

    Science.gov (United States)

    Landreville, Solange; Lupien, Caroline B.; Vigneault, Francois; Gaudreault, Manon; Mathieu, Mélissa; Rousseau, Alain P.; Guérin, Sylvain L.

    2011-01-01

    Purpose Uveal melanoma (UM) is the most common primary cancer of the eye, resulting not only in vision loss, but also in metastatic death. This study attempts to identify changes in the patterns of gene expression that lead to malignant transformation and proliferation of normal uveal melanocytes (UVM) using the Suppressive Subtractive Hybridization (SSH) technique. Methods The SSH technique was used to isolate genes that are differentially expressed in the TP31 cell line derived from a primary UM compared to UVM. The expression level of selected genes was further validated by microarray, semi-quantitative RT–PCR and western blot analyses. Results Analysis of the subtracted libraries revealed that 37 and 36 genes were, respectively, up- and downregulated in TP31 cells compared to UVM. Differential expression of the majority of these genes was confirmed by comparing UM cells with UVM by microarray. The expression pattern of selected genes was analyzed by semi-quantitative RT–PCR and western blot, and was found to be consistent with the SSH findings. Conclusions We demonstrated that the SSH technique is efficient to detect differentially expressed genes in UM. The genes identified in this study represent valuable candidates for further functional analysis in UM and should be informative in studying the biology of this tumor. PMID:21647268

  15. 运用抑制消减杂交构建辐射诱导EST库%Suppression subtractive hybridization in construction of radiation-induced EST library

    Institute of Scientific and Technical Information of China (English)

    铁轶; 罗瑛; 隋建丽; 周平坤

    2001-01-01

    目的 克隆并鉴定低剂量辐射诱导基因,为探讨低剂量辐射生物学效应分子机理提供有益信息。方法 0.5 Gy γ射线照射人胚肺细胞后,用抑制消减杂交技术(Suppression Subtractive Hybridization,SSH)构建低剂量辐射诱导EST库。用反向Northern杂交法对库中的EST片段进行筛选,挑取阳性克隆测序,并在GenBank序列数据库中进行同源性比较。结果 得到受照后表达升高90个EST序列,代表21个基因的转录产物,部分基因的功能已明确。从功能上分析,这些基因产物的生物学作用涵盖了细胞周期、信号转导、细胞骨架、代谢、蛋白合成等几个重要的细胞生物学代谢反应过程,显示出细胞对低剂量辐射反应过程的多样性。另外,实验还获得4个新cDNA序列。结论 利用抑制消减杂交技术,从辐射前后HEL细胞中,得到低剂量辐射诱导EST片段,它们与细胞周期、增殖、代谢、信号转导、应激反应等相关,这些基因可能在细胞的低剂量辐射效应中起到重要作用。%Objective To clone and identify radiation-induced genes from 0.5 Gy γ-ray irradiated human embryo lung cells(HEL).The identification and functional studies of radiation-induced genes will prompt the elucidation of the molecular mechanisms of low dose radiation-induced biological effects.It will also profit the understanding of the basic processes of cellular metabolisms. Methods  A low dose radiation-induced differentially displaying EST library has been constructed by suppression subtractive hybridization from HEL cells irradiated with 0.5 Gy γ-rays.The EST library was screened by reverse Northern hybridization analysis.Positive clones were sequenced and the similarity was searched against the DNA database in GenBank. Results  Altogether 90 positive cDNA clones with increased expression after 0.5 Gy irradiation were identified which corresponded to 21 individual genes

  16. Screening of genes related to ovarian development in the swimming crab, Portunus trituberculatus, by suppression subtractive hybridization.

    Science.gov (United States)

    Yu, Z B; Mu, C K; Song, W W; Li, R H; Chen, Y E; Wang, C L

    2015-12-29

    The swimming crab, Portunus trituberculatus, is an important marine animal and is widely cultured in China. In the present study, suppression subtractive hybridization was applied to identify the differentially expressed genes in the ovaries of mature and immature P. trituberculatus. One hundred and seventy six expressed sequence tag (ESTs) were identified, of which 100 were down-regulated, and 76 up-regulated. BLAST analysis identified 51 unigenes, of which 27 were down-regulated, and 24 up-regulated. Quantitative real-time reverse transcriptase polymerase chain reaction results indicated that the SSH technique is valuable in screening genes related to ovarian development. Genes identified in this study encoded proteins corresponding to a wide range of functions and included immune response protein, transcription initiation factor, metabolic proteins, chromosome, histone h3, ovarian development-related protein, and vitellogenin. In addition, 64 metabolic pathways were annotated in differentially expressed ESTs by using the Kyoto Encyclopedia of Genes and Genomes pathway. Four annotated pathways (oxidative phosphorylation, carbon metabolism, fatty acid degradation, and protein digestion and absorption) appeared to be involved in ovarian development. In ontology analysis, 5.83% of the cellular process genes in reverse subtraction cDNA library are involved in reproduction, and 5.88% involved in developmental process. In up-regulated genes, myosin II-expressed polehole-like protein; histone h3; ovigerous-hair stripping substance; peritrophin 48; and ovarian development-related protein appeared to be involved in ovarian development. Identification of differentially expressed genes in the mature and immature ovary of the swimming crab provides new insights for further studies on the mechanism underlying ovarian development in this species.

  17. Molecular cloning and analysis of functional cDNA and genomic clones encoding bovine cellular retinoic acid-binding protein.

    OpenAIRE

    Shubeita, H E; Sambrook, J F; McCormick, A M

    1987-01-01

    A recombinant cDNA clone, pCRABP-HS1, encoding cellular retinoic acid-binding protein was isolated from a bovine adrenal cDNA library. COS-7 cells transfected with pCRABP-HS1 produced a biologically active retinoic acid-binding protein molecule of the expected molecular mass (15.5 kDa). RNA blot hybridization analysis using pCRABP-HS1 as a probe revealed a single 1050-nucleotide mRNA species in bovine adrenal, uterus, and testis, tissues that contain the highest levels of retinoic acid-bindin...

  18. Requirements in screening cDNA libraries for new genes and solutions offered by SBH technology

    Energy Technology Data Exchange (ETDEWEB)

    Drmanac, R.; Drmanac, S.; Labat, I.; Stavropoulos, N.

    1993-12-31

    Under different assumptions about the total number of genes, the number of housekeeping and tissue-specific genes, and the difference in the number of mRNAs per cell for functional and nonfunctional genes, significantly different results can be expected from screening random cDNA clones. We have developed gene expression models as a guide for interpretation of experimental results. For statistical, biological, and technical reasons, the search for 100,000 plus genes and discrimination between nonfunctional, housekeeping, and tissue-specific genes requires the analysis of up to 10 million clones from 20 to 50 tissues. Oligonucleotide hybridization of dense clone blots is an inexpensive and fast way to screen such large clone sets. Our preliminary results on control clones and thousands of cDNA clones from an infant brain library demonstrate the feasibility of the method. We present several models of gene expression and analyze the main factors which can influence the hunt for new genes via the screening of random cDNA libraries. The basic steps in the preparation and use of dense DNA dot arrays are described, and some results that demonstrate the feasibility and efficiency of gene inventorying by oligonucleotide hybridization are presented. Furthermore, partial SBH and single-pass gel sequencing are compared and a gene analysis scheme that combines the two approaches is discussed.

  19. Construction and analysis of gonad suppression subtractive hybridization library of rice field eel(Monopterus albus)%黄鳝性腺抑制差减文库的构建和分析

    Institute of Scientific and Technical Information of China (English)

    曲宪成; 尚晓莉; 程翠; 曲学伟; 张勇; 张开岳; 蒋骄云

    2011-01-01

    By using suppression subtractive hybridization (SSH), subtracted cDNA libraries were constructed from rice field eel (Monopterus albus) gonad at Ⅳ ovary and ovotestis stage. Two-directional (forward and backward) SSH was performed under cDNAs of rice field eel gonad at Ⅳ ovary and ovotestis stage. The cDNA fragments were inserted into plasmid vectors, and then transformed into Escherichia coli DHSα. Finally, forward and backward subtracted cDNA libraries (461 and 674 clones, respectively) were obtained. By PCR detection, length of the subtractive cDNA fragments cloned into the vector ranged from 200 bp to 2 000 bp. One hundred positive clones were selected and sequenced randomly from the forward and the backward libraries, and ninety gene fragment sequences were obtained. EST analysis of these sequences was performed with Blastx and Blaxtn by comparing sequences with GenBank database. Two sequenced genes were further analyzed by semi-quantitative RT-PCR method. The results showed that the library can enrich differentially expressed genes in the two phases gonad.%应用抑制性差减杂交技术构建了黄鳝(Monopterus albus)Ⅳ期卵巢和间期性腺的差减cDNA文库.正向差减杂交以间期性腺为试验方,Ⅳ期卵巢为驱动方;反向差减杂交以Ⅳ期卵巢为试验方,间期性腺为驱动方.将获得的差减cDNA片段连接质粒载体,然后转化大肠杆菌DH5α,最后获得的正、反向差减文库分别含461、678个重组子.经PCR扩增鉴定插入片段范嗣为200~2000 bp.随机选取正、反文库共100个阳性克隆测序分析,得到90个有效基因片段,与GenBank进行BLASTx和BLASTn同源比对.进一步从文库中选取2个基因用于半定量RT-PCR,对文库质量进行验证.结果表明,所建文库能够达到富集这两期性腺差异表达基因的目的.黄鳝性腺差减文库的构建,为进一步分离、鉴定黄鳝性腺发育和性逆转的相关基因奠定了基础.

  20. Molecular cloning of GA-suppressed G2 pea genes by cDNA RDA

    Institute of Scientific and Technical Information of China (English)

    朱玉贤; 张翼凤; 李慧英

    1997-01-01

    GA-treated and non-treated G2 pea cDNAs were compared using a newly developed method called cDNA representational difference analysis (cDNA-RDA), and several GA-suppressed mRNAs were found. After cloning of the larger fragments PGAS1-3 ( pea GA-suppressed cDNA 1-3), they were demonstrated to be expressed only in pea tissue not treated with GA3 through Northern analysis. Compared with subtractive hybridization and differ-ential display techniques, this method not only can be easily manipulated but also has a relatively low rate of false posi-tive and is highly repetitive. It is the major progress in molecular cloning techniques.

  1. PvuII RFLP detected by a human. beta. ADH cDNA probe

    Energy Technology Data Exchange (ETDEWEB)

    Parsian, A.; Burgess, A.K.; Khan, M.A.; Devor, E.J. (Washington Univ. School of Medicine, St. Louis, MO (USA))

    1989-12-11

    A 0.97 kb cDNA (ADH12) fragment encoding human exons 7, 8, 9 of the ADH{sub 2} gene was isolated from an adult human liver cDNA library. The insert can be excised by Pst I digestion. Pvu II identifies a two-allele polymorphism with bands at 4.4 kb (A{sub 1}) and 3.0 kb (A{sub 2}) and invariant bands at 5.1, 4.0, 2.8, and 2.3 kb. It was localized on Chromosome 4q21-q25 by in situ hybridization. Co-dominant segregation was observed in 18 informative families.

  2. Cloning and expression of full-length cDNA encoding human vitamin D receptor

    Energy Technology Data Exchange (ETDEWEB)

    Baker, A.R.; McDonnell, D.P.; Hughes, M.; Crisp, T.M.; Mangelsdorf, D.J.; Haussler, M.R.; Pike, J.W.; Shine, J.; O' Malley, B.W. (California Biotechnology Inc., Mountain View (USA))

    1988-05-01

    Complementary DNA clones encoding the human vitamin D receptor have been isolated from human intestine and T47D cell cDNA libraries. The nucleotide sequence of the 4605-base pair (bp) cDNA includes a noncoding leader sequence of 115 bp, a 1281-bp open reading frame, and 3209 bp of 3{prime} noncoding sequence. Two polyadenylylation signals, AATAAA, are present 25 and 70 bp upstream of the poly(A) tail, respectively. RNA blot hybridization indicates a single mRNA species of {approx} 4600 bp. Transfection of the cloned sequences into COS-1 cells results in the production of a single receptor species indistinguishable from the native receptor. Sequence comparisons demonstrate that the vitamin D receptor belongs to the steroid-receptor gene family and is closest in size and sequence to another member of this family, the thyroid hormone receptor.

  3. Characterization of a cDNA clone encoding the carboxy-terminal domain of a 90-kilodalton surface antigen of Trypanosoma cruzi metacyclic trypomastigotes.

    OpenAIRE

    1993-01-01

    We have cloned and sequenced a cDNA for a metacyclic trypomastigote-specific glycoprotein with a molecular mass of 90 kDa, termed MTS-gp90. By immunoblotting, antibodies to the MTS-gp90 recombinant protein reacted exclusively with a 90-kDa antigen of metacyclic trypomastigotes. The insert of the MTS-gp90 cDNA clone strongly hybridized with a single 3.0-kb mRNA of metacyclic forms, whereas the hybridization signal with epimastigote mRNA was weak and those with RNAs from other developmental sta...

  4. Isolation of EF1gamma from calli regenerating SSH library in Maize (Zea mays).

    Science.gov (United States)

    Xia, Y L; Ding, J; Zhang, Z M; Rong, T Z; Shi, L Y; Pan, G T

    2007-12-01

    18599Hong, a good Maize (Zea mays) inbred line as well as good transformation acceptor with high regeneration capacity, was used for isolating embryonic callus regeneration genes. Subtractive library was constructed by Suppression subtractive hybridization and screened by Reverse Northern Hybridization. The clones of No. 27 was randomly picked to sequence. NCBI blastx results showed the similarity to elongation factor 1gamma in rice.

  5. RAPID SCREENING OF AN ARRAYED cDNA LIBRARY BY IMPROVED PCR-BASED METHOD

    Institute of Scientific and Technical Information of China (English)

    杜光伟; 潘美辉; 袁建刚; 周彦; 强伯勤; 梁植权

    1998-01-01

    Tbe present study reports an improved PCR-based technique that allows quick and effecfive screening of eDNA libraries. First, the eDNA library was arrayed as follows: about 3×106 cDNA clones were multiplied as individual plsques on solid medium in 24-well culture dishes at 1 200 plaque forming units per well.The phage suspension of each well was transferred to an individual micrccentrifuge tube in 72-tube box. Then,box pool, row pools and column pools were set up that respectively represent a 72-tube box,rows and columns within the box. To screen a specific target cDNA,primers specific for novel ESTs obtained in our laboratory were eznployed to conduct PCR in a fiierarchy mode. PCR began with the box pools, resulting in the identification of some positive box pools. Then PCR went down to the row and column pools of the positive box. Tbe intersection of the positive row(s) and column(s) revealed the candidate positive tubes. The specificity of PCR products were meanwhile checked hy restriction enzyme digestion. Finally, hybridization was carried out to get single specific eDNA clones-from the positive tlabes. This PCR-hased technique features high specificity, high efficiency and Les-cost in large-scale cDNA library screening. Our initial implementation of the technique resulted in the isolation of three longer different cDNA clones from a hnman fetal brain eDNA library. Thus this improved technique can serve as an alternative to the time-consuming and laborious conventional hybridization-hased metfiod for screening cDNA library.

  6. Cloning and expression of a cDNA encoding human sterol carrier protein 2

    Energy Technology Data Exchange (ETDEWEB)

    Yamamoto, Ritsu; Kallen, C.B.; Babalola, G.O.; Rennert, H.; Strauss, J.F. III (Univ. of Pennsylvania School of Medicine, Philadelphia (United States)); Billheimer, J.T. (E.I. DuPont de Nemours, Inc., Wilmington, DE (United States))

    1991-01-15

    The authors report the cloning and expression of a cDNA encoding human sterol carrier protein 2 (SCP{sub 2}). The 1.3-kilobase (kb) cDNA contains an open reading frame which encompasses a 143-amino acid sequence which is 89% identical to the rat SCP{sub 2} amino acid sequence. The deduced amino acid sequence of the polypeptide reveals a 20-residue amino-terminal leader sequence in front of the mature polypeptide, which contains a carboxyl-terminal tripeptide (Ala-Lys-Leu) related to the peroxisome targeting sequence. The expressed cDNA in COS-7 cells yields a 15.3-kDa polypeptide and increased amounts of a 13.2-kDa polypeptide, both reacting with a specific rabbit antiserum to rat liver SCP{sub 2}. The cDNA insert hybridizes with 3.2- and 1.8-kb mRNA species in human liver poly(A){sup +} RNA. In human fibroblasts and placenta the 1.8-kb mRNA was most abundant. Southern blot analysis suggests either that there are multiple copies of the SCP{sub 2} gene in the human genome or that the SCP{sub 2} gene is very large. Coexpression of the SCP{sub 2} cDNA with expression vectors for cholesterol side-chain cleavage enzyme and adrenodoxin resulted in a 2.5-fold enhancement of progestin synthesis over that obtained with expression of the steroidogenic enzyme system alone. These findings are concordant with the notion that SCP{sub 2} plays a role in regulating steroidogenesis, among other possible functions.

  7. Characterization of cDNA encoding human placental anticoagulant protein (PP4): Homology with the lipocortin family

    Energy Technology Data Exchange (ETDEWEB)

    Grundmann, U.; Abel, K.J.; Bohn, H.; Loebermann, H.; Lottspeich, F.; Kuepper, H. (Research Institutes, Postfach (West Germany))

    1988-06-01

    A cDNA library prepared from human placenta was screened for sequences encoding the placental protein 4 (PP4). PP4 is an anticoagulant protein that acts as an indirect inhibitor of the thromboplastin-specific complex, which is involved in the blood coagulation cascade. Partial amino acid sequence information from PP4-derived cyanogen bromide fragments was used to design three oligonucleotide probes for screening the library. From 10{sup 6} independent recombinants, 18 clones were identified that hybridized to all three probes. These 18 recombinants contained cDNA inserts encoding a protein of 320 amino acid residues. In addition to the PP4 cDNA the authors identified 9 other recombinants encoding a protein with considerable similarity (74%) to PP4, which was termed PP4-X. PP4 and PP4-X belong to the lipocortin family, as judged by their homology to lipocortin I and calpactin I.

  8. Cloning and characterization of a novel barley gene,HvORG4,induced by Fusarium graminearum infection

    Institute of Scientific and Technical Information of China (English)

    Theo; Van-Der; Lee

    2007-01-01

    Barley Fusarium head blight(FHB),caused by species of the Fusarium fungus,is a devastating disease that is reemerging worldwide in recent years.In this study,a novel gene,HvORG4,was cloned from barley by using cDNA library and suppression subtractive hybridization(SSH) library strategies.The SSH library and cDNA library were constructed from the Chinese barley cultivar Jing02-461(resistance to FHB) infected by Fusarium graminearum isolate Huanggang-1.For the SSH analysis,more than 120 differentially express...

  9. Construction of cDNA Library from Populus euphratica

    Institute of Scientific and Technical Information of China (English)

    Yu Guangjun; Wang Yiqin; Shen Xin

    2003-01-01

    In order to isolate and clone salt-tolerance involved genes of Populus euphratica, we constructed a cDNA library from salt-treated leaves of P. euphratica. In the experiment, double strand cDNA were synthesized by a beads-based method. The syntheses of the first strand and the second strand cDNA, adapter ligation and restriction reaction for releasing cDNA were all conducted on the beads. The double strand cDNA were released from magnetic beads by digestion with NotI, and cDNA fragments smaller than 500 bp and residual adapters were removed through cDNA size fractionation columns. Finally, double strand cDNA were directionally cloned intoλExcell vector. The results show that the primary titer of the cDNA library is 7.46×106 pfu per mL and the packaging efficiency reaches 1.47×107 recombinants per μg DNA. λDNA extracted from two clones of plaque were digested by EcoR I and NotI, both of the clones contained inserts larger than 900 bp. These results show that the cDNA library of salt-treated P. euphratica leaves has been successfully constructed.

  10. Tle Triangulation Campaign by Japanese High School Students as a Space Educational Project of the Ssh Consortium Kochi

    Science.gov (United States)

    Yamamoto, Masa-Yuki; Okamoto, Sumito; Miyoshi, Terunori; Takamura, Yuzaburo; Aoshima, Akira; Hinokuchi, Jin

    As one of the space educational projects in Japan, a triangulation observation project of TLE (Transient Luminous Events: sprites, elves, blue-jets, etc.) has been carried out since 2006 in collaboration between 29 Super Science High-schools (SSH) and Kochi University of Technol-ogy (KUT). Following with previous success of sprite observations by "Astro High-school" since 2004, the SSH consortium Kochi was established as a national space educational project sup-ported by Japan Science and Technology Agency (JST). High-sensitivity CCD camera (Watec, Neptune-100) with 6 mm F/1.4 C-mount lens (Fujinon) and motion-detective software (UFO-Capture, SonotaCo) were given to each participating team in order to monitor Northern night sky of Japan with almost full-coverage. During each school year (from April to March in Japan) since 2006, thousands of TLE images were taken by many student teams, with considerably large numbers of successful triangulations, i.e., (School year, Numbers of TLE observations, Numbers of triangulations) are (2006, 43, 3), (2007, 441, 95), (2008, 734, 115), and (2009, 337, 78). Note that, school year in Japan begins on April 1 and ends on March 31. The observation campaign began in December 2006, numbers are as of Feb. 28, 2010. Recently, some high schools started wide field observations using multiple cameras, and others started VLF observations using handmade loop antennae and amplifiers. Infomation exchange among the SSH consortium Kochi is frequently communicated with scientific discussion via KUT's mailing lists. Also, interactions with amateur observers in Japan are made through an internet forum of "SonotaCo Network Japan" (http://sonotaco.jp). Not only as an educational project but also as a scientific one, the project is also in success. In February 2008, simultaneous observations of Elves were obtained, in November 2009 a Giant "Graft-shaped" Sprites driven by Jets was clearly imaged with VLF signals. Most recently, ob-servations of Elves

  11. 基于SSH架构的语文教学系统设计%Design of Chinese Teaching System On the basis of SSH Framework

    Institute of Scientific and Technical Information of China (English)

    刘志军

    2012-01-01

    提出基于SSH(Struts+Spring+Hibernate)架构构建语文教学系统,设计了SSH架构Web系统分层设计方案及执行流程.利用MyEclipse平台实现了语文教学平台的软件开发过程,证明了方案的可行性.SSH架构方案具有可扩展性、可维护性和可重用性.%A Chinese teaching system has been proposed based on SSH framework. A hierarchical design and the operating process of Web system of SSH ( Struts + Spring + Hibernate) framework has been designed. The development project of the Chinese teaching system is achieved on the MyEclipse. The SSH framework project is characterized by its feasibility, extensibility, maintainability and reusability.

  12. 基于 SSH 框架的高校学生出勤考核管理系统%Management System of Students Attendance Assessment Based on SSH Framework

    Institute of Scientific and Technical Information of China (English)

    张亚娟; 刘寒冰

    2015-01-01

    现有的学生出勤考核系统由于操作较复杂,响应时间长,并且不能对未出勤现象进行申诉,应用情况并不理想。该文提出基于 SSH 框架设计学生上机考勤系统,让学生通过在线完成实验课的考勤,老师可以集中查看学生的出勤情况,并对未正常出勤的学生进行申诉处理。结果表明,该系统操作简单、签到时间短,极大地减轻了老师的工作量。%The existing student attendance assessment system had many poor characteristics,such as complex operation,long period response,and it can't complain when student can't attend class.Student attendance assessment system based on SSH framework can easily assess students’daily attendance in experiment class and help teachers to check students’attendance and punish students that do not attend class.The results show that it is simple to operate,short to check in time and thus reduces workload of student commis-sioner.

  13. Cloning and expression of cDNA for human poly(ADP-ribose)polymerase

    Energy Technology Data Exchange (ETDEWEB)

    Alkhatib, H.M.; Chen, D.; Cherney, B.; Bhatia, K.; Notario, V.; Giri, C.; Stein, G.; Slattery, E.; Roeder, R.G.; Smulson, M.E.

    1987-03-01

    cDNAs encoding poly(ADP-ribose) polymerase from a human hepatoma lambdagt11 cDNA library were isolated by immunological screening. One insert of 1.3 kilobases (kb) consistently hybridized on RNA gel blots to an mRNA species of 3.6-3.7 kb, which is consistent with the size of RNA necessary to code for the polymerase protein (116 kDa). This insert was subsequently used in both in vitro hybrid selection and hybrid-arrested translation studies. An mRNA species from HeLa cells of 3.6-3.7 kb was selected that was translated into a 116-kDa protein, which was selectively immunoprecipitated with anti-poly(ADP-ribose) polymerase. To confirm that the 1.3-kb insert from lambdagt11 encodes for poly(ADP-ribose) polymerase, the insert was used to screen a 3- to 4-kb subset of a transformed human fibroblast cDNA library in the Okayama-Berg vector. One of these vectors was tested in transient transfection experiments in COS cells. This cDNA insert contained the complete coding sequence for polymerase. Using pcD-p(ADPR)P as probe, it was observed that the level of poly(ADP-ribose) polymerase mRNA was elevated at 5 and 7 hr of S phase of the HeLa cell cycle, but was unaltered when artificial DNA strand breaks are introduced in HeLa cells by alkylating agents.

  14. Correction of chromosomal instability and sensitivity to diverse mutagens by a cloned cDNA of the XRCC3 DNA repair gene

    Energy Technology Data Exchange (ETDEWEB)

    Tebbs, R.S.; Tucker, J.D.; Hwang, M. [Lawrence Livermore National Lab., CA (United States)] [and others

    1995-07-03

    The mutagen-sensitive CHO line irs1SF was previously isolated on the basis of hypersensitivity to ionizing radiation and was found to be chromosomally unstable as well as cross-sensitive to diverse kinds of DNA-damaging agents. The analysis of somatic cell hybrids formed between irs1SF and human lymphocytes implicated a human gene (defined as XRCC3; x-ray repair cross-complementing), which partially restored mitomycin C resistance to the mutant. A functional cDNA that confers mitomycin C resistance was transferred to irs1SF cells by transforming them with an expression cDNA library and obtaining primary and secondary transformants. Functional cDNA clones were recovered from a cosmid library prepared from a secondary transformant. Transformants also showed partial correction of sensitivity to displatin and {gamma}-rays, efficient correction of chromosomal instability, and substantially improved plating efficiency and growth rate. The XRCC3 cDNA insert is {approx} 2.5 kb and detects an {approx} 3.0-kb mRNA on Northern blots. The cDNA was mapped by fluorescence in situ hybridization to human chromosome 14q32.3, which was consistent with the chromosome concordance data of two independent hybrid clone panels. 30 refs., 5 figs., 2 tabs.

  15. Cloning and expression analysis of human reticulon 4c cDNA

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    RTNs (reticulons) is a gene family related to the growth and differentiation of neuroendocrine cell. This family is composed of several members such as RTN1, RTN2 and RTN3. RTN1 and RTN2 have been proved to have 3 transcripts with different length. Because the RTN1c cDNA was involved in the sologenesis of small cell lung carcinoma (SCLC), it was selected as a bioinformatic probe to clone novel members of RTN family with the electric hybridization assistant new-gene cloning method (EHAC). A 1677-bp cDNA was identified from human brain cDNA library. The cDNA contains an intact open reading frame (ORF) which encodes a protein of 199 amino acids. This deduced protein is highly homologous to RTN1c, RTN2c and RTN3 with identities of 64.4%, 45.8% and 50.0% respectively. This new gene was named RTN4c (GenBank accession number: AF087901). Northern hybridization showed that the full length of RTN4c transcript is about 1.8 kb. It is hardly expressed in heart, placenta, lung, spleen, thymus, testis, ovary, small intestine and peripheral blood white cells; but it is highly expressed in the tissues of skeletal muscle, brain, liver and kidney, and less expressed in the pancreas, prostate and colon. Furthermore, Northern results also showed that there is a 2.3 kb transcript expressed in 14 tissues except liver and skeletal muscle; while another 5.0 kb transcript in brain, skeletal muscle and testis. By the electric hybridization walking, we obtained two full-length contigs with a length of 4632 and 2235 bp respectively. The former encodes a protein with 1192 amino acids and was defined as RTN4a; the latter encodes another protein with 373 amino acids, and was named RTN4b. The RTN4 gene was mapped to human chromosome 2p14-p13 region by the radiation hybridization (RH).

  16. Suppression subtractive hybridization and comparative expression of a pore-forming toxin and glycosyl hydrolase genes in Rhizoctonia solani during potato sprout infection.

    Science.gov (United States)

    Chamoun, Rony; Samsatly, Jamil; Pakala, Suman B; Cubeta, Marc A; Jabaji, Suha

    2015-06-01

    Rhizoctonia solani is a plant pathogenic fungus that causes black scurf on tubers and stem and stolon canker on underground parts of potato plant. Early in the season, the fungus attacks germinating sprouts underground before they emerge from the soil. Damage at this stage results in delayed emergence of weakened plants with poor and uneven stands. The mechanism underlying this phenomenon has been investigated in this study by coupling a cDNA-suppression subtractive hybridization (SSH) library to differential screening to identify transcripts of R. solani that are down-regulated during infection of potato sprouts. We report on the identification of 33 unique genes with functions related to carbohydrate binding, vitamin synthesis, pathogenicity, translation, ATP and nucleic acid binding and other categories. RACE-PCR was used to clone and characterize the first full-length cDNA clones, RSENDO1 and RSGLYC1 that encode for an eukaryotic delta-endotoxin CytB protein and an intracellular glycosyl hydrolase, respectively. Quantitative real-time PCR revealed the down-regulation of RSENDO1 during infection of potato sprouts and the up-regulation of RSGLYC1 when the fungus was grown on a cellulose-based nutrient medium. In contrast, additional experiments have highlighted the down-regulation of RSENDO1 when R. solani was co-cultured with the mycoparasite Stachybotrys elegans and the bacterial antagonist Bacillus subtilis B26. These results advance our understanding of R. solani-potato interaction in subterranean parts of the plant. Such approaches could be considered in building an efficient integrated potato disease management program.

  17. The rate parameters for coupled vibration-dissociation in a generalized SSH approximation. [Schwarz, Slawsky, and Herzfeld

    Science.gov (United States)

    Sharma, Surendra P.; Huo, Winifred M.; Park, Chul

    1988-01-01

    A theoretical study of vibrational excitations and dissociations of nitrogen undergoing a nonequilibrium relaxation process upon heating and cooling is reported. The rate coefficients for collisional induced vibrational transitions and transitions from a bound vibrational state into a dissociative state have been calculated using an extension of the theory originally proposed by Schwarz (SSH) et al. (1952). High-lying vibrational states and dissociative states were explicitly included but rotational energy transfer was neglected. The transition probabilities calculated from the SSH theory were fed into the master equation, which was integrated numerically to determine the population distribution of the vibrational states as well as bulk thermodynamic properties. The results show that: (1) the transition rates have a minimum near the middle of the bound vibrational levels, causing a bottleneck in the vibrational relaxation and dissociation rates; (2) high vibrational states are always in equilibrium with the dissociative state; (3) for the heating case, only the low vibrational states relax according to the Landau-Teller theory; (4) for the cooling case, vibrational relaxation cannot be described by a rate equation; (5) Park's (1985, 1988) two-temperature model is approximately valid; and (6) the average vibrational energy removed in dissociation is about 30 percent of the dissociation energy.

  18. Identification of sennoside A as a novel inhibitor of the slingshot (SSH) family proteins related to cancer metastasis.

    Science.gov (United States)

    Lee, Seon Young; Kim, Wooil; Lee, Young Geun; Kang, Hyo Jin; Lee, Sang-Hyun; Park, Sun Young; Min, Jeong-Ki; Lee, Sang-Rae; Chung, Sang J

    2017-03-06

    Phospho-cofilin (p-cofilin), which has a phosphate group on Ser-3, is involved in actin polymerization. Its dephosphorylated form promotes filopodia formation and cell migration by enhancing actin depolymerization. Protein phosphatase slingshot homologs (SSHs), known as dual-specificity phosphatases, catalyze hydrolytic removal of the Ser-3 phosphate group from phospho-cofilin. Aberrant SSH activity results in cancer metastasis, implicating SSHs as potential therapeutic targets for cancer metastasis. In this study, we screened 658 natural products purified from traditional oriental medicinal plants to identify three potent SSH inhibitors with submicromolar or single-digit micromolar Ki values: gossypol, hypericin, and sennoside A. The three compounds were purified from cottonseed, Saint John's wort, and rhubarb, respectively. Sennoside A markedly increased cofilin phosphorylation in pancreatic cancer cells, leading to impaired actin dynamics in pancreatic cancer cells with or without EGF stimulation and reduced motility and invasiveness in vitro and in vivo. Collaboratively, these results demonstrate that sennoside A is a novel inhibitor of SSHs and suggest that it may be valuable in the development of pharmaceutical drugs for treating cancer metastasis.

  19. 申克孢子丝菌酵母相和菌丝相cDNA消减文库的构建%Construction of cDNA subtractive library for the yeast and mycelium phases of Sporothrix Schenckii

    Institute of Scientific and Technical Information of China (English)

    周汛; 杨致邦; 郭丽媛; 肖异珠

    2011-01-01

    Objective To explore molecular mechanism of dimorphic transition of Sporothrix schenckii, differential gene expression in dimorphic transition of Sporothrix schenckii was screened. Methods cDNA subtractive library for the yeast ( Y ) and mycelium ( M ) phases was constructed by suppression subtractive hybridization ( SSH ) . Bioinformatics analysis was performed to profile the relationship between the differently expressed genes and dimorphic transition. Results 751 ESTs were obtained in M + Y library , with the average length of 690. 98bp. Meanwhile . 875 ESTs were obtained in Y + M library, with the average length of 575. 9bp. After splicing of ESTs,101 unigenes were obtained in M + Y library and 249 unigenes in Y + M library. Some structure genes and function-unknown genes in these differentially expressed genes appeared to be related to dimorphic transition as compared with BLASTN. Conclusion It is evident that the subtracted cDNA library for the dimorphic transition of Sporothrix Schenckii was successfully constructed, thereby providing solid foundation for screening the genes involved the dimorphic transition of Sporothrix schenckii.%目的 筛选与申克孢子丝菌酵母相与菌丝相双相转换相关的差异表达基因,为探讨其双相转换的分子的机制奠定基础.方法 应用抑制性消减杂交技术,构建高特异性的申克孢子丝菌菌丝相(mycelium,M)和酵母相(yeast,Y)的正反cDNA消减文库,并对其差异表达的基因进行生物信息学分析.结果 M+Y文库获得751条表达序列标签,平均长度为690.98 bp,经拼接后获得101条非冗余序列.Y+M文库获得875条表达序列标签,平均长度为575.9 bp,拼接获得249条非冗余序列.经BLASTN比对,这些差异表达基因中,某些结构基因和功能不明的细胞分子类基因可能与双相转换有关.结论 成功构建了高特异性的申克孢子丝菌菌丝相和酵母相的正反cDNA 消减文库,为进一步筛选申克孢子丝菌的双相转换基因奠定了基础.

  20. Cross-species hybridisation of human and bovine orthologous genes on high density cDNA microarrays

    OpenAIRE

    2004-01-01

    Abstract Background Cross-species gene-expression comparison is a powerful tool for the discovery of evolutionarily conserved mechanisms and pathways of expression control. The usefulness of cDNA microarrays in this context is that broad areas of homology are compared and hybridization probes are sufficiently large that small inter-species differences in nucleotide sequence would not affect the analytical results. This comparative genomics approach would allow a common set of genes within a s...

  1. Cloning and roles of goldfish maternal factor β-Catenin cDNA in embryonic development

    Institute of Scientific and Technical Information of China (English)

    ZHANG Jingpu; WANG Weixian; ZHU Shaoxia

    2004-01-01

    Interaction between nucleus and cytoplasm has been focused in the field of animal embryonic development, in which study of maternal factors is required positively. Β-Catenin, an important maternal factor in early embryogenesis, has been analyzed in its expression pattern and functions in this paper. We have cloned goldfish β-Catenin cDNA gene and compared it with zebrafish β-Catenin cDNA. High homology was found in cDNA and in amino acid sequences between them, 93% (2227/2384 bp) and 98.5% (768/780 aa) respectively. The expression pattern of β-Catenin by in situ hybridization and the roles of β-Catenin on embryonic development by co-injection of anti-sense RNA and reporter gene, EGFP have been investigated in the whole process of goldfish embryonic development. The results suggest that β-Catenin presents dynamic distribution, mainly locates at body axis, dorsal tissues, head and tail structures after being fertilized. The loss of β-Catenin activity would cause serious destruction of embryo in dorsal tissues and in anteroposterior axes, and leads embryos to die before larva get hatched.

  2. Detection of HIV cDNA Point Mutations with Rolling-Circle Amplification Arrays

    Directory of Open Access Journals (Sweden)

    Zhongwei Wu

    2010-01-01

    Full Text Available In this paper we describe an isothermal rolling-circle amplification (RCA protocol to detect gene point mutations on chips. The method is based on an allele-specific oligonucleotide circularization mediated by a special DNA ligase. The probe is circularized when perfect complementary sequences between the probe oligonucleotide and HIV cDNA gene. Mismatches around the ligation site can prevent probe circularization. The circularized probe (C-probe can be amplified by rolling circle amplification to generate multimeric singlestranded DNA (ssDNA under isothermal conditions. There are four sequence regions to bind respectively with fluorescent probe, RCA primer, solid probe and HIV cDNA template in the C-probe which we designed. These ssDNA products are hybridized with fluorescent probes and solid probes which are immobilized on a glass slide composing a regular microarray pattern. The fluorescence signals can be monitored by a scanner in the presence of HIV cDNA templates, whereas the probes cannot be circularized and signal of fluorescence cannot be found. The RCA array has capability of high-throughput detection of the point mutation and the single-nucleotide polymorphism (SNP.The development of C-probe-based technologies offers a promising prospect for situ detection, microarray, molecular diagnosis, single nucleotide polymorphism, and whole genome amplification.

  3. Coordinate regulation of stromelysin and collagenase genes determined with cDNA probes

    Energy Technology Data Exchange (ETDEWEB)

    Frisch, S.M.; Clark, E.J.; Werb, Z.

    1987-05-01

    Secreted proteinases are required for tumor metastasis, angiogenesis, and tissue remodeling during wound healing and embryonic growth. Thus, the regulation of the genes of secreted proteinases may serve as an interesting model for growth-controlled genes in general. The authors studied the genes of the secreted proteinases stromelysin and collagenase by using molecularly cloned cDNAs from each proteinase. Stromelysin cDNA was cloned by differential screening of a total cDNA library from rabbit synovial cells treated with phorbol 12-myristate 13-acetate, which yielded a clone of 1.2 kilobase pairs; collagenase cDNA was obtained by cloning reverse transcripts of anti-collagenase-immunoadsorbed polysomal mRNA, which yielded a clone of 0.8 kilobase pairs. Stromelysin and collagenase mRNA species of 2.2 and 2.4 kilobases, respectively, were detected on hybridization blots of RNA from phorbol 12-myristate 13-acetate-treated but not untreated rabbit synovial cells. Expression of stromelysin mRNA was also induced in rabbit alveolar macrophages and rabbit brain capillary endothelial cells treated with phorbol 12-myristate 13-acetate. Stromelysin and collagenase mRNA were both induced by phorbol 12-myristate 13-acetate and cytochalasin B at a constant ratio of the two gene products; this suggest coordinate regulation. The fact that induction was blocked after inhibition of protein synthesis by cycloheximide implicates an indirect signal transduction pathway that requires new protein synthesis.

  4. Isolation and sequence analysis of a cDNA clone encoding the fifth complement component

    DEFF Research Database (Denmark)

    Lundwall, Åke B; Wetsel, Rick A; Kristensen, Torsten

    1985-01-01

    obtained further predicted an arginine-rich sequence (RPRR) immediately upstream of the N-terminal threonine of C5a, indicating that the promolecule form of C5 is synthesized with a beta alpha-chain orientation as previously shown for pro-C3 and pro-C4. The C5 cDNA clone was sheared randomly by sonication......We have used available protein sequence data for the anaphylatoxin (C5a) portion of the fifth component of human complement (residues 19-25) to synthesize a mixed-sequence oligonucleotide probe. The labeled oligonucleotide was then used to screen a human liver cDNA library, and a single candidate cDNA...... clone of 1.85 kilobase pairs was isolated. Hybridization of the mixed-sequence probe to the complementary strand of the plasmid insert and sequence analysis by the dideoxy method predicted the expected protein sequence of C5a (positions 1-12), amino-terminal to the anticipated priming site. The sequence...

  5. Isolation of a cDNA Encoding a Protease from Perinereis aibuhitensis Grube

    Institute of Scientific and Technical Information of China (English)

    Rong-Gui LI; Dong-Meng QIAN; Dao-Sen GUO; Gui-Cai DU; Zhi-Yong YAN; Bin WANG

    2006-01-01

    The cDNA encoding a protease of Perinereis aibuhitensis Grube (PPA) was cloned. The deduced amino acid sequence analysis showed that the protein had 49% identity to the C-terminal amino acid 169-246 of serine protease of Heterodera glycines. Northern blotting analysis indicated that the cDNA could hybridize with mRNA of approximately 260 bases isolated from the marine earthworm. The cDNA was amplified by polymerase chain reaction and cloned into pMAL-p2 to construct expression vector pMALPPA. pMAL-PPA was introduced into Escherichia coli BL21(DE3) and overexpression of PPA fused with maltose binding protein was achieved by isopropyl-β-D-thiogalactopyranoside induction. The fusion protein was purified by affinity chromatography on an amylose resin column and ion-exchange chromatography on a diethylaminoethyl-Sepharose 4B column. Rabbits were immunized with the purified protein and antiserum was prepared. The antibody could react with a protein of approximately 9 kDa extracted from the marine earthworm as shown by Western blotting analysis. The activity analysis of the recombinant PPA suggested that it was probably a plasminogen activator.

  6. Detection of HIV cDNA point mutations with rolling-circle amplification arrays.

    Science.gov (United States)

    Wu, Lingwei; Liu, Quanjun; Wu, Zhongwei; Lu, Zuhong

    2010-01-27

    In this paper we describe an isothermal rolling-circle amplification (RCA) protocol to detect gene point mutations on chips. The method is based on an allele-specific oligonucleotide circularization mediated by a special DNA ligase. The probe is circularized when perfect complementary sequences between the probe oligonucleotide and HIV cDNA gene. Mismatches around the ligation site can prevent probe circularization. The circularized probe (C-probe) can be amplified by rolling circle amplification to generate multimeric singlestranded DNA (ssDNA) under isothermal conditions. There are four sequence regions to bind respectively with fluorescent probe, RCA primer, solid probe and HIV cDNA template in the C-probe which we designed. These ssDNA products are hybridized with fluorescent probes and solid probes which are immobilized on a glass slide composing a regular microarray pattern. The fluorescence signals can be monitored by a scanner in the presence of HIV cDNA templates, whereas the probes cannot be circularized and signal of fluorescence cannot be found. The RCA array has capability of high-throughput detection of the point mutation and the single-nucleotide polymorphism (SNP).The development of C-probe-based technologies offers a promising prospect for situ detection, microarray, molecular diagnosis, single nucleotide polymorphism, and whole genome amplification.

  7. Chitinase cDNA cloning and mRNA induction by fungal elicitor, wounding, and infection.

    Science.gov (United States)

    Hedrick, S A; Bell, J N; Boller, T; Lamb, C J

    1988-01-01

    Chitinase, which catalyzes the hydrolysis of beta-1,4 N-acetylglucosamine linkages of the fungal cell wall polymer chitin, is a component of the inducible defenses of plants. We show that chitinase synthesis is stimulated in bean (Phaseolus vulgaris L.) cell suspension cultures treated with fungal cell wall elicitors and in hypocotyls in response to infection with the fungus Colletotrichum lindemuthianum. Chitinase cDNA clones were isolated by antibody screening of a lambdagt11 cDNA library containing sequences complementary to poly A(+) RNA from elicited cells. The identity of these clones was confirmed by nucleotide sequence analysis and comparison of the deduced amino acid sequence with that determined for the amino-terminal sequence of bean chitinase. Elicitor causes a very rapid activation of chitinase transcription with a 10-fold stimulation after 5 minutes and 30-fold increase within 20 minutes. This leads to a marked, transient accumulation of chitinase transcripts with maximum levels 2 hours after elicitor treatment, concomitant with the phase of rapid enzyme synthesis. Chitinase transcripts also markedly accumulate in wounded and infected hypocotyls. Chitinase cDNA sequences hybridize to several genomic fragments suggesting there are several chitinase genes in the bean genome.

  8. Infectious Maize rayado fino virus from cloned cDNA

    Science.gov (United States)

    Maize rayado fino virus (MRFV) is the type member of the marafiviruses within the family Tymoviridae. A cDNA clone from which infectious RNA can be transcribed was produced from a US isolate of MRFV (MRFV-US). Infectivity of transcripts derived from cDNA clones was demonstrated by infection of mai...

  9. Characterization of the porcine carboxypeptidase E cDNA

    DEFF Research Database (Denmark)

    Hreidarsdôttir, G.E.; Cirera, Susanna; Fredholm, Merete

    2007-01-01

    the sequence of the cDNA for the porcine CPE gene including all the coding region and the 3'-UTR region was generated. Comparisons with bovine, human, mouse, and rat CPE cDNA sequences showed that the coding regions of the gene are highly conserved both at the nucleotide and at the amino acid level. A very low...

  10. Generation of EST and cDNA Microarray Resources for the Study of Bovine Immunobiology*

    Directory of Open Access Journals (Sweden)

    Coussens PM

    2003-03-01

    Full Text Available Recent developments in expressed sequence tag (EST and cDNA microarray technology have had a dramatic impact on the ability of scientists to study the responses of thousands of genes to external stimuli, such as infection, nutrient flux, and stress. To date however, these studies have largely been limited to human and rodent systems. Despite the tremendous potential benefit of EST and cDNA microarray technology to studies of complex problems in domestic animal species, a lack of integrated resources has precluded application of these technologies to domestic species. To address this problem, the Center for Animal Functional Genomics (CAFG at Michigan State University has developed a normalized bovine total leukocyte (BOTL cDNA library, generated EST clones from this library, and printed cDNA microarrays suitable for studying bovine immunobiology. Our data revealed that the normalization procedure successfully reduced highly abundant cDNA species while enhancing the relative percentage of clones representing rare transcripts. To date, a total of 932 EST sequences have been generated from this library (BOTL and the sequence information plus BLAST results made available through a web-accessible database http://gowhite.ans.msu.edu. Cluster analysis of the data indicates that a total of 842 unique cDNAs are present in this collection, reflecting a low redundancy rate of 9.7%. For creation of first generation cDNA microarrays, inserts from 720 unique clones in this library were amplified and microarrays were produced by spotting each insert or amplicon 3 times on glass slides in a 48-patch arrangement with 64 total spots (including blanks and positive controls per patch. To test our BOTL microarray, we compared gene expression patterns of concanavalin A stimulated and unstimulated peripheral blood mononuclear cells (PBMCs. In total, hybridization signals on over 90 amplicons showed upregulation (>3× in response to Con A stimulation, relative to

  11. Coral record of southeast Indian Ocean SST, SSH and salinity and their modulation by ENSO and the Western Pacific temperature gradient

    Science.gov (United States)

    Zinke, Jens; Hoell, Andrew; Lough, Janice M.; Feng, Ming; McCulloch, Malcolm T.

    2016-04-01

    Variability of southeastern Indian Ocean (SEIO) sea surface temperatures (SST), sea surface height (SSH) and salinities off Western Australia is a footprint of interannual and decadal climate variations in the tropical Indo-Pacific. La Niña events often result in a strengthened Leeuwin Current, high coastal sea levels, low salinities and unusually warm SSTs, now termed Ningaloo Niño events. The long-term teleconnections of the southeastern Indian Ocean (SEIO) with ENSO and the West Pacific Warm Pool are poorly understood. Here we demonstrate the role of Indo-Pacific coupling in modulating SST, SSH and salinity in the poorly studied SEIO, through a robust 215 year (1795-2010) geochemical coral proxy sea surface temperature (SST), SSH and salinity record. We show that higher SST and SSH accompanied by lower salinities in the SEIO are linked to the behaviour of ENSO and the Western Pacific Warm Pool on decadal to centennial timescales, and are most pronounced when an anomalously strong zonal SST gradient between the western and central Pacific co-occurs with strong La Niña's. Better understanding of the interplay between the zonal SST gradient in the western Pacific, ENSO phase and intrinsic Indian Ocean variability is expected to improve our ability to better predict unusual marine heat waves, sea level surges and important consequences for marine socio-ecological systems in the Future.

  12. A supersulfated low-molecular-weight heparin (IK-SSH) increases plasma levels of free and total tissue factor pathway inhibitor after intravenous and subcutaneous administration in humans.

    Science.gov (United States)

    Kaiser, B; Glusa, E; Hoppensteadt, D A; Breddin, H K; Amiral, J; Fareed, J

    1998-09-01

    Unfractionated as well as low-molecular-weight heparins (LMWH) are known to cause an increase in blood levels of tissue factor pathway inhibitor (TFPI). To study the effect of a newly developed supersulfated LMWH (IK-SSH, Iketon Farmaceutici) on TFPI concentrations in human plasma, the compound was injected into volunteers at doses of 0.14, 0.33 and 0.66 mg/kg intravenously or 0.33, 0.66 and 1.0 mg/kg subcutaneously. At certain known times blood was drawn and plasma levels of both total and free TFPI were measured using enzyme-linked immunosorbent assay methodology. Baseline plasma concentrations of TFPI were 72.2+/-3.1 ng/ml for total and 10.8+/-0.8 ng/ml for free TFPI. Intravenous or subcutaneous injection of IK-SSH led to a strong and long-lasting rise in TFPI levels which were increased more than 5-fold for total TFPI and more than 30-fold for free TFPI. Maximum TFPI levels were reached 5-10 min after intravenous and 60 min after subcutaneous administration. IK-SSH caused prolongation of ex-vivo clotting times in the APTT and Heptest assay, whereas thrombin time was not affected. Anticoagulant actions of IK-SSH showed a significant correlation to plasma concentrations of TFPI and they are thought to be based at least partially on the release of TFPI from vascular sites.

  13. Analysis of gene expression profile of pancreatic carcinoma using CDNA microarray

    Institute of Scientific and Technical Information of China (English)

    ZhiJun Tan; Xian-Gui Hu; Gui-Song Cao; Yan Tang

    2003-01-01

    AIM: To identify new diagnostic markers and drug targets,the gene expression profiles of pancreatic cancer were compared with that of adjacent normal tissues utilizing cDNA microarray analysis.METHODS: cDNA probes were prepared by labeling mRNA from samples of six pancreatic carcinoma tissues with Cy5dUTP and mRNA from adjacent normal tissues with Cy3dUTP respectively through reverse transcription. The mixed probes of each sample were then hybridized with 12 800cDNA arrays (12 648 unique human cDNA sequences), and the fluorescent signals were scanned by ScanArray 3 000scanner (General Scanning, Inc.). The values of CyS-dUTP and Cy3-dUTP on each spot were analyzed and calculated by ImaGene 3.0 software (BioDiscovery, Inc.). Differentially expressed genes were screened according to the criterion that the absolute value of natural logarithm of the ratio of Cy5-dUTP to Cy3-dUTP was greater-than 0.69.RESETS: Among 6 samples investigated, 301 genes, which accounted for 2.38% of genes on the microarry slides,exhibited differentially expression at least in 5. There were 166 over-expressed genes including 136 having been registered in Genebank, and 135 under-expressed genes including 79 in Genebank in cancerous tissues.CONCLUSION: Microarray analysis may provide invaluable information on disease pathology, progression, resistance to treatment, and response to cellular microenvironments of pancreatic carcinoma and ultimately may lead to improving early diagnosis and discovering innovative therapeutic approaches for cancer.

  14. 辣椒细胞质雄性不育系和保持系SSH-cDNA文库的构建%Construction of SSH-cDNA Library for Cytoplasm Male Sterility Lines and Maintainer Lines in Pepper

    Institute of Scientific and Technical Information of China (English)

    王雯; 刘辰; 沈火林

    2011-01-01

    A forward subtracted cDNA library contained expression genes of pepper cytoplasm male sterility maintainer lines,was constructed by using suppression subtractive hybridization technique. The pepper cytoplasm male sterile lines A1 and A5 were used as drivers,and the corresponding maintainer lines B1 and B5 as testers. Based on the 189 positive monoclones selected from the subtracted library, 42 expressed sequence tags (ESTs)were identified by DIG Nonradioactive Nucleic Acid Labeling and Detection System as well as sequencing. The BLAST analysis showed that 35 ESTs had gene potential functions,including 26 ESTs that are homologous to known genes,and 9 ESTs without homology. Most of the homologous gene functions are related to the basic material metabolism, stress defense,signal transduction;the 7 ESTs without BLAST results might imply some new genes.%以辣椒细胞质雄性不育系A1、A5为驱动方(driver),以其相应的保持系B1、B5为测验方(tester),利用抑制差减杂交技术构建了1个辣椒细胞质雄性不育保持系表达基因的正向差减cDNA文库.从差减文库中筛选到189个阳性单克隆,经高密度点阵膜杂交筛选和测序分析,共获得了42条表达序列标签(ESTs).BLAST比对分析后,得到了35条有比对结果的序列,其中包括26条已知功能基因序列和9条未知功能基因序列,已知基因功能多与基础物质代谢、胁迫应答、信号转导等方面有关;有7条ESTs没有比对结果,可能代表一些新基因.

  15. Application of restriction display PCR technique in the preparation of cDNA microarray probes

    Institute of Scientific and Technical Information of China (English)

    Zhao-Hui Sun; Wen-Li Ma; Bao Zhang; Yi-Fei Peng; Wen-Ling Zheng

    2005-01-01

    AIM: To develop a simplified and efficient method for the preparation of hepatitis C virus (HCV) cDNA microarray probes.METHODS: With the technique of restriction display PCR (RD-PCR), restriction enzyme Sau3A I was chosen to digest the full-length HCV cDNAs. The products were classified and re-amplified by RD-PCR. We separated the differential genes by polyacrylamide gel electrophoresis and silver staining. Single bands cut out from the polyacrylamide gel were isolated. The third-round PCR was performed using the single bands as PCR template.The RD-PCR fragments were purified and cloned into the pMD18-T vector. The recombinant plasmids were extracted from positive clones, and the target gene fragments were sequenced. The cDNA microarray was prepared by spotting RD-PCR products to the surface of amino-modified glass slides using a robot. We validated the detection of microarray by hybridization and sequence analysis.RESULTS: A total of 24 different cDNA fragments ranging from 200 to 800 bp were isolated and sequenced,which were the specific gene fragments of HCV. These fragments could be further used as probes in microarray preparation. The diagnostic capability of the microarray was evaluated after the washing and scanning steps. The results of hybridization and sequence analysis showed that the specificity, sensitivity, accuracy, reproducibility,and linearity in detecting HCV RNA were satisfactory.CONCLUSION: The RD-PCR technique is of great value in obtaining a large number of size-comparable gene probes, which provides a speedy protocol in generating probes for the preparation of microarrays. Microarray prepared as such could be further optimized and applied in the clinical diagnosis of HCV.

  16. 基于SSH+jQuery框架的餐饮Web App的设计与实现%Design and implementation of a catering industry Web App based on SSH and jQuery

    Institute of Scientific and Technical Information of China (English)

    张佳佳; 王杨; 韩力英

    2016-01-01

    针对传统方式开发的餐饮Web App平台难以维护和扩展、用户体验不够好等问题,本文提出一种以Windows为开发环境,Eclipse为开发工具,Oracle为数据库,将SSH和jQuery这两种框架整合应用于系统开发的方案。该方案包括视图层、业务逻辑层和数据持久层,分别由SSH+jQuery框架组合实现相应功能。结果表明,该方案将SSH和jQuery这2个框架整合应用于系统开发中,实现了注册登录、订餐、外卖等主要功能,实现了上述三个层面的完全分离,提高了用户体验度。%For solving the Web App platform difficult to maintain and extend and promoting the user experience, a system developing scheme based on integrating framework SSH and jQuery has been proposed in this paper.This scheme adopts Windows as development environment, Eclipse as development tool and Oracle as the database. It contains view layer, business logic layer and date persistence layer, implemented by the JSP+jQuery framework, Struts+Spring framework, and Hibermate+Spring frameworkseparately. The results shows that this scheme has achieved the main function of registration, ordering, take-out, and has realized the complete separation of theview layer, business logic layer and date persistence layer, which also will improved the user experience.

  17. The Application of SSH in Telecommunication Service%SSH协议在电信业务的应用

    Institute of Scientific and Technical Information of China (English)

    李岳梦; 赵绍刚

    2009-01-01

    随着网络的不断发展,网络安全性已成为网络设计的一项重要内容.文章介绍了SSH(Secure Shell)协议体系结构,详细分析了SSH的三层结构和密钥机制,讨论了SSH连接建立过程的各个阶段.在此基础上,重点介绍了SSH协议在电信业务中的应用,包括建立一个SSH安全通道过程和在安全通道内实现数据安全交互的过程.

  18. cDNA cloning and sequencing of ostrich Growth hormone

    Directory of Open Access Journals (Sweden)

    Doosti Abbas

    2012-01-01

    Full Text Available In recent years, industrial breeding of ostrich (Struthio camelus has been widely developed in Iran. Growth hormone (GH is a peptide hormone that stimulates growth and cell reproduction in different animals. The aim of this study was to clone and sequence the ostrich growth hormone gene in E. coli, done for the first time in Iran. The cDNA that encodes ostrich growth hormone was isolated from total mRNA of the pituitary gland and amplified by RT-PCR using GH specific PCR primers. Then GH cDNA was cloned by T/A cloning technique and the construct was transformed into E. coli. Finally, GH cDNA sequence was submitted to the GenBank (Accession number: JN559394. The results of present study showed that GH cDNA was successfully cloned in E. coli. Sequencing confirmed that GH cDNA was cloned and that the length of ostrich GH cDNA was 672 bp; BLAST search showed that the sequence of growth hormone cDNA of the ostrich from Iran has 100% homology with other records existing in GenBank.

  19. Generation and characterisation of a full-length cDNA encoding murine myotonic dystrophy protein kinase from cardiac tissue

    Energy Technology Data Exchange (ETDEWEB)

    Carey, N.; Tongeren, T. van; Winchester, C. [Charing Cross & Wesminster Medical School, London (United Kingdom)] [and others

    1994-09-01

    The mutation underlying myotonic dystrophy (DM) is a CTG trinucleotide expansion in the 3{prime} untranslated region of a putative protein kinase gene (DMPK). We report the isolation of a full-length cDNA clone of the murine (DMPK) gene from a heart cDNA library. Sequence analysis shows that the clone is a splice isoform which has only previously been identified in brain, suggesting that there may be some flexibility of the splicing pattern in some tissues. We are currently analyzing the library for the presence of other isoforms. The full-length cDNA has been cloned into a bacterial expression system and the expressed protein is being used as an immunogen to generate both polyclonal and monoclonal antisera. These reagents will allow the analysis of the intracellular targets of the DMPK. Subclones of the cDNA have been generated for use as in situ hybridization probes, allowing investigation of the normal patterns of expression of the gene and the differential expression of the protein isoforms. These data will be essential for deciding on a rational use of rare patient material and will provide the necessary baseline for the analysis of transgenic and {open_quotes}knock-out{close_quotes} mice.

  20. Molecular cloning and nucleotide sequence of a full-length cDNA for human alpha enolase.

    Science.gov (United States)

    Giallongo, A; Feo, S; Moore, R; Croce, C M; Showe, L C

    1986-01-01

    We previously purified a 48-kDa protein (p48) that specifically reacts with an antiserum directed against the 12 carboxyl-terminal amino acids of the c-myc gene product. Using an antiserum directed against the purified p48, we have cloned a cDNA from a human expression library. This cDNA hybrid-selects an mRNA that translates to a 48-kDa protein that specifically reacts with anti-p48 serum. We have isolated a full-length cDNA that encodes p48 and spans 1755 bases. The coding region is 1299 bases long; 94 bases are 5' noncoding and 359 bases are 3' noncoding. The cDNA encodes a 433 amino acid protein that is 67% homologous to yeast enolase and 94% homologous to the rat non-neuronal enolase. The purified protein has been shown to have enolase activity and has been identified to be of the alpha type by isoenzyme analysis. The transcriptional regulation of enolase expression in response to mitogenic stimulation of peripheral blood lymphocytes and in response to heat shock is also discussed. Images PMID:3529090

  1. Association of differentially expressed genes with activation of mouse hepatic stellate cells by high-density cDNA mircoarray

    Institute of Scientific and Technical Information of China (English)

    Xiao-Jing Liu; Li Yang; Feng-Ming Luo; Hong-Bin Wu; Qu-Qiang

    2004-01-01

    AIM: To characterize the gene expression profiles associated with activation of mouse hepatic stellate cell (HSC) and provide novel insights into the pathogenesis of hepatic fibrosis.METHODS: Mice HSCs were isolated from BALB/c mice by in situ perfusion of collagenase and pronase and singlestep density Nycodenz gradient. Total RNA and mRNA of quiescent HSC and culture-activated HSC were extracted,quantified and reversely transcripted into cDNA. cDNAs from activated HSC were labeled with Cy5 and cDNAs from the quiescent HSC were labeled with Cy3, which were mixed with equal quantity, then hybridized with cDNA chips containing 4 000 genes. Chips were washed, scanned and analyzed. Increased expression of 4 genes and decreased expression of one gene in activated HSC were confirmed by reverse transcription- polymerase chain reaction (RT-PCR).RESULTS: A total of 835 differentially expressed genes were identified by cDNA chip between activated and quiescent HSC, and 465 genes were highly expressed in activated HSC. The differentially expressed genes included those involved in protein synthesis, cell-cycle regulation,apoptosis, and DNA damage response.CONCLUSION: Many genes implicated in intrahepatic inflammation, fibrosis and proliferation were up-regulated in activated HSC. cDNA microarray is an effective technique in screening for differentially expressed genes between two different situations of the HSC. Further analysis of the obtained genes will help understand the molecular mechanism of activation of HSC and hepatic fibrosis.

  2. cDNA microarray in isolation of novel differentially expressed genes related to human glioma and clone of a novel full-length gene

    Institute of Scientific and Technical Information of China (English)

    QI Zhen-yu; HUI Guo-zhen; LI Yao; ZHOU Zong-xiang; GU Shao-hua; YING Kang; XIE Yi

    2005-01-01

    Background This investigation was undertaken to obtain differentially expressed genes related to human glioma using cDNA microarray and the characterization of one novel full-length gene. Methods Total RNA was extracted from human glioma tissues and normal brain tissues, and mRNA was used to make probes. After hybridization and washing, the results were scanned using a computer system. The gene named 681F05 clone was an expressed gene to human glioma through four-time hybridization and scanning. Subsequently northern blot analysis was performed by northern blot, 5'RACE and bioinformatics. Results Fifteen differentially expressed genes to human glioma were obtained through four-time hybridization and scanning. Northern blot analysis confirmed that 681F05 clone was low-expressed in human brain tissues and over-expressed in human glioma tissues. The analysis of BLASTn and BLASTx showed that 681F05 clone is two cDNA clones encoding two novel proteins that are highly identified to the cyclophilin isoform 10 of C. Elgans, respectively. Sequence analysis revealed the two cDNA clones are two different splicing variants of a novel cycophilin-like gene (PPIL3a and PPIL3b).Conclusions cDNA microarray technology can be successfully used to identify differentially expressed genes. The novel full-length gene of human PPIL3 may be correlated with the formation of human glioma.

  3. Cloning and expression of a cDNA coding for the human platelet-derived growth factor receptor: evidence for more than one receptor class.

    OpenAIRE

    Gronwald, R G; Grant, F J; Haldeman, B A; Hart, C E; O'Hara, P J; Hagen, F S; Ross, R.; Bowen-Pope, D F; Murray, M. J.

    1988-01-01

    The complete nucleotide sequence of a cDNA encoding the human platelet-derived growth factor (PDGF) receptor is presented. The cDNA contains an open reading frame that codes for a protein of 1106 amino acids. Comparison to the mouse PDGF receptor reveals an overall amino acid sequence identity of 86%. This sequence identity rises to 98% in the cytoplasmic split tyrosine kinase domain. RNA blot hybridization analysis of poly(A)+ RNA from human dermal fibroblasts detects a major (approximately ...

  4. Construction of differentially expressed genes library of bighead carp (Aristichthys nobilis) exposed to microcystin-lr using ssh and expression profile of related genes.

    Science.gov (United States)

    Cui, Zhihui; Zhang, Kaiyue; Qu, Xiancheng; Liu, Qigen

    2011-12-01

    Microcystins (MCs) are hepatotoxic cyclic heptapeptides produced by cyanobacteria (blue-green algae). There are more than 70 MCs variants of which the most common and widely studied is MC-LR. We screened the hepatocellular differentially expressed genes against MC-LR in the bighead carp (Aristichthys nobilis). Suppression subtractive hybridization was used to construct the forward subtracted and reverse subtracted cDNA libraries, and one hundred and thirty two positive clones (seventy one in forward library and sixty one in reverse library) were randomly selected and sequenced. Finally, fifty five reliable sequences from the forward subtracted library were used in a homology search by BLASTn and BLASTx, as were 57 reliable sequences from the reverse subtracted library. Furthermore, eight analyzed sequences from the forward subtracted cDNA library and seven from the reverse subtracted library were found to be non-homologous sequences. The screening identified genes induced by MC-LR in both libraries that are involved in various processes, such as energy metabolism, immunity, and apoptosis. Some are cytoskeleton- and transportation-related genes, while signal transduction-related genes were also found. Significant genes, such as the apoptosis-related gene p53 and the proto-oncogene c-myc, are involved in inhibition of the MC-LR response in the reverse subtracted library. In addition, several immune-related genes, which play an important role in antioxidation and detoxification of MC-LR, were characterized and identified in both of the subtracted libraries. The study provides the basic data to further identify the genes and molecular mechanism of detoxification of microcystins.

  5. Identification of differentially expressed genes of Xanthomonas axonopodis pv. citri by representational difference analysis of cDNA

    Directory of Open Access Journals (Sweden)

    Angela Mehta

    2005-03-01

    Full Text Available Xanthomonas axonopodis pv. citri is a phytopathogenic bacterium responsible for citrus canker, a serious disease which causes severe losses in citriculture around the world. In this study we report the differential expression of X. axonopodis pv. citri in response to specific treatments by using Representational Difference Analysis of cDNA (cDNA RDA. cDNAs from X. axonopodis pv. citri cultured in the presence of leaf extract of the host plant (Citrus sinensis, in vivo, as well as in the complex medium were hybridized against cDNA of the bacterium grown in the minimal medium. Sequencing of the difference products obtained after the second and third hybridizations revealed a total of 37 distinct genes identified by homology searches in the genome of X. axonopodis pv. citri. These genes were distributed in different functional categories, including genes that encode hypothetical proteins, genes involved in metabolism, cellular processes and pathogenicity, and mobile genetic elements. Most of these genes are likely related to growth and/or acquisition of nutrients in specific treatments whereas others might be important for the bacterium pathogenicity.

  6. Method for construction of normalized cDNA libraries

    Science.gov (United States)

    Soares, Marcelo B.; Efstratiadis, Argiris

    1998-01-01

    This invention provides a method to normalize a directional cDNA library constructed in a vector that allows propagation in single-stranded circle form comprising: (a) propagating the directional cDNA library in single-stranded circles; (b) generating fragments complementary to the 3' noncoding sequence of the single-stranded circles in the library to produce partial duplexes; (c) purifying the partial duplexes; (d) melting and reassociating the purified partial duplexes to appropriate Cot; and (e) purifying the unassociated single-stranded circles, thereby generating a normalized cDNA library. This invention also provides normalized cDNA libraries generated by the above-described method and uses of the generated libraries.

  7. Lectin cDNA and transgenic plants derived therefrom

    Science.gov (United States)

    Raikhel, Natasha V.

    1994-01-04

    Transgenic plants containing cDNA encoding Gramineae lectin are described. The plants preferably contain cDNA coding for barley lectin and store the lectin in the leaves. The transgenic plants, particularly the leaves exhibit insecticidal and fungicidal properties. GOVERNMENT RIGHTS This application was funded under Department of Energy Contract DE-AC02-76ER01338. The U.S. Government has certain rights under this application and any patent issuing thereon.

  8. Constructing and detecting a cDNA library for mites.

    Science.gov (United States)

    Hu, Li; Zhao, YaE; Cheng, Juan; Yang, YuanJun; Li, Chen; Lu, ZhaoHui

    2015-10-01

    RNA extraction and construction of complementary DNA (cDNA) library for mites have been quite challenging due to difficulties in acquiring tiny living mites and breaking their hard chitin. The present study is to explore a better method to construct cDNA library for mites that will lay the foundation on transcriptome and molecular pathogenesis research. We selected Psoroptes cuniculi as an experimental subject and took the following steps to construct and verify cDNA library. First, we combined liquid nitrogen grinding with TRIzol for total RNA extraction. Then, switching mechanism at 5' end of the RNA transcript (SMART) technique was used to construct full-length cDNA library. To evaluate the quality of cDNA library, the library titer and recombination rate were calculated. The reliability of cDNA library was detected by sequencing and analyzing positive clones and genes amplified by specific primers. The results showed that the RNA concentration was 836 ng/μl and the absorbance ratio at 260/280 nm was 1.82. The library titer was 5.31 × 10(5) plaque-forming unit (PFU)/ml and the recombination rate was 98.21%, indicating that the library was of good quality. In the 33 expressed sequence tags (ESTs) of P. cuniculi, two clones of 1656 and 1658 bp were almost identical with only three variable sites detected, which had an identity of 99.63% with that of Psoroptes ovis, indicating that the cDNA library was reliable. Further detection by specific primers demonstrated that the 553-bp Pso c II gene sequences of P. cuniculi had an identity of 98.56% with those of P. ovis, confirming that the cDNA library was not only reliable but also feasible.

  9. Identification of heat stress-responsive genes in heat-adapted thermal Agrostis scabra by suppression subtractive hybridization.

    Science.gov (United States)

    Tian, Jiang; Belanger, Faith C; Huang, Bingru

    2009-04-01

    To gain insights into molecular mechanisms of grass tolerance to heat stress, we constructed a suppression subtractive cDNA library to identify heat-responsive genes for a C(3) grass species, thermal Agrostis scabra adapted to heat stress in geothermal areas in Yellowstone National Park. Plants were exposed to 20 degrees C (control) or 35 degrees C for 12d. The SSH analysis was performed with control samples as the driver and heat-stressed samples as the tester. Differentially expressed cDNA fragments were cloned to screen the heat up-regulated library. The SSH analysis identified 120 non-redundant putative heat-responsive cDNAs out of 1180 clones. Genes with homology to known proteins were categorized into six functional groups, with the largest group of genes involved in stress/defense, followed by the group of genes related to protein metabolism. Immunoblot analysis confirmed increases in transcripts of selected genes under heat stress. Transcripts of seven and eight genes were strongly enhanced or induced in shoots and roots, respectively, while two genes were only induced in roots under heat stress. The heat up-regulated genes in thermal A. scabra adapted to long-term heat stress are potential candidate genes for engineering stress-tolerant grasses and for revealing molecular mechanisms of grass adaptation to heat stress.

  10. High-Throughput Plasmid cDNA Library Screening

    Energy Technology Data Exchange (ETDEWEB)

    Wan, Kenneth H.; Yu, Charles; George, Reed A.; Carlson, JosephW.; Hoskins, Roger A.; Svirskas, Robert; Stapleton, Mark; Celniker, SusanE.

    2006-05-24

    Libraries of cDNA clones are valuable resources foranalysing the expression, structure, and regulation of genes, as well asfor studying protein functions and interactions. Full-length cDNA clonesprovide information about intron and exon structures, splice junctionsand 5'- and 3'-untranslated regions (UTRs). Open reading frames (ORFs)derived from cDNA clones can be used to generate constructs allowingexpression of native proteins and N- or C-terminally tagged proteins.Thus, obtaining full-length cDNA clones and sequences for most or allgenes in an organism is critical for understanding genome functions.Expressed sequence tag (EST) sequencing samples cDNA libraries at random,which is most useful at the beginning of large-scale screening projects.However, as projects progress towards completion, the probability ofidentifying unique cDNAs via EST sequencing diminishes, resulting in poorrecovery of rare transcripts. We describe an adapted, high-throughputprotocol intended for recovery of specific, full-length clones fromplasmid cDNA libraries in five days.

  11. Screening for metronidazole-resistance associated gene fragments of H pylori by suppression subtractive hybridization

    Institute of Scientific and Technical Information of China (English)

    Ai-Qing Li; Ning Dai; Jie Yan; Yong-Liang Zhu

    2007-01-01

    AIM:To screen for metronidazole (MTZ)-resistance associated gene fragments of H pylori by suppression subtractive hybridization (SSH).METHODS:Five MTZ-resistant (tester,T) and 1 MTZ-susceptible (driver,D) clinical H pylori isolates were selected. Genomic DMAs were prepared and submitted to Rsa I digestion. Then two different adaptors were ligated respectively to the 5'-end of two aliquots of the tester DNA fragments and SSH was made between the tester and driver DNAs. The specific inserts of tester strains were screened and MTZ-resistance related gene fragments were identified by dot blotting.RESULTS:Among the randomly selected 120 subtractive colonies,37 DNA fragments had a different number of DNA copies (≥2 times) in resistant and susceptible strains and 17 of them had a significantly different number of DNA copies (≥3 times). Among the sequences obtained from the 17 DNA fragments,new sequences were found in 10 DNA fragments and duplicated sequences in 7 DNA fragments,representing respectively the sequences of depeptide ABC transporter periplasmic dipeptide-binding protein (dppA),permease protein (dppB),ribosomal protein S4 (rps4),ribonuclease in (rnc),protease (pqqE),diaminopimelate epimerase (dapF),acetatekinase (ackA),H pylori plasmid pHPSl and H pylori gene 1334.CONCLUSION:Gene fragments specific to MTZ-resistant H pylori strains can be screened by SSH and may be associated with MTZ-resistant H pylori.

  12. Estabishment of A Human Liver Cancer Cell Line Transfected with IL-2 cDNA and Its Biologic Activity

    Institute of Scientific and Technical Information of China (English)

    孙跃明; 王学浩; 杜竞辉

    2001-01-01

    Objective To obtain IL-2 gene transfected human liver cancer cells and study IL-2 expression and its biologic activity in vivo. Methods Human liver cancer cells SMMC-7721 were cocultured with recombinant retroviral vector LNC-IL-2,and screening was performed in G418 medium.The exogenous IL-2 cDNA at the DNA,RNA,and protein levels were determined by using dot hybridization,PR-PCR and MTT methods respectively.The tumorigenesis and antitumorigenesis of the screened liver cancer cell with subcutaneous injection in nude mice were observed. Results and Conclusion The IL-2 cDNA was successfully integrated into SMMC-7721 cell genomic DNA and continuously expressed for more than 88 days.Subcutaneous vaccination of the nude mice with transfected cells revealed an obvious suppression of its tumorigenicity,and could induce antitumor activity in vivo.

  13. Analysis of differences of gene expressions in keloid and normal skin with the aid of cDNA microarray

    Institute of Scientific and Technical Information of China (English)

    Chen Wei; Fu Xiaobing; Sun Xiaoqing; Sun Tongzhu; Zhao Zhili; Yang Yinhui; Sheng Zhiyong

    2003-01-01

    Background: Microarray analysis is a popular tool to investigate the function of genes that are responsible for the phenotype of the disease. Keloid is a intricate lesion which is probably modulated by interplay of many genes. We ventured to study the differences of gene expressions between keloids and normal skins with the aid of cDNA microarray in order to explore the molecular mechanism underlying keloid formation. Methods: The PCR products of 8400 human genes were spotted on a chip in array. The DNAs were then fixed on the glass plate by a series of treatments. Total RNAs was isolated from freshly excised human keloids and normal skin, and then was purified to mRNA by Oligotex. Both the mRNA from keloids and normal skin was reversely transcribed to cDNAs with the incorporations of fluorescent dUTP, for preparing the hybridization probes. The mixed probes were then hybridized to the cDNA microarray. After highly stringent washing, the cDNA microarray was scanned for the fluorescent signals to display the differences between two kinds of tissues. Results: Among 8400 human genes, there were 402 genes (4.79%) with different expression levels between the keloids and normal skins in all cases, 250were up-regulated (2.98%) and 152 down-regulated (1.81%). Analyses of collagen, fibronectin, proteoglycan,growth factors and apoptosis related molecule gene expression confirmed that our molecular data obtained by cDNA microarray were consistent with published biochemical and clinical observations of keloids. Conclusions: DNA microarray technology is an effective technique in screening for differences in gene expression between keloid and normal skin. Many genes are involved in the formation of keloids. Further analysis of the obtained genes will help understand the molecular mechanism of keloid formation.

  14. ANALYSIS OF GENES ASSOCIATED WITH LYMPHATIC METASTASIS IN PANCREATIC CARCINOMA USING cDNA MICROARRAY

    Institute of Scientific and Technical Information of China (English)

    谭志军; 胡先贵; 曹贵松; 唐岩

    2003-01-01

    Objective: To identify new markers for prediction of lymph node metastasis. Methods: cDNA probes were prepared by labeling mRNA from samples of four pancreatic carcinoma tissues with Cy5-dUTP and mRNA from adjacent normal tissues with Cy3-dUTP respectively through reverse transcription. The mixed probes of each sample were then hybridized with 4,096 cDNA arrays (4,000 unique human cDNA sequences), and the fluorescent signals were scanned by ScanArray 3000 scanner (General Scanning, Inc.). The values of Cy5-dUTP and Cy3-dUTP on each spot were analyzed and calculated by ImaGene 3.0 software (BioDiscovery, Inc.). Genes that differentially expresses in each cancerous tissue were sought out according to the standard that the absolute value of natural logarithm of the ratio of Cy5 to Cy3 is greater than 0.69, i. e., more than 2 times change of gene expression, and the signal value of either Cy3 and Cy5 need to be greater than 600. Then, the genes differently expressed in cancer with and without lymphatic metastasis were screened out for further analysis. Results: Among 2 samples with lymphatic metastasis and 2 samples without metastasis, 56 genes, which accounted for 1.40% of genes on the microarray slides, exhibited differentially expression in cancerous tissues with lymphatic metastasis. There were 32 over-expressed genes including 11 having been registered in Genebank, and 24 under-expressed genes including 3 in Genebank. Conclusion: Microarray analysis may provide invaluable information to identify specific gene expression profile of lymphatic metastasis in pancreatic cancer.

  15. Dissection of the inflammatory bowel disease transcriptome using genome-wide cDNA microarrays.

    Directory of Open Access Journals (Sweden)

    Christine M Costello

    2005-08-01

    Full Text Available BACKGROUND: The differential pathophysiologic mechanisms that trigger and maintain the two forms of inflammatory bowel disease (IBD, Crohn disease (CD, and ulcerative colitis (UC are only partially understood. cDNA microarrays can be used to decipher gene regulation events at a genome-wide level and to identify novel unknown genes that might be involved in perpetuating inflammatory disease progression. METHODS AND FINDINGS: High-density cDNA microarrays representing 33,792 UniGene clusters were prepared. Biopsies were taken from the sigmoid colon of normal controls (n = 11, CD patients (n = 10 and UC patients (n = 10. 33P-radiolabeled cDNA from purified poly(A+ RNA extracted from biopsies (unpooled was hybridized to the arrays. We identified 500 and 272 transcripts differentially regulated in CD and UC, respectively. Interesting hits were independently verified by real-time PCR in a second sample of 100 individuals, and immunohistochemistry was used for exemplary localization. The main findings point to novel molecules important in abnormal immune regulation and the highly disturbed cell biology of colonic epithelial cells in IBD pathogenesis, e.g., CYLD (cylindromatosis, turban tumor syndrome and CDH11 (cadherin 11, type 2. By the nature of the array setup, many of the genes identified were to our knowledge previously uncharacterized, and prediction of the putative function of a subsection of these genes indicate that some could be involved in early events in disease pathophysiology. CONCLUSION: A comprehensive set of candidate genes not previously associated with IBD was revealed, which underlines the polygenic and complex nature of the disease. It points out substantial differences in pathophysiology between CD and UC. The multiple unknown genes identified may stimulate new research in the fields of barrier mechanisms and cell signalling in the context of IBD, and ultimately new therapeutic approaches.

  16. Mink serum amyloid A protein. Expression and primary structure based on cDNA sequences.

    Science.gov (United States)

    Marhaug, G; Husby, G; Dowton, S B

    1990-06-15

    The nucleotide sequences of two mink serum amyloid A (SAA) cDNA clones have been analyzed, one (SAA1) 776 base pairs long and the other (SAA2) 552 base pairs long. Significant differences were discovered when derived amino acid sequences were compared with data for apoSAA isolated from high density lipoprotein. Previous studies of mink protein SAA and amyloid protein A (AA) suggest that only one SAA isotype is amyloidogenic. The cDNA clone for SAA2 defines the "amyloid prone" isotype while SAA1 is found only in serum. Mink SAA1 has alanine in position 10, isoleucine in positions 24, 67, and 71, lysine in position 27, and proline in position 105. Residue 10 in mink SAA2 is valine while arginine and asparagine are at positions 24 and 27, respectively, all characteristics of protein AA isolated from mink amyloid fibrils. Mink SAA2 also has valine in position 67, phenylalanine in position 71, and amino acid 105 is serine. It remains unknown why these six amino acid substitutions render SAA2 more amyloidogenic than SAA1. Eighteen hours after lipopolysaccharide stimulation, mink SAA mRNA is abundant in liver with relatively minor accumulations in brain and lung. Genes encoding both SAA isotypes are expressed in all three organs while no SAA mRNA was detectable in amyloid prone organs, including spleen and intestine, indicating that deposition of AA from locally synthesized SAA is unlikely. A third mRNA species (2.2 kilobases) was identified and hybridizes with cDNA probes for mink SAA1 and SAA2. In addition to a major primary translation product (molecular mass 14,400 Da) an additional product with molecular mass 28,000 Da was immunoprecipitable.

  17. A potato skin SSH library yields new candidate genes for suberin biosynthesis and periderm formation.

    Science.gov (United States)

    Soler, Marçal; Serra, Olga; Fluch, Silvia; Molinas, Marisa; Figueras, Mercè

    2011-05-01

    Potato (Solanum tuberosum) tubers are underground storage organs covered by the skin or periderm, a suberized layer that protects inner flesh from dehydration and pathogens. Understanding the molecular processes associated with periderm formation is of great importance for a better knowledge of this protective tissue and for improving the storage life of tubers. Here, to isolate new candidate genes for potato periderm, a suppression subtractive hybridization library from potato skin was performed. This library yielded a comprehensive list of 108 candidate genes that were manually sorted in functional categories according to the main cellular and metabolic processes in periderm. As expected, the list contains Suberin and wax genes, including some genes with a demonstrated role in the biosynthesis of these cell wall aliphatic compounds. Moreover, Regulation and Stress and defence genes are highly abundant in the library in general agreement with previous potato skin proteomic studies. The putative function of the genes in periderm is discussed.

  18. Development and characterization of a high temperature stress responsive subtractive cDNA library in Pearl Millet Pennisetum glaucum (L.) R.Br.

    Science.gov (United States)

    James, Donald; Tarafdar, Avijit; Biswas, Koushik; Sathyavathi, Tara C; Padaria, Jasdeep Chatrath; Kumar, P Ananda

    2015-08-01

    Pearl millet (Pennisetum glaucum L. R. Br.) is an important cereal crop grown mainly in the arid and semi-arid regions of India known to possess the natural ability to withstand thermal stress. To elucidate the molecular basis of high temperature response in pearl millet, 12 days old seedlings of P. glaucum cv. 841A were subjected to heat stress at 46 degrees C for different time durations ( 30 min, 2, 4, 8, 12 and 24 h) and a forward subtractive cDNA library was constructed from pooled RNA of heat stressed seedlings. A total of 331 high quality Expressed Sequence Tags (ESTs) were obtained from randomly selected 1050 clones. Sequences were assembled into 103 unique sequences consisting of 37 contigs and 66 singletons. Of these, 92 unique sequences were submitted to NCBI dbEST database. Gene Ontology through RGAP data base and BLASTx analysis revealed that about 18% of the ESTs showed homology to genes for "response to abiotic and biotic stimulus". About 2% of the ESTs showed no homology with genes in dbEST, indicating the presence of uncharacterized candidate genes involved in heat stress response in P. glaucum. Differential expression of selected genes (hsp101 and CRT) from the SSH library were validated by qRT-PCR analysis. The ESTs thus generated are a rich source of heat stress responsive genes, which can be utilized in improving thermotolerance of other food crops.

  19. Differential representation of sunflower ESTs in enriched organ-specific cDNA libraries in a small scale sequencing project

    Directory of Open Access Journals (Sweden)

    Heinz Ruth A

    2003-09-01

    Full Text Available Abstract Background Subtractive hybridization methods are valuable tools for identifying differentially regulated genes in a given tissue avoiding redundant sequencing of clones representing the same expressed genes, maximizing detection of low abundant transcripts and thus, affecting the efficiency and cost effectiveness of small scale cDNA sequencing projects aimed to the specific identification of useful genes for breeding purposes. The objective of this work is to evaluate alternative strategies to high-throughput sequencing projects for the identification of novel genes differentially expressed in sunflower as a source of organ-specific genetic markers that can be functionally associated to important traits. Results Differential organ-specific ESTs were generated from leaf, stem, root and flower bud at two developmental stages (R1 and R4. The use of different sources of RNA as tester and driver cDNA for the construction of differential libraries was evaluated as a tool for detection of rare or low abundant transcripts. Organ-specificity ranged from 75 to 100% of non-redundant sequences in the different cDNA libraries. Sequence redundancy varied according to the target and driver cDNA used in each case. The R4 flower cDNA library was the less redundant library with 62% of unique sequences. Out of a total of 919 sequences that were edited and annotated, 318 were non-redundant sequences. Comparison against sequences in public databases showed that 60% of non-redundant sequences showed significant similarity to known sequences. The number of predicted novel genes varied among the different cDNA libraries, ranging from 56% in the R4 flower to 16 % in the R1 flower bud library. Comparison with sunflower ESTs on public databases showed that 197 of non-redundant sequences (60% did not exhibit significant similarity to previously reported sunflower ESTs. This approach helped to successfully isolate a significant number of new reported sequences

  20. Rice bicoid-related cDNA sequence and its expression during early embryogenesis

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    Bicoid is one of the important Drosophila maternal genes involved in the control of embryo polarity and larvae segmentation.To clone and characterize the rice bicoid-related genes,one cDNA clone,Rb24 (EMBL accession number: AJ2771380),was isolated by screening of rice unmature seed cDNA library.Sequence analysis indicates that Rb24 contains a putative amino acid sequence,which is homologous to unique 8 amino acids sequence within Drosophila bicoid homeodomain (50% identity,75% similarity) and involves a lys-9 in putative helix 3.Northern blot analysis of rice RNA has shown that this sequence is expressed in a tissue-specific manner.The transcript was detected strongly in young panicles,but less in young leaves and roots.This results are further confirmed with paraffin section in situ hybridization.The signal is intensive in rice globular embryo and located at the apical tip of the embryo,then,along with the development of embryo,the signal is getting reduced and transfers into both sides of embryo.The existence of bicoid-related sequence in rice embryo and the similarity of polar distribution of bicoid and Rb24 mRNA in early embryo development may implicates a conserved maternal regulation mechanism of body axis presents in Drosophila and in rice.

  1. Rabbit serum amyloid protein A: expression and primary structure deduced from cDNA sequences.

    Science.gov (United States)

    Rygg, M; Marhaug, G; Husby, G; Dowton, S B

    1991-12-01

    Serum amyloid A protein (SAA), the precursor of amyloid protein A (AA) in deposits of secondary amyloidosis, is an acute phase plasma apolipoprotein produced by hepatocytes. The primary structure of SAA demonstrates high interspecies homology. Several isoforms exist in individual species, probably with different amyloidogenic potential. The nucleotide sequences of two different rabbit serum amyloid A cDNA clones have been analysed, one (corresponding to SAA1) 569 base pairs (bp) long and the other (corresponding to SAA2) 513 bp long. Their deduced amino acid sequences differ at five amino acid positions, four of which are located in the NH2-terminal region of the protein. The deduced amino acid sequence of SAA2 corresponds to rabbit protein AA previously described except for one amino acid in position 22. Eighteen hours after turpentine stimulation, rabbit SAA mRNA is abundant in liver, while lower levels are present in spleen. None of the other extrahepatic organs studied showed any SAA mRNA expression. A third mRNA species (1.9 kb) hybridizing with a single-stranded RNA probe transcribed from the rabbit SAA cDNA, was identified. SAA1 and SAA2 mRNA were found in approximately equal amounts in turpentine-stimulated rabbit liver, but seem to be coordinately decreased after repeated inflammatory stimulation.

  2. Nucleotide sequence of cloned cDNA for human pancreatic kallikrein.

    Science.gov (United States)

    Fukushima, D; Kitamura, N; Nakanishi, S

    1985-12-31

    Cloned cDNA sequences for human pancreatic kallikrein have been isolated and determined by molecular cloning and sequence analysis. The identity between human pancreatic and urinary kallikreins is indicated by the complete coincidence between the amino acid sequence deduced from the cloned cDNA sequence and that reported partially for urinary kallikrein. The active enzyme form of the human pancreatic kallikrein consists of 238 amino acids and is preceded by a signal peptide and a profragment of 24 amino acids. A sequence comparison of this with other mammalian kallikreins indicates that key amino acid residues required for both serine protease activity and kallikrein-like cleavage specificity are retained in the human sequence, and residues corresponding to some external loops of the kallikrein diverge from other kallikreins. Analyses by RNA blot hybridization, primer extension, and S1 nuclease mapping indicate that the pancreatic kallikrein mRNA is also expressed in the kidney and sublingual gland, suggesting the active synthesis of urinary kallikrein in these tissues. Furthermore, the tissue-specific regulation of the expression of the members of the human kallikrein gene family has been discussed.

  3. Ultrasensitive cDNA detection of dengue virus RNA using electrochemical nanoporous membrane-based biosensor.

    Directory of Open Access Journals (Sweden)

    Varun Rai

    Full Text Available A nanoporous alumina membrane-based ultrasensitive DNA biosensor is constructed using 5'-aminated DNA probes immobilized onto the alumina channel walls. Alumina nanoporous membrane-like structure is carved over platinum wire electrode of 76 µm diameter dimension by electrochemical anodization. The hybridization of complementary target DNA with probe DNA molecules attached inside the pores influences the pore size and ionic conductivity. The biosensor demonstrates linear range over 6 order of magnitude with ultrasensitive detection limit of 9.55×10(-12 M for the quantification of ss-31 mer DNA sequence. Its applicability is challenged against real time cDNA PCR sample of dengue virus serotype1 derived from asymmetric PCR. Excellent specificity down to one nucleotide mismatch in target DNA sample of DENV3 is also demonstrated.

  4. cDNA cloning, chromosome mapping and expression characterization of human geranylgeranyl pyrophosphate synthase

    Institute of Scientific and Technical Information of China (English)

    赵勇[1; 余龙[2; 高洁[3; 付强[4; 华益民[5; 张宏来[6; 赵寿元[7

    2000-01-01

    Geranylgeranyl pyrophosphate (GGPP) mainly participates in post-translational modification for various proteins including Rho/Rac, Rap and Rab families, as well as in regulation for cell apoptosis. Geranylgeranyl pyrophosphate synthase (GGPPS), which catalyzes the condensation reaction between farnesyl diphosphate and isopentenyl diphosphate, is the key enzyme for synthesizing GGPP. We report the isolation of a gene transcript showing high homology with Drosophila GGPPS cDNA. The transcript is 1 466 bp in length and contains an intact open reading frame (ORF) ranging from nt 239 to 1 138. This ORF encodes a deduced protein of 300 residues with calculated molecular weight of 35 ku. The deduced protein shows 57.5% identity and 75% similarity with Drosophila GGPPS, and contains five characteristic domains of prenyltransferases. Northern hybridization revealed that human GGPPS was expressed highest in heart, and moderately in spleen, testis, brain, placenta, lung, liver, skeletal muscle, kidney and pancreas

  5. Differential screening of mitochondrial cDNA libraries from male-fertile and cytoplasmic male-sterile sugar-beet reveals genome rearrangements at atp6 and atpA loci.

    Science.gov (United States)

    Xue, Y; Collin, S; Davies, D R; Thomas, C M

    1994-04-01

    As part of a strategy to define differences in genome organization and expression between cytoplasmic male-sterile (CMS) and male-fertile (MF) sugar-beet mitochondria, cDNA libraries from both mitochondrial genotypes were constructed. Preliminary screening with ribosomal RNA gene probes identified candidate cDNA clones corresponding to structural genes. In addition, reciprocal hybridization experiments were performed using labelled first-strand cDNA to identify uniquely transcribed sequences. One cDNA clone (pYC700) is unique to CMS mitochondria and is located upstream of the F0F1-ATPase subunit 6 gene (atp6). Another cDNA clone (pYC130), when used as a probe in northern hybridization analysis, revealed novel transcript profiles in CMS sugar-beet mitochondria. Sequence analysis of this cDNA showed strong homology with the F0F1-ATPase subunit alpha (atpA) coding sequences from several higher plants. The atp6 and atpA loci from each genotype were cloned and the genomic organization, DNA sequence and transcription of each locus was studied. Differences in the transcript profiles of each gene are a consequence of genomic rearrangements 5' to the coding sequence.

  6. [Construction of root library by SSH and preliminary analysis of genes responsible for phosphorus deficiency in maize].

    Science.gov (United States)

    Huang, Q; Gao, S B; Zhang, Z M; Lin, H J; Pan, G T; Yang, K C; Rong, T Z

    2010-12-01

    An elite maize inbred line with high tolerance to low phosphorus, 178, was studied for constructing root library and analyzing some genes closely related to phosphorus (P) deficiency using SSH and Semi-quantitative RT-PCR. As a result, 3648 preliminary clones were obtained for root library under stress of P deficiency. By DNA sequencing of 34 random clones, we obtained 23 unique EST sequences which are involved in functions of root cell structure, tolerance and defense, protein modification and composition, transcription regulation, metabolism, and other unknown aspects. Five representative genes were further analyzed for their expression models. The results suggested that the molecular mechanism to adapt P deficiency in maize, performed by multi-genes with different contributions, is similar to rice, Arabidopsis and soybean. The expression order of 5 low P tolerant genes in maize root was PAP, GCS, TOM, PDI and AIP. And it was considered preliminarily that physiological and biochemical changes were prior to morphologic changes in maize root and the essential tolerance to low P may be determined by extending absorption of P to wide soil range through adaption of root architecture and root secretions, which is the greatest difference between tolerant and sensitive maize varieties under low P stress.

  7. Sequence characterization of a human embryonic craniofacial cDNA library

    Energy Technology Data Exchange (ETDEWEB)

    Padanilam, B.J.; Barsel, S.; Solursh, M. [and others

    1994-09-01

    Broad-based sequencing approaches for the characterization of human cDNA libraries have proven successful in identifying large numbers of novel genes of specific tissue or developmental stages. To pursue our interests in human craniofacial development, stages. To pursue our interests in human craniofacial development, we have made use of both subtracted and unsubtracted cDNA libraries constructed from embryonic craniofacial tissue obtained from pooled samples at 42-54 days gestation. Single-pass sequencing was carried out using an ABI automated sequencer and T3 or T7 primers. Sequences were characterized using BLAST and GRAIL, and the identified homologous sequences grouped according to gene class and family. Four genes have been mapped using repeat sequence elements identified in the clones. Using primers developed from sequence data, other genes are being mapped using a panel of somatic cell hybrids. To date, a total of 786 sequences have been returned with 35% identifying no homologies, and 35% with strong homologies to previously identified genes. A number of genes previously identified to play a role in human embryonic development have been returned from the sequence comparisons providing evidence that the library is representative of this tissue and stage of development. Previous characterization of the library has also identified a number of novel embryonically expressed human homeobox genes. Genes felt to be of special relevance based on their homology to characterized genes known to play a role in development or that are members of novel classes but with high scores on GRAIL searches are being characterized using whole mount in situ hybridization with mouse embryos. Characterization of the library with respect to chromosomal mapping, gene types and make-up, and embryonic expression patterns will be presented.

  8. Novel potential ALL low-risk markers revealed by gene expression profiling with new high-throughput SSH-CCS-PCR.

    Science.gov (United States)

    Qiu, J; Gunaratne, P; Peterson, L E; Khurana, D; Walsham, N; Loulseged, H; Karni, R J; Roussel, E; Gibbs, R A; Margolin, J F; Gingras, M C

    2003-09-01

    The current systems of risk grouping in pediatric acute lymphoblastic leukemia (ALL) fail to predict therapeutic success in 10-35% of patients. To identify better predictive markers of clinical behavior in ALL, we have developed an integrated approach for gene expression profiling that couples suppression subtractive hybridization, concatenated cDNA sequencing, and reverse transcriptase real-time quantitative PCR. Using this approach, a total of 600 differentially expressed genes were identified between t(4;11) ALL and pre-B ALL with no determinant chromosomal translocation. The expression of 67 genes was analyzed in different cytogenetic ALL subgroups and B lymphocytes isolated from healthy donors. Three genes, BACH1, TP53BPL, and H2B/S, were consistently expressed as a significant cluster associated with the low-risk ALL subgroups. A total of 42 genes were differentially expressed in ALL vs normal B lymphocytes, with no specific association with any particular ALL subgroups. The remaining 22 genes were part of a specific expression profile associated with the hyperdiploid, t(12;21), or t(4;11) subgroups. Using an unsupervised hierarchical cluster analysis, the discriminating power of these specific expression profiles allowed the clustering of patients according to their subgroups. These genes could help to understand the difference in treatment response and become therapeutical targets to improve ALL clinical outcomes.

  9. PG25, a pineal-specific cDNA, cloned by differential display PCR (DDPCR) and rapid amplification of cDNA ends (RACE).

    Science.gov (United States)

    Wang, X; Brownstein, M J; Young, W S

    1997-05-16

    Synthesis of melatonin in the mammalian pineal gland is regulated by the rhythmic expression of acetyl-CoA: serotonin N-acetyltransferase (SNAT) and other unknown factors. To screen for pineal-specific mRNAs potentially involved in melatonin synthesis and/or regulation, differential display PCR (DDPCR) was employed. We used 80 primer pairs and examined 40 bands of interest. One of the pineal specific clones (relative to brain and eye), PG25, was studied further. Hybridization histochemical and Northern analyses confirmed its tissue specificity. The size of the corresponding mRNA is 2.4 kb. A cDNA (2 kb) containing the coding region was obtained using a long-template PCR-based RACE technique. A data base search indicates that PG25 is highly homologous to a recently identified human lung endothelial cell-specific gene, ESM-1. Interestingly, not only the amino acid sequences but also the cDNA sequences, including the long 3' untranslated regions, are highly similar. This suggests that the conserved 3' untranslated region may carry information to regulate its own expression. Northern analysis revealed that PG25 is also expressed in the rat lung, but at a much lower (10%) level compared to the pineal. Finally, our work shows the feasibility of a fast, integrated PCR-based cloning method for obtaining long, potentially full-length cDNAs with restricted expression in anatomically complex regions of the brain. This protocol combining several existing methodologies is suitable for use with limited tissue sources and uses minimal amounts of isotopes.

  10. Rapid amplification of cDNA ends (RACE).

    Science.gov (United States)

    Yeku, Oladapo; Frohman, Michael A

    2011-01-01

    Rapid Amplification of cDNA ends (RACE) provides an inexpensive and powerful tool to quickly obtain full-length cDNA when the sequence is only partially known. Starting with an mRNA mixture, gene-specific primers generated from the known regions of the gene and non-specific anchors, full-length sequences can be identified in as little as 3 days. RACE can also be used to identify alternative transcripts of a gene when the partial or complete sequence of only one transcript is known. In the following sections, we outline details for rapid amplification of 5(') and 3(') cDNA ends using the "new RACE" technique.

  11. Identification of Differentially Expressed Genes During Ethylene Climacteric of Melon Fruit by Suppression Subtractive Hybridization

    Institute of Scientific and Technical Information of China (English)

    GAO Feng; NIU Yi-ding; HAO Jin-feng; BADE Rengui; ZHANG Li-quan; HASI Agula

    2013-01-01

    Melon (Cucumis melo L.) is an important horticultural crop worldwide. Ethylene regulates the ripening process and affects the ripening rate. To screen genes that are differentially expressed at the burst of ethylene climacteric in melon fruit, we performed suppression subtractive hybridization (SSH) to generate forward and reverse libraries, for which we sequenced 439 and 445 clones, respectively. Our BLAST analysis showed that the genes from the 2 libraries were involved in metabolism, signal transduction, cell structure, transcription, translation, and defense. Six genes were analyzed by qRT-PCR during the differential developmental stage of melon fruit. Our results provide new insight into the understanding of climacteric ripening of melon fruit.

  12. Rainbow trout cytochrome P-450c17 (17 alpha-hydroxylase/17,20-lyase). cDNA cloning, enzymatic properties and temporal pattern of ovarian P-450c17 mRNA expression during oogenesis.

    Science.gov (United States)

    Sakai, N; Tanaka, M; Adachi, S; Miller, W L; Nagahama, Y

    1992-04-13

    A cDNA clone encoding cytochrome P-450c17 (17 alpha-hydroxylase/17,20-lyase) was isolated from a rainbow trout ovarian follicle cDNA library. The cDNA contained an open reading frame of 1,542 nucleotides encoding a protein of 514 amino acid residues. The amino acid sequence of trout P-450c17 shows a much greater homology with chicken P-450c17 than with that of human, bovine and rat. The trout P-450c17 expressed in non-steroidogenic mammalian COS-1 cells showed both 17 alpha-hydroxylase and 17,20-lyase activities. The cDNA only hybridized to a single species of mRNA (2.4 kb) isolated from rainbow trout ovaries; the 2.4 kb transcripts were abundant in trout ovaries during the later stages of oogenesis.

  13. Hybrid Baryons

    CERN Document Server

    Page, P R

    2003-01-01

    We review the status of hybrid baryons. The only known way to study hybrids rigorously is via excited adiabatic potentials. Hybrids can be modelled by both the bag and flux-tube models. The low-lying hybrid baryon is N 1/2^+ with a mass of 1.5-1.8 GeV. Hybrid baryons can be produced in the glue-rich processes of diffractive gamma N and pi N production, Psi decays and p pbar annihilation.

  14. ISOLATION AND IDENTIFICATION OF cDNA FRAGMENTS AND FULL-LENGTH cDNA DIFFERENTIALLY EXPRESSEDIN HUMAN GLIOBLASTOMA CELL LINE BT-325 VERSUS ALL-TRANS RETINOIC ACID INDUCTION

    Institute of Scientific and Technical Information of China (English)

    金虎林; 胡松年; 李光涛; 涂纯; 袁建刚; 强伯勤

    2000-01-01

    Objective. To investigate the differentiation process of the human glioblastoma cells. Methods. Differential display reverse transcribed-PCR(DDRT-PCR) was used to isolate the genes differentially expressed in control and all-trans retinoic acid treated human glioblastoma cell line BT-325. Routine method of cDNA library screening was performed to clone full-length cDNA. Results. Thirty-six RT-PCR reactions were performed and 64 differentially expressed fragments were recovered, amplified and cloned. Of them,46 ESTs were sequenced and delivered into the GenBank. The homology comparison us-ing BLAST algorithm revealed that 22ESFs are highly homologous with the known genes and many of them play impor-tant roles in the cell differentiation progress. A dot-blot hybridization was conducted to certify the differemiation expres-sion. The result showed that 27 EST clones are expressed at different level in control and all-traus retinoic acid treated BT-325 cells. A full-length cDNA was cloned using the EST-HGBB098.Conclusion. DDRT-PCR was a simple and effective method to segally analyze the differentially expressed genes.

  15. ISOLATION AND IDENTIFICATION OF cDNA FRAGMENTS AND FULL-LENGTH cDNA DIFFERENTIALLY EXPRESSED IN HUMAN GLIOBLASTOMA CELL LINE BT-325 VERSUS ALL-TRANS RETINOIC ACID INDUCTION

    Institute of Scientific and Technical Information of China (English)

    金虎林; 胡松年; 李光涛; 涂纯; 袁建刚; 强伯勤

    2000-01-01

    Objective. To investigate the differentiation process of the human glioblastoma cells. Methods. Differential display reverse transcribed-PCR(DDRT-PCR) was used to isolate the genes differentially expressed in control and all-trans retinoic acid treated human glioblastoma cell line BT-325. Routine method of cDNA library screening was performed to clone full-length cDNA. Results. Thirty-six RT-PCR reactions were performed and 64 differentially expressed fragments were recovered, amplified and cloned. Of them,46 ESTs were sequenced and delivered into the GenBank. The homology comparison us ing BLAST algorithm revealed that 22ESTs are highly homologous with the known genes and many of them play impor tant roles in the cell differentiation progress. A dot-blot hybridization was conducted to certify the differentiation expres sion. The result showed that 27 EST clones are expressed at different level in control and all-trans retinoi c acid treated BT-325 cells. A full-length cDNA was cloned using the ES T-HGBB098. Conclusion. DDRT-PCR was a simple and effective method to serially analyze the differentially expressed genes.

  16. Construction of cDNA subtractive library between yeast and mycelium phase of Sporothrix schenckii and screening of differently expressed genes about dimorphic transition%申克孢子丝菌菌丝相和酵母相cDNA消减文库的构建及差异表达基因的筛选

    Institute of Scientific and Technical Information of China (English)

    周汛; 杨致邦; 肖异珠

    2011-01-01

    The purpose of this study was to screen the differentially expressed genes about dimorphic transition of Sporothrix schenckii by construction of eDNA subtractive library between yeast and mycelium phase.The eDNA subtractive library between yeast and mycelium phase was constructed by suppression subtractive hybridization(SSH)and bioinformatics analysis was performed to profile the relationship between those differently expressed genes and dimorphic transition.Of 751 and 875 ESTs were obtained in M+Y and Y+M library, separately.After splicing of ESTs, 101 and 249 unigenes were obtained in M +Y and Y+ M libraries.During the construction of cDNA subtractive libraries, the distribution of differently expressed genes varied with dimorphic transition.The over expressed genes with diversity and complexity were divided into structural genes,metabolic enzymes, molecule on cell surface and molecule with indistinct function.Results disclose that construction of cDNA subtractive libraries for the dimorphic transition and bioinformatics analysis for those differently expressed genes lay the foundation for screening the related genes involved in the pathogenesis of Sporothrix schenckii infection.%目的 构建申克孢子丝菌双相cDNA消减文库,筛选与双相转换相关的差异表达基因.方法 运用抑制性消减杂交技术,构建申克孢子丝菌菌丝相(Mycelium,M)和酵母相(Yeast,Y)的正反cDNA消减文库,并对其差异表达的基因进行生物信息学分析.结果 M+Y文库获得751条表达序列标签(Expressed Sequence Tags,ESTs),经拼接后获得101条unigenes;Y+M文库获得875条ESTs,拼接获得249条unigenes.申克孢子丝菌酵母相菌丝相的转换伴随着不同菌相细胞差异基因的高表达,这些高表达的差异基因可分为结构基因类、代谢酶类、细胞表面分子类及功能不明的细胞分子.结论 成功构建了申克孢子丝菌双相转换相关的cDNA消减文库基础上,筛选出部分差异表达基

  17. Construction and Analyses of SSH cDNA Libraries of Rose Floral Color and Scent Mutant%月季突变体抑制差减杂交cDNA文库构建及分析

    Institute of Scientific and Technical Information of China (English)

    谢吉容; 梁国鲁; 唐开学; 张灏; 程在全; 黄兴奇

    2007-01-01

    为了分析月季花色花香突变机理和揭示花色花香代谢的相关基因,利用抑制性消减杂交技术分离了月季红花无香突变体'往日情怀'(以下简称突变体)与其野生型金黄色浓香品系'金银岛'(以下简称野生型)之间表达差异cDNA片段.分别以突变体作为驱赶子,野生型为检测子,以及以野生型作为驱赶子,突变体为检测子建立了两个差异表达cDNA文库WSSH和JSSH,分别代表在突变体和野生型中特异表达的cDNA;再经文库高密度点阵膜的杂交差示筛选分析,在WSSH库中获得特异表达的27个阳性克隆,在JSSH文库中得到25个阳性克隆.差异表达克隆测序后经BLAST比对分析发现WSSH文库中含有与红花突变体的花青素积累直接相关的CHS、DFR、细胞色素P450加单氧酶、乙二醛酶I、己糖转移因子、MYB1转录因子、S-腺苷蛋氨酸转移酶、ADR等花色相关EST;JSSH文库中含有与野生型花香形成相关的月季甲基间苯二酚O-甲基转移酶、转醛醇酶、Acyl-CoA结合蛋白、钙调素结合蛋白、MYB92转录因子的EST以及导致芽变的Tyl-copia-like逆转座子.

  18. Gene expression of Echinococcus granulosus under H2O2 stress using SSH technology%应用SSH技术研究H2O2胁迫下细粒棘球蚴基因的表达

    Institute of Scientific and Technical Information of China (English)

    侯秋莲; 王慧; 张壮志; 李江伟; 张富春; 张文宝

    2008-01-01

    目的 构建H2O2胁迫下细粒棘球蚴(Echinococcus granulosus)与正常组织差异表达的消减cDNA文库.方法 以H2O2胁迫细粒棘球蚴cDNA为试验方(tester),正常生长的细粒棘球蚴cDNA为驱动方(driver),应用抑制性消减杂交技术(suppression subtractive hybridization,SSH)研究H2O2胁迫下细粒棘球蚴基因的表达.结果 文库扩增后得到124个阳性克隆,菌落PCR分析,均得到200~1 000 bp插入片段.将整个文库克隆进行测序,测得序列结果 利用BLAST在线软件与GenBank数据库进行同源序列比对分析和BlastX分析.结果 获得重要基因的cDNA序列,如氧化还原酶、蛋白激酶、生长因子等.另有部分克隆在GenBank中无法查到对应的同源基因,可能代表了新基因.结论 成功构建了H2O2胁迫与正常组织差异表达的消减cDNA文库,为研究细粒棘球蚴在抗氧化过程中的相关靶基因筛选奠定基础.

  19. Characterization of a novel cDNA encoding a short venom peptide derived from venom gland of scorpion Buthus martensii Karsch: trans-splicing may play an important role in the diversification of scorpion venom peptides.

    Science.gov (United States)

    Zeng, Xian-Chun; Luo, Feng; Li, Wen-Xin

    2006-04-01

    A novel cDNA clone (named BmKT-u) which is a hybrid molecule of the 5'-terminal region of BmKT' cDNA and the 3'-terminal region of an undocumented cDNA (named BmKu), was isolated from a cDNA library made from the venom gland of scorpion Buthus martensii Karsch. BmKT-u codes for a 30 amino acid residue precursor peptide composed of a 20-residue signal sequence, and a putative 10-residue novel mature peptide. Northern blot hybridization showed BmKT-u cDNA is generated from a transcript. RT-PCR experiments excluded the possibility that BmKT-u cDNA is an artifact generated during reverse transcription. Genomic amplifications performed with three pairs of BmKT-u gene-specific primers showed the BmKT-u gene does not exist in the genome of the scorpion as a single transcriptional unit. Genomic cloning for BmKT' showed that the BmKT' gene contains an intron of 509 bp inserted into the region encoding the C-terminal region of the signal peptide. A sequence alignment comparison of the cDNA of BmKT-u with genomic BmKT' revealed that the junction site of the hybrid molecule is located at the 5'-splicing site of the intron. The data suggest that the BmKT-u transcript is a naturally occurring mature mRNA that is generated by trans-splicing. Trans-splicing may contribute to the diversity of venom peptides from venomous animals.

  20. The electronic spectrum of graphene nanoribbons with zigzag edges based on the SSH model%基于 SSH 模型的锯齿型边石墨烯纳米带电子能谱研究磁

    Institute of Scientific and Technical Information of China (English)

    陈琼; 童国平; 李盛

    2014-01-01

    利用SSH( Su-Schrieffer-Heeger)模型研究了2条聚乙炔链键与键平行和非平行结构耦合的电子能谱。两者的电子能谱与耦合强度之间有类似关系,但平行结构受耦合强度的影响更大些。在此基础上,将耦合链模型应用于锯齿型石墨烯纳米带的电子能谱计算,并给出了解析色散关系。同时,还研究了电子能谱与其耦合强度的关系,耦合越强,能带越宽。%The electronic spectrum of two coupled polyacetylene chains with bond-bond parallel and non-paral-lel was studied by using the SSH ( Su-Schrieffer-Heeger) model.Both the spectra were related to the coupling intensity, but the effect of the bond-bond parallel structure on the spectrum was obvious compared to the non-parallel .Based on this model, the electronic spectrum of graphene nanoribbons with zigzag edges was calculat-ed and the energy dispersion relation was derived.The relationship between the spectrum and the coupling in-tensity was also discussed, which showed that the stronger the coupling was the wider the energy band ap-peared.

  1. Cloning and expression of core gene cDNA of Chinese hepatitis C virus in cosmid pTM3

    Institute of Scientific and Technical Information of China (English)

    Rong Long Jiang; Qiao Sheng Lu; Kang Xian Luo

    2000-01-01

    AIM To clone core gene cDNA of Chinese hepatitis C virus ( HCV ) into eukaryotic expression vector cosmid pTM3 and to express HCV core antigen in HepG2 cells. METHODS Core gene cDNA of HCV was introduced into eukaryotic expression vector cosmid pTM3. Using vaccinia virus/bacteriophage T7 hybrid expression system,HepG2 cells were transfected with the recombinant plasmid pTM3-Q534 by lipofectin. RESULTS From the transfected bacteria Top10F′, 2 pTM3-Q534 clones containing the recombinant plasmid were identified from randomly selected 10 ampicillin-resistant colonies. By reverse transcription PCR and indirect immunofluorescence technique, HCV RNA and core protein was identified in HepG2 cells transfected with the recombinant plasmid.CONCLUSION The construction of a recombinant plasmid and the expression of core gene cDNA of HCV in HepG2 was successful.

  2. Human platelet/erythroleukemia cell prostaglandin G/H synthase: cDNA cloning, expression, and gene chromosomal assignment

    Energy Technology Data Exchange (ETDEWEB)

    Funk, C.D.; Funk, L.B.; Kennedy, M.E.; Pong, A.S.; Fitzgerald, G.A. (Vanderbilt Univ., Nashville, TN (United States))

    1991-06-01

    Platelets metabolize arachidonic acid to thromboxane A{sub 2}, a potent platelet aggregator and vasoconstrictor compound. The first step of this transformation is catalyzed by prostaglandin (PG) G/H synthase, a target site for nonsteroidal antiinflammatory drugs. We have isolated the cDNA for both human platelet and human erythroleukemia cell PGG/H synthase using the polymerase chain reaction and conventional screening procedures. The cDNA encoding the full-length protein was expressed in COS-M6 cells. Microsomal fractions from transfected cells produced prostaglandin endoperoxide derived products which were inhibited by indomethacin and aspirin. Mutagenesis of the serine residue at position 529, the putative aspirin acetylation site, to an asparagine reduced cyclooxygenase activity to barely detectable levels, an effect observed previously with the expressed sheep vesicular gland enzyme. Platelet-derived growth factor and phorbol ester differentially regulated the expression of PGG/H synthase mRNA levels in the megakaryocytic/platelet-like HEL cell line. The PGG/H synthase gene was assigned to chromosome 9 by analysis of a human-hamster somatic hybrid DNA panel. The availability of platelet PGG/H synthase cDNA should enhance our understanding of the important structure/function domains of this protein and it gene regulation.

  3. Identification of genes differentially expressed by Metarhizium anisopliae growing on Locusta migratoria wings using suppression subtractive hybridization.

    Science.gov (United States)

    Zhang, Chuanbo; Xia, Yuxian; Li, Zhongyuan

    2011-05-01

    Insect-pathogenic fungi penetrate their hosts directly through the cuticle. To better understand this process, we identified genes that were up-regulated by Metarhizium anisopliae germinating and differentiating on Locusta migratoria wings using suppression subtractive hybridization (SSH). A total of 78 unique expressed sequence tags (ESTs) up-regulated more than twofold during fungal growth on locust wings were identified. Among these 78 ESTs, 30 (38.5%) shared significant similarity with NCBI annotated hypothetical proteins, 16 (20.5%) shared low similarity to known or predicted genes, might represent novel genes, and 32 (41.0%) shared significant similarity with known proteins that are involved in various cell and molecular processes such as cell metabolism, protein metabolism, stress response and defense, and cell structure and function. Semi-quantitative RT-PCR analysis of six randomly selected genes confirmed the SSH results, verifying the fidelity of the SSH data. The results of this study provide novel information on genes expressed during early stages of infection with M. anisopliae, and improve current understanding of fungal pathogenesis.

  4. A BIOINFORMATIC STRATEGY TO RAPIDLY CHARACTERIZE CDNA LIBRARIES

    Science.gov (United States)

    A Bioinformatic Strategy to Rapidly Characterize cDNA LibrariesG. Charles Ostermeier1, David J. Dix2 and Stephen A. Krawetz1.1Departments of Obstetrics and Gynecology, Center for Molecular Medicine and Genetics, & Institute for Scientific Computing, Wayne State Univer...

  5. NORMAL NASAL GENE EXPRESSION LEVELS USING CDNA ARRAY TECHNOLOGY

    Science.gov (United States)

    Normal Nasal Gene Expression Levels Using cDNA Array Technology. The nasal epithelium is a target site for chemically-induced toxicity and carcinogenicity. To detect and analyze genetic events which contribute to nasal tumor development, we first defined the gene expressi...

  6. Rescue of mumps virus from cDNA.

    Science.gov (United States)

    Clarke, D K; Sidhu, M S; Johnson, J E; Udem, S A

    2000-05-01

    A complete DNA copy of the genome of a Jeryl Lynn strain of mumps virus (15,384 nucleotides) was assembled from cDNA fragments such that an exact antigenome RNA could be generated following transcription by T7 RNA polymerase and cleavage by hepatitis delta virus ribozyme. The plasmid containing the genome sequence, together with support plasmids which express mumps virus NP, P, and L proteins under control of the T7 RNA polymerase promoter, were transfected into A549 cells previously infected with recombinant vaccinia virus (MVA-T7) that expressed T7 RNA polymerase. Rescue of infectious virus from the genome cDNA was demonstrated by amplification of mumps virus from transfected-cell cultures and by subsequent consensus sequencing of reverse transcription-PCR products generated from infected-cell RNA to verify the presence of specific nucleotide tags introduced into the genome cDNA clone. The only coding change (position 8502, A to G) in the cDNA clone relative to the consensus sequence of the Jeryl Lynn plaque isolate from which it was derived, resulting in a lysine-to-arginine substitution at amino acid 22 of the L protein, did not prevent rescue of mumps virus, even though an amino acid alignment for the L proteins of paramyxoviruses indicates that lysine is highly conserved at that position. This system may provide the basis of a safe and effective virus vector for the in vivo expression of immunologically and biologically active proteins, peptides, and RNAs.

  7. 应用抑制性消减杂交技术筛选转化生长因子β1刺激LX02细胞的反式调节基因%Screening and cloning genes transactivated by TGF beta 1 in hepatic stellate cells using suppression subtractive hybridization technique

    Institute of Scientific and Technical Information of China (English)

    肖琳; 张跃新; 张建龙; 李燕; 成军; 郭江; 张黎颖; 洪源; 伦永志; 蓝贤勇; 武会娟; 张丽娟

    2008-01-01

    目的 构建转化生长因子(TGF)β1刺激大鼠肝星状细胞(LX02)反式调节基因的cDNA消减文库,筛选并克隆TGF β1反式调节相关基因,以阐明TGF β1介导肝纤维化的分子生物学机制.方法 以TGF β1刺激LX02细胞,同时以磷酸盐缓冲液刺激的LX02细胞作为对照.提取mRNA并逆转录为cDNA,经Rsa Ⅰ酶切后,将实验组cDNA分成两组,分别与两种不同的接头衔接,再与对照组cDNA进行两次消减杂交及两次抑制性多聚酶链反应.将产物与pGEM-Teasy载体连接,构建cDNA消减文库,并转染大肠杆菌进行文库扩增;随机挑选克隆经PCR扩增后进行测序及同源性分析. 结果成功构建了TGF β1刺激LX02细胞反式调节基因的cDNA消减文库.文库扩增后得到146个200~1000bp插入片段的阳性克隆;随机挑取其中35个克隆进行测序,30个列序成功,并通过生物信息学分析发现有28个与已知基因序列和2个与未知功能基因序列高度同源.结论 应用抑制性消减杂交技术成功构建了TGF β1刺激LX02细胞反式调节基因的cDNA消减文库,筛选到一些与细胞生长调节、蛋白质合成,信号传导、细胞外基质代谢、扰脂质过氧化等密切相关的蛋白质编码基因,为进一步阐明TGF β1介导肝纤维化的分子生物学机制提供了线索.%Objectives To construct a eDNA subtractive library of genes transactivated by TGF beta 1 in LX02 hepatic stellate cells (HSC); to screen and to clone the regulated genes transactivated by TGF beta 1; and to elucidate the molecular biological mechanism of hepatic fibrosis mediated by TGF beta 1. Methods mRNA was isolated from HSC treated with TGF beta 1 or with PBS (as controls). Suppression subtractive hybridization (SSH) technique was employed to analyze the differentially expressed DNA sequence between the two groups. After restriction enzyme Rsa I digestion, small size cDNAs were obtained. Then tester cDNA was divided into two groups and ligated

  8. Identifying genes related to choriogenesis in insect panoistic ovaries by Suppression Subtractive Hybridization

    Directory of Open Access Journals (Sweden)

    Bellés Xavier

    2009-04-01

    Full Text Available Abstract Background Insect ovarioles are classified into two categories: panoistic and meroistic, the later having apparently evolved from an ancestral panoistic type. Molecular data on oogenesis is practically restricted to meroistic ovaries. If we aim at studying the evolutionary transition from panoistic to meroistic, data on panoistic ovaries should be gathered. To this end, we planned the construction of a Suppression Subtractive Hybridization (SSH library to identify genes involved in panoistic choriogenesis, using the cockroach Blattella germanica as model. Results We constructed a post-vitellogenic ovary library by SSH to isolate genes involved in choriogenesis in B. germanica. The tester library was prepared with an ovary pool from 6- to 7-day-old females, whereas the driver library was prepared with an ovary pool from 3- to 4-day-old females. From the SSH library, we obtained 258 high quality sequences which clustered into 34 unique sequences grouped in 19 contigs and 15 singlets. The sequences were compared against non-redundant NCBI databases using BLAST. We found that 44% of the unique sequences had homologous sequences in known genes of other organisms, whereas 56% had no significant similarity to any of the databases entries. A Gene Ontology analysis was carried out, classifying the 34 sequences into different functional categories. Seven of these gene sequences, representative of different categories and processes, were chosen to perform expression studies during the first gonadotrophic cycle by real-time PCR. Results showed that they were mainly expressed during post-vitellogenesis, which validates the SSH technique. In two of them corresponding to novel genes, we demonstrated that they are specifically expressed in the cytoplasm of follicular cells in basal oocytes at the time of choriogenesis. Conclusion The SSH approach has proven to be useful in identifying ovarian genes expressed after vitellogenesis in B. germanica. For

  9. cDNA sequence analysis of a 29-kDa cysteine-rich surface antigen of pathogenic Entamoeba histolytica

    Energy Technology Data Exchange (ETDEWEB)

    Torian, B.E.; Stroeher, V.L.; Stamm, W.E. (Univ. of Washington, Seattle (USA)); Flores, B.M. (Louisiana State Univ. Medical Center, New Orleans (USA)); Hagen, F.S. (Zymogenetics Incorporated, Seattle, WA (USA))

    1990-08-01

    A {lambda}gt11 cDNA library was constructed from poly(U)-Spharose-selected Entamoeba histolytica trophozoite RNA in order to clone and identify surface antigens. The library was screened with rabbit polyclonal anti-E. histolytica serum. A 700-base-pair cDNA insert was isolated and the nucleotide sequence was determined. The deduced amino acid sequence of the cDNA revealed a cysteine-rich protein. DNA hybridizations showed that the gene was specific to E. histolytica since the cDNA probe reacted with DNA from four axenic strains of E. histolytica but did not react with DNA from Entamoeba invadens, Acanthamoeba castellanii, or Trichomonas vaginalis. The insert was subcloned into the expression vector pGEX-1 and the protein was expressed as a fusion with the C terminus of glutathione S-transferase. Purified fusion protein was used to generate 22 monoclonal antibodies (mAbs) and a mouse polyclonal antiserum specific for the E. histolytica portion of the fusion protein. A 29-kDa protein was identified as a surface antigen when mAbs were used to immunoprecipitate the antigen from metabolically {sup 35}S-labeled live trophozoites. The surface location of the antigen was corroborated by mAb immunoprecipitation of a 29-kDa protein from surface-{sup 125}I-labeled whole trophozoites as well as by the reaction of mAbs with live trophozoites in an indirect immunofluorescence assay performed at 4{degree}C. Immunoblotting with mAbs demonstrated that the antigen was present on four axenic isolates tested. mAbs recognized epitopes on the 29-kDa native antigen on some but not all clinical isolates tested.

  10. Cloning, tissue expression pattern characterization and chromosome localization of human peptide methionine sulfoxide reductase cDNA

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Oxidation and reduction of some amino acids are one of the molecular mechanisms for regulating the function of proteins. The oxidation of methionine (Met) to methionine sulfoxide (Met(O)) results in decreasing or loss of the biological activity of related proteins. It was found that peptide methionine sulfoxide reductase (msrA) can reduce Met(O) to Met and therefore restored the biological function of the oxidized proteins. To reveal the methionine oxidation-reduction mechanism in human body, in this study, the cDNA sequence of bovine msrA was used as an information-probe to screen the human EST database. Based on a contig assembled from homologous ESTs, a 1 256-bp human MSRA cDNA was cloned from several human cDNA libraries. The cDNA contains an open reading frame (ORF) of 705 bp in length, which encodes 235 amino acid residues. Homology comparison revealed that human MSRA shares 88% and 61% identities with bovine and Escherichia coli msrA protein respectively. Expression pattern analysis revealed a single 1.6-kb transcript of human MSRA in most human tissues and with highest expression in kidney. By radiation hybrid panel mapping, the gene was localized to human chromosome 8p22-23 between markers D8S518 and D8S550. There are 2 human inherited diseases Keratolytic Winter Erythema and Microcephaly related genes in this region, it is inferred that human MSRA might be the candidate of the two diseases.

  11. Existence of homologous sequences corresponding to cDNA of the ver

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    The presence of DNA homologues corresponding to verc203 (vernalization-related cDNA clone) was investigated by molecular hybridization techniques. The genes were detected in 16 plant species that cover 12 subclasses of the Takhtajan system of angiosperms classification including diverse model species. The results of Southern blot analysis showed a low copy number of this gene existed in rice, wheat, barley and Arabidopsis. The hybridization result of PCR products demonstrated the conservation of the gene corresponding to ver203 in diverse plants. The phylogenetic tree of the ver203 gene in tested plants was supported by evolution relationship of species. The ver203 gene expressed in a vernalized plumule winter wheat, instead of the root. And the endosperm before the treatment was essential for the ver203 expression during vernalization in wheat. In Arabidopsis thaliana, the pattern of expression showed that the gene corresponding to ver203 was expressed at low temperature for 14 days. Gibberellin (GA3) may accelerate the expression of ver203 gene in Arabidopsis exposed to low temperature. However, it could not replace vernalization treatment to initiate the gene expression.

  12. cDNA cloning, chromosome mapping and expression characterization of human geranylgeranyl pyrophosphate synthase

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Geranylgeranyl pyrophosphate (GGPP) mainly participates in post-translational modification for various proteins including Rho/Rac, Rap and Rab families, as well as in regulation for cell apoptosis. Geranylgeranyl pyrophosphate synthase (GGPPS), which catalyzes the condensation reaction between farnesyl diphosphate and isopentenyl diphosphate, is the key enzyme for synthesizing GGPP. We report the isolation of a gene transcript showing high homology with Drosophila GGPPS cDNA. The transcript is 1 466 bp in length and contains an intact open reading frame (ORF) ranging from nt 239 to 1 138. This ORF encodes a deduced protein of 300 residues with calculated molecular weight of 35 ku. The deduced protein shows 57.5% identity and 75% similarity with Drosophila GGPPS, and contains five characteristic domains of prenyltransferases. Northern hybridization revealed that human GGPPS was expressed highest in heart, and moderately in spleen, testis, brain, placenta, lung, liver, skeletal muscle, kidney and pancreas. No obvious bands were detected in other examined tissues. The GGPPS gene was located on human chromosome 1q43 by Radiation Hybrid mapping method. It was proved that there was a putative predisposing gene for prostate cancer in this region, and that analogs of GGPP can inhibit the geranylgeranylation of p21rap protein in PC-3 prostate cancer cell lines. These facts suggest that GGPPS may be one of the candidate genes for prostate cancer.

  13. Implementation of Library OPAC System Based on SSH Frame%基于SSH框架技术的图书馆OPAC系统的实现

    Institute of Scientific and Technical Information of China (English)

    陈连和

    2014-01-01

    The process of using the SSH combination frame in Java EE technology to achieve OPAC system is described in detailed. In the general structure of the program, Struts supports the skeleton of program, and Hibernate simplifies database operation, and Spring integrates with Struts and Hibernate, manages dependencies and transaction, simplifies Hibernate code. In the code, using the SSH combination frame, orderly programming is done goodly with the MVC technology and three layers structure and to an interface and according to development order of "view→controller →business logic model →data access model". Therefore, SSH combination framework provides a unified program structure which is enable to code orderly and efficiently and facilitate the expansion and maintenance.%详细地阐述了使用Java EE技术中SSH组合框架实现OPAC系统的过程。程序的总体结构上,以Struts撑起程序的骨架,Hibernate简化数据库操作,Spring与Struts、Hibernate集成,管理依赖关系和管理事务,简化Hibernate编码;编码上,SSH组合框架使得很好地使用MVC技术和基于三层结构、面向接口的编程方式,以“视图→控制器→业务逻辑模型→数据访问模型”的开发顺序有序地进行编程。因此,SSH组合框架提供统一的程序结构,使得有序、高效地编码,也便于扩展和维护。

  14. Using a dynamical advection to reconstruct a part of the SSH evolution in the context of SWOT, application to the Mediterranean Sea

    Science.gov (United States)

    Rogé, Marine; Morrow, Rosemary; Ubelmann, Clément; Dibarboure, Gérald

    2017-08-01

    The main oceanographic objective of the future SWOT mission is to better characterize the ocean mesoscale and sub-mesoscale circulation, by observing a finer range of ocean topography dynamics down to 20 km wavelength. Despite the very high spatial resolution of the future satellite, it will not capture the time evolution of the shorter mesoscale signals, such as the formation and evolution of small eddies. SWOT will have an exact repeat cycle of 21 days, with near repeats around 5-10 days, depending on the latitude. Here, we investigate a technique to reconstruct the missing 2D SSH signal in the time between two satellite revisits. We use the dynamical interpolation (DI) technique developed by Ubelmann et al. (2015). Based on potential vorticity (hereafter PV) conservation using a one and a half layer quasi-geostrophic model, it features an active advection of the SSH field. This model has been tested in energetic open ocean regions such as the Gulf Stream and the Californian Current, and has given promising results. Here, we test this model in the Western Mediterranean Sea, a lower energy region with complex small scale physics, and compare the SSH reconstruction with the high-resolution Symphonie model. We investigate an extension of the simple dynamical model including a separated mean circulation. We find that the DI gives a 16-18% improvement in the reconstruction of the surface height and eddy kinetic energy fields, compared with a simple linear interpolation, and a 37% improvement in the Northern Current subregion. Reconstruction errors are higher during winter and autumn but statistically, the improvement from the DI is also better for these seasons.

  15. Molecular cloning and analysis of functional cDNA and genomic clones encoding bovine cellular retinoic acid-binding protein.

    Science.gov (United States)

    Shubeita, H E; Sambrook, J F; McCormick, A M

    1987-08-01

    A recombinant cDNA clone, pCRABP-HS1, encoding cellular retinoic acid-binding protein was isolated from a bovine adrenal cDNA library. COS-7 cells transfected with pCRABP-HS1 produced a biologically active retinoic acid-binding protein molecule of the expected molecular mass (15.5 kDa). RNA blot hybridization analysis using pCRABP-HS1 as a probe revealed a single 1050-nucleotide mRNA species in bovine adrenal, uterus, and testis, tissues that contain the highest levels of retinoic acid-binding activity. No hybridization was detected in RNA extracted from ovary, spleen, kidney, or liver, which contain relatively low levels of cellular retinoic acid-binding protein activity. Analysis of genomic clones isolated from an EcoRI bovine genomic library demonstrated that the bovine cellular retinoic acid-binding protein gene is composed of four exons and three introns. Two putative promoter sequences were identified in the cloned 5' sequence of the gene.

  16. Isolation and expression of the full-length cDNA encoding CD59 antigen of human lymphocytes.

    Science.gov (United States)

    Sawada, R; Ohashi, K; Anaguchi, H; Okazaki, H; Hattori, M; Minato, N; Naruto, M

    1990-04-01

    To identify the primary structure of CD59 antigen and to elucidate its function, a full-length cDNA clone of CD59 was isolated. The cDNA sequence contained an open reading frame that encodes an 128-amino-acid peptide. The amino-terminal 25 amino acids represented a typical signal peptide sequence and the carboxy-terminal hydrophobic amino acids were characteristic for phosphatidylinositol-anchored proteins. The predicted mature protein sequence showed 35% homology with murine Ly-6C.1 and 31% with Ly-6A.2. The number and the distribution of cysteine residues were conserved, implying that the CD59 represented a human homologue of murine Ly-6. RNA blot hybridization analysis revealed the expression of CD59 mRNA in placental, lung, and pancreatic tissues. The mRNA was not only expressed in T-cell lines but in some of monocytic, myeloid, and B-cell lines. In all of these tissues and cell lines, at least four mRNA species were detected. DNA blot hybridization analysis revealed a rather simple genomic structure, which suggested a single gene as compared with the complex multigene family of murine Ly-6.

  17. Difference in gene expression of macrophage between normal spleen and portal hypertensive spleen idendified by cDNA microarray

    Institute of Scientific and Technical Information of China (English)

    Feng Yan; Xiao-Min Wang

    2007-01-01

    AIM: To identify the difference in gene expression of microphage (Mφ) between normal spleen and portal hypertensive spleen using cDNA microarrays and find new gene functions associated with hypersplenism in portal hypertension.METHODS: The Biostar-H140s chip containing 14112 spots of cDNAs were used to investigate the difference of the expression. The total RNA extracted from macrophages isolated from both normal spleen and portal hypertensive spleen was reversely transcribed to cDNA with the incorporation of fluorescent (cy3 and cy5) labeled dCTP to prepare the hybridization probes.After hybridization, the gene chip was scanned for the fluorescent intensity. The differentially expressed genes were screened. That was repeated three times,and only the genes which had differential expression in all three chips were considered to be associated with hypersplenism in portal hypertension.RESULTS: Eight hundred and ninety-six, 1330 and 898 genes were identified to be differentially expressed in three chips, respectively. One hundred and twenty-one genes (0.86%) were identified to be differentially expressed in all three chips, including 21 up-regulated genes and 73 down-regulated genes. The differentially expressed genes were related to ionic channel and transport protein, cyclin, cytoskeleton, cell receptor, cell signal conduct, metabolism, immune, and so on. These genes might be related to the hypersplenism in portal hypertension.CONCLUSION: The investigations based on cDNA microarray can screen differentially expressed genes of macrophages between normal spleen and portal hypertensive spleen, thus may provide a new idea in studying the pathogenesis of hypersplenism in portal hypertension.

  18. Application Research of J2EE Based SSH Framework%基于SSH框架的J2EE应用研究

    Institute of Scientific and Technical Information of China (English)

    周利江

    2012-01-01

    介绍了SSH (Struts2、Spring、Hibemate)框架的基本特征和优点,介绍了基于SSH的J2EE开发模式及关键技术.提出了基于轻量级Web框架-Struts+Spring+Hibernate的系统结构,介绍了Struts的MVC结构、Spring的基本组成和结构以及Hibernate的基本对象,并将三种框架技术整合起来应用到系统中,随后给出一个设计实例.

  19. Hybrid vehicles

    Energy Technology Data Exchange (ETDEWEB)

    West, J.G.W. [Electrical Machines (United Kingdom)

    1997-07-01

    The reasons for adopting hybrid vehicles result mainly from the lack of adequate range from electric vehicles at an acceptable cost. Hybrids can offer significant improvements in emissions and fuel economy. Series and parallel hybrids are compared. A combination of series and parallel operation would be the ideal. This can be obtained using a planetary gearbox as a power split device allowing a small generator to transfer power to the propulsion motor giving the effect of a CVT. It allows the engine to run at semi-constant speed giving better fuel economy and reduced emissions. Hybrid car developments are described that show the wide range of possible hybrid systems. (author)

  20. CDNA encoding a polypeptide including a hevein sequence

    Science.gov (United States)

    Raikhel, Natasha V.; Broekaert, Willem F.; Chua, Nam-Hai; Kush, Anil

    1995-03-21

    A cDNA clone (HEV1) encoding hevein was isolated via polymerase chain reaction (PCR) using mixed oligonucleotides corresponding to two regions of hevein as primers and a Hevea brasiliensis latex cDNA library as a template. HEV1 is 1018 nucleotides long and includes an open reading frame of 204 amino acids. The deduced amino acid sequence contains a putative signal sequence of 17 amino acid residues followed by a 187 amino acid polypeptide. The amino-terminal region (43 amino acids) is identical to hevein and shows homology to several chitin-binding proteins and to the amino-termini of wound-induced genes in potato and poplar. The carboxyl-terminal portion of the polypeptide (144 amino acids) is 74-79% homologous to the carboxyl-terminal region of wound-inducible genes of potato. Wounding, as well as application of the plant hormones abscisic acid and ethylene, resulted in accumulation of hevein transcripts in leaves, stems and latex, but not in roots, as shown by using the cDNA as a probe. A fusion protein was produced in E. coli from the protein of the present invention and maltose binding protein produced by the E. coli.