Field performance of Populus expressing somaclonal variation in resistance to Septoria musiva

Treesearch

M. E. Ostry; K. T. Ward

2003-01-01

Over 1500 trees from two hybrid poplar clones regenerated from tissue culture and expressing somatic variation in leaf disease resistance in a laboratory leaf disk bioassay were field-tested for 5-11 years to examine their resistance to Septoria leaf spot and canker and to assess their growth characteristics compared with the source clones....

Cloning and expression of the rat homologue of the Huntington disease gene

DOE Office of Scientific and Technical Information (OSTI.GOV)

Schmitt, I.; Epplen, J.T.; Riess, O.

1994-09-01

Huntington`s disease (HD) is an autosomal dominant neurodegenerative disorder which is manifested usually in adult life. The age of onset is variable and leads to progressive symptoms including involuntary choreatic movements and various cognitive and psychiatric disturbances. Recently, a gene (IT15) was cloned containing a (CAG){sub n} repeat which is elongated and unstable in HD patients. IT15 is widely expressed in human tissues but unrelated to any known deduced protein sequence. To further investigate the HD gene, 15 rat cDNA libraries were screened. 24 clones have been identified covering the Huntingtin gene. Comparison of the Huntingtin gene between human andmore » rat revealed homologies between 80% and 87% at the DNA level and about 90% at the protein level. These analyses will help to define biologically important sequence regions, e.g., via evolutionary conservation. One clone contains the (CAG){sub n} repeat which consists of eight triplets compared to seven triplets in the mouse and a median of 17 in human. As in humans there are two transcripts arising from differential 3{prime}-polyadenylation. In the 3{prime}UTR a stretch of about 280 bp is exchanged for a 250 bp fragment with no homology in rodents and man. The cDNA clones are currently used to study Huntingtin gene expression during development in rodent tissues. RNA in situ hybridization of embryonic sections shows predominant signals in all neuronal tissues. In contrast to previously published data Huntingtin mRNA expression in testis is increased in spermatocytes vs. spermatogonia.« less

The Transcriptome of the Reference Potato Genome Solanum tuberosum Group Phureja Clone DM1-3 516R44

PubMed Central

Massa, Alicia N.; Childs, Kevin L.; Lin, Haining; Bryan, Glenn J.; Giuliano, Giovanni; Buell, C. Robin

2011-01-01

Advances in molecular breeding in potato have been limited by its complex biological system, which includes vegetative propagation, autotetraploidy, and extreme heterozygosity. The availability of the potato genome and accompanying gene complement with corresponding gene structure, location, and functional annotation are powerful resources for understanding this complex plant and advancing molecular breeding efforts. Here, we report a reference for the potato transcriptome using 32 tissues and growth conditions from the doubled monoploid Solanum tuberosum Group Phureja clone DM1-3 516R44 for which a genome sequence is available. Analysis of greater than 550 million RNA-Seq reads permitted the detection and quantification of expression levels of over 22,000 genes. Hierarchical clustering and principal component analyses captured the biological variability that accounts for gene expression differences among tissues suggesting tissue-specific gene expression, and genes with tissue or condition restricted expression. Using gene co-expression network analysis, we identified 18 gene modules that represent tissue-specific transcriptional networks of major potato organs and developmental stages. This information provides a powerful resource for potato research as well as studies on other members of the Solanaceae family. PMID:22046362

Cloning and expression of prion protein encoding gene of flounder ( Paralichthys olivaceus)

NASA Astrophysics Data System (ADS)

Zhang, Zhiwen; Sun, Xiuqin; Zhang, Jinxing; Zan, Jindong

2008-02-01

The prion protein (PrP) encoding gene of flounder ( Paralichthys olivaceus) was cloned. It was not interrupted by an intron. This gene has two promoters in its 5' upstream, indicating that its transcription may be intensive, and should have an important function. It was expressed in all 14 tissues tested, demonstrating that it is a house-keeping gene. Its expression in digestion and reproduction systems implies that the possible prions of fish may transfer horizontally.

Cloning and expression analysis of a gene that shows developmental regulation upon tuberization in potato.

PubMed

Jackson, S; Gascón, J; Carrera, E; Monte, E; Prat, S

1997-01-01

Differential screening of a potato leaf cDNA library with cDNA probes made from tuberizing and non-tuberizing Solanum demissum plants led to the identification of a clone that is upregulated in leaves and other tissues upon tuberization. This clone was also shown to have a high level of expression in green tomato fruit, its expression falling off as the fruit turns red. No sucrose or hormonal regulation of the expression of this clone was observed and it did not respond to wounding or heat stress. Clone 32B is 532 bp long and contains an open reading frame encoding a small protein of 98 amino acids. The deduced protein sequence has a putative signal peptide for ER transport and a 10 amino acid domain in the C-terminal region of the protein, both of which are also found in the cotton LEA5, Arabidopsis Di21 and the mungbean Arg2 proteins.

Somatic Donor Cell Type Correlates with Embryonic, but Not Extra-Embryonic, Gene Expression in Postimplantation Cloned Embryos

PubMed Central

Inoue, Kimiko; Ogura, Atsuo

2013-01-01

The great majority of embryos generated by somatic cell nuclear transfer (SCNT) display defined abnormal phenotypes after implantation, such as an increased likelihood of death and abnormal placentation. To gain better insight into the underlying mechanisms, we analyzed genome-wide gene expression profiles of day 6.5 postimplantation mouse embryos cloned from three different cell types (cumulus cells, neonatal Sertoli cells and fibroblasts). The embryos retrieved from the uteri were separated into embryonic (epiblast) and extraembryonic (extraembryonic ectoderm and ectoplacental cone) tissues and were subjected to gene microarray analysis. Genotype- and sex-matched embryos produced by in vitro fertilization were used as controls. Principal component analysis revealed that whereas the gene expression patterns in the embryonic tissues varied according to the donor cell type, those in extraembryonic tissues were relatively consistent across all groups. Within each group, the embryonic tissues had more differentially expressed genes (DEGs) (>2-fold vs. controls) than did the extraembryonic tissues (P<1.0×10–26). In the embryonic tissues, one of the common abnormalities was upregulation of Dlk1, a paternally imprinted gene. This might be a potential cause of the occasional placenta-only conceptuses seen in SCNT-generated mouse embryos (1–5% per embryos transferred in our laboratory), because dysregulation of the same gene is known to cause developmental failure of embryos derived from induced pluripotent stem cells. There were also some DEGs in the extraembryonic tissues, which might explain the poor development of SCNT-derived placentas at early stages. These findings suggest that SCNT affects the embryonic and extraembryonic development differentially and might cause further deterioration in the embryonic lineage in a donor cell-specific manner. This could explain donor cell-dependent variations in cloning efficiency using SCNT. PMID:24146866

Identification of tumor-restricted antigens NY-BR-1, SCP-1, and a new cancer/testis-like antigen NW-BR-3 by serological screening of a testicular library with breast cancer serum.

PubMed

Jäger, Dirk; Unkelbach, Marc; Frei, Claudia; Bert, Florian; Scanlan, Matthew J; Jäger, Elke; Old, Lloyd J; Chen, Yao-Tseng; Knuth, Alexander

2002-06-28

Serological analysis of recombinant cDNA expression libraries (SEREX) has led to the identification of several categories of new tumor antigens. We analyzed a testicular cDNA expression library with serum obtained from a breast cancer patient and isolated 13 genes designated NW-BR-1 through NW-BR-13. Of these, 3 showed tumor-restricted expression (NW-BR-1, -2 and -3), the others being expressed ubiquitously. NW-BR-3, representing 9 of 24 primary clones, showed tissue-restricted mRNA expression, being expressed in normal testis but not in 15 other normal tissues tested by Northern blotting. RT-PCR analysis showed strong NW-BR-3 expression in normal testis, weak expression in brain, kidney, trachea, uterus and normal prostate, and was negative in liver, heart, lung, colon, small intestine, bone marrow, breast, thymus, muscle, spleen, and stomach. NW-BR-3 mRNA expression was found in different tumor tissues and tumor cell lines by RT-PCR, thus showing a 'cancer/testis' (CT)-like mRNA expression pattern. NW-BR-3 shares 71% nucleotide and amino acid homology to a mouse gene cloned from mouse testicular tissue. Based on the mRNA expression pattern, NW-BR-3 represents a new candidate target gene for cancer immunotherapy. NW-BR-1 and NW-BR-2 also showed tumor-restricted mRNA expression. NW-BR-1 is a partial clone of the breast differentiation antigen NY-BR-1 previously identified by SEREX. NY-BR-1 is expressed in normal breast, testis and 80% of breast cancers. NW-BR-2 is identical to the CT antigen SCP-1, initially isolated by SEREX analysis of renal cancer. This study provides further evidence that SEREX is a powerful tool to identify new tumor antigens potentially relevant for immunotherapy approaches.

Characterization and expression of cyp19a gene in the Chinese giant salamander Andrias davidianus.

PubMed

Hu, Qiaomu; Xiao, Hanbing; Tian, HaiFeng; Meng, Yan

2016-02-01

We cloned the full length cyp19a of Chinese giant salamander Andrias davidianus, determined its distribution in tissues and developing gonads, and analyzed the CpG methylation pattern of the cyp19a promoter. The results revealed isoforms of 1706 bp (G arom) and 1698 bp (B arom) in length, differing in the 5' flanking region, both encoding 502 amino acids. The G arom gene was observed mainly in the ovary and kidney, with little in other investigated tissues, while B arom expression was high in the brain, ovary, testis, and pituitary, with low or undetected expression in other examined tissues. Total aromatase expression was high in the ovary; moderate in the kidney, brain, testis, and pituitary; and low in the remaining tissues. G arom expression was significantly higher in the ovary than in the testis and gradually decreased with maturation of the salamander. A single injection of methyltestosterone or letrozole resulted in ovarian G arom expression decreasing over a 12-96 h period. A 1366 bp sequence of the cyp19a promoter was cloned and shown to be conserved in selected species. CpG methylation level was negatively correlated with cyp19a expression in the examined tissues and developing ovaries. Five and three CpG methylation sites positively correlated with DNA methylation levels in tissues and developing ovary, suggesting that they play an important role in regulating cyp19a expression. The aromatase gene showed two isoforms with distinct expression patterns, and the promoter methylation level at specific CpG sites was associated with variation in expression profiles of tissues and developing ovaries. Copyright © 2015 Elsevier Inc. All rights reserved.

Residual Expression of the Reprogramming Factors Prevents Differentiation of iPSC Generated from Human Fibroblasts and Cord Blood CD34+ Progenitors

PubMed Central

Ramos-Mejía, Verónica; Montes, Rosa; Bueno, Clara; Ayllón, Verónica; Real, Pedro J.; Rodríguez, René; Menendez, Pablo

2012-01-01

Human induced pluripotent stem cells (hiPSC) have been generated from different tissues, with the age of the donor, tissue source and specific cell type influencing the reprogramming process. Reprogramming hematopoietic progenitors to hiPSC may provide a very useful cellular system for modelling blood diseases. We report the generation and complete characterization of hiPSCs from human neonatal fibroblasts and cord blood (CB)-derived CD34+ hematopoietic progenitors using a single polycistronic lentiviral vector containing an excisable cassette encoding the four reprogramming factors Oct4, Klf4, Sox2 and c-myc (OKSM). The ectopic expression of OKSM was fully silenced upon reprogramming in some hiPSC clones and was not reactivated upon differentiation, whereas other hiPSC clones failed to silence the transgene expression, independently of the cell type/tissue origin. When hiPSC were induced to differentiate towards hematopoietic and neural lineages those hiPSC which had silenced OKSM ectopic expression displayed good hematopoietic and early neuroectoderm differentiation potential. In contrast, those hiPSC which failed to switch off OKSM expression were unable to differentiate towards either lineage, suggesting that the residual expression of the reprogramming factors functions as a developmental brake impairing hiPSC differentiation. Successful adenovirus-based Cre-mediated excision of the provirus OKSM cassette in CB-derived CD34+ hiPSC with residual transgene expression resulted in transgene-free hiPSC clones with significantly improved differentiation capacity. Overall, our findings confirm that residual expression of reprogramming factors impairs hiPSC differentiation. PMID:22545141

Isolation and expression of homeobox genes from the embryonic chicken eye.

PubMed

Dhawan, R R; Schoen, T J; Beebe, D C

1997-06-11

To identify homeobox-containing genes that may play a role in the differentiation of ocular tissues. Total RNA was isolated from microdissected chicken embryo eye tissues at 3.5 days of development (embryonic day 3.5; E3.5). An "anchor-oligo-dT primer" was used for the synthesis of cDNA. Degenerate oligonucleotides designed from highly-conserved sequences in the third helix of the homeobox and the "anchor-primer" were used to amplify cDNAs by polymerase chain reaction (PCR). PCR products were cloned and sequenced. The spatial and temporal expression of selected transcripts was mapped by whole-mount in situ hybridization and northern blot analysis. After sequencing eighteen clones we identified a member of the distal-less family (dlx-3) in cDNA from presumptive neural retina and three chicken homologs of the Xenopus "anterior neural fold" (Xanf-1) in cDNA from anterior eye tissue. Dlx transcripts were mapped by in situ hybridization. Expression began at Hamburger and Hamilton stage 14 (E2.5) and was widely distributed in embryonic mesenchyme on E3 and E4. Expression increased in the retina during early development and persisted until after hatching. The one anf clone selected for further study was not detected by in situ or northern blot analysis. It is feasible to isolate homeobox cDNAs directly from microdissected embryonic tissues. Chicken dlx-3 mRNA has a wider distribution in the embryo than expected, based on the expression of the mouse homolog. Dlx-3 may play a role in establishing or maintaining the differentiation of the retina.

Re-evaluating TTF-1 immunohistochemistry in diffuse gliomas: Expression is clone-dependent and associated with tumor location.

PubMed

Pratt, Drew; Afsar, Nina; Allgauer, Michael; Fetsch, Patricia; Palisoc, Maryknoll; Pittaluga, Stefania; Quezado, Martha

TTF-1 is widely used as a marker in routine surgical pathology in the work-up of malignancy. Aberrant expression of TTF-1 in extrapulmonary and extrathyroidal malignancies is a frequently reported phenomenon. In addition to the recently characterized pituicyte-derived tumors of the sella, immunoreactivity has been reported in diffuse gliomas with the SPT24 clone. Here, we sought to evaluate TTF-1 expression with three commercially available clones in a large series of gliomas. Expression was compared across the newly defined diagnostic entities in the 2016 WHO Classification of CNS Tumors. Using tissue microarrays (TMA), 212 diffuse gliomas (WHO grades II - IV) were systematically evaluated with TTF-1 immunohistochemistry using three clones: SPT24, 8G7G3/1, and SP141, and results correlated with clinicopathologic features. 14 high-grade diffuse gliomas demonstrated nuclear staining with the SP141 and SPT24 clones. Two tumors showed weak positivity with the 8G7G3/1 clone. All tumors were high grade by histology (WHO grades III and IV). 86% (12/14) of TTF-1-positive gliomas involved the frontal lobes at diagnosis. No relationship with IDH R132H, ATRX, p53, H3K27M, or EGFR immunohistochemistry was identified. TTF-1 expression in gliomas was not independently prognostic of overall survival. TTF-1 expression in diffuse gliomas is a rare but potentially misleading occurrence. In our cohort, staining occurred with both the SPT24 and SP141 clones at equal intensity and frequency. Clustering of TTF-1-positive tumors in the frontal lobe(s) suggests lineage-specific expression. Due to clone-specific expression in diffuse gliomas, caution must be exercised in the work-up of intracranial tumors with TTF-1.
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Production of transgenic cloned pigs expressing the far-red fluorescent protein monomeric Plum.

PubMed

Watanabe, Masahito; Kobayashi, Mirina; Nagaya, Masaki; Matsunari, Hitomi; Nakano, Kazuaki; Maehara, Miki; Hayashida, Gota; Takayanagi, Shuko; Sakai, Rieko; Umeyama, Kazuhiro; Watanabe, Nobuyuki; Onodera, Masafumi; Nagashima, Hiroshi

2015-01-01

Monomeric Plum (Plum), a far-red fluorescent protein with photostability and photopermeability, is potentially suitable for in vivo imaging and detection of fluorescence in body tissues. The aim of this study was to generate transgenic cloned pigs exhibiting systemic expression of Plum using somatic cell nuclear transfer (SCNT) technology. Nuclear donor cells for SCNT were obtained by introducing a Plum-expression vector driven by a combination of the cytomegalovirus early enhancer and chicken beta-actin promoter into porcine fetal fibroblasts (PFFs). The cleavage and blastocyst formation rates of reconstructed SCNT embryos were 81.0% (34/42) and 78.6% (33/42), respectively. At 36-37 days of gestation, three fetuses systemically expressing Plum were obtained from one recipient to which 103 SCNT embryos were transferred (3/103, 2.9%). For generation of offspring expressing Plum, rejuvenated PFFs were established from one cloned fetus and used as nuclear donor cells. Four cloned offspring and one stillborn cloned offspring were produced from one recipient to which 117 SCNT embryos were transferred (5/117, 4.3%). All offspring exhibited high levels of Plum fluorescence in blood cells, such as lymphocytes, monocytes and granulocytes. In addition, the skin, heart, kidney, pancreas, liver and spleen also exhibited Plum expression. These observations demonstrated that transfer of the Plum gene did not interfere with the development of porcine SCNT embryos and resulted in the successful generation of transgenic cloned pigs that systemically expressed Plum. This is the first report of the generation and characterization of transgenic cloned pigs expressing the far-red fluorescent protein Plum.

Establishment and characterization of scleroderma fibroblast clonal cell lines by introduction of the hTERT gene

PubMed Central

Kapanadze, Bagrat; Morris, Erin; Smith, Edwin; Trojanowska, Maria

2010-01-01

Abstract Lack of an adequate experimental model has hindered the ability to fully understand scleroderma (SSc) pathogenesis. Current SSc research is based on the study of cultured fibroblasts from skin biopsies. In depth characterization of the SSc fibroblast phenotype is hindered by the limited lifespan and heterogeneity of these cells. The goal of this study was to isolate high collagen-producing fibroblasts from SSc biopsies and extend their lifespan with hTERT immortalization to enable characterization of their phenotype. Fibroblasts from two pairs of closely matched normal and SSc biopsies were infected with an hTERT lentivirus. Infected colonies were isolated, cultured into clonal cell lines and analysed with respect to profibrotic gene expression. The mRNA levels of nine profibrotic genes were measured by quantitative real-time PCR. Protein levels were assessed by Western blot. The hTERT SSc clones were heterogeneous with regards to expression of the profibrotic genes measured. A subset of the SSc clones showed elevated expression levels of collagen I, connective tissue growth factor and thrombospondin 1 mRNA, while expression of other genes was not significantly changed. Elevated expression of collagen I protein and mRNA was correlative with elevated expression of connective tissue growth factor. Several hTERT clones expressed high levels of pSmad1, Smad1 and TGF-βRI indicative of altered TGF-β signalling. A portion of SSc clones expressed several profibrotic genes. This study demonstrates that select characteristics of the SSc phenotype are expressed in a subset of activated fibroblasts in culture. The clonal SSc cell lines may present a new and useful model to investigate the mechanisms involved in SSc fibrosis. PMID:19432820

Establishment and characterization of scleroderma fibroblast clonal cell lines by introduction of the hTERT gene.

PubMed

Kapanadze, Bagrat; Morris, Erin; Smith, Edwin; Trojanowska, Maria

2010-05-01

Lack of an adequate experimental model has hindered the ability to fully understand scleroderma (SSc) pathogenesis. Current SSc research is based on the study of cultured fibroblasts from skin biopsies. In depth characterization of the SSc fibroblast phenotype is hindered by the limited lifespan and heterogeneity of these cells. The goal of this study was to isolate high collagen-producing fibroblasts from SSc biopsies and extend their lifespan with hTERT immortalization to enable characterization of their phenotype. Fibroblasts from two pairs of closely matched normal and SSc biopsies were infected with an hTERT lentivirus. Infected colonies were isolated, cultured into clonal cell lines and analysed with respect to profibrotic gene expression. The mRNA levels of nine profibrotic genes were measured by quantitative real-time PCR. Protein levels were assessed by Western blot. The hTERT SSc clones were heterogeneous with regards to expression of the profibrotic genes measured. A subset of the SSc clones showed elevated expression levels of collagen I, connective tissue growth factor and thrombospondin 1 mRNA, while expression of other genes was not significantly changed. Elevated expression of collagen I protein and mRNA was correlative with elevated expression of connective tissue growth factor. Several hTERT clones expressed high levels of pSmad1, Smad1 and TGF-betaRI indicative of altered TGF-beta signalling. A portion of SSc clones expressed several profibrotic genes. This study demonstrates that select characteristics of the SSc phenotype are expressed in a subset of activated fibroblasts in culture. The clonal SSc cell lines may present a new and useful model to investigate the mechanisms involved in SSc fibrosis.

Cytokinin oxidase/dehydrogenase genes in barley and wheat: cloning and heterologous expression.

PubMed

Galuszka, Petr; Frébortová, Jitka; Werner, Tomás; Yamada, Mamoru; Strnad, Miroslav; Schmülling, Thomas; Frébort, Ivo

2004-10-01

The cloning of two novel genes that encode cytokinin oxidase/dehydrogenase (CKX) in barley is described in this work. Transformation of both genes into Arabidopsis and tobacco showed that at least one of the genes codes for a functional enzyme, as its expression caused a cytokinin-deficient phenotype in the heterologous host plants. Additional cloning of two gene fragments, and an in silico search in the public expressed sequence tag clone databases, revealed the presence of at least 13 more members of the CKX gene family in barley and wheat. The expression of three selected barley genes was analyzed by RT-PCR and found to be organ-specific with peak expression in mature kernels. One barley CKX (HvCKX2) was characterized in detail after heterologous expression in tobacco. Interestingly, this enzyme shows a pH optimum at 4.5 and a preference for cytokinin ribosides as substrates, which may indicate its vacuolar targeting. Different substrate specificities, and the pH profiles of cytokinin-degrading enzymes extracted from different barley tissues, are also presented.

The Selector Gene apterous and Notch Are Required to Locally Increase Mechanical Cell Bond Tension at the Drosophila Dorsoventral Compartment Boundary

PubMed Central

Michel, Marcus; Aliee, Maryam; Rudolf, Katrin; Bialas, Lisa; Jülicher, Frank; Dahmann, Christian

2016-01-01

The separation of cells with distinct fates and functions is important for tissue and organ formation during animal development. Regions of different fates within tissues are often separated from another along straight boundaries. These compartment boundaries play a crucial role in tissue patterning and growth by stably positioning organizers. In Drosophila, the wing imaginal disc is subdivided into a dorsal and a ventral compartment. Cells of the dorsal, but not ventral, compartment express the selector gene apterous. Apterous expression sets in motion a gene regulatory cascade that leads to the activation of Notch signaling in a few cell rows on either side of the dorsoventral compartment boundary. Both Notch and apterous mutant clones disturb the separation of dorsal and ventral cells. Maintenance of the straight shape of the dorsoventral boundary involves a local increase in mechanical tension at cell bonds along the boundary. The mechanisms by which cell bond tension is locally increased however remain unknown. Here we use a combination of laser ablation of cell bonds, quantitative image analysis, and genetic mutants to show that Notch and Apterous are required to increase cell bond tension along the dorsoventral compartment boundary. Moreover, clonal expression of the Apterous target gene capricious results in cell separation and increased cell bond tension at the clone borders. Finally, using a vertex model to simulate tissue growth, we find that an increase in cell bond tension at the borders of cell clones, but not throughout the cell clone, can lead to cell separation. We conclude that Apterous and Notch maintain the characteristic straight shape of the dorsoventral compartment boundary by locally increasing cell bond tension. PMID:27552097

Cloning and expression of sheep DNA methyltransferase 1 and its development-specific isoform.

PubMed

Taylor, Jane; Moore, Hannah; Beaujean, Nathalie; Gardner, John; Wilmut, Ian; Meehan, Richard; Young, Lorraine

2009-05-01

Unlike the mouse embryo, where loss of DNA methylation in the embryonic nucleus leaves cleavage stage embryos globally hypomethylated, sheep preimplantation embryos retain high levels of methylation until the blastocyst stage. We have cloned and sequenced sheep Dnmt1 and found it to be highly conserved with both the human and mouse homologues. Furthermore, we observed that the transcript normally expressed in adult somatic tissues is highly abundant in sheep oocytes. Throughout sheep preimplantation development the protein is retained in the cytoplasm whereas Dnmt1 transcript production declines after the embryonic genome activation at the 8-16 cell stage. Attempts to clone oocyte-specific 5' regions of Dnmt1, known to be present in the mouse and human gene, were unsuccessful. However, a novel ovine Dnmt1 exon, theoretically encoding 13 amino acids, was found to be expressed in sheep oocytes, preimplantation embryos and early fetal lineages, but not in the adult tissue. RNAi-mediated knockdown of this novel transcript resulted in embryonic developmental arrest at the late morula stage, suggesting an essential role for this isoform in sheep blastocyst formation. (c) 2008 Wiley-Liss, Inc.

CRISPR/Cas9 Editing of Murine Induced Pluripotent Stem Cells for Engineering Inflammation-Resistant Tissues.

PubMed

Brunger, Jonathan M; Zutshi, Ananya; Willard, Vincent P; Gersbach, Charles A; Guilak, Farshid

2017-05-01

Proinflammatory cytokines such as interleukin-1 (IL-1) are found in elevated levels in diseased or injured tissues and promote rapid tissue degradation while preventing stem cell differentiation. This study was undertaken to engineer inflammation-resistant murine induced pluripotent stem cells (iPSCs) through deletion of the IL-1 signaling pathway and to demonstrate the utility of these cells for engineering replacements for diseased or damaged tissues. Targeted deletion of the IL-1 receptor type I (IL-1RI) gene in murine iPSCs was achieved using the RNA-guided, site-specific clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 genome engineering system. Clonal cell populations with homozygous and heterozygous deletions were isolated, and loss of receptor expression and cytokine signaling was confirmed by flow cytometry and transcriptional reporter assays, respectively. Cartilage was engineered from edited iPSCs and tested for its ability to resist IL-1-mediated degradation in gene expression, histologic, and biomechanical assays after a 3-day treatment with 1 ng/ml of IL-1α. Three of 41 clones isolated possessed the IL-1RI +/- genotype. Four clones possessed the IL-1RI -/- genotype, and flow cytometry confirmed loss of IL-1RI on the surface of these cells, which led to an absence of NF-κB transcription activation after IL-1α treatment. Cartilage engineered from homozygous null clones was resistant to cytokine-mediated tissue degradation. In contrast, cartilage derived from wild-type and heterozygous clones exhibited significant degradative responses, highlighting the need for complete IL-1 blockade. This work demonstrates proof-of-concept of the ability to engineer custom-designed stem cells that are immune to proinflammatory cytokines (i.e., IL-1) as a potential cell source for cartilage tissue engineering. © 2016, American College of Rheumatology.

Neuropeptide Y (NPY), cocaine- and amphetamine-regulated transcript (CART) and cholecystokinin (CCK) in winter skate (Raja ocellata): cDNA cloning, tissue distribution and mRNA expression responses to fasting.

PubMed

MacDonald, Erin; Volkoff, Hélène

2009-04-01

cDNAs encoding for neuropeptide Y (NPY), cocaine- and amphetamine-regulated transcript (CART) and cholecystokinin (CCK) were cloned in an elasmobranch fish, the winter skate. mRNA tissue distribution was examined for the three peptides as well as the effects of two weeks of fasting on their expression. Skate NPY, CART and CCK sequences display similarities with sequences for teleost fish but in general the degree of identity is relatively low (50%). All three peptides are present in brain and in several peripheral tissues, including gut and gonads. Within the brain, the three peptides are expressed in the hypothalamus, telencephalon, optic tectum and cerebellum. Two weeks of fasting induced an increase in telencephalon NPY and an increase in CCK in the gut but had no effects on hypothalamic NPY, CART and CCK, or on telencephalon CART. Our results provide basis for further investigation into the regulation of feeding in winter skate.

Molecular cloning and functional characterization of cathepsin D from sea cucumber Apostichopus japonicus.

PubMed

Yu, Cuiping; Cha, Yue; Wu, Fan; Xu, Xianbing; Qin, Lei; Du, Ming

2017-11-01

Cathepsin D (CTSD, EC 3.4.23.5) belongs to aspartic protease family, which is located in lysosomes and is distributed in diverse tissues and cells. CTSD has a wide variety of physiological functions, owing to its proteolytic activity in degradating proteins and peptides. In the current study, the full length cDNA of sea cucumber (Apostichopus japonicus) cathepsin D (AjCTSD) was firstly cloned, then the association between AjCTSD and sea cucumber autolysis was investigated. The full length cDNA of AjCTSD was 2896 bp, with an open reading frame (ORF) for 391 amino acids. AjCTSD was widely expressed in body wall, muscle and intestine; the expression level was the highest in intestine, followed by muscle and body wall. Compared to fresh tissues, AjCTSD expression levels were significantly increased in all examined autolytic tissues. The purified recombinant AjCTSD promoted the degradation of sea cucumber muscle. In conclusion, AjCTSD contributed to sea cucumber muscle autolysis. Copyright © 2017 Elsevier Ltd. All rights reserved.

Changes in WNT signaling-related gene expression associated with development and cloning in bovine extra-embryonic and endometrial tissues during the peri-implantation period.

PubMed

Biase, Fernando H; Rabel, Chanaka; Guillomot, Michel; Sandra, Olivier; Andropolis, Kalista; Olmstead, Colleen; Oliveira, Rosane; Wallace, Richard; Le Bourhis, Daniel; Richard, Christophe; Campion, Evelyne; Chaulot-Talmon, Aurélie; Giraud-Delville, Corinne; Taghouti, Géraldine; Jammes, Hélène; Hue, Isabelle; Renard, Jean Paul; Lewin, Harris A

2013-12-01

We determined if somatic cell nuclear transfer (SCNT) cloning is associated with WNT-related gene expression in cattle development, and if the expression of genes in the WNT pathway changes during the peri-implantation period. Extra-embryonic and endometrial tissues were collected at gestation days 18 and 34 (d18, d34). WNT5A, FZD4, FZD5, LRP5, CTNNB1, GNAI2, KDM1A, BCL2L1, and SFRP1 transcripts were localized in extra-embryonic tissue, whereas SFRP1 and DKK1 were localized in the endometrium. There were no differences in the localization of these transcripts in extra-embryonic tissue or endometrium from SCNT or artificial insemination (AI) pregnancies. Expression levels of WNT5A were 11-fold greater in the allantois of SCNT than AI samples. In the trophoblast, expression of WNT5A, FZD5, CTNNB1, and DKK1 increased significantly from d18 to d34, whereas expression of KDM1A and SFRP1 decreased, indicating that implantation is associated with major changes in WNT signaling. SCNT was associated with altered WNT5A expression in trophoblasts, with levels increasing 2.3-fold more in AI than SCNT conceptuses from d18 to d34. In the allantois, expression of WNT5A increased 6.3-fold more in SCNT than AI conceptuses from d18 to d34. Endometrial tissue expression levels of the genes tested did not differ between AI or SCNT pregnancies, although expression of individual genes showed variation across developmental stages. Our results demonstrate that SCNT is associated with altered expression of specific WNT-related genes in extra-embryonic tissue in a time- and tissue-specific manner. The pattern of gene expression in the WNT pathway suggests that noncanonical WNT signal transduction is important for implantation of cattle conceptuses. © 2013 Wiley Periodicals, Inc.

Cloning and characterization of the murine homolog of the sno proto-oncogene reveals a novel splice variant

NASA Technical Reports Server (NTRS)

Pelzer, T.; Lyons, G. E.; Kim, S.; Moreadith, R. W.; Blomqvist, C. G. (Principal Investigator)

1996-01-01

The cellular function(s) of the SNO protein remain undefined. To gain a better understanding of possible developmental roles of this cellular proto-oncogene, we have cloned two murine sno cDNAs and have investigated their expression patterns in embryonic and postnatal tissues. A single major transcript of 7.5 kb is detected in multiple tissues by Northern blot. However, reverse transcriptase polymerase chain reaction (RT-PCR) and RNAse protection assays revealed a novel splice variant in every tissue examined. Two isoforms, termed sno N and sno-dE3 (dE3, deletion within exon 3), were identified. The sno-dE3 isoform employs a novel 5' splice site located within the coding region of the third exon and deletes potential kinase recognition motifs. Transcripts of both sno isoforms accumulate ubiquitously but are most abundant in the developing central nervous system. The in situ hybridization patterns of sno expression during murine development suggest potential roles in tissues with a high degree of cellular proliferation. Expression in terminally differentiated tissues such as muscle and neurons indicates that SNO may have multiple functional activities.

Molecular cloning and biochemical characterization of three phosphoglycerate kinase isoforms from developing sunflower (Helianthus annuus L.) seeds.

PubMed

Troncoso-Ponce, M A; Rivoal, J; Venegas-Calerón, M; Dorion, S; Sánchez, R; Cejudo, F J; Garcés, R; Martínez-Force, E

2012-07-01

Three cDNAs encoding different phosphoglycerate kinase (PGK, EC 2.7.2.3) isoforms, two cytosolic (HacPGK1 and HacPGK2) and one plastidic (HapPGK), were cloned and characterized from developing sunflower (Helianthus annuus L.) seeds. The expression profiles of these genes showed differences in heterotrophic tissues, such as developing seeds and roots, where HacPGK1 was predominant, while HapPGK was highly expressed in photosynthetic tissues. The cDNAs were expressed in Escherichia coli, and the corresponding proteins purified to electrophoretic homogeneity, using immobilized metal ion affinity chromatography, and biochemically characterized. Despite the high level of identity between sequences, the HacPGK1 isoform showed strong differences in terms of specific activity, temperature stability and pH sensitivity in comparison to HacPGK2 and HapPGK. A polyclonal immune serum was raised against the purified HacPGK1 isoform, which showed cross-immunoreactivity with the other PGK isoforms. This serum allowed the localization of high expression levels of PGK isozymes in embryo tissues. Copyright © 2012 Elsevier Ltd. All rights reserved.

Cloning, characterization, expression and comparative analysis of pig Golgi membrane sphingomyelin synthase 1.

PubMed

Guillén, Natalia; Navarro, María A; Surra, Joaquín C; Arnal, Carmen; Fernández-Juan, Marta; Cebrián-Pérez, Jose Alvaro; Osada, Jesús

2007-02-15

Pig sphingomyelin synthase 1 (SMS1) cDNA was cloned, characterized and compared to the human ortholog. Porcine protein consists of 413 amino acids and displays a 97% sequence identity with human protein. A phylogenic tree of proteins reveals that porcine SMS1 is more closely related to bovine and rodent proteins than to human. Analysis of protein mass was higher than the theoretical prediction based on amino acid sequence suggesting a kind of posttranslational modification. Quantitative representation of tissue distribution obtained by real-time RT-PCR showed that it was widely expressed although important variations in levels were obtained among organs. Thus, the cardiovascular system, especially the heart, showed the highest value of all the tissues studied. Regional differences of expression were observed in the central nervous system and intestinal tract. Analysis of the hepatic mRNA and protein expressions of SMS1 following turpentine treatment revealed a progressive decrease in the former paralleled by a decrease in the protein concentration. These findings indicate the variation in expression in the different tissues might suggest a different requirement of Golgi sphingomyelin for the specific function in each organ and a regulation of the enzyme in response to turpentine-induced hepatic injury.

Recloned dogs derived from adipose stem cells of a transgenic cloned beagle.

PubMed

Oh, Hyun Ju; Park, Jung Eun; Kim, Min Jung; Hong, So Gun; Ra, Jeong Chan; Jo, Jung Youn; Kang, Sung Keun; Jang, Goo; Lee, Byeong Chun

2011-04-15

A number of studies have postulated that efficiency in mammalian cloning is inversely correlated with donor cell differentiation status and may be increased by using undifferentiated cells as nuclear donors. Here, we attempted the recloning of dogs by nuclear transfer of canine adipose tissue-derived mesenchymal stem cells (cAd-MSCs) from a transgenic cloned beagle to determine if cAd-MSCs can be a suitable donor cell type. In order to isolate cAd-MSCs, adipose tissues were collected from a transgenic cloned beagle produced by somatic cell nuclear transfer (SCNT) of canine fetal fibroblasts modified genetically with a red fluorescent protein (RFP) gene. The cAd-MSCs expressed the RFP gene and cell-surface marker characteristics of MSCs including CD29, CD44 and thy1.1. Furthermore, cAd-MSCs underwent osteogenic, adipogenic, myogenic, neurogenic and chondrogenic differentiation when exposed to specific differentiation-inducing conditions. In order to investigate the developmental potential of cAd-MSCs, we carried out SCNT. Fused-couplets (82/109, 75.2%) were chemically activated and transferred into the uterine tube of five naturally estrus-synchronized surrogates. One of them (20%) maintained pregnancy and subsequently gave birth to two healthy cloned pups. The present study demonstrated for the first time the successful production of cloned beagles by nuclear transfer of cAd-MSCs. Another important outcome of the present study is the successful recloning of RFP-expressing transgenic cloned beagle pups by nuclear transfer of cells derived from a transgenic cloned beagle. In conclusion, the present study demonstrates that adipose stem cells can be a good nuclear donor source for dog cloning. Copyright © 2011 Elsevier Inc. All rights reserved.

A multisite gateway-based toolkit for targeted gene expression and hairpin RNA silencing in tomato fruits.

PubMed

Estornell, Leandro Hueso; Orzáez, Diego; López-Peña, Lucas; Pineda, Benito; Antón, María Teresa; Moreno, Vicente; Granell, Antonio

2009-04-01

A collection of fruit promoters, reporter genes and protein tags has been constructed in a triple-gateway format, a recombination-based cloning system that facilitates the tandem assembly of three DNA fragments into plant expression vectors. The new pENFRUIT collection includes, among others, the classical tomato-ripening promoters E8 and 2A11 and a set of six new tomato promoters. The new promoter activities were characterized in both transient assays and stable transgenic plants. The range of expression of the new promoters comprises strong (PNH, PLI), medium (PLE, PFF, PHD) and weak (PSN) promoters driving gene expression preferentially in the fruit, and covering a wide range of tissues and developmental stages. Together, a total of 78 possible combinations for the expression of a gene of interest in the fruit, plus a set of five reporters for new promoter analysis, was made available in the current collection. Moreover, the pENFRUIT promoter collection is adaptable to hairpin RNA strategies aimed at tissue/organ-specific gene silencing with only an additional cloning step. The pENFRUIT toolkit broadens the spectrum of promoter activities available for fruit biotechnology and fundamental research, and bypasses technical difficulties of current ligase-dependent cloning techniques in the construction of fruit expression cassettes. The pENFRUIT vector collection is available for the research community in a plasmid repository, facilitating its accessibility.

Expression Studies of Gibberellin Oxidases in Developing Pumpkin Seeds1

PubMed Central

Frisse, Andrea; Pimenta, Maria João; Lange, Theo

2003-01-01

Two cDNA clones, 3-ox and 2-ox, have been isolated from developing pumpkin (Cucurbita maxima) embryos that show significant amino acid homology to gibberellin (GA) 3-oxidases and 2-oxidases, respectively. Recombinant fusion protein of clone 3-ox converted GA12-aldehyde, GA12, GA15, GA24, GA25, and GA9 to GA14-aldehyde, GA14, GA37, GA36, GA13, and GA4, respectively. Recombinant 2-ox protein oxidized GA9, GA4, and GA1 to GA51, GA34, and GA8, respectively. Previously cloned GA 7-oxidase revealed additional 3β-hydroxylation activity of GA12. Transcripts of this gene were identified in endosperm and embryo of the developing seed by quantitative reverse transcriptase-polymerase chain reaction and localized in protoderm, root apical meristem, and quiescent center by in situ hybridization. mRNA of the previously cloned GA 20-oxidase from pumpkin seeds was localized in endosperm and in tissues of protoderm, ground meristem, and cotyledons of the embryo. However, transcripts of the recently cloned GA 20-oxidase from pumpkin seedlings were found all over the embryo, and in tissues of the inner seed coat at the micropylar end. Previously cloned GA 2β,3β-hydroxylase mRNA molecules were specifically identified in endosperm tissue. Finally, mRNA molecules of the 3-ox and 2-ox genes were found in the embryo only. 3-ox transcripts were localized in tissues of cotyledons, protoderm, and inner cell layers of the root apical meristem, and 2-ox transcripts were found in all tissues of the embryo except the root tips. These results indicate tissue-specific GA-biosynthetic pathways operating within the developing seed. PMID:12644672

Telomerase-immortalized non-malignant human prostate epithelial cells retain the properties of multipotent stem cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Li Hongzhen; Zhou Jianjun; Miki, Jun

2008-01-01

Understanding prostate stem cells may provide insight into the origin of prostate cancer. Primary cells have been cultured from human prostate tissue but they usually survive only 15-20 population doublings before undergoing senescence. We report here that RC-170N/h/clone 7 cells, a clonal cell line from hTERT-immortalized primary non-malignant tissue-derived human prostate epithelial cell line (RC170N/h), retain multipotent stem cell properties. The RC-170N/h/clone 7 cells expressed a human embryonic stem cell marker, Oct-4, and potential prostate epithelial stem cell markers, CD133, integrin {alpha}2{beta}1{sup hi} and CD44. The RC-170N/h/clone 7 cells proliferated in KGM and Dulbecco's Modified Eagle Medium with 10% fetalmore » bovine serum and 5 {mu}g/ml insulin (DMEM + 10% FBS + Ins.) medium, and differentiated into epithelial stem cells that expressed epithelial cell markers, including CK5/14, CD44, p63 and cytokeratin 18 (CK18); as well as the mesenchymal cell markers, vimentin, desmin; the neuron and neuroendocrine cell marker, chromogranin A. Furthermore the RC170 N/h/clone 7 cells differentiated into multi tissues when transplanted into the sub-renal capsule and subcutaneously of NOD-SCID mice. The results indicate that RC170N/h/clone 7 cells retain the properties of multipotent stem cells and will be useful as a novel cell model for studying the mechanisms of human prostate stem cell differentiation and transformation.« less

Confirmation of germ-line transmission in the red fluorescence protein (RFP) transgenic cloned male cat.

PubMed

Cho, Su-Jin; Lee, Young S; Lee, Jae-Ik; Bang, Jae-Il; Yang, Jing; Klassen, Henry; Kong, Il-Keun

2010-12-01

The production of transgenic animals is highly desirable for biotechnology and basic research. We investigated the reproductive ability of a red fluorescence protein (RFP) transgenic cloned male cat (RFP TG cat) by natural mating with a domestic female cat. The RFP expression levels were examined in early embryogenesis, tissues from 45-day-old two fetuses, and four RFP TG cat offspring. The RFP gene was detected in tissue samples from one dead kitten, including several organs and the skin. Also, under a fluorescent light source, we were able to directly detect the RFP expression of in in vitro-produced blastocysts derived with sperm from the RFP TG cat. These results indicate that the RFP TG cat exhibits normal reproductive fertility, stable germ-line transmission of the RFP transgene, and characteristic RFP expression in its offspring. We isolated feline neural progenitor cells from a 45-day-old fetus derived from the natural mating of the RFP TG cat with a domestic female cat. Isolated brain and retinal progenitor cells were successfully passaged at least four times post isolation (day 23), and showed a high RFP expression level. This method of producing genetically modified cloned cats will be important for generating biomedical models of human diseases.

Identification of a mouse synaptic glycoprotein gene in cultured neurons.

PubMed

Yu, Albert Cheung-Hoi; Sun, Chun Xiao; Li, Qiang; Liu, Hua Dong; Wang, Chen Ran; Zhao, Guo Ping; Jin, Meilei; Lau, Lok Ting; Fung, Yin-Wan Wendy; Liu, Shuang

2005-10-01

Neuronal differentiation and aging are known to involve many genes, which may also be differentially expressed during these developmental processes. From primary cultured cerebral cortical neurons, we have previously identified various differentially expressed gene transcripts from cultured cortical neurons using the technique of arbitrarily primed PCR (RAP-PCR). Among these transcripts, clone 0-2 was found to have high homology to rat and human synaptic glycoprotein. By in silico analysis using an EST database and the FACTURA software, the full-length sequence of 0-2 was assembled and the clone was named as mouse synaptic glycoprotein homolog 2 (mSC2). DNA sequencing revealed transcript size of mSC2 being smaller than the human and rat homologs. RT-PCR indicated that mSC2 was expressed differentially at various culture days. The mSC2 gene was located in various tissues with higher expression in brain, lung, and liver. Functions of mSC2 in neurons and other tissues remain elusive and will require more investigation.

MultiSite Gateway-Compatible Cell Type-Specific Gene-Inducible System for Plants1[OPEN

PubMed Central

Siligato, Riccardo; Wang, Xin; Yadav, Shri Ram; Lehesranta, Satu; Ma, Guojie; Ursache, Robertas; Sevilem, Iris; Zhang, Jing; Gorte, Maartje; Prasad, Kalika; Heidstra, Renze

2016-01-01

A powerful method to study gene function is expression or overexpression in an inducible, cell type-specific system followed by observation of consequent phenotypic changes and visualization of linked reporters in the target tissue. Multiple inducible gene overexpression systems have been developed for plants, but very few of these combine plant selection markers, control of expression domains, access to multiple promoters and protein fusion reporters, chemical induction, and high-throughput cloning capabilities. Here, we introduce a MultiSite Gateway-compatible inducible system for Arabidopsis (Arabidopsis thaliana) plants that provides the capability to generate such constructs in a single cloning step. The system is based on the tightly controlled, estrogen-inducible XVE system. We demonstrate that the transformants generated with this system exhibit the expected cell type-specific expression, similar to what is observed with constitutively expressed native promoters. With this new system, cloning of inducible constructs is no longer limited to a few special cases but can be used as a standard approach when gene function is studied. In addition, we present a set of entry clones consisting of histochemical and fluorescent reporter variants designed for gene and promoter expression studies. PMID:26644504

Cloning and expression of a Ca(2+)-inhibitable adenylyl cyclase from NCB-20 cells.

PubMed Central

Yoshimura, M; Cooper, D M

1992-01-01

A cDNA that encodes an adenylyl cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] has been cloned from NCB-20 cells, in which adenylyl cyclase activity is inhibited by Ca2+ at physiological concentrations. The cDNA clone (5.8 kilobases) was isolated by polymerase chain reaction (PCR) using degenerate primers designed by comparison of three adenylyl cyclase sequences (types I, II, and III) and subsequent library screening. Northern analysis revealed expression of mRNA (6.1 kilobases) corresponding to this cDNA in cardiac tissue, which is a prominent source of Ca(2+)-inhibitable adenylyl cyclase. The clone encodes a protein of 1165 amino acids, whose hydrophilicity profile was very similar to those of other mammalian adenylyl cyclases that have recently been cloned. A noticeable difference between this protein and other adenylyl cyclases was a lengthy aminoterminal region before the first transmembrane span. Transient expression of this cDNA in the human embryonic kidney cell line 293 revealed a 3-fold increase in cAMP production in response to forskolin compared with control transfected cells. In purified plasma membranes from transfected cells, increased adenylyl cyclase activity was also detected, which was susceptible to inhibition by submicromolar Ca2+. Thus, this adenylyl cyclase seems to represent the Ca(2+)-inhibitable form that is encountered in NCB-20 cells, cardiac tissue, and elsewhere. Its identification should permit a determination of the structural features that determine the mode of regulation of adenylyl cyclase by Ca2+. Images PMID:1379717

The organization and expression of the mdm2 gene.

PubMed

de Oca Luna, R M; Tabor, A D; Eberspaecher, H; Hulboy, D L; Worth, L L; Colman, M S; Finlay, C A; Lozano, G

1996-05-01

The mdm2 gene encodes a zinc finger protein that negatively regulates p53 function by binding and masking the p53 transcriptional activation domain. Two different promoters control expression of mdm2, one of which is also transactivated by p53. We cloned and characterized the mdm2 gene from a murine 129 library. It contained at least 12 exons and spanned approximately 25 kb of DNA. Sequencing of the mdm2 gene revealed three nucleotide differences that resulted in amino acid substitutions in the previously published mdm2 sequence. Sequencing of normal BalbC/J DNA and the original cosmid clone isolated from the 3T3DM cell line revealed that they are identical, suggesting that the published sequence is in error at these three positions. In addition, we analyzed the expression pattern of mdm2 and found ubiquitous low-level expression throughout embryo development and in adult tissues. Analysis of mRNA from numerous tissues for several mdm2 spliced variants that had been identified in the transformed 3T3DM cell line revealed that these variants could not be detected in the developing embryo or in adult tissues.

Molecular cloning of heat shock protein 60 (PtHSP60) from Portunus trituberculatus and its expression response to salinity stress.

PubMed

Xu, Qianghua; Qin, Ye

2012-09-01

Heat shock protein 60 (HSP60) is a highly conserved and multi-functional molecular chaperone that plays an essential role in both cellular metabolism and stress response. Portunus trituberculatus is an important marine fishery and aquaculture species, and water salinity condition influenced its artificial propagations significantly. In order to investigate the function of P. trituberculatus HSP60 against osmotic stress, P. trituberculatus HSP60 gene was firstly cloned. The full-length cDNA of PtHSP60 contains 1,743 nucleotides encoding 577 amino acids with a calculated molecular weight of 61.25 kDa. Multiple alignments indicated that the deduced amino acid sequences of PtHSP60 shared a high level of identity with invertebrate and vertebrate HSP60 sequence including shrimp, fruit fly, zebrafish, and human. The expression profiles of PtHSP60 at mRNA and protein levels under salinity treatment were investigated by semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blot analysis, respectively. It was found that the mRNA transcripts of PtHSP60 gene varied among different tissues under normal salinity conditions, and the antennal gland showed the highest expression level among the tissues tested. As for low salinity challenge, the mRNA expression of PtHSP60 gene was higher in the gill and appendicular muscle compared with other tissues, and gill and hypodermis represented the higher gene expressions during the hyperosmotic stress, which indicated that those tissues were salinity-sensitive tissues. In addition, salinity challenges significantly altered the expression of PtHSP60 at mRNA and protein level in a salinity- and time-dependent manner in P. trituberculatus gill tissue. The results indicate that PtHSP60 played important roles in mediating the salinity stress in P. trituberculatus.

Three concepts of cloning in human beings.

PubMed

Cui, Ke-Hui

2005-07-01

Human cloning, organ cloning and tissue cloning are various types of cloning that occur at different levels with different methodologies. According to three standards of terminology for an embryo (fertilization through germ cells, development in the uterus and having the potential to produce a human life), tissue cloning and type I organ cloning will not produce an embryo. In contrast, human cloning and type II organ cloning will produce an embryo. Thus, only non-germinal tissue cloning and type I organ cloning are beyond the ethical question and will not change human beings as a species. Using cloned tissues to make new tissues or organs is promising for the future of medicine.

Analysis of the antibody repertoire of lymphoma patients.

PubMed

Huang, Shaoming; Preuss, Klaus-Dieter; Xie, Xiaoxun; Regitz, Evi; Pfreundschuh, Michael

2002-12-01

Cancer testis or cancer germline antigens (CGA) are promising vaccine candidates because they are expressed only in malignant but not in normal tissues, except for germ cells in the testis. Since non-Hodgkin's lymphomas (NHL) express the known CGA at low frequencies, we aimed at increasing the number of CGA with frequent expression in NHL by screening a cDNA expression library derived from normal testis for reactivity with high-titered IgG antibodies in the sera of lymphoma patients using SEREX, the serological identification of antigens by recombinant cDNA expression cloning. The analysis of 1.6x10(6) clones with the sera of 25 lymphoma patients revealed 42 clones which coded for 23 antigens, 12 of which had already been included in the SEREX databank. Four cDNA clones coded for unknown and 19 for known genes. Three antigens reacted only with the serum by which they had been detected, 9 antigens reacted with the sera of several NHL patients, but not with that of healthy controls, and 11 antigens reacted with both normal and NHL sera. Most of the antigens were ubiquitously expressed. Only HOM-NHL-6, HOM-NHL-8, HOM-NHL-21 and HOM-NHL-23 showed a restricted expression pattern. HOM-NHL-6 and HOM-NHL-8 were homologous to the previously described CGA NY-ESO-1 and HOM-TES-14/SCP-1, respectively. HOM-NHL-21 was expressed in rare cases of lymphomas, but not in normal tissues except for testis and brain, while HOM-NHL-23 appeared to be a testis-specific antigen. In summary, using the antibody repertoire of these 25 NHL patients, no new CGA were detected. The number of CGA detectable by the classical SEREX approach appears to be limited, and novel strategies are necessary to identify antigens that can serve as a vaccine target in a broad spectrum of NHL patients.

Highly osteogenic PDL stem cell clones specifically express elevated levels of ICAM1, ITGB1 and TERT.

PubMed

Sununliganon, Laddawun; Singhatanadgit, Weerachai

2012-01-01

Cells derived from the periodontal ligament (PDL) have previously been reported to have stem cell-like characteristics (PDL stem cells; PDLSCs) and play an important part in bone engineering, including that of alveolar bone. However, these populations have been heterogeneous, and thus far no specific marker has yet been established from adult human stem cells derived from PDL tissue. We have previously isolated highly purified single cell-derived PDLSC clones and delineated their phenotypic and functional characteristics. In this report, we further obtained three homogeneous and distinct PDLSC clones demonstrating low, moderate and high mineralized matrix forming ability-namely PC12, PC4 and PC3, respectively, and the expression of mesenchymal stem cell pathway-specific genes in these clones was investigated. PCR array revealed that the expression of intercellular adhesion molecule 1 (ICAM1), integrin beta 1 (ITGB1) and telomerase reverse transcriptase (TERT) was associated with highly osteogenic PDLSC clones, as determined by the expression of key osteoblastic markers and their ability to form alizarin red S positive mineralized matrix in vitro. The present results suggest that these three mesenchymal stem cell-associated markers could potentially be used to isolate PDLSCs with high osteogenic capability for engineering new bone.

Molecular cloning of a small prostate protein, known as beta-microsemenoprotein, PSP94 or beta-inhibin, and demonstration of transcripts in non-genital tissues.

PubMed

Ulvsbäck, M; Lindström, C; Weiber, H; Abrahamsson, P A; Lilja, H; Lundwall, A

1989-11-15

In order to study the gene expression of the seminal plasma protein beta-microseminoprotein, also known as PSP94 and beta-inhibin, clones encoding this protein were isolated from a cDNA library constructed in lambda gt11. Nucleotide sequencing confirmed the structure of a previously cloned cDNA. By northern blot analysis identical sized transcripts were demonstrated in the prostate, the respiratory (tracheal, bronchial and lung) tissues and the antrum part of the gastric mucosa. Thus, the protein is not primarily associated with male reproductive function. Although probably of no physiological significance, a slight structural similarity to the ovarian inhibin beta-chains was identified in the C-terminal half of the molecule.

Characterization of embryo-specific genes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sung, Z.R.

1988-01-01

The objective of the proposed research is to characterize the structure and function of a set of genes whose expression is regulated in embryo development, and that are not expressed in mature tissues -- the embryogenic genes. In order to isolate these genes, we immunized a rabbit with total extracts of somatic embryos of carrot, and enriched the anti-embryo antiserum for antibodies reacting with extracts of carrot somatic embryos. Using this enriched antiserum, we screened a lambda gt11 cDNA library constructed from embryo poly A{sup +} RNA, and isolated 10 cDNA clones that detect embryogenic mRNAs. Monospecific antibodies have beenmore » purified for proteins corresponding to each cDNA sequence. Four cDNA clones were further characterized in terms of the expression of their corresponding mRNA and protein in somatic embryos of carrot. In some cases, comparable gene sequences or products have been detected in somatic and zygotic embryos of other plant species. The characteristics of these 4 cDNA clones -- clone Nos. 8, 59, and 66 -- are described in this report. 3 figs.« less

Cloning and function analysis of an alfalfa (Medicago sativa L.) zinc finger protein promoter MsZPP.

PubMed

Li, Yan; Sun, Yan; Yang, Qingchuan; Kang, Junmei; Zhang, Tiejun; Gruber, Margaret Yvonne; Fang, Feng

2012-08-01

A 1272 bp upstream sequence of MsZFN gene was cloned from alfalfa, which was designed as MsZPP (Genbank accession number: FJ 161979.2) using an adaptor-mediated genome walking method. A sole transcription start site was located 69 bp upstream of the translation start site. Its pattern of expression included roots, stem vascular tissues, floral reproductive organs, and leaves, but the promoter did not express in seeds, petals or sepals. Transcription levels can be stimulated by dark, MeJA, and IAA. However, GUS fusion activities had no change by treatments of GA, ABA, drought and high salt for 3 days. Deletion analysis revealed that all sections of the promoter can drive gus gene expression in the root, stem, leaves and floral reproductive organs; however, only fragments longer than the -460 bp promoter can stimulate strong gus gene expression in these organs. In addition, the -460 bp promoter fragment can drive gus expression not only in the vascular tissue, but also in leaf guard cells. The results suggest that the promoter MsZPP plays roles in the regulation of transgene expression, particularly due to its darkness, MeJA, and IAA responsiveness.

Cloning and expression of calmodulin gene in Scoparia dulcis.

PubMed

Saitoh, Daisuke; Asakura, Yuki; Nkembo, Marguerite Kasidimoko; Shite, Masato; Sugiyama, Ryuji; Lee, Jung-Bum; Hayashi, Toshimitsu; Kurosaki, Fumiya

2007-06-01

A homology-based cloning strategy yielded a cDNA clone, designated Sd-cam, encoding calmodulin protein from Scoparia dulcis. The restriction digests of genomic DNA of S. dulcis showed a single hybridized signal when probed with the fragment of this gene in Southern blot analyses, suggesting that Sd-cam occurs as a sole gene encoding calmodulin in the plant. The reverse-transcription polymerase chain reaction analysis revealed that Sd-cam was appreciably expressed in leaf, root and stem tissues. It appeared that transcription of this gene increased transiently when the leaf cultures of S. dulcis were treated with methyl jasmonate and calcium ionophore A23187. These results suggest that transcriptional activation of Sd-cam is one of the early cellular events of the methyl jasmonate-induced responses of S. dulcis.

cDNA cloning of the murine PEX gene implicated in X-linked hypophosphatemia and evidence for expression in bone

DOE Office of Scientific and Technical Information (OSTI.GOV)

Du, L.; Desbarats, M.; Viel, J.

1996-08-15

The recently identified human PEX g ene apparently encodes for a neutral endopeptidase that is mutated in patients with X-linked hypophosphatemia. The 3{prime} and 5{prime} ends of the coding region of PEX have not been cloned, nor has the tissue expression of the gene been identified. Here we report the isolation and characterization of the complete open reading frame of the mouse Pex gene and the demonstration of its expression in bone. Mouse Pex cDNA is predicted to encode a protein of 749 amino acids with 95% identity to the available human PEX sequence and significant homology to members ofmore » the membrane-bound metalloendopeptidase family. Northern blot analysis revealed a 6.6-kb transcript in bone and in cultured osteoblasts from normal mice that was not detectable in samples from the Hyp mouse, the murine homolog of human X-linked hypophosphatemia. Pex transcripts were, however, detectable in Hyp bone by RT-PCR amplification. Of particular interest, a cDNA clone from rat incisor shows 93% sequence identity to the 5{prime} end of Pex cDNA, suggesting that Pex may be expressed in another calcified tissue, the tooth. The association of impaired mineralization of bone and teeth and disturbed renal phosphate reabsorption with altered expression of Pex suggests that the Pex gene product may play a critical role in these processes. 47 refs., 2 figs., 1 tab.« less

The murine ufo receptor: molecular cloning, chromosomal localization and in situ expression analysis.

PubMed

Faust, M; Ebensperger, C; Schulz, A S; Schleithoff, L; Hameister, H; Bartram, C R; Janssen, J W

1992-07-01

We have cloned the mouse homologue of the ufo oncogene. It encodes a novel tyrosine kinase receptor characterized by a unique extracellular domain containing two immunoglobulin-like and two fibronectin type III repeats. Comparison of the predicted ufo amino acid sequences of mouse and man revealed an overall identity of 87.6%. The ufo locus maps to mouse chromosome 7A3-B1 and thereby extends the known conserved linkage group between mouse chromosome 7 and human chromosome 19. RNA in situ hybridization analysis established the onset of specific ufo expression in the late embryogenesis at day 12.5 post coitum (p.c.) and localized ufo transcription to distinct substructures of a broad spectrum of developing tissues (e.g. subepidermal cells of the skin, mesenchymal cells of the periosteum). In adult animals ufo is expressed in cells forming organ capsules as well as in connective tissue structures. ufo may function as a signal transducer between specific cell types of mesodermal origin.

Analysis of the function of the agouti gene in obesity and diabetes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mynatt, R.L.; Miltenberger, R.J.; Klebig, M.L.

1996-09-01

This chapter discusses the agouti gene and dominant mutations in that gene that lead to agouti-induced obesity, and recent work with transgenic mice to elucidate the role of agouti in obesity. Agouti was cloned in 1992 by the lab of Rick Woychik at Oak Ridge National Laboratory, making it the first of many recently cloned mouse obesity genes. Sequence analysis predicted that mouse agouti is a secreted protein of 131 amino acids. The mature protein has a basic central region (lys57-arg85), a proline-rich domain (pro86-pro91) and a C-terminal region (cys 92-cys 13 1) containing 10 cysteine residues which form 5more » disulfide bonds. The human homologue of agouti has also been cloned by the Woychik lab and maps to human chromosome 20q 11.2. Human agouti is 132 amino acids long and is 85% similar to the mouse agouti protein and is normally expressed in adipose tissue. The researchers have been able to recapitulate obesity, hyperinsulinemia, and hyperglycemia with the ubiquitous expression of agouti. Agouti expression in either liver and adipose tissue alone does not cause obesity, and there`s a dose-dependent effect of agouti on body weight, food efficiency, body temperature, and insulin and glucose levels.« less

Expressed gene sequence of the IFN-gamma-response chemokine CXCL9 of cattle, horses, and swine

USDA-ARS?s Scientific Manuscript database

This report describes the cloning and characterization of expressed gene sequences of bovine, equine, and swine CXCL9 from RNA obtained from peripheral blood mononuclear cell (PBMC) or other tissues. The bovine coding region was 378 nucleotides in length, while the equine and swine coding regions w...

Expression of heavy metal tolerance in pollen and implications for gametophytic selection. [The plants used were clones of Silene dioica and Mimulus guttatus

DOE Office of Scientific and Technical Information (OSTI.GOV)

Searcy, K.B.

Many genes are expressed in both sporophytic and microgametophytic phases of the angiosperm life cycle. Thus, selection in one phase could modify gene frequency in both phases. An attempt was made to investigate microgametophytic selection in response to toxic concentrations of heavy metals and the effect of this selection upon the resultant sporophyte generation. The plants used were clones of a zinc-tolerant Silene dioica, closely related nontolerant S. alba, and copper tolerant and non-tolerant clones of Mimulus guttatus. First, the expression of metal tolerance in pollen was established by in vitro pollen germination and tube growth, and was found tomore » be associated with the tolerance of the pollen source. Second, to test the extent to which the parallel expression of metal tolerance was determined by the gametophytic genotype, tolerant but segregating clones were grown with and without added metals. Finally, selection was applied during pollen germination, tube growth and fertilization. In Silene, neither the tolerance of the pollen nor the metal content of the styles affected pollen tube growth rate. In Mimulus, pollen from the nontolerant source grew faster, but the metal content of the floral tissue had no significant effect on pollen tube growth rate, and only slightly reduced the fertilization ability of pollen from the nontolerant clone.« less

Isolation and expression of three gibberellin 20-oxidase cDNA clones from Arabidopsis.

PubMed

Phillips, A L; Ward, D A; Uknes, S; Appleford, N E; Lange, T; Huttly, A K; Gaskin, P; Graebe, J E; Hedden, P

1995-07-01

Using degenerate oligonucleotide primers based on a pumpkin (Cucurbita maxima) gibberellin (GA) 20-oxidase sequence, six different fragments of dioxygenase genes were amplified by polymerase chain reaction from arabidopsis thaliana genomic DNA. One of these was used to isolate two different full-length cDNA clones, At2301 and At2353, from shoots of the GA-deficient Arabidopsis mutant ga1-2. A third, related clone, YAP169, was identified in the Database of Expressed Sequence Tags. The cDNA clones were expressed in Escherichia coli as fusion proteins, each of which oxidized GA12 at C-20 to GA15, GA24, and the C19 compound GA9, a precursor of bioactive GAs; the C20 tricarboxylic acid compound GA25 was formed as a minor product. The expression products also oxidized the 13-hydroxylated substrate GA53, but less effectively than GA12. The three cDNAs hybridized to mRNA species with tissue-specific patterns of accumulation, with At2301 being expressed in stems and inflorescences, At2353 in inflorescences and developing siliques, and YAP169 in siliques only. In the floral shoots of the ga1-2 mutant, transcript levels corresponding to each cDNA decreased dramatically after GA3 application, suggesting that GA biosynthesis may be controlled, at least in part, through down-regulation of the expression of the 20-oxidase genes.

Molecular Cloning and Characterization of Two Pig Vasoactive Intestinal Polypeptide Receptors (VPAC1-R and VPAC2-R)

PubMed Central

He, Xiaping; Meng, Fengyan; Wang, Yajun

2014-01-01

We here report the cloning, tissue expression, and functional analyses of the two pig vasoactive intestinal polypeptide (VIP) receptors (pVPAC1-R and pVPAC2-R). The cloned full-length pVPAC1-R and pVPAC2-R share high structural similarity with their mammalian counterparts. Functional assay revealed that the full-length pVPAC1-R and pVPAC2-R-expressed Chinese hamster ovary (CHO) cells could be activated by pVIP and pPACAP38 potently, indicating that pVPAC1-R and pVPAC2-R are capable of binding VIP and pituitary adenylate cyclase-activating polypeptide (PACAP). In addition to the identification of the transcripts encoding the two full-length receptors, multiple splice transcript variants were isolated. Comparison with the pig genome database revealed that pVPAC1-R and pVPAC2-R share a unique gene structure with 14 exons different from other vertebrates. Reverse transcription and polymerase chain reaction (RT-PCR) assays further showed that the transcript encoding the full-length pVPAC2-R is widely expressed in all adult tissues whereas the splice variants of pVPAC1-R are predominantly expressed in all tissues instead of the transcript encoding the full-length receptor, hinting that pVPAC2-R may play more important roles than pVPAC1-R in mediating VIP and PACAP actions. Our present findings help to elucidate the important role of VIP and PACAP and promote to rethink of their species-specific physiological roles including their actions in regulation of phenotypic traits in pigs. PMID:24520933

A brain-specific gene cluster isolated from the region of the mouse obesity locus is expressed in the adult hypothalamus and during mouse development

DOE Office of Scientific and Technical Information (OSTI.GOV)

Laig-Webster, M.; Lim, M.E.; Chehab, F.F.

1994-09-01

The molecular defect underlying an autosomal recessive form of genetic obesity in a classical mouse model C57 BL/6J-ob/ob has not yet been elucidated. Whereas metabolic and physiological disturbances such as diabetes and hypertension are associated with obesity, the site of expression and the nature of the primary lesion responsible for this cascade of events remains elusive. Our efforts aimed at the positional cloning of the ob gene by YAC contig mapping and gene identification have resulted in the cloning of a brain-specific gene cluster from the ob critical region. The expression of this gene cluster is remarkably complex owing tomore » the multitude of brain-specific mRNA transcripts detected on Northern blots. cDNA cloning of these transcripts suggests that they are expressed from different genes as well as by alternate splicing mechanisms. Furthermore, the genomic organization of the cluster appears to consist of at least two identical promoters displaying CpG islands characteristic of housekeeping genes, yet clearly involving tissue-specific expression. Sense and anti-sense synthetic RNA probes were derived from a common DNA sequence on 3 cDNA clones and hybridized to 8-16 days mouse embryonic stages and mouse adult brain sections. Expression in development was noticeable as of the 11th day of gestation and confined to the central nervous system mainly in the telencephalon and spinal cord. Coronal and sagittal sections of the adult mouse brain showed expression only in 3 different regions of the brain stem. In situ hybridization to mouse hypothalamus sections revealed the presence of a localized and specialized group of cells expressing high levels of mRNA, suggesting that this gene cluster may also be involved in the regulation of hypothalamic activities. The hypothalamus has long been hypothesized as a primary candidate tissue for the expression of the obesity gene mainly because of its well-established role in the regulation of energy metabolism and food intake.« less

Diurnal oscillation of SBE expression in sorghum endosperm

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sun, Chuanxin; Mutisya, J.; Rosenquist, S.

2009-01-15

Spatial and temporal expression patterns of the sorghum SBEI, SBEIIA and SBEIIB genes, encoding, respectively, starch branching enzyme (SBE) I, IIA and IIB, in the developing endosperm of sorghum (Sorghum bicolor) were studied. Full-length genomic and cDNA clones for sorghum was cloned and the SBEIIA cDNA was used together with gene-specific probes for sorghum SBEIIB and SBEI. In contrast to sorghum SBEIIB, which was expressed primarily in endosperm and embryo, SBEIIA was expressed also in vegetative tissues. All three genes shared a similar temporal expression profile during endosperm development, with a maximum activity at 15-24 days after pollination. This ismore » different from barley and maize where SBEI gene activity showed a significantly later onset compared to that of SBEIIA and SBEIIB. Expression of the three SBE genes in the sorghum endosperm exhibited a diurnal rhythm during a 24-h cycle.« less

Immunotargeting and cloning of two CD34 variants exhibiting restricted expression in adult rat endothelia in vivo.

PubMed

Testa, Jacqueline E; Chrastina, Adrian; Oh, Phil; Li, Yan; Witkiewicz, Halina; Czarny, Malgorzata; Buss, Tim; Schnitzer, Jan E

2009-08-01

Mapping protein expression of endothelial cells (EC) in vivo is fundamental to understanding cellular function and may yield new tissue-selective targets. We have developed a monoclonal antibody, MAb J120, to a protein expressed primarily in rat lung and heart endothelium. The antigen was identified as CD34, a marker of hematopoietic stem cells and global marker of endothelial cells in human and mouse tissues. PCR-based cloning identified two CD34 variant proteins, full length and truncated, both of which are expressed on luminal endothelial cell plasma membranes (P) isolated from lung. Truncated CD34 predominated in heart P, and neither variant was detected in P from kidney or liver. CD34 in lung was readily accessible to (125)I-J120 inoculated intravenously, and immunohistochemistry showed strong CD34 expression in lung EC. Few microvessels stained in heart and kidney, and no CD34 was detected in vessels of other organs or in lymphatics. We present herein the first complete sequence of a rat CD34 variant and show for the first time that the encoded truncated variant is endogenously expressed on EC in vivo. We also demonstrate that CD34 expression in rat EC, unlike mouse and human, is restricted in its distribution enabling quite specific lung targeting in vivo.

Molecular characterization of two ferritins of the scallop Argopecten purpuratus and gene expressions in association with early development, immune response and growth rate.

PubMed

Coba de la Peña, Teodoro; Cárcamo, Claudia B; Díaz, María I; Brokordt, Katherina B; Winkler, Federico M

2016-08-01

Ferritin is involved in several iron homoeostasis processes in molluscs. We characterized two ferritin homologues and their expression patterns in association with early development, growth rate and immune response in the scallop Argopecten purpuratus, a species of economic importance for Chile and Peru. Two ferritin subunits (Apfer1 and Apfer2) were cloned. Apfer1 cDNA is a 792bp clone containing a 516bp open reading frame (ORF) that corresponds to a novel ferritin subunit in A. purpuratus. Apfer2 cDNA is a 681bp clone containing a 522bp ORF that corresponds to a previously sequenced EST. A putative iron responsive element (IRE) was identified in the 5'-untranslated region of both genes. The deduced protein sequences of both cDNAs possessed the motifs and domains characteristic of functional ferritin subunits. Both genes showed differential expression patterns at tissue-specific and early development stage levels. Apfer1 expression level increased 40-fold along larval developmental stages, decreasing markedly after larval settlement. Apfer1 expression in mantle tissue was 2.8-fold higher in fast-growing than in slow-growing scallops. Apfer1 increased 8-fold in haemocytes 24h post-challenge with the bacterium Vibrio splendidus. Apfer2 expression did not differ between fast- and slow-growing scallops or in response to bacterial challenge. These results suggest that Apfer1 and Apfer2 may be involved in iron storage, larval development and shell formation. Apfer1 expression may additionally be involved in immune response against bacterial infections and also in growth; and thus would be a potential marker for immune capacity and for fast growth in A. purpuratus. Copyright © 2016 Elsevier Inc. All rights reserved.

Molecular cloning, characterization, tissue distribution and mRNA expression changes during the hibernation and reproductive periods of estrogen receptor alpha (ESR1) in Chinese alligator, Alligator sinensis.

PubMed

Zhang, Ruidong; Hu, Yuehong; Wang, Huan; Yan, Peng; Zhou, Yongkang; Wu, Rong; Wu, Xiaobing

2016-10-01

Chinese alligator, Alligator sinensis, is a critically endangered reptile species unique to China. Little is known about the mechanism of growth- and reproduction-related hormones gene expression in Chinese alligator. Estrogens play important roles in regulating multiple reproduction- and non-reproduction-related functions by binding to their corresponding receptors. Here, the full-length cDNA of estrogen receptor alpha (ERα/ESR1) was cloned and sequenced from Chinese alligator for the first time, which comprises 1764bp nucleotides and encodes a predicted protein of 587 amino acids. Phylogenetic analysis of ESR1 showed that crocodilians and turtles were the sister-group of birds. The results of real-time quantitative PCR indicated that the ESR1 mRNA was widely expressed in the brain and peripheral tissues. In the brain and pituitary gland, ESR1 was most highly transcribed in the cerebellum. But in other peripheral tissues, ESR1 mRNA expression level was the highest in the ovary. Compared with hibernation period, ESR1 mRNA expression levels were increased significantly in the reproductive period (P<0.05) in cerebellum, pituitary gland, liver, spleen, lung, kidney and ovary, while no significant change in other examined tissues (P>0.05). The ESR1 mRNA expression levels changes during the two periods of different tissues suggested that ESR1 might play an important role in mediation of estrogenic multiple reproductive effects in Chinese alligator. Furthermore, it was the first time to quantify ESR1 mRNA level in the brain of crocodilians, and the distribution and expression of ESR1 mRNA in the midbrain, cerebellum and medulla oblongata was also reported for the first time in reptiles. Copyright © 2016 Elsevier Inc. All rights reserved.

Cloning, Expression, and Characterization of Sorbitol Transporters from Developing Sour Cherry Fruit and Leaf Sink Tissues1

PubMed Central

Gao, Zhifang; Maurousset, Laurence; Lemoine, Remi; Yoo, Sang-Dong; van Nocker, Steven; Loescher, Wayne

2003-01-01

The acyclic polyol sorbitol is a primary photosynthetic product and the principal photosynthetic transport substance in many economically important members of the family Rosaceace (e.g. almond [Prunus dulcis (P. Mill.) D.A. Webber], apple [Malus pumila P. Mill.], cherry [Prunus spp.], peach [Prunus persica L. Batsch], and pear [Pyrus communis]). To understand key steps in long-distance transport and particularly partitioning and accumulation of sorbitol in sink tissues, we have cloned two sorbitol transporter genes (PcSOT1 and PcSOT2) from sour cherry (Prunus cerasus) fruit tissues that accumulate large quantities of sorbitol. Sorbitol uptake activities and other characteristics were measured by heterologous expression of PcSOT1 and PcSOT2 in yeast (Saccharomyces cerevisiae). Both genes encode proton-dependent, sorbitol-specific transporters with similar affinities (Km sorbitol of 0.81 mm for PcSOT1 and 0.64 mm for PcSOT2). Analyses of gene expression of these transporters, however, suggest different roles during leaf and fruit development. PcSOT1 is expressed throughout fruit development, but especially when growth and sorbitol accumulation rates are highest. In leaves, PcSOT1 expression is highest in young, expanding tissues, but substantially less in mature leaves. In contrast, PcSOT2 is mainly expressed only early in fruit development and not in leaves. Compositional analyses suggest that transport mediated by PcSOT1 and PcSOT2 plays a major role in sorbitol and dry matter accumulation in sour cherry fruits. Presence of these transporters and the high fruit sorbitol concentrations suggest that there is an apoplastic step during phloem unloading and accumulation in these sink tissues. Expression of PcSOT1 in young leaves before completion of the transition from sink to source is further evidence for a role in determining sink activity. PMID:12692316

Differential developmental ability of embryos cloned from tissue-specific stem cells.

PubMed

Inoue, Kimiko; Noda, Shinichi; Ogonuki, Narumi; Miki, Hiromi; Inoue, Shinichi; Katayama, Kazufumi; Mekada, Kazuyuki; Miyoshi, Hiroyuki; Ogura, Atsuo

2007-05-01

Although cloning animals by somatic cell nuclear transfer is generally inefficient, the use of certain nuclear donor cell types may significantly improve or deteriorate outcomes. We evaluated whether two multipotent stem cell lines produced in vitro--neural stem cells (NSCs) and mesenchymal stem cells (MSCs)--could serve as nuclear donors for nuclear transfer cloning. Most (76%) NSC-derived embryos survived the two-cell-to-four-cell transition, the stage when the major zygotic gene activation occurs. Consistent with this observation, the expression patterns of zygotically active genes were better in NSC-derived embryos than in fibroblast clone embryos, which arrested at the two-cell stage more frequently. Embryo transfer experiments demonstrated that at least some of these NSC embryos had the ability to develop to term fetuses (1.6%, 3/189). In contrast, embryos reconstructed using MSCs showed a low rate of in vitro development and never underwent implantation in vivo. Chromosomal analysis of the donor MSCs revealed very frequent aneuploidy, which probably impaired the potential for development of their derived clones. This is the first demonstration that tissue-specific multipotent stem cells produced in vitro can serve as donors of nuclei for cloning mice; however, these cells may be prone to chromosomal aberrations, leading to high embryonic death rates. We found previously that hematopoietic stem cells (HSCs) are very inefficient donor cells because of their failure to activate the genes essential for embryonic development. Taken together, our data led us to conclude that tissue-specific stem cells in mice, namely NSCs, MSCs, and HSCs, exhibited marked variations in the ability to produce cloned offspring and that this ability varies according to both the epigenetic and genetic status of the original genomes. Disclosure of potential conflicts of interest is found at the end of this article.

Molecular cloning and expression analysis of jasmonic acid dependent but salicylic acid independent LeWRKY1.

PubMed

Lu, M; Wang, L F; Du, X H; Yu, Y K; Pan, J B; Nan, Z J; Han, J; Wang, W X; Zhang, Q Z; Sun, Q P

2015-11-30

Various plant genes can be activated or inhibited by phytohormones under conditions of biotic and abiotic stress, especially in response to jasmonic acid (JA) and salicylic acid (SA). Interactions between JA and SA may be synergistic or antagonistic, depending on the stress condition. In this study, we cloned a full-length cDNA (LeWRKY1, GenBank accession No. FJ654265) from Lycopersicon esculentum by rapid amplification of cDNA ends. Sequence analysis showed that this gene is a group II WRKY transcription factor. Analysis of LeWRKY1 mRNA expression in various tissues by qRT-PCR showed that the highest and lowest expression occurred in the leaves and stems, respectively. In addition, LeWRKY1 expression was induced by JA and Botrytis cinerea Pers., but not by SA.

Elucidation of the effect of brain cortex tetrapeptide Cortagen on gene expression in mouse heart by microarray.

PubMed

Anisimov, Sergey V; Khavinson, Vladimir Kh; Anisimov, Vladimir N

2004-01-01

Aging is associated with significant alterations in gene expression in numerous organs and tissues. Anti-aging therapy with peptide bioregulators holds much promise for the correction of age-associated changes, making a screening for their molecular targets in tissues an important question of modern gerontology. The synthetic tetrapeptide Cortagen (Ala-Glu-Asp-Pro) was obtained by directed synthesis based on amino acid analysis of natural brain cortex peptide preparation Cortexin. In humans, Cortagen demonstrated a pronounced therapeutic effect upon the structural and functional posttraumatic recovery of peripheral nerve tissue. Importantly, other effects were also observed in cardiovascular and cerebrovascular parameters. Based on these latter observations, we hypothesized that acute course of Cortagen treatment, large-scale transcriptome analysis, and identification of transcripts with altered expression in heart would facilitate our understanding of the mechanisms responsible for this peptide biological effects. We therefore analyzed the expression of 15,247 transcripts in the heart of female 6-months CBA mice receiving injections of Cortagen for 5 consecutive days was studied by cDNA microarrays. Comparative analysis of cDNA microarray hybridisation with heart samples from control and experimental group revealed 234 clones (1,53% of the total number of clones) with significant changes of expression that matched 110 known genes belonging to various functional categories. Maximum up- and down-regulation was +5.42 and -2.86, respectively. Intercomparison of changes in cardiac expression profile induced by synthetic peptides (Cortagen, Vilon, Epitalon) and pineal peptide hormone melatonin revealed both common and specific effects of Cortagen upon gene expression in heart.

Abcb1 in Pigs: Molecular cloning, tissues distribution, functional analysis, and its effect on pharmacokinetics of enrofloxacin

PubMed Central

Guo, Tingting; Huang, Jinhu; Zhang, Hongyu; Dong, Lingling; Guo, Dawei; Guo, Li; He, Fang; Bhutto, Zohaib Ahmed; Wang, Liping

2016-01-01

P-glycoprotein (P-gp) is one of the best-known ATP-dependent efflux transporters, contributing to differences in pharmacokinetics and drug-drug interactions. Until now, studies on pig P-gp have been scarce. In our studies, the full-length porcine P-gp cDNA was cloned and expressed in a Madin-Darby Canine Kidney (MDCK) cell line. P-gp expression was then determined in tissues and its role in the pharmacokinetics of oral enrofloxacin in pigs was studied. The coding region of pig Abcb1 gene was 3,861 bp, encoding 1,286 amino acid residues (Mw = 141,966). Phylogenetic analysis indicated a close evolutionary relationship between porcine P-gp and those of cow and sheep. Pig P-gp was successfully stably overexpressed in MDCK cells and had efflux activity for rhodamine 123, a substrate of P-gp. Tissue distribution analysis indicated that P-gp was highly expressed in brain capillaries, small intestine, and liver. In MDCK-pAbcb1 cells, enrofloxacin was transported by P-gp with net efflux ratio of 2.48 and the efflux function was blocked by P-gp inhibitor verapamil. High expression of P-gp in the small intestine could modify the pharmacokinetics of orally administrated enrofloxacin by increasing the Cmax, AUC and Ka, which was demonstrated using verapamil, an inhibitor of P-gp. PMID:27572343

Potential pathogenetic role of Th17, Th0, and Th2 cells in erosive and reticular oral lichen planus.

PubMed

Piccinni, M-P; Lombardelli, L; Logiodice, F; Tesi, D; Kullolli, O; Biagiotti, R; Giudizi, Mg; Romagnani, S; Maggi, E; Ficarra, G

2014-03-01

The role of Th17 cells and associated cytokines was investigated in oral lichen planus. 14 consecutive patients with oral lichen planus were investigated. For biological studies, tissues were taken from reticular or erosive lesions and from normal oral mucosa (controls) of the same patient. mRNA expression for IL-17F, IL-17A, MCP-1, IL-13, IL-2, IL-10, IL-1β, RANTES, IL-4, IL-12B, IL-8, IFN-γ, TNF-α, IL-1α, IL-18, TGF-β1, IL-23R, IL-7, IL-15, IL-6, MIG, IP-10, LTB, VEGF, IL-5, IL-27, IL-23A, GAPDH, PPIB, Foxp3, GATA3, and RORC was measured using the QuantiGene 2.0. Results showed that Th17-type and Th0-type molecules' mRNAs, when compared with results obtained from tissue controls, were increased in biopsies of erosive lesions, whereas Th2-type molecules' mRNAs were increased in reticular lesions. When the CD4+ T-cell clones, derived from oral lichen planus tissues and tissue controls, were analyzed, a higher prevalence of Th17 (confirmed by an increased CD161 expression) and Th0 CD4+ T clones was found in erosive lesions, whereas a prevalence of Th2 clones was observed in reticular lesions. Our data suggest that Th17, Th0, and Th2 cells, respectively, may have a role in the pathogenesis of erosive and reticular oral lichen planus. © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Yak response to high-altitude hypoxic stress by altering mRNA expression and DNA methylation of hypoxia-inducible factors.

PubMed

Xiong, Xianrong; Fu, Mei; Lan, Daoliang; Li, Jian; Zi, Xiangdong; Zhong, Jincheng

2015-01-01

Hypoxia-inducible factors (HIFs) are oxygen-dependent transcriptional activators, which play crucial roles in tumor angiogenesis and mammalian development, and regulate the transcription of genes involved in oxygen homeostasis in response to hypoxia. However, information on HIF-1α and HIF-2α in yak (Bos grunniens) is scarce. The complete coding region of yak HIF-2α was cloned, its mRNA expression in several tissues were determined, and the expression levels were compared with those of closely related low-altitude cattle (Bos taurus), and the methylation status of promoter regions were analyzed to better understand the roles of HIF-1α and HIF-2α in domesticated yak. The yak HIF-2α cDNA was cloned and sequenced in the present work reveals the evolutionary conservation through multiple sequence alignment, although 15 bases changed, resulting in 8 amino acid substitutions in the translated proteins in cattle. The tissue-specific expression results showed that HIF-1α is ubiquitously expressed, whereas HIF-2α expression is limited to endothelial tissues (kidney, heart, lung, spleen, and liver) and blood in yak. Both HIF-1α and HIF-2α expressions were higher in yak tissues than in cattle. The HIF-1α expression level is much higher in yak than cattle in these organs, except for the lung (P < 0.05), but the HIF-2α gene is significantly different in the heart, spleen, and kidney (P < 0.05). Furthermore, the methylation levels in the 5' flanking regulatory regions of HIF-1α and HIF-2α in yak kidney were significantly decreased than cattle counterparts (P < 0.05). Identifying these genes and the comparison of different expressions facilitates the understanding of the biological high-altitude hypoxic stress response mechanism and may assist current medical research to understand hypoxia-related diseases.

Molecular cloning and expression analysis of five heat shock protein 70 (HSP70) family members in Lateolabrax maculatus with Vibrio harveyi infection.

PubMed

Han, Ying-Li; Hou, Cong-Cong; Du, Chen; Zhu, Jun-Quan

2017-01-01

Heat shock proteins 70 (HSP70s) are molecular chaperones that aid in protection against environmental stress. In this study, we cloned and characterized five members of the HSP70 family (designated as HSPa1a, HSC70-1, HSC70-2, HSPa4 and HSPa14) from Lateolabrax maculatus using rapid amplification cDNA ends (RACE). Multiple sequence alignment and structural analysis revealed that all members of the HSP70 family had a conserved domain architecture, with some distinguishing features unique to each HSP70. Quantitative real-time (qPCR) analysis revealed that all members of the HSP70 family were ubiquitously and differentially expressed in all major types of tissues, including testicular tissue. This indicated that HSP70s have vital and conserved biological functions, and may also function in the development of germinal cells. The expression of mRNA of the five HSP70 family members mRNA expression was significantly increased in the head kidney, intestine and gill after Vibrio harveyi challenge, suggesting that HSP70s play an important role in the immune response. Copyright © 2016 Elsevier Ltd. All rights reserved.

Rapid in silico cloning of genes using expressed sequence tags (ESTs).

PubMed

Gill, R W; Sanseau, P

2000-01-01

Expressed sequence tags (ESTs) are short single-pass DNA sequences obtained from either end of cDNA clones. These ESTs are derived from a vast number of cDNA libraries obtained from different species. Human ESTs are the bulk of the data and have been widely used to identify new members of gene families, as markers on the human chromosomes, to discover polymorphism sites and to compare expression patterns in different tissues or pathologies states. Information strategies have been devised to query EST databases. Since most of the analysis is performed with a computer, the term "in silico" strategy has been coined. In this chapter we will review the current status of EST databases, the pros and cons of EST-type data and describe possible strategies to retrieve meaningful information.

Cloning and expression of hepatic synaptotagmin 1 in mouse.

PubMed

Sancho-Knapik, Sara; Guillén, Natalia; Osada, Jesús

2015-05-15

Mouse hepatic synaptotagmin 1 (SYT1) cDNA was cloned, characterized and compared to the brain one. The hepatic transcript was 1807 bp in length, smaller than the brain, and only encoded by 9 of 11 gene exons. In this regard, 5'-and 3'-untranslated regions were 66 and 476 bp, respectively; the open reading frame of 1266 bp codified for a protein of 421 amino acids, identical to the brain, with a predicted molecular mass of 47.4 kDa and highly conserved across different species. Immunoblotting of protein showed two isoforms of higher molecular masses than the theoretical prediction based on amino acid sequence suggesting posttranslational modifications. Subcellular distribution of protein isoforms corresponded to plasma membrane, lysosomes and microsomes and was identical between the brain and liver. Nonetheless, the highest molecular weight isoform was smaller in the liver, irrespective of subcellular location. Quantitative mRNA tissue distribution showed that it was widely expressed and that the highest values corresponded to the brain, followed by the liver, spleen, abdominal fat, intestine and skeletal muscle. These findings indicate tissue-specific splicing of the gene and posttranslational modification and the variation in expression in the different tissues might suggest a different requirement of SYT1 for the specific function in each organ. Copyright © 2015 Elsevier B.V. All rights reserved.

v-src induces clonal sarcomas and rapid metastasis following transduction with a replication-defective retrovirus

DOE Office of Scientific and Technical Information (OSTI.GOV)

Stoker, A.W.; Sieweke, M.H.

1989-12-01

v-src is an effective carcinogen when expressed from Rous sarcoma virus (RSV) in vivo. Whereas RSV tumors require sustained oncogene expression, their growth is largely a balance between viral recruitment of tissues and host immune destruction of infected cells. The authors have therefore examined the tumorigenic potential of v-src in the absence of viral recruitment and viral antigen expression. v-src was introduced with high efficiency into chicken wing web tissues using replication-defective (rd) retroviral vectors. Clonal sarcomas were induced rapidly, and furthermore, v-src potentiated metastatic progression in {approx} 0.1%-1% of tumor clones with unexpectedly short latency. rd vectors proved effectivemore » not only in transducing v-src into tissues but also as insertional markers of tumor clonality. The rd vector present in most primary and metastatic tumors was a highly truncated form of RSV derived by viral transmission of spliced v-src mRNA; this vector should thus avoid viral recruitment and host anti-viral immune reaction through its complete lack of viral structural genes. Under such conditions v-src maintains strong carcinogenicity in vivo when restricted to clonal tumor growth and can confer rapid metastatic potential on a discrete subset of tumor clones.« less

Hyper-reactive cloned mice generated by direct nuclear transfer of antigen-specific CD4+ T cells.

PubMed

Kaminuma, Osamu; Katayama, Kazufumi; Inoue, Kimiko; Saeki, Mayumi; Nishimura, Tomoe; Kitamura, Noriko; Shimo, Yusuke; Tofukuji, Soichi; Ishida, Satoru; Ogonuki, Narumi; Kamimura, Satoshi; Oikawa, Mami; Katoh, Shigeki; Mori, Akio; Shichijo, Michitaka; Hiroi, Takachika; Ogura, Atsuo

2017-06-01

T-cell receptor (TCR)-transgenic mice have been employed for evaluating antigen-response mechanisms, but their non-endogenous TCR might induce immune response differently than the physiologically expressed TCR Nuclear transfer cloning produces animals that retain the donor genotype in all tissues including germline and immune systems. Taking advantage of this feature, we generated cloned mice that carry endogenously rearranged TCR genes from antigen-specific CD4 + T cells. We show that T cells of the cloned mice display distinct developmental pattern and antigen reactivity because of their endogenously pre-rearranged TCRα (rTα) and TCRβ (rTβ) alleles. These alleles were transmitted to the offspring, allowing us to establish a set of mouse lines that show chronic-type allergic phenotypes, that is, bronchial and nasal inflammation, upon local administrations of the corresponding antigens. Intriguingly, the existence of either rTα or rTβ is sufficient to induce in vivo hypersensitivity. These cloned mice expressing intrinsic promoter-regulated antigen-specific TCR are a unique animal model with allergic predisposition for investigating CD4 + T-cell-mediated pathogenesis and cellular commitment in immune diseases. © 2017 The Authors.

Schizothorax prenanti corticotropin-releasing hormone (CRH): molecular cloning, tissue expression, and the function of feeding regulation.

PubMed

Wang, Tao; Zhou, Chaowei; Yuan, Dengyue; Lin, Fangjun; Chen, Hu; Wu, Hongwei; Wei, Rongbin; Xin, Zhiming; Liu, Ju; Gao, Yundi; Li, Zhiqiong

2014-10-01

Corticotropin-releasing hormone (CRH) is a potent mediator of endocrine, autonomic, behavioral, and immune responses to stress. For a better understanding of the structure and function of the CRH gene and to study its effect on feeding regulation in cyprinid fish, the cDNA of the CRH gene from the brain of Schizothorax prenanti was cloned and sequenced. The full-length CRH cDNA consisted of 1,046 bp with an open reading frame of 489 bp encoding a protein of 162 amino acids. Real-time quantitative PCR analyses revealed that CRH was widely expressed in central and peripheral tissues. In particular, high expression level of CRH was detected in brain. Furthermore, CRH mRNA expression was examined in different brain regions, especially high in hypothalamus. In addition, there was no significant change in CRH mRNA expression in fed group compared with the fasted group in the S. prenanti hypothalamus during short-term fasting. However, CRH gene expression presented significant decrease in the hypothalamus in fasted group compared with the fed group (P < 0.05) on day 7; thereafter, re-feeding could lead to a significant increase in CRH mRNA expression in fasted group on day 9. The results suggest that the CRH may play a critical role in feeding regulation in S. prenanti.

Cloning, Phylogenetic Analysis, and Distribution of Free Fatty Acid Receptor GPR120 Expression along the Gastrointestinal Tract of Housing versus Grazing Kid Goats.

PubMed

Ran, Tao; Li, Hengzhi; Liu, Yong; Zhou, Chuanshe; Tang, Shaoxun; Han, Xuefeng; Wang, Min; He, Zhixiong; Kang, Jinghe; Yan, Qiongxian; Tan, Zhiliang; Beauchemin, Karen A

2016-03-23

G-protein-coupled receptor 120 (GPR120) is reported as a long-chain fatty acid (LCFA) receptor that elicits free fatty acid (FFA) regulation on metabolism homeostasis. The study aimed to clone the gpr120 gene of goats (g-GPR120) and subsequently investigate phylogenetic analysis and tissue distribution throughout the digestive tracts of kid goats, as well as the effect of housing versus grazing (H vs G) feeding systems on GPR120 expression. Partial coding sequence (CDS) of g-GPR120 was cloned and submitted to NCBI (accession no. KU161270 ). Phylogenetic analysis revealed that g-GPR120 shared higher homology in both mRNA and amino acid sequences for ruminants than nonruminants. Immunochemistry, real-time PCR, and Western blot analysis showed that g-GPR120 was expressed throughout the digestive tracts of goats. The expression of g-GPR120 was affected by feeding system and age, with greater expression of g-GPR120 in the G group. It was concluded that the g-GPR120-mediated LCFA chemosensing mechanism is widely present in the tongue and gastrointestinal tract of goats and that its expression can be affected by feeding system and age.

Production of Cloned Miniature Pigs Expressing High Levels of Human Apolipoprotein(a) in Plasma.

PubMed

Ozawa, Masayuki; Himaki, Takehiro; Ookutsu, Shoji; Mizobe, Yamato; Ogawa, Junki; Miyoshi, Kazuchika; Yabuki, Akira; Fan, Jianglin; Yoshida, Mitsutoshi

2015-01-01

High lipoprotein(a) [Lp(a)] levels are a major risk factor for the development of atherosclerosis. However, because apolipoprotein(a) [apo(a)], the unique component of Lp(a), is found only in primates and humans, the study of human Lp(a) has been hampered due to the lack of appropriate animal models. Using somatic cell nuclear transfer (SCNT) techniques, we produced transgenic miniature pigs expressing human apo(a) in the plasma. First, we placed the hemagglutinin (HA)-tagged cDNA of human apo(a) under the control of the β-actin promoter and cytomegalovirus enhancer, and then introduced this construct into kidney epithelial cells. Immunostaining of cells with anti-HA antibody allowed identification of cells stably expressing apo(a); one of the positive clones was used to provide donor cells for SCNT, yielding blastocysts that expressed apo(a). Immunohistochemical analysis of tissue sections and RT-PCR analysis of total RNA from organs of cloned piglet revealed that apo(a) is expressed in various tissues/organs including heart, liver, kidney, and intestine. More importantly, a transgenic line exhibited a high level (>400 mg/dL) of Lp(a) in plasma, and the transgenic apo(a) gene was transmitted to the offspring. Thus, we generated a human apo(a)-transgenic miniature pig that can be used as a model system to study advanced atherosclerosis related to human disease. The anatomical and physiological similarities between the swine and human cardiovascular systems will make this pig model a valuable source of information on the role of apo(a) in the formation of atherosclerosis, as well as the mechanisms underlying vascular health and disease.

Upregulated Genes In Sporadic, Idiopathic Pulmonary Arterial Hypertension

PubMed Central

Edgar, Alasdair J; Chacón, Matilde R; Bishop, Anne E; Yacoub, Magdi H; Polak, Julia M

2006-01-01

Background To elucidate further the pathogenesis of sporadic, idiopathic pulmonary arterial hypertension (IPAH) and identify potential therapeutic avenues, differential gene expression in IPAH was examined by suppression subtractive hybridisation (SSH). Methods Peripheral lung samples were obtained immediately after removal from patients undergoing lung transplant for IPAH without familial disease, and control tissues consisted of similarly sampled pieces of donor lungs not utilised during transplantation. Pools of lung mRNA from IPAH cases containing plexiform lesions and normal donor lungs were used to generate the tester and driver cDNA libraries, respectively. A subtracted IPAH cDNA library was made by SSH. Clones isolated from this subtracted library were examined for up regulated expression in IPAH using dot blot arrays of positive colony PCR products using both pooled cDNA libraries as probes. Clones verified as being upregulated were sequenced. For two genes the increase in expression was verified by northern blotting and data analysed using Student's unpaired two-tailed t-test. Results We present preliminary findings concerning candidate genes upregulated in IPAH. Twenty-seven upregulated genes were identified out of 192 clones examined. Upregulation in individual cases of IPAH was shown by northern blot for tissue inhibitor of metalloproteinase-3 and decorin (P < 0.01) compared with the housekeeping gene glyceraldehydes-3-phosphate dehydrogenase. Conclusion Four of the up regulated genes, magic roundabout, hevin, thrombomodulin and sucrose non-fermenting protein-related kinase-1 are expressed specifically by endothelial cells and one, muscleblind-1, by muscle cells, suggesting that they may be associated with plexiform lesions and hypertrophic arterial wall remodelling, respectively. PMID:16390543

Melatonin receptors in a pleuronectiform species, Solea senegalensis: Cloning, tissue expression, day-night and seasonal variations.

PubMed

Confente, Francesca; Rendón, María Carmen; Besseau, Laurence; Falcón, Jack; Muñoz-Cueto, José A

2010-06-01

Melatonin receptors are expressed in neural and peripheral tissues and mediate melatonin actions on the synchronization of circadian and circannual rhythms. In this study we have cloned three melatonin receptor subtypes (MT1, MT2 and Mel1c) in the Senegalese sole and analyzed their central and peripheral tissue distribution. The full-length MT1 (1452 nt), MT2 (1728 nt) and Mel1c (1980 nt) cDNAs encode different proteins of 345, 373, 355 amino acids, respectively. They were mainly expressed in retina, brain and pituitary, but MT1 was also expressed in gill, liver, intestine, kidney, spleen, heart and skin. At peripheral level, MT2 expression was only evident in gill, kidney and skin whereas Mel1c expression was restricted to the muscle and skin. This pattern of expression was not markedly different between sexes or among the times of day analyzed. The real-time quantitative PCR analyses showed that MT1 displayed higher expression at night than during the day in the retina and optic tectum. Seasonal MT1 expression was characterized by higher mRNA levels in spring and autumn equinoxes for the retina, and in winter and summer solstices for the optic tectum. An almost similar expression profile was found for MT2, but differences were less conspicuous. No day-night differences in MT1 and MT2 expression were observed in the pituitary but a seasonal variation was detected, being mRNA levels higher in summer for both receptors. Mel1c expression did not exhibit significant day-night variation in retina and optic tectum but showed seasonal variations, with higher transcript levels in summer (optic tectum) and autumn (retina). Our results suggest that day-night and seasonal variations in melatonin receptor expression could also be mediating circadian and circannual rhythms in sole. Copyright 2010 Elsevier Inc. All rights reserved.

Molecular cloning, characterization and in vitro expression of SERPIN B1 of bighorn sheep (Ovis canadensis) and domestic sheep (Ovis aries), and comparison with that of other species

USDA-ARS?s Scientific Manuscript database

Mannheimia haemolytica infection results in enhanced PMN-mediated tissue damage in the lungs of bighorn sheep (BHS) compared to that of domestic sheep (DS). SERPIN B1 is an inhibitor of PMN-derived serine proteases. It prevents lung tissue injury by inhibiting the serine proteases released as a resu...

Isoform specificity of progesterone receptor antibodies.

PubMed

Fabris, Victoria; Abascal, María F; Giulianelli, Sebastián; May, María; Sequeira, Gonzalo R; Jacobsen, Britta; Lombès, Marc; Han, Julie; Tran, Luan; Molinolo, Alfredo; Lanari, Claudia

2017-10-01

Progesterone receptors (PR) are prognostic and predictive biomarkers in hormone-dependent cancers. Two main PR isoforms have been described, PRB and PRA, that differ only in that PRB has 164 extra N-terminal amino acids. It has been reported that several antibodies empirically exclusively recognize PRA in formalin-fixed paraffin-embedded (FFPE) tissues. To confirm these findings, we used human breast cancer xenograft models, T47D-YA and -YB cells expressing PRA or PRB, respectively, MDA-MB-231 cells modified to synthesize PRB, and MDA-MB-231/iPRAB cells which can bi-inducibly express either PRA or PRB. Cells were injected into immunocompromised mice to generate tumours exclusively expressing PRA or PRB. PR isoform expression was verified using immunoblots. FFPE samples from the same tumours were studied by immunohistochemistry using H-190, clone 636, clone 16, and Ab-6 anti-PR antibodies, the latter exclusively recognizing PRB. Except for Ab-6, all antibodies displayed a similar staining pattern. Our results indicate that clones 16, 636, and the H-190 antibody recognize both PR isoforms. They point to the need for more stringency in evaluating the true specificity of purported PRA-specific antibodies as the PRA/PRB ratio may have prognostic and predictive value in breast cancer.

PCR amplification and sequences of cDNA clones for the small and large subunits of ADP-glucose pyrophosphorylase from barley tissues.

PubMed

Villand, P; Aalen, R; Olsen, O A; Lüthi, E; Lönneborg, A; Kleczkowski, L A

1992-06-01

Several cDNAs encoding the small and large subunit of ADP-glucose pyrophosphorylase (AGP) were isolated from total RNA of the starchy endosperm, roots and leaves of barley by polymerase chain reaction (PCR). Sets of degenerate oligonucleotide primers, based on previously published conserved amino acid sequences of plant AGP, were used for synthesis and amplification of the cDNAs. For either the endosperm, roots and leaves, the restriction analysis of PCR products (ca. 550 nucleotides each) has revealed heterogeneity, suggesting presence of three transcripts for AGP in the endosperm and roots, and up to two AGP transcripts in the leaf tissue. Based on the derived amino acid sequences, two clones from the endosperm, beps and bepl, were identified as coding for the small and large subunit of AGP, respectively, while a leaf transcript (blpl) encoded the putative large subunit of AGP. There was about 50% identity between the endosperm clones, and both of them were about 60% identical to the leaf cDNA. Northern blot analysis has indicated that beps and bepl are expressed in both the endosperm and roots, while blpl is detectable only in leaves. Application of the PCR technique in studies on gene structure and gene expression of plant AGP is discussed.

Cloning and characterization of a cDNA encoding topoisomerase II in pea and analysis of its expression in relation to cell proliferation.

PubMed

Reddy, M K; Nair, S; Tewari, K K; Mudgil, Y; Yadav, B S; Sopory, S K

1999-09-01

We have isolated and sequenced four overlapping cDNA clones to identify the full-length cDNA for topoisomerase II (PsTopII) from pea. Using degenerate primers, based on the conserved amino acid sequences of other eukaryotic type II topoisomerases, a 680 bp fragment was PCR-amplified with pea cDNA as template. This fragment was used as a probe to screen an oligo-dT-primed pea cDNA library. A partial cDNA clone was isolated that was truncated at the 3' end. RACE-PCR was employed to isolate the remaining portion of the gene. The total size of PsTopII is 4639 bp with an open reading frame of 4392 bp. The deduced amino acid sequence shows a strong homology to other eukaryotic topoisomerase II (topo II) at the N-terminus end. The topo II transcript was abundant in proliferative tissues. We also show that the level of topo II transcripts could be stimulated by exogenous application of growth factors that induced proliferation in vitro cultures. Light irradiation to etiolated tissue strongly stimulated the expression of topo II. These results suggest that topo II gene expression is up-regulated in response to light and hormones and correlates with cell proliferation. Besides, we have also isolated and analysed the 5'-flanking region of the pea TopII gene. This is first report on the isolation of a putative promoter for topoisomerase II from plants.

NEIBank: Genomics and bioinformatics resources for vision research

PubMed Central

Peterson, Katherine; Gao, James; Buchoff, Patee; Jaworski, Cynthia; Bowes-Rickman, Catherine; Ebright, Jessica N.; Hauser, Michael A.; Hoover, David

2008-01-01

NEIBank is an integrated resource for genomics and bioinformatics in vision research. It includes expressed sequence tag (EST) data and sequence-verified cDNA clones for multiple eye tissues of several species, web-based access to human eye-specific SAGE data through EyeSAGE, and comprehensive, annotated databases of known human eye disease genes and candidate disease gene loci. All expression- and disease-related data are integrated in EyeBrowse, an eye-centric genome browser. NEIBank provides a comprehensive overview of current knowledge of the transcriptional repertoires of eye tissues and their relation to pathology. PMID:18648525

Characterization of cDNA encoding molt-inhibiting hormone of the crab, Cancer pagurus; expression of MIH in non-X-organ tissues.

PubMed

Lu, W; Wainwright, G; Olohan, L A; Webster, S G; Rees, H H; Turner, P C

2001-10-31

Synthesis of ecdysteroids (molting hormones) by crustacean Y-organs is regulated by a neuropeptide, molt-inhibiting hormone (MIH), produced in eyestalk neural ganglia. We report here the molecular cloning of a cDNA encoding MIH of the edible crab, Cancer pagurus. Full-length MIH cDNA was obtained by using reverse transcription-polymerase chain reaction (RT-PCR) with degenerate oligonucleotides based upon the amino acid sequence of MIH, in conjunction with 5'- and 3'-RACE. Full-length clones of MIH cDNA were obtained that encoded a 35 amino acid putative signal peptide and the mature 78 amino acid peptide. Of various tissues examined by Northern blot analysis, the X-organ was the sole major site of expression of the MIH gene. However, a nested-PCR approach using non-degenerate MIH-specific primers indicated the presence of MIH transcripts in other tissues. Southern blot analysis indicated a simple gene arrangement with at least two copies of the MIH gene in the genome of C. pagurus. Additional Southern blotting experiments detected MIH-hybridizing bands in another Cancer species, Cancer antennarius and another crab species, Carcinus maenas.

Identification and tissue distribution of mRNAs encoding salmon-type calcitonins-IV and -V in the rainbow trout.

PubMed

Hidaka, Yoshie; Suzuki, Masakazu

2004-06-01

Four types of calcitonin are produced in salmonid fish, although their functional diversity is almost unknown. To explore the significance of these isoforms, we have characterized salmon-type calcitonin (sCT) mRNAs in the rainbow trout (Oncorhynchus mykiss), and examined their tissue distribution. In addition to the previously isolated sCT-I cDNAs, two new forms of sCT cDNA were cloned from the ultimobranchial gland, and one of them (sCT-IV cDNA) was predicted to encode an N-terminal peptide of 80 amino acid residues, a putative cleavage site Lys-Arg, sCT-IV, a cleavage and amidation sequence Gly-Lys-Lys-Arg, and a C-terminal peptide of 18 amino acids. The sCT-IV precursor was 78% identical with the rainbow trout sCT-I precursors. The other cloned cDNA encoded a precursor for a novel CT, sCT-V. The sCT-V peptide was different from sCT-IV by only one amino acid residue: Val at position 8 in the latter was replaced by Met. The sCT-V precursor had 80 and 90% identity with the sCT-I and -IV precursors respectively. No cDNA clones were obtained for sCTs-II or -III.Tissue distribution of sCT-I, -IV and -V mRNAs was examined by RT-PCR and specific cleavage with restriction enzymes. An amplified fragment from sCT-I mRNA was detected not only in the ultimobranchial gland, but also in the gills, testis and ovary. RT-PCR analysis coupled to restriction digestion further revealed that sCT-IV mRNA was expressed in both the testis and the ultimobranchial gland. The expression sites of sCT-IV mRNA were localized to the Leydig cells of the testis and to the parenchymal cells of the ultimobranchial gland, by in situ hybridization histochemistry. Although the amino acid sequence of sCT-V peptide was nearly the same as that of sCT-IV, the sCT-V gene showed a much wider pattern of expression: the band amplified by RT-PCR was detected in all the tissues examined except the kidney, gills and blood cells. The sCT-V mRNA was shown to be localized in the parenchymal cells of the ultimobranchial gland, but not in other tissues at the cellular level, suggesting very low expression of sCT-V mRNA in those tissues. Our results show different patterns of tissue expression of three types of sCT genes in the rainbow trout, suggesting that sCTs-I, -IV and -V might differ in their local actions.

Molecular cloning, tissue expression of gene Muc2 in blunt snout bream Megalobrama amblycephala and regulation after re-feeding

NASA Astrophysics Data System (ADS)

Xue, Chunyu; Xi, Bingwen; Ren, Mingchun; Dong, Jingjing; Xie, Jun; Xu, Pao

2015-03-01

Mucins are important components of mucus, which form a natural, physical, biochemical and semipermeable mucosal layer on the epidermis of fish gills, skin, and the gastrointestinal tract. As the first step towards characterizing the function of Muc2, we cloned a partial Megalobrama amblycephala Muc2 cDNA of 2 175 bp, and analyzed its tissue-specific expression pattern by quantitative real-time PCR (qPCR). The obtained sequence comprised 41 bp 5'-untranslated region (5'-UTR), 2 134 bp open reading frame encoding a protein of 711 amino acids. BLAST searching and phylogenetic analysis showed that the predicted protein contained several common secreted mucin-module domains (VWD-C8-TIL-VWD-C8) and had high homology with mucins from other vertebrates. Among four candidate reference genes ( β- Actin, RPI13α, RPII, 18S) for the qPCR, RPII was chosen as an appropriate reference gene because of its lowest variation in different tissues. M. amblycephala Muc2 was mainly expressed in the intestine, in the order (highest to lowest) middle-intestine > fore-intestine > hind-intestine. Muc2 was expressed relatively poorly in other organs (brain, liver, kidney, spleen, skin and gill). Furthermore, after 20-days of starvation, M. amblycephala Muc2 expressions after refeeding for 0 h, 3 h, 16 h, 3 d, and 10 d were significantly decreased in the three intestinal segments ( P<0.05) at 16 h, and were then upregulated to near the initial level at 10 d.

Studies of the effects of Vilon and Epithalon on gene expression in mouse heart using DNA-microarray technology.

PubMed

Anisimov, S V; Bokheler, K R; Khavinson, V Kh; Anisimov, V N

2002-03-01

Expression of 15,247 clones from a cDNA library in the heart of mice receiving Vilon and Epithalon was studied by DNA-microarray technology. We revealed 300 clones (1.94% of the total count), whose expression changed more than by 2 times. Vilon changed expression of 36 clones, while Epithalon modulated expression of 98 clones. Combined treatment with Vilon and Epithalon changed expression of 144 clones. Vilon alone or in combination with Epithalon activated expression of 157 clones (maximally by 6.13 times) and inhibited expression of 23 clones (maximally by 2.79 times). Epithalon alone or in combination with Vilon activated expression of 194 clones (maximally by 6.61 times) and inhibited expression of 48 clones (maximally by 2.71 times). Our results demonstrate the specific effects of Epithalon and Vilon on gene expression.

Use of electroporation for high-molecular-weight DNA-mediated gene transfer.

PubMed

Jastreboff, M M; Ito, E; Bertino, J R; Narayanan, R

1987-08-01

Electroporation was used to introduce high-molecular-weight DNA into murine hematopoietic cells and NIH3T3 cells. CCRF-CEM cells were stably transfected with SV2NEO plasmid and the genomic DNA from G-418-resistant clones (greater than 65 kb) was introduced into mouse bone marrow and NIH3T3 cells by electroporation. NEO sequences and expression were detected in the hematopoietic tissues of lethally irradiated mice, with 24% of individual spleen colonies expressing NEO. The frequency of genomic DNA transfer into NIH3T3 cells was 0.25 X 10(-3). Electroporation thus offers a powerful mode of gene transfer not only of cloned genes but also of high-molecular-weight DNA into cells.

Cloning, expression analysis, and chromosomal localization of HIP1R, an isolog of huntingtin interacting protein (HIP1).

PubMed

Seki, N; Muramatsu, M; Sugano, S; Suzuki, Y; Nakagawara, A; Ohhira, M; Hayashi, A; Hori, T; Saito, T

1998-01-01

Huntington disease (HD) is an inherited neurodegenerative disorder which is associated with CAG expansion in the coding region of the gene for huntingtin protein. Recently, a huntingtin interacting protein, HIP1, was isolated by the yeast two-hybrid system. Here we report the isolation of a cDNA clone for HIP1R (huntingtin interacting protein-1 related), which encodes a predicted protein product sharing a striking homology with HIP1. RT-PCR analysis showed that the messenger RNA was ubiquitously expressed in various human tissues. Based on PCR-assisted analysis of a radiation hybrid panel and fluorescence in situ hybridization, HIP1R was localized to the q24 region of chromosome 12.

Erasure and reestablishment of random allelic expression imbalance after epigenetic reprogramming

PubMed Central

Jeffries, Aaron Richard; Uwanogho, Dafe Aghogho; Cocks, Graham; Perfect, Leo William; Dempster, Emma; Mill, Jonathan; Price, Jack

2016-01-01

Clonal level random allelic expression imbalance and random monoallelic expression provides cellular heterogeneity within tissues by modulating allelic dosage. Although such expression patterns have been observed in multiple cell types, little is known about when in development these stochastic allelic choices are made. We examine allelic expression patterns in human neural progenitor cells before and after epigenetic reprogramming to induced pluripotency, observing that loci previously characterized by random allelic expression imbalance (0.63% of expressed genes) are generally reset to a biallelic state in induced pluripotent stem cells (iPSCs). We subsequently neuralized the iPSCs and profiled isolated clonal neural stem cells, observing that significant random allelic expression imbalance is reestablished at 0.65% of expressed genes, including novel loci not found to show allelic expression imbalance in the original parental neural progenitor cells. Allelic expression imbalance was associated with altered DNA methylation across promoter regulatory regions, with clones characterized by skewed allelic expression being hypermethylated compared to their biallelic sister clones. Our results suggest that random allelic expression imbalance is established during lineage commitment and is associated with increased DNA methylation at the gene promoter. PMID:27539784

Erasure and reestablishment of random allelic expression imbalance after epigenetic reprogramming.

PubMed

Jeffries, Aaron Richard; Uwanogho, Dafe Aghogho; Cocks, Graham; Perfect, Leo William; Dempster, Emma; Mill, Jonathan; Price, Jack

2016-10-01

Clonal level random allelic expression imbalance and random monoallelic expression provides cellular heterogeneity within tissues by modulating allelic dosage. Although such expression patterns have been observed in multiple cell types, little is known about when in development these stochastic allelic choices are made. We examine allelic expression patterns in human neural progenitor cells before and after epigenetic reprogramming to induced pluripotency, observing that loci previously characterized by random allelic expression imbalance (0.63% of expressed genes) are generally reset to a biallelic state in induced pluripotent stem cells (iPSCs). We subsequently neuralized the iPSCs and profiled isolated clonal neural stem cells, observing that significant random allelic expression imbalance is reestablished at 0.65% of expressed genes, including novel loci not found to show allelic expression imbalance in the original parental neural progenitor cells. Allelic expression imbalance was associated with altered DNA methylation across promoter regulatory regions, with clones characterized by skewed allelic expression being hypermethylated compared to their biallelic sister clones. Our results suggest that random allelic expression imbalance is established during lineage commitment and is associated with increased DNA methylation at the gene promoter. © 2016 Jeffries et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

The platelet-derived growth factor signaling system in snapping turtle embryos, Chelydra serpentina: potential role in temperature-dependent sex determination and testis development.

PubMed

Rhen, Turk; Jangula, Adam; Schroeder, Anthony; Woodward-Bosh, Rikki

2009-05-01

The platelet-derived growth factor (Pdgf) signaling system is known to play a significant role during embryonic and postnatal development of testes in mammals and birds. In contrast, genes that comprise the Pdgf system in reptiles have never been cloned or studied in any tissue, let alone developing gonads. To explore the potential role of PDGF ligands and their receptors during embryogenesis, we cloned cDNA fragments of Pdgf-A, Pdgf-B, and receptors PdgfR-alpha and PdgfR-beta in the snapping turtle, a reptile with temperature-dependent sex determination (TSD). We then compared gene expression profiles in gonads from embryos incubated at a male-producing temperature to those from embryos at a female-producing temperature, as well as between hatchling testes and ovaries. Expression of Pdgf-B mRNA in embryonic gonads was significantly higher at a male temperature than at a female temperature, but there was no difference between hatchling testes and ovaries. This developmental pattern was reversed for Pdgf-A and PdgfR-alpha mRNA: expression of these genes did not differ in embryos, but diverged in hatchling testes and ovaries. Levels of PdgfR-beta mRNA in embryonic gonads were not affected by temperature and did not differ between testes and ovaries. However, expression of both receptors increased at least an order of magnitude from the embryonic to the post-hatching period. Finally, we characterized expression of these genes in several other embryonic tissues. The brain, heart, and liver displayed unique expression patterns that distinguished these tissues from each other and from intestine, lung, and muscle. Incubation temperature had a significant effect on expression of PdgfR-alpha and PdgfR-beta in the heart but not other tissues. Together, these findings demonstrate that temperature has tissue specific effects on the Pdgf system and suggest that Pdgf signaling is involved in sex determination and the ensuing differentiation of testes in the snapping turtle.

The platelet-derived growth factor signaling system in snapping turtle embryos, Chelydra serpentina: potential role in temperature-dependent sex determination and testis development

PubMed Central

Rhen, Turk; Jangula, Adam; Schroeder, Anthony; Woodward-Bosh, Rikki

2009-01-01

The platelet-derived growth factor (Pdgf) signaling system is known to play a significant role during embryonic and postnatal development of testes in mammals and birds. In contrast, genes that comprise the Pdgf system in reptiles have never been cloned or studied in any tissue, let alone developing gonads. To explore the potential role of PDGF ligands and their receptors during embryogenesis, we cloned cDNA fragments of Pdgf-A, Pdgf-B, and receptors PdgfR-α and PdgfR-β in the snapping turtle, a reptile with temperature-dependent sex determination (TSD). We then compared gene expression profiles in gonads from embryos incubated at a male-producing temperature to those from embryos at a female-producing temperature, as well as between hatchling testes and ovaries. Expression of Pdgf-B mRNA in embryonic gonads was significantly higher at a male temperature than at a female temperature, but there was no difference between hatchling testes and ovaries. This developmental pattern was reversed for Pdgf-A and PdgfR-α mRNA: expression of these genes did not differ in embryos, but diverged in hatchling testes and ovaries. Levels of PdgfR-β mRNA in embryonic gonads were not affected by temperature and did not differ between testes and ovaries. However, expression of both receptors increased at least an order of magnitude from the embryonic to the post-hatching period. Finally, we characterized expression of these genes in several other embryonic tissues. The brain, heart, and liver displayed unique expression patterns that distinguished these tissues from each other and from intestine, lung, and muscle. Incubation temperature had a significant effect on expression of PdgfR-α and PdgfR-β in the heart but not other tissues. Together, these findings demonstrate that temperature has tissue specific effects on the Pdgf system and suggest that Pdgf signaling is involved in sex determination and the ensuing differentiation of testes in the snapping turtle. PMID:19523392

Persistence and evolution of allergen-specific IgE repertoires during subcutaneous specific immunotherapy

PubMed Central

Levin, Mattias; King, Jasmine J.; Glanville, Jacob; Jackson, Katherine J. L.; Looney, Timothy J.; Hoh, Ramona A.; Mari, Adriano; Andersson, Morgan; Greiff, Lennart; Fire, Andrew Z.; Boyd, Scott D.; Ohlin, Mats

2016-01-01

Background Specific immunotherapy (SIT) is the only treatment with proven long-term curative potential in allergic disease. Allergen-specific IgE is the causative agent of allergic disease, and antibodies contribute to SIT, but the effects of SIT on aeroallergen-specific B cell repertoires are not well understood. Objective To characterize the IgE sequences expressed by allergen-specific B cells, and track the fate of these B cell clones during SIT. Methods We have used high-throughput antibody gene sequencing and identification of allergen-specific IgE using combinatorial antibody fragment library technology to analyze immunoglobulin repertoires of blood and nasal mucosa of aeroallergen-sensitized individuals before and during the first year of subcutaneous SIT. Results Of 52 distinct allergen-specific IgE heavy chains from eight allergic donors, 37 were also detected by high-throughput antibody gene sequencing of blood, nasal mucosa, or both sample types. The allergen-specific clones had increased persistence, higher likelihood of belonging to clones expressing other switched isotypes, and possibly larger clone size than the rest of the IgE repertoire. Clone members in nasal tissue showed close mutational relationships. Conclusion Combining functional binding studies, deep antibody repertoire sequencing, and information on clinical outcomes in larger studies may in the future aid assessment of SIT mechanisms and efficacy. PMID:26559321

Molecular cloning, characterization and in vitro expression of SERPIN B1 of bighorn sheep (Ovis canadensis) and domestic sheep (Ovis aries), and comparison with that of other species.

PubMed

Subramaniam, Renuka; Dassanayake, Rohana P; Norimine, Junzo; Brown, Wendy C; Knowles, Donald P; Srikumaran, Subramaniam

2010-10-15

Mannheimia haemolytica infection results in enhanced PMN-mediated tissue damage in the lungs of bighorn sheep (BHS) compared to that of domestic sheep (DS). SERPIN B1 is an inhibitor of PMN-derived serine proteases. It prevents lung tissue injury by inhibiting the serine proteases released as a result of PMN lysis and degranulation. It is conceivable that PMNs of BHS exhibit decreased quantity and/or activity of SERPIN B1 which results in enhanced tissue injury and decreased bacterial clearance in pneumonic lungs of BHS. The objective of this study was to clone and express SERPIN B1 of BHS and DS, and develop antibodies to facilitate quantification of SERPIN B1. The 1,134bp cDNA of SERPIN B1 of BHS and DS encodes a polypeptide of 377 amino acids. SERPIN B1 of BHS and DS exhibits 100% identity at the nucleotide and amino acid levels. The amino acid sequence of ovine (BHS/DS) SERPIN B1 displays 69%, 71%, 74%, 78% and 80% identity with that of rats, dogs, mice, humans and horses, respectively. Ovine SERPIN B1 expressed in Escherichia coli was used to develop polyclonal antibodies in mice. Western blot analysis revealed the specificity of these antibodies for ovine rSERPIN B1. (c) 2010 Elsevier B.V. All rights reserved.

Cloning, tissue expression and polymorphisms of chicken Krüppel-like factor 7 gene.

PubMed

Zhang, Zhi-Wei; Wang, Zhi-Peng; Zhang, Kun; Wang, Ning; Li, Hui

2013-07-01

Krüppel-like factor 7 (KLF7) has been extensively studied in mammalian species, but its role in birds is still unclear. In the current study, cloning and sequencing showed that the full-length coding region of chicken KLF7 (Gallus gallus KLF7, gKLF7) was 891 bp long, encoding 296 amino acids. In addition, real-time RT-PCR analysis showed that gKLF7 was broadly expressed in all 15 chicken tissues selected, and its expression was significantly different in spleen, proventriculus, abdominal fat, brain, leg muscle, gizzard and heart between fat and lean broilers at 7 weeks of age. Additionally, one novel single nucleotide polymorphism (SNP), XM_426569.3: c. A141G, was identified in the second exon of gKLF7. Association analysis showed that this locus was significantly associated with fatness traits in Arbor Acres broiler random population and the eighth generation of Northeast Agricultural University broiler lines divergently selected for abdominal fat content (NEAUHLF) population (P < 0.05). These results suggest that gKLF7 might be a candidate gene for chicken fatness traits. © 2013 Japanese Society of Animal Science.

Developmental Regulation of Diacylglycerol Acyltransferase Family Gene Expression in Tung Tree Tissues

PubMed Central

Cao, Heping; Shockey, Jay M.; Klasson, K. Thomas; Chapital, Dorselyn C.; Mason, Catherine B.; Scheffler, Brian E.

2013-01-01

Diacylglycerol acyltransferases (DGAT) catalyze the final and rate-limiting step of triacylglycerol (TAG) biosynthesis in eukaryotic organisms. DGAT genes have been identified in numerous organisms. Multiple isoforms of DGAT are present in eukaryotes. We previously cloned DGAT1 and DGAT2 genes of tung tree (Vernicia fordii), whose novel seed TAGs are useful in a wide range of industrial applications. The objective of this study was to understand the developmental regulation of DGAT family gene expression in tung tree. To this end, we first cloned a tung tree gene encoding DGAT3, a putatively soluble form of DGAT that possesses 11 completely conserved amino acid residues shared among 27 DGAT3s from 19 plant species. Unlike DGAT1 and DGAT2 subfamilies, DGAT3 is absent from animals. We then used TaqMan and SYBR Green quantitative real-time PCR, along with northern and western blotting, to study the expression patterns of the three DGAT genes in tung tree tissues. Expression results demonstrate that 1) all three isoforms of DGAT genes are expressed in developing seeds, leaves and flowers; 2) DGAT2 is the major DGAT mRNA in tung seeds, whose expression profile is well-coordinated with the oil profile in developing tung seeds; and 3) DGAT3 is the major form of DGAT mRNA in tung leaves, flowers and immature seeds prior to active tung oil biosynthesis. These results suggest that DGAT2 is probably the major TAG biosynthetic isoform in tung seeds and that DGAT3 gene likely plays a significant role in TAG metabolism in other tissues. Therefore, DGAT2 should be a primary target for tung oil engineering in transgenic organisms. PMID:24146944

Tissue- and cell-specific expression of metallothionein genes in cadmium- and copper-exposed mussels analyzed by in situ hybridization and RT-PCR

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zorita, I.; Bilbao, E.; Schad, A.

2007-04-15

Metallothioneins (MTs) are metal-inducible proteins that can be used as biomarkers of metal exposure. In mussels two families of MT isoforms (MT10 and MT20) have been characterized. In this study, mussels (Mytilus galloprovincialis) were exposed to 200 ppb Cd and 40 ppb Cu for 2 and 9 days to characterize the tissue and isoform specificity of metal-induced MT expression. Non-radioactive in situ hybridization demonstrated that both MT isoforms were mainly transcribed in digestive tubule epithelial cells, especially in basophilic cells. Weaker MT expression was detected in non-ciliated duct cells, stomach and gill epithelial cells, haemocytes, adipogranular cells, spermatic follicles andmore » oocytes. RT-PCR resulted in cloning of a novel M. galloprovincialis isoform homologous to recently cloned Mytilus edulis intron-less MT10B isoform. In gills, Cd only affected MT10 gene expression after 2 days of exposure while increases in MT protein levels occurred at day 9. In the digestive gland, a marked increase of both isoforms, but especially of MT20, was accompanied by increased levels of MT proteins and basophilic cell volume density (Vv{sub BAS}) after 2 and 9 days and of intralysosomal metal accumulation in digestive cells after 9 days. Conversely, although metal was accumulated in digestive cells lysosomes and the Vv{sub BAS} increased in Cu-exposed mussels, Cu exposure did not produce an increase of MT gene expression or MT protein levels. These data suggest that MTs are expressed in a tissue-, cell- and isoform-specific way in response to different metals.« less

Cinnamate-4-hydroxylase expression in arabidopsis. Regulation in response to development and the environment

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bell-Lelong, D.A.; Cusumano, J.C.; Meyer, K.

1997-03-01

Cinnamate-r-hydroxylase (C4H) is the first Cyt P450-dependent monooxygenase of the phenylpropanoid pathway. To study the expression of this gene in Arabidopsis thaliana, a C4H cDNA clone from the Arabidopsis expressed sequence tag database was identified and used to isolate its corresponding genomic clone. The entire C4H coding sequence plus 2.9 kb of its promoter were isolated on a 5.4-kb HindIII fragment of this cosmid. Inspection of the promoter sequence revealed the presence of a number of putative regulatory motifs previously identified in the promoters of other phenylpropanoid pathway genes. The expression of C4H was analyzed by RNA blot hybridization analysismore » and in transgenic Arabidopsis carrying a C4H-{beta}-glucuronidase transcriptional fusion. C4H message accumulation was light-dependent, but was detectable even in dark-grown seedlings. Consistent with these data, C4H mRNA was accumulated to light-grown levels in etiolated det1-1 mutant seedlings. C4H is widely expressed in various Arabidopsis tissues, particularly in roots and cells undergoing lignification. The C4H-driven {beta}-glucuronidase expression accurately reflected the tissue-specificity and wound-inducibility of the C4H promoter indicated by RNA blot hybridization analysis. A modest increase in C4H expression was observed in the tt8 mutant of Arabidopsis. 77 refs., 5 figs.« less

Isolation, cDNA cloning and gene expression of an antibacterial protein from larvae of the coconut rhinoceros beetle, Oryctes rhinoceros.

PubMed

Yang, J; Yamamoto, M; Ishibashi, J; Taniai, K; Yamakawa, M

1998-08-01

An antibacterial protein, designated rhinocerosin, was purified to homogeneity from larvae of the coconut rhinoceros beetle, Oryctes rhinoceros immunized with Escherichia coli. Based on the amino acid sequence of the N-terminal region, a degenerate primer was synthesized and reverse-transcriptase PCR was performed to clone rhinocerosin cDNA. As a result, a 279-bp fragment was obtained. The complete nucleotide sequence was determined by sequencing the extended rhinocerosin cDNA clone by 5' rapid amplification of cDNA ends. The deduced amino acid sequence of the mature portion of rhinocerosin was composed of 72 amino acids without cystein residues and was shown to be rich in glycine (11.1%) and proline (11.1%) residues. Comparison of the deduced amino acid sequence of rhinocerosin with those of other antibacterial proteins indicated that it has 77.8% and 44.6% identity with holotricin 2 and coleoptrecin, respectively. Rhinocerosin had strong antibacterial activity against E. coli, Streptococcus pyogenes, Staphylococcus aureus but not against Pseudomonas aeruginosa. Results of reverse-transcriptase PCR analysis of gene expression in different tissues indicated that the rhinocerosin gene is strongly expressed in the fat body and the Malpighian tubule, and weakly expressed in hemocytes and midgut. In addition, gene expression was inducible by bacteria in the fat body, the Malpighian tubule and hemocyte but constitutive expression was observed in the midgut.

Molecular cloning of a human Ca2+-dependent cell-cell adhesion molecule homologous to mouse placental cadherin: its low expression in human placental tissues

PubMed Central

1989-01-01

P-cadherin is a subclass of Ca2+-dependent cell-cell adhesion molecules present in mouse placenta, where its localization suggests a function of connecting the embryo to the uterus (Nose, A., and M. Takeichi. 1986. J. Cell Biol. 103:2649-2658). We recently identified a human cadherin detected by an mAb capable of disrupting cell-cell adhesion of A-431 cells, and found that it was closely related immunochemically to mouse P-cadherin. Curiously, this cadherin was undetectable in human placenta by immunohistochemical examination (Shimoyama, Y., S. Hirohashi, S. Hirano, M. Noguchi, Y. Shimosato, M. Takeichi, and O. Abe. 1989. Cancer Res. 49:2128-2133). We here report the cloning and sequencing of cDNA clone encoding the human homologue of mouse P- cadherin. The deduced amino acid sequence of the human P-cadherin consists of 829 amino acid and shows striking homology with mouse P- cadherin. On Northern blot analysis, human P-cadherin was scarcely expressed in human placenta in contrast to mouse P-cadherin, which was abundantly expressed in mouse placenta throughout pregnancy, and it was shown that E-cadherin, but not P-cadherin, was the major cadherin molecule in human placenta. Moreover, NIH3T3 cells transfected with human P-cadherin cDNA expressed the functional cadherin molecule, which was identical to the cadherin we had previously identified using the mAb, showing that this molecule really does mediate cell-cell adhesion and that the cadherin we detected immunochemically is undoubtedly human P-cadherin. The results obtained in this study support the idea that P- cadherin plays little role, if any, in Ca2+-dependent cell-cell binding in human placental tissue at least after several weeks of pregnancy. PMID:2793940

Cloning and expression profile of FLT3 gene during progenitor cell-dependent liver regeneration.

PubMed

Aydin, Iraz T; Tokcaer, Zeynep; Dalgic, Aydin; Konu, Ozlen; Akcali, Kamil C

2007-12-01

The liver has a unique capacity to regenerate upon exposure to viral infections, toxic reactions and cancer formation. Liver regeneration is a complex phenomenon in which several factors participate during its onset. Cellular proliferation is an important component of this process and the factors that regulate this proliferation have a vital role. FLT3, a well-known hematopoietic stem cell and hepatic lineage surface marker, is involved in proliferative events of hematopoietic stem cells. However, its contribution to liver regeneration is not known. Therefore, the aim of this study was to clone and examine the role of FLT3 during liver regeneration in rats. Partial cDNA of rat homolog of FLT3 gene was cloned from thymus and the tissue specific expression of this gene at mRNA and protein levels was examined by RT-PCR and Western blot. After treating with 2-AAF and performing hepatectomy in rats to induce progenitor-dependent liver regeneration, the mRNA and protein expression profile of FLT3 was investigated by real-time PCR and Western blot during liver regeneration. In addition, cellular localization of FLT3 protein was determined by immunohistochemistry. The results indicated that rat FLT3 cDNA has high homology with mouse and human FLT3 cDNA. It was also found that FLT3 is expressed in most of the rat tissues and during liver regeneration. In addition, its intracellular localization is altered during the late stages of liver regeneration. The FLT3 receptor is activated at the late stages of liver regeneration and participates in the proliferation response that is observed during progenitor-dependent liver regeneration.

Human fetal enterocytes in vitro: modulation of the phenotype by extracellular matrix.

PubMed Central

Sanderson, I R; Ezzell, R M; Kedinger, M; Erlanger, M; Xu, Z X; Pringault, E; Leon-Robine, S; Louvard, D; Walker, W A

1996-01-01

The differentiation of small intestinal epithelial cells may require stimulation by microenvironmental factors in vivo. In this study, the effects of mesenchymal and luminal elements in nonmalignant epithelia] cells isolated from the human fetus were studied in vitro. Enterocytes from the human fetus were cultured and microenvironmental factors were added in stages, each stage more closely approximating the microenvironment in vivo. Four stages were examined: epithelial cells derived on plastic from intestinal culture and grown as a cell clone, the same cells grown on connective tissue support, primary epithelial explants grown on fibroblasts with a laminin base, and primary epithelial explants grown on fibroblasts and laminin with n-butyrate added to the incubation medium. The epithelial cell clone dedifferentiated when grown on plastic; however, the cells expressed cytokeratins and villin as evidence of their epithelial cell origin. Human connective tissue matrix from Engelbreth-Holm-Swarm sarcoma cells (Matrigel) modulated their phenotype: alkaline phosphatase activity increased, microvilli developed on their apical surface, and the profile of insulin-like growth factor binding proteins resembled that secreted by differentiated enterocytes. Epithelial cells taken directly from the human fetus as primary cultures and grown as explants on fibroblasts and laminin expressed greater specific enzyme activities in brush border membrane fractions than the cell clone. These activities were enhanced by the luminal molecule sodium butyrate. Thus the sequential addition of connective tissue and luminal molecules to nonmalignant epithelia] cells in vitro induces a spectrum of changes in the epithelial cell phenotype toward full differentiation. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 Fig. 6 PMID:8755542

Cloning of cardiac, kidney, and brain promoters of the feline ncx1 gene.

PubMed

Barnes, K V; Cheng, G; Dawson, M M; Menick, D R

1997-04-25

The Na+-Ca2+ exchanger (NCX1) plays a major role in calcium efflux and therefore in the control and regulation of intracellular calcium in the heart. The exchanger has been shown to be regulated at several levels including transcription. NCX1 mRNA levels are up-regulated in both cardiac hypertrophy and failure. In this work, the 5'-end of the ncx1 gene has been cloned to study the mechanisms that mediate hypertrophic stimulation and cardiac expression. The feline ncx1 gene has three exons that encode 5'-untranslated sequences that are under the control of three tissue-specific promoters. The cardiac promoter drives expression in cardiocytes, but not in mouse L cells. Although it contains at least one enhancer (-2000 to -1250 base pairs (bp)) and one or more negative elements (-1250 to -250 bp), a minimum promoter (-250 to +200 bp) is sufficient for cardiac expression and alpha-adrenergic stimulation.

Goat RSPO1 over-expression rescues sex-reversal in Rspo1-knockout XX mice but does not perturb testis differentiation in XY or sex-reversed XX mice.

PubMed

Buscara, Laurine; Montazer-Torbati, Fatemeh; Chadi, Sead; Auguste, Aurélie; Laubier, Johann; Chassot, Anne-Amandine; Renault, Lauriane; Passet, Bruno; Costa, José; Pannetier, Maëlle; Vilotte, Marthe; Chaboissier, Marie-Christine; Vilotte, Jean-Luc; Pailhoux, Eric; Le Provost, Fabienne

2009-08-01

RSPO1 is a newly discovered gene involved in sex differentiation. Two goat BAC clones encompassing the RSPO1 gene (gRSPO1) were injected into mouse oocytes and several transgenic lines derived. Both clones induced gRSPO1 over-expression in various tissues, including male and female gonads, with no obvious phenotype and normal sex-ratios. Introgression of the gRSPO1 transgene into a mouse RSPO1 knockout genotype resulted in the rescue of the fertility and the disappearance of the masculinized gonadic features of the females, demonstrating the functionality of the goat protein in a mouse context. On the contrary, over-expression of gRSPO1 within a mSRY or a gSRY-XX genotypes did not interfere with the SRY-induced male phenotype.

Molecular cloning, phylogenetic analysis, and expression profiling of endoplasmic reticulum molecular chaperone BiP genes from bread wheat (Triticum aestivum L.).

PubMed

Zhu, Jiantang; Hao, Pengchao; Chen, Guanxing; Han, Caixia; Li, Xiaohui; Zeller, Friedrich J; Hsam, Sai L K; Hu, Yingkao; Yan, Yueming

2014-10-01

The endoplasmic reticulum chaperone binding protein (BiP) is an important functional protein, which is involved in protein synthesis, folding assembly, and secretion. In order to study the role of BiP in the process of wheat seed development, we cloned three BiP homologous cDNA sequences in bread wheat (Triticum aestivum), completed by rapid amplification of cDNA ends (RACE), and examined the expression of wheat BiP in wheat tissues, particularly the relationship between BiP expression and the subunit types of HMW-GS using near-isogenic lines (NILs) of HMW-GS silencing, and under abiotic stress. Sequence analysis demonstrated that all BiPs contained three highly conserved domains present in plants, animals, and microorganisms, indicating their evolutionary conservation among different biological species. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) revealed that TaBiP (Triticum aestivum BiP) expression was not organ-specific, but was predominantly localized to seed endosperm. Furthermore, immunolocalization confirmed that TaBiP was primarily located within the protein bodies (PBs) in wheat endosperm. Three TaBiP genes exhibited significantly down-regulated expression following high molecular weight-glutenin subunit (HMW-GS) silencing. Drought stress induced significantly up-regulated expression of TaBiPs in wheat roots, leaves, and developing grains. The high conservation of BiP sequences suggests that BiP plays the same role, or has common mechanisms, in the folding and assembly of nascent polypeptides and protein synthesis across species. The expression of TaBiPs in different wheat tissue and under abiotic stress indicated that TaBiP is most abundant in tissues with high secretory activity and with high proportions of cells undergoing division, and that the expression level of BiP is associated with the subunit types of HMW-GS and synthesis. The expression of TaBiPs is developmentally regulated during seed development and early seedling growth, and under various abiotic stresses.

Cloning and functional characterization of inward-rectifying potassium (Kir) channels from Malpighian tubules of the mosquito Aedes aegypti

PubMed Central

Piermarini, Peter M.; Rouhier, Matthew F.; Schepel, Matthew; Kosse, Christin; Beyenbach, Klaus W.

2013-01-01

Inward-rectifying K+ (Kir) channels play critical physiological roles in a variety of vertebrate cells/tissues, including the regulation of membrane potential in nerve and muscle, and the transepithelial transport of ions in osmoregulatory epithelia, such as kidneys and gills. It remains to be determined whether Kir channels play similar physiological roles in insects. In the present study, we sought to 1) clone the cDNAs of Kir channel subunits expressed in the renal (Malpighian) tubules of the mosquito Aedes aegypti, and 2) characterize the electrophysiological properties of the cloned Kir subunits when expressed heterologously in oocytes of Xenopus laevis. Here, we reveal that three Kir subunits are expressed abundantly in Aedes Malpighian tubules (AeKir1, AeKir2B, and AeKir3); each of their full-length cDNAs was cloned. Heterologous expression of the AeKir1 or the AeKir2B subunits in Xenopus oocytes elicits inward-rectifying K+ currents that are blocked by barium. Relative to the AeKir2B-expressing oocytes, the AeKir1-expressing oocytes 1) produce larger macroscopic currents, and 2) exhibit a modulation of their conductive properties by extracellular Na+. Attempts to functionally characterize the AeKir3 subunit in Xenopus oocytes were unsuccessful. Lastly, we show that in isolated Aedes Malpighian tubules, the cation permeability sequence of the basolateral membrane of principal cells (Tl+ > K+ > Rb+ > NH4+) is consistent with the presence of functional Kir channels. We conclude that in Aedes Malpighian tubules, Kir channels contribute to the majority of the barium-sensitive transepithelial transport of K+. PMID:23085358

Nuclear receptors HR96 and ultraspiracle from the fall armyworm (Spodoptera frugiperda), developmental expression and induction by xenobiotics.

PubMed

Giraudo, Maeva; Audant, Pascaline; Feyereisen, René; Le Goff, Gaëlle

2013-05-01

The fall armyworm Spodoptera frugiperda is a major polyphagous pest in agriculture and little is known on how this insect can adapt to the diverse and potentially toxic plant allelochemicals that they ingest or to insecticides. To investigate the involvement of nuclear receptors in the response of S. frugiperda to its chemical environment, we cloned SfHR96, a nuclear receptor orthologous to the mammalian xenobiotic receptors, pregnane X receptor (PXR) and constitutive androstane receptor (CAR). We also cloned ultraspiracle (USP), the ortholog of retinoid X receptor (RXR) that serves as partner of dimerization of PXR and CAR. Cloning of SfUSP revealed the presence of two isoforms, SfUSP-1 and SfUSP-2 in this species, that differ in their N-terminal region. The expression of these receptors as well as the ecdysone receptor was studied during specific steps of development in different tissues. SfHR96 was constitutively expressed in larval midgut, fat body and Malpighian tubules throughout the last two instars and pupal stage, as well as in Sf9 cells. EcR and SfUSP-2 showed peaks of expression before larval moults and during metamorphosis, whereas SfUSP-1 was mainly expressed in the pre-pupal stage. Receptor induction was followed after exposure of larvae or cells to 11 chemical compounds. SfHR96 was not inducible by the tested compounds. EcR was significantly induced by the 20-hydroxyecdysone agonist, methoxyfenozide, and SfUSP showed an increase expression when exposed to the juvenile hormone analog, methoprene. The cloning of these nuclear receptors is a first step in understanding the important capacities of adaptation of this insect pest. Copyright © 2013 Elsevier Ltd. All rights reserved.

An oleate 12-hydroxylase from Ricinus communis L. is a fatty acyl desaturase homolog

DOE Office of Scientific and Technical Information (OSTI.GOV)

Van De Loo, F.J.; Broun, P.; Turner, S.

1995-07-18

Recent spectroscopic evidence implicating a binuclear iron site at the reaction center of fatty acyl desaturases suggested to us that certain fatty acyl hydroxylases may share significant amino acid sequence similarity with desaturases. To test this theory, we prepared a cDNA library from developing endosperm of the castor-oil plant (Ricinus communis L.) and obtained partial nucleotide sequences for 468 anonymous clones that were not expressed at high levels in leaves, a tissue deficient in 12-hydroxyoleic acid. This resulted in the identification of several cDNA clones encoding a polypeptide of 387 amino acids with a predicted molecular weight of 44,407 andmore » with {approx}67% sequence homology to microsomal oleate desaturase from Arabidopsis. Expression of a full-length clone under control of the cauliflower mosaic virus 35S promoter in transgenic tobacco resulted in the accumulation of low levels of 12-hydroxyoleic acid in seeds, indicating that the clone encodes the castor oleate hydroxylase. These results suggest that fatty acyl desaturases and hydroxylases share similar reaction mechanisms and provide an example of enzyme evolution. 26 refs., 6 figs., 1 tab.« less

Characterization of a candidate bcl-1 gene.

PubMed Central

Withers, D A; Harvey, R C; Faust, J B; Melnyk, O; Carey, K; Meeker, T C

1991-01-01

The t(11;14)(q13;q32) translocation has been associated with human B-lymphocytic malignancy. Several examples of this translocation have been cloned, documenting that this abnormality joins the immunoglobulin heavy-chain gene to the bcl-1 locus on chromosome 11. However, the identification of the bcl-1 gene, a putative dominant oncogene, has been elusive. In this work, we have isolated genomic clones covering 120 kb of the bcl-1 locus. Probes from the region of an HpaII-tiny-fragment island identified a candidate bcl-1 gene. cDNAs representing the bcl-1 mRNA were cloned from three cell lines, two with the translocation. The deduced amino acid sequence from these clones showed bcl-1 to be a member of the cyclin gene family. In addition, our analysis of expression of bcl-1 in an extensive panel of human cell lines showed it to be widely expressed except in lymphoid or myeloid lineages. This observation may provide a molecular basis for distinct modes of cell cycle control in different mammalian tissues. Activation of the bcl-1 gene may be oncogenic by directly altering progression through the cell cycle. Images PMID:1833629

Isolation and characterization of 21 novel expressed DNA sequences from the distal region of human chromosome 4p

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ishida, Yoshikazu; Hadano, Shinji; Nagayama, Tomiko

1994-07-15

The authors have established an approach to the isolation of expressed DNA sequences from a defined region of the human chromosome. The method relies on the direct screening of cDNA libraries using pooled single-copy microclones generated by a laser chromosome microdissection in conjunction with a single unique primer polymerase chain reaction (SUP-PCR) procedure. They applied this method to the distal region of human chromosome 4p (4p15-4pter), which contains the Huntington disease (HD) and the Wolf-Hirschhorn syndrome (WHS) loci. Twenty-one nonoverlapping and region-specific cDNA clones encoding novel genes were isolated in this manner. Ten of 21 clones were subregionally assigned tomore » 4p16.1-4pter, and the remainder mapped to the region proximal to 4p16.1. Northern blot and reverse transcription followed by the PCR (RT-PCR) analysis revealed that 16 of these 21 clones detected transcripts in total RNA from human tissues. The method is applicable to other chromosomal regions and is a powerful approach to the isolation of region-specific cDNA clones. 44 refs., 3 figs., 3 tabs.« less

BPI-fold (BPIF) containing/plunc protein expression in human fetal major and minor salivary glands.

PubMed

Alves, Daniel Berretta Moreira; Bingle, Lynne; Bingle, Colin David; Lourenço, Silvia Vanessa; Silva, Andréia Aparecida; Pereira, Débora Lima; Vargas, Pablo Agustin

2017-01-16

The aim of this study was to determine expression, not previously described, of PLUNC (palate, lung, and nasal epithelium clone) (BPI-fold containing) proteins in major and minor salivary glands from very early fetal tissue to the end of the second trimester and thus gain further insight into the function of these proteins. Early fetal heads, and major and minor salivary glands were collected retrospectively and glands were classified according to morphodifferentiation stage. Expression of BPI-fold containing proteins was localized through immunohistochemistry. BPIFA2, the major BPI-fold containing protein in adult salivary glands, was detected only in the laryngeal pharynx; the lack of staining in salivary glands suggested salivary expression is either very late in development or is only in adult tissues. Early expression of BPIFA1 was seen in the trachea and nasal cavity with salivary gland expression only seen in late morphodifferentiation stages. BPIFB1 was seen in early neural tissue and at later stages in submandibular and sublingual glands. BPIFA1 is significantly expressed in early fetal oral tissue but BPIFB1 has extremely limited expression and the major salivary BPIF protein (BPIFA2) is not produced in fetal development. Further studies, with more sensitive techniques, will confirm the expression pattern and enable a better understanding of embryonic BPIF protein function.

Pig has no uncoupling protein 1.

PubMed

Hou, Lianjie; Shi, Jia; Cao, Lingbo; Xu, Guli; Hu, Chingyuan; Wang, Chong

2017-06-10

Brown adipose tissue (BAT) is critical for mammal's survival in the cold environment. Uncoupling protein 1 (UCP1) is responsible for the non-shivering thermogenesis in the BAT. Pig is important economically as a meat-producing livestock. However, whether BAT or more precisely UCP1 protein exists in pig remains a controversy. The objective of this study was to ascertain whether pig has UCP1 protein. In this study, we used rapid amplification of cDNA ends (RACE) technique to obtain the UCP1 mRNA 3' end sequence, confirmed only exons 1 and 2 of the UCP1 gene are transcribed in the pig. Then we cloned the pig UCP1 gene exons 1 and 2, and expressed the UCP1 protein from the truncated pig gene using E. coli BL21. We used the expressed pig UCP1 protein as antigen for antibody production in a rabbit. We could not detect any UCP1 protein expression in different pig adipose tissues by the specific pig UCP1 antibody, while our antibody can detect the cloned pig UCP1 as well as the mice adipose UCP1 protein. This result shows although exons 1 and 2 of the pig UCP1 gene were transcribed but not translated in the pig adipose tissue. Furthermore, we detected no uncoupled respiration in the isolated pig adipocytes. Thus, these results unequivocally demonstrate that pig has no UCP1 protein. Our results have resolved the controversy of whether pigs have the brown adipose tissue. Copyright © 2017 Elsevier Inc. All rights reserved.

A Preliminary Study on a Specifically Expressed Arabidopsis Promotor in Vascular Bundle

NASA Astrophysics Data System (ADS)

Yun-hong, Gu; Chuan-xiao, Xie; Li-fang, Wu; Zeng-liang, Yu; Guang-yong, Qin; Yu-ping, Huo

2003-04-01

From a population of about 3500 single plants in Arabidopsis promoter trapping bank, one plant whose GUS-gene had been specifically expressed in vascular bundle, was screened by the method of gus tissue staining. The T-DNA flanking sequence was amplified using TAIL-PCR. This band will be purified and connected to TA cloning vector. After sequencing and searching in the genebank, its function will be demonatrated through transformation.

Generation of Five Human Lactoferrin Transgenic Cloned Goats Using Fibroblast Cells and Their Methylation Status of Putative Differential Methylation Regions of IGF2R and H19 Imprinted Genes

PubMed Central

Sun, Yanyan; Zhang, Yanli; Wang, Ziyu; Song, Yang; Wang, Feng

2013-01-01

Background Somatic cell nuclear transfer (SCNT) is a promising technique to produce transgenic cloned mammalian, including transgenic goats which may produce Human Lactoferrin (hLF). However, success percentage of SCNT is low, because of gestational and neonatal failure of transgenic embryos. According to the studies on cattle and mice, DNA methylation of some imprinted genes, which plays a vital role in the reprogramming of embryo in NT maybe an underlying mechanism. Methodology/Principal Findings Fibroblast cells were derived from the ear of a two-month-old goat. The vector expressing hLF was constructed and transfected into fibroblasts. G418 selection, EGFP expression, PCR, and cell cycle distribution were applied sequentially to select transgenic cells clones. After NT and embryo transfer, five transgenic cloned goats were obtained from 240 cloned transgenic embryos. These transgenic goats were identified by 8 microsatellites genotyping and southern blot. Of the five transgenic goats, 3 were lived after birth, while 2 were dead during gestation. We compared differential methylation regions (DMR) pattern of two paternally imprinted genes (H19 and IGF2R) of the ear tissues from the lived transgenic goats, dead transgenic goats, and control goats from natural reproduction. Hyper-methylation pattern appeared in cloned aborted goats, while methylation status was relatively normal in cloned lived goats compared with normal goats. Conclusions/Significance In this study, we generated five hLF transgenic cloned goats by SCNT. This is the first time the DNA methylation of lived and dead transgenic cloned goats was compared. The results demonstrated that the methylation status of DMRs of H19 and IGF2R were different in lived and dead transgenic goats and therefore this may be potentially used to assess the reprogramming status of transgenic cloned goats. Understanding the pattern of gene imprinting may be useful to improve cloning techniques in future. PMID:24204972

Three isozymes of peptidylarginine deiminase in the chicken: molecular cloning, characterization, and tissue distribution.

PubMed

Shimizu, Akira; Handa, Kenji; Honda, Tomonori; Abe, Naoki; Kojima, Toshio; Takahara, Hidenari

2014-01-01

Peptidylarginine deiminase (PAD; EC 3.5.3.15) is a post-translational modification enzyme that catalyzes the conversion of protein-bound arginine to citrulline (deimination) in a calcium ion dependent manner. Although PADI genes are widely conserved among vertebrates, their function in the chicken is poorly understood. Here, we cloned and sequenced three chicken PADI cDNAs and analyzed the expression of their proteins in various tissues. Immunoblotting analysis showed that chicken PAD1 and PAD3 were present in cells of several central neuron system tissues including the retina; the chicken PAD2 protein was not detected in any tissue. We expressed recombinant chicken PADs in insect cells and characterized their enzymatic properties. The chicken PAD1 and PAD3 recombinant proteins required calcium ions as an essential cofactor for their catalytic activity. The two recombinant proteins showed similar substrate specificities toward synthetic arginine derivatives. By contrast to them, chicken PAD2 did not show any activity. We found that one of the conserved active centers in mammalian PADs had been altered in chicken PAD2; we prepared a reverse mutant but we did not detect an activity. We conclude that chicken PAD1 and PAD3 might play specific roles in the nervous system, but that chicken PAD2 might not be functional under normal physiological conditions. © 2013 Elsevier Inc. All rights reserved.

Molecular Characterization of Ferulate 5-Hydroxylase Gene from Kenaf (Hibiscus cannabinus L.)

PubMed Central

Park, Young-Hwan; Lim, Hyoun-Sub; Natarajan, Savithiry; Park, Sang-Un

2013-01-01

The purpose of this study is to clone and characterize the expression pattern of a F5H gene encoding ferulate 5-hydroxylase in the phenylpropanoid pathway from kenaf (Hibiscus cannabinus L.). Kenaf is a fast-growing dicotyledonous plant valued for its biomass. F5H, a cytochrome P450-dependent monooxygenase (CYP84), is a key enzyme for syringyl lignin biosynthesis. The full length of the F5H ortholog was cloned and characterized. The full-length F5H ortholog consists of a 1,557-bp open reading frame (ORF) encoding 518 amino acids (GenBank Accession number JX524278). The deduced amino acid sequence showed that kenaf F5H had the highest similarity (78%) with that of Populus trichocarpa. Transcriptional analysis of F5H ortholog was conducted using quantitative real-time PCR during the developmental stages of various tissues and in response to various abiotic stresses. The highest transcript level of the F5H ortholog was observed in immature flower tissues and in early stage (6 week-old) of stem tissues, with a certain level of expression in all tissues tested. The highest transcript level of F5H ortholog was observed at the late time points after treatments with NaCl (48 h), wounding (24 h), cold (24 h), abscisic acid (24 h), and methyl jasmonate (24 h). PMID:24204204

Production of cloned transgenic cow expressing omega-3 fatty acids.

PubMed

Wu, Xia; Ouyang, Hongsheng; Duan, Biao; Pang, Daxin; Zhang, Li; Yuan, Ting; Xue, Lian; Ni, Daibang; Cheng, Lei; Dong, Shuhua; Wei, Zhuying; Li, Lin; Yu, Ming; Sun, Qing-Yuan; Chen, Da-Yuan; Lai, Liangxue; Dai, Yifan; Li, Guang-Peng

2012-06-01

n-3 Polyunsaturated fatty acids (n-3 PUFA) are important for human health. Alternative resources of n-3 PUAFs created by transgenic domestic animals would be an economic approach. In this study, we generated a mfat-1 transgenic cattle expressed a Caenorhabditis elegans gene, mfat-1, encoding an n-3 fatty acid desaturase. Fatty acids analysis of tissue and milk showed that all of the examined n-3 PUAFs were greatly increased and simultaneously the n-6 PUAFs decreased in the transgenic cow. A significantly reduction of n-6/n-3 ratios (P<0.05) in both tissue and milk were observed.

Cloning, functional expression, and characterization of a chalcone 3-hydroxylase from Cosmos sulphureus.

PubMed

Schlangen, Karin; Miosic, Silvija; Thill, Jana; Halbwirth, Heidi

2010-07-01

A chalcone 3-hydroxylase (CH3H) cDNA clone was isolated and characterized from Cosmos sulphureus petals accumulating butein (2',3,4,4'-tetrahydroxychalcone) derivatives as yellow flower pigments. The recombinant protein catalyses the introduction of an additional hydroxyl group in the B-ring of chalcones, a reaction with high similarity to the hydroxylation of flavonoids catalysed by the well-studied flavonoid 3'-hydroxylase (F3'H). CH3H shows high specificity for chalcones, but a low F3'H activity was also detected. By contrast, the common F3'H from C. sulphureus does not accept chalcones as substrates and is therefore unlikely to be involved in the creation of the B-ring hydroxylation pattern of the yellow flower pigments. CH3H was primarily expressed in young buds, the main tissue for chalcone pigment formation. Expression levels in open flowers and 3-d-old seedlings were lower and almost no CH3H expression was observed in leaves. F3'H, in contrast, showed the highest expression also in buds, but comparable expression rates in all other tissues tested. Recombinant hybrid proteins constructed from CH3H and F3'H fragments demonstrated that amino acid residues at a substrate recognition site and an insertion of four amino acid residues in a putative loop region have an impact on chalcone acceptance. This is the first identification of a CH3H cDNA from any plant species.

Molecular cloning and expression analysis of Sox3 during gonad and embryonic development in Misgurnus anguillicaudatus.

PubMed

Xia, Xiaohua; Huo, Weiran; Wan, Ruyan; Zhang, Linxia; Xia, Xiaopei; Chang, Zhongjie

2017-01-01

Sox3 is a single-exon gene located on the X chromosome in most vertebrates. It belongs to the SoxB1 subfamily, which is part of the larger Sox family. Previous studies have revealed that Sox3 is expressed in many fish species. However, how Sox3 influences the development of Misgurnus anguillicaudatus remains unknown. In this study, a Sox3 homologue, termed MaSox3, was cloned from the brain of M. anguillicaudatus using homology-based cloning and the rapid amplification of cDNA ends method. Sequence analysis reveals that MaSox3 encodes a hydrophilic protein, which contains a characteristic HMG-box DNA-binding domain of 79 amino acids, and shares high homology with Sox3 in other species. Additionally, quantitative real-time reverse transcription PCR and in situ hybridization showed that MaSox3 is consistently expressed during embryogenesis, with peak expression during the neurula stage and broad expression in the central nervous system. Moreover, tissue distribution analyses have revealed that MaSox3 is abundant in the adult brain, the particle cell layer, and the gonad. Additionally, its expression is observed in primary spermatocyte cells, primary oocytes and previtellogenic oocyte cells. Taken together, all of these results suggest that the expression of the MaSox3 gene is highly conserved during vertebrate evolution and involved in a wide range of developmental processes including embryogenesis, neurogenesis and gonad development.

Isoform specificity of progesterone receptor antibodies

PubMed Central

Fabris, Victoria; Abascal, María F; Giulianelli, Sebastián; May, María; Sequeira, Gonzalo R; Jacobsen, Britta; Lombès, Marc; Han, Julie; Tran, Luan; Molinolo, Alfredo

2017-01-01

Abstract Progesterone receptors (PR) are prognostic and predictive biomarkers in hormone‐dependent cancers. Two main PR isoforms have been described, PRB and PRA, that differ only in that PRB has 164 extra N‐terminal amino acids. It has been reported that several antibodies empirically exclusively recognize PRA in formalin‐fixed paraffin‐embedded (FFPE) tissues. To confirm these findings, we used human breast cancer xenograft models, T47D‐YA and ‐YB cells expressing PRA or PRB, respectively, MDA‐MB‐231 cells modified to synthesize PRB, and MDA‐MB‐231/iPRAB cells which can bi‐inducibly express either PRA or PRB. Cells were injected into immunocompromised mice to generate tumours exclusively expressing PRA or PRB. PR isoform expression was verified using immunoblots. FFPE samples from the same tumours were studied by immunohistochemistry using H‐190, clone 636, clone 16, and Ab‐6 anti‐PR antibodies, the latter exclusively recognizing PRB. Except for Ab‐6, all antibodies displayed a similar staining pattern. Our results indicate that clones 16, 636, and the H‐190 antibody recognize both PR isoforms. They point to the need for more stringency in evaluating the true specificity of purported PRA‐specific antibodies as the PRA/PRB ratio may have prognostic and predictive value in breast cancer. PMID:29085663

Molecular cloning and expression of the calmodulin gene from guinea pig hearts.

PubMed

Feng, Rui; Liu, Yan; Sun, Xuefei; Wang, Yan; Hu, Huiyuan; Guo, Feng; Zhao, Jinsheng; Hao, Liying

2015-06-01

The aim of the present study was to isolate and characterize a complementary DNA (cDNA) clone encoding the calmodulin (CaM; GenBank accession no. FJ012165) gene from guinea pig hearts. The CaM gene was amplified from cDNA collected from guinea pig hearts and inserted into a pGEM®-T Easy vector. Subsequently, CaM nucleotide and protein sequence similarity analysis was conducted between guinea pigs and other species. In addition, reverse transcription-polymerase chain reaction (RT-PCR) was performed to investigate the CaM 3 expression patterns in different guinea pig tissues. Sequence analysis revealed that the CaM gene isolated from the guinea pig heart had ∼90% sequence identity with the CaM 3 genes in humans, mice and rats. Furthermore, the deduced peptide sequences of CaM 3 in the guinea pig showed 100% homology to the CaM proteins from other species. In addition, the RT-PCR results indicated that CaM 3 was widely and differentially expressed in guinea pigs. In conclusion, the current study provided valuable information with regard to the cloning and expression of CaM 3 in guinea pig hearts. These findings may be helpful for understanding the function of CaM3 and the possible role of CaM3 in cardiovascular diseases.

Construction of cDNA expression library of watermelon for isolation of ClWRKY1 transcription factors gene involved in resistance to Fusarium wilt.

PubMed

Yang, Bing-Yan; Huo, Xiu-Ai; Li, Peng-Fei; Wang, Cui-Xia; Duan, Hui-Jun

2014-08-01

Full-length cDNAs are very important for genome annotation and functional analysis of genes. The number of full-length cDNAs from watermelon remains limited. Here we report first the construction of a full-length enriched cDNA library from Fusarium wilt stressed watermelon (Citrullus lanatus Thunb.) cultivar PI296341 root tissues using the SMART method. The titer of primary cDNA library and amplified library was 2.21 x 10(6) and 2.0 x 10(10) pfu/ml, respectively and the rate of recombinant was above 85%. The size of insert fragment ranged from 0.3 to 2.0 kb. In this study, we first cloned a gene named ClWRKY1, which was 1981 bp long and encoded a protein consisting of 394 amino acids. It contained two characteristic WRKY domains and two zinc finger motifs. Quantitative real-time PCR showed that ClWRKY1 expression levels reached maximum level at 12 h after inoculation with Fusarium oxysporum f. sp. niveum. The full-length cDNA library of watermelon root tissues is not only essential for the cloning of genes which are known, but also an initial key for the screening and cloning of new genes that might be involved in resistance to Fusarium wilt.

Molecular Cloning and Sequencing of Channel Catfish, Ictalurus punctatus, Cathepsin H and L cDNA

USDA-ARS?s Scientific Manuscript database

Cathepsin H and L, a lysosomal cysteine endopeptidase of the papain family, are ubiquitously expressed and involve in antigen processing. In this communication, the channel catfish cathepsin H and L transcripts were sequenced and analyzed. Total RNA from tissues was extracted and cDNA libraries we...

Molecular cloning of the myo-inositol oxygenase gene from the kidney of baboons

PubMed Central

González-Álvarez, Rafael; Pérez-Ibave, Diana Cristina; Garza-Rodríguez, María Lourdes; Lugo-Trampe, Ángel; Delgado-Enciso, Iván; Tejero-Barrera, María Elizabeth; Martínez-De-Villarreal, Laura Elia; Garza-Guajardo, Raquel; Sánchez-Chaparro, María Marisela; Ruiz-Ayma, Gabriel; Barboza-Quintana, Oralia; Barrera-Saldaña, Hugo Alberto; Rocha-Pizaña, María Del Refugio; Rodríguez-Sánchez, Irám Pablo

2017-01-01

The enzyme myo-Inositol oxygenase (MIOX) is also termed ALDRL6. It is a kidney-specific member of the aldo-keto reductase family. MIOX catalyzes the first reaction involved in the myo-inositol metabolism signaling pathway and is fully expressed in mammalian tissues. MIOX catalyzes the oxidative cleavage of myo-Inositol and its epimer, D-chiro-Inositol to D-glucuronate. The dioxygen-dependent cleavage of the C6 and C1 bond in myo-Inositol is achieved by utilizing the Fe2+/Fe3+ binuclear iron center of MIOX. This enzyme has also been implicated in the complications of diabetes, including diabetic nephropathy. The MIOX gene was amplified with reverse transcription-polymerase chain reaction from baboon tissue samples, and the product was cloned and sequenced. MIOX expression in the baboon kidney is described in the present study. The percentages of nucleotide and amino acid similarities between baboons and humans were 95 and 96%, respectively. The MIOX protein of the baboon may be structurally identical to that of humans. Furthermore, the evolutionary changes, which have affected these sequences, have resulted from purifying forces. PMID:29085625

Molecular cloning of the myo-inositol oxygenase gene from the kidney of baboons.

PubMed

González-Álvarez, Rafael; Pérez-Ibave, Diana Cristina; Garza-Rodríguez, María Lourdes; Lugo-Trampe, Ángel; Delgado-Enciso, Iván; Tejero-Barrera, María Elizabeth; Martínez-De-Villarreal, Laura Elia; Garza-Guajardo, Raquel; Sánchez-Chaparro, María Marisela; Ruiz-Ayma, Gabriel; Barboza-Quintana, Oralia; Barrera-Saldaña, Hugo Alberto; Rocha-Pizaña, María Del Refugio; Rodríguez-Sánchez, Irám Pablo

2017-10-01

The enzyme myo-Inositol oxygenase (MIOX) is also termed ALDRL6. It is a kidney-specific member of the aldo-keto reductase family. MIOX catalyzes the first reaction involved in the myo-inositol metabolism signaling pathway and is fully expressed in mammalian tissues. MIOX catalyzes the oxidative cleavage of myo-Inositol and its epimer, D-chiro-Inositol to D-glucuronate. The dioxygen-dependent cleavage of the C6 and C1 bond in myo-Inositol is achieved by utilizing the Fe 2+ /Fe 3+ binuclear iron center of MIOX. This enzyme has also been implicated in the complications of diabetes, including diabetic nephropathy. The MIOX gene was amplified with reverse transcription-polymerase chain reaction from baboon tissue samples, and the product was cloned and sequenced. MIOX expression in the baboon kidney is described in the present study. The percentages of nucleotide and amino acid similarities between baboons and humans were 95 and 96%, respectively. The MIOX protein of the baboon may be structurally identical to that of humans. Furthermore, the evolutionary changes, which have affected these sequences, have resulted from purifying forces.

Molecular cloning and responsive expression to injury stimulus of a defender against cell death 1 (DAD1) gene from bay scallops Argopecten irradians.

PubMed

Zhu, Ling; Song, Linsheng; Zhang, Huan; Zhao, Jianmin; Li, Chenghua; Xu, Wei

2008-06-01

Apoptosis is an active process of cell death, which is an integral part of growth and development in multicellular organisms. The defender against cell death 1 (DAD1), the regulatory protein to inhibit the apoptosis process, was first cloned from the bay scallop Argopecten irradians by randomly sequencing a whole tissue cDNA library and rapid amplification of cDNA end (RACE). The full-length cDNA of the A. irradians DAD1 was 607 bp, consist of a 5'-terminal untranslated region (UTR) of 63 bp, a 3'-terminal UTR of 205 bp with a canonical polyadenylation signal sequence AATAAA and a poly (A) tail, and an open reading frame of 339 bp. The deduced amino acid sequence of the A. irradians DAD1 showed 75.5% identity to Araneus ventricosus, 74.5% to Drosophila melanogaster, and 73.6% to Homo sapiens, Sus scrofa, Mesocricetus auratus, Rattus norvegicus and Mus musculus. Excluding the Saccharomyces cerevisiae DAD1 homologue, all animal DAD1 including A. irradians DAD1 homologue formed a subgroup and all plant DAD1 proteins formed another subgroup in the phylogenetic analysis. The A. irradians DAD1 was expressed in all examined tissues including adductor muscle, mantle, gills, digestive gland, gonad and hemolymph, suggesting that A. irradians DAD1 is expressed in most body tissues. Furthermore, the mRNA expression levels of A. irradians DAD1 gene of hemolymph were particularly high after injury, suggesting that the gene is responsive to injury stimuli.

Molecular cloning and characterization of SoxB2 gene from Zhikong scallop Chlamys farreri

NASA Astrophysics Data System (ADS)

He, Yan; Bao, Zhenmin; Guo, Huihui; Zhang, Yueyue; Zhang, Lingling; Wang, Shi; Hu, Jingjie; Hu, Xiaoli

2013-11-01

The Sox proteins play critical roles during the development of animals, including sex determination and central nervous system development. In this study, the SoxB2 gene was cloned from a mollusk, the Zhikong scallop ( Chlamys farreri), and characterized with respect to phylogeny and tissue distribution. The full-length cDNA and genomic DNA sequences of C. farreri SoxB2 ( Cf SoxB2) were obtained by rapid amplification of cDNA ends and genome walking, respectively, using a partial cDNA fragment from the highly conserved DNA-binding domain, i.e., the High Mobility Group (HMG) box. The full-length cDNA sequence of Cf SoxB2 was 2 048 bp and encoded 268 amino acids protein. The genomic sequence was 5 551 bp in length with only one exon. Several conserved elements, such as the TATA-box, GC-box, CAAT-box, GATA-box, and Sox/sry-sex/testis-determining and related HMG box factors, were found in the promoter region. Furthermore, real-time quantitative reverse transcription PCR assays were carried out to assess the mRNA expression of Cf SoxB 2 in different tissues. SoxB2 was highly expressed in the mantle, moderately in the digestive gland and gill, and weakly expressed in the gonad, kidney and adductor muscle. In male and female gonads at different developmental stages of reproduction, the expression levels of Cf SoxB2 were similar. Considering the specific expression and roles of SoxB 2 in other animals, in particular vertebrates, and the fact that there are many pallial nerves in the mantle, cerebral ganglia in the digestive gland and gill nerves in gill, we propose a possible essential role in nervous tissue function for Sox B 2 in C. farreri.

Gene expression in the pulp of ripening bananas. Two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis of in vitro translation products and cDNA cloning of 25 different ripening-related mRNAs.

PubMed Central

Medina-Suárez, R; Manning, K; Fletcher, J; Aked, J; Bird, C R; Seymour, G B

1997-01-01

mRNA was extracted from the pulp and peel of preclimacteric (d 0) bananas (Musa AAA group, cv Grand Nain) and those exposed to ethylene gas for 24 h and stored in air alone for a further 1 (d 2) and 4 d (d 5). Two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis of in vitro translation products from the pulp and peel of these fruits revealed significant up-regulation of numerous transcripts during ripening. The majority of the changes were initiated by d 2, with the level of these messages increasing during the remainder of the ripening period. Pulp tissue from d 2 was used for the construction of a cDNA library. This library was differentially screened for ripening-related clones using cDNA from d-0 and d-2 pulp by a novel microtiter plate method. In the primary screen 250 up- and down-regulated clones were isolated. Of these, 59 differentially expressed clones were obtained from the secondary screen. All of these cDNAs were partially sequenced and grouped into families after database searches. Twenty-five nonredundant groups of pulp clones were identified. These encoded enzymes were involved in ethylene biosynthesis, respiration, starch metabolism, cell wall degradation, and several other key metabolic events. We describe the analysis of these clones and their possible involvement in ripening. PMID:9342865

Molecular Cloning, Characterization, and Expression of MiSOC1: A Homolog of the Flowering Gene SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1 from Mango (Mangifera indica L).

PubMed

Wei, Junya; Liu, Debing; Liu, Guoyin; Tang, Jie; Chen, Yeyuan

2016-01-01

MADS-box transcription factor plays a crucial role in plant development, especially controlling the formation and development of floral organs. Mango ( Mangifera indica L) is an economically important fruit crop, but its molecular control of flowering is largely unknown. To better understand the molecular basis of flowering regulation in mango, we isolated and characterized the MiSOC1, a putative mango orthologs for the Arabidopsis SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1/AGAMOUS-LIKE 20 (SOC1/AGL20) with homology-based cloning and RACE. The full-length cDNA (GenBank accession No.: KP404094) is 945 bp in length including a 74 bp long 5' UTR and a 189 bp long 3' UTR and the open reading frame was 733 bps, encoding 223 amino acids with molecular weight 25.6 kD. Both sequence alignment and phylogenetic analysis all indicated that deduced protein contained a conservative MADS-box and semi-conservative K domain and belonged to the SOC1/TM3 subfamily of the MADS-box family. Quantitative real-time PCR was performed to investigate the expression profiles of MiSOC1 gene in different tissues/organs including root, stem, leaves, flower bud, and flower. The result indicated MiSOC1 was widely expressed at different levels in both vegetative and reproductive tissues/organs with the highest expression level in the stems' leaves and inflorescences, low expression in roots and flowers. The expression of MiSOC1 in different flower developmental stages was different while same tissue -specific pattern among different varieties. In addition, MiSOC1 gene expression was affect by ethephon while high concentration ethephon inhibit the expression of MiSOC1. Overexpression of MiSOC1 resulted in early flowering in Arabidopsis . In conclusion, these results suggest that MiSOC1 may act as induce flower function in mango.

Molecular Cloning, Characterization, and Expression of MiSOC1: A Homolog of the Flowering Gene SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1 from Mango (Mangifera indica L)

PubMed Central

Wei, Junya; Liu, Debing; Liu, Guoyin; Tang, Jie; Chen, Yeyuan

2016-01-01

MADS-box transcription factor plays a crucial role in plant development, especially controlling the formation and development of floral organs. Mango (Mangifera indica L) is an economically important fruit crop, but its molecular control of flowering is largely unknown. To better understand the molecular basis of flowering regulation in mango, we isolated and characterized the MiSOC1, a putative mango orthologs for the Arabidopsis SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1/AGAMOUS-LIKE 20 (SOC1/AGL20) with homology-based cloning and RACE. The full-length cDNA (GenBank accession No.: KP404094) is 945 bp in length including a 74 bp long 5′ UTR and a 189 bp long 3′ UTR and the open reading frame was 733 bps, encoding 223 amino acids with molecular weight 25.6 kD. Both sequence alignment and phylogenetic analysis all indicated that deduced protein contained a conservative MADS-box and semi-conservative K domain and belonged to the SOC1/TM3 subfamily of the MADS-box family. Quantitative real-time PCR was performed to investigate the expression profiles of MiSOC1 gene in different tissues/organs including root, stem, leaves, flower bud, and flower. The result indicated MiSOC1 was widely expressed at different levels in both vegetative and reproductive tissues/organs with the highest expression level in the stems’ leaves and inflorescences, low expression in roots and flowers. The expression of MiSOC1 in different flower developmental stages was different while same tissue –specific pattern among different varieties. In addition, MiSOC1 gene expression was affect by ethephon while high concentration ethephon inhibit the expression of MiSOC1. Overexpression of MiSOC1 resulted in early flowering in Arabidopsis. In conclusion, these results suggest that MiSOC1 may act as induce flower function in mango. PMID:27965680

Cloning of a neonatal calcium atpase isoform (SERCA 1B) from extraocular muscle of adult blue marlin (Makaira nigricans).

PubMed

Londraville, R L; Cramer, T D; Franck, J P; Tullis, A; Block, B A

2000-10-01

Complete cDNAs for the fast-twitch Ca2+ -ATPase isoform (SERCA 1) were cloned and sequenced from blue marlin (Makaira nigricans) extraocular muscle (EOM). Complete cDNAs for SERCA 1 were also cloned from fast-twitch skeletal muscle of the same species. The two sequences are identical over the coding region except for the last five codons on the carboxyl end; EOM SERCA 1 cDNA codes for 996 amino acids and the fast-twitch cDNAs code for 991 aa. Phylogenetic analysis revealed that EOM SERCA 1 clusters with an isoform of Ca2+ -ATPase normally expressed in early development of mammals (SERCA 1B). This is the first report of SERCA 1B in an adult vertebrate. RNA hybridization assays indicate that 1B expression is limited to extraocular muscles. Because EOM gives rise to the thermogenic heater organ in marlin, we investigated whether SERCA 1B may play a role in heat generation, or if 1B expression is common in EOM among vertebrates. Chicken also expresses SERCA 1B in EOM, but rat expresses SERCA 1A; because SERCA 1B is not specific to heater tissue we conclude it is unlikely that it plays a specific role in intracellular heat production. Comparative sequence analysis does reveal, however, several sites that may be the source of functional differences between fish and mammalian SERCAs.

Signatures from Tissue-specific MPSS Libraries Identify Transcripts Preferentially Expressed in the Mouse Inner Ear

PubMed Central

Peters, Linda M.; Belyantseva, Inna A.; Lagziel, Ayala; Battey, James F.; Friedman, Thomas B.; Morell, Robert J.

2007-01-01

Specialization in cell function and morphology is influenced by the differential expression of mRNAs, many of which are expressed at low abundance and restricted to certain cell types. Detecting such transcripts in cDNA libraries may require sequencing millions of clones. Massively parallel signature sequencing (MPSS) is well-suited for identifying transcripts that are expressed in discrete cell types and in low abundance. We have made MPSS libraries from microdissections of three inner ear tissues. By comparing these MPSS libraries to those of 87 other tissues included in the Mouse Reference Transcriptome (MRT) online resource, we have identified genes that are highly enriched in, or specific to, the inner ear. We show by RT-PCR and in situ hybridization that signatures unique to the inner ear libraries identify transcripts with highly specific cell-type localizations. These transcripts serve to illustrate the utility of a resource that is available to the research community. Utilization of these resources will increase the number of known transcription units and expand our knowledge of the tissue-specific regulation of the transcriptome. PMID:17049805

Characterization and expression of the calpastatin gene in Cyprinus carpio.

PubMed

Chen, W X; Ma, Y

2015-07-03

Calpastatin, an important protein used to regulate meat quality traits in animals, is encoded by the CAST gene. The aim of the present study was to clone the cDNA sequence of the CAST gene and detect the expression of CAST in the tissues of Cyprinus carpio. The cDNA of the C. carpio CAST gene, amplified using rapid amplification of cDNA ends PCR, is 2834 bp in length (accession No. JX275386), contains a 2634-bp open reading frame, and encodes a protein with 877 amino acid residues. The amino acid sequence of the C. carpio CAST gene was 88, 80, and 59% identical to the sequences observed in grass carp, zebrafish, and other fish, respectively. The C. carpio CAST was observed to contain four conserved domains with 54 serine phosphorylation loci, 28 threonine phosphorylation loci, 1 tyrosine phosphorylation loci, and 6 specific protein kinase C phosphorylation loci. The CAST gene showed widespread expression in different tissues of C. carpio. Surprisingly, the relative expression of the CAST transcript in the muscle and heart tissues of C. carpio was significantly higher than in other tissues (P < 0.01).

VGLL3 expression is associated with a tumor suppressor phenotype in epithelial ovarian cancer.

PubMed

Gambaro, Karen; Quinn, Michael C J; Wojnarowicz, Paulina M; Arcand, Suzanna L; de Ladurantaye, Manon; Barrès, Véronique; Ripeau, Jean-Sébastien; Killary, Ann M; Davis, Elaine C; Lavoie, Josée; Provencher, Diane M; Mes-Masson, Anne-Marie; Chevrette, Mario; Tonin, Patricia N

2013-06-01

Previous studies have implicated vestigial like 3 (VGLL3), a chromosome 3p12.3 gene that encodes a putative transcription co-factor, as a candidate tumor suppressor gene (TSG) in high-grade serous ovarian carcinomas (HGSC), the most common type of epithelial ovarian cancer. A complementation analysis based on microcell-mediated chromosome transfer (MMCT) using a centric fragment of chromosome 3 (der3p12-q12.1) into the OV-90 ovarian cancer cell line haploinsufficient for 3p and lacking VGLL3 expression was performed to assess the effect on tumorigenic potential and growth characteristics. Genetic characterization of the derived MMCT hybrids revealed that only the hybrid that contained an intact VGLL3 locus exhibited alterations of tumorigenic potential in a nude mouse xenograft model and various in vitro growth characteristics. Only stable OV-90 transfectant clones expressing low levels of VGLL3 were derived. These clones exhibited an altered cytoplasmic morphology characterized by numerous single membrane bound multivesicular-bodies (MVB) that were not attributed to autophagy. Overexpression of VGLL3 in OV-90 was achieved using a lentivirus-based tetracycline inducible gene expression system, which also resulted in MVB formation in the infected cell population. Though there was no significant differences in various in vitro and in vivo growth characteristics in a comparison of VGLL3-expressing clones with empty vector transfectant controls, loss of VGLL3 expression was observed in tumors derived from mouse xenograft models. VGLL3 gene and protein expression was significantly reduced in HGSC samples (>98%, p < 0.05) relative to either normal ovarian surface epithelial cells or epithelial cells of the fallopian tube, possible tissues of origin of HGSC. Also, there appeared to be to be more cases with higher staining levels in stromal tissue component from HGSC cases that had a prolonged disease-free survival. The results taken together suggest that VGLL3 is involved in tumor suppressor pathways, a feature that is characterized by the absence of VGLL3 expression in HGSC samples. Copyright © 2013 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Successful pod infections by Moniliophthora roreri result in differential Theobroma cacao gene expression depending on the clone's level of tolerance.

PubMed

Ali, Shahin S; Melnick, Rachel L; Crozier, Jayne; Phillips-Mora, Wilberth; Strem, Mary D; Shao, Jonathan; Zhang, Dapeng; Sicher, Richard; Meinhardt, Lyndel; Bailey, Bryan A

2014-09-01

An understanding of the tolerance mechanisms of Theobroma cacao used against Moniliophthora roreri, the causal agent of frosty pod rot, is important for the generation of stable disease-tolerant clones. A comparative view was obtained of transcript populations of infected pods from two susceptible and two tolerant clones using RNA sequence (RNA-Seq) analysis. A total of 3009 transcripts showed differential expression among clones. KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway analysis of differentially expressed genes indicated shifts in 152 different metabolic pathways between the tolerant and susceptible clones. Real-time quantitative reverse transcription polymerase chain reaction (real-time qRT-PCR) analyses of 36 genes verified the differential expression. Regression analysis validated a uniform progression in gene expression in association with infection levels and fungal loads in the susceptible clones. Expression patterns observed in the susceptible clones diverged in tolerant clones, with many genes showing higher expression at a low level of infection and fungal load. Principal coordinate analyses of real-time qRT-PCR data separated the gene expression patterns between susceptible and tolerant clones for pods showing malformation. Although some genes were constitutively differentially expressed between clones, most results suggested that defence responses were induced at low fungal load in the tolerant clones. Several elicitor-responsive genes were highly expressed in tolerant clones, suggesting rapid recognition of the pathogen and induction of defence genes. Expression patterns suggested that the jasmonic acid-ethylene- and/or salicylic acid-mediated defence pathways were activated in the tolerant clones, being enhanced by reduced brassinosteroid (BR) biosynthesis and catabolic inactivation of both BR and abscisic acids. Finally, several genes associated with hypersensitive response-like cell death were also induced in tolerant clones. © 2014 BSPP AND JOHN WILEY & SONS LTD.

MsZEP, a novel zeaxanthin epoxidase gene from alfalfa (Medicago sativa), confers drought and salt tolerance in transgenic tobacco.

PubMed

Zhang, Zhiqiang; Wang, Yafang; Chang, Leqin; Zhang, Tong; An, Jie; Liu, Yushi; Cao, Yuman; Zhao, Xia; Sha, Xuyang; Hu, Tianming; Yang, Peizhi

2016-02-01

The zeaxanthin epoxidase gene ( MsZEP ) was cloned and characterized from alfalfa and validated for its function of tolerance toward drought and salt stresses by heterologous expression in Nicotiana tabacum. Zeaxanthin epoxidase (ZEP) plays important roles in plant response to various environment stresses due to its functions in ABA biosynthetic and the xanthophyll cycle. To understand the expression characteristics and the biological functions of ZEP in alfalfa (Medicago sativa), a novel gene, designated as MsZEP (KM044311), was cloned, characterized and overexpressed in Nicotiana tabacum. The open reading frame of MsZEP contains 1992 bp nucleotides and encodes a 663-amino acid polypeptide. Amino acid sequence alignment indicated that deduced MsZEP protein was highly homologous to other plant ZEP sequences. Phylogenetic analysis showed that MsZEP was grouped into a branch with other legume plants. Real-time quantitative PCR revealed that MsZEP gene expression was clearly tissue-specific, and the expression levels were higher in green tissues (leaves and stems) than in roots. MsZEP expression decreased in shoots under drought, cold, heat and ABA treatment, while the expression levels in roots showed different trends. Besides, the results showed that nodules could up-regulate the MsZEP expression under non-stressful conditions and in the earlier stage of different abiotic stress. Heterologous expression of the MsZEP gene in N. tabacum could confer tolerance to drought and salt stress by affecting various physiological pathways, ABA levels and stress-responsive genes expression. Taken together, these results suggested that the MsZEP gene may be involved in alfalfa responses to different abiotic stresses and nodules, and could enhance drought and salt tolerance of transgenic tobacco by heterologous expression.

Cloning and pharmacological characterization of the rabbit bradykinin B2 receptor.

PubMed

Bachvarov, D R; Saint-Jacques, E; Larrivée, J F; Levesque, L; Rioux, F; Drapeau, G; Marceau, F

1995-12-01

Degenerate primers, corresponding to consensus sequences of third and sixth transmembrane domains of G protein-coupled receptor superfamily, were used for the polymerase chain reaction amplification and consecutive characterization of G protein-coupled receptors present in cultured rabbit aortic smooth muscle cells. One of the isolated resulting fragments was highly homologous to the corresponding region of the bradykinin (BK) B2 receptor cloned in other species. The polymerase chain reaction fragment was used to screen a rabbit genomic library, which allowed the identification of an intronless 1101-nucleotide open reading frame which codes for a 367-amino acid receptor protein. The rabbit B2 receptor sequence is more than 80% identical to the ones determined in three other species and retain putative glycosylation, palmitoylation and phosphorylation sites. In the rabbit genomic sequence, an acceptor splice sequence was found 8 base pairs upstream of the start codon. Northern blot analysis showed a high expression of a major transcript (4.2 kilobases) in the rabbit kidney and duodenum, and a less abundant expression in other tissues. Southern blot experiments suggest that a single copy of this gene exists in the rabbit genome. The cloned rabbit B2 receptor expressed in COS-1 cells binds [3H]BK in a saturable manner (KD 2.1 nM) and this ligand competes with a series of kinin agonists and antagonist with a rank order consistent with the B2 receptor identity. The insurmountable character of the antagonism exerted by Hoe 140 against BK on the rabbit B2 receptor, previously shown in pharmacological experiments, was confirmed in binding experiments with the cloned receptor expressed in a controlled manner. By contrast, Hoe 140 competed with [3H]BK in a surmountable manner for the human B2 receptor expressed in COS-1 cells. The cloning of the rabbit B2 receptor will be useful notably for the study of the structural basis of antagonist binding and for studies on receptor regulation in a relatively large animal.

Protein Equilibration through Somatic Ring Canals in Drosophila

PubMed Central

McLean, Peter F.; Cooley, Lynn

2013-01-01

Although intercellular bridges resulting from incomplete cytokinesis were discovered in somatic Drosophila tissues decades ago, the impact of these structures on intercellular communication and tissue biology is largely unknown. In this work, we demonstrate that the ~250 nm diameter somatic ring canals permit diffusion of cytoplasmic contents between connected cells and across mitotic clone boundaries, and enable the equilibration of protein between transcriptionally mosaic follicle cells in the Drosophila ovary. We obtained similar, though more restricted, results in the larval imaginal discs. Our work illustrates the lack of cytoplasmic autonomy in these tissues and suggests a role for somatic ring canals in promoting homogeneous protein expression within the tissue. PMID:23704373

The Drosophila gene collection: Identification of putative full-length cDNAs for 70 percent of D. melanogaster genes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Stapleton, Mark; Liao, Guochun; Brokstein, Peter

2002-08-12

Collections of full-length nonredundant cDNA clones are critical reagents for functional genomics. The first step toward these resources is the generation and single-pass sequencing of cDNA libraries that contain a high proportion of full-length clones. The first release of the Drosophila Gene Collection Release 1 (DGCr1) was produced from six libraries representing various tissues, developmental stages, and the cultured S2 cell line. Nearly 80,000 random 5prime expressed sequence tags (EST) from these libraries were collapsed into a nonredundant set of 5849 cDNAs, corresponding to {approx}40 percent of the 13,474 predicted genes in Drosophila. To obtain cDNA clones representing the remainingmore » genes, we have generated an additional 157,835 5prime ESTs from two previously existing and three new libraries. One new library is derived from adult testis, a tissue we previously did not exploit for gene discovery; two new cap-trapped normalized libraries are derived from 0-22hr embryos and adult heads. Taking advantage of the annotated D. melanogaster genome sequence, we clustered the ESTs by aligning them to the genome. Clusters that overlap genes not already represented by cDNA clones in the DGCr1 were analyzed further, and putative full-length clones were selected for inclusion in the new DGC. This second release of the DGC (DGCr2) contains 5061 additional clones, extending the collection to 10,910 cDNAs representing >70 percent of the predicted genes in Drosophila.« less

Amphiphysin I but not dynamin I nor synaptojanin mRNA expression increased after repeated methamphetamine administration in the rat cerebrum and cerebellum.

PubMed

Hamamura, Mitsuko; Okouchi, Jiro; Ozawa, Hidetoshi; Kimuro, Yoshihiko; Iwaki, Akiko; Fukumaki, Yasuyuki

2013-07-01

Dopamine increases/decreases synaptic vesicle recycling and in schizophrenia the proteins/mRNA is decreased. We isolated cDNA clone, similar to amphiphysin 1 (vesicle protein) mRNA from the neocortex of rats injected repeatedly with methamphetamine using polymerase chain reaction (PCR) differential display. This clone is highly homologous to the 3' region of the human amphiphysin gene. PCR extension study using a primer specific for the rat amphiphysin 1 gene and a primer located within the clone revealed that it is the 3' UTR region of the rat amphiphysin 1 gene. Furthermore, in situ hybridization revealed that amphiphysin 1 mRNA is expressed in the cerebrum, medial thalamus, hippocampus and cerebellum. In the cerebellum, amphiphysin mRNA expression was confined to upper granule cell layer. Repeated methamphetamine administration increased amphiphysin I mRNA expression in both anterior part of the cerebrum, and the cerebellum. However, the repeated administration did not alter mRNA expression of the other vesicle proteins, synaptotagmin I, synapsin I, synaptojanin and dynamin I, we conclude that the repeated administration selectively increased amphiphysin 1 mRNA expression. Thus, amphiphysin 1 does not work as synaptic recycling, but it is suggested, as a part of pathogenesis of brain tissue injury (under Ca²⁺ and Mg²⁺ devoid environment) in repeated methamphetamine-injected states, the gene regulate actin-asssembly, learning, cell stress signaling and cell polarity.

In silico cloning, expression of Rieske-like apoprotein gene and protein subcellular localization in the Pacific oyster, Crassostrea gigas.

PubMed

He, Xiaocui; Zhang, Yang; Yu, Ziniu

2010-10-01

Rieske protein gene in the Pacific oyster Crassostrea gigas was obtained by in silico cloning for the first time, and its expression profiles and subcellular localization were determined, respectively. The full-length cDNA of Cgisp is 985 bp in length and contains a 5'- and 3'-untranslated regions of 35 and 161 bp, respectively, with an open reading frame of 786 bp encoding a protein of 262 amino acids. The predicted molecular weight of 30 kDa of Cgisp protein was verified by prokaryotic expression. Conserved Rieske [2Fe-2S] cluster binding sites and highly matched-pair tertiary structure with 3CWB_E (Gallus gallus) were revealed by homologous analysis and molecular modeling. Eleven putative SNP sites and two conserved hexapeptide sequences, box I (THLGC) and II (PCHGS), were detected by multiple alignments. Real-time PCR analysis showed that Cgisp is expressed in a wide range of tissues, with adductor muscle exhibiting the top expression level, suggesting its biological function of energy transduction. The GFP tagging Cgisp indicated a mitochondrial localization, further confirming its physiological function.

Cloning, tissue distribution and effects of fasting on pituitary adenylate cyclase-activating polypeptide in largemouth bass

NASA Astrophysics Data System (ADS)

Li, Shengjie; Han, Linqiang; Bai, Junjie; Ma, Dongmei; Quan, Yingchun; Fan, Jiajia; Jiang, Peng; Yu, Lingyun

2015-03-01

Pituitary adenylate cyclase activating polypeptide (PACAP) has a wide range of biological functions. We cloned the full-length cDNAs encoding PACAP and PACAP-related peptide (PRP) from the brain of largemouth bass ( Micropterus salmoides) and used real-time quantitative PCR to detect PRP-PACAP mRNA expression. The PRP-PACAP cDNA has two variants expressed via alternative splicing: a long form, which encodes both PRP and PACAP, and a short form, which encodes only PACAP. Sequence analysis results are consistent with a higher conservation of PACAP than PRP peptide sequences. The expression of PACAP-long and PACAP-short transcripts was highest in the forebrain, followed by the medulla, midbrain, pituitary, stomach, cerebellum, intestine, and kidney; however, these transcripts were either absent or were weakly expressed in the muscle, spleen, gill, heart, fatty tissue, and liver. The level of PACAP-short transcript expression was significantly higher than expression of the long transcript in the forebrain, cerebella, pituitary and intestine, but lower than that of the long transcript in the stomach. PACAP-long and PACAP-short transcripts were first detected at the blastula stage of embryogenesis, and the level of expression increased markedly between the muscular contraction stage and 3 d post hatch (dph). The expression of PACAP-long and PACAP-short transcripts decreased significantly in the brain following 4 d fasting compared with the control diet group. The down-regulation effect was enhanced as fasting continued. Conversely, expression levels increased significantly after 3 d of re-feeding. Our results suggest that PRP-PACAP acts as an important factor in appetite regulation in largemouth bass.

Cloning and characterization of murine fanconi anemia group A gene: Fanca protein is expressed in lymphoid tissues, testis, and ovary.

PubMed

van de Vrugt, H J; Cheng, N C; de Vries, Y; Rooimans, M A; de Groot, J; Scheper, R J; Zhi, Y; Hoatlin, M E; Joenje, H; Arwert, F

2000-04-01

Fanconi anemia (FA) is an autosomal recessive disorder in humans characterized by bone marrow failure, cancer predisposition, and cellular hypersensitivity to cross-linking agents such as mitomycin C and diepoxybutane. FA genes display a caretaker function essential for maintenance of genomic integrity. We have cloned the murine homolog of FANCA, the gene mutated in the major FA complementation group (FA-A). The full-length mouse Fanca cDNA consists of 4503 bp and encodes a protein with a predicted molecular weight of 161 kDa. The deduced Fanca mouse protein shares 81% amino acid sequence similarity and 66% identity with the human protein. The nuclear localization signal and partial leucine zipper consensus motifs found in the human FANCA protein were also present in the murine homolog. In spite of the species difference, the murine Fanca cDNA was capable of correcting the cross-linker sensitive phenotype of human FA-A cells, suggesting functional conservation. Based on Northern as well as Western blots, Fanca was mainly expressed in lymphoid tissues, testis, and ovary. This expression pattern correlates with some of the clinical symptoms observed in FA patients. The availability of the murine Fanca cDNA now allows the gene to be studied in experimental mouse models.

Human protoporphyrinogen oxidase: expression, purification, and characterization of the cloned enzyme.

PubMed Central

Dailey, T. A.; Dailey, H. A.

1996-01-01

Protoporphyrinogen oxidase (E.C.1.3.3.4) catalyzes the oxygen-dependent oxidation of protoporphyrinogen IX to protoporphyrin IX. The enzyme from human placenta has been cloned, sequenced, expressed in Escherichia coli, purified to homogeneity, and characterized. Northern blot analysis of eight different human tissues show evidence for only a single transcript in all tissue types and the size of this transcript is approximately 1.8 kb. The human cDNA has been inserted into an expression vector for E. coli and the protein produced at high levels in these cells. The protein is found in both membrane and cytoplasmic fractions. The enzyme was purified to homogeneity in the presence of detergents using a metal chelate affinity column. The purified protein is a homodimer composed of subunits of molecular weight of 51,000. The enzyme contains one noncovalently bound FAD per dimer, has a monomer extinction coefficient of 48,000 at 270 nm and contains no detectable redox active metals. The apparent K(m) and Kcat for protoporphyrinogen IX are 1.7 microM and 10.5 min-1, respectively. The enzyme does not use coproporphyrinogen III as a substrate and is inhibited by micromolar concentrations of the herbicide acifluorfen. Protein database searches reveal significant homology between protoporphyrinogen oxidase and monoamine oxidase. PMID:8771201

Cloning and characterization of mouse extracellular-signal-regulated protein kinase 3 as a unique gene product of 100 kDa.

PubMed

Turgeon, B; Saba-El-Leil, M K; Meloche, S

2000-02-15

MAP (mitogen-activated protein) kinases are a family of serine/threonine kinases that have a pivotal role in signal transduction. Here we report the cloning and characterization of a mouse homologue of extracellular-signal-regulated protein kinase (ERK)3. The mouse Erk3 cDNA encodes a predicted protein of 720 residues, which displays 94% identity with human ERK3. Transcription and translation of this cDNA in vitro generates a 100 kDa protein similar to the human gene product ERK3. Immunoblot analysis with an antibody raised against a unique sequence of ERK3 also recognizes a 100 kDa protein in mouse tissues. A single transcript of Erk3 was detected in every adult mouse tissue examined, with the highest expression being found in the brain. Interestingly, expression of Erk3 mRNA is acutely regulated during mouse development, with a peak of expression observed at embryonic day 11. The mouse Erk3 gene was mapped to a single locus on central mouse chromosome 9, adjacent to the dilute mutation locus and in a region syntenic to human chromosome 15q21. Finally, we provide several lines of evidence to support the existence of a unique Erk3 gene product of 100 kDa in mammalian cells.

T cell receptor (TCR) structure of autologous melanoma-reactive cytotoxic T lymphocyte (CTL) clones: tumor-infiltrating lymphocytes overexpress in vivo the TCR beta chain sequence used by an HLA-A2- restricted and melanocyte-lineage-specific CTL clone

PubMed Central

1993-01-01

HLA-A2+ melanomas express common melanoma-associated antigens (Ags) recognized in vitro by autologous cytotoxic T lymphocytes (CTL). However, it is not known whether tumor Ags can drive in vivo a selective accumulation/expansion of Ag-specific, tumor-infiltrating T lymphocytes (TIL). Therefore, to evaluate this possibility, 39 CTL clones isolated from several independent mixed lymphocyte tumor cultures (MLTC) of TIL and peripheral blood lymphocytes (PBL) of an HLA- A2+ melanoma patient and selected for T cell receptor (TCR)-dependent, HLA-restricted tumor lysis, were used for analysis of TCR alpha and beta chain structure by the cDNA polymerase chain reaction (PCR) technique with variable gene-specific primers followed by sequencing. Despite absence of oligoclonality in fresh TIL and PBL, as well as in T cells of day 28 MLTC (day of cloning), sequence analysis of TCR alpha and beta chains of TIL clones revealed a dominance of a major category of melanoma-specific, HLA-A2-restricted T cells expressing a V alpha 8.2/J alpha AP511/C alpha and V beta 2.1/D beta 1/J beta 1.1/C beta 1 TCR. The same TCR was also found in 2 out of 14 PBL clones. The other PBL clones employed a V alpha 2.1 gene segment associated with either V beta 13.2, 14, or w22. Clones A81 (V alpha 2.1/J alpha IGRJ alpha 04/C alpha and V beta 14/D beta 1/J beta 1.2/C beta 1) and A21 (V alpha 8.2/J alpha AP511/C alpha and V beta 2.1/D beta 1/J beta 1.1/C beta 1), representative of the two most frequent TCR of PBL and TIL, respectively, expressed different lytic patterns, but both were HLA-A2 restricted and lysed only HLA-A2+ melanomas and normal melanocytes, thus indicating recognition of two distinct HLA-A2-associated and tissue-related Ags. Finally, by the inverse PCR technique, the specific TCR beta chain (V beta 2.1/D beta 1/J beta 1.1/C beta 1) expressed by the dominant TIL clone was found to represent 19 and 18.4% of all V beta 2 sequences expressed in the fresh tumor sample and in the purified TIL, respectively, but < 0.19% of V beta 2+ sequences expressed in PBL. These results are consistent with the hypothesis that a clonal expansion/accumulation of a melanocyte-lineage-specific and HLA-A2-restricted T cell clone occurred in vivo at the site of tumor growth. PMID:8376931

Recombinational Cloning Using Gateway and In-Fusion Cloning Schemes

PubMed Central

Throop, Andrea L.; LaBaer, Joshua

2015-01-01

The comprehensive study of protein structure and function, or proteomics, depends on the obtainability of full-length cDNAs in species-specific expression vectors and subsequent functional analysis of the expressed protein. Recombinational cloning is a universal cloning technique based on site-specific recombination that is independent of the insert DNA sequence of interest, which differentiates this method from the classical restriction enzyme-based cloning methods. Recombinational cloning enables rapid and efficient parallel transfer of DNA inserts into multiple expression systems. This unit summarizes strategies for generating expression-ready clones using the most popular recombinational cloning technologies, including the commercially available Gateway® (Life Technologies) and In-Fusion® (Clontech) cloning technologies. PMID:25827088

Molecular cloning and promoter analysis of squalene synthase and squalene epoxidase genes from Betula platyphylla.

PubMed

Zhang, Mengyan; Wang, Siyao; Yin, Jing; Li, Chunxiao; Zhan, Yaguang; Xiao, Jialei; Liang, Tian; Li, Xin

2016-09-01

Betula platyphylla is a rich repository of pharmacologically active secondary metabolites known as birch triterpenoids (TBP). Here, we cloned the squalene synthase (SS) and squalene epoxidase genetic (SE) sequences from B. platyphylla that encode the key enzymes that are involved in triterpenoid biosynthesis and analyzed the conserved domains and phylogenetics of their corresponding proteins. The full-length sequence of BpSS is 1588 bp with a poly-A tail, which contained an open reading frame (ORF) of 1241 bp that encoded a protein of 413 amino acids. Additionally, the BpSE full-length sequence of 2040 bp with a poly-A tail was also obtained, which contained an ORF of 1581 bp encoding a protein of 526 amino acids. Their organ-specific expression patterns in 4-week-old tissue culture seedlings of B. platyphylla were detected by real-time PCR and showed that they were all highly expressed in leaves, as compared to stem and root tissues. Additionaly, both BpSS and BpSE were enhanced following stimulation with ethephon and MeJA. The expression of BpSS was enhanced by ABA, whereas BpSE was not. The SA treatment did not affect the BpSS and BpSE transcripts notably. Using a genome walking approach, promoter sequences of 965 and 1193 bp, respectively, for BpSS and BpSE were isolated, and they revealed several key cis-regulatory elements known to be involved in the response to phytohormone and abiotic plant stress. We also found that the BpSS protein is localized in the cytoplasm. Opening reading frames of BpSS and BpSE were ligated into yeast expression plasmid pYES2 under control of GAL1 promoter and introduced into the yeast INVScl1 strain. The transformants were cultured for 12 h, the squalene content of galactose-induced BpSS expression yeast cells was 13.2 times of control (empty vector control yeast cells) by high-performance liquid chromatography (HPLC) test method. And, the squalene epoxidase activity of induced BpSE expression yeast cell was about 11.8 times of control. These indicated that we cloned birch BpSS and BpSE that were indeed involved in the synthesis of triteropenoids. This is the first report wherein SS and SE from B. platyphylla were cloned and may be of significant interest to understand the regulatory role of SS and SE in the triterpenoids biosynthesis of B. platyphylla. This is the first report wherein SS and SE from B. platyphylla were cloned and may be of significant interest to understand the regulatory role of SS and SE in the biosynthesis of birch triterpenoids.

Characterization of a full-length infectious cDNA clone and a GFP reporter derivative of the oncolytic picornavirus SVV-001.

PubMed

Poirier, John T; Reddy, P Seshidhar; Idamakanti, Neeraja; Li, Shawn S; Stump, Kristine L; Burroughs, Kevin D; Hallenbeck, Paul L; Rudin, Charles M

2012-12-01

Seneca Valley virus (SVV-001) is an oncolytic picornavirus with selective tropism for a subset of human cancers with neuroendocrine differentiation. To characterize further the specificity of SVV-001 and its patterns and kinetics of intratumoral spread, bacterial plasmids encoding a cDNA clone of the full-length wild-type virus and a derivative virus expressing GFP were generated. The full-length cDNA of the SVV-001 RNA genome was cloned into a bacterial plasmid under the control of the T7 core promoter sequence to create an infectious cDNA clone, pNTX-09. A GFP reporter virus cDNA clone, pNTX-11, was then generated by cloning a fusion protein of GFP and the 2A protein from foot-and-mouth disease virus immediately following the native SVV-001 2A sequence. Recombinant GFP-expressing reporter virus, SVV-GFP, was rescued from cells transfected with in vitro RNA transcripts from pNTX-11 and propagated in cell culture. The proliferation kinetics of SVV-001 and SVV-GFP were indistinguishable. The SVV-GFP reporter virus was used to determine that a subpopulation of permissive cells is present in small-cell lung cancer cell lines previously thought to lack permissivity to SVV-001. Finally, it was shown that SVV-GFP administered to tumour-bearing animals homes in to and infects tumours whilst having no detectable tropism for normal mouse tissues at 1×10(11) viral particles kg(-1), a dose equivalent to that administered in ongoing clinical trials. These infectious clones will be of substantial value in further characterizing the biology of this virus and as a backbone for the generation of additional oncolytic derivatives.

Clonal population of adult stem cells: life span and differentiation potential.

PubMed

Seruya, Mitchel; Shah, Anup; Pedrotty, Dawn; du Laney, Tracey; Melgiri, Ryan; McKee, J Andrew; Young, Henry E; Niklason, Laura E

2004-01-01

Adult stem cells derived from bone marrow, connective tissue, and solid organs can exhibit a range of differentiation potentials. Some controversy exists regarding the classification of mesenchymal stem cells as bona fide stem cells, which is in part derived from the limited ability to propagate true clonal populations of precursor cells. We isolated putative mesenchymal stem cells from the connective tissue of an adult rat (rMSC), and generated clonal populations via three rounds of dilutional cloning. The replicative potential of the clonal rMSC line far exceeded Hayflick's limit of 50-70 population doublings. The high capacity for self-renewal in vitro correlated with telomerase activity, as demonstrated by telomerase repeat amplification protocol (TRAP) assay. Exposure to nonspecific differentiation culture medium revealed multilineage differentiation potential of rMSC clones. Immunostaining confirmed the appearance of mesodermal phenotypes, including adipocytes possessing lipid-rich vacuoles, chondrocytes depositing pericellular type II collagen, and skeletal myoblasts expressing MyoD1. Importantly, the spectrum of differentiation capability was sustained through repeated passaging. Furthermore, serum-free conditions that led to high-efficiency smooth muscle differentiation were identified. rMSCs plated on collagen IV-coated surfaces and exposed to transforming growth factor-beta1 (TGF-beta1) differentiated into a homogeneous population expressing alpha-actin and calponin. Hence, clonogenic analysis confirmed the presence of a putative MSC population derived from the connective tissue of rat skeletal muscle. The ability to differentiate into a smooth muscle cell (SMC) phenotype, combined with a high proliferative capacity, make such a connective tissue-derived MSC population ideal for applications in vascular tissue construction.

An alternative method for cDNA cloning from surrogate eukaryotic cells transfected with the corresponding genomic DNA.

PubMed

Hu, Lin-Yong; Cui, Chen-Chen; Song, Yu-Jie; Wang, Xiang-Guo; Jin, Ya-Ping; Wang, Ai-Hua; Zhang, Yong

2012-07-01

cDNA is widely used in gene function elucidation and/or transgenics research but often suitable tissues or cells from which to isolate mRNA for reverse transcription are unavailable. Here, an alternative method for cDNA cloning is described and tested by cloning the cDNA of human LALBA (human alpha-lactalbumin) from genomic DNA. First, genomic DNA containing all of the coding exons was cloned from human peripheral blood and inserted into a eukaryotic expression vector. Next, by delivering the plasmids into either 293T or fibroblast cells, surrogate cells were constructed. Finally, the total RNA was extracted from the surrogate cells and cDNA was obtained by RT-PCR. The human LALBA cDNA that was obtained was compared with the corresponding mRNA published in GenBank. The comparison showed that the two sequences were identical. The novel method for cDNA cloning from surrogate eukaryotic cells described here uses well-established techniques that are feasible and simple to use. We anticipate that this alternative method will have widespread applications.

Inefficient reprogramming of the hematopoietic stem cell genome following nuclear transfer.

PubMed

Inoue, Kimiko; Ogonuki, Narumi; Miki, Hiromi; Hirose, Michiko; Noda, Shinichi; Kim, Jin-Moon; Aoki, Fugaku; Miyoshi, Hiroyuki; Ogura, Atsuo

2006-05-15

In general, cloning undifferentiated preimplantation embryos (blastomeres) or embryonic stem cells is more efficient than cloning differentiated somatic cells. Therefore, there has been an assumption that tissue-specific stem cells might serve as efficient donors for nuclear transfer because of the undifferentiated state of their genome. Here, we show that this is not the case with adult hematopoietic stem cells (HSCs). Although we have demonstrated for the first time that mouse HSCs can be cloned to generate offspring, the birth rates (0-0.7%) were lowest among the clones tested (cumulus, immature Sertoli and fibroblast cells). Only 6% of reconstructed embryos reached the morula or blastocyst stage in vitro (versus 46% for cumulus clones; P < 5 x 10(-10)). Transcription and gene expression analyses of HSC clone embryos revealed that they initiated zygotic gene activation (ZGA) at the appropriate timing, but failed to activate five out of six important embryonic genes examined, including Hdac1 (encoding histone deacetylase 1), a key regulator of subsequent ZGA. These results suggest that the HSC genome has less plasticity than we imagined, at least in terms of reprogrammability in the ooplasm after nuclear transfer.

[Cloning and expression analysis of differentially expressed genes in Chinese fir stems treated by different concentrations of exogenous IAA].

PubMed

Yang, Li-Wei; Shi, Ji-Sen

2012-04-01

To reveal the potential genetic mechanisms of indole-3-acetic acid (IAA) that regulate Chinese fir wood formation, cloned the differentially expressed genes via suppress subtractive hybridization (SSH) using the truncated stems treated by 0 and 3 mg IAA/g lanolin as the driver and tester, respectively. A total of 332 unigenes that were involved in cell organization and biosynthesis, developmental processes control, electron transport, stress response, and signal transduction. To further test the results from SSH, we selected those unigenes, whose putative encoding proteins showed significantly homologous with HIRA, PGY1, SMP1, TCT, TRN2, and ARF4, and analyzed their expressed specificity in the wood formative tissues and their response to the secondary developmental changes of vascular cambium stimulated by 0, 1, and 3 mg.IAA/g.lanolin treatment. The results showed that ClHIRA, ClPGY1, and ClARF4, which were specifically expressed in the adaxial zone of stem, were positively response to the activities of cell division and tracheid differentiation stimulated by exogenous IAA treatment. However, ClSMP1, ClTCTP1, and ClTRN2, which were mainly expressed in the abaxial zones of stems, showed negative correlation with the treated levels of exogenous IAA and activities of vascular cambium secondary development at the transcriptional level. This result showed that the differential response of developmental regulatory genes located in different vascular tissues to the level changes of edogenous IAA in stems is likely to be an important molecular mechanism of auxin regulating wood formation.

Galactinol synthase transcriptional profile in two genotypes of Coffea canephora with contrasting tolerance to drought

PubMed Central

Santos, Tiago Benedito Dos; de Lima, Rogério Barbosa; Nagashima, Getúlio Takashi; Petkowicz, Carmen Lucia de Oliveira; Carpentieri-Pípolo, Valéria; Pereira, Luiz Filipe Protasio; Domingues, Douglas Silva; Vieira, Luiz Gonzaga Esteves

2015-01-01

Increased synthesis of galactinol and raffinose family oligosaccharides (RFOs) has been reported in vegetative tissues in response to a range of abiotic stresses. In this work, we evaluated the transcriptional profile of a Coffea canephora galactinol synthase gene (CcGolS1) in two clones that differed in tolerance to water deficit in order to assess the contribution of this gene to drought tolerance. The expression of CcGolS1 in leaves was differentially regulated by water deficit, depending on the intensity of stress and the genotype. In clone 109A (drought-susceptible), the abundance of CcGolS1 transcripts decreased upon exposure to drought, reaching minimum values during recovery from severe water deficit and stress. In contrast, CcGolS1 gene expression in clone 14 (drought-tolerant) was stimulated by water deficit. Changes in galactinol and RFO content did not correlate with variation in the steady-state transcript level. However, the magnitude of increase in RFO accumulation was higher in the tolerant cultivar, mainly under severe water deficit. The finding that the drought-tolerant coffee clone showed enhanced accumulation of CcGolS1 transcripts and RFOs under water deficit suggests the possibility of using this gene to improve drought tolerance in this important crop. PMID:26273221

Galactinol synthase transcriptional profile in two genotypes of Coffea canephora with contrasting tolerance to drought.

PubMed

Santos, Tiago Benedito Dos; de Lima, Rogério Barbosa; Nagashima, Getúlio Takashi; Petkowicz, Carmen Lucia de Oliveira; Carpentieri-Pípolo, Valéria; Pereira, Luiz Filipe Protasio; Domingues, Douglas Silva; Vieira, Luiz Gonzaga Esteves

2015-05-01

Increased synthesis of galactinol and raffinose family oligosaccharides (RFOs) has been reported in vegetative tissues in response to a range of abiotic stresses. In this work, we evaluated the transcriptional profile of a Coffea canephora galactinol synthase gene (CcGolS1) in two clones that differed in tolerance to water deficit in order to assess the contribution of this gene to drought tolerance. The expression of CcGolS1 in leaves was differentially regulated by water deficit, depending on the intensity of stress and the genotype. In clone 109A (drought-susceptible), the abundance of CcGolS1 transcripts decreased upon exposure to drought, reaching minimum values during recovery from severe water deficit and stress. In contrast, CcGolS1 gene expression in clone 14 (drought-tolerant) was stimulated by water deficit. Changes in galactinol and RFO content did not correlate with variation in the steady-state transcript level. However, the magnitude of increase in RFO accumulation was higher in the tolerant cultivar, mainly under severe water deficit. The finding that the drought-tolerant coffee clone showed enhanced accumulation of CcGolS1 transcripts and RFOs under water deficit suggests the possibility of using this gene to improve drought tolerance in this important crop.

Molecular cloning and characterization of RGA1 encoding a G protein alpha subunit from rice (Oryza sativa L. IR-36).

PubMed

Seo, H S; Kim, H Y; Jeong, J Y; Lee, S Y; Cho, M J; Bahk, J D

1995-03-01

A cDNA clone, RGA1, was isolated by using a GPA1 cDNA clone of Arabidopsis thaliana G protein alpha subunit as a probe from a rice (Oryza sativa L. IR-36) seedling cDNA library from roots and leaves. Sequence analysis of genomic clone reveals that the RGA1 gene has 14 exons and 13 introns, and encodes a polypeptide of 380 amino acid residues with a calculated molecular weight of 44.5 kDa. The encoded protein exhibits a considerable degree of amino acid sequence similarity to all the other known G protein alpha subunits. A putative TATA sequence (ATATGA), a potential CAAT box sequence (AGCAATAC), and a cis-acting element, CCACGTGG (ABRE), known to be involved in ABA induction are found in the promoter region. The RGA1 protein contains all the consensus regions of G protein alpha subunits except the cysteine residue near the C-terminus for ADP-ribosylation by pertussis toxin. The RGA1 polypeptide expressed in Escherichia coli was, however, ADP-ribosylated by 10 microM [adenylate-32P] NAD and activated cholera toxin. Southern analysis indicates that there are no other genes similar to the RGA1 gene in the rice genome. Northern analysis reveals that the RGA1 mRNA is 1.85 kb long and expressed in vegetative tissues, including leaves and roots, and that its expression is regulated by light.

Localization and regulation of a facilitative urea transporter in the kidney of the red-eared slider turtle (Trachemys scripta elegans).

PubMed

Uchiyama, Minoru; Kikuchi, Ryosuke; Konno, Norifumi; Wakasugi, Tatsuya; Matsuda, Kouhei

2009-01-01

Urea is the major excretory end product of nitrogen metabolism in most chelonian reptiles. In the present study, we report the isolation of a 1632 base pair cDNA from turtle kidney with one open reading frame putatively encoding a 403-residue protein, the turtle urea transporter (turtle UT). The first cloned reptilian UT has high homology with UTs (facilitated urea transporters) cloned from vertebrates, and most closely resembles the UT-A subfamily. Injection of turtle UT cRNA into Xenopus oocytes induced a 6-fold increase in [(14)C]urea uptake that was inhibited by phloretin. The turtle UT mRNA expression and tissue distribution were examined by RT-PCR with total RNA from various tissues. Expression of turtle UT mRNA was restricted to the kidney, and no signal was detected in the other tissues, such as brain, heart, alimentary tract and urinary bladder. An approximately 58 kDa protein band was detected in membrane fractions of the kidney by western blot using an affinity-purified antibody that recognized turtle UT expressed in Xenopus oocytes. In an immunohistochemical study using the anti-turtle UT antibody, UT-immunopositive cells were observed along the distal tubule but not in the collecting duct. In turtles under dry conditions, plasma osmolality and urea concentration increased, and using semi-quantitative RT-PCR the UT mRNA expression level in the kidney was found to increase 2-fold compared with control. The present results, taken together, suggest that the turtle UT probably contributes to urea transport in the distal tubule segments of the kidney in response to hyperosmotic stress under dry conditions.

Molecular cloning and characterization of a SID-1-like gene in Plutella xylostella.

PubMed

Wang, Huidong; Gong, Liang; Qi, Jiangwei; Hu, Meiying; Zhong, Guohua; Gong, Liang

2014-11-01

RNA interference (RNAi) signal can spread from the point where the double-stranded RNA (dsRNA) was initially applied to other cells or tissues. SID-related genes in Caenorhabditis elegans help in the spreading of this signal. However, the mechanisms of systemic RNAi are still not unveiled in insects. In this study, we cloned a full-length cDNA of sid-1-like gene, Pxylsid-1, from Plutella xylostella that contains 1,047 bp opening reading frame encoding a putative protein of 348 amino acids. This transcript is very much similar to the sil-1 in Bombyx mori (68.8%). The higher expression levels of Pxylsid-1 were found at the adult and fourth-instar stages compared to the second-instar stage with 21.48- and 10.36-fold increase, respectively. Its expression levels in different tissues were confirmed with the highest expression in the hemolymph, which showed 21.09-fold increase than the midgut; however it was lower in other tissues. The result of RNAi by feeding bacterially expressed dsRNA targeting Pxylace-1, which showed that the mRNA level of Pxylace-1 decreased by 34.52 and 64.04% after 36- and 72-h treatment, respectively. However, the mRNA level of Pxylsid-1 was not significantly induced when the Pxylace-1 was downregulated. Furthermore, we found that downregulation of Pxylsid-1 did not affect the RNAi effect of Pxylace-1. Hence, the Pxylsid-1 may not be involved in absorption of dsRNA from the midgut fluid. A further study is needed to uncover the function of Pxylsid-1. © 2014 Wiley Periodicals, Inc.

Progress in gene targeting and gene therapy for retinitis pigmentosa

DOE Office of Scientific and Technical Information (OSTI.GOV)

Farrar, G.J.; Humphries, M.M.; Erven, A.

1994-09-01

Previously, we localized disease genes involved in retinitis pigmentosa (RP), an inherited retinal degeneration, close to the rhodopsin and peripherin genes on 3q and 6p. Subsequently, we and others identified mutations in these genes in RP patients. Currently animal models for human retinopathies are being generated using gene targeting by homologous recombination in embryonic stem (ES) cells. Genomic clones for retinal genes including rhodopsin and peripherin have been obtained from a phage library carrying mouse DNA isogenic with the ES cell line (CC1.2). The peripherin clone has been sequenced to establish the genomic structure of the mouse gene. Targeting vectorsmore » for rhodopsin and peripherin including a neomycin cassette for positive selection and thymidine kinase genes enabling selection against random intergrants are under construction. Progress in vector construction will be presented. Simultaneously we are developing systems for delivery of gene therapies to retinal tissues utilizing replication-deficient adenovirus (Ad5). Efficacy of infection subsequent to various methods of intraocular injection and with varying viral titers is being assayed using an adenovirus construct containing a CMV promoter LacZ fusion as reporter and the range of tissues infected and the level of duration of LacZ expression monitored. Viral constructs with the LacZ reporter gene under the control of retinal specific promoters such as rhodopsin and IRBP cloned into pXCJL.1 are under construction. An update on developments in photoreceptor cell-directed expression of virally delivered genes will be presented.« less

Genome-wide identification and expression profiling of dehydrin gene family in Malus domestica.

PubMed

Liang, Dong; Xia, Hui; Wu, Shan; Ma, Fengwang

2012-12-01

The family of dehydrin genes has important roles in protecting higher plants against abiotic stress, such as drought, salinity and cold. However, knowledge about apple dehydrin gene family is limited. In the present study, we used a bioinformatics approach to identify members of that family in apple (Malus domestica). A total of 12 apple dehydrin genes (MdDHNs) were identified and located on various chromosomes. All putative proteins from those genes contained a typical K domain. Among 12 MdDHNs, nine were cloned and their expression patterns were investigated. Expression profiling indicated that the these nine dehydrin genes display differential expression patterns in various tissues. Moreover, transcript levels of some MdDHNs were up-regulated significantly under drought, low temperature, or ABA treatment, which indicated their important roles during stress adaptation. These results demonstrate that the apple dehydrin gene family may function in tissue development and plant stress responses.

Characterization and Expression Analysis of a Retinoblastoma-Related Gene from Chinese Wild Vitis pseudoreticulata.

PubMed

Wen, Zhifeng; Gao, Min; Jiao, Chen; Wang, Qian; Xu, Hui; Walter, Monika; Xu, Weirong; Bassett, Carole; Wang, Xiping

2012-01-01

Retinoblastoma-related (RBR) genes, a conserved gene family in higher eukaryotes, play important roles in cell differentiation, development, and mammalian cell death; however, little is known of their function in plants. In this study, a RBR gene was isolated from the Chinese wild grape, Vitis pseudoreticulata W. T. Wang clone "Baihe-35-1", and designated as VpRBR . The cDNA sequence of VpRBR was 3,030 bp and contained an open reading frame of 3,024 bp. Conceptual translation of this gene indicated a composition of 1,007 amino acids with a predicted molecular mass of 117.3 kDa. The predicted protein showed a retinoblastoma-associated protein domain A from amino acid residues 416 to 579, and domain B from residues 726 to 855. The result of expression analysis indicated that VpRBR was expressed in tissues, leaves, stem, tendrils, flower, and grape skin at different expression levels. Further quantitative reverse transcription-PCR (qRT-PCR) data indicated that VpRBR levels were higher in Erysiphe necator-treated "Baihe-35-1" and "Baihe-13-1", two resistant clones of Chinese wild V. pseudoreticulata , than in E. necator-treated "Hunan-1", a susceptible clone of V. pseudoreticulata . Furthermore, the expression of VpRBR in response to salicylic acid (SA), methyl jasmonate (MeJA), and ethylene (Eth) in grape leaves was also investigated. Taken together, these data indicate that VpRBR may contribute to some aspect of powdery mildew resistance in grape.

Cloning and expression analysis of Zmglp1, a new germin-like protein gene in maize.

PubMed

Fan, Zhanmin; Gu, Hongya; Chen, Xiaowei; Song, Hui; Wang, Qian; Liu, Meihua; Qu, Li-Jia; Chen, Zhangliang

2005-06-17

The cDNA and genomic DNA of a green tissue-specific gene were cloned from maize (Zea mays L.) using cDNA-amplified fragment length polymorphism (cDNA-AFLP) and library screening. The deduced protein was highly similar to Hordeum vulgare germin-like protein 1 (HvGLP1), and the maize gene was therefore designated Zmglp1. Northern blot specifically detected the mRNA of Zmglp1 in young whorl leaves at the early-whorl stage. However, at the late-whorl, tassel, and silk stages, Zmglp1 transcripts were highly abundant in young whorl leaves; less abundant in mature leaves, young tassels, and cobs; and not detectable in roots, immature kernels, and stalks. RNA in situ hybridization revealed that Zmglp1 expressed only in mesophyllous, phloem, and guard cells in the young whorl leaves. Deletion analysis of the promoter in transgenic Arabidopsis resulted in the identification of several regions containing important regulatory cis-elements controlling the expression levels and circadian rhythm-oscillated patterns of Zmglp1.

Calmodulin Gene Family in Potato: Developmental and Touch-Induced Expression of the mRNA Encoding a Novel Isoform

NASA Technical Reports Server (NTRS)

Takezawa, D.; Liu, Z. H.; An, G.; Poovaiah, B. W.

1995-01-01

Eight genomic clones of potato calmodulin (PCM1 to 8) were isolated and characterized. Sequence comparisons of different genes revealed that the deduced amino acid sequence of PCM1 had several unique substitutions, especially in the fourth Ca(2+)-binding area. The expression patterns of different genes were studied by northern analysis using the 3'-untranslated regions as probes. The expression of PCM1, 5, and 8 was highest in the stolon tip and it decreased during tuber development. The expression of PCM6 did not vary much in the tissues tested, except in the leaves, where the expression was lower; whereas, the expression of PCM4 was very low in all the tissues. The expression of PCM2 and PCM3 was not detected in any of the tissues tested. Among these genes, only PCM1 showed increased expression following touch stimulation. To study the regulation of PCM1, transgenic potato plants carrying the PCM1 promoter fused to the beta-glucuronidase (GUS) reporter gene were produced. GUS expression was found to be developmentally regulated and touch-responsive, indicating a positive correlation between the expression of PCM1 and GUS mRNAs. These results suggest that the 5'-flanking region of PCM1 controls developmental and touch-induced expression. X-Gluc staining patterns revealed that GUS localization is high in meristematic tissues such as the stem apex, stolon tip, and vascular regions.

Cloning and characterization of a mouse gene with homology to the human von Hippel-Lindau disease tumor suppressor gene: implications for the potential organization of the human von Hippel-Lindau disease gene.

PubMed

Gao, J; Naglich, J G; Laidlaw, J; Whaley, J M; Seizinger, B R; Kley, N

1995-02-15

The human von Hippel-Lindau disease (VHL) gene has recently been identified and, based on the nucleotide sequence of a partial cDNA clone, has been predicted to encode a novel protein with as yet unknown functions [F. Latif et al., Science (Washington DC), 260: 1317-1320, 1993]. The length of the encoded protein and the characteristics of the cellular expressed protein are as yet unclear. Here we report the cloning and characterization of a mouse gene (mVHLh1) that is widely expressed in different mouse tissues and shares high homology with the human VHL gene. It predicts a protein 181 residues long (and/or 162 amino acids, considering a potential alternative start codon), which across a core region of approximately 140 residues displays a high degree of sequence identity (98%) to the predicted human VHL protein. High stringency DNA and RNA hybridization experiments and protein expression analyses indicate that this gene is the most highly VHL-related mouse gene, suggesting that it represents the mouse VHL gene homologue rather than a related gene sharing a conserved functional domain. These findings provide new insights into the potential organization of the VHL gene and nature of its encoded protein.

Molecular cloning and characterization of a cDNA encoding a novel apoplastic protein preferentially expressed in a shikonin-producing callus strain of Lithospermum erythrorhizon.

PubMed

Yamamura, Yoshimi; Sahin, F Pinar; Nagatsu, Akito; Mizukami, Hajime

2003-04-01

A cDNA (LEPS-2) encoding a novel cell wall protein was cloned from shikonin-producing callus tissues of Lithospermum erythrorhizon by differential display between a shikonin-producing culture strain and a non-producing strain. The LEPS-2 cDNA encoded a polypeptide of 184 amino acids. The deduced amino acid sequence exhibited no significant homology with known proteins. Expression of LEPS-2 gene as well as accumulation of LEPS-2 protein was highly correlated with shikonin production in L. erythrorhizon cells in culture. In the intact plant, expression of LEPS-2 was detected only in the roots where shikonin pigments accumulated. Cell fractionation experiments and immunocytochemical analysis showed that the protein was localized in the apoplast fraction of the cell walls. The shikonin pigments were also stored on the cell walls as oil droplets. These results indicate that expression of the LEPS-2 is closely linked with shikonin biosynthesis and the LEPS-2 protein may be involved in the intra-cell wall trapping of shikonin pigments.

Cloning of aged animals: a medical model for tissue and organ regeneration.

PubMed

Tian, X C; Kubota, C; Yang, X

2001-11-01

Cloning by nuclear transfer has great potential application in pharmaceutical protein production, xeno-transplantation, and perhaps most excitingly, therapeutic cloning. In therapeutic cloning a patient's own skin cells can be used to generate cloned embryos from which embryonic stem cells are isolated. Through targeted differentiation, embryonic stem cells can be directed to develop into the desired tissues/organs for replacement. The combination of homologous recombination of genes and nuclear transfer also offers the promise of correcting defective genes in humans. Demonstration of the successful cloning of aged animals is important for these future medical applications because degenerative diseases often afflict older adults. Our studies have demonstrated that skin fibroblast cells from aged adults, even after prolonged culture, provide nuclear donors equally as competent for cloning as cells from young adults or fetuses. These findings have paved the way for medically treating degenerative diseases of aged humans by tissue regeneration technologies made possible through cloning and homologous recombination.

Bone Marrow-derived Mesenchymal Stem Cells (MSCs) as a Selective Delivery Vehicle for a PSA-Activated Protoxin for Advanced Prostate Cancer

DTIC Science & Technology

2013-03-01

tissue have been optimized • Optimization of flow cytometry-based analyses to characterize co- incident expression of multiple MSC-related markers on a...CA). Mouse anti-human vimentin (clone LN-6) was purchased from Sigma-Aldrich (St. Louis, MO). Goat anti-mouse Alexa Fluor 488, Roswell Park

cDNA cloning, tissue distribution, and chromosomal localization of myelodysplasia/myeloid leukemia factor 2 (MLF2).

PubMed

Kuefer, M U; Look, A T; Williams, D C; Valentine, V; Naeve, C W; Behm, F G; Mullersman, J E; Yoneda-Kato, N; Montgomery, K; Kucherlapati, R; Morris, S W

1996-07-15

A fusion gene between nucleophosmin (NPM) and myelodysplasia/myeloid leukemia factor 1 (MLF1) is formed by a recurrent t(3;5)(q25.1;q34) in myelodysplastic syndrome and acute myeloid leukemia. Here we report the identification of a novel gene, MLF2, which contains an open reading frame of 744 bp encoding a 248-amino-acid protein highly related to the previously identified MLF1 protein (63% similarity, 40% identity). In contrast to the tissue-restricted expression pattern of MLF1, the MLF2 messenger RNA is expressed ubiquitously. The MLF2 gene locus was mapped by fluorescence in situ hybridization to human chromosome 12p13, a chromosomal region frequently involved in translocations and deletions in acute leukemias of lymphoid or myeloid lineage. In a physical map of chromosome 12, MLF2 was found to reside on the yeast artificial chromosome clone 765b9. Southern blotting analysis of malignant cell DNAs prepared from a series of acute lymphoblastic leukemia cases with translocations involving chromosome arm 12p, as well as a group of acute myeloid leukemias with various cytogenetic abnormalities, failed to reveal MLF2 gene rearrangements.

A Polymerase Chain Reaction-Based Method for Isolating Clones from a Complimentary DNA Library in Sheep

PubMed Central

Friis, Thor Einar; Stephenson, Sally; Xiao, Yin; Whitehead, Jon

2014-01-01

The sheep (Ovis aries) is favored by many musculoskeletal tissue engineering groups as a large animal model because of its docile temperament and ease of husbandry. The size and weight of sheep are comparable to humans, which allows for the use of implants and fixation devices used in human clinical practice. The construction of a complimentary DNA (cDNA) library can capture the expression of genes in both a tissue- and time-specific manner. cDNA libraries have been a consistent source of gene discovery ever since the technology became commonplace more than three decades ago. Here, we describe the construction of a cDNA library using cells derived from sheep bones based on the pBluescript cDNA kit. Thirty clones were picked at random and sequenced. This led to the identification of a novel gene, C12orf29, which our initial experiments indicate is involved in skeletal biology. We also describe a polymerase chain reaction-based cDNA clone isolation method that allows the isolation of genes of interest from a cDNA library pool. The techniques outlined here can be applied in-house by smaller tissue engineering groups to generate tools for biomolecular research for large preclinical animal studies and highlights the power of standard cDNA library protocols to uncover novel genes. PMID:24447069

Molecular cloning and developmental expression of the catalytic and 65-kDa regulatory subunits of protein phosphatase 2A in Drosophila.

PubMed Central

Mayer-Jaekel, R E; Baumgartner, S; Bilbe, G; Ohkura, H; Glover, D M; Hemmings, B A

1992-01-01

cDNA clones encoding the catalytic subunit and the 65-kDa regulatory subunit of protein phosphatase 2A (PR65) from Drosophila melanogaster have been isolated by homology screening with the corresponding human cDNAs. The Drosophila clones were used to analyze the spatial and temporal expression of the transcripts encoding these two proteins. The Drosophila PR65 cDNA clones contained an open reading frame of 1773 nucleotides encoding a protein of 65.5 kDa. The predicted amino acid sequence showed 75 and 71% identity to the human PR65 alpha and beta isoforms, respectively. As previously reported for the mammalian PR65 isoforms, Drosophila PR65 is composed of 15 imperfect repeating units of approximately 39 amino acids. The residues contributing to this repeat structure show also the highest sequence conservation between species, indicating a functional importance for these repeats. The gene encoding Drosophila PR65 was located at 29B1,2 on the second chromosome. A major transcript of 2.8 kilobase (kb) encoding the PR65 subunit and two transcripts of 1.6 and 2.5 kb encoding the catalytic subunit could be detected throughout Drosophila development. All of these mRNAs were most abundant during early embryogenesis and were expressed at lower levels in larvae and adult flies. In situ hybridization of different developmental stages showed a colocalization of the PR65 and catalytic subunit transcripts. The mRNA expression is high in the nurse cells and oocytes, consistent with a high equally distributed expression in early embryos. In later embryonal development, the expression remains high in the nervous system and the gonads but the overall transcript levels decrease. In third instar larvae, high levels of mRNA could be observed in brain, imaginal discs, and in salivary glands. These results indicate that protein phosphatase 2A transcript levels change during development in a tissue and in a time-specific manner. Images PMID:1320961

Molecular cloning, expression pattern, and 3D structural prediction of the cold inducible RNA-binding protein (CIRP) in Japanese flounder ( Paralichthys olivaceus)

NASA Astrophysics Data System (ADS)

Yang, Xiao; Gao, Jinning; Ma, Liman; Li, Zan; Wang, Wenji; Wang, Zhongkai; Yu, Haiyang; Qi, Jie; Wang, Xubo; Wang, Zhigang; Zhang, Quanqi

2015-02-01

Cold-inducible RNA-binding protein (CIRP) is a kind of RNA binding proteins that plays important roles in many physiological processes. The CIRP has been widely studied in mammals and amphibians since it was first cloned from mammals. On the contrary, there are little reports in teleosts. In this study, the Po CIRP gene of the Japanese flounder was cloned and sequenced. The genomic sequence consists of seven exons and six introns. The putative PoCIRP protein of flounder was 198 amino acid residues long containing the RNA recognition motif (RRM). Phylogenetic analysis showed that the flounder PoCIRP is highly conserved with other teleost CIRPs. The 5' flanking sequence was cloned by genome walking and many transcription factor binding sites were identified. There is a CpGs region located in promoter and exon I region and the methylation state is low. Quantitative real-time PCR analysis uncovered that Po CIRP gene was widely expressed in adult tissues with the highest expression level in the ovary. The mRNA of the Po CIRP was maternally deposited and the expression level of the gene was regulated up during the gastrula and neurula stages. In order to gain the information how the protein interacts with mRNA, we performed the modeling of the 3D structure of the flounder PoCIRP. The results showed a cleft existing the surface of the molecular. Taken together, the results indicate that the CIRP is a multifunctional molecular in teleosts and the findings about the structure provide valuable information for understanding the basis of this protein's function.

Production and Breeding of Transgenic Cloned Pigs Expressing Human CD73.

PubMed

Lee, Seung-Chan; Lee, Haesun; Oh, Keon Bong; Hwang, In-Sul; Yang, Hyeon; Park, Mi-Ryung; Ock, Sun-A; Woo, Jae-Seok; Im, Gi-Sun; Hwang, Seongsoo

2017-06-01

One of the reasons to causing blood coagulation in the tissue of xenografted organs was known to incompatibility of the blood coagulation and anti-coagulation regulatory system between TG pigs and primates. Thus, overexpression of human CD73 (hCD73) in the pig endothelial cells is considered as a method to reduce coagulopathy after pig-to-non-human-primate xenotransplantation. This study was performed to produce and breed transgenic pigs expressing hCD73 for the studies immune rejection responses and could provide a successful application of xenotransplantation. The transgenic cells were constructed an hCD73 expression vector under control porcine Icam2 promoter (pIcam2-hCD73) and established donor cell lines expressing hCD73. The numbers of transferred reconstructed embryos were 127 ± 18.9. The pregnancy and delivery rate of surrogates were 8/18 (44%) and 3/18 (16%). The total number of delivered cloned pigs were 10 (2 alive, 7 mummy, and 1 died after birth). Among them, three live hCD73-pigs were successfully delivered by Caesarean section, but one was dead after birth. The two hCD73 TG cloned pigs had normal reproductive ability. They mated with wild type (WT) MGH (Massachusetts General Hospital) female sows and produced totally 16 piglets. Among them, 5 piglets were identified as hCD73 TG pigs. In conclusion, we successfully generated the hCD73 transgenic cloned pigs and produced their litters by natural mating. It can be possible to use a mate for the production of multiple transgenic pigs such as α-1,3-galactosyltransferase knock-out /hCD46 for xenotransplantation.

Porcine MAP3K5 analysis: molecular cloning, characterization, tissue expression pattern, and copy number variations associated with residual feed intake.

PubMed

Pu, L; Zhang, L C; Zhang, J S; Song, X; Wang, L G; Liang, J; Zhang, Y B; Liu, X; Yan, H; Zhang, T; Yue, J W; Li, N; Wu, Q Q; Wang, L X

2016-08-12

Mitogen-activated protein kinase kinase kinase 5 (MAP3K5) is essential for apoptosis, proliferation, differentiation, and immune responses, and is a candidate marker for residual feed intake (RFI) in pig. We cloned the full-length cDNA sequence of porcine MAP3K5 by rapid-amplification of cDNA ends. The 5451-bp gene contains a 5'-untranslated region (UTR) (718 bp), a coding region (3738 bp), and a 3'-UTR (995 bp), and encodes a peptide of 1245 amino acids, which shares 97, 99, 97, 93, 91, and 84% sequence identity with cattle, sheep, human, mouse, chicken, and zebrafish MAP3K5, respectively. The deduced MAP3K5 protein sequence contains two conserved domains: a DUF4071 domain and a protein kinase domain. Phylogenetic analysis showed that porcine MAP3K5 forms a separate branch to vicugna and camel MAP3K5. Tissue expression analysis using real-time quantitative polymerase chain reaction (qRT-PCR) revealed that MAP3K5 was expressed in the heart, liver, spleen, lung, kidney, muscle, fat, pancrea, ileum, and stomach tissues. Copy number variation was detected for porcine MAP3K5 and validated by qRT-PCR. Furthermore, a significant increase in average copy number was detected in the low RFI group when compared to the high RFI group in a Duroc pig population. These results provide useful information regarding the influence of MAP3K5 on RFI in pigs.

Production of Transgenic Pigs with an Introduced Missense Mutation of the Bone Morphogenetic Protein Receptor Type IB Gene Related to Prolificacy.

PubMed

Zhao, Xueyan; Yang, Qiang; Zhao, Kewei; Jiang, Chao; Ren, Dongren; Xu, Pan; He, Xiaofang; Liao, Rongrong; Jiang, Kai; Ma, Junwu; Xiao, Shijun; Ren, Jun; Xing, Yuyun

2016-07-01

In the last few decades, transgenic animal technology has witnessed an increasingly wide application in animal breeding. Reproductive traits are economically important to the pig industry. It has been shown that the bone morphogenetic protein receptor type IB (BMPR1B) A746G polymorphism is responsible for the fertility in sheep. However, this causal mutation exits exclusively in sheep and goat. In this study, we attempted to create transgenic pigs by introducing this mutation with the aim to improve reproductive traits in pigs. We successfully constructed a vector containing porcine BMPR1B coding sequence (CDS) with the mutant G allele of A746G mutation. In total, we obtained 24 cloned male piglets using handmade cloning (HMC) technique, and 12 individuals survived till maturation. A set of polymerase chain reactions indicated that 11 of 12 matured boars were transgene-positive individuals, and that the transgenic vector was most likely disrupted during cloning. Of 11 positive pigs, one (No. 11) lost a part of the terminator region but had the intact promoter and the CDS regions. cDNA sequencing showed that the introduced allele (746G) was expressed in multiple tissues of transgene-positive offspring of No.11. Western blot analysis revealed that BMPR1B protein expression in multiple tissues of transgene-positive F1 piglets was 0.5 to 2-fold higher than that in the transgene-negative siblings. The No. 11 boar showed normal litter size performance as normal pigs from the same breed. Transgene-positive F1 boars produced by No. 11 had higher semen volume, sperm concentration and total sperm per ejaculate than the negative siblings, although the differences did not reached statistical significance. Transgene-positive F1 sows had similar litter size performance to the negative siblings, and more data are needed to adequately assess the litter size performance. In conclusion, we obtained 24 cloned transgenic pigs with the modified porcine BMPR1B CDS using HMC. cDNA sequencing and western blot indicated that the exogenous BMPR1B CDS was successfully expressed in host pigs. The transgenic pigs showed normal litter size performance. However, no significant differences in litter size were found between transgene-positive and negative sows. Our study provides new insight into producing cloned transgenic livestock related to reproductive traits.

Three Drought-Responsive Members of the Nonspecific Lipid-Transfer Protein Gene Family in Lycopersicon pennellii Show Different Developmental Patterns of Expression1

PubMed Central

Treviño, Marcela B.; Connell, Mary A. O'

1998-01-01

Genomic clones of two nonspecific lipid-transfer protein genes from a drought-tolerant wild species of tomato (Lycopersicon pennellii Corr.) were isolated using as a probe a drought- and abscisic acid (ABA)-induced cDNA clone (pLE16) from cultivated tomato (Lycopersicon esculentum Mill.). Both genes (LpLtp1 and LpLtp2) were sequenced and their corresponding mRNAs were characterized; they are both interrupted by a single intron at identical positions and predict basic proteins of 114 amino acid residues. Genomic Southern data indicated that these genes are members of a small gene family in Lycopersicon spp. The 3′-untranslated regions from LpLtp1 and LpLtp2, as well as a polymerase chain reaction-amplified 3′-untranslated region from pLE16 (cross-hybridizing to a third gene in L. pennellii, namely LpLtp3), were used as gene-specific probes to describe expression in L. pennellii through northern-blot analyses. All LpLtp genes were exclusively expressed in the aerial tissues of the plant and all were drought and ABA inducible. Each gene had a different pattern of expression in fruit, and LpLtp1 and LpLtp2, unlike LpLtp3, were both primarily developmentally regulated in leaf tissue. Putative ABA-responsive elements were found in the proximal promoter regions of LpLtp1 and LpLtp2. PMID:9536064

Molecular Cloning and Characterization of a New C-type Lysozyme Gene from Yak Mammary Tissue

PubMed Central

Jiang, Ming Feng; Hu, Ming Jun; Ren, Hong Hui; Wang, Li

2015-01-01

Milk lysozyme is the ubiquitous enzyme in milk of mammals. In this study, the cDNA sequence of a new chicken-type (c-type) milk lysozyme gene (YML), was cloned from yak mammary gland tissue. A 444 bp open reading frames, which encodes 148 amino acids (16.54 kDa) with a signal peptide of 18 amino acids, was sequenced. Further analysis indicated that the nucleic acid and amino acid sequences identities between yak and cow milk lysozyme were 89.04% and 80.41%, respectively. Recombinant yak milk lysozyme (rYML) was produced by Escherichia coli BL21 and Pichia pastoris X33. The highest lysozyme activity was detected for heterologous protein rYML5 (M = 1,864.24 U/mg, SD = 25.75) which was expressed in P. pastoris with expression vector pPICZαA and it clearly inhibited growth of Staphylococcus aureus. Result of the YML gene expression using quantitative polymerase chain reaction showed that the YML gene was up-regulated to maximum at 30 day postpartum, that is, comparatively high YML can be found in initial milk production. The phylogenetic tree indicated that the amino acid sequence was similar to cow kidney lysozyme, which implied that the YML may have diverged from a different ancestor gene such as cow mammary glands. In our study, we suggest that YML be a new c-type lysozyme expressed in yak mammary glands that plays a role as host immunity. PMID:26580446

Molecular characterization, expression profile of the FSHRgene and its association with egg production traits in muscovy duck.

PubMed

Xu, Jiguo; Gao, Xinfeng; Li, Xing; Ye, Qiao; Jebessa, Endashaw; Abdalla, Bahareldin Ali; Nie, Qinghua

2017-06-01

Follicle-stimulating hormone (FSH) and its receptor play a key role in the follicular development and regulation of steroidogenesis in the ovary and spermatogenesis in the testis. The purpose of this study was to characterize themuscovy duck FSHR gene, identify SNPs and their association with egg production traits in muscovy ducks. Here, we cloned the complementary DNA (cDNA) sequence of FSHR, and examined the expression patterns of FSHR gene in adult female muscovy duck tissues. The cloned cDNA of the muscovy duck FSHR gene shared high similarity to those of pekin duck (Anas platyrhynchos) (95.7%) and chicken (93.2%). Three different muscovy duck FSHR transcripts were identified. Quantitative real-time PCR (RT-qPCR) results showed that the FSHR gene was expressed in all the 14 tested tissues, and the highest expression level was seen in the ovary. A total of 16 SNPs were identified, among which, four SNPs were located in the coding region of FSHR. The SNP C320T is significantly associated with egg production at 59 weeks of age (P < 0.05), whereas the SNP A227G is significantly associated with age at first egg stage (P < 0.05). These results suggest that the two SNPs (A227G and C320T) of FSHR gene are associated with egg production traits and could be potential markers that can be used for marker-assisted selection programmes to increase egg production in muscovy duck.

Molecular cloning of a ripening-specific lipoxygenase and its expression during wild-type and mutant tomato fruit development.

PubMed Central

Kausch, K D; Handa, A K

1997-01-01

A 94-kD protein that accumulates predominately in tomato (Ly-copersicon esculentum) fruit during ripening was purified, and antibodies specific for the purified protein were used to isolate cDNA clones from a red-ripe fruit cDNA library. A sequence analysis of these cDNAs and cross-reactivity of the 94-kD-specific antibodies to the soybean lipoxygenase (LOX) L-1, L-2, and L-3 proteins and soybean LOX L-1-specific antibodies to the 94-kD protein identified it as a member of the LOX gene family. Maximum levels of the 94-kD LOX mRNA and protein are present in breaker to ripe and red-ripe stages, respectively. Expression of 94-kD LOX in different tissues from mature green and red-ripe tomato fruits was found to be greatest in the radial walls of ripe fruit, but immunocytolocalization using tissue printing suggests that the highest accumulation of its protein occurs in locular jelly. None of 94-kD LOX is expressed in nonripening mutant fruits of any age. Never-ripe mutant fruit accumulate the 94-kD LOX mRNA to levels similar to those obtained in wild-type fruit, but fail to accumulate the 94-kD LOX protein. Collectively, the results show that expression of 94-kD LOX is regulated by the ripening process, and ethylene may play a role in its protein accumulation. PMID:9112767

A New Protein Phosphatase 2C (FsPP2C1) Induced by Abscisic Acid Is Specifically Expressed in Dormant Beechnut Seeds1

PubMed Central

Lorenzo, Oscar; Rodríguez, Dolores; Nicolás, Gregorio; Rodríguez, Pedro L.; Nicolás, Carlos

2001-01-01

An abscisic acid (ABA)-induced cDNA fragment encoding a putative protein phosphatase 2C (PP2C) was obtained by means of differential reverse transcriptase-polymerase chain reaction approach. The full-length clone was isolated from a cDNA library constructed using mRNA from ABA-treated beechnut (Fagus sylvatica) seeds. This clone presents all the features of plant type PP2C and exhibits homology to members of this family such as AthPP2CA (61%), ABI1 (48%), or ABI2 (47%), therefore it was named FsPP2C1. The expression of FsPP2C1 is detected in dormant seeds and increases after ABA treatment, when seeds are maintained dormant, but it decreases and tends to disappear when dormancy is being released by stratification or under gibberellic acid treatment. Moreover, drought stress seems to have no effect on FsPP2C1 transcript accumulation. The FsPP2C1 transcript expression is tissue specific and was found to accumulate in ABA-treated seeds rather than in other ABA-treated vegetative tissues examined. These results suggest that the corresponding protein could be related to ABA-induced seed dormancy. By expressing FsPP2C1 in Escherichia coli as a histidine tag fusion protein, we have obtained direct biochemical evidence supporting Mg2+-dependent phosphatase activity of this protein. PMID:11299374

Comparative gene expression analysis of bovine nuclear-transferred embryos with different developmental potential by cDNA microarray and real-time PCR to determine genes that might reflect calf normality.

PubMed

Kato, Yoko; Li, Xiangping; Amarnath, Dasari; Ushizawa, Koichi; Hashizume, Kazuyoshi; Tokunaga, Tomoyuki; Taniguchi, Masanori; Tsunoda, Yukio

2007-01-01

Placental abnormalities are the main factor in the high incidence of somatic cell clone abnormalities. The expression of several trophoblast cell-specific molecules is enhanced during gestational days 7 to 14. To determine the possible genes whose expression patterns might reflect calf normality, we first compared the gene expression profiles on day 15 between in vitro-fertilized (IVF) embryos and two types of somatic cell nuclear-transferred embryos with either a high (FNT) or low (CNT) incidence of neonatal abnormalities using a cDNA microarray containing 16 of 21 placenta-specific genes developed from tissues collected across gestation. To identify significant genes from the screening of day 15 embryos, genes with a less than two-fold difference in expression between IVF and CNT embryos, and those with a greater than two-fold difference between IVF and FNT and between CNT and FNT were considered to contribute to clone abnormalities. These two comparisons revealed 18 down-regulated and 18 upregulated genes of the 1722 genes examined. We then examined the expression levels of 10 genes with known functions in eight-cell and blastocyst-stage embryos by real-time PCR. The mRNA expression pattern of interferon (IFN)-tau, a trophectoderm-related gene, differed between IVF, CNT, and FNT eight-cell embryos; few or none of the IVF or CNT eight-cell embryos expressed IFN-tau mRNA, but all eight-cell FNT embryos expressed IFN-tau. IFN-tau mRNA expression was significantly higher in IVF blastocysts, however, than in nuclear-transferred blastocysts. Average IFN-tau mRNA expression in FNT blastocysts was not different from that in CNT blastocysts, due to one CNT blastocyst with high expression. The precise relation between early expression of IFN-tau mRNA and inferior developmental potential in cloned embryos should be examined further.

Rooting of conifer propagules

Treesearch

R.L. Mott

1977-01-01

An outline of the general problems involved with the propagation of elite conifer clones by rooted cuttings is drawn from published reports. New approaches for resolving these problems can come from studies of clone production through tissue culture methods. Probable extension of tissue culture techniques will permit the establishment of clones from adult, proven trees...

Rat PPAR delta contains a CGG triplet repeat and is prominently expressed in the thalamic nuclei.

PubMed

Xing, G; Zhang, L; Zhang, L; Heynen, T; Yoshikawa, T; Smith, M; Weiss, S; Detera-Wadleigh, S

1995-12-26

We have isolated a new rat sequence containing motifs of a nuclear hormone receptor from a brain cDNA library. The deduced amino acid sequence encoded by the cDNA clone showed a strong homology to the human NUCI and the mouse peroxisome proliferator activated receptor delta (PPAR delta). We therefore refer to this new clone as rat PPAR delta (rPPAR delta). The new feature of rPPAR delta is a 14 CGG triplet repeat on the 5' untranslated region, not previously reported in either NUCI or mPPAR delta. We found that rPPAR delta was expressed as a 3.5-kb transcript which showed a wide distribution in adult rat tissues. Abundant expression was detected in brain, heart, skeletal muscle, kidney and lung. Weaker expression was noted in the liver, spleen and testis. To determine the specific brain localization of rPPAR delta we performed in situ hybridization analysis. Prominent expression was observed in the thalamus, particularly in the posterior part of the ventral medial nucleus, a site responsive to pain and cold stress. These results raise the possibility that PPAR delta might play a role in modulating response to thermal and pain sensations.

Apolipoprotein A-I in Labeo rohita: Cloning and functional characterisation reveal its broad spectrum antimicrobial property, and indicate significant role during ectoparasitic infection.

PubMed

Mohapatra, Amruta; Karan, Sweta; Kar, Banya; Garg, L C; Dixit, A; Sahoo, P K

2016-08-01

Apolipoprotein A-I (ApoA-I) is the most abundant and multifunctional high-density lipoprotein (HDL) having a major role in lipid transport and potent antimicrobial activity against a wide range of microbes. In this study, a complete CDS of 771 bp of Labeo rohita (rohu) ApoA-I (LrApoA-I) encoding a protein of 256 amino acids was amplified, cloned and sequenced. Tissue specific transcription analysis of LrApoA-I revealed its expression in a wide range of tissues, with a very high level of expression in liver and spleen. Ontogenic study of LrApoA-I expression showed presence of transcripts in milt and 3 h post-fertilization onwards in the larvae. The expression kinetics of LrApoA-I was studied upon infection with three different types of pathogens to elucidate its functional significance. Its expression was found to be up-regulated in the anterior kidney of L. rohita post-infection with Aeromonas hydrophila. Similarly following poly I:C (poly inosinic:cytidylic) stimulation, the transcript levels increased in both the anterior kidney and liver tissues. Significant up-regulation of LrApoA-I expression was observed in skin, mucous, liver and anterior kidney of the fish challenged with the ectoparasite Argulus siamensis. Immunomodulatory effect of recombinant LrApoA-I (rApoA-I) produced in Escherichia coli was demonstrated against A. hydrophila challenge in vivo. L. rohita administered with rApoA-I at a dose of 100 μg exhibited significantly higher protection (∼55%) upon challenge with A. hydrophila 12 h post-administration of the protein, in comparison to that observed in control group, along with higher level of expression of immune-related genes. The heightened expression of ApoA-I observed post-infection reflected its involvement in immune responses against a wide range of infections including bacterial, viral as well as parasitic pathogens. Our results also suggest the possibility of using rApoA-I as an immunostimulant, particularly rendering protection against A. hydrophila. Copyright © 2016 Elsevier Ltd. All rights reserved.

CD81 expression is important for the permissiveness of Huh7 cell clones for heterogeneous hepatitis C virus infection.

PubMed

Akazawa, Daisuke; Date, Tomoko; Morikawa, Kenichi; Murayama, Asako; Miyamoto, Michiko; Kaga, Minako; Barth, Heidi; Baumert, Thomas F; Dubuisson, Jean; Wakita, Takaji

2007-05-01

Huh7 cells constitute a permissive cell line for cell culture of hepatitis C virus (HCV) particles. However, our Huh7 line shows limited permissiveness for HCV. Thus, in this study we set out to determine which host factors are important for conferring permissiveness. To analyze the limited permissiveness of our Huh7 cells, 70 clones were obtained after single-cell cloning of parental Huh7 cells. The cloned Huh7 cells exhibited various levels of HCV pseudoparticles and JFH-1 virus infection efficiency, and some clones were not permissive. A subgenomic replicon was then transfected into the cloned Huh7 cells. While the replication efficiencies differed among the cloned Huh7 cells, these efficiencies did not correlate with infectious permissibility. Flow cytometry showed that CD81, scavenger receptor class B type I, and low-density-lipoprotein receptor expression on the cell surfaces of the Huh7 clones differed among the clones. Interestingly, we found that all of the permissive cell clones expressed CD81 while the nonpermissive cell clones did not. To confirm the importance of CD81 expression for HCV permissiveness, CD81 was then transiently and stably expressed on a nonpermissive Huh7 cell clone, which was consequently restored to HCV infection permissiveness. Furthermore, permissiveness was down-regulated upon transfection of CD81 silencing RNA into a CD81-positive cell clone. In conclusion, CD81 expression is an important determinant of HCV permissiveness of Huh7 cell clones harboring different characteristics.

Cloning and Expression Analysis of a UDP-Galactose/Glucose Pyrophosphorylase from Melon Fruit Provides Evidence for the Major Metabolic Pathway of Galactose Metabolism in Raffinose Oligosaccharide Metabolizing Plants1

PubMed Central

Dai, Nir; Petreikov, Marina; Portnoy, Vitaly; Katzir, Nurit; Pharr, David M.; Schaffer, Arthur A.

2006-01-01

The Cucurbitaceae translocate a significant portion of their photosynthate as raffinose and stachyose, which are galactosyl derivatives of sucrose. These are initially hydrolyzed by α-galactosidase to yield free galactose (Gal) and, accordingly, Gal metabolism is an important pathway in Cucurbitaceae sink tissue. We report here on a novel plant-specific enzyme responsible for the nucleotide activation of phosphorylated Gal and the subsequent entry of Gal into sink metabolism. The enzyme was antibody purified, sequenced, and the gene cloned and functionally expressed in Escherichia coli. The heterologous protein showed the characteristics of a dual substrate UDP-hexose pyrophosphorylase (PPase) with activity toward both Gal-1-P and glucose (Glc)-1-P in the uridinylation direction and their respective UDP-sugars in the reverse direction. The two other enzymes involved in Glc-P and Gal-P uridinylation are UDP-Glc PPase and uridyltransferase, and these were also cloned, heterologously expressed, and characterized. The gene expression and enzyme activities of all three enzymes in melon (Cucumis melo) fruit were measured. The UDP-Glc PPase was expressed in melon fruit to a similar extent as the novel enzyme, but the expressed protein was specific for Glc-1-P in the UDP-Glc synthesis direction and did not catalyze the nucleotide activation of Gal-1-P. The uridyltransferase gene was only weakly expressed in melon fruit, and activity was not observed in crude extracts. The results indicate that this novel enzyme carries out both the synthesis of UDP-Gal from Gal-1-P as well as the subsequent synthesis of Glc-1-P from the epimerase product, UDP-Glc, and thus plays a key role in melon fruit sink metabolism. PMID:16829585

Down-regulation of Pax6 is associated with abnormal differentiation of corneal epithelial cells in severe ocular surface diseases

PubMed Central

Li, W; Chen, Y-T; Hayashida, Y; Blanco, G; Kheirkah, A; He, H; Chen, S-Y; Liu, C-Y; Tseng, SCG

2010-01-01

Pax6 is the universal master control gene for eye morphogenesis. Other than retina and lens, Pax6 also expressed in the ocular surface epithelium from early gestation until the postnatal stage, in which little is known about the function of Pax6. In this study, corneal pannus tissues from patients with ocular surface diseases such as Stevens–Johnson syndrome (SJS), chemical burn, aniridia and recurrent pterygium were investigated. Our results showed that normal ocular surface epithelial cells expressed Pax6. However, corneal pannus epithelial cells from the above patients showed a decline or absence of Pax6 expression, accompanied by a decline or absence of K12 keratin but an increase of K10 keratin and filaggrin expression. Pannus basal epithelial cells maintained nuclear p63 expression and showed activated proliferation, evidenced by positive Ki67 and K16 keratin staining. On 3T3 fibroblast feeder layers, Pax6 immunostaining was negative in clones generated from epithelial cells harvested from corneal pannus from SJS or aniridia, but positive in those from the normal limbal epithelium; whereas western blots showed that some epithelial clones expanded from pannus retained Pax6 expression. Transient transfection of an adenoviral vector carrying EGFP–Pax6 transgenes into these Pax6− clones increased both Pax6 and K12 keratin expression. These results indicate that Pax6 helps to maintain the normal corneal epithelial phenotype postnatally, and that down-regulation of Pax6 is associated with abnormal epidermal differentiation in severe ocular surface diseases. Reintroduction of activation of the Pax6 gene might be useful in treating squamous metaplasia of the ocular surface epithelium. PMID:18027901

Cloning and characterization of Sdga gene encoding alpha-subunit of heterotrimeric guanosine 5'-triphosphate-binding protein complex in Scoparia dulcis.

PubMed

Shite, Masato; Yamamura, Yoshimi; Hayashi, Toshimitsu; Kurosaki, Fumiya

2008-11-01

A homology-based cloning strategy yielded Sdga, a cDNA clone presumably encoding alpha-subunit of heterotrimeric guanosine 5'-triphosphate-binding protein complex, from leaf tissues of Scoparia dulcis. Phylogenetic tree analysis of G-protein alpha-subunits from various biological sources suggested that, unlike in animal cells, classification of Galpha-proteins into specific subfamilies could not be applicable to the proteins from higher plants. Restriction digests of genomic DNA of S. dulcis showed a single hybridized signal in Southern blot analysis, suggesting that Sdga is a sole gene encoding Galpha-subunit in this plant. The expression level of Sdga appeared to be maintained at almost constant level after exposure of the leaves to methyl jasmonate as analyzed by reverse-transcription polymerase chain reaction. These results suggest that Sdga plays roles in methyl jasmonate-induced responses of S. dulcis without a notable change in the transcriptional level.

Comprehensive T-cell immunophenotyping and next-generation sequencing of human papillomavirus (HPV)-positive and HPV-negative head and neck squamous cell carcinomas.

PubMed

Poropatich, Kate; Fontanarosa, Joel; Swaminathan, Suchitra; Dittmann, Dave; Chen, Siqi; Samant, Sandeep; Zhang, Bin

2017-11-01

The success of programmed cell death 1 (PD-1) inhibition in achieving a clinical response in a subset of head and neck squamous cell carcinoma (HNSCC) patients emphasizes the need to better understand the immunobiology of HNSCC. Immunophenotyping was performed for 30 HCSCC patients [16 human papillomavirus (HPV)-positive; 14 HPV-negative] on matched tissue from the primary tumour site, locally metastatic cervical lymph nodes (LNs), uninvolved local cervical LNs, and peripheral blood. CD4 + and CD8 + T-cell lymphocytes obtained from tissue were analysed for expression levels of the inhibitory receptors PD-1, TIM-3 and CTLA-4. Next-generation sequencing of the T-cell receptor (TCR) β chain was performed on patients (n = 9) to determine receptor repertoire diversity and for clonality analysis. HPV-negative HNSCC patients, particularly those with stage IV disease, had significantly higher proportions of CD8 + T cells expressing CTLA-4 in tumour tissue (P = 0.0013) and in peripheral blood (P = 0.0344) than HPV-positive patients, as well as higher expression levels of TIM-3 + PD-1 + CD8 + T cells (P = 0.0072) than controls. For all patients, PD-1 expression on CD8 + T cells - particularly in HPV-negative HNSCC cases - strongly correlated (r = 0.63, P = 0.013) with tumour size at the primary site. The top CD8 + TCR clones from tumour tissue significantly overlapped with circulating peripheral blood TCR clones (r = 0.946), and HPV-positive patients had frequently expanded TCR clones that were more hydrophobic - and potentially more immunogenic - than those from HPV-negative patients. Collectively, our findings demonstrate, for the first time, that high-stage HPV-negative HNSCC patients with primary tumours at different sites in the head and neck have elevated peripheral CTLA-4 + CD8 + T-cell levels, that tumour-familiar CD8 + T cells are detectable in peripheral blood from HNSCC patients, and that TCRs from HPV-positive HNSCC patients potentially recognize distinctly immunogenic cognate antigens. However, our findings are preliminary, and need to be further confirmed in a larger patient cohort; also, how these factors affect patient response to immunotherapy needs to be determined. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Cloning and Functional Analysis of Phosphoethanolamine Methyltransferase Promoter from Maize (Zea mays L.)

PubMed Central

Niu, Gai-Li; Gou, Wei; Han, Xiang-Long; Qin, Cheng; Zhang, Li-Xin; Ashraf, Muhammad

2018-01-01

Betaine, a non-toxic osmoprotectant, is believed to accumulate considerably in plants under stress conditions to maintain the osmotic pressure and promote a variety of processes involved in growth and development. Phosphoethanolamine N-methyltransferase (PEAMT), a key enzyme for betaine synthesis, is reported to be regulated by its upstream promoter. In the present investigation, by using the transgenic approach, a 1048 bp long promoter region of ZmPEAMT gene from Zea mays was cloned and functionally characterized in tobacco. Computational analysis affirmed the existence of abiotic stress responsive cis-elements like ABRE, MYC, HST, LST etc., as well as pathogen, wound and phytohormone responsive motifs. For transformation in tobacco, four 5′-deletion constructs of 826 bp (P2), 642 bp (P3), 428 bp (P4) and 245 bp (P5) were constructed from the 1048 bp (P1) promoter fragment. The transgenic plants generated through a single event exhibited a promising expression of GUS reporter protein in the leaf tissues of treated with salt, drought, oxidative and cold stress as well as control plants. The GUS expression level progressively reduced from P1 to P5 in the leaf tissues, whereas a maximal expression was observed with the P3 construct in the leaves of control plants. The expression of GUS was noted to be higher in the leaves of osmotically- or salt-treated transgenic plants than that in the untreated (control) plants. An effective expression of GUS in the transgenic plants manifests that this promoter can be employed for both stress-inducible and constitutive expression of gene(s). Due to this characteristic, this potential promoter can be effectively used for genetic engineering of several crops. PMID:29316727

Cloning and primary immunological study of TGF-β1 and its receptors TβR I /TβR II in tilapia(Oreochromis niloticus).

PubMed

Zhan, Xu-liang; Ma, Tai-yang; Wu, Jin-ying; Yi, Li-yuan; Wang, Jing-yuan; Gao, Xiao-ke; Li, Wen-sheng

2015-07-01

The transforming growth factor β (TGF-β) superfamily plays critical roles in tumor suppression, cell proliferation and differentiation, tissue morphogenesis, lineage determination, cell migration and apoptosis. Recently, TGF-β1, one important member of TGF-β superfamily, is suggested as an immune regulator in the teleost. In this study, we cloned the cDNAs of TGF-β1 and its receptors, TβR I and TβR II (including three isoforms) from tilapia (Genbank accession numbers: KP754231- KP754235). A tissue distribution profile analysis indicated that TGF-β1 was highly expressed in the head kidney, gill, spleen, kidney and PBLs (peripheral blood leukocytes); TβR I only showed considerable expression in the liver; and TβR II-2 was highly expressed in the kidney, gill, liver, head kidney and heart. We determined that the mRNA expressions of TGF-β and TβR I /TβR II-2 were significantly increased in tilapia head kidney and spleen leukocytes by the stimulation of Lipopolysaccharide (LPS) or Poly I: C. We also examined their expressions in the spleen and head kidney of tilapia after IP injection of streptococcus agalactiae. The results showed that the mRNA expressions of these three genes all increased in the head kidney as early as 6 h post infection, and in the spleen 3 d post infection. In addition, the protein level of TGF-β1 was also up-regulated in the head kidney and the spleen after infection. Taken together, our data indicate that the TGF-β1-TβR I /TβR II-2 system functions potentially in tilapia immune system. Copyright © 2015 Elsevier Ltd. All rights reserved.

Molecular cloning and characterization of amh, dax1 and cyp19a1a genes and their response to 17α-methyltestosterone in Pengze crucian carp.

PubMed

Li, Meng; Wang, Lihong; Wang, Houpeng; Liang, Hongwei; Zheng, Yao; Qin, Fang; Liu, Shaozhen; Zhang, Yingying; Wang, Zaizhao

2013-05-01

The proteins encoded by amh, dax1 and cyp19a1a play important roles in gonad differentiation. Their functions have been far less studied in teleosts. In this study, the full-length cDNAs of amh, dax1 and cyp19a1a were cloned and characterized in a triploid gynogenic fish, the Pengze crucian carp. Their expression profilings in juvenile development, adult tissues and juveniles exposed to 100 ng/L 17α-methyltestosterone (MT) were investigated. Results showed that their putative proteins shared high identities to their counterparts in cyprinid fish species, respectively. The tissue distribution results indicated that amh and cyp19a1a were predominantly expressed in the ovary and dax1 was dominantly expressed in the liver. Gene profiling in the developmental stages showed that all the three target genes had a consistent highest expression at 48 days post hatching (dph). The period of 48 dph appeared to be a key time during the process of the gonad development of Pengze crucian carp. 100 ng/L MT significantly increased the mRNA expression of amh at 2- and 4-week exposures and enhanced dax1 and cyp19a1a at 6-week exposure. The present study indicated that MT could influence the gonad development in Pengze crucian carp by disturbing sex-differentiation associated gene expression. Furthermore, the present study will be of great significance to broaden the understanding of molecular mechanisms of the physiological processes of reproduction in fish. Copyright © 2013 Elsevier Inc. All rights reserved.

Zinc and copper tolerance of Agrostis stolonifera L. in tissue culture

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wu, L.; Antonovics, J.

1978-03-01

Callus tissue was induced from shoot meristematic tissue and root tips of a clone of the grass Agrostis stolonifera tolerant to both zinc and copper, and from a control clone tolerant to neither metal. Growth of the callus tissue on media containing zinc and copper showed that tolerance to both metals was maintained in tissue culture. The pattern of metal uptake in tissue culture resembled uptake by whole plants in that tolerant tissue took up more metal than nontolerant tissue. Plants regenerated from callus had the same copper and zinc tolerance as the original parental clones regardless of time ofmore » growth in tissue culture and shoot or root origin of the tissue. The results support previous evidence that metal tolerance is genetically determined and acts at the cellular level.« less

Towards a transcription map spanning a 250 kb area within the DiGeorge syndrome chromosome region

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wong, W.; Emanuel, B.S.; Siegert, J.

1994-09-01

DiGeorge syndrome (DGS) and velocardiofacial syndrome (VCFS) are congenital anomalies affecting predominantly the thymus, parathyroid glands, heart and craniofacial development. Detection of 22q11.2 deletions in the majority of DGS and VCFS patients implicate 22q11 haploinsufficiency in the etiology of these disorders. The VCFS/DGS critical region lies within the proximal portion of a commonly deleted 1.2 Mb region in 22q11. A 250 kb cosmid contig covering this critical region and containing D22S74 (N25) has been established. From this contig, eleven cosmids with minimal overlap were biotinylated by nick translation, and hybridized to PCR-amplified cDNAs prepared from different tissues. The use ofmore » cDNAs from a variety of tissues increases the likelihood of identifying low abundance transcripts and tissue-specific expressed sequences. A DGCR-specific cDNA sublibrary consisting of 670 cDNA clones has been constructed. To date, 49 cDNA clones from this sub-library have been identified with single copy probes and cosmids containing putative CpG islands. Based on sequence analysis, 25 of the clones contain regions of homology to several cDNAs which map within the proximal contig. LAN is a novel partial cDNA isolated from a fetal brain library probed with one of the cosmids in the proximal contig. Using LAN as a probe, we have found 19 positive clones in the DGCR-specific cDNA sub-library (4 clones from fetal brain, 14 from adult skeletal muscle and one from fetal liver). Some of the LAN-positive clones extend the partial cDNA in the 5{prime} direction and will be useful in assembling a full length transcript. This resource will be used to develop a complete transcriptional map of the critical region in order to identify candidate gene(s) involved in the etiology of DGS/VCFS and to determine the relationship between the transcriptional and physical maps of 22q11.« less

Microaspiration of esophageal gland cells and cDNA library construction for identifying parasitism genes of plant-parasitic nematodes.

PubMed

Hussey, Richard S; Huang, Guozhong; Allen, Rex

2011-01-01

Identifying parasitism genes encoding proteins secreted from a plant-parasitic nematode's esophageal gland cells and injected through its stylet into plant tissue is the key to understanding the molecular basis of nematode parasitism of plants. Parasitism genes have been cloned by directly microaspirating the cytoplasm from the esophageal gland cells of different parasitic stages of cyst or root-knot nematodes to provide mRNA to create a gland cell-specific cDNA library by long-distance reverse-transcriptase polymerase chain reaction. cDNA clones are sequenced and deduced protein sequences with a signal peptide for secretion are identified for high-throughput in situ hybridization to confirm gland-specific expression.

Variability in Immunohistochemical Detection of Programmed Death Ligand 1 (PD-L1) in Cancer Tissue Types

PubMed Central

Scognamiglio, Giosuè; De Chiara, Anna; Di Bonito, Maurizio; Tatangelo, Fabiana; Losito, Nunzia Simona; Anniciello, Annamaria; De Cecio, Rossella; D’Alterio, Crescenzo; Scala, Stefania; Cantile, Monica; Botti, Gerardo

2016-01-01

In normal cell physiology, programmed death 1 (PD-1) and its ligand, PD-L1, play an immunoregulatory role in T-cell activation, tolerance, and immune-mediated tissue damage. The PD-1/PD-L1 pathway also plays a critical role in immune escape of tumor cells and has been demonstrated to correlate with a poor prognosis of patients with several types of cancer. However, recent reports have revealed that the immunohistochemical (IHC) expression of the PD-L1 in tumor cells is not uniform for the use of different antibodies clones, with variable specificity, often doubtful topographical localization, and with a score not uniquely defined. The purpose of this study was to analyze the IHC expression of PD-L1 on a large series of several human tumors to correctly define its staining in different tumor tissues. PMID:27213372

Identification of molecular performance from oil palm clones based on SSR markers

NASA Astrophysics Data System (ADS)

Putri, Lollie Agustina P.; Basyuni, M.; Bayu, Eva S.; Arvita, D.; Arifiyanto, D.; Syahputra, I.

2018-03-01

In Indonesia, the oil palms are an important economic crop, producing food and raw materials for the food, confectionary, cosmetics and oleo-chemical industrial demands of oil palm products. Clonal oil palm offers the potential for greater productivity because it is possible to establish uniform tree stands comprising identical copies (clones) of a limited number of highly productive oil palms. Unfortunately, tissue culture sometimes accentuates the expression of detects in oil palm, particularly when embryogenesis is induced in particullar callus for prolonged periods. This research is conducted by taking individual tree sample of clone germplasm two years old. The purpose of this research is to molecular performance analysis of some oil palm clones based on SSR markers. A total of 30 trees oil palm clones were used for analysis. In this experiment, the DNA profile diversity was assessed using five loci of oil palm’s specific SSR markers. The results of the experiment indicated out of 3 SSR markers (FR-0779, FR-3663 and FR-0782) showed monomorphic of PCR product and 2 SSR markers (FR-0783 and FR- 3745) showed polymorphic of PCR product. There are 10 total number of PCR product. These preliminary results demonstrated SSR marker can be used to evaluate genetic relatedness among trees of oil palm clones.

Cloning

MedlinePlus

Cloning describes the processes used to create an exact genetic replica of another cell, tissue or organism. ... named Dolly. There are three different types of cloning: Gene cloning, which creates copies of genes or ...

Molecular cloning, expression analysis and functional characterization of interleukin-22 in So-iny mullet, Liza haematocheila.

PubMed

Qi, Zhitao; Zhang, Qihuan; Wang, Zisheng; Zhao, Weihong; Chen, Shannan; Gao, Qian

2015-02-01

In the present study, interleukin-22 (IL-22) from So-iny mullet (Liza haematocheila) was identified, and its tissue expression in both healthy and Streptococcus dysgalactiae-infected fish was examined. The full length cDNA sequence of mullet IL-22 was 1070bp, containing an open reading frame of 555bp. The deduced amino acid sequence shared high similarity (45.1-67.9%) with IL-22 from other fish species. Mullet IL-22 also contained an IL-10 family signature and four cysteine residues that were well conserved in other vertebrate IL-22 molecules. Mullet IL-22 mRNA was highly expressed in kidney, moderately expressed in liver and gut, and relatively weakly expressed in spleen, and its expression was significantly up-regulated in all the examined tissues following S. dysgalactiae infection. Furthermore, recombinant mullet IL-22 protein was shown to promote the expression of β-defensin in the four tissues and to increase the survival rate of the fish infected with S. dysgalactiae. Our results suggest mullet IL-22 plays an important role in the immune defense against bacterial infection and has the potential to be used to treat bacterial diseases in fish. Copyright © 2014 Elsevier Ltd. All rights reserved.

Vectors for co-expression of an unrestricted number of proteins

PubMed Central

Scheich, Christoph; Kümmel, Daniel; Soumailakakis, Dimitri; Heinemann, Udo; Büssow, Konrad

2007-01-01

A vector system is presented that allows generation of E. coli co-expression clones by a standardized, robust cloning procedure. The number of co-expressed proteins is not limited. Five ‘pQLink’ vectors for expression of His-tag and GST-tag fusion proteins as well as untagged proteins and for cloning by restriction enzymes or Gateway cloning were generated. The vectors allow proteins to be expressed individually; to achieve co-expression, two pQLink plasmids are combined by ligation-independent cloning. pQLink co-expression plasmids can accept an unrestricted number of genes. As an example, the co-expression of a heterotetrameric human transport protein particle (TRAPP) complex from a single plasmid, its isolation and analysis of its stoichiometry are shown. pQLink clones can be used directly for pull-down experiments if the proteins are expressed with different tags. We demonstrate pull-down experiments of human valosin-containing protein (VCP) with fragments of the autocrine motility factor receptor (AMFR). The cloning method avoids PCR or gel isolation of restriction fragments, and a single resistance marker and origin of replication are used, allowing over-expression of rare tRNAs from a second plasmid. It is expected that applications are not restricted to bacteria, but could include co-expression in other hosts such as Bacluovirus/insect cells. PMID:17311810

Two GH3 genes from longan are differentially regulated during fruit growth and development.

PubMed

Kuang, Jian-Fei; Zhang, Yu; Chen, Jian-ye; Chen, Qiu-Jin; Jiang, Yue-Ming; Lin, He-Tong; Xu, Shi-Juan; Lu, Wang-Jin

2011-10-01

In the present work, two full length cDNAs of GH3 genes, named DlGH3.1 and DlGH3.2 were cloned from pericarp and aril tissues of the longan fruit, respectively. Three conserved motifs, SSGTSAGERK, YASSE and YRVGD, as a characteristic of the acyladenylate/thioester forming enzyme superfamily were observed in DlGH3.1 and DlGH3.2 proteins. DlGH3.1 mainly expressed in pericarp tissues while DlGH3.2 accumulated in both the pericarp and aril tissues during fruit growth and development. In addition, NAA treatment induced the expression of DlGH3.1 and DlGH3.2 in the pericarp tissues at 21 and 77days after anthesis (DAA), while only DlGH3.2 in the aril tissues could be induced by NAA at 77DAA. More importantly, ABA and ethrel treatments suppressed the accumulations of DlGH3.1 and DlGH3.2 in the pericarp tissues of longan fruit at 21DAA (a rapid growth stage of pericarp), but enhanced DlGH3.2 expression in the aril tissues at 77DAA (a fruit ripening stage). Furthermore, the expression patterns of DlGH3.1 and DlGH3.2 showed different tissue specificity. Thus, our results suggest that DlGH3.1 gene expression might be associated with pericarp growth, while DlGH3.2 accumulation is likely to be related to both pericarp growth and fruit ripening, and the responses of DlGH3s to plant growth hormones are different and dependent on fruit development stage and fruit tissue. Copyright © 2011 Elsevier B.V. All rights reserved.

Giant Panda (Ailuropoda melanoleuca) Buccal Mucosa Tissue as a Source of Multipotent Progenitor Cells

PubMed Central

Prescott, Hilary M. A.; Manning, Craig; Gardner, Aaron; Ritchie, William A.; Pizzi, Romain; Girling, Simon; Valentine, Iain; Wang, Chengdong; Jahoda, Colin A. B.

2015-01-01

Since the first mammal was cloned, the idea of using this technique to help endangered species has aroused considerable interest. However, several issues limit this possibility, including the relatively low success rate at every stage of the cloning process, and the dearth of usable tissues from these rare animals. iPS cells have been produced from cells from a number of rare mammalian species and this is the method of choice for strategies to improve cloning efficiency and create new gametes by directed differentiation. Nevertheless information about other stem cell/progenitor capabilities of cells from endangered species could prove important for future conservation approaches and adds to the knowledge base about cellular material that can be extremely limited. Multipotent progenitor cells, termed skin-derived precursor (SKP) cells, can be isolated directly from mammalian skin dermis, and human cheek tissue has also been shown to be a good source of SKP-like cells. Recently we showed that structures identical to SKPs termed m-SKPs could be obtained from monolayer/ two dimensional (2D) skin fibroblast cultures. Here we aimed to isolate m-SKPs from cultured cells of three endangered species; giant panda (Ailuropoda melanoleuca); red panda (Ailurus fulgens); and Asiatic lion (Panthera leo persica). m-SKP-like spheres were formed from the giant panda buccal mucosa fibroblasts; whereas dermal fibroblast (DF) cells cultured from abdominal skin of the other two species were unable to generate spheres. Under specific differentiation culture conditions giant panda spheres expressed neural, Schwann, adipogenic and osteogenic cell markers. Furthermore, these buccal mucosa derived spheres were shown to maintain expression of SKP markers: nestin, versican, fibronectin, and P75 and switch on expression of the stem cell marker ABCG2. These results demonstrate that giant panda cheek skin can be a useful source of m-SKP multipotent progenitors. At present lack of sample numbers means that we can only postulate why we were unable to obtain m-SKPs from the lion and red panda cultures. However the giant panda observations point to the value of archiving cells from rare species, and the possibilities for later progenitor cell derivation. PMID:26398672

Giant Panda (Ailuropoda melanoleuca) Buccal Mucosa Tissue as a Source of Multipotent Progenitor Cells.

PubMed

Prescott, Hilary M A; Manning, Craig; Gardner, Aaron; Ritchie, William A; Pizzi, Romain; Girling, Simon; Valentine, Iain; Wang, Chengdong; Jahoda, Colin A B

2015-01-01

Since the first mammal was cloned, the idea of using this technique to help endangered species has aroused considerable interest. However, several issues limit this possibility, including the relatively low success rate at every stage of the cloning process, and the dearth of usable tissues from these rare animals. iPS cells have been produced from cells from a number of rare mammalian species and this is the method of choice for strategies to improve cloning efficiency and create new gametes by directed differentiation. Nevertheless information about other stem cell/progenitor capabilities of cells from endangered species could prove important for future conservation approaches and adds to the knowledge base about cellular material that can be extremely limited. Multipotent progenitor cells, termed skin-derived precursor (SKP) cells, can be isolated directly from mammalian skin dermis, and human cheek tissue has also been shown to be a good source of SKP-like cells. Recently we showed that structures identical to SKPs termed m-SKPs could be obtained from monolayer/ two dimensional (2D) skin fibroblast cultures. Here we aimed to isolate m-SKPs from cultured cells of three endangered species; giant panda (Ailuropoda melanoleuca); red panda (Ailurus fulgens); and Asiatic lion (Panthera leo persica). m-SKP-like spheres were formed from the giant panda buccal mucosa fibroblasts; whereas dermal fibroblast (DF) cells cultured from abdominal skin of the other two species were unable to generate spheres. Under specific differentiation culture conditions giant panda spheres expressed neural, Schwann, adipogenic and osteogenic cell markers. Furthermore, these buccal mucosa derived spheres were shown to maintain expression of SKP markers: nestin, versican, fibronectin, and P75 and switch on expression of the stem cell marker ABCG2. These results demonstrate that giant panda cheek skin can be a useful source of m-SKP multipotent progenitors. At present lack of sample numbers means that we can only postulate why we were unable to obtain m-SKPs from the lion and red panda cultures. However the giant panda observations point to the value of archiving cells from rare species, and the possibilities for later progenitor cell derivation.

Two members of the mouse mdr gene family confer multidrug resistance with overlapping but distinct drug specificities.

PubMed Central

Devault, A; Gros, P

1990-01-01

We report the cloning and functional analysis of a complete clone for the third member of the mouse mdr gene family, mdr3. Nucleotide and predicted amino acid sequence analyses showed that the three mouse mdr genes encode highly homologous membrane glycoproteins, which share the same length (1,276 residues), the same predicted functional domains, and overall structural arrangement. Regions of divergence among the three proteins are concentrated in discrete segments of the predicted polypeptides. Sequence comparison indicated that the three mouse mdr genes were created from a common ancestor by two independent gene duplication events, the most recent one producing mdr1 and mdr3. When transfected and overexpressed in otherwise drug-sensitive cells, the mdr3 gene, like mdr1 and unlike mdr2, conferred multidrug resistance to these cells. In independently derived transfected cell clones expressing similar amounts of either MDR1 or MDR3 protein, the drug resistance profile conferred by mdr3 was distinct from that conferred by mdr1. Cells transfected with and expressing MDR1 showed a marked 7- to 10-fold preferential resistance to colchicine and Adriamycin compared with cells expressing equivalent amounts of MDR3. Conversely, cells transfected with and expressing MDR3 showed a two- to threefold preferential resistance to actinomycin D over their cellular counterpart expressing MDR1. These results suggest that MDR1 and MDR3 are membrane-associated efflux pumps which, in multidrug-resistant cells and perhaps normal tissues, have overlapping but distinct substrate specificities. Images PMID:1969610

Should we clone human beings? Cloning as a source of tissue for transplantation.

PubMed Central

Savulescu, J

1999-01-01

The most publicly justifiable application of human cloning, if there is one at all, is to provide self-compatible cells or tissues for medical use, especially transplantation. Some have argued that this raises no new ethical issues above those raised by any form of embryo experimentation. I argue that this research is less morally problematic than other embryo research. Indeed, it is not merely morally permissible but morally required that we employ cloning to produce embryos or fetuses for the sake of providing cells, tissues or even organs for therapy, followed by abortion of the embryo or fetus. PMID:10226910

Cytokine profiling identifies an interaction of IL-6 and IL-1α to drive PSMA-PSA prostate clones.

PubMed

Jemaa, Awatef Ben; Bouraoui, Yosra; Rais, Nawfel Ben; Nouira, Yassine; Oueslati, Ridha

2016-12-01

Several PSMA-PSA prostate clones have been identified during prostate cancer progression; however, until now, their in situ inflammatory characteristics have remained unclear. We therefore investigated the interplay between proinflammatory cytokines and (PSMA,PSA) sub-groups. 27 benign prostate hyperplasia (BPH) and 18 prostate cancers (PC) were enrolled in this study. Immunohistochemical analysis was performed. Serum levels of PSA were assayed by Immulite autoanalyser. In BPH and PC patients with elevated serum PSA levels, IL-1α was the most proinflammatory cytokine expressed in (PSMA+,PSA-) subgroup. However, most samples of (PSMA+,PSA+) subgroup had positive immunoreaction to IL-6. In samples of PC with PSA serum levels of 4-20ng/mL or >20ng/mL, immunoreaction to TNF-α was seen only in (PSMA+,PSA+) subgroup. Interestingly, several combinations of proinflammatory cytokines (IL-6, IL-1α and TNF-α) showed that coexpression of tissue PSMA and PSA was concomitant with high immunoreactions to (IL-6+,TNF-α-), (IL-6+,IL-1α+) and (IL-1α+,TNFα-) in BPH and PC patients. (PSMA,PSA) subgroup lacking tissue PSA expression showed a high immunoexpression of the profile (IL-6+,TNF-α-). The combinations of (IL-6-, TNF-α-) and (IL-6-, IL-1α-) were absent in (PSMA+,PSA-) and (PSMA+,PSA+) BPH sub-groups. Collectively, these findings underscore the importance of TNF-α and highlight the interaction between IL-6 and IL-1α to generate an inflammatory microenvironment in driving (PSMA,PSA) prostate clones. Copyright © 2016 Elsevier GmbH. All rights reserved.

Rebmab200, a humanized monoclonal antibody targeting the sodium phosphate transporter NaPi2b displays strong immune mediated cytotoxicity against cancer: a novel reagent for targeted antibody therapy of cancer.

PubMed

Lopes dos Santos, Mariana; Yeda, Fernanda Perez; Tsuruta, Lilian Rumi; Horta, Bruno Brasil; Pimenta, Alécio A; Degaki, Theri Leica; Soares, Ibere C; Tuma, Maria Carolina; Okamoto, Oswaldo Keith; Alves, Venancio A F; Old, Lloyd J; Ritter, Gerd; Moro, Ana Maria

2013-01-01

NaPi2b, a sodium-dependent phosphate transporter, is highly expressed in ovarian carcinomas and is recognized by the murine monoclonal antibody MX35. The antibody had shown excellent targeting to ovarian cancer in several early phase clinical trials but being murine the antibody's full therapeutic potential could not be explored. To overcome this impediment we developed a humanized antibody version named Rebmab200, expressed in human PER.C6® cells and cloned by limiting dilution. In order to select a clone with high therapeutic potential clones were characterized using a series of physicochemical assays, flow cytometry, real-time surface plasmon resonance, glycosylation analyses, immunohistochemistry, antibody-dependent cell-mediated cytotoxicity, complement-dependent-cytotoxicity assays and quantitative PCR. Comparative analyses of Rebmab200 and MX35 monoclonal antibodies demonstrated that the two antibodies had similar specificity for NaPi2b by flow cytometry with a panel of 30 cell lines and maintained similar kinetic parameters. Robust and high producer cell clones potentially suitable for use in manufacturing were obtained. Rebmab200 antibodies were assessed by immunohistochemistry using a large panel of tissues including human carcinomas of ovarian, lung, kidney and breast origin. An assessment of its binding towards 33 normal human organs was performed as well. Rebmab200 showed selected strong reactivity with the tested tumor types but little or no reactivity with the normal tissues tested confirming its potential for targeted therapeutics strategies. The remarkable cytotoxicity shown by Rebmab200 in OVCAR-3 cells is a significant addition to the traits of stability and productivity displayed by the top clones of Rebmab200. Antibody-dependent cell-mediated toxicity functionality was confirmed in repeated assays using cancer cell lines derived from ovary, kidney and lung as targets. To explore use of this antibody in clinical trials, GMP production of Rebmab200 has been initiated. As the next step of development, Phase I clinical trials are now planned for translation of Rebmab200 into the clinic.

Rebmab200, a Humanized Monoclonal Antibody Targeting the Sodium Phosphate Transporter NaPi2b Displays Strong Immune Mediated Cytotoxicity against Cancer: A Novel Reagent for Targeted Antibody Therapy of Cancer

PubMed Central

dos Santos, Mariana Lopes; Yeda, Fernanda Perez; Tsuruta, Lilian Rumi; Horta, Bruno Brasil; Pimenta, Alécio A.; Degaki, Theri Leica; Soares, Ibere C.; Tuma, Maria Carolina; Okamoto, Oswaldo Keith; Alves, Venancio A. F.; Ritter, Gerd; Moro, Ana Maria

2013-01-01

NaPi2b, a sodium-dependent phosphate transporter, is highly expressed in ovarian carcinomas and is recognized by the murine monoclonal antibody MX35. The antibody had shown excellent targeting to ovarian cancer in several early phase clinical trials but being murine the antibody's full therapeutic potential could not be explored. To overcome this impediment we developed a humanized antibody version named Rebmab200, expressed in human PER.C6® cells and cloned by limiting dilution. In order to select a clone with high therapeutic potential clones were characterized using a series of physicochemical assays, flow cytometry, real-time surface plasmon resonance, glycosylation analyses, immunohistochemistry, antibody-dependent cell-mediated cytotoxicity, complement-dependent-cytotoxicity assays and quantitative PCR. Comparative analyses of Rebmab200 and MX35 monoclonal antibodies demonstrated that the two antibodies had similar specificity for NaPi2b by flow cytometry with a panel of 30 cell lines and maintained similar kinetic parameters. Robust and high producer cell clones potentially suitable for use in manufacturing were obtained. Rebmab200 antibodies were assessed by immunohistochemistry using a large panel of tissues including human carcinomas of ovarian, lung, kidney and breast origin. An assessment of its binding towards 33 normal human organs was performed as well. Rebmab200 showed selected strong reactivity with the tested tumor types but little or no reactivity with the normal tissues tested confirming its potential for targeted therapeutics strategies. The remarkable cytotoxicity shown by Rebmab200 in OVCAR-3 cells is a significant addition to the traits of stability and productivity displayed by the top clones of Rebmab200. Antibody-dependent cell-mediated toxicity functionality was confirmed in repeated assays using cancer cell lines derived from ovary, kidney and lung as targets. To explore use of this antibody in clinical trials, GMP production of Rebmab200 has been initiated. As the next step of development, Phase I clinical trials are now planned for translation of Rebmab200 into the clinic. PMID:23936189

TA-GC cloning: A new simple and versatile technique for the directional cloning of PCR products for recombinant protein expression.

PubMed

Niarchos, Athanasios; Siora, Anastasia; Konstantinou, Evangelia; Kalampoki, Vasiliki; Lagoumintzis, George; Poulas, Konstantinos

2017-01-01

During the last few decades, the recombinant protein expression finds more and more applications. The cloning of protein-coding genes into expression vectors is required to be directional for proper expression, and versatile in order to facilitate gene insertion in multiple different vectors for expression tests. In this study, the TA-GC cloning method is proposed, as a new, simple and efficient method for the directional cloning of protein-coding genes in expression vectors. The presented method features several advantages over existing methods, which tend to be relatively more labour intensive, inflexible or expensive. The proposed method relies on the complementarity between single A- and G-overhangs of the protein-coding gene, obtained after a short incubation with T4 DNA polymerase, and T and C overhangs of the novel vector pET-BccI, created after digestion with the restriction endonuclease BccI. The novel protein-expression vector pET-BccI also facilitates the screening of transformed colonies for recombinant transformants. Evaluation experiments of the proposed TA-GC cloning method showed that 81% of the transformed colonies contained recombinant pET-BccI plasmids, and 98% of the recombinant colonies expressed the desired protein. This demonstrates that TA-GC cloning could be a valuable method for cloning protein-coding genes in expression vectors.

TA-GC cloning: A new simple and versatile technique for the directional cloning of PCR products for recombinant protein expression

PubMed Central

Niarchos, Athanasios; Siora, Anastasia; Konstantinou, Evangelia; Kalampoki, Vasiliki; Poulas, Konstantinos

2017-01-01

During the last few decades, the recombinant protein expression finds more and more applications. The cloning of protein-coding genes into expression vectors is required to be directional for proper expression, and versatile in order to facilitate gene insertion in multiple different vectors for expression tests. In this study, the TA-GC cloning method is proposed, as a new, simple and efficient method for the directional cloning of protein-coding genes in expression vectors. The presented method features several advantages over existing methods, which tend to be relatively more labour intensive, inflexible or expensive. The proposed method relies on the complementarity between single A- and G-overhangs of the protein-coding gene, obtained after a short incubation with T4 DNA polymerase, and T and C overhangs of the novel vector pET-BccI, created after digestion with the restriction endonuclease BccI. The novel protein-expression vector pET-BccI also facilitates the screening of transformed colonies for recombinant transformants. Evaluation experiments of the proposed TA-GC cloning method showed that 81% of the transformed colonies contained recombinant pET-BccI plasmids, and 98% of the recombinant colonies expressed the desired protein. This demonstrates that TA-GC cloning could be a valuable method for cloning protein-coding genes in expression vectors. PMID:29091919

Molecular cloning and expression of rat and mouse B61 gene: implications on organogenesis.

PubMed

Takahashi, H; Ikeda, T

1995-09-07

ECK is a member of EPH receptor protein-tyrosine kinase subfamily and human B61 has been identified as the ligand for ECK recently. In order to better understand the roles of B61-ECK signalling pathway in mammalian development, we have cloned rat and mouse B61 cDNA and examined the expression pattern during rat development. Sequence analysis has revealed that there is a considerable degree of identity among rat, mouse and human B61 (98.0% between rat and mouse, 86.3% between rat and human in amino acid level). Examination of B61 mRNA expression by in situ hybridization analysis revealed tight association of B61 with endothelial cells at an early stage and epithelial cells in various tissues including lung, kidney, intestine, skin at later stage of organogenesis. In the developing skeletal system, B61 is expressed in periosteum, perichondrium and hypertrophic chondrocytes and osteoblasts. In the developing nervous system, expression of B61 is restricted in the neurons of dorsal root ganglia. These expression profiles of B61 in epithelial cells of various organs, developing skeletal system and dorsal root ganglia match those of ECK. Our data suggest that B61 plays pivotal roles in organogenesis, especially vasculogenesis/angiogenesis and epithelial cell proliferation/differentiation.

Effect of melatonin and tetrapeptide on gene expression in mouse brain.

PubMed

Anisimov, S V; Khavinson, V Kh; Anisimov, V N

2004-11-01

A microchip technique was used to study expression of 16,897 clones from a cDNA library in the brain of mice receiving melatonin or tetrapeptide Epithalon (Ala-Glu-Asp-Gly). Expression of 53 transcripts in mouse brain underwent significant changes after treatment with the preparations. Melatonin and Epithalon modified expression of 38 and 22 transcripts, respectively. These preparations produced similar changes in the expression of 6 transcripts. Expression of 1 transcript (Rp119) was inhibited by melatonin, but induced by Epithalon. The target genes are physiologically related to the cell cycle, apoptosis, biosynthesis, processing, and transport of nucleic acids. Comparative study of gene expression in the brain and heart of CBA mice receiving melatonin and Epithalon suggest that these preparations have a tissue-specific biological effect.

Cloning of peroxisome proliferators activated receptors in the cobia (Rachycentron canadum) and their expression at different life-cycle stages under cage aquaculture.

PubMed

Tsai, Mei-Ling; Chen, Houng-Yung; Tseng, Mei-Cheuh; Chang, Rey-Chang

2008-12-01

We present the cDNA sequences and tissue mRNA expression of peroxisome proliferator-activated receptor (PPAR) alpha, beta and gamma isotypes in the cobia (Rachycentron canadum), a warm water pelagic fish that is becoming a fish of choice for offshore cage farming. RT-PCR and real-time PCR showed that PPARalpha mRNA predominated in red muscle, heart and liver whereas PPARbeta was expressed mainly in liver and pyloric caeca. In contrast, PPARgamma transcripts were detected in all of the tissues examined, with the highest level occurring in visceral fat depot. Our 52-wk time-series investigation showed that while the mRNA expression of PPARgamma in the cobia was positively (P < 0.05) related to its body lipid deposition, a negative (P < 0.05) relationship was found between PPARalpha expression in the liver and body lipid deposition. There was a significant increase in body lipid deposition and hepatic PPARgamma expression as the fish grew. The hepatic PPARgamma expression could be a sufficient parameter describing the bodily expression of PPARgamma because of its positive correlation with PPARgamma expressions in all other tissues. These results showed that PPARgamma and alpha played a pivotal role in the control of lipid metabolic and storage functions in the liver, muscle and visceral fat depot of the cobia.

Characterization and clinical validation of MCM2 and TOP2A monoclonal antibodies in the BD ProEx™ C assay: An immunoassay which detects aberrant S-phase induction in cervical tissue.

PubMed

Dixon, Eric P; King, Lorraine M; Nelson, Ramona; Simkins, Stephen G; Knapp, Steven L; Brough, George H; Lenz, Karen L; Henderson, Dorian T; Whitehead, Clark M; Hessling, Janice; Brown, Charlotte A; Malinowski, Douglas P

2017-03-01

The Papanicolaou (Pap) screen has been successful in reducing cervical cancer; but exhibits low sensitivity when detecting cervical dysplasia. Use of molecular biomarkers in Pap tests may improve diagnostic accuracy. Monoclonal antibodies to Minichromosome Maintenance Protein 2 (MCM2) and DNA Topoisomerase II α (TOP2A) were selected for use in IHC based on their ability to differentiate normal from diseased cervical tissues in tissue microarrays. Enhanced Green Fluorescent Protein Western blot analysis was used to help identify binding epitopes specific to MCM2 and TOP2A antibody clones. Antibody affinity was determined by solution phase affinity measurement and immunohistochemistry was performed using high affinity MCM2 or TOP2A antibodies on serial histological sections. Antibody clones to MCM2 and TOP2A clones were selected based on their ability to detect over expression in abnormal cervical epithelia. In IHC, MCM2-27C5.6 and MCM2-26H6.19 demonstrated superior staining in abnormal cervical tissue over the MCM2-CRCT2.1 antibody. A combination of MCM2 and TOP2A antibodies showed greater staining when compared to staining with any of the antibodies alone on serial histological sections. Distinct linear epitopes were elucidated for each of the MCM2 and TOP2A clones. Affinity values (Kd) for MCM2 or TOP2A antibodies had a similar range. In a research study, the MCM2 and TOP2A (BD ProEx™ C) antibody cocktail showed increased epithelia staining with increasing dysplasia. The use of BD ProEx™ C in combination with H&E staining enhanced immunohistochemical discrimination of dysplastic and non-dysplastic FFPE cervical tissue specimens. BD ProEx™ C containing MCM2 and TOP2A antibodies showed strong specific nuclear staining that correlated with increased dysplasia and lesion severity. Enhanced performance of the antibodies was linked to their unique topography recognition. BD ProEx™ C incorporates antibodies that enhance detection of CIN2+ cervical disease. Copyright © 2017 Elsevier B.V. All rights reserved.

Expression of novel rice gibberellin 2-oxidase gene is under homeostatic regulation by biologically active gibberellins.

PubMed

Sakai, Miho; Sakamoto, Tomoaki; Saito, Tamio; Matsuoka, Makoto; Tanaka, Hiroshi; Kobayashi, Masatomo

2003-04-01

We have cloned two genes for gibberellin (GA) 2-oxidase from rice ( Oryza sativa L.). Expression of OsGA2ox2 was not observed. The other gene, OsGA2ox3, was expressed in every tissue examined and was enhanced by the application of biologically active GA. Recombinant OsGA2ox3 protein catalyzed the metabolism of GA(1) to GA(8) and GA(20) to GA(29)-catabolite. These results indicate that OsGA2ox3 is involved in the homeostatic regulation of the endogenous level of biologically active GA in rice.

Molecular cloning and functional characterization of borneol dehydrogenase from the glandular trichomes of Lavandula x intermedia.

PubMed

Sarker, Lukman S; Galata, Mariana; Demissie, Zerihun A; Mahmoud, Soheil S

2012-12-15

Several varieties of Lavandula x intermedia (lavandins) are cultivated for their essential oils (EOs) for use in cosmetic, hygiene and personal care products. These EOs are mainly constituted of monoterpenes including camphor, which contributes an off odor reducing the olfactory appeal of the oil. We have recently constructed a cDNA library from the glandular trichomes (the sites of EO synthesis) of L. x intermedia plants. Here, we describe the cloning of a borneol dehydrogenase cDNA (LiBDH) from this library. The 780 bp open reading frame of the cDNA encoded a 259 amino acid short chain alcohol dehydrogenase with a predicted molecular mass of ca. 27.5 kDa. The recombinant LiBDH was expressed in Escherichia coli, purified by Ni-NTA agarose affinity chromatography, and functionally characterized in vitro. The bacterially produced enzyme specifically converted borneol to camphor as the only product with K(m) and k(cat) values of 53 μM and 4.0 × 10(-4) s(-1), respectively. The LiBDH transcripts were specifically expressed in glandular trichomes of mature flowers indicating that like other Lavandula monoterpene synthases the expression of this gene is regulated in a tissue-specific manner. The cloning of LiBDH has far reaching implications in improving the quality of Lavandula EOs through metabolic engineering. Copyright © 2012 Elsevier Inc. All rights reserved.

Genetic analyses of Per.C6 cell clones producing a therapeutic monoclonal antibody regarding productivity and long-term stability.

PubMed

Tsuruta, Lilian Rumi; Lopes Dos Santos, Mariana; Yeda, Fernanda Perez; Okamoto, Oswaldo Keith; Moro, Ana Maria

2016-12-01

Genetic characterization of protein-producing clones represents additional value to cell line development. In the present study, ten Per.C6 clones producing a Rebmab100 monoclonal antibody were selected using two cloning methods: six clones originated from limiting dilution cloning and four by the automated colony picker ClonePix FL. A stability program was performed for 50 generations, including 4 batches distributed along the timeframe to determine specific productivity (Qp) maintenance. Four stable clones (two from limiting dilution and two from ClonePix FL) were further evaluated. The relative mRNA expression levels of both heavy chain (HC) and light chain (LC) genes were verified at generations 0, 30-35, and 50-55 of the stability program. At generations 0 and 30-35, LC gene expression level was higher than HC gene, whereas at generation 50-55, the opposite prevailed. A high correlation was observed between Qp and HC or LC mRNA expression level for all clones at each generation analyzed along the continuous culture. The mRNA stability study was performed at steady-state culture. The LC gene displayed a higher half-life and lower decay constant than HC gene, accounting for the higher observed expression level of LC mRNA in comparison to HC mRNA. Clone R6 was highlighted due its high Qp, mRNA expression levels, and mRNA stability. Besides the benefits of applying genetic characterization for the selection of stable and high-producing clones, the present study shows for the first time the correlation between Qp and HC or LC expression levels and also mRNA stability in clones derived from human cell line Per.C6(®).

Molecular cloning, ontogeny and tissue distribution of zebrafish (Danio rerio) prohormone convertases: pcsk1 and pcsk2.

PubMed

Morash, Michael G; MacDonald, Angela B; Croll, Roger P; Anini, Younes

2009-06-01

Prohormone convertase subtilisin/kexin (PCSK) enzymes are a family of nine related serine proteases, found in a multitude of tissues, and responsible for the maturation of a variety of protein and peptide precursors. Pcsk1 and Pcsk2 are found within dense core secretory granules in endocrine and neuroendocrine cells and are responsible for cleaving several hormones and neuropeptide precursors. In this work, we cloned and sequenced the cDNA of pcsk1 and pcsk2 from zebrafish (Danio rerio). pcsk1 is a 2268bp ORF, whose 755 amino acid protein product is identical to that predicted from the genome sequence. pcsk2 is a 1941bp ORF, encoding a 646 amino acid peptide. Both Pcsk1 and Pcsk2 display high degrees of similarity to their counterparts in other species, including the conservation of the catalytic triad and other essential residues. The brain contained the highest expression levels of both pcsk1 (1.49+/-0.21) (displayed as ratio to EF-1a), and pcsk2 (0.23+/-0.04). Both transcripts were also detectable in the fore, mid and distal gut. pcsk1 and 2 were detectable at 4.5h post-fertilization, and while pcsk1 expression increased throughout development (0.12+/-0.01 maximum at 3 days post-fertilization), pcsk2 expression was highest at day 5 post-fertilization (0.03+/-0.01), and decreased prior. For the first time, we have identified and characterized a pcsk1 transcript in fish. We have also identified and characterized the pcsk2 transcript in zebrafish, and have assessed the tissue distribution and ontogeny of both.

Selenoprotein Gene Expression in Thyroid and Pituitary of Young Pigs Is Not Affected by Dietary Selenium Deficiency or Excess1–3

PubMed Central

Zhou, Ji-Chang; Zhao, Hua; Li, Jun-Gang; Xia, Xin-Jie; Wang, Kang-Ning; Zhang, Ya-Jun; Liu, Yan; Zhao, Ying; Lei, Xin Gen

2009-01-01

Expression and function of selenoproteins in endocrine tissues remain unclear, largely due to limited sample availability. Pigs have a greater metabolic similarity and tissue size than rodents as a model of humans for that purpose. We conducted 2 experiments: 1) we cloned 5 novel porcine selenoprotein genes; and 2) we compared the effects of dietary selenium (Se) on mRNA levels of 12 selenoproteins, activities of 4 antioxidant enzymes, and Se concentrations in testis, thyroid, and pituitary with those in liver of pigs. In Experiment 1, porcine Gpx2, Sephs2, Sep15, Sepn1, and Sepp1 were cloned and demonstrated 84–94% of coding sequence homology to human genes. In Experiment 2, weanling male pigs (n = 30) were fed a Se-deficient (0.02 mg Se/kg) diet added with 0, 0.3, or 3.0 mg Se/kg as Se-enriched yeast for 8 wk. Although dietary Se resulted in dose-dependent increases (P < 0.05) in Se concentrations and GPX activities in all 4 tissues, it did not affect the mRNA levels of any selenoprotein gene in thyroid or pituitary. Testis mRNA levels of Txnrd1 and Sep15 were decreased (P < 0.05) by increasing dietary Se from 0.3 to 3.0 mg/kg. Comparatively, expressions of Gpx2, Gpx4, Dio3, and Sep15 were high in pituitary and Dio1, Sepp1, Sephs2, and Gpx1 were high in liver. In conclusion, the mRNA abundances of the 12 selenoprotein genes in thyroid and pituitary of young pigs were resistant to dietary Se deficiency or excess. PMID:19357213

Tissue-specific expression of human CD4 in transgenic mice.

PubMed Central

Gillespie, F P; Doros, L; Vitale, J; Blackwell, C; Gosselin, J; Snyder, B W; Wadsworth, S C

1993-01-01

The gene for the human CD4 glycoprotein, which serves as the receptor for human immunodeficiency virus type 1, along with approximately 23 kb of sequence upstream of the translational start site, was cloned. The ability of 5' flanking sequences to direct tissue-specific expression was tested in cell culture and in transgenic mice. A 5' flanking region of 6 kb was able to direct transcription of the CD4 gene in NIH 3T3 cells but did not result in detectable expression in the murine T-cell line EL4 or in four lines of transgenic mice. A larger 5' flanking region of approximately 23 kb directed high-level CD4 transcription in the murine T-cell line EL4 and in three independent lines of transgenic mice. Human CD4 expression in all tissues analyzed was tightly correlated with murine CD4 expression; the highest levels of human CD4 RNA expression were found in the thymus and spleen, with relatively low levels detected in other tissues. Expression of human CD4 protein in peripheral blood mononuclear cells was examined by flow cytometry in these transgenic animals and found to be restricted to the murine CD4+ subset of lymphocytes. Human CD4 protein, detected with an anti-human CD4 monoclonal antibody, was present on the surface of 45 to 50% of the peripheral blood mononuclear cells from all transgenic lines. Images PMID:8474453

Cell cycle, differentiation and tissue-independent expression of ribosomal protein L37.

PubMed

Su, S; Bird, R C

1995-09-15

A unique human cDNA (hG1.16) that encodes a mRNA of 450 nucleotides was isolated from a subtractive library derived from HeLa cells. The relative expression level of hG1.16 during different cell-cycle phases was determined by Northern-blot analysis of cells synchronized by double-thymidine block and serum deprivation/refeeding. hG1.16 was constitutively expressed during all phases of the cell cycle, including the quiescent phase when even most constitutively expressed genes experience some suppression of expression. The expression level of hG1.16 did not change during terminal differentiation of myoblasts to myotubes, during which cells become permanently post-mitotic. Examination of other tissues revealed that the relative expression level of hG1.16 was constitutive in all embryonic mouse tissues examined, including brain, eye, heart, kidney, liver, lung and skeletal muscle. This was unusual in that expression was not down-modulated during differentiation and did not vary appreciably between tissue types. Analysis by inter-species Northern-blot analysis revealed that hG1.16 was highly conserved among all vertebrates studied (from fish to humans but not in insects). DNA sequence analysis of hG1.16 revealed a high level of similarity to rat ribosomal protein L37, identifying hG1.16 as a new member of this multigene family. The deduced amino acid sequence of hG1.16 was identical to rat ribosomal protein L37 that contained 97 amino acids, many of which are highly positively charged (15 arginine and 14 lysine residues with a predicted M(r) of 11,065). hG1.16 protein has a single C2-C2 zinc-finger-like motif which is also present in rat ribosomal protein L37. Using primers designed from the sequence of hG1.16, unique bovine and rat cDNAs were also isolated by 5'-rapid-amplification of cDNA ends. DNA sequences of bovine and rat G1.16, clones were 92.8% and 92.2% similar to human G1.16 while the deduced amino acid sequences derived from bovine and rat cDNAs each differed by a single amino acid from the sequence of hG1.16 and the published rat L37 sequence. Southern-blot analysis revealed that hG1.16 exists in multiple copies in human, rat and mouse genomes. These G1.16 clones encode unique human, rat and bovine members of the ribosomal protein L37 gene family, which are constitutively expressed even during transitions from quiescence to active cell proliferation or terminal differentiation, in all tissues and all vertebrates investigated.

Identification and validation of quantitative real-time reverse transcription PCR reference genes for gene expression analysis in teak (Tectona grandis L.f.)

PubMed Central

2014-01-01

Background Teak (Tectona grandis L.f.) is currently the preferred choice of the timber trade for fabrication of woody products due to its extraordinary qualities and is widely grown around the world. Gene expression studies are essential to explore wood formation of vascular plants, and quantitative real-time reverse transcription PCR (qRT-PCR) is a sensitive technique employed for quantifying gene expression levels. One or more appropriate reference genes are crucial to accurately compare mRNA transcripts through different tissues/organs and experimental conditions. Despite being the focus of some genetic studies, a lack of molecular information has hindered genetic exploration of teak. To date, qRT-PCR reference genes have not been identified and validated for teak. Results Identification and cloning of nine commonly used qRT-PCR reference genes from teak, including ribosomal protein 60s (rp60s), clathrin adaptor complexes medium subunit family (Cac), actin (Act), histone 3 (His3), sand family (Sand), β-Tubulin (Β-Tub), ubiquitin (Ubq), elongation factor 1-α (Ef-1α), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Expression profiles of these genes were evaluated by qRT-PCR in six tissue and organ samples (leaf, flower, seedling, root, stem and branch secondary xylem) of teak. Appropriate gene cloning and sequencing, primer specificity and amplification efficiency was verified for each gene. Their stability as reference genes was validated by NormFinder, BestKeeper, geNorm and Delta Ct programs. Results obtained from all programs showed that TgUbq and TgEf-1α are the most stable genes to use as qRT-PCR reference genes and TgAct is the most unstable gene in teak. The relative expression of the teak cinnamyl alcohol dehydrogenase (TgCAD) gene in lignified tissues at different ages was assessed by qRT-PCR, using TgUbq and TgEf-1α as internal controls. These analyses exposed a consistent expression pattern with both reference genes. Conclusion This study proposes a first broad collection of teak tissue and organ mRNA expression data for nine selected candidate qRT-PCR reference genes. NormFinder, Bestkeeper, geNorm and Delta Ct analyses suggested that TgUbq and TgEf-1α have the highest expression stability and provided similar results when evaluating TgCAD gene expression, while the commonly used Act should be avoided. PMID:25048176

Molecular cloning of peroxidase cDNAs from dehydration-treated fibrous roots of sweetpotato and their differential expression in response to stress.

PubMed

Kim, Yun-Hee; Yang, Kyoung-Sil; Kim, Cha Young; Ryu, Sun-Hwa; Song, Wan-Keun; Kwon, Suk-Yoon; Lee, Haeng-Soon; Bang, Jae-Wook; Kwak, Sang-Soo

2008-03-31

Three peroxidase (POD) cDNAs were isolated from dehydration-treated fibrous roots of sweetpotato (Ipomoea batatas) plant via the screening of a cDNA library, and their expressions were assessed to characterize functions of each POD in relation to environmental stress. Three PODs were divided into two groups, designated the basic PODs (swpb4, swpb5) and the anionic PODs (swpa7), on the basis of the pI values of mature proteins. Fluorescence microscope analysis indicated that three PODs are secreted into the extracellular space. RTPCR analysis revealed that POD genes have diverse expression patterns in a variety of plant tissues. Swpb4 was abundantly expressed in stem tissues, whereas the expression levels of swpb5 and swpa7 transcripts were high in fibrous and thick pigmented roots. Swpb4 and swpa7 showed abundant expression levels in suspension cultured cells. Three POD genes responded differently in the leaf and fibrous roots in response to a variety of stresses including dehydration, temperature stress, stress-associated chemicals, and pathogenic bacteria.

Molecular cloning and expression analysis of sea bass (Dicentrarchus labrax L.) tumor necrosis factor-alpha (TNF-alpha).

PubMed

Nascimento, Diana S; Pereira, Pedro J B; Reis, Marta I R; do Vale, Ana; Zou, Jun; Silva, Manuel T; Secombes, Christopher J; dos Santos, Nuno M S

2007-09-01

In the search for pro-inflammatory genes in sea bass a TNF-alpha gene was cloned and sequenced. The sea bass TNF-alpha (sbTNF-alpha) putative protein conserves the TNF-alpha family signature, as well as the two cysteines usually involved in the formation of a disulfide bond. The mouse TNF-alpha Thr-Leu cleavage sequence and a potential transmembrane domain were also found, suggesting that sbTNF-alpha exists as two forms: a approximately 28 kDa membrane-bound form and a approximately 18.4 kDa soluble protein. The single copy sbTNF-alpha gene contains a four exon-three intron structure similar to other known TNF-alpha genes. Homology modeling of sbTNF-alpha is compatible with the trimeric quaternary architecture of its mammalian counterparts. SbTNF-alpha is constitutively expressed in several unstimulated tissues, and was not up-regulated in the spleen and head-kidney, in response to UV-killed Photobacterium damselae subsp. piscicida. However, an increase of sbTNF-alpha expression was detected in the head-kidney during an experimental infection using the same pathogen.

Molecular cloning, characterization and expression analysis of coagulation factor VII gene in grass carp (Ctenopharyngodon idella).

PubMed

Liu, Qiaolin; Xu, Baohong; Xiao, Tiaoyi; Su, Jianming; Zhong, Lei

2013-08-01

Coagulation factor VII has been studied in several species but, to date, not in grass carp (Ctenopharyngodon idella), a commercially important freshwater fish found in China. In this study, the full-length cDNA of grass carp coagulation factor VII (GcCFVII) was cloned using a RACE-Ready cDNA Kit, grass carp were challenged with a hemorrhagic virus, and temporal expression profiles of GcCFVII in the thymus, gills, liver, spleen, and head kidney were examined at 0 h, 24 h, 48 h, 72 h, 96 h, and 138 h using fluorescence quantitative PCR. The results showed the 1480 bp GcCFVII to contain three conservative motifs: Gla, EGF-CA, and Tryp-SPc, similar to other species. Phylogenetic analysis showed the evolution of GcCFVII gene to be consistent with the evolution of the species. After viral challenge, GcCFVII expression in five tissues of grass carp showed different patterns of fluctuation. These results provide a solid basis for further investigation of GcCFVII and its relationship with grass carp hemorrhage. Copyright © 2013 Elsevier Ltd. All rights reserved.

Cloning of the cDNA encoding Scg-SPRP, an unusual Ser-protease-related protein from vitellogenic female desert locusts (Schistocerca gregaria).

PubMed

Chiou, S J; Vanden Broeck, J; Janssen, I; Borovsky, D; Vandenbussche, F; Simonet, G; De Loof, A

1998-10-01

The cDNA coding for a Ser-protease-related protein (Scg-SPRP) was cloned from desert locust (Schistocerca gregaria) midgut. The derived amino acid sequence consists of 260 residues and shows strong sequence similarity to insect trypsin-like molecules. It is, however, likely that Scg-SPRP is not a proteolytically active enzyme and that it plays another physiologically relevant role, since two out of three residues which are indispensable for catalytic activity of Ser-proteases are replaced. Northern analysis revealed that the Scg-SPRP gene is expressed in midgut tissue and that this expression is strongly induced in adult female locusts. Moreover, the occurrence of the transcript (1.2 kb) fluctuates during the molting cycle and during the female reproductive cycle. Juvenile hormone (JH III) dependence of transcription was investigated by chemical allatectomy (precocene I) of adult females. This resulted in inhibition of vitellogenesis and in disappearance of the Scg-SPRP transcript. Expression of Scg-SPRP in precocene-treated locusts could be reinduced by additional treatment with JH III or with 20-OH-ecdysone.

Identification and characterisation of DfCHS, a chalcone synthase gene regulated by temperature and ultraviolet in Dryopteris fragrans.

PubMed

Sun, L L; Li, Y; Li, S S; Wu, X J; Hu, B Z; Chang, Y

2014-12-30

Chalcone synthase (CHS) is an enzyme that catalyzes the first committed step in flavonoid biosynthesis, and its transcription level is regulated by light conditions. By using homology cloning and rapid amplification of cDNA ends, we cloned a chalcone synthase gene (DfCHS) from Dryopteris fragrans (L.) Schott. The full-length cDNA of DfCHS is 1,737 bp, with an open reading frame (ORF) of 1,122 bp (deposited in GenBank under Accession Number KF530802) encoding a predicted protein of 373 amino acids. The calculated molecular mass of DfCHS is 41.3 kDa. We studied the expression of DfCHS and total flavonoid contents in tissue culture seedlings cultured under the low temperature at 4ºC, high temperature at 35ºC and UV conditions, respectively. The results show that the expression of DfCHS are not the same, but all present rising trends, then flavonoid contents were increased. Overall, our results imply that the expression of DfCHS gene provide a certain theory basis in the status of evolution among ferns.

Cloning, characterization, and expression of xyloglucan endotransglucosylase/hydrolase and expansin genes associated with petal growth and development during carnation flower opening

PubMed Central

Harada, Taro; Torii, Yuka; Morita, Shigeto; Onodera, Reiko; Hara, Yoshinao; Yokoyama, Ryusuke; Nishitani, Kazuhiko; Satoh, Shigeru

2011-01-01

Growth of petal cells is a basis for expansion and morphogenesis (outward bending) of petals during opening of carnation flowers (Dianthus caryophyllus L.). Petal growth progressed through elongation in the early stage, expansion with outward bending in the middle stage, and expansion of the whole area in the late stage of flower opening. In the present study, four cDNAs encoding xyloglucan endotransglucosylase/hydrolase (XTH) (DcXTH1–DcXTH4) and three cDNAs encoding expansin (DcEXPA1–DcEXPA3) were cloned from petals of opening carnation flowers and characterized. Real-time reverse transcription-PCR analyses showed that transcript levels of XTH and expansin genes accumulated differently in floral and vegetative tissues of carnation plants with opening flowers, indicating regulated expression of these genes. DcXTH2 and DcXTH3 transcripts were detected in large quantities in petals as compared with other tissues. DcEXPA1 and DcEXPA2 transcripts were markedly accumulated in petals of opening flowers. The action of XTH in growing petal tissues was confirmed by in situ staining of xyloglucan endotransglucosylase (XET) activity using a rhodamine-labelled xyloglucan nonasaccharide as a substrate. Based on the present findings, it is suggested that two XTH genes (DcXTH2 and DcXTH3) and two expansin genes (DcEXPA1 and DcEXPA2) are associated with petal growth and development during carnation flower opening. PMID:20959626

Spontaneous establishment of an Epstein-Barr virus-infected fibroblast line from the synovial tissue of a rheumatoid arthritis patient.

PubMed Central

Koide, J; Takada, K; Sugiura, M; Sekine, H; Ito, T; Saito, K; Mori, S; Takeuchi, T; Uchida, S; Abe, T

1997-01-01

An Epstein-Barr virus (EBV)-infected fibroblast line, designated DSEK, was spontaneously established from synovial tissue of a patient with rheumatoid arthritis (RA). DSEK cells expressed EBV nuclear antigens EBNA-1 and EBNA-2 and latent membrane protein LMP-1. Cell surface markers of DSEK cells were similar to those of EBV-negative fibroblast clones derived from synoviocytes and were negative for lymphocyte and macrophage markers. DSEK cells expressed CD44, CD58, and HLA-DR antigens and spontaneously produced interleukin-10 basic fibroblast growth factor and transforming growth factor beta1. These results indicate that rheumatoid synoviocytes can be a target for EBV infection and suggest that EBV may play a role in the pathogenesis of RA. PMID:9032386

Molecular cloning, developmental expression, and cellular localization of the 70-kDa RPA-1 subunit of Drosophila melanogaster.

PubMed

Perdigão, J; Logarinho, E; Avides, M C; Sunkel, C E

1999-12-01

Replication protein A (RPA) is a highly conserved multifunctional heterotrimeric complex, involved in DNA replication, repair, recombination, and possibly transcription. Here, we report the cloning of the gene that codes for the largest subunit of the Drosophila melanogaster RPA homolog, dmRPA70. In situ hybridization showed that dmRPA70 RNA is present in developing embryos during the first 16 cycles. After this point, dm-RPA70 expression is downregulated in cells that enter a G1 phase and exit the mitotic cycle, becoming restricted to brief bursts of accumulation from late G1 to S phase. This pattern of regulated expression is also observed in the developing eye imaginal disc. In addition, we have shown that the presence of cyclin E is necessary and sufficient to drive the expression of dmRPA70 in embryonic cells arrested in G1 but is not required in tissues undergoing endoreduplication. Immunolocalization showed that in early developing embryos, the dmRPA70 protein associates with chromatin from the end of mitosis until the beginning of the next prophase in a dynamic speckled pattern that is strongly suggestive of its association with replication foci.

Molecular cloning and chromosomal mapping of bone marrow stromal cell surface gene, BST2, that may be involved in pre-B-cell growth

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ishikawa, Jun; Kaisho, Tsuneyasu; Tomizawa, Hitoshi

1995-04-10

Bone marrow stromal cells regulate B-cell growth and development through their surface molecules and cytokines. In this study, we generated a mAb, RS38, that recognized a novel human membrane protein, BST-2, expressed on bone marrow stromal cell lines and synovial cell lines. We cloned a cDNA encoding BST-2 from a rheumatoid arthritis-derived synovial cell line. BST-2 is a 30- to 36-kDa type II transmembrane protein, consisting of 180 amino acids. The BST-2 gene (HGMW-approved symbol BST2) is located on chromosome 19p13.2. BST-2 is expressed not only on certain bone marrow stromal cell lines but also on various normal tissues, althoughmore » its expression pattern is different from that of another bone marrow stromal cell surface molecule, BST-1. BST-2 surface expression on fibroblast cell lines facilitated the stromal cell-dependent growth of a murine bone marrow-derived pre-B-cell line, DW34. The results suggest that BST-2 may be involved in pre-B-cell growth. 45 refs., 7 figs., 2 tabs.« less

Cloning, expression profiling, and acetylation identification of alpha-tubulin N-acetyltransferase 1 from Bombyx mori.

PubMed

Zhou, Huaixiang; Cheng, Xusheng; Xu, Xiaoyuan; Jiang, Tianlong; Zhou, Haimeng; Sheng, Qing; Nie, Zuoming

2018-03-22

Alpha-tubulin N-acetyltransferase 1 (ATAT1) is an acetyltransferase specific to α-tubulin and performs important functions in many cellular processes. Bombyx mori is an economic insect and also known as a model lepidoptera insect. In this study, we cloned a B. mori ATAT1 gene (BmATAT1) (Gen Bank accession number: XP_004932777.1). BmATAT1 contained an open reading frame (ORF) of 1,065 bp encoding 355 amino acids (aa). Expression profiling of BmATAT1 protein showed that the expression levels of BmATAT1 at different developmental stages and different tissues in fifth-instar larvae differ. BmATAT1 was highly expressed at the egg stage and in the head of the fifth-instar larvae. Subcellular localization showed that BmATAT1 was distributed in the cytoplasm and nucleus. Furthermore, BmATAT1 may lead to time-dependent induction of cell cycle arrest in the G2/M phase by flow cytometry analysis. Interestingly, using site-specific mutation, immunoprecipitation, and Western blotting, we further found a BmATAT1 acetylated site at K156, suggesting that this acetyltransferase could be regulated by acetylation itself. © 2018 Wiley Periodicals, Inc.

Ectopic expression of pMADS3 in transgenic petunia phenocopies the petunia blind mutant.

PubMed Central

Tsuchimoto, S; van der Krol, A R; Chua, N H

1993-01-01

We cloned a MADS-box gene, pMADS3, from Petunia hybrida, which shows high sequence homology to the Arabidopsis AGAMOUS and Antirrhinum PLENA. pMADS3 is expressed exclusively in stamens and carpels of wild-type petunia plants. In the petunia mutant blind, which shows homeotic conversions of corolla limbs into antheroid structures with pollen grains and small parts of sepals into carpelloid tissue, pMADS3 is expressed in all floral organs as well as in leaves. Ectopic expression of pMADS3 in transgenic petunia leads to phenocopies of the blind mutant, i.e., the formation of antheroid structures on limbs and carpelloid tissue on sepals. Transgenic tobacco plants that overexpress pMADS3 exhibit an even more severe phenotype, with the sepals forming a carpel-like structure encasing the interior floral organs. Our results identify BLIND as a negative regulator of pMADS3, which specifies stamens and carpels during petunia flower development. PMID:8104573

Putative storage root specific promoters from cassava and yam: cloning and evaluation in transgenic carrots as a model system.

PubMed

Arango, Jacobo; Salazar, Bertha; Welsch, Ralf; Sarmiento, Felipe; Beyer, Peter; Al-Babili, Salim

2010-06-01

A prerequisite for biotechnological improvements of storage roots is the availability of tissue-specific promoters enabling high expression of transgenes. In this work, we cloned two genomic fragments, pMe1 and pDJ3S, controlling the expression of a gene with unknown function from cassava (Manihot esculenta) and of the storage protein dioscorin 3 small subunit gene from yam (Dioscorea japonica), respectively. Using beta-glucuronidase as a reporter, the activities of pMe1 and pDJ3S were evaluated in independent transgenic carrot lines and compared to the constitutive CaMV35S and the previously described cassava p15 promoters. Activities of pMe1 and pDJ3S in storage roots were assessed using quantitative GUS assays that showed pDJ3S as the most active one. To determine organ specificities, uidA transcript levels in leaves, stems and roots were measured by real-time RT-PCR analyses showing highest storage root specificity for pDJ3S. Root cross sections revealed that pMe1 was highly active in secondary xylem. In contrast, pDJ3S was active in all root tissues except for the central xylem. The expression patterns caused by the cassava p15 promoter in carrot storage roots were consistent with its previously described activities for the original storage organ. Our data demonstrate that the pDJ3S and, to a lesser extent, the pMe1 regulatory sequences represent feasible candidates to drive high and preferential expression of genes in carrot storage roots.

Cloning of cDNA encoding the nuclear form of chicken sterol response element binding protein-2 (SREBP-2), chromosomal localization, and tissue expression of chicken SREBP-1 and -2 genes.

PubMed

Assaf, S; Hazard, D; Pitel, F; Morisson, M; Alizadeh, M; Gondret, F; Diot, C; Vignal, A; Douaire, M; Lagarrigue, S

2003-01-01

Sterol regulatory element binding protein-1 and -2 (SREBP-1 and -2) are key transcription factors involved in the biosynthesis of cholesterol and fatty adds. The SREBP have mainly been studied in rodents in which lipogenesis is regulated in both liver and adipose tissue. There is, however, a paucity of information on birds, in which lipogenesis occurs essentially in the liver as in humans. As a prelude to the investigation of the role of SREBP in lipid metabolism regulation in chicken, we sequenced the cDNA, encoding the mature nuclear form of chicken SREBP-2 protein, mapped SREBP-1 and -2 genes and studied their tissue expressions. The predicted chicken SREBP-2 amino acid sequence shows a 77 to 79% identity with human, mouse, and hamster homologues, with a nearly perfect conservation in all the important functional motifs, basic, helix-loop-helix, and leucine zipper (bHLH-Zip) region as well as cleavage sites. As in the human genome, SREBP-1 and SREBP-2 chicken genes are located on two separate chromosomes, respectively microchromosome 14 and macrochromosome 1. Tissue expression data show that SREBP-1 and SREBP-2 are expressed in a wide variety of tissues in chicken. However, unlike SREBP-2, SREBP-1 is expressed preferentially in the liver and uropygial gland, suggesting an important role of SREBP-1 in the regulation of lipogenesis in avian species.

A versatile and efficient high-throughput cloning tool for structural biology.

PubMed

Geertsma, Eric R; Dutzler, Raimund

2011-04-19

Methods for the cloning of large numbers of open reading frames into expression vectors are of critical importance for challenging structural biology projects. Here we describe a system termed fragment exchange (FX) cloning that facilitates the high-throughput generation of expression constructs. The method is based on a class IIS restriction enzyme and negative selection markers. FX cloning combines attractive features of established recombination- and ligation-independent cloning methods: It allows the straightforward transfer of an open reading frame into a variety of expression vectors and is highly efficient and very economic in its use. In addition, FX cloning avoids the common but undesirable feature of significantly extending target open reading frames with cloning related sequences, as it leaves a minimal seam of only a single extra amino acid to either side of the protein. The method has proven to be very robust and suitable for all common pro- and eukaryotic expression systems. It considerably speeds up the generation of expression constructs compared to traditional methods and thus facilitates a broader expression screening.

Cloning of rat amelotin and localization of the protein to the basal lamina of maturation stage ameloblasts and junctional epithelium.

PubMed

Moffatt, Pierre; Smith, Charles E; St-Arnaud, René; Simmons, Darrin; Wright, J Timothy; Nanci, Antonio

2006-10-01

Formation of tooth enamel is a very complex process in which a specific set of proteins secreted by ameloblasts play a primordial role. As part of a screening procedure to identify novel proteins secreted by EO (enamel organ) cells of rat incisors, we isolated a partial cDNA fragment (EO-017) that is the homologue of the recently described mouse Amtn (amelotin) gene [Iwasaki, Bajenova, Somogyi-Ganss, Miller, Nguyen, Nourkeyhani, Gao, Wendel and Ganss (2005) J. Dent. Res. 84, 1127-1132]. Presented herein is the cloning of rat and pig full-length cDNAs with their deduced protein sequences. Detailed expression profiling by Northern-blot analysis and RT (reverse transcriptase)-PCR on rat and mouse tissues revealed highest expression in the mandible, more specifically in the maturation stage of the EO. Among all tissues tested, low expression was detected only in periodontal ligament, lung, thymus and gingiva. In silico analyses revealed that the Amtn gene is highly conserved in seven other mammals, but is absent from fish, birds and amphibians. The Amtn protein is enriched in proline, leucine, glutamine and threonine (52% of total) and contains a perfectly conserved protein kinase CK2 phosphorylation site. Transient transfection experiments in HEK-293 cells (human embryonic kidney cells) showed that secreted Amtn is post-translationally modified possibly through O-linked oligosaccharides on threonine residues. In concordance with its predominant expression site, immunofluorescence localization within the rat and mouse mandibles revealed Amtn localized to the basal lamina of maturation stage ameloblasts of incisors and unerupted molars. Intense Amtn protein expression was also detected in the internal basal lamina of junctional epithelium in molars. The peculiar and unique cellular localization of Amtn suggests a role in cell adhesion.

Molecular profiling of tumor progression in head and neck cancer.

PubMed

Belbin, Thomas J; Singh, Bhuvanesh; Smith, Richard V; Socci, Nicholas D; Wreesmann, Volkert B; Sanchez-Carbayo, Marta; Masterson, Jessica; Patel, Snehal; Cordon-Cardo, Carlos; Prystowsky, Michael B; Childs, Geoffrey

2005-01-01

To assess gene expression changes associated with tumor progression in patients with squamous cell carcinoma of the oral cavity. A microarray containing 17 840 complementary DNA clones was used to measure gene expression changes associated with tumor progression in 9 patients with squamous cell carcinoma of the oral cavity. Samples were taken for analysis from the primary tumor, nodal metastasis, and "normal" mucosa from the patients' oral cavity. Tertiary care facility. Patients Nine patients with stage III or stage IV untreated oral cavity squamous cell carcinoma. Our analysis to categorize genes based on their expression patterns has identified 140 genes that consistently increased in expression during progression from normal tissue to invasive tumor and subsequently to metastatic node (in at least 4 of the 9 cases studied). A similar list of 94 genes has been identified that decreased in expression during tumor progression and metastasis. We validated this gene discovery approach by selecting moesin (a member of the ezrin/radixin/moesin [ERM] family of cytoskeletal proteins) and one of the genes that consistently increased in expression during tumor progression for subsequent immunohistochemical analysis using a head and neck squamous cell carcinoma tissue array. A distinct pattern of gene expression, with progressive up- or down-regulation of expression, is found during the progression from histologically normal tissue to primary carcinoma and to nodal metastasis.

Tissue expression analysis, cloning and characterization of the 5'-regulatory region of the bovine FABP3 gene.

PubMed

Li, Anning; Wu, Lijuan; Wang, Xiaoyu; Xin, Yaping; Zan, Linsen

2016-09-01

Fatty acid binding protein 3 (FABP3) is a member of the FABP family which bind fatty acids and have an important role in fatty acid metabolism. A large number of studies have shown that the genetic polymorphisms of FABP3 are positively correlated with intramuscular fat (IMF) content in domestic animals, however, the function and transcriptional characteristics of FABP3 in cattle remain unclear. Real-time PCR analysis revealed that bovine FABP3 was highly expressed in cardiac tissue. The 5'-regulatory region of bovine FABP3 was cloned and its transcription initiation sites were identified. Sequence analysis showed that many transcriptional factor binding sites including TATA-box and CCAAT-box were present on the 5'-flanking region of bovine FABP3, and four CpG islands were found on nucleotides from -891 to +118. Seven serial deletion constructs of the 5'-regulatory region evaluated in dual-luciferase reporter assay indicated that its core promoter was 384 base pairs upstream from the transcription initiation site. The transcriptional factor binding sites RXRα, KLF15, CREB and Sp1 were conserved in the core promoter of cattle, sheep, pigs and dogs. These results provide further understanding of the function and regulation mechanism of bovine FABP3.

Molecular cloning of the heat shock protein 20 gene from Paphia textile and its expression in response to heat shock

NASA Astrophysics Data System (ADS)

Li, Jiakai; Wu, Xiangwei; Tan, Jing; Zhao, Ruixiang; Deng, Lingwei; Liu, Xiande

2015-07-01

P. textile is an important aquaculture species in China and is mainly distributed in Fujian, Guangdong, and Guangxi Provinces. In this study, an HSP20 cDNA designated PtHSP20 was cloned from P. textile. The full-length cDNA of PtHSP20 is 1 090 bp long and contains a 5' untranslated region (UTR) of 93 bp, a 3' UTR of 475 bp, and an open reading frame (ORF) of 522 bp. The PtHSP20 cDNA encodes 173 amino acid residues and has a molecular mass of 20.22 kDa and an isoelectric point of 6.2. Its predicted amino acid sequence shows that PtHSP20 contains a typical α-crystallin domain (residues 77-171) and three polyadenylation signal-sequences at the C-terminus. According to an amino acid sequence alignment, PtHSP20 shows moderate homology to other mollusk sHSPs. PtHSP20 mRNA was present in all of the test tissues including the heart, digestive gland, adductor muscle, gonad, gill, and mantle, with the highest concentration found in the gonad. Under the stress of high temperature, the expression of PtHSP20 mRNA was down-regulated in all of the tissues except the adductor muscle and gonad.

cDNA cloning, tissue distribution, and chromosomal localization of myelodysplasia/Myeloid Leukemia Factor 2 (MLF2)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kuefer, M.U.; Valentine, V.; Behm, F.G.

A fusion gene between nucleophosmin (NPM) and myelodysplasia/myeloid leukemia factor 1 (MLF1) and myelodysplasia/myeloid leukemia factor 1 (MLF1) is formed by a recurrent t(3;5)(q25.1;q34) in myelodysplastic syndrome and acute myeloid leukemia. Here we report the identification of a novel gene, MLF2, which contains an open reading frame of 744 bp encoding a 248-amino-acid protein highly related to the previously identified MLF1 protein (63% similarity, 40% identity). In contrast to the tissue-restricted expression pattern of MLF1, and MLF2 messenger RNA is expressed ubiquitously. The MLF2 gene locus was mapped by fluorescence in situ hybridization to human chromosome 12p13, a chromosomal regionmore » frequently involved in translocations and deletions in acute leukemias of lymphoid or myeloid lineage. In a physical map of chromosome 12, MLF2 was found to reside on the yeast artificial chromosome clone 765b9. Southern blotting analysis of malignant cell DNAs prepared from a series of acute lymphoblastic leukemia cases with translocations involving chromosome arm 12p, as well as a group of acute myeloid leukemias with various cytogenetic abnormalities, failed to reveal MLF2 gene rearrangements. 19 refs., 2 figs.« less

cDNA cloning and characterization of mouse DTEF-1 and ETF, members of the TEA/ATTS family of transcription factors.

PubMed

Yockey, C E; Shimizu, N

1998-02-01

Members of the TEA/ATTS family of transcription factors have been found in most representative eukaryotic organisms. In vertebrates, the TEA family contains at least four members, which share overlapping DNA-binding specificity and have similar transcriptional activation properties. In this article, we describe the cDNA cloning and characterization of the murine TEA proteins DTEF-1 (mDTEF-1) and ETF. Using in situ hybridization analysis of mouse embryos, we found that mDTEF-1 and ETF transcript distributions substantially overlap. ETF is expressed throughout the embryo except in the myocardium early in development, whereas late in development, it is enriched in lung and neuroectoderm. Mouse DTEF-1 is expressed at a much lower level throughout development and is substantially enriched in ectoderm and skin, as well as in the developing pituitary at midgestation. Northern blot analysis of adult mouse tissue total RNA showed that both ETF and mDTEF-1 are abundant in uterus and lung relative to other tissues. Using gel mobility shift assays and GAL4-fusion protein analysis, we demonstrated that the full coding sequences of ETF and mDTEF-1 encode M-CAT/GT-IIC-binding proteins containing activation domains.

Involvement of Ethylene in the Latex Metabolism and Tapping Panel Dryness of Hevea brasiliensis

PubMed Central

Putranto, Riza-Arief; Herlinawati, Eva; Rio, Maryannick; Leclercq, Julie; Piyatrakul, Piyanuch; Gohet, Eric; Sanier, Christine; Oktavia, Fetrina; Pirrello, Julien; Kuswanhadi; Montoro, Pascal

2015-01-01

Ethephon, an ethylene releaser, is used to stimulate latex production in Hevea brasiliensis. Ethylene induces many functions in latex cells including the production of reactive oxygen species (ROS). The accumulation of ROS is responsible for the coagulation of rubber particles in latex cells, resulting in the partial or complete stoppage of latex flow. This study set out to assess biochemical and histological changes as well as changes in gene expression in latex and phloem tissues from trees grown under various harvesting systems. The Tapping Panel Dryness (TPD) susceptibility of Hevea clones was found to be related to some biochemical parameters, such as low sucrose and high inorganic phosphorus contents. A high tapping frequency and ethephon stimulation induced early TPD occurrence in a high latex metabolism clone and late occurrence in a low latex metabolism clone. TPD-affected trees had smaller number of laticifer vessels compared to healthy trees, suggesting a modification of cambial activity. The differential transcript abundance was observed for twenty-seven candidate genes related to TPD occurrence in latex and phloem tissues for ROS-scavenging, ethylene biosynthesis and signalling genes. The predicted function for some Ethylene Response Factor genes suggested that these candidate genes should play an important role in regulating susceptibility to TPD. PMID:26247941

Involvement of Ethylene in the Latex Metabolism and Tapping Panel Dryness of Hevea brasiliensis.

PubMed

Putranto, Riza-Arief; Herlinawati, Eva; Rio, Maryannick; Leclercq, Julie; Piyatrakul, Piyanuch; Gohet, Eric; Sanier, Christine; Oktavia, Fetrina; Pirrello, Julien; Kuswanhadi; Montoro, Pascal

2015-08-04

Ethephon, an ethylene releaser, is used to stimulate latex production in Hevea brasiliensis. Ethylene induces many functions in latex cells including the production of reactive oxygen species (ROS). The accumulation of ROS is responsible for the coagulation of rubber particles in latex cells, resulting in the partial or complete stoppage of latex flow. This study set out to assess biochemical and histological changes as well as changes in gene expression in latex and phloem tissues from trees grown under various harvesting systems. The Tapping Panel Dryness (TPD) susceptibility of Hevea clones was found to be related to some biochemical parameters, such as low sucrose and high inorganic phosphorus contents. A high tapping frequency and ethephon stimulation induced early TPD occurrence in a high latex metabolism clone and late occurrence in a low latex metabolism clone. TPD-affected trees had smaller number of laticifer vessels compared to healthy trees, suggesting a modification of cambial activity. The differential transcript abundance was observed for twenty-seven candidate genes related to TPD occurrence in latex and phloem tissues for ROS-scavenging, ethylene biosynthesis and signalling genes. The predicted function for some Ethylene Response Factor genes suggested that these candidate genes should play an important role in regulating susceptibility to TPD.

Differential gene expression by Moniliophthora roreri while overcoming cacao tolerance in the field.

PubMed

Bailey, Bryan A; Melnick, Rachel L; Strem, Mary D; Crozier, Jayne; Shao, Jonathan; Sicher, Richard; Phillips-Mora, Wilberth; Ali, Shahin S; Zhang, Dapeng; Meinhardt, Lyndel

2014-09-01

Frosty pod rot (FPR) of Theobroma cacao (cacao) is caused by the hemibiotrophic fungus Moniliophthora roreri. Cacao clones tolerant to FPR are being planted throughout Central America. To determine whether M. roreri shows a differential molecular response during successful infections of tolerant clones, we collected field-infected pods at all stages of symptomatology for two highly susceptible clones (Pound-7 and CATIE-1000) and three tolerant clones (UF-273, CATIE-R7 and CATIE-R4). Metabolite analysis was carried out on clones Pound-7, CATIE-1000, CATIE-R7 and CATIE-R4. As FPR progressed, the concentrations of sugars in pods dropped, whereas the levels of trehalose and mannitol increased. Associations between symptoms and fungal loads and some organic and amino acid concentrations varied depending on the clone. RNA-Seq analysis identified 873 M. roreri genes that were differentially expressed between clones, with the primary difference being whether the clone was susceptible or tolerant. Genes encoding transcription factors, heat shock proteins, transporters, enzymes modifying membranes or cell walls and metabolic enzymes, such as malate synthase and alternative oxidase, were differentially expressed. The differential expression between clones of 43 M. roreri genes was validated by real-time quantitative reverse transcription polymerase chain reaction. The expression profiles of some genes were similar in susceptible and tolerant clones (other than CATIE-R4) and varied with the biotrophic/necrotropic shift. Moniliophthora roreri genes associated with stress metabolism and responses to heat shock and anoxia were induced early in tolerant clones, their expression profiles resembling that of the necrotrophic phase. Moniliophthora roreri stress response genes, induced during the infection of tolerant clones, may benefit the fungus in overcoming cacao defense mechanisms. © 2014 BSPP AND JOHN WILEY & SONS LTD.

Nutritional impact on gene expression and competence of oocytes used to support embryo development and livebirth by cloning procedures in goats.

PubMed

Fernandes, C C L; Aguiar, L H; Calderón, C E M; Silva, A M; Alves, J P M; Rossetto, R; Bertolini, L R; Bertolini, M; Rondina, D

2018-01-01

Changes in the nutritional plan have been shown to affect oocyte quality, crucial to oocyte donors animals used in cloning. This study aimed to evaluate the impact of diets with increasing nutritional levels (maintenance diet=M; 1.3M; 1.6M; 1.9M) fed to goats for four weeks on follicular fluid composition, gene expression and oocyte competence used to cloning in goats. Donor females were superovulated for the retrieval of matured oocytes and physical measurements reported. After four weeks, groups receiving diets above maintenance increased thickness of subcutaneous adipose tissue and body weight, with higher values in 1.9M Group (P<0.05). Treatments did not affect follicular density, number of aspirated follicles, retrieved and matured oocytes. Animals from 1.3M group had lower (P<0.05) maturation rate (44.0%) and number of viable oocytes (65.3%) than M (68.8%) and 1.9M (76.0%). Follicular fluid glucose concentrations increased with nutritional levels (P=0.010), with a difference (P<0.05) between groups 1.9M (11.4±2.6mg/dL) and M (2.6±0.5mg/dL). The diet did not affect the expression of GDF9, BMP15, and BAX genes in oocytes, but BCL2 and apoptotic index were significantly higher (P<0.05) in the 1.3M and 1.6M groups than the other groups. Following the transfer of cloned embryos, one fetus was born live of a twin pregnancy in the 1.9M Group. The association between energy intake and oocyte quality suggests better nutritional use by oocytes when the maximum flow was used (1.9M), but the optimal feeding level in cloning still needs refinement. Copyright © 2017 Elsevier B.V. All rights reserved.

Cloning and identification of a cDNA that encodes a novel human protein with thrombospondin type I repeat domain, hPWTSR.

PubMed

Chen, Jin-Zhong; Wang, Shu; Tang, Rong; Yang, Quan-Sheng; Zhao, Enpeng; Chao, Yaoqiong; Ying, Kang; Xie, Yi; Mao, Yu-Min

2002-09-01

A cDNA was isolated from the fetal brain cDNA library by high throughput cDNA sequencing. The 2390 bp cDNA with an open reading fragment (ORF) of 816 bp encodes a 272 amino acids putative protein with a thrombospondin type I repeat (TSR) domain and a cysteine-rich region at the N-terminus, so it is named hPWTSR. We used Northern blot detected two bands with length of about 3 kb and 4 kb respectively, which expressed in human adult tissues with different intensities. The expression pattern was verified by RT-PCR, revealing that the transcripts were expressed ubiquitously in fetal tissues and human tumor tissues too. However, the transcript was detected neither in ovarian carcinoma GI-102 nor in lung carcinoma LX-1. Blast analysis against NCBI database revealed that the new gene contained at least 5 exons and located in human chromosome 6q22.33. Our results demonstrate that the gene is a novel member of TSR supergene family.

In vivo gene expression and immunoreactivity of Leptospira collagenase.

PubMed

Janwitthayanan, Weena; Keelawat, Somboon; Payungporn, Sunchai; Lowanitchapat, Alisa; Suwancharoen, Duangjai; Poovorawan, Yong; Chirathaworn, Chintana

2013-06-12

Pulmonary hemorrhage is an increasing cause of death of leptospirosis patients. Bacterial collagenase has been shown to be involved in lung hemorrhage induced by various infectious agents. According to Leptospira whole genome study, colA, a gene suggested to code for bacterial collagenase has been identified. We investigated colA gene expression in lung tissues of Leptospira infected hamsters. Golden Syrian Hamsters were injected intraperitoneally with Leptospira interrogans serovar Pyrogenes. The hamsters were sacrificed on days 3, 5 and 7 post-infection and lung tissues were collected for histological examination and RNA extraction. Lung pathologies including atelectasis and hemorrhage were observed. Expression of colA gene in lung tissues was demonstrated by both RT-PCR and real time PCR. In addition, ColA protein was cloned and the purified protein could react with sera from leptospirosis patients. Leptospira ColA protein may play a role in Leptospira survival or pathogenesis in vivo. Its reaction with leptospirosis sera suggests that this protein is immunogenic and could be another candidate for vaccine development. Copyright © 2012 Elsevier GmbH. All rights reserved.

Biosynthesis of Polyunsaturated Fatty Acids in the Razor Clam Sinonovacula constricta: Characterization of Δ5 and Δ6 Fatty Acid Desaturases.

PubMed

Ran, Zhaoshou; Xu, Jilin; Liao, Kai; Li, Shuang; Chen, Shubing; Yan, Xiaojun

2018-05-09

To investigate the endogenous long-chain polyunsaturated fatty acid (LC-PUFA) biosynthetic ability in Sinonovacula constricta, fatty acid desaturases (Fads) of this bivalve, namely, Scfad5a, Scfad5b, and Scfad6, were cloned and characterized in the current study. Meanwhile, the tissue distributions of S. constricta Fads and fatty acids (FAs) were examined. Heterologous expression in yeasts confirmed that Scfad5a and Scfad5b were both Δ5 Fads, while Scfad6 was a Δ6 Fad. However, compared with Fads in other organisms, the desaturation activities of S. constricta Fads were relatively low (especially for Scfad6), indicating an adaptation to living conditions. S. constricta Fads were expressed in all tissues examined, and particularly high expressions were found in intestine and gonad. Moreover, FAs were differently distributed among tissues, which might be correlated with their corresponding physiological roles. Taken together, the results provided an insight into LC-PUFA biosynthesis in S. constricta. Notably, Scfad6 was the first functionally characterized Δ6 Fad in marine molluscs to date.

Expression and Localization of Plant Protein Disulfide Isomerase.

PubMed Central

Shorrosh, B. S.; Subramaniam, J.; Schubert, K. R.; Dixon, R. A.

1993-01-01

A cDNA clone encoding a putative protein disulfide isomerase (PDI, EC 5.3.4.1) from alfalfa (Medicago sativa L.) was expressed in Escherichia coli cells, and an antiserum was raised against the expressed PDI-active protein. The antiserum recognized a protein of approximately 60 kD in extracts from alfalfa, soybean, and tobacco roots and stems. Levels of this protein remained relatively constant on exposure of alfalfa cell suspension cultures to the protein glycosylation inhibitor tunicamycin, whereas a slightly lower molecular mass form, also detected by the antiserum, was induced by this treatment. A lower molecular mass form of PDI was also observed in roots of alfalfa seedlings during the first 5 weeks after germination. PDI levels increased in developing soybean seeds up to 17 d after fertilization and then declined. Tissue print immunoblots revealed highest levels of PDI protein in the cambial tissues of soybean stems and petioles and in epidermal, subepidermal, cortical, and pith tissues of stems of alfalfa and tobacco. Immunogold electron microscopy confirmed the localization of PDI to the endoplasmic reticulum in soybean root nodules. PMID:12231974

[TSA improve transgenic porcine cloned embryo development and transgene expression].

PubMed

Kong, Qing-Ran; Zhu, Jiang; Huang, Bo; Huan, Yan-Jun; Wang, Feng; Shi, Yong-Qian; Liu, Zhong-Feng; Wu, Mei-Ling; Liu, Zhong-Hua

2011-07-01

Uncompleted epigenetic reprogramming is attributed to the low efficiency of producing transgenic cloned animals. Histone modification associated with epigenetics can directly influence the embryo development and transgene expression. Trichostatin A (TSA), as an inhibitor of histone deacetylase, can change the status of histone acetylation, improve somatic cell reprogramming, and enhance cloning efficiency. TSA prevents the chromatin structure from being condensed, so that transcription factor could binds to DNA sequence easily and enhance transgene expression. Our study established the optimal TSA treatment on porcine donor cells and cloned embryos, 250 nmol/L, 24 h and 40 nmol/L, 24 h, respectively. Furthermore, we found that both the cloned embryo and the donor cell treated by TSA resulted in the highest development efficiency. Meanwhile, TSA can improve transgene expression in donor cell and cloned embryo. In summary, TSA can significantly improve porcine reconstructed embryo development and transgene expression.

Cloning, cell-type specificity, and regulatory function of the mouse alpha(1B)-adrenergic receptor promoter.

PubMed

Zuscik, M J; Piascik, M T; Perez, D M

1999-12-01

The functionality of a 3422-base pair promoter fragment from the mouse alpha(1B)-adrenergic receptor (alpha(1B)AR) gene was examined. This fragment, cloned from a mouse genomic library, was found to have significant sequence homology to the known human and rat alpha(1B)AR promoters. However, the consensus motif of several key cis-acting elements is not conserved among the rat, human, and mouse genes, suggesting species specificity. Confirming fidelity of the murine promoter, robust in vitro expression of a chloramphenicol acetyltransferase (CAT) reporter was detected in known alpha(1B)AR-expressing BC(3)H1, NB41A3, and DDT(1)MF-2 cells transiently transfected with a promoter-CAT construct. Conversely, minimal CAT expression was detected in known alpha(1B)AR-negative RAT-1 and R3T3 cells. These findings were extended by transfecting the same promoter-CAT construct into various primary cell types. In support of the hypothesis that alpha(1)ARs are differentially expressed in the smooth muscle of the vasculature, primary cultures of superior mesenteric and renal artery vascular smooth muscle cells showed significantly stronger CAT expression than did vascular smooth muscle cells derived from pulmonary, femoral, and iliac arteries. Primary osteoblastic bone-forming cells, which are known to be alpha(1B)AR negative, showed minimal CAT expression. Indicating regulatory function through cis-acting elements, RAT-1, R3T3, NB41A3, BC(3)H1, and DDT(1)MF2 cells transfected with the promoter-CAT construct all showed increased CAT production when challenged with forskolin or hypoxic conditions. Additionally, tissue-specific regulation of the promoter was observed when cells were simultaneously challenged with both forskolin and hypoxia. These results collectively demonstrate that a 3.4-kb PvuII fragment of the murine alpha(1B)AR gene promoter can: 1) drive tissue-specific production of a CAT reporter in both clonal and primary cell lines; and 2) confer tissue-specific regulation of that CAT reporter when induced by challenge with forskolin and/or hypoxic conditions.

Characterization of X-OCRL, a Xenopus laevis homologue of OCRL-1, the Lowe oculocerebrorenal syndrome candidate gene

DOE Office of Scientific and Technical Information (OSTI.GOV)

Reilly, D.S.; Nussbaum, R.L.

1994-09-01

The Lowe oculocerebrorenal syndrome (OCRL) is an X-linked disease characterized by congenital cataract, mental retardation, and renal tubular dysfunction. A candidate cDNA, OCRL-1, was identified by positional cloning and mutations in OCRL-1 have been detected in patients with Lowe syndrome. The OCRL-1 nucleotide sequence encodes a predicted protein of 968 amino acids and shares 51% amino acid identity with a human inositol polyphosphate-5-phosphatase. This suggests that the underlying defect in OCRL may be due to a defect in inositol phosphate metabolism. The isolation of OCRL-1 provides the opportunity to investigate its function through the use of animal model systems. Wemore » have isolated a partial cDNA clone encoding an OCRL-1 homologue, X-OCRL, from the South African clawed frog, Xenopus laevis. We used a portion of the human cDNA to screen a Xenopus laevis embryo cDNA library and isolated four positive clones. One clone, 42-5A, is a 650 bp insert with over 75% amino acid identity to the corresponding region of the human OCRL-1 sequence. 42-5A detects messenger RNA in adult Xenopus brain, stomach, small intestine, skin, muscle, lung, blood, and oviduct. X-OCRL messenger RNA is first detected during late gastrula and continues to be expressed throughout Xenopus development. In situ hybridization studies are underway to identify the cellular localization of X-OCRL expression in Xenopus embryos and adult tissues. We are especially interested in characterizing X-OCRL expression during formation of the amphibian lens since congenital cataracts are a constant feature of the human disease.« less

Insights in KIR2.1 channel structure and function by an evolutionary approach; cloning and functional characterization of the first reptilian inward rectifier channel KIR2.1, derived from the California kingsnake (Lampropeltis getula californiae).

PubMed

Houtman, Marien J C; Korte, Sanne M; Ji, Yuan; Kok, Bart; Vos, Marc A; Stary-Weinzinger, Anna; van der Heyden, Marcel A G

2014-10-03

Potassium inward rectifier KIR2.1 channels contribute to the stable resting membrane potential in a variety of muscle and neuronal cell-types. Mutations in the KIR2.1 gene KCNJ2 have been associated with human disease, such as cardiac arrhythmias and periodic paralysis. Crystal structure and homology modelling of KIR2.1 channels combined with functional current measurements provided valuable insights in mechanisms underlying channel function. KIR2.1 channels have been cloned and analyzed from all main vertebrate phyla, except reptilians. To address this lacuna, we set out to clone reptilian KIR2.1 channels. Using a degenerated primer set we cloned the KCNJ2 coding regions from muscle tissue of turtle, snake, bear, quail and bream, and compared their deduced amino acid sequences with those of KIR2.1 sequences from 26 different animal species obtained from Genbank. Furthermore, expression constructs were prepared for functional electrophysiological studies of ectopically expressed KIR2.1 ion channels. In general, KCNJ2 gene evolution followed normal phylogenetic patterns, however turtle KIR2.1 ion channel sequence is more homologues to avians than to snake. Alignment of all 31 KIR2.1 sequences showed that all disease causing KIR2.1 mutations, except V93I, V123G and N318S, are fully conserved. Homology models were built to provide structural insights into species specific amino acid substitutions. Snake KIR2.1 channels became expressed at the plasmamembrane and produced typical barium sensitive (IC50 ∼6μM) inward rectifier currents. Copyright © 2014 Elsevier Inc. All rights reserved.

Characterization of xanthine dehydrogenase and aldehyde oxidase of Marsupenaeus japonicus and their response to microbial pathogen.

PubMed

Okamura, Yo; Inada, Mari; Elshopakey, Gehad Elsaid; Itami, Toshiaki

2018-05-16

Reactive oxygen species (ROS) play key roles in many physiological processes. In particular, the sterilization mechanism of bacteria using ROS in macrophages is a very important function for biological defense. Xanthine dehydrogenase (XDH) and aldehyde oxidase (AOX), members of the molybdo-flavoenzyme subfamily, are known to generate ROS. Although these enzymes occur in many vertebrates, some insects, and plants, little research has been conducted on XDHs and AOXs in crustaceans. Here, we cloned the entire cDNA sequences of XDH (MjXDH: 4328 bp) and AOX (MjAOX: 4425 bp) from Marsupenaeus japonicus (kuruma shrimp) using reverse transcriptase-polymerase chain reaction (RT-PCR) and random amplification of cDNA ends (RACE). Quantitative real-time RT-PCR transcriptional analysis revealed that MjXDH mRNA is highly expressed in heart and stomach tissues, whereas MjAOX mRNA is highly expressed in the lymphoid organ and intestinal tissues. Furthermore, expression of MjAOX was determined to be up-regulated in the lymphoid organ in response to Vibrio penaeicida at 48 and 72 h after injection; in contrast, hydrogen peroxide (H 2 O 2 ) concentrations increased significantly at 6, 12, 48, and 72 h after injection with white spot syndrome virus (WSSV) and at 72 h after injection with V. penaeicida. To the best of our knowledge, this study is the first to have identified and cloned XDH and AOX from a crustacean species.

Molecular analysis of two cDNA clones encoding acidic class I chitinase in maize.

PubMed Central

Wu, S; Kriz, A L; Widholm, J M

1994-01-01

The cloning and analysis of two different cDNA clones encoding putative maize (Zea mays L.) chitinases obtained by polymerase chain reaction (PCR) and cDNA library screening is described. The cDNA library was made from poly(A)+ RNA from leaves challenged with mercuric chloride for 2 d. The two clones, pCh2 and pCh11, appear to encode class I chitinase isoforms with cysteine-rich domains (not found in pCh11 due to the incomplete sequence) and proline-/glycine-rich or proline-rich hinge domains, respectively. The pCh11 clone resembles a previously reported maize seed chitinase; however, the deduced proteins were found to have acidic isoelectric points. Analysis of all monocot chitinase sequences available to date shows that not all class I chitinases possess the basic isoelectric points usually found in dicotyledonous plants and that monocot class II chitinases do not necessarily exhibit acidic isoelectric points. Based on sequence analysis, the pCh2 protein is apparently synthesized as a precursor polypeptide with a signal peptide. Although these two clones belong to class I chitinases, they share only about 70% amino acid homology in the catalytic domain region. Southern blot analysis showed that pCh2 may be encoded by a small gene family, whereas pCh11 was single copy. Northern blot analysis demonstrated that these genes are differentially regulated by mercuric chloride treatment. Mercuric chloride treatment caused rapid induction of pCh2 from 6 to 48 h, whereas pCh11 responded only slightly to the same treatment. During seed germination, embryos constitutively expressed both chitinase genes and the phytohormone abscisic acid had no effect on the expression. The fungus Aspergillus flavus was able to induce both genes to comparable levels in aleurone layers and embryos but not in endosperm tissue. Maize callus growth on the same plate with A. flavus for 1 week showed induction of the transcripts corresponding to pCh2 but not to pCh11. These studies indicate that the different chitinase isoforms in maize might have different functions in the plant, since they show differential expression patterns under different conditions. PMID:7972490

Cloning and expression profile of ionotropic receptors in the parasitoid wasp Microplitis mediator (Hymenoptera: Braconidae).

PubMed

Wang, Shan-Ning; Peng, Yong; Lu, Zi-Yun; Dhiloo, Khalid Hussain; Zheng, Yao; Shan, Shuang; Li, Rui-Jun; Zhang, Yong-Jun; Guo, Yu-Yuan

2016-07-01

Ionotropic receptors (IRs) mainly detect the acids and amines having great importance in many insect species, representing an ancient olfactory receptor family in insects. In the present work, we performed RNAseq of Microplitis mediator antennae and identified seventeen IRs. Full-length MmedIRs were cloned and sequenced. Phylogenetic analysis of the Hymenoptera IRs revealed that ten MmedIR genes encoded "antennal IRs" and seven encoded "divergent IRs". Among the IR25a orthologous groups, two genes, MmedIR25a.1 and MmedIR25a.2, were found in M. mediator. Gene structure analysis of MmedIR25a revealed a tandem duplication of IR25a in M. mediator. The tissue distribution and development specific expression of the MmedIR genes suggested that these genes showed a broad expression profile. Quantitative gene expression analysis showed that most of the genes are highly enriched in adult antennae, indicating the candidate chemosensory function of this family in parasitic wasps. Using immunocytochemistry, we confirmed that one co-receptor, MmedIR8a, was expressed in the olfactory sensory neurons. Our data will supply fundamental information for functional analysis of the IRs in parasitoid wasp chemoreception. Copyright © 2016 Elsevier Ltd. All rights reserved.

Insertional Mutagenesis for Genes involved in Otic/Vestibular Development and Function in Xenopus Tropicalis

NASA Technical Reports Server (NTRS)

Torrejon, Marcela; Li, Erica; Nguyen, Minh; Winfree, Seth; Wang, Esther; Reinsch, Sigrid; Dalton, Bonnie (Technical Monitor)

2002-01-01

Sensitivity to gravity is essential for spatial orientation. Consequently, the gravity receptor system is one of the phylogenetically oldest sensory systems, and the special adaptations that enhance sensitivity to gravity are highly conserved. The main goal of this project is to use Xenopus (frog) to identify genes expressed during vestibular and auditory development. These studies will lead a better understanding of the molecular mechanisms involved in vestibular and auditory development and function. We are using a gene-trap approach in Xenopus tropicalis with the green fluorescent protein (GFP) gene as the transgene reporter. GFP expression occurs only when the GFP gene is correctly integrated in actively transcribed genes. Using the GFP as a tag we can easily identify and clone the mutated gene. In addition, we can study the function of the mutated gene by analyzing the defects generated by insertion of the GFP transgene. To date we have tissue specific GFP expression in X. tropicalis including expression in ear, neural tube, kidney, muscle, eyes and nose. Our transgenic animals will soon reach maturity so that we can outcross them and analyze their progeny. Our next goal is to isolate RNA from our transgenics and clone the tagged genes using RACE-PCR. Currently we are optimizing the RACE-PCR method using transgenics with crystallin GFP expression.

Transgene expression of green fluorescent protein and germ line transmission in cloned pigs derived from in vitro transfected adult fibroblasts.

PubMed

Brunetti, Dario; Perota, Andrea; Lagutina, Irina; Colleoni, Silvia; Duchi, Roberto; Calabrese, Fiorella; Seveso, Michela; Cozzi, Emanuele; Lazzari, Giovanna; Lucchini, Franco; Galli, Cesare

2008-12-01

The pig represents the xenogeneic donor of choice for future organ transplantation in humans for anatomical and physiological reasons. However, to bypass several immunological barriers, strong and stable human genes expression must occur in the pig's organs. In this study we created transgenic pigs using in vitro transfection of cultured cells combined with somatic cell nuclear transfer (SCNT) to evaluate the ubiquitous transgene expression driven by pCAGGS vector in presence of different selectors. pCAGGS confirmed to be a very effective vector for ubiquitous transgene expression, irrespective of the selector that was used. Green fluorescent protein (GFP) expression observed in transfected fibroblasts was also maintained after nuclear transfer, through pre- and postimplantation development, at birth and during adulthood. Germ line transmission without silencing of the transgene was demonstrated. The ubiquitous expression of GFP was clearly confirmed in several tissues including endothelial cells, thus making it a suitable vector for the expression of multiple genes relevant to xenotransplantation where tissue specificity is not required. Finally cotransfection of green and red fluorescence protein transgenes was performed in fibroblasts and after nuclear transfer blastocysts expressing both fluorescent proteins were obtained.

Molecular cloning and expression of collagenase-3, a novel human matrix metalloproteinase produced by breast carcinomas.

PubMed

Freije, J M; Díez-Itza, I; Balbín, M; Sánchez, L M; Blasco, R; Tolivia, J; López-Otín, C

1994-06-17

A cDNA coding for a new human matrix metalloproteinase (MMP) has been cloned from a cDNA library derived from a breast tumor. The isolated cDNA contains an open reading frame coding for a polypeptide of 471 amino acids. The predicted protein sequence displays extensive similarity to the previously known MMPs and presents all the structural features characteristic of the members of this protein family, including the well conserved PRCGXPD motif, involved in the latency of the enzyme and the zinc-binding domain (HEXGHXXXXXHS). In addition, this novel human MMP contains in its amino acid sequence several residues specific to the collagenase subfamily (Tyr-214, Asp-235, and Gly-237) and lacks the 9-residue insertion present in the stromelysins. According to these structural characteristics, the MMP described herein has been tentatively called collagenase-3, since it represents the third member of this subfamily, composed at present of fibroblast and neutrophil collagenases. The collagenase-3 cDNA was expressed in a vaccinia virus system, and the recombinant protein was able to degrade fibrillar collagens, providing support to the hypothesis that the isolated cDNA codes for an authentic collagenase. Northern blot analysis of RNA from normal and pathological tissues demonstrated the existence in breast tumors of three different mRNA species, which seem to be the result of the utilization of different polyadenylation sites present in the 3'-noncoding region of the gene. By contrast, no collagenase-3 mRNA was detected either by Northern blot or RNA polymerase chain reaction analysis with RNA from other human tissues, including normal breast, mammary fibroadenomas, liver, placenta, ovary, uterus, prostate, and parotid gland. On the basis of the increased expression of collagenase-3 in breast carcinomas and the absence of detectable expression in normal tissues, a possible role for this metalloproteinase in the tumoral process is proposed.

Molecular cloning and characterization of chicken prostaglandin E receptor subtypes 2 and 4 (EP2 and EP4).

PubMed

Kwok, Amy Ho Yan; Wang, Yajun; Wang, Crystal Ying; Leung, Frederick C

2008-06-01

Prostaglandin E(2) (PGE(2)) is an important chemical mediator responsible for regulation of many vital physiological processes. Four receptor subtypes have been identified to mediate its biological actions. Among these subtypes, prostaglandin E receptor subtypes 2 and 4 (EP(2) and EP(4)), both coupled to cAMP-protein kinase A (cAMP-PKA) signaling pathway, are proposed to play crucial roles under both physiological and pathological conditions. Though both receptors were extensively studied in mammals, little is known about their functionality and expression in non-mammalian species including chicken. In present study, the full-length cDNAs for chicken EP(2) and EP(4) receptors were first cloned from adult chicken ovary and testis, respectively. Chicken EP(2) is 356 amino acids in length and shows high amino acid identity to that of human (61%), mouse (63%), and rat (61%). On the other hand, the full-length cDNA of EP(4) gene encodes a precursor of 475 amino acids with a high degree of amino acid identity to that of mammals, including human (87%), mouse (86%), rat (84%), dog (85%), and cattle (83%), and a comparatively lower sequence identity to zebrafish (52%). RT-PCR assays revealed that EP(2) mRNA was expressed in all tissues examined including the oviduct, while EP(4) expression was detected only in a few tissues. Using the pGL3-CRE-luciferase reporter system, we also demonstrated that PGE(2) could induce luciferase activity in DF-1 cells expressing EP(2) and EP(4) in dose-dependent manners (EC(50): <1 nM), confirming that both receptors could be activated by PGE(2) and functionally coupled to the cAMP-PKA signaling pathway. Together, our study establishes a molecular basis to understand the physiological roles of PGE(2) in target tissues of chicken.

Micropropagation of transgenic lettuce containing HBsAg as a method of mass-scale production of standardised plant material for biofarming purposes.

PubMed

Pniewski, Tomasz; Czyż, Marcin; Wyrwa, Katarzyna; Bociąg, Piotr; Krajewski, Paweł; Kapusta, Józef

2017-01-01

Micropropagation protocol of transgenic lettuce bearing S-, M- and L-HBsAg was developed for increased production of uniformised material for oral vaccine preparation. Effective manufacturing of plant-based biopharmaceuticals, including oral vaccines, depends on sufficient content of a protein of interest in the initial material and its efficient conversion into an administrable formulation. However, stable production of plants with a uniformised antigen content is equally important for reproducible processing. This can be provided by micropropagation techniques. Here, we present a protocol for micropropagation of transgenic lettuce lines bearing HBV surface antigens: S-, M- and L-HBsAg. These were multiplied through axillary buds to avoid the risk of somaclonal variation. Micropropagation effectiveness reached 3.5-5.7 per passage, which implies potential production of up to 6600 plant clones within a maximum 5 months. Multiplication and rooting rates were statistically homogenous for most transgenic and control plants. For most lines, more than 90 % of clones obtained via in vitro micropropagation had HBsAg content as high as reference plants directly developed from seeds. Clones were also several times more uniform in HBsAg expression. Variation coefficients of HBsAg content did not exceed 10 % for approximately 40-85 % of clones, or reached a maximum 20 % for 90 % of all clones. Tissue culture did not affect total and leaf biomass yields. Seed production for clones was decreased insignificantly and did not impact progeny condition. Micropropagation facilitates a substantial increase in the production of lettuce plants with high and considerably equalised HBsAg contents. This, together with the previously reported optimisation of plant tissue processing and its long-term stability, constitutes a successive step in manufacturing of a standardised anti-HBV oral vaccine of reliable efficacy.

Oral lipase activities and fat-taste receptors for fat-taste sensing in chickens.

PubMed

Kawabata, Yuko; Kawabata, Fuminori; Nishimura, Shotaro; Tabata, Shoji

2018-01-01

It has been reported that a functional fat-taste receptor, GPR120, is present in chicken oral tissues, and that chickens can detect fat taste in a behavioral test. However, although triglycerides need to be digested to free fatty acids to be recognized by fat-taste receptors such as GPR120, it remains unknown whether lipase activities exist in chicken oral tissues. To examine this question, we first cloned another fat-taste receptor candidate gene, CD36, from the chicken palate. Then, using RT-PCR, we determined that GPR120 and CD36 were broadly expressed in chicken oral and gastrointestinal tissues. Also by RT-PCR, we confirmed that several lipase genes were expressed in both oral and gastrointestinal tissues. Finally, we analyzed the lipase activities of oral tissues by using a fluorogenic triglyceride analog as a lipase substrate. We found there are functional lipases in oral tissues as well as in the stomach and pancreas. These results suggested that chickens have a basic fat-taste reception system that incorporates a triglycerides/oral-lipases/free fatty acids/GPR120 axis and CD36 axis. Copyright © 2017 Elsevier Inc. All rights reserved.

Therapeutic cloning and tissue engineering.

PubMed

Koh, Chester J; Atala, Anthony

2004-01-01

A severe shortage of donor organs available for transplantation in the United States leaves patients suffering from diseased and injured organs with few treatment options. Scientists in the field of tissue engineering apply the principles of cell transplantation, material science, and engineering to construct biological substitutes that will restore and maintain normal function in diseased and injured tissues. Therapeutic cloning, where the nucleus from a donor cell is transferred into an enucleated oocyte in order to extract pluripotent embryonic stem cells, offers a potentially limitless source of cells for tissue engineering applications. The present chapter reviews recent advances that have occurred in therapeutic cloning and tissue engineering and describes applications of these new technologies that may offer novel therapies for patients with end-stage organ failure.

Identification of genes showing differential expression profile associated with growth rate in skeletal muscle tissue of Landrace weanling pig.

PubMed

Komatsu, Yuuta; Sukegawa, Shin; Yamashita, Mai; Katsuda, Naoki; Tong, Bin; Ohta, Takeshi; Kose, Hiroyuki; Yamada, Takahisa

2016-06-01

Suppression subtractive hybridization was used to identify genes showing differential expression profile associated with growth rate in skeletal muscle tissue of Landrace weanling pig. Two subtracted cDNA populations were generated from musculus longissimus muscle tissues of selected pigs with extreme expected breeding values at the age of 100 kg. Three upregulated genes (EEF1A2, TSG101 and TTN) and six downregulated genes (ATP5B, ATP5C1, COQ3, HADHA, MYH1 and MYH7) in pig with genetic propensity for higher growth rate were identified by sequence analysis of 12 differentially expressed clones selected by differential screening following the generation of the subtracted cDNA population. Real-time PCR analysis confirmed difference in expression profiles of the identified genes in musculus longissimus muscle tissues between the two Landrace weanling pig groups with divergent genetic propensity for growth rate. Further, differential expression of the identified genes except for the TTN was validated by Western blot analysis. Additionally, the eight genes other than the ATP5C1 colocalized with the same chromosomal positions as QTLs that have been previously identified for growth rate traits. Finally, the changes of expression predicted from gene function suggested association of upregulation of expression of the EEF1A2, TSG101 and TTN genes and downregulation of the ATP5B, ATP5C1, COQ3, HADHA, MYH1 and MYH7 gene expression with increased growth rate. The identified genes will provide an important insight in understanding the molecular mechanism underlying growth rate in Landrace pig breed.

Molecular Cloning, Characterization, and Functional Analysis of Acetyl-CoA C-Acetyltransferase and Mevalonate Kinase Genes Involved in Terpene Trilactone Biosynthesis from Ginkgo biloba.

PubMed

Chen, Qiangwen; Yan, Jiaping; Meng, Xiangxiang; Xu, Feng; Zhang, Weiwei; Liao, Yongling; Qu, Jinwang

2017-01-02

Ginkgolides and bilobalide, collectively termed terpene trilactones (TTLs), are terpenoids that form the main active substance of Ginkgo biloba . Terpenoids in the mevalonate (MVA) biosynthetic pathway include acetyl-CoA C -acetyltransferase (AACT) and mevalonate kinase (MVK) as core enzymes. In this study, two full-length (cDNAs) encoding AACT ( GbAACT , GenBank Accession No. KX904942) and MVK ( GbMVK , GenBank Accession No. KX904944) were cloned from G. biloba . The deduced GbAACT and GbMVK proteins contain 404 and 396 amino acids with the corresponding open-reading frame (ORF) sizes of 1215 bp and 1194 bp, respectively. Tissue expression pattern analysis revealed that GbAACT was highly expressed in ginkgo fruits and leaves, and GbMVK was highly expressed in leaves and roots. The functional complementation of GbAACT in AACT-deficient Saccharomyces cerevisiae strain Δerg10 and GbMVK in MVK-deficient strain Δerg12 confirmed that GbAACT mediated the conversion of mevalonate acetyl-CoA to acetoacetyl-CoA and GbMVK mediated the conversion of mevalonate to mevalonate phosphate. This observation indicated that GbAACT and GbMVK are functional genes in the cytosolic mevalonate (MVA) biosynthesis pathway. After G. biloba seedlings were treated with methyl jasmonate and salicylic acid, the expression levels of GbAACT and GbMVK increased, and TTL production was enhanced. The cloning, characterization, expression and functional analysis of GbAACT and GbMVK will be helpful to understand more about the role of these two genes involved in TTL biosynthesis.

Quantitative Evaluation of Myostatin Gene in Stably Transfected Caprine Fibroblast Cells by Anti-Myostatin shRNA.

PubMed

Jain, Sudhir Kumar; Jain, Hemlata; Kumar, Dharmendra; Bedekar, Megha Kadam; Pandey, Akhilesh Kumar; Sarkhel, Bikash Chandra

2015-09-01

Skeletal muscle is the major component of lean tissue that is used for consumption, and myostatin is a negative regulator of skeletal muscle growth. Downregulation of this gene therefore offers a strategy for developing superior animals with enhanced muscle growth. Knockdown of myostatin was achieved by RNA interference technology. The anti-myostatin shRNA were designed and stably transfected in caprine fibroblast cells. The reduced expression of target gene was achieved and measured in clonal fibroblast cells by real-time PCR. Two single-cell clones induced significant decrease of myostatin gene expression by 73.96 and 72.66 %, respectively (P < 0.05). To ensure the appropriate growth of transfected cell, seven media were tested. The best suited media was used for transfected fibroblast cell proliferation. The findings suggest that shRNA provides a novel potential tool for gene knockdown and these stably transfected cells can be used as the donor cells for animal cloning.

Cloning of a new member of the insulin gene superfamily (INSL4) expressed in human placenta

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chassin, D.; Laurent, A.; Janneau, J.L.

1995-09-20

A new member of the insulin gene superfamily was identified by screening a subtracted cDNA library of first-trimester human placenta and, hence, was tentatively named early placenta insulin-like peptide (EPIL). In this paper, we report the cloning and sequencing of the EPIL cDNA and the EPIL gene (INSL4). Comparison of the deduced amino acid sequence of the early placenta insulin-like peptide revealed significant overall and structural homologies with members of the insulin-like hormone superfamily. Moreover, the organization of the early placenta insulin-like gene, which is composed of two exons and one intron, is similiar to that of insulin and relaxin.more » By in situ hybridization, the INSL4 gene was assigned to band p24 of the short arm of chromosome 9. RT-PCR analysis of EPIL tissue distribution revealed that its transcripts are expressed in the placenta and uterus. 22 refs., 3 figs.« less

Selective expression of a splice variant of decay-accelerating factor in c-erbB-2-positive mammary carcinoma cells showing increased transendothelial invasiveness

DOE Office of Scientific and Technical Information (OSTI.GOV)

Brandt, Burkhard; Mikesch, Jan-Hendrik; Simon, Ronald

2005-04-01

By differential-display-PCR a subclone of the SK-BR-3 cell line with high in vitro transendothelial invasiveness was identified to express increased levels of a new alternative splice variant of decay-accelerating factor (DAF). DAF seems to play an important role in some malignant tumours since on the one hand the expression of complement inhibitors on the surface of tumour cells prevents the accumulation of complement factors and in consequence cell lysis. On the other hand, DAF has been identified as a ligand for the CD97 surface receptor which induces cell migration. Immunofluorescence procedures, Western blot analyses, and cDNA clone sequencing were employedmore » to confirm the expression of DAF restricted to invasive tumour cells. Using a radioactive RNA-in situ hybridisation on freshly frozen tissue microarrays and RT-PCR on native tumour tissue, the expression of alternative spliced DAF mRNA was demonstrated in invasive breast cancer. Due to the fact that it could thereby not be detected in normal mammary tissues, it has to be confirmed in larger studies that the DAF splice variant might be a specific tumour marker for invasive breast cancer.« less

Identification and characterization of a novel human hepatocellular carcinoma-associated gene

PubMed Central

Wang, Z-X; Wang, H-Y; Wu, M-C

2001-01-01

To investigate liver cancer-associated genes and to explore the molecular basis of liver cancer genesis, we have cloned a novel hepatocellular carcinoma (HCC)-related gene with a transcript of 2520 base pairs in length named HCCA2 by mRNA differential display polymerase chain reaction (DDPCR) and screening a placenta cDNA library. No significant homologous protein with known genes was found. Western blot analysis showed that HCCA2 could be expressed in transfected 293 cells. Northern hybridization analysis showed that HCCA2 mRNA was expressed in 79% (34/43) patients with HCC, most of whom had significantly high expression in HCC tissues, while not expressed in corresponding noncancerous liver tissues. The clinical pathological data showed that the HCCA2 was significantly associated with the invasion of tumour capsule (P= 0.0007) and the expression of ki-67 protein (P= 0.0022). Immunohistochemical staining confirmed that the HCCA2 protein was localized in cytoplasm of liver cancer tissues. According to amino acid analysis of the protein and its localization, it may play a role in a cascade of intracellular signal transduction because the protein was characterized with two Src homology 3 (SH3) binding-domains and several functional motifs of phophorylation. © 2001 Cancer Research Campaign  http://www.bjcancer.com PMID:11710830

Domain selection combined with improved cloning strategy for high throughput expression of higher eukaryotic proteins

PubMed Central

Chen, Yunjia; Qiu, Shihong; Luan, Chi-Hao; Luo, Ming

2007-01-01

Background Expression of higher eukaryotic genes as soluble, stable recombinant proteins is still a bottleneck step in biochemical and structural studies of novel proteins today. Correct identification of stable domains/fragments within the open reading frame (ORF), combined with proper cloning strategies, can greatly enhance the success rate when higher eukaryotic proteins are expressed as these domains/fragments. Furthermore, a HTP cloning pipeline incorporated with bioinformatics domain/fragment selection methods will be beneficial to studies of structure and function genomics/proteomics. Results With bioinformatics tools, we developed a domain/domain boundary prediction (DDBP) method, which was trained by available experimental data. Combined with an improved cloning strategy, DDBP had been applied to 57 proteins from C. elegans. Expression and purification results showed there was a 10-fold increase in terms of obtaining purified proteins. Based on the DDBP method, the improved GATEWAY cloning strategy and a robotic platform, we constructed a high throughput (HTP) cloning pipeline, including PCR primer design, PCR, BP reaction, transformation, plating, colony picking and entry clones extraction, which have been successfully applied to 90 C. elegans genes, 88 Brucella genes, and 188 human genes. More than 97% of the targeted genes were obtained as entry clones. This pipeline has a modular design and can adopt different operations for a variety of cloning/expression strategies. Conclusion The DDBP method and improved cloning strategy were satisfactory. The cloning pipeline, combined with our recombinant protein HTP expression pipeline and the crystal screening robots, constitutes a complete platform for structure genomics/proteomics. This platform will increase the success rate of purification and crystallization dramatically and promote the further advancement of structure genomics/proteomics. PMID:17663785

Genome-wide cloning, identification, classification and functional analysis of cotton heat shock transcription factors in cotton (Gossypium hirsutum).

PubMed

Wang, Jun; Sun, Na; Deng, Ting; Zhang, Lida; Zuo, Kaijing

2014-11-06

Heat shock transcriptional factors (Hsfs) play important roles in the processes of biotic and abiotic stresses as well as in plant development. Cotton (Gossypium hirsutum, 2n=4x=(AD)2=52) is an important crop for natural fiber production. Due to continuous high temperature and intermittent drought, heat stress is becoming a handicap to improve cotton yield and lint quality. Recently, the related wild diploid species Gossypium raimondii genome (2n=2x=(D5)2=26) has been fully sequenced. In order to analyze the functions of different Hsfs at the genome-wide level, detailed characterization and analysis of the Hsf gene family in G. hirsutum is indispensable. EST assembly and genome-wide analyses were applied to clone and identify heat shock transcription factor (Hsf) genes in Upland cotton (GhHsf). Forty GhHsf genes were cloned, identified and classified into three main classes (A, B and C) according to the characteristics of their domains. Analysis of gene duplications showed that GhHsfs have occurred more frequently than reported in plant genomes such as Arabidopsis and Populus. Quantitative real-time PCR (qRT-PCR) showed that all GhHsf transcripts are expressed in most cotton plant tissues including roots, stems, leaves and developing fibers, and abundantly in developing ovules. Three expression patterns were confirmed in GhHsfs when cotton plants were exposed to high temperature for 1 h. GhHsf39 exhibited the most immediate response to heat shock. Comparative analysis of Hsfs expression differences between the wild-type and fiberless mutant suggested that Hsfs are involved in fiber development. Comparative genome analysis showed that Upland cotton D-subgenome contains 40 Hsf members, and that the whole genome of Upland cotton contains more than 80 Hsf genes due to genome duplication. The expression patterns in different tissues in response to heat shock showed that GhHsfs are important for heat stress as well as fiber development. These results provide an improved understanding of the roles of the Hsf gene family during stress responses and fiber development.

A short region of the promoter of the breast cancer associated PLU-1 gene can regulate transcription in vitro and in vivo.

PubMed

Catteau, Aurélie; Rosewell, Ian; Solomon, Ellen; Taylor-Papadimitriou, Joyce

2004-07-01

The recently cloned gene PLU-1 shows restricted expression in adult tissues, with high expression being found in testis, and transiently in the pregnant mammary gland. However, both the gene and the protein product are specifically up-regulated in breast cancer. To investigate the control of expression of the PLU-1 gene, we have cloned and functionally characterised the 5' flanking region of the gene, which was found to contain another putative gene. Two transcription start sites of the PLU-1 gene were mapped by 5' RACE. A short proximal 249 bp region was defined using reporter gene assays, which encompasses the major transcription start site and exhibits a strong constitutive promoter activity in all cell lines tested. However, regions upstream of this sequence repress transcription more effectively in a non-malignant breast cell line as compared to breast cancer cell lines. The 249 bp region is GC-rich and includes consensus Sp1 sites, GC boxes, cAMP-responsive element (CRE) and other putative cis-elements. Mutational analysis showed that two intact conserved Sp1 binding sites (shown here to bind Sp1 and/or Sp3) are critical for constitutive promoter activity, while a negative role for a neighbouring GC box is indicated. The sequence of the core promoter is highly conserved in the mouse and Plu-1 expression in the mouse embryo has been documented. Using transgenesis, we therefore examined the ability of the 249 bp fragment to control expression of a reporter gene during embryogenesis. We found that not only is the core promoter sufficient to activate transcription in vivo, but that the expression of the reporter gene coincides both temporally and spatially with regions where endogenous Plu-1 is highly expressed. This suggests that tissue specific controlling elements are found within the short fragment and are functional in the embryonic environment.

Gene Cloning, Expression and Enzyme Activity of Vitis vinifera Vacuolar Processing Enzymes (VvVPEs)

PubMed Central

Gong, Peijie; Li, Shuxiu; Wang, Yuejin; Zhang, Chaohong

2016-01-01

Vacuolar processing enzymes (VPEs) have received considerable attention due to their caspase-1-like activity and ability to regulate programmed cell death (PCD), which plays an essential role in the development of stenospermocarpic seedless grapes ovules. To characterize VPEs and the relationship between stenospermocarpic grapes and the VPE gene family, we identified 3 Vitis vinifera VPE genes (VvβVPE, VvγVPE, and VvδVPE) from the PN40024 grape genome and cloned the full-length complementary DNAs (cDNAs) from the ‘Vitis vinifera cv. Pinot Noir’ and ‘Vitis vinifera cv. Thompson Seedless’ varietals. Each of the VPEs contained a typical catalytic dyad [His (177), Cys (219)] and substrate binding pocket [Arg (112), Arg (389), Ser (395)], except that Ser (395) in the VvγVPE protein sequence was replaced with alanine. Phylogenetic analysis of 4 Arabidopsis thaliana and 6 Vitis vinifera VPEs revealed that the 10 VPEs form 3 major branches. Furthermore, the 6 grapevine VPEs share a similar gene structure, with 9 exons and 8 introns. The 6 grapevine VPEs are located on 3 different chromosomes. We also tested the enzymatic activity of recombinant VPEs expressed in the Pichia Pastoris expression system and found that the VvVPEs exhibit cysteine peptidase activity. Tissue-specific expression analysis showed that VvδVPE is only expressed in flowers, buds and ovules, that VvγVPE is expressed in various tissues, and that VvβVPE was expressed in roots, flowers, buds and ovules. The results of quantitative real-time PCR (qRT-PCR) suggested that VvβVPE in seeded grapes increased significantly at 30 days after full-bloom (DAF), close to the timing of endosperm abortion at 32 DAF. These results suggested that VvβVPE is related to ovule abortion in seedless grapes. Our experiments provide a new perspective for understanding the mechanism of stenospermocarpic seedlessness and represent a useful reference for the further study of VPEs. PMID:27551866

Dopamine D2-like receptors (DRD2 and DRD4) in chickens: Tissue distribution, functional analysis, and their involvement in dopamine inhibition of pituitary prolactin expression.

PubMed

Lv, Can; Mo, Chunheng; Liu, Haikun; Wu, Chao; Li, Zhengyang; Li, Juan; Wang, Yajun

2018-04-20

Dopamine (DA) D2-like (and D1-like) receptors are suggested to mediate the dopamine actions in the anterior pituitary and/or CNS of birds. However, the information regarding the structure, functionality, and expression of avian D2-like receptors have not been fully characterized. In this study, we cloned two D2-like receptors (cDRD2, cDRD4) from chicken brain using RACE PCR. The cloned cDRD4 is a 378-amino acid receptor, which shows 57% amino acid (a.a.) identity with mouse DRD4. As in mammals, two cDRD2 isoforms, cDRD2L (long isoform, 437 a.a.) and cDRD2S (short isoform, 408 a.a.), which differ in their third intracellular loop, were identified in chickens. Using cell-based luciferase reporter assays or Western blot, we demonstrated that cDRD4, cDRD2L and cDRD2S could be activated by dopamine and quinpirole (a D2-like receptor agonist) dose-dependently, and their activation inhibits cAMP signaling pathway and stimulates MAPK/ERK signaling cascade, indicating that they are functional receptors capable of mediating dopamine actions. Quantitative real-time PCR revealed that cDRD2 and cDRD4 are widely expressed in chicken tissues with abundant expression noted in anterior pituitary, and their expressions are likely controlled by their promoters near exon 1, as demonstrated by dual-luciferase reporter assays in DF-1 cells. In accordance with cDRD2/cDRD4 expression in the pituitary, DA or quinpirole could partially inhibit vasoactive intestinal peptide-induced prolactin expression in cultured chick pituitary cells. Together, our data proves the functionality of DRD2 and DRD4 in birds and aids to uncover the conserved roles of DA/D2-like receptor system in vertebrates, such as its action on the pituitary. Copyright © 2018. Published by Elsevier B.V.

ERP, a new member of the ets transcription factor/oncoprotein family: cloning, characterization, and differential expression during B-lymphocyte development.

PubMed

Lopez, M; Oettgen, P; Akbarali, Y; Dendorfer, U; Libermann, T A

1994-05-01

The ets gene family encodes a group of proteins which function as transcription factors under physiological conditions and, if aberrantly expressed, can cause cellular transformation. We have recently identified two regulatory elements in the murine immunoglobulin heavy-chain (IgH) enhancer, pi and microB, which exhibit striking similarity to binding sites for ets-related proteins. To identify ets-related transcriptional regulators expressed in pre-B lymphocytes that may interact with either the pi or the microB site, we have used a PCR approach with degenerate oligonucleotides encoding conserved sequences in all members of the ets family. We have cloned the gene for a new ets-related transcription factor, ERP (ets-related protein), from the murine pre-B cell line BASC 6C2 and from mouse lung tissue. The ERP protein contains a region of high homology with the ETS DNA-binding domain common to all members of the ets transcription factor/oncoprotein family. Three additional smaller regions show homology to the ELK-1 and SAP-1 genes, a subgroup of the ets gene family that interacts with the serum response factor. Full-length ERP expresses only negligible DNA-binding activity by itself. Removal of the carboxy terminus enables ERP to interact with a variety of ets-binding sites including the E74 site, the IgH enhancer pi site, and the lck promoter ets site, suggesting a carboxy-terminal negative regulatory domain. At least three ERP-related transcripts are expressed in a variety of tissues. However, within the B-cell lineage, ERP is highly expressed primarily at early stages of B-lymphocyte development, and expression declines drastically upon B-cell maturation, correlating with the enhancer activity of the IgH pi site. These data suggest that ERP might play a role in B-cell development and in IgH gene regulation.

Molecular cloning of Pcc-dmrt1s and their specific expression patterns in Pengze crucian carp (Carassius auratus var. Pengze) affected by 17α-methyltestosterone.

PubMed

Zheng, Yao; Liang, Hongwei; Xu, Peng; Li, Meng; Wang, Zaizhao

2014-08-01

Dmrt1, an important transcription factor associated with testicular differentiation, is conserved among teleost, which could also be detected in ovaries. In the present study, three isoforms of Pcc-dmrt1s (Pcc-dmrt1a, Pcc-dmrt1b and Pcc-dmrt1c) resulting from alternative splicing of the dmrt1 gene were cloned and characterized in the triploid gynogenetic fish, the Pengze crucian carp. Their mRNA expression profiling was investigated in juvenile developmental stages, tissues of the adult fish, and the juveniles under 84.2 ng/L 17α-methyltestosterone (MT) treatments. Results showed that their putative proteins shared high identities to Dmrt1 in cyprinid fish species. Gene expression profiling in the developmental stages showed that all the three target genes had a highest/lowest expression at 56/40 days post-hatching (dph), respectively. The period of 40 dph appeared to be a key time during the process of the ovary development of Pengze crucian carp. The tissue distribution results indicated that Pcc-dmrt1s were predominantly expressed in hepatopancreas, brain, spleen and ovary of the female fish. MT significantly increased the mRNA expression of Pcc-dmrt1a (all 4-week exposures) and Pcc-dmrt1b (except for week 2), while repressed Pcc-dmrt1c transcripts at all exposure period except for week 2. MT extremely significant repressed cyp19a1a transcripts for 1 week. The present study indicated that MT could influence the ovary development of Pengze crucian carp by disturbing gene expressions of Pcc-dmrt1s and cyp19a1a. Furthermore, the present study will be of great significance to broaden the understanding of masculinizing pathway during ovary development in gynogenetic teleost.

Gene Cloning, Expression and Enzyme Activity of Vitis vinifera Vacuolar Processing Enzymes (VvVPEs).

PubMed

Tang, Yujin; Wang, Ruipu; Gong, Peijie; Li, Shuxiu; Wang, Yuejin; Zhang, Chaohong

2016-01-01

Vacuolar processing enzymes (VPEs) have received considerable attention due to their caspase-1-like activity and ability to regulate programmed cell death (PCD), which plays an essential role in the development of stenospermocarpic seedless grapes ovules. To characterize VPEs and the relationship between stenospermocarpic grapes and the VPE gene family, we identified 3 Vitis vinifera VPE genes (VvβVPE, VvγVPE, and VvδVPE) from the PN40024 grape genome and cloned the full-length complementary DNAs (cDNAs) from the 'Vitis vinifera cv. Pinot Noir' and 'Vitis vinifera cv. Thompson Seedless' varietals. Each of the VPEs contained a typical catalytic dyad [His (177), Cys (219)] and substrate binding pocket [Arg (112), Arg (389), Ser (395)], except that Ser (395) in the VvγVPE protein sequence was replaced with alanine. Phylogenetic analysis of 4 Arabidopsis thaliana and 6 Vitis vinifera VPEs revealed that the 10 VPEs form 3 major branches. Furthermore, the 6 grapevine VPEs share a similar gene structure, with 9 exons and 8 introns. The 6 grapevine VPEs are located on 3 different chromosomes. We also tested the enzymatic activity of recombinant VPEs expressed in the Pichia Pastoris expression system and found that the VvVPEs exhibit cysteine peptidase activity. Tissue-specific expression analysis showed that VvδVPE is only expressed in flowers, buds and ovules, that VvγVPE is expressed in various tissues, and that VvβVPE was expressed in roots, flowers, buds and ovules. The results of quantitative real-time PCR (qRT-PCR) suggested that VvβVPE in seeded grapes increased significantly at 30 days after full-bloom (DAF), close to the timing of endosperm abortion at 32 DAF. These results suggested that VvβVPE is related to ovule abortion in seedless grapes. Our experiments provide a new perspective for understanding the mechanism of stenospermocarpic seedlessness and represent a useful reference for the further study of VPEs.

Cloning and Functional Characterization of a Gene for Capsanthin-Capsorubin Synthase from Tiger Lily (Lilium lancifolium Thunb. ‘Splendens’)

PubMed Central

Chen, Tony H.H.

2012-01-01

The orange color of tiger lily (Lolium lancifolium ‘Splendens’) flowers is due, primarily, to the accumulation of two κ-xanthophylls, capsanthin and capsorubin. An enzyme, known as capsanthin-capsorubin synthase (CCS), catalyzes the conversion of antheraxanthin and violaxanthin into capsanthin and capsorubin, respectively. We cloned the gene for capsanthin-capsorubin synthase (Llccs) from flower tepals of L. lancifolium by the rapid amplification of cDNA ends (RACE) with a heterologous non-degenerate primer that was based on the sequence of a gene for lycopene β-cyclase (lcyB). The full-length cDNA of Llccs was 1,785 bp long and contained an open reading frame of 1,425 bp that encoded a polypeptide of 474 amino acids with a predicted N-terminal plastid-targeting sequence. Analysis by reverse transcription–PCR (RT–PCR) revealed that expression of Llccs was spatially and temporally regulated, with expression in flower buds and flowers of L. lancifolium but not in vegetative tissues. Stable overexpression of the Llccs gene in callus tissue of Iris germanica, which accumulates several xanthophylls including violaxanthin, the precursor of capsorubin, resulted in transgenic callus whose color had changed from its normal yellow to red-orange. This novel red-orange coloration was due to the accumulation of two non-native κ-xanthophylls, capsanthin and capsorubin, as confirmed by HPLC and ultraperformance liquid chromatography–tandem mass spectrometry (UPLC-MS/MS) analysis with authentic standards. Cloning of the Llccs gene should advance our understanding of the molecular and genetic mechanisms of the biosynthesis of κ-carotenoids in general and in the genus Lilium in particular, and will facilitate transgenic alterations of the colors of flowers and fruits of many plant species. PMID:23008421

Cloning and characterization of full-length mouse thymidine kinase 2: the N-terminal sequence directs import of the precursor protein into mitochondria.

PubMed Central

Wang, L; Eriksson, S

2000-01-01

The subcellular localization of mitochondrial thymidine kinase (TK2) has been questioned, since no mitochondrial targeting sequences have been found in cloned human TK2 cDNAs. Here we report the cloning of mouse TK2 cDNA from a mouse full-length enriched cDNA library. The mouse TK2 cDNA codes for a protein of 270 amino acids, with a 40-amino-acid presumed N-terminal mitochondrial targeting signal. In vitro translation and translocation experiments with purified rat mitochondria confirmed that the N-terminal sequence directed import of the precursor TK2 into the mitochondrial matrix. A single 2.4 kb mRNA transcript was detected in most tissues examined, except in liver, where an additional shorter (1.0 kb) transcript was also observed. There was no correlation between the tissue distribution of TK2 activity and the expression of TK2 mRNA. Full-length mouse TK2 protein and two N-terminally truncated forms, one of which corresponds to the mitochondrial form of TK2 and a shorter form corresponding to the previously characterized recombinant human TK2, were expressed in Escherichia coli and affinity purified. All three forms of TK2 phosphorylated thymidine, deoxycytidine and 2'-deoxyuridine, but with different kinetic efficiencies. A number of cytostatic pyrimidine nucleoside analogues were also tested and shown to be good substrates for the various forms of TK2. The active form of full-length mouse TK2 was a dimer, as judged by Superdex 200 chromatography. These results enhance our understanding of the structure and function of TK2, and may help to explain the mitochondrial disorder, mitochondrial neurogastrointestinal encephalomyopathy. PMID:11023833

Generation of CMAHKO/GTKO/shTNFRI-Fc/HO-1 quadruple gene modified pigs.

PubMed

Kim, Geon A; Lee, Eun Mi; Jin, Jun-Xue; Lee, Sanghoon; Taweechaipaisankul, Anukul; Hwang, Jong Ik; Alam, Zahid; Ahn, Curie; Lee, Byeong Chun

2017-08-01

As an alternative source of organs for transplantation into humans, attention has been directed to pigs due to their similarities in biological features and organ size. However, severe immune rejection has prevented successful xenotransplantation using pig organs and tissues. To overcome immune rejection, recently developed genetic engineering systems such as TALEN coupled with somatic cell nuclear transfer (SCNT) to make embryos could be used to produce pigs compatible with xenotransplantation. We used the TALEN system to target the non-Gal antigen cytidine monophosphate-N-acetylneuraminic acid hydroxylase (CMAH) gene in pigs that is naturally deleted in humans. Gal-deleted cells expressing both soluble human tumor necrosis factor receptor I IgG 1 -Fc (shTNFRI-Fc) and human hemagglutinin -tagged-human heme oxygenase-1 (hHO-1) were transfected with a TALEN target for CMAH. Cells lacking CMAH were negatively selected using N-glyconeuraminic acid (Neu5Gc)/magnetic beads and the level of Neu5Gc expression of isolated cells were analyzed by FACS and DNA sequencing. Cloned embryos using 3 different genetically modified cell clones were respectively transferred into 3 recipients, with 55.6% (5/9) becoming pregnant and three cloned pigs were produced. Successful genetic disruption of the CMAH gene was confirmed by sequencing, showing lack of expression of CMAH in tail-derived fibroblasts of the cloned piglets. Besides decreased expression of Neu5Gc in piglets produced by SCNT, antibody-mediated complement-dependent cytotoxicity assays and natural antibody binding for examining immuno-reactivity of the quadruple gene modified pigs derived from endothelial cells and fibroblasts were reduced significantly compared to those of wild type animals. We conclude that by combining the TALEN system and transgenic cells, targeting of multiple genes could be useful for generating organs for xenotransplantation. We produced miniature pigs with quadruple modified genes CMAHKO/GTKO/shTNFRI-Fc/hHO-1 that will be suitable for xenotransplantation by overcoming hyperacute, acute and anti-inflammatory rejection.

The Identification and Cloning of the Wnt-1 Receptor

DTIC Science & Technology

1996-10-01

examination of embryos with duplicated axes revealed that Xwnt-5A and hFz5 induced a full array of dorsal tissues, including notochord , neural tube...tube, a notochord and somites in both axes. c). Xwnt-5A plus hfz5 induce ectopic goosecoid (gsc) expression in stage 11 embryos, as visualized by whole...Lai CJ, Olson DJ, Kelly GM: Dissecting Wnt signalling pathways and Wnt-sensitive developmental processes through transient misexpression analyses in

Long non-coding RNA BANCR regulates cancer stem cell markers in papillary thyroid cancer via the RAF/MEK/ERK signaling pathway.

PubMed

Wang, Yuanyuan; Lin, Xiangde; Fu, Xinghao; Yan, Wei; Lin, Fusheng; Kuang, Penghao; Luo, Yezhe; Lin, Ende; Hong, Xiaoquan; Wu, Guoyang

2018-06-18

Thyroid cancer is one of the most common malignant tumors of the endocrine system. Among all thyroid cancers, papillary thyroid carcinoma (PTC) is the most common type. The BRAF-activated non-coding RNA (BANCR) is a 693-bp nucleotide transcript which was first identified in melanoma. However, the role of BANCR in the development of thyroid cancer remains unclear. Therefore, the present study investigated the potential involvement of BANCR in the development of thyroid cancer in vitro using patient tissue samples and a panel of thyroid cancer cell lines, and in vivo using a xenograft mouse model. We observed that BANCR was expressed at a higher level in human thyroid tumor tissues than that noted in the adjacent normal tissues. The expression level of BANCR differed between cultured thyroid cancer cell lines; BANCR expression was lower in the BCPAP cell line than that observed in the CAL-62, WRO and FTC-133 cell lines. Western blot analysis and flow cytometry revealed that overexpression of BANCR in the BCPAP cell line resulted in increased expression of the cancer stem cell markers, LGR5 and EpCAM. Single-clone formation experiments showed that upregulated expression of BANCR in the BCPAP cell line promoted an increase in the number of clones formed. Similarly, in microsphere formation experiments, overexpression of BANCR resulted in increased number and size of microspheres compared with the control cell line. Western blotting experiments showed that BANCR overexpression in BCPAP upregulated the expression of phosphorylated c-Raf, MEK1/2 and ERK1/2. Inhibition of c-Raf via U0126 decreased the expression of LGR5 and EpCAM, as well as phosphorylated levels of c-Raf, MEK1/2 and ERK1/2 in the BCPAP cells, compared to levels in the DMSO controls. In the xenograft mouse model, BANCR overexpression in the thyroid cancer cells significantly increased tumor growth. Taken together, these results suggest that BANCR plays a role in PTC development by regulating the expression of cancer stem cell markers LGR5 and EpCAM via the c-Raf/MEK/ERK signaling pathway. Therefore, BANCR may be used as a novel prognostic marker for PTC.

Cloning of transgenic tobacco BY-2 cells; an efficient method to analyse and reduce high natural heterogeneity of transgene expression.

PubMed

Nocarova, Eva; Fischer, Lukas

2009-04-22

Phenotypic characterization of transgenic cell lines, frequently used in plant biology studies, is complicated because transgene expression in individual cells is often heterogeneous and unstable. To identify the sources and to reduce this heterogeneity, we transformed tobacco (Nicotiana tabacum L.) BY-2 cells with a gene encoding green fluorescent protein (GFP) using Agrobacterium tumefaciens, and then introduced a simple cloning procedure to generate cell lines derived from the individual transformed cells. Expression of the transgene was monitored by analysing GFP fluorescence in the cloned lines and also in lines obtained directly after transformation. The majority ( approximately 90%) of suspension culture lines derived from calli that were obtained directly from transformation consisted of cells with various levels of GFP fluorescence. In contrast, nearly 50% of lines generated by cloning cells from the primary heterogeneous suspensions consisted of cells with homogenous GFP fluorescence. The rest of the lines exhibited "permanent heterogeneity" that could not be resolved by cloning. The extent of fluorescence heterogeneity often varied, even among genetically identical clones derived from the primary transformed lines. In contrast, the offspring of subsequent cloning of the cloned lines was uniform, showing GFP fluorescence intensity and heterogeneity that corresponded to the original clone. The results demonstrate that, besides genetic heterogeneity detected in some lines, the primary lines often contained a mixture of epigenetically different cells that could be separated by cloning. This indicates that a single integration event frequently results in various heritable expression patterns, which are probably accidental and become stabilized in the offspring of the primary transformed cells early after the integration event. Because heterogeneity in transgene expression has proven to be a serious problem, it is highly advisable to use transgenes tagged with a visual marker for BY-2 transformation. The cloning procedure can be used not only for efficient reduction of expression heterogeneity of such transgenes, but also as a useful tool for studies of transgene expression and other purposes.

Molecular cloning, characterization, and expression profiles of androgen receptors in spotted scat (Scatophagus argus).

PubMed

Chen, H P; Deng, S P; Dai, M L; Zhu, C H; Li, G L

2016-04-07

Androgen plays critical roles in vertebrate reproductive systems via androgen receptors (ARs). In the present study, the full-length spotted scat (Scatophagus argus) androgen receptor (sAR) cDNA sequence was cloned from testis. The sAR cDNA measured 2448 bp in length with an open-reading frame of 2289 bp, encoding 763 amino acids. Amino acid alignment analyses showed that the sARs exhibited highly evolutionary conserved functional domains. Phylogenetically, the sARs clustered within the ARβ common vertebrate group. Real-time polymerase chain reaction (RT-PCR) revealed that sAR expression varied in level and distribution throughout the tissues of both females and males. sAR expression was detected during testicular development by quantitative RT-PCR. The results showed that the highest transcription of sARs was observed in the mid-testicular stage, and remained at a high expression level until the late-testicular stage. In addition, the effects of 17α-methyltestosterone (MT) and estrogen (E2) on the expression of sARs in ovaries were determined using quantitative RT-PCR. sAR expression increased at 12 and 24 h post-MT treatment and decreased with E2 treatment. The present study provides preliminary evidence indicating gonadal plasticity of spotted scat under exogenous steroidal hormone treatments. It also provides a theoretical basis for sex reversal and production of artificial pseudo-males for female monosex breeding.

Raman spectroscopic study of keratin 8 knockdown oral squamous cell carcinoma derived cells

NASA Astrophysics Data System (ADS)

Singh, S. P.; Alam, Hunain; Dmello, Crismita; Vaidya, Milind M.; Krishna, C. Murali

2012-03-01

Keratins are one of most widely used markers for oral cancers. Keratin 8 and 18 are expressed in simple epithelia and perform both mechanical and regulatory functions. Their expression are not seen in normal oral tissues but are often expressed in oral squamous cell carcinoma. Aberrant expression of keratins 8 and 18 is most common change in human oral cancer. Optical-spectroscopic methods are sensitive to biochemical changes and being projected as novel diagnostic tools for cancer diagnosis. Aim of this study was to evaluate potentials of Raman spectroscopy in detecting minor changes associated with differential level of keratin expression in tongue-cancer-derived AW13516 cells. Knockdown clones for K8 were generated and synchronized by growing under serum-free conditions. Cell pellets of three independent experiments in duplicate were used for recording Raman spectra with fiberoptic-probe coupled HE-785 Raman-instrument. A total of 123 and 96 spectra from knockdown clones and vector controls respectively in 1200-1800 cm-1 region were successfully utilized for classification using LDA. Two separate clusters with classification-efficiency of ~95% were obtained. Leave-one-out cross-validation yielded ~63% efficiency. Findings of the study demonstrate the potentials of Raman spectroscopy in detecting even subtle changes such as variations in keratin expression levels. Future studies towards identifying Raman signals from keratin in oral cells can help in precise cancer diagnosis.

Molecular cloning, expression analysis and transcript localization of testicular orphan nuclear receptor 2 in the male catfish, Clarias batrachus.

PubMed

Murugananthkumar, R; Akhila, M V; Rajakumar, A; Mamta, S K; Sudhakumari, C C; Senthilkumaran, B

2016-12-01

Testicular receptor 2 (TR2; also known as Nr2c1) is one of the first orphan nuclear receptors identified and known to regulate various physiological process with or without any ligand. In this study, we report the cloning of full length nr2c1 and its expression analysis during gonadal development, seasonal testicular cycle and after human chorionic gonadotropin (hCG) induction. In addition, in situ hybridization (ISH) was performed to localize nr2c1 transcripts in adult testis and whole catfish (1day post hatch). Tissue distribution and gonadal ontogeny studies revealed high expression of nr2c1 in developing and adult testis. Early embryonic stage-wise expression of nr2c1 seems to emphasize its importance in cellular differentiation and development. Substantial expression of nr2c1 during pre-spawning phase and localization of nr2c1 transcripts in sperm/spermatids were observed. Significant upregulation after hCG induction indicate that nr2c1 is under the regulation of gonadotropins. Whole mount ISH analysis displayed nr2c1 expression in notochord indicating its role in normal vertebrate development. Taken together, our findings suggest that nr2c1 may have a plausible role in the testicular and embryonic development of catfish. Copyright © 2015. Published by Elsevier Inc.

Molecular Cloning, Bioinformatic Analysis, and Expression of Bombyx mori Lebocin 5 Gene Related to Beauveria bassiana Infection.

PubMed

Lü, Dingding; Hou, Chengxiang; Qin, Guangxing; Gao, Kun; Chen, Tian; Guo, Xijie

2017-01-01

A full-length cDNA of lebocin 5 (BmLeb5) was first cloned from silkworm, Bombyx mori , by rapid amplification of cDNA ends. The BmLeb5 gene is 808 bp in length and the open reading frame encodes a 179-amino acid hydroxyproline-rich peptide. Bioinformatic analysis results showed that BmLeb5 owns an O-glycosylation site and four RXXR motifs as other lebocins. Sequence similarity and phylogenic analysis results indicated that lebocins form a multiple gene family in silkworm as cecropins. Quantitative real-time PCR analysis revealed that BmLeb5 was highest expressed in the fat body. In the silkworm larvae infected by Beauveria bassiana , the expression level of BmLeb5 was upregulated in the fat body and hemolymph which are the most important immune tissues in silkworm. The recombinant protein of BmLeb5 was for the first time successfully expressed with prokaryotic expression system and purified. There are no reports so far that the expression of lebocins could be induced by entomopathogenic fungus. Our study suggested that BmLeb5 might play an important role in the immune response of silkworm to defend B. bassiana infection. The results also provided helpful information for further studying the lebocin family functioned in antifungal immune response in the silkworm.

Cysteine-rich secretory protein 3 plays a role in prostate cancer cell invasion and affects expression of PSA and ANXA1.

PubMed

Pathak, Bhakti R; Breed, Ananya A; Apte, Snehal; Acharya, Kshitish; Mahale, Smita D

2016-01-01

Cysteine-rich secretory protein 3 (CRISP-3) is upregulated in prostate cancer as compared to the normal prostate tissue. Higher expression of CRISP-3 has been linked to poor prognosis and hence it has been thought to act as a prognostic marker for prostate cancer. It is proposed to have a role in innate immunity but its role in prostate cancer is still unknown. In order to understand its function, its expression was stably knocked down in LNCaP cells. CRISP-3 knockdown did not affect cell viability but resulted in reduced invasiveness. Global gene expression changes upon CRISP-3 knockdown were identified by microarray analysis. Microarray data were quantitatively validated by evaluating the expression of seven candidate genes in three independent stable clones. Functional annotation of the differentially expressed genes identified cell adhesion, cell motility, and ion transport to be affected among other biological processes. Prostate-specific antigen (PSA, also known as Kallikrein 3) was the top most downregulated gene whose expression was also validated at protein level. Interestingly, expression of Annexin A1 (ANXA1), a known anti-inflammatory protein, was upregulated upon CRISP-3 knockdown. Re-introduction of CRISP-3 into the knockdown clone reversed the effect on invasiveness and also led to increased PSA expression. These results suggest that overexpression of CRISP-3 in prostate tumor may maintain higher PSA expression and lower ANXA1 expression. Our data also indicate that poor prognosis associated with higher CRISP-3 expression could be due to its role in cell invasion.

Molecular cloning of a novel widely expressed human 80 kDa 17 beta-hydroxysteroid dehydrogenase IV.

PubMed Central

Adamski, J; Normand, T; Leenders, F; Monté, D; Begue, A; Stéhelin, D; Jungblut, P W; de Launoit, Y

1995-01-01

Reactions of oestrogens and androgens at position C-17 are catalysed by 17 beta-hydroxysteroid dehydrogenases (17 beta-HSDs). Cloning of the cDNA of a novel human 17 beta-HSD IV and expression of its mRNA are described. A probe derived from the recently discovered porcine 17 beta-oestradiol dehydrogenase (17 beta-EDH) was used to isolate a 2.6 kb human cDNA encoding a continuous protein of 736 amino acids of high (84%) similarity to the porcine 17 beta-EDH. The calculated molecular mass of the human enzyme is 79,595 Da. Other sequence similarities shared by the two enzymes are: an N-terminal sequence which is similar to that of members of the short-chain alcohol dehydrogenase family; amino acids 343-607 which are similar to the C-terminal domains of a trifunctional Candida tropicalis enzyme and the FOX2 gene product of Saccharomyces cerevisiae; amino acids 596-736 which are similar to human sterol carrier protein 2. The previously cloned human 17 beta-HSD I, II and III are less than 25% identical with 17 beta-HSD IV. mRNA for HSD IV is a single species of 3.0 kb, present in many tissues with highest concentrations in liver, heart, prostate and testes. When over-expressed in mammalian cells, the human 17 beta-HSD IV enzyme displays a specific unidirectional oxidative 17 beta-HSD activity. Images Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 PMID:7487879

Nematicidal protease genes screened from a soil metagenomic library to control Radopholus similis mediated by Pseudomonas fluorescens pf36.

PubMed

Chen, Deqiang; Wang, Dongwei; Xu, Chunling; Chen, Chun; Li, Junyi; Wu, Wenjia; Huang, Xin; Xie, Hui

2018-04-01

Controlling Radopholus similis, an important phytopathogenic nematode, is a challenge worldwide. Herein, we constructed a metagenomic fosmid library from the rhizosphere soil of banana plants, and six clones with protease activity were obtained by functionally screening the library. Furthermore, subclones were constructed using the six clones, and three protease genes with nematicidal activity were identified: pase1, pase4, and pase6. The pase4 gene was successfully cloned and expressed, demonstrating that the protease PASE4 could effectively degrade R. similis tissues and result in nematode death. Additionally, we isolated a predominant R. similis-associated bacterium, Pseudomonas fluorescens (pf36), from 10 R. similis populations with different hosts. The pase4 gene was successfully introduced into the pf36 strain by vector transformation and conjugative transposition, and two genetically modified strains were obtained: p4MCS-pf36 and p4Tn5-pf36. p4MCS-pf36 had significantly higher protease expression and nematicidal activity (p < 0.05) than p4Tn5-pf36 in a microtiter plate assay, whereas p4Tn5-pf36 was superior to p4MCS-pf36 in terms of genetic stability and controlling R. similis in growth pot tests. This study confirmed that R. similis is inhibited by the associated bacterium pf36-mediated expression of nematicidal proteases. Herein, a novel approach is provided for the study and development of efficient, environmentally friendly, and sustainable biocontrol techniques against phytonematodes.

Nucleotide sequence and structural organization of the human vasopressin pituitary receptor (V3) gene.

PubMed

René, P; Lenne, F; Ventura, M A; Bertagna, X; de Keyzer, Y

2000-01-04

In the pituitary, vasopressin triggers ACTH release through a specific receptor subtype, termed V3 or V1b. We cloned the V3 cDNA and showed that its expression was almost exclusive to pituitary corticotrophs and some corticotroph tumors. To study the determinants of this tissue specificity, we have now cloned the gene for the human (h) V3 receptor and characterized its structure. It is composed of two exons, spanning 10kb, with the coding region interrupted between transmembrane domains 6 and 7. We established that the transcription initiation site is located 498 nucleotides upstream of the initiator codon and showed that two polyadenylation sites may be used, while the most frequent is the most downstream. Sequence analysis of the promoter region showed no TATA box but identified consensus binding motifs for Sp1, CREB, and half sites of the estrogen receptor binding site. However comparison with another corticotroph-specific gene, proopiomelanocortin, did not identify common regulatory elements in the two promoters except for a short GC-rich region. Unexpectedly, hV3 gene analysis revealed that a formerly cloned 'artifactual' hV3 cDNA indeed corresponded to a spliced antisense transcript, overlapping the 5' part of the coding sequence in exon 1 and the promoter region. This transcript, hV3rev, was detected in normal pituitary and in many corticotroph tumors expressing hV3 sense mRNA and may therefore play a role in hV3 gene expression.

CLONING AND CHARACTERIZATION OF OSTEOCLAST PRECURSORS FROM THE RAW264.7 CELL LINE

PubMed Central

Cuetara, Bethany L. V.; Crotti, Tania N.; O'Donoghue, Anthony J.

2006-01-01

SUMMARY Osteoclasts are bone-resorbing cells that differentiate from macrophage precursors in response to receptor activator of NF-κB (RANKL). In vitro models of osteoclast differentiation are principally based on primary cell culture, which are poorly suited to molecular and transgene studies due to the limitations associated with the use of primary macrophage. RAW264.7 is a transfectable macrophage cell line with the capacity to form osteoclast-like cells. In the present study we have identified osteoclast precursors among clones of RAW264.7 cells. RAW264.7 cell were cloned by limiting dilution and induced to osteoclast differentiation by treatment with recombinant RANKL. Individual RAW264.7 cell clones formed tartrate resistant acid phosphatase (TRAP) positive multinuclear cells to various degrees with RANKL treatment. All clones tested expressed the RANKL receptor RANK. Each of the clones expressed the osteoclast marker genes TRAP and cathepsin-K mRNA with RANKL treatment. However, we noted that only select clones were able to form large, well-spread, TRAP positive multinuclear cells. Clones capable of forming large TRAP positive multinuclear cells also expressed β3 integrin and calcitonin receptor mRNAs and were capable of resorbing a mineralized matrix. All clones tested activated NF-κB with RANKL treatment. cDNA expression profiling of osteoclast precursor RAW264.7 cell clones demonstrates appropriate expression of a large number of genes before and after osteoclastic differentiation. These osteoclast precursor RAW264.7 cell clones provide a valuable model for dissecting the cellular and molecular regulation of osteoclast differentiation and activation. PMID:16948499

Cloning and localization of MCdef, a defensin from Manila clams (Ruditapes philippinarum).

PubMed

Adhya, Mausumi; Jeung, Hee-Do; Kang, Hyun-Sil; Choi, Kwang-Sik; Lee, Dong Sun; Cho, Moonjae

2012-01-01

A defensin-like peptide was previously detected in hemocytes of Manila clams (Ruditapes philippinarum). In the current study, we cloned and characterized this defensin, designated MCdef. Cloning produced a full-length gene sequence of 201 bp predicted to encode a 66-amino-acid precursor protein maturing to a 44-amino-acid residue. Amino acid sequence analysis showed that MCdef is similar to defensins from marine mollusks and ticks. Phylogenetic analysis suggested that MCdef is closely related to defensins from Mytilus galloprovincialis (Mediterranean mussel) and Crassostrea gigas (Pacific cupped oyster). The three-dimensional structure of MCdef was modeled using the solution structure of C. gigas defensin as a template. With the exception of three variable loop areas, the modeled structure of MCdef was identical to that of C. gigas defensin. MCdef antiserum was raised against a synthetic MCdef peptide and verified by Western blotting using recombinant MCdef. RT-PCR analysis demonstrated high levels of MCdef mRNA in hemocytes and adductor, foot, gill, mantle, palp, and siphon tissues of Vibrio tapetis-infected Manila clams, whereas in V. tapetis-uninfected Manila clams, the level of MCdef mRNA was low in adductor, palp, and siphon tissues and even lower in the other tested tissues. Immunohistochemical analysis revealed high MCdef expression was detected in the gill, the mantle, and the digestive tubules of the diverticulum of V. tapetis-infected Manila clams. Minimum inhibitory concentration (MIC) of the purified rMCdef was determined. MCdef showed highest activity against Streptococcus iniae and Staphylococcus aureus. Copyright © 2011 Elsevier Inc. All rights reserved.

Tissue-specific and hormonally regulated expression of a rat alpha 2u globulin gene in transgenic mice.

PubMed Central

Soares, V da C; Gubits, R M; Feigelson, P; Costantini, F

1987-01-01

To investigate the tissue-specific and hormonal regulation of the rat alpha 2u globulin gene family, we introduced one cloned member of the gene family into the mouse germ line and studied its expression in the resulting transgenic mice. Alpha 2u globulingene 207 was microinjected on a 7-kilobase DNA fragment, and four transgenic lines were analyzed. The transgene was expressed at very high levels, specifically in the liver and the preputial gland of adult male mice. The expression in male liver was first detected at puberty, and no expression was detected in female transgenic mice. This pattern of expression is similar to the expression of endogenous alpha 2u globulin genes in the rat but differs from the expression of the homologous mouse major urinary protein (MUP) gene family in that MUPs are synthesized in female liver and not in the male preputial gland. We conclude that these differences between rat alpha 2u globulin and mouse MUP gene expression are due to evolutionary differences in cis-acting regulatory elements. The expression of the alpha 2u globulin transgene in the liver was abolished by castration and fully restored after testosterone replacement. The expression could also be induced in the livers of female mice by treatment with either testosterone or dexamethasone, following ovariectomy and adrenalectomy. Therefore, the cis-acting elements responsible for regulation by these two hormones, as well as those responsible for tissue-specific expression, are closely linked to the alpha 2u globulin gene. Images PMID:2446121

Tissue-specific expression of silkmoth chorion genes in vivo using Bombyx mori nuclear polyhedrosis virus as a transducing vector.

PubMed Central

Iatrou, K; Meidinger, R G

1990-01-01

A pair of silkmoth chorion chromosomal genes, HcA.12-HcB.12, was inserted into a baculovirus transfer vector, pBmp2, derived from the nuclear polyhedrosis virus of Bombyx mori. This vector, which permits the insertion of foreign genetic material in the vicinity of a mutationally inactivated polyhedrin gene, was used to acquire the corresponding recombinant virus. Injection of mutant silkmoth pupae that lack all Hc chorion genes with the recombinant virus resulted in the infection of all internal organs including follicular tissue. Analysis of RNA from infected tissues has demonstrated that the two chorion genes present in the viral genome are correctly transcribed under the control of their own promoter in follicular cells, the tissue in which chorion genes are normally expressed. The chorion primary transcripts are also correctly processed in the infected follicular cells and yield mature mRNAs indistinguishable from authentic chorion mRNAs present in wild-type follicles. These results demonstrate that recombinant nuclear polyhedrosis viruses can be used as transducing vectors for introducing genetic material of host origin into the cells of the organism and that the transduced genes are transiently expressed in a tissue-specific manner under the control of their resident regulatory sequences. Thus we show the in vivo expression of cloned genes under cellular promoter control in an insect other than Drosophila melanogaster. The approach should be applicable to all insect systems that are subject to nuclear polyhedrosis virus infection. Images PMID:2187186

Different Expression Profiles Suggest Functional Differentiation Among Chemosensory Proteins in Nilaparvata lugens (Hemiptera: Delphacidae)

PubMed Central

Yang, Ke; He, Peng; Dong, Shuang-Lin

2014-01-01

Abstract Chemosensory proteins (CSPs) play various roles in insect physiology including olfaction and development. The brown planthopper, Nilaparvata lugens Stål , is one of the most notorious rice pests worldwide. The wing-from variation and annually long distance migration imply that olfaction would play a key role in N. lugens behavior. In this study, full-length cDNAs of nine CSPs were cloned by the rapid amplification of cDNA ends procedure, and their expression profiles were determined by the quantitative real-time Polymerase Chain Reaction (qPCR), with regard to developmental stage, wing-form, gender, and tissues of short-wing adult. These NlugCSP genes showed distinct expression patterns, indicating different roles they play. In particular, NlugCSP5 was long wing form biased and highly expressed in female wings among tissues; NlugCSP1 was mainly expressed in male adults and abdomen; NlugCSP7 was widely expressed in chemosensory tissues but little in the nonchemosensory abdomen. The function of NlugCSP7 in olfaction was further explored by the competitive fluorescence binding assay using the recombinant protein. However, the recombinant NlugCSP7 showed no obvious binding with all tested volatile compounds, suggesting that it may participate in physiological processes other than olfaction. Our results provide bases and some important clues for the function of NlugCSPs . PMID:25527582

Identification and molecular cloning of novel transcripts of the human kallikrein-related peptidase 10 (KLK10) gene using next-generation sequencing

DOE Office of Scientific and Technical Information (OSTI.GOV)

Adamopoulos, Panagiotis G.; Kontos, Christos K.; Scorilas, Andreas

Tissue kallikrein and kallikrein-related peptidases (KLKs) form the largest group of serine proteases in the human genome, sharing many structural and functional characteristics. Multiple alternative transcripts have been reported for the most human KLK genes, while many of them are aberrantly expressed in various malignancies, thus possessing significant prognostic and/or diagnostic value. Alternative splicing of cancer-related genes is a common cellular mechanism accounting for cancer cell transcriptome complexity, as it affects cell cycle control, proliferation, apoptosis, invasion, and metastasis. In this study, we describe the identification and molecular cloning of eight novel transcripts of the human KLK10 gene using 3′more » rapid amplification of cDNA ends (3′ RACE) and next-generation sequencing (NGS), as well as their expression analysis in a wide panel of cell lines, originating from several distinct cancerous and normal tissues. Bioinformatic analysis revealed that the novel KLK10 transcripts contain new alternative splicing events between already annotated exons as well as novel exons. In addition, investigation of their expression profile in a wide panel of cell lines was performed with nested RT-PCR using variant-specific pairs of primers. Since many KLK mRNA transcripts possess clinical value, these newly discovered alternatively spliced KLK10 transcripts appear as new potential biomarkers for diagnostic and/or prognostic purposes or as targets for therapeutic strategies. - Highlights: • NGS was used to identify novel transcripts of the human KLK10 gene. • 8 novel KLK10 transcripts were identified. • A novel 3′UTR was detected and characterized. • The expression profiles of all 8 novel KLK10 transcripts were identified.« less

The expression of sphingosine-1 phosphate receptor-1 in chronic lymphocytic leukemia cells is impaired by tumor microenvironmental signals and enhanced by piceatannol and R406.

PubMed

Borge, Mercedes; Remes Lenicov, Federico; Nannini, Paula R; de los Ríos Alicandú, María M; Podaza, Enrique; Ceballos, Ana; Fernández Grecco, Horacio; Cabrejo, María; Bezares, Raimundo F; Morande, Pablo E; Oppezzo, Pablo; Giordano, Mirta; Gamberale, Romina

2014-09-15

Chronic lymphocytic leukemia (CLL) is characterized by the progressive accumulation of clonal B lymphocytes. Proliferation occurs in lymphoid tissues upon interaction of leukemic cells with a supportive microenvironment. Therefore, the mobilization of tissue-resident CLL cells into the circulation is a useful therapeutic strategy to minimize the reservoir of tumor cells within survival niches. Because the exit of normal lymphocytes from lymphoid tissues depends on the presence of sphingosine-1 phosphate (S1P) and the regulated expression of S1P receptor-1 (S1PR1), we investigated whether the expression and function of S1PR1 can be modulated by key microenvironment signals. We found that activation of CLL cells with CXCL12, fibroblast CD40L(+), BCR cross-linking, or autologous nurse-like cells reduces their S1PR1 expression and the migratory response toward S1P. Moreover, we found that S1PR1 expression was reduced in the proliferative/activated subset of leukemic cells compared with the quiescent subset from the same patient. Similarly, bone marrow-resident CLL cells expressing high levels of the activation marker CD38 showed a lower expression of S1PR1 compared with CD38(low) counterparts. Finally, given that treatment with BCR-associated kinase inhibitors induces a transient redistribution of leukemic cells from lymphoid tissues to circulation, we studied the effect of the Syk inhibitors piceatannol and R406 on S1PR1 expression and function. We found that they enhance S1PR1 expression in CLL cells and their migratory response toward S1P. Based on our results, we suggest that the regulated expression of S1PR1 might modulate the egress of the leukemic clone from lymphoid tissues. Copyright © 2014 by The American Association of Immunologists, Inc.

Cloning and functional characterization of the guinea pig apoptosis inhibitor protein Survivin.

PubMed

Habtemichael, Negusse; Wünsch, Desiree; Bier, Carolin; Tillmann, Sarah; Unruhe, Britta; Frauenknecht, Katrin; Heinrich, Ulf-Rüdiger; Mann, Wolf J; Stauber, Roland H; Knauer, Shirley K

2010-12-01

The guinea pig is widely used as a model to study (patho)physiological processes, such as neurodegenerative disorders. Survivin's dual function as an apoptosis inhibitor and a mitotic regulator is crucial not only for ordered development but its modulation seems crucial also under disease conditions. However, data on the expression and function of the guinea pig Survivin protein (Survivin(Gp)) are currently lacking. Here, we here report the cloning and functional characterization of Survivin(Gp). The respective cDNA was cloned from spleen mRNA, containing a 426 bp open reading frame encoding for a protein of 142aa. Survivin(Gp) displays a high homology to the human and murine orthologue, especially in domains critical for function, such as binding sites for chromosomal passenger complex (CPC) proteins and the nuclear export signal (NES). Notably, phylogenetic analyses revealed that Survivin(Gp) is more related to humans than to rodents. Ectopic expression studies of a Survivin(Gp)-GFP fusion confirmed its dynamic intracellular localization, analogous to the human and murine counterparts. In interphase cells, Survivin(Gp)-GFP was predominantly cytoplasmic and accumulated in the nucleus following export inhibition with leptomycin B (LMB). A typical CPC protein localization during mitosis was observed for Survivin(Gp)-GFP. Microinjection experiments together with genetic knockout demonstrated that the NES is essential for the anti-apoptotic and regulatory role of Survivin(Gp) during cell division. In vivo protein interaction assays further demonstrated its dimerization with human Survivin and its interaction with human CPC proteins. Importantly, RNAi-depletion studies show that Survivin(Gp) can fully substitute for human Survivin as an apoptosis inhibitor and a mitotic effector. Immunohistochemistry, immunofluorescence, and western blotting were employed to detect Survivin expression in guinea pig tissues. Besides its expression in proliferating tissues, such as spleen and liver, we also found Survivin in terminally differentiated cell types. Importantly, Survivin was detectable also in the cochlea, suggesting a potential role for Survivin in the auditory system. We provide the first experimental evidence for the expression of Survivin in the guinea pig. As Survivin(Gp) can substitute for known functions of human Survivin, the guinea pig model will now also allow investigating Survivin's (patho)physiological role and to test Survivin-directed potential therapeutic strategies. Copyright © 2010 Elsevier B.V. All rights reserved.

Ribosomal Proteins RPS11 and RPS20, Two Stress-Response Markers of Glioblastoma Stem Cells, Are Novel Predictors of Poor Prognosis in Glioblastoma Patients

PubMed Central

Yang, Shuai; Tso, Jonathan L.; Menjivar, Jimmy C.; Wei, Bowen; Lucey, Gregory M.; Mareninov, Sergey; Chen, Zugen; Liau, Linda M.; Lai, Albert; Nelson, Stanley F.; Cloughesy, Timothy F.; Tso, Cho-Lea

2015-01-01

Glioblastoma stem cells (GSC) co-exhibiting a tumor-initiating capacity and a radio-chemoresistant phenotype, are a compelling cell model for explaining tumor recurrence. We have previously characterized patient-derived, treatment-resistant GSC clones (TRGC) that survived radiochemotherapy. Compared to glucose-dependent, treatment-sensitive GSC clones (TSGC), TRGC exhibited reduced glucose dependence that favor the fatty acid oxidation pathway as their energy source. Using comparative genome-wide transcriptome analysis, a series of defense signatures associated with TRGC survival were identified and verified by siRNA-based gene knockdown experiments that led to loss of cell integrity. In this study, we investigate the prognostic value of defense signatures in glioblastoma (GBM) patients using gene expression analysis with Probeset Analyzer (131 GBM) and The Cancer Genome Atlas (TCGA) data, and protein expression with a tissue microarray (50 GBM), yielding the first TRGC-derived prognostic biomarkers for GBM patients. Ribosomal protein S11 (RPS11), RPS20, individually and together, consistently predicted poor survival of newly diagnosed primary GBM tumors when overexpressed at the RNA or protein level [RPS11: Hazard Ratio (HR) = 11.5, p<0.001; RPS20: HR = 4.5, p = 0.03; RPS11+RPS20: HR = 17.99, p = 0.001]. The prognostic significance of RPS11 and RPS20 was further supported by whole tissue section RPS11 immunostaining (27 GBM; HR = 4.05, p = 0.01) and TCGA gene expression data (578 primary GBM; RPS11: HR = 1.19, p = 0.06; RPS20: HR = 1.25, p = 0.02; RPS11+RPS20: HR = 1.43, p = 0.01). Moreover, tumors that exhibited unmethylated O-6-methylguanine-DNA methyltransferase (MGMT) or wild-type isocitrate dehydrogenase 1 (IDH1) were associated with higher RPS11 expression levels [corr (IDH1, RPS11) = 0.64, p = 0.03); [corr (MGMT, RPS11) = 0.52, p = 0.04]. These data indicate that increased expression of RPS11 and RPS20 predicts shorter patient survival. The study also suggests that TRGC are clinically relevant cells that represent resistant tumorigenic clones from patient tumors and that their properties, at least in part, are reflected in poor-prognosis GBM. The screening of TRGC signatures may represent a novel alternative strategy for identifying new prognostic biomarkers. PMID:26506620

Several genes encoding ribosomal proteins are over-expressed in prostate-cancer cell lines: confirmation of L7a and L37 over-expression in prostate-cancer tissue samples.

PubMed

Vaarala, M H; Porvari, K S; Kyllönen, A P; Mustonen, M V; Lukkarinen, O; Vihko, P T

1998-09-25

A cDNA library specific for mRNA over-expressed in prostate cancer was generated by subtractive hybridization of transcripts originating from prostatic hyperplasia and cancer tissues. cDNA encoding ribosomal proteins L4, L5, L7a, L23a, L30, L37, S14 and S18 was found to be present among 100 analyzed clones. Levels of ribosomal mRNA were significantly higher at least in one of the prostate-cancer cell lines, LNCaP, DU-145 and PC-3, than in hyperplastic tissue, as determined by slot-blot hybridization. Furthermore, L23a- and S14-transcript levels were significantly elevated in PC-3 cells as compared with those in the normal prostate epithelial cell line PrEC. Generally, dramatic changes in the mRNA content of the ribosomal proteins were not detected, the most evident over-expression being that of L37 mRNA, which was 3.4 times more abundant in LNCaP cells than in hyperplastic prostate tissue. The over-expression of L7a and L37 mRNA was confirmed in prostate-cancer tissue samples by in situ hybridization. Elevated cancer-related expression of L4 and L30 has not been reported, but levels of the other ribosomal proteins are known to be increased in several types of cancers. These results therefore suggest that prostate cancer is comparable with other types of cancers, in that a larger pool of some ribosomal proteins is gained during the transformation process, by an unknown mechanism.

Molecular Characterization and Expression Analysis of Chloroplast Protein Import Components in Tomato (Solanum lycopersicum)

PubMed Central

Yan, Jianmin; Campbell, James H.; Glick, Bernard R.; Smith, Matthew D.; Liang, Yan

2014-01-01

The translocon at the outer envelope membrane of chloroplasts (Toc) mediates the recognition and initial import into the organelle of thousands of nucleus-encoded proteins. These proteins are translated in the cytosol as precursor proteins with cleavable amino-terminal targeting sequences called transit peptides. The majority of the known Toc components that mediate chloroplast protein import were originally identified in pea, and more recently have been studied most extensively in Arabidopsis. With the completion of the tomato genome sequencing project, it is now possible to identify putative homologues of the chloroplast import components in tomato. In the work reported here, the Toc GTPase cDNAs from tomato were identified, cloned and analyzed. The analysis revealed that there are four Toc159 homologues (slToc159-1, -2, -3 and -4) and two Toc34 homologues (slToc34-1 and -2) in tomato, and it was shown that tomato Toc159 and Toc34 homologues share high sequence similarity with the comparable import apparatus components from Arabidopsis and pea. Thus, tomato is a valid model for further study of this system. The expression level of Toc complex components was also investigated in different tissues during tomato development. The two tomato Toc34 homologues are expressed at higher levels in non-photosynthetic tissues, whereas, the expression of two tomato Toc159 homologues, slToc159-1 and slToc159-4, were higher in photosynthetic tissues, and the expression patterns of slToc159-2 was not significantly different in photosynthetic and non-photosynthetic tissues, and slToc159-3 expression was limited to a few select tissues. PMID:24751891

PCR cloning and characterization of multiple ADP-glucose pyrophosphorylase cDNAs from tomato

NASA Technical Reports Server (NTRS)

Chen, B. Y.; Janes, H. W.; Gianfagna, T.

1998-01-01

Four ADP-glucose pyrophosphorylase (AGP) cDNAs were cloned from tomato fruit and leaves by the PCR techniques. Three of them (agp S1, agp S2, and agp S3) encode the large subunit of AGP, the fourth one (agp B) encodes the small subunit. The deduced amino acid sequences of the cDNAs show very high identities (96-98%) to the corresponding potato AGP isoforms, although there are major differences in tissue expression profiles. All four tomato AGP transcripts were detected in fruit and leaves; the predominant ones in fruit are agp B and agp S1, whereas in leaves they are agp B and agp S3. Genomic southern analysis suggests that the four AGP transcripts are encoded by distinct genes.

Molecular structure of P2X receptors.

PubMed

Egan, Terrance M; Cox, Jane A; Voigt, Mark M

2004-01-01

P2X receptors are ligand-gated ion channels that transduce many of the physiological effects of extracellular ATP. There has been a dramatic increase in awareness of these receptors over the past 5 or so years, in great part due to their molecular cloning and characterization. The availability of cDNA clones for the various subunits has led to rapid progress in identifying their tissue-specific expression, resulting in new ideas concerning the functional roles these receptors might play in physiological and pathophysiological processes. In addition, molecular approaches have yielded much information regarding the structure and function of the receptor proteins themselves. In this review we seek to review recent findings concerning the molecular determinants of receptor-channel function, with particular focus on ligand binding and gating, ion selectivity, and subunit assembly.

Seabream ghrelin: cDNA cloning, genomic organization and promoter studies.

PubMed

Yeung, Chung-Man; Chan, Chi-Bun; Woo, Norman Y S; Cheng, Christopher H K

2006-05-01

Recent studies have indicated that ghrelin stimulates growth hormone release from the pituitary via the growth hormone secretagogue receptor (GHSR). We have previously isolated two GHSR subtypes from the pituitary of the black seabream Acanthopagrus schlegeli. In the present study, we have cloned and characterized ghrelin from the same fish species at both the cDNA and gene levels. The full-length seabream ghrelin cDNA, isolated from sea-bream stomach using a novel approach by exploiting a single conserved region in the coding region, was found to encode a prepropeptide of 107 amino acids, with the predicted mature ghrelin peptide consisting of 20 amino acids (GSSFLSPSQKPQNRGKSSRV). Embedded in this full-length cDNA is a putative fish orthologue of the recently reported mammalian obestatin peptide. The ghrelin gene in black seabream, obtained by genomic PCR, was found to encompass four exons and three introns, possessing the same structural organization as in tilapia and goldfish, but different from that in rainbow trout. In addition, a 2230-bp 5'-flanking region of the seabream ghrelin gene was obtained by genome walking. Sequence analysis revealed that, as in the case of the human ghrelin gene, there is neither a GC box nor a CAAT box present in the isolated 5'-flanking region. However, a number of putative transcription factor-binding sites different from the human counterpart were found in the 5'-flanking region of the seabream ghrelin gene, suggesting that different cis- and trans-acting elements are involved in controlling their gene expression. Functional activity of this 5'-flanking region was examined by cloning it into the pGL3-Basic vector upstream of the luciferase reporter gene and transfected into various cell lines. Positive promoter activity could only be recorded in the colon-derived Caco-2 cells, suggesting that the cloned 5'-flanking region represents the functional promoter of the seabream ghrelin gene, which exhibits tissue-specific promoter activity. Using reverse transcriptase PCR analysis, expression of ghrelin was detected only in the seabream stomach, but not in the other tissues examined, including the brain, gill, intestine, kidney, liver and spleen. This stomach-specific expression of ghrelin in seabream is subject to regulation, as administration of growth hormone or ipamorelin to the fish in vivo was demonstrated to enhance its expression. Reminiscent of the homologous upregulation found in the transcriptional control of the seabream GHSR gene, a similar homologous regulatory mechanism might also exist in controlling the expression of seabream ghrelin. The identification of both GHSR and ghrelin from a single fish species would facilitate our subsequent studies on the elucidation of the physiological functions of the ghrelin/GHSR system in teleost. The possible existence of obestatin in teleost opens up new research avenues on the somatotropic axis in fish.

Molecular cloning and functional analysis of nine cinnamyl alcohol dehydrogenase family members in Populus tomentosa.

PubMed

Chao, Nan; Liu, Shu-Xin; Liu, Bing-Mei; Li, Ning; Jiang, Xiang-Ning; Gai, Ying

2014-11-01

Nine CAD/CAD-like genes in P. tomentosa were classified into four classes based on expression patterns, phylogenetic analysis and biochemical properties with modification for the previous claim of SAD. Cinnamyl alcohol dehydrogenase (CAD) functions in monolignol biosynthesis and plays a critical role in wood development and defense. In this study, we isolated and cloned nine CAD/CAD-like genes in the Populus tomentosa genome. We investigated differential expression using microarray chips and found that PtoCAD1 was highly expressed in bud, root and vascular tissues (xylem and phloem) with the greatest expression in the root. Differential expression in tissues was demonstrated for PtoCAD3, PtoCAD6 and PtoCAD9. Biochemical analysis of purified PtoCADs in vitro indicated PtoCAD1, PtoCAD2 and PtoCAD8 had detectable activity against both coniferaldehyde and sinapaldehyde. PtoCAD1 used both substrates with high efficiency. PtoCAD2 showed no specific requirement for sinapaldehyde in spite of its high identity with so-called PtrSAD (sinapyl alcohol dehydrogenase). In addition, the enzymatic activity of PtoCAD1 and PtoCAD2 was affected by temperature. We classified these nine CAD/CAD-like genes into four classes: class I included PtoCAD1, which was a bone fide CAD with the highest activity; class II included PtoCAD2, -5, -7, -8, which might function in monolignol biosynthesis and defense; class III genes included PtoCAD3, -6, -9, which have a distinct expression pattern; class IV included PtoCAD12, which has a distinct structure. These data suggest divergence of the PtoCADs and its homologs, related to their functions. We propose genes in class II are a subset of CAD genes that evolved before angiosperms appeared. These results suggest CAD/CAD-like genes in classes I and II play a role in monolignol biosynthesis and contribute to our knowledge of lignin biosynthesis in P. tomentosa.

Molecular Cloning, Identification, and Expression Patterns of Myostatin Gene in Water Buffalo (Bubalus Bubalis).

PubMed

Zhu, Peng; Li, Haiyang; Huang, Guiting; Cui, Jiayu; Zhang, Ruimen; Cui, Kuiqing; Yang, Sufang; Shi, Deshun

2018-01-02

Myostatin (MSTN), also named growth differentiation factor 8 (GDF8), is a transforming growth factor-β (TGF-β) family member with a key role in the negative regulation of skeletal muscle growth. However, its role in ovarian folliculogenesis remains unclear. To provide us with a basis for understanding this role, we cloned MSTN and examined its expression patterns in water buffalo (Bubalus bubalis). The complete ORF of the water buffalo MSTN gene is 1,128 nucleotides, which encode a 375 amino acid protein and sharing 99% identity at the deducted amino acid level with that of Bos taurus. Protein sequence analysis showed that MSTN is a weakly acerbic extracellular protein, consisting of signal peptides at 18-19 sites, a TGF-β propeptide, and a TGF-β domain. RT-PCR analyses demonstrated that water buffalo MSTN was expressed in multiple tissues but not limited to muscle. Immunohistochemistry staining confirmed the presence of MSTN in oocytes and granulosal cells. To our knowledge, this is the first study to confirm the expression of MSTN in the water buffalo ovary, suggesting an additional role of MSTN in water buffalo folliculogenesis, along with its role in skeletal muscle growth regulation. Further study of the regulatory mechanism of MSTN in water buffalo reproduction is warranted. MSTN, myostatin; ORF, open reading frame.

Tropine Forming Tropinone Reductase Gene from Withania somnifera (Ashwagandha): Biochemical Characteristics of the Recombinant Enzyme and Novel Physiological Overtones of Tissue-Wide Gene Expression Patterns

PubMed Central

Kushwaha, Amit Kumar; Sangwan, Neelam Singh; Trivedi, Prabodh Kumar; Negi, Arvind Singh; Misra, Laxminarain; Sangwan, Rajender Singh

2013-01-01

Withania somnifera is one of the most reputed medicinal plants of Indian systems of medicine synthesizing diverse types of secondary metabolites such as withanolides, alkaloids, withanamides etc. Present study comprises cloning and E. coli over-expression of a tropinone reductase gene (WsTR-I) from W. somnifera, and elucidation of biochemical characteristics and physiological role of tropinone reductase enzyme in tropane alkaloid biosynthesis in aerial tissues of the plant. The recombinant enzyme was demonstrated to catalyze NADPH-dependent tropinone to tropine conversion step in tropane metabolism, through TLC, GC and GC-MS-MS analyses of the reaction product. The functionally active homodimeric ∼60 kDa enzyme catalyzed the reaction in reversible manner at optimum pH 6.7. Catalytic kinetics of the enzyme favoured its forward reaction (tropine formation). Comparative 3-D models of landscape of the enzyme active site contours and tropinone binding site were also developed. Tissue-wide and ontogenic stage-wise assessment of WsTR-I transcript levels revealed constitutive expression of the gene with relatively lower abundance in berries and young leaves. The tissue profiles of WsTR-I expression matched those of tropine levels. The data suggest that, in W. somnifera, aerial tissues as well possess tropane alkaloid biosynthetic competence. In vivo feeding of U-[14C]-sucrose to orphan shoot (twigs) and [14C]-chasing revealed substantial radiolabel incorporation in tropinone and tropine, confirming the de novo synthesizing ability of the aerial tissues. This inherent independent ability heralds a conceptual novelty in the backdrop of classical view that these tissues acquire the alkaloids through transportation from roots rather than synthesis. The TR-I gene expression was found to be up-regulated on exposure to signal molecules (methyl jasmonate and salicylic acid) and on mechanical injury. The enzyme's catalytic and structural properties as well as gene expression profiles are discussed with respect to their physiological overtones. PMID:24086372

Tropine forming tropinone reductase gene from Withania somnifera (Ashwagandha): biochemical characteristics of the recombinant enzyme and novel physiological overtones of tissue-wide gene expression patterns.

PubMed

Kushwaha, Amit Kumar; Sangwan, Neelam Singh; Trivedi, Prabodh Kumar; Negi, Arvind Singh; Misra, Laxminarain; Sangwan, Rajender Singh

2013-01-01

Withania somnifera is one of the most reputed medicinal plants of Indian systems of medicine synthesizing diverse types of secondary metabolites such as withanolides, alkaloids, withanamides etc. Present study comprises cloning and E. coli over-expression of a tropinone reductase gene (WsTR-I) from W. somnifera, and elucidation of biochemical characteristics and physiological role of tropinone reductase enzyme in tropane alkaloid biosynthesis in aerial tissues of the plant. The recombinant enzyme was demonstrated to catalyze NADPH-dependent tropinone to tropine conversion step in tropane metabolism, through TLC, GC and GC-MS-MS analyses of the reaction product. The functionally active homodimeric ~60 kDa enzyme catalyzed the reaction in reversible manner at optimum pH 6.7. Catalytic kinetics of the enzyme favoured its forward reaction (tropine formation). Comparative 3-D models of landscape of the enzyme active site contours and tropinone binding site were also developed. Tissue-wide and ontogenic stage-wise assessment of WsTR-I transcript levels revealed constitutive expression of the gene with relatively lower abundance in berries and young leaves. The tissue profiles of WsTR-I expression matched those of tropine levels. The data suggest that, in W. somnifera, aerial tissues as well possess tropane alkaloid biosynthetic competence. In vivo feeding of U-[(14)C]-sucrose to orphan shoot (twigs) and [(14)C]-chasing revealed substantial radiolabel incorporation in tropinone and tropine, confirming the de novo synthesizing ability of the aerial tissues. This inherent independent ability heralds a conceptual novelty in the backdrop of classical view that these tissues acquire the alkaloids through transportation from roots rather than synthesis. The TR-I gene expression was found to be up-regulated on exposure to signal molecules (methyl jasmonate and salicylic acid) and on mechanical injury. The enzyme's catalytic and structural properties as well as gene expression profiles are discussed with respect to their physiological overtones.

Tissue engineering applications of therapeutic cloning.

PubMed

Atala, Anthony; Koh, Chester J

2004-01-01

Few treatment options are available for patients suffering from diseased and injured organs because of a severe shortage of donor organs available for transplantation. Therapeutic cloning, where the nucleus from a donor cell is transferred into an enucleated oocyte in order to extract pluripotent embryonic stem cells, offers a potentially limitless source of cells for replacement therapy. Scientists in the field of tissue engineering apply the principles of cell transplantation, material science, and engineering to construct biological substitutes that will restore and maintain normal function in diseased and injured tissues. The present chapter reviews recent advances that have occurred in therapeutic cloning and tissue engineering and describes applications of these new technologies that may offer novel therapies for patients with end-stage organ failure.

Cloning of nitric oxide associated 1 (NOA1) transcript from oil palm (Elaeis guineensis) and its expression during Ganoderma infection.

PubMed

Kwan, Yee-Min; Meon, Sariah; Ho, Chai-Ling; Wong, Mui-Yun

2015-02-01

Nitric oxide associated 1 (NOA1) protein is implicated in plant disease resistance and nitric oxide (NO) biosynthesis. A full-length cDNA encoding of NOA1 protein from oil palm (Elaeis guineensis) was isolated and designated as EgNOA1. Sequence analysis suggested that EgNOA1 was a circular permutated GTPase with high similarity to the bacterial YqeH protein of the YawG/YlqF family. The gene expression of EgNOA1 and NO production in oil palm root tissues treated with Ganoderma boninense, the causal agent of basal stem rot (BSR) disease were profiled to investigate the involvement of EgNOA1 during fungal infection and association with NO biosynthesis. Real-time PCR (qPCR) analysis revealed that the transcript abundance of EgNOA1 in root tissues was increased by G. boninense treatment. NO burst in Ganoderma-treated root tissue was detected using Griess reagent, in advance of the up-regulation of the EgNOA1 transcript. This indicates that NO production was independent of EgNOA1. However, the induced expression of EgNOA1 in Ganoderma-treated root tissues implies that it might be involved in plant defense responses against pathogen infection. Copyright © 2014 Elsevier GmbH. All rights reserved.

Exploiting translational coupling for the selection of cells producing toxic recombinant proteins from expression vectors.

PubMed

Tagliavia, Marcello; Cuttitta, Angela

2016-01-01

High rates of plasmid instability are associated with the use of some expression vectors in Escherichia coli, resulting in the loss of recombinant protein expression. This is due to sequence alterations in vector promoter elements caused by the background expression of the cloned gene, which leads to the selection of fast-growing, plasmid-containing cells that do not express the target protein. This phenomenon, which is worsened when expressing toxic proteins, results in preparations containing very little or no recombinant protein, or even in clone loss; however, no methods to prevent loss of recombinant protein expression are currently available. We have exploited the phenomenon of translational coupling, a mechanism of prokaryotic gene expression regulation, in order to select cells containing plasmids still able to express recombinant proteins. Here we designed an expression vector in which the cloned gene and selection marker are co-expressed. Our approach allowed for the selection of the recombinant protein-expressing cells and proved effective even for clones encoding toxic proteins.

Quantum dot-based molecular imaging of cancer cell growth using a clone formation assay.

PubMed

Geng, Xia-Fei; Fang, Min; Liu, Shao-Ping; Li, Yan

2016-10-01

This aim of the present study was to investigate clonal growth behavior and analyze the proliferation characteristics of cancer cells. The MCF‑7 human breast cancer cell line, SW480 human colon cancer cell line and SGC7901 human gastric cancer cell line were selected to investigate the morphology of cell clones. Quantum dot‑based molecular targeted imaging techniques (which stained pan‑cytokeratin in the cytoplasm green and Ki67 in the cell nucleus yellow or red) were used to investigate the clone formation rate, cell morphology, discrete tendency, and Ki67 expression and distribution in clones. From the cell clone formation assay, the MCF‑7, SW480 and SGC7901 cells were observed to form clones on days 6, 8 and 12 of cell culture, respectively. These three types of cells had heterogeneous morphology, large nuclear:cytoplasmic ratios, and conspicuous pathological mitotic features. The cells at the clone periphery formed multiple pseudopodium. In certain clones, cancer cells at the borderline were separated from the central cell clusters or presented a discrete tendency. With quantum dot‑based molecular targeted imaging techniques, cells with strong Ki67 expression were predominantly shown to be distributed at the clone periphery, or concentrated on one side of the clones. In conclusion, cancer cell clones showed asymmetric growth behavior, and Ki67 was widely expressed in clones of these three cell lines, with strong expression around the clones, or aggregated at one side. Cell clone formation assay based on quantum dots molecular imaging offered a novel method to study the proliferative features of cancer cells, thus providing a further insight into tumor biology.

Expression of an alfalfa (Medicago sativa L.) peroxidase gene in transgenic Arabidopsis thaliana enhances resistance to NaCl and H2O2.

PubMed

Teng, K; Xiao, G Z; Guo, W E; Yuan, J B; Li, J; Chao, Y H; Han, L B

2016-05-23

Peroxidases (PODs) are enzymes that play important roles in catalyzing the reduction of H2O2 and the oxidation of various substrates. They function in many different and important biological processes, such as defense mechanisms, immune responses, and pathogeny. The POD genes have been cloned and identified in many plants, but their function in alfalfa (Medicago sativa L.) is not known, to date. Based on the POD gene sequence (GenBank accession No. L36157.1), we cloned the POD gene in alfalfa, which was named MsPOD. MsPOD expression increased with increasing H2O2. The gene was expressed in all of the tissues, including the roots, stems, leaves, and flowers, particularly in stems and leaves under light/dark conditions. A subcellular analysis showed that MsPOD was localized outside the cells. Transgenic Arabidopsis with MsPOD exhibited increased resistance to H2O2 and NaCl. Moreover, POD activity in the transgenic plants was significantly higher than that in wild-type Arabidopsis. These results show that MsPOD plays an important role in resistance to H2O2 and NaCl.

Cloning and functional analysis of the promoter region of the human Disc large gene.

PubMed

Cavatorta, Ana Laura; Giri, Adriana A; Banks, Lawrence; Gardiol, Daniela

2008-11-15

A number of studies have demonstrated the involvement of human Disc large (DLG1) in the control of both cell polarity and maintenance of tissue architecture. However, the mechanisms controlling DLG1 transcription are not fully understood. This is relevant since DLG1 is lost in many tumours during the later stages of malignant progression. Therefore, we performed the cloning and functional analysis of a genomic 5' flanking region of the DLG1 open reading frame with promoter activity. We analyzed the activity of a series of 5' deletion constructs of the DLG1 promoter and determined the minimal essential sequences that are required for promoter activity as well as cis-elements that regulate transcription. We found, within the DLG1 promoter sequences, consensus-binding sites for the Snail family of transcription factors that repress the expression of epithelial markers and are up-regulated in a variety of tumours. Snail transcription factors repress the transcriptional activity of the DLG1 promoter and, ectopically expressed Snail proteins bind to the native DLG1 promoter. These data suggest a role for Snail transcription factors in the control of DLG1 expression and provide a basis for understanding the transcriptional regulation of DLG1.

Cloning and characterization of a riboflavin-binding hexamerin from the larval fat body of a lepidopteran stored grain pest, Corcyra cephalonica.

PubMed

Rao, V Venkat; Ningshen, Thuirei Jacob; Chaitanya, R K; Senthilkumaran, B; Dutta-Gupta, Aparna

2016-01-01

In the present study, a riboflavin-binding hexamerin (RbHex) was cloned and characterized from the larval fat body of Corcyra cephalonica. The complete cDNA (2121bp) encodes a 706-amino acid protein with a molecular mass ~82kDa. Expression of RbHex 82 was predominant in fat body among larval tissues. Further, it is prominently expressed during the last instar larval development. Homology modeling and docking studies predicted riboflavin binding site of the hexamerin. Spectrofluorimetric analysis further confirmed riboflavin release from the hexamerin fraction. Quantitative RT-PCR studies demonstrated hormonal regulation of RbHex 82. 20-Hydroxyecdysone (20HE) had a stimulatory effect on its transcription whereas JH alone did not show any effect. However, JH in the presence of 20HE maintains the RbHex 82 expression which indicates the JH's role as a status quo factor. This study is the first to report the characterization of riboflavin-binding hexamerin in a lepidopteran pest. Further, the possibility of RbHex 82 as a pest control target is discussed. Copyright © 2016 Elsevier Inc. All rights reserved.

Gambling on a shortcut to genome sequencing

DOE Office of Scientific and Technical Information (OSTI.GOV)

Roberts, L.

1991-06-21

Almost from the start of the Human Genome Project, a debate has been raging over whether to sequence the entire human genome, all 3 billion bases, or just the genes - a mere 2% or 3% of the genome, and by far the most interesting part. In England, Sydney Brenner convinced the Medical Research Council (MRC) to start with the expressed genes, or complementary DNAs. But the US stance has been that the entire sequence is essential if we are to understand the blueprint of man. Craig Venter of the National Institute of Neurological Disorders and Stroke says that focusingmore » on the expressed genes may be even more useful than expected. His strategy involves randomly selecting clones from cDNA libraries which theoretically contain all the genes that are switched on at a particular time in a particular tissue. Then the researchers sequence just a short stretch of each clone, about 400 to 500 bases, to create can expressed sequence tag or EST. The sequences of these ESTs are then stored in a database. Using that information, other researchers can then recreate that EST by using polymerase chain reaction techniques.« less

Analysis and Functional Annotation of an Expressed Sequence Tag Collection for Tropical Crop Sugarcane

PubMed Central

Vettore, André L.; da Silva, Felipe R.; Kemper, Edson L.; Souza, Glaucia M.; da Silva, Aline M.; Ferro, Maria Inês T.; Henrique-Silva, Flavio; Giglioti, Éder A.; Lemos, Manoel V.F.; Coutinho, Luiz L.; Nobrega, Marina P.; Carrer, Helaine; França, Suzelei C.; Bacci, Maurício; Goldman, Maria Helena S.; Gomes, Suely L.; Nunes, Luiz R.; Camargo, Luis E.A.; Siqueira, Walter J.; Van Sluys, Marie-Anne; Thiemann, Otavio H.; Kuramae, Eiko E.; Santelli, Roberto V.; Marino, Celso L.; Targon, Maria L.P.N.; Ferro, Jesus A.; Silveira, Henrique C.S.; Marini, Danyelle C.; Lemos, Eliana G.M.; Monteiro-Vitorello, Claudia B.; Tambor, José H.M.; Carraro, Dirce M.; Roberto, Patrícia G.; Martins, Vanderlei G.; Goldman, Gustavo H.; de Oliveira, Regina C.; Truffi, Daniela; Colombo, Carlos A.; Rossi, Magdalena; de Araujo, Paula G.; Sculaccio, Susana A.; Angella, Aline; Lima, Marleide M.A.; de Rosa, Vicente E.; Siviero, Fábio; Coscrato, Virginia E.; Machado, Marcos A.; Grivet, Laurent; Di Mauro, Sonia M.Z.; Nobrega, Francisco G.; Menck, Carlos F.M.; Braga, Marilia D.V.; Telles, Guilherme P.; Cara, Frank A.A.; Pedrosa, Guilherme; Meidanis, João; Arruda, Paulo

2003-01-01

To contribute to our understanding of the genome complexity of sugarcane, we undertook a large-scale expressed sequence tag (EST) program. More than 260,000 cDNA clones were partially sequenced from 26 standard cDNA libraries generated from different sugarcane tissues. After the processing of the sequences, 237,954 high-quality ESTs were identified. These ESTs were assembled into 43,141 putative transcripts. Of the assembled sequences, 35.6% presented no matches with existing sequences in public databases. A global analysis of the whole SUCEST data set indicated that 14,409 assembled sequences (33% of the total) contained at least one cDNA clone with a full-length insert. Annotation of the 43,141 assembled sequences associated almost 50% of the putative identified sugarcane genes with protein metabolism, cellular communication/signal transduction, bioenergetics, and stress responses. Inspection of the translated assembled sequences for conserved protein domains revealed 40,821 amino acid sequences with 1415 Pfam domains. Reassembling the consensus sequences of the 43,141 transcripts revealed a 22% redundancy in the first assembling. This indicated that possibly 33,620 unique genes had been identified and indicated that >90% of the sugarcane expressed genes were tagged. PMID:14613979

cDNA cloning, expression pattern, and chromosomal localization of Mlf1, murine homologue of a gene involved in myelodysplasia and acute myeloid leukemia.

PubMed

Hitzler, J K; Witte, D P; Jenkins, N A; Copeland, N G; Gilbert, D J; Naeve, C W; Look, A T; Morris, S W

1999-07-01

The NPM-MLF1 fusion protein is expressed in blasts from patients with myelodysplasia/acute myeloid leukemia (MDS/AML) containing the t(3;5) chromosomal rearrangement. Nucleophosmin (NPM), a previously characterized nucleolar phosphoprotein, contributes to two other fusion proteins found in lympho-hematopoietic malignancies, anaplastic large cell lymphoma (NPM-ALK) and acute promyelocytic leukemia (NPM-RARalpha). By contrast, the function of the carboxy-terminal fusion partner, myelodysplasia/myeloid leukemia factor 1 (MLF1), is unknown. To aid in understanding normal MLF1 function, we isolated the murine cDNA, determined the chromosomal localization of Mlf1, and defined its tissue expression by in situ hybridization. Mlf1 was highly similar to its human homologue (86% and 84% identical nucleotide and amino acid sequence, respectively) and mapped to the central region of chromosome 3, within a segment lacking known mouse mutations. Mlf1 tissue distribution was restricted during both development and postnatal life, with high levels present only in skeletal, cardiac, and selected smooth muscle, gonadal tissues, and rare epithelial tissues including the nasal mucosa and the ependyma/choroid plexus in the brain. Mlf1 transcripts were undetectable in the lympho-hematopoietic organs of both the embryonic and adult mouse, suggesting that NPM-MLF1 contributes to the genesis of MDS/AML in part by enforcing the ectopic overexpression of MLF1 within hematopoietic tissues.

Cloning, tissue distribution, functional characterization and nutritional regulation of a fatty acyl Elovl5 elongase in chu's croaker Nibea coibor.

PubMed

Lin, Zhideng; Huang, Yisheng; Zou, Weiguang; Rong, Hua; Hao, Meiling; Wen, Xiaobo

2018-06-15

Enzymes that lengthen the carbon chain of polyunsaturated fatty acids (PUFA) are key to the biosynthesis of the long-chain polyunsaturated fatty acids (LC-PUFA). Here we report on the molecular cloning, tissue distribution, functional characterization and nutritional regulation of a elovl5 gene from Nibea coibor. The full-length cDNA was 1315 bp, including a 5-untranslated region (UTR) of 134 bp, a 3-UTR of 296 bp and an open reading frame of 885 bp, which specified a peptide of 294 amino acids. Bioinformatics analysis showed that the deduced peptide sequence possessed all the characteristic features of microsomal fatty acyl elongases, including the so-called histidine box (HXXHH), the canonical C-terminal endoplasmic reticulum retention signal, several predicted transmembrane regions and other highly conserved motifs. Expression of elovl5 was strongly observed in stomach, and more weakly in kidney, spleen, intestine, brain, eye, liver, gill, muscle and heart. Functional characterization revealed that the chu's croaker Elovl5 was able to elongate both C18 and C20 PUFA substrates. Nutritional study indicated that the hepatic expression of elovl5 could be up-regulated by low dietary n-3 LC-PUFA. These results may contribute to better understanding the LC-PUFA biosynthetic pathway and regulation mechanism in chu's croaker. Copyright © 2018. Published by Elsevier B.V.

Molecular cloning, subcelluar location and expression profile of signal transducer and activator of transcription 2 (STAT2) from turbot, Scophthalmus maximus.

PubMed

Wang, Na; Wang, Xian-Li; Yang, Chang-Geng; Chen, Song-Lin

2013-10-01

Signal transducer and activator of transcription 2 (STAT2) is an important molecule involved in the type I interferon signalling pathway. To date, little STAT2 homologue is available in fish except Atlantic salmon and goldfish. In this paper, STAT2 was firstly cloned and characterized from turbot, a marine flatfish with high economic value. Briefly, turbot STAT2 cDNA is 3206 bp in length encoding a predicted protein of 793 amino acids. The phylogenetic tree shows that turbot STAT2 protein shared the closest relationship with Atlantic salmon. Analysis of subcellular distribution indicates that STAT2 is mainly present in the cytoplasm of TK cells. Stat2 mRNA is constitutively expressed in widespread tissues and induced by several folds in turbot tissues and TK cells after stimulation with Vibrio anguillarum and lymphocystis disease virus (LCDV). Unlike the higher vertebrate STAT2, turbot STAT2 nuclear export signal (NES) exists not in the C-terminal 79 amino acids but in N-terminal 137-312 amino acids (STAT_alpha domain). The nuclear translocation of turbot STAT2 after Poly(I:C) treatment proved its transcription activity in TK cells. All these results suggested that STAT2 may be involved in the immune response in turbot as a transcription factor. Copyright © 2013 Elsevier Ltd. All rights reserved.

Molecular cloning of a C-type lectin with two CRD domains from the banana shrimp Fenneropenaeus merguiensis: early gene up-regulation after Vibrio harveyi infection.

PubMed

Rattanaporn, Onnicha; Utarabhand, Prapaporn

2011-02-01

A diverse class of pattern-recognition proteins called lectins play important roles in shrimp innate immunity. A novel C-type lectin gene (FmLC) was cloned from the hepatopancreas of banana shrimp Fenneropenaeus merguiensis by means of PCR and 5' and 3' rapid amplification of cDNA ends (RACE). The full-length cDNA consists of 1118 bp with one 1002 bp open reading frame, encoding 333 amino acids. Its deduced amino acid sequence contains a putative signal peptide of 20 amino acids. FmLC contains two carbohydrate recognition domains, CRD1 and CRD2, that share only 30% identity with each other. The first CRD comprises a QPD motif with specificity for binding galactose and a single Ca(2+) binding site, while the second CRD consists of an EPN motif for a mannose-specific binding site. FmLC had a close evolutionary relationship to other dual-CRD lectins of penaeid shrimp. Expression results showed that transcripts of FmLC were detected only in the hepatopancreas, none was found in other tissues. After challenging either whole shrimp or hepatopancreas tissue fragments with Vibrioharveyi, the expression of FmLC was up-regulated. This indicates that FmLC is inducible and may be involved in a shrimp immune response to recognize potential bacterial pathogens. Copyright © 2010 Elsevier Inc. All rights reserved.

T-cell epitope analysis using subtracted expression libraries (TEASEL): application to a 38-kDA autoantigen recognized by T cells from an insulin-dependent diabetic patient.

PubMed Central

Neophytou, P I; Roep, B O; Arden, S D; Muir, E M; Duinkerken, G; Kallan, A; de Vries, R R; Hutton, J C

1996-01-01

Studies on circulating T cells and antibodies in newly diagnosed type 1 diabetic patients and rodent models of autoimmune diabetes suggest that beta-cell membrane proteins of 38 kDa may be important molecular targets of autoimmune attack. Biochemical approaches to the isolation and identification of the 38-kDa autoantigen have been hampered by the restricted availability of islet tissue and the low abundance of the protein. A procedure of epitope analysis for CD4+ T cells using subtracted expression libraries (TEASEL) was developed and used to clone a 70-amino acid pancreatic beta-cell peptide incorporating an epitope recognized by a 38-kDa-reactive CD4+ T-cell clone (1C6) isolated from a human diabetic patient. The minimal epitope was mapped to a 10-amino acid synthetic peptide containing a DR1 consensus binding motif. Data base searches did not reveal the identity of the protein, though a weak homology to the bacterial superantigens SEA (Streptococcus pyogenes exotoxin A) and SEB (Staphylococcus aureus enterotoxin B) (23% identity) was evident. The TEASEL procedure might be used to identify epitopes of other autoantigens recognized by CD4+ T cells in diabetes as well as be more generally applicable to the study low-abundance autoantigens in other tissue-specific autoimmune diseases. PMID:8700877

Rapid one-step recombinational cloning

PubMed Central

Fu, Changlin; Wehr, Daniel R.; Edwards, Janice; Hauge, Brian

2008-01-01

As an increasing number of genes and open reading frames of unknown function are discovered, expression of the encoded proteins is critical toward establishing function. Accordingly, there is an increased need for highly efficient, high-fidelity methods for directional cloning. Among the available methods, site-specific recombination-based cloning techniques, which eliminate the use of restriction endonucleases and ligase, have been widely used for high-throughput (HTP) procedures. We have developed a recombination cloning method, which uses truncated recombination sites to clone PCR products directly into destination/expression vectors, thereby bypassing the requirement for first producing an entry clone. Cloning efficiencies in excess of 80% are obtained providing a highly efficient method for directional HTP cloning. PMID:18424799

Identification of differentially expressed genes in pistils from self-incompatible Citrus reticulata by suppression subtractive hybridization.

PubMed

Miao, Hongxia; Qin, Yonghua; da Silva, Jaime A Teixeira; Ye, Zixing; Hu, Guibing

2013-01-01

Self-incompatibility (SI) is one important factor that can result in Citrus seedlessness. However, the molecular mechanism of SI in Citrus is not clear yet. To isolate the pistil's SI-related genes, a suppression subtractive hybridization library was constructed using mature pistils of 'Wuzishatangju' mandarin (SI) as the tester and mature pistils of 'Shatangju' mandarin (self-compatibility, SC) as the driver. 229 differentially expressed cDNA clones from 967 positive clones were sequenced and identified. Differentially expressed ESTs are possibly involved in the SI reaction of 'Wuzishatangju' through a regulating signaling pathway, serine/threonine phosphatase activity, receptor kinase, embryonic development, gibberellin stimulus, or transcription. 11 out of 36 SI candidate genes displayed different expression patterns in various tissues and stages after self- and cross-pollination of 'Wuzishatangju'. The expression of CaBP (WY65), a senescence-protease (WY372), an unknown gene (WY283), and a WRKY (WY17) were up-regulated in the styles of 'Wuzishatangju' while higher expression of WY190 was observed in styles of 'Shatangju'. Highest expression levels of WY65, WY372, an annexin (WY598), the zinc-finger protein (WY376), a C2-protein (WY291), and an unknown gene (WY318) were detected in styles at 3 days after self-pollination of 'Wuzishatangju' while lowest levels were observed in styles at 3 days after cross-pollination of 'Wuzishatangju' × 'Shatangju'. The potential involvement of these genes in the SI reaction is discussed.

Identification of a novel splice variant of human PD-L1 mRNA encoding an isoform-lacking Igv-like domain.

PubMed

He, Xian-hui; Xu, Li-hui; Liu, Yi

2005-04-01

To investigate the expression and regulation of PD-1 ligand 1 (PD-L1) in peripheral blood mononuclear cells (PBMC). The cDNA encoding human PD-L1 precursor was cloned from the total RNA extracted from the resting and phorbol dibutyrate plus ionomycin- or phytohemagglutinin-activated PBMC, by reverse transcription polymerase chain reaction (RT-PCR), and independent clones were sequenced and analyzed. The expression and subcellular localization were examined in transiently transfected cells. The PD-L1 gene expression in different PBMC was also analyzed by RT-PCR. A novel human PD-L1 splice variant was identified from the activated PBMC. It was generated by splicing out exon? encoding an immunoglobulin variable domain (Igv)-like domain but retaining all other exons without a frame-shift. Consequently, the putative translated protein contained all other domains including the transmembrane region except for the Igv-like domain. Furthermore, the conventional isoform was expressed on the plasma surface whereas the novel isoform showed a pattern of intracellular membrane distribution in transiently transfected K562 cells. In addition, the expression pattern of the PD-L1 splice variant was variable in different individuals and in different cellular status. PD-L1 expression may be regulated at the posttranscriptional level through alternative splicing, and modulation of the PD-L1 isoform expression may influence the outcome of specific immune responses in the peripheral tissues.

[Construction of forward and reverse subtracted cDNA libraries between muscle tissue of Meishan and Landrace pigs].

PubMed

Xu, De-Quan; Zhang, Yi-Bing; Xiong, Yuan-Zhu; Gui, Jian-Fang; Jiang, Si-Wen; Su, Yu-Hong

2003-07-01

Using suppression subtractive hybridization (SSH) technique, forward and reverse subtracted cDNA libraries were constructed between Longissimus muscles from Meishan and Landrace pigs. A housekeeping gene, G3PDH, was used to estimate the efficiency of subtractive cDNA. In two cDNA libraries, G3PDH was subtracted very efficiently at appropriate 2(10) and 2(5) folds, respectively, indicating that some differentially expressed genes were also enriched at the same folds and the two subtractive cDNA libraries were very successful. A total of 709 and 673 positive clones were isolated from forward and reverse subtracted cDNA libraries, respectively. Analysis of PCR showed that most of all plasmids in the clones contained 150-750 bp inserts. The construction of subtractive cDNA libraries between muscle tissue from different pig breeds laid solid foundations for isolating and identifying the genes determining muscle growth and meat quality, which will be important to understand the mechanism of muscle growth, determination of meat quality and practice of molecular breeding.

A putative octopamine/tyramine receptor mediating appetite in a hungry fly

NASA Astrophysics Data System (ADS)

Ishida, Yuko; Ozaki, Mamiko

2011-07-01

In the blowfly Phormia regina, experience of simultaneous feeding with d-limonene exposure inhibits proboscis extension reflex (PER) due to decreased tyramine (TA) titer in the brain. To elucidate the molecular mechanism of TA signaling pathway related to the associated feeding behavior, we cloned cDNA encoding the octopamine/TA receptor (PregOAR/TAR). The deduced protein is composed of 607 amino acid residues and has 7 predicted transmembrane domains. Based on homology and phylogenetic analyses, this protein belongs to the OAR/TAR family. The PregOAR/TAR was mainly expressed in head, with low levels of expression in other tissues at adult stages. Gene expression profile is in agreement with a plethora of functions ascribed to TA in various insect tissues. The immunolabeled cell bodies and processes were localized in the medial protocerebrum, outer layer of lobula, antennal lobe, and subesophageal ganglion. These results suggest that decrease of TA level in the brain likely affects neurons expressing PregOAR/TAR, causing mediation of the sensitivity in the sensillum and/or output of motor neurons for PER.

The gene for fibroblast activation protein {alpha} (FAP), a putative cell surface-bound serine protease expressed in cancer stroma and wound healing, maps to chromosome band 2q23

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mathew, S.; Murty, V.V.V.S.; Chaganti, R.S.K.

The human fibroblast activation protein {alpha} (FAP{alpha}) is an inducible cell surface glycoprotein of M{sub r} 95,000 recognized by a number of monoclonal antibodies (mAbs), including the prototype mAb F19. Immunohistochemical studies have shown that FAP{alpha} expression in vivo is tightly regulated, with transient expression in some fetal mesenchymal tissues but absence of expression in most normal adult tissues. Reexpression of FAP{alpha} is observed in the reactive stromal fibroblasts of several common types of epithelial cancers, including >90% of breast, colorectal, and lung carcinomas and healing wounds. Cloning and sequence analysis of an FAP{alpha}-specific cDNA has revealed that the moleculemore » is encoded by a novel gene, FAP, which shows sequence similarity to members of the serine protease family of integral membrane proteins, namely dipeptidyl peptidase IV (DPPIV, also known as lymphocyte activation antigen, CD26, or adenosine dearoinase binding protein) and DPPX, a DPPIV-related molecule of unknown function. 15 refs., 1 fig.« less

Expression of EGFP and NPTII protein is not associated with organ abnormalities in deceased transgenic cloned cattle.

PubMed

Liu, Yan; Wu, Qian; Cui, Huiting; Li, Qinghe; Zhao, Yiqiang; Luo, Juan; Liu, Qiuyue; Sun, Xiuzhu; Tang, Bo; Zhang, Lei; Dai, Yunping; Li, Ning

2008-12-01

Both enhanced green fluorescence protein (EGFP) and neomycin phosphotransferase type II enzyme (NPTII) are widely used in transgenic studies, but their side effects have not been extensively investigated. In this study, we evaluated the expression profiles of the two marker genes and the relationship between their expression and organ abnormalities. Eight transgenic cloned cattle were studied, four harboring both EGFP and NPTII, and four harboring only the NPTII gene. Four age-matched cloned cattle were used as controls. EGFP and NPTII expression were measured and detected by Q-PCR, Western blot, ELISA, and RIA in heart, liver, and lungs, and the values ranged from 0.3 to 5 microg/g. The expression profiles exhibited differential or mosaic pattern between the organs, the pathologic symptoms of which were identified, but were similar to those of age-matched cloned cattle. All data indicated that the expression of EGFP and NPTII is not associated with organ abnormalities in transgenic cloned cattle.

c-MYC independent nuclear reprogramming favors cardiogenic potential of induced pluripotent stem cells

PubMed Central

Martinez-Fernandez, Almudena; Nelson, Timothy J.; Ikeda, Yasuhiro; Terzic, Andre

2010-01-01

Induced pluripotent stem cell (iPS) technology has launched a new platform in regenerative medicine aimed at deriving unlimited replacement tissue from autologous sources through somatic cell reprogramming using stemness factor sets. In this way, authentic cardiomyocytes have been obtained from iPS and recently demonstrated in proof-of-principle studies to repair infarcted heart. Optimizing the cardiogenic potential of iPS progeny would ensure a maximized yield of bioengineered cardiac tissue. Here, we reprogrammed fibroblasts in the presence or absence of c-MYC to determine if the acquired cardiogenicity is sensitive to the method of nuclear reprogramming. Using lentiviral constructs that expressed stemness factors SOX2, OCT4, and KLF4 with or without c-MYC, iPS clones generated through fibroblast reprogramming demonstrated indistinguishable characteristics for 5 days of differentiation with similar cell morphology, growth rates, and chimeric embryo integration. However, 4-factor c-MYC dependent nuclear reprogramming produced iPS progeny that consistently prolonged the expression of pluripotent Oct-4 and Fgf4 genes and repressed cardiac differentiation. In contrast, 3-factor c-MYC-less iPS clones efficiently up-regulated pre-cardiac (CXCR4, Flk-1, and Mesp1/2) and cardiac (Nkx2.5, Mef2c, and Myocardin) gene expression patterns. In fact, 3-factor iPS progeny demonstrated early and robust cardiogenesis during in vitro differentiation with consistent beating activity, sarcomere maturation, and rhythmical intracellular calcium dynamics. Thus, nuclear reprogramming independent of c-MYC enhances production of pluripotent stem cells with innate cardiogenic potential. PMID:20221419

Involvement of clip-domain serine protease in the anti-Vibrio immune response of abalone (Haliotis discus hannai)-Molecular cloning, characterization and functional analysis.

PubMed

Hu, Jian-Jian; Chen, Yu-Lei; Duan, Xue-Kun; Jin, Teng-Chuan; Li, Yue; Zhang, Ling-Jing; Liu, Guang-Ming; Cao, Min-Jie

2018-01-01

Vibrio parahemolyticus (V. parahemolyticus) is a major pathogen for abalone, an important economical shellfish in coastal area of China. There is little known about the abalone innate immune system against pathogen infection. Clip-domain serine proteases (cSPs) are increasingly recognized to play important roles in host immune defense in invertebrates. In this study, we cloned a cSP (Hdh-cSP) from abalone (Haliotis discus hannai). We found out that Hdh-cSP was widely expressed in multiple tissues of abalone, with highest level in the immune-like organ, hepatopancreas. V. parahemolyticus infection induced significantly elevated expression of Hdh-cSP in addition to better-characterized innate immune component genes including Rel/NF-κB, allograft inflammatory factor (ALInFa), macrophage expressed protein (MEP) and caspase-8. Importantly, the silencing of Hdh-cSP reduced the expression of these genes, suggesting that Hdh-cSP was an upstream regulatory factor in V. parahemolyticus infection. Further analysis showed that apoptosis of hemocytes was inhibited when the transcription of Hdh-cSP was knocked down, suggesting that Hdh-cSP participated in cell apoptosis by regulation of caspase 8 expression in V. parahemolyticus infection. Therefore, our study established an important role of cSP in the innate immunity against V. parahemolyticus infection in abalone. Copyright © 2017 Elsevier Ltd. All rights reserved.

Molecular cloning and functional analysis of ESGP, an embryonic stem cell and germ cell specific protein.

PubMed

Chen, Yan-Mei; Du, Zhong-Wei; Yao, Zhen

2005-12-01

Several putative Oct-4 downstream genes from mouse embryonic stem (ES) cells have been identified using the suppression-subtractive hybridization method. In this study, one of the novel genes encoding an ES cell and germ cell specific protein (ESGP) was cloned by rapid amplification of cDNA ends. ESGP contains 801 bp encoding an 84 amino acid small protein and has no significant homology to any known genes. There is a signal peptide at the N-terminal of ESGP protein as predicted by SeqWeb (GCG) (SeqWeb version 2.0.2, http://gcg.biosino.org:8080/). The result of immunofluorescence assay suggested that ESGP might encode a secretory protein. The expression pattern of ESGP is consistent with the expression of Oct-4 during embryonic development. ESGP protein was detected in fertilized oocyte, from 3.5 day postcoital (dpc) blastocyst to 17.5 dpc embryo, and was only detected in testis and ovary tissues in adult. In vitro, ESGP was only expressed in pluripotent cell lines, such as embryonic stem cells, embryonic caoma cells and embryonic germ cells, but not in their differentiated progenies. Despite its specific expression, forced expression of ESGP is not indispensable for the effect of Oct-4 on ES cell self-renewal, and does not affect the differentiation to three germ layers.

Molecular cloning and functional identification of sterol C24-methyltransferase gene from Tripterygium wilfordii.

PubMed

Guan, Hongyu; Zhao, Yujun; Su, Ping; Tong, Yuru; Liu, Yujia; Hu, Tianyuan; Zhang, Yifeng; Zhang, Xianan; Li, Jia; Wu, Xiaoyi; Huang, Luqi; Gao, Wei

2017-09-01

Sterol C24-methyltransferase (SMT) plays multiple important roles in plant growth and development. SMT1, which belongs to the family of transferases and transforms cycloartenol into 24-methylene cycloartenol, is involved in the biosynthesis of 24-methyl sterols. Here, we report the cloning and characterization of a cDNA encoding a sterol C24-methyltransferase from Tripterygium wilfordii ( TwSMT1 ). TwSMT1 (GenBank access number KU885950) is a 1530 bp cDNA with a 1041 bp open reading frame predicted to encode a 346-amino acid, 38.62 kDa protein. The polypeptide encoded by the SMT1 cDNA was expressed and purified as a recombinant protein from Escherichia coli ( E. coli ) and showed SMT activity. The expression of TwSMT1 was highly up-regulated in T. wilfordii cell suspension cultures treated with methyl jasmonate (MeJA). Tissue expression pattern analysis showed higher expression in the phellem layer compared to the other four organs (leaf, stem, xylem and phloem), which is about ten times that of the lowest expression in leaf. The results are meaningful for the study of sterol biosynthesis of T. wilfordii and will further lay the foundations for the research in regulating both the content of other main compounds and growth and development of T. wilfordii.

Molecular cloning, expression analysis, and potential food intake attenuation effect of peptide YY in grass carp (Ctenopharyngodon idellus).

PubMed

Chen, Yong; Shen, Yubang; Pandit, Narayan Prasad; Fu, Jianjun; Li, Da; Li, Jiale

2013-06-15

The peptide YY (PYY) is a 36 amino acid peptide involved in the food intake control in vertebrates. We have cloned and characterized a PYY gene from grass carp Ctenopharyngodon idellus. The full-length cDNA encodes a precursor protein of grass carp PYY (gcPYY) that consists of a putative 28-amino acid signal peptide, a 36-amino acid mature peptide, an amidation-proteolytic site, and a 30-amino acid carboxy-terminal extension. The gcPYY gene is comprised of 4 exons interspaced by 3 introns as seen in PYYs from other species. Amino acid alignment and gene structure comparison indicate that the structure of PYY is well preserved throughout vertebrate phylogeny. The tissue distribution and postprandial changes in gcPYY mRNA expression were evaluated by real-time PCR, which showed that the gcPYY is expressed abundantly in the central nervous system, with significantly increased expression following a single meal. During embryogenesis, the presence of gcPYY mRNA was detected in early developing embryos, and high expression levels were observed when most larvae completed their switch from endogenous nourishment to exogenous feeding. Reduced food intake by juveniles during a single meal after giving perpheral injection of gcPYY1-36 suggests a potentially important role of PYY in the food intake attenuation in grass carp. Copyright © 2013 Elsevier Inc. All rights reserved.

Homologue gene of bile acid transporters ntcp, asbt, and ost-alpha in rainbow trout Oncorhynchus mykiss: tissue expression, effect of fasting, and response to bile acid administration.

PubMed

Murashita, Koji; Yoshiura, Yasutoshi; Chisada, Shin-Ichi; Furuita, Hirofumi; Sugita, Tsuyoshi; Matsunari, Hiroyuki; Iwashita, Yasuro; Yamamoto, Takeshi

2014-04-01

Bile acid transporters belonging to the SLC10A protein family, Na+ taurocholate cotransporting polypeptide (NTCP or SLC10A1), apical sodium-dependent bile salt transporter (ASBT or SLC10A2), and organic solute transporter alpha (Ost-alpha) have been known to play critical roles in the enterohepatic circulation of bile acids in mammals. In this study, ntcp, asbt, and ost-alpha-1/-2 cDNA were cloned, their tissue distributions were characterized, and the effects of fasting and bile acid administration on their expression were examined in rainbow trout Oncorhynchus mykiss. The structural characteristics of Ntcp, Asbt, and Ost-alpha were well conserved in trout, and three-dimensional structure analysis showed that Ntcp and Asbt were similar to each other. Tissue distribution analysis revealed that trout asbt was primarily expressed in the hindgut, while ntcp expression occurred in the brain, and ost-alpha-1/-2 was mainly expressed in the liver or ovary. Although asbt and ost-alpha-1 mRNA levels in the gut increased in response to fasting for 4 days, ost-alpha-1 expression in the liver decreased. Similarly, bile acid administration increased asbt and ost-alpha-1 expression levels in the gut, while those of ntcp and ost-alpha-2 in the liver decreased. These results suggested that the genes asbt, ntcp, and ost-alpha are involved in bile acid transport in rainbow trout.

Molecular cloning, polymorphisms, and expression analysis of the RERG gene in indigenous Chinese goats.

PubMed

Sui, M X; Wang, H H; Wang, Z W

2015-11-24

The current study aimed to investigate the coding sequence, polymorphisms, and expression of the RERG gene in indigenous Chinese goats. cDNA of RERG, obtained through reverse transcription PCR was analyzed using bioinformatic techniques. Polymorphisms in the exon regions of the RERG gene were identified and their associations with growth traits in three varieties of indigenous Chinese goats were investigated. Expression of the RERG gene in three goat breeds of the same age was detected using real-time quantitative PCR. The results revealed that the cDNA of RERG, which contained a complete open reading frame of 20-620 bp, was 629 bp in length. The associated accession numbers in GenBank are JN672576, JQ917222, and JN580309 for the QianBei Ma goat, the GuiZhou white goat, and the GuiZhou black goat, respectively. Four consistent SNP sites were found in the exon regions of the RERG gene for the three goat breeds. mRNA expression of the RERG gene differed between different tissues in adult goats of same age. The highest expression was observed in lung and spleen tissues, while the lowest expression was recorded in thymus gland tissue. In addition, the expression of the RERG gene in the muscle of Guizhou white goat, GuiZhou black goat, and QianBei Ma goat decreased sequentially. Our results lay the foundations for further investigation into the role of the RERG gene in goat growth traits.

Expression analysis of sox3 during testicular development, recrudescence, and after hCG induction in catfish, Clarias batrachus.

PubMed

Rajakumar, Anbazhagan; Senthilkumaran, Balasubramanian

2014-01-01

In teleosts, the expression of steroidogenic enzymes and related transcription factor genes occurs in a stage- and tissue-specific manner causing sexual development. The role of sox3, an evolutionary ancestor of SRY, has not been studied in detail. Therefore, the full-length cDNA of sox3 (1,197 kb) was cloned from catfish testis, and mRNA expression was analyzed during gonadal development, during the seasonal reproductive cycle, and after human chorionic gonadotropin (hCG) induction. Tissue distribution analysis showed that sox3 expression was higher in testis, ovary, and brain compared to other tissues analyzed. Developing and mature testis showed higher sox3 expression than ovary of corresponding stages, and more sox3 transcripts were found during the spawning phase of the seasonal reproductive cycle. Expression of sox3 was upregulated by hCG after in vivo and in vitro induction, suggesting that gonadotropins might stimulate it. In situ hybridization and immunohistochemistry showed the presence of sox3 mRNA and protein in somatic and interstitial cell layers of the testis. Sox3 could also be found in the zona radiata of developing and mature oocytes. Exposure of methyltestosterone (1 µg/l) and ethinylestradiol (1 µg/l) for 21 days during testicular development showed lower sox3 expression levels in the testis and brain, indicating a certain feedback intervention. These results suggest a possible role for Sox3 in the regulation of testicular development and function. © 2014 S. Karger AG, Basel.

Molecular cloning, expression pattern of follistatin gene and association analysis with growth traits in bighead carp (Hypophthalmichthys nobilis).

PubMed

Pang, Meixia; Tong, Jingou; Yu, Xiaomu; Fu, Beide; Zhou, Ying

2018-04-01

Follistatin (FST) is a single-chain gonadal protein involving in various biological effects. FST plays important roles in not only ovary development but also body growth, whereas myostatin (MSTN) negatively regulates muscle growth. In this study, FST gene in bighead carp (HynFST) was cloned and characterized. A 5797 bp genomic sequence of HynFST, consisting six exons and five introns were cloned. The full-length cDNA of HynFST (2134 bp) has an open reading fragment encoding a polypeptide of 349 amino acids. Sequence comparison and phylogenetic analysis confirmed that FSTs are conserved throughout the vertebrates and HynFST belongs to FST-1 isoform. Nine single nucleotide polymorphisms (SNPs) of the HynFST were identified and three of them (g.2443 T > C, g.2852 T > C and g.5483A > G) were significantly associated with four growth-related traits. The average body weight of those fish with the combined genotype (CC CC GG) was 12.15-22.63% higher than that of triplotype (TT TT AA) in two bighead carp populations. HynFST was expressed in most of the development stages and various tissues with highest level in ovary. The co-expression results for FST and MSTN in brain and muscle of divergent weight groups showed that FST may inhibit MSTN expression, thus enhancing growth in bighead carp. Our results suggest that FST has significant genetic effects on the regulation of early growth in bighead carp. This study would facilitate the elucidation of multiple functions of FST gene in fish and exploration of the potentials as a gene marker in selective breeding programs for growth of bighead carp. Copyright © 2018 Elsevier Inc. All rights reserved.

Significant expression of thyroid transcription factor-1 in pulmonary squamous cell carcinoma detected by SPT24 monoclonal antibody and CSA-II system.

PubMed

Kashima, Kenji; Hashimoto, Hisashi; Nishida, Haruto; Arakane, Motoki; Yada, Naomi; Daa, Tsutomu; Yokoyama, Shigeo

2014-01-01

In contrast to the usefulness of thyroid transcription factor-1 (TTF-1) in distinguishing primary adenocarcinoma of the lung from metastatic lesions, TTF-1 expression in pulmonary squamous cell carcinoma is reported to be at low level and not a suitable immunohistochemical marker. We hypothesized that the highly sensitive detection system, CSA-II, can visualize even faint expression of TTF-1 in pulmonary squamous cell carcinoma. In this study, 2 commercially available clones of TTF-1 monoclonal antibody, 8G7G3/1 and SPT24, were used for staining 38 cases of pulmonary squamous cell carcinoma, in combination with the CSA-II and the conventional detection system, EnVision. The combined use of the 8G7G3/1 clone with EnVision and CSA-II showed a positive reaction in only 1 and 4 cases, respectively. The use of SPT24 clone showed positive staining in 5 cases with EnVision and in 20 of 38 cases (52.6%) with the CSA-II. Interestingly, positive staining by the SPT24-CSA-II technique of samples from tissue blocks preserved for <2 years was 73.6% compared with only 31.5% in those preserved for >2 years. In addition, a 6-month preservation of the cut sections resulted in stain fading and decreased positivity (50%), compared with freshly cut sections. We conclude that the use of the SPT24 monoclonal antibody with the CSA-II system can detect even weak expression of TTF-1 in pulmonary squamous cell carcinoma. This staining technique can potentially allow the discrimination of primary squamous cell carcinoma of the lung from metastatic lesions, especially in freshly prepared paraffin sections.

Reproductive performance and expression of imprinted genes in somatic cell cloned boars.

PubMed

Kawarasaki, Tatsuo; Enya, Satoko; Otake, Masayoshi; Shibata, Masatoshi; Mikawa, Satoshi

2017-11-01

To assess the performance of boars derived by somatic cell cloning, we analyzed various aspects of their reproductive characteristics and the expression of two imprinted genes. Cloned boars (cloned Duroc × Jinhua) were analyzed for birth weight, growth rate, age at first ejaculation, semen characteristics and fertility, in comparison with naturally bred control boars of the same strain. The expression of imprinted genes was analyzed using the microsatellite marker SWC9 for the paternally expressed gene insulin-like growth factor -2 (IGF2) and with single nucleotide polymorphisms (SNPs) for the gene maternally expressed 3 (MEG3). The cloned boars had high production of semen and were nearly equal in level of fertility to conventional pigs; they showed similar characteristics as naturally bred boars of the same strains. The expression of IGF2 was partially disturbed, but this disturbed expression was not linked to a change in developmental fate or reproductive performance. These results indicate that use of cloned boars could be highly effective for proliferation of pigs with desirable characteristics, preservation of genetic resources and risk reduction against epidemic diseases, such as foot-and-mouth disease, through storage of somatic cells as a precautionary measure for use in regenerating pig populations after a future pandemic. © 2017 Japanese Society of Animal Science.

SPLASH (PLA2IID), a novel member of phospholipase A2 family, is associated with lymphotoxin deficiency.

PubMed

Shakhov, A N; Rubtsov, A V; Lyakhov, I G; Tumanov, A V; Nedospasov, S A

2000-02-01

Lymphotoxin (LT) deficient mice have profound defects in the splenic microarchitecture associated with defective expression on certain gene products, including chemokines. By using subtraction cloning of splenic cDNA from wild-type and LT alpha or TNF/LT alpha double deficient mice we isolated a novel murine gene encoding a secretory type phospholipase A2, called SPLASH. The two major alternative transcripts of SPLASH gene are predominantly expressed in lymphoid tissues, such as spleen and lymph nodes. SPLASH maps to the distal part of chromosome 4, to which several cancer-related loci have been also mapped.

Isolation and expression analysis of FTZ-F1 encoding gene of black rock fish ( Sebastes schlegelii)

NASA Astrophysics Data System (ADS)

Shafi, Muhammad; Wang, Yanan; Zhou, Xiaosu; Ma, Liman; Muhammad, Faiz; Qi, Jie; Zhang, Quanqi

2013-03-01

Sex related FTZ-F1 is a transcriptional factor regulating the expression of fushi tarazu (a member of the orphan nuclear receptors) gene. In this study, FTZ-F1 gene ( FTZ-F1) was isolated from the testis of black rockfish ( Sebastes schlegeli) by homology cloning. The full-length cDNA of S. schlegeli FTZ-F1 ( ssFTZ-F1) contained a 232bp 5' UTR, a 1449bp ORF encoding FTZ-F1 (482 amino acid residules in length) with an estimated molecular weight of 5.4kD and a 105bp 3' UTR. Sequence, tissue distribution and phylogenic analysis showed that ssFTZ-F1 belonged to FTZ group, holding highly conserved regions including I, II and III FTZ-F1 boxes and an AF-2 hexamer. Relatively high expression was observed at different larva stages. In juveniles (105 days old), the transcript of ssFTZ-F1 can be detected in all tissues and the abuncance of the gene transcript in testis, ovary, spleen and brain was higher than that in other tissues. In mature fish, the abundance of gene transcript was higher in testis, ovary, spleen and brain than that in liver (trace amount), and the gene was not transcribed in other tissues. The highest abundance of gene transcript was always observed in gonads of both juvenile and mature fish. In addition, the abundance of gene transcript in male tissues were higher than that in female tissue counterparts ( P<0.05).

Cloning, expression and nuclear localization of human NPM3, a member of the nucleophosmin/nucleoplasmin family of nuclear chaperones

PubMed Central

Shackleford, Gregory M; Ganguly, Amit; MacArthur, Craig A

2001-01-01

Background Studies suggest that the related proteins nucleoplasmin and nucleophosmin (also called B23, NO38 or numatrin) are nuclear chaperones that mediate the assembly of nucleosomes and ribosomes, respectively, and that these activities are accomplished through the binding of basic proteins via their acidic domains. Recently discovered and less well characterized members of this family of acidic phosphoproteins include mouse nucleophosmin/nucleoplasmin 3 (Npm3) and Xenopus NO29. Here we report the cloning and initial characterization of the human ortholog of Npm3. Results Human genomic and cDNA clones of NPM3 were isolated and sequenced. NPM3 lies 5.5 kb upstream of FGF8 and thus maps to chromosome 10q24-26. In addition to amino acid similarities, NPM3 shares many physical characteristics with the nucleophosmin/nucleoplasmin family, including an acidic domain, multiple potential phosphorylation sites and a putative nuclear localization signal. Comparative analyses of 14 members of this family from various metazoans suggest that Xenopus NO29 is a candidate ortholog of human and mouse NPM3, and they further group both proteins closer with the nucleoplasmins than with the nucleophosmins. Northern blot analysis revealed that NPM3 was strongly expressed in all 16 human tissues examined, with especially robust expression in pancreas and testis; lung displayed the lowest level of expression. An analysis of subcellular fractions of NIH3T3 cells expressing epitope-tagged NPM3 revealed that NPM3 protein was localized solely in the nucleus. Conclusions Human NPM3 is an abundant and widely expressed protein with primarily nuclear localization. These biological activities, together with its physical relationship to the chaparones nucleoplasmin and nucleophosmin, are consistent with the proposed function of NPM3 as a molecular chaperone functioning in the nucleus. PMID:11722795

Tissue expression pattern of ABCG transporter indicates functional roles in reproduction of Toxocara canis.

PubMed

Luo, Yong-Li; Ma, Guang-Xu; Luo, Yong-Fang; Kuang, Ce-Yan; Jiang, Ai-Yun; Li, Guo-Qing; Zhou, Rong-Qiong

2018-03-01

Toxocara canis is a zoonotic parasite with worldwide distribution. ATP-binding cassette (ABC) transporters are integral membrane proteins which involve in a range of biological processes in various organisms. In present study, the full-length coding sequence of abcg-5 gene of T. canis (Tc-abcg-5) was cloned and characterized. A 633 aa polypeptide containing two conserved Walker A and Walker B motifs was predicted from a continuous 1902 nt open reading frame. Quantitative real-time PCR was employed to determine the transcriptional levels of Tc-abcg-5 gene in adult male and female worms, which indicated high mRNA level of Tc-abcg-5 in the reproductive tract of adult female T. canis. Tc-abcg-5 was expressed to produce rabbit polyclonal antiserum against recombinant TcABCG5. Indirect-fluorescence immunohistochemical assays were carried out to detect the tissue distribution of TcABCG5, which showed predominant distribution of TcABCG5 in the uterus (especially in the germ cells) of adult female T. canis. Tissue transcription and expression pattern of Tc-abcg-5 indicated that Tc-abcg-5 might play essential roles in the reproduction of this parasitic nematode.

Capsicum annuum dehydrin, an osmotic-stress gene in hot pepper plants.

PubMed

Chung, Eunsook; Kim, Soo-Yong; Yi, So Young; Choi, Doil

2003-06-30

Osmotic stress-related genes were selected from an EST database constructed from 7 cDNA libraries from different tissues of the hot pepper. A full-length cDNA of Capsicum annuum dehydrin (Cadhn), a late embryogenesis abundant (lea) gene, was selected from the 5' single pass sequenced cDNA clones and sequenced. The deduced polypeptide has 87% identity with potato dehydrin C17, but very little identity with the dehydrin genes of other organisms. It contains a serine-tract (S-segment) and 3 conserved lysine-rich domains (K-segments). Southern blot analysis showed that 2 copies are present in the hot pepper genome. Cadhn was induced by osmotic stress in leaf tissues as well as by the application of abscisic acid. The RNA was most abundant in green fruit. The expression of several osmotic stress-related genes was examined and Cadhn proved to be the most abundantly expressed of these in response to osmotic stress.

Expression of cardiac neural crest and heart genes isolated by modified differential display.

PubMed

Martinsen, Brad J; Groebner, Nathan J; Frasier, Allison J; Lohr, Jamie L

2003-08-01

The invasion of the cardiac neural crest (CNC) into the outflow tract (OFT) and subsequent outflow tract septation are critical events during vertebrate heart development. We have performed four modified differential display screens in the chick embryo to identify genes that may be involved in CNC, OFT, secondary heart field, and heart development. The screens included differential display of RNA isolated from three different axial segments containing premigratory cranial neural crest cells; of RNA from distal outflow tract, proximal outflow tract, and atrioventricular tissue of embryonic chick hearts; and of RNA isolated from left and right cranial tissues, including the early heart fields. These screens have resulted in the identification of the five cDNA clones presented here, which are expressed in the cardiac neural crest, outflow tract and developing heart in patterns that are unique in heart development.

Drought-induced gene expression in Atriplex canescens (salt bush): Transcriptional and post transcriptional response

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cairney, J.; Hays, D.; Stockand, J.D.

1991-05-01

The rangeland shrub Atriplex canescens (saltbush) is extremely drought-tolerant and is capable of growing at water potentials below {minus}40 bar. To discover the molecular basis of this tolerance, the authors have isolated a number of cDNA clones of drought-stress induced genes. Analysis of the nucleotide sequence and expression of these genes in different tissues and in response to different stresses reveals the diversity of the stress response. Members of a drought-induced, multi-gene family, have been sequenced. Although 95% homologous, non-conservative substitutions result in proteins of different tertiary structure. Additionally, the genes are expressed through a number of mature forms ofmore » mRNA which may arise by alternative RNA processing.« less

The contribution of 700,000 ORF sequence tags to the definition of the human transcriptome

PubMed Central

Camargo, Anamaria A.; Samaia, Helena P. B.; Dias-Neto, Emmanuel; Simão, Daniel F.; Migotto, Italo A.; Briones, Marcelo R. S.; Costa, Fernando F.; Aparecida Nagai, Maria; Verjovski-Almeida, Sergio; Zago, Marco A.; Andrade, Luis Eduardo C.; Carrer, Helaine; El-Dorry, Hamza F. A.; Espreafico, Enilza M.; Habr-Gama, Angelita; Giannella-Neto, Daniel; Goldman, Gustavo H.; Gruber, Arthur; Hackel, Christine; Kimura, Edna T.; Maciel, Rui M. B.; Marie, Suely K. N.; Martins, Elizabeth A. L.; Nóbrega, Marina P.; Paçó-Larson, Maria Luisa; Pardini, Maria Inês M. C.; Pereira, Gonçalo G.; Pesquero, João Bosco; Rodrigues, Vanderlei; Rogatto, Silvia R.; da Silva, Ismael D. C. G.; Sogayar, Mari C.; Sonati, Maria de Fátima; Tajara, Eloiza H.; Valentini, Sandro R.; Alberto, Fernando L.; Amaral, Maria Elisabete J.; Aneas, Ivy; Arnaldi, Liliane A. T.; de Assis, Angela M.; Bengtson, Mário Henrique; Bergamo, Nadia Aparecida; Bombonato, Vanessa; de Camargo, Maria E. R.; Canevari, Renata A.; Carraro, Dirce M.; Cerutti, Janete M.; Corrêa, Maria Lucia C.; Corrêa, Rosana F. R.; Costa, Maria Cristina R.; Curcio, Cyntia; Hokama, Paula O. M.; Ferreira, Ari J. S.; Furuzawa, Gilberto K.; Gushiken, Tsieko; Ho, Paulo L.; Kimura, Elza; Krieger, José E.; Leite, Luciana C. C.; Majumder, Paromita; Marins, Mozart; Marques, Everaldo R.; Melo, Analy S. A.; Melo, Monica; Mestriner, Carlos Alberto; Miracca, Elisabete C.; Miranda, Daniela C.; Nascimento, Ana Lucia T. O.; Nóbrega, Francisco G.; Ojopi, Élida P. B.; Pandolfi, José Rodrigo C.; Pessoa, Luciana G.; Prevedel, Aline C.; Rahal, Paula; Rainho, Claudia A.; Reis, Eduardo M. R.; Ribeiro, Marcelo L.; da Rós, Nancy; de Sá, Renata G.; Sales, Magaly M.; Sant'anna, Simone Cristina; dos Santos, Mariana L.; da Silva, Aline M.; da Silva, Neusa P.; Silva, Wilson A.; da Silveira, Rosana A.; Sousa, Josane F.; Stecconi, Daniella; Tsukumo, Fernando; Valente, Valéria; Soares, Fernando; Moreira, Eloisa S.; Nunes, Diana N.; Correa, Ricardo G.; Zalcberg, Heloisa; Carvalho, Alex F.; Reis, Luis F. L.; Brentani, Ricardo R.; Simpson, Andrew J. G.; de Souza, Sandro J.

2001-01-01

Open reading frame expressed sequences tags (ORESTES) differ from conventional ESTs by providing sequence data from the central protein coding portion of transcripts. We generated a total of 696,745 ORESTES sequences from 24 human tissues and used a subset of the data that correspond to a set of 15,095 full-length mRNAs as a means of assessing the efficiency of the strategy and its potential contribution to the definition of the human transcriptome. We estimate that ORESTES sampled over 80% of all highly and moderately expressed, and between 40% and 50% of rarely expressed, human genes. In our most thoroughly sequenced tissue, the breast, the 130,000 ORESTES generated are derived from transcripts from an estimated 70% of all genes expressed in that tissue, with an equally efficient representation of both highly and poorly expressed genes. In this respect, we find that the capacity of the ORESTES strategy both for gene discovery and shotgun transcript sequence generation significantly exceeds that of conventional ESTs. The distribution of ORESTES is such that many human transcripts are now represented by a scaffold of partial sequences distributed along the length of each gene product. The experimental joining of the scaffold components, by reverse transcription–PCR, represents a direct route to transcript finishing that may represent a useful alternative to full-length cDNA cloning. PMID:11593022

The contribution of 700,000 ORF sequence tags to the definition of the human transcriptome.

PubMed

Camargo, A A; Samaia, H P; Dias-Neto, E; Simão, D F; Migotto, I A; Briones, M R; Costa, F F; Nagai, M A; Verjovski-Almeida, S; Zago, M A; Andrade, L E; Carrer, H; El-Dorry, H F; Espreafico, E M; Habr-Gama, A; Giannella-Neto, D; Goldman, G H; Gruber, A; Hackel, C; Kimura, E T; Maciel, R M; Marie, S K; Martins, E A; Nobrega, M P; Paco-Larson, M L; Pardini, M I; Pereira, G G; Pesquero, J B; Rodrigues, V; Rogatto, S R; da Silva, I D; Sogayar, M C; Sonati, M F; Tajara, E H; Valentini, S R; Alberto, F L; Amaral, M E; Aneas, I; Arnaldi, L A; de Assis, A M; Bengtson, M H; Bergamo, N A; Bombonato, V; de Camargo, M E; Canevari, R A; Carraro, D M; Cerutti, J M; Correa, M L; Correa, R F; Costa, M C; Curcio, C; Hokama, P O; Ferreira, A J; Furuzawa, G K; Gushiken, T; Ho, P L; Kimura, E; Krieger, J E; Leite, L C; Majumder, P; Marins, M; Marques, E R; Melo, A S; Melo, M B; Mestriner, C A; Miracca, E C; Miranda, D C; Nascimento, A L; Nobrega, F G; Ojopi, E P; Pandolfi, J R; Pessoa, L G; Prevedel, A C; Rahal, P; Rainho, C A; Reis, E M; Ribeiro, M L; da Ros, N; de Sa, R G; Sales, M M; Sant'anna, S C; dos Santos, M L; da Silva, A M; da Silva, N P; Silva, W A; da Silveira, R A; Sousa, J F; Stecconi, D; Tsukumo, F; Valente, V; Soares, F; Moreira, E S; Nunes, D N; Correa, R G; Zalcberg, H; Carvalho, A F; Reis, L F; Brentani, R R; Simpson, A J; de Souza, S J; Melo, M

2001-10-09

Open reading frame expressed sequences tags (ORESTES) differ from conventional ESTs by providing sequence data from the central protein coding portion of transcripts. We generated a total of 696,745 ORESTES sequences from 24 human tissues and used a subset of the data that correspond to a set of 15,095 full-length mRNAs as a means of assessing the efficiency of the strategy and its potential contribution to the definition of the human transcriptome. We estimate that ORESTES sampled over 80% of all highly and moderately expressed, and between 40% and 50% of rarely expressed, human genes. In our most thoroughly sequenced tissue, the breast, the 130,000 ORESTES generated are derived from transcripts from an estimated 70% of all genes expressed in that tissue, with an equally efficient representation of both highly and poorly expressed genes. In this respect, we find that the capacity of the ORESTES strategy both for gene discovery and shotgun transcript sequence generation significantly exceeds that of conventional ESTs. The distribution of ORESTES is such that many human transcripts are now represented by a scaffold of partial sequences distributed along the length of each gene product. The experimental joining of the scaffold components, by reverse transcription-PCR, represents a direct route to transcript finishing that may represent a useful alternative to full-length cDNA cloning.

Cloning and characterization of three suppressors of cytokine signaling (SOCS) genes from the Pacific oyster, Crassostrea gigas.

PubMed

Li, Jun; Zhang, Yang; Zhang, Yuehuan; Liu, Ying; Xiang, Zhiming; Qu, Fufa; Yu, Ziniu

2015-06-01

Members of the suppressor of cytokine signaling (SOCS) family are crucial for the control of a variety of signal transduction pathways that are involved in the immunity, growth and development of organisms. However, in mollusks, the identity and function of SOCS proteins remain largely unclear. In the present study, three SOCS genes, CgSOCS2, CgSOCS5 and CgSOCS7, have been identified by searching and analyzing the Pacific oyster genome. Structural analysis indicated that the CgSOCS share conserved functional domains with their vertebrate counterparts. Phylogenetic analysis showed that the three SOCS genes clustered into two distinct groups, the type I and II subfamilies, indicating that these subfamilies had common ancestors. Tissue-specific expression results showed that the three genes were constitutively expressed in all examined tissues and were highly expressed in immune-related tissues, such as the hemocytes, gills and digestive gland. The expression of CgSOCS can also be induced to varying degrees in hemocytes after challenge with pathogen-associated molecular patterns (PAMPs). Moreover, dual-luciferase reporter assays showed that the over-expression of CgSOCS2 and CgSOCS7, but not CgSOC5, can activate an NF-κB reporter gene. Collectively, these results demonstrated that the CgSOCS might play an important role in the innate immune responses of the Pacific oyster. Copyright © 2015 Elsevier Ltd. All rights reserved.

Light controls phospholipase A2α and β gene expression in Citrus sinensis

PubMed Central

Liao, Hui-Ling; Burns, Jacqueline K.

2010-01-01

The low-molecular weight secretory phospholipase A2α (CssPLA2α) and β (CsPLA2β) cloned in this study exhibited diurnal rhythmicity in leaf tissue of Citrus sinensis. Only CssPLA2α displayed distinct diurnal patterns in fruit tissues. CssPLA2α and CsPLA2β diurnal expression exhibited periods of approximately 24 h; CssPLA2α amplitude averaged 990-fold in the leaf blades from field-grown trees, whereas CsPLA2β amplitude averaged 6.4-fold. Diurnal oscillation of CssPLA2α and CsPLA2β gene expression in the growth chamber experiments was markedly dampened 24 h after transfer to continuous light or dark conditions. CssPLA2α and CsPLA2β expressions were redundantly mediated by blue, green, red and red/far-red light, but blue light was a major factor affecting CssPLA2α and CsPLA2β expression. Total and low molecular weight CsPLA2 enzyme activity closely followed diurnal changes in CssPLA2α transcript expression in leaf blades of seedlings treated with low intensity blue light (24 μmol m−2 s−1). Compared with CssPLA2α basal expression, CsPLA2β expression was at least 10-fold higher. Diurnal fluctuation and light regulation of PLA2 gene expression and enzyme activity in citrus leaf and fruit tissues suggests that accompanying diurnal changes in lipophilic second messengers participate in the regulation of physiological processes associated with phospholipase A2 action. PMID:20388744

Alu sequence involvement in transcriptional insulation of the keratin 18 gene in transgenic mice.

PubMed Central

Thorey, I S; Ceceña, G; Reynolds, W; Oshima, R G

1993-01-01

The human keratin 18 (K18) gene is expressed in a variety of adult simple epithelial tissues, including liver, intestine, lung, and kidney, but is not normally found in skin, muscle, heart, spleen, or most of the brain. Transgenic animals derived from the cloned K18 gene express the transgene in appropriate tissues at levels directly proportional to the copy number and independently of the sites of integration. We have investigated in transgenic mice the dependence of K18 gene expression on the distal 5' and 3' flanking sequences and upon the RNA polymerase III promoter of an Alu repetitive DNA transcription unit immediately upstream of the K18 promoter. Integration site-independent expression of tandemly duplicated K18 transgenes requires the presence of either an 825-bp fragment of the 5' flanking sequence or the 3.5-kb 3' flanking sequence. Mutation of the RNA polymerase III promoter of the Alu element within the 825-bp fragment abolishes copy number-dependent expression in kidney but does not abolish integration site-independent expression when assayed in the absence of the 3' flanking sequence of the K18 gene. The characteristics of integration site-independent expression and copy number-dependent expression are separable. In addition, the formation of the chromatin state of the K18 gene, which likely restricts the tissue-specific expression of this gene, is not dependent upon the distal flanking sequences of the 10-kb K18 gene but rather may depend on internal regulatory regions of the gene. Images PMID:7692231

Influence of cloning by chromatin transfer on placental gene expression at Day 45 of pregnancy in cattle.

PubMed

Mesquita, Fernando S; Machado, Sergio A; Drnevich, Jenny; Borowicz, Pawel; Wang, Zhongde; Nowak, Romana A

2013-01-30

Poor success rates in somatic cell cloning are often attributed to abnormal early embryonic development as well as late abnormal fetal growth and placental development. Although promising results have been reported following chromatin transfer (CT), a novel cloning method that includes the remodeling of the donor nuclei in vitro prior to their transfer into enucleated oocytes, animals cloned by CT show placental abnormalities similar to those observed following conventional nuclear transfer. We hypothesized that the placental gene expression pattern from cloned fetuses was ontologically related to the frequently observed placental phenotype. The aim of the present study was to compare global gene expression by microarray analysis of Day 44-47 cattle placentas derived from CT cloned fetuses with those derived from in vitro fertilization (i.e. control), and confirm the altered mRNA and protein expression of selected molecules by qRT-PCR and immunohistochemistry, respectively. The differentially expressed genes identified in the present study are known to be involved in a range of activities associated with cell adhesion, cell cycle control, intracellular transport and proteolysis. Specifically, an imprinted gene, involved with cell proliferation and placentomegaly in humans (CDKN1C) and a peptidase that serves as a marker for non-invasive trophoblast cells in human placentas (DPP4), had mRNA and protein altered in CT placentas. It was concluded that the altered pattern of gene expression observed in CT samples may contribute to the abnormal placental development phenotypes commonly identified in cloned offspring, and that expression of imprinted as well as trophoblast invasiveness-related genes is altered in cattle cloned by CT. Copyright © 2012 Elsevier B.V. All rights reserved.

Tissue-Culture Method of Cloning Rubber Plants

NASA Technical Reports Server (NTRS)

Ball, E. A.

1983-01-01

Guayule plant, a high-yield rubber plant cloned by tissue-culture method to produce multiple new plants that mature quickly. By adjusting culture medium, excised shoot tip produces up to 50 identical guayule plants. Varying concentration of cytokinin, single excised tip produces either 1 or several (up to 50) new plants.

Therapeutic cloning applications for organ transplantation.

PubMed

Koh, Chester J; Atala, Anthony

2004-04-01

A severe shortage of donor organs available for transplantation in the United States leaves patients suffering from diseased and injured organs with few treatment options. Scientists in the field of tissue engineering apply the principles of cell transplantation, material science, and engineering to construct biological substitutes that will restore and maintain normal function in diseased and injured tissues. Therapeutic cloning, where the nucleus from a donor cell is transferred into an enucleated oocyte in order to extract pluripotent embryonic stem cells, offers a potentially limitless source of cells for tissue engineering applications. The present chapter reviews recent advances that have occurred in therapeutic cloning and tissue engineering and describes applications of these new technologies that may offer novel therapies for patients with end-stage organ failure. Copyright 2004 Elsevier B.V.

Identification and expression patterns of adipokine genes during adipocyte differentiation in the Tibetan goat (Capra hircus).

PubMed

Li, Xueying; Wang, Yan; Guo, Jiazhong; Zhong, Tao; Li, Li; Zhang, Hongping; Wang, Linjie

2018-02-15

Adipokines are secreted by adipose tissue and play an important role in the regulation of lipid metabolism. However, the information regarding adipokines in goats is limited. PPARγ is a key gene in adipocyte differentiation and activates adipokine genes. Rosiglitazone is a PPARγ agonist and can promote the expression of PPARγ to increase the expression of lipogenesis-related genes. Therefore, investigation of the relationship between rosiglitazone and adipokines will help us to better understand the function of PPARγ in lipid metabolism in Tibetan goats. In this study, we cloned the resistin (RETN), apelin (APLN), fibroblast growth factor 21 (FGF21), and visfatin (NAMPT) genes from non-pregnant female Tibetan goat adipose tissue. APLN and NAMPT were predominantly expressed in the kidney, and FGF21 was expressed at the highest levels in the liver in vivo. In fat tissues, the highest expression levels of FGF21 and RETN were detected in omental fat, whereas their expression in perirenal and subcutaneous fat was extremely weak. APLN and NAMPT were abundantly expressed in omental and subcutaneous fat in vivo. In addition, the four adipokines had different expression profiles during goat adipocyte differentiation in vitro. Oil red O staining showed that rosiglitazone could promote adipocyte differentiation and lipid droplet formation. In addition, rosiglitazone significantly increased the expression of FGF21 and RETN (p<0.05) but decreased the expression of APLN and NAMPT (p<0.05). These results suggest that the four adipocytokine genes may have different roles during goat adipocyte differentiation. And PPARγ could regulate the expression of the four adipokines, but the detailed regulatory mechanism still needs to be elucidated. Copyright © 2017 Elsevier B.V. All rights reserved.

EpCAM expression in primary tumour tissues and metastases: an immunohistochemical analysis.

PubMed

Spizzo, Gilbert; Fong, Dominic; Wurm, Martin; Ensinger, Christian; Obrist, Peter; Hofer, Carina; Mazzoleni, Guido; Gastl, Guenther; Went, Philip

2011-05-01

Epithelial cell adhesion molecule (EpCAM) is a cell surface protein with oncogenic features that is expressed on healthy human epithelia and corresponding malignant tumours. EpCAM expression frequently correlates with more aggressive tumour behaviour and new EpCAM-specific therapeutic agents have recently been approved for clinical use in patients with cancer. However, no consensus exists on how and when to evaluate EpCAM expression in patients with cancer. EpCAM expression was assessed by a well-established immunohistochemical staining protocol in 2291 primary tumour tissues and in 108 metastases using the EpCAM-specific antibody clone VU1D9. A total immunostaining score was calculated as the product of a proportion score and an intensity score. Four expression subgroups (no, weak, moderate and intense) were defined. As described previously, the term 'EpCAM overexpression' was reserved for tissues showing a total immunostaining score >4. EpCAM was highly expressed in most tumours of gastrointestinal origin and in some carcinomas of the genitourinary tract. However, hepatocellular carcinomas, clear cell renal cell cancer, urothelial cancer and squamous cell cancers were frequently EpCAM negative. EpCAM expression in breast cancer depended on the histological subtype; lobular histology usually showed no or weak expression. Most metastases were EpCAM positive and they frequently reflected the expression phenotype of the primary tumour. EpCAM expression was detected on adenocarcinomas of various primary sites. If EpCAM-specific antibodies are intended to be used in patients with cancer, we recommend prior immunohistochemical evaluation of EpCAM expression, particularly in patients with renal cell cancer, hepatocellular carcinoma, urothelial carcinoma, breast cancer and squamous cell carcinomas.

Molecular cloning and expression profiles of IGFBP-1a in common carp (Cyprinus carpio) and its expression regulation by growth hormone in hepatocytes.

PubMed

Chen, Wenbo; Lin, Haoran; Li, Wensheng

2018-04-23

In this study, we cloned and determined IGFBP-1a cDNA from common carp (Cyprinus carpio) liver. The 1655 bp full-length cDNA consisted of a 96 bp 5-untranslated region (UTR), a 789 bp open reading frame encoding 262 amino acid residues and a 770 bp 3-UTR containing seven mRNA instability motifs. Northern blot revealed a 1.8 kb IGFBP-1a transcript. IGFBP-1a mRNA was widely distributed in all tissues examined and predominantly expressed in the liver. During embryogenesis, IGFBP-1a mRNA was firstly observed in blastula stage, and significant increases were observed in body segment stage, lens formation stage and blood cycling stage. After hatching, its expression increased more than twenty times. Furthermore, hypoxia could significantly up-regulate IGFBP-1a expression in the liver and brain. IGFBP-1a expression increased with ovarian maturation and decreased at regressed stage. In testis, IGFBP-1a mRNA maintained relatively higher levels at recrudescing and matured stages, while it sharply declined at regressed stage. In primary cultured hepatocytes, IGFBP-1a gene was greatly down-regulated by growth hormone via the MAPK and PI3 kinase signaling pathways. These results suggest that IGFBP-1a may be involved in the IGF system regulating growth, development and reproduction in common carp. Copyright © 2018. Published by Elsevier Inc.

Cloning and expression of follistatin gene in half-smooth tongue sole Cynoglossus semilaevis during the reproduction cycle

NASA Astrophysics Data System (ADS)

Wen, Haishen; Si, Yufeng; Zhang, Yuanqing; He, Feng; Li, Jifang

2015-03-01

Follistatin (FST) is a monomeric glycoprotein highly enriched in cysteines and belongs to TGF-β superfamily. FST can suppress the secretion of follicle-stimulating hormone and plays a vital role in the reproduction of vertebrates. We used rapid amplification of cDNA ends technology to clone the FST gene of half-smooth tongue sole, Cynoglossus semilaevis. We characterized its phylogenetic context and expression patterns to elucidate its function in the breeding season. The full-length sequence of FST is 1 455 bp and encodes a protein of 321 amino acids. We investigated the expression pattern of FST in C. semilaevis at different stages of reproduction using reverse transcription-polymerase chain reaction (RTPCR). FST mRNA was expressed in all 13 tissues analyzed, and was expressed at high levels in gonad and at slightly lower levels in gill and brain. During the reproductive cycle of C. semilaevis, the transcript level of FST was the highest in the perinucleolus stage, decreased in the primary yolk stage, slightly increased in the tertiary yolk stage, and then reduced to a minimal level in the atretic follicles stage of the ovary. We concluded that FST suppressed follicle-stimulating hormone, which stimulated oocyte development. However, no significant variation was observed across all stages of testis development, although the expression level in the spermatogenesis stage was relatively low, which may result from the regulation of FST by aromatase.

Identification of a cluster IV pleiotropic drug resistance transporter gene expressed in the style of Nicotiana plumbaginifolia.

PubMed

Trombik, Tomasz; Jasinski, Michal; Crouzet, Jérome; Boutry, Marc

2008-01-01

ATP-binding cassette transporters of the pleiotropic drug resistance (PDR) subfamily are composed of five clusters. We have cloned a gene, NpPDR2, belonging to the still uncharacterized cluster IV from Nicotiana plumbaginifolia. NpPDR2 transcripts were found in the roots and mature flowers. In the latter, NpPDR2 expression was restricted to the style and only after pollination. A 1.5-kb genomic sequence containing the putative NpPDR2 transcription promoter was fused to the beta-glucuronidase reporter gene. The GUS expression pattern confirmed the RT-PCR results that NpPDR2 was expressed in roots and the flower style and showed that it was localized around the conductive tissues. Unlike other PDR genes, NpPDR2 expression was not induced in leaf tissues by none of the hormones typically involved in biotic and abiotic stress response. Moreover, unlike NpPDR1 known to be involved in biotic stress response, NpPDR2 expression was not induced in the style upon Botrytis cinerea infection. In N. plumbaginifolia plants in which NpPDR2 expression was prevented by RNA interference, no unusual phenotype was observed, including at the flowering stage, which suggests that NpPDR2 is not essential in the reproductive process under the tested conditions.

Dynamic Proteomic Characteristics and Network Integration Revealing Key Proteins for Two Kernel Tissue Developments in Popcorn.

PubMed

Dong, Yongbin; Wang, Qilei; Zhang, Long; Du, Chunguang; Xiong, Wenwei; Chen, Xinjian; Deng, Fei; Ma, Zhiyan; Qiao, Dahe; Hu, Chunhui; Ren, Yangliu; Li, Yuling

2015-01-01

The formation and development of maize kernel is a complex dynamic physiological and biochemical process that involves the temporal and spatial expression of many proteins and the regulation of metabolic pathways. In this study, the protein profiles of the endosperm and pericarp at three important developmental stages were analyzed by isobaric tags for relative and absolute quantification (iTRAQ) labeling coupled with LC-MS/MS in popcorn inbred N04. Comparative quantitative proteomic analyses among developmental stages and between tissues were performed, and the protein networks were integrated. A total of 6,876 proteins were identified, of which 1,396 were nonredundant. Specific proteins and different expression patterns were observed across developmental stages and tissues. The functional annotation of the identified proteins revealed the importance of metabolic and cellular processes, and binding and catalytic activities for the development of the tissues. The whole, endosperm-specific and pericarp-specific protein networks integrated 125, 9 and 77 proteins, respectively, which were involved in 54 KEGG pathways and reflected their complex metabolic interactions. Confirmation for the iTRAQ endosperm proteins by two-dimensional gel electrophoresis showed that 44.44% proteins were commonly found. However, the concordance between mRNA level and the protein abundance varied across different proteins, stages, tissues and inbred lines, according to the gene cloning and expression analyses of four relevant proteins with important functions and different expression levels. But the result by western blot showed their same expression tendency for the four proteins as by iTRAQ. These results could provide new insights into the developmental mechanisms of endosperm and pericarp, and grain formation in maize.

Dynamic Proteomic Characteristics and Network Integration Revealing Key Proteins for Two Kernel Tissue Developments in Popcorn

PubMed Central

Du, Chunguang; Xiong, Wenwei; Chen, Xinjian; Deng, Fei; Ma, Zhiyan; Qiao, Dahe; Hu, Chunhui; Ren, Yangliu; Li, Yuling

2015-01-01

The formation and development of maize kernel is a complex dynamic physiological and biochemical process that involves the temporal and spatial expression of many proteins and the regulation of metabolic pathways. In this study, the protein profiles of the endosperm and pericarp at three important developmental stages were analyzed by isobaric tags for relative and absolute quantification (iTRAQ) labeling coupled with LC-MS/MS in popcorn inbred N04. Comparative quantitative proteomic analyses among developmental stages and between tissues were performed, and the protein networks were integrated. A total of 6,876 proteins were identified, of which 1,396 were nonredundant. Specific proteins and different expression patterns were observed across developmental stages and tissues. The functional annotation of the identified proteins revealed the importance of metabolic and cellular processes, and binding and catalytic activities for the development of the tissues. The whole, endosperm-specific and pericarp-specific protein networks integrated 125, 9 and 77 proteins, respectively, which were involved in 54 KEGG pathways and reflected their complex metabolic interactions. Confirmation for the iTRAQ endosperm proteins by two-dimensional gel electrophoresis showed that 44.44% proteins were commonly found. However, the concordance between mRNA level and the protein abundance varied across different proteins, stages, tissues and inbred lines, according to the gene cloning and expression analyses of four relevant proteins with important functions and different expression levels. But the result by western blot showed their same expression tendency for the four proteins as by iTRAQ. These results could provide new insights into the developmental mechanisms of endosperm and pericarp, and grain formation in maize. PMID:26587848

Diagnostic utility of aP2/FABP4 expression in soft tissue tumours.

PubMed

Kashima, T G; Turley, H; Dongre, A; Pezzella, F; Athanasou, N A

2013-04-01

Adipocyte P2 (aP2), also known as fatty acid-binding protein 4 (FABP4), is a fatty acid-binding protein found in the cytoplasm of cells of adipocyte differentiation. In this study, we examined a large number of soft tissue tumours with a commercial polyclonal anti-aP2/FABP4 antibody and a newly developed mouse monoclonal antibody raised against this protein to determine the diagnostic utility of aP2/FABP4 as a marker of tumours of adipose differentiation. A mouse monoclonal antibody, clone 175d, was raised against a mixture of synthetic peptides corresponding to the amino acid sequence of residues 10-28 and 121-132 of the human aP2/FABP4 protein. Antigen expression with polyclonal and monoclonal antibodies was immunohistochemically determined in paraffin sections of normal adipose tissue and a wide range of benign and malignant primary soft tissue tumours (n = 200). aP2/FABP4 was expressed around the cytoplasmic lipid vacuole in white and brown fat cells in benign lipomas and hibernomas. Immature fat cells and lipoblasts in spindle cell/pleomorphic lipoma, atypical lipomatous tumour/well-differentiated liposarcoma, myxoid/round cell liposarcoma and pleomorphic liposarcoma also reacted strongly for aP2/FABP4. No specific staining was seen in non-adipose benign and malignant mesenchymal and non-mesenchymal tumours. aP2/FABP4 is expressed by mature and immature fat cells and is a marker of tumours of adipose differentiation. Immunophenotypic aP2/FABP4 expression is useful in identifying lipoblasts, and immunohistochemistry with polyclonal/monoclonal anti-aP2/FABP4 antibodies should be useful in distinguishing liposarcoma from other malignancies, particularly round cell, myxoid and pleomorphic soft tissue sarcomas.

The CesA Gene Family of Barley. Quantitative Analysis of Transcripts Reveals Two Groups of Co-Expressed Genes1

PubMed Central

Burton, Rachel A.; Shirley, Neil J.; King, Brendon J.; Harvey, Andrew J.; Fincher, Geoffrey B.

2004-01-01

Sequence data from cDNA and genomic clones, coupled with analyses of expressed sequence tag databases, indicate that the CesA (cellulose synthase) gene family from barley (Hordeum vulgare) has at least eight members, which are distributed across the genome. Quantitative polymerase chain reaction has been used to determine the relative abundance of mRNA transcripts for individual HvCesA genes in vegetative and floral tissues, at different stages of development. To ensure accurate expression profiling, geometric averaging of multiple internal control gene transcripts has been applied for the normalization of transcript abundance. Total HvCesA mRNA levels are highest in coleoptiles, roots, and stems and much lower in floral tissues, early developing grain, and in the elongation zone of leaves. In most tissues, HvCesA1, HvCesA2, and HvCesA6 predominate, and their relative abundance is very similar; these genes appear to be coordinately transcribed. A second group, comprising HvCesA4, HvCesA7, and HvCesA8, also appears to be coordinately transcribed, most obviously in maturing stem and root tissues. The HvCesA3 expression pattern does not fall into either of these two groups, and HvCesA5 transcript levels are extremely low in all tissues. Thus, the HvCesA genes fall into two general groups of three genes with respect to mRNA abundance, and the co-expression of the groups identifies their products as candidates for the rosettes that are involved in cellulose biosynthesis at the plasma membrane. Phylogenetic analysis allows the two groups of genes to be linked with orthologous Arabidopsis CesA genes that have been implicated in primary and secondary wall synthesis. PMID:14701917

A new set of ESTs and cDNA clones from full-length and normalized libraries for gene discovery and functional characterization in citrus

PubMed Central

Marques, M Carmen; Alonso-Cantabrana, Hugo; Forment, Javier; Arribas, Raquel; Alamar, Santiago; Conejero, Vicente; Perez-Amador, Miguel A

2009-01-01

Background Interpretation of ever-increasing raw sequence information generated by modern genome sequencing technologies faces multiple challenges, such as gene function analysis and genome annotation. Indeed, nearly 40% of genes in plants encode proteins of unknown function. Functional characterization of these genes is one of the main challenges in modern biology. In this regard, the availability of full-length cDNA clones may fill in the gap created between sequence information and biological knowledge. Full-length cDNA clones facilitate functional analysis of the corresponding genes enabling manipulation of their expression in heterologous systems and the generation of a variety of tagged versions of the native protein. In addition, the development of full-length cDNA sequences has the power to improve the quality of genome annotation. Results We developed an integrated method to generate a new normalized EST collection enriched in full-length and rare transcripts of different citrus species from multiple tissues and developmental stages. We constructed a total of 15 cDNA libraries, from which we isolated 10,898 high-quality ESTs representing 6142 different genes. Percentages of redundancy and proportion of full-length clones range from 8 to 33, and 67 to 85, respectively, indicating good efficiency of the approach employed. The new EST collection adds 2113 new citrus ESTs, representing 1831 unigenes, to the collection of citrus genes available in the public databases. To facilitate functional analysis, cDNAs were introduced in a Gateway-based cloning vector for high-throughput functional analysis of genes in planta. Herein, we describe the technical methods used in the library construction, sequence analysis of clones and the overexpression of CitrSEP, a citrus homolog to the Arabidopsis SEP3 gene, in Arabidopsis as an example of a practical application of the engineered Gateway vector for functional analysis. Conclusion The new EST collection denotes an important step towards the identification of all genes in the citrus genome. Furthermore, public availability of the cDNA clones generated in this study, and not only their sequence, enables testing of the biological function of the genes represented in the collection. Expression of the citrus SEP3 homologue, CitrSEP, in Arabidopsis results in early flowering, along with other phenotypes resembling the over-expression of the Arabidopsis SEPALLATA genes. Our findings suggest that the members of the SEP gene family play similar roles in these quite distant plant species. PMID:19747386

Molecular cloning and tissue-specific transcriptional regulation of the first peroxidase family member, Udp1, in stinging nettle (Urtica dioica).

PubMed

Douroupi, Triantafyllia G; Papassideri, Issidora S; Stravopodis, Dimitrios J; Margaritis, Lukas H

2005-12-05

A full-length cDNA clone, designated Udp1, was isolated from Urtica dioica (stinging nettle), using a polymerase chain reaction based strategy. The putative Udp1 protein is characterized by a cleavable N-terminal signal sequence, likely responsible for the rough endoplasmic reticulum entry and a 310 amino acids mature protein, containing all the important residues, which are evolutionary conserved among different members of the plant peroxidase family. A unique structural feature of the Udp1 peroxidase is defined into the short carboxyl-terminal extension, which could be associated with the vacuolar targeting process. Udp1 peroxidase is differentially regulated at the transcriptional level and is specifically expressed in the roots. Interestingly, wounding and ultraviolet radiation stress cause an ectopic induction of the Udp1 gene expression in the aerial parts of the plant. A genomic DNA fragment encoding the Udp1 peroxidase was also cloned and fully sequenced, revealing a structural organization of three exons and two introns. The phylogenetic relationships of the Udp1 protein to the Arabidopsis thaliana peroxidase family members were also examined and, in combination with the homology modelling approach, dictated the presence of distinct structural elements, which could be specifically involved in the determination of substrate recognition and subcellular localization of the Udp1 peroxidase.

Immunolocalization of type X collagen in normal fetal and adult osteoarthritic cartilage with monoclonal antibodies.

PubMed

Girkontaite, I; Frischholz, S; Lammi, P; Wagner, K; Swoboda, B; Aigner, T; Von der Mark, K

1996-09-01

For studies on processing and tissue distribution of type X collagen, monoclonal antibodies were prepared against human recombinant collagen type X (hrCol X) and tested by ELISA, immunoblotting and immunohistology. Forty-two clones were obtained which were grouped into four different subsets based on their reactivity against native and denatured hrCol X, pepsin-treated hrCol X, and the C-terminal NC-1 domain. Here we present results obtained with four monoclonal antibodies: Clone X 53, a representative of group I, binds with high affinity to both native and pepsin-digested hrCol X but with low affinity to the NC-1 dimer; monoclonal antibodies of group II and III recognized native and denatured hrCol X but not NC-1; antibodies of group II, but not III, reacted to some extent with pepsin treated hrCol X; one antibody (X 34) was obtained that reacted strongly with the isolated NC-1 dimer and native hrCol X but not with the NC-1 monomer or pepsin-digested hrCol X (group IV). Antibodies of all groups stained specifically the hypertrophic zone of fetal human epiphyseal cartilage. Mab X 53 stained the peri- and extracellular matrix of hypertrophic chondrocytes in the lower hypertrophic zone and in the calcified cartilage core in endochondral bone trabecules, while clone X 34 stained intracellularly and the pericellular matrix. All other tissues or cells of the epiphysis were negative. Antibody X 53 reacted also with canine, murine and guinea pig hypertrophic cartilage in tissue sections, but not with bovine or porcine type X collagen. In sections of osteoarthritic cartilage, clusters of hypertrophic chondrocytes in the deep zone were stained, confirming previous observations on enhanced chondrocyte hypertrophy and type X collagen expression in osteoarthritic articular cartilage.

Monoclonal antibody against human ovarian tumor-associated antigens.

PubMed

Poels, L G; Peters, D; van Megen, Y; Vooijs, G P; Verheyen, R N; Willemen, A; van Niekerk, C C; Jap, P H; Mungyer, G; Kenemans, P

1986-05-01

Mouse monoclonal antibodies (OV-TL 3) were raised against human ovarian tumor-associated antigens for diagnostic purposes. A cloned hybridoma cell line was obtained by fusion of murine myeloma cells with spleen lymphocytes from BALB/c mice immunized with a tumor cell suspension prepared from an ovarian endometrioid carcinoma. The antibodies were initially screened for their ability to bind on frozen sections of human ovarian carcinoma tissue and a negative reaction on gastric carcinoma tissue by indirect immunofluorescence. The reactivity of the selected OV-TL 3 clone (IgG1 subclass) was studied on normal and neoplastic tissues as well as on a cell line derived from the original tumor cell suspension used for immunization. OV-TL 3 antibodies stained frozen sections of human ovarian carcinomas of the following histological types: serous, mucinous, endometrioid, and clear cell. No reaction was found with breast cancers or other nongynecological tumors. No differences in staining pattern were observed between primary and metastatic ovarian carcinomas. OV-TL 3 antibodies brightly stained ovarian carcinoma cell clusters in ascitic fluids and left unstained mesothelial cells and peripheral blood cells. The OV-TL 3-defined antigen also remained strongly expressed on a cell line derived from the endometrioid ovarian carcinoma originally used for generation of OV-TL 3 clone. Reactivity was weak and irregular in a few ovarian cysts, while traces of fluorescence were sometimes detected in epithelial cells lining the female genital tract. In only 3 specimens of 15 endometrium carcinomas was weak focal reactivity with OV-TL 3 antibodies observed. The results of the immunofluorescence study were confirmed by the more sensitive avidin-biotin method and by 125I-labeled OV-TL 3 antibodies. Thus OV-TL 3 recognizes a common antigen for most ovarian carcinomas and may be a useful tool for rapid diagnosis of ovarian carcinomas.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Poels, L.G.; Peters, D.; van Megen, Y.

Mouse monoclonal antibodies (OV-TL 3) were raised against human ovarian tumor-associated antigens for diagnostic purposes. A cloned hybridoma cell line was obtained by fusion of murine myeloma cells with spleen lymphocytes from BALB/c mice immunized with a tumor cell suspension prepared from an ovarian endometrioid carcinoma. The antibodies were initially screened for their ability to bind on frozen sections of human ovarian carcinoma tissue and a negative reaction on gastric carcinoma tissue by indirect immunofluorescence. The reactivity of the selected OV-TL 3 clone (IgG1 subclass) was studied on normal and neoplastic tissues as well as on a cell line derivedmore » from the original tumor cell suspension used for immunization. OV-TL 3 antibodies stained frozen sections of human ovarian carcinomas of the following histological types: serous, mucinous, endometrioid, and clear cell. No reaction was found with breast cancers or other nongynecological tumors. No differences in staining pattern were observed between primary and metastatic ovarian carcinomas. OV-TL 3 antibodies brightly stained ovarian carcinoma cell clusters in ascitic fluids and left unstained mesothelial cells and peripheral blood cells. The OV-TL 3-defined antigen also remained strongly expressed on a cell line derived from the endometrioid ovarian carcinoma originally used for generation of OV-TL 3 clone. Reactivity was weak and irregular in a few ovarian cysts, while traces of fluorescence were sometimes detected in epithelial cells lining the female genital tract. In only 3 specimens of 15 endometrium carcinomas was weak focal reactivity with OV-TL 3 antibodies observed. The results of the immunofluorescence study were confirmed by the more sensitive avidin-biotin method and by /sup 125/I-labeled OV-TL 3 antibodies.« less

Pit-1/growth hormone factor 1 splice variant expression in the rhesus monkey pituitary gland and the rhesus and human placenta.

PubMed

Schanke, J T; Conwell, C M; Durning, M; Fisher, J M; Golos, T G

1997-03-01

We have examined the expression of Pit-1 messenger RNA (mRNA) splice variants in the nonhuman primate pituitary and in rhesus and human placenta. Full-length complementary DNAs (cDNAs) representing Pit-1 and the Pit-1 beta splice variants were cloned from a rhesus monkey pituitary cDNA library and were readily detectable by RT-PCR with rhesus pituitary gland RNA. The Pit-1T variant previously reported in mouse pituitary tumor cell lines was not detectable in normal rhesus pituitary tissue, although two novel splice variants were detected. A cDNA approximating the rat Pit-1 delta 4 variant was cloned but coded for a truncated and presumably nonfunctional protein. Only by using a nested RT-PCR approach were Pit-1 and Pit-1 beta variants consistently detectable in both human and rhesus placental tissue. The Pit-1 beta variant mRNA was not detectable in JEG-3 choriocarcinoma cells unless the cells were stimulated with 8-Br-cAMP. Immunoblot studies with nuclear extracts from primary rhesus syncytiotrophoblast cultures or JEG-3 choriocarcinoma cells indicated that although mRNA levels were very low, Pit-1 protein was detectable in differentiated cytotrophoblasts, and levels increased after treatment with 8-Br-cAMP. Two major species of Pit-1 protein were detected that corresponded to the two major bands in rat pituitary GH3 cell nuclear extracts. Low levels of slightly larger bands also were seen, which may represent Pit-1 beta protein or phosphorylated species. We conclude that Pit-1 splice variants expressed in the primate pituitary gland differ from those in the rodent gland and that the Pit-1 and Pit-1 beta mRNAs expressed in the placenta give rise to a pattern of protein expression similar to that seen in pituitary cells, which is inducible by treatment with 8-Br-cAMP.

Molecular cloning and characterization of Siamese crocodile (Crocodylus siamensis) copper, zinc superoxide dismutase (CSI-Cu,Zn-SOD) gene.

PubMed

Sujiwattanarat, Penporn; Pongsanarakul, Parinya; Temsiripong, Yosapong; Temsiripong, Theeranan; Thawornkuno, Charin; Uno, Yoshinobu; Unajak, Sasimanas; Matsuda, Yoichi; Choowongkomon, Kiattawee; Srikulnath, Kornsorn

2016-01-01

Superoxide dismutase (SOD, EC 1.15.1.1) is an antioxidant enzyme found in all living cells. It regulates oxidative stress by breaking down superoxide radicals to oxygen and hydrogen peroxide. A gene coding for Cu,Zn-SOD was cloned and characterized from Siamese crocodile (Crocodylus siamensis; CSI). The full-length expressed sequence tag (EST) of this Cu,Zn-SOD gene (designated as CSI-Cu,Zn-SOD) contained 462bp encoding a protein of 154 amino acids without signal peptides, indicated as intracellular CSI-Cu,Zn-SOD. This agreed with the results from the phylogenetic tree, which indicated that CSI-Cu,Zn-SOD belonged to the intracellular Cu,Zn-SOD. Chromosomal location determined that the CSI-Cu,Zn-SOD was localized to the proximal region of the Siamese crocodile chromosome 1p. Several highly conserved motifs, two conserved signature sequences (GFHVHEFGDNT and GNAGGRLACGVI), and conserved amino acid residues for binding copper and zinc (His(47), His(49), His(64), His(72), His(81), Asp(84), and His(120)) were also identified in CSI-Cu,Zn-SOD. Real-time PCR analysis showed that CSI-Cu,Zn-SOD mRNA was expressed in all the tissues examined (liver, pancreas, lung, kidney, heart, and whole blood), which suggests a constitutively expressed gene in these tissues. Expression of the gene in Escherichia coli cells followed by purification yielded a recombinant CSI-Cu,Zn-SOD, with Km and Vmax values of 6.075mM xanthine and 1.4×10(-3)mmolmin(-1)mg(-1), respectively. This Vmax value was 40 times lower than native Cu,Zn-SOD (56×10(-3)mmolmin(-1)mg(-1)), extracted from crocodile erythrocytes. This suggests that cofactors, protein folding properties, or post-translational modifications were lost during the protein purification process, leading to a reduction in the rate of enzyme activity in bacterial expression of CSI-Cu,Zn-SOD. Copyright © 2015 Elsevier Inc. All rights reserved.

Highly repressible expression system for cloning genes that specify potentially toxic proteins.

PubMed Central

O'Connor, C D; Timmis, K N

1987-01-01

A highly repressible expression vector system that allows the cloning of potentially deleterious genes has been constructed. Undesired expression of a cloned gene was prevented (i) at the level of initiation of transcription, by the presence of the strong but highly repressible leftward promoter of bacteriophage lambda, lambda pL, and (ii) at the level of transcript elongation or translation, through synthesis of antisense RNA complementary to the mRNA of the cloned gene. The system was tested by measuring the inhibition of expression of traT, the gene for the TraT major outer membrane lipoprotein. Direct detection and functional assays indicated that an essentially complete inhibition of traT expression was obtained. As a further test of the system, the gene encoding the EcoRI restriction endonuclease was cloned in the absence of the gene of the corresponding protective EcoRI modification methylase. Transformants harboring this construct were only viable when both repression controls were operational. Images PMID:2443481

Embryonic rather than extraembryonic tissues have more impact on the development of placental hyperplasia in cloned mice.

PubMed

Miki, H; Wakisaka, N; Inoue, K; Ogonuki, N; Mori, M; Kim, J-M; Ohta, A; Ogura, A

2009-06-01

Somatic cell cloning by nuclear transfer (NT) in mice is associated with hyperplastic placentas at term. To dissect the effects of embryonic and extraembryonic tissues on this clone-associated phenotype, we constructed diploid (2n) fused with (<-->) tetraploid (4n) chimeras from NT- and fertilization-derived (FD) embryos. Generally, the 4n cells contributed efficiently to all the extraembryonic tissues but not to the embryo itself. Embryos constructed by 2n NT<-->4n FD aggregation developed hyperplastic placentas (0.33+/-0.22 g) with a predominant contribution by NT-derived cells. Even when the population of FD-derived cells in placentas was increased using multiple FD embryos (up to four) for aggregation, most placentas remained hyperplastic (0.36+/-0.13 g). By contrast, placentas of the reciprocal combination, 2n FD<-->4n NT, were less hyperplastic (0.15+/-0.02 g). These nearly normal-looking placentas had a large proportion of NT-derived cells. Thus, embryonic rather than extraembryonic tissues had more impact on the onset of placental hyperplasia, and that the abnormal placentation in clones occurs in a noncell-autonomous manner. These findings suggest that for improvement of cloning efficiency we should understand the mechanisms regulating placentation, especially those of embryonic origin that might control the proliferation of trophoblastic lineage cells.

A novel protein involved in heart development in Ambystoma mexicanum is localized in endoplasmic reticulum.

PubMed

Jia, P; Zhang, C; Huang, X P; Poda, M; Akbas, F; Lemanski, S L; Erginel-Unaltuna, N; Lemanski, L F

2008-11-01

The discovery of the naturally occurring cardiac non-function (c) animal strain in Ambystoma mexicanum (axolotl) provides a valuable animal model to study cardiomyocyte differentiation. In homozygous mutant animals (c/c), rhythmic contractions of the embryonic heart are absent due to a lack of organized myofibrils. We have previously cloned a partial sequence of a peptide cDNA (N1) from an anterior-endoderm-conditioned-medium RNA library that had been shown to be able to rescue the mutant phenotype. In the current studies we have fully cloned the N1 full length cDNA sequence from the library. N1 protein has been detected in both adult heart and skeletal muscle but not in any other adult tissues. GFP-tagged expression of the N1 protein has revealed localization of the N1 protein in the endoplasmic reticulum (ER). Results from in situ hybridization experiments have confirmed the dramatic decrease of expression of N1 mRNA in mutant (c/c) embryos indicating that the N1 gene is involved in heart development.

Human retina-specific amine oxidase (RAO): cDNA cloning, tissue expression, and chromosomal mapping

DOE Office of Scientific and Technical Information (OSTI.GOV)

Imamura, Yutaka; Kubota, Ryo; Wang, Yimin

In search of candidate genes for hereditary retinal disease, we have employed a subtractive and differential cDNA cloning strategy and isolated a novel retina-specific cDNA. Nucleotide sequence analysis revealed an open reading frame of 2187 bp, which encodes a 729-amino-acid protein with a calculated molecular mass of 80,644 Da. The putative protein contained a conserved domain of copper amine oxidase, which is found in various species from bacteria to mammals. It showed the highest homology to bovine serum amine oxidase, which is believed to control the level of serum biogenic amines. Northern blot analysis of human adult and fetal tissuesmore » revealed that the protein is expressed abundantly and specifically in retina as a 2.7-kb transcript. Thus, we considered this protein a human retina-specific amine oxidase (RAO). The RAO gene (AOC2) was mapped by fluorescence in situ hybridization to human chromosome 17q21. We propose that AOC2 may be a candidate gene for hereditary ocular diseases. 38 refs., 4 figs.« less

Complementary DNA cloning and functional characterization of cytochrome P450 3A138 in common carp (Cyprinus carpio L.).

PubMed

Ma, Junguo; Bu, Yanzhen; Li, Yao; Niu, Daichun; Li, Xiaoyu

2014-06-01

The full-length sequence of a cytochrome P450 3A 138 (CYP3A138) cDNA in common carp was cloned and sequenced. The transcriptional and microsome enzyme activities of CYP3A138 in the fish liver after rifampicin exposure were also determined in this study. The results showed that the full-length CYP3A138 cDNA is 1912 base pairs (bp) long and contains an open reading frame of 1551 bp encoding a protein of 517 amino acids. Sequence analysis revealed that CYP3A138 is highly conserved in fish. Furthermore, the results of quantitative real-time PCR revealed that CYP3A138 in common carp is constitutively expressed in all tissues, but mainly in the liver and intestine. Additionally, rifampicin exposure promoted both the expression of CYP3A138 at the transcriptional level and the activity of the protein, suggesting that CYP3A138 is a member of the CYP3A subfamily. © 2014 Wiley Periodicals, Inc.

Cloning and characterization of the rat HIF-1 alpha prolyl-4-hydroxylase-1 gene.

PubMed

Cobb, Ronald R; McClary, John; Manzana, Warren; Finster, Silke; Larsen, Brent; Blasko, Eric; Pearson, Jennifer; Biancalana, Sara; Kauser, Katalin; Bringmann, Peter; Light, David R; Schirm, Sabine

2005-08-01

Prolyl-4-hydroxylase domain-containing enzymes (PHDs) mediate the oxygen-dependent regulation of the heterodimeric transcription factor hypoxia-inducible factor-1 (HIF-1). Under normoxic conditions, one of the subunits of HIF-1, HIF-1alpha, is hydroxylated on specific proline residues to target HIF-1alpha for degradation by the ubiquitin-proteasome pathway. Under hypoxic conditions, the hydroxylation by the PHDs is attenuated by lack of the oxygen substrate, allowing HIF-1 to accumulate, translocate to the nucleus, and mediate HIF-mediated gene transcription. In several mammalian species including humans, three PHDs have been identified. We report here the cloning of a full-length rat cDNA that is highly homologous to the human and murine PHD-1 enzymes and encodes a protein that is 416 amino acids long. Both cDNA and protein are widely expressed in rat tissues and cell types. We demonstrate that purified and crude baculovirus-expressed rat PHD-1 exhibits HIF-1alpha specific prolyl hydroxylase activity with similar substrate affinities and is comparable to human PHD-1 protein.

A new approach for cloning hLIF cDNA from genomic DNA isolated from the oral mucous membrane.

PubMed

Cui, Y H; Zhu, G Q; Chen, Q J; Wang, Y F; Yang, M M; Song, Y X; Wang, J G; Cao, B Y

2011-11-25

Complementary DNA (cDNA) is valuable for investigating protein structure and function in the study of life science, but it is difficult to obtain by traditional reverse transcription. We employed a novel strategy to clone human leukemia inhibitory factor (hLIF) gene cDNA from genomic DNA, which was directly isolated from the mucous membrane of mouth. The hLIF sequence, which is 609 bp long and is composed of three exons, can be acquired within a few hours by amplifying each exon and splicing all of them using overlap-PCR. This new approach developed is simple, time- and cost-effective, without RNA preparation or cDNA synthesis, and is not limited to the specific tissues for a particular gene and the expression level of the gene.

Expression pattern conferred by a glutamic acid-rich protein gene promoter in field-grown transgenic cassava (Manihot esculenta Crantz).

PubMed

Beltrán, J; Prías, M; Al-Babili, S; Ladino, Y; López, D; Beyer, P; Chavarriaga, P; Tohme, J

2010-05-01

A major constraint for incorporating new traits into cassava using biotechnology is the limited list of known/tested promoters that encourage the expression of transgenes in the cassava's starchy roots. Based on a previous report on the glutamic-acid-rich protein Pt2L4, indicating a preferential expression in roots, we cloned the corresponding gene including promoter sequence. A promoter fragment (CP2; 731 bp) was evaluated for its potential to regulate the expression of the reporter gene GUSPlus in transgenic cassava plants grown in the field. Intense GUS staining was observed in storage roots and vascular stem tissues; less intense staining in leaves; and none in the pith. Consistent with determined mRNA levels of the GUSPlus gene, fluorometric analyses revealed equal activities in root pulp and stems, but 3.5 times less in leaves. In a second approach, the activity of a longer promoter fragment (CP1) including an intrinsic intron was evaluated in carrot plants. CP1 exhibited a pronounced tissue preference, conferring high expression in the secondary phloem and vascular cambium of roots, but six times lower expression levels in leaf vascular tissues. Thus, CP1 and CP2 may be useful tools to improve nutritional and agronomical traits of cassava by genetic engineering. To date, this is the first study presenting field data on the specificity and potential of promoters for transgenic cassava.

Informatic selection of a neural crest-melanocyte cDNA set for microarray analysis

PubMed Central

Loftus, S. K.; Chen, Y.; Gooden, G.; Ryan, J. F.; Birznieks, G.; Hilliard, M.; Baxevanis, A. D.; Bittner, M.; Meltzer, P.; Trent, J.; Pavan, W.

1999-01-01

With cDNA microarrays, it is now possible to compare the expression of many genes simultaneously. To maximize the likelihood of finding genes whose expression is altered under the experimental conditions, it would be advantageous to be able to select clones for tissue-appropriate cDNA sets. We have taken advantage of the extensive sequence information in the dbEST expressed sequence tag (EST) database to identify a neural crest-derived melanocyte cDNA set for microarray analysis. Analysis of characterized genes with dbEST identified one library that contained ESTs representing 21 neural crest-expressed genes (library 198). The distribution of the ESTs corresponding to these genes was biased toward being derived from library 198. This is in contrast to the EST distribution profile for a set of control genes, characterized to be more ubiquitously expressed in multiple tissues (P < 1 × 10−9). From library 198, a subset of 852 clustered ESTs were selected that have a library distribution profile similar to that of the 21 neural crest-expressed genes. Microarray analysis demonstrated the majority of the neural crest-selected 852 ESTs (Mel1 array) were differentially expressed in melanoma cell lines compared with a non-neural crest kidney epithelial cell line (P < 1 × 10−8). This was not observed with an array of 1,238 ESTs that was selected without library origin bias (P = 0.204). This study presents an approach for selecting tissue-appropriate cDNAs that can be used to examine the expression profiles of developmental processes and diseases. PMID:10430933

In vitro resistance to 5-nitroimidazoles and benzimidazoles in Giardia duodenalis: variability and variation in gene expression.

PubMed

Argüello-García, Raúl; Cruz-Soto, Maricela; Romero-Montoya, Lydia; Ortega-Pierres, Guadalupe

2009-12-01

The susceptibility of Giardia duodenalis trophozoites exposed in vitro to sublethal concentrations of metronidazole (MTZ) and albendazole (ABZ) may exhibit inter-culture (variability) and intra-culture (variation) differences in drug susceptibility. It was previously reported that MTZ-resistant trophozoites may display changes in pyruvate:ferredoxin oxidoreductase (PFOR) expression while changes at the beta-tubulin molecule are apparently absent in ABZ-resistant cultures. To assess the levels of gene expression of these molecules, we obtained cloned cultures growing at concentrations up to 23 microM MTZ (WBRM23) and up to 8muM ABZ (WBRA8) and gene sequence and expression of pfor and beta-tubulin loci were compared with these of drug-susceptible clone WB1. Neither the pfor nor the beta-tubulin genes showed changes at sequence level but the MTZ-resistant clones WBRM21 and WBRM23 showed up-regulation of the pfor RNA using the gdh gene as reference. By using WB1 and WBRA8 clones in representational difference analyses of gene expression (RDA) an insert referred to as ARR-VSP was selected and sequenced. It showed the highest homology to one VSP molecule in the Giardia Genome Database (orf GL50803_101765). This isogene was up-regulated in five ABZ-resistant clones and the clone WBRA8 exhibited the highest RNA expression level. When successive progenies of clones WB1, WBRM23 and WBRA8 were analyzed in Northern blot assays to detect pfor and ARR-VSP RNAs respectively, the expression patterns showed variation for both genes but it was much lower in the clone WBRA8. These results suggest that G. duodenalis cultures either susceptible or resistant to MTZ and ABZ may display variability and variation at RNA expression levels albeit these were more marked in the MTZ-resistant parasites. These data might have further implications defining major mechanisms involved in drug resistance of Giardia.

Sequence of the cDNA of a human dihydrodiol dehydrogenase isoform (AKR1C2) and tissue distribution of its mRNA.

PubMed Central

Shiraishi, H; Ishikura, S; Matsuura, K; Deyashiki, Y; Ninomiya, M; Sakai, S; Hara, A

1998-01-01

Human liver contains three isoforms (DD1, DD2 and DD4) of dihydrodiol dehydrogenase with 20alpha- or 3alpha-hydroxysteroid dehydrogenase activity; the dehydrogenases belong to the aldo-oxo reductase (AKR) superfamily. cDNA species encoding DD1 and DD4 have been identified. However, four cDNA species with more than 99% sequence identity have been cloned and are compatible with a partial amino acid sequence of DD2. In this study we have isolated a cDNA clone encoding DD2, which was confirmed by comparison of the properties of the recombinant and hepatic enzymes. This cDNA showed differences of one, two, four and five nucleotides from the previously reported four cDNA species for a dehydrogenase of human colon carcinoma HT29 cells, human prostatic 3alpha-hydroxysteroid dehydrogenase, a human liver 3alpha-hydroxysteroid dehydrogenase-like protein and chlordecone reductase-like protein respectively. Expression of mRNA species for the five similar cDNA species in 20 liver samples and 10 other different tissue samples was examined by reverse transcriptase-mediated PCR with specific primers followed by diagnostic restriction with endonucleases. All the tissues expressed only one mRNA species corresponding to the newly identified cDNA for DD2: mRNA transcripts corresponding to the other cDNA species were not detected. We suggest that the new cDNA is derived from the principal gene for DD2, which has been named AKR1C2 by a new nomenclature for the AKR superfamily. It is possible that some of the other cDNA species previously reported are rare allelic variants of this gene. PMID:9716498

A Recombinant Potato virus Y Infectious Clone Tagged with the Rosea1 Visual Marker (PVY-Ros1) Facilitates the Analysis of Viral Infectivity and Allows the Production of Large Amounts of Anthocyanins in Plants

PubMed Central

Cordero, Teresa; Mohamed, Mohamed A.; López-Moya, Juan-José; Daròs, José-Antonio

2017-01-01

Potato virus Y (PVY) is a major threat to the cultivation of potato and other solanaceous plants. By inserting a cDNA coding for the Antirrhinum majus Rosea1 transcription factor into a PVY infectious clone, we created a biotechnological tool (PVY-Ros1) that allows infection by this relevant plant virus to be tracked by the naked eye with no need for complex instrumentation. Rosea1 is an MYB-type transcription factor whose expression activates the biosynthesis of anthocyanin pigments in a dose-specific and cell-autonomous manner. Our experiments showed that the mechanical inoculation of solanaceous plants with PVY-Ros1 induced the formation of red infection foci in inoculated tissue and solid dark red pigmentation in systemically infected tissue, which allows disease progression to be easily monitored. By using silver nanoparticles, a nanomaterial with exciting antimicrobial properties, we proved the benefits of PVY-Ros1 to analyze novel antiviral treatments in plants. PVY-Ros1 was also helpful for visually monitoring the virus transmission process by an aphid vector. Most importantly, the anthocyanin analysis of infected tobacco tissues demonstrated that PVY-Ros1 is an excellent biotechnological tool for molecular farming because it induces the accumulation of larger amounts of anthocyanins, antioxidant compounds of nutritional, pharmaceutical and industrial interest, than those that naturally accumulate in some fruits and vegetables well known for their high anthocyanin content. Hence these results support the notion that the virus-mediated expression of regulatory factors and enzymes in plants facilitates easy quick plant metabolism engineering. PMID:28428782

Isolation of a candidate human telomerase catalytic subunit gene, which reveals complex splicing patterns in different cell types.

PubMed

Kilian, A; Bowtell, D D; Abud, H E; Hime, G R; Venter, D J; Keese, P K; Duncan, E L; Reddel, R R; Jefferson, R A

1997-11-01

Telomerase is a multicomponent reverse transcriptase enzyme that adds DNA repeats to the ends of chromosomes using its RNA component as a template for synthesis. Telomerase activity is detected in the germline as well as the majority of tumors and immortal cell lines, and at low levels in several types of normal cells. We have cloned a human gene homologous to a protein from Saccharomyces cerevisiae and Euplotes aediculatus that has reverse transcriptase motifs and is thought to be the catalytic subunit of telomerase in those species. This gene is present in the human genome as a single copy sequence with a dominant transcript of approximately 4 kb in a human colon cancer cell line, LIM1215. The cDNA sequence was determined using clones from a LIM1215 cDNA library and by RT-PCR, cRACE and 3'RACE on mRNA from the same source. We show that the gene is expressed in several normal tissues, telomerase-positive post-crisis (immortal) cell lines and various tumors but is not expressed in the majority of normal tissues analyzed, pre-crisis (non-immortal) cells and telomerase-negative immortal (ALT) cell lines. Multiple products were identified by RT-PCR using primers within the reverse transcriptase domain. Sequencing of these products suggests that they arise by alternative splicing. Strikingly, various tumors, cell lines and even normal tissues (colonic crypt and testis) showed considerable differences in the splicing patterns. Alternative splicing of the telomerase catalytic subunit transcript may be important for the regulation of telomerase activity and may give rise to proteins with different biochemical functions.

Antibody repertoire development in fetal and neonatal piglets. XVII. IgG subclass transcription revisited with emphasis on new IgG3.

PubMed

Butler, John E; Wertz, Nancy

2006-10-15

Fetal piglets offer an in vivo model for determining whether Ag-independent IgG subclass transcription proceeds in a manner that differs from subclass transcription in pigs exposed to environmental Ags and TLR ligands. Our data from approximately 12,000 Cgamma clones from > 60 piglets provide the first report on the relative usage of all known porcine Cgamma genes in fetal and young pigs. Studies revealed that among the six Cgamma genes, allelic variants of IgG1 comprised 50-80% of the repertoire, and IgG2 alleles comprised < 10% in nearly all tissues. However, relative transcription of allelic variants of IgG1 randomly deviate from the 1:1 ratio expected in heterozygotes. Most surprising was the finding that IgG3 accounted for half of all Cgamma transcripts in the ileal Peyer's patches (IPPs) and mesenteric lymph nodes but on average only approximately 5% of the clones from the thymus, tonsil, spleen, peripheral blood, and bone marrow of newborns. Lymphoid tissues from late term fetuses revealed a similar expression pattern. Except for IgG3 in the IPPs and mesenteric lymph nodes, no stochastic pattern of Cgamma expression during development was seen in animals from mid-gestation through 5 mo. The age and tissue dependence of IgG3 transcription paralleled the developmental persistence of the IPP, and its near disappearance corresponds to the diversification of the preimmune VDJ repertoire in young piglets. We hypothesize that long-hinged porcine IgG3 may be important in preadaptive responses to T cell-independent Ags similar to those described for its murine namesake.

Human Hrs, a tyrosine kinase substrate in growth factor-stimulated cells: cDNA cloning and mapping of the gene to chromosome 17.

PubMed

Lu, L; Komada, M; Kitamura, N

1998-06-15

Hrs is a 115kDa zinc finger protein which is rapidly tyrosine phosphorylated in cells stimulated with various growth factors. We previously purified the protein from a mouse cell line and cloned its cDNA. In the present study, we cloned a human Hrs cDNA from a human placenta cDNA library by cross-hybridization, using the mouse cDNA as a probe, and determined its nucleotide sequence. The human Hrs cDNA encoded a 777-amino-acid protein whose sequence was 93% identical to that of mouse Hrs. Northern blot analysis showed that the Hrs mRNA was about 3.0kb long and was expressed in all the human adult and fetal tissues tested. In addition, we showed by genomic Southern blot analysis that the human Hrs gene was a single-copy gene with a size of about 20kb. Furthermore, the human Hrs gene was mapped to chromosome 17 by Southern blotting of genomic DNAs from human/rodent somatic cell hybrids. Copyright 1998 Elsevier Science B.V. All rights reserved.

Cloning and expression characterization of peroxisome proliferator-activated receptors (PPARs) with their agonists, dietary lipids, and ambient salinity in rabbitfish Siganus canaliculatus.

PubMed

You, Cuihong; Jiang, Danli; Zhang, Qinghao; Xie, Dizhi; Wang, Shuqi; Dong, Yewei; Li, Yuanyou

2017-04-01

Rabbitfish Siganus canaliculatus is the first marine teleost reported to have the ability of biosynthesizing C 20-22 long-chain polyunsaturated fatty acids (LC-PUFA) from C 18 precursors, and thus provides a model for studying the regulatory mechanisms of LC-PUFA biosynthesis in teleosts. To investigate the possible roles of peroxisome proliferator-activated receptors (PPARs), critical transcription factors involved in the regulation of lipid metabolism, in the regulation of LC-PUFA biosynthesis in rabbitfish, the PPAR genes were cloned and their expression characterization with PPAR agonists, dietary lipid resource, and ambient salinity were examined. Three cDNA sequences respectively encoding 477, 516 and 519 amino acids of PPARα, PPARβ, and PPARγ isoforms were obtained. PPARα exhibited a wide tissue expression with its highest levels in the heart and brain; PPARβ was predominantly expressed in the gills, while PPARγ was highly expressed in the intestine and gills. In rabbitfish primary hepatocytes, both the PPAR agonists 2-bromopalmitate (2-Bro) and fenofibrate (FF) increased the expression of PPARγ, SREBP1c and Elovl5, whereas FF depressed the expression of Δ6/Δ5 Fad. Moreover, a higher hepatic PPARβ expression was observed in fish fed diets with vegetable oils (VO) than that with fish oil (FO), in the former the expression of PPARα, PPARβ, and PPARγ were increased at the low ambient salinity (10ppt), where an increasing expression of Δ5/Δ6 Fad, Δ4 Fad and Elovl5 genes was previously reported. These results suggest that PPARs might be involved in the upregulation of LC-PUFA biosynthesis with dietary VO and low ambient salinity in rabbitfish. Copyright © 2017 Elsevier Inc. All rights reserved.

Immortalization of normal human embryonic fibroblasts by introduction of either the human papillomavirus type 16 E6 or E7 gene alone.

PubMed

Yamamoto, Akito; Kumakura, Shin-ichi; Uchida, Minoru; Barrett, J Carl; Tsutsui, Takeki

2003-09-01

The ability of the human papillomavirus type 16 (HPV-16) E6 or E7 gene to induce immortalization of normal human embryonic fibroblast WHE-7 cells was examined. WHE-7 cells at 9 population doublings (PD) were infected with retrovirus vectors encoding either HPV-16 E6 or E7 alone or both E6 and E7 (E6/E7). One of 4 isolated clones carrying E6 alone became immortal and is currently at >445 PD. Four of 4 isolated clones carrying E7 alone escaped from crisis and are currently at >330 PD. Three of 5 isolated clones carrying E6/E7 were also immortalized and are currently at >268 PD. The immortal clone carrying E6 only and 2 of the 3 immortal clones carrying E6/E7 expressed a high level of E6 protein, and all the immortal clones carrying E7 alone and the other immortal clone carrying E6/E7 expressed a high level of E7 protein when compared to their mortal or precrisis clones. The immortal clones expressing a high level of E6 or E7 protein were positive for telomerase activity or an alternative mechanism of telomere maintenance, respectively, known as ALT (alternative lengthening of telomeres). All the mortal or precrisis clones were negative for both phenotypes. All the immortal clones exhibited abrogation of G1 arrest after DNA damage by X-ray irradiation. The expression of INK4a protein (p16(INK4a)) was undetectable in the E6-infected mortal and immortal clones, whereas Rb protein (pRb) was hyperphosphorylated only in the immortal clone. The p16(INK4a) protein was overexpressed in all the E7-infected immortal clones and their clones in the pre-crisis period as well as all the E6/E7-infected mortal and immortal clones, but the pRb expression was downregulated in all of these clones. These results demonstrate for the first time to our knowledge that HPV-16 E6 or E7 alone can induce immortalization of normal human embryonic fibroblasts. Inactivation of p16(INK4a)/pRb pathways in combination with activation of a telomere maintenance mechanism is suggested to be necessary for immortalization of normal human embryonic fibroblasts by these viral oncogenes. The susceptibility of human cells to immortalization may be related to the state of differentiation of the cells. Copyright 2003 Wiley-Liss, Inc.

Molecular cloning, characterization and expression of heat shock protein 70 gene from the oyster Crassostrea hongkongensis responding to thermal stress and exposure of Cu(2+) and malachite green.

PubMed

Zhang, Zhanhui; Zhang, Qizhong

2012-04-15

Heat shock protein 70 (HSP70) acts mostly as a molecular chaperone and plays a key role in the process of protecting cells by facilitating the folding of nascent peptides and the cellular stress response. The cDNA of the oyster Crassostrea hongkongensis hsp70 (designated chhsp70) was cloned with the techniques of homological cloning and rapid amplification of cDNA ends (RACE). The full-length chhsp70 cDNA was 2251bp, consisting of a 130bp 5'-UTR, 216bp 3'-UTR with a canonical polyadenylation signal sequence AATAAA and a poly (A) tail, and an open reading frame of 1905bp, which encoded a polypeptide of 634 amino acids. Three classical HSP signature motifs were detected in ChHSP70, i.e., DLGTT-S-V, IFDLGGGTFDVSIL and VVLVGGSTRIPKIQK. BLAST analysis revealed that the ChHSP70 shared high identity with other bivalve HSP70. The phylogenetic analysis indicated that the ChHSP70 was a member of the HSP70 family. The chhsp70 mRNA transcripts were quantified by fluorescent real time RT-PCR under both unstressed and stressed conditions, i. e., heat shock and exposure to Cu(2+) and malachite green. Basal expression level was similar in mantle, gill, digestive gland, and heart, but higher in muscle than that in the others. A similar trend showed that the chhsp70 mRNA expression significantly increased at 3-6h, then dropped and returned to control level at 24h in the five tissues and organs mentioned above after heat shock. A clearly time-dependent expression pattern of chhsp70 mRNA in digestive gland and gill of the oyster was observed after exposure of Cu(2+) and malachite green. In the two tissues, the chhsp70 mRNA level reached the maximum at 6h after malachite green exposure and on day 4 after Cu(2+) exposure, and then decreased progressively to the control level. The results indicated that ChHSP70 of the oyster is an inducible protein, and plays an important role in response to the Cu(2+) and malachite green polluted stress, so chhsp70 might be used as a potential molecular biomarker of above pollutants. Copyright © 2012 Elsevier B.V. All rights reserved.

Establishing a human pancreatic stem cell line and transplanting induced pancreatic islets to reverse experimental diabetes in rats.

PubMed

Xiao, Mei; An, LiLong; Yang, XueYi; Ge, Xin; Qiao, Hai; Zhao, Ting; Ma, XiaoFei; Fan, JingZhuang; Zhu, MengYang; Dou, ZhongYing

2008-09-01

The major obstacle in using pancreatic islet transplantation to cure type I and some type II diabetes is the shortage of the donors. One of ways to overcome such obstacle is to isolate and clone pancreatic stem cells as "seed cells" and induce their differentiation into functional islets as an abundant transplantation source. In this study, a monoclonal human pancreatic stem cell (mhPSC) line was obtained from abortive fetal pancreatic tissues. Pancreatic tissues were taken from abortive fetus by sterile procedures, and digested into single cells and cell clusters with 0.1% type IV collagenase. Cultured in modified glucose-low DMEM with 10% fetal bovine serum (FBS), these single cells and cell clusters adhered to culture dishes, and then primary epidermal-like pancreatic stem cells started to clone. After digesting with 0.25% trypsin and 0.04% EDTA, fibroblasts and other cells were gradually eliminated and epithelioid pancreatic stem cells were gradually purified during generations. Using clone-ring selection, the mhPSCs were obtained. After addition of 10 ng/mL epidermal growth factor (EGF) in cell culture medium, the mhPSCs quickly grew and formed a gravelstone-like monolayer. Continuously proliferated, a mhPSC line, which was derived from a male abortive fetus of 4 months old, has been passed through 50 generations. More than 1 x 10(9) mhPSCs were cryo-preserved in liquid nitrogen. Karyotype analysis showed that the chromosome set of the mhPSC line was normal diploid. Immunocytochemistry results demonstrated that the mhPSC line was positive for the pdx1, glucagon, nestin and CK19, and negative for the insulin, CD34, CD44 and CD45 protein expression. RT-PCR revealed further that the mhPSCs expressed transcription factors of the pdx1, glucagon, nestin and CK19. Also, in vitro induced with beta-mercaptoethanol, the mhPSCs differentiated into nerve cells that expressed the NF protein. Induced with nicotinamide, the mhPSCs differentiated into functional islet-like clusters, as identified by dithizone staining, which expressed the transcription factor of the insulin and secreted the insulin and C-peptide. Furthermore, the transplantation of mhPSCs-induced pancreatic islets into the subcapsular region of the kidney in streptozotocin-induced diabetic rats could reduce blood glucose levels and prolong the life time.

Hypoexpression and epigenetic regulation of candidate tumor suppressor gene CADM-2 in human prostate cancer.

PubMed

Chang, Guimin; Xu, Shuping; Dhir, Rajiv; Chandran, Uma; O'Keefe, Denise S; Greenberg, Norman M; Gingrich, Jeffrey R

2010-11-15

Cell adhesion molecules (CADM) comprise a newly identified protein family whose functions include cell polarity maintenance and tumor suppression. CADM-1, CADM-3, and CADM-4 have been shown to act as tumor suppressor genes in multiple cancers including prostate cancer. However, CADM-2 expression has not been determined in prostate cancer. The CADM-2 gene was cloned and characterized and its expression in human prostatic cell lines and cancer specimens was analyzed by reverse transcription-PCR and an immunohistochemical tissue array, respectively. The effects of adenovirus-mediated CADM-2 expression on prostate cancer cells were also investigated. CADM-2 promoter methylation was evaluated by bisulfite sequencing and methylation-specific PCR. We report the initial characterization of CADM-2 isoforms: CADM-2a and CADM-2b, each with separate promoters, in human chromosome 3p12.1. Prostate cancer cell lines, LNCaP and DU145, expressed negligible CADM-2a relative to primary prostate tissue and cell lines, RWPE-1 and PPC-1, whereas expression of CADM-2b was maintained. Using immunohistochemistry, tissue array results from clinical specimens showed statistically significant decreased expression in prostate carcinoma compared with normal donor prostate, benign prostatic hyperplasia, prostatic intraepithelial neoplasia, and normal tissue adjacent to tumor (P < 0.001). Adenovirus-mediated CADM-2a expression suppressed DU145 cell proliferation in vitro and colony formation in soft agar. The decrease in CADM-2a mRNA in cancer cell lines correlated with promoter region hypermethylation as determined by bisulfite sequencing and methylation-specific PCR. Accordingly, treatment of cells with the demethylating agent 5-aza-2'-deoxycytidine alone or in combination with the histone deacetylase inhibitor trichostatin A resulted in the reactivation of CADM-2a expression. CADM-2a protein expression is significantly reduced in prostate cancer. Its expression is regulated in part by promoter methylation and implicates CADM-2 as a previously unrecognized tumor suppressor gene in a proportion of human prostate cancers. ©2010 AACR.

Cloning and tissue distribution of rat hear fatty acid binding protein mRNA: identical forms in heart and skeletal muscle

DOE Office of Scientific and Technical Information (OSTI.GOV)

Claffey, K.P.; Herrera, V.L.; Brecher, P.

1987-12-01

A fatty acid binding protein (FABP) as been identified and characterized in rat heart, but the function and regulation of this protein are unclear. In this study the cDNA for rat heart FABP was cloned from a lambda gt11 library. Sequencing of the cDNA showed an open reading frame coding for a protein with 133 amino acids and a calculated size of 14,776 daltons. Several differences were found between the sequence determined from the cDNA and that reported previously by protein sequencing techniques. Northern blot analysis using rat heart FABP cDNA as a probe established the presence of an abundantmore » mRNA in rat heart about 0.85 kilobases in length. This mRNA was detected, but was not abundant, in fetal heart tissue. Tissue distribution studies showed a similar mRNA species in red, but not white, skeletal muscle. In general, the mRNA tissue distribution was similar to that of the protein detected by Western immunoblot analysis, suggesting that heart FABP expression may be regulated at the transcriptional level. S1 nuclease mapping studies confirmed that the mRNA hybridized to rat heart FABP cDNA was identical in heart and red skeletal muscle throughout the entire open reading frame. The structural differences between heart FABP and other members of this multigene family may be related to the functional requirements of oxidative muscle for fatty acids as a fuel source.« less

Identification and characterization of a novel family of mammalian ependymin-related proteins (MERPs) in hematopoietic, nonhematopoietic, and malignant tissues.

PubMed

Apostolopoulos, J; Sparrow, R L; McLeod, J L; Collier, F M; Darcy, P K; Slater, H R; Ngu, C; Gregorio-King, C C; Kirkland, M A

2001-10-01

Evidence is presented for a family of mammalian homologs of ependymin, which we have termed the mammalian ependymin-related proteins (MERPs). Ependymins are secreted glycoproteins that form the major component of the cerebrospinal fluid in many teleost fish. We have cloned the entire coding region of human MERP-1 and mapped the gene to chromosome 7p14.1 by fluorescence in situ hybridization. In addition, three human MERP pseudogenes were identified on chromosomes 8, 16, and X. We have also cloned the mouse MERP-1 homolog and an additional family member, mouse MERP-2. Then, using bioinformatics, the mouse MERP-2 gene was localized to chromosome 13, and we identified the monkey MERP-1 homolog and frog ependymin-related protein (ERP). Despite relatively low amino acid sequence conservation between piscine ependymins, toad ERP, and MERPs, several amino acids (including four key cysteine residues) are strictly conserved, and the hydropathy profiles are remarkably alike, suggesting the possibilities of similar protein conformation and function. As with fish ependymins, frog ERP and MERPs contain a signal peptide typical of secreted proteins. The MERPs were found to be expressed at high levels in several hematopoietic cell lines and in nonhematopoietic tissues such as brain, heart, and skeletal muscle, as well as several malignant tissues and malignant cell lines. These findings suggest that MERPs have several potential roles in a range of cells and tissues.

Cloning, characterization and tissue specific expression of Amur tiger (Panthera tigris altaica) IGF-I.

PubMed

Hu, Xi-Lian; Zhu, Mu-Yuan; Zhang, Zhi-He; Hou, Rong; Shen, Fu-Jun; Li, Fu-Zhen; Zhang, An-Ju

2006-08-01

Insulin-like growth factor I (IGF-I) plays an important role in regulating gonad function, which is essential for normal reproduction in animals, especially in sexual receptivity and reproductive behavior. In this study, a cDNA encoding Amur tiger (Panthera tigris altaica) IGF-I was isolated from liver total RNA using RT-PCR. The IGF-I cDNA of Amur tiger (ATIGF-I) was highly homologous to that of other animals, 84.8% to rat, 93.7% to human and horse. Alignment analysis showed that the cysteine residues and many amino acid residues of putative mature ATIGF-I are highly conserved in mammalian species, confirming the high sequence homology observed in other species. DNA encoding the mature ATIGF-I peptide was ligated with pET-DsbA expression vector and highly expressed in Escherichia coli BL21 with IPTG induction. The recombinant proteins expressed existed mostly in the soluble protein fraction, and were purified with metal affinity resins. Western blotting confirmed that the recombinant proteins reacted with antibodies against IGF-I. The results obtained here should be useful for large-scale production of biological active ATIGF-I protein, as well as for further research on growth, development, and reproduction in the Amur tiger. Tissue specific expression of ATIGF-I mRNA in the Amur tiger was examined by reverse transcription-polymerase chain reaction (RT-PCR), The major ATIGF-I mRNA expression tissue was the liver, while medium signals were found in the uterus, ovary, and pituitary, and minor signals were detected in various tissues including the heart, spleen, pancreas, and kidney. The results indicate that IGF-I might play an important role in the reproductive system and in cub development in the Amur tiger.

Cloning of growth hormone, somatolactin, and their receptor mRNAs, their expression in organs, during development, and on salinity stress in the hermaphroditic fish, Kryptolebias marmoratus.

PubMed

Rhee, Jae-Sung; Kim, Bo-Mi; Seo, Jung Soo; Kim, Il-Chan; Lee, Young-Mi; Lee, Jae-Seong

2012-04-01

Salinity is an important parameter that affects survival and metabolism in fish. In fish, pituitary growth hormone (GH) regulates physiological functions including adaptation to different salinity as well as somatic growth. GH is stimulated by growth hormone-releasing hormone (GHRH) and exerts its function via binding to growth hormone receptor (GHR). As Kryptolebias marmoratus is a euryhaline fish, this species would be a useful model species for studying the adaptation to osmotic stress conditions. Here, we cloned GH, -GHR, somatolactin (SL), and somatolactin receptor (SLR) genes, and analyzed their expression patterns in different tissues and during early developmental stages by using real-time RT-PCR. We also further examined expression of them after acclimation to different salinity. Tissue distribution studies revealed that Km-GH and -SL mRNAs were remarkably expressed in brain and pituitary, whereas Km-GHR and -SLR mRNAs were predominantly expressed in liver, followed by gonad, muscle, pituitary, and brain. During embryonic developmental stages, the expression of their mRNA was increased at stage 3 (9 dpf). The Km-GH and -SL mRNA transcripts were constantly elevated until stage 5 (5h post hatch), whereas Km-GHR and -SLR mRNA levels decreased at this stage. After we transferred K. marmoratus from control (12 psu) to hyper-osmotic condition (hyperseawater, HSW; 33 psu), Km-GH, -SL, and GHR mRNA levels were enhanced. In hypo-osmotic conditions like freshwater (FW), Km-GH and -SL expressions were modulated 24 h after exposure, and Km-SLR transcripts were significantly upregulated. This finding suggests that Km-GH and -SL may be involved in the osmoregulatory mechanism under hyper-osmotic as well as hypo-osmotic stress. This is the first report on transcriptional modulation and relationship of GH, GHR, SL, and SLR during early development and after salinity stress. This study will be helpful to a better understanding on molecular mechanisms of adaptation response to salt stress in euryhaline fish. Copyright © 2012 Elsevier Inc. All rights reserved.

Cloning and expression of Pectobacterium carotovorum endo-polygalacturonase gene in Pichia pastoris for production of oligogalacturonates

USDA-ARS?s Scientific Manuscript database

A bacterial endo-polygalacturonase (endo-PGase) gene from the plant pathogen Pectobacterium carotovorum was cloned into pGAPZaA vector and constitutively expressed in Pichia pastoris. The recombinant endo-PGase secreted by the Pichia clone showed a 1.7 fold increase when the culture medium included ...

Cloning of the transgenic pigs expressing human decay accelerating factor and N-acetylglucosaminyltransferase III.

PubMed

Fujimura, Tatsuya; Kurome, Mayuko; Murakami, Hiroshi; Takahagi, Yoichi; Matsunami, Katsuyoshi; Shimanuki, Shinichi; Suzuki, Kohei; Miyagawa, Shuji; Shirakura, Ryota; Shigehisa, Tamotsu; Nagashima, Hiroshi

2004-01-01

The present paper describes production of cloned pigs from fibroblast cells of transgenic pigs expressing human decay accelerating factor (DAF, CD55) and N-acetylglucosaminyltransferase III (GnT-III) that remodels sugar-chain biosynthesis. Two nuclear transfer protocols were used: a two-step activation (TA) method and a delayed activation (DA) method. Enucleated in vitro-matured oocytes and donor cells were electrically fused in a calcium-containing medium by TA method or in a calcium-free medium by DA method, followed by electrical activation 1-1.5 h later, respectively. In vitro blastocyst formation rates of nuclear transferred embryos reconstructed by TA and DA method were 8% and 14%, respectively. As a result of embryo transfer of the reconstructed embryos made by each method into recipient pigs, both gave rise to cloned piglets. These cloned pigs expressed transgene as much as their nuclear donor cells. In conclusions, (1) pig cloning can be carried out by TA or DA nuclear transfer methods, (2) expression of transgenes can be maintained to cloned pigs from the nuclear donor cells derived from transgenic animals.

cDNA Cloning, Expression Pattern, and Chromosomal Localization of Mlf1, Murine Homologue of a Gene Involved in Myelodysplasia and Acute Myeloid Leukemia

PubMed Central

Hitzler, Johann K.; Witte, David P.; Jenkins, Nancy A.; Copeland, Neal G.; Gilbert, Debra J.; Naeve, Clayton W.; Look, A. Thomas; Morris, Stephan W.

1999-01-01

The NPM-MLF1 fusion protein is expressed in blasts from patients with myelodysplasia/acute myeloid leukemia (MDS/AML) containing the t(3;5) chromosomal rearrangement. Nucleophosmin (NPM), a previously characterized nucleolar phosphoprotein, contributes to two other fusion proteins found in lympho-hematopoietic malignancies, anaplastic large cell lymphoma (NPM-ALK) and acute promyelocytic leukemia (NPM-RARα). By contrast, the function of the carboxy-terminal fusion partner, myelodysplasia/myeloid leukemia factor 1 (MLF1), is unknown. To aid in understanding normal MLF1 function, we isolated the murine cDNA, determined the chromosomal localization of Mlf1, and defined its tissue expression by in situ hybridization. Mlf1 was highly similar to its human homologue (86% and 84% identical nucleotide and amino acid sequence, respectively) and mapped to the central region of chromosome 3, within a segment lacking known mouse mutations. Mlf1 tissue distribution was restricted during both development and postnatal life, with high levels present only in skeletal, cardiac, and selected smooth muscle, gonadal tissues, and rare epithelial tissues including the nasal mucosa and the ependyma/choroid plexus in the brain. Mlf1 transcripts were undetectable in the lympho-hematopoietic organs of both the embryonic and adult mouse, suggesting that NPM-MLF1 contributes to the genesis of MDS/AML in part by enforcing the ectopic overexpression of MLF1 within hematopoietic tissues. PMID:10393836

Tissue-Specific Transcriptomic Profiling of Sorghum propinquum using a Rice Genome Array

PubMed Central

Zhang, Ting; Zhao, Xiuqin; Huang, Liyu; Liu, Xiaoyue; Zong, Ying; Zhu, Linghua; Yang, Daichang; Fu, Binying

2013-01-01

Sorghum (Sorghum bicolor) is one of the world's most important cereal crops. S. propinquum is a perennial wild relative of S. bicolor with well-developed rhizomes. Functional genomics analysis of S. propinquum, especially with respect to molecular mechanisms related to rhizome growth and development, can contribute to the development of more sustainable grain, forage, and bioenergy cropping systems. In this study, we used a whole rice genome oligonucleotide microarray to obtain tissue-specific gene expression profiles of S. propinquum with special emphasis on rhizome development. A total of 548 tissue-enriched genes were detected, including 31 and 114 unique genes that were expressed predominantly in the rhizome tips (RT) and internodes (RI), respectively. Further GO analysis indicated that the functions of these tissue-enriched genes corresponded to their characteristic biological processes. A few distinct cis-elements, including ABA-responsive RY repeat CATGCA, sugar-repressive TTATCC, and GA-responsive TAACAA, were found to be prevalent in RT-enriched genes, implying an important role in rhizome growth and development. Comprehensive comparative analysis of these rhizome-enriched genes and rhizome-specific genes previously identified in Oryza longistaminata and S. propinquum indicated that phytohormones, including ABA, GA, and SA, are key regulators of gene expression during rhizome development. Co-localization of rhizome-enriched genes with rhizome-related QTLs in rice and sorghum generated functional candidates for future cloning of genes associated with rhizome growth and development. PMID:23536906

The disparity between human cell senescence in vitro and lifelong replication in vivo.

PubMed

Rubin, Harry

2002-07-01

Cultured human fibroblasts undergo senescence (a loss of replicative capacity) after a uniform, fixed number of approximately 50 population doublings, commonly termed the Hayflick limit. It has been long known from clonal and other quantitative studies, however, that cells decline in replicative capacity from the time of explantation and do so in a stochastic manner, with a half-life of only approximately 8 doublings. The apparent 50-cell doubling limit reflects the expansive propagation of the last surviving clone. The relevance of either figure to survival of cells in the body is questionable, given that stem cells in some renewing tissues undergo >1,000 divisions in a lifetime with no morphological sign of senescence. Oddly enough, these observations have had little if any effect on general acceptance of the Hayflick limit in its original form. The absence of telomerase in cultured human cells and the shortening of telomeres at each population doubling have suggested that telomere length acts as a mitotic clock that accounts for their limited lifespan. This concept assumed an iconic character with the report that ectopic expression of telomerase by a vector greatly extended the lifespan of human cells. That something similar might occur in vivo seemed consistent with initial reports that most human somatic tissues lack telomerase activity. More careful study, however, has revealed telomerase activity in stem cells and some dividing transit cells of many renewing tissues and even in dividing myocytes of repairing cardiac muscle. It now seems likely that telomerase is active in vivo where and when it is needed to maintain tissue integrity. Caution is recommended in applying telomerase inhibition to kill telomerase-expressing cancer cells, because it would probably damage stem cells in essential organs and even increase the likelihood of secondary cancers. The risk may be especially high in sun-exposed skin, where there are usually thousands of p53-mutant clones of keratinocytes predisposed to cancer.

Cloning, biochemical characterization and expression of a sunflower (Helianthus annuus L.) hexokinase associated with seed storage compounds accumulation.

PubMed

Troncoso-Ponce, M A; Rivoal, J; Dorion, S; Moisan, M-C; Garcés, R; Martínez-Force, E

2011-03-01

A full-length hexokinase cDNA, HaHXK1, was cloned and characterized from Helianthus annuus L. developing seeds. Based on its sequence and phylogenetic relationships, HaHXK1 is a membrane-associated (type-B) hexokinase. The predicted structural model resembles known hexokinase structures, folding into two domains of unequal size: a large and a small one separated by a deep cleft containing the residues involved in the enzyme active site. A truncated version, without the 24 N-terminal residues, was heterologously expressed in Escherichia coli, purified to electrophoretic homogeneity using immobilized metal ion affinity chromatography and biochemically characterized. The purified enzyme behaved as a monomer on size exclusion chromatography and had a specific activity of 19.3 μmol/min/mg protein, the highest specific activity ever reported for a plant hexokinase. The enzyme had higher affinity for glucose and mannose relative to fructose, but the enzymatic efficiency was higher with glucose. Recombinant HaHXK1 was inhibited by ADP and was insensitive either to glucose-6-phosphate or to trehalose-6-phosphate. Its expression profile showed higher levels in heterotrophic tissues, developing seeds and roots, than in photosynthetic ones. A time course of HXK activity and expression in seeds showed that the highest HXK levels are found at the early stages of reserve compounds, lipids and proteins accumulation. Copyright © 2010 Elsevier GmbH. All rights reserved.

Molecular cloning and expression profile of an abiotic stress and hormone responsive MYB transcription factor gene from Panax ginseng.

PubMed

Afrin, Sadia; Zhu, Jie; Cao, Hongzhe; Huang, Jingjia; Xiu, Hao; Luo, Tiao; Luo, Zhiyong

2015-04-01

The v-myb avian myeloblastosis viral oncogene homolog (MYB) family constitutes one of the most abundant groups of transcription factors and plays vital roles in developmental processes and defense responses in plants. A ginseng (Panax ginseng C.A. Meyer) MYB gene was cloned and designated as PgMYB1. The cDNA of PgMYB1 is 762 base pairs long and encodes the R2R3-type protein consisting 238 amino acids. Subcellular localization showed that PgMYB1-mGFP5 fusion protein was specifically localized in the nucleus. To understand the functional roles of PgMYB1, we investigated the expression patterns of PgMYB1 in different tissues and under various conditions. Quantitative real-time polymerase chain reaction and western blot analysis showed that PgMYB1 was expressed at higher level in roots, leaves, and lateral roots than in stems and seeds. The expression of PgMYB1 was up-regulated by abscisic acid, salicylic acid, NaCl, and cold (chilling), and down-regulated by methyl jasmonate. These results suggest that PgMYB1 might be involved in responding to environmental stresses and hormones. © The Author 2015. Published by ABBS Editorial Office in association with Oxford University Press on behalf of the Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences.

A potyvirus vector efficiently targets recombinant proteins to chloroplasts, mitochondria and nuclei in plant cells when expressed at the amino terminus of the polyprotein.

PubMed

Majer, Eszter; Navarro, José-Antonio; Daròs, José-Antonio

2015-09-01

Plant virus-based expression systems allow quick and efficient production of recombinant proteins in plant biofactories. Among them, a system derived from tobacco etch virus (TEV; genus potyvirus) permits coexpression of equimolar amounts of several recombinant proteins. This work analyzed how to target recombinant proteins to different subcellular localizations in the plant cell using this system. We constructed TEV clones in which green fluorescent protein (GFP), with a chloroplast transit peptide (cTP), a nuclear localization signal (NLS) or a mitochondrial targeting peptide (mTP) was expressed either as the most amino-terminal product or embedded in the viral polyprotein. Results showed that cTP and mTP mediated efficient translocation of GFP to the corresponding organelle only when present at the amino terminus of the viral polyprotein. In contrast, the NLS worked efficiently at both positions. Viruses expressing GFP in the amino terminus of the viral polyprotein produced milder symptoms. Untagged GFPs and cTP and NLS tagged amino-terminal GFPs accumulated to higher amounts in infected tissues. Finally, viral progeny from clones with internal GFPs maintained the extra gene better. These observations will help in the design of potyvirus-based vectors able to coexpress several proteins while targeting different subcellular localizations, as required in plant metabolic engineering. Copyright © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Cloning and characterization of the Cerasus humilis sucrose phosphate synthase gene (ChSPS1)

PubMed Central

Du, Junjie; Mu, Xiaopeng; Wang, Pengfei

2017-01-01

Sucrose is crucial to the growth and development of plants, and sucrose phosphate synthase (SPS) plays a key role in sucrose synthesis. To understand the genetic and molecular mechanisms of sucrose synthesis in Cerasus humilis, ChSPS1, a homologue of SPS, was cloned using RT-PCR. Sequence analysis showed that the open reading frame (ORF) sequence of ChSPS1 is 3174 bp in length, encoding a predicted protein of 1057 amino acids. The predicted protein showed a high degree of sequence identity with SPS homologues from other species. Real-time RT-PCR analysis showed that ChSPS1 mRNA was detected in all tissues and the transcription level was the highest in mature fruit. There is a significant positive correlation between expression of ChSPS1 and sucrose content. Prokaryotic expression of ChSPS1 indicated that ChSPS1 protein was expressed in E. coli and it had the SPS activity. Overexpression of ChSPS1 in tobacco led to upregulation of enzyme activity and increased sucrose contents in transgenic plants. Real-time RT-PCR analysis showed that the expression of ChSPS1 in transgenic tobacco was significantly higher than in wild type plants. These results suggested that ChSPS1 plays an important role in sucrose synthesis in Cerasus humilis. PMID:29036229

Molecular cloning and tissue expression of the fatty acid-binding protein (Es-FABP) gene in female Chinese mitten crab (Eriocheir sinensis).

PubMed

Gong, Ya-Nan; Li, Wei-Wei; Sun, Jiang-Ling; Ren, Fei; He, Lin; Jiang, Hui; Wang, Qun

2010-09-16

Fatty acid-binding proteins (FABPs), small cytosolic proteins that function in the uptake and utilization of fatty acids, have been extensively studied in higher vertebrates while invertebrates have received little attention despite similar nutritional requirements during periods of reproductive activity. Therefore, a cDNA encoding Eriocheir sinensis FABP (Es-FABP) was cloned based upon EST analysis of a hepatopancreas cDNA library. The full length cDNA was 750 bp and encoded a 131 aa polypeptide that was highly homologous to related genes reported in shrimp. The 9108 bp Es-FABP gene contained four exons that were interrupted by three introns, a genomic organization common among FABP multigene family members in vertebrates. Gene expression analysis, as determined by RT-PCR, revealed the presence of Es-FABP transcripts in hepatopancreas, hemocytes, ovary, gills, muscle, thoracic ganglia, heart, and intestine, but not stomach or eyestalk. Real-time quantitative RT-PCR analysis revealed that Es-FABP expression in ovary, hemocytes, and hepatopancreas was dependent on the status of ovarian development, with peak expression observed in January. Evidence provided in the present report supports a role of Es-FABP in lipid transport during the period of rapid ovarian growth in E. sinensis, and indirectly confirms the participation of the hepatopancreas, ovary, and hemocytes in lipid nutrient absorption and utilization processes.

A plasmid toolkit for cloning chimeric cDNAs encoding customized fusion proteins into any Gateway destination expression vector

PubMed Central

2013-01-01

Background Valuable clone collections encoding the complete ORFeomes for some model organisms have been constructed following the completion of their genome sequencing projects. These libraries are based on Gateway cloning technology, which facilitates the study of protein function by simplifying the subcloning of open reading frames (ORF) into any suitable destination vector. The expression of proteins of interest as fusions with functional modules is a frequent approach in their initial functional characterization. A limited number of Gateway destination expression vectors allow the construction of fusion proteins from ORFeome-derived sequences, but they are restricted to the possibilities offered by their inbuilt functional modules and their pre-defined model organism-specificity. Thus, the availability of cloning systems that overcome these limitations would be highly advantageous. Results We present a versatile cloning toolkit for constructing fully-customizable three-part fusion proteins based on the MultiSite Gateway cloning system. The fusion protein components are encoded in the three plasmids integral to the kit. These can recombine with any purposely-engineered destination vector that uses a heterologous promoter external to the Gateway cassette, leading to the in-frame cloning of an ORF of interest flanked by two functional modules. In contrast to previous systems, a third part becomes available for peptide-encoding as it no longer needs to contain a promoter, resulting in an increased number of possible fusion combinations. We have constructed the kit’s component plasmids and demonstrate its functionality by providing proof-of-principle data on the expression of prototype fluorescent fusions in transiently-transfected cells. Conclusions We have developed a toolkit for creating fusion proteins with customized N- and C-term modules from Gateway entry clones encoding ORFs of interest. Importantly, our method allows entry clones obtained from ORFeome collections to be used without prior modifications. Using this technology, any existing Gateway destination expression vector with its model-specific properties could be easily adapted for expressing fusion proteins. PMID:23957834

A plasmid toolkit for cloning chimeric cDNAs encoding customized fusion proteins into any Gateway destination expression vector.

PubMed

Buj, Raquel; Iglesias, Noa; Planas, Anna M; Santalucía, Tomàs

2013-08-20

Valuable clone collections encoding the complete ORFeomes for some model organisms have been constructed following the completion of their genome sequencing projects. These libraries are based on Gateway cloning technology, which facilitates the study of protein function by simplifying the subcloning of open reading frames (ORF) into any suitable destination vector. The expression of proteins of interest as fusions with functional modules is a frequent approach in their initial functional characterization. A limited number of Gateway destination expression vectors allow the construction of fusion proteins from ORFeome-derived sequences, but they are restricted to the possibilities offered by their inbuilt functional modules and their pre-defined model organism-specificity. Thus, the availability of cloning systems that overcome these limitations would be highly advantageous. We present a versatile cloning toolkit for constructing fully-customizable three-part fusion proteins based on the MultiSite Gateway cloning system. The fusion protein components are encoded in the three plasmids integral to the kit. These can recombine with any purposely-engineered destination vector that uses a heterologous promoter external to the Gateway cassette, leading to the in-frame cloning of an ORF of interest flanked by two functional modules. In contrast to previous systems, a third part becomes available for peptide-encoding as it no longer needs to contain a promoter, resulting in an increased number of possible fusion combinations. We have constructed the kit's component plasmids and demonstrate its functionality by providing proof-of-principle data on the expression of prototype fluorescent fusions in transiently-transfected cells. We have developed a toolkit for creating fusion proteins with customized N- and C-term modules from Gateway entry clones encoding ORFs of interest. Importantly, our method allows entry clones obtained from ORFeome collections to be used without prior modifications. Using this technology, any existing Gateway destination expression vector with its model-specific properties could be easily adapted for expressing fusion proteins.

Molecular cloning and characterisation of banana fruit polyphenol oxidase.

PubMed

Gooding, P S; Bird, C; Robinson, S P

2001-09-01

Polyphenol oxidase (PPO; EC 1.10.3.2) is the enzyme thought to be responsible for browning in banana [Musa cavendishii (AAA group, Cavendish subgroup) cv. Williams] fruit. Banana flesh was high in PPO activity throughout growth and ripening. Peel showed high levels of activity early in development but activity declined until ripening started and then remained constant. PPO activity in fruit was not substantially induced after wounding or treatment with 5-methyl jasmonate. Banana flowers and unexpanded leaf roll had high PPO activities with lower activities observed in mature leaves, roots and stem. Four different PPO cDNA clones were amplified from banana fruit (BPO1, BPO11, BPO34 and BPO35). Full-length cDNA and genomic clones were isolated for the most abundant sequence (BPO1) and the genomic clone was found to contain an 85-bp intron. Introns have not been previously found in PPO genes. Northern analysis revealed the presence of BPO1 mRNA in banana flesh early in development but little BPO1 mRNA was detected at the same stage in banana peel. BPO11 transcript was only detected in very young flesh and there was no detectable expression of BPO34 or BPO35 in developing fruit samples. PPO transcripts were also low throughout ripening in both flesh and peel. BPO1 transcripts were readily detected in flowers, stem, roots and leaf roll samples but were not detected in mature leaves. BPO11 showed a similar pattern of expression to BPO1 in these tissues but transcript levels were much lower. BPO34 and BPO35 mRNAs were only detected at a low level in flowers and roots and BPO34 transcript was detected in mature leaves, the only clone to do so. The results suggest that browning of banana fruit during ripening results from release of pre-existing PPO enzyme, which is synthesised very early in fruit development.

[Expression of human-mouse chimeric antibody directed against Chikungunya virus with site-specific integration system].

PubMed

Li, Jian-min; Chen, Wei; Jia, Xiu-jie; An, Xiao-ping; Li, Bing; Fan, Ying-ru; Tong, Yi-gang

2005-05-01

To obtain CHO/dhfr(-) cells line with integrated FRT sequence in the chromosome transcription active site and to express human-mouse chimeric antibody directed against Chikungunya Virus by using the cell line. The fusion gene of FRT and HBsAg was constructed by PCR and cloned into the MCS of pCI-neo to construct pCI-FRT-HBsAg. The pCI-FRT-HBsAg was transfected into CHO/dhfr(-) cells and cell clones with high expression of HBsAg were screened by detecting the amount of HBsAg with ELISA. A CHO cell clone with the highest expression was chosen and named as CHO/dhfr(-) FRT(+). pAFRT HFLF, a expression plasmid of chimeric antibody with RFT sequence was transfected into CHO/dhfr(-) FRT(+) cells and cell clones with high expression of the chimeric antibody were screened by increasing concentration of MTX. A CHO cell clone with high expression of the chimeric antibody was cultured in large scale and supernatant was collected from which the chimeric antibody was purified. The purified chimeric antibody was analyzed by SDS-PAGE, Western blot and IFA. A CHO/dhfr(-) cells line with integrated FRT sequence in the chromosome transcription active site was obtained successfully. A cell clone with yield of 5 mg/L of chimeric antibody was obtained, as compared with routine CHO cell expression system with a yield of 2 mg/L. A cell line with integrated FRT sequence in the chromosome transcription active site was obtained and with it human-mouse chimeric antibody directed against Chikungunya virus was expressed. This system lays a solid foundation which can be used for expressing antibodies and other proteins.

Mammalian cDNA Library from the NIH Mammalian Gene Collection (MGC) | Office of Cancer Genomics

Cancer.gov

The MGC provides the research community full-length clones for most of the defined (as of 2006) human and mouse genes, along with selected clones of cow and rat genes. Clones were designed to allow easy transfer of the ORF sequences into nearly any type of expression vector. MGC provides protein ‘expression-ready’ clones for each of the included human genes. MGC is part of the ORFeome Collaboration (OC).

Expression of proteinase inhibitor II proteins during floral development in Solanum americanum.

PubMed

Sin, Suk-Fong; Chye, Mee-Len

2004-10-01

The heterologous expression of serine proteinase inhibitor II (PIN2) proteins confers insect resistance in transgenic plants, but little is known of their endogenous roles. We have cloned two cDNAs encoding Solanum americanum PIN2 proteins, SaPIN2a and SaPIN2b. SaPIN2a is highly expressed in stem, particularly in the phloem, suggesting it could possibly regulate proteolysis in the sieve elements. When SaPIN2a was expressed in transgenic lettuce, we observed an inhibition of endogenous trypsin- and chymotrypsin-like activities. Here, we demonstrate that both SaPIN2a and SaPIN2b are expressed in floral tissues that are destined to undergo developmental programmed cell death (PCD), suggesting possible endogenous roles in inhibiting trypsin- and chymotrypsin-like activities during flower development. Northern and western blot analyses revealed that SaPIN2a and SaPIN2b mRNAs and proteins show highest expression early in floral development. In situ hybridization analysis and immunolocalization on floral sections, localized SaPIN2a and SaPIN2b mRNAs and their proteins to tissues that would apparently undergo PCD: the ovules, the stylar transmitting tissue, the stigma and the vascular bundles. Detection of PCD in floral sections was achieved using terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL) analysis. Examination of the mid-style before, and 1 day after, pollination revealed that high expression of SaPIN2a and SaPIN2b in the style was inversely correlated with PCD.

Cloning, localization and expression analysis of vacuolar sugar transporters in the CAM plant Ananas comosus (pineapple).

PubMed

Antony, Edna; Taybi, Tahar; Courbot, Mikaël; Mugford, Sam T; Smith, J Andrew C; Borland, Anne M

2008-01-01

In photosynthetic tissues of the CAM plant pineapple (Ananas comosus), storage of soluble sugars in the central vacuole during the daytime and their remobilization at night is required to provide carbon skeletons for nocturnal CO(2) fixation. However, soluble sugars produced photosynthetically must also be exported to support growth processes in heterotrophic tissues. To begin to address how vacuolar sugar storage and assimilate partitioning are regulated in A. comosus, degenerate PCR and cDNA library screening were used to clone three candidate sugar transporters from the leaves of this species. Subcellular localization of the three transporters was investigated via expression of YFP-fusion proteins in tobacco epidermal cells and their co-localization with subcellular markers by confocal microscopy. Using this strategy, a putative hexose transporter (AcMST1) and a putative inositol transporter (AcINT1) were identified that both localized to the tonoplast, whereas a putative sucrose transporter (AcSUT1) was found to localize to prevacuolar compartments. A cDNA (AcMST2) with high similarity to a recently characterized tonoplast hexose transporter in Arabidopsis was also identified from an A. comosus fruit EST database. Analyses of transcript abundance indicated that AcMST1 was more highly expressed in fruits compared to leaves of A. comosus, whilst transcripts of AcINT1, AcSUT1, and AcMST2 were more abundant in leaves. Transcript abundance of AcINT1, the putative inositol transporter, showed day-night changes comparable to those of other CAM-related transcripts described in Mesembryanthemum crystallinum. The results are discussed in terms of the role of vacuolar sugar transporters in regulating carbon flow during the diel cycle in CAM plants.

Cloning and characterization of the SERK1 gene in triploid Pingyi Tiancha [Malus hupehensis (Pamp.) Rehd. var. pingyiensis Jiang] and a tetraploid hybrid strain.

PubMed

Zhang, L J; Dong, W X; Guo, S M; Wang, Y X; Wang, A D; Lu, X J

2015-11-19

This study aims to explore the roles of somatic embryogenesis receptor-like kinase (SERK) in Malus hupehensis (Pingyi Tiancha). The full-length sequences of SERK1 in triploid Pingyi Tiancha (3n) and a tetraploid hybrid strain 33# (4n) were cloned, sequenced, and designated as MhSERK1 and MhdSERK1, respectively. Multiple alignments of amino acid sequences were conducted to identify similarity between MhSERK1 and MhdSERK1 and SERK sequences in other species, and a neighbor-joining phylogenetic tree was constructed to elucidate their phylogenetic relations. Expression levels of MhSERK1 and MhdSERK1 in different tissues and developmental stages were investigated using quantitative real-time PCR. The coding sequence lengths of MhSERK1 and MhdSERK1 were 1899 bp (encoding 632 amino acids) and 1881 bp (encoding 626 amino acids), respectively. Sequence analysis demonstrated that MhSERK1 and MhdSERK1 display high similarity to SERKs in other species, with a conserved intron/exon structure that is unique to members of the SERK family. Additionally, the phylogenetic tree showed that MhSERK1 and MhdSERK1 clustered with orange CitSERK (93%). Furthermore, MhSERK1 and MhdSERK1 were mainly expressed in the reproductive organs, in particular the ovary. Their expression levels were highest in young flowers and they differed among different tissues and organs. Our results suggest that MhSERK1 and MhdSERK1 are related to plant reproduction, and that MhSERK1 is related to apomixis in triploid Pingyi Tiancha.

DNA methylation profiles of donor nuclei cells and tissues of cloned bovine fetuses.

PubMed

Kremenskoy, Maksym; Kremenska, Yuliya; Suzuki, Masako; Imai, Kei; Takahashi, Seiya; Hashizume, Kazuyoshi; Yagi, Shintaro; Shiota, Kunio

2006-04-01

Methylation of DNA in CpG islands plays an important role during fetal development and differentiation because CpG islands are preferentially located in upstream regions of mammalian genomic DNA, including the transcription start site of housekeeping genes and are also associated with tissue-specific genes. Somatic nuclear transfer (NT) technology has been used to generate live clones in numerous mammalian species, but only a low percentage of nuclear transferred animals develop to term. Abnormal epigenetic changes in the CpG islands of donor nuclei after nuclear transfer could contribute to a high rate of abortion during early gestation and increase perinatal death. These changes have yet to be explored. Thus, we investigated the genome-wide DNA methylation profiles of CpG islands in nuclei donor cells and NT animals. Using Restriction Landmark Genomic Scanning (RLGS), we showed, for the first time, the epigenetic profile formation of tissues from NT bovine fetuses produced from cumulus cells. From approximately 2600 unmethylated NotI sites visualized on the RLGS profile, at least 35 NotI sites showed different methylation statuses. Moreover, we proved that fetal and placental tissues from artificially inseminated and cloned cattle have tissue-specific differences in the genome-wide methylation profiles of the CpG islands. We also found that possible abnormalities occurred in the fetal brain and placental tissues of cloned animals.

Optimization of in vitro inhibition of HT-29 colon cancer cell cultures by Solanum tuberosum L. extracts.

PubMed

Zuber, T; Holm, D; Byrne, P; Ducreux, L; Taylor, M; Kaiser, M; Stushnoff, C

2015-01-01

Secondary metabolites in potato have been reported to possess bioactive properties, including growth inhibition of cancer cells. Because potatoes are widely consumed globally, potential health benefits may have broad application. Thus we investigated growth inhibition of HT-29 colon cancer cell cultures by extracts from 13 diverse genetic breeding clones. Extracts from three pigmented selections (CO97226-2R/R, CO97216-1P/P, CO04058-3RW/RW) inhibited growth of in vitro HT-29 cell cultures more effectively than other clones tested. While inhibition was highest from pigmented selections and pigmented tuber tissue sectors, not all pigmented breeding lines tested had appreciable inhibitory properties. Thus, inhibition was not uniquely linked to pigmentation. Immature tubers had the highest inhibitory properties, and in most cases mature tubers retained very low inhibition properties. Flowers and skins inhibited strongly at lower extract concentrations. An extract consisting of 7.2 mg mL⁻¹ cell culture medium was the lowest effective concentration. While raw tuber extracts inhibited most effectively, a few clones at higher concentrations retained inhibition after cooking. Heated whole tubers retained higher inhibition than heated aqueous extracts. While all aqueous extracts from the two tuber selections (CO97216-1P/P and CO97226-2R/R) inhibited HT-29 cell cultures, inhibition was significantly enhanced in purple pigmented tubers of CO97216-1P/P prepared cryogenically as liquid nitrogen powders compared to extracts from freeze dried samples. Upregulation of caspase-3 protease activity, indicative of apoptosis, was highest among the most inhibitory clone samples. The unique sectorial red pigment expressing selection (CO04058-3RW/RW) provided a model system that isolated expression in pigmented sectors, and thus eliminated developmental, environmental and genetic confounding.

Genistein promotes DNA demethylation of the steroidogenic factor 1 (SF-1) promoter in endometrial stromal cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Matsukura, Hiroshi, E-mail: hmatsukura.epi@mri.tmd.ac.jp; Aisaki, Ken-ichi; Igarashi, Katsuhide

2011-08-26

Highlights: {yields} Genistein (GEN) is a phytoestrogen found in soy products. {yields} GEN demethylated/unsilenced the steroidogenic factor 1 gene in endometrial tissue. {yields} GEN thus altered mRNA expression in uteri of ovariectomized (OVX) mice. {yields} A high-resolution melting assay was used to screen for epigenetic change. {yields} We isolated an endometrial cell clone that was epigenetically modulated by GEN. -- Abstract: It has recently been demonstrated that genistein (GEN), a phytoestrogen in soy products, is an epigenetic modulator in various types of cells; but its effect on endometrium has not yet been determined. We investigated the effects of GEN onmore » mouse uterine cells, in vivo and in vitro. Oral administration of GEN for 1 week induced mild proliferation of the endometrium in ovariectomized (OVX) mice, which was accompanied by the induction of steroidogenic factor 1 (SF-1) gene expression. GEN administration induced demethylation of multiple CpG sites in the SF-1 promoter; these sites are extensively methylated and thus silenced in normal endometrium. The GEN-mediated promoter demethylation occurred predominantly on the luminal side, as opposed to myometrium side, indicating that the epigenetic change was mainly shown in regenerated cells. Primary cultures of endometrial stromal cell colonies were screened for GEN-mediated alterations of DNA methylation by a high-resolution melting (HRM) method. One out of 20 colony-forming cell clones showed GEN-induced demethylation of SF-1. This clone exhibited a high proliferation capacity with continuous colony formation activity through multiple serial clonings. We propose that only a portion of endometrial cells are capable of receiving epigenetic modulation by GEN.« less

Simple cloning strategy using GFPuv gene as positive/negative indicator.

PubMed

Miura, Hiromi; Inoko, Hidetoshi; Inoue, Ituro; Tanaka, Masafumi; Sato, Masahiro; Ohtsuka, Masato

2011-09-15

Because construction of expression vectors is the first requisite in the functional analysis of genes, development of simple cloning systems is a major requirement during the postgenomic era. In the current study, we developed cloning vectors for gain- or loss-of-function studies by using the GFPuv gene as a positive/negative indicator of cloning. These vectors allow us to easily detect correct clones and obtain expression vectors from a simple procedure by means of the combined use of the GFPuv gene and a type IIS restriction enzyme. Copyright © 2011 Elsevier Inc. All rights reserved.

Cloning of two individual cDNAS encoding 9-cis-epoxycarotenoid dioxygenase from Gentiana lutea, their tissue-specific expression and physiological effect in transgenic tobacco.

PubMed

Zhu, Changfu; Kauder, Friedrich; Römer, Susanne; Sandmann, Gerhard

2007-02-01

Two 9-cis-epoxycarotenoid dioxygenase (NCED) cDNAs have been cloned from a petal library of Gentiana lutea. Both cDNAs carry a putative transit sequence for chloroplast import and differ mainly in their length and the 5'-flanking regions. GlNCED1 was evolutionary closely related to Arabidopsis thaliana NCED6 whereas GlNCED2 showed highest homology to tomato NCED1 and A. thaliana NCED3. The amounts of GlNCED2 transcript were below Northern detection in G. lutea. In contrast, GlNCED1 was specifically expressed at higher levels in developing flowers when petals start appearing. By genetic engineering of tobacco with coding regions of either gene under a constitutive promoter, their function was further analyzed. Although mRNA of both genes was detectable in the corresponding transgenic plants, a physiological effect was only found for GlNCED1 but not for GlNCED2. In germination experiments of GlNCED1 transgenic lines, delayed radicle formation and cotyledon appearance were observed. However, the transformants exhibited no improved tolerance against desiccation stress. In contrast to other plants with over-expressed NCEDs, prolonged delay of seed germination is the only abscisic-acid-related phenotypic effect in the GlNCED1 transgenic lines.

Cloning of a MADS box gene (GhMADS3) from cotton and analysis of its homeotic role in transgenic tobacco.

PubMed

Guo, Yulong; Zhu, Qinlong; Zheng, Shangyong; Li, Mingyang

2007-06-01

A MADS box gene (GhMADS3) was cloned from cotton (Gossypium hirsutum L.) based on EST sequences. The predicted protein sequence of GhMADS3 showed 85%, 73%, and 62% identity with Theobroma cacao TcAG, Antirrhinum majus FAR, and Arabidopsis thaliana AG, respectively, and was grouped with AG homologues when the full length sequences excluding N-extensions were compared. GhMADS3 expressed in the wild type cotton flower primarily in stamens and carpels, which was comparable to AG in Arabidopsis. However, it was not expressed in floral buds of a homeotic cotton variant chv1. Ectopic expression of GhMADS3 in tobacco (Nicotiana tabacum L.) resulted in flowers with sepal-to-carpel and petal-to-stamen transformation. The carpelloid first whorl organs, with stigmatic tissue on their upper edges, had a white appearance when compared with the dark green color of the wild type sepals. At times, long filaments were observed at the fusion site of the first carpelloid oranges. The second whorl organs in staminoid were usually smaller than the wild type and the color was changed from pink to white. These results suggest that GhMADS3 has a homeotic role in flower development.

Molecular Cloning and Characterization of G Alpha Proteins from the Western Tarnished Plant Bug, Lygus hesperus

PubMed Central

Hull, J. Joe; Wang, Meixian

2014-01-01

The Gα subunits of heterotrimeric G proteins play critical roles in the activation of diverse signal transduction cascades. However, the role of these genes in chemosensation remains to be fully elucidated. To initiate a comprehensive survey of signal transduction genes, we used homology-based cloning methods and transcriptome data mining to identity Gα subunits in the western tarnished plant bug (Lygus hesperus Knight). Among the nine sequences identified were single variants of the Gαi, Gαo, Gαs, and Gα12 subfamilies and five alternative splice variants of the Gαq subfamily. Sequence alignment and phylogenetic analyses of the putative L. hesperus Gα subunits support initial classifications and are consistent with established evolutionary relationships. End-point PCR-based profiling of the transcripts indicated head specific expression for LhGαq4, and largely ubiquitous expression, albeit at varying levels, for the other LhGα transcripts. All subfamilies were amplified from L. hesperus chemosensory tissues, suggesting potential roles in olfaction and/or gustation. Immunohistochemical staining of cultured insect cells transiently expressing recombinant His-tagged LhGαi, LhGαs, and LhGαq1 revealed plasma membrane targeting, suggesting the respective sequences encode functional G protein subunits. PMID:26463065

A protein that interacts with members of the nuclear hormone receptor family: identification and cDNA cloning.

PubMed Central

Zeiner, M; Gehring, U

1995-01-01

In search of proteins which interact with activated steroid hormone receptors, we screened a human liver lambda gt11 expression library with the glucocorticoid receptor. We identified and cloned a cDNA sequence of 1322 bp that encodes a protein of 274 aa. This protein consists predominantly of hydrophilic amino acids and contains a putative bipartite nuclear localization signal. The in vitro translated receptor-associating protein runs in SDS/polyacrylamide gels with an apparent molecular mass of 46 kDa. By use of the bacterially expressed fusion protein with glutathione S-transferase we have found that interaction is not limited to the glucocorticoid receptor but included other nuclear receptors--most notably, the estrogen and thyroid receptors. Binding also occurs with the glucocorticoid receptor complexed with the antiglucocorticoid RU 38486, with the estrogen receptor complexed with the antiestrogen 4-hydroxytamoxifen or ICI 164,384, and even with receptors not complexed with ligand. Association with steroid hormone receptors depends on prior receptor activation--i.e., release from heat shock proteins. The sequence identified here appears to be a general partner protein for nuclear hormone receptors, with the gene being expressed in a variety of mammalian tissues. Images Fig. 2 Fig. 3 Fig. 4 PMID:8524784

Molecular cloning and functional characterization of duck nucleotide-binding oligomerization domain 1 (NOD1).

PubMed

Li, Huilin; Jin, Hui; Li, Yaqian; Liu, Dejian; Foda, Mohamed Frahat; Jiang, Yunbo; Luo, Rui

2017-09-01

Nucleotide-binding oligomerization domain 1 (NOD1) is an imperative cytoplasmic pattern recognition receptor (PRR) and considered as a key member of the NOD-like receptor (NLR) family which plays a critical role in innate immunity through sensing microbial components derived from bacterial peptidoglycan. In the current study, the full-length of duck NOD1 (duNOD1) cDNA from duck embryo fibroblasts (DEFs) was cloned. Multiple sequence alignment and phylogenetic analysis demonstrated that duNOD1 exhibited a strong evolutionary relationship with chicken and rock pigeon NOD1. Tissue-specific expression analysis showed that duNOD1 was widely distributed in various organs, with the highest expression observed in the liver. Furthermore, duNOD1 overexpression induced NF-κB activation in DEFs and the CARD domain is crucial for duNOD1-mediated NF-κB activation. In addition, silencing the duNOD1 decreased the activity of NF-κB in DEFs stimulated by iE-DAP. Overexpression of duNOD1 significantly increased the expression of TNF-α, IL-6, and RANTES in DEFs. These findings highlight the crucial role of duNOD1 as an intracellular sensor in duck innate immune system. Copyright © 2017 Elsevier Ltd. All rights reserved.

The smallest natural high-active luciferase: cloning and characterization of novel 16.5-kDa luciferase from copepod Metridia longa.

PubMed

Markova, Svetlana V; Larionova, Marina D; Burakova, Ludmila P; Vysotski, Eugene S

2015-01-30

Coelenterazine-dependent copepod luciferases containing natural signal peptide for secretion are a very convenient analytical tool as they enable monitoring of intracellular events with high sensitivity, without destroying cells or tissues. This property is well suited for application in biomedical research and development of cell-based assays for high throughput screening. We report the cloning of cDNA gene encoding a novel secreted non-allelic 16.5-kDa isoform (MLuc7) of Metridia longa luciferase, which, in fact, is the smallest natural luciferase of known for today. Despite the small size, isoform contains 10 conservative Cys residues suggesting the presence of up to 5 SS bonds. This hampers the efficient production of functionally active recombinant luciferase in bacterial expression systems. With the use of the baculovirus expression system, we produced substantial amounts of the proper folded MLuc7 luciferase with a yield of ∼3 mg/L of a high purity protein. We demonstrate that MLuc7 produced in insect cells is highly active and extremely thermostable, and is well suited as a secreted reporter when expressed in mammalian cells ensuring higher sensitivity of detection as compared to another Metridia luciferase isoform (MLuc164) which is widely employed in real-time imaging. Copyright © 2014 Elsevier Inc. All rights reserved.

Cloning, Expression, and Purification of Brucella suis Outer Membrane Proteins

DTIC Science & Technology

2005-01-01

13-09-20061 4. TITLE AND SUBTITLE 5a. CONTRACT NUMBER Cloning, expression and purification of Brucella suis outer membrane proteins 5b. GRANT NUMBER...attractive for this purpose. In this study, we cloned, expressed and purified seven predicted OMPs of Brucella suis . The recombinant proteins were...fused with 6-his and V5 epitope tags at their C termini to facilitate detection and purification. The B. suis surface genes were PCR synthesized based

A reassessment of IgM memory subsets in humans

PubMed Central

Bagnara, Davide; Squillario, Margherita; Kipling, David; Mora, Thierry; Walczak, Aleksandra M.; Da Silva, Lucie; Weller, Sandra; Dunn-Walters, Deborah K.; Weill, Jean-Claude; Reynaud, Claude-Agnès

2015-01-01

From paired blood and spleen samples from three adult donors we performed high-throughput V-h sequencing of human B-cell subsets defined by IgD and CD27 expression: IgD+CD27+ (“MZ”), IgD−CD27+(“memory”, including IgM (“IgM-only”), IgG and IgA) and IgD−CD27− cells (“double-negative”, including IgM, IgG and IgA). 91,294 unique sequences clustered in 42,670 clones, revealing major clonal expansions in each of these subsets. Among these clones, we further analyzed those shared sequences from different subsets or tissues for Vh-gene mutation, H-CDR3-length, and Vh/Jh usage, comparing these different characteristics with all sequences from their subset of origin, for which these parameters constitute a distinct signature. The IgM-only repertoire profile differed notably from that of MZ B cells by a higher mutation frequency, and lower Vh4 and higher Jh6 gene usage. Strikingly, IgM sequences from clones shared between the MZ and the memory IgG/IgA compartments showed a mutation and repertoire profile of IgM-only and not of MZ B cells. Similarly, all IgM clonal relationships (between MZ, IgM-only, and double-negative compartments) involved sequences with the characteristics of IgM-only B cells. Finally, clonal relationships between tissues suggested distinct recirculation characteristics between MZ and switched B cells. The “IgM-only” subset (including cells with its repertoire signature but higher IgD or lower CD27 expression levels) thus appear as the only subset showing precursor-product relationships with CD27+ switched memory B cells, indicating that they represent germinal center-derived IgM memory B cells, and that IgM memory and MZ B cells constitute two distinct entities. PMID:26355154

A Reassessment of IgM Memory Subsets in Humans.

PubMed

Bagnara, Davide; Squillario, Margherita; Kipling, David; Mora, Thierry; Walczak, Aleksandra M; Da Silva, Lucie; Weller, Sandra; Dunn-Walters, Deborah K; Weill, Jean-Claude; Reynaud, Claude-Agnès

2015-10-15

From paired blood and spleen samples from three adult donors, we performed high-throughput VH sequencing of human B cell subsets defined by IgD and CD27 expression: IgD(+)CD27(+) ("marginal zone [MZ]"), IgD(-)CD27(+) ("memory," including IgM ["IgM-only"], IgG and IgA) and IgD(-)CD27(-) cells ("double-negative," including IgM, IgG, and IgA). A total of 91,294 unique sequences clustered in 42,670 clones, revealing major clonal expansions in each of these subsets. Among these clones, we further analyzed those shared sequences from different subsets or tissues for VH gene mutation, H-CDR3-length, and VH/JH usage, comparing these different characteristics with all sequences from their subset of origin for which these parameters constitute a distinct signature. The IgM-only repertoire profile differed notably from that of MZ B cells by a higher mutation frequency and lower VH4 and higher JH6 gene usage. Strikingly, IgM sequences from clones shared between the MZ and the memory IgG/IgA compartments showed a mutation and repertoire profile of IgM-only and not of MZ B cells. Similarly, all IgM clonal relationships (among MZ, IgM-only, and double-negative compartments) involved sequences with the characteristics of IgM-only B cells. Finally, clonal relationships between tissues suggested distinct recirculation characteristics between MZ and switched B cells. The "IgM-only" subset (including cells with its repertoire signature but higher IgD or lower CD27 expression levels) thus appear as the only subset showing precursor-product relationships with CD27(+) switched memory B cells, indicating that they represent germinal center-derived IgM memory B cells and that IgM memory and MZ B cells constitute two distinct entities. Copyright © 2015 by The American Association of Immunologists, Inc.

Members of the aquaporin family in the developing pea seed coat include representatives of the PIP, TIP, and NIP subfamilies.

PubMed

Schuurmans, Jolanda A M J; van Dongen, Joost T; Rutjens, Bas P W; Boonman, Alex; Pieterse, Corné M J; Borstlap, Adrianus C

2003-11-01

Water and nutrients required by developing seeds are mainly supplied by the phloem and have to be released from a maternal parenchyma tissue before being utilized by the filial tissues of embryo and endosperm. To identify aquaporins that could be involved in this process four full-length cDNAs were cloned and sequenced from a cDNA library of developing seed coats of pea (Pisum sativum L.). The cDNA of PsPIP1-1 appeared to be identical to that of clone 7a/TRG-31, a turgor-responsive gene cloned previously from pea roots. PsPIP1-1, PsPIP2-1, and PsTIP1-1, or their possible close homologues, were also expressed in cotyledons of developing and germinating seeds, and in roots and shoots of seedlings, but transcripts of PsNIP-1 were only detected in the seed coat. In mature dry seeds, high hybridization signals were observed with the probe for PsPIP1-1, but transcripts of PsPIP2-1, PsTIP1-1, and PsNIP-1 were not detected. Functional characterization after heterologous expression in Xenopus oocytes showed that PsPIP2-1 and PsTIP1-1 are aquaporins whereas PsNIP-1 is an aquaglyceroporin. PsNIP-1, like several other NIPs, contains a tryptophan residue corresponding with Trp-48 in GlpF (the glycerol facilitator of Escherichia coli) that borders the selectivity filter in the permeation channel. It is suggested that PsPIP1-1 and/or its possible close homologues could play a role in water absorption during seed imbibition, and that PsPIP2-1, possibly together with PsPIP1-1, could be involved in the release of phloem water from the seed coat symplast, which is intimately connected with the release of nutrients for the embryo.

cDNA cloning and expression of carotenogenic genes during flower development in Gentiana lutea.

PubMed

Zhu, Changfu; Yamamura, Saburo; Koiwa, Hiroyuki; Nishihara, Masashiro; Sandmann, Gerhard

2002-02-01

All cDNAs involved in carotenoid biosynthesis leading to lycopene in yellow petals of Gentiana lutea have been cloned from a cDNA library. They encode a geranylgeranyl pyrophosphate synthase, a phytoene synthase, a phytoene desaturase and a zeta-carotene desaturase. The indicated function of all cDNAs was established by heterologous complementation in Escherichia coli. The amino acid sequences deduced from the cDNAs were between 47.5% and 78.9% identical to those reported for the corresponding enzymes from other higher plants. Southern analysis suggested that the genes for each enzyme probably represent a small multi-gene family. Tissue-specific expression of the genes and expression during flower development was investigated. The expression of the phytoene synthase gene, psy, was enhanced in flowers but transcripts were not detected in stems and leaves by northern blotting. Transcripts of the genes for geranylgeranyl pyrophosphate (ggpps), phytoene desaturase (pds) and zeta-carotene desaturase (zds) were detected in flowers and leaves but not in stems. Analysis of the expression of psy and zds in petals revealed that levels of the transcripts were lowest in young buds and highest in fully open flowers, in parallel with the formation of carotenoids. Obviously, the transcription of these genes control the accumulation of carotenoids during flower development in G. lutea. For pds only a very slight increase of mRNA was found whereas the transcripts of ggpps decreased during flower development.

Orange-spotted grouper (Epinephelus coioides) orexin: molecular cloning, tissue expression, ontogeny, daily rhythm and regulation of NPY gene expression.

PubMed

Yan, Aifen; Zhang, Lingjuang; Tang, Zhiguo; Zhang, Yanhong; Qin, Chaobin; Li, Bo; Li, Wensheng; Lin, Haoran

2011-07-01

Orexin-A and -B, collectively called orexins, are hypothalamic neuropeptides involved in the regulation of food intake, sleep and energy balance. In this study, the full-length cDNA of prepro-orexin was isolated from the hypothalamus of orange-spotted grouper (Epinephelus coioides) using RT-PCR and RACE. The grouper prepro-orexin cDNA is 711 bp in length and encodes a 149-amino acid precursor protein that contains a 46-amino acid signal peptide, a 43-amino acid mature orexin-A peptide, a 27-amino acid mature orexin-B peptide and a 33-amino acid C terminus of unknown function. The tissue distribution and ontogeny of prepro-orexin were examined by quantitative real-time PCR. We found that the prepro-orexin mRNA is widely expressed in brain and peripheral tissues, with abundant expression in the hypothalamus. During the embryonic development, prepro-orexin mRNA was first detected in neurula stage embryos, and its expression gradually increased during the remainder of embryogenesis. Our analysis of grouper hypothalamic prepro-orexin expression showed that prepro-orexin mRNA levels were greater in the light phase than in the dark phase and increased significantly at meal-time. Intraperitoneal injection of orexin-A caused a dose-related increase in hypothalamus NPY mRNA expression level after 4h. Orexin-A also increased NPY mRNA expression level from static hypothalamic fragments incubation. Our results imply that orexin may be involved in feeding in the orange-spotted grouper and orexin-A is a stimulator of NPY mRNA expression in vivo and in vitro. Copyright © 2011 Elsevier Inc. All rights reserved.

Comparative study of angiostatic and anti-invasive gene expressions as prognostic factors in gastric cancer.

PubMed

Lee, J H; Koh, J T; Shin, B A; Ahn, K Y; Roh, J H; Kim, Y J; Kim, K K

2001-02-01

Genes involving angiogenesis and metastasis play an important role in the progression and infiltration of cancer. We examined the expressions of various angiostatic and potential invasion/metastasis suppressor genes through RT-PCR analyses in 32 gastric cancer specimens with or without distant metastasis. The expressions of the invasion/metastasis suppressor, nm23 and E-cadherin increased much more in the cancer tissue (CT) and metastatic lymph node (MLN) than in the extraneoplastic mucosa (EM) and non-metastatic lymph node (NLN), respectively. The expressions of the angiostatic factor, angiopoietin 2 and thrombospondin 2 increased in the CT and MLN as compared with the EM and NLN, respectively. The newly cloned angiostatic factor, brain-specific angiogenesis inhibitor 1 (BAI1) decreased much more in the CT and MLN than the EM and NLN, respectively. However, BAI1 increased in the CT compared with the EM among the patients with poor prognosis and distant metastasis, such as liver or peritoneum. The expressions of the invasive factor, matrix metalloproteinase-2 and its suppressor, tissue inhibitor metalloproteinase-2 (TIMP-2) increased in the CM as compared with the EM, but the increased expression pattern of these genes in the CT became blunted among the patients with good prognosis. Our results indicate that BAI1 and TIMP-2 expressions in the extraneoplastic mucosa and non-metastatic lymph nodes were not suppressed in the patients with good prognosis, but increased expressions of angiopoietin 2, thrombospondin 2, TIMP-2, nm23 and E-cadherin in the tumor tissue did not lead to a long survival after operation. It is suggested that the extent of BAI1 and TIMP-2 expression in the gastric mucosa may be an important prognostic factor for predicting survival in gastric cancer.

Expression of spicule matrix protein gene SM30 in embryonic and adult mineralized tissues of sea urchin Hemicentrotus pulcherrimus

NASA Technical Reports Server (NTRS)

Kitajima, T.; Tomita, M.; Killian, C. E.; Akasaka, K.; Wilt, F. H.

1996-01-01

We have isolated a cDNA clone for spicule matrix protein, SM30, from sea urchin Hemicentrotus pulcherrimus and have studied the expression of this gene in comparison with that of another spicule matrix protein gene, SM50. In cultured micromeres as well as in intact embryos transcripts of SM30 were first detectable around the onset of spicule formation and rapidly increased with the growth of spicules, which accompanied accumulation of glycosylated SM30 protein(s). When micromeres were cultured in the presence of Zn2+, spicule formation and SM30 expression were suppressed, while both events resumed concurrently after the removal of Zn2+ from the culture medium. Expression of SM50, in contrast, started before the appearance of spicules and was not sensitive to Zn2+. Differences were also observed in adult tissues; SM30 mRNA was detected in spines and tube feet but not in the test, while SM50 mRNA was apparent in all of these mineralized tissues at similar levels. These results strongly suggest that the SM30 gene is regulated by a different mechanism to that of the SM50 gene and that the products of these two genes are differently involved in sea urchin biomineralization. A possible role of SM30 protein in skeleton formation is discussed.

Expression of spicule matrix protein gene SM30 in embryonic and adult mineralized tissues of sea urchin Hemicentrotus pulcherrimus.

PubMed

Kitajima, T; Tomita, M; Killian, C E; Akasaka, K; Wilt, F H

1996-12-01

We have isolated a cDNA clone for spicule matrix protein, SM30, from sea urchin Hemicentrotus pulcherrimus and have studied the expression of this gene in comparison with that of another spicule matrix protein gene, SM50. In cultured micromeres as well as in intact embryos transcripts of SM30 were first detectable around the onset of spicule formation and rapidly increased with the growth of spicules, which accompanied accumulation of glycosylated SM30 protein(s). When micromeres were cultured in the presence of Zn2+, spicule formation and SM30 expression were suppressed, while both events resumed concurrently after the removal of Zn2+ from the culture medium. Expression of SM50, in contrast, started before the appearance of spicules and was not sensitive to Zn2+. Differences were also observed in adult tissues; SM30 mRNA was detected in spines and tube feet but not in the test, while SM50 mRNA was apparent in all of these mineralized tissues at similar levels. These results strongly suggest that the SM30 gene is regulated by a different mechanism to that of the SM50 gene and that the products of these two genes are differently involved in sea urchin biomineralization. A possible role of SM30 protein in skeleton formation is discussed.

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

PubMed

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.

Cloning of human cDNA encoding a novel heptahelix receptor expressed in Burkitt's lymphoma and widely distributed in brain and peripheral tissues.

PubMed

Owman, C; Blay, P; Nilsson, C; Lolait, S J

1996-11-12

Using PCR with degenerate primers and screening of a human B-cell lymphoblast cDNA library, a full-length cDNA encoding a 375-amino-acid protein was isolated. It contains seven regions of hydrophobic amino acids probably representing membrane-spanning domains of a novel heptahelix receptor, tentatively named CMKRL2. It shows nearly 30% overall identity with the high-affinity IL8 receptor and similar degree of homology with other chemoattractant receptors, including the "fusin" coreceptors for HIV1. Measurements of various transduction pathways following application of a panel of chemokines to transfected cells failed to evoke any reproducible response. Although the natural ligand for CMKRL2 could, thus, not be identified, receptor expression in spleen and lymph nodes as well as in Burkitt's lymphoma (irrespective of EBV status) supports a functional role in activated B-cells. Receptor message was ubiquitously distributed in normal peripheral tissues and CNS, suggesting that CMKRL2 is expressed in widespread cell populations, such as macrophages and neuroglia.

Maintenance of mitochondrial DNA copy number and expression are essential for preservation of mitochondrial function and cell growth.

PubMed

Jeng, Jaan-Yeh; Yeh, Tien-Shun; Lee, Jing-Wen; Lin, Shyh-Hsiang; Fong, Tsorng-Han; Hsieh, Rong-Hong

2008-02-01

To examine whether a reduction in the mtDNA level will compromise mitochondrial biogenesis and mitochondrial function, we created a cell model with depleted mtDNA. Stable transfection of small interfering (si)RNA of mitochondrial transcription factor A (Tfam) was used to interfere with Tfam gene expression. Selected stable clones showed 60-95% reduction in Tfam gene expression and 50-90% reduction in cytochrome b (Cyt b) gene expression. Tfam gene knockdown clones also showed decreased mtDNA-encoded cytochrome c oxidase subunit I (COX I) protein expression. However, no significant differences in protein expression were observed in nuclear DNA (nDNA)-encoded mitochondrial respiratory enzyme subunits. The cell morphology changed from a rhombus-like to a spindle-like form as determined in clones with decreased expressions of Tfam, mtRNA, and mitochondrial proteins. The mitochondrial respiratory enzyme activities and ATP production in such clones were significantly lower. The proportions of mtDNA mutations including 8-hydroxy-2'-deoxyguanosine (8-OHdG), a 4,977-bp deletion, and a 3,243-point mutation were also examined in these clones. No obvious increase in mtDNA mutations was observed in mitochondrial dysfunctional cell clones. The mitochondrial respiratory activity and ATP production ability recovered in cells with increased mtDNA levels after removal of the specific siRNA treatment. These experimental results provide direct evidence to substantiate that downregulation of mtDNA copy number and expression may compromise mitochondrial function and subsequent cell growth and morphology. (c) 2007 Wiley-Liss, Inc.

An atlas of B-cell clonal distribution in the human body.

PubMed

Meng, Wenzhao; Zhang, Bochao; Schwartz, Gregory W; Rosenfeld, Aaron M; Ren, Daqiu; Thome, Joseph J C; Carpenter, Dustin J; Matsuoka, Nobuhide; Lerner, Harvey; Friedman, Amy L; Granot, Tomer; Farber, Donna L; Shlomchik, Mark J; Hershberg, Uri; Luning Prak, Eline T

2017-09-01

B-cell responses result in clonal expansion, and can occur in a variety of tissues. To define how B-cell clones are distributed in the body, we sequenced 933,427 B-cell clonal lineages and mapped them to eight different anatomic compartments in six human organ donors. We show that large B-cell clones partition into two broad networks-one spans the blood, bone marrow, spleen and lung, while the other is restricted to tissues within the gastrointestinal (GI) tract (jejunum, ileum and colon). Notably, GI tract clones display extensive sharing of sequence variants among different portions of the tract and have higher frequencies of somatic hypermutation, suggesting extensive and serial rounds of clonal expansion and selection. Our findings provide an anatomic atlas of B-cell clonal lineages, their properties and tissue connections. This resource serves as a foundation for studies of tissue-based immunity, including vaccine responses, infections, autoimmunity and cancer.

Characterization and pharmacological properties of in vitro propagated clones of Echinacea tennesseensis (Beadle) small

USDA-ARS?s Scientific Manuscript database

Tissue culture techniques have been used to establish and maintain a repository of medicinal Echinacea. In vitro clones obtained from hypocotyls of germinated seeds, varied macroscopically, microscopically and exhibited variation in immune enhancing activity. Two in vitro produced clones of Echinace...

Ribosomal Binding Site Switching: An Effective Strategy for High-Throughput Cloning Constructions

PubMed Central

Li, Yunlong; Zhang, Yong; Lu, Pei; Rayner, Simon; Chen, Shiyun

2012-01-01

Direct cloning of PCR fragments by TA cloning or blunt end ligation are two simple methods which would greatly benefit high-throughput (HTP) cloning constructions if the efficiency can be improved. In this study, we have developed a ribosomal binding site (RBS) switching strategy for direct cloning of PCR fragments. RBS is an A/G rich region upstream of the translational start codon and is essential for gene expression. Change from A/G to T/C in the RBS blocks its activity and thereby abolishes gene expression. Based on this property, we introduced an inactive RBS upstream of a selectable marker gene, and designed a fragment insertion site within this inactive RBS. Forward and reverse insertions of specifically tailed fragments will respectively form an active and inactive RBS, thus all background from vector self-ligation and fragment reverse insertions will be eliminated due to the non-expression of the marker gene. The effectiveness of our strategy for TA cloning and blunt end ligation are confirmed. Application of this strategy to gene over-expression, a bacterial two-hybrid system, a bacterial one-hybrid system, and promoter bank construction are also verified. The advantages of this simple procedure, together with its low cost and high efficiency, makes our strategy extremely useful in HTP cloning constructions. PMID:23185557

Molecular cloning, expression pattern, and chemical analysis of heat shock protein 70 (HSP70) in the mudskipper Boleophthalmus pectinirostris: Evidence for its role in regulating spermatogenesis.

PubMed

Han, Ying-Li; Yang, Wan-Xi; Long, Ling-Li; Sheng, Zhang; Zhou, Yang; Zhao, Yong-Qiang; Wang, You-Fa; Zhu, Jun-Quan

2016-01-10

Heat shock protein 70 (HSP70) is molecular chaperone that is important for reproductive biological processes. In this study, a full length HSP70 from the mudskipper (Boleophthalmus pectinirostris) was characterized. It was found to contain: a 108 bp 5'-untranslated region, a 208 bp 3'-untranslated region, and a 1953 bp open reading frame, which encodes a protein of 650 amino acids with a theoretical molecular weight of 71.1 kDa and an isoelectric point of 5.17. RT-PCR analysis revealed that HSP70 was ubiquitously expressed in all major tissues with differential expression levels. This suggests that HSP70 has vital and conserved biological functions. HSP70 was localized mainly in the cytoplasm of germinal cells, indicating an important role of this protein during spermatogenesis. In response to heat stress, the testes presented abnormal morphology in connective tissues, in which HSP70 immunoreactivity was not observed. HSP70 mRNA expression in the gill, liver, and testes was significantly increased, which suggests that HSP70 plays an important role in protection against heat stress. Copyright © 2015 Elsevier B.V. All rights reserved.

Dedifferentiation of Human Primary Thyrocytes into Multilineage Progenitor Cells without Gene Introduction

PubMed Central

Saenko, Vladimir; Suzuki, Masatoshi; Matsuse, Michiko; Ohtsuru, Akira; Kumagai, Atsushi; Uga, Tatsuya; Yano, Hiroshi; Nagayama, Yuji; Yamashita, Shunichi

2011-01-01

While identification and isolation of adult stem cells have potentially important implications, recent reports regarding dedifferentiation/reprogramming from differentiated cells have provided another clue to gain insight into source of tissue stem/progenitor cells. In this study, we developed a novel culture system to obtain dedifferentiated progenitor cells from normal human thyroid tissues. After enzymatic digestion, primary thyrocytes, expressing thyroglobulin, vimentin and cytokeratin-18, were cultured in a serum-free medium called SAGM. Although the vast majority of cells died, a small proportion (∼0.5%) survived and proliferated. During initial cell expansion, thyroglobulin/cytokeratin-18 expression was gradually declined in the proliferating cells. Moreover, sorted cells expressing thyroid peroxidase gave rise to proliferating clones in SAGM. These data suggest that those cells are derived from thyroid follicular cells or at least thyroid-committed cells. The SAGM-grown cells did not express any thyroid-specific genes. However, after four-week incubation with FBS and TSH, cytokeratin-18, thyroglobulin, TSH receptor, PAX8 and TTF1 expressions re-emerged. Moreover, surprisingly, the cells were capable of differentiating into neuronal or adipogenic lineage depending on differentiating conditions. In summary, we have developed a novel system to generate multilineage progenitor cells from normal human thyroid tissues. This seems to be achieved by dedifferentiation of thyroid follicular cells. The presently described culture system may be useful for regenerative medicine, but the primary importance will be as a tool to elucidate the mechanisms of thyroid diseases. PMID:21556376

Isolation of Xenopus frizzled-10A and frizzled-10B genomic clones and their expression in adult tissues and embryos.

PubMed

Moriwaki, J; Kajita, E; Kirikoshi, H; Koike, J; Sagara, N; Yasuhiko, Y; Saitoh, T; Hirai, M; Katoh, M; Shiokawa, K

2000-11-19

Frizzled genes, encoding WNT receptors, play key roles in cell fate determination. Here, we isolated two Xenopus frizzled genes (Xfz10A and Xfz10B), probably reflecting pseudotetraploidy in Xenopus. Xfz10A (586 amino acids) and Xfz10B (580 amino acids) both encoded by a single exon, consisted of the N-terminal cysteine-rich domain, seven transmembrane domains, and the C-terminal Ser/Thr-X-Val motif. Xfz10A and Xfz10B were 97.0% identical at the amino acid level, and Xfz10B was 100% identical to previously reported Xfz9, yet Xfz10A was 85.3% and 62.4% identical to FZD10 and FZD9, respectively. Xfz10 mRNA appeared as 3.4 kb in adult tissues and embryos. RT-PCR analyses revealed the expression of more Xfz10A mRNA in stomach, kidney, eye, skeletal muscle, and skin, and more Xfz10B mRNA in heart and ovary, but in embryos, two mRNAs were equally expressed from the blastula stage with their peak expression at the late gastrula stage. The main site of Xfz10 mRNA expression was neural fold at the neurula stage and the dorsal region of midbrain, hindbrain, and spinal cord at the tadpole stage. These results suggest that Xfz10 has important roles in neural tissue formation. Copyright 2000 Academic Press.

Olfactomedin-like 2 A and B (OLFML2A and OLFML2B) expression profile in primates (human and baboon).

PubMed

Pérez-Ibave, Diana Cristina; González-Alvarez, Rafael; de La Luz Martinez-Fierro, Margarita; Ruiz-Ayma, Gabriel; Luna-Muñoz, Maricela; Martínez-De-Villarreal, Laura Elia; De Lourdes Garza-Rodríguez, María; Reséndez-Pérez, Diana; Mohamed-Noriega, Jibran; Garza-Guajardo, Raquel; Bautista-De-Lucío, Víctor Manuel; Mohamed-Noriega, Karim; Barboza-Quintana, Oralia; Arámburo-De-La-Hoz, Carlos; Barrera-Saldaña, Hugo Alberto; Rodríguez-Sánchez, Irám Pablo

2016-11-08

The olfactomedin-like domain (OLFML) is present in at least four families of proteins, including OLFML2A and OLFML2B, which are expressed in adult rat retina cells. However, no expression of their orthologous has ever been reported in human and baboon. The aim of this study was to investigate the expression of OLFML2A and OLFML2B in ocular tissues of baboons (Papio hamadryas) and humans, as a key to elucidate OLFML function in eye physiology. OLFML2A and OLFML2B cDNA detection in ocular tissues of these species was performed by RT-PCR. The amplicons were cloned and sequenced, phylogenetically analyzed and their proteins products were confirmed by immunofluorescence assays. OLFML2A and OLFML2B transcripts were found in human cornea, lens and retina and in baboon cornea, lens, iris and retina. The baboon OLFML2A and OLFML2B ORF sequences have 96% similarity with their human's orthologous. OLFML2A and OLFML2B evolution fits the hypothesis of purifying selection. Phylogenetic analysis shows clear orthology in OLFML2A genes, while OLFML2B orthology is not clear. Expression of OLFML2A and OLFML2B in human and baboon ocular tissues, including their high similarity, make the baboon a powerful model to deduce the physiological and/or metabolic function of these proteins in the eye.

Novel murine clonal cell lines either express slow or mixed (fast and slow) muscle markers following differentiation in vitro.

PubMed

Peltzer, J; Colman, L; Cebrian, J; Musa, H; Peckham, M; Keller, A

2008-05-01

We have investigated whether the phenotype of myogenic clones derived from satellite cells of different muscles from the transgenic immortomouse depended on muscle type origin. Clones derived from neonatal, or 6- to 12-week-old fast and slow muscles, were analyzed for myosin and enolase isoforms as phenotypic markers. All clones derived from slow-oxidative muscles differentiated into myotubes with a preferentially slow contractile phenotype, whereas some clones derived from rapid-glycolytic or neonatal muscles expressed both fast and slow myosin isoforms. Thus, muscle origin appears to bias myosin isoform expression in myotubes. The neonatal clone (WTt) was cultivated in various medium and substrate conditions, allowing us to determine optimized conditions for their differentiation. Matrigel allowed expressions of adult myosin isoforms, and an isozymic switch from embryonic alpha- toward muscle-specific beta-enolase, never previously observed in vitro. These cells will be a useful model for in vitro studies of muscle fiber maturation and plasticity.

Assessing the potential for AAV vector genotoxicity in a murine model

PubMed Central

Li, Hojun; Malani, Nirav; Hamilton, Shari R.; Schlachterman, Alexander; Bussadori, Giulio; Edmonson, Shyrie E.; Shah, Rachel; Arruda, Valder R.; Mingozzi, Federico; Fraser Wright, J.; Bushman, Frederic D.

2011-01-01

Gene transfer using adeno-associated virus (AAV) vectors has great potential for treating human disease. Recently, questions have arisen about the safety of AAV vectors, specifically, whether integration of vector DNA in transduced cell genomes promotes tumor formation. This study addresses these questions with high-dose liver-directed AAV-mediated gene transfer in the adult mouse as a model (80 AAV-injected mice and 52 controls). After 18 months of follow-up, AAV-injected mice did not show a significantly higher rate of hepatocellular carcinoma compared with controls. Tumors in mice treated with AAV vectors did not have significantly different amounts of vector DNA compared with adjacent normal tissue. A novel high-throughput method for identifying AAV vector integration sites was developed and used to clone 1029 integrants. Integration patterns in tumor tissue and adjacent normal tissue were similar to each other, showing preferences for active genes, cytosine-phosphate-guanosine islands, and guanosine/cysteine-rich regions. Gene expression data showed that genes near integration sites did not show significant changes in expression patterns compared with genes more distal to integration sites. No integration events were identified as causing increased oncogene expression. Thus, we did not find evidence that AAV vectors cause insertional activation of oncogenes and subsequent tumor formation. PMID:21106988

Cloning, sequencing, and expression of cDNA for human. beta. -glucuronidase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Oshima, A.; Kyle, J.W.; Miller, R.D.

1987-02-01

The authors report here the cDNA sequence for human placental ..beta..-glucuronidase (..beta..-D-glucuronoside glucuronosohydrolase, EC 3.2.1.31) and demonstrate expression of the human enzyme in transfected COS cells. They also sequenced a partial cDNA clone from human fibroblasts that contained a 153-base-pair deletion within the coding sequence and found a second type of cDNA clone from placenta that contained the same deletion. Nuclease S1 mapping studies demonstrated two types of mRNAs in human placenta that corresponded to the two types of cDNA clones isolated. The NH/sub 2/-terminal amino acid sequence determined for human spleen ..beta..-glucuronidase agreed with that inferred from the DNAmore » sequence of the two placental clones, beginning at amino acid 23, suggesting a cleaved signal sequence of 22 amino acids. When transfected into COS cells, plasmids containing either placental clone expressed an immunoprecipitable protein that contained N-linked oligosaccharides as evidenced by sensitivity to endoglycosidase F. However, only transfection with the clone containing the 153-base-pair segment led to expression of human ..beta..-glucuronidase activity. These studies provide the sequence for the full-length cDNA for human ..beta..-glucuronidase, demonstrate the existence of two populations of mRNA for ..beta..-glucuronidase in human placenta, only one of which specifies a catalytically active enzyme, and illustrate the importance of expression studies in verifying that a cDNA is functionally full-length.« less

Expressed sequence tag analysis of adult human optic nerve for NEIBank: Identification of cell type and tissue markers

PubMed Central

Bernstein, Steven L; Guo, Yan; Peterson, Katherine; Wistow, Graeme

2009-01-01

Background The optic nerve is a pure white matter central nervous system (CNS) tract with an isolated blood supply, and is widely used in physiological studies of white matter response to various insults. We examined the gene expression profile of human optic nerve (ON) and, through the NEIBANK online resource, to provide a resource of sequenced verified cDNA clones. An un-normalized cDNA library was constructed from pooled human ON tissues and was used in expressed sequence tag (EST) analysis. Location of an abundant oligodendrocyte marker was examined by immunofluorescence. Quantitative real time polymerase chain reaction (qRT-PCR) and Western analysis were used to compare levels of expression for key calcium channel protein genes and protein product in primate and rodent ON. Results Our analyses revealed a profile similar in many respects to other white matter related tissues, but significantly different from previously available ON cDNA libraries. The previous libraries were found to include specific markers for other eye tissues, suggesting contamination. Immune/inflammatory markers were abundant in the new ON library. The oligodendrocyte marker QKI was abundant at the EST level. Immunofluorescence revealed that this protein is a useful oligodendrocyte cell-type marker in rodent and primate ONs. L-type calcium channel EST abundance was found to be particularly low. A qRT-PCR-based comparative mammalian species analysis reveals that L-type calcium channel expression levels are significantly lower in primate than in rodent ON, which may help account for the class-specific difference in responsiveness to calcium channel blocking agents. Several known eye disease genes are abundantly expressed in ON. Many genes associated with normal axonal function, mRNAs associated with axonal transport, inflammation and neuroprotection are observed. Conclusion We conclude that the new cDNA library is a faithful representation of human ON and EST data provide an initial overview of gene expression patterns in this tissue. The data provide clues for tissue-specific and species-specific properties of human ON that will help in design of therapeutic models. PMID:19778450

Cloning and Expression of Genes for Dengue Virus Type-2 Encoded-Antigens for Rapid Diagnosis and Vaccine Development

DTIC Science & Technology

1988-10-31

00 0 Cloning and Expression of Genes for Dengue Virus (Type-2 Encoded-Antigens for Rapid ODiagnosis and Vaccine DevelopmentN| ANNUAL PROGRESS REPORT...11. TITLE (include Security Classification) Cloning and Expression of Genes f or Dengue Virus Type 2 Fncoded Antigens for Rapid Diagnosis and Vaccine ...epidemics in Central and South Americas and the Caribbean is a cause of major concern. An effective vaccine is not available to protect individuals

Neuropeptide B in Nile tilapia Oreochromis niloticus: molecular cloning and its effects on the regulation of food intake and mRNA expression of growth hormone and prolactin.

PubMed

Yang, Lu; Sun, Caiyun; Li, Wensheng

2014-05-01

Neuropeptide B (NPB) regulates food intake, energy homeostasis and hormone secretion in mammals via two G-protein coupled receptors, termed as GPR 7 and GPR 8. However, there is no study that reports the function of NPB in teleosts. In this study, the full-length cDNA of prepro-NPB with the size of 663bp was cloned from the hypothalamus of Nile tilapia. The CDS of the prepro-NPB is 387bp which encodes a precursor protein with the size of 128a.a. This precursor contains a mature peptide with the size of 29a.a, and it was named as NPB29. Tissue distribution study showed that this gene was mainly expressed in different parts of brain, especially in the diencephalon as well as hypothalamus, and the spinal cord in Nile tilapia. Fasting significantly stimulated the mRNA expression of NPB in the brain area without hypothalamus, and refeeding after fasting for 3 and 14days also showed similar effects on NPB expression. While, only short-term fasting (3days) and refeeding after fasting for 7 and 14days induced mRNA expression of NPB in the hypothalamus. Intraperitoneal (i.p.) injection of NPB remarkably elevated the mRNA expression of hypothalamic neuropeptide Y (NPY), cholecystokinin 1 (CCK1) and pituitary prolactin (PRL), whereas significantly inhibited growth hormone (GH) expression in pituitary. These observations in the present study suggested that NPB may participate in the regulation of feeding and gene expression of pituitary GH and PRL in Nile tilapia. Copyright © 2014 Elsevier Inc. All rights reserved.

Protein structure controls the processing of the N-linked oligosaccharides and glycosylphosphatidylinositol glycans of variant surface glycoproteins expressed in bloodstream form Trypanosoma brucei.

PubMed

Zitzmann, N; Mehlert, A; Carrouée, S; Rudd, P M; Ferguson, M A; Carroué, S

2000-03-01

The variant surface glycoproteins (VSGs) of Trypanosoma brucei are a family of homodimeric glycoproteins that adopt similar shapes. An individual trypanosome expresses one VSG at a time in the form of a dense protective mono-layer on the plasma membrane. VSG genes are expressed from one of several polycistronic transcription units (expression sites) that contain several expression site associated genes. We used a transformed trypanosome clone expressing two different VSGs (VSG121 and VSG221) from the same expression site (that of VSG221) to establish whether the genotype of the trypanosome clone or the VSG structure itself controls VSG N-linked oligosaccharide and GPI anchor glycan processing. In-gel release and fluorescent labeling of N-linked oligosaccharides and on-blot fluorescent labeling and release of GPI anchor glycans were employed to compare the carbohydrate structures of VSG121 and VSG221 when expressed individually in wild-type trypanosome clones and when expressed together in the transformed trypanosome clone. The data indicate that the genotype of the trypanosome clone has no effect on the N-linked oligosaccharide structures present on a given VSG variant and only a minor effect on the GPI anchor glycans. The latter is most likely an effect of changes in inter-VSG packing when two VGSs are expressed simultaneously. Thus, N-linked oligosaccharide and GPI anchor processing enzymes appear to be constitutively expressed in bloodstream form African trypanosomes and the tertiary and quaternary structures of the VSG homodimers appear to dictate the processing and glycoform microheterogeneity of surface-expressed VSGs.

Expression of proteins in Escherichia coli as fusions with maltose-binding protein to rescue non-expressed targets in a high-throughput protein-expression and purification pipeline

PubMed Central

Hewitt, Stephen N.; Choi, Ryan; Kelley, Angela; Crowther, Gregory J.; Napuli, Alberto J.; Van Voorhis, Wesley C.

2011-01-01

Despite recent advances, the expression of heterologous proteins in Escherichia coli for crystallization remains a nontrivial challenge. The present study investigates the efficacy of maltose-binding protein (MBP) fusion as a general strategy for rescuing the expression of target proteins. From a group of sequence-verified clones with undetectable levels of protein expression in an E. coli T7 expression system, 95 clones representing 16 phylogenetically diverse organisms were selected for recloning into a chimeric expression vector with an N-terminal histidine-tagged MBP. PCR-amplified inserts were annealed into an identical ligation-independent cloning region in an MBP-fusion vector and were analyzed for expression and solubility by high-throughput nickel-affinity binding. This approach yielded detectable expression of 72% of the clones; soluble expression was visible in 62%. However, the solubility of most proteins was marginal to poor upon cleavage of the MBP tag. This study offers large-scale evidence that MBP can improve the soluble expression of previously non-expressing proteins from a variety of eukaryotic and prokaryotic organisms. While the behavior of the cleaved proteins was disappointing, further refinements in MBP tagging may permit the more widespread use of MBP-fusion proteins in crystallographic studies. PMID:21904041

Universality of clone dynamics during tissue development

NASA Astrophysics Data System (ADS)

Rulands, Steffen; Lescroart, Fabienne; Chabab, Samira; Hindley, Christopher J.; Prior, Nicole; Sznurkowska, Magdalena K.; Huch, Meritxell; Philpott, Anna; Blanpain, Cedric; Simons, Benjamin D.

2018-05-01

The emergence of complex organs is driven by the coordinated proliferation, migration and differentiation of precursor cells. The fate behaviour of these cells is reflected in the time evolution of their progeny, termed clones, which serve as a key experimental observable. In adult tissues, where cell dynamics is constrained by the condition of homeostasis, clonal tracing studies based on transgenic animal models have advanced our understanding of cell fate behaviour and its dysregulation in disease1,2. But what can be learnt from clonal dynamics in development, where the spatial cohesiveness of clones is impaired by tissue deformations during tissue growth? Drawing on the results of clonal tracing studies, we show that, despite the complexity of organ development, clonal dynamics may converge to a critical state characterized by universal scaling behaviour of clone sizes. By mapping clonal dynamics onto a generalization of the classical theory of aerosols, we elucidate the origin and range of scaling behaviours and show how the identification of universal scaling dependences may allow lineage-specific information to be distilled from experiments. Our study shows the emergence of core concepts of statistical physics in an unexpected context, identifying cellular systems as a laboratory to study non-equilibrium statistical physics.

Ethylene biosynthesis in detached young persimmon fruit is initiated in calyx and modulated by water loss from the fruit.

PubMed

Nakano, Ryohei; Ogura, Emi; Kubo, Yasutaka; Inaba, Akitsugu

2003-01-01

Persimmon (Diospyros kaki Thunb.) fruit are usually classified as climacteric fruit; however, unlike typical climacteric fruits, persimmon fruit exhibit a unique characteristic in that the younger the stage of fruit detached, the greater the level of ethylene produced. To investigate ethylene induction mechanisms in detached young persimmon fruit, we cloned three cDNAs encoding 1-aminocyclopropane-1-carboxylic acid (ACC) synthase (DK-ACS1, 2, and -3) and two encoding ACC oxidase (DK-ACO1 and -2) genes involved in ethylene biosynthesis, and we analyzed their expression in various fruit tissues. Ethylene production was induced within a few days of detachment in all fruit tissues tested, accompanied by temporally and spatially coordinated expression of all the DK-ACS and DK-ACO genes. In all tissues except the calyx, treatment with 1-methylcyclopropene, an inhibitor of ethylene action, suppressed ethylene production and ethylene biosynthesis-related gene expression. In the calyx, one ACC synthase gene (DK-ACS2) exhibited increased mRNA accumulation accompanied by a large quantity of ethylene production, and treatment of the fruit with 1-methylcyclopropene did not prevent either the accumulation of DK-ACS2 transcripts or ethylene induction. Furthermore, the alleviation of water loss from the fruit significantly delayed the onset of ethylene production and the expression of DK-ACS2 in the calyx. These results indicate that ethylene biosynthesis in detached young persimmon fruit is initially induced in calyx and is modulated by water loss through transcriptional activation of DK-ACS2. The ethylene produced in the calyx subsequently diffuses to other fruit tissues and acts as a secondary signal that stimulates autocatalytic ethylene biosynthesis in these tissues, leading to a burst of ethylene production.

Ethylene Biosynthesis in Detached Young Persimmon Fruit Is Initiated in Calyx and Modulated by Water Loss from the Fruit1

PubMed Central

Nakano, Ryohei; Ogura, Emi; Kubo, Yasutaka; Inaba, Akitsugu

2003-01-01

Persimmon (Diospyros kaki Thunb.) fruit are usually classified as climacteric fruit; however, unlike typical climacteric fruits, persimmon fruit exhibit a unique characteristic in that the younger the stage of fruit detached, the greater the level of ethylene produced. To investigate ethylene induction mechanisms in detached young persimmon fruit, we cloned three cDNAs encoding 1-aminocyclopropane-1-carboxylic acid (ACC) synthase (DK-ACS1, 2, and -3) and two encoding ACC oxidase (DK-ACO1 and -2) genes involved in ethylene biosynthesis, and we analyzed their expression in various fruit tissues. Ethylene production was induced within a few days of detachment in all fruit tissues tested, accompanied by temporally and spatially coordinated expression of all the DK-ACS and DK-ACO genes. In all tissues except the calyx, treatment with 1-methylcyclopropene, an inhibitor of ethylene action, suppressed ethylene production and ethylene biosynthesis-related gene expression. In the calyx, one ACC synthase gene (DK-ACS2) exhibited increased mRNA accumulation accompanied by a large quantity of ethylene production, and treatment of the fruit with 1-methylcyclopropene did not prevent either the accumulation of DK-ACS2 transcripts or ethylene induction. Furthermore, the alleviation of water loss from the fruit significantly delayed the onset of ethylene production and the expression of DK-ACS2 in the calyx. These results indicate that ethylene biosynthesis in detached young persimmon fruit is initially induced in calyx and is modulated by water loss through transcriptional activation of DK-ACS2. The ethylene produced in the calyx subsequently diffuses to other fruit tissues and acts as a secondary signal that stimulates autocatalytic ethylene biosynthesis in these tissues, leading to a burst of ethylene production. PMID:12529535

A dual host vector for Fab phage display and expression of native IgG in mammalian cells.

PubMed

Tesar, Devin; Hötzel, Isidro

2013-10-01

A significant bottleneck in antibody discovery by phage display is the transfer of immunoglobulin variable regions from phage clones to vectors that express immunoglobulin G (IgG) in mammalian cells for screening. Here, we describe a novel phagemid vector for Fab phage display that allows expression of native IgG in mammalian cells without sub-cloning. The vector uses an optimized mammalian signal sequence that drives robust expression of Fab fragments fused to an M13 phage coat protein in Escherichia coli and IgG expression in mammalian cells. To allow the expression of Fab fragments fused to a phage coat protein in E.coli and full-length IgG in mammalian cells from the same vector without sub-cloning, the sequence encoding the phage coat protein was embedded in an optimized synthetic intron within the immunoglobulin heavy chain gene. This intron is removed from transcripts in mammalian cells by RNA splicing. Using this vector, we constructed a synthetic Fab phage display library with diversity in the heavy chain only and selected for clones binding different antigens. Co-transfection of mammalian cells with DNA from individual phage clones and a plasmid expressing the invariant light chain resulted in the expression of native IgG that was used to assay affinity, ligand blocking activity and specificity.

Cloning and expression analysis of a novel G-protein-coupled receptor selectively expressed on granulocytes.

PubMed

Yousefi, S; Cooper, P R; Potter, S L; Mueck, B; Jarai, G

2001-06-01

The migration of neutrophils into sites of acute and chronic inflammation is mediated by chemokines. We used degenerate-primer reverse transcriptase-polymerase chain reaction (RT-PCR) to analyze chemokine receptor expression in neutrophils and identify novel receptors. RNA was isolated from human peripheral blood neutrophils and from neutrophils that had been stimulated for 5 h with granulocyte-macrophage colony-stimulating factor or by coculturing with primary human bronchial epithelial cells. Amplification products were cloned, and clone redundancy was determined. Seven known G-protein-coupled receptors were identified among 38 clones-CCR1, CCR4, CXCR1, CXCR2, CXCR4, HM63, and FPR1-as well as a novel gene, EX33. The full-length EX33 clone was obtained, and an in silico approach was used to identify the putative murine homologue. The EX33 gene encodes a 396-amino-acid protein with limited sequence identity to known receptors. Expression studies of several known chemokine receptors and EX33 revealed that resting neutrophils expressed higher levels of CXCRs and EX33 compared with activated neutrophils. Northern blot experiments revealed that EX33 is expressed mainly in bone marrow, lung, and peripheral blood leukocytes. Using RT-PCR analysis, we showed more abundant expression of EX33 in neutrophils and eosinophils, in comparison with that in T- or B-lymphocytes, indicating cell-specific expression among leukocytes.

A sugar transporter from Medicago truncatula: altered expression pattern in roots during vesicular-arbuscular (VA) mycorrhizal associations.

PubMed

Harrison, M J

1996-04-01

A cDNA clone encoding a hexose transporter has been isolated from a library prepared from Medicago truncatula roots colonized by the mycorrhizal fungus Glomus versiforme. The clone (Mtst1) represents a M. truncatula gene and expression studies in yeast indicate that the encoded protein transports glucose and fructose but not sucrose. Transcripts corresponding to Mtst1 are expressed in leaves, stems and roots of M. truncatula, with the highest levels of expression in roots. In the roots, Mtst1 transcripts were detected in two distinct locations; the phloem fiber cells of the vascular tissue, and the cells of the root tip. Mtst1 expression in the roots is regulated in response to colonization by G. versiforme; transcript levels increased two- to fourfold in both M. truncatula and M. sativa following colonization by G. versiforme but did not increase during the unsuccessful interaction between G. versiforme and a M. sativa myc- mutant, suggesting that the increase in Mtst1 transcripts in the successful mycorrhizal interaction is correlated with internal growth of the fungus and potentially with a functioning symbiosis. Mtst1 transcripts were also detected in the cortical cells of the mycorrhizal root, specifically in areas of the root that were highly colonized by the mycorrhizal fungus. Thus, the formation of a symbiotic association with a VA mycorrhizal fungus is accompanied by a change in the cell type-specific expression of a transporter that potentially functions to supply sugars to root cells critically involved in the symbiotic association.

Cloning, expression pattern, and potential role of apoptosis inhibitor 5 in the termination of embryonic diapause and early embryo development of Artemia sinica.

PubMed

Zhang, Shuang; Yao, Feng; Jing, Ting; Zhang, Mengchen; Zhao, Wei; Zou, Xiangyang; Sui, Linlin; Hou, Lin

2017-09-10

During the embryonic development of Artemia sinica, the diapause phenomenon can be induced by high salinity or low temperature conditions. The diapause embryo at the gastrula stage is maintained under the threat of apoptosis to guarantee the embryo's normal development. In this process, apoptosis inhibitor proteins play vital roles in protecting embryos against apoptosis. Apoptosis inhibitor5 (API5) plays a pivotal role in regulating the cell cycle and preventing programmed cell death after growth factor starvation. In the present study, we cloned the full-length cDNA representing the api5 gene from A. sinica (As-api5), which encodes a 372-amino acid protein. In situ hybridization experiments revealed that As-api5 expression is not tissue or organ specific. Quantitative real-time PCR analyses of the developmental expression of As-api5 showed that it reached its highest level at 10h, after which its expression decreased. High salinity and low temperature treatments increased the expression of As-api5. Western blotting was used to assess the abundance of As-API5 and related proteins (As-CyclinA, As-CyclinE, As-E2F1, As-CDK2, As-APAF1, and As-Caspase9). Downregulation of As-api5 expression using a short interfering RNA resulted in increased mortality and embryo malformation of A. sinica. Taken together, the results indicated that API5 plays a crucial role in embryonic diapause termination and early embryo development of A. sinica. Copyright © 2017. Published by Elsevier B.V.

Functional characterization of a vacuolar invertase from Solanum lycopersicum: post-translational regulation by N-glycosylation and a proteinaceous inhibitor.

PubMed

Tauzin, Alexandra S; Sulzenbacher, Gerlind; Lafond, Mickael; Desseaux, Véronique; Reca, Ida Barbara; Perrier, Josette; Bellincampi, Daniela; Fourquet, Patrick; Lévêque, Christian; Giardina, Thierry

2014-06-01

Plant vacuolar invertases, which belong to family 32 of glycoside hydrolases (GH32), are key enzymes in sugar metabolism. They hydrolyse sucrose into glucose and fructose. The cDNA encoding a vacuolar invertase from Solanum lycopersicum (TIV-1) was cloned and heterologously expressed in Pichia pastoris. The functional role of four N-glycosylation sites in TIV-1 has been investigated by site-directed mutagenesis. Single mutations to Asp of residues Asn52, Asn119 and Asn184, as well as the triple mutant (Asn52, Asn119 and Asn184), lead to enzymes with reduced specific invertase activity and thermostability. Expression of the N516D mutant, as well as of the quadruple mutant (N52D, N119D, N184D and N516D) could not be detected, indicating that these mutations dramatically affected the folding of the protein. Our data indicate that N-glycosylation is important for TIV-1 activity and that glycosylation of N516 is crucial for recombinant enzyme stability. Using a functional genomics approach a new vacuolar invertase inhibitor of S. lycopersicum (SolyVIF) has been identified. SolyVIF cDNA was cloned and heterologously expressed in Escherichia coli. Specific interactions between SolyVIF and TIV-1 were investigated by an enzymatic approach and surface plasmon resonance (SPR). Finally, qRT-PCR analysis of TIV-1 and SolyVIF transcript levels showed a specific tissue and developmental expression. TIV-1 was mainly expressed in flowers and both genes were expressed in senescent leaves. Copyright © 2013 Elsevier Masson SAS. All rights reserved.

Molecular analysis of the mouse agouti gene and the role of dominant agouti-locus mutations in obesity and insulin resistance

DOE Office of Scientific and Technical Information (OSTI.GOV)

Klebig, M.L.; Woychik, R.P.; Wilkinson, J.E.

1994-09-01

The lethal yellow (A{sup y/-}) and viable yellow (A{sup vy/-}) mouse agouti mutants have a predominantly yellow pelage and display a complex syndrome that includes obesity, hyperinsulinemia, and insulin resistance, hallmark features of obesity-associated noninsulin-dependent diabetes mellitus (NIDDM) in humans. A new dominant agouti allele, A{sup iapy}, has recently been identified; like the A{sup vy} allele, it is homozygous viable and confers obesity and yellow fur in heterozygotes. The agouti gene was cloned and characterized at the molecular level. The gene is expressed in the skin during hair growth and is predicted to encode a 131 amino acid protein, thatmore » is likely to be a secreted factor. In both Ay/- and A{sup iapy}/- mice, the obesity and other dominant pleiotropic effects are associated with an ectopic expression of agouti in many tissues where the gene product is normally not produced. In Ay, a 170-kb deletion has occurred that causes an upstream promoter to drive the ectopic expression of the wild-type agouti coding exons. In A{sup iapy}, the coding region of the gene is expressed from a cryptic promoter within the LTR of an intracisternal A-particle (IAP), which has integrated within the region just upstream of the first agouti coding exon. Transgenic mice ubiquitously expressing the cloned agouti gene under the influence of the beta-actin and phosphoglycerate kinase promoters display obesity, hyperinsulinemia, and yellow coat color. This demonstrates unequivocally that ectopic expression of agouti is responsible for the yellow obese syndrome.« less

Cloning of a Gene Whose Expression is Increased in Scrapie and in Senile Plaques in Human Brain

NASA Astrophysics Data System (ADS)

Wietgrefe, S.; Zupancic, M.; Haase, A.; Chesebro, B.; Race, R.; Frey, W.; Rustan, T.; Friedman, R. L.

1985-12-01

A complementary DNA library was constructed from messenger RNA's extracted from the brains of mice infected with the scrapie agent. The library was differentially screened with the objectives of finding clones that might be used as markers of infection and finding clones of genes whose increased expression might be correlated with the pathological changes common to scrapie and Alzheimer's disease. A gene was identified whose expression is increased in scrapie. The complementary DNA corresponding to this gene hybridized preferentially and focally to cells in the brains of scrapie-infected animals. The cloned DNA also hybridized to the neuritic plaques found with increased frequency in brains of patients with Alzheimer's disease.

Molecular Cloning and mRNA Expression of Heat Shock Protein Genes and Their Response to Cadmium Stress in the Grasshopper Oxya chinensis.

PubMed

Zhang, Yuping; Liu, Yaoming; Zhang, Jianzhen; Guo, Yaping; Ma, Enbo

2015-01-01

Heat shock proteins (Hsps) are highly conserved molecular chaperones that are synthesized in response to stress. In this study, we cloned the full-length sequences of the Grp78 (glucose-regulated protein 78), Hsp70, Hsp90, and Hsp40 genes from the Chinese rice grasshopper Oxya chinensis. The full-length cDNA sequences of OcGrp78, OcHsp70, OcHsp90, and OcHsp40 contain open reading frames of 1947, 1920, 2172, and 1042 bp that encode proteins of 649, 640, 724, and 347 amino acids, respectively. Fluorescent real-time quantitative PCR (RT-qPCR) was performed to quantify the relative transcript levels of these Hsp genes in different tissues and developmental stages. The mRNAs encoding these four Hsp genes were present at all developmental stages and in all tissues examined but were expressed at varying levels. Additionally, we investigated the mRNA expression profiles of these four Hsps in O. chinensis subjected to Cadmium (Cd) stress. OcGrp78, OcHsp70, OcHsp90, and OcHsp40 mRNA expression was induced under acute Cd stress; the levels reached a maximum within a short time (6 h), were reduced significantly at 12 h, and were lowered to or below control levels by 48 h. Regarding induction efficiency, OcHsp70 was the most sensitive gene to acute Cd stress. Chronic Cd exposure showed that dietary Cd treatment induced increased OcGrp78, OcHsp90, and OcHsp40 expression. However, dietary Cd induced a significant reduction of OcHsp70 expression. In the period tested, no significant difference in the mortality of the grasshoppers was observed. Our results suggest that these four Hsps genes, especially OcHsp70, are sensitive to acute Cd stress and could be used as molecular markers for toxicology studies. However, our results also indicate that OcHsp70 is not suitable for use as a molecular marker of chronic Cd contamination.

Peptide-independent Recognition by Alloreactive Cytotoxic T Lymphocytes (CTL)

PubMed Central

Smith, Pamela A.; Brunmark, Anders; Jackson, Michael R.; Potter, Terry A.

1997-01-01

We have isolated several H-2Kb–alloreactive cytotoxic T cell clones and analyzed their reactivity for several forms of H-2Kb. These cytotoxic T lymphocytes (CTL) were elicited by priming with a skin graft followed by in vitro stimulation using stimulator cells that express an H-2Kb molecule unable to bind CD8. In contrast to most alloreactive T cells, these CTL were able to recognize H-2Kb on the surface of the antigen processing defective cell lines RMA-S and T2. Furthermore, this reactivity was not increased by the addition of an extract containing peptides from C57BL/6 (H-2b) spleen cells, nor was the reactivity decreased by treating the target cells with acid to remove peptides bound to MHC molecules. The CTL were also capable of recognizing targets expressing the mutant H-2Kbm8 molecule. These findings suggested that the clones recognized determinants on H-2Kb that were independent of peptide. Further evidence for this hypothesis was provided by experiments in which H-2Kb produced in Drosophila melanogaster cells and immobilized on the surface of a tissue culture plate was able to stimulate hybridomas derived from these alloreactive T cells. Precursor frequency analysis demonstrated that skin graft priming, whether with skin expressing the wild-type or the mutant H-2Kb molecule, is a strong stimulus to elicit peptide-independent CTL. Moreover, reconstitution experiments demonstrated that the peptide-independent CTL clones were capable of mediating rapid and complete rejection of H-2–incompatible skin grafts. These findings provide evidence that not all allorecognition is peptide dependent. PMID:9091576

A cloning and expression analysis of pregnancy-associated glycoproteins expressed in trophoblasts of the white-tail deer placenta.

PubMed

Brandt, Gretchen A; Parks, Tina E; Killian, Gary; Ealy, Alan D; Green, Jonathan A

2007-11-01

The pregnancy-associated glycoproteins (PAGs) are placental proteins that have been cloned from swine, sheep, goats, and cattle, but never from animals within the Cervidae family. The goal of this work was to characterize PAGs in white-tailed deer. Placenta and uterine tissues were collected from pregnant does at days 85 and 90 of pregnancy. RNA from cotyledons was used to amplify deer PAGs by RT-PCR. Ten distinct cDNAs were cloned and sequenced. Some normally conserved amino acids comprising the catalytic site were found to be altered in deer PAGs 4, 5, and 8; another PAG, (PAG-9) was a splice variant that lacked exon 7. In each case, these mutations would likely preclude proteolytic activity for these proteins. A phylogenetic analysis revealed that most of the deer PAGs fell within the ancient PAG grouping. The remainder fell within the more modern (BNC-specific) PAG group. Western blotting was performed with anti-PAG antibodies and this analysis revealed that deer PAGs comprise a heterogeneous group based on different antigenicities and electrophoretic mobilities. Immunohistochemistry and in situ hybridization revealed some unique localization patterns of PAGs in the deer placentome compared to those in other ruminants. Most notably, deer PAGs 4 and 5, which according to the phylogeny, are "ancient PAGs," were expected to be present in all trophoblasts; instead, they were localized to the BNC. Although many of the PAGs identified here are very similar to those in Bovidae, some are clearly distinct in their expression pattern and probably possess functional roles unique to cervid reproduction. (c) 2007 Wiley-Liss, Inc.

[Hydroxyproline: Rich glycoproteins of the plant and cell wall]. Annual technical progress report, 1993

DOE Office of Scientific and Technical Information (OSTI.GOV)

Varner, J.E.

1993-06-01

Since xylem tissue includes the main cell types which are lignified, we are interested in gene expression of glycine-rich proteins and proline-rich proteins, and other proteins which are involved in secondary cell wall thickening during xylogenesis. Since the main feature of xylogenesis is the deposition of additional wall components, study of the mechanism of xylogenesis will greatly advance our knowledge of the synthesis and assembly of wall macromolecules. We are using the in vitro xylogenesis system from isolated Zinnia mesophyll cells to isolate genes which are specifically expressed during xylogenesis. We have used subtractive hybridization methods to isolate a numbermore » of cDNA clones for differentially regulated genes from the cells after hormonal induction. So far, we have partially characterized 18 different cDNA clones from 239 positive clones. These differentially regulated genes can be divided into three sets according to the characteristics of gene expression in the induction medium and the control medium. The first set is induced in both the induction medium and the control medium without hormones. The second set is induced mainly in the induction medium and in the control medium with the addition of NAA alone. Two of thesegenes are exclusively induced by auxin. The third set of genes is induced mainly in the induction medium. Since these genes are not induced by either auxin or cytokinin alone, they may be directly involved in the process of xylogenesis. Our experiments on the localization of H{sub 2}O{sub 2} production reinforce the earlier ideas of others that H{sub 2}O{sub 2} is involved in normal lignification.« less

[Hydroxyproline: Rich glycoproteins of the plant and cell wall

DOE Office of Scientific and Technical Information (OSTI.GOV)

Varner, J.E.

1993-01-01

Since xylem tissue includes the main cell types which are lignified, we are interested in gene expression of glycine-rich proteins and proline-rich proteins, and other proteins which are involved in secondary cell wall thickening during xylogenesis. Since the main feature of xylogenesis is the deposition of additional wall components, study of the mechanism of xylogenesis will greatly advance our knowledge of the synthesis and assembly of wall macromolecules. We are using the in vitro xylogenesis system from isolated Zinnia mesophyll cells to isolate genes which are specifically expressed during xylogenesis. We have used subtractive hybridization methods to isolate a numbermore » of cDNA clones for differentially regulated genes from the cells after hormonal induction. So far, we have partially characterized 18 different cDNA clones from 239 positive clones. These differentially regulated genes can be divided into three sets according to the characteristics of gene expression in the induction medium and the control medium. The first set is induced in both the induction medium and the control medium without hormones. The second set is induced mainly in the induction medium and in the control medium with the addition of NAA alone. Two of thesegenes are exclusively induced by auxin. The third set of genes is induced mainly in the induction medium. Since these genes are not induced by either auxin or cytokinin alone, they may be directly involved in the process of xylogenesis. Our experiments on the localization of H[sub 2]O[sub 2] production reinforce the earlier ideas of others that H[sub 2]O[sub 2] is involved in normal lignification.« less

Multiplex Real-Time PCR for Monitoring Heterobasidion annosum Colonization in Norway Spruce Clones That Differ in Disease Resistance

PubMed Central

Hietala, Ari M.; Eikenes, Morten; Kvaalen, Harald; Solheim, Halvor; Fossdal, Carl G.

2003-01-01

A multiplex real-time PCR assay was developed to monitor the dynamics of the Picea abies-Heterobasidion annosum pathosystem. Tissue cultures and 32-year-old trees with low or high resistance to this pathogen were used as the host material. Probes and primers were based on a laccase gene for the pathogen and a polyubiquitin gene for the host. The real-time PCR procedure was compared to an ergosterol-based quantification method in a tissue culture experiment, and there was a strong correlation (product moment correlation coefficient, 0.908) between the data sets. The multiplex real-time PCR procedure had higher resolution and sensitivity during the early stages of colonization and also could be used to monitor the host. In the tissue culture experiment, host DNA was degraded more rapidly in the clone with low resistance than in the clone with high resistance. In the field experiment, the lesions elicited were not strictly proportional to the area colonized by the pathogen. Fungal colonization was more restricted and localized in the lesion in the clone with high resistance, whereas in the clone with low resistance, the fungus could be detected until the visible end of the lesion. Thus, the real-time PCR assay gives better resolution than does the traditionally used lesion length measurement when screening host clones for resistance. PMID:12902224

Rapid modification of the pET-28 expression vector for ligation independent cloning using homologous recombination in Saccharomyces cerevisiae

PubMed Central

Gay, Glen; Wagner, Drew T.; Keatinge-Clay, Adrian T.; Gay, Darren C.

2014-01-01

The ability to rapidly customize an expression vector of choice is a valuable tool for any researcher involved in high-throughput molecular cloning for protein overexpression. Unfortunately, it is common practice to amend or neglect protein targets if the gene that encodes the protein of interest is incompatible with the multiple-cloning region of a preferred expression vector. To address this issue, a method was developed to quickly exchange the multiple-cloning region of the popular expression plasmid pET-28 with a ligation-independent cloning cassette, generating pGAY-28. This cassette contains dual inverted restriction sites that reduce false positive clones by generating a linearized plasmid incapable of self-annealing after a single restriction-enzyme digest. We also establish that progressively cooling the vector and insert leads to a significant increase in ligation-independent transformation efficiency, demonstrated by the incorporation of a 10.3 kb insert into the vector. The method reported to accomplish plasmid reconstruction is uniquely versatile yet simple, relying on the strategic placement of primers combined with homologous recombination of PCR products in yeast. PMID:25304917

Macrophage migration inhibitory factor (MIF) family in arthropods: Cloning and expression analysis of two MIF and one D-dopachrome tautomerase (DDT) homologues in mud crabs, Scylla paramamosain.

PubMed

Huang, Wen-Shu; Duan, Li-Peng; Huang, Bei; Wang, Ke-Jian; Zhang, Cai-Liang; Jia, Qin-Qin; Nie, Pin; Wang, Tiehui

2016-03-01

The macrophage migration inhibitory factor (MIF) family, consisting of MIF and D-dopachrome tautomerase (DDT) in vertebrates, is evolutionarily ancient and has been found across Kingdoms including vertebrates, invertebrates, plants and bacteria. The mammalian MIF family are chemokines at the top of the inflammatory cascade in combating infections. They also possess enzymatic activities, e.g. DDT catalysis results in the production of 5,6-dihydroxyindole (DHI), a precursor of eumelanin. MIF-like genes are widely distributed, but DDT-like genes have only been described in vertebrates and a nematode. In this report, we cloned a DDT-like gene, for the first time in arthropods, and a second MIF in mud crab. The mud crab MIF family have a three exon/two intron structure as seen in vertebrates. The identification of a DDT-like gene in mud crab and other arthropods suggests that the separation of MIF and DDT preceded the divergence of protostomes and deuterostomes. The MIF family is differentially expressed in tissues of adults and during embryonic development and early life. The high level expression of the MIF family in immune tissues, such as intestine and hepatopancreas, suggests an important role in mud crab innate immunity. Mud crab DDT is highly expressed in early embryos, in megalops and crablets and this coincides with the requirement for melanisation in egg chorion tanning and cuticular hardening in arthropods, suggesting a potential novel role of DDT in melanogenesis via its tautomerase activity to produce DHI in mud crab. The clarification of the presence of both MIF and DDT in this report paves the way for further investigation of their functional roles in immunity and in melanogenesis in mud crab and other arthropods. Copyright © 2016 Elsevier Ltd. All rights reserved.

Reverse genetics with a full-length infectious cDNA of the Middle East respiratory syndrome coronavirus.

PubMed

Scobey, Trevor; Yount, Boyd L; Sims, Amy C; Donaldson, Eric F; Agnihothram, Sudhakar S; Menachery, Vineet D; Graham, Rachel L; Swanstrom, Jesica; Bove, Peter F; Kim, Jeeho D; Grego, Sonia; Randell, Scott H; Baric, Ralph S

2013-10-01

Severe acute respiratory syndrome with high mortality rates (~50%) is associated with a novel group 2c betacoronavirus designated Middle East respiratory syndrome coronavirus (MERS-CoV). We synthesized a panel of contiguous cDNAs that spanned the entire genome. Following contig assembly into genome-length cDNA, transfected full-length transcripts recovered several recombinant viruses (rMERS-CoV) that contained the expected marker mutations inserted into the component clones. Because the wild-type MERS-CoV contains a tissue culture-adapted T1015N mutation in the S glycoprotein, rMERS-CoV replicated ~0.5 log less efficiently than wild-type virus. In addition, we ablated expression of the accessory protein ORF5 (rMERS•ORF5) and replaced it with tomato red fluorescent protein (rMERS-RFP) or deleted the entire ORF3, 4, and 5 accessory cluster (rMERS-ΔORF3-5). Recombinant rMERS-CoV, rMERS-CoV•ORF5, and MERS-CoV-RFP replicated to high titers, whereas MERS-ΔORF3-5 showed 1-1.5 logs reduced titer compared with rMERS-CoV. Northern blot analyses confirmed the associated molecular changes in the recombinant viruses, and sequence analysis demonstrated that RFP was expressed from the appropriate consensus sequence AACGAA. We further show dipeptidyl peptidase 4 expression, MERS-CoV replication, and RNA and protein synthesis in human airway epithelial cell cultures, primary lung fibroblasts, primary lung microvascular endothelial cells, and primary alveolar type II pneumocytes, demonstrating a much broader tissue tropism than severe acute respiratory syndrome coronavirus. The availability of a MERS-CoV molecular clone, as well as recombinant viruses expressing indicator proteins, will allow for high-throughput testing of therapeutic compounds and provide a genetic platform for studying gene function and the rational design of live virus vaccines.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Li, YanHua, E-mail: liyanhua.1982@aliyun.com; Li, AiHua; Yang, Z.Q.

Cell death-inducing DNA fragmentation factor-α-like effector b (CIDEb) is a member of the CIDE family of apoptosis-inducing factors, CIDEa and CIDEc have been reported to be Lipid droplets (LDs)-associated proteins that promote atypical LD fusion in adipocytes, and responsible for liver steatosis under fasting and obese conditions, whereas CIDEb promotes lipid storage under normal diet conditions [1], and promotes the formation of triacylglyceride-enriched VLDL particles in hepatocytes [2]. Here, we report the gene cloning, chromosome mapping, tissue distribution, genetic expression analysis, and identification of a novel splicing variant of the porcine CIDEb gene. Sequence analysis shows that the open readingmore » frame of the normal porcine CIDEb isoform covers 660bp and encodes a 219-amino acid polypeptide, whereas its alternative splicing variant encodes a 142-amino acid polypeptide truncated at the fourth exon and comprised of the CIDE-N domain and part of the CIDE-C domain. The deduced amino acid sequence of normal porcine CIDEb shows an 85.8% similarity to the human protein and 80.0% to the mouse protein. The CIDEb genomic sequence spans approximately 6KB comprised of five exons and four introns. Radiation hybrid mapping demonstrated that porcine CIDEb is located at chromosome 7q21 and at a distance of 57cR from the most significantly linked marker, S0334, regions that are syntenic with the corresponding region in the human genome. Tissue expression analysis indicated that normal CIDEb mRNA is ubiquitously expressed in many porcine tissues. It was highly expressed in white adipose tissue and was observed at relatively high levels in the liver, lung, small intestine, lymphatic tissue and brain. The normal version of CIDEb was the predominant form in all tested tissues, whereas the splicing variant was expressed at low levels in all examined tissues except the lymphatic tissue. Furthermore, genetic expression analysis indicated that CIDEb mRNA levels were significantly higher in the white adipose tissue of lean pigs than their obese counterparts, in contrast to porcine CIDEa and CIDEc [3]. We therefore speculate that CIDEb may play a contrary role to the other CIDEs. The basic molecular information we provide here will be useful for further investigations of the physiological function of the gene, which will be helpful in better understanding the role of the CIDE family in lipid metabolism in pig models.« less

Ultrastructural characterization of atrial natriuretic peptide receptors (ANP-R) mRNA expression in rat kidney cortex.

PubMed

Grandclément, B; Morel, G

1998-06-01

Atrial natriuretic peptide (ANP) and two complementary peptides named brain natriuretic peptide and C-type natriuretic peptide are involved in diuresis, natriuresis, hypotension and vasorelaxation. Their actions are mediated by highly selective and specific ANP receptors. Three subtypes have been characterized and cloned: ANP receptor A, -B and -C. In the present study, the mRNA for each subtype was detected by ultrastructural in situ hybridization on ultrathin sections of Lowicryl-embedded tissue and frozen tissue. The distribution of mRNA (visualized by gold particles) for each subtype was found to differ in different cells of the nephron. The three subtypes of this receptor family were expressed in all the parts of the nephron, but their expression levels were different. The ANPR-A mRNA was the most abundant in cells of glomerulus, proximal and distal tubules. The subtype C was the least expressed mRNA in glomerulus. In contrast, the subcellular localization of the three mRNAs was similar; they were found in the cytoplasmic matrix and the euchromatin of the nucleus. In conclusion, the differential expression of these mRNAs in kidney cortex indicates that these three peptides act directly in differing parts of nephron regions which are the glomerulus, the proximal and distal tubules.

Molecular Cloning and Tissue-Specific Expression of an Anionic Peroxidase in Zucchini1

PubMed Central

Carpin, Sabine; Crèvecoeur, Michèle; Greppin, Hubert; Penel, Claude

1999-01-01

A calcium-pectate-binding anionic isoperoxidase (APRX) from zucchini (Cucurbita pepo) was purified and subjected to N-terminal amino acid microsequencing. The cDNA encoding this enzyme was obtained by reverse transcriptase polymerase chain reaction from a cDNA library. It encoded a mature protein of 309 amino acids exhibiting all of the sequence characteristics of a plant peroxidase. Despite the presence of a C-terminal propeptide, APRX was found in the apoplast. APRX protein and mRNA were found in the root, hypocotyls, and cotyledons. In situ hybridization showed that the APRX-encoding gene was expressed in many different tissues. The strongest expression was observed in root epidermis and in some cells of the stele, in differentiating tracheary elements of hypocotyl, in the lower and upper epidermis, in the palisade parenchyma of cotyledons, and in lateral and adventitious root primordia. In the hypocotyl hook there was an asymmetric expression, with the inner part containing more transcripts than the outer part. Treatment with 2,3,5-triiodobenzoic acid reduced the expression of the APRX-encoding gene in the lower part of the hypocotyl. Our observations suggest that APRX could be involved in lignin formation and that the transcription of its gene was related to auxin level. PMID:10398715

Targeted expression of suicide gene by tissue-specific promoter and microRNA regulation for cancer gene therapy.

PubMed

Danda, Ravikanth; Krishnan, Gopinath; Ganapathy, Kalaivani; Krishnan, Uma Maheswari; Vikas, Khetan; Elchuri, Sailaja; Chatterjee, Nivedita; Krishnakumar, Subramanian

2013-01-01

In order to realise the full potential of cancer suicide gene therapy that allows the precise expression of suicide gene in cancer cells, we used a tissue specific Epithelial cell adhesion molecule (EpCAM) promoter (EGP-2) that directs transgene Herpes simplex virus-thymidine kinase (HSV-TK) expression preferentially in EpCAM over expressing cancer cells. EpCAM levels are considerably higher in retinoblastoma (RB), a childhood eye cancer with limited expression in normal cells. Use of miRNA regulation, adjacent to the use of the tissue-specific promoter, would provide the second layer of control to the transgene expression only in the tumor cells while sparing the normal cells. To test this hypothesis we cloned let-7b miRNA targets in the 3'UTR region of HSV-TK suicide gene driven by EpCAM promoter because let-7 family miRNAs, including let-7b, were found to be down regulated in the RB tumors and cell lines. We used EpCAM over expressing and let-7 down regulated RB cell lines Y79, WERI-Rb1 (EpCAM (+ve)/let-7b(down-regulated)), EpCAM down regulated, let-7 over expressing normal retinal Müller glial cell line MIO-M1(EpCAM (-ve)/let-7b(up-regulated)), and EpCAM up regulated, let-7b up-regulated normal thyroid cell line N-Thy-Ori-3.1(EpCAM (+ve)/let-7b(up-regulated)) in the study. The cell proliferation was measured by MTT assay, apoptosis was measured by probing cleaved Caspase3, EpCAM and TK expression were quantified by Western blot. Our results showed that the EGP2-promoter HSV-TK (EGP2-TK) construct with 2 or 4 copies of let-7b miRNA targets expressed TK gene only in Y79, WERI-Rb-1, while the TK gene did not express in MIO-M1. In summary, we have developed a tissue-specific, miRNA-regulated dual control vector, which selectively expresses the suicide gene in EpCAM over expressing cells.

Rapid stalk elongation in tulip (Tulipa gesneriana L. cv. Apeldoorn) and the combined action of cold-induced invertase and the water-channel protein gammaTIP.

PubMed

Balk, P A; de Boer, A D

1999-09-01

Many bulbous plants need a low-temperature treatment for flowering. Cold, for example, affects the elongation of the stalk, thereby influencing the quality of the cut flower. How the elongation of the stalk is promoted by cold and which physiological and biochemical mechanisms are involved have remained obscure. As invertase has been shown to be involved in the cold-induced elongation of the flower stalks of tulips (Lambrechts et al., 1994, Plant Physiol 104: 515-520), we further characterized this enzyme by cloning the cDNA and analysing its expression in various tissues of the tulip (Tulipa gesneriana L. cv. Apeldoorn) stalk. In addition, the role of sucrose synthase was investigated. Since turgor pressure is an important force driving cell elongation, the role of a water-channel protein (gammaTIP) was studied in relation to these two enzymes. The mRNA level of the invertase found was substantially up-regulated as a result of cold treatment. Analysis of the amino acid sequence of this invertase revealed the presence of a vacuolar targeting signal. Two different forms of sucrose synthase were found, the expression of one of them appeared to be restricted to the vascular tissue while the other form was present in the surrounding tissue. Both sucrose synthases were present in the stalk during the entire period of bulb storage and after planting, but their activities declined during stalk elongation. The expression of the gammaTIP gene was restricted mainly to the vascular tissue and its expression profile was identical to that of invertase. Simultaneous expression of invertase and gammaTIP possibly leads to an increase in osmotic potential and vacuolar water uptake, thus providing a driving force for stretching the stalk cells.

Microarray Analysis of microRNA Expression during Axolotl Limb Regeneration

PubMed Central

Holman, Edna C.; Campbell, Leah J.; Hines, John; Crews, Craig M.

2012-01-01

Among vertebrates, salamanders stand out for their remarkable capacity to quickly regrow a myriad of tissues and organs after injury or amputation. The limb regeneration process in axolotls (Ambystoma mexicanum) has been well studied for decades at the cell-tissue level. While several developmental genes are known to be reactivated during this epimorphic process, less is known about the role of microRNAs in urodele amphibian limb regeneration. Given the compelling evidence that many microRNAs tightly regulate cell fate and morphogenetic processes through development and adulthood by modulating the expression (or re-expression) of developmental genes, we investigated the possibility that microRNA levels change during limb regeneration. Using two different microarray platforms to compare the axolotl microRNA expression between mid-bud limb regenerating blastemas and non-regenerating stump tissues, we found that miR-21 was overexpressed in mid-bud blastemas compared to stump tissue. Mature A. mexicanum (“Amex”) miR-21 was detected in axolotl RNA by Northern blot and differential expression of Amex-miR-21 in blastema versus stump was confirmed by quantitative RT-PCR. We identified the Amex Jagged1 as a putative target gene for miR-21 during salamander limb regeneration. We cloned the full length 3′UTR of Amex-Jag1, and our in vitro assays demonstrated that its single miR-21 target recognition site is functional and essential for the response of the Jagged1 gene to miR-21 levels. Our findings pave the road for advanced in vivo functional assays aimed to clarify how microRNAs such as miR-21, often linked to pathogenic cell growth, might be modulating the redeployment of developmental genes such as Jagged1 during regenerative processes. PMID:23028429

Molecular cloning and mRNA expression analysis of interleukin-8 gene in Japanese sea perch (Lateolabrax japonicus).

PubMed

Qiu, Lihua; Zhang, Hanhua; Yang, Keng; Jiang, Shigui

2009-05-01

Interleukin-8 (IL-8), the first known chemokine, is a CXC chemokine, which is cable of attracting neutrophils and inducing them to release lysozomal enzymes, triggering the respiratory burst. In the present study, the cDNA of an IL-8 was cloned from Japanese sea perch Lateolabrax japonicus (designated LjIL-8) by homology cloning and rapid amplification of cDNA ends (RACE) approaches. The full-length cDNA of LjIL-8 consisted of 803 nucleotides with a canonical polyadenylation signal sequence AATAAA and a poly(A) tail, and an open reading frame (ORF) of 300 bp encoding a polypeptide of 99 amino acid residues with a predicted molecular weight of 6.6 kDa. The high identity of LjIL-8 with IL-8 in other organisms indicated that LjIL-8 should be a new member of the IL-8 family. By fluorescent quantitative real-time PCR, mRNA transcript of LjIL-8 was detectable in all the examined tissues with higher level in spleen and head-kidney. The temporal expression of LjIL-8 mRNA in the spleen was up-regulated by lipopolyssacharide (LPS) stimulation and reached the maximum level at 6 h post-stimulation, and then dropped back to the original level gradually. These results indicated that LjIL-8 was a constitutive and inducible acute-phase protein that perhaps involved in the immune defense of L. japonicus.

Molecular cloning and sequence analysis of stearoyl-CoA desaturase in milkfish, Chanos chanos.

PubMed

Hsieh, S L; Liao, W L; Kuo, C M

2001-12-01

Stearoyl-CoA desaturase (EC 1.14.99.5) is a key enzyme in the biosynthesis of polyunsaturated fatty acids and the maintenance of the homeoviscous fluidity of biological membranes. The stearoyl-CoA desaturase cDNA in milkfish (Chanos chanos) was cloned by RT-PCR and RACE, and it was compared with the stearoyl-CoA desaturase in cold-tolerant teleosts, common carp and grass carp. Nucleotide sequence analysis revealed that the cDNA clone has a 972-bp open reading frame encoding 323 amino acid residues. Alignments of the deduced amino acid sequence showed that the milkfish stearoyl-CoA desaturase shares 79% and 75% identity with common carp and grass carp, and 63%-64% with other vertebrates such as sheep, hamsters, rats, mice, and humans. Like common carp and grass carp, the deduced amino acid sequence in milkfish well conserves three histidine cluster motifs (one HXXXXH and two HXXHH) that are essential for catalysis of stearoyl-CoA desaturase activity. However, RT-PCR analysis showed that stearoyl-CoA desaturase expression in milkfish is detected in the tissues of liver, muscle, kidney, brain, and gill, and more expression sites were found in milkfish than in common carp and grass carp. Phylogenic relationships among the deduced stearoyl-CoA desaturase amino acid sequence in milkfish and those in other vertebrates showed that the milkfish stearoyl-CoA desaturase amino acid sequence is phylogenetically closer to those of common carp and grass carp than to other higher vertebrates.

The expression analysis of ICOS-L on activated T cells and immature dendritic cells as well as malignant B cells and Grave's-disease-derived thyroid tissues by two novel mAbs against human ICOS-L.

PubMed

Wang, F; Zhu, W; Liu, T; Sun, Z; Ju, S; Ju, S; Yu, G; Xie, W; Deng, Z; Lu, B; Zhang, X

2007-01-01

ICOS-L, a newly identified member of B7 superfamily, plays an important role in immune responses. In this article, we report on two novel mouse anti-human ICOS-L monoclonal antibodies (mAbs) named as 11C4 and 12B11, whose specificities were verified by methods of flow cytometry, western blotting, and epitope competition assay. The two mAbs bound to distinct ICOS-L epitopes on B cells. Interestingly, mAb 11C4 could well recognize ICOS-L molecule on activated T cells and Jurkat cell lines, which is different from commercial anti-ICOS-L mAb (clone number MIH12) and the other mAb 12B11. In addition, we found that the expression of ICOS-L molecule was only detected on the surface of immature monocyte-derived dendritic cells (Mo-DCs) and was sharply decreased after induction of mature Mo-DCs activated by tumor necrosis factor-alpha or CD40. Furthermore, we showed that 11C4 could effectively suppress the maturation of Mo-DCs in vitro as evidenced by the low expression of CD80, CD86, CD83, and human leukocyte antigen-DR, which suggested that ICOS-L may be involved in the maturation of Mo-DCs. Using immunohistochemistry staining with mAb 11C4, the expression of ICOS-L was found in B lymphoma tissues and thyroid tissues from the Grave's disease but not in thyroid adenoma and normal thyroid tissues.

Green tissue-specific co-expression of chitinase and oxalate oxidase 4 genes in rice for enhanced resistance against sheath blight.

PubMed

Karmakar, Subhasis; Molla, Kutubuddin Ali; Chanda, Palas K; Sarkar, Sailendra Nath; Datta, Swapan K; Datta, Karabi

2016-01-01

Green tissue-specific simultaneous overexpression of two defense-related genes ( OsCHI11 & OsOXO4 ) in rice leads to significant resistance against sheath blight pathogen ( R. solani ) without distressing any agronomically important traits. Overexpressing two defense-related genes (OsOXO4 and OsCHI11) cloned from rice is effective at enhancing resistance against sheath blight caused by Rhizoctonia solani. These genes were expressed under the control of two different green tissue-specific promoters, viz. maize phosphoenolpyruvate carboxylase gene promoter, PEPC, and rice cis-acting 544-bp DNA element, immediately upstream of the D54O translational start site, P D54O-544 . Putative T0 transgenic rice plants were screened by PCR and integration of genes was confirmed by Southern hybridization of progeny (T1) rice plants. Successful expression of OsOXO4 and OsCHI11 in all tested plants was confirmed. Expression of PR genes increased significantly following pathogen infection in overexpressing transgenic plants. Following infection, transgenic plants exhibited elevated hydrogen peroxide levels, significant changes in activity of ROS scavenging enzymes and reduced membrane damage when compared to their wild-type counterpart. In a Rhizoctonia solani toxin assay, a detached leaf inoculation test and an in vivo plant bioassay, transgenic plants showed a significant reduction in disease symptoms in comparison to non-transgenic control plants. This is the first report of overexpression of two different PR genes driven by two green tissue-specific promoters providing enhanced sheath blight resistance in transgenic rice.

Regulation of c- and N-myc expression during induced differentiation of murine neuroblastoma cells.

PubMed

Larcher, J C; Vayssière, J L; Lossouarn, L; Gros, F; Croizat, B

1991-04-01

Using clones N1E-115 and N1A-103 from mouse neuroblastoma C1300, a comparative analysis of c- and N-myc gene expression was undertaken both in proliferating cells and in cultures exposed to conditions which induce differentiation. Under the latter conditions, while N1E-115 cells extend abundant neurites and express many biochemical features of mature neurons, clone N1A-103 stops dividing and expresses certain neurospecific markers but is unable to differentiate morphologically. In both clones, chemical agents, i.e. 1-methyl cyclohexane carboxylic acid (CCA) or dimethyl sulfoxide (DMSO), induce a decrease in c-myc expression. Similar results were found for N-myc gene in N1E-115 cells, but in contrast, in clone N1A-103, N-myc expression is increased with CCA and not modified with DMSO. Globally, this study favours the hypothesis that changes in c-myc expression would correspond to cell division blockade and differentiation, while modulations in N-myc are more closely related to an early phase of terminal differentiation.

Cloning of Russian sturgeon (Acipenser gueldenstaedtii) growth hormone and insulin-like growth factor I and their expression in male and female fish during the first period of growth.

PubMed

Yom Din, S; Hurvitz, A; Goldberg, D; Jackson, K; Levavi-Sivan, B; Degani, G

2008-03-01

In this study, the GH and IGF-I of the Russian sturgeon (rs), Acipenser gueldenstaedtii, were cloned and sequenced, and their mRNA gene expression determined. In addition, to improve our understanding of the GH function, the expression of this hormone was assessed in young males and females. Moreover, IGF-I expression was quantified in young males and compared to that in older ones. The nucleotide sequence of the rsGH cDNA was 980 bp long and had an open reading frame of 642 bp, beginning with the first ATG codon at position 39 and ending with the stop codon at position 683. A putative polyadenylation signal, AATAAA, was recognized 42 bp upstream of the poly (A) tail. The position of the signal- peptide cleavage site was predicted to be at position 111, yielding a signal peptide of 24 amino-acids (aa) and a mature peptide of 190 aa. When the rsGH aa sequence was compared with other species, the highest degree of identity was found to be with mammalians (66-70% identity), followed by anguilliformes and amphibia (61%) and other fish (39-47%). The level of rsGH mRNA was discovered to be similar in pituitaries of females and males of 5 age groups (1, 2, 3, 4, and 5- yr-old). In females and males, the levels did not change dramatically during the first 5 yr of growth. The partial nucleotide sequence of the rsIGF-I was 445 bp long and had an open reading frame of 396 bp, beginning with the ATG codon at position 50. The position of the signal-peptide cleavage site was predicted to be at position 187, yielding a signal peptide of 44 aa. The highest level of IGF-I mRNA expression was recorded in the kidney of adult sturgeons. The IGF-I mRNA expression levels in the intestine, pituitary gland, and liver were not significantly different. Low levels of expression were found in the brain, heart, and muscle. In most tissues, there was no significant difference between mRNA levels of one and 5-yr-old fish. In conclusion, based on the GH-sequence analysis, A. gueldenstaedtii is genetically distant from other teleosts. The expression of the GH mRNA was similar in males and females, and its level remained constant during the first 5 yr of growth. While the IGF-I mRNA expression differed amongst various tissues, the level in each tissue was similar in 1 and 5-yr-old fish.

Frame-Insensitive Expression Cloning of Fluorescent Protein from Scolionema suvaense.

PubMed

Horiuchi, Yuki; Laskaratou, Danai; Sliwa, Michel; Ruckebusch, Cyril; Hatori, Kuniyuki; Mizuno, Hideaki; Hotta, Jun-Ichi

2018-01-26

Expression cloning from cDNA is an important technique for acquiring genes encoding novel fluorescent proteins. However, the probability of in-frame cDNA insertion following the first start codon of the vector is normally only 1/3, which is a cause of low cloning efficiency. To overcome this issue, we developed a new expression plasmid vector, pRSET-TriEX, in which transcriptional slippage was induced by introducing a DNA sequence of (dT) 14 next to the first start codon of pRSET. The effectiveness of frame-insensitive cloning was validated by inserting the gene encoding eGFP with all three possible frames to the vector. After transformation with one of these plasmids, E. coli cells expressed eGFP with no significant difference in the expression level. The pRSET-TriEX vector was then used for expression cloning of a novel fluorescent protein from Scolionema suvaense . We screened 3658 E. coli colonies transformed with pRSET-TriEX containing Scolionema suvaense cDNA, and found one colony expressing a novel green fluorescent protein, ScSuFP. The highest score in protein sequence similarity was 42% with the chain c of multi-domain green fluorescent protein like protein "ember" from Anthoathecata sp. Variations in the N- and/or C-terminal sequence of ScSuFP compared to other fluorescent proteins indicate that the expression cloning, rather than the sequence similarity-based methods, was crucial for acquiring the gene encoding ScSuFP. The absorption maximum was at 498 nm, with an extinction efficiency of 1.17 × 10⁵ M -1 ·cm -1 . The emission maximum was at 511 nm and the fluorescence quantum yield was determined to be 0.6. Pseudo-native gel electrophoresis showed that the protein forms obligatory homodimers.

Preparation of Proper Immunogen by Cloning and Stable Expression of cDNA coding for Human Hematopoietic Stem Cell Marker CD34 in NIH-3T3 Mouse Fibroblast Cell Line

PubMed Central

Shafaghat, Farzaneh; Abbasi-Kenarsari, Hajar; Majidi, Jafar; Movassaghpour, Ali Akbar; Shanehbandi, Dariush; Kazemi, Tohid

2015-01-01

Purpose: Transmembrane CD34 glycoprotein is the most important marker for identification, isolation and enumeration of hematopoietic stem cells (HSCs). We aimed in this study to clone the cDNA coding for human CD34 from KG1a cell line and stably express in mouse fibroblast cell line NIH-3T3. Such artificial cell line could be useful as proper immunogen for production of mouse monoclonal antibodies. Methods: CD34 cDNA was cloned from KG1a cell line after total RNA extraction and cDNA synthesis. Pfu DNA polymerase-amplified specific band was ligated to pGEMT-easy TA-cloning vector and sub-cloned in pCMV6-Neo expression vector. After transfection of NIH-3T3 cells using 3 μg of recombinant construct and 6 μl of JetPEI transfection reagent, stable expression was obtained by selection of cells by G418 antibiotic and confirmed by surface flow cytometry. Results: 1158 bp specific band was aligned completely to reference sequence in NCBI database corresponding to long isoform of human CD34. Transient and stable expression of human CD34 on transfected NIH-3T3 mouse fibroblast cells was achieved (25% and 95%, respectively) as shown by flow cytometry. Conclusion: Cloning and stable expression of human CD34 cDNA was successfully performed and validated by standard flow cytometric analysis. Due to murine origin of NIH-3T3 cell line, CD34-expressing NIH-3T3 cells could be useful as immunogen in production of diagnostic monoclonal antibodies against human CD34. This approach could bypass the need for purification of recombinant proteins produced in eukaryotic expression systems. PMID:25789221

Human Bronchial Epithelial Cell Response to Heavy Particle Exposure

NASA Astrophysics Data System (ADS)

Story, Michael; Ding, Liang-Hao; Minna, John; Park, Seong-mi; Peyton, Michael; Larsen, Jill

2012-07-01

A battery of non-oncogenically immortalized human bronchial epithelial cells (HBECs) are being used to examine the molecular changes that lead to lung carcinogenesis after exposure to heavy particles found in the free space environment. The goal is to ultimately identify biomarkers of radioresponse that can be used for prediction of carcinogenic risk for fatal lung cancer. Our initial studies have focused on the cell line HBEC3 KT and the isogenic variant HBEC3 KTR53, which overexpresses the RASv12 mutant and where p53 has been knocked down by shRNA, and is considered to be a more oncogenically progressed variant. We have previously described the response of HBEC3 KT at the cellular and molecular level, however, the focus here is on the rate of cellular transformation after HZE radiation exposure and the molecular changes in transformed cells. When comparing the two cell lines we find that there is a maximum rate of cellular transformation at 0.25 Gy when cells are exposed to 1 GeV Fe particles, and, for the HBEC3 KTR53 there are multiple pathways upregulated that promote anchorage independent growth including the mTOR pathway, the TGF-1 pathway, RhoA signaling and the ERK/MAPK pathway as early as 2 weeks after radiation. This does not occur in the HBEC3 KT cell line. Transformed HBEC3 KT cells do not show any morphologic or phenotypic changes when grown as cell cultures. HBEC3 KTR53 cells on the other hand show substantial changes in morphology from a cobblestone epithelial appearance to a mesenchymal appearance with a lack of contact inhibition. This epithelial to mesenchymal change in morphology is accompanied by the expression of vimentin and a reduction in the expression of E-cadherin, which are hallmarks of epithelial to mesenchymal transition. Interestingly, for HBEC3 KT transformed cells there are no mutations in the p53 gene, 2 of 15 clones were found to be heterozygous for the RASV12 mutation, and 3 of 15 clones expressed high levels of BigH3, a TGFB-responsive gene associated with loss of cell anchorage. There is also a range of aneuploidy amongst the transformed clones and ongoing chromosomal analysis by array-based comparative genomic hybridization has identified single or two copy loss of the tumor suppressor gene FHIT, in 8 of 15 transformed clones. This is accompanied by a 6-fold reduction, overall, in FHIT gene expression amongst the 15 clones under examination. Interestingly, in spite of these changes at the molecular level, when implanted subcutaneously into immune-compromised mice, the transformed clones from the HBEC3 KT cell line do not form tumors. This suggests that additional hits are required for oncogenesis, at least in a subcutaneous model, and/or, 2-D tissue culture models to not adequately reflect the underlying biology. We have therefore, begun to examine transformation in a 3-D tissue culture model, bronchocysts, where HBEC cells ultimately differentiate and stop cycling. We have shown that cells in 3-D have reduced gene expression of key DNA repair genes, and are less effective at repairing complex damage. We are now irradiating at dose rates as low as 0.2 cGy/min to test the notion of an inverse dose rate effect for carcinogenesis by HZE particles. In our early experiments we have shown that as the dose rate dropped from 20 cGy/min to 0.2 cGy/min, for the same total dose (0.25 and 0.50 Gy) an increasing percentage of bronchocysts become mis-shapen, suggesting that some cells within the cyst have de-differentiated and have reentered the cell cycle. We are now testing whether those cells are, in fact, cycling and wherther they are transformed by disaggregating the cyst and placing the cells into soft agar culture.

Multipotential differentiation of human urine-derived stem cells: potential for therapeutic applications in urology.

PubMed

Bharadwaj, Shantaram; Liu, Guihua; Shi, Yingai; Wu, Rongpei; Yang, Bin; He, Tongchuan; Fan, Yuxin; Lu, Xinyan; Zhou, Xiaobo; Liu, Hong; Atala, Anthony; Rohozinski, Jan; Zhang, Yuanyuan

2013-09-01

We sought to biologically characterize and identify a subpopulation of urine-derived stem cells (USCs) with the capacity for multipotent differentiation. We demonstrated that single USCs can expand to a large population with 60-70 population doublings. Nine of 15 individual USC clones expressed detectable levels of telomerase and have long telomeres. These cells expressed pericyte and mesenchymal stem cell markers. Upon induction with appropriate media in vitro, USCs differentiated into bladder-associated cell types, including functional urothelial and smooth muscle cell lineages. When the differentiated USCs were seeded onto a scaffold and subcutaneously implanted into nude mice, multilayered tissue-like structures formed consisting of urothelium and smooth muscle. Additionally, USCs were able to differentiate into endothelial, osteogenic, chondrogenic, adipogenic, skeletal myogenic, and neurogenic lineages but did not form teratomas during the 1-month study despite telomerase activity. USCs may be useful in cell-based therapies and tissue engineering applications, including urogenital reconstruction. © AlphaMed Press.

Cloning and characterization of the hamster and guinea pig nicotinic acid receptors.

PubMed

Torhan, April Smith; Cheewatrakoolpong, Boonlert; Kwee, Lia; Greenfeder, Scott

2007-09-01

In this study, we present the identification and characterization of hamster and guinea pig nicotinic acid receptors. The hamster receptor shares approximately 80-90% identity with the nucleotide and amino acid sequences of human, mouse, and rat receptors. The guinea pig receptor shares 76-80% identity with the nucleotide and amino acid sequences of these other species. [(3)H]nicotinic acid binding affinity at guinea pig and hamster receptors is similar to that in human (dissociation constant = 121 nM for guinea pig, 72 nM for hamster, and 74 nM for human), as are potencies of nicotinic acid analogs in competition binding studies. Inhibition of forskolin-stimulated cAMP production by nicotinic acid and related analogs is also similar to the activity in the human receptor. Analysis of mRNA tissue distribution for the hamster and guinea pig nicotinic acid receptors shows expression across a number of tissues, with higher expression in adipose, lung, skeletal muscle, spleen, testis, and ovary.