Identification and functional characterization of the soybean GmaPPO12 promoter conferring Phytophthora sojae induced expression.

PubMed

Chai, Chunyue; Lin, Yanling; Shen, Danyu; Wu, Yuren; Li, Hongjuan; Dou, Daolong

2013-01-01

Identification of pathogen-inducible promoters largely lags behind cloning of the genes for disease resistance. Here, we cloned the soybean GmaPPO12 gene and found that it was rapidly and strongly induced by Phytophthorasojae infection. Computational analysis revealed that its promoter contained many known cis-elements, including several defense related transcriptional factor-binding boxes. We showed that the promoter could mediate induction of GUS expression upon infection in both transient expression assays in Nicotianabenthamiana and stable transgenic soybean hairy roots. Importantly, we demonstrated that pathogen-induced expression of the GmaPPO12 promoter was higher than that of the soybean GmaPR1a promoter. A progressive 5' and 3' deletion analysis revealed two fragments that were essential for promoter activity. Thus, the cloned promoter could be used in transgenic plants to enhance resistance to phytophthora pathogens, and the identified fragment could serve as a candidate to produce synthetic pathogen-induced promoters.

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.

High-throughput cloning and expression library creation for functional proteomics.

PubMed

Festa, Fernanda; Steel, Jason; Bian, Xiaofang; Labaer, Joshua

2013-05-01

The study of protein function usually requires the use of a cloned version of the gene for protein expression and functional assays. This strategy is particularly important when the information available regarding function is limited. The functional characterization of the thousands of newly identified proteins revealed by genomics requires faster methods than traditional single-gene experiments, creating the need for fast, flexible, and reliable cloning systems. These collections of ORF clones can be coupled with high-throughput proteomics platforms, such as protein microarrays and cell-based assays, to answer biological questions. In this tutorial, we provide the background for DNA cloning, discuss the major high-throughput cloning systems (Gateway® Technology, Flexi® Vector Systems, and Creator(TM) DNA Cloning System) and compare them side-by-side. We also report an example of high-throughput cloning study and its application in functional proteomics. This tutorial is part of the International Proteomics Tutorial Programme (IPTP12). © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Expression cloning of human B cell immunoglobulins.

PubMed

Wardemann, Hedda; Kofer, Juliane

2013-01-01

The majority of lymphomas originate from B cells at the germinal center stage or beyond. Preferential selection of B cell clones by a limited set of antigens has been suggested to drive lymphoma development. However, little is known about the specificity of the antibodies expressed by lymphoma cells, and the role of antibody-specificity in lymphomagenesis remains elusive. Here, we describe a strategy to characterize the antibody reactivity of human B cells. The approach allows the unbiased characterization of the human antibody repertoire on a single cell level through the generation of recombinant monoclonal antibodies from single primary human B cells of defined origin. This protocol offers a detailed description of the method starting from the flow cytometric isolation of single human B cells, to the RT-PCR-based amplification of the expressed Igh, Igκ, and Igλ chain genes, and Ig gene expression vector cloning for the in vitro production of monoclonal antibodies. The strategy may be used to obtain information on the clonal evolution of B cell lymphomas by single cell Ig gene sequencing and on the antibody reactivity of human lymphoma B cells.

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

High-Throughput Cloning and Expression Library Creation for Functional Proteomics

PubMed Central

Festa, Fernanda; Steel, Jason; Bian, Xiaofang; Labaer, Joshua

2013-01-01

The study of protein function usually requires the use of a cloned version of the gene for protein expression and functional assays. This strategy is particular important when the information available regarding function is limited. The functional characterization of the thousands of newly identified proteins revealed by genomics requires faster methods than traditional single gene experiments, creating the need for fast, flexible and reliable cloning systems. These collections of open reading frame (ORF) clones can be coupled with high-throughput proteomics platforms, such as protein microarrays and cell-based assays, to answer biological questions. In this tutorial we provide the background for DNA cloning, discuss the major high-throughput cloning systems (Gateway® Technology, Flexi® Vector Systems, and Creator™ DNA Cloning System) and compare them side-by-side. We also report an example of high-throughput cloning study and its application in functional proteomics. This Tutorial is part of the International Proteomics Tutorial Programme (IPTP12). Details can be found at http://www.proteomicstutorials.org. PMID:23457047

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.

Leptospira borgpetersenii hybrid leucine-rich repeat protein: Cloning and expression, immunogenic identification and molecular docking evaluation.

PubMed

Sritrakul, Tepyuda; Nitipan, Supachai; Wajjwalku, Worawidh; La-Ard, Anchalee; Suphatpahirapol, Chattip; Petkarnjanapong, Wimol; Ongphiphadhanakul, Boonsong; Prapong, Siriwan

2017-11-01

Leptospirosis is an important zoonotic disease, and the major outbreak of this disease in Thailand in 1999 was due largely to the Leptospira borgpetersenii serovar Sejroe. Identification of the leucine-rich repeat (LRR) LBJ_2271 protein containing immunogenic epitopes and the discovery of the LBJ_2271 ortholog in Leptospira serovar Sejroe, KU_Sej_R21_2271, led to further studies of the antigenic immune properties of KU_Sej_LRR_2271. The recombinant hybrid (rh) protein was created and expressed from a hybrid PCR fragment of KU_Sej_R21_2271 fused with DNA encoding the LBJ_2271 signal sequence for targeting protein as a membrane-anchoring protein. The fusion DNA was cloned into pET160/GW/D-TOPO® to form the pET160_hKU_R21_2271 plasmid. The plasmid was used to express the rhKU_Sej_LRR_2271 protein in Escherichia coli BL21 Star™ (DE3). The expressed protein was immunologically detected by Western blotting and immunoreactivity detection with hyperimmune sera, T cell epitope prediction by HLA allele and epitope peptide binding affinity, and potential T cell reactivity analysis. The immunogenic epitopes of the protein were evaluated and verified by HLA allele and epitope peptide complex structure molecular docking. Among fourteen best allele epitopes of this protein, binding affinity values of 12 allele epitopes remained unchanged compared to LBJ_2271. Two epitopes for alleles HLA-A0202 and -A0301 had higher IC 50 values, while T cell reactivity values of these peptides were better than values from LBJ_2271 epitopes. Eight of twelve epitope peptides had positive T-cell reactivity scores. Although the molecular docking of two epitopes, 3FPLLKEFLV11/47FPLLKEFLV55 and 50KLSTVPEGV58, into an HLA-A0202 model revealed a good fit in the docked structures, 50KLSTVPEGV58 and 94KLSTVPEEV102 are still considered as the proteins' best epitopes for allele HLA-A0202. The results of this study showed that rhKU_Sej_LRR_2271 protein contained natural immunological properties that should

Echinostoma caproni: identification of enolase in excretory/secretory products, molecular cloning, and functional expression.

PubMed

Marcilla, Antonio; Pérez-García, Ana; Espert, Ana; Bernal, Dolores; Muñoz-Antolí, Carla; Esteban, José Guillermo; Toledo, Rafael

2007-09-01

In order to investigate molecules that could be involved in host-trematode relationships, we have analysed the excretory/secretory products (ESP) of Echinostoma caproni following a proteomic approach. Actin, Gluthathione S-transferase (GST) and enolase have been identified in the ESP. Enolase, observed to be one of the most abundant proteins, was further characterized. The molecular cloning and in vitro expression in Escherichia coli of E. caproni enolase allowed us to determine that the protein contains 431 amino acids and a theoretical MW of 46272 Da. E. caproni enolase shows high homology to other trematode enolases. The recombinant protein binds specifically to human plasminogen in vitro, as observed for the native protein, confirming its properties as a host-interacting molecule.

[Cloning of human CD45 gene and its expression in Hela cells].

PubMed

Li, Jie; Xu, Tianyu; Wu, Lulin; Zhang, Liyun; Lu, Xiao; Zuo, Daming; Chen, Zhengliang

2015-11-01

To clone human CD45 gene PTPRC and establish Hela cells overexpressing recombinant human CD45 protein. The intact cDNA encoding human CD45 amplified using RT-PCR from the total RNA extracted from peripheral blood mononuclear cells (PBMCs) of a healthy donor was cloned into pMD-18T vector. The CD45 cDNA fragment amplified from the pMD-18T-CD45 by PCR was inserted to the coding region of the PcDNA3.1-3xflag vector, and the resultant recombinant expression vector PcDNA3.1-3xflag-CD45 was transfected into Hela cells. The expression of CD45 in Hela cells was detected by flow cytometry and Western blotting, and the phosphastase activity of CD45 was quantified using an alkaline phosphatase assay kit. The cDNA fragment of about 3 900 bp was amplified from human PBMCs and cloned into pMD-18T vector. The recombinant expression vector PcDNA3.1-3xflag-CD45 was constructed, whose restriction maps and sequence were consistent with those expected. The expression of CD45 in transfected Hela cells was detected by flow cytometry and Western blotting, and the expressed recombinant CD45 protein in Hela cells showed a phosphastase activity. The cDNA of human CD45 was successfully cloned and effectively expressed in Hela cells, which provides a basis for further exploration of the functions of CD45.

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.

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.

Expression of Innate Immune Response Genes in Liver and Three Types of Adipose Tissue in Cloned Pigs

PubMed Central

Rødgaard, Tina; Skovgaard, Kerstin; Stagsted, Jan

2012-01-01

Abstract The pig has been proposed as a relevant model for human obesity-induced inflammation, and cloning may improve the applicability of this model. We tested the assumptions that cloning would reduce interindividual variation in gene expression of innate immune factors and that their expression would remain unaffected by the cloning process. We investigated the expression of 40 innate immune factors by high-throughput quantitative real-time PCR in samples from liver, abdominal subcutaneous adipose tissue (SAT), visceral adipose tissue (VAT), and neck SAT in cloned pigs compared to normal outbred pigs. The variation in gene expression was found to be similar for the two groups, and the expression of a small number of genes was significantly affected by cloning. In the VAT and abdominal SAT, six out of seven significantly differentially expressed genes were downregulated in the clones. In contrast, most differently expressed genes in both liver and neck SAT were upregulated (seven out of eight). Remarkably, acute phase proteins (APPs) dominated the upregulated genes in the liver, whereas APP expression was either unchanged or downregulated in abdominal SAT and VAT. The general conclusion from this work is that cloning leads to subtle changes in specific subsets of innate immune genes. Such changes, even if minor, may have phenotypic effects over time, e.g., in models of long-term inflammation related to obesity. PMID:22928970

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.

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.

Clones identification of Sequoia sempervirens (D. Don) Endl. in Chile by using PCR-RAPDs technique.

PubMed

Toral Ibañez, Manuel; Caru, Margarita; Herrera, Miguel A; Gonzalez, Luis; Martin, Luis M; Miranda, Jorge; Navarro-Cerrillo, Rafael M

2009-02-01

A protocol of polymerase chain reaction-random amplified polymorphic DNAs (PCR-RAPDs) was established to analyse the gene diversity and genotype identification for clones of Sequoia sempervirens (D. Don) Endl. in Chile. Ten (out of 34) clones from introduction trial located in Voipir-Villarrica, Chile, were studied. The PCR-RAPDs technique and a modified hexadecyltrimethylammonium bromide (CTAB) protocol were used for genomic DNA extraction. The PCR tests were carried out employing 10-mer random primers. The amplification products were detected by electrophoresis in agarose gels. Forty nine polymorphic bands were obtained with the selected primers (BG04, BF07, BF12, BF13, and BF14) and were ordered according to their molecular size. The genetic similarity between samples was calculated by the Jaccard index and a dendrogram was constructed using a cluster analysis of unweighted pair group method using arithmetic averages (UPGMA). Of the primers tested, 5 (out of 60) RAPD primers were selected for their reproducibility and high polymorphism. A total of 49 polymorphic RAPD bands were detected out of 252 bands. The genetic similarity analysis demonstrates an extensive genetic variability between the tested clones and the dendrogram depicts the genetic relationships among the clones, suggesting a geographic relationship. The results indicate that the RAPD markers permitted the identification of the assayed clones, although they are derived from the same geographic origin.

Clones identification of Sequoia sempervirens (D. Don) Endl. in Chile by using PCR-RAPDs technique*

PubMed Central

Toral Ibañez, Manuel; Caru, Margarita; Herrera, Miguel A.; Gonzalez, Luis; Martin, Luis M.; Miranda, Jorge; Navarro-Cerrillo, Rafael M.

2009-01-01

A protocol of polymerase chain reaction-random amplified polymorphic DNAs (PCR-RAPDs) was established to analyse the gene diversity and genotype identification for clones of Sequoia sempervirens (D. Don) Endl. in Chile. Ten (out of 34) clones from introduction trial located in Voipir-Villarrica, Chile, were studied. The PCR-RAPDs technique and a modified hexadecyltrimethylammonium bromide (CTAB) protocol were used for genomic DNA extraction. The PCR tests were carried out employing 10-mer random primers. The amplification products were detected by electrophoresis in agarose gels. Forty nine polymorphic bands were obtained with the selected primers (BG04, BF07, BF12, BF13, and BF14) and were ordered according to their molecular size. The genetic similarity between samples was calculated by the Jaccard index and a dendrogram was constructed using a cluster analysis of unweighted pair group method using arithmetic averages (UPGMA). Of the primers tested, 5 (out of 60) RAPD primers were selected for their reproducibility and high polymorphism. A total of 49 polymorphic RAPD bands were detected out of 252 bands. The genetic similarity analysis demonstrates an extensive genetic variability between the tested clones and the dendrogram depicts the genetic relationships among the clones, suggesting a geographic relationship. The results indicate that the RAPD markers permitted the identification of the assayed clones, although they are derived from the same geographic origin. PMID:19235269

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

Identification of human antibody fragment clones specific for tetanus toxoid in a bacteriophage. lambda. immunoexpression library

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

Mullinax, R.L.; Gross, E.A.; Amberg, J.R.

1990-10-01

The authors have applied a molecular biology approach to the identification of human monoclonal antibodies. Human peripheral blood lymphocyte mRNA was converted to cDNA and a select subset was amplified by the polymerase chain reaction. These products, containing coding sequences for numerous immunoglobulin heavy- and {kappa} light-chain variable and constant region domains, were inserted into modified bacteriophase {lambda} expression vectors and introduced into Escherichia coli by infection to yield a combinatorial immunoexpression library. Clones with binding activity to tetanus toxoid were identified by filter hybridization with radiolabeled antigen and appeared at a frequency of 0.2{percent} in the library. These humanmore » antigen binding fragments, consisting of a heavy-chain fragment covalently linked to a light chain, displayed high affinity of binding to tetanus toxoid with equilibrium constants in the nanomolar range but did not cross-react with other proteins tested. They estimate that this human immunoexpression library contains 20,000 clones with high affinity and specificity to our chosen antigen.« less

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.

Development and characterization of K562 cell clones expressing BCL11A-XL: Decreased hemoglobin production with fetal hemoglobin inducers and its rescue with mithramycin

PubMed Central

Finotti, Alessia; Gasparello, Jessica; Breveglieri, Giulia; Cosenza, Lucia Carmela; Montagner, Giulia; Bresciani, Alberto; Altamura, Sergio; Bianchi, Nicoletta; Martini, Elisa; Gallerani, Eleonora; Borgatti, Monica; Gambari, Roberto

2015-01-01

Induction of fetal hemoglobin (HbF) is considered a promising strategy in the treatment of β-thalassemia, in which production of adult hemoglobin (HbA) is impaired by mutations affecting the β-globin gene. Recent results indicate that B-cell lymphoma/leukemia 11A (BCL11A) is a major repressor of γ-globin gene expression. Therefore, disrupting the binding of the BCL11A transcriptional repressor complex to the γ-globin gene promoter provides a novel approach for inducing expression of the γ-globin genes. To develop a cellular screening system for the identification of BCL11A inhibitors, we produced K562 cell clones with integrated copies of a BCL11A-XL expressing vector. We characterized 12 K562 clones expressing different levels of BCL11A-XL and found that a clear inverse relationship does exist between the levels of BCL11A-XL and the extent of hemoglobinization induced by a panel of HbF inducers. Using mithramycin as an inducer, we found that this molecule was the only HbF inducer efficient in rescuing the ability to differentiate along the erythroid program, even in K562 cell clones expressing high levels of BCL11A-XL, suggesting that BCL11A-XL activity is counteracted by mithramycin. PMID:26342260

[Cloning of Clostridium perfringens alpha-toxin gene and extracellular expression in Escherichia coli].

PubMed

Inoue, Masaharu; Kikuchi, Maho; Komoriya, Tomoe; Watanabe, Kunitomo; Kouno, Hideki

2007-01-01

Clostridium perfringens (C. perfringens) is a Gram-positive bacterial pathogen that widely propagets in the soil and the gastrointestinal tract of human and animals. This bacteria causes food poisoning, gas gangrene and other various range of infectious diseases. But there is no standard diagnosis method of C. perfringens. In order to develop a new type of immunoassay for clinical purpose, we studied expression and extracellular secretion of recombinant alpha-toxin having enzyme activity in E. coli expression system. Cloning was carried out after PCR amplification from C. perfringens GAI 94074 which was clinical isolate. Three kinds of fragment were cloned using pET100/D-TOPO vector. These fragments coded for ribosome binding site, signal peptide, and alpha-toxin gene respectively. Recombinant pET100 plasmid transformed into TOP 10 cells and the obtained plasmids were transformed into BL21 (DE3) cells. Then, the transformants were induced expression with IPTG. In conclusion, we successfully cloned, expressed and exteracellular secreted C. perfringens alpha-toxin containing signal peptide. Biologically, the obtained recombinant protein was positive for phospholipase C activity.

Purification, cloning, expression and immunological analysis of Scylla serrata arginine kinase, the crab allergen.

PubMed

Shen, Yuan; Cao, Min-Jie; Cai, Qiu-Feng; Su, Wen-Jin; Yu, Hui-Lin; Ruan, Wei-Wei; Liu, Guang-Ming

2011-05-01

Although crustaceans have been reported to be one of the most common causes of IgE-mediated allergic reactions, there are no reports about the characterization and identification of arginine kinase (AK) from the mud crab (Scylla serrata) as allergen. In the present study, the purification, molecular cloning, expression and immunological analyses of the IgE allergen AK from the mud crab were investigated. The results showed that cloned DNA fragments of AK from the mud crab had open reading frames of 1021 bp, predicted to encode proteins with 356 amino acid residues. Sequence alignment revealed that mud crab AK shares high homology with other crustacean species. Mud crab AK gene was further recombined with the vector of pGEX-4T-3 and expressed in Escherichia coli BL 21. 2-D electrophoresis suggested that native AK (nAK) and recombinant AK (rAK) shared the same molecular weight of 40 kDa, and the pI is 6.5 and 6.3, respectively. The nAK and rAK were further confirmed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Immunoblotting analysis and colloidal gold immunochromatographic assay (GICA) using sera from subjects with crustacean allergy confirmed that the nAK and rAK reacted positively with these sera, indicating AK is a specific allergen of mud crab. Both of purified nAK and rAK reacted positively with sera from subjects with crustacean allergy in immunoblotting and GICA analysis, indicating AK is a common allergen of mud crab. In vitro expressed AK is proposed as a source of the protein for immunological or clinical studies. Copyright © 2011 Society of Chemical Industry.

Cloning, Expression, and Purification of Histidine-Tagged Escherichia coli Dihydrodipicolinate Reductase.

PubMed

Trigoso, Yvonne D; Evans, Russell C; Karsten, William E; Chooback, Lilian

2016-01-01

The enzyme dihydrodipicolinate reductase (DHDPR) is a component of the lysine biosynthetic pathway in bacteria and higher plants. DHDPR catalyzes the NAD(P)H dependent reduction of 2,3-dihydrodipicolinate to the cyclic imine L-2,3,4,5,-tetrahydropicolinic acid. The dapB gene that encodes dihydrodipicolinate reductase has previously been cloned, but the expression of the enzyme is low and the purification is time consuming. Therefore the E. coli dapB gene was cloned into the pET16b vector to improve the protein expression and simplify the purification. The dapB gene sequence was utilized to design forward and reverse oligonucleotide primers that were used to PCR the gene from Escherichia coli genomic DNA. The primers were designed with NdeI or BamHI restriction sites on the 5'and 3' terminus respectively. The PCR product was sequenced to confirm the identity of dapB. The gene was cloned into the expression vector pET16b through NdeI and BamHI restriction endonuclease sites. The resulting plasmid containing dapB was transformed into the bacterial strain BL21 (DE3). The transformed cells were utilized to grow and express the histidine-tagged reductase and the protein was purified using Ni-NTA affinity chromatography. SDS/PAGE gel analysis has shown that the protein was 95% pure and has approximate subunit molecular weight of 28 kDa. The protein purification is completed in one day and 3 liters of culture produced approximately 40-50 mgs of protein, an improvement on the previous protein expression and multistep purification.

Development and characterization of K562 cell clones expressing BCL11A-XL: Decreased hemoglobin production with fetal hemoglobin inducers and its rescue with mithramycin.

PubMed

Finotti, Alessia; Gasparello, Jessica; Breveglieri, Giulia; Cosenza, Lucia Carmela; Montagner, Giulia; Bresciani, Alberto; Altamura, Sergio; Bianchi, Nicoletta; Martini, Elisa; Gallerani, Eleonora; Borgatti, Monica; Gambari, Roberto

2015-12-01

Induction of fetal hemoglobin (HbF) is considered a promising strategy in the treatment of β-thalassemia, in which production of adult hemoglobin (HbA) is impaired by mutations affecting the β-globin gene. Recent results indicate that B-cell lymphoma/leukemia 11A (BCL11A) is a major repressor of γ-globin gene expression. Therefore, disrupting the binding of the BCL11A transcriptional repressor complex to the γ-globin gene promoter provides a novel approach for inducing expression of the γ-globin genes. To develop a cellular screening system for the identification of BCL11A inhibitors, we produced K562 cell clones with integrated copies of a BCL11A-XL expressing vector. We characterized 12 K562 clones expressing different levels of BCL11A-XL and found that a clear inverse relationship does exist between the levels of BCL11A-XL and the extent of hemoglobinization induced by a panel of HbF inducers. Using mithramycin as an inducer, we found that this molecule was the only HbF inducer efficient in rescuing the ability to differentiate along the erythroid program, even in K562 cell clones expressing high levels of BCL11A-XL, suggesting that BCL11A-XL activity is counteracted by mithramycin. Copyright © 2015 ISEH - International Society for Experimental Hematology. Published by Elsevier Inc. All rights reserved.

Identification, cloning, and expression analysis of three putative Lymantria dispar nuclear polyhedrosis virus immediate early genes

Treesearch

James M. Slavicek; Nancy Hayes-Plazolles

1991-01-01

Viral immediate early gene products are usually regulatory proteins that control expression of other viral genes at the transcriptional level or are proteins that are part of the viral DNA replication complex. The identification and functional characterization of the immediate early gene products of Lymantria dispar nuclear polyhedrosis virus (LdNPV...

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.
.

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.

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.

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.

Identification and Cloning of Centaurin-α

PubMed Central

Hammonds-Odie, Latanya P.; Jackson, Trevor R.; Profit, Adam A.; Blader, Ira J.; Turck, Christoph W.; Prestwich, Glenn D.; Theibert, Anne B.

2015-01-01

Using an affinity resin and photoaffinity label based on phospholipid analogs of inositol 1,3,4,5-tetrakisphosphate (InsP4), we have isolated, characterized, and cloned a 46-kDa protein from rat brain, which we have named centaurin-α. Binding specificity was determined using displacement of 1-O-[3H](3-[4-benzoyldihydrocinnamidyl]propyl)-InsP4 photoaffinity labeling. Centaurin-α displayed highest affinity for phosphatidylinositol 3,4,5-trisphosphate (PtdInsP3) (IC50 = 120 nm), whereas InsP4, PtdInsP2, and InsP3 bound with 5-, 12-, and >50-fold lower affinity, respectively. Screening a rat brain cDNA library with a polymerase chain reaction product, generated using partial amino acid sequence from tryptic peptides, yielded a full-length clone. The 2,450-base pair cDNA contained an open reading frame (ORF) encoding a novel protein of 419 amino acids. Northern analysis revealed a 2.5-kilobase transcript that is highly expressed in brain. The deduced sequence contains a novel putative zinc finger motif, 10 ankyrin-like repeats, and shows homology to recently identified yeast and mammalian Arf GTPase-activating proteins. Given the specificity of binding and enrichment in brain, centaurin-α is a candidate PtdInsP3 receptor that may link the activation of phosphoinositide 3-kinase to downstream responses in the brain. PMID:8702546

Cloning, Expression, and Purification of Histidine-Tagged Escherichia coli Dihydrodipicolinate Reductase

PubMed Central

Trigoso, Yvonne D.; Evans, Russell C.; Karsten, William E.; Chooback, Lilian

2016-01-01

The enzyme dihydrodipicolinate reductase (DHDPR) is a component of the lysine biosynthetic pathway in bacteria and higher plants. DHDPR catalyzes the NAD(P)H dependent reduction of 2,3-dihydrodipicolinate to the cyclic imine L-2,3,4,5,-tetrahydropicolinic acid. The dapB gene that encodes dihydrodipicolinate reductase has previously been cloned, but the expression of the enzyme is low and the purification is time consuming. Therefore the E. coli dapB gene was cloned into the pET16b vector to improve the protein expression and simplify the purification. The dapB gene sequence was utilized to design forward and reverse oligonucleotide primers that were used to PCR the gene from Escherichia coli genomic DNA. The primers were designed with NdeI or BamHI restriction sites on the 5’and 3’ terminus respectively. The PCR product was sequenced to confirm the identity of dapB. The gene was cloned into the expression vector pET16b through NdeI and BamHI restriction endonuclease sites. The resulting plasmid containing dapB was transformed into the bacterial strain BL21 (DE3). The transformed cells were utilized to grow and express the histidine-tagged reductase and the protein was purified using Ni-NTA affinity chromatography. SDS/PAGE gel analysis has shown that the protein was 95% pure and has approximate subunit molecular weight of 28 kDa. The protein purification is completed in one day and 3 liters of culture produced approximately 40–50 mgs of protein, an improvement on the previous protein expression and multistep purification. PMID:26815040

Rapid high-throughput cloning and stable expression of antibodies in HEK293 cells.

PubMed

Spidel, Jared L; Vaessen, Benjamin; Chan, Yin Yin; Grasso, Luigi; Kline, J Bradford

2016-12-01

Single-cell based amplification of immunoglobulin variable regions is a rapid and powerful technique for cloning antigen-specific monoclonal antibodies (mAbs) for purposes ranging from general laboratory reagents to therapeutic drugs. From the initial screening process involving small quantities of hundreds or thousands of mAbs through in vitro characterization and subsequent in vivo experiments requiring large quantities of only a few, having a robust system for generating mAbs from cloning through stable cell line generation is essential. A protocol was developed to decrease the time, cost, and effort required by traditional cloning and expression methods by eliminating bottlenecks in these processes. Removing the clonal selection steps from the cloning process using a highly efficient ligation-independent protocol and from the stable cell line process by utilizing bicistronic plasmids to generate stable semi-clonal cell pools facilitated an increased throughput of the entire process from plasmid assembly through transient transfections and selection of stable semi-clonal cell pools. Furthermore, the time required by a single individual to clone, express, and select stable cell pools in a high-throughput format was reduced from 4 to 6months to only 4 to 6weeks. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

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.

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

[Cloning and characterization of genes differentially expressed in human dental pulp cells and gingival fibroblasts].

PubMed

Wang, Zhong-dong; Wu, Ji-nan; Zhou, Lin; Ling, Jun-qi; Guo, Xi-min; Xiao, Ming-zhen; Zhu, Feng; Pu, Qin; Chai, Yu-bo; Zhao, Zhong-liang

2007-02-01

To study the biological properties of human dental pulp cells (HDPC) by cloning and analysis of genes differentially expressed in HDPC in comparison with human gingival fibroblasts (HGF). HDPC and HGF were cultured and identified by immunocytochemistry. HPDC and HGF subtractive cDNA library was established by PCR-based modified subtractive hybridization, genes differentially expressed by HPDC were cloned, sequenced and compared to find homogeneous sequence in GenBank by BLAST. Cloning and sequencing analysis indicate 12 genes differentially expressed were obtained, in which two were unknown genes. Among the 10 known genes, 4 were related to signal transduction, 2 were related to trans-membrane transportation (both cell membrane and nuclear membrane), and 2 were related to RNA splicing mechanisms. The biological properties of HPDC are determined by the differential expression of some genes and the growth and differentiation of HPDC are associated to the dynamic protein synthesis and secretion activities of the cell.

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.

Cloning and Expression of cDNA for Rat Heme Oxygenase

NASA Astrophysics Data System (ADS)

Shibahara, Shigeki; Muller, Rita; Taguchi, Hayao; Yoshida, Tadashi

1985-12-01

Two cDNA clones for rat heme oxygenase have been isolated from a rat spleen cDNA library in λ gt11 by immunological screening using a specific polyclonal antibody. One of these clones has an insert of 1530 nucleotides that contains the entire protein-coding region. To confirm that the isolated cDNA encodes heme oxygenase, we transfected monkey kidney cells (COS-7) with the cDNA carried in a simian virus 40 vector. The heme oxygenase was highly expressed in endoplasmic reticulum of transfected cells. The nucleotide sequence of the cloned cDNA was determined and the primary structure of heme oxygenase was deduced. Heme oxygenase is composed of 289 amino acids and has one hydrophobic segment at its carboxyl terminus, which is probably important for the insertion of heme oxygenase into endoplasmic reticulum. The cloned cDNA was used to analyze the induction of heme oxygenase in rat liver by treatment with CoCl2 or with hemin. RNA blot analysis showed that both CoCl2 and hemin increased the amount of hybridizable mRNA, suggesting that these substances may act at the transcriptional level to increase the amount of heme oxygenase.

Handmade Cloned Transgenic Piglets Expressing the Nematode Fat-1 Gene

PubMed Central

Zhang, Peng; Zhang, Yidi; Dou, Hongwei; Yin, Jingdong; Chen, Yu; Pang, Xinzhi; Vajta, Gabor; Bolund, Lars

2012-01-01

Abstract Production of transgenic animals via somatic cell nuclear transfer (SCNT) has been adapted worldwide, but this application is somewhat limited by its relatively low efficiency. In this study, we used handmade cloning (HMC) established previously to produce transgenic pigs that express the functional nematode fat-1 gene. Codon-optimized mfat-1 was inserted into eukaryotic expression vectors, which were transferred into primary swine donor cells. Reverse transcriptase PCR (RT-PCR), gas chromatography, and chromosome analyses were performed to select donor clones capable of converting n-6 into n-3 fatty acids. Blastocysts derived from the clones that lowered the n-6/n-3 ratio to approximately 1:1 were transferred surgically into the uteri of recipients for transgenic piglets. By HMC, 37% (n=558) of reconstructed embryos developed to the blastocyst stage after 7 days of culture in vitro, with an average cell number of 81±36 (n=14). Three recipients became pregnant after 408 day-6 blastocysts were transferred into four naturally cycling females, and a total of 14 live offspring were produced. The nematode mfat-1 effectively lowered the n-6/n-3 ratio in muscle and major organs of the transgenic pig. Our results will help to establish a reliable procedure and an efficient option in the production of transgenic animals. PMID:22686479

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

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.

Molecular transformation, gene cloning, and gene expression systems for filamentous fungi

USGS Publications Warehouse

Gold, Scott E.; Duick, John W.; Redman, Regina S.; Rodriguez, Rusty J.

2001-01-01

This chapter discusses the molecular transformation, gene cloning, and gene expression systems for filamentous fungi. Molecular transformation involves the movement of discrete amounts of DNA into cells, the expression of genes on the transported DNA, and the sustainable replication of the transforming DNA. The ability to transform fungi is dependent on the stable replication and expression of genes located on the transforming DNA. Three phenomena observed in bacteria, that is, competence, plasmids, and restriction enzymes to facilitate cloning, were responsible for the development of molecular transformation in fungi. Initial transformation success with filamentous fungi, involving the complementation of auxotrophic mutants by exposure to sheared genomic DNA or RNA from wt isolates, occurred with low transformation efficiencies. In addition, it was difficult to retrieve complementing DNA fragments and isolate genes of interest. This prompted the development of transformation vectors and methods to increase efficiencies. The physiological studies performed with fungi indicated that the cell wall could be removed to generate protoplasts. It was evident that protoplasts could be transformed with significantly greater efficiencies than walled cells.

Cloning and expression of autogenes encoding RNA polymerases of T7-like bacteriophages

DOEpatents

Studier, F. William; Dubendorff, John W.

1998-01-01

This invention relates to the cloning and expression of autogenes encoding RNA polymerases of T7 and T7-like bacteriophages, in which the RNA polymerase gene is transcribed from a promoter which is recognized by the encoded RNA polymerase. Cloning of T7 autogenes was achieved by reducing the activity of the RNA polymerase sufficiently to permit host cell growth. T7 RNA polymerase activity was controlled by combining two independent methods: lac-repression of the recombinant lac operator-T7 promoter in the autogene and inhibition of the polymerase by T7 lysozyme. Expression systems for producing the RNA polymerases of T7 and other T7-like bacteriophages, and expression systems for producing selected gene products are described, as well as other related materials and methods.

Cloning and expression of autogenes encoding RNA polymerases of T7-like bacteriophages

DOEpatents

Studier, F.W.; Dubendorff, J.W.

1998-10-20

This invention relates to the cloning and expression of autogenes encoding RNA polymerases of T7 and T7-like bacteriophages, in which the RNA polymerase gene is transcribed from a promoter which is recognized by the encoded RNA polymerase. Cloning of T7 autogenes was achieved by reducing the activity of the RNA polymerase sufficiently to permit host cell growth. T7 RNA polymerase activity was controlled by combining two independent methods: lac-repression of the recombinant lac operator-T7 promoter in the autogene and inhibition of the polymerase by T7 lysozyme. Expression systems for producing the RNA polymerases of T7 and other T7-like bacteriophages, and expression systems for producing selected gene products are described, as well as other related materials and methods. 12 figs.

Cloning and expression of autogenes encoding RNA polymerases of T7-like bacteriophages

DOEpatents

Studier, F.W.; Dubendorff, J.W.

1998-11-03

This invention relates to the cloning and expression of autogenes encoding RNA polymerases of T7 and T7-like bacteriophages, in which the RNA polymerase gene is transcribed from a promoter which is recognized by the encoded RNA polymerase. Cloning of T7 autogenes was achieved by reducing the activity of the RNA polymerase sufficiently to permit host cell growth. T7 RNA polymerase activity was controlled by combining two independent methods: lac-repression of the recombinant lac operator-T7 promoter in the autogene and inhibition of the polymerase by T7 lysozyme. Expression systems for producing the RNA polymerases of T7 and other T7-like bacteriophages, and expression systems for producing selected gene products are described, as well as other related materials and methods. 12 figs.

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.

Chapter 7. Cloning and analysis of natural product pathways.

PubMed

Gust, Bertolt

2009-01-01

The identification of gene clusters of natural products has lead to an enormous wealth of information about their biosynthesis and its regulation, and about self-resistance mechanisms. Well-established routine techniques are now available for the cloning and sequencing of gene clusters. The subsequent functional analysis of the complex biosynthetic machinery requires efficient genetic tools for manipulation. Until recently, techniques for the introduction of defined changes into Streptomyces chromosomes were very time-consuming. In particular, manipulation of large DNA fragments has been challenging due to the absence of suitable restriction sites for restriction- and ligation-based techniques. The homologous recombination approach called recombineering (referred to as Red/ET-mediated recombination in this chapter) has greatly facilitated targeted genetic modifications of complex biosynthetic pathways from actinomycetes by eliminating many of the time-consuming and labor-intensive steps. This chapter describes techniques for the cloning and identification of biosynthetic gene clusters, for the generation of gene replacements within such clusters, for the construction of integrative library clones and their expression in heterologous hosts, and for the assembly of entire biosynthetic gene clusters from the inserts of individual library clones. A systematic approach toward insertional mutation of a complete Streptomyces genome is shown by the use of an in vitro transposon mutagenesis procedure.

Molecular cloning, sequencing, and expression of Eimeria tenella HSP70 partial gene.

PubMed

Bogado, A L G; Martins, G F; Sasse, J P; Guimarães, J da S; Garcia, J L

2017-03-15

Members of the Eimeria genus are protozoan parasites of the subphylum Apicomplexa (Eimeriidae family), and belong to the coccidia group. Eimeria tenella is one of the most pathogenic species owing to its ability to penetrate the mucosa, and cause inflammation and damage. It is an obligate intracellular parasite that causes disease by destroying the host cells during multiplication. Heat shock protein 70 (HSP70) is a molecular chaperone that prevents cellular stress. The objective of this study was to clone, sequence, and express E. tenella HSP70 protein. After selecting the region of highest hydrophilicity in the hsp70 gene, we cloned complementary DNA (cDNA) into a pTrcHis2-TOPO vector and transformed it into TOP10 Escherichia coli cells; after induction, the bacteria expressed a 23-kDa protein with insoluble expression levels of approximately 5 mg/L. In summary, the partial hsp70 gene was successfully expressed in E. coli, producing a 23-kDa protein under insoluble conditions, and the antigen characteristics predicted by hydrophilicity analysis suggest the development of a vaccine for use in avian coccidiosis.

Altered imprinted gene expression and methylation patterns in mid-gestation aborted cloned porcine fetuses and placentas.

PubMed

Zhang, Xiaoyang; Wang, Dongxu; Han, Yang; Duan, Feifei; Lv, Qinyan; Li, Zhanjun

2014-11-01

To determine the expression patterns of imprinted genes and their methylation status in aborted cloned porcine fetuses and placentas. RNA and DNA were prepared from fetuses and placentas that were produced by SCNT and controls from artificial insemination. The expression of 18 imprinted genes was determined by quantitative real-time PCR (q-PCR). Bisulfite sequencing PCR (BSP) was conducted to determine the methylation status of PRE-1 short interspersed repetitive element (SINE), satellite DNA and H19 differentially methylated region 3 (DMR3). The weight, imprinted gene expression and genome-wide DNA methylation patterns were compared between the mid-gestation aborted and normal control samples. The results showed hypermethylation of PRE-1 and satellite sequences, the aberrant expression of imprinted genes, and the hypomethylation of H19 DMR3 occurred in mid-gestation aborted fetuses and placentas. Cloned pigs generated by somatic cell nuclear transfer (SCNT) showed a greater ratio of early abortion during mid-gestation than did normal controls because of the incomplete epigenetic reprogramming of the donor cells. Altered expression of imprinted genes and the hypermethylation profile of the repetitive regions (PRE-1 and satellite DNA) may be associated with defective development and early abortion of cloned pigs, emphasizing the importance of epigenetics during pregnancy and implications thereof for patient-specific embryonic stem cells for human therapeutic cloning and improvement of human assisted reproduction.

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

Dihydroneopterin triphosphate epimerase of Escherichia coli: purification, genetic cloning, and expression.

PubMed Central

Haussmann, C; Rohdich, F; Lottspeich, F; Eberhardt, S; Scheuring, J; Mackamul, S; Bacher, A

1997-01-01

The enzyme catalyzing the epimerization at position 2' of dihydroneopterin triphosphate was purified by a factor of about 10,000 from cell extract of Escherichia coli. The cognate gene was cloned, sequenced, expressed, and mapped to kb 2427 on the E. coli chromosome. PMID:9006053

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

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

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.

Cloning and expression of Tenebrio molitor antifreeze protein in Escherichia coli.

PubMed

Yue, Chang-Wu; Zhang, Yi-Zheng

2009-03-01

A novel antifreeze protein cDNA was cloned by RT-PCR from the larva of the yellow mealworm Tenebrio molitor. The coding fragment of 339 bp encodes a protein of 112 amino acid residues and was fused to the expression vectors pET32a and pTWIN1. The resulted expression plasmids were transformed into Escherischia coli strains BL21 (DE3), ER2566, and Origami B (DE3), respectively. Several strategies were used for expression of the highly disulfide-bonded beta-helix-contained protein with the activity of antifreeze in different expression systems. A protocol for production of refolded and active T. molitor antifreeze protein in bacteria was obtained.

[Cloning, expression and identification of functional fragment rC3B of human complement C3 in E. Coli].

PubMed

Gan, Hui; Zhou, Yong; Sun, Ping; Zhu, Xiao-Xia; Wang, Quan-Li; Zhan, Lin-Sheng

2007-08-01

This study was purposed to verify the binding part of human complement C3 to complement receptor III (CRIII) in monocytes, the peptide rC3B, including the binding-site, was expressed, purified and identified. rC3B, the binding part of human complement C3 to CRIII, was selected by computer-aided modeling and summarizing researches published. Then, rC3B gene fragment was amplified by PCR, and cloned into prokaryotic vector pQE30a. The fusion protein rC3B was expressed in E.coli M15 and purified by Ni(2+)-chelating affinity chromatography. The activity of rC3B was identified by Western blot and adherence assay with monocytes. The results showed that rC3B fragment was obtained, and a prokaryotic expression vector pQE30-rC3B was constructed. rC3B was efficiently expressed and purified. In Western blot, the target protein showed the activity of binding with C3 antibody, while the purified protein showed the activity of adherence with monocytes. It is concluded that the recombinant C3B was obtained and identified, and this study lay the basis for the further functional analysis of C3.

Cloning and expression of autogenes encoding RNA poly,erases of T7-like bacteriophages

DOEpatents

Studier, F. William; Dubendorff, John W.

1998-01-01

This invention relates to the cloning and expression of autogenes encoding RNA polymerases of T7 and T7-like bacteriophages, in which the RNA polymerase gene is transcribed from a promoter which is recognized by the encoded RNA polymerase. Cloning of T7 autogenes was achieved by reducing the activity of the RNA polymerase sufficiently to permit host cell growth. T7 RNA polymerase activity was controlled by combining two independent methods: lac-repression of the recombinant lac operator-T7 promoter in the autogene and inhibition of the polymerase by T7 lysozyme. Expression systems for producing the RNA polymerases of T7 and other T7-like bacteriophages, and expression systems for producing selected gene products are described, as well as other related materials and methods.

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 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.

Cloning of a yeast alpha-amylase promoter and its regulated heterologous expression

DOEpatents

Gao, Johnway [Richland, WA; Skeen, Rodney S [Pendleton, OR; Hooker, Brian S [Kennewick, WA; Anderson, Daniel B [Pasco, WA

2003-04-01

The present invention provides the promoter clone discovery of an alpha-amylase gene of a starch utilizing yeast strain Schwanniomyces castellii. The isolated alpha-amylase promoter is an inducible promoter, which can regulate strong gene expression in starch culture medium.

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

Cloning and Expressing Recombinant Protective Antigen Domains of B. anthracis

DTIC Science & Technology

2011-09-01

future predictive modeling toolkits. 1 1. Introduction The use of Bacillus anthracis as a bio - weapon in the United States in 2001 affirmed the need...for improved sensing and detection of biological weapons of mass destruction (WMD). Protective Antigen (PA) protein of Bacillus anthracis is the...Cloning and Expressing Recombinant Protective Antigen Domains of B. anthracis by Deborah A. Sarkes, Joshua M. Kogot, Irene Val-Addo

Cloning, Codon Optimization, and Expression of Yersinia intermedia Phytase Gene in E. coli.

PubMed

Mirzaei, Maryam; Saffar, Behnaz; Shareghi, Behzad

2016-06-01

Phytate is an anti-nutritional factor in plants, which catches the most phosphorus contents and some vital minerals. Therefore, Phytase is added mainly as an additive to the monogastric animals' foods to hydrolyze phytate and increase absorption of phosphorus. Y. intermedia phytase is a new phytase with special characteristics such as high specific activity, pH stability, and thermostability. Our aim was to clone, express, and characterizea codon optimized Y. intermedia phytase gene in E. coli . The Y. intermedia phytase gene was optimized according to the codon usage in E. coli . The sequence was synthesized and sub-cloned in pET-22b (+) vector and transformed into E. coli Bl21 (DE3). The protein was expressed in the presence of IPTG at a final concentration of 1 mM at 30°C. The purification of recombinant protein was performed by Ni 2+ affinity chromatography. Phytase activity and stability were determined in various pH and temperatures. The codon optimized Y. intermedia phytase gene was sub-cloned successfully.The expression was confirmed by SDS-PAGE and Western blot analysis. The recombinant enzyme (approximately 45 kDa) was purified. Specific activity of enzyme was 3849 (U.mg -1 ) with optimal pH 5 and optimal temperature of 55°C. Thermostability (80°C for 15 min) and pH stability (3-6) of the enzyme were 56 and more than 80%, respectively. The results of the expression and enzyme characterization revealed that the optimized Y. intermedia phytase gene has a good potential to be produced commercially andto be applied in animals' foodsindustry.

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

Isolation, molecular cloning and expression of cellobiohydrolase B (CbhB) from Aspergillus niger in Escherichia coli

NASA Astrophysics Data System (ADS)

Woon, J. S. K.; Murad, A. M. A.; Abu Bakar, F. D.

2015-09-01

A cellobiohydrolase B (CbhB) from Aspergillus niger ATCC 10574 was cloned and expressed in E. coli. CbhB has an open reading frame of 1611 bp encoding a putative polypeptide of 536 amino acids. Analysis of the encoded polypeptide predicted a molecular mass of 56.2 kDa, a cellulose binding module (CBM) and a catalytic module. In order to obtain the mRNA of cbhB, total RNA was extracted from A. niger cells induced by 1% Avicel. First strand cDNA was synthesized from total RNA via reverse transcription. The full length cDNA of cbhB was amplified by PCR and cloned into the cloning vector, pGEM-T Easy. A comparison between genomic DNA and cDNA sequences of cbhB revealed that the gene is intronless. Upon the removal of the signal peptide, the cDNA of cbhB was cloned into the expression vector pET-32b. However, the recombinant CbhB was expressed in Escherichia coli Origami DE3 as an insoluble protein. A homology model of CbhB predicted the presence of nine disulfide bonds in the protein structure which may have contributed to the improper folding of the protein and thus, resulting in inclusion bodies in E. coli.

Molecular cloning and characterization of ADP-glucose pyrophosphorylase cDNA clones isolated from pea cotyledons.

PubMed

Burgess, D; Penton, A; Dunsmuir, P; Dooner, H

1997-02-01

Three ADP-glucose pyrophosphorylase (ADPG-PPase) cDNA clones have been isolated and characterized from a pea cotyledon cDNA library. Two of these clones (Psagps1 and Psagps2) encode the small subunit of ADPG-PPase. The deduced amino acid sequences for these two clones are 95% identical. Expression of these two genes differs in that the Psagps2 gene shows comparatively higher expression in seeds relative to its expression in other tissues. Psagps2 expression also peaks midway through seed development at a time in which Psagps1 transcripts are still accumulating. The third cDNA isolated (Psagp11) encodes the large subunit of ADPG-PPase. It shows greater selectivity in expression than either of the small subunit clones. It is highly expressed in sink organs (seed, pod, and seed coat) and undetectable in leaves.

Arsenic resistance in Pteris vittata L.: identification of a cytosolic triosephosphate isomerase based on cDNA expression cloning in Escherichia coli.

PubMed

Rathinasabapathi, Bala; Wu, Shan; Sundaram, Sabarinath; Rivoal, Jean; Srivastava, Mrittunjai; Ma, Lena Q

2006-12-01

Arsenic hyperaccumulator Pteris vittata L. (Chinese brake fern) grows well in arsenic-contaminated media, with an extraordinary ability to tolerate high levels of arsenic. An expression cloning strategy was employed to identify cDNAs for the genes involved in arsenic resistance in P. vittata. Excised plasmids from the cDNA library of P. vittata fronds were introduced into Escherichia coli XL-1 Blue and plated on medium containing 4 mM of arsenate, a common form of arsenic in the environment. The deduced amino acid sequence of an arsenate-resistant clone, PV4-8, had cDNA highly homologous to plant cytosolic triosephosphate isomerases (cTPI). Cell-free extracts of PV4-8 had 3-fold higher level of triosephosphate isomerase (TPI) specific activities than that found in E. coli XL-1 Blue and had a 42 kD fusion protein immunoreactive to polyclonal antibodies raised against recombinant Solanum chacoense cTPI. The PV4-8 cDNA complemented a TPI-deficient E. coli mutant. PV4-8 expression improved arsenate resistance in E. coli WC3110, a strain deficient in arsenate reductase but not in AW3110 deficient for the whole ars operon. This is consistent with the hypothesis that PV4-8 TPI increased arsenate resistance in E. coli by directly or indirectly functioning as an arsenate reductase. When E. coli tpi gene was expressed in the same vector, bacterial arsenate resistance was not altered, indicating that arsenate tolerance was specific to P. vittata TPI. Paradoxically, P. vittata TPI activity was not more resistant to inhibition by arsenate in vitro than its bacterial counterpart suggesting that arsenate resistance of conventional TPI reaction was not the basis for the cellular arsenate resistance. P. vittata TPI activity was inhibited by incubation with reduced glutathione while bacterial TPI was unaffected. Consistent with cTPI's role in arsenate reduction, bacterial cells expressing fern TPI had significantly greater per cent of cellular arsenic as arsenite compared to cells

A thermolabile aspartic proteinase from Mucor mucedo DSM 809: gene identification, cloning, and functional expression in Pichia pastoris.

PubMed

Yegin, Sirma; Fernandez-Lahore, Marcelo

2013-06-01

In this study, the cDNA encoding the aspartic proteinase of Mucor mucedo DSM 809 has been identified by RNA ligased-mediated and oligo-capping rapid amplification of cDNA ends (RACE) technique. The gene contained an open reading frame of 1,200 bp and encoded for a signal peptide of 21 amino acid residues. Two N-glycosylation sites were observed within the identified sequence. The proteinase gene was cloned into the vector pGAPZαA and expressed in Pichia pastoris X-33 for the first time. The protein has been secreted in functionally active form into the culture medium. The expression system does not require any acid activation process. The factors affecting the expression level were optimized in shaking flask cultures. Maximum enzyme production was observed with an initial medium pH of 3.5 at 20 °C and 220 rpm shaking speed utilizing 4 % glucose as a carbon and energy source. The enzyme was purified with cation exchange chromatography and further studies revealed that the enzyme was secreted in glycosylated form. The purified enzyme exhibited remarkable sensitivity to thermal treatment and became completely inactivated after incubation at 55 °C for 10 min. These results indicated that the recombinant proteinase could be considered as a potential rennet candidate for the cheese-making industry.

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 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

Quick and clean cloning.

PubMed

Thieme, Frank; Marillonnet, Sylvestre

2014-01-01

Identification of unknown sequences that flank known sequences of interest requires PCR amplification of DNA fragments that contain the junction between the known and unknown flanking sequences. Since amplified products often contain a mixture of specific and nonspecific products, the quick and clean (QC) cloning procedure was developed to clone specific products only. QC cloning is a ligation-independent cloning procedure that relies on the exonuclease activity of T4 DNA polymerase to generate single-stranded extensions at the ends of the vector and insert. A specific feature of QC cloning is the use of vectors that contain a sequence called catching sequence that allows cloning specific products only. QC cloning is performed by a one-pot incubation of insert and vector in the presence of T4 DNA polymerase at room temperature for 10 min followed by direct transformation of the incubation mix in chemo-competent Escherichia coli cells.

Isolation, molecular cloning and expression of cellobiohydrolase B (CbhB) from Aspergillus niger in Escherichia coli

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

Woon, J. S. K., E-mail: jameswoon@siswa.ukm.edu.my; Murad, A. M. A., E-mail: munir@ukm.edu.my; Abu Bakar, F. D., E-mail: fabyff@ukm.edu.my

A cellobiohydrolase B (CbhB) from Aspergillus niger ATCC 10574 was cloned and expressed in E. coli. CbhB has an open reading frame of 1611 bp encoding a putative polypeptide of 536 amino acids. Analysis of the encoded polypeptide predicted a molecular mass of 56.2 kDa, a cellulose binding module (CBM) and a catalytic module. In order to obtain the mRNA of cbhB, total RNA was extracted from A. niger cells induced by 1% Avicel. First strand cDNA was synthesized from total RNA via reverse transcription. The full length cDNA of cbhB was amplified by PCR and cloned into the cloning vector, pGEM-Tmore » Easy. A comparison between genomic DNA and cDNA sequences of cbhB revealed that the gene is intronless. Upon the removal of the signal peptide, the cDNA of cbhB was cloned into the expression vector pET-32b. However, the recombinant CbhB was expressed in Escherichia coli Origami DE3 as an insoluble protein. A homology model of CbhB predicted the presence of nine disulfide bonds in the protein structure which may have contributed to the improper folding of the protein and thus, resulting in inclusion bodies in E. coli.« less

Functional cDNA expression cloning: Pushing it to the limit

PubMed Central

OKAYAMA, Hiroto

2012-01-01

The 1970s and the following decade are the era of the birth and early development of recombinant DNA technologies, which have entirely revolutionized the modern life science by providing tools that enable us to know the structures of genes and genomes and to dissect their components and understand their functions at the molecular and submolecular levels. One major objective of the life sciences is to achieve molecular and chemical understandings of the functions of genes and their encoded proteins, which are responsible for the manifestation of all biological phenomena in organisms. In the early 1980s, I developed, together with Paul Berg, a new technique that enables the cloning of full-length complementary DNAs (cDNAs) on the basis of their functional expression in a given cell of interest. I review the development, application and future implications in the life sciences of this gene-cloning technique. PMID:22450538

Cloning and high level expression of gene encoding ES antigen from Trichinella spiralis muscle larvae.

PubMed

Yan, Y; Xu, W; Chen, H; Ma, Z; Zhu, Y; Cai, S

1994-01-01

The partial structure gene encoding ES antigen derived from Trichinella spiralis (TSP) muscle larvae was cloned, characterized, and expressed in E. coli. The target DNA (0.7 kb) was directly obtained from the TSP total RNA by using RNA PCR technique. Based on the analysis with the RE digestion, the fragment was cloned into the fusion expression vector pEX31C. It was shown that a kind of 37kDa fusion protein was expressed in E. coli containing the recombinant plasmid by SDS-PAGE electrophoresis. The expressed protein was over 22% of the total cell protein, and it was aggregated in the form of inclusion bodies in E. coli. The purified protein could be recognized in ELISA both by sera from swine-infected with TSP and by the monoclonal antibody against TSP. These findings suggest that the recombinant protein is a potentially valuable antigen both for immunodiagnosis and vaccine development of trichinellosis.

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.

Expression and regulation of the penicillin G acylase gene from Proteus rettgeri cloned in Escherichia coli.

PubMed

Daumy, G O; Williams, J A; McColl, A S; Zuzel, T J; Danley, D

1986-10-01

The penicillin G acylase genes from the Proteus rettgeri wild type and from a hyperproducing mutant which is resistant to succinate repression were cloned in Escherichia coli K-12. Expression of both wild-type and mutant P. rettgeri acylase genes in E. coli K-12 was independent of orientation in the cloning vehicle and apparently resulted from recognition in E. coli of the P. rettgeri promoter sequences. The P. rettgeri acylase was secreted into the E. coli periplasmic space and was composed of subunits electrophoretically identical to those made in P. rettgeri. Expression of these genes in E. coli K-12 was not repressed by succinate as it is in P. rettgeri. Instead, expression of the enzymes was regulated by glucose catabolite repression.

Cloning and characterization of a novel α-amylase from a fecal microbial metagenome.

PubMed

Xu, Bo; Yang, Fuya; Xiong, Caiyun; Li, Junjun; Tang, Xianghua; Zhou, Junpei; Xie, Zhenrong; Ding, Junmei; Yang, Yunjuan; Huang, Zunxi

2014-04-01

To isolate novel and useful microbial enzymes from uncultured gastrointestinal microorganisms, a fecal microbial metagenomic library of the pygmy loris was constructed. The library was screened for amylolytic activity, and 8 of 50,000 recombinant clones showed amylolytic activity. Subcloning and sequence analysis of a positive clone led to the identification a novel gene (amyPL) coding for α-amylase. AmyPL was expressed in Escherichia coli BL21 (DE3) and the purified AmyPL was enzymatically characterized. This study is the first to report the molecular and biochemical characterization of a novel α-amylase from a gastrointestinal metagenomic library.

Production of cloned embryos from caprine mammary epithelial cells expressing recombinant human β-defensin-3.

PubMed

Liu, Jun; Luo, Yan; Liu, Qingqing; Zheng, Liming; Yang, Zhongcai; Wang, Yongsheng; Su, Jianmin; Quan, Fusheng; Zhang, Yong

2013-03-01

Transgenic animals that express antimicrobial agents in their milk can inhibit bacterial pathogens that cause mastitis. Our objective was to produce human β-defensin-3 (HBD3) transgenic embryos by nuclear transfer using goat mammary epithelial cells (GMECs) as donor cells. Three GMEC lines (GMEC1, GMEC2, and GMEC3) were transfected with a HBD3 mammary-specific expression vector by electroporation. There was a difference (P < 0.05) in the rate of geneticin-resistant colony formation among cell lines GMEC1, GMEC2, and GMEC3 (39 and 47 vs. 19 colonies per 3 × 10(6) cells, respectively). After inducing expression, the mRNA and protein of HBD3 were detected by reverse transcription polymerase chain reaction and Western blot analysis in transgenic cells. Transgenic clonal cells expressing HBD3 were used as donor cells to investigate development of cloned embryos. There were no significant differences in rates of cleavage or blastocyst formation of cloned embryos from transgenic (GMEC1T2 and GMEC2T3) and nontransgenic (GMEC1 and GMEC2) GMECs (72.3 ± 5.0%, 69.5 ± 2.3%, 61.8 ± 4.8%, and 70.0 ± 2%; and 16.8 ± 0.5%, 17.5 ± 0.7%, 16.7 ± 0.9%, and 17.5 ± 0.6%, respectively). However, the fusion rate, cleavage rate, and blastocyst formation rate of cloned embryos from a transgenic clonal cell line (GMEC2T6, 50.7 ± 2.1%, 55.5 ± 2.0%, and 11.1 ± 0.6%) were lower than those of other groups (P < 0.05). We concluded that genetic modification of GMECs might not influence the in vitro development of cloned embryos, but that some of the transgenic clonal cells were not suitable for nuclear transfer to produce transgenic goats, because of low developmental rates. However, transgenic GMECs expressing HBD3 might be used as donor cells for producing transgenic goats that express increased concentrations of β-defensins in their milk. Copyright © 2013 Elsevier Inc. All rights reserved.

Retrotransposon expression and incorporation of cloned human and mouse retroelements in human spermatozoa.

PubMed

Lazaros, Leandros; Kitsou, Chrysoula; Kostoulas, Charilaos; Bellou, Sofia; Hatzi, Elissavet; Ladias, Paris; Stefos, Theodoros; Markoula, Sofia; Galani, Vasiliki; Vartholomatos, Georgios; Tzavaras, Theodore; Georgiou, Ioannis

2017-03-01

To investigate the expression of long interspersed element (LINE) 1, human endogenous retrovirus (HERV) K10, and short interspersed element-VNTR-Alu element (SVA) retrotransposons in ejaculated human spermatozoa by means of reverse-transcription (RT) polymerase chain reaction (PCR) analysis as well as the potential incorporation of cloned human and mouse active retroelements in human sperm cell genome. Laboratory study. University research laboratories and academic hospital. Normozoospermic and oligozoospermic white men. RT-PCR analysis was performed to confirm the retrotransposon expression in human spermatozoa. Exogenous retroelements were tagged with a plasmid containing a green fluorescence (EGFP) retrotransposition cassette, and the de novo retrotransposition events were tested with the use of PCR, fluorescence-activated cell sorting analysis, and confocal microscopy. Retroelement expression in human spermatozoa, incorporation of cloned human and mouse active retroelements in human sperm genome, and de novo retrotransposition events in human spermatozoa. RT-PCR products of expressed human LINE-1, HERV-K10, and SVA retrotransposons were observed in ejaculated human sperm samples. The incubation of human spermatozoa with either retrotransposition-active human LINE-1 and HERV-K10 or mouse reverse transcriptase-deficient VL30 retrotransposons tagged with an EGFP-based retrotransposition cassette led to EGFP-positive spermatozo; 16.67% of the samples were positive for retrotransposition. The respective retrotransposition frequencies for the LINE-1, HERV-K10, and VL30 retrotransposons in the positive samples were 0.34 ± 0.13%, 0.37 ± 0.17%, and 0.30 ± 0.14% per sample of 10,000 spermatozoa. Our results show that: 1) LINE-1, HERV-K10, and SVA retrotransposons are transcriptionally expressed in human spermatozoa; 2) cloned active retroelements of human and mammalian origin can be incorporated in human sperm genome; 3) active reverse transcriptases exist in human

Molecular cloning and expression in streptomyces lividans of a hygromycin B phosphotransferase gene from Streptomyces hygroscopicus.

PubMed

Malpartida, F; Zalacaín, M; Jiménez, A; Davies, J

1983-11-30

The gene encoding the phosphotransferase enzyme that modifies hygromycin B in its producing organism Streptomyces hygroscopicus, has been cloned in the Streptomyces vector pIJ41. Two plasmids, pFM4 and pFM6, containing 2.1 and 19.6 kb inserts of Streptomyces hygroscopicus DNA, respectively, which express the modifying enzyme, have been isolated. A 3.1 kb PstI restriction fragment from pFM4 was inserted in the Streptomyces vector pIJ350 and the resulting plasmids, pMZ11.1 and pMZ11.2, express the hygromycin B-resistance phenotype. The utility of this dominant marker for cloning experiments is discussed in the text.

[Cloning, Expression and Immunodiagnostic Evaluation of the Fasciola gigantica Thioredoxin Peroxidase].

PubMed

Wang, Yue-qi; Zhou, Yan; Cheng, Na; Chen, Mu-xin; Ai, Lin; Liu, Yu-hua; Zhang, Jian-guo; Luo, Jia-jun; Xu, Xue-nian

2015-04-01

To immunoscreen the gene encoding thioredoxin peroxidase (TPx) from a cDNA library made from adult Fasciola gigantica worms, clone and express the gene, and evaluate the immunodiagnostic value of TPx recombinant protein. The A ZAP cDNA library was immunoscreened with pooled serum of fascioliasis gigantica patients. The obtained positive clones were sequenced and analyzed by multiple sequence alignment. The full-length (rFgTPx) and N-termianal truncated (rFgTPx_nt) sequence of FgTPx was subcloned into prokaryotic plasmid pET28a(+) with a non-fusion expression technique, respectively. The recombinant proteins of rFgTPx and rFgTPx_nt were purified by His-bind affinity column (Ni-NTA). rFgTPx and rFgTPx_nt were used in indirect ELISA to test the antibody response of the serum samples. Sera of 27 fascioliasis gigantica patients, 15 patients with schistosomaisis japonica, 15 clonorchiasis sinensis patients, and 32 healthy donors were tested by using the recombinant protein based ELISA. The TPx recombinant proteins were obtained through expression, purification and renaturation, the relative molecular mass of rFgTPx and rFgTPx_nt were Mr 30,000 and Mr 26,000, respectively. The total diagnostic coincidence rate, sensitivity and specificity of rFgTPx_nt-based ELISA was 87.6% (78/89), 66.7% (18/27), and 96.8% (60/62), respectively. The cross reaction with Schistosoma japonicum and Clonorchis sinensis was 0 and 1/15 for rFgTPx_nt, respectively. Before and after treatment, A450 value of the serum samples from fascioliasis patients was 0.233 ± 0.088 and 0.129 ± 0.072, respectively (t = 4.27, P < 0.01). The gene encoding TPx is expressed in the prokaryotic expression system. The recombinant protein shows proper sensitivity and high specificity for the serodiagnosis of Fasciola gigantica infection.

Lipid transfer proteins from fruit: cloning, expression and quantification.

PubMed

Zuidmeer, Laurian; van Leeuwen, W Astrid; Budde, Ilona Kleine; Cornelissen, Jessica; Bulder, Ingrid; Rafalska, Ilona; Besolí, Noèlia Telléz; Akkerdaas, Jaap H; Asero, Riccardo; Fernandez Rivas, Montserrat; Rivas, Montserrat Fernandez; Gonzalez Mancebo, Eloina; Mancebo, Eloina Gonzalez; van Ree, Ronald

2005-08-01

Lipid transfer proteins (LTP) are stable, potentially life-threatening allergens in fruits and many other vegetable foods. The aim of this study was to clone and express recombinant apple LTP (Mal d 3), as has previously been done for peach LTP (Pru p 3) and set up quantitative tests for measuring fruit LTPs. cDNA for Mal d 3 and Pru p 3 was cloned, expressed in the yeast Pichia pastoris and the resulting proteins were purified via cation exchange chromatography. The immune reactivity of rMal d 3 was compared to nMal d 3 by RAST (inhibition), immunoblotting and basophil histamine release testing. To obtain monoclonal and monospecific polyclonal antibodies, mice and rabbits were immunized with purified nMal d 3. The deduced amino acid sequence of Mal d 3 was identical to the published sequence, Pru p 3 differed at two positions (S9A and S76H). The rMal d 3 had an IgE-binding potency and biological activity close to its natural counterpart. One sandwich ELISA selectively detecting apple LTP and another cross-reactive with cherry, nectarine and hazelnut LTP were developed. In addition, a competitive RIA was developed with polyclonal rabbit antiserum and labeled nMal d 3. rMal d 3 (as shown before for rPru p 3) may be a useful tool for application in component-resolved diagnosis of food allergy. Assays for the measurement of LTP will increase the traceability of this potentially dangerous allergen. Copyright 2005 S. Karger AG, Basel.

Cloning and heterologous expression of genes from the kinamycin biosynthetic pathway of Streptomyces murayamaensis.

PubMed

Gould, S J; Hong, S T; Carney, J R

1998-01-01

The genes for most of the biosynthesis of the kinamycin antibiotics have been cloned and heterologously expressed. Genomic DNA of Streptomyces murayamaensis was partially digested with MboI and a library of approximately 40 kb fragments in E. coli XL1-BlueMR was prepared using the cosmid vector pOJ446. Hybridization with the actI probe from the actinorhodin polyketide synthase genes identified two clusters of polyketide genes. After transferal of these clusters to S. lividans ZX7, expression of one cluster was established by HPLC with photodiode array detection. Peaks were identified from the kin cluster for dehydrorabelomycin, kinobscurinone, and stealthin C, which are known intermediates in kinamycin biosynthesis. Two shunt metabolites, kinafluorenone and seongomycin were also identified. The structure of the latter was determined from a quantity obtained from large-scale fermentation of one of the clones.

Identification of downy mildew resistance gene candidates by positional cloning in maize (Zea mays subsp. mays; Poaceae)1

PubMed Central

Kim, Jae Yoon; Moon, Jun-Cheol; Kim, Hyo Chul; Shin, Seungho; Song, Kitae; Kim, Kyung-Hee; Lee, Byung-Moo

2017-01-01

Premise of the study: Positional cloning in combination with phenotyping is a general approach to identify disease-resistance gene candidates in plants; however, it requires several time-consuming steps including population or fine mapping. Therefore, in the present study, we suggest a new combined strategy to improve the identification of disease-resistance gene candidates. Methods and Results: Downy mildew (DM)–resistant maize was selected from five cultivars using a spreader row technique. Positional cloning and bioinformatics tools were used to identify the DM-resistance quantitative trait locus marker (bnlg1702) and 47 protein-coding gene annotations. Eventually, five DM-resistance gene candidates, including bZIP34, Bak1, and Ppr, were identified by quantitative reverse-transcription PCR (RT-PCR) without fine mapping of the bnlg1702 locus. Conclusions: The combined protocol with the spreader row technique, quantitative trait locus positional cloning, and quantitative RT-PCR was effective for identifying DM-resistance candidate genes. This cloning approach may be applied to other whole-genome-sequenced crops or resistance to other diseases. PMID:28224059

Cloning and expression of a cDNA coding for catalase from zebrafish (Danio rerio).

PubMed

Ken, C F; Lin, C T; Wu, J L; Shaw, J F

2000-06-01

A full-length complementary DNA (cDNA) clone encoding a catalase was amplified by the rapid amplication of cDNA ends-polymerase chain reaction (RACE-PCR) technique from zebrafish (Danio rerio) mRNA. Nucleotide sequence analysis of this cDNA clone revealed that it comprised a complete open reading frame coding for 526 amino acid residues and that it had a molecular mass of 59 654 Da. The deduced amino acid sequence showed high similarity with the sequences of catalase from swine (86.9%), mouse (85.8%), rat (85%), human (83.7%), fruit fly (75.6%), nematode (71.1%), and yeast (58.6%). The amino acid residues for secondary structures are apparently conserved as they are present in other mammal species. Furthermore, the coding region of zebrafish catalase was introduced into an expression vector, pET-20b(+), and transformed into Escherichia coli expression host BL21(DE3)pLysS. A 60-kDa active catalase protein was expressed and detected by Coomassie blue staining as well as activity staining on polyacrylamide gel followed electrophoresis.

Sodium butyrate improves the cloned yak embryo viability and corrects gene expression patterns.

PubMed

Xiong, Xian-rong; Lan, Dao-liang; Li, Jian; Wang, Yong; Zhong, Jin-cheng

2015-02-01

Interspecies somatic cell nuclear transfer (iSCNT), a powerful tool in basic scientific research, has been used widely to increase and preserve the population of endangered species. Yak (Bos grunniens) is one of these species. Development to term of interspecies cloned yak embryos has not been achieved, possibly due to abnormal epigenetic reprogramming. Previous studies have demonstrated that treatment of intraspecies cloned embryos with (NaBu) significantly improves nuclear-cytoplasmic reprogramming and viability in vitro. Therefore, in this study, we evaluated the effect of optimal NaBu concentration and exposure time on preimplantation development of yak iSCNT embryos and on the expression patterns of developmentally important genes. The results showed that 8-cell rate, blastocyst formation rate and total cell number increased significantly compared with their untreated counterparts when yak iSCNT embryos were treated with 5 nM NaBu for 12 h after activation, but that the 2-cell stage embryo rate was not significantly different. The treatment of NaBu also increased significantly the expression levels of Oct-4 and decreased the expression levels of HDAC-2, Dnmt-1 and IGF-1; the expression patterns of these genes were more similar to that of their bovine-yak in vitro fertilization (BY-IVF) counterparts. The results described above indicated that NaBu treatment improved developmental competence in vitro and 'corrected' the gene expression patterns of yak iSCNT embryos.

Molecular cloning and expression of Cro s 1: an occupational allergen from saffron pollen (Crocus sativus)

PubMed Central

Varasteh, Abdol-Reza; Sankian, Mojtaba; Midoro-Horiuti, Terumi; Moghadam, Malihe; Shakeri, Mohamad Taghi; Brooks, Edward G.; Goldblum, Randall M.; Chapman, Martin D.; Pomés, Anna

2012-01-01

Background: The cultivation of saffron is expanding through the southeast of Iran, and allergy to saffron pollen occurs in workers involved in processing this plant. We aimed to clone, sequence and express a major allergen involved in saffron pollen allergy, and to compare the recombinant with the natural allergen. Methods: The N-terminal amino acid sequence of Cro s 1, an allergen from saffron pollen, was determined after immunoblotting. The cDNA encoding for this allergen was cloned by PCR utilizing a primer based on the N-terminal amino acid sequence. Recombinant Cro s 1 (rCro s 1) was expressed as a soluble protein in Pichia pastoris and purified to homogeneity by gel filtration. Inhibition of IgE binding to rCro s 1 by pollen extract was analyzed by ELISA. Section Title The allergen Cro s 1 was identified from saffron pollen extracts and cloned by PCR. Cro s 1 cDNA defined an acidic polypeptide with homology to pollen proteins from Chenopodium album and Ligastrum vulgaris. The rCro s 1 was expressed in P. pastoris at 28 mg/l. Saffron pollen extract inhibited the binding of patient serum IgE to rCro s 1. Conclusion: We identified and cloned the first Crocus sativus pollen allergen. rCro s 1 cDNA shows a very high homology with Che a 1, the major allergen of lamb's-quarter, Chenopodium album, Caryophyllales, pollen (97%). Cro s 1 is a useful tool for specific diagnosis and structural studies of occupational allergy to saffron. PMID:26989701

Molecular cloning and expression of Cro s 1: an occupational allergen from saffron pollen (Crocus sativus).

PubMed

Varasteh, Abdol-Reza; Sankian, Mojtaba; Midoro-Horiuti, Terumi; Moghadam, Malihe; Shakeri, Mohamad Taghi; Brooks, Edward G; Goldblum, Randall M; Chapman, Martin D; Pomés, Anna

2012-10-01

The cultivation of saffron is expanding through the southeast of Iran, and allergy to saffron pollen occurs in workers involved in processing this plant. We aimed to clone, sequence and express a major allergen involved in saffron pollen allergy, and to compare the recombinant with the natural allergen. The N-terminal amino acid sequence of Cro s 1, an allergen from saffron pollen, was determined after immunoblotting. The cDNA encoding for this allergen was cloned by PCR utilizing a primer based on the N-terminal amino acid sequence. Recombinant Cro s 1 (rCro s 1) was expressed as a soluble protein in Pichia pastoris and purified to homogeneity by gel filtration. Inhibition of IgE binding to rCro s 1 by pollen extract was analyzed by ELISA. The allergen Cro s 1 was identified from saffron pollen extracts and cloned by PCR. Cro s 1 cDNA defined an acidic polypeptide with homology to pollen proteins from Chenopodium album and Ligastrum vulgaris. The rCro s 1 was expressed in P. pastoris at 28 mg/l. Saffron pollen extract inhibited the binding of patient serum IgE to rCro s 1. We identified and cloned the first Crocus sativus pollen allergen. rCro s 1 cDNA shows a very high homology with Che a 1, the major allergen of lamb's-quarter, Chenopodium album, Caryophyllales, pollen (97%). Cro s 1 is a useful tool for specific diagnosis and structural studies of occupational allergy to saffron.

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.

Cloning and Expression of Yak Active Chymosin in Pichia pastoris.

PubMed

Luo, Fan; Jiang, Wei Hua; Yang, Yuan Xiao; Li, Jiang; Jiang, Ming Feng

2016-09-01

Rennet, a complex of enzymes found in the stomachs of ruminants, is an important component for cheese production. In our study, we described that yak chymosin gene recombinant Pichia pastoris strain could serve as a novel source for rennet production. Yaks total RNA was extracted from the abomasum of an unweaned yak. The yak preprochymosin, prochymosin, and chymosin genes from total RNA were isolated using gene specific primers based on cattle chymosin gene sequence respectively and analyzed their expression pattern byreal time-polymerase chain reaction. The result showed that the chymosin gene expression level of the sucking yaks was 11.45 times higher than one of adult yaks and yak chymosin belongs to Bovidae family in phylogenetic analysis. To express each, the preprochymosin, prochymosin, and chymosin genes were ligated into the expression vector pPICZαA, respectively, and were expressed in Pichia pastoris X33. The results showed that all the recombinant clones of P. pastoris containing the preprochymosin, prochymosin or chymosin genes could produce the active form of recombinant chymosin into the culture supernatant. Heterologous expressed prochymosin (14.55 Soxhlet unit/mL) had the highest enzyme activity of the three expressed chymosin enzymes. Therefore, we suggest that the yak chymosin gene recombinant Pichia pastoris strain could provide an alternative source of rennet production.

Cloning and expression of cDNA coding for bouganin.

PubMed

den Hartog, Marcel T; Lubelli, Chiara; Boon, Louis; Heerkens, Sijmie; Ortiz Buijsse, Antonio P; de Boer, Mark; Stirpe, Fiorenzo

2002-03-01

Bouganin is a ribosome-inactivating protein that recently was isolated from Bougainvillea spectabilis Willd. In this work, the cloning and expression of the cDNA encoding for bouganin is described. From the cDNA, the amino-acid sequence was deduced, which correlated with the primary sequence data obtained by amino-acid sequencing on the native protein. Bouganin is synthesized as a pro-peptide consisting of 305 amino acids, the first 26 of which act as a leader signal while the 29 C-terminal amino acids are cleaved during processing of the molecule. The mature protein consists of 250 amino acids. Using the cDNA sequence encoding the mature protein of 250 amino acids, a recombinant protein was expressed, purified and characterized. The recombinant molecule had similar activity in a cell-free protein synthesis assay and had comparable toxicity on living cells as compared to the isolated native bouganin.

Identification and Cloning of gusA, Encoding a New β-Glucuronidase from Lactobacillus gasseri ADH†

PubMed Central

Russell, W. M.; Klaenhammer, T. R.

2001-01-01

The gusA gene, encoding a new β-glucuronidase enzyme, has been cloned from Lactobacillus gasseri ADH. This is the first report of a β-glucuronidase gene cloned from a bacterial source other than Escherichia coli. A plasmid library of L. gasseri chromosomal DNA was screened for complementation of an E. coli gus mutant. Two overlapping clones that restored β-glucuronidase activity in the mutant strain were sequenced and revealed three complete and two partial open reading frames. The largest open reading frame, spanning 1,797 bp, encodes a 597-amino-acid protein that shows 39% identity to β-glucuronidase (GusA) of E. coli K-12 (EC 3.2.1.31). The other two complete open reading frames, which are arranged to be separately transcribed, encode a putative bile salt hydrolase and a putative protein of unknown function with similarities to MerR-type regulatory proteins. Overexpression of GusA was achieved in a β-glucuronidase-negative L. gasseri strain by expressing the gusA gene, subcloned onto a low-copy-number shuttle vector, from the strong Lactobacillus P6 promoter. GusA was also expressed in E. coli from a pET expression system. Preliminary characterization of the GusA protein from crude cell extracts revealed that the enzyme was active across an acidic pH range and a broad temperature range. An analysis of other lactobacilli identified β-glucuronidase activity and gusA homologs in other L. gasseri isolates but not in other Lactobacillus species tested. PMID:11229918

[Cloning, expression and characterization of a novel esterase from marine sediment microbial metagenomic library].

PubMed

Xu, Shiqing; Hu, Yongfei; Yuan, Aihua; Zhu, Baoli

2010-07-01

To clone, express and characterize a novel esterase from marine sediment microbial metagenomic library. Using esterase segregation agar containing tributyrin, we obtained esterase positive fosmid clone FL10 from marine sediment microbial metagenomic library. This fosmid was partially digested with Sau3A I to construct the sublibrary, from which the esterase positive subclone pFLS10 was obtained. The full length of the esterase gene was amplified and cloned into the expressing vector pET28a, and the recombinant plasmid was transformed into E. coli BL21 cells. We analyse the enzyme activity and study the characterization of the esterase after its expression and purification. An ORF (Open Reading Frame) of 924 bp was identified from the subclone pFLS10. Sequence analysis indicated that it showed 71% amino acid identity to esterase (ADA70030) from a marine sediment metagenomic library. The esterase is a novel low-temperature-active esterase and had highest lipolytic activity to the substrate of 4-nitrophenyl butyrate (C4). The optimum temperature of the esterase was 20 degrees C, the optimum pH was 7.5. The esterase in this study had good thermostability at 20 degrees C and good pH stability at pH8 -10. Significant increase in lipolytic activity was observed with addition of K+ and Mg2+, while decrease with Mn2+ etc. We obtained the novel esterase gene fls10 from the marine sediment microbial metagenomic library. The esterase had good thermostability and high lipolytic activity at low temperature and under basic conditions, which laid a basis for industrial application.

Cloning, functional expression, and characterization of a PKA-activated gastric Cl- channel.

PubMed

Malinowska, D H; Kupert, E Y; Bahinski, A; Sherry, A M; Cuppoletti, J

1995-01-01

cDNA encoding a Cl- channel was isolated from a rabbit gastric library, sequenced, and expressed in Xenopus oocytes. The predicted protein (898 amino acids, relative molecular mass 98,433 Da) was overall 93% similar to the rat brain ClC-2 Cl- channel. However, a 151-amino acid stretch toward the COOH-terminus was 74% similar to ClC-2 with six amino acids deleted. Two new potential protein kinase A (PKA) phosphorylation sites (also protein kinase C phosphorylation sites) were introduced. cRNA-injected Xenopus oocytes expressed a Cl- channel that was active at pHtrans 3 and had a linear current-voltage (I-V) curve and a slope conductance of 29 +/- 1 pS at 800 mM CsCl. A fivefold Cl- gradient caused a rightward shift in the I-V curve with a reversal potential of +30 +/- 3 mV, indicating anion selectivity. The selectivity was I- > Cl- > NO3-. The native and recombinant Cl- channel were both activated in vitro by PKA catalytic subunit and ATP. The electrophysiological and regulatory properties of the cloned and the native channel were similar. The cloned protein may be the Cl- channel involved in gastric HCl secretion.

Identification of Genes Differentially Expressed During Heat Shock Treatment in Aedes aegypti

DTIC Science & Technology

2009-01-01

Chan, C. W. Cheng, and R. S. Wu. 2003. Cloning of theHSP70 gene in barnacle larvae and its expression under hypoxic conditions. Mar. Pollut. Bull. 46...665Ð671. Chuang, K. H., S. H. Ho, and Y. L. Song. 2007. Cloning and expression analysis of heat shock cognate 70 gene pro- moter in tiger shrimp ...in larvae , but not adults, of a polar insect. Proc. Natl. Acad. Sci. U.S.A. 103: 14223Ð14227. Robich, R. M., J. P. Rinehart, L. J. Kitchen, and D. L

Cloning and expression of Bartonella henselae sucB gene encoding an immunogenic dihydrolipoamide succinyltransferase homologous protein.

PubMed

Kabeya, Hidenori; Maruyama, Soichi; Hirano, Kouji; Mikami, Takeshi

2003-01-01

Immunoscreening of a ZAP genomic library of Bartonella henselae strain Houston-1 expressed in Escherichia coli resulted in the isolation of a clone containing 3.5 kb BamHI genomic DNA fragment. This 3.5 kb DNA fragment was found to contain a sequence of a gene encoding a protein with significant homology to the dihydrolipoamide succinyltransferase of Brucella melitensis (sucB). Subsequent cloning and DNA sequence analysis revealed that the deduced amino acid sequence from the cloned gene showed 66.5% identity to SucB protein of B. melitensis, and 43.4 and 47.2% identities to those of Coxiella burnetii and E. coli, respectively. The gene was expressed as a His-Nus A-tagged fusion protein. The recombinant SucB protein (rSucB) was shown to be an immunoreactive protein of about 115 kDa by Western blot analysis with sera from B. henselae-immunized mice. Therefore the rSucB may be a candidate antigen for a specific serological diagnosis of B. henselae infection.

Identification of Actinobacillus pleuropneumoniae Genes Preferentially Expressed During Infection Using In Vivo-Induced Antigen Technology (IVIAT).

PubMed

Zhang, Fei; Zhang, Yangyi; Wen, Xintian; Huang, Xiaobo; Wen, Yiping; Wu, Rui; Yan, Qigui; Huang, Yong; Ma, Xiaoping; Zhao, Qin; Cao, Sanjie

2015-10-01

Porcine pleuropneumonia is an infectious disease caused by Actinobacillus pleuropneumoniae. The identification of A. pleuropneumoniae genes, specially expressed in vivo, is a useful tool to reveal the mechanism of infection. IVIAT was used in this work to identify antigens expressed in vivo during A. pleuropneumoniae infection, using sera from individuals with chronic porcine pleuropneumonia. Sequencing of DNA inserts from positive clones showed 11 open reading frames with high homology to A. pleuropneumoniae genes. Based on sequence analysis, proteins encoded by these genes were involved in metabolism, replication, transcription regulation, and signal transduction. Moreover, three function-unknown proteins were also indentified in this work. Expression analysis using quantitative real-time PCR showed that most of the genes tested were up-regulated in vivo relative to their expression levels in vitro. IVI (in vivoinduced) genes that were amplified by PCR in different A. pleuropneumoniae strains showed that these genes could be detected in almost all of the strains. It is demonstrated that the identified IVI antigen may have important roles in the infection of A. pleuropneumoniae.

Cloning, bacterial expression and crystallization of Fv antibody fragments

NASA Astrophysics Data System (ADS)

E´, Jean-Luc; Boulot, Ginette; Chitarra, V´ronique; Riottot, Marie-Madeleine; Souchon, H´le`ne; Houdusse, Anne; Bentley, Graham A.; Narayana Bhat, T.; Spinelli, Silvia; Poljak, Roberto J.

1992-08-01

The variable Fv fragments of antibodies, cloned in recombinant plasmids, can be expressed in bacteria as functional proteins having immunochemical properties which are very similar or identical with those of the corresponding parts of the parent eukaryotic antibodies. They offer new possibilities for the study of antibody-antigen interactions since the crystals of Fv fragments and of their complexes with antigen reported here diffract X-rays to a higher resolution that those obtained with the cognate Fab fragments. The Fv approach should facilitate the structural study of the combining site of antibodies and the further characterization of antigen-antibody interactions by site-directed mutagenesis experiments.

kappa-Opioid receptor in humans: cDNA and genomic cloning, chromosomal assignment, functional expression, pharmacology, and expression pattern in the central nervous system.

PubMed Central

Simonin, F; Gavériaux-Ruff, C; Befort, K; Matthes, H; Lannes, B; Micheletti, G; Mattéi, M G; Charron, G; Bloch, B; Kieffer, B

1995-01-01

Using the mouse delta-opioid receptor cDNA as a probe, we have isolated genomic clones encoding the human mu- and kappa-opioid receptor genes. Their organization appears similar to that of the human delta receptor gene, with exon-intron boundaries located after putative transmembrane domains 1 and 4. The kappa gene was mapped at position q11-12 in human chromosome 8. A full-length cDNA encoding the human kappa-opioid receptor has been isolated. The cloned receptor expressed in COS cells presents a typical kappa 1 pharmacological profile and is negatively coupled to adenylate cyclase. The expression of kappa-opioid receptor mRNA in human brain, as estimated by reverse transcription-polymerase chain reaction, is consistent with the involvement of kappa-opioid receptors in pain perception, neuroendocrine physiology, affective behavior, and cognition. In situ hybridization studies performed on human fetal spinal cord demonstrate the presence of the transcript specifically in lamina II of the dorsal horn. Some divergences in structural, pharmacological, and anatomical properties are noted between the cloned human and rodent receptors. Images Fig. 3 Fig. 4 PMID:7624359

Identification of the propionicin F bacteriocin immunity gene (pcfI) and development of a food-grade cloning system for Propionibacterium freudenreichii.

PubMed

Brede, Dag Anders; Lothe, Sheba; Salehian, Zhian; Faye, Therese; Nes, Ingolf F

2007-12-01

This report describes the first functional analysis of a bacteriocin immunity gene from Propionibacterium freudenreichii and its use as a selection marker for food-grade cloning. Cloning of the pcfI gene (previously orf5 [located as part of the pcfABC propionicin F operon]) rendered the sensitive host 1,000-fold more tolerant to the propionicin F bacteriocin. The physiochemical properties of the 127-residue large PcfI protein resemble those of membrane-bound immunity proteins from bacteriocin systems found in lactic acid bacteria. The high level of immunity conferred by pcfI allowed its use as a selection marker for plasmid transformation in P. freudenreichii. Electroporation of P. freudenreichii IFO12426 by use of the pcfI expression plasmid pSL102 and propionicin F selection (200 bacteriocin units/ml) yielded 10(7) transformants/microg DNA. The 2.7-kb P. freudenreichii food-grade cloning vector pSL104 consists of the pLME108 replicon, a multiple cloning site, and pcfI expressed from the constitutive P(pampS) promoter for selection. The pSL104 vector efficiently facilitated cloning of the propionicin T1 bacteriocin in P. freudenreichii. High-level propionicin T1 production (640 BU/ml) was obtained with the IFO12426 strain, and the food-grade propionicin T1 expression plasmid pSL106 was maintained by approximately 91% of the cells over 25 generations in the absence of selection. To the best of our knowledge this is the first report of an efficient cloning system that facilitates the generation of food-grade recombinant P. freudenreichii strains.

Cloning, expression, and crystallization of Cpn60 proteins from Thermococcus litoralis.

PubMed

Osipiuk, J; Sriram, M; Mai, X; Adams, M W; Joachimiak, A

2000-01-01

Two genes of the extreme thermophilic archaeon Thermococcus litoralis homologous to those that code for Cpn60 chaperonins were cloned and expressed in Escherichia coli. Each of the Cpn60 subunits as well as the entire Cpn60 complex crystallize in a variety of morphological forms. The best crystals diffract to 3.6 A resolution at room temperature and belong to the space group 1422 with unit cell parameters a = b = 193.5 A, c = 204.2 A.

Molecular cloning and functional expression of the guinea pig alpha(1a)-adrenoceptor.

PubMed

González-Espinosa, C; Romero-Avila, M T; Mora-Rodríguez, D M; González-Espinosa, D; García-Sáinz, J A

2001-08-31

In the present paper, the cloning and expression of the guinea pig alpha(1A)-adrenoceptor is presented. The nucleotide sequence had an open reading frame of 1401 bp that encoded a 466 amino-acid protein with an estimated molecular mass of approximately 51.5 kDa. When the clone was expressed in Cos-1 cells, specific high-affinity binding of [(3)H]prazosin and [(3)H]tamsulosin was observed. Chloroethylclonidine treatment of membranes slightly decreased the total binding with both radioligands. Binding competition experiments using [(3)H]tamsulosin showed the following potency order: (a) for agonists: oxymetazoline >epinephrine>norepinephrine>methoxamine, and (b) for antagonists: prazosin> or 5-methyl-urapidil=benoxathian>phentolamine>BMY 7378 (8-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-8-azaspiro[4,5]decane-7,9-dione). Photoaffinity labeling using [(125)I-aryl]azido-prazosin revealed a major broad band with a molecular mass between 70 and 80 kDa. The receptor was functional, as evidenced by an epinephrine-increased production of [(3)H]inositol phosphates that was blocked by prazosin.

CLONING, EXPRESSION, AND MUTATIONAL ANALYSIS OF RAT S-ADENOSYL-1-METHIONINE: ARSENIC (III) METHYLTRANSFERASE

EPA Science Inventory

CLONING, EXPRESSION, AND MUTATIONAL ANALYSIS OF RAT
S-ADENOSYL-L-METHIONINE: ARSENIC(III) METHYLTRANSFERASE

Stephen B. Waters, Ph.D., Miroslav Styblo, Ph.D., Melinda A. Beck, Ph.D., University of North Carolina at Chapel Hill; David J. Thomas, Ph.D., U.S. Environmental...

Identification, expression and tissue distribution of a renalase homologue from mouse.

PubMed

Wang, Jian; Qi, Shaoling; Cheng, Wei; Li, Li; Wang, Fu; Li, Ying-Zi; Zhang, Shu-Ping

2008-12-01

FAD (flavin adenine dinucleotide)-dependent monoamine oxidases play very important roles in many biological processes. A novel monoamine oxidase, named renalase, has been identified in human kidney recently and is found to be markedly reduced in patients with end-stage renal disease (ESRD). Here, we reported the identification of a renalase homologue from mouse, termed mMAO-C (mouse monoamine oxidase-C) after the monoamine oxidase-A and -B (MAO-A and -B). This gene locates on the mouse chromosome 19C1 and its coding region spans 7 exons. The deuced amino acid sequences were predicted to contain a typical secretive signal peptide and a conserved amine oxidase domain. Phylogenetic analysis and multiple sequences alignment indicated that mMAO-C-like sequences exist in all examined species and share significant similarities. This gene has been submitted to the NCBI GenBank database (Accession number: DQ788834). With expression vectors generated from the cloned mMAO-C gene, exogenous protein was effectively expressed in both prokaryotic and eukaryotic cells. Recombinant mMAO-C protein was secreted out of human cell lines, indicating the biological function of its signal peptide. Moreover, tissue expression pattern analysis revealed that mMAO-C gene is predominantly expressed in the mouse kidney and testicle, which implies that kidney and testicle are the main sources of renalase secretion. Shortly, this study provides an insight into understanding the physiological and biological functions of mMAO-C and its homologues in endocrine.

Molecular cloning and expression in Saccharomyces cerevisiae of two Aspergillus nidulans xylanase genes.

PubMed Central

Pérez-Gonzalez, J A; De Graaff, L H; Visser, J; Ramón, D

1996-01-01

Two Aspergillus nidulans genes, xlnA and xlnB, encoding the X22 and X24 xylanases from this fungus, respectively, have been cloned and sequenced. Their cDNAs have been expressed in a laboratory Saccharomyces cerevisiae strain under the control of a constitutive yeast promoter, resulting in the construction of recombinant xylanolytic yeast strains. PMID:8787417

Cloning and Expression of Yak Active Chymosin in Pichia pastoris

PubMed Central

Luo, Fan; Jiang, Wei Hua; Yang, Yuan Xiao; Li, Jiang; Jiang, Ming Feng

2016-01-01

Rennet, a complex of enzymes found in the stomachs of ruminants, is an important component for cheese production. In our study, we described that yak chymosin gene recombinant Pichia pastoris strain could serve as a novel source for rennet production. Yaks total RNA was extracted from the abomasum of an unweaned yak. The yak preprochymosin, prochymosin, and chymosin genes from total RNA were isolated using gene specific primers based on cattle chymosin gene sequence respectively and analyzed their expression pattern byreal time-polymerase chain reaction. The result showed that the chymosin gene expression level of the sucking yaks was 11.45 times higher than one of adult yaks and yak chymosin belongs to Bovidae family in phylogenetic analysis. To express each, the preprochymosin, prochymosin, and chymosin genes were ligated into the expression vector pPICZαA, respectively, and were expressed in Pichia pastoris X33. The results showed that all the recombinant clones of P. pastoris containing the preprochymosin, prochymosin or chymosin genes could produce the active form of recombinant chymosin into the culture supernatant. Heterologous expressed prochymosin (14.55 Soxhlet unit/mL) had the highest enzyme activity of the three expressed chymosin enzymes. Therefore, we suggest that the yak chymosin gene recombinant Pichia pastoris strain could provide an alternative source of rennet production. PMID:27004812

Cloning and expression of L-asparaginase gene in Escherichia coli.

PubMed

Wang, Y; Qian, S; Meng, G; Zhang, S

2001-08-01

The L-asparaginase (ASN) from Escherichia coli AS1.357 was cloned as a DNA fragment generated using polymerase chain reaction technology and primers derived from conserved regions of published ASN gene sequences. Recombinant plasmid pASN containing ASN gene and expression vector pBV220 was transformed in different E. coli host strains. The activity and expression level of ASN in the engineering strains could reach 228 IU/mL of culture fluid and about 50% of the total soluble cell protein respectively, more than 40-fold the enzyme activity of the wild strain. The recombinant plasmid in E. coli AS1.357 remained stable after 72 h of cultivation and 5 h of heat induction without selective pressure. The ASN gene of E. coli AS1.357 was sequenced and had high homology compared to the reported data.

Identification of amino acid residues of mammalian mitochondrial phosphate carrier important for its functional expression in yeast cells, as achieved by PCR-mediated random mutation and gap-repair cloning.

PubMed

Yamagoshi, Ryohei; Yamamoto, Takenori; Hashimoto, Mitsuru; Sugahara, Ryohei; Shiotsuki, Takahiro; Miyoshi, Hideto; Terada, Hiroshi; Shinohara, Yasuo

2017-01-01

The mitochondrial phosphate carrier (PiC) of mammals, but not the yeast one, is synthesized with a presequence. The deletion of this presequence of the mammalian PiC was reported to facilitate the import of the carrier into yeast mitochondria, but the question as to whether or not mammalian PiC could be functionally expressed in yeast mitochondria was not addressed. In the present study, we first examined whether the defective growth on a glycerol plate of yeast cells lacking the yeast PiC gene could be reversed by the introduction of expression vectors of rat PiCs. The introduction of expression vectors encoding full-length rat PiC (rPiC) or rPiC lacking the presequence (ΔNrPiC) was ineffective in restoring growth on the glycerol plates. When we examined the expression levels of individual rPiCs in yeast mitochondria, ΔNrPiC was expressed at a level similar to that of yeast PiC, but that of rPiC was very low. These results indicated that ΔNrPiC expressed in yeast mitochondria is inert. Next, we sought to isolate "revertants" viable on the glycerol plate by expressing randomly mutated ΔNrPiC, and obtained two clones. These clones carried either of two mutations, F267S or F282S; and these mutations restored the transport function of ΔNrPiC in yeast mitochondria. These two Phe residues were conserved in human carrier (hPiC), and the transport function of ΔNhPiC expressed in yeast mitochondria was also markedly improved by their substitutions. Thus, substitution of F267S or F282S was concluded to be important for functional expression of mammalian PiCs in yeast mitochondria. Copyright © 2016 Elsevier B.V. and Mitochondria Research Society. All rights reserved.

Cloning and expression of Clostridium perfringens type D vaccine strain epsilon toxin gene in E. coli as a recombinant vaccine candidate.

PubMed

Aziminia, Parastoo; Pilehchian-Langroudi, Reza; Esmaeilnia, Kasra

2016-08-01

Clostridium perfringens, a Gram-positive obligate anaerobic bacterium, is able to form resistant spores which are widely distributed in the environment. C. perfringens is subdivided into five types A to E based on its four major alpha, beta, epsilon and iota toxins. The aim of the present study was cloning and expression of C. perfringens type D vaccine strain epsilon toxin gene. Genomic DNA was extracted and the epsilon toxin gene was amplified using Pfu DNA polymerase. The PCR product was cloned into pJET1.2/blunt cloning vector. The recombinant vector (pJETε) was sequenced using universal primers. At the next step epsilon toxin gene was subcloned into pET22b(+) expression vector and transformed into E. coli Rosetta (DE3) host strain. The recombinant protein has been expressed in E. coli Rosetta (DE3) cells after subcloning of C. perfringens etx gene (1008 bp) into the expression vector. We concluded that E. coli Rosetta strain was suitable for the expression of recombinant C. perfringens epsilon toxin protein from pET22ε expression vector. This recombinant cell can be used for further research on recombinant vaccine development.

Cloning, expression and crystallisation of SGT1 co-chaperone protein from Glaciozyma antarctica

NASA Astrophysics Data System (ADS)

Yusof, Nur Athirah; Bakar, Farah Diba Abu; Beddoe, Travis; Murad, Abdul Munir Abdul

2013-11-01

Studies on psycrophiles are now in the limelight of today's post genomic era as they fascinate the research and development industries. The discovery from Glaciozyma antarctica, an extreme cold adapted yeast from Antarctica shows promising future to provide cost effective natural sustainable energy and create wider understanding of the property that permits this organisms to adapt to extreme temperature downshift. In plants and yeast, studies show the interaction between SGT1 and HSP90 are essential for disease resistance and heat stress by activating a number of resistance proteins. Here we report for the first time cloning, expression and crystallization of the recombinant SGT1 protein of G. antarctica (rGa_SGT1), a highly conserved eukaryotic protein that interacts with the molecular chaperones HSP90 (heat shock protein 90) apparently associated in a role of co-chaperone that may play important role in cold adaptation. The sequence analysis of rGa_SGT1 revealed the presence of all the characteristic features of SGT1 protein. In this study, we present the outlines and results of protein structural study of G. antarctica SGT1 protein. We validate this approach by starting with cloning the target insert into Ligation Independent Cloning system proceeded with expression using E. coli system, and crystallisation of the target rGA_SGT1 protein. The work is still on going with the target subunit of the complex proteins yielded crystals. These results, still ongoing, open a platform for better understanding of the uniqueness of this crucial molecular machine function in cold adaptation.

Tissue Gene Expression Analysis Using Arrayed Normalized cDNA Libraries

PubMed Central

Eickhoff, Holger; Schuchhardt, Johannes; Ivanov, Igor; Meier-Ewert, Sebastian; O'Brien, John; Malik, Arif; Tandon, Neeraj; Wolski, Eryk-Witold; Rohlfs, Elke; Nyarsik, Lajos; Reinhardt, Richard; Nietfeld, Wilfried; Lehrach, Hans

2000-01-01

We have used oligonucleotide-fingerprinting data on 60,000 cDNA clones from two different mouse embryonic stages to establish a normalized cDNA clone set. The normalized set of 5,376 clones represents different clusters and therefore, in almost all cases, different genes. The inserts of the cDNA clones were amplified by PCR and spotted on glass slides. The resulting arrays were hybridized with mRNA probes prepared from six different adult mouse tissues. Expression profiles were analyzed by hierarchical clustering techniques. We have chosen radioactive detection because it combines robustness with sensitivity and allows the comparison of multiple normalized experiments. Sensitive detection combined with highly effective clustering algorithms allowed the identification of tissue-specific expression profiles and the detection of genes specifically expressed in the tissues investigated. The obtained results are publicly available (http://www.rzpd.de) and can be used by other researchers as a digital expression reference. [The sequence data described in this paper have been submitted to the EMBL data library under accession nos. AL360374–AL36537.] PMID:10958641

Cloning and expression of phosphoglycerate mutase from the psychrophilic yeast, Glaciozyma antarctica PI12

NASA Astrophysics Data System (ADS)

Jaafar, Nardiah Rizwana; Bakar, Farah Diba Abu; Murad, Abdul Munir Abdul; Mahadi, Nor Muhammad

2015-09-01

The conversion of 3-phosphoglycerate to 2-phosphoglycerate during glycolysis and gluconeogenesis is catalyzed by phosphoglycerate mutase (PGM). Better understanding of metabolic reactions performed by this enzyme has been studied extensively in prokaryotes and eukaryotes. Here, we report a phosphoglycerate mutase from the psychrophilic yeast, Glaciozyma antarctica. cDNA encoding for PGM from G. antarctica PI12, a psychrophilic yeast isolated from sea ice at Casey Station, Antarctica was amplified. The gene was then cloned into a cloning vector and sequenced, which verified its identity as the gene putatively encoding for PGM. The recombinant protein was expressed in Escherichia coli BL21 (DE3) as inclusion bodies and this was confirmed by SDS-PAGE and Western blot.

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.

Generating an Open Reading Frame (ORF) Entry Clone and Destination Clone.

PubMed

Reece-Hoyes, John S; Walhout, Albertha J M

2018-01-02

This protocol describes using the Gateway recombinatorial cloning system to create an Entry clone carrying an open reading frame (ORF) and then to transfer the ORF into a Destination vector. In this example, BP recombination is used to clone an ORF from a cDNA source into the Donor vector pDONR 221. The ORF from the resulting Entry clone is then transferred into the Destination vector pDEST-15; the product (the Destination clone) will express the ORF as an amino-terminal GST-fusion. The technique can be used as a guide for cloning any other DNA fragment of interest-a promoter sequence or 3' untranslated region (UTR), for example-with substitutions of different genetic material such as genomic DNA, att sites, and vectors as required. The series of constructions and transformations requires 9-15 d, not including time that may be required for sequence confirmation, if desired/necessary. © 2018 Cold Spring Harbor Laboratory Press.

Cloning and heterologous expression of chlorophyll a synthase in Rhodobacter sphaeroides.

PubMed

Ipekoğlu, Emre M; Göçmen, Koray; Öz, Mehmet T; Gürgan, Muazzez; Yücel, Meral

2017-03-01

Rhodobacter sphaeroides is a purple non-sulfur bacterium which photoheterotrophically produces hydrogen from organic acids under anaerobic conditions. A gene coding for putative chlorophyll a synthase (chlG) from cyanobacterium Prochlorococcus marinus was amplified by nested polymerase chain reaction and cloned into an inducible-expression plasmid which was subsequently transferred to R. sphaeroides for heterologous expression. Induced expression of chlG in R. sphaeroides led to changes in light absorption spectrum within 400-700 nm. The hydrogen production capacity of the mutant strain was evaluated on hydrogen production medium with 15 mM malate and 2 mM glutamate. Hydrogen yield and productivity were increased by 13.6 and 22.6%, respectively, compared to the wild type strain. The results demonstrated the feasibility of genetic engineering to combine chlorophyll and bacteriochlorophyll biosynthetic pathways which utilize common intermediates. Heterologous expression of key enzymes from biosynthetic pathways of various pigments is proposed here as a general strategy to improve absorption spectra and yield of photosynthesis and hydrogen gas production in bacteria. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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

Molecular Cloning of an Immunogenic Protein of Baylisascaris procyonis and Expression in Escherichia coli for Use in Developing Improved Serodiagnostic Assays▿

PubMed Central

Dangoudoubiyam, Sriveny; Vemulapalli, Ramesh; Hancock, Kathy; Kazacos, Kevin R.

2010-01-01

Larva migrans caused by Baylisascaris procyonis is an important zoonotic disease. Current serological diagnostic assays for this disease depend on the use of the parasite's larval excretory-secretory (ES) antigens. In order to identify genes encoding ES antigens and to generate recombinant antigens for use in diagnostic assays, construction and immunoscreening of a B. procyonis third-stage larva cDNA expression library was performed and resulted in identification of a partial-length cDNA clone encoding an ES antigen, designated repeat antigen 1 (RAG1). The full-length rag1 cDNA contained a 753-bp open reading frame that encoded a protein of 250 amino acids with 12 tandem repeats of a 12-amino-acid long sequence. The rag1 genomic DNA revealed a single intron of 837 bp that separated the 753-bp coding sequence into two exons delimited by canonical splice sites. No nucleotide or amino acid sequences present in the GenBank databases had significant similarity with those of RAG1. We have cloned, expressed, and purified the recombinant RAG1 (rRAG1) and analyzed its diagnostic potential by enzyme-linked immunosorbent assay. Anti-Baylisascaris species-specific rabbit serum showed strong reactivity to rRAG1, while only minimal to no reactivity was observed with sera against the related ascarids Toxocara canis and Ascaris suum, strongly suggesting the specificity of rRAG1. On the basis of these results, the identified RAG1 appears to be a promising diagnostic antigen for the development of serological assays for specific detection of B. procyonis larva migrans. PMID:20926699

CLONING, EXPRESSION, AND CHARACTERIZATION OF RAT S-ADENOSYL-L-METHIONINE: ARSENIC (III) METHYLTRANSFERASE (CYT19)

EPA Science Inventory

CLONING, EXPRESSION, AND CHARACTERIZATION OF RAT S-ADENOSYL-L-METHIONINE: ARSENIC(III) METHYLTRANSFERASE (cyt19)

Stephen B. Waters1 , Felicia Walton1 , Miroslav Styblo1 , Karen Herbin-Davis2, and David J. Thomas2 1 School of Medicine, University of North Carolina at Chape...

Cloning, expression, crystallization and preliminary X-ray data analysis of norcoclaurine synthase from Thalictrum flavum

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

Pasquo, Alessandra; Bonamore, Alessandra; Franceschini, Stefano

The cloning, expression, crystallization and preliminary X-ray data analysis of norcoclaurine synthase from T. flavum, a protein which catalyzes the first committed step in the biosynthesis of benzylisoquinoline alkaloids, are reported. Norcoclaurine synthase (NCS) catalyzes the condensation of 3,4-dihydroxyphenylethylamine (dopamine) and 4-hydroxyphenylacetaldehyde (4-HPAA) as the first committed step in the biosynthesis of benzylisoquinoline alkaloids in plants. The protein was cloned, expressed and purified. Crystals were obtained at 294 K by the hanging-drop vapour-diffusion method using ammonium sulfate and sodium chloride as precipitant agents and diffract to better than 3.0 Å resolution using a synchrotron-radiation source. The crystals belong to themore » trigonal space group P3{sub 1}21, with unit-cell parameters a = b = 86.31, c = 118.36 Å. A selenomethionine derivative was overexpressed, purified and crystallized in the same space group. A complete MAD data set was collected at 2.7 Å resolution. The model is under construction.« less

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.

Identification of the Propionicin F Bacteriocin Immunity Gene (pcfI) and Development of a Food-Grade Cloning System for Propionibacterium freudenreichii▿ †

PubMed Central

Brede, Dag Anders; Lothe, Sheba; Salehian, Zhian; Faye, Therese; Nes, Ingolf F.

2007-01-01

This report describes the first functional analysis of a bacteriocin immunity gene from Propionibacterium freudenreichii and its use as a selection marker for food-grade cloning. Cloning of the pcfI gene (previously orf5 [located as part of the pcfABC propionicin F operon]) rendered the sensitive host 1,000-fold more tolerant to the propionicin F bacteriocin. The physiochemical properties of the 127-residue large PcfI protein resemble those of membrane-bound immunity proteins from bacteriocin systems found in lactic acid bacteria. The high level of immunity conferred by pcfI allowed its use as a selection marker for plasmid transformation in P. freudenreichii. Electroporation of P. freudenreichii IFO12426 by use of the pcfI expression plasmid pSL102 and propionicin F selection (200 bacteriocin units/ml) yielded 107 transformants/μg DNA. The 2.7-kb P. freudenreichii food-grade cloning vector pSL104 consists of the pLME108 replicon, a multiple cloning site, and pcfI expressed from the constitutive PpampS promoter for selection. The pSL104 vector efficiently facilitated cloning of the propionicin T1 bacteriocin in P. freudenreichii. High-level propionicin T1 production (640 BU/ml) was obtained with the IFO12426 strain, and the food-grade propionicin T1 expression plasmid pSL106 was maintained by ∼91% of the cells over 25 generations in the absence of selection. To the best of our knowledge this is the first report of an efficient cloning system that facilitates the generation of food-grade recombinant P. freudenreichii strains. PMID:17933941

Cloning and expression of clt genes encoding milk-clotting proteases from Myxococcus xanthus 422.

PubMed

Poza, M; Prieto-Alcedo, M; Sieiro, C; Villa, T G

2004-10-01

The screening of a gene library of the milk-clotting strain Myxococcus xanthus 422 constructed in Escherichia coli allowed the description of eight positive clones containing 26 open reading frames. Only three of them (cltA, cltB, and cltC) encoded proteins that exhibited intracellular milk-clotting ability in E. coli, Saccharomyces cerevisiae, and Pichia pastoris expression systems.

Literature and patent analysis of the cloning and identification of human functional genes in China.

PubMed

Xia, Yan; Tang, LiSha; Yao, Lei; Wan, Bo; Yang, XianMei; Yu, Long

2012-03-01

The Human Genome Project was launched at the end of the 1980s. Since then, the cloning and identification of functional genes has been a major focus of research across the world. In China too, the potentially profound impact of such studies on the life sciences and on human health was realized, and relevant studies were initiated in the 1990s. To advance China's involvement in the Human Genome Project, in the mid-1990s, Committee of Experts in Biology from National High Technology Research and Development Program of China (863 Program) proposed the "two 1%" goal. This goal envisaged China contributing 1% of the total sequencing work, and cloning and identifying 1% of the total human functional genes. Over the past 20 years, tremendous achievement has been accomplished by Chinese scientists. It is well known that scientists in China finished the 1% of sequencing work of the Human Genome Project, whereas, there is no comprehensive report about "whether China had finished cloning and identifying 1% of human functional genes". In the present study, the GenBank database at the National Center of Biotechnology Information, the PubMed search tool, and the patent database of the State Intellectual Property Office, China, were used to retrieve entries based on two screening standards: (i) Were the newly cloned and identified genes first reported by Chinese scientists? (ii) Were the Chinese scientists awarded the gene sequence patent? Entries were retrieved from the databases up to the cut-off date of 30 June 2011 and the obtained data were analyzed further. The results showed that 589 new human functional genes were first reported by Chinese scientists and 159 gene sequences were patented (http://gene.fudan.sh.cn/introduction/database/chinagene/chinagene.html). This study systematically summarizes China's contributions to human functional genomics research and answers the question "has China finished cloning and identifying 1% of human functional genes?" in the affirmative.

Cloning and expression of soluble truncated variants of Borrelia OspA, OspB and Vmp7

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

Dunn, J.J.; Barbour, A.G.

1996-11-05

A method is provided for preparing soluble recombinant variations of Borrelia lipoproteins such as Borrelia burgdorferi outer surface protein A (OspA) and outer surface protein B (OspB), and B. hermsii variable major protein 7 (Vmp7). The method includes synthesizing a set of oligonucleotide primers, amplifying the template DNA utilizing the PCR, purifying the amplification products, cloning the amplification products into a suitable expression vector, transforming a suitable host utilizing the cloned expression vector, cultivating the transformed host for protein production and subsequently isolating and purifying the resulting protein. Also provided are soluble, recombinant variations of Borrelia burgdorferi outer surface proteinmore » A (OspA), outer surface protein B (OspB), and B. hermsii variable major protein 7 (Vmp7). The expression vectors harboring DNA encoding the recombinant variations, pET9-OspA, pET9-OspB and pET9-Vmp7, as well as the E. coli host BL21(DE3)/pLysS transformed with each of these vectors, are also disclosed. 38 figs.« less

Cloning and expression of soluble truncated variants of Borrelia OspA, OspB and Vmp7

DOEpatents

Dunn, John J.; Barbour, Alan G.

1996-11-05

A method is provided herein for preparing soluble recombinant variations of Borrelia lipoproteins such as Borrelia burgdorferi outer surface protein A (OspA) and outer surface protein B (OspB), and B. hermsii variable major protein 7 (Vmp7). The method includes synthesizing a set of oligonucleotide primers, amplifying the template DNA utilizing the PCR, purifying the amplification products, cloning the amplification products into a suitable expression vector, transforming a suitable host utilizing the cloned expression vector, cultivating the transformed host for protein production and subsequently isolating and purifying the resulting protein. Also provided are soluble, recombinant variations of Borrelia burgdorferi outer surface protein A (OspA), outer surface protein B (OspB), and B. hermsii variable major protein 7 (Vmp7). The expression vectors harboring DNA encoding the recombinant variations, pET9-OspA, pET9-OspB and pET9-Vmp7, as well as the E. coli host BL21(DE3)/pLysS transformed with each of these vectors, are also disclosed.

Cloning and expression of soluble truncated variants of Borrelia OspA, OspB and Vmp7

DOEpatents

Dunn, J.J.; Barbour, A.G.

1996-11-05

A method is provided for preparing soluble recombinant variations of Borrelia lipoproteins such as Borrelia burgdorferi outer surface protein A (OspA) and outer surface protein B (OspB), and B. hermsii variable major protein 7 (Vmp7). The method includes synthesizing a set of oligonucleotide primers, amplifying the template DNA utilizing the PCR, purifying the amplification products, cloning the amplification products into a suitable expression vector, transforming a suitable host utilizing the cloned expression vector, cultivating the transformed host for protein production and subsequently isolating and purifying the resulting protein. Also provided are soluble, recombinant variations of Borrelia burgdorferi outer surface protein A (OspA), outer surface protein B (OspB), and B. hermsii variable major protein 7 (Vmp7). The expression vectors harboring DNA encoding the recombinant variations, pET9-OspA, pET9-OspB and pET9-Vmp7, as well as the E. coli host BL21(DE3)/pLysS transformed with each of these vectors, are also disclosed. 38 figs.

Cloning, expression, and characterization of cadmium and manganese uptake genes from Lactobacillus plantarum

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

Hao, Z.; Chen, S.; Wilson, D.B.

1999-11-01

An Mn{sup 2+} and Cd{sup 2+} uptake gene, mntA, was cloned from Lactobacillus plantarum ATCC 14917 into Escherichia coli. Its expression conferred on E. coli cells increased Cd{sup 2+} sensitivity as well as energy-dependent Cd{sup 2+} uptake activity. Both transcription and translation of mntA were induced by Mn{sup 2+} starvation in L. plantarum, as indicated by reverse transcriptase PCR and immunoblotting. Two Cd{sup 2+} uptake systems have been identified in L. plantarum: one is a high-affinity Mn{sup 2+} and Cd{sup 2+} uptake system that is expressed in Mn{sup 2+}-starved cells, and the other is a nonsaturable Cd{sup 2+} uptake systemmore » that is expressed in Cd{sup 2+}-sufficient cells. MntA was not detected in an Mn{sup 2+}-dependent mutant of L. plantarum which had lost high-affinity Mn{sup 2+} and Cd{sup 2+} uptake activity. The results suggest that mntA is the gene encoding the high-affinity Mn{sup 2+} and Cd{sup 2+} transporter. On the basis of its predicted amino acid sequence, MntA belongs to the family of P-type cation-translocating ATPases. The topology and potential Mn{sup 2+}- and Cd{sup 2+}-binding sites of MntA are discussed. A second clone containing a low-affinity Cd{sup 2+} transport system was also isolated.« less

The GA5 locus of Arabidopsis thaliana encodes a multifunctional gibberellin 20-oxidase: molecular cloning and functional expression.

PubMed

Xu, Y L; Li, L; Wu, K; Peeters, A J; Gage, D A; Zeevaart, J A

1995-07-03

The biosynthesis of gibberellins (GAs) after GA12-aldehyde involves a series of oxidative steps that lead to the formation of bioactive GAs. Previously, a cDNA clone encoding a GA 20-oxidase [gibberellin, 2-oxoglutarate:oxygen oxidoreductase (20-hydroxylating, oxidizing), EC 1.14.11.-] was isolated by immunoscreening a cDNA library from liquid endosperm of pumpkin (Cucurbita maxima L.) with antibodies against partially purified GA 20-oxidase. Here, we report isolation of a genomic clone for GA 20-oxidase from a genomic library of the long-day species Arabidopsis thaliana Heynh., strain Columbia, by using the pumpkin cDNA clone as a heterologous probe. This genomic clone contains a GA 20-oxidase gene that consists of three exons and two introns. The three exons are 1131-bp long and encode 377 amino acid residues. A cDNA clone corresponding to the putative GA 20-oxidase genomic sequence was constructed with the reverse transcription-PCR method, and the identity of the cDNA clone was confirmed by analyzing the capability of the fusion protein expressed in Escherichia coli to convert GA53 to GA44 and GA19 to GA20. The Arabidopsis GA 20-oxidase shares 55% identity and > 80% similarity with the pumpkin GA 20-oxidase at the derived amino acid level. Both GA 20-oxidases share high homology with other 2-oxoglutarate-dependent dioxygenases (2-ODDs), but the highest homology was found between the two GA 20-oxidases. Mapping results indicated tight linkage between the cloned GA 20-oxidase and the GA5 locus of Arabidopsis. The ga5 semidwarf mutant contains a G-->A point mutation that inserts a translational stop codon in the protein-coding sequence, thus confirming that the GA5 locus encodes GA 20-oxidase. Expression of the GA5 gene in Ara-bidopsis leaves was enhanced after plants were transferred from short to long days; it was reduced by GA4 treatment, suggesting end-product repression in the GA biosynthetic pathway.

Identification of candidate vaccine antigens of bovine hemoparasites Theileria parva and Babesia bovis by use of helper T cell clones.

PubMed

Brown, W C; Zhao, S; Logan, K S; Grab, D J; Rice-Ficht, A C

1995-03-01

Current vaccines for bovine hemoparasites utilize live attenuated organisms or virulent organisms administered concurrently with antiparasitic drugs. Although such vaccines can be effective, for most hemoparasites the mechanisms of acquired resistance to challenge infection with heterologous parasite isolates have not been clearly defined. Selection of potentially protective antigens has traditionally made use of antibodies to identify immunodominant proteins. However, numerous studies have indicated that induction of high antibody titers neither predicts the ability of an antigen to confer protective immunity nor correlates with protection. Because successful parasites have evolved antibody evasion tactics, alternative strategies to identify protective immunogens should be used. Through the elaboration of cytokines, T helper 1-(Th1)-like T cells and macrophages mediate protective immunity against many intracellular parasites, and therefore most likely play an important role in protective immunity against bovine hemoparasites. CD4+ T cell clones specific for soluble or membrane antigens of either Theileria parva schizonts or Babesia bovis merozoites were therefore employed to identify parasite antigens that elicit strong Th cell responses in vitro. Soluble cytosolic parasite antigen was fractionated by gel filtration, anion exchange chromatography or hydroxylapatite chromatography, or a combination thereof, and fractions were tested for the ability to induce proliferation of Th cell clones. This procedure enabled the identification of stimulatory fractions containing T. parva proteins of approximately 10 and 24 kDa. Antisera raised against the purified 24 kDa band reacted with a native schizont protein of approximately 30 kDa. Babesia bovis-specific Th cell clones tested against fractionated soluble Babesia bovis merozoite antigen revealed the presence of at least five distinct antigenic epitopes. Proteins separated by gel filtration revealed four patterns of

Identification of Small Molecules Targeting the Posttranscriptional Control of ERG Expression

DTIC Science & Technology

2012-10-01

ied. To establish a cell line expressing lucife rase-ERG fusion protein, the vector along pRL-CMV-Rluc expressing Renilla luciferase gene was...expanded, and examined for the e xpression of t wo different luciferases. A clone expressing both Firefly luciferase and Renilla luciferase was selected...treated with the individual chemical at 10 μM for 24 h. The dual luciferase activities were measured. The ratio of Firefly to Renilla lu ciferase

Identification of antigens by monoclonal antibody PD4 and its expression in Escherichia coli

PubMed Central

Ning, Jin-Ying; Sun, Guo-Xun; Huang, Su; Ma, Hong; An, Ping; Meng, Lin; Song, Shu-Mei; Wu, Jian; Shou, Cheng-Chao

2003-01-01

AIM: To clone and express the antigen of monoclonal antibody (MAb) PD4 for further investigation of its function. METHODS: MGC803 cDNA expression library was constructed and screened with PD4 as probes to clone the antigen. After failed in the library screening, immunoprecipitation and SDS-polyacrylamide gel electrophoresis were applied to purify the antigen for sequence analysis. The antigen coming from Mycoplasma hyorhinis (M. hyorhinis) was further confirmed with Western blot analysis by infecting M. hyorhinis -free HeLa cells and eliminating the M. hyorhinis from MGC803 cells. The full p37 gene was cloned by PCR and expressed successfully in Escherichia coli after site-directed mutations. Immunofluorescence assay was used to demonstrate if p37 protein could directly bind to gastric tumor cell AGS. RESULTS: The cDNA library constructed with MGC803 cells was screened by MAb PD4 as probes. Unfortunately, the positive clones identified with MAb PD4 were also reacted with unrelated antibodies. Then, immunoprecipitation was performed and the purified antigen was identified to be a membrane protein of Mycoplasma hyorhinis (M. hyorhinis) by sequencing of N-terminal amino acid residues. The membrane protein was intensively verified with Western blot by eliminating M. hyorhinis from MGC803 cells and by infecting M. hyorhinis-free HeLa cells. The full p37 gene was cloned and expressed successfully in Escherichia coli after site-directed mutations. Immunofluorescence demonstrated that p37 protein could directly bind to gastric tumor cell AGS. CONCLUSION: The antigen recognized by MAb PD4 is from M. hyorhinis, which suggests the actions involved in MAb PD4 is possibly mediated by p37 protein or M. hyorhinis. As p37 protein can bind directly to tumor cells, the pathogenic role of p37 involved in tumorigenesis justifies further investigation. PMID:14562370

[Cloning, prokaryotic expression and antibacterial assay of Tenecin gene encoding an antibacterial peptide from Tenebrio molitor].

PubMed

Liu, Ying; Jiang, Yu-xin; Li, Chao-pin

2011-12-01

To clone tenecin gene, an antibacterial peptide gene, from Tenebrio molitor for its prokaryotic expression and explore the molecular mechanism for regulating the expression of antibacterial peptide in Tenebrio molitor larvae. The antibacterial peptide was induced from the larvae of Tenebrio molitor by intraperitoneal injection of Escherichia coli DH-5α (1×10(8)/ml). RT-PCR was performed 72 h after the injection to clone Tenecin gene followed by sequencing and bioinformatic analysis. The recombinant expression vector pET-28a(+)-Tenecin was constructed and transformed into E. coli BL21(DE3) cells and the expression of tenecin protein was observed after IPTG induction. Tenecin expression was detected in transformed E.coli using SDS-PAGE after 1 mmol/L IPTG induction. Tenecin gene, which was about 255 bp in length, encoded Tenecin protein with a relative molecular mass of 9 kD. Incubation of E.coli with 80, 60, 40, and 20 µg/ml tenecin for 18 h resulted in a diameter of the inhibition zone of 25.1∓0.03, 20.7∓0.06, 17.2∓0.11 and 9.3∓0.04 mm, respectively. Tenecin protein possesses strong antibacterial activity against E. coli DH-5α, which warrants further study of this protein for its potential as an antibacterial agent in clinical application.

Expression, Identification and Purification of Dictyostelium Acetoacetyl-CoA Thiolase Expressed in Escherichia coli

PubMed Central

Tanaka, Takeshi; Shima, Yasuyuki; Ogawa, Naoki; Nagayama, Koki; Yoshida, Takashi; Ohmachi, Tetsuo

2011-01-01

Acetoacetyl-CoA thiolase (AT) is an enzyme that catalyses the CoA-dependent thiolytic cleavage of acetoacetyl-CoA to yield 2 molecules of acetyl-CoA, or the reverse condensation reaction. A full-length cDNA clone pBSGT-3, which has homology to known thiolases, was isolated from Dictyostelium cDNA library. Expression of the protein encoded in pBSGT-3 in Escherichia coli, its thiolase enzyme activity, and the amino acid sequence homology search revealed that pBSGT-3 encodes an AT. The recombinant AT (r-thiolase) was expressed in an active form in an E. coli expression system, and purified to homogeneity by selective ammonium sulfate fractionation and two steps of column chromatography. The purified enzyme exhibited a specific activity of 4.70 mU/mg protein. Its N-terminal sequence was (NH2)-Arg-Met-Tyr-Thr-Thr-Ala-Lys-Asn-Leu-Glu-, which corresponds to the sequence from positions 15 to 24 of the amino acid sequence deduced from pBSGT-3 clone. The r-thiolase in the inclusion body expressed highly in E. coli was the precursor form, which is slightly larger than the purified r-thiolase. When incubated with the cell-free extract of Dictyostelium cells, the precursor was converted to the same size to the purified r-thiolase, suggesting that the presequence at the N-terminus is removed by a Dictyostelium processing peptidase. PMID:21209787

Use of in vivo Expression Technology for the Identification of Putative Host Adaptation Factors of the Lyme Disease Spirochete.

PubMed

Casselli, Timothy; Bankhead, Troy

2015-01-01

The causative agent of Lyme disease, Borrelia burgdorferi, is an obligate parasite that requires either a tick vector or a mammalian host for survival. Identification of the bacterial genes that are specifically expressed during infection of the mammalian host could provide targets for novel therapeutics and vaccines. In vivo expression technology (IVET) is a reporter-based promoter trap system that utilizes selectable markers to identify promoters of bacterial host-specific genes. Using previously characterized genes for in vivo and in vitro selection, this study utilized an IVET system that allows for selection of B. burgdorferi sequences that act as active promoters only during murine infection. This promoter trap system was able to successfully distinguish active promoter sequences both in vivo and in vitro from control sequences and a library of cloned B. burgdorferi genomic fragments. However, a bottleneck effect during the experimental mouse infection limited the utility for genome-wide promoter screening. Overall, IVET was demonstrated as a tool for the identification of in vivo-induced promoter elements of B. burgdorferi, and the observed infection bottleneck apparent using a polyclonal infection pool provides insight into the dynamics of experimental infection with B. burgdorferi. © 2015 S. Karger AG, Basel.

Development of two bacterial artificial chromosome shuttle vectors for a recombination-based cloning and regulated expression of large genes in mammalian cells.

PubMed

Hong, Y K; Kim, D H; Beletskii, A; Lee, C; Memili, E; Strauss, W M

2001-04-01

Most conditional expression vectors designed for mammalian cells have been valuable systems for studying genes of interest by regulating their expressions. The available vectors, however, are reliable for the short-length cDNA clones and not optimal for relatively long fragments of genomic DNA or long cDNAs. Here, we report the construction of two bacterial artificial chromosome (BAC) vectors, capable of harboring large inserts and shuttling among Escherichia coli, yeast, and mammalian cells. These two vectors, pEYMT and pEYMI, contain conditional expression systems which are designed to be regulated by tetracycline and mouse interferons, respectively. To test the properties of the vectors, we cloned in both vectors the green fluorescence protein (GFP) through an in vitro ligation reaction and the 17.8-kb-long X-inactive-specific transcript (Xist) cDNA through homologous recombination in yeast. Subsequently, we characterized their regulated expression properties using real-time quantitative RT-PCR (TaqMan) and RNA-fluorescent in situ hybridization (FISH). We demonstrate that these two BAC vectors are good systems for recombination-based cloning and regulated expression of large genes in mammalian cells. Copyright 2001 Academic Press.

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.

Identification of apoptosis-related PLZF target genes

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

Bernardo, Maria Victoria; Yelo, Estefania; Gimeno, Lourdes

2007-07-27

The PLZF gene encodes a BTB/POZ-zinc finger-type transcription factor, involved in physiological development, proliferation, differentiation, and apoptosis. In this paper, we investigate proliferation, survival, and gene expression regulation in stable clones from the human haematopoietic K562, DG75, and Jurkat cell lines with inducible expression of PLZF. In Jurkat cells, but not in K562 and DG75 cells, PLZF induced growth suppression and apoptosis in a cell density-dependent manner. Deletion of the BTB/POZ domain of PLZF abrogated growth suppression and apoptosis. PLZF was expressed with a nuclear speckled pattern distinctively in the full-length PLZF-expressing Jurkat clones, suggesting that the nuclear speckled localizationmore » is required for PLZF-induced apoptosis. By microarray analysis, we identified that the apoptosis-inducer TP53INP1, ID1, and ID3 genes were upregulated, and the apoptosis-inhibitor TERT gene was downregulated. The identification of apoptosis-related PLZF target genes may have biological and clinical relevance in cancer typified by altered PLZF expression.« less

Cloning and expression of delta-1-pyrroline-5-carboxylate dehydrogenase in Escherichia coli DH5α improves phosphate solubilization.

PubMed

Gong, Mingbo; Tang, Chaoxi; Zhu, Changxiong

2014-11-01

A primary cDNA library of Penicillium oxalicum I1 was constructed using the switching mechanism at the 5' end of the RNA transcript (SMART) technique. A total of 106 clones showed halos in tricalcium phosphate (TCP) medium, and clone I-40 showed clear halos. The full-length cDNA of clone I-40 was 1355 bp with a complete open reading frame (ORF) of 1032 bp, encoding a protein of 343 amino acids. Multiple alignment analysis revealed a high degree of homology between the ORF of clone I-40 and delta-1-pyrroline-5-carboxylate dehydrogenase (P5CDH) of other fungi. The ORF expression vector was constructed and transformed into Escherichia coli DH5α. The transformant (ORF-1) with the P5CDH gene secreted organic acid in medium with TCP as the sole source of phosphate. Acetic acid and α-ketoglutarate were secreted in 4 and 24 h, respectively. ORF-1 decreased the pH of the medium from 6.62 to 3.45 and released soluble phosphate at 0.172 mg·mL(-1) in 28 h. Expression of the P. oxalicum I1 p5cdh gene in E. coli could enhance organic acid secretion and phosphate-solubilizing ability.

Molecular cloning and expression in Saccharomyces cerevisiae and Neurospora crassa of the invertase gene from Neurospora crassa.

PubMed

Carú, M; Cifuentes, V; Pincheira, G; Jiménez, A

1989-10-01

A plasmid (named pCN2) carrying a 7.6 kb BamHI DNA insert was isolated from a Neurospora crassa genomic library raised in the yeast vector YRp7. Saccharomyces cerevisiae suco and N. crassa inv strains transformed with pNC2 were able to grow on sucrose-based media and expressed invertase activity. Saccharomyces cerevisiae suco (pNC2) expressed a product which immunoreacted with antibody raised against purified invertase from wild type N. crassa, although S. cerevisiae suc+ did not. The cloned DNA hybridized with a 7.6 kb DNA fragment from BamHI-restricted wild type N. crassa DNA. Plasmid pNC2 transformed N. crassa Inv- to Inv+ by integration either near to the endogenous inv locus (40% events) or at other genomic sites (60% events). It appears therefore that the cloned DNA piece encodes the N. crassa invertase enzyme. A 3.8 kb XhoI DNA fragment, derived from pNC2, inserted in YRp7, in both orientation, was able to express invertase activity in yeast, suggesting that it contains an intact invertase gene which is not expressed from a vector promoter.

Identification of positional candidates for bovine placental genes responsible for early embryonic death during cloning-attempted pregnancy.

PubMed

Yamada, Takahisa; Muramatsu, Youji; Taniguchi, Yukio; Sasaki, Yoshiyuki

Our previous study detected 291 and 77 genes showing early embryonic death-associated elevation and reduction of expression, respectively, in the fetal placenta of the cow carrying somatic nuclear transfer-derived cloned embryo. In this study, we mapped the 10 genes showing the elevation and the 10 genes doing the reduction most significantly, using somatic cell hybrid and bovine draft genome sequence. We then compared the mapped positions for these genes with the genomic locations of bovine quantitative trait loci for still-birth and/or abortion. Among the mapped genes, peptidylglycine alpha-amidating monooxygenase (PAM), spectrin, beta, nonerythrocytic 1 (SPTBNI), and an unknown novel gene containing AU277832 expressed sequence tag were intriguing, in that the mapped positions were consistent with the genomic locations of bovine still-birth and/or abortion quantitative trait loci, and thus identified as positional candidates for bovine placental genes responsible for the early embryonic death during the pregnancy attempted by somatic nuclear transfer-derived cloning.

[Molecular cloning and expression of the severe acute respiratory syndrome-associated coronavirus nucleocapsid protein and its clinical application].

PubMed

Lu, Jian; Zhou, Bai-ping; Zhou, Yu-sen; Jiang, Xiao-ling; Wen, Li-xia; Le, Xiao-hua; Li, Bing; Xu, Liu-mei; Li, Li-xiong

2005-03-01

To clone and express nucleocapsid (N) protein of the severe acute respiratory syndrome (SARS)-associated coronavirus, and to evaluate its antigenicity and application value in the development of serological diagnostic test for SARS. SARS-associated coronavirus N protein gene was amplified from its genomic RNA by reverse transcript nested polymerase chain reaction (RT-nested-PCR) and cloned into pBAD/Thio-TOPO prokaryotic expression vector. The recombinant N fusion protein was expressed and purified, and its antigenicity and specificity was analyzed by Western Blot, to establish the recombinant N protein-based ELISA for detection of IgG antibodies to SARS-associated coronavirus, and SARS-associated coronavirus lysates-based ELISA was compared parallelly. The recombinant expression vector produced high level of the N fusion protein after induction, and that protein was purified successfully by affinity chromatography and displayed higher antigenicity and specificity as compared with whole virus lysates. The recombinant SARS-associated coronavirus N protein possessed better antigenicity and specificity and could be employed to establish a new, sensitive, and specific ELISA for SARS diagnosis.

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, expression, and characterization of an insoluble glucan-producing glucansucrase from Leuconostoc mesenteroides NRRL B-1118

USDA-ARS?s Scientific Manuscript database

We have cloned a glucansucrase from the type strain of Leuconostoc mesenteroides (NRRL B-1118; ATCC 8293) and successfully expressed the enzyme in Escherichia coli. The recombinant processed enzyme has a putative sequence identical to the predicted secreted native enzyme (1,473 amino acids; 161,468...

WebPrInSeS: automated full-length clone sequence identification and verification using high-throughput sequencing data.

PubMed

Massouras, Andreas; Decouttere, Frederik; Hens, Korneel; Deplancke, Bart

2010-07-01

High-throughput sequencing (HTS) is revolutionizing our ability to obtain cheap, fast and reliable sequence information. Many experimental approaches are expected to benefit from the incorporation of such sequencing features in their pipeline. Consequently, software tools that facilitate such an incorporation should be of great interest. In this context, we developed WebPrInSeS, a web server tool allowing automated full-length clone sequence identification and verification using HTS data. WebPrInSeS encompasses two separate software applications. The first is WebPrInSeS-C which performs automated sequence verification of user-defined open-reading frame (ORF) clone libraries. The second is WebPrInSeS-E, which identifies positive hits in cDNA or ORF-based library screening experiments such as yeast one- or two-hybrid assays. Both tools perform de novo assembly using HTS data from any of the three major sequencing platforms. Thus, WebPrInSeS provides a highly integrated, cost-effective and efficient way to sequence-verify or identify clones of interest. WebPrInSeS is available at http://webprinses.epfl.ch/ and is open to all users.

WebPrInSeS: automated full-length clone sequence identification and verification using high-throughput sequencing data

PubMed Central

Massouras, Andreas; Decouttere, Frederik; Hens, Korneel; Deplancke, Bart

2010-01-01

High-throughput sequencing (HTS) is revolutionizing our ability to obtain cheap, fast and reliable sequence information. Many experimental approaches are expected to benefit from the incorporation of such sequencing features in their pipeline. Consequently, software tools that facilitate such an incorporation should be of great interest. In this context, we developed WebPrInSeS, a web server tool allowing automated full-length clone sequence identification and verification using HTS data. WebPrInSeS encompasses two separate software applications. The first is WebPrInSeS-C which performs automated sequence verification of user-defined open-reading frame (ORF) clone libraries. The second is WebPrInSeS-E, which identifies positive hits in cDNA or ORF-based library screening experiments such as yeast one- or two-hybrid assays. Both tools perform de novo assembly using HTS data from any of the three major sequencing platforms. Thus, WebPrInSeS provides a highly integrated, cost-effective and efficient way to sequence-verify or identify clones of interest. WebPrInSeS is available at http://webprinses.epfl.ch/ and is open to all users. PMID:20501601

Identification of a nucleoside analog active against adenosine kinase–expressing plasma cell malignancies

PubMed Central

Sadek, Jouliana; Hernandez-Hopkins, Denise; Akar, Gunkut; Barelli, Peter J.; Sahai, Michelle A.; Zhou, Hufeng; Totonchy, Jennifer; Jayabalan, David; Niesvizky, Ruben; Guasparri, Ilaria; Liu, Yifang; Sei, Shizuko; Shoemaker, Robert H.; Elemento, Olivier; Kaye, Kenneth M.

2017-01-01

Primary effusion lymphoma (PEL) is a largely incurable malignancy of B cell origin with plasmacytic differentiation. Here, we report the identification of a highly effective inhibitor of PEL. This compound, 6-ethylthioinosine (6-ETI), is a nucleoside analog with toxicity to PEL in vitro and in vivo, but not to other lymphoma cell lines tested. We developed and performed resistome analysis, an unbiased approach based on RNA sequencing of resistant subclones, to discover the molecular mechanisms of sensitivity. We found different adenosine kinase–inactivating (ADK-inactivating) alterations in all resistant clones and determined that ADK is required to phosphorylate and activate 6-ETI. Further, we observed that 6-ETI induces ATP depletion and cell death accompanied by S phase arrest and DNA damage only in ADK-expressing cells. Immunohistochemistry for ADK served as a biomarker approach to identify 6-ETI–sensitive tumors, which we documented for other lymphoid malignancies with plasmacytic features. Notably, multiple myeloma (MM) expresses high levels of ADK, and 6-ETI was toxic to MM cell lines and primary specimens and had a robust antitumor effect in a disseminated MM mouse model. Several nucleoside analogs are effective in treating leukemias and T cell lymphomas, and 6-ETI may fill this niche for the treatment of PEL, plasmablastic lymphoma, MM, and other ADK-expressing cancers. PMID:28504647

Identification of a nucleoside analog active against adenosine kinase-expressing plasma cell malignancies.

PubMed

Nayar, Utthara; Sadek, Jouliana; Reichel, Jonathan; Hernandez-Hopkins, Denise; Akar, Gunkut; Barelli, Peter J; Sahai, Michelle A; Zhou, Hufeng; Totonchy, Jennifer; Jayabalan, David; Niesvizky, Ruben; Guasparri, Ilaria; Hassane, Duane; Liu, Yifang; Sei, Shizuko; Shoemaker, Robert H; Warren, J David; Elemento, Olivier; Kaye, Kenneth M; Cesarman, Ethel

2017-06-01

Primary effusion lymphoma (PEL) is a largely incurable malignancy of B cell origin with plasmacytic differentiation. Here, we report the identification of a highly effective inhibitor of PEL. This compound, 6-ethylthioinosine (6-ETI), is a nucleoside analog with toxicity to PEL in vitro and in vivo, but not to other lymphoma cell lines tested. We developed and performed resistome analysis, an unbiased approach based on RNA sequencing of resistant subclones, to discover the molecular mechanisms of sensitivity. We found different adenosine kinase-inactivating (ADK-inactivating) alterations in all resistant clones and determined that ADK is required to phosphorylate and activate 6-ETI. Further, we observed that 6-ETI induces ATP depletion and cell death accompanied by S phase arrest and DNA damage only in ADK-expressing cells. Immunohistochemistry for ADK served as a biomarker approach to identify 6-ETI-sensitive tumors, which we documented for other lymphoid malignancies with plasmacytic features. Notably, multiple myeloma (MM) expresses high levels of ADK, and 6-ETI was toxic to MM cell lines and primary specimens and had a robust antitumor effect in a disseminated MM mouse model. Several nucleoside analogs are effective in treating leukemias and T cell lymphomas, and 6-ETI may fill this niche for the treatment of PEL, plasmablastic lymphoma, MM, and other ADK-expressing cancers.

The identification of unfolding facial expressions.

PubMed

Fiorentini, Chiara; Schmidt, Susanna; Viviani, Paolo

2012-01-01

We asked whether the identification of emotional facial expressions (FEs) involves the simultaneous perception of the facial configuration or the detection of emotion-specific diagnostic cues. We recorded at high speed (500 frames s-1) the unfolding of the FE in five actors, each expressing six emotions (anger, surprise, happiness, disgust, fear, sadness). Recordings were coded every 10 frames (20 ms of real time) with the Facial Action Coding System (FACS, Ekman et al 2002, Salt Lake City, UT: Research Nexus eBook) to identify the facial actions contributing to each expression, and their intensity changes over time. Recordings were shown in slow motion (1/20 of recording speed) to one hundred observers in a forced-choice identification task. Participants were asked to identify the emotion during the presentation as soon as they felt confident to do so. Responses were recorded along with the associated response times (RTs). The RT probability density functions for both correct and incorrect responses were correlated with the facial activity during the presentation. There were systematic correlations between facial activities, response probabilities, and RT peaks, and significant differences in RT distributions for correct and incorrect answers. The results show that a reliable response is possible long before the full FE configuration is reached. This suggests that identification is reached by integrating in time individual diagnostic facial actions, and does not require perceiving the full apex configuration.

Heterologous expression of laccase cDNA from Ceriporiopsis subvermispora yields copper-activated apoprotein and complex isoform patterns

Treesearch

Luis F. Larrondo; Marcela Avila; Loreto Salas; Dan Cullen; Rafael Vicuna

2003-01-01

Analysis of genomic clones encoding a putative laccase in homokaryon strains of Ceriporiopsis subvermispora led to the identification of an allelic variant of the previously described lcs-1 gene. A cDNA clone corresponding to this gene was expressed in Aspergillus nidulans and in Aspergillus niger. Enzyme assays and Western blots showed that both hosts secreted active...

The Dermatophagoides farinae group 22 allergen: cloning and expression in Escherichia coli.

PubMed

Cui, Yu-bao; Cai, Hong-xing; Zhou, Ying; Wang, Nan; Yu, Li-li; Yang, Li; Zhang, Cheng-bo

2015-09-01

Dermatophagoides farinae (Hughes) (Acari: Pyroglyphidae) and other domestic mites produce allergens that affect people worldwide. Here, the complementary DNA (cDNA) coding for group 22 allergen of D. farinae (Der f 22) from China was cloned, sequenced, and expressed successfully. The cDNA encoding Der f 22 was synthesized by reverse transcription polymerase chain reaction (RT-PCR), then ligated to the pCold-TF for expression in Escherichia coli BL21 cells. The purified recombinant fusion protein was identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), Western-blotting, and tandem matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF/TOF). The full-length cDNA comprised 468 nucleotides and was 99.57% (466/468) identical with the reference sequence (GenBank: DQ643992). After the plasmid pCold-TF-Der f 22 was transformed into E. coli BL21 and expressed with the induction of IPTG, SDS-PAGE showed a specific band for the recombinant fusion protein. The recombinant fusion protein, which was purified by chromatography, bound with a His-tagged antibody by Western blotting. MALDI-TOF/TOF mass spectrometry revealed that the structure of the recombinant protein was identical to the predicted Der f 22 structure. The hydrophilic protein contains a signal peptide of 20 amino acids, and the mature Der f 22 consists of 135 amino acid residues with a molecular weight of 14.7 kDa and theoretical isoelectric points (pI) of 6.38. Its secondary structure comprises an alpha helix (38.5%), beta-sheet (45.9%), random coils (11.85%), and beta-turns (11.1%). This work represents the first reported full-length sequence and successful cloning of Der f 22 from D. farinae in China; bioinformatics analysis can be used to further study the allergenicity and clinical utility of the recombinant Der f 22. © 2015 ARS-AAOA, LLC.

Cloning, expression, and purification of recombinant protein MPT-64 from a virulent strain of Mycobacterium bovis in a prokaryotic system.

PubMed

Tashakkori, Maryam Mohammadi; Tebianian, Majid; Tabatabaei, Mohammad; Mosavari, Nader

2016-12-01

Tuberculosis (TB) is a zoonotic infectious disease common to humans and animals that is caused by the rod-shaped acid-fast bacterium Mycobacterium bovis. Rapid and sensitive detection of TB is promoted by specific antigens. Virulent strains of the TB complex from M. bovis contain 16 regions of difference (RD) in their genome that encode important proteins, including major protein of Mycobacterium Tuberculosis 64 (MBT-64, which is a primary immune-stimulating antigen encoded by RD-2. In this study, we cloned, expressed, and purified MPT-64 as a potent M. bovis antigen in a prokaryotic system for use in future diagnostic studies. The antigenic region of the Mpt64 gene was investigated by bioinformatics methods, cloned into the PQE-30 plasmid, and expressed in Escherichia coli M15 cells, followed by isopropyl β-d-1-thiogalactopyranoside induction. The expressed protein was analyzed sodium dodecyl sulfate polyacrylamide gel electrophoresis and purified using a nickel-affinity column. Biological activity was confirmed by western blot using specific antibodies. Our data verified the successful cloning of the Mpt64 gene (687-bp segment) via the expression vector and purification of recombinant MPT-64 as a 24-kDa protein. These results indicated successful expression and purification of recombinant MPT-64 protein in a prokaryotic system. This protein can be used for serological diagnosis, improved detection of pathogenicity and non-pathogenicity between infected cattle, and for verification of suspected cases of bovine TB. Copyright © 2016.

Cloning and expression patterns of two Smad genes during embryonic development and shell formation of the Pacific oyster Crassostrea gigas

NASA Astrophysics Data System (ADS)

Liu, Gang; Huan, Pin; Liu, Baozhong

2014-11-01

Increasing evidence indicates that transforming growth factor β (TGF-β) signaling pathways play many important roles in the early development of mollusks. However, limited information is known concerning their detailed mechanisms. Here, we describe the identification, cloning and characterization of two Smad genes, the key components of TGF-β signaling pathways, from the Pacific oyster Crassostrea gigas. Sequence analysis of the two genes, designated as cgi-smad1/ 5/ 8 and cgi-smad4, revealed conserved functional characteristics. The two genes were widely expressed in embryos and larvae, suggesting multiple roles in the early development of C. gigas. The mRNA of the two genes aggregated in the D quadrant and cgi-smad4 was highly expressed on the dorsal side of the gastrula, indicating that TGF-β signaling pathways may be involved in dorsoventral patterning in C. gigas. Furthermore, high expression levels of the two genes in the shell fields of embryos at different stages suggested important roles for TGF-β signaling pathways in particular phases of shell development, including the formation of the initial shell field and the biomineralization of larval shells. The results of this study provide fundamental support for elucidating how TGF-β signaling pathways participate in the early development of bivalve mollusks, and suggest that further work is warranted to this end.

[Cloning and expressing of cyclophilin B gene from Schistosoma japonnicum and the analysis of immunoprotective effect].

PubMed

Peng, Jinbiao; Han, Hongxiao; Hong, Yang; Wang, Yan; Guo, Fanji; Shi, Yaojun; Fu, Zhiqiang; Liu, Jinming; Cheng, Guofeng; Lin, Jiaojiao

2010-03-01

The present study was intend to clone and express the cDNA encoding Cyclophilin B (CyPB) of Schistosoma japonicum, its preliminary biological function and further immunoprotective effect against schistosome infection in mice. RT-PCR technique was applied to amplify a full-length cDNA encoding protein Cyclophilin B (Sj CyPB) from schistosomula cDNA. The expression profiles of Sj CyPB were determined by Real-time PCR using the template cDNAs isolated from 7, 13, 18, 23, 32 and 42 days parasites. The cDNA containing the Open Reading Frame of CyPB was then subcloned into a pGEX-6P-1 vector and transformed into competent Escherichia coli BL21 for expressing. The recombinant protein was renaturated, purified and its antigenicity were detected by Western blotting, and the immunoprotective effect induced by recombinant Sj CyPB was evaluated in Balb/C mice. The cDNA containing the ORF of Sj CyPB was cloned with the length of 672 base pairs, encoding 223 amino acids. Real-time PCR analysis revealed that the gene had the highest expression in 18-day schistosomula, suggesting that Sj CyPB was schistosomula differentially expressed gene. The recombinant protein showed a good antigenicity detected by Western blotting. Animal experiment indicated that the vaccination of recombinant CyPB protein in mice led to 31.5% worm and 41.01% liver egg burden reduction, respectively, compared with those of the control. A full-length cDNA differentially expressed in schistosomula was obtained. The recombinant Sj CyPB protein could induce partial protection against schistosome infection.

The GA5 locus of Arabidopsis thaliana encodes a multifunctional gibberellin 20-oxidase: Molecular cloning and functional expression

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

Xu, Yun-Ling; Li, Li; Wu, Keqiang

1995-07-03

The biosynthesis of gibberellins (GAs) after GA{sub 12}-aldehyde involves a series of oxidative steps that lead to the formation of bioactive GAs. Previously, a cDNA clone encoding a GA 20-oxidase [gibberellin, 2-oxoglutarate:oxygen oxidoreductase (20-hydroxylating, oxidizing), EC 1.14.11-] was isolated by immunoscreening a cDNA library from liquid endosperm of pumpkin (Cucurbita maxima L.) with antibodies against partially purified GA 20-oxidase. Here, we report isolation of a genomic clone for GA 20-oxidase from a genomic library of the long-day species Arabidopsis thaliana Heynh., strain Columbia, by using the pumpkin cDNA clone as a heterologous probe. This genomic clone contains a GA 20-oxidasemore » gene that consists of three exons and two introns. The three exons are 1131-bp long and encode 377 amino acid residues. A cDNA clone corresponding to the putative GA 20-oxidase genomic sequence was constructed with the reverse transcription-PCR method, and the identity of the cDNA clone was confirmed by analyzing the capability of the fusion protein expressed in Escherichia coli to convert GA{sub 53} to GA{sub 44} and GA{sub 19} to GA{sub 20}. The Arabidopsis GA 20-oxidase shares 55% identity and >80% similarity with the pumpkin GA 20-oxidase at the derived amino acid level. Both GA 20-oxidases share high homology with other 2-oxoglutarate-dependent dioxygenases (2-ODDs), but the highest homology was found between the two GA 20-oxidases. Mapping results indicated tight linkage between the cloned GA 20-oxidase and the GA locus of Arabidopsis. The ga5 semidwarf mutant contains a G {yields} A point mutation that inserts a translational stop codon in the protein-coding sequence, thus confirming that the GA5 locus encodes GA 20-oxidase. Expression of the GA5 gene in Arabidopsis leaves was enhanced after plants were transferred from short to long days; it was reduced by GA{sub 4} treatment, suggesting end-product repression in the GA biosynthetic pathway. 28 refs., 6

Survival of Skin Graft between Transgenic Cloned Dogs and Non-Transgenic Cloned Dogs

PubMed Central

Kim, Geon A; Oh, Hyun Ju; Kim, Min Jung; Jo, Young Kwang; Choi, Jin; Park, Jung Eun; Park, Eun Jung; Lim, Sang Hyun; Yoon, Byung Il; Kang, Sung Keun; Jang, Goo; Lee, Byeong Chun

2014-01-01

Whereas it has been assumed that genetically modified tissues or cells derived from somatic cell nuclear transfer (SCNT) should be accepted by a host of the same species, their immune compatibility has not been extensively explored. To identify acceptance of SCNT-derived cells or tissues, skin grafts were performed between cloned dogs that were identical except for their mitochondrial DNA (mtDNA) haplotypes and foreign gene. We showed here that differences in mtDNA haplotypes and genetic modification did not elicit immune responses in these dogs: 1) skin tissues from genetically-modified cloned dogs were successfully transplanted into genetically-modified cloned dogs with different mtDNA haplotype under three successive grafts over 63 days; and 2) non-transgenic cloned tissues were accepted into transgenic cloned syngeneic recipients with different mtDNA haplotypes and vice versa under two successive grafts over 63 days. In addition, expression of the inserted gene was maintained, being functional without eliciting graft rejection. In conclusion, these results show that transplanting genetically-modified tissues into normal, syngeneic or genetically-modified recipient dogs with different mtDNA haplotypes do not elicit skin graft rejection or affect expression of the inserted gene. Therefore, therapeutically valuable tissue derived from SCNT with genetic modification might be used safely in clinical applications for patients with diseased tissues. PMID:25372489

Improved production of genetically modified fetuses with homogeneous transgene expression after transgene integration site analysis and recloning in cattle.

PubMed

Bressan, Fabiana Fernandes; Dos Santos Miranda, Moyses; Perecin, Felipe; De Bem, Tiago Henrique; Pereira, Flavia Thomaz Verechia; Russo-Carbolante, Elisa Maria; Alves, Daiani; Strauss, Bryan; Bajgelman, Marcio; Krieger, José Eduardo; Binelli, Mario; Meirelles, Flavio Vieira

2011-02-01

Animal cloning by nuclear transfer (NT) has made the production of transgenic animals using genetically modified donor cells possible and ensures the presence of the gene construct in the offspring. The identification of transgene insertion sites in donor cells before cloning may avoid the production of animals that carry undesirable characteristics due to positional effects. This article compares blastocyst development and competence to establish pregnancies of bovine cloned embryos reconstructed with lentivirus-mediated transgenic fibroblasts containing either random integration of a transgene (random integration group) or nuclear transfer derived transgenic fibroblasts with known transgene insertion sites submitted to recloning (recloned group). In the random integration group, eGFP-expressing bovine fetal fibroblasts were selected by fluorescence activated cell sorting (FACS) and used as nuclei donor cells for NT. In the recloned group, a fibroblast cell line derived from a transgenic cloned fetus was characterized regarding transgene insertion and submitted to recloning. The recloned group had higher blastocyst production (25.38 vs. 14.42%) and higher percentage of 30-day pregnancies (14.29 vs. 2.56%) when compared to the random integration group. Relative eGFP expression analysis in fibroblasts derived from each cloned embryo revealed more homogeneous expression in the recloned group. In conclusion, the use of cell lines recovered from transgenic fetuses after identification of the transgene integration site allowed for the production of cells and fetuses with stable transgene expression, and recloning may improve transgenic animal yields.

Cloning and expression analysis of FaPR-1 gene in strawberry

NASA Astrophysics Data System (ADS)

Mo, Fan; Luo, Ya; Ge, Cong; Mo, Qin; Ling, Yajie; Luo, Shu; Tang, Haoru

2018-04-01

The FaPR-1 gene was cloned by RT-PCR from `Benihoppe' strawberry and its bioinformatics analysis was conducted. The results showed that the open reading frame was 483 bp encoding encoding l60 amino acids which protein molecular weight and theoretical isoelectricity were 17854.17 and 8.72 respectively. Subcellular localization prediction shows that this gene is located extracellularly. By comparing strawberry FaPR-l and other plant Pathogenesis-related protein, homology and phylogenetic tree construction showed that the homology with grapes, peach is relatively close. In the treatments of ABA, sucrose and the mixture of the two, the expression of FaPR-1 in strawberry fruit were significantly increased.

Identification and expression profiles of nine glutathione S-transferase genes from the important rice phloem sap-sucker and virus vector Laodelphax striatellus (Fallén) (Hemiptera: Delphacidae).

PubMed

Zhou, Wen-Wu; Li, Xi-Wang; Quan, Yin-Hua; Cheng, Jiaan; Zhang, Chuan-Xi; Gurr, Geoff; Zhu, Zeng-Rong

2012-09-01

Glutathione S-transferases (GSTs) have received considerable attention in insects for their roles in insecticide resistance. Laodelphax striatellus (Fallén) is a serious rice pest. L. striatellus outbreaks occur frequently throughout eastern Asia. A key problem in controlling this pest is its rapid adaptation to numerous insecticides. In this research, nine cDNAs encoding GSTs in L. striatellus were cloned and characterised. The cloned GSTs of L. striatellus belonged to six cytosolic classes and a microsomal subgroup. Exposure to sublethal concentrations of each of the six insecticides, DDT, chlorpyrifos, fipronil, imidacloprid, buprofezin and beta-cypermethrin, quickly induced (6 h) up-expression of LsGSTe1. The expression of LsGSTs2 was increased by chlorpyrifos, fipronil and beta-cypermethrin. Furthermore, exposure of L. striatellus to fipronil, imidacloprid, buprofezin and beta-cypermethrin increased the expression of the LsGSTm gene after 24 or 48 h. This work is the first identification of GST genes from different GST groups in Auchenorrhyncha species and their induction characteristics with insecticide types and time. The elevated expression of GST genes induced by insecticides might be related to the enhanced tolerance of this insect to insecticides and xenobiotics. Copyright © 2012 Society of Chemical Industry.

Molecular cloning and expression analysis of annexin A2 gene in sika deer antler tip.

PubMed

Xia, Yanling; Qu, Haomiao; Lu, Binshan; Zhang, Qiang; Li, Heping

2018-04-01

Molecular cloning and bioinformatics analysis of annexin A2 ( ANXA2 ) gene in sika deer antler tip were conducted. The role of ANXA2 gene in the growth and development of the antler were analyzed initially. The reverse transcriptase polymerase chain reaction (RT-PCR) was used to clone the cDNA sequence of the ANXA2 gene from antler tip of sika deer ( Cervus Nippon hortulorum ) and the bioinformatics methods were applied to analyze the amino acid sequence of Anxa2 protein. The mRNA expression levels of the ANXA2 gene in different growth stages were examined by real time reverse transcriptase polymerase chain reaction (real time RT-PCR). The nucleotide sequence analysis revealed an open reading frame of 1,020 bp encoding 339 amino acids long protein of calculated molecular weight 38.6 kDa and isoelectric point 6.09. Homologous sequence alignment and phylogenetic analysis indicated that the Anxa2 mature protein of sika deer had the closest genetic distance with Cervus elaphus and Bos mutus . Real time RT-PCR results showed that the gene had differential expression levels in different growth stages, and the expression level of the ANXA2 gene was the highest at metaphase (rapid growing period). ANXA2 gene may promote the cell proliferation, and the finding suggested Anxa2 as an important candidate for regulating the growth and development of deer antler.

Gene expression complex networks: synthesis, identification, and analysis.

PubMed

Lopes, Fabrício M; Cesar, Roberto M; Costa, Luciano Da F

2011-10-01

Thanks to recent advances in molecular biology, allied to an ever increasing amount of experimental data, the functional state of thousands of genes can now be extracted simultaneously by using methods such as cDNA microarrays and RNA-Seq. Particularly important related investigations are the modeling and identification of gene regulatory networks from expression data sets. Such a knowledge is fundamental for many applications, such as disease treatment, therapeutic intervention strategies and drugs design, as well as for planning high-throughput new experiments. Methods have been developed for gene networks modeling and identification from expression profiles. However, an important open problem regards how to validate such approaches and its results. This work presents an objective approach for validation of gene network modeling and identification which comprises the following three main aspects: (1) Artificial Gene Networks (AGNs) model generation through theoretical models of complex networks, which is used to simulate temporal expression data; (2) a computational method for gene network identification from the simulated data, which is founded on a feature selection approach where a target gene is fixed and the expression profile is observed for all other genes in order to identify a relevant subset of predictors; and (3) validation of the identified AGN-based network through comparison with the original network. The proposed framework allows several types of AGNs to be generated and used in order to simulate temporal expression data. The results of the network identification method can then be compared to the original network in order to estimate its properties and accuracy. Some of the most important theoretical models of complex networks have been assessed: the uniformly-random Erdös-Rényi (ER), the small-world Watts-Strogatz (WS), the scale-free Barabási-Albert (BA), and geographical networks (GG). The experimental results indicate that the inference

Three urocortins in medaka: identification and spatial expression in the central nervous system.

PubMed

Hosono, K; Yamashita, J; Kikuchi, Y; Hiraki-Kajiyama, T; Okubo, K

2017-05-01

The urocortin (UCN) group of neuropeptides includes urocortin 1/sauvagine/urotensin 1 (UTS1), urocortin 2 (UCN2) and urocortin 3 (UCN3). In recent years, evidence has accumulated showing that UCNs play pivotal roles in mediating stress response and anxiety in mammals. Evidence has also emerged regarding the evolutionary conservation of UCNs in vertebrates, but very little information is available about UCNs in non-mammalian vertebrates. Indeed, at present, there are no reports of the empirical identification of ucn2 in non-mammalian vertebrates or of the distribution of ucn2 and ucn3 expression in the adult central nervous system (CNS) of these animals. To gain insight into the evolutionary nature of UCNs in vertebrates, we cloned uts1, ucn2 and ucn3 in a teleost fish, medaka and examined the spatial expression of these genes in the adult brain and spinal cord. Although all known UCN2 genes except those in rodents have been reported to likely lack the necessary structural features to produce a functional pre-pro-protein, all three UCN genes in medaka, including ucn2, displayed all of these features, suggesting their functionality. The three UCN genes exhibited distinct spatial expression patterns in the medaka brain: uts1 was primarily expressed in broad regions of the dorsal telencephalon, ucn2 was expressed in restricted regions of the thalamus and brainstem and ucn3 was expressed in discrete nuclei throughout many regions of the brain. We also found that these genes were all expressed throughout the medaka spinal cord, each with a distinct spatial pattern. Given that many of these regions have been implicated in stress responses and anxiety, the three UCNs may serve distinct physiological roles in the medaka CNS, including those involved in stress and anxiety, as shown in the mammalian CNS. © 2017 British Society for Neuroendocrinology.

Identification and cloning of a gene encoding tannase (tannin acylhydrolase) from Lactobacillus plantarum ATCC 14917(T).

PubMed

Iwamoto, Kazuaki; Tsuruta, Hiroki; Nishitaini, Yosuke; Osawa, Ro

2008-09-01

The gene tanLpl, encoding a novel tannase enzyme (TanLpl), has been cloned from Lactobacillus plantarum ATCC 14917(T). This is the first report of a tannase gene cloned from a bacterial source other than from Staphylococcus lugdunensis, which has been reported elsewhere. The open reading frame of tanLpl, spanning 1410 bp, encoded a 469-amino-acid protein that showed 28.8% identity to the tannase of S. lugdunensis with several commonly conserved sequences. These sequences could not be found in putative tannases reported for other bacteria and fungi. TanLpl was expressed in Escherichia coli DH5alpha from a pGEM-T expression system and purified. SDS-PAGE analysis indicated that purified TanLpl was a monomer polypeptide of approximately 50 kDa in size. Subsequent enzymatic characterization revealed that TanLpl was most active in an alkaline pH range at 40 degrees C, which was quite different from that observed for a fungal tannase of Aspergillus oryzae. In addition, the Michaelis-Menten constant of TanLpl was markedly lower than that of A. oryzae tannase. The evidence suggests that TanLpl should be classified into a novel family of tannases.

Expression of the cloned ColE1 kil gene in normal and Kilr Escherichia coli.

PubMed Central

Altieri, M; Suit, J L; Fan, M L; Luria, S E

1986-01-01

The kil gene of the ColE1 plasmid was cloned under control of the lac promoter. Its expression under this promoter gave rise to the same pattern of bacterial cell damage and lethality as that which accompanies induction of the kil gene in the colicin operon by mitomycin C. This confirms that cell damage after induction is solely due to expression of kil and is independent of the cea or imm gene products. Escherichia coli derivatives resistant to the lethal effects of kil gene expression under either the normal or the lac promoter were isolated and found to fall into several classes, some of which were altered in sensitivity to agents that affect the bacterial envelope. PMID:2946661

Cloning, Expression Analysis and Enzyme Activity Assays of the α-Carbonic Anhydrase Gene from Chlamydomonas sp. ICE-L.

PubMed

Qu, Changfeng; He, Yingying; Zheng, Zhou; An, Meiling; Li, Lulu; Wang, Xixi; He, Xiaodong; Wang, Yibin; Liu, Fangming; Miao, Jinlai

2018-01-01

The α-carbonic anhydrase (α-CA) is a zinc ion-containing enzyme that catalyzes the hydration of carbon dioxide. In this paper, a full-length α-CA gene was cloned from Chlamydomonas sp. ICE-L using RT-PCR and RACE-PCR for bioinformatic analysis. The α-CA open reading frame obtained by PCR was cloned into a vector and transformed into Escherichia coli to generate α-CA-producing bacteria. The α-CA was highly expressed upon induction with isopropyl-β-d-thiogalactoside (IPTG) at a final concentration of 0.8 mM. A single band with a molecular weight of approximate 40 kDa expressed in the recombinant E. coli strain harboring the α-CA vector was observed in SDS-PAGE analysis. The carbon dioxide hydration activity and esterase activity of α-CA expressed by the recombinant strain were 0.404 U/mg and 0.319 U, respectively. In addition, three conditions, temperature, salinity and UVB radiation exposure, were selected to analyze α-CA transcription levels by qRT-PCR. The results suggested UVB exposure increased the expression of relative mRNA; meanwhile, the α-CA mRNA expression was rapidly induced by temperature and salinity stress, indicating that Chlamydomonas sp. ICE-L might modulate the α-CA mRNA expression to adapt to the extreme environments.

Expression cloning of a gibberellin 20-oxidase, a multifunctional enzyme involved in gibberellin biosynthesis.

PubMed Central

Lange, T; Hedden, P; Graebe, J E

1994-01-01

In the biosynthetic pathway to the gibberellins (GAs), carbon-20 is removed by oxidation to give the C19-GAs, which include the biologically active plant hormones. We report the isolation of a cDNA clone encoding a GA 20-oxidase [gibberellin, 2-oxoglutarate:oxygen oxidoreductase (20-hydroxylating, oxidizing) EC 1.14.11.-] by screening a cDNA library from developing cotyledons of pumpkin (Cucurbita maxima L.) for expression of this enzyme. When mRNA from either the cotyledons or the endosperm was translated in vitro using rabbit reticulocyte lysates, the products contained GA12 20-oxidase activity. A polyclonal antiserum was raised against the amino acid sequence of a peptide released by tryptic digestion of purified GA 20-oxidase from the endosperm. A cDNA expression library in lambda gt11 was prepared from cotyledon mRNA and screened with the antiserum. The identity of positive clones was confirmed by the demonstration of GA12 20-oxidase activity in single bacteriophage plaques. Recombinant protein from a selected clone catalyzed the three-step conversions of GA12 to GA25 and of GA53 to GA17, as well as the formation of the C19-GAs, GA1, GA9, and GA20, from their respective aldehyde precursors, GA23, GA24, and GA19. The nucleotide sequence of the cDNA insert contains an open reading frame of 1158 nt encoding a protein of 386 amino acid residues. The predicted M(r) (43,321) and pI (5.3) are similar to those determined experimentally for the native GA 20-oxidase. Furthermore, the derived amino acid sequence includes sequences obtained from the N terminus and two tryptic peptides from the native enzyme. It also contains regions that are highly conserved in a group of non-heme Fe-containing dioxygenases. Images PMID:8078921

Cloning, expression, purification, and activity assay of proteins related to D-lactic acid formation in Lactobacillus rhamnosus.

PubMed

Wang, Xiuwen; Zheng, Zhaojuan; Dou, Peipei; Qin, Jiayang; Wang, Xiaochen; Ma, Cuiqing; Tang, Hongzhi; Xu, Ping

2010-08-01

Two proteins that might be responsible for D-lactic acid (D-LA) formation were screened from the genome database of Lactobacillus rhamnosus GG. The coding genes of the two proteins in L. rhamnosus CASL, ldhD1 and ldhD2, were cloned and expressed in Escherichia coli Rosetta with an inducible expression vector pETDuet-1 (Novagen, Darmstadt, Germany), respectively. The two purified proteins, LdhD-1 and LdhD-2, migrated as a single protein band separately, both corresponding to an apparent molecular mass between 35 kDa and 45 kDa on sodium dodecyl sulfate polyacrylamide gel electrophoresis. The specific activities of LdhD-1 and LdhD-2 catalyzing pyruvate to LA were 0.02 U/mg and 0.21 U/mg, respectively. The configuration of LA converted from pyruvate was determined using high-performance liquid chromatography equipped with a chiral column. Only D-LA was detected when LdhD-1 and LdhD-2 were tested. In summary, the two proteins cloned and expressed in this study were most probably responsible for D-LA formation during fermentation of L. rhamnosus CASL.

Biosynthesis of aminoglycoside antibiotics: cloning, expression and characterisation of an aminotransferase involved in the pathway to 2-deoxystreptamine.

PubMed

Huang, Fanglu; Li, Yanyan; Yu, Jinquan; Spencer, Jonathan B

2002-12-07

The gene btrR from Bacillus circulans has been cloned and expressed and shown to produce a protein which catalyses the transamination of 2-deoxy-scyllo-inosose to give 2-deoxy-scyllo-inosamine, an intermediate in the biosynthesis of 2-deoxystreptamine.

An Aspergillus oryzae acetyl xylan esterase: molecular cloning and characteristics of recombinant enzyme expressed in Pichia pastoris.

PubMed

Koseki, Takuya; Miwa, Yozo; Akao, Takeshi; Akita, Osamu; Hashizume, Katsumi

2006-02-10

We screened 20,000 clones of an expressed sequence tag (EST) library from Aspergillus oryzae (http://www.nrib.go.jp/ken/EST/db/index.html) and obtained one cDNA clone encoding a protein with similarity to fungal acetyl xylan esterase. We also cloned the corresponding gene, designated as Aoaxe, from the genomic DNA. The deduced amino acid sequence consisted of a putative signal peptide of 31-amino acids and a mature protein of 276-amino acids. We engineered Aoaxe for heterologous expression in P. pastoris. Recombinant AoAXE (rAoAXE) was secreted by the aid of fused alpha-factor secretion signal peptide and accumulated as an active enzyme in the culture medium to a final level of 190 mg/l after 5 days. Purified rAoAXEA before and after treatment with endoglycosidase H migrated by SDS-PAGE with a molecular mass of 31 and 30 kDa, respectively. Purified rAoAXE displayed the greatest hydrolytic activity toward alpha-naphthylacetate (C2), lower activity toward alpha-naphthylpropionate (C3) and no detectable activity toward acyl-chain substrates containing four or more carbon atoms. The recombinant enzyme catalyzed the release of acetic acid from birchwood xylan. No activity was detectable using methyl esters of ferulic, caffeic or sinapic acids. rAoAXE was thermolabile in comparison to other AXEs from Aspergillus.

Hybrid sequencing approach applied to human fecal metagenomic clone libraries revealed clones with potential biotechnological applications.

PubMed

Džunková, Mária; D'Auria, Giuseppe; Pérez-Villarroya, David; Moya, Andrés

2012-01-01

Natural environments represent an incredible source of microbial genetic diversity. Discovery of novel biomolecules involves biotechnological methods that often require the design and implementation of biochemical assays to screen clone libraries. However, when an assay is applied to thousands of clones, one may eventually end up with very few positive clones which, in most of the cases, have to be "domesticated" for downstream characterization and application, and this makes screening both laborious and expensive. The negative clones, which are not considered by the selected assay, may also have biotechnological potential; however, unfortunately they would remain unexplored. Knowledge of the clone sequences provides important clues about potential biotechnological application of the clones in the library; however, the sequencing of clones one-by-one would be very time-consuming and expensive. In this study, we characterized the first metagenomic clone library from the feces of a healthy human volunteer, using a method based on 454 pyrosequencing coupled with a clone-by-clone Sanger end-sequencing. Instead of whole individual clone sequencing, we sequenced 358 clones in a pool. The medium-large insert (7-15 kb) cloning strategy allowed us to assemble these clones correctly, and to assign the clone ends to maintain the link between the position of a living clone in the library and the annotated contig from the 454 assembly. Finally, we found several open reading frames (ORFs) with previously described potential medical application. The proposed approach allows planning ad-hoc biochemical assays for the clones of interest, and the appropriate sub-cloning strategy for gene expression in suitable vectors/hosts.

Cloning and expression of cyclophilin from Platanus orientalis pollens in Escherichia coli

PubMed Central

Sankian, Mojtaba; Vahedi, Fatemeh; Pazouki, Nazanin; Moghadam, Malihe; Jabbari Azad, Farahzad; Varasteh, Abdol-Reza

2012-01-01

Background: Allergy is a clinical disorder affecting the human population with wide geographical distribution. Platanus orientalis (P. orientalis) trees are planted in many countries and their pollen causes allergic reactions. Cyclophilin has recently been identified as one of the most important allergens of P. orientalis pollen. We aimed to clone and purify this allergen in Escherichia coli for further studies and therapeutic and diagnostic purposes for allergy to P. orientalis. Methods: RNA was extracted from P. orientalis. A full-length fragment encoding cyclophilin was prepared by polymerase chain reaction amplification of the first-strand cDNA synthesized from P. orientalis RNA. The cDNA was inserted into the pET32b (+) vector, and the construct transformed into E. coli Top10 and BL21 cells. The expressed protein was purified by the CuSO4 method. Results: The cDNA for the cyclophilin of P. orientalis pollen was cloned, and a specific reactivity of recombinant cyclophin was confirmed by immunoblotting using sera from patients allergic to P. orientalis pollen. Conclusion: The recombinant cyclophilin has a potential for immunologic assays for evaluation of allergy to P. orientalis pollen. PMID:26989705

The Associations between Visual Attention and Facial Expression Identification in Patients with Schizophrenia.

PubMed

Lin, I-Mei; Fan, Sheng-Yu; Huang, Tiao-Lai; Wu, Wan-Ting; Li, Shi-Ming

2013-12-01

Visual search is an important attention process that precedes the information processing. Visual search also mediates the relationship between cognition function (attention) and social cognition (such as facial expression identification). However, the association between visual attention and social cognition in patients with schizophrenia remains unknown. The purposes of this study were to examine the differences in visual search performance and facial expression identification between patients with schizophrenia and normal controls, and to explore the relationship between visual search performance and facial expression identification in patients with schizophrenia. Fourteen patients with schizophrenia (mean age=46.36±6.74) and 15 normal controls (mean age=40.87±9.33) participated this study. The visual search task, including feature search and conjunction search, and Japanese and Caucasian Facial Expression of Emotion were administered. Patients with schizophrenia had worse visual search performance both in feature search and conjunction search than normal controls, as well as had worse facial expression identification, especially in surprised and sadness. In addition, there were negative associations between visual search performance and facial expression identification in patients with schizophrenia, especially in surprised and sadness. However, this phenomenon was not showed in normal controls. Patients with schizophrenia who had visual search deficits had the impairment on facial expression identification. Increasing ability of visual search and facial expression identification may improve their social function and interpersonal relationship.

Cloning and functional expression of a plant voltage-dependent chloride channel.

PubMed Central

Lurin, C; Geelen, D; Barbier-Brygoo, H; Guern, J; Maurel, C

1996-01-01

Plant cell membrane anion channels participate in basic physiological functions, such as cell volume regulation and signal transduction. However, nothing is known about their molecular structure. Using a polymerase chain reaction strategy, we have cloned a tobacco cDNA (CIC-Nt1) encoding a 780-amino acid protein with several putative transmembrane domains. CIC-Nt1 displays 24 to 32% amino acid identity with members of the animal voltage-dependent chloride channel (CIC) family, whose archetype is CIC-0 from the Torpedo marmorata electric organ. Injection of CIC-Nt1 complementary RNA into Xenopus oocytes elicited slowly activating inward currents upon membrane hyperpolarization more negative than -120 mV. These currents were carried mainly by anions, modulated by extracellular anions, and totally blocked by 10 mM extracellular calcium. The identification of CIC-Nt1 extends the CIC family to higher plants and provides a molecular probe for the study of voltage-dependent anion channels in plants. PMID:8624442

Cloning and identification of a novel thyroid hormone receptor β isoform expressed in the pituitary gland.

PubMed

Zhao, Rong-Lan; Sun, Bei; Liu, Ying; Li, Jing-Hua; Xiong, Wei-Li; Liang, Dong-Chun; Guo, Gang; Zuo, Ai-Jun; Zhang, Jing-Yu

2014-04-01

We have previously identified a novel Trβ isoform (TrβΔ) in the rat, in which a novel exon N (108 bps) was found between exon 3 and exon 4 of TrβΔ, which represents the only difference between TrβΔ and Trβ1. In this study, we searched for an elongated Trβ2-like subtype with one additional exon N. We successfully isolated the entire mRNA/cDNA of a novel elongated Trβ2 isoform via PCR in the rat pituitary gland. The mRNA/cDNA was only 108 bps (exon N) longer than that Trβ2, and the extension of the sequence was between exon 3 and 4 of Trβ. The whole sequence of this novel Trβ isoform has been published in NCBI GenBank (HM043807.1); it is named TRbeta2Delta (Trβ2Δ). In adult rat pituitary tissue, quantitative real-time RT-PCR analysis showed that the mRNA levels of Trβ2Δ and Trβ2 were roughly equal (P > 0.05). We cloned, expressed, and purified the His-Trβ2Δ protein [recombinant TRβ2Δ (rTRβ2Δ)]. SDS-PAGE and western blotting revealed that the molecular weight of rTRβ2Δ was 58.2 kDa. Using a radioligand binding assay and an electrophoretic mobility shift assay, rTRβ2Δ-bound T3 with high affinity and recognized thyroid hormone response element (TRE) binding sites. Finally, in vitro transfection experiments further confirmed that rTRβ2Δ binding T3 significantly promotes the transcription of target genes via the TRE. Here, we have provided evidence suggesting that rTRβ2Δ is a novel functional TR isoform.

Molecular cloning and mRNA expression pattern of Sox10 in Paramisgurnus dabryanus.

PubMed

Xia, Xiaohua; Chen, Jianjun; Zhang, Linxia; Du, Qiyan; Sun, Jinsheng; Chang, Zhongjie

2013-04-01

A number of genetic studies have established that Sox10 involved in a wide range of developmental processes including sex differentiation and neurogenesis in vertebrates. A Sox10 homologue was cloned from brain of Paramisgurnus dabryanus by using homologous cloning and RACE method, designated as PdSox10. The full-length cDNA of PdSox10 contains a 312 bp 5' UTR, a 1,476 bp open reading frame (ORF) encoding 492 amino acids and a 262 bp 3' UTR (Accession no.: JQ217143). The overall topology of the phylogenetic tree shows that the PdSox10 fits within the Sox10 clade. During embryogenesis, PdSox10 gene seemed to be de novo synthesized in the embryos from gastrulae stage. From the somitogenesis stage and thereafter, distinct expression of PdSox10 was observed in the medial neural tube, extending from the hindbrain through the posterior trunk. In adult, PdSox10 mRNA was detected primarily in the gonads, as well as in brain and heart by RT-PCR. In situ hybridization on gonadal sections further demonstrated that PdSox10 is expressed especially in premature germ cells, in early perinucleolus stage oocytes and cortical-alveolar stage oocytes in ovaries and in spermatogonia and spermatocytes in testes. These preliminary findings suggested that PdSox10 is highly conserved during vertebrate evolution and involved in a wide range of developmental processes including neurogenesis and sex differentiation in vertebrates.

Cloning and expression studies of the Dunaliella salina UDP-glucose dehydrogenase cDNA.

PubMed

Qinghua, He; Dairong, Qiao; Qinglian, Zhang; Shunji, He; Yin, Li; Linhan, Bai; Zhirong, Yang; Yi, Cao

2005-06-01

The enzyme UDP-glucose dehydrogenase (EC 1.1.1.22) converts UDP-glucose to UDP-glucuronate. Plant UDP-glucose dehydrogenase (UGDH) is an important enzyme in the formation of hemicellulose and pectin, the components of primary cell walls. A cDNA, named DsUGDH, (GeneBank accession number: AY795899) corresponding to UGDH was cloned by RT-PCR approach from Dunaliella salina. The cDNA is 1941-bp long and has an open reading frame encoded a protein of 483 amino acids with a calculated molecular weight of 53 kDa. The derived amino acids sequence shows high homology with reported plants UGDHs, and has highly conserved amino acids motifs believed to be NAD binding site and catalytic site. Although UDP-glucose dehydrogenase is a comparatively well characterized enzyme, the cloning and characterization of the green alga Dunaliella salina UDP-glucose dehydrogenase gene is very important to understand the salt tolerance mechanism of Dunaliella salina. Northern analyses indicate that NaCl can induce the expression the DsUGDH.

Cloning and Expression Analysis of Phenylalanine Ammonia-Lyase Gene in the Mycelium and Fruit Body of the Edible Mushroom Flammulina velutipes

PubMed Central

Yun, Yeo Hong; Koo, Ja Sun

2015-01-01

Phenylalanine ammonia-lyase (PAL) gene is known to be expressed in plants, and is involved in the differentiation, growth and synthesis of secondary metabolites. However, its expression in fungi remains to be explored. To understand its expression in mushroom fungi, the PAL gene of the edible mushroom Flammulina velutipes (Fvpal) was cloned and characterized. The cloned Fvpal consists of 2,175 bp, coding for a polypeptide containing 724 amino acids and having 11 introns. The translated amino acid sequence of Fvpal shares a high identity (66%) with that of ectomycorrhizal fungus Tricholoma matsutake. Distinctively, the Fvpal expression in the mycelium was higher in minimal medium supplemented with L-tyrosine than with other aromatic amino acids. During cultivation of the mushroom on sawdust medium, Fvpal expression in the fruit body correspondingly increased as the mushroom grew. In the fruiting body, Fvpal was expressed more in the stipe than in the pileus. These results suggest that F. velutipes PAL activity differs in the different organs of the mushroom. Overall, this is first report to show that the PAL gene expression is associated with mushroom growth in fungi. PMID:26539050

Molecular cloning, sequence characterization and recombinant expression of Nanog gene in goat fibroblast cells using lentiviral based expression system.

PubMed

Singhal, Dinesh K; Singhal, Raxita; Malik, Hruda N; Kumar, Surender; Kumar, Sudarshan; Mohanty, Ashok K; Kaushik, Jai K; Malakar, Dhruba

2014-01-01

Nanog is a homeodomain containing protein which plays important roles in regulation of signaling pathways for maintenance and induction of pluripotency in stem cells. Because of its unique expression in stem cells it is also regarded as pluripotency marker. In this study goat Nanog (gNanog) gene has been amplified, cloned and characterized at sequence level with successful over-expression in CHO-K1 cell line using a lentiviral based system. gNanog ORF is 903 bp long which codes for Nanog protein of size 300 amino acids (aas). Complete nucleotide sequence shows some evolutionary mutation in goat in comparision to other species. Protein sequence of goat is highly similar to other species. Overall, gNanog nucleotide sequence and predicted protein sequence showed high similarity and minimum divergence with cattle (96 % identity/4 % divergence) and buffalo (94/5 %) while low similarity and high divergence with pig (84/15 %), human (81/23 %) and mouse (69/40 %) indicating evolutionary closeness of gNanog to cattle and buffalo. gNanog lentiviral expression construct was prepared for over-expression of Nanog gene in adult goat fibroblast cells. Lentiviral expression construct of Nanog enabled continuous protein expression for induction and maintenance of pluripotency. Western blotting revealed the expression of Nanog gene at protein level which supported that the lentiviral expression system is highly promising for Nanog protein expression in differentiated goat cell.

Molecular Cloning, Expression and Characterization of Pectin Methylesterase (CtPME) from Clostridium thermocellum.

PubMed

Rajulapati, Vikky; Goyal, Arun

2017-05-01

Many phytopathogenic micro-organisms such as bacteria and fungi produce pectin methylesterases (PME) during plant invasion. Plants and insects also produce PME to degrade plant cell wall. In the present study, a thermostable pectin methylesterase (CtPME) from Clostridium thermocellum belonging to family 8 carbohydrate esterase (CE8) was cloned, expressed and purified. The amino acid sequence of CtPME exhibited similarity with pectin methylesterase from Erwinia chrysanthemi with 38% identity. The gene encoding CtPME was cloned into pET28a(+) vector and expressed using Escherichia coli BL21(DE3) cells. The recombinant CtPME expressed as a soluble protein and exhibited a single band of molecular mass approximately 35.2 kDa on SDS-PAGE gels. The molecular mass, 35.5 kDa of the enzyme, was also confirmed by MALDI-TOF MS analysis. Notably, highest protein concentration (11.4 mg/mL) of CtPME was achieved in auto-induction medium, as compared with LB medium (1.5 mg/mL). CtPME showed maximum activity (18.1 U/mg) against citrus pectin with >85% methyl esterification. The optimum pH and temperature for activity of CtPME were 8.5 and 50 °C, respectively. The enzyme was stable in pH range 8.0-9.0 and thermostable between 45 and 70 °C. CtPME activity was increased by 40% by 5 mM Ca 2+ or Mg 2+ ions. Protein melting curve of CtPME gave a peak at 80 °C. The peak was shifted to 85 °C in the presence of 5 mM Ca 2+ ions, and the addition of 5 mM EDTA shifted back the melting peak to 80 °C. CtPME can be potentially used in food and textile industry applications.

[Cloning of Chinese Banna minipig inbred-line alpha1,3-galactosyltransferase gene and construction of its recombinant eukaryotic expression vector].

PubMed

Zhu, Shengming; Wang, Yanping; Zheng, Hong; Cheng, Jingqiu; Lu, Yanrong; Zeng, Yangzhi; Wang, Yu; Wang, Zhu

2009-04-01

This study sought to clone Chinese Banna minipig inbred-line (BMI) alpha1,3-galactosyltransferase (alpha1,3-GT) gene and construct its recombinant eukaryotic expression vector. Total RNA was isolated from BMI liver. Full length cDNA of alpha1,3-GT gene was amplified by RT-PCR and cloned into pMD18-T vector to sequence. Subsequently, alpha1,3-GT gene was inserted into pEGFP-N1 to construct eukaryotic expression vector pEGFP-N1-GT. Then the reconstructed plasmid pEGFP-N1-GT was transiently transfected into human lung cancer cell line A549. The expression of alpha1,3-GT mRNA in transfected cells was detected by RT-PCR. FITC-BS-IB4 lectin was used in the direct immunofluorescence method, which was performed to observe the alpha-Gal synthesis function of BMI alpha1,3-GT in transfected cells. The results showed that full length of BMI alpha1,3-GT cDNA was 1116 bp. BMI alpha1,3-GT cDNA sequence was highly homogenous with those of mouse and bovine, and was exactly the same as the complete sequence of those of swine, pEGFP-N1-GT was confirmed by enzyme digestion and PCR. The expression of alpha1,3-GT mRNA was detected in A549 cells transfected by pEGFP-N1-GT. The expression of alpha-Gal was observed on the membrane of A549 cells transfected by pEGFP-N1-GT. Successful cloning of BMI alpha1,3-GT cDNA and construction of its eukaryotic expression vector have established a foundation for further research and application of BMI alpha1,3-GT in the fields of xenotransplantation and immunological therapy of cancer.

Cloning and baculovirus expression of a desiccation stress gene from the beetle, Tenebrio molitor.

PubMed

Graham, L A; Bendena, W G; Walker, V K

1996-02-01

The cDNA sequence encoding a novel desiccation stress protein (dsp28) found in the hemolymph of the common yellow mealworm beetle, Tenebrio molitor, has been determined. The sequence encodes a 225 amino acid protein containing a 20 amino acid signal peptide. Dsp28 shows no significant similarity to any known nucleic acid or protein sequence. Levels of dsp28 mRNA were found to increase approx 5-fold following desiccation. Dsp28 cDNA has been cloned into a baculovirus expression vector and the expressed protein was compared to native dsp28. Both dsp28 expressed by recombinant baculovirus and native dsp28 are glycosylated and N-terminally processed. Although dsp28 is induced by cold in addition to desiccation stress, it does not contribute to the freezing point depression (thermal hysteresis) observed in Tenebrio hemolymph.

CD4+ T-cell clones obtained from cattle chronically infected with Fasciola hepatica and specific for adult worm antigen express both unrestricted and Th2 cytokine profiles.

PubMed Central

Brown, W C; Davis, W C; Dobbelaere, D A; Rice-Ficht, A C

1994-01-01

The well-established importance of helper T (Th)-cell subsets in immunity and immunoregulation of many experimental helminth infections prompted a detailed study of the cellular immune response against Fasciola hepatica in the natural bovine host. T-cell lines established from two cattle infected with F. hepatica were characterized for the expression of T-cell surface markers and proliferative responses against F. hepatica adult worm antigen. Parasite-specific T-cell lines contained a mixture of CD4+, CD8+, and gamma/delta T-cell-receptor-bearing T cells. However, cell lines containing either fewer than 10% CD8+ T cells or depleted of gamma/delta T cells proliferated vigorously against F. hepatica antigen, indicating that these T-cell subsets are not required for proliferative responses in vitro. Seventeen F. hepatica-specific CD4+ Th-cell clones were examined for cytokine expression following concanavalin A stimulation. Biological assays to measure interleukin-2 (IL-2) or IL-4, gamma interferon (IFN-gamma), and tumor necrosis factor and Northern (RNA) blot analysis to verify the expression of IL-2, IL-4, and IFN-gamma revealed that the Th-cell clones expressed a spectrum of cytokine profiles. Several Th-cell clones were identified as Th2 cells by the strong expression of IL-4 but little or no IL-2 or IFN-gamma mRNA. The majority of Th-cell clones were classified as Th0 cells by the expression of either all three cytokines or combinations of IL-2 and IL-4 or IL-4 and IFN-gamma. No Th1-cell clones were obtained. All of the Th-cell clones expressed a typical memory cell surface phenotype, characterized as CD45Rlow, and all expressed the lymph node homing receptor (L selectin). These results are the first to describe cytokine responses of F. hepatica-specific T cells obtained from infected cattle and extend our previous analysis of Th0 and Th1 cells from cattle immune to Babesia bovis (W. C. Brown, V. M. Woods, D. A. E. Dobbelaere, and K. S. Logan, Infect. Immun. 61

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

Production of rhesus monkey cloned embryos expressing monomeric red fluorescent protein by interspecies somatic cell nuclear transfer

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

Zhu, Hai-Ying; Kang, Jin-Dan; Li, Suo

2014-02-21

Highlights: • Rhesus monkey cells were electroporated with a plasmid containing mRFP1, and an mRFP1-expressing cell line was generated. • For the first time, mRFP1-expressing rhesus monkey cells were used as donor cells for iSCNT. • The effect of VPA on the development of embryos cloned using iSCNT was determined. - Abstract: Interspecies somatic cell nuclear transfer (iSCNT) is a promising method to clone endangered animals from which oocytes are difficult to obtain. Monomeric red fluorescent protein 1 (mRFP1) is an excellent selection marker for transgenically modified cloned embryos during somatic cell nuclear transfer (SCNT). In this study, mRFP-expressing rhesusmore » monkey cells or porcine cells were transferred into enucleated porcine oocytes to generate iSCNT and SCNT embryos, respectively. The development of these embryos was studied in vitro. The percentage of embryos that underwent cleavage did not significantly differ between iSCNT and SCNT embryos (P > 0.05; 71.53% vs. 80.30%). However, significantly fewer iSCNT embryos than SCNT embryos reached the blastocyst stage (2.04% vs. 10.19%, P < 0.05). Valproic acid was used in an attempt to increase the percentage of iSCNT embryos that developed to the blastocyst stage. However, the percentages of embryos that underwent cleavage and reached the blastocyst stage were similar between untreated iSCNT embryos and iSCNT embryos treated with 2 mM valproic acid for 24 h (72.12% vs. 70.83% and 2.67% vs. 2.35%, respectively). These data suggest that porcine-rhesus monkey interspecies embryos can be generated that efficiently express mRFP1. However, a significantly lower proportion of iSCNT embryos than SCNT embryos reach the blastocyst stage. Valproic acid does not increase the percentage of porcine-rhesus monkey iSCNT embryos that reach the blastocyst stage. The mechanisms underling nuclear reprogramming and epigenetic modifications in iSCNT need to be investigated further.« less

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.

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, expression and purification of d-tagatose 3-epimerase gene from Escherichia coli JM109.

PubMed

He, Xiaoliang; Zhou, Xiaohui; Yang, Zi; Xu, Le; Yu, Yuxiu; Jia, Lingling; Li, Guoqing

2015-10-01

An unknown d-tagatose 3-epimerase (DTE) containing a IoIE domain was identified and cloned from Escherichia coli. This gene was subcloned into the prokaryotic expression vector pET-15b, and induced by IPTG in E. coli BL21 expression system. Through His-select gel column purification and fast-protein liquid chromatography, highly purified and stable DTE protein was produced. The molecular weight of the DTE protein was estimated to be 29.8kDa. The latest 83 DTE sequences from public database were selected and analyzed by molecular clustering, multi-sequence alignment. DTEs were roughly divided into five categories. Copyright © 2015 Elsevier Inc. All rights reserved.

Cloning, expression, purification and crystallization of saccharopine reductase from Magnaporthe grisea.

PubMed

Johansson, E; Steffens, J J; Emptage, M; Lindqvist, Y; Schneider, G

2000-05-01

The gene coding for saccharopine reductase (E.C. 1.5.1.10), an enzyme of the alpha-aminoadipic pathway of lysine biosynthesis in the pathogenic fungus Magnaporthe grisea, was cloned and expressed in Escherichia coli. The purified enzyme was crystallized in space groups C2 and C222(1) using ammonium sulfate pH 4.8 or PEG 6000 pH 4. 1 as precipitants. The unit-cell parameters are a = 115.0, b = 56.6, c = 74.3 A, beta = 111.1 degrees for space group C2, and a = 89.3, b = 119.0, c = 195.9 A for space group C222(1). The crystals diffract to resolutions of 2.0 A (C2) and 2.4 A (C222(1)) at synchrotron sources.

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.

Cloning and Expression of Major Surface Antigen 1 Gene of Toxoplasma gondii RH Strain Using the Expression Vector pVAX1 in Chinese Hamster Ovary Cells

PubMed Central

Abdizadeh, Rahman; Maraghi, Sharif; Ghadiri, Ata A.; Tavalla, Mehdi; Shojaee, Saeedeh

2015-01-01

Background: Toxoplasmosis is an opportunistic protozoan infection with a high prevalence in a broad range of hosts infecting up to one-third of the world human population. Toxoplasmosis leads to serious medical problems in immunocompromised individuals and fetuses and also induces abortion and mortality in domestic animals. Therefore, there is a huge demand for the development of an effective vaccine. Surface Antigen 1 (SAG1) is one of the important immunodominant surface antigens of Toxoplasma gondii, which interacts with host cells and primarily involved in adhesion, invasion and stimulation of host immune response. Surface antigen 1 is considered as the leading candidate for development of an effective vaccine against toxoplasmosis. Objectives: The purpose of this study was to clone the major surface antigen1 gene (SAG1) from the genotype 1 of T. gondii, RH strain into the eukaryotic expression vector pVAX1 in order to use for a DNA vaccine. Materials and Methods: Genomic DNA was extracted from tachyzoite of the parasite using the QIAamp DNA mini kit. After designing the specific primers, SAG1 gene was amplified by Polymerase Chain Reaction (PCR). The purified PCR products were then cloned into a pPrime plasmid vector. The aforementioned product was subcloned into the pVAX1 eukaryotic expression vector. The recombinant pVAX1-SAG1 was then transfected into Chinese Hamster Ovary (CHO) cells and expression of SAG1 antigen was evaluated using Reverse Transcriptase Polymerase Chain Reaction (RT-PCR), Immunofluorescence Assay (IFA) and Western Blotting (WB). Results: The cloning and subcloning products (pPrime-SAG1 and pVAX1-SAG1 plasmid vectors) of SAG1 gene were verified and confirmed by enzyme digestion and sequencing. A 30 kDa recombinant protein was expressed in CHO cells as shown by IFA and WB methods. Conclusions: The pVAX1 expression vector and CHO cells are a suitable system for high-level recombinant protein production for SAG1 gene from T. gondii parasites

Cloning of zebrafish Mustn1 orthologs and their expression during early development.

PubMed

Camarata, Troy; Vasilyev, Aleksandr; Hadjiargyrou, Michael

2016-11-15

Mustn1 is a small nuclear protein that is involved in the development and regeneration of the musculoskeletal system. Previous work established a role for Mustn1 in myogenic and chondrogenic differentiation. In addition, recent evidence suggests a potential role for Mustn1 in cilia function in zebrafish. A detailed study of Mustn1 expression has yet to be conducted in zebrafish. As such, we report herein the cloning of the zebrafish Mustn1 orthologs, mustn1a and mustn1b, and their expression during zebrafish embryonic and larval development. Results indicate a 44% nucleotide identity between the two paralogs. Phylogenetic analysis further confirmed that the Mustn1a and 1b predicted proteins were highly related to other vertebrate members of the Mustn1 protein family. Whole mount in situ hybridization revealed expression of both mustn1a and 1b at the 7-somite stage through 72hpf in structures such as Kupffer's vesicle, segmental mesoderm, head structures, and otic vesicle. Additionally, in 5day old larva, mustn1a and 1b expression is detected in the neurocranium, otic capsule, and the gut. Although both were expressed in the neurocranium, mustn1a was localized in the hypophyseal fenestra whereas mustn1b was found near the posterior basicapsular commissure. mustn1b also displayed expression in the ceratohyal and ceratobranchial elements of the pharyngeal skeleton. These expression patterns were verified temporally by q-PCR analysis. Taken together, we conclude that Mustn1 expression is conserved in vertebrates and that the variations in expression of the two zebrafish paralogs suggest different modes of molecular regulation. Copyright © 2016 Elsevier B.V. All rights reserved.

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

Clone tag detection in distributed RFID systems

PubMed Central

Kamaludin, Hazalila; Mahdin, Hairulnizam

2018-01-01

Although Radio Frequency Identification (RFID) is poised to displace barcodes, security vulnerabilities pose serious challenges for global adoption of the RFID technology. Specifically, RFID tags are prone to basic cloning and counterfeiting security attacks. A successful cloning of the RFID tags in many commercial applications can lead to many serious problems such as financial losses, brand damage, safety and health of the public. With many industries such as pharmaceutical and businesses deploying RFID technology with a variety of products, it is important to tackle RFID tag cloning problem and improve the resistance of the RFID systems. To this end, we propose an approach for detecting cloned RFID tags in RFID systems with high detection accuracy and minimal overhead thus overcoming practical challenges in existing approaches. The proposed approach is based on consistency of dual hash collisions and modified count-min sketch vector. We evaluated the proposed approach through extensive experiments and compared it with existing baseline approaches in terms of execution time and detection accuracy under varying RFID tag cloning ratio. The results of the experiments show that the proposed approach outperforms the baseline approaches in cloned RFID tag detection accuracy. PMID:29565982

Clone tag detection in distributed RFID systems.

PubMed

Kamaludin, Hazalila; Mahdin, Hairulnizam; Abawajy, Jemal H

2018-01-01

Although Radio Frequency Identification (RFID) is poised to displace barcodes, security vulnerabilities pose serious challenges for global adoption of the RFID technology. Specifically, RFID tags are prone to basic cloning and counterfeiting security attacks. A successful cloning of the RFID tags in many commercial applications can lead to many serious problems such as financial losses, brand damage, safety and health of the public. With many industries such as pharmaceutical and businesses deploying RFID technology with a variety of products, it is important to tackle RFID tag cloning problem and improve the resistance of the RFID systems. To this end, we propose an approach for detecting cloned RFID tags in RFID systems with high detection accuracy and minimal overhead thus overcoming practical challenges in existing approaches. The proposed approach is based on consistency of dual hash collisions and modified count-min sketch vector. We evaluated the proposed approach through extensive experiments and compared it with existing baseline approaches in terms of execution time and detection accuracy under varying RFID tag cloning ratio. The results of the experiments show that the proposed approach outperforms the baseline approaches in cloned RFID tag detection accuracy.

Cloning and expression of a CYP720B orthologue involved in the biosynthesis of diterpene resin acids in Pinus brutia.

PubMed

Semiz, Asli; Sen, Alaattin

2015-03-01

Cytochrome P450 monooxygenases mediate a broad range of oxidative reactions involved in the biosynthesis of both primary and secondary metabolites in plants. Until now, only two P450 genes, CYP720B1 from Pinus taeda and CYP720B4 from Picea sitchensis, have been functionally characterised and described in the literature. The purpose of this study was to describe the cloning and expression of CYP720B from Pinus brutia due to its suggested role in the synthesis of bioactive compounds used for chemical defence against insects. A PCR product of the P. brutia CYP720B gene was cloned into the pCR8/GW/TOPO cloning vector. After optimising the sequence for codon usage in yeast, it was transferred into the inducible expression vector pYES-DEST52 and transfected into the S. cerevisiae INVSc1 strain. Sequence analysis showed that the P. brutia CYP720B gene contains an open reading frame of 1,464 nucleotides, which encodes a 53,570 Da putative protein of 487 amino acid residues. The putative protein contains the classic heme-binding sequence motif that is conserved in all P450 enzymes. It shares 99 and 61% identity with the deduced amino acid sequences of CYP720B1 from Pinus taeda and CYP720B4 from Picea sitchensis, respectively. Recombinant CYP720B protein expression was confirmed using western blot analysis. Furthermore, recombinant CYP720B was functionally active, showing a Soret peak at approximately 448 nm in the reduced CO difference spectra. These data suggest that the cloned gene is an orthologue of CYP720B in P. brutia and might be involved in DRA biosynthesis.

Development of the gateway recycling cloning system for multiple linking of expression cassettes in a defined order, and direction on gateway compatible binary vectors.

PubMed

Kimura, Tetsuya; Nakao, Akihide; Murata, Sachiko; Kobayashi, Yasuyuki; Tanaka, Yuji; Shibahara, Kenta; Kawazu, Tetsu; Nakagawa, Tsuyoshi

2013-01-01

We developed the Gateway recycling cloning system, which allows multiple linking of expression cassettes by multiple rounds of the Gateway LR reaction. Employing this system, the recycling donor vector pRED419 was subjected to the first LR reaction with an attR1-attR2 type destination vector. Then conversion vector pCON was subjected to an LR reaction to restore the attR1-attR2 site on the destination vector for the next cloning cycle. By repetition of these two simple steps, we linked four expression cassettes of a reporter gene in Gateway binary vector pGWB1, introduced the constructs into tobacco BY-2 cells, and observed the expression of transgenes.

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

cDNA Cloning, expression and characterization of an allergenic 60s ribosomal protein of almond (prunus dulcis).

PubMed

Abolhassani, Mohsen; Roux, Kenneth H

2009-06-01

Tree nuts, including almond (prunus dulcis) are a source of food allergens often associated with life-threatening allergic reactions in susceptible individuals. Although the proteins in almonds have been biochemically characterized, relatively little has been reported regarding the identity of the allergens involved in almond sensitivity. The present study was undertaken to identify the allergens of the almond by cDNA library approach. cDNA library of almond seeds was constructed in Uni-Zap XR lamda vector and expressed in E. coli XL-1 blue. Plaques were immunoscreened with pooled sera of allergic patients. The cDNA clone reacting significantly with specific IgE antibodies was selected and subcloned and subsequently expressed in E. coli. The amino acids deducted from PCR product of clone showed homology to 60s acidic ribosomal protein of almond. The expressed protein was 11,450 Dalton without leader sequence. Immunoreactivity of the recombinant 60s ribosomal protein (r60sRP) was evaluated with dot blot analysis using pooled and individual sera of allergic patients. The data showed that r60sRP and almond extract (as positive control) possess the ability to bind the IgE antibodies. The results showed that expressed protein is an almond allergen.Whether this r60sRP represents a major allergen of almond needs to be further studied which requires a large number of sera from the almond atopic patients and also need to determine the IgE-reactive frequencies of each individual allergen.

Mutation of the XIST gene upregulates expression of X-linked genes but decreases the developmental rates of cloned male porcine embryos.

PubMed

Yang, Yang; Wu, Dan; Liu, Dewu; Shi, Junsong; Zhou, Rong; He, Xiaoyan; Quan, Jianping; Cai, Gengyuan; Zheng, Enqin; Wu, Zhenfang; Li, Zicong

2017-06-01

XIST is an X-linked, non-coding gene responsible for the cis induction of X-chromosome inactivation (XCI). Knockout of the XIST allele on an active X chromosome abolishes erroneous XCI and enhances the in vivo development of cloned mouse embryos by more than 10-fold. This study aimed to investigate whether a similar manipulation would improve cloning efficiency in pigs. A male, porcine kidney cell line containing an EGFP insert in exon 1 of the XIST gene, resulting in a knockout allele (XIST-KO), was generated by homologous recombination using transcription activator-like effector nucleases (TALENs). The expression of X-linked genes in embryos cloned from the XIST-KO kidney cells was significantly higher than in male embryos cloned from wild-type (WT) kidney cells, but remained lower than that of in vivo fertilization-produced counterparts. The XIST-KO cloned embryos also had a significantly lower blastocyst rate and a reduced full-term development rate compared to cloned WT embryos. These data suggested that while mutation of a XIST gene can partially rescue abnormal XCI, it cannot improve the developmental efficiency of cloned male porcine embryos-a deficiency that may be caused by incomplete rescue of abnormal XCI and/or by long-term drug selection of the XIST-KO nuclear donor cells, which might adversely affect the developmental efficiency of embryos created from them. © 2017 Wiley Periodicals, Inc.

m-carboxycinnamic acid bishydroxamide improves developmental competence, reduces apoptosis and alters epigenetic status and gene expression pattern in cloned buffalo (Bubalus bubalis) embryos.

PubMed

Agrawal, H; Selokar, N L; Saini, M; Singh, M K; Chauhan, M S; Palta, P; Singla, S K; Manik, R S

2018-05-07

Incomplete or aberrant reprogramming of nuclear genome is one of the major problems in somatic cell nuclear transfer. In this study, we studied the effect of histone deacetylase inhibitor m-carboxycinnamic acid bishydroxamide (CBHA) on in vitro development of buffalo embryos produced by Hand-made cloning. Cloned embryos were treated with CBHA (0, 5, 10, 20 or 50 μM) for 10 hr from the start of reconstruction till activation. At 10 μM, but not at other concentrations examined, CBHA increased (p < .05) the blastocyst rate (63.77 ± 3.97% vs 48.63 ± 3.55%) and reduced (p < .05) the apoptotic index of the cloned blastocysts (8.91 ± 1.94 vs 4.36 ± 1.08) compared to untreated controls, to levels similar to those in IVF blastocysts (4.78 ± 0.74). CBHA treatment, at all the concentrations examined, increased (p < .05) the global level of H3K9ac in cloned blastocysts than in untreated controls to that observed in IVF blastocysts. Treatment with CBHA (10 μM) decreased (p < .05) the global level of H3K27me3 in cloned blastocysts than in untreated controls but it was still higher (p < .05) than in IVF blastocysts. CBHA (10 μM) treatment increased (p < .05) the relative expression level of pluripotency-related genes OCT-4 and NANOG, and anti-apoptotic gene BCL-XL, and decreased (p < .05) that of pro-apoptotic gene BAX than in untreated controls but did not affect the relative expression level of apoptosis-related genes p53 and CASPASE3 and epigenetics-related genes DNMT1, DNMT3a and HDAC1. These results suggest that treatment of cloned embryos with 10 μM CBHA improves the blastocyst rate, reduces the level of apoptosis and alters the epigenetic status and gene expression pattern. © 2018 Blackwell Verlag GmbH.

Cloning, expression and activation of a truncated 92-kDa gelatinase minienzyme.

PubMed

Kröger, M; Tschesche, H

1997-09-01

The matrix metalloproteinases (MMPs) are a family of highly homologous zinc-endopeptidases that degrade extracellular matrix components. Human 92-kDa gelatinase (MMP-9) represents one of the MMPs that cleaves native collagen type IV. As a basis for structural investigations, the short form (catalytic domain, amino acid residues 113-450) of the 92-kDa gelatinase cDNA was cloned and expressed in E. coli as a minienzyme. By combination of reverse transcription (RT) and polymerase chain reaction (PCR), the truncated 92-kDa gelatinase-cDNA was amplified from the corresponding mRNA derived from ovarian carcinoma cells. The cDNA fragment obtained was cloned in E. coli and sequenced. With the exception of one nucleotide inversion at position 745 (gt-->tg) the cDNA sequence was identical to the nucleotide sequence of the 92-kDa gelatinase as has been previously reported. The protein was expressed in E. coli using the vector pET-12b. The recombinant protein was stored in inclusion bodies and extracted as a 38 kDa species from the inclusion bodies by solubilization in 8 M urea. The product was purified by affinity chromatography and gel filtration. Amino-terminal sequence analysis confirmed the identity with the catalytic domain of 92-kDa gelatinase. The recombinant protein was refolded in the presence of Ca2+ and Zn2+ and yielded an active minienzyme with gelatinolytic activity. It degrades the native substrate collagen type IV and the synthetic substrate Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH2 x AcOH like the full-length 92-kDa gelatinase. The catalytic activity could be inhibited by the specific MMP inhibitors TIMP-1 and TIMP-2.

Molecular cloning and gene expression analysis of Ercc6l in Sika deer (Cervus nippon hortulorum).

PubMed

Yin, Yupeng; Tang, Lina; Zhang, Jiabao; Tang, Bo; Li, Ziyi

2011-01-01

One important protein family that functions in nucleotide excision repair (NER) factors is the SNF2 family. A newly identified mouse ERCC6-like gene, Ercc6l (excision repair cross-complementing rodent repair deficiency, complementation group 6-like), has been shown to be another developmentally related member of the SNF2 family. In this study, Sika deer Ercc6l cDNA was first cloned and then sequenced. The full-length cDNA of the Sika deer Ercc6l gene is 4197 bp and contains a 3732 bp open reading frame that encodes a putative protein of 1243 amino acids. The similarity of Sika deer Ercc6l to Bos taurus Ercc6l is 94.05% at the amino acid sequence level. The similarity, however, is reduced to 68.42-82.21% when compared to Ercc6l orthologs in other mammals and to less than 50% compared to orthologs in Gallus gallus and Xenopus. Additionally, the expression of Ercc6l mRNA was investigated in the organs of fetal and adult Sika deer (FSD and ASD, respectively) by quantitative RT-PCR. The common expression level of Ercc6l mRNA in the heart, liver, spleen, lung, kidney, and stomach from six different developmental stages of 18 Sika deer were examined, though the expression levels in each organ varied among individual Sika deer. During development, there was a slight trend toward decreased Ercc61 mRNA expression. The highest Ercc6l expression levels were seen at 3 months old in every organ and showed the highest level of detection in the spleen of FSD. The lowest Ercc6l expression levels were seen at 3 years old. We are the first to successfully clone Sika deer Ercc6l mRNA. Ercc6l transcript is present in almost every organ. During Sika deer development, there is a slight trend toward decreased Ercc61 mRNA expression. It is possible that Ercc6l has other roles in embryonic development and in maintaining the growth of animals.

The transgenic cloned pig population with integrated and controllable GH expression that has higher feed efficiency and meat production

PubMed Central

Ju, Huiming; Zhang, Jiaqing; Bai, Lijing; Mu, Yulian; Du, Yutao; Yang, Wenxian; Li, Yong; Sheng, Anzhi; Li, Kui

2015-01-01

Sustained expression of the GH gene has been shown to have detrimental effects on the health of animals. In the current study, transgenic founder pigs, with controllable pig growth hormone (pGH) expression, were cloned via the handmade cloning method (HMC), and pGH expression levels were examined at the cellular and organismal levels. The serum pGH levels in 3 founder male pigs were found to be significantly higher after induction with intramuscular injection of doxycycline (DOX) compared to baseline. A daily dose of DOX was administered via feed to these animals for a period of 65 to 155 days. The growth rate, feed efficiency and pGH serum concentration increased in the DOX-induced transgenic group compared with the other groups. 8 numbers of animals were euthanized and the dressing percentage, loin muscle and lean meat percentage were significantly higher in the DOX-induced F1 transgenic group compared with the other groups. In this study a large population of transgenic pigs, with integrated controllable expression of a transgene, was obtained. The transgenic pigs were healthy and normal in terms of reproductive capability. At the same time, feed efficiency was improved, production processes were accelerated and meat yield was increased. PMID:25959098

The transgenic cloned pig population with integrated and controllable GH expression that has higher feed efficiency and meat production.

PubMed

Ju, Huiming; Zhang, Jiaqing; Bai, Lijing; Mu, Yulian; Du, Yutao; Yang, Wenxian; Li, Yong; Sheng, Anzhi; Li, Kui

2015-05-11

Sustained expression of the GH gene has been shown to have detrimental effects on the health of animals. In the current study, transgenic founder pigs, with controllable pig growth hormone (pGH) expression, were cloned via the handmade cloning method (HMC), and pGH expression levels were examined at the cellular and organismal levels. The serum pGH levels in 3 founder male pigs were found to be significantly higher after induction with intramuscular injection of doxycycline (DOX) compared to baseline. A daily dose of DOX was administered via feed to these animals for a period of 65 to 155 days. The growth rate, feed efficiency and pGH serum concentration increased in the DOX-induced transgenic group compared with the other groups. 8 numbers of animals were euthanized and the dressing percentage, loin muscle and lean meat percentage were significantly higher in the DOX-induced F1 transgenic group compared with the other groups. In this study a large population of transgenic pigs, with integrated controllable expression of a transgene, was obtained. The transgenic pigs were healthy and normal in terms of reproductive capability. At the same time, feed efficiency was improved, production processes were accelerated and meat yield was increased.

Cloning and heterologous expression of the entire gene clusters for PD 116740 from Streptomyces strain WP 4669 and tetrangulol and tetrangomycin from Streptomyces rimosus NRRL 3016.

PubMed Central

Hong, S T; Carney, J R; Gould, S J

1997-01-01

The genes for the complete pathways for two polycyclic aromatic polyketides of the angucyclinone class have been cloned and heterologously expressed. Genomic DNAs of Streptomyces rimosus NRRL 3016 and Streptomyces strain WP 4669 were partially digested with MboI, and libraries (ca. 40-kb fragments) in Escherichia coli XL1-Blue MR were prepared with the cosmid vector pOJ446. Hybridization with the actI probe from the actinorhodin polyketide synthase genes identified two clusters of polyketide genes from each organism. After transfer of the four clusters to Streptomyces lividans TK24, expression of one cluster from each organism was established through the identification of pathway-specific products by high-performance liquid chromatography with photodiode array detection. Peaks were identified from the S. rimosus cluster (pksRIM-1) for tetrangulol, tetrangomycin, and fridamycin E. Peaks were identified from the WP 4669 cluster (pksWP-2) for tetrangulol, 19-hydroxytetrangulol, 8-O-methyltetrangulol, 19-hydroxy-8-O-methyltetrangulol, and PD 116740. Structures were confirmed by 1H nuclear magnetic resonance spectroscopy and high-resolution mass spectrometry. PMID:8990300

Cloning and heterologous expression of the entire gene clusters for PD 116740 from Streptomyces strain WP 4669 and tetrangulol and tetrangomycin from Streptomyces rimosus NRRL 3016.

PubMed

Hong, S T; Carney, J R; Gould, S J

1997-01-01

The genes for the complete pathways for two polycyclic aromatic polyketides of the angucyclinone class have been cloned and heterologously expressed. Genomic DNAs of Streptomyces rimosus NRRL 3016 and Streptomyces strain WP 4669 were partially digested with MboI, and libraries (ca. 40-kb fragments) in Escherichia coli XL1-Blue MR were prepared with the cosmid vector pOJ446. Hybridization with the actI probe from the actinorhodin polyketide synthase genes identified two clusters of polyketide genes from each organism. After transfer of the four clusters to Streptomyces lividans TK24, expression of one cluster from each organism was established through the identification of pathway-specific products by high-performance liquid chromatography with photodiode array detection. Peaks were identified from the S. rimosus cluster (pksRIM-1) for tetrangulol, tetrangomycin, and fridamycin E. Peaks were identified from the WP 4669 cluster (pksWP-2) for tetrangulol, 19-hydroxytetrangulol, 8-O-methyltetrangulol, 19-hydroxy-8-O-methyltetrangulol, and PD 116740. Structures were confirmed by 1H nuclear magnetic resonance spectroscopy and high-resolution mass spectrometry.

Cloning of rat MLH1 and expression analysis of MSH2, MSH3, MSH6, and MLH1 during spermatogenesis.

PubMed

Geeta Vani, R; Varghese, C M; Rao, M R

1999-12-15

The mismatch repair system has been highly conserved in various species. In eukaryotic cells, the Mut S and Mut L homologues play crucial roles in both DNA mismatch repair and meiotic recombination. A full-length rat cDNA clone for rat MLH1 has been constructed using the RT-PCR method. The cDNA has an open reading frame of 2274 nucleotides for a protein of 757 amino acids. We have also obtained partial cDNA clones for MSH3 and MSH6. Northern blot analysis of rat MLH1, MSH2, MSH3, and MSH6 in the testes of rats of different ages showed differential expression of these genes as a function of developmental maturation of the testes. The expression analysis suggests that MSH3 may have a more predominant role in the meiotic recombination process. Copyright 1999 Academic Press.

Cloning and expression of N-glycosylation-related glucosidase from Glaciozyma antarctica

NASA Astrophysics Data System (ADS)

Yajit, Noor Liana Mat; Kamaruddin, Shazilah; Hashim, Noor Haza Fazlin; Bakar, Farah Diba Abu; Murad, Abd. Munir Abd.; Mahadi, Nor Muhammad; Mackeen, Mukram Mohamed

2016-11-01

The need for functional oligosaccharides in various field is ever growing. The enzymatic approach for synthesis of oligosaccharides is advantageous over traditional chemical synthesis because of the regio- and stereo- selectivity that can be achieved without the need for protection chemistry. In this study, the α-glucosidase I protein sequence from Saccharomyces cerevisiae (UniProt database) was compared using Basic Local Alignment Search Tool (BLAST) with Glaciozyma antarctica genome database. Results showed 33% identity and an E-value of 1 × 10-125 for α-glucosidase I. The gene was amplified, cloned into the pPICZα C vector and used to transform Pichia pastoris X-33 cells. Soluble expression of α-Glucosidase I (˜91 kDa) was achieved at 28 °C with 1.0 % of methanol.

Cloning and expression analysis of carboxyltransferase of acetyl-coA carboxylase from Jatropha curcas.

PubMed

Xie, Wu-Wei; Gao, Shun; Wang, Sheng-Hua; Zhu, Jin-Qiu; Xu, Ying; Tang, Lin; Chen, Fang

2010-01-01

A full-length cDNA of the carboxyltransferase (accA) gene of acetyl-coenzym A (acetyl-CoA) carboxylase from Jatropha curcas was cloned and sequenced. The gene with an open reading frame (ORF) of 1149 bp encodes a polypeptide of 383 amino acids, with a molecular mass of 41.9 kDa. Utilizing fluorogenic real-time polymerase chain reaction (RT-PCR), the expression levels of the accA gene in leaves and fruits at early, middle and late stages under pH 7.0/8.0 and light/darkness stress were investigated. The expression levels of the accA gene in leaves at early, middle and late stages increased significantly under pH 8.0 stress compared to pH 7.0. Similarly, the expression levels in fruits showed a significant increase under darkness condition compared to the control. Under light stress, the expression levels in the fruits at early, middle and late stages showed the largest fluctuations compared to those of the control. These findings suggested that the expression levels of the accA gene are closely related to the growth conditions and developmental stages in the leaves and fruits of Jatropha curcas.

Cloning and expression of recombinant, functional ricin B chain

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

Chang, M.S.; Russell, D.W.; Uhr, J.W.

1987-08-01

The cDNA encoding the B chain of the plant toxin ricin has been cloned and expressed in monkey kidney COS-M6 cells. The recombinant B chain was detected by labeling the transfected cells with (/sup 35/S)methionine and (/sup 35/S)-cysteine and demonstrating the secretion of a protein with a M/sub r/ of 30,000-32,000 that was not present in the medium of mock-transfected COS-M6 cells. This protein was specifically immunoprecipitated by an anti-ricin or anti-B-chain antibody and the amount of recombinant B chain secreted by the COS-M6 cells was determined by a radioimmunoassay. Virtually all of the recombinant B chain formed active ricinmore » when mixed with native A chain; it could also bind to the galactose-containing glycoprotein asialofetuin as effectively as native B chain.These results indicate that the vast majority of recombinant B chains secreted into the medium of the COS-M6 cells retain biological function« less

Cloning, expression and functional characterization of Schizosaccharomyces pombe TFIIB.

PubMed

Tamayo, Evelyn; Maldonado, Edio

2002-09-27

The transcription factor TFIIB has been identified and cloned from the yeast Schizosaccharomyces pombe. The cloned polypeptide is highly homologous to human TFIIB and to Saccharomyces cerevisiae TFIIB. S. pombe TFIIB is a 340-amino-acid-long protein and it possesses a repeated motif of 75 amino acids near the carboxy-terminal region. The purified recombinant protein is able to bind to the TBP-DNA promoter complex in gel retardation experiments. Recombinant S. pombe TFIIB is active in in vitro transcription assays, since it can complement the transcription activity of a S. pombe cell extract in which TFIIB was depleted by using antibodies.

Cloning and expression of SgCYP450-4 from Siraitia grosvenorii.

PubMed

Tu, Dongping; Ma, Xiaojun; Zhao, Huan; Mo, Changming; Tang, Qi; Wang, Liuping; Huang, Jie; Pan, Limei

2016-11-01

CYP450 plays an essential role in the development and growth of the fruits of Siraitia grosvenorii . However, little is known about the SgCYP450-4 gene in S. grosvenorii . Here, based on transcriptome data, a full-length cDNA sequence of SgCYP450-4 was cloned by reverse transcriptase-polymerase chain reaction (RT-PCR) and rapid-amplification of cDNA ends (RACE) strategies. SgCYP450-4 is 1677 bp in length (GenBank accession No. AEM42985.1) and contains a complete open reading frame (ORF) of 1422 bp. The deduced protein was composed of 473 amino acids, the molecular weight is 54.01 kDa, the theoretical isoelectric point (PI) is 8.8, and the protein was predicted to possess cytochrome P450 domains. SgCYP450-4 gene was highly expressed in root, diploid fruit and fruit treated with hormone and pollination. At 10 days after treatment with pollination and hormones, the expression of Sg CYP450-4 had the highest level and then decreased over time, which was consistent with the development of fruits of S. Grosvenorii . Hormonal treatment could significantly induce the expression of SgCYP450-4 . These results provide a reference for regulation of fruit development and the use of parthenocarpy to generate seedless fruit, and provide a scientific basis for the production of growth regulator application agents.

Identification of new modes of Dd-STATa regulation of gene expression in Dictyostelium by in situ hybridisation.

PubMed

Shimada, Nao; Maeda, Mineko; Urushihara, Hideko; Kawata, Takefumi

2004-09-01

Signal Transducers and Activators of Transcription (STATs) are transcription factors which lie at the end of cytokine and growth signal transduction pathways. Dictyostelium Dd-STATa is a functional homologue of metazoan STATs. It is activated by cAMP and, at the slug stage, it translocates into the nuclei of the tip cells, which are a subset of the anterior, prestalk A (pstA) cells. Here we searched for novel Dd-STATa regulated genes by in situ hybridisation. A set of 54 cDNA clones whose gene expression patterns are known to be prestalk-specific (Maeda et al., 2003), were chosen as probes and we compared their expression patterns in parental and Dd-STATa-null strains. We identified 13 genes which are candidates for direct induction by Dd-STATa. In the parental strain, most of these genes are expressed in the cone shaped mass of pstAB cells which is located within the prestalk region. These cDNAs show little or no expression in the Dd-STATa-null strain. This contrasts markedly with the paradigmatic ecmB gene which is expressed in pstAB cells in parental cells, but which is expressed throughout the prestalk zone in the Dd-STATa-null strain. We also identified several genes which are normally expressed in pstA cells, or throughout the prestalk region, but whose expression is markedly down-regulated in the null mutant. Again, this contrasts with markers derived from the paradigmatic, ecmA gene which are expressed normally in the Dd-STATa-null strain. The identification of these novel genes provides valuable tools to investigate the role of Dd-STATa.

Homologous cloning, characterization and expression of a new halophyte phytochelatin synthase gene in Suaeda salsa

NASA Astrophysics Data System (ADS)

Cong, Ming; Zhao, Jianmin; Lü, Jiasen; Ren, Zhiming; Wu, Huifeng

2016-09-01

The halophyte Suaeda salsa can grow in heavy metal-polluted areas along intertidal zones having high salinity. Since phytochelatins can eff ectively chelate heavy metals, it was hypothesized that S. salsa possessed a phytochelatin synthase (PCS) gene. In the present study, the cDNA of PCS was obtained from S. salsa (designated as SsPCS) using homologous cloning and the rapid amplification of cDNA ends (RACE). A sequence analysis revealed that SsPCS consisted of 1 916 bp nucleotides, encoding a polypeptide of 492 amino acids with one phytochelatin domain and one phytochelatin C domain. A similarity analysis suggested that SsPCS shared up to a 58.6% identity with other PCS proteins and clustered with PCS proteins from eudicots. There was a new kind of metal ion sensor motif in its C-terminal domain. The SsPCS transcript was more highly expressed in elongated and fibered roots and stems ( P<0.05) than in leaves. Lead and mercury exposure significantly enhanced the mRNA expression of SsPCS ( P<0.05). To the best of our knowledge, SsPCS is the second PCS gene cloned from a halophyte, and it might contain a diff erent metal sensing capability than the first PCS from Thellungiella halophila. This study provided a new view of halophyte PCS genes in heavy metal tolerance.

Interferon modulation of c-myc expression in cloned Daudi cells: relationship to the phenotype of interferon resistance.

PubMed

Dron, M; Modjtahedi, N; Brison, O; Tovey, M G

1986-05-01

Treatment of interferon-sensitive Daudi cell with electrophoretically pure human interferon alpha markedly reduced the level of c-myc mRNA, increased the level of class I histocompatibility antigen (HLA) mRNA, and did not affect the level of actin mRNA within the same cells. In contrast, the level of c-myc mRNA or HLA mRNA did not change significantly following interferon treatment in different clones of Daudi cells selected for resistance to the antiproliferative action of interferon. These cells possessed interferon receptors, however, and responded to interferon modulation of other genes, including 2',5' oligoisoadenylate synthetase (M. G. Tovey, M. Dron, K. E. Mogensen, B. Lebleu, N. Metchi, and J. Begon-Lours, Guymarho, J. Gen. Virol., 64:2649-2653, 1983; M. Dron, M. G. Tovey, and P. Eid, J. Gen. Virol., 66:787-795, 1985). A clone of interferon-resistant Daudi cells which had reverted to almost complete sensitivity to both the antiproliferative action of interferon and the interferon-enhanced expression of HLA mRNA remained refractory, however, to interferon modulation of c-myc expression, suggesting that a reduced level of c-myc mRNA may not be a prerequisite for inhibition of cell proliferation in interferon-treated cells. Our results do not exclude the possibility, however, that posttranscriptional modification(s) of c-myc expression may precede an inhibition of cell proliferation in interferon-treated cells.

Exploration of panviral proteome: high-throughput cloning and functional implications in virus-host interactions.

PubMed

Yu, Xiaobo; Bian, Xiaofang; Throop, Andrea; Song, Lusheng; Moral, Lerys Del; Park, Jin; Seiler, Catherine; Fiacco, Michael; Steel, Jason; Hunter, Preston; Saul, Justin; Wang, Jie; Qiu, Ji; Pipas, James M; LaBaer, Joshua

2014-01-01

Throughout the long history of virus-host co-evolution, viruses have developed delicate strategies to facilitate their invasion and replication of their genome, while silencing the host immune responses through various mechanisms. The systematic characterization of viral protein-host interactions would yield invaluable information in the understanding of viral invasion/evasion, diagnosis and therapeutic treatment of a viral infection, and mechanisms of host biology. With more than 2,000 viral genomes sequenced, only a small percent of them are well investigated. The access of these viral open reading frames (ORFs) in a flexible cloning format would greatly facilitate both in vitro and in vivo virus-host interaction studies. However, the overall progress of viral ORF cloning has been slow. To facilitate viral studies, we are releasing the initiation of our panviral proteome collection of 2,035 ORF clones from 830 viral genes in the Gateway® recombinational cloning system. Here, we demonstrate several uses of our viral collection including highly efficient production of viral proteins using human cell-free expression system in vitro, global identification of host targets for rubella virus using Nucleic Acid Programmable Protein Arrays (NAPPA) containing 10,000 unique human proteins, and detection of host serological responses using micro-fluidic multiplexed immunoassays. The studies presented here begin to elucidate host-viral protein interactions with our systemic utilization of viral ORFs, high-throughput cloning, and proteomic technologies. These valuable plasmid resources will be available to the research community to enable continued viral functional studies.

cDNA cloning, expression, and mutagenesis of a PR-10 protein SPE-16 from the seeds of Pachyrrhizus erosus.

PubMed

Wu, Fang; Yan, Ming; Li, Yikun; Chang, Shaojie; Song, Xiaomin; Zhou, Zhaocai; Gong, Weimin

2003-12-19

SPE-16 is a new 16kDa protein that has been purified from the seeds of Pachyrrhizus erosus. It's N-terminal amino acid sequence shows significant sequence homology to pathogenesis-related class 10 proteins. cDNA encoding 150 amino acids was cloned by RT-PCR and the gene sequence proved SPE-16 to be a new member of PR-10 family. The cDNA was cloned into pET15b plasmid and expressed in Escherichia coli. The bacterially expressed SPE-16 also demonstrated ribonuclease-like activity in vitro. Site-directed mutation of three conserved amino acids E95A, E147A, Y150A, and a P-loop truncated form were constructed and their different effects on ribonuclease activities were observed. SPE-16 is also able to bind the fluorescent probe 8-anilino-1-naphthalenesulfonate (ANS) in the native state. The ANS anion is a much-utilized "hydrophobic probe" for proteins. This binding activity indicated another biological function of SPE-16.

Identification of Genes Expressed in Premalignant Breast Disease by Microscopy-Directed Cloning

NASA Astrophysics Data System (ADS)

Jensen, Roy A.; Page, David L.; Holt, Jeffrey T.

1994-09-01

Histopathologic study of human breast biopsy samples has identified specific lesions which are associated with a high risk of development of invasive breast cancer. Presumably, these lesions (collectively termed premalignant breast disease) represent the earliest recognizable morphologic expression of fundamental molecular events that lead to the development of invasive breast cancer. To study molecular events underlying premalignant breast disease, we have developed a method for isolating RNA from histologically identified lesions from frozen human breast tissue. This method specifically obtains mRNA from breast epithelial cells and has identified three genes which are differentially expressed in premalignant breast epithelial lesions. One gene identified by this method is overexpressed in four of five noncomedo ductal carcinoma in situ lesions and appears to be the human homologue of the gene encoding the M2 subunit of ribonucleotide reductase, an enzyme involved in DNA synthesis.

Critical Factors Affecting the Success of Cloning, Expression, and Mass Production of Enzymes by Recombinant E. coli.

PubMed

Fakruddin, Md; Mohammad Mazumdar, Reaz; Bin Mannan, Khanjada Shahnewaj; Chowdhury, Abhijit; Hossain, Md Nur

2013-01-01

E. coli is the most frequently used host for production of enzymes and other proteins by recombinant DNA technology. E. coli is preferable for its relative simplicity, inexpensive and fast high-density cultivation, well-known genetics, and large number of compatible molecular tools available. Despite all these advantages, expression and production of recombinant enzymes are not always successful and often result in insoluble and nonfunctional proteins. There are many factors that affect the success of cloning, expression, and mass production of enzymes by recombinant E. coli. In this paper, these critical factors and approaches to overcome these obstacles are summarized focusing controlled expression of target protein/enzyme in an unmodified form at industrial level.

Cloning of endangered mammalian species: any progress?

PubMed

Loi, Pasqualino; Galli, Cesare; Ptak, Grazyna

2007-05-01

Attempts through somatic cell nuclear transfer to expand wild populations that have shrunk to critical numbers is a logical extension of the successful cloning of mammals. However, although the first mammal was cloned 10 years ago, nuclear reprogramming remains phenomenological, with abnormal gene expression and epigenetic deregulation being associated with the cloning process. In addition, although cloning of wild animals using host oocytes from different species has been successful, little is known about the implication of partial or total mitochondrial DNA heteroplasmy in cloned embryos, fetuses and offspring. Finally, there is a need for suitable foster mothers for inter-intra specific cloned embryos. Considering these issues, the limited success achieved in cloning endangered animals is not surprising. However, optimism comes from the rapid gain in the understanding of the molecular clues underlying nuclear reprogramming. If it is possible to achieve a controlled reversal of the differentiated state of a cell then it is probable that other issues that impair the cloning of endangered animals, such as the inter-intra species oocyte or womb donor, will be overcome in the medium term.

High level expression of bioactive recombinant human growth hormone in the milk of a cloned transgenic cow.

PubMed

Salamone, Daniel; Barañao, Lino; Santos, Claudio; Bussmann, Leonardo; Artuso, Jorge; Werning, Carlos; Prync, Aida; Carbonetto, Cesar; Dabsys, Susana; Munar, Carlos; Salaberry, Roberto; Berra, Guillermo; Berra, Ignacio; Fernández, Nahuel; Papouchado, Mariana; Foti, Marcelo; Judewicz, Norberto; Mujica, Ignacio; Muñoz, Luciana; Alvarez, Silvina Fenández; González, Eliseo; Zimmermann, Juan; Criscuolo, Marcelo; Melo, Carlos

2006-07-13

Transgenic farm animals have been proposed as an alternative to current bioreactors for large scale production of biopharmaceuticals. However, the efficiency of both methods in the production of the same protein has not yet been established. Here we report the production of recombinant human growth hormone (hGH) in the milk of a cloned transgenic cow at levels of up to 5 g l(-1). The hormone is identical to that currently produced by expression in E. coli. In addition, the hematological and somatometric parameters of the cloned transgenic cow are within the normal range for the breed and it is fertile and capable of producing normal offspring. These results demonstrate that transgenic cattle can be used as a cost-effective alternative for the production of this hormone.

Cloning and expression in Escherichia coli of isopenicillin N synthetase genes from Streptomyces lipmanii and Aspergillus nidulans.

PubMed Central

Weigel, B J; Burgett, S G; Chen, V J; Skatrud, P L; Frolik, C A; Queener, S W; Ingolia, T D

1988-01-01

beta-Lactam antibiotics such as penicillins and cephalosporins are synthesized by a wide variety of microbes, including procaryotes and eucaryotes. Isopenicillin N synthetase catalyzes a key reaction in the biosynthetic pathway of penicillins and cephalosporins. The genes encoding this protein have previously been cloned from the filamentous fungi Cephalosporium acremonium and Penicillium chrysogenum and characterized. We have extended our analysis to the isopenicillin N synthetase genes from the fungus Aspergillus nidulans and the gram-positive procaryote Streptomyces lipmanii. The isopenicillin N synthetase genes from these organisms have been cloned and sequenced, and the proteins encoded by the open reading frames were expressed in Escherichia coli. Active isopenicillin N synthetase enzyme was recovered from extracts of E. coli cells prepared from cells containing each of the genes in expression vectors. The four isopenicillin N synthetase genes studied are closely related. Pairwise comparison of the DNA sequences showed between 62.5 and 75.7% identity; comparison of the predicted amino acid sequences showed between 53.9 and 80.6% identity. The close homology of the procaryotic and eucaryotic isopenicillin N synthetase genes suggests horizontal transfer of the genes during evolution. Images PMID:3045077

Purification and characterization of recombinant human bile salt-stimulated lipase expressed in milk of transgenic cloned cows.

PubMed

Wang, Yuhang; Ding, Fangrong; Wang, Tao; Liu, Wenjie; Lindquist, Susanne; Hernell, Olle; Wang, Jianwu; Li, Jing; Li, Ling; Zhao, Yaofeng; Dai, Yunping; Li, Ning

2017-01-01

Bile salt-stimulated lipase (BSSL) is a lipolytic digestive enzyme with broad substrate specificity secreted from exocrine pancreas into the intestinal lumen in all species and from the lactating mammary gland into the milk of some species, notably humans but not cows. BSSL in breast milk facilitates digestion and absorption of milk fat and promotes growth of small for gestational age preterm infants. Thus, purified recombinant human BSSL (rhBSSL) can be used for treatment of patients with fat malabsorption and expressing rhBSSL in the milk of transgenic cloned cows would therefore be a mean to meet a medical need. In the present study, a vector pBAC-hLF-hBSSL was constructed, which efficiently expressed active rhBSSL in milk of transgenic cloned cows to a concentration of 9.8 mg/ml. The rhBSSL purified from cow milk had the same enzymatic activity, N-terminal amino acid sequence, amino acid composition and isoelectric point and similar physicochemical characteristics as human native BSSL. Our study supports the use of transgenic cattle for the cost-competitive, large-scale production of therapeutic rhBSSL.

Purification and characterization of recombinant human bile salt-stimulated lipase expressed in milk of transgenic cloned cows

PubMed Central

Ding, Fangrong; Wang, Tao; Liu, Wenjie; Lindquist, Susanne; Hernell, Olle; Wang, Jianwu; Li, Jing; Li, Ling; Zhao, Yaofeng; Dai, Yunping; Li, Ning

2017-01-01

Bile salt-stimulated lipase (BSSL) is a lipolytic digestive enzyme with broad substrate specificity secreted from exocrine pancreas into the intestinal lumen in all species and from the lactating mammary gland into the milk of some species, notably humans but not cows. BSSL in breast milk facilitates digestion and absorption of milk fat and promotes growth of small for gestational age preterm infants. Thus, purified recombinant human BSSL (rhBSSL) can be used for treatment of patients with fat malabsorption and expressing rhBSSL in the milk of transgenic cloned cows would therefore be a mean to meet a medical need. In the present study, a vector pBAC-hLF-hBSSL was constructed, which efficiently expressed active rhBSSL in milk of transgenic cloned cows to a concentration of 9.8 mg/ml. The rhBSSL purified from cow milk had the same enzymatic activity, N-terminal amino acid sequence, amino acid composition and isoelectric point and similar physicochemical characteristics as human native BSSL. Our study supports the use of transgenic cattle for the cost-competitive, large-scale production of therapeutic rhBSSL. PMID:28475629

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.

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 and characterization of a functional GSTmu class from the cattle tick Boophilus annulatus.

PubMed

Shahein, Yasser Ezzat; El Sayed El-Hakim, Amr; Abouelella, Amira Mohamed Kamal; Hamed, Ragaa Reda; Allam, Shaimaa Abdul-Moez; Farid, Nevin Mahmoud

2008-03-25

A full-length cDNA of a glutathione S-transferase (GST) was cloned from a cDNA library of the local Egyptian cattle tick Boophilus annulatus. The 672 bp cloned fragment was sequenced and showed an open reading frame encoding a protein of 223 amino acids. Comparison of the deduced amino acid sequence with GSTs from other species revealed that the sequence is closely related to the mammalian mu-class GST. The cloned gene was expressed in E. coli under T7 promotor of pET-30b vector, and purified under native conditions. The purified enzyme appeared as a single band on 12% SDS-PAGE and has a molecular weight of 30.8 kDa including the histidine tag of the vector. The purified enzyme was assayed upon the chromogenic substrate 1-chloro-2,4-dinitrobenzene (CDNB) and the recombinant enzyme showed high level of activity even in the presence of the beta-galactosidase region on its 5' end and showed maximum activity at pH 7.5. The Km values for CDNB and GSH were 0.57 and 0.79 mM, respectively. The over expressed rBaGST showed high activity toward CDNB (121 units/mg protein) and less toward DCNB (29.3 units/mg protein). rBaGST exhibited peroxidatic activity on cumene hydroperoxide sharing this property with GSTs belonging to the GST alpha class. I50 values for cibacron blue and bromosulfophthalein were 0.22 and 8.45 microM, respectively, sharing this property with the mammalian GSTmu class. Immunoblotting revealed the presence of the GST molecule in B. annulatus protein extracts; whole tick, larvae, gut, salivary gland and ovary. Homologues to the GSTmu were also detected in other tick species as Hyalomma dromedarii and Rhipicephalus sp. while in Ornithodoros moubata, GSTmu homologue could not be detected.

Development of a gene cloning system in a fast-growing and moderately thermophilic Streptomyces species and heterologous expression of Streptomyces antibiotic biosynthetic gene clusters

PubMed Central

2011-01-01

Background Streptomyces species are a major source of antibiotics. They usually grow slowly at their optimal temperature and fermentation of industrial strains in a large scale often takes a long time, consuming more energy and materials than some other bacterial industrial strains (e.g., E. coli and Bacillus). Most thermophilic Streptomyces species grow fast, but no gene cloning systems have been developed in such strains. Results We report here the isolation of 41 fast-growing (about twice the rate of S. coelicolor), moderately thermophilic (growing at both 30°C and 50°C) Streptomyces strains, detection of one linear and three circular plasmids in them, and sequencing of a 6996-bp plasmid, pTSC1, from one of them. pTSC1-derived pCWH1 could replicate in both thermophilic and mesophilic Streptomyces strains. On the other hand, several Streptomyces replicons function in thermophilic Streptomyces species. By examining ten well-sporulating strains, we found two promising cloning hosts, 2C and 4F. A gene cloning system was established by using the two strains. The actinorhodin and anthramycin biosynthetic gene clusters from mesophilic S. coelicolor A3(2) and thermophilic S. refuineus were heterologously expressed in one of the hosts. Conclusions We have developed a gene cloning and expression system in a fast-growing and moderately thermophilic Streptomyces species. Although just a few plasmids and one antibiotic biosynthetic gene cluster from mesophilic Streptomyces were successfully expressed in thermophilic Streptomyces species, we expect that by utilizing thermophilic Streptomyces-specific promoters, more genes and especially antibiotic genes clusters of mesophilic Streptomyces should be heterologously expressed. PMID:22032628

PanGEA: identification of allele specific gene expression using the 454 technology.

PubMed

Kofler, Robert; Teixeira Torres, Tatiana; Lelley, Tamas; Schlötterer, Christian

2009-05-14

Next generation sequencing technologies hold great potential for many biological questions. While mainly used for genomic sequencing, they are also very promising for gene expression profiling. Sequencing of cDNA does not only provide an estimate of the absolute expression level, it can also be used for the identification of allele specific gene expression. We developed PanGEA, a tool which enables a fast and user-friendly analysis of allele specific gene expression using the 454 technology. PanGEA allows mapping of 454-ESTs to genes or whole genomes, displaying gene expression profiles, identification of SNPs and the quantification of allele specific gene expression. The intuitive GUI of PanGEA facilitates a flexible and interactive analysis of the data. PanGEA additionally implements a modification of the Smith-Waterman algorithm which deals with incorrect estimates of homopolymer length as occuring in the 454 technology To our knowledge, PanGEA is the first tool which facilitates the identification of allele specific gene expression. PanGEA is distributed under the Mozilla Public License and available at: http://www.kofler.or.at/bioinformatics/PanGEA

PanGEA: Identification of allele specific gene expression using the 454 technology

PubMed Central

Kofler, Robert; Teixeira Torres, Tatiana; Lelley, Tamas; Schlötterer, Christian

2009-01-01

Background Next generation sequencing technologies hold great potential for many biological questions. While mainly used for genomic sequencing, they are also very promising for gene expression profiling. Sequencing of cDNA does not only provide an estimate of the absolute expression level, it can also be used for the identification of allele specific gene expression. Results We developed PanGEA, a tool which enables a fast and user-friendly analysis of allele specific gene expression using the 454 technology. PanGEA allows mapping of 454-ESTs to genes or whole genomes, displaying gene expression profiles, identification of SNPs and the quantification of allele specific gene expression. The intuitive GUI of PanGEA facilitates a flexible and interactive analysis of the data. PanGEA additionally implements a modification of the Smith-Waterman algorithm which deals with incorrect estimates of homopolymer length as occuring in the 454 technology Conclusion To our knowledge, PanGEA is the first tool which facilitates the identification of allele specific gene expression. PanGEA is distributed under the Mozilla Public License and available at: PMID:19442283

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.

Cloning and expression of codon-optimized recombinant darbepoetin alfa in Leishmania tarentolae T7-TR.

PubMed

Kianmehr, Anvarsadat; Golavar, Raziyeh; Rouintan, Mandana; Mahrooz, Abdolkarim; Fard-Esfahani, Pezhman; Oladnabi, Morteza; Khajeniazi, Safoura; Mostafavi, Seyede Samaneh; Omidinia, Eskandar

2016-02-01

Darbepoetin alfa is an engineered and hyperglycosylated analog of recombinant human erythropoietin (EPO) which is used as a drug in treating anemia in patients with chronic kidney failure and cancer. This study desribes the secretory expression of a codon-optimized recombinant form of darbepoetin alfa in Leishmania tarentolae T7-TR. Synthetic codon-optimized gene was amplified by PCR and cloned into the pLEXSY-I-blecherry3 vector. The resultant expression vector, pLEXSYDarbo, was purified, digested, and electroporated into the L. tarentolae. Expression of recombinant darbepoetin alfa was evaluated by ELISA, reverse-transcription PCR (RT-PCR), Western blotting, and biological activity. After codon optimization, codon adaptation index (CAI) of the gene raised from 0.50 to 0.99 and its GC% content changed from 56% to 58%. Expression analysis confirmed the presence of a protein band at 40 kDa. Furthermore, reticulocyte experiment results revealed that the activity of expressed darbepoetin alfa was similar to that of its equivalent expressed in Chinese hamster ovary (CHO) cells. These data suggested that the codon optimization and expression in L. tarentolae host provided an efficient approach for high level expression of darbepoetin alfa. Copyright © 2015 Elsevier Inc. 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.

New methods for tightly regulated gene expression and highly efficient chromosomal integration of cloned genes for Methanosarcina species

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

Guss, Adam M.; Rother, Michael; Zhang, Jun Kai

A highly efficient method for chromosomal integration of cloned DNA into Methanosarcina spp. was developed utilizing the site-specific recombination system from the Streptomyces phage φC31. Host strains expressing the φC31 integrase gene and carrying an appropriate recombination site can be transformed with non-replicating plasmids carrying the complementary recombination site at efficiencies similar to those obtained with self-replicating vectors. We have also constructed a series of hybrid promoters that combine the highly expressed M. barkeri P mcrB promoter with binding sites for the tetracycline-responsive, bacterial TetR protein. These promoters are tightly regulated by the presence or absence of tetracycline in strainsmore » that express the tetR gene. The hybrid promoters can be used in genetic experiments to test gene essentiality by placing a gene of interest under their control. Thus, growth of strains with tetR -regulated essential genes becomes tetracycline-dependent. A series of plasmid vectors that utilize the site-specific recombination system for construction of reporter gene fusions and for tetracycline regulated expression of cloned genes are reported. These vectors were used to test the efficiency of translation at a variety of start codons. Fusions using an ATG start site were the most active, whereas those using GTG and TTG were approximately one half or one fourth as active, respectively. The CTG fusion was 95% less active than the ATG fusion.« less

New methods for tightly regulated gene expression and highly efficient chromosomal integration of cloned genes for Methanosarcina species

DOE PAGES

Guss, Adam M.; Rother, Michael; Zhang, Jun Kai; ...

2008-01-01

A highly efficient method for chromosomal integration of cloned DNA into Methanosarcina spp. was developed utilizing the site-specific recombination system from the Streptomyces phage φC31. Host strains expressing the φC31 integrase gene and carrying an appropriate recombination site can be transformed with non-replicating plasmids carrying the complementary recombination site at efficiencies similar to those obtained with self-replicating vectors. We have also constructed a series of hybrid promoters that combine the highly expressed M. barkeri P mcrB promoter with binding sites for the tetracycline-responsive, bacterial TetR protein. These promoters are tightly regulated by the presence or absence of tetracycline in strainsmore » that express the tetR gene. The hybrid promoters can be used in genetic experiments to test gene essentiality by placing a gene of interest under their control. Thus, growth of strains with tetR -regulated essential genes becomes tetracycline-dependent. A series of plasmid vectors that utilize the site-specific recombination system for construction of reporter gene fusions and for tetracycline regulated expression of cloned genes are reported. These vectors were used to test the efficiency of translation at a variety of start codons. Fusions using an ATG start site were the most active, whereas those using GTG and TTG were approximately one half or one fourth as active, respectively. The CTG fusion was 95% less active than the ATG fusion.« less

Cloning, expression and characterization of phenylalanine ammonia-lyase from Rhodotorula glutinis.

PubMed

Zhu, Longbao; Cui, Wenjing; Fang, Yueqin; Liu, Yi; Gao, Xinxing; Zhou, Zhemin

2013-05-01

The industrial-scale production of phenylalanine ammonia-lyase (PAL) mainly uses strains of Rhodotorula. However, the PAL gene from Rhodotorula has not been cloned. Here, the full-length gene of PAL from Rhodotorula glutinis was isolated. It was 2,121 bp, encoding a polypeptide with 706 amino acids and a calculated MW of 75.5 kDa. Though R. glutinis is an anamorph of Rhodosporium toruloides, the amino acid sequences of PALs them are not the same (about 74 % identity). PAL was expressed in E. coli and characterized. Its specific activity was 4.2 U mg(-1) and the k cat/K m was 1.9 × 10(4) mM(-1) s(-1), exhibiting the highest catalytic ability among the reported PALs. The genetic and biochemical information reported here should facilitate future application in industry.

Molecular cloning and expression analysis of KIN10 and cold-acclimation related genes in wild banana 'Huanxi' (Musa itinerans).

PubMed

Liu, Weihua; Cheng, Chunzhen; Lai, Gongti; Lin, Yuling; Lai, Zhongxiong

2015-01-01

Banana cultivars may experience chilling or freezing injury in some of their cultivated regions, where wild banana can still grow very well. The clarification of the cold-resistant mechanism of wild banana is vital for cold-resistant banana breeding. In this study, the central stress integrator gene KIN10 and some cold-acclimation related genes (HOS1 and ICE1s) from the cold-resistant wild banana 'Huanxi' (Musa itinerans) were cloned and their expression patterns under different temperature treatments were analyzed. Thirteen full-length cDNA transcripts including 6 KIN10s, 1 HOS1 and 6 ICE1s were successfully cloned. Quantitative real-time PCR (qRT-PCR) results showed that all these genes had the highest expression levels at the critical temperature of banana (13 °C). Under chilling temperature (4 °C), the expression level of KIN10 reduced significantly but the expression of HOS1 was still higher than that at the optimal temperature (28 °C, control). Both KIN10 and HOS1 showed the lowest expression levels at 0 °C, the expression level of ICE1, however, was higher than control. As sucrose plays role in plant cold-acclimation and in regulation of KIN10 and HOS1 bioactivities, the sucrose contents of wild banana under different temperatures were detected. Results showed that the sucrose content increased as temperature lowered. Our result suggested that KIN10 may participate in cold stress response via regulating sucrose biosynthesis, which is helpful in regulating cold acclimation pathway in wild banana.

Molecular cloning, expression and immunological properties of LiD1, a protein from the dermonecrotic family of Loxosceles intermedia spider venom.

PubMed

Kalapothakis, Evanguedes; Araujo, Simone Costa; de Castro, Cibele Soares; Mendes, Thais Melo; Gomez, Marcus Vinícius; Mangili, Oldemir C; Gubert, Ida C; Chávez-Olórtegui, Carlos

2002-12-01

The present report describes the identification and molecular characterization of LiD1, a protein expressed in the venom gland of the brown spider Loxosceles intermedia. LiD1 belongs to a family of proteins with dermonecrotic activity and members of this family have been found in spiders from the genus Loxosceles. The necrotic lesions caused by this group of proteins may lead to serious socio-economic problems such as surgical tissue reconstitution and even patient death. LiD1 was cloned using a cDNA library constructed from the venom gland of L. intermedia and antibodies against proteins with dermonecrotic activity isolated from the crude venom of this spider. The amino acid sequence deduced from the cDNA revealed a mature protein of approximately 31 kDa, with a pI of 7.37. The cDNA also revealed the existence of a signal peptide, a propeptide and also an untranslated 3' region with 218 nucleotides. LiD1 was expressed as a protein fused with beta-galactoside protein using the vector pBK-CMV, resulting in the recombinant protein recLiD1 with important immunological properties. recLiD1 was strongly recognised by anti-dermonecrotic antibodies and was also able to generate reactive antibodies against native dermonecrotic proteins isolated from the venom of L. intermedia.

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.

Cloning, analysis and functional annotation of expressed sequence tags from the Earthworm Eisenia fetida

PubMed Central

Pirooznia, Mehdi; Gong, Ping; Guan, Xin; Inouye, Laura S; Yang, Kuan; Perkins, Edward J; Deng, Youping

2007-01-01

Background Eisenia fetida, commonly known as red wiggler or compost worm, belongs to the Lumbricidae family of the Annelida phylum. Little is known about its genome sequence although it has been extensively used as a test organism in terrestrial ecotoxicology. In order to understand its gene expression response to environmental contaminants, we cloned 4032 cDNAs or expressed sequence tags (ESTs) from two E. fetida libraries enriched with genes responsive to ten ordnance related compounds using suppressive subtractive hybridization-PCR. Results A total of 3144 good quality ESTs (GenBank dbEST accession number EH669363–EH672369 and EL515444–EL515580) were obtained from the raw clone sequences after cleaning. Clustering analysis yielded 2231 unique sequences including 448 contigs (from 1361 ESTs) and 1783 singletons. Comparative genomic analysis showed that 743 or 33% of the unique sequences shared high similarity with existing genes in the GenBank nr database. Provisional function annotation assigned 830 Gene Ontology terms to 517 unique sequences based on their homology with the annotated genomes of four model organisms Drosophila melanogaster, Mus musculus, Saccharomyces cerevisiae, and Caenorhabditis elegans. Seven percent of the unique sequences were further mapped to 99 Kyoto Encyclopedia of Genes and Genomes pathways based on their matching Enzyme Commission numbers. All the information is stored and retrievable at a highly performed, web-based and user-friendly relational database called EST model database or ESTMD version 2. Conclusion The ESTMD containing the sequence and annotation information of 4032 E. fetida ESTs is publicly accessible at . PMID:18047730

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

Cloning and expression of the gene for bacteriophage T7 RNA polymerase

DOEpatents

Studier, F.W.; Davanloo, P.; Rosenberg, A.H.

1984-03-30

This application describes a means to clone a functional gene for bacteriophage T7 RNA polymerase. Active T7 RNA polymerase is produced from the cloned gene, and a plasmid has been constructed that can produce the active enzyme in large amounts. T7 RNA polymerase transcribes DNA very efficiently and is highly selective for a relatively long promoter sequence. This enzyme is useful for synthesizing large amounts of RNA in vivo or in vitro, and is capable of producing a single RNA selectively from a complex mixture of DNAs. The procedure used to obtain a clone of the T7 RNA polymerase gene can be applied to other T7-like phages to obtain clones that produce RNA polymerases having different promoter specificities, different bacterial hosts, or other desirable properties.

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.

Cloning and High-Level Expression of α-Galactosidase cDNA from Penicillium purpurogenum

PubMed Central

Shibuya, Hajime; Nagasaki, Hiroaki; Kaneko, Satoshi; Yoshida, Shigeki; Park, Gwi Gun; Kusakabe, Isao; Kobayashi, Hideyuki

1998-01-01

The cDNA coding for Penicillium purpurogenum α-galactosidase (αGal) was cloned and sequenced. The deduced amino acid sequence of the α-Gal cDNA showed that the mature enzyme consisted of 419 amino acid residues with a molecular mass of 46,334 Da. The derived amino acid sequence of the enzyme showed similarity to eukaryotic αGals from plants, animals, yeasts, and filamentous fungi. The highest similarity observed (57% identity) was to Trichoderma reesei AGLI. The cDNA was expressed in Saccharomyces cerevisiae under the control of the yeast GAL10 promoter. Almost all of the enzyme produced was secreted into the culture medium, and the expression level reached was approximately 0.2 g/liter. The recombinant enzyme purified to homogeneity was highly glycosylated, showed slightly higher specific activity, and exhibited properties almost identical to those of the native enzyme from P. purpurogenum in terms of the N-terminal amino acid sequence, thermoactivity, pH profile, and mode of action on galacto-oligosaccharides. PMID:9797312

Twins: A cloning experience.

PubMed

Prainsack, Barbara; Spector, Tim D

2006-11-01

Drawing upon qualitative interviews with monozygotic (identical) twins sharing 100% of their genes, and with dizygotic (fraternal) twins and singletons as control groups, this paper explores what it means to be genetically identical. (The twins interviewed were from the TwinsUK register in London.) In the context of the ongoing debate on human reproductive cloning, it examines questions such as: To what extent do identical twins perceive their emotional and physical bond to be a result of their genetic makeup? What would they think if they had been deliberately created genetically identical? How would they feel about being genetically identical to a person who was born a few years earlier or later? First, our respondents ascribed no great significance to the role of genes in their understanding of what it means to be identical twins. Second, the opinion that human reproductive cloning would "interfere with nature", or "contradict God's will", was expressed by our respondents exclusively on the abstract level. The more our respondents were able to relate a particular invented cloning scenario to their own life-worlds, the lower the prevalence of the argument. Third, for all three groups of respondents, the scenario of having been born in one of the other groups was perceived as strange. Fourth, the aspect that our respondents disliked about cloning scenarios was the potential motives of the cloners. Without equating monozygotic twins directly with "clones", these results from "naturally" genetically identical individuals add a new dimension to what a future cloning situation could entail: The cloned person might possibly (a) perceive a close physical and emotional connection to the progenitor as a blessing; (b) suffer from preconceptions of people who regard physical likeness as a sign of incomplete individuality; and (c) perceive the idea of not having been born a clone of a particular person as unpleasant.

Molecular cloning of the Coch gene of guinea pig inner ear and its expression analysis in cultured fibrocytes of the spiral ligament.

PubMed

Li, Lishu; Ikezono, Tetsuo; Sekine, Kuwon; Shindo, Susumu; Matsumura, Tomohiro; Pawankar, Ruby; Ichimiya, Issei; Yagi, Toshiaki

2010-08-01

We have cloned guinea pig Coch cDNA and the sequence information will be useful for future molecular study combined with physiological experiments. Proper Coch gene expression appears to be dependent on the unique extracellular micro-environment of the inner ear in vivo. These results provide insight into the Coch gene expression and its regulation. To characterize the guinea pig Coch gene, we performed molecular cloning and expression analysis in the inner ear and cultured fibrocytes of the spiral ligament. The Coch cDNA was isolated using RACE. Cochlin isofoms were studied by Western blot using three different types of mammalian inner ear. The cochlear fibrocytes were cultured and characterized by immunostaining. Coch gene expression in the fibrocytes was investigated and the influence of cytokine stimulation was evaluated. The full-length 1991 bp Coch cDNA that encodes a 553 amino acid protein was isolated. The sequence had significant homology with other mammals, and the sizes of the Cochlin isoforms were identical. In the cultured fibrocytes, Coch mRNA was expressed in a very small amount and the isoform production was different, compared with the results in vivo. Cytokine stimulation did not alter the level of mRNA expression or isoform formation.

Cloning and characterization of an RNase-related protein gene preferentially expressed in rice stems.

PubMed

Wei, Jun-Ya; Li, An-Ming; Li, Yin; Wang, Jing; Liu, Xiao-Bin; Liu, Liang-Shi; Xu, Zeng-Fu

2006-04-01

RNase-related proteins (RRPs) are S- and S-like RNase homologs lacking the active site required for RNase activity. Here we describe the cloning and characterization of the rice (Oryza sativa) RRP gene (OsRRP). A single copy of OsRRP occurs in the rice genome. OsRRP contains three introns and an open reading frame encoding 252 amino acids, with the replacement of two histidines involved in the active site of RNase by lysine and tyrosine respectively. OsRRP is preferentially expressed in stems of wild-type rice and is significantly down-regulated in an increased tillering dwarf mutant ext37.

Expansion of the gateway multisite recombination cloning toolkit.

PubMed

Shearin, Harold K; Dvarishkis, Alisa R; Kozeluh, Craig D; Stowers, R Steven

2013-01-01

Precise manipulation of transgene expression in genetic model organisms has led to advances in understanding fundamental mechanisms of development, physiology, and genetic disease. Transgene construction is, however, a precondition of transgene expression, and often limits the rate of experimental progress. Here we report an expansion of the modular Gateway MultiSite recombination-cloning platform for high efficiency transgene assembly. The expansion includes two additional destination vectors and entry clones for the LexA binary transcription system, among others. These new tools enhance the expression levels possible with Gateway MultiSite generated transgenes and make possible the generation of LexA drivers and reporters with Gateway MultiSite cloning. In vivo data from transgenic Drosophila functionally validating each novel component are presented and include neuronal LexA drivers, LexAop2 red and green fluorescent synaptic vesicle reporters, TDC2 and TRH LexA, GAL4, and QF drivers, and LexAop2, UAS, and QUAS channelrhodopsin2 T159C reporters.

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.

Facial expressions of emotion (KDEF): identification under different display-duration conditions.

PubMed

Calvo, Manuel G; Lundqvist, Daniel

2008-02-01

Participants judged which of seven facial expressions (neutrality, happiness, anger, sadness, surprise, fear, and disgust) were displayed by a set of 280 faces corresponding to 20 female and 20 male models of the Karolinska Directed Emotional Faces database (Lundqvist, Flykt, & Ohman, 1998). Each face was presented under free-viewing conditions (to 63 participants) and also for 25, 50, 100, 250, and 500 msec (to 160 participants), to examine identification thresholds. Measures of identification accuracy, types of errors, and reaction times were obtained for each expression. In general, happy faces were identified more accurately, earlier, and faster than other faces, whereas judgments of fearful faces were the least accurate, the latest, and the slowest. Norms for each face and expression regarding level of identification accuracy, errors, and reaction times may be downloaded from www.psychonomic.org/archive/.

Cloning, expressing, and hemolysis of tdh, trh and tlh genes of Vibrio parahaemolyticus

NASA Astrophysics Data System (ADS)

Zhao, Yonggang; Tang, Xiaoqian; Zhan, Wenbin

2011-09-01

Vibrio parahaemolyticus (VP) is one of the pathogenic vibrios endangering net-cage cultured Pseudosciaena crocea, Fennerpenaeus chinensis, and shellfish in coastal areas of China. Several types of hemolysins produced by Vp have been characterized as major virulence factors. They are thermostable direct hemolysin (TDH), TDH-related hemolysin (TRH) and thermolabile hemolysin (TLH). In this study, we cloned tdh, trh, and tlh genes from the genome DNA of VP by polymerase chain reaction (PCR). We ligated the three genes into prokaryotic expression vector pET-28a (+), and transformed the recombinant plasmids into Escherichia coli BL21 (DE3). The expression of recombinant proteins was induced by isopropyl-β-D-thiogalacto-pyranoside (IPTG). The recombinant proteins were expressed in a form of inclusion bodies and thus purified with Ni-NTA affinity chromatography. Western blotting results showed that recombinant proteins, TDH, TRH and TLH, could be recognized by rabbit anti-VP serum. The three purified proteins were renatured by gradient dialysis. The renatured proteins exhibited hemolytic activity except for TLH in the presence of phosphatidylcholine. These results not only are helpful for better understanding these genes' functions under a single factor level, but also provide evidence for VP vaccine engineering.

Animal cloning: problems and prospects.

PubMed

Wells, D N

2005-04-01

An efficient animal cloning technology would provide many new opportunities for livestock agriculture, human medicine, and animal conservation. Nuclear cloning involves the production of animals that are genetically identical to the donor cells used in a technique known as nuclear transfer (NT). However, at present it is an inefficient process: in cattle, only around 6% of the embryos transferred to the reproductive tracts of recipient cows result in healthy, longterm surviving clones. Of concern are the high losses throughout gestation, during birth and in the post-natal period through to adulthood. Many of the pregnancy losses relate to failure of the placenta to develop and function correctly. Placental dysfunction may also have an adverse influence on postnatal health. These anomalies are probably due to incorrect epigenetic reprogramming of the donor genome following NT, leading to inappropriate patterns of gene expression during the development of clones. Whilst some physiological tests on surviving clones suggest normality, other reports indicate a variety of post-natal clone-associated abnormalities. This variability in outcome may reflect species-specific and/or cloning methodological differences. Importantly, to date it appears that these clone-associated phenotypes are not transmitted to offspring following sexual reproduction. This indicates that they represent epigenetic errors, rather than genetic errors, which are corrected during gametogenesis. Whilst this needs confirmation at the molecular level, it provides initial confidence in the first application of NT in agriculture, namely, the production of small numbers of cloned sires from genetically elite bulls, for natural mating, to effectively disseminate genetic gain. In addition to the animal welfare concerns with the technology, the underlying health of the animals and the consequential effect on food safety are critical aspects that require investigation to gain regulatory and consumer

Molecular Cloning and Gene Expression Analysis of Ercc6l in Sika Deer (Cervus nippon hortulorum)

PubMed Central

Zhang, Jiabao; Tang, Bo; Li, Ziyi

2011-01-01

Background One important protein family that functions in nucleotide excision repair (NER) factors is the SNF2 family. A newly identified mouse ERCC6-like gene, Ercc6l (excision repair cross-complementing rodent repair deficiency, complementation group 6-like), has been shown to be another developmentally related member of the SNF2 family. Methodology/Principal Findings In this study, Sika deer Ercc6l cDNA was first cloned and then sequenced. The full-length cDNA of the Sika deer Ercc6l gene is 4197 bp and contains a 3732 bp open reading frame that encodes a putative protein of 1243 amino acids. The similarity of Sika deer Ercc6l to Bos taurus Ercc6l is 94.05% at the amino acid sequence level. The similarity, however, is reduced to 68.42–82.21% when compared to Ercc6l orthologs in other mammals and to less than 50% compared to orthologs in Gallus gallus and Xenopus. Additionally, the expression of Ercc6l mRNA was investigated in the organs of fetal and adult Sika deer (FSD and ASD, respectively) by quantitative RT-PCR. The common expression level of Ercc6l mRNA in the heart, liver, spleen, lung, kidney, and stomach from six different developmental stages of 18 Sika deer were examined, though the expression levels in each organ varied among individual Sika deer. During development, there was a slight trend toward decreased Ercc61 mRNA expression. The highest Ercc6l expression levels were seen at 3 months old in every organ and showed the highest level of detection in the spleen of FSD. The lowest Ercc6l expression levels were seen at 3 years old. Conclusions/Significance We are the first to successfully clone Sika deer Ercc6l mRNA. Ercc6l transcript is present in almost every organ. During Sika deer development, there is a slight trend toward decreased Ercc61 mRNA expression. It is possible that Ercc6l has other roles in embryonic development and in maintaining the growth of animals. PMID:21695076

Interferon modulation of c-myc expression in cloned Daudi cells: relationship to the phenotype of interferon resistance.

PubMed Central

Dron, M; Modjtahedi, N; Brison, O; Tovey, M G

1986-01-01

Treatment of interferon-sensitive Daudi cell with electrophoretically pure human interferon alpha markedly reduced the level of c-myc mRNA, increased the level of class I histocompatibility antigen (HLA) mRNA, and did not affect the level of actin mRNA within the same cells. In contrast, the level of c-myc mRNA or HLA mRNA did not change significantly following interferon treatment in different clones of Daudi cells selected for resistance to the antiproliferative action of interferon. These cells possessed interferon receptors, however, and responded to interferon modulation of other genes, including 2',5' oligoisoadenylate synthetase (M. G. Tovey, M. Dron, K. E. Mogensen, B. Lebleu, N. Metchi, and J. Begon-Lours, Guymarho, J. Gen. Virol., 64:2649-2653, 1983; M. Dron, M. G. Tovey, and P. Eid, J. Gen. Virol., 66:787-795, 1985). A clone of interferon-resistant Daudi cells which had reverted to almost complete sensitivity to both the antiproliferative action of interferon and the interferon-enhanced expression of HLA mRNA remained refractory, however, to interferon modulation of c-myc expression, suggesting that a reduced level of c-myc mRNA may not be a prerequisite for inhibition of cell proliferation in interferon-treated cells. Our results do not exclude the possibility, however, that posttranscriptional modification(s) of c-myc expression may precede an inhibition of cell proliferation in interferon-treated cells. Images PMID:3785169

Cloning, Expression, and Characterization of a Novel Thermophilic Monofunctional Catalase from Geobacillus sp. CHB1

PubMed Central

2016-01-01

Catalases are widely used in many scientific areas. A catalase gene (Kat) from Geobacillus sp. CHB1 encoding a monofunctional catalase was cloned and recombinant expressed in Escherichia coli (E. coli), which was the first time to clone and express this type of catalase of genus Geobacillus strains as far as we know. This Kat gene was 1,467 bp in length and encoded a catalase with 488 amino acid residuals, which is only 81% similar to the previously studied Bacillus sp. catalase in terms of amino acid sequence. Recombinant catalase was highly soluble in E. coli and made up 30% of the total E. coli protein. Fermentation broth of the recombinant E. coli showed a high catalase activity level up to 35,831 U/mL which was only lower than recombinant Bacillus sp. WSHDZ-01 among the reported catalase production strains. The purified recombinant catalase had a specific activity of 40,526 U/mg and K m of 51.1 mM. The optimal reaction temperature of this recombinant enzyme was 60°C to 70°C, and it exhibited high activity over a wide range of reaction temperatures, ranging from 10°C to 90°C. The enzyme retained 94.7% of its residual activity after incubation at 60°C for 1 hour. High yield and excellent thermophilic properties are valuable features for this catalase in industrial applications. PMID:27579320

Cloning, Expression, and Characterization of a Novel Thermophilic Monofunctional Catalase from Geobacillus sp. CHB1.

PubMed

Jia, Xianbo; Chen, Jichen; Lin, Chenqiang; Lin, Xinjian

2016-01-01

Catalases are widely used in many scientific areas. A catalase gene (Kat) from Geobacillus sp. CHB1 encoding a monofunctional catalase was cloned and recombinant expressed in Escherichia coli (E. coli), which was the first time to clone and express this type of catalase of genus Geobacillus strains as far as we know. This Kat gene was 1,467 bp in length and encoded a catalase with 488 amino acid residuals, which is only 81% similar to the previously studied Bacillus sp. catalase in terms of amino acid sequence. Recombinant catalase was highly soluble in E. coli and made up 30% of the total E. coli protein. Fermentation broth of the recombinant E. coli showed a high catalase activity level up to 35,831 U/mL which was only lower than recombinant Bacillus sp. WSHDZ-01 among the reported catalase production strains. The purified recombinant catalase had a specific activity of 40,526 U/mg and K m of 51.1 mM. The optimal reaction temperature of this recombinant enzyme was 60°C to 70°C, and it exhibited high activity over a wide range of reaction temperatures, ranging from 10°C to 90°C. The enzyme retained 94.7% of its residual activity after incubation at 60°C for 1 hour. High yield and excellent thermophilic properties are valuable features for this catalase in industrial applications.

Mere social categorization modulates identification of facial expressions of emotion.

PubMed

Young, Steven G; Hugenberg, Kurt

2010-12-01

The ability of the human face to communicate emotional states via facial expressions is well known, and past research has established the importance and universality of emotional facial expressions. However, recent evidence has revealed that facial expressions of emotion are most accurately recognized when the perceiver and expresser are from the same cultural ingroup. The current research builds on this literature and extends this work. Specifically, we find that mere social categorization, using a minimal-group paradigm, can create an ingroup emotion-identification advantage even when the culture of the target and perceiver is held constant. Follow-up experiments show that this effect is supported by differential motivation to process ingroup versus outgroup faces and that this motivational disparity leads to more configural processing of ingroup faces than of outgroup faces. Overall, the results point to distinct processing modes for ingroup and outgroup faces, resulting in differential identification accuracy for facial expressions of emotion. PsycINFO Database Record (c) 2010 APA, all rights reserved.

Cloning, expression, purification, crystallization and preliminary X-ray crystallographic analysis of bacterioferritin A from Mycobacterium tuberculosis

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

Gupta, Vibha; Gupta, Rakesh K.; Ram Lal Anand College, University of Delhi, Benito Juarez Road, New Delhi 110021

2008-05-01

The cloning, purification and crystallization of a bacterioferritin from M. tuberculosis together with preliminary X-ray characterization of its crystals are reported. Bacterioferritins (Bfrs) comprise a subfamily of the ferritin superfamily of proteins that play an important role in bacterial iron storage and homeostasis. Bacterioferritins differ from ferritins in that they have additional noncovalently bound haem groups. To assess the physiological role of this subfamily of ferritins, a greater understanding of the structural details of bacterioferritins from various sources is required. The gene encoding bacterioferritin A (BfrA) from Mycobacterium tuberculosis was cloned and expressed in Escherichia coli. The recombinant protein productmore » was purified by affinity chromatography on a Strep-Tactin column and crystallized with sodium chloride as a precipitant at pH 8.0 using the vapour-diffusion technique. The crystals diffracted to 2.1 Å resolution and belonged to space group P4{sub 2}, with unit-cell parameters a = 123.0, b = 123.0, c = 174.6 Å.« less

[Cloning, subcellular localization, and heterologous expression of ApNAC1 gene from Andrographis paniculata].

PubMed

Wang, Jian; Qi, Meng-Die; Guo, Juan; Shen, Ye; Lin, Hui-Xin; Huang, Lu-Qi

2017-03-01

Andrographis paniculata is widely used as medicinal herb in China for a long time and andrographolide is its main medicinal constituent. To investigate the underlying andrographolide biosynthesis mechanisms, RNA-seq for A. paniculata leaves with MeJA treatment was performed. In A. paniculata transcriptomic data, the expression pattern of one member of NAC transcription factor family (ApNAC1) matched with andrographolide accumulation. The coding sequence of ApNAC1 was cloned by RT-PCR, and GenBank accession number was KY196416. The analysis of bioinformatics showed that the gene encodes a peptide of 323 amino acids, with a predicted relative molecular weight of 35.9 kDa and isoelectric point of 6.14. To confirm the subcellular localization, ApNAC1-GFP was transiently expressed in A. paniculata protoplast. The results indicated that ApNAC1 is a nucleus-localized protein. The analysis of real-time quantitative PCR revealed that ApNAC1 gene predominantly expresses in leaves. Compared with control sample, its expression abundance sharply increased with methyl jasmonate treatment. Based on its expression pattern, ApNAC1 gene might involve in andrographolide biosynthesis. ApNAC1 was heterologously expressed in Escherichia coli and recombinant protein was purified by Ni-NTA agarose. Further study will help us to understand the function of ApNAC1 in andrographolide biosynthesis. Copyright© by the Chinese Pharmaceutical Association.

Cloning and strong expression of a Bacillus subtilis WL-3 mannanase gene in B. subtilis.

PubMed

Yoon, Ki-Hong; Lim, Byung-Lak

2007-10-01

A gene encoding the mannanase of Bacillus subtilis WL-3, which had been isolated from Korean soybean paste, was cloned into Escherichia coli and the nucleotide sequence of a 2.7-kb DNA fragment containing the mannanase gene was subsequently determined. The mannanase gene, designated manA, consisted of 1,080 nucleotides encoding polypeptide of 360 amino acid residues. The deduced amino acid sequence was highly homologous to those of mannanases belonging to glycosyl hydrolase family 26. The manA gene was strongly expressed in B. subtilis 168 by cloning the gene downstream of a strong B. subtilis promoter of plasmid pJ27Delta 88U. In flask cultures, the production of mannanase by recombinant B. subtilis 168 reached maximum levels of 300 units/ml and 450 units/ml in LB medium and LB medium containing 0.3% locust bean gum, respectively. Based on the zymogram of the mannanase, it was found that the mannanase produced by recombinant B. subtilis could be maintained stably without proteolytic degradation during the culture time.

Construction of broad-host-range cosmid cloning vectors: identification of genes necessary for growth of Methylobacterium organophilum on methanol

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

Allen, L.N.; Hanson, R.S.

Four new cloning vectors have been constructed from the broad-host-range cloning vector pRK290. These vectors, pLA2901, pLA2905, pLA2910, and pLA2917, confer resistance to kanamycin and tetracycline. The latter two are cosmid derivatives of pLA2901. The new vectors can be mobilized into, and are stably maintained in, a variety of gram-negative bacteria. A Sau3A genomic bank of Methylobacterium organophilum strain xx DNA has been constructed in pLA2917, and complementation analysis, with a variety of mutants unable to grow on methanol, revealed at least five separate regions necessary for growth on methanol. Complementation analysis and Tn5 mutagenesis data suggest that at leastmore » three genes are responsible for expression of active methanol dehydrogenase.« less

Cloning, expression and purification of a glycosylated form of the DNA-binding protein Dps from Salmonella enterica Typhimurium.

PubMed

Hanna, Ebert Seixas; Roque-Barreira, Maria-Cristina; Mendes, Guilherme Martines Teixeira; Soares, Sandro Gomes; Brocchi, Marcelo

2008-06-01

Dps, found in many eubacterial and archaebacterial species, appears to protect cells from oxidative stress and/or nutrient-limited environment. Dps has been shown to accumulate during the stationary phase, to bind to DNA non-specifically, and to form a crystalline structure that compacts and protects the chromosome. Our previous results have indicated that Dps is glycosylated at least for a certain period of the bacterial cell physiology and this glycosylation is thought to be orchestrated by some factors not yet understood, explaining our difficulties in standardizing the Dps purification process. In the present work, the open reading frame of the dps gene, together with all the upstream regulatory elements, were cloned into a PCR cloning vector. As a result, the expression of dps was also controlled by the plasmid system introduced in the bacterial cell. The gene was then over-expressed regardless of the growth phase of the culture and a glycosylated fraction was purified to homogeneity by lectin-immobilized chromatography assay. Unlike the high level expression of Dps in Salmonella cells, less than 1% of the recombinant protein was purified by affinity chromatography using jacalin column. Sequencing and mass spectrometry data confirmed the identity of the dps gene and the protein, respectively. In spite of the low level of purification of the jacalin-binding Dps, this work shall aid further investigations into the mechanism of Dps glycosylation.

Identification of Abundantly Expressed Novel and Conserved Genes from the Infective Larval Stage of Toxocara canis by an Expressed Sequence Tag Strategy

PubMed Central

Tetteh, Kevin K. A.; Loukas, Alex; Tripp, Cindy; Maizels, Rick M.

1999-01-01

Larvae of Toxocara canis, a nematode parasite of dogs, infect humans, causing visceral and ocular larva migrans. In noncanid hosts, larvae neither grow nor differentiate but endure in a state of arrested development. Reasoning that parasite protein production is orientated to immune evasion, we undertook a random sequencing project from a larval cDNA library to characterize the most highly expressed transcripts. In all, 266 clones were sequenced, most from both 3′ and 5′ ends, and similarity searches against GenBank protein and dbEST nucleotide databases were conducted. Cluster analyses showed that 128 distinct gene products had been found, all but 3 of which represented newly identified genes. Ninety-five genes were represented by a single clone, but seven transcripts were present at high frequencies, each composing >2% of all clones sequenced. These high-abundance transcripts include a mucin and a C-type lectin, which are both major excretory-secretory antigens released by parasites. Four highly expressed novel gene transcripts, termed ant (abundant novel transcript) genes, were found. Together, these four genes comprised 18% of all cDNA clones isolated, but no similar sequences occur in the Caenorhabditis elegans genome. While the coding regions of the four genes are dissimilar, their 3′ untranslated tracts have significant homology in nucleotide sequence. The discovery of these abundant, parasite-specific genes of newly identified lectins and mucins, as well as a range of conserved and novel proteins, provides defined candidates for future analysis of the molecular basis of immune evasion by T. canis. PMID:10456930

Activation of an ATP-dependent K(+) conductance in Xenopus oocytes by expression of adenylate kinase cloned from renal proximal tubules.

PubMed

Brochiero, E; Coady, M J; Klein, H; Laprade, R; Lapointe, J Y

2001-02-09

In rabbit proximal convoluted tubules, an ATP-sensitive K(+) (K(ATP)) channel has been shown to be involved in membrane cross-talk, i.e. the coupling (most likely mediated through intracellular ATP) between transepithelial Na(+) transport and basolateral K(+) conductance. This K(+) conductance is inhibited by taurine. We sought to isolate this K(+) channel by expression cloning in Xenopus oocytes. Injection of renal cortex mRNA into oocytes induced a K(+) conductance, largely inhibited by extracellular Ba(2+) and intracellular taurine. Using this functional test, we isolated from our proximal tubule cDNA library a unique clone, which induced a large K(+) current which was Ba(2+)-, taurine- and glibenclamide-sensitive. Surprisingly, this clone is not a K(+) channel but an adenylate kinase protein (AK3), known to convert NTP+AMP into NDP+ADP (N could be G, I or A). AK3 expression resulted in a large ATP decrease and activation of the whole-cell currents including a previously unknown, endogenous K(+) current. To verify whether ATP decrease was responsible for the current activation, we demonstrated that inhibition of glycolysis greatly reduces oocyte ATP levels and increases an inwardly rectifying K(+) current. The possible involvement of AK in the K(ATP) channel's regulation provides a means of explaining their observed activity in cytosolic environments characterized by high ATP concentrations.

Cloning, sequencing, and expression of interferon-γ from elk in North America

USGS Publications Warehouse

Sweeney, Steven J.; Emerson, Carlene; Eriks, Inge S.

2001-01-01

Eradication of Mycobacterium bovis relies on accurate detection of infected animals, including potential domestic and wildlife reservoirs. Available diagnostic tests lack the sensitivity and specificity necessary for accurate detection, particularly in infected wildlife populations. Recently, an in vitro diagnostic test for cattle which measures plasma interferon-gamma (IFN-γ) levels in blood following in vitro incubation with M. bovis purified protein derivative has been enveloped. This test appears to have increased sensitivity over traditional testing. Unfortunately, it does not detect IFN-γ from Cervidae. To begin to address this problem, the IFN-γ gene from elk (Cervus elaphus) was cloned, sequenced, expressed, and characterized. cDNA was cloned from mitogen stimulated peripheral blood mononuclear cells. The predicted amino acid (aa) sequence was compared to known sequences from cattle, sheep, goats, red deer (Cervus elaphus), humans, and mice. Biological activity of the recombinant elk IFN-γ (rElkIFN-γ) was confirmed in a vesicular stomatitis virus cytopathic effect reduction assay. Production of monoclonal antibodies to IFN-γ epitopes conserved between ruminant species could provide an important tool for the development of reliable, practical diagnostic assays for detection of a delayed type hypersensitivity response to a variety of persistent infectious agents in ruminants, including M. bovis and Brucella abortus. Moreover, development of these reagents will aid investigators in studies to explore immunological responses of elk that are associated with resistance to infectious diseases.

Exploration of Panviral Proteome: High-Throughput Cloning and Functional Implications in Virus-host Interactions

PubMed Central

Yu, Xiaobo; Bian, Xiaofang; Throop, Andrea; Song, Lusheng; Moral, Lerys Del; Park, Jin; Seiler, Catherine; Fiacco, Michael; Steel, Jason; Hunter, Preston; Saul, Justin; Wang, Jie; Qiu, Ji; Pipas, James M.; LaBaer, Joshua

2014-01-01

Throughout the long history of virus-host co-evolution, viruses have developed delicate strategies to facilitate their invasion and replication of their genome, while silencing the host immune responses through various mechanisms. The systematic characterization of viral protein-host interactions would yield invaluable information in the understanding of viral invasion/evasion, diagnosis and therapeutic treatment of a viral infection, and mechanisms of host biology. With more than 2,000 viral genomes sequenced, only a small percent of them are well investigated. The access of these viral open reading frames (ORFs) in a flexible cloning format would greatly facilitate both in vitro and in vivo virus-host interaction studies. However, the overall progress of viral ORF cloning has been slow. To facilitate viral studies, we are releasing the initiation of our panviral proteome collection of 2,035 ORF clones from 830 viral genes in the Gateway® recombinational cloning system. Here, we demonstrate several uses of our viral collection including highly efficient production of viral proteins using human cell-free expression system in vitro, global identification of host targets for rubella virus using Nucleic Acid Programmable Protein Arrays (NAPPA) containing 10,000 unique human proteins, and detection of host serological responses using micro-fluidic multiplexed immunoassays. The studies presented here begin to elucidate host-viral protein interactions with our systemic utilization of viral ORFs, high-throughput cloning, and proteomic technologies. These valuable plasmid resources will be available to the research community to enable continued viral functional studies. PMID:24955142

Molecular cloning of toxins expressed by the venom gland of Lasiodora sp.

PubMed

Vieira, A L G; Moura, M B; Babá, E H; Chávez-Olórtegui, C; Kalapothakis, E; Castro, I M

2004-12-15

The present work describes the identification of toxins expressed by the venom gland of the spider Lasiodora sp. The toxins LTx1, LTx2 and LTx3 were identified by the screening of a cDNA library. These toxins showed significant similarity at the amino acid level with spider toxins from Lasiodora parahybana, Eurypelma californicum, Brachypelma smithii, Selenocosmia huwena.

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

Molecular cloning and expression of the CRISP family of proteins in the boar.

PubMed

Vadnais, Melissa L; Foster, Douglas N; Roberts, Kenneth P

2008-12-01

The family of mammalian cysteine-rich secretory proteins (CRISP) have been well characterized in the rat, mouse, and human. Here we report the molecular cloning and expression analysis of CRISP1, CRISP2, and CRISP3 in the boar. A partial sequence published in the National Center for Biotechnology Information (NCBI) database was used to derive the full-length sequences for CRISP1 and CRISP2 using rapid amplification of cDNA ends. RT-PCR confirmed the expression of these mRNAs in the boar reproductive tract, and real time RT-PCR showed CRISP1 to be highly expressed throughout the epididymis, with CRISP2 highly expressed in the testis. A search of the porcine genomic sequence in the NCBI database identified a BAC (CH242-199E6) encoding the CRISP1 gene. This BAC is derived from porcine Chromosome 7 and is syntenic with the regions of the mouse, rat, and human genomes encoding the CRISP gene family. This BAC was found to encode a third CRISP protein with a predicted amino acid sequence of high similarity to human CRISP3. Using RT-PCR we show that CRISP3 expression in the boar reproductive tract is confined to the prostate. Recombinant porcine (rp) CRISP2 protein was produced and purified. When incubated with capacitated boar sperm, rpCRISP2 induced an acrosome reaction, consistent with its demonstrated ability to alter the activity of calcium channels.

The ura5 gene of the ascomycete Sordaria macrospora: molecular cloning, characterization and expression in Escherichia coli.

PubMed

Le Chevanton, L; Leblon, G

1989-04-15

We cloned the ura5 gene coding for the orotate phosphoribosyl transferase from the ascomycete Sordaria macrospora by heterologous probing of a Sordaria genomic DNA library with the corresponding Podospora anserina sequence. The Sordaria gene was expressed in an Escherichia coli pyrE mutant strain defective for the same enzyme, and expression was shown to be promoted by plasmid sequences. The nucleotide sequence of the 1246-bp DNA fragment encompassing the region of homology with the Podospora gene has been determined. This sequence contains an open reading frame of 699 nucleotides. The deduced amino acid sequence shows 72% similarity with the corresponding Podospora protein.

Cloning and expression of trehalose-6-phosphate synthase 1 from Rhizopus oryzae.

PubMed

Ozer Uyar, Ebru; Yücel, Meral; Hamamcı, Haluk

2016-05-01

Trehalose is a reducing disaccharide acting as a protectant against environmental stresses in many organisms. In fungi, Trehalose-6-phosphate synthase 1 (TPS1) plays a key role in the biosynthesis of trehalose. In this study, a full-length cDNA from Rhizopus oryzae encoding TPS1 (designated as RoTPS1) was isolated. The RoTPS1 cDNA is composed of 2505 nucleotides and encodes a protein of 834 amino acids with a molecular mass of 97.8 kDa. The amino acid sequence of RoTPS1 has a relatively high homology with the TPS1s in several other filamentous fungi. RoTPS1 was cloned into Saccharomyces cerevisiae and secretively expressed. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

[Cloning of new acylamidase gene from Rhodococcus erythropolis and its expression in Escherichia coli].

PubMed

Lavrov, K V; Ianenko, A S

2013-10-01

The gene for new Rhodococcus erythropolis TA37 acylamidase, which possesses unique substrate specificity, has been cloned and expressed in E. coli. Substrates for this enzyme are not only simple amides, such as acetamide and propionamide, but also N-substituted amides, such as 4'-nitroacetanilide. The 1431-bp gene was expressed in E. coli BL21 (DE3) cells on pET16b plasmid under the control of a promoter of the φ 10 gene from the T7 phage. The molecular mass of recombinant acylamidase in E. coli was 55 kDa, which corresponded to that of native acylamidase from Rhodococcus erythropolis TA37. Recombinant acylamidase was able to hydrolize N-substituted amides. A search of a nucleotide database and multiple alignment revealed that acylamidase belonged to the Amidase protein family PF01425, but its nucleotide and amino acid sequences differed significantly from those of the described amidases.

[Cloning and expression of Micrococcus luteus IAM 14879 Rpf and its role in the recovery of the VBNC state in Rhodococcus sp. DS471].

PubMed

Ding, Linxian; Zhang, Pinghua; Hong, Huachang; Lin, Hongjun; Yokota, Akira

2012-01-01

The purpose of the present study was to produce the Rpf (resuscitation promoting factor) protein by cloning and expressing the rpf gene, secreted by Micrococcus luteus IAM 14879, in Escherichia coli and to evaluate its role in the recovery of the VBNC (viable but non-culturable) state in high-GC Gram-positive bacteria. Genomic DNA was extracted from Micrococcus luteus IAM 14879 and the rpf gene was amplified by PCR using specific primers. The PCR products was purified, cloned into a pET15b expression vector, and transformed into Escherichia coli BL21 (DE3). Then the pET15b plasmid expression vector was used to confirm the purification of the recombinant proteins via SDS-PAGE. The VBNC state cells from the high-GC Gram-positive bacteria, Rhodococcus sp. DS471, were used to confirm the promotion and recovery of growth capacity. Rhodococcus sp. DS471 were isolated from soil and closely related to Micrococcus luteus IAM 14879. The gene sequences confirmed that the rpf gene from Micrococcus luteus IAM 14879 that was expressed in Escherichia coli, was 672 bp. SDS-PAGE analysis showed that the recombinant Rpf protein was obtained successfully, and further studies showed it capable of promoting the recovery of the VBNC state by about 100-fold relative to the control. Rpf of Micrococus luteus IAM 14879 can be successfully cloned and expressed in Escherichia coli and shows a strong ability to promote the recovery of the VBNC state of cells of Rhodococcus sp. DS471.

[Clone, construct, expression and verification of lactoferricin B gene and several sequence mutations in yeast].

PubMed

Feng, Yong-qian; Zha, Xiao-jun; Zhai, Chao-yang

2007-07-01

To construct the eucaryotic recombinant plasmid of pYES2/LactoferricinB expressing in yeast of S. cerevisiae, of which the expressed protein antibacterial activity was verified in preliminary. By self-template PCR method, the gene of Lactoferricin B and its several sequence mutations were amplified with the parts of the pre-synthesized single chains. And then Lactoferricin B gene and its mutants were cloned into the vector of pYES2 to construct the recombined expression plasmid pYES2/Lactoferricin B etc. extracted and used to transform the yeast S. cerevisiae. The expressions of proteins were determined after induced by galactose. The expression proteins were collected and purified by hydronium-exchange column, and the bacterial inhibited test was applied to identify the protein antibacterial activities. The PCR amplifying and DNA sequencing tests indicated that the purpose plasmid contained the Lactoferricin B gene and several mutations. The induced target proteins were confirmed by SDS-PAGE electrophoresis and mass spectrum test. The protein antibacterial activities of mutations were verified in preliminary. The recombined plasmid pYES2/Lactoferricin B etc. are successfully constructed and induced to express in yeast cell of S. cerevisiae; the obtained recombined protein of Lactoferricin B provides a basis for further research work on the biological function and antibacterial activity.

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.

Identification of genes differentially expressed in B16 murine melanoma sublines with different metastatic potentials.

PubMed

Ishiguro, T; Nakajima, M; Naito, M; Muto, T; Tsuruo, T

1996-02-15

B16-F10 and B16-BL6 are B16 mouse melanoma sublines that preferentially metastasize to the lung following i.v. and s.c. injections, respectively. To study molecular mechanisms underlying the different metastatic behaviors exhibited by the B16 melanoma sublines, we performed differential hybridization of the genes transcribed in these cells and compared their expression levels. We isolated four genes that were highly expressed in B16-F10 cells but not in B16-BL6 cells: TI-225 (polyubiquitin), TI-229 (pyruvate kinase), TI-241 (LRF-1 homologue), and TI-227 (novel gene). Triosephosphate isomerase, 10-formyltetrahydrofolate dehydrogenase, tyrosinase-related protein 2, cytochrome c oxidase, ATP synthetase alpha subunit, RNA helicase, and ribosomal protein (L37, J1, acidic phosphoprotein), however, showed higher expression in B16-BL6 cells than in B16-F10 cells. Among these clones, transfection of TI-241 into the low metastatic clone F1 converted the parental cells from low- into high-metastatic cells. TI-241 may regulate the expression of various genes as a transcription factor in the complex process of metastasis.

Melibiose permease and alpha-galactosidase of Escherichia coli: Identification by selective labeling using a T7 RNA polymerase/promoter expression system

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

Pourcher, T.; Bassilana, M.; Sarkar, H.K.

1990-01-23

Identification and selective labeling of the melibiose permease and alpha-galactosidase in Escherichia coli, which are encoded by the melB and melA genes, respectively, have been accomplished by selectively labeling the two gene products with a T7 RNA polymerase expression system. Following generation of a novel EcoRI restriction site in the intergenic sequence between the two genes of the mel operon by oligonucleotide-directed, site-specific mutagenesis, melA and melB were separately inserted into plasmid pT7-6 of the T7 expression system. Expression of melB was markedly enhanced by placing a strong, synthetic ribosome binding site at an optimal distance upstream from the initiationmore » codon of melB. Expression of cloned gene products was characterized functionally and by performing autoradiographic analysis on total cell, inner membrane, and cytoplasmic proteins from cells pulse labeled with (35S)methionine in the presence of rifampicin and resolved by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. The results first confirm that alpha-galactosidase is a cytoplasmic protein with an Mr of 50K; in contrast, the membrane-bound melibiose permease is identified as a protein with an apparent Mr of 39K, a value significantly higher than that of 30K previously suggested.« less

Stable MSAP markers for the distinction of Vitis vinifera cv Pinot noir clones.

PubMed

Ocaña, Juan; Walter, Bernard; Schellenbaum, Paul

2013-11-01

Grapevine is one of the most economically important fruit crops. Molecular markers have been used to study grapevine diversity. For instance, simple sequence repeats are a powerful tool for identification of grapevine cultivars, while amplified fragment length polymorphisms have shown their usefulness in intra-varietal diversity studies. Other techniques such as sequence-specific amplified polymorphism are based on the presence of mobile elements in the genome, but their detection lies upon their activity. Relevant attention has been drawn toward epigenetic sources of variation. In this study, a set of Vitis vinifera cv Pinot noir clones were analyzed using the methylation-sensitive amplified polymorphism technique with isoschizomers MspI and HpaII. Nine out of fourteen selective primer combinations were informative and generated two types of polymorphic fragments which were categorized as "stable" and "unstable." In total, 23 stable fragments were detected and they discriminated 92.5 % of the studied clones. Detected stable polymorphisms were either common to several clones, restricted to a few clones or unique to a single clone. The identification of these stable epigenetic markers will be useful in clonal diversity studies. We highlight the relevance of stable epigenetic variation in V. vinifera clones and analyze at which level these markers could be applicable for the development of forthright techniques for clonal distinction.

Cloning and expression of the gene for bacteriophage T7 RNA polymerase

DOEpatents

Studier, F.W.; Davanloo, P.; Rosenberg, A.H.; Moffatt, B.A.; Dunn, J.J.

1997-12-02

This application describes a means to clone a functional gene for bacteriophage T7 RNA polymerase. Active T7 RNA polymerase is produced from the cloned gene, and a plasmid has been constructed that can produce the active enzyme in large amounts. T7 RNA polymerase transcribes DNA very efficiently and is highly selective for a relatively long promoter sequence. This enzyme is useful for synthesizing large amounts of RNA in vivo or in vitro, and is capable of producing a single RNA selectively from a complex mixture of DNAs. The procedure used to obtain a clone of the R7 RNA polymerase gene can be applied to other T7-like phages to obtain clones that produce RNA polymerases having different promoter specificities, different bacterial hosts, or other desirable properties. T7 RNA polymerase is also used in a system for selective, high-level synthesis of RNAs and proteins in suitable host cells. 10 figs.

Cloning and expression of the gene for bacteriophage T7 RNA polymerase

DOEpatents

Studier, F. William; Davanloo, Parichehre; Rosenberg, Alan H.; Moffatt, Barbara A.; Dunn, John J.

1999-02-09

This application describes a means to clone a functional gene for bacteriophage T7 RNA polymerase. Active T7 RNA polymerase is produced from the cloned gene, and a plasmid has been constructed that can produce the active enzyme in large amounts. T7 RNA polymerase transcribes DNA very efficiently and is highly selective for a relatively long promoter sequence. This enzyme is useful for synthesizing large amounts of RNA in vivo or in vitro, and is capable of producing a single RNA selectively from a complex mixture of DNAs. The procedure used to obtain a clone of the R7 RNA polymerase gene can be applied to other T7-like phages to obtain clones that produce RNA polymerases having different promoter specificities, different bacterial hosts, or other desirable properties. T7 RNA polymerase is also used in a system for selective, high-level synthesis of RNAs and proteins in suitable host cells.

Cloning and expression of the gene for bacteriophage T7 RNA polymerase

DOEpatents

Studier, F. William; Davanloo, Parichehre; Rosenberg, Alan H.; Moffatt, Barbara A.; Dunn, John J.

1997-12-02

This application describes a means to clone a functional gene for bacteriophage T7 RNA polymerase. Active T7 RNA polymerase is produced from the cloned gene, and a plasmid has been constructed that can produce the active enzyme in large amounts. T7 RNA polymerase transcribes DNA very efficiently and is highly selective for a relatively long promoter sequence. This enzyme is useful for synthesizing large amounts of RNA in vivo or in vitro, and is capable of producing a single RNA selectively from a complex mixture of DNAs. The procedure used to obtain a clone of the R7 RNA polymerase gene can be applied to other T7-like phages to obtain clones that produce RNA polymerases having different promoter specificities, different bacterial hosts, or other desirable properties. T7 RNA polymerase is also used in a system for selective, high-level synthesis of RNAs and proteins in suitable host cells.

Cloning and expression of the gene for bacteriophage T7 RNA polymerase

DOEpatents

Studier, F. William; Davanloo, Parichehre; Rosenberg, Alan H.; Moffatt, Barbara A.; Dunn, John J.

1990-01-01

This application describes a means to clone a functional gene for bacteriophage T7 RNA polymerase. Active T7 RNA polymerase is produced from the cloned gene, and a plasmid has been constructed that can produce the active enzyme in large amounts. T7 RNA polymerase transcribes DNA very efficiently and is highly selective for a relatively long promoter sequence. This enzyme is useful for synthesizing large amounts of RNA in vivo or in vitro, and is capable of producing a single RNA selectively from a complex mixture of DNAs. The procedure used to obtain a clone of the T7 RNA polymerase gene can be applied to other T7-like phages to obtain clones that produce RNA polymerases having different promoter specificities, different bacterial hosts, or other desirable properties. T7 RNA polymerase is also used in a system for selective, high-level synthesis of RNAs and proteins in suitable host cells.

Cloning and expression of the gene for bacteriophage T7 RNA polymerase

DOEpatents

Studier, F.W.; Davanloo, P.; Rosenberg, A.H.; Moffatt, B.A.; Dunn, J.J.

1999-02-09

This application describes a means to clone a functional gene for bacteriophage T7 RNA polymerase. Active T7 RNA polymerase is produced from the cloned gene, and a plasmid has been constructed that can produce the active enzyme in large amounts. T7 RNA polymerase transcribes DNA very efficiently and is highly selective for a relatively long promoter sequence. This enzyme is useful for synthesizing large amounts of RNA in vivo or in vitro, and is capable of producing a single RNA selectively from a complex mixture of DNAs. The procedure used to obtain a clone of the R7 RNA polymerase gene can be applied to other T7-like phages to obtain clones that produce RNA polymerases having different promoter specificities, different bacterial hosts, or other desirable properties. T7 RNA polymerase is also used in a system for selective, high-level synthesis of RNAs and proteins in suitable host cells. 10 figs.

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

Cloning of the citrate permease gene of Lactococcus lactis subsp. lactis biovar diacetylactis and expression in Escherichia coli.

PubMed Central

Sesma, F; Gardiol, D; de Ruiz Holgado, A P; de Mendoza, D

1990-01-01

The citrate plasmid (Cit+ plasmid) from Lactococcus lactis subsp. lactis biovar diacetylactis was cloned into the EcoRI site of plasmid pUC18. This recombinant plasmid enabled Escherichia coli K-12 to transport and utilize citrate as a source of energy, indicating expression of the citrate permease from L. lactis biovar diacetylactis. The citrate permease was under the control of the lac promoter of pUC18. Genetic expression of the Cit+ plasmid in maxicells revealed that the plasmid encoded two polypeptides of 47 and 32 kilodaltons, determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Images PMID:2117878

Molecular cloning and photoperiod-regulated expression of gibberellin 20-oxidase from the long-day plant spinach.

PubMed

Wu, K; Li, L; Gage, D A; Zeevaart, J A

1996-02-01

Spinach (Spinacia oleracea L.) is a long-day (LD) rosette plant in which stem growth under LD conditions is mediated by gibberellins (GAs). Major control points in spinach are the later steps of sequential oxidation and elimination of C-20 of C20-GAs. Degenerate oligonucleotide primers were used to obtain a polymerase chain reaction product from spinach genomic DNA that has a high homology with GA 20-oxidase cDNAs from Cucurbita maxima L. and Arabidopsis thaliana Heynh. This polymerase chain reaction product was used as a probe to isolate a full-length cDNA clone with an open reading frame encoding a putative 43-kD protein of 374 amino acid residues. When this cDNA clone was expressed in Escherichia coli, the fusion protein catalyzed the biosynthetic sequence GA53-->GA44-->GA19-->GA20 and GA19-->GA17. This establishes that in spinach a single protein catalyzes the oxidation and elimination of C-20. Transfer of spinach plants from short day (SD) to LD conditions caused an increase in the level of all GAs of the early-13-hydroxylation pathway, except GA53, with GA20, GA1, and GA8 showing the largest increases. Northern blot analysis indicated that the level of GA 20-oxidase mRNA was higher in plants in LD than in SD conditions, with highest level of expression in the shoot tips and elongating stems. This expression pattern of GA 20-oxidase is consistent with the different levels of GA20, GA1, and GA8 found in spinach plants grown in SD and LD conditions.

Back to BAC: The Use of Infectious Clone Technologies for Viral Mutagenesis

PubMed Central

Hall, Robyn N.; Meers, Joanne; Fowler, Elizabeth; Mahony, Timothy

2012-01-01

Bacterial artificial chromosome (BAC) vectors were first developed to facilitate the propagation and manipulation of large DNA fragments in molecular biology studies for uses such as genome sequencing projects and genetic disease models. To facilitate these studies, methodologies have been developed to introduce specific mutations that can be directly applied to the mutagenesis of infectious clones (icBAC) using BAC technologies. This has resulted in rapid identification of gene function and expression at unprecedented rates. Here we review the major developments in BAC mutagenesis in vitro. This review summarises the technologies used to construct and introduce mutations into herpesvirus icBAC. It also explores developing technologies likely to provide the next leap in understanding these important viruses. PMID:22470833

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, 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.

Cloning and expression of synthetic genes encoding angiotensin-I converting enzyme (ACE)-inhibitory bioactive peptides in Bifidobacterium pseudocatenulatum.

PubMed

Losurdo, Luca; Quintieri, Laura; Caputo, Leonardo; Gallerani, Raffaele; Mayo, Baltasar; De Leo, Francesca

2013-03-01

A wide range of biopeptides potentially able to lower blood pressure through inhibition of the angiotensin-I converting enzyme (ACE) is produced in fermented foods by proteolytic starter cultures. This work applies a procedure based on recombinant DNA technologies for the synthesis and expression of three ACE-inhibitory peptides using a probiotic cell factory. ACE-inhibitory genes and their pro-active precursors were designed, synthesized by PCR, and cloned in Escherichia coli; after which, they were cloned into the pAM1 E. coli-bifidobacteria shuttle vector. After E. coli transformation, constructs carrying the six recombinant clones were electrotransferred into the Bifidobacterium pseudocatenulatum M115 probiotic strain. Interestingly, five of the six constructs proved to be stable. Their expression was confirmed by reverse transcription PCR. Furthermore, transformed strains displayed ACE-inhibitory activity linearly correlated to increasing amounts of cell-free cellular lysates. In particular, 50 μg of lysates from constructs pAM1-Pro-BP3 and pAM1-BP2 showed a 50% higher ACE-inhibitory activity than that of the controls. As a comparison, addition of 50 ng of Pro-BP1 and Pro-BP3 synthetic peptides to 50 μg of cell-free extracts of B. pseudocatenulatum M115 wild-type strain showed an average of 67% of ACE inhibition; this allowed estimating the amount of the peptides produced by the transformants. Engineering of bifidobacteria for the production of biopeptides is envisioned as a promising cell factory model system. © 2012 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

Characterization of bioactive recombinant human lysozyme expressed in milk of cloned transgenic cattle.

PubMed

Yang, Bin; Wang, Jianwu; Tang, Bo; Liu, Yufang; Guo, Chengdong; Yang, Penghua; Yu, Tian; Li, Rong; Zhao, Jianmin; Zhang, Lei; Dai, Yunping; Li, Ning

2011-03-16

There is great potential for using transgenic technology to improve the quality of cow milk and to produce biopharmaceuticals within the mammary gland. Lysozyme, a bactericidal protein that protects human infants from microbial infections, is highly expressed in human milk but is found in only trace amounts in cow milk. We have produced 17 healthy cloned cattle expressing recombinant human lysozyme using somatic cell nuclear transfer. In this study, we just focus on four transgenic cattle which were natural lactation. The expression level of the recombinant lysozyme was up to 25.96 mg/L, as measured by radioimmunoassay. Purified recombinant human lysozyme showed the same physicochemical properties, such as molecular mass and bacterial lysis, as its natural counterpart. Moreover, both recombinant and natural lysozyme had similar conditions for reactivity as well as for pH and temperature stability during in vitro simulations. The gross composition of transgenic and non-transgenic milk, including levels of lactose, total protein, total fat, and total solids were not found significant differences. Thus, our study not only describes transgenic cattle whose milk offers the similar nutritional benefits as human milk but also reports techniques that could be further refined for production of active human lysozyme on a large scale.

Characterization of Bioactive Recombinant Human Lysozyme Expressed in Milk of Cloned Transgenic Cattle

PubMed Central

Yang, Bin; Wang, Jianwu; Tang, Bo; Liu, Yufang; Guo, Chengdong; Yang, Penghua; Yu, Tian; Li, Rong; Zhao, Jianmin; Zhang, Lei; Dai, Yunping; Li, Ning

2011-01-01

Background There is great potential for using transgenic technology to improve the quality of cow milk and to produce biopharmaceuticals within the mammary gland. Lysozyme, a bactericidal protein that protects human infants from microbial infections, is highly expressed in human milk but is found in only trace amounts in cow milk. Methodology/Principal Findings We have produced 17 healthy cloned cattle expressing recombinant human lysozyme using somatic cell nuclear transfer. In this study, we just focus on four transgenic cattle which were natural lactation. The expression level of the recombinant lysozyme was up to 25.96 mg/L, as measured by radioimmunoassay. Purified recombinant human lysozyme showed the same physicochemical properties, such as molecular mass and bacterial lysis, as its natural counterpart. Moreover, both recombinant and natural lysozyme had similar conditions for reactivity as well as for pH and temperature stability during in vitro simulations. The gross composition of transgenic and non-transgenic milk, including levels of lactose, total protein, total fat, and total solids were not found significant differences. Conclusions/Significance Thus, our study not only describes transgenic cattle whose milk offers the similar nutritional benefits as human milk but also reports techniques that could be further refined for production of active human lysozyme on a large scale. PMID:21436886

Expansion of the Gateway MultiSite Recombination Cloning Toolkit

PubMed Central

Shearin, Harold K.; Dvarishkis, Alisa R.; Kozeluh, Craig D.; Stowers, R. Steven

2013-01-01

Precise manipulation of transgene expression in genetic model organisms has led to advances in understanding fundamental mechanisms of development, physiology, and genetic disease. Transgene construction is, however, a precondition of transgene expression, and often limits the rate of experimental progress. Here we report an expansion of the modular Gateway MultiSite recombination-cloning platform for high efficiency transgene assembly. The expansion includes two additional destination vectors and entry clones for the LexA binary transcription system, among others. These new tools enhance the expression levels possible with Gateway MultiSite generated transgenes and make possible the generation of LexA drivers and reporters with Gateway MultiSite cloning. In vivo data from transgenic Drosophila functionally validating each novel component are presented and include neuronal LexA drivers, LexAop2 red and green fluorescent synaptic vesicle reporters, TDC2 and TRH LexA, GAL4, and QF drivers, and LexAop2, UAS, and QUAS channelrhodopsin2 T159C reporters. PMID:24204935

Molecular cloning and functional expression of allergenic sarcoplasmic calcium-binding proteins from Penaeus shrimps.

PubMed

Mita, Hajime; Koketsu, Aiko; Ishizaki, Shoichiro; Shiomi, Kazuo

2013-05-01

Sarcoplasmic calcium-binding proteins (SCPs) have recently been identified as crustacean allergens. However, information on their primary structures is very limited and no recombinant SCP (rSCP) as an alternative of natural SCP (nSCP) is available. This study was aimed to elucidate primary structures of SCPs from two species of Penaeus shrimp (black tiger shrimp and kuruma shrimp) by cDNA cloning and to produce a black tiger shrimp rSCP preparation that is comparable in IgE reactivity to nSCP. The full-length cDNAs encoding black tiger shrimp and kuruma shrimp SCPs were successfully cloned. Both SCPs are composed of 193 amino acid residues and share more than 80% sequence identity with the known crustacean SCPs. The black tiger shrimp SCP was then expressed in Escherichia coli using the pFN6A (HQ) Flexi vector system. Enzyme-linked immunosorbent assay (ELISA) and inhibition ELISA experiments demonstrated that rSCP has the same IgE reactivity as nSCP. Our results provide further evidence for the high sequence identity among crustacean SCPs. In addition, rSCP will be a useful tool in studying crustacean allergens and also in the diagnosis of crustacean allergy. © 2012 Society of Chemical Industry.

[Prokaryotic expression, purification and antigenicity identification of recombinant human survivin protein].

PubMed

Yin, Xiaotao; Wang, Wei; Tian, Renli; Xu, Yuanji; Yan, Jinqi; Zhang, Wei; Gao, Jiangping; Yu, Jiyun

2013-08-01

To construct a prokaryotic expression plasmid pET28a-survivin, optimize the recombinant protein expression conditions in E.coli, and purify the survivin recombinant protein and identify its antigenicity. Survivin cDNA segment was amplified by PCR and cloned into prokaryotic expression vector pET28a(+) to construct the recombinant expression vector pET28a-survivin. The expression vector was transformed into BL21 (DE3) and the fusion protein survivin/His was induced by IPTG. The fusion protein was purified through Ni affinity chromatography. The antigenicity of the purified survivin protein was identified by Western blotting and ELISA. The recombinant expression vector was verified successfully by BamHI and HindIII. The fusion protein induced by IPTG was obtained with Mr; about 24 000. The purity of the purified protein reached 90% by SDS-PAGE analysis. And the antigenicity of the survivin protein was validated by Western blotting and ELISA. The prokaryotic expression plasmid pET28a-survivin was successfully constructed and the survivin protein was expressed and purified in E.coli. The antigenicity of the purified survivin protein was demonstrated desirable.

Ebbie: automated analysis and storage of small RNA cloning data using a dynamic web server

PubMed Central

Ebhardt, H Alexander; Wiese, Kay C; Unrau, Peter J

2006-01-01

Background DNA sequencing is used ubiquitously: from deciphering genomes[1] to determining the primary sequence of small RNAs (smRNAs) [2-5]. The cloning of smRNAs is currently the most conventional method to determine the actual sequence of these important regulators of gene expression. Typical smRNA cloning projects involve the sequencing of hundreds to thousands of smRNA clones that are delimited at their 5' and 3' ends by fixed sequence regions. These primers result from the biochemical protocol used to isolate and convert the smRNA into clonable PCR products. Recently we completed a smRNA cloning project involving tobacco plants, where analysis was required for ~700 smRNA sequences[6]. Finding no easily accessible research tool to enter and analyze smRNA sequences we developed Ebbie to assist us with our study. Results Ebbie is a semi-automated smRNA cloning data processing algorithm, which initially searches for any substring within a DNA sequencing text file, which is flanked by two constant strings. The substring, also termed smRNA or insert, is stored in a MySQL and BlastN database. These inserts are then compared using BlastN to locally installed databases allowing the rapid comparison of the insert to both the growing smRNA database and to other static sequence databases. Our laboratory used Ebbie to analyze scores of DNA sequencing data originating from an smRNA cloning project[6]. Through its built-in instant analysis of all inserts using BlastN, we were able to quickly identify 33 groups of smRNAs from ~700 database entries. This clustering allowed the easy identification of novel and highly expressed clusters of smRNAs. Ebbie is available under GNU GPL and currently implemented on Conclusion Ebbie was designed for medium sized smRNA cloning projects with about 1,000 database entries [6-8].Ebbie can be used for any type of sequence analysis where two constant primer regions flank a sequence of interest. The reliable storage of inserts, and their

Gene cloning and prokaryotic expression of recombinant flagellin A from Vibrio parahaemolyticus

NASA Astrophysics Data System (ADS)

Yuan, Ye; Wang, Xiuli; Guo, Sheping; Liu, Yang; Ge, Hui; Qiu, Xuemei

2010-11-01

The Gram-negative Vibrio parahaemolyticus is a common pathogen in humans and marine animals. Bacteria flagellins play an important role during infection and induction of the host immune response. Thus, flagellin proteins are an ideal target for vaccines. We amplified the complete flagellin subunit gene ( flaA) from V. parahaemolyticus ATCC 17802. We then cloned and expressed the gene into Escherichia coli BL21 (DE3) cells. The gene coded for a protein that was 62.78 kDa. We purified and characterized the protein using Ni-NTA affinity chromatography and Anti-His antibody Western blotting, respectively. Our results provide a basis for further studies into the utility of the FlaA protein as a vaccine candidate against infection by Vibrio parahaemolyticus. In addition, the purified FlaA protein can be used for further functional and structural studies.

Phenolic Compounds and Expression of 4CL Genes in Silver Birch Clones and Pt4CL1a Lines

PubMed Central

Sutela, Suvi; Hahl, Terhi; Tiimonen, Heidi; Aronen, Tuija; Ylioja, Tiina; Laakso, Tapio; Saranpää, Pekka; Chiang, Vincent; Julkunen-Tiitto, Riitta; Häggman, Hely

2014-01-01

A small multigene family encodes 4-coumarate:CoA ligases (4CLs) catalyzing the CoA ligation of hydroxycinnamic acids, a branch point step directing metabolites to a flavonoid or monolignol pathway. In the present study, we examined the effect of antisense Populus tremuloides 4CL (Pt4CL1) to the lignin and soluble phenolic compound composition of silver birch (Betula pendula) Pt4CL1a lines in comparison with non-transgenic silver birch clones. The endogenous expression of silver birch 4CL genes was recorded in the stems and leaves and also in leaves that were mechanically injured. In one of the transgenic Pt4CL1a lines, the ratio of syringyl (S) and guaiacyl (G) lignin units was increased. Moreover, the transcript levels of putative silver birch 4CL gene (Bp4CL1) were reduced and contents of cinnamic acid derivatives altered. In the other two Pt4CL1a lines changes were detected in the level of individual phenolic compounds. However, considerable variation was found in the transcript levels of silver birch 4CLs as well as in the concentration of phenolic compounds among the transgenic lines and non-transgenic clones. Wounding induced the expression of Bp4CL1 and Bp4CL2 in leaves in all clones and transgenic lines, whereas the transcript levels of Bp4CL3 and Bp4CL4 remained unchanged. Moreover, minor changes were detected in the concentrations of phenolic compounds caused by wounding. As an overall trend the wounding decreased the flavonoid content in silver birches and increased the content of soluble condensed tannins. The results indicate that by reducing the Bp4CL1 transcript levels lignin composition could be modified. However, the alterations found among the Pt4CL1a lines and the non-transgenic clones were within the natural variation of silver birches, as shown in the present study by the clonal differences in the transcripts levels of 4CL genes, soluble phenolic compounds and condensed tannins. PMID:25502441

Cloning and expression of antibacterial goat lactoferricin from Escherichia coli AD494(DE3)pLysS expression system.

PubMed

Chen, Gen-Hung; Yin, Li-Jung; Chiang, I-Hua; Jiang, Shann-Tzong

2008-12-01

Goat lactoferricin (GLfcin), an antibacterial peptide, is released from the N terminus of goat lactoferrin by pepsin digestion. Two GLfcin-related cDNAs, GLfcin L and GLfcin S, encoding Ala20-Ser60 and Ser36-Ser60 of goat lactoferrin, respectively, were cloned into the pET-23a(+) expression vector upstream from (His)6-Tag gene and transformed into Escherichia coli AD494(DE3)pLysS expression host. After being induced by isopropyl-beta-D-thiogalactopyranoside (IPTG), two (His)6-Tag fused recombinant lactoferricins, GLfcin L-His*Tag and GLfcin S-His*Tag, were expressed in soluble form within the E. coli cytoplasm. The GLfcin L-His*Tag and GLfcin S-His*Tag were purified using HisTrap affinity chromatography. According to an antibacterial activity assay using the agar diffusion method, GLfcin L-His*Tag had antibacterial activity against E. coli BCRC 11549, Staphylococcus aureus BCRC 25923, and Propionibacterium acnes BCRC 10723, while GLfcin S-His*Tag was able to inhibit the growth of E. coli BCRC 11549 and P. acnes BCRC 10723. These two recombinant lactoferricins behaved as thermostable peptides, which could retain their activity for up to 30 min of exposure at 100 degrees C.

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 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

Cloning and expression of N22 region of Torque Teno virus (TTV) genome and use of peptide in developing immunoassay for TTV antibodies

PubMed Central

2014-01-01

Background Torque Teno Virus (TTV) is a DNA virus with high rate of prevalence globally. Since its discovery in 1997, several studies have questioned the role of this virus in causing disease. However, it still remains an enigma. Although methods are available for detection of TTV infection, there is still a need for simple, rapid and reliable method for screening of this virus in human population. Present investigation describes the cloning and expression of N22 region of TTV-genome and the use of expressed peptide in development of immunoassay to detect anti-TTV antibodies in serum. Since TTV genotype-1 is more common in India, the serum positive for genotype-1 was used as source of N22 for expression purpose. Methods Full length N22 region of ORF1 from TTV genotype-1 was amplified and cloned in pGEM®-T Easy vector. After cloning, the amplicon was transformed and expressed as a fusion protein containing hexa-histidine tag in pET-28a(+) vector using BL21 E. coli cells as host. Expression was conducted both in LB medium as well as ZYP-5052 auto-induction medium. The expressed peptide was purified using metal-chelate affinity chromatography and used as antigen in developing a blot immunoassay. Results Analysis of translated product by SDS-PAGE and western blotting demonstrated the presence of 25 kDa polypeptide produced after expression. Solubility studies showed the polypeptide to be associated with insoluble fraction. The use of this peptide as antigen in blot assay produced prominent spot on membrane treated with sera from TTV-infected patients. Analysis of sera from 75 patients with liver and renal diseases demonstrated a successful implication of N22 polypeptide based immunoassay in screening sera for anti-TTV antibodies. Comparison of the immunoassay developed using expressed N22 peptide with established PCR method for TTV-DNA detection showed good coherence between TTV-DNA and presence of anti-TTV antibodies in the sera analysed. Conclusions This concludes

Cloning and expression of N-glycosylation-related mannosidase from Glaciozyma antarctica for the production of a mannosynthase

NASA Astrophysics Data System (ADS)

Elangovan, Dharshini; Kamaruddin, Shazilah; Hashim, Noor Haza Fazlin; Bakar, Farah Diba Abu; Murad, Abd. Munir Abd.; Mahadi, Nor Muhammad; Allman, Sarah Ann; Mackeen, Mukram Mohamed

2016-11-01

The controlled synthesis of oligosaccharides is of growing interest due to the important roles of oligosaccharides in various biological processes. Enzymatic synthesis enables regio- and stereo-selective control during synthesis which still remains a challenge using total chemical synthesis. In this study, endoplasmic reticulum 1,2-α-mannosidase from Glaciozyma antractica was recombinantly expressed in Pichia pastoris. The gene sequence for ER mannosidase was obtained from the Glaciozyma antractica database. The BLAST (Basic Local Alignment Search Tool) results from bioinformatics screening showed that ER mannosidase had 41 % identity with the equivalent mannosidases from Sacchromyces cerevesiae. ER mannosidase from G. antartica was then cloned into the pPICZαC expression vector and used to transform in the host Pichia pastoris X33 cells. The ER mannosidase (MW˜58 kDa) was successfully expressed at 25 °C with 1.0 % methanol induction.

Differences in Flower Transcriptome between Grapevine Clones Are Related to Their Cluster Compactness, Fruitfulness, and Berry Size

PubMed Central

Grimplet, Jérôme; Tello, Javier; Laguna, Natalia; Ibáñez, Javier

2017-01-01

Grapevine cluster compactness has a clear impact on fruit quality and health status, as clusters with greater compactness are more susceptible to pests and diseases and ripen more asynchronously. Different parameters related to inflorescence and cluster architecture (length, width, branching, etc.), fruitfulness (number of berries, number of seeds) and berry size (length, width) contribute to the final level of compactness. From a collection of 501 clones of cultivar Garnacha Tinta, two compact and two loose clones with stable differences for cluster compactness-related traits were selected and phenotyped. Key organs and developmental stages were selected for sampling and transcriptomic analyses. Comparison of global gene expression patterns in flowers at the end of bloom allowed identification of potential gene networks with a role in determining the final berry number, berry size and ultimately cluster compactness. A large portion of the differentially expressed genes were found in networks related to cell division (carbohydrates uptake, cell wall metabolism, cell cycle, nucleic acids metabolism, cell division, DNA repair). Their greater expression level in flowers of compact clones indicated that the number of berries and the berry size at ripening appear related to the rate of cell replication in flowers during the early growth stages after pollination. In addition, fluctuations in auxin and gibberellin signaling and transport related gene expression support that they play a central role in fruit set and impact berry number and size. Other hormones, such as ethylene and jasmonate may differentially regulate indirect effects, such as defense mechanisms activation or polyphenols production. This is the first transcriptomic based analysis focused on the discovery of the underlying gene networks involved in grapevine traits of grapevine cluster compactness, berry number and berry size. PMID:28496449

Differences in Flower Transcriptome between Grapevine Clones Are Related to Their Cluster Compactness, Fruitfulness, and Berry Size.

PubMed

Grimplet, Jérôme; Tello, Javier; Laguna, Natalia; Ibáñez, Javier

2017-01-01

Grapevine cluster compactness has a clear impact on fruit quality and health status, as clusters with greater compactness are more susceptible to pests and diseases and ripen more asynchronously. Different parameters related to inflorescence and cluster architecture (length, width, branching, etc.), fruitfulness (number of berries, number of seeds) and berry size (length, width) contribute to the final level of compactness. From a collection of 501 clones of cultivar Garnacha Tinta, two compact and two loose clones with stable differences for cluster compactness-related traits were selected and phenotyped. Key organs and developmental stages were selected for sampling and transcriptomic analyses. Comparison of global gene expression patterns in flowers at the end of bloom allowed identification of potential gene networks with a role in determining the final berry number, berry size and ultimately cluster compactness. A large portion of the differentially expressed genes were found in networks related to cell division (carbohydrates uptake, cell wall metabolism, cell cycle, nucleic acids metabolism, cell division, DNA repair). Their greater expression level in flowers of compact clones indicated that the number of berries and the berry size at ripening appear related to the rate of cell replication in flowers during the early growth stages after pollination. In addition, fluctuations in auxin and gibberellin signaling and transport related gene expression support that they play a central role in fruit set and impact berry number and size. Other hormones, such as ethylene and jasmonate may differentially regulate indirect effects, such as defense mechanisms activation or polyphenols production. This is the first transcriptomic based analysis focused on the discovery of the underlying gene networks involved in grapevine traits of grapevine cluster compactness, berry number and berry size.

The dominant expression of functional human lactoferrin in transgenic cloned goats using a hybrid lactoferrin expression construct.

PubMed

Yu, Huiqing; Chen, Jianquan; Sun, Wei; Liu, Siguo; Zhang, Aimin; Xu, Xujun; Wang, Xuebin; He, Zhuzi; Liu, Guohui; Cheng, Guoxiang

2012-10-31

Human Lactoferrin (hLF) is an iron-binding protein with multiple physiological functions. As the availability of natural hLF is limited, alternative means of producing this biopharmaceutical protein have been extensively studied. Here we report on the dominant expression of recombinant human lactoferrin (rhLF) in transgenic cloned goats using a novel optimised construct made by fusing a 3.3 kb hLF minigene to the regulatory elements of the β-casein gene. The transgenic goat produced more than 30 mg/ml rhLF in its milk, and rhLF expression was stable during the entire lactation cycle. The rhLF purification efficiency from whole goat milk is approximately 70%, and its purity is above 98%. Compared with natural hLF, the rhLF from transgenic goats has similar biological characteristics including molecular mass, N-terminal sequence, isoelectric point, immunoreactivity and digestive stability. More importantly, the purified rhLF showed specific anti-tumour activity in the mouse model of melanoma experimental metastasis. Therefore, our study shows that the large-scale production of functional rhLF in transgenic goat milk could be an economical and promising source of human therapeutic use in the future. Copyright © 2012. Published by Elsevier B.V.

Cloning and Identification of Recombinant Argonaute-Bound Small RNAs Using Next-Generation Sequencing.

PubMed

Gangras, Pooja; Dayeh, Daniel M; Mabin, Justin W; Nakanishi, Kotaro; Singh, Guramrit

2018-01-01

Argonaute proteins (AGOs) are loaded with small RNAs as guides to recognize target mRNAs. Since the target specificity heavily depends on the base complementarity between two strands, it is important to identify small guide and long target RNAs bound to AGOs. For this purpose, next-generation sequencing (NGS) technologies have extended our appreciation truly to the nucleotide level. However, the identification of RNAs via NGS from scarce RNA samples remains a challenge. Further, most commercial and published methods are compatible with either small RNAs or long RNAs, but are not equally applicable to both. Therefore, a single method that yields quantitative, bias-free NGS libraries to identify small and long RNAs from low levels of input will be of wide interest. Here, we introduce such a procedure that is based on several modifications of two published protocols and allows robust, sensitive, and reproducible cloning and sequencing of small amounts of RNAs of variable lengths. The method was applied to the identification of small RNAs bound to a purified eukaryotic AGO. Following ligation of a DNA adapter to RNA 3'-end, the key feature of this method is to use the adapter for priming reverse transcription (RT) wherein biotinylated deoxyribonucleotides specifically incorporated into the extended complementary DNA. Such RT products are enriched on streptavidin beads, circularized while immobilized on beads and directly used for PCR amplification. We provide a stepwise guide to generate RNA-Seq libraries, their purification, quantification, validation, and preparation for next-generation sequencing. We also provide basic steps in post-NGS data analyses using Galaxy, an open-source, web-based platform.

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.

Developmental Competence and Epigenetic Profile of Porcine Embryos Produced by Two Different Cloning Methods.

PubMed

Liu, Ying; Lucas-Hahn, Andrea; Petersen, Bjoern; Li, Rong; Hermann, Doris; Hassel, Petra; Ziegler, Maren; Larsen, Knud; Niemann, Heiner; Callesen, Henrik

2017-06-01

The "Dolly" based cloning (classical nuclear transfer, [CNT]) and the handmade cloning (HMC) are methods that are nowadays routinely used for somatic cloning of large domestic species. Both cloning protocols share several similarities, but differ with regard to the required in vitro culture, which in turn results in different time intervals until embryo transfer. It is not yet known whether the differences between cloned embryos from the two protocols are due to the cloning methods themselves or the in vitro culture, as some studies have shown detrimental effects of in vitro culture on conventionally produced embryos. The goal of this study was to unravel putative differences between two cloning methods, with regard to developmental competence, expression profile of a panel of developmentally important genes and epigenetic profile of porcine cloned embryos produced by either CNT or HMC, either with (D5 or D6) or without (D0) in vitro culture. Embryos cloned by these two methods had a similar morphological appearance on D0, but displayed different cleavage rates and different quality of blastocysts, with HMC embryos showing higher blastocyst rates (HMC vs. CNT: 35% vs. 10%, p < 0.05) and cell numbers per blastocyst (HMC vs. CNT: 31 vs. 23 on D5 and 42 vs. 18 on D6, p < 0.05) compared to CNT embryos. With regard to histone acetylation and gene expression, CNT and HMC derived cloned embryos were similar on D0, but differed on D6. In conclusion, both cloning methods and the in vitro culture may affect porcine embryo development and epigenetic profile. The two cloning methods essentially produce embryos of similar quality on D0 and after 5 days in vitro culture, but thereafter both histone acetylation and gene expression differ between the two types of cloned embryos.

Sequencing and characterization of asclepain f: the first cysteine peptidase cDNA cloned and expressed from Asclepias fruticosa latex.

PubMed

Trejo, Sebastián A; López, Laura M I; Caffini, Néstor O; Natalucci, Claudia L; Canals, Francesc; Avilés, Francesc X

2009-07-01

Asclepain f is a papain-like protease previously isolated and characterized from latex of Asclepias fruticosa. This enzyme is a member of the C1 family of cysteine proteases that are synthesized as preproenzymes. The enzyme belongs to the alpha + beta class of proteins, with two disulfide bridges (Cys22-Cys63 and Cys56-Cys95) in the alpha domain, and another one (Cys150-Cys201) in the beta domain, as was determined by molecular modeling. A full-length 1,152 bp cDNA was cloned by RT-RACE-PCR from latex mRNA. The sequence was predicted as an open reading frame of 340 amino acid residues, of which 16 residues belong to the signal peptide, 113 to the propeptide and 211 to the mature enzyme. The full-length cDNA was ligated to pPICZalpha vector and expressed in Pichia pastoris. Recombinant asclepain f showed endopeptidase activity on pGlu-Phe-Leu-p-nitroanilide and was identified by PMF-MALDI-TOF MS. Asclepain f is the first peptidase cloned and expressed from mRNA isolated from plant latex, confirming the presence of the preprocysteine peptidase in the latex.

[Identification of Env-specific monoclonal antibodies from Chinese HIV-1 infected person by magnetic beads separating B cells and single cell RT-PCR cloning].

PubMed

Huang, Xiang-Ying; Yu, Shuang-Qing; Cheng, Zhan; Ye, Jing-Rong; Xu, Ke; Feng, Xia; Zeng, Yi

2013-04-01

To establish a simple and practical method for screening of Env-specific monoclonal antibodies from HIV-1 infected individuals. Human B cells were purified by negative sorting from PBMCs and memory B cells were further enriched using anti-CD27 microbeads. Gp120 antigen labbled with biotin was incubated with memory B cells to specifically bind IgG on cells membrane. The memory B cells expressing the Env-specific antibody were harvested by magnetic beads separating, counted and diluted to the level of single cell in each PCR well that loading with catch buffer containing RNase inhibitor to get RNAs. The antibody genes were amplified by single cell RT-PCR and nested PCR, cloned into eukaryotic expression vectors and transfected into 293T cells. The binding activity of recombinant antibodies to Env were tested by ELISA. Three monocolonal Env-specific antibodies were isolated from one HIV-1 infected individual. We can obtain Env-specific antibody by biotin labbled antigen, magnetic beads separating technique coupled with single cell RT-PCR and expression cloning.

Molecular cloning, characterization, and expression of Cuc m 2, a major allergen in Cucumis melo

PubMed Central

Sankian, Mojtaba; Mahmoudi, Mahmoud; Varasteh, Abdol-Reza

2013-01-01

Background: Several studies reported the clinical features of IgE-mediated hypersensitivity after ingestion of melon. Melon allergy is a common IgE-mediated fruit allergy in Iran. This prompted us to investigate immunochemical and molecular properties of the major allergen in melon fruit, to compare the IgE-binding capacity of the natural protein with the recombinant allergen, and to determine cross-reactivity of the major allergen with closely-related allergens from other plants displaying clinical cross-reactivity with melon. Methods: Identification and molecular characterization of the major melon allergen were performed using IgE immunoblotting, allergen-specific ELISA, affinity-based purifications, cross-inhibition assays, cloning, and expression of the allergen in Escherichia coli. Results: Melon profilin was identified and isolated as a major IgE-binding component and designated as Cuc m 2. Sequencing corresponding cDNA revealed an open reading frame of 363 bp coding for 131 amino acid residues and two fragments of 171 bp and 383 bps for the 5’and 3’ UTRs, respectively. Significant cross-reactivity was found between melon profilin and Cynodon dactylon, tomato, peach, and grape profilins in cross-inhibition assays. Although the highest degree of amino acid identity was revealed with watermelon profilin, there was no significant cross-reactivity between melon and watermelon profilins. Conclusion: Melon profilin is the major IgE-binding component in melon extract, and the recombinant and natural forms exhibited similar IgE-binding capacities. A part of the fruit-fruit and pollen-fruit cross-reactions could be explained by the presence of this conserved protein; however, sequence homology provides insufficient information to predict IgE cross-reactivity of profilins. PMID:26989709

Grizzly bear corticosteroid binding globulin: Cloning and serum protein expression.

PubMed

Chow, Brian A; Hamilton, Jason; Alsop, Derek; Cattet, Marc R L; Stenhouse, Gordon; Vijayan, Mathilakath M

2010-06-01

Serum corticosteroid levels are routinely measured as markers of stress in wild animals. However, corticosteroid levels rise rapidly in response to the acute stress of capture and restraint for sampling, limiting its use as an indicator of chronic stress. We hypothesized that serum corticosteroid binding globulin (CBG), the primary transport protein for corticosteroids in circulation, may be a better marker of the stress status prior to capture in grizzly bears (Ursus arctos). To test this, a full-length CBG cDNA was cloned and sequenced from grizzly bear testis and polyclonal antibodies were generated for detection of this protein in bear sera. The deduced nucleotide and protein sequences were 1218 bp and 405 amino acids, respectively. Multiple sequence alignments showed that grizzly bear CBG (gbCBG) was 90% and 83% identical to the dog CBG nucleotide and amino acid sequences, respectively. The affinity purified rabbit gbCBG antiserum detected grizzly bear but not human CBG. There were no sex differences in serum total cortisol concentration, while CBG expression was significantly higher in adult females compared to males. Serum cortisol levels were significantly higher in bears captured by leg-hold snare compared to those captured by remote drug delivery from helicopter. However, serum CBG expression between these two groups did not differ significantly. Overall, serum CBG levels may be a better marker of chronic stress, especially because this protein is not modulated by the stress of capture and restraint in grizzly bears. Copyright 2010 Elsevier Inc. All rights reserved.

Cloning of the Pichia anomala SEC61 gene and its expression in a Saccharomyces cerevisiae sec61 mutant.

PubMed

Ruíz, Teresa; De la Rosa, José M; Domínguez, Angel; Rodríguez, Luis

2003-05-01

In several organisms, including Saccharomyces cerevisiae and other yeast species, the product encoded by the SEC61 gene is considered to be the core element of the translocation apparatus within the endoplasmic reticulum membrane through which translocation of secretory and membrane proteins occurs. In this study, we have cloned and characterized the homolog of the SEC61 gene from the yeast Pichia anomala. The cloned gene includes an ORF, interrupted after the first ten nucleotides by an intron of 131 bp, encoding a 479-amino acid putative polypeptide exhibiting homology to the products encoded by different eukaryotic SEC61 genes, particularly to those from other yeast species. We show that the P. anomala SEC61 gene is correctly processed (intron splicing) when expressed in S. cerevisiae and that it is able to complement the thermosensitive phenotype associated with a mutation in the S. cerevisiae SEC61 gene.

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.

Positional cloning in mice and its use for molecular dissection of inflammatory arthritis.

PubMed

Abe, Koichiro; Yu, Philipp

2009-02-01

One of the upcoming next quests in the field of genetics might be molecular dissection of the genetic and environmental components of human complex diseases. In humans, however, there are certain experimental limitations for identification of a single component of the complex interactions by genetic analyses. Experimental animals offer simplified models for genetic and environmental interactions in human complex diseases. In particular, mice are the best mammalian models because of a long history and ample experience for genetic analyses. Forward genetics, which includes genetic screen and subsequent positional cloning of the causative genes, is a powerful strategy to dissect a complex phenomenon without preliminarily molecular knowledge of the process. In this review, first, we describe a general scheme of positional cloning in mice. Next, recent accomplishments on the patho-mechanisms of inflammatory arthritis by forward genetics approaches are introduced; Positional cloning effort for skg, Ali5, Ali18, cmo, and lupo mutants are provided as examples for the application to human complex diseases. As seen in the examples, the identification of genetic factors by positional cloning in the mouse have potential in solving molecular complexity of gene-environment interactions in human complex diseases.

Probabilistic quantum cloning of a subset of linearly dependent states

NASA Astrophysics Data System (ADS)

Rui, Pinshu; Zhang, Wen; Liao, Yanlin; Zhang, Ziyun

2018-02-01

It is well known that a quantum state, secretly chosen from a certain set, can be probabilistically cloned with positive cloning efficiencies if and only if all the states in the set are linearly independent. In this paper, we focus on probabilistic quantum cloning of a subset of linearly dependent states. We show that a linearly-independent subset of linearly-dependent quantum states {| Ψ 1⟩,| Ψ 2⟩,…,| Ψ n ⟩} can be probabilistically cloned if and only if any state in the subset cannot be expressed as a linear superposition of the other states in the set {| Ψ 1⟩,| Ψ 2⟩,…,| Ψ n ⟩}. The optimal cloning efficiencies are also investigated.

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.

Expression of recombinant human α-lactalbumin in milk of transgenic cloned pigs is sufficient to enhance intestinal growth and weight gain of suckling piglets.

PubMed

Ma, Jin; Li, Qiuyan; Li, Yan; Wen, Xiao; Li, Zhiyuan; Zhang, Zaihu; Zhang, Jiuming; Yu, Zhengquan; Li, Ning

2016-06-10

Human α-lactalbumin (HLA) has very high nutritional value and important physiological functions during the neonatal period. The peptides derived from HLA provide diverse health benefits including antimicrobial, antiviral, immune-modulating, and antihypertensive effects. Thus, it is worth investigating the effects on offspring development of increasing HLA in milk. In this study, we found that recombinant human α-lactalbumin (rHLA) exhibits efficient inhibition of dipeptidyl peptidase-IV (DPP-IV) activity in an in vitro simulated gastrointestinal digestion system. Using a BAC clone containing the complete HLA gene as a candidate vector, we generated two lines of transgenic cloned sows via somatic cell nuclear transfer that over-expressed rHLA. The average concentrations of rHLA in milk from the two lines of transgenic cloned sows were 2.24 ± 0.71 mg/ml and 2.67 ± 1.29 mg/ml. The feeding experiments revealed that rHLA represses dipeptidyl peptidase-IV (DPP-IV) activity in vivo. Furthermore, the piglets reared by rHLA transgenic cloned sows exhibit better performance in gain of body weight and intestine growth than the control piglets reared by non-transgenic sows. Therefore, these findings indicate that rHLA could serve as a natural precursor for a DPP-IV inhibitor, and the transgenic technology that produced the over-expression of rHLA could be a useful method for pig breeders to improve lactation performance. Copyright © 2016. Published by Elsevier B.V.

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 ...

What is Cloning?

MedlinePlus

Donate Home Cloning What is Cloning What is Cloning Clones are organisms that are exact genetic copies. ... clones made through modern cloning technologies. How Is Cloning Done? Many people first heard of cloning when ...

Scriptaid and 5-aza-2'deoxycytidine enhanced expression of pluripotent genes and in vitro developmental competence in interspecies Black-footed cat cloned embryos

USGS Publications Warehouse

Gómez, M. C.; Biancardi, M.N.; Jenkins, J.A.; Dumas, C.; Galiguis, J.; Wang, G.; Earle Pope, C.

2012-01-01

Somatic cell nuclear transfer offers the possibility of preserving endangered species including the black-footed cat, which is threatened with extinction. The effectiveness and efficiency of somatic cell nuclear transfer (SCNT) depends on a variety of factors, but 'inappropriate epigenetic reprogramming of the transplanted nucleus is the primary cause of the developmental failure of cloned embryos. Abnormal epigenetic events such as DNA methylation and histone modifications during SCNT perturb the expression of imprinted and pluripotent-related genes that, consequently, may result in foetal and neonatal abnormalities. We have demonstrated that pregnancies can be established after transfer of black-footed cat cloned embryos into domestic cat recipients, but none of the implanted embryos developed to term and the foetal failure has been associated to aberrant reprogramming in cloned embryos. There is growing evidence that modifying the epigenetic pattern of the chromatin template of both donor cells and reconstructed embryos with a combination of inhibitors of histone deacetylases and DNA methyltransferases results in enhanced gene reactivation and improved in vitro and in vivo developmental competence. Epigenetic modifications of the chromatin template of black-footed cat donor cells and reconstructed embryos with epigenetic-modifying compounds enhanced in vitro development, and regulated the expression of pluripotent genes, but these epigenetic modifications did not improve in vivo developmental competence.

Expression from cloned DNA of biologically active glycoprotein C of herpes simplex virus type 1 in mammalian cells.

PubMed

Ghosh-Choudhury, N; Butcher, M; Ghosh, H P

1990-03-01

A DNA fragment of the herpes simplex virus type 1 genome encoding glycoprotein C (gC-1) has been cloned into different eukaryotic expression vectors for transient and stable expression of the glycoprotein in a number of cell lines. All of these expression vectors use a non-HSV promoter, such as the adenovirus major late promoter or murine leukemia virus long terminal repeat promoter to express gC-1 in COS and CHO cells or 3T3 cells. The gC-1 protein synthesized was fully glycosylated with both N- and O-linked oligosaccharides. Synthesis of the mature 120K gC-1 glycoprotein involved partially glycosylated 100K and 105K proteins and the non-glycosylated 70K protein as intermediate molecules. Immunofluorescence studies showed that the expressed gC-1 was localized intracellularly in the nuclear envelope as well as on the cell surface. The expressed gC-1 was biologically active and could act as a receptor for the complement component C3b in the absence of other HSV proteins.

Molecular cloning, expression, and in silico structural analysis of guinea pig IL-17.

PubMed

Dirisala, Vijaya R; Jeevan, Amminikutty; Ramasamy, Suresh K; McMurray, David N

2013-11-01

Interleukin-17A (IL-17A) is a potent proinflammatory cytokine and the signature cytokine of Th17 cells, a subset which is involved in cytokine and chemokine production, neutrophil recruitment, promotion of T cell priming, and antibody production. IL-17 may play an important role in tuberculosis and other infectious diseases. In preparation for investigating its role in the highly relevant guinea pig model of pulmonary tuberculosis, we cloned guinea pig IL-17A for the first time. The complete coding sequence of the guinea pig IL-17A gene (477 nucleotides; 159 amino acids) was subcloned into a prokaryotic expression vector (pET-30a) resulting in the expression of a 17 kDa recombinant guinea pig IL-17A protein which was confirmed by mass spectrometry analysis. Homology modeling of guinea pig IL-17A revealed that the three-dimensional structure resembles that of human IL-17A. The secondary structure predicted for this protein showed the presence of one extra helix in the N-terminal region. The expression profile of IL-17A was analyzed quantitatively in spleen, lymph node, and lung cells from BCG-vaccinated guinea pigs by real-time PCR. The guinea pig IL-17A cDNA and its recombinant protein will serve as valuable tools for molecular and immunological studies in the guinea pig model of pulmonary TB and other human diseases.

Paramyosin from the parasitic mite Sarcoptes scabiei: cDNA cloning and heterologous expression.

PubMed

Mattsson, J G; Ljunggren, E L; Bergström, K

2001-05-01

The burrowing mite Sarcoptes scabiei is the causative agent of the highly contagious disease sarcoptic mange or scabies. So far, there is no in vitro propagation system for S. scabiei available, and mites used for various purposes must be isolated from infected hosts. Lack of parasite-derived material has limited the possibilities to study several aspects of scabies, including pathogenesis and immunity. It has also hampered the development of high performance serological assays. We have now constructed an S. scabiei cDNA expression library with mRNA purified from mites isolated from red foxes. Immunoscreening of the library enabled us to clone a full-length cDNA coding for a 102.5 kDa protein. Sequence similarity searches identified the protein as a paramyosin. Recombinant S. scabiei paramyosin expressed in Escherichia coli was recognized by sera from dogs and swine infected with S. scabiei. We also designed a small paramyosin construct of about 17 kDa that included the N-terminal part, an evolutionary variable part of the helical core, and the C-terminal part of the molecule. The miniaturized protein was efficiently expressed in E. coli and was recognized by sera from immunized rabbits. These data demonstrate that the cDNA library can assist in the isolation of important S. scabiei antigens and that recombinant proteins can be useful for the study of scabies.

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

cDNA cloning, functional expression and antifungal activities of a dimeric plant defensin SPE10 from Pachyrrhizus erosus seeds.

PubMed

Song, Xiaomin; Wang, Jing; Wu, Fang; Li, Xu; Teng, Maikun; Gong, Weimin

2005-01-01

SPE10 is an antifungal protein isolated from the seeds of Pachyrrhizus erosus. cDNA encoding a 47 amino acid peptide was cloned by RT-PCR and the gene sequence proved SPE10 to be a new member of plant defensin family. The synthetic cDNA with codons preferred in yeast was cloned into the pPIC9 plasmid directly in-frame with the secretion signal alpha-mating factor, and highly expressed in methylotrophic Pichia pastoris. Activity assays showed the recombinant SPE10 inhibited specifically the growth of several pathogenic fungi as native SPE10. Circular dichroism and fluorescence spectroscopy analysis indicated that the native and recombinant protein should have same folding, though there are eight cystein residues in the sequence. Several evidence suggested SPE10 should be the first dimeric plant defensin reported so far.

Latrunculin A treatment prevents abnormal chromosome segregation for successful development of cloned embryos.

PubMed

Terashita, Yukari; Yamagata, Kazuo; Tokoro, Mikiko; Itoi, Fumiaki; Wakayama, Sayaka; Li, Chong; Sato, Eimei; Tanemura, Kentaro; Wakayama, Teruhiko

2013-01-01

Somatic cell nuclear transfer to an enucleated oocyte is used for reprogramming somatic cells with the aim of achieving totipotency, but most cloned embryos die in the uterus after transfer. While modifying epigenetic states of cloned embryos can improve their development, the production rate of cloned embryos can also be enhanced by changing other factors. It has already been shown that abnormal chromosome segregation (ACS) is a major cause of the developmental failure of cloned embryos and that Latrunculin A (LatA), an actin polymerization inhibitor, improves F-actin formation and birth rate of cloned embryos. Since F-actin is important for chromosome congression in embryos, here we examined the relation between ACS and F-actin in cloned embryos. Using LatA treatment, the occurrence of ACS decreased significantly whereas cloned embryo-specific epigenetic abnormalities such as dimethylation of histone H3 at lysine 9 (H3K9me2) could not be corrected. In contrast, when H3K9me2 was normalized using the G9a histone methyltransferase inhibitor BIX-01294, the Magea2 gene-essential for normal development but never before expressed in cloned embryos-was expressed. However, this did not increase the cloning success rate. Thus, non-epigenetic factors also play an important role in determining the efficiency of mouse cloning.

Latrunculin A Treatment Prevents Abnormal Chromosome Segregation for Successful Development of Cloned Embryos

PubMed Central

Terashita, Yukari; Yamagata, Kazuo; Tokoro, Mikiko; Itoi, Fumiaki; Wakayama, Sayaka; Li, Chong; Sato, Eimei; Tanemura, Kentaro; Wakayama, Teruhiko

2013-01-01

Somatic cell nuclear transfer to an enucleated oocyte is used for reprogramming somatic cells with the aim of achieving totipotency, but most cloned embryos die in the uterus after transfer. While modifying epigenetic states of cloned embryos can improve their development, the production rate of cloned embryos can also be enhanced by changing other factors. It has already been shown that abnormal chromosome segregation (ACS) is a major cause of the developmental failure of cloned embryos and that Latrunculin A (LatA), an actin polymerization inhibitor, improves F-actin formation and birth rate of cloned embryos. Since F-actin is important for chromosome congression in embryos, here we examined the relation between ACS and F-actin in cloned embryos. Using LatA treatment, the occurrence of ACS decreased significantly whereas cloned embryo-specific epigenetic abnormalities such as dimethylation of histone H3 at lysine 9 (H3K9me2) could not be corrected. In contrast, when H3K9me2 was normalized using the G9a histone methyltransferase inhibitor BIX-01294, the Magea2 gene—essential for normal development but never before expressed in cloned embryos—was expressed. However, this did not increase the cloning success rate. Thus, non-epigenetic factors also play an important role in determining the efficiency of mouse cloning. PMID:24205216

Identification of a DNA sequence motif required for expression of iron-regulated genes in pseudomonads.

PubMed

Rombel, I T; McMorran, B J; Lamont, I L

1995-02-20

Many bacteria respond to a lack of iron in the environment by synthesizing siderophores, which act as iron-scavenging compounds. Fluorescent pseudomonads synthesize strain-specific but chemically related siderophores called pyoverdines or pseudobactins. We have investigated the mechanisms by which iron controls expression of genes involved in pyoverdine metabolism in Pseudomonas aeruginosa. Transcription of these genes is repressed by the presence of iron in the growth medium. Three promoters from these genes were cloned and the activities of the promoters were dependent on the amounts of iron in the growth media. Two of the promoters were sequenced and the transcriptional start site were identified by S1 nuclease analysis. Sequences similar to the consensus binding site for the Fur repressor protein, which controls expression of iron-repressible genes in several gram-negative species, were not present in the promoters, suggesting that they are unlikely to have a high affinity for Fur. However, comparison of the promoter sequences with those of iron-regulated genes from other Pseudomonas species and also the iron-regulated exotoxin gene of P. aeruginosa allowed identification of a shared sequence element, with the consensus sequence (G/C)CTAAAT-CCC, which is likely to act as a binding site for a transcriptional activator protein. Mutations in this sequence greatly reduced the activities of the promoters characterized here as well as those of other iron-regulated promoters. The requirement for this motif in the promoters of iron-regulated genes of different Pseudomonas species indicates that similar mechanisms are likely to be involved in controlling expression of a range of iron-regulated genes in pseudomonads.

Cloning and recombinant expression of a cellulase from the cellulolytic strain Streptomyces sp. G12 isolated from compost

PubMed Central

2012-01-01

��Menten kinetics with a KM of 9.13 mg/ml and a vmax of 3469 μM min-1. The enzyme exhibits a half life of around 24 h and 96 h at 60°C and 50°C, respectively and shows a retention of around 80% of activity after 96 h at 40°C. Conclusions In this manuscript, we describe the isolation of a new cellulolytic strain, Streptomyces sp. G12, from industrial waste based compost, the identification of the enzymes putatively responsible for its cellulolytic activity, the cloning and the recombinant expression of the gene coding for the Streptomyces sp. G12 cellulase CelStrep, that was characterized showing to exhibit a relevant thermoresistance increasing its potential for cellulose conversion. PMID:23267666

Cloning, optimization of induction conditions and purification of Mycobacterium tuberculosis Rv1733c protein expressed in Escherichia coli

PubMed Central

Ashayeri-Panah, Mitra; Eftekhar, Fereshteh; Kazemi, Bahram; Joseph, Joan

2017-01-01

Background and Objectives: Rv1733c is a latency antigen from Mycobacterium tuberculosis, a probable integral-membrane protein with promiscuous T-cell and B-cell epitopes, making it a potential vaccine candidate against tuberculosis. This study aimed to clone and optimize the expression of recombinant Rv1733c in Escherichia coli for purification. Materials and Methods: Chemically synthesized rv1733c coding sequence was cloned in pET-23a(+) followed by transforming E. coli BL21 (DE3) cells. To evaluate the induction conditions for optimized expression, factorial design of experiments was employed using four different media as well as four levels of isopropyl-b-D-thiogalactopyranosid [IPTG] concentration and duration of induction. The recombinant protein was then purified using a His-tag purification kit and detected through western blotting. Results: Recombinant Rv1733c (> 24 kDa) was expressed and accumulated in the cytoplasm of the E. coli cells. Medium composition showed the most significant effect on the yield of the recombinant protein (P = 0.000). The highest yield of recombinant Rv1733c occurred in the presence of 0.4 mM of IPTG in Terrific Broth medium (containing 1.2% tryptone, 2.4% yeast extract, 72 mM K 2 HPO 4 , 17 mM KH 2 PO 4 and 0.4% glycerol) after 10 h at 37°C. Under these conditions, the expression level was around 0.5 g/L of culture medium. Purified Rv1733c was detected by an anti-polyhistidine antibody and a tuberculosis patient’s serum. Systematic optimization of induction conditions gave us high yield of recombinant polyhistidine-tagged Rv1733c in E. coli which was successfuly purified. Conclusion: We believe that the purified Rv1733c recombinant protein from M. tuberculosis might be a good candidate for vaccine production against tuberculosis. PMID:29213997

Cloning, expression, purification, crystallization and preliminary X-ray studies of argininosuccinate lyase (Rv1659) from Mycobacterium tuberculosis

PubMed Central

Paul, A.; Mishra, A.; Surolia, A.; Vijayan, M.

2013-01-01

The last enzyme in the arginine-biosynthesis pathway, argininosuccinate lyase, from Mycobacterium tuberculosis has been cloned, expressed, purified and crystallized, and preliminary X-ray studies have been carried out on the crystals. The His-tagged tetrameric enzyme with a subunit molecular weight of 50.9 kDa crystallized with two tetramers in the asymmetric unit of the orthorhombic unit cell, space group P212121. Molecular-replacement calculations and self-rotation calculations confirmed the space group and the tetrameric nature of the molecule. PMID:24316845

New ligation independent cloning vectors for expression of recombinant proteins with a self-cleaving CPD/6xHis-tag.

PubMed

Biancucci, Marco; Dolores, Jazel S; Wong, Jennifer; Grimshaw, Sarah; Anderson, Wayne F; Satchell, Karla J F; Kwon, Keehwan

2017-01-05

Recombinant protein purification is a crucial step for biochemistry and structural biology fields. Rapid robust purification methods utilize various peptide or protein tags fused to the target protein for affinity purification using corresponding matrices and to enhance solubility. However, affinity/solubility-tags often need to be removed in order to conduct functional and structural studies, adding complexities to purification protocols. In this work, the Vibrio cholerae MARTX toxin Cysteine Protease Domain (CPD) was inserted in a ligation-independent cloning (LIC) vector to create a C-terminal 6xHis-tagged inducible autoprocessing enzyme tag, called "the CPD-tag". The pCPD and alternative pCPD/ccdB cloning vectors allow for easy insertion of DNA and expression of the target protein fused to the CPD-tag, which is removed at the end of the purification step by addition of the inexpensive small molecule inositol hexakisphosphate to induce CPD autoprocessing. This process is demonstrated using a small bacterial membrane localization domain and for high yield purification of the eukaryotic small GTPase KRas. Subsequently, pCPD was tested with 40 proteins or sub-domains selected from a high throughput crystallization pipeline. pCPD vectors are easily used LIC compatible vectors for expression of recombinant proteins with a C-terminal CPD/6xHis-tag. Although intended only as a strategy for rapid tag removal, this pilot study revealed the CPD-tag may also increase expression and solubility of some recombinant proteins.

Molecular cloning, expression and characterization of Pekin duck interferon-λ.

PubMed

Yao, Qingxia; Fischer, Karl P; Arnesen, Karina; Tyrrell, D Lorne; Gutfreund, Klaus S

2014-09-10

Interferons (IFNs) are the first line of defense against viral infections in vertebrates. Type III interferon (IFN-λ) is recognized for its key role in innate immunity of tissues of epithelial origin. Here we describe the identification of the Pekin duck IFN-λ ortholog (duIFN-λ). The predicted duIFN-λ protein has an amino acid identity of 63%, 38%, 37% and 33% with chicken IFN-λ and human IFN-λ3, IFN-λ2 and IFN-λ1, respectively. The duck genome contains a single IFN-λ gene that is comprised of five exons and four introns. Recombinant duIFN-λ up-regulated OASL and Mx-1 mRNA in primary duck hepatocytes. Our observations suggest evolutionary conservation of genomic organization and structural features implicated in receptor binding and antiviral activity. The identification and expression of duIFN-λ will facilitate further study of the role of type III IFN in antiviral defense and inflammatory responses of the Pekin duck, a non-mammalian vertebrate and pathogen host with relevance for human and animal health. Copyright © 2014 Elsevier B.V. All rights reserved.

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.

Identification of a testis-expressed creatine transporter gene at 16p11.2 and confirmation of the X-linked locus to Xq28

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

Iyer, G.S.; Funanage, V.L.; Proujansky, R.

1996-05-15

Creatine and creatine phosphate act as a buffer system for the regeneration of ATP in tissues with fluctuating energy demands. Following reports of the cloning of a creatine transporter in rat, rabbit, and human, we cloned and sequenced a creatine transporter from a human intestinal cDNA library. PCR amplification of genomic DNAs from somatic cell hybrid panels localized two creatine transporter (CT) genes: CT1 to Xq26-q28 and CT2 to 16p11.2. Refinement of CT1 to Xq28 was confirmed by FISH. Identification of CT2 sequences in YACs and cosmid contigs that had been ordered on human chromosome 16 enabled its assignment tomore » the proximal end of 16p11.2. Sequencing of the CT2 gene identified sequence differences between CT1 and CT2 transcripts that were utilized to determine that CT2 is expressed in testis only. CT2 is the most proximally identified gene on chromosome 16p to date. The existence of an autosomal, testis-specific form of the human creatine transporter gene suggests that creatine transporter activity is critical for normal function of spermatazoa following meiosis. 17 refs., 2 figs., 2 tabs.« less

Molecular cloning, structural analysis, and expression of a human IRLB, MYC promoter-binding protein: new DENN domain-containing protein family emerges.

PubMed

Semova, Natalia; Kapanadze, Bagrat; Corcoran, Martin; Kutsenko, Alexei; Baranova, Ancha; Semov, Alexandre

2003-09-01

IRLB was originally identified as a partial cDNA clone, encoding a 191-aa protein binding the interferon-stimulated response element (ISRE) in the P2 promoter of human MYC. Here, we cloned the full-size IRLB using different bioinformatics tools and an RT-PCR approach. The full-size gene encompasses 131 kb within chromosome 15q22 and consists of 32 exons. IRLB is transcribed as a 6.6-kb mRNA encoding a protein of 1865 aa. IRLB is ubiquitously expressed and its expression is regulated in a growth- and cell cycle-dependent manner. In addition to the ISRE-binding domain IRLB contains a tripartite DENN domain, a nuclear localization signal, two PPRs, and a calmodulin-binding domain. The presence of DENN domains predicts possible interactions of IRLB with GTPases from the Rab family or regulation of growth-induced MAPKs. Strongly homologous proteins were identified in all available vertebrate genomes as well as in Caenorhabditis elegans and Drosophila melanogaster. In human and mouse a family of IRLB proteins exists, consisting of at least three members.

Cloning of Plasmodium falciparum by single-cell sorting

PubMed Central

Miao, Jun; Li, Xiaolian; Cui, Liwang

2010-01-01

Malaria parasite cloning is traditionally carried out mainly by using the limiting dilution method, which is laborious, imprecise, and unable to distinguish multiply-infected RBCs. In this study, we used a parasite engineered to express green fluorescent protein (GFP) to evaluate a single-cell sorting method for rapidly cloning Plasmodium falciparum. By dividing a two dimensional scattergram from a cell sorter into 17 gates, we determined the parameters for isolating singly-infected erythrocytes and sorted them into individual cultures. Pre-gating of the engineered parasites for GFP allowed the isolation of almost 100% GFP-positive clones. Compared with the limiting dilution method, the number of parasite clones obtained by single-cell sorting was much higher. Molecular analyses showed that parasite isolates obtained by single-cell sorting were highly homogenous. This highly efficient single-cell sorting method should prove very useful for cloning both P. falciparum laboratory populations from genetic manipulation experiments and clinical samples. PMID:20435038

Cloning of Plasmodium falciparum by single-cell sorting.

PubMed

Miao, Jun; Li, Xiaolian; Cui, Liwang

2010-10-01

Malaria parasite cloning is traditionally carried out mainly by using the limiting dilution method, which is laborious, imprecise, and unable to distinguish multiply-infected RBCs. In this study, we used a parasite engineered to express green fluorescent protein (GFP) to evaluate a single-cell sorting method for rapidly cloning Plasmodium falciparum. By dividing a two-dimensional scattergram from a cell sorter into 17 gates, we determined the parameters for isolating singly-infected erythrocytes and sorted them into individual cultures. Pre-gating of the engineered parasites for GFP allowed the isolation of almost 100% GFP-positive clones. Compared with the limiting dilution method, the number of parasite clones obtained by single-cell sorting was much higher. Molecular analyses showed that parasite isolates obtained by single-cell sorting were highly homogenous. This highly efficient single-cell sorting method should prove very useful for cloning both P. falciparum laboratory populations from genetic manipulation experiments and clinical samples. Copyright 2010 Elsevier Inc. All rights reserved.

Cloning changes the response to obesity of innate immune factors in blood, liver, and adipose tissues in domestic pigs.

PubMed

Rødgaard, Tina; Skovgaard, Kerstin; Stagsted, Jan; Heegaard, Peter M H

2013-06-01

The objective of this study was to evaluate the usefulness of cloned pigs as porcine obesity models reflecting obesity-associated changes in innate immune factor gene expression profiles. Liver and adipose tissue expression of 43 innate immune genes as well as serum concentrations of six immune factors were analyzed in lean and diet-induced obese cloned domestic pigs and compared to normal domestic pigs (obese and lean). The number of genes affected by obesity was lower in cloned animals than in control animals. All genes affected by obesity in adipose tissues of clones were downregulated; both upregulation and downregulation were observed in the controls. Cloning resulted in a less differentiated adipose tissue expression pattern. Finally, the serum concentrations of two acute-phase proteins (APPs), haptoglobin (HP) and orosomucoid (ORM), were increased in obese clones as compared to obese controls as well as lean clones and controls. Generally, the variation in phenotype between individual pigs was not reduced in cloned siblings as compared to normal siblings. Therefore, we conclude that cloning limits both the number of genes responding to obesity as well as the degree of tissue-differentiated gene expression, concomitantly with an increase in APP serum concentrations only seen in cloned, obese pigs. This may suggest that the APP response seen in obese, cloned pigs is a consequence of the characteristic skewed gene response to obesity in cloned pigs, as described in this work. This should be taken into consideration when using cloned animals as models for innate responses to obesity.

Cloning Changes the Response to Obesity of Innate Immune Factors in Blood, Liver, and Adipose Tissues in Domestic Pigs

PubMed Central

Rødgaard, Tina; Skovgaard, Kerstin; Stagsted, Jan

2013-01-01

Abstract The objective of this study was to evaluate the usefulness of cloned pigs as porcine obesity models reflecting obesity-associated changes in innate immune factor gene expression profiles. Liver and adipose tissue expression of 43 innate immune genes as well as serum concentrations of six immune factors were analyzed in lean and diet-induced obese cloned domestic pigs and compared to normal domestic pigs (obese and lean). The number of genes affected by obesity was lower in cloned animals than in control animals. All genes affected by obesity in adipose tissues of clones were downregulated; both upregulation and downregulation were observed in the controls. Cloning resulted in a less differentiated adipose tissue expression pattern. Finally, the serum concentrations of two acute-phase proteins (APPs), haptoglobin (HP) and orosomucoid (ORM), were increased in obese clones as compared to obese controls as well as lean clones and controls. Generally, the variation in phenotype between individual pigs was not reduced in cloned siblings as compared to normal siblings. Therefore, we conclude that cloning limits both the number of genes responding to obesity as well as the degree of tissue-differentiated gene expression, concomitantly with an increase in APP serum concentrations only seen in cloned, obese pigs. This may suggest that the APP response seen in obese, cloned pigs is a consequence of the characteristic skewed gene response to obesity in cloned pigs, as described in this work. This should be taken into consideration when using cloned animals as models for innate responses to obesity. PMID:23668862

Molecular cloning and expression in mammalian cells of ricin B chain

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

Chang, M.

1987-01-01

In these studies, the cDNA encoding the B chain of ricin has been cloned and expressed in monkey kidney COS-M6 cells. The recombinant B chain was detected by labeling the transfected cells with {sup 35}S-methionine and {sup 35}S-cysteine and demonstrating secretion of a protein with a Mr of 30-32,000 which was not present in the medium of mock-transfected COS-M6 cells. This protein was specifically immunoprecipitated by an anti-ricin or anti-B chain antibody. The amount of recombinant B chain secreted by the COS-M6 cells was determined by radioimmunoassay to be 1-10 ng/ml of media. Virtually all the recombinant B chain formedmore » active ricin when mixed with native A chain; it could also bind as effectively as native B chain to the galactose-containing glycoprotein, asialofetuin. These results indicate that the vast majority of recombinant B chains secreted into the medium of the COS-M6 cells retain biological function.« less

Genetic Identification of an Enzymatic Se(VI) Reduction Pathway

NASA Astrophysics Data System (ADS)

Yee, N.; Kobayashi, D. Y.

2006-12-01

Enterobacter cloacae is a biofilm-forming organism that colonizes the subterranean portions of plants. Because of its ability to catalyze the reduction of selenium oxyanions, this bacterium plays an important role in Se(0) biomineralization and Se cycling in soils. Identification of the genes that regulate selenate reductase activity is needed to elucidate the mechanisms employed by this organism to reduce Se(VI). However, the genes in E. cloacae involved in selenium reduction are currently unknown. In this study, transposon mutagenesis and direct cloning techniques were used to identify genetic regions in E. cloacae SLD1a-1 associated with selenate reductase activity. The mini-Tn5 transposon system was used to produce mutants that have lost the ability to reduce selenate. E. cloacae mutants and genomic library clones heterologously expressed in E. coli S17-1 were screened for activity on LB agar supplemented with sodium selenate. The rate of selenate reduction by the clones was measured in liquid minimal media, and the Se(0) minerals formed by the clones were examined using EXAFS, TEM, and XRD. The transposon mutagenesis experiments revealed that mutation of menaquinone biosynthesis genes inhibits selenate reduction. The direct cloning experiments showed that heterologous expression of the global anaerobic regulatory gene fnr from Enterobacter cloacae in the non Se-reducing strain E. coli S17-1 activated selenate reductase activity and the ability to precipitate Se(0) particles. Se(VI) reduction by E. coli S17-1 containing the fnr gene occurred at similar rates as E. cloacae and produced elemental selenium particles with identical morphologies and short range atomic order. These findings indicate that Se(VI) reduction by facultative anaerobes is regulated by anaerobic electron carriers and oxygen sensing transcription factors.

Transcript profiling of Wilms tumors reveals connections to kidney morphogenesis and expression patterns associated with anaplasia.

PubMed

Li, Wenliang; Kessler, Patricia; Williams, Bryan R G

2005-01-13

Anaplasia (unfavorable histology) is associated with therapy resistance and poor prognosis of Wilms tumor, but the molecular basis for this phenotype is unclear. Here, we used a cDNA array with 9240 clones relevant to cancer biology and/or kidney development to examine the expression profiles of 54 Wilms tumors, five normal kidneys and fetal kidney. By linking genes differentially expressed between fetal kidney and Wilms tumors to kidney morphogenesis, we found that genes expressed at a higher level in Wilms tumors tend to be expressed more in uninduced metanephrogenic mesenchyme or blastema than in their differentiated structures. Conversely, genes expressed at a lower level in Wilms tumors tend to be expressed less in uninduced metanephrogenic mesenchyme or blastema. We also identified 97 clones representing 76 Unigenes or unclustered ESTs that clearly separate anaplastic Wilms tumors from tumors with favorable histology. Genes in this set provide insight into the nature of the abnormal nuclear morphology of anaplastic tumors and may facilitate identification of molecular targets to improve their responsiveness to treatment.

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

Interferon-gamma of the giant panda (Ailuropoda melanoleuca): complementary DNA cloning, expression, and phylogenetic analysis.

PubMed

Tao, Yaqiong; Zeng, Bo; Xu, Liu; Yue, Bisong; Yang, Dong; Zou, Fangdong

2010-01-01

Interferon-gamma (IFN-gamma) is the only member of type II IFN and is vital in the regulation of immune and inflammatory responses. Herein we report the cloning, expression, and sequence analysis of IFN-gamma from the giant panda (Ailuropoda melanoleuca). The open reading frame of this gene is 501 base pair in length and encodes a polypeptide consisting of 166 amino acids. All conserved N-linked glycosylation sites and cysteine residues among carnivores were found in the predicted amino acid sequence of the giant panda. Recombinant giant panda IFN-gamma with a V5 epitope and polyhistidine tag was expressed in HEK293 host cells and confirmed by Western blotting. Phylogenetic analysis of mammalian IFN-gamma-coding sequences indicated that the giant panda IFN-gamma was closest to that of carnivores, then to ungulates and dolphin, and shared a distant relationship with mouse and human. These results represent a first step into the study of IFN-gamma in giant panda.

Cloning and heterologous expression of the antibiotic peptide (ABP) genes from Rhizopus oligosporus NBRC 8631.

PubMed

Yamada, Osamu; Sakamoto, Kazutoshi; Tominaga, Mihoko; Nakayama, Tasuku; Koseki, Takuya; Fujita, Akiko; Akita, Osamu

2005-03-01

We carried out protein sequencing of purified Antibiotic Peptide (ABP), and cloned two genes encoding this peptide as abp1 and abp2, from Rhizopus oligosporus NBRC 8631. Both genes contain an almost identical 231-bp segment, with only 3 nucleotide substitutions, encoding a 77 amino acid peptide. The abp gene product comprises a 28 amino acid signal sequence and a 49 amino acid mature peptide. Northern blot analysis showed that at least one of the abp genes is transcribed in R. oligosporus NBRC 8631. A truncated form of abp1 encoding only the mature peptide was fused with the alpha-factor signal peptide and engineered for expression in Pichia pastoris SMD1168H. Culture broth of the recombinant Pichia displayed ABP activity against Bacillus subtilis NBRC 3335 after induction of heterologous gene expression. This result indicates that mature ABP formed the active structure without the aid of other factors from R. oligosporus, and was secreted.

Clone DB: an integrated NCBI resource for clone-associated data

PubMed Central

Schneider, Valerie A.; Chen, Hsiu-Chuan; Clausen, Cliff; Meric, Peter A.; Zhou, Zhigang; Bouk, Nathan; Husain, Nora; Maglott, Donna R.; Church, Deanna M.

2013-01-01

The National Center for Biotechnology Information (NCBI) Clone DB (http://www.ncbi.nlm.nih.gov/clone/) is an integrated resource providing information about and facilitating access to clones, which serve as valuable research reagents in many fields, including genome sequencing and variation analysis. Clone DB represents an expansion and replacement of the former NCBI Clone Registry and has records for genomic and cell-based libraries and clones representing more than 100 different eukaryotic taxa. Records provide details of library construction, associated sequences, map positions and information about resource distribution. Clone DB is indexed in the NCBI Entrez system and can be queried by fields that include organism, clone name, gene name and sequence identifier. Whenever possible, genomic clones are mapped to reference assemblies and their map positions provided in clone records. Clones mapping to specific genomic regions can also be searched for using the NCBI Clone Finder tool, which accepts queries based on sequence coordinates or features such as gene or transcript names. Clone DB makes reports of library, clone and placement data on its FTP site available for download. With Clone DB, users now have available to them a centralized resource that provides them with the tools they will need to make use of these important research reagents. PMID:23193260

Cloning, expression, and sequence analysis of the Bacillus methanolicus C1 methanol dehydrogenase gene.

PubMed Central

de Vries, G E; Arfman, N; Terpstra, P; Dijkhuizen, L

1992-01-01

The gene (mdh) coding for methanol dehydrogenase (MDH) of thermotolerant, methylotroph Bacillus methanolicus C1 has been cloned and sequenced. The deduced amino acid sequence of the mdh gene exhibited similarity to those of five other alcohol dehydrogenase (type III) enzymes, which are distinct from the long-chain zinc-containing (type I) or short-chain zinc-lacking (type II) enzymes. Highly efficient expression of the mdh gene in Escherichia coli was probably driven from its own promoter sequence. After purification of MDH from E. coli, the kinetic and biochemical properties of the enzyme were investigated. The physiological effect of MDH synthesis in E. coli and the role of conserved sequence patterns in type III alcohol dehydrogenases have been analyzed and are discussed. Images PMID:1644761

Optimized paired-sgRNA/Cas9 cloning and expression cassette triggers high-efficiency multiplex genome editing in kiwifruit.

PubMed

Wang, Zupeng; Wang, Shuaibin; Li, Dawei; Zhang, Qiong; Li, Li; Zhong, Caihong; Liu, Yifei; Huang, Hongwen

2018-01-13

Kiwifruit is an important fruit crop; however, technologies for its functional genomic and molecular improvement are limited. The clustered regulatory interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) system has been successfully applied to genetic improvement in many crops, but its editing capability is variable depending on the different combinations of the synthetic guide RNA (sgRNA) and Cas9 protein expression devices. Optimizing conditions for its use within a particular species is therefore needed to achieve highly efficient genome editing. In this study, we developed a new cloning strategy for generating paired-sgRNA/Cas9 vectors containing four sgRNAs targeting the kiwifruit phytoene desaturase gene (AcPDS). Comparing to the previous method of paired-sgRNA cloning, our strategy only requires the synthesis of two gRNA-containing primers which largely reduces the cost. We further compared efficiencies of paired-sgRNA/Cas9 vectors containing different sgRNA expression devices, including both the polycistronic tRNA-sgRNA cassette (PTG) and the traditional CRISPR expression cassette. We found the mutagenesis frequency of the PTG/Cas9 system was 10-fold higher than that of the CRISPR/Cas9 system, coinciding with the relative expressions of sgRNAs in two different expression cassettes. In particular, we identified large chromosomal fragment deletions induced by the paired-sgRNAs of the PTG/Cas9 system. Finally, as expected, we found both systems can successfully induce the albino phenotype of kiwifruit plantlets regenerated from the G418-resistance callus lines. We conclude that the PTG/Cas9 system is a more powerful system than the traditional CRISPR/Cas9 system for kiwifruit genome editing, which provides valuable clues for optimizing CRISPR/Cas9 editing system in other plants. © 2018 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons

Cloning and expression of a small heat and salt tolerant protein (Hsp22) from Chaetomium globosum.

PubMed

Aggarwal, Rashmi; Gupta, Sangeeta; Sharma, Sapna; Banerjee, Sagar; Singh, Priyanka

2012-11-01

The present study reports molecular characterization of small heat shock protein gene in Indian isolates of Chaetomium globosum, C. perlucidum, C. reflexum, C. cochlioides and C. cupreum. Six isolates of C. globosum and other species showed a band of 630bp using specific primers. Amplified cDNA product of C. globosum (Cg 1) cloned and sequenced showed 603bp open reading frame encoding 200 amino-acids. The protein sequence had a molecular mass of 22 kDa and was therefore, named Hsp22. BlastX analysis revealed that the gene codes for a protein homologous to previously characterized Hsp22.4 gene from C. globosum (AAR36902.1, XP 001229241.1) and shared 95% identity in amino acid sequence. It also showed varying degree of similarities with small Hsp protein from Neurospora spp. (60%), Myceliophthora sp. (59%), Glomerella sp. (50%), Hypocrea sp. (52%), and Fusarium spp. (51%). This gene was further cloned into pET28a (+) and transformed E. coli BL21 cells were induced by IPTG, and the expressed protein of 30 kDa was analyzed by SDS-PAGE. The IPTG induced transformants displayed significantly greater resistance to NaCl and Na2CO3 stresses.

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

[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.

oPOSSUM: identification of over-represented transcription factor binding sites in co-expressed genes

PubMed Central

Ho Sui, Shannan J.; Mortimer, James R.; Arenillas, David J.; Brumm, Jochen; Walsh, Christopher J.; Kennedy, Brian P.; Wasserman, Wyeth W.

2005-01-01

Targeted transcript profiling studies can identify sets of co-expressed genes; however, identification of the underlying functional mechanism(s) is a significant challenge. Established methods for the analysis of gene annotations, particularly those based on the Gene Ontology, can identify functional linkages between genes. Similar methods for the identification of over-represented transcription factor binding sites (TFBSs) have been successful in yeast, but extension to human genomics has largely proved ineffective. Creation of a system for the efficient identification of common regulatory mechanisms in a subset of co-expressed human genes promises to break a roadblock in functional genomics research. We have developed an integrated system that searches for evidence of co-regulation by one or more transcription factors (TFs). oPOSSUM combines a pre-computed database of conserved TFBSs in human and mouse promoters with statistical methods for identification of sites over-represented in a set of co-expressed genes. The algorithm successfully identified mediating TFs in control sets of tissue-specific genes and in sets of co-expressed genes from three transcript profiling studies. Simulation studies indicate that oPOSSUM produces few false positives using empirically defined thresholds and can tolerate up to 50% noise in a set of co-expressed genes. PMID:15933209

Cloning, over-expression and purification of Pseudomonas aeruginosa murC encoding uridine diphosphate N-acetylmuramate: L-alanine ligase.

PubMed

El Zoeiby, A; Sanschagrin, F; Lamoureux, J; Darveau, A; Levesque, R C

2000-02-15

We cloned and sequenced the murC gene from Pseudomonas aeruginosa encoding a protein of 53 kDa. Multiple alignments with 20 MurC peptide sequences from different bacteria confirmed the presence of highly conserved regions having sequence identities ranging from 22-97% including conserved motifs for ATP-binding and the active site of the enzyme. Genetic complementation was done in Escherichia coli (murCts) suppressing the lethal phenotype. The murC gene was subcloned into the expression vector pET30a and overexpressed in E. coli BL21(lambdaDE3). Three PCR cloning strategies were used to obtain the three recombinant plasmids for expression of the native MurC, MurC His-tagged at N-terminal and at C-terminal, respectively. MurC His-tagged at C-terminal was chosen for large scale production and protein purification in the soluble form. The purification was done in a single chromatographic step on an affinity nickel column and obtained in mg quantities at 95% homogeneity. MurC protein was used to produce monoclonal antibodies for epitope mapping and for assay development in high throughput screenings. Detailed studies of MurC and other genes of the bacterial cell cycle will provide the reagents and strain constructs for high throughput screening and for design of novel antibacterials.

Isolation of human hexosaminidase. cap alpha. cDNA and expression of. cap alpha. chains in E. coli

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

Wiktorowicz, J.E.; Whitman, J.M.

1986-05-01

Pooled antisera against homogeneous, glutaraldehyde cross-linked hexosaminidase (hex) A was adsorbed with E. coli lysate insolubilized on Sepharose 4B. Aliquots of a human liver lambdagtll cDNA library (50,000-100,000 pfu) were plated on E. coli Y1090. Expression of cloned cDNA, after sufficient plaque growth at 42/sup 0/, was accomplished by induction with isopropylthiogalactoside soaked nitrocellulose filters. Identification of hex cDNA clones was performed by incubation of the filters with purified antisera. Protein A labelled with I-125 was used to develop the reactive plaques. Positive plaques, identified by autoradiography, were picked, replated at a lower density, and rescreened. This was repeated severalmore » more times until all plaques yielded positive signals. Identification of the clones as containing ..cap alpha.. or ..beta.. cDNA was accomplished by replating the purified phage and rescreening the plaques with anti-hex B antiserum preadsorbed with E. coli lysate. According to this protocol several hex ..cap alpha.. clones have been identified. While these clones generate ..beta..-galactosidase: hex ..cap alpha.. fusion proteins, these findings suggest that in the future it may be possible to obtain large quantities of unmodified hex ..cap alpha.. and ..beta.. polypeptides from E. coli for the study of the structural and enzymatic properties of these polypeptides and for diagnostic purposes in the GM2 gangliosidoses.« less

Development of Transgenic Cloned Pig Models of Skin Inflammation by DNA Transposon-Directed Ectopic Expression of Human β1 and α2 Integrin

PubMed Central

Staunstrup, Nicklas Heine; Madsen, Johannes; Primo, Maria Nascimento; Li, Juan; Liu, Ying; Kragh, Peter M.; Li, Rong; Schmidt, Mette; Purup, Stig; Dagnæs-Hansen, Frederik; Svensson, Lars; Petersen, Thomas K.; Callesen, Henrik; Bolund, Lars; Mikkelsen, Jacob Giehm

2012-01-01

Integrins constitute a superfamily of transmembrane signaling receptors that play pivotal roles in cutaneous homeostasis by modulating cell growth and differentiation as well as inflammatory responses in the skin. Subrabasal expression of integrins α2 and/or β1 entails hyperproliferation and aberrant differentiation of keratinocytes and leads to dermal and epidermal influx of activated T-cells. The anatomical and physiological similarities between porcine and human skin make the pig a suitable model for human skin diseases. In efforts to generate a porcine model of cutaneous inflammation, we employed the Sleeping Beauty DNA transposon system for production of transgenic cloned Göttingen minipigs expressing human β1 or α2 integrin under the control of a promoter specific for subrabasal keratinocytes. Using pools of transgenic donor fibroblasts, cloning by somatic cell nuclear transfer was utilized to produce reconstructed embryos that were subsequently transferred to surrogate sows. The resulting pigs were all transgenic and harbored from one to six transgene integrants. Molecular analyses on skin biopsies and cultured keratinocytes showed ectopic expression of the human integrins and localization within the keratinocyte plasma membrane. Markers of perturbed skin homeostasis, including activation of the MAPK pathway, increased expression of the pro-inflammatory cytokine IL-1α, and enhanced expression of the transcription factor c-Fos, were identified in keratinocytes from β1 and α2 integrin-transgenic minipigs, suggesting the induction of a chronic inflammatory phenotype in the skin. Notably, cellular dysregulation obtained by overexpression of either β1 or α2 integrin occurred through different cellular signaling pathways. Our findings mark the creation of the first cloned pig models with molecular markers of skin inflammation. Despite the absence of an overt psoriatic phenotype, these animals may possess increased susceptibility to severe skin damage

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.

Gene cloning and prokaryotic expression of recombinant outer membrane protein from Vibrio parahaemolyticus

NASA Astrophysics Data System (ADS)

Yuan, Ye; Wang, Xiuli; Guo, Sheping; Qiu, Xuemei

2011-06-01

Gram-negative Vibrio parahaemolyticus is a common pathogen in humans and marine animals. The outer membrane protein of bacteria plays an important role in the infection and pathogenicity to the host. Thus, the outer membrane proteins are an ideal target for vaccines. We amplified a complete outer membrane protein gene (ompW) from V. parahaemolyticus ATCC 17802. We then cloned and expressed the gene into Escherichia coli BL21 (DE3) cells. The gene coded for a protein that was 42.78 kDa. We purified the protein using Ni-NTA affinity chromatography and Anti-His antibody Western blotting, respectively. Our results provide a basis for future application of the OmpW protein as a vaccine candidate against infection by V. parahaemolyticus. In addition, the purified OmpW protein can be used for further functional and structural studies.

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, expression, purification, crystallization and preliminary X-ray crystallographic investigations of a unique editing domain from archaebacteria.

PubMed

Dwivedi, Shweta; Kruparani, Shobha P; Sankaranarayanan, Rajan

2004-09-01

Threonyl-tRNA synthetase (ThrRS) faces a crucial double-discrimination problem during the translation of genetic code. Most ThrRSs from the archaeal kingdom possess a unique editing domain that differs from those of eubacteria and eukaryotes. In order to understand the structural basis of the editing mechanism in archaea, the editing module of ThrRS from Pyrococcus abyssi comprising of the first 183 amino-acid residues was cloned, expressed, purified and crystallized. The crystals belong to the trigonal space group P3(1(2))21, with one molecule in the asymmetric unit.

Cloning and expression of a conjugated bile acid hydrolase gene from Lactobacillus plantarum by using a direct plate assay.

PubMed

Christiaens, H; Leer, R J; Pouwels, P H; Verstraete, W

1992-12-01

The conjugated bile acid hydrolase gene from the silage isolate Lactobacillus plantarum 80 was cloned and expressed in Escherichia coli MC1061. For the screening of this hydrolase gene within the gene bank, a direct plate assay developed by Dashkevicz and Feighner (M. P. Dashkevicz and S. D. Feighner, Appl. Environ. Microbiol. 53:331-336, 1989) was adapted to the growth requirements of E. coli. Because of hydrolysis and medium acidification, hydrolase-active colonies were surrounded with big halos of precipitated, free bile acids. This phenomenon was also obtained when the gene was cloned into a multicopy shuttle vector and subsequently reintroduced into the parental Lactobacillus strain. The cbh gene and surrounding regions were characterized by nucleotide sequence analysis. The deduced amino acid sequence was shown to have 52% similarity with a penicillin V amidase from Bacillus sphaericus. Preliminary characterization of the gene product showed that it is a cholylglycine hydrolase (EC 3.5.1.24) with only slight activity against taurine conjugates. The optimum pH was between 4.7 and 5.5. Optimum temperature ranged from 30 to 45 degrees C. Southern blot analysis indicated that the cloned gene has similarity with genomic DNA of bile acid hydrolase-active Lactobacillus spp. of intestinal origin.

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

Molecular cloning and expression analysis of the sucrose transporter gene family from Theobroma cacao L.

PubMed

Li, Fupeng; Wu, Baoduo; Qin, Xiaowei; Yan, Lin; Hao, Chaoyun; Tan, Lehe; Lai, Jianxiong

2014-08-10

In this study, we performed cloning and expression analysis of six putative sucrose transporter genes, designated TcSUT1, TcSUT2, TcSUT3, TcSUT4, TcSUT5 and TcSUT6, from the cacao genotype 'TAS-R8'. The combination of cDNA and genomic DNA sequences revealed that the cacao SUT genes contained exon numbers ranging from 1 to 14. The average molecular mass of all six deduced proteins was approximately 56 kDa (range 52 to 66 kDa). All six proteins were predicted to exhibit typical features of sucrose transporters with 12 trans-membrane spanning domains. Phylogenetic analysis revealed that TcSUT2 and TcSUT4 belonged to Group 2 SUT and Group 4 SUT, respectively, and the other TcSUT proteins were belonging to Group 1 SUT. Real-time PCR was conducted to investigate the expression pattern of each member of the SUT family in cacao. Our experiment showed that TcSUT1 was expressed dominantly in pods and that, TcSUT3 and TcSUT4 were highly expressed in both pods and in bark with phloem. Within pods, TcSUT1 and TcSUT4 were expressed more in the seed coat and seed from the pod enlargement stage to the ripening stage. TcSUT5 expression sharply increased to its highest expression level in the seed coat during the ripening stage. Expression pattern analysis indicated that TcSUT genes may be associated with photoassimilate transport into developing seeds and may, therefore, have an impact on seed production. Copyright © 2014 Elsevier B.V. All rights reserved.

Molecular cloning, expression and purification of L-amino acid oxidase from the Malayan pit viper Calloselasma rhodostoma.

PubMed

Kommoju, Phaneeswara Rao; Macheroux, Peter; Ghisla, Sandro

2007-03-01

A cDNA encoding LAAO from the Malayan pit viper (Calloselasma rhodostoma) was cloned into an expression vector of the methylotropic yeast Pichia pastoris. The LAAO open reading frame was inserted after the alpha-MF-signal sequence. Upon induction soluble and active LAAO is produced and exported into the culture supernatant at a concentration of up to 0.4 mg/L. Recombinant LAAO was purified from this by ion exchange and molecular sieve chromatography to yield apparently homogeneous protein in quantities of approximately 0.25 mg/L growth medium. Expressed LAAO exhibits the same electrophoretic mobility as native LAAO (62 kDa) and exhibits approximately the same extent of glycosylation as authentic LAAO from snake venom. Catalytic properties and substrate specificity of recombinant LAAO are similar to those of native enzyme.

Identification and characterization of a novel fumarase gene by metagenome expression cloning from marine microorganisms

PubMed Central

2010-01-01

Background Fumarase catalyzes the reversible hydration of fumarate to L-malate and is a key enzyme in the tricarboxylic acid (TCA) cycle and in amino acid metabolism. Fumarase is also used for the industrial production of L-malate from the substrate fumarate. Thermostable and high-activity fumarases from organisms that inhabit extreme environments may have great potential in industry, biotechnology, and basic research. The marine environment is highly complex and considered one of the main reservoirs of microbial diversity on the planet. However, most of the microorganisms are inaccessible in nature and are not easily cultivated in the laboratory. Metagenomic approaches provide a powerful tool to isolate and identify enzymes with novel biocatalytic activities for various biotechnological applications. Results A plasmid metagenomic library was constructed from uncultivated marine microorganisms within marine water samples. Through sequence-based screening of the DNA library, a gene encoding a novel fumarase (named FumF) was isolated. Amino acid sequence analysis revealed that the FumF protein shared the greatest homology with Class II fumarate hydratases from Bacteroides sp. 2_1_33B and Parabacteroides distasonis ATCC 8503 (26% identical and 43% similar). The putative fumarase gene was subcloned into pETBlue-2 vector and expressed in E. coli BL21(DE3)pLysS. The recombinant protein was purified to homogeneity. Functional characterization by high performance liquid chromatography confirmed that the recombinant FumF protein catalyzed the hydration of fumarate to form L-malate. The maximum activity for FumF protein occurred at pH 8.5 and 55°C in 5 mM Mg2+. The enzyme showed higher affinity and catalytic efficiency under optimal reaction conditions: Km= 0.48 mM, Vmax = 827 μM/min/mg, and kcat/Km = 1900 mM/s. Conclusions We isolated a novel fumarase gene, fumF, from a sequence-based screen of a plasmid metagenomic library from uncultivated marine microorganisms. The

[Expression and identification of eukaryotic expression vectors of Brucella melitensis lipoprotein OMP19].

PubMed

He, Zuoping; Luo, Peifang; Hu, Feihuan; Weng, Yunceng; Wang, Wenjing; Li, Chengyao

2016-04-01

To construct eukaryotic expression vectors carrying Brucella melitensis outer membrane protein 19 (OMP19), express them in transfected Huh7.5.1 and JEG-3 cells, and analyze their role in cell apoptosis. Brucella melitensis lipidated OMP19 (L-OMP19) gene and unlipidated OMP19 (U-OMP19) gene were amplified by PCR and inserted into the vector pZeroBack/blunt. The correct L-OMP19 and U-OMP19 genes verified by XbaI and BamHI double digestion and sequencing were cloned into the lentivirus expression vector pHAGE-CMV-MCS-IZsGreen to construct vectors pHAGE-L-OMP19 and pHAGE-U-OMP19, which were separately transfected into 293FT cells, Huh7.5.1 and JEG-3 cells. L-OMP19 and U-OMP19 in the cells were detected by Western blotting and immunofluorescence technique. Flow cytometry combined with annexin V-PE/7-AAD staining was used to detect the cell apoptosis. The lentiviral vectors pHAGE-L-OMP19 and pHAGE-U-OMP19 were constructed correctly and the recombinant lipoproteins L-OMP19 and U-OMP19 expressed in the above cells were well recognized by the specific antibodies against L-OMP19 in Western blotting and immunofluorescence technique. L-OMP19 and U-OMP19 induced JEG-3 cell death, but did not induce the apoptosis of Huh7.5.1 cells. The eukaryotic expression vectors of L-OMP19 and U-OMP19 have been constructed successfully. Recombinant lipoproteins L-OMP19 and U-OMP19 expressed in cells have a good antigenicity, which could be used as experimental materials for the research on the relationship between host cells and lipoproteins in Brucella infection.

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.

CLONING AND EXPRESSING TRYPSIN MODULATING OOSTATIC FACTOR IN Chlorella desiccata TO CONTROL MOSQUITO LARVAE.

PubMed

Borovsky, Dov; Sterner, Andeas; Powell, Charles A

2016-01-01

The insect peptide hormone trypsin modulating oostatic factor (TMOF), a decapeptide that is synthesized by the mosquito ovary and controls the translation of the gut's trypsin mRNA was cloned and expressed in the marine alga Chlorella desiccata. To express Aedes aegypti TMOF gene (tmfA) in C. desiccata cells, two plasmids (pYES2/TMOF and pYDB4-tmfA) were engineered with pKYLX71 DNA (5 Kb) carrying the cauliflower mosaic virus (CaMV) promoter 35S(2) and the kanamycin resistant gene (neo), as well as, a 8 Kb nitrate reductase gene (nit) from Chlorella vulgaris. Transforming C. desiccata with pYES2/TMOF and pYDB4-tmfA show that the engineered algal cells express TMOF (20 ± 4 μg ± SEM and 17 ± 3 μg ± SEM, respectively in 3 × 10(8) cells) and feeding the cells to mosquito larvae kill 75 and 60% of Ae. aegypti larvae in 4 days, respectively. Southern and Northern blots analyses show that tmfA integrated into the genome of C. desiccata by homologous recombination using the yeast 2 μ circle of replication and the nit in pYES2/TMOF and pYDB4-tmfA, respectively, and the transformed algal cells express tmfA transcript. Using these algal cells it will be possible in the future to control mosquito larvae in the marsh. © 2015 Wiley Periodicals, Inc.

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 cloning and developmental expression of Tlx (Hox11) genes in zebrafish (Danio rerio).

PubMed

Langenau, D M; Palomero, T; Kanki, J P; Ferrando, A A; Zhou, Y; Zon, L I; Look, A T

2002-09-01

Tlx (Hox11) genes are orphan homeobox genes that play critical roles in the regulation of early developmental processes in vertebrates. Here, we report the identification and expression patterns of three members of the zebrafish Tlx family. These genes share similar, but not identical, expression patterns with other vertebrate Tlx-1 and Tlx-3 genes. Tlx-1 is expressed early in the developing hindbrain and pharyngeal arches, and later in the putative splenic primordium. However, unlike its orthologues, zebrafish Tlx-1 is not expressed in the cranial sensory ganglia or spinal cord. Two homologues of Tlx-3 were identified: Tlx-3a and Tlx-3b, which are both expressed in discrete regions of the developing nervous system, including the cranial sensory ganglia and Rohon-Beard neurons. However, only Tlx-3a is expressed in the statoacoustic cranial ganglia, enteric neurons and non-neural tissues such as the fin bud and pharyngeal arches and Tlx-3b is only expressed in the dorsal root ganglia. Copyright 2002 Elsevier Science Ireland Ltd.

Cloning and expression of 3-deoxy-d-manno-oct-2-ulosonic acid α-ketoside hydrolase from oyster hepatopancreas†

PubMed Central

Nakagawa, Tetsuto; Shimada, Yoshimi; Pavlova, Nadejda V; Li, Su-Chen; Li, Yu-Teh

2015-01-01

We have previously reported that oyster hepatopancreas contained three unusual α-ketoside hydrolases: (i) a 3-deoxy-d-manno-oct-2-ulosonic acid α-ketoside hydrolase (α-Kdo-ase), (ii) a 3-deoxy-d-glycero-d-galacto-non-2-ulosonic acid α-ketoside hydrolase and (iii) a bifunctional ketoside hydrolase capable of cleaving both the α-ketosides of Kdn and Neu5Ac (Kdn-sialidase). After completing the purification of Kdn-sialidase, we proceeded to clone the gene encoding this enzyme. Unexpectedly, we found that instead of expressing Kdn-sialidase, our cloned gene expressed α-Kdo-ase activity. The full-length gene, consisting of 1176-bp (392 amino acids, Mr 44,604), expressed an active recombinant α-Kdo-ase (R-α-Kdo-ase) in yeast and CHO-S cells, but not in various Escherichia coli strains. The deduced amino acid sequence contains two Asp boxes (S277PDDGKTW and S328TDQGKTW) commonly found in sialidases, but is devoid of the signature FRIP-motif of sialidase. The R-α-Kdo-ase effectively hydrolyzed the Kdo in the core-oligosaccharide of the structurally defined lipopolysaccharide (LPS), Re-LPS (Kdo2-Lipid A) from Salmonella minnesota R595 and E. coli D31m4. However, Rd-LPS from S. minnesota R7 that contained an extra outer core phosphorylated heptose was only slowly hydrolyzed. The complex type LPS from Neisseria meningitides A1 and M992 that contained extra 5–6 sugar units at the outer core were refractory to R-α-Kdo-ase. This R-α-Kdo-ase should become useful for studying the structure and function of Kdo-containing glycans. PMID:26362869

Identification, Characterization, and Developmental Expression Pattern of Type III Interferon Receptor Gene in the Chinese Goose

PubMed Central

Zhou, Qin; Chen, Shun; Qi, Yulin; Zhou, Hao; Wang, Mingshu; Jia, Renyong; Zhu, Dekang; Liu, Mafeng; Liu, Fei; Chen, Xiaoyue; Zhou, Xue; Cheng, Anchun

2015-01-01

Interferons, as the first line of defense against the viral infection, play an important role in innate immune responses. Type III interferon (IFN-λ) was a newly identified member of IFN family, which plays IFN-like antiviral activity. Towards a better understanding of the type III interferon system in birds, type III interferon lambda receptor (IFNLR1) was first identified in the Chinese goose. In this paper, we had cloned 1952 bp for goose IFNLR1 (goIFNLR1), including an ORF of 1539 bp, encoding a 512-amino acid protein with a 20 aa predict signal peptide at its N terminal and a 23 aa transmembrane region. The predicted amino acid sequence of goIFNLR1 has 90%, 73%, and 34% identity with duck IFNLR1 (predicted sequence), chicken IFNLR1, and human IFNLR1, respectively. And the age-related tissue distribution of goIFNLR1 was identified by Real Time quantitative PCR (RT-qPCR), we found that the goIFNLR1 has a mainly expression in epithelium-rich tissues similar to other species', such as small intestinal, lung, liver, and stomach. Moreover, a relatively high expression of goIFNLR1 was also observed in the secondary immune tissues (harderian gland and cecal tonsil). The identification and tissue distribution of goIFNLR1 will facilitate further study of the role of IFN-λ in goose antiviral defense. PMID:26064884

Molecular cloning, recombinant expression, and antifungal functional characterization of the lipid transfer protein from Panax ginseng.

PubMed

Cai, Kexin; Wang, Jiawen; Wang, Min; Zhang, Hui; Wang, Siming; Zhao, Yu

2016-07-01

To establish an efficient expression system for a fusion protein GST-pgLTP (Lipid Transfer Protein) and to test its antifungal activity. The nucleotide sequence of LTP gene was obtained from Panax ginseng using RT-PCR. The ORF of the cDNA is 363 bp, codING for a protein OF 120 amino acids with a calculated MW of 12.09 kDa. The pgLTP gene with a His6-tag at the C-terminus was cloned into the pGEX-6p1 vector to generate a GST-fusion pgLTP protein construct that was expressed in Escherichia coli Rosetta. Following purification by Ni-NTA, the fusion protein exhibited antifungal activity against five fungi found in ginseng. The fusion protein GST-pgLTP has activity against a broad spectrum of phytopathogenic fungi, and can potentially be adapted for production to combat fungal diseases that affect P. ginseng.

High-Throughput Protein Expression Using a Combination of Ligation-Independent Cloning (LIC) and Infrared Fluorescent Protein (IFP) Detection

PubMed Central

Dortay, Hakan; Akula, Usha Madhuri; Westphal, Christin; Sittig, Marie; Mueller-Roeber, Bernd

2011-01-01

Protein expression in heterologous hosts for functional studies is a cumbersome effort. Here, we report a superior platform for parallel protein expression in vivo and in vitro. The platform combines highly efficient ligation-independent cloning (LIC) with instantaneous detection of expressed proteins through N- or C-terminal fusions to infrared fluorescent protein (IFP). For each open reading frame, only two PCR fragments are generated (with three PCR primers) and inserted by LIC into ten expression vectors suitable for protein expression in microbial hosts, including Escherichia coli, Kluyveromyces lactis, Pichia pastoris, the protozoon Leishmania tarentolae, and an in vitro transcription/translation system. Accumulation of IFP-fusion proteins is detected by infrared imaging of living cells or crude protein extracts directly after SDS-PAGE without additional processing. We successfully employed the LIC-IFP platform for in vivo and in vitro expression of ten plant and fungal proteins, including transcription factors and enzymes. Using the IFP reporter, we additionally established facile methods for the visualisation of protein-protein interactions and the detection of DNA-transcription factor interactions in microtiter and gel-free format. We conclude that IFP represents an excellent reporter for high-throughput protein expression and analysis, which can be easily extended to numerous other expression hosts using the setup reported here. PMID:21541323

Isolation and bacterial expression of a sesquiterpene synthase CDNA clone from peppermint(mentha .chi. piperita, L.) that produces the aphid alarm pheromone (E)-.beta.-farnesene

DOEpatents

Croteau, Rodney Bruce; Wildung, Mark Raymond; Crock, John E.

1999-01-01

A cDNA encoding (E)-.beta.-farnesene synthase from peppermint (Mentha piperita) has been isolated and sequenced, and the corresponding amino acid sequence has been determined. Accordingly, an isolated DNA sequence (SEQ ID NO:1) is provided which codes for the expression of (E)-.beta.-farnesene synthase (SEQ ID NO:2), from peppermint (Mentha piperita). In other aspects, replicable recombinant cloning vehicles are provided which code for (E)-.beta.-farnesene synthase, or for a base sequence sufficiently complementary to at least a portion of (E)-.beta.-farnesene synthase DNA or RNA to enable hybridization therewith. In yet other aspects, modified host cells are provided that have been transformed, transfected, infected and/or injected with a recombinant cloning vehicle and/or DNA sequence encoding (E)-.beta.-farnesene synthase. Thus, systems and methods are provided for the recombinant expression of the aforementioned recombinant (E)-.beta.-farnesene synthase that may be used to facilitate its production, isolation and purification in significant amounts. Recombinant (E)-.beta.-farnesene synthase may be used to obtain expression or enhanced expression of (E)-.beta.-farnesene synthase in plants in order to enhance the production of (E)-.beta.-farnesene, or may be otherwise employed for the regulation or expression of (E)-.beta.-farnesene synthase, or the production of its product.

A full-length cDNA infectious clone of North American type 1 porcine reproductive and respiratory syndrome virus: expression of green fluorescent protein in the Nsp2 region.

PubMed

Fang, Ying; Rowland, Raymond R R; Roof, Michael; Lunney, Joan K; Christopher-Hennings, Jane; Nelson, Eric A

2006-12-01

The recent emergence of a unique group of North American type 1 porcine reproductive and respiratory syndrome virus (PRRSV) in the United States presents new disease control problems for a swine industry that has already been impacted seriously by North American type 2 PRRSV. In this study, a full-length cDNA infectious clone was generated from a low-virulence North American type 1 PRRSV isolate, SD01-08. In vitro studies demonstrated that the cloned virus maintained growth properties similar to those of the parental virus. Virological, pathological, and immunological observations from animals challenged with cloned viruses were similar to those from animals challenged with the parental virus and a modified live virus vaccine. To further explore the potential use as a viral backbone for expressing foreign genes, the green fluorescent protein (GFP) was inserted into a unique deletion site located at amino acid positions 348 and 349 of the predicted Nsp2 region in the virus, and expression of the Nsp2-GFP fusion protein was visualized by fluorescent microscopy. The availability of this North American type 1 infectious clone provides an important research tool for further study of the basic viral biology and pathogenic mechanisms of this group of type 1 PRRSV in the United States.

A Full-Length cDNA Infectious Clone of North American Type 1 Porcine Reproductive and Respiratory Syndrome Virus: Expression of Green Fluorescent Protein in the Nsp2 Region▿

PubMed Central

Fang, Ying; Rowland, Raymond R. R.; Roof, Michael; Lunney, Joan K.; Christopher-Hennings, Jane; Nelson, Eric A.

2006-01-01

The recent emergence of a unique group of North American type 1 porcine reproductive and respiratory syndrome virus (PRRSV) in the United States presents new disease control problems for a swine industry that has already been impacted seriously by North American type 2 PRRSV. In this study, a full-length cDNA infectious clone was generated from a low-virulence North American type 1 PRRSV isolate, SD01-08. In vitro studies demonstrated that the cloned virus maintained growth properties similar to those of the parental virus. Virological, pathological, and immunological observations from animals challenged with cloned viruses were similar to those from animals challenged with the parental virus and a modified live virus vaccine. To further explore the potential use as a viral backbone for expressing foreign genes, the green fluorescent protein (GFP) was inserted into a unique deletion site located at amino acid positions 348 and 349 of the predicted Nsp2 region in the virus, and expression of the Nsp2-GFP fusion protein was visualized by fluorescent microscopy. The availability of this North American type 1 infectious clone provides an important research tool for further study of the basic viral biology and pathogenic mechanisms of this group of type 1 PRRSV in the United States. PMID:16971421

[Molecular cloning, expression and characterization of lysine decarboxylase gene of endophytic fungus Shiraia sp. Slf14 from Huperzia serrata].

PubMed

Peng, Silu; Yang, Huilin; Zhu, Du; Zhang, Zhibin; Yan, Riming; Wang, Ya

2016-04-14

Huperzine A (HupA) was approved as a drug for the treatment of Alzheimer's disease. The HupA biosynthetic pathway was started from lysine decarboxylase (LDC), which catalyzes lysine to cadaverine. In this study, we cloned and expressed an LDC gene from a HupA-producing endophytic fungus, and tested LDC activities. An endophytic fungus Shiraia sp. Slf14 from Huperzia serrata was used. LDC gene was obtained by RT-PCR, and cloned into pET-22b(+) and pET-32a(+) vectors to construct recombinant plasmids pET- 22b-LDC and pET-32a-LDC. These two recombinant plasmids were transformed into E. coli BL21, cultured for 8 h at 24 °C, 200 r/min with 1×10–3 mol/L IPTG into medium to express the LDC proteins, respectively. LDC proteins were purified by Ni2+ affinity chromatography. Catalytic activities were measured by Thin Layer Chromatography. At last, the physicochemical properties and structures of these two LDCs were obtained by bioinformatics software. LDC and Trx-LDC were expressed in E. coli BL21 successfully. SDS-PAGE analysis shows that the molecular weight of LDC and Trx-LDC were 24.4 kDa and 42.7 kDa respectively, which are consistent with bioinformatics analysis. In addition, TLC analysis reveals that both LDC and Trx-LDC had catalytic abilities. This work can provide fundamental data for enriching LDC molecular information and reveal the HupA biosynthetic pathway in endophytic fungi.

Cloning and functional expression of the small subunit of acetolactate synthase from Nicotiana plumbaginifolia.

PubMed

Hershey, H P; Schwartz, L J; Gale, J P; Abell, L M

1999-07-01

Acetolactate synthase (ALS) is the first committed step of branched-chain amino acid biosynthesis in plants and bacteria. The bacterial holoenzyme has been well characterized and is a tetramer of two identical large subunits (LSUs) of 60 kDa and two identical small subunits (SSUs) ranging in molecular mass from 9 to 17 kDa depending on the isozyme. The enzyme from plants is much less well characterized. Attempts to purify the protein have yielded an enzyme which appears to be an oligomer of LSUs, with the potential existence of a SSU for the plant enzyme remaining a matter of considerable speculation. We report here the discovery of a cDNA clone that encodes a SSU of plant ALS based upon the homology of the encoded peptide with various bacterial ALS SSUs. The plant ALS SSU is more than twice as large as any of its prokaryotic homologues and contains two domains that each encode a full-length copy of the prokaryotic SSU polypeptide. The cDNA clone was used to express Nicotiana plumbaginifolia SSU in Escherichia coli. Mixing a partially purified preparation of this SSU with the LSU of ALS from either N. plumbaginifolia or Arabidopsis thaliana results in both increased specific activity and increased stability of the enzymic activity. These results are consistent with those observed for the bacterial enzyme in similar experiments and represent the first functional demonstration of the existence of a SSU for plant ALS.

Cloning, expression and characterization of a pectate lyase from Paenibacillus sp. 0602 in recombinant Escherichia coli

PubMed Central

2014-01-01

Background Biotechnological applications of microbial pectate lyases (Pels) in plant fiber processing are considered as environmentally friendly. As such, they become promising substitutes for conventional chemical degumming process. Since applications of Pels in various fields are widening, it is necessary to explore new pectolytic microorganisms and enzymes for efficient and effective usage. Here, we describe the cloning, expression, characterization and application of the recombinant Pel protein from a pectolytic bacterium of the genus Paenibacillus in Escherichia coli. Results A Pel gene (pelN) was cloned using degenerate PCR and inverse PCR from the chromosomal DNA of Paenibacillus sp. 0602. The open reading frame of pelN encodes a 30 amino acid signal peptide and a 445 amino acid mature protein belonging to the polysaccharide lyase family 1. The maximum Pel activity produced by E. coli in shake flasks reached 2,467.4 U mL−1, and the purified recombinant enzyme exhibits a specific activity of 2,060 U mg−1 on polygalacturonic acid (PGA). The maximum activity was observed in a buffer with 5 mM Ca2+ at pH 9.8 and 65°C. PelN displays a half-life of around 9 h and 42 h at 50°C and 45°C, respectively. The biochemical treatment achieved the maximal reduction of percentage weight (30.5%) of the ramie bast fiber. Conclusions This work represents the first study that describes the extracellular expression of a Pel gene from Paenibacillus species in E. coli. The high yield of the extracellular overexpression, relevant thermostability and efficient degumming using combined treatments indicate its strong potential for large-scale industrial production. PMID:24612647

Cloning, expression, purification and preliminary crystallographic characterization of a shikimate dehydrogenase from Corynebacterium glutamicum

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

Schoepe, Jan, E-mail: jschoepe@smail.uni-koeln.de; Niefind, Karsten; Chatterjee, Shivani

2006-07-01

The crystallization and preliminary X-ray characterization of a shikimate dehydrogenase from C. glutamicum is presented. The shikimate dehydrogenase from Corynebacterium glutamicum has been cloned into an Escherichia coli expression vector, overexpressed and purified. Native crystals were obtained by the vapour-diffusion technique using 2-methyl-2,4-pentanediol as a precipitant. The crystals belong to the centred monoclinic space group C2, with unit-cell parameters a = 118.77, b = 63.17, c = 35.67 Å, β = 92.26° (at 100 K), and diffract to 1.64 Å on a synchrotron X-ray source. The asymmetric unit is likely to contain one molecule, corresponding to a packing density ofmore » 2.08 Å{sup 3} Da{sup −1} and a solvent content of about 41%.« less

6-phospho-alpha-D-glucosidase from Fusobacterium mortiferum: cloning, expression, and assignment to family 4 of the glycosylhydrolases.

PubMed Central

Bouma, C L; Reizer, J; Reizer, A; Robrish, S A; Thompson, J

1997-01-01

The Fusobacterium mortiferum malH gene, encoding 6-phospho-alpha-glucosidase (maltose 6-phosphate hydrolase; EC 3.2.1.122), has been isolated, characterized, and expressed in Escherichia coli. The relative molecular weight of the polypeptide encoded by malH (441 residues; Mr of 49,718) was in agreement with the estimated value (approximately 49,000) obtained by sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the enzyme purified from F. mortiferum. The N-terminal sequence of the MalH protein obtained by Edman degradation corresponded to the first 32 amino acids deduced from the malH sequence. The enzyme produced by the strain carrying the cloned malH gene cleaved [U-14C]maltose 6-phosphate to glucose 6-phosphate (Glc6P) and glucose. The substrate analogs p-nitrophenyl-alpha-D-glucopyranoside 6-phosphate (pNP alphaGlc6P) and 4-methylumbelliferyl-alpha-D-glucopyranoside 6-phosphate (4MU alphaGlc6P) were hydrolyzed to yield Glc6P and the yellow p-nitrophenolate and fluorescent 4-methylumbelliferyl aglycons, respectively. The 6-phospho-alpha-glucosidase expressed in E. coli (like the enzyme purified from F. mortiferum) required Fe2+, Mn2+, Co2+, or Ni2+ for activity and was inhibited in air. Synthesis of maltose 6-phosphate hydrolase from the cloned malH gene in E. coli was modulated by addition of various sugars to the growth medium. Computer-based analyses of MalH and its homologs revealed that the phospho-alpha-glucosidase from F. mortiferum belongs to the seven-member family 4 of the glycosylhydrolase superfamily. The cloned 2.2-kb Sau3AI DNA fragment from F. mortiferum contained a second partial open reading frame of 83 residues (designated malB) that was located immediately upstream of malH. The high degree of sequence identity of MalB with IIB(Glc)-like proteins of the phosphoenol pyruvate dependent:sugar phosphotransferase system suggests participation of MalB in translocation of maltose and related alpha-glucosides in F. mortiferum. PMID:9209025

Cloning, characterization and functional expression of Taenia solium 17 beta-hydroxysteroid dehydrogenase.

PubMed

Aceves-Ramos, A; de la Torre, P; Hinojosa, L; Ponce, A; García-Villegas, R; Laclette, J P; Bobes, R J; Romano, M C

2014-07-01

The 17β-hydroxysteroid dehydrogenases (17β-HSD) are key enzymes involved in the formation (reduction) and inactivation (oxidation) of sex steroids. Several types have been found in vertebrates including fish, as well as in invertebrates like Caenorhabditis elegans, Ciona intestinalis and Haliotis diversicolor supertexta. To date limited information is available about this enzyme in parasites. We showed previously that Taenia solium cysticerci are able to synthesize sex steroid hormones in vitro when precursors are provided in the culture medium. Here, we identified a T. solium 17β-HSD through in silico blast searches in the T. solium genome database. This coding sequence was amplified by RT-PCR and cloned into the pcDNA 3.1(+) expression vector. The full length cDNA contains 957bp, corresponding to an open reading frame coding for 319 aa. The highest identity (84%) at the protein level was found with the Echinococcus multilocularis 17β-HSD although significant similarities were also found with other invertebrate and vertebrate 17β-HSD sequences. The T. solium Tsol-17βHSD belongs to the short-chain dehydrogenase/reductase (SDR) protein superfamily. HEK293T cells transiently transfected with Tsol17β-HSD induced expression of Tsol17β-HSD that transformed 3H-androstenedione into testosterone. In contrast, 3H-estrone was not significantly transformed into estradiol. In conclusion, T. solium cysticerci express a 17β-HSD that catalyzes the androgen reduction. The enzyme belongs to the short chain dehydrogenases/reductase family and shares motifs and activity with the type 3 enzyme of some other species. Copyright © 2014 Elsevier Inc. All rights reserved.

Cloning, clones and clonal disease.

PubMed

Luzzatto, L

2000-01-01

In the past, cloning has been familiar to plant breeders because many plants can be easily reproduced in this way, bypassing the lengthy process of cross-fertilisation. Recently, the concept of cloning has become popular in human biology and medicine on two accounts. First, individual genes can be cloned from the enormous complexity of the DNA that makes up the human genetic material. It is expected that, within a few years, all the estimated 100,000 human genes will be isolated by this approach. This should make it possible to identify all the genes that determine the individual characteristics of human beings, including those responsible for causing human diseases or for making people more or less susceptible to pick up diseases from the environment. Cloned genes made into pharmaceutical products are already in use for treating a variety of diseases, from hormonal deficiencies to certain types of anaemia.

Walleye dermal sarcoma virus: expression of a full-length clone or the rv-cyclin (orf a) gene is cytopathic to the host and human tumor cells.

PubMed

Xu, Kun; Zhang, Ting Ting; Wang, Ling; Zhang, Cun Fang; Zhang, Long; Ma, Li Xia; Xin, Ying; Ren, Chong Hua; Zhang, Zhi Qiang; Yan, Qiang; Martineau, Daniel; Zhang, Zhi Ying

2013-02-01

Walleye dermal sarcoma virus (WDSV) is etiologically associated with a skin tumor, walleye dermal sarcoma (WDS), which develops in the fall and regresses in the spring. WDSV genome contains, in addition to gag, pol and env, three open reading frames (orfs) designated orf a (rv-cyclin), orf b and orf c. Unintegrated linear WDSV provirus DNA isolated from infected tumor cells was used to construct a full-length WDSV provirus clone pWDSV, while orf a was cloned into pSVK3 to construct the expression vector porfA. Stable co-transfection of a walleye cell line (W12) with pWDSV and pcDNA3 generated fewer and smaller G418-resistant colonies compared to the control. By Northern blot analysis, several small transcripts (2.8, 1.8, 1.2, and 0.8 kb) were detected using a WDSV LTR-specific probe. By RT-PCR and Southern blot analysis, three cDNAs (2.4, 1.6 and 0.8 kb) were identified, including both orf a and orf b messenger. Furthermore stable co-transfection of both a human lung adenocarcinoma cell line (SPC-A-1) and a cervical cancer cell line (HeLa) with pcDNA3 and ether porfA or pWDSV also generated fewer and smaller G418-resistant colonies. We conclude that expression of the full-length WDSV clone or the orf a gene inhibits the host fish and human tumor cell growth, and Orf A protein maybe a potential factor which contributes to the seasonal tumor development and regression. This is the first fish provirus clone that has been expressed in cell culture system, which will provide a new in vitro model for tumor research and oncotherapy study.

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.

Screening Fusion Tags for Improved Recombinant Protein Expression in E. coli with the Expresso® Solubility and Expression Screening System.

PubMed

Steinmetz, Eric J; Auldridge, Michele E

2017-11-01

The simplicity, speed, and low cost of bacterial culture make E. coli the system of choice for most initial trials of recombinant protein expression. However, many heterologous proteins are either poorly expressed in bacteria, or are produced as incorrectly folded, insoluble aggregates that lack the activity of the native protein. In many cases, fusion to a partner protein can allow for improved expression and/or solubility of a difficult target protein. Although several different fusion partners have gained favor, none are universally effective, and identifying the one that best improves soluble expression of a given target protein is an empirical process. This unit presents a strategy for parallel screening of fusion partners for enhanced expression or solubility. The Expresso® Solubility and Expression Screening System includes a panel of seven distinct fusion partners and utilizes an extremely simple cloning strategy to enable rapid screening and identification of the most effective fusion partner. © 2017 by John Wiley & Sons, Inc. Copyright © 2017 John Wiley & Sons, Inc.

Single-cell analysis of HIV-1 transcriptional activity reveals expression of proviruses in expanded clones during ART.

PubMed

Wiegand, Ann; Spindler, Jonathan; Hong, Feiyu F; Shao, Wei; Cyktor, Joshua C; Cillo, Anthony R; Halvas, Elias K; Coffin, John M; Mellors, John W; Kearney, Mary F

2017-05-02

Little is known about the fraction of human immunodeficiency virus type 1 (HIV-1) proviruses that express unspliced viral RNA in vivo or about the levels of HIV RNA expression within single infected cells. We developed a sensitive cell-associated HIV RNA and DNA single-genome sequencing (CARD-SGS) method to investigate fractional proviral expression of HIV RNA (1.3-kb fragment of p6, protease, and reverse transcriptase) and the levels of HIV RNA in single HIV-infected cells from blood samples obtained from individuals with viremia or individuals on long-term suppressive antiretroviral therapy (ART). Spiking experiments show that the CARD-SGS method can detect a single cell expressing HIV RNA. Applying CARD-SGS to blood mononuclear cells in six samples from four HIV-infected donors (one with viremia and not on ART and three with viremia suppressed on ART) revealed that an average of 7% of proviruses (range: 2-18%) expressed HIV RNA. Levels of expression varied from one to 62 HIV RNA molecules per cell (median of 1). CARD-SGS also revealed the frequent expression of identical HIV RNA sequences across multiple single cells and across multiple time points in donors on suppressive ART consistent with constitutive expression of HIV RNA in infected cell clones. Defective proviruses were found to express HIV RNA at levels similar to those proviruses that had no obvious defects. CARD-SGS is a useful tool to characterize fractional proviral expression in single infected cells that persist despite ART and to assess the impact of experimental interventions on proviral populations and their expression.

A Novel mRNA Level Subtraction Method for Quick Identification of Target-Orientated Uniquely Expressed Genes Between Peanut Immature Pod and Leaf

PubMed Central

2010-01-01

Subtraction technique has been broadly applied for target gene discovery. However, most current protocols apply relative differential subtraction and result in great amount clone mixtures of unique and differentially expressed genes. This makes it more difficult to identify unique or target-orientated expressed genes. In this study, we developed a novel method for subtraction at mRNA level by integrating magnetic particle technology into driver preparation and tester–driver hybridization to facilitate uniquely expressed gene discovery between peanut immature pod and leaf through a single round subtraction. The resulting target clones were further validated through polymerase chain reaction screening using peanut immature pod and leaf cDNA libraries as templates. This study has resulted in identifying several genes expressed uniquely in immature peanut pod. These target genes can be used for future peanut functional genome and genetic engineering research. PMID:21406066

Cloning, sequencing, and expression of the Zymomonas mobilis phosphoglycerate mutase gene (pgm) in Escherichia coli.

PubMed Central

Yomano, L P; Scopes, R K; Ingram, L O

1993-01-01

Phosphoglycerate mutase is an essential glycolytic enzyme for Zymomonas mobilis, catalyzing the reversible interconversion of 3-phosphoglycerate and 2-phosphoglycerate. The pgm gene encoding this enzyme was cloned on a 5.2-kbp DNA fragment and expressed in Escherichia coli. Recombinants were identified by using antibodies directed against purified Z. mobilis phosphoglycerate mutase. The pgm gene contains a canonical ribosome-binding site, a biased pattern of codon usage, a long upstream untranslated region, and four promoters which share sequence homology. Interestingly, adhA and a D-specific 2-hydroxyacid dehydrogenase were found on the same DNA fragment and appear to form a cluster of genes which function in central metabolism. The translated sequence for Z. mobilis pgm was in full agreement with the 40 N-terminal amino acid residues determined by protein sequencing. The primary structure of the translated sequence is highly conserved (52 to 60% identity with other phosphoglycerate mutases) and also shares extensive homology with bisphosphoglycerate mutases (51 to 59% identity). Since Southern blots indicated the presence of only a single copy of pgm in the Z. mobilis chromosome, it is likely that the cloned pgm gene functions to provide both activities. Z. mobilis phosphoglycerate mutase is unusual in that it lacks the flexible tail and lysines at the carboxy terminus which are present in the enzyme isolated from all other organisms examined. Images PMID:8320209

Heritable gene expression differences between apomictic clone members in Taraxacum officinale: Insights into early stages of evolutionary divergence in asexual plants.

PubMed

Ferreira de Carvalho, Julie; Oplaat, Carla; Pappas, Nikolaos; Derks, Martijn; de Ridder, Dick; Verhoeven, Koen J F

2016-03-08

Asexual reproduction has the potential to enhance deleterious mutation accumulation and to constrain adaptive evolution. One source of mutations that can be especially relevant in recent asexuals is activity of transposable elements (TEs), which may have experienced selection for high transposition rates in sexual ancestor populations. Predictions of genomic divergence under asexual reproduction therefore likely include a large contribution of transposable elements but limited adaptive divergence. For plants empirical insight into genome divergence under asexual reproduction remains limited. Here, we characterize expression divergence between clone members of a single apomictic lineage of the common dandelion (Taraxacum officinale) to contribute to our knowledge of genome evolution under asexuality. Using RNA-Seq, we show that about one third of heritable divergence within the apomictic lineage is driven by TEs and TE-related gene activity. In addition, we identify non-random transcriptional differences in pathways related to acyl-lipid and abscisic acid metabolisms which might reflect functional divergence within the apomictic lineage. We analyze SNPs in the transcriptome to assess genetic divergence between the apomictic clone members and reveal that heritable expression differences between the accessions are not explained simply by genome-wide genetic divergence. The present study depicts a first effort towards a more complete understanding of apomictic plant genome evolution. We identify abundant TE activity and ecologically relevant functional genes and pathways affecting heritable within-lineage expression divergence. These findings offer valuable resources for future work looking at epigenetic silencing and Cis-regulation of gene expression with particular emphasis on the effects of TE activity on asexual species' genome.

Identification of immunogenic polypeptides from a Mycoplasma hyopneumoniae genome library by phage display.

PubMed

Kügler, Jonas; Nieswandt, Simone; Gerlach, Gerald F; Meens, Jochen; Schirrmann, Thomas; Hust, Michael

2008-09-01

The identification of immunogenic polypeptides of pathogens is helpful for the development of diagnostic assays and therapeutic applications like vaccines. Routinely, these proteins are identified by two-dimensional polyacrylamide gel electrophoresis and Western blot using convalescent serum, followed by mass spectrometry. This technology, however, is limited, because low or differentially expressed proteins, e.g. dependent on pathogen-host interaction, cannot be identified. In this work, we developed and improved a M13 genomic phage display-based method for the selection of immunogenic polypeptides of Mycoplasma hyopneumoniae, a pathogen causing porcine enzootic pneumonia. The fragmented genome of M. hyopneumoniae was cloned into a phage display vector, and the genomic library was packaged using the helperphage Hyperphage to enrich open reading frames (ORFs). Afterwards, the phage display library was screened by panning using convalescent serum. The analysis of individual phage clones resulted in the identification of five genes encoding immunogenic proteins, only two of which had been previously identified and described as immunogenic. This M13 genomic phage display, directly combining ORF enrichment and the presentation of the corresponding polypeptide on the phage surface, complements proteome-based methods for the identification of immunogenic polypeptides and is particularly well suited for the use in mycoplasma species.

Production in stirred-tank bioreactor of recombinant bovine chymosin B by a high-level expression transformant clone of Pichia pastoris.

PubMed

Noseda, Diego Gabriel; Recúpero, Matías; Blasco, Martín; Bozzo, Joaquín; Galvagno, Miguel Ángel

2016-07-01

An intense screening of Pichia pastoris clones transformed with the gene of bovine chymosin under methanol-inducible AOX1 promoter was performed, obtaining a transformant clone with a higher milk-clotting activity value in comparison with our previous studies. The scaling of recombinant-chymosin production was carried out by a fed-batch strategy in a stirred-tank bioreactor using biodiesel-byproduct crude glycerol as the carbon source and pure methanol for the induction of chymosin expression, achieving a biomass concentration of 158 g DCW/L and a maximum coagulant activity of 192 IMCU/ml after 120 h of methanol induction. Recombinant bovine chymosin was purified from bioreactor-fermentation culture by a procedure including anion-exchange chromatography which allowed obtaining heterologous chymosin with high level of purity and activity; suggesting that this downstream step could be scaled up in a successful manner for chymosin purification. Thermoestability assay permitted to establish that unformulated recombinant chymosin could be stored at 5 °C without decrease of enzyme activity throughout at least 120 days. Finally, reiterative methanol-inductions of recombinant chymosin expression in bioreactor demonstrated that the reutilization of cell biomass overcame the low enzyme productivity usually reached by P. pastoris system. Copyright © 2016 Elsevier Inc. All rights reserved.

Cloning and Expression of Synthetic Genes Encoding the Broad Antimicrobial Spectrum Bacteriocins SRCAM 602, OR-7, E-760, and L-1077, by Recombinant Pichia pastoris

PubMed Central

Jiménez, Juan J.; Gútiez, Loreto; Cintas, Luis M.; Herranz, Carmen; Hernández, Pablo E.

2015-01-01

We have evaluated the cloning and functional expression of previously described broad antimicrobial spectrum bacteriocins SRCAM 602, OR-7, E-760, and L-1077, by recombinant Pichia pastoris. Synthetic genes, matching the codon usage of P. pastoris, were designed from the known mature amino acid sequence of these bacteriocins and cloned into the protein expression vector pPICZαA. The recombinant derived plasmids were linearized and transformed into competent P. pastoris X-33, and the presence of integrated plasmids into the transformed cells was confirmed by PCR and sequencing of the inserts. The antimicrobial activity, expected in supernatants of the recombinant P. pastoris producers, was purified using a multistep chromatographic procedure including ammonium sulfate precipitation, desalting by gel filtration, cation exchange-, hydrophobic interaction-, and reverse phase-chromatography (RP-FPLC). However, a measurable antimicrobial activity was only detected after the hydrophobic interaction and RP-FPLC steps of the purified supernatants. MALDI-TOF MS analysis of the antimicrobial fractions eluted from RP-FPLC revealed the existence of peptide fragments of lower and higher molecular mass than expected. MALDI-TOF/TOF MS analysis of selected peptides from eluted RP-FPLC samples with antimicrobial activity indicated the presence of peptide fragments not related to the amino acid sequence of the cloned bacteriocins. PMID:25821820

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

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.

Cloning, expression, crystallization and preliminary X-ray analysis of the XMT and DXMT N-methyltransferases from Coffea canephora (robusta)

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

McCarthy, Andrew A., E-mail: andrewmc@embl.fr; Biget, Laurent; Lin, Chenwei

2007-04-01

The genes encoding XMT and DXMT, the enzymes from Coffea canephora (robusta) that catalyse the three independent N-methyl transfer reactions in the caffeine-biosynthesis pathway, have been cloned and the proteins have been expressed in Escherichia coli. Both proteins have been crystallized in the presence of the demethylated cofactor S-adenosyl-l-cysteine (SAH) and substrate (xanthosine for XMT and theobromine for DXMT). Caffeine is a secondary metabolite produced by a variety of plants including Coffea canephora (robusta) and there is growing evidence that caffeine is part of a chemical defence strategy protecting young leaves and seeds from potential predators. The genes encoding XMTmore » and DXMT, the enzymes from Coffea canephora (robusta) that catalyse the three independent N-methyl transfer reactions in the caffeine-biosynthesis pathway, have been cloned and the proteins have been expressed in Escherichia coli. Both proteins have been crystallized in the presence of the demethylated cofactor S-adenosyl-l-cysteine (SAH) and substrate (xanthosine for XMT and theobromine for DXMT). The crystals are orthorhombic, with space group P2{sub 1}2{sub 1}2{sub 1} for XMT and C222{sub 1} for DXMT. X-ray diffraction to 2.8 Å for XMT and to 2.5 Å for DXMT have been collected on beamline ID23-1 at the ESRF.« less

Cloning and expression of colonization factor antigen I (CFA/I) epitopes of enterotoxigenic Escherichia coli (ETEC) in Salmonella flagellin.

PubMed

Luna, M G; Martins, M M; Newton, S M; Costa, S O; Almeida, D F; Ferreira, L C

1997-01-01

Oligonucleotides coding for linear epitopes of the fimbrial colonization factor antigen I (CFA/I) of enterotoxigenic Escherichia coli (ETEC) were cloned and expressed in a deleted form of the Salmonella muenchen flagellin fliC (H1-d) gene. Four synthetic oligonucleotide pairs coding for regions corresponding to amino acids 1 to 15 (region I), amino acids 11 to 25 (region II), amino acids 32 to 45 (region III) and amino acids 88 to 102 (region IV) were synthesized and cloned in the Salmonella flagellin-coding gene. All four hybrid flagellins were exported to the bacterial surface where they produced flagella, but only three constructs were fully motile. Sera recovered from mice immunized with intraperitoneal injections of purified flagella containing region II (FlaII) or region IV (FlaIV) showed high titres against dissociated solid-phase-bound CFA/I subunits. Hybrid flagellins containing region I (FlaI) or region III (FlaIII) elicited a weak immune response as measured in enzyme-linked immunosorbent assay (ELISA) with dissociated CFA/I subunits. None of the sera prepared with purified hybrid flagella were able to agglutinate or inhibit haemagglutination promoted by CFA/I-positive strains. Moreover, inhibition ELISA tests indicated that antisera directed against region I, II, III or IV cloned in flagellin were not able to recognize surface-exposed regions on the intact CFA/I fimbriae.

Biological properties of Beet soil-borne mosaic virus and Beet necrotic yellow vein virus cDNA clones produced by isothermal in vitro recombination: Insights for reassortant appearance.

PubMed

Laufer, Marlene; Mohammad, Hamza; Maiss, Edgar; Richert-Pöggeler, Katja; Dall'Ara, Mattia; Ratti, Claudio; Gilmer, David; Liebe, Sebastian; Varrelmann, Mark

2018-05-01

Two members of the Benyviridae family and genus Benyvirus, Beet soil-borne mosaic virus (BSBMV) and Beet necrotic yellow vein virus (BNYVV), possess identical genome organization, host range and high sequence similarity; they infect Beta vulgaris with variable symptom expression. In the US, mixed infections are described with limited information about viral interactions. Vectors suitable for agroinoculation of all genome components of both viruses were constructed by isothermal in vitro recombination. All 35S promoter-driven cDNA clones allowed production of recombinant viruses competent for Nicotiana benthamiana and Beta macrocarpa systemic infection and Polymyxa betae transmission and were compared to available BNYVV B-type clone. BNYVV and BSBMV RNA1 + 2 reassortants were viable and spread long-distance in N. benthamiana with symptoms dependent on the BNYVV type. Small genomic RNAs were exchangeable and systemically infected B. macrocarpa. These infectious clones represent a powerful tool for the identification of specific molecular host-pathogen determinants. Copyright © 2018 Elsevier Inc. All rights reserved.