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Sample records for full length recombinant

  1. Crystallization and X-ray structure of full-length recombinant human butyrylcholinesterase

    SciTech Connect

    Ngamelue, Michelle N.; Homma, Kohei; Lockridge, Oksana; Asojo, Oluwatoyin A.

    2007-09-01

    The first crystals and the 2.8 Å X-ray structure of full-length recombinant human butyrylcholinesterase are reported. Human butyrylcholinesterase (BChE) has been shown to function as an endogenous scavenger of diverse poisons. BChE is a 340 kDa tetrameric glycoprotein that is present in human serum at a concentration of 5 mg l{sup −1}. The well documented therapeutic effects of BChE on cocaine toxicity and organophosphorus agent poisoning has increased the need for effective methods of producing recombinant therapeutic BChE. In order to be therapeutically useful, BChE must have a long circulatory residence time or associate as tetramers. Full-length recombinant BChE produced in Chinese hamster ovary (CHO) cells or human embryonic kidney cells has been shown to associate as monomers, with a shorter circulatory residence time than the naturally occurring tetrameric serum protein. Based on the preceding observation as well as the need to develop novel methodologies to facilitate the mass production of therapeutic recombinant BChE, studies have been initiated to determine the structural basis of tetramer formation. Towards these ends, full-length monomeric recombinant BChE has been crystallized for the first time. A 2.8 Å X-ray structure was solved in space group P42{sub 1}2, with unit-cell parameters a = b = 156, c = 146 Å.

  2. Recombinant production and film properties of full-length hornet silk proteins.

    PubMed

    Kambe, Yusuke; Sutherland, Tara D; Kameda, Tsunenori

    2014-08-01

    Full-length versions of the four main components of silk cocoons of Vespa simillima hornets, Vssilk1-4, were produced as recombinant proteins in Escherichia coli. In shake flasks, the recombinant Vssilk proteins yielded 160-330mg recombinant proteinl(-1). Films generated from solutions of single Vssilk proteins had a secondary structure similar to that of films generated from native hornet silk. The films made from individual recombinant hornet silk proteins had similar or enhanced mechanical performance compared with films generated from native hornet silk, possibly reflecting the homogeneity of the recombinant proteins. The pH-dependent changes in zeta (ζ) potential of each Vssilk film were measured, and isoelectric points (pI) of Vssilk1-4 were determined as 8.9, 9.1, 5.0 and 4.2, respectively. The pI of native hornet silk, a combination of the four Vssilk proteins, was 4.7, a value similar to that of Bombyx mori silkworm silk. Films generated from Vssilk1 and 2 had net positive charge under physiological conditions and showed significantly higher cell adhesion activity. It is proposed that recombinant hornet silk is a valuable new material with potential for cell culture applications. PMID:24862540

  3. Cocrystallization studies of full-length recombinant butyrylcholinesterase (BChE) with cocaine

    SciTech Connect

    Asojo, Oluwatoyin Ajibola; Asojo, Oluyomi Adebola; Ngamelue, Michelle N.; Homma, Kohei; Lockridge, Oksana

    2011-09-16

    Human butyrylcholinesterase (BChE; EC 3.1.1.8) is a 340 kDa tetrameric glycoprotein that is present in human serum at about 5 mg l{sup -1} and has well documented therapeutic effects on cocaine toxicity. BChE holds promise as a therapeutic that reduces and finally eliminates the rewarding effects of cocaine, thus weaning an addict from the drug. There have been extensive computational studies of cocaine hydrolysis by BChE. Since there are no reported structures of BChE with cocaine or any of the hydrolysis products, full-length monomeric recombinant wild-type BChE was cocrystallized with cocaine. The refined 3 {angstrom} resolution structure appears to retain the hydrolysis product benzoic acid in sufficient proximity to form a hydrogen bond to the active-site Ser198.

  4. Production, purification and characterization of recombinant, full-length human claudin-1.

    PubMed

    Bonander, Nicklas; Jamshad, Mohammed; Oberthür, Dominik; Clare, Michelle; Barwell, James; Hu, Ke; Farquhar, Michelle J; Stamataki, Zania; Harris, Helen J; Dierks, Karsten; Dafforn, Timothy R; Betzel, Christian; McKeating, Jane A; Bill, Roslyn M

    2013-01-01

    The transmembrane domain proteins of the claudin superfamily are the major structural components of cellular tight junctions. One family member, claudin-1, also associates with tetraspanin CD81 as part of a receptor complex that is essential for hepatitis C virus (HCV) infection of the liver. To understand the molecular basis of claudin-1/CD81 association we previously produced and purified milligram quantities of functional, full-length CD81, which binds a soluble form of HCV E2 glycoprotein (sE2). Here we report the production, purification and characterization of claudin-1. Both yeast membrane-bound and detergent-extracted, purified claudin-1 were antigenic and recognized by specific antibodies. Analytical ultracentrifugation demonstrated that extraction with n-octyl-β-d-glucopyranoside yielded monodispersed, dimeric pools of claudin-1 while extraction with profoldin-8 or n-decylphosphocholine yielded a dynamic mixture of claudin-1 oligomers. Neither form bound sE2 in line with literature expectations, while further functional analysis was hampered by the finding that incorporation of claudin-1 into proteoliposomes rendered them intractable to study. Dynamic light scattering demonstrated that claudin-1 oligomers associate with CD81 in vitro in a defined molar ratio of 1∶2 and that complex formation was enhanced by the presence of cholesteryl hemisuccinate. Attempts to assay the complex biologically were limited by our finding that claudin-1 affects the properties of proteoliposomes. We conclude that recombinant, correctly-folded, full-length claudin-1 can be produced in yeast membranes, that it can be extracted in different oligomeric forms that do not bind sE2 and that a dynamic preparation can form a specific complex with CD81 in vitro in the absence of any other cellular components. These findings pave the way for the structural characterization of claudin-1 alone and in complex with CD81. PMID:23704991

  5. Characterization of full-length recombinant human Proteoglycan 4 as an ocular surface boundary lubricant.

    PubMed

    Samsom, Michael L; Morrison, Sheila; Masala, Nemanja; Sullivan, Benjamin D; Sullivan, David A; Sheardown, Heather; Schmidt, Tannin A

    2014-10-01

    Proteoglycan 4 (PRG4, or lubricin) is a lubricating mucin-like glycoprotein recently discovered at the ocular surface, where it functions as a boundary lubricant and appears to play a protective role. Recent technological advances have enabled abundant expression of full-length recombinant human PRG4 (rhPRG4). The objectives of this study were to 1) biochemically characterize the gross structure and glycosylations of full-length rhPRG4, and 2) assess the ocular surface boundary lubricating ability of rhPRG4 at both human cornea-eyelid and human cornea-polydimethylsiloxane (PDMS) biointerfaces. rhPRG4 expressed by a Chinese hamster ovary cell line was characterized and compared to native bovine PRG4 by SDS-PAGE western blotting, and protein identity was assessed by tandem mass spectrometry (MS/MS). Human corneas were articulated against PDMS or human eyelids, at effective sliding velocities of 0.3-30 mm/s under physiological loads of ∼15 kPa, to assess and compare the ocular lubricating ability of rhPRG4 to PRG4. Samples were tested serially in PRG4, rhPRG4 (both 300 μg/ml), then saline. Western blotting indicated that rhPRG4 had immunoreactivity at the appropriate apparent molecular weight, and possessed O-linked glycosylation consistent with that of PRG4. rhPRG4 protein identity was confirmed by MS/MS. Both PRG4 and rhPRG4 significantly, and similarly, reduced friction compared to saline at both human cornea - PDMS and human cornea-eyelid biointerfaces. In conclusion, the rhPRG4 studied here demonstrated appropriate higher order structure, O-linked glycosylations, and ocular surface boundary lubricating. Purified rhPRG4 may have clinical utility as a topical treatment of dry eye disease or contact lens biomaterial coating to promote more comfortable wear. PMID:24997456

  6. Near full-length genomic characterization of a HIV type 1 BC recombinant strain from Manipur, India.

    PubMed

    Sarkar, Roni; Sarkar, Kamalesh; Singh, N Brajachand; Singh, Y Manihar; Chakrabarti, Sekhar

    2012-10-01

    Genetic complexity of HIV-1 is brought about by recombination between HIV-1 subtypes which leads to the development of epidemiologically significant founder strains. In the present study, the near full-length genome sequence of an HIV-1 isolate from an injecting drug user of Manipur (India) was determined, which evidenced the presence of a novel HIV-1 BC recombinant strain. Near full-length genome was amplified by polymerase chain reaction using primer walking approach. The recombination break points were detected using bootscan and simplot analyses. This isolate exhibited a mosaic structure consisting of subtype C backbone with subtype B insertions at the upstream of pol gene (3026-3259) and the downstream of env gene which spanned till the nef gene (8183-8961). Phylogenetic relationships determined with neighbor-joining trees, revealed that the subtype C sequences clustered with sequences from Indian subtype C HIV-1 strains, and the subtype B sequences clustered with HIV-1 subtype B strains from Thailand. This finding may create a complex scenario of HIV-1 epidemic among the injecting drug users of Manipur in near future. PMID:22710995

  7. Functional Recombinant Extra Membrane Loop of Human CD20, an Alternative of the Full Length CD20 Antigen

    PubMed Central

    Anbouhi, Mahdi Habibi; Baraz, Aida Feiz; Bouzari, Saeid; Abolhassani, Mohsen; Khanahmad, Hossein; Golkar, Majid; Aghasadeghi, Mohammad Reza; Behdani, Mahdi; Najafabadi, Ali Jahanian; Shokrgozar, Mohammad Ali

    2012-01-01

    Background: Targeting of CD20 antigen with monoclonal antibodies has become the mainstay in the treatment of non-Hodgkin's lymphomas and immunotherapeutic depletion of malignant B cells. Accessibility of antigen is one of the crucial factors in development of monoclonal antibodies against this antigen. One major problem in expression of full length CD20 is aggregation and misfolding. Therefore, production of an alternative polypeptide is easer and favorable comparing to that of a full length transmembrane protein CD20. Methods: In this study, we expressed the extra membrane loop of hCD20 (exCD20) consisting of a non-glycosylated 47-amino acids region. The exCD20 coding sequence was amplified by PCR and cloned in pET32a(+) expression vector. The desired protein was expressed in fusion with thioredoxin and 6× His tag in E. coli Origami strain. ELISA and Western-blotting data were performed to indicate the functionality of this protein. Results: We have obtained the exCD20 recombinant protein which can be detected in ELISA and Western-blot experiments. This recombinant fusion protein was soluble and stable without aggregation and misfolding problems. Conclusion: The recombinant extra membrane loop of human CD20 protein in fusion with thioredoxin (exCD20) can be used in function assays and some applications such as ELISA, immuneblotting, affinity purification, immunization, screening, and development of anti-CD20 antibodies. PMID:23023212

  8. Milligram Quantities of Homogeneous Recombinant Full-Length Mouse Munc18c from Escherichia coli Cultures

    PubMed Central

    Rehman, Asma; Jarrott, Russell J.; Whitten, Andrew E.; King, Gordon J.; Hu, Shu-Hong; Christie, Michelle P.; Collins, Brett M.; Martin, Jennifer L.

    2013-01-01

    Vesicle fusion is an indispensable cellular process required for eukaryotic cargo delivery. The Sec/Munc18 protein Munc18c is essential for insulin-regulated trafficking of glucose transporter4 (GLUT4) vesicles to the cell surface in muscle and adipose tissue. Previously, our biophysical and structural studies have used Munc18c expressed in SF9 insect cells. However to maximize efficiency, minimize cost and negate any possible effects of post-translational modifications of Munc18c, we investigated the use of Escherichia coli as an expression host for Munc18c. We were encouraged by previous reports describing Munc18c production in E. coli cultures for use in in vitro fusion assay, pulldown assays and immunoprecipitations. Our approach differs from the previously reported method in that it uses a codon-optimized gene, lower temperature expression and autoinduction media. Three N-terminal His-tagged constructs were engineered, two with a tobacco etch virus (TEV) or thrombin protease cleavage site to enable removal of the fusion tag. The optimized protocol generated 1–2 mg of purified Munc18c per L of culture at much reduced cost compared to Munc18c generated using insect cell culture. The purified recombinant Munc18c protein expressed in bacteria was monodisperse, monomeric, and functional. In summary, we developed methods that decrease the cost and time required to generate functional Munc18c compared with previous insect cell protocols, and which generates sufficient purified protein for structural and biophysical studies. PMID:24391775

  9. A full-length Plasmodium falciparum recombinant circumsporozoite protein expressed by Pseudomonas fluorescens platform as a malaria vaccine candidate.

    PubMed

    Noe, Amy R; Espinosa, Diego; Li, Xiangming; Coelho-Dos-Reis, Jordana G A; Funakoshi, Ryota; Giardina, Steve; Jin, Hongfan; Retallack, Diane M; Haverstock, Ryan; Allen, Jeffrey R; Vedvick, Thomas S; Fox, Christopher B; Reed, Steven G; Ayala, Ramses; Roberts, Brian; Winram, Scott B; Sacci, John; Tsuji, Moriya; Zavala, Fidel; Gutierrez, Gabriel M

    2014-01-01

    The circumsporozoite protein (CSP) of Plasmodium falciparum is a major surface protein, which forms a dense coat on the sporozoite's surface. Preclinical research on CSP and clinical evaluation of a CSP fragment-based RTS, S/AS01 vaccine have demonstrated a modest degree of protection against P. falciparum, mediated in part by humoral immunity and in part by cell-mediated immunity. Given the partial protective efficacy of the RTS, S/AS01 vaccine in a recent Phase 3 trial, further improvement of CSP-based vaccines is crucial. In this report, we describe the preclinical development of a full-length, recombinant CSP (rCSP)-based vaccine candidate against P. falciparum malaria suitable for current Good Manufacturing Practice (cGMP) production. Utilizing a novel high-throughput Pseudomonas fluorescens expression platform, we demonstrated greater efficacy of full-length rCSP as compared to N-terminally truncated versions, rapidly down-selected a promising lead vaccine candidate, and developed a high-yield purification process to express immunologically active, intact antigen for clinical trial material production. The rCSP, when formulated with various adjuvants, induced antigen-specific antibody responses as measured by enzyme-linked immunosorbent assay (ELISA) and immunofluorescence assay (IFA), as well as CD4+ T-cell responses as determined by ELISpot. The adjuvanted rCSP vaccine conferred protection in mice when challenged with transgenic P. berghei sporozoites containing the P. falciparum repeat region of CSP. Furthermore, heterologous prime/boost regimens with adjuvanted rCSP and an adenovirus type 35-vectored CSP (Ad35CS) showed modest improvements in eliciting CSP-specific T-cell responses and anti-malarial protection, depending on the order of vaccine delivery. Collectively, these data support the importance of further clinical development of adjuvanted rCSP, either as a stand-alone product or as one of the components in a heterologous prime/boost strategy

  10. A Novel Drug-Resistant HIV-1 Circulating Recombinant Form CRF76_01B Identified by Near Full-Length Genome Analysis.

    PubMed

    Ogawa, Satoko; Hachiya, Atsuko; Hosaka, Masumi; Matsuda, Masakazu; Ode, Hirotaka; Shigemi, Urara; Okazaki, Reiko; Sadamasu, Kenji; Nagashima, Mami; Toyokawa, Takao; Tateyama, Masao; Tanaka, Yasuhito; Sugiura, Wataru; Yokomaku, Yoshiyuki; Iwatani, Yasumasa

    2016-03-01

    HIV-1 CRF01_AE and subtype B (B) have dominated and their different circulating recombinant forms (CRFs) have emerged in East and Southeast Asian countries. Here, we report a novel drug-resistant HIV-1 CRF. Five independent recombinant specimens exhibiting discordant subtype results for the gag, pol, and env sequences were isolated. These recombinants had the CRF01_AE (gag p17)/B (pol PR-RT and IN)/CRF01_AE (env C2-V3) pattern similar to CRF69_01B. Sequence analysis of four near full-length HIV-1 genomes revealed a unique phylogenetic cluster distinct from previously reported CRFs. Of the four recombinants, three shared an identical mosaic structure including seven breakpoints in the gag, pol, vif, and env regions, designated CRF76_01B. The one remaining recombinant had additional recombination breakpoints in the vpu region and exhibited another unique recombinant form composed of CRF76_01B and B. These findings provide important insight into the transmission dynamics of HIV-1 in Asia that may be important for its effective prevention. PMID:26528581

  11. Potency of Full- Length MGF to Induce Maximal Activation of the IGF-I R Is Similar to Recombinant Human IGF-I at High Equimolar Concentrations

    PubMed Central

    Janssen, Joseph A. M. J. L.; Hofland, Leo J.; Strasburger, Christian J.; van den Dungen, Elisabeth S. R.; Thevis, Mario

    2016-01-01

    Aims To compare full-length mechano growth factor (full-length MGF) with human recombinant insulin-like growth factor-I (IGF-I) and human recombinant insulin (HI) in their ability to activate the human IGF-I receptor (IGF-IR), the human insulin receptor (IR-A) and the human insulin receptor-B (IR-B), respectively. In addition, we tested the stimulatory activity of human MGF and its stabilized analog Goldspink-MGF on the IGF-IR. Methods The effects of full-length MGF, IGF-I, human mechano growth factor (MGF), Goldspink-MGF and HI were compared using kinase specific receptor activation (KIRA) bioassays specific for IGF-I, IR-A or IR-B, respectively. These assays quantify activity by measuring auto-phosphorylation of the receptor upon ligand binding. Results IGF-IR: At high equimolar concentrations maximal IGF-IR stimulating effects generated by full-length MGF were similar to that of IGF-I (89-fold vs. 77-fold, respectively). However, EC50 values of IGF-I and full-length MGF for the IGF-I receptor were 0.86 nmol/L (95% CI 0.69–1.07) and 7.83 nmol/L (95% CI: 4.87–12.58), respectively. No IGF-IR activation was observed by human MGF and Goldspink-MGF, respectively. IR-A/IR-B: At high equimolar concentrations similar maximal IR-A stimulating effects were observed for full -length MGF and HI, but maximal IR-B stimulation achieved by full -length MGF was stronger than that by HI (292-fold vs. 98-fold). EC50 values of HI and full-length MGF for the IR-A were 1.13 nmol/L (95% CI 0.69–1.84) and 73.11 nmol/L (42.87–124.69), respectively; for IR-B these values were 1.28 nmol/L (95% CI 0.64–2.57) and 35.10 nmol/L (95% 17.52–70.33), respectively. Conclusions Full-length MGF directly stimulates the IGF-IR. Despite a higher EC50 concentration, at high equimolar concentrations full-length MGF showed a similar maximal potency to activate the IGF-IR as compared to IGF-I. Further research is needed to understand the actions of full-length MGF in vivo and to define the

  12. Near Full-Length Genome Identification of a Novel HIV-1 Recombinant Form (CRF01_AE/CRF07_BC) in Zhejiang, China.

    PubMed

    Peng, Xiaorong; Li-Jun, Xu; Xie, Tiansheng; Liu, Fumin; Wu, Nanping

    2016-09-01

    CRF01_AE and CRF07_BC are the two major circulating recombinant forms (CRFs) in China. Furthermore, many kinds of unique recombinant forms (URFs) between CRF01_AE and CRF07_BC were recently identified in China. Here we detected a novel recombinant of CRF07_BC/CRF01_AE, whose genome structure is distinctly different from other URFs reported before. The phylogenetic analysis of the near full-length sequence of 15zj032 reveals that three regions of CRF01_AE insert into the CRF07_BC backbone. Recently, the continued emergence of novel URFs implies that super infections of different subtypes of HIV-1 are common in China and should be given enough importance. PMID:27353182

  13. Near Full-Length Genomic Characterization of a Novel CRF 01_AE/C Recombinant from Western India.

    PubMed

    Karade, Santosh; Pandey, Sudhanshu; Gianchandani, Sheetal; Kurle, Swarali N; Ghate, Manisha; Gaikwad, Nitin S; Rewari, Bharat B; Gangakhedkar, Raman R

    2015-12-01

    HIV is known for its genetic variability across the globe. The HIV epidemic in India is primarily driven by subtype C, although sporadic circulating and unique recombinant forms are also reported from a few metropolitan cities in which genotyping facilities are available. Here we report a novel CRF01_AE/C recombinant from a multicenter study on the effectiveness of antiretroviral therapy (ART), 12 months after its initiation. Our subject is a 32-year-old heterosexual female, a native of Pune city in western India. Identification and analyses of recombination breakpoints using jpHMM@Gobics and SimPlot bootscanning revealed six recombination breakpoints, indicating insertion of the CRF01_AE genome at three points in the backbone of subtype C. Both subtype C and CRF01_AE are commonly seen in the population at risk of heterosexual HIV transmission, thereby providing an opportunity for cocirculation and recombination. The emergence of a novel recombinant of CRF01_AE/C is indicative of the increasing genetic diversity of the HIV epidemic in India. PMID:26323027

  14. First full-length genomic sequence of a hepatitis A virus isolated in Argentina shows recombination between subgenotypes IA and IB.

    PubMed

    Aguirre, Sebastian; Malirat, Viviana; Scodeller, Eduardo; Mattion, Nora

    2011-01-01

    A hepatitis A virus (HAV) recovered in Argentina from a stool sample of a sick child in the year 2006 (HAV-Arg/06) was entirely sequenced. Phylogenetic analysis included the HAV-Arg/06 sequence in subgenotype IA, either considering the usual VP1-2A variable junction fragment or the full length nucleotide sequence. Interestingly, a recombination event with subgenotype IB, involving a portion of the 2C-3A nonstructural proteins coding region (nucleotides 4961-5140) was detected using specific software. Only subgenotype IA strains have been detected in Argentina or Uruguay, whereas subgenotype IA and IB strains have been reported to circulate in Brazil. Although recombination has been given an important role in the evolution of picornaviruses, there have been only a few reports of its involvement in the evolution of HAV, probably due to the limited number of complete HAV sequences available. This study constitutes the first report of a full-length HAV sequence in Argentina and the third in South America, after the sequence of the IA isolate HAV5 from Uruguay and the IB isolate HAF-203 from Brazil. The availability of new sequence data covering the complete HAV genome will help establish a more consistent genetic relatedness among HAV isolates and the role of recombination in its evolution. PMID:21056065

  15. Efficient assembly of full-length infectious clone of Brazilian IBDV isolate by homologous recombination in yeast.

    PubMed

    Silva, J V J; Arenhart, S; Santos, H F; Almeida-Queiroz, S R; Silva, A N M R; Trevisol, I M; Bertani, G R; Gil, L H V G

    2014-01-01

    The Infectious Bursal Disease Virus (IBDV) causes immunosuppression in young chickens. Advances in molecular virology and vaccines for IBDV have been achieved by viral reverse genetics (VRG). VRG for IBDV has undergone changes over time, however all strategies used to generate particles of IBDV involves multiple rounds of amplification and need of in vitro ligation and restriction sites. The aim of this research was to build the world's first VRG for IBDV by yeast-based homologous recombination; a more efficient, robust and simple process than cloning by in vitro ligation. The wild type IBDV (Wt-IBDV-Br) was isolated in Brazil and had its genome cloned in pJG-CMV-HDR vector by yeast-based homologous recombination. The clones were transfected into chicken embryo fibroblasts and the recovered virus (IC-IBDV-Br) showed genetic stability and similar phenotype to Wt-IBDV-Br, which were observed by nucleotide sequence, focus size/morphology and replication kinetics, respectively. Thus, IBDV reverse genetics by yeast-based homologous recombination provides tools to IBDV understanding and vaccines/viral vectors development. PMID:25763067

  16. Efficient assembly of full-length infectious clone of Brazilian IBDV isolate by homologous recombination in yeast

    PubMed Central

    Silva, J.V.J.; Arenhart, S.; Santos, H.F.; Almeida-Queiroz, S.R.; Silva, A.N.M.R.; Trevisol, I.M.; Bertani, G.R.; Gil, L.H.V.G.

    2014-01-01

    The Infectious Bursal Disease Virus (IBDV) causes immunosuppression in young chickens. Advances in molecular virology and vaccines for IBDV have been achieved by viral reverse genetics (VRG). VRG for IBDV has undergone changes over time, however all strategies used to generate particles of IBDV involves multiple rounds of amplification and need of in vitro ligation and restriction sites. The aim of this research was to build the world’s first VRG for IBDV by yeast-based homologous recombination; a more efficient, robust and simple process than cloning by in vitro ligation. The wild type IBDV (Wt-IBDV-Br) was isolated in Brazil and had its genome cloned in pJG-CMV-HDR vector by yeast-based homologous recombination. The clones were transfected into chicken embryo fibroblasts and the recovered virus (IC-IBDV-Br) showed genetic stability and similar phenotype to Wt-IBDV-Br, which were observed by nucleotide sequence, focus size/morphology and replication kinetics, respectively. Thus, IBDV reverse genetics by yeast-based homologous recombination provides tools to IBDV understanding and vaccines/viral vectors development. PMID:25763067

  17. Sequencing and Phylogenetic Analysis of Near Full-Length HIV-1 Subtypes A, B, G and Unique Recombinant AC and AD Viral Strains Identified in South Africa

    PubMed Central

    Wilkinson, Eduan; Holzmayer, Vera; Jacobs, Graeme B.; de Oliveira, Tulio; Brennan, Catherine A.; Hackett, John; van Rensburg, Estrelita Janse

    2015-01-01

    Abstract By the end of 2012, more than 6.1 million people were infected with HIV-1 in South Africa. Subtype C was responsible for the majority of these infections and more than 300 near full-length genomes (NFLGs) have been published. Currently very few non-subtype C isolates have been identified and characterized within the country, particularly full genome non-C isolates. Seven patients from the Tygerberg Virology (TV) cohort were previously identified as possible non-C subtypes and were selected for further analyses. RNA was isolated from five individuals (TV047, TV096, TV101, TV218, and TV546) and DNA from TV016 and TV1057. The NFLGs of these samples were amplified in overlapping fragments and sequenced. Online subtyping tools REGA version 3 and jpHMM were used to screen for subtypes and recombinants. Maximum likelihood (ML) phylogenetic analysis (phyML) was used to infer subtypes and SimPlot was used to confirm possible intersubtype recombinants. We identified three subtype B (TV016, TV047, and TV1057) isolates, one subtype A1 (TV096), one subtype G (TV546), one unique AD (TV101), and one unique AC (TV218) recombinant form. This is the first NFLG of subtype G that has been described in South Africa. The subtype B sequences described also increased the NFLG subtype B sequences in Africa from three to six. There is a need for more NFLG sequences, as partial HIV-1 sequences may underrepresent viral recombinant forms. It is also necessary to continue monitoring the evolution and spread of HIV-1 in South Africa, because understanding viral diversity may play an important role in HIV-1 prevention strategies. PMID:25492033

  18. Near-Full-Length Genome Sequences of a Novel HIV-1 Circulating Recombinant Form, CRF01_AE/B'/C (CRF78_cpx), in Yunnan, China.

    PubMed

    Song, Yindi; Feng, Yue; Miao, Zhijiang; Wang, Binghui; Yang, Ming; Zhang, A-Mei; Liu, Li; Xia, Xueshan

    2016-06-01

    We report a novel HIV circulating recombinant form (CRF78_cpx) composed mainly of CRF01_AE with inserts from subtypes B and C identified from three epidemiologically unlinked individuals in Yunnan province, China. Two of the subjects are heterosexual men and one is a male intravenous drug user. Sequencing and analyzing the near-full-length genome of these three isolates (YNTC88, YNTC19, and YNTC35) revealed identical recombination breakpoints in all three viruses, but considerable genetic diversity between them, across the genomes, indicating that this is not a newly created CRF, only newly detected. CRF78_cpx differs from previously documented CRF01-AE/B'/C forms in its distinct backbone, inserted fragment size, and breakpoints, and is not related to other described recombinants in the region such as CRF07_BC or CRF65_cpx (also composed of CRF01_AE, B', and C). Our present findings further enrich the diversity of the prevalent HIV-1 CRFs in Yunnan, which is considered as an epicenter of HIV-1 infections in China. PMID:26885715

  19. Amyloid Core Formed of Full-Length Recombinant Mouse Prion Protein Involves Sequence 127–143 but Not Sequence 107–126

    PubMed Central

    Chatterjee, Biswanath; Lee, Chung-Yu; Lin, Chen; Chen, Eric H.-L.; Huang, Chao-Li; Yang, Chien-Chih; Chen, Rita P.-Y.

    2013-01-01

    The principal event underlying the development of prion disease is the conversion of soluble cellular prion protein (PrPC) into its disease-causing isoform, PrPSc. This conversion is associated with a marked change in secondary structure from predominantly α-helical to a high β-sheet content, ultimately leading to the formation of aggregates consisting of ordered fibrillar assemblies referred to as amyloid. In vitro, recombinant prion proteins and short prion peptides from various species have been shown to form amyloid under various conditions and it has been proposed that, theoretically, any protein and peptide could form amyloid under appropriate conditions. To identify the peptide segment involved in the amyloid core formed from recombinant full-length mouse prion protein mPrP(23–230), we carried out seed-induced amyloid formation from recombinant prion protein in the presence of seeds generated from the short prion peptides mPrP(107–143), mPrP(107–126), and mPrP(127–143). Our results showed that the amyloid fibrils formed from mPrP(107–143) and mPrP(127–143), but not those formed from mPrP(107–126), were able to seed the amyloidogenesis of mPrP(23–230), showing that the segment residing in sequence 127–143 was used to form the amyloid core in the fibrillization of mPrP(23–230). PMID:23844138

  20. BAY 81-8973, a full-length recombinant factor VIII: Human heat shock protein 70 improves the manufacturing process without affecting clinical safety.

    PubMed

    Maas Enriquez, Monika; Thrift, John; Garger, Stephen; Katterle, Yvonne

    2016-11-01

    BAY 81-8973 is a full-length, unmodified recombinant human factor VIII (FVIII) approved for the treatment of hemophilia A. BAY 81-8973 has the same amino acid sequence as the currently marketed sucrose-formulated recombinant FVIII (rFVIII-FS) product and is produced using additional advanced manufacturing technologies. One of the key manufacturing advances for BAY 81-8973 is introduction of the gene for human heat shock protein 70 (HSP70) into the rFVIII-FS cell line. HSP70 facilitates proper folding of proteins, enhances cell survival by inhibiting apoptosis, and potentially impacts rFVIII glycosylation. HSP70 expression in the BAY 81-8973 cell line along with other manufacturing advances resulted in a higher-producing cell line and improvements in the pharmacokinetics of the final product as determined in clinical studies. HSP70 protein is not detected in the harvest or in the final BAY 81-8973 product. However, because this is a new process, clinical trial safety assessments included monitoring for anti-HSP70 antibodies. Most patients, across all age groups, had low levels of anti-HSP70 antibodies before exposure to the investigational product. During BAY 81-8973 treatment, 5% of patients had sporadic increases in anti-HSP70 antibody levels above a predefined threshold (cutoff value, 239 ng/mL). No clinical symptoms related to anti-HSP70 antibody development occurred. In conclusion, addition of HSP70 to the BAY 81-8973 cell line is an innovative technology for manufacturing rFVIII aimed at improving protein folding and expression. Improved pharmacokinetics and no effect on safety of BAY 81-8973 were observed in clinical trials in patients with hemophilia A. PMID:27436242

  1. Full-Length Recombinant Plasmodium falciparum VAR2CSA Binds Specifically to CSPG and Induces Potent Parasite Adhesion-Blocking Antibodies

    PubMed Central

    Khunrae, Pongsak; Dahlbäck, Madeleine; Nielsen, Morten A.; Andersen, Gorm; Ditlev, Sisse B.; Resende, Mafalda; Pinto, Vera V.; Theander, Thor G.; Higgins, Matthew K.; Salanti, Ali

    2010-01-01

    Plasmodium falciparum malaria remains one of the world's leading causes of human suffering and poverty. Each year, the disease takes 1–3 million lives, mainly in sub-Saharan Africa. The adhesion of infected erythrocytes (IEs) to vascular endothelium or placenta is the key event in the pathogenesis of severe P. falciparum infection. In pregnant women, the parasites express a single and unique member of the P. falciparum erythrocyte membrane protein 1 (PfEMP1) family named VAR2CSA, which is associated with the ability of the IEs to adhere specifically to chondroitin sulphate A (CSA) in the placenta. Several Duffy-binding-like domains from VAR2CSA molecules have been shown in vitro to bind to CSA, but it has also been demonstrated that Duffy-binding-like domains from PfEMP1 proteins other than VAR2CSA can bind CSA. In addition, the specificity of the binding of VAR2CSA domains to glycosaminoglycans does not match that of VAR2CSA-expressing IEs. This has led to speculation that the domains of native VAR2CSA need to come together to form a specific binding site or that VAR2CSA might bind to CSA through a bridging molecule. Here, we describe the expression and purification of the complete extracellular region of VAR2CSA secreted at high yields from insect cells. Using surface plasmon resonance, we demonstrate that VAR2CSA alone binds with nanomolar affinity to human chondroitin sulphate proteoglycan and with significantly weaker affinity to other glycosaminoglycans, showing a specificity similar to that observed for IEs. Antibodies raised against full-length VAR2CSA completely inhibit recombinant VAR2CSA binding, as well as parasite binding to chondroitin sulphate proteoglycan. This is the first study to describe the successful production and functionality of a full-length PfEMP1. The specificity of the binding and anti-adhesion potency of induced IgG, together with high-yield production, encourages the use of full-length PfEMP1 in vaccine development strategies. PMID

  2. Expression, purification, refolding and in vitro recovery of active full length recombinant human gelatinase MMP-9 in Escherichia coli.

    PubMed

    Mohseni, Sara; Moghadam, Tahereh Tohidi; Dabirmanesh, Bahareh; Khajeh, Khosro

    2016-10-01

    Human gelatinase (MMP-9) is a member of matrix metalloproteinases family (MMPs), which has been associated with malignant tumor progression and metastasis by matrix degradation. Herein, active full length recombinant human MMP-9 (amino acid residues 107-707) has been expressed in the form of inclusion bodies in Escherichia coli BL21, using pET21a vector. Solubilization of inclusion bodies was carried out in Tris-HCl buffer with 6 M urea, and refolding was performed using dilution and urea gradient methods. Tris-HCl buffer with 5 mM CaCl2 and 1 μM ZnCl2 at pH 7.8 was used as a refolding buffer. Analysis of the structure by fluorescence and far-UV circular dichroism showed a well-formed structure by urea gradient method. Kinetic parameters in refolding conditions of rhMMP-9 were also analyzed, depicting increase in the enzyme's activity without any aggregation. PMID:27164034

  3. Therapeutic effects of recombinant forms of full-length and truncated human surfactant protein D in a murine model of invasive pulmonary aspergillosis.

    PubMed

    Singh, Mamta; Madan, Taruna; Waters, Patrick; Sonar, Sanchaita; Singh, Shiv K; Kamran, Mohammad F; Bernal, Andrés López; Sarma, P Usha; Singh, Vijay K; Crouch, Erika C; Kishore, Uday

    2009-07-01

    Aspergillus fumigatus (Afu) is an opportunistic fungal pathogen that can cause fatal invasive pulmonary aspergillosis (IPA) in immunocompromised individuals. Previously, surfactant protein D (SP-D), a surfactant-associated innate immune molecule, has been shown to enhance phagocytosis and killing of Afu conidia by phagocytic cells in vitro. An intranasal treatment of SP-D significantly increased survival in a murine model of IPA. Here we have examined mechanisms via which recombinant forms of full-length (hSP-D) or truncated human SP-D (rhSP-D) offer protection in a murine model of IPA that were immunosuppressed with hydrocortisone and challenged intranasally with Afu conidia prior to the treatment. SP-D or rhSP-D treatment increased the survival rate to 70% and 80%, respectively (100% mortality on day 7 in IPA mice), with concomitant reduction in the growth of fungal hyphae in the lungs, and increased levels of TNF-alpha and IFN-gamma in the lung suspension supernatants, as compared to untreated IPA mice. The level of macrophage inflammatory protein-1 alpha (MIP-1 alpha) in the lung cell suspension was also raised considerably following treatment with SP-D or rhSP-D. Our results appear to reaffirm the notion that under immunocompromised conditions, human SP-D or its truncated form can offer therapeutic protection against fatal challenge with Afu conidia challenge. Taken together, the SP-D-mediated protective mechanisms include enhanced phagocytosis by recruited macrophages and neutrophils and fungistatic properties, suppression of the levels of pathogenic Th2 cytokines (IL-4 and IL-5), enhanced local production of protective Th1 cytokines, TNF-alpha and IFN-gamma, and that of protective C-C chemokine, MIP-1 alpha. PMID:19403176

  4. Effect of the electrostatic surface potential on the oligomerization of full-length human recombinant prion protein at single-molecule level

    NASA Astrophysics Data System (ADS)

    Wang, Bin; Lou, Zhichao; Zhang, Haiqian; Xu, Bingqian

    2016-03-01

    The electrostatic surface potential (ESP) of prion oligomers has critical influences on the aggregating processes of the prion molecules. The atomic force microscopy (AFM) and structural simulation were combined to investigate the molecular basis of the full-length human recombinant prion oligomerization on mica surfaces. The high resolution non-intrusive AFM images showed that the prion oligomers formed different patterns on mica surfaces at different buffer pH values. The basic binding units for the large oligomers were determined to be prion momoners (Ms), dimers (Ds), and trimers (Ts). The forming of the D and T units happened through the binding of hydrophobic β-sheets of the M units. In contrast, the α-helices of these M, D, and T units were the binding areas for the formation of large oligomers. At pH 4.5, the binding units M, D, and T showed clear polarized ESP distributions on the surface domains, while at pH 7.0, they showed more evenly distributed ESPs. Based on the conformations of oligomers observed from AFM images, the D and T units were more abundantly on mica surface at pH 4.5 because the ESP re-distribution of M units helped to stabilize these larger oligomers. The amino acid side chains involved in the binding interfaces were stabilized by hydrogen bonds and electrostatic interactions. The detailed analysis of the charged side chains at pH 4.5 indicated that the polarized ESPs induced the aggregations among M, D, and T to form larger oligomers. Therefore, the hydrogen bonds and electrostatic interactions worked together to form the stabilized prion oligomers.

  5. Effect of the electrostatic surface potential on the oligomerization of full-length human recombinant prion protein at single-molecule level.

    PubMed

    Wang, Bin; Lou, Zhichao; Zhang, Haiqian; Xu, Bingqian

    2016-03-21

    The electrostatic surface potential (ESP) of prion oligomers has critical influences on the aggregating processes of the prion molecules. The atomic force microscopy (AFM) and structural simulation were combined to investigate the molecular basis of the full-length human recombinant prion oligomerization on mica surfaces. The high resolution non-intrusive AFM images showed that the prion oligomers formed different patterns on mica surfaces at different buffer pH values. The basic binding units for the large oligomers were determined to be prion momoners (Ms), dimers (Ds), and trimers (Ts). The forming of the D and T units happened through the binding of hydrophobic β-sheets of the M units. In contrast, the α-helices of these M, D, and T units were the binding areas for the formation of large oligomers. At pH 4.5, the binding units M, D, and T showed clear polarized ESP distributions on the surface domains, while at pH 7.0, they showed more evenly distributed ESPs. Based on the conformations of oligomers observed from AFM images, the D and T units were more abundantly on mica surface at pH 4.5 because the ESP re-distribution of M units helped to stabilize these larger oligomers. The amino acid side chains involved in the binding interfaces were stabilized by hydrogen bonds and electrostatic interactions. The detailed analysis of the charged side chains at pH 4.5 indicated that the polarized ESPs induced the aggregations among M, D, and T to form larger oligomers. Therefore, the hydrogen bonds and electrostatic interactions worked together to form the stabilized prion oligomers. PMID:27004887

  6. Recombinant production of functional full-length and truncated human TRAM/TICAM-2 adaptor protein involved in Toll-like receptor and interferon signaling.

    PubMed

    Ullah, M Obayed; Valkov, Eugene; Ve, Thomas; Williams, Simon; Mas, Caroline; Mansell, Ashley; Kobe, Bostjan

    2015-02-01

    TRAM/TICAM-2 is used by Toll-like receptor 4 (TLR4) as a bridging adaptor during the mammalian innate immune response. It recruits TRIF, another TIR domain-containing adaptor protein, to TLR4 via TIR domain interactions, which leads to the activation of transcription factors responsible for the production of type-1 interferon and cytokines. The molecular mechanisms of these dual interactions mediated by the TRAM TIR domain are not clear. To understand the molecular basis of TIR:TIR domain interactions, structural and biochemical studies of TRAM TIR domain are necessary, and require a functional soluble protein. In this paper, we report a successful purification and characterization of full-length TRAM. Because full-length TRAM likely contains unstructured regions that may be disadvantageous for structural studies, we also carried out a systematic construct design to determine the boundaries of the TRAM TIR domain. The truncated TRAM constructs were designed based on secondary structure predictions and screened by small-scale expression. Selected constructs were subjected to biophysical analyses. We show that the expressed TRAM TIR domain is functional using in vitro GST pull-down assays that demonstrate a physical interaction with the TLR4 TIR domain. We further show, by site-directed mutagenesis, that the "BB loop" regions of both the TRAM TIR domain and the TLR4 TIR domain are crucial for this physical interaction. PMID:25306876

  7. Near Full-Length Genomic Characterization of a Novel HIV Type 1 CRF01_AE/CRF07_BC Recombinant Form Transmitted Between a Heterosexual Couple in Guangxi, China.

    PubMed

    Dong, Zhe; Shen, Zhiyong; Xiong, Runsong; Liang, Fuxiong; Liang, Shujia; Li, Jianjun; Tan, Guangjie; Jiang, He; Yang, Xiaoyi; Li, Fan; Liao, Lingjie; Shao, Yiming; Feng, Yi; Zhu, Qiuying

    2016-07-01

    In this research, we reported a new second generation recombinant form (GXDY460B) between circulating recombinant form (CRF)01_AE and CRF07_BC in a seroconversion couple who obtained the virus from her husband by heterosexual behavior. The analysis result of the near full-length genomic characterization showed that the genome comprises at least 12 interlaced segments, including six CRF07_BC and six CRF01_AE segments, with CRF07_BC as the main framework. Cocirculation of multiple virus subtypes and multiple infection routes have existed for a long time in Guangxi, but the recombinant strain was rarely reported among heterosexual transmission population because of its lower crowd confounding degree than men who have sex with men and injecting drug user population. It is the first time that the unique recombinant form (URF) between CRF01_AE and CRF07_BC was identified among heterosexual transmission in Guangxi. The emergence of the novel recombinant helps to understand the pattern of the URF virus. PMID:26892263

  8. Analysis of ORF5 and Full-Length Genome Sequences of Porcine Reproductive and Respiratory Syndrome Virus Isolates of Genotypes 1 and 2 Retrieved Worldwide Provides Evidence that Recombination Is a Common Phenomenon and May Produce Mosaic Isolates

    PubMed Central

    Martín-Valls, G. E.; Kvisgaard, L. K.; Tello, M.; Darwich, L.; Cortey, M.; Burgara-Estrella, A. J.; Hernández, J.; Larsen, L. E.

    2014-01-01

    ABSTRACT Recombination is currently recognized as a factor for high genetic diversity, but the frequency of such recombination events and the genome segments involved are not well known. In the present study, we initially focused on the detection of recombinant porcine reproductive and respiratory syndrome virus (PRRSV) isolates by examining previously published data sets of ORF5 sequences (genotypes 1 and 2) obtained worldwide. We then examined full-length genome sequences in order to determine potential recombination breakpoints along the viral genome. For ORF5, 11 sets of genotype 1 sequences from different geographical areas, including 2 Asian, 1 American, and 7 European regions, and three sets of genotype 2, including sets from China, Mexico, and the United States, were analyzed separately. Potential recombination breakpoints were detected in 10/11 genotype 1 sets, including 9 cases in which the clustering of at least one isolate was different before and after the breakpoints. In genotype 2, potential breakpoints and different tree clustering of at least one strain before and after the breakpoint were observed in 2 out of 3 sets. The results indicated that most of the ORF5 data sets contained at least one recombinant sequence. When the full-length genome sequences were examined, both genotype 1 and 2 sets presented breakpoints (10 and 9, respectively), resulting in significantly different topologies before and after the breakpoints. Mosaic genomes were detected in genotype 1 sequences. These results may have significant implications for the understanding of the molecular epidemiology of PRRSV. IMPORTANCE PRRSV is one of the most important viruses affecting swine production worldwide, causing big economic losses and sanitary problems. One of the key questions on PRRSV arises from its genetic diversity, which is thought to have a direct impact on immunobiology, epidemiology, diagnosis, and vaccine efficacy. One of the causes of this genetic diversity is

  9. Recombinant production and characterization of full-length and truncated β-1,3-glucanase PglA from Paenibacillus sp. S09

    PubMed Central

    2013-01-01

    of non-catalytic modules on enzymatic properties of β-1,3-glucanase. Activity comparison of full-length PglA and truncated forms revealed the negative effect of C-terminal region on thermal stability of the enzyme. Both the N-and C-terminal domains exerted strong binding activity toward insoluble β-1,3-glucan, and could be classified into CBM families. PMID:24283345

  10. Near full-length genome sequence of a novel HIV-1 recombinant form (CRF01_AE/B) detected among men who have sex with men in Jilin Province, China.

    PubMed

    Li, Xingguang; Feng, Yi; Yang, Yao; Chen, Yanli; Guo, Qi; Sun, Liuyan; Zang, Xihui; Xing, Hui; Shao, Yiming

    2014-07-01

    We report here a novel HIV-1 recombinant form (CRF01_AE/B) detected from a comprehensive HIV-1 molecular epidemiologic study among men who have sex with men (MSM) in Jilin province of northeastern China. The near full-length genome (NFLG) analyses showed that the novel HIV-1 recombinant isolate (JL.RF07) was composed of CRF01_AE cluster 5 (northeastern China origin) and subtype B (U.S. and European origin), with six recombinant breakpoints observed in the pol, vif, tat, rev, and env gene regions. To the best of our knowledge, this is the first detection of a novel HIV-1 recombinant form (CRF01_AE/B) in Jilin, which may indicate an active transmission network of HIV-1 infection among MSM in the region. Further studies of the molecular epidemiology of the HIV-1 epidemic among MSM in northeastern China are necessary to gain a fuller understanding of the transmission network and potential public health impact of HIV-1 among MSM in this region. PMID:24521207

  11. A Method to Produce and Purify Full-Length Recombinant Alpha Dystroglycan: Analysis of N- and O-Linked Monosaccharide Composition in CHO Cells with or without LARGE Overexpression

    PubMed Central

    Yoon, Jung Hae; Xu, Rui; Martin, Paul

    2013-01-01

    α dystroglycan (αDG) is part of the dystrophin-associated glycoprotein (DAG) complex, a series of cytoskeletal, transmembrane, and membrane-associated proteins that serve to link the extracellular matrix (ECM) surrounding individual skeletal myofibers to the intracellular F-actin cytoskeleton. Glycosylation and ECM protein binding to αDG are regulated by a number of genes that, when defective, give rise to congenital or limb-girdle forms of muscular dystrophy termed dystroglycanopathies. One such dystroglycanopathy gene is LARGE. Here, we describe a method to produce and purify full-length, furin-resistant, recombinant αDG from CHO cells and CHO cells overexpressing LARGE (CHO-LARGE). In addition, we analyze the O- and N-linked monosaccharide composition of such proteins. αDG purified from CHO-LARGE cells had increased molar content of xylose and fucose relative to CHO, while no significant changes were found in N-linked monosaccharides. Glucuronic acid could not be quantified by the methods used. These studies describe a method to produce and purify the milligram amounts of αDG needed for certain biochemical methods, including monosaccharide analysis. Key words: Dystroglycan, muscular dystrophy, xylose, fucose, laminin, LARGE Correspondence: Paul.Martin@nationwidechildrens.org PMID:23390591

  12. Development of a full-length human protein production pipeline

    PubMed Central

    Saul, Justin; Petritis, Brianne; Sau, Sujay; Rauf, Femina; Gaskin, Michael; Ober-Reynolds, Benjamin; Mineyev, Irina; Magee, Mitch; Chaput, John; Qiu, Ji; LaBaer, Joshua

    2014-01-01

    There are many proteomic applications that require large collections of purified protein, but parallel production of large numbers of different proteins remains a very challenging task. To help meet the needs of the scientific community, we have developed a human protein production pipeline. Using high-throughput (HT) methods, we transferred the genes of 31 full-length proteins into three expression vectors, and expressed the collection as N-terminal HaloTag fusion proteins in Escherichia coli and two commercial cell-free (CF) systems, wheat germ extract (WGE) and HeLa cell extract (HCE). Expression was assessed by labeling the fusion proteins specifically and covalently with a fluorescent HaloTag ligand and detecting its fluorescence on a LabChip® GX microfluidic capillary gel electrophoresis instrument. This automated, HT assay provided both qualitative and quantitative assessment of recombinant protein. E. coli was only capable of expressing 20% of the test collection in the supernatant fraction with ≥20 μg yields, whereas CF systems had ≥83% success rates. We purified expressed proteins using an automated HaloTag purification method. We purified 20, 33, and 42% of the test collection from E. coli, WGE, and HCE, respectively, with yields ≥1 μg and ≥90% purity. Based on these observations, we have developed a triage strategy for producing full-length human proteins in these three expression systems. PMID:24806540

  13. Near full-length genome sequence of a novel HIV type 1 second-generation recombinant form (CRF01_AE/CRF07_BC) identified among men who have sex with men in Jilin, China.

    PubMed

    Li, Xingguang; Ning, Chuanyi; He, Xiang; Yang, Yao; Xing, Hui; Hong, Kunxue; Shao, Yiming; Yang, Rongge

    2013-12-01

    We report here a novel HIV-1 second-generation recombinant form (CRF01_AE/CRF07_BC) composed of CRF01_AE and CRF07_BC, identified among men who have sex with men (MSM) in Jilin, with four breakpoints observed in the pol, vif, and vpr genes. The CRF01_AE regions of the recombinant were clustered with the CRF01_AE lineage, which is mainly circulating among MSM in northern China, with the support of 100% bootstrap value, indicating that the parental origin of the CRF01_AE regions was from MSM, in which recombination events may be more likely to occur. To the best of our knowledge, this is the first detection of a novel HIV-1 second-generation recombinant form (CRF01AE/CRF07_BC) in Jilin, which indicates active transmission networks of HIV-1 infection among MSM in the region. Therefore, it is necessary to continue monitoring the molecular epidemiology of HIV-1 among MSM in Jilin to obtain a better understanding of the transmission and potential public health impact of HIV-1 among MSM in the region. PMID:23809010

  14. The recombinant C-terminus of the human MUC2 mucin forms dimers in Chinese-hamster ovary cells and heterodimers with full-length MUC2 in LS 174T cells.

    PubMed Central

    Lidell, Martin E; Johansson, Malin E V; Mörgelin, Matthias; Asker, Noomi; Gum, James R; Kim, Young S; Hansson, Gunnar C

    2003-01-01

    The entire cDNA corresponding to the C-terminal cysteine-rich domain of the human MUC2 apomucin, after the serine- and threonine-rich tandem repeat, was expressed in Chinese-hamster ovary-K1 cells and in the human colon carcinoma cell line, LS 174T. The C-terminus was expressed as a fusion protein with the green fluorescent protein and mycTag sequences and the murine immunoglobulin kappa-chain signal sequence to direct the protein to the secretory pathway. Pulse-chase studies showed a rapid conversion of the C-terminal monomer into a dimer in both Chinese-hamster ovary-K1 and LS 174T cells. Disulphide-bond-stabilized dimers secreted into the media of both cell lines had a higher apparent molecular mass compared with the intracellular forms. The MUC2 C-terminus was purified from the spent culture medium and visualized by molecular electron microscopy. The dimer nature of the molecule was visible clearly and revealed that each monomer was attached to the other by a large globular domain. Gold-labelled antibodies against the mycTag or green fluorescent protein revealed that these were localized to the ends opposite to the parts responsible for the dimerization. The C-terminus expressed in LS 174T cells formed heterodimers with the full-length wild-type MUC2, but not with the MUC5AC mucin, normally expressed in LS 174T cells. The homodimers of the MUC2 C-termini were secreted continuously from the LS 174T cells, but no wild-type MUC2 secretion has been observed from these cells. This suggests that the information for sorting the MUC2 mucin into the regulated secretory pathway in cells having this ability is present in parts other than the C-terminus of MUC2. PMID:12597771

  15. Nanoscale insights into full-length prion protein aggregation on model lipid membranes.

    PubMed

    Pan, Yangang; Wang, Bin; Zhang, Tong; Zhang, Yanan; Wang, Hongda; Xu, Bingqian

    2016-06-30

    The aggregates of the full-length human recombinant prion protein (PrP) (23-231) on model membranes were investigated by combining the atomic force microscopy (AFM) measurements and theoretical calculations at pH 5.0, showing the great effect of PrP concentration on its supramolecular assemblies on the lipid bilayer. PMID:27284592

  16. Management of full-length complete ureteral avulsion

    PubMed Central

    Tang, Kaifa; Sun, Fa; Tian, Yuan; Zhao, Yili

    2016-01-01

    ABSTRACT Introduction Complete ureteral avulsion is one of the most serious complications of ureteroscopy. The aim of this report was to look for a good solution to full-length complete ureteral avulsion. Case presentation A 40-year-old man underwent ureteroscopic management. Full-length complete avulsion of ureter occurred during ureteroscopy. Pyeloureterostomy plus greater omentum investment outside the avulsed ureter and ureterovesical anastomosis were performed 6 hours after ureteral avulsion. The patient was followed-up during 34 months. Double-J tube was removed at 3 months after operation. Twenty three months after the first operation, the patient developed hydronephrosis because of a new ureter upside stone, then rigid ureteroscopy and holmium laser lithotripsy were used successfully. Conclusion Pyeloureterostomy plus greater omentum investment outside the avulsed ureter and ureterovesical anastomosis may be a good choice for full-length complete ureteral avulsion. PMID:27136483

  17. Full-length fuel rod behavior under severe accident conditions

    SciTech Connect

    Lombardo, N J; Lanning, D D; Panisko, F E

    1992-12-01

    This document presents an assessment of the severe accident phenomena observed from four Full-Length High-Temperature (FLHT) tests that were performed by the Pacific Northwest Laboratory (PNL) in the National Research Universal (NRU) reactor at Chalk River, Ontario, Canada. These tests were conducted for the US Nuclear Regulatory Commission (NRC) as part of the Severe Accident Research Program. The objectives of the test were to simulate conditions and provide information on the behavior of full-length fuel rods during hypothetical, small-break, loss-of-coolant severe accidents, in commercial light water reactors.

  18. Full-length infectious clone of a low passage dengue virus serotype 2 from Brazil

    PubMed Central

    Santos, Jefferson José da Silva; Magalhães, Tereza; Silva, José Valter Joaquim; da Silva, Andréa Nazaré Monteiro Rangel; Cordeiro, Marli Tenório; Gil, Laura Helena Vega Gonzales

    2015-01-01

    Full-length dengue virus (DENV) cDNA clones are an invaluable tool for many studies, including those on the development of attenuated or chimeric vaccines and on host-virus interactions. Furthermore, the importance of low passage DENV infectious clones should be highlighted, as these may harbour critical and unique strain-specific viral components from field-circulating isolates. The successful construction of a functional Brazilian low passage DENV serotype 2 full-length clone through homologous recombination reported here supports the use of a strategy that has been shown to be highly useful by our group for the development of flavivirus infectious clones and replicons. PMID:26200712

  19. Isolation of full-length RNA from a thermophilic cyanobacterium.

    PubMed

    Luo, X Z; Stevens, S E

    1997-11-01

    Isolation of full-length mRNA without degradation is critical in the study of in vivo gene regulation and transcription, cDNA synthesis and reverse transcription (RT)-PCR. It is particularly difficult to isolate full-length mRNA from thermophiles, which have higher turnover rates of mRNA degradation. Mastigocladus laminosus is a thermophilic heterocystous cyanobacterium. The assay of M. laminosus cell lysates showed that RNase activity was high and was resistant to the conventional guanidine thiocyanate and 2-mercaptoethanol denaturation methods. The mRNA isolated by several conventional methods was completely degraded. A method was developed to purify full-length mRNA by a combination of fast cooling, vanadyl-ribonucleoside-complex inhibition, phenol-chloroform-isoamyl alcohol extraction, lithium chloride precipitation and the lysing of cells with the French Press. This method produced high-quality, full-length mRNA in high yield. Purified mRNA was suitable for Northern blotting, cDNA synthesis and RT-PCR. This method could be applicable to other thermophiles in which the RNase activity is high and/or is resistant to guanidine thiocyanate. PMID:9383558

  20. Recovering full-length viral genomes from metagenomes

    PubMed Central

    Smits, Saskia L.; Bodewes, Rogier; Ruiz-González, Aritz; Baumgärtner, Wolfgang; Koopmans, Marion P.; Osterhaus, Albert D. M. E.; Schürch, Anita C.

    2015-01-01

    Infectious disease metagenomics is driven by the question: “what is causing the disease?” in contrast to classical metagenome studies which are guided by “what is out there?” In case of a novel virus, a first step to eventually establishing etiology can be to recover a full-length viral genome from a metagenomic sample. However, retrieval of a full-length genome of a divergent virus is technically challenging and can be time-consuming and costly. Here we discuss different assembly and fragment linkage strategies such as iterative assembly, motif searches, k-mer frequency profiling, coverage profile binning, and other strategies used to recover genomes of potential viral pathogens in a timely and cost-effective manner. PMID:26483782

  1. Renal Agenesis with Full Length Ipsilateral Refluxing Ureter.

    PubMed

    Pal, Dilip Kumar; Chandra, Vipin; Banerjee, Manju

    2016-01-01

    Unilateral renal agenesis with vesicoureteral reflux in the ipsilateral full length ureter is a rare phenomenon. Herein we report a case of 10-year old boy who presented with recurrent urinary tract infections. No renal tissue was identified on left side in various imaging studies. Micturating cystourethrogram (MCUG) showed left sided refluxing and blind ending ureter. Left ureterectomy was done because of recurrent UTI in the refluxing system. PMID:27170916

  2. Renal Agenesis with Full Length Ipsilateral Refluxing Ureter

    PubMed Central

    Chandra, Vipin; Banerjee, Manju

    2016-01-01

    Unilateral renal agenesis with vesicoureteral reflux in the ipsilateral full length ureter is a rare phenomenon. Herein we report a case of 10-year old boy who presented with recurrent urinary tract infections. No renal tissue was identified on left side in various imaging studies. Micturating cystourethrogram (MCUG) showed left sided refluxing and blind ending ureter. Left ureterectomy was done because of recurrent UTI in the refluxing system. PMID:27170916

  3. Technology development for gene discovery and full-length sequencing

    SciTech Connect

    Marcelo Bento Soares

    2004-07-19

    In previous years, with support from the U.S. Department of Energy, we developed methods for construction of normalized and subtracted cDNA libraries, and constructed hundreds of high-quality libraries for production of Expressed Sequence Tags (ESTs). Our clones were made widely available to the scientific community through the IMAGE Consortium, and millions of ESTs were produced from our libraries either by collaborators or by our own sequencing laboratory at the University of Iowa. During this grant period, we focused on (1) the development of a method for preferential cloning of tissue-specific and/or rare transcripts, (2) its utilization to expedite EST-based gene discovery for the NIH Mouse Brain Molecular Anatomy Project, (3) further development and optimization of a method for construction of full-length-enriched cDNA libraries, and (4) modification of a plasmid vector to maximize efficiency of full-length cDNA sequencing by the transposon-mediated approach. It is noteworthy that the technology developed for preferential cloning of rare mRNAs enabled identification of over 2,000 mouse transcripts differentially expressed in the hippocampus. In addition, the method that we optimized for construction of full-length-enriched cDNA libraries was successfully utilized for the production of approximately fifty libraries from the developing mouse nervous system, from which over 2,500 full-ORF-containing cDNAs have been identified and accurately sequenced in their entirety either by our group or by the NIH-Mammalian Gene Collection Program Sequencing Team.

  4. A drosophila full-length cDNA resource

    SciTech Connect

    Stapleton, Mark; Carlson, Joseph; Brokstein, Peter; Yu, Charles; Champe, Mark; George, Reed; Guarin, Hannibal; Kronmiller, Brent; Pacleb, Joanne; Park, Soo; Rubin, Gerald M.; Celniker, Susan E.

    2003-05-09

    Background: A collection of sequenced full-length cDNAs is an important resource both for functional genomics studies and for the determination of the intron-exon structure of genes. Providing this resource to the Drosophila melanogaster research community has been a long-term goal of the Berkeley Drosophila Genome Project. We have previously described the Drosophila Gene Collection (DGC), a set of putative full-length cDNAs that was produced by generating and analyzing over 250,000 expressed sequence tags (ESTs) derived from a variety of tissues and developmental stages. Results: We have generated high-quality full-insert sequence for 8,921 clones in the DGC. We compared the sequence of these clones to the annotated Release 3 genomic sequence, and identified more than 5,300 cDNAs that contain a complete and accurate protein-coding sequence. This corresponds to at least one splice form for 40 percent of the predicted D. melanogaster genes. We also identified potential new cases of RNA editing. Conclusions: We show that comparison of cDNA sequences to a high-quality annotated genomic sequence is an effective approach to identifying and eliminating defective clones from a cDNA collection and ensure its utility for experimentation. Clones were eliminated either because they carry single nucleotide discrepancies, which most probably result from reverse transcriptase errors, or because they are truncated and contain only part of the protein-coding sequence.

  5. Structural photoactivation of a full-length bacterial phytochrome

    PubMed Central

    Björling, Alexander; Berntsson, Oskar; Lehtivuori, Heli; Takala, Heikki; Hughes, Ashley J.; Panman, Matthijs; Hoernke, Maria; Niebling, Stephan; Henry, Léocadie; Henning, Robert; Kosheleva, Irina; Chukharev, Vladimir; Tkachenko, Nikolai V.; Menzel, Andreas; Newby, Gemma; Khakhulin, Dmitry; Wulff, Michael; Ihalainen, Janne A.; Westenhoff, Sebastian

    2016-01-01

    Phytochromes are light sensor proteins found in plants, bacteria, and fungi. They function by converting a photon absorption event into a conformational signal that propagates from the chromophore through the entire protein. However, the structure of the photoactivated state and the conformational changes that lead to it are not known. We report time-resolved x-ray scattering of the full-length phytochrome from Deinococcus radiodurans on micro- and millisecond time scales. We identify a twist of the histidine kinase output domains with respect to the chromophore-binding domains as the dominant change between the photoactivated and resting states. The time-resolved data further show that the structural changes up to the microsecond time scales are small and localized in the chromophore-binding domains. The global structural change occurs within a few milliseconds, coinciding with the formation of the spectroscopic meta-Rc state. Our findings establish key elements of the signaling mechanism of full-length bacterial phytochromes. PMID:27536728

  6. Conformational states of the full-length glucagon receptor

    NASA Astrophysics Data System (ADS)

    Yang, Linlin; Yang, Dehua; de Graaf, Chris; Moeller, Arne; West, Graham M.; Dharmarajan, Venkatasubramanian; Wang, Chong; Siu, Fai Y.; Song, Gaojie; Reedtz-Runge, Steffen; Pascal, Bruce D.; Wu, Beili; Potter, Clinton S.; Zhou, Hu; Griffin, Patrick R.; Carragher, Bridget; Yang, Huaiyu; Wang, Ming-Wei; Stevens, Raymond C.; Jiang, Hualiang

    2015-07-01

    Class B G protein-coupled receptors are composed of an extracellular domain (ECD) and a seven-transmembrane (7TM) domain, and their signalling is regulated by peptide hormones. Using a hybrid structural biology approach together with the ECD and 7TM domain crystal structures of the glucagon receptor (GCGR), we examine the relationship between full-length receptor conformation and peptide ligand binding. Molecular dynamics (MD) and disulfide crosslinking studies suggest that apo-GCGR can adopt both an open and closed conformation associated with extensive contacts between the ECD and 7TM domain. The electron microscopy (EM) map of the full-length GCGR shows how a monoclonal antibody stabilizes the ECD and 7TM domain in an elongated conformation. Hydrogen/deuterium exchange (HDX) studies and MD simulations indicate that an open conformation is also stabilized by peptide ligand binding. The combined studies reveal the open/closed states of GCGR and suggest that glucagon binds to GCGR by a conformational selection mechanism.

  7. Conformational states of the full-length glucagon receptor

    PubMed Central

    Yang, Linlin; Yang, Dehua; de Graaf, Chris; Moeller, Arne; West, Graham M.; Dharmarajan, Venkatasubramanian; Wang, Chong; Siu, Fai Y.; Song, Gaojie; Reedtz-Runge, Steffen; Pascal, Bruce D.; Wu, Beili; Potter, Clinton S.; Zhou, Hu; Griffin, Patrick R.; Carragher, Bridget; Yang, Huaiyu; Wang, Ming-Wei; Stevens, Raymond C.; Jiang, Hualiang

    2015-01-01

    Class B G protein-coupled receptors are composed of an extracellular domain (ECD) and a seven-transmembrane (7TM) domain, and their signalling is regulated by peptide hormones. Using a hybrid structural biology approach together with the ECD and 7TM domain crystal structures of the glucagon receptor (GCGR), we examine the relationship between full-length receptor conformation and peptide ligand binding. Molecular dynamics (MD) and disulfide crosslinking studies suggest that apo-GCGR can adopt both an open and closed conformation associated with extensive contacts between the ECD and 7TM domain. The electron microscopy (EM) map of the full-length GCGR shows how a monoclonal antibody stabilizes the ECD and 7TM domain in an elongated conformation. Hydrogen/deuterium exchange (HDX) studies and MD simulations indicate that an open conformation is also stabilized by peptide ligand binding. The combined studies reveal the open/closed states of GCGR and suggest that glucagon binds to GCGR by a conformational selection mechanism. PMID:26227798

  8. Full-length minor ampullate spidroin gene sequence.

    PubMed

    Chen, Gefei; Liu, Xiangqin; Zhang, Yunlong; Lin, Senzhu; Yang, Zijiang; Johansson, Jan; Rising, Anna; Meng, Qing

    2012-01-01

    Spider silk includes seven protein based fibers and glue-like substances produced by glands in the spider's abdomen. Minor ampullate silk is used to make the auxiliary spiral of the orb-web and also for wrapping prey, has a high tensile strength and does not supercontract in water. So far, only partial cDNA sequences have been obtained for minor ampullate spidroins (MiSps). Here we describe the first MiSp full-length gene sequence from the spider species Araneus ventricosus, using a multidimensional PCR approach. Comparative analysis of the sequence reveals regulatory elements, as well as unique spidroin gene and protein architecture including the presence of an unusually large intron. The spliced full-length transcript of MiSp gene is 5440 bp in size and encodes 1766 amino acid residues organized into conserved nonrepetitive N- and C-terminal domains and a central predominantly repetitive region composed of four units that are iterated in a non regular manner. The repeats are more conserved within A. ventricosus MiSp than compared to repeats from homologous proteins, and are interrupted by two nonrepetitive spacer regions, which have 100% identity even at the nucleotide level. PMID:23251707

  9. Full-Length Minor Ampullate Spidroin Gene Sequence

    PubMed Central

    Chen, Gefei; Liu, Xiangqin; Zhang, Yunlong; Lin, Senzhu; Yang, Zijiang; Johansson, Jan; Rising, Anna; Meng, Qing

    2012-01-01

    Spider silk includes seven protein based fibers and glue-like substances produced by glands in the spider's abdomen. Minor ampullate silk is used to make the auxiliary spiral of the orb-web and also for wrapping prey, has a high tensile strength and does not supercontract in water. So far, only partial cDNA sequences have been obtained for minor ampullate spidroins (MiSps). Here we describe the first MiSp full-length gene sequence from the spider species Araneus ventricosus, using a multidimensional PCR approach. Comparative analysis of the sequence reveals regulatory elements, as well as unique spidroin gene and protein architecture including the presence of an unusually large intron. The spliced full-length transcript of MiSp gene is 5440 bp in size and encodes 1766 amino acid residues organized into conserved nonrepetitive N- and C-terminal domains and a central predominantly repetitive region composed of four units that are iterated in a non regular manner. The repeats are more conserved within A. ventricosus MiSp than compared to repeats from homologous proteins, and are interrupted by two nonrepetitive spacer regions, which have 100% identity even at the nucleotide level. PMID:23251707

  10. Simulations of The Dalles Dam Proposed Full Length Spillwall

    SciTech Connect

    Rakowski, Cynthia L.; Perkins, William A.; Richmond, Marshall C.; Serkowski, John A.

    2008-02-25

    This report presents results of a computational fluid dynamics (CFD) modeling study to evaluatethe impacts of a full-length spillwall at The Dalles Dam. The full-length spillwall is being designed and evaluated as a structural means to improve tailrace egress and thus survival of juvenile fish passing through the spillway. During the course of this study, a full-length spillwall at Bays 6/7 and 8/9 were considered. The U.S. Army Corps of Engineers (USACE) has proposed extending the spillwall constructed in the stilling basin between spillway Bays 6 and 7 about 590 ft farther downstream. It is believed that the extension of the spillwall will improve egress conditions for downstream juvenile salmonids by moving them more rapidly into the thalweg of the river hence reducing their exposure to predators. A numerical model was created, validated, and applied the The Dalles Dam tailrace. The models were designed to assess impacts to flow, tailrace egress, navigation, and adult salmon passage of a proposed spill wall extension. The more extensive model validation undertaken in this study greatly improved our confidence in the numerical model to represent the flow conditions in The Dalles tailrace. This study used these validated CFD models to simulate the potential impacts of a spillwall extension for The Dalles Dam tailrace for two locations. We determined the following: (1)The construction of an extended wall (between Bays 6/7) will not adversely impact entering or exiting the navigation lock. Impact should be less if a wall were constructed between Bays 8/9. (2)The construction of a wall between Bays 6/7 will increase the water surface elevation between the wall and the Washington shore. Although the increased water surface elevation would be beneficial to adult upstream migrants in that it decreases velocities on the approach to the adult ladder, the increased flow depth would enhance dissolved gas production, impacting potential operations of the project because of

  11. Quantifying elongation rhythm during full-length protein synthesis.

    PubMed

    Rosenblum, Gabriel; Chen, Chunlai; Kaur, Jaskiran; Cui, Xiaonan; Zhang, Haibo; Asahara, Haruichi; Chong, Shaorong; Smilansky, Zeev; Goldman, Yale E; Cooperman, Barry S

    2013-07-31

    Pauses regulate the rhythm of ribosomal protein synthesis. Mutations disrupting even minor pauses can give rise to improperly formed proteins and human disease. Such minor pauses are difficult to characterize by ensemble methods, but can be readily examined by single-molecule (sm) approaches. Here we use smFRET to carry out real-time monitoring of the expression of a full-length protein, the green fluorescent protein variant Emerald GFP. We demonstrate significant correlations between measured elongation rates and codon and isoacceptor tRNA usage, and provide a quantitative estimate of the effect on elongation rate of replacing a codon recognizing an abundant tRNA with a synonymous codon cognate to a rarer tRNA. Our results suggest that tRNA selection plays an important general role in modulating the rates and rhythms of protein synthesis, potentially influencing simultaneous co-translational processes such as folding and chemical modification. PMID:23822614

  12. Analysis and Optimization of "Full-Length" Diodes

    SciTech Connect

    Schock, Alfred

    2012-01-19

    A method of analyzing the axial variation of the heat generation rate, temperature, voltage, current density and emitter heat flux in a thermionic converter is described. The method is particularly useful for the case of "long" diodes, each extending over the full length of the reactor core. For a given diode geometry and fuel distribution, the analysis combines a nuclear solution of the axial fission density profile with the iterative solution of four differential equations representing the thermal, electrical, and thermionic interactions within the diode. The digital computer program developed to solve these equations can also perform a design optimization with respect to lead resistance, load voltage, and emitter thickness, for a specified maximum emitter temperature. Typical results are presented, and the use of this analysis for predicting the diode operating characteristics is illustrated.

  13. Full length talin stimulates integrin activation and axon regeneration

    PubMed Central

    Tan, Chin Lik; Kwok, Jessica C.F.; Heller, Janosch P.D.; Zhao, Rongrong; Eva, Richard; Fawcett, James W.

    2015-01-01

    Integrin function is regulated by activation involving conformational changes that modulate ligand-binding affinity and downstream signaling. Activation is regulated through inside-out signaling which is controlled by many signaling pathways via a final common pathway through kindlin and talin, which bind to the intracellular tail of beta integrins. Previous studies have shown that the axon growth inhibitory molecules NogoA and chondroitin sulfate proteoglycans (CSPGs) inactivate integrins. Overexpressing kindlin-1 in dorsal root ganglion (DRG) neurons activates integrins, enabling their axons to overcome inhibitory molecules in the environment, and promoting regeneration in vivo following dorsal root crush. Other studies have indicated that expression of the talin head alone or with kindlin can enhance integrin activation. Here, using adult rat DRG neurons, we investigate the effects of overexpressing various forms of talin on axon growth and integrin signaling. We found that overexpression of the talin head activated axonal integrins but inhibited downstream signaling via FAK, and did not promote axon growth. Similarly, co-expression of the talin head and kindlin-1 prevented the growth-promoting effect of kindlin-1, suggesting that the talin head acts as a form of dominant negative for integrin function. Using full-length talin constructs in PC12 cells we observed that neurite growth was enhanced by the expression of wild-type talin and more so by two ‘activated’ forms of talin produced by point mutation (on laminin and aggrecan–laminin substrates). Nevertheless, co-expression of full-length talin with kindlin did not promote neurite growth more than either molecule alone. In vivo, we find that talin is present in PNS axons (sciatic nerve), and also in CNS axons of the corticospinal tract. PMID:25771432

  14. Generation of a Mouse Full-length Balancer with Versatile Cassette-shuttling Selection Strategy

    PubMed Central

    Ye, Zhisheng; Sun, Lei; Li, Rongbo; Han, Min; Zhuang, Yuan; Wu, Xiaohui; Xu, Tian

    2016-01-01

    Balancer chromosomes are important tools for a variety of genetic manipulations in lower model organisms, owing to their ability to suppress recombination. In mouse, however, such effort has not been accomplished, mostly due to the size of the chromosomes and the complexity of multiple step chromosomal engineering. We developed an effective and versatile cassette-shuttling selection (CASS) strategy involving only two selection markers to achieve the sequential production of multiple large inversions along the chromosome. Using this strategy, we successfully generated the first full-length balancer in mice and showed that Balancer 17M-GFP can efficiently suppress recombination. Our study has not only generated a useful genetic resource, but also provided a strategy for constructing mammalian balancer chromosomes. PMID:27489495

  15. Blueprint for a High-Performance Biomaterial: Full-Length Spider Dragline Silk Genes

    PubMed Central

    Ayoub, Nadia A.; Garb, Jessica E.; Tinghitella, Robin M.; Collin, Matthew A.; Hayashi, Cheryl Y.

    2007-01-01

    Spider dragline (major ampullate) silk outperforms virtually all other natural and manmade materials in terms of tensile strength and toughness. For this reason, the mass-production of artificial spider silks through transgenic technologies has been a major goal of biomimetics research. Although all known arthropod silk proteins are extremely large (>200 kiloDaltons), recombinant spider silks have been designed from short and incomplete cDNAs, the only available sequences. Here we describe the first full-length spider silk gene sequences and their flanking regions. These genes encode the MaSp1 and MaSp2 proteins that compose the black widow's high-performance dragline silk. Each gene includes a single enormous exon (>9000 base pairs) that translates into a highly repetitive polypeptide. Patterns of variation among sequence repeats at the amino acid and nucleotide levels indicate that the interaction of selection, intergenic recombination, and intragenic recombination governs the evolution of these highly unusual, modular proteins. Phylogenetic footprinting revealed putative regulatory elements in non-coding flanking sequences. Conservation of both upstream and downstream flanking sequences was especially striking between the two paralogous black widow major ampullate silk genes. Because these genes are co-expressed within the same silk gland, there may have been selection for similarity in regulatory regions. Our new data provide complete templates for synthesis of recombinant silk proteins that significantly improve the degree to which artificial silks mimic natural spider dragline fibers. PMID:17565367

  16. Crystal Structure of a Full-Length [beta]-Catenin

    SciTech Connect

    Xing, Yi; Takemaru, Ken-Ichi; Liu, Jing; Berndt, Jason D.; Zheng, Jie J.; Moon, Randall T.; Xu, Wenqing

    2008-08-19

    {beta}-catenin plays essential roles in cell adhesion and Wnt signaling, while deregulation of {beta}-catenin is associated with multiple diseases including cancers. Here, we report the crystal structures of full-length zebrafish {beta}-catenin and a human {beta}-catenin fragment that contains both the armadillo repeat and the C-terminal domains. Our structures reveal that the N-terminal region of the C-terminal domain, a key component of the C-terminal transactivation domain, forms a long {alpha} helix that packs on the C-terminal end of the armadillo repeat domain, and thus forms part of the {beta}-catenin superhelical core. The existence of this helix redefines our view of interactions of {beta}-catenin with some of its critical partners, including ICAT and Chibby, which may form extensive interactions with this C-terminal domain {alpha} helix. Our crystallographic and NMR studies also suggest that the unstructured N-terminal and C-terminal tails interact with the ordered armadillo repeat domain in a dynamic and variable manner.

  17. Crystal Structure of a Full-Length Autotransporter

    SciTech Connect

    van den Berg, B.

    2010-01-01

    The autotransporter (AT) secretion mechanism is the most common mechanism for the secretion of virulence factors across the outer membrane (OM) from pathogenic Gram-negative bacteria. In addition, ATs have attracted biotechnological and biomedical interest for protein display on bacterial cell surfaces. Despite their importance, the mechanism by which passenger domains of ATs pass the OM is still unclear. The classical view is that the {beta}-barrel domain provides the conduit through which the unfolded passenger moves, with the energy provided by vectorial folding of the {beta}-strand-rich passenger on the extracellular side of the OM. We present here the first structure of a full-length AT, the esterase EstA from Pseudomonas aeruginosa, at a resolution of 2.5 {angstrom}. EstA has a relatively narrow, 12-stranded {beta}-barrel that is covalently attached to the passenger domain via a long, curved helix that occupies the lumen of the {beta}-barrel. The passenger has a structure that is dramatically different from that of other known passengers, with a globular fold that is dominated by {alpha}-helices and loops. The arrangement of secondary-structure elements suggests that the passenger can fold sequentially, providing the driving force for passenger translocation. The esterase active-site residues are located at the apical surface of the passenger, at the entrance of a large hydrophobic pocket that contains a bound detergent molecule that likely mimics substrate. The EstA structure provides insight into AT mechanism and will facilitate the design of fusion proteins for cell surface display.

  18. Phylogenetic Analysis of Near Full-Length HIV Type 1 Genomic Sequences from 21 Korean Individuals

    PubMed Central

    Kim, Jung-Eun; Foley, Brian T.

    2013-01-01

    Abstract The Korean subclade of subtype B (KSB) is the most prevalent HIV-1 strain found in Korea. To date, only two near full-length HIV-1 sequences from Korean patients have been reported. Here, we analyzed a total of 24 near full-length genomes of HIV-1 strains that were isolated from 17 antiretroviral therapy (ART)-naive patients and four ART-exposed patients. Proviral DNA from peripheral blood mononuclear cells was PCR amplified and directly sequenced. Phylogenetic analyses were used to classify viruses from 19 patients as KSB, from one patient as subtype B, from one patient as subtype D, and three viruses from one patient as CRF02_AG. All KSB viruses demonstrated TAAAA instead of TATAA at the TATA box in the LTR. Of the 19 KSB patients, their sequence identities at the nucleotide level ranged from 89.8% to 97.1% from the lowest env gene to the highest pol gene. Other than the CRF02_AG viruses, no recombination events were noted in any of the 19 KSB patients, which is consistent with our previous studies on the pol, vif, and nef genes. Except for one strain, all of the strains were classified as non-syncytium-inducing strains. This is the first report to describe near full-length KSB. PMID:23199052

  19. Synaptonemal complex extension from clustered telomeres mediates full-length chromosome pairing in Schmidtea mediterranea

    PubMed Central

    Xiang, Youbin; Miller, Danny E.; Ross, Eric J.; Sánchez Alvarado, Alejandro; Hawley, R. Scott

    2014-01-01

    In the 1920s, József Gelei proposed that chromosome pairing in flatworms resulted from the formation of a telomere bouquet followed by the extension of synapsis from telomeres at the base of the bouquet, thus facilitating homolog pairing in a processive manner. A modern interpretation of Gelei’s model postulates that the synaptonemal complex (SC) is nucleated close to the telomeres and then extends progressively along the full length of chromosome arms. We used the easily visible meiotic chromosomes, a well-characterized genome, and RNAi in the sexual biotype of the planarian Schmidtea mediterranea to test that hypothesis. By identifying and characterizing S. mediterranea homologs of genes encoding synaptonemal complex protein 1 (SYCP1), the topoisomerase-like protein SPO11, and RAD51, a key player in homologous recombination, we confirmed that SC formation begins near the telomeres and progresses along chromosome arms during zygotene. Although distal regions pair at the time of bouquet formation, pairing of a unique interstitial locus is not observed until the formation of full-length SC at pachytene. Moreover, neither full extension of the SC nor homologous pairing is dependent on the formation of double-strand breaks. These findings validate Gelei’s speculation that full-length pairing of homologous chromosomes is mediated by the extension of the SC formed near the telomeres. S. mediterranea thus becomes the first organism described (to our knowledge) that forms a canonical telomere bouquet but does not require double-strand breaks for synapsis between homologous chromosomes. However, the initiation of SC formation at the base of the telomere bouquet, which then is followed by full-length homologous pairing in planarian spermatocytes, is not observed in other species and may not be conserved. PMID:25404302

  20. Characterization of full-length HIV-1 CRF17_BF genomes and comparison to the prototype CRF12_BF strains.

    PubMed

    Aulicino, Paula C; Gómez-Carrillo, Manuel; Bello, Gonzalo; Rocco, Carlos; Mangano, Andrea; Carr, Jean; Sen, Luisa; Foley, Brian

    2012-03-01

    The aim of this work is to characterize the full-length intersubtype recombinant structure of the HIV-1 Circulating Recombinant Form CRF17_BF. A single genome of CRF17_BF was originally described in 2001 as being largely similar to CRF12_BF. Since then, more genomes of CRF17_BF have been sequenced but not adequately described in publications. Here we describe CRF17_BF as a genuine CRF, and analyze its recombination pattern based on bootscan analyses, subtype signature patterns, and phylogenetic reconstruction of subtype-delimited segments. We show that CRF17_BF can be distinguished from CRF12_BF in several regions of the genome, including vpu, pol, env and nef. A complete and accurate characterization and description of recombination breakpoints in CRFs is required for a proper surveillance of HIV-1 genotypes, and important for epidemiological purposes. PMID:22266022

  1. The correlation between intron length and recombination in drosophila. Dynamic equilibrium between mutational and selective forces.

    PubMed

    Comeron, J M; Kreitman, M

    2000-11-01

    Intron length is negatively correlated with recombination in both Drosophila melanogaster and humans. This correlation is not likely to be the result of mutational processes alone: evolutionary analysis of intron length polymorphism in D. melanogaster reveals equivalent ratios of deletion to insertion in regions of high and low recombination. The polymorphism data do reveal, however, an excess of deletions relative to insertions (i.e., a deletion bias), with an overall deletion-to-insertion events ratio of 1.35. We propose two types of selection favoring longer intron lengths. First, the natural mutational bias toward deletion must be opposed by strong selection in very short introns to maintain the minimum intron length needed for the intron splicing reaction. Second, selection will favor insertions in introns that increase recombination between mutations under the influence of selection in adjacent exons. Mutations that increase recombination, even slightly, will be selectively favored because they reduce interference among selected mutations. Interference selection acting on intron length mutations must be very weak, as indicated by frequency spectrum analysis of Drosophila intron length polymorphism, making the equilibrium for intron length sensitive to changes in the recombinational environment and population size. One consequence of this sensitivity is that the advantage of longer introns is expected to decrease inversely with the rate of recombination, thus leading to a negative correlation between intron length and recombination rate. Also in accord with this model, intron length differs between closely related Drosophila species, with the longest variant present more often in D. melanogaster than in D. simulans. We suggest that the study of the proposed dynamic model, taking into account interference among selected sites, might shed light on many aspects of the comparative biology of genome sizes including the C value paradox. PMID:11063693

  2. Full length SSC R and D dipole magnet test results

    SciTech Connect

    Strait, J.; Bleadon, M.; Brown, B.C.; Hanft, R.; Kuchnir, M.; Lamm, M.; Mantsch, P.; Mazur, P.O.; Orris, D.; Peoples, J.

    1989-03-01

    Four full scale SSC development dipole magnets have been tested for mechanical and quench behavior. Two are of a design similar to previous magnets but contain a number of improvements, including more uniform coil size, higher pre-stress and a redesigned inner-outer coil splice. One exceeds the SSC operating current on the second quench but the other appears to be limited by damaged superconductor to a lower current. The other two magnets are of alternate designs. One trains erratically and fails to reach a plateau and the other reaches plateau after four quenches. 12 refs., 4 figs.

  3. Unambiguous distinction between diffusion length and surface recombination velocity of solar cells at different excitation levels

    NASA Astrophysics Data System (ADS)

    Wawer, P.; Rochel, M.; Wagemann, H.-G.

    1999-06-01

    In this work we present a conclusive separation of bulk and surface recombination properties of solar cells. For this purpose, bifacial silicon solar cells were fabricated. The backside differential spectral response of the cells has been measured in the presence of bias light, both with and without backside passivation by means of corona charging on top of a thermal oxide. Employing the common one-dimensional Shockley model, the measurement curves have been simulated. This enables the base diffusion length to be distinguished from the backside surface recombination velocity. As such, their values have been determined individually. Repeating this procedure for different intensities of bias light has yielded the nonlinear behavior of the recombination mechanisms. By applying the Schockley-Read-Hall recombination theory, it was deduced that Fe interstitials presumably are the predominant bulk recombination centers.

  4. Construction, characterization and expression of full length cDNA clone of sheep YAP1 gene.

    PubMed

    Sun, Wei; Li, Da; Su, Rui; Musa, Hassan H; Chen, Ling; Zhou, Hong

    2014-02-01

    RT-PCR, 5'RACE, 3'RACE were used to clone sheep full length cDNA sequence of YAP1 (Yes-associated protein 1), eukaryotic expression plasmid and a mutant that cannot be phosphorylated at Ser42 was successfully constructed. The amino acid sequence analysis revealed that sheep YAP1 gene encoded water-soluble protein and its relative molecular weight and isoelectric point was 44,079.0 Da and 4.91, respectively. Sub-cellular localization of YAP1 was in the nucleus, it is hydrophilic non-transmembrane and non-secreted protein. YAP1 protein contained 33 phosphorylation sites, seven glycosylation sites and two WW domains. The secondary structure of YAP1 was mainly composed of random coil, while the tertiary structure of domain area showed a forniciform helix structure. YAP1 gene was expressed in different tissues, the highest expression was in kidney and the lowest was in hypothalamus. The CDS of sheep YAP1was amplified by RT-PCR from healthy sheep longissimus dorsi muscle, cloned into pMD19-T simple vector by T/A ligation. YAP1 coding region was further sub-cloned into pEGFP-C1 vector by T4 Ligase to construct a eukaryotic expression plasmid and then make the eukaryotic expression vector as the template to construct the phosphorylation site mutant. PCR, restriction enzyme and sequencing were used to confirm the recombinant plasmid. The sheep full-length YAP1 cDNA sequence is 1712 in length encoding 403 amino acids. It was confirmed that the sheep YAP1 CDS was correctly inserted into eukaryotic expression vector and serine had been mutated to alanine by PCR, restriction digestion and sequencing. The result showed that the recombinant plasmid pEGFP-C1-YAP1 and pEGFP-C1-YAP1 S42A was constructed correctly, this will help for further studies on the YAP1 protein expression and its biological activities. PMID:24381103

  5. Three-Body Recombination of {sup 6}Li Atoms with Large Negative Scattering Lengths

    SciTech Connect

    Braaten, Eric; Kang, Daekyoung; Platter, Lucas; Hammer, H.-W.

    2009-08-14

    The three-body recombination rate at threshold for distinguishable atoms with large negative pair scattering lengths is calculated in the zero-range approximation. The only parameters in this limit are the 3 scattering lengths and the Efimov parameter, which can be complex-valued. We provide semianalytic expressions for the cases of 2 or 3 equal scattering lengths, and we obtain numerical results for the general case of 3 different scattering lengths. Our general result is applied to the three lowest hyperfine states of {sup 6}Li atoms. Comparisons with recent experiments provide indications of loss features associated with Efimov trimers near the 3-atom threshold.

  6. Surface recombination velocity and diffusion length of minority carriers in heavily doped silicon layers

    NASA Technical Reports Server (NTRS)

    Gatos, H. C.; Watanabe, M.; Actor, G.

    1977-01-01

    Quantitative analysis of the electron beam-induced current and the dependence of the effective diffusion length of the minority carriers on the penetration depth of the electron beam were employed for the analysis of the carrier recombination characteristics in heavily doped silicon layers. The analysis is based on the concept of the effective excitation strength of the carriers which takes into consideration all possible recombination sources. Two dimensional mapping of the surface recombination velocity of P-diffused Si layers will be presented together with a three dimensional mapping of minority carrier lifetime in ion implanted Si. Layers heavily doped with As exhibit improved recombination characteristics as compared to those of the layers doped with P.

  7. Stable preparations of tyrosine hydroxylase provide the solution structure of the full-length enzyme

    PubMed Central

    Bezem, Maria T.; Baumann, Anne; Skjærven, Lars; Meyer, Romain; Kursula, Petri; Martinez, Aurora; Flydal, Marte I.

    2016-01-01

    Tyrosine hydroxylase (TH) catalyzes the rate-limiting step in the biosynthesis of catecholamine neurotransmitters. TH is a highly complex enzyme at mechanistic, structural, and regulatory levels, and the preparation of kinetically and conformationally stable enzyme for structural characterization has been challenging. Here, we report on improved protocols for purification of recombinant human TH isoform 1 (TH1), which provide large amounts of pure, stable, active TH1 with an intact N-terminus. TH1 purified through fusion with a His-tagged maltose-binding protein on amylose resin was representative of the iron-bound functional enzyme, showing high activity and stabilization by the natural feedback inhibitor dopamine. TH1 purified through fusion with a His-tagged ZZ domain on TALON is remarkably stable, as it was partially inhibited by resin-derived cobalt. This more stable enzyme preparation provided high-quality small-angle X-ray scattering (SAXS) data and reliable structural models of full-length tetrameric TH1. The SAXS-derived model reveals an elongated conformation (Dmax = 20 nm) for TH1, different arrangement of the catalytic domains compared with the crystal structure of truncated forms, and an N-terminal region with an unstructured tail that hosts the phosphorylation sites and a separated Ala-rich helical motif that may have a role in regulation of TH by interacting with binding partners. PMID:27462005

  8. Stable preparations of tyrosine hydroxylase provide the solution structure of the full-length enzyme.

    PubMed

    Bezem, Maria T; Baumann, Anne; Skjærven, Lars; Meyer, Romain; Kursula, Petri; Martinez, Aurora; Flydal, Marte I

    2016-01-01

    Tyrosine hydroxylase (TH) catalyzes the rate-limiting step in the biosynthesis of catecholamine neurotransmitters. TH is a highly complex enzyme at mechanistic, structural, and regulatory levels, and the preparation of kinetically and conformationally stable enzyme for structural characterization has been challenging. Here, we report on improved protocols for purification of recombinant human TH isoform 1 (TH1), which provide large amounts of pure, stable, active TH1 with an intact N-terminus. TH1 purified through fusion with a His-tagged maltose-binding protein on amylose resin was representative of the iron-bound functional enzyme, showing high activity and stabilization by the natural feedback inhibitor dopamine. TH1 purified through fusion with a His-tagged ZZ domain on TALON is remarkably stable, as it was partially inhibited by resin-derived cobalt. This more stable enzyme preparation provided high-quality small-angle X-ray scattering (SAXS) data and reliable structural models of full-length tetrameric TH1. The SAXS-derived model reveals an elongated conformation (Dmax = 20 nm) for TH1, different arrangement of the catalytic domains compared with the crystal structure of truncated forms, and an N-terminal region with an unstructured tail that hosts the phosphorylation sites and a separated Ala-rich helical motif that may have a role in regulation of TH by interacting with binding partners. PMID:27462005

  9. Infectious full-length clones of Calibrachoa Mottle Virus (CbMV)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Full-length cDNA clones derived from genomic RNA (gRNA) and subgenomic RNAs (sgRNAs) of Calibrachoa mottle virus (CbMV) were constructed under the control of the T7 RNA promoter and ligated into plasmid pUC-18. The capped and uncapped in vitro transcripts, synthesized from full length genomic cDNA...

  10. Development of a full-length cDNA-derived enterovirus A71 vaccine candidate using reverse genetics technology.

    PubMed

    Yang, Ya-Ting; Chow, Yen-Hung; Hsiao, Kuang-Nan; Hu, Kai-Chieh; Chiang, Jen-Ron; Wu, Suh-Chin; Chong, Pele; Liu, Chia-Chyi

    2016-08-01

    Enterovirus A71 (EV-A71) is responsible for epidemics of hand, foot and mouth disease (HFMD) in young children. To circumvent difficulties in obtaining clinical enterovirus isolates that might be contaminated with other viruses, a platform technology was developed to quickly generate vaccine virus strains based on the published enterovirus genomic sequences. A recombinant plasmid containing the full-length infectious cDNA clone of EV-A71 vaccine strain E59 was directly generated after transfecting the recombinant plasmid into Vero, RD or HEK293A cells, and phenotypic characteristics similar to the parental strain were observed. The cDNA-derived infectious EV-A71 virus grown in Vero cells produced relatively stable virus titers in both T-flasks and microcarrier culture systems. To evaluate the genetic stability of the cDNA-derived EV-A71 viruses, the immunodominant structural proteins, VP1 and VP2, of the recombinant EV-A71 viruses were sequenced and analyzed. The cDNA-derived EV-A71 virus showed weak pathogenicity in a human SCARB2 mouse model. These results show the successful generation of a recombinant virus derived from a published viral genomic sequence that demonstrated good genetic stability and viral yields, which could represent an efficient and safe vaccine strain for cGMP-grade manufacturing. PMID:27387826

  11. Minority carrier diffusion length and edge surface-recombination velocity in InP

    NASA Technical Reports Server (NTRS)

    Hakimzadeh, Roshanak; Bailey, Sheila G.

    1993-01-01

    A scanning electron microscope was used to obtain the electron-beam-induced current (EBIC) profiles in InP specimens containing a Schottky barrier perpendicular to the scanned (edge) surface. An independent technique was used to measure the edge surface-recombination velocity. These values were used in a fit of the experimental EBIC data with a theoretical expression for normalized EBIC (Donolato, 1982) to obtain the electron (minority carrier) diffusion length.

  12. Full-Length Semaphorin-3C Is an Inhibitor of Tumor Lymphangiogenesis and Metastasis.

    PubMed

    Mumblat, Yelena; Kessler, Ofra; Ilan, Neta; Neufeld, Gera

    2015-06-01

    Semaphorins play important regulatory roles in diverse processes such as axon guidance, angiogenesis, and immune responses. We find that semaphorin-3C (sema3C) induces the collapse of the cytoskeleton of lymphatic endothelial cells (LEC) in a neuropilin-2-, plexin-D1-, and plexin-A1-dependent manner, while most other semaphorins, including antiangiogenic semaphorins such as sema3A do not. Sema3C is cleaved, like other class-3 semaphorins, by furin-like pro-protein convertases (FPPC). Cleaved sema3C (p65-Sema3C) was unable to induce the collapse of the cytoskeleton of LEC. FPPC are strongly upregulated in tumor cells. In order to examine the effects of full-length sema3C on tumor progression, we therefore generated an active point mutated furin cleavage-resistant sema3C (FR-sema3C). FR-sema3C inhibited potently proliferation of LEC and to a lesser extent proliferation of human umbilical vein-derived endothelial cells. FR-sema3C also inhibited VEGF-C-induced phosphorylation of VEGFR-3, ERK1/2, and AKT. Expression of recombinant FR-sema3C in metastatic, triple-negative LM2-4 breast cancer cells did not affect their migration or proliferation in vitro. However, tumors derived from FR-sema3C-expressing LM2-4 cells implanted in mammary fat pads developed at a slower rate, contained a lower concentration of blood vessels and lymph vessels, and metastasized much less effectively to lymph nodes. Interestingly, p65-Sema3C, but not FR-sema3C, rendered A549 lung cancer cells resistant to serum deprivation, suggesting that previously reported protumorigenic activities of sema3C may be due to p65-Sema3C produced by tumor cells. Our observations suggest that FR-sema3C may be further developed into a novel antitumorigenic drug. PMID:25808871

  13. Construction and characterization of bacterial artificial chromosomes (BACs) containing herpes simplex virus full-length genomes.

    PubMed

    Nagel, Claus-Henning; Pohlmann, Anja; Sodeik, Beate

    2014-01-01

    Bacterial artificial chromosomes (BACs) are suitable vectors not only to maintain the large genomes of herpesviruses in Escherichia coli but also to enable the traceless introduction of any mutation using modern tools of bacterial genetics. To clone a herpes simplex virus genome, a BAC replication origin is first introduced into the viral genome by homologous recombination in eukaryotic host cells. As part of their nuclear replication cycle, genomes of herpesviruses circularize and these replication intermediates are then used to transform bacteria. After cloning, the integrity of the recombinant viral genomes is confirmed by restriction length polymorphism analysis and sequencing. The BACs may then be used to design virus mutants. Upon transfection into eukaryotic cells new herpesvirus strains harboring the desired mutations can be recovered and used for experiments in cultured cells as well as in animal infection models. PMID:24671676

  14. Retrotransposon mdg3 of Drosophila: General structure and functional domains of the full-length copy

    SciTech Connect

    Avedisov, S.N.; Ilyin, Yu.V.

    1995-09-01

    A full-length copy of the transposable element mdg3 from the plasmid clone Dm38 of Drosophila melanogaster was obtained by screening the DNA library of the cell culture 67J25D. Previous work demonstrated that only full-length copies of mdg3 (5.5 kb) are amplified in this culture, whereas the number of deleted copies probably has not changed since the cell line was established. We sequenced the full-length copy of mdg3 from cDm38 by the method described by Sanger. 10 refs., 2 figs., 2 tabs.

  15. Infectious RNA transcripts from full-length dengue virus type 2 cDNA clones made in yeast.

    PubMed Central

    Polo, S; Ketner, G; Levis, R; Falgout, B

    1997-01-01

    The dengue virus type 2 genomic RNA was amplified by reverse transcription-PCR and cloned as four cDNA fragments. We could not assemble these four fragments into full-length cDNA in Escherichia coli. The full-length dengue virus cDNA was constructed by homologous recombination in yeast, either as part of a yeast artificial chromosome or in a yeast-E. coli shuttle vector. Full-length cDNA clones were propagated once in E. coli to prepare useful quantities of DNA. In vitro transcription of these clones produced full-length RNA transcripts. Introduction of these transcripts into LLC-MK2 cells produced typical dengue infection, as judged by cytopathic effects and indirect immunofluorescence. Infectivity was sensitive to RNase digestion and was dependent on the presence of cap analog in the transcription reaction mixture. Virus in the medium was passaged on C6-36 cells to produce stocks, and these stocks had titers and plaque morphologies similar to those of the parental dengue virus type 2. Intracellular dengue virus RNA from cells infected with transcript-derived virus contained an introduced BstEII site, proving that infectivity was derived from RNA transcripts and not from contamination with parental dengue virus. Transcript-derived virus was comparable to dengue virus type 2 for growth and protein expression in tissue culture cells. Sequence analysis of the dengue virus cDNA in one full-length clone revealed only one unexpected silent mutation. By using yeast technology, it will be easy to introduce specific mutations into the dengue virus cDNA, allowing analysis of the virus phenotype in cells transfected with mutant transcripts. PMID:9188607

  16. Effective-range corrections to three-body recombination for atoms with large scattering length

    SciTech Connect

    Hammer, H.-W.; Laehde, Timo A.; Platter, L.

    2007-03-15

    Few-body systems with large scattering length a have universal properties that do not depend on the details of their interactions at short distances. The rate constant for three-body recombination of bosonic atoms of mass m into a shallow dimer scales as ({Dirac_h}/2{pi})a{sup 4}/m times a log-periodic function of the scattering length. We calculate the leading and subleading corrections to the rate constant, which are due to the effective range of the atoms, and study the correlation between the rate constant and the atom-dimer scattering length. Our results are applied to {sup 4}He atoms as a test case.

  17. [Comparison of methods to construct a full-length cDNA library].

    PubMed

    Mao, Xin-Guo; Jing, Rui-Lian; Kong, Xiu-Ying; Zhao, Guang-Yao; Jia, Ji-Zeng

    2006-07-01

    The use of full-length cDNA libraries is an effective tool to obtain complete gene information in a high-efficiency, high-throughput manner, especially in organisms with huge genomes that are not amenable to whole genome sequencing. In this review, we outlined several methods of full-length cDNA library construction and compared their advantages and disadvantages based on their respective principles. Drawing on our own experience, we described the Cap-trapper method in detail, with an emphasis on its application in wheat full-length cDNA library construction as well as the determination of the ratio of full-length cDNA in a library. PMID:16825176

  18. Fabrication and Testing of Full-Length Single-Cell Externally Fueled Converters for Thermionic Reactors

    SciTech Connect

    Schock, Alfred

    1995-08-01

    Paper presented at the 29th IECEC in Monterey, CA in August 1994. The present paper describes the fabrication and testing of full-length prototypcial converters, both unfueled and fueled, and presents parametric results of electrically heated tests.

  19. Characteristic lengths for three-carrier transport with spin-flip and electron-hole recombination

    NASA Astrophysics Data System (ADS)

    Krcmar, Maja; Saslow, Wayne M.

    2016-05-01

    The exact solution of the linearized, steady-state transport equation for three-carrier systems, such as can occur for semiconductors and ionic conductors, is constructed starting from the near-equilibrium entropy-production requirements of irreversible thermodynamics. Three characteristic modes are found, one associated with electrostatic screening (which is often neglected), and two modes associated with diffusion and "reactions." For a spintronics model with up and down electrons and unpolarized holes, the "reactions" are spin-flip and electron-hole recombination. We discuss how the variations in carrier density, diffusivity, recombination rate, and spin relaxation time affect the characteristic lengths. We apply these modes to study spin-polarized surface photoabsorption.

  20. A systematic approach for testing expression of human full-length proteins in cell-free expression systems

    PubMed Central

    Langlais, Claudia; Guilleaume, Birgit; Wermke, Nadja; Scheuermann, Tina; Ebert, Lars; LaBaer, Joshua; Korn, Bernhard

    2007-01-01

    Background The growing field of proteomics and systems biology is resulting in an ever increasing demand for purified recombinant proteins for structural and functional studies. Here, we show a systematic approach to successfully express a full-length protein of interest by using cell-free and cell-based expression systems. Results In a pre-screen, we evaluated the expression of 960 human full-length open reading frames in Escherichia coli (in vivo and in vitro). After analysing the protein expression rate and solubility, we chose a subset of 87 plasmids yielding no protein product in E. coli in vivo. These targets were subjected to a more detailed analysis comparing a prokaryotic cell-free E. coli system with an eukaryotic wheat germ system. In addition, we determined the expression rate, yield and solubility of those proteins. After sequence optimisation for the E. coli in vitro system and generating linear templates for wheat germ expression, the success rate of cell-free protein expression reached 93%. Conclusion We have demonstrated that protein expression in cell-free systems is an appropriate technology for the successful expression of soluble full-length proteins. In our study, wheat germ expression using a two compartment system is the method of choice as it shows high solubility and high protein yield. PMID:17915018

  1. In vitro translation of the full-length RNA transcript of figwort mosaic virus (Caulimovirus).

    PubMed

    Ranu, R S; Gowda, S; Scholthof, H; Wu, F C; Shepherd, R J

    1996-01-01

    The circular DNA genome of FMV consists of seven tandemly arranged genes placed successively on a full-length RNA transcript that spans the entire circular viral genome. This transcript is a tentative mRNA for at least five of the six major conserved genes of this virus (genes I-V) that are positioned on this transcript. The sixth major gene (gene VI) is expressed as a separate monocistronic transcript. A long 5'-nontranslated leader (598 nucleotides), a small nonconserved gene (VII), and a short intergenic region (57 nucleotides) precede the five major conserved genes (I through V) on the full-length transcript. A reporter gene (CAT), as a separate cistron or fused in-frame, to viral cistrons in various downstream positions in cloned versions of the viral genome was used in a transcription vector to generate artificial full-length transcripts of FMV. When these mRNAs were translated in vitro (rabbit reticulocyte lysate system), the reporter gene was translated efficiently in all positions. Translation of internal native viral gene positioned on the full-length transcript of FMV was also determined (the gene VI product). These observations suggest that the full-length FMV transcript functions as a polycistronic mRNA in plants. Results are best explained on the basis of translational coupling/relay race model. PMID:8882638

  2. Chinese hamster ovary cells contain transcriptionally active full-length type C proviruses.

    PubMed Central

    Lie, Y S; Penuel, E M; Low, M A; Nguyen, T P; Mangahas, J O; Anderson, K P; Petropoulos, C J

    1994-01-01

    We have isolated a genomic locus from Chinese hamster ovary (CHO) cells that contains a full-length provirus. Nucleotide sequence analysis indicates that it is a defective member of the rodent type C retrovirus family with an env region that is similar to those of mouse amphotropic retrovirus and subgroup B feline leukemia virus. We were able to demonstrate that this provirus is a member of a closely related family of full-length proviruses in CHO cells and Chinese hamster liver. Hybridization probes generated from this genomic clone were used to characterize type C retrovirus RNA expression in CHO cells. Full-length genomic RNA and subgenomic envelope mRNA were detected in CHO cell lines but not in the human-derived 293 cell line. Interestingly, we discovered that the site of retrovirus integration lies within a G repeat sequence belonging to the short interspersed element family of retroposons. Images PMID:7966574

  3. Chinese hamster ovary cells contain transcriptionally active full-length type C proviruses.

    PubMed

    Lie, Y S; Penuel, E M; Low, M A; Nguyen, T P; Mangahas, J O; Anderson, K P; Petropoulos, C J

    1994-12-01

    We have isolated a genomic locus from Chinese hamster ovary (CHO) cells that contains a full-length provirus. Nucleotide sequence analysis indicates that it is a defective member of the rodent type C retrovirus family with an env region that is similar to those of mouse amphotropic retrovirus and subgroup B feline leukemia virus. We were able to demonstrate that this provirus is a member of a closely related family of full-length proviruses in CHO cells and Chinese hamster liver. Hybridization probes generated from this genomic clone were used to characterize type C retrovirus RNA expression in CHO cells. Full-length genomic RNA and subgenomic envelope mRNA were detected in CHO cell lines but not in the human-derived 293 cell line. Interestingly, we discovered that the site of retrovirus integration lies within a G repeat sequence belonging to the short interspersed element family of retroposons. PMID:7966574

  4. Internalization of the Extracellular Full-Length Tau Inside Neuro2A and Cortical Cells Is Enhanced by Phosphorylation.

    PubMed

    Wauters, Mathilde; Wattiez, Ruddy; Ris, Laurence

    2016-01-01

    Tau protein is mainly intracellular. However, several studies have demonstrated that full-length Tau can be released into the interstitial fluid of the brain. The physiological or pathological function of this extracellular Tau remains unknown. Moreover, as evidence suggests, extracellular Tau aggregates can be internalized by neurons, seeding Tau aggregation. However, much less is known about small species of Tau. In this study, we hypothesized that the status of phosphorylation could alter the internalization of recombinant Tau in Neuro2A and cortical cells. Our preliminary results revealed that the highly phosphorylated form of Tau entered the cells ten times more easily than a low phosphorylated one. This suggests that hyperphosphorylated Tau protein could spread between neurons in pathological conditions such as Alzheimer's disease. PMID:27548242

  5. Conditional expression of full-length humanized anti-prion protein antibodies in Chinese hamster ovary cells.

    PubMed

    Mueller, Daniel A; Heinig, Lars; Ramljak, Sanja; Krueger, Astrid; Schulte, Reiner; Wrede, Arne; Stuke, Andreas W

    2010-12-01

    Because of their high antigen specificity and metabolic stability, genetically engineered human monoclonal antibodies are on the way to becoming one of the most promising medical diagnostics and therapeutics. In order to establish an in vitro system capable of producing such biosimilar antibodies, we used human constant chain sequences to design the novel human antibody expressing vector cassette pMAB-ABX. A bidirectional tetracycline (tet)-controllable promotor was used for harmonized expression of immunoglobulin type G (IgG) heavy and light chains. As an example we used anti-prion protein (anti-PrP) IgGs. Therefore, the variable heavy (V(H)) and light chain (V(L)) sequences of anti-PrP antibodies, previously generated in our laboratory by DNA immunization of prion protein knock-out mice, were isolated from murine hybridoma cell lines and inserted into pMAB-ABX vector. After transfection of Chinese hamster ovary (CHO) cells, a number of stable antibody producing cell clones were selected. One cell line (pMAB-ABX-13F10/3B5) stably expressing the recombinant humanized antibody (rechuAb) 13F10/3B5 was selected for detailed characterization by Western blot, immunofluorescence, and flow cytometric analyses. The full-length recombinant humanized IgG antibody showed a high level of expression in the cytoplasm. In conclusion, the new cell system described here is a suitable tool to produce functional intact full-length humanized IgG antibodies. PMID:21087094

  6. piggyBac transposons expressing full-length human dystrophin enable genetic correction of dystrophic mesoangioblasts

    PubMed Central

    Loperfido, Mariana; Jarmin, Susan; Dastidar, Sumitava; Di Matteo, Mario; Perini, Ilaria; Moore, Marc; Nair, Nisha; Samara-Kuko, Ermira; Athanasopoulos, Takis; Tedesco, Francesco Saverio; Dickson, George; Sampaolesi, Maurilio; VandenDriessche, Thierry; Chuah, Marinee K.

    2016-01-01

    Duchenne muscular dystrophy (DMD) is a genetic neuromuscular disorder caused by the absence of dystrophin. We developed a novel gene therapy approach based on the use of the piggyBac (PB) transposon system to deliver the coding DNA sequence (CDS) of either full-length human dystrophin (DYS: 11.1 kb) or truncated microdystrophins (MD1: 3.6 kb; MD2: 4 kb). PB transposons encoding microdystrophins were transfected in C2C12 myoblasts, yielding 65±2% MD1 and 66±2% MD2 expression in differentiated multinucleated myotubes. A hyperactive PB (hyPB) transposase was then deployed to enable transposition of the large-size PB transposon (17 kb) encoding the full-length DYS and green fluorescence protein (GFP). Stable GFP expression attaining 78±3% could be achieved in the C2C12 myoblasts that had undergone transposition. Western blot analysis demonstrated expression of the full-length human DYS protein in myotubes. Subsequently, dystrophic mesoangioblasts from a Golden Retriever muscular dystrophy dog were transfected with the large-size PB transposon resulting in 50±5% GFP-expressing cells after stable transposition. This was consistent with correction of the differentiated dystrophic mesoangioblasts following expression of full-length human DYS. These results pave the way toward a novel non-viral gene therapy approach for DMD using PB transposons underscoring their potential to deliver large therapeutic genes. PMID:26682797

  7. piggyBac transposons expressing full-length human dystrophin enable genetic correction of dystrophic mesoangioblasts.

    PubMed

    Loperfido, Mariana; Jarmin, Susan; Dastidar, Sumitava; Di Matteo, Mario; Perini, Ilaria; Moore, Marc; Nair, Nisha; Samara-Kuko, Ermira; Athanasopoulos, Takis; Tedesco, Francesco Saverio; Dickson, George; Sampaolesi, Maurilio; VandenDriessche, Thierry; Chuah, Marinee K

    2016-01-29

    Duchenne muscular dystrophy (DMD) is a genetic neuromuscular disorder caused by the absence of dystrophin. We developed a novel gene therapy approach based on the use of the piggyBac (PB) transposon system to deliver the coding DNA sequence (CDS) of either full-length human dystrophin (DYS: 11.1 kb) or truncated microdystrophins (MD1: 3.6 kb; MD2: 4 kb). PB transposons encoding microdystrophins were transfected in C2C12 myoblasts, yielding 65±2% MD1 and 66±2% MD2 expression in differentiated multinucleated myotubes. A hyperactive PB (hyPB) transposase was then deployed to enable transposition of the large-size PB transposon (17 kb) encoding the full-length DYS and green fluorescence protein (GFP). Stable GFP expression attaining 78±3% could be achieved in the C2C12 myoblasts that had undergone transposition. Western blot analysis demonstrated expression of the full-length human DYS protein in myotubes. Subsequently, dystrophic mesoangioblasts from a Golden Retriever muscular dystrophy dog were transfected with the large-size PB transposon resulting in 50±5% GFP-expressing cells after stable transposition. This was consistent with correction of the differentiated dystrophic mesoangioblasts following expression of full-length human DYS. These results pave the way toward a novel non-viral gene therapy approach for DMD using PB transposons underscoring their potential to deliver large therapeutic genes. PMID:26682797

  8. Full-Length cDNA, Prokaryotic Expression, and Antimicrobial Activity of UuHb-F-I from Urechis unicinctus

    PubMed Central

    Niu, Rongli; Chen, Xiang

    2016-01-01

    Hemoglobin, which widely exists in all vertebrates and in some invertebrates, is possibly a precursor of antimicrobial peptides (AMPs). However, AMPs in the hemoglobin of invertebrates have been rarely investigated. This study is the first to report the full-length cDNA, prokaryotic expression, and antimicrobial activity of UuHb-F-I from Urechis unicinctus. The full-length cDNA sequence of UuHb-F-I was 780 bp with an open-reading frame of 429 bp encoding 142 amino acids. MALDI-TOF-MS suggested that the recombinant protein of UuHb-F-I (rUuHb-F-I) yielded a molecular weight of 15,168.01 Da, and its N-terminal amino acid sequence was MGLTGAQIDAIK. rUuHb-F-I exhibited different antimicrobial activities against microorganisms. The lowest minimum inhibitory concentration against Micrococcus luteus was 2.78–4.63 μM. Our results may help elucidate the immune defense mechanism of U. unicinctus and may provide insights into new AMPs in drug discovery. PMID:27471730

  9. Full-Length cDNA, Prokaryotic Expression, and Antimicrobial Activity of UuHb-F-I from Urechis unicinctus.

    PubMed

    Niu, Rongli; Chen, Xiang

    2016-01-01

    Hemoglobin, which widely exists in all vertebrates and in some invertebrates, is possibly a precursor of antimicrobial peptides (AMPs). However, AMPs in the hemoglobin of invertebrates have been rarely investigated. This study is the first to report the full-length cDNA, prokaryotic expression, and antimicrobial activity of UuHb-F-I from Urechis unicinctus. The full-length cDNA sequence of UuHb-F-I was 780 bp with an open-reading frame of 429 bp encoding 142 amino acids. MALDI-TOF-MS suggested that the recombinant protein of UuHb-F-I (rUuHb-F-I) yielded a molecular weight of 15,168.01 Da, and its N-terminal amino acid sequence was MGLTGAQIDAIK. rUuHb-F-I exhibited different antimicrobial activities against microorganisms. The lowest minimum inhibitory concentration against Micrococcus luteus was 2.78-4.63 μM. Our results may help elucidate the immune defense mechanism of U. unicinctus and may provide insights into new AMPs in drug discovery. PMID:27471730

  10. Progress towards large gain-length products on the Li-like recombination scheme

    SciTech Connect

    Zeitoun, P.; Jamelot, G.; Carillon, A.; Goedtkindt, P.; Guennou, H.; Jaegle, P.; Klisnick, A.; Moeller, C.; Rus, B.; Sureau, A.

    1995-05-01

    Investigating possibilities of attaining large gain-length products on the recombination scheme using lithium-like ions, we have examined two approaches aimed at overcoming the problem of plasma non-uniformity susceptible to destroy gain by a number of processes. In the first approach we studied amplification on the transitions 5f-3d and 4f-3d in Li-like Al{sup 10+} plasma column produced by smoothing optics using lens arrays. Employing this device resulted in the gain holding up significantly longer than when no smoothing optics was used. Second, we have investigated numerically and experimentally the 5g-4f transition in Li-like S{sup 13+}, as the gain should be barely affected by the plasma nonuniformities. Encouraging results were obtained and their various aspects are discussed.

  11. Recombination within a Subclass of Restriction Fragment Length Polymorphisms May Help Link Classical and Molecular Genetics

    PubMed Central

    Meagher, R. B.; McLean, M. D.; Arnold, J.

    1988-01-01

    Restriction fragment length polymorphisms (RFLPs) are being used to construct complete linkage maps for many eukaryotic genomes. These RFLP maps can be used to predict the inheritance of important phenotypic loci and will assist in the molecular cloning of linked gene(s) which affect phenotypes of scientific, medical and agronomic importance. However, genetic linkage implies very little about the actual physical distances between loci. An assay is described which uses genetic recombinants to measure physical distance from a DNA probe to linked phenotypic loci. We have defined the subset of all RFLPs which have polymorphic restriction sites at both ends as class II RFLPs. The frequency of class II RFLPs is computed as a function of sequence divergence and total RFLP frequency for highly divergent genomes. Useful frequencies exist between organisms which differ by more than 7% in DNA sequence. Recombination within class II RFLPs will produce fragments of novel sizes which can be assayed by pulsed field electrophoresis to estimate physical distance in kilobase pairs between linked RFLP and phenotypic loci. This proposed assay should have particular applications to crop plants where highly divergent and polymorphic species are often genetically compatible and thus, where class II RFLPs will be most frequent. PMID:2906304

  12. The structure of full-length LysR-type transcriptional regulators. Modeling of the full-length OxyR transcription factor dimer

    PubMed Central

    Zaim, Jolanta; Kierzek, Andrzej M.

    2003-01-01

    The LysR-type transcriptional regulators (LTTRs) comprise the largest family of prokaryotic transcription factors. These proteins are composed of an N-terminal DNA binding domain (DBD) and a C-terminal cofactor binding domain. To date, no structure of the DBD has been solved. According to the SUPERFAMILY and MODBASE databases, a reliable homology model of LTTR DBDs may be built using the structure of the Escherichia coli ModE transcription factor, containing a winged helix– turn–helix (HTH) motif, as a template. The remote, but statistically significant, sequence similarity between ModE and LTTR DBDs and an alignment generated using SUPERFAMILY and MODBASE methods was independently confirmed by alignment of sequence profiles representing ModE and LTTR family DBDs. Using the crystal structure of the E.coli OxyR C-terminal domain and the DBD alignments we constructed a structural model of the full-length dimer of this LTTR family member and used it to investigate the mode of protein–DNA interaction. We also applied the model to interpret, in a structural context, the results of numerous biochemical studies of mutated LTTRs. A comparison of the LTTR DBD model with the structures of other HTH proteins also provides insights into the interaction of LTTRs with the C-terminal domain of the RNA polymerase α subunit. PMID:12595552

  13. Measurement of diffusion length and surface recombination velocity in Interdigitated Back Contact (IBC) and Front Surface Field (FSF) solar cells

    NASA Astrophysics Data System (ADS)

    Verlinden, Pierre; Van de Wiele, Fernand

    1985-03-01

    A method is proposed for measuring the diffusion length and surface recombination velocity of Interdigitated Back Contact (IBC) solar cells by means of a simple linear regression on experimental quantum efficiency values versus the inverse of the absorption coefficient. This method is extended to the case of Front Surface Field (FSF) solar cells. Under certain conditions, the real or the effective surface recombination velocity may be measured.

  14. Loss of GATA-1 full length as a cause of Diamond-Blackfan anemia phenotype.

    PubMed

    Parrella, Sara; Aspesi, Anna; Quarello, Paola; Garelli, Emanuela; Pavesi, Elisa; Carando, Adriana; Nardi, Margherita; Ellis, Steven R; Ramenghi, Ugo; Dianzani, Irma

    2014-07-01

    Mutations in the hematopoietic transcription factor GATA-1 alter the proliferation/differentiation of hemopoietic progenitors. Mutations in exon 2 interfere with the synthesis of the full-length isoform of GATA-1 and lead to the production of a shortened isoform, GATA-1s. These mutations have been found in patients with Diamond-Blackfan anemia (DBA), a congenital erythroid aplasia typically caused by mutations in genes encoding ribosomal proteins. We sequenced GATA-1 in 23 patients that were negative for mutations in the most frequently mutated DBA genes. One patient showed a c.2T > C mutation in the initiation codon leading to the loss of the full-length GATA-1 isoform. PMID:24453067

  15. Structure of the full-length TRPV2 channel by cryo-EM

    NASA Astrophysics Data System (ADS)

    Huynh, Kevin W.; Cohen, Matthew R.; Jiang, Jiansen; Samanta, Amrita; Lodowski, David T.; Zhou, Z. Hong; Moiseenkova-Bell, Vera Y.

    2016-03-01

    Transient receptor potential (TRP) proteins form a superfamily Ca2+-permeable cation channels regulated by a range of chemical and physical stimuli. Structural analysis of a `minimal' TRP vanilloid subtype 1 (TRPV1) elucidated a mechanism of channel activation by agonists through changes in its outer pore region. Though homologous to TRPV1, other TRPV channels (TRPV2-6) are insensitive to TRPV1 activators including heat and vanilloids. To further understand the structural basis of TRPV channel function, we determined the structure of full-length TRPV2 at ~5 Å resolution by cryo-electron microscopy. Like TRPV1, TRPV2 contains two constrictions, one each in the pore-forming upper and lower gates. The agonist-free full-length TRPV2 has wider upper and lower gates compared with closed and agonist-activated TRPV1. We propose these newly revealed TRPV2 structural features contribute to diversity of TRPV channels.

  16. [Isolation, identification and full-length genome sequence analysis of encephalomyocarditis virus from local aardvarks].

    PubMed

    Chang, Hong-Tao; Liu, Hui-Min; He, Xiu-Yuan; Zhao, Jun; Chen, Lu; Wang, Xin-Wei; Yang, Xia; Yao, Hui-Xia; Wang, Chuan-Qing

    2014-07-01

    Encephalomyocarditis virus (EMCV) is a natural epidemic zoonotic pathogen. However, no reports have been published regarding the isolation, identification and full-length genome of EMCV from a local aardvark population. In present study, an EMCV isolate HNXX13 was isolated from aardvarks named Huainan-pig in Henan Province. The systematic identification, full-length genome sequencing and molecular characteristic analysis of the isolate HNXX13 were conducted. The result showed that the isolate was spherical with a diameter of 24-30 nm, neither heat- nor acid-resistant, sensitive to trypsin, insensitive to chloroform, not protected by bivalent cationic, and the specific fluorescence was observed in the cytoplasm of BHK-21 cells infected with the isolate by using indirect fluorescence assay. The full-length genome of EMCV HNXX13 generated a 7 725bp sequence (GenBank: F771002), with 81.0%-99.9% nucleotide identity to reference strains from different animals, and 99.5% with a Chinese reference strain isolated earlier from a commercial pig herd. The phylogenetic tree based on the full-length genome and ORF sequences identified that all EMCV strains were divided into three groups G1, G2 and G3, and strain HNXX13 belonging to the G1 group with other Chinese reference strains. The result also identified that this EMCV infection could cause severe clinical signs in a local aardvark population, and enriches the molecular epidemiological data of EMCV in China. Regional differences exist in EMCV genome and transmission is limited within a certain area. However, the cross-infection and transmission of EMCV between aardvark and mice appears most likely. Mutations have occurred in some amino acids of EMCV strain HNXX13 during the transmission in local aardvark herd and these mutations might make the virus easier to infect the aardvark. PMID:25272589

  17. Full-length high-temperature severe fuel damage test No. 2. Final safety analysis

    SciTech Connect

    Hesson, G.M.; Lombardo, N.J.; Pilger, J.P.; Rausch, W.N.; King, L.L.; Hurley, D.E.; Parchen, L.J.; Panisko, F.E.

    1993-09-01

    Hazardous conditions associated with performing the Full-Length High- Temperature (FLHT). Severe Fuel Damage Test No. 2 experiment have been analyzed. Major hazards that could cause harm or damage are (1) radioactive fission products, (2) radiation fields, (3) reactivity changes, (4) hydrogen generation, (5) materials at high temperature, (6) steam explosion, and (7) steam pressure pulse. As a result of this analysis, it is concluded that with proper precautions the FLHT- 2 test can be safely conducted.

  18. Detection and Full-Length Genome Characterization of Novel Canine Vesiviruses

    PubMed Central

    Pinto, Pierfrancesco; Lorusso, Eleonora; Di Martino, Barbara; Wang, Qiuhong; Larocca, Vittorio; Cavalli, Alessandra; Camero, Michele; Decaro, Nicola; Bányai, Krisztián; Saif, Linda J.; Buonavoglia, Canio

    2015-01-01

    Vesiviruses have been detected in several animal species and as accidental contaminants of cells. We detected vesiviruses in asymptomatic kennel dogs (64.8%) and symptomatic (1.1%) and asymptomatic (3.5%) household dogs in Italy. The full-length genome of 1 strain, Bari/212/07/ITA, shared 89%–90% nt identity with vesiviruses previously detected in contaminated cells. PMID:26196075

  19. [Construction and sequencing of full-length cDNA of peste des petits ruminants virus].

    PubMed

    Zhai, Jun-Jun; Dou, Yong-Xi; Zhang, Hai-Rui; Mao, Li; Meng, Xue-Lian; Luo, Xuo-Nong; Cai, Xue-Peng

    2010-07-01

    To develop a reverse genetics system of Peste des petits ruminants virus(PPRV), five pairs of oligonucleotide primers were designed on the basis of the full-length genomic sequence of PPRV Nigeria 75/ 1 strain. Using RT-PCR technique, five over-lapping cDNA fragments, designated as JF1, JF2, JF3, JF4 and JF5, respectively, were amplified, followed by cloning into pcDNA3.1(+)vector. An AscI restriction enzyme site and a T7 promoter sequence were introduced immediately upstream of 5'-end, while a PacI restriction enzyme site was engineered downstream of 3'-end. Using pok12 as a plasmid vector, the full-length cDNA clone pok12-PPRV of Nigeria 75/1 was assembled by connecting the five cDNA fragments via the unique restriction endonuclease site of PPRV genome. The resultant nucleotide sequence of the PPRV Nigeria 75/1 strain in the study was compared with other members of genus morbillivirus, and phylogenetic analysis was used to examine the evolutionary relationships. The results showed that PPRV Nigeria 75/ 1 was antigenically closely related to Rinderpest virus and Measles virus. Successful construction of full-length cDNA clone of PPRV Nigeria 75/1 strain lays the basis rescuing PPRV effectively and enables further research of PPRV at molecular level. PMID:20836386

  20. cDNA Library Enrichment of Full Length Transcripts for SMRT Long Read Sequencing

    PubMed Central

    Hartwig, Benjamin; Reinhardt, Richard; Schneeberger, Korbinian

    2016-01-01

    The utility of genome assemblies does not only rely on the quality of the assembled genome sequence, but also on the quality of the gene annotations. The Pacific Biosciences Iso-Seq technology is a powerful support for accurate eukaryotic gene model annotation as it allows for direct readout of full-length cDNA sequences without the need for noisy short read-based transcript assembly. We propose the implementation of the TeloPrime Full Length cDNA Amplification kit to the Pacific Biosciences Iso-Seq technology in order to enrich for genuine full-length transcripts in the cDNA libraries. We provide evidence that TeloPrime outperforms the commonly used SMARTer PCR cDNA Synthesis Kit in identifying transcription start and end sites in Arabidopsis thaliana. Furthermore, we show that TeloPrime-based Pacific Biosciences Iso-Seq can be successfully applied to the polyploid genome of bread wheat (Triticum aestivum) not only to efficiently annotate gene models, but also to identify novel transcription sites, gene homeologs, splicing isoforms and previously unidentified gene loci. PMID:27327613

  1. Cognitive flexibility deficits in a mouse model for the absence of full-length dystrophin.

    PubMed

    Remmelink, E; Aartsma-Rus, A; Smit, A B; Verhage, M; Loos, M; van Putten, M

    2016-07-01

    Duchenne muscular dystrophy (DMD) is a progressive muscle-wasting disorder, caused by mutations in the DMD gene and the resulting lack of dystrophin. The DMD gene has seven promoters, giving rise to multiple full-length and shorter isoforms. Besides the expression of dystrophin in muscles, the majority of dystrophin isoforms is expressed in brain and dystrophinopathy can lead to cognitive deficits, including intellectual impairments and deficits in executive function. In contrast to the muscle pathology, the impact of the lack of dystrophin on the brain is not very well studied. Here, we study the behavioral consequences of a lack of full-length dystrophin isoforms in mdx mice, particularly with regard to domains of executive functions and anxiety. We observed a deficit in cognitive flexibility in mdx mice in the absence of motor dysfunction or general learning impairments using two independent behavioral tests. In addition, increased anxiety was observed, but its expression depended on the context. Overall, these results suggest that the absence of full-length dystrophin in mice has specific behavioral effects that compare well to deficits observed in DMD patients. PMID:27220066

  2. Full-length genomic characterizations of two canine parvoviruses prevalent in Northwest China.

    PubMed

    Han, Shi-Chong; Guo, Hui-Chen; Sun, Shi-Qi; Shu, Long; Wei, Yan-Quan; Sun, De-Hui; Cao, Sui-Zhong; Peng, Guang-Neng; Liu, Xiang-Tao

    2015-05-01

    Canine parvovirus (CPV) can cause acute hemorrhagic diarrhea and fatal myocarditis in young dogs. Currently, most studies have focused on the evolution of the VP2 gene, whereas the full-length genome of CPV has been rarely reported. In this study, the whole genomes of CPV-LZ1 and CPV-LZ2 strains prevalent in Northwest China were determined and analyzed in comparison with those of the reference CPVs. The genome sequences of both LZ strains consisted of 5053 nucleotides. CPV-LZ1 and CPV-LZ2 strains were designated as new CPV-2a and CPV-2b, respectively. Sequence alignment analysis results revealed that these two new strains underwent specific unique variations during the process of local adaption. The left non-translated regions of these strains formed a Y-shaped hairpin structure, whereas the right non-translated regions lacked the reiteration of DNA sequence. A phylogenetic tree constructed from 33 whole coding regions of CPVs showed a strong spatial clustering, and these two strains belonged to the Chinese strain cluster lineage. This study provides a method to obtain the full-length genome of CPV. The isolation and characterization of these viruses adds incrementally to the knowledge of the full-length genome of CPV. The results from this study also provide insight into the molecular epidemiology and genetic diversity of the CPV field isolates from Northwest China and can be useful in preventing and controlling CPV infection in this region. PMID:25690604

  3. Infectious foot-and-mouth disease virus derived from a cloned full-length cDNA.

    PubMed Central

    Zibert, A; Maass, G; Strebel, K; Falk, M M; Beck, E

    1990-01-01

    A full-length cDNA plasmid of foot-and-mouth disease virus has been constructed. RNA synthesized in vitro by means of a bacteriophage SP6 promoter inserted in front of the cDNA led to the production of infectious particles upon transfection of BHK-21 cells. These particles were also found to be highly infectious for primary bovine kidney cells as well as for baby mice. The difficulty in cloning the foot-and-mouth disease virus cytidyl tract in Escherichia coli was circumvented by joining two separate cloned parts, representing the S and L fragments of the genome, and, in a second step, inserting a dC-dG homopolymer. Homopolymeric sequences of up to 25 cytidyl residues did not lead to the production of virus. Replicons containing poly(C) tracts long enough to permit virus replication were first established in yeast cells. One of these constructs could also be maintained in E. coli and was used to produce infectious RNA in vitro. The length of the poly(C) sequence in this cDNA plasmid was 32 nucleotides. However, the poly(C) tracts of two recombinant viruses found in transfected BHK-21 cells were 60 and 80 nucleotides long, respectively. Possible mechanisms leading to the enlargement of the poly(C) tract during virus replication are discussed. Images PMID:2159523

  4. Characterisation of a new infectious full-length cDNA clone of BVDV genotype 2 and generation of virus mutants.

    PubMed

    Mischkale, Katrin; Reimann, Ilona; Zemke, J; König, P; Beer, Martin

    2010-04-21

    Based on their genomic sequences, two genotypes of Bovine viral diarrhea virus (BVDV) can be differentiated, BVDV type 1 (BVDV-1) and BVDV type 2 (BVDV-2). The complete genomic sequence of the highly virulent BVDV-2 strain 890 was cloned as cDNA to establish the infectious cDNA clone p890FL. In vitro-synthesised full-length RNA of p890FL was transfected into bovine cells and infectious virus could be recovered (v890FL). In vitro, recombinant v890FL showed similar growth characteristics as wild type virus v890WT. However, infection experiments in calves revealed an attenuation of recombinant v890FL in comparison to the parental isolate. Both leukocytopenia and fever were less pronounced in v890FL-infected calves. Nevertheless, viremia and virus shedding were comparable between recombinant and parental BVDV 890. Furthermore, mutants with partial deletions of the genomic region encoding for the autoprotease N(pro) (p890DeltaN(pro)) or the capsid protein (p890DeltaC) were constructed and characterised. In order to generate pseudovirions, replicon p890DeltaC was efficiently trans-complemented on a helper cell line. In summary, the newly developed construct p890FL represents the first infectious full-length cDNA clone for the BVDV-2 strain 890 and offers a useful tool for further studies on the pathogenesis of BVDV-2 and the development of novel recombinant BVDV-2 specific vaccine candidates. PMID:19875251

  5. In vivo length changes of wrist ligaments at full wrist extension.

    PubMed

    Tan, J; Chen, J; Tang, J B

    2014-05-01

    The aim of this study was to investigate the length changes of carpal ligaments when loaded in full extension in vivo. We obtained computed tomography scans of the right wrists in three positions for six volunteers: neutral; 75° extension; and 75° extension with a further 10° of radial deviation. Nine ligaments were measured and analysed with computer modelling. The results showed that the radioscaphocapitate, long radiolunate, and ulnolunate ligaments lengthened the most at full wrist extension, suggesting that they were under greatest load. The radioscapholunate, ulnocapitate, and ulnotriquetral ligaments lengthened further with the addition of wrist radial deviation. At full extension, the dorsal intercarpal ligament inserting on the scaphoid was lengthened. The dorsal radiocarpal and dorsal intercarpal ligaments inserting on the trapezoid were shortened, suggesting reduced loading. In conclusion, a number of volar carpal ligaments lengthened significantly in full wrist extension and the ulnar carpal ligaments were further lengthened at wrist radial deviation. PMID:23571488

  6. Decomposition, lookup, and recombination: MEG evidence for the full decomposition model of complex visual word recognition.

    PubMed

    Fruchter, Joseph; Marantz, Alec

    2015-04-01

    There is much evidence that visual recognition of morphologically complex words (e.g., teacher) proceeds via a decompositional route, first involving recognition of their component morphemes (teach + -er). According to the Full Decomposition model, after the visual decomposition stage, followed by morpheme lookup, there is a final "recombination" stage, in which the decomposed morphemes are combined and the well-formedness of the complex form is evaluated. Here, we use MEG to provide evidence for the temporally-differentiated stages of this model. First, we demonstrate an early effect of derivational family entropy, corresponding to the stem lookup stage; this is followed by a surface frequency effect, corresponding to the later recombination stage. We also demonstrate a late effect of a novel statistical measure, semantic coherence, which quantifies the gradient semantic well-formedness of complex words. Our findings illustrate the usefulness of corpus measures in investigating the component processes within visual word recognition. PMID:25797098

  7. Electronic structure of cuprate superconductors in a full charge-spin recombination scheme

    NASA Astrophysics Data System (ADS)

    Feng, Shiping; Kuang, Lülin; Zhao, Huaisong

    2015-10-01

    A long-standing unsolved problem is how a microscopic theory of superconductivity in cuprate superconductors based on the charge-spin separation can produce a large electron Fermi surface. Within the framework of the kinetic-energy driven superconducting mechanism, a full charge-spin recombination scheme is developed to fully recombine a charge carrier and a localized spin into a electron, and then is employed to study the electronic structure of cuprate superconductors in the superconducting-state. In particular, it is shown that the underlying electron Fermi surface fulfills Luttinger's theorem, while the superconducting coherence of the low-energy quasiparticle excitations is qualitatively described by the standard d-wave Bardeen-Cooper-Schrieffer formalism. The theory also shows that the observed peak-dip-hump structure in the electron spectrum and Fermi arc behavior in the underdoped regime are mainly caused by the strong energy and momentum dependence of the electron self-energy.

  8. Design, fabrication, and testing of an external fuel (UO2), full-length thermionic converter

    NASA Technical Reports Server (NTRS)

    Schock, A.; Raab, B.

    1971-01-01

    The development of a full-length external-fuel thermionic converter for in-pile testing is described. The development program includes out-of-pile performance testing of the fully fueled-converter, using RF-induction heating, before its installation in the in-pile test capsule. The external-fuel converter is cylindrical in shape, and consists of an inner, centrally cooled collector, and an outer emitter surrounded by nuclear fuel. The term full-length denotes that the converter is long enough to extend over the full height of the reactor core. Thus, the converter is not a scaled-down test device, but a full-scale fuel element of the thermionic reactor. The external-fuel converter concept permits a number of different design options, particularly with respect to the fuel composition and shape, and the collector cooling arrangement. The converter described was developed for the Jet Propulsion Laboratory, and is based on their concept for a thermionic reactor with uninsulated collector cooling as previously described. The converter is double-ended, with through-flow cooling, and with ceramic seals and emitter and collector power take-offs at both ends. The design uses a revolver-shaped tungsten emitter body, with the central emitter hole surrounded by six peripheral fuel holes loaded with cylindrical UO2 pellets.

  9. The full-length form of the Drosophila amyloid precursor protein is involved in memory formation.

    PubMed

    Bourdet, Isabelle; Preat, Thomas; Goguel, Valérie

    2015-01-21

    The APP plays a central role in AD, a pathology that first manifests as a memory decline. Understanding the role of APP in normal cognition is fundamental in understanding the progression of AD, and mammalian studies have pointed to a role of secreted APPα in memory. In Drosophila, we recently showed that APPL, the fly APP ortholog, is required for associative memory. In the present study, we aimed to characterize which form of APPL is involved in this process. We show that expression of a secreted-APPL form in the mushroom bodies, the center for olfactory memory, is able to rescue the memory deficit caused by APPL partial loss of function. We next assessed the impact on memory of the Drosophila α-secretase kuzbanian (KUZ), the enzyme initiating the nonamyloidogenic pathway that produces secreted APPLα. Strikingly, KUZ overexpression not only failed to rescue the memory deficit caused by APPL loss of function, it exacerbated this deficit. We further show that in addition to an increase in secreted-APPL forms, KUZ overexpression caused a decrease of membrane-bound full-length species that could explain the memory deficit. Indeed, we observed that transient expression of a constitutive membrane-bound mutant APPL form is sufficient to rescue the memory deficit caused by APPL reduction, revealing for the first time a role of full-length APPL in memory formation. Our data demonstrate that, in addition to secreted APPL, the noncleaved form is involved in memory, raising the possibility that secreted and full-length APPL act together in memory processes. PMID:25609621

  10. Fabrication and Testing of Full-Length Single-Cell Externally Fueled Converters for Thermionic Reactors

    SciTech Connect

    Schock, Alfred

    1994-06-01

    The preceding paper described designs and analyses of thermionic reactors employing full-core-length single-cell converters with their heated emitters located on the outside of their internally cooled collectors, and it presented results of detailed parametric analyses which illustrate the benefits of this unconventional design. The present paper describes the fabrication and testing of full-length prototypical converters, both unfueled and fueled, and presents parametric results of electrically heated tests. The unfueled converter tests demonstrated the practicality of operating such long converters without shorting across a 0.3-mm interelectrode gap. They produced a measured peak output of 751 watts(e) from a single diode and a peak efficiency of 15.4%. The fueled converter tests measured the parametric performance of prototypic UO(subscript 2)-fueled converters designed for subsequent in-pile testing. They employed revolver-shaped tungsten elements with a central emitter hole surrounded by six fuel chambers. The full-length converters were heated by a water-cooled RF-induction coil inside an ion-pumped vacuum chamber. This required development of high-vacuum coaxial RF feedthroughs. In-pile test rules required multiple containment of the UO (subscript 2)-fuel, which complicated the fabrication of the test article and required successful development of techniques for welding tungsten and other refractory components. The test measured a peak power output of 530 watts(e) or 7.1 watts/cm (superscript 2) at an efficiency of 11.5%. There are three copies in the file. Cross-Reference a copy FSC-ESD-217-94-529 in the ESD files with a CID #8574.

  11. Performance of initial full-length RHIC (Relativistic Heavy Ion Collider) dipoles

    SciTech Connect

    Dahl, P.; Cottingham, J.; Garber, M.; Ghosh, A.; Goodzeit, C.; Greene, A.; Herrera, J.; Kahn, S.; Kelly, E.; Morgan, G.

    1987-01-01

    The first four full-length (9.7 m) R and D dipoles for the proposed Relativistic Heavy Ion Collider (RHIC) have been successfully tested. The magnets reached a quench plateau of approximately 4.5 T with very reasonable training - a field level comfortably above the design field of 3.45 T required for operation with beams of 100 GeV/amu gold nuclei. Measured field multipoles are considered to be quite acceptable for this series of R and D magnets.

  12. High-quality full-length immunoglobulin profiling with unique molecular barcoding.

    PubMed

    Turchaninova, M A; Davydov, A; Britanova, O V; Shugay, M; Bikos, V; Egorov, E S; Kirgizova, V I; Merzlyak, E M; Staroverov, D B; Bolotin, D A; Mamedov, I Z; Izraelson, M; Logacheva, M D; Kladova, O; Plevova, K; Pospisilova, S; Chudakov, D M

    2016-09-01

    High-throughput sequencing analysis of hypermutating immunoglobulin (IG) repertoires remains a challenging task. Here we present a robust protocol for the full-length profiling of human and mouse IG repertoires. This protocol uses unique molecular identifiers (UMIs) introduced in the course of cDNA synthesis to control bottlenecks and to eliminate PCR and sequencing errors. Using asymmetric 400+100-nt paired-end Illumina sequencing and UMI-based assembly with the new version of the MIGEC software, the protocol allows up to 750-nt lengths to be sequenced in an almost error-free manner. This sequencing approach should also be applicable to various tasks beyond immune repertoire studies. In IG profiling, the achieved length of high-quality sequence covers the variable region of even the longest chains, along with the fragment of a constant region carrying information on the antibody isotype. The whole protocol, including preparation of cells and libraries, sequencing and data analysis, takes 5 to 6 d. PMID:27490633

  13. Full-Length cDNA Cloning, Molecular Characterization and Differential Expression Analysis of Lysophospholipase I from Ovis aries.

    PubMed

    Liu, Nan-Nan; Liu, Zeng-Shan; Hu, Pan; Zhang, Ying; Lu, Shi-Ying; Li, Yan-Song; Yang, Yong-Jie; Zhang, Dong-Song; Zhou, Yu; Ren, Hong-Lin

    2016-01-01

    Lysophospholipase I (LYPLA1) is an important protein with multiple functions. In this study, the full-length cDNA of the LYPLA1 gene from Ovis aries (OaLypla1) was cloned using primers and rapid amplification of cDNA ends (RACE) technology. The full-length OaLypla1 was 2457 bp with a 5'-untranslated region (UTR) of 24 bp, a 3'-UTR of 1740 bp with a poly (A) tail, and an open reading frame (ORF) of 693 bp encoding a protein of 230 amino acid residues with a predicted molecular weight of 24,625.78 Da. Phylogenetic analysis showed that the OaLypla1 protein shared a high amino acid identity with LYPLA1 of Bos taurus. The recombinant OaLypla1 protein was expressed and purified, and its phospholipase activity was identified. Monoclonal antibodies (mAb) against OaLypla1 that bound native OaLypla1 were generated. Real-time PCR analysis revealed that OaLypla1 was constitutively expressed in the liver, spleen, lung, kidney, and white blood cells of sheep, with the highest level in the kidney. Additionally, the mRNA levels of OaLypla1 in the buffy coats of sheep challenged with virulent or avirulent Brucella strains were down-regulated compared to untreated sheep. The results suggest that OaLypla1 may have an important physiological role in the host response to bacteria. The function of OaLypla1 in the host response to bacterial infection requires further study in the future. PMID:27483239

  14. GST-His purification: a two-step affinity purification protocol yielding full-length purified proteins.

    PubMed

    Maity, Ranjan; Pauty, Joris; Krietsch, Jana; Buisson, Rémi; Genois, Marie-Michelle; Masson, Jean-Yves

    2013-01-01

    Key assays in enzymology for the biochemical characterization of proteins in vitro necessitate high concentrations of the purified protein of interest. Protein purification protocols should combine efficiency, simplicity and cost effectiveness. Here, we describe the GST-His method as a new small-scale affinity purification system for recombinant proteins, based on a N-terminal Glutathione Sepharose Tag (GST) and a C-terminal 10xHis tag, which are both fused to the protein of interest. The latter construct is used to generate baculoviruses, for infection of Sf9 infected cells for protein expression. GST is a rather long tag (29 kDa) which serves to ensure purification efficiency. However, it might influence physiological properties of the protein. Hence, it is subsequently cleaved off the protein using the PreScission enzyme. In order to ensure maximum purity and to remove the cleaved GST, we added a second affinity purification step based on the comparatively small His-Tag. Importantly, our technique is based on two different tags flanking the two ends of the protein, which is an efficient tool to remove degraded proteins and, therefore, enriches full-length proteins. The method presented here does not require an expensive instrumental setup, such as FPLC. Additionally, we incorporated MgCl2 and ATP washes to remove heat shock protein impurities and nuclease treatment to abolish contaminating nucleic acids. In summary, the combination of two different tags flanking the N- and the C-terminal and the capability to cleave off one of the tags, guaranties the recovery of a highly purified and full-length protein of interest. PMID:24193370

  15. Full-length cDNA cloning, molecular characterization and differential expression analysis of peroxiredoxin 6 from Ovis aries.

    PubMed

    Liu, Nan-Nan; Liu, Zeng-Shan; Lu, Shi-Ying; Hu, Pan; Li, Yan-Song; Feng, Xiao-Li; Zhang, Shou-Yin; Wang, Nan; Meng, Qing-Feng; Yang, Yong-Jie; Tang, Feng; Xu, Yun-Ming; Zhang, Wen-Hui; Guo, Xing; Chen, Xiao-Feng; Zhou, Yu; Ren, Hong-Lin

    2015-04-15

    Peroxiredoxin 6 (Prdx6), an important antioxidant enzyme that can eliminate reactive oxygen species (ROS) to maintain homeostasis, is a bifunctional protein that possesses the activities of both glutathione peroxidase and phospholipase A2. In this study, a novel full-length Prdx6 cDNA (OaPrdx6) was cloned from Sheep (Ovis aries) using rapid amplification of cDNA ends (RACE). The full-length cDNA of OaPrdx6 was 1753bp containing a 5'-untranslated region (UTR) of 93bp, a 3'-UTR of 985bp with a poly(A) tail, and an open reading frame (ORF) of 675bp encoding a protein of 224 amino acid residues with a predicted molecular weight of 25.07kDa. The recombinant protein OaPrdx6 was expressed and purified, and its DNA protection activity was identified. In order to analyze the Prdx6 protein expression in tissues from O. aries, monoclonal antibodies against OaPrdx6 were prepared. Western blotting results indicated that OaPrdx6 protein could be detected in heart, liver, spleen, lung, kidney, stomach, intestine, muscle, lymph node and white blood cells, and the highest expression was found in lung while the lowest expression in muscle. Compared to the normal sheep group, the mRNA transcription level of Prdx6 in buffy coat was up-regulated in the group infected with a virulent field strain of Brucella melitensis, and down-regulated in the group inoculated with a vaccine strain S2 of brucellosis. The results indicated that Prdx6 was likely to be involved in the host immune responses against Brucella infection, and probably regarded as a molecular biomarker for distinguishing between animals infected with virulent Brucella infection and those inoculated with vaccine against brucellosis. PMID:25712755

  16. Full-Length cDNA Cloning, Molecular Characterization and Differential Expression Analysis of Lysophospholipase I from Ovis aries

    PubMed Central

    Liu, Nan-Nan; Liu, Zeng-Shan; Hu, Pan; Zhang, Ying; Lu, Shi-Ying; Li, Yan-Song; Yang, Yong-Jie; Zhang, Dong-Song; Zhou, Yu; Ren, Hong-Lin

    2016-01-01

    Lysophospholipase I (LYPLA1) is an important protein with multiple functions. In this study, the full-length cDNA of the LYPLA1 gene from Ovis aries (OaLypla1) was cloned using primers and rapid amplification of cDNA ends (RACE) technology. The full-length OaLypla1 was 2457 bp with a 5′-untranslated region (UTR) of 24 bp, a 3′-UTR of 1740 bp with a poly (A) tail, and an open reading frame (ORF) of 693 bp encoding a protein of 230 amino acid residues with a predicted molecular weight of 24,625.78 Da. Phylogenetic analysis showed that the OaLypla1 protein shared a high amino acid identity with LYPLA1 of Bos taurus. The recombinant OaLypla1 protein was expressed and purified, and its phospholipase activity was identified. Monoclonal antibodies (mAb) against OaLypla1 that bound native OaLypla1 were generated. Real-time PCR analysis revealed that OaLypla1 was constitutively expressed in the liver, spleen, lung, kidney, and white blood cells of sheep, with the highest level in the kidney. Additionally, the mRNA levels of OaLypla1 in the buffy coats of sheep challenged with virulent or avirulent Brucella strains were down-regulated compared to untreated sheep. The results suggest that OaLypla1 may have an important physiological role in the host response to bacteria. The function of OaLypla1 in the host response to bacterial infection requires further study in the future. PMID:27483239

  17. Methionine oxidation induces amyloid fibril formation by full-length apolipoprotein A-I

    PubMed Central

    Wong, Yuan Qi; Binger, Katrina J.; Howlett, Geoffrey J.; Griffin, Michael D. W.

    2010-01-01

    Apolipoprotein A-I (apoA-I) is the major protein component of HDL, where it plays an important role in cholesterol transport. The deposition of apoA-I derived amyloid is associated with various hereditary systemic amyloidoses and atherosclerosis; however, very little is known about the mechanism of apoA-I amyloid formation. Methionine residues in apoA-I are oxidized via several mechanisms in vivo to form methionine sulfoxide (MetO), and significant levels of methionine oxidized apoA-I (MetO-apoA-I) are present in normal human serum. We investigated the effect of methionine oxidation on the structure, stability, and aggregation of full-length, lipid-free apoA-I. Circular dichrosim spectroscopy showed that oxidation of all three methionine residues in apoA-I caused partial unfolding of the protein and decreased its thermal stability, reducing the melting temperature (Tm) from 58.7 °C for native apoA-I to 48.2 °C for MetO-apoA-I. Analytical ultracentrifugation revealed that methionine oxidation inhibited the native self association of apoA-I to form dimers and tetramers. Incubation of MetO-apoA-I for extended periods resulted in aggregation of the protein, and these aggregates bound Thioflavin T and Congo Red. Inspection of the aggregates by electron microscopy revealed fibrillar structures with a ribbon-like morphology, widths of approximately 11 nm, and lengths of up to several microns. X-ray fibre diffraction studies of the fibrils revealed a diffraction pattern with orthogonal peaks at spacings of 4.64 Å and 9.92 Å, indicating a cross-β amyloid structure. This systematic study of fibril formation by full-length apoA-I represents the first demonstration that methionine oxidation can induce amyloid fibril formation. PMID:20133843

  18. Effects of Full-Length Kisspeptin Administration on Follicular Development in Japanese Black Beef Cows

    PubMed Central

    NANIWA, Yousuke; NAKATSUKASA, Keisuke; SETSUDA, Shohei; OISHI, Shinya; FUJII, Nobutaka; MATSUDA, Fuko; UENOYAMA, Yoshihisa; TSUKAMURA, Hiroko; MAEDA, Kei-ichiro; OHKURA, Satoshi

    2013-01-01

    Abstract Kisspeptin is a key molecule that stimulates gonadotropin secretion via release of gonadotropin-releasing hormone (GnRH). In the present study, our aim was to investigate whether kisspeptin has stimulatory effects on follicular development via GnRH/gonadotropin secretion in cows. Japanese Black beef cows were intravenously injected with full-length bovine kisspeptin [Kp-53 (0.2 or 2 nmol/kg)] or vehicle 5 days after they exhibited standing estrus (Day 0). In cows injected with Kp-53 at 2 nmol/kg, the follicular sizes of the first dominant follicles increased on Day 6 and thereafter. Ovulation of the first dominant follicle occurred in 1 out of 4 cows treated with Kp-53 at 2 nmol/kg. Injection of Kp-53 at 2 nmol/kg increased the concentration of plasma luteinizing hormone (LH) but not follicle-stimulating hormone, over a 4-h period following injection in all cows. The present study suggests that administration of full-length kisspeptin causes LH secretion, which is sustained for a few hours, and it is capable of stimulating follicular development and/or ovulation. PMID:24107742

  19. Structure of the full-length TRPV2 channel by cryo-EM

    PubMed Central

    Huynh, Kevin W.; Cohen, Matthew R.; Jiang, Jiansen; Samanta, Amrita; Lodowski, David T.; Zhou, Z. Hong; Moiseenkova-Bell, Vera Y.

    2016-01-01

    Transient receptor potential (TRP) proteins form a superfamily Ca2+-permeable cation channels regulated by a range of chemical and physical stimuli. Structural analysis of a ‘minimal' TRP vanilloid subtype 1 (TRPV1) elucidated a mechanism of channel activation by agonists through changes in its outer pore region. Though homologous to TRPV1, other TRPV channels (TRPV2–6) are insensitive to TRPV1 activators including heat and vanilloids. To further understand the structural basis of TRPV channel function, we determined the structure of full-length TRPV2 at ∼5 Å resolution by cryo-electron microscopy. Like TRPV1, TRPV2 contains two constrictions, one each in the pore-forming upper and lower gates. The agonist-free full-length TRPV2 has wider upper and lower gates compared with closed and agonist-activated TRPV1. We propose these newly revealed TRPV2 structural features contribute to diversity of TRPV channels. PMID:27021073

  20. Full-length soluble urokinase plasminogen activator receptor down-modulates nephrin expression in podocytes.

    PubMed

    Alfano, Massimo; Cinque, Paola; Giusti, Guido; Proietti, Silvia; Nebuloni, Manuela; Danese, Silvio; D'Alessio, Silvia; Genua, Marco; Portale, Federica; Lo Porto, Manuela; Singhal, Pravin C; Rastaldi, Maria Pia; Saleem, Moin A; Mavilio, Domenico; Mikulak, Joanna

    2015-01-01

    Increased plasma level of soluble urokinase-type plasminogen activator receptor (suPAR) was associated recently with focal segmental glomerulosclerosis (FSGS). In addition, different clinical studies observed increased concentration of suPAR in various glomerular diseases and in other human pathologies with nephrotic syndromes such as HIV and Hantavirus infection, diabetes and cardiovascular disorders. Here, we show that suPAR induces nephrin down-modulation in human podocytes. This phenomenon is mediated only by full-length suPAR, is time-and dose-dependent and is associated with the suppression of Wilms' tumor 1 (WT-1) transcription factor expression. Moreover, an antagonist of αvβ3 integrin RGDfv blocked suPAR-induced suppression of nephrin. These in vitro data were confirmed in an in vivo uPAR knock out Plaur(-/-) mice model by demonstrating that the infusion of suPAR inhibits expression of nephrin and WT-1 in podocytes and induces proteinuria. This study unveiled that interaction of full-length suPAR with αvβ3 integrin expressed on podocytes results in down-modulation of nephrin that may affect kidney functionality in different human pathologies characterized by increased concentration of suPAR. PMID:26380915

  1. Structural Organization of a Full-Length Gp130/LIF-R Cytokine Receptor Transmembrane Complex

    SciTech Connect

    Skiniotis, G.; Lupardus, P.J.; Martick, M.; Walz, T.; Garcia, K.C.

    2009-05-26

    gp130 is a shared receptor for at least nine cytokines, and can signal either as a homodimer, or as a heterodimer with Leukemia Inhibitory Factor Receptor (LIF-R). Here we biophysically and structurally characterize the full-length, transmembrane form of a quaternary cytokine receptor complex consisting of gp130, LIF-R, the cytokine Ciliary Neurotrophic Factor (CNTF), and its alpha receptor (CNTF-R{alpha}). Thermodynamic analysis indicates that, unlike the cooperative assembly of the symmetric gp130/Interleukin-6/IL-6R{alpha} hexameric complex, CNTF/CNTF-R{alpha} heterodimerizes gp130 and LIF-R via non-cooperative energetics to form an asymmetric 1:1:1:1 complex. Single particle electron microscopic (EM) analysis of the full-length gp130/LIF-R/CNTF-R{alpha}/CNTF quaternary complex elucidates an asymmetric structural arrangement, in which the receptor extracellular and transmembrane segments join as a continuous, rigid unit, poised to sensitively transduce ligand engagement to the membrane-proximal intracellular signaling regions. These studies also enumerate the organizing principles for assembly of the 'tall' class of gp130-family cytokine receptor complexes including LIF, IL-27, IL-12, and others.

  2. Full-length genomic characterization and molecular evolution of canine parvovirus in China.

    PubMed

    Zhou, Ling; Tang, Qinghai; Shi, Lijun; Kong, Miaomiao; Liang, Lin; Mao, Qianqian; Bu, Bin; Yao, Lunguang; Zhao, Kai; Cui, Shangjin; Leal, Élcio

    2016-06-01

    Canine parvovirus type 2 (CPV-2) can cause acute haemorrhagic enteritis in dogs and myocarditis in puppies. This disease has become one of the most serious infectious diseases of dogs. During 2014 in China, there were many cases of acute infectious diarrhoea in dogs. Some faecal samples were negative for the CPV-2 antigen based on a colloidal gold test strip but were positive based on PCR, and a viral strain was isolated from one such sample. The cytopathic effect on susceptible cells and the results of the immunoperoxidase monolayer assay, PCR, and sequencing indicated that the pathogen was CPV-2. The strain was named CPV-NY-14, and the full-length genome was sequenced and analysed. A maximum likelihood tree was constructed using the full-length genome and all available CPV-2 genomes. New strains have replaced the original strain in Taiwan and Italy, although the CPV-2a strain is still predominant there. However, CPV-2a still causes many cases of acute infectious diarrhoea in dogs in China. PMID:27038801

  3. Full-length high-temperature severe fuel damage test No. 5

    SciTech Connect

    Lanning, D.D.; Lombardo, N.J.; Hensley, W.K.; Fitzsimmons, D.E.; Panisko, F.E.; Hartwell, J.K.

    1993-09-01

    This report describes and presents data from a severe fuel damage test that was conducted in the National Research Universal (NRU) reactor at Chalk River Nuclear Laboratories (CRNL), Ontario, Canada. The test, designated FLHT-5, was the fourth in a series of full-length high-temperature (FLHT) tests on light-water reactor fuel. The tests were designed and performed by staff from the US Department of Energy`s Pacific Northwest Laboratory (PNL), operated by Battelle Memorial Institute. The test operation and test results are described in this report. The fuel bundle in the FLHT-5 experiment included 10 unirradiated full-length pressurized-water reactor (PWR) rods, 1 irradiated PWR rod and 1 dummy gamma thermometer. The fuel rods were subjected to a very low coolant flow while operating at low fission power. This caused coolant boilaway, rod dryout and overheating to temperatures above 2600 K, severe fuel rod damage, hydrogen generation, and fission product release. The test assembly and its effluent path were extensively instrumented to record temperatures, pressures, flow rates, hydrogen evolution, and fission product release during the boilaway/heatup transient. Post-test gamma scanning of the upper plenum indicated significant iodine and cesium release and deposition. Both stack gas activity and on-line gamma spectrometer data indicated significant ({approximately}50%) release of noble fission gases. Post-test visual examination of one side of the fuel bundle revealed no massive relocation and flow blockage; however, rundown of molten cladding was evident.

  4. Full-length Dysferlin Transfer by the Hyperactive Sleeping Beauty Transposase Restores Dysferlin-deficient Muscle.

    PubMed

    Escobar, Helena; Schöwel, Verena; Spuler, Simone; Marg, Andreas; Izsvák, Zsuzsanna

    2016-01-01

    Dysferlin-deficient muscular dystrophy is a progressive disease characterized by muscle weakness and wasting for which there is no treatment. It is caused by mutations in DYSF, a large, multiexonic gene that forms a coding sequence of 6.2 kb. Sleeping Beauty (SB) transposon is a nonviral gene transfer vector, already used in clinical trials. The hyperactive SB system consists of a transposon DNA sequence and a transposase protein, SB100X, that can integrate DNA over 10 kb into the target genome. We constructed an SB transposon-based vector to deliver full-length human DYSF cDNA into dysferlin-deficient H2K A/J myoblasts. We demonstrate proper dysferlin expression as well as highly efficient engraftment (>1,100 donor-derived fibers) of the engineered myoblasts in the skeletal muscle of dysferlin- and immunodeficient B6.Cg-Dysf(prmd) Prkdc(scid)/J (Scid/BLA/J) mice. Nonviral gene delivery of full-length human dysferlin into muscle cells, along with a successful and efficient transplantation into skeletal muscle are important advances towards successful gene therapy of dysferlin-deficient muscular dystrophy. PMID:26784637

  5. Full-length dysferlin expression driven by engineered human dystrophic blood derived CD133+ stem cells.

    PubMed

    Meregalli, Mirella; Navarro, Claire; Sitzia, Clementina; Farini, Andrea; Montani, Erica; Wein, Nicolas; Razini, Paola; Beley, Cyriaque; Cassinelli, Letizia; Parolini, Daniele; Belicchi, Marzia; Parazzoli, Dario; Garcia, Luis; Torrente, Yvan

    2013-12-01

    The protein dysferlin is abundantly expressed in skeletal and cardiac muscles, where its main function is membrane repair. Mutations in the dysferlin gene are involved in two autosomal recessive muscular dystrophies: Miyoshi myopathy and limb-girdle muscular dystrophy type 2B. Development of effective therapies remains a great challenge. Strategies to repair the dysferlin gene by skipping mutated exons, using antisense oligonucleotides (AONs), may be suitable only for a subset of mutations, while cell and gene therapy can be extended to all mutations. AON-treated blood-derived CD133+ stem cells isolated from patients with Miyoshi myopathy led to partial dysferlin reconstitution in vitro but failed to express dysferlin after intramuscular transplantation into scid/blAJ dysferlin null mice. We thus extended these experiments producing the full-length dysferlin mediated by a lentiviral vector in blood-derived CD133+ stem cells isolated from the same patients. Transplantation of engineered blood-derived CD133+ stem cells into scid/blAJ mice resulted in sufficient dysferlin expression to correct functional deficits in skeletal muscle membrane repair. Our data suggest for the first time that lentivirus-mediated delivery of full-length dysferlin in stem cells isolated from Miyoshi myopathy patients could represent an alternative therapeutic approach for treatment of dysferlinopathies. PMID:24028392

  6. Two methods for full-length RNA sequencing for low quantities of cells and single cells

    PubMed Central

    Pan, Xinghua; Durrett, Russell E.; Zhu, Haiying; Tanaka, Yoshiaki; Li, Yumei; Zi, Xiaoyuan; Marjani, Sadie L.; Euskirchen, Ghia; Ma, Chao; LaMotte, Robert H.; Park, In-Hyun; Snyder, Michael P.; Mason, Christopher E.; Weissman, Sherman M.

    2013-01-01

    The ability to determine the gene expression pattern in low quantities of cells or single cells is important for resolving a variety of problems in many biological disciplines. A robust description of the expression signature of a single cell requires determination of the full-length sequence of the expressed mRNAs in the cell, yet existing methods have either 3′ biased or variable transcript representation. Here, we report our protocols for the amplification and high-throughput sequencing of very small amounts of RNA for sequencing using procedures of either semirandom primed PCR or phi29 DNA polymerase-based DNA amplification, for the cDNA generated with oligo-dT and/or random oligonucleotide primers. Unlike existing methods, these protocols produce relatively uniformly distributed sequences covering the full length of almost all transcripts independent of their sizes, from 1,000 to 10 cells, and even with single cells. Both protocols produced satisfactory detection/coverage of the abundant mRNAs from a single K562 erythroleukemic cell or a single dorsal root ganglion neuron. The phi29-based method produces long products with less noise, uses an isothermal reaction, and is simple to practice. The semirandom primed PCR procedure is more sensitive and reproducible at low transcript levels or with low quantities of cells. These methods provide tools for mRNA sequencing or RNA sequencing when only low quantities of cells, a single cell, or even degraded RNA are available for profiling. PMID:23267071

  7. EFFECTS OF PHOSPHORYLATION ON THE SELF-ASSEMBLY OF NATIVE FULL-LENGTH PORCINE AMELOGENIN AND ITS REGULATION OF CALCIUM PHOSPHATE FORMATION IN VITRO

    PubMed Central

    Wiedemann-Bidlack, Felicitas B.; Kwak, Seo-Young; Beniash, Elia; Yamakoshi, Yasuo; Simmer, James P.; Margolis, Henry C.

    2010-01-01

    The self-assembly of the predominant extracellular enamel matrix protein amelogenin plays an essential role in regulating the growth and organization of enamel mineral during early stages of dental enamel formation. The present study describes the effect of the phosphorylation of a single site on the full-length native porcine amelogenin P173 on self-assembly and on the regulation of spontaneous calcium phosphate formation in vitro. Studies were also conducted using recombinant non-phosphorylated (rP172) porcine amelogenin, along with the most abundant amelogenin cleavage product (P148) and its recombinant form (rP147). Amelogenin self-assembly was assessed using dynamic light scattering (DLS) and transmission electron microscopy (TEM). Using these approaches, we have shown that self-assembly of each amelogenin is very sensitive to pH and appears to be affected by both hydrophilic and hydrophobic interactions. Furthermore, our results suggest that the phosphorylation of the full-length porcine amelogenin P173 has a small but potentially important effect on its higher-order self-assembly into chain-like structures under physiological conditions of pH, temperature, and ionic strength. Although phosphorylation has a subtle effect on the higher-order assembly of full-length amelogenin, native phosphorylated P173 was found to stabilize amorphous calcium phosphate for extended periods of time, in sharp contrast to previous findings using non-phosphorylated rP172. The biological relevance of these findings is discussed. PMID:21074619

  8. Mechanism of activation gating in the full-length KcsA K[superscript +] channel

    SciTech Connect

    Uysal, Serdar; Cuello, Luis G.; Cortes, D. Marien; Koide, Shohei; Kossiakoff, Anthony A.; Perozo, Eduardo

    2012-10-25

    Using a constitutively active channel mutant, we solved the structure of full-length KcsA in the open conformation at 3.9 {angstrom}. The structure reveals that the activation gate expands about 20 {angstrom}, exerting a strain on the bulge helices in the C-terminal domain and generating side windows large enough to accommodate hydrated K{sup +} ions. Functional and spectroscopic analysis of the gating transition provides direct insight into the allosteric coupling between the activation gate and the selectivity filter. We show that the movement of the inner gate helix is transmitted to the C-terminus as a straightforward expansion, leading to an upward movement and the insertion of the top third of the bulge helix into the membrane. We suggest that by limiting the extent to which the inner gate can open, the cytoplasmic domain also modulates the level of inactivation occurring at the selectivity filter.

  9. Full-length apolipoprotein E protects against the neurotoxicity of an apoE-related peptide

    PubMed Central

    Crutcher, K.A.; Lilley, H.N.; Anthony, S. R.; Zhou, W.; Narayanaswami, V.

    2009-01-01

    Apolipoprotein E was found to protect against the neurotoxic effects of a dimeric peptide derived from the receptor-binding region of this protein (residues 141–149). Both apoE3 and apoE4 conferred protection but the major N-terminal fragment of each isoform did not. Nor was significant protection provided by bovine serum albumin or apoA-I. Full-length apoE3 and apoE4 also inhibited the uptake of a fluorescent-labeled derivative of the peptide, suggesting that the mechanism of inhibition might involve competition for cell surface receptors/proteoglycans that mediate endocytosis and/or signaling pathways. These results might bear on the question of the role of apoE in neuronal degeneration, such as occurs in Alzheimer’s disease where apoE4 confers a significantly greater risk of pathology. PMID:19836363

  10. On the Minimum Weight of Simple Full-Length Array LDPC Codes

    NASA Astrophysics Data System (ADS)

    Sugiyama, Kenji; Kaji, Yuichi

    We investigate the minimum weights of simple full-length array LDPC codes (SFA-LDPC codes). The SFA-LDPC codes are a subclass of LDPC codes, and constructed algebraically according to two integer parameters p and j. Mittelholzer and Yang et al. have studied the minimum weights of SFA-LDPC codes, but the exact minimum weights of the codes are not known except for some small p and j. In this paper, we show that the minimum weights of the SFA-LDPC codes with j=4 and j=5 are upper-bounded by 10 and 12, respectively, independent from the prime number p. By combining the results with Yang's lower-bound limits, we can conclude that the minimum weights of the SFA-LDPC codes with j=4 and p>7 are exactly 10 and those of the SFA-LDPC codes with j=5 are 10 or 12.

  11. The first detection and full-length genome sequence of porcine deltacoronavirus isolated in Lao PDR.

    PubMed

    Lorsirigool, Athip; Saeng-Chuto, Kepalee; Temeeyasen, Gun; Madapong, Adthakorn; Tripipat, Thitima; Wegner, Matthew; Tuntituvanont, Angkana; Intrakamhaeng, Manakant; Nilubol, Dachrit

    2016-10-01

    Porcine deltacoronavirus (PDCoV) has been reported in many countries, including Hong Kong, the United States, South Korea, China and Thailand. In January 2016, clinical diarrhea similar to that of porcine epidemic diarrhea virus (PEDV) with a lower mortality rate was reported on a swine farm in Lao PDR. Intestine samples were collected from 3-day-old pigs with clinical diarrhea and assayed for the presence of swine enteric coronaviruses. The PCR results were positive for PDCoV but negative for PEDV and TGEV. A phylogenetic tree demonstrated that PDCoV from Lao PDR was grouped separately from PDCoV isolates from China and the USA, but was more closely related to the Chinese isolates than to the US isolates. The full-length genome sequence of the novel PDCoV isolate P1_16_BTL_0116 was determined. PMID:27424024

  12. Efficient expression of full-length antibodies in the cytoplasm of engineered bacteria

    PubMed Central

    Robinson, Michael-Paul; Ke, Na; Lobstein, Julie; Peterson, Cristen; Szkodny, Alana; Mansell, Thomas J.; Tuckey, Corinna; Riggs, Paul D.; Colussi, Paul A.; Noren, Christopher J.; Taron, Christopher H.; DeLisa, Matthew P.; Berkmen, Mehmet

    2015-01-01

    Current methods for producing immunoglobulin G (IgG) antibodies in engineered cells often require refolding steps or secretion across one or more biological membranes. Here, we describe a robust expression platform for biosynthesis of full-length IgG antibodies in the Escherichia coli cytoplasm. Synthetic heavy and light chains, both lacking canonical export signals, are expressed in specially engineered E. coli strains that permit formation of stable disulfide bonds within the cytoplasm. IgGs with clinically relevant antigen- and effector-binding activities are readily produced in the E. coli cytoplasm by grafting antigen-specific variable heavy and light domains into a cytoplasmically stable framework and remodelling the fragment crystallizable domain with amino-acid substitutions that promote binding to Fcγ receptors. The resulting cytoplasmic IgGs—named ‘cyclonals'—effectively bypass the potentially rate-limiting steps of membrane translocation and glycosylation. PMID:26311203

  13. First structure of full-length mammalian phenylalanine hydroxylase reveals the architecture of an autoinhibited tetramer.

    PubMed

    Arturo, Emilia C; Gupta, Kushol; Héroux, Annie; Stith, Linda; Cross, Penelope J; Parker, Emily J; Loll, Patrick J; Jaffe, Eileen K

    2016-03-01

    Improved understanding of the relationship among structure, dynamics, and function for the enzyme phenylalanine hydroxylase (PAH) can lead to needed new therapies for phenylketonuria, the most common inborn error of amino acid metabolism. PAH is a multidomain homo-multimeric protein whose conformation and multimerization properties respond to allosteric activation by the substrate phenylalanine (Phe); the allosteric regulation is necessary to maintain Phe below neurotoxic levels. A recently introduced model for allosteric regulation of PAH involves major domain motions and architecturally distinct PAH tetramers [Jaffe EK, Stith L, Lawrence SH, Andrake M, Dunbrack RL, Jr (2013) Arch Biochem Biophys 530(2):73-82]. Herein, we present, to our knowledge, the first X-ray crystal structure for a full-length mammalian (rat) PAH in an autoinhibited conformation. Chromatographic isolation of a monodisperse tetrameric PAH, in the absence of Phe, facilitated determination of the 2.9 Å crystal structure. The structure of full-length PAH supersedes a composite homology model that had been used extensively to rationalize phenylketonuria genotype-phenotype relationships. Small-angle X-ray scattering (SAXS) confirms that this tetramer, which dominates in the absence of Phe, is different from a Phe-stabilized allosterically activated PAH tetramer. The lack of structural detail for activated PAH remains a barrier to complete understanding of phenylketonuria genotype-phenotype relationships. Nevertheless, the use of SAXS and X-ray crystallography together to inspect PAH structure provides, to our knowledge, the first complete view of the enzyme in a tetrameric form that was not possible with prior partial crystal structures, and facilitates interpretation of a wealth of biochemical and structural data that was hitherto impossible to evaluate. PMID:26884182

  14. Quasispecies Analyses of the HIV-1 Near-full-length Genome With Illumina MiSeq

    PubMed Central

    Ode, Hirotaka; Matsuda, Masakazu; Matsuoka, Kazuhiro; Hachiya, Atsuko; Hattori, Junko; Kito, Yumiko; Yokomaku, Yoshiyuki; Iwatani, Yasumasa; Sugiura, Wataru

    2015-01-01

    Human immunodeficiency virus type-1 (HIV-1) exhibits high between-host genetic diversity and within-host heterogeneity, recognized as quasispecies. Because HIV-1 quasispecies fluctuate in terms of multiple factors, such as antiretroviral exposure and host immunity, analyzing the HIV-1 genome is critical for selecting effective antiretroviral therapy and understanding within-host viral coevolution mechanisms. Here, to obtain HIV-1 genome sequence information that includes minority variants, we sought to develop a method for evaluating quasispecies throughout the HIV-1 near-full-length genome using the Illumina MiSeq benchtop deep sequencer. To ensure the reliability of minority mutation detection, we applied an analysis method of sequence read mapping onto a consensus sequence derived from de novo assembly followed by iterative mapping and subsequent unique error correction. Deep sequencing analyses of aHIV-1 clone showed that the analysis method reduced erroneous base prevalence below 1% in each sequence position and discarded only < 1% of all collected nucleotides, maximizing the usage of the collected genome sequences. Further, we designed primer sets to amplify the HIV-1 near-full-length genome from clinical plasma samples. Deep sequencing of 92 samples in combination with the primer sets and our analysis method provided sufficient coverage to identify >1%-frequency sequences throughout the genome. When we evaluated sequences of pol genes from 18 treatment-naïve patients' samples, the deep sequencing results were in agreement with Sanger sequencing and identified numerous additional minority mutations. The results suggest that our deep sequencing method would be suitable for identifying within-host viral population dynamics throughout the genome. PMID:26617593

  15. Isolation and characterization of full-length putative alcohol dehydrogenase genes from polygonum minus

    NASA Astrophysics Data System (ADS)

    Hamid, Nur Athirah Abd; Ismail, Ismanizan

    2013-11-01

    Polygonum minus, locally named as Kesum is an aromatic herb which is high in secondary metabolite content. Alcohol dehydrogenase is an important enzyme that catalyzes the reversible oxidation of alcohol and aldehyde with the presence of NAD(P)(H) as co-factor. The main focus of this research is to identify the gene of ADH. The total RNA was extracted from leaves of P. minus which was treated with 150 μM Jasmonic acid. Full-length cDNA sequence of ADH was isolated via rapid amplification cDNA end (RACE). Subsequently, in silico analysis was conducted on the full-length cDNA sequence and PCR was done on genomic DNA to determine the exon and intron organization. Two sequences of ADH, designated as PmADH1 and PmADH2 were successfully isolated. Both sequences have ORF of 801 bp which encode 266 aa residues. Nucleotide sequence comparison of PmADH1 and PmADH2 indicated that both sequences are highly similar at the ORF region but divergent in the 3' untranslated regions (UTR). The amino acid is differ at the 107 residue; PmADH1 contains Gly (G) residue while PmADH2 contains Cys (C) residue. The intron-exon organization pattern of both sequences are also same, with 3 introns and 4 exons. Based on in silico analysis, both sequences contain "classical" short chain alcohol dehydrogenases/reductases ((c) SDRs) conserved domain. The results suggest that both sequences are the members of short chain alcohol dehydrogenase family.

  16. Molecular cloning and characterization of the full-length Hsp90 gene from Matricaria recutita.

    PubMed

    Ling, S P; Su, S S; Zhang, H M; Zhang, X S; Liu, X Y; Pan, G F; Yuan, Y

    2014-01-01

    Heat shock protein 90 (Hsp90) is one of the most abundant and conserved chaperone proteins and plays important roles in plant growth and responses to environmental stimuli. However, little is known regarding the sequence and function of Hsp90s in Matricaria recutita. In the present study, we cloned the full-length cDNA sequence of the hsp90 gene from this species. Using rapid amplification of cDNA ends technologies with 2 degenerate primers that were designed based on the hsp90 gene sequence from other members of Asteraceae, we isolated and characterized an Hsp90 homolog gene from M. recutita (Mr-Hsp90). The full-length Mr-hsp90 cDNA sequence, containing 2097 base pairs, encodes a protein of 698 amino acids. Based on amino acid sequence identity, Mr-Hsp90 showed high similarity to other cloned Hsp90 proteins. The Mr-Hsp90 protein was closely clustered with the Lactuca sativa in a phylogenetic tree. These results indicate that the cloned sequence of Mr-Hsp90 is a member of the Hsp90 family, which is reported for the first time in M. recutita. Next, we conducted a salt stress experiment to determine the protein's function under salt stress conditions. Survival of chamomile seedlings subjected to heat-shock pretreatment was significantly increased compared with groups that had not undergone heat-shock pretreatment in a salt stress environment. This indicates that Mr-Hsp90 plays an important role in the salt resistance of chamomile seedlings. PMID:25526220

  17. Quasispecies Analyses of the HIV-1 Near-full-length Genome With Illumina MiSeq.

    PubMed

    Ode, Hirotaka; Matsuda, Masakazu; Matsuoka, Kazuhiro; Hachiya, Atsuko; Hattori, Junko; Kito, Yumiko; Yokomaku, Yoshiyuki; Iwatani, Yasumasa; Sugiura, Wataru

    2015-01-01

    Human immunodeficiency virus type-1 (HIV-1) exhibits high between-host genetic diversity and within-host heterogeneity, recognized as quasispecies. Because HIV-1 quasispecies fluctuate in terms of multiple factors, such as antiretroviral exposure and host immunity, analyzing the HIV-1 genome is critical for selecting effective antiretroviral therapy and understanding within-host viral coevolution mechanisms. Here, to obtain HIV-1 genome sequence information that includes minority variants, we sought to develop a method for evaluating quasispecies throughout the HIV-1 near-full-length genome using the Illumina MiSeq benchtop deep sequencer. To ensure the reliability of minority mutation detection, we applied an analysis method of sequence read mapping onto a consensus sequence derived from de novo assembly followed by iterative mapping and subsequent unique error correction. Deep sequencing analyses of aHIV-1 clone showed that the analysis method reduced erroneous base prevalence below 1% in each sequence position and discarded only < 1% of all collected nucleotides, maximizing the usage of the collected genome sequences. Further, we designed primer sets to amplify the HIV-1 near-full-length genome from clinical plasma samples. Deep sequencing of 92 samples in combination with the primer sets and our analysis method provided sufficient coverage to identify >1%-frequency sequences throughout the genome. When we evaluated sequences of pol genes from 18 treatment-naïve patients' samples, the deep sequencing results were in agreement with Sanger sequencing and identified numerous additional minority mutations. The results suggest that our deep sequencing method would be suitable for identifying within-host viral population dynamics throughout the genome. PMID:26617593

  18. How to obtain a non-digital Full-length lower limb Radiographs using regular X-ray plates

    PubMed Central

    Parihar, Mangal; Singh, Tarandeep; Shah, Lalitkumar

    2011-01-01

    Full-length hip-to-ankle radiographs are an important part of planning and execution of any realignment procedure of the lower limb. This technical note describes a simple and inexpensive technique to obtain plain, standing, full-length hip-to-ankle radiograph using regular x-ray plates.

  19. Factors Influencing the Production of MFSV Full-Length Clone: Maize Fine Streak Virus Proteins in Drosophila S2 Cells

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Maize fine streak virus (MFSV) is negative-sense RNA virus member of the genus Nucleorhabdovirus. Our goal is to determine whether Drosophila S2 cells can support the production of a full-length clone of MFSV. We have previously demonstrated that the full-length MFSV nucleoprotein (N) and phosphopro...

  20. Effect of systemically increasing human full-length Klotho on glucose metabolism in db/db mice.

    PubMed

    Forsberg, E A; Olauson, H; Larsson, T; Catrina, S B

    2016-03-01

    The metabolic effects of antiaging Klotho were previously investigated in vivo by genetic manipulation. We have here studied the metabolic effect of physiologic levels of circulating full length Klotho in db/db mice. Increasing the full-length human Klotho levels has a positive effect on blood glucose through increasing insulin secretion. PMID:26806457

  1. Full-length cDNA cloning and structural characterization of preproinsulin in Alligator sinensis.

    PubMed

    Zhang, R; Zhang, S Z; Li, E; Wang, C; Wang, C L; Wu, X B

    2014-01-01

    Insulin is an important endocrine hormone that plays a critical physiological role in regulating metabolism and glucostasis in vertebrates. In this study, the complete cDNA of Alligator sinensis preproinsulin gene was cloned for the first time by reverse transcription-polymerase chain reaction and rapid amplification of cDNA ends methods; the amino acid sequence encoded and protein structure were analyzed. The full-length of preproinsulin cDNA sequence consists of 528 base pairs (bp), comprising a 34-bp 5'-untranslated region, a 170-bp 3'-untranslated region and an open reading frame that is 324 bp in length. The open reading frame encodes a 107-amino acid preproinsulin with a molecular weight of approximately 12,153.8 Da, theoretical isoelectric point of 5.68, aliphatic index of 92.06, and grand average of hydropathicity of -0.157, from which a signal peptide, a B-chain, a C-peptide, and an A-chain are derived. Online analysis suggested that the deduced preproinsulin amino acid sequence contains a transmembrane region, and that it has a signal peptide whose cleavage site occurs between alanine 24 and alanine 25. Comparative analysis of preproinsulin amino acid sequences indicated that the A-chain and B-chain sequences of preproinsulins are highly conserved between reptiles and birds, and that the preproinsulin amino acid sequence of Alligator sinensis shares 89% similarity to that of Chelonia mydas, but low similarity of 48-63% to those of mammals and fishes. The phylogenetic tree constructed using the neighbor-joining method revealed that preproinsulin of Alligator sinensis had high homology with reptiles and birds, such as Chelonia mydas, Gallus gallus, and Columba livia. PMID:25366775

  2. Structure and Dynamics of Full Length HIV-1 Capsid Protein in Solution

    PubMed Central

    Deshmukh, Lalit; Schwieters, Charles D.; Grishaev, Alexander; Ghirlando, Rodolfo; Baber, James L.; Clore, G. Marius

    2013-01-01

    The HIV-1 capsid protein plays a crucial role in viral infectivity, assembling into a cone that encloses the viral RNA. In the mature virion, the N-terminal domain of the capsid protein forms hexameric and pentameric rings, while C-terminal domain homodimers connect adjacent N-terminal domain rings to one another. Structures of disulfide-linked hexamer and pentamer assemblies, as well as structures of the isolated domains have been solved previously. The dimer configuration in C-terminal domain constructs differs in solution (residues 144–231) and crystal (residues 146–231) structures by ~30°, and it has been postulated that the former connects the hexamers while the latter links pentamers to hexamers. Here we study the structure and dynamics of full-length capsid protein in solution, comprising a mixture of monomeric and dimeric forms in dynamic equilibrium, using ensemble simulated annealing driven by experimental NMR residual dipolar couplings and X-ray scattering data. The complexity of the system necessitated the development of a novel computational framework that should be generally applicable to many other challenging systems that currently escape structural characterization by standard application of mainstream techniques of structural biology. We show that the orientation of the C-terminal domains in dimeric full-length capsid and isolated C-terminal domain constructs is the same in solution, and obtain a quantitative description of the conformational space sampled by the N-terminal domain relative to the C-terminal domain on the nano- to millisecond time-scale. The positional distribution of the N-terminal domain relative to the C-terminal domain is large and modulated by the oligomerization state of the C-terminal domain. We also show that a model of the hexamer/pentamer assembly can be readily generated with a single configuration of the C-terminal domain dimer, and that capsid assembly likely proceeds via conformational selection of sparsely

  3. Construction of a Full-Length Enriched cDNA Library and Preliminary Analysis of Expressed Sequence Tags from Bengal Tiger Panthera tigris tigris

    PubMed Central

    Liu, Changqing; Liu, Dan; Guo, Yu; Lu, Taofeng; Li, Xiangchen; Zhang, Minghai; Ma, Jianzhang; Ma, Yuehui; Guan, Weijun

    2013-01-01

    In this study, a full-length enriched cDNA library was successfully constructed from Bengal tiger, Panthera tigris tigris, the most well-known wild Animal. Total RNA was extracted from cultured Bengal tiger fibroblasts in vitro. The titers of primary and amplified libraries were 1.28 × 106 pfu/mL and 1.56 × 109 pfu/mL respectively. The percentage of recombinants from unamplified library was 90.2% and average length of exogenous inserts was 0.98 kb. A total of 212 individual ESTs with sizes ranging from 356 to 1108 bps were then analyzed. The BLASTX score revealed that 48.1% of the sequences were classified as a strong match, 45.3% as nominal and 6.6% as a weak match. Among the ESTs with known putative function, 26.4% ESTs were found to be related to all kinds of metabolisms, 19.3% ESTs to information storage and processing, 11.3% ESTs to posttranslational modification, protein turnover, chaperones, 11.3% ESTs to transport, 9.9% ESTs to signal transducer/cell communication, 9.0% ESTs to structure protein, 3.8% ESTs to cell cycle, and only 6.6% ESTs classified as novel genes. By EST sequencing, a full-length gene coding ferritin was identified and characterized. The recombinant plasmid pET32a-TAT-Ferritin was constructed, coded for the TAT-Ferritin fusion protein with two 6× His-tags in N and C-terminal. After BCA assay, the concentration of soluble Trx-TAT-Ferritin recombinant protein was 2.32 ± 0.12 mg/mL. These results demonstrated that the reliability and representativeness of the cDNA library attained to the requirements of a standard cDNA library. This library provided a useful platform for the functional genome and transcriptome research of Bengal tigers. PMID:23708105

  4. Construction of a full-length enriched cDNA library and preliminary analysis of expressed sequence tags from Bengal Tiger Panthera tigris tigris.

    PubMed

    Liu, Changqing; Liu, Dan; Guo, Yu; Lu, Taofeng; Li, Xiangchen; Zhang, Minghai; Ma, Jianzhang; Ma, Yuehui; Guan, Weijun

    2013-01-01

    In this study, a full-length enriched cDNA library was successfully constructed from Bengal tiger, Panthera tigris tigris, the most well-known wild Animal. Total RNA was extracted from cultured Bengal tiger fibroblasts in vitro. The titers of primary and amplified libraries were 1.28 × 106 pfu/mL and 1.56 × 109 pfu/mL respectively. The percentage of recombinants from unamplified library was 90.2% and average length of exogenous inserts was 0.98 kb. A total of 212 individual ESTs with sizes ranging from 356 to 1108 bps were then analyzed. The BLASTX score revealed that 48.1% of the sequences were classified as a strong match, 45.3% as nominal and 6.6% as a weak match. Among the ESTs with known putative function, 26.4% ESTs were found to be related to all kinds of metabolisms, 19.3% ESTs to information storage and processing, 11.3% ESTs to posttranslational modification, protein turnover, chaperones, 11.3% ESTs to transport, 9.9% ESTs to signal transducer/cell communication, 9.0% ESTs to structure protein, 3.8% ESTs to cell cycle, and only 6.6% ESTs classified as novel genes. By EST sequencing, a full-length gene coding ferritin was identified and characterized. The recombinant plasmid pET32a-TAT-Ferritin was constructed, coded for the TAT-Ferritin fusion protein with two 6× His-tags in N and C-terminal. After BCA assay, the concentration of soluble Trx-TAT-Ferritin recombinant protein was 2.32 ± 0.12 mg/mL. These results demonstrated that the reliability and representativeness of the cDNA library attained to the requirements of a standard cDNA library. This library provided a useful platform for the functional genome and transcriptome research of Bengal tigers. PMID:23708105

  5. Single-Molecule Studies of Unlabeled Full-Length p53 Protein Binding to DNA.

    PubMed

    Nuttall, Philippa; Lee, Kidan; Ciccarella, Pietro; Carminati, Marco; Ferrari, Giorgio; Kim, Ki-Bum; Albrecht, Tim

    2016-03-10

    p53 is an antitumor protein that plays an important role in apoptosis, preserving genomic stability and preventing angiogenesis, and it has been implicated in a large number of human cancers. For this reason it is an interesting target for both fundamental studies, such as the mechanism of interaction with DNA, and applications in biosensing. Here, we report a comprehensive study of label-free, full length p53 (flp53) and its interaction with engineered double-stranded DNA in vitro, at the single-molecule level, using atomic force microscopy (AFM) imaging and solid-state nanopore sensing. AFM data show that dimeric and tetrameric p53 bind to the DNA in a sequence-specific manner, confirming previously reported relative binding affinities. The statistical significance is tested using both the Grubbs test and stochastic simulations. For the first time, ultralow noise solid-state nanopore sensors are employed for the successful differentiation between bare DNA and p53/DNA complexes. Furthermore, translocation statistics reflect the binding affinities of different DNA sequences, in accordance with AFM data. Our results thus highlight the potential of solid-state nanopore sensors for single-molecule biosensing, especially when labeling is either not possible or at least not a viable option. PMID:26855037

  6. Improving the diffraction of full-length human selenomethionyl metavinculin crystals by streak-seeding

    SciTech Connect

    Rangarajan, Erumbi S.; Izard, Tina

    2012-06-28

    Metavinculin is an alternatively spliced isoform of vinculin that has a 68-residue insert in its tail domain (1134 total residues) and is exclusively expressed in cardiac and smooth muscle tissue, where it plays important roles in myocyte adhesion complexes. Mutations in the metavinculin-specific insert are associated with dilated cardiomyopathy (DCM) in man. Crystals of a DCM-associated mutant of full-length selenomethionine-labeled metavinculin grown by hanging-drop vapor diffusion diffracted poorly and were highly sensitive to radiation, preventing the collection of a complete X-ray diffraction data set at the highest possible resolution. Streak-seeding markedly improved the stability, crystal-growth rate and diffraction quality of DCM-associated mutant metavinculin crystals, allowing complete data collection to 3.9 {angstrom} resolution. These crystals belonged to space group P4{sub 3}2{sub 1}2, with two molecules in the asymmetric unit and unit-cell parameters a = b = 170, c = 211 {angstrom}, {alpha} = {beta} = {gamma} = 90{sup o}.

  7. Complete sequencing and characterization of 21,243 full-length human cDNAs.

    PubMed

    Ota, Toshio; Suzuki, Yutaka; Nishikawa, Tetsuo; Otsuki, Tetsuji; Sugiyama, Tomoyasu; Irie, Ryotaro; Wakamatsu, Ai; Hayashi, Koji; Sato, Hiroyuki; Nagai, Keiichi; Kimura, Kouichi; Makita, Hiroshi; Sekine, Mitsuo; Obayashi, Masaya; Nishi, Tatsunari; Shibahara, Toshikazu; Tanaka, Toshihiro; Ishii, Shizuko; Yamamoto, Jun-ichi; Saito, Kaoru; Kawai, Yuri; Isono, Yuko; Nakamura, Yoshitaka; Nagahari, Kenji; Murakami, Katsuhiko; Yasuda, Tomohiro; Iwayanagi, Takao; Wagatsuma, Masako; Shiratori, Akiko; Sudo, Hiroaki; Hosoiri, Takehiko; Kaku, Yoshiko; Kodaira, Hiroyo; Kondo, Hiroshi; Sugawara, Masanori; Takahashi, Makiko; Kanda, Katsuhiro; Yokoi, Takahide; Furuya, Takako; Kikkawa, Emiko; Omura, Yuhi; Abe, Kumi; Kamihara, Kumiko; Katsuta, Naoko; Sato, Kazuomi; Tanikawa, Machiko; Yamazaki, Makoto; Ninomiya, Ken; Ishibashi, Tadashi; Yamashita, Hiromichi; Murakawa, Katsuji; Fujimori, Kiyoshi; Tanai, Hiroyuki; Kimata, Manabu; Watanabe, Motoji; Hiraoka, Susumu; Chiba, Yoshiyuki; Ishida, Shinichi; Ono, Yukio; Takiguchi, Sumiyo; Watanabe, Susumu; Yosida, Makoto; Hotuta, Tomoko; Kusano, Junko; Kanehori, Keiichi; Takahashi-Fujii, Asako; Hara, Hiroto; Tanase, Tomo-o; Nomura, Yoshiko; Togiya, Sakae; Komai, Fukuyo; Hara, Reiko; Takeuchi, Kazuha; Arita, Miho; Imose, Nobuyuki; Musashino, Kaoru; Yuuki, Hisatsugu; Oshima, Atsushi; Sasaki, Naokazu; Aotsuka, Satoshi; Yoshikawa, Yoko; Matsunawa, Hiroshi; Ichihara, Tatsuo; Shiohata, Namiko; Sano, Sanae; Moriya, Shogo; Momiyama, Hiroko; Satoh, Noriko; Takami, Sachiko; Terashima, Yuko; Suzuki, Osamu; Nakagawa, Satoshi; Senoh, Akihiro; Mizoguchi, Hiroshi; Goto, Yoshihiro; Shimizu, Fumio; Wakebe, Hirokazu; Hishigaki, Haretsugu; Watanabe, Takeshi; Sugiyama, Akio; Takemoto, Makoto; Kawakami, Bunsei; Yamazaki, Masaaki; Watanabe, Koji; Kumagai, Ayako; Itakura, Shoko; Fukuzumi, Yasuhito; Fujimori, Yoshifumi; Komiyama, Megumi; Tashiro, Hiroyuki; Tanigami, Akira; Fujiwara, Tsutomu; Ono, Toshihide; Yamada, Katsue; Fujii, Yuka; Ozaki, Kouichi; Hirao, Maasa; Ohmori, Yoshihiro; Kawabata, Ayako; Hikiji, Takeshi; Kobatake, Naoko; Inagaki, Hiromi; Ikema, Yasuko; Okamoto, Sachiko; Okitani, Rie; Kawakami, Takuma; Noguchi, Saori; Itoh, Tomoko; Shigeta, Keiko; Senba, Tadashi; Matsumura, Kyoka; Nakajima, Yoshie; Mizuno, Takae; Morinaga, Misato; Sasaki, Masahide; Togashi, Takushi; Oyama, Masaaki; Hata, Hiroko; Watanabe, Manabu; Komatsu, Takami; Mizushima-Sugano, Junko; Satoh, Tadashi; Shirai, Yuko; Takahashi, Yukiko; Nakagawa, Kiyomi; Okumura, Koji; Nagase, Takahiro; Nomura, Nobuo; Kikuchi, Hisashi; Masuho, Yasuhiko; Yamashita, Riu; Nakai, Kenta; Yada, Tetsushi; Nakamura, Yusuke; Ohara, Osamu; Isogai, Takao; Sugano, Sumio

    2004-01-01

    As a base for human transcriptome and functional genomics, we created the "full-length long Japan" (FLJ) collection of sequenced human cDNAs. We determined the entire sequence of 21,243 selected clones and found that 14,490 cDNAs (10,897 clusters) were unique to the FLJ collection. About half of them (5,416) seemed to be protein-coding. Of those, 1,999 clusters had not been predicted by computational methods. The distribution of GC content of nonpredicted cDNAs had a peak at approximately 58% compared with a peak at approximately 42%for predicted cDNAs. Thus, there seems to be a slight bias against GC-rich transcripts in current gene prediction procedures. The rest of the cDNAs unique to the FLJ collection (5,481) contained no obvious open reading frames (ORFs) and thus are candidate noncoding RNAs. About one-fourth of them (1,378) showed a clear pattern of splicing. The distribution of GC content of noncoding cDNAs was narrow and had a peak at approximately 42%, relatively low compared with that of protein-coding cDNAs. PMID:14702039

  8. Full-length RNA structure prediction of the HIV-1 genome reveals a conserved core domain.

    PubMed

    Sükösd, Zsuzsanna; Andersen, Ebbe S; Seemann, Stefan E; Jensen, Mads Krogh; Hansen, Mathias; Gorodkin, Jan; Kjems, Jørgen

    2015-12-01

    A distance constrained secondary structural model of the ≈10 kb RNA genome of the HIV-1 has been predicted but higher-order structures, involving long distance interactions, are currently unknown. We present the first global RNA secondary structure model for the HIV-1 genome, which integrates both comparative structure analysis and information from experimental data in a full-length prediction without distance constraints. Besides recovering known structural elements, we predict several novel structural elements that are conserved in HIV-1 evolution. Our results also indicate that the structure of the HIV-1 genome is highly variable in most regions, with a limited number of stable and conserved RNA secondary structures. Most interesting, a set of long distance interactions form a core organizing structure (COS) that organize the genome into three major structural domains. Despite overlapping protein-coding regions the COS is supported by a particular high frequency of compensatory base changes, suggesting functional importance for this element. This new structural element potentially organizes the whole genome into three major domains protruding from a conserved core structure with potential roles in replication and evolution for the virus. PMID:26476446

  9. Molecular cloning and properties of a full-length putative thyroid hormone receptor coactivator.

    PubMed

    Takeshita, A; Yen, P M; Misiti, S; Cardona, G R; Liu, Y; Chin, W W

    1996-08-01

    Thyroid hormone receptors (TRs) are ligand-dependent transcription factors that regulate target gene transcription. The conserved carboxy-terminal region of the ligand-binding domain (AF-2) has been thought to play a critical role in mediating ligand-dependent transactivation by the interaction with coactivator(s). Using bacterially-expressed TR as a probe, far-Western-based expression cDNA library screening identified cDNAs that encode, in part, the recently reported partial steroid receptor coactivator-1 (SRC-1) sequence. Additional work, including 5' RACE, has characterized a full-length cDNA that encodes a approximately 160 kD protein as a putative thyroid hormone receptor coactivator (F-SRC-1). In vitro binding studies show that F-SRC-1 binds to a variety of nuclear hormone receptors in a ligand-dependent manner, along with TBP and TFIIB, suggesting that F-SRC-1 may play a role as a bridging molecule between nuclear hormone receptors and general transcription factors. Interestingly, AF-2 mutants also retain ligand-dependent interaction with F-SRC-1. Although F-SRC-1 recognizes the ligand-induced conformational changes of nuclear hormone receptors, our observations suggest that F-SRC-1 may bind directly with subregion(s) in nuclear hormone receptors other than the AF-2 region. PMID:8754792

  10. Comprehensive Analysis of the Green-to-Blue Photoconversion of Full-Length Cyanobacteriochrome Tlr0924

    PubMed Central

    Hardman, Samantha J.O.; Hauck, Anna F.E.; Clark, Ian P.; Heyes, Derren J.; Scrutton, Nigel S.

    2014-01-01

    Cyanobacteriochromes are members of the phytochrome superfamily of photoreceptors and are of central importance in biological light-activated signaling mechanisms. These photoreceptors are known to reversibly convert between two states in a photoinitiated process that involves a basic E/Z isomerization of the bilin chromophore and, in certain cases, the breakage of a thioether linkage to a conserved cysteine residue in the bulk protein structure. The exact details and timescales of the reactions involved in these photoconversions have not been conclusively shown. The cyanobacteriochrome Tlr0924 contains phycocyanobilin and phycoviolobilin chromophores, both of which photoconvert between two species: blue-absorbing and green-absorbing, and blue-absorbing and red-absorbing, respectively. Here, we followed the complete green-to-blue photoconversion process of the phycoviolobilin chromophore in the full-length form of Tlr0924 over timescales ranging from femtoseconds to seconds. Using a combination of time-resolved visible and mid-infrared transient absorption spectroscopy and cryotrapping techniques, we showed that after photoisomerization, which occurs with a lifetime of 3.6 ps, the phycoviolobilin twists or distorts slightly with a lifetime of 5.3 μs. The final step, the formation of the thioether linkage with the protein, occurs with a lifetime of 23.6 ms. PMID:25418104

  11. The full-length transcripts and promoter analysis of intergenic microRNAs in Drosophila melanogaster.

    PubMed

    Qian, Jinjun; Zhang, Zan; Liang, Jingdong; Ge, Qiongqiong; Duan, Xuchu; Ma, Fei; Li, Fei

    2011-05-01

    MicroRNA (miRNA) transcription is still not well understood until now. To increase the miRNA abundance, we stimulated miRNA transcription with CuSO(4) and knocked down Drosha enzyme using dsRNA in Drosophila S2 cells. The full length transcripts of bantam, miR-276a and miR-277, the 5'-end of miR-8, the 3'-end of miR-2b and miR-10 were obtained. We also conducted a series of miRNA promoter analysis to prove the reliability of RACE results. Luciferase-reporter assays proved that both bantam and miR-276a promoters successfully drove the expressions of downstream luciferase genes. The promoter activities were impaired by introducing one or multiple mutations at predicted transcription factor binding sites. Chromatin immunoprecipitation analysis confirmed that hypophosphorylated RNA polymerase II and transcription factor c-Myc physically bind at miRNA promoter. RNA interference of transcription factors Mad and Prd led to down-expression of bantam, miR-277 and miR-2b but not miR-276a, whereas RNAi of Dorsal had the opposite effect. PMID:21333734

  12. Expression, Purification, and Identification of Associated Proteins of the Full-length hCDK12/CyclinK Complex*

    PubMed Central

    Bartkowiak, Bartlomiej; Greenleaf, Arno L.

    2015-01-01

    The coupling of transcription and associated processes has been shown to be dependent on the RNA polymerase II (RNAPII) C-terminal repeat domain (CTD) and the phosphorylation of the heptad repeats of which it is composed (consensus sequence Y1S2P3T4S5P6S7). Two primary S2 position CTD kinases have been identified in higher eukaryotes: P-TEFb and CDK12/CyclinK. The more recently discovered CDK12 appears to act at the 3′-end of the transcription unit and has been identified as a tumor suppressor for ovarian cancer; however much is still unknown about the in vivo roles of CDK12/CyclinK. In an effort to further characterize these roles we have purified to near homogeneity and characterized, full-length, active, human CDK12/CyclinK, and identified hCDK12-associated proteins via mass spectrometry. We find that employing a “2A” peptide-linked multicistronic construct containing CDK12 and CyclinK results in the efficient production of active, recombinant enzyme in the baculovirus/Sf9 expression system. Using GST-CTD fusion protein substrates we find that CDK12/CyclinK prefers a substrate with unmodified repeats or one that mimics prephosphorylation at the S7 position of the CTD; also the enzyme is sensitive to the inhibitor flavopiridol at higher concentrations. Identification of CDK12-associating proteins reveals a strong enrichment for RNA-processing factors suggesting that CDK12 affects RNA processing events in two distinct ways: Indirectly through generating factor-binding phospho-epitopes on the CTD of elongating RNAPII and directly through binding to specific factors. PMID:25429106

  13. Fate of prions in soil: trapped conformation of full-length ovine prion protein induced by adsorption on clays.

    PubMed

    Revault, M; Quiquampoix, H; Baron, M H; Noinville, S

    2005-08-01

    Studying the mechanism of retention of ovine prion protein in soils will tackle the environmental aspect of potential dissemination of scrapie infectious agent. We consider the surface-induced conformational changes that the recombinant ovine prion protein (ovPrP) may undergo under different pH conditions when interacting with soil minerals of highly adsorptive capacities such as montmorillonite. The conformational states of the full-length ovine prion protein adsorbed on the electronegative clay surface are compared to its solvated state in deuterated buffer in the pD range 3.5-9, using FTIR spectroscopy. The in vitro pH-induced conversion of the alpha-helical monomer of ovPrP into oligomers of beta-like structure prone to self-aggregation does not occur when the protein is adsorbed on the clay surface. The conformation of the trapped ovPrP molecules on montmorillonite is pH-independent and looks like that of the ovPrP solvated state at pD higher than 7, suggesting the major role of Arg and Lys residues in the electrostatic origin of adsorption. The uneven distribution of positively and negatively charged residues of the ovPrP protein would promote a favored orientation of the protein towards the clay, so that not only the basic residues embedded in the N-terminal flexible part but also external basic residues in the globular part of the protein might participate to the attractive interaction. From these results, it appears unlikely that the interaction of normal prions (PrP(C)) with soil clay surfaces could induce a change of conformation leading to the pathogenic form of prions (PrP(Sc)). PMID:15950385

  14. Generation and analysis of a large-scale expressed sequence tags from a full-length enriched cDNA library of Siberian tiger (Panthera tigris altaica).

    PubMed

    Guo, Yu; Liu, Changqing; Lu, Taofeng; Liu, Dan; Bai, Chunyu; Li, Xiangchen; Ma, Yuehui; Guan, Weijun

    2014-05-15

    In this study, a full-length enriched cDNA library was successfully constructed from Siberian tiger, the world's most endangered species. The titers of primary and amplified libraries were 1.28×10(6)pfu/mL and 1.59×10(10)pfu/mL respectively. The proportion of recombinants from unamplified library was 91.3% and the average length of exogenous inserts was 1.06kb. A total of 279 individual ESTs with sizes ranging from 316 to 1258bps were then analyzed. Furthermore, 204 unigenes were successfully annotated and involved in 49 functions of the GO classification, cell (175, 85.5%), cellular process (165, 80.9%), and binding (152, 74.5%) are the dominant terms. 198 unigenes were assigned to 156 KEGG pathways, and the pathways with the most representation are metabolic pathways (18, 9.1%). The proportion pattern of each COG subcategory was similar among Panthera tigris altaica, P. tigris tigris and Homo sapiens, and general function prediction only cluster (44, 15.8%) represents the largest group, followed by translation, ribosomal structure and biogenesis (33, 11.8%), replication, recombination and repair (24, 8.6%), and only 7.2% ESTs classified as novel genes. Moreover, the recombinant plasmid pET32a-TAT-COL6A2 was constructed, coded for the Trx-TAT-COL6A2 fusion protein with two 6× His-tags in N and C-terminal. After BCA assay, the concentration of soluble Trx-TAT-COL6A2 recombinant protein was 2.64±0.18mg/mL. This library will provide a useful platform for the functional genome and transcriptome research of for the P. tigris and other felid animals in the future. PMID:24630959

  15. Crystal structure of the full-length bacterial selenocysteine-specific elongation factor SelB

    PubMed Central

    Itoh, Yuzuru; Sekine, Shun-ichi; Yokoyama, Shigeyuki

    2015-01-01

    Selenocysteine (Sec), the 21st amino acid in translation, uses its specific tRNA (tRNASec) to recognize the UGA codon. The Sec-specific elongation factor SelB brings the selenocysteinyl-tRNASec (Sec-tRNASec) to the ribosome, dependent on both an in-frame UGA and a Sec-insertion sequence (SECIS) in the mRNA. The bacterial SelB binds mRNA through its C-terminal region, for which crystal structures have been reported. In this study, we determined the crystal structure of the full-length SelB from the bacterium Aquifex aeolicus, in complex with a GTP analog, at 3.2-Å resolution. SelB consists of three EF-Tu-like domains (D1–3), followed by four winged-helix domains (WHD1–4). The spacer region, connecting the N- and C-terminal halves, fixes the position of WHD1 relative to D3. The binding site for the Sec moiety of Sec-tRNASec is located on the interface between D1 and D2, where a cysteine molecule from the crystallization solution is coordinated by Arg residues, which may mimic Sec binding. The Sec-binding site is smaller and more exposed than the corresponding site of EF-Tu. Complex models of Sec-tRNASec, SECIS RNA, and the 70S ribosome suggest that the unique secondary structure of tRNASec allows SelB to specifically recognize tRNASec and characteristically place it at the ribosomal A-site. PMID:26304550

  16. Purification and Activity Testing of the Full-Length YycFGHI Proteins of Staphylococcus aureus

    PubMed Central

    Türck, Michael; Bierbaum, Gabriele

    2012-01-01

    Background The YycFG two-component regulatory system (TCS) of Staphylococcus aureus represents the only essential TCS that is almost ubiquitously distributed in Gram-positive bacteria with a low G+C-content. YycG (WalK/VicK) is a sensor histidine-kinase and YycF (WalR/VicR) is the cognate response regulator. Both proteins play an important role in the biosynthesis of the cell envelope and mutations in these proteins have been involved in development of vancomycin and daptomycin resistance. Methodology/Principal Findings Here we present high yield expression and purification of the full-length YycG and YycF proteins as well as of the auxiliary proteins YycH and YycI of Staphylococcus aureus. Activity tests of the YycG kinase and a mutated version, that harbours an Y306N exchange in its cytoplasmic PAS domain, in a detergent-micelle-model and a phosholipid-liposome-model showed kinase activity (autophosphorylation and phosphoryl group transfer to YycF) only in the presence of elevated concentrations of alkali salts. A direct comparison of the activity of the kinases in the liposome-model indicated a higher activity of the mutated YycG kinase. Further experiments indicated that YycG responds to fluidity changes in its microenvironment. Conclusions/Significance The combination of high yield expression, purification and activity testing of membrane and membrane-associated proteins provides an excellent experimental basis for further protein-protein interaction studies and for identification of all signals received by the YycFGHI system. PMID:22276191

  17. Isolation, characterization and functional analysis of full length p53 cDNA from Bubalus bubalis.

    PubMed

    Singh, Minu; Aggarwal, Suruchi; Mohanty, Ashok K; Mukhopadhyay, Tapas

    2015-09-01

    p53 plays a pivotal role in maintaining the genomic integrity of the cell and has an important role in cellular transformation. We isolated and cloned a full length p53 cDNA (Bp53) from water buffalo in expression vectors designed to generate tagged proteins with FLAG or GFP. Bp53 was found to be 1161 nucleotide long and codes for 386 amino acid residues with 79% homology with human p53 containing 393 amino acids. Although Bp53 has some inherent differences in amino acid composition in different functional domains as compared to human p53 but the total electrostatic charge of amino acids has been maintained. Bp53 cDNA was transiently transfected in a p53 null human NSCLC cell line and as expected, it was predominantly localized in the nucleus. Besides, Bp53 effectively transactivates a number of target genes similar to human p53 and exerts most of its anti-tumorigenic potential in culture as observed in clonogenic and cell viability assays. Like human p53 mutants, core domain mutant version of Bp53 was found to be mis-localized to cytoplasm with diminished tumor suppressor activity. However, Bp53 appeared to be more sensitive to mdm2 mediated degradation and as a result, this protein was less stable as compared to human p53. For the first time we have characterized a functionally efficient wild-type p53 from buffalo having lower stability than human p53 and thus, buffalo p53 could be used as a model system for further insight to the molecular basis of wild-type p53 instability. PMID:26003295

  18. Modelling the structure of full-length Epstein-Barr virus nuclear antigen 1.

    PubMed

    Hussain, Mushtaq; Gatherer, Derek; Wilson, Joanna B

    2014-12-01

    Epstein-Barr virus is a clinically important human virus associated with several cancers and is the etiologic agent of infectious mononucleosis. The viral nuclear antigen-1 (EBNA1) is central to the replication and propagation of the viral genome and likely contributes to tumourigenesis. We have compared EBNA1 homologues from other primate lymphocryptoviruses and found that the central glycine/alanine repeat (GAr) domain as well as predicted cellular protein (USP7 and CK2) binding sites are present in homologues in the Old World primates, but not the marmoset, suggesting that these motifs may have co-evolved. Using the resolved structure of the C-terminal one-third of EBNA1 (homodimerization and DNA binding domain), we have gone on to develop monomeric and dimeric models in silico of the full-length protein. The C-terminal domain is predicted to be structurally highly similar between homologues, indicating conserved function. Zinc could be stably incorporated into the model, bonding with two N-terminal cysteines predicted to facilitate multimerisation. The GAr contains secondary structural elements in the models, while the protein binding regions are unstructured, irrespective of the prediction approach used and sequence origin. These intrinsically disordered regions may facilitate the diversity observed in partner interactions. We hypothesize that the structured GAr could mask the disordered regions, thereby protecting the protein from default degradation. In the dimer conformation, the C-terminal tails of each monomer wrap around a proline-rich protruding loop of the partner monomer, providing dimer stability, a feature which could be exploited in therapeutic design. PMID:25011696

  19. Cognitive defects are reversible in inducible mice expressing pro-aggregant full-length human Tau

    PubMed Central

    Sydow, Astrid; Hofmann, Anne; Wu, Dan; Messing, Lars; Balschun, Detlef; D'Hooge, Rudi; Mandelkow, Eva-Maria

    2016-01-01

    Neurofibrillary lesions of abnormal Tau are hallmarks of Alzheimer´s disease and frontotemporal dementias. Our regulatable (Tet-OFF) mouse models of tauopathy express variants of human full-length Tau in the forebrain (CaMKIIα promoter) either with mutation ΔK280 (pro-aggregant) or ΔK280/I277P/I308P (anti-aggregant). Co-expression of luciferase enables in vivo quantification of gene expression by bioluminescence imaging. Pro-aggregant mice develop synapse loss and Tau pathology including missorting, phosphorylation and early pretangle formation, whereas anti-aggregant mice do not. We correlated hippocampal Tau pathology with learning/memory performance and synaptic plasticity. Pro-aggregant mice at 16 months of gene expression exhibited severe cognitive deficits in Morris water-maze and in passive-avoidance paradigms, whereas anti-aggregant mice were comparable to controls. Cognitive impairment of pro-aggregant mice was accompanied by loss of hippocampal LTP in CA1 and CA3 areas and by a reduction of synaptic proteins and dendritic spines, although no neuronal loss was observed. Remarkably, memory and LTP recovered when pro-aggregant Tau was switched-OFF for ∼4 months, Tau phosphorylation and missorting were reversed, and synapses recovered. Moreover soluble and insoluble pro-aggregant hTau40 disappeared while insoluble mouse Tau was still present. This study links early Tau pathology without neurofibrillary tangles and neuronal death to cognitive decline and synaptic dysfunction. It demonstrates that Tau-induced impairments are reversible after switching-OFF pro-aggregant Tau. Therefore our mouse model may mimic an early phase of AD when the hippocampus does not yet suffer from irreversible cell death but cognitive deficits are already striking. It offers potential to evaluate drugs with regard to learning and memory performance. PMID:22532069

  20. Structural Influences: Cholesterol, Drug, and Proton Binding to Full-Length Influenza A M2 Protein.

    PubMed

    Ekanayake, E Vindana; Fu, Riqiang; Cross, Timothy A

    2016-03-29

    The structure and functions of the M2 protein from Influenza A are sensitive to pH, cholesterol, and the antiinfluenza drug Amantadine. This is a tetrameric membrane protein of 97 amino-acid residues that has multiple functions, among them as a proton-selective channel and facilitator of viral budding, replacing the need for the ESCRT proteins that other viruses utilize. Here, various amino-acid-specific-labeled samples of the full-length protein were prepared and mixed, so that only interresidue (13)C-(13)C cross peaks between two differently labeled proteins representing interhelical interactions are observed. This channel is activated at slightly acidic pH values in the endosome when the His(37) residues in the middle of the transmembrane domain take on a +2 or +3 charged state. Changes observed here in interhelical distances in the N-terminus can be accounted for by modest structural changes, and no significant changes in structure were detected in the C-terminal portion of the channel upon activation of the channel. Amantadine, which blocks proton conductance by binding in the aqueous pore near the N-terminus, however, significantly modifies the tetrameric structure on the opposite side of the membrane. The interactions between the juxtamembrane amphipathic helix of one monomer and its neighboring monomer observed in the absence of drug are disrupted in its presence. However, the addition of cholesterol prevents this structural disruption. In fact, strong interactions are observed between cholesterol and residues in the amphipathic helix, accounting for cholesterol binding adjacent to a native palmitoylation site and near to an interhelix crevice that is typical of cholesterol binding sites. The resultant stabilization of the amphipathic helix deep in the bilayer interface facilitates the bilayer curvature that is essential for viral budding. PMID:27028648

  1. Identification and quantification of full-length BK channel variants in the developing mouse cochlea.

    PubMed

    Sakai, Yoshihisa; Harvey, Margaret; Sokolowski, Bernd

    2011-11-01

    Maxi-K(+) (BK) channel diversity is attributed to alternative splicing in the kcnma1 gene. The resultant variants manifest themselves in different cell types, tissues, and functions, such as excitation, metabolism, and signaling. Immunoelectron microscopy revealed immunogold particle labeling of BK in apical and basal regions of inner and outer hair cells, respectively. Additional labeling occurs in Deiters' cells and the inner mitochondrial membrane. Identification of full-length sequences reveals 27 BK variants from embryonic and postnatal mouse inner ear, per classification by tail motif, VYR, DEC, and ERL, and by exon usage. Three predicted start codons are found encoding MAN, MSS, and MDA, of which MDA shows the greatest expression through all stages in development, whereas MAN is undetectable. Complex splice sites occur between exons 9 and 10 and between 21 and 23. Spliced-in/out exons between 8 and 10 reveal a short fragment composed of exons 8 + 10, detectable on postnatal day (PD) 14 and PD30, and a longer fragment composed of exons 8 + 9 + 10 that is upregulated on embryonic day (ED) 14. Spliced-in exons 22 or 23 are expressed on ED14 but decrease over time; however, exon 22 increases again on PD34. Using tail-specific primers, qRT-PCR from ED14, PD4, -14, and -30 shows that BK-VYR and -ERL dominate expression on ED14, whereas DEC dominates after birth in all cochlear regions. The localization of BK and the changes in expression of its exons and tail types, by alternative splicing during development, may contribute to cochlear organization, acquisition of hearing, and intracellular signaling. PMID:21800349

  2. Sequence Variability, Gene Structure, and Expression of Full-Length Human Endogenous Retrovirus H

    PubMed Central

    Jern, Patric; Sperber, Göran O.; Ahlsén, Göran; Blomberg, Jonas

    2005-01-01

    Recently, we identified and classified 926 human endogenous retrovirus H (HERV-H)-like proviruses in the human genome. In this paper, we used the information to, in silico, reconstruct a putative ancestral HERV-H. A calculated consensus sequence was nearly open in all genes. A few manual adjustments resulted in a putative 9-kb HERV-H provirus with open reading frames (ORFs) in gag, pro, pol, and env. Long terminal repeats (LTRs) differed by 1.1%, indicating proximity to an integration event. The gag ORF was extended upstream of the normal myristylation start site. There was a long leader (including a “pre-gag” ORF) region positioned like the N terminus of murine leukemia virus (MLV) “glyco-Gag,” potentially encoding a proline- and serine-rich domain remotely similar to MLV pp12. Another ORF, starting inside the 5′ LTR, had no obvious similarity to known protein domains. Unlike other hitherto described gammaretroviruses, the reconstructed Gag had two zinc finger motifs. Alternative splicing of sequences related to the HERV-H consensus was confirmed using dbEST data. env transcripts were most prevalent in colon tumors, but also in normal testis. We found no evidence for full length env transcripts in the dbEST. HERV-H had a markedly skewed nucleotide composition, disfavoring guanine and favoring cytidine. We conclude that the HERV-H consensus shared a gene arrangement common to gammaretroviruses with gag separated by stop codon from pro-pol in the same reading frame, while env resides in another reading frame. There was also alternative splicing. HERV-H consensus yielded new insights in gammaretroviral evolution and will be useful as a model in studies on expression and function. PMID:15858016

  3. Mapping Full-Length Porcine Endogenous Retroviruses in a Large White Pig

    PubMed Central

    Herring, C.; Quinn, G.; Bower, R.; Parsons, N.; Logan, N. A.; Brawley, A.; Elsome, K.; Whittam, A.; Fernandez-Suarez, X. M.; Cunningham, D.; Onions, D.; Langford, G.; Scobie, L.

    2001-01-01

    Xenotransplantation may bridge the widening gap between the shortage of donor organs and the increasing number of patients waiting for transplantation. However, a major safety issue is the potential cross-species transmission of porcine endogenous retroviruses (PERV). This problem could be resolved if it is possible to produce pigs that do not contain replication-competent copies of this virus. In order to determine the feasibility of this, we have determined the number of potentially replication-competent full-length PERV proviruses and obtained data on their integration sites within the porcine genome. We have screened genomic DNA libraries from a Large White pig for potentially intact proviruses. We identified six unique PERV B proviruses that were apparently intact in all three genes, while the majority of isolated proviruses were defective in one or more genes. No intact PERV A proviruses were found in this pig, despite the identification of multiple defective A proviruses. Genotyping of 30 unrelated pigs for these unique proviruses showed a heterogeneous distribution. Two proviruses were uncommon, present in 7 of 30 and 3 of 30 pigs, while three were each present in 24 of 30 pigs, and one was present in 30 of 30 animals examined. Our data indicate that few PERV proviruses in Large White pigs are capable of productive infection and suggest that many could be removed by selective breeding. Further studies are required to determine if all potentially functional proviruses could be removed by breeding or whether gene knockout techniques will be required to remove the residuum. PMID:11711616

  4. Crystal structure of the full-length bacterial selenocysteine-specific elongation factor SelB.

    PubMed

    Itoh, Yuzuru; Sekine, Shun-Ichi; Yokoyama, Shigeyuki

    2015-10-15

    Selenocysteine (Sec), the 21(st) amino acid in translation, uses its specific tRNA (tRNA(Sec)) to recognize the UGA codon. The Sec-specific elongation factor SelB brings the selenocysteinyl-tRNA(Sec) (Sec-tRNA(Sec)) to the ribosome, dependent on both an in-frame UGA and a Sec-insertion sequence (SECIS) in the mRNA. The bacterial SelB binds mRNA through its C-terminal region, for which crystal structures have been reported. In this study, we determined the crystal structure of the full-length SelB from the bacterium Aquifex aeolicus, in complex with a GTP analog, at 3.2-Å resolution. SelB consists of three EF-Tu-like domains (D1-3), followed by four winged-helix domains (WHD1-4). The spacer region, connecting the N- and C-terminal halves, fixes the position of WHD1 relative to D3. The binding site for the Sec moiety of Sec-tRNA(Sec) is located on the interface between D1 and D2, where a cysteine molecule from the crystallization solution is coordinated by Arg residues, which may mimic Sec binding. The Sec-binding site is smaller and more exposed than the corresponding site of EF-Tu. Complex models of Sec-tRNA(Sec), SECIS RNA, and the 70S ribosome suggest that the unique secondary structure of tRNA(Sec) allows SelB to specifically recognize tRNA(Sec) and characteristically place it at the ribosomal A-site. PMID:26304550

  5. 78 FR 13071 - Guidance for Industry: Implementation of an Acceptable Full-Length and Abbreviated Donor History...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-02-26

    ... (76 FR 44013), FDA announced the availability of the draft guidance of the same title dated July 2011...- Length and Abbreviated Donor History Questionnaires and Accompanying Materials for Use in Screening... ``Guidance for Industry: Implementation of an Acceptable Full-Length and Abbreviated Donor...

  6. Substrate and target sequence lengths influence RecTEPsy recombineering efficiency in Pseudomonas syringae

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We are developing a new recombineering system to assist experimental manipulation of the Pseudomonas syringae genome. P. syringae is a globally dispersed plant pathogen and an important model species used to study the molecular biology of bacteria-plant interactions. We have previously identified ...

  7. [Rapid construction of full-length MnSOD cDNA of chickens by one-step 3'RACE].

    PubMed

    Bu, You-Quan; Luo, Xu-Gang; Liu, Bin; Li, Su-Fen

    2004-07-01

    RACE (rapid amplification of cDNA ends) is a popular technique to rapidly obtain the full-length cDNA. After obtaining the 3' cDNA and 5' cDNA fragments with a overlapped region by 3' RACE and 5' RACE, the full-length cDNA could be generated by end-to-end PCR or subcloning. In this study, 3' RACE combined with touch-down PCR was successfully used for the rapid construction of full-length MnSOD cDNA of chickens. Compared with the conventional end-to-end PCR or subcloning, this method, called one-step 3' RACE, is fast, economical and highly specific. It especially fits the rapid construction of full-length cDNA by RACE method. PMID:15640053

  8. A novel copper(II) coordination at His186 in full-length murine prion protein

    SciTech Connect

    Watanabe, Yasuko; Hiraoka, Wakako; Igarashi, Manabu; Ito, Kimihito; Shimoyama, Yuhei; Horiuchi, Motohiro; Yamamori, Tohru; Yasui, Hironobu; Kuwabara, Mikinori; Inagaki, Fuyuhiko; Inanami, Osamu

    2010-04-09

    To explore Cu(II) ion coordination by His{sup 186} in the C-terminal domain of full-length prion protein (moPrP), we utilized the magnetic dipolar interaction between a paramagnetic metal, Cu(II) ion, and a spin probe introduced in the neighborhood of the postulated binding site by the spin labeling technique (SDSL technique). Six moPrP mutants, moPrP(D143C), moPrP(Y148C), moPrP(E151C), moPrP(Y156C), moPrP(T189C), and moPrP(Y156C,H186A), were reacted with a methane thiosulfonate spin probe and a nitroxide residue (R1) was created in the binding site of each one. Line broadening of the ESR spectra was induced in the presence of Cu(II) ions in moPrP(Y148R1), moPrP(Y151R1), moPrP(Y156R1), and moPrP(T189R1) but not moPrP(D143R1). This line broadening indicated the presence of electron-electron dipolar interaction between Cu(II) and the nitroxide spin probe, suggesting that each interspin distance was within 20 A. The interspin distance ranges between Cu(II) and the spin probes of moPrP(Y148R1), moPrP(Y151R1), moPrP(Y156R1), and moPrP(T189R1) were estimated to be 12.1 A, 18.1 A, 10.7 A, and 8.4 A, respectively. In moPrP(Y156R1,H186A), line broadening between Cu(II) and the spin probe was not observed. These results suggest that a novel Cu(II) binding site is involved in His186 in the Helix2 region of the C-terminal domain of moPrP{sup C}.

  9. Soluble expression, purification and characterization of the full length IS2 Transposase

    PubMed Central

    2011-01-01

    Background The two-step transposition pathway of insertion sequences of the IS3 family, and several other families, involves first the formation of a branched figure-of-eight (F-8) structure by an asymmetric single strand cleavage at one optional donor end and joining to the flanking host DNA near the target end. Its conversion to a double stranded minicircle precedes the second insertional step, where both ends function as donors. In IS2, the left end which lacks donor function in Step I acquires it in Step II. The assembly of two intrinsically different protein-DNA complexes in these F-8 generating elements has been intuitively proposed, but a barrier to testing this hypothesis has been the difficulty of isolating a full length, soluble and active transposase that creates fully formed synaptic complexes in vitro with protein bound to both binding and catalytic domains of the ends. We address here a solution to expressing, purifying and structurally analyzing such a protein. Results A soluble and active IS2 transposase derivative with GFP fused to its C-terminus functions as efficiently as the native protein in in vivo transposition assays. In vitro electrophoretic mobility shift assay data show that the partially purified protein prepared under native conditions binds very efficiently to cognate DNA, utilizing both N- and C-terminal residues. As a precursor to biophysical analyses of these complexes, a fluorescence-based random mutagenesis protocol was developed that enabled a structure-function analysis of the protein with good resolution at the secondary structure level. The results extend previous structure-function work on IS3 family transposases, identifying the binding domain as a three helix H + HTH bundle and explaining the function of an atypical leucine zipper-like motif in IS2. In addition gain- and loss-of-function mutations in the catalytic active site define its role in regional and global binding and identify functional signatures that are common

  10. High avidity antibodies to full-length VAR2CSA correlate with absence of placental malaria.

    PubMed

    Tutterrow, Yeung Lo; Salanti, Ali; Avril, Marion; Smith, Joseph D; Pagano, Ian S; Ako, Simon; Fogako, Josephine; Leke, Rose G F; Taylor, Diane Wallace

    2012-01-01

    VAR2CSA mediates sequestration of Plasmodium falciparum-infected erythrocytes in the placenta, increasing the risk of poor pregnancy outcomes. Naturally acquired antibodies (Ab) to placental parasites at delivery have been associated with improved pregnancy outcomes, but Ab levels and how early in pregnancy Ab must be present in order to eliminate placental parasites before delivery remains unknown. Antibodies to individual Duffy-binding like domains of VAR2CSA have been studied, but the domains lack many of the conformational epitopes present in full-length VAR2CSA (FV2). Thus, the purpose of this study was to describe the acquisition of Ab to FV2 in women residing in high and low transmission areas and determine how Ab levels during pregnancy correlate with clearance of placental parasites. Plasma samples collected monthly throughout pregnancy from pregnant women living in high and low transmission areas in Cameroon were evaluated for Ab to FV2 and the proportion of high avidity Ab (i.e., Ab that remain bound in the presence of 3M NH(4)SCN) was assessed. Ab levels and proportion of high avidity Ab were compared between women with placental malaria (PM(+)) and those without (PM(-)) at delivery. Results showed that PM(-) women had significantly higher Ab levels (p = 0.0047) and proportion of high avidity Ab (p = 0.0009) than PM(+) women throughout pregnancy. Specifically, women with moderate to high Ab levels (>5,000 MFI) and those with ≥ 35% high avidity Ab at 5-6 months were found to have 2.3 (95% CI, 1.0-4.9) and 7.6-fold (p = 0.0013, 95% CI: 1.2-50.0) reduced risk of placental malaria, respectively. These data show that high levels of Ab to FV2, particularly those with high avidity for FV2, produced by mid-pregnancy are important in clearing parasites from the placenta. Both high Ab levels and proportion of high avidity Ab to FV2 may serve as correlates of protection for assessing immunity against placental malaria. PMID:22761948

  11. Identification of HBsAg-specific antibodies from a mammalian cell displayed full-length human antibody library of healthy immunized donor.

    PubMed

    Li, Chang-Zheng; Liang, Zhong-Kun; Chen, Zhen-Rui; Lou, Hai-Bo; Zhou, Ye; Zhang, Zhe-Huan; Yu, Fei; Liu, Shuwen; Zhou, Yuanping; Wu, Shuguang; Zheng, Wenling; Tan, Wanlong; Jiang, Shibo; Zhou, Chen

    2012-03-01

    Hepatitis B immunoglobulin (HBIG) is important in the management of hepatitis B virus (HBV) infection. Aiming to develop recombinant monoclonal antibodies as an alternative to HBIG, we report the successful identification of HBV surface antigen (HBsAg)-specific antibodies from a full-length human antibody library displayed on mammalian cell surface. Using total RNA of peripheral blood mononuclear cells of a natively immunized donor as template, the antibody repertoire was amplified. Combining four-way ligation and the Flp recombinase-mediated integration (Flp-In) system, we constructed a mammalian cell-based, fully human, full-length antibody display library in which each cell displayed only one kind of antibody molecule. By screening the cell library using fluorescence-activated cell sorting (FACS), eight cell clones that displayed HBsAg-specific antibodies on cell surfaces were identified. DNA sequence analysis of the antibody genes revealed three unique antibodies. FACS data indicated that fluorescent strength of expression (FSE), fluorescent strength of binding (FSB) and relative binding ability (RBA) were all different among them. These results demonstrated that by using our antibody mammalian display and screening platform, we can successfully identify antigen-specific antibodies from an immunized full-length antibody library. Therefore, this platform is very useful for the development of therapeutic antibodies. PMID:22179672

  12. Measurement of the minority carrier diffusion length and edge surface-recombination velocity in InP

    NASA Technical Reports Server (NTRS)

    Bailey, Sheila G.; Hakimzadeh, Roshanak

    1993-01-01

    A scanning electron microscope (SEM) was used to measure the electron (minority carrier) diffusion length (L(sub n)) and the edge surface-recombination velocity (V(sub s)) in zinc-doped Czochralski-grown InP wafers. Electron-beam-induced current (EBIC) profiles were obtained in specimens containing a Schottky barrier perpendicular to the scanned (edge) surface. An independent technique was used to measure V(sub s), and these values were used in a theoretical expression for normalized EBIC. A fit of the experimental data with this expression enabled us to determine L(sub n).

  13. Analysis of 4,664 high-quality sequence-finished poplar full-length

    SciTech Connect

    Ralph, S.; Gunter, Lee E; Tuskan, Gerald A; Douglas, Carl; Holt, Robert A.; Jones, Steven; Marra, Marco; Bohlmann, J.

    2008-01-01

    The genus Populus includes poplars, aspens and cottonwoods, which will be collectively referred to as poplars hereafter unless otherwise specified. Poplars are the dominant tree species in many forest ecosystems in the Northern Hemisphere and are of substantial economic value in plantation forestry. Poplar has been established as a model system for genomics studies of growth, development, and adaptation of woody perennial plants including secondary xylem formation, dormancy, adaptation to local environments, and biotic interactions. As part of the poplar genome sequencing project and the development of genomic resources for poplar, we have generated a full-length (FL)-cDNA collection using the biotinylated CAP trapper method. We constructed four FLcDNA libraries using RNA from xylem, phloem and cambium, and green shoot tips and leaves from the P. trichocarpa Nisqually-1 genotype, as well as insect-attacked leaves of the P. trichocarpa x P. deltoides hybrid. Following careful selection of candidate cDNA clones, we used a combined strategy of paired end reads and primer walking to generate a set of 4,664 high-accuracy, sequence-verified FLcDNAs, which clustered into 3,990 putative unique genes. Mapping FLcDNAs to the poplar genome sequence combined with BLAST comparisons to previously predicted protein coding sequences in the poplar genome identified 39 FLcDNAs that likely localize to gaps in the current genome sequence assembly. Another 173 FLcDNAs mapped to the genome sequence but were not included among the previously predicted genes in the poplar genome. Comparative sequence analysis against Arabidopsis thaliana and other species in the non-redundant database of GenBank revealed that 11.5% of the poplar FLcDNAs display no significant sequence similarity to other plant proteins. By mapping the poplar FLcDNAs against transcriptome data previously obtained with a 15.5 K cDNA microarray, we identified 153 FLcDNA clones for genes that were differentially expressed in

  14. Construction of a full-length cDNA library of Solen grandis dunker and identification of defense- and immune-related genes

    NASA Astrophysics Data System (ADS)

    Sun, Guohua; Liu, Xiangquan; Ren, Lihua; Yang, Jianmin; Wei, Xiumei; Yang, Jialong

    2013-11-01

    The basic genetic characteristics, important functional genes, and entire transcriptome of Solen grandis Dunker were investigated by constructing a full-length cDNA library with the `switching mechanism at the 5'-end of the RNA transcript' (SMART) technique. Total RNA was isolated from the immune-relevant tissues, gills and hemocytes, using the Trizol reagent, and cDNA fragments were digested with Sfi I before being ligated to the pBluescript II SK* vector. The cDNA library had a titer of 1048 cfu μL-1 and a storage capacity of 1.05×106 cfu. Approximately 98% of the clones in the library were recombinants, and the fragment lengths of insert cDNA ranged from 0.8 kb to 3.0 kb. A total of 2038 expressed sequence tags were successfully sequenced and clustered into 965 unigenes. BLASTN analysis showed that 240 sequences were highly similar to the known genes (E-value < 1e -5; percent identity >80%), accounting for 25% of the total unigenes. According to the Gene Ontology, these unigenes were related to several biological processes, including cell structure, signal transport, protein synthesis, transcription, energy metabolism, and immunity. Fifteen of the identified sequences were related to defense and immunity. The full-length cDNA sequence of HSC70 was obtained. The cDNA library of S. grandis provided a useful resource for future researches of functional genomics related to stress tolerance, immunity, and other physiological activities.

  15. Characterization of full-length and polymerase chain reaction-derived partial-length Gottfried and OSU gene 4 probes for serotypic differentiation of porcine rotaviruses.

    PubMed

    Rosen, B I; Parwani, A V; Gorziglia, M; Larralde, G; Saif, L J

    1992-10-01

    To determine the VP4 (P type) specificity of porcine rotaviruses, full- and partial-length gene 4 probes were produced from cloned Gottfried and OSU porcine rotavirus genomic segment 4 cDNAs. The gene 4 segments from the prototype Gottfried (VP7 serotype 4) and OSU (VP7 serotype 5) porcine rotavirus strains were selected for study because of their distinct P types and the occurrence of rotaviruses with similar serotypes among swine. Partial-length gene 4 cDNAs were produced and amplified by the polymerase chain reaction (PCR) and encompassed portions of the variable region (nucleotides 211 to 612) of VP8 encoded by genomic segment 4. The hybridization stringency conditions necessary for optimal probe specificity and sensitivity were determined by dot or Northern (RNA) blot hybridizations against a diverse group of human and animal rotaviruses of heterologous group A serotypes and against representative group B and C porcine rotaviruses. The PCR-derived gene 4 probes were more specific than the full-length gene 4 probes but demonstrated equivalent sensitivity. The Gottfried PCR-derived probe hybridized with Gottfried, SB2, SB3, and SB5 G serotype 4 porcine rotaviruses. The OSU PCR-derived probe hybridized with OSU, EE, A580, and SB-1A porcine rotaviruses and equine H1 rotavirus. Results of the hybridization reactions of the PCR-derived gene 4 probes with selected porcine rotavirus strains agreed with previous serological or genetic analyses, indicating their suitability as diagnostic reagents. PMID:1328281

  16. From selective full-length genes isolation by TAR cloning in yeast to their expression from HAC vectors in human cells.

    PubMed

    Kouprina, Natalay; Lee, Nicholas C O; Kononenko, Artem V; Samoshkin, Alexander; Larionov, Vladimir

    2015-01-01

    Transformation-associated recombination (TAR) cloning allows selective isolation of full-length genes and genomic loci as large circular Yeast Artificial Chromosomes (YACs) in yeast. The method has a broad application for structural and functional genomics, long-range haplotyping, characterization of chromosomal rearrangements, and evolutionary studies. In this paper, we describe a basic protocol for gene isolation by TAR as well as a method to convert TAR isolates into Bacterial Artificial Chromosomes (BACs) using a retrofitting vector. The retrofitting vector contains a 3' HPRT-loxP cassette to allow subsequent gene loading into a unique loxP site of the HAC-based (Human Artificial Chromosome) gene delivery vector. The benefit of combining the TAR gene cloning technology with the HAC gene delivery system for gene expression studies is discussed. PMID:25239739

  17. [Rescue of bovine Asia 1 serotype foot-and-mouth disease virus from a full-length cDNA clone].

    PubMed

    Li, Shuang; Zhang, Runxiang; Song, Ge; Gao, Mingchun; Liu, Xiangtao; Wang, Junwei

    2009-11-01

    After sequencing the Asia 1 foot-and-mouth disease virus (FMDV) (As01 strain), we amplified the two fragments covering the whole genome by overlapping PCR and long PCR. The 5' fragment was 1.8 kb in length including 15Cs, and the 3' fragment was 6.7 kb in length. The two fragments were cloned into the pBluescript SK vector to construct recombinant plasmid pBSAs carrying the full-length cDNA of FMDV As01 strain. The RNA transcript was synthesized in vitro using T7 polymerase and transfected into BHK-21 cells. We observed the typical CPE caused by rescued FMDV. The harvested virus was confirmed to be Asia 1 FMDV by RT-PCR, indirect immunofluorescence assay (IFA) and electron microscope observation. The rescued virus showed a similar pathogenicity in suckling mouse (LD50) compared to its wild-type virus. The infectious cDNA clone of the FMDV As01 strain laid a new ground for further investigation of FMDV virulence determinants and development of novel vaccines against FMD. PMID:20222458

  18. Giardia canis: ultrastructural analysis of G. canis trophozoites transfected with full length G. canis virus cDNA transcripts

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Giardia canis virus (GCV) is a double-stranded RNA (dsRNA) virus of the family Totiviridae. In this study, the full-length cDNA of the G. canis virus was constructed in pPoly2/sfinot vector and RNA was transcribed in vitro. Virus-free G. canis trophozoites were transfected with in vitro transcribed ...

  19. Crystallization and preliminary X-ray diffraction analysis of full-length and proteolytically activated pyruvate oxidase from Escherichia coli

    SciTech Connect

    Weidner, Annett; Neumann, Piotr; Wille, Georg; Stubbs, Milton T.; Tittmann, Kai

    2008-03-01

    The peripheral membrane flavoprotein pyruvate oxidase from E. coli has been crystallized in the full-length form and as a proteolytically activated truncation variant lacking the last 23 amino acids at the C-terminus. The thiamine diphosphate- and flavin-dependent peripheral membrane enzyme pyruvate oxidase from Escherichia coli (EcPOX) has been crystallized in the full-length form and as a proteolytically activated C-terminal truncation variant which lacks the last 23 amino acids (Δ23 EcPOX). Crystals were grown by the hanging-drop vapour-diffusion method using either protamine sulfate (full-length EcPOX) or 2-methyl-2,4-pentanediol (Δ23 EcPOX) as precipitants. Native data sets were collected at a X-ray home source to a resolution of 2.9 Å. The two forms of EcPOX crystallize in different space groups. Whereas full-length EcPOX crystallizes in the tetragonal space group P4{sub 3}2{sub 1}2 with two monomers per asymmetric unit, the crystals of Δ23 EcPOX belong to the orthorhombic space group P2{sub 1}2{sub 1}2{sub 1} and contain 12 monomers per asymmetric unit.

  20. Generation of Arabidopsis mutants by heterologous expression of a full length cDNA library from tomato fruits

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Heterologous expression of cDNA libraries in Arabidopsis and other plants has been used for gene identifications. To identify functions of tomato genes, we expressed a tomato full-length cDNA library in Arabidopsis thaliana and generated over 7,000 mutants. We constructed a tomato cDNA library with ...

  1. A combined computational and structural model of the full-length human prolactin receptor

    NASA Astrophysics Data System (ADS)

    Bugge, Katrine; Papaleo, Elena; Haxholm, Gitte W.; Hopper, Jonathan T. S.; Robinson, Carol V.; Olsen, Johan G.; Lindorff-Larsen, Kresten; Kragelund, Birthe B.

    2016-05-01

    The prolactin receptor is an archetype member of the class I cytokine receptor family, comprising receptors with fundamental functions in biology as well as key drug targets. Structurally, each of these receptors represent an intriguing diversity, providing an exceptionally challenging target for structural biology. Here, we access the molecular architecture of the monomeric human prolactin receptor by combining experimental and computational efforts. We solve the NMR structure of its transmembrane domain in micelles and collect structural data on overlapping fragments of the receptor with small-angle X-ray scattering, native mass spectrometry and NMR spectroscopy. Along with previously published data, these are integrated by molecular modelling to generate a full receptor structure. The result provides the first full view of a class I cytokine receptor, exemplifying the architecture of more than 40 different receptor chains, and reveals that the extracellular domain is merely the tip of a molecular iceberg.

  2. A combined computational and structural model of the full-length human prolactin receptor

    PubMed Central

    Bugge, Katrine; Papaleo, Elena; Haxholm, Gitte W.; Hopper, Jonathan T. S.; Robinson, Carol V.; Olsen, Johan G.; Lindorff-Larsen, Kresten; Kragelund, Birthe B.

    2016-01-01

    The prolactin receptor is an archetype member of the class I cytokine receptor family, comprising receptors with fundamental functions in biology as well as key drug targets. Structurally, each of these receptors represent an intriguing diversity, providing an exceptionally challenging target for structural biology. Here, we access the molecular architecture of the monomeric human prolactin receptor by combining experimental and computational efforts. We solve the NMR structure of its transmembrane domain in micelles and collect structural data on overlapping fragments of the receptor with small-angle X-ray scattering, native mass spectrometry and NMR spectroscopy. Along with previously published data, these are integrated by molecular modelling to generate a full receptor structure. The result provides the first full view of a class I cytokine receptor, exemplifying the architecture of more than 40 different receptor chains, and reveals that the extracellular domain is merely the tip of a molecular iceberg. PMID:27174498

  3. Llama immunization with full-length VAR2CSA generates cross-reactive and inhibitory single-domain antibodies against the DBL1X domain.

    PubMed

    Nunes-Silva, Sofia; Gangnard, Stéphane; Vidal, Marta; Vuchelen, Anneleen; Dechavanne, Sebastien; Chan, Sherwin; Pardon, Els; Steyaert, Jan; Ramboarina, Stephanie; Chêne, Arnaud; Gamain, Benoît

    2014-01-01

    VAR2CSA stands today as the leading vaccine candidate aiming to protect future pregnant women living in malaria endemic areas against the severe clinical outcomes of pregnancy associated malaria (PAM). The rational design of an efficient VAR2CSA-based vaccine relies on a profound understanding of the molecular interactions associated with P. falciparum infected erythrocyte sequestration in the placenta. Following immunization of a llama with the full-length VAR2CSA recombinant protein, we have expressed and characterized a panel of 19 nanobodies able to recognize the recombinant VAR2CSA as well as the surface of erythrocytes infected with parasites originating from different parts of the world. Domain mapping revealed that a large majority of nanobodies targeted DBL1X whereas a few of them were directed towards DBL4ε, DBL5ε and DBL6ε. One nanobody targeting the DBL1X was able to recognize the native VAR2CSA protein of the three parasite lines tested. Furthermore, four nanobodies targeting DBL1X reproducibly inhibited CSA adhesion of erythrocytes infected with the homologous NF54-CSA parasite strain, providing evidences that DBL1X domain is part or close to the CSA binding site. These nanobodies could serve as useful tools to identify conserved epitopes shared between different variants and to characterize the interactions between VAR2CSA and CSA. PMID:25487735

  4. Directed Evolution of a Secretory Leader for the Improved Expression of Heterologous Proteins and Full-Length Antibodies in S. cerevisiae

    PubMed Central

    Rakestraw, J. Andy; Sazinsky, Stephen L.; Piatesi, Andrea; Antipov, Eugene; Wittrup, K. Dane

    2010-01-01

    Because of its eukaryotic nature, simple fermentation requirements, and pliable genetics, there have been many attempts at improving recombinant protein production in S. cerevisiae. These strategies typically involve altering the expression of a native protein thought to be involved in heterologous protein trafficking. Usually, these approaches yield three to ten-fold improvements over wild-type strains and are almost always specific to one type of protein. In this study, a library of mutant alpha mating factor 1 leader peptides (MFα1pp) is screened for the enhanced secretion of a single-chain antibody. One of the isolated mutants is shown to enhance the secretion of the scFv up to sixteen-fold over wild-type. These leaders also confer a secretory improvement to two other scFvs as well as two additional, structurally unrelated proteins. Moreover, the improved leader sequences, combined with strain engineering, allow for a one-hundred eighty fold improvement over previous reports in the secretion of full length, functional, glycosylated human IgG1. The production of full-length IgG1 at milligram per liter titers in a simple, laboratory-scale system will significantly expedite drug discovery and reagent synthesis while reducing antibody cloning, production, and characterization costs. PMID:19459139

  5. A cryogenic test stand for full length SSC magnets with superfluid capability

    SciTech Connect

    Peterson, T.J.; Mazur, P.O.

    1989-02-01

    The Fermilab Magnet Test Facility performs testing of the full scale SSC magnets on test stands capable of simulating the cryogenic environment of the SSC main ring. One of these test stands, Stand 5, also has the ability to operate the magnet under test at temperatures from 1.8K to 4.5K with either supercritical helium or subcooled liquid, providing at least 25 Watts of refrigeration. At least 50 g/s flow is available from 2.3K to 4.5K, whereas superfluid operation occurs with zero flow. Cooldown time from 4.5K to 1.8K is 1.5 hours. A maximum current capability of 10,000 amps is provided, as is instrumentation to monitor and control the cryogenic conditions. This paper describes the cryogenic design of this test stand. 8 refs., 6 figs.

  6. Recombinant 1F9 spidroin microgels for murine full-thickness wound repairing.

    PubMed

    Moisenovich, M M; Malyuchenko, N V; Arkhipova, A Yu; Goncharenko, A V; Kotlyarova, M S; Davydova, L I; Vasil'eva, T V; Bogush, V G; Agapov, I I; Debabov, V G; Kirpichnikov, M P

    2016-01-01

    The study of the stimulating effect of the microgels (MGs) based on recombinant 1F9 spidroin on the regeneration of the deep skin wound in mice was carried out. The use of spidroin MGs was shown to increase significantly the quality of healing compared to the control. The introduction of the MG in the wound edges led to recovery of all the structural elements of the skin: the epidermis, the dermis, including vascular and nervous network, in the periphery of the wound underlying muscles, and skin appendages (sebaceous and sweat glands and hair follicles) was revealed. PMID:27025477

  7. The C-Terminal Region Mesd Peptide Mimics Full-Length Mesd and Acts as an Inhibitor of Wnt/β-Catenin Signaling in Cancer Cells

    PubMed Central

    Lin, Cuihong; Lu, Wenyan; Zhang, Wei; Londoño-Joshi, Angelina I.; Buchsbaum, Donald J.; Bu, Guojun; Li, Yonghe

    2013-01-01

    While Mesd was discovered as a specialized molecular endoplasmic reticulum chaperone for the Wnt co-receptors LRP5 and LRP6, recombinant Mesd protein is able to bind to mature LRP5 and LRP6 on the cell surface and acts as a universal antagonist of LRP5/6 modulators. In our previous study, we found that the C-terminal region of Mesd, which is absent in sequences from invertebrates, is necessary and sufficient for binding to mature LRP6 on the cell surface. In the present studies, we further characterized the interaction between the C-terminal region Mesd peptide and LRP5/6. We found that Mesd C-terminal region-derived peptides block Mesd binding to LRP5 at the cell surface too. We also showed that there are two LRP5/6 binding sites within Mesd C-terminal region which contain several positively charged residues. Moreover, we demonstrated that the Mesd C-terminal region peptide, like the full-length Mesd protein, blocked Wnt 3A- and Rspodin1-induced Wnt/β-catenin signaling in LRP5- and LRP6- expressing cells, suppressed Wnt/β-catenin signaling in human breast HS578T cells and prostate cancer PC-3 cells, and inhibited cancer cell proliferation, although the full-length Mesd protein is more potent than its peptide. Finally, we found that treatment of the full-length Mesd protein and its C-terminal region peptide significantly increased chemotherapy agent Adriamycin-induced cytotoxicity in HS578T and PC-3 cells. Together, our results suggest that Mesd C-terminal region constitutes the major LRP5/6-binding domain, and that Mesd protein and its C-terminal region peptide have a potential therapeutic value in cancer. PMID:23469146

  8. Reversal of a full-length mutant huntingtin neuronal cell phenotype by chemical inhibitors of polyglutamine-mediated aggregation

    PubMed Central

    Wang, Jin; Gines, Silvia; MacDonald, Marcy E; Gusella, James F

    2005-01-01

    Background Huntington's disease (HD) is an inherited neurodegenerative disorder triggered by an expanded polyglutamine tract in huntingtin that is thought to confer a new conformational property on this large protein. The propensity of small amino-terminal fragments with mutant, but not wild-type, glutamine tracts to self-aggregate is consistent with an altered conformation but such fragments occur relatively late in the disease process in human patients and mouse models expressing full-length mutant protein. This suggests that the altered conformational property may act within the full-length mutant huntingtin to initially trigger pathogenesis. Indeed, genotype-phenotype studies in HD have defined genetic criteria for the disease initiating mechanism, and these are all fulfilled by phenotypes associated with expression of full-length mutant huntingtin, but not amino-terminal fragment, in mouse models. As the in vitro aggregation of amino-terminal mutant huntingtin fragment offers a ready assay to identify small compounds that interfere with the conformation of the polyglutamine tract, we have identified a number of aggregation inhibitors, and tested whether these are also capable of reversing a phenotype caused by endogenous expression of mutant huntingtin in a striatal cell line from the HdhQ111/Q111 knock-in mouse. Results We screened the NINDS Custom Collection of 1,040 FDA approved drugs and bioactive compounds for their ability to prevent in vitro aggregation of Q58-htn 1–171 amino terminal fragment. Ten compounds were identified that inhibited aggregation with IC50 < 15 μM, including gossypol, gambogic acid, juglone, celastrol, sanguinarine and anthralin. Of these, both juglone and celastrol were effective in reversing the abnormal cellular localization of full-length mutant huntingtin observed in mutant HdhQ111/Q111 striatal cells. Conclusions At least some compounds identified as aggregation inhibitors also prevent a neuronal cellular phenotype caused

  9. Integrating de novo transcriptome assembly and cloning to obtain chicken Ovocleidin-17 full-length cDNA.

    PubMed

    Zhang, Quan; Liu, Long; Zhu, Feng; Ning, ZhongHua; Hincke, Maxwell T; Yang, Ning; Hou, ZhuoCheng

    2014-01-01

    Efficiently obtaining full-length cDNA for a target gene is the key step for functional studies and probing genetic variations. However, almost all sequenced domestic animal genomes are not 'finished'. Many functionally important genes are located in these gapped regions. It can be difficult to obtain full-length cDNA for which only partial amino acid/EST sequences exist. In this study we report a general pipeline to obtain full-length cDNA, and illustrate this approach for one important gene (Ovocleidin-17, OC-17) that is associated with chicken eggshell biomineralization. Chicken OC-17 is one of the best candidates to control and regulate the deposition of calcium carbonate in the calcified eggshell layer. OC-17 protein has been purified, sequenced, and has had its three-dimensional structure solved. However, researchers still cannot conduct OC-17 mRNA related studies because the mRNA sequence is unknown and the gene is absent from the current chicken genome. We used RNA-Seq to obtain the entire transcriptome of the adult hen uterus, and then conducted de novo transcriptome assembling with bioinformatics analysis to obtain candidate OC-17 transcripts. Based on this sequence, we used RACE and PCR cloning methods to successfully obtain the full-length OC-17 cDNA. Temporal and spatial OC-17 mRNA expression analyses were also performed to demonstrate that OC-17 is predominantly expressed in the adult hen uterus during the laying cycle and barely at immature developmental stages. Differential uterine expression of OC-17 was observed in hens laying eggs with weak versus strong eggshell, confirming its important role in the regulation of eggshell mineralization and providing a new tool for genetic selection for eggshell quality parameters. This study is the first one to report the full-length OC-17 cDNA sequence, and builds a foundation for OC-17 mRNA related studies. We provide a general method for biologists experiencing difficulty in obtaining candidate gene full-length

  10. Caspase 3 inactivates biologically active full length interleukin-33 as a classical cytokine but does not prohibit nuclear translocation

    SciTech Connect

    Ali, Shafaqat; Nguyen, Dang Quan; Falk, Werner; Martin, Michael Uwe

    2010-01-15

    IL-33 is a member of the IL-1 family of cytokines with dual function which either activates cells via the IL-33 receptor in a paracrine fashion or translocates to the nucleus to regulate gene transcription in an intracrine manner. We show that full length murine IL-33 is active as a cytokine and that it is not processed by caspase 1 to mature IL-33 but instead cleaved by caspase 3 at aa175 to yield two products which are both unable to bind to the IL-33 receptor. Full length IL-33 and its N-terminal caspase 3 breakdown product, however, translocate to the nucleus. Finally, bioactive IL-33 is not released by cells constitutively or after activation. This suggests that IL-33 is not a classical cytokine but exerts its function in the nucleus of intact cells and only activates others cells via its receptor as an alarm mediator after destruction of the producing cell.

  11. [Full-length cDNA cloning of flavonol synthase genes of Carthamus tinctorius and construction plant expression vector].

    PubMed

    Yang, Wen-ting; Liu, Xiu-ming; Wan, Qiu; Yao, Na; Wang, Nan; Zhang, Xue-meng; Jiao, Zhong-da; Li, Hai-yan; Li, Xiao-kun

    2015-02-01

    Flavonol synthase (FLS) is one of the key enzymes in flavonoids metabolic pathways. In this study, middle sequence was obtained from Carthamus tinctorius transcriptome sequencing results. Full-length cDNAs of FLS was cloned from petals of C. tinctorius to FLS by using RT-PCR and RACE technology. Its full-length cDNA was 1,201 bp, with an open reading frame of 1,101 bp and 336 encoded amino acids. The phylogenetic analysis showed that, FLS gene encoded amino acids in C. tinctorius were highly homologous with amino acids in congeneric Compositae species, especially Rudbeckia laciniata. The pBASTA-FLS plant expression vector was successfully built by the molecular biology method, which lays a foundation for further studying biology functions of the gene and biosynthesis mechanism of flavonoids. PMID:26137682

  12. Increased proteolytic processing of full-length Gli2 transcription factor reduces the Hedgehog pathway activity in vivo

    PubMed Central

    Li, Juan; Wang, Chengbing; Pan, Yong; Bai, Zengliang; Wang, Baolin

    2011-01-01

    The proteolytic processing of Gli2 and Gli3 full-length transcription factors into repressors is a key step of the regulation in Hedgehog (Hh) signaling. The differential Gli2 and Gli3 processing is controlled by the processing determinant domain or PDD, but its significance is not clear. We generated a Gli2 mutant allele, Gli23PDD, in which the Gli3PDD substitutes for the Gli2PDD. As expected, Gli23PDD is processed more efficiently and at the different position as compared to Gli2, indicating that PDD also determines the extent and site of Gli2 and Gli3 processing in vivo. The increase in levels of the Gli2 repressor in Gli23PDD mutant reduces the Hh pathway activity. Gli23PDD processing is still regulated by Hh signaling. These results indicate that the proper balance between the Gli2 full-length activator and repressor is essential for Hh signaling. PMID:21337666

  13. Construction and characterization of a full-length cDNA library for the wheat stripe rust pathogen (Puccinia striiformis f. sp. tritici)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A full-length cDNA library with complete genomic coverage is a powerful tool for functional genomic studies. We have constructed a full-length cDNA library from urediniospores of race PST-78 of Puccinia striiformis f. sp. tritici, the fungal pathogen causing wheat stripe rust. The full-length cDNA l...

  14. Massive Collection of Full-Length Complementary DNA Clones and Microarray Analyses:. Keys to Rice Transcriptome Analysis

    NASA Astrophysics Data System (ADS)

    Kikuchi, Shoshi

    2009-02-01

    Completion of the high-precision genome sequence analysis of rice led to the collection of about 35,000 full-length cDNA clones and the determination of their complete sequences. Mapping of these full-length cDNA sequences has given us information on (1) the number of genes expressed in the rice genome; (2) the start and end positions and exon-intron structures of rice genes; (3) alternative transcripts; (4) possible encoded proteins; (5) non-protein-coding (np) RNAs; (6) the density of gene localization on the chromosome; (7) setting the parameters of gene prediction programs; and (8) the construction of a microarray system that monitors global gene expression. Manual curation for rice gene annotation by using mapping information on full-length cDNA and EST assemblies has revealed about 32,000 expressed genes in the rice genome. Analysis of major gene families, such as those encoding membrane transport proteins (pumps, ion channels, and secondary transporters), along with the evolution from bacteria to higher animals and plants, reveals how gene numbers have increased through adaptation to circumstances. Family-based gene annotation also gives us a new way of comparing organisms. Massive amounts of data on gene expression under many kinds of physiological conditions are being accumulated in rice oligoarrays (22K and 44K) based on full-length cDNA sequences. Cluster analyses of genes that have the same promoter cis-elements, that have similar expression profiles, or that encode enzymes in the same metabolic pathways or signal transduction cascades give us clues to understanding the networks of gene expression in rice. As a tool for that purpose, we recently developed "RiCES", a tool for searching for cis-elements in the promoter regions of clustered genes.

  15. Hibiscus latent Fort Pierce virus in Brazil and synthesis of its biologically active full-length cDNA clone.

    PubMed

    Gao, Ruimin; Niu, Shengniao; Dai, Weifang; Kitajima, Elliot; Wong, Sek-Man

    2016-10-01

    A Brazilian isolate of Hibiscus latent Fort Pierce virus (HLFPV-BR) was firstly found in a hibiscus plant in Limeira, SP, Brazil. RACE PCR was carried out to obtain the full-length sequences of HLFPV-BR which is 6453 nucleotides and has more than 99.15 % of complete genomic RNA nucleotide sequence identity with that of HLFPV Japanese isolate. The genomic structure of HLFPV-BR is similar to other tobamoviruses. It includes a 5' untranslated region (UTR), followed by open reading frames encoding for a 128-kDa protein and a 188-kDa readthrough protein, a 38-kDa movement protein, 18-kDa coat protein, and a 3' UTR. Interestingly, the unique feature of poly(A) tract is also found within its 3'-UTR. Furthermore, from the total RNA extracted from the local lesions of HLFPV-BR-infected Chenopodium quinoa leaves, a biologically active, full-length cDNA clone encompassing the genome of HLFPV-BR was amplified and placed adjacent to a T7 RNA polymerase promoter. The capped in vitro transcripts from the cloned cDNA were infectious when mechanically inoculated into C. quinoa and Nicotiana benthamiana plants. This is the first report of the presence of an isolate of HLFPV in Brazil and the successful synthesis of a biologically active HLFPV-BR full-length cDNA clone. PMID:27139727

  16. RT-PCR and sequence analysis of the full-length fusion protein of Canine Distemper Virus from domestic dogs.

    PubMed

    Romanutti, Carina; Gallo Calderón, Marina; Keller, Leticia; Mattion, Nora; La Torre, José

    2016-02-01

    During 2007-2014, 84 out of 236 (35.6%) samples from domestic dogs submitted to our laboratory for diagnostic purposes were positive for Canine Distemper Virus (CDV), as analyzed by RT-PCR amplification of a fragment of the nucleoprotein gene. Fifty-nine of them (70.2%) were from dogs that had been vaccinated against CDV. The full-length gene encoding the Fusion (F) protein of fifteen isolates was sequenced and compared with that of those of other CDVs, including wild-type and vaccine strains. Phylogenetic analysis using the F gene full-length sequences grouped all the Argentinean CDV strains in the SA2 clade. Sequence identity with the Onderstepoort vaccine strain was 89.0-90.6%, and the highest divergence was found in the 135 amino acids corresponding to the F protein signal-peptide, Fsp (64.4-66.7% identity). In contrast, this region was highly conserved among the local strains (94.1-100% identity). One extra putative N-glycosylation site was identified in the F gene of CDV Argentinean strains with respect to the vaccine strain. The present report is the first to analyze full-length F protein sequences of CDV strains circulating in Argentina, and contributes to the knowledge of molecular epidemiology of CDV, which may help in understanding future disease outbreaks. PMID:26611227

  17. Kinetics of Hedgehog-Dependent Full-Length Gli3 Accumulation in Primary Cilia and Subsequent Degradation ▿ † ‡

    PubMed Central

    Wen, Xiaohui; Lai, Cary K.; Evangelista, Marie; Hongo, Jo-Anne; de Sauvage, Frederic J.; Scales, Suzie J.

    2010-01-01

    Hedgehog (Hh) signaling in vertebrates depends on intraflagellar transport (IFT) within primary cilia. The Hh receptor Patched is found in cilia in the absence of Hh and is replaced by the signal transducer Smoothened within an hour of Hh stimulation. By generating antibodies capable of detecting endogenous pathway transcription factors Gli2 and Gli3, we monitored their kinetics of accumulation in cilia upon Hh stimulation. Localization occurs within minutes of Hh addition, making it the fastest reported readout of pathway activity, which permits more precise temporal and spatial localization of Hh signaling events. We show that the species of Gli3 that accumulates at cilium tips is full-length and likely not protein kinase A phosphorylated. We also confirmed that phosphorylation and βTrCP/Cul1 are required for endogenous Gli3 processing and that this is inhibited by Hh. Surprisingly, however, Hh-dependent inhibition of processing does not lead to accumulation of full-length Gli3, but instead renders it labile, leading to its proteasomal degradation via the SPOP/Cul3 complex. In fact, full-length Gli3 disappears with faster kinetics than the Gli3 repressor, the latter not requiring SPOP/Cul3 or βTrCP/Cul1. This may contribute to the increased Gli3 activator/repressor ratios found in IFT mutants. PMID:20154143

  18. Species-Specific Expression of Full-Length and Alternatively Spliced Variant Forms of CDK5RAP2.

    PubMed

    Park, John S Y; Lee, Marie-Katrina; Kang, SungMyung; Jin, Yan; Fu, Songbin; Rosales, Jesusa L; Lee, Ki-Young

    2015-01-01

    CDK5RAP2 is one of the primary microcephaly genes that are associated with reduced brain size and mental retardation. We have previously shown that human CDK5RAP2 exists as a full-length form (hCDK5RAP2) or an alternatively spliced variant form (hCDK5RAP2-V1) that is lacking exon 32. The equivalent of hCDK5RAP2-V1 has been reported in rat and mouse but the presence of full-length equivalent hCDK5RAP2 in rat and mouse has not been examined. Here, we demonstrate that rat expresses both a full length and an alternatively spliced variant form of CDK5RAP2 that are equivalent to our previously reported hCDK5RAP2 and hCDK5RAP2-V1, repectively. However, mouse expresses only one form of CDK5RAP2 that is equivalent to the human and rat alternatively spliced variant forms. Knowledge of this expression of different forms of CDK5RAP2 in human, rat and mouse is essential in selecting the appropriate model for studies of CDK5RAP2 and primary microcephaly but our findings further indicate the evolutionary divergence of mouse from the human and rat species. PMID:26550838

  19. Species-Specific Expression of Full-Length and Alternatively Spliced Variant Forms of CDK5RAP2

    PubMed Central

    Park, John S. Y.; Lee, Marie-Katrina; Kang, SungMyung; Jin, Yan; Fu, Songbin; Rosales, Jesusa L.; Lee, Ki-Young

    2015-01-01

    CDK5RAP2 is one of the primary microcephaly genes that are associated with reduced brain size and mental retardation. We have previously shown that human CDK5RAP2 exists as a full-length form (hCDK5RAP2) or an alternatively spliced variant form (hCDK5RAP2-V1) that is lacking exon 32. The equivalent of hCDK5RAP2-V1 has been reported in rat and mouse but the presence of full-length equivalent hCDK5RAP2 in rat and mouse has not been examined. Here, we demonstrate that rat expresses both a full length and an alternatively spliced variant form of CDK5RAP2 that are equivalent to our previously reported hCDK5RAP2 and hCDK5RAP2-V1, repectively. However, mouse expresses only one form of CDK5RAP2 that is equivalent to the human and rat alternatively spliced variant forms. Knowledge of this expression of different forms of CDK5RAP2 in human, rat and mouse is essential in selecting the appropriate model for studies of CDK5RAP2 and primary microcephaly but our findings further indicate the evolutionary divergence of mouse from the human and rat species. PMID:26550838

  20. Identification and Functional Analyses of 11 769 Full-length Human cDNAs Focused on Alternative Splicing

    PubMed Central

    Wakamatsu, Ai; Kimura, Kouichi; Yamamoto, Jun-ichi; Nishikawa, Tetsuo; Nomura, Nobuo; Sugano, Sumio; Isogai, Takao

    2009-01-01

    We analyzed diversity of mRNA produced as a result of alternative splicing in order to evaluate gene function. First, we predicted the number of human genes transcribed into protein-coding mRNAs by using the sequence information of full-length cDNAs and 5′-ESTs and obtained 23 241 of such human genes. Next, using these genes, we analyzed the mRNA diversity and consequently sequenced and identified 11 769 human full-length cDNAs whose predicted open reading frames were different from other known full-length cDNAs. Especially, 30% of the cDNAs we identified contained variation in the transcription start site (TSS). Our analysis, which particularly focused on multiple variable first exons (FEVs) formed due to the alternative utilization of TSSs, led to the identification of 261 FEVs expressed in the tissue-specific manner. Quantification of the expression profiles of 13 genes by real-time PCR analysis further confirmed the tissue-specific expression of FEVs, e.g. OXR1 had specific TSS in brain and tumor tissues, and so on. Finally, based on the results of our mRNA diversity analysis, we have created the FLJ Human cDNA Database. From our result, it has been understood mechanisms that one gene produces suitable protein-coding transcripts responding to the situation and the environment. PMID:19880432

  1. Predicting the strength of UP-elements and full-length E. coli σE promoters

    PubMed Central

    Rhodius, Virgil A.; Mutalik, Vivek K.; Gross, Carol A.

    2012-01-01

    Predicting the location and strength of promoters from genomic sequence requires accurate sequenced-based promoter models. We present the first model of a full-length bacterial promoter, encompassing both upstream sequences (UP-elements) and core promoter modules, based on a set of 60 promoters dependent on σE, an alternative ECF-type σ factor. UP-element contribution, best described by the length and frequency of A- and T-tracts, in combination with a PWM-based core promoter model, accurately predicted promoter strength both in vivo and in vitro. This model also distinguished active from weak/inactive promoters. Systematic examination of promoter strength as a function of RNA polymerase (RNAP) concentration revealed that UP-element contribution varied with RNAP availability and that the σE regulon is comprised of two promoter types, one of which is active only at high concentrations of RNAP. Distinct promoter types may be a general mechanism for increasing the regulatory capacity of the ECF group of alternative σ's. Our findings provide important insights into the sequence requirements for the strength and function of full-length promoters and establish guidelines for promoter prediction and for forward engineering promoters of specific strengths. PMID:22156164

  2. Predicting the strength of UP-elements and full-length E. coli σE promoters.

    PubMed

    Rhodius, Virgil A; Mutalik, Vivek K; Gross, Carol A

    2012-04-01

    Predicting the location and strength of promoters from genomic sequence requires accurate sequenced-based promoter models. We present the first model of a full-length bacterial promoter, encompassing both upstream sequences (UP-elements) and core promoter modules, based on a set of 60 promoters dependent on σ(E), an alternative ECF-type σ factor. UP-element contribution, best described by the length and frequency of A- and T-tracts, in combination with a PWM-based core promoter model, accurately predicted promoter strength both in vivo and in vitro. This model also distinguished active from weak/inactive promoters. Systematic examination of promoter strength as a function of RNA polymerase (RNAP) concentration revealed that UP-element contribution varied with RNAP availability and that the σ(E) regulon is comprised of two promoter types, one of which is active only at high concentrations of RNAP. Distinct promoter types may be a general mechanism for increasing the regulatory capacity of the ECF group of alternative σ's. Our findings provide important insights into the sequence requirements for the strength and function of full-length promoters and establish guidelines for promoter prediction and for forward engineering promoters of specific strengths. PMID:22156164

  3. Effect of Full Correction Versus Partial Correction of Elevated Blood Glucose in the Emergency Department on Hospital Length of Stay.

    PubMed

    Johnson-Clague, Michaela; DiLeo, Jessica; Katz, Michael D; Patanwala, Asad E

    2016-01-01

    There is limited information to guide the extent to which asymptomatic hyperglycemia needs to be corrected in patients presenting to the emergency department (ED) with unrelated complaints. The objective of this study was to compare full correction (FC) versus partial correction (PC) of elevated blood glucose in the ED on hospital length of stay. This was a retrospective cohort study conducted in an academic ED in the United States. Adult diabetic patients with hyperglycemia (blood glucose >200 mg/dL) in the ED who were treated with subcutaneous insulin were included. Patients were categorized based on the level of blood glucose control achieved within the first 24 hours from triage: (1) FC group for whom blood glucose <200 mg/dL was achieved or (2) PC group for whom blood glucose remained ≥200 mg/dL. The primary outcome measure was a comparison of hospital length of stay between groups. A total of 161 patients were included in this study (FC = 81, PC = 80). There was no significant difference between hospital length of stay in the FC [3 days (interquartile range, 1-5 days)] and PC [3 days (interquartile range, 2-6 days)] groups (P = 0.159). In the multivariate analysis, after adjusting for potential confounders, there was no significant association between level of correction and hospital length of stay (log-transformed) (coefficient 0.238; 95% confidence interval, -0.062 to 0.537; P = 0.119; R = 13%). The extent of glucose correction was not associated with a decrease in hospital length of stay in diabetic patients with hyperglycemia in the ED. PMID:25187094

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

    SciTech Connect

    Stapleton, Mark; Liao, Guochun; Brokstein, Peter; Hong, Ling; Carninci, Piero; Shiraki, Toshiyuki; Hayashizaki, Yoshihide; Champe, Mark; Pacleb, Joanne; Wan, Ken; Yu, Charles; Carlson, Joe; George, Reed; Celniker, Susan; Rubin, Gerald M.

    2002-08-12

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

  5. Identification of 48 full-length MHC-DAB functional alleles in miiuy croaker and evidence for positive selection.

    PubMed

    Liu, Jiang; Sun, Yueyan; Xu, Tianjun

    2016-07-01

    Major histocompatibility complex (MHC) molecules play a vital role in the immune response and are a highly polymorphic gene superfamily in vertebrates. As the molecular marker associated with polymorphism and disease susceptibility/resistance, the polymorphism of MHC genes has been investigated in many tetrapods and teleosts. Most studies were focused on the polymorphism of the second exon, which encodes the peptide-binding region (PBR) in the α1- or β1-domain, but few studies have examined the full-length coding region. To comprehensive investigate the polymorphism of MHC gene, we identified 48 full-length miiuy croaker (Miichthys miiuy) MHC class IIB (Mimi-DAB) functional alleles from 26 miiuy croaker individuals. All of the alleles encode 34 amino acid sequences, and a high level of polymorphism was detected in Mimi-DAB alleles. The rate of non-synonymous substitutions (dN) occurred at a significantly higher frequency than that of synonymous substitutions (dS) in the PBR, and this result suggests that balancing selection maintains polymorphisms at the Mimi-DAB locus. Phylogenetic analysis based on the full-length and exon 2 sequences of Mimi-DAB alleles both showed that the Mimi-DAB alleles were clustered into two major groups. A total of 19 positive selected sites were identified on the Mimi-DAB alleles after testing for positive selection, and 14 sites were predicted to be associated with antigen-binding sites, which suggests that most of selected sites are significant for disease resistance. The polymorphism of Mimi-DAB alleles provides an important resource for analyzing the association between the polymorphism of MHC gene and disease susceptibility/resistance, and for researching the molecular selective breeding of miiuy croaker with enhanced disease resistance. PMID:27164216

  6. The Juxtamembrane Linker of Full-length Synaptotagmin 1 Controls Oligomerization and Calcium-dependent Membrane Binding*

    PubMed Central

    Lu, Bin; Kiessling, Volker; Tamm, Lukas K.; Cafiso, David S.

    2014-01-01

    Synaptotagmin 1 (Syt1) is the calcium sensor for synchronous neurotransmitter release. The two C2 domains of Syt1, which may mediate fusion by bridging the vesicle and plasma membranes, are connected to the vesicle membrane by a 60-residue linker. Here, we use site-directed spin labeling and a novel total internal reflection fluorescence vesicle binding assay to characterize the juxtamembrane linker and to test the ability of reconstituted full-length Syt1 to interact with opposing membrane surfaces. EPR spectroscopy demonstrates that the majority of the linker interacts with the membrane interface, thereby limiting the extension of the C2A and C2B domains into the cytoplasm. Pulse dipolar EPR spectroscopy provides evidence that purified full-length Syt1 is oligomerized in the membrane, and mutagenesis indicates that a glycine zipper/GXXXG motif within the linker helps mediate oligomerization. The total internal reflection fluorescence-based vesicle binding assay demonstrates that full-length Syt1 that is reconstituted into supported lipid bilayers will capture vesicles containing negatively charged lipid in a Ca2+-dependent manner. Moreover, the rate of vesicle capture increases with Syt1 density, and mutations in the GXXXG motif that inhibit oligomerization of Syt1 reduce the rate of vesicle capture. This work demonstrates that modifications within the 60-residue linker modulate both the oligomerization of Syt1 and its ability to interact with opposing bilayers. In addition to controlling its activity, the oligomerization of Syt1 may play a role in organizing proteins within the active zone of membrane fusion. PMID:24973220

  7. Characterization of expressed sequence tags from a full-length enriched cDNA library of Cryptomeria japonica male strobili

    PubMed Central

    Futamura, Norihiro; Totoki, Yasushi; Toyoda, Atsushi; Igasaki, Tomohiro; Nanjo, Tokihiko; Seki, Motoaki; Sakaki, Yoshiyuki; Mari, Adriano; Shinozaki, Kazuo; Shinohara, Kenji

    2008-01-01

    Background Cryptomeria japonica D. Don is one of the most commercially important conifers in Japan. However, the allergic disease caused by its pollen is a severe public health problem in Japan. Since large-scale analysis of expressed sequence tags (ESTs) in the male strobili of C. japonica should help us to clarify the overall expression of genes during the process of pollen development, we constructed a full-length enriched cDNA library that was derived from male strobili at various developmental stages. Results We obtained 36,011 expressed sequence tags (ESTs) from either one or both ends of 19,437 clones derived from the cDNA library of C. japonica male strobili at various developmental stages. The 19,437 cDNA clones corresponded to 10,463 transcripts. Approximately 80% of the transcripts resembled ESTs from Pinus and Picea, while approximately 75% had homologs in Arabidopsis. An analysis of homologies between ESTs from C. japonica male strobili and known pollen allergens in the Allergome Database revealed that products of 180 transcripts exhibited significant homology. Approximately 2% of the transcripts appeared to encode transcription factors. We identified twelve genes for MADS-box proteins among these transcription factors. The twelve MADS-box genes were classified as DEF/GLO/GGM13-, AG-, AGL6-, TM3- and TM8-like MIKCC genes and type I MADS-box genes. Conclusion Our full-length enriched cDNA library derived from C. japonica male strobili provides information on expression of genes during the development of male reproductive organs. We provided potential allergens in C. japonica. We also provided new information about transcription factors including MADS-box genes expressed in male strobili of C. japonica. Large-scale gene discovery using full-length cDNAs is a valuable tool for studies of gymnosperm species. PMID:18691438

  8. Full-Length GB Virus C (Hepatitis G Virus) RNA Transcripts Are Infectious in Primary CD4-Positive T Cells

    PubMed Central

    Xiang, Jinhua; Wünschmann, Sabina; Schmidt, Warren; Shao, Jianqiang; Stapleton, Jack T.

    2000-01-01

    GB virus C (GBV-C or hepatitis G virus) is a recently described flavivirus which frequently leads to chronic viremia in humans. Although GBV-C is associated with acute posttransfusion hepatitis, it is not clear if the virus is pathogenic for humans. We constructed a full-length cDNA from the plasma of a person with chronic GBV-C viremia. Peripheral blood mononuclear cells (PBMCs) transfected with full-length RNA transcripts from this GBV-C clone resulted in viral replication. This was demonstrated by serial passage of virus from cell culture supernatants, detection of increasing concentrations of positive- and negative-sense GBV-C RNA over time, and the detection of the GBV-C E2 antigen by confocal microscopy. In addition, two types of GBV-C particles were identified in cell lysates; these particles had buoyant densities of 1.06 and 1.12 to 1.17 g/ml in sucrose gradients. PBMCs sorted for expression of CD4 contained 100-fold-more GBV-C RNA than CD4-negative cells. Taken together, these data demonstrate that RNA transcripts from GBV-C full-length cDNA are infectious in primary CD4-positive T cells. In contrast, RNA transcripts from an infectious hepatitis C virus clone did not replicate in the same cell culture system. Infectious RNA transcripts from GBV-C cDNA should prove useful for studying viral replication and may allow identification of differences between GBV-C and hepatitis C virus cultivation in vitro. PMID:10982359

  9. Binding of Full-Length HIV-1 gp120 to CD4 Induces Structural Reorientation around the gp120 Core

    SciTech Connect

    Ashish,F.; Garg, R.; Anguita, J.; Krueger, J.

    2006-01-01

    Small-angle x-ray scattering data on the unliganded full-length fully glycosylated HIV-1 gp120, the soluble CD4 (domains 1-2) receptor and their complex in solution are presented. Ab initio structure restorations using these data provides the first look at the envelope shape for the unliganded and the complexed gp120 molecule. Fitting known crystal structures of the unliganded SIV and the complexed HIV gp120 core regions within our resultant shape constraints reveals movement of the V3 loop upon binding.

  10. Cloning and expression of full-length human insulin-like growth factor binding protein 3 (IGFBP3) in the Escherichia coli

    PubMed Central

    Khodadadi, Emad; Panjepour, Mojtaba; Abbasian, Mahdi; Broujeni, Zahra Khalili; Mofid, Mohammad Reza

    2015-01-01

    Background: The effect of the growth hormone on target cells is mediated by the insulin-like growth factor 1 (IGF-1). IGF-1 binds to the insulin-like growth factor binding proteins (IGFBPs) in blood and biological fluids. Considering the important application of IGBP3 as a drug component, in this research we cloned and expressed the full-length IGFBP3 in the pET-11a vector and BL21 (DE3) expression host. Materials and Methods: First the sequence encoding of IGFBP3 was designed based on the amino acid sequence of the protein and then by codon optimization, in order to ensure the maximum expression in Escherichia coli. In the next step, the synthetic DNA encoding IGFBP3 was inserted into the pUC57 vector, at the appropriate restriction sites and then subcloned in the pET-11a expression vector in the same restriction sites. The constructed vector was transformed to E. coli BL21 as an expression host and induced in the presence of IPTG for expression of the IGFBP3 protein. Protein expression was evaluated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Results: Double digestion of the new plasmid (pET-11a -IGBP3) with NdeI and BamHI showed two bands in 873 bp and 5700 bp. To study the accurate cloning procedure, the plasmid was sequenced and its authenticity was confirmed. Also the expected protein band (31.6 kDa) was observed in SDS-PAGE analysis. Conclusion: DNA fragment encoding the full-length IGFBP3 protein was accurately cloned in the pET-11a expression vector and the recombinant plasmid transformed to E. coli BL21 (DE3) expression host. Results of the SDS-PAGE analysis verified that recombinant IGFBP3 (31.6 kDa) are successfully expressed under the control of T7 promoter. As we shown pET-11a can be successfully used for expression of the IGFBP3 protein. PMID:25878991

  11. Mesenchymal stromal cell delivery of full-length tumor necrosis factor–related apoptosis-inducing ligand is superior to soluble type for cancer therapy

    PubMed Central

    Yuan, ZhengQiang; Kolluri, Krishna K.; Sage, Elizabeth K.; Gowers, Kate H.C.; Janes, Sam M.

    2015-01-01

    Background aims Mesenchymal stromal cell (MSC) delivery of pro-apoptotic tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is an attractive strategy for anticancer therapy. MSCs expressing full-length human TRAIL (flT) or its soluble form (sT) have previously been shown to be effective for cancer killing. However, a comparison between the two forms has never been performed, leaving it unclear which approach is most effective. This study addresses the issue for the possible clinical application of TRAIL-expressing MSCs in the future. Methods MSCs were transduced with lentiviruses expressing flT or an isoleucine zipper-fused sT. TRAIL expression was examined and cancer cell apoptosis was measured after treatment with transduced MSCs or with MSC-derived soluble TRAIL. Results The transduction does not adversely affect cell phenotype. The sT-transduced MSCs (MSC-sT) secrete abundant levels of soluble TRAIL but do not present the protein on the cell surface. Interestingly, the flT-transduced MSCs (MSC-flT) not only express cell-surface TRAIL but also release flT into medium. These cells were examined for inducing apoptosis in 20 cancer cell lines. MSC-sT cells showed very limited effects. By contrast, MSC-flT cells demonstrated high cancer cell-killing efficiency. More importantly, MSC-flT cells can overcome some cancer cell resistance to recombinant TRAIL. In addition, both cell surface flT and secreted flT are functional for inducing apoptosis. The secreted flT was found to have higher cancer cell-killing capacity than either recombinant TRAIL or MSC-secreted sT. Conclusions These observations demonstrate that MSC delivery of flT is superior to MSC delivery of sT for cancer therapy. PMID:25888191

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

    PubMed

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

    2012-12-01

    Seneca Valley virus (SVV-001) is an oncolytic picornavirus with selective tropism for a subset of human cancers with neuroendocrine differentiation. To characterize further the specificity of SVV-001 and its patterns and kinetics of intratumoral spread, bacterial plasmids encoding a cDNA clone of the full-length wild-type virus and a derivative virus expressing GFP were generated. The full-length cDNA of the SVV-001 RNA genome was cloned into a bacterial plasmid under the control of the T7 core promoter sequence to create an infectious cDNA clone, pNTX-09. A GFP reporter virus cDNA clone, pNTX-11, was then generated by cloning a fusion protein of GFP and the 2A protein from foot-and-mouth disease virus immediately following the native SVV-001 2A sequence. Recombinant GFP-expressing reporter virus, SVV-GFP, was rescued from cells transfected with in vitro RNA transcripts from pNTX-11 and propagated in cell culture. The proliferation kinetics of SVV-001 and SVV-GFP were indistinguishable. The SVV-GFP reporter virus was used to determine that a subpopulation of permissive cells is present in small-cell lung cancer cell lines previously thought to lack permissivity to SVV-001. Finally, it was shown that SVV-GFP administered to tumour-bearing animals homes in to and infects tumours whilst having no detectable tropism for normal mouse tissues at 1×10(11) viral particles kg(-1), a dose equivalent to that administered in ongoing clinical trials. These infectious clones will be of substantial value in further characterizing the biology of this virus and as a backbone for the generation of additional oncolytic derivatives. PMID:22971818

  13. Development of three full-length infectious cDNA clones of distinct brassica yellows virus genotypes for agrobacterium-mediated inoculation.

    PubMed

    Zhang, Xiao-Yan; Dong, Shu-Wei; Xiang, Hai-Ying; Chen, Xiang-Ru; Li, Da-Wei; Yu, Jia-Lin; Han, Cheng-Gui

    2015-02-01

    Brassica yellows virus is a newly identified species in the genus of Polerovirus within the family Luteoviridae. Brassica yellows virus (BrYV) is prevalently distributed throughout Mainland China and South Korea, is an important virus infecting cruciferous crops. Based on six BrYV genomic sequences of isolates from oilseed rape, rutabaga, radish, and cabbage, three genotypes, BrYV-A, BrYV-B, and BrYV-C, exist, which mainly differ in the 5' terminal half of the genome. BrYV is an aphid-transmitted and phloem-limited virus. The use of infectious cDNA clones is an alternative means of infecting plants that allows reverse genetic studies to be performed. In this study, full-length cDNA clones of BrYV-A, recombinant BrYV5B3A, and BrYV-C were constructed under control of the cauliflower mosaic virus 35S promoter. An agrobacterium-mediated inoculation system of Nicotiana benthamiana was developed using these cDNA clones. Three days after infiltration with full-length BrYV cDNA clones, necrotic symptoms were observed in the inoculated leaves of N. benthamiana; however, no obvious symptoms appeared in the upper leaves. Reverse transcription-PCR (RT-PCR) and western blot detection of samples from the upper leaves showed that the maximum infection efficiency of BrYVs could reach 100%. The infectivity of the BrYV-A, BrYV-5B3A, and BrYV-C cDNA clones was further confirmed by northern hybridization. The system developed here will be useful for further studies of BrYV, such as host range, pathogenicity, viral gene functions, and plant-virus-vector interactions, and especially for discerning the differences among the three genotypes. PMID:25499296

  14. Purification, crystallization and preliminary crystallographic analysis of the full-length cystathionine β-synthase from Apis mellifera

    PubMed Central

    Oyenarte, Iker; Majtan, Tomas; Ereño, June; Corral-Rodríguez, María Angeles; Klaudiny, Jaroslav; Majtan, Juraj; Kraus, Jan P.; Martínez-Cruz, Luis Alfonso

    2012-01-01

    Cystathionine β-synthase (CBS) is a pyridoxal-5′-phosphate-dependent enzyme that catalyzes the first step of the transsulfuration pathway, namely the condensation of serine with homocysteine to form cystathionine. Mutations in the CBS gene are the single most common cause of hereditary homocystinuria, a multisystemic disease affecting to various extents the vasculature, connective tissues and central nervous system. At present, the crystal structure of CBS from Drosophila melanogaster is the only available structure of the full-length enzyme. Here we describe a cloning, overexpression, purification and preliminary crystallographic analysis of a full-length CBS from Apis mellifera (AmCBS) which maintains 51 and 46% sequence identity with its Drosophila and human homologs, respectively. The AmCBS yielded crystals belonging to space group P212121, with unit-cell parameters a = 85.90, b = 95.87, c = 180.33 Å. Diffraction data were collected to a resolution of 3.0 Å. The crystal structure contained two molecules in the asymmetric unit which presumably correspond to the dimeric species observed in solution. PMID:23143241

  15. Characterization of 40 full-length MHC class IIA functional alleles in miiuy croaker: Polymorphism and positive selection.

    PubMed

    Xu, Tianjun; Liu, Jiang; Sun, Yueyan; Zhu, Zhihuang; Liu, Tianxing

    2016-02-01

    The major histocompatibility complex is a highly polymorphic gene superfamily in vertebrates that plays an important role in adaptive immune response. In the present study, we identified 40 full-length miiuy croaker MHC class IIA (Mimi-DAA) functional alleles from 26 miiuy croaker individuals and found that the alleles encode 30 amino acid sequences. A high level of polymorphism in Mimi-DAA was detected in miiuy croaker. The rate of non-synonymous substitutions (d(N)) occurred at a significantly higher frequency than that of synonymous substitutions (d(S)) in the peptide-binding region (PBR) and non-PBR. This result suggests that balancing selection maintains polymorphisms at the Mimi-DAA locus. Phylogenetic analysis based on the full-length sequences showed that the Mimi-DAA alleles clustered into three groups. However, the phylogenetic tree constructed using the exon 2 sequences indicated that the Mimi-DAA alleles clustered into two groups. A total of 22 positively selected sites were identified on the Mimi-DAA alleles after testing for positive selection, and five sites were predicted to be associated with the binding of peptide antigen, suggesting that a few selected residues may play a significant role in immune function. PMID:26598111

  16. Global Identification of the Full-Length Transcripts and Alternative Splicing Related to Phenolic Acid Biosynthetic Genes in Salvia miltiorrhiza

    PubMed Central

    Xu, Zhichao; Luo, Hongmei; Ji, Aijia; Zhang, Xin; Song, Jingyuan; Chen, Shilin

    2016-01-01

    Salvianolic acids are among the main bioactive components in Salvia miltiorrhiza, and their biosynthesis has attracted widespread interest. However, previous studies on the biosynthesis of phenolic acids using next-generation sequencing platforms are limited with regard to the assembly of full-length transcripts. Based on hybrid-seq (next-generation and single molecular real-time sequencing) of the S. miltiorrhiza root transcriptome, we experimentally identified 15 full-length transcripts and four alternative splicing events of enzyme-coding genes involved in the biosynthesis of rosmarinic acid. Moreover, we herein demonstrate that lithospermic acid B accumulates in the phloem and xylem of roots, in agreement with the expression patterns of the identified key genes related to rosmarinic acid biosynthesis. According to co-expression patterns, we predicted that six candidate cytochrome P450s and five candidate laccases participate in the salvianolic acid pathway. Our results provide a valuable resource for further investigation into the synthetic biology of phenolic acids in S. miltiorrhiza. PMID:26904067

  17. [Rapid site-directed mutagenesis on full-length plasmid DNA by using designed restriction enzyme assisted mutagenesis].

    PubMed

    Zhang, Baozhong; Ran, Duoliang; Zhang, Xin; An, Xiaoping; Shan, Yunzhu; Zhou, Yusen; Tong, Yigang

    2009-02-01

    To use the designed restriction enzyme assisted mutagenesis technique to perform rapid site-directed mutagenesis on double-stranded plasmid DNA. The target amino acid sequence was reversely translated into DNA sequences with degenerate codons, resulting in large amount of silently mutated sequences containing various restriction endonucleases (REs). Certain mutated sequence with an appropriate RE was selected as the target DNA sequence for designing mutation primers. The full-length plasmid DNA was amplified with high-fidelity Phusion DNA polymerase and the amplified product was 5' phosphorylated by T4 polynucleotide kinase and then self-ligated. After transformation into an E. coli host the transformants were rapidly screened by cutting with the designed RE. With this strategy we successfully performed the site-directed mutagenesis on an 8 kb plasmid pcDNA3.1-pIgR and recovered the wild-type amino acid sequence of human polymeric immunoglobulin receptor (pIgR). A novel site-directed mutagenesis strategy based on DREAM was developed which exploited RE as a rapid screening measure. The highly efficient, high-fidelity Phusion DNA polymerase was applied to ensure the efficient and faithful amplification of the full-length sequence of a plasmid of up to 8 kb. This rapid mutagenesis strategy avoids using any commercial site-directed mutagenesis kits, special host strains or isotopes. PMID:19459340

  18. Functional and expression analyses of transcripts based on full-length cDNAs of Sorghum bicolor

    PubMed Central

    Shimada, Setsuko; Makita, Yuko; Kuriyama-Kondou, Tomoko; Kawashima, Mika; Mochizuki, Yoshiki; Hirakawa, Hideki; Sato, Shusei; Toyoda, Tetsuro; Matsui, Minami

    2015-01-01

    Sorghum bicolor is one of the most important crops for food and bioethanol production. Its small diploid genome and resistance to environmental stress make sorghum an attractive model for studying the functional genomics of the Saccharinae and other C4 grasses. We analyzed the domain-based functional annotation of the cDNAs using the gene ontology (GO) categories for molecular function to characterize all the genes cloned in the full-length cDNA library of sorghum. The sorghum cDNA library successfully captured a wide range of cDNA-encoded proteins with various functions. To characterize the protein function of newly identified cDNAs, a search of their deduced domains and comparative analyses in the Oryza sativa and Zea mays genomes were carried out. Furthermore, genes on the sense strand corresponding to antisense transcripts were classified based on the GO of molecular function. To add more information about these genes, we have analyzed the expression profiles using RNA-Seq of three tissues (spikelet, seed and stem) during the starch-filling phase. We performed functional analysis of tissue-specific genes and expression analysis of genes of starch biosynthesis enzymes. This functional analysis of sorghum full-length cDNAs and the transcriptome information will facilitate further analysis of the Saccharinae and grass families. PMID:26546227

  19. Penalized likelihood for sparse contingency tables with an application to full-length cDNA libraries

    PubMed Central

    Dahinden, Corinne; Parmigiani, Giovanni; Emerick, Mark C; Bühlmann, Peter

    2007-01-01

    Background The joint analysis of several categorical variables is a common task in many areas of biology, and is becoming central to systems biology investigations whose goal is to identify potentially complex interaction among variables belonging to a network. Interactions of arbitrary complexity are traditionally modeled in statistics by log-linear models. It is challenging to extend these to the high dimensional and potentially sparse data arising in computational biology. An important example, which provides the motivation for this article, is the analysis of so-called full-length cDNA libraries of alternatively spliced genes, where we investigate relationships among the presence of various exons in transcript species. Results We develop methods to perform model selection and parameter estimation in log-linear models for the analysis of sparse contingency tables, to study the interaction of two or more factors. Maximum Likelihood estimation of log-linear model coefficients might not be appropriate because of the presence of zeros in the table's cells, and new methods are required. We propose a computationally efficient ℓ1-penalization approach extending the Lasso algorithm to this context, and compare it to other procedures in a simulation study. We then illustrate these algorithms on contingency tables arising from full-length cDNA libraries. Conclusion We propose regularization methods that can be used successfully to detect complex interaction patterns among categorical variables in a broad range of biological problems involving categorical variables. PMID:18072965

  20. Responses of Nontransformed Human Hepatocytes to Conditional Expression of Full-Length Hepatitis C Virus Open Reading Frame

    PubMed Central

    Tang, Weiliang; Lázaro, Catherine A.; Campbell, Jean S.; Parks, W. Tony; Katze, Michael G.; Fausto, Nelson

    2007-01-01

    Hepatitis C virus (HCV) is a major cause of chronic hepatitis that can lead to cirrhosis and hepatocellular carcinoma. To study the effects of HCV protein expression on host cells, we established conditional expression of the full-length open reading frame (ORF) of an infectious cDNA clone of HCV (genotype 1a, H77 strain) in the nontransformed human hepatocyte line cell HH4 using the ecdysone receptor regulatory system. Treatment with the ecdysone analog ponasterone-A induced tightly regulated and dose-dependent full-length HCV ORF expression and properly processed HCV proteins. HCV Core, NS3, and NS5A colocalized in perinuclear regions and associated with the early endosomal protein EEA1. HCV ORF expression caused marked growth inhibition, increased intracellular reactive oxygen species, up-regulation of glutamate-l-cysteine ligase activity, increased glutathione level, and activation of nuclear factor κB. Although it was not directly cytotoxic, HCV ORF expression sensitized HH4 cells to Fas at certain concentrations but not to tumor necrosis factor-related apoptosis-inducing ligand. HCV ORF expression in HH4 cells up-regulated genes involved in innate immune response/inflammation and oxidative stress responses and down-regulated cell growth-related genes. Expression of HCV ORF in host cells may contribute to HCV pathogenesis by producing oxidative stress and increasing the expression of genes related to the innate immune response and inflammation. PMID:17991716

  1. Full-length sequence analysis of chloroquine resistance transporter gene in Plasmodium falciparum isolates from Sabah, Malaysia.

    PubMed

    Tan, Lii Lian; Lau, Tiek Ying; Timothy, William; Prabakaran, Dhanaraj

    2014-01-01

    Chloroquine resistance (CQR) in falciparum malaria was identified to be associated with several mutations in the chloroquine resistance transporter gene (pfcrt) that encodes the transmembrane transporter in digestive vacuole membrane of the parasite. This study aimed to investigate the point mutations across the full-length pfcrt in Plasmodium falciparum isolates in Sabah, Malaysia. A total of 31 P. falciparum positive samples collected from Keningau, Kota Kinabalu, and Kudat, Sabah, were analyzed. pfcrt was PCR amplified and cloned prior to sequence analysis. This study showed that all the previously described 10 point mutations associated with CQR at codons 72, 74, 75, 76, 97, 220, 271, 326, 356, and 371 were found with different prevalence. Besides, two novel point mutations, I166V and H273N, were identified with 22.5% and 19.3%, respectively. Three haplotypes, namely, CVMNK (29%), CVIET (3.2%), and SVMNT (67.7%), were identified. High prevalence of SVMNT among P. falciparum isolates from Sabah showed that these isolates are closer to the P. falciparum isolates from Papua New Guinea rather than to the more proximal Southeast Asian CVIET haplotype. Full-length analysis of pfcrt showed that chloroquine resistant P. falciparum in Sabah is still prevalent despite the withdrawal of chloroquine usage since 1979. PMID:25574497

  2. An efficient full-length cDNA amplification strategy based on bioinformatics technology and multiplexed PCR methods.

    PubMed

    Chen, Nan; Wang, Wei-Min; Wang, Huan-Ling

    2016-01-01

    A novel strategy for amplification full-length cDNA and promoter sequences has been developed using bioinformatics technology and multiplexed PCR methods in this study. The amplification of 3' ends of cDNA is performed according to the modified classic 3' RACE techniques, therein the more efficient and effective oligo(dT)-anchor primer with hairpin structure is specially designed. For the amplification of 5' ends of cDNA, two or three-round TAIL-PCR or touch-down PCR using arbitrary degenerate (AD) and sequence-specific reverse (SPR) primers is performed until the 5' sequence of multi-assembled fragment reaches the exon1 region identified by aligning this fragment to reference genome database. Then another TAIL-PCR or touch-down PCR using genomic DNA as template is conducted to obtain the remaining 5' and promoter sequences. The 5' end sites of cDNA are predicted by aligning finally assembled fragment to homologous reference genes of other species, and screening the relative locations of common characteristic cis-elements in silico on promoter. The putative 5' ends are further validated by primers corresponding to these predicted sites in cDNAs. This method is suitable for researchers to isolate limited full-length cDNA sequences due to its operability, inexpensiveness, efficiency and speediness. PMID:26758040

  3. An efficient full-length cDNA amplification strategy based on bioinformatics technology and multiplexed PCR methods

    PubMed Central

    Chen, Nan; Wang, Wei-Min; Wang, Huan-Ling

    2016-01-01

    A novel strategy for amplification full-length cDNA and promoter sequences has been developed using bioinformatics technology and multiplexed PCR methods in this study. The amplification of 3′ ends of cDNA is performed according to the modified classic 3′ RACE techniques, therein the more efficient and effective oligo(dT)-anchor primer with hairpin structure is specially designed. For the amplification of 5′ ends of cDNA, two or three-round TAIL-PCR or touch-down PCR using arbitrary degenerate (AD) and sequence-specific reverse (SPR) primers is performed until the 5′ sequence of multi-assembled fragment reaches the exon1 region identified by aligning this fragment to reference genome database. Then another TAIL-PCR or touch-down PCR using genomic DNA as template is conducted to obtain the remaining 5′ and promoter sequences. The 5′ end sites of cDNA are predicted by aligning finally assembled fragment to homologous reference genes of other species, and screening the relative locations of common characteristic cis-elements in silico on promoter. The putative 5′ ends are further validated by primers corresponding to these predicted sites in cDNAs. This method is suitable for researchers to isolate limited full-length cDNA sequences due to its operability, inexpensiveness, efficiency and speediness. PMID:26758040

  4. Analysis of the mouse gut microbiome using full-length 16S rRNA amplicon sequencing

    PubMed Central

    Shin, Jongoh; Lee, Sooin; Go, Min-Jeong; Lee, Sang Yup; Kim, Sun Chang; Lee, Chul-Ho; Cho, Byung-Kwan

    2016-01-01

    Demands for faster and more accurate methods to analyze microbial communities from natural and clinical samples have been increasing in the medical and healthcare industry. Recent advances in next-generation sequencing technologies have facilitated the elucidation of the microbial community composition with higher accuracy and greater throughput than was previously achievable; however, the short sequencing reads often limit the microbial composition analysis at the species level due to the high similarity of 16S rRNA amplicon sequences. To overcome this limitation, we used the nanopore sequencing platform to sequence full-length 16S rRNA amplicon libraries prepared from the mouse gut microbiota. A comparison of the nanopore and short-read sequencing data showed that there were no significant differences in major taxonomic units (89%) except one phylotype and three taxonomic units. Moreover, both sequencing data were highly similar at all taxonomic resolutions except the species level. At the species level, nanopore sequencing allowed identification of more species than short-read sequencing, facilitating the accurate classification of the bacterial community composition. Therefore, this method of full-length 16S rRNA amplicon sequencing will be useful for rapid, accurate and efficient detection of microbial diversity in various biological and clinical samples. PMID:27411898

  5. Analysis of the mouse gut microbiome using full-length 16S rRNA amplicon sequencing.

    PubMed

    Shin, Jongoh; Lee, Sooin; Go, Min-Jeong; Lee, Sang Yup; Kim, Sun Chang; Lee, Chul-Ho; Cho, Byung-Kwan

    2016-01-01

    Demands for faster and more accurate methods to analyze microbial communities from natural and clinical samples have been increasing in the medical and healthcare industry. Recent advances in next-generation sequencing technologies have facilitated the elucidation of the microbial community composition with higher accuracy and greater throughput than was previously achievable; however, the short sequencing reads often limit the microbial composition analysis at the species level due to the high similarity of 16S rRNA amplicon sequences. To overcome this limitation, we used the nanopore sequencing platform to sequence full-length 16S rRNA amplicon libraries prepared from the mouse gut microbiota. A comparison of the nanopore and short-read sequencing data showed that there were no significant differences in major taxonomic units (89%) except one phylotype and three taxonomic units. Moreover, both sequencing data were highly similar at all taxonomic resolutions except the species level. At the species level, nanopore sequencing allowed identification of more species than short-read sequencing, facilitating the accurate classification of the bacterial community composition. Therefore, this method of full-length 16S rRNA amplicon sequencing will be useful for rapid, accurate and efficient detection of microbial diversity in various biological and clinical samples. PMID:27411898

  6. Functional and expression analyses of transcripts based on full-length cDNAs of Sorghum bicolor.

    PubMed

    Shimada, Setsuko; Makita, Yuko; Kuriyama-Kondou, Tomoko; Kawashima, Mika; Mochizuki, Yoshiki; Hirakawa, Hideki; Sato, Shusei; Toyoda, Tetsuro; Matsui, Minami

    2015-12-01

    Sorghum bicolor is one of the most important crops for food and bioethanol production. Its small diploid genome and resistance to environmental stress make sorghum an attractive model for studying the functional genomics of the Saccharinae and other C4 grasses. We analyzed the domain-based functional annotation of the cDNAs using the gene ontology (GO) categories for molecular function to characterize all the genes cloned in the full-length cDNA library of sorghum. The sorghum cDNA library successfully captured a wide range of cDNA-encoded proteins with various functions. To characterize the protein function of newly identified cDNAs, a search of their deduced domains and comparative analyses in the Oryza sativa and Zea mays genomes were carried out. Furthermore, genes on the sense strand corresponding to antisense transcripts were classified based on the GO of molecular function. To add more information about these genes, we have analyzed the expression profiles using RNA-Seq of three tissues (spikelet, seed and stem) during the starch-filling phase. We performed functional analysis of tissue-specific genes and expression analysis of genes of starch biosynthesis enzymes. This functional analysis of sorghum full-length cDNAs and the transcriptome information will facilitate further analysis of the Saccharinae and grass families. PMID:26546227

  7. The feline oral microbiome: a provisional 16S rRNA gene based taxonomy with full-length reference sequences.

    PubMed

    Dewhirst, Floyd E; Klein, Erin A; Bennett, Marie-Louise; Croft, Julie M; Harris, Stephen J; Marshall-Jones, Zoe V

    2015-02-25

    The human oral microbiome is known to play a significant role in human health and disease. While less well studied, the feline oral microbiome is thought to play a similarly important role. To determine roles oral bacteria play in health and disease, one first has to be able to accurately identify bacterial species present. 16S rRNA gene sequence information is widely used for molecular identification of bacteria and is also useful for establishing the taxonomy of novel species. The objective of this research was to obtain full 16S rRNA gene reference sequences for feline oral bacteria, place the sequences in species-level phylotypes, and create a curated 16S rRNA based taxonomy for common feline oral bacteria. Clone libraries were produced using "universal" and phylum-selective PCR primers and DNA from pooled subgingival plaque from healthy and periodontally diseased cats. Bacteria in subgingival samples were also cultivated to obtain isolates. Full-length 16S rDNA sequences were determined for clones and isolates that represent 171 feline oral taxa. A provisional curated taxonomy was developed based on the position of each taxon in 16S rRNA phylogenetic trees. The feline oral microbiome curated taxonomy and 16S rRNA gene reference set will allow investigators to refer to precisely defined bacterial taxa. A provisional name such as "Propionibacterium sp. feline oral taxon FOT-327" is an anchor to which clone, strain or GenBank names or accession numbers can point. Future next-generation-sequencing studies of feline oral bacteria will be able to map reads to taxonomically curated full-length 16S rRNA gene sequences. PMID:25523504

  8. Integrative Annotation of 21,037 Human Genes Validated by Full-Length cDNA Clones

    SciTech Connect

    Imanishi, Tadashi; Itoh, Takeshi; Suzuki, Yutaka; O'Donovan, Claire; Fukuchi, Satoshi; Koyanagi, Kanako O.; Barrero, Roberto A.; Tamura, Takuro; Yamaguchi-Kabata, Yumi; Tanino, Motohiko; Yura, Kei; Miyazaki, Satoru; Ikeo, Kazuho; Homma, Keiichi; Kasprzyk, Arek; Nishikawa, Tetsuo; Hirakawa, Mika; Thierry-Mieg, Jean; Thierry-Mieg, Danielle; Ashurst, Jennifer; Jia, Libin; Nakao, Mitsuteru; Thomas, Michael A.; Mulder, Nicola; Karavidopoulou, Youla; Jin, Lihua; Kim, Sangsoo; Yasuda, Tomohiro; Lenhard, Boris; Eveno, Eric; Suzuki, Yoshiyuki; Yamasaki, Chisato; Takeda, Jun-ichi; Gough, Craig; Hilton, Phillip; Fujii, Yasuyuki; Sakai, Hiroaki; Tanaka, Susumu; Amid, Clara; Bellgard, Matthew; de Fatima Bonaldo, Maria; Bono Hidemasa; Bromberg, Susan K.; Brookes, Anthony J.; Bruford, Elspeth; Carninci Piero; Chelala, Claude; Couillault, Christine; de Souza, Sandro J.; Debily, Marie-Anne; Devignes, Marie-Dominique; Dubchak, Inna; Endo, Toshinori; Estreicher, Anne; Eyras, Eduardo; Fukami-Kobayashi, Kaoru; Gopinath, Gopal R.; Graudens, Esther; Hahn, Yoonsoo; Han, Michael; Han, Ze-Guang; Hanada, Kousuke; Hanaoka, Hideki; Harada, Erimi; Hashimoto, Katsuyuki; Hinz, Ursula; Hirai, Momoki; Hishiki, Teruyoshi; Hopkinson, Ian; Imbeaud, Sandrine; Inoko, Hidetoshi; Kanapin, Alexander; Kaneko, Yayoi; Kasukawa, Takeya; Kelso, Janet; Kersey, Paul; Kikuno Reiko; Kimura, Kouichi; Korn, Bernhard; Kuryshev, Vladimir; Makalowska, Izabela; Makino Takashi; Mano, Shuhei; Mariage-Samson, Regine; Mashima, Jun; Matsuda, Hideo; Mewes, Hans-Werner; Minoshima, Shinsei; Nagai, Keiichi; Nagasaki, Hideki; Nagata, Naoki; Nigam, Rajni; Ogasawara, Osamu; Ohara, Osamu; Ohtsubo, Masafumi; Okada, Norihiro; Okido, Toshihisa; Oota, Satoshi; Ota, Motonori; Ota, Toshio; Otsuki, Tetsuji; Piatier-Tonneau, Dominique; Poustka, Annemarie; Ren, Shuang-Xi; Saitou, Naruya; Sakai, Katsunaga; Sakamoto, Shigetaka; Sakate, Ryuichi; Schupp, Ingo; Servant, Florence; Sherry, Stephen; Shiba Rie; et al.

    2004-01-15

    The human genome sequence defines our inherent biological potential; the realization of the biology encoded therein requires knowledge of the function of each gene. Currently, our knowledge in this area is still limited. Several lines of investigation have been used to elucidate the structure and function of the genes in the human genome. Even so, gene prediction remains a difficult task, as the varieties of transcripts of a gene may vary to a great extent. We thus performed an exhaustive integrative characterization of 41,118 full-length cDNAs that capture the gene transcripts as complete functional cassettes, providing an unequivocal report of structural and functional diversity at the gene level. Our international collaboration has validated 21,037 human gene candidates by analysis of high-quality full-length cDNA clones through curation using unified criteria. This led to the identification of 5,155 new gene candidates. It also manifested the most reliable way to control the quality of the cDNA clones. We have developed a human gene database, called the H-Invitational Database (H-InvDB; http://www.h-invitational.jp/). It provides the following: integrative annotation of human genes, description of gene structures, details of novel alternative splicing isoforms, non-protein-coding RNAs, functional domains, subcellular localizations, metabolic pathways, predictions of protein three-dimensional structure, mapping of known single nucleotide polymorphisms (SNPs), identification of polymorphic microsatellite repeats within human genes, and comparative results with mouse full-length cDNAs. The H-InvDB analysis has shown that up to 4 percent of the human genome sequence (National Center for Biotechnology Information build 34 assembly) may contain misassembled or missing regions. We found that 6.5 percent of the human gene candidates (1,377 loci) did not have a good protein-coding open reading frame, of which 296 loci are strong candidates for nonprotein-coding RNA

  9. Full-Length Fibronectin Drives Fibroblast Accumulation at the Surface of Collagen Microtissues during Cell-Induced Tissue Morphogenesis

    PubMed Central

    Foolen, Jasper; Shiu, Jau-Ye; Mitsi, Maria; Zhang, Yang; Chen, Christopher S.; Vogel, Viola

    2016-01-01

    Generating and maintaining gradients of cell density and extracellular matrix (ECM) components is a prerequisite for the development of functionality of healthy tissue. Therefore, gaining insights into the drivers of spatial organization of cells and the role of ECM during tissue morphogenesis is vital. In a 3D model system of tissue morphogenesis, a fibronectin-FRET sensor recently revealed the existence of two separate fibronectin populations with different conformations in microtissues, i.e. ‘compact and adsorbed to collagen’ versus ‘extended and fibrillar’ fibronectin that does not colocalize with the collagen scaffold. Here we asked how the presence of fibronectin might drive this cell-induced tissue morphogenesis, more specifically the formation of gradients in cell density and ECM composition. Microtissues were engineered in a high-throughput model system containing rectangular microarrays of 12 posts, which constrained fibroblast-populated collagen gels, remodeled by the contractile cells into trampoline-shaped microtissues. Fibronectin’s contribution during the tissue maturation process was assessed using fibronectin-knockout mouse embryonic fibroblasts (Fn-/- MEFs) and floxed equivalents (Fnf/f MEFs), in fibronectin-depleted growth medium with and without exogenously added plasma fibronectin (full-length, or various fragments). In the absence of full-length fibronectin, Fn-/- MEFs remained homogenously distributed throughout the cell-contracted collagen gels. In contrast, in the presence of full-length fibronectin, both cell types produced shell-like tissues with a predominantly cell-free compacted collagen core and a peripheral surface layer rich in cells. Single cell assays then revealed that Fn-/- MEFs applied lower total strain energy on nanopillar arrays coated with either fibronectin or vitronectin when compared to Fnf/f MEFs, but that the presence of exogenously added plasma fibronectin rescued their contractility. While collagen

  10. Full-Length Fibronectin Drives Fibroblast Accumulation at the Surface of Collagen Microtissues during Cell-Induced Tissue Morphogenesis.

    PubMed

    Foolen, Jasper; Shiu, Jau-Ye; Mitsi, Maria; Zhang, Yang; Chen, Christopher S; Vogel, Viola

    2016-01-01

    Generating and maintaining gradients of cell density and extracellular matrix (ECM) components is a prerequisite for the development of functionality of healthy tissue. Therefore, gaining insights into the drivers of spatial organization of cells and the role of ECM during tissue morphogenesis is vital. In a 3D model system of tissue morphogenesis, a fibronectin-FRET sensor recently revealed the existence of two separate fibronectin populations with different conformations in microtissues, i.e. 'compact and adsorbed to collagen' versus 'extended and fibrillar' fibronectin that does not colocalize with the collagen scaffold. Here we asked how the presence of fibronectin might drive this cell-induced tissue morphogenesis, more specifically the formation of gradients in cell density and ECM composition. Microtissues were engineered in a high-throughput model system containing rectangular microarrays of 12 posts, which constrained fibroblast-populated collagen gels, remodeled by the contractile cells into trampoline-shaped microtissues. Fibronectin's contribution during the tissue maturation process was assessed using fibronectin-knockout mouse embryonic fibroblasts (Fn-/- MEFs) and floxed equivalents (Fnf/f MEFs), in fibronectin-depleted growth medium with and without exogenously added plasma fibronectin (full-length, or various fragments). In the absence of full-length fibronectin, Fn-/- MEFs remained homogenously distributed throughout the cell-contracted collagen gels. In contrast, in the presence of full-length fibronectin, both cell types produced shell-like tissues with a predominantly cell-free compacted collagen core and a peripheral surface layer rich in cells. Single cell assays then revealed that Fn-/- MEFs applied lower total strain energy on nanopillar arrays coated with either fibronectin or vitronectin when compared to Fnf/f MEFs, but that the presence of exogenously added plasma fibronectin rescued their contractility. While collagen decoration of

  11. Interaction of the Full-length Bax Protein with Biomimetic Mitochondrial Liposomes: A Small-Angle Neutron Scattering and Fluorescence Study

    SciTech Connect

    Satsoura, D; Kucerka, Norbert; Shivakumar, S; Pencer, J; Griffiths, C; Leber, B; Andrews, D.W; Katsaras, John; Fradin, C

    2012-01-01

    In response to apoptotic stimuli, the pro-apoptotic protein Bax inserts in the outer mitochondrial membrane, resulting in the formation of pores and the release of several mitochondrial components, and sealing the cell's fate. To study the binding of Bax to membranes, we used an in vitro system consisting of 50 nm diameter liposomes prepared with a lipid composition mimicking that of mitochondrial membranes in which recombinant purified full-length Bax was inserted via activation with purified tBid. We detected the association of the protein with the membrane using fluorescence fluctuation methods, and found that it could well be described by an equilibrium between soluble and membrane-bound Bax and that at a high protein-toliposome ratio the binding seemed to saturate at about 15 Bax proteins per 50 nm diameter liposome. We then obtained structural data for samples in this saturated binding regime using small-angle neutron scattering under different contrast matching conditions. Utilizing a simple model to fit the neutron data, we observed that a significant amount of the protein mass protrudes above the membrane, in contrast to the conjecture that all of the membrane-associated Bax states are umbrella-like. Upon protein binding, we also observed a thinning of the lipid bilayer accompanied by an increase in liposome radius, an effect reminiscent of the action of antimicrobial peptides on membranes.

  12. A truncated fragment of Ov-ASP-1 consisting of the core pathogenesis-related-1 (PR-1) domain maintains adjuvanticity as the full-length protein.

    PubMed

    Guo, Jingjing; Yang, Yi; Xiao, Wenjun; Sun, Weilai; Yu, Hong; Du, Lanying; Lustigman, Sara; Jiang, Shibo; Kou, Zhihua; Zhou, Yusen

    2015-04-15

    The Onchocerca volvulus activation-associated secreted protein-1 (Ov-ASP-1) has good adjuvanticity for a variety of antigens and vaccines, probably due to its ability activate antigen-processing cells (APCs). However, the functional domain of Ov-ASP-1 as an adjuvant is not clearly defined. Based on the structural prediction of this protein family, we constructed a 16-kDa recombinant protein of Ov-ASP-1 that contains only the core pathogenesis-related-1 (PR-1) domain (residues 10-153), designated ASPPR. We found that ASPPR exhibits adjuvanticity similar to that of the full-length Ov-ASP-1 (residues 10-220) for various antigens, including ovalbumin (OVA), HBsAg protein antigen, and the HIV peptide 5 (Pep5) antigen, but it is more suitable for vaccine design in ASPPR-antigen fusion proteins, and more stable in PBS than Ov-ASP-1 stored at -70 °C. These results suggest that ASPPR might be the functional region of Ov-ASP-1 as an adjuvant, and therefore could be developed as an adjuvant for human use. PMID:25736195

  13. Strategies to facilitate the development of uncloned or cloned infectious full-length viral cDNAs: Apple chlorotic leaf spot virus as a case study

    PubMed Central

    2011-01-01

    Background Approaches to simplify and streamline the construction of full-length infectious cDNA clones (FL-cDNAs) are needed. Among desirable improvements are the ability to use total nucleic acids (TNA) extracts from infected hosts (to bypass viral purification limitations) for the direct one-step amplification of large FL-cDNAs, the possibility to inoculate plants with uncloned FL-cDNAs and the simplified cloning of these large molecules. Results Using the 7.55 kb genome of Apple chlorotic leaf spot trichovirus (ACLSV) approaches allowing the rapid generation from TNA extracts of FL-cDNAs under the control of the T7 promoter and the successful inoculation of plants using in vitro transcripts obtained from these uncloned amplification products have been developed. We also show that the yeast homologous recombination system permits efficient cloning of FL-cDNAs and the simultaneous one-step tailoring of a ternary Yeast-Escherichia coli-Agrobacterium tumefaciens shuttle vector allowing efficient inoculation of both herbaceous and woody host plants by agroinfiltration. Conclusions The fast and efficient strategies described here should have broad applications, in particular for the study of "difficult" plant viruses, such as those infecting woody hosts, and potentially for other, non plant-infecting viral agents. PMID:22040379

  14. Modular structure of the full-length DNA gyrase B subunit revealed by small-angle X-ray scattering.

    PubMed

    Costenaro, Lionel; Grossmann, J Günter; Ebel, Christine; Maxwell, Anthony

    2007-03-01

    DNA gyrase, the only topoisomerase able to introduce negative supercoils into DNA, is essential for bacterial transcription and replication; absent from humans, it is a successful target for antibacterials. From biophysical experiments in solution, we report a structural model at approximately 12-15 A resolution of the full-length B subunit (GyrB). Analytical ultracentrifugation shows that GyrB is mainly a nonglobular monomer. Ab initio modeling of small-angle X-ray scattering data for GyrB consistently yields a "tadpole"-like envelope. It allows us to propose an organization of GyrB into three domains-ATPase, Toprim, and Tail-based on their crystallographic and modeled structures. Our study reveals the modular organization of GyrB and points out its potential flexibility, needed during the gyrase catalytic cycle. It provides important insights into the supercoiling mechanism by gyrase and suggests new lines of research. PMID:17355868

  15. Molecular characterization of full-length MLV-related endogenous retrovirus ChiRV1 from the chicken, Gallus gallus.

    PubMed

    Borysenko, Leonid; Stepanets, Volodymir; Rynditch, Alla V

    2008-06-20

    We report the first full-length sequence of an endogenous retrovirus from the genome of domestic chicken, that is not related to the Avian leukemia viruses (ALV). This retrovirus, designated ChiRV1, clusters with Murine leukemia virus (MLV)-related retroviruses and hence is the first complete gammaretrovirus from the genome of a bird. Nevertheless it is not related to exogenous MLV-related retroviruses infecting chicken. The provirus is 9133 bp long and contains 90%-identical LTRs as well as reading frames for the gag, pol and env genes, interrupted by in-frame stop codons. Expression analysis showed that ChiRV1 is a transcribed provirus. Screening of the chicken genome database revealed 100 ChiRV1-related sequences that are grouped into three classes based upon LTR alignment and subsequent phylogenetic analysis. PMID:18440041

  16. Structure of full-length human anti-PD1 therapeutic IgG4 antibody pembrolizumab.

    PubMed

    Scapin, Giovanna; Yang, Xiaoyu; Prosise, Winifred W; McCoy, Mark; Reichert, Paul; Johnston, Jennifer M; Kashi, Ramesh S; Strickland, Corey

    2015-12-01

    Immunoglobulin G4 antibodies exhibit unusual properties with important biological consequences. We report the structure of the human full-length IgG4 S228P anti-PD1 antibody pembrolizumab, solved to 2.3-Å resolution. Pembrolizumab is a compact molecule, consistent with the presence of a short hinge region. The Fc domain is glycosylated at the CH2 domain on both chains, but one CH2 domain is rotated 120° with respect to the conformation observed in all reported structures to date, and its glycan chain faces the solvent. We speculate that this new conformation is driven by the shorter hinge. The structure suggests a role for the S228P mutation in preventing the IgG4 arm exchange. In addition, this unusual Fc conformation suggests possible structural diversity between IgG subclasses and shows that use of isolated antibody fragments could mask potentially important interactions, owing to molecular flexibility. PMID:26595420

  17. Full-Length Enriched cDNA Libraries and ORFeome Analysis of Sugarcane Hybrid and Ancestor Genotypes

    PubMed Central

    Becker, Scott; Pörtner-Taliana, Antje; Souza, Glaucia Mendes

    2014-01-01

    Sugarcane is a major crop used for food and bioenergy production. Modern cultivars are hybrids derived from crosses between Saccharum officinarum and Saccharum spontaneum. Hybrid cultivars combine favorable characteristics from ancestral species and contain a genome that is highly polyploid and aneuploid, containing 100–130 chromosomes. These complex genomes represent a huge challenge for molecular studies and for the development of biotechnological tools that can facilitate sugarcane improvement. Here, we describe full-length enriched cDNA libraries for Saccharum officinarum, Saccharum spontaneum, and one hybrid genotype (SP803280) and analyze the set of open reading frames (ORFs) in their genomes (i.e., their ORFeomes). We found 38,195 (19%) sugarcane-specific transcripts that did not match transcripts from other databases. Less than 1.6% of all transcripts were ancestor-specific (i.e., not expressed in SP803280). We also found 78,008 putative new sugarcane transcripts that were absent in the largest sugarcane expressed sequence tag database (SUCEST). Functional annotation showed a high frequency of protein kinases and stress-related proteins. We also detected natural antisense transcript expression, which mapped to 94% of all plant KEGG pathways; however, each genotype showed different pathways enriched in antisense transcripts. Our data appeared to cover 53.2% (17,563 genes) and 46.8% (937 transcription factors) of all sugarcane full-length genes and transcription factors, respectively. This work represents a significant advancement in defining the sugarcane ORFeome and will be useful for protein characterization, single nucleotide polymorphism and splicing variant identification, evolutionary and comparative studies, and sugarcane genome assembly and annotation. PMID:25222706

  18. Characterization of Full-Length Genomes of Hepatitis B Virus Quasispecies in Sera of Patients at Different Phases of Infection.

    PubMed

    Yang, Zhi-Tao; Huang, Su-Yuan; Chen, Li; Liu, Feng; Cai, Xiao-Hui; Guo, Yang-Fan; Wang, Ming-Jie; Han, Yue; Yu, De-Min; Jiang, Jie-Hong; Zhang, Dong-Hua; Gong, Qi-Ming; Zhang, Guo-Qing; Zang, Guo-Qing; Lu, Zhong-Hua; Huang, Li-Hua; Zhang, Xin-Xin

    2015-07-01

    Hepatitis B virus (HBV) infection results in different clinical presentation due to different levels of immune response. Our study aimed to characterize HBV full-length genome quasispecies (QS) in patients with different phases of infection to better understand its pathogenesis. Forty treatment-naive HBV-infected patients were enrolled, including 10 cases of acute hepatitis B (AHB), 9 cases of immunotolerant (IT) HBV carriers, 11 cases of chronic hepatitis B (CHB), and 10 cases of acute-on-chronic liver failure (ACLF). The present study was conducted by clone-based sequencing. QS heterogeneity within each open reading frame was calculated. The mutation frequency index (MFI) and amino acid variations within the large HBsAg, HBcAg, and HBxAg regions were analyzed based on the different infection phases. In total, 606 HBV full-length sequences were obtained. HBV QS had higher heterogeneity in ACLF and CHB than that in IT among chronically infected individuals. AHB patients had the lower QS heterogeneity at onset than those with chronic infection. ACLF patients had the highest frequency of mutations in the core promoter and precore region. A triple mutation (A1762T/G1764A/G1896A) was observed more frequently in genotype C than in genotype B. The MFI indicated that specific peptides of the studied regions had more frequent mutations in ACLF. Furthermore, several amino acid variations, known as T- and B-cell epitopes, were potentially associated with the immunoactive phase of infection. More HBV genome mutations and deletions were observed in patients with more severe diseases, particularly in specific regions of the core and preS regions, the clinical significance and mechanism of which need to be further investigated. PMID:25926495

  19. A Novel mouse model of enhanced proteostasis: Full-length human heat shock factor 1 transgenic mice

    SciTech Connect

    Pierce, Anson; Wei, Rochelle; Halade, Dipti; Yoo, Si-Eun; Ran, Qitao; Richardson, Arlan

    2010-11-05

    Research highlights: {yields} Development of mouse overexpressing native human HSF1 in all tissues including CNS. {yields} HSF1 overexpression enhances heat shock response at whole-animal and cellular level. {yields} HSF1 overexpression protects from polyglutamine toxicity and favors aggresomes. {yields} HSF1 overexpression enhances proteostasis at the whole-animal and cellular level. -- Abstract: The heat shock response (HSR) is controlled by the master transcriptional regulator heat shock factor 1 (HSF1). HSF1 maintains proteostasis and resistance to stress through production of heat shock proteins (HSPs). No transgenic model exists that overexpresses HSF1 in tissues of the central nervous system (CNS). We generated a transgenic mouse overexpressing full-length non-mutant HSF1 and observed a 2-4-fold increase in HSF1 mRNA and protein expression in all tissues studied of HSF1 transgenic (HSF1{sup +/0}) mice compared to wild type (WT) littermates, including several regions of the CNS. Basal expression of HSP70 and 90 showed only mild tissue-specific changes; however, in response to forced exercise, the skeletal muscle HSR was more elevated in HSF1{sup +/0} mice compared to WT littermates and in fibroblasts following heat shock, as indicated by levels of inducible HSP70 mRNA and protein. HSF1{sup +/0} cells elicited a significantly more robust HSR in response to expression of the 82 repeat polyglutamine-YFP fusion construct (Q82YFP) and maintained proteasome-dependent processing of Q82YFP compared to WT fibroblasts. Overexpression of HSF1 was associated with fewer, but larger Q82YFP aggregates resembling aggresomes in HSF1{sup +/0} cells, and increased viability. Therefore, our data demonstrate that tissues and cells from mice overexpressing full-length non-mutant HSF1 exhibit enhanced proteostasis.

  20. Short-Read Assembly of Full-Length 16S Amplicons Reveals Bacterial Diversity in Subsurface Sediments

    PubMed Central

    Miller, Christopher S.; Handley, Kim M.; Wrighton, Kelly C.; Frischkorn, Kyle R.; Thomas, Brian C.; Banfield, Jillian F.

    2013-01-01

    In microbial ecology, a fundamental question relates to how community diversity and composition change in response to perturbation. Most studies have had limited ability to deeply sample community structure (e.g. Sanger-sequenced 16S rRNA libraries), or have had limited taxonomic resolution (e.g. studies based on 16S rRNA hypervariable region sequencing). Here, we combine the higher taxonomic resolution of near-full-length 16S rRNA gene amplicons with the economics and sensitivity of short-read sequencing to assay the abundance and identity of organisms that represent as little as 0.01% of sediment bacterial communities. We used a new version of EMIRGE optimized for large data size to reconstruct near-full-length 16S rRNA genes from amplicons sheared and sequenced with Illumina technology. The approach allowed us to differentiate the community composition among samples acquired before perturbation, after acetate amendment shifted the predominant metabolism to iron reduction, and once sulfate reduction began. Results were highly reproducible across technical replicates, and identified specific taxa that responded to the perturbation. All samples contain very high alpha diversity and abundant organisms from phyla without cultivated representatives. Surprisingly, at the time points measured, there was no strong loss of evenness, despite the selective pressure of acetate amendment and change in the terminal electron accepting process. However, community membership was altered significantly. The method allows for sensitive, accurate profiling of the “long tail” of low abundance organisms that exist in many microbial communities, and can resolve population dynamics in response to environmental change. PMID:23405248

  1. Sequencing and analysis of 10967 full-length cDNA clones from Xenopus laevis and Xenopus tropicalis

    SciTech Connect

    Morin, R D; Chang, E; Petrescu, A; Liao, N; Kirkpatrick, R; Griffith, M; Butterfield, Y; Stott, J; Barber, S; Babakaiff, R; Matsuo, C; Wong, D; Yang, G; Smailus, D; Brown-John, M; Mayo, M; Beland, J; Gibson, S; Olson, T; Tsai, M; Featherstone, R; Chand, S; Siddiqui, A; Jang, W; Lee, E; Klein, S; Prange, C; Myers, R M; Green, E D; Wagner, L; Gerhard, D; Marra, M; Jones, S M; Holt, R

    2005-10-31

    Sequencing of full-insert clones from full-length cDNA libraries from both Xenopus laevis and Xenopus tropicalis has been ongoing as part of the Xenopus Gene Collection initiative. Here we present an analysis of 10967 clones (8049 from X. laevis and 2918 from X. tropicalis). The clone set contains 2013 orthologs between X. laevis and X. tropicalis as well as 1795 paralog pairs within X. laevis. 1199 are in-paralogs, believed to have resulted from an allotetraploidization event approximately 30 million years ago, and the remaining 546 are likely out-paralogs that have resulted from more ancient gene duplications, prior to the divergence between the two species. We do not detect any evidence for positive selection by the Yang and Nielsen maximum likelihood method of approximating d{sub N}/d{sub S}. However, d{sub N}/d{sub S} for X. laevis in-paralogs is elevated relative to X. tropicalis orthologs. This difference is highly significant, and indicates an overall relaxation of selective pressures on duplicated gene pairs. Within both groups of paralogs, we found evidence of subfunctionalization, manifested as differential expression of paralogous genes among tissues, as measured by EST information from public resources. We have observed, as expected, a higher instance of subfunctionalization in out-paralogs relative to in-paralogs.

  2. Isolation and characterization of a full length cDNA for dentatorubral-pallidoluysian atrophy (DRPLA) gene

    SciTech Connect

    Oyake, M.; Onodera, O.; Ikeuchi, T.

    1994-09-01

    Hereditary dentatorubral-pallidoluysian atrophy (DRPLA) is an autosomal dominant spinocerebellar degeneration characterized by anticipation and variable combination of symptoms including myoclonus, epilepsy, cerebellar ataxia, choleoathetosis, and dementia. Recently, we discovered that DRPLA is caused by unstable expansion of a CAG repeat of a B37 gene on chromosome 12. To characterize functions of the DRPLA gene product, we isolated several cDNA clones for the DRPLA gene from human adult and fetus brain cDNA libraries, using an oligonucleotide flanking the CAG repeat. The cDNA spans 4247 bp in length and there is only an open reading frame coding for 986 amino acids. The CAG repeat, which is expanded in DRPLA, is located 291 bp downstream from the initiation methionine and encodes a polyglutamine tract. The deduced amino acid sequence from amino acids residues 582 to 707 has a high homology to published human hippocampus derived expressed sequence (M78755) located at chromosome 1p (63.8% identity), and 3{prime}-untranslated region of the DRPLA cDNA revealed homology to the mouse small nuclear RNA U7 gene (X54165). Northern blot analysis revealed a 4.7 knt transcript which is widely expressed in various tissues including heart, lung, kidney, placenta, skeletal muscle, and brain. In human adult brain, the transcript was broadly expressed including amygdala, caudate nucleus, corpus callosum, hippocampus, hypothalamus, substantia nigra, subthalamic nucleus and thalamus, and was not specific to the dentatorubral-pallidoluysian system. The availability of a full length cDNA will be highly useful for analyzing the pathogenesis of this unique neurodegenerative disease as well as for analyzing other CAG repeat related neurodegenerative diseases.

  3. Complex Mosaic Composition of Near Full-Length Genomes of Two NED (NIH-ENVA-DOD) Subtype Panel HIV Type 1 Strains, BCF-Dioum and BCF-Kita, Originating from the Democratic Republic of Congo (DRC)

    PubMed Central

    Foley, Brian T.; Tolzmann, Catlin A.; Ouma, Annastasia; Bremer, James W.

    2009-01-01

    Abstract Sequence characterization of the near full-length genomes of HIV-1 isolates BCF-Dioum and BCF-Kita, originating from the Democratic Republic of Congo (DRC), was continued. These NED panel isolates, contributed by F. Brun-Vezinet (ENVA-France), were first identified as subtypes G and H, respectively. Our earlier analyses of portions of their pol genes showed that both were likely to be intersubtype recombinants of different composition. This study analyzed the remainder of each genome, confirming them to be complex recombinants. The BCF-Dioum genome resembles CRF06_cpx strains found in West Africa, composed of subtypes A/G/J/K. The BCF-Kita genome is a unique complex recombinant A–F–G–H–K–U strain. These data support previous observations of the complexity of strains originating from the DRC. BCF-Dioum may be a suitable strain for standards and reagents since it matches a defined circulating recombinant form. Studies and reagents made from BCF-Kita should take into account its complex genome. PMID:19795987

  4. Construction of phosphorylation interaction networks by text mining of full-length articles using the eFIP system

    PubMed Central

    Tudor, Catalina O.; Ross, Karen E.; Li, Gang; Vijay-Shanker, K.; Wu, Cathy H.; Arighi, Cecilia N.

    2015-01-01

    Protein phosphorylation is a reversible post-translational modification where a protein kinase adds a phosphate group to a protein, potentially regulating its function, localization and/or activity. Phosphorylation can affect protein–protein interactions (PPIs), abolishing interaction with previous binding partners or enabling new interactions. Extracting phosphorylation information coupled with PPI information from the scientific literature will facilitate the creation of phosphorylation interaction networks of kinases, substrates and interacting partners, toward knowledge discovery of functional outcomes of protein phosphorylation. Increasingly, PPI databases are interested in capturing the phosphorylation state of interacting partners. We have previously developed the eFIP (Extracting Functional Impact of Phosphorylation) text mining system, which identifies phosphorylated proteins and phosphorylation-dependent PPIs. In this work, we present several enhancements for the eFIP system: (i) text mining for full-length articles from the PubMed Central open-access collection; (ii) the integration of the RLIMS-P 2.0 system for the extraction of phosphorylation events with kinase, substrate and site information; (iii) the extension of the PPI module with new trigger words/phrases describing interactions and (iv) the addition of the iSimp tool for sentence simplification to aid in the matching of syntactic patterns. We enhance the website functionality to: (i) support searches based on protein roles (kinases, substrates, interacting partners) or using keywords; (ii) link protein entities to their corresponding UniProt identifiers if mapped and (iii) support visual exploration of phosphorylation interaction networks using Cytoscape. The evaluation of eFIP on full-length articles achieved 92.4% precision, 76.5% recall and 83.7% F-measure on 100 article sections. To demonstrate eFIP for knowledge extraction and discovery, we constructed phosphorylation

  5. Full-length transcriptome analysis of human retina-derived cell lines ARPE-19 and Y79 using the vector-capping method.

    PubMed

    Oshikawa, Mio; Tsutsui, Chihiro; Ikegami, Tomoko; Fuchida, Yuki; Matsubara, Maki; Toyama, Shigeru; Usami, Ron; Ohtoko, Kuniyo; Kato, Seishi

    2011-08-01

    PURPOSE. To collect an entire set of full-length cDNA clones derived from human retina-derived cell lines and to identify full-length transcripts for retinal preferentially expressed genes. METHODS. The full-length cDNA libraries were constructed from a retinoblastoma cell line, Y79, and a retinal pigment epithelium cell line, ARPE-19, using the vector-capping method, which generates a genuine full-length cDNA. By single-pass sequencing of the 5'-end of cDNA clones and subsequent mapping to the human genome, the authors determined their transcriptional start sites and annotated the cDNA clones. RESULTS. Of the 23,616 clones isolated from Y79-derived cDNA libraries, 19,229 full-length cDNA clones were identified and classified into 4808 genes, including genes of >10 kbp. Of the 7067 genes obtained from the Y79 and ARPE-19 libraries, the authors selected 72 genes that were preferentially expressed in the eye, of which 131 clones corresponding to 57 genes were fully sequenced. As a result, we discovered many variants that were produced by different transcriptional start sites, alternative splicing, and alternative polyadenylation. CONCLUSIONS. The bias-free, full-length cDNA libraries constructed using the vector-capping method were shown to be useful for collecting an entire set of full-length cDNA clones for these retinal cell lines. Full-length transcriptome analysis of these cDNA libraries revealed that there were, unexpectedly, many transcript variants for each gene, indicating that obtaining the full-length cDNA for each variant is indispensable for analyzing its function. The full-length cDNA clones (approximately 80,000 clones each for ARPE-19 and Y79) will be useful as a resource for investigating the human retina. PMID:21697133

  6. Ultra-Deep Sequencing of HIV-1 near Full-Length and Partial Proviral Genomes Reveals High Genetic Diversity among Brazilian Blood Donors

    PubMed Central

    Pessôa, Rodrigo; Loureiro, Paula; Esther Lopes, Maria; Carneiro-Proietti, Anna B. F.; Sabino, Ester C; Busch, Michael P.; Sanabani, Sabri S

    2016-01-01

    Background Here, we aimed to gain a comprehensive picture of the HIV-1 diversity in the northeast and southeast part of Brazil. To this end, a high-throughput sequencing-by-synthesis protocol and instrument were used to characterize the near full length (NFLG) and partial HIV-1 proviral genome in 259 HIV-1 infected blood donors at four major blood centers in Brazil: Pro-Sangue foundation (São Paulo state (SP), n 51), Hemominas foundation (Minas Gerais state (MG), n 41), Hemope foundation (Recife state (PE), n 96) and Hemorio blood bank (Rio de Janeiro (RJ), n 70). Materials and Methods A total of 259 blood samples were obtained from 195 donors with long-standing infections and 64 donors with a lack of stage information. DNA was extracted from the peripheral blood mononuclear cells (PBMCs) to amplify the HIV-1 NFLGs from five overlapping fragments. The amplicons were molecularly bar-coded, pooled, and sequenced by Illumina paired-end protocol. Results Of the 259 samples studied, 208 (80%) NFLGs and 49 (18.8%) partial fragments were de novo assembled into contiguous sequences and successfully subtyped. Of these 257 samples, 183 (71.2%) were pure subtypes consisting of clade B (n = 167, 65%), C (n = 10, 3.9%), F1 (n = 4, 1.5%), and D (n = 2, 0.7%). Recombinant viruses were detected in 74 (28.8%) samples and consist of unique BF1 (n = 41, 15.9%), BC (n = 7, 2.7%), BCF1 (n = 4, 1.5%), CF1 and CDK (n = 1, 0.4%, each), CRF70_BF1 (n = 4, 1.5%), CRF71_BF1 (n = 12, 4.7%), and CRF72_BF1 (n = 4, 1.5%). Evidence of dual infection was detected in four patients coinfected with the same subtype (n = 3) and distinct subtype (n = 1). Conclusion Based on this work, subtype B appears to be the prevalent subtype followed by a high proportion of intersubtype recombinants that appeared to be arising continually in this country. Our study represents the largest analysis of the viral NFLG ever undertaken worldwide and provides insights into the understanding the genesis of the HIV-1

  7. Immunization of malignant melanoma patients with full-length NY-ESO-1 protein using Toll-like receptor 7 agonist imiquimod as vaccine adjuvant

    PubMed Central

    Adams, Sylvia; O'Neill, David W.; Nonaka, Daisuke; Hardin, Elizabeth; Chiriboga, Luis; Siu, Kimberly; Cruz, Crystal M.; Angiulli, Angelica; Angiulli, Francesca; Ritter, Erika; Holman, Rose Marie; Shapiro, Richard L.; Berman, Russell S.; Berner, Natalie; Shao, Yongzhao; Manches, Olivier; Pan, Linda; Venhaus, Ralph R.; Hoffman, Eric W.; Jungbluth, Achim; Gnjatic, Sacha; Old, Lloyd; Pavlick, Anna C.; Bhardwaj, Nina

    2008-01-01

    T cell-mediated immunity to microbes and to cancer can be enhanced by the activation of dendritic cells (DCs) via Toll-like receptors (TLRs). In this study, we evaluated the safety and feasibility of topical imiquimod, a TLR7 agonist, in a series of vaccinations (26) proteins,(27) and DNA, (28, 29) as well as in vaccines using recombinant Listeria(30) or DCs.(31) In humans, it was shown that topical imiquimod treatment may enhance the immunogenicity of a melanoma peptide vaccine when given with systemic FLT3 ligand. (32) In addition, injection of immature DCs into imiquimod pretreated skin lead to DC activation in situ and enhanced migratory capacity to draining lymph nodes in cancer patients. (33) In this study, we test the safety and feasibility of imiquimod in a vaccine against the cancer/testis antigen NY-ESO-1, and evaluate the immunogenicity of the combination. NY-ESO-1 is detectable in approximately 30% of metastatic melanomas. (34-36) It is against the cancer/testis antigen NY-ESO-1 in patients with malignant melanoma. Recombinant, full-length NY-ESO-1 protein was administered intradermally into imiquimod pre-conditioned sites followed by additional topical applications of imiquimod. The regimen was very well-tolerated with only mild and transient local reactions and constitutional symptoms. Secondarily, we examined the systemic immune response induced by the imiquimod/NY-ESO-1 combination, and show that it elicited both humoral and cellular responses in a significant fraction of patients. Skin biopsies were assessed for imiquimod's in situ immunomodulatory effects. Compared with untreated skin, topical imiquimod induced dermal mononuclear cell infiltrates in all patients composed primarily of T cells, monocytes, macrophages, myeloid DCs and natural killer (NK) cells, and to a lesser extent plasmacytoid DCs. DC activation was evident. This study demonstrates the feasibility and excellent safety profile of a topically applied TLR7 agonist utilized as a

  8. A Glutamine/Asparagine-Rich Fragment of Gln3, but not the Full-Length Protein, Aggregates in Saccharomyces cerevisiae.

    PubMed

    Antonets, K S; Sargsyan, H M; Nizhnikov, A A

    2016-04-01

    The amino acid sequence of protein Gln3 in yeast Saccharomyces cerevisiae has a region enriched with Gln (Q) and Asn (N) residues. In this study, we analyzed the effects of overexpression of Gln3 and its Q/N-rich fragment fused with yellow fluorescent protein (YFP). Being overexpressed, full-length Gln3-YFP does not form aggregates, inhibits vegetative growth, and demonstrates nuclear localization, while the Q/N-rich fragment (Gln3QN) fused with YFP forms aggregates that do not colocalize with the nucleus and do not affect growth of the cells. Although detergent-resistant aggregates of Gln3QN are formed in the absence of yeast prions, the aggregation of Gln3QN significantly increases in the presence of [PIN(+)] prion, while in the presence of two prions, [PSI(+)] and [PIN(+)], the percentage of cells with Gln3QN aggregates is significantly lower than in the strain bearing only [PIN(+)]. Data on colocalization demonstrate that this effect is mediated by interaction between Gln3QN aggregates and [PSI(+)] and [PIN(+)] prions. PMID:27293098

  9. Improved yields of full-length functional human FGF1 can be achieved using the methylotrophic yeast Pichia pastoris.

    PubMed

    Fantoni, Adele; Bill, Roslyn M; Gustafsson, Lena; Hedfalk, Kristina

    2007-03-01

    We have produced human fibroblast growth factor 1 (hFGF1) in the methylotrophic yeast Pichia pastoris in order to obtain the large amounts of active protein required for subsequent functional and structural characterization. Four constructs were made to examine both intracellular and secreted expression, with variations in the location of the His6 tag at either end of the peptide. hFGF1 could be produced from all four constructs in shake flasks, but production was optimized by growing only the highest-yielding of these strains, which produced hFGF1 intracellularly, under tightly controlled conditions in a 3 L fermentor. One hundred and eight milligrams of pure protein was achieved per liter culture (corresponding to 0.68 mg of protein per gram of wet cells), the function of which was verified using NIH 3T3 cell cultures. This is a 30-fold improvement over previously reported yields of full-length hFGF1. PMID:17134911

  10. Computational Analysis of Full-length cDNAs Reveals Frequent Coupling Between Transcriptional and Splicing Programs

    PubMed Central

    Chern, Tzu-Ming; Paul, Nicodeme; van Nimwegen, Erik; Zavolan, Mihaela

    2008-01-01

    High-throughput sequencing studies revealed that the majority of human and mouse multi-exon genes have multiple splice forms. High-density oligonucleotide array-based measurements have further established that many exons are expressed in a tissue-specific manner. The mechanisms underlying the tissue-dependent expression of most alternative exons remain, however, to be understood. In this study, we focus on one possible mechanism, namely the coupling of (tissue specific) transcription regulation with alternative splicing. We analyzed the FANTOM3 and H-Invitational datasets of full-length mouse and human cDNAs, respectively, and found that in transcription units with multiple start sites, the inclusion of at least 15% and possibly up to 30% of the ‘cassette’ exons correlates with the use of specific transcription start sites (TSS). The vast majority of TSS-associated exons are conserved between human and mouse, yet the conservation is weaker when compared with TSS-independent exons. Additionally, the currently available data only support a weak correlation between the probabilities of TSS association of orthologous exons. Our analysis thus suggests frequent coupling of transcriptional and splicing programs, and provides a large dataset of exons on which the molecular basis of this coupling can be further studied. PMID:18276623