Relevant bibliographies by topics / Cloning and sequencing

Contents

  1. Journal articles
  2. Dissertations / Theses
  3. Books
  4. Book chapters
  5. Conference papers
  6. Reports

Academic literature on the topic 'Cloning and sequencing'

Author: Grafiati
Published: 4 June 2021
Last updated: 11 February 2022

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Journal articles on the topic "Cloning and sequencing"

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Haak, Wolfgang, Joachim Burger, and Kurt Werner Alt. "ABO genotyping by PCR-RFLP and cloning and sequencing." Anthropologischer Anzeiger 62, no. 4 (December 16, 2004): 397–410. http://dx.doi.org/10.1127/anthranz/62/2004/397.

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Moradas-Ferreira, Pedro. "Gene cloning and sequencing." Biochemical Education 17, no. 4 (October 1989): 215. http://dx.doi.org/10.1016/0307-4412(89)90153-2.

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Šimková, H., J. Janda, E. Hřibová, J. Šafář, and J. Doležel. "Cot-based cloning and sequencing of the short arm of wheat chromosome 1B." Plant, Soil and Environment 53, No. 10 (January 7, 2008): 437–41. http://dx.doi.org/10.17221/2195-pse.

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Sequencing of cereal genomes is not a feasible task due to their large size and high content of repetitive DNA sequences. There are two basic approaches to simplify analysis of such genomes: reduced representation approaches, such as EST sequencing, methyl filtration and Cot-based cloning and sequencing; on the other side there is analysis of genomes in a step-wise manner, e.g. through creation of chromosome-specific genomic resources. Combination of both approaches – i.e. Cot-based cloning and sequencing of DNA obtained from a chromosome-arm-specific BAC library – was tested in this work.
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Linder, Jodell E., Tatyana E. Plachco, Romina Libster, and E. Kathryn Miller. "Sequencing human rhinoviruses: Direct sequencing versus plasmid cloning." Journal of Virological Methods 211 (January 2015): 64–69. http://dx.doi.org/10.1016/j.jviromet.2014.09.020.

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Tang, D., H. Jiang, Y. Zhang, Y. Li, X. Zhang, and T. Zhou. "Cloning and sequencing of the complicated rDNA gene family of Bos taurus." Czech Journal of Animal Science 51, No. 10 (December 5, 2011): 425–28. http://dx.doi.org/10.17221/3960-cjas.

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The rDNA genes coding for ribosomal RNA (rRNA) in animals are repeat sequences with high GC content and complicated structure. Based on the sequences of human ribosomal DNA repeat unit and transcription unit and the long and accurate PCR method with LA Taq DNA polymerase and GC buffer, we were able to amplify the complicated repeat sequences of rDNA genes in Bos taurus. Three rDNA genes and 2 internal transcribed spacer (ITS) fragments were cloned and confirmed by sequencing. The conditions for the cloning of complicated DNA sequences such as special rules of primer design, improvement of the reaction system, selection of DNA polymerase and adjustment of cycle parameters were discussed.  
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Nakahama, Kazuo, Koji Yoshimura, Ryuji Marumoto, Masakazu Kikuchi, In Sook Lee, Toshiharu Hase, and Hiroshi Matsubara. "Cloning and sequencing ofSerratiaprotease gene." Nucleic Acids Research 14, no. 14 (1986): 5843–55. http://dx.doi.org/10.1093/nar/14.14.5843.

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Lawler, J., M. Duquette, and P. Ferro. "Cloning and sequencing of chicken thrombospondin." Journal of Biological Chemistry 266, no. 13 (May 1991): 8039–43. http://dx.doi.org/10.1016/s0021-9258(18)92936-4.

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Darrah, P. M., S. A. Kay, G. R. Teakle, and W. T. Griffiths. "Cloning and sequencing of protochlorophyllide reductase." Biochemical Journal 265, no. 3 (February 1, 1990): 789–98. http://dx.doi.org/10.1042/bj2650789.

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Putative protochlorophyllide reductase cDNA clones (252 and 113) were isolated from an etiolated-oat (Avena sativa) cDNA library. These were used to indirectly characterize a further clone, p127, isolated from a lambda-phage gt11 cDNA library. The latter (1.15 kb in length) was sequenced, and the derived amino acid sequence was shown to be remarkably similar to that derived from chemical analysis of a CNBr-cleavage fragment of the purified reductase, p127 codes for more than 95% of the reductase protein.
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Svoboda, P. "Cloning and Sequencing an Inverted Repeat." Cold Spring Harbor Protocols 2009, no. 1 (January 1, 2009): pdb.ip64. http://dx.doi.org/10.1101/pdb.ip64.

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Fang, Guo-Hua, Paul Kenigsberg, Milton J. Axley, Mark Nuell, and Lowell P. Hager. "Cloning and sequencing of chloroperoxidase cDNA." Nucleic Acids Research 14, no. 20 (1986): 8061–71. http://dx.doi.org/10.1093/nar/14.20.8061.

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Dissertations / Theses on the topic "Cloning and sequencing"

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Elliott, Gillian Daphne. "The cloning and sequencing of mumps virus." Thesis, Queen's University Belfast, 1988. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.356913.

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Kourdi, Zerikly Malek. "Cloning, sequencing and analysis of spirotetronate biosynthetic gene clusters." Thesis, University of Warwick, 2011. http://wrap.warwick.ac.uk/38545/.

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Spirotetronate antibiotics are natural products that have been shown to have a broad spectrum of biological activities, including antiviral, antibacterial, antitumor and antimalarial activities. Two representatives of this group are quartromicins and chrolactomycin, which are produced by Amycolatopsis orientalis and Streptomyces sp. 569N-3 respectively. Based on other related natural products with known biosynthetic pathways, the biosynthetic routes for quartromicins and chrolactomycin were proposed and several strategies for locating the putative gene clusters directing the biosynthesis of these natural products were used. An FkbH-like protein was proposed to be specifically involved in the biosynthetic incorporation of a glycerol-derived three-carbon unit into the tetronate moieties of quartromicins and chrolactomycin. It was selected as a target to locate the biosynthetic gene clusters of tetronate natural products. After aligning sequences of known FkbH-like proteins, a set of degenerate oligonucleotide primers for amplification of fkbH-like genes was designed and tested. Fragments of fkbH-like sequences were amplified from several available strains and sequenced. Several genomic libraries were created and screened by PCR using the degenerate fkbH primers. After validating the library, clones containing the putative biosynthetic gene cluster were identified and sequenced. The obtained sequence data was then assembled and analysed. Coding sequences were identified, protein functions assigned and a biosynthetic route was proposed for the biosynthesis of quartromicins and chrolactomycin.
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Vipond, I. Barry. "Cloning, sequencing and expression of Newcastle disease virus genes." Thesis, University of Newcastle Upon Tyne, 1990. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.315647.

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Self, Susan Jane. "Cloning, sequencing and expression cytochrome C2 from Rhodospirillum rubrum." Thesis, Imperial College London, 1990. http://hdl.handle.net/10044/1/46542.

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Antic, Dragana. "Molecular genetics of hantaviruses: Cloning, sequencing, expression, and morphogenesis." Thesis, University of Ottawa (Canada), 1993. http://hdl.handle.net/10393/7653.

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Molecular characterization of the Seoul 80-39 virus, a prototype of the Seoul serotype, was carried out by cloning and sequencing. The large (L) genomic RNA segment is 6530 nucleotides long. The viral complementary-sense RNA contains a single open reading frame (ORF) from the AUG codon at nucleotide positions 37-39 to the UAA stop codon at nucleotide positions 6490-6492. This ORF could encode a polypeptide of 2151 amino acids (246,662 D) which likely corresponds to the L protein detected in purified viral particles (Elliot et al., 1984) and is assumed to be an RNA-dependent RNA polymerase molecule (Schmaljohn and Dalrymple, 1983). The M RNA segment is 3651 nucleotides long. A single ORF was detected in the viral complementary-sense RNA. This ORF could encode a polypeptide of 1133 amino acids which is cotranslationally processed into viral glycoproteins G1 and G2. The S genomic RNA segment was characterized from the 3$\sp\prime$ end to nucleotide 1332. One long ORF was detected from the AUG codon at nucleotide positions 43-45 to the UAA stop codon at nucleotide positions 1330-1332. This ORF has a capacity to encode a 48 kD protein which corresponds to the viral nucleocapsid (N) protein. A comparison of coding properties of the tripartite genomes of various hantaviruses allowed construction of the evolutionary tree for the Hantavirus genus. The second part of the study was focused on the maturation of Hantaan 76-118 virus glycoproteins G1 and G2 virus morphogenesis. Glycoproteins G1 and G2 form a complex. Experiments with the glycosylation inhibitor brefeldin A indicate that complex formation between the two glycoproteins occurs in the ER. Resistance of both G1 and G2 to endoglycosidase D suggests a structure with more than five mannose residues. Processing of a high mannose structures ((Man)$\sb8$(GlcNAc)$\sb2$ to (Man)$\sb7$(GlcNAc)$\sb2$ or (Man)$\sb6$(GlcNAc)$\sb2$) may take place in the cis-Golgi by the enzyme $\alpha$-mannosidase I. Virus budding was observed only in Hantaan virus-infected cells, in the trans-Golgi network. When the Golgi apparatus was destroyed by brefeldin A treatment, assembly of virus particles was not observed. The third project was to express nucleocapsid proteins of Seoul 80-39 virus and Hantaan 76-118 virus using baculovirus recombinants. Analysis of recombinant virus-infected Sf9 cell lysates by SDS-PAGE showed that N proteins were expressed in high levels and were identical to virion associated N proteins in size and antigenicity. (Abstract shortened by UMI.)
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Cekic, Caglar. "Cloning, Expression And Sequencing Of Citrate Synthase From Thermoplasma Volcanium." Master's thesis, METU, 2004. http://etd.lib.metu.edu.tr/upload/3/1260432/index.pdf.

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In this study first time, we have cloned and sequenced the citrate synthase gene from a thermoacidophilic archaeon Thermoplasma (Tp.) volcanium (Optimum growth temperature of Tp.volcanium is 60oC and optimum pH is 2.0.). For cloning we have followed a PCR based approach. Amplification of citrate synthase gene from chromosomal DNA of Tp.volcanium yielded a product of 1476 bp containing an open reading frame of 1161 bp comprising the structural gene. After ligation of the PCR amplicon to pDrive vector through AU complementation, recombinant plasmids were transferred into E.coli TG-1 competent cells. Out of three recombinants, E.coli pDriveCS-31 was selected for further characterization by restriction mapping and DNA sequencing. Southern Blotting and Hybridization using the membrane blot of pDriveCS-31 plasmid and DIG-labeled PCR amplified citrate synthase gene probe, also confirmed the cloning of Tp.volcanium citrate synthase gene in E.coli. Clustal W Version 1.82 was used for alignment of aminoacid sequence of Tp.volcanium citrate synthase with that of other archaeal, bacterial and eukaryotic citrate synthases. The highest sequence similarity (87%) was found between Tp.volcanium and Tp.acidophilum enzymes. Despite low sequence homology (18%) with the pig enzyme, of the 11 residues implicated in catalytic activity of the pig citrate synthase 9 were conserved in the Tp.volcanium enzyme. Heterologous expression of this citrate synthase gene in E.coli has been achieved under the control of its promoter sequences. The recombinant enzyme (386 aa) has been purified to homogeneity by affinity chromatography on Reactive Red 120 column. The subunit molecular size was estimated as 43 kDa. The purified enzyme followed classical Michaelis-Menten kinetics. The Km values of 5.15 &
#956
M and 5.60 &
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M, and Vmax values of 1.74 &
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moles/ml/min and 1.60 &
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moles/ml/min were calculated from Lineweaver-Burk plots for acetyl-CoA and oxaloacetate, respectively. The recombinant enzyme was thermostable and retained about 80% of the activity at 85oC after 1 hour.
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Lloyd, Amanda Lian. "Cloning, characterisation and sequencing of promoters of Helicobacter pylori 4187E." University of Western Australia. Microbiology Discipline Group, 2005. http://theses.library.uwa.edu.au/adt-WU2005.0112.

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Published information on the structure and regulation of H. pylori promoters is limited. The work presented in this thesis describes the cloning and characterisation of promoter regions from a clinical isolate of H. pylori, and the development of an alternative, non-radioactive method for verifying the location of transcriptional start sites of bacterial promoters. H. pylori 4187E promoters were randomly cloned into the promoter-trap vector pKK232-8 in Escherichia coli DH5α using two sets of restriction enzymes. Vector pKK232-8 contains a promoterless chloramphenicol acetyltransferase (CAT) gene. Seventy-four promoter-containing clones were isolated from selective media based on their resistance to chloramphenicol. The strength of each promoter was analysed qualitatively, using chloramphenicol minimum inhibitory concentrations, and quantitatively, using CAT assays following exposure of the clones to pH 4 and pH 7. Selected promoter fragments were subcloned into the GFP reporter vector pFPV25, containing a promoterless gfp gene. The subclones were exposed to buffered LB broth at pH 4, 5, 6, 7 and 8, for varying lengths of time, to study acid-induced regulation of gene expression. Subclones were examined qualitatively, using visual examination of GFP fluorescence and fluorescence microscopy, and quantitatively, using flow cytometry following acid shock. DNA sequences were determined for 61 of the 74 H. pylori promoters, and sequence alignments with the published H. pylori strains (26695 and J99) were performed. The transcriptional start site of 27 H. pylori promoter fragments was experimentally mapped using a fluorescence-based primer extension protocol developed by our group. Potential -35 and -10 sequences were identified for each promoter, and a new consensus sequence for H. pylori promoters was proposed based upon these results. This study has considerably expanded knowledge of H. pylori promoter sequences and transcriptional start sites based on those which also function in E. coli. It has also revealed several H. pylori promoters which appear to respond to acid stress
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Xu, Jing. "Cloning and sequencing of genes that control lung branching morphogenesis." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1997. http://www.collectionscanada.ca/obj/s4/f2/dsk2/tape16/PQDD_0002/MQ28788.pdf.

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Lloyd, Amanda Lian. "Cloning, characterisation and sequencing of promoters of Helicobacter pylori 4187E /." Connect to this title, 2004. http://theses.library.uwa.edu.au/adt-WU2005.0112.

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Bright, Jeremy R. "Cloning, sequencing and expression of glucose dehydrogenase from Thermoplasma acidophilum." Thesis, University of Bath, 1991. https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.292614.

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Books on the topic "Cloning and sequencing"

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Firman, Keith. DNA cloning/sequencing workshop: A short course. New York: E. Horwood, 1991.

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Xu, Jing. Cloning and sequencing of genes that control lung branching morphogenesis. Ottawa: National Library of Canada = Bibliothèque nationale du Canada, 1999.

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Clark, D. Cloning and sequencing of genes involved in thiophene oxidation by Escherichia coli. S.l: s.n, 1990.

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Zouganelis, George D. Cloning, sequencing and characterization of a chitin synthase gene fragment from the fungus Phascolomyces articulosus. St. Catharines, Ont: Brock University, Dept. of Biological Sciences, 1997.

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Taylor, Sally Catriona. Cloning and sequencing pea early browning virus RNA-1 and studies on the 30K gene. Norwich: University of East Anglia, 1990.

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Lang, Walter H. CDNA cloning and sequencing of Octopus dofleini hemocyanin. 1990.

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Chamblin, Caroline C. Cloning and partial sequencing of tobacco ringspot virus RNA 1. 1989.

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McDonald, Jeffrey Dean. Cloning, sequencing, and evolutionary analysis of the mouse erythropoietin gene. 1986.

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White, Michael Kenneth. Sequencing and comparison of Drosophila melanogaster vitelline membrane cDNA clones. 1988.

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(Editor), Wilhelm Ansorge, Hartmut Voss (Editor), and Jürgen Zimmermann (Editor), eds. DNA Sequencing Strategies: Automated and Advanced Approaches (Embo Practical Course). Wiley-Liss, 1996.

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Book chapters on the topic "Cloning and sequencing"

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Jankevics, Eriks. "DNA Sequencing." In Basic Cloning Procedures, 22–42. Berlin, Heidelberg: Springer Berlin Heidelberg, 1998. http://dx.doi.org/10.1007/978-3-642-71965-3_2.

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Babu, Bandamaravuri Kishore, Anu Sharma, and Hari Kishan Sudini. "DNA Cloning and Sequencing." In Springer Protocols Handbooks, 291–302. Berlin, Heidelberg: Springer Berlin Heidelberg, 2012. http://dx.doi.org/10.1007/978-3-642-34410-7_19.

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Wong, Dominic W. S. "Whole Genome and Next Generation Sequencing." In The ABCs of Gene Cloning, 219–30. Cham: Springer International Publishing, 2018. http://dx.doi.org/10.1007/978-3-319-77982-9_24.

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Gausson, Valérie, and Maria-Carla Saleh. "Viral Small RNA Cloning and Sequencing." In Antiviral RNAi, 107–22. Totowa, NJ: Humana Press, 2011. http://dx.doi.org/10.1007/978-1-61779-037-9_6.

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Saluz, Hanspeter, and Jean-Pierre Jost. "Cloning of DNA Probe in M13." In A Laboratory Guide to Genomic Sequencing, 99–105. Basel: Birkhäuser Basel, 1987. http://dx.doi.org/10.1007/978-3-0348-9302-2_12.

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Humphreys-Pereira, Danny A., Taeho Kim, and Joong-Ki Park. "Characterization of nematode mitochondrial genomes." In Techniques for work with plant and soil nematodes, 250–64. Wallingford: CABI, 2021. http://dx.doi.org/10.1079/9781786391759.0250.

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Abstract This chapter presents procedures on polymerase chain reaction (PCR) amplification, protocols for PCR, cloning and sequencing, and mitochondrial genome annotation and gene identification for the characterization of nematodes.
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Humphreys-Pereira, Danny A., Taeho Kim, and Joong-Ki Park. "Characterization of nematode mitochondrial genomes." In Techniques for work with plant and soil nematodes, 250–64. Wallingford: CABI, 2021. http://dx.doi.org/10.1079/9781786391759.0014.

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Abstract This chapter presents procedures on polymerase chain reaction (PCR) amplification, protocols for PCR, cloning and sequencing, and mitochondrial genome annotation and gene identification for the characterization of nematodes.
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Fulton, Tara L., and Mathias Stiller. "PCR Amplification, Cloning, and Sequencing of Ancient DNA." In Methods in Molecular Biology, 111–19. Totowa, NJ: Humana Press, 2011. http://dx.doi.org/10.1007/978-1-61779-516-9_15.

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Walsh, David. "Cloning PCR Products for Sequencing in M13 Vectors." In PCR Protocols, 385–91. Totowa, NJ: Humana Press, 2003. http://dx.doi.org/10.1007/978-1-4612-0055-0_56.

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Voit, R., and H. J. Schneider. "Cloning and Sequencing of Eurypelma Californicum Hemocyanin cDNA." In Invertebrate Oxygen Carriers, 495–98. Berlin, Heidelberg: Springer Berlin Heidelberg, 1986. http://dx.doi.org/10.1007/978-3-642-71481-8_86.

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Conference papers on the topic "Cloning and sequencing"

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Leksin, I. Yu, C. C. Gorina, and F. V. Minibaeva. "Lichen peroxidase genes: cloning, sequencing, analysis activity." In IX Congress of society physiologists of plants of Russia "Plant physiology is the basis for creating plants of the future". Kazan University Press, 2019. http://dx.doi.org/10.26907/978-5-00130-204-9-2019-260.

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Priambodo, Rizky, Irvan Faizal, Abinawanto, Wibowo Mangunwardoyo, and Anom Bowolaksono. "Transferrin gene cloning and sequencing analysis from Indonesian Nile Tilapia (Oreochromis niloticus)." In INTERNATIONAL SYMPOSIUM ON CURRENT PROGRESS IN MATHEMATICS AND SCIENCES 2015 (ISCPMS 2015): Proceedings of the 1st International Symposium on Current Progress in Mathematics and Sciences. Author(s), 2016. http://dx.doi.org/10.1063/1.4946964.

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Youssoufiän, H., A. Patel, D. Phillips, H. H. Kazazian, and S. E. Antonarakis. "RECURRENT MUTATIONS AND AN UNUSUAL DELETION IN HEMOPHILIA A." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1644014.

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We have identified 15 mutations of the factor VIII (F8) gene from a panel of 107 patients with hemophilia A and have characterized these gene defects byrestriction analysis, oligonucleotide hybridization, cloning and DNA sequencing. Recurrent point mutations that involve CG to TG transitions were identified in exon 18, exon 22, and exon 24; a single CG to TG transition was identified in exon 23; and a CG to CA transition was identified in exon 24. In addition, a Taq I site alteration in intron 4 was identified in a patient with mild hemophilia, which arose dg. S23&in a grandpaternal germ cell. Cloning and sequencing of this region suggests the generation of a newsplice donor site. These data suggest that CG to TG transition is a prominent mechanism of mutation in hemophilia A. Six different deletions were also characterized. In one family, the deletion involved exon 26. However, the deletion endpoints in the male proband were different from those in his carrier mother, suggesting either gonadal mosaicism or a second deletion event in maternal meiosis.Of the 15 mutations, 6 occurred de novo within 2 generations: 4 in males and 2 in females. In these djg.novo mutations paternal age at conception was 35 (range = 32-38) and maternal age was 24 and 27. The ability to discover a sizable number of mutations in the F8 gene producing hemophilia A enables us to determine the frequency and nature of de novo mutations in man.
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Qiao, Na-na, Qiang Gao, Qin Zhao, De-pei Wang, Wen-yi Zhao, and Chang-yan Yu. "The Cloning and Analysis of a Partial Lipase Gene Sequence of Staphylococcus hominis GIMT1.079: Partial Lipase Gene Sequencing of S. hominis." In 2010 4th International Conference on Bioinformatics and Biomedical Engineering (iCBBE 2010). IEEE, 2010. http://dx.doi.org/10.1109/icbbe.2010.5518078.

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Smith, Duane, Payal Watchmaker, Guido Stadler, Natalie Marks, Yelena Bronevetsky, Keviin Chapman, and Hideho Okada. "Abstract 5773: Sequencing and cloning IDH1 R132H-targeted monoclonal T cell receptors from CD4+T cells facilitated by opto-electric-positioning technology." In Proceedings: AACR Annual Meeting 2018; April 14-18, 2018; Chicago, IL. American Association for Cancer Research, 2018. http://dx.doi.org/10.1158/1538-7445.am2018-5773.

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Wicki, A. N., A. Walz, and K. J. Clemetson. "IDENTIFICATION OF GLYCOPROTEIN lb IN “IN VITRO” TRANSLATES FROM ISOLATED HUMAN PLATELET mRNA." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1643628.

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Platelet membrane glycoproteins play a crucial role in platelet adhesion and activation. To understand how they function it is of great importance to know their amino acid sequences and structures. It is rather difficult to purify membrane glycoproteins in amounts that are sufficient to determine their amino acid sequences by protein sequencing techniques. The easier way seems to be molecular cloning of the genes for these proteins.Metabolically stable mRNA derived from nucleated megakaryocytes is known to be present in the anuclear human platelets. We have developed a purification method for the isolation of platelet mRNA. Starting with 100 units of blood platelets we isolated 0.5 mg of mRNA by guanidine chloride/lithium chloride/phenol extraction. Crude mRNA as well as oligo-dT purified, polyadenylated mRNA, was assayed for protein synthesis in a reticulocyte lysate translation system in the presence of different labelled amino acids. SDS-PAGE of nonreduced and reduced samples of the translation products showed molecular masses up to at least 200 kDa. Immunoprecipitation and affinity column chromatography with poly- and monoclonal anti platelet glycoprotein lb antibodies showed that a protein with a molecular mass of 60 kDa crossreacting immunologically with glycocalicin is specifically recognized by these antibodies. The 60 kDa protein seems to be the nonglycosylated form of GPIbケ.
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Yi-peng, Chen, Gao Qiang, Qiao Na-na, Wang De-pei, Wang Bin-zhe, and Zhao Wen-yi. "Notice of Retraction: Cloning and homologous analysis of a partial lipase gene in Staphylococcus caprae TCCC 11546: Partial lipase gene sequencing of S. caprae." In 2010 2nd International Conference on Future Computer and Communication (ICFCC 2010). IEEE, 2010. http://dx.doi.org/10.1109/icfcc.2010.5497690.

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Ichinose, A., R. E. Bottenus, K. R. Loeb, and E. W. Davie. "ISOLATION AND CHARACTERIZATION OF THE GENES FOR THE a AND b SUBUNITS OF HUMAN COAGULATION FACTOR XIII." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1644652.

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Factor XIII (plasma transglutaminase, fibrin stabilizing factor) is a plasma protein that plays an important role in the final stages of blood coagulation and fibrinolysis. The molecule occurs in blood as a tetramer (a2b2) consisting of two a. subunits and two b subunits. Recently, we have determined the amino acid sequences for both the a. and b subunits of human factor XIII by a combination of cDNA cloning and amino acid sequence analysis. cDNAs coding for the a (3.8 Kb) and b (2.2 Kb) subunits were used for the screening of human genomic DNA libraries. Among 12 × 106 recombinant phage, ∼30 have been shown to contain the sequences for the a subunit and ∼10 have been shown to contain the gene for the b subunit of factor XIII. The clones coding for the a. subunit span ∼90 Kb and have been characterized by restriction mapping. Southern blotting, and DNA sequencing. Both 5’ and 3’ ends of the genomic clones correspond to the 5’ and 3’portions of the cDNA for the a.subunit of factor XIII. The DNA sequence revealed that the activation peptide released ^thrombin (amino acid residues 137), the first putative Ca2+ binding region (around residue 251), the active Site Cys (amino acid residue 314), and the second putative Ca2+ binding region (around residue 473) are encoded by separate exons. Accordingly, the intervening sequences may separate the a subunit into functional and structural domains. The gene organization for the b subunit will also be presented. (Supported by NIH Grant HL 16919.)
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Homes, W. E., H. R. Lijnen, L. Nelles, C. Kluft, and D. Collen. "AN ALANINE INSERTION IN α2-ANTIPLASMIN ‘ENSCHEDE’ ABOLISHES ITS PLASM IN INHIBITORY ACTIVITY." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1642897.

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Congenital deficiencies of the fibrinolytic inhibitor α2antiplasmin (α2AP) may result in bleeding disorders. An abnormal a AP (α2AP‘Enschede’) is known. 2 siblings with 3% functional activity and normal antigen level have parents with 50% activity and normal antigen. The protein interacts normally with the lysine-binding site(s) of plasmin(ogen) but does not inhibit plasmin irreversibly. α2AP Enschede is a plasmin substrate that like the normal protein releases a M 8,000 peptide upon reaction with plasmin. In the present study, Southern blot analysis, using an α2AP cDNA probe showed a restriction fragment length polymorphism within a small genomic DNA fragment of the Enschede family members. Cloning and sequencing of these fragments revealed a GCG inframe insertion that results in an alanine addition between amino acids 353 and 357, 7-10 positions NH -terminal to the reactive site PI residue, Arg364. This area is homologous to the A4 B-sheet of reactive site cleaved a -antitrypsin. Clones from each individual confirm the parents as true heterozygotes and the children as true homozygotes. A cloned genomic DNA sequence containing the insertion (V ) was exchanged for the normal sequence in a eukaryotic a AP expression plasmid. Recombinant α2AP‘Enschede’ (ra AfVAla) purified from the conditioned media of transfected Chinese Hamster Ovary Cells is analogous to plasma a α2AP‘Enschede’ with respect to interactions with plasmin and plasminogen. Preliminary analysis of the released Mr 8,000 recombinant peptide shows that its NH -terminus is the same as the peptide cleaved from normal a AP. Although ra α2APVAla does not inhibit plasmin irreversibly it does, however, act as a competitive inhibitor of hydrolysis of the chromogenic substrate S-2251 by plasmin.The K for this interaction is 25 nM. Thus, α2APAla retains a high affinity for the active center of plasmin. In conclusion, an Ala insertion near the reactive site of α2AP must have resulted in a structural perturbation that has abolished the plasmin inhibitory activity of a α2AP‘Enschede’. This variant may provide a model for further investigation of structure-function relationships in the serpins which determine the relative inhibitor vs. substrate properties.
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10

Antonarakis, E. "The Molecular Genetics of Hemophilia A Stylianos." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1643980.

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Hemophilia A is a common X linked hereditary disorder of blood coagulation due to deficiency of factor 8. The gene for factor 8 has been cloned and characterized (Nature 312:326-342, 1984). It is divided into 26 exons and 25 introns and spans 186 kb of DNA. The CGNA is 9 kb and codes for 2351 amino acids. The first 19 amino acids comprise the secretory leader peptide and the mature excreted polypeptide consists of 2332 amino acids. The nucleotide sequence of the exons and the exon-intron junctions is known and the complete amino acid sequence has been deducedSeveral laboratories have used cloned factor 8 DNA sequences as probes to characterized mutations that are responsible for hemophilia A in certain pedigrees. These mutations have been characterized by restriction analysis, oligonucleotide hybridization, cloning and sequencing of DNA from appropriate patientsIn about 500 patients with hemophilia A examined, the molecular defect has been recognized in 39. Both gross alterations (mainly deletions) and point mutations of the factor 8 gene have been found.A total of 19 different deletions have been observed. No two unrelated pedigrees share the same exact deletion.The size of the deleted DNA varies from 1.5 kb to more than 210 kb. All but one of these deletions are associated with severe hemophilia A. A deletion of 6 kb that contains exon 22 only is associated with moderate hemophilia. Some deletions are present in patients with inhibitors to factor 8. No correlation of the size or the position of the deletions can be found with the presence of inhibitors to factor 8.A total of 20 point mutations have been characterized. All are recognized by restriction analysis and involve Taq I sites. All are mutations of CpG dinucleotides and generate nonsense or missence codons. Unrelated pedigrees have the same single nucleotide change because of independent origin of the same mutation. In many instances de novo occurrence of a point mutation has been observed. CpG dinucleotides are hot spots for mutation to TG or CA presumably because of spontaneous deamination of methylcytosine. Some point mutations are present in patients with inhibitors but no correlation of the site of mutation and inhibitor formation has been found. The nonsense mutations are present in patients with severe hemophilia A. A missense mutation (Arg Gin) in exon 26 was found in a patient with mild hemophilia while another Arg Gin mutation in exon 24 has been observed in a patient with severe disease. The creation of a donor splice site in IVS 4 of factor 8 gene has been observed in a patient with mild hemophilia.Few DNA polymorphisms within the factor 8 gene and two other closely linked polymorphisms have been used for carrier detection and prenatal diagnosis of hemophilia A. These DNA markers are useful in more than 90% of families at risk for hemophilia A.The author thanks Drs. Gitschier, Din, Olek, Pirastou, Lawn for communication of their data prior to publication.The hemophilia project at Johns Hopkins was supported by an Institutional grant and NIH grant to S.S.A. and Haig H. Kazazian, Jr.
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Reports on the topic "Cloning and sequencing"

1

Chernova, T., V. K. Viswanathan, N. Austria, and B. P. Nichols. Cloning and sequencing of the trpE gene from Arthrobacter globiformis ATCC 8010 and several related subsurface Arthrobacter isolates. Office of Scientific and Technical Information (OSTI), September 1998. http://dx.doi.org/10.2172/663530.

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