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Molecular cloning

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https://www.readbyqxmd.com/read/28527998/molecular-cloning-and-structural-modelling-of-gamma-phospholipase-a2-inhibitors-from-bothrops-atrox-and-micrurus-lemniscatus-snakes
#1
Carina G Picelli, Rafael J Borges, Carlos A H Fernandes, Fabio M Matioli, Carla F C Fernandes, Juliana C Sobrinho, Rudson J Holanda, Luiz S Ozaki, Anderson M Kayano, Leonardo A Calderon, Marcos R M Fontes, Rodrigo G Stábeli, Andreimar M Soares
Phospholipases A2 inhibitors (PLIs) produced by venomous and non-venomous snakes play essential role in this resistance. These endogenous inhibitors may be classified by their fold in PLIα, PLIβ and PLIγ. Phospholipases A2 (PLA2s) develop myonecrosis in snake envenomation, a consequence that is not efficiently neutralized by antivenom treatment. This work aimed to identify and characterize two PLIs from Amazonian snake species, Bothrops atrox and Micrurus lemniscatus. Liver tissues RNA of specimens from each species were isolated and amplified by RT-PCR using PCR primers based on known PLIγ gene sequences, followed by cloning and sequencing of amplified fragments...
May 17, 2017: International Journal of Biological Macromolecules
https://www.readbyqxmd.com/read/28527895/suppression-of-pmrab11-inhibits-yhv-infection-in-penaeus-monodon
#2
Akechai Kongprajug, Sakol Panyim, Chalermporn Ongvarrasopone
Yellow head virus (YHV) is one of the most serious pathogens that causes worldwide shrimp production loss. It enters the cells via clathrin-mediated endocytosis and utilizes small GTPase Rab proteins such as PmRab5 and PmRab7 for intracellular trafficking. In this study, molecular cloning and functional analysis of Rab11 during YHV infection were investigated. PmRab11 cDNA was cloned by Rapid amplification of cDNA ends (RACEs). It contained two forms of sizes 1200 and 1050 bp distinct at the 5' UTR. The coding region of PmRab11 was 645 bp, encoding 214 amino acids...
May 17, 2017: Fish & Shellfish Immunology
https://www.readbyqxmd.com/read/28527099/molecular-diversity-among-viroids-infecting-chrysanthemum-in-india
#3
C R Adkar-Purushothama, G Chennappa, K Poornachandra Rao, M Y Sreenivasa, P K Maheshwar, M N Nagendra Prasad, T Sano
Association of Chrysanthemum stunt viroid (CSVd) and Chrysanthemum chlorotic mottle viroid (CChMVd) with the Chrysanthemum plants exhibiting severe stunting, distinct yellow leaf mottling, and chlorosis was detected in the main chrysanthemum-growing regions of India. Sequence analysis of 90 cDNA clones obtained for CSVd and CChMVd, representing the chrysanthemum-growing regions of India, revealed the high degree of sequence variation throughout the genome under natural conditions. Additionally, all the analyzed CChMVd clones revealed the presence of UUUC in the tetraloop, a signature of symptomatic variants in susceptible cultivars...
May 19, 2017: Virus Genes
https://www.readbyqxmd.com/read/28526948/an-innovative-strategy-to-clone-positive-modifier-genes-of-defects-caused-by-mtdna-mutations-mrps18c-as-suppressor-gene-of-m-3946g-a-mutation-in-mt-nd1-gene
#4
María Elena Rodríguez-García, Francisco Javier Cotrina-Vinagre, Patricia Carnicero-Rodríguez, Francisco Martínez-Azorín
We have developed a new functional complementation approach to clone modifier genes which overexpression is able to suppress the biochemical defects caused by mtDNA mutations (suppressor genes). This strategy consists in transferring human genes into respiratory chain-deficient fibroblasts, followed by a metabolic selection in a highly selective medium. We used a normalized expression cDNA library in an episomal vector (pREP4) to transfect the fibroblasts, and a medium with glutamine and devoid of any carbohydrate source to select metabolically...
May 19, 2017: Human Genetics
https://www.readbyqxmd.com/read/28523377/mining-lipolytic-enzymes-in-community-dna-from-high-andean-soils-using-a-targeted-approach
#5
Daniel Borda-Molina, José Salvador Montaña, María Mercedes Zambrano, Sandra Baena
Microbial enrichments cultures are a useful strategy to speed up the search for enzymes that can be employed in industrial processes. Lipases have gained special attention because they show unique properties such as: broad substrate specificity, enantio- and regio-selectivity and stability in organic solvents. A major goal is to identify novel lipolytic enzymes from microorganisms living in cold extreme environments such as high Andean soils, of relevance to our study being their capability be used in industrial processes...
May 18, 2017: Antonie Van Leeuwenhoek
https://www.readbyqxmd.com/read/28522527/lineage-dependent-spatial-and-functional-organization-of-the-mammalian-enteric-nervous-system
#6
Reena Lasrado, Werend Boesmans, Jens Kleinjung, Carmen Pin, Donald Bell, Leena Bhaw, Sarah McCallum, Hui Zong, Liqun Luo, Hans Clevers, Pieter Vanden Berghe, Vassilis Pachnis
The enteric nervous system (ENS) is essential for digestive function and gut homeostasis. Here we show that the amorphous neuroglia networks of the mouse ENS are composed of overlapping clonal units founded by postmigratory neural crest-derived progenitors. The spatial configuration of ENS clones depends on proliferation-driven local interactions of ENS progenitors with lineally unrelated neuroectodermal cells, the ordered colonization of the serosa-mucosa axis by clonal descendants, and gut expansion. Single-cell transcriptomics and mutagenesis analysis delineated dynamic molecular states of ENS progenitors and identified RET as a regulator of neurogenic commitment...
May 19, 2017: Science
https://www.readbyqxmd.com/read/28521948/genomics-of-interaction-between-the-brown-planthopper-and-rice
#7
REVIEW
Shengli Jing, Yan Zhao, Bo Du, Rongzhi Chen, Lili Zhu, Guangcun He
Rice (Oryza sativa L.) and the brown planthopper (Nilaparvata lugens (Stål)) form a model system for dissection of the mechanism of interaction between insect pest and crop. In this review, we focus on the genomics of BPH-rice interaction. On the side of rice, a number of BPH-resistance genes have been identified genetically. Thirteen of these genes have been cloned which shed a light on the molecular basis of the interaction. On the aspect of BPH, a lot of salivary proteins have been identified using transcriptome and proteome techniques...
February 2017: Current Opinion in Insect Science
https://www.readbyqxmd.com/read/28521208/molecular-cloning-and-characterization-of-two-%C3%AE-ketoacyl-acyl-carrier-protein-synthase-i-genes-from-jatropha-curcas-l
#8
Wangdan Xiong, Qian Wei, Pingzhi Wu, Sheng Zhang, Jun Li, Yaping Chen, Meiru Li, Huawu Jiang, Guojiang Wu
The β-ketoacyl-acyl carrier protein synthase I (KASI) is involved in de novo fatty acid biosynthesis in many organisms. Two putative KASI genes, JcKASI-1 and JcKASI-2, were isolated from Jatropha curcas. The deduced amino acid sequences of JcKASI-1 and JcKASI-2 exhibit around 83.8% and 72.5% sequence identities with AtKASI, respectively, and both contain conserved Cys-His-Lys-His-Phe catalytic active sites. Phylogenetic analysis indicated that JcKASI-2 belongs to a clade with several KASI proteins from dicotyledonous plants...
May 4, 2017: Journal of Plant Physiology
https://www.readbyqxmd.com/read/28520983/genomic-epidemiology-of-global-vim-producing-enterobacteriaceae
#9
Yasufumi Matsumura, Gisele Peirano, Rebekah Devinney, Patricia A Bradford, Mary R Motyl, Mark D Adams, Liang Chen, Barry Kreiswirth, Johann D D Pitout
Background: : International data on the molecular epidemiology of Enterobacteriaceae with VIM carbapenemases are limited. Methods: We performed short read (Illumina) WGS on a global collection of 89 VIM-producing clinical Enterobacteriaceae (2008-14). Results: VIM-producing (11 varieties within 21 different integrons) isolates were mostly obtained from Europe. Certain integrons with bla VIM were specific to a country in different species and clonal complexes (CCs) (In 87 , In 624 , In 916 and In 1323 ), while others had spread globally among various Enterobacteriaceae species (In 110 and In 1209 )...
May 16, 2017: Journal of Antimicrobial Chemotherapy
https://www.readbyqxmd.com/read/28520787/development-of-an-r4-dual-site-r4ds-gateway-cloning-system-enabling-the-efficient-simultaneous-cloning-of-two-desired-sets-of-promoters-and-open-reading-frames-in-a-binary-vector-for-plant-research
#10
Mostafa Aboulela, Yuji Tanaka, Kohji Nishimura, Shoji Mano, Mikio Nishimura, Sumie Ishiguro, Tetsuya Kimura, Tsuyoshi Nakagawa
Vast numbers of proteins work cooperatively to exert their functions in various cells. In order to understand the functions and molecular mechanisms of these proteins in plants, analyses of transgenic plants that concomitantly express two protein-coding genes are often required. We developed a novel Gateway cloning technology-compatible binary vector system, the R4 dual-site (R4DS) Gateway cloning system, which enables the easy and efficient cloning of two desired sets of promoters and open reading frames (ORFs) into a binary vector using promoter and ORF entry clones...
2017: PloS One
https://www.readbyqxmd.com/read/28518103/structure-function-studies-in-mouse-embryonic-stem-cells-using-recombinase-mediated-cassette-exchange
#11
Tim Pieters, Lieven Haenebalcke, Kenneth Bruneel, Niels Vandamme, Tino Hochepied, Jolanda van Hengel, Dagmar Wirth, Geert Berx, Jody J Haigh, Frans van Roy, Steven Goossens
Gene engineering in mouse embryos or embryonic stem cells (mESCs) allows for the study of the function of a given protein. Proteins are the workhorses of the cell and often consist of multiple functional domains, which can be influenced by posttranslational modifications. The depletion of the entire protein in conditional or constitutive knock-out (KO) mice does not take into account this functional diversity and regulation. An mESC line and a derived mouse model, in which a docking site for FLPe recombination-mediated cassette exchange (RMCE) was inserted within the ROSA26 (R26) locus, was previously reported...
April 27, 2017: Journal of Visualized Experiments: JoVE
https://www.readbyqxmd.com/read/28513878/gene-cloning-expression-and-characterization-of-trehalose-6-phosphate-synthase-from-pleurotus-ostreatus
#12
Min Lei, Xiangli Wu, Jinxia Zhang, Hexiang Wang, Chenyang Huang
Trehalose-6-phosphate synthase (TPS; EC2.4.1.15) catalyzes the first step in trehalose synthesis, which involves transfer of glucose from uridine diphosphate glucose (UDPG) to glucose 6-phosphate (G6P) to form trehalose-6-phosphate. To determine the gene and enzymatic characteristics of TPS in Pleurotus ostreatus, we cloned and sequenced the cDNA of PoTPS1, which contains a 1665 bp open reading frame that encodes a 554-amino acid protein with a predicted molecular weight of 62.01 kDa. This gene was expressed in Escherichia coli BL21 and then the recombinant protein was purified and characterized...
May 17, 2017: Journal of Basic Microbiology
https://www.readbyqxmd.com/read/28512468/molecular-identification-and-karyological-analysis-of-a-rampant-aspen-populus-tremula-l-salicaceae-clone
#13
Dmitry V Politov, Yuri S Belokon, Anna V Shatokhina, Maryana M Belokon, Nail A Khanov, Elena A Mudrik, Tatyana A Polyakova, Anna B Azarova, Konstantin A Shestibratov
A rampant highly heterozygous aspen (Populus tremula L.) clone "Meshabash" has been revealed in course of population genetic diversity analysis in a native stand in the Republic of Tatarstan, Russia. Here we report the results of karyological analysis showing that this highly vigorous clone is diploid (2n = 38) while typically triploid aspen demonstrates increased growth rate and resistance to aspen trunk rot caused by fungus Phellinus tremulae. By means of DNA identification of a series of model trees using 14 SSR loci we outlined the area occupied by this clone (at least 1...
2017: International Journal of Plant Genomics
https://www.readbyqxmd.com/read/28512093/population-genomic-analysis-of-1-777-extended-spectrum-beta-lactamase-producing-klebsiella-pneumoniae-isolates-houston-texas-unexpected-abundance-of-clonal-group-307
#14
S Wesley Long, Randall J Olsen, Todd N Eagar, Stephen B Beres, Picheng Zhao, James J Davis, Thomas Brettin, Fangfang Xia, James M Musser
Klebsiella pneumoniae is a major human pathogen responsible for high morbidity and mortality rates. The emergence and spread of strains resistant to multiple antimicrobial agents and documented large nosocomial outbreaks are especially concerning. To develop new therapeutic strategies for K. pneumoniae, it is imperative to understand the population genomic structure of strains causing human infections. To address this knowledge gap, we sequenced the genomes of 1,777 extended-spectrum beta-lactamase-producing K...
May 16, 2017: MBio
https://www.readbyqxmd.com/read/28511091/molecular-cloning-and-characterization-of-dngr-1-in-rhesus-macaques
#15
Wen-Rong Yao, Lei Yu, Dong Li, Gui-Bo Yang
DC, NK lectin group receptor-1 (DNGR-1), also known as C-type lectin domain family 9 member A (CLEC9A), is a promising target for immunological therapeutics and vaccination against tumors and viruses. However, little is known about its property in rhesus macaques. In this study, we cloned rhesus macaque DNGR-1 cDNA, and found that its coding region could encode a 241-amino acid polypeptide with 91.7% sequence identity and similar antigenicity to that of humans. Both free and cell surface rhesus macaque DNGR-1 expressed in vitro could bind to apoptotic/dead cells induced by serum deprivation or freeze-thaw, and to pyroptotic cells stimulated with PMA and LPS...
May 13, 2017: Molecular Immunology
https://www.readbyqxmd.com/read/28510252/assessing-non-additive-effects-in-gblup-model
#16
I C Vieira, J P R Dos Santos, L P M Pires, B M Lima, F M A Gonçalves, M Balestre
Understanding non-additive effects in the expression of quantitative traits is very important in genotype selection, especially in species where the commercial products are clones or hybrids. The use of molecular markers has allowed the study of non-additive genetic effects on a genomic level, in addition to a better understanding of its importance in quantitative traits. Thus, the purpose of this study was to evaluate the behavior of the GBLUP model in different genetic models and relationship matrices and their influence on the estimates of genetic parameters...
May 10, 2017: Genetics and Molecular Research: GMR
https://www.readbyqxmd.com/read/28510246/molecular-cloning-and-differential-expression-of-the-glucocorticoid-receptorgene-in-the-estuarine-tapertail-anchovy-coilia-nasus
#17
F K Du, G C Xu, Y Li, Z J Nie, P Xu
To understand the regulation of the glucocorticoid receptor gene (Gcr) during loading and transport stress in fish, the Gcr gene of Coilia nasus was cloned. Gcr in C. nasus is expressed strongly in the liver and muscle, and less stronglyin the gills, brain, spleen, intestine, trunk kidney, and head kidney. Gcr expression in both the liver and muscle was increased by loading and transport stress. NaCl reduced the death rate caused by loading and transport stress, and the expression of Gcr in liver and muscle differed significantly between the NaCl and non-NaCl groups...
May 10, 2017: Genetics and Molecular Research: GMR
https://www.readbyqxmd.com/read/28509615/mining-of-a-phospholipase-d-and-its-application-in-enzymatic-preparation-of-phosphatidylserine
#18
Wen-Bin Zhou, Jin-Song Gong, Hai-Juan Hou, Heng Li, Zhen-Ming Lu, Hong-Yu Xu, Zheng-Hong Xu, Jin-Song Shi
Phosphatidylserine (PS) is useful as the additive in industries for memory improvement, mood enhancement and drug delivery. Conventionally, PS was extracted from soybeans, vegetable oils, egg yolk, and biomass; however, their low availability and high extraction cost were limiting factors. Phospholipase D (PLD) is a promising tool for enzymatic synthesis of PS due to its transphosphatidylation activity. In this contribution, a new and uncharacterized PLD was first obtained from GenBank database via genome mining strategy...
May 16, 2017: Bioengineered
https://www.readbyqxmd.com/read/28508225/plasmid-based-reverse-genetics-of-influenza-a-virus
#19
Daniel R Perez, Matthew Angel, Ana Silvia Gonzalez-Reiche, Jefferson Santos, Adebimpe Obadan, Luis Martinez-Sobrido
Influenza A viruses have broad host range with a recognized natural reservoir in wild aquatic birds. From this reservoir, novel strains occasionally emerge with the potential to establish stable lineages in other avian and mammalian species, including humans. Understanding the molecular changes that allow influenza A viruses to change host range is essential to better assess their animal and public health risks. Reverse genetics systems have transformed the ability to manipulate and study negative strand RNA viruses...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28508223/reverse-genetics-of-influenza-b-viruses
#20
Aitor Nogales, Daniel R Perez, Jefferson Santos, Courtney Finch, Luis Martínez-Sobrido
Annual influenza epidemics are caused not only by influenza A viruses but also by influenza B viruses. Initially established for the generation of recombinant influenza A viruses, plasmid-based reverse genetics techniques have allowed researchers the generation of wild type and mutant viruses from full-length cDNA copies of the influenza viral genome. These reverse genetics approaches have allowed researchers to answer important questions on the biology of influenza viruses by genetically engineering infectious recombinant viruses...
2017: Methods in Molecular Biology
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