scholarly journals Antibody Upstream Sequence Diversity and Its Biological Implications Revealed by Repertoire Sequencing

2021 ◽  
Author(s):  
Yan Zhu ◽  
Xiujia Yang ◽  
Cuiyu Ma ◽  
Haipei Tang ◽  
Qilong Wang ◽  
...  
Keyword(s):  
Sequence Diversity ◽  
Upstream Sequence ◽  
Repertoire Sequencing

scholarly journals Antibody Upstream Sequence Diversity and Its Biological Implications Revealed by Repertoire Sequencing

2020 ◽  
Author(s):  
Yan Zhu ◽  
Xiujia Yang ◽  
Jiaqi Wu ◽  
Haipei Tang ◽  
Qilong Wang ◽  
...  
Keyword(s):  
Cynomolgus Macaque ◽  
Pcr Amplification ◽  
Mrna Translation ◽  
Variable Region ◽  
Antibody Engineering ◽  
Upstream Sequence ◽  
Nucleotide Polymorphisms ◽  
Bioinformatics Pipeline ◽  
Repertoire Sequencing ◽  
Rapid Amplification

AbstractThe sequence upstream of antibody variable region (Antibody Upstream Sequence, or AUS) consists of 5’ untranslated region (5’ UTR) and two leader regions, L-PART1 and L-PART2. The sequence variations in AUS affect the efficiency of PCR amplification, mRNA translation, and subsequent PCR-based antibody quantification as well as antibody engineering. Despite their importance, the diversity of AUSs has long been neglected. Utilizing the rapid amplification of cDNA ends (5’RACE) and high-throughput antibody repertoire sequencing (Rep-Seq) technique, we acquired full-length AUSs for human, rhesus macaque (RM), cynomolgus macaque (CM), mouse, and rat. We designed a bioinformatics pipeline and discovered 2,957 unique AUSs, corresponding to 2,786 and 1,159 unique sequences for 5’ UTR and leader, respectively. Comparing with the leader records in the international ImMunoGeneTics (IMGT), while 529 were identical, 313 were with single nucleotide polymorphisms (SNPs), 280 were totally new, and 37 updated the incomplete records. The diversity of AUSs’ impact on related antibody biology was also probed. Taken together, our findings would facilitate Rep-Seq primer design for capturing antibodies comprehensively and efficiently as well as provide a valuable resource for antibody engineering and the studies of antibody at the molecular level.

Cloning of the Rat Tissue Factor cDNA and Promoter: Identification of a Serum-response Region

1996 ◽  
Vol 76 (05) ◽  
pp. 697-702 ◽  
Author(s):  
Olivier Taby ◽  
Claire-Lise Rosenfield ◽  
Vladimir Bogdanov ◽  
Yale Nemerson ◽  
Mark B Taubman
Keyword(s):  
Tissue Factor ◽  
General Structure ◽  
Amino Acid Sequences ◽  
Proximal Promoter ◽  
Striking Similarity ◽  
Upstream Sequence ◽  
Tf Gene ◽  
Rat Tissue ◽  
High Degree ◽  
Transcriptional Elements

SummaryTissue factor (TF) initiates coagulation and its expression in vascular smooth muscle cells (VSMC) likely plays a role in the propagation of arterial thrombosis. We report cloning the cDNA and proximal promoter region of the rat TF gene. While maintaining the general structure and organization of the TF molecule, there is a surprising divergence (≈ 18%) between the derived amino acid sequences of the rat and mouse TF. In contrast, there is striking similarity (90%) in the 5’ untranslated regions. High levels of basal promoter activity were seen in rat VSMC with constructs containing 106 bp of sequence downstream from the putative transcription start site and 426 to 103 bp of upstream sequence. Deletion of the sequence from −103 to −79, containing a single SP1 site, removed virtually all of the basal and serum-induced activity. Removal of the NFkB site or two additional upstream SP1 sites had little effect on serum responsiveness. Removal of the 5’ untranslated region abolished most of the basal activity of the TF promoter, suggesting that its high degree of conservation may be due to the presence of transcriptional elements critical for TF expression in rodent VSMC.

scholarly journals PLASTID DNA SEQUENCE DIVERSITY IN A WORLDWIDE SET OF GRAPEVINE CULTIVARS (VITIS VINIFERA L. SUBSP. VINIFERA)

2014 ◽  
pp. 609-616 ◽  
Author(s):  
T. Beridze ◽  
I. Pipia ◽  
J. Beck ◽  
S.-C. Hsu ◽  
B. Schaal ◽  
...  
Keyword(s):  
Vitis Vinifera ◽  
Dna Sequence ◽  
Plastid Dna ◽  
Sequence Diversity ◽  
Vitis Vinifera L

scholarly journals A 1232 bp upstream sequence of glutamine synthetase 1b from Eichhornia crassipes is a root-preferential promoter sequence

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Yanshan Zhong ◽  
Xiaodan Lu ◽  
Zhiwei Deng ◽  
Ziqing Lu ◽  
Minghui Fu
Keyword(s):  
Glutamine Synthetase ◽  
Eichhornia Crassipes ◽  
Invasive Plant ◽  
Promoter Sequence ◽  
Regulatory Mechanisms ◽  
Pcr Analysis ◽  
Regulation Mechanism ◽  
Upstream Sequence ◽  
Specific Expression ◽  
N Metabolism

Abstract Background Glutamine synthetase (GS) acts as a key enzyme in plant nitrogen (N) metabolism. It is important to understand the regulation of GS expression in plant. Promoters can initiate the transcription of its downstream gene. Eichhornia crassipes is a most prominent aquatic invasive plant, which has negative effects on environment and economic development. It also can be used in the bioremediation of pollutants present in water and the production of feeding and energy fuel. So identification and characterization of GS promoter in E. crassipes can help to elucidate its regulation mechanism of GS expression and further to control its N metabolism. Results A 1232 bp genomic fragment upstream of EcGS1b sequence from E. crassipes (EcGS1b-P) has been cloned, analyzed and functionally characterized. TSSP-TCM software and PlantCARE analysis showed a TATA-box core element, a CAAT-box, root specific expression element, light regulation elements including chs-CMA1a, Box I, and Sp1 and other cis-acting elements in the sequence. Three 5′-deletion fragments of EcGS1b upstream sequence with 400 bp, 600 bp and 900 bp length and the 1232 bp fragment were used to drive the expression of β-glucuronidase (GUS) in tobacco. The quantitative test revealed that GUS activity decreased with the decreasing of the promoter length, which indicated that there were no negative regulated elements in the EcGS1-P. The GUS expressions of EcGS1b-P in roots were significantly higher than those in leaves and stems, indicating EcGS1b-P to be a root-preferential promoter. Real-time Quantitative Reverse Transcription-Polymerase Chain Reaction (qRT-PCR) analysis of EcGS1b gene also showed higher expression in the roots of E.crassipes than in stems and leaves. Conclusions EcGS1b-P is a root-preferential promoter sequence. It can specifically drive the transcription of its downstream gene in root. This study will help to elucidate the regulatory mechanisms of EcGS1b tissue-specific expression and further study its other regulatory mechanisms in order to utilize E.crassipes in remediation of eutrophic water and control its overgrowth from the point of nutrient metabolism.

scholarly journals Single-component multilayered self-assembling nanoparticles presenting rationally designed glycoprotein trimers as Ebola virus vaccines

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Linling He ◽  
Anshul Chaudhary ◽  
Xiaohe Lin ◽  
Cindy Sou ◽  
Tanwee Alkutkar ◽  
...  
Keyword(s):  
Neutralizing Antibodies ◽  
Ebola Virus ◽  
Heptad Repeat ◽  
T Cell Epitopes ◽  
Vaccine Strategy ◽  
Cell Responses ◽  
Self Assembling ◽  
Protein Nanoparticles ◽  
Main Target ◽  
Repertoire Sequencing

AbstractEbola virus (EBOV) glycoprotein (GP) can be recognized by neutralizing antibodies (NAbs) and is the main target for vaccine design. Here, we first investigate the contribution of the stalk and heptad repeat 1-C (HR1C) regions to GP metastability. Specific stalk and HR1C modifications in a mucin-deleted form (GPΔmuc) increase trimer yield, whereas alterations of HR1C exert a more complex effect on thermostability. Crystal structures are determined to validate two rationally designed GPΔmuc trimers in their unliganded state. We then display a modified GPΔmuc trimer on reengineered protein nanoparticles that encapsulate a layer of locking domains (LD) and a cluster of helper T-cell epitopes. In mice and rabbits, GP trimers and nanoparticles elicit cross-ebolavirus NAbs, as well as non-NAbs that enhance pseudovirus infection. Repertoire sequencing reveals quantitative profiles of vaccine-induced B-cell responses. This study demonstrates a promising vaccine strategy for filoviruses, such as EBOV, based on GP stabilization and nanoparticle display.

scholarly journals Massive clonal expansion of polycytotoxic skin and blood CD8+ T cells in patients with toxic epidermal necrolysis

2021 ◽  
Vol 7 (12) ◽  
pp. eabe0013
Author(s):  
Axel Patrice Villani ◽  
Aurore Rozieres ◽  
Benoît Bensaid ◽  
Klara Kristin Eriksson ◽  
Amandine Mosnier ◽  
...  
Keyword(s):  
T Cells ◽  
Toxic Epidermal Necrolysis ◽  
Clonal Expansion ◽  
Cell Receptor ◽  
Effector Memory ◽  
Skin Symptoms ◽  
Tcr Repertoire ◽  
Repertoire Sequencing ◽  
Life Threatening ◽  
Cutaneous Adverse Drug Reaction

Toxic epidermal necrolysis (TEN) is a life-threatening cutaneous adverse drug reaction. To better understand why skin symptoms are so severe, we conducted a prospective immunophenotyping study on skin and blood. Mass cytometry results confirmed that effector memory polycytotoxic CD8+ T cells (CTLs) are the main leucocytes in TEN blisters at the acute phase. Deep T cell receptor (TCR) repertoire sequencing identified massive expansion of unique CDR3 clonotypes in blister cells. The same clones were highly expanded in patient’s blood, and the degree of their expansion showed significant correlation with disease severity. By transducing α and β chains of the expanded clonotypes into a TCR-defective cell line, we confirmed that those cells were drug specific. Collectively, these results suggest that the relative clonal expansion and phenotype of skin-recruited CTLs condition the clinical presentation of cutaneous adverse drug reactions.

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