scholarly journals Sequence-Dependent Dynamics of Synthetic and Endogenous RSSs in V(D)J Recombination

2019 ◽  
Author(s):  
Soichi Hirokawa ◽  
Griffin Chure ◽  
Nathan M. Belliveau ◽  
Geoffrey A. Lovely ◽  
Michael Anaya ◽  
...  
Keyword(s):  
Single Molecule ◽  
Large Scale ◽  
Antigen Receptor ◽  
Quantitative Information ◽  
Dna Dynamics ◽  
Endogenous Gene ◽  
Quantitative Studies ◽  
Recombination Signal Sequences ◽  
Sequence Dependent ◽  
Dna Nicking

Developing lymphocytes in the immune system of jawed vertebrates assemble antigen-receptor genes by undergoing large-scale reorganization of spatially separated V, D, and J gene segments through a process known as V(D)J recombination. The RAG protein initiates this process by binding and cutting recombination signal sequences (RSSs) composed of conserved heptamer and nonamer sequences flanking less well-conserved 12- or 23-bp spacers. Little quantitative information is known about the contributions of individual RSS positions over the course of the RAG-RSS interaction. We employ a single-molecule method known as tethered particle motion to quantify the formation, stability, and cleavage of the RAG-12RSS-23RSS paired complex (PC) for numerous synthetic and endogenous 12RSSs. We thoroughly investigate the sequence space around a RSS by making 40 different single-bp changes and characterizing the reaction dynamics. We reveal that single-bp changes affect RAG function based on their position: loss of cleavage function (first three positions of the heptamer); reduced propensity for forming the PC (the nonamer and last four bp of the heptamer); or variable effects on PC formation (spacer). We find that the rare usage of some endogenous gene segments can be mapped directly to their adjacent 12RSSs to which RAG binds weakly. The 12RSS, however, cannot explain the high-frequency usage of other gene segments. Finally, we find that RSS nicking, while not required for PC formation, substantially stabilizes the PC. Our findings provide detailed insights into the contribution of individual RSS positions to steps of the RAG-RSS re-action that previously have been difficult to assess quantitatively.SummaryV(D)J recombination is a genomic cut-and-paste process for generating diverse antigen-receptor repertoires. The RAG enzyme brings separate gene segments together by binding the neighboring sequences called RSSs, forming a paired complex (PC) before cutting the DNA. There are limited quantitative studies of the sequence-dependent dynamics of the crucial inter-mediate steps of PC formation and cleavage. Here, we quantify individual RAG-DNA dynamics for various RSSs. While RSSs of frequently-used segments do not comparatively enhance PC formation or cleavage, the rare use of some segments can be explained by their neighboring RSSs crippling PC formation and/or cleavage. Furthermore, PC lifetimes reveal DNA-nicking is not required for forming the PC, but PCs with nicks are more stable.

scholarly journals Sequence-dependent dynamics of synthetic and endogenous RSSs in V(D)J recombination

2020 ◽  
Vol 48 (12) ◽  
pp. 6726-6739 ◽  
Author(s):  
Soichi Hirokawa ◽  
Griffin Chure ◽  
Nathan M Belliveau ◽  
Geoffrey A Lovely ◽  
Michael Anaya ◽  
...  
Keyword(s):  
Single Molecule ◽  
Particle Motion ◽  
Antigen Receptor ◽  
Quantitative Information ◽  
Lifetime Distributions ◽  
Recombination Signal ◽  
Endogenous Gene ◽  
Distinct Gene ◽  
Recombination Signal Sequences ◽  
Tethered Particle Motion

Abstract Developing lymphocytes of jawed vertebrates cleave and combine distinct gene segments to assemble antigen–receptor genes. This process called V(D)J recombination that involves the RAG recombinase binding and cutting recombination signal sequences (RSSs) composed of conserved heptamer and nonamer sequences flanking less well-conserved 12- or 23-bp spacers. Little quantitative information is known about the contributions of individual RSS positions over the course of the RAG–RSS interaction. We employ a single-molecule method known as tethered particle motion to track the formation, lifetime and cleavage of individual RAG–12RSS–23RSS paired complexes (PCs) for numerous synthetic and endogenous 12RSSs. We reveal that single-bp changes, including in the 12RSS spacer, can significantly and selectively alter PC formation or the probability of RAG-mediated cleavage in the PC. We find that some rarely used endogenous gene segments can be mapped directly to poor RAG binding on their adjacent 12RSSs. Finally, we find that while abrogating RSS nicking with Ca2+ leads to substantially shorter PC lifetimes, analysis of the complete lifetime distributions of any 12RSS even on this reduced system reveals that the process of exiting the PC involves unidentified molecular details whose involvement in RAG–RSS dynamics are crucial to quantitatively capture kinetics in V(D)J recombination.

scholarly journals Rag1D600A, a novel catalytically inactive RAG mouse model

2019 ◽  
Author(s):  
Jason B Wong ◽  
Jane A Skok
Keyword(s):  
Mouse Model ◽  
Signal Sequence ◽  
Antigen Receptor ◽  
Model System ◽  
Transgenic Expression ◽  
Recombination Signal ◽  
Recombination Signal Sequences ◽  
Acidic Residues ◽  
Dna Nicking ◽  
Mouse Model System

AbstractThe RAG complex (RAG1 and RAG2) can bind to recombination signal sequences of antigen receptor loci gene segments and coordinate V(D)J recombination which is the primary method of generating antigen receptor diversity. Previous biochemistry studies discovered RAG1 D600, D708 and E962 residues as essential for catalytic DNA nicking and hairpin forming activity of the RAG complex. Neutralization of each of the acidic residues does not impair DNA binding to recombination signal sequence containing DNA substrates, but cleavage of the substrates is severely compromised. These three acidic residues are thought to comprise a DDE motif that is responsible for binding to a divalent cation that is necessary for cleavage activity. Although a Rag1-/-; RAG1-D708A transgenic mouse model system has been used to study dynamics of RAG activity, transgenic expression may not precisely mimic expression from the endogenous locus. In order to improve upon this model, we created Rag1D600A mice that lack B and T cells and demonstrate a developmental block at the pro-B and DN stages, respectively. Thus, Rag1D600A mice provide a novel mouse model system for studying the poorly understood noncanonical functions of RAG1.

scholarly journals 3D DNA structural barcode copying and random access

2020 ◽  
Author(s):  
Filip Bošković ◽  
Alexander Ohmann ◽  
Ulrich F. Keyser ◽  
Kaikai Chen
Keyword(s):  
Data Storage ◽  
Single Molecule ◽  
Self Assembly ◽  
Large Scale ◽  
Three Dimensional ◽  
Random Access ◽  
Scale Production ◽  
Digital Information ◽  
Dna Nanostructures ◽  
Large Scale Production

AbstractThree-dimensional (3D) DNA nanostructures built via DNA self-assembly have established recent applications in multiplexed biosensing and storing digital information. However, a key challenge is that 3D DNA structures are not easily copied which is of vital importance for their large-scale production and for access to desired molecules by target-specific amplification. Here, we build 3D DNA structural barcodes and demonstrate the copying and random access of the barcodes from a library of molecules using a modified polymerase chain reaction (PCR). The 3D barcodes were assembled by annealing a single-stranded DNA scaffold with complementary short oligonucleotides containing 3D protrusions at defined locations. DNA nicks in these structures are ligated to facilitate barcode copying using PCR. To randomly access a target from a library of barcodes, we employ a non-complementary end in the DNA construct that serves as a barcode-specific primer template. Readout of the 3D DNA structural barcodes was performed with nanopore measurements. Our study provides a roadmap for convenient production of large quantities of self-assembled 3D DNA nanostructures. In addition, this strategy offers access to specific targets, a crucial capability for multiplexed single-molecule sensing and for DNA data storage.

scholarly journals Total workflows of the single-molecule imaging analysis in living cells: a tutorial guidance to the measurement of the drug effects on a GPCR

2020 ◽  
Author(s):  
Masataka Yanagawa ◽  
Yasushi Sako
Keyword(s):  
Single Molecule ◽  
Metabotropic Glutamate Receptor ◽  
Particle Density ◽  
Drug Effects ◽  
Quantitative Information ◽  
Single Molecule Imaging ◽  
Single Molecule Tracking ◽  
Metabotropic Glutamate ◽  
Protein Protein Interaction ◽  
Diffusion Dynamics

AbstractSingle-molecule imaging (SMI) is a powerful method to measure the dynamics of membrane proteins on the cell membrane. The single-molecule tracking (SMT) analysis provides information about the diffusion dynamics, the oligomer size distribution, and the particle density change. The affinity and on/off-rate of a protein—protein interaction can be estimated from the dual-color SMI analysis. However, it is difficult for trainees to determine quantitative information from the SMI movies. The present protocol guides the detailed workflows to measure the drug-activated dynamics of a G protein-coupled receptor (GPCR) and metabotropic glutamate receptor 3 (mGluR3), by using the total internal reflection fluorescence microscopy (TIRFM). This tutorial guidance comprises an open-source software named smDynamicsAnalyzer, with which one can easily analyze the SMT dataset by just following the workflows after building a designated folder structure (https://github.com/masataka-yanagawa/IgorPro8-smDynamicsAnalyzer).

Diverse developmental mutants revealed in an activation-tagged population of poplarThis article is one of a selection of papers published on the Special Issue of Poplar Research in Canada.

2007 ◽  
Vol 85 (11) ◽  
pp. 1071-1081 ◽  
Author(s):  
Edward J. Harrison ◽  
Michael Bush ◽  
Jonathan M. Plett ◽  
Daniel P. McPhee ◽  
Robin Vitez ◽  
...  
Keyword(s):  
Large Scale ◽  
Insertional Mutagenesis ◽  
Hybrid Poplar ◽  
Activation Tagging ◽  
Developmental Abnormalities ◽  
Endogenous Gene ◽  
Developmental Mutants ◽  
Poplar Trees ◽  
Wide Range ◽  
Mutant Lines

We have produced the largest population of activation-tagged poplar trees to date, approximately 1800 independent lines, and report on phenotypes of interest that have been identified in tissue culture and greenhouse conditions. Activation tagging is an insertional mutagenesis technique that results in the dominant upregulation of an endogenous gene. A large-scale Agrobacterium -mediated transformation protocol was used to transform the pSKI074 activation-tagging vector into Populus tremula × Populus alba hybrid poplar. We have screened the first 1000 lines for developmental abnormalities and have a visible mutant frequency of 2.4%, with alterations in leaf and stem structure as well as overall stature. Most of the phenotypes represent new phenotypes that have not previously been identified in poplar and, in some cases, not in any other plant either. Molecular analysis of the T-DNA inserts of a subpopulation of mutant lines reveal both single and double T-DNA inserts with double inserts more common in lines with visible phenotypes. The broad range of developmental mutants identified in this pilot screen of the population reveals that it will be a valuable resource for gene discovery in poplar. The full value of this population will only be realized as we screen these lines for a wide range of phenotypes.

Research Challenges and Opportunities in Conducting Quantitative Studies on Large-Scale Agile Methodology

2020 ◽  
Vol 31 (2) ◽  
pp. 64-73
Author(s):  
Dinesh Batra
Keyword(s):  
Large Scale ◽  
Empirical Literature ◽  
Adoption Studies ◽  
Agile Methodology ◽  
Quantitative Studies ◽  
Balanced Approach ◽  
Research Challenges ◽  
Time Period ◽  
Dependent Variables ◽  
Challenges And Opportunities

This research note suggests five research challenges when conducting quantitative studies on large-scale agile methodology (LSAM). First, the LSAM empirical literature, which is mainly characterized by qualitative studies primarily focusing on coordination issues, provides limited background. Second, the notion of “large” in LSAM needs to be clarified because the existing research seems to have focused on “very large” or outlier projects. Third, the popular LSAM methods suggest broad and general maxims that may result in difficulty in operationalizing dependent variables, especially in innovation adoption studies. Fourth, the researcher may get overwhelmed when selecting independent variables from the plethora of suggested constructs. Finally, some of the problems associated with large-scale agile are mostly challenges of using conventional agile during a time-period when LSAM had not formally emerged. Researchers should take a balanced approach considering both benefits and challenges of using LSAM and focusing on project-level dependent measures such as success and acceptance.

scholarly journals Conducting quantitative studies with the participation of political elites: best practices for designing the study and soliciting the participation of political elites

2020 ◽  
Author(s):  
Barbara Vis ◽  
Sjoerd Stolwijk
Keyword(s):  
Decision Making ◽  
Best Practices ◽  
Quantitative Research ◽  
Large Scale ◽  
Response Rate ◽  
Response Rates ◽  
Political Elite ◽  
Political Elites ◽  
Future Studies ◽  
Quantitative Studies

Abstract Conducting quantitative research (e.g., surveys, a large number of interviews, experiments) with the participation of political elites is typically challenging. Given that a population of political elites is typically small by definition, a particular challenge is obtaining a sufficiently high number of observations and, thus, a certain response rate. This paper focuses on two questions related to this challenge: (1) What are best practices for designing the study? And (2) what are best practices for soliciting the participation of political elites? To arrive at these best practices, we (a) examine which factors explain the variation in response rates across surveys within and between large-scale, multi-wave survey projects by statistically analyzing a newly compiled dataset of 342 political elite surveys from eight projects, spanning 30 years and 58 countries, (b) integrate the typically scattered findings from the existing literature and (c) discuss results from an original expert survey among researchers with experience with such research (n = 23). By compiling a comprehensive list of best practices, systematically testing some widely held believes about response rates and by providing benchmarks for response rates depending on country, survey mode and elite type, we aim to facilitate future studies where participation of political elites is required. This will contribute to our knowledge and understanding of political elites’ opinions, information processing and decision making and thereby of the functioning of representative democracies.

scholarly journals The sequencing and de novo assembly of the Larimichthys crocea genome using PacBio and Hi-C technologies

2019 ◽  
Vol 6 (1) ◽  
Author(s):  
Baohua Chen ◽  
Zhixiong Zhou ◽  
Qiaozhen Ke ◽  
Yidi Wu ◽  
Huaqiang Bai ◽  
...  
Keyword(s):  
Marine Fish ◽  
Single Molecule ◽  
Large Scale ◽  
Reference Genome ◽  
De Novo ◽  
Larimichthys Crocea ◽  
Chromosome Conformation ◽  
Protein Coding ◽  
Total Length ◽  
Chromosome Level

Abstract Larimichthys crocea is an endemic marine fish in East Asia that belongs to Sciaenidae in Perciformes. L. crocea has now been recognized as an “iconic” marine fish species in China because not only is it a popular food fish in China, it is a representative victim of overfishing and still provides high value fish products supported by the modern large-scale mariculture industry. Here, we report a chromosome-level reference genome of L. crocea generated by employing the PacBio single molecule sequencing technique (SMRT) and high-throughput chromosome conformation capture (Hi-C) technologies. The genome sequences were assembled into 1,591 contigs with a total length of 723.86 Mb and a contig N50 length of 2.83 Mb. After chromosome-level scaffolding, 24 scaffolds were constructed with a total length of 668.67 Mb (92.48% of the total length). Genome annotation identified 23,657 protein-coding genes and 7262 ncRNAs. This highly accurate, chromosome-level reference genome of L. crocea provides an essential genome resource to support the development of genome-scale selective breeding and restocking strategies of L. crocea.

scholarly journals Energetic dependencies dictate folding mechanism in a complex protein

2019 ◽  
Vol 116 (51) ◽  
pp. 25641-25648 ◽  
Author(s):  
Kaixian Liu ◽  
Xiuqi Chen ◽  
Christian M. Kaiser
Keyword(s):  
Optical Tweezers ◽  
Single Molecule ◽  
Large Scale ◽  
Elongation Factor ◽  
Multidomain Protein ◽  
Domain Interactions ◽  
The Stability ◽  
Domain Iii ◽  
And Function ◽  
Terminal Domains

Large proteins with multiple domains are thought to fold cotranslationally to minimize interdomain misfolding. Once folded, domains interact with each other through the formation of extensive interfaces that are important for protein stability and function. However, multidomain protein folding and the energetics of domain interactions remain poorly understood. In elongation factor G (EF-G), a highly conserved protein composed of 5 domains, the 2 N-terminal domains form a stably structured unit cotranslationally. Using single-molecule optical tweezers, we have defined the steps leading to fully folded EF-G. We find that the central domain III of EF-G is highly dynamic and does not fold upon emerging from the ribosome. Surprisingly, a large interface with the N-terminal domains does not contribute to the stability of domain III. Instead, it requires interactions with its folded C-terminal neighbors to be stably structured. Because of the directionality of protein synthesis, this energetic dependency of domain III on its C-terminal neighbors disrupts cotranslational folding and imposes a posttranslational mechanism on the folding of the C-terminal part of EF-G. As a consequence, unfolded domains accumulate during synthesis, leading to the extensive population of misfolded species that interfere with productive folding. Domain III flexibility enables large-scale conformational transitions that are part of the EF-G functional cycle during ribosome translocation. Our results suggest that energetic tuning of domain stabilities, which is likely crucial for EF-G function, complicates the folding of this large multidomain protein.

Impact of irrigation on rural poverty in India: an aggregate panel-data analysis

Water Policy ◽  
2003 ◽  
Vol 5 (5-6) ◽  
pp. 443-458 ◽  
Author(s):  
Madhusudan Bhattarai ◽  
A. Narayanamoorthy
Keyword(s):  
Large Scale ◽  
Panel Data Analysis ◽  
Rural Poverty ◽  
Quantitative Information ◽  
Poverty Level ◽  
Road Density ◽  
Literacy Rate ◽  
Canal Irrigation ◽  
Groundwater Irrigation ◽  
The Impact

The main objective of this study is to quantify the marginal impacts of irrigation and selected input factors on spatial (across 14 states) and temporal (from 1970–1993) variation in the rural poverty level in India. The study uses the head count ratio measure (percent of population below the poverty line) of poverty to evaluate how the poverty level is affected by input factors: irrigation, adoption of HYVs, fertilizer application, rural literacy rate and rural road density. It was found that marginal (incremental) impacts of irrigation followed by the rural literacy rate were larger in explaining the variation of rural poverty level in India than those of other factor-inputs selected. The marginal impact of groundwater irrigation on poverty reduction was larger than that of canal irrigation, which is due to greater control in the application and widespread use of groundwater irrigation than of canal irrigation. Despite mixed findings about the impact of irrigation on poverty from past studies, we have found large-scale marginal impacts of irrigation on rural poverty in India. This quantitative information is expected to be useful for designing targeted poverty alleviation and rural development strategies that also enhance agricultural-productivity growth.

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