scholarly journals Insertion Sequences

1998 ◽  
Vol 62 (3) ◽  
pp. 725-774 ◽  
Author(s):  
Jacques Mahillon ◽  
Michael Chandler
Keyword(s):  
Open Reading Frames ◽  
Insertion Sequences ◽  
Bacterial Genomes ◽  
Or Groups ◽  
Direct Target ◽  
Transposition Mechanism ◽  
The Individual ◽  
And Function

SUMMARY Insertion sequences (ISs) constitute an important component of most bacterial genomes. Over 500 individual ISs have been described in the literature to date, and many more are being discovered in the ongoing prokaryotic and eukaryotic genome-sequencing projects. The last 10 years have also seen some striking advances in our understanding of the transposition process itself. Not least of these has been the development of various in vitro transposition systems for both prokaryotic and eukaryotic elements and, for several of these, a detailed understanding of the transposition process at the chemical level. This review presents a general overview of the organization and function of insertion sequences of eubacterial, archaebacterial, and eukaryotic origins with particular emphasis on bacterial elements and on different aspects of the transposition mechanism. It also attempts to provide a framework for classification of these elements by assigning them to various families or groups. A total of 443 members of the collection have been grouped in 17 families based on combinations of the following criteria: (i) similarities in genetic organization (arrangement of open reading frames); (ii) marked identities or similarities in the enzymes which mediate the transposition reactions, the recombinases/transposases (Tpases); (iii) similar features of their ends (terminal IRs); and (iv) fate of the nucleotide sequence of their target sites (generation of a direct target duplication of determined length). A brief description of the mechanism(s) involved in the mobility of individual ISs in each family and of the structure-function relationships of the individual Tpases is included where available.

A Search for Ribonucleic Antiterminator Sites in Bacterial Genomes: Not Only Antitermination

2015 ◽  
Vol 25 (2-3) ◽  
pp. 143-153 ◽  
Author(s):  
Noa Gordon ◽  
Ronen Rosenblum ◽  
Anat Nussbaum-Shochat ◽  
Elad Eliahoo ◽  
Orna Amster-Choder
Keyword(s):  
Rna Stability ◽  
Open Reading Frames ◽  
Structural Constraints ◽  
Bacterial Genomes ◽  
Rna Motifs ◽  
E Coli ◽  
Bioinformatic Tools ◽  
Intergenic Regions

BglG/LicT-like proteins are transcriptional antiterminators that prevent termination of transcription at intrinsic terminators by binding to ribonucleic antiterminator (RAT) sites and stabilizing an RNA conformation which is mutually exclusive with the terminator structure. The known RAT sites, which are located in intergenic regions of sugar utilization operons, show low sequence conservation but significant structural analogy. To assess the prevalence of RATs in bacterial genomes, we employed bioinformatic tools that describe RNA motifs based on both sequence and structural constraints. Using descriptors with different stringency, we searched the genomes of <i>Escherichia</i><i>coli</i> K12, uropathogenic <i>E. coli</i> and <i>Bacillus subtilis</i> for putative RATs. Our search identified all known RATs and additional putative RAT elements. Surprisingly, most putative RATs do not overlap an intrinsic terminator and many reside within open reading frames (ORFs). The ability of one of the putative RATs, which is located within an antiterminator-encoding ORF and does not overlap a terminator, to bind to its cognate antiterminator protein in vitro and in vivo was confirmed experimentally. Our results suggest that the capacity of RAT elements has been exploited during evolution to mediate activities other than antitermination, for example control of transcription elongation or of RNA stability.

scholarly journals Posttranslational Modifications and the Immunogenicity of Biotherapeutics

2016 ◽  
Vol 2016 ◽  
pp. 1-15 ◽  
Author(s):  
Roy Jefferis
Keyword(s):  
Posttranslational Modifications ◽  
Drug Product ◽  
Molecular Forms ◽  
Phagocytic Cells ◽  
Production Platform ◽  
The Individual ◽  
And Function ◽  
Antigen Presenting

Whilst the amino acid sequence of a protein is determined by its gene sequence, the final structure and function are determined by posttranslational modifications (PTMs), including quality control (QC) in the endoplasmic reticulum (ER) and during passage through the Golgi apparatus. These processes are species and cell specific and challenge the biopharmaceutical industry when developing a production platform for the generation of recombinant biologic therapeutics. Proteins and glycoproteins are also subject to chemical modifications (CMs) bothin vivoandin vitro. The individual is naturally tolerant to molecular forms of self-molecules but nonself variants can provoke an immune response with the generation of anti-drug antibodies (ADA); aggregated forms can exhibit enhanced immunogenicity and QC procedures are developed to avoid or remove them. Monoclonal antibody therapeutics (mAbs) are a special case because their purpose is to bind the target, with the formation of immune complexes (ICs), a particular form of aggregate. Such ICs may be removed by phagocytic cells that have antigen presenting capacity. These considerations may frustrate the possibility of ameliorating the immunogenicity of mAbs by rigorous exclusion of aggregates from drug product. Alternate strategies for inducing immunosuppression or tolerance are discussed.

Structure and function of ribosomal RNA

1995 ◽  
Vol 73 (11-12) ◽  
pp. 997-1009 ◽  
Author(s):  
Harry F. Noller ◽  
Rachel Green ◽  
Gabriele Heilek ◽  
Vernita Hoffarth ◽  
Alexander Hüttenhofer ◽  
...  
Keyword(s):  
16S Rrna ◽  
Hydroxyl Radical ◽  
Ribosomal Proteins ◽  
23S Rrna ◽  
Active Population ◽  
30S Subunit ◽  
Radical Cleavage ◽  
The Individual ◽  
And Function

A refined model has been developed for the folding of 16S rRNA in the 30S subunit, based on additional constraints obtained from new experimental approaches. One set of constraints comes from hydroxyl radical footprinting of each of the individual 30S ribosomal proteins, using free Fe2+–EDTA complex. A second approach uses localized hydroxyl radical cleavage from a single Fe2+tethered to unique positions on the surface of single proteins in the 30S subunit. This has been carried out for one position on the surface of protein S4, two on S17, and three on S5. Nucleotides in 16S rRNA that are essential for P-site tRNA binding were identified by a modification interference strategy. Ribosomal subunits were partially inactivated by chemical modification at a low level. Active, partially modified subunits were separated from inactive ones by binding 3′-biotin-derivatized tRNA to the 30S subunits and captured with streptavidin beads. Essential bases are those that are unmodified in the active population but modified in the total population. The four essential bases, G926, 2mG966, G1338, and G1401 are a subset of those that are protected from modification by P-site tRNA. They are all located in the cleft of our 30S subunit model. The rRNA neighborhood of the acceptor end of tRNA was probed by hydroxyl radical probing from Fe2+tethered to the 5′ end of tRNA via an EDTA linker. Cleavage was detected in domains IV, V, and VI of 23S rRNA, but not in 5S or 16S rRNA. The sites were all found to be near bases that were protected from modification by the CCA end of tRNA in earlier experiments, except for a set of E-site cleavages in domain IV and a set of A-site cleavages in the α-sarcin loop of domain VI. In vitro genetics was used to demonstrate a base-pairing interaction between tRNA and 23S rRNA. Mutations were introduced at positions C74 and C75 of tRNA and positions 2252 and 2253 of 23S rRNA. Interaction of the CCA end of tRNA with mutant ribosomes was tested using chemical probing in conjunction with allele-specific primer extension. The interaction occurred only when there was a Watson–Crick pairing relationship between positions 74 of tRNA and 2252 of 23S rRNA. Using a novel chimeric in vitro reconstitution method, it was shown that the peptidyl transferase reaction depends on this same Watson–Crick base pair.Key words: rRNA, ribosome, tRNA, hydroxyl radical, ribosomal protein.

scholarly journals The Lyt-2 molecule recognizes residues in the class I alpha 3 domain in allogeneic cytotoxic T cell responses.

1988 ◽  
Vol 168 (1) ◽  
pp. 325-341 ◽  
Author(s):  
J M Connolly ◽  
T A Potter ◽  
E M Wormstall ◽  
T H Hansen
Keyword(s):  
Primary Response ◽  
Class I ◽  
Single Amino Acid ◽  
Cytotoxic T Cell ◽  
Mhc Class I Molecule ◽  
Cytotoxic T Cell Responses ◽  
The Individual ◽  
And Function

The involvement of the different domains of the MHC class I molecule in CTL recognition was investigated. mAbs specific for the alpha 1/alpha 2 domains of H-2Ld interfered with both the primary and secondary generation and effector function of in vitro Ld-specific CTL. mAbs specific for the alpha 3 domain of H-2Ld interfered with the generation and function of primary in vitro Ld-specific CTL; however, there was no effect on the in vitro generation of secondary CTL and only partial inhibition of their function. In vivo treatment with graft-specific antibodies to both the alpha 3 domain and the alpha 1/alpha 2 domains together resulted in a dramatic enhancement of Ld- or Dd-disparate skin grafts, whereas the individual mAbs showed minimal effects. This suggested that the class I alpha 3 domain is recognized by alloreactive CTL. Several approaches were undertaken to examine whether recognition of the alpha 3 domain determinants is mediated by the Lyt-2 molecule. When mAbs specific for the alpha 3 domain of either H-2Ld or H-2Dd were used in vivo and in vitro, the resulting CTL population was not inhibited by antibody to the alpha 3 domain and was only partially inhibited by antibody to Lyt-2. We therefore observed a correlation between the effects of antibody to the class I alpha 3 domain of the target molecule and antibody to the Lyt-2 molecule on the CTL. To further test the relationship between CTL recognition of the alpha 3 domain and the involvement of Lyt-2, we used a cell expressing a mutation in the alpha 3 domain of the Dd molecule. The mutation resulted in a single amino acid substitution of glu to lys at residue 227 of the alpha 3 domain. Consistent with an earlier report, cells expressing the mutant Dd lys molecule were not lysed by CTL from a primary stimulation against the wild-type Dd glu molecule. However, this same cell line was killed by the Lyt-2-independent secondary Dd-specific CTL generated in the presence of antibody to the alpha 3 domain in vivo and in vitro. Furthermore, cells expressing the mutant Dd lys molecule failed to stimulate a primary response. In conclusion, several independent lines of evidence indicate that residues in the alpha 3 domain of the class I molecule are involved in recognition by the Lyt-2 molecule, and that Lyt-2-mediated recognition can be specifically blocked using mAb to determinants in the alpha 3 domain.

scholarly journals Positive Selection on Transposase Genes of Insertion Sequences in the Crocosphaera watsonii Genome

2006 ◽  
Vol 188 (20) ◽  
pp. 7176-7185 ◽  
Author(s):  
Ted H. M. Mes ◽  
Marije Doeleman
Keyword(s):  
Amino Acid ◽  
Positive Selection ◽  
Mobile Elements ◽  
Open Reading Frames ◽  
Data Sets ◽  
Insertion Sequences ◽  
Bacterial Genomes ◽  
Transposase Gene ◽  
Genomic Locations ◽  
Crocosphaera Watsonii

ABSTRACT Insertion sequences (ISs) are mobile elements that are commonly found in bacterial genomes. Here, the structural and functional diversity of these mobile elements in the genome of the cyanobacterium Crocosphaera watsonii WH8501 is analyzed. The number, distribution, and diversity of nucleotide and amino acid stretches with similarity to the transposase gene of this IS family suggested that this genome harbors many functional as well as truncated IS fragments. The selection pressure acting on full-length transposase open reading frames of these ISs suggested (i) the occurrence of positive selection and (ii) the presence of one or more positively selected codons. These results were obtained using three data sets of transposase genes from the same IS family that were collected based on the level of amino acid similarity, the presence of an inverted repeat, and the number of sequences in the data sets. Neither recombination nor ribosomal frameshifting, which may interfere with the selection analyses, appeared to be important forces in the transposase gene family. Some positively selected codons were located in a conserved domain, suggesting that these residues are functionally important. The finding that this type of selection acts on IS-carried genes is intriguing, because although ISs have been associated with the adaptation of the bacterial host to new environments, this has typically been attributed to transposition or transformation, thus involving different genomic locations. Intragenic adaptation of IS-carried genes identified here may constitute a novel mechanism associated with bacterial diversification and adaptation.

scholarly journals Molecular Characterization, Phylogenetic Relationships, and Specific Detection of Peach mosaic virus

2006 ◽  
Vol 96 (2) ◽  
pp. 137-144 ◽  
Author(s):  
D. James ◽  
A. Varga ◽  
H. Croft ◽  
H. Rast ◽  
D. Thompson ◽  
...  
Keyword(s):  
Mosaic Virus ◽  
Open Reading Frames ◽  
Nucleic Acid Binding ◽  
Specific Detection ◽  
Deletion Event ◽  
Polymerase Chain ◽  
Similar Genome Organization ◽  
Leaf Virus

Peach mosaic virus (PcMV) and Cherry mottle leaf virus (CMLV) are serologically related viruses that cause distinct diseases, have a different host range, and are vectored by different eriophyid mites. Sequence analysis of the genome of PcMV indicates that it is closely related genetically to CMLV but distinct, with similar genome organization and a member of the genus Trichovirus. The genome of PcMV consists of 7,988 nucleotides, excluding a poly(A) tail at the 3′ end of the genome. Four putative open reading frames (ORF1 to 4) were identified coding for proteins of 216.3, 47.2, 21.7, and 15.7 kDa, respectively. Also, three noncoding regions were identified, including an intergenic region separating ORF3 and ORF4. The complete nucleotide sequence of PcMV shares 73% identity with CMLV. The CP amino acid sequence identity between isolates of PcMV ranged from 97 to 99% versus 83% identity when compared with the CP of CMLV. In vitro expression and subsequent western blot analysis confirmed ORF3 as encoding the CP gene of PcMV. Phylogenetic analysis supports classification of PcMV and CMLV as members of the genus Trichovirus. They are unique members of this genus with an extra ORF (ORF4). PcMV ORF4 appears to code for a putative nucleic acid-binding (NB) protein which has identity with the NB protein of CMLV and members of the genera Allexivirus, Carlavirus, and Vitivirus. PcMV and CMLV appear to be the products of recombination between members of the genus Trichovirus and a virus group containing the putative NB protein. Alternatively, PcMV and CMLV may represent the intact genome, with a deletion event producing members that lack ORF4. A reverse transcription-polymerase chain reaction procedure was developed for reliable and specific detection of PcMV. This will be an asset for stone fruit virus certification.

scholarly journals Aptamers that bind filovirus GP : a new frontier for detection and neutralization of Ebola and Marburg virus

2020 ◽  
Author(s):  
◽  
Kwaku Dwumah Tawiah
Keyword(s):  
Point Of Care ◽  
Low Cost ◽  
Marburg Virus ◽  
Whole Cells ◽  
Affinity Enrichment ◽  
Disease Diagnostics ◽  
Individual Sequences ◽  
The Individual ◽  
And Function

Aptamers are single chained, nucleic acid-based affinity probes that bind to their targets with strong affinity and specificity. They are made through an in vitro combinatorial selection method, wherein large libraries of nucleic acids with randomized sequences are subjected to an iterative process of affinity enrichment, partitioning, and amplification. Evolved libraries are sequenced, and the individual sequences are screened and characterized for their structure and function. Aptamers have been developed to target many molecules, including small molecules, purified proteins, whole cells, bacteria, and viruses. They have been developed for therapeutics and as research and diagnostic probes. Aptamers that have an affinity for virus surfaces are excellent probes for developing low-cost biosensors and potentially antiviral therapeutics. In this work, I present the development of aptamers that have an affinity for filovirus surfaces. I first describe the development of an improved method for purifying highly lytic vesicular stomatitis virus-based filovirus GP displayed surrogate viruses. Filoviruses are highly pathogenic and thus require highly secured containment facilities for their studies. The use of attenuated surrogates facilitates filovirus research at biosafety level 2 facilities. This work outlines the steps required to propagate and generate pure virus particles to be used as selection targets. I then describe the development of aptamer probes that differentially recognize GPs from MARV and EBOV. This work represents the first step in the development of aptamer based-low-cost point of care devices for filovirus disease diagnostics. Finally, this work describes the use of a hybrid selection approach that combines two different selection platforms to generate aptamers that bind to EBOV surfaces.

scholarly journals Unraveling the mechanism of the cadherin-catenin-actin catch bond

2018 ◽  
Author(s):  
Shishir Adhikari ◽  
Jacob Moran ◽  
Christopher Weddle ◽  
Michael Hinczewski
Keyword(s):  
Protein Complexes ◽  
Optical Tweezer ◽  
Force Transducer ◽  
Salt Bridges ◽  
Lifetime Distributions ◽  
Catch Bond ◽  
The Individual ◽  
And Function

The adherens junctions between epithelial cells involve a protein complex formed by E-cadherin, β-catenin, α-catenin and F-actin. The stability of this complex was a puzzle for many years, since in vitro studies could reconstitute various stable subsets of the individual proteins, but never the entirety. The missing ingredient turned out to be mechanical tension: a recent experiment that applied physiological forces to the complex with an optical tweezer dramatically increased its lifetime, a phenomenon known as catch bonding. However, in the absence of a crystal structure for the full complex, the microscopic details of the catch bond mechanism remain mysterious. Building on structural clues that point to α-catenin as the force transducer, we present a quantitative theoretical model for how the catch bond arises, fully accounting for the experimental lifetime distributions. The model allows us to predict the energetic changes induced by tension at the interface between α-catenin and F-actin. It also identifies a significant energy barrier due to a network of salt bridges between two conformational states of β-catenin. By stabilizing one of these states, this barrier could play a role in how the complex responds to additional in vivo binding partners like vinculin. Since significant conformational energy barriers are a common feature of other adhesion systems that exhibit catch bonds, our model can be adapted into a general theoretical framework for integrating structure and function in a variety of force-regulated protein complexes.

Wheelchairs and seating

2019 ◽  
pp. 305-318
Author(s):  
Manoj Sivan ◽  
Margaret Phillips ◽  
Ian Baguley ◽  
Melissa Nott
Keyword(s):  
Health Condition ◽  
International Classification Of Functioning ◽  
Personal Factors ◽  
Freedom Of Movement ◽  
Health Model ◽  
The Individual ◽  
And Function ◽  
Life Roles

Freedom of movement is an essential component of independence and mobility is closely associated with quality of life. People who have difficulty with independent walking often require a wheelchair to assist with mobility. The International Classification of Functioning, Disability and Health model can be used to guide the provision of wheelchairs and seating through consideration of the individual personal factors and preferences, environmental limitations, the person’s desired activities and participation in life roles, as well as the person’s health condition or impairments of body structure and function. This chapter describes the steps in prescribing a wheelchair, types of manual and powered wheelchairs, and specialized seating and positioning components.

scholarly journals DNGR-1 is a specific and universal marker of mouse and human Batf3-dependent dendritic cells in lymphoid and nonlymphoid tissues

Blood ◽  
2012 ◽  
Vol 119 (25) ◽  
pp. 6052-6062 ◽  
Author(s):  
Lionel F. Poulin ◽  
Yasmin Reyal ◽  
Heli Uronen-Hansson ◽  
Barbara U. Schraml ◽  
David Sancho ◽  
...  
Keyword(s):  
Dendritic Cells ◽  
Transcription Factor ◽  
Human Blood ◽  
High Expression ◽  
Humanized Mice ◽  
Evolutionarily Conserved ◽  
And Function ◽  
Different Tissues

Abstract Mouse CD8α+ dendritic cells (DCs) in lymphoid organs and CD103+ CD11b− DCs in nonlymphoid tissues share phenotypic and functional similarities, as well as a unique shared developmental dependence on the transcription factor Batf3. Human DCs resembling mouse CD8α+ DCs in phenotype and function have been identified in human blood, spleen, and tonsil. However, it is not clear whether such cells are also present in human nonlymphoid organs, and their equivalence to mouse CD8α+ DC has recently been questioned. Furthermore, the identification of “CD8α+ DC-like” cells across different tissues and species remains problematic because of the lack of a unique marker that can be used to unambiguously define lineage members. Here we show that mouse CD8α+ DCs and CD103+ CD11b− DCs can be defined by shared high expression of DNGR-1 (CLEC9A). We further show that DNGR-1 uniquely marks a CD11b− human DC population present in both lymphoid and nonlymphoid tissues of humans and humanized mice. Finally, we demonstrate that knockdown of Batf3 selectively prevents the development of DNGR-1+ human DCs in vitro. Thus, high expression of DNGR-1 specifically and universally identifies a unique DC subset in mouse and humans. Evolutionarily conserved Batf3 dependence justifies classification of DNGR-1hi DCs as a distinct DC lineage.

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