scholarly journals RNA Multimerisation in the DNA Packaging Motor of Bacteriophage φ29

2005 ◽  
Vol 6 (2) ◽  
pp. 127-134 ◽  
Author(s):  
Jonathan P. A. Wood ◽  
Stephanie A. Capaldi ◽  
Mark A. Robinson ◽  
Andrew J. Baron ◽  
Nicola J. Stonehouse
Keyword(s):  
Analytical Ultracentrifugation ◽  
Molecular Level ◽  
Viral Assembly ◽  
Partial Specific Volume ◽  
Wild Type ◽  
Rna Molecules ◽  
Dna Packaging Motor ◽  
Self Association

The use of bacteriophages as experimental tools allows the investigation of interactions between components at the molecular level that are often not possible in more complex virus systems. The bacteriophage φ29 acts as a molecular machine to package its own genomic DNA during viral assembly. Self-associating RNA molecules, called pRNA, have an essential role in the function of this machine. This paper reports the characterization of this self-association (which leads to multimerisation of wild-type and truncated variant pRNAs) by analytical ultracentrifugation (including determination of the partial specific volume of the pRNA), together with an investigation into the domains of the molecule important for multimerisation by the use of complementary DNA probes.

scholarly journals Characterization of recombinant-derived granulocyte-colony stimulating factor (G-CSF)

1988 ◽  
Vol 256 (1) ◽  
pp. 213-218 ◽  
Author(s):  
P Wingfield ◽  
R Benedict ◽  
G Turcatti ◽  
B Allet ◽  
J J Mermod ◽  
...  
Keyword(s):  
Coli Strain ◽  
Granulocyte Colony Stimulating Factor ◽  
Colony Stimulating Factor ◽  
Wild Type ◽  
Disulphide Bonds ◽  
Disulphide Bond Formation ◽  
Stimulating Factor ◽  
Factor G

Human granulocyte colony-stimulating factor (G-CSF), and a mutant having a Ser for Cys substitution at residue 18 were produced in Escherichia coli strain W3110. About 60 mg of pure protein was obtained from 50 g of wet cells with a recovery of about 20%. The proteins were characterized physically and chemically, including determination of disulphide bonds, which were found to exist between residues 37-43 and 65-75. Cys-18 is not involved in disulphide bond formation and was substituted by Ser with no effects on gross protein conformation or biological activity. Both the wild-type and the mutant recombinant-derived proteins, although not glycosylated, possess colony-stimulating activities. In a bioassay using the murine myelomonocytic leukaemic cell line WEH1 3B D+, activities were obtained which were similar to those of natural G-CSF and of a glycosylated recombinant-derived human G-CSF produced in monkey cells.

scholarly journals A Statistical Approach for Determination of Disk Diffusion-Based Cutoff Values for Systematic Characterization of Wild-Type and Non-Wild-Type Bacterial Populations in Antimicrobial Susceptibility Testing

2015 ◽  
Vol 53 (6) ◽  
pp. 1812-1822 ◽  
Author(s):  
Giorgia Valsesia ◽  
Malgorzata Roos ◽  
Erik C. Böttger ◽  
Michael Hombach
Keyword(s):  
Resistance Mechanisms ◽  
Beta Lactamase ◽  
Wild Type ◽  
Beta Lactam ◽  
Bacterial Populations ◽  
Content Type ◽  
Extended Spectrum Beta Lactamase ◽  
E Coli

In this study, we introduce a new approach for determination of epidemiologic cutoffs (ECOFFs) and resistant-population cutoffs (RCOFFs) based on receiver operating characteristic (ROC) curves. As an example, the method was applied for determination of ECOFFs for seven different beta-lactam antibiotics and wild-type populations ofEscherichia coli,Klebsiella pneumoniae, andEnterobacter cloacae. In addition, RCOFFs were determined for bacterial populations with defined resistance mechanisms (“resistotypes”), i.e., extended-spectrum beta-lactamase (ESBL)-positiveE. coli, ESBL-positiveK. pneumoniae, and ESBL-positiveE. cloacae; AmpC cephalosporinase-positiveE. coliand AmpC-positiveK. pneumoniae; and broad-spectrum beta-lactamase (BSBL)-positiveE. coli. RCOFFs and ECOFFs are instrumental for a systematic characterization of associations between resistotypes and wild-type populations.

scholarly journals Structure and inhibitor binding characterization of oncogenic MLLT1 mutants

2021 ◽  
Author(s):  
Xiaomin Ni ◽  
Allyn T. Londregan ◽  
Dafydd R. Owen ◽  
Stefan Knapp ◽  
Apirat Chaikuad
Keyword(s):  
Conformational Changes ◽  
Structural Basis ◽  
Wild Type ◽  
Structural Comparison ◽  
Inhibitor Binding ◽  
Binding Interface ◽  
Association Behavior ◽  
Self Association ◽  
Potential Strategy

AbstractDysfunction of YEATS-domain-containing MLLT1, an acetyl/acyl-lysine dependent epigenetic reader domain, has been implicated in the development of aggressive cancers. Mutations in the YEATS domain have been recently reported as a cause of MLLT1 aberrant reader function. However, structural basis for the reported alterations in affinity for acetyled/acylated histone has remained elusive. Here, we report the crystal structures of both insertion and substitution present in cancer, revealing significant conformational changes of the YEATS-domain loop 8. Structural comparison demonstrates that such alteration not only altered the binding interface for acetylated/acylated histones, but the sequence alterations in the T1 loop may enable dimeric assembly consistent inducing self-association behavior. Nevertheless, we show that also the MLLT1 mutants can be targeted by developed acetyllysine mimetic inhibitors with affinities similarly to wild type. Our report provides a structural basis for the altered behaviors and potential strategy for targeting oncogenic MLLT1 mutants.

The development of chromatography for the characterization of protein interactions: a personal perspective

2003 ◽  
Vol 31 (5) ◽  
pp. 1010-1014 ◽  
Author(s):  
D.J. Winzor
Keyword(s):  
Ligand Binding ◽  
Protein Interactions ◽  
Equilibrium Constants ◽  
Gel Filtration ◽  
Personal Perspective ◽  
Self Association ◽  
Quantitative Procedure ◽  
Insight Into

This article reviews the progress of a personal endeavour to develop chromatography as a quantitative procedure for the determination of reaction stoichiometries and equilibrium constants governing protein interactions. As well as affording insight into an aspect of chromatography with which many protein chemists are unfamiliar, it shows the way in which minor adaptations of conventional chromatographic practices have rendered the technique one of the most powerful methods available for the characterization of interactions. That pathway towards quantification is followed from the introduction of frontal gel filtration for the study of protein self-association to the characterization of ligand binding by the biosensor variant of quantitative affinity chromatography.

scholarly journals Characterization of the solution properties and conformation of pneumolysin, the membrane-damaging toxin of Streptococcus pneumoniae

1993 ◽  
Vol 296 (3) ◽  
pp. 671-674 ◽  
Author(s):  
P J Morgan ◽  
P G Varley ◽  
A J Rowe ◽  
P W Andrew ◽  
T J Mitchell
Keyword(s):  
Streptococcus Pneumoniae ◽  
Tertiary Structure ◽  
Analytical Ultracentrifugation ◽  
Sedimentation Coefficient ◽  
Spectroscopic Methods ◽  
Solution Conformation ◽  
Structure Information ◽  
Self Association ◽  
Secondary And Tertiary Structure

Pneumolysin is a membrane-damaging toxin produced by Streptococcus pneumoniae. In order to understand fully the mode of action of this toxin, it is necessary to have an appreciation of the size, self-association behaviour and solution conformation of pneumolysin. A combination of analytical ultracentrifugation methodologies has shown that pneumolysin lacks self-association behaviour in solution and has provided a weight-average M(r) (M omega) of 52,000 +/- 2000, which was in agreement with that derived from the amino acid sequence. By determining a sedimentation coefficient (S20,w0) of 3.35 +/- 0.10 S, it was possible to suggest a model for the gross solution conformation of pneumolysin monomers. Spectroscopic methods provide additional secondary and tertiary structure information.

scholarly journals Characterization of the Self Association ofAvian SarcomaVirus Integrase by Analytical Ultracentrifugation

1999 ◽  
Vol 274 (46) ◽  
pp. 32842-32846 ◽  
Author(s):  
Jacqueline Coleman ◽  
Steven Eaton ◽  
George Merkel ◽  
Anna Marie Skalka ◽  
Thomas Laue
Keyword(s):  
Analytical Ultracentrifugation ◽  
The Self ◽  
Self Association

scholarly journals Molecular characterization of the mitochondrial DNA of a new stopper mutant ER-3 of Neurospora crassa.

Genetics ◽  
1988 ◽  
Vol 120 (4) ◽  
pp. 935-945
Author(s):  
A Almasan ◽  
N C Mishra
Keyword(s):  
Mitochondrial Dna ◽  
Neurospora Crassa ◽  
Molecular Level ◽  
Wild Type ◽  
Type Molecule ◽  
Nd5 Gene ◽  
Nucleotide Repeats ◽  
Two Populations ◽  
Mitochondrial Chromosome

Abstract An ethidium bromide-induced stopper mutant of Neurospora crassa is characterized at the molecular level. The mutant has two populations of mitochondrial DNA: a defective predominant mutant molecule and a basal level of the wild-type molecule. The aberrant DNA resulted after a 25-kbp deletion from the wild-type mitochondrial chromosome, which included major genes such as cytb, co1 and oli2. The deletion endpoints are located in the second intron of the ND5 gene, and in a sequence 250 nucleotides upstream of the co2 gene. The recombination has taken place between two nine nucleotide repeats CCCCGCCCC, one of which is close to a PstI palindrome at its 5' end. Thus the mutant ER-3 differs from all the other stopper mutants described previously in the extent and location of the deletions in the mtDNA.

Electron Microscope Study of RNA's of Paramyxoviruses

1972 ◽  
Vol 30 ◽  
pp. 196-197
Author(s):  
Ruchama Baum ◽  
J.T. Seto
Keyword(s):  
Electron Microscopy ◽  
Electron Microscope ◽  
Electron Microscope Study ◽  
Sendai Virus ◽  
Sodium Dodecyl ◽  
Rna Molecules ◽  
Virus Particles ◽  
Molecular Weights ◽  
Length Measurements

The ribonucleic acid (RNA) of paramyxoviruses has been characterized by biochemical and physiochemical methods. However, paramyxovirus RNA molecules have not been studied by electron microscopy. The molecular weights of these single-stranded viral RNA molecules are not known as yet. Since electron microscopy has been found to be useful for the characterization of single-stranded RNA, this investigation was initiated to examine the morphology and length measurements of paramyxovirus RNA's.Sendai virus Z strain and Newcastle disease virus (NDV), Milano strain, were used. For these studies it was necessary to develop a method of extracting RNA molecules from purified virus particles. Highly purified Sendai virus was treated with pronase (300 μg/ml) at 37°C for 30 minutes and the RNA extracted by the sodium dodecyl sulfate (SDS)-phenol procedure.

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