Determinants of adenine-mutagenesis in diversity-generating retroelements

Abstract Diversity-generating retroelements (DGRs) vary protein sequences to the greatest extent known in the natural world. These elements are encoded by constituents of the human microbiome and the microbial ‘dark matter’. Variation occurs through adenine-mutagenesis, in which genetic information in RNA is reverse transcribed faithfully to cDNA for all template bases but adenine. We investigated the determinants of adenine-mutagenesis in the prototypical Bordetella bacteriophage DGR through an in vitro system composed of the reverse transcriptase bRT, Avd protein, and a specific RNA. We found that the catalytic efficiency for correct incorporation during reverse transcription by the bRT-Avd complex was strikingly low for all template bases, with the lowest occurring for adenine. Misincorporation across a template adenine was only somewhat lower in efficiency than correct incorporation. We found that the C6, but not the N1 or C2, purine substituent was a key determinant of adenine-mutagenesis. bRT-Avd was insensitive to the C6 amine of adenine but recognized the C6 carbonyl of guanine. We also identified two bRT amino acids predicted to nonspecifically contact incoming dNTPs, R74 and I181, as promoters of adenine-mutagenesis. Our results suggest that the overall low catalytic efficiency of bRT-Avd is intimately tied to its ability to carry out adenine-mutagenesis.

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Determinants of Adenine-mutagenesis in Diversity-Generating Retroelements

10.1101/2020.04.29.068544 ◽

2020 ◽

Author(s):

Sumit Handa ◽

Andres Reyna ◽

Timothy Wiryaman ◽

Partho Ghosh

Keyword(s):

Amino Acids ◽

Dark Matter ◽

Reverse Transcriptase ◽

Reverse Transcription ◽

Genetic Information ◽

Human Microbiome ◽

Protein Sequences ◽

Catalytic Efficiency ◽

Natural World

ABSTRACTDiversity-generating retroelements (DGRs) vary protein sequences to the greatest extent known in the natural world. These elements are encoded by constituents of the human microbiome and the microbial ‘dark matter’. Variation occurs through adenine-mutagenesis, in which genetic information in RNA is reverse transcribed faithfully to cDNA for all template bases but adenine. We investigated the determinants of adenine-mutagenesis in the prototypical Bordetella bacteriophage DGR through an in vitro system composed of the reverse transcriptase bRT, Avd protein, and a specific RNA. We found that the catalytic efficiency for correct incorporation during reverse transcription by the bRT-Avd complex was strikingly low for all template bases, with the lowest occurring for adenine. Misincorporation across a template adenine was only somewhat lower in efficiency than correct incorporation. We found that the C6, but not the N1 or C2, purine substituent was a key determinant of adenine-mutagenesis. bRT-Avd was insensitive to the C6 amine of adenine but recognized the C6 carbonyl of guanine. We also identified two bRT amino acids predicted to nonspecifically contact incoming dNTPs, R74 and I181, as promoters of adenine-mutagenesis. Our results suggest that the overall low catalytic efficiency of bRT-Avd is intimately tied to its ability to carry out adenine-mutagenesis.

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Computational approach for selection of epitope-based dengue vaccine targets

Vietnam Journal of Biotechnology ◽

10.15625/1811-4989/14/4/12244 ◽

2018 ◽

Vol 14 (4) ◽

pp. 605-618

Author(s):

Phuc Nguyen ◽

Ly Le

Keyword(s):

Amino Acids ◽

T Cell ◽

Dengue Virus ◽

Rational Design ◽

Protein Sequences ◽

Computational Approach ◽

Amino Acid Sequences ◽

Universal Vaccine ◽

E Protein

High antigenic variability in the envelope (E) protein of different virus strains has been a major obstacle in designing effective vaccines for Dengue virus (DENV). To maintain their biological function, some parts of viral proteins remain stable during evolution thus one possible approach to solve this problem is to recognize specific regions within different protein sequences of E that have the tendency to stay constant through evolution. These regions may possess some special attributes to become a vaccine candidate against dengue virus. In this study, a computational approach was utilized to identify and analyze highly conserved amino acid sequences of the DENV E protein. Sequences of 9 amino acids or more were specifically focused due to their immune-relevant as T-cell determinants. Different bioinformatics tools were responsible for revealing conserved regions in the DENV E protein and constructing the phylogenetic tree from the sequence database. The tools also predicted immunogenicity of the identified vaccine targets. Ultimately, two peptide regions of at least 9 amino acids were chosen due to their high conserved attribute in more than 95% of all collected DENV sequences. Moreover, both of them was found to be immune-relevant by their correspondence to known or putative HLA-restricted T cell determinants. The conserved attribute of these sequences through the entire analysis of this study supports their potential as candidates for further in vitro experiments for rational design a universal vaccine which has longer and broader impact.

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Inducible degradation of I kappa B alpha in vitro and in vivo requires the acidic C-terminal domain of the protein.

Molecular and Cellular Biology ◽

10.1128/mcb.15.5.2413 ◽

1995 ◽

Vol 15 (5) ◽

pp. 2413-2419 ◽

Cited By ~ 66

Author(s):

M S Rodriguez ◽

I Michalopoulos ◽

F Arenzana-Seisdedos ◽

R T Hay

Keyword(s):

Amino Acids ◽

Proteolytic Activity ◽

Cell Activation ◽

Nf Kappa B ◽

Free System ◽

In Vitro System ◽

I Kappa B Alpha ◽

Terminal Domain

After exposure of cells to tumor necrosis factor (TNF), I kappa B alpha is rapidly degraded by a proteolytic activity that is required for nuclear localization and activation of transcription factor NF-kappa B. To investigate this problem, we have developed a cell-free system to study the degradation of I kappa B alpha initiated in vivo. In this in vitro system, characteristics of endogenous I kappa B alpha degradation were comparable to those observed in vivo. Recombinant I kappa B alpha, when added to lysates from cells exposed to TNF, was specifically degraded by a cellular proteolytic activity; however, it was stable in extracts from unstimulated cells. Inhibition characteristics of the proteolytic activity responsible for I kappa B alpha degradation suggest the involvement of a serine protease. Analysis of mutated forms of I kappa B alpha in the in vitro system demonstrated that an I kappa B alpha species which was unable to interact with NF-kappa B was still efficiently degraded. In contrast, deletion of the C-terminal 61 amino acids from I kappa B alpha rendered the protein resistant to proteolytic degradation. Expression of I kappa B alpha mutated forms in COS-7 cells confirmed the importance of the C-terminal domain for the degradation of the protein in vivo following cell activation. Thus, it is likely that the acidic, negatively charged region represented by the C-terminal 61 amino acids of the protein contains residues critical for TNF-inducible degradation of I kappa B alpha.

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THE BIOSYNTHESIS OF THYROXINE: INCORPORATION OF [U-14C]TYROSINE INTO THYROGLOBULIN BY MOUSE THYROID GLANDS IN VIVO AND IN VITRO

Journal of Endocrinology ◽

10.1677/joe.0.0560173 ◽

1973 ◽

Vol 56 (2) ◽

pp. 173-185 ◽

Cited By ~ 3

Author(s):

W. H. WAIN

Keyword(s):

Amino Acids ◽

Thyroid Lobe ◽

In Vitro System ◽

Electrophoretic Separations ◽

Thyroid Glands ◽

Mouse Thyroid

SUMMARY The incorporation in vivo of [U-14C]tyrosine into 19S thyroglobulin by mice was achieved to a level of 2·85 c.p.m./μg. This level of incorporation was insufficient to permit the isolation of 14C-labelled iodinated tyrosines or residues. Isolated mouse thyroid lobes were used as an in-vitro system for the synthesis of 19S thyroglobulin. The lobes continued to incorporate 131I into 19S thyroglobulin for at least 48 h and this incorporation of iodine was specifically inhibited by propylthiouracil. The isolated mouse thyroid lobe in-vitro system was used to incorporate 14C-labelled amino acids into 19S thyroglobulin. [U-14C]Tyrosine was incorporated to a level of 1150 c.p.m./μg. Electrophoretic separations of enzymic hydrolysates of [14C]tyrosine-labelled 19S thyroglobulin showed the presence of [14C]tyrosine, [14C]monoiodotyrosine, [14C]di-iodotyrosine and [14C]thyroxine. The presence of [14C]tyrosine, [14C]monoiodotyrosine and [14C]thyroxine was demonstrated by chromatography of the eluates from the electrophoretic separations. The results provide evidence for the utilization of tyrosyl residues within the thyroglobulin molecule for iodination and subsequent coupling to form thyroxine.

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In Vitro Assay of Platelet Adhesiveness with Washed and Tanned Human Red Cells

Thrombosis and Haemostasis ◽

10.1055/s-0038-1651281 ◽

1968 ◽

Vol 20 (03/04) ◽

pp. 384-396 ◽

Cited By ~ 1

Author(s):

G Zbinden ◽

S Tomlin

Keyword(s):

Platelet Adhesion ◽

Sodium Citrate ◽

Blood Platelets ◽

In Vitro Assay ◽

Red Cells ◽

Platelet Adhesiveness ◽

Vitro Assay ◽

In Vitro System ◽

Human Red Cells

SummaryAn in vitro system is described in which adhesion of blood platelets to washed and tannic acid-treated red cells was assayed quantitatively by microscopic observation. ADP, epinephrine and TAME produced a reversible increase in platelet adhesiveness which was antagonized by AMP. With Evans blue, polyanetholsulfonate, phthalanilide NSC 38280, thrombin and heparin at concentrations above 1-4 u/ml the increase was irreversible. The ADP-induced increase in adhesiveness was inhibited by sodium citrate, EDTA, AMP, ATP and N-ethylmaleimide. EDTA, AMP and the SH-blocker N-ethylmaleimide also reduced spontaneous platelet adhesion to red cells. No significant effects were observed with adenosine, phenprocoumon, 5-HT, phthalanilide NSC 57155, various estrogens, progestogens and fatty acids, acetylsalicylic acid and similarly acting agents, hydroxylamine, glucose and KCN. The method may be useful for the screening of thrombogenic and antithrombotic properties of drugs.

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The Mesothelial Cell as a Non-Thrombogenic Surface

Thrombosis and Haemostasis ◽

10.1055/s-0038-1661149 ◽

1984 ◽

Vol 52 (02) ◽

pp. 102-104 ◽

Cited By ~ 25

Author(s):

L J Nicholson ◽

J M F Clarke ◽

R M Pittilo ◽

S J Machin ◽

N Woolf

Keyword(s):

Electron Microscopy ◽

Scanning Electron Microscopy ◽

Blood Flow ◽

Cell Surface ◽

Mesothelial Cell ◽

Mesothelial Cells ◽

Plastic Film ◽

In Vitro System ◽

Scanning Electron

SummaryA technique for harvesting mesothelial cells is described. This entails collagenase digestion of omentum after which the cells can be cultured. The technique has been developed using the rat, but has also been successfully applied to human tissue. Cultured rat mesothelial cells obtained in this way have been examined by scanning electron microscopy. Rat mesothelial cells grown on plastic film have been exposed to blood in an in vitro system using a Baumgartner chamber and have been demonstrated to support blood flow. No adhering platelets were observed on the mesothelial cell surface. Fibroblasts similarily exposed to blood as a control were washed off the plastic.

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