scholarly journals Disruption of hmgA by DNA Duplication is Responsible for Hyperpigmentation in a Vibrio anguillarum Strain

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Veronica Batallones ◽  
Jennifer Fernandez ◽  
Brett Farthing ◽  
Jordan Shoemaker ◽  
Keizen Li Qian ◽  
...  
Keyword(s):  
Vibrio Anguillarum ◽  
Early Termination ◽  
Whole Genome Sequence ◽  
Brown Pigment ◽  
Insertion Sequences ◽  
Genome Sequence Analysis ◽  
Recombinant Clone ◽  
Tyrosine Metabolism ◽  
Intergenic Regions ◽  
Diseased Fish

Abstract Vibrio anguillarum 531A, isolated from a diseased fish in the Atlantic Ocean, is a mixture composed of about 95 and 5% of highly pigmented cells (strain 531Ad) and cells with normal levels of pigmentation (strain 531Ac), respectively. Analysis of the V. anguillarum 531Ad DNA region encompassing genes involved in the tyrosine metabolism showed a 410-bp duplication within the hmgA gene that results in a frameshift and early termination of translation of the homogentisate 1,2-dioxygenase. We hypothesized that this mutation results in accumulation of homogentisate that is oxidized and polymerized to produce pyomelanin. Introduction in E. coli of recombinant clones carrying the V. anguillarum hppD (4-hydroxyphenylpyruvate-dioxygenase), and a mutated hmgA produced brown colored colonies. Complementation with a recombinant clone harboring hmgA restored the original color to the colonies confirming that in the absence of homogentisate 1,2-dioxygenase the intermediary in tyrosine catabolism homogentisate accumulates and undergoes nonenzymatic oxidation and polymerization resulting in high amounts of the brown pigment. Whole-genome sequence analysis showed that V. anguillarum 531 Ac and 531Ad differ in the hmgA gene mutation and 23 mutations, most of which locate to intergenic regions and insertion sequences.

scholarly journals Non-Cleavage Site Gag Mutations in Amprenavir-Resistant Human Immunodeficiency Virus Type 1 (HIV-1) Predispose HIV-1 to Rapid Acquisition of Amprenavir Resistance but Delay Development of Resistance to Other Protease Inhibitors

2009 ◽  
Vol 83 (7) ◽  
pp. 3059-3068 ◽  
Author(s):  
Manabu Aoki ◽  
David J. Venzon ◽  
Yasuhiro Koh ◽  
Hiromi Aoki-Ogata ◽  
Toshikazu Miyakawa ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Wild Type ◽  
Recombinant Clone ◽  
Rapid Acquisition ◽  
Development Of Resistance ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT In an attempt to determine whether mutations in Gag in human immunodeficiency virus type 1 (HIV-1) variants selected with a protease inhibitor (PI) affect the development of resistance to the same or a different PI(s), we generated multiple infectious HIV-1 clones carrying mutated Gag and/or mutated protease proteins that were identified in amprenavir (APV)-selected HIV-1 variants and examined their virological characteristics. In an HIV-1 preparation selected with APV (33 passages, yielding HIVAPVp33), we identified six mutations in protease and six apparently critical mutations at cleavage and non-cleavage sites in Gag. An infectious recombinant clone carrying the six protease mutations but no Gag mutations failed to replicate, indicating that the Gag mutations were required for the replication of HIVAPVp33. An infectious recombinant clone that carried wild-type protease and a set of five Gag mutations (rHIVWTpro 12/75/219/390/409gag) replicated comparably to wild-type HIV-1; however, when exposed to APV, rHIVWTpro 12/75/219/390/409gag rapidly acquired APV resistance. In contrast, the five Gag mutations significantly delayed the acquisition of HIV-1 resistance to ritonavir and nelfinavir (NFV). Recombinant HIV-1 clones containing NFV resistance-associated mutations, such as D30N and N88S, had increased susceptibilities to APV, suggesting that antiretroviral regimens including both APV and NFV may bring about favorable antiviral efficacy. The present data suggest that the preexistence of certain Gag mutations related to PI resistance can accelerate the emergence of resistance to the PI and delay the acquisition of HIV resistance to other PIs, and these findings should have clinical relevance in the therapy of HIV-1 infection with PI-including regimens.

scholarly journals Simian-Human Immunodeficiency Virus Containing a Human Immunodeficiency Virus Type 1 Subtype-E Envelope Gene: Persistent Infection, CD4+ T-Cell Depletion, and Mucosal Membrane Transmission in Macaques

2000 ◽  
Vol 74 (17) ◽  
pp. 7851-7860 ◽  
Author(s):  
Sunee Himathongkham ◽  
Nancy S. Halpin ◽  
Jinling Li ◽  
Michael W. Stout ◽  
Christopher J. Miller ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
T Cell ◽  
Nonhuman Primate ◽  
Coreceptor Usage ◽  
Nonhuman Primate Model ◽  
Primate Model ◽  
Recombinant Clone ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT The envelope (env) glycoprotein of human immunodeficiency virus type 1 (HIV-1) determines several viral properties (e.g., coreceptor usage, cell tropism, and cytopathicity) and is a major target of antiviral immune responses. Most investigations on env have been conducted on subtype-B viral strains, prevalent in North America and Europe. Our study aimed to analyze env genes of subtype-E viral strains, prevalent in Asia and Africa, with a nonhuman primate model for lentivirus infection and AIDS. To this end, we constructed a simian immunodeficiency virus/HIV-1 subtype-E (SHIV) recombinant clone by replacing the env ectodomain of the SHIV-33 clone with theenv ectodomain from the subtype-E strain HIV-1CAR402, which was isolated from an individual in the Central African Republic. Virus from this recombinant clone, designated SHIV-E-CAR, replicated efficiently in macaque peripheral blood mononuclear cells. Accordingly, juvenile macaques were inoculated with cell-free SHIV-E-CAR by the intravenous or intravaginal route; virus replicated in these animals but did not produce hematological abnormalities. In an attempt to elicit the pathogenic potential of the recombinant clone, we serially passaged this viral clone via transfusion of blood and bone marrow through juvenile macaques to produce SHIV-E-P4 (fourth-passage virus). The serially passaged virus established productive infection and CD4+ T-cell depletion in juvenile macaques inoculated by either the intravenous or the intravaginal route. Determination of the coreceptor usage of SHIV-E-CAR and serially passaged SHIV-E-P4 indicated that both of these viruses utilized CXCR4 as a coreceptor. In summary, the serially passaged SHIV subtype-E chimeric virus will be important for studies aimed at developing a nonhuman primate model for analyzing the functions of subtype-E env genes in viral transmission and pathogenesis and for vaccine challenge experiments with macaques immunized with HIV-1 env antigens.

A noncommercial polymerase chain reaction–based method to approach one hundred percent recombinant clone selection efficiency

2008 ◽  
Vol 382 (1) ◽  
pp. 75-76 ◽  
Author(s):  
Mohammed M. Shareef ◽  
Horatiu C. Dancea ◽  
Jessica L. Gross ◽  
Tamara T. Myers ◽  
Wendy W. Griggs ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Selection Efficiency ◽  
Chain Reaction ◽  
Recombinant Clone ◽  
Clone Selection ◽  
Polymerase Chain

scholarly journals Constitutively expressed rat mRNA encoding a 70-kilodalton heat-shock-like protein

1985 ◽  
Vol 5 (12) ◽  
pp. 3476-3483
Author(s):  
K O'Malley ◽  
A Mauron ◽  
J D Barchas ◽  
L Kedes
Keyword(s):  
Heat Shock ◽  
Cdna Clone ◽  
Sequence Similarity ◽  
Normal Growth ◽  
Antigenic Determinants ◽  
Related Sequence ◽  
Recombinant Clone ◽  
Hsp70 Protein ◽  
Pheochromocytoma Cell ◽  
Catecholamine Synthesizing Enzymes

A nearly full-length cDNA clone isolated from the rat pheochromocytoma cell line, PC12, revealed extensive nucleotide sequence similarity between the rat cDNA and the Drosophila melanogaster hsp70 gene. The rat recombinant clone encodes a 71,000-dalton protein that is 70% identical with the dipteran hsp70 protein. Remarkably, a truncated segment of this cDNA clone was originally isolated by immunoreactivity with antisera raised to catecholamine-synthesizing enzymes, suggesting that this heat shock protein and these catecholamine enzymes shared antigenic determinants. The rat hsp70-related mRNA is responsible for the production of a constitutive hsp70 protein, because it is present in abundant amounts in various tissues at normal growth temperatures and is only minimally induced by hyperthermia. The rat hsp70-related sequence is part of a multigene family that extends across species to mice and humans.

Cloning, over-expression and evaluation of a recombinant fusion protein of Wuchereria bancrofti towards its application as a diagnostic agent for bancroftian filariasis

Parasitology ◽  
1993 ◽  
Vol 106 (4) ◽  
pp. 413-420 ◽  
Author(s):  
J. G. Theodore ◽  
P. Kaliraj ◽  
S. Jayachandran ◽  
K. Jayaraman
Keyword(s):  
Fusion Protein ◽  
Genomic Library ◽  
Wuchereria Bancrofti ◽  
Specific Ige ◽  
Western Blots ◽  
Setaria Digitata ◽  
Recombinant Clone ◽  
Over Expression ◽  
Specific Igg ◽  
Diagnostic Agent

SUMMARYA low molecular weight (15 kDa) surface antigen of the cattle filarial nematode, Setaria digitata, was earlier shown to be specifically recognized by the antibodies from human bancroftian filarial (Mf positive) patients' sera (Theodore & Kaliraj, 1990). The filarial specific antibodies bound to a 15 kDa peptide in preparative Western blots were eluted and employed in screening of candidate antigens expressed in the genomic library of Wuchereria bancrofti at the IgG4 subclass antibody level. A recombinant clone (λ WbG7) reacting strongly with filarial sera but poorly with sera from patients infected with non-filarial helminths was selected for further studies. The 2 kb DNA insert of the clone λ WbG7 was recloned into a pMAL vector and the recombinant clone pGT7 thus obtained was over-expressed and affinity purified. The purified 105 kDa fusion protein of the clone pGT7 was specific and was not recognized by the non-filarial sera at the IgG4 level. All microfilaraemic individuals were positive by IgG4 assay. However, similar attempts to diagnose by filarial-specific IgE assays failed to recognize microfilaraemic individuals. Moreover, by filarial-specific IgG4 assays, the endemic normals were distinctly divided into two groups, showing higher and lower recognition for this antigen indicating current and past/no infection. Among the filarial-IgG4 (assay)-positive ‘endemic normals’, 14% showed ‘microfilariae’ during repeated peripheral night blood examination, confirming the validity of the recombinant antigen, pGT7 based assay.

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