Tangier disease is characterized by low serum high density lipoproteins and a biochemical defect in the cellular efflux of lipids to high density lipoproteins. ABC1, a member of the ATP-binding cassette family, recently has been identified as the defective gene in Tangier
A two-step gene replacement procedure was developed that generates infectious adenoviral genomes through homologous recombination in Escherichia coli. As a prerequisite, a human adenovirus serotype 5 (Ad5)-derived genome was first introduced as a PacI restriction fragment into an incP-derived replicon which, in contrast to ColE1-derivatives (e.g., pBR322 or pUC plasmids), is functional in a polA mutant of E. coli. Any modification can be introduced at will following two consecutive homologous recombinations between the incP͞Ad5 replicon and the ColE1 plasmid. The overall procedure requires only the in vitro engineering of the ColE1-derivative by f lanking the desired modification with small stretches of identical sequences. In the first step, a cointegrate between the tetracycline-resistant incP͞Ad5 replicon and the kanamycin-resistant ColE1-derivative is selected by growing the polA host in the presence of both antibiotics. Resolution of this cointegrate is further selected in sucrose growth conditions due to the loss of a conditional suicide marker (the sacB gene of Bacillus subtilis) present in the ColE1 plasmid, leading to unmodified and modified incP͞Ad5 replicons that can be differentiated upon restriction analysis. Consecutive rounds of this two-step cloning procedure allowed the introduction of multiple independent modifications within the virus genome, with no requirement for an intermediate virus. The potential of this procedure is demonstrated by the recovery of several E1E3E4-deleted adenoviruses following transfection of the corresponding E. coli-derived genomes in IGRP2 cells.
Two genes involved in the biosynthesis of the depsipeptide antibiotics pristinamycins I (PI) produced by Streptomyces pristinaespiralis were cloned and sequenced. The 1.7-kb snbA gene encodes a 3-hydroxypicolinic acid:AMP ligase, and the 7.7-kb snbC gene encodes PI synthetase 2, responsible for incorporating L-threonine and L-aminobutyric acid in the PI macrocycle. snbA and snbC, which encode the two first structural enzymes of PI synthesis, are not contiguous. Both genes are located in PI-specific transcriptional units, as disruption of one gene or the other led to PI-deficient strains producing normal levels of the polyunsaturated macrolactone antibiotic pristinamycin II, also produced by S. pristinaespiralis. Analysis of the deduced amino acid sequences showed that the SnbA protein is a member of the adenylate-forming enzyme superfamily and that the SnbC protein contains two amino acid-incorporating modules and a C-terminal epimerization domain. A model for the initiation of PI synthesis analogous to the established model of initiation of fatty acid synthesis is proposed.Pristinamycins I (PI), cyclohexadepsipeptide antibiotics produced by Streptomyces pristinaespiralis, are members of the streptogramin B group. They are coproduced with the polyunsaturated macrolactone antibiotics pristinamycins II (PII), members of the streptogramin A group (6, 52). Like many other small metabolites with a peptide structure, PI are synthesized nonribosomally by large multifunctional enzymes (49). More than 25 years ago, Lipmann emphasized the analogy between nonribosomal peptide synthesis and fatty acid synthesis (25). In his model, large multifunctional enzymes called peptide synthetases (PPSs) catalyze elongation of a peptide covalently linked to a phosphopantetheinyl arm by a thioester bond (for a review, see reference 23). The original version of the thiotemplate multienzymatic mechanism was revised recently, and a multiple-carrier model was proposed (39,43,46,53). According to this model, each amino acid is activated as an aminoacyl adenylate and linked to the enzyme as a thioester with a phosphopantetheinyl group. Elongation then occurs by transfer of the activated carboxyl to the amino group of the next amino acid, thus effecting N-to-C stepwise condensation. Primary structure analysis of several PPS genes resulted in the identification of approximately 1,000-aminoacid (aa)-long modules selectively catalyzing activation and condensation of one amino or hydroxy acid (30, 51). The modules are organized such that they are colinear with the sequence of the oligopeptide. Each module can be subdivided in domains. The activation domain (500 aa) belongs to the large family of adenylate-forming enzymes that includes firefly luciferase and acyl coenzyme A (acyl-CoA) synthetases and contains nine core sequences (named boxes A to I by Pfeifer et al.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.