The final two steps of de novo uridine 5-monophosphate (UMP) biosynthesis are catalyzed by orotate phosphoribosyltransferase (OPRT) and orotidine 5-monophosphate decarboxylase (OMPDC). In most prokaryotes and simple eukaryotes these two enzymes are encoded by separate genes, whereas in mammals they are expressed as a bifunctional gene product called UMP synthase (UMPS), with OPRT at the N terminus and OMPDC at the C terminus. Leishmania and some closely related organisms also express a bifunctional enzyme for these two steps, but the domain order is reversed relative to mammalian UMPS. In this work we demonstrate that L. donovani UMPS (LdUMPS) is an essential enzyme in promastigotes and that it is sequestered in the parasite glycosome. We also present the crystal structure of the LdUMPS in complex with its product, UMP. This structure reveals an unusual tetramer with two head to head and two tail to tail interactions, resulting in two dimeric OMPDC and two dimeric OPRT functional domains. In addition, we provide structural and biochemical evidence that oligomerization of LdUMPS is controlled by product binding at the OPRT active site. We propose a model for the assembly of the catalytically relevant LdUMPS tetramer and discuss the implications for the structure of mammalian UMPS.
Targeted gene replacement is a powerful tool in Leishmania genetics that can be time-consuming to implement. One tedious aspect that delays progress is the multi-step construction of gene targeting vectors. To accelerate this process, we developed a streamlined method that allows the assembly of a complete targeting vector from all its constituent parts in a single-step multifragment ligation. The individual components to be assembled are flanked by sites for the restriction endonuclease SfiI that generates nonidentical, non-palindromic three base 3'-overhangs designed to allow annealing and ligation of the parts only in the proper order. The method was optimized by generating constructs for targeting the Leishmania donovani inosine monophosphate dehydrogenase gene (LdIMPDH) encoding six different drug resistance markers, and was found to be rapid and efficient. These constructs were successfully employed to generate heterozygous LdIMPDH gene replacement mutants. This method is adaptable for generating targeting vectors for a variety of species. KeywordsLeishmania; gene targeting; gene knockout; homologous recombination; multi-fragment ligation; SfiI restriction endonuclease Targeted gene replacement via homologous recombination has been an invaluable tool for the genetic dissection of important metabolic and virulence pathways in Leishmania species [1], as well as for many other protozoan parasites [2]. The general experimental approach for the genetic manipulation of model organisms is essentially the same: DNA sequences of sufficient length to direct homologous recombination flanking the gene to be targetedreferred to herein as 5'-and 3'-targeting sequences (TS) -are independently isolated and joined to an alternative gene (i.e., drug resistance gene) that allows selection of cells in which the appropriate integration event has occurred. The most common method for generating leishmanial and other parasite gene targeting constructs involves the sequential cloning of 5'-TS and 3'-TS DNAs into a vector encoding a drug resistance cassette flanked by restriction sites [3]. This multi-step process can be time consuming and is complicated by the fact that commonly used vectors have limited restriction sites for TS insertion, exchange of drug resistance markers, and excision of the targeting cassette from the vector backbone * Corresponding Author. 3181 SW Sam Jackson Park Road, 7320 Richard T. Jones Hall, Portland,; yatesp@ohsu.edu. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. To overcome these many impediments to gene replacement in Leishmania, we have developed a...
embolization results in longer time-to-progression and reduced toxicity compared with chemoembolization in patients with hepatocellular carcinoma. Gastroenterology 2011;140:497-507. 12. Moreno-Luna LE, Yang JD, Sanchez W, et al. Efficacy and safety of transarterial radioembolization versus chemoembolization in patients with hepatocellular carcinoma. Cardiovasc Intervent Radiol 2013;36:714-723. 13. Salem R, Gordon AC, Mouli S, et al. Y90 Radioembolization significantly prolongs time to progression compared with chemoembolization in patients with hepatocellular carci
Kupffer cells (KC) are the resident macrophages that reside within the lumen of the liver sinusoid. Based on the origin there are two different sub sets of liver kupffer cells. The long-lived, radio-resistant subset also known as the sessile KC, derives from the progenitor cells generated during development in the yolk sac and maintains through self-renewal. The short-lived, radio-sensitive subset originates from hematopoietic stem cells. KCs play a major role in liver homeostasis, hepatic injury and induction of fibro-genesis as well as resolution of inflammation and fibrosis. Determination of the exact role of those ontogenetically different KC subpopulations in those critical liver functions is eluded by the inability to distinguish between the two subsets. Sessile KCs cannot be isolated and hence need an in situ method to study their gene expression. We propose to use the Ribo tag approach combined with Cre recombinase driven by a macrophage specific promoter (Emr1) to isolate actively translating polyribosomes using a ribo immunoprecipitation technique. The isolated KC subset-specific RNA will be used to analyze the transcriptome during both steady state conditions and liver inflammation.
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.