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Molecular Genetic Approaches to Obesity
Published in Claude Bouchard, The Genetics of Obesity, 2020
Streamson C. Chua, Rudolph L. Leibel
YACs are generated by the ligation of large fragments (0.3 to 2 Mb) of genomic DNA to two vector arms which contain elements needed for DNA replication in yeast cells. These elements include a centromere, two telomeres, and a sequence equivalent to an origin of replication. Also included in the vector arms are genes (URA3, TRP1 [auxotrophic mutations which confer necessity of uracil and tryptophan, respectively, for growth]) for selection in transformed yeast and an ampicillin resistance cassette for selection in Escherichia coli. Yeast spheroplasts are transformed with these YACs, and the URA3 and TRP1 genes are used to enrich the resulting yeast pool for transformants by growth in appropriate media. The YAC replicates in synchrony with the normal chromosomes of Saccharomyces cerevisiae so that one copy is made per yeast cell.
Generation and engineering of potent single domain antibody-based bispecific IL-18 mimetics resistant to IL-18BP decoy receptor inhibition
Published in mAbs, 2023
Britta Lipinski, Laura Unmuth, Paul Arras, Stefan Becker, Christina Bauer, Lars Toleikis, Simon Krah, Achim Doerner, Desislava Yanakieva, Ammelie Svea Boje, Katja Klausz, Matthias Peipp, Vanessa Siegmund, Andreas Evers, Harald Kolmar, Lukas Pekar, Stefan Zielonka
For antibody surface display, yeast Saccharomyces cerevisiae strain EBY100 (MATa URA3–52 trp1 leu2Δ1 his3Δ200 pep4:HIS3 prb1Δ1.6 R can1 GAL (pIU211:URA3)) (Thermo Fisher Scientific) was used. Cells were cultivated in yeast extract – peptone–dextrose (YPD) medium composed of 10 g/L yeast extract, 20 g/L peptone and 20 g/L dextrose, additionally supplemented with 10 mg/mL penicillin – streptomycin (Gibco). Cells harboring the library plasmids (pDisp) after gap repair cloning were cultivated in minimal synthetic defined (SD)-base (Takara Bio) medium supplemented with 5.4 g/L Na2HPO4 and 8.6 g/L NaH2PO4 H2O, also comprising the corresponding dropout mix (Takara Bio) composed of all essential amino acids except for tryptophan (−Trp) for selection. To induce antibody gene expression, dextrose was replaced by galactose as carbon source. Thus, cells were transferred into SG dropout medium (−Trp) consisting of SG-base medium (Takara Bio) and 10% (w/v) polyethylene glycol 8000 (PEG 8000).
High iron-mediated increased oral fungal burden, oral-to-gut transmission, and changes to pathogenicity of Candida albicans in oropharyngeal candidiasis
Published in Journal of Oral Microbiology, 2022
Aparna Tripathi, Anubhav Nahar, Rishabh Sharma, Trevor Kanaskie, Nezar Al-Hebshi, Sumant Puri
C. albicans CAI4 (Ura+) strain (Δura3::imm434/Δura3::imm434RPS1/Δrps1::Clp10-URA3) [2] and WT SC5314 strain (ATCC, MYA-2876™) were used, along with C. albicans oral isolates. These clinical isolates were collected over the past 2 years from the dental clinics at the Temple University, and strain identification was performed at the Oral Microbiome Research Laboratory, initially using Chrom AgarTM (BD), followed by molecular characterization, as described in the Supplemental section (Figure S2 and Supplemental methods). Isolates from de-identified yeast extract-peptone-dextrose (YPD; Difco) plates were regrown in YPD and stored at −80°C as glycerol stocks. C. albicans exponential-phase cells were prepared as described previously [16] in yeast nitrogen base (YNB; 4027–112; MP Biomedicals) limited iron medium as low iron medium or the same with the addition of 50 µM bathophenanthrolinedisulfonic acid (iron-chelator) and 100 µM FeCl3.6H2O as replete iron medium (or high iron medium) [18]. For antifungal sensitivity assays, uridine (50 μg ml−1) containing YNB or YPD agar plates were used. For experiments involving animals, 4 to 6 weeks old C57BL/6 female mice from Jackson Labs, Bar Harbor, ME, were used.
Effect of low frequency magnetic field on efficiency of chromosome break repair
Published in Electromagnetic Biology and Medicine, 2020
Antonio M. Burgos-Molina, Silvia Mercado-Sáenz, Francisco Sendra-Portero, Miguel J. Ruiz-Gómez
S. cerevisiae strain AF2082 contains a 40 bp sequence from the mating type locus, MAT, including the cutting site for the HO endonuclease (HOcs), inserted into de URA3 gene on chromosome V. It also contains the gene encoding the HO endonuclease under control of a galactose-inducible promoter, inserted into the ADE3 locus. Upon growth of the cells in galactose-containing media, this endonuclease is expressed and introduces a staggered DSB at the HOcs. Because of the absence of a homologous donor sequence, this DSB has to be repaired by non-homologous end joining. Pho91 and Rmd5 derivatives of AF2082 contain a second DSB site generated by a pair of HOcs sequences flanking a selectable marker gene (TRP1) which confers tryptophane prototrophy. Successful cutting can thus be verified by loss of this marker gene and a tryptophane auxotrophic phenotype.