Extensive loss of cell-cycle and DNA repair genes in an ancient lineage of bipolar budding yeasts

Cell-cycle checkpoints and DNA repair processes protect organisms from potentially lethal mutational damage. Compared to other budding yeasts in the subphylum Saccharomycotina, we noticed that a lineage in the genus Hanseniaspora exhibited very high evolutionary rates, low Guanine–Cytosine (GC) content, small genome sizes, and lower gene numbers. To better understand Hanseniaspora evolution, we analyzed 25 genomes, including 11 newly sequenced, representing 18/21 known species in the genus. Our phylogenomic analyses identify two Hanseniaspora lineages, a faster-evolving lineage (FEL), which began diversifying approximately 87 million years ago (mya), and a slower-evolving lineage (SEL), which began diversifying approximately 54 mya. Remarkably, both lineages lost genes associated with the cell cycle and genome integrity, but these losses were greater in the FEL. E.g., all species lost the cell-cycle regulator WHIskey 5 (WHI5), and the FEL lost components of the spindle checkpoint pathway (e.g., Mitotic Arrest-Deficient 1 [MAD1], Mitotic Arrest-Deficient 2 [MAD2]) and DNA-damage–checkpoint pathway (e.g., Mitosis Entry Checkpoint 3 [MEC3], RADiation sensitive 9 [RAD9]). Similarly, both lineages lost genes involved in DNA repair pathways, including the DNA glycosylase gene 3-MethylAdenine DNA Glycosylase 1 (MAG1), which is part of the base-excision repair pathway, and the DNA photolyase gene PHotoreactivation Repair deficient 1 (PHR1), which is involved in pyrimidine dimer repair. Strikingly, the FEL lost 33 additional genes, including polymerases (i.e., POLymerase 4 [POL4] and POL32) and telomere-associated genes (e.g., Repressor/ activator site binding protein-Interacting Factor 1 [RIF1], Replication Factor A 3 [RFA3], Cell Division Cycle 13 [CDC13], Pbp1p Binding Protein [PBP2]). Echoing these losses, molecular evolutionary analyses reveal that, compared to the SEL, the FEL stem lineage underwent a burst of accelerated evolution, which resulted in greater mutational loads, homopolymer instabilities, and higher fractions of mutations associated with the common endogenously damaged base, 8-oxoguanine. We conclude that Hanseniaspora is an ancient lineage that has diversified and thrived, despite lacking many otherwise highly conserved cell-cycle and genome integrity genes and pathways, and may represent a novel, to our knowledge, system for studying cellular life without them.Fil: Steenwyk, Jacob L.. Vanderbilt University; Estados UnidosFil: Opulente, Dana A.. University of Wisconsin; Estados UnidosFil: Kominek, Jacek. University of Wisconsin; Estados UnidosFil: Shen, Xing-Xing. Vanderbilt University; Estados UnidosFil: Zhou, Xiaofan. South China Agricultural University; ChinaFil: Labella, Abigail L.. Vanderbilt University; Estados UnidosFil: Bradley, Noah P.. Vanderbilt University; Estados UnidosFil: Eichman, Brandt F.. Vanderbilt University; Estados UnidosFil: Cadez, Neza. University of Ljubljana; EsloveniaFil: Libkind Frati, Diego. Universidad Nacional del Comahue. Centro Regional Universitario Bariloche; ArgentinaFil: DeVirgilio, Jeremy. United States Department of Agriculture. Agricultural Research Service; ArgentinaFil: Hulfachor, Amanda Beth. University of Wisconsin; Estados UnidosFil: Kurtzman, Cletus P.. United States Department of Agriculture. Agricultural Research Service; ArgentinaFil: Hittinger, Chris Todd. University of Wisconsin; Estados UnidosFil: Rokas, Antonis. Vanderbilt University; Estados Unido

Recombinant human GAD65 accumulates to high levels in transgenic tobacco plants when expressed as an enzymatically inactive mutant.

Summary The 65-kDa isoform of glutamic acid decarboxylase (GAD65) is the major autoantigen implicated in the development of type 1 diabetes mellitus (T1DM). The bulk manufacture of GAD65 is a potential issue in the fight against T1DM but current production platforms are expensive. We show that a catalytically inactive form of GAD65 (GAD65mut) accumulates at up to 2.2\% total soluble protein in transgenic tobacco leaves, which is more than 10-fold the levels achieved with active GAD65, yet the protein retains the immunogenic properties required to treat T1DM. This higher yield was found to be a result of a higher rate of protein synthesis and not transcript availability or protein stability. We found that targeting GAD65 to the endoplasmic reticulum, a strategy that increases the accumulation of many recombinant proteins expressed in plants, did not improve production of GAD65mut. The production of a catalytically inactive autoantigen that retains its immunogenic properties could be a useful strategy to provide high-quality therapeutic protein for treatment of autoimmune T1DM

The proteomics of quiescent and nonquiescent cell differentiation in yeast stationary-phase cultures

Starved yeast cultures differentiate into quiescent (Q) and nonquiescent (NQ) cell fractions. The yeast GFP-fusion library (4159 strains) and high-throughput flow cytometry were used to study this process. This showed significant metabolic and physiologic differences between Q/NQ cells and provided new tools for studying their differentiation