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      Modeling in yeast how rDNA introns slow growth and increase desiccation tolerance in lichens

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          Abstract

          We connect ribosome biogenesis to desiccation tolerance in lichens, widespread symbioses between specialized fungi (mycobionts) and unicellular phototrophs. We test whether the introns present in the nuclear ribosomal DNA of lichen mycobionts contribute to their anhydrobiosis. Self-splicing introns are found in the rDNA of several eukaryotic microorganisms, but most introns populating lichen rDNA are unable to self-splice, being either catalytically impaired group I introns, or spliceosomal introns ectopically present in rDNA. Although the mycobiont’s splicing machinery removes all introns from rRNA, Northern analysis indicates delayed post-transcriptional removal during rRNA processing, suggesting interference with ribosome assembly. To study the effects of lichen introns in a model system, we used CRISPR to introduce a spliceosomal rDNA intron from the lichen fungus Cladonia grayi into all nuclear rDNA copies of Saccharomyces cerevisiae, which lacks rDNA introns. Three intron-bearing yeast mutants were constructed with the intron inserted either in the 18S rRNA genes, the 25S rRNA genes, or in both. The mutants removed the introns correctly but had half the rDNA genes of the wildtype, grew 4.4–6 times slower, and were 40–1700 times more desiccation tolerant depending on intron position and number. Intracellular trehalose, a disaccharide implicated in desiccation tolerance, was detected at low concentration. Our data suggest that the interference of the splicing machinery with ribosome assembly leads to fewer ribosomes and proteins and to slow growth and increased desiccation tolerance in the yeast mutants. The relevance of these findings for slow growth and desiccation tolerance in lichens is discussed.

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          A new mathematical model for relative quantification in real-time RT-PCR.

          M. Pfaffl (2001)
          Use of the real-time polymerase chain reaction (PCR) to amplify cDNA products reverse transcribed from mRNA is on the way to becoming a routine tool in molecular biology to study low abundance gene expression. Real-time PCR is easy to perform, provides the necessary accuracy and produces reliable as well as rapid quantification results. But accurate quantification of nucleic acids requires a reproducible methodology and an adequate mathematical model for data analysis. This study enters into the particular topics of the relative quantification in real-time RT-PCR of a target gene transcript in comparison to a reference gene transcript. Therefore, a new mathematical model is presented. The relative expression ratio is calculated only from the real-time PCR efficiencies and the crossing point deviation of an unknown sample versus a control. This model needs no calibration curve. Control levels were included in the model to standardise each reaction run with respect to RNA integrity, sample loading and inter-PCR variations. High accuracy and reproducibility (<2.5% variation) were reached in LightCycler PCR using the established mathematical model.
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            Genomic Expression Programs in the Response of Yeast Cells to Environmental Changes

            We explored genomic expression patterns in the yeast Saccharomyces cerevisiae responding to diverse environmental transitions. DNA microarrays were used to measure changes in transcript levels over time for almost every yeast gene, as cells responded to temperature shocks, hydrogen peroxide, the superoxide-generating drug menadione, the sulfhydryl-oxidizing agent diamide, the disulfide-reducing agent dithiothreitol, hyper- and hypo-osmotic shock, amino acid starvation, nitrogen source depletion, and progression into stationary phase. A large set of genes (approximately 900) showed a similar drastic response to almost all of these environmental changes. Additional features of the genomic responses were specialized for specific conditions. Promoter analysis and subsequent characterization of the responses of mutant strains implicated the transcription factors Yap1p, as well as Msn2p and Msn4p, in mediating specific features of the transcriptional response, while the identification of novel sequence elements provided clues to novel regulators. Physiological themes in the genomic responses to specific environmental stresses provided insights into the effects of those stresses on the cell.
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              A ten-minute DNA preparation from yeast efficiently releases autonomous plasmids for transformation of Escherichia coli.

              A procedure for the rapid isolation of DNA from the yeast Saccharomyces cerevisiae is described. To release plasmid DNA for the transformation of Escherichia coli, cells are subjected to vortex mixing in the presence of acid-washed glass beads, Triton X-100, sodium dodecyl sulfate, phenol and chloroform. Centrifugation of this mixture separates the DNA from cellular debris. E. coli can be efficiently transformed with plasmid present in the aqueous layer without further purification of the plasmid DNA. This procedure also releases chromosomal DNA. Following two ethanol precipitations, the chromosomal DNA can be digested by restriction endonucleases and analysed by Southern blot analysis.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                G3 (Bethesda)
                Genetics
                g3journal
                G3: Genes|Genomes|Genetics
                Oxford University Press
                2160-1836
                November 2021
                10 August 2021
                10 August 2021
                : 11
                : 11
                : jkab279
                Affiliations
                [1 ] Department of Biology, Duke University , Durham, NC 27708, USA
                [2 ] Curriculum in Genetics and Molecular Biology, University of North Carolina at Chapel Hill , Chapel Hill, NC 27599, USA
                Author notes
                Corresponding author: Department of Biology, Duke University, PO Box 90338, 062 Biological Sciences Bldg. 130 Science Drive, Durham, NC 27708, USA. Email: darmaleo@ 123456duke.edu
                Author information
                https://orcid.org/0000-0002-2862-6086
                Article
                jkab279
                10.1093/g3journal/jkab279
                8527467
                74eca1ca-f1b7-480a-833f-b7f25e8fa63e
                © The Author(s) 2021. Published by Oxford University Press on behalf of Genetics Society of America.

                This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( https://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.

                History
                : 03 May 2021
                : 16 July 2021
                : 14 October 2021
                Page count
                Pages: 15
                Funding
                Funded by: Duke University Undergraduate Research Support;
                Categories
                Investigation
                AcademicSubjects/SCI01180
                AcademicSubjects/SCI01140
                AcademicSubjects/SCI00010
                AcademicSubjects/SCI00960

                Genetics
                desiccation tolerance,growth inhibition,rdna introns,rrna processing,ribosome assembly,lichen fungi,cladonia grayi,saccharomyces cerevisiae,trehalose,crispr

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