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      Plant cholesterol biosynthetic pathway overlaps with phytosterol metabolism

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          A multicolored set of in vivo organelle markers for co-localization studies in Arabidopsis and other plants.

          Brook K Nelson, Xue Cai, Andreas Nebenführ (2007)
          Genome sequencing has resulted in the identification of a large number of uncharacterized genes with unknown functions. It is widely recognized that determination of the intracellular localization of the encoded proteins may aid in identifying their functions. To facilitate these localization experiments, we have generated a series of fluorescent organelle markers based on well-established targeting sequences that can be used for co-localization studies. In particular, this organelle marker set contains indicators for the endoplasmic reticulum, the Golgi apparatus, the tonoplast, peroxisomes, mitochondria, plastids and the plasma membrane. All markers were generated with four different fluorescent proteins (FP) (green, cyan, yellow or red FPs) in two different binary plasmids for kanamycin or glufosinate selection, respectively, to allow for flexible combinations. The labeled organelles displayed characteristic morphologies consistent with previous descriptions that could be used for their positive identification. Determination of the intracellular distribution of three previously uncharacterized proteins demonstrated the usefulness of the markers in testing predicted subcellular localizations. This organelle marker set should be a valuable resource for the plant community for such co-localization studies. In addition, the Arabidopsis organelle marker lines can also be employed in plant cell biology teaching labs to demonstrate the distribution and dynamics of these organelles.
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            High efficiency transformation of intact yeast cells using single stranded nucleic acids as a carrier.

            R H Schiestl, R Gietz (1989)
            A method, using LiAc to yield competent cells, is described that increased the efficiency of genetic transformation of intact cells of Saccharomyces cerevisiae to more than 1 X 10(5) transformants per microgram of vector DNA and to 1.5% transformants per viable cell. The use of single stranded, or heat denaturated double stranded, nucleic acids as carrier resulted in about a 100 fold higher frequency of transformation with plasmids containing the 2 microns origin of replication. Single stranded DNA seems to be responsible for the effect since M13 single stranded DNA, as well as RNA, was effective. Boiled carrier DNA did not yield any increased transformation efficiency using spheroplast formation to induce DNA uptake, indicating a difference in the mechanism of transformation with the two methods.
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              Selection of internal control genes for quantitative real-time RT-PCR studies during tomato development process

              Marino Expósito-Rodríguez, Andres A Bazan-Borges, Andrés Borges-Pérez … (2008)
              Background The elucidation of gene expression patterns leads to a better understanding of biological processes. Real-time quantitative RT-PCR has become the standard method for in-depth studies of gene expression. A biologically meaningful reporting of target mRNA quantities requires accurate and reliable normalization in order to identify real gene-specific variation. The purpose of normalization is to control several variables such as different amounts and quality of starting material, variable enzymatic efficiencies of retrotranscription from RNA to cDNA, or differences between tissues or cells in overall transcriptional activity. The validity of a housekeeping gene as endogenous control relies on the stability of its expression level across the sample panel being analysed. In the present report we describe the first systematic evaluation of potential internal controls during tomato development process to identify which are the most reliable for transcript quantification by real-time RT-PCR. Results In this study, we assess the expression stability of 7 traditional and 4 novel housekeeping genes in a set of 27 samples representing different tissues and organs of tomato plants at different developmental stages. First, we designed, tested and optimized amplification primers for real-time RT-PCR. Then, expression data from each candidate gene were evaluated with three complementary approaches based on different statistical procedures. Our analysis suggests that SGN-U314153 (CAC), SGN-U321250 (TIP41), SGN-U346908 ("Expressed") and SGN-U316474 (SAND) genes provide superior transcript normalization in tomato development studies. We recommend different combinations of these exceptionally stable housekeeping genes for suited normalization of different developmental series, including the complete tomato development process. Conclusion This work constitutes the first effort for the selection of optimal endogenous controls for quantitative real-time RT-PCR studies of gene expression during tomato development process. From our study a tool-kit of control genes emerges that outperform the traditional genes in terms of expression stability.
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                Author and article information

                Journal
                Title: Nature Plants
                Abbreviated Title: NPLANTS
                Publisher: Springer Nature
                ISSN (Electronic): 2055-0278
                Publication date Created: December 22 2016
                Publication date (Print): December 22 2016
                Volume: 3
                Issue: 1
                Page: 16205
                Article
                DOI: 10.1038/nplants.2016.205
                PubMed ID: 28005066
                SO-VID: 8d89db4b-280c-4baa-8337-f7dbfb2054a1
                Copyright © © 2016
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