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      Promoter analysis by saturation mutagenesis

      research-article
      1 ,
      Biological Procedures Online
      Biological Procedures Online
      promoter regions (genetics), mutagenesis, polymerase chain reaction

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          Abstract

          Gene expression and regulation are mediated by DNA sequences, in most instances, directly upstream to the coding sequences by recruiting transcription factors, regulators, and a RNA polymerase in a spatially defined fashion. Few nucleotides within a promoter make contact with the bound proteins. The minimal set of nucleotides that can recruit a protein factor is called a cis-acting element. This article addresses a powerful mutagenesis strategy that can be employed to define cis-acting elements at a molecular level. Technical details including primer design, saturation mutagenesis, construction of promoter libraries, phenotypic analysis, data analysis, and interpretation are discussed.

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          Most cited references13

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          Direct amplification of the entire ITS region from poorly preserved plant material using recombinant PCR.

          Sequences of the internal transcribed spacers (ITS) of the nuclear ribosomal DNA are important molecular markers in phylogenetic analyses. To obtain sequences from herbarium material in which DNA often is severely degraded, the ITS region has to be amplified in two steps. Two methods that reduce bench time and reagents used are described. (i) Separately amplified preparations of subunits ITS-1 and ITS-2 are combined before purification. The presence of two fragments in the sequencing reaction does not impair the quality of sequences. (ii) Newly designed internal primers amplify partly overlapping regions of the two subunits. A combination of these internal primers with the external primers in one PCR allows the amplification of the entire ITS region even when degraded DNAs are used. This recombinant PCR approach, taking into account the +A bases added by several Taq DNA polymerases, will also be useful with other marker regions used in molecular phylogenetics.
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            Regulation of a carotenoid biosynthesis gene promoter during plant development.

            Carotenoids are terpenoid pigments which are accumulated in the chloroplasts of leaves and in the chromoplasts of many flowers and fruits. Phytoene desaturase (Pds), the second dedicated enzyme in carotenoid biosynthesis, is encoded in tomato by a single copy gene. A 2 kb fragment from the tomato Pds gene, comprising 1.5 kb from the promoter and 0.5 kb from the 5' non-translated region, is able to drive developmentally regulated expression of the GUS reporter gene in transgenic tomato and tobacco plants. In tomato, high levels of Pds/GUS expression are found in organs and at stages of development where chromoplasts are formed: petals, anthers and ripening fruits. Tobacco petals and fruits, which do not contain chromoplasts, show instead low levels of Pds/GUS expression. Transgenic tobacco seedlings were subjected to treatment with a range of inhibitors of carotenoid and chlorophyll biosynthesis. The results indicate that, in green tissues, carotenoid and chlorophyll levels are tightly co-regulated and that a chemically induced arrest in pigment biosynthesis results in activation of the Pds promoter. The promoter is also induced in etiolated seedlings, which contain much lower carotenoid levels than light-grown seedlings. These data suggest that in green tissues Pds gene transcription may respond to end-product regulation.
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              Characterization of the distal promoter element of halobacteria in vivo using saturation mutagenesis and selection.

              The sequence and spacing requirements of the archaeal "distal promoter element' (DPE) were examined by randomizing positions -19 to -32 upstream of the transcriptional start site of the ferredoxin (fdx) promoter of Halobacterium salinarium. This randomized promoter library containing 4(14) entries was cloned in front of the dihydrofolate reductase (DHFR) reporter gene and transformed into Haloferax volcanii. Two approaches were used to characterize these synthetic promoters. First, 1040 independent clones were randomly chosen and their degrees of trimethoprim resistance were determined. The sequences of 20 clones that were either sensitive, partially resistant or very resistant, respectively, were determined. Secondly, the transformed library was screened by direct selection for high-activity promoters by growing transformants in the presence of trimethoprim. Both approaches produced the following consensus sequence for a halobacterial promoter: (Formula: see text) (where R = A or G; Y = C or T; W = A or T; S = G or C; N = A, C, G or T). Further characterization of two sensitive, two partially resistant, and two very resistant clones verified that DHFR activity and cell phenotype are directly correlated. Sensitive clones did not contain detectable dhfr mRNA, whereas partially resistant clones contained a 700 nucleotide (nt)-long transcript, and very resistant clones contained both the 700nt-long transcript and a second, more abundant, 500nt-long truncated transcript. Quantification of the dhfr mRNA and DHFR enzyme activity suggests that the 3'-untranslated region of the dhfr transcript, missing from the shorter transcript, functions as a negative regulator of translation.
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                Author and article information

                Journal
                Biol Proced Online
                Biological Procedures Online
                Biological Procedures Online
                1480-9222
                May 2001 - April 2002
                22 December 2001
                : 3
                : 64-69
                Affiliations
                [1 ]Institute for Systems Biology. 4225 Roosevelt way NE, Suite 200, Seattle, WA 98105. USA. Phone: 206-732-1266 Fax: 206-732-1299
                Author notes
                Nitin Baliga, Institute for Systems Biology. 4225 Roosevelt way NE, Suite 200, Seattle, WA 98105. USA. Phone: 206-732-1266. Fax: 206-732-1299. nbaliga@ 123456systemsbiology.org
                Article
                m24
                10.1251/bpo24
                145547
                12734578
                d0ad0b65-3a86-42c7-80c5-504c2e7d37fa
                Copyright © December 12, 2001, NS Baliga. Published in Biological Procedures Online under license from the author. Copying, printing, redistribution and storage permitted.
                History
                : 30 August 2001
                : 15 December 2001
                : 17 December 2001
                Categories
                Research Article

                Life sciences
                polymerase chain reaction,promoter regions (genetics),mutagenesis
                Life sciences
                polymerase chain reaction, promoter regions (genetics), mutagenesis

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