Journal: BMC Microbiology

Article Title: The promoter and transcribed regions of the Leishmania tarentolae spliced leader RNA gene array are devoid of nucleosomes

doi: 10.1186/1471-2180-7-44

Figure Lengend Snippet: Detection of nucleosome ladders and positioning on the SL RNA gene repeat . A) Negative image of an ethidium-bromide stained agarose gel for a typical MNase digestion. Digestion of the L. tarentolae chromatin produced a characteristic ladder of DNA fragments. M = 100-bp size marker; 0 = mock treated chromatin. Increasing units of MNase are indicated, ranging from 5 – 500 units/ml. The arrowhead indicates the monosome. B) Southern blot mapping reveals strong preferential protection of an intergenic region between the T tract and the Proximal Sequence Element (PSE). DNA size marker positions are indicated on the left. Lane1, Sac II digest of the SL RNA gene in mock-treated DNA; lane 2, MNase digested chromatin. The blot was hybridized with 32 P-labelled oligonucleotide probes. The letter below each blot refers to the specific oligonucleotide probe, as listed in Table 1. The probe location is indicated by the lines above the schematic. The histogram represents the ratios of the monosome signal: genomic signal.

Article Snippet: The extension products were separated on a 1× TBE/6% polyacrylamide/8 M urea sequencing gel (Whatman/Biometra).

Techniques: Staining, Agarose Gel Electrophoresis, Produced, Marker, Southern Blot, Sequencing

Journal: BMC Microbiology

Article Title: The promoter and transcribed regions of the Leishmania tarentolae spliced leader RNA gene array are devoid of nucleosomes

doi: 10.1186/1471-2180-7-44

Figure Lengend Snippet: Primer extension mapping of the nucleosomes on the SL array . A) The panel on the left represents the 5' end of the nucleosome, and the right panel the 3' end. The schematics along the top edge of the extensions depict the sequence as it relates to the SL RNA gene map. Oligonucleotide C and C' were used to generate the results. The light gray and dark ovals represent total range of nucleosome protection along the sequence. Lane 1, MNase-digested chromatin; lane2, MNase-digested purified DNA; lane 3, Bss HII digested or Dra III-digested DNA, respectively; lane 4, Mock treated DNA. Arrowheads in lane 1 indicate bands resulting from nucleosome protection. The sequences below the extensions map the major 5' and 3' ends of the protected DNA. *: anomalous Bss HII digestion product. B) Schematic of nucleosome organization on the SL RNA gene array. The genomic SL RNA gene array has a regular arrangement of nucleosomes confined to the non-transcribed spacer regions. The top schematic highlights the regular ~164-bp phasing between the major PE stops. Four potential positions of the nucleosome are presented relative to the DNA sequence of the spacer region. The bottom diagram depicts the conformation of the nucleosome in the SL RNA gene array. ~180 bp encompassing the upstream promoter sequences and the transcribed region are free of nucleosome binding.

Article Snippet: The extension products were separated on a 1× TBE/6% polyacrylamide/8 M urea sequencing gel (Whatman/Biometra).

Techniques: Sequencing, Purification, Binding Assay

Journal: BMC Microbiology

Article Title: The promoter and transcribed regions of the Leishmania tarentolae spliced leader RNA gene array are devoid of nucleosomes

doi: 10.1186/1471-2180-7-44

Figure Lengend Snippet: Southern blot mapping of nucleosomes on the episomal pX-tSL cassette . A) Unlike the results for the genomic array, the pX-tSL cassette shows no preferential protection. The letter below each blot refers to the specific oligonucleotide hybridization probe, as listed in Table 1. The probe location is indicated by the lines above the schematic. The schematic shows the location of the SL RNA gene, the upstream promoter elements, and the borders of the cassette (thick black lines). The tag sequence is represented as a white box. The histogram represents the ratios of the genomic signal:monosome signal. Lane1, Sac II digest of mock-treated DNA; lane 2, Bst EII/ Not I digest of the pX-tSL mock-treated DNA; lane 3, MNase-digested chromatin. B) Three hypothetical episomal nucleosome array conformations, to which only the first is able to bind the SL RNA gene pre-initiation complex. Assuming the periodicity of the nucleosomes on the drug-selectable marker-carrying episome are regular for L. tarentolae , the tSL RNA gene promoter has at most a 26% chance of exposure to cognate transcription factors.

Article Snippet: The extension products were separated on a 1× TBE/6% polyacrylamide/8 M urea sequencing gel (Whatman/Biometra).

Techniques: Southern Blot, Hybridization, Sequencing, Marker