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First published online 15 May 2007
doi: 10.1242/jcs.03457

Journal of Cell Science 120, 1935-1943 (2007)
Published by The Company of Biologists 2007
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The Clr4 methyltransferase determines the subnuclear localization of the mating-type region in fission yeast

Jenny Alfredsson-Timmins, Frida Henningson and Pernilla Bjerling*

University of Uppsala, Department of Medical Biochemistry and Microbiology (IMBIM), Box 582, SE-751 23 Uppsala, Sweden


Figure 1
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  		Fig. 1. Schematic representation of the mating-type region that is located on the right arm of chromosome II in S. pombe. The mating-type region consists of three linked loci; mat1 (checkered/black box), mat2-P (checkered box) and mat3-M (black box). mat2/3 is surrounded by two inverted repeats, IR-L and IR-R, with perfect sequence identity (block arrows). In the K-region separating mat2-P and mat3-M there is a 4.3 kb sequence, denoted cenH, with 96% homology to the repeats at cen2 (white box). The inserted ade6+ reporter gene is shown as a grey box.

 

Figure 2
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  		Fig. 2. Cis- and trans-acting mutations cause derepression of an ade6+ reporter gene inserted next to mat3-M. (A) Fivefold serial dilutions of each strain were spotted onto non-selective media (left panel) and on selective media lacking adenine (right panel). Strain PJ78 with a truncated allele at the endogenous ade6 locus, denoted ade6-DN/N, and strain PJ282 with a wild-type allele, ade6+, were used as reference strains. The strains shown are: PJ644 (w.t. ade6-M210), PJ654 (w.t. ade6-DN/N), PJ579 (IR-L{Delta}/IR-R{Delta}), PJ577 (hK{Delta} repressed) and PJ586 (hK{Delta} derepressed), PJ653 (clr4{Delta}), PJ668 (swi6{Delta}) and PJ651 (dcr1{Delta}). (B) RT-PCR analysis of the transcriptional levels from the ade6+ reporter gene at mat3-M performed on total RNA from the same strains as in Fig. 3A, except for the control strains PJ78, PJ282. The lower bands in the top panel are the transcripts from the endogenous ade6-DN/N, and served as an internal control for all strains. The top bands are transcripts from the ade6+ reporter gene inserted at mat-3M amplified using the same primers. The numbers below the top panel denote the level of ade6+ gene expression in the mating-type region calculated as a ratio between the ade6+ reporter gene PCR-product and the ade6-DN/N PCR-product. The lower panel shows control reactions in the absence of reverse transcription (labelled –).

 

Figure 3
Figure 3
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  		Fig. 3. The mating-type region is localized close to the SPB at the nuclear periphery in the wild-type strains and is delocalized in strains with cis- and trans-acting silencing mutations. (A) Confocal microscopy images of wild-type and mutant cells. Shown in the different panels are: the NM labelled with DsRed (red, first column), the SPB labelled with CFP (blue, second column), the mating-type region or (in the bottom row) the cut3 locus, labelled with lacR-GFP (green, third column), and merged images (fourth column). Bars, 2 µm. Histograms with the distributions of observed distances between the mating-type region (or, in the bottom row, the cut3 locus) and the SPB are shown to the right of the panels. The distribution of the observed distances between the GFP and CFP signals were binned in groups spanning 0.2 µm. The strains analysed were PJ644 (w.t. ade6-M210), PJ654 (w.t. ade6-DN/N), PJ579 (IR-L{Delta}/IR-R{Delta}), PJ577 (hK{Delta} repressed) and PJ586 (hK{Delta} derepressed), PJ653 (clr4{Delta}), PJ668 (swi6{Delta}), PJ651 (dcr1{Delta}) and PJ575 (cut3::lacO). (B) The nucleus divided into three zones of equal surface area. Zone I corresponded to a distance of 0-0.22 µm, zone II to a distance of 0.23-0.51 µm and zone III to a distance of 0.52-1.20 µm from the nuclear periphery. (C) Distribution of mat2/3 localization into zone I, zone II or zone III for each strain. The significance of the differences between each mutant strain and the PJ644 wild type was tested statistically (Mann-Whitney rank sum test, P<0.05). no, no significant difference; yes, a significant difference; n.a., not applicable. (D) The distribution of cells having the mat2/3 region localized into zone I, zone II or zone III plotted as pie charts. Black, zone I; light grey, zone II; dark grey, zone III.

 

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© The Company of Biologists Ltd 2007