PMC

The nessy point mutation, alternative splicing, and sequence conservation in kleisin β. (A) Predicted wild-type and nessy N-terminal sequence of the three hypothetical proteins encoded by alternative exon 1 splicing. The causative mutation and altered amino acids are boxed. (B) Clustal W alignment of kleisin β sequences from different organisms, showing sequence conservation of the Ile residue mutated in nessy. Numbers in brackets refer to National Center for Biotechnology Information accession numbers. Asterisks, colons, and periods indicate identity, conservation, or semiconservation of amino acids at each position.

A) Schematic representation of the structure of condensin complexes. In metazoans, there are two types of condensins, condensin I and condensin II. The SMC2/SMC4 heterodimer is shared by both complexes, while the non-SMC subunits differ: CAP-D2, CAP-G, and CAP-H (Kleisin γ) for condensin I and CAP-D3, CAP-G2, and CAP-H2 (Kleisin β) for condensin II. B) Possible models for the condensins’ role in DNA compaction include DNA supercoiling, loop-holder, and topological linker.

The nessy phenotype is due to a T cell-intrinsic defect and can be rescued by expression of the wild-type kleisin β sequence. (A) The hemopoietic systems of lethally irradiated B6.SJL.Ly5^a mice were reconstituted with bone marrow cells from a wild-type or nes/nes C57BL/6 (Ly5^b) mouse or a 50/50 mixture of bone marrow from nes/nes C57BL/6 (Ly5^b) and wild-type B6.SJL.Ly5^a mice. The left three dot plots are gated on Ly5^b spleen cells, showing the subsets of CD44^high and CD44^low CD8 cells. The rightmost dot plot is gated on Ly5^a spleen cells in the 50/50 chimeras, showing normal CD8 subsets derived from wt precursors, which contrast with the absence of CD44^low CD8 cells from nes/nes precursors. (B) Hemopoietic chimeras reconstituted with B6 nes/nes marrow as in A, except that the hemopoietic stem cells were first transduced in vitro with a bicistronic retrovirus expressing the wild-type long cDNA from ENSMUSG00000008690 and GFP. Profiles are gated on Ly5^b+ CD8⁺ nes/nes splenic lymphocytes and display CD44^low and CD44^high subsets of CD8 cells differentiated from transduced (GFP⁺) and nontransduced (GFP⁻) nes/nes stem cells. The dot plots show data from three independently reconstituted mice in two separate transduction experiments. Note that the GFP⁻ cells in the left-hand quadrants of each plot show the typical CD44^high nessy profile and serve as internal controls in each case.

Mitotic chromosome structure. Axial localizations of condensins I and II in mitotic chromosomes in live mammalian cells. For DNA staining, DM (Indian Muntjac cells) cells stably expressing EGFP-Kleisin γ (condensin I) and EGFP-Kleisin β (condensing II) were stained with Hoechst 33342. Live-cell imaging was performed using a Delta Vision microscope (applied precision). a Clear axial signals of EGFP-Kleisin γ in mitotic chromosomes are shown. b End-on-view of mitotic chromosomes. The upper panel shows DM cells expressing EGFP-Kleisin γ, whereas the lower panel shows DM cells expressing EGFP-Kleisin β stably. Restricted dot signals from two types of EGFP-Kleisin in the cross-section of a chromosome body (DNA staining) are shown. c Quantitative data using line-profile analysis (blue line, DNA; red line, Kleisin signals) is shown. There is clear axial localization of condensins I and II in mitotic chromosomes in live mammalian cells. d Chromosomes consist of irregularly folded 10-nm nucleosome fibers. Condensins (blue) hold the nucleosome fibers (red) around the center of the chromosome. Locally, the nucleosome fibers are folded in an irregular or disordered manner, forming loop structures that collapse towards the center of the chromosome center (blue). The collapsed fiber (red) then forms a domain