Localization of Mlh1 during conjugation stage.

<p>(A) Localization of HA-Mlh1 during the conjugation stage. The cell was induced by Cd²⁺ in the growing stage. HA-Mlh1 was detected by anti-HA antibody and DNA was stained with DAPI. a, pair formation; b, early crescent; c, crescent; d, meiosis I; e, meiosis II; f, pronuclear selection. Arrowheads indicate Mics. Arrows show spindle-shaped micronuclei. Scale bar, 10 μm. (B) Mlh1 co-localized with spindle structures during the crescent stage. HA-Mlh1 was detected using anti-HA antibody, spindle structure was detected with anti α-tubulin antibody, and DNA was stained with DAPI. Scale bar, 10 μm.</p

Functional compensation of <i>MLH1</i> and <i>HMGB3</i>.

<p>(A) RT-PCR analysis of <i>HMGB3</i> in the Δ<i>MLH1</i> strains. (B) RT-PCR analysis of <i>MLH1</i> in the Δ<i>HMGB3</i> strains. Total RNA was isolated from conjugating cells after mixing for 4.5 h. 17S rRNA was used as the internal control. Each reaction was performed in triplicate.</p

Knocking out <i>MLH1</i> affects DNA stability.

<p>(A) Schematic drawings of the <i>MLH1</i> locus and the knock-out construct used to disrupt it. <i>MLH1</i> was replaced by Bsr cassette by homologous recombination. (B) Identification of Δ<i>MLH1</i>. Total DNA was isolated from wild-type CU428 and somatic <i>MLH1</i> knock-out cells. Δ<i>MLH1-</i>B and Δ<i>MLH1-</i>C are different mating type mutants from B2086 and CU428 respectively. Different lanes represent different clones. The target gene was amplified by PCR. The arrow indicates recombination band (~1.4 kb). (C) Mic-specific sequences were amplified by PCR with 5 set of primers. Primers I to V designed for five chromosomes in Mic, respectively. The loci on chromosomes III, IV, and V appeared to be missing in Δ<i>MLH1</i>–B cells. I, 1.1 kb; II, 0.35 kb; III, 0.4 kb; IV, 1.2 kb; V, 0.9 kb. Arrow indicates band of Mic specific sequences.</p

Overexpression of Mlh1 leads to defects during conjugation.

<p>(A) Percentage of different developmental stages during conjugation. I: single cell; II: pair formation; III: crescent; IV: meiosis I; V: meiosis II; VI: pronuclear selection; VII: post-meiotic mitosis; VIII: postzygotic mitosis I; IX: postzygotic mitosis II; X: anlagen; XI: 2Mac-1Mic. i: abnormal pairs; ii: abnormal signal cells (n>200). (B) Schematic representation of the developmental cells during conjugation. (C) A representative nuclear abnormal cell: (a) looser Mics; (b) irregular Mics; (c) loss of Mics; (d) separated cell with only one Mac; (e) one cell with three Mics; (f) one cell with four Mics. Scale bar, 10 μm.</p

Micronucleus-specific histone H1 is required for micronuclear chromosome integrity in <i>Tetrahymena thermophila</i>

<div><p>Histone H1 molecules play a key role in establishing and maintaining higher order chromatin structures. They can bind to linker DNA entering and exiting the nucleosome and regulate transcriptional activity. <i>Tetrahymena thermophila</i> has two histone H1, namely, macronuclear histone H1 and micronuclear histone H1 (Mlh1). Mlh1 is specifically localized at micronuclei during growth and starvation stages. Moreover, Mlh1 is localized around micronuclei and forms a specific structure during the conjugation stage. It co-localizes partially with spindle apparatus during micronuclear meiosis. Analysis of <i>MLH1</i> knock-out revealed that Mlh1 was required for the micronuclear integrity and development during conjugation stage. Overexpression of Mlh1 led to abnormal conjugation progression. RT-PCR analysis indicated that the expression level of <i>HMGB3</i> increased in Δ<i>MLH1</i> strains, while the expression level of <i>MLH1</i> increased in Δ<i>HMGB3</i> cells during conjugation. These results indicate that micronuclear integrity and sexual development require normal expression level of Mlh1 and that HmgB3 and Mlh1 may functionally compensate each other in regulating micronuclear structure in <i>T</i>. <i>thermophila</i>.</p></div