Depletion of UBC9 Causes Nuclear Defects during the Vegetative and Sexual Life Cycles in Tetrahymena thermophila.

Eukaryotic Cell Pub Date : 2015-12-01 Epub Date: 2015-10-09 DOI:10.1128/EC.00115-15
Qianyi Yang, Amjad M Nasir, Robert S Coyne, James D Forney
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引用次数: 3

Abstract

Ubc9p is the sole E2-conjugating enzyme for SUMOylation, and its proper function is required for regulating key nuclear events such as transcription, DNA repair, and mitosis. In Tetrahymena thermophila, the genome is separated into a diploid germ line micronucleus (MIC) that divides by mitosis and a polyploid somatic macronucleus (MAC) that divides amitotically. This unusual nuclear organization provides novel opportunities for the study of SUMOylation and Ubc9p function. We identified the UBC9 gene and demonstrated that its complete deletion from both MIC and MAC genomes is lethal. Rescue of the lethal phenotype with a GFP-UBC9 fusion gene driven by a metallothionein promoter generated a cell line with CdCl2-dependent expression of green fluorescent protein (GFP)-Ubc9p. Depletion of Ubc9p in vegetative cells resulted in the loss of MICs, but MACs continued to divide. In contrast, expression of catalytically inactive Ubc9p resulted in the accumulation of multiple MICs. Critical roles for Ubc9p were also identified during the sexual life cycle of Tetrahymena. Cell lines that were depleted for Ubc9p did not form mating pairs and therefore could not complete any of the subsequent stages of conjugation, including meiosis and macronuclear development. Mating between cells expressing catalytically inactive Ubc9p resulted in arrest during macronuclear development, consistent with our observation that Ubc9p accumulates in the developing macronucleus.

Abstract Image

在嗜热四膜虫的营养和有性生命周期中,UBC9的缺失导致核缺陷。
Ubc9p是唯一用于SUMOylation的e2偶联酶,其正常功能是调节转录、DNA修复和有丝分裂等关键核事件所必需的。嗜热四膜虫的基因组分为有丝分裂的二倍体生殖系微核(MIC)和无丝分裂的多倍体体细胞大核(MAC)。这种不寻常的核组织为研究SUMOylation和Ubc9p功能提供了新的机会。我们鉴定了UBC9基因,并证明其从MIC和MAC基因组中完全缺失是致命的。用金属硫蛋白启动子驱动的GFP- ubc9融合基因拯救致死表型,产生了依赖cdcl2表达绿色荧光蛋白(GFP)-Ubc9p的细胞系。营养细胞中Ubc9p的缺失导致mic的丢失,但mac继续分裂。相反,不具有催化活性的Ubc9p的表达导致了多个mic的积累。Ubc9p在四膜虫的性生命周期中也发挥了关键作用。缺乏Ubc9p的细胞系不能形成交配对,因此不能完成任何后续的接合阶段,包括减数分裂和大核发育。表达无催化活性Ubc9p的细胞之间的交配导致大核发育过程中的阻滞,这与我们观察到的Ubc9p在发育中的大核中积累一致。
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来源期刊
Eukaryotic Cell
Eukaryotic Cell 生物-微生物学
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审稿时长
1 months
期刊介绍: Eukaryotic Cell (EC) focuses on eukaryotic microbiology and presents reports of basic research on simple eukaryotic microorganisms, such as yeasts, fungi, algae, protozoa, and social amoebae. The journal also covers viruses of these organisms and their organelles and their interactions with other living systems, where the focus is on the eukaryotic cell. Topics include: - Basic biology - Molecular and cellular biology - Mechanisms, and control, of developmental pathways - Structure and form inherent in basic biological processes - Cellular architecture - Metabolic physiology - Comparative genomics, biochemistry, and evolution - Population dynamics - Ecology
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