Functional Roles for Tetrahymena RAD51 During Conjugation and the Cell Cycle

四膜虫 RAD51 在接合和细胞周期中的功能作用

基本信息

  • 批准号:
    0091194
  • 负责人:
  • 金额:
    $ 10万
  • 依托单位:
  • 依托单位国家:
    美国
  • 项目类别:
    Standard Grant
  • 财政年份:
    2001
  • 资助国家:
    美国
  • 起止时间:
    2001-03-01 至 2002-06-30
  • 项目状态:
    已结题

项目摘要

The ciliated protozoa possess an unusual genome organization that effectively divides the labor of somatic and germline genetic functions between two distinct nuclei. The micronucleus is the germline nucleus, divides mitotically during vegetative growth, is diploid (2N = 10 for Tetrahymena), and is transcriptionally silent. In contrast, the macronucleus is the somatic nucleus, divides amitotically, contains multiple copies of all genes, and is transcriptionally active. Sexual reproduction, or conjugation, is a tightly controlled process in Tetrahymena whose progression follows a predictable pattern over approximately twelve hours. Soon after two Tetrahymena cells of opposite mating type form a conjugating pair, the micronuclei undergo a series of meiotic and mitotic divisions. Following these nuclear divisions, conjugants exchange haploid nuclei, which then fuse to produce a zygotic, diploid nucleus. Late in conjugation the parental macronuclei are destroyed, and a new macronucleus develops from a copy of the zygotic micronucleus. Macronuclear development proceeds in a pre-programmed and controlled manner that involves a series of site-specific chromosome breakage and DNA deletion events.A functional study of the highly conserved DNA strand transfer protein Rad51 (the eukaryotic homolog to the bacterial recA recombinase) from Tetrahymena thermophila has been initiated. Tetrahymena RAD51 expression varies under a number of different environmental conditions and developmental stages. RAD51 expression increases following exposure to DNA damaging agents. In addition, RAD51 levels vary during both the vegetative cell cycle and conjugation. Disruption of the somatic RAD51 locus results in failure of conjugating cells to complete meiosis, and in vegetative cell cycle defects that lead to an accumulation of hypodiploid cells. Conjugation of germline, homozygous rad51 null strains lead to a block of the first vegetative cell division following conjugation, resulting in an arrest at the exconjugant developmental stage. In order to more precisely decipher the role of Rad51p during conjugation, a conditional, temperature sensitive (t.s.) allele of Tetrahymena RAD51 will be identified. Plasmid-encoded Tetrahymena Rad51p will be tested for complementation of a yeast rad51 null strain. Successful complementation will make it possible to directly screen for Tetrahymena RAD51 t.s. alleles in yeast. If the Tetrahymena RAD51 homolog fails to complement a yeast rad51 null strain, plasmid-encoded yeast RAD51 will be mutagenized, introduced into a rad51 null strain, and screened for a t.s. allele. Missense mutations of conserved amino acid(s) that confer a t.s. phenotype to the yeast Rad51p will be introduced to the Tetrahymena homolog, which will subsequently be expressed in Tetrahymena rad51 null exconjugants. A RAD51 conditional mutant that can be inactivated during conjugation at the restrictive temperature will provide insight into how developmentally controlled genome rearrangements in Tetrahymena, including those leading to rDNA palindrome formation, are mediated.A more complete understanding of these developmentally controlled recombination will provide insight into how similar processes are achieved in other organisms, such as the rearrangement of vertebrate immunoglobulin genes. Mechanisms by which DNA sequences are rearranged can be more easily explored and understood using a single-celled model organism such as Tetrahymena.
纤毛虫原生动物拥有一个不寻常的基因组组织,有效地划分两个不同的细胞核之间的体细胞和生殖系遗传功能的劳动。 微核是生殖细胞核,在营养生长期间有丝分裂,是二倍体(四膜虫2N = 10),并且是转录沉默的。 相比之下,大核是体细胞核,无丝分裂,包含所有基因的多个拷贝,并且转录活跃。 有性生殖或接合是四膜虫中严格控制的过程,其进展遵循约12小时的可预测模式。 在两个交配型相反的四膜虫细胞形成接合对后不久,微核经历一系列减数分裂和有丝分裂。 在这些核分裂之后,接合子交换单倍体核,然后融合产生合子二倍体核。 接合后期,亲本大核被破坏,新的大核从合子微核的副本发育而来。 嗜热四膜虫(Tetrahymena thermophila)大核的发育是一个预先设定好的程序和受控的过程,其中包括一系列的染色体断裂和DNA缺失事件。 四膜虫RAD51的表达在许多不同的环境条件和发育阶段下变化。 暴露于DNA损伤剂后,RAD51表达增加。 此外,RAD51水平在营养细胞周期和接合过程中变化。 体细胞RAD51基因座的破坏导致接合细胞不能完成减数分裂,以及导致亚二倍体细胞积累的营养细胞周期缺陷。 种系纯合rad51无效菌株的缀合导致缀合后第一次营养细胞分裂的阻断,导致在接合外发育阶段的停滞。 为了更精确地破译Rad51p在缀合期间的作用,使用条件性温度敏感(t.s.)将鉴定四膜虫RAD 51的等位基因。 将检测质粒编码的四膜虫Rad51 p是否与酵母rad51缺失菌株互补。 成功的互补将使直接筛选四膜虫RAD 51 t.s.成为可能。酵母中的等位基因 如果四膜虫RAD 51同源物不能补充酵母rad 51缺失菌株,则将质粒编码的酵母RAD 51诱变,引入到rad 51缺失菌株中,并筛选t.s.等位基因 保守氨基酸的错义突变,赋予t.s.将酵母Rad51 p的表型引入到四膜虫同源物中,其随后将在四膜虫rad51无效接合子中表达。 一个在限制性温度下接合过程中失活的RAD 51条件突变体将有助于深入了解四膜虫中发育控制的基因组重排,包括那些导致rDNA回文序列形成的基因组重排是如何介导的。例如脊椎动物免疫球蛋白基因的重排。 使用单细胞模式生物如四膜虫,可以更容易地探索和理解DNA序列重排的机制。

项目成果

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Daniel Romero其他文献

Elevated mutation rates are unlikely to evolve in sexual species, not even under rapid environmental change
即使在快速的环境变化下,有性物种也不太可能进化出高突变率
  • DOI:
    10.1186/s12862-019-1494-0
  • 发表时间:
    2019
  • 期刊:
  • 影响因子:
    3.4
  • 作者:
    Daniel Romero;F. Jeltsch;R. Tiedemann
  • 通讯作者:
    R. Tiedemann
Evolution of the Mutation Rate in Sexual Species
有性物种突变率的演变
  • DOI:
  • 发表时间:
    2021
  • 期刊:
  • 影响因子:
    0
  • 作者:
    Daniel Romero;R. Tiedemann
  • 通讯作者:
    R. Tiedemann
Generalized matched filter detector for fast fading channels
用于快速衰落信道的广义匹配滤波器检测器
Fast Graph Filters for Decentralized Subspace Projection
用于分散子空间投影的快速图形过滤器
Prospective evaluation of the association between varicocele and benign prostatic hyperplasia in men over 40 years of age
40 岁以上男性精索静脉曲张与良性前列腺增生之间关系的前瞻性评估
  • DOI:
  • 发表时间:
    2021
  • 期刊:
  • 影响因子:
    2.4
  • 作者:
    J. Gaona;F. Gonzalez;Daniel Sánchez;César González;R. Rueda;J. Ortiz;Daniel Romero;Pablo Robles;Andres Osma;Daniela Martínez;H. Díaz;Fernando Monterroza;Margarita M. Zuluaga;Marlio Vanegas;E. Rueda
  • 通讯作者:
    E. Rueda

Daniel Romero的其他文献

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{{ truncateString('Daniel Romero', 18)}}的其他基金

Collaborative Research: HCC: Small: Science communication in the ecosystem of digital media platforms
合作研究:HCC:小型:数字媒体平台生态系统中的科学传播
  • 批准号:
    2133964
  • 财政年份:
    2022
  • 资助金额:
    $ 10万
  • 项目类别:
    Standard Grant
CHS: Small: Large-Scale Examination of the Impact of Shocks on Crowd Attributes and Performance in Collaborative Volunteering Systems
CHS:小型:大规模检查冲击对协作志愿服务系统中人群属性和绩效的影响
  • 批准号:
    1617820
  • 财政年份:
    2016
  • 资助金额:
    $ 10万
  • 项目类别:
    Standard Grant
Functional Roles for Tetrahymena RAD51 During Conjugation and the Cell Cycle
四膜虫 RAD51 在接合和细胞周期中的功能作用
  • 批准号:
    0220085
  • 财政年份:
    2002
  • 资助金额:
    $ 10万
  • 项目类别:
    Standard Grant
In vitro Mutagenesis of E. coli Ribosomal Proteins
大肠杆菌核糖体蛋白的体外诱变
  • 批准号:
    9302014
  • 财政年份:
    1993
  • 资助金额:
    $ 10万
  • 项目类别:
    Standard Grant

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