Ty Element Retrotransposition in Saccharomyces cerevisia

酿酒酵母中的 Ty 元件逆转录转座

• 批准号: 6951650
• 负责人: David J. Garfinkel
• 金额: --
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6951650
• 关键词: Saccharomyces cerevisiae; complementary DNA; functional /structural genomics; fungal genetics; gene mutation; genetic transcription; genetic translation; genome; helicase; microorganism culture; nuclear membrane; transposon /insertion element

Our research concerns the mechanism and consequences of Ty (Transposon yeast) element retrotransposition in the budding yeast Saccharomyces cerevisiae. Ty elements comprise five related families of long terminal repeat retrotransposons that transpose via an RNA intermediate. The Ty genome contains two genes, TYA and TYB, which correspond to the gag and pol genes of retroviruses, respectively. The retrotransposon is transcribed into a nearly genome-length RNA, which is the template for reverse transcription by the self-encoded reverse transcriptase protein and for translation. Ty protein maturation and reverse transcription take place within Ty virus-like particles (Ty-VLPs), which appear to be essential for the transposition process. Although Ty-VLPs accumulate in the cytoplasm, a Ty preintegration complex containing Ty cDNA, the element-encoded integrase and perhaps other proteins return to the nucleus, where integration takes place at different chromosome locations. We are particularly interested in the biology of Ty1 elements because these elements are the most abundant, competent for transposition, and their RNA transcripts accumulate to an exceptionally high level. Despite the abundance of Ty1 RNA, however, mature Ty1 proteins and VLPs are present at low levels, and Ty1 transposition events are also very rare. Although Ty1 elements preferentially integrate upstream of genes transcribed by RNA polymerase III, Ty1 insertions can mutate essentially any yeast gene, form large complex multimeric insertions of 100 kb or more, and can also initiate chromosomal deletions, inversions and translocations by homologous recombination with other Ty1 elements in the genome. Information gained from studying Ty elements has been successfully applied to several other areas of biomedical research. For example, understanding how Ty elements transpose in yeast has led to a greater understanding of how retroelements in other organisms including humans function, because many of these elements are related. Over 30% of the human genome is comprised of retroelement sequences, such as LINE and SINE, intracisternal A-type particle, and endogenous retroviral elements. Most importantly, genome rearrangements and insertional events involving these elements have been implicated in human disease and cancer. Completion of the human genome sequence coupled with further genomic analyses of cancerous cells will likely reveal new roles for retroelements that can be modeled in yeast using Ty elements or their mammalian counterparts. In addition, many aspects of the retrotransposon replication cycle are similar to those of retroviruses, including HIV. Therefore, steps in the process of retrotransposition can be compared and contrasted with similar processes in retroviruses to learn more about both classes of elements. Over the past year, we have made progress in the following areas. We, in collaboration with Dr. Robert Fisher (SAIC Frederick), have utilized a novel method for cleaving proteins with formic acid that is suitable for mass spectroscopy. Cleavage with formic acid is efficient and specific for aspartyl residues, and this specificity of cleavage lends itself easily to database searches. Parallel digests with trypsin suggest that formic acid cleavage generated comparable or better results than tryptic digestion for protein identification. We are currently using this technique to search for cofactors that associate with Ty-VLPs. We, in collaboration with an international consortium of yeast researchers headed by Dr. Mark Johnston (Washington University), have developed a near complete set (95% of all ORFs) of single gene deletions to systematically survey gene function. These mutations are currently being screened for their affects on Ty1 retrotransposition. In our continuing effort to identify cellular genes that modulate Ty1 retrotransposition, we have surveyed all members of the RAD2 family of nucleases for their affects on Ty1 retrotransposition. We have shown that only Rad27/Fen1, a highly conserved structure-specific nuclease important for DNA replication and genome stability, inhibits Ty1 mobility by affecting the fate of unincorporated cDNA.

我们的研究涉及Ty（转座酵母）元素在酿酒酵母中发芽的机理和后果。 TY元素包括通过RNA中间体转置的五个相关的长期重复返回转座子的家族。 TY基因组包含两个基因Tya和Tyb，它们分别对应于逆转录病毒的GAG和POL基因。逆转录座子被转录为几乎基因组长度的RNA，这是通过自我调节的逆转录酶蛋白和翻译进行逆转录的模板。 Ty蛋白成熟和逆转录发生在Ty病毒样颗粒（TY-VLP）内，这对于转置过程似乎是必不可少的。尽管TY-VLP积聚在细胞质中，这是一种含有Ty cDNA的TY预融合复合物，但元素编码的整合酶以及其他蛋白质也可能返回到核，在不同的染色体位置进行整合。 我们对TY1元素的生物学特别感兴趣，因为这些元素是最丰富，有能力进行换位的生物学，其RNA转录本积累到异常高的水平。尽管有丰富的TY1 RNA，但是成熟的TY1蛋白和VLP仍存在低水平，而TY1换位事件也非常罕见。尽管TY1元素优先地整合了由RNA聚合酶III转录的基因上游，但TY1插入可以突变基本上任何酵母基因，形成100 kb或更多的大型复杂多聚体插入，并且还可以启动染色体缺失，染色体缺失，逆转和转换，通过与其他Ty1元素进行同源重组。 从研究元素中获得的信息已成功应用于生物医学研究的其他几个领域。例如，了解酵母中Ty元素的转置如何使人们对包括人类在内的其他生物的重新元素进行更深入的了解，因为这些元素中的许多是相关的。超过30％的人类基因组由追溯序列（例如线和正弦，肠内A型颗粒和内源性逆转录病毒元件）组成。最重要的是，涉及这些元素的基因组重排和插入事件与人类疾病和癌症有关。结合癌细胞的进一步基因组分析的人类基因组序列的完成可能会揭示可以使用TY元素或其哺乳动物对应物在酵母中建模的恢复元素的新作用。此外，逆转录盆地复制周期的许多方面与包括HIV在内的逆转录病毒相似。因此，可以比较逆转录置换过程中的步骤，并与逆转录病毒中的类似过程形成鲜明对比，以更多地了解这两种类别的元素。 在过去的一年中，我们在以下领域取得了进展。我们与罗伯特·费舍尔（Robert Fisher）博士（SAIC FREDERICK）合作，使用了一种新方法将适用于质谱法的甲酸切割蛋白质。用甲酸切割是有效的，并且对天冬氨酸残基有效，并且这种裂解的特异性很容易适合数据库搜索。与胰蛋白酶的平行消化表明，甲酸裂解产生的结果比胰蛋白酶鉴定的胰蛋白酶消化产生了可比的或更好的结果。我们目前正在使用此技术来搜索与TY-VLP相关联的辅助因子。 我们与由华盛顿大学马克·约翰斯顿（Mark Johnston）博士领导的国际酵母研究人员合作，已经开发了几乎完整的单个基因缺失（占所有ORF的95％），以系统地调查基因功能。目前，这些突变正在筛选它们对TY1逆转录置的影响。在我们继续努力鉴定调节TY1逆转录置位的细胞基因的努力中，我们对RAD2核酸酶家族的所有成员进行了调查，以便它们对TY1逆转转化的影响。我们已经表明，仅RAD27/FEN1，这是一种高度保守的结构特异性核酸酶，对DNA复制和基因组稳定性很重要，可以通过影响未合并的cDNA的命运来抑制TY1迁移率。