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.

我们的研究涉及芽殖酵母酿酒酵母中 Ty（转座子酵母）元件逆转座的机制和后果。 Ty 元件包含五个相关的长末端重复反转录转座子家族，这些家族通过 RNA 中间体转座。 Ty基因组包含两个基因TYA和TYB，分别对应逆转录病毒的gag和pol基因。逆转录转座子被转录成接近基因组长度的RNA，它是自编码逆转录酶蛋白进行逆转录和翻译的模板。 Ty 蛋白成熟和逆转录发生在 Ty 病毒样颗粒 (Ty-VLP) 内，这似乎对于转座过程至关重要。尽管 Ty-VLP 在细胞质中积累，但含有 Ty cDNA、元件编码整合酶和可能其他蛋白质的 Ty 预整合复合物返回细胞核，整合发生在不同的染色体位置。 我们对 Ty1 元件的生物学特别感兴趣，因为这些元件最丰富，能够转座，并且它们的 RNA 转录物积累到异常高的水平。然而，尽管 Ty1 RNA 丰富，但成熟的 Ty1 蛋白和 VLP 的含量较低，而且 Ty1 转座事件也非常罕见。尽管Ty1元件优先整合RNA聚合酶III转录的基因上游，但Ty1插入基本上可以突变任何酵母基因，形成100 kb或更长的大型复杂多聚体插入，并且还可以通过与其他Ty1同源重组启动染色体缺失、倒位和易位基因组中的元素。 从 Ty 元素研究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 Ty 元件如何在酵母中转座可以更好地了解逆转录元件在包括人类在内的其他生物体中如何发挥作用，因为其中许多元件都是相关的。超过 30% 的人类基因组由逆转录元件序列组成，例如 LINE 和 SINE、脑池内 A 型颗粒和内源逆转录病毒元件。最重要的是，涉及这些元件的基因组重排和插入事件与人类疾病和癌症有关。人类基因组序列的完成加上对癌细胞的进一步基因组分析可能会揭示逆转录元件的新作用，可以使用 Ty 元件或其哺乳动物对应物在酵母中建模。此外，逆转录转座子复制周期的许多方面与逆转录病毒相似，包括HIV。因此，可以将逆转录转座过程中的步骤与逆转录病毒中的类似过程进行比较和对比，以更多地了解这两类元件。 一年来，我们在以下方面取得了进展。我们与 Robert Fisher 博士（SAIC Frederick）合作，利用了一种适用于质谱法的甲酸裂解蛋白质的新方法。使用甲酸进行切割对于天冬氨酰残基是高效且特异的，并且这种切割的特异性很容易进行数据库搜索。胰蛋白酶的平行消化表明，在蛋白质鉴定中，甲酸裂解产生的结果与胰蛋白酶消化相当或更好。我们目前正在使用这种技术来寻找与 Ty-VLP 相关的辅助因子。 我们与由 Mark Johnston 博士（华盛顿大学）领导的国际酵母研究人员联盟合作，开发了一套近乎完整的单基因缺失集（占所有 ORF 的 95%），以系统地调查基因功能。目前正在筛选这些突变对 Ty1 逆转录转座的影响。在我们不断努力识别调节 Ty1 逆转录转座的细胞基因的过程中，我们调查了 RAD2 核酸酶家族的所有成员，了解它们对 Ty1 逆转录转座的影响。我们已经证明，只有 Rad27/Fen1（一种对 DNA 复制和基因组稳定性非常重要的高度保守的结构特异性核酸酶）能够通过影响未掺入 cDNA 的命运来抑制 Ty1 的移动性。