Ty Element Retrotransposition in Saccharomyces cerevisia

我们的研究关注Ty（转座子酵母）元件在芽殖酵母酿酒酵母中的反转录转座的机制和后果。Ty元件包括通过RNA中间体转座的长末端重复反转录转座子的五个相关家族。Ty基因组包含两个基因TYA和TYB，分别对应于逆转录病毒的gag和pol基因。逆转录转座子被转录成接近基因组长度的RNA，其是通过自身编码的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，这似乎是转座过程所必需的。虽然Ty-VLP在细胞质中积累，但含有Ty cDNA、元件编码的整合酶和可能的其他蛋白质的Ty整合前复合物返回到细胞核，其中整合发生在不同的染色体位置。 我们对Ty 1元件的生物学特别感兴趣，因为这些元件是最丰富的，能够进行转座，并且它们的RNA转录物积累到非常高的水平。然而，尽管Ty 1 RNA丰富，但成熟的Ty 1蛋白和VLP以低水平存在，并且Ty 1转座事件也非常罕见。尽管Ty 1元件优先整合在RNA聚合酶III转录的基因上游，但Ty 1插入可以使基本上任何酵母基因突变，形成100 kb或更大的大的复杂多聚体插入，并且还可以通过与基因组中的其他Ty 1元件同源重组来引发染色体缺失、倒位和易位。 从研究Ty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Ty元件如何在酵母中转座，可以更好地了解包括人类在内的其他生物体中的逆转录元件如何发挥作用，因为这些元件中的许多都是相关的。超过30%的人类基因组由逆转录病毒元件序列（如LINE和SINE）、脑池内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癌细胞的进一步基因组分析可能会揭示新的作用，可以在酵母中使用Ty元件或其哺乳动物对应物建模的retroelements。此外，逆转录转座子复制周期的许多方面与逆转录病毒（包括HIV）相似。因此，逆转录转座过程中的步骤可以与逆转录病毒中的类似过程进行比较和对比，以了解更多关于这两类元件的信息。 过去一年，我们在以下方面取得了进展。我们与Robert Fisher博士（SAIC Frederick）合作，利用一种适用于质谱的甲酸裂解蛋白质的新方法。用甲酸切割是有效的，并且对乙酰基残基具有特异性，并且这种切割的特异性本身容易用于数据库搜索。与胰蛋白酶的平行酶切表明，对于蛋白质鉴定，甲酸裂解产生与胰蛋白酶消化相当或更好的结果。我们目前正在使用这种技术来搜索与Ty-VLP相关的辅因子。 我们与由Mark约翰斯顿博士（华盛顿大学）领导的酵母研究人员国际联盟合作，开发了一套近乎完整的单基因缺失（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Ty 1逆转录转座的影响。在我们继续努力，以确定细胞基因，调节Ty 1逆转录转座，我们调查了所有成员的RAD 2家族的核酸酶对Ty 1逆转录转座的影响。我们已经表明，只有Rad 27/Fen 1，一个高度保守的结构特异性核酸酶的DNA复制和基因组的稳定性，抑制Ty 1的流动性，通过影响未纳入的cDNA的命运。

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.