Selective autophagy regulates insertional mutagenesis by the Ty1 retrotransposon in Saccharomyces cerevisiae

Macroautophagy (autophagy) is a bulk degradation system for cytoplasmic components and is ubiquitously found in eukaryotic cells. Autophagy is induced under starvation conditions and plays a cytoprotective role by degrading unwanted cytoplasmic materials. The Ty1 transposon, a member of the Ty1/copia superfamily, is the most abundant retrotransposon in the yeast Saccharomyces cerevisiae and acts to introduce mutations in the host genome via Ty1 virus-like particles (VLPs) localized in the cytoplasm. Here we show that selective autophagy downregulates Ty1 transposition by eliminating Ty1 VLPs from the cytoplasm under nutrient-limited conditions. Ty1 VLPs are targeted to autophagosomes by an interaction with Atg19. We propose that selective autophagy safeguards genome integrity against excessive insertional mutagenesis caused during nutrient starvation by transposable elements in eukaryotic cells.     

裸燕麦Ty1-copia类反转录转座子反转录酶序列的分离、鉴定与分析

本研究根据Ty1-copia类反转录转座子反转录酶的保守区设计简并引物,通过PCR扩增,从裸燕麦(Avena nuda L.)品种‘品燕1号’基因组中分离获得23条Ty1-copia类反转录转座子序列,并对序列特征、系统发育关系及其转录活性进行分析。结果显示,23条Ty1-copia类反转录转座子存在较高的异质性,序列间的一致性为45%～98%,存在插入、移码和终止密码突变,但频率不高;系统发育分析结果表明,燕麦Ty1-copia类反转录转座子在进化过程中主要为垂直传递。本研究通过检索燕麦基因表达数据库,发现了5个有转录活性的Ty1-copia类反转录转座子。     

The Role of Interelement Selection in Saccharomyces cerevisiae Ty Element Evolution

Retrotransposons are mobile genetic elements that are ubiquitous components of eukaryotic genomes. The evolutionary success of retrotransposons is explained by their ability to replicate faster than the host genomes in which they reside. Elements with higher rates of genomic replication possess a selective advantage over less active elements. Retrotransposon populations, therefore, are shaped largely by selective forces acting at the genomic level between elements. To evaluate rigorously the effects of selective forces acting on retrotransposons, detailed information on the patterns of molecular variation within and between retrotransposon families is needed. The sequencing of the Saccharomyces cerevisiae genome, which includes the entire genomic complement of yeast retrotransposons, provides an unprecedented opportunity to access and analyze such data. In this study, we analyzed in detail the patterns of nucleotide variation within the open reading frames of two parental (Ty1 and Ty2) and one hybrid (Ty1/2) family of yeast retrotransposons. The pattern and distribution of nucleotide changes on the phylogenetic reconstructions of the three families of Ty elements reveal evidence of negative selection on both internal and external branches of the Ty phylogenies. These results indicate that most, if not all, Ty elements examined represent active or recently active retrotransposon lineages. We discuss the relevance of these findings with respect to the coevolutionary dynamic operating between genomic element populations and the host organisms in which they reside. Received: 5 November 1998 / Accepted: 17 March 1999     

Ty1 Gag Enhances the Stability and Nuclear Export of Ty1 mRNA

Retrotransposon and retroviral RNA delivery to particle assembly sites is essential for their replication. mRNA and Gag from the Ty1 retrotransposon colocalize in cytoplasmic foci, which are required for transposition and may be the sites for virus‐like particle (VLP) assembly. To determine which Ty1 components are required to form mRNA/Gag foci, localization studies were performed in a Ty1‐less strain expressing galactose‐inducible Ty1 plasmids (pGTy1) containing mutations in GAG or POL. Ty1 mRNA/Gag foci remained unaltered in mutants defective in Ty1 protease (PR) or deleted for POL. However, Ty1 mRNA containing a frameshift mutation (Ty1fs) that prevents the synthesis of all proteins accumulated in the nucleus. Ty1fs RNA showed a decrease in stability that was mediated by the cytoplasmic exosome, nonsense‐mediated decay (NMD) and the processing body. Localization of Ty1fs RNA remained unchanged in an nmd2Δ mutant. When Gag and Ty1fs mRNA were expressed independently, Gag provided in trans increased Ty1fs RNA level and restored localization of Ty1fs RNA in cytoplasmic foci. Endogenously expressed Gag also localized to the nuclear periphery independent of RNA export. These results suggest that Gag is required for Ty1 mRNA stability, efficient nuclear export and localization into cytoplasmic foci.     

PGU1 gene natural deletion is responsible for the absence of endo-polygalacturonase activity in some wine strains of Saccharomyces cerevisiae

The PGU1 gene encodes an endo-polygalacturonase enzyme in Saccharomyces cerevisiae. The literature reports that most S. cerevisiae strains possess this gene, despite a wide range of enzyme activity levels. Nevertheless, a few wine strains lack the PGU1 gene. We investigated the PGU1 locus sequence in these strains. The results indicated that the gene had been replaced by a partial Ty mobile element, whereas the gene promoter was still at the expected location. As all the strains lacking the PGU1 gene experienced the same phenomenon, it was tempting to hypothesize a common phylogenetic origin. However, fingerprints only allowed grouping of a few of them within one cluster.     

Species-Wide Transposable Element Repertoires Retrace the Evolutionary History of the Saccharomyces cerevisiae Host

Transposable elements (TE) are an important source of genetic variation with a dynamic and content that greatly differ in a wide range of species. The origin of the intraspecific content variation is not always clear and little is known about the precise nature of it. Here, we surveyed the species-wide content of the Ty LTR-retrotransposons in a broad collection of 1,011 Saccharomyces cerevisiae natural isolates to understand what can stand behind the variation of the repertoire that is the type and number of Ty elements. We have compiled an exhaustive catalog of all the TE sequence variants present in the S. cerevisiae species by identifying a large set of new sequence variants. The characterization of the TE content in each isolate clearly highlighted that each subpopulation exhibits a unique and specific repertoire, retracing the evolutionary history of the species. Most interestingly, we have shown that ancient interspecific hybridization events had a major impact in the birth of new sequence variants and therefore in the shaping of the TE repertoires. We also investigated the transpositional activity of these elements in a large set of natural isolates, and we found a broad variability related to the level of ploidy as well as the genetic background. Overall, our results pointed out that the evolution of the Ty content is deeply impacted by clade-specific events such as introgressions and therefore follows the population structure. In addition, our study lays the foundation for future investigations to better understand the transpositional regulation and more broadly the TE–host interactions.     

节瓜Ty1-copia类逆转座子逆转录酶序列的克隆及比对

对8个节瓜（Benincasa hispida var.chieh-qua How）品系基因组DNA中的Ty1-copia类逆转座子逆转录酶核苷酸序列进行扩增,并对品系A39FA的29个克隆产物的核苷酸序列及翻译的氨基酸序列的系统进化和同源性进行了分析,还对29条氨基酸序列进行了比对。扩增结果表明：8个节瓜品系的基因组DNA中均包含长度约260 bp的逆转录酶核苷酸片段;从品系A39FA中获得的29条Ty1-copia类逆转座子逆转录酶核苷酸序列（CqRt1至CqRt29）的长度为247~267 bp,同源率为46.2%~98.1%,而它们的氨基酸序列同源率为26.7%~98.8%。序列分析结果表明：节瓜Ty1-copia类逆转座子逆转录酶核苷酸序列中碱基A、T、G和C的数量分别为65~96、47~92、45~74和32~49,所有序列均富含碱基A和T,AT/GC比为1.35~2.33;缺失突变是造成节瓜Ty1-copia类逆转座子逆转录酶核苷酸序列长度差异的主要因素,在序列长度和碱基组成方面的明显差异表明节瓜Ty1-copia类逆转座子逆转录酶核苷酸序列具有高度异质性。翻译后的氨基酸序列中有21条序列存在终止密码子突变、12条序列存在移框突变,表明Ty1-copia类逆转座子是节瓜基因组内序列重组的热点。通过聚类分析可将29个逆转录酶核苷酸序列分为5个家族（Family）,分别包括16、4、4、4和1条序列,其中Family 1可能是具有转座活性的逆转座子家族,但存在转录活性的逆转录酶序列仅占全部序列数量的20.69%。将每一家族中的1~2条序列与其他15种植物的Ty1-copia类逆转座子逆转录酶的氨基酸序列进行比对,显示出较高的同源性。研究结果表明：节瓜与其他植物的Ty1-copia类逆转座子可能有相同起源,而且Ty1-copia类逆转座子可在不同类群间横向传递。     

酵母细胞中Pil1的磷酸化与细胞压力抵抗的关系

在外界因素处理下,细胞将启动一系列保护措施以适应各种环境改变,磷酸化调节是蛋白功能调节的主要方式. 为了探讨酵母细胞中Pil1的磷酸化与细胞压力抵抗的关系,实验应用Pil1突变细胞检测在过氧化氢或热处理后细胞的生长情况,用免疫印记法检测热处理后Pil1的表达. 结果表明,相比野生细胞,Pil1突变细胞对抗过氧化氢和热的能力强,热处理后 Pil1的磷酸化水平增高, Pil1的丝氨酸273对于其磷酸化发生至关重要.     

苹果Ty1-copia类逆转座子家族鉴定及特性分析

根据逆转座子RT保守序列设计引物,利用PCR方法从苹果'嘎拉'中克隆了20条RT片段,分析苹果基因组内Ty1-copia类逆转座子家族特性及进化关系.结果显示,20条逆转录酶保守序列表现出了高度的异质性.结合已报道的37条苹果Ty1-copia类逆转座子RT片段,构建了系统发育树,发现家族1、3和4中具有转座活性的逆转座子的可能性较大;序列分析表明,Ty1-copia类逆转座子是苹果基因组内序列重组的热点.用RT序列为探针进行Southern杂交,发现苹果基因组内Ty1-copia类逆转座子拷贝数高、分布广泛.研究结果为进一步分离具有转座活性的苹果Ty1-copia类逆转座子及其人工诱导芽变奠定了基础.     

Ty1 transposition induced by carcinogens in Saccharomyces cerevisiae yeast depends on mitochondrial function

The transposition of the Ty mobile genetic element of Saccharomyces cerevisiae is induced by carcinogens. While the molecular background of spontaneous Ty1 transposition is well understood, the detailed mechanism of carcinogen induced Ty1 transposition is not clear. We found that mitochondrial functions participate in the Ty induced transposition induced by carcinogens. Contrary to the parental rho(+) cells rho(-) mutants (spontaneous or induced by ethidium bromide) do not increase the rate of Ty1 transposition upon treatment with carcinogens. Preliminary results strongly suggest that the absence of oxidative phosphorylation in rho(-) mutants is the reason for the inhibited Ty transposition. The lack of carcinogen induced Ty1 transposition in rho(-) cells is not specific for a particular carcinogen and represents a general feature of different carcinogenic substances inducing rho(-). It is concluded that carcinogen induced Ty1 transposition depends on the functional state of mitochondria and cannot take place in cells with compromised mitochondrial function (rho(-)).     

Multistress resistance of Saccharomyces cerevisiae is generated by insertion of retrotransposon Ty into the 5'' coding region of the adenylate cyclase gene.

Heat shock-resistant mutants, which were isolated by their ability to withstand lethal heat treatment, were characterized. Resistance was demonstrated to be a consequence of insertion of retrotransposon Ty into either the 5' coding or noncoding region, close to the putative initiation codon of the adenylate cyclase gene CYR1 (or CDC35). These heat shock-resistant mutants contained about threefold lower adenylate cyclase activity than wild-type strains. The mutants were also observed to be resistant to other stresses such as UV light and ethanol. These results demonstrate that multistress resistance, which may confer a survival advantage to yeast cells, can be generated by transposition of a Ty element into CYR1.     

Evidence for the Role of Recombination in the Regulatory Evolution of Saccharomyces cerevisiae Ty Elements

The recent completion of the sequencing of the Saccharomyces cerevisiae genome provides a unique opportunity to analyze the evolutionary relationships existing among the entire complement of retrotransposons residing within a single genome. In this article we report the results of such an analysis of two closely related families of yeast long terminal repeat (LTR) retrotransposons, Ty1 and Ty2. In our study, we analyzed the molecular variation existing among the 32 Ty1 and 13 Ty2 elements present within the S. cerevisiae genome recently sequenced within the context of the yeast genome project. Our results indicate that while the Ty1 family is most likely ancestral to Ty2 elements, both families of elements are relatively recent components of the S. cerevisiae genome. Our results also indicate that both families of elements have been subject to purifying selection within their protein coding regions. Finally, and perhaps most interestingly, our results indicate that a relatively recent recombination event has occurred between Ty2 and a subclass of Ty1 elements involving the LTR regulatory region. We discuss the possible biological significance of these findings and, in particular, how they contribute to a better overall understanding of LTR retrotransposon evolution. Received: 30 September 1997 / Accepted: 3 February 1998