滑动序列对游仆虫中识别+1位和+2位编程性核糖体移码具有关键作用

编程性核糖体移码(programmed ribosomal frameshifting,PRF)广泛存在于生命进化谱系的各个分支,是一种翻译水平上的基因表达调控方式。单细胞真核生物游仆虫(Euplotes)中不仅PRF基因比例高,而且移码类型有+1和+2位两种。本研究从基因组水平对八肋游仆虫(E.octocarinatus)中的+2 PRF基因进行了鉴定,比较分析+1及+2 PRF基因中可能的调控元件。为了探讨游仆虫中滑动序列对移码类型的影响,克隆了八肋游仆虫的+1 PRF基因——η微管蛋白基因,将其构建到含绿色荧光蛋白报告基因的游仆虫大核人工染色体中,转染游仆虫细胞,通过检测GFP的表达来确定不同滑动序列突变体对应的移码类型。结果表明,滑动序列的改变能使游仆虫+1 PRF转变为+2 PRF,且这种移码类型的改变与滑动序列第1个密码子编码何种氨基酸无关。本研究揭示了滑动序列对游仆虫中识别+1和+2位的编程性核糖体移码具有关键作用。     

Macronuclear Genome Sequence of the Ciliate Tetrahymena thermophila, a Model Eukaryote

The ciliate Tetrahymena thermophila is a model organism for molecular and cellular biology. Like other ciliates, this species has separate germline and soma functions that are embodied by distinct nuclei within a single cell. The germline-like micronucleus (MIC) has its genome held in reserve for sexual reproduction. The soma-like macronucleus (MAC), which possesses a genome processed from that of the MIC, is the center of gene expression and does not directly contribute DNA to sexual progeny. We report here the shotgun sequencing, assembly, and analysis of the MAC genome of T. thermophila, which is approximately 104 Mb in length and composed of approximately 225 chromosomes. Overall, the gene set is robust, with more than 27,000 predicted protein-coding genes, 15,000 of which have strong matches to genes in other organisms. The functional diversity encoded by these genes is substantial and reflects the complexity of processes required for a free-living, predatory, single-celled organism. This is highlighted by the abundance of lineage-specific duplications of genes with predicted roles in sensing and responding to environmental conditions (e.g., kinases), using diverse resources (e.g., proteases and transporters), and generating structural complexity (e.g., kinesins and dyneins). In contrast to the other lineages of alveolates (apicomplexans and dinoflagellates), no compelling evidence could be found for plastid-derived genes in the genome. UGA, the only T. thermophila stop codon, is used in some genes to encode selenocysteine, thus making this organism the first known with the potential to translate all 64 codons in nuclear genes into amino acids. We present genomic evidence supporting the hypothesis that the excision of DNA from the MIC to generate the MAC specifically targets foreign DNA as a form of genome self-defense. The combination of the genome sequence, the functional diversity encoded therein, and the presence of some pathways missing from other model organisms makes T. thermophila an ideal model for functional genomic studies to address biological, biomedical, and biotechnological questions of fundamental importance.     

Macronuclear genome sequence of the ciliate Tetrahymena thermophila, a model eukaryote

The ciliate Tetrahymena thermophila is a model organism for molecular and cellular biology. Like other ciliates, this species has separate germline and soma functions that are embodied by distinct nuclei within a single cell. The germline-like micronucleus (MIC) has its genome held in reserve for sexual reproduction. The soma-like macronucleus (MAC), which possesses a genome processed from that of the MIC, is the center of gene expression and does not directly contribute DNA to sexual progeny. We report here the shotgun sequencing, assembly, and analysis of the MAC genome of T. thermophila, which is approximately 104 Mb in length and composed of approximately 225 chromosomes. Overall, the gene set is robust, with more than 27,000 predicted protein-coding genes, 15,000 of which have strong matches to genes in other organisms. The functional diversity encoded by these genes is substantial and reflects the complexity of processes required for a free-living, predatory, single-celled organism. This is highlighted by the abundance of lineage-specific duplications of genes with predicted roles in sensing and responding to environmental conditions (e.g., kinases), using diverse resources (e.g., proteases and transporters), and generating structural complexity (e.g., kinesins and dyneins). In contrast to the other lineages of alveolates (apicomplexans and dinoflagellates), no compelling evidence could be found for plastid-derived genes in the genome. UGA, the only T. thermophila stop codon, is used in some genes to encode selenocysteine, thus making this organism the first known with the potential to translate all 64 codons in nuclear genes into amino acids. We present genomic evidence supporting the hypothesis that the excision of DNA from the MIC to generate the MAC specifically targets foreign DNA as a form of genome self-defense. The combination of the genome sequence, the functional diversity encoded therein, and the presence of some pathways missing from other model organisms makes T. thermophila an ideal model for functional genomic studies to address biological, biomedical, and biotechnological questions of fundamental importance.     

艾美游仆虫大核基因组与转录组测序及结构特征

为获得艾美游仆虫(Euplotes amieti)大核基因组结构特征、分析基因功能及其表达调控方式,研究采用高通量测序技术对艾美游仆虫进行了大核基因组与转录组测序,结果显示基因组测序最终得到原始reads数据为10.92 Gb,过滤后得到50287条Contigs。GC含量较低,为31%;其中两端同时具有端粒的微染色体数量为27542条,占54.76%,只含有一端端粒的基因数量为6118条。Contigs进行基因结构分析, 96.5%的基因能够被预测出功能,最终得到27650条基因,平均外显子长度为311.69 bp;内含子较短,平均长度为150 bp。转录组测序结果为76219898条,拼接后获得38588条转录组Unigenes,其平均长度为1189 bp。将转录组的38588条Unigenes比对发现有2%—3%基因发生了编程性移码,其中,绝大多数为+1PRF;除此之外,艾美游仆虫的终止密码子还存在重配现象,其终止密码子为UAA和UAG,而UGA编码半胱氨酸或硒代半胱氨酸。这与游仆虫属的编程性移码及终止密码子重配的特点一致。将27650条基因组Contigs与38588条转录组U...     

Massive colonization of protein-coding exons by selfish genetic elements in Paramecium germline genomes

Ciliates are unicellular eukaryotes with both a germline genome and a somatic genome in the same cytoplasm. The somatic macronucleus (MAC), responsible for gene expression, is not sexually transmitted but develops from a copy of the germline micronucleus (MIC) at each sexual generation. In the MIC genome of Paramecium tetraurelia, genes are interrupted by tens of thousands of unique intervening sequences called internal eliminated sequences (IESs), which have to be precisely excised during the development of the new MAC to restore functional genes. To understand the evolutionary origin of this peculiar genomic architecture, we sequenced the MIC genomes of 9 Paramecium species (from approximately 100 Mb in Paramecium aurelia species to >1.5 Gb in Paramecium caudatum). We detected several waves of IES gains, both in ancestral and in more recent lineages. While the vast majority of IESs are single copy in present-day genomes, we identified several families of mobile IESs, including nonautonomous elements acquired via horizontal transfer, which generated tens to thousands of new copies. These observations provide the first direct evidence that transposable elements can account for the massive proliferation of IESs in Paramecium. The comparison of IESs of different evolutionary ages indicates that, over time, IESs shorten and diverge rapidly in sequence while they acquire features that allow them to be more efficiently excised. We nevertheless identified rare cases of IESs that are under strong purifying selection across the aurelia clade. The cases examined contain or overlap cellular genes that are inactivated by excision during development, suggesting conserved regulatory mechanisms. Similar to the evolution of introns in eukaryotes, the evolution of Paramecium IESs highlights the major role played by selfish genetic elements in shaping the complexity of genome architecture and gene expression.

A comparative genomics study of nine Paramecium species reveals successful invasion of genes by transposable elements in their germline genomes, showing that the internal eliminated sequences (IESs) followed an evolutionary trajectory remarkably similar to that of spliceosomal introns.     

Time-course analysis of nuclear events during conjugation in the marine ciliate Euplotes vannus and comparison with other ciliates (Protozoa,Ciliophora)

Ciliates represent a morphologically and genetically distinct group of single-celled eukaryotes that segregate germline and somatic functions into two types of nuclei and exhibit complex cytogenetic events during the sexual process of conjugation, which is under the control of the so-called “mating type systems”. Studying conjugation in ciliates may provide insight into our understanding of the origins and evolution of sex and fertilization. In the present work, we studied in detail the sexual process of conjugation using the model species Euplotes vannus, and compared these nuclear events with those occurring in other ciliates. Our results indicate that in E. vannus: 1) conjugation requires about 75 hours to complete: the longest step is the development of the new macronucleus (ca. 64h), followed by the nuclear division of meiosis I (5h); the mitotic divisions usually take only 2h; 2) there are three prezygotic divisions (mitosis and meiosis I and II), and two of the eight resulting nuclei become pronuclei; 3) after the exchange and fusion of the pronuclei, two postzygotic divisions occur; two of the four products differentiate into the new micronucleus and macronucleus, respectively, and the parental macronucleus degenerates completely; 4) comparison of the nuclear events during conjugation in different ciliates reveals that there are generally three prezygotic divisions while the number of postzygotic divisions is highly variable. These results can serve as reference to investigate the mating type system operating in this species and to analyze genes involved in the different steps of the sexual process.     

Tetrahymena macronuclear genome mapping: colinearity Of macronuclear coassortment groups and the micronuclear map on chromosome 1l

The genetics of the ciliate Tetrahymena thermophila are richer than for most other eukaryotic cells, because Tetrahymena possesses two genomes: a germline (micronuclear) genome that follows a Mendelian model of genetic transmission and a somatic (macronuclear) genome, derived from the micronuclear genome by fragmentation, which follows a different genetic transmission model called phenotypic assortment. While genetic markers in the micronucleus fall into classical linkage groups under meiotic recombination and segregation, the same markers in the macronucleus fall into coassortment groups (CAGs) under phenotypic assortment by the random distribution of MAC chromosome pieces. We set out to determine whether genomic mapping in the macronucleus by genetic means is feasible. To investigate the relationship between the micronuclear map and coassortment groups, we systematically placed into CAGs all of the markers lying on chromosome 1L that are also found in the macronucleus. Sixteen CAGs were identified, 7 of which contain at least two loci. We have concluded that CAGs represent a fundamental genetic feature of the MAC. The MIC and MAC maps on 1L are colinear; that is, CAGs consist exclusively of markers that map to a continuous segment in a given region of the micronuclear map, with no intervening markers from other CAGs. These findings provide a solid foundation for exploiting the MAC chromosome pieces to build a physical map of the Tetrahymena genome.     

UAA and UAG may Encode Amino Acid in Cathepsin B Gene of Euplotes octocarinatus

The ciliate Euplotes deviates from the universal genetic code by translating UGA as cysteine and using UAA and UAG as the termination codon. Here, we cloned and sequenced the Cathepsin B gene of Euplotes octocarinatus (Eo‐CTSB) which containing several in‐frame stop codons throughout the coding sequence. We provide evidences, based on 3′‐RACE method and Western blot, that the Eo‐CTSB gene is actively expressed. Comparison of the derived amino acid sequence with the homologs in other eukaryotes revealed that UAA and UAG may code for glutamine in Eo‐CTSB. These findings imply an evolutionary complexity of stop codon reassignment in eukaryotes.     

New contribution to epigenetic studies: Isolation of micronuclei with high purity and DNA integrity in the model ciliated protist,Tetrahymena thermophila

The ciliated protist Tetrahymena thermophila is a well-known model organism with typical nuclear dimorphism containing a somatic macronucleus (MAC) and a germline micronucleus (MIC). The presence in the same cell compartment of two nuclei with distinctly different structural and functional properties provides an ideal model system to explore mechanisms of genome maintenance. Although methods for the isolation of MIC have been available for many years, cross-contamination and DNA degradation remain unresolved. Here, we describe a reliable and quick method to isolate MIC with high purity and DNA integrity in T. thermophila. Different factors are examined to optimize the MIC purification. The MAC contamination ratio in purified MIC is about 0.19% and DNA integrity of purified MIC is maintained. We also establish a more accurate method to detect the contamination rate of nuclei including microscopic observation and PCR detection. This study will facilitate further epigenetic research in Tetrahymena.     

八肋游仆虫氨酰-tRNA合成酶基因序列分析

氨酰-tRNA合成酶 (aminoacyl-tRNA synthetase, aaRS) 是蛋白质生物合成中的关键酶,能够催化特定的氨基酸和相应tRNA结合。为了研究八肋游仆虫氨酰 tRNA合成酶(Euplotes octocarinatus aminoacyl-tRNA synthetase, EoaaRS)基因的种类、数目、结构及起源,本研究利用生物信息学方法,对八肋游仆虫大核基因组编码的aaRS进行了系统分析。结果表明,八肋游仆虫大核基因组共包含45个aaRS基因,可编码20种不同的aaRS蛋白。其中,EoGlnRS和EoAlaRS仅由1个基因编码,其余EoaaRS均由多个基因编码。亚细胞定位分析显示,仅8个EoaaRS具有线粒体导肽,对应于6种EoaaRS。此外,基于核酸序列分析显示,多个EoaaRS在翻译过程中需要发生编程性核糖体移码,才能形成结构完整的蛋白质产物。结构域分析表明,部分EoaaRS存在特殊结构域,暗示其可能具有氨酰化以外的新功能。进化分析揭示,2个EoGlyRS起源于古菌,而2个EoLysRS起源于细菌。本研究为后续探讨低等真核生物aaRS的结构与功能奠定了基础。     

Intraclonal Dimorphism of Caudal Cirri in Euplotes vannus: Cortical Determination*

SYNOPSIS. Intraclonal variation in number of right caudal cirri (RCC) occurs within some species of the hypotrichous genus Euplotes. Euplotes vannus, a marine species, may have either 2 or 3 RCC. A single clone always contains individuals of both types. The frequency of individuals of each type within a clone was found to be 0.5. This fact suggested that during division each parental cell gives rise to one daughter having 3 RCC and one having 2. Formation of RCC during division was studied in E. vannus and in E. plumipes, a fresh-water form which always has 2 RCC. The studies were made on living animals and on fixed animals stained with protargol or by the Chatton-Lwoff method. In both species, the new RCC first appear in the right dorsal kineties and later migrate to the ventral surface. The RCC for the proter develop near the parental equator while those for the opisthe form near the posterior end of the parent cell, both sets developing in close proximity to kinetosomes of the kineties. In both species the 2 dorsal kineties furthest to the right each give rise to 2 RCC, one for the proter and one for the opisthe. In E. vannus, however, the third-from-the-right dorsal kinety also produces one right caudal cirrus for the proter. Therefore, in E. vannus it is the proter which always receives 3 caudal cirri and the opisthe which gets only 2. The role of the cortex in determining these events is discussed. Two cases of abnormal caudal cirrus formation are also described. Other aspects of morphogenesis during division, not previously reported, are also presented and discussed.     

Comparative analysis of single-cell genome sequencing techniques toward the characterization of germline and somatic genomes in ciliated protists

Ciliated protists contain both germline micronucleus (MIC) and somatic macronucleus (MAC) in a single cytoplasm. Programmed genome rearrangements occur in ciliates during sexual processes, and the extent of rearrangements varies dramatically among species, which lead to significant differences in genomic architectures. However, genomic sequences remain largely unknown for most ciliates due to the difficulty in culturing and in separating the germline from the somatic genome in a single cell. Single-cell whole genome amplification (WGA) has emerged as a powerful technology to characterize the genomic heterogeneity at the single-cell level. In this study, we compared two single-cell WGA, multiple displacement amplification (MDA) and multiple annealing and looping-based amplification cycles (MALBAC) in characterizing the germline and somatic genomes in ciliates with different genomic architectures. Our results showed that: 1) MALBAC exhibits strong amplification bias towards MAC genome while MDA shows bias towards MIC genome of ciliates with extensively fragmented MAC genome; 2) both MDA and MALBAC could amplify MAC genome more efficiently in ciliates with moderately fragmented MAC genome. Moreover, we found that more sample replicates could help to obtain more genomic data. Our work provides a reference for selecting the appropriate method to characterize germline and somatic genomes of ciliates.     

Reprogramming the genetic code: The emerging role of ribosomal frameshifting in regulating cellular gene expression

Reading frame maintenance is a critical property of ribosomes. However, a number of genetic elements have been described that can induce ribosomes to shift on mRNAs, the most well understood of which are a class that directs ribosomal slippage by one base in 5' (‐1) direction. This is referred to as programmed ‐1 ribosomal frameshifting (‐1 PRF). Recently, a new ‐1 PRF promoting element was serendipitously discovered in a study examining the effects of stretches of adenosines in the coding sequences of mRNAs. Here, we discuss this finding, recent studies describing how ‐1 PRF is used to control gene expression in eukaryotes, and how ‐1 PRF is itself regulated. The implications of dysregulation of ‐1 PRF on human health are examined, as are possible new areas in which novel ‐1 PRF promoting elements might be discovered. Also watch the Video Abstract .

     

FSscan: a mechanism-based program to identify +1 ribosomal frameshift hotspots

In +1 programmed ribosomal frameshifting (PRF), ribosomes skip one nucleotide toward the 3′-end during translation. Most of the genes known to demonstrate +1 PRF have been discovered by chance or by searching homologous genes. Here, a bioinformatic framework called FSscan is developed to perform a systematic search for potential +1 frameshift sites in the Escherichia coli genome. Based on a current state of the art understanding of the mechanism of +1 PRF, FSscan calculates scores for a 16-nt window along a gene sequence according to different effects of the stimulatory signals, and ribosome E-, P- and A-site interactions. FSscan successfully identified the +1 PRF site in prfB and predicted yehP, pepP, nuoE and cheA as +1 frameshift candidates in the E. coli genome. Empirical results demonstrated that potential +1 frameshift sequences identified promoted significant levels of +1 frameshifting in vivo. Mass spectrometry analysis confirmed the presence of the frameshifted proteins expressed from a yehP-egfp fusion construct. FSscan allows a genome-wide and systematic search for +1 frameshift sites in E. coli. The results have implications for bioinformatic identification of novel frameshift proteins, ribosomal frameshifting, coding sequence detection and the application of mass spectrometry on studying frameshift proteins.     

Evolution of the Germline Actin Gene in Hypotrichous Ciliates: Multiple Nonscrambled IESs at Extremely Conserved Locations in Two Urostylids

In hypotrichous ciliates, macronuclear chromosomes are gene‐sized, and micronuclear genes contain short, noncoding internal eliminated segments (IESs) as well as macronuclear‐destined segments (MDSs). In the present study, we characterized the complete macronuclear gene and two to three types of micronuclear actin genes of two urostylid species, i.e. Pseudokeronopsis rubra and Uroleptopsis citrina. Our results show that (1) the gain/loss of IES happens frequently in the subclass Hypotrichia (formerly Stichotrichia), and high fragmentation of germline genes does not imply for gene scrambling; and (2) the micronuclear actin gene is scrambled in the order Sporadotrichida but nonscrambled in the orders Urostylida and Stichotrichida, indicating the independent evolution of MIC‐actin gene patterns in different orders of hypotrichs; (3) locations of MDS–IES junctions of micronuclear actin gene in coding regions are conserved among closely related species.