Cloning and sequence analysis of the micronuclear and macronuclear gene encoding Rab protein of Euplotes octocarinatus

The DNA in a micronucleus undergoes remarkable rearrangements when it develops into a macronucleus after cell mating in the hypotrichous ciliate. A Rab gene was isolated from the macronuclear plasmid mini-library of Euplotes octocarinatus. A micronuclear version of the Rab gene was amplified by polymerase chain reaction (PCR). The macronuclear DNA molecule carrying the Rab gene is 767 bp long and shows characteristics typical of macronuclear chromosomes of hypotrichous ciliates. Three of the five cysteines are encoded by the opal codon UGA. The deduced protein is a 207-amino acid (aa) with a molecular mass of 23 kDa. The protein shares 36% identity with Rab 1 protein of Plasmodium and yeast. Analysis of the sequences indicated that the micronuclear version of the Rab gene contains two internal eliminated sequences, internal eliminated sequence (IES)1 and IES2. IES1 is flanked by a pair of hepta-nucleotide 5'-AAATTTT-3' direct repeats, and IES2 is flanked by 5'-TA-3' direct repeats.     

八肋游仆虫一种富含三核苷酸重复序列的新基因GARP的克隆与序列分析

人类基因中三核苷酸重复序列拷贝数的异常扩增, 可导致多种神经系统疾病。一种富含GAA三核苷酸的GARP (glutamic acid-rich protein)基因从八肋游仆虫(Euplotes octocarinatus)大核文库中筛选获得。大核中该基因的染色体全长460 bp, 基因两端具有下毛类纤毛虫大核特有的端粒序列(C₄A₄C₄A₄C₄A₄C₄), 开放读框内含有一个TGA_(88-99)密码子, 在游仆虫中编码为半胱氨酸。经DNA Star 软件分析, 该基因编码的蛋白质由112个氨基酸组成, 预测其分子量为13 kDa, 等电点为3.82, 含有四个 [[alpha]] 螺旋和一个 [[beta]] 折叠。小核中对应的该基因含有两个内部删除序列, IES1 和IES2。IES1和IES2分别长41 bp, IES1以GA二核苷酸直接重复为删除信号, IES2以TA二核苷酸直接重复为删除信号。RT- PCR 证明该基因具有转录活性。     

Analysis of micronuclear, macronuclear and cDNA sequences encoding the regulatory subunit of cAMP-dependent protein kinase of Euplotes octocarinatus: evidence for a ribosomal frameshift

We have isolated and characterized the micronuclear gene encoding the regulatory subunit of cAMP-dependent protein kinase of the ciliated protozoan Euplotes octocarinatus, as well as its macronuclear version and the corresponding cDNA. Analyses of the sequences revealed that the micronuclear gene contains one small 69-bp internal eliminated sequence (IES) that is removed during macronuclear development. The IES is located in the 5'-noncoding region of the micronuclear gene and is flanked by a pair of tetranucleotide 5'-TACA-3' direct repeats. The macronuclear DNA molecule carrying this gene is approximately 1400 bp long and is amplified to about 2000 copies per macronucleus. Sequence analysis suggests that the expression of this gene requires a +1 ribosomal frameshift. The deduced protein shares 31% identity with the cAMP-dependent protein kinase type I regulatory subunit of Homo sapiens, and 53% identity with the regulatory subunit R44 of one of the two cAMP-dependent protein kinases of Paramecium. In addition, it contains two highly conserved cAMP binding sites in the C-terminal domain. The putative autophosphorylation site ARTSV of the regulatory subunit of E. octocarinatus is similar to that of the regulatory subunit R44 of Paramecium but distinct from the consensus motif RRXSZ of other eukaryotic regulatory subunits of cAMP-dependent protein kinases.     

Micronuclear and macronuclear forms of beta-tubulin genes in the ciliate Chilodonella uncinata reveal insights into genome processing and protein evolution

Chilodonella uncinata, like all ciliates, contains two distinct nuclei in every cell: a germline micronucleus and a somatic macronucleus. During development of the macronucleus from a zygotic nucleus, the genome is processed in several ways, including elimination of internal sequences. In this study, we analyze micronuclear and macronuclear copies of beta-tubulin in C. uncinata and find at least four divergent paralogs of beta-tubulin in the macronucleus. We characterize the micronuclear version of one paralog and compare its internally eliminated sequences (IESs) with previously described IESs in this species. These comparisons reveal the presence of a conserved sequence motif within IESs. In addition, we compare the sequences of beta-tubulin from C. uncinata with other ciliates and to other alveolates in order to test the hypothesis that the mode of molecular evolution in ciliates obscures phylogenetic signal in protein-coding genes. We find that heterogeneous rates of substitution in beta-tubulin across ciliates result in unstable genealogies that are inconsistent with phylogenies based on small subunit rDNA genes and on ultrastructure. We discuss the implications of our findings for genome processing and protein evolution in ciliates.     

Volatility of internal eliminated segments in germ line genes of hypotrichous ciliates.

Germ line micronuclear genes in ciliated protozoa contain two types of interrupting sequences. Some genes contain introns, but internal eliminated segments (IESs) are much more prevalent. IESs are AT-rich DNA segments that separate macronucleus-destined segments (MDSs) in micronuclear genes. All IESs are excised and destroyed when a micronucleus develops into a macronucleus after each cell mating. IESs have no discernible function. Therefore, an investigation of the behavior of IESs in evolution has been undertaken to assess their possible significance. The IESs in the micronuclear gene encoding the beta-subunit of the telomere-binding protein (beta-TP) are not conserved in number, position, sequence, or length during the evolution of four oxytrichid ciliates. In contrast, the scrambled pattern of MDSs and IESs of the micronuclear actin I gene has been conserved during evolution; however, the precise positions, sequences, and lengths of the IESs differ among species, and in some organisms the actin I gene contains an additional IES and MDS. Corresponding IESs in the actin I genes among the different organisms have shifted positions by 1 to 14 bp, presumably by a mutation-shifting mechanism, creating differences in the repeat sequences flanking IESs. Thus, conservation of a particular repeat sequence among species is not required for IES excision. The changes in IES number and position in the beta-TP genes among ciliates are in sharp contrast to the stability of the intron position. Therefore, IESs are volatile, hypermutable elements that are inserted, removed, shifted, and modified continuously in the germ line through evolutionary time.     

Template-guided recombination for IES elimination and unscrambling of genes in stichotrichous ciliates

The micronuclear versions of genes in stichotrichous ciliates are interrupted by multiple, short, non-coding DNA segments called internal eliminated segments, or IESs. IESs divide a gene into macronuclear destined segments, or MDSs. In some micronuclear genes MDSs are in a scrambled disorder. During development of a micronucleus into a macronucleus after cell mating the IESs are excised from micronuclear genes and the MDSs are spliced in the sequentially correct order. Pairs of short repeat sequences in the ends of MDSs undergo homologous recombination to excise IESs and splice MDSs. However, the repeat sequences are too short to guide unambiguously their own alignment in preparation for recombination. Based on experiments by others on the distantly related ciliate, Paramecium, we propose a molecular model of template-guided recombination to explain the excision of the 100,000-150,000 IESs and splicing of MDSs, including unscrambling, in the genome of stichotrichous ciliates. The model solves the problem of correct pairing of pointers, precisely identifies MDS-IES junctions, and provides for irreversible recombination.     

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.     

Tec3, a new developmentally eliminated DNA element in Euplotes crassus

More than 100,000 interstitial segments of DNA (internal eliminated sequences [IESs]) are excised from the genome during the formation of a new macronucleus in Euplotes crassus. IESs include unique sequence DNA as well as two related families of transposable elements, Tec1 and Tec2. Here we describe a new class of E. crassus transposons, Tec3, which is present in 20 to 30 copies in the micronuclear genome. Tec3 elements have long inverted terminal repeats and contain a degenerate open reading frame encoding a tyrosine-type recombinase. One characterized copy of Tec3 (Tec3-1) is 4.48 kbp long, has 1.23-kbp inverted terminal repeats, and resides within the micronuclear copy of the ribosomal protein L29 gene (RPL29). The 23 bp at the extreme ends of this element are very similar to those in other E. crassus IESs and, like these other IESs, Tec3-1 is excised during the polytene chromosome stage of macronuclear development to generate a free circular form with an unusual junction structure. In contrast, a second cloned element, Tec3-2, is quite similar to Tec3-1 but lacks the terminal 258 bp of the inverted repeats, so that its ends do not resemble the other E. crassus IES termini. The Tec3-2 element appears to reside in a large segment of the micronuclear genome that is subject to developmental elimination. Models for the origins of these two types of Tec3 elements are presented, along with a discussion of how some members of this new transposon family may have come to be excised by the same machinery that removes other E. crassus IESs.     

Conserved DNA sequences adjacent to chromosome fragmentation and telomere addition sites in Euplotes crassus.

During the formation of a new macronucleus in the ciliate Euplotes crassus, micronuclear chromosomes are reproducibly broken at approximately 10 000 sites. This chromosome fragmentation process is tightly coupled with de novo telomere synthesis by the telomerase ribonucleoprotein complex, generating short linear macronuclear DNA molecules. In this study, the sequences of 58 macronuclear DNA termini and eight regions of the micronuclear genome containing chromosome fragmentation/telomere addition sites were determined. Through a statistically based analysis of these data, along with previously published sequences, we have defined a 10 bp conserved sequence element (E-Cbs, 5'-HATTGAAaHH-3', H = A, C or T) near chromosome fragmentation sites. The E-Cbs typically resides within the DNA destined to form a macronuclear DNA molecule, but can also reside within flanking micronuclear DNA that is eliminated during macronuclear development. The location of the E-Cbs in macronuclear-destined versus flanking micronuclear DNA leads us to propose a model of chromosome fragmentation that involves a 6 bp staggered cut in the chromosome. The identification of adjacent macronuclear-destined sequences that overlap by 6 bp provides support for the model. Finally, our data provide evidence that telomerase is able to differentiate between newly generated ends that contain partial telomeric repeats and those that do not in vivo.     

Conservation of sequences adjacent to the telomeric C4A2 repeats of ciliate macronuclear ribosomal RNA gene molecules.

We sequenced and compared the telomeric regions of linear rDNAs from vegetative macronuclei of several ciliates in the suborder Tetrahymenina. All telomeres consisted of tandemly repeated C4A2 sequences, including the 5' telomere of the 11 kb rDNA from developing macronuclei of Tetrahymena thermophila. Our sequence of the 11 kb 5' telomeric region shows that each one of a previously described pair of inverted repeats flanking the micronuclear rDNA (Yao et al., Mol. Cell. Biol. 5: 1260-1267, 1985) is 29 bp away from the positions to which telomeric C4A2 repeats are joined to the ends of excised 11 kb rDNA. In general we found that the macronuclear rDNA sequences adjacent to C4A2 repeats are not highly conserved. However, in the non-palindromic rDNA of Glaucoma, we identified a single copy of a conserved sequence, repeated in inverted orientation in Tetrahymena spp., which all form palindromic rDNAs. We propose that this sequence is required for a step in rDNA excision common to both Tetrahymena and Glaucoma.     

尖毛虫属Actin Ⅰ、α-TBP和DNA pol α乱序基因的模式研究

研究旨在对尖毛虫属内现有物种的3种乱序小核基因结构进行比较,探讨其乱序模式。于湛江湖光红树林水域中采集到一个尖毛虫属物种Oxytricha sp.（ZJ）,成功扩增了其肌动蛋白Ⅰ（ActinⅠ）、端粒结合蛋白（α-TBP）、DNA聚合酶α（DNA pol α）3个乱序基因的完整大核基因序列和完整/部分小核基因序列,并结合已有资料对比研究了尖毛虫属这3个乱序基因的进化。结果表明:（1）Oxytricha sp.（ZJ）与O.nova的小核Actin Ⅰ基因具有相同的乱序模式,区别于其余的尖毛虫属物种;在增加尖毛虫属物种的基础上,对前人推测提出了质疑,我们认为MDS-IES接合处移动现象在乱序MDSs之间并非比非乱序MDSs之间更保守。（2）Oxytricha sp.（ZJ）与O.nova的小核α-TBP基因具有相同乱序模式和相似长度的IESs。（3）Oxytricha sp.（ZJ）的小核DNA pol α基因乱序模式,区别于任一已报道物种,与属内O. trifallax最为相近。基于序列分析,在DNA pol α基因中发现了一例IES转换为MDS的痕迹,以及由此导致原先MDS的丢失。研究发现在编码区内IES向MDS的转变,使得本应删除的序列成为基因组永久保留的一部分。     

The scrambled Actin I gene in<Emphasis Type="Italic"> Uroleptus pisces</Emphasis>

The micronuclear gene encoding actin I in Uroleptus pisces occurs in two segments. Segment I contains 638 bp divided into six macronuclear destined subsegments, or MDSs, by five internal eliminated segments, or IESs. The MDSs in segment 1 are in the scrambled disorder, 1-2-4-8-6-15, with MDSs 8 and 6 inverted. Segment II contains 2452 bp divided into ten MDSs by nine IESs in the scrambled disorder, 3-5-7-10-13-12-9-14-16-11, with MDSs 12, 9, and 11 inverted. Extensive attempts by polymerase chain reaction to connect the two segments failed. We conclude that the two segments are separated by a very long IES or are in different loci. The pattern of the 16 scrambled MDSs is entirely different from the scrambled pattern observed for the actin I gene in six other stichotrichs. We conclude that the actin I gene became scrambled on two separate occasions during stichotrich evolution: once in the lineage leading to the group of six stichotrichs, which includes, among others, Sterkiella species and Stylonychia lemnae, and once in the lineage leading to Uroleptus pisces. Repeated sequence pairs (pointers) of three to 14 bases at the ends of MDSs appear to be essential for correct splicing of MDSs during macronuclear development. However, the micronuclear actin gene also contains 40 matches of eight or more bases between IESs and MDSs that do not function as pointers. To prevent these ectopic repeats from causing improper processing of the micronuclear gene appears to demand a template of DNA or RNA from the old macronucleus to guide splicing of MDSs in the orthodox order.Communicated by A. SpradlingAccession numbers: AY373659, AY382825, AY382826     

The Micronuclear Gene Encoding β-Telomere Binding Protein in Oxytricha nova

ABSTRACT The micronuclear version of the gene encoding β-telomere binding protein (β-TBP) in Oxytricha nova has been sequenced and compared to the macronuclear β-TBP gene, previously described. The micronuclear gene contains three AT-rich internal eliminated sequences (IES) of 37, 40, and 43 bp and four macronuclear destined sequences (MDS). The IES interrupt the gene once near the 5′ end of the coding region and twice in the 3′ trailer downstream from the TGA stop codon. The sequences of the micronuclear and macronuclear genes are colinear. Thus, the micronuclear β-TBP gene is not scrambled, which contrasts with the highly scrambled state among the 14 MDS in the micronuclear α;-TBP gene.     

Internal Eliminated Segments (IESs) of Oxytrichidae1

ABSTRACT Internal eliminated segments (IESs) are sequences that interrupt coding and noncoding regions of germline (micronuclear) genes of ciliated protozoa. IESs are flanked by short, unique repeat sequences, which are presumably required for precise IES excision during macronuclear development. Coding and noncoding segments of genes separated by IESs are called macronuclear-destined segments, or MDSs. We have compiled the characteristics of 89 individual IESs in 12 micronuclear genes in the Oxytricha and Stylonychia genera to define the IES phenomenon precisely, a first step in determining the origin, function and significance of IESs. Although all 89 IESs among the 12 different genes are AT-rich, they show no other similarity in sequence, length, position or number. Two main types of IESs are present. IESs that separate scrambled MDSs are significantly shorter and more frequent and have longer flanking repeat sequences than IESs that intervene between nonscrambled MDSs. A comparison of the nonscrambled gene encoding β-telomere binding protein in three species of hypotrichs shows that even in the same gene IESs are not conserved in sequence, length, position, or number from species to species. A comparison of IESs in the scrambled gene encoding actin I in the three species shows that the evolutionary behavior of IESs in a scrambled gene may be more constrained. However, IESs in the scrambled actin I gene have shifted along the DNA molecule during evolution. In total, the various studies show that IESs are hypermutable in sequence and length. They insert, excise, and shift along DNA molecules more or less randomly during evolution, with no discernible function or consequences.     

Internal sequences are eliminated from genes during macronuclear development in the ciliated protozoan Oxytricha nova

During the life cycle of the hypotrichous ciliate Oxytricha nova, a macronucleus containing short, gene-sized DNA molecules is produced from a copy of the chromosomal micronuclear genome. In order to characterize the process of macronuclear development, we have isolated and determined the DNA sequence of a particular macronuclear gene and its micronuclear precursor. The results of this analysis indicate that macronuclear telomeric sequences (5'C4A4(3') repeats) are not present at the ends of the gene in its micronuclear chromosomal location and must be added during development. In addition, the micronuclear copy of the gene contains three short blocks of sequence that must be removed during development, implying the involvement of a nucleic acid-splicing process in generating mature macronuclear genes.     

A mutation in Paramecium tetraurelia reveals functional and structural features of developmentally excised DNA elements.

The excision of internal eliminated sequences (IESs) from the germline micronuclear DNA occurs during the differentiation of a new macronuclear genome in ciliated protozoa. In Paramecium, IESs are generally short (28-882 bp), AT rich DNA elements that show few conserved sequence features with the exception of an inverted-terminal-repeat consensus sequence that has similarity to the ends of mariner/Tcl transposons (KLOBUTCHER and HERRICK 1995). We have isolated and analyzed a mutant cell line that cannot excise a 370-bp IESs (IES2591) from the coding region of the 51A variable surface protein gene. A single micronuclear C to T transition within the consensus sequence prevents excision. The inability to excise IES259 I has revealed a 28-bp IES inside the larger IES, suggesting that reiterative integration of these elements can occur. Together, the consensus sequence mutation and the evidence for reiterative integration support the theory that Paramecium IESs evolved from transposable elements. Unlike a previously studied Paramecium IES, the presence of this IES in the macronucleus does not completely inhibit excision of its Mild-type micronuclear copy through multiple sexual generations.     

Rare internal C4A4 repeats in the micronuclear genome of Oxytricha fallax.

Approximately 20,000 different short, linear, macronuclear DNA molecules are derived from micronuclear sequences of Oxytricha fallax after conjugation. These macronuclear DNAs are terminated at both ends by 20 base pairs of the sequence 5'-dC4A4-3'. Sequences homologous to this repeat (C4A4+) are also abundant in the micronuclear chromosomes, but most reside at their telomeres. Here we show that nontelomeric C4A4 clusters of 20 base pairs or longer exist in only a few hundred copies per micronuclear genome. This demonstrates that nearly none of the 20,000 sequence blocks of micronuclear DNA destined to be macronuclear DNA molecules can be flanked by full-length (20-base pair) C4A4 clusters, and therefore C4A4 repeats must be added to most, if not all, macronuclear telomeres during macronuclear development. Six internal micronuclear C4A4+ loci were cloned, and their structural relationships with macronuclear and micronuclear sequences were examined. The possible origins and functions of these rare, micronuclear internal C4A4 loci are discussed.     

The germline gene encoding DNA polymerase alpha in the hypotrichous ciliate Oxytricha nova is extremely scrambled.

We report the structure of the micronuclear (germline) gene encoding the large catalytic subunit of DNA polymerase alpha (DNA pol alpha) in the ciliate Oxytricha nova. It contains 44 internal eliminated segments (IESs) that divide the gene into 45 macronuclear-destined segments (MDSs) that are in a non-randomly scrambled order with an inversion near the gene center. Odd numbered MDSs 29-43, containing 230 bp out of a total of 4938 bp of macronuclear sequence, are missing from the 14 kb cloned gene. The missing MDSs have not been located but are at least several kilobases from the main body of the gene. The remarkably scrambled DNA pol alpha gene must be extensively cut, re-ordered and spliced and an inversion must occur to produce an unscrambled, functional version of the gene during development of a new macronucleus. Unscrambling is hypothesized to occur by a homologous recombination mechanism guided by repeat sequences at MDS ends.     

Macronuclear and micronuclear configurations of a gene encoding the protein synthesis elongation factor EF 1α in Stylonychia lemnae

The micronuclear and macronuclear configurations of a gene encoding the protein synthesis elongation factor EF 1 alpha in the hypotrich ciliate Stylonychia lemnae were compared. The two sequences are generally colinear. The coding sequence of the micronuclear gene is, however, interrupted by a 64 bp insert flanked by a 2 bp direct repeat in a gene region which is moderately conserved among EF 1 alpha genes of different organisms. The insertion site is distinct from known intron positions in eukaryotic EF 1 alpha genes. The insert sequence shows inverted repeats at its ends and thus exhibits typical features of an internal eliminated sequence (IES). Comparison with other such sequences in the related organism Oyxtricha nova shows that the IES falls into a new group of such elements. The macronuclear gene exhibits a strikingly limited codon usage, which cannot be simply explained by the overall base composition of the DNA but probably also relates to the very high copy number of the macronuclear gene and the putative high amount of the gene product.     

Evolution of internal eliminated segments and scrambling in the micronuclear gene encoding DNA polymerase alpha in two Oxytricha species.

To learn about the evolution of internal eliminated segments (IESs) and gene scrambling in hypotrichous ciliates we determined the structure of the micronuclear (germline) gene encoding DNA polymerasealpha(DNA polalpha) in Oxytricha trifallax and compared it to the previously published structure of the germline DNA polalphagene in Oxytricha nova . The DNA polalphagene of O.trifallax contains 51 macronuclear-destined segments (MDSs) separated by 50 IESs, compared to 45 MDSs and 44 IESs in the O.nova gene. This means that IESs and MDSs have been gained and/or lost during evolutionary divergence of the two species. Most of the MDSs are highly scrambled in a similar non-random pattern in the two species. We present a model to explain how IESs, non-scrambled MDSs and scrambled MDSs may be added and/or eliminated during evolution. Corresponding IESs in the two species differ totally in sequence, and junctions between MDSs and IESs are shifted by 1-18 bp in O.trifallax compared to the O.nova gene. In both species a short region of the gene is distantly separated from the main part of the gene. Comparison of the gene in the two species shows that IESs and scrambling are highly malleable over evolutionary time.