Micronuclear Genome Research Articles

Deletion of the Tetrahymena thermophila rDNA replication fork barrier region disrupts macronuclear rDNA excision and creates a fragile site in the micronuclear genome

During macronuclear development the Tetrahymena thermophila ribosomal RNA gene is excised from micronuclear chromosome 1 by site-specific cleavage at chromosome breakage sequence (Cbs) elements, rearranged into a ‘palindromic’ 21 kb minichromosome and extensively amplified. Gene amplification initiates from origins in the 5′ non-transcribed spacer, and forks moving toward the center of the palindrome arrest at a developmentally regulated replication fork barrier (RFB). The RFB is inactive during vegetative cell divisions, suggesting a role in the formation or amplification of macronuclear rDNA. Using micronuclear (germline) transformation, we show that the RFB region facilitates Cbs-mediated excision. Deletion of the RFB inhibits chromosome breakage in a sub-population of developing macronuclei and promotes alternative processing by a Cbs-independent mechanism. Remarkably, the RFB region prevents spontaneous breakage of chromosome 1 in the diploid micronucleus. Strains heterozygous for ΔRFB and wild-type rDNA lose the ΔRFB allele and distal left arm of chromosome 1 during vegetative propagation. The wild-type chromosome is subsequently fragmented near the rDNA locus, and both homologs are progressively eroded, suggesting that broken micronuclear chromosomes are not ‘healed’ by telomerase. Deletion of this 363 bp segment effectively creates a fragile site in the micronuclear genome, providing the first evidence for a non-telomere cis-acting determinant that functions to maintain the structural integrity of a mitotic eukaryotic chromosome.

A new scrambled gene in the ciliate Uroleptus

In the germline micronucleus of spirotrichous ciliates, the gene segments, or macronuclear destined sequences (MDSs), that give rise to the somatic macronucleus are interrupted by internal eliminated sequences (IESs). For some genes, the MDSs are not arranged sequentially, but rather are scrambled, in the micronucleus. Three scrambled genes have been extensively studied in many species: actin I, α -telomere binding protein, and DNA polymerase α. However, in the past decade, no new scrambled genes have been reported, and the prevalence of scrambled genes is still an important question. To screen for scrambled genes, we completely sequenced 11 macronuclear chromosomes in the spirotrich Uroleptus sp., and then pursued their micronuclear organization. This allowed us to identify new scrambled genes, which also display novel features. In this study we describe one of these newly discovered scrambled genes. This gene, tentatively named USG1 ( Unknown Scrambled Gene 1), encodes a putative protein of 1016 aa. While the function of this protein product is not clear, d N/d S calculated from the two alleles suggests the encoded protein is under purifying selection. USG1 consists of 16 germline MDSs, of which 14 are located on one locus. The other locus, which is at least 3 kb away from the main locus, contains two scrambled MDSs separated by a nonscrambled IES. Curiously, one MDS and its outgoing (3′) pointer (direct repeat) overlap intron splice sites, indicating that these DNA sequences may be under dual (or multiple) constraints. Our findings identify a new scrambled gene in the micronuclear genome of a spirotrichous ciliate, and suggest that even more complicated structures may be present.

Complex Germline Architecture: Two Genes Intertwined on Two Loci

The germline micronuclear genome of some ciliated protists can be scrambled, with coding segments disordered relative to the expressed macronuclear genome. Here, we report a surprisingly complex pair of genes that assemble from interwoven segments on two germline loci in the ciliate Uroleptus. This baroque organization requires two scrambled genes to be disentangled from each other from two clusters in the genome, one containing segments 1-2-4-5-6-8-11-13-15-16 and the other 7-9-3-10-12-14, with pieces 1-5 comprising the first gene and 6-16 the second gene. Both genes remain linked in the somatic genome on a 1.5-kb "nanochromosome." This study is the first to reveal that two genes can become scrambled during evolution with their coding segments intertwined. These twin scrambled genes underscore the beauty and exceptions of protist genome architecture, pointing to the critical need for evolutionary biologists to survey protist genomes broadly.

The Tetrahymena thermophila genome sequence: 3. Completing macronuclear chromosome sequence scaffolds and ordering and aligning them along the micronuclear genome

In Ciliates, the diploid, mitotic, genetically silent micronucleus (MIC) is the cell's germline, while the macronucleus (MAC) is the exclusive site of gene expression. During (postzygotic) MAC differentiation in T. thermophila, several hundred chromosomes are generated by site specific fragmentation of the five germline‐derived chromosomes at highly conserved chromosome breakage sites. Telomeres are added to every newly generated end, and chromosomes are amplified to ∼45 N. The Tetrahymena thermophila macronuclear genome was recently whole‐genome‐shotgun sequenced to nine‐fold redundancy at TIGR. The sequence (publicly available at http://www.tigr.org/tdb/e2k1/ttg/) is assembled into contigs (contiguous segments of overlapping sequence reads) linked into scaffolds. Here, we report here the completion of the assembly of many scaffold ends by linking them to their corresponding telomere, using diverse bioinformatic or experimental approaches. 120 chromosomes are now assembled from telomere to telomere and 125 additional scaffolds are telomere capped at one end. The 120 complete scaffolds encompass ∼45% of the ∼105‐Mb assembly, suggesting a total of ∼290 MAC chromosomes. By using paired sequence tags flanking chromosome breakage sites, we are linking unambiguously ordered and oriented MAC chromosome DNA sequence into MIC genome assemblies.Supported by NIH grant RR02931. Preliminary sequence data were obtained from The Institute for Genomic Research website.

Elimination of Foreign DNA during Somatic Differentiation in Tetrahymena thermophila Shows Position Effect and Is Dosage Dependent

In the ciliate Tetrahymena thermophila, approximately 15% of the germ line micronuclear DNA sequences are eliminated during formation of the somatic macronucleus. The vast majority of the internal eliminated sequences (IESs) are repeated in the micronuclear genome, and several of them resemble transposable elements. Thus, it has been suggested that DNA elimination evolved as a means for removing invading DNAs. In the present study, bacterial neo genes introduced into the germ line micronuclei were eliminated from the somatic genome. The efficiency of elimination from two different loci increased dramatically with the copy number of the neo genes in the micronuclei. The timing of neo elimination is similar to that of endogenous IESs, and they both produce bidirectional transcripts of the eliminated element, suggesting that the deletion of neo occurred by the same mechanism as elimination of endogenous IESs. These results indicate that repetition of an element in the micronucleus enhances the efficiency of its elimination from the newly formed somatic genome of Tetrahymena thermophila. The implications of these data in relation to the function and mechanism of IES elimination are discussed.

Epigenetic control of chromosome breakage at the 5' end of Paramecium tetraurelia gene A.

Macronuclei and micronuclei of ciliates have related genomes, with macronuclei developing from zygotic micronuclei through programmed DNA rearrangements. While Paramecium tetraurelia wild-type strain 51 and mutant strain d48 have the same micronuclear genome, qualitative differences between their macronuclear genomes have been described, demonstrating that programmed DNA rearrangements could be epigenetically controlled in ciliates. Macronuclear chromosomes end downstream of gene A (A51 Mac ends) and at the 5' end of gene A (Ad48 Mac ends) in strains 51 and d48, respectively. To gain further insight into the process of chromosome end formation, we performed an extensive analysis of locus A rearrangement in strains d48 and 51, in strain d12, which harbors a gene A deletion, and in interstrain cross progeny. We show that (i) allele Ad12 harbors a deletion of >16 kb, (ii) A51 Mac ends distribute over four rather than three DNA regions, (iii) strains d48 and 51 display only quantitative differences (rare Ad48 and A51 Mac ends do form in strains 51 and d48, respectively), (iv) the level of A51 Mac ends is severalfold enhanced in d12- and d48-derived progeny, and (v) this level inversely correlates with the level of Ad48 Mac ends in the d48 parent. Together, these data lead to a model in which the formation of Ad48 Mac ends is epigenetically controlled by a d48 factor(s). We propose that the d48 factor(s) may be derived from RNA molecules transcribed from the Ad48 Mac ends and encompassing the truncated A gene and telomeric repeats.

A Micronuclear Locus Containing Three Protein-Coding Genes Remains Linked During Macronuclear Development in the Spirotrichous Ciliate Holosticha

We have discovered a three-gene macronuclear chromosome in a spirotrichous ciliate of the genus Holosticha. From 5' to 3', this chromosome contains genes encoding a member of the small G-protein family, an NAD kinase domain-containing protein, and the large subunit of DNA polymerase alpha. These three genes are separated by 16 and 38 nucleotides, respectively, and are oriented in the same direction in both the macronuclear and the micronuclear genomes. Probes made to these genes all hybridize to a single, strong band of size 7.0 kbp on a Southern Blot of Holosticha sp. macronuclear DNA, corresponding to the size of the three-gene macronuclear chromosome. Mapping the 5' and 3' ends of each of these genes using RACE showed that the transcripts of these genes exist as discrete mRNAs that are capped and polyadenylated. No nucleotides appeared to be added at the 5' ends of these transcripts, indicating that these transcripts are not generated by alternative or trans-splicing, but rather that each gene is transcribed from its own distinct promoter. Analysis of these linked genes may help define the evolutionary pressures leading to the extensive chromosome fragmentation seen in spirotrichous ciliates.

Homing endonucleases encoded by germ line-limited genes in Tetrahymena thermophila have APETELA2 DNA binding domains.

Three insertion elements were previously found in a family of germ line-limited mobile elements, the Tlr elements, in the ciliate Tetrahymena. Each of the insertions contains an open reading frame (ORF). Sequence analysis of the deduced proteins encoded by the elements suggests that they are homing endonucleases. The genes are designated TIE1-1, TIE2-1, and TIE3-1 for Tetrahymena insertion-homing endonuclease. The endonuclease motif occupies the amino terminal half of each TIE protein. The C-terminal regions of the proteins are similar to the APETELA2 DNA binding domain of plant transcription factors. The TIE1 and TIE3 elements belong to families of repeated sequences in the germ line micronuclear genome. Comparison of the genes and the deduced proteins they encode suggests that there are at least two distinct families of homing endonuclease genes, each of which appears to be preferentially associated with a specific region of the Tlr elements. The TIE1 and TIE3 elements and their cognates undergo programmed elimination from the developing somatic macronucleus of Tetrahymena. The possible role of homing endonuclease-like genes in the DNA breakage step in developmentally programmed DNA elimination in Tetrahymena is discussed.

Diverse sequences within Tlr elements target programmed DNA elimination in Tetrahymena thermophila.

Tlr elements are a novel family of approximately 30 putative mobile genetic elements that are confined to the germ line micronuclear genome in Tetrahymena thermophila. Thousands of diverse germ line-limited sequences, including the Tlr elements, are specifically eliminated from the differentiating somatic macronucleus. Macronucleus-retained sequences flanking deleted regions are known to contain cis-acting signals that delineate elimination boundaries. It is unclear whether sequences within deleted DNA also play a regulatory role in the elimination process. In the current study, an in vivo DNA rearrangement assay was used to identify internal sequences required in cis for the elimination of Tlr elements. Multiple, nonoverlapping regions from the approximately 23-kb Tlr elements were independently sufficient to stimulate developmentally regulated DNA elimination when placed within the context of flanking sequences from the most thoroughly characterized family member, Tlr1. Replacement of element DNA with macronuclear or foreign DNA abolished elimination activity. Thus, diverse sequences dispersed throughout Tlr DNA contain cis-acting signals that target these elements for programmed elimination. Surprisingly, Tlr DNA was also efficiently deleted when Tlr1 flanking sequences were replaced with DNA from a region of the genome that is not normally associated with rearrangement, suggesting that specific flanking sequences are not required for the elimination of Tlr element DNA.

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.

Spatiotemporal sites of DNA replication in macro- and micronuclei of the ciliate Paramecium caudatum.

Spatiotemporal sites of DNA replication in macro- and micronuclei of the ciliated protozoan Paramecium caudatum were analyzed by confocal laser scanning microscopy following incorporation of the thymidine analogue BrdU and indirect immunofluorescence. In the macronucleus, replication sites were localized to numerous small domains and scattered throughout the nucleoplasm. This pattern persisted during all periods of the S phase. A single constant pattern with discrete replication foci was also observed in the micronucleus. No obvious differences were seen between the two kinds of nuclei. Pulse-chase-pulse double-labeling experiments with two thymidine analogues (CldU and IdU) revealed that dispersed sites of replication were activated at different times during the S phase and a replication site takes about 2h to complete replication in the macronucleus. When cells were labeled by BrUTP to examine transcriptional activity in the two kinds of nuclei, incorporation of BrUTP into the macronucleus occurred throughout the cell cycle, whereas there was no detectable RNA synthesis in the micronucleus. From these findings, we conclude that, despite large differences in structure and function of macro- and micronuclear genomes, both nuclei show a similar replication pattern with discrete subnuclear foci scattered throughout the nucleoplasm at all times during the S phase.

Evolution of IESs and scrambling in the actin I gene in hypotrichous ciliates.

Germ-line (micronuclear) genes in hypotrichous ciliates are interrupted by numerous, short, noncoding, AT-rich segments called internal eliminated segments, or IESs. IESs divide a gene into macronuclear destined segments, or MDSs. IESs are excised from micronuclear genes, and the MDSs are spliced when a micronuclear genome is processed into a macronuclear genome after cell mating. In the micronuclear version of the actin I gene intramolecular recombination between IESs during evolution has put MDSs into a scrambled disorder in some but not all hypotrichs. Studies using rDNA sequences to define phylogenetic relationships among eight hypotrichs suggests that evolution of the micronuclear actin I gene proceeds by successive addition of IESs in earlier diverging species, without MDS scrambling. Continued addition of IESs and recombination among IESs in later diverging species produced actin I genes with scrambled MDSs. Subsequent to MDS scrambling, additional IESs were inserted into the more recently evolved species. Thus, IES insertions and gene scrambling occur in a progressive manner during species evolution to produce micronuclear actin I genes of increasing structural complexity.

Genome gymnastics: unique modes of DNA evolution and processing in ciliates.

In some ciliates, the DNA sequences of the germline genomes have been profoundly modified during evolution, providing unprecedented examples of germline DNA malleability. Although the significance of the modifications and malleability is unclear, they may reflect the evolution of mechanisms that facilitate evolution. Because of the modifications, these ciliates must perform remarkable feats of cutting, splicing, rearrangement and elimination of DNA sequences to convert the chromosomal DNA in the germline genome (micronuclear genome) into gene-sized DNA molecules in the somatic genome (macronuclear genome). How these manipulations of DNA are guided and carried out is largely unknown. However, the organization and manipulation of ciliate DNA sequences are new phenomena that expand a general appreciation for the flexibility of DNA in evolution and development.

Programmed DNA under-amplification in Paramecium primaurelia.

Ciliates are unicellular eukaryotic organisms that contain two types of nuclei throughout their vegetative life, transcriptionally active macronuclei governing the cell phenotype, and transcriptionally inert micronuclei. Following sexual reproduction, new macronuclear genomes regularly develop from micronuclear genomes through programmed DNA rearrangements that include DNA splicing, DNA fragmentation and DNA amplification. In the course of characterization of the micronuclear version of the 9.0 kb G gene, which encodes the G surface antigen in Paramecium primaurelia, we characterized a G gene duplicate. Compared with the G gene, the G gene duplicate displays features identifying it as a psi G pseudogene. About 1.6 kb upstream from the G gene, we characterized a new gene, the P gene. A related psi P putative pseudogene lies 1.6 kb upstream from the psi G pseudogene, showing that the duplicated region extends over > 15 kb and putatively defining it as a pseudogene region. Within macronuclear genomes, this region is highly under-amplified; its level never exceeds 20% of that of the corresponding G gene/P gene region in the 11 cell clones we tested. Under-amplification of the psi G pseudogene/psi P putative pseudogene region could be due to its distal position on macronuclear chromosomes, the use of alternative DNA fragmentation domains being frequent in Paramecium species, or to intrinsically lower amplification of a large genomic region. Therefore, the psi G pseudogene/psi P putative pseudogene region presented in this study provides a useful tool for the analysis of DNA fragmentation and/or amplification in eukaryotic genomes.

A family of developmentally excised DNA elements in Tetrahymena is under selective pressure to maintain an open reading frame encoding an integrase-like protein.

Tlr1 is a member of a family of approximately 20-30 DNA elements that undergo developmentally regulated excision during formation of the macronucleus in the ciliated protozoan TETRAHYMENA: Analysis of sequence internal to the right boundary of Tlr1 revealed the presence of a 2 kb open reading frame (ORF) encoding a deduced protein with similarity to retrotransposon integrases. The ORFs of five unique clones were sequenced. The ORFs have 98% sequence conservation and align without frameshifts, although one has an additional trinucleotide at codon 561. Nucleotide changes among the five clones are highly non-random with respect to the position in the codon and 93% of the nucleotide changes among the five clones encode identical or similar amino acids, suggesting that the ORF has evolved under selective pressure to preserve a functional protein. Nineteen T/C transitions in T/CAA and T/CAG codons suggest selection has occurred in the context of the TETRAHYMENA: genome, where TAA and TAG encode Gln. Similarities between the ORF and those encoding retrotransposon integrases suggest that the Tlr family of elements may encode a polynucleotide transferase. Possible roles for the protein in transposition of the elements within the micronuclear genome and/or their developmentally regulated excision from the macronucleus are discussed.

The Tetrahymena p80/p95 Complex Is Required for Proper Telomere Length Maintenance and Micronuclear Genome Stability

The telomerase enzyme adds simple sequence repeats to chromosome ends. Telomerases share two essential subunits, telomerase RNA and telomerase reverse transcriptase, that associate with species-specific proteins of predominantly unknown functions. The Tetrahymena p80/p95 complex can coimmunopurify active telomerase from cell extract, and recombinant p80/p95 can interact directly with telomerase RNA and single-stranded telomeric DNA in vitro. Here, we test the functions of p80/p95 in vivo. Surprisingly, telomerase RNA accumulation and telomerase activity in cell extract are unaffected by loss of the genes encoding p80/p95. However, in the absence of p80/p95, telomeres become elongated in both macronuclei and micronuclei. Micronuclear chromosome maintenance is also compromised. These findings suggest that p80/p95 functions to maintain appropriate telomere length and micronuclear genomic stability but does so in a manner different than previously anticipated.

The Tetrahymena p80/p95 Complex Is Required for Proper Telomere Length Maintenance and Micronuclear Genome Stability

The Tetrahymena p80/p95 Complex Is Required for Proper Telomere Length Maintenance and Micronuclear Genome Stability

Toward sequencing the Tetrahymena genome: exploiting the gift of nuclear dimorphism.

Important scientific discoveries have utilized the unique advantages of Tetrahymena thermophila as a research organism. Recently developed molecular genetic manipulations allow full exploitation of the many scientific dividends that would result from having its genome sequenced. As a typical ciliated protozoan, Tetrahymena exhibits "nuclear dimorphism". It possesses two differentiated forms of its nuclear genome: a globally repressed, diploid germline or micronuclear genome, and a polyploid, site-specifically fragmented somatic or macronuclear genome. The macronuclear genome is, in effect, a natural, large-insert library of the micronuclear genome. This presentation describes how the gifts of nuclear dimorphism are being exploited in the experimental analysis of molecular and cell biology. Mechanisms present in humans that are either absent in other eukaryotic microbial model systems, or not as readily accessible in them as in Tetrahymena, are especially relevant. This presentation also reviews unique tools generated by nuclear dimorphism that are being used for genetically and physically mapping the Tetrahymena genome.

A novel chromodomain protein, pdd3p, associates with internal eliminated sequences during macronuclear development in Tetrahymena thermophila.

Conversion of the germ line micronuclear genome into the genome of a somatic macronucleus in Tetrahymena thermophila requires several DNA rearrangement processes. These include (i) excision and subsequent elimination of several thousand internal eliminated sequences (IESs) scattered throughout the micronuclear genome and (ii) breakage of the micronuclear chromosomes into hundreds of DNA fragments, followed by de novo telomere addition to their ends. Chromosome breakage sequences (Cbs) that determine the sites of breakage and short regions of DNA adjacent to them are also eliminated. Both processes occur concomitantly in the developing macronucleus. Two stage-specific protein factors involved in germ line DNA elimination have been described previously. Pdd1p and Pdd2p (for programmed DNA degradation) physically associate with internal eliminated sequences in transient electron-dense structures in the developing macronucleus. Here, we report the purification, sequence analysis, and characterization of Pdd3p, a novel developmentally regulated, chromodomain-containing polypeptide. Pdd3p colocalizes with Pdd1p in the peripheral regions of DNA elimination structures, but is also found more internally. DNA cross-linked and immunoprecipitated with Pdd1p- or Pdd3p-specific antibodies is enriched in IESs, but not Cbs, suggesting that different protein factors are involved in elimination of these two groups of sequences.

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.